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[ { "id": "pmcA1805739__text", "type": "Article", "text": [ "Embryonic sympathoblasts transiently express TrkB in vivo and proliferate in response to brain-derived neurotrophic factor in vitro\nAbstract\nBackground\nNerve growth factor and neurotrophin-3 are involved in the development of sympathetic neurons; however, whether brain derived neurotrophic factor also plays a role is not known. The purpose of this study was to determine whether BDNF and its receptor, TrkB, are expressed during the development of paravertebral sympathetic ganglia in vivo and to determine the effect of BDNF in vitro.\n\nResults\nAs neural crest cells coalesce to form sympathetic ganglia, TrkB-positive cells are seen in both chicken and mouse embryos. In chicken embryos, TrkB-expressing cells first appear at Hamburger-Hamilton Stage (St) 27 and they co-express HNK-1, confirming that they are migrating neural crest cells. The TrkB-positive cells lack neural markers at this stage; however, they migrate with other neurally differentiating cells that are TrkA and TrkC-positive. By St. 29/30, TrkB-positive cells begin to express the neural specific markers Hu C/D and Islet-1; eventually, all TrkB positive cells commence neural differentiation. By St. 34, TrkB and TrkC staining are lost. BDNF transcript expression parallels that of TrkB. In the mouse, TrkB-positive cells surround newly formed sympathetic ganglia and a small number of TrkB positive cells that co-express tyrosine hydroxylase are seen within ganglia between E13.5-15. In cell culture, many cells from St. 29–30 chicken lumbar sympathetic ganglia express neural markers and are dividing, indicating that they are sympathoblasts. Sympathoblasts and neurons require both nerve growth factor and neurotrophin-3 for survival. BDNF increases the number of cells expressing neural markers in culture by increasing number of cells that incorporate bromodeoxyuridine. In contrast, most TrkB-positive sympathetic cells in vivo are not actively proliferating between E6–E8.\n\nConclusion\nDeveloping paravertebral sympathetic ganglia in avian and murine embryos contain a subpopulation of sympathoblasts that transiently express TrkB and ultimately commence neuronal differentiation. These TrkB expressing sympathoblasts are not actively dividing in vivo; yet, when placed in vitro, will divide in response to BDNF. This suggests that the availability of BDNF in vivo fails to reach a threshold necessary to induce proliferation. We suggest that excess TrkB stimulation of sympathoblasts in vivo may lead to the genesis of neuroblastoma.\n\n\n\nBackground\nNeural crest cells destined to become paravertebral sympathetic neurons proliferate and differentiate during migration and gangliogenesis. In chicken embryos, migrating neural crest cells express catecholamines at Hamburger/Hamilton Stage (St.) 19, and these cells form the primary sympathetic chain dorsolateral to the aorta at St. 22 (E3.5) [1]. Between St. 23 (E4) and St. 28 (E6), these cells disperse and undergo a secondary migration to form the paravertebral sympathetic chain that resides ventral to the spinal cord and dorsal root ganglion [1]. After ganglia coalesce, sympathoblasts express markers of neuronal differentiation, such as Q211 and tyrosine hydroxylase (TH), at a time when they also incorporate [3H]-thymidine [2]. Time lapse photography has shown that cultured E15.5–E16.5 sympathetic neurons from rat embryos extend axons while they divide [3-5]. Although proliferation appears to be an important process to expand the sympathetic neuron population during differentiation, the mechanisms that guide sympathoblast proliferation have not been identified.\nThe development of sympathetic neurons is guided by neurotrophins. Neurotrophin-3 (NT-3) binds to its receptor, TrkC, to promote the survival of cultured sympathoblasts from early lumbar paravertebral ganglia [6]. Nerve growth factor (NGF) signals through its receptor, TrkA, to promote the survival of sympathetic neurons upon target innervation [7]. There are severe sympathetic defects in the superior cervical ganglion of individual NT-3 and NGF knockout mice [8-10]. Furthermore, there is no additional cell death in the superior cervical ganglion of NT-3 and NGF double knockout mouse embryos, suggesting that all of the neurons are dependent on both neurotrophins for survival [11]. There is also an increase in sympathetic neuron cell death in TrkA knockout mice [12]. However, in TrkB and BDNF knockout mice, there is no apparent phenotype in the superior cervical ganglion, and there is little evidence that TrkB or BDNF is expressed in sympathetic ganglia. Thus, it is generally thought that TrkB and BDNF have little or no roles in guiding the development of sympathetic neurons.\nIn addition to their developmental functions, neurotrophin receptors regulate cell behavior in neuroblastoma, a tumor found in sympathetic ganglia and adrenal medulla. Tumors that express TrkA often spontaneously regress, while those that express TrkB and its ligand, brain-derived neurotrophic factor (BDNF), grow aggressively, are invasive, and fail to respond to chemotherapeutic agents [13]. The presence of TrkA in neuroblastoma tumors is consistent with its expression in developing sympathetic neurons, and suggests that regressive neuroblastoma tumors arise from early sympathetic neurons that express TrkA. The function of TrkB in early sympathetic development is unknown, which makes understanding the etiology of aggressive neuroblastoma tumors difficult. Based on its function in neuroblastoma tumors, we hypothesize that BDNF and TrkB expression in differentiating sympathoblasts is responsible for expanding the neuronal population through proliferation.\nWe sought to determine whether BDNF and TrkB are involved in sympathetic development. We report that during early embryonic development, TrkB is expressed in a subset of differentiating sympathoblasts in both avian and murine embryos. We also find that BDNF promotes the proliferation of TrkB-positive sympathoblasts in cell culture. However, the majority of TrkB positive cells in vivo fail to take up bromodeoxyuridine (BrdU) over a 24 hr period, suggesting that endogenous BDNF concentrations do not reach a threshold necessary to stimulate proliferation of sympathoblasts. Shortly after all of the TrkB positive cells commence neuronal differentiation, TrkB immunoreactivity is lost. These results suggest that prolonged expression and/or activation of TrkB signaling at these early stages may be an early event triggering the formation of neuroblastoma.\n\nResults\nTrkB is expressed during migration of neural crest cells to sympathetic ganglia\nWe first determined whether TrkB is expressed in neural crest-derived cells in the region ventral to the spinal cord and dorsal root ganglia where sympathetic ganglia coalesce between Hamburger/Hamilton Stages (St.) 25–28/29. To identify cells that have commenced neuronal differentiation, transverse sections of the lumbar spinal column region were stained with antibodies against Hu C/D [14], a neuronal-specific RNA-binding protein, or Islet-1, a transcription factor found in sympathetic neurons [15]. We found that Hu C/D and Islet-1 are expressed in the same cells both in vivo and in vitro throughout sympathetic development. In experiments done between St. 25 and 28, Islet-1 staining appeared weaker than Hu C/D staining, and thus we used Hu C/D to identify differentiating neurons at these stages. At later stages, Islet-1 was used to facilitate the identification of neurons because of the nuclear location of its immunoreactivity.\nCells expressing Hu C/D are first detected at St. 25 ventral to the spinal cord and dorsal root ganglion and lateral to the dorsal aorta (Figure 1A, 1B). By St. 26, the number of cells that express Hu C/D in this region increases dramatically (Figure 1C). TrkB-expressing cells first appear at St. 27 in the same region and are adjacent to Hu C/D-positive cells (Figure 1D, 2A, 2H). TrkB-positive cells co-localize with a neural crest marker, HNK-1 (Figures 2B, 2C, 2D). The Hu C/D-positive cells in this region are likely to be sympathetic neurons, since they appear in the region where sympathetic ganglia form and express tyrosine hydroxylase, a rate-limiting enzyme in the synthesis of catecholamines (Figures 2E, 2F, 2G). At St. 28/29, the cells begin to coalesce ventral to the dorsal root ganglion and the Hu C/D-positive cells and TrkB-positive cells remain as two separate cell populations (Figure 1E); however, shortly afterwards, all of the TrkB-positive cells begin to express Islet-1 (Figure 3B) and Hu C/D (data not shown).\n\nDevelopmental regulation of TrkA, TrkB, TrkC, and BDNF expression\nIn contrast to TrkB, the other neurotrophin receptors, TrkA and TrkC, are co-expressed in both Hu C/D-positive and Hu C/D-negative cells at St. 27 (Figures 2I, 2J). We find that approximately 30% of the Islet-1-positive cells express TrkA (Figure 3A), while 50% express TrkB (Figure 3B) and 100% express TrkC (Figure 3C) at St. 29/30 (E6.5). Thus, all developing neurons express TrkC in combination with either TrkA or TrkB. By St. 31 (E7), the number of TrkA-positive, Islet-1-positive cells increases to 100% (Figure 3D) and immunoreactivities for both TrkB and TrkC appear dispersed (Figure 3E, 3F). By St. 34 (E8), TrkA expression is well-sustained (Figure 3G) and TrkB and TrkC immunoreactivities are lost (Figure 3H, 3I). We also examined the early development of murine sympathetic ganglia (Figure 4). At E13, the newly formed lumbar sympathetic ganglia can be observed ventral to the spinal cord and notochord by their staining for Hu C/D and TH (Figure 4A). TrkB-positive cells can be seen surrounding developing ganglia, as well as in occasional cells within the ganglia (Figure 4C, D). These TrkB-positive cells within the ganglia co-express TH and are seen at a frequency of 1–2 cells per section starting at E13 (Figure 4A–D) and are still present at E15.5 (data not shown).\nIn neuroblastoma cells, BDNF is co-expressed with TrkB, suggesting that autocrine stimulation is a means by which proliferation is sustained in the transformed cells. To test whether BDNF, the ligand for TrkB, was present in embryonic chick sympathetic ganglia, we used quantitative real-time PCR with TaqMan probes to determine the relative abundance of BDNF transcripts in total RNA extracted from lumbar sympathetic ganglia at St. 29/30 (E6.5), St. 31 (E7), St. 34 (E8), and E9. BDNF expression within the ganglia parallels that of TrkB: BDNF mRNA expression levels are highest at St 29/30 (E6.5), and these levels decrease 2-fold at St. 31 (E7) and St. 34 (E8; Figure 5). By E9, BDNF levels are 7 times lower than at St. 29/30 (E6.5; Figure 5).\n\nNT3 and NGF promote survival of differentiating sympathetic neurons in culture\nTo determine the effect of neurotrophins, we cultured cells dispersed from lumbar sympathetic ganglia at St. 29/30 (E6.5) because, at this stage, ganglion formation is complete, the number of TrkA-, TrkB-, and TrkC-positive cells have peaked, and all Trk-expressing cells have initiated neural differentiation. First, we identified markers expressed by acutely isolated cells. As shown in Table 1, 80–91% of the cells are p75 neurotrophin receptor (NTR)-positive, indicating that most of the cells are neural crest-derived and little mesenchymal contamination is introduced by the isolation procedure. In addition, 28–33% of the cultured cells express the neural marker Hu C/D. Approximately half of these Hu C/D-positive cells express TrkB. Conversely, all of the TrkB positive cells express Hu C/D. These TrkB-positive cells comprise approximately 14–17% of the total cell population.\nWe then determined how many of the acutely isolated cells were proliferating by incubating them for 12 hrs in BrdU-containing medium. For these experiments, we identified differentiating neurons with the transcription factor Islet-1 because this marker labels nuclei, thus it co localizes with any BrdU that has been incorporated into the DNA, allowing us to determine whether the cell had undergone S-phase of the cell cycle. After 12 hrs in BrdU, 59% of Islet-1-positive nuclei stain for BrdU immunoreactivity. Thus, cultures of St. 29/30 sympathetic ganglia contain many cells that proliferate while exhibiting markers of neuronal differentiation, confirming previous observations [2]. We call these dividing neuronal precursors sympathoblasts. The remaining non-BrdU incorporating, Islet-1 positive cells are likely to be post mitotic neurons.\nFinally, we determined the trophic requirements of St. 29/30 (E6.5) sympathetic neurons and sympathoblasts. We monitored cultures over a three day period after plating and counted the number of phase bright cells with neurites, a morphological feature of both neurons and sympathoblasts. In the absence of trophic factors, more than 2/3 of the cells die by 24 hours in culture and BDNF, NT-3, or NGF alone is not sufficient to promote survival (Figure 6). However, NGF together with NT-3 supports the survival of a significantly larger number of cells (Figure 6). For the subsequent experiments, all neurons were cultured with 25 ng/ml NT-3 and 1 μg/ml 7S NGF to optimize survival.\n\nBDNF promotes proliferation of TrkB-positive sympathetic neurons in culture\nTo determine the effects of BDNF, cultures of cells from St. 29/30 (E6.5) sympathetic ganglia were supplemented with 200 ng/ml BDNF and the number of neurons and sympathoblasts were counted at 24, 48, and 72 hours using phase microscopy (Figure 7A). A 1.6-fold increase in the number of neurons due to BDNF is observed by 24 hours and this number does not increase further at 48 or 72 hours. This effect of BDNF is concentration-dependent with an EC50 of 75 ng/ml (Figure 7B).\nTo test whether the increase in the number of neurons and sympathoblasts caused by BDNF is due to the differentiation of pluripotent neural crest cells, we quantified the effects of BDNF on the number of neurally differentiating cells (Hu C/D-positive) versus the number of non-neuronal cells (Hu C/D-negative) after identifying all neural crest-derived cells by staining for p75NTR in St. 29/30 (E6.5) cultures. If BDNF increases the number of neurons and sympathoblasts by inducing a non-neuronal cell to express Hu C/D, then we expected that the total cell number would remain the same and that there would be a decrease in the number of non-neuronal cells as well as a corresponding increase in the number of neurons. After 24 hours, BDNF significantly increases the number of p75NTR-positive cells as well as the number of Hu C/D-positive cells (Figure 8A). However, there was no statistically significant change in the number of non-neuronal cells. Thus, it is unlikely that BDNF increases the number of neurons and sympathoblasts by inducing differentiation of non-neuronal cells.\nTo determine whether the increase in the total number of neurons and sympathoblasts is caused by BDNF-induced proliferation, control and BDNF-treated sympathetic cultures were exposed to BrdU for 12 hours after plating, and the number of cells that incorporated BrdU into their DNA was determined after 24 hours in culture. Even in the control condition, a number of cells in the culture are dividing, giving a high baseline of BrdU incorporation (Figure 8B). When BDNF is added, the total number of BrdU positive cells increases approximately 1.6-fold (Figure 8B). This BDNF-induced increase in the total number of BrdU positive cells occurs in sympathoblasts because the number of Islet-1-positive nuclei from control cultures that label with BrdU is 268 ± 59 and BDNF treatment raises this number to 424 ± 80, which corresponds to an increase of 1.6-fold. This accounts for the 1.6-fold increase in total neuron number and total BrdU-positive cells described above. We then confirmed that BDNF acts on TrkB-positive cells: BDNF increases the number of TrkB-expressing cells that incorporate BrdU 2.6 – 4-fold over control (Table 2) and it also increases the overall number of TrkB-positive cells 2 – 2.5-fold over control (Table 2). BDNF does not increase the number of BrdU-positive, TrkB-negative cells or the overall number of TrkB-negative cells (Table 2). In further support that BDNF acts directly on TrkB-expressing cells, an antibody directed against the extracellular domain of TrkB completely prevents the effect of BDNF in promoting proliferation of TrkB-positive, but not TrkB-negative cells (compare Figure 9A to 9B). Thus, the effect of BDNF is restricted to the population that expresses TrkB, which are developing sympathoblasts.\nTo determine whether TrkB-positive cells are actively proliferating in vivo, embryos were injected with BrdU at St. 27 and harvested at St. 29, approximately 24 hrs later. The majority (85–90%) of TrkB-positive cells do not incorporate BrdU into their nuclei under basal conditions in vivo (Figure 10), although a few TrkB positive cells with labeled nuclei could be observed (arrows). This contrasts with our observation that 71–76% of TrkB-positive cells incorporate BrdU in culture after treatment with BDNF (Table 2), suggesting that endogenous BDNF does not achieve a threshold sufficient to support a high level of sympathoblast proliferation in vivo.\n\n\nDiscussion\nWe report that the neurotrophin receptor TrkB is expressed in a subset of embryonic sympathoblasts during the early development of lumbar paravertebral sympathetic ganglia in chicken and mouse embryos. In the chicken, TrkB expression is transient, and completely lost by St 34 (E8). Since BDNF induces the proliferation of sympathoblasts in cell culture, yet in vivo there is little proliferation observed in TrkB-positive cells in nascent ganglia, we propose that if TrkB activation becomes unregulated by excess BDNF or constitutive phosphorylation of TrkB [16], this transient population of TrkB-positive sympathoblasts may trigger the genesis of neuroblastoma, a childhood tumor found in the paravertebral chain and adrenal medulla.\nThe two populations of sympathoblasts that we observe support previous findings of heterogeneity among developing sympathetic neurons and neural crest cells. Early sympathetic ganglia contain at least two subpopulations: early differentiating neurons that lack TrkB expression and express TrkA and TrkC, and late differentiating sympathoblasts that express TrkB. Explant cultures of sympathetic ganglia from E16 chick embryos give rise to two neuronal populations: one that remains close to the explant, and one that migrates away from the explant [1]. In addition, early neuronal subpopulations have been observed in cultures of neural crest cells from St. 13/14 quail embryos as evidenced by the expression of neuronal cell type-specific gangliosides [17]. Perhaps these different subpopulations will ultimately give rise to the two neurochemically distinct populations found in lumbar sympathetic ganglia: the noradrenergic, NPY-containing neurons that innervate internal organs and enteric ganglia and the cholinergic, VIP-containing neurons that innervate vasculature in the hind limbs.\nThe effects of BDNF and TrkB deletion and over expression have been studied on superior cervical ganglion and preganglionic neurons in thoracic segments of the spinal column, but not on paravertebral sympathetic neurons. In the superior cervical ganglion, an increase in the number of neurons of BDNF null mice is likely due to apoptosis induced by BDNF via p75NTR [18]. In contrast, the responses of paravertebral sympathetic neurons to BDNF are complex and subtype dependent. Over expression of BDNF leads to an increase in the number of noradrenergic fibers innervating the erector pilli muscles of hair follicles, while noradrenergic fibers innervating blood vessels were unaffected [19]. If our results indicating that BDNF promotes proliferation of TrkB-positive sympathoblasts in the chicken embryo can be extrapolated to the subset of TrkB-positive sympathoblasts in murine ganglia, then these TrkB-positive cells may be neurons destined to innervate the erector pilli. In other studies, TrkB null mice showed no changes in morphology or cell number in superior cervical ganglia [12] or in the intermediolateral column [20]; but this may not be predictive of a phenotype in the lumbar paravertebral chain. It is thus possible that BDNF/TrkB signaling could play a specific role in other regions of the paravertebral sympathetic chain, such as the lumbar region. However, if TrkB-positive cells are not normally actively proliferating in vivo, then it would not be surprising that the development of the paravertebral sympathetic chain is not disrupted in TrkB or BDNF null mice. It may be more informative to examine mice that over express BDNF on a promoter that targets expression to embryonic lumbar ganglia. Unfortunately, such mice do not exist.\nOur findings that the St. 29/30 (E6.5) sympathoblasts are dependent on both NT-3 and NGF for survival in culture are consistent with previous work on mouse sympathoblasts from the superior cervical ganglion [11]. In these studies, NT-3 and NGF deletion separately led to a decrease in the number of sympathetic neurons at E17.5 compared to control. Deletion of both NT-3 and NGF together did not enhance cell death. In contrast, cultured rat superior cervical ganglion sympathetic neurons respond to NT-3 at E14.5 and then to NGF at E19.5, although time points in between were not analyzed [6].\nIn addition to promoting survival, NT-3, NGF, and BDNF also induce proliferation of various neuronal precursors at different stages of development. NT-3 can promote the incorporation of [3H]-thymidine into cultured quail neural crest cells from the trunk region [21,22], Later in rat sympathetic development, NT-3 supports survival of neurons, but does not promote proliferation [6], which is consistent with our results. NGF promotes an increase in BrdU incorporation from 25% to 35% in the DRG cervical segment 2 in the chick embryo [23]. In chicken embryos that are treated with NGF in ovo at St. 18 and 21, there is an increase in BrdU uptake after formation of the primary sympathetic chain at St. 23 [24]. Since NGF does not appear to affect proliferation of St. 29/30 (E6.5) chick sympathoblasts, NGF may only promote proliferation in primary, but not secondary chain sympathoblasts. Motor neuron progenitors in the ventral neural tube from the chick embryo express TrkB and when ventral neural tube explants are treated with BDNF, there is an increase in the number of motor neurons produced and BrdU incorporation [25]. BDNF also promotes the proliferation of cultured neuroblastoma cells [13]. Taken together, these results are consistent with our findings that NT-3 and NGF do not promote proliferation of St. 29/30 (E6.5) sympathoblasts, and support the assertion that BDNF promotes proliferation of TrkB-positive sympathoblasts in culture.\nOur observations suggest a transient function of TrkB during early sympathetic development in supporting proliferation of this early subpopulation of sympathoblasts. However, the in vivo labeling suggests that only a minority (10–20%) of this population is dividing during the window that TrkB is expressed. In light of the very strong proliferative effect produced in cell culture, these TrkB expressing cells could respond more strongly if endogenous BDNF rises to higher levels, or if the mechanism that down regulates TrkB expression becomes nonfunctional. Such events could trigger an early proliferative event that leads to a cascade of changes that initiates transformation of cells to neuroblastoma. Thus, these early TrkB expressing cells help solve the puzzle as to why TrkB is expressed in aggressive and invasive forms of neuroblastoma, particularly because BDNF induces cultured neuroblastoma cells to become more proliferative, invasive, angiogenic, and resistant to chemotherapeutic reagents than untreated cultures [13]. Future studies will determine whether constitutive expression of BDNF and TrkB in the chick embryo sustains proliferation of differentiating sympathoblasts.\n\nConclusion\nWe have identified a time point during development when differentiating lumbar sympathetic neurons transiently express TrkB and proliferate in response to high concentrations of BDNF in culture. These studies suggest that elevated BDNF expression above basal levels and signaling through TrkB may be a mechanism that contributes to the onset of neuroblastoma. A further understanding of the two populations of sympathetic neurons and the fate of the TrkB-positive cells will provide additional insight into the development of paravertebral sympathetic ganglia and the genesis of neuroblastoma.\n\nMethods\nPreparation of tissue for immunohistochemistry\nThe lumbar spinal column and surrounding tissues were dissected from chicken embryos at the indicated stages and placed in Zamboni's fixative (4% (w/v) paraformaldehyde, 15% (v/v) picric acid in 0.1 M sodium phosphate buffer, pH 7.4) for two hours at room temperature. Mouse embryos at 13–15 days post-coitus were collected according to an IACUC-approved protocol to Dr. L. Sherman at the Oregon Health and Science University. The mouse embryos were immersion-fixed in Zamboni's fixative overnight at 4 degrees C then washed with phosphate buffered saline (PBS; 130 mM NaCl, 20 mM sodium phosphate buffer, pH 7.4). Fixed tissues were equilibrated in 30% sucrose in 1× phosphate-buffered saline (PBS). Fixed mouse embryos were shipped to Vermont in sucrose. Transverse 30 μM sections of the spinal columns were cut at on a Microm HM cryostat (knife temperature: 16°C; object temperature: 23°C) and collected on Superfrost Plus slides (Fisher). Sections were dried at room temperature, washed in 1× PBS and incubated overnight in blocking buffer (1× PBS consisting of 10% (v/v) heat-inactivated horse serum (Invitrogen/Gibco), 0.5% Triton X-100 (Sigma), and 0.1% sodium azide (Fisher)).\n\nImmunohistochemistry\nSections were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 2 hours at room temperature. Primary antibodies used were: rabbit anti-p75 (1:1500, generous gift from Louis Reichardt, UCSF [26]), mouse IgG2b anti-Hu C/D, (1:250, Molecular Probes); mouse IgG1 anti-Islet-1, (1:10, Developmental Studies Hybridoma Bank); rabbit anti-chicken TrkA (1:500); rabbit anti-chicken TrkB (1:500); rabbit anti-chicken TrkC (1:500) (all Trk antibodies were generous gifts of Dr. Louis Reichardt, UCSF [26-28]); mouse anti-HNK-1 (1:50, Developmental Studies Hybridoma Bank); mouse IgG2a anti-tyrosine hydroxylase (1:10, Developmental Studies Hybridoma Bank), sheep anti-BrdU (1:100, Biodesign International), rabbit anti-tyrosine hydroxylase (1:100, Chemicon), and goat anti-TrkB (1:1000, R&D Systems). Immunofluorescence was imaged using a Nikon C1 confocal mounted on a Nikon Eclipse E800 microscope with a 10× Plan Apo (NA 0.785) air objective or a 60× Plan Apo (NA 1.4) oil objective lens, E7-C1 software, and UV, Argon, and He/Ne lasers exciting at 408, 488, and 543 nm and emitting at 404 500–530, and 555–615 nm, respectively. A Nikon Eclipse E800 microscope in the nearby COBRE Molecular/Cellular Core Facility was used for counting immunofluorescent cells at 200× using epifluorescence optics.\n\nRNA Extraction/cDNA synthesis\nSympathetic ganglia were removed from chick embryos and RNA was isolated using TriReagent (Molecular Research Center), an acidified guanidinium with phenol extraction method [29]. RNA was transcribed to cDNA using oligo-dT with Superscript II Reverse Transcriptase (Invitrogen) at 42°C for 1 hour.\n\nReal-time PCR\nRelative RNA levels were determined using quantitative real-time PCR with an ABI 7500 Fast Real Time PCR System. TaqMan probes were used to quantify the progression of the PCR reaction and reactions were normalized using the constitutively expressed gene chick ribosomal binding protein s17 (CHRPS). The sequences were used for primer/probes sets: for BDNF: forward: 5'-AGCCCAGTGAGGAAAACAAG-3', reverse: 5'-ACTCCTCGAGCAGAAAGAGC-3', probe: 5'-[6-FAM]-TACACATCCCGAGTCATGCTGAGCA-[BHQ]-3'; for CHRPS (chick ribosomal binding protein S-17): 5'AACGACTTCCACACCAACAA3', reverse: 5'CTTCATCAGGTGGGTGACAT3', probe: 5'-[6-FAM]-CGCCATCATCCCCAGCAAGA [BHQ]-3'. Primers and probes were synthesized by Operon Technologies, Inc (Alameda, CA). The primers for BDNF were validated against primers for CHRPS according to an Applied BioSystems protocol by serially diluting the target cDNA 1:10, determining the cycle threshold (Ct) for each reaction, and plotting the Ct versus log concentration. Slopes of the resulting lines were calculated and primers were accepted if their Ct slopes were between -3.2 and -3.4 (a perfect efficiency of 1.0 yields a slope of -3.3). To analyze the data, the delta Ct method of relative quantification was used, where the Ct of Chrps was subtracted from the Ct of the gene of interest (Delta Ct) and the arbitrary units of mRNA were expressed as 10000/2^(Delta Ct).\n\nCell culture\nSympathetic neurons were cultured as previously described [30] with a few modifications. Sympathetic ganglia were removed from the lumbar region of the paravertebral chain of St. 29/30 (E6.5) chick embryos and placed in Modified Puck's solution with glucose (MPG). The cells were dissociated by incubation of sympathetic ganglia with 0.1% trypsin in MPG at 37°C for 10 minutes followed by triturating with a fire polished 9\" Pasteur pipette. Cells were then resuspended in Dulbecco's Modified Eagle Medium (DMEM) consisting of 10% horse serum, 2% fetal calf serum, and 10 mg/ml penicillin/streptomycin. For neurotrophin studies, the culture medium was supplemented with 25 ng/ml NT-3 (R & D Systems) and 1 μg/ml 7S NGF (Alomone Labs) upon plating, and 50 ng/ml, 100 ng/ml, or 200 ng/ml BDNF (R & D Systems) once the cells adhered to the wells. Cells were plated on poly-D-lysine/laminin coated wells or cover slips (Fisher) as previously described [30].\n\nQuantification of neurons and sympathoblasts using phase microscopy\nEmbryonic sympathoblasts and neurons are small, phase bright cells with neurites. The total number of cells with neurites the length of two cell bodies were counted in 10 non-overlapping fields of view evenly spaced in a grid-like pattern across the bottom of a well from a 24 well plate at 200× using a Nikon Eclipse TE200 microscope.\n\nBrdU labeling\nFor in vitro studies, approximately 2 hours after plating cells from St. 29/30 (E6.5) sympathetic ganglia, cells were labeled with 10 μM bromodeoxyuridine (BrdU, Sigma) for 12 hours at 37°C. Following this labeling period, cells were incubated in complete medium without BrdU for an additional 10 hrs. Cells were then fixed in Zamboni's fixative for 30 min at room temperature and rinsed with 1× PBS. For in vivo studies, 25 μg BrdU was injected into the amnion of chick embryos at St. 27. The cells and sections were denatured with 2 N HCl at 37°C for 1 hr, and were then neutralized with 0.1 M borate buffer, pH 8.5, for 10 min at room temperature. Immunochemistry was performed as described above.\n\n\nAbbreviations\nBDNF, brain-derived neurotrophic factor; BrdU, Bromodeoxyuridine; DA, dorsal aorta; DMEM, Dulbecco's Modified Eagle's Medium; DRG, dorsal root ganglion; E, embryonic day; HS, horse serum; MPG, Modified Puck's solution with glucose; NGF, nerve growth factor; NC, notochord; NT, neural tube; NT-3, neurotrophin-3; NTR, neurotrophin receptor; PBS, phosphate-buffered saline; SC, spinal cord; SCG, superior cervical ganglion; SEM, standard error of the mean; SG, sympathetic ganglion; St., stage; w/v, weight/volume; v/v, volume/volume.\n\nAuthors' contributions\nJAS designed the experiments, performed the experiments, analyzed the data, and wrote the manuscript. GLSS contributed intellectually to the conception and design of this study, and assisted in the interpretation of the results. RN supervised the study, participated in the design of experiments, edited the manuscript, and obtained funding for the project. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 32104 ] ] } ]

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[ { "id": "pmcA1779441__text", "type": "Article", "text": [ "Identification of a human peripheral blood monocyte subset that differentiates into osteoclasts\nAbstract\nIncreased bone resorption mediated by osteoclasts causes various diseases such as osteoporosis and bone erosion in rheumatoid arthritis (RA). Osteoclasts are derived from the monocyte/macrophage lineage, but the precise origin remains unclear. In the present study, we show that the purified CD16- human peripheral blood monocyte subset, but not the CD16+ monocyte subset, differentiates into osteoclast by stimulation with receptor activator of NF-κB ligand (RANKL) in combination with macrophage colony-stimulating factor (M-CSF). Integrin-β3 mRNA and the integrin-αvβ3 heterodimer were only expressed on CD16- monocytes, when they were stimulated with RANKL + M-CSF. Downregulation of β3-subunit expression by small interfering RNA targeting β3 abrogated osteoclastogenesis from the CD16- monocyte subset. In contrast, the CD16+ monocyte subset expressed larger amounts of tumor necrosis factor alpha and IL-6 than the CD16- subset, which was further enhanced by RANKL stimulation. Examination of RA synovial tissue showed accumulation of both CD16+ and CD16- macrophages. Our results suggest that peripheral blood monocytes consist of two functionally heterogeneous subsets with distinct responses to RANKL. Osteoclasts seem to originate from CD16- monocytes, and integrin β3 is necessary for osteoclastogenesis. Blockade of accumulation and activation of CD16- monocytes could therefore be a beneficial approach as an anti-bone resorptive therapy, especially for RA.\n\nIntroduction\nRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and proliferation of the synovium in multiple joints. A large number of inflammatory cells, including T cells, B cells, macrophages and dendritic cells, accumulate in the affected synovium, and these inflammatory cells, together with fibroblast-like synoviocytes, express various cytokines, such as tumor necrosis factor alpha (TNFα), IL-6 and receptor activator of NF-κB ligand (RANKL), which are known to induce differentiation and activation of osteoclasts. The inflammatory synovial tissue, known as pannus, invades the articular bone and causes focal bone erosion, which is the hallmark of RA. Histopathologically, osteoclasts are present at the interface of the pannus and bone. Interestingly, the deletion of RANKL or c-Fos gene, which is important for osteoclastogenesis, results in minimal bone destruction in mouse models of arthritis [1,2]. Furthermore, other studies indicated that inhibition of osteoclastogenesis by osteoprotegerin, a decoy receptor for RANKL, limits bone destruction in experimental models of arthritis. These studies suggest that osteoclasts are involved in focal bone erosion in RA [3].\nOsteoclasts are derived from the monocyte/macrophage lineage. It is reported that osteoclast precursors reside in human peripheral blood monocytes [4,5]. A marked increase of the circulating osteoclast precursors was demonstrated in patients with erosive psoriatic arthritis as well as in arthritic TNFα transgenic mice [6,7]. It was also shown that peripheral monocytes differentiate into osteoclasts when seeded on RANKL/osteoclast differentiation factor-producing RA synovial fibroblasts [8]. In addition, RA synovial macrophages thought to originate from peripheral blood monocytes were shown to differentiate into osteoclasts [9,10]. Monocytes are therefore involved not only in synovial inflammation, but also in bone remodeling as potential precursors for synovial macrophages and osteoclasts.\nHuman peripheral blood monocytes consist of two major subsets, CD16+ and CD16-, comprising 5–10% and 90–95% of the monocytes, respectively. These two subsets exhibit different chemotaxis activities and potential of cytokine production [11,12]. Moreover, activation of the Toll-like receptor induces distinct subsets, CD1b+ dendritic cells and DC-SIGN+ (dendritic cell-specific C-type lectin ICAM-3-grabbing nonintegrin) macrophages from CD16+ and CD16- monocytes, respectively [13]. It has not been revealed, however, which monocyte subset develops into osteoclasts.\nIn the present study, we determined the human peripheral blood monocyte subset that differentiates into osteoclasts, and revealed that each subset exhibits a different response for osteoclastogenic stimuli.\n\nMaterials and methods\nPurification of peripheral blood monocytes\nPeripheral blood monocytes from healthy donors were collected using Ficoll-Conray (Imuuno-Biological Laboratories, Gunma, Japan) gradient centrifugation. Negative selection of monocytes was performed using MACS microbeads (Miltenyi Biotec, Auburn, CA, USA) according to the protocol supplied by the manufacturer.\nThe purified monocytes were separated into two subsets, CD16+ and CD16- monocytes, using CD16 MicroBeads (Miltenyi Biotec). Flow cytometry analysis using FITC-conjugated mouse anti-CD14 mAb (MY4; Bechman Coulter, Fullerton, CA, USA) and phycoerythin-conjugated mouse anti-CD16 mAb (3G8; BD Biosciences, San Jose, CA, USA) showed that the purities of the CD16+ and CD16- monocytes were more than 90% and 92%, respectively.\nFor the other experiment, monocytes were purified using CD14 MicroBeads (Miltenyi Biotec), and then stained either with FITC-conjugated mouse anti-CD33 mAb (MY9; Bechman Coulter) or phycoerythin-conjugated mouse anti-CD16 mAb (3G8). Cell sorting of the stained cells was performed using a FACS Vantage cytometer (BD Biosciences) or a MoFlo cell sorter (Dako, Glostrup, Denmark).\n\nOsteoclast differentiation\nPurified CD16+ and CD16- monocytes (5 × 104 cells/well) were incubated in 96-well plates in αMEM (Sigma, St Louis, MO, USA) with heat-inactivated 10% fetal bovine serum (FBS) (Sigma) or with Ultra-Low IgG FBS (IgG < 5 μg/ml; Invitrogen, Carlsbad, CA, USA), and where indicated with M-CSF + RANKL (Peprotech, Rocky Hill, NJ, USA).\nFor the other experiments, varied numbers of CD16+ monocytes (1 × 103, 2.5 × 103, 5 × 103) were mixed with CD16- monocytes (5 × 104 cells/well), and were cultured in 96-well plates in αMEM with heat-inactivated 10% FBS. The medium was replaced with fresh medium 3 days later, and after incubation for 7 days the cells were stained for tartrate-resistant acid phosphatase (TRAP) expression using a commercial kit (Hokudo, Sapporo, Japan). The number of TRAP-positive multinucleated cells (MNC) in three randomly selected fields examined at 100× magnification or the total number of TRAP-positive MNC per well was counted under light microscopy.\n\nResorption assay\nMonocytes were seeded onto plates coated with calcium phosphate thin films (Biocoat Osteologic; BD Biosciences) and were incubated with RANKL (40 ng/ml) + M-CSF (25 ng/ml) for 7 days. The cells were then lysed in bleach solution (6% NaOCl, 5.2% NaCl). The resorption lacunae were examined under light microscopy.\n\nEnzyme-linked immunosorbent assay\nPurified monocytes were cultured in 96-well plates where indicated either with RANKL or M-CSF for 24 hours. Concentrations of TNFα and IL-6 in the culture supernatant were measured with an ELISA kit (BioSourse International, Camarillo, CA, USA). For experiments of matrix metalloproteinase (MMP)-9 and TRAP-5b, culture supernatants were collected on day 7 and the concentrations of these enzymes were measured using an MMP-9 ELISA kit (Amersham Biosciences, Piscataway, NJ, USA) or a TRAP-5b ELISA kit (Suomen, Turku, Finland).\n\nReverse transcriptase-polymerase chain reaction\nMonocytes (1 × 106 cells/well) were cultured in six-well plates with M-CSF alone or with M-CSF + RANKL for 3 days. Total RNA was extracted using RNeasy Micro (Qiagen, Valencia, CA, USA). The RNA was then treated with DNase I (Qiagen). The oligo(dT)-primed cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). The amount of cDNA for amplification was adjusted by the amount of RNA measured by an optical density meter and also by β-actin or GAPDH PCR products. One microliter of cDNA was amplified in a 50 μl final volume containing 25 pmol appropriate primer pair, 10 pmol each of the four deoxynucleotide triphosphates, and 5 units FastStart Taq DNA Polymerase (Roche, Manheim, Germany) in a thermal cycler (PTC-200; MJ GeneWorks, Waltham, MA, USA).\nThe PCR conditions were 25–40 cycles of denaturation (95°C for 30 s), annealing (60–62°C for 1 min) and extension (72°C for 1 min). The sequences of the primers are presented in Table 1. The PCR products were separated by electrophoresis through 2% agarose gel.\n\nWestern immunoblot analysis\nPurified monocytes were cultured for 3 days in the presence of 40 ng/ml M-CSF with or without 25 ng/ml RANKL. Cells were lysed in RIPA Lysis buffer (upstate, Lake Placid, NY, USA) containing protease inhibitors (Roche) for 15 min at 4°C. A total of 20 μg protein was boiled in the presence of 6 × sodium dodecyl sulfate sample buffer, and was separated on 7.5% or 10% sodium dodecyl sulfate-polyacrylamide gel (ATTO, Tokyo, Japan). Proteins were then electrotransferred to a polyvinylidene fluoride microporous membrane (Millipore, Billerica, MA, USA) in a semidry system. Membranes were incubated in 10% skim milk prepared in phosphate-buffered saline (PBS) containing 0.1% Tween 20, and were subjected to immunoblotting. Antibodies used were goat anti-RANK antibody (Techne Corporation, Minneapolis, MN, USA), goat anti-c-fms antibody (R&D systems, Minneapolis, MN, USA), and mouse anti-β-actin mAb (AC-15; Sigma). Peroxidase-conjugated rabbit anti-goat IgG antibody (Dako) or peroxidase-conjugated rabbit anti-mouse IgG antibody (Dako) was used as the second antibody. The signals were visualized by chemiluminescence reagent (ECL; Amersham Biocsiences, Little Chalfont, UK).\n\nCell surface expression of c-fms\nThe following mAbs were used for analysis of c-fms expression: Alexa 647-conjugated anti-CD14 mAb (UCHM1; Serotec, Oxford, UK), FITC-conjugated anti-CD16 mAb (3G8; Bechman Coulter) and phycoerythin-conjugated anti-c-fms mAb (61708; R&D systems). Alexa 647-conjugated mouse IgG2a (Serotec), FITC-conjugated mouse IgG1 (BD Biosciences) and phycoerythin-conjugated mouse IgG1 (Bechman Coulter) were used as isotype controls. Peripheral blood monocytes (1 × 105 cells) were incubated with 1 μg human IgG for 15 minutes, and were then stained with three fluorochrome-labeled mAbs for 45 minutes on ice. The stained cells were analyzed with a FACS Calibur (BD Biosciences).\n\nImmunofluorescent staining\nMonocytes (8 × 104 cells/well) were allowed to adhere on 96-well plates overnight or were cultured with M-CSF and RANKL for 2–4 days. The cells were fixed in acetone and then stained with anti-αvβ3 mAb (LM609; Chemicon, Temecula, CA, USA) or mouse IgG1 (11711; R&D Systems) as an isotype-matched control. Alexa fluor546-conjugated goat anti-mouse IgG1 antibody (Molecular Probes, Eugene, OR, USA) was used as the second antibody. TOTO-3 (Molecular Probes) was used for nuclear staining.\n\nFlow cytometric analysis of p38 MAPK and ERK1/2 phosphorylation\nPurified monocytes were cultured in the presence of 25 ng/ml M-CSF for 3 days, and were either left unstimulated or were stimulated with 40 ng/ml RANKL at 37°C. Stimulations were stopped by adding an equal volume of PhosFlow Fix Buffer I solution (BD Biosciences) to the cell culture. After incubation for 10 minutes at 37°C, the cells were permeabilized by washing twice at room temperature in PhosFlow Perm/Wash Buffer I (BD Biosciences). A total of 1 × 105 cells was then Fc blocked with 1 μg human IgG for 15 minutes, and was stained with Alexa Fluor 647-conjugated mAb either to phospho-p38 MAPK (T180/Y182) or to phospho-ERK1/2 (T202/Y204) (BD Biosciences) for 30 minutes at room temperature. Alexa Fluor 647-conjugated mouse IgG1 (BD Biosciences) was used as an isotype control. The cells were washed in PhosFlow Perm/Wash Buffer I, and were analyzed by flow cytometry, as already described.\n\nRNA interference\nRNA oligonucleotides (iGENE, Tsukuba, Japan) were designed based on the algorithm that incorporates single nucleotide polymorphism and homology screening to ensure a target-specific RNA interference effect. The following sense and antisense oligonucleotides were used: integrin β3, 5'-GCU UCA AUG AGG AAG UGA AGA AGC A-AG and 3'-UA-CGA AGU UAC UCC UUC ACU UCU UCG U; randomized control, 5'-CGA UUC GCU AGA CCG GCU UCA UUG C-AG and 3'-UA-GCU AAG CGA UCU GGC CGA AGU AAC G; and lamin, 5'-GAG GAA CUG GAC UUC CAG AAG AAC A-AG and 3'-UA-CUC CUU GAC CUG AAG GUC UUC UUG U.\nCD16- monocytes (8 × 104 cells/well) were incubated in 96-well plates in optimem (Invitrogen). After 1 hour, siRNAs were transfected into the cells using oligofectamine (Qiagen) based on the method recommended by the manufacturer. After 2 hours, the cells were washed once with PBS, followed by the addition of αMEM supplemented with 10% FBS, M-CSF and RANKL. After a 2-day incubation, the β3 mRNA expression was analyzed by RT-PCR with different PCR cycles, as described earlier. Immunofluorescent staining for the αvβ3 heterodimer was also performed as described above, and numbers of αvβ3-positive cells were counted in randomly selected three fields at 100× magnification. Seven days after the transfection of siRNAs, the number of TRAP-positive MNC in five fields examined at 100× magnification was counted under light microscopy.\n\nInhibition of osteoclastogenesis with cyclic RGDfV peptide\nCD16- monocytes were incubated in 96-well plates with M-CSF + RANKL for 2 days. A medium containing either cyclic RGDfV peptide (Arg-Gly-Asp-D-Phe-Val) (Calbiochem, San Diego, CA, USA) or dimethyl sulfoxide was then added. After incubation for a further 5 days, the number of TRAP-positive MNC in five fields examined at 100× magnification was counted under light microscopy.\n\nImmunohistochemistry\nSynovial tissue samples were obtained during total knee joint replacement surgery from four RA patients. Signed consent forms were obtained before the operation. The experimental protocol was approved by the ethics committee of the Tokyo Medical and Dental University. RA was diagnosed according to the American College of Rheumatology criteria [14].\nDouble immunofluorescent staining for CD68 and CD16 antigens was conducted on optimal cutting temperature-embedded sections of frozen synovial samples. Eight-micrometer-thick cryostat sections of RA synovium were fixed in acetone for 3 minutes and were then rehydrated in PBS for 5 minutes. The samples were incubated in 5 μg/ml proteinase K (Roche), 50 mM ethylenediamine tetraacetic acid, 100 mM Tris–HCl, pH 8.0, for 15 minutes at room temperature followed by a wash in PBS. The samples were then blocked with 10% goat serum in PBS for 60 minutes at room temperature, and were incubated with anti-CD16 mAb (3G8; Immunotech, Marseille, France) or mouse IgG1 (11711) as an isotype-matched control in 1% bovine serum albumin/PBS for 60 minutes at room temperature. The samples were then washed three times in PBS, for 5 minutes each, and incubated with Alexa fluor546-conjugated goat anti-mouse IgG1 antibody (Molecular Probes) in 1% bovine serum albumin/PBS for 60 minutes at room temperature. The samples were then sequentially stained for CD68 antigen in a manner similar to that used for CD16 staining. The samples were stained with anti-CD68 mAb (PGM1; Immunotech) or mouse IgG3 (6A3; MBL, Nagoya, Japan) followed by labeling with Alexa fluor488-conjugated goat anti-mouse IgG3 antibody (Molecular Probes). The samples were examined by confocal laser scanning microscope (Olympus, Tokyo, Japan).\n\nStatistical analysis\nData are expressed as the mean ± standard error of the mean. A nonpaired Student's t test was used for comparison, using the StatView program (Abacus Concepts, Berkeley, CA, USA). P < 0.05 was considered statistically significant.\n\n\nResults\nInduction of osteoclasts from CD16- peripheral blood monocytes\nTo identify the monocyte subset that differentiates into osteoclasts, we examined osteoclast formation from CD16+ and CD16- human peripheral blood monocytes. The monocyte subsets were purified using magnetic beads. Incubation with M-CSF alone did not induce osteoclast formation from either subset (Figure 1a). Culture with M-CSF + RANKL induced a significant number of TRAP-positive MNC from the CD16- subset, whereas only few CD16+ monocytes differentiated into TRAP-positive MNC (Figure 1a,b). We then assessed the bone resorptive ability by culturing cells on calcium phosphate-coated plates with M-CSF + RANKL. Resorption lacunae were detected only in the CD16- subset (Figure 1c), indicating the TRAP-positive CD16--derived MNC possessed the osteoclast phenotype.\nSimilar results were obtained using purified monocytes by FACS sorting (purities: CD16+, 96%; CD16-, 97%). The number of TRAP-positive MNC induced were 36 ± 3 cells/well and 348 ± 13 cells/well from CD16+ and CD16- monocytes, respectively. To exclude the possibility that the anti-CD16 antibody used for isolation of CD16+ monocytes inhibits osteoclast formation, we separated the two subsets, CD33low monocytes and CD33high monocytes, using anti-CD33 mAb and a fluorescent cell sorter, since it was reported that CD33low monocytes correspond to CD16+, and that CD33high correspond to CD16- monocytes [15]. On average, the CD33low population contained CD16- (10.2%)/CD16+ (89.8%) monocytes, and the CD33high population contained CD16- (86.3%)/CD16+ (13.7%) monocytes.\nCulture with M-CSF + RANKL induced TRAP-positive MNC from CD33high monocytes, whereas no or few CD33low monocytes differentiated into TRAP-positive MNC (CD33low vs CD33high, 2 ± 1 vs 192 ± 71 cells/well; n = 3). TRAP-5b and MMP-9 in the culture supernatants, both of which are known to be produced by osteoclasts, were measured by ELISA. The concentrations of both enzymes were significantly higher in the culture supernatant of CD16- monocytes than in that of CD16+ monocytes (Figure 1d). These results suggest that the CD16- peripheral blood monocyte subset, but not the CD16+ subset, differentiate into osteoclasts by incubation with RANKL + M-CSF.\n\nCD16+ monocytes do not affect the osteoclastogenesis from CD16- monocytes\nTo examine whether CD16+ monocytes affect osteoclastogenesis from CD16- monocytes, varied numbers of CD16+ monocytes were mixed with CD16- monocytes (5 × 104 cells/well), and were cultured for 7 days in the presence of M-CSF + RANKL. The number of TRAP-positive MNC was not altered by the presence of CD16+ monocytes (Figure 2). The results indicated that CD16+ monocytes did not hamper or enhance the osteoclastogenesis from CD16- monocytes.\n\nDifferences in cytokine production by RANKL-stimulated or M-CSF-stimulated CD16+ and CD16- monocytes\nTo compare the biological response of CD16+ and CD16- subsets with either RANKL or M-CSF stimulation, we measured the amount of TNFα and IL-6 production after exposure of cells to various concentrations of RANKL or M-CSF with an ELISA. Without RANKL the CD16+ subset produced a significant amount of TNFα and IL-6, whereas the CD16- subset produced undetectable levels (Figure 3a). RANKL stimulation increased TNFα production from both subsets in a dose-dependent manner, although the CD16+ subset produced more TNFα than did the CD16- subset. RANKL stimulation also enhanced IL-6 production from the CD16+ subset, but not from the CD16- subset. M-CSF stimulation increased TNFα and IL-6 production from both subsets, although the CD16+ subset produced more than the CD16- subset (Figure 3b).\nThese results suggest that CD16+ monocytes also respond both to RANKL and M-CSF stimulation, although such stimulation does not result in differentiation into osteoclasts. CD16+ monocytes were also noted to express higher amounts of inflammatory cytokines compared with CD16- monocytes with or without RANKL or M-CSF stimulation.\n\nComparison of expression levels of molecules involved in osteoclastogenesis between CD16+ and CD16- monocytes\nDiverse molecules are involved in RANKL/RANK and its costimulatory signal transduction pathways [16]. The different response to RANKL + M-CSF stimulation between the CD16+ monocyte subset and the CD16- monocytes subset might be explained by the expression profiles of these molecules. We therefore examined the mRNA levels of the following molecules: receptor activator of NF-κB (RANK), the receptor for RANKL; c-fms, the receptor for M-CSF; tumor necrosis factor receptor-associated factor 6 (TRAF-6), the adaptor protein for RANK; c-Fos and nuclear factor of activated T cells c1 (NFATc1), transcription factors that are essential for osteoclastogenesis; DNAX-activation protein 12 (DAP12) and Fc receptor γ chain (FcRγ), adaptor proteins known to deliver costimulatory signals in RANKL-induced osteoclastogenesis; signal regulatory protein β1 (SIRP-β1), triggering receptor expressed on myeloid cells 2 (TREM-2) and osteoclast-associated receptor (OSCAR), transmembrane receptors that associate with either DAP12 or FcRγ; and αv and β3, integrins known to be expressed as the αvβ3 heterodimer on osteoclasts.\nThe mRNA levels of RANK, c-fms, TRAF-6, DAP12 and SIRP-β1 under the baseline condition (no stimulation) varied between the donors; however, we did not find consistent differences in the mRNA levels of these molecules between the CD16+ monocyte subset and the CD16- monocyte subset among three to six donors (Figure 4a). The mRNA levels of other molecules, apart from integrin β3, were similar between the two subsets under the no-stimulation condition. Although the mRNA levels of RANK, c-fms, DAP12, FcRγ, TREM-2 and OSCAR increased in response to M-CSF alone or M-CSF + RANKL in both subsets, the expression levels were not significantly different between the two subsets. Expressions of TRAF-6, c-Fos and SIRP-β1 mRNA did not change following stimulation with M-CSF + RANKL. Of note, the expression of NFATc1 mRNA was enhanced by M-CSF + RANKL treatment only in the CD16- subset. Furthermore, expression of integrin αv in both subsets was enhanced by M-CSF with or without RANKL; however, the expression level was greater in the CD16- subset. It was noted that integrin-β3 mRNA was detected only in the CD16- subset and was increased by M-CSF + RANKL stimulation, but not by M-CSF alone. The protein expression of RANK under the baseline condition was weakly detected in both subsets, and the levels were varied between donors by western immunoblotting.\nThe protein expression of c-fms was weakly detected in unstimulated CD16+ monocytes, but not in CD16- monocytes (Figure 4b). Flow cytometry analysis of c-fms in fresh monocytes, however, showed that both subsets express the molecule on the cell surface (Figure 4c). Expressions of both RANK and c-fms were upregulated by M-CSF alone and by M-CSF + RANKL, and we did not find consistent differences in the protein levels of these molecules between the two monocyte subsets. The profiles of expression levels of molecules involved in RANKL/RANK and its costimulatory pathways are similar between the two subsets, except for NFATc1, integrin αv and integrin β3. We therefore assumed that the distinct induction of NFATc1, integrin αv and integrin β3 in response to RANKL stimulation among the two monocyte subsets might explain the differences in their abilities to differentiate into osteoclasts.\n\nRANKL stimulation induces αvβ3 expression on CD16- monocytes\nThe integrin-β3 subunit binds to integrin αv only and is expressed as the heterodimeric protein αvβ3 on monocytes and osteoclasts [17]. We examined the expression of αvβ3 on CD16+ and CD16- monocytes by immunofluorescent staining. Neither unstimulated nor M-CSF-stimulated monocyte subsets expressed αvβ3 (Figure 4d and data not shown). After 48 and 72 hours of treatment with M-CSF + RANKL, αvβ3-positive mononuclear cells were observed in CD16- monocyte cultures but not in CD16+ monocyte cultures. At 96 hours, both αvβ3-positive mononuclear cells and multinucleated cells were present in the CD16- monocyte culture. The results indicated that αvβ3 was selectively expressed on CD16- monocytes in the presence of M-CSF + RANKL, and the expression was revealed before the cells differentiate into typical multinucleated osteoclasts.\n\nRANKL activates ERK and p38 kinases only in CD16- monocytes\nSince ERK and p38 MAPK are essential in RANKL-induced osteoclastogenesis [18-20], we next examined whether these kinases were activated differently in CD16+ monocytes and in CD16- monocytes. Purified monocytes were precultured with 25 ng/ml M-CSF for 3 days to enhance RANK expression, and were then treated with RANKL. The RANKL treatment induced phosphorylation of both ERK and p38 MAPK in CD16- monocytes at 5 minutes postexposure, although the p38 MAPK phosphorylation was weak. Both phosphorylations declined to a basal level within 20 minutes (Figure 5). In contrast, ERK and p38 MAPK were not detectably phosphorylated in CD16+ monocytes with RANKL.\n\nsiRNA targeting integrin β3 inhibits osteoclastogenesis from CD16- monocytes\nThe integrin-β3 cytoplasmic domain is essential for activation of intracellular signals from αvβ3 heterodimers [17]. We therefore examined the involvement of αvβ3 in RANKL + M-CSF-induced osteoclastogenesis in human CD16- monocytes using siRNA targeting the integrin-β3 subunit. The integrin-β3 siRNA or control randomized siRNA were transfected into CD16- monocytes. At 48 hours post-transfection, we determined the integrin-β3 mRNA level and αvβ3 heterodimer protein expression. The integrin-β3 mRNA level was reduced in the integrin-β3 siRNA-transfected monocytes compared with control siRNA-transfected monocytes (Figure 6a). The αvβ3 heterodimer expression was evaluated by immunofluorescent staining. The number of αvβ3-positive cells was significantly decreased in integrin-β3 siRNA-transfected monocytes compared with that in control siRNA (Figure 6b).\nAfter 7 days of incubation, the number of TRAP-positive MNC was counted. Transfection with integrin-β3 siRNA significantly reduced the number of TRAP-positive MNC in a dose-dependent manner compared with control siRNA transfection (Figure 6c). In addition, the use of siRNA directed toward a different site of integrin-β3 mRNA also inhibited osteoclast formation from CD16- monocytes (data not shown). On the other hand, siRNA that targeted lamin, which was used as a negative control, did not inhibit the induction of osteoclasts (data not shown). These results indicate the importance of integrin β3 in RANKL-induced osteoclast formation from CD16- peripheral blood monocytes.\n\nCyclic RGDfV peptide inhibits the osteoclastogenesis from CD16- monocytes\nIntegrin αvβ3 recognizes a common tripeptide sequence, RGD (Arg-Gly-Asp), which is present in bone matrix proteins such as vitronectin and fibronectin [21]. Cyclic RGDfV peptide (Arg-Gly-Asp-D-Phe-Val) inhibits binding of the RGD-containing molecules to αvβ3 [22]. To investigate the role of ligand binding to the αvβ3 heterodimer in the osteoclastogenesis, we examined whether cyclic RGDfV peptide inhibits the formation of osteoclasts. Cyclic RGDfV peptide significantly reduced the number of TRAP-positive MNC in a dose-dependent manner (Figure 6d). The results imply possible involvement of ligand bindings to αvβ3 in the osteoclastogenesis.\n\nKnockdown of integrin β3 did not affect the expression of NFATc1 mRNA\nIn the next step, we determined whether integrin-β3-siRNA-induced inhibition of the osteoclastogenesis reflects downregulation of NFATc1, which is a key transcription factor in osteoclastogenesis [23]. For this purpose, we compared NFATc1 mRNA levels between integrin β3 and control siRNA-transfected monocytes. Interestingly, integrin-β3 knockdown did not alter the NFATc1 mRNA level (Figure 7), suggesting that signal transduction mediated by integrin β3 does not affect the expression of NFATc1.\n\nDetection of CD16+ and CD16- macrophages in synovium of RA patients\nRA synovial macrophages are derived from peripheral blood monocytes, and their recruitment into the synovium is facilitated by various adhesion molecules and chemokines [24]. To analyze CD16 expression on synovial macrophages, RA synovial tissues were double-stained for CD16 and a macrophage marker, CD68. CD16-/CD68+ macrophages were widespread in the synovium. Although less frequent, CD16+/CD68+ macrophages were also observed both in the synovial intima and subintima (Figure 8). The presence of two subsets of macrophages, CD16+ and CD16-, in RA synovium indicates that both CD16+ and CD16- peripheral blood monocytes are recruited into the synovium.\n\n\nDiscussion\nHuman peripheral blood monocytes are a heterogeneous population, and they are divided into two subsets based on the expression of CD16. The CD16+ and CD16- monocyte subsets show functional differences in migration, cytokine production and differentiation into macrophages or dendritic cells [11-13,15]. We focused on the heterogeneity of the monocytes, and the primary question addressed in this study was which monocyte subset could be the source of osteoclasts. The results demonstrated that CD16- peripheral blood monocytes, but not CD16+ monocytes, differentiated in vitro into osteoclasts by treatment with RANKL + M-CSF.\nTo investigate the molecular mechanisms of the different response to RANKL and the differentiation into osteoclasts between CD16+ and CD16- monocytes, we examined the expression of molecules known to be involved in osteoclastogenesis. The expression profiles of integrin αv, integrin β3 and NFATc1 were different between the two subsets. Integrin αvβ3 heterodimer was expressed only on RANKL and M-CSF-stimulated CD16- monocytes. It is known that αvβ3 expressed on osteoclasts is important in bone resorption as well as in attachment of osteoclasts to the bone matrix [25].\nIt was recently reported that bone marrow macrophages of integrin-β3-deficient mice could not differentiate into mature osteoclasts in vitro, suggesting that αvβ3 is involved not only in activation, but also in differentiation, of osteoclasts in mice [26,27]. The authors also showed that αvβ3 and c-fms share a common intracellular signaling pathway, including the activation of ERK and the induction of c-Fos [27], both of which are essential for osteoclastogenesis [28,29]. In addition, it was reported that echistatin, an αvβ3 antagonist, inhibited osteoclast formation of mouse bone marrow cells [30].\nIn accordance with these reports, our data showed that knockdown of integrin-β3 expression resulted in downregulation of the αvβ3 heterodimer, and abrogated osteoclastogenesis from human peripheral blood CD16- monocytes. We also showed that blocking of adhesive ligands to bind to αvβ3 by RGDfV peptide inhibited osteoclast formation from CD16- monocytes. Taken together, the process of ligand binding to αvβ3 may be involved in the osteoclastogenesis. Blockade of αvβ3 could therefore be a therapeutically beneficial approach to modulate osteoclastogenesis. Indeed, integrin αvβ3 antagonists effectively treated osteoporosis in mice, rats and humans, and protected bone destruction in rat adjuvant-induced arthritis in vivo [31-34]. Of note, it is reported that patients with Iraqi-Jewish-type Glanzmann thrombasthenia who are deficient in integrin β3 do not develop osteopetrosis because of the upregulation of α2β1 expression on osteoclasts, although the bone-resorptive ability of the osteoclasts was decreased in vitro [35]. The function of αvβ3 in vivo in osteoclast formation and resorptive function could therefore be partially compensated by other integrins.\nAlthough all the multinucleated osteoclasts expressed αvβ3 (Figure 4d) [36], a small number of M-CSF + RANKL-stimulated mononuclear CD16- monocytes expressed αvβ3 (Figure 4d). Multinucleated osteoclasts are formed by fusion of osteoclast precursor cells [37]. It was reported that αvβ3 is involved in the migration of osteoclast precursors [30]. The αvβ3-positive cells could therefore be forced to migrate by the ligands and may fuse with closed αvβ3-negative cells. Alternatively, only αvβ3-positive cells may be fused with each other.\nIt is possible to consider that signaling from CD16 by anti-CD16 mAb-coated magnetic beads, which were used for the cell separation, or by IgG contained in FBS might inhibit osteoclastogenesis from CD16+ monocytes. We therefore separated the two subsets using anti-CD33 mAb and a fluorescent cell sorter, and stimulated the cells with M-CSF + RANKL. The results showed that CD33low monocytes, which correspond to CD16+ monocytes, still could not differentiate into osteoclasts. CD16 is a heterodimer consisting of FcγIIIa and Fcγ, and has low affinity for the Fc region of IgG. Aggregation of CD16 by immune complexes leads to transmission of activating signals via the immunoreceptor tyrosine-based activation motif in the γ chain [38]. We also assessed osteoclastogenesis from the two monocyte subsets using IgG-depleted bovine serum. Even in the IgG-free medium, CD16- monocytes but not CD16+ monocytes differentiated into osteoclasts (data not shown). We could therefore exclude the possibility that signal transduction through CD16 inhibits osteoclastogenesis from CD16+ monocytes.\nNFATc1 is a key transcription factor in osteoclastogenesis [16]. In the present study, stimulation with M-CSF + RANKL increased the NFATc1 mRNA expression in the CD16- subset only, similar to integrin αv and integrin β3. The differences in NFATc1 induction might therefore also explain the difference in osteoclastogenesis between the two monocyte subsets. It is of interest that knockdown of integrin β3 did not lower the mRNA level of NFATc1. This result supports the notion that NFATc1 is located upstream of integrin-β3 expression [39]. It is also possible that parallel activation of two signaling pathways mediated by integrin β3 and NFATc1 contributes to osteoclastogenesis independently or cooperatively. Further studies are needed to determine the mechanisms of integrin β3 involvement in RANKL/RANK-mediated osteoclast differentiation.\nIt has been demonstrated that MAPK families, ERK and p38 MAPK, were activated by RANKL-induced intracellular signalings in osteoclasts and osteoclast precursors [18,19]. In addition, these kinases are involved in the differentiation of osteoclasts [20]. We showed that RANKL stimulation induced phosphorylation of ERK and p38 MAPK only in CD16- monocytes. It is suggested that differential activation of these kinases may partially explain the distinct properties of the two monocyte subsets upon RANKL stimulation.\nOur results showed that CD16+ monocytes produce higher levels of inflammatory cytokines including TNFα and IL-6 compared with CD16- monocytes. These results are consistent with the previous report showing that CD16+ monocytes produced larger amounts of TNFα upon lipopolysaccharide or lipopeptide stimulation than did CD16- monocytes [40]. Interestingly, we showed that stimulation either with RANKL or M-CSF upregulated the TNFα and IL-6 production by CD16+ monocytes. A marked increase of CD16+ monocytes in peripheral blood is reported in inflammatory diseases, such as infection, malignancy, Kawasaki disease and RA [41-44]. Taken together, CD16+ monocytes may be an important source of inflammatory cytokines.\nIn mice, peripheral blood Ly-6Chigh monocytes, which are thought to correspond to human CD16- monocytes, increase in inflammatory conditions, and these cells are recruited into sites of inflammation [45]. In contrast, Ly-6Clow monocytes, which are thought to correspond to human CD16+, migrate into noninflamed tissues [12]. These data on mouse monocytes seem to be in contrast to the data on human monocytes, which show expansion of CD16+ monocytes in inflammatory conditions where they produce larger amounts of inflammatory cytokines. At present, it is not clear whether mouse monocyte subsets, Ly-6Clow/Ly-6Chigh, represent human monocyte subsets, CD16+/CD16- monocytes, and whether the biologic functions of mouse monocytes are analogous to those of human monocytes.\nIn mice, blood monocytes newly released from the bone marrow are exclusively Ly-6Chigh and the level of Ly-6C is downregulated while in circulation [45]. It is thus suggested that in mice the two monocyte subsets differing in Ly-6C expression represent different stages in the maturation pathway. In the human, transition from CD16- monocytes to CD16+ monocytes is observed upon culture with IL-10, M-CSF and transforming growth factor beta in vitro [42,46]. Similar to mouse monocytes, therefore, human peripheral blood CD16- monocytes may also maturate into CD16+ monocytes.\nIt is reported that a significant number of RA synovial cells in the intima express CD16, suggesting that CD16+ cells are synovial macrophages [47]. We confirmed that both CD16+ and CD16- macrophages accumulate in the RA synovium by double-color immunohistochemical staining for CD68 and CD16. A number of chemokines are abundantly expressed in the RA synovium [24,48]. Among these cytokines, MCP-1, MIP-1α, SDF-1, RANTES and fractalkine can induce migration of CD16- monocytes in vitro ([11,12] and unpublished data). On the other hand, migration of CD16+ monocytes is induced only by fractalkine. These chemokines therefore seem to play an important role in recruitment of CD16+ and CD16- monocytes from the circulating pool into the RA synovium.\nThe osteoclast inducers are also produced in the RA synovium. RANKL is expressed by synovial fibroblasts and activated T cells [49-51], while M-CSF is expressed on RA synovial macrophages and fibroblasts [52,53].\nTNFα and IL-6, which are mainly expressed on RA synovial macrophages and fibroblasts, respectively, could also enhance osteoclast differentiation [54]. Collectively, it is probable that the recruited CD16- monocytes/macrophages differentiate into osteoclasts in the RA synovium, and contribute to bone destruction. On the other hand, CD16+ monocytes/macrophages might also be involved in RA pathogenesis by producing inflammatory cytokines including TNFα and IL-6. Since TNFα and IL-6 enhance osteoclast formation [54,55], CD16+ monocytes/macrophages may also contribute to osteoclastogenesis in RA synovium.\n\nConclusion\nWe have shown that human peripheral blood monocytes consist of two functionally heterogeneous subsets with distinct response to osteoclastogenic stimuli. Osteoclasts seem to originate from CD16- monocytes, and integrin β3 is necessary for the osteoclastogenesis. The blockade of accumulation and activation of CD16- monocytes could therefore be a beneficial approach as an anti-bone resorptive therapy, especially for RA.\n\nAbbreviations\nDAP = DNAX-activation protein; ELISA = enzyme-linked immunosorbent assay; FBS = fetal bovine serum; FcRγ = Fc receptor γ chain; IL = interleukin; FITC = fluorescein isothiocianate; mAb, monoclonal antibody; M-CSF = macrophage colony-stimulating factor; MEM = modified Eagle's medium; MMP = matrix metalloproteinase; MNC = multinucleated cells; NF = nuclear factor; OSCAR = osteoclast-associated receptor; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; RA = rheumatoid arthritis; RANK = receptor activator of NF-κB; RANKL = receptor activator of NF-κB ligand; RT = reverse transcriptase; siRNA = small interfering RNA; SIRP-β1 = signal regulatory protein-β1; TNFα = tumor necrosis factor alpha; TRAF = tumor necrosis factor receptor-associated factor; TRAP = tartrate-resistant acid phosphatase; TREM = triggering receptor expressed on myeloid cells.\n\nCompeting interests\nThe authors declare that they have no competing interests.\n\nAuthors' contributions\nYK participated in the design of the study, carried out the experiments and statistical analysis, and drafted the manuscript. KH and KT participated in the design of the study and its coordination. TN and NM conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 39944 ] ] } ]

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[ { "id": "pmcA2556924__text", "type": "Article", "text": [ "Are there sensitive subgroups for the effects of airborne particles?\nAbstract\nRecent studies have shown that particulate air pollution is a risk factor for hospitalization for heart and lung disease; however, little is known about what subpopulations are most sensitive to this pollutant. We analyzed Medicare hospital admissions for heart disease, chronic obstructive pulmonary disorders (COPD) and pneumonia in Chicago, Cook County, Illinois, between 1985 and 1994. We examined whether previous admissions or secondary diagnoses for selected conditions predisposed persons to having a greater risk from air pollution. We also considered effect modification by age, sex, and race. We found that the air-pollution-associated increase in hospital admissions for cardiovascular diseases was almost doubled in subjects with concurrent respiratory infections. The risk was also increased by a previous admission for conduction disorders. For COPD and pneumonia admissions, diagnosis of conduction disorders or dysrhythmias increased the risk of particulate matter < 10 microm in aerodynamic diameter (PM(10))-associated admissions. Persons with asthma had twice the risk of a PM(10)-associated pneumonia admission and persons with heart failure had twice the risk of PM(10)-induced COPD admissions. The PM(10) effect did not vary by sex, age, and race. These results suggest that patients with acute respiratory infections or defects in the electrical control of the heart are a risk group for particulate matter effects.\nArticles \n\n Are There Sensitive Subgroups for the Effects of Airborne Particles? Antonella Zanobetti,1 Joel Schwartz,1,2 and Diane Gold1,2 1Environmental \n\n Epidemiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts, USA; 2Channing Laboratory, Department of Medicine, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts, USA \n\n Recent studies have shown that particulate air pollution is a risk factor for hospitalization for heart and lung disease; however, little is known about what subpopulations are most sensitive to this pollutant. We analyzed Medicare hospital admissions for heart disease, chronic obstructive pulmonary disorders (COPD) and pneumonia in Chicago, Cook County, Illinois, between 1985 and 1994. We examined whether previous admissions or secondary diagnoses for selected conditions predisposed persons to having a greater risk from air pollution. We also considered effect modification by age, sex, and race. We found that the air-pollution-associated increase in hospital admissions for cardiovascular diseases was almost doubled in subjects with concurrent respiratory infections. The risk was also increased by a previous admission for conduction disorders. For COPD and pneumonia admissions, diagnosis of conduction disorders or dysrhythmias increased the risk of particulate matter < 10 �m in aerodynamic diameter (PM10)-associated admissions. Persons with asthma had twice the risk of a PM10-associated pneumonia admission and persons with heart failure had twice the risk of PM10-induced COPD admissions. The PM10 effect did not vary by sex, age, and race. These results suggest that patients with acute respiratory infections or defects in the electrical control of the heart are a risk group for particulate matter effects. Key words: effect modification, hospital admissions, particulate air pollution. Environ Health Perspect 108:841�845 (2000). [Online 28 July 2000] http://ehpnet1.niehs.nih.gov/docs/2000/108p841-845zanobetti/abstract.html \n\n Particulate air pollution has been associated with increases in daily deaths and hospital admissions in studies all over the world (1�15). These associations are now well documented but little is known, as yet, of the characteristics of persons that put them at increased risk of adverse events related to particulate air pollution. This has been identified as a key data gap (16). Schwartz and Dockery (17) reported that persons older than 65 years of age had a somewhat increased risk of death, and this has been confirmed in other studies (18). A more detailed examination of particulate matter-related risk by deciles of age (19) showed the risk beginning to increase at approximately 40 years of age and reaching its maximum for those 75 years of age and older. In addition to age, several studies suggest that persons with respiratory illness are at increased risk for cardiovascular effects associated with air pollution. An examination of death certificates on high- and low-air pollution days reported a substantial difference in the proportion of deaths from cardiovascular causes that had respiratory disease as a contributing cause of death (19). A recent follow-up study of a cohort of persons with chronic obstructive pulmonary disease (COPD) in Barcelona, Spain, found an association between particulate air pollution and all-cause mortality in the cohort (20). The magnitude of the risk per microgram per cubic meter of exposure was substantially greater than that for the general population. Environmental Health Perspectives \n\n Controlled exposure of animals with chronic bronchitis and control animals to concentrated air particles also demonstrated a potentiating effect of chronic lung disease in the response to airborne particles (21). This has led to the hypothesis that the cardiovascular effects of air pollution are predominantly in persons with chronic lung disease. There has been even less done to examine potential modifiers of the effects of airborne particles on hospital admissions. The existing literature on comorbidity shows that comorbidity per se seems to increase the risk of adverse outcomes (22�30). Little is known about the role of these comorbidities as effect modifiers for the effects of air pollution. This study uses data from the Medicare system to examine potential short-term and long-term medical conditions that may increase a person's risk of hospital admissions associated with particulate air pollution. In addition, we examine potential effect modification by age, race, and sex. \n\n Materials and Methods Health data. The Health Care Financing Administration (Baltimore, MD) maintains records of every hospital admission for Medicare participants in the United States. Persons in this database have a unique identifier. Using this identifier, we traced every hospital admission for heart and lung disease for each person in Cook County, Illinois, between 1985 and 1994. We chose Cook County because it is the most populous \n\n county in the United States with daily monitoring for particulate matter with aerodynamic diameter < 10 �m (PM10). The data were then analyzed to look at effect modification by concurrent and preexisting conditions as well as by age, race, and sex. To establish a baseline risk, we computed daily counts of hospital admissions for cardiovascular disease (CVD) [International Classification of Disease, 9th edition, World Health Organization, Geneva (ICD-9) code 390�429], pneumonia (ICD-9 code 480�487), and COPD (ICD-9 code 490�496, excluding 493). The association between these daily counts and PM10 was examined for the years 1988�1994, when daily PM10 monitoring data were available in Chicago. Once our baseline risks were established, we examined three classes of potential effect modifiers. First, we looked at whether previous admissions for selected conditions predisposed persons to having a greater risk from air pollution. For each of the three admission categories (CVD, pneumonia, and COPD), we considered 10 causes (defined by a previous admission) as effect modifiers: COPD (ICD-9 code 490�496 except 493), asthma (ICD-9 code 493), acute bronchitis (ICD-9 code 466), acute respiratory illness (ICD-9 code 460�466), pneumonia (ICD-9 code 480�487), CVD (ICD-9 code 390�429), myocardial infarction (ICD-9 code 410), congestive heart failure (ICD-9 code 428), conduction disorders (ICD-9 code 426), and dysrhythmias (ICD9 code 427). To test the hypothesis that persons with these conditions had higher risks of subsequent PM10-related admissions, we computed separate daily counts of admissions for our three target causes, stratified by whether or not the person admitted had been previously admitted for the hypothesized predisposing condition. Separate analyses were then performed within each strata to see if the effects of PM10 differed by strata. Address correspondence to A. Zanobetti, Department of Environmental Health, Environmental Epidemiology Program, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115 USA. Telephone: (617) 4324642. Fax: (617) 277-2382. E-mail: azanob@ sparc6a.harvard.edu Supported by NIEHS grant ES07937. Received 18 January 2000; accepted 18 April 2000. \n\n � VOLUME 108 | NUMBER 9 | September 2000 \n\n 841 \n\n \fArticles \n\n � \n\n Zanobetti et al. \n\n The second set of potential predisposing conditions included secondary diagnoses associated with the index admission. These could represent the presence of a chronic condition (e.g., COPD) that has not resulted in a previous hospital admission. They could also represent acute conditions that may have increased the subjects' sensitivity to air pollution. For example, if respiratory infections modified the effect of particulate matter on the cardiovascular health of persons with underlying heart disease, then the risk of a hospital admission for heart disease might be different in persons with infections. If this were true, then the risk ratio of a 10-�g/m3 increase of PM10 on cardiovascular admissions of persons with a concurrent respiratory infection would be different from the ratio in persons without respiratory infection. To test these hypotheses, we computed separate daily counts of admissions for events with and without the concurrent conditions hypothesized to increase sensitivity to air pollution. These were taken as the same 10 conditions in the first analysis with certain exclusions for pairing that would be illogical. That is, the concurrent diagnosis of a specific cardiac condition was not treated as an effect modifier for admissions for any cardiovascular condition. Likewise, pneumonia and COPD were not possible concurrent conditions for each other. The third set of predisposing conditions considered was being older than 75 years of age, nonwhite, and female. These were examined for all three outcomes. We obtained weather data for O'Hare Airport from the EarthInfo CD-ROM (EarthInfo CD NCDC Surface Airways, EarthInfo Inc., Boulder, CO), and we obtained air pollution data from the U.S. Environmental Protection Agency Aerometric Information Retrieval System network (31). \n\n running-line smoother, loess (35), was chosen to estimate the smooth function. To control for weather variables and day of the week, we chose the smoothing parameter that minimized the Akaike's information criterion (36). To model seasonality we chose the smoothing parameter that minimized the sum of the autocorrelation of the residuals while removing seasonal patterns. Two autoregressive terms (37) were added in the model to eliminate the remaining serial correlation from the residuals. We used the mean of PM10 on the day of the admission and the day before the admission as our exposure variable. This gives results that are similar to those obtained fitting a full distributed lag model (38). PM10 was treated linearly. Our baseline models used the daily counts of CVD, pneumonia, and COPD admissions as outcomes. We then subdivided those counts by the presence or absence of the potential effect modifier and reestimated our regressions on those subgroups. We considered effect modification to be indicated when the estimates of PM10 in the group with the condition was outside of the 95% confidence interval (CI) of the effect estimate in persons without the condition. \n\n Results Table 1 shows the mean daily admissions for COPD, cardiovascular, and pneumonia both overall and in the presence of the potential effect modifiers. For some effect modifiers such as conduction disorders or myocardial infarctions, the counts in conjunction with our respiratory outcomes are \n\n low, which limits power. In general, the numbers are lower for examining effect modification by previous admissions than for effect modification by concurrent diagnosis. This is as expected because many clinically relevant comorbidities may never have resulted in a hospital admission. Table 2 shows the 25th, 50th, and 75th percentile values for the environmental variables. The mean value for PM10 is 33 �g/m3. The daily values for PM10 were computed as the average of 10 monitors, two of which measured PM10 almost every day and the others less frequently (38). Table 3 shows the mean daily counts of CVD, COPD, and pneumonia by sex, age groups, and race. The distribution by sex is almost even, although the counts of admissions for males are generally lower (approximately 10%) than for females, particularly for cardiovascular diseases. The counts of CVD, COPD, and pneumonia admissions were similar for people 65�75 or 75 years of age and older. Tables 4�6 show the results for the effect PM10 overall and stratifying by concurrent diagnosis and previous admissions. These are expressed as the percentage increase for 10 �g/m3 PM10. Table 4 shows the results for CVD. A 10-�g/m3 increase in PM10 was associated with a 1.31% (5% CI, 0.97%; 95% CI, 1.66%) increase in hospital admissions for heart disease in all elderly persons. A concurrent (not previous) diagnosis of COPD modified the risk of PM10-associated admissions for heart disease. However, significant associations were still seen between PM10 \n\n Table 1. Mean daily counts of admissions, Chicago 1986�1994, for COPD, CVD, and pneumonia overall and by concurrent diagnosis and by previous admissions. By concurrent diagnosis COPD CVD Pneumonia Overall Respiratory disease Acute bronchitis Acute respiratory infections Pneumonia Asthma COPD Cardiovascular disease CVD Conduction disorders Cardiac dysrhythmias Congestive heart failure Myocardial infarction NA, not applicable. \n\n By previous admissions COPD CVD Pneumonia 7.8 0.8 0.9 1.6 0.9 2.7 2.1 0.0 0.4 0.9 0.3 102.1 1.6 1.8 7.3 1.5 2.0 54.7 1.0 9.9 24.2 11.4 26.5 0.9 1.0 6.4 0.7 1.4 7.2 0.2 1.5 3.1 1.0 \n\n Methods We analyzed the data with a generalized additive robust Poisson regression model (32). This approach has become the norm in such studies (14,33,34). In the generalized additive model the outcome is assumed to depend on a sum of nonparametric smooth functions for each variable that models the potential nonlinear dependence of daily admission on weather and season. The model is of the form: log[E(Yt)] = 0+ S1 (X1 )+... + Sp (Xp) where E(Yt) is the expected value of the daily count of admissions Yt and Si are the smooth functions of the covariates Xi. We examined temperature, previous day's temperature, relative humidity, barometric pressure, and day of week covariates. The locally weighted \n\n 7.8 0.1 0.3 0.4 0.1 NA 4.7 0.2 1.4 1.8 0.1 \n\n 102.1 0.9 1.3 4.0 1.8 13.4 NA NA NA NA NA \n\n 26.5 0.3 0.3 NA 0.9 6.9 14.7 0.6 4.6 7.3 0.4 \n\n Table 2. 25th, 50th, and 75th percentile values for the environmental variables in Chicago, 1988�1994. Temperature (�F) 35 51 67 Relative humidity 62 70 79 Barometric pressure 29.2 29.3 29.4 PM10 (�g/m3) 23 33 46 \n\n Table 3. Mean daily counts of admissions by sex, race, and age groups, Chicago, 1986�1994. Group Overall Female Nonwhite Age > 75 years COPD 7.8 4.2 1.6 3.7 CVD 102.1 59.4 21.0 55.1 Pneumonia 26.5 14.7 5.2 17.4 \n\n 842 \n\n VOLUME \n\n 108 | NUMBER 9 | September 2000 � Environmental Health Perspectives \n\n \fArticles \n\n � \n\n Effects of particles on sensitive subgroups \n\n and heart disease admissions in persons without COPD listed as either a comorbidity or a cause of previous admission (Table 4). A significant association was also seen in persons without any respiratory disease as a concurrent diagnosis, although the risk is much lower than in persons with respiratory disease. However, the risk associated with PM10 was roughly doubled in subjects with concurrent respiratory infections and the risk estimates in those subjects were outside the 95% CI of the risk in patients without concurrent respiratory infections. A previous admission for conduction disorders (e.g., heart block) increased the risk of a PM10-related subsequent admission for any heart condition, and a weaker indication of effect modification was seen for persons with previous admission for dysrhythmias. In contrast heart failure and previous myocardial infarctions were highly insignificant as effect modifiers. Table 5 shows the results for COPD. Overall, there is a 1.89% (95% CI, 0.8�3.0) increase in COPD admissions for a 10�g/m3 increase in PM10. The results of the stratified analysis suggest that preexisting heart disease modifies Table 4. Percentage increase in hospital admissions for CVD in all persons and by concurrent diagnosis and previous admissions. PM10 2.5% CI 97.5% CI All persons 1.31 By concurrent diagnosis Respiratory disease All respiratory disease With 1.65 Without 0.98 Acute bronchitis With 2.50 Without 1.07 Acute respiratory infections With 2.71 Without 1.06 Pneumonia With 1.95 Without 1.03 COPD With 1.59 Without 1.08 By previous admissions Respiratory disease All respiratory disease With 1.18 Without 1.08 COPD With 1.48 Without 1.09 Asthma With 1.71 Without 1.08 Cardiovascular disease Conduction disorders With 2.89 Without 1.07 Cardiac dyshrethmias With 1.61 Without 1.04 0.97 1.66 \n\n the risk of COPD admissions on high particle days. Previous admissions for any cardiovascular disease increased the risk of a PM10associated COPD admission approximately 2.5-fold. A previous heart failure admission caused an even more striking increase in the PM10 effect. Previous admissions for dysrhythmias and conduction defects were rare (Table 1) with no power to examine effect modifications. Listings as concurrent diagnoses were more common and here they joined heart failure in increasing the risk of PM 10 -associated COPD admissions. For COPD there was also some indication that concurrent pneumonia or an acute respiratory infection admission in the last year increased risk. The low numbers made these estimates less precise, however. The percentage increase in pneumonia admission (Table 6) for 10 �g/m3 PM10 is higher than for COPD or CVD with an increase of 2.34% (95% CI, 1.66�3.0). As with COPD, persons with heart disease appeared at higher risk of pneumonia hospital admissions associated with particulate air pollution. Here diagnoses suggestive of impaired autonomic control of the heart, such as conduction disorders or dysrhythmias, were associated with increased risk for PM 10 effects on pneumonia admissions. Unlike COPD, no difference was seen for congestive heart failure. Persons with asthma Table 5. Percentage increase in hospital admissions for COPD in all persons and by concurrent diagnosis and previous admissions. PM10 2.5% CI 97.5% CI All persons 1.89 By concurrent diagnosis Respiratory disease Pneumonia With 4.00 Without 1.51 Cardiovascular disease Conduction disorders With 2.34 Without 1.60 Cardiac dysrhythmias With 3.09 Without 1.43 Congestive heart failure With 2.90 Without 1.39 By previous admissions Respiratory disease Acute respiratory infections With 3.20 Without 1.70 Cardiovascular disease CVD With 2.90 Without 1.18 Congestive heart failure With 4.37 Without 1.14 Within 1 year 6.04 0.80 2.99 \n\n had twice the risk of a PM10-induced pneumonia admission as persons without asthma. Table 7 shows the results by sex, age, and race. None of the effect size estimates for any of the stratification variables were outside of the 95% CI for the opposite strata. There was a tendency for the effect of PM10 on CVD admissions to be higher for females, whereas the effect on pneumonia admissions was higher for males. In general, we found somewhat larger effects on whites compared to nonwhites, and for persons older than 75 years of age compared to younger persons. \n\n Discussion In this analysis we examined whether the effect of PM10 on the risk of hospital admission for heart and lung disease was different depending on the presence of comorbidities. We found that PM10 was associated with hospital admissions for all three causes (CVD, COPD, and pneumonia) and we found not a general increase in PM10 related risk with comorbidities, but a specific pattern that is suggestive of potential mechanisms and consistent with other recent epidemiologic and toxicologic findings. One major finding of this study is that preexisting cardiovascular disease, particularly impaired autonomic control (conduction defects and dysrhythmias) and heart failure, substantially increased the risk of respiratory admissions associated with airborne particles. In fact, recent human studies have shown that exposure to particulate air pollution is a risk factor for reduced heart rate variability (39�41). Reduced heart rate variability is an adverse response and a risk factor for arrhythmia. A new study of defibrillator discharges in patients with implanted cardioverter defibrillators found that discharges were associated with air pollution (42). Exposure to combustion Table 6. Percentage increase in hospital admissions for pneumonia in all persons and by concurrent diagnosis and previous admissions. PM10 All persons By concurrent diagnosis Respiratory disease Asthma With Without Cardiovascular disease Conduction disorders With Without Cardiac dysrhythmias With Without By previous admissions Cardiovascular disease Cardiac dysrhythmias With Without 2.34 2.5% CI 97.5% CI 1.66 3.02 \n\n 1.10 0.64 �0.47 0.76 0.18 0.76 0.55 0.72 0.85 0.75 \n\n 2.20 1.33 5.55 1.37 5.30 1.37 3.36 1.35 2.34 1.41 \n\n �0.45 0.47 �4.42 0.58 0.64 0.33 0.77 0.24 \n\n 8.65 2.57 9.59 2.64 5.60 2.55 5.08 2.55 \n\n 0.45 0.76 �0.40 0.78 �0.43 0.77 0.22 0.76 0.75 0.72 \n\n 1.91 1.41 3.40 1.40 3.89 1.39 5.63 1.38 2.48 1.36 \n\n 4.18 2.07 7.92 1.99 � � \n\n 1.01 1.46 4.28 1.37 � � \n\n 7.46 2.69 11.69 2.61 � � \n\n �1.38 0.66 0.99 �0.01 1.43 0.05 2.10 \n\n 8.01 2.76 4.85 2.39 7.40 2.24 10.14 \n\n 3.47 2.08 \n\n 1.21 1.45 \n\n 5.79 2.71 \n\n Increases are for a 10-�g/m3 increase in PM10. \n\n Increases are for a 10-�g/m3 increase in PM10. \n\n Increases are for a 10-�g/m3 increase in PM10. \n\n Environmental Health Perspectives \n\n � VOLUME 108 | NUMBER 9 | September 2000 \n\n 843 \n\n \fArticles \n\n � \n\n Zanobetti et al. \n\n Table 7. Effect modification by sex, race, and age groups for 10 �g/m3 PM10. % All persons Male Female White Non-white Age > 75 Age 75 1.89 1.34 2.19 1.65 1.07 2.20 1.33 COPD (95% CI) (0.80, 2.99) (�0.14, 2.84) (0.81, 3.59) (0.51, 2.81) (�1.11, 3.3) (0.72, 3.69) (0.03, 2.65) % 1.31 1.07 1.21 1.20 0.70 1.28 0.93 CVD (95% CI) (0.97, 1.66) (0.62, 1.51) (0.83, 1.6) (0.86, 1.55) (0.1, 1.3) (0.88, 1.69) (0.51, 1.35) % 2.34 2.65 1.91 2.45 1.91 2.12 2.52 Pneumonia (95% CI) (1.66, 3.02) (1.81, 3.5) (1.11, 2.72) (1.77, 3.14) (0.69, 3.14) (1.38, 2.86) (1.57, 3.48) \n\n Figures shown are the percentage increase in admissions (95% CI). \n\n particles has also been associated with arrhythmia in an animal model (43) and changes in ST segments have been noted as well (44). This is the first study to suggest persons with defects in the electrical control of the heart are also at higher risk of respiratory illness after exposure to airborne particles. These data also suggest that persons admitted to hospitals for pneumonia during an air pollution episode may be at high risk for clinically significant conduction disorders during that hospital admission. Patients with congestive heart failure were at greater risk of hospital admissions for COPD in association with airborne particles. Heart failure and COPD is not an uncommon combination. The finding that these patients are at higher risk for admissions associated with particulate air pollution is new but is also consistent with several other recent reports. The spontaneous hypertensive rat develops a model of heart failure, and recent studies have reported greater sensitivity to particulate air pollution in these rats. These include both electrocardiogram abnormalities (44) and pulmonary toxicity (45,46). Similarly, in an epidemiologic study, Hoek et al. (47), found a higher relative risk of death with an increase in PM10 for congestive heart failure deaths than other deaths. The potential role of COPD in those heart failure deaths was not examined. Another consistent pattern in our data is of acute respiratory infections increasing susceptibility to airborne particles. Acute bronchitis, or more generally acute upper respiratory illnesses, as well as pneumonia, increased susceptibility to particle-associated admissions for CVD and COPD. The notion that air pollution exacerbates acute respiratory infections is well supported by studies which report associations between airborne particles and hospital admissions for respiratory infections (48,49). Zelikoff et al. (50) exposed rats infected with streptococcus to concentrated air particles and reported a significant increase in bacterial burdens and in the extent of pneumonia compared to animals exposed to filtrated air. This suggests an impaired immune response. Similarly, exposure to combustion \n\n particles enhances influenza infections in mice (51). An impaired defense to respiratory infection is a major reason that persons with COPD require hospital admission. If airborne particles result in further impairment the effect modification we observe makes good sense. The effect modification for heart disease admissions is more relevant. This modification is consistent with the earlier report of Schwartz (19), who found greater reports of respiratory complications on death certificates with an underlying cause of heart disease if the death occurred on a day with high levels of airborne particles. Although airborne particle exposure has been associated with increased exacerbation of asthma (2,12,48,52�59), this paper is the first to suggest that asthmatics are more susceptible to PM10-induced pneumonia exacerbation or to cardiovascular effects. The effects on pneumonia admissions are plausible, given the impaired ability to fight off infections in asthmatics with mucus plugs and the evidence the airborne particles impair the lungs' ability to fight off bacterial and viral infections, as noted earlier. The increased cardiovascular sensitivity, albeit weaker, is interesting. If airborne particles affect the cardiovascular system via the role of the lung in autonomic control, it is possible that asthmatics would be more sensitive to those effects. Animal models of asthma showed that combustion particles enhance the asthmatic response to aeroallergen challenges (59). This suggests an enhancement of pulmonary response in asthmatics. On the other hand, the diagnosis of asthma is problematic in the elderly, and crossover with COPD is possible. The possibility that this explains our results is reduced by our failure to find previous hospital admission for COPD was an effect modifier for the effect of particles on cardiovascular admissions. We must acknowledge several potential limitations of this study. First, we considered only previous admissions that occurred within Cook County. Hence persons with previous admissions elsewhere would be misclassified to our reference group. The effect of this would be to reduce the difference in PM 10 effect between the two groups. VOLUME \n\n Nevertheless, we identified some interesting interactions. We cannot exclude the possibility that there are areas we missed for this reason. We also examined interactions in a log relative risk model, which is inherently multiplicative. Although we believe this is justified because doubling the population exposed would be expected to double the pollution associated admissions, it results in a more conservative definition of interaction than would an additive risk model. Finally, our exposure is clearly measured with error. Most of this error is Berkson error (60) and hence will introduce no bias, and Zeger et al. (60) showed that the remaining error would have to have pathologic correlations with other variables to result in an upward bias. Another important result from this study, of course, is an estimate of the magnitude of the effect of airborne particles on public health. The PM10 concentrations in Chicago during this period were associated with approximately 1,600 additional admissions per year for heart disease, 740 additional admissions per year for pneumonia, and 170 additional admissions per year for COPD. These are not trivial increases in serious morbidity. 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[ { "id": "pmcA1474674__text", "type": "Article", "text": [ "Physiological and chemical characterization of cyanobacterial metallothioneins.\nAbstract\nTechniques have been developed for detection, quantitation, and isolation of bacterial metallothioneins (MTs) from cyanobacterial species. These methods involve differential pulse polarography and reverse-phase high-performance liquid chromatography (HPLC) and have allowed detection of picomole quantities of these high sulfhydryl content proteins. The prokaryotic molecule was found to be induced in the presence of Cd or Zn salts with regulation at the level of transcription. Cu was not found to induce synthesis of the prokaryotic MT. Exposure to the former metals resulted in a growth lag followed by simultaneous induction of MT synthesis and onset of growth. Amino acid analysis and N-terminal sequence analysis indicated that the bacterial MTs from cyanobacteria are unique, having many aromatic and aliphatic residues and no apparent association of hydroxylated or basic amino acids with cysteines. Although the characteristic Cys-X-Cys sequences were present, no apparent amino acid sequence homology with the eukaryotic MTs was found in the first 42 residues.\n\n\n\n\n Environmental Health Perspectives Vol. 65, pp. 71-75, 1986 \n\n Physiological and Chemical \n\n Characterization of Cyanobacterial Metallothioneins \n\n by Robert W. Olafson* \n\n Techniques have been developed for detection, quantitation, and isolation of bacterial metallothioneins (MTs) from cyanobacterial species. These methods involve differential pulse polarography and reverse-phase high-performance liquid chromatography (HPLC) and have allowed detection of picomole quantities of these high sulfhydryl content proteins. The prokaryotic molecule was found to be induced in the presence of Cd or Zn salts with regulation at the level of transcription. Cu was not found to induce synthesis of the prokaryotic MT. Exposure to the former metals resulted in a growth lag followed by simultaneous induction of MT synthesis and onset of growth. Amino acid analysis and N-terminal sequence analysis indicated that the bacterial MTs from cyanobacteria are unique, having many aromatic and aliphatic residues and no apparent association of hydroxylated or basic amino acids with cysteines. Although the characteristic CysX-Cys sequences were present, no apparent amino acid sequence homology with the eukaryotic MTs was found in the first 42 residues. \n\n Introduction \n\n Metallothioneins have now been isolated and characterized from a large variety of eukaryotic organisms (1) and shown to be involved in heavy metal detoxification and/or homeostasis (2-4). These proteins are highly homologous with respect to amino acid sequence and complex metals in characteristic metal-thiolate cluster arrangements, the structures of which have been recently \n\n investigated by 113Cd-NMR (6). \n\n Although metallothionein (MT) is found throughout the eukaryotic world, few reports of the presence of this type of high sulfhydryl content metal-binding protein exist for prokaryotes. We earlier reported the presence of a prokaryotic MT in cyanobacteria (7,8) and have recently provided primary sequence evidence substantiating these data. This manuscript is intended to summarize the present state of knowledge regarding the physiological and chemical characterization of these bacterial MTs, with an emphasis on the techniques employed in such studies. \n\n Analytical Procedure \n\n In order to facilitate rapid detection, isolation, and quantitation of MT from various sources, we have routinely employed a differential pulse polarographic tech\n\n *Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada. \n\n nique first described by Brdicka (9-11). This procedure avoids use of radioisotopes and circumvents problems such as species specificity and metal stoichiometric assumptions associated with radioimmunoassays and metal binding assays. Figure 1 shows a typical polarographic wave for MT using the Brdicka procedure. Measurement of the wave height can be performed from either the \n\n 0.20\n\n glJ MT-1 \n\n O 0.15\n\n E \n\n 0 0.10 0 \n\n E 0.05 _ _ \n\n -1.35 -1.45 -1.55 -1.65 \n\n V vs Agt/AgC1 \n\n FIGURE 1. Differential pulse polarographic wave for murine MT-1. \n\n Wave heights were calculated from the broken tangent line between minima rather than the supporting electrolyte baseline at the bottom of the figure. \n\n R. W OLAFSON \n\n supporting electrolyte baseline or a tangent to the minima as shown in the preceding figure. In the cobalt hexaminechloride supporting electrolyte, the half-wave potential for all MTs studied to date is approximately -1.45 V versus a Ag/AgCl reference electrode. This electrochemical reaction results in highly reproducible and linear current responses which can be used with samples containing picomole levels of MT (Fig. 2). Since this is an exceedingly sensitive instrument, attention to detail is particularly important for successful use of the Brdicka procedure (11). Routine use of a reference standard MT is highly recommended to compensate for small day-today variations in response. For absolute values, standards must be identical to the species of protein quantitated; otherwise commercially available standard is adequate. Although certain tissues and cellular types can be shown to have no other polarographically active material other than MT, with samples from new species, it is necessary to evaluate this potential problem by gelperneation chromatography and, if necessary, take steps to remove contaminants before assaying. In most cases, \n\n 0 \n\n E i \n\n m 0 \n\n 0 E \n\n 0.31 \n\n 0.11 \n\n O 0.1 0.3 0.5 \n\n MT (nanomoles) \n\n FIGURE 2. Standard curve for polarographic determination of me\n\n tallothionein. \n\n FIGURE 3. Cyclic voltamogram obtained by equilibration at -1.0 V \n\n for 4 min followed by cyclic scanning to OV and -1.4 V at a scan rate of 200 mV/sec with a PAR Model 303 static mercury drop electrode in the hanging drop mode. The mercury drop electrode was set in the large drop position producing a drop of 0.0226 cm3. The supporting electrolyte was 20 mM HEPES buffer, pH 7.3. The dotted line is the equilibrium trace. Adapted Olafson and Sim (10). \n\n this is easily managed by heat denaturation, if care is taken to assess the degree of MT losses due to coprecipitation. Such losses later can usually be minimized to less than 10% by adjustment of the tissue homogenate density prior to denaturation. \n\n It should be indicated that additional utility can be found with a polarographic analyzer beyond application of the Brdicka procedure. For example, metal levels can be readily determined by using differential pulse or anodic stripping voltametry procedures (10), while Figure 3 shows a cyclic voltamogram of bacterial MT. The latter technique is potentially useful in assessing metal speciation and integrity of the protein tertiary structure near metal clusters, particularly after metal reconstitution studies. \n\n E \n\n 'C2A- E 24 \n\n 0 30 40 E0 \n\n *~~~~~~~~~~~~ \n\n o C~~~~~~~~~~~~~~~~~~~~C \n\n Fraction Number (18ml) \n\n FIGURE 4. Sephadex G-75 chromatography profile of bacterial cell \n\n lysate applied to a 5 x 100 cm column and eluted with 10 mM anunoium bicarbonate, 5 mM mercaptoethanol (7). \n\n Elution Time (min) \n\n FIGURE 5. Reverse-phase high-performance chromatography profile \n\n of Sephadex G-75 fractionated bacterial MT. The 300 A pore size propyl column was eluted with 20 mM triethylamine phosphate, pH 7.0, by using an acetonitrile organic modifier (phase B). Column effluent was monitored at 250 nm with a sensitivity of 1.0 AUFS (12). \n\n 72 \n\n CYANOBACTERIAL METALLOTHIONEINS \n\n Isolation of Cyanobacterial MT \n\n Synechococcus strain Tx-20 cyanobacteria were maintained in axenic culture on BG-11 medium (7) and harvested from aerated 20 L cultures grown at 280C under fluorescent light. Cells were broken in a French pressure cell at 0?C in 0.5 M Tris-HCI, pH 8.6, and the lysate exposed to 10 ,uCi of 109CdCl. The 40,000g supernatant was applied to a Sephadex G-75 column developed with 10 mM ammonium bicarbonate and 5 mM mercaptoethanol resulting in the profile shown in Figure 4. Polarographic activity and radionuclide binding were coincident with a 10,000 MW ultraviolet absorbing fraction. Further purification of this material could be undertaken by DEAE-cellulose chromatography or isoelectric focusing but maximum resolution of isoproteins was best attained by reverse-phase high-performance liquid chromatogra\n\n 0 5 \n\n DAYS \n\n I0 \n\n 5 \n\n phy. Figure 5 shows such a separation performed on a 300 A pore size C-3 column (Beckman Instruments). Four baseline resolved MT peaks appear between 40 and 70 min. Peaks eluting at 80 min contain at least four additional MT components resolvable by adjustment of the applied organic modifier ramp. Preliminary evidence indicates that these isoproteins are microheterogeneous with respect to both amino acid composition and metal speciation resulting in the observed reverse-phase separation. \n\n Physiological Characteristics of Cyanobacterial MT \n\n Using the Brdicka polarographic procedure, it is possible to measure basal MT levels in Synechococcus strain \n\n 5 \n\n DAYS \n\n 10 \n\n 15 \n\n FIGURE 6. Cell numbers and MT concentrations as a function of time after inoculation of wild-type cyanobacteria in the presence and absence \n\n of (A) 22.5 ,uM CdCl2; (B) control; (C) 50 ,uM ZnSO4; (D) 50 ,uM CuCl2 (8). \n\n 73 \n\n 74 ~~~~~~~~~~R. W OLAFSON \n\n Table 1. Amino acid compositions of cyanobacterial \n\n metallothioneins compared with eukaryotic metallothioneins. \n\n Nearest integer residues per molecule Human (MT-2) Crab (MT-2) Tx-20t CysA 20 18 9.5 Asx 4 6 5.7 Thr 2 3 4.8 Ser 8 5 3.8 Glx 2 5 2.0 Gly 5 3 8.0 Ala 7 1 4.0 Val 1 - 2.3 Met 1 - \n\n Ile 1 -1.1 \n\n Leu Tyr Phe His Lys Arg Pro \n\n 8 2 \n\n 8 2 6 \n\n 2.9 2.3 \n\n 2.8 2.6 1.0 1.9 \n\n RRIMP NI cells cultured in the absence of added metal, as shown in Figure 6B (8). Comparison with cultures exposed to cadmium chloride (Fig. 6A) or zinc sulfate (Fig. 6C) showed that introduction of metal salts resulted in a growth lag and that resumption of growth occurred coincident with an increase in cellular levels of MT. In addition, it should be noted that, like mammalian MT, cadmium appears to be a more potent inducer than zinc. Twice as much cadmium-thionein was synthesized on exposure to half as much metal as was used in the zinc induction experiment. A further interesting finding was that copper exposure resulted in an even greater growth lag than observed with cadmium, but on resumption of growth in the presence copper, MT synthesis was not noted (Fig. 6D). Recent results suggest that this copper resistance is manifest via a membrane exclusion mechanism (unpublished results). \n\n HOURS \n\n FIGURE 7. Inhibition of cadmium induced synthesis by actinomycin \n\n D (hatched bar) and chloramphenicol (solid bar). Control cells are shown by unmarked bars. \n\n In order that the level of regulation of metal induction be determined for cyanobacteria, logarithmically growing cultures were exposed to cadmium and MT concentrations measured at several time intervals thereafter. Two cultures were exposed to either chloramphenicol or actinomycin D 30 min prior to introduction of metal, while a third culture was used as a control. The results of this experiment are shown in Figure 7 and indicate induction at the level of transcription, as was found in eukaryotic organisms. \n\n The assumption at this stage in these investigations was that the growth lag observed in cultures exposed to \n\n Table 2. Amino acid sequence analysis. \n\n Amino acid \n\n 1 10 20 30 Cyanobacteria T S T T L V K C A C E P C L C N V D P S K A I D R N G L Y Y Scylla P DP C C NDK C DC K EG EC KT GC K CT S C Human Ac MD P NC S C AA G DS CT C AG S C KC KE C KC T S C \n\n Neurospora \n\n G D C G C S G A S S C N C G S G C S C S N \n\n C G S K \n\n Table 2. Continued. \n\n Amino acid \n\n 40 50 60 C C EAC A H GHT G G \n\n R CP PC EQ C S SGC K CA NK E DC RK TC SK PC SCC P K K SCC S CCP V GC A KCA Q BC I C K GA SDK C S CC A \n\n 74 \n\n I \n\n II \n\n I I \n\n CYANOBACTERIAL METALLOTHIONEINS 75 \n\n cadmium or zinc was due to a rather protracted time of MT induction, perhaps associated with toxic burden. If the above cadmium-resistant cells were transferred into fresh media in the absence of cadmium and allowed to grow up between three successive transfers, levels of MT dropped to near basal values as synthesis was repressed in the absence of metal. However, when these cells were now transferred into cadmium-containing media, instead of the predicted growth lag, they grew immediately. Thus, these cells were truly cadmium resistant. Since 50 tubes of wild-type cells at a dilution of 104 grew, after the usual lag on exposure to cadmium-containing media, the acquisition of cadmium resistance in this strain of Synechococcus was therefore considered unlikely to be related to a chromosomal mutational event. Such a mutation frequency would be unreasonably high. Although no direct evidence exists at this time, this metal resistance phenomenon is best explained by the amplification of an extrachromosomal gene, especially since this strain is known to have plasmids. \n\n Structure of Cyanobacterial MT \n\n The structural determination of cyanobacterial MT is not yet complete, although a substantial amount of information is now known. For example, Table 1 compares the amino acid composition of a Synechococcus TX-20 isoprotein with two eukaryotic MTs. These data indicate that the bacterial form is unique. While cysteine was still the predominant amino acid in this protein, levels of the amino acid were half that seen in the eukaryotes. In addition, the bacterial protein had two tyrosines, three histidines, and five long-chain aliphatics-all residues rarely found in eukaryotic MTs. Thus, the amino acid composition indicates that prokaryotic MTs are not as structurally homologous with eukaryotes as they are physiologically homologous. This was further exemplified on amino terminal amino acid sequence analysis (12). With the exception of the typical Cys-X-Cys sequences, the first 42 residues of the prokaryotic MT were essentially without homology on comparison with the crab (Scylla), human, or Neurospora crassa MTs (Table 2). Of the above-mentioned uncommon MT residues, two tyrosines were found together in positions 29 and 30, all five of the long-chain aliphatic amino acids were located in the first 28 residues, and two histidines were situated at positions 37 and 39. Perhaps of greater interest was the complete lack of association of basic residues or hydroxylated residues with cysteines, as is found in eukaryotes (11). Instead, a block of four hydroxylated amino acids was found at the amino terminus. \n\n The sequence of this unique MT is now nearing completion, opening the way for an X-ray crystallographic investigation. However, the degree of similarity between the metal cluster arrangement of these bacterial molecules with the eukaryotic structures will initially be undertaken spectroscopically. Reliable interpretation of the 113Cd-NMR data for this molecule will be dependent upon adequate isolation of isoproteins using RP-HPLC and assessment of conformational integrity of metal reconstituted molecules, using procedures such as cyclic voltametry. Studies along these lines are presently underway in this laboratory \n\n The author gratefully acknowledges the financial support of the Australian Institute of Marine Science and the National Science and Engineering Research Council of Canada. Excellent technical assistance was provided by R. G. Sim and S. Kielland in carrying out the polarographic and sequence analyses, respectively. \n\n REFERENCES \n\n 1. Kagi, J. H. R., and Nordberg, M. (Eds.). Metallothionein. Birk\n\n hauser Verlag, Basel, 1979, pp. 5-55. \n\n 2. Olafson, R. W Differential pulse polarographic determination of \n\n murine metallothionein induction kinetics. J. Biol. Chem. 256:12631268 (1981). \n\n 3. Olafson, R. W Intestinal metallothionein: effect of parenteral and \n\n enteral zinc exposure on tissue levels of mice on controlled zinc diets. J. Nutr. 113: 268-275 (1983). \n\n 4. McCarter, J.A., and Roch, M. Chronic exposure of Coho salmon \n\n to sublethal concentrations of copper. III. Kinetics of metabolism of metallothionein. Comp. Biochem. Physiol. 77C: 83-87 (1984). \n\n 5. Lerch, K., Ammer, D., and Olafson, R. W Crab metallothionein\n\n primary structures of metallothioneins 1 and 2. J. Biol. Chem. 257: 2420-2426 (1982). \n\n 6. Otvos, J. D., Olafson, R.W, and Armitage, I. M. Structure of an \n\n invertebrate metallothionein from Scylla serrata. J. Biol. Chem. 257: 2427-2431 (1982). \n\n 7. Olafson, R. W, Abel, K., and Sim, R. G. Prokaryotic metallothi\n\n onein: preliminary characteristics of a blue-green alga heavy metalbinding protein. Biochem. Biophys. Res. Commun. 89: 36-40 (1979). 8. Olafson, R. W, Loya, S., and Sim, R. G. Physiological parameters \n\n of prokaryotic metallothionein induction. Biochem. Biophys. Res. Commun. 95: 1495-1503 (1980). \n\n 9. Brdicka, R. Pblarographic studies with the dropping mercury ka\n\n thode.-Part XXXI.-A new test for proteins in the presence of cobalt salts in ammoniacal solutions of ammonium chloride. Collect. Czech. Chem. Commun. 5: 112-128 (1933). \n\n 10. Olafson, R. W, and Sim, R. G. An electrochemical approach to \n\n quantitation and characterization of metallothionein. Anal. Biochem. 100: 343-351 (1979). \n\n 11. Olafson, R. W Differential pulse polarographic determination of \n\n murine metallothionein induction kinetics. J. Biol. Chem. 256: 12631268 (1981). \n\n 12. Olafson, R. W Prokaryotic metallothionein: amino terminal se\n\n quence analysis of a unique metallothionein. Int. J. Peptide Protein Res. 24: 303-308 (1984). " ], "offsets": [ [ 0, 18513 ] ] } ]

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[ { "id": "pmcA2652712__text", "type": "Article", "text": [ "Neurogenin2 Directs Granule Neuroblast Production and Amplification while NeuroD1 Specifies Neuronal Fate during Hippocampal Neurogenesis\nAbstract\nThe specification and differentiation of dentate gyrus granule neurons in the hippocampus require temporally and spatially coordinated actions of both intrinsic and extrinsic molecules. The basic helix-loop-helix transcription factor Neurogenin2 (Ngn2) and NeuroD1 are key regulators in these processes. Based on existing classification, we analyzed the molecular events occurring during hippocampal neurogenesis, primarily focusing on juvenile animals. We found that Ngn2 is transiently expressed by late type-2a amplifying progenitors. The Ngn2 progenies mature into hippocampal granule neurons. Interestingly, the loss of Ngn2 at early stages of development leads to a robust reduction in neurogenesis, but does not disturb granule neuron maturation per se. We found that the role of Ngn2 is to maintain progenitors in an undifferentiated state, allowing them to amplify prior to their maturation into granule neurons upon NeuroD1 induction. When we overexpressed Ngn2 and NeuroD1 in vivo, we found NeuroD1 to exhibit a more pronounced neuron-inductive effect, leading to granule neuron commitment, than that displayed by Ngn2. Finally, we observed that all markers expressed during the transcriptional control of hippocampal neurogenesis in rodents are also present in the human hippocampus. Taken together, we demonstrate a critical role of for Ngn2 and NeuroD1 in controlling neuronal commitment and hippocampal granule neuroblast formation, both during embryonic development and in post-natal hippocampal granule neurogenesis.\n\nIntroduction\nNeurons are born not only during development of the central nervous system, but neurogenesis also continues into adulthood. In both rodent and human adult brain, neurogenesis is active in two distinct zones of the forebrain: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) [1]–[5]. The molecular mechanism underlying neurogenesis in the DG is not fully understood. Clearly, a cascade of transcriptional events controls the specification of neuronal identity in the DG [3], [6]–[8], but details of the expression pattern and function of each transcription factor remain elusive.\nThe paired-box homeodomain transcription factor Pax6 and the bHLH transcription factors Ngn2 and NeuroD1 are important when cells acquire a pan-neuronal character and a specific neuronal subtype [9], [10]. In the developing neocortex, Pax6 is expressed in dividing radial glial cells at the ventricular surface [9]. In the adult hippocampus, Pax6 is expressed in astrocytes in the SGZ and are considered to be the true stem cells [9], [11]. Loss- and gain-of-function studies indicate that Pax6 is involved in regulating the proliferation of neocortical and hippocampal progenitors [9], [12]–[16]. During neocorticogenesis, high concentrations of Pax6 induce the expression of the bHLH transcription factor Neurogenin2 (Ngn2) [17]. In turn, Ngn2 causes cell cycle exit [18], an event that takes place when NeuroD1 starts to be expressed [19]. In the developing hippocampus, the absence of Ngn2 leads to a reduced number of granule neurons [20]. NeuroD1, on the other hand, is essential for the differentiation and survival of hippocampal granule neurons [10]. Interestingly, in the absence of Ngn2, NeuroD1 can still be activated and neuronal differentiation can still take place [20]. This has led to the idea that the primary role of Ngn2 is not to direct neuronal differentiation.\nOnly a small number of studies have addressed the role of Ngn2 and NeuroD1 in hippocampal neurogenesis [10], [20], [21]. Therefore, we now re-examine their roles in hippocampal neurogenesis in detail, using gain- and loss-of-function experiments. We first establish a hierarchy of transcriptional events that occur during neurogenesis in the DG and then define the place that Ngn2, NeuroD1 and other transcription factors have in this cascade. We find that a lack of Ngn2 expression result in a markedly smaller hippocampus and an almost complete absence of the DG. We show that Ngn2 is required for granule neuroblasts production/amplification. Gain-of-function of NeuroD1 during development of the DG results in an efficient generation of granule neuroblasts, an effect that we do not observe when we overexpress Ngn2. Finally, we demonstrate that the same transcription factors and cellular markers seen in mouse and rat tissue are also present in the human hippocampus.\n\nResults\nExpression of transcription factors and cellular markers define different phases of postnatal hippocampal granule neuron maturation\nWe first analyzed in detail the chronology of expression of different transcription factors and cell-specific markers during DG granule neuron formation and correlated our findings to the previously established classification of adult hippocampal neurogenesis [3]. The current classification describes hippocampal stem cells and progenies into three categories (type-1, -2 and -3 cells) depending on the markers they express as they mature. In the adult mouse DG, only a very small proportion of cells undergo mitosis at any one given time [22]. Consequently, in adult animals it is difficult to analyze which transcription factors are involved in the transition phase from one cell category to the next. Therefore, we studied 2 week-old rodents which have higher numbers of maturing granule neurons in the DG. We performed triple immunohistochemistry for all markers of interest and compared results from 2 week- and 2 month-old mice (Figure 1 and figure S1).\nWe first confirmed that stem cells in the SGZ exhibit characteristics of radial glia [3], [11], [16]. They extend radial processes from the SGZ to the apex of the subgranular layer (SGL) and the basal molecular layer. Moreover, they express the intermediate filament protein nestin, glial fibrillary acidic protein (GFAP) and glial glutamate transporter (GLAST) [16] (Figure 1A and B and figure S1A and S1B). The GFAP- and GLAST+ cells are defined as type-1 cells [3]. They also express the paired-homeodomain transcription factor Pax6 (Figure 1B). These radial glia-like stem cells divide relatively infrequently, and are believed to undergo symmetric division (giving rise to two identical stem cells) under some conditions. They can also divide asymmetrically and give rise to a new stem cell and one neuronal progenitor, which usually is defined as a type-2 cell (Figure 2B) [3].\nType-2 cells are divided into two different populations: type-2a expressing nestin and type-2b co-expressing nestin and doublecortin (Dcx) [23]. Based on a previous report [24], we examined the expression pattern of the T-domain transcription factors Tbr1 and Tbr2. We found that Tbr2 is expressed in type-2a cells, in agreement with a recent report [25]. In the SGZ of 2 week-old mice, we found an average of 50–70 Tbr2+ cells per 30 µm-thick section throughout the rostro-caudal axis of the dorsal hippocampus. This number was reduced by 35% in 2 month-old mice (Figure 1C with figure S1C and data not shown). As opposed to Tbr1 (Figure 1D and figure S1D), Tbr2+ cells did not co-express Dcx or PSA-NCAM (Figure 1C and figure S1C). We observed that Tbr1-immuoreactivity gradually decreased as neurons matured and started to express NeuN (Figure 1H and figure S1H). Thus, Tbr2 labels type-2 cells, while Tbr1 is expressed by immature granule neurons.\nWe then examined the molecular phenotype of type-3 cells, a cell type that transiently expresses Calretinin [26]. We hypothesized that hippocampal type-3 cells might also express the bHLH transcription factor NeuroD1, known to partially overlap with Tbr2 and Tbr1 in different brain regions [24]. We found that NeuroD1 expression in the hippocampus starts in Tbr2+ cells and extends to post-mitotic Tbr1+ cells, which also express the hippocampal granule identity transcription factor Prox1 (Figure 1G and 1I and figure S1G and S1I). While Tbr1 expression ceases during granule maturation, that of NeuroD1 is weakly maintained when NeuN expression starts (Figure 1G and 1H and figure S1G and S1H). We found that calretinin expression decreased during granule neuron maturation, before NeuroD1 was reduced (Figure 1F). Thus NeuroD1 is expressed in type-2b and type-3 cells, as well as immature granule neurons. Finally, we found that NeuroD2 starts to be expressed just after NeuroD1 and, unlike NeuroD1, continues to be highly expressed in mature neurons (Figure 1J and data not shown).\n\nMash1 and Ngn2 define the early versus late type-2a stage in 2 weeks old mice\nHaving established the hierarchy of transcription factors and cellular markers appearing during hippocampal neurogenesis, we set out to clarify the position of the two bHLH transcription factors Ngn2 and Mash1, in this process. We found that more than 90% of Ngn2-immunoreactive cells are Pax6 positive (Figure 2A), and almost all co-express Tbr2 (Figure 2B). Thus, Ngn2 appears to be expressed by type-2a cells. As our antibodies against Ngn2 and NeuroD1 were made in the same species, we could not determine if there was an overlap between these two proteins. However, by comparing their overlapping expression pattern with that of Tbr2, we propose that some Ngn2+ cells co-express NeuroD1, and that they appear at the onset of NeuroD1 expression and downregulation of Ngn2. We base this assumption on the facts that more than 50% of the Tbr2+ cells are Ngn2+ and more than 50% of Tbr2+ cells also express NeuroD1. The expression of Ngn2 in adult hippocampus was hard to detect using immunohistochemistry due to the low expression of the protein (data not shown). However, it is present, as previously described using reporter mice and as revealed by in situ hybridization [21].\nWe next examined the expression of Mash1 and compared it with that of Ngn2, Pax6 and Tbr2. We found some Mash1+ cells (10–20 cells per section) in the dorsal hippocampus. Almost all of them co-expressed Pax6 (Figure 2C). Only few cells co-expressed Tbr2 (Figure 2D) or Ngn2 (Figure 2E), suggesting that Mash1 is downregulated when these two proteins are expressed. None of the Mash1-expressing cells were positive for NeuroD1 (Figure 2F). Mash1+ cells were still undergoing mitosis (Figure 2C) and consequently over 90% of them co-labeled with the cell-cycle marker Ki67 (Figure S1J).\nTaken together, we show that Mash1 and Ngn2 are expressed in early and late stages of maturation of type2a progenitors, respectively, and that both transcription factors are co-expressed briefly when cells transit from early to late phase type2a cells.\n\nNgn2 progenies become hippocampal granule neurons\nPrevious work has shown that in Mash1 null mutant mice hippocampal neurogenesis is not reduced and the hippocampus is not malformed [20]. Therefore we focused our initial analysis on the role of Ngn2 in hippocampal neurogenesis. We characterized the fate of Ngn2-expressing cells using Ngn2 Knock-in green fluorescent protein (GFP) mice (Ngn2+/GFP) [27]. In the SGZ of 2 week-old hippocampi, we found bright GFP-expressing cells that were positive for Tbr2 (Figure 2H), the immature neuronal marker PSA-NCAM (Figure 2I) and NeuroD1 (Figure 2J). Furthermore, Prox1+, NeuroD2+ and calretinin+ cells were also weakly positive for GFP. We made similar observations in 2 month-old mice (data not shown) confirming earlier published results [21]). We did not observe Mash1+/GFP+ and NeuN+/GFP+ cells (Figure 2G). These data from Ngn2+/GFP mice are entirely consistent with our earlier observations using immunocytochemistry to label Ngn2-expressing cells and with GFP undergoing slow degradation after the expression of Ngn2 has ceased. Importantly, none of the GFP-expressing cells, including those only weakly fluorescent, residing in the SGL expressed the astrocytic marker GFAP or the oligodendrocytic markers CNPase (data not shown). Taken together, the data show that Ngn2 progenies become neurons and never generate astrocytes or oligodendrocytes.\n\nMarked reduction of hippocampal granule neurons in absence of Ngn2\nTo determine the role of Ngn2 during the initiation of hippocampal granule neurogenesis, we analyzed the hippocampus of mice lacking Ngn2 (Ngn2GFP/GFP). In contrast to the mice use by Galichet and coworkers, our mice have a lifespan of only 2 weeks after birth. Therefore, we analyzed 2 week-old and younger mice. Consistent with previous observations [20], the hippocampus in Ngn2GFP/GFP mutants is clearly malformed (Figure 3A). In two week-old Ngn2GFP/GFP mice, the ventral blade of the DG is completely absent. Already on postnatal day 1–2, the ventral blade of the DG is malformed along its whole rostro-caudal axis (Figure 3B).\nAs neither migration defects nor cell death cause the reduced number of hippocampal neurons in Ngn2 null mice [20], we asked if Ngn2 is necessary for production of hippocampal granule neuroblasts. We first injected 2 day-old mouse pups with BrdU 2 hours prior to sacrifice, allowing us to evaluate cell proliferation and ongoing neurogenesis. We also injected BrdU into female mice on their final day of pregnancy and examined the brains of their pups 48 hours later (corresponding to P1–P2 old pups). This allowed us to assess the number of neurons derived from the last day of intrauterine development. Regardless of whether the mice were WT or hetero/homozygous mutant pups, we found cells that had incorporated BrdU in the hippocampal subventricular zone (hSVZ), fimbria and the DG (Figure 4A and B; [28]), suggesting that progenitors originating in the hSVZ divide while migrating towards the DG. Most of them were organized in chains typical of migrating cells (Figure 4A and B and figure S2). In Ngn2GFP/GFP mutant mice injected with BrdU 2 hours prior to sacrifice, we observed a decrease in the number of BrdU+ cells compared to heterozygous littermates. Thus, the numbers of newborn cells were reduced in the hSVZ/fimbria (133.3±7.5 for Ngn2+/GFP vs 69.7±5.0 for Ngn2GFP/GFP) and DG (132.0±9.1 for Ngn2+/GFP vs 42.6±2.2 for Ngn2GFP/GFP)(Figure 4C and D). We also examined the number of newborn cells differentiating into neurons, and identified them by the co-expression of BrdU and NeuroD1. As expected, they were relatively few in numbers because the short delay (2 hours) between BrdU administration and sacrifice in this first experimental paradigm. In Ngn2GFP/GFP null mutants, we found the number of cells differentiating into neurons to be reduced by 60% and 71% in the hSVZ/fimbria (8.6±1.4 for Ngn2+/GFP vs 3.4±0.5 for Ngn2GFP/GFP) and DG (9.7±1.1 for Ngn2+/GFP vs 2.8±0.6 for Ngn2GFP/GFP), respectively, (Figure 4E and F). As a result, the number of NeuroD1+ cells in the DG was reduced by 80% (580.3±27.7 for Ngn2+/GFP vs 116.2±7.3 for Ngn2GFP/GFP; Figure 4G).\nWe obtained similar results in the second paradigm, i.e. in mice that we sacrificed 48 hours after BrdU administration and in which a larger number of the newborn cells had time to mature into neurons. In this case, the number of BrdU+/NeuroD1+ cells in the DG was reduced by 73% of Ngn2GFP/GFP mutant animals (91.5±24.1 for Ngn2+/GFP vs 26.4±3.3 for Ngn2GFP/GFP) (Figure 4J). We found no differences in the proportion of BrdU+ cells that expressed NeuroD1 in Ngn2+/GFP and Ngn2GFP/GFP mice (14.2±1.0% and 17.4±1.8% respectively; Figure 4K). This shows that the few cells that manage to proliferate in the Ngn2 null mice have the same ability to differentiate into neurons as those in mice with one Ngn2 allele.\nInterestingly, the absence of Ngn2 did not alter the identity of neurons in the DG granule layer in 2 week-old Ngn2 null mice. The maturing granule progenitors sequentially expressed Pax6, Tbr2 (Figure S3A and S3B), NeuroD1 and Calretinin (Figure 4L), PSA-NCAM (Figure 4N), Tbr1 (data not shown) and Prox1 (Figure 4M). We confirmed that the same transcriptional cascade is active in the 20% (compared to mice with one Ngn2 allele) residual granule neurons that are formed two day-old in Ngn2GFP/GFP mice (Figure S3D–G and data not shown).\nWe next investigated if the cells that failed to develop into neurons in Ngn2GFP/GFP mutant mice, became glial cells. We found that cells expressing GFP never co-labeled with the astrocyte marker GFAP (Figure 4N) or the oligodendrocyte marker CNPase (data not shown).\nAltogether, our results show that Ngn2 plays an important role during the production/amplification of hippocampal granule neuroblasts, but not the acquisition of the granule neuron identity (Figure S7).\n\nNgn2 controls the amplification of granule neuron progenitors\nWe next monitored the mitogenic activity of Ngn2+ cells in the DG of 2 week-old WT mice. We observed that over 50% of Ngn2+ cells in the DG of WT mice co-expressed the mitosis marker Ki67 (Figure 5A). Inspired by this finding, we injected BrdU into 2 week-old WT and Ngn2GFP/GFP mice and compared cell proliferation in the DG. We observed 92% reduction in number of proliferating cells in the SGZ of Ngn2 null mutant mice (6.7±0.5) compared to WT (83.6±1.2) and heterozygotic (data not shown) littermates (Figure 5B–C). This data confirm the importance of Ngn2 during the amplification of granule progenitor.s.\nTo further explore whether Ngn2+ cells become post-mitotic or still proliferate after Ngn2 is downregulated, we performed immunohistochemistry on sections through the DG of Ngn2+/GFP mice. We stained them with the mitosis marker Phospho-histone 3 (PH3) and NeuroD1, which is downstream of Ngn2 in the transcriptional cascade controlling neurogenesis. Thus, in the same sections we could identify whether cells that had initiated Ngn2 expression (GFP labeled), continued to divide (PH3+) or committed to neuronal differentiation (NeuroD1+). We observed some GFP+ cells that co-expressed PH3. They all exhibited morphological characteristics of one of the five mitotic phases: prophase, metaphase, anaphase, telophase and cytokinesis (Figure 5D and data not shown). Unexpectedly, cells that colabeled for GFP, PH3 and NeuroD1 (Figure 5E and figure S4A and S4B) were rare. This indicates that Ngn2+ cells undergo division/amplification and that they mature into post-mitotic neurons upon NeuroD1 expression. In mice lacking Ngn2, we found the cells expressing GFP localized to the malformed ventral blade of the DG (Figure 5F and G). All of these cells were Ki67+ and they only very rarely expressed PH3 (Figure 5F and G, and figure S4C) in the absence of Ngn2 most of the cells are arrested in the cell cycle prior to entering the M phase and their mitosis is impaired. These data suggest that the mechanism of action of Ngn2 is conserved from development of the DG to postnatal hippocampal neurogenesis.\nOur findings suggest that Ngn2 regulates amplification and cell cycle exit of DG granule progenitors. To examine this hypothesis, we compared the effects of Ngn2 and NeuroD1 on mitotic activity in embryonic cortico-hippocampal neurosphere-derived progenitors, 5 days upon transduction, in vitro. In cultures transduced with Ngn2 retrovirus, we found that 27.2% (±4.1%) of the PH3+ cells had been transduced (Figure I1–J). These cells were immunopositive for MAP2 (Figure 5I1 and I2) and therefore represented dividing neuroblasts. By contrast, in NeuroD1 transduced cultures, only 7.3% of PH3+ cells were GFP+ (Figure 5H1, H2 and J). All of the NeuroD1-transduced cells became MAP2+ (Figure 5H). To confirm this data we pulse-labeled transduced cultures with chlorodeoxyuridine (CldU) for 48 hours, five days after differentiation. In contrast to NeuroD1-transduced cultures, we observed dividing cells transduced with Ngn2 retrovirus that were positive for PH3 and that had incorporated CldU (Figure 5K–L2). As the number of cells PH3+/CldU+/GFP+ we observed was low, we did not quantify this finding.\nCollectively, we have shown that Ngn2 is required for granule neuroblast production and amplification and that in the absence of Ngn2 the progenitors arrest in the cell cycle. NeuroD1, on the other hand, induces cell cycle exit and promotes rapid neuronal maturation.\n\nNeuroD1 directs neuronal differentiation and maturation\nBased on our previous observations we proposed that Ngn2 primarily controls amplification of granule neuroblasts and NeuroD1 directs neuronal differentiation. To test this hypothesis, we overexpressed Ngn2 or NeuroD1 in E14.5 cortico-hippocampal neurospheres and compared their effects after 5 days of differentiation of the progenitors. All cortico-hippocampal progenitors expressed Pax6 prior to differentiation (Figure S5). After 5 days, both factors suppressed Pax6 and Sox2 (Figure S5, figure 6C) and induced expression of Tbr1, Map2, NeuroD1 and PSA-NCAM (Figure 6D and E).\nInterestingly, all Ngn2-overexpressing cells co-expressed NeuroD1. Based on these findings, we next explored whether Ngn2 overexpression induces NeuroD1 expression, and if NeuroD1 in turn directs neuronal differentiation. We compared the effects of both transcription factors in progenitors that either do or do not normally express them. Thus, we expressed Ngn2 in E14.5 neural progenitors isolated from three different brain regions: cortex/hippocampus, lateral ganglionic eminence (LGE) and ventral mesencephalon (VM). Ngn2 expression in the developing forebrain is normally limited to the neocortex, and it does not appear in LGE tissue [29]. In the developing VM, both Ngn2 and NeuroD1 are expressed, but not when neural progenitors from this region are cultured in vitro [30], [31]. In our experiments, cortico-hippocampal progenitors could differentiate into neurons, although they did not normally express NeuroD1 (Figure 6E). When we overexpressed Ngn2 in VM and LGE progenitors, the cells started to express both NeuroD1 and PSA-NCAM (Figure 6F and G). We then overexpressed NeuroD1 in the same types of cultured progenitors and found that the cells became immunoreactive for PSA-NCAM (Figure 6G and data not shown). Thus, NeuroD1 is sufficient to direct neuronal differentiation in cortico-hippocampal-, LGE- and VM-derived progenitors.\nOur results from in vitro cultures and the analysis of the DG of Ngn2 mutant animals collectively show the neuron-inducing effect of NeuroD1 in hippocampal granule cell progenitors [32].\n\nNeuroD1 directs exclusive neuronal differentiation of hippocampal granule neuron progenitors\nAs the next step, we tested the effects of NeuroD1 in vivo and compared them with those of Ngn2. We injected retroviruses carrying the gene for either Ngn2 [33] or NeuroD1, and the reporter gene eGFP into the ventricles E15.5 rat embryos, in utero (Figure 7A). Three weeks later, when the rats were about two weeks old, we examined their hippocampi and analyzed the eGFP+ cells. The rats injected with control vector exhibited eGFP+ cells within the hippocampus that were either star-shaped, progenitor/glial-like cells (14.8±3.4%) or neuron-like cells with long neurites (85.3±3.4%) (Figure 7B). In rats that we had injected with the vector encoding Ngn2, the eGFP-labeled, the transduced hippocampal cells were composed of 31.9% (±5.7%) progenitor/glia-like cells and 69.4% (±6.1%) neuron-like cells (Figure 7B). The glia-like subpopulation was immunopositive for the astrocytic marker GFAP (Figure 7C–D). In contrast, virtually all of the transduced cells in rats injected with the NeuroD1 vector became neuron-like cells (99.9%±0.1; Figure 7B). None of these cells stained for GFAP (Figure 7E and F). They were positioned in the external layers of both the ventral and dorsal blades of the DG (Figure 7B3, E and I and figure S6A).\nWhen we examined neuronal maturation of Ngn2- and NeuroD1-transduced cells, we observed that only a few of those transduced with the NeuroD1 vector expressed the early mature neuronal marker Tbr1 (3.1±1.4%; Figure 7I and J). They were located within the basal layers, near the SGL, of the ventral and dorsal blades of the DG. In contrast, a greater proportion of the cells transduced with Ngn2 still expressed Tbr1 (19.6%±3.3%; Figure 7H and J), indicating that they were less mature than the vast majority of the NeuroD1-transduced cells. Immunohistochemistry for NeuN confirmed these data (Figure 7M and O, and figure S6A). Indeed, when we overexpressed Ngn2 the proportion of hippocampal progenitors that became NeuN+ was no greater than in rats transduced with the control virus (Figure 7F).\nAs a whole, our in utero injection experiments confirm that NeuroD1 has a stronger neuron-inducing effect than Ngn2 when overexpressed in hippocampal progenitors.\n\nInvolvement of Mash1, Ngn2, Tbr and NeuroD proteins during human hippocampal neurogenesis\nFinally, we examined whether the transcription factors and cellular markers that are expressed in rodents are also expressed in human hippocampus. We performed immunohistochemistry on aged human hippocampal DG and found GFAP-, Sox2-, Pax6-, Nestin-, Prox1- and NeuN-immunopositive cells. This indicates that radial glia-like stem cells, neural progenitors and mature granule neurons are present in the aged human hippocampus (Figure 8A–E). While Prox1 is found mainly in granule neurons, Sox2 and Pax6 are exclusively localized to cells in the SGZ (Figure 8D and E).\nAs for aged rodents, we did not observe the presence of Mash1, Ngn2, Tbr2, Tbr1 and NeuroD1 proteins in aged human hippocampus, using immunohistochemistry. However, we could identify the presence of the transcripts, using RT-PCR (Figure 8F). In addition, we detected mRNA for Prox1, Calbindin1 and Calbindin2/Calretinin (Figure 8F). Interestingly, we also observed Sonic Hedgehog and Wnt-3A transcripts, indicating that they are present in the human DG and supporting previous claims that they are important for the regulation of adult hippocampal neurogenesis (Figure 8F; [34], [35]). Finally, we also found mRNA for GLAST, Pax6 and GFAP which suggests that radial glia-like stem cells are present in the adult human hippocampus, and may play a role in adult human hippocampal neurogenesis (Figure 8F; [8]).\nOverall, our data show that different proteins known to play key roles in rodent hippocampal granule neurogenesis are present in the adult human hippocampus.\n\n\nDiscussion\nIn this study we determine the functions of Ngn2 and NeuroD1 during hippocampal neurogenesis. First, we map the hierarchy of molecular markers of neurogenesis in the DG. Second, we describe that Ngn2 is necessary for granule progenitor production/amplification. Third, we demonstrate that NeuroD1 directs neuronal differentiation of granule progenitors. Finally, we show that different cellular markers expressed during hippocampal neurogenesis in rodents are present in human.\nSequential expression of different transcription factors and cellular markers during hippocampal neurogenesis\nWe clarified the pattern of expression of various markers expressed during postnatal and adult hippocampal granule neurogenesis in detail. We found that the transcription factor Pax6 is initially expressed by both radial glia stem cells (type-1) and early amplifying progenitors (type-2a). The next transcription factor to appear in chronology is Mash1. Mash1 expression characterizes the early stage of hippocampal progenitor amplification (early type-2a). The role of Mash1 during hippocampal granule neurogenesis is still unclear. Mash1 null mutant mice do not display any clear malformation of the DG [20]. However, overexpression of Mash1 alone in the DG leads to the generation of oligodendrocytes [36]. Based on these observations, we hypothesize that Mash1-expressing cells are not yet committed towards a granule cell fate. They may still have the potential to generate both neurons and oligodendrocytes, as is the case in the SVZ [37].\nIn agreement with different developing brain regions, we found that Ngn2 starts to be expressed in Mash1-positive transiently amplifying progenitors, i.e. “late type-2a cells” according to the classification we propose. We observed that Pax6 expression persisted longer than that of Mash1, in Ngn2-expressing cells (late type-2a cells), in juvenile but not adult DG (Fig. 1 and figure S1). Later on, Ngn2 is downregulated, whilst Tbr2 expression persists (type-2b cells). The transition from amplifying progenitor to neuroblast is defined by the expression of NeuroD1. Thus, Ngn2 is expressed at the beginning of the transiently amplifying progenitor phase while NeuroD1 marks the end of that period. This applies both to the juvenile and adult rat brain [21], [25]. After NeuroD1 is turned on the progenitors leave the cell cycle, gradually mature, express PSA-NCAM, NeuroD2, Calretinin, Prox1, Tbr1 and, finally, NeuN. NeuroD1 expression persists at low levels in mature neurons (Figure 9; [10]). We found the sequential expression of these markers conserved in juvenile (P2 and 2 weeks) and adult rodent brains. Importantly, we observed that the same transcription factors and neuronal markers are also present in the adult human hippocampus and arranged spatially in a manner reminiscent with what we saw in rodents. It is possible, however, that some of these markers are expressed for longer or shorter periods at postnatal and adult stages of hippocampal progenitor maturation. Moreover, one can ask to which extent the number of divisions occurring during neuroblasts maturation is conserved at postnatal and adult stages of neurogenesis.\n\nA new function for Ngn2 in maintenance of a progenitor state for granule neuron production/amplification during embryonic and postnatal hippocampal neurogenesis\nIn agreement with earlier work, we confirm that Ngn2 is indispensable for hippocampal development and plays a vital role in postnatal hippocampal granule neurogenesis. Ngn2 null mutant animals display a reduced size of the cornu ammonis and a malformed DG (Figure 3 and 4; [20]). We show that the number of newborn neurons (incorporated BrdU) is decreased in absence of Ngn2, but hippocampal granule neuron subtype specification is not affected, post-nataly. Likewise, neurons from other brain regions, e.g. ventral midbrain dopamine neurons, can be generated in the absence of Ngn2 [30], [31]. The reduced numbers of mature neurons in the hippocampus and ventral midbrain of Ngn2 null mutant animals does not appear to be due to cell death or migration defects [20], [31]. Instead it may be due to a defect in the generation and/or amplification of neuronal progenitors. Indeed, in the hippocampus of postnatal animals, we observed that cells lacking Ngn2 arrest in the cell cycle, maintain Pax6 expression and cease to proliferate.\nWhen overexpressed in neural progenitors in vitro, Ngn2 lead to up-regulation of NeuroD1 and caused neuronal differentiation (Figure 6), which was not always the case when we overexpressed the same factor in vivo (Figure 7L, N and O). We also saw that Ngn2 does not always efficiently induce cell cycle exit. Typically some mitotically active eGFP+ cells were present in the DG of rats injected with the Ngn2 retrovirus (Figure 7P), and we observed that cultured Ngn2-transduced cells are still capable of dividing. Thus, Ngn2 does not always promote cell cycle exit and neuronal commitment, but depending on the state of the cells, Ngn2 may instead promote alternate cellular fates [38]–[40]. Indeed, an earlier study has shown that Ngn2-overexpressing progenitors generate oligodendrocytes when grafted to the adult spinal cord [40]. We have also seen that Ngn2-overexpressing neural progenitors from the embryonic midbrain form astrocytes when grafted to the striatum (unpublished observations). Probably, Ngn2 cannot influence already committed cells, but rather would control neuroblasts production. One could speculate that Ngn2 oscillates during granule neuron formation, as recently demonstrated during neocorticogenesis [41], [42].\n\nNeuroD1, but not Ngn2, is obligatory for granule neuron progenitor differentiation\nHippocampal granule progenitors can mature and express Tbr1 and Prox1, both during the development and postnatally, even in the absence of Ngn2. Because neuronal differentiation still occurs in Ngn2 null mice, it is clear that compensatory, Ngn2-independent mechanisms induce NeuroD1 expression. One candidate is Ngn1, which is expressed during neocorticogenesis, the specification of olfactory sensory neurons and during embryonic rat hippocampal development [43]–[47]. Both Ngn1 and Ngn2 regulate NeuroD1 [19], [48]. The introduction of two mutant forms of Ngn1, a deletion of the basic region of Ngn1 and a substitution of two amino acids in the C-terminal basic region, prevents NeuroD1 expression and neuronal differentiation [48]. The double null Ngn1/Ngn2 mutant displays a more severe phenotype than single gene (Ngn1 or Ngn2) mutant mice. For example, the total brain size of the double mutants is much smaller [10], [32]. If the role of Ngn1 during hippocampal neurogenesis is to activate NeuroD1 and that of Ngn2 is to control granule neuroblasts production/amplification, the DG of double Ngn1/Ngn2 knockout mice should resemble that of NeuroD1 null mutant mice.\nWe demonstrated that NeuroD1 directs neuronal differentiation both in vitro and in vivo. In progenitors isolated from different embryonic brain regions and cultured in vitro, we found that overexpression of NeuroD1 induced neuronal differentiation. The neurons generated expressed PSA-NCAM, Dcx and MAP2. After in utero retroviral vector-mediated gene delivery, virtually all cells transduced with NeuroD1 became neurons. In contrast, progenitors transduced with Ngn2 or control retroviruses adopted a neuron-like morphology in only 70–85% of cases. Under in vitro cell culture conditions, when we overexpressed Ngn2 in progenitors derived from the LGE we observed robust expression of NeuroD1 and neuronal differentiation. These in vitro results differ from those we obtained when we transduced the embryonic brain with a viral vector expressing Ngn2. In this latter case, Ngn2 did not induce neurons and the transduced cells did not express NeuroD1. Therefore, Ngn2 appears to direct non-neuronal cell type specification in vivo [38]–[40]. Taken together, we have confirmed that NeuroD1 plays a key role in neuronal differentiation in the hippocampus, both during development and in the adult brain.\n\nConcluding remark\nWe present a detailed classification of different stages in hippocampal neurogenesis. Our detailed molecular mapping of hippocampal neurogenesis allows for a more accurate analysis of how new factors stimulate neurogenesis at different steps in the development of granule neurons. Thereby we hope to facilitate the development of new agents, which stimulate endogenous progenitors in the treatment of diseases.\n\n\nMaterials and Methods\nAnimal tissue preparation\nThe creation of the Ngn2 transgenic mice was reported elsewhere (ref guillemot). Heterozygote male and female mice were crossed to obtain WT, heterozygote (Ngn2+/GFP) and null mutant (Ngn2GFP/GFP) animals. Tails of the Ngn2 offspring were used to obtain DNA for determination of the genotype using a polymerase chain reaction (PCR) assay as previously reported [31]. As null mutant Ngn2GFP/GFP do not survive longer than 2.5–3 weeks after birth, Ngn2GFP/GFP and their littermates were sacrificed at the postnatal ages of two days (P2) or two weeks for this study. Neurogenesis was assessed in two weeks or two months old WT mice. Sprague Dawley pregnant rats were ordered from B&K Universal Ltd, Sollentuna, Sweden (hppt://www.bku.com). All animals were housed in groups with ad libitum access to food and water at a 12-h light/dark cycle. All experimental procedures conducted in this study had been approved by the Ethical Committee at Lund University.\nFor immunohistochemical analysis, mice (from two weeks old and adult stage) and juvenile rats were sacrificed by transcardial perfusion with saline for 5–10 minutes, followed by 4% paraformaldehyde (PFA) for 10 minutes. Brains were kept in PFA overnight at 4°C and subsequently cryopreserved in a 20–30% sucrose/0.1 M phosphate buffer solution until sectioning on a microtome apparatus (30 µm thickness sections, Microm Zeiss). Seven series of coronal sections were cut throughout the brain. Free-floating sections were preserved in antifreeze solution until immunohistochemistry was performed. Heads of postanatal two days old mice were decapitated and soaked in PFA 4% for 24 hours, at 4°C and transferred into sucrose solution until sectioning on cryostat apparatus (16 µm thickness; Leica CM3000). Sections were mounted on Superfrost glass slides and stored at −80°C until immunohistochemistry was performed.\n\nCloning, subcloning, virus production and titer measurement\nThe Moloney leukemia-derived retroviral vectors used in this study, pCMMP-IRES2eGFP-WPRE and pCMMP-Ngn2-IRES2eGFP-WPRE were previously described [40], [49]. To generate the construct pCMMP-NeuroD1-IRES2eGFP-WPRE, mouse NeuroD1 cDNA was amplified from a pCS2+mtNeuroD1 plasmid (kindly provided by Professor Jackie Lee, Denver university, Boulder, USA) by PCR to introduce the restriction sites PmeI in 5′ and XhoI in 3′. Amplification of cDNA was performed as previously described [49]. The construction was verified by enzymatic restriction and by DNA sequencing using BigDye 3.1 (ABI). All infectious particles were produced using the producer cell line 293VSV-G and as previously described [49]. The titer of each retrovirus was measured by flow-cytometry based on eGFP expression, four days following infection of HT1080 cells and ranged from 0.5×109−2.1×109 TU/ml (All details on how to produce infectious particles can be provided upon request).\n\nNeurosphere generation, transduction and differentiation\nPregnant female Sprague-Dawley rats (B & K Universal, Sollentuna, Sweden) were terminally anesthetized by an overdose of sodium pentobarbital (i.p., 60 ng/ml). Embryos at stage embryonic day E14.5 (Plug day as day 0) were collected and cortical-hippocampal neurospheres were generated following dissection of the dorso-posterior part of the cortical tissue, and generated as previously described [49]. In this study, second passage (P2) neurospheres were used to study the effect of the overexpression of Ngn2 and NeuroD1 on neuronal differentiation. Each well, containing an equal starting population of 200,000 cells/ml, corresponding to 15–25 neurospheres, was transduced independently with each retrovirus at a multiplicity of infection (MOI) of 1, in proliferation medium supplemented by protamine-sulfate (4 mg/ml, Sigma). To induce differentiation, the medium was replaced with normal basic differentiation medium two days post-transduction and subsequently changed every other day until fixation.\n\nImmunocytochemistry, immunohistochemistry and microscopy\nThe antibodies used in this study are: rabbit anti-GFAP (1∶1000; DAKO), mouse anti-Nestin (1∶100; BD PharMingen), guinea pig anti-Glast (1∶500; Chemicon), rabbit anti-Prox1 (1∶1000; Covance), goat anti-Ngn2 (1∶20; Santa Cruz), goat anti-NeuroD1 (1∶200; Santa Cruz), rabbit anti-Pax6 (1∶150; Covance), rabbit anti-Trb2 (1∶500; Chemicon), rabbit anti-Trb1: (1∶1000; Chemicon), goat anti-Dcx: (1∶500; Santa Cruz), mouse anti-PSA-NCAM: (1∶500; Chemicon), mouse anti-NeuN (1∶300; Chemicon), rabbit anti-Calretinin (1∶500; Swant); mouse anti-Sox2 (1∶100; R&D systems), mouse anti-Mash1 (1∶100; BD PharMingen), rabbit anti-NeuroD2 (1∶300; ABCAM), mouse anti-MAP2 (1∶500; Sigma), rabbit anti-Ki67 (1∶150; NovaCastra) and rabbit anti-phospho-Histone H3. The secondary antibodies (1∶200) Cy2, FITC, Cy3 and Cy5 were from Jackson IR laboratories, Alexa-fluor 488, 568, 595 and 647 from Invitrogen-Molecular Probes. DAPI (1∶1000) was purchased from Sigma.\nFor immunocytochemistry, cultures were fixed in 4% paraformaldehyde at day five, rinsed with PBS three times prior to pre-incubation with a blocking solution (10% donkey serum, 0.25% TritonX100 in PBS) for 1 hour. The remainder of the procedure was performed as previously described [49]. Specimen analyses were performed using a Leica confocal microscope (Leica software, equipped with a GreNe and a HeNe laser, using the following lines of excitation: 488 nm, 594 nm and 647 nm). Samples were analyzed using 20×, 40× and 63× objectives, sometimes zoomed. Figures were composed in CANVAS-X software.\n\nBromo-deoxyuridine (BrdU) and Cloro-deoxyuridine (CldU) pulse labeling and immunohisto- and immunocyto-chemistry\nTo assess cell proliferation and ongoing neurogenesis in vivo, animals were injected with BrdU (100 mg/kg, Sigma), two hours prior to sacrifice (for both P2 and two weeks old Ngn2 mice). To assess neurogenesis, BrdU (100 mg/kg) was injected 48 hours prior to sacrifice (for P2 animals, BrdU was injected in pregnant dams half day prior to give birth). Immunohistochemistry was carried on as described above using a rat anti-CldU/BrdU primary antibody (1∶200, monoclonal, Immunologicalsdirect, Oxfordshire, UK), with an additional denaturation in 1 M HCl for 30 minutes prior to pre-incubation with serum.\nTo assess the neuronal-inducing activity of Ngn2 and NeuroD1, transduced cultures were incubated with CldU (20 µM, Sigma) for 2 days, after a period of differentiation of five days. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde at day 7, rinsed with PBS three times, treated with 1 M HCl at 65°C for 5–10 min, pre-incubated and then incubated with a rat anti-CldU/BrdU antibody and other primary antibodies. The remainder of the procedure was performed according to the protocol for immunohistochemistry already mentioned.\n\nIn utero surgery\nTimed pregnant female Sprague Dawley rats with embryos at gestational age E15.5 were anesthetized with halothane. The mother was placed in the lower level of a two-level wooden stage. The abdomen was shaved with an electric razor and then cleaned with 70% alcohol. A 2–3 cm midline laparotomy was performed. Each uterine horn was carefully taken out individually and the number of embryos recorded. One horn was then placed back inside the mother, whilst the other horn was prepared for injection. The embryos were kept moist with constant application of warm saline to prevent dehydration. Approximately 2 µl of viral suspension (1×10∧9TU/ml) was injected into the lateral ventricle of each embryo, except for the embryo closest to the vagina. After the injections, the uterine horns were placed back into the abdomen. The abdominal wall and the overlying skin were then sutured. Care was taken not to damage abdominal muscles so that normal delivery of the pups was possible at term. The entire surgery generally took about 45 minutes to one hour. Each mother was allowed to recover in her cage before being returned to the animal stable. Shredded paper was added to each cage to encourage nesting and special care was taken not to stress the mothers. Following normal delivery, the pups were allowed to develop to adulthood up to two weeks.\n\nQuantification\nFor the in vivo experiment, manual cell counting was performed on 18 and 30 µm thick brain sections for two days and two weeks old animals, respectively. The brain of P2 animals was cut into 10 series of coronal sections; the brain of two-weeks old animals was cut into 7–8 series of coronal sections. For each staining, one-two series from three to five different individuals were analyzed per genotype using a confocal microscope with 1, 2 or 3 lines of excitation, in sequential scanning in order to avoid false positives. When two series were analyzed, only one series was counted. The mean number of cells per hippocampus expressing the markers of interest, was calculated for each brain, based on the analysis of 6–8 consecutive dorsal hippocampal sections. The final mean number of cells per section was calculated by adding the mean number from each individual. We expressed the data as the mean number of positive cells±standard error of the mean (SEM). In this study, more than 25 Ngn2 null, >45 Ngn2 heterozygotes, and >60 WT individuals (including rats injected with retroviruses) were analyzed, in total. Statistical comparison was performed using one-factor analysis of variance (ANOVA) with transcription factor, cellular marker, genotype or time as variables, followed by post-hoc analysis when significant differences were observed, using Statview 5.0 software (SAS institute Inc.). For cell culture experiments, three independent experiments were performed, each in duplicate. The counting was based on seven randomly chosen different fields of view. For each diagram, the level of significance (p-value) is represented as follows: P<0.05 = *; P<0.001 = **; P<0.0001 = ***. All data are expressed as±standard error of the mean (SEM).\n\nHuman sample and RT-PCR\nThe brain from a 64 year-old male was provided by the Harvard Brain Tissue Resource Center. The individual whose brain tissue was being analyzed gave written consent for storage and use of his tissue for research. Five millimeters thick fresh human hippocampal tissue sample was snap frozen. The frozen tissue block was cut into 20 series of coronal sections, using a cryostat. Every 10 sections, one section was placed in an Eppendorf tube, on dry ice. Pooled sections were used for RT-PCR. Total RNA was prepared using Trizol and RNAeasy, supplemented with RNAGuard as previously described [49]. The RNA was digested with shrimp DNAse before cDNA synthesis, which was performed using a mix of oligo-dT and random hexamer primers and SuperscriptII reverse transcriptase. Advantage2 polymerase mix (Clontech/BRL) was used for PCR, with the following cycling conditions: 10 cycles of 94° C for 30 seconds, 68°C for 2 minutes, 30 cycles of 94°C for 30 seconds, 60°C for 1 minute, 68°C for 1 minute and 30 seconds, one cycle of 68° C for 2 minutes, soak at 16°C. Primers marked * were designed by PrimerBank. hRPS18 (sense) 5′-GCCTTTGCCATCACTGCCATT and hRPS18 (antisense) 5′-GCCAGTGGTCTTGGTGTGCT, hPAX6 (sense) 5′-GCCCTGGAGAAAGAGTTTGAGAGAACCCATT hPAX6 (antisense) 5′-GGGGAAATGAGTCCTGTTGAAGTGGTGC, hTBR1 (sense) 5′-GCGGACACCAATGTGCAAGGAAATCG and hTBR1 (antisense) 5′-CGAGGGGGTCAGGCGGTCCATGTCACAGC, hTBR2 (sense) 5′-GACCTGTGGCAAAGCCGACAATAACATGC and hTBR2 (antisense) 5′-GGGGGTGTCTCTATCCAAGAAGAGCCAAT, hPDHX ( = PROX1) (sense) 5′-GGGACACTACGGTTCCGTTTAAGTCCAGC and hPDHX (antisense) 5′-CTCTCCATCCCAGCTTACATTAACATCTGGCATTTGT, hNGN2 (sense) * 5′-CATCAAGAAGACCCGTAGACTGA and hNGN2 (antisense)* 5′-CAACACTGCCTCGGAGAAG, hMASH1 (sense) 5′-CCTGGATCCGCATGGAAAGCTCTGCCAAGATGGAG and hMASH1 (antisense) 5′-CCTGGATCCCCCCTCAGAACCAGTTGGTGAAGTCGA, hNEUROD1 (sense) 5′-GCTCAGGACCTACTAACAACAAAGGAAATCGAAACATG and hNEUROD1 (antisense) 5′-CAAAGCGTCTGAACGAAGGAGACCAGGT, hGLAST (sense) 5′-CATCAGGGAAGATGGGAATGCGAGCTGTAGTCTATTAT and hGLAST (antisense) 5′-CCACGGGGGCATACCACATTATTACTGCTACC, hNEST (sense) 5′-CAGGAGCGGCTGCGGGCTACTGAAAAGTTCC and hNEST (antisense) 5′-CAGGGCTGAGGGGTGGTGCCAAGGAGG, hGFAP (sense) 5′-CCACGAGGAGGAGGTTCGGGAACTCCAGGAGC and hGFAP (antisense) 5-GGAATGGTGATCCGGTTCTCCTCGCCCTCTAGC, hCALB2 ( = Calretinin) (sense) 5′-GGCTCTGGCATGATGTCAAAGAGTGACAACTTT and hCALB2 (antisense) 5′-GGGCATCCAGCTCATGCTCGTCAATGTAGCC, hCALB1 (sense) 5′-GCGAAAGAAGGCTGGATTGGAGTTATCACC and hCALB1 (antisense) 5′-CCCTCCATCCGACAAAGCCATTATGTTCTTCTTGTATG, hSOX2 (sense) 5′-GGAGAACCCCAAGATGCACAACTCGGAGAT and hSOX2 (antisense) 5′-GAGGAAGAGGTAACCACAGGGGGGCTGGAGC, hWNT3A (sense) 5′-CCGAGGGCATCAAGATTGGCATCCAGGAGTGC and hWNT3A (antisense) 5′-TCGGGTTGCGACCACCAGCATGTCTTCACCTC, hSHH* (sense) 5′-ACTCCGAGCGATTTAAGGAACT and hSHH* (antisense) 5′-CAGACGTGGTGATGTCCACTG.\n\n\nSupporting Information\n\n\n" ], "offsets": [ [ 0, 47655 ] ] } ]

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[ { "id": "pmcA2365090__text", "type": "Article", "text": [ "Carbonic Anhydrase Inhibitors. Part 541: Metal Complexes of Heterocyclic Sulfonamides: A New Class of Antiglaucoma Agents\nAbstract\nMetal complexes of heterocyclic sulfonamides possessing carbonic anhydrase (CA) inhibitory properties were recently shown to be useful as intraocular pressure (IOP) lowering agents in experimental animals, and might be developed as a novel class of antiglaucoma drugs. Here we report the synthesis of a heterocyclic sulfonamide CA inhibitor and of the metal complexes containing main group metal ions, such as Be(II), Mg(II), Al(III), Zn(II), Cd(II) and Hg(II) and the new sulfonamide as well as 5-amino-1,3,4-thiadiazole-2-sulfonamide as ligands. The new complexes were characterized by standard physico-chemical procedures, and assayed as inhibitors of three CA isozymes, CA I, II and IV. Some of them (but not the parent sulfonamides) strongly lowered IOP in rabbits when administered as a 2% solution into the eye.\nCARBONIC ANHYDRASE INHIBITORS. Part 54 METAL COMPLEXES OF HETEROCYCLIC SULFONAMIDES: A NEW CLASS OF ANTIGLAUCOMA AGENTS Claudiu T. \n\n Supuran*, Andrea Scozzafava \n\n and Andrei Jitianu 2 \n\n Universit& degli Studi, Dipartimento di Chimica, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, 1-50121, Florence, Italy 2 \"I.G. Murgulescu\" Institute of Physical Chemistry, Academia Romana, Spl. Independentei 202, R-77208 Bucharest, Roumania Abstract: Metal complexes of heterocyclic sulfonamides possessing carbonic anhydrase (CA) inhibitory properties were recently shown to be useful as intraocular pressure (IOP) lowering agents in experimental animals, and might be developed as a novel class of antiglaucoma drugs. Here we report the synthesis of a heterocyclic sulfonamide CA inhibitor and of the metal complexes containing main group metal ions, such as Be(II), Mg(II), AI(III), Zn(II), Cd(II) and Hg(II) and the new sulfonamide as well as 5-amino-l,3,4thiadiazole-2-sulfonamide as ligands. The new complexes were characterized by standard physico-chemical procedures, and assayed as inhibitors of three CA isozymes, CA I, II and IV. Some of them (but not the parent sulfonamides) strongly lowered IOP in rabbits when administered as a 2% solution into the eye. Introduction Sulfonamides possessing carbonic anhydrase (CA, EC 4.2.1.1) inhibitory properties [2] such as acetazolamide 1, methazolamide 2, ethoxzolamide 3 and dichlorophenamide 4 have been used for more than 40 years as pressure lowering systemic drugs in the treatment of open-angle glaucoma [3,4]. Their effect is due to inhibition of at least two CA isozymes present within cilliary processes of the eye, ie, CA II and CA IV, which is followed by lowered bicarbonate formation and reduction of aqueous humor secretion [5-7]. Their main drawback is constituted by side effects such as fatigue, augmented diuresis, or paresthesias, due to CA inhibition in other tissues/organs than the target one, ie, the eye [8]. \n\n N--N \n\n XN--N EtO S \n\n NH 2 \n\n O \n\n NH O:S----O \n\n NHEt \n\n The above-mentioned side effects are absent in the case in which the inhibitor has topical activity, and is applied directly into the eye. This route has been demonstrated only in 1983 by Maren's group [9] and was followed by the development of the first clinical agent of this type, dorzolamide 5 [10,11 ]. Dorzolamide (Trusopt) has been introduced in clinical use in 1995 in USA and Europe and it constituted the beginning of a radically new treatment of glaucoma, devoid of the severe side effects observed with the systemic inhibitors [4-6]. The success of topical antiglaucoma CA inhibitors fostered much research in the synthesis and clinical evaluation of other types of such compounds [12-15]. 307 \n\n \fVol. 4, No. 6, 1997 \n\n Metal Complexes of Heterocyclic Sulfonamides: A New Class ofAntiglaucoma Agents \n\n On the other hand, metal complexes of heterocyclic sulfonamides of type 1-5 have been recently prepared by two groups 16-20], and it was proved that they possess much stronger CA inhibitory properties than the sulfonamides from which they were prepared 18-22]. Although the mechanism of CA inhibition of the metal complexes is presently unknown, it was hypothesized that their increased inhibitory power might be due to two processes, occurring separately or in concert, ie, (i) dissociation of the complex inhibitor in sulfonamide anions and metal ions (in diluted solution), which in turn both interact thereafter with the enzyme, at different binding sites, and (ii) direct interaction of the undissociated complex with the enzyme, and more specifically with the hydrophilic patch at the entrance of CA II active site [23], this being the isozyme most susceptible to inhibition with this class of compounds [2,22]. Whether initially the first mechanism of action mentioned above was favored by us [22], recent evidences suggested that the undissociated complex might be the inhibitory species, at least for some isozymes [24]. Since metal complexes are much more inhibitory than the parent sulfonamide from which they were prepared, it appeared of interest to test whether this property might be useful for their use in lowering IOP in experimental animals and whence as a possible glaucoma therapy. Recently we proved [25, 26] that some metal complexes of heterocyclic sulfonamides (which themselves do not possess IOP lowering properties) act as very powerful such agents when administered as diluted solutions directly into the eye of experimental animals, and would thus offer the possibility of developing such totally novel drugs. Here we report the synthesis of a heterocyclic sulfonamide possessing strong CA inhibitory properties, ie, 5-(chloroacetamido)-l,3,4-thiadiazole-2-sulfonamide, and of the metal complexes of this sulfonamide and of 5-amino-1,3,4-thiadiazole-2-sulfonamide, containing some main group metal ions. The new compounds have been characterized by standard physico-chemical procedures, and were assayed as inhibitors of three CA isozymes, hCA I, hCA II and bCA IV (h human; b bovine; these are the isozymes considered to play a critical role in aqueous humour secretion within the eye of higher vertebrates [2-5]). Materials and Methods \n\n Melting points were recorded with a heating plate microscope and are not corrected. IR spectra were recorded in KBr pellets with a Carl Zeiss IR-80 instrument. 1H-NMR spectra were recorded in DMSO-d6 as solvent, with a Bruker CPX200 instrument. Chemical shifts are reported as values, relative to Me4Si as internal standard. Conductimetric measurements were done at room temperature (1 mM concentration of complex) in DMSO solution with a Fisher conductimeter. Elemental analyses were done by combustion for C, H, N with an automated Carlo Erba analyzer, and gravimetrically for the metal ions, and were 0.4% of the theoretical values. Thermogravimetric measurements were done in air, at a heating rate of 10C/min., with a Perkin Elmer 3600 thermobalance. Sulfonamides used as standards in the enzymatic assay (except for 5), acetazolamide, pyridine, and chloroacetyl chloride used for the preparation of compound 7, solvents as well as inorganic reagents were from Sigma, Merck and Carlo Erba. 5-Amino-l,3,4-thiadiazole-2-sulfonamide 6 was prepared from acetazolamide by literature procedures [27], by desacetylation with concentrated hydrochloric acid, followed by neutralization with sodium bicarbonate of the corresponding hydrochloride (Scheme 1). Dorzolamide hydrochloride 5 was from Merck, Sharp and Dohme or was prepared as described by Ponticello et al 10,11 ]. Human CA and CA II cDNAs were expressed in Escherichia coli strain BL21 (DE3) from the plasmids pACA/hCA I and pACAdaCA II described by Forsman et al. [28] (the two plasmids were a gift from Prof. Sven Lindskog, Umea University, Sweden). Cell growth conditions were those described by Lindskog's group [29], and enzymes were purified by affinity chromatography according to the method of Khalifah et al [30]. Enzyme concentrations were determined spectrophotometrically at 280 nm, utilizing a molar absorptivity of 49 mM-l.cm-1 for hCA and 54 mM-l.cm-1 for hCA II, respectively, based on M 28.85 kDa for hCA I, and 29.3 kDa for hCA II, respectively [31,32]. bCA IV was isolated from bovine lung microsomes as described by Maren et al, and its concentration has been determined by titration with ethoxzolamide [33]. Synthesis of 5-(chloroacetamido)- l, 3, 4-thiadiazole-2-sulfonamide 7 An amount of 1.80 g (10 mmol) of 5-amino-1,3,4-thiadiazole-2-sulfonamide 6 was suspended in 20 mL of anhydrous acetonitrile and 0.9 mL (0.87g, 11 mmol) of pyridine added. The mixture was magnetically stirred at 4 C for 10 minutes, then 10.5 mmol of monochloroacetyl chloride, dissolved in 3 mL acetonitrile, were added dropwise for 5 min, and stirring was continued for other 2 hours at room temperature. After an additional 30 min of refluxation, followed by cooling, the precipitated crystals were filtered and recrystallized from ethanol. Yield of 62% white crystals, mp 246-248 o lit [34] mp IR (KBr), cm-l: 590, 610, 660, 790, 935, 1090, 1115, 1170, 1350, 1400, 1550, 1650, 1720, 2870, 3280 3370 (broad); UV spectrum, ,max, nm (lg): 255 (3.50); 288 (4.37) H-NMR (DMSO-d6), i, ppm: 2.96 (s, 2H, CH2); 8.20 (s, 2H, SOzNH2); 12.22 (s, 1H, CONH). Analysis, found: C, 18.56; H, 1.88; N, 21.76; S, 24.62 %; C4HsC1N403S2 requires\" C, 18.72; H, 1.96; N, 21.83; S, 24.98 %. \n\n General procedure for the preparation of compounds 8-20 An amount of 6 mmol of sodium salt of sulfonamides 6 or 7 was prepared by reacting the corresponding sulfonamide with the required amount of an alcoholic 1N NaOH solution, in ethanol as solvent. To this 308 \n\n \fClaudiu T. Supuran et al. \n\n Metal-Based Drugs \n\n solution was added the aqueous metal salt (Zn(II), Mg(II), AI(III), Cd(II) chlorides, and Be(II), Pb(II) and Hg(II) nitrate) solution, working in molar ratios RSOzNH- Mn+ of 2:1 for the divalent cations and 3:1 for the trivalent cation, respectively. The aqueous-alcoholic reaction mixture was heated on a steam bath for one hour, adjusting the pH at 7 if necessary, and after being cooled at 0 C the precipitated complexes were filtered and thoroughly washed with alcohol-water 1:1 (v/v) and air dried. Yields were in the range of 85-90 %. The obtained white powders of compounds 8-20 melted with decomposition at temperatures higher than 350 C, and were poorly soluble in water and alcohol, but had good solubilities in DMSO, DMF as well as mixtures of DMSO-water, DMF-water. \n\n Pharmacology Carbonic anhydrase inhibition Initial rates of 4-nitrophenyl acetate hydrolysis catalysed by different CA isozymes were monitored spectrophotometrically, at 400 rim, with a Cary 3 instrument interfaced with an IBM compatible PC [35]. Solutions of substrate were prepared in anhydrous acetonitrile; the substrate concentrations varied between 2.10-2 and 1.10-6 M, working at 25C. A molar absorption coefficient of 18,400 M-l.cm-1 was used for the 4-nitrophenolate formed by hydrolysis, in the conditions of the experiments (pH 7.40), as reported in the literature [35]. Non-enzymatic hydrolysis rates were always subtracted from the observed rates. Duplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results. Stock solutions of inhibitor (1 mM) were prepared in distilled-deionized water with 1020% (v/v) DMSO (which is not inhibitory at these concentrations [2]) and dilutions up to 0.01 nM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constant KI was determined as described by Pocket and Stone [35]. Enzyme concentrations were 3.3 nM for hCA II, l0 nM for hCA and 34 nM for bCA IV (this isozyme has a decreased esterase activity [36] and higher concentrations had to be used for the-measurements). \n\n Measurement of tonometric lOP Adult male New Zealand albino rabbits weighing 2-3 kg were used in the experiments (three animals were used for each inhibitor studied). The experimental procedures conform to the Association for Research in Vision and Ophthalmology Resolution on the use of animals. The rabbits were kept in individual cages with food and water provided ad libitum. The animals were maintained on a 12 h: 12 h light/dark cycle in a temperature controlled room, at 22-26 C. Solutions of inhibitors (2 %, by weight) were obtained in DMSOwater (2:3, v/v) due to the low water solubility of some of these derivatives. Control experiments with DMSO (at the same concentration as that used for obtaining the inhibitors solutions showed that it does not possess IOP lowering or increasing effects. IOP was measured using a Digilab 30R pneumatonometer (BioRad, Cambridge, MA, USA) as described by Maren's group [37-39]. The pressure readings were matched with two-point standard pressure measurements at least twice each day using a Digilab Calibration verifier. All IOP measurements were done by the same investigator with the same tonometer. One drop of 0.2 % oxybuprocaine hydrochloride (novesine, Sandoz) diluted 1\"1 with saline was instilled in each eye immediately before each set of pressure measurements. IOP was measured three times at each time interval, and the means reported. IOP was measured first immediately before drug administration, then at 30 min after the instillation of the pharmacological agent, and then each 30 minutes for a period of several hours. For all IOP experiments drug was administered to only one eye, leaving the contralateral eye as an untreated control. The ocular hypotensive activity is expressed as the average difference in IOP between the treated and control eye, in this way minimizing the diurnal, seasonal and interindividual variations commonly observed in the rabbit [37-39]. All data are expressed as mean SE, using a one-tailed test. Results and Discussion Reaction of 5-amino-l,3,4-thiadiazole-2-sulfonamide 6 [19b] with chloroacteyl chloride in the presence of pyridine afforded 5-(chloroacetamido)-l,3,4-thiadiazole-2-sulfonamide 7, by the procedure already reported by Young et al. [34] (Scheme 1). The sulfonamide 7 has been characterized by elemental analysis and spectroscopic methods which confirmed its structure (only its m.p. has been reported in ref. [34]). The sodium salt of sulfonamides 6 and 7, obtained in situ from the corresponding sulfonamide and sodium hydroxide, were then used for the preparation of coordination compounds, containing the following metal ions: Be(II), Mg(II), Al(III), Zn(II), Cd(II) and Hg(II). Mention should be made that although 5-amino-l,3,4-thiadiazole-2-sulfonamide 6 is the parent compound of important sulfonamide CA inhibitors, such as acetazolamide, benzolamide, methazolamide, etc., its coordination chemistry has been scarcely investigated up to now [22, 40]. The new complexes prepared in this work are shown in Table I. Both compounds containing the sulfonamide-deprotonated species of sulfonamide 7 (LH), as well as complexes in which the anion of 5amino-1,3,4-thiadiazole-2-sulfonamide (tda) act as ligands, have been prepared. In fact in another work [40] it was documented that in some cases, sulfonamides derived from this ring system may undergo hydrolysis to \n\n 309 \n\n \fVol. 4, No. 6, 1997 \n\n Metal Complexes of Heterocyclic Sulfonamides.\" A New Class ofAntiglaucoma Agents \n\n the moiety substituting the 5 position, with the formation of 5-amino-l,3,4-thiadiazole-2-sulfonamide 6, which thereafter coordinates metal ions present in solution. \n\n N CIH N \n\n I \n\n N +NaHCO 2 \n\n N 2 \n\n S' \n\n -NaCl \n\n I S \n\n N \n\n II \n\n SO2NH \n\n 1_12 O Py/MeCN \n\n 6Htda +CIOCCH2CI \n\n 0 Cl CH 2 Scheme 1 \n\n HN \n\n .I \n\n N \n\n N \n\n S 7: LH \n\n II \n\n SqNH \n\n Thus, the X-ray crystal structure of the complex [Zn(tda)2(NH3)].H20 prepared in this way has recently been reported by this group [40]. On the other hand, when the ligand 7 has not been hydrolyzed (during the preparation of the coordination compounds) in the presence of the metal ion to 5-amino-l,3,4-thiadiazole-2sulfonamide and chloroacetate, the metal complexes contining 6 as ligand have been prepared from the last (pure) compound (as sodium salt) and the corresponding metal salt, by the general procedure described in the Experimental part. Table I: Prepared complexes 8-20, containing the conjugate bases of sulfonamides 6 and 7 as ligands and their elemental analysis data. L stands for the sulfonamide deprotonated species of 7, whereas tda for the sulfonamide deprotonated species of 5-amino-1,3,4-thiadiazole-2-sulfonamide 6. \n\n No. 8 9 10 11 12 13 14 15 16 17 18 19 20 \n\n Complex \n\n Yield \n\n (%) \n\n %Ma \n\n Analysis (calculated/found) %H b %C b 13.0/13.1 11.0/10.8 11.3/11.4 10.2/10.1 8.5/8.1 7.9/7.9 12.7/12.8 18.4/18.1 18.1/17.9 16.6/16.2 15.3/14.9 13.4/13.3 12.7/12.5 1.6/1.3 2.7/2.3 1.4/1.1 1.2/1.2 1.0/1.2 1.6/1.3 1.6/1.6 1.5/1.5 1.5/1.3 1.3/1.4 1.2/1.1 1.1/1.2 1.0/1.0 \n\n %N b 30.4/30.2 25.6/25.5 26.4/26.4 13.7/23.3 20.0/19.8 18.6/18.5 29.7/29.6 21.5/21.3 21.1/20.8 19.4/19.3 17.9/17.8 15.7/15.6 14.8/14.6 \n\n [Be(tda)2] [Mg(tda)2].3 H20 [Zn(tda)2] [Cd(tda)2] [Hg(tda)2] [Pb(tda)z(OH2)2] [Al(tda)3] [BeL2] [ALL3] [ZnL2] [CdL2] [HgL2] [PbLz(OH2)2] \n\n 78 76 83 90 95 84 72 75 59 87 88 92 95 \n\n 2.4/2.5 5.5/5.1 15.4/15.0 23.8/24.1 35.8/35.7 34.4/34.7 4.7/4.4 1.7/1.6 3.4/3.5 11.3/11.5 18.0/18.1 28.1/28.3 27.4/27.2 \n\n aBy gravimetry; bBy combustion. The new complexes have also been characterized by spectroscopic, conductimetric and thermogravimetric measurements (Table II). By comparing the IR spectra of the complexes and the corresponding ligands, the following observations should be made: (i) the shift of the two sulfonamido vibrations (both the symmetric as well as the the assymetric one), towards lower wavenumbers in the spectra of the complexes, as compared to the spectra of the corresponding ligand (Table II), as already documented previously for similar complexes [13-22]. This is a direct indication that the deprotonated sulfonamido 310 \n\n \fClaudiu T. Supuran et al. \n\n Metal-Based Drugs \n\n moieties of the ligands interacts with the metal ions in the newly prepared coordination compounds; (ii) the amide vibrations (the most intense such bands at 1670-1680 cm-1) of ligand 7 appear unchanged in the IR spectra of complexes 15-20 (data not shown), suggesting that these moieties do not participate in coordination of the metal ions; (iii) the C-N stretching vibration in the spectra of the prepared complexes is shifted with 5-20 cm-1 towards lower wavenumbers, as compared to the same vibration in the spectra of sulfonamides 6 and 7, indicating that one of the endocyclic nitrogens of the thiadiazolic ring (presumably N3) acts as donor atom, as already documented by X-ray crystallographic and spectroscopic determinations on complexes of other sulfonamides (such as 1-3) with divalent metal ions [13-22] (Table II); (iv) changes in the region 3100-3160 cm-, as the bands present in the spectra of sulfonamides 6, 7 are present in the spectra of complexes 8-20 too, but they are not well resolved, and have a smaller intensity. This is probably due to deprotonation of the SO2NH2 moiety and participation in the binding of cations; (v) the amino vibrations from 3320 cm- in the spectra of 6 appear unchanged in the spectra of its complexes 8-14 (data not s.hown). In the 1H-NMR spectra of compound 6 and its metal complexes, the signal of the amino group has been evidenced as a broad singlet centered at 4.54 ppm (Table II), which is not exchangeable by addition of D20 into the NMR tube, in contrast to the sulfonamido NH2 protons (which readily exchange). This proves that the 5-amino moiety is not involved in binding the metal ions, as already shown in the X-ray crystallographic work of the complex [Zn(tda)z(NH3)].H20 previously reported [40]. For sulfonamide 7 the CONH proton resonates as a singlet at 12.22 ppm. In complexes 15-20 only very minor shifts of this signal were evidenced (Table II), proving basically that the CONH moiety does not interact with the metal ions in these complexes. \n\n Table II: Spectroscopic, thermogravimetric and conductimetric data for compounds 6-20. \n\n 1H-NMR Spectrab Comp. IR Spectraa ,cm-1 as v(C=N) CONH, i (ppm) v(SO2)S; v(SO2) 6 8 9 10 11 \n\n TG analysisc calc./found \n\n Conductimetryd AM (-1 x cm2x mol-) 2 7 4 5 3 2 2 6 3 4 2 9 3 2 8 \n\n 12 13 14 7 15 16 17 18 19 20 \n\n 1170; 1150; 1150; 1145; 1145; 1140; 1145; 1150; 1170; 1130; 1140; 1140; 1150; 1140; 1145; \n\n 1350 1300 1305 1300 1305 1300 1300 1300 1350 1335 1330 1330 1330 1335 1330 \n\n 1610 1600 1600 1600 1600 1590 1605 1605 1610 1605 1610 1610 1605 1600 1600 \n\n A A A A A A A \n\n e e \n\n 12.3/12.1f e e e 5.9/5.7g \n\n A 12.22 (1H) 12.19 (2H) 12.18 (3H) 12.20 (2H) 12.18 (2H) 12.19 (2H) 12.21 (2H) \n\n e e e e e e e 4.7/4.8g \n\n a In KBr; bin DMSO-d6; A the signal of the 5-amino group of 6 (appearing in the ligand at 4.54 ppm as a broad singlet) appears at the same chemical shift (4.50 4.55 ppm) in complexes 8-14; cWeight loss between 70-250 C; d mM solution, in DMF, at 25C; e No weight loss seen under 250 C; fCorresponding to three lattice water molecules lost at 70-110C, and gCorresponding to two coordinated water molecules, lost at 160-180 C. \n\n Thermogravimetric analysis showed the presence of uncoordinated water molecules in the molecule of complex 9 (the three waters were lost in a single step, between 70-110 C) and of coordinated water in the molecules of the lead(II) derivatives 13 and 20. All these compounds behaved as non-electrolytes in DMF as solvent (Table II). Mention should be made that the Mg(II) complex of sulfonamide 7 could not be isolated. Instead, only the correponding complex of 5-amino-l,3,4-thiadiazole has been obtained from reaction mixtures containing magnesium salts and the sodium salt of 7, probably due to a metal ion assisted hydrolysis of 7 to 6 and chloroacetate. Generally such hydrolytic processes involve highly acidic conditions and prolonged heating of the 5-alkylamido-1,3,4-thiadiazole-2-sulfonamide derivatives [41 ], but they might become milder by taking into account the putative catalytic effect of Mg2+ ions reported here. The data shown above lead to the conclusion that ligand 7 shares a common coordination chemistry with acetazolamide 1 with which it is structurally related, whereas 6 probably also behaves similarly to acetazolamide in the sense that the 5-amino group seems not to be involved in coordinating metal ions, at 311 \n\n \fVol. 4, No. 6, 1997 \n\n Metal Complexes of Heterocyclic Sulfonamides: A New Class ofAntiglaucoma Agents \n\n least in the complexes reported by us here (and also in the compound characterized by X-ray crystallography mentioned above [40]). Thus, in all complexes reported here these sulfonamides (as monodeprotonated species at the SO2NH 2 moieties) act as bidentate ligands, through the endocyclic N-3 and the NH- groups. The proposed formulae of the new complxes are shown below. Except for the two Pb(II) complexes 13 and 20, as well as the AI(III) derivatives 14 and 16, which presumably are pseudo-octahedral, the other derivatives are supposed to contain tetrahedral M(II) ions. \n\n R \n\n R 13\"R = 20\" R= \n\n H2N \n\n CICH2CONH \n\n 14: R = 16: R = \n\n H2N CICH2CONH \n\n The compounds 6-20 together with the standard CA inhibitors 1-5 were assayed for inhibition against three isozymes, hCA I, hCA II and bCA IV (Table III). As seen from the above data, the chloracetamido derivative is more inhibitory than acetazolamide, methazolamide and dichlorophenamide, whereas the unacylated compound 6 is less inhibitory than the above sulfonamides. The metal complexes 820 are much more inhibitory than the sulfonamides from which they derive 6, 7 and than all other simple sulfonamides assayed. They behave similarly to the metal complexes of acetazolamide, methazolamide or dorzolamide previously reported by this group, which were all more inhibitory than the parent sulfonamide from which were prepared [16-22, 40]. Particularly strong inhibition was observed for the Zn(II), Hg(II), Pb(II) and Cd(II) complexes, especially against CA II and CA IV, the isozymes critical for aqueous humor formation. In vivo IOP lowering experiments were done in rabbits with some of the new compounds prepared in the present work, such as the sulfonamides 6 and 7, and their Zn(II) complexes, which were among the strong CA II and CA IV inhibitors in the obtained series. Some of the IOP lowering data at half an hour and one hour after the instillation of one drop of 2 % solution of inhibitor within the rabbit eye are shown in 312 \n\n \fClaudiu T. Supuran et al. \n\n Metal-Based Drugs \n\n Table IV, with dorzolamide (at the same concentration) as standard. In Fig. the time dependence of IOP lowering with dorzolamide 5 and the two Zn(II) complexes 10 and 17 is presented. Table III. CA inhibition data with the standard inhibitors 1-5, the sulfonamides 6 and 7, and their metal complexes 8-20. \n\n No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 a \n\n Inhibitor \n\n KI hCA Ia 900 780 25 1200 >50,000 1550 640 1050 350 50 40 12 80 240 120 80 40 40 9 15 \n\n (nM) bCA IVb 220 240 13 380 43 780 24 540 220 25 19 10 26 110 12 16 9 10 5 10 \n\n hCA IIa 12 14 8 38 9 230 5 190 110 15 14 7 10 76 5 4 3 3 2 5 \n\n Acetazolamide Methazolamide Ethoxzolamide \n\n Dichlorophenamide Dorzolamide \n\n Human (cloned) isozymes; b From bovine lung microsomes. \n\n Table IV: IOP lowering following topical application of CA inhibitors, half an hour and one hour after instillation into the eye of a drop (50 L) of 2 % solution of inhibitor. Inhibitor \n\n lOP SE 1/2 h \n\n a \n\n (mm Hg) lh 4.1 0.15 0 0.09 0 0.09 5.0 0.12 8.1 0.21 Dorzolamide 5 6 7 10 17 a \n\n 2.2 0.10 0 0.10 0 0.10 2.00.09 8.0 0.14 \n\n IOP \n\n IOP control eye\" IOP treated eye (N 3). \n\n As seen from the above data, the sulfonamides 6 and 7 are totally ineffective as lOP lowering agents, similarly to the classical clinically used inhibitors of type 1-5 [2,3]. On the other hand, dorzolamide, the first topical sulfonamide used clinically in the treatment of glaucoma is an effective such agent, with a decrease of lOP of around 4 mm Hg, one hour after administration directly into the eye (Table IV). From the data of this table, it is obvious that the metal complexes of heterocyclic sulfonamides investigated by us behave as much more effective IOP lowering agents than dorzolamide, and their effect is generally longerlasting (Fig. 1). A last remark should be made about the possible mechanism of action of the new class of IOP lowering agents. Obviously, their activity is due to inhibition of CA isozymes present in the cilliary processes within the eye, similarly to other topically active sulfonamides [2-6]. The fact that the sulfonamide per se is inactive via the topical route, whereas the metal complexes result much better than the drug \n\n 313 \n\n \fVol. 4, No. 6, 1997 \n\n Metal Complexes of Heterocyclic Sulfonamides.\" A New Class ofAntiglaucoma Agents \n\n dorzolamide, indicates that the presence of metal ions in the molecules of these CA inhibitors is essential and confers them completely new properties. \n\n Lowering of IOP (ram Hg) \n\n \"||, \n\n / \n\n \", , \n\n '\",., \n\n ,< \n\n .7, / \n\n / \n\n .6\n\n -0 \n\n -10 0 \n\n 2 \n\n Time (hours) \n\n Fig l\" Time dependence of IOP lowering with dorzolamide (curve 1); the zinc complex 10 (curve 3) and the zinc complex 17 (curve 3), after topical administration of one drop of 2 % solution of inhibitor in rabbit. \n\n Preliminary results from this laboratory indicate that the metal complexes of topically active sulfonamides show also increased lOP lowering effects with respect to the complexes prepared in the present study [42]. Our hypothesis is that the presence of the metal ion in the molecules of these complex inhibitors induces a dramatic change in their physico-chemical properties as compared to those of the parent sulfonamide. This phenomenon is certainly governed by the strong polarization induced by the metal ions. In this way, it is quite probable that the right balance between the lipo- and hydrosolubility of these compounds is achieved, which has been considered to be the critical factor for not observing topical activity in the classical CA inhibitors, such as acetazolamide, methazolamide and ethoxzolamide, which were either too lipophilic or too hy.drosoluble [2,3]. So, by choosing different metal ions and diverse sulfonamides, much larger possibilities arise to finely tune the pharmacological properties which strongly influence the value of a drug. In conclusion we describe here a novel class of lOP lowering agents, ie, the metal complexes of sulfonamide CA inhibitors. These derivatives appear to be very active and longer lasting than the drug dorzolamide, and might constitute the premises for a new generation of antiglaucoma drugs. \n\n Acknowledgments. We are grateful to Prof. S. Lindskog (Umea Univ., Sweden) for the gift of the hCA and II plasmids. References Preceding part of this series\" Supuran CT, Scozzafava A, Ilies MA, Iorga B, Cristea T, Chiraleu F, Banciu MD (1997) Eur J Med Chem, submitted. 2 Supuran CT (1994) \"Carbonic anhydrase inhibitors\" In Carbonic Anhydrase and Modulation of Physiologic and Pathologic Processes in the Organism, (Puscas I, Ed) Helicon, Timisoara, pp. 29-111. 3 Maren TH (1991) \"The links among biochemistry, physiology and pharmacology in carbonic anhydrase mediated systems\". 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[ { "id": "pmcA2327290__text", "type": "Article", "text": [ "Borrelia burgdorferi membranes are the primary targets of reactive oxygen species\nAbstract\nSpirochetes living in an oxygen-rich environment or when challenged by host immune cells are exposed to reactive oxygen species (ROS). These species can harm/destroy cysteinyl residues, iron-sulphur clusters, DNA and polyunsaturated lipids, leading to inhibition of growth or cell death. Because Borrelia burgdorferi contains no intracellular iron, DNA is most likely not a major target for ROS via Fenton reaction. In support of this, growth of B. burgdorferi in the presence of 5 mM H2O2 had no effect on the DNA mutation rate (spontaneous coumermycin A1 resistance), and cells treated with 10 mM t-butyl hydroperoxide or 10 mM H2O2 show no increase in DNA damage. Unlike most bacteria, B. burgdorferi incorporates ROS-susceptible polyunsaturated fatty acids from the environment into their membranes. Analysis of lipoxidase-treated B. burgdorferi cells by Electron Microscopy showed significant irregularities indicative of membrane damage. Fatty acid analysis of cells treated with lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (50-fold) in these cells, with a corresponding increase in the levels of malondialdehyde by-product (fourfold). These data suggest that B. burgdorferi membrane lipids are targets for attack by ROS encountered in the various stages of the infective cycle.\n\nIntroduction\nBorrelia burgdorferi, the causative agent of Lyme disease, survives and proliferates in distinctly different niches, including its arthropod vector and various mammalian hosts. These ‘micro’ environments provide their own distinct sets of advantages and challenges to B. burgdorferi. For example, during the initial stages of infection of the mammalian host, immune cells attempt to prevent B. burgdorferi from establishing an infection using several systems including those generating bacteriocidal reactive oxygen species (ROS) [e.g. superoxide radicals (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH)] and reactive nitrogen species (RNS) [e.g. nitric oxide (NO) and peroxynitrite] (Storz and Imlay, 1999). In order for B. burgdorferi to successfully colonize a new host and cause disease, they must overcome the challenges posed by the innate immune system including the deleterious effects of ROS/RNS compounds.\nThe effects of ROS/RNS on cells have been extensively investigated. These highly reactive compounds have been shown to damage cellular macromolecules including DNA, proteins and cellular membranes. The damage to membranes can arise through either lipid or membrane protein damage. In eukaryotes, membrane lipids are a major target of ROS. Free radicals attack polyunsaturated fatty acids in membranes and initiate lipid peroxidation. A primary effect of this is a decrease in membrane fluidity which affects the physical properties of the membrane altering the function of membrane-associated proteins. Once lipid peroxides form, they react with adjacent polyunsaturated lipids causing an amplification of the damage. Lipid peroxides undergo further oxidation to a variety of products, including aldehydes, which subsequently react with and damage membrane proteins. However, in bacteria, it is assumed that lipids are not subject to the oxidative damage observed in eukaryotic cells. Only certain polyunsaturated lipids, such as linoleic acid and linolenic acid, are susceptible to oxidation (Gutteridge and Halliwell, 1990), and it is clear that most bacteria do not synthesize or incorporate these types of lipids in their cell membrane. Two notable exceptions are the photosynthetic bacteria which synthesize and incorporate significant levels of linoleic acid in their membrane (Tasaka et al., 1996) and Helicobacter pylori membranes which contain between 0.5% and 3% linoleic acid (Ursini and Bindoli, 1987).\nInstead, it has been shown that the most damaging effects of ROS in bacteria result from the interactions of H2O2 with ‘free’ Fe2+ (Imlay, 2003), generating very reactive OH (Fenton reaction). Because of this reactivity, the effect on any given biomolecule will depend largely upon proximity to the target. Because Fe2+ localizes along the phosphodiester backbone of nucleic acid, DNA is a major target of OH. This reactive species can pull electrons from either the base or sugar moieties, producing a variety of lesions including single- and double-stranded breaks in the backbone and chemical cross-links to other molecules. These strand breaks and other lesions block DNA replication and contribute to OH toxicity and cell death. Other base damage, which does not hinder replication, may result in a significant increase in mutation rates.\nThe intracellular biochemistry of B. burgdorferi suggests that the primary cellular target of ROS may be distinct from that described in other bacteria such as Escherichia coli. In E. coli, the extent of DNA damage due to H2O2 and Fenton chemistry is directly proportional to Fe metabolism and the free Fe concentration within the cell (10 µM) (Keyer and Imlay, 1996). As the intracellular Fe concentrations of B. burgdorferi are estimated to be < 10 atoms per cell (Posey and Gherardini, 2000), it seems unlikely that DNA is a primary target for ROS. Therefore, the purpose of this study is to determine the biochemical effects of ROS on B. burgdorferi, including growth effects and biological/physical damage.\n\nResults\nEffect of ROS on B. burgdorferi cells\nIn order to determine what the cellular targets of ROS in B. burgdorferi are, we first needed to determine the sensitivity of Borrelia cells to various oxidants. Microaerobic cultures of B. burgdorferi strain B31A3 were grown to a cell density of 5 × 107 cells ml−1, treated with varying concentrations of H2O2 (0–50 mM) or t-butyl hydroperoxide (0–50 mM) and the number of surviving cells determined by plating. The results are shown in Fig. 1. When cells were exposed to 10 mM t-butyl hydroperoxide, approximately 50% of the cells survive (Fig. 1A). In contrast, when E. coli cells were exposed to 1 mM t-butyl hydroperoxide, approximately 1% of the cells survive. B. burgdorferi cells were much more resistant to t-butyl hydroperoxide than E. coli cells, with a survival rate of approximately 100% at 1 mM t-butyl hydroperoxide. This trend was observed when cells were exposed to H2O2. E. coli cells exposed to 1 mM H2O2 have approximately 10% survivability, while 100% of the B. burgdorferi cells survive at this concentration, and approximately 80% survive when exposed to 10 mM H2O2 (Fig. 1B). Similar results were obtained when cells were exposed in HN (Hepes-NaCl) buffer, suggesting that the high-level resistance to ROS was not due to the interaction of ROS with components of Barbour-Stoenner-Kelly (BSK-II) growth medium. Taken together, these data indicated that B. burgdorferi strain B31A3 was highly resistant to exposure to both t-butyl peroxide and H2O2.\n\nBorrelia burgdorferi DNA is not the primary target of ROS\nFor most bacteria, DNA is the major target of ROS causing a wide variety of DNA lesions. This is in part due to the localization of ‘free’ Fe2+ along the phosphodiester backbone of nucleic acids, putting the DNA in close proximity to the active species formed via the Fenton reaction. However, B. burgdorferi has been shown to harbour few genes encoding orthologues of known iron-containing proteins, does not require Fe for growth and has intracellular Fe concentrations estimated to be < 10 atoms per cell (Posey and Gherardini, 2000). Taken together, these observations strongly suggest that B. burgdorferi DNA is not a major target for ROS. To test this hypothesis, different techniques were used to measure DNA damage in B. burgdorferi cells after exposure to ROS.\nOne reliable indicator of DNA damage by ROS in a cell is an increase in the spontaneous mutation rate. In B. burgdorferi, mutations that confer resistance to coumermycin A1, which targets the β subunit of DNA gyrase, have been mapped to gyrB, the gene encoding DNA gyrase B (Samuels et al., 1994). In each case, a single point mutation correlated with this drug resistance. To determine if exposure to oxidants increases DNA damage by increasing point mutations, B. burgdorferi B31A3 cells grown under microaerobic conditions were treated with 5 mM H2O2 and plated in the presence and absence of 250 ng ml−1 coumermycin A1. The mutation frequency was calculated as the number of colonies that are CouR per total number of cells plated. The spontaneous resistance frequency of treated cells was approximately equivalent to that of untreated cells, 8.8 × 10−8 and 1.33 × 10−7, respectively, indicating no increase in the number of point mutations (Fig. 2A). Also, no increase in point mutations was observed when cells were treated with higher concentrations of H2O2 (10 mM) or when treated with t-butyl hydroperoxide (5 and 10 mM) (data not shown).\nAnother effective way to determine DNA damage is by measuring the number of apurinic/apyrimidinic sites (AP). AP sites in DNA, where the DNA base is lost, can be generated spontaneously under physiological conditions by hydrolysis of the N-glycosylic bond, or can also be formed by DNA-damaging agents (Lindahl and Nyberg, 1972), such as UV, alkylating agents or OH. They are also intermediates in the base excision repair pathway (BER) (Weiss and Grossman, 1987; Friedberg and Hanawalt, 1988; Wallace, 1988). Thus, the cellular steady state level of AP sites would increase as a consequence of base modifications and their subsequent repair. As AP sites are bypassed inefficiently by DNA polymerase in bacterial cells, DNA lesions can result in a significant block in DNA replication. Therefore, the number of AP sites can serve as a sensitive indicator of DNA damage resulting from oxidative stress (Kubo et al., 1992). To determine whether ROS can damage B. burgdorferi DNA, strain B31A3 cells grown under microaerobic conditions were treated with H2O2 (1 or 10 mM), t-butyl hydroperoxide (1 or 10 mM) or lipoxidase (an enzyme which specifically catalyses the hydroperoxidation of lipids containing a cis, cis-pentadiene structure, such as linoleic acid). The DNA was isolated and assayed for AP sites. The results are shown in Fig. 2B. In all cases, the numbers of AP sites per 105 bp DNA were equivalent, indicating that the addition of oxidants did not increase the number of DNA base lesions. In contrast, when E. coli strain TA4315 cells (ahpCF) (Storz et al., 1989) were treated with 100 µM H2O2, the number of AP sites increased ∼10-fold over untreated cells. These data suggest that B. burgdorferi DNA was not a major target for oxidative damage under these conditions.\nIt is of note to mention that the total number of AP sites per 105 bp DNA is approximately 10-fold higher in untreated Borrelia B31A3 DNA than in untreated E. coli DNA. The Borrelia genome consists of a linear chromosome and multiple linear and circular plasmids. Numerous observations indicate that functional telomeres require interaction with DNA damage repair proteins, suggesting that the DNA damage repair machinery, including the BER, is involved in replication of telomeres and protection of functional chromosome ends (Verdun and Karlseder, 2007). An important intermediate in BER is the apurinic or abasic site. Therefore, it was possible that the higher numbers of AP sites in untreated Borrelia DNA was due to the number of telomeres present in the genome. The B. burgdorferi strain B31A3 harbours 11 linear plasmids and a linear chromosome with ∼100 unpaired bases in the telomere loops per genome or 10 bases per 105 bp DNA (Hinnebusch and Barbour, 1991; Chaconas, 2005). Untreated Borrelia B31A3 contains 11 ± 3.8 AP sites per 105 bp DNA, suggesting that the elevated number of AP sites in Borrelia was due to the number of telomeres. To test this hypothesis, the AP sites were measured in DNA isolated from untreated B. burgdorferi strain B314 which harbours no linear plasmids (Sadziene et al., 1993). The number of unpaired bases in the telomere loops per genome was estimated to be 10, which corresponds to 1 base per 105 bp DNA, and only 3.6 ± 0.2 AP sites per 105 bp DNA were detected, supporting the hypothesis that the high number of AP sites in untreated Borrelia DNA was due to the number of unpaired bases in the telomeres. The analyses of the AP sites strongly suggested that B. burgdorferi DNA was not the major target for oxidative damage.\nIn addition to the generation of abasic sites, oxygen radicals often damage DNA through the formation of 8-oxoguanine lesions (Nakamura et al., 2000). 8-Oxoguanine, through its ability to mispair with bases other than cytosine, likely plays a role in DNA mutagenesis. Consequently, 8-oxoguanine is often used as a marker of oxidized DNA damage. To determine whether ROS can damage B. burgdorferi DNA and cause 8-oxoguanine lesions, strain B31A3 cells grown under microaerobic conditions were treated with H2O2 (5 or 10 mM), t-butyl hydroperoxide (5 or 10 mM) or lipoxidase. The DNA was isolated and assayed for 8-oxoguanine using an Enzyme-Linked ImmunoSorbent Assay. The results are shown in Fig. 2C. In all cases, the amount of 8-oxoguanine in Borrelia cells was below the detection limit of the assay. In contrast, when a MutM (the specific glycosylase that removes the 8-oxoguanine)-deficient E. coli strain was treated with 100 µM H2O2, the amount of 8-oxoguanine sites increased ∼fivefold over untreated cells. It is important to point out that no MutM homologue has been identified in the genome of B. burgdorferi. Taken together, these data suggested that B. burgdorferi DNA was not a major target for oxidative damage under these conditions.\n\nThe membranes of B. burgdorferi are targeted during oxidative stress\nIn eukaryotes, membrane lipids are a major target of ROS. Free radicals can attack polyunsaturated fatty acids, such as linoleic acid and linolenic acid, in membranes and initiate lipid peroxidation. This reaction can cascade throughout the membrane to adjacent polyunsaturated fatty acids, decreasing membrane fluidity and generating more toxic products such as aldehydes (Imlay, 2003). Because most bacterial membranes contain saturated and monounsaturated fatty acids rather than ‘reactive’ polyunsaturated lipids, peroxidation of lipids in bacterial membranes is not considered a problem. As B. burgdorferi cannot synthesize their own lipids, they must instead scavenge them. Therefore, it seems likely that their membrane composition would reflect the host's lipid profile or that of their growth medium (Barbour, 1984; Fraser et al., 1997) and would contain some polyunsaturated fatty acids. To determine if B. burgdorferi contains polyunsaturated lipids, B. burgdorferi strain B31A3 was grown under anaerobic conditions and analysed for fatty acid composition by Lipid Technologies (Austin, MN). The results are shown in Table 1 and are reported as percentage of total fatty acid content. Linoleic acid comprised ∼10% of the total lipid content and the linolenic acid content was measured to be ∼1%, indicating that Borrelia cells do contain lipids that are susceptible to ROS damage. The amount of linoleic acid and linolenic acid present in Borrelia reflected the amount present in the media. Therefore, these results suggested that the amount of these fatty acids in the membranes would vary as availability varies.\nTo determine if Borrelia polyunsaturated lipids can undergo lipid peroxidation, B. burgdorferi B31A3 cells grown microaerobically were treated with 1 mM t-butyl hydroperoxide or 250 mg of lipoxidase, and the cell pellets were analysed for fatty acid composition (Industrial Laboratory). The results are shown in Table 2 and are reported as the percentage of total cell mass. The per cent of linoleic acid in the total cell mass decreased with treatment, while the levels of oleic acid (c18:1n9) and pentadecanoic acid (c15:0) remained relatively constant. Untreated cells contained 0.04% linoleic acid, while cells treated with t-butyl hydroperoxide contained 10-fold less (0.004%) linoleic acid and cells treated with lipoxidase had no detectable linoleic acid present in the sample. These data indicated that the linoleic acid present in B. burgdorferi membranes can be oxidized by ROS.\nMalondialdehyde (MDA) is generated as a relatively stable end-product from the oxidative degradation of polyunsaturated fatty acids. MDA has thus been used as an indicator of lipid peroxidation (Gutteridge and Halliwell, 1990; Esterbauer et al., 1991). To further demonstrate that Borrelia lipids can undergo lipid peroxidation, B31A3 cells grown microaerobically were treated with 5 mM AAPH (free radical generator) or 250 mg of lipoxidase and MDA measured (Seljeskog et al., 2006). The results are shown in Fig. 3. Untreated cells contained ∼16.5 µM of MDA per mg of protein. When the cells were treated with AAPH or lipoxidase, the amount of MDA increased ∼1.5-fold (27.3 µM of MDA per mg of protein) and approximately twofold (33 µM of MDA per mg of protein) respectively (Fig. 3A). As a control, eukaryotic cells (mouse myeloma cells SP2) were treated with AAPH and MDA measured (Fig. 3B). Mouse myeloma cells have been used as a model system for the determination of phospholipid hydroperoxides (Chotimarkorn et al., 2005). In this case, the amount of MDA increased approximately threefold (36.3 µM of MDA per mg of protein) in the treated cells versus untreated cells (11.7 µM of MDA per mg of protein). Taken together, these data suggested that, like eukaryotic membranes, Borrelia membrane lipids were capable of undergoing lipid peroxidation.\nAs shown in Fig. 3A, there is a measurable quantity of MDA present even in untreated cells, suggesting that the membrane lipids are damaged without the addition of exogenous ROS. Oxidative damage is an unavoidable by-product of growth in an oxygen environment because superoxide anions and H2O2 are formed whenever molecular oxygen chemically oxidizes electron carriers. To determine if the Borrelia lipids are damaged from growth in an oxygen environment, B. burgdorferi B31A3 cells were grown under anaerobic, microaerobic and aerobic conditions and MDA measured. Figure 3C demonstrates that as the oxygen concentration increased, the amount of MDA increased. Cells grown under anaerobic conditions contained the lowest amount of MDA (7.6 µM of MDA per mg of protein), approximately twofold less than the measured amount in microaerobic cells (16.5 µM of MDA per mg of protein). Aerobically grown cells contained the highest amount of MDA, ∼1.5-fold greater than that observed in microaerobic cells (28.8 µM of MDA per mg of protein) and ∼3.7-fold greater than that observed in anaerobically grown cells. These data suggested that Borrelia lipids can be damaged during aerobic growth.\nOur High Performance Liquid Chromatography analyses of the MDA present in anaerobically grown B. burgdorferi cells showed a small peak with a retention time similar to that of the MDA standard. This was puzzling as little or no lipid peroxidation should occur under these conditions. To further characterize this ‘MDA’ peak, a three-dimensional diode array spectra was generated by scanning each sample during the elution of the peak (Fig. 4, lower sections). An authentic MDA standard was also scanned as a control (Fig. 4A, lower section). In the anaerobically grown cells, the spectrum shows that two compounds with different absorbance maximums (Fig. 4B, lower section) comprised the single retention time peak from the HPLC chromatogram (Fig. 4B, upper section). Based on this spectrum, the amount of actual MDA contributed < 15% of the total amount of material detected in the HPLC peak while the second contaminating peak contributed > 85%. Therefore, the amount of MDA in untreated anaerobically grown cells was considerably less than the 7.6 µM of MDA per mg of protein actually measured. However, in cells grown under microaerobic conditions, the MDA peak contributed more to the overall retention time peak when compared with the anaerobic spectrum, while the second contaminating peak stays relatively constant (Fig. 4C, lower section). These spectra demonstrated that the increase in the MDA retention time peak between anaerobically and aerobically grown cells was due to the increase in the amount of authentic MDA present.\nThe fluorescent probe diphenyl-1-pyrenylphophine (DPPP) has been used for detection of lipid hydroperoxides in cell membranes (Okimoto et al., 2000; Takahashi et al., 2001). In this method, the hydroperoxides are reduced with DPPP, resulting in the formation of the fluorescent arylphosphine oxide. To visualize the lipid hydroperoxides, B. burgdorferi B31A3 cells were labelled with DPPP and observed by fluorescence microscopy (Fig. 5B). E. coli Top10 cells and mouse myeloma SP2 cells were also labelled and visualized to serve as negative and positive controls respectively (Fig. 5A and C). Red Fluorescent dye was used to visualize the cell membranes. A strong fluorescence of DPPP was observed for B31A3 cells (Fig. 5B) and for the mouse myeloma cells (Fig. 5C), but not for the E. coli cells (Fig. 5A). An overlay of the two dyes demonstrates that the DPPP fluorescence of both the Borrelia and myeloma cells corresponds to the areas of red fluorescence. This indicated that lipid hydroperoxides were present on the Borrelia cell membranes and suggested that the membranes were damaged.\nTo further demonstrate Borrelia membrane damage, B. burgdorferi B31A3 cells were grown under anaerobic and microaerobic conditions and visualized by negative stain using an electron microscope. Additionally, cells grown under microaerobic conditions were treated with 250 mg of lipoxidase and visualized by Electron Microscopy. Intact membranes were observed in cultures of B31A3 grown under anaerobic and microaerobic conditions (Fig. 6A and B respectively). However, in cultures treated with lipoxidase (Fig. 6C), a significant number of membrane blebs were seen surrounding the spirochetes, indicating membrane damage. Taken together, these data indicated that Borrelia membranes were a target for oxidative stress.\n\n\nDiscussion\nMost bacterial pathogens are faced with the challenge of overcoming ROS generated by the host immune system. These radicals can cause a great deal of damage to biological molecules both in vitro and in vivo. The potential cellular targets for ROS damage in bacteria include DNA, RNA, proteins and lipids, and extensive work has been done to determine the cellular targets of ROS that affect bacterial survival. To date, the most definitive work has been done on E. coli. It seems clear from several studies that the most physiologically relevant target of ROS in E. coli is DNA. The exposure to µM concentrations of ROS (i.e. H2O2) is sufficient to cause DNA damage, inhibit DNA replication, increase the mutation rate and often lead to cell death. This process leading to DNA damage begins with formation of O2- and H2O2 from the oxidation of flavoproteins and/or the diffusion of these reactive species from the extracellular milieu. The subsequent oxidation of Fe-S proteins by ROS (e.g. O2-) leads to an increase in intracellular ‘free’ Fe2+ which triggers the reduction of H2O2 to OH (Fenton reaction). The highly reactive nature of OH limits its diffusion so that it generally reacts with molecules in close proximity to its origin (e.g. the Fe2+ associated with DNA). In DNA, OH oxidizes sugar and base moieties, producing radicals which ultimately generate lesions, including base modifications, strand breaks and chemical cross-links to other molecules. Base modifications lead to mismatching and increased mutation frequencies while more severe damage, such as strand breaks, prevents DNA replication, contributing to OH toxicity and cell death. Critical to this chemical process in vivo is the presence of iron. No other metal or non-metal electron carrier appears to be able to univalently reduce H2O2 to OH in vivo (Macomber et al., 2007).\nThese observations are critical in beginning to understand the oxidative damage/targets in B. burgdorferi. It has been shown in E. coli that the free-iron pool size determines the rate of oxidative DNA damage. For example, in wild-type E. coli, free iron levels are estimated to be 10 µM, yet H2O2 is only mildly genotoxic (Keyer and Imlay, 1996). However, in E. coli Fur- mutants, intracellular iron concentration increases eightfold while survival is 10-fold lower when cells are exposed to H2O2. Because B. burgdorferi cells do not contain detectable levels of intracellular Fe, it seems unlikely that DNA is a major target for damage via the Fenton reaction in this bacterium (Posey and Gherardini, 2000). The experimental data present in this report suggested that this was the case. When B. burgdorferi cells were exposed to high concentrations of ROS (e.g. H2O2), there was no effect on the spontaneous mutation rate (Fig. 2A) or the number of DNA lesions (AP sites or 8-oxoguanine) (Fig. 2B and C). It should be noted that in these experiments, a wild-type strain of B. burgdorferi was used and presumably all of the endogenous oxidative stress enzymes were expressed. Therefore, it is possible that no DNA damage was observed because these enzymes are capable of detoxifying the cell and protecting nucleic acids from oxidation via the Fenton reaction. However, we do not believe this to be the case as the concentration of oxidants used in these experiments were significantly higher than concentrations known to cause DNA damage in E. coli and other pathogenic bacteria.\nThe lack of detectable DNA damage in B. burgdorferi cells under the conditions tested could be the result of very efficient DNA repair systems. Bacteria, such as E. coli, harbour genes encoding repair enzymes (i.e. MutM, MutY, Ung, AlkA, MutS, MutL, ExoIII, EndoVIII, PolI, RecJ). Key enzymes in the repair of 8-oxoguanine lesions resulting from the oxidation of DNA are the bifunctional glycosylases, such as MutM, MutY or EndoVIII. The first activity of these enzymes is to remove oxidized or ring-saturated bases while the second activity is to remove the resulting deoxyribose residue, generating a 3′-phosphate end (Krwawicz et al., 2007). This 3′-P is converted to a 3′-OH by various enzymes/pathways and the lesion is repaired by enzymes (e.g. ExoIII, PolI, LigI etc.) in the short or long BER pathways (S-BER, L-BER). Interestingly, the B. burgdorferi genome does not contain genes encoding homologues of MutM, MutY or EndoVIII, suggesting that it would be difficult for the cells to efficiently repair 8-oxoguanine sites using this pathway. B. burgdorferi does harbour the genes encoding the enzymes Ung (monofunctional gylcosylase, BB0053), MutS (BB0797, BB0098), MutL (BB0211), ExoIII (BB0114), PolI (BB0548), LigI (BB0552) and RecJ (BB0254) (Fraser et al., 1997) which are involved in excision and repair, via S-BER or L-BER, of deaminated, alkylated, methylated or mismatched bases. Clearly, these repair systems in B. burgdorferi do not seem as robust at defending against oxidation of DNA as those described in E. coli. The lack of key repair enzymes in this system may indicate that B. burgdorferi DNA is not subjected to the same challenge from ROS as is E. coli.\nIn most bacteria, ROS-mediated damage to lipids (lipid peroxidation) is very unlikely because of the lack of polyunsaturated fatty acids (e.g. linoleic acid) (Imlay, 2003). When it does occur, lipid peroxidation is initiated by the attack of free radicals on polyunsaturated fatty acids which decreases the membrane fluidity and, if these reactions propagate, lipid peroxides and their degradation products (e.g. aldehydes) in turn could damage proteins (Gutteridge and Halliwell, 1990). This would dramatically affect the function of transmembrane proteins and membrane-bound lipoproteins involved in the maintenance of membrane potential and solute transport, decreasing cell survivability. In contrast to most bacteria, B. burgdorferi membranes contained significant levels of unsaturated fatty acids, such as linoleic acid and linolenic acid, which were derived from the growth media (Table 1). Thus, it seemed possible that lipids and/or proteins rather than DNA are the primary targets of ROS in B. burgdorferi. When Borrelia cells are treated with oxidants, the levels of linoleic acid decreased while other fatty acids remain unaffected (Table 2). In addition, HPLC-based assays demonstrated that, as linoleic acid concentrations decrease, MDA (a toxic lipid peroxide intermediate) increased (Fig. 3). When these cells were examined by electron microscopy, damage to the membranes (membrane ‘blebs’) was observed (Fig. 6). These data indicated that unlike most other bacteria, Borrelia membranes were damaged by oxygen radicals.\nAs B. burgdorferi may be exposed to potentially harmful oxygen species at different stages of the infective cycle, the ability to protect its membrane lipids from ROS should be required for survival in the different host environments. In eukaryotic cells, where lipid peroxidation is a major consequence of oxidative attack, proteins which protect membranes have been well studied. For example, phospholipid hydroperoxide glutathione peroxidase, a member of the glutathione peroxidase family, has been identified in a variety of higher organisms. These enzymes are capable of reducing an assortment of hydroperoxy lipids, including oxidized phospholipids and cholesterol esters (Ursini and Bindoli, 1987), and protect complex membranes from oxidative damage. Much less is known about the enzyme(s) responsible for protecting unsaturated lipids from ROS in prokaryotes. In H. pylori, lipid hydroperoxide levels in ahpC mutants are approximately three times higher than in wild-type cells, suggesting a role for AhpC in reducing organic peroxides (Wang et al., 2006a,b). In addition, purified AhpC has been shown to reduce linoleic acid hydroperoxide in vitro (Baker et al., 2001). Because these antioxidant enzymes promote the in vivo survival of cells when challenged with ROS, enzymes for the reduction of lipid peroxides in Borrelia need to be identified.\nInterestingly, Borrelia cells grown aerobically showed signs of membrane damage similar to those observed in cells exposed to various oxidants. The amount of MDA in untreated aerobically grown cells was equivalent to that observed in treated cells (Fig. 3). Also, EM indicated that cells grown microaerobically or aerobically had significantly more membrane damage than cells grown anaerobically (Fig. 6). This indicated that Borrelia membranes can be damaged simply by exposure to physiologically relevant concentrations of dissolved oxygen. In exponentially growing E. coli, Imlay and Fridovich (1991) have shown that both O2- and H2O2 are generated by the autoxidation of components of the respiratory chain during oxygen metabolism. In contrast, B. burgdorferi is very metabolically limited and has no enzymes for the TCA cycle or respiration. Therefore, it seems more likely that sources of ROS are exogenous (e.g. innate immune response in the mammalian host) rather than endogenous. In addition, the current practice of growing B. burgdorferi under atmospheric oxygen could itself be unintentionally compromising cell integrity during in vitro manipulations.\nAnalyses of the B. burgdorferi genome indicates that only a few genes encoding putative oxidative stress/intracellular redox proteins (SodA, NapA, BosR, CoADR, Trx and TrxR) (Fraser et al., 1997) are present, compared with other bacterial pathogens, including other pathogenic spirochetes (e.g. Treponema, Leptospira). Despite this apparent ‘deficiency’ in the number of ROS-protective enzymes, B. burgdorferi cells appear to be able to cope with physiologically relevant levels of ROS. There are several factors that would contribute to this phenomenon: (i) as B. burgdorferi does not harbour the genes encoding respiratory enzymes nor metabolize oxygen, it seems unlikely that significant levels of ROS are generated via the incomplete reduction of O2 during cellular metabolism. This would suggest that potential ROS challenges to B. burgdorferi would come almost completely from extracellular sources with little contribution from intracellularly generated ROS; (ii) owing to a lack of understanding of the physiological conditions in vector and host tissues infected with B. burgdorferi, it is difficult to assess the levels and/or sites of the potential ROS challenge during the infective cycle and (iii) most importantly, analyses of the potential targets for ROS in B. burgdorferi strongly suggested that the major targets of oxidative damage are different and perhaps less extensive in this bacterium than in other bacterial pathogens (e.g. E. coli). Taken together, this suggests that B. burgdorferi would be innately more resistant to ROS and require a less extensive repertoire of enzymes to protect the cells from oxidative damage.\n\nExperimental procedures\nStrains, growth conditions and reagents\nBorrelia burgdorferi strain B31A3 and strain B314 (Sadziene et al., 1993) were grown in modified BSK-II (Barbour, 1984) medium at 34°C under an atmosphere of 0–20% O2 with 5% CO2 and the balance N2. Cells density was determined using a dark-field microscope. All reagents were purchased from Sigma Chemicals, St. Louis, MO unless stated otherwise. E. coli strain Top10, strain TA4315 (ahpCF) (Storz et al., 1989) and CM1319 (mutM) (Bridges et al., 1996) were grown in Luria–Bertani (LB) at 37°C with shaking.\n\nPer cent survivability assays\nBorrelia burgdorferi strain B31A3 was grown to a cell density of 5 × 107 cells ml−1 in BSK II under microaerobic (3% O2) conditions, the culture split and the cells treated with varying concentrations of oxidants (0–50 mM t-butyl hydroperoxide or H2O2) at 34°C for 4 h. After the incubation, cells were diluted in fresh BSK II, plated on BSK plates and incubated 7–14 days at 34°C. Per cent survivability was calculated as the number of colonies on the treated plates versus the number of colonies on the untreated plates.\n\nDetermination of the spontaneous mutation rate\nTo determine spontaneous resistance to coumermycin A1, B. burgdorferi B31A3 cells were grown under microaerobic conditions to a cell density of 5 × 107 cells ml−1 and treated with 5 mM H2O2 for 1 h at 34°C. The cells were plated on BSK plates containing 0 or 250 ng ml−1 coumermycin A1 and incubated 7–14 days at 34°C. The resistance frequency was calculated as the number of colonies that are CouR per total number of cells plated.\n\nMeasurement of DNA base lesions\nBorrelia burgdorferi B31A3 cells were grown in BSK-II under microaerobic conditions to a cell density of 5 × 107 cells ml−1, treated with various oxidants (1 or 10 mM t-butyl hydroperoxide, 1 or 10 mM H2O2, 10 mM paraquat or lipoxidase) for 4 h and total DNA was isolated using Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI). The number of base lesions was determined using the DNA Damage Quantification Colorimetric Assay kit (Oxford Biomedical Research, Oxford, MI) following the manufacturer's protocol. Briefly, 500 ng of DNA was mixed with an equal volume of 10 mM biotinylated aldehyde reactive probe (ARP) reagent and incubated for 1 h at 37°C. The DNA-ARP product was ethanol-precipitated using glycogen as a carrier, washed three times with 70% ethanol and resuspended in Tris-EDTA to give a final concentration of 0.5 µg ml−1. The DNA-ARP product was allowed to bind to the wells of 96-well microplate overnight at 37°C. After the binding, the wells were washed four times with TPBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na3HPO4, 2 mM KH2PO4, 0.5% Tween 20, pH 7.4). The HRP-streptavidin conjugate was diluted to 0.5 µg ml−1 in Assay Buffer (0.15 M NaCl, 10 mM NasHPO4, 1.5 mM KH2PO4, 2.5 mM KCl, 5 mg ml−1 BSA, 0.1% Tween, pH 7.5), 100 µl was added to each well and the plate incubated at 100 r.p.m. for 1 h at room temperature (RT). After incubation, the wells were washed four times with TPBS, 100 µl of substrate was added to each well and incubated for 1 h at 37°C. The reaction was then quenched with 100 µl of 1 M sulphuric acid and the reaction was monitored at 450 nm. The number of aldehyde reactive probe (DNA base lesions) per 105 bp DNA was determined using a standard curve. As a control, E. coli TA4315 cells were grown in minimal media to OD600 of 0.4, treated with 0 or 100 µM H2O2 for 30 min and DNA isolated. The number of base lesions was determined as described above. B. burgdorferi strain B314 cells were grown under microaerobic conditions and DNA isolated. The number of DNA lesions was determined as described.\nTo determine the amount of 8-oxoguanine in B. burgdorferi DNA, cells were grown and treated as described above and DNA isolated. The DNA was converted to single-stranded DNA by boiling the sample for 5 min followed by rapid chill on ice. The DNA was then digested with nuclease P1 for 2 h and then treated with alkaline phosphatase for 1 h following manufacturer's protocols. The resultant mixture was then centrifuged for 5 min at 6000 g and the supernatant used for the 8-oxoguanine ELISA assay (Oxford Biomedical Research, Oxford, MI). As a control, E. coli CM1319 (mutM) cells were grown in LB to OD600 of 0.4, treated with 0 or 100 µM H2O2 for 1 h and DNA isolated. The concentration of 8-oxoguanine was determined as above.\n\nLipid analyses\nTo determine fatty acid content in B. burgdorferi total membranes, B. burgdorferi strain B31A3 was grown under anaerobic conditions to a cell density of 5 × 107 cells ml−1, harvested by centrifugation (5000 g, 15 min, 4°C) and washed two times with HN (20 mM NaCl, 50 mM Hepes, pH 7.6) buffer. Cell pellets were analysed for fatty acid composition by fatty acid methyl ester (FAME) gas chromatography (Lipid Technologies, Austin, MN) and results are reported as percentage of total fatty acid content. To determine the effects of oxidants on fatty acid composition, a 1.5 l culture of B. burgdorferi strain B31A3 was grown under microaerobic conditions to a cell density of 5 × 107 cells ml−1. The culture was split into 500 ml aliquots, the first was treated with 1 mM t-butyl hydroperoxide, the second with 0.25 mg of lipoxidase (17 700 units) and the third was untreated. All cultures were incubated for 12 h at 34°C. Cells were harvested by centrifugation (5000 g, 4°C, 15 min) and washed three times with HN buffer. The fatty acids present in the cell pellets were analysed by FAME gas chromatography (Industrial Laboratory, Wheat Ridge, CO). Fatty acids were reported as percentage of total cell mass.\n\nMeasurement of MDA\nBorrelia burgdorferi strain B31A3 was grown aerobically, microaerobically and anaerobically as described above to a cell density of 5 × 107 cells ml−1. The microaerobic culture was split and treated with either 5 mM AAPH [2,2′-azobis(2-methylpropionamidine) dihydrochloride] or 250 mg of lipoxidase for 4 h at 34°C. To measure the amount of MDA, the cells were derivatized with thiobarbituric acid and analysed by HPLC as described by Seljeskog et al. (2006). After the incubation, all cells were harvested by centrifugation (1000 g, 5 min, 4°C) and washed three times with HN buffer. Each sample was resuspended in 50 µl of HN, mixed with 150 µl of 0.1 N perchloric acid, 150 µl of 40 mM thiobarbituric acid and 35 µl of 20% SDS, vortexed and heated at 97°C for 60 min. After cooling at −20°C for 20 min, 300 µl of methanol and 100 µl of 20% trichloroacetic acid was added and the samples were mixed vigorously and centrifuged (13 000 g, 6 min). The samples (10 µl) were then analysed with an Agilent Technologies 1200 series HPLC system using a C18 4.6 × 150 mm column with mobile phase 72:17:11 (50 mM KPO4, pH 6.8 : methanol : acetonitrile). Absorbance was monitored at 532 nm. Pure MDA standards (0–10 µM) were prepared in methanol for comparison. As a negative control, E. coli Top10 cells were grown to OD600 of 0.4, treated with 0 or 5 mM AAPH for 30 min and MDA measured as above. As a positive control, mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere, treated with 1 mM AAPH at 37°C for 4 h (Chotimarkorn et al., 2005) and MDA measured as above.\n\nIdentification of lipid damage using Diphenyl-1-pyrenylphosphine fluorescent stain\nBorrelia burgdorferi strain B31A3 cells were grown microaerobically as described above until a cell density of 5 × 107 cells ml−1 was obtained. The culture was divided into two equal aliquots and the cells treated with 5 mM AAPH for 4 h at 34°C. After the incubation, all cells were harvested by centrifugation (1000 g, 5 min, 4°C) and washed three times with HN buffer. To visualize Borrelia cells, the cells were stained with PKH26 Red Fluorescent Cell Linker Dye (Sigma Aldrich) following the manufacturer's protocol. To visualize the lipid hydroperoxides in the cell membrane, the cells were counterstained with DPPP (Cayman Chemicals, MI) (Okimoto et al., 2000). Briefly, after cells were stained with the Red Fluorescent dye, 2 ml of rabbit serum was added to stop the reaction and the mixture incubated for 1 min at RT. Next, 4 ml of HN buffer was added, the cells harvested by centrifugation (1000 g, 10 min, RT) and washed three times with HN buffer. The cells were then resuspended in 1 ml of HN buffer, incubated at 34°C for 5 min and 30 µl of 2.5 mM DPPP was added. The incubation was then continued at 34°C for 5 min in the dark. After incubation, the mixture was centrifuged (1000 g, 10 min, RT) and the cells washed three times with HN buffer. The cells were then resuspended in 100 µl HN buffer and observed by fluorescence microscopy with excitation wavelengths 551 nm (Red Fluorescent) and 351 nm (DPPP), and emission wavelengths 567 nm (Red Fluorescent) and 380 nm (DPPP). As a positive control, mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere, treated with 1 mM AAPH at 37°C for 4 h (Chotimarkorn et al., 2005) and stained as described above. As a negative control, E. coli Top10 cells were grown to OD600 of 0.4, treated with 0 or 5 mM AAPH for 30 min and stained as described above.\n\nElectron microscopy\nBorrelia burgdorferi cells were grown under microaerobic or anaerobic conditions to a cell density of 5 × 107 cells ml−1. The microaerobic cultures were split and treated with lipoxidase for 4 h at 34°C. Cells were harvested by centrifugation (5000 g, 10 min, 4°C), washed and resuspended in HBSS (Lonza Group Ltd, Switzerland). The cells were fixed with Karnovsky's phosphate for 5 min at RT adsorbed to Formvar/carbon-coated grids (Ted Pella, Redding, CA) for 5 min and washed three times in H2O. The grids were stained with 1% ammonium molybdate and allowed to air-dry. Samples were examined using a Hitachi H7500 electron microscope (Hitachi High Technologies America, Pleasanton, CA).\n\n\n\n" ], "offsets": [ [ 0, 43000 ] ] } ]

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[ { "id": "pmcA1257442__text", "type": "Article", "text": [ "Identification of kinectin as a novel Behçet's disease autoantigen\nAbstract\nThere has been some evidence that Behçet's disease (BD) has a significant autoimmune component but the molecular identity of putative autoantigens has not been well characterized. In the initial analysis of the autoantibody profile in 39 Chinese BD patients, autoantibodies to cellular proteins were uncovered in 23% as determined by immunoblotting. We have now identified one of the major autoantibody specificities using expression cloning. Serum from a BD patient was used as a probe to immunoscreen a λZAP expression cDNA library. Candidate autoantigen cDNAs were characterized by direct nucleotide sequencing and their expressed products were examined for reactivity to the entire panel of BD sera using immunoprecipitation. Reactivity was also examined with normal control sera and disease control sera from patients with lupus and Sjögren's syndrome. Six independent candidate clones were isolated from the cDNA library screen and were identified as overlapping partial human kinectin cDNAs. The finding that kinectin was an autoantigen was verified in 9 out of 39 (23%) BD patient sera by immunoprecipitation of the in vitro translation products. Sera from controls showed no reactivity. The significance of kinectin as a participant in autoimmune pathogenesis in BD and the potential use of autoantibody to kinectin in serodiagnostics are discussed.\n\nIntroduction\nBehçet's disease (BD) is a systemic vasculitic disease typified by a triad of symptoms including recurrent oral ulcers, genital ulcers and uveitis. In addition, skin, joint, large vessels, nervous system and gastrointestinal systems may be involved. BD is a global disease but has the highest prevalence in the region along the ancient 'Silk Road' in China. The etiopathogenesis of the disease remains unclear but microbial agent triggers, environmental factors, genetic predisposition, neutrophil hyperfunction, endothelial cell dysfunction and immunological abnormalities involving both T and B cells have been implicated. Increasing amounts of research evidence supports the possibility that it is an immune-mediated vasculitis, and that abnormal T-cell and B-cell reactions and autoantigen-driven autoimmunity play pivotal roles [1]. Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune rheumatic disease with autoantibodies against cellular (particularly nuclear) antigens, some of which are critically implicated in the autoimmune pathology while others provide valuable serodiagnostic markers for the disease. Unlike the picture in SLE and other related rheumatic diseases, in BD, antinuclear antibodies and antibodies to neutrophil cytoplasmic antigens etc. are not present. To date, since neither a specific autoantibody nor pathognomonic pathological index is available to help establish the diagnosis of BD, it is largely or solely based on clinical manifestations [2], and a dilemma in diagnosis is not a rare occurrence in clinical practice. Nevertheless, since the 1960s, there have been reports of autoantibodies against certain unknown components of human oral mucosa in sera of patients with BD. Since then, sporadic reports on findings of autoantibodies in this disease have been described, such as antibodies to retinal antigen(s), heat shock protein (HSP) of some strains of Streptococcus sanguis cross-reactive with human HSP polypeptide [3], antibodies to endothelial cell antigens (AECA) and antibodies to α-tropomyosin [4,5], attesting to the complicated humoral immune disorders in this disease.\nThis investigation was aimed at defining target cellular autoantigens using time-tested and well-established molecular techniques. Immunoscreening of expression libraries using BD sera was used since this approach has been successfully employed in the characterization of many clinically relevant antigens in systemic rheumatic diseases such as SS-A/Ro [6-9] and SS-B/La [10] antigens in Sjögren's syndrome (SjS) and centromere antigen CENP-B [11] in scleroderma. In addition, we have been successful in using this strategy to identify interesting autoantigens that have other biological significance. Examples of these include NOR90/hUBF [12], p80-coilin [13], Golgi autoantigens [14-16] and, more recently, GW182 [17].\n\nMaterials and methods\nPatients and sera\nThe currently used empirical criteria for the diagnosis of BD in this study were the criteria proposed by the International Study Group for BD (abbreviated as 'International Criteria') [2]. The study subjects of 39 Chinese BD patients comprised 17 males and 22 females, mean age 37 ± 11.3 years old, who were divided into two subgroups: 25 typical BD patients (Group I, satisfying the International Criteria) and 14 clinically diagnosed BD patients who had recurrent oral ulcers and one of the symptoms of genital ulcers, eye symptoms or skin lesions as defined by the International Criteria, as well as additional symptom(s) closely related to BD as listed in the International Criteria, that is, gastrointestinal ulcerations, deep vein thrombosis or arthralgia/arthritis without evidence that the latter symptoms might be related to any other disease (Group II, defined as 'probable BD' in this study). Disease controls included 10 patients with SLE and 10 with SjS, all satisfying corresponding international classification criteria. All BD patients and disease controls involved in the study were patients treated at the Rheumatology Department of Ren Ji Hospital, Shanghai, China, where their clinical data and serum samples were collected. Twenty normal control sera were randomly selected from healthy blood donors working in the same hospital. This study was approved by the institution review board of Ren Ji Hospital which is affiliated with Shanghai Second Medical University, and each patient involved gave informed consent. All serum samples were preserved at -20°C or -70°C until use.\n\nCell lines and cell extracts\nHeLa (ATCC CCL 2.2) and T24 (human transitional cell bladder carcinoma) were obtained from the American Type Culture Collection (Rockville, MD, USA). A bovine aortic endothelial cell line was kindly provided by Dr Eugene G Levin from the Scripps Research Institute (La Jolla, CA, USA). Cells were cultured in DMEM containing 10% calf serum, harvested and extracted in Buffer A (150 mM NaCl, 10 mM Tris-HCl, pH7.2, 0.5% Nonidet P-40) with protease inhibitor (Complete™; Boehringer Mannheim, Indianapolis, IN, USA). For the preparation of whole cell extract, 10 volumes of Laemmli gel sample buffer [18] were added to the cell pellet, boiled for 3 min and stored at -20°C until use.\n\nWestern blot\nWhole cell lysates from bovine aortic endothelial cell, HeLa and T24 cells were resolved individually by discontinuous 7.5% gel SDS-PAGE according to Laemmli's method [18]. Immunoblotting was performed as described by Towbin et al. [19] with modifications. Nitrocellulose strips were blocked with 3% nonfat milk in PBS containing 0.05% Tween-20 (PBS-T) and then incubated with BD patient sera and normal control sera (1:100 dilution) at room temperature for 1 h. Filters were washed extensively with PBS-T to remove any unbound antibodies. Bound antibodies were detected with polyvalent, peroxidase-conjugated goat anti-human Ig and visualized by incubating the nitrocellulose strips in chemiluminescent reagents (NEN Life Science Products Inc., Boston, MA, USA) and exposing to Kodak XAR-5 films.\n\nScreening of phage cDNA expression library with antibody probes\nSerum from a BD patient showing the highest antibody titer in immunoblotting was selected as a probe and used at a dilution of 1:300 for initial immunoscreening of approximately 106 recombinants from a T24 cDNA expression library. The latter was constructed in λZAPExpress vector (Stratagene, La Jolla, CA, USA) and screened as previously described [20-22]. All screenings were performed on duplicate isopropyl β-D-thiogalactoside (IPTG) pre-impregnated nitrocellulose filters, and immunoreactive clones were detected by chemiluminescence. Positive phages were subsequently plaque purified to 100% by two repeated rounds of screening at low plaque densities. Before screening the cDNA library, the BD serum was extensively adsorbed against bacteria and wild-type λZAP phage mixture to reduce background binding.\n\nAnalysis of candidate cDNAs\nPurified candidate plaques were subcloned in vivo into pBK-CMV plasmids using ExAssist™ helper phage as recommended in the manufacturer's instructions (Stratagene). The recombinant pBK-CMV plasmids were then purified using QIAprep Spin Minprep Kit (Qiagen, Valencia, CA, USA). Restriction enzyme digestion of plasmids with EcoRI and XhoI and electrophoresis in a standard 1.0% agarose gel was used to analyze the length of cDNA insert of each candidate plasmid. The complete nucleotide sequence was determined using Bigdye terminator sequencing and a semi-automated sequencer model 377 (ABI, Foster City, CA, USA). Both nucleotide and deduced amino acid sequences were analyzed for similarity with known sequences using BLAST search [23] and ExPASy Proteomics tools . Secondary structure analysis for coiled-coil motifs was conducted with the software program COILS [24].\n\nImmunoprecipitation of in vitro translation products\nCandidate cDNA clones were used as templates for in vitro transcription and translation and the products were used as substrates for immunoprecipitation to confirm the specificity of reaction with BD sera. In brief, 1 μg of the pBK-CMV plasmid identified in the screening outlined above was added as template in a 50-μl reaction for the coupled in vitro transcription and translation reaction with a rabbit reticulocyte lysate system (Promega, Madison, WI, USA) in the presence of 35S-methionine (Trans-35S label; ICN Biochemicals, Costa Mesa, CA, USA) and RNasin® Ribonuclease Inhibitor (Stratagene) as recommended by the manufacturer (Promega). Translation was carried out at 30°C for 1.5 h. Products were analyzed in a 12.5% gel SDS-PAGE and stored at -80°C for further immunoprecipitation analysis. The in vitro translation proteins were examined for reactivity by sera using immunoprecipitation described [8,25].\n\n\nResults and discussion\nAutoantibody detection in sera from BD patients\nInitial examination of a group of 39 BD patients using indirect immunofluorescence (IIF) on a HEp-2 cell substrate did not yield any characteristic nuclear or cytoplasmic staining patterns. BD is thought by some to be a vasculitic disease involving pathophysiology of endothelial cells, and antibody to endothelial cell antigen (AECA) has been reported. Reports on the prevalence of AECA have varied largely and alpha-enolase was reported as one of the putative target antigens [26]. In this study, the use of bovine aortic endothelial cells as substrate for IIF did not provide any additional data. However, Western blot analysis of the BD sera began to show some interesting autoreactivity using cell lysates from both HeLa and bovine aortic endothelial cells. HeLa cells were initially used for this analysis because they are commonly used in the laboratory as Western blot substrate. Fig. 1 illustrates the common reactivity to 49 kDa and 120 kDa proteins in the endothelial cell lysates. These antigens were also detected in HeLa and T24 cells; the latter cell line was analyzed because our laboratory at The Scripps Research Institute has produced an excellent expression cDNA library from the T24 line and the positive result with the T24 cell extracts allowed us to screen the T24 library. Ig isotype analysis showed that all reactivity was largely IgG antibodies. Since the 49 kDa and 120 kDa bands were observed in cell extracts from bovine as well as human cell lines, these autoantigens might be evolutionarily conserved.\nIn total, nine out of 39 BD sera (23%) had autoantibody to the 49 kDa antigen and eight (20%) to the 120 kDa antigen. Four BD sera (10%) reacted with both proteins. Additionally, sera that showed common reactivity to the 120 kDa protein also demonstrated a common band that migrated at ~150 kDa, although it appeared weaker than the 120 kDa band. These antigens appeared to have different molecular weights than those of the known autoantigens in systemic rheumatic diseases. In addition, other reactive bands were detected but they were not as commonly shared as the 49 kDa and 120 kDa bands. The 49 kDa protein was shown to be distinct from 48 kDa SS-B/La or 50 kDa Jo-1 proteins (Fig. 1). The 120 kDa antigen was also shown to migrate differently from alanyl tRNA synthetase in another Western blot analysis (data not shown) and did not share any apparent crossreactive epitopes with the 49 kDa antigen. Western blot analyses of 20 normal control sera did not show the reactivities observed with BD sera. In order to further characterize these autoreactivities, a serum sample from the Group I definitive BD patients with the strongest reactivity to 49 kDa and 120 kDa antigens (Fig. 1, lane 2) was selected as antibody probe for expression library screening.\n\nKinectin identified as a novel BD autoantigen\nAfter screening 500,000 clones from the T24 cell λZAPExpress expression library, seven immunoreactive clones were isolated and plaque purified in two to three rounds to achieve 100% homogeneity. The cDNA inserts were subcloned in vivo into pBK-CMV plasmids, analyzed by restriction digestion using EcoRI and XhoI enzymes, and submitted to direct nucleotide sequencing across the polylinker arms. The cDNA inserts represented six independent clones designated BD41 (identical to BD44), BD481, BD42, BD47, BD482 and BD49. Their identities were established as overlapping partial cDNAs of human kinectin, ranging from 1.9 kb to 3 kb (Fig. 2a). The full-length human kinectin (GenBank accession number NM_182926[27]) has 4,816 bases containing an open reading frame coding 1,357 amino acid residues with molecular mass 156 kDa. All six cDNAs lacked the 5' portion of the kinectin sequence to different degrees but spanned a sequence of kinectin that extended to the 3'-untranslated region. Secondary structure analysis of kinectin protein using the program COILS identified a long region of α-helical coiled-coil domain that extended from amino acid residue 327 to the C-terminus (Fig. 2a, hatched boxes). In vitro coupled transcription and translation of BD44 and BD42 clones directed the synthesis of [35S]-methionine-labeled polypeptides that migrated at 95 and 60 kDa, respectively, in addition to smaller polypeptides (Fig. 2b). These products had predicted molecular weights of 103 kDa and 75 kDa.\nKinectin was initially identified in chick embryo brain microsome as an integral membrane protein anchored in endoplasmic reticulum and involved in kinesin-driven vesicle motility along microtubules [28,29]. Kinectin consists of a 120-kDa and a 160-kDa polypeptide interacting through the α-helical coiled-coil domain to form a heterodimer [30]. The full-length kinectin is the 160 kDa polypeptide containing an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two C-terminal leucine zipper motifs. We presume that the 120 kDa polypeptide detected in Western blot (Fig. 1) is the truncated version of the 160-kDa polypeptide, lacking the N-terminal first 232 amino acids [30]. The N-terminus of the 160-kDa polypeptide consists of a transmembrane domain that anchors kinectin to endoplasmic reticulum [30,31]. This 120 kDa polypeptide is probably the predominant form detected in the Western blot analysis (Fig. 1) because of its preferential solubility due to the omission of the N-terminal transmembrane domain.\nOther functions for kinectin have been reported. Yeast two-hybrid screen studies from several laboratories have revealed the interaction of the Rho family of GTPase with kinectin, and have shown the functional links among RhoG, kinectin and kinesin, with kinectin as a key effector of RhoG microtubule-dependent cellular activity [32]. Kinectin was also identified as an important constituent of integrin-based adhesion complexes, which link integrins to the cytoskeleton and recruit signaling molecules [33]. A new study reported that a kinectin isoform lacking a major portion of the kinesin-binding domain is very probably the most conservative form of kinectin; it does not bind kinesin but act as a membrane anchor for the translation elongation factor-1 delta in the endoplasmic reticulum [34].\n\nPrevalence and specificity of anti-kinectin autoantibodies\nThe in vitro [35S]-methionine-labeled translation product of BD44, representing the largest recombinant kinectin fragment available, was used as the antigen substrate in an immunoprecipitation assay. Out of 39 BD patient sera, nine (23%) recognized the BD44 translation product (Fig. 3), whereas sera from 20 normal controls, 10 SLE and 10 SjS patients did not show reactivity. Among the nine anti-kinectin positive patients, six (6/25, 24%) were from Group I (definitive BD) including the BD patient whose serum was used in the immunoscreening of expression cDNA library, and three (3/14, 21.4%) patients were from the Group II (probable BD) in this study. According to the Fisher Exact Probability calculation (P = 1.00), there is no statistically significant difference for antibody to kinectin between the two groups. The combined data substantiated the finding that kinectin is an autoantigen that can be recognized by sera from 23% of Chinese BD patients in this study with at least one immunoreactive region or autoepitope residing within the BD44 encoded polypeptide.\nCurrently, there are more than six diagnostic/classification criteria for BD, among which the International Criteria have been applied most extensively due to its relatively high sensitivity (91%) and specificity (96%) [2]. As discussed above, differential diagnosis of BD might be confusing in clinical practice since no specific laboratory test is available, and some patients may have symptoms and signs strongly suggestive of BD but do not fully satisfy the International Criteria, as in the Group II (probable BD) patients in our study group. A number of investigators have pointed out that a comprehensive analysis of the clinical data for a given patient is very important for correct clinical diagnosis of BD, and that classification/diagnosis criteria, including the International Criteria, should be followed but should not be exclusive. The observation that three out of 14 patients in the probable BD group also had antibody to kinectin and the similar percentage of positive reactors between this group and Group I (21.4% versus 24%) supports this notion. The further use of non-clinical parameters such as immunological biomarkers as adjuncts to identify BD patients could be of help in the classification of this disease entity\nWhile our work was ongoing, anti-kinectin antibodies were reported in sera from patients with hepatocellular carcinoma (HCC) [35,36] and aplastic anemia [37,38]. The first HCC report [35] identified kinectin as a tumor-associated antigen from the screening of an autologous cDNA library constructed from the cancer of a 30-year-old patient from Guangxi, China. This report stated that four out of five HCC patients tested were positive for anti-kinectin antibody [35]. In 2004, another laboratory also reported the cloning of kinectin as a tumor-associated antigen from a (presumably) different 30-year-old Chinese HCC patient [36]. In contrast, anti-kinectin antibodies were not detected in other studies of HCC patients associated with our laboratory [39,40]. The reports of anti-kinectin antibodies in aplastic anemia are also very interesting [37,38]. The initial report by Hirano et al. identified kinectin by screening an aplastic anemia patient for candidate antigens using a Clontech human fetal liver cDNA expression library and it was concluded that seven out of 18 aplastic anemia patients were positive for anti-kinectin while none of the normal or disease controls had this antibody [37]. In their recent report, Hirano et al. reported that anti-kinectin antibodies were found in 39% of aplastic anemia patients from the United States but only in three out of 30 (10%) cases in Japan [38]. In our study reported here, kinectin antibodies were only detected in BD patients and not in normal controls and SLE and SjS disease controls. None of the BD patients with anti-kinectin had signs of HCC or aplastic anemia at the time of diagnosis and at up to 4 years of follow-up. Mapping of epitope(s) recognized by anti-kinectin antibodies may shed light on the question of whether different autoepitopes reside within the kinectin molecule recognized by sera from different diseases.\n\nKinectin – a new member of coiled-coil cytoplasmic autoantigens\nWe have recently reviewed the literature on the growing number of cytoplasmic autoantigens rich in α-helical coiled-coil domains as typified from our study of Golgi autoantigens [41]. Golgi autoantigens are generally high molecular weight proteins between 100 and 350 kDa and rich in coiled-coil domains in the central region with non-coiled-coil or globular domains at both N and C termini. Golgi autoantigens are displayed on the cytoplasmic face of the Golgi complex and are not localized to apoptotic blebs during apoptosis [42]. Giantin, the highest molecular weight Golgi autoantigen reported, is the predominant target of human anti-Golgi complex antibodies and multiple non-cross-reactive epitopes have been mapped spanning the 350 kDa protein [43]. Other high molecular weight autoantigens with similar features have been reported in cytoplasmic and mitotic organelles suggesting that these selected proteins become autoimmunogenic based on their subcellular association and molecular features [41]. For example, in the endosomal compartment, the two known autoantigens are early endosomal protein EEA1 (180 kDa) [44] and CLIP-170 (170 kDa) [45]. There is also a series of centrosomal autoantigens identified as coiled-coil-rich proteins including pericentrin, a 220 kDa protein [46], ninein, a protein with alternatively spliced products of 245 and 249 kDa [47], Cep250 (250 kDa) and Cep110 (110 kDa) [48]. Centromere autoantigens have been described but the two interesting ones related to this discussion are CENP-E [49] and CENP-F [50]; both are high molecular weight proteins (312 to 400 kDa) and have the same type of overall structure as discussed above. NuMA is another large coiled-coil protein located at the mitotic spindle pole and is the most common target autoantigen in sera with mitotic spindle apparatus staining [51]. Non-muscle myosin (~200 kDa) is a cytoskeletal autoantigen [52] that falls in the same group of high molecular weight and coiled-coil-rich autoantigens. These endosomal, centrosomal, mitotic apparatus and intracellular autoantigens are, like the golgins, proteins with high molecular weights and an overall high content of coiled-coil domains. The combination of these two physical features in autoantigens may contribute to the induction and production of autoimmune antibodies in certain disease states. Kinectin is an integral membrane protein largely confined to the endoplasmic reticulum [28,31] and it fits into this new category of autoantigens that are large coiled-coil rich proteins (≥100 kDa) in the cytoplasm.\n\n\nConclusion\nHere we report the detection of kinectin autoantibody in 23% of Chinese patients with BD. The identity of kinectin as a BD-related autoantigen has not been reported to date. Autoantibody reaction against kinectin in BD observed in this study further confirms the autoimmune involvement in BD and may provide new inroads into elucidating the immunopathogenesis of the disease. In an effort to clarify the association of BD with antibody to kinectin, it is essential to measure antibody to kinectin in larger patient populations including both BD, probable BD and important autoimmune rheumatic diseases such as SLE, SjS, rheumatoid arthritis etc., as well as those diseases not easily differentiated from BD, such as recurrent aphthous oral ulcer, Reiter's syndrome, inflammatory bowel diseases etc. On the other hand, further analysis of the association of anti-kinectin antibody with different manifestations or disease 'subtypes' of BD is another important project. Anti-kinectin is clearly only one of the antigen-antibody systems identified because there were many other antibodies observed in the Western blot analysis of BD sera. Using other sera for immunoscreening would probably lead to the identification of other potentially important antigen-antibody systems.\n\nAbbreviations\nAECA = antibody to endothelial cell antigen; BD = Behçet's disease; DMEM = Dulbecco's modified Eagle's medium; HCC = hepatocellular carcinoma; HSP = heat shock protein; IIF = indirect immunofluorescence; PBS = phosphate buffered saline; SjS = Sjögren's syndrome; SLE = systemic lupus erythematosus.\n\nCompeting interests\nThe authors declare that they have no competing interests.\n\nAuthors' contributions\nYL performed the study and drafted the manuscript. PY provided technical help throughout the study. SLC and EMT conceived the study, participated in the design and helped in the analysis of the data. EKLC participated in the design of the study, interpreted data and helped to draft the manuscript. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 25454 ] ] } ]

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[ { "id": "pmcA2658668__text", "type": "Article", "text": [ "A novel DNA-binding protein modulating methicillin resistance in Staphylococcus aureus\nAbstract\nBackground\nMethicillin resistance in Staphylococcus aureus is conferred by the mecA-encoded penicillin-binding protein PBP2a. Additional genomic factors are also known to influence resistance levels in strain specific ways, although little is known about their contribution to resistance phenotypes in clinical isolates. Here we searched for novel proteins binding to the mec operator, in an attempt to identify new factor(s) controlling methicillin resistance phenotypes.\n\nResults\nAnalysis of proteins binding to a DNA fragment containing the mec operator region identified a novel, putative helix-turn-helix DNA-binding protein, SA1665. Nonpolar deletion of SA1665, in heterogeneously methicillin resistant S. aureus (MRSA) of different genetic backgrounds, increased methicillin resistance levels in a strain dependent manner. This phenotype could be fully complemented by reintroducing SA1665 in trans. Northern and Western blot analyses, however, revealed that SA1665 had no visible influence on mecA transcription or amounts of PBP2a produced.\n\nConclusion\nSA1665 is a new chromosomal factor which influences methicillin resistance in MRSA. Although SA1665 bound to the mecA promoter region, it had no apparent influence on mecA transcription or translation, suggesting that this predicted DNA-binding protein modulates resistance indirectly, most likely through the control of other genomic factors which contribute to resistance.\n\n\n\nBackground\nMethicillin resistant S. aureus (MRSA) are an ever increasing threat, both in clinical settings and more recently as an emerging community acquired pathogen. Their invasiveness and pathogenesis relies on a variable arsenal of virulence factors, paired with resistance to virtually all β-lactams and their derivatives. Their ability to rapidly generate resistance to other unrelated classes of antibiotics, or to take up additional resistance determinants, severely hampers therapy and eradication.\nIn S. aureus, methicillin resistance is conferred by an acquired, β-lactam-insensitive penicillin-binding protein (PBP), PBP2a [1-4]. PBP2a is encoded by mecA, which is divergently transcribed from its cognate regulators, mecR1 (sensor/signal transducer) and mecI (repressor). If mecR1-mecI are absent or truncated, transcriptional control of mecA is taken over by the structurally similar blaZ (penicillinase) regulatory elements blaR1/blaI, if present. In the absence of both regulatory loci, mecA is constitutively transcribed [5,6]. In the presence of β-lactams, the transmembrane sensor/signal transducers BlaR1/MecR1, undergo a conformational change, followed by autoproteolytic cleavage of the n-terminal cytoplasmic domain, leading to the activation of the cytoplasmic peptidase and subsequent dissociation of the repressor due to proteolytic degradation [7-9]. However, the signal transduction cascade of this regulatory system has still not been completely elucidated.\nOxacillin resistance levels conferred by mecA are strain specific and can vary greatly, with oxacillin minimal inhibitory concentrations (MICs) of different strains ranging from phenotypically susceptible levels, as low as 1 μg/ml up to extremely high values of > 500 μg/ml. Methicillin resistance is also generally expressed heterogeneously. Heterogeneously resistant MRSA, when exposed to β-lactam antibiotics, segregate highly resistant subpopulations, which are much more resistant than the majority of the cells [10]. The frequency of highly resistant subclones generated is often well above the spontaneous mutation frequency, and once selected high level resistance often remains stable, even in the absence of selective pressure. There is currently no satisfactory genetic model which explains how these higher resistance levels are triggered or selected and exactly what factors are functionally responsible for the increased resistance in clinical isolates. Methicillin resistance levels are known to not directly correlate with mecA transcription or levels of PBP2a produced [11,12]. However, resistance levels can be manipulated by environmental conditions, such as temperature, pH, osmolarity, and medium composition [13,14].\nIt has been shown experimentally, that in addition to mecA, methicillin resistance depends on the correct interplay of a multitude of genomic factors, termed fem/aux factors, including genes involved in peptidoglycan precursor formation, composition and turnover; teichoic acid synthesis; and genes of unknown or poorly characterised functions [15-18]. In addition to structural genes, many regulatory loci have also been shown to influence resistance levels, including global regulators of virulence factor production such as the quorum sensing agr system, the staphylococcal accessory regulator SarA and the alternate sigma factor σB [19,20]; regulators of metabolism, such as the catabolite control protein A (CcpA) [21]; and the VraSR two-component sensor transducer, which induces the cell wall stress stimulon in response to cell wall active antibiotic challenge [22].\nThe vast MIC differences between MRSA strains, the population heterogeneity within single strains and the dependence of resistance levels on external factors are reflected in these many structural genes and global regulators, which can influence resistance levels.\nWhile typically considered nosocomial pathogens, new faster growing and apparently more virulent MRSA have begun spreading in the community. Interestingly, these emerging strains often express very low methicillin resistance, e.g. the MRSA clone spreading amongst intravenous drug users in the Zurich area, which has an in vitro doubling time of 25 min, but oxacillin MICs of only 0.5 to 4 μg/ml [23]. This particular clone's low-level resistance is partially due to a promoter mutation, leading to tight repression of mecA, but resistance levels appear to be mainly restricted by unknown factors within its genomic background [12].\nTo identify potential factors involved in mecA regulation or methicillin resistance levels in such an extremely low level resistant MRSA, we performed DNA-binding protein purification assays, using the mecA operator region as bait. A novel, uncharacterized protein, SA1665, was found to bind to this DNA fragment, and shown to increase methicillin resistance levels when deleted.\n\nResults\nIdentification of SA1665\nMRSA strain CHE482 is the type strain for the so-called \"drug clone\" spreading amongst intravenous drug users in the Zurich area [12,23]. This strain carries mecA and expresses PBP2a, but appears phenotypically methicillin susceptible by conventional phenotypic tests. However, like most other low-level resistant MRSA, it can segregate a small proportion of higher resistant subclones in the presence of β-lactams. We hypothesized that regulation of methicillin resistance in such low-level resistant clonal lineages may differ qualitatively from classical heterogeneously- or highly-resistant MRSA.\nA DNA-binding protein purification assay was performed to identify new potential factors involved in the regulation of mecA/PBP2a. The mecA/mecR1 intergenic DNA region, including the 5' 9 bp of mecR1 and the first 52 bp of mecA, was used as bait against crude protein extract from strain CHE482. Proteins binding to this DNA fragment were analysed by SDS-PAGE. Even though CHE482 contained BlaI, which is known to bind to the mec operator, this band could not be identified on gels due to co-migrating, non-specific bands the same size as BlaI (14.9 KDa) that bound to both the DNA-coated and uncoated control beads. The most prominent protein band of ~16–20 kDa, isolated from DNA-labelled but not from control beads, was identified as the hypothetical protein SA1665 (N315 genome annotation [BA000018]) (Figure 1A). SA1665 encodes a predicted 17-kDa protein with an n-terminal helix-turn-helix (HTH) motif characteristic of DNA-binding transcriptional regulators. The amino acid sequence of SA1665 showed 100% identity amongst S. aureus database sequences and 97–98% identity amongst other staphylococci, including S. haemolyticus, S. epidermidis and S. saprophyticus, indicating that SA1665 is highly conserved. Conversely, there were no orfs highly similar to SA1665 found in other bacterial species, with the most similar sequences found in Bacillus licheniformis DSM13 and Desulfitobacterium hafniense Y51, which shared only 64% and 59% similarity, respectively.\n\nElectro mobility shift assays (EMSA)\nEMSA was used to confirm binding of SA1665 to the mec operator region. Crude protein extracts of E. coli strain BL21, carrying the empty plasmid (pET28nHis6) or pME20 (pET28nHis6-SA1665) which expressed nHis6-SA1665 upon induction with IPTG, were incubated with the 161-bp biotinylated-DNA fragment previously used as bait in the DNA-binding protein assay. A band shift was observed with extracts from the strain expressing recombinant nHis6-SA1665 but not from the control strain carrying the empty plasmid. Several bands resulted from the shift, which is most likely due to protein oligomerisation (Figure 2A). The specificity of the gel shift was also demonstrated by the addition of increasing concentrations of purified nHis6-SA1665 protein to the biotinylated-DNA fragment (Figure 2B). Band-shift of the biotinylated DNA was inhibited in the presence of specific competitor DNA but not by the presence of the non-specific competitor DNA, confirming that nHis6-SA1665 had a specific binding affinity for the 161-bp DNA fragment.\n\nEffect of SA1665 deletion on β-lactam resistance\nTo analyse the effect of SA1665 inactivation on methicillin resistance, nonpolar markerless deletions of SA1665 (Figure 1B) were constructed in a selection of clinical MRSA isolates, which varied in their genetic background, SCCmec type, and mecA regulation [24]. Strain CHE482, belongs to clonal complex CC45 and sequence type ST45, and contains a novel SCCmec (SCCmecN1 [23]); while strains ZH37 (CC45/ST45) and ZH73 (CC22/ST22) contain type IV SCCmecs. All three of these strains have truncated mecI/mecR1 regulatory loci but intact BlaI/BlaR1 loci controlling mecA expression. Strain ZH44 (CCT8/ST8) contained a type A mec complex (mecI-mecR1-mecA) within a type II SCCmec, and had no β-lactamase locus; so mecA was only under the control of its cognate regulators MecI/MecR1.\nDeletion of SA1665 increased oxacillin resistance in all mutants compared to their corresponding parent strains, as demonstrated on oxacillin gradient plates (Figure 3A); with mutants ΔCHE482 and ΔZH37 approximately doubling in resistance and ΔZH44 and ΔZH73 expressing considerably higher resistance. Population analysis resistance profiles of the mutants showed a distinct shift at the top of the curve, indicating that the higher resistance was due to increased basal oxacillin resistance levels (Figure 3B). Strains CHE482/ΔCHE482 and ZH37/ΔZH37 had very similar resistance profiles, despite having different SCCmec elements, suggesting that it was their common clonal background (CC45) that determined their resistance levels and the extent of resistance increase upon SA1665 deletion.\nGrowth curve analyses showed that deletion of SA1665 slightly reduced the growth rate of all strains tested (Figure 3C). Wild type growth rates were restored upon complementation (data not shown).\n\nResistance complementation\nPlasmids pME26 and pME27 were constructed for complementation of the deletion mutants. Both plasmids contained the SA1665 orf along with its own promoter and transcriptional terminator. Strains ΔCHE482, ΔZH37, and ΔZH73 were complemented with pME26, and intrinsically kanamycin resistant strain ΔZH44 was complemented with pME27. Wild type-like resistance levels were restored in all mutants by introduction of the complementing plasmids, as shown by gradient plates (Figure 3A).\n\nTranscriptional analyses\nPrimer extension, using the 5'-biotinylated primer me97, identified two potential SA1665 transcriptional start sites (TSS), 76-nt and 139-nt upstream of the SA1665 ATG start codon (Figure 4A). Predicted σA promoter consensus -10/-35 box sequences were located upstream of both TSS (Figure 4B). Identical TSS were also identified using the downstream primer me98 (data not shown).\nNorthern blot analysis was used to investigate SA1665 expression and the influence of SA1665 deletion on mecA and mecR1 transcription. RNA samples taken from different time points over the growth curve of CHE482 showed that SA1665 was expressed strongly in early exponential phase at OD600 nm 0.25 and 0.5, then transcript levels decreased and were almost undetectable in early stationary phase at OD600 nm 4.0 (Figure 5A). In addition to the main transcript of ~0.46 kb, a weaker, larger transcript of ~0.6 kb was also visible, especially at later growth stages. Figure 5B shows the transcriptional behaviour of SA1665 when CHE482 cells were challenged with sub-inhibitory (4 μg/ml) and inhibitory (120 μg/ml) concentrations of cefoxitin. These results showed that low levels of cefoxitin, such as those used to induce mecA/mecR1 transcription, appeared to slightly decrease SA1665 transcription after 30 min exposure, while larger, inhibitory concentrations caused even more significant alterations in the SA1665 transcriptional profile, making it similar to that normally seen in stationary phase growth. These results indicate that transcription of SA1665 may respond in some way to cell wall stress, rather than in direct response to the presence of β-lactams. This observation is based on relatively subtle changes in SA1665 transcription, especially at low concentrations of cefoxitin such as those required for mecA/mecR1 induction. Since deletion of SA1665 has been shown to increase β-lactam resistance, reduced SA1665 transcription in the presence of β-lactams may also provide some protection against β-lactam exposure.\nNortherns also showed that, as expected, the SA1665 transcripts were absent from the deletion mutant (Figure 5C), and additional experiments demonstrated that wild type SA1665 transcription patterns were restored by complementation of ΔCHE482 with pME26 (data not shown). The effects of SA1665 deletion on directly up- and down-stream genes were also investigated. Northern blots of the neighbouring genes SA1664, SA1666 and SA1667, showed that expression of all three genes was very weak compared to that of SA1665. A weak transcript of about 3 kb was present in hybridizations probed with orfs SA1665-SA1667. This band decreased in size in the SA1665 mutant when probed with SA1666 and SA1667. One of the transcripts hybridising to the SA1664 probe also decreased in size by ~0.5 kb in the SA1665 mutant, suggesting that SA1665 was present on several transcripts of different lengths, including a high abundance monocistronic transcript and low abundance polycistronic transcripts (Figure 5C). Transcript abundance of both the upstream SA1666-SA1667 operon and the downstream SA1664-specific transcript all appeared to increase slightly in ΔCHE482. The significance of these subtle increases in transcription are unknown, however, polar effects from SA1665 deletion seem unlikely, based on the facts that all genes were still transcribed, their transcription levels all remained extremely low and the transcriptional terminator of SA1665 remained intact in the deletion mutant (Figure 1B).\nExpression of mecR1 and mecA were analysed from RNA of uninduced and induced cultures of CHE482 and ΔCHE482. Cells were induced at OD600 nm 0.25 (Figure 5D) and 1.0 (data not shown) with sub-inhibitory concentrations of cefoxitin, to relieve BlaI-repression of mecA. mecR1, although truncated in CHE482, was still transcribed and had the same expression pattern as mecA, as both became derepressed over time and had the highest transcript levels after 30 min of induction. In the mutant ΔCHE482, transcripts of both mecA and mecR1' were unaffected by SA1665 deletion, indicating that SA1665 had no influence on their expression at either OD 0.25 (Figure 5D) or OD 1.0 (data not shown). SA1665 deletion also had no effect on mecA transcription or induction in strains ZH37, ZH44 and ZH73 (data not shown).\n\nWestern blot analysis\nMutants of CHE482 and of ZH44 and ZH73, which had the largest differences in oxacillin resistance levels, were analysed by Western blot analysis to determine if SA1665 affected production of PBP2a from mecA. As shown in Figure 5E, all pairs of wild type and mutant strains had similar amounts of PBP2a present both before and after induction with cefoxitin, indicating that SA1665 deletion did not alter amounts of PBP2a produced. Therefore it seems that SA1665 exerts no direct control over mecA or PBP2a expression.\n\n\nDiscussion\nMethicillin resistance in MRSA is primarily dependent on the presence of the mecA gene, however, resistance levels are generally governed by strain-specific factors including mecA regulatory elements and other chromosomal fem/aux factors which either enhance or repress the expression of resistance. For instance, the very low-level methicillin resistance of the Zurich drug clone CHE482, was shown to be controlled by its genetic background [12] suggesting that it either contained or lacked certain fem/aux factors involved in controlling resistance expression. Many of the currently known fem/aux factors are directly or indirectly involved in cell wall synthesis and turnover, or envelope biogenesis, however there still remain factors of unknown function. Most of the currently known fem/aux factors reduce methicillin resistance levels when inactivated. A few genes, such as lytH, dlt, norG, sarV and cidA increase resistance levels upon inactivation or mutation. All of these genes, except norG, which is an efflux pump regulator, play a role in either autolysis or are important for cell physiology and growth [25-30]. Other genes increase β-lactam resistance upon overexpression, such as hmrA coding for a putative amidohydrolase, hmrB coding for a putative acyl carrier protein [31], or the NorG-controlled abcA multidrug efflux pump [28].\nSA1665, a predicted DNA-binding transcriptional regulator, was found to bind to a DNA fragment containing the mecA promoter region. However, although this protein shifted the mecA operator/5' coding sequence, it did not appear to directly control mecA or mecR1 transcription or PBP2a production. Therefore its binding to the mecA region may have no specific regulatory function. Such interactions have been noted before, such as the HTH protein NorG, which was shown to bind specifically to norA, norB and norC promoters, but only transcription of norB was increased when NorG was overexpressed [28]. We have to postulate therefore that SA1665 may modulate β-lactam resistance in a mecA-independent manner, by controlling cellular functions affecting resistance levels. Experiments to determine the SA1665 regulon are ongoing. The impact of deleting SA1665 in MRSA was extremely strain specific, underlining the importance of the genetic background in governing the final methicillin resistance levels of MRSA, and demonstrating the large genomic variability between different strain lineages.\n\nConclusion\nSA1665 is a previously uncharacterised DNA-binding protein that has a negative effect on β-lactam resistance in MRSA. The SA1665 protein was identified in a DNA-binding protein purification assay, in which it bound to a DNA fragment covering the mec operator region. However, while nonpolar deletion of SA1665 was shown to increase oxacillin resistance levels in several heterogeneously resistant MRSA, its deletion had no effect on mecA transcription or PBP2a production. Therefore the negative impact of SA1665 on methicillin resistance is most likely to be through the regulation of other chromosomal factors or cellular functions required for methicllin resistance.\n\nMethods\nStrains and growth conditions\nStrains and plasmids used in this study are listed in Table 1. Clinical isolates are from the IMM collection in Zurich, Switzerland. Strains were grown at 37°C in Luria Bertani (LB) broth, shaking at 180 rpm, or on LB agar. Media were supplemented with the following antibiotics when appropriate: 25 or 50 μg/ml kanamycin, 10 μg/ml chloramphenicol, 5 or 10 μg/ml tetracycline, 100 μg/ml ampicillin. Concentrations of cefoxitin used for transcriptional induction were either sub-inhibitory (4 μg/ml) or inhibitory (120 μg/ml).\n\nSusceptibility testing\nOxacillin resistance levels were compared by swabbing 0.5 McFarland cell suspensions across agar plates containing appropriate concentration gradients of oxacillin. For population analysis profiles, appropriate dilutions of an overnight culture, ranging from 100 to 108, were plated on increasing concentrations of oxacillin. Plates were incubated at 35°C and colony forming units per ml (cfu/ml) were determined after 48 h.\n\nBinding-protein purification\nCrude protein extracts were isolated from CHE482, grown under normal culture conditions until OD600 nm 1.5. Cells were harvested, resuspended in PBS (pH 7.4) and mechanically lysed using Lysing Matrix B (BIO 101 Systems) tubes and a FastPrep FP120 (BIO 101 Systems). Suspensions were clarified by centrifugation and supernatants, containing soluble cytoplasmic proteins, were transferred to Amicon Ultra centrifugal filter devices (Millipore) with a pore cut-off size of 10 kDa. Proteins were then washed and concentrated in 1× binding buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1 mM DTT, 0.5 M NaCl). Protein concentrations were measured by Bradford assay (BioRad Laboratories GmbH) [32]. Primer pair me36F/me36Rbiot (Table 2) were used to amplify a biotinylated mecA promoter/operator fragment, which was bound to streptavidin coated magnetic beads (Dynabeads M-280 Streptavidin, DYNAL BIOTECH) according to the manufacturer's instructions. Binding reactions, containing DNA-coated beads mixed with 100 μg of crude protein extract in 1× protein binding buffer (20 mM Hepes, pH 7.6, 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM DTT, 0.2% Tween 20 (w/v), 30 mM KCl), 0.02 μg/μl poly d(I-C) and 2 ng/μl poly L-lysine, were incubated at room temperature for 30 min with constant rotation. Beads were then washed and binding-proteins eluted in elution buffer (1× protein binding buffer containing 2 M KCl). Eluted proteins were dialysed against water, concentrated by evaporation, and run on 15% SDS polyacrylamide gels. Gels were silver stained using the Protein Silver Staining kit (Amersham Biosciences AB) without the addition of glutaraldehyde. Protein bands were excised from gels and analysed by mass spectrometry (LC/ESI/MS/MS) at the Functional Genomics Centre, Zurich. The SA1665 protein sequence [BAB42933] was analysed by Blast search and motif scan .\n\nExpression of recombinant SA1665 protein\nSA1665 was amplified using primer pair me65BamHI/me65XhoI (Table 2) and cloned in-frame into pET28nHis6 (unpublished, D. Frey). The resulting plasmid, pME20, was transformed into E. coli BL21 for expression of recombinant nHis6-SA1665 protein. To maximise the abundance of soluble protein produced, cultures were grown in osmotic shock medium at 37°C (1 g/l NaCl, 16 g/l tryptone, 10 g/l yeast, 1 M sorbitol, 10 mM betaine, modified from [33]) to an OD600 nm of 0.5, cooled briefly on ice, then induced by adding 100 μM IPTG and growing overnight at 22°C. Crude soluble proteins were extracted using CelLyticB 2× cell lysis reagent (SIGMA). HIS-Select Cobalt Affinity Gel (SIGMA) was used to purify recombinant nHis6-SA1665 according to the manufacturer's instructions.\n\nElectro mobility shift assay\nFor gel shift assays, 6 ng aliquots of the biotinylated-DNA fragment used for binding-protein purification were incubated with 0–250 ng of purified nHis6-SA1665 protein in 1× binding buffer (20 mM Hepes pH 7.6, 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM DTT, 0.2% Tween 20 (w/v), 30 mM KCl) containing 0.05 μg/μl poly d(I-C) (Roche) and 5 ng/μl poly L-lysine (Roche). For control binding reactions, 130 × unlabelled mec operator DNA (amplified using primers me36F/me36R, Table 2) was used as a specific binding competitor and 6 ng of herring sperm DNA was used as unspecific competitor DNA. Binding was carried out at 22°C for 30 min. Samples were run on 6% native polyacrylamide gels, contact blotted onto positively charged nylon membrane and detected with the Biotin Chromogenic Detection Kit (Fermentas).\n\nPrimer extension\nRNA was extracted from CHE482 cultures that were grown to OD600 nm 0.5, as previously described [12]. Primer extension reactions were performed using 20 μg of total RNA and 3 pmol of the 5'-biotin-labelled primers me97 and me98 (Table 2) using Superscript II reverse transcriptase (Invitrogen), according to the manufacturers instructions. Sequencing reactions were performed using the Thermo Sequenase cycle sequencing kit (U.S. Biochemicals). The Biotin Chromogenic Detection Kit (Fermentas) was used for biotin detection.\n\nMarkerless deletion of SA1665\nIn frame markerless deletions of SA1665, from the chromosomes of CHE482, ZH37, ZH44, and ZH73, were constructed using the pKOR1 allelic replacement system, as described by Bae et al. [34]. Primer pairs used to amplify the DNA fragments flanking SA1665, for recombination into pKOR1 were: me62attB1/me51BamHI and me62BamHI/me62attB2 (Table 2). All deletion mutants were confirmed by nucleotide sequencing over the deleted region, as well as by Southern blot analysis [35] and pulsed field gel electrophoresis (PFGE) [36].\n\nCloning of SA1665 for complementation\nA 1533-bp DNA fragment, containing SA1665 together with 690-bp of upstream and 379-bp of downstream DNA, was amplified from strain CHE482 using primers me94BamHI/me94Asp718 (Table 2) and cloned into the E. coli/S. aureus shuttle vectors pAW17 and pBUS1 [37], creating the complementing plasmids pME26 and pME27, respectively. Plasmids were electroporated into RN4220 [38] and then transduced into different strains using phage 80α.\n\nNorthern blot analysis\nStrains were grown overnight in LB (Difco), diluted 1:200 and grown for another 3 h. This preculture was used to inoculate 150 ml (1:1000) of fresh prewarmed LB. Cells were then grown to OD600 nm 0.25 or 1.0 and either left uninduced or induced with cefoxitin 4 or 120 μg/ml. Cultures were sampled from both uninduced and induced cells at time point 0' before induction and at 10' and 30' (min) after induction. To monitor SA1665 expression over growth, separate cultures were also sampled at different growth stages corresponding to OD600 nm 0.25, 0.5, 1, 2, and 4. Total RNA was extracted as described by Cheung et al. [39]. RNA samples (10 μg) were separated in a 1.5% agarose-20 mM guanidine thiocyanate gel in 1× TBE running buffer [40], then transferred and detected as described previously [41]. Digoxigenin (DIG) labelled-probes were amplified using the PCR DIG Probe synthesis kit (Roche). Table 2 contains the list of primer pairs used for the amplification of SA1664, SA1665, SA1666, SA1667, mecR1 and mecA [42] probes. All Northern's were repeated at least two times, using independently isolated RNA samples.\n\nWestern blot analysis\nCells were cultured, as described for Northern blot analysis, to OD600 nm 1.0, then induced with cefoxitin 4 μg/ml. Samples were collected at time 0 (before induction), 10 and 30 min (after induction). Cells were harvested by centrifugation, resuspended in PBS pH 7.4 containing DNase, lysostaphin and lysozyme (150 μg/ml of each) and incubated for 1 h at 37°C. Suspensions were then sonicated and protein aliquots (15 μg) were separated on 7.5% SDS-polyacrylamide gels, blotted onto nitrocellulose membranes (Hybond) and stained with Ponceau to confirm equal protein loading. PBP2a detection was performed using monoclonal PBP2a antibody (1:20000) from the MRSA-screen kit (Denka Seiken).\n\n\nAuthors' contributions\nME carried out molecular genetic and microbiological studies and drafted the manuscript. BB participated in the design of the study and helped to draft the manuscript. NM participated in the design and coordination of the study, carried out molecular biological studies and helped to draft the manuscript. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 28283 ] ] } ]

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8185, 8199 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1282" } ] }, { "id": "pmcA2658668__T19", "type": "species", "text": [ "S. saprophyticus" ], "offsets": [ [ 8204, 8220 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "29385" } ] }, { "id": "pmcA2658668__T20", "type": "species", "text": [ "Bacillus licheniformis" ], "offsets": [ [ 8397, 8419 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1402" } ] }, { "id": "pmcA2658668__T21", "type": "species", "text": [ "Desulfitobacterium hafniense Y51" ], "offsets": [ [ 8430, 8462 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "138119" } ] }, { "id": "pmcA2658668__T22", "type": "species", "text": [ "E. coli strain BL21" ], "offsets": [ [ 8655, 8674 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "511693" } ] }, { "id": "pmcA2658668__T23", "type": "species", "text": [ "MRSA" ], "offsets": [ [ 9810, 9814 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1280" } ] }, { "id": "pmcA2658668__T24", "type": "species", "text": [ "MRSA" ], "offsets": [ [ 16834, 16838 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1280" } ] }, { "id": "pmcA2658668__T25", "type": "species", "text": [ "MRSA" ], "offsets": [ [ 19018, 19022 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1280" } ] }, { "id": "pmcA2658668__T26", "type": "species", "text": [ "MRSA" ], "offsets": [ [ 19163, 19167 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1280" } ] }, { "id": "pmcA2658668__T27", "type": "species", "text": [ "MRSA" ], "offsets": [ [ 19376, 19380 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1280" } ] }, { "id": "pmcA2658668__T28", "type": "species", "text": [ "MRSA" ], "offsets": [ [ 19661, 19665 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1280" } ] }, { "id": "pmcA2658668__T29", "type": "species", "text": [ "E. coli BL21" ], "offsets": [ [ 23053, 23065 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "511693" } ] }, { "id": "pmcA2658668__T30", "type": "species", "text": [ "yeast" ], "offsets": [ [ 23262, 23267 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4932" } ] }, { "id": "pmcA2658668__T31", "type": "species", "text": [ "E. coli" ], "offsets": [ [ 25810, 25817 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "562" } ] }, { "id": "pmcA2658668__T32", "type": "species", "text": [ "S. aureus" ], "offsets": [ [ 25818, 25827 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1280" } ] }, { "id": "pmcA2658668__T33", "type": "species", "text": [ "MRSA" ], "offsets": [ [ 27866, 27870 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1280" } ] } ]

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[ { "id": "pmcA335194__text", "type": "Article", "text": [ "The Caenorhabditis elegans genome contains monomorphic minisatellites and simple sequences.\nAbstract\nMany species have been shown to contain tandemly repeated short sequence DNA known as minisatellites and simple sequence motifs. Due to allelic variation in the copy number of the repeat unit these loci are usually highly polymorphic. Here we demonstrate the presence of sequences in the genome of the nematode Caenorhabditis elegans which are homologous to two sets of short sequence DNA. However, when two independent strains were compared no polymorphism for these sequences could be detected.Images\n\n\n\n\n\n\n Volume 17 Number 23 1989 Nucleic Acids Research \n\n The Caenorhabds elegans genome contains monomorphic minisatellites and simple sequences Andrd G.Uitterlinden, P.Eline Slagboom, Thomas E.Johnsonl and Jan Vijg \n\n Department of Molecular Biology, TNO Institute for Experimental Gerontology, PO Box 5815, 2280 HV Rijswijk, The Netherlands and lInstitute for Behavioral Genetics, University of Colorado, Boulder, CO 80309, USA \n\n Received October 27, 1989; Accepted November 3, 1989 \n\n ABSTRACT \n\n Many species have been shown to contain tandemly repeated short sequence DNA kinown as minisatellites and simple sequence motifs. Due to allelic variation in the copy number of the repeat unit these loci are usually highly polymorphic. Here we demonstrate the presence of sequences in the genome of the nematode Caenorhabditis elegans which are homologous to two sets of short sequence DNA. However, when two independent strains were compared no polymorphism for these sequences could be detected. \n\n INTRODUCTION \n\n The genome of many species, including lower organisms, contain minisatellite sequences and so-called simple sequence motifs (1,2,3). Due to extensive variation in the number of repeat units, many of these loci have been shown useful as polymorphic markers, e.g. for genetic linkage studies and identification purposes. \n\n Here we demonstrate that the genome of the nematode Caenorhabditis elegans contains sequences that are homologous to the minisatellite core sequence 33.6 (1) and to the simple sequence motif (AGC)n. Surprisingly, these sequences did not display polymorphism when two independent C. elegans strains, Bristol and Bergerac, were compared. \n\n MATERIALS AND METHODS Genomic DNA from nematodes \n\n Genomic DNA was isolated according to standard procedures from the two strains Bergerac (BO) and Bristol (strain N2). These strains are derived from two different individual worms isolated in France and England, respectively. Southern blotting \n\n Genomic DNA digests (5 Ag) were separated in a 1 % agarose gel in 1 x TAE (40 mM Tris.HCl, pH 7.4/20 mM sodium acetate/I mM NaEDTA) by electrophoresis at 75 V for 16 h. Separation patterns were transferred to Zetaprobe membrane (BIORAD) in 0.4 N NaOH, 0.6 M NaCl in a vacu-blot apparatus (LKB) according to the manufacturer's instructions. The minisatellite and simple sequence probes used were prepared essentially as described (5). The Tcl probe is a plasmid containing an insert of the transposable element Tcl (4). All probes were labelled by random-priming. Hybridization was performed for 12 h in 7 % SDS, 0.5 M phosphate buffer, 1 mM Na2EDTA at 650C. Blots were washed twice in 2.5 x SSC at 650C and exposed to Kodak XAR-5 film with intensifying screens. Exposure times are indicated in Fig. 1. \n\n ( IRL Press \n\n Nucleic Acids Research \n\n Volume 17 Number 23 1989 \n\n 9527 \n\n Nucleic Acids Research \n\n C. elegans \n\n N 0$ 8 \n\n _ _ \n\n : , s , ..e :*. ,~ :::::\n\n . 4i \n\n .t_: -::?J?.: \n\n i- \n\n .a \n\n E?:< . :: y : \n\n *01 \n\n 4?j* \n\n .-I \n\n kb - 27 \n\n - 9A - \n\n : .::....... \n\n Mi .... - 4v3 \n\n IC .3 \n\n If.0r \n\n MW \n\n !5 c6 \n\n I \n\n \" R 1d~ ! ;,\n\n Figure 1. Southern hybridization analysis of Hae III, Rsa I and Eco RI digested genomic DNA isolated from the two C. elegans strains Bristol (N) and Bergerac (B). The probes used and the exposure times of the autoradiograph are indicated below the figure. kb=kilo basepairs. 9528 \n\n *:..?.;. .*. i... :\". \n\n ANN& \n\n 'ROW :. lwmwww. \n\n .Ammkw.l \n\n 4w, \n\n 9 \n\n _ i ob \n\n Ill a \n\n Nucleic Acids Research \n\n RESULTS \n\n In Figure 1 the hybridization patterns are shown of C. elegans genomic DNA digested with Hae II, Rsa I and Eco RI, and subsequently hybridized to minisatellite core probe 33.6, the simple sequence probe (AGC)n and Tcl. The latter probe contains a member of the Tcl family of transposable elements which is present in different copy numbers in the two different strains (4). The hybridization patterns of probes 33.6 and (AGC)n obtained after Hae Im and Rsa I digestion and the differences in intensities of the hybridizing bands is reminiscent of DNA-fingerprint patterns obtained with these probes in other species (1,2,3). However, identical hybridization patterns for the two strains with the three enzymes tested indicated that in C. elegans these sequences are monomorphic (Fig. 1). Based on the number of hybridizing bands we estimate the C. elegans genome to contain about 30 33.6 homologous loci and about 20 (AGC)n homologous loci. \n\n Rehybridization of the same blot with a Tcl probe revealed extensive RFLPs due to the different copy number of the transposon in the two strains, a phenomenon which has been described previously by others (4). \n\n DISCUSSION \n\n The findings presented in this paper indicate that polymorphism of minisatellite sequences and simple sequence motifs, is not a general phenomenon in animal species. So far, only some species of whales have displayed similar high levels of monomorphism (6). In other species thus far tested both minisatellites and simple sequences display high to very high levels of polymorphism. It should be noted, however, that at least in humans, a substantial part of the minisatellites detected by core probes also displays high levels of monomorphism as analysed by cloning (1) or by two-dimensional DNA fingerprinting (5). \n\n The fact that nematodes are hermaphrodites, and thus inbred, might be a contributing factor to the observed lack of polymorphism. However, the high levels of polymorphism detected by the Tcl probe do not indicate a general absence of events causing genetic variation. Indeed, Eide and Anderson (7) showed that tandemly repeated duplications in the unc-54 gene of C. elegans revert at high frequencies. Since in all cases the revertants had the normal genomic configuration this suggests that unequal crossing-over does occur in the nematode. \n\n A more likely explanation for the monomorphic nature of the sequences detected with 33.6 and (AGC)n in C. elegans is selection against sequence variants at these loci. This might be the result of the presence of a particular subset of minisatellites and/or simple sequences at sites in the genome of this organism, e.g. in coding sequences, in which variation in copy number of repeat units cannot be tolerated. An example of such a coding sequence could be the High Mobility Group proteins which usually have stretches of identical (acidic) amino acids (2,8). An interesting observation in this respect is the demonstration of the absence of any protein polymorphisms in electrophoretic comparisons for 24 different enzymes between the Bristol and Bergerac strains (9). An important step in understanding this phenomenon will therefore be the isolation and analysis of individual homologous minisatellite and simple sequence loci from a genomic library of C. elegans. \n\n REFERENCES \n\n 1. Jeffreys, A.J., Wilson, V., and Thein, S.L. (1985) Nature 314, 67-73. 2. Tautz, D., Trick M., and Dover, G.A. (1986) Nature 322, 652-656. \n\n 3. Rogstad, S.H., Herwaldt, B.L., Schlesinger, P.H., and Krogstad, D.J. (1989) Nucleic Acids Res. 17, 9, 3610. 4. Emmons, S.W., Yesner, L., Ruan, K. and Katzenberg, D. (1983) Cell 32, 55-65. \n\n 9529 \n\n Nucleic Acids Research \n\n 5. Uitterlinden, A.G., Slagboom, P.E., Knook, D.L., and Vijg, J. (1989) Proc. Natl. Acad. USA 86, 2742-2746. 6. Rus Hoelzel, A., and Amos, W. (1988) Nature 333, 305. 7. Eide, D., and Anderson, P. (1985) Genetics 109, 67-79. \n\n 8. Pentecost, B.T., Wright, J.M., and Dixon, G.H. (1985) Nucleic Acids Res. 13, 4871-4888. 9. Butler et al. (1981) J. Molec. Evolution 18, 18-23. \n\n 9530 \n\n This article, submitted on disc, has been automatically \n\n converted into this typeset format by the publisher. " ], "offsets": [ [ 0, 8402 ] ] } ]

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[ { "id": "pmcA2412862__text", "type": "Article", "text": [ "Minocycline attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia\nAbstract\nBackground\nActivation of the peripheral innate immune system stimulates the secretion of CNS cytokines that modulate the behavioral symptoms of sickness. Excessive production of cytokines by microglia, however, may cause long-lasting behavioral and cognitive complications. The purpose of this study was to determine if minocycline, an anti-inflammatory agent and purported microglial inhibitor, attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia.\n\nMethods\nIn the first set of experiments the effect of minocycline pretreatment on LPS-induced microglia activation was assessed in BV-2 microglia cell cultures. In the second study, adult (3–6 m) BALB/c mice received an intraperitoneal (i.p.) injection of vehicle or minocycline (50 mg/kg) for three consecutive days. On the third day, mice were also injected (i.p.) with saline or Escherichia coli LPS (0.33 mg/kg) and behavior (i.e., sickness and anhedonia) and markers of neuroinflammation (i.e., microglia activation and inflammatory cytokines) were determined. In the final study, adult and aged BALB/c mice were treated with the same minocycline and LPS injection regimen and markers of neuroinflammation were determined. All data were analyzed using Statistical Analysis Systems General Linear Model procedures and were subjected to one-, two-, or three-way ANOVA to determine significant main effects and interactions.\n\nResults\nMinocycline blocked LPS-stimulated inflammatory cytokine secretion in the BV-2 microglia-derived cell line and reduced LPS-induced Toll-like-receptor-2 (TLR2) surface expression on brain microglia. Moreover, minocycline facilitated the recovery from sickness behavior (i.e., anorexia, weight loss, and social withdrawal) and prevented anhedonia in adult mice challenged with LPS. Furthermore, the minocycline associated recovery from LPS-induced sickness behavior was paralleled by reduced mRNA levels of Interleukin (IL)-1β, IL-6, and indoleamine 2, 3 dioxygenase (IDO) in the cortex and hippocampus. Finally, in aged mice, where exaggerated neuroinflammation was elicited by LPS, minocycline pretreatment was still effective in markedly reducing mRNA levels of IL-1β, TLR2 and IDO in the hippocampus.\n\nConclusion\nThese data indicate that minocycline mitigates neuroinflammation in the adult and aged brain and modulates the cytokine-associated changes in motivation and behavior.\n\n\n\nBackground\nThe bi-directional communication between the immune system and the central nervous system (CNS) is necessary for mounting the appropriate immunological, physiological, and behavioral responses to immune stimulation [1]. CNS innate immune cells including microglia and macrophages play integral roles in receiving and propagating inflammatory signals that are initiated at the periphery. Activation of peripheral innate immune cells elicits the secretion of inflammatory cytokines, including interleukin (IL)-1, IL-6, and tumor necrosis factor-α (TNFα.), that use neural [2,3], humoral [4] and blood brain barrier pathways [5] to relay this signal to the CNS. This inflammatory signal, in turn, induces CNS macrophages and microglia to produce the same cytokines [6], which target neuronal substrates and elicit a sickness behavior syndrome that is normally adaptive and beneficial to the host [1]. An amplified or excessive inflammatory cytokine response in the brain, however, is associated with a myriad of complications including cognitive dysfunction [7-10], prolonged sickness behavior [11-14], and depressive-like behavior [15].\nMicroglia are primarily involved in immune surveillance [16,17], but when activated have macrophage-like capabilities including phagocytosis, inflammatory cytokine production, and antigen presentation [18]. Normally these neuroinflammatory changes are transient with microglia returning to a resting state as the immune stimulus is resolved. Aging or neurological disease, however, may provide a brain environment where microglia are more \"reactive or primed\" to a peripheral immune challenge [19]. Recent findings indicate that several markers of glial activation such as major histocompatibility complex (MHC) class II, complement receptors, and scavenger receptors are increased in brain during normal aging [13,20-26]. Furthermore, we and others have reported that a biological consequence of this reactive glial profile is an exaggerated neuroinflammatory response to innate immune challenge [9,10,12-14,27,28].\nActive microglia and CNS macrophages also contribute to the production of oxidative and neuroactive mediators that may influence behavior. For instance, inflammatory cytokines in the CNS upregulate the enzyme IDO [29,30], which metabolizes tryptophan (TRP) into L-kynurenine (KYN) [31]. TRP degradation to KYN can reduce TRP levels that are required for serotonin synthesis [32] and can lead to the production of neuroactive mediators including 3-hydroxykynurenine (3HK) and quinolinic acid (QUIN) [31]. High levels of 3HK and QUIN induce neuronal damage through oxidative stress [33] and over stimulation of N-methyl-D-aspartate (NMDA) receptors [34,35]. A recent study indicates that while several cell types in the CNS express IDO, only microglia maintain all the enzymes required to produce 3HK and QUIN [36]. Because IDO mediated TRP degradation impacts both serotonergic and glutamatergic pathways, this may be an important mechanism underlying mood and behavior complications concomitant with inflammation [37-39].\nBecause activated microglia are suspected to cause or exacerbate several neurodegenerative diseases, pharmacological strategies to suppress microglial activity are being explored as therapies. Minocycline is a tetracycline derived antibiotic that has anti-inflammatory properties in the CNS that are separate from its antimicrobial action [40]. Minocycline readily crosses the blood brain barrier and attenuates inflammation associated with microglial activation. For example, minocycline blocks the deleterious effects of neuroinflammation on neurogenesis, long-term potentiation, and neuronal survival [41-43]. The mechanism of action is unclear, but recent studies indicate that minocycline abrogates MAPkinase and NFκB dependent signaling pathways in primary microglia and microglia cell cultures [44]. Moreover, in the brain of rats, minocycline abrogates microglial expression of CD11b and MHC II through a protein kinase-c dependent mechanism [45]. This is relevant because minocycline attenuates neuroinflammation in several rodent models of disease including Amyotrophic Lateral Sclerosis [46], Experimental Autoimmune Encephalomyelitis (EAE) [45] and MPTP-induced Parkinson's disease [47]. However, the extent to which minocycline facilitates the recovery from cytokine-mediated sickness behavior is unknown.\nThe present study investigated the degree to which minocycline–an anti-inflammatory agent and purported microglial inhibitor–reduced LPS-induced neuroinflammation and sickness behavior. We show that minocycline blocked LPS-stimulated inflammatory cytokine secretion in the BV-2 microglia-derived cell line and reduced LPS-induced Toll-like-receptor-2 (TLR2) surface expression on brain microglia. Moreover, our data show that minocycline pretreatment attenuated LPS-induced weight loss, social withdrawal, and anhedonia in adult mice. The attenuation of sickness behavior was paralleled with minocycline dependent decrease in markers of neuroinflammation (IL-1β, TLR2, and IDO) in adult and aged mice. These findings support our hypothesis that the ability to mitigate cytokine expression in the brain during systemic inflammatory events may be useful in preventing cognitive and behavioral deficits.\n\nMethods\nAnimals\nMale BALB/c mice, adults (3 month old) and juvenile (3–4 week old) were purchased from Harlan (Indianapolis, IN). For age comparisons, male BALB/c mice (3–4 and 20–22 month old) were purchased from the National Institute on Aging specific pathogen free colony. Upon arrival, mice were individually housed in polypropylene cages and maintained at 21°C under a 12 h light: 12 h dark cycle with ad libitum access to water and rodent chow. At the end of each study, mice were examined postmortem for gross signs of disease (e.g., splenomeglia or tumors). Data from mice determined to be unhealthy were excluded from analysis (< 5%). All procedures were in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee.\n\nCell culture\nBV-2 microglia cell lines were cultured in growth medium (DMEM supplemented with 10% FBS, sodium bicarbonate 3.7 g/l, 200 mM glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin, 0.25 μg/ml fungizone) as previously described [12]. Cultures were maintained at 37°C with 95% humidity and 5% CO2 and growth medium was replenished every third day until confluence. Cultures were washed twice and supplemented with warm growth medium containing experimental treatments. Cell viability was measured by the MTS cell proliferation assay according to the manufacturer's instructions (Promega, Madison, WI).\n\nCNS macrophage/microglia isolation\nCNS macrophages and microglia were collected from whole brain homogenates as described previously [48], but with several modifications. Mice were euthanized by CO2 asphyxiation and whole brains were collected. Brains were homogenized in Hank's balanced salt solution (HBSS) pH 7.4. Brain homogenates were passed through a 70 μm nylon cell strainer and centrifuged at 400 × g for 10 min. Supernatants were removed and cell pellets were re-suspended in 70% isotonic Percoll (GE-healthcare, Uppsala, Sweden) at room temperature. A discontinuous Percoll density gradient was set up as follows: 70%, 35%, and 0% isotonic Percoll. This suspension was centrifuged for 30 minutes at 400 × g. A mixed population of CNS macrophages and microglia was collected from the interphase between the 70% and 35% Percoll layers. Cells were washed and then re-suspended in sterile HBSS. The number of viable cells was determined using a hemacytometer and 0.2% trypan blue staining.\n\nFlow cytometry\nFlow cytometric analysis of microglial cell surface markers was performed as described previously, but with a few modifications [48]. In brief, Fc receptors on macrophages and microglia were blocked with anti-CD16/CD32 antibody (eBiosciences, CA). Next, cells were incubated with either Panel-1 (anti-CD11b APC, anti-CD45 FITC, and anti-MHC II PE from eBiosciences, CA) or Panel-2 antibodies (anti-CD11b APC, anti-CD45 FITC, and anti-TLR2 PE from eBiosciences, CA). Expression of these surface receptors was determined by flow cytometry using a Becton-Dickinson FACSCaliber four color Cytometer. Thirty thousand events were collected and microglia were differentiated from macrophages based on the levels of CD11b and CD45 surface expression. Microglia stain CD11b+/CD45low and macrophages stain CD11b+/CD45high [48,49]. Flow data were analyzed using FlowJo software (Tree Star, San Carlos, CA).\n\nBehavior tests\nSocial exploratory behavior\nTo assess the motivation to engage in social exploratory behavior, a novel juvenile conspecific was introduced into the test subject's home cage for a 10-min period. Behavior was video taped and the cumulative amount of time the subject engaged in social investigation was determined from the video records by a trained observer who was blind to the experimental treatments. Baseline social behavior was measured at time 0 for all experimental treatments. Social behavior was determined as the amount of time that the experimental subject spent investigating (e.g., anogenital sniffing, trailing) the juvenile. Results are expressed as percent decrease in time engaged in social behavior compared to respective baseline measures.\n\nSucrose preference\nTo assess sucrose preference, mice were provided two solutions, water or water supplemented with 2% sucrose, in 50 ml conical tubes with stoppers fitted with ball-type sipper tubes. Prior to testing conditions, all mice were acclimated to the two bottle test choice. All mice drank both the water and the 2% sucrose solution, but preferred drinking the sucrose over the water (data not shown). On the day of testing, mice were fluid and food deprived for 2 h prior to testing [50]. At the start of the dark phase of the photoperiod, drinking water and the 2% sucrose solution were placed in the home cage overnight (15 h). At the end of each testing period the fluid content of the conical tubes was measured and sucrose preference was determined using the equation: Sucrose intake/Total fluid intake (water + sucrose intake) × 100 [51].\n\n\nPlasma cytokine measurement\nIL-6 and IL-1β were measured in the plasma as previously described [52]. In brief, mice were euthanized by CO2 asphyxiation and blood was collected by cardiac puncture into EDTA coated syringes. Samples were centrifuged (6000 × g for 15 min at 4°C) and plasma was collected and stored frozen (-80°C) until assaying. Plasma samples were assayed for IL-6 using a customized ELISA that we have described in detail [52] and for IL-1β using a commercial ELISA kit (R&D Systems, Minneapolis, MN). Assays were sensitive to 8 pg/ml of IL-6 and 1.5 pg/ml of IL-1β, and inter- and intra-assay coefficients of variation were less than 10%.\n\nReal time PCR\nTotal RNA was isolated from brain using the Tri Reagent protocol (Sigma, St. Louis, MO). RNA samples were subjected to a DNase I digestion procedure and then reverse transcribed to cDNA using a RT RETROscript kit (Ambion, Austin, TX). Quantitative real time PCR was performed using the Applied Biosystems (Foster, CA) Assay-on Demand Gene Expression protocol as previously described [13]. In brief, cDNA was amplified by real time PCR where a target cDNA (IL-1β, IL-6, MHC II, TLR2, or IDO) and a reference cDNA (glyceraldehyde-3-phosphate dehydrogenase) were amplified simultaneously using an oligonucleotide probe with a 5' fluorescent reporter dye (6-FAM) and a 3' quencher dye (NFQ). Fluorescence was determined on an ABI PRISM 7300-sequence detection system (Applied Biosystems, CA). Data were analyzed using the comparative threshold cycle (Ct) method and results are expressed as fold difference.\n\nExperimental protocols\nFor the cell culture studies, minocycline was prepared in dimethyl sulfoxide (DMSO) and BV-2 cells were washed and replenished with growth mediumsupplemented with 0, 25, 50, 100, 200, or 400 μg/ml minocycline. After 30 min, LPS at 10 ng/ml was added to the culture medium. Supernatants were collected 4 h later and IL-6 and IL-1β concentrations were determined by ELISA. Total proteins were determined from cell culture homogenates by the Bio-Rad Dc protein assay according to the manufacturer's instructions (Bio-Rad Lboratories, Hercules, CA). Each treatment was replicated a minimum of four times. Cell viability was confirmed by the MTS cell proliferation assay according to the manufacturer's instructions (Promega, Madison, WI).\nFor all mouse studies, minocycline (Sigma, St. Louis, MO) was dissolved in sterile water and sonicated to ensure complete solubilization. In the first mouse study, adult male BALB/c mice received an intraperitoneal (i.p.) injection of vehicle or minocycline (50 mg/kg) for three consecutive days. On the 3rd day, mice were also injected i.p. with saline or Escherichia coli LPS (0.33 mg/kg; serotype 0127:B8, Sigma, St. Louis, MO) and were euthanized by CO2 asphyxiation 24 h later (n = 6). The LPS dosage was selected because it elicits a proinflammatory cytokine response in the brain resulting in mild transient sickness behavior in adult mice [13,53]. Macrophage/microglial cells were isolated from whole brain homogenates and TLR2 and MHC II surface expression were determined by flow cytometry. The minocycline injection regimen and dosage was selected because a repeated pretreatment course with minocycline is necessary to attenuate neuroinflammation [41-43,45].\nIn the second study, adult male BALB/c mice received an i.p. injection with vehicle or minocycline for three consecutive days. On the third day, motivation to engage in social behavior was determined immediately before i.p. injection of saline or LPS (0.33 mg/kg) and again 2, 4, 8, 12, and 24 h later (n = 8). Body weight and food intake were measured at each time point over the 24 h period. In a related, but separate study, adult mice were treated with minocycline and LPS as described and anhedonia was assessed 24–39 h following i.p. injection of saline or LPS (0.33 mg/kg) (n = 15). Body weight, food intake, water intake, and sucrose intake were determined over the testing period.\nIn the third study, adult BALB/c mice were treated with minocycline and then LPS as described. Mice were euthanized by CO2 asphyxiation 4 later. Brains were removed and dissected to collect different brain regions. Brain regions were stored at -20°C in RNAlater (Qiagen, CA). Total RNA was isolated from brain samples and assayed using quantitative PCR (n = 8). Plasma was also collected and stored (-80°C) until assaying.\nIn a final study, adult (3–4 month old) or aged (20–22 month old) male BALB/c mice were treated with minocycline and LPS as described and euthanized 4 h later. Brains were dissected to collect different brain regions and were stored at -20°C in RNAlater (Qiagen, CA). Total RNA was isolated from the hippocampus and assayed using quantitative PCR (n = 8).\n\nStatistical analysis\nAll data were analyzed using Statistical Analysis Systems (SAS) General Linear Model procedures. Data were subjected to one, two- (Mino × LPS, Age × LPS, Mino × Age) or three-way (Mino × LPS × Time, Mino × LPS × Age) ANOVA to determine significant main effects and interactions between main factors. When appropriate, differences between treatment means were evaluated by an F-protected t-test using the Least-Significant Difference procedure of SAS. All data are expressed as treatment means ± standard error of the mean (SEM).\n\n\nResults\nMinocycline attenuates LPS-induced cytokine production in BV-2 microglia\nMinocycline is a tetracycline-type antibiotic that has anti-inflammatory properties in the CNS [41-43,45]. To determine the degree to which minocycline suppresses microglia activation, BV-2 microglia-derived cell lines were used. In the first experiment, BV-2 cells were treated with LPS and IL-6 production was determined 4 h later. Fig. 1A shows that LPS increased IL-6 production in a dose dependent manner F(5, 23) = 101, P < 0.001). In the second experiment, BV-2 cells were incubated with DMSO or minocycline and then stimulated with LPS. Minocycline reduced LPS-induced IL-6 secretion in a dose dependent manner (Mino × LPS interaction, F(4, 23) = 16.87, P < 0.001, Fig. 1B). Minocycline pretreatment had a similar anti-inflammatory effect on LPS-stimulated IL-1β secretion (Fig. 1C). In a third experiment, minocycline suppressed LPS-induced MHC II, TLR2, IL-1β, and IL-6 mRNA levels (P < 0.05, for each, Fig. 1D). The MTS assay verified that neither cell survival nor proliferation was affected by the experimental treatments (data not shown).\n\nLPS-induced TLR2 surface expression on microglia is reduced by minocycline\nBecause minocycline attenuated LPS-induced cytokine secretion and TLR2 mRNA expression in BV-2 cells we next sought to determine if minocycline suppresses markers of microglial activation in the brain of mice. Mice were injected i.p. with vehicle or minocycline for 3 consecutive days then challenged with saline or LPS i.p. Markers of activation, TLR2 and MHC II, were determined on microglia collected 24 h later. The representative bivariate density plot in Fig. 2A shows that there were two populations of CD11b/CD45 positive cells and that more cells stained CD11b+/CD45low (microglia) than CD11b+/CD45high (CNS macrophages). ANOVA revealed that LPS injection increased TLR2 surface expression on microglia (F(1, 20) = 17.6, P < 0.004, Fig. 2B&D), but this induction was abrogated by minocycline pretreatment (Tendency for Mino × LPS interaction, F(1, 20) = 2.66, P = 0.10, Fig. 2C&D). It is important to note that because minocycline and saline controls did not differ in their TLR2 expression, these data were grouped together as the Control group (Fig. 2B&C). In addition, neither minocycline nor LPS treatment had a significant main effect on MHC class II surface expression on microglia (data not shown). These data indicate that minocycline attenuated LPS-induced TLR2 expression on microglia.\n\nMinocycline facilitates the recovery from LPS-induced sickness behavior\nCNS macrophages and microglia produce inflammatory cytokines and secondary messengers that modulate behavioral responses. Therefore, we next investigated if minocycline reduced the sickness response associated with peripheral LPS injection. In this experiment, adult mice were treated with minocycline and LPS as described. Social exploratory behavior was measured before i.p. LPS injection and again 2, 4, 8, and 24 h later. Fig. 3A shows that LPS injection caused a reduction in social exploratory behavior (F(1,57) = 218, P < 0.001) that was time dependent (F(4,57) = 66.5, P < 0.001). Moreover, the LPS-associated reduction in social exploration was attenuated by minocycline (Mino × LPS interaction, F(1,57) = 7.5, P < 0.007). For example, at 8 h post LPS, social exploration was reduced by 35% in minocycline pretreated mice given LPS compared to a 67% reduction in vehicle pretreated mice given LPS (P < 0.001). While minocycline administration alone reduced food intake and body weight in control mice (P < 0.05, for each), it also protected against LPS-associated anorexia (Mino × LPS interaction, F(1, 60) = 70.0, P < 0.001, Fig. 3B) and weight loss (Mino × LPS interaction, F(1, 60) = 29.7, P < 0.001, Fig. 3C).\nBecause sickness can also be associated with longer lasting changes in motivation [38], we next sought to determine if minocycline abrogated LPS-induced anhedonia [54,55]. In this experiment, mice were subjected to the same minocycline injection regimen and LPS challenge as above and sucrose preference was assessed 24–39 h post LPS injection. By 24 h post LPS injection, food and water intake returned to baseline and LPS treated mice still exhibited a marked reduction in sucrose preference from 24–39 h (F(1,59) = 14.3, P < 0.003). Moreover, this LPS-dependent reduction in sucrose preference was prevented by minocycline pretreatment (Mino × LPS interaction, F(1, 59) = 9.9, P < 0.004, Fig. 4). For example, minocycline pretreated mice injected with LPS maintained the same strong preference for sucrose as saline and minocycline controls (i.e., approximately 85% preference). These data can be interpreted to indicate that minocycline blocks anhedonia associated with peripheral LPS challenge.\n\nMinocycline reduces LPS-induced neuroinflammation\nPro-inflammatory cytokines in the CNS are partially responsible for the behavioral symptoms of sickness (e.g., anorexia, social withdrawal, and anhedonia) [1]. Therefore, we investigated the degree to which minocycline reduces neuroinflammation (IL-1β, IL-6, and IDO) after peripheral injection of LPS. In this experiment, mice were subjected to the minocycline injection regimen and LPS challenge as above and cytokine mRNA levels were determined in the cortex and hippocampus 4 h after LPS injection. In mice pretreated with vehicle, LPS markedly increased IL-1β mRNA levels in the hippocampus (F(1,31) = 62, P < 0.0001) and cortex (F(1,31) = 17.25, P < 0.0003). The LPS-induced IL-1β mRNA expression was reduced in both brain regions in mice receiving minocycline prior to LPS injection: (hippocampus, F(1,31) = 9.63, P < 0.01) and cortex, F(1,31) = 7.23, P = 0.08, Fig. 5A). LPS caused a similar induction of IL-6 mRNA levels in the hippocampus (F(1,31) = 37.2, P < 0.001) and cortex (F(1,31) = 22.5, P < 0.001), but minocycline pretreatment only significantly attenuated LPS-induced IL-6 mRNA levels in the hippocampus (F(1,31) = 10.27, P < 0.004, Fig. 5B).\nIDO mRNA levels were determined from the same RNA pool. Fig. 6D shows that LPS injection increased IDO mRNA expression in the hippocampus (F(1,31) = 11.69, P < 0.002) and cortex (F(1,31) = 5.26, P < 0.03). This LPS-induced IDO mRNA expression was attenuated by minocycline in the hippocampus (F(1,31) = 11.69, P < 0.002) and cortex (F(1,31) = 5.26, P < 0.03). It is important to note that IDO mRNA was undetected in saline treated mice. Therefore, the fold IDO change was relative to the IDO mRNA levels in mice receiving minocycline prior to LPS.\n\nMinocycline reduces LPS-induced IL-6, but not IL-1β, in the plasma\nBecause cytokine signals can be relayed from the periphery to the brain by humoral pathways [56], plasma cytokine levels of IL-6 and IL-1β were determined 4 h post LPS injection. As expected, LPS injection caused a marked increase in plasma IL-1β (F(1,36) = 52.5, P < 0.001) and IL-6 levels (F(1,36) 34.01, P < 0.01). Minocycline pretreatment reduced LPS-induced IL-6 levels in the plasma (F(1,36) 6.68, P < 0.01) but had no significant main effect on LPS-induced IL-1β levels (Fig. 6).\n\nMinocycline attenuates LPS-induced exaggerated neuroinflammation in aged mice\nAged BALB/c mice (22–24 m) have an exaggerated neuroinflammatory response to LPS injection [10,13,14]. Therefore, we next sought to determine if the heightened inflammatory response in the brain of aged mice was reduced by minocycline. In this experiment, adult and aged mice were subjected to the minocycline injection regimen and LPS challenge as above. As we have reported previously, MHC II mRNA expression was increased by age (P < 0.03, Fig. 7A)[13,14], but MHC II levels were unaffected by either LPS or minocycline treatment (not shown). Consistent with the data presented in Fig. 2, ANOVA revealed a significant main effect of LPS injection on TLR2 mRNA expression in the hippocampus (F(1,63) = 85.5, P < 0.001). Moreover, LPS caused a greater increase in TLR2 mRNA in the hippocampus of aged mice compared to adults (LPS × Age interaction, F(1,63) = 12.70, P < 0.01). Furthermore, minocycline pretreatment attenuated LPS-induced TLR2 mRNA levels in both adult and aged mice (Mino × LPS interaction, F(1,63) = 9.02, P < 0.004).\nParallel to the results for TLR2, LPS caused a greater increase in IL-1β and IDO mRNA levels in hippocampus of aged mice compared to adults (Age × LPS, F(1,60) = 8.64, P < 0.01 for IL-1β and F(1,60) = 4.0, P < 0.05 for IDO). Minocycline pretreatment attenuated LPS-induced mRNA levels of IL-1β (Mino × LPS, F(1,60) = 8.76, P < 0.01, Fig. 7C) and IDO (Mino × LPS, F(1,60) = 9.7, P < 0.003, Fig. 7D). While LPS induced higher IL-6 mRNA levels in the hippocampus of both adult and aged mice (F(1,59) = 44.5, P < 0.001), there was not an Age × LPS interaction. Minocycline pretreatment attenuated the LPS-induced increase in hippocampal IL-6 mRNA (Mino × LPS, F(1,59) = 5.4, P < 0.02, Fig. 7E). Taken together these data indicate that minocycline pretreatment was effective in attenuating the exaggerated neuroinflammation in aged mice.\n\n\nDiscussion\nIn the elderly, systemic infection is associated with an increased frequency of behavioral and cognitive complications [57,58]. We have reported that stimulation of the peripheral immune system in older (20–24 m) BALB/c mice causes exaggerated neuroinflammation that is paralleled by prolonged sickness [13], impaired working memory [10], and depressive-like behaviors [15]. Therefore, it is important to understand the mechanisms that can modulate cytokine-mediated pathways in the brain. Here we show that minocycline treatment reduced LPS-induced TLR2 expression in BV-2 cells and on microglia isolated from adult mice. Moreover, we demonstrate that minocycline was effective in facilitating the recovery from LPS-induced sickness and preventing anhedonia in adult mice. Furthermore, we show that minocycline attenuated LPS-induced neuroinflammation in adults and normalized the exaggerated neuroinflammation in aged mice.\nOur findings, using cell culture and animal experiments, support the notion that minocycline attenuates microglial activation and limits production of inflammatory mediators. For instance, minocycline pretreatment of BV-2 cultures decreased LPS-stimulated cytokine production in a dose dependent manner (Fig. 1A). In BV-2 cells, minocycline also attenuated mRNA expression of inflammatory genes including IL-6, IL-1β, MHC II, and TLR2 (Fig. 1D). These data are consistent with other studies using minocycline and LPS in BV-2 cells [44,59]. Based on these data we next investigated if microglial activation could be attenuated in the brain. Because LPS increases brain cytokine production we expected that MHC II expression would also be increased. Contrary to our predictions, neither MHC II mRNA levels (Fig. 7) in the brain nor MHC II surface expression on microglia (CD11b+/CD45low) (data not shown) were increased by LPS injection. In an EAE model, minocycline reduced microglial expression of MHC II [45], but one key difference from our study is that the induction of EAE pathology requires functional antigen presentation on MHC II [60]. It is postulated that microglia have several activation states that depend on the specific inflammatory stimulus [61]. Thus, in situations of transient peripheral innate immune stimulation, markers in the CNS such as Toll-Like receptors [6] may be indicative of microglia activation. In support of this premise, our data show that LPS injection increases TLR2 surface expression on microglia (CD11b+/CD45low), which is inhibited by minocycline pretreatment (Fig. 2). These data are consistent with other studies showing that central or peripheral LPS challenge increases TLR2 mRNA in the brain [6,14]. Taken together our findings can be interpreted to suggest that minocycline attenuates pathways associated with microglia activation following peripheral LPS challenge.\nOne of the important findings of this study was that reduction of neuroinflammation by minocycline was associated with facilitated recovery from LPS-induced sickness behavior. These results are akin to our previous work with the anti-oxidant, α-tocopherol [52], and an NFKB decoy inhibitor [62]. Consistent with our previous studies [52,53,62,63], reductions in neuroinflammatory cytokines (Fig. 5) did not prevent the induction of the LPS-induced sickness response (2–4 h), but rather facilitated the recovery from sickness (8–24 h) (Fig. 3A). Recovery may be a critical issue because brain cytokines and the corresponding physiological and behavioral responses are beneficial to the host [1]. The potential risk for a maladaptive response occurs when the normally transient neuroinflammatory response is amplified or protracted [64]. Therefore pharmacological agents, similar to minocycline, that attenuate neuroinflammatory responses, but do not completely inhibit them, may be important in preventing the development of more severe and long-lasting cognitive and behavioral complications.\nThe results of the sucrose preference experiments support the idea that limiting exposure to neuroinflammation decreases the duration of behavioral responses. For example, while minocycline did not inhibit cytokine expression (Fig. 5) or the induction of sickness (Fig. 3A), minocycline pretreatment completely reversed the reduction in sucrose preference (i.e., anhedonia) associated with LPS injection (Fig. 4). It is also important to mention that while LPS-associated sickness and anhedonia are interrelated, these behaviors can be differentiated from one another. For instance, reduced social exploration was evident 2–24 h post injection (Fig. 3A), but only decreased sucrose preference was exhibited 24 to 39 h later (Fig. 4). This separation between behaviors is consistent with other studies investigating sickness and longer-lasting changes in motivation [15,65,66].\nIDO mediated TRP metabolism represents a potential connection between activation of CNS innate immune cells and longer lasting behavioral responses. IDO mediated TRP metabolism in the brain may affect behavior by impacting both serotonin and glutamate pathways [39]. We have reported that IDO induction and activity is amplified in the brain of aged mice and is associated with prolonged depressive-like behavior [15]. Here we show that IDO mRNA induction is blocked by minocycline in the brain of both adult and aged mice (Figs. 5&7). These data are consistent with a recent report showing a causal relationship between IDO activity and acute depressive effects in adult CD-1 mice. In this study, O'Connor et al. report that both 1-methyl tryptophan (a competitive inhibitor of IDO) and minocycline blocked IDO induction and prevented depressive-like immobility in the tail suspension and forced swimming tests [66]. Thus, in the present study, the minocycline blockade of IDO induction may explain the abrogation of LPS-induced anhedonia.\nAnother interesting finding was that while minocycline pretreatment in adult mice attenuated LPS-induced brain IL-1β at 4 h (Fig. 5), it had no effect on plasma IL-1β levels (Fig. 6). Because IL-1β signals can be relayed from the periphery to the brain by humoral pathways [5], these findings suggest that minocycline has anti-inflammatory properties within the brain. These data are consistent with related findings that minocycline readily crosses the blood brain barrier to elicit an anti-inflammatory effect [41-43]. With regard to IL-6, minocycline pretreatment attenuated both brain and plasma levels at 4 h post LPS injection. Because circulating IL-6 levels can be increased by CNS mediated pathways including activation of the hypothalamus-pituitary-adrenal (HPA) axis [67] and the sympathetic nervous system [68], the specific reduction in plasma IL-6 by minocycline may reflect the reduction in brain inflammation at 4 h (Fig. 5). In support of this notion, we and others have reported that i.c.v. injection of LPS or IL-1β increase plasma IL-6 levels, but not IL-1β levels [14,68,69].\nThe final critical finding of this study was that minocycline was effective in attenuating neuroinflammation independent of age. Consistent with other aging and neuroinflammation studies, our data show that LPS caused exaggerated neuroinflammation in aged mice compared to adults [10,13-15]. It is important to mention that while there was an age-related difference in MHC II expression in the hippocampus of saline treated mice (Fig. 7A) there was not an age-related difference in IL-1β and IL-6 mRNA levels. These data differ from a previous report using BALB/c mice showing an increase in IL-6 in older mice [70]. This may be because the mice used in the present study were approximately 4 months younger than the mice used previously. Nonetheless, microglia can be primed or reactive with increased MHC II expression, but do not necessarily produce inflammatory cytokines in this state [19]. The key results are that peripheral LPS injection causes a greater induction of TLR2, IL-1β, and IDO mRNA in the aged brain than in the adult brain and that minocycline pretreatment normalizes this age-related exaggerated neuroinflammation (Fig. 7). These findings are also important because an amplified neuroinflammatory response in the aged brain is a precursor to complications including deficits in working memory, memory consolidation, and depressive-like behavior [9,10,15]. Based on the biochemical and behavioral data obtained from this study, we predict that minocycline will abrogate the prolonged LPS-induced sickness [13] and depressive-like behavior exhibited by aged BALB/c mice [15]. We acknowledge, however, that future studies are necessary to test these predictions.\n\nConclusion\nThe present study demonstrates that minocycline reduces LPS-induced microglial activation, CNS cytokine production, and behavioral symptoms of sickness (e.g., social withdrawal and anhedonia). These findings are potentially important because they indicate that minocycline can be used to mitigate cytokine expression in the brain and have a beneficial affect on behavioral responses. Taken together, these data support the idea that pharmacological strategies aimed at decreasing neuroinflammation associated with microglial activation are important for improving recovery from sickness and reducing the frequency of neurobehavioral complications.\n\nList of abbreviations\n3-Hydroxy-L-Kynuriene (3HK), Allophycocyanin (APC), Analysis of variance (ANOVA), Central Nervous System (CNS), Dulbecco's Modified Eagle's Medium (DMEM), Dimethyl Sulfoxide (DMSO), Experimental Autoimmune Encephalomyelitis (EAE), Enzyme Linked Immmunosorbent Assay (ELISA), Fluorescein Isothiocyanate (FITC), Fetal Bovine Serum (FBS), Hank's Balanced Salt Solution (HBSS), Indoleamine 2, 3 dioxygenase (IDO), Intraperitoneal (i.p.), Intracerebroventricular (i.c.v.), Interleukin (IL), Kynurenine (KYN), Lipopolysaccharide (LPS), Major Histocompatibility Complex class II (MHC II), Mitogen Activated Protein Kinase (MAP-kinase), Nuclear factor kappa B (NFκB), N-methyl-D-aspartate (NMDA), R-Phycoerthrin (PE), Quinolinic acid (QUIN), Statistical Analysis Systems (SAS), Standard Error of the Mean (SEM), Toll-like Receptor-2 (TLR2), and Tryptophan (TRP).\n\nCompeting interests\nThe authors of this manuscript declare that there are no actual or potential conflicts of interest. The authors affirm that there are no financial, personal or other relationships with other people or organizations that have inappropriately influenced or biased their research.\n\nAuthors' contributions\nCJH was involved in research experimentation, completion of statistical analysis, and writing of the manuscript. YH, AW, MH and JH assisted with experimentation and data analysis. MB and JFS contributed to the design of the experiments and assisted in editing the manuscript. JPG directed all aspects of this research project including the experimental design, research experimentation, completion of statistical analysis, and writing of the manuscript.\n\n\n" ], "offsets": [ [ 0, 38238 ] ] } ]

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[ { "id": "pmcA1190194__text", "type": "Article", "text": [ "Structural organization and interactions of transmembrane domains in tetraspanin proteins\nAbstract\nBackground\nProteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed.\n\nResults\nAmong 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)n. In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80.\n\nConclusion\nOur results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that defines TM1-TM2 interaction in tetraspanins is the specific packing of bulky residues against small residues.\n\n\n\nBackground\nTetraspanins constitute a large family of integral membrane proteins, characteristically containing 4, 6 or 8 conserved cysteine residues in the large extracellular loop (including the CCG and PxxCC motifs), which form disulfide bonds, and several conserved polar residues in the intracellular loop and transmembrane regions [1,2]. There are 32 putative tetraspanin family members in mammals, 37 in Drosophila melanogaster and 20 in Caenorhabditis elegans. Tetraspanins play diverse roles in cell adhesion, migration and fusion processes, cellular activation and signaling (reviewed in refs. [2-4]). Mammalian tetraspanins such as CD9, CD63, CD81, CD82, CD151, rds/peripherin, and uroplakins Ia and Ib have been most extensively studied, with mouse knock-out models available for CD9 [5-7], CD81 [8,9], CD151 [10] and a few others. However, the majority of tetraspanins are characterized very little, if at all, at genetic, biochemical or structural levels.\nThe large extracellular loop (LEL) of tetraspanins has received most attention, since it contains functionally important sites. Sequence QRD (194–196) in CD151 is important for association with integrins, which has functional consequences for integrin-dependent cell spreading and multicellular cable formation [11]. A site in the LEL of CD9, SFQ (residues 173–175), is essential for CD9 function in sperm-egg fusion [12]. The crystal structure of tetraspanin CD81 LEL revealed five α-helixes, A-E [13]. Helices A, B and E form a relatively conserved region in tetraspanins, whereas the region between helices B and E is the most variable [14]. Interestingly, the variable region contains most of the functionally important sites involved in tetraspanin protein-protein interactions.\nA remarkable biochemical property of tetraspanin molecules is their ability to associate with a large number of other transmembrane proteins, including integrins, membrane-associated growth factors and receptors, MHC class II molecules, Ig superfamily proteins, and each other [2,3,15]. Several of these lateral associations of tetraspanins are detected in \"mild\" detergents (Brij series, CHAPS), but are disrupted by \"strong\" detergents such as Triton X-100 or SDS. Multiprotein complexes of tetraspanins and associated molecules, also called the \"tetraspanin web\" [16], may represent a distinct tetraspanin-enriched membrane microdomain [17,18]. The formation of this microdomain is influenced by palmitoylation of several conserved juxtamembrane cysteine residues in tetraspanins [19-21].\nThe transmembrane domains, encompassing nearly half of a tetraspanin protein, are the most conserved part of the molecule (Stipp et al. [1] and this study). However, very little functional information is available on these domains. The differential detergent sensitivity of tetraspanin-tetraspanin associations suggests that hydrophobic interactions between TM helices may play a role. Indeed, when the large extracellular loop (LEL) of CD151 is deleted, the molecule is still able to associate with other tetraspanins [22]. Thus, TM domains are strong candidates for mediating tetraspanin-tetraspanin interactions.\nThe importance of TM domain interactions in intramolecular organization was demonstrated in a study showing that CD82 fragment TM2-4, lacking TM1, was retained in the endoplasmic reticulum, but was transported to the cell surface upon co-expression of TM1 [23]. This in vivo reconstitution experiment demonstrated a strong interaction between TM1 and the rest of the molecule. Expression of a truncated CD9 molecule (TM3-LEL-TM4) results in intracellular accumulation of the protein and significant misfolding of the LEL, as judged by inappropriate disulfide formation and diminished antibody reactivity (our unpublished data). Similarly, a CD9 epitope in the LEL is lost in molecules lacking either TM2+TM3 or just TM4 [24]. Thus, TM domain interactions and packing are crucial for proper folding, stability and transport of tetraspanin molecules.\nIn a previous study, we showed that covalent cross-linking of membrane-proximal cysteine residues can be used as a tool for detection of tetraspanin-tetraspanin associations [25]. Inhibition of cysteine palmitoylation by 2-bromopalmitate (2-BP) made cysteines available for cross-linking and enabled demonstration of specific tetraspanin homodimerization and low levels of heterodimerization. We concluded that tetraspanin homodimers, formed in the Golgi, may be a fundamental structural unit within tetraspanin microdomains.\nIn this study, we carried out detailed sequence analysis of human tetraspanin TM domains. We show that a heptad repeat containing conserved glycine, asparagine and large hydrophobic residues occurs in TM1 and TM2 domains, and predict tight intramolecular association of these two domains by packing of the large residues against the small residues. Moreover, by using cysteine cross-linking we map a dimerization interface in the human CD9 protein, and show that conserved heptad motif glycine residues are also important for intermolecular CD9 associations.\n\nResults\nSequence analysis of tetraspanin transmembrane domains: presence of the heptad repeat motif\nWe focused our attention on 28 human tetraspanins identified from the SWISS-PROT and GenBank databases. All tetraspanins have in common four hydrophobic stretches (TM domains) of 20–25 residues, and contain highly conserved residues in the second extracellular loop, in particular the Cys-Cys-Gly (CCG) motif. Detailed analysis of the large extracellular loop sequences [14], and dendrograms based on full-length alignment can be found in earlier studies [26,27]. The length of each transmembrane domain was established based on previous sequence analysis of tetraspanin sequences [27,28], and on annotations to the database entries. Manual adjustments based on sequence homology and hydrophobicity profiles were done to fully delineate the TM domains. The resulting lengths of TM domains were: TM1 – 23 residues; TM2 – 21 residues; TM3 – 25 residues; TM4 – 25 residues. Two more residues could be added onto the N-terminal part of TM2; however, relatively small sequence conservation of these residues among tetraspanins and occurrence of polar/charged side chains in some tetraspanins precluded us from doing so for the global alignment.\nFigures 1 and 2 show a multiple sequence alignment of four TM domains of 28 human tetraspanins. For each position within the domains, consensus residues were determined and classified (with individual color code) in 4 categories: 1) large hydrophobic residues (including Val, Met, Leu, Ile, Phe, Tyr, Trp), 2) small residues (Gly, Ala, Ser and Thr), 3) Cys, and 4) Asn. When more than two types of residues occupied a given position in a TM, a dual-color pattern that reflected the prevalence of the particular residue type was used (Figure 1). Cysteine residues were shown separately due to their importance as palmitoylation target sites. The highly conserved asparagine residue in TM1 was considered separately. No proline residues are found in TM domains 1–3 of human tetraspanins.\nAn inspection of the multiple sequence alignment reveals a repeating heptad amino acid pattern, (abcdefg)n, in TM1, 2 and 3 (Figure 1, 2). Heptad repeats promote helical coiled coil interactions in multiple soluble and membrane-spanning proteins [29-31]. In the heptad repeat, hydrophobic residues in positions a and d are of special importance, as they directly mediate interhelical contacts by creating a tight knobs-into-holes packing in the coiled coil structure [32]. For instance, in the leucine zipper of the yeast transcription factor GCN4, positions a and d contain Val and Leu residues, respectively, with an Asn residue in a single a position forming a hydrogen bond across the GCN4 dimer interface [33].\nIn TM1 of tetraspanins, highly conserved Asn, Gly and Gly residues (numbers 18, 25 and 32 in the CD9 sequence) appear at d positions of the heptad repeats, and residues 14, 21 and 28 are at a positions (Figure 1). In TM2, residues 67, 74 and 81 (consensus Gly, Gly and Ala, respectively) occupy a positions, whereas residues 63, 70 and 77 are at d positions. Another highly conserved glycine, Gly80, occupies the 3rd g position in TM2. In TM3, the conserved pattern consists of two leucine residues (Leu89 and Leu96) and a glutamate/glutamine residue (Glu/Gln103) in a positions (Figure 2). Two d positions are also conserved – Phe/Tyr92 and Ile/Val/Leu99. TM4 lacks a conserved heptad pattern and has only a single conserved position, Glu/Gln209 (with four exceptions). These features of TM1-4 of tetraspanins are displayed on helical wheel diagrams (Figure 3).\n\nAnalysis of TM1 sequences\nThe conserved Asn-Gly-Gly motif, occupying designated d positions of the heptad repeat, is the most prominent structural feature of TM1. We also compared sequences of CD9 orthologs from 10 different organisms (the most available for any tetraspanin) to gain further insight into conservation and variability of the TM1 sequence. As shown in Figure 4, positions a, d and g in TM1 are among the most conserved (0, 1 and 1 substitution, respectively), while interspecies variability tends to occur in other positions: b (5 substitutions), c (4 substitutions), e (4 substitutions) and f (4 substitutions). Thus, the positions typically involved in coiled coil interactions (a and d) are the most conserved.\nWhen residues of TM1 are plotted as a helical wheel, additional structural features are revealed (Figure 3). There are highly conserved aliphatic and aromatic residues in the first three a positions of the heptad motif (Phe15, Trp22 and Leu29 in CD9), as well as in g positions (Leu14, Phe21, Val28 in CD9). The \"ridges\" formed by these bulky residues are flanking the \"groove\"-forming Gly residues of the Asn-Gly-Gly position d motif. In contrast, b, c, e and f positions show an overall higher variability among tetraspanins, as also seen in the comparison of CD9 orthologs described above.\n\nAnalysis of TM2 sequences\nA landmark feature of TM2 in tetraspanins is the presence of highly conserved glycine residues (Gly67, 74, 77 and 80 in CD9, Figure 1). Other substitutions at these positions are almost exclusively small residues, such as Ala or Ser. In addition, Ala, Ser or Thr occupy position 81. This residue, together with Gly67 and Gly74, forms face a of the helix. Residue Gly77 (position d) is preceded by conserved, chiefly large hydrophobic residues on the same helical face (Leu63 and Met70 in CD9). Extremely conserved Gly80 falls into heptad position g (Figure 3). Among CD9 orthologs, heptad positions a and d are absolutely conserved, whereas other positions have the following number of substitutions: b – 3; c – 2; e – 1; f – 3; g – 1 (Figure 4). Two of the f position residues in TM2 (65 and 79) also show higher variability among different tetraspanins (Figures 1, 3). Cysteine residues are frequently found near the cytoplasmic end of TM2 helix at positions 78 and 79; these cysteines are likely to be palmitoylated.\n\nAnalysis of TM3 and TM4 sequences\nThe TM3 domain provides another example of the heptad repeat pattern. Position a is occupied by two highly conserved leucine and a glutamate/glutamine residue (Leu89, Leu96 and Glu/Gln103 in CD9). Furthermore, two d positions are conserved – Phe/Tyr92 (aromatic residue) and Ile/Val99 (β-branched aliphatic residue; Figures 2, 3). In addition, residue 100 in position e is generally Phe or Leu. Among CD9 orthologs, position a has 1 substitution, positions b, c and f each have 6, positions d and e each have 2, and g has 4. Thus, as for TM1 and TM2, positions a and d are among the most conserved, but overall TM3 has more variable positions than TM1 or TM2 (Figure 3). Less than half of TM3 sequences contain cysteine residues, and those tend to occur at the internal positions of the helix (Figure 2).\nTM4 shows less conservation among various tetraspanin family members than the other TM domains (Figures 2, 3). The only highly conserved feature is the glutamate/glutamine residue in position 209. In addition, one or two cysteine residues can be found at the C-terminal end of TM4 in some tetraspanins (e.g. CD9, CD81, CD151), and many sequences contain additional polar residues (Arg, Lys, His, Asn, Gln). No conserved heptad motif was identified in TM4, as also confirmed by analysis of substitutions in CD9 orthologs (data not shown).\n\nMutational analysis of conserved glycine residues in TM1 and TM2\nThe conserved nature of the Asn and Gly residues in TM1 and TM2 prompted an analysis of their functional role. To this end, we have probed whether mutations of these residues destabilize the protein molecule. We expressed a construct of the first and second TMs of CD9, connected by the small extracellular loop, and tagged with a C-terminal green fluorescent protein (TM(1+2)-GFP molecule). In human rhabdomyosarcoma RD cells, the wild-type fusion protein localized mostly in a reticular, intracellular pattern, without forming any large aggregates (Figure 5, panel A). Remarkably, when double mutants Gly25Leu + Gly32Leu and Gly67Leu + Gly74Leu were expressed, the protein formed distinct large aggregates in a high proportion of cells (Figure 5, panels C and E). In contrast, double mutant Gly77Leu + Gly80Leu did not form such aggregates (Figure 5, panel G). Results with respective single mutants were similar to that with double mutants, with the aggregation being somewhat more pronounced for Leu substitutions of Gly67 and Gly74 compared to Gly25 and Gly32 mutations. No aggregation was observed for Asn18Ser and Asn18Tyr mutants (data not shown). Also, nearly identical results were obtained with human HT1080 cells (data not shown).\nWe interpret these results as an indication that aggregating mutants are destabilized or misfolded while non-aggregating mutants retain the wild-type conformation. Intriguingly, mutations to the conserved GG7 motifs caused protein aggregation while the mutation of other glycines had no detectable effect. These results also suggest that wild-type GFP, which has weak tendency to self-associate, could enhance non-specific interactions of destabilized mutant TM(1+2) CD9 moieties, leading to their aggregation. Consistent with this hypothesis, the aggregation of Gly25Leu + Gly32Leu and Gly67Leu + Gly74Leu double mutants was suppressed when monomeric GFP molecule, Leu221Lys [34] was used (Figure 5, panels D and F). The use of monomeric GFP did not affect intercellular localization of wild-type CD9 TM(1+2) (Figure 5, panel B), or a Gly77Leu + Gly80Leu double mutant (Figure 5, panel H).\nIn summary, Leu substitutions of Gly residues that are part of the Asn-Gly-Gly (NGG7) motif in TM1, or Gly-Gly-Ala (GGA7) motif in TM2, resulted in destabilization and aggregation of GFP-fused TM(1+2) proteins, whereas substitutions of Gly77 or Gly80, which are not part of these motifs (Figure 3), failed to show such aggregation.\n\nPrediction and modelling of interaction between TM1 and TM2\nConsecutive helices in polytopic membrane proteins frequently interact [35]. Sequence analysis of TM1 and TM2 helices of tetraspanins reveals a remarkable complementarity in the distribution of large and small residues at heptad positions a and d along the helical axis (Figure 3), suggesting that these residues may interact. To further elucidate the potential for TM1-TM2 interaction, the putative interface was modeled using a novel algorithm that considers mutational data during each step of a Monte Carlo simulated annealing cycle (see Methods for details). Specifically, Gly25Leu, Gly32Leu, Gly67Leu and Gly74Leu were scored as disruptive mutations, while Asn18Ser, Gly77Leu and Gly80Leu were scored as silent mutations, based on their effects on protein stability (Figure 5 and data not shown).\nThe resulting model predicts left-handed crossing of TM1 and TM2 helices at an angle of +28°. The key element of the structure is the apposition of bulky and small heptad position a and d residues, as follows: Gly32-Leu63; Gly67-Leu29; Gly25-Met70; Gly74-Trp22; Asn18-Gly77; Ala81-Phe15 (Figure 6). Our model predicts that each of these residue pairs are in van der Waals contact. Additionally, two potential H-bonds are predicted in this model, indicating close packing: Gly67 Cα to Gly25 carbonyl oxygen, and Trp22 Cα to Met70 carbonyl oxygen. The packing is tighter in the ectodomain-proximal portion of the helices (Figure 6, panel B), as determined by Cα-Cα distances between interacting residue pairs.\nThe key elements of the model are corroborated by the presence of apparently complementary substitutions in TM1 and TM2 sequences of different tetraspanins (Figure 1, boxed residues). For example, Gly74 is predicted to interact with Trp22. In 8 of the 10 tetraspanins that contain a substitution for Gly74, a compensatory substitution occurs at the Trp22 position (Figure 1). Thus, a larger non-glycine side chain at position 74 may necessitate a less bulky non-Trp side chain in position 22. Likewise, the presence of a Cβ at position 25, typically occupied by glycine, necessitates a non-β-branched amino acid at position 70, which is occupied by a β-branched residue in nearly half of all cases. Indeed, we find that in each of 5 cases in which position 25 contains a Cβ, a leucine residue occurs in position 70. This analysis is consistent with our molecular model that suggests Leu70 will pack most favorably against a Cβ at position 25 than a β-branched residue or a methionine.\n\nRole of TM1 and TM2 heptad motif residues in CD9 dimerization\nTo probe CD9 dimerization, we used a cysteine-mediated cross-linking approach. We established previously a simple and efficient method for cysteine-mediated cross-linking [25]. After cells are pre-treated with 2-BP for 16–24 hours to expose normally palmitoylated cysteines, the cysteines can be cross-linked using any of the following methods: a) Spontaneous oxidation in Brij97 lysates (a condition that preserves tetraspanin-tetraspanin associations), b) In situ cross-linking, by pre-lysis oxidation of cells with Cu2+-phenanthroline (CuP) to promote disulfide bond formation. c) In situ cross-linking with thiol-reactive cross-linking agents of defined length (e.g. DTME, BMB). The first two approaches produce in essence \"zero-length\" disulfides, indicative of close proximity of target cysteines and presumably high specificity of interaction. In contrast, chemical cross-linkers with 6–20 Å spacer arm may cross-link with higher efficiency, but not necessarily higher specificity. However, they provide advantages such as variable membrane permeability, and potential linkage cleavability. For tetraspanins such as CD9, membrane-permeable cross-linker DTME (13.3 Å-long, reducible) provides highly specific and efficient cross-linking [25]. Here we have used a cysteine cross-linking strategy, in combination with cysteine-scanning mutagenesis, to map the residues from TM1 and TM2 contributing to the CD9 dimerization interface.\nFor subsequent cross-linking experiments using CD9 TM(1+2)-GFP protein, the non-dimerizing form of GFP was used. This avoids potential GFP-dependent dimerization and aggregation that can be observed with wild-type GFP, especially when fusions with transmembrane proteins are studied [36]. Importantly, the Leu221Lys mutation in GFP prevented aggregation of mutant forms of CD9 TM(1+2), which was observed with wild-type GFP fusion (Figure 5). The TM(1+2) fragment of CD9 contains three native cysteines – Cys9, Cys78 and Cys79. Single-cysteine mutants of TM(1+2) were constructed, in which a cysteine was placed at various faces of TM1 or TM2 while all of the wild-type cysteines were simultaneously replaced by serines. The mutant proteins were transiently expressed in RD cells (having little endogenous CD9), which were then treated for 16–18 hours with 2-BP. To achieve maximal specificity in cross-linking we used a \"zero-length\" agent, CuP.\nFirst, single-cysteine replacements were constructed for residues Leu14, Phe15, Gly16, Phe17 and Asn18, covering just over one complete helical turn at the beginning of TM1. While residue Asn18 is highly conserved, positions 14, 15 and 17 are occupied by bulky hydrophobic residues in most tetraspanins, whereas position 16 shows less conservation (Figures 1, 4). All of the single-cysteine mutants showed diffused pattern of protein localization, without any signs of aggregation. As shown in Figure 7A, the highest level of intermolecular cross-linking was observed for Leu14Cys and Phe17Cys mutants, a lower level for Phe15Cys and Gly16Cys mutants, and very little cross-linking for Asn18Cys substitution. These results indicate that: a) the first two transmembrane domains of CD9 alone can mediate its dimerization, and b) the g and c residues of TM1 (e.g. Leu14 and Phe17, Figure 3) are likely to be part of the intermolecular interface.\nSimilarly, single-cysteine substitutions were made for residues Gly77, Gly80 and Ala81 in TM2; in addition, proteins carrying a single wild-type cysteine, Cys9, Cys78 or Cys79, were tested. No protein aggregation was observed for any of these single-cysteine mutants. As shown in Figure 7B, the relatively low level of intermolecular cross-linking of wild-type CD9 TM(1+2)-GFP protein was enhanced dramatically in single-cysteine TM2 mutants Gly80Cys and Ala81Cys. The Gly77Cys mutant also had an elevated level of cross-linking. In contrast, any of the three native cysteines (9, 78 and 79) produced level of cross-linking not much greater than the wild-type TM(1+2) protein. Similar results were obtained with cysteine-reactive cross-linker BMB (data not shown). Likewise, comparable results were obtained with single-cysteine mutants of untagged, full-length CD9, using CuP (Figure 7C) as well as DTME cross-linker (data not shown).\nThese cross-linking results for TM1 and TM2 are consistent with our model that places residues Leu14, Phe17 and Gly80 on the same side of the TM1-TM2 pair (Figure 6, panel C). The strong cross-linking with Leu14Cys, Phe17Cys and Gly80Cys places the intermolecular interface toward the c and g phases of the TM1 helix, and the g phase of the TM2 helix, away from its e and f faces containing wild-type cysteines 78 and 79.\nCritical residues at the TM1-TM2 interface also affect dimerization indirectly. To assess specific CD9 dimerization, we used a Gly80Cys substitution at the intermolecular interface for cross-linking. As shown in Figure 8A, single replacements of conserved heptad residues in positions 18, 25, 32, 67 and 74 (Asn18Ser, Gly25/32/67/74→Leu) strongly decreased the cross-linking mediated by Cys80. The effect was most pronounced for mutations of residues, Gly32 and Gly67, located in the tightly packed extracellular end of TM helices (Figure 6). In contrast, mutations of residues closer to the cytoplasmic end of TM2 (Gly74 and especially Ala81) had only modest to very little effect on cross-linking.\nRelatively low efficiency of intermolecular cross-linking via native residues Cys9, 78, and 79 (Figures 7B,C) correlates well with the predicted location of Cys78 and 79 away from the dimeric interface (Figure 3), and suggests that the extramembrane N-terminal part of CD9 (residues 1–13) does not self-associate. We next examined whether mutations of conserved Asn and Gly residues in TM1 and TM2 decreased low-level background cross-linking via native cysteines. As expected, these mutations had virtually no effect on dimer formation of CD9 TM(1+2)-GFP (Figure 8B). The level of covalent dimer formed was not diminished for triple Asn18Ser + Gly25Leu + Gly32Leu and double Gly67Leu + Gly74Leu mutants, compared to wild-type TM(1+2) CD9 molecule. Similarly, the same triple and double mutations in the context of full-length CD9-GFP protein (with six cysteines) produced wild-type levels of cross-linking (Figure 8C). We interpret these findings as evidence for at least two types of associations between CD9 molecules: primary, involving residues 14, 17 and 80, and dependent on integrity of conserved heptad residues in TM1 and TM2, and less efficient secondary interactions, probably representing random collision events, and independent of the heptad residues (see Discussion for more details).\n\nTM3 and TM4 cysteine residues in CD9 dimerization\nAfter identifying the roles of conserved TM1 and TM2 residues in CD9 dimerization, we next probed whether residues proximal to TM domains 3 and 4 are also involved. To this end, disulfide cross-linking of full-length CD9 molecules containing 3 C-terminal cysteines (87, just before TM3; 218 and 219 in TM4) or 3 N-terminal cysteines (9, 78 and 79) was compared (Figure 9). We found that the C-terminal cysteines were only slightly better than N-terminal cysteines with respect to detection of CD9 dimers. However, markedly more trimers and tetramers were detected using C-terminal cysteines. Thus, residues 87, 218 and 219 at TM3 and TM4 in CD9 can together form contacts across the dimeric interface and also additional contacts with other neighboring CD9 molecules.\n\n\nDiscussion\nHere we provide the first detailed analysis of tetraspanin protein transmembrane domains. First, we show 1) the presence of a heptad repeat motif in TM1 and TM2, containing highly conserved Asn and Gly residues, 2) a leucine and glutamate/glutamine-containing heptad motif in TM3, and 3) high variability and absence of heptad repeats in TM4 sequences. Second, we provide evidence for a specific, intramolecular interaction between TM1 and TM2 domains, in which bulky hydrophobic residues pack against GG7 motif, and present a molecular model for this interaction. Third, experimental mapping of the CD9 dimerization interface firmly establishes an additional role for conserved TM1 and TM2 residues in dimeric intermolecular interactions. Fourth, preliminary evidence is provided to suggest that TM3 and TM4 domains contribute to expansion of CD9 dimers into higher order multimers.\nConserved residues in TM1 and TM2 of tetraspanins: role in intramolecular packing\nWe hypothesized that the first two transmembrane domains of tetraspanins might interact with each other because: a) consecutive TM domains frequently associate in known protein 3D structures [35], and b) they both contain a series of highly conserved amino acids – several Gly residues and an Asn residue (Figure 1). Conserved Gly residues are a frequent theme in the organization of interacting transmembrane domains. Analysis of 3D helix packing in polytopic membrane proteins reveals that Gly residues tend to localize in buried positions, especially at the helix-helix interfaces and helix crossing points [37,38]. Due to the absence of a side chain, Gly provides a flat surface for packing of a side chain from another residue, without loss of side-chain entropy upon interaction. The most common Gly-containing motif is GxxxG [39,40]. In glycophorin A (GpA), the major glycoprotein in erythrocyte cell membranes, Gly79 and Gly83 are part of the LIxxGVxxGVxxT sequence that promotes homodimerization of parallel transmembrane α-helixes [41,42]. In the GpA dimerization motif, Gly residues allow for tight packing in the right-handed helical crossing [43]. There are also examples of left-handed helical crossing in the context of a GxxxG motif [44]. Other membrane proteins that use the GxxxG motif for homo- or heterodimerization include bacteriophage M13 coat proteins [45], yeast alpha factor receptor [46], integrin α IIb subunit [47], and ErbB1 receptor tyrosine kinase [48]. Other small residues, such as Ala and Ser, can substitute for Gly in this motif [49].\nA protein motif in which Gly residues are separated by 6 other residues (GG7) is also common in transmembrane helices, especially in transporter/channel-like membrane proteins [50]. However, the structural features associated with this motif are not well known. In particular, it is unclear whether left-handed GG7 heptad repeat motif (as opposed to the \"classic\" right-handed GxxxG motif) can drive membrane helix association. In a recent work addressing this issue, Lear et al. [51] showed that a synthetic peptide containing Gly at heptad positions a and d could self-associate in vitro, likely in an antiparallel orientation. Heptad repeats containing conserved Gly residues occur in TM domains of α and β chains of MHC class II proteins, and mutations of the Gly residues disrupt the αβ heterodimer [52]. These examples demonstrate that Gly-based heptad motifs may be used for both intra- and intermolecular associations.\nIn this work, we identified a highly conserved GG7 motif in the first two tetraspanin TM domains. The GG7 sequence in tetraspanins is a part of a larger motif that also includes a conserved Asn residue in TM1 (NGG7) and an Ala/Ser/Thr residue in TM2 (GGA7). The seven-residue periodicity of these motifs strongly suggests their involvement in left-handed coiled coil packing reminiscent of the leucine zipper, rather than right-handed packing of the GpA-like GxxxG motif. For antiparallel helices, the left-handed crossing is in fact predominant over the right-handed in known TM domain structures [44].\nIn our model, heptad Gly residues in NGG7 and GGA7 sequences provide specific packing between antiparallel tetraspanin TM1 and TM2 helices by allowing tight van der Waals interactions with large hydrophobic residues (Figure 6). Highly efficient packing of bulky side chains against glycine residues is observed in known transmembrane protein 3D structures [38,53,54]. An example includes packing of helices M1 and M2 in potassium channel KcsA, where Val91 in M2 is paired with Gly43 in M1, and Leu36 in M1 contacts Ala98 and Gly99 in helix M2 [54,55]. In addition to facilitating helix-helix packing, Gly residues frequently provide additional CαH...O hydrogen bonds between two helices [44]. In our model, two Cα-backbone carbonyl H-bonds are predicted – between residues Gly27-Gly67, and Trp22-Met70.\nAlthough polar and charged amino acid residues (such as Asn in the TM1 heptad motif) are infrequent in transmembrane domains, they are functionally important. Polar residues such as glutamine, glutamic acid, aspartic acid and asparagine can promote strong oligomerization of model membrane-associated helices [56-58]. Ruan et al. [59] used asparagine scanning mutagenesis to probe the interface of self-associating polyleucine helices by detecting their enhanced self-interaction in vitro and in the E. coli-based ToxR assay. Thus, a hydrogen bond in an apolar environment can result in strong, though not necessarily specific, association of transmembrane helices. In fact, mutations to polar residues in transmembrane proteins are commonly associated with disease [60]. Because of this potential for non-specific interactions, polar residues tend to localize at buried positions in TM domains.\nIn our case, the conserved Asn18 residue in CD9 is predicted to be a part of the TM1-TM2 interface, though our model does not predict any electrostatic interaction between Asn18 and TM2 (Figure 6). Consistently, substitution such as Asn18Tyr (and Gly77Leu) in TM(1+2)-GFP protein was not destabilizing as analyzed by protein aggregation. Curiously, the full-length Asn18Ser CD9 migrated slightly slower on SDS-PAGE gel (data not shown), suggesting that Asn18 does play a role in maintaining conformation of the molecule. The Asn18Cys single-cysteine mutant shows very little intermolecular cross-linking (Figure 7A), supporting the proposed location of this residue at the intramolecular interface. It is tempting to speculate that the \"pocket\" between TM1 and TM2 lined by Asn18 and Gly77 might be important for accommodating palmitate moieties that target Cys78 and Cys79 residues, and/or important for access by the putative palmitoyl transferase to those residues. Understanding the exact role of these highly conserved Asn18 and Gly77 residues in tetraspanins awaits further investigation.\nIn summary, we identified conserved glycine residues of TM1 and TM2 of tetraspanins as key elements required for intramolecular packing. Mutations of these key residues (Gly25, Gly32, Gly67 and Gly74 in CD9) resulted in protein destabilization and aggregation. There is ample evidence in the literature for mutations in transmembrane proteins that lead to protein destabilization, misassembly and pathologic conditions [61]. Thus, we have identified conserved heptad Gly residues in TM1 and TM2 of tetraspanins as plausible targets of destabilizing mutations with potential functional consequences.\n\nIntermolecular interactions in tetraspanins\nTetraspanin CD9 forms mostly homodimers, but also a low level of heterodimers with CD81 and CD151 [25]. Thus, mapping the dimerization interface is an important next step in structure-function analysis of tetraspanins. Disulfide-mediated cross-linking, often in combination with cysteine-scanning mutagenesis, is a common strategy to probe oligomerization or intersubunit interactions of transmembrane proteins such as histidine kinase EnvZ [62], M(3) muscarinic acetylcholine receptor [63], E. coli lactose permease [64], synaptobrevin [65], integrins [66] and many others. In tetraspanins such as CD9, membrane-proximal cysteine residues are especially useful targets for disulfide trapping, as their linkage can be enhanced by pre-treating cells with 2-BP. While the ability of wild-type cysteines in CD9 to be cross-linked may indicate that they are close to the dimerization interface, more precise mapping was achieved here using cysteine-scanning mutagenesis.\nOur data clearly identify regions, near the cytoplasmic face of TM1 and TM2, important for dimerization. Intermolecular zero-length cross-linking was highest when single cysteines were placed in positions 14, 17, 77, 80 and 81 in TM(1+2)-GFP molecule, or at positions 77, 80 or 81 in the full-length CD9 protein. Positions 14, 17 and 80 are distinct from the intramolecular interface and are on the same side of the TM1-TM2 pair (Figure 6). Thus, they are well-positioned to participate in an interaction with another molecule. At the same time, the model predicts that wild-type cysteines (Cys78, 79), which do not yield very efficient zero-length cross-linking, are on the other side of TM1-TM2 pair.\nWhile using the cysteine at position 80 as the dimeric interface probe, mutations of conserved residues in TM1 and TM2 (especially Gly32 and Gly67 to Leu) clearly reduced intermolecular cross-linking. We do not suggest that those residues are directly involved in intermolecular interaction. Rather, we propose that destabilization of the intramolecular TM1-TM2 interaction by Gly to Leu substitutions (discussed above) causes an overall conformational change that reduces dimer formation.\nAn Ala81Leu mutation did not reduce cross-linking via Cys80, even though single-cysteine Ala81Cys molecules themselves produced a high level of cross-linking. These results, together with data on Gly32Leu and Gly67Leu mutations, are consistent with our model predicting that helices 1 and 2 interact more tightly near the extracellular end and less at the cytoplasmic end. This would give more flexibility to a cysteine at position 81 and also limit the effect of an Ala81Leu mutation. Location of this residue at the membrane/cytoplasmic border could also make it more accessible to CuP reagent as compared to residues buried in TM domain, thus elevating the efficiency of disulfide formation of the Ala81Cys mutant.\n\nMultiple interfaces in tetraspanin molecules\nIn the full-length CD9 molecules, the 3 C-terminal cysteines (Cys87, 218 and 219) located at or in TM3 and TM4 promoted efficient dimer and even more efficient oligomer formation compared to the 3 N-terminal cysteines (Figure 9). Cys87 alone can be used to capture CD9 dimers [25]. These results suggest the existence of two dimeric interfaces in CD9 molecule – the TM 1-2/1-2 interface and the TM 3-4/3-4 interface (Figure 10). In a TM(1+2) molecule, the destabilization of 1–2 interaction, e.g. by Gly→Leu mutations, would affect the 1-2/1-2 interface, as discussed above. However, these mutations would not interfere with the 3-4/3-4 interface in a full-length molecule, which includes Cys87, 218 and 219. Thus, cross-linking of full-length molecules, containing all 6 cysteine residues, would be unaffected, as seen in Figure 8C. Furthermore, wild-type Cys9, Cys78 and Cys79 are apparently not at the primary 1-2/1-2 interface. Their relative inefficiency in cross-linking CD9 TM(1+2) protein likely reflects weak secondary contacts between the molecules, or possibly random collision events. Such events should be independent of mutations in the conserved Gly residues in TM1 and TM2, as was demonstrated in Figure 8B. The potential existence of two interfaces in tetraspanin molecules, 1-2/1-2 and 3-4/3-4, should provide enhanced flexibility for forming additional intermolecular contacts. Current understanding of tetraspanin microdomains assumes a few strong, primary homotypic and heterotypic tetraspanin complexes (e.g. CD9-CD9, CD9-CD81, CD151-α3 integrin, CD81-EWI2) that help bring together various other proteins, forming secondary-type associations. Such properties of tetraspanins may bring signaling molecules such as protein kinase C or phosphatidylinositol 4-kinase to the vicinity of integrins [67,68].\nThe organization of the TM3 domain points to a potential role in protein-protein interactions. A motif of Leu-Leu-Glu(Gln) spaced 7 residues apart (heptad positions a), with highly conserved residues in two consecutive positions d, poses as a likely interaction module. If responsible for heterologous protein-protein interactions, it would form another distinct interface of tetraspanin molecule. Our preliminary data indicate that replacing the Leu and Glu residues in TM3 of CD9 with Ala has no effect on cell surface expression of the protein and its dimerization (data not shown). It remains to be tested if interactions with other proteins will be affected. Similarly, the TM4 domain may provide additional contributions to lateral tetraspanin associations. Much higher sequence variability, and the lack of a distinct heptad pattern suggests that TM4 is a major contributor to diversity among tetraspanin complexes. Structure-function analysis of TM3 and TM4 domains in tetraspanins is the subject of ongoing investigation.\n\n\nConclusion\nWe have defined the TM1-TM2 intramolecular interface in tetraspanin CD9, providing evidence for glycines (Gly25 and Gly32 in TM1, Gly67 and Gly74 in TM2) packing against apposing bulky aliphatic residues. Second, we mapped an intermolecular CD9 interface (involved in CD9 homodimer formation) to the vicinity of residues Leu14 and Phe17 in TM1 and Gly77, Gly80 and Ala81 in TM2. Finally, we provide preliminary evidence that TM3 and TM4 in CD9 may contribute to a second intermolecular interface. Key CD9 residues involved in intra- and intermolecular interactions are highly conserved throughout the tetraspanin family, thus suggesting that our findings will apply to most tetraspanins.\n\nMethods\nMaterials\nCell culture reagents were from Invitrogen (Carlsbad, CA). 2-bromopalmitate was from Fluka (Milwaukee, WI), N-ethylmaleimide (NEM) and 1,10-phenanthroline were from Sigma-Aldrich (St. Louis, MO), and chemical cross-linkers dithio-bis-maleimidoethane (DTME) and 1,4-Bis-maleimidobutane (BMB) were purchased from Pierce Endogen (Rockford, IL). Triton X-100, protease inhibitor cocktail and FuGENE 6 transfection reagent were obtained from Roche (Indianapolis, IN). Restriction endonucleases and Pfu DNA polymerase were obtained New England Biolabs (Beverly, MA) and Stratagene (Carlsbad, CA), respectively. All other chemicals were purchased from Sigma-Aldrich or Fisher Scientific (Pittsburg, PA).\n\nSequence analysis\nTetraspanin sequences were obtained from SWISS-PROT and GenBank databases. Locus designations, accession numbers and the most commonly used protein names are summarized in Tables 1 and 2. TM segments were delineated by inspection of hydrophobicity profiles, using database annotations and previous analyses of TM sequences as a guide ([28], M. Hemler, unpublished), and aligned manually. Residue numbers in human CD9 sequence are used a reference point throughout the study.\n\nDNA cloning and mutagenesis\nSequence encoding CD9 protein was cloned into vector pcDNA3 (Invitrogen, Carlsbad, CA) and pEGFP-N1 (Clontech, Palo Alto, CA), for expression of untagged and C-terminally GFP-tagged CD9, respectively. pEGFP-N1 encoding CD9 TM(1+2) -GFP fusion protein was constructed by subcloning DNA for residues 1–83 of CD9 into HindIII and PstI sites of the vector; to introduce the PstI site, codon GTG for Val82 was changed to CTG (coding for Ala). In the resulting fusion protein, there is a 13-amino acid linker (with no cysteines) between CD9 and GFP. To minimize the low inherent ability of GFP to homodimerize, which could potentially influence the results of CD9 cross-linking, we used a monomeric GFP mutant, Leu221→Lys [34], for cross-linking experiments.\nMutations were introduced in full-length and TM(1+2) CD9 proteins by a PCR-based strategy using mutagenic primers and Pfu DNA polymerase. All mutations were confirmed by DNA sequencing.\n\nProtein expression, microscopy, cysteine disulfide cross-linking and Western blotting\nDNA constructs encoding TM(1+2)-GFP or full-length CD9 proteins were transfected into human rhabdomyosarcoma RD cells using the FuGENE 6 reagent. Cells expressing GFP fusion proteins were analysed by fluorescence microscopy 18–28 hours post-transfection. Images were captured using Spot 1.4.0 camera (Diagnostic Instruments, Sterling Heights, MI) attached to Nikon Eclipse TE300 microscope.\nFor experiments involving cysteine-mediated cross-linking, cells were treated with 50 μM 2-BP starting 24–26 hours post-transfection and continuing for 16–18 hours. Cross-linking was carried out by incubating cells in HBSM buffer (25 mM Hepes-NaOH, pH 7.2, 150 mM NaCl, 2 mM MgCl2) containing either a) 0.6 mM CuSO4 and 1.8 mM 1,10-phenanthroline (CuP complex) or b) 0.2 mg/ml homobifunctional cysteine-reactive cross-linker (e.g. DTME), diluted from fresh 10 mg/ml solution in DMSO. After incubation for 10–15 minutes (with CuP) or 30–45 minutes (with cross-linker), cells were washed twice for 10 minutes with HBSM containing 10 mM NEM to block residual free cysteines. Cells were lysed in HBSM containing 1% Triton X-100, 0.1% SDS and a cocktail of protease inhibitors with 1 mM EDTA at 4°C for 45–60 minutes. Cell lysate was clarified by centrifugation at 14,000 × g for 15 minutes, an aliquot was removed, and proteins from it were precipitated by addition of trichloroacetic acid to 10% on ice followed by centrifugation at 14,000 × g for 10 minutes. After two washes with ice-cold acetone, protein pellet was solubilized in SDS-PAGE sample buffer without a reducing agent (50 mM Tris-HCl, pH 6.8, 1% SDS, 8% glycerol).\nIn some experiments, CD9 protein was immunoprecipitated using monoclonal antibody Alb6 (Immunotech, Marseille, France). Proteins were separated by SDS-PAGE and analyzed by Western blotting using monoclonal antibody JL-8 (Clontech) for GFP or Alb6 for CD9. Bands from X-ray films were quantitated using GeneTools™ software from Syngene (Frederick, MD).\n\nModeling of TM1-TM2 interaction\nAn atomic model of the CD9 TM1-TM2 dimer was constructed with a Monte Carlo-simulated annealing (MCSA) algorithm [69]. Two idealized α-helices corresponding to TM1 residues Tyr12 through Leu35 and TM2 residues Gly59 through Val82 were docked with six orthogonal parameters: three rigid body translations and three rotations. During each step of a MCSA cycle, there was an equal probability of changing either one parameter or all six parameters to random values. A conformation's energy was calculated in vacuo with the AMBER united-atom force field for van der Waals interactions [70]. The van der Waals term was modified as described by Kuhlman and Baker [71]. If a structure had favorable dimerization energy, the energies of select mutants were calculated. Structures were selected with a novel scoring function that maximizes the Boltzmann probability of dimerization for silent mutations while minimizing the probability for disruptive mutations. Asn18Ser, Gly77Leu, and Gly80Leu were scored as silent mutations while Gly25Leu, Gly32Leu, Gly67Leu, and Gly74Leu were considered to be disruptive. Each MCSA cycle consisted of 50,000 steps with an exponential temperature decay from 10,000 to 10 K.\nTen MCSA cycles through global sample space were used to restrict the search area. Parameters were restricted to ± 2 standard deviations from their mean value for structures within 10 kcal of the best structure. MCSA cycles were repeated as described above with additional optimization of χ values: rotamers at the protein-protein interface were optimized with Dead End Elimination [72], and χ values were further optimized with Monte Carlo. All MCSA cycles converged upon structures that were within a root mean squared deviation (RMSD) of 1.5 Å with the best structure, and structures that scored within 5 kcal of the best score had an RMSD of less than 0.5 Å with the best structure.\n\n\nList of abbreviations\n2-BP, 2-bromopalmitate; CuP, Cu2+-phenanthroline; DTME, dithio-bis-maleimidoethane; LEL, large extracellular loop; NEM, N-ethylmaleimide; TM, transmembrane (domain).\n\nAuthors' contributions\nOVK carried out sequence comparisons, mutational analysis and cross-linking experiments, and drafted the manuscript. DGM built the TM1-TM2 interaction model and contributed to the manuscript. WFD supervised DGM's work. MEH coordinated the whole study and prepared the final manuscript.\n\n\n" ], "offsets": [ [ 0, 47976 ] ] } ]

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[ { "id": "pmcA1036069__text", "type": "Article", "text": [ "In spite of medical help: the puzzle of an eighteenth-century Prime Minister's illness.\nAbstract\n\n\n\n\n\n Medical History, 1990, 34: 178-184. \n\n IN SPITE OF MEDICAL HELP: THE PUZZLE OF AN \n\n EIGHTEENTH-CENTURY PRIME MINISTER'S \n\n ILLNESS \n\n by \n\n MARJORIE BLOY * \n\n Charles Watson Wentworth, second Marquis of Rockingham, died suddenly and unexpectedly on 1 July 1782, when he was only 52 years old. He had suffered-or enjoyed-ill health all his life but in 1782 appeared to be no worse than he had ever been. His death in London terminated his second period of office as Prime Minister, to which he had been appointed only 14 weeks earlier. In May he had reported to the Duke of Portland that he had \"for some weeks past undergone much Pain and much inconvenience from something similar to my old Complaint in my Side and Stomach\" but that he felt much better than he had.' By 17 June he was recovering from both influenza and his \"old complaint\".2 On 1 July he died and on the 20th he was interred in York Minster. \n\n The first recorded bout of illness suffered by the marquis, then Lord Higham, was in July 1741 when the 11-year-old was \"a little indisposed, something Feaverish I guess it proceeds from Worms and will Soon be removed\".3 He also had a rash and it was thought that the cause of the problem was that the boy had overheated himself.4 He was still ill at the beginning of August: he had been \"much out of order\" for a long time but had been recommended to take warm baths by Dr Wilmot and Mr Ranby when they were consulted in London.5 Charles's aunt, Lady Isabella Finch, was sure that the baths \"and other Things They'll prescribe will in a short Time entirely Cure his Complaints w[hic]h neither of Them thought proceed from any dangerous Causes\".6 In spite of Lady Isabella's hopes, Charles did not greatly improve, even though his mother believed that he continued mending every day. The main reason that Higham and his mother had gone to London to consult Dr Wilmot and Mr \n\n *Marjorie Bloy, Ph.D., 18 Farm View Road, Kimberworth, Rotherham, S. Yorks. S61 2BA. \n\n The Rockingham Papers are in the holdings of the Wentworth Woodhouse Muniments at Sheffield City Archives Department, Sheffield City Library. I am grateful to Dr R. S. Morton for his advice and help with the diagnostic sections of this essay. \n\n 'WWM, RI-2094. Rockingham to Portland, 25 May 1782. \n\n 2WWM, RI-2094. Rockingham to Charlemont, 17 June 1782. 3 WWM, M8-25. Malton to Nottingham, after 16 June 1741. 4 WWM, M8-26. Lady Finch to Lady Malton, 30 July 1741. 5 WWM, M8-28. Winchelsea to Malton, 7 August 1741. 6 WWM, M8-29. Lady Finch to Malton, 7 August 1741. \n\n 178 \n\n An eighteenth-century Prime Minister's illness \n\n Ranby was his mother's concern about a \"swelling in a certain part which was larger than when we left Wentworth\". The doctors hoped that it would burst outwards \"which they assure me will be the safest way and give the poor Monkey but very little pain' 7 \n\n On 20 August Lord Winchelsea, Higham's uncle, surprised to see the boy so well and brisk, hoped that Charles was \"now safe from this complaint\"-the same one from which he had suffered in 1738-39-but thought that he would never be safe \"if he continues the practice of overheating himself and then drinking Cold Water\". He said that Charles was of a \"pretty healthy strong Constitution\";8 Lady Malton was not so sure. The same day she wrote a progress report to her husband saying that Charles's swelling continued to grow, as did the pain \"in that part (but not the lease [sic] trouble in making Water or going to Stool) & less Fever than c[oulJd be imagined where Matter is as they now imagine certainly gathering and must end in an operation\". In spite of it all, Charles was in fine spirits.9 Lady Malton dosed the boy with cinchona bark, which removed the pains in his legs and reduced his fever, and she was convinced that they would soon have \"a clear Stage to act in a proper manner a[bou]t his other Complaints w[hic]h the Learned assure me are to be conquered also\". 10 Charles was soon allowed to eat meat and Mr Ranby still assured her that the swelling would break outwards. 1 \" Three days later he decided to lance it, even though Dr Bourne disagreed. The boy's mother was puzzled because the swelling \"sometimes pushes forward very fast then retires a little\" but the doctor and Ranby seemed happy with his condition.'2 At this point the letters cease, presumably because Malton arrived in London with his daughters, to have them inoculated against smallpox, but a later letter states that surgery to open the swelling was not undertaken. 13 \n\n By the end of October the correspondence had recommenced. Charles was ill again. He was just the same as when he left Kensington, so John Bourne had bled him and the child had started on Sir Edward Hulse's prescription, unfortunately not defined in the letter, but which was apparently as bad as the last one, if not worse. Lady Malton thought that \"with such a State of Blood the Continuation of Health cannot be expected\" but was hopeful that the \"Cinnabar may prove a more Efficacious remedie than any than has been tryed yet\"\".\"4 That night she applied \"a Blister ... without the least Symptom or tendency to anything like Strangury\". He bore the treatment well, as he had done three years previously, and it seemed so successful that Lady Malton was \"determined to keep it running full as long as I did last time by the help of John Borne [sic] with much ease to the Dear Child\".'5 She \n\n 7 WWM, M7-51. Lady to Lord Malton, 18 August 1741. 8 WWM, M2-84. Winchelsea to Malton, 20 August 1741. 9 WWM, M7-52. Lady to Lord Malton, 20 August 1741. '0 WWM, M7-53. Lady to Lord Malton, 25 August 1741. \n\n WWM, M7-54. Lady to Lord Malton, 29 August 1741. \n\n WWM, M7-55. Lady to Lord Malton, 1 September 1741. \n\n 3 WWM, R170-20. Nicol6 Scanagati of Padua, 20 July 1750. I am grateful to Fr John McMahon and Dr Stephen Bemrose for their translations of this letter. \n\n 4 WWM, M7-14. Lady to Lord Malton, 31 October 1741. \n\n 5 WWM, M7-19. Lady Malton to Lady Finch, 2 November 1741. \n\n 179 \n\n Marjorie Bloy \n\n continued with the blister and applied \"ointment with flyes\", apparently some sort of irritant potion, with no sign of strangury. Charles found her treatment \"not near the pain he expected\" and she was \"full of hopes that he will rec[eiv]e great benefit from it\".1 \n\n Apart from his other troubles, one of Charles's knees had swollen but this had much abated since the application of the blisters which Lady Malton believed \"must be acting upon the whole Mass of Blood\" since it had \"reached the remote part\". She thought that Sir Edward Hulse's powders were too slow in taking effect although the boy took them very quietly.'7 Sir Edward did not \"apprehend any great danger from the Siziness [thickness] of Charles' blood\"; Lady Malton thought that the condition was the cause of all the child's problems, which would not end until it was set to rights. At any rate, he was fit enough to go hunting.'8 Charles continued in the same state of health. He slept well at night, ate more than his mother thought was good for him, and was able to exercise strenuously without tiring. He put on no weight though, and \"as for them swellings at his throat, they are almost gone one day and rise the next\". His mother did not expect a speedy recovery and \"if the D[octo]rs think him in a good state of health now, I s[houl]d be glad to see him in a better\".'9 He began to improve and by the end of November even she thought he was on the mend and gaining weight.20 Unfortunately, Lady Malton again had cause for concern over his health in January 1742 when he began to suffer from an intermittent hoarseness.2' Otherwise he was as well as one could expect, with no other complaints.22 It was not to last. \n\n In May 1742 Charles and his mother were again in Bristol, taking the waters because he had been indisposed. Lady Malton thought the waters were doing them good because they were both being violently sick.23 However, Charles had had no dinner on 25 or 26 May and was hot, lazy, and inclined to stir, \"from which I conclude he is not well, . . . and therefore Intend to give him a gentle Vomit ... and to let him take his old Remedie the Salt Draughts for a few Daies which I dare say will set him quite to rights\".24 By 29 May Dr Boume had bled the boy \"which succeeded very well but ... found it [his blood] as bad as ever\". The waters were not working \"but there is a great deal for them to do which grant God they may effect\". The weather had turned warm so Lady Malton had \"shorn him ... which has display'd a most scabby head and indeed several other untoward Blotches he has out upon other parts of his Body\", which made her uneasy.25 The blotches on his head were not numerous \"yet they made up in quality for so virulent a Corrosive Humour is not easily conceived without seeing it\". The pustules on his body were of the same sort \n\n 16 WWM, M7-17. Lady to Lord Malton, 4 November 1741. 7 WWM, M7-18. Lady to Lord Malton, 4 November 1741. 18 WWM, M7-16. Lady to Lord Malton, 7 November 1741. '9WWM, M7-15. Lady to Lord Malton, 9 November 1741. \n\n 20 WWM, M7-22. Lady to Lord Malton, 25 November 1741. 21 WWM, M7-1. Lady to Lord Malton, 25 January 1742. \n\n 22 WWM, M7-4. Lady to Lord Malton, 8 February 1742 and WWM, M7-9. Lady to Lord Malton, 22 February 1742. \n\n 23 WWM, M7-29. Lady to Lord Malton, 12 May 1742. 24 WWM, M7-35. Lady to Lord Malton, 26 May 1742. 25 WWM, M7-36. Lady to Lord Malton, 29 May 1742. \n\n 180 \n\n An eighteenth-century Prime Minister's illness \n\n and his mother intended to put plasters on them to prevent them from spreading. Charles was also feverish; his glands were swollen and his pulse was erratic \"but out of compassion to you I must tell you that he is with me as Brisk and lively as you ever Saw him\". Lady Malton had called in two eminent Bristol men, Dr Logan and Mr Pye, to treat the boy; Mr Pye prescribed \"the Precipitate Per se\" as the cure for the \"hectic\". Pye made it himself and said that it was the only remedy that would work. Clearly Charles was impatient to be cured because he told his mother to give him the medicine \"to cure me which I am sure it will do or shoot me through the head at once\". She thought that this attitude was \"odd from one of his Age and [it] does not a little disturb\".26 The blotches began to burst and indent but the doctor thought that all would be well in the end.27 Meanwhile, Charles was still losing weight even though \"he had none to spare before\" and he was inclined to be lazy which was not his natural turn. His father recommended some unknown cure which he called Gascoin's Powder-a dose of five grains made up with syrup into a pill-every night. \n\n To make matters worse, the doctors disagreed about the treatment. \"Dr Pye is Vehemently for the P. Per se, Dr Logan saies that it is a Medicine that may prove too rough in its operation for his Constitution & therefore begs a tryal of Beazor mineral [gall stones from a goat] and Viper Broth\". The Bristol water had not yet acted \"because his case is of too obstinate a Nature\" and Lady Malton herself was satisfied that since nothing else had worked to cure the boy, the time had come to try mercurials, even though she knew that they were \"powerful and perhaps in some cases hazardous medicines\".28 She wanted to see some remedy succeed but was \"afraid of violent ones and at the same time vastly distrustfull [sic] of mild ones\". It would appear that the \"precipitate Per se\", probably mercury-based, could be a kill-or-cure remedy. Her \"terrors\" did not arise from any immediate danger to her son, and her \"perfect Knowledge\" of his disorder convinced her that whatever remedies he took, the cure was in the hands of God.29 \n\n To add to Charles' disorders, on 12 June he developed a \"very inflamed bad Eye ... the same Eye that ... he did not see so well of [as] the other ... He sais [sic] that from that eye Alone he can Scarcely distinguish anything\". The doctors suggested bathing the eye: Lady Malton knew that the \"frightful symptoms\" which were \"shocking to behold\" were a result of \"the Same as produces all the rest of his complaints in whichever Shape they appear\". 30 The eye was very bloodshot and inflamed; the eyelid was swollen so he could hardly open it. The other eye was dull and \"he had very little sight of it\".31 By 14 June the eye problem had eased somewhat but Lady Malton could find no cause to attribute the improvement to any of the \"cures\". Charles was still being subjected to Bristol water, Beazor mineral, Viper broth, cinnabar and the precipitate per se.32 She decided to take the boy home to Wentworth because he was \n\n 26 WWM, M7-38. Lady to Lord Malton, 1 June 1742. 27 WWM, M7-39. Lady to Lord Malton, 2 June 1742. 28 WWM, M7-41. Lady to Lord Malton, 5 June 1742. 29 WWM, M7-43. Lady to Lord Malton, 7 June 1742. \n\n 30 WWM, M7-45. Lady to Lord Malton, 12 June 1742. \n\n 31 WWM, M7-56. Lady to Lord Malton, undated: 12 June? 1742. 32 WWM, M7-46. Lady to Lord Malton, 14 June? 1742. \n\n 181 \n\n Marjorie Bloy \n\n more likely to recover there than anywhere else.33 He still ate and slept well and was \"pretty cheerful but his looks are bitter bad still. The flesh he lost in the Accidental Feavour he has not Recover'd and his complexion is of the most sickly sort his hands of the same Hue his legs are tollerable [sic] well\".34 \n\n They returned to Wentworth in short stages and by 6 September Higham was \"perfectly recovered . . . after the long and successful Care that Lady Malton has taken\" of him.35 In May 1743 he was inoculated against smallpox and made a perfect recovery after which he caught cold \"by stripping when He was hot\".36 Lord Higham does not seem to have been seriously ill after that until, at the age of 19, he undertook his Grand Tour in 1749. \n\n In July 1750, by then Lord Malton, he had cause to consult Nicolo Scanagati in Padua for the treatment of gonorrhoea. Scanagati produced a lengthy medical report of Malton's treatment, presumably for his English doctor's enlightenment.37 The initial treatment was an \"electuary, consisting of three ounces of emollient, three drams of powdered jalap, a half [dram] of purified nitre, bound together with lemon juice taken twice a day\". The result was satisfactory: \"The dark greenish poison was oozing slowly from his penis, which was all contracted and the sharp and constant pain extended from the perineum up to the urinary bladder, producing small swellings now in this place, now in that.\" There was a fierce burning sensation in the glands, which prevented him from sleeping. Because of this, it seemed reasonable to bathe that part in tepid water and milk, and to apply poultices to the areas affected by swelling and contractions, together with cold drinks and a few grains of laudanum at night. \n\n Malton was blooded regularly besides being given purgatives; the treatment then moved on to the administration of mercury, both internal and on the gums, since it was widely believed at the time that gonorrhoea and syphilis were steps of the same disease, \"the Venereal\". Scanagati at this point ruled out the suggestion of syphilitic chancre because Malton's urine was fine and light with a pungent odour. Scanagati did ask if Malton had previously ever had a similar peculiarity of his urine. Malton replied that when he was very young and still inexperienced sexually, for some time following a fever he had had the same unusual urine, and indeed that on one occasion this symptom coincided with certain tumours on the testicles. It was only by chance that he had not had recourse to surgery, the reason being that he was also afflicted with a throat infection-to which he was prone-and therefore had his vein opened four times. Thereupon the inflammation subsided, and equally the tumours and sediment disappeared. \n\n He told me that as a youth he had sometimes experienced some difficulty and a burning sensation when urinating, which subsided when his blood was let and with the application of poultices. I observed that from time to time his face and body were covered with purplish spots, which, having produced a little fluid, would disappearas indeed happened in the course of the cure, at the end of which his face was entirely \n\n 33 WWM, M7-47. Lady to Lord Malton, 15 June 1742. 34 WWM, M7-48. Lady to Lord Malton, 16 June 1742. \n\n 35 WWM, M2-104/5. Lady Finch to Lady Malton, 6 September 1742. 36 WWM, M2-135. Lady Finch to Malton, 18 June 1742. 37 WWM, R170-20. Nicol6 Scanagati's report. \n\n 182 \n\n An eighteenth-century Prime Minister's illness \n\n free from these spots. From this observation, it seemed to me simple to deduce both the original cause and the more immediate cause of the said sediment: namely a natural complexion of humours which are exacerbated by muriatic [i.e., acidic] sourness, together with the marked inflammation of the blood and the motion of the contracted poison. \n\n Scanagati then recommended the continuation of the electuary made of emollient, guaiacum resin, balsam, rhubarb, and nitre. \n\n What does all this add up to in terms of diagnosis? The early illness is a mystery. Did he have an inguinal hernia; or perhaps mumps or epididymitis? His was a childless marriage. He seems to have been too fit for the illness to have been rheumatic fever. Cystitis was not uncommon and this could certainly lead to \"strangury\". Perhaps Rockingham suffered from a congenital defect of his urinogenitary system which would result in recurrent attacks of cystitis and might cause long-term damage to the urinary tract and eventual destruction of the kidneys, precipitating sudden and unexpected death. \n\n Another possibility might be diabetes. Rockingham had a urinary infection and certainly suffered from recurrent skin infections, although it appears from Scanagati's report that these cleared up when mercury was administered. \n\n Certainly the marquis complained often about pains in his side and stomach and made no secret of his \"old complaint\". He was noticeably less physically active as he moved into his thirties and only occasionally exerted himself by riding any distance, even though he had enjoyed hunting when he was younger. He found the pains caused by his \"old complaint\" made him feel so ill that he was unable to concentrate on \"any Manner of Business\" and on occasion it seemed likely to prevent him from attending Parliament.38 He may well have suffered from a problem with gallstones from an early age.39 He appears to have suffered from a nervous disorder which manifested itself in severe palpitations, trembling, and other types of physical discomforts such as boils and headaches with which he was frequently afflicted.40 He probably had constipation too, since he was always dosing himself with purgatives. In fact in April 1772 the Duke of Richmond decided that Rockingham's real problem was a \"surfeit of physick\"41 although Edmund Burke noted in June 1772 that the marquis had had a long and severe illness.42 Whatever his many and varied ailments, Rockingham survived until he was 52 in spite of the attentions of both doctors and quacks and that, for the mid-eighteenth century, was a good age. The mystery still remains, however. Contemporary opinion had it that he died of pneumonia but it must have struck \n\n 38 WWM, R153-1. Rockingham to Burke, 31 October 1767; WWM, RI-1238. Rockingham to Dowdeswell, 20 October 1769; WWM, RI-1928. Rockingham to Savile, September 1780. \n\n 39 Ross J. S. Hoffman, The Marquis. A study of Lord Rockingham, 1730-1782, New York, Fordham University Press, 1973, p. 35. This is the only recent biography of the second Marquis of Rockingham, and does not deal with his illnesses. Although he does not give the source of his information, Hoffman asserts that Rockingham was in Bath between March and August 1761 suffering from gallstones. The marquis would then have been 31 years old. \n\n 40 Historical Manuscripts Commission, Lindley Wood, p. 184. \n\n 41 WWM, RI-1403. Richmond to Rockingham, 26 April 1772. \n\n 42 Burke to James de Lancey, 30 June 1772. The correspondence of Edmund Burke, vol. 2, ed. T. W. Copeland and others, Cambridge University Press, 1958-1978, p. 311. \n\n 183 \n\n Marjorie Bloy \n\n suddenly: it was only two weeks from him \"recovering\" to dying. Another almost contemporary account of the marquis's death came from the Earl of Albemarle. He noted that Rockingham had \"for some time past been afflicted with water on the chest: and to this well-known malady was superadded the then novel disease of influenza\".43 This conceivably could be an uninformed account, handed down orally by surviving members of the marquis's family, of emphysema. More fascinating than the diagnosis of the cause of death, however, is - what was wrong with him during his lifetime? Or is it yet another example of the \"English disease\": hypochondria? \n\n 43Albemarle, George Thomas, Earl of, Memoirs of the Marquis of Rockingham and his contemporaries, London, Richard Bentley, 2 vols., 1852, vol. 2, p. 483. This is the only contemporary work concerning Rockingham, but does not mention his early life and illnesses. \n\n 184 " ], "offsets": [ [ 0, 21199 ] ] } ]

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[ { "id": "pmcA2636785__text", "type": "Article", "text": [ "Identification of recruitment and retention strategies for rehabilitation professionals in Ontario, Canada: results from expert panels\nAbstract\nBackground\nDemand for rehabilitation services is expected to increase due to factors such as an aging population, workforce pressures, rise in chronic and complex multi-system disorders, advances in technology, and changes in interprofessional health service delivery models. However, health human resource (HHR) strategies for Canadian rehabilitation professionals are lagging behind other professional groups such as physicians and nurses. The objectives of this study were: 1) to identify recruitment and retention strategies of rehabilitation professionals including occupational therapists, physical therapists and speech language pathologists from the literature; and 2) to investigate both the importance and feasibility of the identified strategies using expert panels amongst HHR and education experts.\n\nMethods\nA review of the literature was conducted to identify recruitment and retention strategies for rehabilitation professionals. Two expert panels, one on Recruitment and Retention and the other on Education were convened to determine the importance and feasibility of the identified strategies. A modified-delphi process was used to gain consensus and to rate the identified strategies along these two dimensions.\n\nResults\nA total of 34 strategies were identified by the Recruitment and Retention and Education expert panels as being important and feasible for the development of a HHR plan for recruitment and retention of rehabilitation professionals. Seven were categorized under the Quality of Worklife and Work Environment theme, another seven in Financial Incentives and Marketing, two in Workload and Skill Mix, thirteen in Professional Development and five in Education and Training.\n\nConclusion\nBased on the results from the expert panels, the three major areas of focus for HHR planning in the rehabilitation sector should include strategies addressing Quality of Worklife and Work Environment, Financial Incentives and Marketing and Professional Development.\n\n\n\nBackground\nDemand for rehabilitation services is expected to increase within the next decade primarily due to factors such as an aging population, workforce pressures, rise in chronic and complex multi-system disorders, advances in technology, and changes in health service delivery models [1-4]. In Canada, rehabilitation personnel constitute the third largest health professional group after nurses and physicians. Despite the size of this workforce, studies have consistently reported ongoing shortages of physiotherapists (PTs), occupational therapists (OTs) and speech-language pathologists (SLPs) across all jurisdictions [5-7]. Similarly, recruitment and retention of rehabilitation professionals has been considered a challenge internationally, nationally and provincially. At the international level, the literature reports recruitment and retention difficulties of rehabilitation therapists in countries such as Australia, New Zealand, United Kingdom and the United States [8-13]. Provinces across Canada face similar issues; with Ontario projected to face the most difficulty due to its population growth rate [14].\nBased on the Canadian Institute for Health Information's Health Personnel Trends in Canada from 1993 to 2002 report, numerous factors have been suggested to influence demand for physiotherapy and occupational therapy services. Factors that may influence increase demand for physiotherapy include: shift in health service delivery models from hospital to community care; earlier patient discharge; increased expectations from aging Canadians concerning more active lifestyles; growing private practice sector and continued shortages for PTs in both private and public sectors in rural, remote and urban settings across Canada [2]. In 1993, an Ontario study stated that in order to meet demands of changing health care policy, medical technology and demographic changes in the population, the PT profession required an annual growth rate of 4.4% until the year 2000 [5]. However, the national health personnel databases revealed that the actual average annual growth rate of active PTs in Canada from 1995 to 2004 was only 2.5%, approximately half of the projected requirement suggested to meet demand [15].\nSimilarly, in Ontario in the early 1990s an increase in demand for OTs was projected because of the reported shortage in OTs and high attrition rate [16]. The shortage of OTs was explained by another Ontario study to be the result of the changing philosophies of care and management for the disabled, and a clearer understanding of the role of OT in the physical and mental well-being of the disabled [17]. In terms of actual shortages, some authors have reported ongoing vacancies and recruitment difficulties for OTs [11,18] while others have reported an increase in demand for both PTs [19] and OTs [20].\nSpeech language pathology is facing similar service demands. A report released in March 2003 by the College of Audiologists and Speech-Language Pathologists of Ontario (CASLPO) concluded that based on prevalence rates for Ontario residents with speech, language and related disorders, the demand for service would increase by 13% while the number of SLPs would decrease by 4% resulting in an overall reduction in service of 15% [21]. The American Speech-Language-Hearing Association (ASHA) has been tracking SLP vacancies. In their 2005 ASHA Speech-Language Pathology Health Care survey, 48% of respondents indicated that they had funded unfilled positions for SLPS in their agency [22]. The same survey also reported that 65% of respondents in home care indicated that job openings were more numerous than job seekers in their geographic area.\nWhile labour market demand and supply are influential factors on recruitment and retention decisions, the development of strategies requires an understanding of conceptual frameworks or theories to categorise and explain how other underlying factors impact health worker's mobility. For example, Lehmann et al.'s model described that health worker's decisions to accept and stay in remote areas in the public sector depends on two interrelated aspects: the impact of the different environments (i.e. individual, local, work, national and international) and the location of decision-makers (i.e. local government, Ministry of Health, HR directorate, public service and other ministries)[23]. Behavioural and social science theories, such as those explained by Tett and Meyer, found that job satisfaction and organizational commitment each contribute independently to the prediction of the intention to resign (turnover), however job satisfaction was a stronger predictor than organizational commitment[24]. Based on this notion, considerable research has been devoted to identifying factors that affect job satisfaction among rehabilitation professionals. While there is no single, agreed upon model of job satisfaction, a variety of theoretical models have been studied to explain concepts and relationships associated with overall job satisfaction. The two most commonly used theories of job satisfaction for rehabilitation professionals are the Herzberg's Motivation-Hygiene Theory[25] and Mottaz's concepts of work values and work rewards [26].\nA number of rehabilitation studies have used the Herzberg's Motivation-Hygiene Theory, also known as the two-factor theory of motivation to explain associations between motivation, job satisfaction and retention factors among OTs, PTs and SLPs [27-31]. Frederick Herzberg et al. explained that there were two independent incidents occurring at peoples' jobs: one that made them feel good or satisfied, and another that made them feel bad or dissatisfied at work [25]. Intrinsic factors that motivate people such as achievement, recognition, work itself, responsibility, advancement and personal growth were called the \"motivators\" which lead to feelings of satisfaction. Extrinsic factors such as work conditions, company policies, supervision, interpersonal work relations, salary and job security, known as \"hygiene\" factors, were claimed to prevent dissatisfaction. \"Motivators\" directly affect a person's motivational drive to do a good job, therefore they are believed to be more important than hygiene factors.\nMottaz on the other hand, accounted for individual differences in job satisfaction among workers and based his study on two dimensions: \"work rewards\" and \"work values\"[26]. \"Work rewards\" are perceived characteristics of the job and have three conceptual clusters which include task, social and organizational rewards [26]. Mottaz describes \"task rewards\" (intrinsic) as having five independent characteristics including: skill variety, task identity, task significance, autonomy and feedback. Examples include interesting and challenging work, self-direction and responsibility, creativity, opportunities to use one's skills and feedback. In the same study, Mottaz stated that \"social rewards\" (extrinsic) are derived from the interpersonal relationships established with others at work. Having supportive colleagues and supervisors is an example of this dimension. Lastly, \"organizational rewards\" (extrinsic) are tangible rewards that are provided by the employer/organization to facilitate performance. Such factors include working conditions, pay and fringe benefits, career advancement and security. The second dimension of job satisfaction is based on \"work values\", which is the importance that individuals place on their work rewards [26]. For example, some rehabilitation therapists may value extrinsic rewards such as pay and benefits as more important than intrinsic factors like clinical autonomy and challenging work. Although the Herzberg and Mottaz conceptual frameworks are organized differently, their job satisfaction variables are very similar (i.e. work conditions, pay, interpersonal relationships, etc.) and they both classify these factors as having intrinsic or extrinsic elements.\nDespite the growing body of literature on recruitment and retention factors in various industries, there is a minimal amount of research studying these factors specifically among rehabilitation professionals. One published study however, did look at extrinsic and intrinsic job satisfaction factors on recruitment and retention of rehabilitation professionals (OTs, PTs and SLPs)[32]. Results from this study showed that intrinsic factors such as professional growth and having a work environment in line with personal values are more significant in predicting career satisfaction than extrinsic factors such as pay and continuing education. These same intrinsic factors are also significant in predicting retention in rehabilitation professionals. Another study looking at recruitment and retention of allied health professionals in the rural areas in New South Wales identified that the main reasons why people liked working in rural areas were because of the attractive environment and helpful team members[33]. However 82% of employees reported that having their partner move away was the number one reason for leaving a rural job. A similar study was conducted among OTs and PTs in Northwestern Ontario[34]. Findings from this study indicated that factors contributing to initial decision on location of practice include availability of leisure/recreation activities, proximity of family origin and influences of spouse/partners. Study results also showed that the main reasons therapists left their job were to be closer to their family, lack of job opportunity and spousal influence.\nSolely understanding factors that influence recruitment and retention decisions is not sufficient in the development of a HHR plan for rehabilitation professionals. In order for the plan to be effective and sustainable in addressing these factors, the most important and feasible workforce strategies needs to be identified.\nThere have been a number of reports on health human resources (HHR) planning, recruitment and retention strategies for physicians [35,36] and nurses [37], however information regarding rehabilitation professionals is lacking. Canadian reports indicate that the main reason for significant gaps in this field is the absence of current and reliable data available on supply, demand and labour force participation trends for rehabilitation therapists [38-40]. There is some research that has investigated theoretical models of job satisfaction on recruitment and retention [28,32]; however few studies have looked at how these models have been implemented. Other studies have examined the relationship of gender, workplace setting (i.e. hospital, ambulatory, rehabilitation, acute and long-term care) and geographical location (i.e. rural or urban) on job satisfaction and retention among rehabilitation professionals [41-43]. Furthermore, no empirical studies have examined conceptual frameworks for organizing recruitment and retention strategies for rehabilitation therapists. To address this gap, this research identified recruitment and retention strategies from the literature for rehabilitation professionals and determined their importance and feasibility using expert panels.\n\nMethods\nPhase 1: Literature Review\nIdentification of recruitment and retention strategies\nA review of the literature was conducted to identify recruitment and retention strategies for rehabilitation therapists. In this study, rehabilitation professionals were defined as physical therapists (PTs), occupational therapists (OTs) and speech-language pathologists (SLPs). Both published and non-empirical literature was accessed in this review. Keywords used to search for relevant published studies in the Consolidated International Nursing and Allied Health Sciences Library (CINAHL) (1982 to 2005) and Medline (1996 to 2005) included: \"health human resources or health manpower\", \"rehabilitation or rehabilitation professionals or vocational\", \"allied health professionals or personnel\", \"recruitment strategies\", \"retention strategies\", \"physical therapist or physiotherapist\", \"occupational therapy or occupational therapist\", \"speech-language pathologist or speech-language pathology\". Non-empirical literature searches were made on international and national on-line catalogues and publications from health organizations, professional associations, and hospital and home care organizations. International reports were limited to developed countries since the purpose of this study was to identify strategies appropriate to the Ontario setting.\n\nOrganization and consolidation of strategies\nThere was a paucity of peer-reviewed studies obtained exploring rehabilitation HHR strategies, therefore the majority of strategies were selected from grey literature reports from international, national and provincial health organizations. From the literature review, 107 potential strategies were identified according to their relevance to HHR issues for rehabilitation in Ontario. These strategies were then categorized into two broad groups: A) Recruitment and Retention (n = 73), and B) Education (n = 34). The majority of strategies were not specific to rehabilitation professionals and they were reviewed by a group of three individuals collectively (rehabilitation researcher, manager, and clinician) for duplication, clarity, action focused properties and appropriateness to the Canadian or Ontario setting. When necessary, a small number of strategies were re-worded to be relevant to a rehabilitation context. This analysis resulted in the selection of 40 Recruitment and Retention and 24 Education strategies. Only 14 recruitment and retention and six education strategies were obtained from peer-reviewed articles. Since the majority of strategies were identified from the grey literature, it was not surprising that there was no apriori peer-reviewed conceptual framework that reflected the breadth of the strategies obtained from the literature review. As a result, the themes used by the Health and Community Services Human Resources Sector Study in Newfoundland and Labrador [44] formed the basis for the organizational framework for this study since they aligned with the identified strategies. Each group was further categorized into the five themes (three for Recruitment and Retention and two for Education). The three Recruitment and Retention strategy themes were: (1) Quality of Worklife and Work Environment [n = 19]; (2) Workload and Skill Mix [n = 6]; and (3) Financial Incentives and Marketing [n = 15]. The two Education strategy themes were: (1) Education and Training [n = 11] and (2) Professional Development (n = 13).\n\n\nPhase 2: Expert Panel\nParticipant Selection\nOnce this study was approved by the Research Ethics Board at the University of Toronto, key informants who participated in a previous study regarding rehabilitation supply and demand at the University of Toronto [21] were asked to nominate potential participants for the panels. The selection criteria considered were acknowledged leadership in the panel member's specialty, expertise in recruitment and retention or education and training of rehabilitation professionals. Absence of conflicts of interest, geographic diversity, and diversity of practice setting were also considered. After purposefully selecting the initial list of candidates from among the nominations, each nominee was contacted to establish their interest and availability. Those who expressed an interest in participating were asked to send their curriculum vitae to help the research team evaluate their contributions to their field of expertise. Once candidate panelists were selected, each received a letter explaining the expert panel process and consent form. Two separate panels were constructed: one for Recruitment and Retention (n = 8) and the other for Education (n = 9) (Table 1). The size of the panel was large enough to permit diversity of representation while still being small enough to allow all participants to be involved in the group discussion [45].\n\nExpert Panel Process: Round 1 Survey\nA modified-delphi technique was then used for the expert panel process, [46,47] based on the RAND/UCLA appropriateness method [45]. In round 1, members of the Recruitment and Retention panel were sent an electronic survey containing the 40 strategies identified from the literature review and the Education panelists were also sent an electronic survey with 24 strategies. For Round 1, each panel was asked to rate the strategies using a nine-point Likert-type rating scale that ranged from \"none\" (1) to \"maximum\" (9), on two key dimensions: Feasibility and Importance. Feasibility was defined as the practicality and cost implications of the strategy and was rated from the respondents' perspective. Importance was defined as how valuable, appropriate and useful the strategy could be for rehabilitation HHR planning in Ontario. At the end of the survey, panelists were given the opportunity to suggest additional strategies that they felt were appropriate to consider. Once completed, panelists were asked to return the survey to the study office by email or fax prior to the expert panel meeting in Round 2. Data from each questionnaire were entered into a spreadsheet and tabulated. Descriptive statistics were calculated for each strategy using frequency distributions and proportional percentages of respondents. Importance and feasibility rankings were based on the percentage of expert panelists' low, medium and high ratings.\n\nExpert Panel Process Round 2: Expert Panel one-day meeting\nAfter the independent completion of the survey, each panel was convened separately for a one-day meeting for final discussions, debates and consensus voting to decide on strategies [48]. A strategy that had been scored 7, 8 or 9 for both feasibility and importance by two-thirds of the panel was considered a high rating. Strategies that had a combination of medium and high scores between 4 and 9 in either of the two dimensions were considered medium rated strategies, while low rated strategies had scores between 1 and 3 for both dimensions.\nOn the day of the meeting, the panelists were given a copy of the aggregated survey results indicating the ratings of all of the strategies. High and low rated strategies were not discussed as there was already consensus, whereas all medium rated strategies were subject to discussion. Using a nominal group process [47], each strategy was discussed in turn, and panelists were given an opportunity to raise any issues or concerns regarding the clarity and wording of each strategy. Each of the strategies discussed were then individually rated a second time by each panelist in an attempt to reach further consensus.\n\n\n\nResults\nSelection of strategies for Round 1: Modified Delphi process\nFollowing Round 1 rating of the 40 identified strategies, the Recruitment and Retention panel reached consensus on 12 strategies. However, 14 had a combination of high/medium importance and feasibility ratings and 14 had medium ratings on both dimensions, therefore it required further discussion. An additional strategy regarding family relocation programs was added by this panel.\nThe Education Panel ranked 16 of 24 strategies with high importance and feasibility after Round 1. Since there were only eight strategies with medium ratings, this expert panel decided to review all the strategies at the face-to-face meeting to discuss the rationale that would explain why some of the highly rated strategies were not already implemented and to come to a consensus on the other eight medium rated strategies. They also added an additional strategy for career paths.\n\nSelection of strategies for Round 2: Face-to-face meeting\nA total of 34 strategies were identified by both the Recruitment and Retention and Education expert panels as being important and feasible for the development of a HHR plan for recruitment and retention of rehabilitation professionals. Under the Recruitment and Retention theme, seven were categorized as Quality of Worklife and Work Environment; two were Workload and Skill Mix, and another seven were Financial Incentives and Marketing. As for the Education panel, five were categorized as Education and Training strategies while the other thirteen were related to Professional Development.\nAs indicated in Table 2, at the end of the second round of voting, the Recruitment and Retention panel had a total of 16 highly important and feasible strategies, 8 high/medium importance and feasibility, 8 medium and 9 low ratings for both dimensions. The Education panel on the other hand had a total of 18 high, 1 high/medium, 3 medium and 3 low rating strategies.\n\nRecruitment and Retention Strategy Rankings\nTable 3 provides a detailed description and ranking of each of the recruitment and retention strategies that were rated highly important and feasible. The importance and feasibility rankings were based on the largest proportion of panel members rating a strategy a 7, 8 or 9. The overall combined ranking was based on the average of the proportion of these two dimensions. Among these selected strategies, the majority were classified under Quality of Worklife and Work Environment (44%) and Financial Incentives and Marketing (44%), followed by Workload and Skill Mix (12%). It should be noted that some strategies had equal rankings; therefore the total number of rankings did not equal the total number of strategies.\nRecruitment and retention strategies that had a combination of high and medium ratings in either of the two dimensions included: sense of empowerment in promoting healthy work environments; team-building exercises; developing participatory decision-making systems; improving rural working conditions; recognizing work-life balance; creating an environment where staff are valued; optimizing scope of practice and work-management autonomy. Strategies that had medium importance and feasibility ratings included: resolving concerns about liability and accountability in collaborative practice; recruiting international trained therapists; opportunity to work in different settings; interprofessional payment schemes; family leave; staff recognition and creating a position for a provincial health professional recruiter. Low importance and feasibility strategies included: word of mouth references; bursaries and retention bonuses; exchange employment opportunities; health promotion; retention workshop; 80–20 staffing model (80% clinical and 20% learning new skills or training others); using recruitment agencies and providing recruitment bonuses (Table 4).\n\nEducation Strategy Rankings\nEducation strategies that were rated highly important and feasible are described in Table 5. The majority of strategies in this group tend to be in the area of Professional Development (72%), more so than Education and Training (28%).\nThe medium rated education strategies included: expand interprofessional education; provide incentives for students interested in rural practice; summer mentorship programs for high school students; and aboriginal student support program. The strategies that were considered neither feasible nor important included: using return of service contracts after professional development; create a tiered pathway approach through modular education and laddered credentialing and in accreditation standards allow greater use of rural practice sites (Table 6).\nSince the purpose of the panel was to identify recruitment and retention and education strategies that could inform the development of a HHR plan for rehabilitation professionals, there was also discussion about contextual factors that would influence a plan. Panelists commented that key factors to consider prior to implementation of these strategies should include workplace setting, geographical location (i.e. urban and rural) and gender issues.\n\n\nDiscussion\nThe purpose of this study was to identify recruitment and retention strategies to inform the development of HHR planning for rehabilitation professionals in Ontario. This study highlights that Quality of Worklife and Work Environment, Financial Incentives and Marketing and Professional Development are the three major areas of focus when developing a competitive HR plan in the rehabilitation sector.\nQuality of Worklife and Work Environment\nQuality of Worklife and Work Environment strategies ranked among the top category for recruitment and retention of rehabilitation therapists. This has also been found among nurses [44] where it was reported that addressing such factors can affect the overall success of the program [49]. Specifically, our findings showed that the top ranked strategy for both importance and feasibility was improving and maintaining the safety of rehabilitation professionals in the workplace. Specific strategies that could reduce aggression, abuse and violence in the workplace include: zero tolerance policies, access to employee support programs and providing assistance to rehabilitation professionals who work alone (i.e. home care and rural and remote areas). Since there is less control over the environment in the home care setting, safety may become a greater concern in one practice setting over another. This might suggest that because there is less control in environments such as home care, remote areas, and psychiatric settings, maintaining safety will be more difficult and that solutions will need to be tailored to these settings in order to ensure retention of providers. Although no studies have looked at implementing personal safety strategies for home care therapists, written policies and procedures for home care nurses during inclement weather and for dealing with abusive or dangerous patients, families and neighbourhoods have been reported [50].\nIn addition to the above, ensuring open and timely communication between employer and worker was also ranked highly among the recruitment and retention strategies. Examples of strategies include: open door policies, employee advisory committees and regular staff meetings and evaluations. Although these strategies were reported to be used among organizations providing services to persons with developmental disabilities in Alberta, there was no description of the organizations, sample size or methodology [51]. Similar findings were found in a qualitative study among 16 nurses working from diverse practice settings (acute, long-term care, rehabilitation and community; from both urban and rural areas) in a health region in western Canada. From the semi-structured interviews, study participants expressed a desire for improved consultation and communication with nurses regarding changes to the health care system [52].\n\nFinancial Incentives and Marketing\nAnother area that was ranked highly was marketing strategies to increase high school student and public awareness of rehabilitation careers. These specific strategies were also recommended by the Ontario Hospital Association (OHA) in order to establish a competitive position for Ontario hospitals in respect of recruitment and retention of health care professionals [53]. Similar strategies have been developed by the American Physical Therapy Association (APTA) in response to the declining number of students applying to Physical Therapy Education Programs [54]. To address this trend, APTA developed a campaign to promote Physical Therapy as the profession of choice to high school and college students across the United States. The potential components of their plan included: developing a \"Recruiting Kit\" for educators, students and various APTA members to be used in the high school and college settings to introduce Physical Therapy as a career; establishing public relations initiatives that demonstrate the role of Physical Therapy in the public arena targeting minority groups that are underrepresented in the profession; and creating alliances with professional associations of high school guidance counsellors and educators. Based on our finding, the APTA model may have applicability in Ontario.\n\nWorkload and Skill Mix\nOf the six Workload and Skill Mix strategies only two were highly rated: implementing a caseload management database and using support personnel. Caseload management has been identified in the literature as an issue affecting all three rehabilitation professions. For example, in physiotherapy, Christie's study [55] found that caseload expectations tended to be significantly higher than the reality and that caseload varies across different programs. Similarly, the Canadian Association of Speech-Language Pathologists and Audiologists (CASPLA) survey indicated that factors affecting the workload of SLPs include delivery models, client disorder, severity and work setting [56]. A literature review and environmental scan undertaken by the Canadian Association of Occupational Therapists (CAOT) proposed that guiding principles for caseload management should include: evidence-based occupational therapy, cost-effectiveness, accountability, professional leadership and expert judgment, comprehensiveness and flexibility [57]. Therefore, upon implementation of a caseload management database for rehabilitation, key factors to consider include workplace setting and client service delivery models.\nThe other highly rated Workload and Skill Mix strategy was the use of support personnel (i.e. physiotherapy assistants or exercise therapists) to increase efficiency of utilization of scarcer and higher order rehabilitation competencies. Considerations for implementing this strategy include addressing key issues such supply, standards of education, standards of practice and accreditation. These are highlighted in an article by Salvatori [58] who reported that the actual number of OT personnel delivering OT services in Canada remains unknown and that there are no national standards of education nor accreditation process for OT assistants. CAOT believes that in order to utilize support personnel appropriately, studies on human resource needs for occupational therapy and support personnel are first needed with input from OTs, stakeholders, funders, decision makers and health policy planners [59]. Since there is a lack of competency profiles related to the role, responsibilities, and supervision of assistants, particularly with regards to delivering services in unsupervised community-based settings, the type of workplace setting where this strategy may be implemented should be considered [58].\n\nEducation and Training\nGiven that 60% of highly rated Education and Training strategies targeted rural and remote practices underscores the importance of specific strategies for rural and remote areas in the development of a HHR plan. The need to build on existing mechanisms to expand the availability of rural and remote clinical placements by providing financial and accommodation support was ranked among the top two most important and feasible education strategies for rehabilitation professionals. Not only has this strategy been used as a recruitment tool for rehabilitation students, it has also been reported by Solomon et al. [34] to be effective in retaining OTs and PTs in underserviced Northwestern Ontario communities. Respondents from Solomon et al.'s study reported that the top three benefits of supervising students were that it stimulates thinking, it provides opportunity to contribute to the profession and that it provides access to current information. The reported disadvantages however was that it was time-consuming and students contributed stress to the working environment. Similarly, a two-part study found substantial gaps between financial incentives students deem important in the creation of an appealing clinical placement opportunity and the actual provisions offered to them by Southeastern Ontario communities [60,61]. Although OT and PT students reported that they were more willing to complete a clinical placement in an underserviced community if provided travel stipends, rent-free housing and interprofessional education opportunities, the majority of these incentives were only available to medical students. In addition to training students for rural and remote practice, a longitudinal study reported that perceived opportunity for career development was the most significant factor related to job turnover and regional attrition among physiotherapists working in Northern Ontario [5]. Therefore developing workforce strategies for rehabilitation therapists working in these areas should be among one of the priority areas in HHR planning.\n\nProfessional Development\nOur findings indicate that the theme with the largest number of strategies that were considered important and feasible to implement as part of HHR planning was professional development. Many were specific to rural and remote areas. Although the importance of continuous professional development (CPD) in recruitment and retention is well recognized, a Canadian study reported several barriers to its implementation [62]. In the case of OTs employed in public settings in Nova Scotia, Townsend et al. (2006) found that the most powerful deterrent for CPD was the lack of support from workplace policies. Based on their study results, the use of CPD as a recruitment and retention strategy was highly influenced by gender issues, work-life balance, career advancement, working conditions, geographical location, professional versus employer responsibility, and employee benefits. Although occupational therapy is a female-dominated profession, workplace policies did not address issues of gender. For example, therapists in this study indicated that CPD competes with family commitments, therefore these activities are \"done largely during personal time, mainly at their own cost, and on top of childcare, eldercare, homemaking and other family responsibilities\" [62]. In addition, heavy workloads, lack of salary and career incentives, and lack of policy and funding support are all barriers to CPD. These issues become more pronounced in rural and remote settings because smaller communities often only have one therapist; hence the systemic pressure of workload demands makes it difficult for the therapist to leave patient care. OTs from this study also questioned who was responsible for CPD. Some felt that it was the professional's responsibility while others felt that it was the responsibility of the employer to provide CPD opportunities. The primary limitation employers faced was the lack of financial resources, however giving employees time off without pay was an alternative strategy utilized instead of funding professional development activities. Although there are professional and provincial variations in funding for CPD across Canada, these results are informative in that it highlights the need for employers to consider how workplace policies can affect recruitment and retention strategies.\nLimitations of this study should be noted. First, the majority of the strategies were obtained from the grey literature that is not subject to the same scrutiny as the peer-reviewed literature. Second, almost none of the strategies were specifically developed for rehabilitation professionals and in many cases had to be re-worded to fit the rehabilitation context. There is a lack of research on rehabilitation clinicians' perspectives on recruitment and retention strategies; therefore future research should focus on investigating this area. Third, during the face-to-face meeting, bias could have resulted from panelists whose opinion may have influenced others significantly, especially if members came from similar workplace settings. The facilitator of the expert panels however, followed a strict process for managing the discussion and ensured that all panelists were given the opportunity to express their opinions.\nFinally, although some strategies such as competitive wage packages, training/growth opportunities and professional development are viewed as both a recruitment and retention incentive, other strategies do not overlap and are appropriate for only one of the two tasks. For example, increasing public awareness of rehabilitation careers, providing rural and remote orientation packages and family relocation programs are only appropriate for attracting a worker while ensuring open and timely communication may be seen as a strategy only for retention. Future research should therefore consider studying recruitment and retention strategies separately so that a distinction between the two can be made.\n\n\nConclusion\nThis study identified 34 strategies that should be considered as important and feasible for implementation as part of HHR planning for rehabilitation professionals. Although the highest ranked strategies focused on areas of Quality of Worklife and Work Environment, Financial Incentives and Marketing and Professional Development, key factors that need to be considered in the context of implementation include: workplace setting, geographical location and gender issues. While this is the first study to our knowledge that provides a comprehensive list of recruitment and retention strategies relevant to rehabilitation professionals, more information is needed for the development of a HHR plan. Information on trends in labour force participation as well as knowledge regarding the use and effectiveness of recruitment and retention strategies for rehabilitation professionals is needed. More importantly, the success of implementing and sustaining such strategies requires future research to validate these strategies from the perspective of rehabilitation clinicians and human resource decisions makers (i.e. local government, stakeholders, etc.) so that specific barriers and challenges can be identified.\n\nCompeting interests\nThe authors declare that they have no competing interests.\n\nAuthors' contributions\nDT participated in the design of the study, conducted the literature review, analyzed and interpreted the results and drafted the manuscript.\nLMH, AD, DB and KB were involved in providing feedback and editing the content of the manuscript.\nMDL recommended participants for the expert panel and was involved in providing feedback and editing the content of the manuscript.\nSJ participated in the design of the study, recommended participants for the expert panel, organized and consolidated strategies, interpreted the data and edited the content of the manuscript.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\n\n" ], "offsets": [ [ 0, 40637 ] ] } ]

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[ { "id": "pmcA2589670__text", "type": "Article", "text": [ "The ineffectiveness of tobramycin combination therapy in Streptococcus faecium endocarditis.\nAbstract\nA patient required mitral valve replacement following ineffective antibiotic treatment of enterococcal endocarditis caused by Streptococcus faecium. Endocarditis had relapsed despite therapy with ampicillin and tobramycin for six weeks. A second relapse had occurred following treatment with penicillin and gentamicin. Initial failure of antibiotic therapy may be related to the known lack of in vitro and in vivo synergy between penicillin and tobramycin against S. faecium. Effective therapy of enterococcal endocarditis requires considerations of bacterial speciation, determination of high-level aminoglycoside resistance, and preferably adequate antibiotic synergy studies to assure effective therapy.\n\n\n\n\n THE YALE JOURNAL OF BIOLOGY AND MEDICINE 56 (1983), 243-249 \n\n The Ineffectiveness of Tobramycin Combination Therapy \n\n in Streptococcus Faecium Endocarditis \n\n JUDITH A. GOLDSTEIN, M.D.,a HOWARD COHEN, M.D.,a AND \n\n FRANK J. BIA, M.D., M.P.H.a,b \n\n aInfectious Disease Section of the Department of Medicine, and bThe Department of \n\n Laboratory Medicine, Veterans Administration Medical Center, \n\n West Haven, and Yale University School of Medicine, \n\n New Haven, Connecticut \n\n Received July 11, 1983 \n\n A patient required mitral valve replacement following ineffective antibiotic treatment of enterococcal endocarditis caused by Streptococcusfaecium. Endocarditis had relapsed despite therapy with ampicillin and tobramycin for six weeks. A second relapse had occurred following treatment with penicillin and gentamicin. Initial failure of antibiotic therapy may be related to the known lack of in vitro and in vivo synergy between penicillin and tobramycin against S. faecium. Effective therapy of enterococcal endocarditis requires considerations of bacterial speciation, determination of high-level aminoglycoside resistance, and preferably adequate antibiotic synergy studies to assure effective therapy. \n\n INTRODUCTION \n\n Enterococcal endocarditis requires special therapeutic considerations because the responsible organisms are relatively penicillin-resistant streptococci which require synergistic combinations of antibiotics to achieve acceptable cure rates [1,2]. The group D enterococci include three main species S. faecalis, S. faecium, and S. durans. S. faecium cause a minority of all cases of enterococcal endocarditis, in those instances in which enterococci have been speciated [3], but they have been more resistant both to penicillin and penicillin-aminoglycoside combinations than S. faecalis [4,5,6]. \n\n We describe a 64-year-old man with S. faecium endocarditis in whom a six-week course of ampicillin and tobramycin, followed by additional courses of penicillin and other aminoglycosides, failed to eradicate the organism from the patient's mitral valve. This case is of interest because therapeutic failure of ampicillin and tobramycin in S. faecium endocarditis has not been reported previously, but might have been predicted on the basis of previous in vitro and in vivo studies [6]. Although the need for both a penicillin derivative and an aminoglycoside in the therapy of enterococcal endocarditis is widely known, it is important to distinguish between the differing efficacies of penicillin-aminoglycoside combinations for treating various species of enterococci such as S. faecium. \n\n 243 \n\n Address reprint requests to: Judith A. Goldstein, M.D., Section of Infectious Diseases, Department of Medicine, Long Island Jewish-Hillside Medical Center, Queen's Hosp. Center Affiliate, 82-68 164th Street, Jamaica, NY 11432 \n\n Copyright c 1983 by The Yale Journal of Biology and Medicine, Inc. All rights of reproduction in any form reserved. \n\n GOLDSTEIN ET AL. \n\n CASE REPORT \n\n A 64-year-old male was in good health until December 1980, when he noted intermittent night sweats, malaise, fever, and fatigue. He received oral erythromycin for 14 days with transient improvement of symptoms. However, after completing therapy, symptoms reappeared and he noted a 15-pound weight loss with low-grade fever (99-100.5?F) during the two months preceding admission. There was no previous history of rheumatic or congenital heart disease. \n\n In March 1981, the patient was admitted to his community hospital where evaluation revealed a new apical systolic murmur radiating to the axilla. There were no petechiae, Janeway lesions, Osler's nodes, Roth spots, or splenomegaly. The hematocrit was 34.8 percent, WBC count 9,300 cells per cu mm with a differential count of 67 segmented forms, 9 bands, 15 lymphocytes, 7 monocytes, 1 eosinophil, and 1 basophil. The erythrocyte sedimentation rate (ESR) was 62 mm per hour (nl < 10 mm per hour) and the serum rheumatoid factor titer was 1:320. Chest films and electrocardiogram were reportedly normal. Group D streptococci grew from three sets of blood cultures. Enterococcal endocarditis was diagnosed and he was treated with six weeks of parenteral ampicillin (12 grams per day) and tobramycin (3 mg per kg per day). Resolution of symptoms occurred within several days after antibiotics were begun. Serum bactericidal titers against the organism, obtained during peak antibiotic levels, were 1:8 or greater on several occasions, and blood cultures were negative while the patient was receiving antibiotics. M-mode echocardiography demonstrated left atrial enlargement but no definable abnormalities of the mitral or aortic valves. Intravenous pyelogram, oral cholecystogram, cystoscopy, liver-spleen scan, and upper and lower gastrointestinal series were normal except for a few sigmoid diverticuli. Flexible signoidoscopy demonstrated both a small perianal fissure and hemorrhoids. Blood cultures two weeks after therapy were negative. \n\n Second Admission (June 19-August 4, 1981) \n\n In June 1981, he again noted intermittent fever, night sweats, and fatigue. Group D streptococci grew from three sets of blood cultures and he was admitted to the West Haven VA Medical Center for recurrent endocarditis. On examination a somewhat louder apical systolic murmur was noted. The ESR was 50 mm/hour and rheumatoid factor titer was 1:320. Serum complement levels were normal and cryoglobulins were not detectable. Chest films, EKG, and urinalysis were normal. Cardiac M-mode echocardiogram revealed a globular mass attached to the posterior mitral valve leaflet with prolapse into a slightly enlarged left atrium. A twodimensional echocardiogram confirmed mitral valve prolapse and suggested a posterior leaflet vegetation. Gallium citrate scan was negative. \n\n Three separate morphologic colony variants were isolated from blood, each identified as S. faecium by Dr. R.R. Facklam (Center for Disease Control, Atlanta, Georgia). The patient received intravenous penicillin (20 million units per day) and gentamicin (3 mg per kg per day) for six weeks with improvement. Peak serum bactericidal titers of 1:8 or greater were achieved against two of the colony variants; however, against the large colony morphotype, a titer of only 1:2 was obtained. An enlarged left atrium with intermittent fluttering and prolapse of the mitral valve was noted on echocardiography three weeks into therapy. Multiple blood cultures taken while the patient was receiving antibiotics were negative, as were those obtained 48 and 72 hours after discontinuation of antibiotics. \n\n 244 \n\n S. FAECIUM ENDOCARDITIS \n\n Third Admission (August 12-October 4, 1981) \n\n Withing a week following discharge, the patient again developed fever, nocturnal sweats, and malaise. S. faecium (large colony morphotype, and poorly growing small colony morphotype) grew from six sets of blood cultures obtained on admission. Penicillin (30 million units/day) and gentamicin (3 mg/kg/day) were again administered, initially achieving peak serum inhibitory and bactericidal dilutions of 1:16 and 1:8 against the organism, respectively. However, the organism had a lowlevel resistance to streptomycin (MIC < 125 /Ag/ml), and streptomycin (2 grams per day) was substituted for gentamicin two weeks into antibiotic therapy. Repeat echocardiograms showed irregular and shaggy densities of both mitral valve leaflets with partial prolapse. Cardiac catheterization demonstrated marked mitral valve prolapse with mitral regurgitation. A radiolucent filling defect was noted, suggesting a coronary artery embolus at the origin of the left anterior descending (LAD) artery, causing 75 percent occlusion of the lumen. The patient underwent mitral valve replacement, receiving a number 31 porcine Hancock bioprosthesis and bypass graft to the midportion of the LAD. The mitral valve was thickened with several ruptured chordae of the posterior leaflet noted, but no vegetations. The aortic valve appeared normal, with no visible septal or ring abscesses. The occlusion in the LAD was not approached to avoid embolizing distal fragments. \n\n Histopathologically the mitral valve showed mild fibrosis and myxoid degeneration without inflammatory changes. Bacterial and fungal stains were negative but S. faecium grew from fragments of the resected valve. Following surgery the patient received six additional weeks parenteral penicillin and streptomycin. Repeat blood cultures on this regimen and following therapy were negative. Evaluation six months following discontinuation of antibiotics showed no evidence of recurrent endocarditis. \n\n LABORATORY EVALUATION \n\n The minimum inhibitory and bactericidal concentrations of penicillin, ampicillin, and tobramycin against the S. faecium isolated from the patient's blood cultures \n\n TABLE 1 \n\n Minimum Inhibitory and Bactericidal Concentrations of Antibiotics, \n\n Against Streptococcus faecium Isolates from Blood Cultures \n\n Date Penicillin Ampicillin Tobramycin \n\n Organism Isolated MIC MIC MBC MIC MBC S. faecium, prior to ampicillin/ \n\n tobramycin therapy 3/17/81 2 1 2 >32 >32 S. faecium, after ampicillin/ \n\n tobramycin therapy \n\n a. Large colony \n\n variant 6/16/81 2 1 1 >32 >32 b. Medium colony \n\n variant 6/16/81 2 1 2 32 32 c. Small colony \n\n variant 6/16/81 4 2 4 >32 >32 aMIC and MBC are minimum inhibitory and bactericidal concentrations of antibiotics, respectively, in tig/ml. \n\n 245 \n\n GOLDSTEIN ET AL. \n\n FIG. 1. Time-kill curve demonstrat8 3- \\ \"'' ing the effects of various penicillinO aminoglycoside combinations on S. {73 2- \\ >, faecium obtained from the patient's \n\n blood cultures immediately prior to mitral valve excision. Note the lack of 4u, synergy between penicillin and tobra25kg/rm Streporrrychn t mycin against this organism when \n\n /0 units/mI Penicillin _ compared to synergistic combinations 0 4 8 12 6 20 24 of penicillin-gentamicin and penicillin\n\n HOURS streptomycin. \n\n after initial oral erythromycin therapy, and prior to therapy for endocarditis, are shown in Table 1. Following combined therapy with ampicillin and tobramycin and relapse of endocarditis, three morphological variants were isolated from blood cultures and also evaluated. \n\n Twenty-four hour time-kill curves for penicillin in combination with various aminoglycosides were performed by Dr. Robert Moellering on the S. faecium isolated from the patient's blood immediately prior to mitral valve excision and replacement (Fig. 1). Synergy was readily demonstrable against this organism by penicillin-streptomycin and penicillin-gentamicin combinations in vitro, but not by penicillin-tobramycin. \n\n DISCUSSION \n\n Among streptococci, the enterococci are unusual in their relative resistance to a broad spectrum of antimicrobial agents, and single-agent therapy is rarely bactericidal against them [2]. Since Hunter's original observations in 1947, it has become increasingly clear that effective synergistic combinations of antibiotics are necessary to successfully treat enterococcal endocarditis [1]. \n\n Although S. faecalis represents the majority of clinical enterococcal isolates, S. faecium nonetheless comprises 5-10 percent of these isolates in some series [3,4]. Moreover, major differences exist in antimicrobial susceptibility and resistance to penicillin-aminoglycoside synergism between these two enterococcal species. The MIC of penicillin against S. faecium is higher and this organism is more resistant to a number of different combinations of penicillin and various aminoglycosides than is S. faecalis [6]. \n\n The mechanisms of resistance exhibited by enterococci to penicillin-aminoglycoside synergy have been investigated. Clinically achievable levels of amino\n\n 246 \n\n S. FAECIUM ENDOCARDITIS \n\n glycosides are generally ineffective against enterococci. This intrinsic low-level resistance (MIC c 250 isg/ml) is thought to be the result of poor antibiotic penetration of the bacterial cell wall. However, in the presence of antibiotics that interfere with cell wall synthesis, there is enhanced aminoglycoside uptake [7]. In concert, these events are the basis for penicillin-aminoglycoside synergism. Ribosomal resistance of the 30S subunit to streptomycin and defective uptake of gentamicin in the presence of penicillin have been reported mechanisms of resistance among enterococcal isolates [8,9]. However, in the majority of instances, failure of synergy involves plasmid-mediated production of aminoglycoside-modifying enzymes. For streptomycin and kanamycin, plasmid-mediated enzymatic inactivation confers high-level resistance (MIC > 2,000 Ag/ml) and correlates with failure of these aminoglycosides to exert a synergistic effect when combined with penicillin [10,11]. Plasmid-mediated modifying enzymes have been found in both S. faecalis and S. faecium' [12]. Currently, approximately one-half of clinical enterococcal isolates demonstrate high-level resistance to streptomycin and kanamycin [13]. \n\n Combinations of penicillin with kanamycin, tobramycin, sisomicin, and netilmicin have consistently failed to demonstrate synergistic killing of S. faecium in vitro [6]. This failure of synergy occurs even when high-level resistance to these aminoglycosides is not present. The mechanism of resistance appears to be related to the production of an inactivating enzyme that acetylates the aminoglycoside substrate at the 6' position1 [14]. The genetic basis for production of this enzyme has not been well-defined and plasmid transfer experiments have thus far been unsucessful in demonstrating the encodement of this enzyme by extrachromosomal DNA [14]. \n\n Moellering et al. demonstrated in vivo, utilizing the rabbit model of endocarditis, that penicillin and netilmicin were not efficacious in the treatment of endocarditis caused by a low-level aminoglycoside-resistant strain of S. faecium [6]. Although combinations of penicillin with tobramycin, kanamycin, or sisomicin were not evaluated, the authors postulated that the same ineffectual result would have occurred. In the present case, the recurrence of S. faecium endocarditis after six weeks of therapy with ampicillin and tobramycin confirms the therapeutic and clinical significance of their data, and emphasizes that tobramycin is not an aminoglycoside to be used for treatment of serious S. faecium infections. \n\n Bacterial tolerance has been suggested as a possible basis for therapeutic failures, particularly in the treatment of infections caused by Staphylococcus aureus with defects in the production of autolysins. MBCs are generally several-fold higher than MICs and this phenomenon appears to be associated with the autolysin defect [5]. Lorian has also described the formation of numerous aberrant cross-walls by S. faecalis grown in the presence of subinhibitory concentrations of penicillin [15]. MBCs were only slightly higher than MICs for the three S. faecium variants obtained from our patient, and they did not appear to be tolerant strains of S. faecium. There was no evidence that any of the morphological variants isolated were unusually resistant to antibiotics. Therefore, subsequent failure of therapy could not be explained on the basis of antibiotic resistance patterns. The initial failure of ampicillin and tobramycin therapy may have allowed the infecting organism to become better established and more difficult to eradicate from the valve. Alternatively, a \n\n 'Wennersten C, Moellering RC Jr: Mechanism of resistance to penicillin-aminoglycoside synergism in Streptococcus faecium. Proceedings of the 1th International Congress of Chemotherapy and 19th Interscience Conference on Antimicrobial Agents and Chemotherapy 1:710-711, 1979 \n\n 247 \n\n 248 GOLDSTEIN ET AL. \n\n persistent focus of infection causing the LAD lesion seen by arteriography may have slowly resolved and accounted for the apparent failure to respond to synergistic combinations of antibiotics. \n\n In summary, our patient was treated for Streptococcusfaecium endocarditis with both ampicillin and the aminoglycoside antibiotic, tobramycin. Relapse of endocarditis might have been anticipated on the basis of previous experimental data showing lack of synergy when tobramycin is used against this organism. This case graphically illustrates the relevance of synergy studies to therapeutic considerations in the treatment of endocarditis. However, subsequent therapy with synergistic combinations of antibiotics did not result in cure. The data do not implicate tolerant organisms as a cause for relapses. Failure of therapy may have been related to the presence of protected organisms in vegetations which were seen on echocardiograms, and also suggested by the presence of a possible coronary artery embolus seen on coronary arteriograms. \n\n In conclusion, speciation of isolates suspected of causing endocarditis and adequate synergy studies of antibiotic combinations are indicated before a long and expensive course of therapy with antimicrobial agents is undertaken for this disease. However, as this case also illustrates, demonstration of synergism in vitro does not assure clinical cure. \n\n ACKNOWLEDGEMENTS \n\n Dr. Goldstein was supported by training grant Al 07033-05 from the National Institute of Allergy and Infectious Diseases. \n\n The authors gratefully acknowledge the advice, laboratory assistance, and careful review of this manuscript by Dr. Robert C. Moellering, Jr. \n\n The authors also wish to thank Dr. Howard S. Forster for referring this patient to us, Ms. Mary Murray and Deborah Beauvais for manuscript preparation, and Ms. Gertrude Barden, MT (ASCP), MHS, for technical assistance and advice in evaluation of this patient. \n\n REFERENCES \n\n 1. Hunter TH: Use of streptomycin in the treatment of bacterial endocarditis. Am J Med 2:436-442, \n\n 1947 \n\n 2. Moellering RC Jr, Krogstad DJ: Antibiotic resistance in enterococci. Microbiology 293-298, 1979 3. Facklam RR: Recognition of group D streptococcal species of human origin by biochemical and \n\n physiological tests. Appl Microbiol 23:1131-1139, 1972 \n\n 4. Toala P, McDonald A, Wilcox C, et al: Susceptibility of group D streptococcus (enterococcus) to 21 \n\n antibiotics in vitro, with special reference to species differences. Am J Med Sci 258:416-430, 1969 5. Shungu DL, Cornett JB, Shockman GD: Morphological and physiological study of autolytic\n\n defective Streptococcus faecium strains. J Bacteriol 138:598-608, 1979 \n\n 6. Moellering RC Jr, Korzeniowski OM, Sande MA, et al: Species-specific resistance to antimicrobial \n\n synergism in Streptococcusfaecium and Streptococcusfaecalis. J Infect Dis 140:203-208, 1979 \n\n 7. Moellering RC Jr, Weinberg AN: Studies on antibiotic synergism against enterococci. 11. Effect of \n\n various antibiotics on the uptake of '4C-labelled streptomycin by enterococci. J Clin Invest 50:2580-2584, 1971 \n\n 8. Zimmermann RA, Moellering RC Jr, Weinberg AN: Mechanisms of resistance to antibiotic \n\n synergism in enterococci. J Bacteriol 105:873-879, 1971 \n\n 9. Moellering RC Jr, Murray BE, Schoenbaum SC, et al: A novel mechanism of resistance to penicillin\n\n gentamicin synergism in S. faecalis. J Infect Dis 141:81-86, 1980 \n\n 10. Krogstad DJ, Korfhagen TR, Moellering RC Jr, et al: Plasmid-mediated resistance to antibiotic \n\n synergism in enterococci. J Clin Invest 61:1645-1653, 1978 \n\n 11. Krogstad DJ, Korfhagen TR, Moellering RC Jr, et al: Aminoglycoside-inactivating enzymes in \n\n clinical isolates of Streptococcusfaecalis: An explanation for resistance to antibiotic synergism. J Clin Invest 62:480-486, 1978 \n\n S. FAECIUM ENDOCARDITIS 249 \n\n 12. Courvalin PM, Shaw WV, Jacob AE: Plasmid-mediated mechanisms of resistance to \n\n aminoglycoside-aminocyclitol antibiotics and to chloramphenicol in group D streptococci. Antimicrob Agents Chemother 13:716-725, 1978 \n\n 13. Calderwood SA, Wennersten C, Moellering RC Jr, et al: Resistance to six aminoglycosidic \n\n aminocyclitol antibiotics among enterococci: Prevalence, evolution, and relationship to synergism with penicillin. Antimicrob Agents Chemother 12:401-405, 1977 \n\n 14. Bisno AL: Treatment of infective endocarditis. New York, Grune and Stratton, 1981, p 90 \n\n 15. Lorian V: Effects of subminimum inhibitory concentrations of antibiotics on bacteria. In Antibiotics \n\n in Laboratory Medicine. Edited by V Lorian. Baltimore, Williams and Wilkins, 1980, p 342 " ], "offsets": [ [ 0, 21701 ] ] } ]

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[ { "id": "pmcA324075__text", "type": "Article", "text": [ "Structural organization of a 17 KB segment of the alpha 2 collagen gene: evaluation by R loop mapping.\nAbstract\nA recombinant phage, SpC3, containing a 17 kb genomic DNA insert representing approximately 60% of the 3' portion of the sheep collagen alpha 2 gene, was evaluated by electron microscopic R loop analysis. A minimum of 17 intervening sequences (introns) and 18 alpha 2 coding sequences (exons) were mapped. With the exception of the 850 base pair exon located at the extreme 3' end of the insert, all exons contained 250 base pairs or less. The total length of all the exons in SpC3 was 3,014 base pairs. The length distribution of the 17 introns ranged from 300 to 1600 base pairs; together, all of the introns comprised 14,070 base pairs of SpC3 DNA. Thus, the DNA region required for coding the interspersed 3 kb of alpha 2 collagen genetic information was 5.6 fold longer than the corresponding alpha 2 mRNA coding sequences.Images\n\n\n\n\n\n\n Nucleic Acids Research \n\n Structural organization of a 17 KB segment of the a2 collagen gene: evaluation by R loop mapping Millie P.Schafer, Charles D.Boyd, Paul Tolstoshev and Ronald G.Crystal \n\n Pulmonary Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20205, USA \n\n Received 13 February 1980 \n\n ABSTRACT \n\n A recombinant phage, SpC3, containing a 17 kb genomic DNA insert rep\n\n resenting approximately 60% of the 3' portion of the sheep collagen a2 gene, was evaluated by electron microscopic R loop analysis. A minimum of 17 intervening sequences (introns) and 18 a2 coding sequences (exons) were \n\n mapped. With the exception of the 850 base pair exon located at the extreme 3' end of the insert, all exons contained 250 base pairs or less. The total length of all the exons in SpC3 was 3,014 base pairs. The length distribution of the 17 introns ranged from 300 to 1600 base pairs; together, all of the introns comprised 14,070 base pairs of SpC3 DNA. Thus, the DNA region \n\n required for coding the interspersed 3 kb of a2 collagen genetic information was 5.6 fold longer than the corresponding a2 mRNA coding sequences. \n\n INTRODUCTION \n\n Type I collagen, composed of two al(I) and one a2 polypeptide chains, is the most abundant of the five known mammalian collagen types. It is a major extracellular constituent of tissues such as bone, tendon, skin and lung where, because of its great tensile strength, it plays an important \n\n role in tissue structure and function. The polypeptides comprising type I \n\n collagen are synthesized in long, precursor forms, referred to as pro al(I) and pro a2 chains,1 each containing approximately 1500 amino acid residues (1). \n\n 1 The terminology for the primary translation products of the various \n\n collagen messenger RNAs is still in a state of flux. It is known that al(I) and a2 chains are synthesized in precursor forms; these are currently termed pro al(I) and pro a2 chains (1). However, recent studies (43,44) have shown that the actual primary translation product of al(I) mRNA is somewhat larger \n\n than the pro al(I) chain; this translation product has been termed a \"pre-pro al(I) chain.\" Although the corresponding \"pre-pro a2 chain\" likely exists, it has not yet been characterized. For simplicity, therefore, until the \n\n proper terminology for the primary translation product is clarified, we have \n\n chosen to use the terms \"al(I)\" and \"a2\" to refer to the type I collagen mRNAs and collagen structural gene sequences. \n\n ?) IRL Press Umited, 1 Falconberg Court, London W1V 5FG, U.K. \n\n Volurne 8 Number 10 1980 \n\n 2241 \n\n Nucleic Acids Research \n\n There is increasing evidence that the quantities of al(I) and a2 \n\n chains produced by cells may be regulated, in part, at the genomic level \n\n (1). However, to understand the processes which mediate their expression, \n\n it is necessary to understand the structural organization of these collagen genes and the various enzymes that modify the collagen chains after its \n\n synthesis (1). As an initial approach to this problem, we have recently \n\n isolated a recombinant bacteriophage, termed SpC3, containing approximately 60% of the a2 gene for type I collagen. Partial characterization of this \n\n a2 gene demonstrated that, like most other structurally evaluated eukaryotic genes, the coding sequences are interspersed with intervening sequences (2). The present study further characterizes this portion of the a2 \n\n gene by utilizing electron microscopic R loop analysis to map the coding regions (exons) and intervening sequences (introns) contained within the sheep genomic insert of SpC3. The data suggests that the structural \n\n organization of this portion of the a2 collagen gene represents 3 kb of coding information that is interspersed in a complex fashion with 17 introns over 17 kb of genome. \n\n MATERIALS AND METHODS \n\n Isolation and characterization of a2 collagen recombinant clone, SpC3. \n\n The sheep a2 collagen recombinant clone, SpC3, containing a portion of the sheep a2 gene, was isolated as previously described (2). Briefly, \n\n high molecular weight fetal sheep liver DNA was extracted by the method of Blin and Stafford (3), and 15-20 kb DNA fragments, resulting from partial Eco RI digestion, were isolated and ligated to the left and right arms of Charon 4A. The recombinant DNA was then packaged in vitro and the sheep \n\n genomic library amplified (4-6). Screening of the sheep genomic library was \n\n conducted utilizing [32 P]-labeled fetal sheep tendon type I collagen cDNA and \n\n several positive recombinant phages were isolated. One recombinant, containing a 17 kb sheep genomic insert, was demonstrated to have coding sequences corresponding to 60% of the 3' end of a2 mRNA. Prior studies showed that the insert in SpC3 was not due to ligation of noncontiguous restriction fragments and/or genetic rearrangement during the cloning process (2). Electron mi croscopy. \n\n To visualize the recombinant bacteriophage DNA structure complementary to a2 collagen mRNA, 1 iig of SpC3 DNA was lyophilized with 1 ig or 100 ng of mRNA \n\n to give DNA:mRNA ratios of 1:1 and 10:1, respectively. These were then dissolved in 10 i1 of R loop buffer [70% (v/v) deionized formamide, 0.1 N-[tris (hydroxy\n\n 2242 \n\n Nucleic Acids Research \n\n methyl)methyl] glycine (tricine)-NaOH, pH 8.0, 0.5 M NaCl, and 0.01 M ethyl\n\n enediaminetetraacetate (EDTA)], incubated in sealed capillary tubes at 520 for 14 hr (7), and then diluted 1:10 with R loop buffer. Two spreading methods were used to subsequently prepare the hybrids for visualization in the electron microscope. \n\n 70% formamide method. Immediately before spreading onto a deionized \n\n water hypophase, the nucleic acid mixtures were diluted an additional 10-fold in R loop buffer and cytochrome c, 100 vg/ml, was added. \n\n Urea-formamide method. The nucleic acid solutions were spread using a modification of the method of Westphal and Lai (8,9). The hyperphase contained 55% formamide, 2.6 M urea, 0.009 M EDTA, 0.09 M tricine-NaOH, pH 8.0, and the nucleic acid mixture (approximately 0.1 to 1.0 ig/ml). \n\n The mixture was heated at 40? for 30 sec, placed in ice water, and then allowed to reach room temperature. Cytochrome c, 100 pg/ml, was added immediately before spreading onto a deionized water hypophase. \n\n The nucleic acid-protein films from both methods were absorbed onto \n\n parlodion-coated grids, stained with uranyl acetate (10), dehydrated in 90% ethanol and rotary-shadowed with platinum-palladium (80:20) at an angle of 50. Micrographs were taken with a Siemens Elmiskop 101 electron microscope at an original magnification of 10,000 and an accelerating voltage of 60 kv. Nucleic acid lengths were measured at a final magnification of 43,000 with a \n\n Hewlett-Packard 9810A calculator equipped with a 9864A digitizer using pBR322 and fX-174 double and single stranded DNA, respectively, as internal length standards. \n\n Approximately 10,000 hybrid molecules were screened for these studies; approximately 200 represented molecules containing unambiguous regions \n\n appropriate for quantitative analysis. The mean lengths of each intron and exon were determined for each method and the data expressed as mean ? \n\n standard deviation. Nucleic acid lengths of < 50 base pairs could not be \n\n accurately determined using the methods outlined above. Thus, in the cases \n\n where introns or exons were of this length or less, accurate error estimates could not be made. \n\n Bi ohazard precauti ons. \n\n The construction and screening of the sheep genomic library together with amplification of pCg45 and preparation of high titre lysates of chimaeric \n\n Charon 4A were performed under the physical and biological containment levels specified by the NIH guidelines for recombinant DNA research (11). \n\n 2243 \n\n Nucleic Acids Research \n\n RESULTS \n\n Location and orientation of the intervening and a2 mRNA coding sequences in the SpC3 recombinant DNA. \n\n Hybridization of fetal sheep tendon type I collagen mRNA to SpC3 DNA \n\n yielded a complex R loop pattern in the region where the sheep genomic insert was expected to be ligated to the arms of the lambda vector, Charon 4A. To \n\n confirm that these hybridization events were restricted to the inserted sheep DNA fragment, duplex DNA strands on both sides of this hybridization area were measured. Using pBR322 as an internal length standard, the left arm of the phage was found to contain 21,780 ? 1500 bp, and the right arm, 11,950 ? \n\n 1030 bp, corresponding to the report values for the left and right arms, respectively, of Charon 4A DNA (5,6). \n\n The hybridization of SpC3 to a2 mRNA resulted in the destabilization of \n\n the inserted helical duplex DNA fragment such that one DNA strand was displaced (Figure 1). Occasionally, the displaced DNA strand was mostly absent, probably due to fragmentation induced by accidental mechanical shearing. This event considerably enhanced the visualization and ordering of the introns. \n\n As the a2 mRNA annealed to complementary DNA regions present in one \n\n DNA strand, numerous single-stranded loops resulted. These loops, referred to as introns, represented DNA sequences not complementary to the mRNA. Although, by convention, introns are usually sequentially labeled 5' to 3', an opposite order had to be employed in this case as SpC3 does not contain the sequences coding for the 5' end of sheep a2 mRNA. Infrequently, all introns \n\n were clearly observed within the same hybrid molecule (Figure 1). The largest introns were towards the left arm of Charon 4A and were more clustered than the introns at the opposite end of the hybrid. Thus, much of the genetic \n\n information coding for the a2 mRNA was contained in that half of the hybrid attached to the right arm. \n\n A small non-hybridized tail was consistently found in the region where \n\n the right arm of the phage was ligated to the sheep genomic insert (Figure 1). This tail likely corresponded to the 3' poly A sequence of the a2 mRNA. At \n\n the extreme 5' end of the insert, a variable length was often observed for the remaining unhybridized mRNA strand. This was most likely due to the size heterogeneity of the a2 mRNA used in these studies. \n\n Size determination and comparative analysis of SpC3 introns. \n\n Although localization of exon and intron sequences was possible with both the 70% formamide and urea-formamide spreading methods, the probability of \n\n detecting unambiguous regions in a hybrid molecule was far greater with the \n\n 2244 \n\n Nucleic Acids Research \n\n A B ',: '14 .0. \n\n 13 \n\n 7 1 \n\n -5 13 \n\n . . . . ,4 x2%. N1 \n\n 9 \n\n > 10 R 4 \n\n Figure 1. Electron microscopic visualization of all 17 introns and 18 exons \n\n from hybrid molecules formed between recombinant clone SpC3 DNA and fetal sheep \n\n tendon a2 mRNA. (A,B) Shown are the double-stranded segment of the right arm CR) of Charon 4A; double-stranded segment of the left arm CL) of Charon 4A; the \n\n displaced single-stranded DNA segment of the insert (5); single-stranded DNA loops representing introns sequentially labeled 1 through 17 in the 3' to 5' direction; and 18 regions of insert DNA sequences hybridized to ct2 mRNA \n\n --- - ). (C,D) Similar to (A,B) but the single-stranded DNA segment (5) of the insert is mostly absent. The urea-formamide spreading method, as described in Materials and Methods, was employed. \n\n 2245 \n\n Nucleic Acids Research \n\n urea-formamide method than with the 70% formamide procedure. For this reason, most measurements were made with the urea-formamide method (Tables 1,2). For \n\n example, the lowest probability event, the detection of intron 17 and the surrounding small exons, \"q\" and \"r\", was only observed by way of the ureaformamide spreading procedure. \n\n The smallest introns, 7 and 12, were found to contain approximately \n\n the same number of bases. Introns 1 through 8, representing the DNA sequences which split the a2 coding sequence corresponding to the 3' end of the a2 mRNA, varied in length over approximately a 2.4-fold range. With the exception of \n\n Table 1. Electron microscopic R loop analysis \n\n present in SpC3 DNA1. \n\n of the sheep a2 introns \n\n SpC3 was hybridized to sheep a2 mRNA as described in Materials and Methods; intron lengths were determined by comparison with *X-174 DNA lengths. \n\n 2 Introns are designated sequentially 3' to 5'. \n\n 3 All data is presented as mean ? standard deviation. \n\n 4 Intron 17 was visualized only by the urea-formamide method. \n\n 2246 \n\n 70% Formamide Method Urea-Formamide Method \n\n Intron2 Number of Intron length Number of Intron length \n\n introns analyzed (bases) introns analyzed (bases) \n\n 1 10 819 t 063 35 806 t 76 2 12 771 ? 89 46 757 t 78 3 11 458 ? 58 47 464 ? 66 4 6 627? 53 41 611 ? 73 5 5 554 ? 51 34 562 ? 76 6 6 675 ? 19 40 684 ? 76 7 5 386 ? 48 50 342 ? 54 8 7 771 ? 84 49 757 ? 82 9 8 1,181 ? 94 52 1,124 ?122 10 8 1,133 ?106 53 1,124 ?137 11 10 964 t?79 61 952 ?105 12 9 362 ? 77 62 342 ? 71 13 4 723 ?222 53 806 ?105 14 4 1,085 ?142 53 1,075 ?105 15 3 795 ? 60 47 806 ?100 16 2 1,229 ? 48 43 1,319 ?146 174 - - 12 1,539 ?166 \n\n 1 \n\n Nucleic Acids Research \n\n Table 2. Electron microscopic R loop analysis of the sheep a2 exons \n\n present in SpC3 DNA1. \n\n 70% Formamide Method Urea-Formamide Method \n\n Exon2 Number of Exon length Number of Exon length \n\n exons analyzed (base pairs) exons analyzed (base pairs) \n\n a 7 807 ? 883 26 883 ?132 b 10 252 ? 35 41 234 ? 49 c 11 151 ? 25 48 182 ? 42 d 6 252 ? 60 44 234 ? 62 e 5 126 ? 45 37 129 ? 47 f 5 126 ? 38 35 104 ? 41 9 5 176 ? 48 40 182 ? 54 h 5 100 ? 43 47 104 ? 31 \n\n 7 100 ? 35 50 104 ? 28 j 7 126 ? 48 45 130 ? 47 k 8 50 ? 48 51 52 ? 39 1 9 75? 10 60 52 ? 44 m 6 126 ? 43 51 182 ? 54 n 4 100 ? 45 53 52 ? 42 o 3 <504 50 78 ? 36 p 2 126 ? 71 47 104 ? 36 q5 - _ 33 130?47 r5 r 6 78?36 \n\n SpC3 was hybridized to sheep a2 mRNA as described in M4aterials and \n\n Methods; exon lengths were determined by comparison with pBR322 DNA lengths. \n\n 2 Exons are designated sequentially 3' to 5'. \n\n 3 All data is presented as mean ? standard deviation. \n\n 4 5 \n\n No statistical evaluation was possible because of the size of the exon (see (Materials and Methods). \n\n Exons q and r were only analyzed by the urea-formamide method. \n\n intron 1, the nucleotide size of these introns was less than 800 bases. In contrast, introns 9 through 17 varied over a 4.5-fold range. Except for intron 12, all of the introns 9 through 17 contained 800 bases or more. \n\n Summation of introns 1 through 16 for the 70% formamide and urea\n\n formamide spreading methods yielded 12,533 and 12,531 bases, respectively. \n\n However, detection of intron 17 by the urea-formamide method indicates that \n\n the actual summed value for the intervening sequences of SpC3 is 14,070 bases. \n\n 2247 \n\n 1 \n\n Nucleic Acids Research \n\n Size determination and the comparative analysis of SpC3 exons. \n\n Eighteen exons, \"a\" through \"r\", were mapped (Table 2). The largest exon, \"a\", located on the extreme 3' end of the sheep genomic insert, contained \n\n approximately 800 bp. With the exception of exon \"m\", exons larger than 150 \n\n base pairs were confined to the genetic region corresponding to the far 3' end of the a2 mRNA. Exon \"a\" and exon \"b\", the two exons situated on either side of intron 1, coded for around 1000 bases of a2 collagen information; these two \n\n exons contained one-third of the mapped 3,014 DNA bases complementary to a2 mRNA Length distribution and organization of the introns and exons in the sheep genomi c i nsert. \n\n There was a sharp contrast in the size distribution of the exons and introns contained within SpC3 (Figure 2). While the intron sizes varied \n\n considerably, the exons, in general, showed a very narrow distribution of \n\n lengths, and thus tended to cluster at the lowest end of the length distri\n\n A. \n\n (I) \n\n z 6 0 \n\n z \n\n ZL4 0 \n\n 021 \n\n z \n\n 10 \n\n B. \n\n 8 \n\n z 0 \n\n 16 0 z \n\n 2 \n\n 20 400 600 800 1000 1200 1400 1600 \n\n LENGTH IN NUCLEOTIDES \n\n Figure 2. Length distribution of the intron and exon mean lengths measured from micrographs prepared by the urea-formamide method. (A) Introns; (b) Exons. See Tables 1 and 2 for details. \n\n 2248 \n\n Nucleic Acids Research bution graph. The exon in the 800-900 bp region was unique and was the only exon which exhibited length overlap with introns. \n\n The maps of the a2 collagen gene obtained with both electron micro\n\n scopic spreading methods were almost identical (Figure 3), except that the \n\n region at the extreme 5' end of the insert could only be mapped with the ureaformamide method. From these measurements we concluded that the length of the a2 collagen genetic regi?on in SpC3 was 17,084 bases long with only 3,014 bases actually coding for a2 mRNA. Therefore, only 18% of the mapped 17 kb sheep genetic insert actually contained a2 structural information. \n\n DISCUSSION \n\n The organizational structure of the a2 collagen gene is complex. A minimum of 17 introns interrupt the 3 kb of ca2 genetic information interspersed as 18 exons over 17 kb of sheep DNA. With the exception of exon \"a\" located at the extreme 3' end of the inserted a2 gene, all other mapped exons are rather small, exhibiting a size distribution range of 50 to 250 bp. In \n\n contrast, the intron sizes range from 340 to 1540 bases occupying a total \n\n of 14 kb. Thus, the DNA region containing the 3 kb of a2 collagen genetic information is 5.6 fold longer than the corresponding co2 mRNA sequences. \n\n Although the total number of introns present within the 60% 3' portion \n\n of the a2 sheep gene was determined to be 17, variation in the precise number \n\n TRANSRIPTlO \n\n A 5w r \n\n 1 \"2a4 133 112s 11.17 0. 3O 2 715 6.2 313 04 4.2B 3.51 2.2 12 \n\n EXON0 p o n 5 Ik h f d 4 . b \n\n WPM300 16 16 14 13 12 11 10 9 6 7 6 5 4 3 2 1 \n\n B \n\n 110 16 4.2 13111 112n 1113104 3.6 gm74 2 6. 4 4 2 3.2 22 in \n\n 1 a1m03 133 1.2 11 10 01 341 6. 1 117 6.2 4 12 417 3I5'm 2 \n\n DM00 1 q5 p 0 n m I k. j i h g f d c b a 147306 17 16 15 14 13 12 11 10 9 a 7 6 5 4 3 2 1 \n\n 175 17.0 16.6& 16'5.5 15.0 14.5 14. 13.5 13.0 12.5 12.0 11.5 11 10.5M 10.0 9.5 9.0 8. 5.0 71 7.0 6.5 6. 5.5 50 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 \n\n KILOBASE \n\n Fiue3 Organization of 60% of the 3' end of the sheep a2 collagen gene. (A)}The exon ( ) and intron (CDJ) order within the a2 gene as determined by R loop analysis of SpC3 DNA-a2 mRNA hybrid molecules visualized by the electron microscopic 70% formamide spreading method. (B) The exon Um) \n\n and intron (Efl) order within the a2 gene as determined by R loop analysis of SpC3 DNA-a2 mRNA hybrid molecules evaluated by the electron microscopic \n\n urea-formamide spreading method. Arrows represent the approximate location of intron-exon junctions. All values are presented as kilobase pairs. \n\n 2249 \n\n Nucleic Acids Research \n\n of introns present in individual hybrid molecules was observed. The reason \n\n for this may be due to size heterogeneity of the a2 mRNA used as a complementary probe. Although the a2 mRNA was extensively purified (2), some degradation was likely present. It is also possible that additional introns exist that are too small to be detected by electron microscopic R loop analysis. The most likely a2 genetic regions containing such introns are exons \"g\" and \"m\". Small \n\n \"protrusions\" were sometimes observed within these exons that could represent small introns. Very small introns have been reported for tRNA genes (12,13), \n\n and it will be necessary to sequence these regions of SpC3 to determine if such introns are present within the a2 sheep genome. \n\n The existence of mRNAs comprised of sequences complementary to noncontiguous regions in a DNA genome was first described for adenovirus (14,15) and \n\n simian virus (16,17) genes. Similar observations were soon reported for eukaryotic genes, including serum albumin (18), conalbumin (19), fibroin (20), s\n\n globin (21,22), growth hormones (23,24), immunoglobulin (25), lysozyme (26), \n\n ovalbumin (27-29), ovomucoid (30), rRNA (31-34), tRNA (12,13), and vitellogenin (35). The precise number or occurrence of introns within these genes does not seem to follow any clear pattern. However, the genes for albumin, conalbumin, \n\n lysozyme, ovalbumin, and ovomucoid exhibit a structural organization comparable to the a2 collagen gene. All of these genes are composed of numerous small \n\n exons, usually less than 250 base pairs, and introns which show a large size variation within an individual gene. Within all of these genomes, the DNA \n\n regions required for coding structural formation are 4 to 7 times longer than the corresponding mRNA coding sequences. \n\n As with the a2 collagen gene, a large unique exon at the extreme 3' end \n\n is also present in the ovalbumin gene (36,37). In the a2 gene, this exon was 800 to 900 nucleotides in length, while in the ovalbumin gene the exon contained 1,030 base pairs. Interestingly, this ovalbumin exon contained 634-650 base pairs ot DNA sequence to the 3' side of the terminator \n\n triplet, suggesting the ovalbumin mRNA has a large untranslated region at the 3' end. \n\n The consistent appearance of a small non-hybridized tail, approximately 100 nucleotides in length, in the region where the right arm of the phage was ligated to the sheep genomic insert of SpC3 very likely corresponded to the \n\n 3' poly A sequence of the a2 mRNA. This would suggest that the a2 mRNA coding sequence terminates very near or at this point in the genome. Previously published biochemical evidence (2) obtained from restriction mapping and Southern blot analysis using [32P]-labeled chick a2 cDNA probe supports \n\n 2250 \n\n Nucleic Acids Research \n\n this observation. \n\n It is unclear what role introns play in genetic expression. However, \n\n it is known that these sequences are tranncribed as part of larger precursor mRNAs and the introns are then excised and the fragmented mRNA chains \n\n covalently rejoined (38). Some evidence has recently accumulated demonstrating the existence of precursor a2 mRNAs (39,40). However, if the introns in the a2 gene are transcribed in their entirety, as has been \n\n demonstrated for the introns in ovalbumin and globin (41,42), much larger precursor a2 mRNAs may exist than those reported to date. For if the re\n\n mainder of the a2 collagen gene has a structural organization comparable to the 60% 3' portion that has been analyzed, it is probable that the a2 \n\n collagen gene in its entirety is dispersed throughout a 30 kb DNA segment. \n\n ACKNOWLEDGMENTS \n\n We thank Dr. John Dahlberg and Frances Loebenstein, Laboratory of Cellular and Molecular Biology, National Cancer Institute, for the generous use of a \n\n Siemens Elmiskop 101 electron microscope; Dr. Ursula Heine and Benjamin Elliott, Laboratory of Viral Carcinogenesis, National Cancer Institute, for the use of a Hewlett-Packard calculator equipped with a digitizer and a Denton vacuum \n\n evaporator, and Drs. Victor Ferrans and Oichi Kawanami, Laboratory of Pathology, \n\n National Heart, Lung, and Blood Institute, for expert assistance with photography. 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[ { "id": "pmcA102792__text", "type": "Article", "text": [ "Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast\nAbstract\nScp160p is a 160 kDa protein in the yeast Saccharomyces cerevisiae that contains 14 repeats of the hnRNP K-homology (KH) domain, and demonstrates significant sequence homology to a family of proteins collectively known as vigilins. As a first step towards defining the function of Scp160p, we have characterized the subcellular distribution and in vivo interactions of this protein. Using sucrose gradient fractionation studies we have demonstrated that Scp160p in cytoplasmic lysates is predominantly associated with polyribosomes. Furthermore, we have found that Scp160p is released from polyribosomes by EDTA in the form of a large complex of ≥1300 kDa that is sensitive both to RNase and NaCl. Using affinity-chromatography to isolate these complexes, we have identified two protein components other than Scp160p: poly(A) binding protein, Pab1p, and Bfr1p. The presence of Pab1p confirms these complexes to be mRNPs. The presence of Bfr1p is intriguing because the null phenotype for this gene is essentially the same as that reported for scp160-null cells: increased cell size and aberrant DNA content. These results demonstrate that Scp160p associates with polyribosome-bound mRNP complexes in vivo, implicating a role for this protein in one or more levels of mRNA metabolism in yeast.\n\nINTRODUCTION\nScp160p is a 1222 amino acid Saccharomyces cerevisiae protein that contains 14 copies of the hnRNP K homology (KH) domain, a highly conserved motif found in many proteins involved in RNA metabolism (1). KH domain-containing proteins appear to have diverse functions and have been identified in all kingdoms of life, including the ribosomal protein S3 from Escherichia coli (1), Mer1p from S.cerevisiae, a meiosis-specific splicing factor (1), MEX-3 from Caenorhabditis elegans, presumably involved in mRNA localization during development (2), and FMRP, the fragile-X mental retardation protein in humans (3). A partial clone of Scp160p, known as HX, was one of the first multiple-KH proteins identified (1).\nWhole cell immunofluorescence studies have demonstrated that Scp160p localizes to the cytoplasm, with enrichment around the nuclear envelope, and what appears to be the endoplasmic reticulum (3). Deletion of the SCP160 locus in yeast is not lethal, but results in a complex phenotype, including increased DNA content per cell, missegregation of genetic markers during sporulation, and abnormal cell morphology, including increased size and irregular shape (4). Observation of this phenotype led to the hypothesis that Scp160p may function in regulating ploidy during cell division. More recently, Weber and colleagues demonstrated in vitro RNA binding activity of Scp160p using northwestern blot analyses; the protein was found to bind efficiently to ribohomopolymers and rRNA, but not to tRNA (5). Cell fractionation studies revealed that a large percentage of Scp160p associates with membrane-pellets, and is released by treatment with either 10 mM EDTA or 500 mM NaCl (5). While these authors interpreted these results to suggest that the nuclear envelope/ER localization of Scp160p was due primarily to interactions of the protein with membrane-bound polyribosomes, clear evidence of this association has not been reported (5). Currently, the relationship between the phenotype of scp160 null mutants and the RNA-binding activity of Scp160p remains unclear.\nScp160p demonstrates significant sequence homology (~23% identity and ~40% similarity at the amino acid level) to a class of vertebrate KH-domain proteins collectively known as vigilins. First identified in chicken, vigilin homologues have now been found in human (6), Xenopus laevis (7), Drosophila melanogaster (8), C.elegans (5) and Schizosaccharomyces pombe. While all of the vigilin proteins studied to date are reported to bind nucleic acid, both the type of nucleic acid bound and the functional significance of these interactions remain unclear. For example, Kruse and colleagues reported from their work with human cells in culture that vigilin may be involved in the binding and transport of tRNA (9–11). In contrast, Dodson and Shapiro concluded from their work with Xenopus vigilin that, in response to estrogen, the protein bound specifically to the 3′ UTR of vitellogenin mRNA (7,12), potentially stabilizing the message (13). Lastly, DDP1, the Drosophila homolog of vigilin, was reported recently to interact with the dodeca-satellite repeat regions of centromeric heterochromatin in embryonic and larval cell nuclei, suggesting a possible role for this protein in heterochromatin structure (8).\nThe goal of the present study was to begin elucidating the function of Scp160p in yeast by characterizing the subcellular distribution and macromolecular interactions of this protein. We have demonstrated that Scp160p in cytoplasmic lysates is associated predominantly with polyribosomes, and that following treatment with EDTA, Scp160p remains in an RNase/NaCl-sensitive complex of apparent molecular weight approximately ≥1300 kDa. Affinity purification of this complex revealed the presence of poly(A)-binding protein (Pab1p), a well-characterized component of eukaryotic mRNPs (14,15). Finally, a third abundant protein component of this complex was identified as Bfr1p, a protein not previously reported to associate with mRNPs. While the function of Bfr1p remains unknown, gene deletion reportedly leads to a phenotype remarkably similar to that of scp160 deletion (16). These results indicate a role for Scp160p in mRNA metabolism in yeast, and by extension, support results seen with Xenopus vigilin in its interactions with mRNA. To our knowledge, the data reported here demonstrate Scp160p to be the first example of a KH-domain protein that functions as a component of polyribosome-associated mRNP complexes in the yeast, S.cerevisiae.\n\nMATERIALS AND METHODS\nPlasmids, yeast strains and culture conditions\nAll recombinant DNA manipulations were performed according to standard techniques and utilized E.coli strain XL1-Blue (Stratagene). The yeast strain JFy1511, expressing FLAG–Scp160p, was derived by two-step gene replacement from strain yJJ52 (MATα gal7Δ102 ura3-52 trp1-289 ade1 lys1 leu2-3, 112; generously donated by Drs Mark Parthun and Judith Jaehning, University of Colorado Health Sciences Center), and confirmed by PCR. The wild-type SCP160 coding sequence was obtained by PCR amplification from a genomic DNA preparation from yJFK1 (MATα gal7Δ102 ura3-52 trp1-289 ade1 lys1 leu2-3, 112 ΔGAL80::URA3), using a 16:1 mixture of Taq (Fisher Biotech) and Pfu (Stratagene) DNA polymerases and the following primers: Scp160F1 (5′-GCCGGTCGA-CTAACTGCAATGTCTGAAGAACAAACCGCTATTG-3′) and Scp160R1 (5′-GCGCGTCGACGAGCTTGTCTATCTT-CTTAAGG-3′). A wild-type SCP160 genomic clone, containing 1 kb upstream and 300 bp downstream sequence was PCR amplified from yJFK1 genomic DNA using the primers Scp160F0 (5′-GCCGAGCTCACACCAGCTTTGTCCTGG-3′) and Scp160R2 (5′-GCGCAAGCTTGTGCGGTA-TCCCAGTCTATG-3′). The resultant clone was confirmed by dideoxy sequencing. The N-terminal FLAG and HA tags were added using PCR with the primers ScpFLAGF1(5′-CCAT-TATAACTGCAATGGACTACAAGGACGACGACGACGAC-AAGATGTCTGAAGAACAAACCGCTATTG-3′) and ScpHAF1 (5′-CCCCCTCCTGTCGACATTATAACTGCAATGCACCA-TCACCATCACCATTCTGAAGAACAAACCGCTATTG-3′) respectively. For integration, constructs containing the 1 kb upstream and 300 bp downstream sequence were subcloned into the plasmid YIplac211 (17) using SacI and HindIII restriction sites. The HA-tagged construct was subcloned into the low copy number plasmid, YCplac22 using SacI and HindIII restriction sites. The SCP160 deletion construct was made by removal of a 3.4 kb ApaI–KpnI fragment from the coding region of this construct, followed by treatment with Klenow fragment and re-ligation following attachment of BamHI linker oligonucleotides. Genomic integration of the plasmid sequences and subsequent removal of the endogenous SCP160 allele were achieved by standard two-step gene replacement techniques (18) and confirmed by PCR. The N-terminally HA-tagged allele of BFR1 was generated by PCR-amplifiction of the BFR1 locus from wild-type (W303) yeast genomic DNA using the primers BFR1HAF1 (5′-CCGCGGATCCATGTACC-CATACGACGTCCCAGACTACGCTATGTCCTCCCAAC-AACACAA-3′) and BFR1HINDR1 (5′-CCGCAAGCTTGTCG-ACTATTTCATATGCCACAGGAAACAG-3′), and subcloned into YIPlac211. The BFR1 promoter region was PCR-amplified in a similar manner using the primers BFR1SACF1 (5′-CCGCGAG-CTCAGCATTAAGCATTCACGAGC-3′) and BFR1BAMR1 (5′-CCGCGGATCCGGCAATGGCTGTGTTGTTAGA-3′) and subcloned into the appropriate position upstream of the HA–Bfr1p open reading frame in the plasmid backbone. The entire open reading frame was confirmed by dideoxy sequencing. Finally, the HA–BFR1 allele was substituted into the yeast genome in place of the native allele using linearization with SphI and standard two-step gene replacement techniques. All yeast transformations and culture manipulations were performed according to standard protocols as described elsewhere (19).\n\nConfirmation of genomic integrations\nAll genomic integrations were confirmed by PCR amplifications from purified yeast genomic DNA. For scp160 deletion mutants, the primers Scp160PF (5′-GATTTCCTAACTTTCC-GTCTA-3′) and Scp160R5 (5′-GCGCAAGCTTCACCGCCTTATAACGAAGAC-3′) that flanked the deleted region were used.\nSimilarly, epitope tags on Scp160p were confirmed by PCR using primers that flanked the tag sequence. Positive clones were further confirmed by western blot analysis of crude cell lysates using the appropriate anti-tag antibodies. The presence of HA–Bfr1p in cells was confirmed by western blot analysis of soluble cell lysates using 12CA5 mAb (Boehringer Mannheim).\n\nPolyribosome isolation\nPolyribosomes were isolated using a combination of protocols described by Stansfield and colleagues (20) and by Dr Maurice Swanson (personal communication). In brief, a 100 ml culture of yeast was grown in either YEPD or Hartwell synthetic medium, to early-log phase (OD600 = 1.0), at which time cyclohexamide (Sigma) was added directly to the culture to a final concentration of 100 µg/ml. The culture was incubated on ice for 15 min, and cells were harvested by centrifugation (4000 r.p.m./10 min). Following two washes in 10 ml of water containing 100 µg/ml cyclohexamide, cells were lysed by vortex agitation with an equal volume of glass beads in 1 ml lysis buffer (25 mM Tris pH 7.2, 50 mM KCl, 30 mM MgCl2, 5 mM β-mercaptoethanol, 200 µg/ml cyclohexamide, 2 µg/ml aprotonin, 1 mM PMSF, 0.5 µg/ml leupeptin, 2.9 µg/ml E64, 1 µg/ml antipain, 0.2 µg/ml chymostatin). Lysate was transferred to a clean microfuge tube, and centrifuged 10 min at 3000 g at 4°C. The supernatant was again transferred to a clean microfuge tube and centrifuged at 12 000 g for 15 min at 4°C, and then assayed for absorbance at 260 nm.\nApproximately 12 OD260 units were loaded onto a 11 ml 15–45% sucrose gradient made in 10 mM Tris, pH 7.4, 70 mM NH4Cl, 4 mM MgOAc, using a Gradient Master automatic system, and the gradient was centrifuged in a SW41ti rotor (Beckman) at 39 000 r.p.m. for 2.5 h. 0.5 ml fractions were collected using an Isco gradient fractionator, and gradient profiles were determined by monitoring absorbance at 254 nm. For EDTA controls, lysis buffer containing 5 mM MgCl2 was used, and 30 mM EDTA was added to the sample before loading onto the gradient. Where indicated, addition of 50 U/ml of RNase One (Promega) was performed prior to loading the sample onto the gradient.\n\nGel filtration chromatography\nGel filtration chromatography was performed using a 120 ml Hi-Prep S-300 Sephacryl column (Pharmacia) with a cut-off of 1300 kDa, attached to an FPLC system (Pharmacia). The column had been calibrated previously using the following molecular weight standards (Sigma): blue dextran (2000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), and bovine serum albumin (66 kDa). Yeast were lysed as described for polyribosome analysis, with an additional clarification by passage through a 0.2 µm syringe-tip filter (Acrodisc) and run over the column at a rate of 0.5 ml/min in polyribosome lysis buffer following treatment with the indicated reagents. Fractions (2.0 ml) were collected, from which 12 µl were combined with sample buffer (2% SDS, 10% glycerol, 100 mM dithiothreitol, 60 mM Tris pH 6.8, 0.001% bromophenol blue) and analyzed by western blot using the indicated antibodies.\n\nα-FLAG affinity chromatography\nFor most experiments, one liter yeast cultures were grown to early log-phase and harvested by centrifugation. Cells were washed twice in T75 buffer (25 mM Tris pH 7.5, 75 mM NaCl) and then were lysed by vortex agitation with an equal volume of glass beads in 4 ml T75 buffer containing 30 mM EDTA. Lysate was transferred to a clean microfuge tube, and centrifuged for 10 min at 3000 g at 4°C. The supernatant was again transferred to a clean microfuge tube and centrifuged at 12 000 g for 15 min at 4°C, and finally passed through a 0.2 µm syringe filter. Lysate was then pre-purified by running over the S-300 gel-filtration column in T75 buffer, with pooling of the void volume fractions (~10 ml total). For several experiments (Fig. 5A, B, E and F), a low concentration (<10 µg/ml) of FLAG peptide (N-DYKDDDDK-C) was added to this sample. This void material was then loaded onto a 1 ml M2 α-FLAG column (Sigma); column flow-through was passed over the column a second time. The column was then washed extensively with 75 ml T75 buffer; material bound to the column was eluted with 5 ml T75 containing 184 mg/ml FLAG peptide. The peptide solution was allowed to incubate on the column for 20 min prior to collection of the first 1 ml fraction; subsequent fractions were collected every 10 min. Samples of crude material, S-300 void, α-FLAG flow-through, first and last wash, and eluate fractions were analyzed by western blot using the indicated antibodies. For Figure 5A and E, samples of first and last wash and eluate fractions were concentrated, run on SDS–PAGE and stained with colloidal G250 Coomassie.\n\nConcentration of samples\nWhere indicated, samples were concentrated using the method of Traub et al. (21). 400 µl of sample were transferred to a microfuge tube, and combined with 400 µl of methanol, and 100 µl of chloroform. Samples were then vortexed vigorously, and centrifuged for 5 min at 12 000 g. The supernatant was discarded, leaving the interface intact, and an additional 400 µl of methanol was added to each tube. Samples were then inverted several times, and centrifuged again for 5 min. The supernatant was discarded, and the protein pellet was air-dried and resuspended in 1× sample buffer.\n\nWestern blot analysis\nWestern blot analysis was performed essentially as described previously (19). Briefly, samples to be analyzed were mixed with sample buffer, boiled, electrophoresed through a 10% SDS–polyacrylamide gel, and electro-blotted onto nitrocellulose (Bio-Rad). FLAG–Scp160p fusion protein was detected by incubation of the filter with mouse M2 anti-FLAG monoclonal antibody (10 µg/ml final concentration), followed by HRP-conjugated sheep anti-mouse secondary antibody (Amersham), diluted 1:5000 as per the manufacturer’s instructions, and ECL reagent (Amersham), followed by exposure to X-ray film. HA-tagged Scp160p and HA-tagged Bfr1p were detected using the 12CA5 mAb (Boehringer Mannheim) at a final concentration of 0.8 µg/ml; Pab1p and Pub1p were detected using 1G1 mAb at 1:5000, and 4C3 mAb at 1:1000, respectively, both generous gifts from Dr Maurice Swanson (22,23). Where appropriate, films were analyzed by scanning densitometry (Molecular Dynamics), and quantitated using ImageQuant software (Molecular Dynamics).\n\nColloidal G250 coomassie staining\nProcedure used was that of Neuhoff (24). Briefly, a one liter stock of staining solution was prepared containing 1 g Coomassie brilliant blue G-250 (Sigma), 100 g ammonium sulfate (Sigma), and 11.76 ml 85% phosphoric acid (Fisher). Following SDS–PAGE, gels were fixed in 40% methanol, 10% acetic acid for 10 min. Gels were then rinsed several times in water, then stained using 40 ml of the stock staining solution mixed with 10 ml methanol, for 2 h at room temperature. Stain was then poured off, and residual stain was removed by rinsing in water. By performing a standard analysis using bovine serum albumin, the stain was able to detect ~15 ng total protein per lane.\n\nPichia expression system\nAn N-terminally HIS6/FLAG-tagged allele of the SCP160 coding sequence was blunt-end sub-cloned into the BamHI/SnaBI sites of the Pichia expression vector pPIC3.5K (Invitrogen). The construct was then linearized using SalI and integrated into the genome of the Pichia strain GS115 (Invitrogen). High expression transformants were selected initially on histidine-deficient medium followed by selection on increasing concentrations of G418 (US Biological). Crude lysates of the resultant transformants were then confirmed by western blot analysis with the anti-FLAG antibody M2, and the best expressing strain was cultured in a fermenter, harvested, and lysed by agitation with glass beads. To purify Scp160p, 2 ml of crude lysate was first diluted to 10 ml with 25 mM Tris, pH 7.5, 1 M NaCl, then twice passed over a 1 ml α-FLAG affinity column. The column was then washed with 75 ml of 25 mM Tris, pH 7.5, 1 M NaCl, and then eluted with 184 µg/ml FLAG peptide prepared in the same buffer.\n\n\nRESULTS\nExpression of tagged Scp160p in yeast\nAn N-terminal FLAG-tagged form of Scp160p was created to facilitate detection of the protein in cells and extracts (Fig. 1A). To probe functionality of this fusion protein, we used two-step gene replacement (18) to substitute the modified allele into the SCP160 locus of haploid yeast, and then tested the morphological phenotype of the resultant cells. All strains were confirmed by PCR analysis of the SCP160 locus with appropriate primers (Materials and Methods), and expression of the tagged protein was confirmed by western blot analysis with the appropriate antibody (M2αFLAG) (Fig. 1B). In all cases, yeast expressing tagged Scp160p in place of the native protein appeared indistinguishable from the corresponding wild-type strains (data not shown). As a negative control, we also deleted almost the entire SCP160 coding region from the genomes of these yeast strains (Fig. 1A), and confirmed the expected mutant phenotype (data not shown).\n\nScp160p associates with polyribosomes\nTo test the hypothesis that Scp160p associates with polyribosomes we used sucrose gradient ultracentrifugation to size-fractionate subcellular components of lysates prepared from yeast expressing the N-terminal FLAG-tagged Scp160p protein. Western blot analyses of gradient fractions with an α-FLAG antibody (M2, Boehringer Mannheim), revealed a 160 kDa band that was most abundant in the denser fractions, consistent with the location of polyribosomes (>80S) (Fig. 2A, solid arrow). A convenient internal control for these gradients was provided by an unknown endogenous yeast cross-reacting protein at ~100 kDa, that exemplified the migration pattern of a free protein, appearing only in the upper fractions of the gradient (Fig. 2A, open arrow).\nTo determine whether the migration pattern of Scp160p in these sucrose gradients truly reflected association with yeast polyribosomes, lysates were pre-treated with either 30 mM EDTA or 50 U/ml RNase One (Promega) immediately prior to sucrose gradient fractionation. EDTA chelates Mg2+ cations, resulting in the dissociation of the small and large ribosomal subunits, reflected in gradient profiles (OD254) by the disappearance of 80S monosomes and polyribosomes along with a marked increase in the abundance of free ribosomal subunits (Fig. 2B). Under these conditions, the greatest intensity of FLAG–Scp160p signal was seen only in the upper-most fractions of the gradient (Fig. 2B). Alternatively, pre-treatment of lysates with RNase, which results in inter-ribosomal severing of translating messages, gave rise to a large pool of single 80S ribosomes (Fig. 2C). Again, FLAG–Scp160p was shifted to the uppermost fractions of these gradients. Although some of the Scp160p signal was detected in fractions larger than 40S, the migration pattern of the 100 kDa cross-reacting protein in these experiments indicated that RNase treatment caused an apparent diffusion of material in the upper portion of the gradient.\n\nCharacterization of Scp160p-containing complexes following EDTA and RNase treatment\nGel-filtration chromatography was used to determine the apparent molecular weight of Scp160p following its release from polyribosomes by both EDTA and RNase treatment. Lysates were prepared as described above for sucrose gradient analysis, but were instead size fractionated over a Sephacryl S-300 Hi-Prep column, with an inclusion cut-off of 1300 kDa. As expected, Scp160p from untreated lysates eluted in the void volume, confirming its association with large complexes, ostensibly polyribosomes (Fig. 3A, solid arrow). In EDTA treated lysates, all Scp160p signal was still detected in the void volume, indicating it remained in a complex of >1300 kDa (Fig. 3B, solid arrow). In contrast, limited RNase treatment (10 min) resulted in the appearance of an Scp160p-containing species of ~450 kDa, in addition to a fraction still visible in the void (Fig. 3C, solid arrow). More extensive RNase treatment (30 min) led to complete conversion to the 450 kDa species (Fig. 3D, solid arrow). Sequential treatment, first with RNase, then with EDTA, also resulted in a 450 kDa species (data not shown), suggesting that all components of this apparent 450 kDa complex were also present following EDTA treatment alone. As before, the 100 kDa, endogenous α-FLAG cross-reacting protein (Fig. 3, open arrows) served as a convenient internal control, eluting from the column at a volume consistent with its expected monomeric size.\nTo characterize further the stability of the large (apparent molecular weight >1300 kDa), EDTA-resistant complex, lysates were treated with increasing concentrations of NaCl prior to size fractionation (Fig. 3, panels E, F and G). As shown in Figure 3E, at 75 mM NaCl, the large complex remained intact. However, at a NaCl concentration of 150 mM, the Scp160p complex was partially reduced to 450 kDa, with some signal still remaining in the void (Fig. 3F, solid arrow). Following treatment with 1 M NaCl, Scp160p was only visible as the 450 kDa species (Fig. 3G, solid arrow). The fact that RNase treatment and high salt both generated Scp160p species of similar apparent molecular weight suggests that these high salt concentrations resulted in the disassociation of RNA, and perhaps other components, from Scp160p.\n\nPab1p and Bfr1p are present in EDTA-resistant Scp160p-containing complexes\nTo determine if the >1300 kDa Scp160p-containing complexes remaining after EDTA treatment were mRNPs, we assayed for the presence of the yeast poly(A) binding protein, Pab1p, following α-FLAG affinity purification as illustrated in Figure 4. Pab1p is an abundant and well-characterized component of mRNP complexes in yeast, as well as higher eukaryotes (14,15,25). As seen in Figure 5A and B, Pab1p did co-purify with FLAG–Scp160p; moreover, treatment with RNase immediately prior to FLAG-purification completely abolished this interaction (Fig. 5C), indicating an RNA-dependant association between these two proteins.\nWe also probed the isolated complexes for the presence of another abundant mRNP-component protein, Pub1p (23,26). Pub1p, as opposed to Pab1p, is not reported to associate with polyribosomes, and is hypothesized to bind a pool of non-translatable mRNAs (23). As predicted, Pub1p did not co-purify with Scp160p (Fig. 5D). The absence of Pub1p in Scp160p-containing complexes served as a negative control, demonstrating that abundant RNA-binding proteins did not co-purify non-specifically by this protocol. In addition, we performed mock purifications using lysates from yeast expressing the wild-type allele of SCP160 without the FLAG epitope (Fig. 5F), or with an unrelated FLAG-tagged protein, human galactose 1-phosphate uridylyltransferase (GALT) (Fig. 5G, lower panel). In neither case did Pab1p bind and elute from the FLAG affinity column. Finally, no bands were visible by colloidal G250 Coomassie staining of samples from the mock (wild-type) preparations (Fig. 5E), further demonstrating the specificity of this procedure.\nBy colloidal G250 Coomassie staining (Materials and Methods) of the FLAG-purified complex, we observed in addition to Scp160p, two major bands, at ~70 kDa and ~55 kDa, which ostensibly represent co-purifying proteins (Fig. 5A, bottom panel). Western blot analysis suggested that the ~70 kDa species is Pab1p. To identify the 55 kDa protein, that band was excised from the gel, and sent to the Keck microchemical facility at Yale University for analysis by in gel tryptic digestion, followed by MALDI-mass spectrometry. The results of these analyses clearly identified the unknown protein as Bfr1p. To confirm this result, an N-terminal HA tag was engineered onto the genomic BFR1 locus in strains expressing FLAG-tagged or wild-type Scp160p. Scp160p complexes were then isolated from both of these strains using the protocol described above. As seen in Figure 6, an HA-tagged protein of ~55 kDa (center panel) appears in α-FLAG column elutions only when FLAG–Scp160p is also present (top panel). No signal is visible in mock purifications of extracts where Scp160p is not FLAG-tagged (bottom panel).\n\nMonomeric Scp160p migrates as a ~450 kDa protein under native conditions\nAs a first step to characterize the post RNase/NaCl 450 kDa Scp160p species we compared it with Scp160p over-expressed and purified from an exogenous host, the yeast Pichia pastoris. The purified N-terminal FLAG/hexahistidine-tagged Scp160p was run over an S300 gel filtration column; 2 ml fractions were collected and analyzed by α-FLAG western blot. As seen in Figure 7A, purified Scp160p eluted at a volume consistent with a >443 kDa protein. To confirm that no other proteins were associated with the purified Scp160p, 12 µl of the protein was run on SDS–PAGE and subjected to colloidal G250 Coomassie staining before loading on the S-300 column (Fig. 7B). As shown, no bands other than Scp160p were visible.\nSeveral KH-domain proteins, including Sam68 and FMRP, have been reported to form homodimers in vivo (27). Therefore, we explored the possibility that the ~450 kDa Scp160p species might contain two or more copies of Scp160p. To test this hypothesis, yeast expressing FLAG–Scp160p were transfected with a centromeric plasmid, YCplac22–HA–Scp160p, encoding a distinct, HA-tagged allele of the protein. The functionality of this tagged protein had previously been demonstrated by polyribosome association as well as complementation of the null morphological phenotype in cells expressing only HA–Scp160p (data not shown). As seen in Figure 7C, when yeast co-expressing the two alleles were lysed and the FLAG-tagged protein was isolated as described above, no HA–Scp160p co-purified with the FLAG–Scp160p. These data strongly suggest that Scp160p does not form homo-dimers or higher-order multimers in vivo, and that the ~450 kDa species of Scp160p observed is simply the monomeric protein.\n\n\nDISCUSSION\nThe data presented here demonstrate two main points regarding the biochemical associations and function of Scp160p in yeast. First, the sucrose gradient fractionation data clearly demonstrate that Scp160p exists primarily associated with large complexes, likely to be polyribosomes. To our knowledge, this is the first time a vigilin family member has been shown to associate with polyribosomes in this manner. Interestingly, Weber and colleagues did not observe sucrose gradient fractionation data consistent with polyribosome association of Scp160p (5). These authors hypothesized that Scp160p associates only with membrane-bound polyribosomes, and that Scp160p is not found complexed with cytosolic polyribosomes (5). Our data do not support this hypothesis, since unlike Weber and colleagues, we were able to detect significant amounts of Scp160p associated with polyribosomes without using detergents during preparation. There are several possible explanations for this discrepancy. First, the lysate buffer utilized by that group contained 100 mM NaCl, whereas our buffer contained 50 mM KCl. Our data shown in Figure 3 suggest that 100 mM NaCl may have caused an instability in the Scp160p–RNA interaction, leading to release from polyribosomes during their procedure. Secondly, Weber’s lysate buffer reportedly contained heparin to inhibit ribonucleases; a number of reports (28,29) suggest that heparin can disrupt RNA–protein interactions. Although we have not in the past included heparin in any of our experiments, we have observed disruption of the polyribosome association in buffer containing residual diethylpyrocarbonate, another inhibitor of ribonucleases (data not shown).\nSecond, our data provide compelling evidence that Scp160p is released from polyribosomes as a component of an mRNP complex. Since Scp160p does not remain associated with either single ribosomes or ribosomal subunits following treatment with EDTA or RNase, it seems unlikely that Scp160p is a constitutive component of the translational machinery. Partial purification of the EDTA-resistant Scp160p complexes allowed us to demonstrate the presence of Pab1p but not Pub1p in these preparations, which is consistent with the idea that Scp160p associates with polyribosomes as a component of mRNP complexes. Previously, Scp160p had been shown to bind ribohomopolymers and rRNA in vitro, although the in vivo nucleic acid targets of Scp160p were unknown (5). The presence of Pab1p in RNase-sensitive Scp160p complexes indicates that Scp160p is primarily bound to polyadenylated RNAs. Whether or not Scp160p binds only specific sets of mRNAs will be the subject of future studies.\nIn addition to Scp160p and Pab1p, we have identified a third component of the complex, the protein Bfr1p. The gene, BFR1, was originally identified in a screen for high-copy suppressors of Brefeldin-A induced lethality, suggesting a role in the secretory pathway (16). Interestingly, however, bfr1 null mutants do not demonstrate any defects in the secretory pathway, but rather display similar phenotypes to scp160 null mutants, most notably increased ploidy and increased cell size (16). It is therefore unclear whether Bfr1p is capable of functioning in both RNA metabolism and secretion directly, or if the Scp160p–Bfr1p complex regulates the expression of one or more secretory genes at the post-transcriptional level. Additionally, two-hybrid analysis indicated an interaction of Bfr1p with the protein Bbp1p, a component of the mitotic spindle apparatus (30). While Bbp1p is an essential gene, overexpression leads to a phenotype similar to both bfr1 and scp160 null strains (30). Future work will address the functional relationship between Scp160p and Bfr1p including the possibility that these proteins represent a regulatory mechanism connecting the translational machinery with cell division and/or the secretory pathway.\nFollowing treatment with either RNase or >150 mM NaCl, Scp160p remained as an apparent ~450 kDa species. Considering that purified Scp160p alone also migrates at this size, we conclude that this apparent complex may consist only of Scp160p, although the presence of other small components cannot be ruled out at this time. Furthermore, by utilizing yeast co-expressing two distinct epitope-tagged versions of Scp160p, we have ruled out the possibility of self-association of Scp160p. Although both tagged proteins appear functional, it remains a formal possibility that the tags somehow prevented formation of FLAG–HA hetero-complexes. Nonetheless, it seems most likely that native Scp160p monomers may simply migrate aberrantly under native conditions due to an unusual shape or some other physical property.\nIn conclusion we have shown convincing evidence that Scp160p exists in yeast cytoplasmic extracts primarily associated with polyribosomes. We have purified Scp160p following disruption of polyribosomes with EDTA, and identified two associated proteins: Pab1p and Bfr1p. The presence of Pab1p suggests that Scp160p associates with polyribosomes as a component of an mRNP, demonstrating Scp160p to be the first S.cerevisiae multiple-KH domain protein characterized to function in this way.\n\n\n" ], "offsets": [ [ 0, 32706 ] ] } ]

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[ { "id": "pmcA2257940__text", "type": "Article", "text": [ "Discovery of genes implicated in whirling disease infection and resistance in rainbow trout using genome-wide expression profiling\nAbstract\nBackground\nWhirling disease, caused by the pathogen Myxobolus cerebralis, afflicts several salmonid species. Rainbow trout are particularly susceptible and may suffer high mortality rates. The disease is persistent and spreading in hatcheries and natural waters of several countries, including the U.S.A., and the economic losses attributed to whirling disease are substantial. In this study, genome-wide expression profiling using cDNA microarrays was conducted for resistant Hofer and susceptible Trout Lodge rainbow trout strains following pathogen exposure with the primary objective of identifying specific genes implicated in whirling disease resistance.\n\nResults\nSeveral genes were significantly up-regulated in skin following pathogen exposure for both the resistant and susceptible rainbow trout strains. For both strains, response to infection appears to be linked with the interferon system. Expression profiles for three genes identified with microarrays were confirmed with qRT-PCR. Ubiquitin-like protein 1 was up-regulated over 100 fold and interferon regulating factor 1 was up-regulated over 15 fold following pathogen exposure for both strains. Expression of metallothionein B, which has known roles in inflammation and immune response, was up-regulated over 5 fold in the resistant Hofer strain but was unchanged in the susceptible Trout Lodge strain following pathogen exposure.\n\nConclusion\nThe present study has provided an initial view into the genetic basis underlying immune response and resistance of rainbow trout to the whirling disease parasite. The identified genes have allowed us to gain insight into the molecular mechanisms implicated in salmonid immune response and resistance to whirling disease infection.\n\n\n\nBackground\nWhirling disease was first described among farmed rainbow trout (Oncorhynchus mykiss), a native North American salmonid species, introduced to Germany as a food fish in the late 1800s [1]. Whirling disease is associated with systemic infections by the myxozoan Myxobolus cerebralis, a parasite with presumed origins among salmonid fish in Eurasia [2,3]. Rainbow trout are highly susceptible to whirling disease and the introduction of the parasite to the U.S.A. in the 1950s had immediate economic impacts on salmonid hatcheries in both eastern and western states [2]. The parasite has a broad worldwide distribution and has been identified in 25 states in the U.S.A. where salmonid fish are present [4]. Salmonid hatcheries throughout the U.S.A have suffered drastic economic losses due to whirling disease outbreaks. The disease has more recently been recognized as the principal cause of major population declines among wild rainbow trout populations in the intermountain region of the U.S.A with serious negative impacts on sportfishing and allied industries [5-7]. Additionally, concerns continue over the potential negative ecologic impacts of whirling disease on wild salmonid populations, particularly threatened or endangered salmonids such as bull trout (Salvelinus confluentus), cutthroat trout (Oncorhynchus clarki), and steelhead (Oncorhynchus mykiss) [8,9].\nMyxobolus cerebralis has a complex life cycle that includes two alternate hosts, a salmonid fish and an oligochaete worm, Tubifex tubifex [10,11]. Infection in the salmonid host begins when microscopic waterborne actinospore stages of M. cerebralis are released from the worm and contact the skin of the fish host. Actinospores, also referred to as triactinomyxons for M. cerebralis, attach preferentially to fins and the buccal cavity where they release one or more of three coiled polar filaments which penetrate and then anchor them to the epidermis[12]. Within minutes the sporoplasm, which contains 64 internal cells, migrates from the triactinomxyon to deeper layers of the epidermis, an action that may be facilitated by parasite proteases [13-15]. Aggregates and single cells from the sporoplasm then begin mitotic replication within two h of initial infection, alternating between inter and intracellular locations, a process that may also depend upon parasite coded protease activity [15,16]. Over the next 10 h at water temperatures of 15°C, parasites within host epithelial cells further divide by the process of endogeny or cell within cell replication prior to release and then penetration of new host cells. Between 12 and 20 h post infection, the number of parasite cells present in the epidermis steadily declines until new stages are observed in the subcutis at 48 h. Degenerative stages observed in the epidermis between 12 and 20 h are suspected to be a result of the action of the host immune response, although the cellular and or humoral factors involved are not currently known. After a brief residence in the subcutis, parasite stages are presumed to migrate to proximal nervous tissues, initially in peripheral and then more central locations [16]. Migration and potential replication of parasite stages in nervous tissue ensues over the next 16 d with the first parasites exiting to invade cartilage observed at 20 d post infection [16]. Feeding on cartilage may induce a host inflammatory response that constricts the spinal cord, brain stem, and caudal nerves resulting in the erratic swimming behavior (whirling) and black tail observed among fish with acute whirling disease [17]. An additional impact of cartilage destruction are permanent deformities to the skeletal system that may increase vulnerability to predation and impair ability to forage for food [1]. The final developmental stages of the parasite in the fish are environmentally resistant spore stages (myxospores) which remained trapped in cartilage or bone [18]. Death of infected fish or ingestion by fish or avian predators releases myxospores from the fish tissues and they may be ingested by the second host, the benthic dwelling oligochaete T. tubifex [19]. A second developmental cycle then occurs under the mucosal lining of the intestine that results in the release of thousands of the actinospore (triactinomyxon) stages potentially over the entire lifetime of the individual oligochaete [20].\nSusceptibility to whirling disease in U.S. rainbow trout strains is pervasive with only two of the tested native strains displaying any degree of resistance, which may be inconsistent and relatively moderate [21,22]. Hatchery rainbow trout in Germany (Hofer strain), however, have acquired a degree of resistance to whirling disease that is consistently much higher than any domestic rainbow strains and comparable to that of brown trout (Salmon trutta), which are native to Europe and typically asymptomatic following infection [23]. Laboratory tests comparing rainbow trout strains under the same environmental conditions and pathogen exposure indicate that the Hofer strain's ability to combat M. cerebralis infection has a genetic basis. Recently, controlled crosses of the Hofer strain and a susceptible strain (Colorado River rainbow trout [CRR]) demonstrated that resistance to whirling disease was inherited by progeny [24] and heritability estimates are currently underway.\nThe discovery of the resistant Hofer strain allowed us to conduct an intraspecific comparison of susceptible and resistant rainbow trout in order to gain insight into the genetic basis underlying whirling disease susceptibility for this species. Gene expression profiling, through the use of microarrays, is an extremely high-throughput method to discover specific genes and pathways involved in a disease phenotype without the bias of a candidate gene approach.\nIn this study, microarray analysis was used to examine expression changes in a resistant and susceptible strain of rainbow trout following exposure to M. cerebralis, the pathogen causing whirling disease. We have found several genes significantly up-regulated in both the resistant and susceptible strain that appear to be involved in host response to infection. We have also found a gene which is significantly up-regulated in the resistant but remains unchanged in the susceptible rainbow trout strain following pathogen exposure that is a likely candidate gene for involvement in conferring whirling disease resistance.\n\nResults and Discussion\nQuantitative PCR conducted on caudal fin tissues at two hours post exposure to M. cerebralis demonstrated each fish strain had similar initial pathogen loads, although there was substantial variation between individual fish within each strain. The mean parasite copy numbers per host cell were 1.20 × 106 (SD 1.53 × 106) for the Hofer and 1.04 × 106 (SD 0.91 × 106) for the Trout Lodge. These mean values and standard deviations are similar to those obtained in additional studies of susceptible rainbow trout when examined at early time points post TAM exposure (unpublished data).\nIn order to study genes involved in whirling disease response, resistant and susceptible rainbow trout strains were exposed to M. cerebralis and RNA from skin tissue was converted to cDNA and hybridized onto microarrays. Relative gene expression for exposed and unexposed controls for each strain was compared and the list of differentially expressed genes for both strains is found in Table 1. A combined total of 17 genes or features (14 annotated genes, 3 unknown features) were differentially expressed in one or both strains following pathogen exposure and are involved with rainbow trout infection response to whirling disease exposure. Several of these genes were found in different locations on the array as unique expressed sequence tag (EST) clones and their repeated presence on the significance gene lists provides additional support for their involvement in the whirling disease phenotype. The small number of genes found potentially indicates that only a few genes contribute to the phenotypic differences found between resistant Hofer and susceptible Trout Lodge, at least in terms of differential gene expression, during early disease progression in the skin. In the microarray statistical analysis, when the delta value was adjusted even slightly lower, the FDR estimate increases from 0% to ~78%. Since increasing the FDR cut-off to such a high percentage would dramatically reduce power, we chose to leave the gene list small with an estimated FDR of 0%. This type of dramatic increase in FDR estimation is additional support that there are not many genes differentially expressed in response to whirling disease infection for our chosen tissue and time points.\nDifferent salmonid microarray platforms, such as those available from Oregon State University and Michigan State University, or different tissues and time points may produce additional candidate genes. A recent time course study used a candidate gene approach to identify four genes (TGF-β, IL-1β1, IL-1β2, and COX-2) that were significantly up-regulated by both Hofer and Trout Lodge in response to whirling disease infection [25]. These genes and their downstream effectors were not identified in the current microarray study, likely due to many differences in experimental design between the two studies (e.g., pathogen exposure levels, tissue types, water temperatures, age of fish at exposure, etc.). While downstream effectors of these genes were present on the microarray, only one of the four genes (COX-2) was actually present on the microarray. It is our hope that future genome sequencing will enable the construction of more comprehensive microarray platforms for economically important aquaculture species, such as Atlantic salmon and rainbow trout.\nAll significant genes identified by the current microarray study were up-regulated following pathogen exposure for one or both strains. Therefore, it appears that both strains are undergoing transcriptional activation to defend against whirling disease infection and thus, are exclusively employing positive regulation for the genes examined in skin during early disease progression.\nThe normal caveats that apply for microarray studies (gene discovery is limited by transcripts on arrays, differences at transcriptional level may not cause phenotypic differences, results are dependent upon tissue type and time point chosen, etc.) apply for this study. Additionally, the comparison of two rainbow trout strains (i.e., resistant versus susceptible) added another layer of complexity to the analysis. We chose to not directly compare the two strains because there could be expression differences between them, due to divergence following strain isolation, that are unrelated to the whirling disease phenotype. With that in mind, the two strains were first compared entirely separately from each other to discover expression differences in response to pathogen exposure for each strain. Only the genes responding to infection, and therefore implicated in the whirling disease phenotype, were compared between the two strains for differential gene expression (Figure 1). A limitation of this approach to our study is that constitutively expressed transcripts which are differentially expressed between the two strains that contribute to the whirling disease phenotype cannot be identified.\nMicroarray analysis of genes differentially expressed in the resistant Hofer strain in response to pathogen exposure\nA total of 16 genes or features (13 annotated genes, 3 unknown features) were up-regulated in the resistant Hofer strain following pathogen exposure. All 13 annotated genes have been previously implicated in host immune response for other infectious diseases. Viral Hemorrhagic Septicemia Virus (VHSV) induced protein and neighbor of COX-4 are the only annotated genes without known molecular functions.\nA common link between the majority of annotated genes with known molecular functions is an involvement in the interferon system. The interferon system is one of the first lines of host defense against invading pathogens for vertebrates (for review see [26]), including teleost fish (for review see [27]). Other economically important salmonid pathogens, such as infectious pancreatic necrosis virus and infectious salmon anaemia virus have been found to activate both type I and type II interferon (IFN) responses in the Atlantic salmon host following infection [28]. Interferons are cytokine proteins that are secreted following infection and play a critical role in both innate and adaptive immunity. The IFN system has been most widely researched in mammals and studies have found that type I IFN (mammalian IFN-α/β) are secreted by the pathogen-infected cells as part of a rapid initial immune response while Type II IFN (mammalian IFN-γ) is secreted by natural killer (NK) and T cells and plays a more central role in the second wave of immune response. To cope with the myriad of host infections, the interferon system is highly complex and involves the regulation of hundreds of genes [29,30]. Specifically, type I IFN acts to increase MHC class I expression for antigen presentation [31], promote T cell survival [32], inhibit cell proliferation [33], mediate apoptosis [26], and increase NK cell activity [34]. Type II IFN acts to increase both MHC class I and II expression for antigen presentation [29], stimulate macrophages to kill engulfed pathogens [35], induce apoptosis [36], and regulate leukocyte-endothelium interactions [37] in addition to many other immune-related activities.\nIt is informative to examine the functional roles of each gene's encoded protein specifically to better understand the part each plays, both individually and as interconnected components, in host immune response. Expression of the interferon-induced 35 kDa protein is induced by IFN and it is involved in cytokine signalling [38]. Interferon regulatory factor 1 (IRF-1) and interferon regulatory factor 7 (IRF-7) are transcription factors that induce expression of IFN responsive genes [39,40]. Additionally, IRF-1 is involved in apoptosis and cell cycle regulation related to tumor suppression [41]. Similarly, cyclin-dependent kinase 4 inhibitor B (p15-INK4b) plays a role in apoptosis[42], cell cycle regulation [42], and tumor suppression [43] and can be induced by the cytokine TGF-β [44]. Gig2 is an interferon-inducible protein that is likely part of the JAK-STAT signal transduction pathway [45]. Ubiquitin and the proteasome subunit beta type 8 precursor are both members of the ubiquitin-proteasome system (for review see [26]), which serves to degrade proteins via proteolysis. These degraded proteins can originate from an invading pathogen and are displayed on MHC class I proteins. The beta-2-microglobulin is an integral component of MHC class I proteins and is therefore involved in antigen processing and presentation to cytotoxic T cells [46]. Haptoglobin binds hemoglobin and limits its availability to infectious bacteria, thus preventing bacterial proliferation in a wound [47]. The PPAR-α-interacting complex protein 285 is a transcriptional co-activator with helicase activity [48] and has sequence similarity to a rainbow trout VHSV-induced protein. Gene expression of metallothionein B (MT-B) is induced by several metal ions [49], cytokines [50-52], and stress hormones [53-55]. MT proteins are believed to play diverse functional roles in inflammation, immune response, apoptosis, tumor suppression, and detoxification (for reviews see [55,56]).\n\nMicroarray analysis of genes differentially expressed in the susceptible Trout Lodge strain in response to pathogen exposure\nA total of six genes or features (five annotated genes, one unknown feature) were up-regulated in the susceptible Trout Lodge strain following pathogen exposure. Only one of the significant genes for Trout Lodge, which has sequence similarity to CC chemokine SCYA113, was not also differentially expressed in Hofer in response to pathogen exposure. The CC chemokine SCYA113 gene is a member of the CC chemokine family, which guides leukocytes to sites of infection and inflammation (for review see [57]). The fewer number of significant genes found for Trout Lodge relative to Hofer may indicate a decrease in transcriptional activation for this susceptible strain. There is, however, likely some degree of overlap in both strains' response to pathogen exposure due to the fact that several genes were up-regulated in both Hofer and Trout Lodge (i.e., ubiquitin-like protein 1, IRF-1, and PPAR-α-interacting protein Gig2). A critical phase in the early stages of M. cerebralis infection in trout is invasion and intracellular replication, processes that begin as early as one hour post exposure to triactinomyxons [16]. A role for accumulated ubiquinated proteins in the lysosome in the killing of Mycobacterium tuberculosis has recently been described that has implications for a range of intracellular infections [58] and some similar responses to infection may be occurring for both resistant and susceptible strains.\n\nMicroarray analysis of genes differentially expressed between resistant and susceptible strains in response to pathogen exposure\nOf the genes differentially expressed in response to pathogen exposure for both strains, only metallothionein B shows a statistically significant difference in expression between the two strains (Table 2). MT-B was found to be up-regulated in the resistant Hofer strain following pathogen exposure but remained unchanged in the susceptible Trout Lodge strain.\nAs previously noted, metallothionein has been implicated in a broad range of functional capacities, including inflammatory and immune responses. Several cytokines can induce metallothionein expression including IFN [59-61], interleukin-1 [50], interleukin-6 [51], and tumor necrosis factor-α [52]. Metallothionein has been shown to mediate leukocyte chemotaxis and has been hypothesized to serve as an early \"danger signal\" during times of stress or infection to activate an immune response [62]. The functional similarities between metallothionein and CC chemokine SCYA113, at least in terms of leukocyte chemotaxis, are certainly of interest since these genes displayed quite distinct expression profiles. Metallothionein was up-regulated in the resistant Hofer strain and CC chemokine SCYA113 was up-regulated in the susceptible Trout Lodge strain (although CC chemokine SCYA113 did not pass the significance cut-off to be considered differentially expressed between the two strains). This distinction between two genes, capable of similar biological roles, may indicate that leukocyte movements to, and their activities once at, the infection site are key factors in determining resistance versus susceptibility to whirling disease. Evaluations by light microscopy and qPCR for M. cerebralis genomic DNA of Hofer and Trout Lodge rainbow trout exposed to triactinomyxons demonstrates Hofer more efficiently eliminates invading parasites in the skin (M. Adkison, pers. comm.). While the parasite effectively penetrates the epidermis in both strains, significantly fewer parasites survive the migration from the skin to the nerves as evaluated at 10 d post exposure. A role for host immune factors in the elimination of invading parasites, even in susceptible rainbow trout strains, is suggested by several prior light and electron microscopy studies that demonstrate an increase in degenerative stages in the skin beginning as early as 12 h and then their elimination by 24 h post-exposure to triactinomyxons [12,16,63].\nThe difference in metallothionein expression may be due to an alternative immune response pathway since the protein has known involvement in diverse functional capacities. For instance, metallothionein's role as a zinc-finger transcriptional regulator [64] may dramatically alter the expression profiles between resistant and susceptible rainbow trout. All biological roles of this diverse protein should be considered when examining the complexities of host immune response. Additionally, upstream regulators of metallothionein expression could be the true underlying cause of the whirling disease phenotype since a gene expression study alone cannot determine if a gene is directly contributing to a phenotype (i.e., cause versus downstream effect).\n\nValidation of microarray results by qRT-PCR\nQuantitative RT-PCR (qRT-PCR) confirmed the microarrays results for two of the genes up-regulated in both Hofer and Trout Lodge following infection, ubiquitin and IRF-1, and the metallothionein gene (MT-B), which was up-regulated in Hofer but remained unchanged in Trout Lodge following infection (Figure 2). The qRT-PCR results for IRF-1 and metallothionein were quite similar to the microarray results for each gene, in terms of relative expression changes in response to infection. MT-B was found to once again be significantly up-regulated in the resistant Hofer strain following pathogen exposure but remained unchanged in the susceptible Trout Lodge. This difference in MT-B gene expression between the two strains was statistically significant (P ~ 0.001). The relative degree of up-regulation for ubiquitin following pathogen exposure was considerably higher in the qRT-PCR (~9 – 17 fold greater up-regulation in qRT-PCR versus microarrays). Many other studies have also observed this pattern of greater sensitivity in qRT-PCR versus microarray results (for examples see [65,66], which is often attributed to the more gene-specific optimized conditions of the qRT-PCR approach.\nGiven the high degree of statistical support and biological relevance of the candidate genes, we believe this study provides initial insight into rainbow trout genes and pathways responding to whirling disease infection and identifies the first candidate genes for whirling disease resistance.\n\nPotential future studies\nWhile the interferon system appears to be a likely candidate system for further study, many of the significant genes are found in alternative pathways and have distinct roles and functions in other systems. Furthermore, it is increasingly apparent that epistatic interactions and the interplay between pathways/networks previously classified as discrete can have enormous phenotypic effects on quantitative traits [67]. Multiple avenues of research should be examined in future studies, using the candidate genes presented here as an initial guide, due to the complex relationships between hosts and pathogens. For instance, the migration of leukocytes and their subsequent activity in the skin are likely a critical part of the early immune and inflammatory host response after pathogen infection. Additionally, it is quite feasible that the difference in metallothionein expression is due to an alternative immune response pathway since the protein has known involvement in diverse functional capacities. For instance, metallothionein's role as a zinc-finger transcriptional regulator [64] may dramatically alter the expression profiles between resistant and susceptible rainbow trout. All biological roles of this diverse protein should be considered when examining the complexities of host immune response. Finally, upstream regulators of metallothionein expression could be the true underlying cause of the whirling disease phenotype since a gene expression study alone cannot determine if a gene is directly contributing to a phenotype (i.e., cause versus downstream effect). The expression profiles of a variety of metallothionein upstream regulators, such as cytokines and metal transcription factor (MTF-1), could be evaluated in a time course study during early disease progression to identify additional candidate genes. A QTL mapping approach could also be used to identify particular chromosomal regions directly contributing to the disease phenotype.\n\n\nConclusion\nThe present study has provided the first examination into the genetic basis underlying rainbow trout's immune response and resistance to the whirling disease pathogen. Several genes were significantly up-regulated in skin following pathogen exposure for both the resistant Hofer and susceptible Trout Lodge rainbow trout strains. For both strains, response to infection appears to be linked with the interferon system. Metallothionein B is differentially expressed between the resistant and susceptible strains and is a good candidate for future whirling disease resistance studies. The identified genes have allowed us to gain initial insight into the molecular mechanisms involved in a salmonid host's immune response and resistance to whirling disease infection.\n\nMethods\nAnimal care, pathogen exposure, and RNA preparation\nHofer and Trout Lodge rainbow trout strains were reared in 35 gallon aquaria with 15°C flow-through well water for nine weeks post-hatch, with each fish weighing approximately 6.5 grams prior to pathogen exposure. Individuals from each strain (n = 60) were exposed to 2,000 triactinomyxons (TAMs) per fish for one hour. Additional fish (n = 60) from both strains served as unexposed controls, which were treated identically to exposed fish at all experimental stages other than their lack of pathogen exposure. Fish were then kept under standard aquaculture conditions until euthanized. TaqMan PCR for the quantitative evaluation of genomic parasite DNA was employed to confirm that fish in both the Hofer and Trout Lodge groups received equal amounts of parasite exposure. At two hours post TAM exposure, 6 fish in each exposed group were removed and euthanized with an overdose of benzocaine at a concentration of 500 mg/L. Caudal fins were removed posterior to the peduncle and used as the tissue for a quantiative TaqMan assay following procedures described by Kelley et al. [68].\nMicroarray studies examining skin four hours after pathogen exposure did not identify any genes differentially expressed between Trout Lodge and Hofer strains (data not shown). Therefore, we chose a later time point (24 hours after exposure) so that early host immune response was more likely to be fully underway and significant expression changes could be detected. After the 24 hour incubation period, all fish were euthanized with an overdose of benzocaine at a concentration of 500 mg/L. Each fish was euthanized individually and the caudal fin (largely comprised of skin tissue) was removed posterior to the peduncle. The fin was immediately placed into 2× Nucleic Acid Purification Lysis Solution supplied with ABI's TransPrep Chemistry kit (Applied Biosystems, Foster City, CA) to stop further gene expression changes. Total RNA was extracted from the fin of each individual using the ABI Prism™ TransPrep system with the ABI Prism™ 6100 Nucleic Acid PrepStation according to manufacturer instructions. RNA quality was assessed by agarose gel electrophoresis and RNA concentrations were measured using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).\nStarting total RNA yields were not sufficient for microarray hybridizations due to the small amount of caudal fin tissue present on these young fish. Therefore, 250 – 1000 ng of total RNA was used as the starting material to create amplified RNA (aRNA) indirectly labeled with Cy3 or Cy5 fluorescent dyes (GE Healthcare, Buckinghamshire, UK) using the Amino Allyl MessageAmp™ II aRNA Amplification kit according to manufacturer instructions (Ambion, Austin, TX).\n\nMicroarray hybridization and data analysis\nSalmonid cDNA microarrays (GRASP16k v2.0) were obtained from consortium for Genomic Research on Atlantic Salmon (cGRASP) and details of microarray development and fabrication can be found in von Schalburg et al. [69]. These arrays contain 13,421 Atlantic salmon and 2,576 rainbow trout cDNA features and have been successfully used for several previous rainbow trout gene expression studies [70-73]. For each rainbow trout strain, competitive hybridization was conducted on every array using equal amounts (8 μg) of differentially labeled aRNA from one control fish and one exposed fish. Four biological replicates were performed for each experimental condition and dye-sample coupling was swapped between biological replicates in a balanced block design.\nPrehybridization washes for all microarrays included: 2 × 5 min in 0.1% SDS, 5 × 1 min in NANOpure H2O with 0.5 mM dithiothreotol, 1 min in near boiling nanopure H2O, centrifugation for 2 min at 1500 RPM. To reduce background, the microarrays were next incubated for 90 min in 5 × SSC, 0.1% SDS, 3% BSA (Fraction V) at 49°C, washed 3 × 20 s in nanopure H2O, and dried by centrifugation for 5 min at 1500 RPM. The labeled aRNA samples were competitively hybridized to microarrays prewarmed to 49°C for 16 hours in a formamide-based buffer (Genisphere, Hatfield, PA) with LNA dT blocker (Genisphere). Posthybridization washes for all microarrays included: 1 × 10 min in 2 × SSC, 0.1% SDS prewarmed to 49°C, 2 × 5 min in 2 × SSC, 0.1% SDS at room temperature, 2 × 5 min 1 × SSC at room temperature, 2 × 5 min 0.1 × SSC at room temperature. Slides were then dried by centrifugation and immediately scanned using an Agilent G2565BA Microarray Scanner (Agilent Technologies, Santa Clara, CA).\nData underwent local background subtraction and LOWESS normalization using Agilent's Feature Extraction software. Raw and processed gene expression data have been deposited into the NCBI Gene Expression Omnibus [74] (series GSE8631) and are in compliance with MIAME guidelines. The Significance Analysis of Microarrays (SAM) software package [75] was used to identify differentially expressed genes between exposed and unexposed control fish for each rainbow trout strain. Both a Wilcoxon rank sum and a modified t-test were conducted with 1,000 permutations and the minimum fold change cut-off was set to 2.0 up- or down-regulated. A false discovery rate (FDR) of 0.00% was estimated for both strains. To determine statistically significant differences between the Hofer and Trout Lodge strains, a Welch's t-test (P-value < 0.01) was implemented in Microsoft Excel between the log ratios (exposed/control) for each strain for all genes that were significant for at least one strain in the SAM program.\n\nQuantitative RT-PCR\nMicroarray expression results were validated by qRT-PCR for several identified genes. Prior to qRT-PCR, 80 ng of total RNA was reverse transcribed from each biological replicate used for the microarray study along with two additional samples (total n = 6 per experimental condition) using the QuantiScript Reverse Transcriptase kit (Qiagen, Valencia, CA) according to manufacturer instructions. In contrast to the microarray experiments, the template RNA was not amplified before cDNA synthesis. EST clone sequences from the cGRASP microarray were used to design primers for genes undergoing validation, along with a β-actin reference gene used for normalization, with Primer3 software [76] and the sequence for each primer pair is shown in Table 3. The Quantitect™ SYBR® Green RT-PCR kit (Qiagen) was used according to the manufacturer's instructions except the final PCR volume was reduced to 25 μl. The PCR conditions used on a Chromo4 Real Time PCR Detection System (Bio-Rad, Hercules, CA) were as follows: HotStarTaq DNA polymerase activation at 95°C for 15 min, 45 cycles of 15 s denaturation at 94°C, 30 s annealing at 58°C, 30 s extension at 72°C, followed by a melting curve to ensure that a single PCR product was produced for each reaction. For each gene, the relative amount of gene expression was calculated using the ΔΔCT method [77] and significance was determined using a nonparametric Mann-Whitney U test and multiple linear regression in JMP.\n\n\nAuthors' contributions\nMRB participated in study conception and design, conducted gene expression experiments and data analysis, and drafted the manuscript. ABW participated in study design, data analysis, and manuscript revision. RPH and BPM participated in study conception and design, supervision of research activities, and manuscript revision. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 34041 ] ] } ]

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"normalized": [ { "db_name": "ncbi", "db_id": "8022" } ] }, { "id": "pmcA2257940__T36", "type": "species", "text": [ "M. cerebralis" ], "offsets": [ [ 9071, 9084 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "59783" } ] }, { "id": "pmcA2257940__T37", "type": "species", "text": [ "rainbow trout" ], "offsets": [ [ 9524, 9537 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "8022" } ] }, { "id": "pmcA2257940__T38", "type": "species", "text": [ "Atlantic salmon" ], "offsets": [ [ 11653, 11668 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "8030" } ] }, { "id": "pmcA2257940__T39", "type": "species", "text": [ "rainbow trout" ], "offsets": [ [ 11673, 11686 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "8022" } ] }, { "id": "pmcA2257940__T40", "type": "species", "text": [ "rainbow trout" ], "offsets": [ [ 12379, 12392 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "8022" } ] }, { "id": "pmcA2257940__T41", "type": "species", "text": [ "Viral Hemorrhagic Septicemia Virus" ], 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[ { "id": "pmcA2377325__text", "type": "Article", "text": [ "Purinergic signalling in the subretinal space: a role in the communication between the retina and the RPE\nAbstract\nThe retinal pigment epithelium (RPE) is separated from the photoreceptor outer segments by the subretinal space. While the actual volume of this space is minimal, the communication that occurs across this microenvironment is important to the visual process, and accumulating evidence suggests the purines ATP and adenosine contribute to this communication. P1 and P2 receptors are localized to membranes on both the photoreceptor outer segments and on the apical membrane of the RPE which border subretinal space. ATP is released across the apical membrane of the RPE into this space in response to various triggers including glutamate and chemical ischemia. This ATP is dephosphorylated into adenosine by a series of ectoenzymes on the RPE apical membrane. Regulation of release and ectoenzyme activity in response to light-sensitive signals can alter the balance of purines in subretinal space, and thus coordinate communication across subretinal space with the visual process.\n\nIntroduction\nThe retinal pigment epithelium (RPE) lies between the outer segments of the photoreceptors and the choroidal blood supply (Fig. 1). The RPE combines the functions of epithelial and glial cells, providing a barrier while also supporting the neural photoreceptors and modulating their function. Tight communication between photoreceptors and the RPE is critical to coordinate the multiple levels of interaction, and the purinergic contribution to this communication is becoming apparent. The relevance of this purinergic input is emphasized by the many functional effects of P1 and P2 receptor stimulation and by the multiple mechanisms in place to regulate subretinal levels of purine agonists. As the dynamics of ATP release and extracellular conversion into adenosine will modify agonist availability, the modulation of these processes can exert a temporal control on purinergic signaling. The following review will outline the main interactions between the RPE and photoreceptors, describe the effects of stimulating purinergic receptors on both sides of subretinal space, and summarize how levels of ATP, ADP, and adenosine are manipulated in this microenvironment.\nFig. 1Schematic illustration of the key components of purinergic signaling in the subretinal microenvironment. Stimulation of P2 receptors on the RPE can enhance transepithelial fluid absorption while P1 receptors can modulate phagocytosis. ATP released through CFTR and other Cl− channels can stimulate P2 receptors or be converted to ADP, AMP, and adenosine (Ado) by a series of ectonucleotidases present on the apical membrane of the RPE. By controlling the balance of extracellular purines available to stimulate these receptors these mechanisms can control levels of endogenous purines available to activate the receptors. While theoretically possible, it remains to be determined whether these subretinal purines can actually stimulate photoreceptors\nPurines and subretinal space\nRPE-photoreceptor interactions across the subretinal space\nThe outer segments of the rods and cones are responsible for the initial stages of vision, converting photon energy into a series of enzymatic reactions that close the light-sensitive channels on the photoreceptor plasma membrane, hyperpolarize the cells, and reduce the release of glutamate from the synaptic terminals [1, 2]. Efficient photoreceptor function depends upon both short-term and long-term support from the RPE. The critical nature of these interactions is evident from the rapid degeneration of photoreceptors in the absence of a healthy RPE layer and by the RPE localization of defective gene product in some forms of hereditary photoreceptor degeneration [3].\nThe apical membrane of the RPE is separated from the plasma membrane of the outer segments by an extracellular space of only 10–20 nm [4]. Although small, this subretinal space contains a highly structured matrix which ensheathes the outer segments and extends to the RPE [5, 6]. The presence of enzymes within this interphotoreceptor matrix emphasizes that this extracellular space itself is functionally active [7, 8]. This intimate anatomical relationship between photoreceptors and the RPE reflects multiple functional interactions. For example, the RPE accepts, recycles, and exports central components of the phototransduction process [9]. The outer segments are continuously resynthesized, and the phagocytosis, degradation, and processing of shed outer segment tips by the RPE cells is central to this renewal [10]. The ion channels and transporters on the apical membrane of the RPE help regulate the ionic composition of the subretinal space [11]. As extracellular levels of ions can modify the ionic driving forces across the photoreceptor plasma membrane, these RPE transporters can influence the state of neural activity. The transport of fluid and ions from the apical membrane to basolateral membrane of the RPE is also one of the main forces keeping the retina attached [12].\nThe control of photoreceptor activity by light gives a rapid temporal dependence to some interactions between the photoreceptors and the RPE. The volume of subretinal space increases in response to light [13], linking small changes in the ionic composition of the subretinal space with activity of the RPE transport mechanisms which maintain this volume [14, 15]. Other processes are controlled on a diurnal cycle. The shed tips of the outer segments are phagocytosed by the RPE soon after the onset of light [16, 17]. These processes can both be modulated by purine levels in subretinal space, indicating purinergic regulation is important over multiple time scales.\n\nPurinergic receptors on photoreceptors\nA2 adenosine receptors were localized to both the inner and outer segments of photoreceptor outer segments over a decade ago by Blazynski and colleagues [18], with more recent reports emphasizing their functional role. A2 agonists inhibit the L-type Ca2+ channel on rod outer segments [19] and can inhibit the synaptic release of glutamate from rods, suggesting changes in adenosine levels in subretinal space could modulate light sensitivity [20]. The A2 agonist DPMA and the adenosine deaminase inhibitor EHNA reduce mRNA for opsin in rods, suggesting that endogenous levels of adenosine can downregulate opsin message at night [21]. EHNA and the A2A receptor agonist CGS21680 also increase the survival of chick embryonic photoreceptors in culture [22], indicating a long-term neuroprotective role for adenosine.\nP2 receptors are also present in the photoreceptors. mRNA for the P2X2 receptor is expressed in the photoreceptor cell bodies, with immunohistochemical localization of the protein to outer segments [23]. In situ hybridization indicates the photoreceptor layer has the highest level of P2Y2 receptor of any region in the rabbit retina, although staining was not pronounced in monkey [24]. P2X7 receptors have recently been localized to photoreceptor synaptic terminals, with evidence for ecto-ATPase activity in the synapse, and functional evidence suggesting ATP augments transmission of the light response by rods [25]. It was suggested that ATP might be co-released from photoreceptors with glutamate, although this remains to be tested directly.\n\nPurinergic receptors on the RPE\nStimulation of P1 receptors can have a considerable impact on RPE cells. A2 receptors have been recognized on cultured and fresh RPE cells for some time [26, 27], with in situ hybridization confirming the presence of A2A receptors in rat RPE [28]. Stimulation of A2 receptors reduces the rate of rod outer segment phagocytosis by RPE cells [29], while application of adenosine to the apical membrane of chick RPE cells increases the basolateral Cl- conductance, the transepithelial potential, and the c-wave, and decreases the hyperpolarization of the basal membrane in response to light [30]. Although adenosine alone does not increase intracellular Ca2+ levels [31], adenosine acts synergistically with ATP to elevate Ca2+ levels in human RPE cells by stimulating both A1 and A2A receptors [32, 33]. Stimulation of A1 receptors with high doses of NECA increases the active transport of fluorescein across the RPE, while activation of A2A receptors decreases this transport, and by extension, transport of the ions that underlie fluid movement [34]. Stimulation of A1 and A2A receptors produces analogous increases and decreases, respectively, in the absorption of subretinal fluid blebs. This is consistent with the negative coupling of the A1 receptor and the positive coupling of the A2 receptors to adenylate cyclase, as increasing cAMP inhibits the transport of fluid across the RPE towards the choroid [35–37]. The agonist 2-Cl adenosine reverses the deficit in phosphoinositide metabolism found in diabetic RPE cells [38], suggesting effects on metabolism in addition to transport and phagocytosis.\nMultiple P2 receptors have been localized to the RPE. The P2Y2 receptor was initially characterized in cultured human RPE [31], with subsequent reports localizing transcript for P2Y1, P2Y2, P2Y4, and P2Y6 in the rat RPE/choroid [39] and for P2Y1 and P2Y12 receptors in ARPE-19 cells [40], and functionally identifying a P2X receptor in rat RPE cells [41]. ATP, ADP, and UTP induce numerous effects on RPE physiology [32, 33, 42, 43]. While these effects likely involve multiple different receptor types, the contributions of the P2Y2 receptor have been explored in most detail to date. The P2Y2 receptor has been specifically localized to the apical membrane of fresh bovine RPE cells, and addition of ATP to this membrane transiently elevates Ca2+, activates a basolateral Cl- conductance, inhibits an apical K+ conductance, and increases the apical to basolateral flow of fluid [43]. This increased absorption of fluid from the subretinal space suggests P2Y2 receptor stimulation could reduce the excess fluid that accumulates in retinal edemas, and several reports have supported this theory. ATP, UTP, and the P2Y2 receptor agonist INS37217 decrease the size of subretinal fluid blebs when injected into subretinal space of rats [44]. In both normal and rds +/- mice with experimentally induced detachment, INS31217 improves the ERG recovery and decreased cell death [45]. INS37217 also reduces subretinal blebs in rabbits [46]. Injection of another P2Y2 agonist, INS542, increases the active transport of fluorescein across the RPE, consistent with this upregulation of ion and fluid transport across the tissue [47]. Together these experiments emphasize the clinical potential of treating retinal edema with P2Y2 agonists.\n\n\nRegulation of purine levels in subretinal space\nWhile synthesized purinergic agonists may prove useful in treating some ocular disorders, the endogenous activation of P1 and P2 receptors in the subretinal microenvironment will ultimately be determined by availability of agonists. These levels are largely controlled by the release of ATP into the subretinal space, its conversion into other purines including adenosine, and the manipulation of adenosine by enzymes or transporters. Recent work has increased our understanding of both the stimuli that initiate changes in subretinal purine levels and the mechanisms that mediate these changes.\nRelease of ATP by the RPE\nAt least some of the ATP capable of stimulating the purinergic receptors on RPE cells is released from the RPE itself. The resulting autocrine stimulation ensures local delivery, and control, of purines to initiate the physiologic changes in the RPE. The release of ATP by RPE cells is triggered by multiple stimuli including osmotic stress, bFGF, UTP, NMDA, glutamate, and ATP [39, 40, 48–51]. The ATP release following activation of NMDA receptors by glutamate may have the most interesting implications for communication across subretinal space, given that glutamate confers the light signal from photoreceptors to the rest of the visual system. Glutamate and the specific receptor agonist NMDA triggers ATP release from ARPE-19 cells, with the release inhibited by NMDA antagonist MK-801, and by DCKA, which inhibits the glycine B binding site on NMDA receptors [51, 52]. Although NMDA raises intracellular Ca2+ levels, this increase is prevented by eliminating ATP with apyrase, indicating autostimulation through released ATP is responsible for this Ca2+ signal. NMDA also triggers a release of ATP when applied to the intact bovine RPE eyecup [51]. The NMDA receptors and the ATP release sites have been functionally identified to the apical membrane of the bovine RPE, suggesting the neurotransmitter interactions could amplify the signal from any glutamate reaching subretinal space.\nThe ability of both UTP and ATP to stimulate release of ATP from the RPE supports the theory that the system acts to amplify signals. When applied at greater than 1 μM, ATP triggers a secondary release of ATP peaking 10 min after the initial stimuli [40]. UTP also initiates a release in extracellular ATP with a similar delay [48]. The rise in ATP triggered by UTP is inhibited by the Cl- channel blocker NPPB, and UDP is much less effective at triggering release than UTP; both observations suggest the P2Y2 receptor contributes to the increase in ATP more than diphosphokinase, although influence from the enzyme cannot be ruled out [53].\nRecent evidence suggests that ischemia may lead to the release of ATP from RPE cells. Chemical ischemia triggers a substantial ATP release from cardiac myocytes [54], while changes in oxygen levels trigger ATP release in central chemoreceptors [55]. We found that exposure to sodium cyanide led to a rapid release of ATP from ARPE-19 cells (Fig. 2). As hypoxic and/or ischemic challenge may lead to changes in the expression of growth factors in RPE cells during certain ocular disorders such as macular degeneration [56], and as purines can induce expression of VEGF in other cells [57], this ATP release may contribute to growth factor signaling by the diseased RPE.\nFig. 2Chemical ischemia triggers ATP release from ARPE-19 cells. ATP release was measured in the bath directly from cells plated in 96-well plates to which the luciferin- luciferase reaction mixture was added [51]. Left Levels of ATP in the bath after addition of 5 mM NaCN to the cells. Measurement began 1 min after addition of NaCN or control solution to wells. Right Levels of ATP measured at the peak, 3 min after addition of NaCN (n = 12). Levels were normalized to concurrent levels in control (n = 14). Symbols and bars represent mean ± SE, *p < 0.001\nThe particular conduit for ATP release varies with the stimuli. The release in response to hypotonic challenge is largely dependent upon CFTR, as it was prevented by the specific CFTR inhibitor CFTR172 in addition to the more general blocker glybenclamide [50]. While the precise mechanisms by which CFTR contributes to this release are not yet known, a role for CFTR in ATP release into subretinal space is consistent with the reduction of certain ERG components in cftr -/- mice [58] and with the ability of apical ATP to activate conductances associated with these ERG components [43]. The release of ATP is also largely blocked by the vesicular transport inhibitor brefeldin A, suggesting the two processes occurred in series whereby ATP efflux follows the insertion of vesicles containing CFTR into the plasma membrane. Although the Ca2+ chelator BAPTA blocks this ATP release [50], raising Ca2+ alone with ionophore ionomycin does not itself initiate release [48]. This necessary but not sufficient contribution of Ca2+ also supports a role for vesicular insertion.\nIn contrast to the release following hypotonic challenge, the ATP release in response to NMDA does not involve CFTR [51]. Release is blocked by NPPB, however, suggesting another type of anion channel could serve as a conduit for ATP release. The presence of parallel mechanisms coexisting on the same cell for ATP release triggered by either agonists or by cell swelling has also been reported in astrocytes [59] and may reflect the multiple roles of purinergic signaling within a given tissue. As both stimuli lead to release across the apical membrane into subretinal space, both are expected to influence signaling in the microenvironment.\n\nInterconversion of purines in subretinal space\nThe interconversion of nucleotides and nucleosides each capable of stimulating distinct receptors makes the purinergic signaling system of particular interest in a confined region such as the subretinal space. The main enzymes responsible for dephosphorylating extracellular ATP on the RPE cells have been analyzed and a basic understanding of their regulation has begun. This section first describes the enzymes that act on ATP and ADP, followed by enzymes which convert AMP into adenosine.\nThe dephosphorylation of extracellular ATP by RPE cells involves enzymes from multiple families [40], as found in airway epithelial cells [60]. Degradation of ATP by the apical membrane of the fresh bovine eyecup and by ARPE-19 cells is inhibited by ARL67156 or βγmATP. Message for eNPP1, eNPP2, and eNPP3 is present in ARPE-19 cells, consistent with the preference of βγmATP for members of the eNPP family [61]. The cells also express NTPDase2, and NTPDase3, although the intermittent presence of NTPDase1 likely reflects a regulated process [40]. Ecto-alkaline phosphatase has no effect on ATP degradation in RPE cells, in contrast to its considerable contribution in airway epithelium [62]. The putative contribution from diphosphokinases to interconversion of subretinal purines is presently unknown.\nExtracellular AMP is rapidly dephosphorylated into adenosine in subretinal space. The production of adenosine from ATP at the apical membrane of the bovine RPE eyecup is inhibited by the ecto-5′-nucleotidase inhibitor αβmADP, confirming a role for this enzyme [63]. The enzyme is localized to rat RPE and ARPE-19 cells immunohistochemically. Degradation of 5′AMP is highest near the subretinal space of rat retina [63], although localization in mouse indicated larger amounts of ecto-5′-nucleotidase at the tips of adjacent Müller cells [64]. Levamisole does not inhibit the dephosphorylation of 5′AMP by the RPE, consistent with the absence of substantial ecto-alkaline phosphatase in subretinal space.\nThe presence of light may alter the levels of adenosine in subretinal space. Epinephrine is released at the onset of light [65] and stimulation of the RPE with epinephrine can decrease activity of ecto-5′-nucleotidase [63]. While norepinephrine and phenylephrine lead to similar decreases in enzyme activity, prazosin and corynanthine block the effects of norepinephrine, implicating the α1 epinephrine receptor in the inhibition of ecto-5′-nucleotidase [63]. The kinetics of inhibition are consistent with cleavage of the nucleotidase from its GPI anchor. The phagocytosis of rod outer segments is maximal shortly after light onset [16], and this phagocytosis is inhibited by adenosine [29]. The ability of epinephrine released by the illuminated retina to reduce ecto-5′-nucleotidase activity and consequently adenosine levels may relieve this inhibition and enhance the rate of phagocytosis at light onset.\n\n\nPhysiologic effects of subretinal purines on the RPE and photoreceptors\nThe number of purinergic receptors on both photoreceptor and RPE membranes suggests purines make multiple contributions to the physiology of the outer retina. Our increased understanding of how agonist levels in subretinal space are controlled has begun to indicate how and when this contribution may occur. Future research will involve applying these findings from isolated systems to intact RPE-photoreceptor models, and pursuing the role of defective purinergic regulation in ocular disease. While it is unlikely that ATP released across the apical membrane of the RPE can diffuse to these P2 receptors in the outer plexiform layer given the ecto-ATPase activity in the synaptic clef [25], stimulation of receptors elsewhere on the photoreceptor membrane is possible. It would be interesting to determine whether ATP released from the RPE and converted to adenosine by ecto-nucleotidases can actually modulate the response to light by stimulating the A2A receptors on photoreceptor outer segments. The impact of purinergic signaling on chronic ocular diseases is also of interest, such as the role of ischemia-driven ATP release in VEGF production. While the small size of subretinal space can complicate pharmacologic manipulation within the intact RPE-photoreceptor complex, molecular approaches may provide new insight into how endogenous purines in subretinal space affect the physiology, and pathophysiology, of both RPE and photoreceptors.\n\n\n\n" ], "offsets": [ [ 0, 20870 ] ] } ]

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[ { "id": "pmcA1312363__text", "type": "Article", "text": [ "Gene expression studies in isolated mitochondria: Solanum tuberosum rps10 is recognized by cognate potato but not by the transcription, splicing and editing machinery of wheat mitochondria\nAbstract\nThe complex gene expression mechanisms that occur in plant mitochondria, such as RNA editing and splicing, are not yet well understood. RNA editing in higher plant mitochondria is a highly specific process which modifies mRNA sequences by C-to-U conversions. It has been suggested that in some cases this process is required for splicing. Here, we use an experimental model based on the introduction of DNA into isolated mitochondria by electroporation to study organellar gene expression events. Our aim was to compare processing and editing of potato small ribosomal protein 10 gene (rps10) transcripts in heterologous (wheat mitochondria) and homologous (potato mitochondria) contexts. rps10 is a suitable model because it contains a group II intron, is absent in wheat mitochondria but is actively expressed in potato mitochondria, where transcripts are spliced and undergo five C-to-U editing events. For this purpose, conditions for electroporating isolated potato mitochondria were established. rps10 was placed under the control of either potato or wheat cox2 promoters. We found that rps10 was only transcribed under the control of a cognate promoter. In wheat mitochondria, rps10 transcripts were neither spliced nor edited while they are correctly processed in potato mitochondria. Interestingly, a wheat editing site grafted into rps10 was not recognized by wheat mitochondria but was correctly edited in potato mitochondria. Taken together, these results suggest that editing might occur only when the transcripts are engaged in processing and that they would not be available to editing factors outside of a putative RNA maturation machinery complex.\n\nINTRODUCTION\nGene expression in plant mitochondria is a complex process involving multiple steps such as transcription, cis- and trans-splicing, RNA trimming and RNA editing on the way to translation (1). RNA editing has been found in a variety of organisms and occurs through different mechanisms such as insertion or deletion of nucleotides, or base conversions [for details see reference (2)]. In higher plant organelles, RNA editing is an important post-transcriptional event characterized by C-to-U changes via a deamination mechanism (3–5). In Arabidopsis thaliana 456 C-to-U editing events have been described (6). RNA editing occurs mostly in the coding regions which alter the identity of the encoded amino acid, but some editing events occur in highly structured regions of introns (7,8). Like other maturation processes, RNA editing is an essential post-transcriptional event in plant mitochondrial gene expression (9–11) required for the synthesis of functional proteins (12,13).\nThe introduction of foreign DNA into isolated mitochondria is a novel experimental model which has provided important information on RNA editing and RNA splicing enabling the use of a site-directed mutagenesis approach (14,15). Using a cognate cox2 chimeric gene construct, the cis-recognition elements required for plant mitochondria RNA editing have been determined (16,17). Using the same approach, Staudinger and Kempken (18) have reported that transcripts from A.thaliana cox2, but not Sorghum bicolor atp6, are edited when the genes are introduced into maize mitochondria.\nInterestingly, higher plant mitochondrial genomes differ in their gene contents due to an evolutionary information transfer from the organelle to the nucleus (19). Of particular interest is the situation of the small ribosomal protein 10 gene (rps10), a group II intron-bearing gene which is encoded in Solanum tuberosum mitochondrial DNA but is absent from the wheat mitochondrial genome (20). Higher plant mitochondria contain group II introns either in cis- or trans- configuration (1) which can be folded in a characteristic secondary structure (21). Intron removal in plant mitochondrial mRNAs is not well documented because such introns are unable to self-splice. Previously, we described that electroporated cox2 constructs were a good model for the study of the splicing process. Using this model, we found that editing and splicing of cox2 transcripts were not linked in wheat mitochondria (15).\nTo challenge the ability of mitochondrial gene expression machinery to recognize genetic information which has been lost during evolution, we decided to introduce the S.tuberosum non-cognate rps10 gene into wheat mitochondria. Five C residues are changed to U in potato mitochondria rps10 transcripts by editing. Two of them have been postulated as being necessary for acquisition of a proper secondary and/or tertiary structure for splicing. To test this hypothesis, it was necessary to set up the conditions for electroporation of foreign DNA into S.tuberosum mitochondria. Here, we show that a potato rps10 construct is transcribed when introduced into wheat mitochondria, but transcripts are not recognized by the post-transcriptional processing machinery. In contrast, a rps10 construct is correctly expressed and processed in cognate potato mitochondria. Moreover, we present evidence that transcript editing might be linked to overall RNA processing. This is the first report on DNA electroporation into potato mitochondria.\n\nMATERIALS AND METHODS\nPlasmids\nAll plasmids used in this study are based on the previously described pCOXII vector (14). An NsiI restriction site was introduced at the initiation codon of the wheat cox2 open reading frame (ORF). Then, NsiI was used in combination with a SpeI restriction site present in the original vector after the stop codon, to produce the chimeric vectors.\nS.tuberosum cox2 gene, including a 727 bp non-coding upstream region, was isolated by PCR from total DNA using the primer A designed from partial sequences reported by Löessl et al. (22), accession no. AF096321, containing the KpnI restriction sequence, and primer B derived from Triticum timopheevi cox2 (AF336134). A fragment of 2888 bp was obtained and cloned on pGEM-T vector. The complete sequence of the S.tuberosum cox2 was determined (accession no. DQ18064). To generate the plasmid pCOXIISt containing the S.tuberosum cox2 gene, a KpnI/SpeI fragment containing the 727 bp upstream region and the complete coding region was used to replace the wheat gene from pCOXII. As for pCOXII, a NsiI site was inserted at the ATG start codon and a 23 bp fragment was inserted at position −20. This 23 bp insertion provides a specific sequence allowing isolation of potato cox2 transcripts originating from the introduced DNA by RT–PCR. The chimeric wheat/potato cox2 gene was constructed by replacing 727 bp KpnI/NsiI promoter sequence from pCOXIISt with the 880 bp wheat upstream region from pCOXII.\nThe vectors pRPS10W and the pRPS10St derivative, containing wheat and potato cox2 promoters, respectively, were constructed by inserting the 1178 bp rps10 sequence containing two exons separated by a 777 bp intronic sequence [(23), accession no. X74826]. The coding region was isolated from total S.tuberosum DNA by PCR using primers D1 and D2 containing the restriction sites NsiI and SpeI, respectively. The fragment NsiI/SpeI was purified and cloned into pCOXII and pCOXIISt, replacing the respective cox2 coding regions.\nSince all vectors used here were based on pCOXII, they contain the downstream region from the wheat cob gene (Ir-cob) (accession no. AF337547) (14). This sequence, combined with the 23 bp upstream insert sequence served to distinguish, using primers 1a and 1b, foreign from endogenous transcripts. All mutants were obtained using QuickChange® Site-Directed Mutagenesis kit (Stratagene). PCR product purifications were carried out with the Wizard® Clean-Up System (Promega).\n\nPCR primers used\nOligonucleotides sequences are in 5′ to 3′ orientation. (1a), GCGGTGCAGTCATACAGATCTGC; (1b), TATCCAGATTTGGTACCAAAC; (2a), GCAGTCATACAGATCTGCCAAAG; (2b), AGATTTGGTACCAAACCCGGG; (A), TATAGGTACCTCTCAGGTGTCAAAGTGTGGATTT; (B), TATAACTAGTTTAAGCTTCCCCG; (D1), TATAATGCATAGACAAAGGAGAGCACTTA; (D2), TATAACTAGTTCAGGAAAGGGTCAACGCAA. Restriction sites are underlined.\n\nMutagenesis primers (only sense primers in 5′–3′ orientation are indicated)\nSingle C2, C3 and C2+C3 double mutant plasmids were constructed with primers (C2) AAGAAGTTCTTTTGGTTAAAACGCC and (C3) CGCCGTGCGACTTGGAGGACATAAG. NsiI-pCOXII: GGAAATCCAATGCATCTTCGTTCATT and NsiI-pCOXIISt: CCAAACCAAATGCATGTTCTAGAATG. Construct pCOXIISt containing a 23 nt insertion in the promoter region, was carried out into two consecutive insertions using primers: TGGGGGGAGCAGAGCAGTGCGGTGCAGTCACAAAGAATGAACCAAACC and GCAGTGCGGTGCAGTCATACAGATCTGCCAAAGAATGAACCAAACC. For constructs MA and MAB containing the wheat cox2 C259 editing site, two consecutive insertions were carried out using primers: GGAAGATTGGATTACTATCGAAATTGCCCTGAATCA and TACTATCGAAATTATTCGGACCATGCCCTGAATCA. For ME6 and ME6b constructs, primers CCGCGAGGAATCAACTACTATCGAAATTATTGCCGGTGCTGAC and CAACTACTATCGAAATTATTCGGACCATATTGCCGGTGCTGAC were used. Inserted nucleotides are underlined and the modified residues are indicated in bold.\n\nMitochondria purification\nS.tuberosum cv. Rosevalt tubers and T.aestivum var Fortal seeds were used. Potato mitochondria were prepared from 2 kg of tubers in batches of 200 g with 200 ml of a homogenization buffer containing 0.4 M mannitol, 25 mM MOPS (pH 7.8), 1 mM EGTA, 8 mM cysteine and 1 mg/ml fatty acid-free BSA. Homogenization was carried out for 15 s in a Waring blendor at full speed. Homogenate was centrifuged in a Sorvall GSA rotor at 1500 g for 10 min at 4°C. Supernatant was centrifuged in a GSA rotor at 12 000 g for 15 min. The mitochondrial pellet was resuspended in 50 ml of homogenization buffer and centrifuged at 1500 g. The supernatant was centrifuged in a Sorvall SS-34 rotor at 15 000 g for 10 min, the pellet was resuspended in 12 ml of homogenization buffer and mitochondria were purified by centrifugation on a sucrose gradient essentially as described for wheat embryo mitochondria (14).\n\nElectroporation\nElectrotransfer experiments were carried out with 1 mg of purified wheat embryo or potato tuber mitochondria in 50 µl of 0.33 M sucrose and 1 µg of recombinant plasmid as described (14). The electroporated mitochondria were incubated for 18 h at 25°C, then recovered by centrifugation in a Sigma N° 12024 rotor (Sigma 1K15 refrigerated centrifuge) at 15 000 g for 15 min at 4°C. In the case of potato mitochondria the incubation mixture was supplemented with 1 mg/ml of fatty acid-free BSA. RNA was purified with 200 µl of TRIzol™ reagent (Gibco-BRL) according to the supplier's protocol.\n\nDNAse I protection assay and DNA purification\nAfter electroporation and centrifugation, the mitochondrial pellet was resuspended in 100 µl of buffer [10 mM Tris–HCl (pH 7.5), 2 mM magnesium acetate and 0.33 M sucrose] containing 60 U of DNase I (Gibco-BRL) and incubated for 1 h at room temperature. The DNase reaction was stopped by adding 4 µl of 0.5 M EDTA and then heating for 10 min at 65°C. Mitochondria were incubated with 100 µg of Proteinase K (Merck) for 4 h at 37°C. One microliter of 20% SDS was added to achieve mitochondrial lysis and the DNA was extracted with phenol/chloroform, precipitated with 0.1 vol of 3 M Sodium Acetate (pH 5.2), 3 vol of 100% ethanol and 100 ng of carrier yeast tRNA and left overnight at −20°C. After centrifugation, the DNA pellet was resuspended in TE buffer [10 mM Tris–HCl (pH 8) and 1 mM EDTA].\n\nRT–PCR\nRNA (1 µg) was treated with 2 U of Amplification grade DNase I (Promega). cDNA synthesis was performed with 200 U of Superscript II RT using 100 ng of random hexamers. The PCR were performed with primers 1a and 1b using Advantage® 2 Polymerase Mix (Clontech) as follows: 95°C, 1 min; 5 cycles at 95°C for 30 s and 68°C for 1 min; 30 cycles at 95°C for 30 s, 58°C for 30 s and 68°C for 30 s, and finally 68°C for 1 min. Primers 2a and 2b were used for nested PCR from 1 µl of the first PCR. No RT–PCR amplification products were obtained with RNA from non-electroporated mitochondria.\n\nDNA sequencing\nSequence analyses were performed directly on the RT–PCR product using an automatic DNA sequencing equipment with the BigDye® Terminator Cycle Sequencing Kit (Applied Biosystem).\n\n\nRESULTS\nS.tuberosum rps10 transcripts are not processed in wheat mitochondria\nThe S.tuberosum rps10 construct under control of a wheat cox2 promoter (Figure 1A) was incorporated into purified wheat mitochondria by electroporation as indicated in Materials and Methods. After electroporation and incubation in expression medium, mitochondrial RNA was extracted and analysed by RT–PCR. The primers used allowed detection of the transcripts generated by the constructs introduced and excluded any product from endogenous cox2 (or rps10 in the potato system). Two bands of 1204 and 427 bp were expected from precursor and mature rps10 mRNAs, respectively. Only precursor rps10 molecules were detected (Figure 1B). As a control, a cognate cox2 construct (16) was used. In this case, the 2048 and 827 bp RT–PCR bands representing the precursor and spliced cox2 products, respectively were observed (Figure 1B). In all cases, the PCR bands actually represent transcription products since when PCR was performed without the RT step we observed no amplification products.\nPreviously, five C residues, two in exon 1, one in the intron and two in exon 2 have been reported to be changed to U by editing in potato mitochondria (23). To determine if the non-cognate transcript could be recognized by the wheat RNA editing machinery, the 1204 bp RT–PCR product was sequenced. While wheat cox2 editing sites were correctly edited in control as expected (Figure 1C, only site C77 is shown), all five editable residues in potato rps10 transcript remain unchanged. cox2 C77 editing sites are identical in potato and wheat.\n\nFailure of rps10 transcript splicing in wheat mitochondria is not linked to the absence of editing\nIt has been suggested that residues C2 and C3 participate in the secondary structure of the intron necessary for splicing (23). A possible explanation to the failure observed in precursor rps10 splicing could be that the absence of edition of C2 and C3 prevent the intron from organizing itself in a catalytically competent conformation. To test this hypothesis, single C2 and C3 mutants and a C2+C3 double mutant were constructed by changing the C residues to T. Neither single nor double mutants were able to undergo splicing (Figure 2).\n\nElectroporation of S.tuberosum mitochondria\nSince two important post-transcriptional processes, RNA editing and splicing, were inoperative when S.tuberosum rps10 was expressed in wheat mitochondria, we decided to verify whether the negative results were inherent to the rps10 chimeric constructs or due to the lack of trans-recognition elements in wheat mitochondria. For this purpose, it was necessary to set up an electroporation protocol adapted to S.tuberosum mitochondria. Purified organelles were prepared from potato tubers as indicated in Materials and Methods. Electric pulses in the range between 8 and 20 kV were performed. Internalization of exogenous DNA was measured by DNAse protection assays (14). Potato mitochondria show a broad range response with a maximum around 13 kV (Figure 3). This voltage setting was therefore used for further experiments.\n\nS.tuberosum mitochondria does not recognize the wheat cox2 promoter\nTo ascertain the ability of electroporated mitochondria to perform expression of the exogenous gene construct, we used a plasmid containing the potato cox2 gene controlled either by T.aestivum or S.tuberosum promoters. As shown in Figure 4, the construct containing the wheat or potato promoters was transcribed only in cognate mitochondria. In potato, the mature product is barely detectable. In contrast to wheat mitochondria, the 821 nt mature transcript was only detected when the electroporated potato mitochondria were incubated in the presence of fatty acid-free BSA.\n\nA cognate rps10 construct is correctly expressed, edited and processed in potato mitochondria\nThe construct expressing potato rps10 under the control of potato cox2 promoter was introduced into S.tuberosum isolated mitochondria. After incubation, the precursor and mature transcripts were amplified by RT–PCR (Figure 5A) and sequenced. As shown in Figure 5B, the four editing sites C1, C2, C4 and C5, described previously in endogenous transcripts, were found edited in mature mRNA. The presence of spliced molecules containing unedited residues (Figure 5B) indicates that rps10 transcripts could be spliced before editing. To confirm this observation, the PCR product was cloned and sequenced. One half of the individual clones were found edited at sites C1 and C2; 65% were edited at site C4 and 25% were edited at site C5.\n\nA cognate editing site in a non-native context is not recognized by wheat mitochondria but is recognized in heterologous mitochondria\nTo test whether wheat mitochondria are able to edit a cognate site when placed in the context of a potato transcript, we introduced the C259 −16/+6 region from wheat cox2 into potato rps10 exon 1 or intron (Figure 6A, construct MA and ME6, respectively). As reported previously, the 23 nt region forming the C259 editing site from wheat cox2 was efficiently edited when grafted into a different context in a wheat transcript (17). Wild-type rps10 (pRPS10W) and the MA and ME6 mutants were expressed in wheat mitochondria but no splicing was observed (Figure 6B). Unexpectedly, wheat mitochondria appear to be unable to edit the cognate C259 site when the −16/+6 region is located on a potato rps10 precursor. The control CM construct, containing the C259 site grafted in a different context but in its own cox2 gene, was expressed, spliced and edited as expected [(17), Figure 6B and D]. To compare the editing status of RNAs at the same stage of processing, the editing status of the inserted C259 site in precursor cox2 transcripts is shown (Figure 6D). The analogous MAb and ME6b constructs, containing the S.tuberosum cox2 promoter and the C259 region inserted into rps10 exon 1 and intron were used to electroporate potato mitochondria. As shown in Figure 6C, MAb and ME6b were expressed and spliced at the same level as the control in the homologous context, indicating that insertion does not affect the splicing process. Interestingly, while C259 insertion was not recognized in wheat mitochondria (Figure 6D), the potato RNA editing machinery edited the inserted C259 region (Figure 6E). The C259 region inserted in the rps10 intron (construct ME6b) was also edited, although at a very low level, showing that editing may precede intron removal (not shown).\n\n\nDISCUSSION\nPlant mitochondria undergo complex expression mechanisms which are poorly understood. Most studies are based on analysis of in vivo mature or intermediate gene products giving clues on possible mechanisms and suggesting pathways operating in gene expression. Direct tests using in vitro approaches have been fruitful (4,5,24), but are hampered by the difficulty of obtaining active mitochondrial extracts. Previously, we devised an experimental model based on electroporation of isolated mitochondria that allows us to test gene expression of wild-type and mutant genes. This approach has been very useful in elucidating the process of RNA maturation in plant mitochondria, in particular splicing and editing (14,15). The aim of this work was to analyse different gene expression events when a foreign gene was expressed in a heterologous context. rps10 was chosen precisely because this genetic information is lacking in wheat mitochondria but is active in potato mitochondria (20,23,25). Moreover, rps10 transcripts undergo five C-to-U editing events in potato mitochondria and rps10 contains an intron, thus facilitating the analysis of post-transcriptional processing.\nTo best evaluate the expression of rps10 in the non-cognate mitochondria, it was necessary to set up electroporation conditions for introducing foreign DNA into S.tuberosum mitochondria. Potato tuber mitochondria were able to incorporate DNA essentially under the same conditions described for wheat embryo organelles (14), except that the optimal voltage range was larger. A major difference to wheat was that isolated potato mitochondria were viable for 3–4 h as measured by oxygen consumption (data not shown). Thus, to observe transcript maturation after electroporation the incubation mixture needed to be supplemented with fatty acid-free BSA (see Figure 4). This behaviour probably reflects the uncoupling of oxidative phosphorylation by fatty acids during incubation of potato mitochondria (26). In fact, previously we found that proper gene expression in isolated mitochondria requires a functional organelle able to generate ATP from ADP and succinate (14).\nAn interesting observation was that neither the wheat nor the potato cox2 promoters were recognized in a heterologous context (Figure 4A and B). This situation might by explained by the fact that potato and wheat promoter sequences have no recognizable homologous motifs. Plant mitochondria promoters are characterized by a conserved core CRTA sequence with differences in the extent and the composition of sequences around the consensus motif (27,28). While the transcription initiation site in wheat mitochondria is located at position −170 (29), the potato promoter has not yet been described. Transcription may be initiated at numerous sites suggesting a relaxed promoter recognition by the transcription machinery (28). However, the lack of crossed recognition of cox2 promoters in wheat and potato is not a general situation since the A.thaliana cox2 gene is expressed and spliced when introduced into maize mitochondria, indicating that the Arabidopsis gene shares some signals with maize cox2 promoter that are sufficient for transcription (18). The sites required for transcript initiation have been recently described in A.thaliana mitochondrial genes (28). Three regions were described as important for transcription initiation. We found no such homologous sequences in the potato cox2 upstream region indicating that the two dicot promoters do not have the same origin. This in turn may reflect the natural history of this particular mitochondrial gene evolving in its own context. It should be mentioned that the presence of a conserved sequence is not sufficient for expression since a region that acts as promoter in Arabidopsis, potato and Oenothera is inactive in vivo in pea (30). Further studies will be required to understand the transcriptional events in plant mitochondria. Electroporation of foreign DNA into isolated mitochondria provides an interesting functional model, complementary to in vitro transcription assay, for answering these questions.\nThe potato rps10 gene controlled by a T.aestivum promoter (Figure 1A) was transcribed as a 1204 nt precursor in wheat mitochondria, but no traces of mature RNA were observed. Moreover, the five C residues reported as RNA editing targets in vivo (23) remain unchanged, indicating that rps10 transcript was not recognized by the wheat splicing and editing machinery. A control using the cognate cox2 construct demonstrates that electroporated organelles were competent for splicing and editing (Figure 1B and C).\nSome editing events occur in highly structured domains of introns. Because in some cases, editing corrects A-C mispairing improving conformation of the intron, it has been proposed that the C-to-U change might be necessary for efficient splicing (8,9,23,31). Based on the canonical structure of group II mitochondrial introns (21), two editing sites, C2 located at the Intron Binding Site 2 (IBS2) and C3 located in the intron, nine residues downstream from the end of exon 1 in S.tuberosum rps10 are of particular interest. It has been predicted that both edited residues participate in base-pair interactions in the putative secondary structure. Of particular interest is the site C3 located in intron domain I (23). This position may be crucial for splicing as inferred from mutants of a yeast mitochondrial intron (32). We addressed this question by introducing rps10 mutant genes presenting C2, C3 or both positions in the edited form into mitochondria. As shown in Figure 2, transcription was as efficient as for the cognate cox2 construct but neither wild-type rps10 nor the C-U mutants underwent splicing. These results clearly demonstrate that C2 and C3 editing is not sufficient for splicing of rps10 precursor in wheat mitochondria and lead us to conclude that splicing failure is probably linked to the lack of trans-recognition elements. One may speculate that these trans-acting factors, for instance nuclear-encoded maturases, were lost after rps10 was transferred to the nucleus in monocots (20,25). The C-to-U mutants were also tested in potato mitochondria; no significant differences were detected in splicing efficiency when compared to the unedited construct (D. Choury, unpublished data). It should be noted that the lack of splicing in wheat mitochondria is not a general feature of potato introns, since the potato cox2 intron is removed efficiently (Figure 4B). This is consistent with the hypothesis that potato and wheat mitochondria have similar trans-acting factors for cox2 intron removal.\nA possible link between intron removal and editing has been proposed for splicing of nad1e and nad5III trans-introns where domain six may require editing to be structured in a catalytic competent secondary conformation (8). In rice, failure of RNA maturation and editing has been correlated with cytoplasmic male sterile phenotype. In this model, the absence of the nuclear gene Rf-1 affects B-atp6 RNA cleavage and editing (33). In organello studies have shown that S.bicolor atp6-1 gene was transcribed but not edited when introduced into maize mitochondria (18). However, partially edited molecules were detected in sorghum-maize atp6 chimeric transcripts when they included the maize atp6 5′-untranslated sequence, suggesting that the 5′ non-coding region provides a structural motif or binding site for a transcript-specific editing factor (34). Unfortunately, no data on atp6 processing was reported to determine whether this region is directly involved in editing or some other maturation event.\nSince wheat mitochondria do not have rps10 encoded in the mitochondrial genome (20), it was interesting to test whether wheat mitochondria were able to recognize editing sites which had been lost during evolution. In other words, whether the mitochondrial trans-recognition elements are specific for each site or whether they are operating on a subset of editing sites. This question is important since to date the factors responsible for RNA editing in plant mitochondria remain unknown. Solving this issue may provide clues to uncover such factors. As described above, the rps10 transcript was not processed in wheat but it was correctly spliced in cognate mitochondria (Figure 5A). Indeed, the mature transcript had identical exon1 and exon2 junction as found in endogenous rps10 mRNA (data not shown). More importantly, all editing sites, C1, C2, C4 and C5 were significantly converted to Us (Figure 5B). The fact that the four editing sites were found either edited or unedited in spliced rps10 mRNA is an indication that editing does not precede splicing as was previously found for cox2 in wheat (15).\nIt may be argued that the absence of splicing precluded editing. This possibility can be discarded since potato rps10 precursor mRNA was found to be edited in vivo (23). These data clearly show that splicing is not required for rps10 editing, similar to previous findings for cox2 mRNA and also in cox2 mutant derivatives unable to remove the intron (15). This led us to postulate that the inability of wheat mitochondria to recognize rps10 editing sites is likely due to the fact that wheat mitochondria have lost the editing trans-recognition elements which become dispensable after transfer of rps10 to the nucleus. To test this possibility, rps10 chimeric plasmids containing editing site C259 from wheat cox2 inserted either in exon1 or the intron were constructed. Site C259 is formed by 23 nt corresponding to the −16/+6 sequence embedding the target C. Previously we found that this small region could be recognized by the RNA editing machinery when placed outside of its natural context (16,17). The wheat C259 editing site in the chimeric construct was correctly edited by potato mitochondria. This result is not unexpected since the corresponding region in endogenous potato cox2 mRNA, which presents two differences at positions −3 (C instead of A) and −7 (G instead of A), is edited. Furthermore, these positions were not crucial for editing of C259 (17). Surprisingly, the C259 editing site grafted in rps10 was not recognized by the wheat editing machinery. We cannot exclude the possibility that editing efficiency in precursor molecules is very low and so is undetectable when sequencing RT–PCR products representing a pool of transcripts. However, analysis of the same region in cox2 precursors indicates that this was not the case since significant C-to-U conversion was observed (Figure 6D). These observations lead us to postulate that editing is occurring only when the transcript is engaged in post-transcriptional processing, suggesting that rps10 transcripts are not available to editing factors independently of the RNA maturation machinery. The fact that the wheat editing site C259 inserted in chimeric rps10 transcripts was not recognized by wheat mitochondria is a strong argument for this hypothesis. One might speculate that in plant mitochondria, transcripts have to be engaged in a kind of multiprotein processing complex. A failure to be recognized at some early stage will lead to their accumulation as unmodified precursors.\n\n\n" ], "offsets": [ [ 0, 30076 ] ] } ]

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"text": [ "wheat" ], "offsets": [ [ 965, 970 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4565" } ] }, { "id": "pmcA1312363__T7", "type": "species", "text": [ "potato" ], "offsets": [ [ 1013, 1019 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4113" } ] }, { "id": "pmcA1312363__T8", "type": "species", "text": [ "potato" ], "offsets": [ [ 1162, 1168 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4113" } ] }, { "id": "pmcA1312363__T9", "type": "species", "text": [ "potato" ], "offsets": [ [ 1245, 1251 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4113" } ] }, { "id": "pmcA1312363__T10", "type": "species", "text": [ "wheat" ], "offsets": [ [ 1255, 1260 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4565" } ] }, { "id": "pmcA1312363__T11", "type": "species", "text": [ "wheat" ], "offsets": [ [ 1362, 1367 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4565" } ] }, { "id": "pmcA1312363__T12", "type": "species", "text": [ "potato" ], "offsets": [ [ 1470, 1476 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[ { "id": "pmcA2504341__text", "type": "Article", "text": [ "A novel, non-invasive, online-monitoring, versatile and easy plant-based probe for measuring leaf water status\nAbstract\nA high-precision pressure probe is described which allows non-invasive online-monitoring of the water relations of intact leaves. Real-time recording of the leaf water status occurred by data transfer to an Internet server. The leaf patch clamp pressure probe measures the attenuated pressure, Pp, of a leaf patch in response to a constant clamp pressure, Pclamp. Pp is sensed by a miniaturized silicone pressure sensor integrated into the device. The magnitude of Pp is dictated by the transfer function of the leaf, Tf, which is a function of leaf patch volume and ultimately of cell turgor pressure, Pc, as shown theoretically. The power function Tf=f(Pc) theoretically derived was experimentally confirmed by concomitant Pp and Pc measurements on intact leaflets of the liana Tetrastigma voinierianum under greenhouse conditions. Simultaneous Pp recordings on leaflets up to 10 m height above ground demonstrated that changes in Tf induced by Pc changes due to changes of microclimate and/or of the irrigation regime were sensitively reflected in corresponding changes of Pp. Analysis of the data show that transpirational water loss during the morning hours was associated with a transient rise in turgor pressure gradients within the leaflets. Subsequent recovery of turgescence during the afternoon was much faster than the preceding transpiration-induced water loss if the plants were well irrigated. Our data show the enormous potential of the leaf patch clamp pressure probe for leaf water studies including unravelling of the hydraulic communication between neighbouring leaves and over long distances within tall plants (trees).\n\nIntroduction\nOptimum water supply, particularly at peak needs of the plants, is an important issue for greenhouse and field vegetable production. In a greenhouse on a sunny day, evaporation and transpiration (so-called evapotranspiration) can occur so rapidly that water loss can cause plant damage before wilting symptoms are visible. Even at lower temperatures the restricted rooting in greenhouses leads frequently to plant water deficiency. Thus, no matter how slight, drought stress will result in a significant reduction in growth and, in turn, of harvest yield. On the other hand, over-irrigation must be avoided and the appropriate method of watering must be selected because wet soils and permanent moisture on plant organs promote the incidence of root mould, grey mould (Botrytis blight), formation of leaf oedema, and other plant diseases (Sherf and MacNab, 1986). Conservation of water is another important issue in semi-arid and arid regions where water is scarce. The advent of (subsurface) drip irrigation has considerably reduced water consumption of agricultural, horticultural, and greenhouse crops, but has also highlighted the need for sensors monitoring water deficiency of plants directly and online (Jones, 2004). In plant physiology and agriculture the pressure bomb technique pioneered by Scholander et al. (1965) is a widely accepted reference technique for measuring leaf water status. However, the method is massively invasive, slow, labour-intensive (and therefore expensive), unsuitable for automation, and gives only spot measurements. Furthermore, interpretation of the data is still a matter of debate (Zimmermann U et al., 2004; Zimmermann D et al., 2007). Leaf thickness is also sometimes used as an indicator for water stress (Burquez, 1987; McBurney, 1992; Malone, 1993). Leaf thickness monitoring devices are commercially available. They are non-invasive and suitable for online measurements, but have the disadvantage that changes in water status are frequently not reflected sensitively in changes of leaf thickness. The pressure probe technique pioneered by Zimmermann, Tomos, and others (Zimmermann et al., 1969, 2004; Tomos, 1988; Balling and Zimmermann, 1990), on the other hand, is a well established technology for measuring turgor and xylem pressure on the levels of individual cells and xylem vessels, respectively. This technique is not suitable for automation. Moreover, the probe technique requires rather sophisticated equipment and a high level of technical skill. These disadvantages are also shared by the ball tonometry, a method which allows non-invasive monitoring of cell turgor pressure by application of an external pressure (Lintilhac et al., 2000; Geitmann, 2006). Other water stress monitoring devices are described in the literature, but all of them have various disadvantages which prevented their implementation for leaf water status measurements in greenhouses or in the field (see, for example, the review article of Jones, 2004, and also Grant et al., 2007).\nIn this communication a novel, non-invasive, online-monitoring plant-based probe is described that meets the demands of controlling horticultural and agricultural water applications. The probe is characterized by high precision, operating convenience, minimum costs, and by automation suitability. Data can be transferred wireless to a personal computer or to an Internet server via a mobile phone network for real-time evaluation and for the automatic regulation of irrigation. The technology includes a miniaturized silicone pressure sensor integrated into a spring clamp that is clamped to a patch of an intact plant leaf. The patch clamp pressure probe measures the attenuated pressure response of the leaf patch upon the application of a constant, clamped pressure. The attenuation of the applied pressure depends on the transfer function of the leaf. The magnitude of the transfer function depends on two terms, a turgor pressure-independent term (related to the compression of the cuticle, cell walls, and other structural elements) and a turgor pressure-dependent term. Theory shows that the turgor pressure-dependent part of the transfer function Tf is a power function of the cell turgor pressure, Pc, and predicts that Tf assumes values close to zero if Pc is high and, vice versa, values close to unity if Pc is low. This could be verified by combined turgor pressure probe and leaf patch clamp pressure probe measurements on leaflets of the liana Tetrastigma voinierianum. The 10-m tall liana growing in a tropical greenhouse was selected for the first studies because a comprehensive data set about diurnal changes in xylem and turgor pressure gradients under various environmental conditions existed for this greenhouse plant (Benkert et al., 1995; Thürmer et al., 1999; see also Zimmermann et al., 2004). Measurements of diurnal changes of the patch clamp pressure performed here yielded results which were consistent with the previous pressure probe work on the liana. The data also demonstrated that changes in the irrigation regime and in microclimate can be sensed by this novel probe very sensitively and that use of several probes allowed the study of the diurnal water transport at multiple scales from single leaves to the whole organism (Meinzer et al., 2001). This can be done without knowledge of the turgor pressure because the transfer function is a direct measure of leaf water status.\n\nMaterials and methods\nPlant material\nExperiments were performed on two specimens of the liana Tetrastigma voinierianum, growing in the c. 12 m tall tropical greenhouse of the University of Salzburg, Austria. The ground ascended by about 5 m towards the backstage of the greenhouse. The height of the plant in this area was about 4.5 m, whereas the height of the plant in the front area reached 10 m. However, the stem and the branches of the two plants were considerably longer, because the plants had grown vertically upwards and then part of the way downwards. First measurements were performed during the last week of May, 2007. This week was very sunny and warm. Experiments were repeated during the last week of August and the first week of September, 2007. At this time of the year the weather conditions were quite poor. The weeks were rainy, sunshine occurred only occasionally. On average, the sky was very cloudy. Due to the variable weather conditions the ambient temperature, T, in the greenhouse usually varied between 19 °C and 27 °C. By contrast, during the hot week in May temperatures at the top of the plants could reach up to 44 °C. The greenhouse was illuminated between 07.30 h and 19.00 h (CET=Central European Time). During the day-time, the artificial illumination was switched off automatically once the natural irradiance exceeded about 45 μmol photons m−2 s−1 at ground level. At full sunshine, the light intensity was limited by automatically operating blinds. The relative humidity (r.h.) of the air was regulated by overhead misters. Despite attempts to keep the r.h. above 60–70% large axial r.h. gradients along the stem of the liana were observed during sunny days (see below).\nThe ambient temperature and relative humidity at the measuring sites were determined by using thermistors (Tinytag; RS Components GmbH, Mörfelden-Walldorf, Germany).\n\nLeaf patch clamp pressure probe and data acquisition\nThe pressure sensor chip allows pressure measurements up to 100 kPa. The sensor consists of a miniature piezoresistive Wheatstone bridge. The sensors used in this work were purchased from the company ‘Raumedic’ (Helmbrechts, Germany) and from the company ‘Keller’ (AG, Druckmesstechnik; Winterthur, Switzerland). However, it has to be pointed out that any other miniaturized pressure or force sensor could be used. The sensor chip was integrated into one of the planar circular pads of a spring clamp shown schematically in Fig. 1. The spring clamp consisted of two curved arms bridged by a spring in the middle (Wolfcraft GmbH; microfix S (B3630Fz60), Kempenich, Germany). The clamp pressure exerted by the spring on the leaf, Pclamp, could be varied by a stiff strap (made of synthetic material) spanned between the arms of the spring clip at the handhold site (Fig. 1). The length of the strap and thus the spring load acting on the leaflet between the two pads could be varied by regularly punched holes (as in a belt).\nCalibration of the sensor chips was performed by pressurization in a pressure chamber equipped with an integrated manometer (LEO 1, Keller AG, Winterthur, Switzerland). The readings of all sensors used in this study increased linearly with pressure in the range between 0 kPa and 100 kPa. The pressure sensitivity of the various sensors was found to be almost identical for all probes. Before use the probes were also tested for temperature-sensitivity in an accessible climate chamber because it is well-known that silicone-embedded sensors tend to become temperature-sensitive during storage. Probes were subject to temperature regimes ranging from 10 °C to 35 °C. Probes were not used if the temperature treatment induced a pressure change larger than 0.5 kPa.\nThe signals of the leaf patch clamp pressure probe were transmitted by a telemetry system (teleBITcom gmbh, Teltow, Germany). The operating distance between the battery-powered wireless telemetric transmitter and the receiver base station was up to 400 m. Each transmitter read, amplified, and converted the analogue signals of the pressure sensor into digitalized signals. The data were sent together with the transmitter ID-code every 90 s via the ISM band of 433 MHz to the receiver base station which logged and transferred the data to a personal computer or to a GPRS modem linked to an Internet server (NTBB Systemtechnik GmbH, Zeuthen, Germany) for analysis and archival storage. For sensor calibration and data recording the SENBIT software (teleBITcom gmbh, Teltow, Germany) was used. The software allowed communication with up to 32 sensors at regular short intervals (90 s to 5 min). In this study, up to 10 sensor/transmitter units were installed at different heights on the two lianas. Data transfer to the Internet server operated without any problem during the entire experimental period. However, for safety reasons hard-wired conventional data acquisition was also performed using dataloggers (Raumedic, Helmbrechts, Germany). The dataloggers were controlled by a computer program that provided functions for data storage and sensor calibration. Data were transferred regularly from the dataloggers to a personal computer.\n\nCell turgor pressure probe\nTurgor pressure measurements were performed on leaflets of the 10-m tall liana at c. 0.2 m height above the roots. The construction and function of the cell turgor pressure probe has been described elsewhere (Zimmermann et al., 1969, 2004). Briefly, the microcapillary was filled with oil up to the very tip and was then inserted into the upper leaflet surface between the main vein and the leaflet edge. Insertion was made under a small overpressure (about 20 kPa) achieved by appropriate displacement of the metal rod in the probe in order to avoid tip clogging. Penetration was stopped upon reaching the mesophyll cell layer and formation of a stable oil/sap meniscus within the tip region of the microcapillary of the probe. During the measurements it was regularly proved whether the tip was clogged or not by appropriate displacement of the metal rod.\n\nTheoretical background\nThe input pressure, Pin, experienced by the cells in a leaf patch is equal to the clamp pressure, Pclamp, only if the pressure signal is transferred lossless to the cells in the leaves. However, losses usually occur due to the compressibility and the deformability of the silicone of the pressure sensor as well as of the compressibility of the cuticle and other structural elements of the leaf. Therefore, theory shows that only a fraction of Pclamp may arrive at the cells, i.e. that the attenuation factor, Fa=Pin/Pclamp, is smaller than unity. Fa depends on the individual leaf properties. Fa can be assumed to be constant (and leaf thickness changes are negligible) if the structural elements are completely pre-compressed by application of an appropriate Pclamp. If Pclamp is kept constant during the following experimental period, Pin is constant and the output pressure, Pp, is only determined by the cell transfer function, Tf(V), where V is the leaf patch volume. In other words, Tf(V) determines the fraction of Pin sensed by the probe. It is dimensionless and assumes values between zero and unity:(1)Tf depends on the volume of the leaf patch, V, at constant ambient temperature, T:(2)δV depends on changes in cell turgor pressure, δPc, as follows:(3)where op is the average volumetric elastic modulus of the clamped tissue (Philip, 1958). op is a very complex parameter and will be dictated by the turgor pressure in the turgescent state. For a first approximation it is assumed that op increases linearly with Pc (support for this assumption is given by Zimmermann and Steudle, 1978; Zimmermann and Hüsken, 1980; Wendler et al., 1983):(4)where a and b are constants for individual leaf properties. Because of the viscoelastic properties of the cell wall, the magnitude of the constants depends on the duration of the external pressure application (Zimmermann and Hüsken, 1980). The constants are relatively large if rapid turgor pressure changes are induced (e.g. by using the cell turgor pressure probe), whereas slow turgor pressure changes (e.g. under transpirational conditions) result in small values.\nCombining equations 2–4 leads to equation 5.(5)\nEquation 5 can be integrated by assuming for a first approximation that at Pc=0 Tf=1 and that the internal osmotic pressure of the cells remained constant. After appropriate re-arrangements equation 6 is obtained:(6)and, respectively, if the denominator is replaced by equation 4:(7)\nIntroducing equation 6 into equation 1 yields the wanted relationship between the parameters Pp and Pin:(8)\nEquation 8 can be verified experimentally. Inspection of the equation shows that the patch clamp pressure, Pp, is a power function of the turgor pressure, Pc. The exponent of the function is equal to or smaller than unity. If a=1 and b <<Pc, equation 8 turns into Pp=b/Pc, i.e. both parameters are directly reciprocally coupled with each other. Thus Tf assumes low values if Pc is high and, vice versa, a value close to unity if Pc is close to zero. Using appropriate values for a and b for a given leaf (see below) it can be shown that below Pc=100 kPa, Pp increases dramatically. This means that the transfer function responds very sensitively upon loss of complete turgor pressure.\nIn the derivation of equation 8 it was assumed that op is temperature-independent. However, temperature effects on cell elasticity are well-known, although they are not very large in the temperature range investigated here (see, for example, Petersen et al., 1982; Niklas, 1992; Hogan and Niklas, 2004; Edelmann et al., 2005). Temperature effects on op cannot be excluded if different values for the Pclamp, and, in turn, for the input pressure, Pin, are selected, as well as if significantly different values for the constants a and b are assumed for optimum fitting of the Pp=f(Pc) curves. Therefore, in the case of large temperature gradients as observed here (see Figs 2 and 3) only the relative, but not the absolute changes in Pp measured at the different heights can be compared with each other. However, Pp values measured at the same height are still comparable and give information about the turgescence, i.e. about the water status of the leaf cells.\n\n\nResults\nThe leaf patch clamp pressure probe was clamped about 2 cm away from the edge of a leaflet of the compound leaves. Leaflets of similar size (176±58 cm2, n=12) were used. Inspection of the leaf patches after removal of the probes under the microscope revealed no changes in leaflet appearance beneath the pads, even after several weeks. Lesions on the leaves were never found. Only occasionally were very slight impressions of the pads on the leaflet surface observed.\nClamping of the leaves could be performed at any time of the day. Clamp pressures, Pclamp, applied to the leaves were of the order of 80–200 kPa. The optimum value for Pclamp of a given leaf has to be found empirically. Experience shows that Pclamp values could be considered as optimal if the output pressure Pp assumed values between 10 kPa and 25 kPa upon clamping in the early morning (when turgor pressure was high) or between 50 kPa and 70 kPa upon clamping around noon or towards early afternoon (when turgor pressure was usually low). The magnitude of Pp depended on the compressibility and deformability properties of the individual leaves which may vary considerably due to age, morphology, leaf thickness, abiotic factors etc. The magnitude of Pclamp had no effect on the diurnal profiles of Pp, in response to changes in microclimate and/or changes in the irrigation regime. Probes clamped on the same leaflet or nearby leaflets of leaves responded almost identically upon temporary changes in transpiration and/or water supply even if the Pclamp values were different due to different leaf thickness as well as local compressibility and deformability properties (data not shown). This was found over the entire height of the plants.\nScreening experiments proved further that a high surface roughness prevented a uniform contact between the pads of the probe and the leaf. Non-uniform contact resulted in a reduced pressure response of the probe (data not shown). Thus, in order to obtain maximum resolution of microclimate-induced and/or plant-based effects on the Pp values, it was crucial that the probe was placed on relatively smooth areas, i.e. preferentially between the veins rather than on the veins in order to guarantee a uniform pressure transmission. It also turned out that areas with lesions or covered with dust should be avoided. Probably due to the absence of dust, high resolution results were obtained when the sensor-containing pad faced the abaxial side, but not the adaxial side of the leaf.\nDiurnal changes in the patch clamp pressure\nFigure 2A represents Pp recordings conducted on the 10 m tall liana on 24 May and 25 May, 2007. The plant was well-watered before the beginning of the measurements (22 May) and was also watered regularly during the experimental period. The sufficient supply of the leaves with water was indicated by high turgor pressure values of c. 500 kPa at predawn and guttation up to a height of 6 m around sunrise (see also Thürmer et al., 1999). Diurnal changes of the Pp values were recorded simultaneously at 0.2 m, 6 m, and 10 m height.\nThe corresponding diurnal changes in the ambient temperature, T, and relative humidity, r.h., measured close to the sites of the patch clamp pressure probes, are given in Fig. 2B and C. It was very sunny on 24 May resulting in a rapid heating-up of the greenhouse, particularly in the upper part. This led to the development of large vertical gradients in T and r.h. along the stem of the liana (Fig. 2B, C). Because sunlight was dimmed by operation of the automatic blinds at noon (see Materials and methods) the gradients did not reach maximum values before the afternoon. Between 15.30 h and 16.30 h an ambient temperature of 44 °C and a relative humidity of 20% were recorded at the top of the liana, whereas at 0.2 m height T and r.h. were 27 °C and 70%, respectively. The gradients in T and r.h. disappeared at c. 22.00 h. Due to the dense foliage of the plant above c. 7 m and due to light dimming by the blinds, leaves closer to the ground only became exposed to direct sunshine towards the early afternoon. In contrast to 24 May, the following day was cloudy until noon. Thus, leaves in the upper part of the liana became fully exposed to bright sunshine first around 14.00 h as indicated by the rapid increase in T and the corresponding decrease of r.h. above a height of 6 m (Fig. 2B, C). Despite the strongly delayed exposure of the leaves to sunshine, comparable maximum values for T and r.h. at the three measuring sites, as well as vertical gradients in T and r.h. of comparable size, were measured between 15.30 h and 16.30 h as on the day before.\nInspection of Fig. 2 shows that the diurnal changes of T and r.h. at the three measuring sites are reflected in corresponding changes of Pp. During the nights the Pp values were low. After sunrise at 04.24 h the Pp values remained low for further 4–5 h, except at 6 m height where a slight and continuous increase in the Pp values was very often recorded. After about 09.00 h the Pp values increased over the entire height of the liana. Peaking of the Pp values occurred between 15.30 h and 16.30 h at all heights when the T and r.h. gradients reached maximum values. Towards the evening the Pp values decreased again in order to reach the original low values at c. 22.00 h. Until peaking in the afternoon, more or less pronounced fluctuations of the Pp values were seen. A part of these fluctuations seemed to be correlated with changes in the local ambient temperature, relative humidity and/or with a temporary exposure of the clamped leaflet to sunshine (compare Fig. 2A with Fig. 2B, C). However, there was also a considerable number of fluctuations which could not be traced back to changes in the microclimate. An example can be found in Fig. 2A (arrow). The peaking of the Pp value measured at a height of 0.2 m around 14.00 h is obviously not induced by changes in the local temperature and relative humidity or by exposure to sunshine. Similar diurnal changes in Pp, T, and r.h. were also found for the 4.5 m tall liana in the backstage of the greenhouse (data not shown).\nFigure 3 shows long-term Pp, T, and r.h. measurements performed at 0.2 m, 6 m, and 10 m height in late summer 2007. In contrast to the experimental conditions in late spring (Fig. 2) the liana had been watered only sporadically during the weeks before the measurements. The soil was extremely dry and no guttation was observed at predawn, even at 0.2 m height. Turgor pressure measurements performed at 0.2 m height yielded values below 250 kPa at predawn. Continuous irrigation was started at 10.00 h on 28 August (c. 400 l d−1). Guttation occurred at 0.2 m height, but not at 6 m height on 1 September. At sunrise (05.25 h), turgor pressures of c. 500 kPa were recorded at the base of the liana. Guttation at 6 m height was still not observed on 3 September indicating that the hydration state of the plant was still less than in the last week of May.\nThe T and r.h. profiles during the end of summer were also different to those in late spring (compare Fig. 2B, C with Fig. 3B, C). On 28 August when irrigation started it was sunny. Similarly to May, peak values of T and r.h. were reached at c. 15.30 h. However, temperatures at the top of the plant did not exceed 35 °C and r.h. did not drop below 40%. Accordingly, the vertical T and r.h. gradients were smaller than those measured at the end of May. On 29 August, when it became very rainy and light irradiance dropped dramatically, gradients in T and r.h. of slightly decreased size were still recorded. Peaking of T and r.h. occurred at c. 14.30 h. T and r.h. gradients did not develop in the following three days which were very cloudy and rainy. An almost constant temperature of c. 25 °C and a relative humidity of 80% were measured on the ground and at the top of the greenhouse throughout the day; light irradiance did not exceed 40 μmol photons m−2 s−1. Only on 31 August, did the rain stop and the upper part of the liana was exposed to sunshine for 1–2 h and thus small T and r.h. gradients developed. The following days of 2 and 3 September were partly cloudy and sunny and the peaking of T and r.h. occurred at c. 13.00 h. The upper, but not the lower part of the liana was exposed to sunshine for several hours. This resulted in the formation of T and r.h. gradients. The size of the gradients was similar to that measured on 29 August.\nInspection of Fig. 3A indicates that peaking of the Pp values coincided with peaking of r.h. and T in Fig. 3B and C. Figure 3A shows further that the peak amplitude of the Pp values recorded at the three measuring sites decreased continuously from 28 August whereas the low Pp values at the nights remained unaltered. The peak amplitude measured on 29 August was only slightly lower than that measured on the day before, the peak amplitude was rather low on 30 August. It remained at this low level until 2 September, except that on 31 August the peak amplitude at the three measuring sites increased slightly, presumably because of the short-time exposure of the upper leaves to sun (see above). Comparison of the diurnal changes in the Pp values with the corresponding T and r.h. changes in Fig. 3B and C indicates that part of the changes in the peak amplitude of the Pp values were induced by changes in the environmental parameters. However, part of the changes must obviously be attributed to irrigation. This conclusion can be drawn if, for example, the peaking of Pp on 2 and 3 September is compared with the peaking of Pp on 29 August. Despite the comparable size of the T and r.h. gradients, the peak Pp values were much smaller, particularly at 0.2 m height, on 2 and 3 September compared with 29 August. Plots of the Pp values measured at 6 m and 10 m height on 24 May, 28 August, and 2 September against r.h. (or against T since both parameters were usually closely related) also support the view that the local microclimate is not the only factor which determined the magnitude of the Pp values at the three measuring sites. Figure 4 shows that the Pp values recorded at 6 m and 10 m height increased with decreasing r.h. from c. 09.00 h until r.h. reached the minimum value (and T the maximum value, respectively), when with the progression of the day r.h. increased and T decreased, Pp decreased again. This decrease was much faster than expected in the light of the preceding r.h.-induced increase of Pp if the plant was well-watered (24 May; Fig. 4A, B). Refilling of the leaf cells was apparently enhanced by the high hydration state of the plant. By contrast, when the liana had not been irrigated well (28 August, Fig. 4C, D) both kinetics of the changes of Pp with r.h. were comparable. The low hydration state of the plant apparently delayed cell refilling. After 4 d irrigation (2 September), the low value of Pp recovered at relatively low r.h. as in May at 10 m height (Fig. 4F), but not at 6 m height (Fig. 4E). Recovery of the low Pp values at this height occurred, however, at significantly lower r.h. than on 28 August, indicating that rehydration of the leaves at this height had not been completed. Evaluation of other data sets yielded similar results (not shown).\n\nPatch clamp pressure versus cell turgor pressure measurements\nThe leaf patch clamp pressure probe measures the pressure transfer function of a defined leaflet area. The above theoretical considerations have shown that the transfer function is mainly determined by turgor pressure provided that the structural elements do not contribute or constantly contribute to the pressure signal transfer. In order to prove the theory parallel measurements of leaf patch clamp pressure (Pp) and cell turgor pressure (Pc) were performed on a leaflet at 0.2 m height (Fig. 5A). Simultaneously, the Pp values were recorded at 6 m and 10 m height (Fig. 5B, C). Measurements were performed after several days of irrigation. In these experiments, the microcapillary of the turgor pressure probe was inserted into parenchymal cells located close to the main vein on the abaxial side of a leaflet, i.e. c. 10 cm away from the leaf patch clamp pressure probe near the leaflet periphery.\nTurgor pressure measurements on leaf cells of T. voinierianum were difficult to perform because of the presence of mucilage. Mucilage resulted in clogging of the tip of the microcapillary of the cell turgor pressure probe. For this reason, measurements with the Scholander pressure chamber were not very reliable. It was extremely difficult to determine exactly the balancing pressure at which water appears at the cut end of the petiole of the leaf. In Fig. 5A the average turgor pressure values are plotted which were calculated from 3–8 min long measurements.\nInspection of Fig. 5A–C shows that Pc exhibited similar diurnal changes as the Pp values at 0.2 m, 6 m, and 10 m height. Changes in Pc after sunrise preceded changes in Pp, most likely due to the different measuring sites on the leaflets. After the onset of transpiration the loss of turgor pressure of cells located at the periphery of the leaflets (where Pp is recorded) is apparently immediately compensated by water shifting from the xylem and the cells located close to the main vein (where Pc is measured). Consistent with this explanation, towards the afternoon when all cells throughout the leaflets exhibit low turgor pressure, the increase in Pc and the decrease in Pp occurred nearly concomitantly.\nInterestingly, the delay between the response of Pc and Pp upon changes in T and r.h. in the early morning hours decreased from about 2.5 h at 0.2 m height to 1.5 h at 6 m height, and to 1 h at 10 m height. In Fig. 5D the Pp values measured at the three heights are plotted against the corresponding Pc values (i.e. neglecting the delayed response of Pp). The curves could be fitted quite well by equation 8. Fitting of the curves required the assumption of different values for the constants a and b in equation 4 (see legend to Fig. 5) indicating that the elastic properties of the leaves were different, presumably due to changes of the ambient temperature with height (see above).\nTurgor pressure measurements at 5 m height failed because of abundant mucilage in the leaflet tissue at this time of the year. Mucilage in the cells and xylem vessels was less abundant when turgor pressure measurements were performed during the summer of 1999. Plot of the Pc data measured by Thürmer et al. (1999) at 5 m height against online recordings of Pp in May 2007 revealed very good agreement between both parameters, even though the microcapillary was inserted into cells located on the upper leaflet surface between the main vein and the leaflet edge (Fig. 6). On both measuring days in 1999 and 2007, similar environmental conditions existed. Inspection of Fig. 6 shows that Pp measured at 6 m height was delayed by c. 1.5 h. A plot of the Pp values against the Pc values according to Fig. 5D could also be fitted very well by equation 8 (see inset of Fig. 6) indicating the validity of equation 8 to describe properly the relationship between Pp and Pc.\n\n\nDiscussion\nThe results presented here demonstrate that the novel patch clamp pressure probe is a very sensitive, versatile, and easy-to-handle plant-based tool for online monitoring of the effects of evapotranspiration and/or of irrigation regimes on the water relations of leaves (Figs 2, 3). The probe measures the output signal upon application of a constantly clamped pressure, i.e. the pressure transfer function of leaf patches. Since the application of the probe is not restricted to special leaves, patch clamp pressure measurements will readily show which crop varieties can be grown with the least water for the most yield. It has also been demonstated here that the plant-based data can be sent by small telemetric units connected to the patch clamp pressure probes to a receiver base station which logs and stores the data pending transfer to a GPRS modem linked to an Internet server. The telemetric systems operated faultlessly. This is a very important result because there is agreement between scientists and growers (Jones, 2004) that control of irrigation must be tied to remote sensing devices in order to reduce manpower and to conserve water. The patch clamp pressure probe technique introduced here meets these demands.\nA further advantage is that the probe is cheap and easy to handle. Some care is required only when clamping the leaf. Leaves must be clean and any rough surface topography must be avoided. This implies that the dimensions of the pads of the spring clamp must be adjusted to the size of the intercostal area since non-uniform contact between leaf surface and pads lowered the output pressure signal. Experience collected with patch clamp pressure measurements on T. voinierianum and on preliminary results of other plants (e.g. grapevines, bananas, Eucalyptus, and Arabidopsis) showed that a smooth intercostal leaf area for clamping can readily be found. Equally, optimum values for the clamp pressure can also be relatively quickly selected empirically if the guidelines mentioned above are followed. For many purposes calibration of the leaf patch clamp pressure probe against the cell turgor pressure probe is not stringent. It may be sufficient to know that the transfer function assumes small values at full turgescence and is nearly unity at low turgescence.\nHowever, for scientific reasons and for the application of the probe in basic research, the dependency of the transfer function on cell turgor pressure might be of great interest. The theoretical considerations have demonstrated that the relationship between the transfer function and the turgor pressure can obviously be described by equation 8, i.e. by a power function. Concomitant measurements of turgor pressure and patch clamp pressure have confirmed the theoretical considerations (Fig. 5). Analogous recordings of turgor pressure and patch clamp pressure on grapevines, bananas, and Eucalyptus trees also yielded results (D Zimmermann, unpublished results) which were consistent with the predictions of equation 8. Patch clamp pressure measurements allow, therefore, calculation of turgor pressures if the constants a and b defined by equation 4 are known. These parameters can be obtained by numerical fitting of Pp versus Pc curves.\nOnline patch clamp pressure measurements open up new possibilities to unravel the hydraulic communication among leaves, including tall trees. It is shown here that the patch clamp pressure can simultaneously be monitored in shaded or sun-exposed leaflets at different heights under greenhouse conditions by using several probes. It was even possible to measure the turgor pressure at different sites on the leaf. Therefore, water shifting between leaf cells via the apoplastic and symplastic pathways can be studied. Physically sound theories are available (Molz and Ikenberry, 1974; Molz et al., 1979), but experimental facts are not available or are mainly based on speculations (Steudle and French, 1996). The first evidence for the dynamics of the hydraulic communication between leaflets at different height and within leaflets is given here. Thürmer et al. (1999) have shown by using the xylem and turgor pressure probe that the decrease in local xylem pressure after daybreak from positive, sub-atmospheric or slightly above-atmospheric values to negative values was associated with an almost 1:1 decrease in turgor pressure of the cells located close to the main vein of the leaflets (see also Zimmermann et al., 2004). Similarly, in the afternoon, the increase of xylem pressure in non-cavitated vessels towards more positive values correlated with a 1:1 increase in cell turgor pressure. Obviously, changes in transpiration resulted in a very rapid establishment of a new local water equilibrium state between the xylem and the turgor pressure of the adjacent cells. The turgor pressure measurements of Thürmer et al. (1999) were performed at 1 m and 5 m height. No significant differences in the turgor pressure values were found over the day. The turgor pressure measurements at 0.2 m height reported here confirmed the finding of Thürmer et al. (1999) that the turgor pressure in the cells located near the main vein dropped immediately upon daybreak. However, our patch clamp pressure measurements at the periphery of the leaflets imply that the turgor pressure of these cells apparently had a significantly delayed response upon the onset of transpiration. This finding can be taken as evidence that turgor pressure gradients are temporarily generated within the leaf during the early morning hours.\nThis delay in the response of patch clamp pressure after daybreak was c. 1 h for leaves at the top, but c. 2.5 h for leaves at the base. The data suggest that with progression of the day the gradients collapsed first at the top of the liana, where transpiration was high, before it occurred in leaves further down. During the afternoon hours the increase in the turgor pressure values measured close to the main vein coincided with the values measured by the patch clamp pressure probe (Fig. 5). The unbalanced osmotic pressure throughout the leaflets obviously favoured simultaneous turgor pressure regeneration in all leaflet cells. The process seemed to be very similar to the refilling of the cells of the resurrection plant Myrothamnus flabellifolia with water after desiccation (Wagner et al., 2000). Uniform radial refilling of cells with water in this species is also driven by the unbalanced osmotic pressure throughout the tissue. As indicated by Fig. 4A and B, restoration of full turgescence in T. voinierianum was faster than the preceding turgor pressure loss if the lianas were well-watered. By contrast, limitations in water supply resulted in the coincidence of the phases of turgor pressure loss and regeneration.\nInterestingly, watering of the lianas after non-irrigation seemed to lead preferentially to water shifting to the top of the plant, because, after 3 d of irrigation, turgor pressure regeneration during the afternoon was very fast again at 10 m height (Fig. 4F), but not at 5 m height (Fig. 4E). Rapid water movement to the top is expected if hydrostatic pressure, osmotic pressure, and/or gel-based gradients throughout the vessels exist or are generated rapidly in the afternoon when cavitation has occurred. This was demonstrated by Benkert et al. (1995) and Thürmer et al. (1999) by using the xylem pressure probe. Continuous xylem pressure gradients during the morning hours can also explain why changes in Pp at lower heights were frequently found which could not be correlated with changes in local temperature or relative humidity. Water loss at the top of the plant induces a drop in xylem pressure which is transferred rapidly to the basal leaves where it induces a temporary turgor pressure loss that is soon compensated by water uptake from the roots (arrow in Fig. 2).\nEven though more work is required, the present study shows that the leaf patch clamp pressure probe is a promising tool to elucidate short- and long-distance water transport in T. voinierianum and other plants.\n\n\n" ], "offsets": [ [ 0, 40704 ] ] } ]

[ { "id": "pmcA2504341__T0", "type": "species", "text": [ "Tetrastigma voinierianum" ], "offsets": [ [ 900, 924 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345143" } ] }, { "id": "pmcA2504341__T1", "type": "species", "text": [ "Tetrastigma voinierianum" ], "offsets": [ [ 6252, 6276 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345143" } ] }, { "id": "pmcA2504341__T2", "type": "species", "text": [ "Tetrastigma voinierianum" ], "offsets": [ [ 7303, 7327 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345143" } ] }, { "id": "pmcA2504341__T3", "type": "species", "text": [ "T. voinierianum" ], "offsets": [ [ 29732, 29747 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345143" } ] }, { "id": "pmcA2504341__T4", "type": "species", "text": [ "T. voinierianum" ], "offsets": [ [ 34317, 34332 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345143" } ] }, { "id": "pmcA2504341__T5", "type": "species", "text": [ "bananas" ], "offsets": [ [ 34394, 34401 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4641" } ] }, { "id": "pmcA2504341__T6", "type": "species", "text": [ "bananas" ], "offsets": [ [ 35498, 35505 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4641" } ] }, { "id": "pmcA2504341__T7", "type": "species", "text": [ "Eucalyptus" ], "offsets": [ [ 35511, 35521 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "3932" } ] }, { "id": "pmcA2504341__T8", "type": "species", "text": [ "Myrothamnus flabellifolia" ], "offsets": [ [ 38907, 38932 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "83223" } ] }, { "id": "pmcA2504341__T9", "type": "species", "text": [ "T. voinierianum" ], "offsets": [ [ 39185, 39200 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345143" } ] }, { "id": "pmcA2504341__T10", "type": "species", "text": [ "T. voinierianum" ], "offsets": [ [ 40668, 40683 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345143" } ] } ]

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[ { "id": "pmcA2599109__text", "type": "Article", "text": [ "Suggestions Concerning the Use of the Subclavian which Arises from the Aorta in the Treatment of the Tetralogy of Fallot *\nAbstract\nImages\n\n\n\n\n\n\n SUGGESTIONS CONCERNING THE USE OF THE SUBCLAVIAN \n\n WHICH ARISES FROM THE AORTA IN THE TREATMENT \n\n OF THE TETRALOGY OF FALLOT* \n\n HARRIS B. SHUMACKER, JR.** \n\n In Blalock's early experience with the operative treatment of the tetralogy of Fallot, the two subclavian, the innominate, and carotid arteries were all used for anastomosis to the pulmonary artery. Soon, however, it became evident that use of the innominate or carotid was followed by a relatively high incidence of complications resulting from cerebral ischemia. Blalock suggested, therefore, that the subclavian be used by preference. The subclavian branch of the innominate does not become kinked or badly angulated when it is turned down for the anastomosis, and a good functional result almost invariably follows the completion of a satisfactory anastomosis. The subclavian which arises directly from the aorta, on the other hand, tends to form a bad angle when it is brought down for the anastomosis, and, indeed, near its point of origin it may be so flattened out against the relatively rigid aortic wall as to obstruct all blood flow through it. These considerations led Blalock to recommend the use of the former except in infants under two years of age in whom this artery may be too small and in adults or children over twelve years old or five feet tall with a left aortic arch in whom it is often too short to permit a satisfactory anastomosis.7' \n\n In contrast, Paine and Varco,' Lam,6 Holman,' Olim,7 and others have preferred to use the subclavian which arises from the aorta and have obtained generally excellent results. They point out that the tendency to kinking of the artery is more a theoretical than a practical disadvantage, that the exposure and dissection of both the pulmonary artery and the subclavian are easier on the side of the aortic arch, that both vessels are generally longer than on the opposite side and that, as a general rule, the anastomosis can be accomplished more readily. The practical usefulness of a third systemic vessel, namely the aorta, was demonstrated by the development of a technique for side-to-side aortic pulmonary anastomosis by Potts and his associates.\"0 In spite of the preferences held by individual operators for use of one vessel or another, the fact remains, as Blalock has emphasized, that there is always present a usable systemic vessel provided a suitable pulmonary artery is available. Nevertheless, the choice of the systemic artery in each individual patient is important since in certain cases one or another is unsuitable. \n\n * From the Department of Surgery, the Indiana University Medical Center, Indianapolis, Indiana. Aided by a contract between the Office of Naval Research, Department of the Navy, and Indiana University. \n\n ** Resident Surgeon, New Haven Hospital, 1937-1938. Received for publication April 26, 1951. \n\n TREATMENT OF TETRALOGY OF FALLOT \n\n It has been my custom to follow a policy rather similar to that outlined by Blalock. The subclavian branch of the innominate has generally been preferred in patients ranging between two and twelve years of age and in older individuals in whom there is a right aortic arch. In others the thoracotomy is usually made on the side of the aortic arch, and either the aorta itself or the subclavian arising from the aorta is used for the anastomosis. My experience with the use of the subclavian originating from the aorta is small and the results have not been as good as those which others have reported. One eighteen-month-old child died the day after operation and was found at post-mortem examination to have an occluded anastomosis. In one three-year-old child and one twenty-year-old woman a poor result was obtained and a second operation upon the other side was necessary. In two additional cases the result was only fair. Excluding the patients mentioned in the present report, only in two was an unquestionably good result obtained. \n\n Recently, in several cases in which failure seemed evident a modification has been required in order to obtain a functioning shunt. Since these modifications proved successful and since these threats of failure tax one's ingenuiity at the time of operation, I have thought it might be helpful to describe the procedures employed and to illustrate them with case reports. One is a method which obviously has very limited applicability, while the otlher would seem to be rather generally applicable. \n\n The first consists of the transplantation of the origin of the subclavian to a more suitable portion of the aorta. \n\n Case report \n\n The patient was a 19-year-old girl who had been cyanotic since the age of six months. She had developed normally but was always limited in exercise capacity. In her early years of school she was taken to school by her parents and carried to and from her seat in the classroom. She did reasonably well in high school, being driven to and from the school, but she got into severe difficulty when she attempted to attend college. The longer distances between classes and the necessity for climbing stairs precipitated a downhill course, with increasing dyspnea, more marked cyanosis and fatigue, and the onset of bouts of loss of consciousness. The latter occurred several times daily. One which was witnessed by her physician lasted 45 minutes; he feared it would prove fatal. Ordinarily, she could not walk more than half a block. \n\n There was marked cyanosis of the nails and mucous membranes and to a lesser extent of the skin. Clubbing was very prominent. All the results of physical and laboratory examination fitted in with a diagnosis of tetralogy of Fallot. The hematocrit ranged between 83 and 90. Oxygen saturation of arterial blood was 65 per cent at complete rest. The aortic arch was on the left side. On October 26, 1949 a left lateral thoracotomy was performed, the pleural cavity being entered through the bed of the fifth rib. There was evident greatly increased collateral circulation in the mediastinum and hilum. The pulmonary artery appeared to have markedly reduced blood flow, was very short, small, and thin-walled. Its diameter was less than 5 mm. The aorta seemed to be bowed out laterally in an unusual fashion and the short pulmonary artery could not possibly be brought out over it. The subclavian artery was relatively small, having \n\n 487 \n\n YALE JOURNAL OF BIOLOGY AND MEDICINE \n\n a diameter of only about 4 mm. but it seemed rather long. It was apparent that a Pott's procedure was impossible and that an end-to-side subclavian pulmonary artery anastomosis would be doomed to failure. Hence, the subclavian artery was divided at the level of its first branch, the pulmonary artery was divided proximally, and an end-to-end subclavian-pulmonary anastomosis was carried out. This was done with ease. When the clamps were removed, however, no blood flowed through it. The softwalled subclavian was completely flattened out against the relatively rigid aorta (Fig. 1). When the hilar structures were forcibly elevated in a cephalad direction the subclavian immediately filled and pulsated normally and a thrill could be felt. Simple expansion of the lungs, however, failed to achieve this result and I could conceive of no way in which the hilar region could be held in a more cephalad direction. The subclavian was then ligated at the point where it came off the aorta and its proximal end was anastomosed end-to-side to the descending aorta. It now pulsated vigorously as did the pulmonary artery. In spite of the good pulsation no thrill was palpable. The patient had an uneventful but disappointing convalescence. She remained markedly cyanotic and no continuous bruit was audible. When she left the hospital 14 days after operation there was no improvement in her appearance, hematocrit, or arterial oxygen saturation. \n\n Surprisingly enough she reported progress on each follow-up examination. By the end of seven weeks she was obviously less cyanotic, her hematocrit was 77, and she was able to walk a number of blocks and to climb a flight of stairs without difficulty. She reported that she had danced and roller-skated without much trouble. Shortly thereafter a continuous murmur was audible in the left chest. She continued to improve and in January entered a southern college. She did well. At least once a day, often twice, she walked without difficulty from the campus into town and back, a distance of ten blocks each way. She played a little tennis, learned to swim, and began to dance, including jitterbugging. When she xvas seen in June, her color was good, although there was still slight cyanosis of lips and nail beds. There was a very loud, continuous murmur in the left chest. The following fall she transferred to a midwestern university. She got along reasonably well but not as well as she had in a warmer climate and on more level terrain. She walked four or five blocks up and down hills between classes without difficulty in good weather but complained of some dyspnea and fatigue on cold, windy days. She attended dances and often danced each number throughout the evening without trouble. When seen in December she looked well. Her color was good and clubbing was definitely less marked than it had been previously. She had a severe cold at the time. Her oxygen saturation of arterial blood was 75 per cent at rest and it did not fall when she stood or still-walked. The hematocrit was 69. There was audible the same loud continuous murmur in the left chest. \n\n Though the patient has been markedly improved, it is recognized that the result is not as good as is commonly obtained when patients with more adequate pulmonary arteries are treated by the conventional anastomosis of a systemic artery to the side of the pulmonary artery. Nevertheless, the patient has thus far been given such good health and relatively normal capacity for ordinary activity that further operation has seemed unwarranted, though the possibility of some future attempt at creation of an additional shunt is being kept in mind. \n\n The second procedure embodies the cephalad transplantation of the pulmonary artery by a plastic repair of the incision in the hilar and mediastinal pleural structures. \n\n 488 \n\n FIG. 1. Drawing illustrating the condition which existed in the first case after completion of the anastomosis (A) and its correction by transplantation of the origin of the subclavian (B). \n\n FIG. 2. Drawing illustrating correction of obstruction to blood flow through the subclavian artery by inverted T or L plastic closure of the defect in the hilar and mediastinal structures. \n\n TREATMENT OF TETRALOGY OF FALLOT \n\n Case reports \n\n The first patient was a fully grown young man of 17 with tetralogy of Fallot which caused considerable incapacity. He was admitted to the hospital on July 18, 1950 and was operated upon two days later. He was known to have a left aortic arch, and a left lateral thoracotomy was performed. The aorta was freed for a side-to-side anastomosis with the pulmonary artery. So much difficulty was encountered, however, in placing the aortic clamp in proper position for making a satisfactory incision in the aorta that the procedure was abandoned and an end-to-side subclavian-pulmonary artery anastomosis accomplished instead. The pulmonary artery was of fair size, having an estimated diameter of 1 cm. The subclavian artery appeared to be quite long and it was of very satisfactory size, having a diameter of about 6 mm. To my dismay, the first portion of the soft-walled subclavian artery was completely flattened out against the rather rigid wall of the aorta and no blood flow through it could be demonstrated, there being no subclavian pulsation nor thrill in the pulmonary artery. If one forcefully elevated the hilum of the lung in a cephalad direction, the obstruction disappeared, the subclavian artery began to pulsate, and a thrill could be palpated. When the lung was allowed to assume its usual position, the subclavian obstruction was again evident and could not be prevented by full expansion of the lung. It was found that the hilar region and the pulmonary artery could be maintained in a satisfactory cephalad position by traction upward upon the cuff of hilar pleura and the adjacent vascular sheath and that these structures were sufficiently strong so that traction could be maintained upon them with a small hemostat. The defect in the mediastinal and hilar tissues was then repaired using a sort of inverted T-plastic closure. By this maneuver success was achieved in elevating the pulmonary artery in a cephalad direction so that the subclavian obstruction was relieved and excellent pulsation was evident (Fig. 2B). A fairly good continuous thrill was palpable. The patient had an uneventful convalescence. When last seen on January 16, 1951 he had excellent color without any visible cyanosis. The clubbing seemed to have decreased somewhat. A continuous murmur was audible. He stated that he noted no limitation of exercise capacity. In outlining his activities he said that, among other things, he was doing a great deal of ice-skating and was playing ice hockey regularly. He was planning to start college work the following month. \n\n The second patient was a 16-year-old boy who had been cyanotic since birth. His physical development was somewhat retarded and he was slow in learning to sit and walk. Until he had grown old enough to be self-conscious about it he had always squatted when he was tired. By perseverance he had managed to do more than one would have suspected he could from the degree of his cyanosis. He could walk as much as five or six blocks at a slow pace. He was fond of drums and managed to play occasionally with an orchestra in a somewhat restricted fashion. For the past few months he had had more dyspnea, fatigability, and seemed to be going downhill generally. \n\n The results of physical and laboratory studies were rather typical of the tetralogy of Fallot. Clubbing was marked, the nailbeds and mucous membranes were a rather deep purplish-blue color, and the skin had a dusky cyanotic tint. The aortic arch was determined to be on the left. On July 21, 1950 a left lateral thoracotomy was performed, the pleural cavity being entered through the bed of the fourth rib. There was a rather marked increase in the collateral circulation in the mediastinum and the hilar region. The pulmonary artery was easily dissected free. It was fairly long and was about 1 cm. in diameter. The subclavian artery seemed quite long and was of adequate size, having a diameter of about 5 mm. An end-to-side sub9lavian pulmonary anastomosis was performed. Again in this case, however, the first part of the subclavian was acutely angulated and completely flattened out against the aortic wall. There was no \n\n 489 \n\n YALE JOURNAL OF BIOLOGY AND MEDICINE \n\n pulsation in the subclavian and no thrill in the pulmonary. In this instance also, good blood flow through the subclavian artery was evident whenever the hilar structures were elevated in a cephalad direction but no pulsation was demonstrable simply by expanding fully the lung. Again, a sort of inverted T-plastic closure of the defect in the hilum and mediastinum brought about a cephalad elevation of the pulmonary artery so that tension was released and there was excellent pulsation in the subclavian artery. A thrill was now palpable. Convalescence was uneventful. Improvement in color was evident within a few days and his color was excellent by the time he was discharged from the hospital on the thirteenth postoperative day. He rapidly found that he was now able to lead a quite normal sort of life. When he was seen on December 9 he stated that he had no limitation in exercise capacity. He could walk rapidly without fatigue. He was going to school and was playing the drums in a professional orchestra three or four nights each week. There was some diminution in the clubbing and his color was excellent. There was a loud continuous bruit audible in the chest. On March 27 the arterial oxygen saturation was 88.2 per cent. \n\n When I first used this procedure I was rather surprised that such tissues would hold sutures and serve satisfactorily to elevate the hilum. On occasions I had previously toyed with the idea of suturing hilar pleura to the mediastinal pleura but had abandoned it as impractical because the sutures seemed to pull out whenever there was any tension whatsoever. If the procedure is to be successful, it is essential that the sutures encompass any adjacent areolar and fibrous tissue and especially the so-called vascular sheath which surrounds both pulmonary artery and aorta and is dissected free during the course of the operation. Fortunately, in its proximal portion the sheath about the pulmonary artery gains added strength from the extension into it of a reflection of the fibrous pericardium. Sutures through the mediastinal pleura in the region of the aortic arch purposely include the perivascular tissues which have been stripped off the aorta and subclavian artery and also any other available tissue which may lend strength, such as the divided ends of the supreme intercostal vein or other vessels which lhave been transected and ligated. The exact method of repair will vary from case to case. By placing the sutures properly it would seem possible sometimes to displace the pulmonary artery laterally as well as in a cephalad direction if such a maneuver was thought to be desirable (Fig. 2C). On occasions one would close the pleural defect fairly snugly, on others leave it wide open in places. \n\n Discussion \n\n Though there is always available some suitable systemic vessel and though the major concern in the operative treatment of the tetralogy of Fallot is the adequacy of the pulmonary artery, from time to time one may find the achievement of a satisfactory result thwarted by the local anatomical characteristics regardless of one's choice of procedure. Consequently, those modifications which may add to the likelihood of a successful outcome are important. Blalock\"' pointed out the practicability of performing an end-toend subclavian-pulmonary artery anastomosis whenever the pulmonary artery is judged too small or the subclavian too short for a satisfactory end\n\n 490 \n\n TREATMENT OF TETRALOGY OF FALLOT 491 \n\n to-side anastomosis. Holman' feels that a poorly functioning shunt after end-to-side anastomosis can usually be corrected by proximal division of the subclavian artery, thus in effect converting the procedure into an end-to-end anastomosis. If a satisfactory end-to-side suture of subclavian and pulmonary arteries or side-to-side aortic pulmonary anastomosis seems difficult to achieve, one may occasionally find it useful to divide the upper lobe branch of the pulmonary artery and carry out an end-to-end suture of the subclavian and the proximal end of the upper lobe branch. Potts and Smith' performed an anastomosis between the proximal end of the upper lobe branch and the side of the aorta in a case in which complete temporary occlusion of the main pulmonary artery was withstood poorly. I have found this principle of division of the upper lobe branch and use of its proximal end of value in obtaining a suitable subclavian pulmonary anastomosis when the subclavian seemed to have inadequate length. On occasions when the systemic vessel seems too short, one may elect to interpose a free vascular transplant,3\"' a modification I first employed in 1946 though unfortunately not with success in this instance. \n\n The operation performed in my first patient constitutes in reality the use of the subclavian artery as an autogenous graft between the aorta and pulmonary artery. It will obviously not often be the procedure of choice but sometimes may be found a useful measure in converting an apparently inadequate functional shunt into a good one. If my initial experiences with the plastic repair of the defect in the mediastinal and hilar structures are characteristic of what may be expected of this procedure, it would seem to have wide applicability whenever a poorly functioning subclavian-pulmonary shunt seems correctable by cephalad transplantation of the hilar structures and the consequent release of tension. I was unaware of any reference in the literature to its use until belatedly I discovered that I had overlooked a statement in the legend of one of the excellent drawings in Blalock's paper on surgical procedures in pulmonic stenosis.' Here he states that suture of the pleura of the superior aspect of the hilum to the mediastinal pleura may effectively elevate a little the pulmonary artery. Perhaps our more detailed consideration of this maneuver may add to its general usefulness. \n\n REFERENCES \n\n 1 Blalock, A.: The technique of creation of an artificial ductus arteriosus in the \n\n treatment of pulmonic stenosis. J. Thorac. Surg., 1947, 16, 244. \n\n 2 Blalock, A.: Surgical procedures employed and anatomical variations encountered \n\n in the treatment of congenital pulmonic stenosis. Surg., Gyn. Obst., 1948, 87, 385. \n\n 3 Gross, R. E., Bill, A. H., Jr., and Pierce, E. C.: Methods for preservation and \n\n transplantation of aortic grafts. Observation on arterial grafts in dogs. Report of transplantation of preserved arterial grafts in nine human cases. Surg., Gyn. Obst., 1949, 88, 689. \n\n 4 Holman, E.: The surgery of pulmonary stenosis. Experiences with left subclavian \n\n to left pulmonary artery anastomosis. J. Thorac. Surg., 1949, 18, 827. \n\n 5 Johnson, J., Kirby, C. K., Greifenstein, F. E., and Costillo, A.: The experimental \n\n and clinical use of vein grafts to replace defects of large arteries. Surgery, 1949, 26, 945. \n\n 492 YALE JOURNAL OF BIOLOGY AND MEDICINE \n\n 6 Lam, C. R.: The choice of the side for approach in operations for pulmonary \n\n stenosis. J. Thorac. Surg., 1949, 18, 661. \n\n 7 Olim, C. B.: Experiences in the surgical treatment of congenital pulmonary \n\n stenosis. American Surgeon, 1951, 17, 245. \n\n 8 Paine, J. R. and Varco, R. C.: Experiences with the surgical treatment of pul\n\n monic stenosis. Surgery, 1948, 24, 355. \n\n 9 Potts, W. J. and Smith, S.: New surgical procedures in certain cases of congenital \n\n pulmonary stenosis. Arch. Surg., 1949, 59, 491. \n\n 10 Potts, W. J., Smith S., and Gibson, S.: Anastomosis of the aorta to a pulmonary \n\n artery; certain types in congenital heart disease. J. Am. M. Ass., 1946, 132, 627. " ], "offsets": [ [ 0, 22605 ] ] } ]

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[ { "id": "pmcA320464__text", "type": "Article", "text": [ "Organization and structure of the mouse interleukin-2 gene.\nAbstract\nWe have cloned a chromosomal DNA segment which covers the entire sequence for the murine interleukin-2 gene and analysed the structure of the gene. The coding regions are separated into four blocks by three introns each of which is located similarly to the corresponding human gene. The exon sequences can be aligned perfectly with the previously cloned cDNA sequence. Of particular interests is the presence of sequences within the 5'-flanking region which are highly conserved between mouse and man. The conserved region which spans more than 400 base pairs may play a role in the regulation of IL-2 gene expression.Images\n\n\n\n\n\n\n Volume 12 Number 24 1984 Nucleic Acids Research \n\n Organization and structure of the mouse interleukin-2 gene \n\n Akira Fuse*, Takashi Fujita, Hidetaro Yasumitsu, Nobukazu Kashima+, Katsushige Hasegawa and Tadatsugu Taniguchi? \n\n Department of Biochemistry, Cancer Institute, Japanese Foundation for Cancer Research, Toshimaku, Tokyo 170 and Institute for Molecular and Cellular Biology, Osaka University, Suita-shi, Osaka 565, Japan \n\n Received 10 October 1984; Revised and Accepted 20 November 1984 \n\n ABSTRACT \n\n We have cloned a chromosomal DNA segment which covers the entire sequence for the murine interleukin-2 gene and analysed the structure of the gene. The coding regions are separated into four blocks by three introns each of which is located similarly to the corresponding human gene. The exon sequences can be aligned perfectly with the previously cloned cDNA sequence. Of particular interests is the presence of sequences within the 5'flanking region which are highly conserved between mouse and man. The conserved region which spans more than 400 base pairs may play a role in the regulation of IL-2 gene expression. \n\n INTRODUCTION \n\n Interleukin-2 (IL-2) is a lymphokine produced by T cells upon antigenic or mitogenic stimulation and is required for the proliferation of T cells (1,2). Several other biological activities of IL-2 which appear to be crucial in the immune regulation have also been reported (3, 4. 5. 6. 7.) . We previously reported isolation and sequence analysis of the cDNA for human IL-2 (8), as well as the chromosomal gene (9). More recently, we have isolated a cDNA which encodes murine IL-2 (Kashima et al., submitted for publication). The cDNA contains a unique tandem repeat of CAG sequence which would encode 12 consecutive glutamine residues in the active IL-2 molecule. \n\n In order to study the structure of the murine IL-2 chromosomal gene and its controlling region, we isolated and analysed a A phage clone containing the gene and its flanking sequences. \n\n MATERIALS AND METHODS \n\n Southern blotting of total mouse DNA \n\n Mouse chromosomal DNA was extracted from liver of BALB/c6 \n\n ? I R L Press Limited, Oxford, England. \n\n Nucleic Acids Research \n\n Volume 12 Number 24 1984 \n\n 9323 \n\n Nucleic Acids Research \n\n mouse as described before (10). High molecular genomic DNA was digested with various restriction enzymes and electrophoresed on 0.8% agarose gel. Blotting analysis of DNA was carried out by the method of Southern (11). Hybridization was carried out as described previously and filters were washed either in 3 x SSC at 650C (lower stringent condition) or in 0.1 x SSC at 650C (higher stringent condition). \n\n Screening of genomic DNA library \n\n A bacteriophage XCharon 4A/mouse genomic DNA library prepared with partial EcoRI digests of mouse DNA from MPC 11 plasmacytoma cells was kindly provided by Dr. T. Honjo. Mouse IL-2-specific clones were screened by the method of Benton and Davis (12), using 700 bp PstI-AccI fragment of a cDNA clone, pMIL2-45 as the probe (Kashima et al., submitted for publication). Hybridization was performed as described previously(13). Positive clones were rescreened at least twice. Subcloninq and sequencing of the mouse IL-2 gene \n\n Two EcoRI fragments of 3.3 Kbp and 2.8 Kbp from the positive recombinant X phage were subcloned into EcoRI site of plasmid pBR322. DNA segments derived from subcloned 3.3 Kbp and 2.8 Kbp fragments were labelled at either 3' end or 5' end, and subjected to sequence analysis by the chemical degradation method (14). The 0.8 Kbp EcoRI fragment from the same X phage clone was directly subjected to sequence analysis by the dideoxy chain termination method (15). \n\n RESULTS \n\n Total DNA blotting analysis \n\n In order to study structural organization of the mouse IL-2 gene, we first subjected total mouse DNA to the blotting analysis by using various probes specific for IL-2 gene. When mouse DNA was digested with various restriction endonucleases and then probed with a 7 kb human chromosomal DNA segment which contains the human IL-2 gene and its flanking region (Fig. 1 lane 1-8, ref. 9.), single positive band appeared at lower but not at higher stringent condition for washing the filters (see Figure legend). Additional bands corresponding to those observed by using mouse IL-2 cDNA probes (lane 13-17) also appeared by longer \n\n 9324 \n\n Nucleic Acids Research \n\n M 1 2 3 4 M 5 6 7 8 M 9 101112 M 13 141516 17 \n\n a~ ~ 3 .. \n\n I pMIL2-45 SacI Acc I \n\n CZI~~1 1t L2- 20 \n\n Acc I \n\n 1 00 tp lX i \n\n Fig. 1. Blot hybridization analysis of mouse chromosomal DNA. High molecular DNA prepared from Liver BALB/C6 mouse was digested with various restriction endonucleases (BamHI for lanes 1, 5, 9, 13 ; EcoRI for lanes 2, 6, 10, 14, 17 ; HindIII for lanes 3, 7, 11, 15 ; XbaI for lanes 4, 8, 12, 16 ). The resulting digests were fractionated on 0.8 % agarose gel and transferred to a nitrocellulose filter. Filters were hybridized by the published procedure (13) either with the nick-translated chromosomal DNA containing human IL-2 gene and its flanking region (total length, 7.0 kb, ref. 9) (lane 1-8) or with the nick-translated cDNA for mouse IL-2 (see figure). Filters were then washed either in 3 x SSC at 65 % (lower stringent condition) (lane 1-4, 9-12) or 0.1 x SSC at 65 OC (higher stringent condition) (lane 5-8, 13-17). Lane M each contains 7 size markers with their size being 23.7 kb, 9.5 kb, 6.7 kb, 4.3 kb, 2.3 kb, 2.0 kb and 0.6 kb, respectively. Brief restriction endonuclease cleavage map for the mouse IL-2 cDNAs is presented in the lower part of the figure. \n\n exposure of the film (data not shown). Those results suggest the presence of highly conserved sequences between human and mouse DNA either in the flanking regions or in the introns of the IL-2 gene, since the coding regions apparently show lower degree of sequence homology as evidenced in this series of blotting analysis (see below). When the PstI insert of a mouse IL-2 cDNA clone, pMIL2-20, was used as the probe, a simple pattern was \n\n 9325 \n\n Nucleic Acids Research \n\n obtained at higher stringent condition (lane 13-17). While the EcoRI-digested DNA gave rise to two positive bands (2.8 kb and 0.8 kb)(Fig. 1, lane 14) by this analysis, one additional band of 3.3 kb also appeared when the same DNA was probed with a longer cDNA insert from another clone, pMIL2-45 (Fig. 1, lane 17). The 3.3 kb band was similar in its size with the positive band which became detectable by probing the same DNA with the 7.0 kb human DNA probe (Fig. 1, lane 2). Since this band appeared with the cDNA probe extending further upstream, it is likely that the 5' region of the gene is located within this DNA segment (see below). Indeed, this 3.3 kb band did not appear even after longer exposure of lane 14 (result not shown). BamHI digest of the mouse DNA (Fig. 1, lane 1, 13) constantly gave a very faint signal which would correspond to a DNA larger than 15 kb. Taken together, the results suggested the presence of a single copy gene for murine IL-2. On the other hand appearance of the multiple positive bands at lower stringent washing condition (lane 9-12) indicates the presence of IL-2 related sequences within the mouse genome. \n\n Screening of recombinant phaqe libraries \n\n We next screened a gene library from partial EcoRIdigested DNA from MPC 11 cells and by using 0.8 Kbp SacI-AccI cDNA fragment as the probe and isolated 14 positive clones containing sequences specific to the mouse IL-2 gene. Three of the clones analysed all contained three EcoRI fragments whose size is in agreement with the result of blotting analysis of the chromosomal DNA as shown in Fig. 1 (lane 17). One of these is designated MIL-2G70. \n\n Nucleotide sequence analysis \n\n Two DNA fragments of 3.3 Kbp and 2.8 Kbp were excised from the phage clone MIL-2G70 by EcoRI digestion and they were subcloned into pBR322. DNA sequences were determined for selected regions of both inserts and compared to the known cDNA sequences (Kashima et al., submitted for publication). The strategy used for sequence analysis of the genomic DNA is presented in Fig. 2. Comparison of the mouse genomic IL-2 sequence with mouse IL-2 cDNA sequence revealed that, like the human gene, the gene is divided into four exons. A putative \n\n 9326 \n\n Nucleic Acids Research \n\n E E E E E \n\n 500bp s 7S P HBH H \\A \n\n 200 bp * , - 4\n\n I 4-4----* *- --4 \n\n Fig. 2. Restriction map and sequencing strategy of mouse IL-2 gene. Horizontal lines indicate the length of mouse DNA inserted into the X phage Charon 4A or plasmid subclones. Filled blocks, dashed blocks and open blocks indicate protein coding regions, untranslated regions and introns, respectively. Horizontal arrows indicate the direction and extent of sequence determination without ambiguity. Dashed arrows: determination was done by the chain termination method (15) after subcloning the 0.8 kb fragment into M13. Rest of the sequence determination was carried out by the method of Maxam and Gilbert (14). A AccI site, B; BamHI site, E; EcoRI site, H; HindIII site, P; PstI site, S; SacI site. \n\n capping site or the transcription initiation site was located 32 bp downstream from a TATAAA consensus promotor sequence (Fig. 3). The first ATG triplet was located 79 bp downstream from the TATA box. As seen also in the murine IL-2 cDNA, there is an unusual repeat of CAG triplet coding for 12 glutamine residues in a row in the first exon. The second exon (60 bp) is separated from the first exon by a short intron consisting of 97 bp. The second, the third and the fourth exons are interrupted by longer introns whose size is about 2.3 Kbp and 1.6 Kbp, respectively. As far as the available sequence data are concerned, it seems that, despite their identical location, intron sequences are distinctly dissimilar except for the junction regions between the human and mouse IL-2 genes. There are two potential poly (A) addition signals within the mouse gene (nucleotide positions 793 - 798 and 924 - 929 in Fig. 3 ) and, based on our sequence data for various cDNA clones, both signals seem to function and give rise to heterogeneous termini of the mRNA in the LBRM-33 cells (16). \n\n We have also determined the sequence of about 500 bp of 5'-flanking region of mouse IL-2 gene, since (i) promoter/regulatory sequences are located in this region in many other genes of eukaryotes and (ii) this region appeared to contain sequences which show strongest cross-hybridization between human and mouse DNA around the IL-2 gene (Fig. 1). Comparison of the nucleotide sequences for the 5' flanking region \n\n 9327 \n\n Nucleic Acids Research \n\n -4.0 \n\n TAGGAGGTAAACCATCTCGAAACGGAAACCAATATCCTTCCTGTCTAATCAACAAATCTAAAG -400 -350 \n\n GATTTATTCTTTTCATCTATCTCCTCTTGCGCCCGTCCACCACAACAGGCTGCTTACAGGTTCAGGATGGTTTTGACAAAGAGAACATTTT \n\n -300 -250 \n\n CATGAGTTACTTTTGTGTCTCCACCCCAAAGAGGAAAATTTTGTTCATACGAAAGGCGTTCATTGTATGAATTAAAACTGCCACCTAAGAG \n\n -200 -150 \n\n TGGGCTAACCCGACCAAGAGGGATTTCACCTAAATCCATTCAGTCAGTATATGGGGTTTAAACAAATTCCAGAGAGTCATCAGAAGAGGAA \n\n -100 -50 \n\n AAACAAAGGTAATACTTTCTGCCACACAGGTAGACTCTTTTGAAAATATGTGTAATATGTAAAACATCGTGACACCCCCATATTATTTTTC CAGCATTAACAGTATAAATTGCCTCCCATGCTGAAGAGCTGCC TCACCCTTGCTAATCACTCCTCACAGTGACCTCAAGTCCTGCAGGC \n\n 50 100 \n\n ATG TAC AGC ATG CAG CTC GCA TCC TGT GTC ACA TTG ACA CTT GTG CTC CTT GTC AAC AGC GCA CCC ACT Met Tyr Ser Met Gln Leu Ala Ser Cys Val Thr Leu Thr Leu Val Leu Leu Val Asn Ser Ala Pro Thr \n\n 150 \n\n TCA AGC TCC ACT TCA AGC TCT ACA GCG GAA GCA CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln \n\n 200 \n\n CAC CTG GAG CAG CTG TTG ATG GAC CTA CAG GAG CTC CTG AGC AGG ATG GAG GTAAGTGCACAGCCATCCCATC His Leu Glu Gln Leu Leu Met Asp Leu Gln Glu Leu Leu Ser Arg Met Glu \n\n TATAGGCAATACCTTTAGCTTTCTTGCCAAAGGCTGTGTTTAATAACCTTTAATAATAATGTTACGCTTTCTCAG AAT TAC AGG AAC \n\n lAsn Tyr Arg Asn 250 \n\n CTG AAA CTC CCC AGG ATG CTC ACC TTC AAA TTT TAC TTG CCC AAG CAG GTGAGTGAGTTTCTGTTTAACTGGTGC Leu Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys Gln \n\n TCTAATG----------------------------------------------------------__ \n\n ?_______________----------------------- 2000 bp --------------------?---------------------------------------------------------- AATGTGAACCTTGTAGTTTCTTTGTAGATTGGAACAATAGTCTGAACTTGTGT TATGCATTGGTAGAGAAACACAGACTTTTTACAAAGTCTAATGAAGCCAGAAGAGCTCACTAGAGTGGCCTAACAGTTAAGGTGACCCTTA TGGTTGTGAGCTCTTGCTCTTCTAGATTTATGGCATCGATTACCTCAGTCCCCCTTTACAGAGGACAGGGAGTGGTAAAAGCTATGTGCTG CCTTCTCTTGATTAGAGAGACTGCAGACTAACTTTCTGGCTCTTCAGTATGTGGTGAGCTGAGCTGATGGTTAAGCTTATTACTCCTCTAG \n\n 300 350 \n\n GCC ACA GAA TTG AAA GAT CTT CAG TGC CTA GAA GAT GAA CTT GGA CCT CTG CGG CAT GTT CTG GAT TTG Ala Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu Leu Gly Pro Leu Arg His Val Leu Asp Leu \n\n 490 \n\n ACT CAA AGC AAA AGC TTT CAA TTG GAA GAT GCT GAG AAT TTC ATC AGC AAT ATC AGA GTA ACT GTT GTA Thr Gln Ser Lys Ser Phe Gln Leu Glu Asp Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val AAA CTA AAG GTAAGGTGTTGCTTTATTTGCTAATCTGGAAATAAAATAGAGAAGAAATGCATTTTTAAGTGGCTTGCCATTTCTGGT Lys Leu Lys \n\n CTTTGATGGGTTCTGTGCATTTAGTCAACCAAAGTTTAAAGTCACTGTGCAAGTGAATCC -------------------------------------------------------------------- -1000 bp ------ --------------------------------------------------------------- GAATTCTACAGAAGTGTTCAGTGTTCCCATCAAATGCTCGTTGGTAATCAACTCTGGAGAGCTT TATTTTTTATGCTTTACCATTTGATATCATAATAAAACATAACCCAATAATTGTGATCATTTCAGAAATGTGAATGTTCAATATATTTAAC \n\n CAGTGTTAAAATAAATGCCTAAAAGCTCCGTTGAAGAATAATTATATGCAAAGTAAGCTACCTTAGCCTACAATTTTATATTCTTTTTTAG \n\n 450 500 \n\n GGC TCT GAC AAC ACA TTT GAG TGC CAA TTC GAT GAT GAG TCA GCA ACT GTG GTG GAC TTT CTG AGG AGA Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp GlU Ser Ala Thr Val Val Asp Phe Leu Arg Arg \n\n 550 \n\n TGG ATA GCC TTC TGT CAA AGC ATC ATC TCA ACA AGC CCT CAA TAACTATGTACCTCCTGCTTACAACACATAAGGC Trp Ile Ala Phe Cys Gln Ser Ile Ile Ser Thr Ser Pro Gln \n\n 600 650 \n\n TCTCTATTTAT4TAAATATTTAACTTTAATTTATTTTTGGATGTATTGTTTACTATCTTTTGTAACTACTAGTCTTCAGATGATAAATATG \n\n 700 750 \n\n GATCTTTAAAGATTCTTTTTGTAAGCCCCAAGGGCTCAAAAATGTTTTAAACTATTTATCTGAAATTATT+ATTATATTGAATTGTTAAAT \n\n < 4>800 850 \n\n ATCATGTGTAGGTAGACTcATTCA TAAAAGTAmAGATGATTCAAATATAAATAAGCTCAGATGTCTGTCATTTTTAGGACAGCACAAAG \n\n 900 0\" \n\n TAAGCGCTAAAATAACTTCTCAGTTATTCCTGTGAACTcTATGTTAATCAGTGTTTTCAAGAAATAAAGCTCTCCTCT AAA GATGGCTTGTGGGAAAAGATCTCCTCTCCAGGGAGCTAACATCAGCTCAGAGTTTACTCAAGAATTC \n\n Fig. 3. Nucleotide sequence of mouse IL-2 gene. Four exons are framed. Numbers refer to nucleotide positions of exons from the presumed cap site. Dots and open circles indicate TATA box and poly A additional signals, respectively. \n\n 9328 \n\n Nucleic Acids Research \n\n -4 5 0 -400 \n\n TAGGAGGT AAACCA T CTCGAAAC GGAAACCAATATCCTTCCTGTCTAATCAACAAATCTAAAGGATTTATTCTTTTCATCTATCTCC \n\n .0 * .0 .- *. * ..* --. -- .*-@ .. ............ -- -*-* * \n\n TAAAAAGGTAAAACCAGTTCT GAAACAGGAAACCAATACACTTCCTGTTTAATCAACAAATCTAAA CATTTATTCTTTTCATCTGTTTAC \n\n -350 -300 \n\n TCTTGCGCCCGTCCACCACAACAGGCTGCT T ACAGGTTCAGGATGGTTTTGACAAAGAGAACATTTTCATGAGTTACTTTTGTGTCTC TCTTGCTCTTGTTCACCACAATA TGCTATTCACATGTTCAGTGTAGTTTTATGACAAAGAAAATTTTC TGAGTTACTTTTGTATCCC \n\n -250 \n\n CA CCCC AAAG AGGAAAATTTGTTTCATACGAAAGGCGTTCATTGTATGAATTAAAACT CCACCTAAGAGTGGGCTAACCCGA \n\n *~~~ ~~ ..... * .-- *..- * .-- .** . *-. --. *- *- - - *- 0 @ *-- 0 .. . \n\n CACCCCCTTAAAGAAAGGAGGAAAAACTGTTTCATACAGAAGGCGTTAATTGCATGAATTAGAGCTATCACCTAAGTGTGGGCTAATGTAA \n\n -200 -150 \n\n CCAAGAGGGATTTCACCTAAATCCATTCAGTCAGTATAT GGGGTTTAAACAAATTCCAGAGAGTCATCAGAAGAGGAAAAACAAAGGTAA CAAAGAGGGATTTCACCTACATCCATTCAGTCAGTCTTTGGGGGTTTAAA AAATTCCAAAGAGTCATCAGAAGAGGAAAAATGAAGGTAA \n\n -100 -50 \n\n TACTTTCTGCCACACAGGTAGACTCTTTTGAAAATATGTGTXATATTAAAACATCGTGACACCCCCATATiXrTCCAGCATTAACAG TGTTTTTT CAGACAGGTAAAGTC TTTGAAAATATGTGTAATATGTAAAACATTTTGACACCCCCATAATATTTTTCCAGAATTAACAG ATAAGCCTCCCATGCTGAAGAGCTGCCTATCACCCTTGCTAATCACTCCTCACAGTGACCTCAAGTCCTGCAGGCATG Mouse rTATGCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACACACAGTAACCTCAACTCCTGCCACTATG Human \n\n Fig. 4. Comparison of 5'-flanking regions of mouse and human IL-2 genes. In aligning the sequences for both genes, gaps were introduced to maximize homology. Dots indicate identical nucleotide sequences. Number 1 indicates putative transcription initiation site. Inverted repeats are indicated by bars and dashed lines. TATA box is framed. \n\n of both genes is illustrated in Fig. 4. The nucleotide sequence homology from the TATA box to -470 is 85%. The highest region of homology was observed between the TATA box and position -97 (60 bp matches out of 64 bp) of both genes. DISCUSSION \n\n We have isolated recombinant clones for mouse IL-2 gene from a phage Charon 4A/mouse genomic DNA library and determined the entire sequence of the gene except for the sequence of the internal portion of the second and third introns. The mouse IL-2 cDNA sequence was aligned with the genomic sequence and both sequences matched completely each other. The unusual CAG repeats encoding 12 glutamines which was found previously in the cloned cDNA was shown to be present also in the chromosomal gene. This finding further excludes the possibility that the unique repeat is generated by artifacts during the cDNA cloning process. Although we can not rule out the possibility for the deletion of this sequence in the human IL-2 gene, it is more likely that the CAG repeat has been generated in the mouse genome rather recently. The repeat could have been generated either by a direct insertion of the sequence or by the duplication after insertion of a unit sequence. \n\n 9329 \n\n Nucleic Acids Research \n\n Organization of the mouse IL-2 gene resembles to that of the human gene (Fig. 2., ref. 9). There seems to be little sequence homology between corresponding introns of mouse and human IL-2 genes, except for the intron-exon junctions part of which is thought to be necessary for the RNA splicing (17, 18). Dissimilarity of the intron sequences among the genes which are derived from a common ancestor has been reported in other genes (19, 20). In spite of the divergence in sequence of introns, the size and position of the introns are very similar between the murine and human IL-2 genes. \n\n Of particular interests is the presence of highly conserved sequences in the 5' -flanking region of the human and mouse IL-2 gene (Fig. 4). Whereas the coding region shows nucleotide sequence homology of 72% between the two genes, the 5' upstream region spanning about 500 bp (Fig. 4) shows 85% homology which was readily detectable by the blotting analysis (Fig. 1, lane 1-4). Since we have not yet determined the nucleotide sequence further upstream of the mouse gene, we do not know whether or not this similarity extends further. It is likely that such sequences are involved in the controlled expression of the IL-2 genes in activated T-lymphocytes. Work is in progress to identify such DNA sequences by introducing the cloned genes into various lymphocytic cell lines. Our preliminary results indicate that the 5 '-flanking sequence of the human IL-2 gene mediates mitogen induced expression of the gene in T-lymphocytic cells (Fujita & Taniguchi, unpublished observation). \n\n ACKNOWLEDGEMENTS \n\n We thank Dr. T. Honjo for mouse gene library. We are also indepted to Ms. M. Nagatsuka for typing the manuscript. This work was supported in part by Grant-in-Aid for Special Project Research, Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan. \n\n ?To whom correspondence should be addressed \n\n *Present address: Department of Microbiology, School of Medicine, Chiba University, Chiba 280, Japan \n\n +Present address: Central Research Laboratory, Ajinomoto Co.Inc., Totsuka-ku, Yokohama 244, Japan \n\n 9330 \n\n Nucleic Acids Research \n\n REFERENCES \n\n 1. Morgan, D. A., Ruscetti, F. W. and Gallo, R. (1976) Science, \n\n 193, 1007-1008. \n\n 2. Gil lis, S. Ferm, M. M., Ou, W. and Smith, K. (1 978) J. \n\n Immunol., 120, 2023-2027. \n\n 3. Chen, B. M. and Di Sabato, G. (1976) Cell. Immunol., 22, 211\n\n 224. \n\n 4. Henney, C. S., Kuribayashi, K., Kern, D. E. and Gillis, S. \n\n (1981) Nature, 291, 335-338. \n\n 5. Wagner, H., Hardt, C., Heeg, K., Rollinghoff, M. and \n\n Pfizenmaier, K. (1980) Nature, 284, 278-280. \n\n 6. Farrar, J. J., Benjamin, W. R., Hilfikr M. L., Howard, M., \n\n Farrar,W. L. and Fuller-Farrar, J. (1982) Immunol. Rev. 63, 129-166. \n\n 7. Pearlstein, K., Palladino, M. A., Welte, K. and Vilcek, J. \n\n (1983) Cell. Immunol., 80, 1-9. \n\n 8. Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, \n\n N., Yoshimoto, R. and Hamuro, J. (1983) Nature 302, 305-310. \n\n 9. Fujita,T.,Takaoka,C., Matsui, H. and Taniguchi T. (1983) \n\n Proc. Natl. Acad. Sci. USA 80, 7437-7411. \n\n 10. Ohno, S. and Taniguchi, T. 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[ { "id": "pmcA1538864__text", "type": "Article", "text": [ "QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences\nAbstract\nThe quadruplex structures formed by guanine-rich nucleic acid sequences have received significant attention recently because of growing evidence for their role in important biological processes and as therapeutic targets. G-quadruplex DNA has been suggested to regulate DNA replication and may control cellular proliferation. Sequences capable of forming G-quadruplexes in the RNA have been shown to play significant roles in regulation of polyadenylation and splicing events in mammalian transcripts. Whether quadruplex structure directly plays a role in regulating RNA processing requires investigation. Computational approaches to study G-quadruplexes allow detailed analysis of mammalian genomes. There are no known easily accessible user-friendly tools that can compute G-quadruplexes in the nucleotide sequences. We have developed a web-based server, QGRS Mapper, that predicts quadruplex forming G-rich sequences (QGRS) in nucleotide sequences. It is a user-friendly application that provides many options for defining and studying G-quadruplexes. It performs analysis of the user provided genomic sequences, e.g. promoter and telomeric regions, as well as RNA sequences. It is also useful for predicting G-quadruplex structures in oligonucleotides. The program provides options to search and retrieve desired gene/nucleotide sequence entries from NCBI databases for mapping G-quadruplexes in the context of RNA processing sites. This feature is very useful for investigating the functional relevance of G-quadruplex structure, in particular its role in regulating the gene expression by alternative processing. In addition to providing data on composition and locations of QGRS relative to the processing sites in the pre-mRNA sequence, QGRS Mapper features interactive graphic representation of the data. The user can also use the graphics module to visualize QGRS distribution patterns among all the alternative RNA products of a gene simultaneously on a single screen. QGRS Mapper can be accessed at .\n\nINTRODUCTION\nThe quadruplex structures formed by guanine-rich nucleic acid sequences have received significant attention recently because of increasing evidence for their role in important biological processes and as therapeutic targets (1–5). The G-quadruplex structure, also known as a G-quartet, is formed by repeated folding of either the single polynucleotide molecule or by association of two or four molecules. The structure consists of stacked G-tetrads, which are square co-planar arrays of four guanine bases each (6). G-quadruplex is stabilized with cyclic Hoogsteen hydrogen bonding between the four guanines within each tetrad. The present work focuses only on the unimolecular quadruplexes, since it is more likely to be encountered in physiological conditions (7,8).\nGuanine-rich sequences capable of forming G-quadruplexes are found in telomeres, promoter regions, transcribed and other biologically important regions of the mammalian genomes. G-quadruplex DNA has been suggested to regulate DNA replication in retinoblastoma susceptibility gene (Rb) region (9). This structure may control cellular proliferation at telomeric level and by transcriptional regulation of oncogenes like c-myc (2,10,11) and c-kit (12). Formation of G-quadruplex seems to be regulated through interactions with cellular proteins. While some proteins help stabilize the structure (13), others are known to resolve it (1,4,14,15). Proteins and chemicals that stabilize the G-quadruplex structure can inhibit telomerase action and, therefore, are being evaluated as anticancer therapeutic agents (16–20). Chemical compounds that inhibit G-quadruplex helicase activity may also be capable of regulating cellular proliferation (4). G-quadruplexes are also being eyed as potential antimicrobial agents due to their ability to transport monovalent anions (21).\nG-quadruplex motifs in the RNA have been shown to play significant roles in mRNA turnover (1), FMRP binding (22), translation initiation (23) as well as repression (24). We have shown previously that a G-rich sequence (GRS) can mediate 3′ end processing of mammalian pre-mRNAs by interacting with DSEF-1/hnRNPH/H′ protein (25–27). Members of the hnRNP H protein family recognize G-rich motifs capable of forming G-quadruplexes and are known to regulate polyadenylation and splicing events in mammalian transcripts (28–30). Regulated RNA processing is an essential component of differential gene expression which is central to many important biologically processes. More than half of human genes are known to have alternative polyadenylation (31). Over two-thirds of human genes are thought to undergo alternative splicing (32). Sequences capable of forming G-quadruplexes found in the vicinity of polyadenylation and splice sites act as regulators by interacting with hnRNP F and H proteins (25–27,30,33). Whether quadruplex structure directly plays a role in regulating RNA processing events requires investigation.\nComputational approaches to study G-quadruplexes in the mammalian genomes allow large-scale and detailed analysis of mammalian genes. Although, G-quadruplexes have been surveyed in the human genome with such techniques (34,35), there are no known user-friendly computational tools easily accessible to the public. We had previously built a database of mapped G-quadruplex sequences in selected alternatively processed human and mouse genes (36). Our preliminary analysis of the database suggests prevalence of such motifs near alternative splice and poly(A) sites. We have now developed a web-based server, QGRS Mapper, that generates detailed information on composition and distribution of putative quadruplex forming G-rich sequences (QGRS) in any NCBI nucleotide sequence identified or provided by the user. The program is also designed to handle the analysis of mammalian pre-mRNA sequences, including those that are alternatively processed (alternatively spliced or alternatively polyadenylated). Researchers interested in predicting the ability of a nucleotide sequence to form G-quadruplex structure will find QGRS Mapper to be a user-friendly application that provides many options for analysis. The user can define the minimum number of tetrads, maximum length of the G-quadruplex motif, and size as well as composition of the loops. The program can map unimolecular QGRS in the entire nucleotide sequence provided in the raw or FASTA format by the user. This method can be used for the analysis of genomic sequences, e.g. promoter and telomeric regions, as well as RNA sequences. It is also useful for predicting G-quadruplex structures in oligonucleotides. Alternatively, the program provides options to search the entire NCBI Gene Entrez, RefSeq and GenBank databases in order to retrieve the desired gene/nucleotide sequence entries for analysis of their transcribed regions. Furthermore, QGRS Mapper is a unique tool for mapping G-quadruplex forming sequences in the context of RNA processing sites. This feature is very useful for investigating the functional relevance of G-quadruplex structure, in particular its role in regulating the gene expression by alternative processing. In addition to providing data on composition and locations of QGRS relative to the processing sites in the pre-mRNA sequence, QGRS Mapper offers interactive graphic representation of the data. The user can also use a graphics module to visualize QGRS distribution patterns among all the alternative RNA products of a gene simultaneously on a single screen.\n\nMETHODS\nQGRS definition\nThe main goal of the QGRS Mapper program is to predict the presence of QGRS in nucleotide entries. These putative G-quadruplexes are identified using the following motif.\nGxNy1GxNy2GxNy3Gx\nHere x = number of guanine tetrads in the G-quadruplex and y1,y2,y3 = length of gaps (i.e. the length of the loops connecting the guanine tetrads). The motif consists of four equal length sets of guanines (which we call G-groups), separated by arbitrary nucleotide sequences, with the following restrictions.\nThe sequence must contain at least two tetrads (i.e. x ≥ 2). Although structures with three or more G-tetrads are considered to be more stable, many nucleotide sequences are known to form quadruplexes with two G-tetrads (37,38). QGRS Mapper is meant to be a flexible and comprehensive tool for investigating G-quadruplexes; hence it considers sequences with two tetrads.By default, only QGRS of maximum length of 30 bases are considered. However, the program gives the user the option to search for sequences up to 45 bases. This restriction on the length of the sequences being considered is in agreement with recent literature (34,35). The maximum length of 30 bases restricts G-groups to a maximum size of 6.The gaps or loops between the G-groups may be arbitrary in composition or length (within the overall restrictions on the length of QGRS). The program gives the user the option to search for QGRS having loops with a specified length range (e.g. the user can search for QGRS with loops of lengths between 1 and 4). The user can also specify a string that one or more loops of each QGRS must contain. This string can be given as a regular expression. For example, entering the regular expression ‘T{3,5}’ will search for QGRS having one or more loops that contain three to five consecutive T's.Also, at most one of the gaps is allowed to be of zero length\nTable 1 shows some examples of valid QGRS. The guanine groups which form the tetrads are underlined.\nThe first sequence has four tetrads and equal length gaps. This would seem to provide a G-quadruplex that is the most stable of the three sequences. The second sequence is notable for the significant differences in the size of its loops. The third sequence has two tetrads, even though three of the G-groups could have included another G (since all G-groups must be equal in size).\n\nG-scores\nWe have devised a scoring system that evaluates a QGRS for its likelihood to form a stable G-quadruplex. Higher scoring sequences will make better candidates for G-quadruplexes. The scoring method uses the following principles which are based on previous studies (34,35,39–42).\nShorter loops are more common than longer loops.G-quadruplexes tend to have loops roughly equal in size.The greater the number of guanine tetrads, the more stable the quadruplex.\nThe computed G-scores are dependent on the user selected maximum QGRS length. The highest possible G-score, using the default maximum QGRS length of 30, is 105. Here is a sequence attaining that score:\nGGGGGGTGGGGGGTGGGGGGTGGGGGG.\n\nEliminating QGRS overlaps\nTwo QGRS are said to overlap if their positions in the nucleotide sequence overlap. QGRS Mapper will start with a nucleotide sequence, find all QGRS occurring in the sequence and then produce a non-overlapping set of QGRS. Overlaps are eliminated by selecting the higher scoring QGRS. In the non-overlapping view, only this sequence will be displayed, although the user can request that all overlapping sequences be displayed.\nPlease see supplementary materials at NAR online for more details on the elimination of overlapping QGRS.\n\n\nFEATURES\nDesign and implementation\nQGRS Mapper is a web-based program, written in PHP, with Java being used for some of its graphics. The program takes a nucleotide sequence from NCBI (or as provided by the user) and analyzes it for the presence of putative G-quadruplexes. The structure of QGRS Mapper is summarized in Table 2.\n\nSearch and analysis\nQGRS Mapper allows the user to search for putative G-quadruplexes in a variety of ways. It is possible to enter a nucleotide sequence in raw or FASTA format for analysis. One can search and analyze gene sequences by Gene ID, Gene name or symbol, accession number or GI number for an NCBI nucleotide sequence entry. The user can opt to change the maximum length of QGRS that will be searched for (the default maximum length being 30) and change the minimum sized G-group (which is two by default). Also, the user can specify that the loops in the QGRS fall within a given size range and that one or more loops of the QGRS contain a given string (for which the user may enter a regular expression). The web page for QGRS analysis can be seen in Figure 1.\nAfter entering a sequence in raw or FASTA format, QGRS Mapper will search the sequence for occurrences of QGRS. The user may enter any combination of the letters A, C, T, G, U, N.\nThe Gene ID field allows the user to search the NCBI Entrez Gene database. QGRS Mapper will connect to NCBI, download and parse the gene entry, and then analyze the transcribed region of its nucleotide sequence for the presence of QGRS. For example, entering the gene ID 403437 results in downloading the Brca1 gene sequence for Canis familiaris. Using the default QGRS search parameters, QGRS Mapper finds 156 non-overlapping QGRS and 3394 overlapping QGRS in the transcribed region of this gene.\nThe Gene Name or Gene Symbol field also allows the user to search the NCBI databases for all such genes. Entering the gene name Bcl2 results in nine different hits which are displayed in Table 3.\nAll nine of these entries can be analyzed for the occurrence of QGRS. Clicking on the Gene ID takes the user to the respective Entrez Gene entry. Clicking on the last column initiates analysis of the selection by QGRS Mapper.\nSimilarly, the user can also enter an NCBI accession number to search for gene sequences. For example, searching the accession number AF312033 results in 12 hits being displayed for this GenBank nucleotide sequence entry.\nThe search phase of the program is followed by an analysis of the QGRS contained in the query sequence. In this phase of QGRS Mapper, the sequence data downloaded previously is analyzed to identify and map all QGRS relative to locations such as splice sites in exons/introns, and poly(A) site (if these locations are known). Furthermore the QGRS are scored by the method described above. The computed G-score is used to eliminate overlapping QGRS.\nAt times, QGRS Mapper must analyze a considerable amount of data. For example, the mouse version of the gene PTPRU, which is 69822 bases long, contains 94681 QGRS of length up to 45 bases. QGRS Mapper will find, analyze and map all of these sequences. During this analysis a message is displayed indicating the estimated time left to completion.\n\nQGRS Mapper output\nAfter the analysis of overlaps is completed, QGRS Mapper displays a summary of its findings, in the Gene View. This summary includes basic gene information such as the gene ID, gene symbol, gene name, a link to the NCBI entry, organism name, chromosome number and number of products and poly(A) signals. Information is also given for each product, such as the number of exons and introns, number of QGRS (non-overlapping and overlapping), number of QGRS found near RNA processing sites, and a visual map of the product.\nAs an example, the Gene View for the human GREB1 is displayed in Figure 2, showing the table of gene information and product information for the first product (the output for all products may be seen in the supplementary material).\nAt this stage in the analysis the user can choose among three further displays: ‘Data View’, ‘Data View (with overlaps)’ and ‘Graphics View’. This can be done for the entire gene or for any particular product.\nIn the Data View, a table is displayed showing information for each of the set of non-overlapping QGRS. This table displays the position of the QGRS, which exon/intron it appears in, its distance from 3′ and 5′ splice sites, the QGRS sequence (with each G-group underlined) and the corresponding G-score. Similar display is also shown for each QGRS mapped to poly(A) region in the product. If the user requests the Data View for the entire gene, then the QGRS information is shown for each product. The ‘Data View (with overlaps)’ gives the same information but shows the locations of all QGRS. Figure 3 shows the Data View for product 1 of the GREB1 gene.\nThe user can also choose the Graphics View to give a visual display of the location of QGRS. This allows the user to see the location of QGRS relative to exons and introns (if that information is available). The Graphics View has the following components.\nA graphic display of the entire gene (showing the location of the exons). This display includes a sliding window that can be used to focus on any particular segment of the gene. This window may be dragged to the left or right to change position within the gene.A magnified view of the fragment of the gene within the sliding window.A graph showing the location of QGRS within the fragment, with each QGRS being displayed by a bar whose height represents its G-score.A vertical slider that allows the user to change the size of the window. This allows the user to zoom in or out on any part of the gene. The sliding window on the gene expands or contracts as one zooms in or out. It is possible to see the nucleotide sequence of the product at maximum zoom levels.\nThe Graphics View for the entire gene shows the G-score graph together with an exon/intron map for each product. This allows the user to visually compare the location of QGRS for each product relative to that of splice sites. The Graphics View for the first product of the GREB1 gene is represented in Figure 4. The Graphics View for the entire GREB1 gene may be seen in the Supplementary Data.\n\n\nCONCLUSIONS\nQGRS Mapper is a user-friendly web-based server that provides computational tools for prediction of Quadruplex forming G-rich sequences in the nucleotide sequences identified or provided by the user. The program offers many options, including user-defined composition of the quadruplex. It can analyze DNA or RNA sequence provided by the user in the raw or FASTA format. The application also provides tools for searching and retrieving gene/nucleotide entries from a variety of NCBI databases. There are several options for data output format, including an interactive graphic module.\nResearchers interested in evaluating the ability of nucleotide sequences to form unimolecular G-quadruplexes will find QGRS Mapper to be very useful. Owing to the flexible and comprehensive nature of the design, it is expected to serve a variety of scientists. The application will be especially attractive to individuals interested in exploring the role of G-quadruplexes in regulated RNA processing. We are using the server to perform a large-scale analysis of alternatively processed mammalian transcripts. We are particularly interested in studying the composition and distribution patterns of G-quadruplexes in the transcribed regions of mammalian genes.\n\nSUPPLEMENTARY DATA\nSupplementary Data are available at NAR online.\n\n\n" ], "offsets": [ [ 0, 18860 ] ] } ]

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[ { "id": "pmcA140510__text", "type": "Article", "text": [ "Enp1, a yeast protein associated with U3 and U14 snoRNAs, is required for pre-rRNA processing and 40S subunit synthesis\nAbstract\nENP1 is an essential Saccharomyces cerevisiae gene encoding a 483 amino acid polypeptide. Enp1 protein is localized in the nucleus and concentrated in the nucleolus. An enp1-1 temperature-sensitive mutant inhibited 35S pre-rRNA early processing at sites A0, A1 and A2 as shown by northern analysis of steady state levels of rRNA precursors. Pulse-chase analysis further revealed that the enp1-1 strain was defective in the synthesis of 20S pre-rRNA and hence 18S rRNA, which led to reduced formation of 40S ribosomal subunits. Co-precipitation analysis revealed that Enp1 was associated with Nop1 protein, as well as with U3 and U14 RNAs, two snoRNAs implicated in early pre-rRNA processing steps. These results suggest a direct role for Enp1 in the early steps of rRNA processing.\n\nINTRODUCTION\nRibosome biogenesis is one of the major cellular activities in eukaryotic cells. It takes place primarily in a specialized subnuclear compartment, the nucleolus (1,2). In the yeast Saccharomyces cerevisiae, each rRNA gene is transcribed by RNA polymerase I into a 35S rRNA precursor, consisting of 18S, 5.8S and 25S rRNA sequences flanked by two external transcribed spacers (ETS) and separated by two internal transcribed spacers (ITS) (Fig. 1). This 35S precursor goes through a series of modifications and processing steps to generate the mature 18S, 5.8S and 25S rRNAs. The processing occurs first at sites A0, A1 and A2, resulting in the 20S pre-rRNA and 27SA2 pre-rRNA. The 20S pre-rRNA is then cleaved, leading to the mature 18S rRNA found in the 40S ribosomal subunit. The 27SA2 pre-rRNA is processed through two alternative pathways. The majority of 27SA2 pre-rRNA is cleaved at sites A3 and B2 to form the 27SA3 pre-rRNA, which is subsequently processed to produce 27SBS. Alternatively, 27SA2 pre-rRNA can be processed at sites B1L and B2 to generate 27SBL pre-rRNA. Both 27SBS and 27SBL pre-rRNAs are then cleaved at sites C1 and C2 to generate the mature 25S rRNA, and 7SS or 7SL intermediates, which are then processed to mature 5.8SS or 5.8SL rRNAs (Fig. 1). The 25S rRNA and 5.8S rRNA are the RNA components of the 60S ribosomal subunit (3). During the course of rRNA modification and processing, many of the ribosomal proteins are assembled onto the rRNA molecules to form the ribosome complex.\nRibosome biogenesis needs a large number of trans-acting factors, including small nucleolar RNAs (snoRNAs), protein components of the snoRNP complexes, rRNA modifying enzymes, endo- and exonucleases, putative RNA helicases and other protein factors (3,4). In yeast cells there are more than 100 different snoRNAs playing important roles in rRNA modification and processing. On the basis of their structure, the snoRNAs can be divided into two groups: the box C/D family and box H/ACA family. Only one snoRNA, MRP RNA, belongs to neither family (5–7). While the majority of the snoRNAs participate in RNA pseudouridylation and 2′-O-ribose methylation, a few of them, including MRP snoRNA, box C/D snoRNAs U3 and U14, and box H/ACA snoRNAs snR10 and snR30, are required for processing of the pre-rRNA (8–12). Not only are U3, U14, snR10 and snR30 essential for early cleavages of 35S pre-rRNA to 18S rRNA, the proteins associated with them, including Nop1, Nop5, Gar1 and Nop10, have also been shown to be required for 18S rRNA synthesis (13–16).\nThe ENP1 (Essential Nuclear Protein 1) gene was identified in a genetic screen for suppressors of an ost4 mutation (oligosaccharide transferase 4) (17). However, in subsequent studies it was found not to be involved in OST4 function (18). ENP1 is an essential gene encoding a 483 amino acid polypeptide. The Enp1 protein is highly conserved and homologs are found in all eukaryotes. The yeast protein was localized to the nucleus in a previous study (18). On the other hand, an Enp1 human homolog, called bystin, was reported to localize to the cytoplasm and was proposed to be involved in cell adhesion (19).\nIn this study, we report that the Enp1 protein not only is nuclear but is enriched in the nucleolus. We also found that Enp1 is required for the synthesis of 40S ribosomal subunits, and its presence is necessary for the pre-rRNA processing to form 20S pre-rRNA and 18S rRNA. An association between Enp1 and U3 and U14 snoRNAs, and with the nucleolar protein Nop1, was established. We also found that human Enp1, expressed in yeast, was located in the nucleus and the nucleolus, suggesting that the function of this protein is conserved.\n\nMATERIALS AND METHODS\nYeast strains and media\nThe S.cerevisiae strains used in this study are all derivatives of a wild-type diploid strain W303 (MATa/MATα ura3-1/ura3-1 leu2-3,112/leu2-3,112 trp1-1/trp1-1 his3-11,15/his3-11,15 ade2-1/ade2-1 can1-100/can1-100) except for strain RS1938. Strain JBY45 (MATa/MATα ENP1/Δenp1::his5+) was constructed by replacing one copy of the ENP1 open reading frame (ORF) with the Schizosaccharomyces pombe his5+ gene (18). Strain JBY46 [MATa Δenp1::his5+/pJB23 (ENP1, URA3, CEN6)] is a haploid strain derived from JBY45 with wild-type ENP1 on a low copy number plasmid. Strain JBY48 [MATa Δenp1::his5+/pJB19 (enp1-1, TRP1, CEN6)] and strain JBY49 [MATa Δenp1::his5+/pJB39 (enp1-2, TRP1, CEN6)] have plasmids with enp1 temperature-sensitive (ts) mutations. Strain JBY51 (MATa Δenp1::his5+ TRP1::enp1-1) is an enp1 ts strain generated by integrating the enp1-1 gene at the chromosomal trp1-1 locus. Strain CWY13 [MATa Δenp1::his5+/pJB24 (pMET25-GFP-ENP1, URA3, CEN6)] has GFP-ENP1 under control of the MET25 promoter. Strain CWY14 (MATa ENP1-TAP) was created by fusing sequences encoding a Tandem Affinity Purification (TAP) tag (20,21) to the 3′ end of ENP1. Strain YRH39 (MATα HST2-TAP) was created by fusing sequences encoding a TAP tag to the 3′end of HST2. Strain RS1938 (Δnop1::URA3 pUN100-ProtA-NOP1) was created by transforming RS1935 (MATa/α leu2/leu2 ura3/ura3 lys2/lys2 ade2/ade2 Δnop1::URA3/NOP1) with pUN100-ProtA-NOP1 (strain and plasmid supplied by T. Schafer), followed by tetrad dissection. Strain CWY15 [MATa/pCW109 (pMET25-GFP-hENP1, URA3, CEN6)] has a human homolog of Enp1 expressed in W303-1a. The media used were prepared as described (22).\n\nCloning of ENP1\npRS426-MEG1 (The original name of ENP1) was a gift from Dr William J. Lennarz. It contains the ENP1 gene within a 2.6 kb EcoRI genomic fragment cloned into pRS426 (18). The EcoRI fragment was subsequently cloned into pRS314 (TRP1, CEN6), pRS316 (URA3, CEN6) and pRS424 (TRP1, 2µ) to create pJB20, pJB23 and pJB21, respectively. pGFP-N-FUS is a centromeric plasmid for fusing green fluorescence protein (GFP) to a polypeptide’s N-terminus under control of the MET25 promoter (23). Cloning the ENP1 ORF into pGFP-N-FUS via XbaI and SalI sites generated plasmid pJB24. pJB19 (enp1-1, TRP1, CEN6) contains the enp1-1 mutant gene. The human homolog of yeast Enp1 was PCR amplified from a human cDNA clone BC007340 (Research Genetics), then cloned into pGFP-N-FUS, p415-ADH, p415-GPD and p415-TEF (24) via XbaI and SalI sites to generate pCW109, pCW113, pCW115 and pCW117, respectively. The fragment of the human Enp1 homolog (amino acids 152–437) was also cloned into these vectors to generate pCW110, pCW114, pCW116 and pCW118.\n\nRandom mutagenesis of ENP1 to generate enp1 ts mutants\nThe enp1 ts mutants were generated with a protocol introducing random mutations by PCR (25). The PCR was performed with oligonucleotides ENP1-MUT5′ (5′-GGTGGTGTCAGT AGGGGA-3′), ENP1-MUT3′ (5′-CAGTCTGCAATATA TGGAC-3′) and plasmid template pJB20 (see above). The nucleotide concentrations in the reaction were 1 mM each for dATP, dGTP, dCTP and dTTP. After strain JBY46 was transformed with the PCR product and with pJB20 gapped by NheI and NsiI, the transformants were replica-plated onto two plates containing 5-FOA synthetic medium without tryptophan; one replica was incubated at 23°C and the other at 37°C. Colonies growing at 23°C but not at 37°C were picked as candidates. Since approximately 10% of the colonies were inviable at both temperatures, we knew that 10% of the ENP1 PCR products lost their function after the PCR mutagenesis. This confirmed the effectiveness of the mutagenesis procedure. Two candidates showed good growth at 23°C but no growth at 37°C. They were named strains JBY48 and JBY49 and contained plasmids with the enp1-1 and enp1-2 mutations, respectively. The mutated enp1-1 gene on pJB19 was cloned into an integration vector, pRS304, as an EcoRI fragment. The plasmid was subsequently linearized with SnaBI within the TRP1 marker and was integrated at the chromosomal trp1-1 locus of strain JBY46. Selection on 5-FOA was used to remove plasmid pJB23, creating strain JBY51.\n\nSucrose gradient analysis\nPolyribosome preparation and analysis were carried out essentially as described (26). Cells grown in YPD were collected at mid-log phase (OD600 0.8–1.0) and were broken with glass beads. The lysate was frozen immediately in liquid N2 and was stored at –80°C. Lysate (30 U of absorbance at OD260) was layered over a 7–47% (w/v) sucrose gradient, which was centrifuged at 28 000 r.p.m. for 5 h at 4°C in a SW28 rotor and was analyzed with an ISCO UA-5 gradient UV detection system on absorbency at 254 nm.\n\nImmunofluorescence\nImmunofluorescence analysis was carried out essentially as described (27). Cells were fixed with 3.7% formaldehyde at room temperature for 1.5 h. Antibodies included a mouse monoclonal anti-Nop1 (a gift from John P. Aris, University of Florida, Gainesville, Florida) and a Texas-red-conjugated donkey-anti-mouse antibody (Jackson Lab), both used at 1:500 dilution. Images were taken on a Zeiss Axioplan2 microscope equipped with a Zeiss AxioCam camera.\n\nPulse-chase labeling analysis of rRNA\nFor [methyl-3H]methionine pulse-chase analysis, cells were grown in synthetic medium without methionine at room temperature or 37°C for 2 h. When the OD600 reached 1.0, 6 ml of the culture were pulse-labeled with 250 µCi [methyl-3H]methionine (Amersham Pharmacia) for 3 min and chased with cold methionine (500 µg/ml) for 2, 4 or 12 min. For each time point of the chase 1.25 ml of culture was mixed with ice and collected. The pellets were frozen immediately in liquid N2 and stored at –80°C before total RNA was purified using a hot phenol method (28). The RNAs were separated on a 1.2% agarose formaldehyde gel and transferred onto Hybond-N+ nylon membranes (Amersham Pharmacia). After being sprayed with EN3HANCE (Du Pont), the membranes were exposed to film at –80°C (29).\n\nNorthern analysis\nCells were grown inYPD at room temperature or 37°C for 2–4 h. When the OD600 reached 1.0, 10 ml of cells were collected and frozen immediately. Total RNA was extracted as described (28). Five micrograms of RNA were separated on 1.2% agarose formaldehyde gels (for high molecular weight RNA) or on 6% polyacrylamide 7 M urea denaturing gels (for low molecular weight RNA) for each sample. The RNA was then transferred to Zeta-Probe GT nylon membranes (Bio-Rad). Probes for hybridyzation were specific oligonucleotides end-labeled using [γ-32P]ATP. After hybridization and washes, membranes were exposed to phosphorimager screens or X-ray films (29). Oligonucleotides specific for various regions of 35S pre-rRNA are: 1, GGTCTCTCTGCTGCCG GAAATG; 3, AATGAGCCATTCGCAGTTTCACTG; 4, GCTCTCATGCTCTTGCCAAAAC; 5, TGTTTGTTACCT CTGGGCCCCG; 6, TCCAGTTACGAAAATTCTTG; 7, CGTATCGCATTTCGCTGCGTTC; 8, GTTCGCCTAGAC GCTCTCTCTTC; 9, GCGAGATTCCCCTACCCAC. The regions complementary to these oligonucleotides are shown in Figure 6A. The oligonucleotides probing box C/D snoRNAs are: U3, TTCGGTTTCTCACTCACTCTGGGGTAC; U14, GGAACCAGTCTTTCATCACCGTG. The oligonucleotides probing box H/ACA snoRNAs are: snoRNA10, CCTTG CAACGGTCCTCATCCGGG; snoRNA30, GTCCGAAGC GCCATCTAGATGA.\n\nRNA and protein precipitation analysis\nCells were grown in YPD (1 l) to OD600 1.0, then collected, washed once in ice-cold PBS and broken using glass beads in 10 ml IPP150 buffer (10 mM Tris pH 8.0, 150 mM NaCl and 0.1% NP-40) with protease inhibitors. The lysates were mixed with 200 µl IgG agarose beads for 2 h at 4°C. After several washes with 50 ml IPP150 buffer, the IgG beads were collected and the RNA associated with the beads extracted by the hot phenol method (28). One-tenth of the precipitated RNA was then separated on 6% polyacrylamide 7 M urea denaturing gels and electro-transferred to Zeta-Probe GT nylon membranes. They were probed with [γ-32P]ATP labeled oligonucleotides. For the lanes showing total RNA, 2 µg RNA prepared from exponentially growing cultures were loaded. For co-precipitation analysis of proteins, the IgG beads were resuspended in 2× Laemmli sample buffer, boiled for 5 min and separated by SDS–polyacrylamide gel electrophoresis. Approximately 1% of the precipitate was loaded onto each lane. For the total protein, 0.1% of the extract prior to precipitation was loaded onto each lane. Following electrophoresis, the proteins were transferred to nitrocellulose membranes and detected using anti-Nop1 antibody at 1:3000 dilution (provided by J. Aris) and anti-L3 antibody (provided by J. Warner) at 1:3000 dilution followed by peroxidase-conjugated anti-mouse secondary antibody at 1:5000 dilution. The antibody complexes were detected using ECL-Plus reagents (Amersham Pharmacia) as specified by the manufacturer.\n\n\nRESULTS\nConstruction and analysis of ENP1 temperature-sensitive alleles\nENP1 is an essential yeast gene conserved among eukaryotes (18). To study its functions, we first created a diploid strain, JBY45, heterozygous for the Δenp1 mutation. Then two enp1 ts mutant alleles (enp1-1 and enp1-2) were generated by random PCR mutagenesis, as described in Materials and Methods. Both mutant alleles were recessive to the wild-type ENP1 gene. The enp1-1 gene was integrated into the chromosomal trp1-1 locus to create strain JBY51 and all subsequent experiments were done with this enp1 allele. At 23°C the growth of JBY51 was comparable to that of W303-1a (ENP1), but at 37°C it did not grow (Fig. 2). Sequence analysis revealed that enp1-1 contains two point mutations, resulting in substitutions of two amino acids: W242→G and V415→A. No attempt was made to determine whether both mutations were required for the ts phenotype. Flow cytometry of strain JBY51 showed DNA content profiles similar to those of the wild-type strain at the non-permissive temperature (data not shown), suggesting that ENP1 is not involved in cell cycle regulation.\n\nEnp1 is enriched in the nucleolus\nEnp1 tagged at the C-terminus with the myc epitope was previously found to localize to the nucleus (18). To expand this analysis, we fused GFP to the N-terminus of Enp1, and expressed the tagged Enp1 protein under control of the MET25 promoter as the sole source of Enp1 protein in the cells. Cells of strain CWY13 (pMET25-GFP-ENP1) were grown in media with methionine (transcription partially repressed) or without methionine (transcription not repressed). Growth of CWY13 was comparable to that of wild-type cells in both media (data not shown), indicating that the GFP tagged Enp1 protein was functional. In cells cultured in medium without methionine, in which the ENP transcription was not repressed, the GFP-Enp1 protein was expressed at a high level and showed strong green fluorescence distributed throughout the nucleus (data not shown), consistent with the original observation (18). With methionine added to the medium, however, the fluorescent signal of GFP-Enp1 was weaker and surprisingly showed crescent or cap-like patterns typical of nucleolar proteins (Fig. 3A). This nucleolar enrichment was confirmed by its co-localization with the nucleolar protein Nop1 (Fig. 3B).\n\nENP1 mutation leads to reduced levels of 40S ribosomal subunits\nThe nucleolar enrichment suggested that Enp1 might play some role in ribosome synthesis. To test this possibility, cells of W303-1a (ENP1), JBY51 (enp1-1) and a top2 ts strain RS191 (30) were grown in YPD at 23 or 37°C and their ribosome profiles analyzed after separation on sucrose gradients. At 23°C, enp1-1 cells showed polysome profiles similar to those of the wild-type and top2-1 strains (Fig. 4A–C). In contrast, after incubation at the non-permissive temperature (37°C) for 40 min (data not shown) and 2 h, enp1-1 cells had reduced levels of 40S ribosomal subunits, 80S monosomes and polysomes, along with a dramatic increase in the free 60S subunit peak (Fig. 4F), while the wild-type and top2-1 cells showed little change in polysome profile at 37°C (Fig. 4D and E). These results demonstrate that the changes of the enp1-1 polysome profile were due to specific defects caused by the enp1-1 mutation, and not due simply to the shift to 37°C for a wild-type or ts strain.\n\nThe processing of pre-rRNA for 18S rRNA is impaired in enp1 mutants\nIn most cases reductions in ribosomal subunit levels are the results of defects in pre-rRNA processing or ribosome assembly or both (3). To study the mechanism by which Enp1 affects the 40S subunit, we analyzed the effects of enp1 mutations on processing of the pre-rRNA using pulse-chase labeling. [methyl-3H]methionine is preferred for labeling rRNAs in pulse-chase analysis because rRNAs are specifically methylated during the early steps of the processing. Cells of W303-1a (ENP1) and JBY51 (enp1-1) were grown at 23 or 37°C for 2 h before they were labeled with [methyl-3H]methionine for 3 min and chased with cold methionine for 2, 4 and 12 min. In wild-type cells, the labeled 35S rRNA precursor, 27S and 20S rRNA intermediates were rapidly chased into mature 25S and 18S rRNAs (Fig. 5), at 23 or 37°C. In contrast, enp1-1 cells showed dramatic changes after incubation at 37°C. Although at 23°C the synthesis and processing were comparable to those of wild-type cells, cells cultured at 37°C for 2 h had neither 20S pre-rRNA nor 18S rRNA, while 25S rRNA was generated at normal levels. The 37°C grown enp1-1 cells also had low levels of aberrant 23S and possibly 21S intermediates (Fig. 5). The defect in 18S rRNA synthesis was further supported by pulse-chase experiments carried out using [5,6-3H]uracil. The results obtained were essentially the same; no 20S pre-rRNA or 18S rRNA was produced, while processing to 25S rRNA was not affected (data not shown).\nTaken together, these results suggested that the enp1 mutation leads to specific defects in the processing pathway for 20S pre-rRNA and 18S rRNA, resulting in reduced 40S subunit synthesis and a lowered level of the 40S subunit.\n\nEnp1 is required for pre-rRNA processing at A0, A1 and A2 sites\nTo define the processing steps affected by the enp1 mutation, the steady state levels of rRNA precursors and mature RNAs were analyzed by northern blotting. Total RNAs were isolated from cells of W303-1a (ENP1) and JBY51 (enp1-1) grown at 37°C for 2 or 4 h. After separation on formaldehyde agarose, RNAs were transferred onto nylon membranes and probed with radiolabeled oligonucleotides specific to various regions of the 35S pre-rRNA (Fig. 6A). After incubation at 37°C, enp1-1 cells had wild-type levels of 25S rRNA, but reduced levels of 18S rRNA (Fig. 6B). A probe specific to ITS1 (P5) further revealed that enp1-1 cells incubated at the non-permissive temperature accumulated two aberrant rRNAs, 23S and 21S (Fig. 6C). Aberrant rRNA processing products of similar sizes have been described in numerous studies (14,31–34) and result from defects in processing at A0, A1 and A2. Additional probes were used to verify the origins of the 23S and 21S RNAs in the enp1-1 strain. The 23S product was detected by oligos 1, 2, 4 and 5, but not by oligos 6, 7 or 8 (Fig. 6D, E, C, F, G and data not shown). Thus this RNA extends from the 5′ end of the 35S rRNA to the A3 site. The 21S product was detected by oligos 4 and 5, but not by oligos 1, 2, 6, 7 and 8 (Fig. 6E, C, D, F, G and data not shown), indicating that it extends from the A1 to the A3 site. At 37°C the enp1-1 cells also had less 32S and 20S rRNA intermediates (Fig. 6B and E). These results suggest that the enp1 mutation led to complete or nearly complete inhibition of processing at site A0 and A2, and partial inhibition of processing at site A1. As a consequence, the 35S pre-rRNA was cleaved at site A3 instead, producing 23S and 21S rRNA products. Consistent with this theory, the 27SA2 rRNA intermediate level was greatly reduced in enp1 cells at 37°C (Fig. 6C) due to the inhibition of processing at A2, while the 27SB pre-rRNA level was similar to wild-type cells (Fig. 6G). Moreover, no difference in the 5.8 rRNA level was observed between the wild-type cells and enp1 cells (Fig. 6H).\n\nEnp1 is associated with U3 and U14 snoRNAs and with Nop1\nIt has previously been shown that mutations in the U3, U14, snR10 and snR30 snoRNAs and in their associated proteins affect processing of 35S pre-rRNA at A0, A1 and A2. Because of the similarity with the processing defects of enp1-1 strains, we tested the association between Enp1 and snoRNAs. To do this, we created a strain in which a TAP tag was fused to the C-terminus of Enp1. The TAP tag contains Staphylococcus aureus Protein A as well as calmodulin-binding peptide sequences, so the tagged Enp1 binds IgG beads with high specificity. Cells grew well with the TAP-tagged Enp1 as the only source of Enp1 protein, showing that it was functional. As a control, we used a TAP tag on an unrelated cytoplasmic protein, Hst2. Strain CWY14 (ENP1-TAP) and the Hst2-TAP tagged strain were grown in YPD to mid-exponential phase before being harvested. Extracts were prepared and mixed with IgG agarose beads to precipitate Enp1-TAP and associated RNAs. Total RNAs were extracted from washed IgG beads, separated on 6% polyacrylamide denaturing gels, transferred to nylon membranes and probed with radiolabeled oligonucleotides. Northern analysis revealed that Enp1-TAP precipitates were enriched with U3 and U14 snoRNAs relative to precipitates from the Hst2-tagged strain (Fig. 7A). The Enp1-TAP samples were not enriched with snR30 snoRNA (Fig. 7A), nor with U24 or U18 snoRNAs (data not shown). We found no change in the levels of U3, U14, snR10 and snR30 snoRNAs in the enp1 mutant, indicating that the mutation did not affect snoRNAs levels (Fig. 6I). The results indicate that Enp1 associates in vivo with U3 and U14 snoRNAs. Much less U3 RNA co-immunoprecipitated with Enp1-TAP than with Protein A-tagged Nop1, which is known to associate with U3 and U14 snoRNAs (Fig. 7A). However, Protein A-Nop1 was much more efficiently precipitated than was the Enp1-TAP protein (data not shown). Therefore, it is likely that similar amounts of RNAs are associated with the two proteins and that the proteins are part of the same complex. To address this further, we checked for the presence of Nop1 in the Enp1-TAP immunoprecipitates. Figure 7B shows that Nop1 indeed is in the Enp1 precipitate but not in the Hst2 control, whereas an abundant ribosomal protein, L3, is in neither precipitate.\nU3 and U14 snoRNAs have been shown to interact with rRNA precursors and these interactions are required for pre-rRNA processing (35–37). Using an in vitro system, U3 was also shown to be associated with its rRNA substrate and processing product (38). This raised the possibility that Enp1 might also be associated with rRNAs since its function in rRNA processing appeared linked to those of U3 and U14 snoRNAs. To test this possibility, we further analyzed the RNAs that co-precipiate with TAP-tagged Enp1. The RNAs were separated on 1.2% agarose formaldehyde gels or on polyacrylamide denaturing gels, transferred to nylon membranes and probed with radiolabeled oligonucleotides for pre-rRNAs. A probe (P4; see Fig. 6) specific to 20s and its precursors revealed that pre-rRNAs 35S, 33S, 32S and 20S specifically co-precipitated with Enp1-TAP (Fig. 7C). No such enrichment was found using a probe (P8; see Fig. 6) specific for 27S RNAs or a probe for 5.8S RNA (Fig. 7C). These results clearly demonstrated that Enp1 is associated with substrates and products of the early steps of 18S rRNA processing.\n\nComparison of Enp1 with homologs in other organisms\nHomologs of Enp1 protein in human, Drosophila and Caenorhabditis elegans have been reported (18). A previously described human homolog, bystin, was only 306 amino acids in length, and lacked sequences corresponding to the N-terminal 163 amino acids of yeast Enp1 (19). In contrast, we identified a human expressed sequence tag (EST), BC007340 in a Blast search that revealed an ORF of 1311 nucleotides encoding a 437 amino acid polypeptide (39). Comparison of this 437 amino acid polypeptide with human bystin (19) showed that they were encoded by the same gene on human chromosome 6. The reported sequence for human bystin is a fragment of the 437 amino acid human Enp1 protein, due to a truncated cDNA sequence. Also, a sequence discrepancy at the C-terminus is due to inaccurate DNA sequence of the human bystin, as confirmed by available human genome and EST sequences. Searches of the database of other organisms identified Enp1 homologs in S.pombe, Arabidopsis thaliana, C.elegans, Drosophila melanogaster and mouse. The alignment of the homologs showed that they shared homology from the N-terminus to the C-terminus, with the C-terminal half extremely well conserved, with close to 90% similarity (Fig. 8). One interesting observation is that the two amino acids changed in the enp1-1 mutant, W242 and V415, are conserved among all homologs.\nThe human Enp1 homolog was cloned and expressed in yeast and tested for function. Expressed under the control of ADH, TEF or GPD promoter, the human Enp1 homolog did not complement a yeast enp1 null mutant. However, a GFP fusion to the N-terminus of human Enp1 homolog localized to the nucleus and was enriched in the nucleolus (data not shown).\n\n\nDISCUSSION\nENP1 is a yeast gene first identified in a genetic screen for complementation of mutations in ost4, which encodes a subunit of oligosaccharide transferase (17), although subsequent work showed that it is unlikely that Enp1 has any connection to oligosaccharide transferase (18). ENP1 was shown to be essential for viability and the Enp1 protein localized to the nucleus (18).\nWhen we re-examined the localization of Enp1, we observed that the protein was enriched in the nucleolus. This finding led us to test for a role of Enp1 in ribosome synthesis. Using an enp1 ts mutant, we found that depletion of Enp1 caused a 40S ribosomal subunit deficiency. Further analysis revealed that this deficiency was not due to a change of the subunit’s stability, but to a defect in the synthesis of the subunit’s 18S rRNA component. Pulse-chase analysis of RNA synthesis in an enp1-1 strain revealed that no 20S pre-rRNA or 18S rRNA was made at the non-permissive temperature, while levels of 25S rRNA appeared normal. Low levels of precursors to 18S rRNA were detected in the mutants, and they were extremely unstable. Northern analysis of steady state RNA levels demonstrated that the enp1 mutation specifically inhibited the pre-rRNA early cleavages at sites A0, A1 and A2, which are required for the production of the 20S pre-rRNA and the 18S rRNA. These defects of the enp1 mutation on rRNA processing are very similar to those caused by mutation of KRR1, another essential nucleolar gene required for synthesis of the 40S, but not the 60S subunit (40).\nCo-precipitation analyses provided strong evidence that Enp1 is directly involved in pre-rRNA processing. In these experiments, TAP-tagged Enp1 bound to IgG beads specifically co-precipitated with two snoRNAs, U3 and U14, and with the 35S, 33S, 32S and 20S pre-rRNAs. Among the more than 100 snoRNAs in yeast cells, U3, U14, snR10, snR30 and MRP RNA are the only ones required for rRNA processing. It has been shown that mutations in U3, U14, snR10 and snR30 snoRNAs and protein components of the snoRNPs affect processing at sites A0, A1 or A2 (15,16,41,42). Enp1’s interaction with U3 and U14 snoRNAs suggests that Enp1’s function in rRNA processing involves these two snoRNAs. The fact that the nucleolar protein, Nop1, known to bind to U3 and U14 RNAs, also was found in the Enp1 precipitate, provides additional evidence that Enp1 is part of a complex involved in processing of rRNA. Recently, a genome-wide study of yeast protein complexes, using a TAP tag method similar to ours, reported a number of proteins that co-immunoprecipitated with Enp1 (43). These proteins included Imp4, Kre31, Kre33, Mpp10, Nop1, Nop14 and Sof1, all of which have been implicated in 18S RNA processing or 40S biogenesis. Very recently (after the studies in this manuscript were concluded) Grandi et al. published an analysis of components of the 90s preribosomal particle, which include the 35S pre-rRNA, U3 snoRNP and rRNA processing factors for the 40S subunit (44). In agreement with our findings, they found that Enp1 is a component of this 90s complex and also of a smaller complex containing the 20S pre-rRNA. Surprisingly, none of the other proteins involved in processing of pre-rRNAs that were tested associated with the 20S rRNA, suggesting that those proteins, but not Enp1, are released prior to 20S pre-rRNA formation (44). The authors also showed co-precipitation of Enp1 with dimethylated 20S pre-rRNA, which is formed after export of 20S to the cytoplasm. These results of Grandi et al. suggest that Enp1 may also be involved in later steps of processing or nuclear export.\nA previous study on the Enp1 human homolog, bystin, reported that the protein was localized in the cytoplasm of mammalian cells and might be involved in cell adhesion (19). Our discovery of the 437 amino acid human Enp1 homolog revealed that the bystin studied previously was from a truncated library cDNA encoding only the C-terminal 306 amino acids. Although the human homolog of Enp1 did not complement a yeast Δenp1 mutant, we did show that it was localized to the nucleus and enriched in the nucleolus (data not shown). This strongly suggests that the conserved function of Enp1 is in rRNA processing. The cytoplasmic localization of bystin and its proposed function in cell adhesion are unlikely to reflect the actual function of the human Enp1 homolog.\nIt will be important to study the nature of the associations between Enp1 and U3 and U14 snoRNPs, and to learn more about the exact role of Enp1 in ribosomal RNA processing.\n\n\n" ], "offsets": [ [ 0, 30540 ] ] } ]

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[ { "id": "pmcA1239921__text", "type": "Article", "text": [ "Epigenetic inactivation and aberrant transcription of CSMD1 in squamous cell carcinoma cell lines\nAbstract\nBackground\nThe p23.2 region of human chromosome 8 is frequently deleted in several types of epithelial cancer and those deletions appear to be associated with poor prognosis. Cub and Sushi Multiple Domains 1 (CSMD1) was positionally cloned as a candidate for the 8p23 suppressor but point mutations in this gene are rare relative to the frequency of allelic loss. In an effort to identify alternative mechanisms of inactivation, we have characterized CSMD1 expression and epigenetic modifications in head and neck squamous cell carcinoma cell lines.\n\nResults\nOnly one of the 20 cell lines examined appears to express a structurally normal CSMD1 transcript. The rest express transcripts which either lack internal exons, terminate abnormally or initiate at cryptic promoters. None of these truncated transcripts is predicted to encode a functional CSMD1 protein. Cell lines that express little or no CSMD1 RNA exhibit DNA methylation of a specific region of the CpG island surrounding CSMD1's first exon.\n\nConclusion\nCorrelating methylation patterns and expression suggests that it is modification of the genomic DNA preceding the first exon that is associated with gene silencing and that methylation of CpG dinucleotides further 3' does not contribute to inactivation of the gene. Taken together, the cell line data suggest that epigenetic silencing and aberrant splicing rather than point mutations may be contributing to the reduction in CSMD1 expression in squamous cancers. These mechanisms can now serve as a focus for further analysis of primary squamous cancers.\n\n\n\nBackground\nCUB and Sushi Multiple Domains 1 (CSMD1) was cloned as a candidate tumor suppressor or progression gene from a region of human chromosome 8 deleted in tumors of the upper aerodigestive tract, prostate, ovary and bladder [1-7]. Deletion of 8p23.2 or reduced expression of CSMD1 has been associated with poor prognosis in head and neck squamous cell carcinomas and in prostate cancers [2,5,8].\nCSMD1, consisting of 70 exons spread over two megabases of 8p23.2, encodes a rare 11.5 kb transcript most abundantly expressed in the brain [1]. It is the founding member of a novel, evolutionarily highly conserved gene family whose proteins contain multiple domains thought to be sites of protein-protein or protein-ligand interactions and whose structure suggests that they may be transmembrane receptors or adhesion proteins [9,10].\nTumor suppressor genes are expected to be inactivated in cancers either genetically by mutations or epigenetically by modification of their promoters. While CSMD1 transcripts are detectable in upper aerodigestive tract epithelium, preliminary analysis of several head and neck squamous cell carcinoma cell lines suggested that CSMD1 expression was lost in these lines [1]. Although the region containing CSMD1 is frequently deleted in head and neck squamous cell carcinomas and prostatic adenocarcinomas [3,11-15], point mutations in the gene are relatively rare in primary squamous cancers [16] and in squamous cell carcinoma cell lines (Schmidt, Richter and Scholnick, unpublished). Nonsense or splice junction mutations in CSMD1 have not been reported and not enough is known about the function of the protein to accurately assess the effect of the few missense mutations that have been detected. Thus, if CSMD1 is inactivated in tumors, alternative mechanisms for gene silencing must be operating.\nIn this paper, we demonstrate that while most squamous cell carcinoma cell lines do not express full length CSMD1 transcripts, nearly all produce abnormal transcripts unlikely to encode functional CSMD1 proteins. Methylation of the DNA preceding CSMD1's first exon is correlated with reduction in the level of expression and cell lines expressing at low levels do not appear to elongate the full 11.5 kb transcript. Other anomalies of expression include incorrect splicing and the use of cryptic promoters. Our data suggest that activation of these promoters may result from the global demethylation of the genome associated with tumorigenesis (reviewed by Ehrlich [17]).\nTaken together these data demonstrate that mechanisms other than point mutation are responsible for the aberrant CSMD1 expression in head and neck squamous cell carcinoma cell lines, and these data suggest potential targets for further investigation in primary tumors.\n\nResults\nCSMD1 promoter methylation in HNSCC cell lines is correlated with expression levels\nPreliminary evidence suggested that CSMD1 expression is lost in head and neck squamous cell carcinomas [1] but that point mutations were rare [[16], and Schmidt, Richter and Scholnick, unpublished]. To date, only two of the 20 cell lines we have tested for CSMD1 expression, UPCI:SCC066 and PCI-13, express large transcripts initiated at the normal CSMD1 promoter. These data suggest that a mechanism(s) other than point mutation must be responsible for the loss of expression. CSMD1's first exon is embedded in a 3.7 kb CpG island (data from the UCSC genome browser [18]) suggesting that promoter methylation might epigenetically silence the gene. To test this hypothesis, we surveyed 32 head and neck cancer cell lines for CSMD1 promoter methylation using the Combined Bisulfite Restriction Analysis (COBRA) technique described by Xiong and Laird (Methods) [19]. COBRA analysis of the three amplicons diagrammed in Figure 1 suggested that 28 of the cell lines (87%) had more promoter methylation than did normal upper aerodigestive epithelium (data not shown).\nWe selected nine of these cell lines for high resolution analysis of promoter methylation by sequencing of clones from bisulfite converted genomic DNA. This approach has the distinct advantage of allowing determination of the state of all the CpG dinucleotides within an amplicon on an allele by allele basis. Amplicons 1 and 2 have 19 and 20 CpG dinucleotides, respectively. Amplicon 3 could not be examined by this technique because it is unclonable after bisulfite conversion. The methylation data were correlated to CSMD1 expression levels as measured by quantitative RT-PCR using an amplicon spanning exons 1 and 2 (Methods). A pool of cDNA from five normal oropharyngeal epithelium specimens served as a basis for comparison to the cell lines.\nOur data from amplicon 1 demonstrate a clear relationship between methylation and the level of expression (Figure 2). The bisulfite sequencing data confirm that there is relatively little promoter methylation in normal tissue (clones 1–20, Figure 2A). This is also the case in cell line UPCI:SCC066 which expresses a large CSMD1 transcript from the normal promoter at a level approximately 33% of that of normal tissue (clones 32–39, Figure 2B). PCI-13, our highest expressing line at 125% of normal epithelium, displays two distinct patterns of promoter methylation with some clones heavily methylated (clones 21–24, and 30) and others with no methylation (clones 25–39 and 31; Figure 2B). This pattern is consistent with either heterozygosity for methylation or the co-existence of 2 distinct populations within the cell line, one heavily methylated and one unmethylated. We cannot distinguish between these two possibilities using the currently available data.\nThe remaining cell lines express CSMD1 at a level half that of UPCI:SCC066 or less (ranging from 17% to 1% of normal epithelium) and they exhibit considerably greater methylation of amplicon 1 (clones 40–76, Figure 2C). Cell lines with more amplicon 1 methylation tend to express the gene at lower levels but the relationship is not strictly quantitative (Figure 2C).\nIn contrast, our data revealed no relationship between expression level and methylation of amplicon 2 (located towards the 3' end of exon 1, Figure 1). For example, all of the 10 clones of amplicon 2 sequenced from PCI-13 were methylated at 19 or 20 of their 20 CpG dinucleotides. UPCI:SCC066, on the other hand, has nearly no methylation in amplicon 2 with only a single methylated CpG dinucleotide detected in one clone out of the nine sequenced. Amplicon 2 ranges from completely unmethylated to heavily methylated in the seven remaining cell lines (data not shown).\n\nLow transcript levels are accompanied by a failure to elongate the full CSMD1 transcript\nOn the surface, the quantitative RT-PCR data presented in Figure 2 suggest that the cell lines we consider low expressing might still have up to 17% of the normal level of CSMD1 transcript. A survey of 20 cell lines using a battery of RT-PCR primer pairs located throughout the 11.5 kb transcript reveals that this is not the case. These lines included OKF6-TERT1, a TERT immortalized, p16 deficient but untransformed oral keratinocyte cell line [20].\nOur data suggest that the low expressing cell lines shown in Figure 2C express considerably more of the 5' end of the 11.5 kb transcript than they do exons further 3', a phenomenon well illustrated by cell line PCI-100. This line expresses the exon 1/exon 2 amplicon at approximately 15% of the level of normal epithelium. In contrast, we had previously reported that CSMD1 transcripts were not detectable in this line by combined RT-PCR and Southern blotting using three sets of intron spanning primers [1]. The most 5' of those amplicons spans exons 9 through 26.\nAnalysis with additional primer pairs resolves this apparent paradox by demonstrating that the amount of transcript declines sharply and reproducibly as one examines progressively more 3' exons (Figure 3). The same effect is seen using either oligo-dT or random hexamer primed cDNA. No transcript of this structure has been detected in normal epithelium nor have we detected any sequence alterations in PCI-100 that would explain why the full transcript is not expressed. It is not clear whether PCI-100 produces a small number of discrete size classes of transcript, if individual transcripts terminate at random points within the very large introns in this part of the gene (the first 10 introns average ~150 kb in length), or if the short transcripts result from the elevated activity of a previously undetected posttranscriptional control mechanism.\n\nInactivation of CSMD1 by aberrant splicing\nTwo cell lines, UPCI:SCC066 and PCI-13, express large CSMD1 transcripts initiated at the normal promoter. Subsequent finer scale analysis demonstrates that PCI-13's transcript lacks exons 4 and 5, resulting in a frameshift-induced nonsense codon in exon 6 (Figure 4). Sequencing of the PCI-13 RT-PCR product demonstrates the direct juxtaposition of wildtype exons 3 and 6 and that the transcript contains no novel sequences or splices that would prevent the frameshift. UPCI:SCC066 produces two transcripts, a normal one that includes exons 4 and 5 and another that lacks them (Figure 4). RT-PCR of human fetal brain cDNA reveals very low levels of an RT-PCR product corresponding in size to that expected from the internally deleted transcript. This band is not readily visible at the exposure used for Figure 4A. We have not detected a similar sized PCR product in RNA from oropharyngeal epithelium but this may reflect the fact that CSMD1 transcripts are ~10x less abundant in oropharyngeal epithelium than they are in fetal brain (data not shown).\nThe transcripts lacking exons 4 and 5 appear to result from aberrant splicing rather than somatic deletion of these two exons or mutations of their splicing consensus sequences. Exons 4 and 5 can be amplified from PCI-13 genomic DNA and sequencing of those PCR products demonstrates that both their coding sequences and consensus splice sites are wildtype.\n\nActivation of cryptic promoters in cancer cell lines\nThe RT-PCR survey revealed a second transcriptional anomaly exhibited by 4 cell lines: SCC9, 041, PCI-1 and PCI-2. Like PCI-100, these lines express low levels of the very 5' end of the transcript and even lower levels of more 3' exons within the first half of the transcript. However, these lines are distinct in expressing higher levels of the 3' half of the transcript, suggesting that alternative promoters in the middle of the gene may be used. SCC9 was chosen for further study because it expresses the 3' half of the transcript at a level dramatically higher than normal for oropharyngeal epithelium. Northern blotting detects a comparatively abundant 6.4 kb truncated transcript as well as smaller amounts of an 8.7 kb transcript in SCC9 (Figure 5A). The other three cell lines express their truncated CSMD1 transcripts at lower levels (data not shown). Sequence analysis of CSMD1 cDNA clones from SCC9 demonstrates that many transcripts are improperly spliced, resulting in retention of intronic sequences and/or deletion of exonic sequences (data not shown). In particular, retention of sequences from intron 40 is common. The high frequency of faulty splicing may explain the broadness of the 6.4 kb CSMD1 band in Figure 5A and suggests that the 8.7 kb transcript may also be incompletely or improperly spliced.\n5' RACE [21] reveals that the SCC9 message is initiated just upstream of an Alu element in intron 36 (Figure 5B). Only the 5'-most 120 base pairs of the Alu element are present in the genome. RT-PCR using a forward primer specific for the novel sequences of the SCC9 transcript (prm1904, cgtttagttcgacacacttcatgt) demonstrates that cell lines 041, PCI-1 and PCI-2 do not initiate their CSMD1 transcripts at the same point, suggesting that other cryptic promoters are active in these lines. The sequence of this novel exon has been entered in Genbank as accession number DQ093422.\n\nDNA methyltransferase inhibitors activate the same cryptic promoter used in cell line SCC9\nExpression from epigenetically silenced promoters can sometimes be restored by treatment with inhibitors of DNA methyltransferase or histone deacetylase activity ([22]). We selected two low expressing cell lines with promoter methylation, UPCI:SCC104 and 094, for treatment with various concentrations of 5-azacytidine or 5-aza-2'-deoxycytidine (5-aza-dC) as well as combinations of either of those drugs with the histone deacetylase inhibitor trichostatin A. These treatments did not reactivate the silenced CSMD1 promoter. COBRA analysis of genomic DNA from the treated cells suggested that the drugs did not robustly affect methylation of the CSMD1 promoter even at levels high enough to be toxic to the cells. These experiments did however shed light on the cryptic promoter used in SCC9 cells and on the interpretation of experiments using methyltransferase inhibitors.\nTreatment of cell line 094 with relatively high doses of 5aza-dC results in the expression of the 3' end of the CSMD1 transcript. This transcript was not detected in control 094 cells undergoing mock drug treatment (Figure 6) nor was it detected in 094 cells growing under normal culture conditions (data not shown). RT-PCR of cDNA from drug-treated 094 cells using the primer developed from the novel 5' exon of SCC9's truncated transcript (prm1904, see above) yielded a product identical in size to that amplified from SCC9 cDNA (Figure 6). The identity of the product was confirmed by DNA sequencing which also revealed that the drug-induced 094 transcript was more faithfully spliced than the transcript expressed in SCC9 (data not shown).\n\n\nDiscussion\nOur data clearly demonstrate that expression of normal CSMD1 transcripts is rare in head and neck squamous cell carcinoma cell lines. Of the HNSCC cell lines examined, only UPCI:SCC066 appears to express a normal transcript from the expected promoter. Even that cell line produces a second species of aberrantly spliced transcript lacking internal exons. Our data suggest that epigenetic modification of the DNA 5' of the transcription start site may contribute to the down-regulation of CSMD1. In addition, a low level of expression appears to be associated with production of prematurely terminated transcripts. This degree of complexity might be expected from a 2 megabase, 70 exon gene.\nMethylation of a specific region of the CpG island, -395 to -112 bp relative to the transcriptional start site (amplicon 1), appears to be correlated with the activity of the normal CSMD1 promoter. In contrast, methylation of amplicon 2, located within the first exon, shows no such relationship. Our data suggest that the relationship between the amount of methylation in amplicon 1 and the level of expression may not be strictly quantitative. Differences between cell lines with amplicon 1 methylation could arise through a number of mechanisms, for example, variations in the levels of transcription factors between cell lines. In cases where there is considerable heterogeneity in the methylation pattern within a cell line like PCI-100, alleles with less methylation may be expressed at higher levels than those more heavily methylated (compare clones 51 and 52 to clone 58 in Figure 2C). Alternatively, the presence of methylation in amplicon 1 could be a qualitative but not strictly quantitative indicator of methylation of a critical segment of the promoter not discovered in this study.\nThe normal CSMD1 promoter was not reactivated by drugs that inhibit DNA methyltransferases and histone deacetylases, nor did the drugs abolish CSMD1 promoter methylation, even at toxic doses. Not all genes with promoter methylation respond to such treatments [23]. These drug treatments did, however, provide a potential explanation for the use of a normally cryptic promoter by cell line SCC9. The CSMD1 transcript in this line is initiated near a partial Alu element. 5-aza-dC treatment of cell line 094 activates the same cryptic promoter. This suggests that cryptic promoters may be naturally activated by general hypomethylation of the genome in cancer cells and the subsequent release of repetitive elements from epigenetic repression (reviewed by Ehrlich [17]). The SCC9 transcript does not appear to encode a functional protein but, with a very large gene like CSMD1, there is a potential for some abnormally initiated transcripts to encode truncated proteins with dominant negative properties.\nThe second ramification of this finding is for the interpretation of data obtained by treating cells with methyltransferase inhibitors. Detection of CSMD1 transcripts solely with primers mapping to the 3' end of the gene could have been erroneously interpreted as representing reactivation of the normal promoter. It seems imperative that such experiments demonstrate that transcripts detected after drug treatment are actually initiated at the normal promoter.\nAberrant splicing also appears to play a role in the production of defective CSMD1 transcripts. Loss of splicing fidelity has been proposed as a characteristic of cancer cells [24,25] and this would be consistent with the variety of misspliced transcripts we detected from SCC9. However, the removal of exons 4 and 5 from the CSMD1 transcript in PCI-13 may reflect a more specific phenomenon than a general inability to splice large introns; this line is still capable of splicing large introns as evidenced by its successful splicing of exon 3 to exon 6, eliminating an intron of over 666 kb. The failure to include exons 4 and 5 may be due to inactivation of a splicing enhancer in intron 3, or to less efficient splicing due to the fact that exons 4 and 5 do not begin with the consensus G residue (Figure 4B) [26].\n\nConclusion\nTaken together, our data suggest that CSMD1 function is lost in head and neck squamous cell carcinoma cell lines through a variety of mechanisms other than point mutagenesis. Epigenetic modifications of amplicon 1 and defective splicing appear to be fruitful areas to explore in primary head and neck squamous cancers.\n\nMethods\nCell lines and tissue samples\nDNA from HNSCC cell lines UMSCC9, UMSCC35, UMSCC37, UMSCC38, UMSCC45, UMSCC49, UMSCC65, UMSCC68, and UMSCC76 was provided by Dr. Thomas Carey, University of Michigan [27]. Dr. Ruud Brakenhoff, Vrije Universitat, provided cell lines 040, 041, and 094 [28]; Dr. Theresa Whiteside, University of Pittsburgh, provided cell lines PCI-1, PCI-2, PCI-4B, PCI-13, PCI-30, PCI-50, PCI-51, PCI-52, PCI-100 [29], SCC4, SCC9 [30,31], and UPCI:SCC068, UPCI:SCC74, UPCI:SCC104, UPCI:SCC182, UPCI:SCC203, and UPCI:SCC220 [developed by Dr. Susanne Gollin, University of Pittsburgh, 32]. Dr. Gollin provided cell lines UPCI:SCC056, UPCI:SCC066, and UPCI:SCC114 [14,16,32]. The immortal but untransformed keratinocyte line OKF6-TERT1 was obtained from Dr. James Rheinwald, Harvard University [20].\nNormal oropharyngeal epithelium was isolated from discarded tissue from uvulopalatopharyngoplasties (UPPP) collected anonymously with the approval of the Washington University Human Studies Committee.\n\nCell Culture and Tissue Preparation\nSquamous cell carcinoma cell lines were grown in DMEM or DMEM:F-12, 1:1 Mixture (BioWhittaker) containing 10% fetal bovine serum (Sigma). DMEM medium was supplemented with 1X MEM Nonessential Amino Acids (BioWhittaker). Upper aerodigestive tract epithelium was separated from the rest of the UPPP specimen by digestion with Dispase II (Roche) using a protocol adapted from Oda and Watson [33].\n\nNucleic acid preparation and bisulfite conversion\nGenomic DNA was isolated by using either Nucleospin Tissue kits (Clontech), QIAamp DNA Blood Mini kits (Qiagen) or Trizol (Invitrogen) according to the manufacturers' instructions. Total RNA isolation, synthesis of first strand cDNA, RT-PCR and 5' RACE PCR were performed essentially as previously described [1]. Poly-A+ RNA for Northern blotting was selected from total RNA using Oligotex beads (Qiagen). Northern blotting and hybridization were performed as previously described [1]. cDNA synthesis was primed using oligo dT or random primers and extended by either Thermoscript or Superscript III reverse transcriptase (Invitrogen). PCR was run in Perkin-Elmer 480 or Applied Biosystems 9700 thermal cyclers for 35 cycles unless otherwise noted. Images of ethidium bromide stained gels were captured with a Gel-Doc imaging station (Biorad). Quantitative PCR was run in an Applied Biosystems 5700 thermal cycler using SYBR Green Master Mix (Applied Biosystems). Primers prm2426 (gtgtggagtatctgcagacatga) and prm2427 (ctggactaagcctccacagttct) were used to amplify a 132 base segment spanning CSMD1's first and second exons. An amplicon from human 18S RNA was used as a basis for comparisons across cell lines (primers prm2396, ttcggaactgaggccatgat and prm2397, tttcgctctggtccgtcttg). Calculations were performed using the ΔΔCt method in GeneAmp 5700 SDS software (version 1.3) and Microsoft Excel. Quantitation was based on the average values obtained from duplicate reactions. The level of CSMD1 expression in normal oropharyngeal epithelium was determined from pooled cDNA from five UPPP specimens.\nWe used the CpGenome DNA Modification kit (Intergen) for bisulfite conversion of the genomic DNA according to the manufacturer's protocol, with the following exception. Incubation of the conversion reaction was carried out in a thermal cycler for six cycles each consisting of three minutes at 94°C followed by three hours at 50°C (Christina Menke and Paul Goodfellow, personal communication).\n\nAnalysis of CSMD1 Promoter Methylation\nMethylation of three segments of the CSMD1 CpG island was examined using the Combined Bisulfite Restriction Analysis technique (COBRA) [19]. All three segments were amplified by using the nested primers and PCR conditions listed in Table 1. Amplicon 1 extends from -395 to -112 bp, amplicon 2 from +175 to +396 bp, and amplicon 3 from +398 to +718 bp relative to the first base of the transcript. A small region surrounding the transcription start site (-111 to +174 bp) could not be examined because no PCR primers could be designed from its extremely GC-rich sequence. The first round PCR used 2 μl of bisulfite converted genomic DNA in a final volume of 10 μl. Subsequent amplifications with nested primers used 4 μl of first round product as template in reactions with a final volume of 20 μl. All PCR was carried out for 35 cycles. A portion of the second round PCR product was run on a 1.5% agarose gel, stained with ethidium bromide, and quantified using the ImageQuant software package (v1.2 for Macintosh, Molecular Dynamics) so that equal amounts of each could be used in restriction digests.\nRestriction digests for COBRA were performed with either BstU I or Taqα I (5 or 10 units per reaction, respectively; New England Biolabs) for 4 hours in a final volume of 10 μl. Taqα I digests were performed only when no methylation was detected with BstU I. BstU I digests also included an internal control DNA fragment to confirm complete digestion. This DNA fragment contains a single BstU I site and was amplified from a cloned CSMD1 cDNA using primers prm2020 (agatcccccagtgtctccctgtgt) and prm2021 (actgctggtgccgtggtaatgact). The control PCR product is 1019 bp long and is digested to two fragments of 605 and 414 bp by BstU I. Digestion products were fractionated on a 10% polyacrylamide gel, stained with ethidium bromide, and visualized with a Gel-Doc video imaging workstation (Bio-Rad).\nHigh resolution analysis of methylation was performed by sequence analysis of individual clones from amplicons 1 and 2. DNA from amplicon 3 proved unclonable and gel electrophoresis suggests that its very AT rich sequence results in a bent DNA configuration. PCR products were purified using the Nucleospin Extraction columns (Clontech) and inserted into the pCR2.1-TOPO vector using the TOPO TA Cloning kit (Invitrogen) according to the manufacturer's instructions. Plasmid DNA from individual colonies was isolated using the Nucleospin Plus Plasmid Miniprep kit (Clontech) and sequenced with a reverse vector primer (agcggataacaatttcacacagga) using fluorescence based sequencing with Big Dye Terminator mix (Applied Biosystems).\n\nTreatment of cultured cells with DNA methyltransferase inhibitors\nCell line 094 was treated with 5-aza-2'-deoxycytidine (5aza-dC) (Sigma) dissolved in DMSO. Two 100 mm cell culture dishes containing 5 × 105 cells were established for each of the drug concentrations tested. Cells were grown for 72 hours in media containing DMEM:F-12, 1:1 Mixture (BioWhittaker) with 1X MEM Nonessential Amino Acids (BioWhittaker) and 10% fetal bovine serum and then switched to media containing 5aza-dC at concentrations of 0 μm, 5 μM, 25 μM, or 100 μM. Cells were fed daily for 4–5 days and then both plates were harvested in 3 ml of Trizol (Invitrogen) for isolation of RNA and DNA according to the manufacturer's instructions. RT-PCR used for detection of CSMD1 transcripts in these treated cells was run for 40 cycles.\n\n\nAbbreviations\n5aza-dC = 5-aza-2'deoxycytidine, COBRA = Combined Bisulfite Restriction Analysis, CSMD1 = Cub and sushi multiple domains 1, HNSCC = head & neck squamous cell carcinoma, RT-PCR = reverse transcription – polymerase chain reaction, SCC = squamous cell carcinoma, UPPP = uvulopalatopharyngoplasty.\n\nCompeting interests\nThe author(s) declare that they have no competing interests\n\nAuthors' contributions\nTMR performed the DNA methylation analysis, and parts of the transcript survey. BDT cloned and characterized the novel first exon expressed in cell line SCC9. SBS performed parts of the transcript survey and characterized the deletion of exons 4 and 5 in PCI-13. All three authors participated in the analysis of the data and in the writing of the manuscript.\n\n\n" ], "offsets": [ [ 0, 27525 ] ] } ]

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[ { "id": "pmcA1859974__text", "type": "Article", "text": [ "Human growth hormone (GH1) gene polymorphism map in a normal-statured adult population\nAbstract\nObjective\nGH1 gene presents a complex map of single nucleotide polymorphisms (SNPs) in the entire promoter, coding and noncoding regions. The aim of the study was to establish the complete map of GH1 gene SNPs in our control normal population and to analyse its association with adult height.\n\nDesign, subjects and measurements\nA systematic GH1 gene analysis was designed in a control population of 307 adults of both sexes with height normally distributed within normal range for the same population: −2 standard deviation scores (SDS) to +2 SDS. An analysis was performed on individual and combined genotype associations with adult height.\n\nResults\nTwenty-five SNPs presented a frequency over 1%: 11 in the promoter (P1 to P11), three in the 5′UTR region (P12 to P14), one in exon 1 (P15), three in intron 1 (P16 to P18), two in intron 2 (P19 and P20), two in exon 4 (P21 and P22) and three in intron 4 (P23 to P25). Twenty-nine additional changes with frequencies under 1% were found in 29 subjects. P8, P19, P20 and P25 had not been previously described. P6, P12, P17 and P25 accounted for 6·2% of the variation in adult height (P = 0·0007) in this population with genotypes A/G at P6, G/G at P6 and A/G at P12 decreasing height SDS (−0·063 ± 0·031, −0·693 ± 0·350 and −0·489 ± 0·265, Mean ± SE) and genotypes A/T at P17 and T/G at P25 increasing height SDS (+1·094 ± 0·456 and +1·184 ± 0·432).\n\nConclusions\nThis study established the GH1 gene sequence variation map in a normal adult height control population confirming the high density of SNPs in a relatively small gene. Our study shows that the more frequent SNPs did not significantly contribute to height determination, while only one promoter and two intronic SNPs contributed significantly to it. Studies in larger populations will have to confirm the associations and in vitro functional studies will elucidate the mechanisms involved. Systematic GH1 gene analysis in patients with growth delay and suspected GH deficiency/insufficiency will clarify whether different SNP frequencies and/or the presence of different sequence changes may be associated with phenotypes in them.\n\n\n\nIntroduction\nHuman skeletal growth and final height attainment are a result of a multifactorial regulation involving systemic and local hormones, growth and nutritional factors, lifestyle and genetic factors. Heritability estimates1 and genome-wide linkage analysis2 have shown that genetic factors play a major role in determining stature. Among these factors, the GH-IGF-I axis plays an important role during postnatal life, and associations between structural variations in its genes and height are currently under study.3 Although growth hormone (GH) deficiency is a well-known cause of growth retardation, which responds to GH replacement therapy, the diagnosis and physiopathological mechanisms for the so-called ‘idiopathic isolated GH deficiency’ (IIGHD) require further clarification. In addition, GH secretion levels and markers of GH biological activity have been demonstrated to be specific and sensitive only in major deficiency states.4,5 Genetic causes of GH deficiency within the GH1 gene have been established; however, they are rarely recognized and only sought in major GH deficiency states during childhood and in family studies.3 GH1 gene, located at 17q22–24, is a component of the GH gene cluster in which five genes evolving from a common ancestor are 91–99% sequence conserved (paralogues).6 GH1 is more abundantly expressed in pituitary cells, while the other four genes are expressed in placental tissue. Large deletions within the GH1 gene cluster were described first followed by point mutations, the majority of which affect introns 3 or 4, provoke skipping of exon 3 product and exert a dominant effect.3,7,8 More recently, the presence of single nucleotide polymorphic points (SNPs) in the promoter region or in intron 4 of the GH1 gene have been described9–12 and associations with promoter allele activities or with GH secretion efficacy and circulating IGF-I levels in growth-retarded patients have also been described.11,12 Other studies have analysed several GH1 gene SNP genotypes as related to the incidence of neoplasia, with a positive association with colorectal neoplasia for intron 4 SNP,13 a negative result for breast carcinoma14,15 or a positive one for breast cancer risk.16,17 In addition, a recent study in a cohort of adults over ages 60 years detected a significant association between genotypes at one SNP in the GH1 gene promoter region and at the intron 4 SNP described by Hasegawa et al.11 with baseline bone density and accelerated bone loss together with an interaction with weight at 1 year.18 Intron 4 SNP described by Hasegawa et al.11 has also been associated, in women, with shorter body height and reduced mortality,19 whereas another intron 4 SNP (T1169A) has been associated in both sexes with a favourable metabolic profile.20 A systematic SNP study was conducted by Adkins et al.21 in GH1 promoter, coding and noncoding regions in DNAs from placental tissues, and analysis of associations between genotypes and birth weight revealed an association between an alternate nucleotide at −1 and +3 of translation initiation site and fetal growth restriction. However, no systematic GH1 gene analysis in the entire promoter, coding and noncoding regions has been conducted in adults to establish the map of structural variation and its possible association with height. The relatively short size of the entire gene permits a complete analysis which is, nevertheless, hampered by the need to avoid amplification of any other of the GH cluster genes (paralogues) and the high density of sequence variations.\nTo obtain normative data for subsequent analysis of GH1 gene contribution to IIGHD in children, a systematic GH1 gene structural analysis was designed in a normal adult control population to establish the GH1 gene SNP map in adults from our population with heights within the normal range, determine the genotype frequencies and analyse possible associations between individual and combined SNPs with height.\n\nSubjects and methods\nSubjects\nA total of 307 adult subjects of both sexes (164 women and 143 men) were recruited from hospital personnel and parents of patients with no history of growth retardation. Subjects had to fulfil the following criteria: Iberian Peninsular (except Basque) family origin and no family history of pathological short stature. A single subject per family was included. The protocol was approved by the Hospital Vall d’Hebron Ethics Committee and written informed consent was obtained from each participant. Height standard deviation scores (height SDS) were calculated according to sex-specific reference growth charts for the Spanish population (Carrascosa et al.22 charts were used for subjects under ages 30 years and Hernández et al.23 for subjects aged 30–50 years). Only individuals with height SDS between −2 and +2 SDS were included in the study (mean −0·016; 32 women and 28 men between −2·000 and −1·010; 99 women and 80 men between −1·000 and +0·910; 33 women and 35 men between +1·010 and +1·980) and sample size was adjusted for normal sex and height SDS distribution. Height and weight were recorded in the morning by a single observer. Height was measured with a Harpenden stadiometer. Four millilitres of peripheral venous blood were drawn into EDTA-containing tubes for molecular genetic analysis.\n\nGenomic DNA study\nGenomic DNA was obtained from peripheral blood following the method described by Lahiri and Nurnberger.24 DNA was amplified by polymerase chain reaction (PCR) using a nested strategy. Briefly, 50 ng of genomic DNA were added to a 10 µl reaction mixture of 1 mm Mg(OAc)2, 0·6 mm dNTPs, 0·3 µm of each primer, and 0·4 U r Tth DNA polymerase XL (Applied Biosystems, Foster City, CA, USA). The sense and antisense primers used corresponded to nucleotides 4156–5′ACGGTCCGCCACTACGCCCAGC-3′ and the complement of 6948–5′TGCAGTGAGCCAAGATTGTGCC-3′ of the GH gene cluster.6 The PCR reaction mix was denatured for 5 min at 94 °C and cycled 40 times (94 °C, 1 min; 72 °C, 3 min 30 s) followed by a 7-min extension at 72 °C. The resulting GH1 PCR products (2893 bp) were used as templates for five nested reactions (AN, BL, CK, DI, FP), carried out as follows: 1 µl of each GH1 PCR product was added to a 20 µl reaction mixture of 1·5 mm MgCl2, 0·2 mm dNTPs, 0·3 µm of each primer and 0·4 U Eco Taq DNA polymerase (Ecogen S.R.L., Barcelona, Spain). Reaction mixtures were denatured for 5 min at 94 °C, cycled 40 times (94 °C, 1 min; 58 °C, 1 min; and 72 °C, 1 min), followed by a 7-min extension at 72 °C. Sense and antisense primers were as follows:\nSequencing from both ends was performed by the dideoxy method using ABI PRISM BigDye Terminator version 3·1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). GH1 gene nucleotide sequence published by Chen et al.6 was used as control. For each DNA, the five segments from the nested PCR were assembled with the SeqEscape programme (Applied Biosystems) and interpretation was made visually and simultaneously by two observers. Antisense sequencing was performed to confirm each nucleotide sequence change up to the establishment of the more frequent SNP map (frequency over 1%), whereas less frequent single or multiple nucleotide changes were reconfirmed in each DNA by antisense sequence and resequencing after a new nested PCR from original DNA was performed.\n\nSingle nucleotide polymorphism (SNP) genotyping\nThe sequences for the five genes of the GH cluster identified by Chen et al.6 and reported as the GI sequence 183148 were aligned using the Multalin program.25 SNPs and other sequence changes identified were indicated using their position corresponding to GH1. Genotypes were deduced by the combination of genetic variation at the polymorphic positions.\n\nStatistical analysis\nStandardized height was investigated for normal distribution (Kolmogorov-Smirnov test: c2 = 2·882, P = 0·4733). Hardy–Weinberg equilibrium was tested for SNPs presenting three alternate genotypes according to standard procedures using χ2-analysis. anova test was applied to investigate individual and combined SNP association with adult height SDS; significance assessment was adjusted for multiple testing using Fisher's PLSD test setting Pcritical = 0·05 or the Bonferroni–Dunn test setting Pcritical = 0·05/n (n = number of comparisons carried out). Stepwise regression analysis was applied to predict the contribution of SNPs to adult height SDS. Statview 4·5 program (Abacus Concepts Inc., Berkeley, CA, USA) was used for statistical analyses.\n\n\nResults\nGH1 gene sequence variation\nGH1 gene sequence comparison with the GI-183148 sequence published by Chen et al.6 yielded a total of 54 single or multiple nucleotide changes. Twenty-five SNPs presented a frequency over 1% (genotypes and frequencies are listed in Table 1). SNPs which presented the three alternate genotypes (P2 to P4, P6, P7, P10 and P24) were in Hardy–Weinberg equilibrium (data not shown). Twenty-nine additional changes were found with a frequency under 1% or involving more than one nucleotide and thus could be considered as rare variant SNPs (R1 to R29) (Table 2). These changes were found in 29 of 307 subjects (9·4%), all in heterozygosity.\n\nGH1-paralogue alignment\nA sequence alignment was performed to study possible sequence recombinations among paralogues of the five GH1-gene cluster (Fig. 1). This alignment showed that 9 of 25 SNPs (36%) in the GH1 gene did not correspond to any of the paralogues.\nAmong the 29 rare SNPs found, six (20·7%) did not correspond to any of the paralogues: two were located in the 5′UTR region (R9 and R10), two in intron 1 (positions 5300 = R14 and 5302 = R17), one in intron 2 (position 5679 = R21) and one in intron 4 (position 6344 = R23) (Table 2).\n\nEquivalence with previously reported GH1 changes\nEquivalence with changes and SNPs previously reported by other authors are shown in Table 3. The majority found a high density of SNPs in the promoter and 5′UTR regions in control populations.10,12,21 Several sequence changes have been reported in patients with familial or idiopathic short stature,11,26,27 whereas P8, P19, P20 and P25 (at positions 5165, 5681, 5686 and 6358, respectively, in the Genebank accession GI 183148) located in the promoter, intron 2 and intron 4 regions, respectively, had not been previously described.\n\nGH1 genotypes and associations with height SDS\nAssociations between genotypes and standardized height were first studied in the subpopulation of 278 controls carrying only the 25 most frequent SNPs in the GH1 gene (c2 = 2·59; P = 0·5458 for normality of height distribution).\nThree individual SNPs showed a statistically significant association with height SDS: at positions 5286 (P16), 5290 (P17) and 6358 (P25). Subjects with heterozygous genotypes presented statistically significant taller stature than the corresponding homozygous genotypes (P = 0·016 for P16, P = 0·015 for P17 and P = 0·023 for P25) (Fig. 2a,b,c). P16 and P17 were in linkage disequilibrium (LD) (r2 = 0·831), while P25 was carried by six subjects homozygous at P16 and P17.\nGH1 gene genotypes were defined by genetic variation in the 25 polymorphic positions. We found 163 different combinations. Only two genotypes presented a frequency over 5% (Table 4). Height SDS in the two more frequent genotypes did not differ significantly and covered the whole height range. Genotype 1 presented four heterozygous variations and Genotype 2 was the corresponding homozygous genotype. Heterozygous positions corresponded to SNPs with the highest frequency variation (4886 (P4), 5107 (P7), 5157 (P10) and 6331 (P24)). In addition, DNAs exhibited a different genotype in each of 129 subjects (46·4%).\nThe 11 SNPs found in the promoter region (Table 1) were grouped in 94 genotypes and analysed for association with adult height SDS. The four more frequent combinations are listed in Table 4: height SDS of these four genotypes did not differ statistically although Genotype 3 tended to have a shorter height. Genotype 3 differed from Genotype 1 in the SNP located at position 5089 (P6), corresponding to Pit 1 proximal responsive element for GH1 gene promoter. Genotype 4 is heterozygous at positions 4856 (P2), 4863 (P3) and 5107 (P7). In addition, 19% of cases exhibited a genotype in the promoter region carried by only one subject.\nCombination of the three SNPs in the 5′UTR region of GH1 gene resulted in five different genotypes. SNPs at positions 5178 (P12) and 5187 (P13) were in LD (r2 = 0·88). Mean height SDS comparison among these genotypes was not statistically significant, although mean height SDS of alternate nucleotide carriers at position 5178 (P12) tended to be shorter (Table 4).\nAn anova analysis was conducted to investigate the interaction between two or more SNPs and height SDS. SNPs at positions 5286 (P16) and 5290 (P17) were in LD (r2 = 0·83): the heterozygous genotype AG/AT for these SNPs was associated with taller stature (shown above). SNP at position 6358 (P25) increased the expected height SDS for individual carriers of the G allele at 5089 (P6) SNP as shown in Fig. 2(d): subjects heterozygous at 6358 (P25) were taller than the mean, and mean height SDS of subjects with GG/TG combined genotype was significantly higher than the corresponding GG/TT genotype (P = 0·0021), suggesting an interaction between these two SNPs as they were not in LD.\nAnalysis of height SDS association with the most frequent single and combined SNPs and with rare variant SNPs was performed in the 29 individuals carrying the rare SNPs (Table 2). None of them carried any of the three SNPs (P16, P17 and P25) related to taller stature in the population of 278 controls with only the frequent SNPs. In these 29, mean height SDS (0·000 ± 0·987, from −1·930 to +1·870) did not differ from that of the 278 controls (−0·018 ± 1·041, from −2·000 to +1·980). Analysis of associations between individual SNP genotypes and height SDS revealed that SNPs at positions 5089 (P6), 5178 (P12) and 5187 (P13) were associated with significantly shorter stature (Fig. 2e). Only two sequence changes considered as rare SNPs were carried by individuals in the lower normal height range (between −1·500 and −2·000 SDS) (Table 2): R4 (4979 C > T) in the promoter region and R14 (5300 C > T) in intron 1. Predicted single amino acid changes located in exon 5 (R25 to R27) were not associated with short stature.\nIn the entire population of 307 controls, stepwise regression analysis between height SDS and genotypes at the 25 SNPs showed that genotypes at 5089 (P6), 5178 (P12), 5290 (P17) and 6358 (P25) were significantly correlated with height SDS (r2 = 0·062, P = 0·0007) with genotypes A/G at P6, G/G at P6 and A/G at P12 decreasing height SDS (−0·063 ± 0·031, −0·693 ± 0·350 and −0·489 ± 0·265, respectively, Mean ± SE) and genotypes A/T at P17 and T/G at P25 increasing height SDS (+ 1·094 ± 0·456 and +1·184 ± 0·432, respectively).\n\n\nDiscussion\nGenetic variations within human GH1 gene have been described by several authors.9–12,21 The populations described to date comprised small numbers of normal-stature individuals,9 male adults with narrow height range12 or growth-retarded patients with/without GHD before achievement of adult height.9,11,12 Our study was designed to characterize the GH1 gene sequence variation in individuals within the whole range of normal adult height (between −2 and +2 SDS) according to the standards for our population. Height was normally distributed, both sexes were equally represented and the GI-183148 homozygous sequence6 was used for comparison. A nested PCR with specific primers for GH1 gene was designed, thus avoiding amplification of any other GH gene paralogue of the GH gene cluster.\nOur results establish a map of 25 SNPs as present in over 1% of individuals, whereas 29 other sequence changes (single or multiple nucleotide) are present in less than 1% of subjects. More than 50% (n = 14) of SNPs are located in the promoter and 5′UTR regions, thus confirming previous reports: Giordano et al.9 reported eight SNPs in the promoter and 5′UTR regions, Wagner et al.10 16 SNPs from the promoter to intron 1 and Horan et al.12 identified 36 haplotypes in control subjects of the British population, which would result from the combination of 15 of the previously reported SNPs. Our results confirm the presence of 13 of those points; SNP at 5165 (R11 in the present study and +3 in references9,12) was present in less than 1% of subjects and a new SNP is described (P8 at 5116 in the VDR/RA/T3 responsive element sequence). The remaining SNPs (from P15 to P25, n = 11) are distributed in introns 1, 2 and 4, and among coding regions only exon 1 and exon 4 bear a total of three SNPs, two of which predict an amino acid change (P15 and P21). These two latter SNPs had been described by Millar et al.26 and the more frequent SNP in intron 4 (P24) has been described by Hasegawa et al.11 together with the two more frequent SNPs in the promoter region (P4 and P7 in our map). Three new SNPs are described (P19 and P20 in intron 2 and P25 in intron 4), all outside the splice sites. Only SNPs presenting high frequency are present in homozygous alternate state and this accounts mostly for the majority of the promoter and 5′UTR SNPs and in the intron 4 more frequent SNP (P24) described by Hasegawa et al.11 In conclusion, in the entire coding and noncoding GH1 gene sequence, only P24 is present in homozygous alternate state. Our results show that the GI 183148 homozygous sequence is present in our population except for SNP P14 in the 5′UTR region which is only present as the alternate nucleotide in homozygous or heterozygous states.\nAs described by several authors9,10,12,21 several promoter SNPs affected functional sequences and P6 is located in the Pit 1 proximal responsive element, P7 and P8 in the VDR/RA/T3 responsive element and P9 (G del) in the TATA box.\nThe mechanisms by which the high density of SNPs in the GH1 gene is generated has been proposed to be recombination and gene conversion with any other(s) of the GH cluster genes.9,12,28 Alignment of the 25 SNPs with the other GH1 gene paralogues demonstrated in our results that this mechanism is possible for 64% of SNPs. Familial SNP transmission pattern analysis will be of interest to support the hypothesis of GH gene recombination.\nIn addition, 29 of 307 individuals (9·4%) bore additional GH1 sequence changes with frequencies under 1%. As for SNPs, they are located along the whole gene with higher density in the promoter and 5′UTR regions. Interestingly, intron 3 and exon 5 present several of these less frequent changes. Intron 3 has been shown to carry the majority of single nucleotide mutations causing the dominant form of GH deficiency.3 The two single nucleotide changes detected in intron 3 (R15 at 6056 and R16 at 6061) are in perfect LD (r2 = 1·0) and located within the enhancer splice site element (ESE) described by Ryther et al.29,30 Studies in additional normal or growth-retarded populations will permit description of their possible clinical implications. Five single nucleotide changes are located in exon 5; of these five, three predict an amino acid change, and one of the three (Ile179Met) has been described by Lewis et al.27 in a paediatric patient with familial short stature and the other two, as yet undescribed, are contiguous in a single individual (Pro133Hys and Arg134Leu). Polynucleotide changes are mostly located in the promoter region corresponding to the VDR/RA/T3 response element. As for frequent SNPs, the majority of the sequence changes with frequencies under 1% may have been generated by recombination within the GH gene cluster as 19 of 24 (79%) may correspond to one or more of the GH1 gene paralogues.\nOur results now show the diversity and complexity of SNP genotypes, as previously highlighted by other authors9,10,12,21 in a normal adult height control population. Our initial aim when designing the present study was to establish the map of GH1 gene SNPs in our adult control population with heights normally distributed within the entire normal range for further comparison with genotypes in our paediatric population with growth delay, variable response to GH secretion tests and adequate response to GH therapy. Analysis of SNP association with adult height was subsequently performed to establish a body of knowledge useful for comparing patient genotypes and phenotypes. This analysis was first performed in controls bearing only frequent SNPs (90·5% of the total population). We demonstrate that the four SNPs with the highest allelic variation frequencies (P4, P7, P10 and P24) do not significantly contribute to adult height determination, with the heterozygous genotype being the most frequent followed by the corresponding homozygous genotype in the whole sequence, and heights are normally distributed over the entire height range. The third most frequent combined genotype in the promoter region in our population presented, in addition, in heterozygosity, the SNP at P6 in the sequence regulated by Pit 1 and although mean height of individuals (6·1%) bearing this genotype was around −0·5 SDS, this was not statistically significant.\nAnalysis of single SNP genotype association with adult height yielded few clues as to the contribution of GH1 gene variation to adult height determination. Only three SNPs (P16, P17 and P25), present with low frequency and only in heterozygous state, were individually significantly associated with taller stature and none was individually associated with shorter stature. P16 and P17 (in LD, r2 = 0·83) are located in intron 1 and P25 in intron 4. The resulting sequence for the presence of P16 and P17 corresponded to the paralogue GH2 and generated a responsive element for a core-binding protein (Matinspector Programme, Geometrix Software GmbH, München, Germany) with three Kruppel-type zinc fingers which could increase the efficacy of GH1 gene transcription;31,32 moreover, Kruppel-like proteins have recently been described in the brain.33 Stepwise regression analysis demonstrated that P17 and P25 contribute, separately, to an increase of almost 1·0 height SDS. P16 and P17 had been described by Adkins et al.21 although they found no association with fetal growth, whereas P25 had not previously been described. The mechanisms by which they may determine taller final height should be established by in vitro studies analysing GH1 gene transcription and GH protein translation efficiencies.\nAnalysis of interaction effect between SNPs detected that variation at P25 masked an effect of P6. Individuals homozygous at P25 (TT) present a significant association between P6 genotype and height with the homozygous alternate genotype at P6 (GG) being associated with shorter stature. This was further confirmed in the subpopulation of 29 individuals bearing rare SNPs who, in the absence of heterozygous change at P25, presented significantly shorter stature in the heterozygous alternate nucleotide change at P6 (AG). P6, located at Pit 1 proximal responsive element of the GH1 gene promoter, was first described by Wagner et al.10 and Giordano et al.9 and further by Horan et al.12 Six of nine GH1 gene promoter haplotypes bearing the alternate G at P6 presented lower transcriptional activities and electrophoretic mobility shift assays (EMSA) detected differential protein binding strength, although in vitro studies were unable to identify this SNP as a major determinant of GH1 gene expression level.12 A recent study from Giordano et al.34 has shown a twofold reduced luciferase activity for the G nucleotide bearing promoter haplotype in transfected rat pituitary cells. Genotypes at P6 had also been associated with decreased breast cancer risk through its association with lower GH secretion and IGF-I circulating levels.16,17\nIn our results, GH1 gene polymorphic structural variation accounted for only 6·2% of adult height determination in the entire adult population studied and genome-wide linkage analysis of stature in multiple populations revealed no linkage with chromosome 17 GH gene cluster.2,35 As only some of the less frequent SNPs are statistically associated with height, and in view of the high density of SNPs, our study may be hampered by selection bias36 and would ideally have required a wider sampling of some 2 000 individuals; however, this was a highly laborious strategy when the complete sequencing technique is applied. The high density of SNPs and their proximity hamper other genotyping strategies for rapid determination of the complete GH1 SNP map in large control and patient populations. Individual SNP associations with height or other GH secretion-related phenotypic traits will require further confirmation by studies in larger populations and by in vitro functional studies.\nIn conclusion, our study established the GH1 gene sequence variation map in an adult control population with heights normally distributed within the normal range. SNPs and other sequence change contributions to skeletal growth as observed at adult height demonstrated that, despite the high frequency of variation and diversity and complexity of combinations, only some of the less frequent SNPs were associated with taller stature (P17 in intron 1 and P25 in intron 4), even masking the SNP contribution to a shorter one (P6 in the promoter and P12 in the 5′UTR regions, respectively). Systematic GH1 gene analysis in patients with growth delay and suspected GH deficiency/insufficiency will clarify whether different SNP frequencies and/or the presence of different sequence changes may be associated with phenotypes in them.\n\n\n" ], "offsets": [ [ 0, 27917 ] ] } ]

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[ { "id": "pmcA411032__text", "type": "Article", "text": [ "Down-regulation of the M6P/IGF-II receptor increases cell proliferation and reduces apoptosis in neonatal rat cardiac myocytes\nAbstract\nBackground\nThe mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) is a multi-functional protein that has been implicated in regulation of cell growth and apoptosis. Cardiac myocytes express relatively high levels of M6P/IGF2R, and cardiomyocyte apoptosis has been identified in a variety of cardiovascular disorders, such as myocardial infarction and heart failure. However, involvement of M6P/IGF2R in the pathogenesis of these conditions has not been determined. Thus, the objective of this study was to determine the role of M6P/IGF2R in regulation of cardiac myocyte growth and apoptosis.\n\nResults\nWe down-regulated the expression of M6P/IGF2R in neonatal rat cardiac myocytes and examined the effect on cell proliferation and apoptosis. Infection of neonatal cardiomyocytes with an adenovirus expressing a ribozyme targeted against the M6P/IGF2R significantly reduced the level of M6P/IGF2R mRNA, as determined by RT-PCR and Ribonuclease Protection Assay (RPA). M6P-containing protein binding and endocytosis as well as the M6P/IGF2R-mediated internalization of 125I-IGF-II were lower in the ribozyme-treated cells than the control myocytes, indicating that the number of functional M6P/IGF2R in the ribozyme treated cells was reduced. Accordingly, a marked increase in cell proliferation and a reduced cell susceptibility to hypoxia- and TNF-induced apoptosis were observed in the ribozyme-treated cells.\n\nConclusions\nThese findings suggest that M6P/IGF2R may play a role in regulation of cardiac myocyte growth and apoptosis. Down regulation of this gene in cardiac tissues might be a new approach to prevention of cell death or promotion of mitogenesis for certain heart diseases.\n\n\n\nBackground\nThe mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) is a unique protein that interacts with multiple ligands, some of which are important growth regulatory factors [1]. The M6P/IGF2R participates in internalization and lysosomal degradation of IGF-II, a mitogen normally acting through the IGF-I receptor to stimulate cell proliferation [2]. The M6P/IGF2 receptor is required for the activation of TGF-β [3], a potent growth inhibitor for many cell types. This receptor is also involved in the binding, transport and activation of newly-synthesized lysosomal enzymes, such as cathepsins [4,5], which have been recently implicated in the induction of apoptosis [6]. On the basis of these functions, the M6P/IGF2R has been proposed to play a significant role in regulation of cell growth and apoptosis [7].\nApoptosis, or programmed cell death, is a tightly regulated process used to remove excess, hazardous or damaged somatic cells, and is crucial for the development, maintenance and survival of an organism. However, alterations in the control of apoptosis have also been shown to contribute to human diseases. In fact, morphological and biochemical markers of apoptosis have been identified in a wide variety of cardiovascular disorders, including myocardial infarction and heart failure. This suggests that activation of apoptotic pathways contributes to cardiomyocyte loss and subsequent cardiac dysfunction in these conditions. A number of factors involved in cardiomyocyte apoptosis are currently known and include insulin-like growth factor-I (IGF-I), stress-activated protein kinases (SAPKs) and the anti-apoptotic Bcl-2 family [8]. There are indications that other factors may be involved in induction and regulation of cardiac apoptosis. However, these potential factors and their corresponding mechanisms have not been identified.\nSeveral lines of evidence point to the potential involvement of M6P/IGF2R in cardiac myocyte proliferation and apoptosis. Cardiac myocytes express relatively high levels of M6P/IGF2R and transgenic mice containing a homologous deletion of the M6P/IGF2R gene manifest ventricular hyperplasia due to an increase in cell number [9,10], suggesting that the M6P/IGF2R normally acts to suppress cardiac myocyte cell growth. It has also been shown that TGF-β, a potent growth suppressor whose activation requires the binding of latent TGF-β to M6P/IGF2R [3], is commonly upregulated in chronic heart failure [11]. Additional evidence for the involvement of M6P/IGF2R in regulation of apoptosis comes from studies of tumorigenesis. It has been shown that M6P/IGF2R expression is significantly reduced in a variety of tumors and loss of heterozygocity (LOH) at the M6P/IGF2R gene locus 6q26 have been found in breast, liver cancers and squamous cell carcinoma of the lung [12-15]. Although several studies have examined the effect of M6P/IGF2R over-expression on cell growth [7], it is not known whether down-regulation of this receptor protein leads to cellular protection against apoptosis.\nRibozymes are catalytic RNA molecules that cleave a complementary mRNA sequence [16], thereby inactivating specific mRNAs and suppressing gene expression in vitro and in vivo [17,18]. Ribozymes have been shown to be highly specific, efficient and stable. They can be packaged into viral vectors to enhance transfer into cells and to achieve longer expression compared with naked oligonucleotides. In the present study, we employed ribozyme technology to study the role of M6P/IGF2R in regulation of cardiac myocyte cell growth. A hammerhead ribozyme against the M6P/IGF2R mRNA was constructed and packaged in an adenoviral vector. We then examined the effect of ribozyme-mediated down-regulation of M6P/IGF2R expression on cell growth and hypoxia- and TNF-induced apoptosis.\n\nResults\nCleavage reaction of the ribozyme in vitro\nThe M6P/IGF2R ribozyme we constructed has 13-bp binding arms complementary to the target site of M6P/IGF2R mRNA, and a catalytic core (Fig. 1A). To evaluate the bioactivity of the ribozyme and the accessibility of the target site, a cleavage reaction was performed in vitro. The substrates, [α-32P] labeled RNA transcripts containing 45 bp of M6P/IGF2R mRNA or an unmatched sequence, were incubated with the ribozyme as described (see Materials and Methods). The ribozyme cleaved only the specific M6P/IGF2R mRNA into the expected products. In the assay of time course, the hammerhead ribozyme was able to cleave 24.2% of the M6P/IGF2R target within 10 minutes of incubation, 50.3% of the M6P/IGF2R target within 40 minutes of incubation, and by 640 minutes, 80.8% of the M6P/IGF2R target was converted to the expected products (Fig. 1B). This ribozyme did not digest the unmatched sequence (Fig. 1B). These results indicate a high efficiency and specificity of the ribozyme in vitro.\n\nRibozymes down-regulate M6P/IGF2R expression in cardiac myocytes\nTo examine the ability of the ribozyme to reduce levels of M6P/IGF2R mRNA in cultured cardiac myocytes, total RNA was extracted from cells infected with Ad-GFP/IGF2R-Rz or Ad-GFP, and subjected to RT-PCR using M6P/IGF2R-specific primers. Primers specific for β-actin were added to a parallel reaction to serve as an internal standard. Cells were used 4 days after infection, with average infection efficiency of 70–80% (for which a viral dose used had minimal cytotoxicity). The RT-PCR product of M6P/IGF2R was 856 bp, and the β-actin product was 285 bp. As shown in Fig. 2A, the Ad-GFP/IGF2R-Rz-infected cells exhibited a significantly lower level of M6P/IGF2R mRNA than Ad-GFP-infected cells, with a reduction of about 50%. This result was confirmed by ribonuclease protection assay (RPA), in which GAPDH was used as a control (Fig. 2C &2D). There was no significant difference in the level of M6P/IGF2R mRNA between Ad-GFP-infected cells and uninfected cells (data not shown), indicating that infection with the adenovirus itself did not alter the endogenous M6P/IGF2R mRNA level. The results demonstrated that the ribozyme was highly effective in suppressing M6P/IGF2R expression in cultured cardiac myocytes.\n\nEffect of ribozyme expression on the functional activity of M6P/IGF2R\nTo determine the effect of the ribozyme on the functional activity of M6P/IGF2R, binding and internalization of exogenous 125I-IGF-II was measured in cells infected with Ad-GFP/IGF2R-Rz. As shown in Fig. 3A, cells infected with Ad-GFP/IGF2R-Rz showed a 54% reduction in 125I-IGF-II internalization when compared with the control cells (infected with Ad-GFP). We also examined the effect of the ribozyme on the M6P-binding activity of the M6P/IGF2R using the M6P-bearing lysosomal enzyme, β-glucuronidase, as a probe. The results showed that the maximal M6P-binding capacity of cells treated with the ribozyme was about 50% less than that of controls (Fig. 3B). Furthermore, we assessed the ability of cells to internalize exogenous β-glucuronidase after treatment with ribozyme. Similarly, the M6P-inhibitable endocytosis of β-glucuronidase by ribozyme-treated cells was about 52% less than that of control cells (Fig. 3C). These results confirm that the number of functional M6P/IGF2R in ribozyme-treated cells was reduced.\n\nAdenoviral delivery of ribozymes increases the proliferation of cardiac myocytes\nWe examined the effects of the ribozyme on the growth of cultured neonatal rat cardiac myocytes. Morphological evaluation showed a remarkable difference in growth pattern between Ad-GFP/IGF2R-Rz-infected cells and the control cells: the ribozyme-expressing cells formed larger and more spread colonies (Fig. 4). Assessment of cell proliferative activity by the MTT assay and counts of viable cells showed that the number of cardiac myocytes in ribozyme-expressing cultures was significantly higher than in control cultures (Fig. 5). These results indicate that treatment with M6P/IGF2R-ribozyme can promote cardiac myocyte proliferation.\n\nEffect of M6P/IGF2R-ribozyme expression on apoptosis of cardiac myocytes\nWe examined the effects of ribozyme expression on TNF-α and hypoxia-induced apoptosis of cultured cardiac myocytes. After a 24 hr challenge with hypoxia, the number of apoptotic cells in M6P/IGF2R-Rz expressing cultures was 38% lower than in control cultures as determined by Hoechst staining (which highlights the nuclei of apoptotic cells) and ELISA (Fig. 6A, 7A). MTT analysis showed that the number of viable cells in ribozyme-treated cultures was 40% higher than in control cultures (Fig. 7A).\nAfter treatment with TNF-α, as shown in Fig. 6B, a large number of control cells underwent apoptosis, as indicated by morphological changes (small round shape) and bright blue nuclear staining. There were significantly more apoptotic cells in control cultures than in cultures expressing the Ad-GFP/IGF2R-Rz. The number of apoptotic cells, as measured by the cell death ELISA assay, in cultures infected with Ad-GFP/IGF2R-Rz was significantly (about 40%) lower than in cultures infected with Ad-GFP (Fig. 7B). Accordingly, the number of viable cells, as measured by MTT analysis, in cultures infected with Ad-GFP/IGF2R-Rz was significantly (about 45%) higher than in cultures infected with Ad-GFP (Fig. 7B). These results are consistent with the hypothesis that decreasing M6P/IGF2R expression by ribozyme treatment can reduce cell apoptosis.\n\n\nDiscussion\nSome 62,000,000 Americans have one or more types of cardiovascular disease (CVD) and CVD is the leading cause (40.1%) of death in the United States. Myocardial infarction and heart failure, conditions accompanied by cardiac myocyte apoptosis, represent 23% of all CVDs and are a growing clinical challenge in need of novel therapeutic strategies. In this study, we investigated the M6P/IGF2R as a potential new therapeutic target for reduction of cardiac apoptosis and cardiac injury in these conditions.\nUsing ribozyme technology we down-regulated the expression of the M6P/IGF2R in neonatal cardiac myocytes. We then examined cell proliferation and apoptosis under normal conditions and post challenge with either hypoxia, a model of ischemia-reperfusion, or TNF-α, a cytokine implicated in the pathogenesis of chronic heart failure [19]. Our results demonstrate an association of a decrease in the expression and function of the M6P/IGF2R with increased cell proliferation and decreased cell susceptibility to hypoxia- and TNF-induced apoptosis. Expression of the ribozyme targeted against the M6P/IGF2R in cardiomyocytes resulted in down-regulation of M6P/IGF2R expression, as measured by RT-PCR and RPA, and of M6P/IGF2R function, as indicated by a decrease in internalization of 125I-IGF-II, and β-glucuronidase binding and endocytosis.\nMTT analysis and viable cell counts showed that ribozyme-mediated down-regulation of M6P/IGF2R resulted in a marked increase in cell proliferation of cardiomyocytes, which normally express high levels of M6P/IGF2R [20] and have limited proliferative capabilities [21]. These results are consistent with the findings of previous knockout studies [9,10]. Since the M6P/IGF2R has multiple actions on cell growth, its proliferative effect on the heart cells observed in this study might involve multiple mechanisms. However, it is likely that unchecked IGF-II stimulation plays a key role in the effect. Because the M6P/IGF2R is believed to sequester and degrade IGF-II [2], a decrease in M6P/IGF2R expression and function could result in decreased degradation and hence increased bioavailability of IGF-II to the IGF-I receptor, which mediates the growth-promoting effect of IGF-II. Supporting evidence for the involvement of IGF-II in the proliferative effect resulting from loss of M6P/IGF2R function comes from studies of M6P/IGF2R knock-out mice. M6P/IGF2R-null mice display global hyperplasia that coincides with elevated levels of IGF-II. Most importantly, however, the lethal nature of an M6P/IGF2R-null phenotype is reversed in an IGF-II-null background [9]. Our results showing that ribozyme-mediated down-regulation of M6P/IGF2R lead to a decrease in IGF-II internalization support the above possibility. However, further investigation to confirm this mechanism is warranted.\nMore importantly, our results also showed that M6P/IGF2R down-regulation resulted in decreased sensitivity of cardiomyocytes to hypoxia- and TNF-induced apoptosis. There is evidence that lysosomal enzymes, such as cathepsins B and D contribute to hypoxia- and TNF-induced apoptosis in vitro [22-25] and in vivo [26,27]. The M6P/IGF2R has been shown to be involved in binding, transport and activation of lysosomal enzymes, including cathepsins [4,5]. Therefore, it is possible that down-regulation of the M6P/IGF2R results in improper trafficking and activation of cathepsins. This, in turn would eliminate the apoptotic cascades triggered by these enzymes under hypoxia and TNF stimulation and result in decreased sensitivity of cardiomyocytes to apoptosis.\nIt has also been shown that TNF stimulation involves the activation of TGF-β [28-30], a ligand of M6P/IGF2R that has been implicated in the progression of chronic heart failure [11,31]. Therefore, down-regulation of M6P/IGF2R expression could also lead to a decreased bioavailability of activated TGF-β, thereby decreasing the sensitivity of cardiomyocytes to the TNF/TGF-β apoptotic pathway. The detailed mechanism of the observed effects is unknown and requires further investigation.\n\nConclusions\nThe present study demonstrates that ribozyme-mediated down-regulation of expression and functional activity of the M6P/IGF2R results in a decrease in the susceptibility of cardiac myocytes to apoptotic stimuli. These findings suggest that this receptor might be involved in cardiac cell growth and apoptosis. The ability of the M6P/IGF2R ribozyme to reduce M6P/IGF2R expression and function in transfected cells verifies the utility of the ribozyme in studying the role of M6P/IGF2R in cardiomyocyte growth and apoptosis. In addition to its utility as a research tool, the ribozyme, with further exploration and development, might have potential application as a therapeutic agent to prevent cell death or promote mitogenesis for certain clinical conditions, such as, myocardial infarction and chronic heart failure.\n\nMethods\nConstruction of recombinant M6P/IGF2R-RZ adenoviral vector\nThe nucleotide numbers of the rat M6P/IGF2R sequence targeted by the hammerhead ribozyme is 1147–1160 after coding site (exon 9). The structure of the M6P/IGF2R hammerhead ribozyme is shown in Fig. 1. A 49 bp M6P/IGF2R ribozyme oligonucleotide, 5'-GAATTCCCC ACACTG ATGAGCCGCTTCGGCGGCGAAACATTCAAC GCGT-3' and the corresponding reverse complementary strand were synthesized. The fragments were subcloned to produce a plasmid containing a ribozyme against M6P/IGF2R. For construction of the recombinant adenovirus containing the M6P/IGF2R-ribozyme (pAd-GFP/IGF2R-Rz), the segments containing the ribozymes were amplified by PCR and cloned into a pAdTrack-CMV vector and then recombined homologously with an adenoviral backbone pAdEasy 1 vector to generate (pAd-GFP/IGF2R-Rz), following the protocol described by He et al. [32]. The pAd-GFP/IGF2R-Rz carries both the IGF2R-Rz and GFP (as reporter) genes, each under the control of separate cytomegalovirus (CMV) promoters. Another viral vector, pAd-GFP, which carries the GFP gene only under the control of the CMV promoter, was generated and used as a control vector. The adenoviral vector DNA were linerized with Pac I and transfected into the replication-permissive 293 cells (E1A transcomplementing cell line) by using Lipofectamine (Life Technologies) to produce E1-deleted, replication-defective recombinant adenovirus as described previously [33]. Large-scale amplification of recombinant adenovirus in 293 cells was followed by purification using a discontinuous CsCl gradient. The constructs were confirmed by enzymatic digestion and DNA sequencing.\n\nTranscription and cleavage reaction of ribozyme in vitro\nPlasmids containing the ribozyme or the substrate (either 45 bp of M6P/IGF2R mRNA or an unmatched sequence 5'-GTGCTGTCTGTATG-3') were linearized with MluI, respectively. All transcripts were generated with T7 RNA polymerase (Promega). Substrate transcripts were labeled by incorporation of [α-32P] UTP (NEN Life Science Products, Inc.). Specific activity of the [α-32P] UTP (10 μCi/μl) and the base composition of each substrate molecule were used to calculate the substrate concentration. Ribozyme transcripts were quantified spectrophotometrically. (The half-life of the M6P/IGF2R target is about 280 minutes).\nCleavage reaction mixture contained substrate RNA (40 nM), increasing amounts of ribozyme (60 nM), 20 mM MgCl2 and 20 mM Tris-HCl, pH8.0, in a final volume of 10 μl. The mixture was incubated at 37°C for a time-course of cleavage reaction from 0, 5, 10, 20, 40, 80, 160, 320, to 640 minutes and the cleavage reaction was stopped by addition of loading buffer (80% formamide, 10 mM Na2EDTA, pH 8.0, and 1 mg/ml each bromophenol blue and xylene cyanol). Cleavage products were analyzed on a 15% polyacrylamide and 8M urea gel. Product and substrate fragments were quantitated by using NIH Imager.\n\nCell cultures and infection with Ad-GFP/Rz-IGF2R and Ad-GFP\nCardiac myocytes were isolated from 1-day-old newborn rats using the Neonatal Cardiomyocyte Isolation System (Worthington). The isolated cells were plated in 6-well plates and cultured in F-10 medium containing 5% (vol/vol) FBS and 10% (vol/vol) horse serum at 37°C in a tissue culture incubator with 5% CO2 and 98% relative humidity. Cells were used for experiments after 2–3 days of culture. Viral infections were carried out by adding viral particles at various concentrations (usually, 2 × 108 virus particles/ml) to culture medium containing 2% (vol/vol) FBS. Initially, optimal viral concentration was determined by using Ad-GFP to achieve an optimal balance of high gene expression and low viral titer to minimize cytotoxicity. After 24 hours of incubation, the infection medium was replaced with normal (15% vol/vol serum) culture medium. For treatment with IGF-II, cells were incubated with 50 ng/ml IGF-II after 24 hours infection with Ad-GFP/IGF2R-Rz or Ad-GFP. Four days after infection, cells were used for analysis of gene expression of M6P/IGF2R and its effect on cell growth and apoptosis.\n\nAnalysis of gene expression in cardiac myocytes\nThe M6P/IGF2R transcripts were determined by both RT-PCR and Ribonuclease Protection Assay (RPA). RT-PCR was performed using the GeneAmp EZ rTth RNA PCR kit (Roche). Total RNA was extracted from cultured cells using an RNA isolation kit (Qiagen,), according to the manufacturer's protocol. M6P/IGF2R transcripts were amplified using the primers (5'-GACAGGCTCGTTCTGACTTA-3') and (5'-CTTCCACTCTTATCCACAGC-3') specific to the M6P/IGF2R. Each RT-PCR assay was performed in triplicate and product levels varied by less than 3.2% for each RNA sample. Primers specific for β-actin cDNA were added to a parallel reaction to standardize for variations in PCR between samples. PCR products were resolved on a 1.0% agarose gel, visualized under UV light and quantitated using NIH Imager.\nRPA was performed using the RPA III kit (Ambion, Austin, TX). Briefly, total RNA was extracted from cultured cells using a total RNA isolation reagent (TRIzol, Gibco BRL) according to the manufacturer's protocol. The plasmid containing the rat M6P/IGF2R gene was linearized and used as a transcription template. Antisense RNA probes were transcribed in vitro using [33P]-UTP, T7 polymerase (Riboprobea System T7 kit, Promega), hybridized with the total RNA extracted from the rat cardiomyocytes, and digested with ribonuclease to remove non-hybridized RNA and probe. The protected RNA·RNA was resolved on a denaturing 5% sequence gel and subjected to autoradiography. A probe targeting the GAPDH gene was used as an internal control.\n\nMeasurement of 125I-IGF-II internalization\nCells were incubated at 37°C for 2 hrs in serum-free F-10 culture medium containing 125I-labeled IGF-II (0.5 ng/ml) with or without excess unlabeled IGF-II (2 μg/ml). Following the incubation, the cells were washed three times with ice-cold PBS, and cell-associated radioactivity was determined by a γ counter. Specific internalized 125I-IGF-II was calculated by subtracting the count of samples with excessive unlabeled IGF-II from that without unlabeled IGF-II, and normalized to protein contents.\n\nBeta-glucuronidase binding assay\nBinding of β-glucuronidase was assayed as described previously [34,35]. Briefly, cells were permeabilized with 0.25% saponin in 50 mM Hepes (pH 7.0), 150 mM NaCl, 5 mM β-glycerophosphate, 0.5% human serum albumin, and 10 mM mannose-6-phosphate (M6P) for 30 minutes on ice. The cells were washed three times with ice-cold PBS containing 0.05% saponin. They were incubated with 20,000 units/ml β-glucuronidase from bovine liver (Sigma) in 50 mM Hepes (pH 7.5) containing 150 mM NaCl, 5 mM β-glycerophosphate, 0.5% human serum albumin, 0.5% saponin with or without 10 mM M6P overnight on ice. Cells were washed five times with ice-cold PBS containing 0.05% saponin and sonicated in 100 mM sodium acetate (pH 4.6). The protein concentration of solubilized cell extract was measured and enzyme activity was assayed as follows: for each reaction 50 ul cell extract were added to 500 ul of 100 mM sodium acetate (pH 4.0) containing 1 mM paranitrophenyl (PNP)-β-glucuronide (Sigma) as substrate. After an incubation period of 3 hours at 37°C, 500 ul 1 M Na2CO3 were added to each reaction and the absorbance was measured at 400 nm. Experimental values were compared to a standard curve that was constructed using 1–100 nM solutions of PNP (Sigma) in 500 ul 100 mM sodium acetate and 500 u1 1 M Na2CO3. Specific activity was calculated as nM of PNP produced/hour/mg of protein.\n\nBeta-glucuronidase endocytosis assay\nBeta-glucuronidase endocytosis assay was carried out as described previously [36]. Briefly, confluent cell cultures were washed twice with pre-warmed serum-free DMEM followed by incubation with DMEM containing 5 mg/ml human serum albumin and 10 mM M6P for 20 minutes. Following incubation cells were washed 3 times with pre-warmed DMEM. Cells were then incubated in DMEM containing 5 mg/ml human serum albumin alone or 4000 units β-glucuronidase with or without 10 mM M6P for 2 hours at 37°C. Following the incubation, the cells were washed 5 times with ice-cold PBS and subjected to enzyme activity assay as described above.\n\nCell proliferation assay (MTT assay and cell counts)\nCardiac myocytes were grown in culture plates (tissue culture grade, 12 wells, flat bottom) in a final volume of 1 ml serum-containing culture medium per well, in a humidified atmosphere (37°C and 5% C02) for 3 days. After infection with Ad-GFP/IGF2R-Rz or Ad-GFP, cells were incubated with or without 50 ng/ml IGF-II for 4 days. Following supplementation with IGF-II, 100 μl MTT labeling reagent (Roche) were added to each well and cells were incubated for 4 hours, followed by addition of 1 ml solubilization solution into each well. The plate was placed in an incubator at 37°C overnight. Spectrophotometrical absorbency of the samples was measured using an UV-visible Recording Spectrophotometer with wavelength of 550–690 nm. In addition, the total number of viable cells in each treatment was counted by trypan blue exclusion method using a hemocytometer.\n\nInduction and analysis of cell apoptosis\nCells were infected with Ad-GFP or Ad-GFP/IGF2R-Rz. Seventy-two hours post infection, cells were treated with TNF (0.1 ng/ml) for 24 hrs or subjected to hypoxia. For induction of apoptosis by hypoxia, cell culture medium was changed to serum-free F-10 saturated with 95% N2/5% CO2 and cells were placed in a 37°C airtight box saturated with 95% N2/5% CO2 for 24 hrs. For normoxic controls, culture medium was changed to F-10/5%F BS/10% HS and cells were placed in a 37°C/5% CO2 incubator for 24 hrs before analysis.\nApoptotic cells were identified by Hoechst staining using the Vybrant™ Apoptosis Kit #5 (Molecular Probes) according to the manufacturer's protocol. In addition, after infection with Ad-GFP or Ad-GFP/IGF2R-Rz and challenge with either TNF or hypoxia, cell viability was assessed using the MTT assay Kit (Roche Molecular Biochemicals) and cell apoptosis was determined using the Cell Death Detection ELISA Kit assay (Roche Molecular Biochemicals) according to the manufacturer's protocol.\n\nStatistical analysis\nStudents' t-test was used to evaluate the difference between two values. Each experiment was repeated at least three times. Statistical significance was accepted at the level of p < 0.05.\n\n\nList of abbreviations used\nAd-GFP, adenovirus carrying GFP gene; Ad-GFP/IGF2R-Rz, adenovirus carrying both the ribozyme against M6P/IGF2R and the GFP gene; GFP, green fluorescent protein; IGF-II, insulin-like growth factor II; M6P/IGF2R, mannose 6-phosphate/insulin-like growth factor II receptor; Rz, ribozyme.\n\nAuthors' contributions\nZC carried out construction of the ribozyme, production of the viruses, cellular experiments, biochemical assays and data analysis.\nYG carried out the RPA assay and participated in the molecular biological studies.\nJXK conceived of the study, participated in its design and coordination, and drafted the manuscript.\n\n\n" ], "offsets": [ [ 0, 27252 ] ] } ]

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[ { "id": "pmcA544950__text", "type": "Article", "text": [ "Thumb force deficit after lower median nerve block\nAbstract\nPurpose\nThe purpose of this study was to characterize thumb motor dysfunction resulting from simulated lower median nerve lesions at the wrist.\n\nMethods\nBupivacaine hydrochloride was injected into the carpal tunnel of six healthy subjects to locally anesthetize the median nerve. Motor function was subsequently evaluated by measuring maximal force production in all directions within the transverse plane perpendicular to the longitudinal axis of the thumb. Force envelopes were constructed using these measured multidirectional forces.\n\nResults\nBlockage of the median nerve resulted in decreased force magnitudes and thus smaller force envelopes. The average force decrease around the force envelope was 27.9%. A maximum decrease of 42.4% occurred in a direction combining abduction and slight flexion, while a minimum decrease of 10.5% occurred in a direction combining adduction and slight flexion. Relative decreases in adduction, extension, abduction, and flexion were 17.3%, 21.2%, 41.2% and 33.5%, respectively. Areas enclosed by pre- and post-block force envelopes were 20628 ± 7747 N.N, and 10700 ± 4474 N.N, respectively, representing an average decrease of 48.1%. Relative decreases in the adduction, extension, abduction, and flexion quadrant areas were 31.5%, 42.3%, 60.9%, and 52.3%, respectively.\n\nConclusion\nLower median nerve lesion, simulated by a nerve block at the wrist, compromise normal motor function of the thumb. A median nerve block results in force deficits in all directions, with the most severe impairment in abduction and flexion. From our results, such a means of motor function assessment can potentially be applied to functionally evaluate peripheral neuropathies.\n\n\n\nIntroduction\nThe thumb has unique anatomical and biomechanical characteristics that are required to perform many manipulative tasks. Thumb motor dysfunction resulting from neuromuscular and musculoskeletal pathologies severely hinders the performance of these daily tasks. Clinical treatment, prevention protocols, and rehabilitation efficacy requires a thorough understanding of thumb motor capabilities, as well as its associated functional deficit. Investigations of underlying pathological mechanism of the thumb help advance clinical treatments such as tendon transfers [1], functional electrical stimulation [2] and plasticity suppression [3].\nMeasurement of strength during maximum voluntary contraction is a simple and direct means of assessing neuromuscular function. Popular instruments used for quantitative assessment of thumb strength are pinch dynamometers. The pinch output, however, provides limited information about thumb motor function in that it offers a single generic force in one specific direction. Each muscle/tendon within the thumb has a distinct anatomical origin and insertion, suggesting its external force potential in a particular direction [4-6]. Hence, evaluation of strengths in multiple directions offers insight concerning the motor capacity of individual muscles. Force production of a digit has been measured in various directions such as flexion/extension [7,8], abduction/adduction [9-14], or in combined directions [15,16]. Bourbonnais et al. developed an apparatus to measure thumb force production in eight directions in the transverse plane of the thumb and investigated force dependence on the direction of effort [15]. Yokogawa and Hara measured index fingertip forces in various directions within the flexion/extension plane [8]. Recently, we developed experimental apparatuses to measure multi-directional forces of a digit in its transverse plane [17-19]. From these multi-directional forces we constructed force envelopes representative of the characteristic force output pattern of a digit [17-19].\nDisorders resulting from traumatic injuries to and various diseases of these nerves are common in clinical practice. Clinical manifestations of hand dysfunction are distinctive depending on the nerve involved. For example, thenar atrophy is a major clinical observation affecting thumb function at the later stages of compression neuropathy of the median nerve. Several studies have been conducted to investigate the effects of simulated peripheral neuropathies using local anesthetization [5,16,20,21]. Kozin et al. [21] studied the effects of median and ulnar nerve blocks on grip and pinch strength and showed significant decreases following nerve blockage [21]. Boatright and Kiebzak [20] investigated the effects of median nerve block on thumb abduction strength. Kaufman et al. [5] measured isometric thumb forces in eight directions together with electromyographic signals of thumb muscles after block of the median nerve. Labosky and Waggy [22] studied the strength related to grip, pinch, thumb adduction, thumb abduction, and finger flexion after radial nerve block [22]. Kuxhaus studied the three dimensional feasible force set at the thumb-tip before and after ulnar nerve block and reported this to be a reproducible and sensitive means to detect impairment.\nThe purpose of this study was to utilize our developed apparatus and protocols to investigate the effects of lower median nerve lesion on thumb motor function. The lesion was simulated by blocking the median nerve at the wrist using an anesthetic. We hypothesized that a median nerve block would cause (1) a decrease in force production, which would be direction-dependent with the most severe reduction in the abduction direction, and (2) a decrease in the force envelope area and force quadrant area, with the greatest decrease in the abduction quadrant.\n\nMethods\nSubjects\nSix healthy male subjects (mean age: 26.9 ± 5.1 years) participated in this study. The subjects had no previous history of neuromuscular or musculoskeletal disorders of the upper extremities. Each subject signed an informed consent form approved by the Institutional Review Board prior to participating in the experiment.\n\nMedian nerve block\nInjections were performed under aseptic conditions while the subjects sat with the forearm supinated and the wrist slightly extended. After the skin at the palmer area of the wrist was cleaned with alcohol, 4 mL of 0.5% bupivacaine hydrochloride (Astra Pharmaceuticals, Westborough, MA, USA) was injected into the carpal tunnel with a sterile 25-gauge short-bevel needle. The needle was inserted through the transverse carpal ligament in line with the radial border of the fourth digit slightly ulnar to the palmaris longus tendon at the level of the distal wrist crease. Forty minutes was allowed for the median nerve block to reach complete effectiveness [23] and was verified using the Semmes-Weinstein monofilament test. The average monofilament score was 2.85 across the five digits before nerve block. About 40 minutes after nerve injection, little sensory impairment occurred in the ulnar distribution (score = 3.22), while the sensory score in the median distribution was greater than 6.15. The effects of nerve block lasted more than 6 hours with all subjects regaining normal hand function within 12 hours.\n\nTesting apparatus\nThe experimental apparatus was designed and constructed to measure maximum voluntary contraction forces of any digit at any point along the digit. Force application was possible in any direction within the transverse plane of the longitudinal axis of the digit. The apparatus consisted of position control accessories, a force transducer, and a custom fitted aluminum ring attached to the transducer (Figure 1A,1B). The transducer (Mini40, ATI Industrial Automation, NC, USA), capable of measuring 6 degrees of freedom forces and moments, was attached to a mounting clamp via an aluminum adapter plate while the aluminum ring was secured to the tool side of the transducer using a custom adapter. The ring served as a connection anchor for the transducer and the digit. The force transducer and ring attachment were positioned in a desired orientation using an aluminum slide rail, tubing, and lockable mounting clamps (80/20 Inc., Columbia City, IN, USA). The slide rail was secured to an aluminum base plate. Foam padded wooden blocks with two locking straps secured the arm to the base plate.\nThe analog outputs from the transducer were digitized using a 16-bit analog-to-digital converter (PCI-6031, National Instruments, TX, USA). The X (abduction/adduction) and Y (flexion/extension) force components in the transverse plane were displayed on the screen while the subject performed a force production task. The resolutions of the force transducer in its axial (flexion/extension) and horizontal (abduction/adduction) directions were 0.16 N and 0.08 N, respectively. A personal computer equipped with LabVIEW (National Instrument, TX, USA) was used for force data acquisition, display, and processing.\n\nExperimental procedures\nEach subject was tested before and after median nerve block. The nerve block procedures were performed immediately after the completion of the first testing session. Post-block testing started after the verification of complete median nerve block, approximately 40 minutes after the injection. During each test, the subject was seated in a chair adjacent to the testing station modified with a wooden board to align their back vertically throughout the trials. The subjects rested their forearm on padded wooden blocks positioning their shoulder in approximately 60° of frontal plane abduction. Nylon straps fitted with plastic snap locking mechanisms secured the forearm and minimized the intervention of the elbow and shoulder during thumb force application. Subjects grasped a vertical dowel secured to the distal end of the wooden blocks in a midprone position. Formable thermoplastic braces were used to fix the elbow in 90° of flexion, and the wrist in 20° of extension and 0° of ulnar deviation. A metallic brace was used to fix the interphalangeal joint of the thumb in full extension. The aluminum ring was placed around the middle of the proximal phalanx and oriented to accommodate comfortable thumb position with the metacarpophalangeal joint flexed approximately 15°. Prior to testing, a line was drawn on the proximal phalange at the midpoint between the interphalangeal and metacarpophalangeal joints. The alignment of the ring with the circumferential line standardized the location of force application within and between subjects. As force application was at the middle of the proximal phalanx, mechanical action pertains to both the metacarpophalangeal and carpometacarpal joints. We chose the terminology of flexion/extension and adduction/adduction based on the mechanical action with respect to the metacarpophalangeal joint. With the thumb in the ring (Figure 1B), extension and flexion occurred in parallel with the palm, and abduction and adduction occurred in a plane perpendicular to the palm.\nEach subject performed 15 circumferential MVC trials with randomized starting directions (Figure 1C). The subject was allotted 15 seconds to complete each circumferential trial, and was instructed to use the entire time allotted to traverse the perimeter of the ring once. A dot generated on the computer screen was programmed to traverse a circle within 15 seconds to provide the subject with directional feedback of their force application. Subjects were given 60 seconds of rest between each circumferential trial. Each subject was familiarized with the task with a few practice trials. Data were collected from each subject at 100 samples per second producing a total of 22,500 pairs of force components from the 15 circumferential trials. Our previous study [19] indicated that the testing protocol did not cause noticeable fatigue.\n\nForce envelope and quadrants\nData from multiple circumferential trials were accumulated to construct a force envelope. The procedures to generate a force envelope were as follows:\nCartesian force coordinates (Xi, Yi) were transformed into polar coordinates (Rα, α), where Rα was the force magnitude at an angular position α. Each α was rounded to the nearest integer ranging from 0 to 359 degrees.\nThe maximum, Fα, was determined from a string of N data points along each radial line defined by α. At the completion of the 15 trials, there were, on average, N = 63 data points on each radial line of α based on the distribution off the 22,500 data points around 360°.\nA moving average with an interval of 10° was applied to the maximal series data Fα (α = 0, 1, 2,..., 359) to obtain filtered maximal forces. These forces formed a force envelope.\nThe area formed by a force envelope was divided into adduction-extension, extension-abduction, abduction-flexion, and flexion-adduction quadrants by radial lines oriented at 0°, 90°, 180°, and 270° A quadrant force was represented using the mean magnitude of the forces in that quadrant. The areas of the entire envelope and each quadrant were calculated by summing the areas of individual arc sections formed by the polar coordinates of the force envelope. (Figure 2).\n\nStatistical Analyses\nOne- and two-factor repeated measures analyses of variance (ANOVA) were used to analyze outcome measures. The independent variables were testing SESSION (n = 2, i.e., pre- and post-block), force DIRECTION (n = 16), and force QUADRANT (n = 4), with SESSION as a repeated variable. Dependent variables were directional force, individual quadrant area and force envelope area. Statistical analyses were performed using SPSS 11 (SPSS Inc., Illinois) with statistical significance set at α = 0.05.\n\n\nResults\nForce envelope and directional forces\nFigure 3 shows the force envelopes produced by each subject (A to F) before and after median nerve block. The post-block force envelope was inside the pre-block envelope for each subject, indicating a decrease in force magnitude in all directions after nerve block. Figure 4 shows the average pre- and post-block force envelope across all subjects. Force magnitudes were significantly reduced after nerve block (p < 0.001) resulting in significantly smaller force envelopes. The average decrease across all directions was 27.9%. A maximum decrease of 42.4% occurred at 199°, corresponding to a combined direction of abduction and slight flexion, while a minimum decrease of 10.5% occurred at 328° corresponding to a combined direction of adduction and slight flexion. Relative decreases at 0° (adduction), 90° (extension), 180° (abduction), and 270° (flexion) directions were 17.3%, 21.2%, 41.2% and 33.5%, respectively.\nA single force in each quadrant was represented using the mean magnitude of the forces in that quadrant (see description in the Methods). The average quadrant forces were significantly decreased after nerve block (p < 0.001; Figure 5). The amount of decrease was also different between quadrants (p < 0.005). Relative decreases in mean quadrant forces were 24.5%, 38.7%, 32.1%, and 18.1% for extension, abduction, flexion, and adduction, respectively. The maximal decreases in mean quadrant force, 38.7%, occurred in the abduction quadrant.\n\nForce envelope areas and quadrant areas\nAreas enclosed by the post-block envelopes were significantly smaller than the pre-block envelopes (p < 0.001; Figure 4). Post-block force envelope area, 10700 ± 4474 N.N, was 51.9% of pre-block force envelope area, 20628 ± 7747 N.N. Quadrant area decreased significantly (p < 0.001; Figure 6). The maximal percentage decrease in area after nerve block was 60.9% in the abduction quadrant, followed by a 52.3% area decrease in the flexion quadrant.\n\n\nDiscussion\nIn this study we simulated a lower median nerve lesion and evaluated the resultant thumb motor function deficit. Our internal control via pre- and post-block design offered a particular advantage of investigating the mechanical role of muscles innervated by a targeted nerve. The testing and analytical methods employed have provided advanced quantification of thumb motor function. The results have confirmed our initial hypotheses that greatest force decreases occurred in directions related to abduction, and that the post-block thumb force envelope area was smaller than the pre-block force envelope area.\nPreferential force attenuation in the quadrants of abduction and flexion after median nerve block are in agreement with anatomical and neuromuscular features of the thumb. The median nerve innervates the abductor pollicis brevis, the opponens pollicis and superficial head of the flexor pollicis brevis, all of which contribute to the abduction and flexion of the thumb [4]; therefore, denervation of these muscles after median nerve block would cause the greatest force deficit related to median nerve function [5]. Additionally, as force application moved towards adduction, the force deficit decreased as neuromuscular control shifted from the median nerve to the ulnar nerve via the first dorsal interosseous and adductor pollicis brevis. Force deficit in extension was also comparably small as extension forces are mainly produced by the extensors pollicis brevis and longus originating in the forearm.\nOur reported force decreases following a median nerve block (40.9% in abduction, 34.1% in flexion) were smaller than those reported in the literature. Kozin et al. [21] reported a 60% decrease in pinch strength after a median nerve block using mepivicaine hydrochloride [21]. Boatright and Kiebzak [20] reported an approximate 70% decrease in thumb abduction strength after median nerve block using Lidocaine [20]. Kaufman et al. [5] stated that a median nerve block with Lidocaine almost completely diminished force production in the abduction direction [5]. The discrepancy may be due to the anesthetic used and strength testing method. Although the sensory block appeared to be complete for each method, the motor capabilities of the muscles associated with the median nerve might or might not be completely eliminated. Such a result is largely dependent on a particular anesthetic, its concentration and dosage, as well as the efficacy of the injection technique at immersing the nerve. The methods of strength testing may also help explain the different magnitudes of strength deficit after the nerve block. All previous results were based on forces obtained in discrete direction(s), and focused exertions, while the current study utilized a method of force production in a continuous, circumferential and dynamic manner. Furthermore, thumb motor performance can be maintained despite the absence of certain individual muscles. For example, Britto and Elliot reported that the loss of abductor pollicis longus and extensor pollicis brevis in their two patients did not show functional compromise of strength and grip strength [24]. In a broader sense, the neuromuscular system has remarkable capabilities to accomplish the same motor function goal using different effectors and different goals using the same effectors, a phenomenon so called \"motor equivalence\" [25].\nAn unexpected finding from this study was that the force deficit occurred in all directions (Figure 4). In other words, the median nerve block caused reduced force production by those muscles not associated with the median nerve. Several potential explanations exist to describe such a phenomenon. First, the injection into the carpal tunnel at the wrist, although localized, potentially diffused into the intrinsic fascia of the hand partially compromising function of the ulnar nerve, which innervates the adductor pollicis. Although Semmes-Weinstein monofilament testing confirmed the continued sensation of the digits within the ulnar nerve distribution, it is not inconceivable that the injection could have contaminated the ulnar innervated muscles, the first dorsal interosseous and deep head of the flexor pollicis brevis [20]. Secondly, thumb force in any direction is produced by synergistic activation of the many intrinsic muscles, and as a result, the muscular deficiency associated with one direction may hinder the force production in other directions by other muscles [5,22]. For example, Kaufman et al. demonstrated that thumb muscles not innervated by the median nerve displayed lower electromyographical activation and shifted the direction of maximum activation after a median nerve block [5]. Labosky and Waggy showed that a radial nerve block caused a 53% decrease in thumb abduction strength because of the lack of stabilization of the radial innervated extensor muscles [22]. Consequently, deficiency of median innervated muscles inherently limits force production in other directions as neuromuscular switching is necessary to produce force in changing directions.\nThe median innervated muscles are the dominant abductors of the thumb metacarpophalangeal and carpometacarpal joint. The more than 50% residual abduction force found in this study suggests that the injection did not totally block the motor function of these muscles, even though a complete sensory loss was verified. This concurs with clinical observations of median compression neuropathy. Individuals with carpal tunnel syndrome complain of sensory dysfunction early in the disease process (at the beginning), while motor signs of thenar wasting and thumb weakness occur as the disease advances. The concept that the motor deficit is more resistant to peripheral median neuropathy than sensory loss has been well documented [23,26,27]. Butterworth et al. studied the temporal effects on sensory and motor blockade after injection of bupivacaine or mepivacaine, and found that sensory loss was complete but about a 20% compound motor action potential remained after 40 minutes [23].\nIn conclusion, we have incorporated a method for assessing thumb motor deficit based on strength measurement with a standard local anesthetic to investigate the effects of a simulated median neuropathy on thumb motor function. Median nerve block results in force deficits in all directions, with the most severe impairment in abduction and flexion. Future endeavors using this methodology can potentially further elucidate underlying pathomechanisms of peripheral neuropathies in all digits of the hand.\n\n\n" ], "offsets": [ [ 0, 22150 ] ] } ]

[ { "id": "pmcA544950__T0", "type": "species", "text": [ "patients" ], "offsets": [ [ 18653, 18661 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] } ]

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[ { "id": "pmcA2398786__text", "type": "Article", "text": [ "Genomic-Bioinformatic Analysis of Transcripts Enriched in the Third-Stage Larva of the Parasitic Nematode Ascaris suum\nAbstract\nDifferential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ‘biological process’ (n = 68), ‘cellular component’ (n = 50), or ‘molecular function’ (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free-living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein–protein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%). A genetic interaction network was predicted for these 17 C. elegans orthologues, revealing highly significant interactions for nine molecules associated with embryonic and larval development (66.9%), information storage and processing (5.1%), cellular processing and signaling (15.2%), metabolism (6.1%), and unknown function (6.7%). The potential roles of these molecules in development are discussed in relation to the known roles of their homologues/orthologues in C. elegans and some other nematodes. The results of the present study provide a basis for future functional genomic studies to elucidate molecular aspects governing larval developmental processes in A. suum and/or the transition to parasitism.\n\nIntroduction\nParasitic nematodes are of major socio-economic importance in animals. For example, hundreds of millions of people are infected with geohelminths (soil-transmitted worms), such as blood-feeding hookworms Ancylostoma duodenale and/or Necator americanus, Trichuris trichiura and Ascaris spp. [1], causing serious adverse effects on human health, particularly in children. Similarly, parasitic nematodes of livestock, such as pigs, also cause substantial economic losses due to subclinical and clinical diseases, with billions of dollars spent annually on the treatment and control of gastro-intestinal nematodes. In addition to the socioeconomic impact that these parasites have, there is potential for the emergence of resistance in them against all of the main classes of (nematocidal) compounds used to treat the diseases they cause [2]–[5]. Therefore, there is a significant need to work toward discovering new compounds to control these parasites. Gaining an improved understanding of the molecular basis of parasite development provides such an avenue.\nCompared with the free-living nematode Caenorhabditis elegans, there is very little information on fundamental molecular aspects of development in parasitic nematodes [6]–[8]. Since the genome sequence of C. elegans was published in 1998 [9], many aspects of the molecular biology of this nematode have been elucidated. For instance, microarray analyses have been used to examine developmental and gender-enriched gene expression [10],[11], and the functions of more than 96% of the C. elegans genes have been assessed by double-stranded RNA interference (RNAi, or gene silencing; [12]) [13]–[18]. Comparative analyses of genetic data sets have shown that parasitic nematodes usually share ∼50–70% of genes with C. elegans (e.g., [19],[20]). There is similarity in other features (such as basic body plan and moulting) between C. elegans and parasitic nematodes, suggesting that some molecular pathways are relatively conserved [8],[21]. Understanding the pathways linked to basic nematode biology and development could have important implications for finding new ways of disrupting these pathways and thus facilitate the identification of new drug targets.\nDespite the advances in genomic technologies [7], [22]–[29] and the study of C. elegans, there is a paucity of information on the genomics of parasitic nematodes of animals, particularly in relation to development. Also considering the major socioeconomic impact of Ascaris and ascariasis in humans and pigs [30]–[32], several characteristics, including the large size of the adult worm (providing the opportunity of investigating individual organ systems and tissues), the ability to maintain Ascaris in the pig, store eggs and culture larvae in vitro for relatively long periods of time (months to years) [32] as well as the discovery that RNAi achieves “cross-species” gene silencing for a selected number of genes [33],[34] and the imminent genome sequence (http://www.sanger.ac.uk/Projects/Helminths/) all indicate that Ascaris could serve as a powerful model system for investigating reproductive and developmental processes in nematodes.\nIn the present study, Ascaris from pigs was used to study molecules abundantly transcribed in the infective third-stage larva (L3). Following the oral ingestion of Ascaris eggs by the host (human or pig), L3s are released and then invade/penetrate predominantly the caecal wall [35] to then undergo hepato-pulmonary migration, after which ultimately the adult females and males establish and develop in the small intestine [36],[37]. The molecular mechanisms linked to host invasion and parasite development are largely unknown. Here, we constructed an L3-enriched cDNA library using the method of suppressive-subtractive hybridization (SSH), explored transcription of a representative subset of molecules by microarray analysis and conducted bioinformatic analyses to characterize these molecules, map them to biochemical pathways and predict genetic interactions based on comparisons with C. elegans and/or other organisms.\n\nMaterials and Methods\nProduction of Different Developmental Stages of Ascaris\nExperimental pigs (8–12 weeks of age) were purchased from and maintained in the Experimental Animal Center of South China Agricultural University. These pigs were treated humanely, according to the Animal Ethics procedures and guidelines of the People's Republic of China. Adult worms (males and females) of A. suum were collected from the small intestines of pigs from an abattoir in Shenzhen, China. Infective eggs and infective L3s of A. suum were produced according to the methods described previously [38]. In brief, eggs from the uteri of adult females of A. suum were collected and incubated at 28°C for 28 days to allow them to develop to infective eggs (containing infective L3s). To obtain pure infective L3s, 7.5% v/v sodium hypochlorite was used to treat the larvated eggs at 37°C overnight and then the eggs were shaken with glass-beads; then, the exsheathed L3s and shells were separated by density gradient centrifugation using lymphocyte separating medium (LSM) [38]. Following the experimental infection of helminth-free pigs with infective Ascaris eggs as described previously [39], the L3s from livers and in lungs as well as L4s in intestines were isolated according to an established method [40]. All parasite materials were snap-frozen in liquid nitrogen prior to storage at −70°C.\n\nConstruction of the cDNA Library by Subtractive-Suppressive Hybridization (SSH)\nTotal RNA was isolated from adult females and males, different larval stages or eggs of A. suum using TriPure reagent (Roche) as recommended by the manufacturer. Equal amounts of total RNA from each stage or sex were pooled. The mRNA was isolated using the Oligotex mRNA Kit (Qiagen), following the manufacturer's protocol. SSH was carried out using the PCR-Select cDNA Subtraction kit (Clontech), according to the manufacturer's protocol. In brief, cDNA synthesized from mRNAs from infective L3s was subtracted against cDNA synthesized from the pooled mRNA from all other stages included herein. The SSH library was constructed using infective L3s as the tester and pooled cDNAs from all other stages as the driver. The effectiveness of this subtraction process has already been demonstrated in previous studies [41],[42]. The cDNA obtained following SSH was cloned into the pGEM-T Easy plasmid vector (Promega) and competent Escherichia coli (JM109) transformed. Positive clones, picked randomly (based on blue/white selection), were grown overnight in Luria Bertani (LB) medium (shaking, 37°C). Individual inserts were PCR-amplified using “nested primers” 1 and 2R from the Subtraction kit (Clontech) and examined by agarose electrophoresis.\n\nPreparation of Microarray Slides\nClones (n = 3075) from the subtracted library were picked and cultured overnight in LB containing ampicillin (1000 IU/ml) in sealed 96-well blocks. Five µl of culture suspension from each well were transferred into individual wells thermocycling (96-well) plates and the inserts PCR-amplified using primers 1 and 2R. Following a 10 min denaturation step at 94°C, the amplification proceeded for 25 cycles of 10 s at 94°C, 30 s at 68°C and 1.5 min at 72°C, with a final extension for 5 min at 72°C. Products were resolved in agarose gels, ethanol precipitated, re-suspended in 16 µl of “spotting solution” (Shanghai BioStar Genechip, Inc) to a final concentration of ∼500 ng per µl, before being printed on to glass slides (in duplicate) using a robotic arrayer. Sixteen blanks (using spotting solution only) and the same number of negative (irrelevant cDNAs with no relationship to Ascaris) were also printed on to slides and served as negative controls; β-actin of A. suum served as a positive control to assess the efficiency of labeling and hybridization. The slides were air-dried for 2 h, and cDNA in the spots were cross-linked at 254 mJ. The printed slides were stored at 4°C.\n\nLabeling of cDNA Probes with Fluorescent Dyes, and Microarray Analysis\nThe cDNAs produced from total RNA from A. suum eggs, infective L3s, L3s isolated from pig liver or lung, fourth-stage larvae (L4s), adult males or females [as described in the section ‘Construction of the cDNA Library by Subtractive-Suppressive Hybridization (SSH)’] were labeled with cyanine dyes. Cy3 or Cy5-dCTP was incorporated into cDNA produced from 30 µg of total RNA by direct labeling in a reverse transcription reaction using an oligo (dT) primer. Labeled cDNA was purified using DyeEx columns (Qiagen).\nMicroarray slides were incubated with a pre-hybridization solution [5×SSC, 1% bovine serum albumin (BSA), 0.1% sodium dodecyl-sulphate (SDS)] for 6 h at 42°C. After pre-hybridization, the microarray slides were incubated with ‘pooled’ Cy3 and Cy5-labeled probes in hybridization solution (5×SSC, 1% BSA, 0.1% SDS), in the dark at 42°C for 18 h, and then washed in solution I (1×SSC, 0.2% SDS) for 10 min, followed by solution II (0.1×SSC, 0.2% SDS) for 10 min at 60°C, according to the protocols provided by Shanghai BioStar Genechip, Inc. A “dye flip” was carried out to control for any bias in hybridization signal between the Cy-labeled cDNA probes (produced for two distinct mRNA populations). The slides were dried and scanned (ScanArray 4000 scanner) using image acquisition software (Shanghai BioStar Genechip Inc.) and a range of laser power and photo-multiplier tube intensities. The mean hybridization signal (derived from four replicates of the same array) were corrected for background, normalized [43], log2-transformed and then subjected to statistical analysis employing the students t-test in a spreadsheet (Excel, Microsoft, USA). The microarray data were analysed for differential cDNA hybridization (>2.0-fold to 114.3-fold) between L3 and each of the other stages (eggs, lung and liver L3s, L4, adult female and adult male).\n\nVerification of Differential Hybridization by Reverse Transcription-Coupled Polymerase Chain Reaction (RT-PCR) Analysis\nFor a subset (n = 17) of representative ESTs (rESTs), RT-PCR was used to verify the differential transcription recorded by microarray analysis. Double-stranded cDNA was synthesized from total RNA (separately) from each stage or sex of A. suum using reverse transcriptase (Superscript III, Invitrogen). Briefly, 5 µg of total RNA were added to 14 µl of H2O and 1 µl of oligo d(T)n = 12–18 primer (0.5 µg/µl), heated to 70 °C for 10 min and chilled on ice. First- and second-strand cDNAs were synthesized via the addition of 4 µl of first-strand cDNA buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl and 15 mM MgCl2), 2 µl of 0.1 M dithiothreitol, and 1 µl of 10 mM of each dNTP, followed by an incubation at 25 °C (10 min), 42 °C (50 min) and 70 °C (15 min). One-tenth of each double-stranded cDNA produced was then used as a template in the PCR. The transcripts were amplified from individual cDNAs by PCR using oligonucleotide primers (sequences available upon request) designed to each EST. The PCR amplification of a portion (209 bp) of the β-actin gene (accession no. BI594141) using forward primer (5′-CTCGAAACAAGAATACGATG-3′) and reverse primer (5′- ACATGTGCCGTTGTATGATG-3′), previously determined to be present in all developmental stages and both sexes of A. suum [44], served as a positive control. Samples without template (no-DNA controls) were included in each PCR run. The following cycling conditions were employed: one cycle at 94 °C (5 min), 94 °C (30 s), 60 °C (30 s) and 72 °C (30 s) for 30 cycles, followed by a final extension of 70 °C (7 min). Following the PCR, 5 µl of individual amplicons were resolved in ethidium bromide-stained agarose gels (2%) and then photographed upon transillumination. The relative band intensities were analyzed using UVIsoft Image Acquisition and Analysis software (UVITEC). The specificity and identity of individual amplicons were confirmed by direct sequencing using the same primers (separately) as employed for their amplification.\n\nSequencing and Bioinformatics Analyses\nClones from the SSH cDNA library with increased hybridization in microarray analysis to the infective L3 compared with other stages were sequenced using standard technology [45]. The nucleotide sequences have been deposited in the GenBank database under accession numbers ES290984-ES291074. Following the processing of the sequences (i.e., removal of vector sequences, quality assurance and clustering), contigs or singletons from individual clusters were subjected to BLASTx (NCBI: www.ncbi.nlm.nih.gov) and BLASTn (EMBL-EBI Parasite Genome Blast Server: www.ebi.ac.uk) analysis to identify putative homologues in C. elegans, other nematodes and other organisms (e-value of ≤1e-05). Peptides inferred from ESTs were classified functionally using Interproscan (available at http://www.ebi.ac.uk/InterProScan/) employing the default search parameters. WormBase (www.wormbase.org) was interrogated extensively for relevant information on C. elegans homologues/orthologues, including RNAi phenotypic, transcriptomic, proteomic and interactomic data. ESTs with homologues/orthologues in C. elegans and other nematodes were also subjected to analysis employing the KEGG Orthology-Based Annotation System (KOBAS) (www.kobas.cbi.pku.edu.cn), which predicts the biochemical pathways in which molecules are involved. The open reading frames (ORFs) inferred from selected ESTs with orthologues in C. elegans were also subjected to “secretome analysis” using the program SignalP v.2.0 www.cbs.dtu.dk/services/SignalP/), employing both the neural network and hidden Markov models to predict signal peptides and/or anchors [46]–[48]. Also, transmembrane domains were predicted using the program TMHMM (www.cbs.dtu.dk/services/TMHMM/; [49]–[51]), and subcellular localization inferred employing the program WoLF PSORT (http://wolfpsort.org/; [52]).\nThe method established by Zhong and Sternberg [53] was used to predict the interactions for C. elegans orthologues of the L3-enriched molecules from Ascaris. In brief, interaction, phenotypic, expression and gene ontology data from fruitfly, yeast, mouse and human were integrated using a naïve Bayesian model to predict genetic interactions among C. elegans genes ([45],[53]; Zhong and Sternberg, unpublished). The predicted networks resulting from the analyses were saved in a graphic display file (gdf) format and examined using the graph exploration system available at http://graphexploration.cond.org/. Images were labeled and saved in the joint photographic experts group (jpeg) format.\n\n\nResults\nTo identify molecules transcribed abundantly in the L3 of A. suum, an enriched cDNA library was constructed by SSH. From a total of 3075 clones from this library, 2921 (95%) were shown to contain an insert (which could be amplified by PCR). From 2671 (92%) of these clones, amplicons representing single bands of ∼400 to 600 bp in size were produced. These latter amplicons were arrayed (in duplicate) on to slides and then hybridized with Cy3-labeled L3-cDNA or with Cy5-labeled cDNA from eggs, liver/lung L3s, L4s, adult female or adult male of Ascaris. Dye flip was conducted to verify the hybridization data. Of the 2671 (duplicate) spots, 1526 had a significant difference in hybridization between infective L3 cDNA and cDNAs from all other stages or sexes of A. suum, of which 515 had a >2.0-fold increased hybridization for the L3.\nIn order to independently verify the hybridization results in the microarray, a PCR-based analysis of a selected subset (n = 17) clones was conducted using specific primer pairs. Having verified the specificity and identity of individual amplicons by sequencing, PCR results were reproducible (based on multiple runs on different days) and ∼94% (16 of 17) concordant with those of the microarray analysis (not shown). There was complete concordance for representative clones associated with a differential signal of ≥3.0-fold in the microarray.\nThe clones linked to the 515 spots representing increased transcription (>2.0-fold) in infective L3 compared with the other developmental stages or sexes included were subjected to sequencing. The 498 sequences (length: 550±115 bp) determined were then subjected to detailed bioinformatic analyses. There were 91 unique clusters (accession numbers ES290984-ES291074), of which 55 were singletons (sequences determined once). Of 56 molecules (61.5%) with significant similarity to sequences other than A. suum in the databases interrogated, 50 (54.9%) had C. elegans or C. briggsae homologues, and six had similarity to ESTs already sequenced from ascaridoid and/or other parasitic nematodes, and/or other organisms (Table 1). A significant proportion (38.4%) did not have any similarity in sequence to any organisms for which data are presently available. Comparative analysis specifically against A. suum EST data sets (n∼42,000) available in public databases confirmed independently that the majority of molecules (>60%) were present exclusively in the infective L3 stage or were orphans.\nAs gene ontology (GO) provides a hierarchy that unifies the descriptions of biological, cellular and molecular functions [54], this approach was employed to predict the classification and gene function of molecules enriched in infective L3 of A. suum. A summary of the GO categories of these molecules is displayed in Fig. 1. Of the 91 contigs, 32 (35%) could be functionally assigned to ‘biological process’ (n = 38), ‘cellular component’ (n = 17) and ‘molecular function’ (n = 64). The most common subcategories were gluconeogenesis (13%) and metabolic process (13%) within ‘biological process’, extracellular region (24%) within ‘cellular component’, and catalytic activity (11%) and phosphoenolpyruvate carboxykinase activity (8%) within ‘molecular function’ (Table S1).\nA focused KOBAS analysis inferred the 50 C. elegans orthologues/homologues to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%) or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway (Table 2). Of these 50 molecules, small numbers were predicted to be secreted (10%), anchored (2%) and/or transmembrane (12%) proteins (Table 2). Functionally, 17 (34%) of the 50 molecules were associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority displaying embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%) (Table 2).\nExtending this analysis, a relatively complex genetic interaction network was predicted for the 17 C. elegans orthologues (i.e., with non-wild-type RNAi phenotypes) (see Table S2). Statistically highly significant interactions were predicted for nine of the C. elegans genes; the top five interactors are displayed in Fig. 2. The gene ontology categories for eight selected C. elegans genes (F33D11.10, F55A12.8, kin-2, mec-12, mup-2, pab-1, rpl-22 and T21B10.2) included: embryonic development, egg hatching, larval development and/or growth. The other categories included: positive regulation of growth rate (F55A12.8, kin-2, mup-2, pab-1, rpl-22 and T21B10.2) and gamete generation and locomotory behaviour (kin-2, mup-2, pab-1 and F55A12.8, kin-2, mup-2, respectively). The C. elegans homologue egl-3 was predicted to be involved in proteolysis (see www.wormbase.org). All nine C. elegans orthologues were predicted to interact directly with a total of 296 (range: 5–75) other genes and, in particular, a direct genetic interaction was predicted between pab-1 and T21B10.2 (Fig. 2). The 296 interactors were associated with embryonic and larval development (n = 198; 66.9%), information storage and processing (n = 15; 5.1%), cellular processes and signalling (n = 45; 15.2%) and metabolism (n = 18; 6.1%); the precise function of some of the interactors (n = 20; 6.7%) is presently unknown (Table S2).\n\nDiscussion\nThe present study investigated transcripts in infective L3s of A. suum using a genomic-bioinformatic platform. The focus was on comparisons with C. elegans homologues/orthologues, because the entire genome sequence of this nematode is known [9] and because there is a wealth of information on the localization and functionality of its molecules (www.wormbase.org; http://elegans.bcgsc.bc.ca/knockout.shtml). The functions of most genes in C. elegans have been assessed using RNAi (e.g., [14],[15],[17],[55],[56]) in the hermaphroditic stage, whereas there is a paucity of functional information available for Ascaris and other parasitic nematodes of animals [57],[58].\nFollowing the microarray analysis of >2500 ESTs from the SSH library, 498 cDNAs inferred to be enriched in the L3, based on hybridization signal, were sequenced and subjected to comprehensive in silico analyses. Of the 91 clusters of molecules categorized, 50 (54.9%) had C. elegans homologues/orthologues with loss-of-function phenotypes could be mapped to key pathways. The statistically significant genetic interactions predicted for 9 of the 50 C. elegans orthologues [namely egl-3, F33D11.10, F55A12.8, kin-2, mec-12, mup-2, pab-1, rpl-22 and T21B10.2 ( = enol-1)] and the interaction network included genes encoding kinases, alpha-tubulins, enolases, troponin and other named and unnamed proteins. Eight of these molecules (enol-1, pab-1, F33D11.10, rpl-22, F55A12.8 mec-12, mup-2 and kin-2) have known or predicted roles in embryonic and larval growth and development, gamete generation, locomotory behaviour or other biological processes in C. elegans (see www.wormbase.org).\nThe enolase encoded by enol-1 is predicted to play a role in glycolysis, gluconeogenesis, phenylalanine, tyrosine and tryptophan biosynthesis (cf. [59]). Since glucose is the main source for ATP production, the alteration in these key glycolytic enzymes may lead to cellular dysfunction, such as impaired ion-motive ATPase required to maintain potential gradients, operate pumps and maintain membrane lipid asymmetry [60]. Bioinformatic analysis for transmembrane helices (TMHMM) and peptide signal sequences (SignalP) predicted ENOL-1 to be a non-secreted protein localized to the cytoplasm (cf. Table 2). Nonetheless, enolases are often detected in the excretory/secretory (ES) products of parasitic helminths, including adult A. suum [61], and appear to play a role in the triggering of nitric oxide production by host cells. The enol-1 orthologue of C. elegans has been predicted to interact specifically with the polyadenylate binding protein gene, pab-1, inferred to be involved in coordinated gene transcription and expression during normal larval development [16]. Poly(A)-binding proteins (PABPs) are recognized to be central to the regulation of mRNA translation and stability [62]. Present evidence suggests that the expression of PAB-1 is regulated by an oligo-pyrimidine tract in response to cell growth and relates to coordinated growth regulation in C. elegans [62]. Furthermore, gene silencing of pab-1 and its selected interactors (see Fig. 2) leads to embryonic lethal (Emb), slow growth (Slo) and sterile progeny (Stp) phenotypes (see www.wormbase.org).\nAnother gene (F33D11.10; EST code 4F10; see Table 2) which encodes an ATP-dependent RNA helicase and is associated with embryonic lethal (Emb) and larval lethal (Lvl) RNAi phenotypes, was shown to be highly transcribed in infective L3s of A. suum. Helicases are involved in a variety of RNA metabolic processes, including translation initiation, pre-mRNA splicing, pre-rRNA processing, rRNA maturation and RNA degradation [63], and are crucial for life cycle progression, sex determination and early embryogenesis in C. elegans [60]. The high transcription levels of a homologue/orthologue in the L3 of A. suum might suggest a similar role in this ascaridoid. Similarly, the coordination of the expression of a large number of genes is required for normal growth and cell proliferation during larval development. The high transcription level for the ribosomal protein gene homologue rpl-22 (large subunit family member; EST code 26G12, see Table 2) in the infective L3 of A. suum compared with other developmental stages is likely to reflect the substantial rate of cell growth in this stage [64].\nThe gene (F55A12.8, EST code 4G11; see Table 2) encoding an acetyl-transferase with a putative ATPase domain, shown to be enriched in the L3 of A. suum, was predicted to interact with 75 other genes all involved in energy production and/or RNA processing (see Table S2). Several molecules involved in ATP synthesis and mitochondrial pathways (e.g., cytochrome oxidase c subunits 1, 2 and 3, ADP/ATP translocases, NADH dehydrogenases, ATPases and ATP synthetases) have been reported previously to be highly represented in the L3 stage of Anisakis simplex [65], thus supporting the proposal that substantial energy is required for larval development as well as the transition from the free-living to the parasitic stage and the invasion of the host. There is also likely to be a substantial energy requirement for muscle contraction linked to larval motility in A. suum, as the L3s penetrate the caecal wall in the porcine host, before undergoing hepato-pulmonary migration [35]. Accordingly, genes encoding a specialized tubulin expressed in mechanoreceptors (mec-12, EST code 13E09) and a troponin (mup-2, EST code 01G03; see Table 2), both predicted to interact with a total number of 32 tubulin- and myosin-encoding genes, also supported a link to extensive muscle contraction and motility in A. suum L3s. Also, neuroactive peptides are required to regulate the responsiveness of nematode larvae to mechanical stimuli [66]. A homologue encoded by egl-3 was shown to be highly transcribed in the L3 of A. suum; EGL-3 is predicted to be a pro-hormone convertase involved in the maturation of neuropeptides and could be associated with mechano-sensory responses and touch sensitivity linked to the host invasion.\nA regulatory subunit of a cAMP-dependent protein kinase (kin-2, EST code 22H01; see Table 2) was predicted to interact with 72 other genes all involved in diverse cellular processes, such as nuclear trafficking, and DNA replication and repair (see Table S2). Based on gene ontology terms, kin-2 is implicated in gamete generation, growth, larval development, post-embryonic body morphogenesis, signal transduction and/or protein amino acid phosphorylation (see Table S2). Gene silencing of kin-2 in C. elegans leads to phenotypes, such as larval lethal (Lvl), larval arrest (Lva), body morphology defect (Bmd), dumpy (Dpy), uncoordinated (Unc) and sterile progeny (Stp) (www.wormbase.org), suggesting that its homologue in A. suum is central to larval maturation. The KOBAS analysis predicted the protein KIN-2 to be involved in the insulin-signaling pathway, previously implicated in controlling the exit from dauer in C. elegans and the activation of L3s of the canine hookworm, Ancylostoma caninum, following exsheathment [67]. In a recent study, Brand and Hawdon [68] were able to inhibit (with a phosphoinositide-3-OH-kinase inhibitor) the activation of infective L3s of both of the hookworms Ancylostoma caninum and Ancylostoma ceylanicum via the insulin signaling pathway, thus lending some credence to the hypothesis that this pathway plays an critical role in regulating the transition from the free-living to the parasitic stage [68]. Recently, it has been proposed that transcriptional and feeding responses to serum-stimulation in Ancylostoma caninum are regulated by parallel systems, with the insulin signaling pathway playing a significant role in the ‘resumption of feeding’ in activated larvae [69].\nProtein kinases are also likely to be involved in pathways linked to sexual maturation in developing larvae. As already proposed for adult stages of H. contortus [45], the protein kinase gene cdk-1 is predicted to play a pivotal role in the germline, oogenesis and spermiogenesis pathways of this parasitic nematode. Other protein kinases, such as PEPCK, and phosphatases, were shown herein to be transcribed at high levels in the L3 stage compared with other developmental stages of A. suum (see Table 2), which is in accordance to findings reported recently for Anisakis simplex [65]. Due to their major regulatory effects in eukaryotic signaling events and regulatory and sensory functions, protein kinases have been considered interesting targets for anti-parasitic drugs [70].\nIn conclusion, this study has given some interesting insights into early molecular processes in the L3 of A. suum. Approximately 60% of the transcripts enriched in the L3 stage of A. suum have homologues/orthologues in C. elegans. The bioinformatic analyses of selected molecules suggest that a complex genetic network regulates or controls larval growth and development in A. suum L3s, and some of these might be involved in or regulate the switch from the free-living to the parasitic stage. Some caution is warranted in drawing conclusions regarding molecular mechanisms regulating the transition to parasitism in parasitic nematodes from information on C. elegans, as latter is a free-living nematode. Also, while the method of data integration is essential for the reliable prediction of genetic interactions, it might limit the capacity of the approach somewhat to infer nematode-specific interactions. As additional datasets of genes and gene functions become available for various parasitic nematodes, more informed inferences can be made regarding the functions of nematode-specific genes, particularly those involved in the transition to parasitism. The imminent genome sequence of A. suum (http://www.sanger.ac.uk/Projects/Helminths/) should all assist in this endeavour. Also, functional analysis of selected molecules representing selected ESTs identified herein, utilizing gene silencing approaches established recently [33],[34], could provide some insights into developmental processes in Ascaris and related ascaridoid nematodes and provide avenues for the development of novel approaches for their control.\n\nSupporting Information\n\n\n" ], "offsets": [ [ 0, 33143 ] ] } ]

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[ { "id": "pmcA2276505__text", "type": "Article", "text": [ "Port site herniation of the small bowel following laparoscopy-assisted distal gastrectomy: a case report\nAbstract\nIntroduction\nPort-site herniation is a rare but potentially dangerous complication after laparoscopic surgery. Closure of port sites, especially those measuring 10 mm or more, has been recommended to avoid such an event.\n\nCase presentation\nWe herein report the only case of a port site hernia among a series 52 consecutive cases of laparoscopy-assisted distal gastrectomy (LADG) carried out by our unit between July 2002 and March 2007. In this case the small bowel herniated and incarcerated through the port site on day 4 after LADG despite closure of the fascia. Initial manifestations experienced by the patient, possibly due to obstruction, and including mild abdominal pain and nausea, occurred on the third day postoperatively. The definitive diagnosis was made on day 4 based on symptoms related to leakage from the duodenal stump, which was considered to have developed after severe obstruction of the bowel. Re-operation for reduction of the incarcerated bowel and tube duodenostomy with peritoneal drainage were required to manage this complication.\n\nConclusion\nWe present this case report and review of literature to discuss further regarding methods of fascial closure after laparoscopic surgery.\n\n\n\nIntroduction\nBowel herniation through the fascial defect created by the entry of trocars is now recognized as a rare but potentially serious complication of laparoscopic surgery [1]. Although port site herniation is an infrequent complication, there are still some reports of port site herniation after these procedures, even with closure of trocar sites[1,2]. The following report describes a case of a trocar site hernia that evolved into leakage from the duodenal stump after laparoscopy-assisted distal gastrectomy (LADG). Progression occurred because of complete obstruction of the incarcerated bowel after a Roux-en-Y reconstruction. We describe the significance of complete closure of the fascial defect at the trocar site including the peritoneum in the prevention of this condition, as well as the importance of early diagnosis to avoid serious subsequent events.\n\nCase presentation\nAn 80-year-old man was found to have early gastric cancer during his yearly check-up by gastrointestinal endoscopy. He was 158 cm in height and weighed 62 kg. Gastrointestinal endoscopy showed a depressed lesion that was diagnosed as early gastric cancer by pathological examination of biopsy specimens. He underwent LADG with regional lymph node dissection (D1 including the nodes surrounding the origin of left gastric artery). A 12-mm trocar for the laparoscope was placed in the umbilicus. Pneumoperitoneum was then established with carbon dioxide and the intraperitoneal pressure was maintained at 10 mm Hg. Two more 12-mm trocars were inserted in the midclavicular line below the costal margin and 2 cm above the umbilicus on each of both flanks and were used for active surgical instruments. All trocars used were the non-bladed type. The specimen was removed through a small medial incision which was 55 mm in length placed after resection of the stomach, and then Roux-en-Y reconstruction (RY) was carried out (Fig. 1). A tubular shaped drainage tube 10 mm in diameter was inserted and placed through the upper trocar site made on the right flank. Wound defect at the umbilical port site was sutured completely including the peritoneum with 0 absorbable suture and fascial incisions at all other trocar insertion sites were closed with 2-0 absorbable sutures. Surgical duration was 263 min, and the volume of blood loss was less than 50 mL with no blood transfusion.\nPostoperatively, the patient complained of an acidic feeling in his stomach; however, there were no remarkable abnormalities on biochemical examination of serum. Radiological findings did not suggest bowel obstruction until 3 days postoperatively, although mild symptoms such as general malaise and vague abdominal pain were reported on day three. However, on day 4, the patient started to complain of upper abdominal pain and developed a high grade fever (38°C). Complete obstruction of the small bowel and leakage of contrast media were demonstrated by Gastrografin swallow and subsequent abdominal computed tomography (CT). CT also showed a mass lesion at the trocar insertion site on the upper left flank, suggesting herniation through the port site (Fig. 2). Marked dilatation of the duodenum including the horizontal part and second portion was observed. A diagnosis of staple failure of the stump of the duodenum and port-site herniation of the small bowel was made, and exploratory laparotomy was carried out. A small medial incision that had been made at the initial surgery was extended downward to the umbilicus to open the peritoneal cavity. As we expected, the small bowel was incarcerated into the peritoneal defect in the abdominal wall created by the trocar placed in the left upper flank leading to complete obstruction of the bowel (Fig. 3). Part of the jejunum 30 cm distal from the ligament of Treitz herniated around the fascial stitch, which still existed at the time of the re-exploration. The peritoneal cavity was contaminated with intestinal juice. Close examination after reduction of the incarcerated bowel did not demonstrate necrosis of the intestine, and thus, we decided not to resect this lesion. Leakage of intestinal juice through a pinhole fistula at the duodenal stump was also observed. Tube duodenostomy was performed with an omental patch used for closure of the fistula. The peritoneal defect was also closed. The postoperative course was fairly good without high output of the intestinal juice leakage or sepsis. The patient remained in the intensive care unit for 5 days after re-operation, and was then transferred to the general ward.\n\nDiscussion\nPort-site herniation, which is one of the major complications after laparoscopic procedures [1], sometimes develops into serious complications, such as bowel obstruction due to incarceration into the fascial defect at the port site. Boughey et al. have reported four cases of Richter's hernia that occurred at a port site after laparoscopic surgery [1]. They reviewed previous reports and found the incidence to be 0.2 to 3%. A report describes the incidence of hernia as 0.23% for 10-mm trocar use, rising to 3.1% for the 12-mm trocar [2] suggesting that the wound created by a larger port carries a greater risk of herniation. Most surgeons now routinely close the fascia of port sites to prevent this complication [2]. According to previous reports, port site herniation apparently happens more often with the use of bladed type trocars than non-bladed type trocars [3]. Indeed, Kolata demonstrated that the wounds made by the non-bladed trocar were narrower than those created by cutting tip trocars in a pig experimental model [4]. Several reports even concluded that port sites created by non-bladed trocars do not require fascial closure [3]. However, the current case suggests that thick preperitoneum is a potential space that allows for the development of bowel herniation even with the use of non-bladed type trocars. A previous report also described port-site herniation, despite the closure of the superficial layer of the fascial defect [5]. The current case did not demonstrate any of the risk factors suggested previously [6]; 1) enlargement of a port site to remove specimen; 2) glucose intolerance; 3) obesity; or 4) extensive manipulation of the trocar during relatively prolonged surgical duration, which might have enlarged the trocar site and thus induced bowel herniation. Therefore, we recommend closing the fascial defect, including the peritoneum, especially if the trocar size is more than 10-mm and in the presence of any of the risk factors described above. However, it is sometimes difficult to completely close the defect, including the peritoneum, especially in obese patients. Shaher reviewed different wound closure techniques by a literature search [7]. In this review, old methods using classical instruments including Deschamps needle are also useful as well as special wound devices designed for port site closure. Elashry et al. described a prospective randomized study demonstrating that the Carter-Thomason device was faster and resulted in fewer port-closure-related complications among eight different techniques tested [8]. Insertion of a SURGICEL plug into the muscular layer of trocar wounds has also been proposed by Chiu et al [9]. Alternatively, tangential insertion of a trocar through the abdominal wall might be effective in reducing the size of fascial defects. Moreover, recent publications have demonstrated that radially expanding type trocars could be useful to avoid the necessity of closing the fascial defect [10].\nSymptoms of trocar-site herniation vary depending on the severity of bowel obstruction. Mild symptoms such as slight nausea and vague abdominal pain, both of which are most frequently seen in the early normal postoperative course after abdominal surgery, could be the first and only complaints at the early stage of this complication. Thus, the diagnosis may be delayed. In our case, mild abdominal pain with general malaise might have been symptoms related to the early stage of the onset. Abdominal CT showing the enlarged duodenum also suggested that leakage from the duodenal stump occurred due to the obstruction of the distal bowel. Thus, severe complication might have been avoided, if early diagnosis had been made. Although the benefit of Roux-en-Y is apparent [11], the duodenal stump could be vulnerable to leakage due to increased intrabowel pressure. Therefore, careful management of the postoperative course is warranted, especially after procedures involving division of the bowel such as LADG. Moreover, special attention should be paid in patients with risk factors for port site hernia such as obesity, aggressive manipulation through the port sites, and prolonged surgery.\n\nConclusion\nPort-site herniation is a potentially dangerous complication after laparoscopic procedures. Careful management of the postoperative course is recommended especially for patients with risk factors such as obesity and extensive manipulation of the trocar during prolonged surgical duration.\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\nAuthors' contributions\nTI, NF, HT and TK performed the first and second operation. TI and KK were responsible for the postoperative management. TI, HT, TW, and KN were involved in editing the manuscript. All authors read and approved the final manuscript.\n\nConsent\nWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.\n\n\n" ], "offsets": [ [ 0, 10919 ] ] } ]

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[ { "id": "pmcA2435603__text", "type": "Article", "text": [ "Potentiation of Nerve Growth Factor-Induced Neurite Outgrowth by Fluvoxamine: Role of Sigma-1 Receptors, IP3 Receptors and Cellular Signaling Pathways\nAbstract\nBackground\nSelective serotonin reuptake inhibitors (SSRIs) have been widely used and are a major therapeutic advance in psychopharmacology. However, their pharmacology is quite heterogeneous. The SSRI fluvoxamine, with sigma-1 receptor agonism, is shown to potentiate nerve-growth factor (NGF)-induced neurite outgrowth in PC 12 cells. However, the precise cellular and molecular mechanisms underlying potentiation by fluvoxamine are not fully understood. In this study, we examined the roles of cellular signaling pathways in the potentiation of NGF-induced neurite outgrowth by fluvoxamine and sigma-1 receptor agonists.\n\nMethods and Findings\nThe effects of three SSRIs (fluvoxamine, sertraline, paroxetine) and three sigma-1 receptor agonists (SA4503, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP), and dehydroepiandrosterone (DHEA)-sulfate) on NGF-induced neurite outgrowth in PC12 cells were examined. Also examined were the effects of the sigma-1 receptor antagonist NE-100, inositol 1,4,5-triphosphate (IP3) receptor antagonist, and specific inhibitors of signaling pathways in the potentiation of NGF-induced neurite outgrowth by selective sigma-1 receptor agonist SA4503. Fluvoxamine (but not sertraline or paroxetine) and the sigma-1 receptor agonists SA4503, PPBP, and DHEA-sulfate significantly potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. The potentiation by fluvoxamine and the three sigma-1 receptor agonists was blocked by co-administration of the selective sigma-1 receptor antagonist NE-100, suggesting that sigma-1 receptors play a role in blocking the enhancement of NGF-induced neurite outgrowth. Moreover, the potentiation by SA4503 was blocked by co-administration of the IP3 receptor antagonist xestospongin C. In addition, the specific inhibitors of phospholipase C (PLC-γ), phosphatidylinositol 3-kinase (PI3K), p38MAPK, c-Jun N-terminal kinase (JNK), and the Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways blocked the potentiation of NGF-induced neurite outgrowth by SA4503.\n\nConclusion\nThese findings suggest that stimulation of sigma-1 receptors and subsequent interaction with IP3 receptors, PLC-γ, PI3K, p38MAPK, JNK, and the Ras/Raf/MAPK signaling pathways are involved in the mechanisms of action of sigma-1 receptor agonists such as fluvoxamine and SA4503.\n\n\n\nIntroduction\nSelective serotonin (5-HT; 5-hydroxytryptamine) reuptake inhibitors (SSRIs) have emerged as a major therapeutic advance in psychopharmacology. SSRIs are the treatment of choice for many indications, including major depressive disorder, dysthymia, panic disorder, obsessive-compulsive disorder, eating disorders, and premenstrual dysphoric disorder. In contrast, it is well known that their pharmacology is quite heterogeneous, although all of them block 5-HT transporters, thus increasing 5-HT levels throughout the central nervous system (CNS) [1]–[9].\nAccumulating evidence suggests that sigma-1 receptors, which are intracellular endoplasmic reticulum (ER) proteins, are involved in both the neuroplasticity and pathophysiology of neuropsychiatric diseases such as major depressive disorder, anxiety, schizophrenia, and Alzheimer's disease [10]–[18]. Previously, we reported that some SSRIs possess high to moderate affinities for sigma-1 receptors in the rat brain. The rank order of SSRIs affinities for sigma-1 receptors is fluvoxamine (Ki = 36 nM)>sertraline (Ki = 57 nM)>>paroxetine (Ki = 1893 nM) [19]. Recently, we reported that fluvoxamine, but not paroxetine, significantly ameliorated cognitive deficits in mice after repeated phencyclidine administration, and that the effects of fluvoxamine were antagonized by co-administration of the selective sigma-1 receptor antagonist NE-100 [20], suggesting that sigma-1 receptors are involved in the mechanism of action of fluvoxamine [9]. Interestingly, it has been demonstrated that sigma-1 receptor agonists including fluvoxamine could potentiate nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells, and that NE-100 blocked the potentiation by sigma-1 receptor agonists, suggesting sigma-1 receptors are involved in neuroplasticity [21]. However, the precise cellular mechanisms underlying the potentiation by sigma-1 receptor agonists are not fully understood [13], [21].\nIt is therefore of great interest to study the precise cellular mechanisms underlying the enhancement by fluvoxamine on NGF-induced neurite sprouting in PC12 cells. In the present study, we examined the effects of three SSRIs (fluvoxamine, sertraline, paroxetine), as well as the effects of a sigma-1 receptor agonist (4-phenyl-1-(4-phenylbutyl) piperidine (PPBP), dehydroepiandrosterone-sulphate (DHEA)-sulfate) [9], [22]–[28] and the selective sigma-1 receptor agonist SA4503 [29], [30], on NGF-induced neurite outgrowth in PC12 cells. Furthermore, it is also known that sigma-1 receptors have been shown to interact with IP3 receptors (17,18). Therefore, we examined the effects of NE-100 and xestospongin C (a selective inositol 1,4,5-triphosphate (IP3) receptor antagonist) [31] in order to investigate the roles of sigma-1 receptors and IP3 receptors in the mechanisms underlying the enhancement of NGF-induced neurite outgrowth by SA4503. Moreover, we examined the effects of specific inhibitors of several cellular signaling targets on the enhancement of NGF-induced neurite outgrowth by SA4503, since several signal transduction molecules have been implicated in NGF-induced neurite outgrowth [32].\n\nMaterials and Methods\nDrugs\nThe drugs were obtained from the following sources: fluvoxamine maleate (Solvay Seiyaku K.K., Tokyo, Japan); paroxetine hydrochloride, dehydroepiandosterone-sulfate (DHEA-sulfate), LY294002 (Sigma-Aldrich, St Louis, MO, USA); sertraline (Toronto Research Chemicals Inc., North York, ON, Canada); SA4503 (M's Science Corporation, Kobe, Japan); NGF (Promega, Madison, WI); xestospongin C, lovastatin, PD98059, GW5074, SB203580, MEK 1/2 inhibitor (SL327), and SP600125 (Calbiochem-Novabiochem, San Diego, CA). The selective sigma-1 receptor antagonists NE-100 and 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) were synthesized in our laboratory. Other drugs were purchased from commercial sources.\n\nCell culture\nPC12 sells (RIKEN Cell Bank, Tsukuba, Japan) were cultured at 37°C, 5% CO2 with Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 10% heat-inactivated horse serum, and 1% penicillin. The medium was changed two or three times a week. PC12 cells were plated onto 24-well tissue culture plates coated with poly-D-lysine/laminin. Cells were plated at relatively low density (0.25×104 cells/cm2) in DMEM medium containing 0.5% FBS, 1% penicillin streptomycin. Medium containing a minimal level of serum (0.5% FBS) was used, since serum is known to contain steroid hormones that bind to sigma-1 receptors [21]. First, we examined the optimal concentration of NGF for NGF-induced neurite outgrowth in PC12 cells. NGF (2.5, 5, 10, 20, 40 ng/ml) increased the number of cells with neurite outgrowth in PC12 cells in a concentration-dependent manner (Figure 1). In the subsequent studies, 2.5 ng/ml of NGF was used to study the potentiating effects of sigma-1 receptor agonists on NGF-induced neurite outgrowth. Twenty-four hours after plating, the medium was replaced with DMEM medium containing 0.5% FBS and 1% penicillin streptomycin with NGF (2.5 ng/ml) with or without several drugs.\n\nQuantification of neurite sprouting\nFive days after incubation with NGF (2.5 ng/ml) with or without the several drugs, morphometric analysis was performed on digitized images of live cells taken under phase-contrast illumination with an inverted microscope linked to a camera. Images of three fields per well were taken, with an average of 100 cells per field. Differentiated cells were counted by visual examination of the field; only cells that had at least one neurite with a length equal to the cell body diameter were counted, and were then expressed as a percentage of the total cells in the field. The counting was performed in a blinded manner.\n\nImmunocytochemistry\nCells were fixed for 30 min at room temperature with 4% paraformaldehyde then permeabilized with 0.2% Triton and blocked with 1.5% normal goat serum, 0.1% bovine serum albumin (BSA) in 0.1 M phosphate-buffer saline for 1 h to reduce nonspecific binding. Cells were incubated overnight at 4°C with anti-microtubule-associated protein 2 (MAP-2) antibodies (1∶1000 dilution in blocking solution, Chemicon International, Temecula, CA, USA). The immunolabeling was visualized with secondary antibodies conjugated to Alexa-488 (1∶1000; Invitrogen, Carlsbad, CA, USA). MAP-2 immuncytochemistry was visualized with a fluorescence microscope (Axiovert 200, Carl Zeiss, Oberkocken, Germany).\n\nStatistical analysis\nData are expressed as means±S.E.M. Statistical analysis was performed by using one-way analysis of variance (ANOVA) and the post hoc Bonferroni/Dunn test. P values less than 0.05 were considered statistically significant.\n\n\nResults\nEffects of SSRIs on NGF-induced neurite outgrowth\nFluvoxamine (0.1, 1.0, or 10 µM) significantly increased the number of cells with neurite outgrowth by NGF (2.5 ng/ml) in PC12 cells, in a concentration-dependent manner (Figure 2 and 4). In contrast, sertraline (0.1, 1.0, or 10 µM) and paroxetine (0.1, 1.0, or 10 µM) did not increase the number of cells with neurite outgrowth by NGF (2.5 ng/ml). A higher concentration (10 µM) of paroxetine and sertraline significantly decreased the number of cells with NGF-induced neurite outgrowth, suggesting the cellular toxicity of these drugs in PC12 cells (Figure 2).\n\nRole of sigma-1 receptors in the potentiation of NGF-induced neurite outgrowth by fluvoxamine and SA4503\nThe selective and potent sigma-1 receptor agonist SA4503 (0.01, 0.1, or 1.0 µM) significantly increased the number of cells with neurite outgrowth by NGF (2.5 ng/ml) in PC12 cells, in a concentration-dependent manner (Figure 3 and 4). In addition, other sigma-1 receptor agonists–PPBP (0.1, 1.0, or 10 µM) [22]–[26] and DHEA-sulphate (0.1, 1.0, or 10 µM) [9], [27], [28]–significantly increased the number of such cells, also in a concentration-dependent manner (Figure 3). In the absence of NGF, sigma-1 receptor agonists did not produce neurite outgrowth in PC12 cells (data not shown).\nTo investigate the role of sigma-1 receptors, we examined the effects of NE-100 (a selective sigma-1 receptor antagonist) on the potentiation of NGF-induced neurite outgrowth by fluvoxamine, SA4503, PPBP, and DHEA-sulphate. Co-administration of NE-100 (1.0 µM) significantly blocked such potentiation by fluvoxamine (10 µM), SA4503 (1.0 µM), PPBP (10 µM), or DHEA-sulphate (10 µM) (Figure 2 and 3). Furthermore, administration of NE-100 (1.0 µM) alone did not alter NGF-induced neurite outgrowth in PC12 cells (Figure 2).\n\nRole of IP3 receptors in the potentiation of NGF-induced neurite outgrowth by SA4503\nSigma-1 receptors have been shown to interact with IP3 receptors (17,18,33–35). To investigate the role of IP3 receptors in the effects of SA4503 on NGF-induced neurite outgrowth, we examined the effects of xestospongin C (a selective, reversible, and membrane-permeable inhibitor of IP3 receptors) [31] on the effects of SA4503 on NGF-induced neurite outgrowth. Co-administration of xestospongin C (1.0 µM) significantly blocked the potentiation of NGF-induced neurite outgrowth by SA4503 (1.0 µM) (Figure 5). Furthermore, administration of xestospongin C (1.0 µM) alone did not alter NGF-induced neurite outgrowth in PC12 cells (Figure 5).\n\nRole of signaling molecules proximal to TrkA in the potentiation of NGF-induced neurite outgrowth by SA4503\nNext, we examined the effects of the specific inhibitors of PLC-γ, PI3K, p38 MAPK, and c-Jun N-terminal kinase (JNK), since these signaling molecules are activated upon the addition of NGF [36]. The PLC-γ inhibitor (U73122; 1.0 µM), PI3K inhibitor (LY294002; 1.0 µM), p38 MAPK inhibitor (SB203580; 1.0 µM), and JNK inhibitor (SP600125; 1.0 µM) significantly blocked the potentiation of NGF-induced neurite outgrowth by SA4503 (1.0 µM) (Figure 6). In contrast, these inhibitors alone did not alter NGF-induced neurite outgrowth in PC12 cells (Figure 6).\n\nRole of Raf/Ras/ERK/MAPK pathway in the potentiation of NGF-induced neurite outgrowth by SA4503\nThe Raf/Ras/ERK/MAPK pathway is known to be involved in NGF-induced outgrowth [32]. Therefore, we examined the effects of this pathway's specific inhibitors. The Ras inhibitor (GW5074; 1.0 µM), Raf inhibitor (lovastatin; 1.0 µM), MEK1/2 inhibitor (SL327; 1.0 µM), and MAPK inhibitor (PD98059; 1.0 µM) significantly blocked the potentiation of NGF-induced neurite outgrowth by SA4503 (1.0 µM) (Figure 7). In contrast, these inhibitors alone did not alter NGF-induced neurite outgrowth in PC12 cells (Figure 7).\n\n\nDiscussion\nIn the present study, we found that fluvoxamine (but not sertraline or paroxetine) and the sigma-1 receptor agonists (SA4503, PPBP, DHEA-sulfate) could potentiate NGF-induced neurite outgrowth in PC12 cells, and that the effects of these drugs were blocked by co-incubation with the selective sigma-1 receptor antagonist NE-100. These findings suggest that agonism at sigma-1 receptors for these drugs is involved in the mechanisms underlying the drugs' potentiation of NGF-induced neurite outgrowth.\nAs shown in Figure 1, NGF increased the number of cells with neurite outgrowth in PC12, in a concentration dependent manner. To examine whether or not the NGF levels are altered by incubation with sigma-1 receptor agonists, we measured the levels of NGF in PC12 cells with or without sigma-1 receptor agonist SA4503. No differences of NGF levels were shown in the between control group and SA4503-treated group (data not shown). It is, therefore, unlikely that the potentaition of NGF-induced neurite outgrowth by sigma-1 receptor agonists might be due to increased levels of NGF.\nUnlike fluvoxamine, sertraline, which has a moderate affinity for sigma-1 receptors, did not alter NGF-induced neurite outgrowth. The reasons underlying this discrepancy between these two SSRIs are currently unclear. One possibility may involve the difference in pharmacological actions (agonist vs. antagonist) between these SSRIs at sigma-1 receptors. Interestingly, we recently found that, in a novel object recognition test, phencyclidine-induced cognitive deficits could be significantly improved by subsequent subchronic (14 days) administration of fluvoxamine, but not of sertraline, suggesting that sigma-1 receptor agonism is involved in fluvoxamine's mechanism of action [9], Ishima et al., submitted. Taken together, these findings suggest that fluvoxamine and sertraline may function as an agonist and an antagonist at sigma-1 receptors, respectively, although further study is necessary. Another possibility may be that other pharmacological activities of sertraline mask the effects of sigma-1 receptor agonism. In this study, we also found that high concentration (10 µM) of paroxetine and sertraline, but not fluvoxamine, showed cytotoxicity in PC12 cells, suggesting that fluvoxamine may be a safe drug than paroxetine and sertraline.\nSigma-1 receptors have been shown to affect intracellular Ca2+ signaling, although the precise molecular and cellular mechanisms underlying this effect are unknown. Sigma-1 receptors bind to IP3 receptors in ER, and sigma-1 receptors regulate Ca2+ release from intracellular Ca2+ storage sites [12]. Very recently, Hayashi and Su [17] reported that sigma-1 receptors function as novel ligand-operated chaperones that specifically target mitochondrion-associated ER membrane. Furthermore, sigma-1 receptors form Ca2+-sensitive chaperone machinery with another chaperone, BiP, and prolong Ca2+ signaling from ER into mitochondria by stabilizing IP3 receptors at mitochondrion-associated ER membrane [17]. In this study, we found that the IP3 receptor antagonist xestospongin C significantly blocked the potentiation of NGF-induced neurite outgrowth by SA4503, suggesting the role of IP3 receptors on sigma-1 receptor-mediated potentiation of NGF-induced neurite outgrowth. Therefore, it is likely that stimulation at sigma-1 receptors by sigma-1 receptor agonists and subsequent interaction with IP3 receptors are involved in the mechanism underlying the potentiation of NGF-induced neurite outgrowth by sigma-1 receptor agonists.\nNGF binds to the high-affinity tyrosine receptor TrkA, initiating several signaling pathways affecting both morphological and transcriptional targets [32], [36], [37]. The signaling molecules, including PLC-γ, PI3K, p38 MAPK, and JNK, are activated upon the addition of NGF [38]. PLC-γ catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol triphosphate (IP3). DAG activates protein kinase C, and IP3 promotes transient release of Ca2+ from the ER [39]. The pathway via PLC-γ is responsible for NGF-induced cell differentiation [40] and neurite outgrowth [41]. Furthermore, stimulation of PI3K is reported to be involved in the promotion of neurite outgrowth in PC12 cells [42]. In this study, we found that the PLC-γ inhibitor U73122 and the PI3K inhibitor LY294002 significantly blocked the potentiation of NGF-induced neurite outgrowth by SA4503. Moreover, we found that both the p38MAPK inhibitor SB203580 and the JNK inhibitor SP600125 significantly blocked the potentiation of NGF-induced neurite outgrowth by SA4503. In addition, it is also interesting that neurite outgrowth induced by low concentration (2.5 ng/ml) of NGF was not blocked by these inihibitors, consistent with a previous report [43], [44], suggesting the existence of novel pathway(s) for NGF-induced neurite outgrowth. These findings suggest that the PLC-γ, PI3K, p38MAPK, and JNK signaling pathways are involved in the potentiation of NGF-induced neurite outgrowth by sigma-1 receptor agonists (Figure 8). In addition, we found that the specific inhibitors for the Raf/Ras/MEK/MAPK pathways significantly blocked the potentiation of NGF-induced neurite outgrowth by SA4503, suggesting that these pathways are involved in the potentiation of NGF-induced neurite outgrowth by sigma-1 receptor agonists (Figure 8).\nOur recent positron emission tomography (PET) study demonstrated that, after a single oral administration, fluvoxamine bound to sigma-1 receptors in the living human brain [45]. This finding suggests that sigma-1 receptors are involved in the mechanism of action of fluvoxamine in the human brain [45]. Taken together, the past and present findings suggest that, unlike paroxetine and sertraline, the SSRI fluvoxamine, with its sigma-1 receptor agonistic activity, might be a unique therapeutic drug for neuropsychiatric diseases.\nIn conclusion, the present results suggest that, as a sigma-1 receptor agonist, fluvoxamine could potentiate the NGF-induced neurite outgrowth in PC12 cells. Furthermore, it is likely that interaction with IP3 receptors and several subsequent signaling molecules are involved in the mechanism underlying the pharmacological action of sigma-1 receptor agonists. Therefore, it is likely that sigma-1 receptor agonists such as fluvoxamine and DHEA-sulfate, which are available around the world, would be unique therapeutic drugs for neuropsychiatric diseases.\n\n\n" ], "offsets": [ [ 0, 19608 ] ] } ]

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[ { "id": "pmcA1562423__text", "type": "Article", "text": [ "Alexithymia and anxiety in female chronic pain patients\nAbstract\nObjectives\nAlexithymia is highly prevalent among chronic pain patients. Pain is a remarkable cause for high levels of chronic anxiety. The purpose of this study was to investigate the prevalence of alexithymia and to determine anxiety levels among DSM-IV somatoform pain disorder (chronic pain) female patients and to examine the relationship between alexithymia and the self-reporting of pain.\n\nMethods\nThirty adult females (mean age: 34,63 ± 10,62 years), who applied to the outpatient psychiatry clinic at a public hospital with the diagnosis of chronic pain disorder (DSM-IV), were included in the study. Thirty seven healthy females (mean age: 34,46 ± 7,43 years), who matched for sociodemographic features with the patient group, consisted the control group. A sociodemographic data form, 26-item Toronto Alexithymia Scale (TAS-26), Spielberger Trait Anxiety Inventory (STAI) were administered to each subject and information was obtained on several aspects of the patients' pain, including intensity (measured by VAS), and duration.\n\nResults\nChronic pain patients were found significantly more alexithymic than controls. There was a positive correlation between TAS-26 scores and the duration of pain. The alexithymic and nonalexithymic group did not differ in their perception of pain. Neither positive correlation nor significant difference was found between alexithymia and trait anxiety in pain patients.\n\nDiscussion\nAlexithymia may be important in addressing the diversity of subjective factors involved in pain. The conceptualization of alexithymia as a personality trait as well as a secondary state reaction is underlined by our data.\n\n\n\nBackground\nThe original definition of alexithymia is the inability to identify and use verbal language to describe feelings [1,2]. Alexithymia has been associated with a variety of psychiatric disorders as well as physical illness [3-10]. As a measure, Toronto Alexithymia Scale was significantly correlated with the measures of the tendency to experience and report physical signs and symptoms [11].\nSeveral studies have found a high prevalence of alexithymia in pain patients. Chronic pain patients frequently exhibit many of the core features of alexithymia, such as problems in identifying and describing subjective feelings, impoverished imaginative abilities, and excessive preoccupation with physical symptoms and external events. Although several studies have found a high prevalence of alexithymia in pain patients, the way alexithymia may possibly influence pain experience is still unclear [12,13].\nDSM-IV-TR defines pain disorder as the presence of pain that is \"the predominant focus of clinical attention\" [14]. In chronic pain disorder, patients complain of chronic pain, for which no physical etiology could be found or the underlying disorder is insufficient in explaining the symptoms. The pain causes clinically significant distress or impairment in social, occupational, or other important areas of functioning. Psychological factors are judged to have an important role in the onset, severity, exacerbation, or maintenance of the pain [15].\nThe alexithymic person's difficulty in identifying and describing feelings may increase symptom reporting by several mechanisms. Consequently, due to the difficulty to experience and express emotions, alexithymia has been linked with somatosensory amplification, which is the tendency to focus on benign somatic sensations. Alexithymic subjects are considered to focus on somatic manifestations of emotional arousal, resulting in misinterpretation of somatic sensations as signs of physical illness [12,13,16]. Accordingly, previous studies have found evidence of an association between alexithymia and the development of functional somatic symptoms, as seen in patients with somatoform disorders. On the other hand, alexithymia may also occur as a secondary state reaction in response to severe and chronic medical illness [17-21].\nBased on previous findings, these factors are worth receiving more attention in terms of clinical research. The purpose of the present study was to investigate the prevalence of alexithymia among DSM-IV somatoform pain disorder (chronic pain) female patients and to examine the relationship between alexithymia and the self-reporting of pain in this group of patients. Besides, the study searched for the anxiety levels of chronic pain patients with or without alexithymia.\n\nMaterials and methods\nSample\nThe sample consisted of 30 females who applied to the outpatient psychiatry clinic at a public hospital and who met DSM-IV diagnostic criteria for chronic pain disorder. Patients with concomitant psychiatric disorders, such as major depression, anxiety disorders and somatoform disorders other than pain disorder were excluded.\nPatients either directly applied to the psychiatry clinic themselves or were referred for psychiatric assessment from another outpatient clinic, mainly physical medicine and rehabilitation. After complete description of the study, written informed consent was obtained from each subject.\nThe control group was 37 healthy females, who matched for age, and education with the subjects. All subjects participated voluntarily in the study and gave consent after the procedure had been fully explained to them.\nThe mean age of the patients and the healthy controls was 34,63 ± 10,62 (range: 16–62) and 34,46 ± 7,43 (range: 22–57) years and the educational level was 6,13 ± 3,03 (range: 5–11) and 6,59 ± 2,9 (range: 5–14) years, respectively. There were no significant differences between the two groups with respect to age (t = 0,79, df = 65, P > 0,05), educational level (t = 1,02, df = 65, P > 0,05), and marital status (x2 = 0,51, df = 1, P > 0,05).\n\nMeasures\nA detailed sociodemographic data form was used for all subjects. All participants were applied Structured Clinical Interview for DSM-IV (SCID-I) [22], Turkish version [23]. Regarding the pain assessment, information was first obtained on several aspects of the patients' pain, such as intensity, and duration. Pain intensity was measured by Visual Analogue Scale (VAS), using a horizontal 10-cm line with the statement 'no pain at all' at the extreme left-hand end and 'the worst possible pain' or 'unbearable' at the right-hand extreme. VAS is scored by measuring the distance from the end of the scale indicating absence of pain (or no distress or no pain relief) to the place marked by the patient [24].\nThe psychometric scales used in the study were the 26-item Toronto Alexithymia Scale (TAS-26] and the Trait Anxiety Inventory (STAI), which were both validated in Turkish population studies [25-28]. TAS is a psychometrically well validated and reliable instrument in the assessment of alexithymia. TAS has been validated in Turkish studies as a true or false scale. Twenty-six items are scored either as 1 or 0 and the higher scores indicate higher degrees of alexithymia. TAS has an interval consistency of 0.65 [Kuder-Richardson) and test-retest reliability is r = 0.71, p < 0.01 in Turkish reliability and validity study. The sample was divided into nonalexithymic and alexityhmic groups, with the recommended cut-off score of 11 [27]. Spielberger Trait Anxiety Inventory (STAI) is one of the two sections of the Spielberger Anxiety Inventory (the other, measuring state anxiety). 'Trait anxiety' has been defined as anxiety proneness, that is, the tendency to respond to situations perceived as threatening with elevations in the intensity of state anxiety [26].\n\nStatistical analysis\nIn order to determine the relative importance of a number of factors in pain disorders, we used both correlation analyses. The alexithymic and nonalexithymic groups were compared using the independent sample t-tests on scores of psychological tests. The statistical procedure, which was carried out by a SPSS package program for Windows using Chi-square, Fisher's exact test, two tailed t test and Pearson correlation coefficients, was also used to determine group differences (alexithymics versus nonalexithymics) in sociodemographic variables and various aspects of pain.\n\n\nResults\nIn the chronic pain group, 56.7% of patients (n = 17) had a score greater than 11 on the TAS-26, and were considered alexithymic. The mean TAS-26 score of the alexithymic group (n = 17) was 17.88 ± 3.43 and the nonalexithymic group (n = 13) was 8.39 ± 2.02. Age (t = 1,38, df = 28, p > 0,18), education (t = -0,21, df = 28, p > 0,16) and marital status (x2 = 0,27, df = 1, p > 0,87) were not associated with alexithymia (Table 1).\nIn the control group, 24,3% of patients (n = 9) were alexithymic according to TAS-26. The mean TAS-26 score of the alexithymic group (n = 9) was 13,82 ± 1,93 and the nonalexithymic group (n = 28) was 10,33 ± 0,86. Alexithymia was not associated with age (t = -1,08, df = 35, p > 0,29), educational level (t = 1,1, df = 35, p > 0,28), or marital status (x2 = 0,74, df = 1, p > 0,79) or anxiety levels in the control subjects (Table 1).\nThe duration and severity of pain, TAS-26 scores, and STAI scores of the female pain patients are shown in Table 2. Comparison of the alexithymics with nonalexithymics on either the severity of pain or pain duration showed no statistical significance (t = 0,64, df = 28, p > 0,52, t = 2,05, df = 28, p > 0,05, respectively).\nTAS-26 score and duration of pain were found positively correlated (r = 0,50, n = 30, p > 0,005). STAI (trait) scores of the alexithymics in the pain group did not significantly differ from the nonlalexithymics (t = 0,06, df = 28, p > 0,95) and besides, TAS-26 and STAI scores were not correlated (r = 0,06, p > 0,72).\nIn summary, there are three points to be emphasized. First, chronic pain patients were found significantly more alexithymic than controls (56,7% to 24,3%). Second, a positive correlation was observed between TAS-26 scores and duration of pain. Third, neither positive correlation nor significant difference was found between alexithymia and trait anxiety in pain patients.\n\nDiscussion\nThe results of the present study suggest that patients with chronic pain disorder are more alexithymic than individuals with no pain. This finding is consistent with results obtained with earlier measures of alexithymia [11-13]. Although they may share common clinical features, alexithymia and somatoform pain are independent constructs. Alexithymia may be a consequence to the effects of severe physical symptoms, such as a reduced quality of life and limitations in daily activities. Besides, alexithymia may be conceptualized as a personality trait as well as a secondary state reaction [2,3,15-17]. In this study, the question investigated was whether alexithymia has any correlation with the duration or severity of the pain itself.\nThere were no significant differences between alexithymic and nonalexitymic patients on self reports of current pain severity. This is in accordance with Cox's study [1994] in which it was further pointed out that alexithymic patients were found to use significantly more verbal descriptors of pain compared to nonalexithymic patients [13]. In our study, pain intensity was only evaluated by using VAS. One problem in trying to measure the intensity of pain is the lack of an objective way. Pain is a subjective experience and each patient may communicate in a different way, verbally or nonverbally [29]. Patients in this sample were sufferers of chronic pain, who had already chosen an approved way of expressing their distress. Since this is true regardless of alexithymia, alexithymic groups and nonalexithymic groups in this sample showed no difference on pain severity.\nThe positive correlation between alexithymia and the duration of pain in this sample supports the assumption of a two-way hypothesis. It is often assumed that pain can be caused by alexithymic personality traits and also that severe and chronic pain may cause emotional change. One of the limitations of this study is that because of the cross-sectional design, we are unable to draw conclusions about the direction of causality between alexithymia and pain. The duration of the patients' pain could approximately be determined, yet the preexisting level of alexithymia was not known. In the usual absence of internal stimuli, alexithymic person may be expected to maintain an external focus of attention, such as pain. Symptom chronicity may force the alexithymic person to attent to and amplify this somatic sensation.\nDifficulties in the ability to identify and differentiate emotions and somatic experiences are core features of the alexithymic construct. Therefore, alexithymic patients might be expected to differ from nonalexithymic ones in their anxiety levels. Yet, in our pain group alexithymic patients showed no significant difference from the nonalexithymics on trait anxiety. Besides, alexithymia and anxiety were not correlated at all. The reasons may be lying in the specific characteristics of this patient group itself.\nThe study included patients suffering from chronic symptoms; with an average of 7,44 ± 6,82 years of pain in the alexithymic and 3,31 ± 2,79 years in the nonalexithymic groups. Persistency of any physical symptom may bring along alexithymia as a coping strategy. In their paper, Crook and Tunks (1988) examined the types of coping strategies used by persistent pain sufferers and addressed to the importance to alter their attitudes and behavior that tend toward catastrophizing, avoidance and withdrawal, rather than simply concentrate on trying to teach them techniques for 'coping with stress' to help persistent pain sufferers [30]. Sufferers of chronic symptoms in this sample were members of a subgroup who have been seeking medical care for a long time and besides given the chance of being referred to a psychiatrist. Therefore, alexithymic or not, their anxiety might have induced unique coping strategies and illness behavior.\nAlexithymia may be important in addressing the diversity of subjective factors involved in pain [31]. It is not known whether it should be addressed in the treatment of pain patients, but a high level of alexithymia may effect the nature of assessment. In summary, the conceptualization of alexithymia as a personality trait as well as a secondary state reaction is underlined by our data. However, regarding the cross-sectional design of this study, only limited conclusions can be drawn about the nature of the causal relationship between alexithymia and chronic pain. Therefore, future longitudinal studies assessing the cause of alexithymic characteristics are required to fully elucidate the concepts of primary and secondary alexithymia.\n\n\n" ], "offsets": [ [ 0, 14714 ] ] } ]

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[ { "id": "pmcA2238978__text", "type": "Article", "text": [ "SuperCAT: a supertree database for combined and integrative multilocus sequence typing analysis of the Bacillus cereus group of bacteria (including B. cereus, B. anthracis and B. thuringiensis)\nAbstract\nThe Bacillus cereus group of bacteria is an important group including mammalian and insect pathogens, such as B. anthracis, the anthrax bacterium, B. thuringiensis, used as a biological pesticide and B. cereus, often involved in food poisoning incidents. To characterize the population structure and epidemiology of these bacteria, five separate multilocus sequence typing (MLST) schemes have been developed, which makes results difficult to compare. Therefore, we have developed a database that compiles and integrates MLST data from all five schemes for the B. cereus group, accessible at http://mlstoslo.uio.no/. Supertree techniques were used to combine the phylogenetic information from analysis of all schemes and datasets, in order to produce an integrated view of the B. cereus group population. The database currently contains strain information and sequence data for 1029 isolates and 26 housekeeping gene fragments, which can be searched by keywords, MLST scheme, or sequence similarity. Supertrees can be browsed according to various criteria such as species, isolate source, or genetic distance, and subtrees containing strains of interest can be extracted. Besides analysis of the available data, the user has the possibility to enter her/his own sequences and compare them to the database and/or include them into the supertree reconstructions.\n\nINTRODUCTION\nMultilocus sequence typing (MLST) is a tool that is widely used for phylogenetic typing of bacteria. MLST is based on polymerase chain reaction (PCR) amplification and sequencing of internal fragments of usually seven essential or housekeeping genes spread around the bacterial chromosome. The genetic relatedness among isolates is then determined by comparison of the nucleotide sequence types (1,2). MLST is thus a method that is unambiguous and truly portable among laboratories. Since the initial development of this technique for Neisseria meningitidis in 1998, MLST schemes have been developed for about 30 species including some of the most important bacterial pathogens, e.g. Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, Staphylococcus aureus, Campylobacter jejuni, Enterococcus faecium, Burkholderia pseudomallei, Escherichia coli, Salmonella enterica and the Bacillus cereus group (see (1) for a recent review). These MLST schemes have been used successfully to explore the population structure of bacteria, to study the evolution of their virulence properties, to identify antibiotic-resistant strains and epidemic clones, and for epidemiological surveillance.\nThe B. cereus group includes bacterial species that are of medical and/or economic importance, such as B. anthracis, an obligate mammalian pathogen causing the lethal disease anthrax, B. cereus, an opportunistic human pathogen involved in food-poisoning incidents and contaminations in hospitals, B. thuringiensis, an insect pathogen and one of the world's most widely used biopesticide and B. weihenstephanensis, a cold-tolerant species known for contaminating dairies. These species are genetically very closely related and may be considered as one species based on genetic and genomic evidence (3–5). Unlike other bacterial species that are typed using a single MLST scheme, five separate schemes have been developed for the B. cereus group, based on different sets of genes and isolates (5–10). The Priest scheme (8) is currently the most widely used. Studies with the various schemes have independently indicated that the B. cereus group population is divided into three main phylogenetic clusters and that species are usually intermixed within the groups. One cluster contains the monomorphic B. anthracis isolates and a number of B. cereus and B. thuringiensis strains, many of which are from clinical sources. A second heterogeneous cluster includes B. cereus and B. thuringiensis isolates from various origins, while cold-tolerant B. weihenstephanensis and B. cereus isolates belong to the third group. The separate MLST analyses have also revealed that the B. cereus group population is weakly clonal overall due to numerous clinical and virulent isolates emerging from different phylogenetic positions (5–8,11–14), with the exception of the ‘cold-tolerant’ cluster that seems to exhibit a panmictic (or sexual) population structure, i.e. with frequent genetic exchanges between strains (9).\nDespite the overall congruence between the various MLST studies, the use of separate schemes with no gene overlap and very little strain overlap has produced a confusing situation and makes the results difficult to compare directly. Therefore, we recently proposed a combined scheme based on genes taken from three of the four schemes available by then and for which we created a web-based database accessible at the University of Oslo's MLST server, http://mlstoslo.uio.no/ (5). Here, in order to provide the B. cereus group research community with a common MLST resource, we have developed on the same website a database, SuperCAT, that compiles and integrates MLST data from all the published B. cereus group schemes. In addition, we applied supertree reconstruction methods to build an integrated view of the B. cereus group population and phylogeny. Below we describe the content and main features of the new database as well as the process of supertree building.\n\nDATABASE CONTENT AND IMPLEMENTATION\nThe SuperCAT database provides information, sequence and phylogenetic data for all bacterial isolates that have been typed using any of the five published MLST schemes for the B. cereus group (Table 1). Strain information, when known, includes isolate description, source and geographical location of isolation, and the scheme(s) used for typing. The sequence data include the nucleotide sequences of the MLST loci examined in a given strain. SuperCAT also contains the phylogenetic supertree of the B. cereus group reconstructed by combining the sequence data from all five schemes, as well as supertrees built for individual schemes. Information and sequences for isolates typed by the Priest and Tourasse–Helgason schemes were retrieved from the databases devoted to these schemes at http://pubmlst.org/bcereus and http://mlstoslo.uio.no/, respectively. MLST data for additional strains not available in the pubmlst.org repository (strains from (15) are missing therein) and for the Helgason, Ko, and Candelon–Sorokin schemes were taken from the published literature and the Genbank nucleotide sequence database (Table 1). In addition, sequences of all MLST loci were extracted from the complete genomes of the 21 sequenced B. cereus group strains available in Genbank. Altogether, SuperCAT currently contains data for 1029 isolates and 26 gene fragments from 25 different genes. However, since most strains have been typed using only 6 or 7 of the 26 loci, about one-third of the complete set of sequences are included. The 26 loci, only available for the completely sequenced strains, sum up to 10 619 bp. All these genes are located on the chromosome, thus the database provides no information about extrachromosomal plasmids even though most of the strains do carry one or several small and/or large plasmids. Unlike scheme-specific MLST databases, SuperCAT does not contain allele and sequence type (ST) numbers. Since isolates in SuperCAT have been typed by different subsets of loci, complete allelic profiles are unavailable and therefore STs cannot be assigned for most strains, except the fully sequenced ones.\nTable 1.The five MLST schemes designed for typing bacteria of the B. cereus groupSchemeGenesTotal sequence length (bp)Total number of isolateseUsed in (references)Helgasonadk, ccpA, ftsA, glpT, pyre, recF and sucC2 938120(6,12,46)Candelon–Sorokina,cclpC, dinB, gdpD, panC, purF and yhfL2 850149(9,10)Kob,cgyrB, mbl, mdh, mutS, pycA(1) and rpoB2 00265(7)Priesta,bglpF, gmk, ilvD, pta, purH, pycA(2) and tpi2 829721(8,11,13–15,46–48)Tourasse–Helgasona,b,dadk, ccpA, glpF, glpT, panC, pta and pycA(2)2 658172(5)aSpecific databases for the Priest and Tourasse–Helgason schemes are accessible at http://pubmlst.org/bcereus/ and http://mlstoslo.uio.no/, respectively. A BLAST database for the Candelon–Sorokin scheme is available at http://spock.jouy.inra.fr/cgi-bin/bacilliMLSopen.cgi.bWhile the Tourasse–Helgason and Priest schemes use the same gene fragment for the pycA gene, the Ko scheme is based on a different and non-overlapping gene region.cThe B. cereus group-specific transcriptional regulator plcR was originally included in the Candelon–Sorokin and Ko schemes. However, plcR follows a phylogeny different from the other MLST loci (7,10) and is no longer used for MLST; therefore, it is not included in SuperCAT.dThe Tourasse–Helgason scheme is a combined scheme based on 3 genes from the Helgason scheme (adk, ccpA, and glpT), 3 genes from the Priest scheme (glpF, pta and pycA(2)), and the panC gene from the Candelon–Sorokin scheme.eIncluding strains with fully sequenced genomes.\nSuperCAT is built as a relational database using the PostgreSQL management system, and data are accessible through a graphical web interface. User queries and results pages are processed and created on-the-fly via a highly modified version of the mlstdbNet software (16) written in PERL and based on the DataBase Interface (DBI) and Common Gateway Interface (CGI) modules. The database is implemented on a Linux Apache web server maintained through the facilities and support provided by the Norwegian EMBnet node. Some large supertree computations are run on a Linux supercomputer at the University of Oslo. The ATV (A Tree Viewer) Java applet is used for phylogenetic tree display (17). ATV notably supports horizontal and vertical zooming capabilities that are suitable for browsing large trees. The Jalview editor Java applet is also implemented in SuperCAT for advanced multiple sequence alignment display (18).\n\nSUPERTREE RECONSTRUCTION\nSupertree techniques allow to combine the phylogenetic information from different datasets into a common phylogenetic tree and several studies have shown that meaningful supertrees can be obtained even when taxon overlap is very sparse (see (19,20) for reviews). Supertree analysis has thus become increasingly popular for taking advantage and combining the massive amount of sequence data available in public databases for reconstructing large-scale organismal phylogenies with the ultimate goal of building the tree of life (21–24). In this study, the 21 B. cereus group strains that have been completely sequenced, and for which the sequences at all 26 MLST loci are thus available, can be used to join all five schemes and provide the strain overlap necessary for supertree analysis. The global B. cereus group supertree, containing 1029 isolates, was reconstructed according to the widely used matrix representation by parsimony (MRP) procedure (Figure 1; (19,25,26)). Scheme-specific supertrees were also reconstructed for each of the five MLST schemes by the same technique. Briefly, a phylogenetic tree is built for every gene separately by the maximum likelihood method with the PHYML_aLRT program (27). Then, each gene tree is recoded into a binary matrix representing the branching order (i.e. the phylogenetic groupings) following standard MRP coding using the SuperMRP.pl script (28). All gene tree matrices are concatenated into a supermatrix, in which isolates missing from a particular tree are coded using the ‘?’ character representing unknown data. In this supermatrix, the sequence of 0's, 1's and ?'s defines the branching profile of a strain. Closely related strains have similar branching profiles. Supertrees are then generated from the supermatrix by the maximum parsimony technique using the program MIX from the PHYLIP package (29) run with default parameters. The maximum parsimony step infers the trees that would require the minimum number of changes between the branching profiles of all isolates, where the unknown characters can take any of the two possible states 0 or 1 (they are not treated as missing gaps). As many trees were equally parsimonious, the final supertree was taken as the strict consensus of all parsimony trees with the CONSENSE program of PHYLIP. In order to obtain branch lengths that are proportional to the amount of nucleotide changes, we added an additional step in which branch lengths and statistical support for groupings are estimated from the concatenated sequences by the maximum likelihood method employing approximate likelihood-ratio tests (aLRTs) for branches using PHYML_aLRT with Shimodaira-Hasegawa-like support values (27,30). aLRTs provide a fast way of testing branch support without requirement of multiple replicates like traditional bootstrap procedures. The Felsenstein-1984 nucleotide substitution model supplemented with a gamma distribution (F84+Γ) was used in maximum likelihood computations for individual gene trees and the supertree (31). This model allows for unequal base frequencies, transition/transversion rate bias, and gamma-distributed substitution rate variation among sites. It was empirically chosen as a consensus from exploratory model testing using ModelTest (32,33), which indicated that models including these three factors were most appropriate for the MLST loci studied, although models for individual loci differed slightly. Note that the maximum likelihood technique also allows for uneven rates of nucleotide substitution between strains, which allows to accommodate slow- and fast-evolving isolates. To reduce the size of the binary supermatrix and speed up computations, individual gene trees and the supertree were built using only one representative from a set of strains having identical sequences. The remaining identical isolates were graphically added to the tree afterwards when drawing the final supertree.\nFigure 1.Schematic overview of the B. cereus group supertree reconstruction procedure using Matrix Representation by Parsimony (MRP). See text for details.\nIt should be noted that the global 1029-strain supertree retains the phylogenetic signals from the individual schemes and contains the three main clusters of the B. cereus group population described in the section ‘Introduction’. The integrated SuperCAT system may also allow to infer new relationships between strains that were analyzed with different gene sets. Even though the 26 loci sequences are available for only 21 isolates, they apparently provide enough overlap information for building the main branches of the supertree. These 21 isolates cover all three clusters, although the majority of them are B. anthracis strains or clinical strains closely related to B. anthracis due to the focus of genome sequencing projects, making the part of the supertree containing these isolates likely to be more accurate than the rest of the tree. Furthermore, 111 other isolates have been typed by 10 genes or more, providing additional overlap (see the ‘Gene Distribution’ page). Although about two-thirds of the sequence data are missing overall, it has been shown for other organisms that relevant supertrees could be reconstructed with datasets containing more than 90% of missing data, especially when the characters that are present are informative (20,22,23,34). Empirical and simulation studies have indicated that this behavior may be due to the fact that the characters which are present are more important for the tree-building process than those which are absent (see (20,34) and references therein). Precise within-cluster groupings may contain more uncertainty, as indicated by the large number of unresolved multifurcations in the B. cereus group supertree. Finally, it is also worth mentioning that the branching orders of the scheme-specific MRP supertrees are highly correlated to those of the published trees built with concatenated sequences and other phylogenetic algorithms.\n\nDATA ACCESS AND MANIPULATION\nThe complete list of isolates included in SuperCAT (currently 1029) with strain description, source and country of origin is available at the database home page. By default all isolates in the database are used in the analysis tools provided, but the user can select strains of interest by keywords, MLST scheme, entering a list of strain identifiers, or choosing isolates individually via checkboxes. All subsequent analyses will be based on the selected strain subset and their loci. The keyword search will look for matches in any of the strain, description, source, location and scheme fields. Complex keyword queries with several logical operators can be formulated in the ‘advanced search’ page. Note that many isolates were referred by alternative names in different MLST schemes and publications, therefore synonyms have been included in the strain descriptions that allow a particular isolate to be looked up using any of its alternative identifiers. A sequence search is also possible using BLASTN (35), in order to select isolates that have allele sequences identical to user-entered query sequences.\nThroughout SuperCAT, clicking on a strain name will pop up an isolate-specific window showing all relevant information and giving access to the nucleotide sequences of individual loci for that isolate. Detailed information about the MLST schemes (e.g. loci names and lengths, genomic coordinates, literature references) and their overlap, the distribution of available loci among the isolates, and the supertree reconstruction procedure can be obtained by clicking the relevant links in the header line present at the top of every page.\nApart from the basic functions for selecting and accessing strain information and sequence data for all five B. cereus group MLST schemes, the main features of SuperCAT relate to the manipulation of the supertrees constructed by the MRP approach. The global supertree based on the combination of all five B. cereus group MLST schemes as well as the five scheme-specific supertrees can be browsed according to various user-chosen criteria (Figure 2). Isolates in the supertrees can be colored by species or source of isolation. It is also possible to specifically mark in red the current subset of strains that has been selected by the user and to extract from the supertrees the subtree containing only those isolates. In the case of the multi-scheme supertree highlighting of the strains can be based on genetic distance. With this option the user can mark on and/or extract from the tree the isolates that are genetically closely related to strains of her/his choice. The user can either select strains that share one or several identical allele sequences with her/his query isolate(s) or that are at a specified genetic distance. Distances between isolates are computed by summing up the lengths of the branches (in average number of nucleotide substitutions per site) connecting the isolates in the supertree (known as patristic distances; (36)). The genetic relatedness search functions are also available in a separate page for the user to find closely related isolates without tree manipulation. SuperCAT allows to compare the scheme-specific MLST supertrees with each other and with the global supertree by using the subset of isolates that are common to all selected schemes. Common isolates can either be highlighted in red or be extracted as subtrees from each supertree, which can be used for comparing the positions of the common strains in the various MLST trees. For all supertree-related options, detailed tree navigation can be achieved using the various functions in the ATV tree window when the trees are displayed (17).\nFigure 2.Examples of supertree browsing and manipulation in SuperCAT. A, supertree colored by species; B, specific highlighting of user-selected strains (in red); C, extracted subtree containing only the strains highlighted in B. Trees are displayed using ATV (17).\nBesides the manipulation of the precomputed supertrees, SuperCAT offers the user the possibility to compute new supertrees by MRP using any combination of strains, schemes and genes. Supertree computations may be extremely time consuming, ranging from a few minutes to 2–3 days with the complete database. Users are therefore requested to enter their e-mail addresses and will receive a notification containing a link to the results page when the supertree is ready. Note that when building a supertree for a user-selected subset of strains, the computation will first include all database isolates. A subtree containing only the user-selected isolates will then be extracted from the supertree of all strains. Although more time consuming, this strategy allows: (i) to avoid sampling artefacts as phylogenies built with different isolate sets may vary and (ii) to obtain relationships even if the selected isolates have been typed using non-overlapping gene sets, as the supertree of all isolates can always be built owing to the completely sequenced strains that are common to all schemes.\nAnother main feature of the SuperCAT database is that the user can enter her/his own private sequences and conduct several sequence analyses (Figure 3). These analyses include: (a) building new supertrees containing user isolates and sequences; (b) finding database isolates having sequences most similar to the user's query sequences using an on-line BLASTN (35) service; and (c) aligning user sequences to database genes using the multiple sequence alignment program CLUSTALW (37). For the last option, the Jalview editor (18) is provided for advanced multiple alignment display. All user-entered data must be in FASTA format and can be either copied and pasted into the query forms or uploaded from text files stored locally on the user's computer.\nFigure 3.Examples of query results in SuperCAT. A, multi-scheme BLAST search with sequence alignment; B, multi-scheme genetic search showing the list of isolates sharing one or more sequences with a query strain; C, multiple sequence alignment using Jalview (18).\nAll strain information data, sequences and phylogenetic trees, including user-made supertrees and extracted subtrees, can be saved and downloaded freely from the database. Users wishing to have their MLST data included as part of the SuperCAT release (and/or the Tourasse–Helgason scheme-specific database) are welcome to contact N.J.T. or A.-B.K. at the e-mail addresses given on the Oslo MLST server front page.\n\nDISCUSSION AND FUTURE DEVELOPMENTS\nSuperCAT is a newly created database devoted to the B. cereus group of bacteria whose main objectives are to provide a common MLST repository and means for building a comprehensive genetic analysis of the group that has been typed by five separate schemes. The database is publicly and freely available at http://mlstoslo.uio.no/, along with the database specific for the combined scheme of (5). We plan to update SuperCAT quarterly.\nFuture developments of the database may deal with refining the supertree-building procedure. In particular, a new improved method has recently been developed for taking into account both nucleotide substitutions and recombination events in phylogenies, as part of the ClonalFrame software, which has been applied to the B. cereus group and the Priest scheme (38). It would therefore be of interest to examine the suitability of ClonalFrame in the supertree context. It is also tempting to extend the supertree analysis beyond MLST data, by incorporating the phylogenies obtained previously from large-scale multilocus enzyme electrophoresis (MLEE; (3,39–41)) and amplified fragment length polymorphism (AFLP; (42–45)) studies. The MRP framework is ideal since it allows to integrate trees that can be built from different methods and data. As MLEE, AFLP and MLST have different levels of resolution, one can hope that combining them might provide an even more robust supertree for the B. cereus group.\n\n\n" ], "offsets": [ [ 0, 24100 ] ] } ]

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[ { "id": "pmcA2365205__text", "type": "Article", "text": [ "Cytotoxic Activity of Silyl- and Germyl-Substituted 4,4-Dioxo-3a,6a-Dihydrothieno[2,3−d]isoxazolines-2\nAbstract\nThe [2+3] dipolar cycloaddition of nitrile oxides to the double C = C bonds of thiophene-1, 1-dioxides leads to formation of the fused isoxazolines-2 (1, 2). Tumor growth inhibition of these compounds strongly depends on the nature of group IV A element increasing from slightly active tert-butyl derivatives to silicon and germanium containing analogues. The products of benzonitrile oxide cycloaddition have greater cytotoxic effect than the compounds obtained from the cycloaddition reaction of 2, 5-disubstituted thiophene-1, 1-dioxides with acetonitrile oxide. Fused silyl substituted isoxazolines-2 are stronger NO-inducers than their germyl and tert-butyl analogues.\nCYTOTOXIC ACTIVITY OF SILYL- AND GERMYL-SUBSTITUTED 4,4-DIOXO-3a,6a-DIHYDROTHIENO [2,3-d] ISOXAZOLINES-2 \n\n E. Lukevics*, P. Arsenyan, I. Shestakova, O. Zharkova, I. Kanepe, R. Mezapuke, and O. Pudova Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga, LV-1006, Latvia \n\n ABSTRACT The [2+3] dipolar cycloaddition of nitrile oxides to the double (3=(3 bonds of thiophene-l,l-dioides leads to formation of the fused isoxazolines-2 (1, 2). Tumor growth inhibition of these compounds strongly depends on the nature of group IV A element increasing from slightly active ert-butyl derivatives to silicon and germanium containing analogues. The products of benzonitdle oxide cycloaddition have greater cytotoic effect than the compounds obtained from he cycloaddition reaction of 2,5-disubstituted thiophene-l,l-dioxides with acetonitrile oxide. Fused silyl substituted isoxazolines-2 are stronger NO-inducers than their germyl and tert-butyl analogues. \n\n INTRODUCTION The interest in silyl substituted thiophene-l,l-dioxides stems from the fact that they are useful synthetic intermediates for the preparation of various types of organic compounds by Diels-Alder cycloaddition [1], amine induced ring-opening reaction [2], or coupling of bromothiophene-l,l-dioxides with thienyl stannanes in the presence of a palladium (0) catalyst [3]. It has been shown that unsubstituted thiophene-l,l-dioxide prepared in situ is a quite reactive dipolarophile in the [2+3] cycloaddition reactions with N,o-diphenylnitrone [4], benzonitrile [4, 5] and mesitonitrile [4, 5] oxides yielding mono- and diisoxazolines-2 and N-substituted isoxazolidines. Moreover, our recent studies indicate that silyl- and germylcontaining isoxazolines have gained a great deal of attention as compounds possessing a wide spectrum of the biological properties. The vasodilating, anticoagulant and cardioprotective activity of 5-Si-(Ge)substituted isoxazolines-2 has been studied in vitro and in vivo [6, 7]. The most active isoxazoline -3-(5\"-triethylgermyl-3\"-isoxazolino)pyridine hydrochloride protected the heart from rhythm disturbances and lethality during ischemiareperfusion [7]. It has been shown that silylisoxazolines-2 are more potent in protection against hypoxia and corazole convulsions than germanium analogues. However, germylisoxazolines-2 are stronger tumor growth inhibitors and NO-inducers than their silicon analogue [8]. This work presents the results of cytotoxic activity for fused isoxazolines-2 bearing a group 14 element as substituent (1, 2) in function of the nature of the group 14 element. \n\n M \n\n e3M.CH3 N \n\n -0\\ R \n\n O\\ M \n\n e3M S \n\n N \n\n 02 \n\n 02 2 \n\n 1 \n\n MATERIALS AND METHODS CHEMISTRY Seven tert-butyl-, trimethylsilyl-, and trimethylgermyl-substituted 4,4-dioxo-3a,6adihydrothieno[2,3-d]isoxazoline-2 1 and 2 (Table 1)were prepared by the [2+3] dipolar 63 \n\n \fVol. 7, No. 2, 2000 \n\n Cytotoxic Activity ofSilyl-and Germyl-Substituted 4, 4-dioxo-3a ,6a-Dihydrothieno[2, 3-d]Isoxazolines-2 \n\n cycloaddition of aceto- and benzonitrile oxides to 2,5-disubstituted thiophene-l,l-dioxides. Their synthesis and characterization are given in ref. [9]. \n\n _,,/0\\N Me3M M'Me3 \n\n Me3M \n\n 02 M, M'=C, Si, Ge; R=Me, Ph; R'=H, Me3Ge Table 1. Me3C, MeSi, Investigated dihydrothieno[2,3-d]isoxazolines-2 \n\n 02 Me3Ge substituted \n\n %, \\R 4,4-dioxo-3a,6a\n\n Compound \n\n 'Type la \n\n M \n\n R H Me3Ge Me3Ge \n\n Yield \n\n 4,4-dioxo-3 methyl\"5-tert-butyl-3a,6adihydrothieno[2,3-d]isoxazoline-2 \n\n (3' C Si \n\n (%) 80 \n\n 4,4-dioxo-3-methyl-3a-trimethylgermyl-5-tert-butyl3a, 6a-d ihyd rothieno[2,3-d]isoxazoli ne-2 4,4-dioxo-3-methyl-3a-trimethyigermyl-5tri methylsilyl-3a, 6a-d hyd roth ien o[2,3d]isoxazoline-2 4,4-d io x o-3- meth y I-3a, 5- b s tri m ethy Ig e rmy I)3a, 6a-d hyd rothieno[2,3-d] isoxazo ne-2 4,4-d ioxo-3-ph en y I-5-tert-b uty I-3a, 6adihydrothieno[2,3-d]isoxazoline-2 4,4-d ioxo-3-p h e ny I-5-tri meth y si ly I-3a, 6adihydrothieno[2,3-d]isoxazoline-2 4,4-d ioxo-3-p h e n y I-5-tri methy Ig e rmy I-3a, 6adi,hydr0thieno[2,3, d]isoxazoline-2 \n\n lb lc \n\n 58 45 67 84 \n\n ld 2a 2b \n\n Ge \n\n Me3Ge \n\n C Si \n\n 77 85 \n\n 2c \n\n Ge \n\n IN VITRO CYTOTOXITY ASSAY Monolayer cells lines were cultivated for 72 h in DMEM standard medium without an indicator and antibiotics. After the ampoule was defreezed not more than four passa4ges were performed. The control cells and cells with tested substances in the range of 2-5 10 cell/mL concentration (depending on line nature)were placed on a separate 96 wells plates. Solutions containing test compounds were diluted and added in wells to give the final concentrations of 50, 25, 12.5, and 6.25 #g/mL Control cells were treated in the same manner only in the absence of test compounds. Plates were cultivated for 72 h. A quantity of survived cells was determined using crystal violet (CV) or 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolinium bromide (MTT) coloration which was assayed by multiscan spectrofotometer. The quantity of alive cells on control plate was taken in calculations for 100% [10, 11]. Concentration of NO was determined according to [10]. RESULTS AND DISCUSSION Potential cytotoxic activity of synthesized fused isoxazolines 1 and 2 was tested in vitro on four monolayer tumor cell lines: MG-22A (mouse hepatoma), HT-1080 (human fibroblastoma), B16 (mouse melanoma), Neuro 2A (mouse neiroblastoma). Concentrations providing 50% of tumor death effect were determined according to the known procedure [12] using 96 well plates. The experimental evaluation of cytotoxicity properties is presented in Table 2. A preliminary analysis of the structure-activity relationship for the cytotoxic action clearly indicates the strong influence of the MeM (M=C, Si, Ge) group in position 5 of fused isoxazolines 1 and 2. Derivatives bearing tert-butyl substituent (la,b and 2a) have a slight cytotoxic effect (> 10 #g/mL). The substitution of the tert-butyl group by trimethylsilyl or 64 \n\n \fE. Lukevics et al. \n\n Metal-Based Drugs \n\n A \n\n oO \n\n {D \n\n 0 \n\n 0 \n\n 65 \n\n \fVol. 7, No. 2, 2000 \n\n Cytotoxic Activity ofSilyl-and Germyl-Substituted 4, 4-dioxo-3a ,6a-Dihydrothieno[2, 3-d]Isoxazolines-2 \n\n trimethylgermyl ones leads to considerable increase of cytotoxicity. It must be noted that the activity of silicon- and germanium-containing compounds (1 and l d) depends on the tumor type. 5-Trimethylsilyl-substituted fused isoxazoline le is more active than the germanium analogue l d in tests on HP-1080 and MG-22A cell lines. However, the germanium compound ld has greater cytotoxic effect on Neuro 2A and B16 cell lines than the silicon derivative lc. Comparison of the tumor growth inhibition for derivatives 1 and 2 shows a higher activity of the condensed isoxazolines 2 containing a phenyl group in position 3 with respect to 4,4-dioxo-3-methyl-3a-trimethylgermyl-5-MeM-3a,6a-dihyd rothieno[2,3d]isoxazolines l b-d. Silyl- and germyl-substituted fused isoxazolines have a medium NOinduction ability, 4,4-dioxo-3-phenyl-5-trimethylsilyl-3a,6a-dihydrothieno[2,3-d]isoxazoline-2 (2b) being the most active (250% in the MG-22A test). \n\n ACKNOWLEGMENT We are grateful to Latvian Taiho Foundation for financial support. REFERENCES 1. A. R. M. Donovan, M. K. Shepherd, Tetrahedron Lett., 35 (1994)4425. 2. S. Gronowitz, A.-B. H0rnfeldt, E. Lukevics, O. Pudova, Synthesis, (1994)40. 3. G. Barbarella, L. Favaretto, G. Sotgiu, M. Zambianchi, L. Antolini, O. Pudova, A. Bongini, J. Org. Chem., 63 (1998) 5497. 4. A. Bened, R. Durand, D. Pioch, P. Geneste, J. P. Declerq, G. Germain, J. Rambaud, R. Roques, J. Org. Chem., 46 (1981) 3502. 5. F. M. Albini, P. Ceva, A. Mascherpa, E. Albini, P. Caramella, Tetrahedron, 38 (1982) \n\n 6. 7. 8. \n\n 9. 10. 11. 12. \n\n 3629. E. Lukevics, M. Veveris, V. Dirnens, Appl. Organomet. Chem., 11 (1997) 805. E. Lukevics, P. Arsenyan, M. Veveris, Metal Based Drugs, 5 (1998) 251. E. Lukevics, P. Arsenyan, S. Germane, I. Shestakova, Applied Organomet. Chem., 13 (1999) 795. E. Lukevics, P. Arsenyan, S. Belyakov, J. Popelis, O. Pudova, Organometallics, 18 (1999) 3187. D.J. Fast, R.C. Lynch, R.W. Leu, J. Leuckocyt. Biol., 52 (1992) 255. P.J. Freshney, Culture of Animal Cells (A Manual of Basic Technique), Wiley-Liss, New York, 1994, pp. 296-297. R. J. Riddell, R. H. Clothier, M. Fd. Balls, Chem. Toxicol., 24 (1986) 469. \n\n Received: January 21, 2000 Accepted: February 1, 2000 Received in revised camera-ready format\" February 2, 2000 \n\n 66 \n\n \f " ], "offsets": [ [ 0, 9219 ] ] } ]

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[ { "id": "pmcA2065882__text", "type": "Article", "text": [ "Identification of Two Independent Risk Factors for Lupus within the MHC in United Kingdom Families\nAbstract\nThe association of the major histocompatibility complex (MHC) with SLE is well established yet the causal variants arising from this region remain to be identified, largely due to inadequate study design and the strong linkage disequilibrium demonstrated by genes across this locus. The majority of studies thus far have identified strong association with classical class II alleles, in particular HLA-DRB1*0301 and HLA-DRB1*1501. Additional associations have been reported with class III alleles; specifically, complement C4 null alleles and a tumor necrosis factor promoter SNP (TNF-308G/A). However, the relative effects of these class II and class III variants have not been determined. We have thus used a family-based approach to map association signals across the MHC class II and class III regions in a cohort of 314 complete United Kingdom Caucasian SLE trios by typing tagging SNPs together with classical typing of the HLA-DRB1 locus. Using TDT and conditional regression analyses, we have demonstrated the presence of two distinct and independent association signals in SLE: HLA-DRB1*0301 (nominal p = 4.9 × 10−8, permuted p < 0.0001, OR = 2.3) and the T allele of SNP rs419788 (nominal p = 4.3 × 10−8, permuted p < 0.0001, OR = 2.0) in intron 6 of the class III region gene SKIV2L. Assessment of genotypic risk demonstrates a likely dominant model of inheritance for HLA-DRB1*0301, while rs419788-T confers susceptibility in an additive manner. Furthermore, by comparing transmitted and untransmitted parental chromosomes, we have delimited our class II signal to a 180 kb region encompassing the alleles HLA-DRB1*0301-HLA-DQA1*0501-HLA-DQB1*0201 alone. Our class III signal importantly excludes independent association at the TNF promoter polymorphism, TNF-308G/A, in our SLE cohort and provides a potentially novel locus for future genetic and functional studies.\nSystemic lupus erythematosus (SLE/lupus) is a complex autoimmune disease in which the body's immune system attacks its own tissues, causing inflammation in a variety of different organs such as the skin, joints, and kidneys. The cause of lupus is not known, but genes play a significant role in the predisposition to disease. The major histocompatibility complex (MHC) on Chromosome 6 contains at least 100 different genes that affect the immune system, including the genes with the strongest effect on lupus susceptibility. Despite the importance of the MHC in SLE, the identity of the actual genes in the MHC region that cause SLE has remained elusive. In the present study, we used the latest set of genetic markers present at the MHC in lupus families to identify the actual genes that affect the disease. To our knowledge, we have shown for the first time that two separate groups of genes are involved in SLE. One group of genes alters how the immune system may inappropriately target its own tissues in the disease. How the second set of genes predisposes to SLE is the subject of ongoing study.\n\n\n\nIntroduction\nSince the early 1970s, the human major histocompatibility complex (MHC) has been shown to be associated with a number of autoimmune, inflammatory, and infectious diseases, and it continues to be the focus of intense research [1]. The recently defined extended MHC (xMHC) encompasses 7.6 Mb of genome on 6p21.3 and is divided into five subregions from telomere to centromere: extended class I, classical class I, classical class III, classical class II, and extended class II. In addition, the MHC contains two hypervariable regions, the RCCX module in class III (spanning complement C4) and the HLA-DRB genes in class II, that both exhibit copy number polymorphism. Examination of the sequence across the extended MHC reveals the presence of 421 genes, and over 252 (60%) are thought to be expressed [2]. Around 40% of genes expressed within the classical MHC encode proteins with putative immunomodulatory function [3]. The classical class I and class II loci encode the human leucocyte antigen (HLA) proteins involved in antigen presentation to T cells, initiating the adaptive immune response. The class III region contains the greatest density of genes in the genome (58 expressed genes), which are often found in functionally related clusters [2].\nA major obstacle in the identification of disease-specific causal variants within the MHC has been the strong linkage disequilibrium (LD) exhibited by certain alleles in this region, resulting in the existence of long-range, conserved, extended haplotypes [4], also known as ancestral haplotypes [5], sometimes spanning more than 2 Mb [6]. Thus, for many MHC-associated diseases, it has only been possible to delimit association signals to a particular extended haplotype or segment of one.\nSystemic lupus erythematosus (SLE/lupus, [Online Mendelian Inheritance in Man 152700, http://www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM&TabCmd=Limits]) is a chronic, multi-system autoimmune disease affecting young women ten times more commonly than men. The worldwide prevalence of SLE is estimated at between 12 and 124 cases per 100,000 individuals [7]. SLE is characterized by the presence of pathogenic autoantibodies to nuclear and cell-surface antigens that show affinity maturation. The consequent immune complexes deposit in tissues, causing inflammation and damage. It is well established that there is a complex genetic component to lupus aetiology, with hormonal and environmental influences also contributing to disease susceptibility [8,9].\nThe MHC has been the most consistently confirmed genetic risk factor for SLE, and multiple different genes within the region have been significantly implicated with disease susceptibility. For example, hereditary and acquired deficiencies of the early classical complement component C4, located within the MHC class III locus, leads to a lupus-like syndrome. A role for another class III gene, tumour necrosis factor alpha (TNF), in SLE was suggested following the observation that the lupus-prone New Zealand F1 mouse hybrid exhibits constitutively low TNF expression [10]. Recently, the development of antinuclear antibodies in patients treated with TNF antagonists has also stimulated interest in the possible role of TNF in SLE [11–13]. Murine and human candidate gene studies, together with genome-wide linkage screens, provide further support that multiple genetic loci, including the mouse MHC complex H2 and the human MHC locus, contribute to disease susceptibility [14–17].\nIt should be noted that the human MHC was first associated with SLE in 1971, when studies demonstrated that lupus probands were enriched for the class I alleles HL-A8 (now known as HLA-B8) and HLA-W15 (now known as HLA-B15) when compared with healthy controls [18,19]. Further case control association studies were small, performed in ethnically diverse populations, and tested only a small number of the classical HLA and complement C4 alleles. The most consistent findings reported to date are associations with the class II alleles HLA-DR2 (DRB1*1501) and HLA-DR3 (DRB1*0301) and their respective haplotypes in Caucasian populations. The complement C4A null allele (C4A*Q0) has shown inconsistent association with lupus in a number of studies—a situation that may reflect genetic heterogeneity in disease susceptibility [20–23]. In addition, a recent study has demonstrated that low C4A copy number is a risk factor for lupus in a European American cohort [24]. However, the C4A null allele lies on the lupus-associated DR3 “autoimmune” extended haplotype (AH8.1), which exhibits extremely strong LD [6]. It therefore remains to be definitively established whether this locus constitutes a distinct susceptibility allele to that of the class II association or is merely in LD with it. Similarly, certain TNF promoter polymorphisms, including the much-studied SNP TNF-308G/A, have shown association with SLE; but again, many of these variants are carried on the highly conserved 8.1 ancestral haplotype, thus restricting interpretation of these data.\nIn 2002, a family-based study employing microsatellites as surrogate markers for HLA-DRB1 haplotypes in Caucasian lupus families demonstrated association with DR3-, DR2-, and DR8 (DRB1*0801)-containing haplotypes. In that study, Graham and colleagues reported that, taking advantage of recombinant chromosomes, the disease risk region could be limited to a 1 Mb region encompassing classical class II and class III [25].\nWe have performed a medium resolution association mapping study of the MHC in lupus families, utilizing a combination of SNPs and four-digit typing at the HLA-DRB1 locus in order to anchor haplotypes. Sixty-eight SNPs were successfully genotyped across a 2.4 Mb region of the MHC, from the class I locus KIAA1949 to the class II gene HLA-DPB2, in 314 UK Caucasian SLE trios. We used these data to perform a family-based association study in an attempt to distinguish the relative effects of the class II and class III regions of the MHC in lupus susceptibility. In addition, we employed the long-range haplotype test to search for the presence of high-frequency, extended haplotypes indicative of recent positive selection [26]. We have also used family-based and case-control strategies to examine genotypic risk at HLA-DRB1 and rs419788.\n\nResults\nAssociation Testing of HLA-DRB1 and MHC Region SNPs\nIn order to define the causal variation within the MHC region, we typed 314 complete SLE trios for the HLA-DRB1 gene as well as for 86 SNPs across a 2.4 Mb region encompassing the HLA class I locus HLA-B to HLA-DPB2. High-quality genotype data was obtained for HLA-DRB1 and 68 MHC SNPs (see Table S1 for quality control data). Association testing of the HLA-DRB1 gene revealed a significant association with HLA-DRB1*0301 (nominal p = 4.9 × 10−8, permuted p < 0.0001, T:U = 129:55) in our lupus cohort (Table 1). There was also a trend for under transmission of the HLA-DRB1*0701 allele (nominal p = 0.0013, T:U 42:77); however, this association was no longer significant after correction for multiple testing as determined by 10,000 permutations of the dataset (permuted p = 0.09). Furthermore, we did not find evidence of association with HLA-DRB1*1501 (nominal p = 1.0, T:U 70:70) or HLA-DRB1*0801 (nominal p = 1.0, T:U = 11:11) in our cohort (see Table S2 for complete HLA-DRB1 association data); alleles previously suggested by microsatellite typing of a US lupus cohort [25].\nAssociation testing of the MHC region SNPs also identified significant evidence of association to SLE (Table 1 for associated markers and Table S3 for all MHC SNPs). The SNP with the most significant association, rs419788 (nominal p = 4.3 × 10−8, permuted p < 0.0001) was of similar strength to that of the HLA-DRB1*0301 allele, with odds ratios (ORs) and 95% confidence intervals (CIs) of 2.0 (1.6–2.6) and 2.3 (1.7–3.2), respectively. This SNP is located within intron 6 of the class III gene, superkiller viralicidic activity 2-like (Saccharomyces cerevisiae) (SKIV2L), and is located approximately 500 kb telomeric to the HLA-DRB1 gene. Of the other 12 SNPs that were significantly associated with SLE (nominal p = 4.0 × 10−4 to 2.5 × 10−7; permuted p = 0.03 to <0.0001), one was located in the class I region between HLA-B and MICA, seven were located in the class III region, and four were situated in the class II region (Table 1; Figure S1). Specifically, the seven associated class III SNPs were located in or close to the following genes: the TNF promoter, BAT3, SLC44A4, EHMT2, TNXB, GPSM3, and NOTCH4. One of the four class II associated SNPs was close to HLA-DRA, two were between HLA-DRB1 and HLA-DQA1 and one was in intron 1 of HLA-DQA1. The correlation between all 68 SNPs and HLA-DRB1 in our UK SLE cohort is illustrated in Figure 1. The markers showing significant association are highlighted.\n\nConditional Analyses Identify Two Independent Association Signals in the MHC\nIn order to establish whether the two most associated signals identified in this association-mapping experiment are likely to represent a single causal allele or independent risk factors, we first examined the association data conditioned upon the presence of the HLA-DRB1*0301 allele. We found that four of the 13 associated SNPs showed evidence of signals independent of HLA-DRB1*0301 in our dataset, the strongest of which was rs419788 (Table 1). We therefore conditioned the three remaining SNPs (rs2523589, rs1052486, and rs605203) on rs419788 to assess whether these signals are truly independent of each other or show association due to LD with rs419788. In addition, we included HLA-DRB1 in stepwise conditional regression analyses performed on the SNPs showing association independent of HLA-DRB1 (unpublished data). These analyses demonstrated that the observed association signals at rs2523589, rs1052486, and rs605203 were predominantly dependent upon the association at rs419788, and suggested that there are two major independent association signals in the MHC in UK SLE: HLA-DRB1 and rs419788. The independence of the association signals at HLA-DRB1 and rs419788 is further supported by the observation that there is only modest LD between these two (r2 = 0.24). There was no association with any other HLA-DRB1 allele and the four SNPs independent of HLA-DRB1*0301 (TRANSMIT, unpublished data).\nThe association of the tumour necrosis factor gene promoter SNP TNF-308G/A with SLE is lost after conditioning for HLA-DRB1*0301 in our cohort. If we perform the reverse analysis and condition HLA-DRB1*0301 on the presence of the TNF promoter SNP, we find that the association remains, confirming that our TNF association is secondary to that of HLA-DRB1*0301.\n\nGenotypic Risk for Class II and Class III Association Signals\nHaving established independent association at the allelic level with HLA-DRB1*0301 and rs419788-T in our UK SLE cohort, we wanted to further determine the genotypic risk conferred by these variants and hence gain insight into their underlying mode of inheritance in lupus. We used case-control and family-based analyses to assess genotypic risk at HLA-DRB1, while the family-based test alone was used for rs419788. Common family-based tests of LD, such as those used in this study (Genehunter), measure transmission distortion based on allele counts rather than genotype counts; the former has been shown to be more powerful under additive models, while the latter has greater power under recessive or dominant genetic models [27]. The genotype-pedigree disequilibrium test (geno-PDT) determines LD between a locus genotype and disease by comparing genotypes that are transmitted from parent to proband with those that are not [27]. We used the geno-PDT to assess genotypic risk for our class II and class III association signals: HLA-DRB1 and the SNP rs419788. In the case-control analysis for HLA-DRB1, ORs with 95% CI were calculated and Fisher's exact test employed to assess statistically significant differences between HLA-DRB1 genotypes in lupus probands and healthy controls. For HLA-DRB1, the alleles were coded as follows: HLA-DRB1*0301, HLA-DRB1*1501, HLA-DRB1*X where X represents all HLA-DRB1 alleles other than HLA-DRB1*0301, and HLA-DRB1*1501. We included HLA-DRB1*1501 in the analysis, even though we find no allelic association in our cohort, because previous studies have shown a greater risk for lupus in individuals who are compound heterozygotes for HLA-DRB1*0301- and HLA-DRB1*1501-containing haplotypes [25,28]. Overall the results are consistent with a dominant effect from HLA-DRB1*0301 (Table 2) and a dose-dependent (additive) effect from rs419788-T (Table 3). Specifically, both case-control and geno-PDT demonstrate that there is no dose-dependent increase in disease risk for HLA-DRB1*0301. Rather, it appears that the presence of a single copy of HLA-DRB1*0301 alone is sufficient to increase susceptibility to disease. Moreover the 0301/X genotypes constitute the greatest risk in our cohort rather than the 0301/1501 heterozygotes. Genotypes containing HLA-DRB1*1501 in the absence of HLA-DRB1*0301 revealed no significant association in our cohort.\nAll three rs419788 genotypes demonstrated significant association in our lupus families (Table 3). The common CC genotype was significantly under transmitted, while the rare T allele displayed dose-dependent over transmission to lupus probands.\n\nCharacterization of HLA-DRB1*0301 Risk Haplotype\nNext, we wanted to further delimit the MHC class II association signal that we have detected at HLA-DRB1. We used phased parental genotype data to compare the allelic composition of HLA-DRB1*0301-bearing haplotypes that were transmitted (T) to affected probands to those that were not transmitted (or untransmitted, UT) with the aim of identifying differences that could delineate the lupus susceptibility interval(s) arising from this haplotype (summarized in Figures 2A, 2B, and S2). We observed a striking difference between transmitted and untransmitted chromosomes within the class II region: nearly all transmitted HLA-DRB1*0301 haplotypes (99%) are identical across a 180 kb region defined by eight SNPs, whereas the corresponding region within untransmitted HLA-DRB1*0301 haplotypes exhibits significant recombination. These data strongly suggest the existence of a risk haplotype that, interestingly, contains only three expressed genes: HLA-DRB1, HLA-DQA1, and HLA-DQB1. Furthermore, we can confidently define the allelic composition of this risk haplotype, as these three genes are in strong LD and occur in one common haplotype in Caucasians: HLA-DRB1*0301-HLA-DQA1*0501-HLA-DQB1*0201. Thus, we hypothesize that the specific combination of all three alleles is required to confer disease risk in lupus or that disease susceptibility lies with either HLA-DRB1*0301 or the HLA-DQ alleles. We do not have sufficient numbers of recombinant chromosomes in this risk region to further delimit this signal: 2/176 (1.1%) transmitted HLA-DRB1*0301 haplotypes are recombinant at HLA-DQA1-HLA-DQB1; 3/178 (1.7%) transmitted haplotypes identical across HLA-DQA1-HLA-DQB1 do not possess HLA-DRB1*0301.\nThe composite relative extended haplotype homozygosity (REHH) versus frequency plot for UK SLE; Utah residents with ancestry from northern and western Europe (CEPH); and Yoruba in Ibadan, Nigeria (Yoruba) populations is shown in Figure 3A. We can only comment on evidence for positive selection in CEPH individuals, as we have used this population alone to assess background variation on Chromosome 6. The SLE and Yoruba cohorts are shown for comparative purposes. We find no evidence of positive selection for HLA-DRB1*0301 in the CEPH population. However, this allele is enriched in our lupus cohort (21% of parental chromosomes) and displays greater extended homozygosity when compared with HLA-DRB1*0301-bearing haplotypes in CEPH and Yoruba. Hence, the HLA-DRB1*0301 allele in lupus is observed as an outlier on the plot when compared to background variation in CEPH. These data support our previous observations (outlined above) of the highly conserved nature of HLA-DRB1*0301 haplotypes in lupus. In addition, the haplotype bifurcation plots centered on HLA-DRB1*0301 for UK SLE, CEPH, and Yoruba populations in Figure 3B illustrate preservation of the common HLA-DRB1*0301 haplotype in CEPH and UK SLE, while that seen in the Yoruba is significantly different. The class II regions of all three populations are essentially identical across our chosen SNPs; the main differences lie in class III. The difference in African populations in the class III region is one possible explanation for the lack of evidence for an association between HLA-DRB1*0301 and SLE in African or African American populations. However, HLA-DRB1*0301 has a lower frequency (∼7%–10%) in African populations compared with Europeans (∼13%), and the number of HLA association studies conducted in African populations is very limited.\n\nCharacterization of Class III Region Risk Haplotype\nOur data reveal a second independent signal at the MHC in SLE arising from the T allele of SNP rs419788 in intron 6 of the class III gene, SKIV2L. Further evidence supporting the independence of the rs419788-T and HLA-DRB1*0301 alleles is provided by the moderate LD between these two variants (r2 = 0.24) coupled with our data demonstrating that only 47% of rs419788-T allele-bearing haplotypes contain HLA-DRB1*0301.\nThe structure and composition of T and UT haplotypes anchored at rs419788-T were essentially identical (Figures 2C, 2D, and S2), and hence not informative in delimiting our class III signal. Therefore, we examined the LD structure around our associated class III SNP to better define our disease risk interval. In our lupus dataset the rs419788-T allele resides on three of seven haplotypes present within a large block of six SNPs exhibiting strong LD. This haplotype block encompasses roughly 270 kb containing class III genes from SLC44A4 to AGER, including the RCCX module. Next, we analyzed the haplotype block structure of this region in CEPH families using SNP data dumped from the International HapMap Project (http://www.hapmap.org/). The greater density of SNP typing available in the HapMap CEPH population compared to our current UK SLE map allowed us to potentially refine our signal by exploring correlations between our associated SNP and those surrounding it. Analysis of these data (Figure 4) suggests the presence of short-range LD around our associated variant, rs419788, in CEPH families, encompassing approximately 40 kb of the genome which includes the five genes: complement factor B (CFB), RD RNA binding protein (RDBP), SKIV2L, dom-3 homolg Z (C. elegans) (DOM3Z), and serine/threonine kinase 19 (STK19), and does not include the complement C4 locus. Furthermore, assessment of marker association in our lupus dataset demonstrates that after conditioning for HLA-DRB1*0301, the only markers that retain association signals are telomeric of SKIV2L, suggesting that complement C4, which is centromeric to this gene, may not be responsible for our independent class III signal.\n\nSubphenotype Analysis\nIn order to gain further insight into disease pathogenesis, we examined common lupus subphenotypes. Such subsets are more homogeneous than lupus per se and thus maybe enriched for specific predisposing variants. In addition, one might expect a close association between MHC class II alleles and autoantibody subsets in lupus if these are indeed causal variants, given their role in antigen presentation and subsequent humoral immunity. We therefore tested our two main MHC association signals, HLA-DRB1*0301 and rs419788, for association with renal disease and autoantibody subsets in our lupus cohort.\nWe found that HLA-DRB1*0301 was associated with the presence of anti-Ro and anti-La antibodies in our UK SLE cohort, with the latter showing the greatest evidence of association (anti-La nominal p < 0.001 compared with anti-Ro nominal p < 0.025). We found no association of HLA-DRB1*0301 with renal disease or any other autoantibody subsets in our dataset (see Table S4 for detailed results).\nGenotypes of the SNP rs419788 were not associated with any of the tested lupus subphenotypes after controlling for the effect of HLA-DRB1*0301 (unpublished data).\n\n\nDiscussion\nWe present the first family-based SNP association study of the MHC in SLE. We have genotyped 69 markers (HLA-DRB1 and 68 SNPs) across 2.4 Mb of the MHC, encompassing class III and class II, in a cohort of 314 UK Caucasian SLE trios. Transmission disequilibrium testing of these data has shown predominant association with the alleles HLA-DRB1*0301 and rs419788-T, together with 12 other MHC SNPs. Moreover, using conditional analyses, we have shown that the two primary signals of association at the MHC are independent of each other. Specifically, one signal arises from HLA-DRB1*0301 in class II and the other from the T allele of SNP rs419788 in the class III gene SKIV2L.\nExamination of bifurcation plots for T and UT HLA-DRB1*0301-containing haplotypes has enabled delineation of our class II association signal to a 180 kb region encompassing HLA-DRB1*0301-HLA-DQA1*0501-HLA-DQB1*0201. These data substantially refine that previously published by Graham et al. in 2002 [25], where the lupus susceptibility interval within HLA-DRB1*0301-containing haplotypes could only be delimited to a 1 Mb region encompassing class II and class III. The precise causal variant(s) within this region remains to be determined, as the three implicated alleles exhibit strong LD with few recombination events separating them (two out of 176 transmitted HLA-DRB1*0301 chromosomes in our dataset). However, all three allelic variants represent attractive functional candidates in lupus susceptibility for their role in antigen presentation and stimulation of the adaptive immune response.\nOur association of HLA-DRB1*0301 with lupus concurs with published data in Caucasian cohorts and is well established [16]. While our lack of association with HLA-DRB1*1501 and HLA-DRB1*0801 is consistent with previous data from the UK [29], Spain [30], the Netherlands [31], Sweden [32], Mexico [33], and the US [34], it conflicts with that of other US groups [25,35]. Interestingly, we demonstrate a trend, though not statistically significant, for undertransmission of HLA-DRB1*0701—a result also observed in prior UK and Canadian lupus studies [29,36]. Moreover, a negative association of HLA-DRB1*0701 has been reported in other autoimmune diseases including Graves disease [37,38], type 1 diabetes [39], and rheumatoid arthritis [40].\nIt appears that the conflicting results between UK SLE and previous US (Minnesota [MN]) [25] SLE data stem from differences in HLA-DRB1 allele frequency in the probands of each cohort. The reason for this is unclear. A comparison between UK and MN SLE cohorts (Table 4) reveals that UK SLE cases are enriched for HLA-DRB1*0301 but not HLA-DRB1*0801 or HLA-DRB1*1501 when compared to a UK control population. In contrast, MN SLE cases are enriched for HLA-DRB1*0301-DQB1*0201, DRB1*0801-DQB1*0402, and DRB1*1501-DQB1*0602 inferred haplotypes when compared to MN controls [25]. There is no statistically significant difference in the aforementioned HLA class II alleles/haplotypes between UK and MN control populations that could account for the disparity seen in the respective lupus cohorts. Differences in disease severity and subphenotype frequency between the two populations could account for the observed discrepancy. From the limited data available we found that the presence of renal disease appears to be similar in both cohorts (UK SLE 36% compared with MN SLE 40%), while the gender ratios are significantly different (female: male UK SLE 11:1 compared with MN SLE 57:1, Chi square p value < 0.001). We were unable to compare other lupus subphenotypes. Furthermore, closer inspection of these data reveals that microsatellite-inference of HLA-DRB1 alleles in the MN SLE dataset may underestimate the frequency of HLA-DRB1*0301 and overestimate that of HLA-DRB1*1501, thus diminishing the effect of the former and enhancing that of the latter. It is also possible that the MN SLE cohort shows greater racial heterogeneity in comparison to our UK SLE cohort, despite both being characterized as Caucasian.\nPrevious studies have demonstrated increased risk for lupus in individuals carrying particular combinations of microsatellite-inferred HLA-DRB1-HLA-DQB1 haplotypes [25,28]. The highest risk genotype was found to be the compound heterozygote HLA-DRB1*0301-DQB1*0201/HLA-DRB1*1501-DQB1*0602, while HLA-DRB1*0301-DQB1*0201-containing genotypes demonstrated a dose-dependent effect in increasing lupus susceptibility [25,28]. In the present study, we have examined genotypic risk at the classically typed HLA-DRB1 locus and in contrast to the aforementioned data of Graham et al. [25,28] we have shown a likely dominant effect of the associated allele, HLA-DRB1*0301. The case-control and family-based analyses for HLA-DRB1 also show the greater power of the former to detect significant association (Table 2). Specifically, all genotypes containing HLA-DRB1*0301 show increased transmission to lupus probands; however, homozygotes show no greater risk compared with heterozygotes, as would be expected under additive or multiplicative models. Thus, a dominant model of inheritance, requiring the presence of a single copy of the disease-predisposing variant alone, likely underlies the susceptibility conferred by HLA-DRB1*0301 in UK SLE. Such a model would fit an antigen presentation hypothesis where susceptible individuals carrying an HLA-DRB1*0301 allele are able to present auto-antigens to CD4+ lymphocytes, thus stimulating an autoimmune response. The differences between our UK SLE and the previously published US SLE data may reflect disease, ethnic, and haplotypic heterogeneity.\nInterestingly, analysis of genotypic risk at the associated class III marker, rs419788, suggests an additive (dose-dependent) pattern of inheritance for the rare T allele, where one copy confers a low risk of disease and two copies results in greater susceptibility. The different inheritance patterns for our class II and class III association signals provide further evidence for their independence.\nA variety of HLA-DR and HLA-DQ alleles have been associated with autoantibody subsets in ethnically diverse populations of lupus. The strongest associations have been demonstrated between anti-Ro and anti-La antibodies and HLA-DR3 and HLA-DQ2 (HLA-DQB1*0201), which are in strong LD [41–45] in case-control studies. Here, we confirm the association of HLA-DRB1*0301 with anti-Ro and anti-La antibody production in our family-based cohort.\nExamination of LD structure around our second independent association, rs419788-T in class III, coupled with the results of our conditional analysis, suggests that this signal could also be delimited to a relatively narrow genomic interval of about 40 kb given further SNP mapping in our cohort. This region includes the genes CFB, RDBP, SKIV2L, DOM3Z, and STK19, but does not include complement C4. Thus, complement C4 null alleles, which have been implicated in lupus pathogenesis, may not be responsible for our class III signal. We conclude, therefore, that our family-based mapping study has potentially revealed a hitherto unknown lupus susceptibility interval in the class III region of the MHC. However, we cannot conclusively exclude association at complement C4/RCCX without direct determination of C4 polymorphism/copy number in our cohort.\nWith respect to the genes implicated in our study, CFB is a vital component of the alternate complement pathway and disregulation may clearly affect the inflammatory response [46]. RD and Skiv2l are proteins potentially involved in RNA processing. The RD protein forms part of a negative elongation factor (NELF) complex that represses RNA polymerase II transcript elongation, while Skiv2l is a DEAD box protein with possible function as an RNA helicase. The function of Dom3z is currently unknown, although the homologous yeast protein binds nuclear exoribonuclease. Moreover, its ubiquitous expression suggests a housekeeping role. STK19 is a protein kinase of unknown function with primary nuclear localization [47]. Interestingly, RDBP and SKIV2L are found to be highly expressed in T lymphocytes, B lymphocytes, and dendritic cells (SymAtlas, http://symatlas.gnf.org/SymAtlas/).\nA number of studies have demonstrated conflicting evidence for and against association with various TNF locus polymorphisms in SLE [48]. A recent meta-analysis of the TNF-308G/A promoter polymorphism in SLE [48] revealed evidence of association for the minor allele (A) in European populations; however, this study did not account for LD with class II alleles. On conditioning our dataset for HLA-DRB1*0301, we find that the TNF promoter signal is lost, suggesting that this association is not independent and is due to LD with HLA-DRB1*0301 (or another variant in LD with HLA-DRB1*0301).\nIn summary, we have found association with two distinct and independent variants within the class II (HLA-DRB1*0301) and class III (SKIV2L) regions of the MHC in UK SLE trios. We can delimit our class II signal in lupus to three genetic variants (HLA-DRB1*0301-HLA-DQA1*0501-HLA-DQB1*0201) that may confer disease risk in combination or as separate signals. Our class III signal importantly excludes independent association at the TNF promoter polymorphism TNF-308G/A and potentially provides a novel locus for further study.\n\nMaterials and Methods\nStudy cohorts.\nSLE families. The cohort comprises 314 complete SLE trios (that is, mother, father, and affected lupus proband) collected as previously described [49]. All study participants are European Caucasian on the basis of grandparental origin. All 314 lupus probands (288 female, 26 male) fulfill the revised American College of Rheumatology (ACR) criteria for SLE [50], 36% of whom have a diagnosis of lupus nephritis. Written consent was obtained from all study participants and ethical approval for this study was obtained from the Multi-Centre Research Ethics Committee (MREC 2 June 1998).\n\nHealthy controls.\nThe control population for the HLA-DRB1 genotypic risk case-control analysis constitutes 1,667 healthy males of Northern European origin. The individuals are potential hematopoietic stem cell donors and were typed to four digits for HLA-DRB1 at the Anthony Nolan Trust, UK for this purpose. The level of resolution used for the typing of HLA-DRB1*15 alleles in these healthy controls resulted in the ambiguous allele string HLA-DRB1*1501/1502/1504/1506. However, it is likely that the great majority are HLA-DRB1*1501. There is no gender bias in HLA-DRB1 allele frequencies, so although we have used a male control cohort, the frequencies would be expected to be the same in a similar female cohort (Steven Marsh, personal communication).\n\nSNP genotyping.\nEighty-six SNPs were chosen for genotyping in our mapping study. Specifically, we selected 40 MHC class II and class III haplotype tagging SNPs from a preliminary MHC SNP map [51] that had previously shown robust genotyping efficacy. In addition, we typed 36 MHC class II tag SNPs from a subsequent high-resolution MHC study [52]. We also included the TNF-308G/A promoter SNP, together with nine further SNPs in the region of HLA-B and MICA obtained from the database, dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/). The latter SNPs had not been well characterized. All variants were typed in the entire cohort (n = 942). The SNPs span approximately 2.4 Mb of the MHC from the class I gene, KIAA1949 to the class II pseudogene, HLA-DPB2 and thus encompass MHC class III and class II. SNP genotyping was performed at the Broad Institute of MIT and Harvard and at Imperial College London by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry using the Sequenom MassARRAY platform as previously described [53]. SNPs that failed Sequenom typing were retyped by KBiosciences (http://www.kbioscience.co.uk/) using their in-house SNP genotyping methodology, KASPar (http://www.kbioscience.co.uk/genotyping/index.htm), a competitive allele-specific PCR technique.\n\nHLA-DRB1 genotyping.\nHLA-DRB1 typing was performed at the Anthony Nolan Trust, UK. All samples (n = 942 UK SLE trios and n = 1,667 controls) were genotyped using LABType SSO (sequence-specific oligonucleotide) typing technology according to the manufacturer's written recommendations (http://www.onelambda.com). Briefly, a locus-specific biotinylated PCR amplicon is produced, denatured, and rehybridized to complementary oligonucleotide probes conjugated to fluorescently coded beads. The bound biotinylated PCR product can be detected using R-phycoerythrin-conjugated streptavidin. A flow analyzer identifies the fluorescent intensity of phycoerythrin on each bead. The assignment of HLA type is based on the reaction pattern compared to patterns associated with known sequences.\nHigh resolution testing was performed using the Dynal AllSet+ SSP (sequence-specific primers) DRB1 assay according to the manufacturer's protocol (Invitrogen) for SLE families only. The presence or absence of PCR amplification was detected in a gel electrophoresis step using visualization by ethidium bromide incorporation. Genotypes were determined using SSPTool software.\nSamples that could not be resolved to four digits using SSO and PCR-SSP were analyzed by DNA sequencing of exon 2 of HLA-DRB1. Primers, reagents, and protocols were provided by The Anthony Nolan Trust, UK (primer sequences are available on request). Specific HLA-DRB1 alleles were assigned by comparing the resultant sequence with reference sequence from the IMGT/HLA Database [54].\n\nData analysis.\nMendelian inconsistencies were removed using PedCheck [55]. Families in which more than eight markers demonstrated Mendel errors were removed from further analysis. Markers with less than 80% genotyping efficiency and markers where more than eight families showed Mendel errors were also excluded from analysis. Two markers located within the SLE associated class II region (rs2239802 in intron 4 of HLA-DRA and rs6457594 in the region between HLA-DRB9 and HLA-DRB5) show deviation from Hardy–Weinberg equilibrium (HWE), which may reflect an undetected SLE association or systematic genotyping error. HWE was assessed in parental samples in our cohort. There is currently no uniform opinion in the community regarding the inclusion or exclusion of SNPs that show deviation from HWE, hence we elected to include these markers in the final analysis.\nSixty-eight out of the total 86 SNPs passed our quality-control measures (see Table S1 for details). In summary, one SNP was monomorphic in our dataset, four SNPs yielded low genotyping efficiency, and 13 SNPs were excluded for unacceptable Mendel error rate. The mean call rate for all markers post-quality control was 94% (range 83% to 99%).\nFamily-based association testing was performed using Genehunter TDT (version 2.1) [56] and TRANSMIT (version 2.5) [57]. Haplotypes were constructed and permutation testing performed using Haploview (version 3.32) [58]. Significance of association signals in all analyses was based on permutation testing (10,000 permutations). The data are represented both as nominal and permuted p values. ORs with 95% CI for family-based analyses were calculated in PLINK (http://pngu.mgh.harvard.edu/purcell/plink/) [59]. Conditional regression analyses were undertaken using WHAP [60].\nThe geno-PDT was performed using PDT version 5.1 with default settings [27]. The HLA-DRB1 alleles were coded into three groups for the geno-PDT and the case-control analysis: HLA-DRB1*0301, HLA-DRB1*1501 and HLA-DRX where HLA-DRX includes all HLA-DRB1 alleles other than HLA-DRB1*0301 or HLA-DRB1*1501. The HLA-DRB1*1501 code in the healthy controls represents the allele string HLA-DRB1*1501/1502/1504/1506, as described previously. The HLA-DRB1*1501 code in the lupus probands represents the alleles HLA-DRB1*1501 (84 out of 91 *1501 and *1502 alleles) and HLA-DRB1*1502 (7/91 *1501 and *1502 alleles), as *1504 and *1506 were not present in this population. Fisher's exact test was used to assess significance of association in the case-control analysis.\n\nSubphenotype analysis.\nWe looked for association of the HLA-DRB1*0301 allele with autoantibody subsets and renal disease in our cohort using the Chi-square test. We compared cases with and without the subphenotype of interest with DRB1*0301 homozygosity, heterozygosity, combined homozygosity and heterozygosity, and non-DRB1*0301 status. We performed the same analyses for homozygous and heterozygous genotypes of the associated SNP rs419788. The autoantibody subsets compared were anti-C1q, IgG, and IgM anti-cardiolipin antibodies (ACLG and ACLM), anti-Ro, anti-La, anti-RNP, anti-Sm, and anti-dsDNA.\n\nDelineation of associated MHC haplotypes and evidence for positive selection.\nWe looked for positively selected alleles in our dataset using the long-range haplotype test as measured by extended haplotype homozygosity (EHH), previously described by Sabeti et al. [26]. Essentially, such an analysis allows assessment of positive selection by mining datasets for high frequency extended haplotypes in comparison to the other core haplotypes at a locus.\nEHH is defined as the probability that two randomly chosen chromosomes carrying the core haplotype of interest will be identical by descent (homozygosity at all SNPs) for the entire interval from the core to a distance x. The REHH is the ratio of the EHH on the tested core haplotype compared with the combined EHH of all the other core haplotypes at the region excluding the tested core; as such, REHH accounts for local variation in recombination rate while EHH does not [26].\nThe program emphase was employed to assign the phase of parental genotype data and reconstruct missing information. Emphase is a simple phaser similar to the phaser of Excoffier and Slatkin [61]. It is very fast, especially on large datasets, and sufficiently accurate for most genetic applications. EHH analysis was performed on the phased parental data using the software program SWEEP (http://www.broad.mit.edu/mpg/sweep/index.html).\n\nHaplotype bifurcation plots.\nWe represent the breakdown of LD on core haplotypes using haplotype bifurcation diagrams generated in the program TREE [62] (also explained in [52]).\n\nREHH versus frequency plots.\nFifty-three SNPs (identified in Figure 1) are common to our dataset and the CEPH and Yoruba HapMap [63] populations. These three datasets, together with CEPH SNP data for Chromosome 6 in its entirety, were used to generate separate REHH versus frequency plots in SWEEP. The plots from the four cohorts were combined for visual, not statistical, comparison. Evidence for positive selection was quantitatively assessed in CEPH individuals, as this population alone was used to assess background variation on Chromosome 6. The UK SLE and Yoruba cohort data are shown for comparison. The 95th percentile based on total CEPH Chromosome 6 SNP data is shown.\n\n\nSupporting Information\nAccession Numbers\nThe Online Mendelian Inheritance in Man (OMIM, http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim) accession numbers for the genes described in this study are as follows: AGER, 600214; BAT3, 142590; C4A, 120810; C4B, 120820; CFB, 138470; DOM3Z, 605996; DOM3Z, 605996; EHMT2, 604599; HLA-B, 142830; HLA-DPB2, 120290; HLA-DQA1, 146880; HLA-DQB1, 604305; HLA-DRA, 142860; HLA-DRB1, 142857; KIAA1949, 610990; MICA, 600169; NOTCH4, 164951; RDBP, 154040; RDBP, 154040; SKIV2L, 600478; SLC44A4, 606107; STK19, 604977; STK19, 604977; TNF, 191160; and TNXB, 600985.\n\n\n\n" ], "offsets": [ [ 0, 42952 ] ] } ]

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[ { "id": "pmcA1851709__text", "type": "Article", "text": [ "Estrogen receptor alpha (ERα) mRNA copy numbers in immunohistochemically ERα-positive-, and negative breast cancer tissues\nAbstract\nBackground\nThe presence of ERα is the basis for treating breast cancer patients with targeted molecular therapies that block estrogen stimulation of breast cancer cell division. To select patients for the above therapies, currently, the ERα presence in breast cancer tissues is determined in clinical laboratories by microscopically scoring the slides subjected to immunohistochemistry (IHC). This method is not quantitative, highly subjective and requires large amount of tumor tissue, therefore, cannot be applied to sterotactic and ultrasound guided biopsy samples. To circumvent these problems, we previously developed quantitative real-time PCR based molecular assay that can be applied to determine mRNA copies of ERα in picogram amounts of total RNA from tumor samples. However, it is not known how the mRNA copy numbers correlate to IHC positive and negative status.\n\nMethods\nIn the current study we determined the copy numbers of ERα mRNA by Q RTPCR in breast cancer tissues that were graded as ERα-positive and negative by 1) IHC and 2) functional estrogen binding assay and statistically analyzed the data.\n\nResults\nWe demonstrate here that ERα mRNA copy numbers are not significantly different in tissues that are graded as positive by IHC and ligand binding assays. We establish here a cut of value of 5 × 106 copies per 1010 mRNA copies of GAPDH with an Odds Radio of 39.4, Sensitivity of 0.81 and Specificity of 0.90 in breast cancer tissues that are negative for ERα protein by IHC and estrogen binding assays. ROC analysis of the data gave an area of 0.8967 under the curve.\n\nConclusion\nWe expect that the cut off values determined here will be highly significant for applying molecular assay in the place of IHC in clinical laboratories for evaluating the presence of ERα for prognostic and therapeutic purposes.\n\n\n\nBackground\nBreast cancer is the most diagnosed and the second leading cause of cancer deaths for women in the United States striking about 300,000 and killing about 40,000 women a year [1]. A substantial body of epidemiological, experimental and clinical evidence indicated that unopposed stimulation of breast epithelial cells by the natural hormone, estrogen, plays a major role in the progression of breast cancers [2]. Because endogenous estrogens directly promote the growth of breast cancer cells, estrogen deprivation either by inhibiting its biosynthesis (aromatase inhibitors) or blocking estrogen-mediated gene transcription (tamoxifen) through its high affinity receptor, the estrogen receptor alpha (ERα), are the primary lines of therapy for breast cancer patients. In most cases, the efficacy of the above treatments has correlated with the presence of ERα in the tumor tissues. Currently, only those patients who express ERα in their tumors are chosen for aromatase inhibitor or tamoxifen therapies. In addition to being a therapeutic target, ERα was also shown to be the most important factor to predict breast cancer prognosis. The patients who express ERα in their tumors have an overall longer cancer-free survival and lower recurrence rates than patients who do not express this receptor [3].\nTo predict prognosis and identify patients for the above two anti-estrogen therapies, every breast cancer tissue is currently screened for the presence of ERα before a treatment regimen is selected for any breast cancer patient. The presence of ERα in breast tumors was originally determined in clinical labs by estrogen binding assay for about 20 years. However, when the tumors were detected at comparatively smaller sizes and highly specific monoclonal antibodies were developed that could detect ERα both in the fresh frozen as well as formalin fixed paraffin-embedded tissues, the clinical labs switched to immunohistochemistry (IHC) from estrogen binding assay for determining the presence of ERα. Currently ERα is determined in the clinical laboratories from rough estimates yielded by microscopically scoring the slides subjected to IHC technique using antibodies against the N-terminal A/B region of ERα. Although this procedure is used for over ten years, it has several limitations including not quantitative, highly subjective, variations due to antibody preparations, variations from one clinical lab to other and comparatively large sample size requirement.\nIn recent times, due to increased awareness and substantially improved screening methods, breast cancers are detected at very early stages and excised, in a large majority of cases, by stereotactic and ultrasound guided techniques. In these cases the limited amount of tumor tissue that remains after histological testing restricts determining ERα status for prognostic and therapeutic purposes by IHC. In many cases, ERα status is not determined due to insufficient amount of tumor tissue. For these reasons there is an urgent need to switch to a procedure that can detect ERα in a very small amount of tumor tissue obtained by the above methods. There is a general consensus that ERα mRNA quantification is a more suited technique for detecting its presence in tumor tissues. Several PCR based approaches have been described for detecting the presence of ERα in breast cancer tissues [4,5]. We recently developed a highly sensitive real-time PCR based quantitative molecular assay that can detect and quantify as low as 50–100 copies of ERα mRNA from as small as 40 picograms of total RNA from breast cancer tissues. Because quantitative real-time PCR is a high through-put method, it could be automated to apply in clinical laboratories. However, it is not known how the ERα mRNA copy numbers correlate to ERα positivity and negativity by IHC assay. Establishing a cut off value in IHC negative tissues is required for the application of molecular assay in the place of IHC assay. To determine the cut off value, we have profiled ERα mRNA copy numbers in breast cancer tissues which have been graded as ERα positive and negative by IHC and estrogen binding assays. We demonstrate here that ERα mRNA copy numbers are not significantly different in tissues that were graded as positive by IHC and ligand binding assays. However, ERα positive tissues, either by IHC or estrogen binding assays, express significantly higher mRNA copy numbers than the negative tissues. We have determined the cut off values of ERα mRNA copy numbers by molecular assay that correlate to ERα negativity by both IHC and ligand binding assays using CART program (Classification And Regression Tree). We expect that the cut off values determined here will be highly significant for applying molecular assay in the place of IHC in clinical laboratories for determining the ERα status for prognostic and therapeutic purposes.\n\nMethods\nAll the primers used in the current study were synthesized by Gibco-BRL Life Technologies. TaqMan Universal PCR Master Mix (Cat # 4304437) was from Applied Biosystems. 5'FAM and 3'TAMARA labeled oligonucleotide probes were synthesized by Applied Biosystems and available from previous studies. PCR quality water and Tris-EDTA buffer were from BioWhittaker.\nBreast tumor samples\nBreast cancer tissues with known ERα status by IHC and ligand binding assay were available from previous studies [6-9]. Briefly, the tumor samples were collected from either biopsy or mastectomies immediately after surgery and stored at -80°C until use. Fresh tumor tissue samples for ERα quantification were routinely harvested immediately adjacent to the histologic/diagnostic sections and considered to be representative of the tissue used for diagnosis. All the samples were examined by a pathologist and tissues containing > 80% cancer cells were excised and used for ERα mRNA quantification. The ERα-status for the samples used in this study was determined either by IHC using monoclonal antibodies against NH2-terminal portion of the molecule at Oncotech Laboratories, Irwine, CA, or by ligand binding assay as described [10]. The tumor tissues were considered positive for ERα by IHC if > 5% of cancer cells showed positive nuclear staining. The tumor tissues that were diagnosed as ERα positive by estrogen binding assay had > 3 fmol of ER/mg of total tissue extract. A total of 70 samples positive by IHC, 33 positive by estrogen binding assay, 43 negative by IHC and 20 negative by estrogen binding assay were included in the current study (Tables 1, 2, 3 and 4 respectively). The tumor tissues were processed to isolate total RNA and cDNAs prepared as previously described [6-9]. Howard University Institutional Review Board granted the ethical approval of Tumor collection procedures for the study.\n\nAbsolute quantification of ERα mRNA copy numbers by quantitative real-time PCR\nAbsolute quantification of ERα transcript copy numbers was achieved by quantitative real-time PCR in ABI Prism GeneAmp 7900 HT Sequence Detection System as described previously (9). Briefly, a typical real-time PCR reaction mixture contained cDNA prepared from reverse transcription of 0.5 – 5 nanograms of tumor tissue total RNA, 0.04 micromolar each of sense and anti-sense primers, 0.05 micromolar 5'FAM and 3'TAMARA labeled oligonucleotide probe and 1 × Taqman Universal PCR Mix in a total volume of 25 μl. PCR conditions were initial hold at 50°C for two minutes, followed by denaturation for ten minutes at 95°C, and denaturation for 15 seconds at 95°C in the subsequent cycles and annealing and extension for 1 min at 60°C for 40 cycles. The ERα mRNA copy numbers in tumor tissues were determined in comparison with a standard graph constructed simultaneously using 102, 103, 104, 105, 106, 107, 108, and 109 copies of reverse transcribed cRNA of ERα. All the samples were amplified in triplicate and real-time PCRs were repeated four times. The ERα mRNA copy numbers in tumor tissues were normalized to mRNA copy numbers of the house keeping gene, glyceraldehyde 3- phosphate dehydrogenase (GAPDH). GAPDH copy numbers were determined as previously described [11,12]. The sense, and anti-sense primers and probe for quantifying the mRNA copy numbers of ERα were 5' caagcccgctcatgatcaa 3' (position, exon 4, bp 1110–1128), 5'ctgatcatggagggtcaaatccac3' (position, exon 5, bp 1358–1338) and FAM 5'agaacagcctggccttgtccctg3'TAMARA (position, exon 4, bp 1140–1162) respectively. The sense and anti-sense primers and probe for quantifying GAPDH mRNA copy numbers were 5'ttccagg agcgag atccct3' (position, bp 304–322), 5'ggctgttgtcatacttctcatgg3' (position, bp 483–505) and FAM 5' tgctggcgctgagtacgtcgtg3' TAMARA (position, bp 342–363) respectively. Primer positions of ERα and GAPDH nucleotide sequences were as described [13,14].\n\nStatistical analysis\nWilcoxon-rank-sum test and standard two-sample t-test were used to determine whether the mRNA copy numbers were significantly different in breast cancer tissues that were 1) ERα-positive and ERα-negative by IHC, 2) ERα-positive and ERα-negative by estrogen binding assays, 3) ERα positive by IHC and estrogen binding assays, and 4) ERα-negative by IHC and estrogen binding assays. Test results were considered significant if P ≤ 0. 05. To determine the cut-off value/maximum level of mRNA copy numbers in the samples which were negative by IHC and estrogen binding assays, we used CART (Classification Regression Tree) [15,16] program. The data consisting of 103 ERα positive and 63 ERα negative samples which had a predictive variable, mRNA copy numbers, were partitioned into two groups using CART program. This program determines the best cut-off value copy numbers in the sense that the OR (Odds Ratio, ERα positive to ERα negative in our case) of the two groups (with copy number greater than the cut-off value in one group and less or equal to the cut-off value in the other) is maximized. Receiver Operating Characteristic (ROC) analysis was performed to determine the sensitivity and specificity of the RNA based molecular assay. The ROC curves were generated by connecting all the points determined by the copy numbers in all the samples in an increasing order. Since data on IHC grading as percent positive cells were available on some of the samples, the correlation between the IHC grading and the mRNA copy number was determined both in the original scale and in logarithmic transformations scale using S-PLUS software.\n\n\nResults\nWe have undertaken the current study to determine ERα mRNA copy numbers in breast cancer tissues that were positive and negative by two conventional methods of assaying ERα protein, IHC and estrogen binding. The rational for undertaking this study is that once we establish a threshold value in IHC negative tissues, then the molecular assay could be applied in clinical laboratories in the place of currently used IHC assay for determining the status of ERα for prognostic and therapeutic purposes. Our rational for establishing a cut off value is that any patient who expresses above the cut off level could be selected as a candidate for anti-estrogen therapies and could be considered to have good prognosis.\nWe first profiled ERα mRNA copy numbers in 70 samples positive by IHC, 43 negative by IHC, 33 positive by estrogen binding assay and 20 negative by estrogen binding assay. The data are presented in Tables 1, 3, 2 and 4 respectively. A box plot drawn for the copy numbers (logarithm base 2 scale) in the four groups (positive and negative by IHC and by estrogen binding assay) using S-PLUS software is shown in Figure 1.\nWe next compared the quantitative data on mRNA copy numbers among samples as described below. 1) We tested whether the two conventional assays, the IHC and estrogen binding assays, correlate in terms of mRNA copy numbers in 70 and 33 positive tissues (Tables 1 and 2 respectively) using Wilcoxon-rank-sum test and standard two-sample t-tests. By these two tests, we did not find significant differences in the ERα mRNA copy numbers in samples that were ERα positive by IHC and estrogen binding assays (p > 0.28 by both tests). 2) We also compared the mRNA copy numbers in 43 samples negative by IHC with 20 samples negative by estrogen binding assay and did not find significant differences (Tables 3 and 4 respectively) (p > 0.25 by the above two tests). However, 3) we found significant differences in mRNA copy numbers in the breast tumors that were IHC positive from those which were IHC negative (Tables 1 and 3 respectively) (p = 1.3e-6 by standard two-sample t-test and p = 2.7e-18 by Wilcoxon-rank-sum test). And 4) we also found significant differences in the samples that were positive and negative by estrogen binding assay (Tables 2 and 4 respectively) (p = 7.6e-3 by standard two-sample t-test and p = 3.6e-7 by Wilcoxon-rank-sum test).\nAfter establishing that ERα-positive tissues express significantly higher levels of mRNA copy numbers compared to negative tumor samples, we next determined the maximum level of expression in ERα-negative samples using CART program. By using this program, we found the maximum level of expression of mRNA copy numbers/cut-off value to be 5 × 106 per 1010copies of GAPDH mRNA. Of the total 106 positive (70 by IHC and 33 by estrogen binding assay) samples in our study, 83 samples showed higher level of expression than 5 × 106 copies per 1010 copies of GAPDH. It is possible that the samples that showed less than the above cut off value copy numbers could be due to false positivity by the above methods. In a total of 63 negative samples (43 by IHC and 20 by estrogen binding assay) only 6 samples showed higher expression than 5 × 106 copies per 1010GAPDH copies. It is also possible that the samples that showed higher than the cut off copy numbers could be false negative. The OR (Odds Ratio) in the two groups is about 39.4, an extremely high OR value. The counts of the ERα-positive (80, 23) and ERα-negative (6, 66) in the two groups produce a chi-square value of 88.2544 with 1 degree of freedom, which is consistent with our T-test and Wilcoxon-rank-sum test results.\nWe applied the above cut off value and determined the Sensitivity (percentage of samples that showed higher copy numbers than the cut off value of 5 × 106 copies per 1010 GAPDH copies in IHC positive tissues) and Specificity (percentage of samples that showed less than 5 × 106 copies per 1010 GAPDH copies in IHC negative tissues) and the values obtained were 0.81 and 0.90 respectively. We also determined Receiver Operating Characteristics using S-PLUS software and the ROC curve generated is shown in Figure 2. The area under the ROC curve, 0.89675, shows that the molecular assay clearly distinguishes the positives by IHC or estrogen binding from the negative tissues.\nSince we have the grading score as percent positive cells for about 50 samples (Table 1), we tested if a correlation exits between the percent positive cells by IHC and the mRNA copy numbers using S-PLUS software. We obtained a correlation coefficient of 0.02. When we used the logarithmic transformations scale, we obtained a correlation coefficient of 0.037. These results indicated that there is no correlation between the percent positive cells and the level of ERα mRNA copy numbers. These observations could be due to qualitative nature of IHC assay. The IHC data only show the number of positive cells but not quantitative to determine the level of ERα expression. The molecular assay based on RNA is quantitative to determine the level of ERα expression.\n\nDiscussion and conclusion\nPreviously ERα mRNA levels in immunohistochemically positive and negative tissues were evaluated in breast cancer tissues by several groups using conventional RT PCR. Cullen et al [17] determined mRNA levels by conventional PCR in 107 breast cancer tissues. They reported that ERα mRNA was more frequently detected in ERα protein positive tissues than ERα protein negative tissues. Jarzabek et al [18] studied ERα mRNA levels and protein levels in 41 primary breast cancer tissues. They reported the presence of ERα mRNA in all the tissues, where as the protein was present only in 70% of tumor tissues by Western blotting and 67% showed positive by immunohistochemistry. They concluded that lack of ERα protein is not due to lack of ERα gene expression or methylation of its promoter, but may be due to post-transcriptional or post-translational mechanisms. Alkarain et al [19] reported the presence of ERα mRNA in immunohistochemically ERα-negative tissues. However, none of the above studies has evaluated the threshold levels of ERα mRNA levels in immunohistochemically negative tissues or those negative by ligand binding assays. Our quantitative analysis of ERα mRNA copy numbers demonstrate that breast cancer tissues that are negative by both IHC and ligand binding express significant levels of ERα mRNA. The reasons why the mRNA is not translated to detectable protein are not clear. Our previous studies on ERβ mRNA copy numbers [9] in breast cancer tissues have shown that at the 5 × 106 copies per 1010 mRNA copies of GAPDH levels ERβ protein is translated. It is possible that either ERα mRNA is not translated or the translated protein is degraded to undetectable levels in these tissues.\nThe results and the analysis presented above clearly demonstrate that the ERα positive tissues by IHC or estrogen binding assay express significantly higher mRNA copy numbers than 5 × 106 copies per 1010 GAPDH copies. An extremely high Odds Ratio, high sensitivity and specificity demonstrate that the molecular assay could be used in the place of currently used IHC in the clinical laboratories. Based on our data above any patient who has more than 5 × 106 copies per 1010 GAPDH copies in her tumor tissue could be considered positive for ERα, could be selected for anti-estrogen therapies and considered to have good prognosis. However, the above described approach has some limitations in that it needs to be verified on a defined set of biopsy samples and with reference to another house keeping gene. Therefore, the results should be interpreted with caution and undoubtedly will require confirmation by a larger prospective multi-centered clinical study with a more accurate design to bring the technology to the clinic. The current study is a first step in that direction. We expect that the cost effective, extremely sensitive, high though-put molecular assay which requires only a few cancer cells could be an assay of choice to replace IHC in clinical labs for determining ERα status in breast cancer tissues once established in a multi-centered prospective clinical study.\n\nAbbreviations\nFAM, carboxy-fluorescein; TAMARA, 6-carboxy tetraethyl-rhodamine; GAPDH, Glyceraldehyde-3 phosphate dehydrogenase; ERα, estrogen receptor alpha; IHC, Immunohistochemistry; CART, Classification And Regression Tree; and OR, Odds Ratio and ROC, Receiver Operating Characteristics.\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\nAuthors' contributions\nI. P conceived the study, participated in the design of the study, performed the real-time PCR quantifications of ERalph mRNA copy numbers and drafted the manuscript. Q.Y participated in performing the statistical analysis of the data. Both authors read and approved the final manuscript.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\n\n" ], "offsets": [ [ 0, 21423 ] ] } ]

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[ { "id": "pmcA327937__text", "type": "Article", "text": [ "Further studies on partially purified calf thymus DNA polymerase a.\nAbstract\nAttempts to prevent the urea conversion of a 200-230,000 molecular weight DNA polymerase alpha to a 150-170,000 molecular weight form by the inclusion of protease inhibitors have not been successful. No other method has been found capable of dissociating a 50-70,000 fragment or subunit from the DNA polymerase subunit. Addition of this 50-70,000 subunit to the polymerase subunit does not aid the binding of the enzyme to DNA, but does have an effect on the utilisation of synthetic template-initiator complexes by the polymerase subunit.\n\n\n\n\n Volume 6 Number 10 1979 \n\n Nucleic Acids Research \n\n Further studies on partially purified calf thymus DNA polymerase a Keith McKune and Andrew M.Holmes* \n\n Biochemistry Department, Strathclyde University, The Todd Centre, 31 Taylor Street, Glasgow, G4 ONR, UK \n\n Received I June 1979 ABSTRACT \n\n Attempts to prevent the urea conversion of a 200-230, 000 molecular weight DNA polymerase a to a 150-170, 000 molecular weight form by the \n\n inclusion of protease inhibitors have not been successful. No other method has been found capable of dissociating a 50-70, 000 fragment or subunit from the DNA polymerase subunit. Addition of this 50-70, 000 subunit to the polymerase subunit does not aid the binding of the enzyme to DNA, but does have an effect on the utilisation of synthetic template-initiator complexes by the polymerase subunit. \n\n INTRODUCTION \n\n In mammals DNA polymerase a is thought to be the replicative \n\n enzyme, but due to low levels of activity, even in tissues actively making DNA, and to enzyme heterogeneity it has been found difficult to purify \n\n However, small samples of DNA polymerase a have been highly purified \n\n 2, 3, 4, 5 from several sources and partially characterised \n\n Heterogeneity has been observed in DNA polymerase a from a var\n\n 6 7 \n\n iety of sources, including rat liver and spleen , Hela cells, baby hamster \n\n 8 9, 10 11 \n\n kidney cells , mouse myeloma , Drosophila embryos and calf thymus \n\n 1,6, 12, 13 . We have previously observed several species of calf thymus \n\n DNA polymerase a differing in size and charge . In order of elution from DEAE cellulose they are enzyme A1 (200-230, 000 molecular weight), A2 (200-230, 000), B (100-110, 000) and C (150-170, 000). A poly (dA). oligo \n\n (dT)10 preferring enzyme, enzyrrme D (140-150, 000 molecular weight) elutes just after enzyme B. The A enzymes seem identical in all properties except their charge. 5.0-5. 5S enzymes, analagous to B, have been observed to \n\n C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England \n\n 3341 \n\n Nucleic Acids Research \n\n arise as the result of proteolytic action ' , similarly the B enzyme above 14 \n\n ,but the relationship of the other observed species is not clear, nor is \n\n the problem of which, if any, of these species is the replicative enzyme, \n\n although circumstantial evidence has implicated a DNA polymerase a to be \n\n 15 the replicative enzyme in adenovirus infected KB cells \n\n We have previously shown that several of these enzymes are inter\n\n 16 \n\n convertible . In particular, mild urea treatment can convert both A \n\n enzymes to C enzyme, with the loss of a subunit or fragment of 50-70, 000 molecular weight. The C enzyme retains DNA polymerase activity, but \n\n does show differences compared to A enzyme in heat sensitivity and sensitivity to N-ethylmaleimide. \n\n Highly purified samples of A and C enzymes when subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis have shown a corre\n\n lation of DNA polymerase activity with a polypeptide band 155, 000 molecular weight . Contaminating material of 50-70, 000 molecular weight appeared \n\n to obscure the putative 50-70, 000 subunit in gels of A enzyme. The conclusions drawn were that DNA polymerase a is a 155, 000 molecular weight \n\n polypeptide (C enzyrrme) which can and does associated with material of 50\n\n 70, 000 molecular weight to give A enzyme, and that enzyme B is a proteol\n\n 14 \n\n ytic degradation product . The relationship of enzyme D to the others is \n\n not clear. \n\n It is possible that during the enzyme isolation procedure proteolytic action may have taken place on a 200-230, 000 molecular weight enzyme releasing 155, 000 and 50-70, 000 fragments which remain in association \n\n until urea treatment separates them. It is also possible that urea treatment itself renders the enzyme susceptible to contaminating proteases. Further experiments to ascertain whether or not this is the case and, if so, to prevent it, have been carried out. \n\n A enzyme can be reconstituted from C enzyme formed by the action \n\n of urea on A enzyme by concentrating it with the 50-70, 000 molecular weight material. The A enzyrme obtained in this manner is highly purified, as is \n\n the C enzyme formed by the urea treatment, and has been used in attempts to show differences in synthetic template-initiator complex utilisation \n\n 3342 \n\n Nucleic Acids Research \n\n b y the se two enzyme s . \n\n MATERIALS AND METHODS \n\n Calf thymus was obtained from 10-16 week old calves and frozen at \n\n -20?C until required. Chromatographic media and chemicals were obtained \n\n 6 \n\n from sources previously referred to . Radioactive deoxynucleoside tri\n\n phosphates were obtained from the Radiochemical Centre, Amersham, Bucks. Synthetic oligo and polynucleotides were obtained from P. L. Biochemicals \n\n Inc., except for poly (dC) which was a gift from Dr. I.R. Johnston and pre\n\n 17 \n\n pared from oligo d(C)5 as described . N-a-p Tosyl-L-lysine chloromethy\n\n lketone HCl and phenylmethylsulphonylfluoride were obtained from Sigma and Trasylol from Bayer. \n\n Except where indicated all buffers contained 20% w/v glycerol and \n\n 1 mM dithiothreitol. Standard linear phosphate gradients were run between 0.03 M and 0.25 M potassium phosphate, pH 7.8. Gradient salt concentr\n\n 2 \n\n ations were measured using a conductivity meter as described . Urea was \n\n prepared as a 4. 8 M solution in 20% w/v glycerol, stirred with Amberlite \n\n MB3 and filtered. Dithiothreitol was added to a final concentration of 1 mM before use. \n\n 6 DNA polymerase was assayed using activated DNA as described \n\n except that the buffer was 50 mM tris HC1, pH 7.8. One unit of DNA polymerase activity incorporates ln mol [ H] dTMP into an acid insoluble form in one hour at 370C. Assays using synthetic template-initiator complexes were carried out at 30?C in 0. 12 ml. The template-initiator complexes \n\n 6 \n\n were prepared and assays processed as described . All assays contained \n\n 1 mM dithiothreitol, 62. 5 p.g bovine serum albumin, 1 ,ug template-initiator complex, enzyme protein and the relevant [ H] deoxynucleoside triphosphate at 0.1 mM and 12-15 cpm/pmol. These assays were carried out at either pH 6.4 in 20 mM sodium-potassium phosphate, or at pH 7.8 in 50 mM tris HCl and contained either 10 mM MgCl2 or 1 mM MnCl2 as indicated. \n\n Preliminary purification of DNA polymerase a to Fraction IV was as \n\n 6 \n\n described , the purification steps being phosphocellulose chromatography, \n\n ammonium sulphate precipitation and gel filtration on Sepharose 6B. \n\n 3343 \n\n Nucleic Acids Research \n\n Enzyme obtained from the DEAE cellulose step is referred to as Fraction \n\n V enzyme. Samples were prepared for sodium dodecyl sulphate polyacrylamide gel electrophoresis and scanned as previously described2 \n\n RESULTS AND DISCUSSION \n\n (a) Interconversion Studies \n\n A enzyme was routinely converted to C enzyme by incubating 200500 units/ml of Fraction V A enzyme in 2.4 M urea in 0. 02 M potassium \n\n phosphate, pH 7.8, for 60 minutes at 0?C. The mixture was then loaded on to a DEAE cellulose, washed with 0. 03 M potassium phosphate pH 7. 8, and enzyrme eluted either with the standard phosphate gradient or batchwise. Under these conditions usually about 50-60% of recovered activity was \n\n enzyme C (Fig 1 a). Overall recovery was 70-80%. A enzyme was recon\n\n 14 \n\n stituted essentially as described . The flow through material from the \n\n DEAE cellulose column after the urea treatment was loaded on to a 1 x 0. 8 cm phosphocellulose column, washed with 0.03 M potassium phosphate, pH 7.8, and the protein eluted with 0.25 M potassium phosphate, pH 7. 8. This material, the putative subunit, was vacuum dialysed with the C enzyme produced by the urea treatment, rechromatographed on DEAE cellulose and enzyme eluted batchwise (Fig 1 b). Recovery from this procedure was \n\n usually 50-70% A enzyme. Overall recovery was 30-40% of the C enzyme dialysed. If the material eluted from the phosphocellulose by the 0. 25 M potassium phosphate was heated to 90?C for 5 minutes prior to vacuum \n\n dialysis with the C enzyme the recovery from the DEAE cellulose column \n\n was significantly higher (60-70% of the original C enzyme activity), but all recoverable DNA polymerase activity was C enzyme. This, together with the fact that the 60 minute treatment with urea has, on occasions, given \n\n rise to a 50% increase in DNA polymerase activity prior to loading on to the DEAE cellulose column, would indicate that A enzyme is less active on \n\n activated DNA than is C enzyme. If the DEAE cellulose flow through material came from urea treatment of A2 enzyme then A2 was produced on reconstitution; if from A1 then A1 was produced. \n\n Although the mild urea treatment of A has been used to prepare C of \n\n 3344 \n\n Nucleic Acids Research \n\n b \n\n 120 180 C \n\n I I ^ \n\n Figure la DEAE cellulose chromatography after 2.4 M urea treatment. \n\n 4, 5000 units, 4.4 mg of Fraction II A2 enzyme were incubated with urea at a final concentration of 2.4 M for 60 minutes at 0?C, loaded on to a 5 x 1.4 cm DEAE cellulose column, the column was washed with 0. 03 M potassium phosphate, pH 7.8 and a 200 ml standard phosphate gradient applied. 2.3 ml fractions were collected and 10 Rl assayed for 5 minutes. (o-o) no \n\n phenylmethylsulphonylfluoride (*-*) 3 mM phenylmethylsulphonylfluoride ( - ) phosphate gradient. \n\n Figure lb The reconstitution of A2 enzyme. 3000 units for C enzyme \n\n derived by urea treatment of A2 were vacuum dialysed with the DEAE cellulose flow through material after phosphocellulose chromatography and \n\n chromatographed on a 2 x 1. 2 cm DEAE cellulose column. After washing with 0.03 M potassium phosphate, pH 7.7, the enzymes were eluted batchwise with the above concentrations of potassium phosphate, pH 7.8. 1 ml fractions were collected and 10 .l assayed for 10 minutes. \n\n 3345 \n\n Nucleic Acids Research \n\n high specific activity it is possible that conversion of the 200-230, 000 molecular weight enzymes to a 155- 170, 000 species maybe due to unfolding of the molecule to allow limited attack by contaminating proteases. Accord\n\n ingly the urea conversion of A to C was investigated in the presence of certain protease inhibitors. The presence of the serine protease inhibitor \n\n phenylmethylsulphonyl-fluoride in the incubation and chromatography buffers did not affect the conversion of A2to C (Fig 1 a). Likewise trasylol and Na-p Tosyl-L-lysine chloromethylketone HC1 had no effect. However, protease action could have occurred earlier in the purification procedure and the urea could be separating two fragments. Usually the DEAE cellulose profile shows that the majority of the enzyme activity is present as A \n\n enzyme (Fig 2). In this instance A1 and A2 have not been separated. When the temperature of the material in the original blending procedure was kept below 0?C or phenylmethylsulphonylfluoride, N-a-p Tosyl-L-lysine chloromethylketone HCl or trasylol was included in the isolation buffers the DEAE cellulose profile was similar. The A enzymes were still capable of conversion by mild urea treatment to C enzyme, indicating that if protease activity is involved then it is not susceptible to these inhibitors. When the calf \n\n 20FI: Now ~ ~ ~ 20 20Fr Na 06 \n\n Figure 2 DEAE cellulose elution profile of calf thymus DNA polymerase a. 47, 000 units, 97 mg of Fraction IV enzyme prepared from 415 g of calf \n\n thymus were loaded on to a 10 x 1.8 cm DEAE cellulose column, after washing with 0. 03 M potassium phosphate, pH 7. 8 a 400 ml standard phosphate gradient was applied. 5 ml fractions were collected and 10 ,ul assayed for 5 minutes. \n\n 3346 \n\n Nucleic Acids Research \n\n thymus was allowed to warm up during the blending procedure, or the supernatant prior to phosphocellulose chromatography was heated to 37 ?C for 30 minutes, there was a marked decrease in the amount of A enzyme with a concomitant increase in the amount of B and C (unpublished observation). The presence of phenylmethylsulphonylfluoride, N- a-p T osyl- L-lysine \n\n chloromethylketone HCl or trasylol under these conditions only had a marginal effect on the appearance of C enzyme, but did reduce the amount of B \n\n enzyme. Heating the enzyme to 37?C after the phosphocellulose step had no effect on the DEAE cellulose elution profile. Attempts to convert A enzyme to C using trypsin have not been successful. A enzyme activity is lost without the appearance of any other species (unpublished observation), although \n\n 14 the action of trypsin on C enzyme can give rise to small amounts of B \n\n Although the conversion of A enzyme to C does not appear to be the result of serine protease action, proteases other than serine proteases \n\n 18 \n\n could have been responsible . Also, the fact that A enzyme can be recon\n\n stituted from C plus the flow through material from the DEAE cellulose after urea treatment does not necessarily mean we are dealing with two subunits as fragments produced by proteases mray be reassembled to give active \n\n enzyme 9 . However, the fact that a 200-230, 000 molecular weight polypeptide band has never been observed in sodium dodecylsulphate polyacrylamride gels of highly purified A enzyme , or even cruder fractions of A \n\n enzyrre (unpublished observation), may be significant. One might expect \n\n some of the enzyme not to have been attacked by whatever is responsible for cleaving the molecule, if, indeed, this does happen. It would appear, therefore, that the A enzyme consists of subunits of 155, 000 and 50-70, 000 molecular weight with the small subunit having a slightly different charge in the \n\n case of A and A2. It has also been concluded that the heterogeneity in the \n\n 1 ~~~~~~~~~~~~~~~~~3 mouse myeloma DNA polymerase a fraction is not due to proteolysis . However, the 50-70, 000 molecular weight subunit has not yet been identified. \n\n Sodiurrm dodecylsulphate polyacrylamide gels of reconstituted A enzyme have shown polypeptide bands at 150-160, 000 and 50-70, 000 molecular weight \n\n (Fig. 3), but the ratio of staining intensity of the bands does not correspond to a 1: 1 relationship. The ratio of the two bands is variable but is usually between 1:2 and 1:3, indicating, perhaps, that more than one subunit of 50\n\n 3347 \n\n Nucleic Acids Research \n\n TOP 123 4 5 6 \n\n Figure 3 Scan of a 5% sodium dodecyl sulphate polyacrylamide gel of \n\n reconstituted A2 enzyme. 1500 units of reconstituted A2 enzyme were subjected to polyacrylamide gel electrophoresis under non-denaturing conditions. The gels were sliced, enzyme extracted and assayed and the peak \n\n fraction of DNA polymerase activity from three gels were pooled, subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis on a single \n\n gel, stained and scanned at 2 volt sensitivity as described2. The molecular weight standards were: (1) bovine serum albumin dimer (134, 000), (2) ,3 galactosidase (130, 000), (3) phosphorylase a (94, 000), (4) bovine serum \n\n albumin (68, 000), (5) pyruvate kinase (57, 000) and (6) lactate dehydrogenase (35, 000). \n\n 70, 000 molecular weight can associate with the 155, 000 subunit. The proportion of the lower molecular weight polypeptide band is higher in the reconstituted A enzyrrme than in the C enzyme preparation from which it was formed, but even the C enzyme contained some material in the region. \n\n Breakdown of material from 155, 000 to 50-70, 000 may be partly responsible for the contamination, but the presence of phenylmethylsulphonylfluoride in samples in preparation for sodium dodecylsulphate polyacrylamide gel electrophoresis has not been successful in preventing it. The human KB cell \n\n DNA polymerase a, equivalent to the C enzyme, has been reported to consist \n\n 4 \n\n of subunits of 76, 000 and 66, 000 molecular weight , but at no time have we \n\n observed polypeptide bands at 76, 000 and 66, 000 rising and falling with \n\n enzyme activity in any gels of calf thyrrmus enzymes A1, A and C (and \n\n 2 unpublished observations). (b) Template Studies \n\n Use has been made of the urea conversion of A to C and of the reconstitution of A enzyme to obtain samples of highly purified DNA polymerase, \n\n specific activity in excess of 50, 000 units/mg, in order to study the effect of this 50-70, 000 molecular weight subunit on the DNA polymerase subunit. Previous results have indicated that A enzyrrme is stabler to heat and less \n\n 3348 \n\n Nucleic Acids Research \n\n 14, 21 \n\n susceptible to N-ethylmaleimide than C enzyme . It has been reported \n\n that highly purified DNA polymerase a can be associated with a protein capable of binding to DNA containing no 3' OH ends and capable of being released during the DNA polymerase assay . Attempts to dissociate the 50\n\n 70, 000 molecular weight subunit from the polymerase subunit by incubating A2 with DNA polymerase reaction mixes containing activated DNA, poly \n\n (dA-T) and poly (dT). oligo (A)10 followed by ultracentrifugation in high salt have been unsuccessful. Similarly A absorbed and eluted from either native or denatured DNA cellulose remained A enzyme. Both A and C enzymes \n\n were eluted from the DNA celluloses by less than 0. 1 M NaCl so it does not appear that the subunit enhances the binding of the DNA polymerase subunit to DNA. However, there are differences in the response of A and C \n\n enzymes to synthetic template-initiator complexes (Table 1). Even if one takes into account the fact that A enzyme is less active on activated DNA \n\n than C enzyme (the addition of the 50-70, 000 subunit to the polymerase subunit appears to result in a decrease of about 30% of polymerase activity on activated DNA) the A enzyme is still more active on these templates. \n\n Although there is a variation in activity on these template-initiator comp\n\n lexes each time assays are carried out on them depending on the method of preparing the complexes and the base ratio of template to initiator, A enzyme always seems to be significantly more active than C. The A2 \n\n enzyme at pH 7.8, with extra subunit(s) is clearly more effective on the \n\n oligoribonucleotide initiator, oligo(A)10 than is enzyme C. In view of the proposed RNA initiation of Okazaki pieces this may indicate a role of this subunit in Okazaki piece synthesis, in that it may aid the DNA polymerase to'take over' from the RNA polymerase. Using poly (dA). oligo (dT) 10 \n\n (A:T=20: 1) and following the incorporation of [ H] dTMP as a function of time a short lag was observed for C enzyme, but not for A2 (Fig. 4). \n\n Similar re sult s we re obtained when poly (dC). oligo (dG) 1 0 (C: G= 5: 1) wa s \n\n used as temrrplate-initiator, but not when poly (dT). oligo (A)10 (T:A=1: 1) was used. Neither enzyme showed a lag on this template-initiator complex or on activated DNA. Incorporation versus enzyme concentration also showed this lag for C enzyme on poly (dA). oligo (dT)10. It is not certain what causes \n\n 3349 \n\n Nucleic Acids Research \n\n TABLE 1 Template utilisation by reconstituted A and C derived from A. \n\n 3H] dNTP Divalent cation A2 at pH C at pH \n\n Template _ 6.4 7.8 6.4 7.8 Activated DNA dTTP Mg++ 100 100 Activated DNA dATP Mg++ 91 85 Activated DNA dGTP Mg++ 85 82 poly(dA).oligo(dT)10 dTTP Mg 66.0 4.0 20 91 \n\n (A:T = 20: 1) \n\n poly(dA). oligo(dT) dTTP Mu 19.0 4. 5 6. 5 2.5 \n\n poly(dA. oig~10 (A:T = 20:1) \n\n poly(dT). oligo(dA)10 dATP Mg 2.5 <1 (1 (1 \n\n (T:A = 5:1) \n\n poly(dT) .oligo(dA)1 dATP Mn ++ 15.5 23.0 9. 0 3. 0 \n\n poly(dT). oligo(A)10 dATP Mg 15.0 230.0 10.0 77. 5 \n\n (T:A = 1:1) \n\n poly(dT) * oligo(A) 10 dATP Mn 41.5 75. 5 19.5 21.5 \n\n (T:A = 1:1) \n\n poly(dC). oligo(dG) dGTP Mg++ 6. 0 15.5 4. 5 7. 5 \n\n (C:G = 5:1) 10 \n\n poly(dC). oligo(dG)10 dGTP Mn + 6. 0 29.5 5. 0 6. 5 \n\n (C:G = 5: 1) . . \n\n Values given are relative to incorporation of [ H] dTMP at pH 7. 8 on activated DNA. For A2 this was 176.5 pmol, for C 232 pmol. dATP, dCTP, dGTP and dTTP were included for assays using activated DNA, only the deoxynucleoside triphosphate stated was used in the synthetic template\n\n initiator complex assays. Assays were for 10 minutes. The buffers and concentrations of the divalent cations were as in Materials and Methods. \n\n this lag, but the annealing of template to initiator is only transient ' and the DNA polymerase subunit may have difficulty in stabilising the complex and the 50-70, 000 molecular weight subunit may be able to help the poly\n\n merase subunit to overcome this. Addition of the 50-70, 000 subunit to the polymerase subunit prior to assaying with these templates had no effect on activity and it may be that a preincubation period is required before the two 3350 \n\n Nucleic Acids Research \n\n Figure 4 Activity of A2 and C enzymes on poly (dA). oligo (dT)jO (A:T = \n\n 20:1) as a function of time. 50 ,ul samples were withdrawn at various times from a 0. 6 inl incubation mix at 30?C and added to 0. 5 ml 0. 1 M sodium \n\n pyrophosphate containing 100 ,ug/ml native calf thymus DNA and processed for counting in the usual manner6. The mix contained 1 mM dithiothreitol, 50 mM tris HC1, pH 7.8 10 mM MgC12, 0. 1 mM [3H] dTTP (15 cpm/pmol), 312.5 ,ug bovine serum albumin, 5 Fg poly (dA). oligo (dT) (A:T = 20:1) and enzyme protein (*-*) 3.3 units reconstituted A2, (o-o) 16. 0 units urea derived C enzyme. \n\n subunits become fully associated. After the lag phase is over the C enzyme is still less active on these template-initiators than A enzyme . That is the A enzyme seems capable of elongating the initiator faster than the C enzyme. Experiments to determine whether the differences in rates of elongation of these template-initiator complexes are differences in processivity of the \n\n enzymes under the different pH and divalent cation conditions are under way. \n\n ACKNOWLEDGEMENTS \n\n We thank the Medical Research Council for a research grant. \n\n Communications concerning the paper should be sent to:\n\n Dr. A. M. Holmes, Department of Biochemistry, University of Strathclyde, The Todd Centre, 31 Taylor Street, Glasgow G4 ONR, U.K. \n\n 3351 \n\n Nucleic Acids Research \n\n REFERENCES \n\n (1) Bollum, F.J. (1975). Prog. Nucleic Acid Res. Mol. Biol. 15, \n\n 109-144. \n\n (2) Holmes, A.M., Hesslewood, I.P. and Johnston, I.R. (1976). Eur. \n\n J. Biochem. 62, 229-235. \n\n (3) Matsukage, A., Sivarajan, M. and Wilson, S.H. (1976). Biochem\n\n istry 15, 5305-5314. \n\n (4) Fisher, P.A. and Korn, D. (1977). J. Biol. Chem. 252, 6528\n\n 6535. \n\n (5) Fichot, O., Pascal, M., Mechali, M. and De Recondo, A, -M. \n\n (1979). Biochim. Biophys. Acta, 561, 28-41. \n\n (6) Holmes, A.M., Hesslewood, I.R. and Johnston, I.R. (1974). Eur. \n\n J. Biochemn. 43, 487-499. \n\n (7) Noy, G.P. and Weissbach, A. (1977). Biochim. Biophys. Acta, \n\n 447, 70-83. \n\n (8) Craig, R.K. and Keir, H. M. (1975). Biochem. J. 145, 225-232. \n\n (9) Matsukage, A., Bohn, E.W. and Wilson, S.H. (1974). Proc. Natl. \n\n Acad. Sci. U.S.A. 71, 578-582. \n\n 110) Hachmann, H.J. and Lezius, A.G. (1975). Eur. J. Biochem. 50, \n\n 357- 366. \n\n (11) Brakel, C.L. and Blumenthal, A.B. (1977). Biochemistry, 16, \n\n 3137-3143. \n\n (12) Momparler, R.L., Rossi, M. and Labitan, A. (1973). J. Biol. \n\n Chem. 248, 285-293. \n\n (13) Yoshida, S., Konda, T. and Ando, T. (1974). Biochim. Biophys. \n\n Acta, 353, 463-474. \n\n (14) Holmes, A.M., Hesslewood, I.P., Wickremasinghe, R.G. and \n\n Johnston, I.R. (1977). Biochem. Soc. Symp. 42, 17-36. \n\n (15) De Jong, A., Van der Vliet, P. and Jansz, H.S. (1977). Biochim. \n\n Biophys. Acta, 476, 156-165. \n\n (16) Holmes, A.M., Hesslewood, I.P. and Johnston, I.R. (1975). \n\n Nature (London) 255, 420-422. \n\n (17) Bollum, F. J. (1966). Procedures in Nucleic Acid Research \n\n (Cantoni, G.L. and Davis, D.R. eds). pp 577-583, Harper and Row, New York. \n\n (18) Barrett, A.J. (1975). Proteases and Biological Control pp 467-482, \n\n Cold Spring Harbor Laboratory, Cold Spring Harbor. \n\n (19) Richards, F. M. and Vithayathil, P.J. (1960). Brookhaven Symp. \n\n Biol. 13, 115-134. \n\n (20) Lowe, P.A., and Malcolm, A.B.D. (1976). Eur. J. Biochem. 64, \n\n 177- 188. \n\n (21) Hesslewood, I.P., Holmes, A.M., Wakeling, W.F. and Johnston, \n\n I.R. (1978). Eur. J. Biochenr.. 84, 123-131. \n\n (22) Mechali, M. and De Recondo, A. -M. (1978). Biochim. Biophys. \n\n Res. Comm, 82, 255-264. \n\n (23) Chang, L.M.S., Cassani, G.R. and Bollum, F.J. (1972). J. Biol. \n\n Che. 247, 7718-7723. \n\n (24) Wickremasinghe, R.G. and Johnston, I.R. (1974). Biochim. \n\n Biophys. Acta, 361, 37-52. \n\n 3352 " ], "offsets": [ [ 0, 25566 ] ] } ]

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[ { "id": "pmcA1931593__text", "type": "Article", "text": [ "Integration in primary community care networks (PCCNs): examination of governance, clinical, marketing, financial, and information infrastructures in a national demonstration project in Taiwan\nAbstract\nBackground\nTaiwan's primary community care network (PCCN) demonstration project, funded by the Bureau of National Health Insurance on March 2003, was established to discourage hospital shopping behavior of people and drive the traditional fragmented health care providers into cooperate care models. Between 2003 and 2005, 268 PCCNs were established. This study profiled the individual members in the PCCNs to study the nature and extent to which their network infrastructures have been integrated among the members (clinics and hospitals) within individual PCCNs.\n\nMethods\nThe thorough questionnaire items, covering the network working infrastructures – governance, clinical, marketing, financial, and information integration in PCCNs, were developed with validity and reliability confirmed. One thousand five hundred and fifty-seven clinics that had belonged to PCCNs for more than one year, based on the 2003–2005 Taiwan Primary Community Care Network List, were surveyed by mail. Nine hundred and twenty-eight clinic members responded to the surveys giving a 59.6 % response rate.\n\nResults\nOverall, the PCCNs' members had higher involvement in the governance infrastructure, which was usually viewed as the most important for establishment of core values in PCCNs' organization design and management at the early integration stage. In addition, it found that there existed a higher extent of integration of clinical, marketing, and information infrastructures among the hospital-clinic member relationship than those among clinic members within individual PCCNs. The financial infrastructure was shown the least integrated relative to other functional infrastructures at the early stage of PCCN formation.\n\nConclusion\nThere was still room for better integrated partnerships, as evidenced by the great variety of relationships and differences in extent of integration in this study. In addition to provide how the network members have done for their initial work at the early stage of network forming in this study, the detailed surveyed items, the concepts proposed by the managerial and theoretical professionals, could be a guide for those health care providers who have willingness to turn their business into multi-organizations.\n\n\n\nBackground\nTaiwan's National Health Insurance (NHI) under the control of the Bureau of National Health Insurance (BNHI), was launched in March 1995 to replace its social insurance system that was covering 59% of its population: government employees, labourers, farmers and servicemen [1]. By June 2003 the number of people insured had reached 21,956,729 (99%). There were 17,259 medical providers (92%), including 575 hospitals and 16,684 clinics contracted with the BNHI for serving the enrolled population. The unique phenomenon characterized in Taiwan health care industry different from those in the western countries is the freedom of patients to choose the health care providers they want, no matter what their disease severity is. Furthermore, Taiwan people favor the larger scales of facilities and this fallacy leads to the phenomenon of big-hospital shopping. For example, people choose the medical centers which are accredited as the highest level of medical science in Taiwan when they only suffer from a common cold.\nIn the spring of 2003, the SARS epidemic viciously attacked the health of Taiwan's people. The people's freedom to choose medical providers caused the national health authority to barely control and traced the flow of epidemic. This event made Taiwan national health authorities rethink what happened and how it damaged under the traditional fragmented health care providers in Taiwan. One health reform launched was named the \"Primary Community Care Network (PCCN) demonstration project\", a nationwide health care financing program funded by the Bureau of National Health Insurance (BNHI) in March 2003 and it was a new model for the Taiwan government to redefine the role of family physicians in the health care delivery system. A PCCN in Taiwan consists of a group of clinic physicians whose medical jobs are viewed as family care and at least one hospital for secondary or tertiary care. The idea of member component design in PCCNs was aimed to lead the Taiwan citizens to choose one clinic physician as their personal family physician for health maintenance and this family physician also would have the responsibility of referring the patients to specialty care if necessary. From a national health authority perspective, they expected the Taiwan people to put an end to their fallacy that \"bigger is better\" for health care organizations and establish the idea of \"human health\", starting with prevention and primary care, followed by secondary or tertiary care, emphasizing health promotion and maintenance instead of disease curing. Furthermore, it could decrease the inappropriateness of medical usage, i.e., over-uses of secondary and tertiary medical services in the high-tech hospitals. In addition, the national health authority was expected to drive the traditional fragmented heath care providers into coordinated medical multidisciplinary teams and share the limited medical resources through the PCCN demonstration project. In summary, the PCCN demonstration project was aimed to: 1) change the traditional patients' customs of freely choosing health care organizations and establish referral channels along the continuum of care, and 2) establish partnerships among the primary care clinics and hospitals to provide a continuum of health care services. It was also expected to establish the primary care system of family physicians to provide whole-people health care and improve care quality [1].\nPartnership structures in the PCCNs represent the virtual vertical (i.e., between the member clinics and hospitals) and virtual horizontal (i.e., among the member clinics) aspects of organizing, which designate the formal relationships between individuals and the total network and include organizational design to ensure effective communication, coordination, and integration across the total network. Each PCCN consists of five to ten clinics: half of them should offer the services of general medicine, internal medicine, surgery, obstetrics and gynecology, pediatric, or family medicine. And each PCCN has a central headquarters, usually in one of the clinic facilities, to coordinate and integrate the network. All the clinic physicians in a PCCN are assigned the roles of \"family physicians\" or \"gatekeepers\" who recruit people from the local community, keep background and medical files on them, certify family physician education training programs, and hold office hours in the member hospital, where they serve as joint faculty members for further medical consultations or medical utilizations of labs and tests, if necessary. In addition, the hospital member is asked to help clinic physicians in their network to set up a medical information system, share hospital resources (medical equipment and library literature) with the clinic physicians in their network and establish referral channels among the network members. Furthermore, this new demonstration model tries to minimize the barriers to patient access by setting up 24-hour a day, 7-day a week medical consultation telephone lines for providing urgent services onsite and for taking care of the patients whose family physicians' practices are closed to assure seamless care channels. The BNHI funded these extra demonstration actions, at around one hundred thousand US dollars (i.e., NT$3,500,000) for each PCCN under the current fee-for-service payment system [1].\nFigure 1 describes the organizational structure of individual PCCNs introduced in the demonstration project in Taiwan.\nTo date, the PCCN demonstration project has been in operation for more than three years. There have been 268 PCCNs formed in the period of 2003 to 2005 around Taiwan. The geographical distributions of PCCNs and their members were described in Table 1. Analyzing all 1,557 participating clinic members in the demonstration project in terms of medical specialties, they cover general medicine, internal medicine, surgeries, obstetrics and gynecology, pediatrics, family medicines, otolaryngology, ophthalmology, rehabilitation medicine, dermatology, and psychiatry, with 237 clinics providing more than two specialties. On the other hand, each PCCN recruits at least one district or regional accredited hospital for acute care demands (required for network members) and a medical center for tertiary care support (not required for network members). There are 6 medical centers, 52 regional hospitals, and 71 district hospitals joining in the demonstration project. See Table 1 for more detailed information about the PCCN members.\nTo date, there have been few empirical studies of the working relationships that have developed between members of the PCCN program. Partnership needs a method to determine at an early stage, to make sure whether they are making the most of collaboration [2] and the acceptance of the contracting networks in Taiwan as an organizational innovation worthy of greater diffusion deserves to be explored. Therefore, this study used a structured questionnaire to characterize the relationship among the members in the individual PCCNs, with regard to governance, clinical, marketing, financing, as well as information integration infrastructures. The results of this study provide descriptive analyses in detail to map the partnership developments, to enrich the body of knowledge of the partner relationships and to help policy makers understand the coordinated efforts of these health care providers which have developed under this system. It also provides the recommendations for heath policy decision-making and management of networks of health care providers for the future involvement.\n\nMethods\nThis study was aimed at providing descriptive analyses to map the partnership development. To understand the actual integration actions done by network members, the theoretical concept employed by network partnerships were described and then the derived survey instrument was developed.\nTheoretical framework for organization design of network integration\nThe rapid organizational changes in the health care industry have driven theorists from every discipline and across the world to seek an approach that allows organizations to flourish. Organization theory allows investigators to profile an organization from the aspect of patterns and regularities in organizational design and behavior. In the early 20th century, classical management theorists claimed that an organization has \"a best way\" to be organized and managed [3]. That implied that all organizations would own the \"same\" organizational styles or structures. In the 1960s, several theorists [4-8] challenged this assumption by applying a \"contingency approach\" to propose that there is no best way to organize an organization, and that the effectiveness of an organizational structure varies with the situation of an organization. Furthermore, it is proposed that the best way to organize an organization depends on the nature of the environment to which the organization relates.\nContingency theory delineates the concepts \"organization's internal features,\" \"the demands of organizational environments,\" \"best adaptation,\" and, the most important and difficult of all, \"best match\" [9]. Lawrence & Lorsch [7] argued that environments characterized by uncertainty and rapid rates of change in market conditions or technology impose different demands, including constraints and opportunities, on organizations than do placid and stable environments. Similarly to Lawrence and Lorsch's views mentioned above, Galbraith [10,11] stressed the contingency perspective on information processing. The information-processing approach emphasizes that environment, size, and technology impose different information-processing requirements on organizations, and thus an organization must be designed to encourage information flow in both vertical and horizontal directions to achieve the overall tasks of the organization and, finally, organizational effectiveness [11-14].\nSome theorists have criticized conventional contingency theorists who presume that organizational structure is driven by the environment. Child [15], Miller [16], Van de Ven and Drazin [17], and Tushman and Romanelli [18] raised such criticisms; they argued that organizations become what they are not only because of the environment, but also because of choices made by members, especially choices about strategy and organizational design. As Thompson's words in the book Organizations in Action [8] put it, \"organizations are not determined simply by their environments (p.27).\" He also pointed out that \"administration may innovate on any or all of the necessary dimensions, but only to the extent that innovations are acceptable to those on whom the organization can and must depend.\" Instead of assuming that administrators are highly constrained in their decisions, strategic contingency theorists emphasized \"the importance of choice,\" that is, \"the freedom of agency\" [15]. Furthermore, Pfeffer [19] explicitly pointed out that \"organizational structures are the outcomes of political contests within organizations (p.38).\"\nDaft [14] proposed a top management model to delineate how \"a strategy is a plan for interacting with the competitive environment to achieve organizational goals.\" He stated that the major responsibility of top management is to determine the goals, strategy, and design of an organization to adapt to a changing environment. To assess the external and internal environments of an organization seems to be the first task for top managers in defining an organization's goals and missions. Then, guided by the goals and missions of the organization, top managers shape the design of the organization, including structural forms, information system, technology, human resources, organizational culture, and inter-organizational linkages, to achieve the final organizational performance.\nIntegration refers to the mechanisms of coordination, the ways guided to partnership goals to fit internal and external conditions [7,20,21]. In the early 1990s, proposals for US national health care reform recognized the need for integrating mechanisms to achieve both financial success and quality of care of a well-organized system of care [22,23]. Several researchers also viewed inter-organizational cooperation as resource exchanges, including client referrals, money, and staff [24-27]. From practical ways of viewing integration, the success of integration lies in the coordinative mechanisms and partnership working that support it [28], including an administrative organization that coordinates the operations of various health care services; a management information system that integrates clinical, utilization, and financial data and follows clients across different settings; a care coordination program such as case management or disease management that works with clients to arrange health care services; and a financial mechanism that enables pooling of funds across services [29-35]. Fox [36] suggested the success of integrated health networks should ensure that the new business link such aspects as technology, functional skills, customer access, management, or products that can be shared across both the core and the new business; to conduct market financial evaluation; to share the risk of vertical integration with outside entities, to develop the management structure that can reflect the degree of coordination necessary to support the core business activities; to ensure that the integration strategy meets the needs of customers, including medical treatment, the use of medical technology, and the preferred methods of purchase; and to measure the new business by its value to the enterprise as a whole, rather than by its profitability as a stand-alone entity.\nIn summary, the effects that integration in inter-organizational designs has on network management were substantial from a managerial perspective. Borrowing the ideas of strategic contingency perspective [8,15,19] and top management model [14], it could be imply that success (organization performance) in reengineering a network lies in the integration of process and services (see Figure 1), including leadership/governing structure, teamwork between disciplines and patient care, financial planning, and information systems, characterized as the constructs of governance, clinical, financial, and information infrastructures, respectively, in this study. In addition, another construct, marketing infrastructure, was especially important and designed to explore for PCCNs in this study because of patients' freedom of making healthcare choice and the traditional fragmented health care systems by individual health care organizations in Taiwan. One major reason for Taiwan people's hospital shopping preferences was that Taiwan people usually believe the bigger the facility, the better capacities a facility has no matter on any aspect from medical professionals to tangible medical equipment and plants. And this fallacy made the public want to overuse the facility with high-tech medical services no matter if it fits their needs. From the health policy and management perspectives, therefore, the health care providers were encouraged to market their services as a new corporate identity and brand strategy [37], including offering tangible resources such as books, libraries, medical equipment, and intangible resources such as knowledge and information exchanges (education) and reputation sharing one another among PCCN members. Furthermore, through the process of marketing resource exchanges, therefore, each PCCN could establish the images of \"one system, one brand and quality\" for the public and for the health care providers. It also makes it be more visible to the public.\nThe five integration infrastructures of network management were constructed as a conceptual framework in this study to help to portray how the PCCN members have done. The survey instrument development was described in the following.\n\nSurvey instrument development: integration infrastructures and measurements of partnerships\nBased on the five integration infrastructures of network management, the structured questionnaire were derived from extensive literature reviews.\nGovernance infrastructure\nGovernance assumes the broad responsibility for organizational goals and survival and involves the series process of setting and monitoring organizational goals and strategy development through a board of representatives [38]. Governance or administrative integration infrastructure in establishing network partnerships refers to administrative structures (or responsibilities) created to facilitate communication, clear lines of authority, accountability, and responsibility for patient care services; to negotiate budgets and financial trade-offs; and to present a cohesive, consistent message in interactions with external agencies and the community [29,39-41] and most important for members in contract agreements, to manage participation [33]. From a multidisciplinary perspective, Mitchell and Shortell [42] applied the concepts of governance and management characteristics in effective community health partnerships. The construct of governance involved several tasks, including setting priorities for strategic goals, choosing the membership composition, obtaining the necessary financial resources, and setting up the accountability systems, and so on. The construct of the management refers to the tasks of engaging and maintaining organizational members' interest in a shared vision and mission, providing appropriate structures and coordination mechanisms for the specified strategies, promoting constructive conflicts and managing destructive conflicts, implementing information systems to monitor the dynamics, adjusting the leadership in the overall membership, and so on. The issues of governance and administrative integration in the PCCNs could include [2,38,40,41,43-47]:\n• planning the shared visions and missions\n• determining the shared service strategies, cooperation priorities, policies and principles\n• identifying the information needed and how to get it\n• organizing the network dynamics and member roles\n• leading and managing the conflicts and communication\n• designing and controlling the shared network performance systems, including indicator settings, feedbacks, and accountability.\n\nClinical infrastructure\nThe idea of care integration begins through such public programs that include social workers in public welfare departments, caseworkers in mental health, or nurses in public health departments. In the late 1980s, care integration was deemed necessary for the streamlining of care and negotiating the maze of long-term care services. At that time, it was referred to as service coordination or case management, or in other related terms [29]. The purpose of care integration is to work directly with patients and their families over time to help them arrange and manage the complex resources that patients may need to maintain health and independent functioning. At the same time, care integration is used to achieve the most cost-effective use possible of scarce resources, by steering patients to the health, social, and support services most appropriate for them at a given time [29]. Conrad and Dowling [33] pointed out that to coordinate and integrate patient care relies on connecting patient services at the different stages of the patient care processes. Care coordination in integrated networks can be achieved through integration of training programs and some clinical services, provision of complementary clinical capabilities, clinical geographic proximity design, clear role definition of each institution, commitment and flexibility of leaderships and medical staffs, and the support of a large referring physician groups embracing the affiliation concepts [48]. The issues of clinical integration in the PCCNs could include [48-50]:\n• planning and differentiating target markets based on the clinical services of the network members\n• uniting individual clinical professionals for clinical project planning\n• designing patient-centered care or case management teams\n• establishing committees responsible for patient-centered case report meetings, case referral, transfer, and tracing, file management (record and information exchanges), clinical quality management (quality assurance, improvement, risk and malpractice management, and utilization review), and medical continuing education and on-job education.\n\nMarketing infrastructure\nMarketing integration refers to how to work together as a whole both from the provider and patient perspectives. One of the case reports interviewing developing integrated delivery system or networks realized that the most important thing is how an integrated system or network is promoted and what is promoted for the consumers [51], including focusing on product development, making sure the branding holds together, marketing directly to consumers, demonstrating values to consumers, and even conducting marketing research to make efforts for the long term. In a health care network with several organizational members and target patients, the marketing infrastructure in PCCNs here refers to provider members' marketing, meaning the resource sharing and market development in a PCCN as a whole. The issues of the marketing integration in the PCCNs could include [37,52-54]:\n• sharing the literature and facility publications among the network members\n•uniting public promotions such as united activities, electronic and paper media for enhancing the network reputation as \"one system, one brand and quality\"\n• differentiating target markets of the network for competing in the medical industry.\n\nFinancial infrastructure\nComprehensive, flexible, and adequate financing is a goal of the ideal continuum of care. That component is the most critical and challenging to manage under the changes in the health care delivery environment. Gillies et al. [30] suggested that integrating financial management across operating units adds the greatest value to systems or organizations. In one case study, Bramson et al. [55] also showed that reducing costs through joint purchasing by the radiology departments of a vertically integrated health system could yield substantial savings. The issues of the financial integration in the PCCNs could include:\n• budgeting\n• uniting equipment, medical materials, and drug purchasing and routine administrative stuff management\n• pooling recruitment funds\n• designing a financial risk and sharing mechanism.\n\nInformation infrastructure\nInformation is an essential component of an organization. A complete information system can help an organization to integrate its individual units and efficiently manage the continuum. The ideal information system for a continuum of care was conceived of and formed in the mid-1980s [56]. During the late 1980s, computer technology began to make an information system feasible and affordable through new computer chips with expanded capability and networking technology. In the 1990s, the individual services of the continuum upgraded their information systems to combine clinical, financial, and utilization data [29]. Some studies have argued that the quality of information systems can drive costs down, because a good information system can give physicians easy electronic access to complete the documentation of the patients' clinical records, better inform them about reimbursement and capitation issues, help them easily associate and manage cases together, and achieve a higher level of professional satisfaction [57,58]. Using Inova Health System, an integrated delivery system in northern Virginia, as an example, Wager, Heda, and Austin [59] showed that by developing a health information network within an integrated delivery system, Inova can have a clinical transaction system for hospitals and other entities, a data repository for decision support and outcome management, a managed care information system to support managed care and capitation contracts, and greater capability to acquire physicians. The issues of information coordination include [60-68]:\n• establishing an electronic medical record system, regional information network for patient clinical and administrative data, clinical service arrangements and administrative work\n• uniting the system information management and web pages.\nThe structured questionnaire was developed with the wording of practical managerial actions based on the five concepts just mentioned. There were 19 survey items on governance infrastructure, 25 on clinical infrastructure, 13 on marketing infrastructure, 20 on financial infrastructure, and 7 on information infrastructure. All 84 items were, simultaneously, applied to examine the relationships of the clinic's peer members and the relationship of clinic and hospital members in a PCCN, and it resulted in a total of 168 survey questions. The detailed information of the item questions was listed in Table 2, 3, 4, 5, 6. The structured questionnaires were drafted from previous literatures and then examined by two academic professors for theoretical accuracy. Then one pilot study was pre-tested for the PCCN pioneers (i.e., 92 network clinic members) and 116 hospital providers which have partner relationships with other health care organizations (i.e., hospitals, clinics, long-term care facilities). The wordings and meanings of each question item were revised to assure content validity. The Cronbach α values for the five integration constructs – governance, clinical, marketing, finance, and information infrastructure were 0.946, 0.958, 0.932, 0.944, and 0.898 for the measures of clinic-clinic member relationships; and 0.945, 0.949, 0.916, 0.948, and 0.896 for the measures of clinic-hospital member relationships.\n\n\nStudy subjects\nTo find the member partnership, we sent questionnaires to 1,557 individual clinics which had belonged to PCCNs for at least one year, based on information contained in the Taiwan Primary Community Care Network List (Bureau of National Health Insurance 2003, 2004 and 2005).\nWe let clinic members in all PCCNs point out how they coordinate with their peer clinic members and hospital members within a PCCN because individual clinic members could be better informants than hospital members, which need to deal with multiple clinic relationships and therefore might find it hard to describe the coordination involvement one by one with clinic members. Moreover, networks form for various reasons and it might lead to the various involvements by individual network members (i.e., hospital and clinic members). Therefore, using the participating clinics as individual survey units, the results could portray the overall dynamics and processes more authentically and detailed throughout all PCCNs in the demonstration project.\nNine hundred and twenty-eight clinics responded (59.6 %), with 239 clinics in the Taipei region, 165 in the northern region, 241 in the central region, 108 in the southern region, 150 in the Kao-Ping region, and 15 in the eastern region of Taiwan. Ten clinics had not mentioned their practicing locations. There is no statistically significant difference in geographical distribution between the respondents and the study population (χ2 = 4.208, p > 0.05).\n\nAnalytical techniques\nThe data was first analyzed descriptively with frequency counts (percentage) for each survey item, instead of using mean as a statistical method, because the variation among the respondents may not represent the normal distribution and it might ignore the extreme values for the respondents' answers. To compare how the respondents perceived the strength of integration existing in clinic-clinic and clinic-hospital relationships, paired t-tests were performed for individual survey items, using the original numerical scores.\n\n\nResults\nProfiling the partnerships in Taiwan PCCNs: governance infrastructure\nWith regard to the governance infrastructures, the frequency was counted for each survey item with recalculated scales: disagree (Likert scale 1 and 2), fair (Likert scale 3), and agree (Likert scale 4 and 5) with individual items. In clinic-clinic relationship (Table 2), the majority of clinic members agree that the determined deals were obeyed (Table 2, item 1: 88.69%), the goals and strategies of members were well-understood (Table 2, item 16: 79.74%), and the united principals for individual members were developed (Table 2, item 15: 79.42%). The higher percentages were also found in clinic-hospital relationship in the same items (Table 2). On the other hand, establishing fair coordination mechanism (Table 2, item 11: 27.91%), designing and employing the network performance indicators (Table 2, item 3: 21.23%), and establishing communication models and channels (Table 2, item 12: 19.94%) still occupied higher percentages not developed and deserved to been made the focus of more efforts in the future. Paired t-test analyses for all individual survey items of governance infrastructure showed that the deals obeyed (Table 2, item1) and plans and goals controlled (Table 2, item 2) were achieved more in clinic-clinic relationships than those in clinic-hospital relationships; however, the design of network performance indicators (Table 2, item 3), development of disintegration policy and principals (Table 2, item 8), and the establishment of fair coordination mechanism (Table 2, item 11) were reached more in clinic-hospital relationships than those in clinic-clinic relationships.\n\nProfiling the partnerships in Taiwan PCCNs: clinical infrastructure\nExamining the extent of clinical infrastructure for network members, establishing two-directed patient referral systems and patient referral information files (Table 3, items 35 & 37) and uniting medical continuing education and on-job education (Table 3, item 34) were shown at a highly implemented rate in clinic-clinic (more than 70%) and clinic-hospital (more than 80%) relationships. On the other hand, network members had higher percentages (more than 40%) not to think about the possible integration mechanisms including establishing committees to deal with medical malpractice (Table 3, item 44), planning and differentiating clinical market areas (Table 3, item 20), and designing patient-centered case management teams (Table 3, item 22). Overall, there was better clinical integration involvement for all the described items in clinic-hospital relationships than those in clinic-clinic relationships within a network in this study (see Table 3, paired t-tests, p < 0.05).\n\nProfiling the partnerships in Taiwan PCCNs: marketing infrastructure\nFor marketing planning, the clinics had better integrated marketing activities with their respective hospitals than with peer clinic members within PCCNs for all studied items (Table 4, paired t-test, p < 0.05). Examining the clinic-clinic relationships, uniting social activities (Table 4, item 53), sharing the individual facility reports for updated services (Table 4, item 46), public promotion (Table 4, item 54), and uniting and joining the facility activities (Table 4, item 51) were the top four marketing works done among clinic members (more than 60% implemented rate); and those items also showed a higher implemented rate (more than 70%) between clinic and hospital members.\nOn the other hand, facility assets such as reports (Table 4, item 49), and professional literatures and books (Table 4, item 45) were not well-shared among clinic members (\"never-thinking\" rate: 42.13%). In addition, uniting the network publication could make more efforts in the future (\"never-thinking\" rate in item 50: 39.98%). The room for clinic-hospital partnership to think about acting was kind of different from those in the clinic-clinic relationship. In addition to the uniting publication that can be encouraged to improve the clinic-hospital relationship (Table 4, item 50: 27.48%), cooperating in research projects (Table 4, item 52: 25.00%) and identifying and differentiating target markets (Table 4, item 57: 24.57%) had still more opportunities to be focused on in the future.\n\nProfiling the partnerships in Taiwan PCCNs: financial infrastructure\nThe PCCN members were found to have a lower extent of financial integration as evidenced by higher percentage of ''never thinking'' scale about the survey items on almost all items (see Table 5). Slightly more integration (that is, ''acting'' rate) was found in only four items both in clinic-clinic relationships and in clinic-hospital relationships, including uniting budget planning (Table 5: item 58), sharing places, materials, and equipment (Table 5: item 68), uniting budgeting for certain services (Table 5: items 72), and designing the resource distribution principals based on the whole network goals (Table 5: item 77).\nFurther examining the financial infrastructure in clinic-clinic relationship and clinic-hospital relationship, paired-t tests revealed that clinic-hospital partnerships were involved more in places, materials, and equipment sharing and maintenance (Table 5, items 62 and 68) (p < 0.05) and higher financial infrastructure coordination exists in clinic-clinic relationships (Table 5, items 58, 59, 63–65, 72, 74, 75, and 77) (p < 0.05).\n\nProfiling the partnerships in Taiwan PCCNs: information infrastructure\nThere was significantly greater integration of information in clinic-hospital than clinic-clinic relationships in all items in this category (Table 6, paired t-tests, p < 0.001). The greatest integration was found in electronic patient records (Table 6: item 78), followed by information integration for patient data (Table 6: item 79) and clinical service arrangements (Table 6: item 81). The lowest level of integration in information infrastructure was found in administrative works such as registration, billing and so on (Table 6: item 82, ''never-thinking'' rate more than 50%) within network members.\n\n\nDiscussion\nIn this study, we surveyed 943 clinics that had belonged to Taiwan PCCNs for more than a year to understand the nature and extent of integration to which they and their associated PCCN members (clinics and hospitals) had in governance, clinical, marketing, financial, and information infrastructures. It was found a wide variance in the kind and degree of integration among them and a lot of room for better integration (Table 2, 3, 4, 5, 6).\nFrom the governance perspective, we found lower integration was found in the establishment of fair coordination mechanism (Table 2: item 11) among member clinics and member hospitals. Coordination could be viewed from different perspectives, including the use of standardized languages and forms, organizational rules and procedures, the establishment of common rules, policies, and procedures, and the monitoring through memos, reports, and a computerized information system [69,70]. Facing the cumbersome integration processes, it suggests that each PCCN's headquarters should become actively involved and clarify the authority, responsibility and accountability of individual members, identify the potential conflict sources, and publicize the rules and regulation of network integration dynamics covering decision making processes, market planning, clinical teamwork designs, and financial reports of individual network members. These actions could enhance the trust and respect of network members one another and could improve the small extent of integration found in this study about the mechanisms for communication models and channels in the PCCNs (Table 2: item 12). From the network management perspective, communication could occur between the various entities such as between hospital and clinics, primary care physicians and specialists, managers and clinical professionals, and even among the clinical professionals in the network. To develop effective and timely communication channels was the key for the management of integrated organizations [30-32] and could alleviate the tensions that sometimes occur in the dynamics of the multi-organizations. In this study, it was found a low level of involvement of medical teams in medical projects, patient-centered case management, and case report meetings among the network members (Table 3: item 21, 22, and 23) from a clinical integration perspective. Several researchers have addressed that clinical integration providing a process of medical management, care management, case management, and patient management designed to transform the traditionally fragmented delivery system into a more cohesive system [71], and lead to higher service quality and assure financial objectives [72,73]. More attention could be paid to these activities in the future. In addition, it was also found less integration in planning and differentiating clinical market areas (Table 3, item 20) among the network members in the category of clinical infrastructure. This may result from the existing specialty diversities in individual PCCNs, which might not need to involve planning and differentiating market area based on the members' clinical services at the early stage of network development.\nThere was more involvement in marketing efforts in clinic-hospital relationships than in clinic-clinic relationships. Generally speaking, hospitals have more resources (i.e., money, human resources, materials, and physical assets) than clinics, which might explain the stronger marketing involvements between the clinic and hospital members, including the library sharing (books and literatures), facility brochure dissemination, public promoting, and medical research cooperation. These integration efforts also meet the expectation of the national health authority for resource sharing and medical quality image enhancement among the health care providers.\nFinancial infrastructure was found to be the least integrated, with most items never considered. Perhaps the only reason for the higher score of budget planning activities (Table 5: items 58 and 77) was that BHNI required each PCCN to design and determine its budgeting arrangement in advance before joining the demonstration project. While slightly more financial involvement was made among network members (Table 5: items 68, 72 & 73), possibly due to similar needs, there remains a lot of room for financial integration in the future.\nThere was a need for networks to develop electronic information systems, though creating and managing an integrated information system involves very detailed work. Most of the clinics surveyed have focused more on the individual public members' administrative works such as filing patient medical records, collecting and managing network patient clinical data, and scheduling clinical services, which were required by the BNHI. The factors for the health care managers to adopt the integrated clinical information systems include the decision of make or buy, adoption leadership, adoption objectives, implementation leadership, phased versus simultaneous implementation, parallel systems, information technology implementation policies and practices, use levels and resistance, and realized benefits and return on investment calculation [66], which might be very cumbersome and time-consuming. It suggests that the network partners might be engaged, firstly, more in simpler network cooperation such as the administrative systems for patient admission to the network members and establishing united web pages for patients to access their family physicians and network members for medical and public promotion purposes. And for further integrated information investments, efforts must be redirected for network members to work together to define the approach to specific classes of integration for the long term [74].\n\nConclusion\nThis study tried to portray and trace how the facility participants were involved in the Taiwan PCCNs. It was found that Taiwan PCCNs' members had higher involvement in the governance infrastructure, which was usually viewed as the most important for establishment of core values in PCCNs' organization design and management. There existed a higher extent of integration of clinical, marketing, and information infrastructures among the hospital-clinic member relationship than those among clinic members within individual PCCNs. The financial infrastructure was shown the least integrated relative to other functional infrastructures at the early stage of PCCN formation. Page [43] argued that networks form and grow for various reasons, however, only some of them could be compatible with the iterative processes of collaboration. Some participants in the PCCNs may simply seek short-term economic gains and have little interest in joint learning and continuous improvement. From an organizational design perspective, the old phrase proposed by the wisdom of the saying about developing the integrated organizations (networks) should be – \"coming together is the beginning, and working together is the success.\" Page [43] examined the virtual provider organizations such as physician-hospital organizations and pointed out the issue of the provider attitudes and behaviors as the critically successful continuous improvements in the health care environments. A wide variance of degree of network integration in Taiwan PCCNs still leaves room to improve.\nIn this study, the thoroughly surveyed items, that is, the potential network design content, were employed. In addition to provide how the network members have done their initial work at the early stage of network forming in this study, the detailed surveyed items, the concepts proposed by the managerial and theoretical professionals, could be also a guide for those health care providers who have a willingness to join multi-organizations. It suggests that health care providers could take more detailed looks about those surveyed items and give some possible opportunities to create the potential actions. Further research could be empirically done to explore the relative influence of these integration mechanisms on the effectiveness of organizational partnerships.\nThe partnerships within each PCCN represent various relationships that depend on how much the members are engaged in the projects. In addition to the macro concepts including governance, clinical, marketing, financial, and information infrastructures explored in this study, other managerial issues for integrated organizations were also suggested such as formation of an integrated cultural atmosphere, human resources management, physician involvement, mission and commitment establishment, from micro organizational behavior perspective [30-32,34,36,75,76]. Micro managerial and longitudinal research designs could be employed to more precisely catch the never completing integration efforts in the future.\n\nAbbreviations\nprimary community care network (PCCN); Bureau of National Health Insurance (BNHI)\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\nAuthors' contributions\nBYJL independently designed and conducted this study.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\n\n" ], "offsets": [ [ 0, 45661 ] ] } ]

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[ { "id": "pmcA1636465__text", "type": "Article", "text": [ "Expression of C-terminal deleted p53 isoforms in neuroblastoma\nAbstract\nThe tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and functional analysis of p53 expressed in eight cell lines: three wild-type cell lines and five cell lines harboring mutations. We identified two transcription-inactive p53 variants truncated in the C-terminus, one of which corresponded to the p53β isoform recently identified in normal tissue by Bourdon et al. [J. C. Bourdon, K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D. P. Xirodimas, M. K. Saville and D. P. Lane (2005) Genes Dev., 19, 2122–2137]. Our results show, for the first time, that the p53β isoform is the only p53 species to be endogenously expressed in the human NB cell line SK-N-AS, suggesting that the C-terminus truncated p53 isoforms may play an important role in NB tumor development.\n\nINTRODUCTION\nThe p53 tumor suppressor gene remains the most frequently altered gene in human tumors. Several p53 mutation databases have been reported previously (1–3), and to date, more than 1500 different p53 mutants have been described (4). Functional inactivation of p53 is usually due to gene mutation, deletion or protein degradation. In general, the majority of p53 mutations in human neoplasia are missense mutations affecting the DNA-binding domain (DBD). Unlike other human cancers, p53 in neuroblastoma (NB) is rarely mutated in the primary tumor at diagnosis but high levels of wild-type p53 (wt p53) protein expression have been found in the cytoplasm of undifferentiated tumors (5,6). More recently, in normal unstressed cells, wt p53 protein was found to be retained in the cytoplasm as a latent form, in huge, p53-associated protein complexes known as ‘Parc’ (7). The steady-state concentration of p53 in normal unstressed cells is usually very low because of the short half-life of the wild-type (wt) protein. Overexpression of p53 in most of the transformed cells containing a missense mutation within the p53 gene appears to be due to the increased stability of mutated p53 (8). In unstressed NB cells, high wt p53 expression may reflect the embryonic origin of NBs, in which precursor cells fail to mature (9).\np53 mutations are unusual in human NB but, when they do occur, are found in post-chemotherapy tumors. In this respect, Tweddle et al. (10) described how two NB cell lines derived from the same patient can elicit a different p53 status: wt p53 for SK-N-BE(1a) established before treatment, and mutated p53 for SK-N-BE(2c) established after relapse of the patient under treatment with cytotoxic agents such as cyclophosphamide, doxorubicin, vincristine and radiotherapy. In NB cell lines, p53 mutation has been found in multidrug-resistant cells (11). Various types of p53 mutation have been detected in NB cells and can lead to inactivation either by shut-down of protein expression or production of aberrant p53 products. Indeed, in LAN-1 cells, p53 nonsense mutation at cysteine 182 in exon 5 leads to the absence of protein (9), whereas in SK-N-BE(2) cells, missense mutation at codon 135 (C135F) leads to stable overexpressed protein (11). By analyzing IGR-N-91, a cell line established in our laboratory from the bone marrow of a patient with metastatic NB after unsuccessful Adriamycin–vincristine chemotherapy (12), we identified another type of aberrant protein that arises from the duplication of exons 7-8-9. This duplication spans from amino acids 225 to 331, which represent part of the DBD and part of the oligomerization domain (13). However, each p53 mutant has been described in the literature as a case report, and so far, no comparative study has been undertaken to link their biochemical features with functional properties.\nIn the present study we report two novel p53 C-terminus mutants identified in SK-N-AS and IGR-NB8 human NB cell lines. The biological properties of these two new variants were analyzed in comparison with p53 isolated from six other human NB lines: three [LAN-1, SK-N-BE(2) and IGR-N-91] expressing mutant p53 and three (SH-SY5Y, LAN-5 and IMR-32) expressing the wt protein. This characterization was done by using a range of functional assays: (i) the ability of the protein to bind with p53 consensus sequence using the functional analysis of separated allele in yeast (FASAY); (ii) the ability of the protein to transactivate the p53-responsive element (RE) identified either in the promoter of p21/WAF1 or in the first intron of BAX, using luciferase reporter assay; (iii) the induction of endogenous p21/WAF1 gene expression under stress conditions.\n\nMATERIALS AND METHODS\nNeuroblastoma cell lines, culture and drug treatments\nThe parental human NB SH-SY5Y, SK-N-AS, IMR-32 and SK-N-BE(2) cell lines were purchased from the European Collection of Cell Cultures (ECACC, Wiltshire, UK). The human IGR-NB8 cells (a gift of Prof. Gilles Vassal, UPRES EA 3535, Institut Gustave Roussy, Villejuif) were derived from a previously untreated localized NB (14). The LAN-1 and LAN-5 cell lines were provided by Dr Nicole Gross (Pediatric Oncology Research, Lausanne, Switzerland). The human IGR-N-91 cell line was established in our laboratory from the bone marrow of a patient with metastatic NB after unsuccessful adriamycin–vincristin chemotherapy (12). LAN-1, LAN-5 and IMR-32 were grown in RPMI medium supplemented with 2 mM l-glutamine and 10% fetal calf serum and gentamicine 10 μg/ml. Others cell lines were cultured in DMEM.\nFor activation of endogenous p53, cells were treated with cis-platinum (Sigma) (10 μg/ml) for 24 h then lysed for western blot analysis.\n\nWestern blot analysis\nThis procedure was carried out as described previously (13). Protein lysates (50 μg) were submitted to 10% SDS–PAGE, and then transferred onto nitrocellulose filters. After saturation, the membranes were incubated with primary antibody diluted in 0.1% phosphate-buffered saline, Tween-20 and 3% skim milk. The primary antibodies used were anti-p53 monoclonal antibody (clone DO-7, 1/1000, DAKO), anti-p21/WAF1 monoclonal antibody (Ab-1, 1/200, Oncogene Research) and anti-β-actin monoclonal antibody (1/1000; Chemicon) as internal control. Protein bands were detected by ECL system (Amersham).\n\nPCR, plasmids cloning\nGenomic DNA was extracted using lysis buffer containing 20 mM Tris–HCl, pH 7.5; 0.4 M NaCl; 0.5% SDS; 10 mM EDTA, treated with proteinase K (200 μg/μl), purified with phenol/chloroform, precipitated with ethanol and dissolved in DNase free water. Total RNA were purified using RNAble reagent (Eurobio), precipitated with isopropanol and dissolved in RNase free water. cDNA was obtained by reverse transcription of 1 μg of total RNA using Superscript II™ RNase H-Reverse transcriptase (Invitrogen) and Oligo-d(T)16 in conditions specified by the manufacturer. Amplification of full-length p53 coding region from SH-SY5Y, IGR-N-91, IGR-NB8 and SK-N-BE(2) cell lines was performed using forward primer at position 152 and reverse primer at position 1583 (F1 and R7, respectively, Table 1; GenBank accession no. K03199). p53 cDNA from SK-N-AS cells for cloning was obtained from RT–PCR using F1 and reverse primer i9+: 5′-GCAAAGTCATAGAACCATTTTCAT-3′ (nucleotide position 14989, GenBank accession no. X54156) primers which encompass from exon 1 to exon i9+ first identified by Flaman et al. (15) included. The PCR was done in the presence of pfu Hotstart DNA polymerase (Stratagene) for 30 cycles of 1 min at 90°C, 1 min at 65°C and 2 min 30 s at 72°C using PTC-100 thermocycler (MJ-Research).\nThe p53 cDNA from SH-SY5Y, SK-N-AS, IGR-N-91, IGR-NB8 and SK-N-AS cells were then cloned into pcDNA3.1/V5-His-Topo vector (Invitrogen) according to the manufacturer's instruction. The p53 sequence of each cell line was investigated by sequencing of plasmids after cloning. Sequencing was performed by Genome Express (Meylan, France).\nThe pDDm-TO harboring p53 dominant negative form (p53DD) pGL3-E1bTATA and the pE1B-hWAF1 firefly luciferase reporter containing the p53-responsive element of the p21/WAF1 promoter were described previously (16,17) pE1B-BAXi contains the p53RE identified in the intron 1 of the BAX gene [(18) and D. Munsch, personal communication]. Oligonucleotides TCGAGGGCAGGCCCGGGCTTGTCG and CTAGCGACAAGCCCGGGCCTGCCC were annealed and cloned into pGL3-E1bTATA digested with NheI and XhoI to obtain pE1B-BAXi. The pcDNA3-ΔNp73α expression plasmid was a gift of Dr Daniel Caput (SANOFI, Labèges, France).\n\nFluorescent in situ hybridization (FISH)\nCytogenetic preparations\nMetaphase spreads from healthy human male lymphocytes and tumor cell lines were prepared as described previously (19). BAC probe RP11-199F11, containing a 167 kb region spanning TP53 gene, was labeled by random priming in the presence of Alexa 594-dUTP (Molecular Probes). A commercial probe specific for chromosome 17 centromere, and labeled with spectrum green, was obtained from Vysis. After over-night cohybridization of the probes in the presence of Cot-1 DNA, the slides were washed and DNA counterstained with DAPI. The preparations were observed with an epifluorescence microscope and images captured with a Vysis imaging station. Between 3 and 14 metaphases spreads and 30–200 nuclei were examined for each cell line.\n\n\nLuciferase reporter assays\nLAN-1 or SH-SY5Y cells were seeded in duplicates onto 6-well plates at a density of 2 × 104 cells per cm2 and cotransfected 24 h later with 0.5 μg (2.5 μg/ml) of pGL3 firefly luciferase reporter gene plasmid under the control of either pE1B-hWAF1 or pE1B-BAX using lipofectamine 2000 and 1 μg of either a p53 expressing plasmid or an empty vector. At 24 h after transfection, cells were lysed with 200 μl/well of passive lysis buffer provided with the ‘Luciferase assay kit’ (Promega). Luciferase activity was measured using Microlumat LB96P luminometer (EG & G Berthold Instrument).\n\nFunctional assay in yeast\ncDNA was obtained by RT of 1 μg of total RNA using Superscript II™ RNase H-Reverse transcriptase (Invitrogen) and random hexamers to prime the synthesis in conditions specified by the manufacturer. p53 cDNA was amplified by PCR and cotransformed into yeast, IG397 Ade2 strain, together with either pRDI-22 vector for p53-standard assay or pFW35 and pFW34 plasmid for 5′ or 3′ split assay, respectively, carrying the ADE2 open reading frame under the control of a p53-responsive promoter (20). In a selective medium lacking leucine, wt-p53 activates transcription of ADE2 gene that encodes enzyme—phosphoribosylimidazole carboxylase—implicated in adenine biosynthesis. Therefore, a colony of cells that expresses ADE2 gene is white whereas the one composed of cells where ADE2 gene is not expressed owing p53 mutation is red.\n\n\nRESULTS\nP53 status in SK-N-AS and IGR-NB8 cells\nWe first compared the migration profiles of p53 expressed in SK-N-AS and IGR-NB8 with those expressed in the other six NB cells, SH-SY5Y, LAN-5, LAN-1, IMR-32, SK-N-BE(2) and IGR-N-91. Western blots from 50 μg of total protein extracts were revealed with p53 monoclonal antibody (DO-7). A range of profiles was identified as shown in Figure 1A. As expected, p53 extracted from the three cell lines, SH-SY5Y, LAN-5 and IMR-32, expressing wt protein (13,21) migrated at the wt position. Of particular note in these three wt p53 cell lines was an additional faint band that migrated faster than the full-length protein. The LAN-1 cells were found to be p53 deficient (9). The SK-N-BE(2) cell line showed an intense band reflecting p53 stability due to a missense mutation at codon 135 (11). As expected, due to the previously identified duplication of exons 7-8-9 (13), p53 protein migration was delayed in the IGR-N-91 cells. In contrast, the p53 protein in the SK-N-AS cell line migrated noticeably faster than the wt protein, indicating that it was smaller in size. The p53 protein in the IGR-NB8 cell line was even smaller than that in SK-N-AS.\nTo analyze the coding region of each of the p53 variants, RT–PCR was performed using p53-specific F1-R7 primers (Table 1 and Figure 1B). The expected 1430 bp for full-length p53 was amplified from wt p53-expressing SH-SY5Y, LAN-5 and IMR-32 cell lines. As SK-N-BE(2) harbors a single point mutation at codon 135 (C135F), the amplified fragment analyzed by electrophoresis migrated as wt p53 (Figure 1B). The longer RT–PCR fragment from the mutated IGR-N-91 cell line resulted from the duplication of exons 7-8-9, as shown in our previous data (13), which corresponds to extra nucleic material of 321 nt. For the LAN-1 cells, no amplified fragment was observed in accordance with published data, which demonstrates the extremely low levels of p53 mRNA and the undetectable level of protein (9). An amplified fragment of the same length as the wt protein was observed in the IGR-NB8 cell line (Figure 1B). Indeed, complete gene sequencing revealed a point mutation E326STOP leading to a truncated protein at C-terminus. No fragment, however, was amplified from SK-N-AS with F1-R7 primers.\nTo further map the p53 mRNA transcribed in these cells, series of RT–PCR tests were performed using the forward primer, F2 (exon 8 position 1008th according to GenBank accession no. K03199), matched with different reverse primers, R3 (at the junction of exon 8/9, nt position 1124), R4, R5 (in exon 9 at positions 1154 and 1184, respectively), and R6 (in exon 10, at position 1230). The sequences of these primers are given in Table 1 and the results are presented in Figure 2A. SK-N-AS cDNA gave an amplified fragment of the same size as SH-SY5Y cDNA with the three primer pairs, F2/R3, F2/R4 and F3/R5. However, in contrast to SH-SY5Y, no fragment was obtained with SK-N-AS cDNA using the F2/R6 primer pair, which suggests the absence of exon 10 in SK-N-AS mRNA.\n\nThe p53 protein expressed in SK-N-AS is the p53β isoform\nAn alternatively spliced form of human p53 mRNA with an additional 133 bp exon derived from intron 9 has been detected in normal human lymphocytes (15). This spliced variant named ‘i9+’ encodes a truncated protein of 341 amino acids including 10 new amino acids derived from the novel exon, the p53β isoform according to Bourdon et al. nomenclature (22). This led us to hypothesize that the shorter protein expressed by the SK-N-AS cell line could be the p53β isoform. To test this hypothesis, RT–PCRs were performed using primer sets designed to amplify the 3′ region of p53 mRNA encoding either the specific C-terminal part of the wt protein (wt C-ter) or the specific C-terminal part of the β isoform (β C-ter). In parallel amplification with a primer pair amplifying the DBD was used as control. The sequences of these oligonucleotides are given in Table 1. The results presented in Figure 2B are consistent with the only expression of the p53β isoform in SK-N-AS as no band was observed in lane using specific C-ter primer located in exon 10. Interestingly, RT–PCR using SH-SY5Y (SH) gave an amplified fragment not only with the primer pair specific to the C-ter domain of wt p53 but also with the primer pair specific to the p53β isoform. This result, combined with the presence of an additional faint band migrating faster than wt p53 in denaturing polyacrylamide gel (Figure 1A), strongly suggests that both the p53 full-length protein and the β isoform were expressed in the SH-SY5Y cells.\nThe full-length SK-N-AS p53 cDNA was then amplified with the forward primer F1 and a reverse primer located within the novel exon i9+ (Table 1). This amplified fragment was cloned in pcDNA3/V5-His-Topo, as described in Materials and Methods. Its sequence analysis confirmed that the truncated p53 expressed in SK-N-AS was encoded by the i9+ splice variant described previously by Flaman et al. (15) that encodes the p53β isoform characterized by Bourdon et al. (22).\nA series of genomic amplifications were performed to identify a possible deletion within the intron 9 that could account for the absence of normal size p53 in SK-N-AS cells. The primer sequences are given in Table 1. Amplifications were performed in parallel with total DNA extracted from SH-SY5Y and SK-N-AS cells. Results are presented in Figure 3. Normal size fragments that encompass the acceptor site of intron 9 were amplified with SH-SY5Y as well as with SK-N-AS DNA. On the contrary amplification fragments that encompass the intron 9 donor site were obtained only with SH-SY5Y but not with SK-N-AS DNA. These results identify a deletion of the intron 9/exon 10 junction within the SK-N-AS p53 gene.\n\nA yeast functional assay confirmed the absence of p53 full-length expression in SK-N-AS and IGR-NB8\nIt is possible to detect p53 mutation using a simple yeast colony color assay as described by Flaman et al. (23). When the strain is transformed with a plasmid-encoding wt p53, the cells express the ADE2 gene and produce white colonies (Figure 4A, a, b2 and c1, and Figure 4B, dish a). Cells containing mutant p53 fail to express ADE2 and form small red colonies (Figure 4A, b and b1, and Figure 4B, dish c). When the p53 cDNA fragment is deleted, cells are unable to form a colony (Figure 4A, c and c2). As shown in Table 2, FASAY was performed as a p53-standard test with full-length cDNA or with the split version at the 5′ and 3′ end (15). The background of FASAY experiments is around 10%. p53 wt expressing SH-SY5Y and LAN-5, 2 wt cell lines, yielded ∼92–97% of white colonies (Table 2).\nOne hundred percent of the colonies carrying SK-N-BE(2) p53, which is homozygous for the C135F mutation, turned red with the standard or 5′ split assay, whereas 94% of the colonies turned white with the 3′ split assay since the missense mutation does not extend to the C′-terminus of the gene (Table 2 and Figure 4B, dish c). No colonies were observed with p53-deficient LAN-1 cells, (see also Figure 4A, c and c2). With SK-N-AS cells, the split 5′ assay gave 96% white colonies, while the p53-standard and split 3′ assay did not produce any colonies (Table 2). This means that the 5′ terminus was intact whereas the 3′ terminus had been deleted, as was confirmed by nucleotide sequence analysis. The IGR-NB8 colonies, however, were red both with the p53-standard assay and the split 3′ assay. In the IGR-N-91 cell line, where p53 harbors two contiguous sets of exons 7–9, spanning the DBD and oligomerization domain, it is interesting to note that the yeast colonies were predominantly white (Figure 4B, dish b) with the split 5′ and the split 3′ assay (96 and 87% of white colonies, respectively) This suggests that the cells express a binding ability that is specific to wild-type p53 rather than mutated p53 (Table 2).\n\nTranscription activity of SH-SY5Y, IGR-N-91, SK-N-BE(2), SK-N-AS and IGR-NB8 p53 variants in mammalian cells\nTo determine the transactivation ability of p53 variants in mammalian cells, we used a reporter gene strategy. The p53RE located within either the human p21/WAF1 promoter or the intron 1 of the mouse and human BAX gene [(18) and D. Munsch, personnal communication) were cloned in a luciferase reporter gene plasmid upstream of the E1B minimal promoter as described in Materials and Methods. The p53-negative LAN-1 cells were cotransfected with the p53 vectors expressing the p53 cloned from either SH-SY5Y, IGR-N-91, SK-N-BE(2), SK-N-AS or IGR-NB8 and the luciferase reporter plasmids. Both p53RE were strongly stimulated in cells cotransfected with wt p53 cloned from SH-SY5Y, as compared to cells cotransfected with an empty plasmid. In contrast, none of the p53 variants was able to transactivate the expression of luciferase driven by either p21/WAF1 or BAX p53RE (Figure 5).\nTo test transactivation capability at the protein level, each variant was transfected into p53-negative LAN-1 cells and the stimulation of endogenous p21/Waf1 gene expression was analyzed by western blotting. As shown in Figure 6, in contrast to wt p53, none of the p53 variants was able to induce p21 protein accumulation.\nWe then tested for a possible dominant negative effect of these various mutants on wt p53-dependent transcriptional activity. To this end, SH-SY5Y cells were cotransfected with constructs encoding the luciferase gene driven by either the p21/Waf1 or BAX p53RE and the constructs expressing the various p53s cloned from IGR-NB8, SK-N-BE(2), IGR-N-91, SK-N-AS and IGR-NB8 NB cells or p53DD, a dominant negative mutant of wt p53 (24). The stress induced by transfection activated the transcriptional activity of the wt p53 expressed in SH-SY5Y, leading to a p53-dependent expression of luciferase as illustrated by the fact that coexpression of p53DD led to a substantial decrease in luciferase activity when compared to the luciferase activity of cells cotransfected with an empty plasmid (Figure 5). Compared to p53DD, mutants within the DBD isolated from IGR-N-91 or SK-N-BE(2) had only a moderate dominant negative effect on endogenous wt p53 transcriptional activity. More surprisingly, the transfection of the C-terminal truncated variant IGR-NB8 enhanced both BAX and p21/WAF1 p53RE activity. The coexpression of p53β cloned from SK-N-AS also enhanced BAX p53RE activity. A similar effect has been reported already for p53β by Bourdon et al. (22).\nWhen combined, these results show that all the p53 variants isolated from the NB cells had lost the ability to specifically transactivate the p53 target genes. Their effect on the transcriptional activity of endogenous wt p53 expressed in SH-SY5Y cells, however, largely depended on the p53 domain affected by the modification.\n\nAll the identified p53 variants inhibited the induction of endogenous p21/WAF1 gene expression under stress conditions\nWe further examined whether the four p53 variants, SK-N-BE(2), IGR-N-91, SK-N-AS and IGR-NB8, had loss their ability to stimulate the endogenous expression of the p21/WAF1 gene, the archetypical cell cycle inhibitor and the true target of p53. To this end, cellular response to genotoxic stress was analyzed by western blot following treatment of the various cell lines with cisplatin, one of the most potent antitumor agents used in neuroblastoma. Results are presented in Figure 7. None of the mutant cells, regardless of the type of mutation, was able to induce p21/WAF1 protein accumulation, unlike the 3 p53 wild-type cells (SH-SY5Y, IMR-32 and LAN-5).\n\nGenomic status of TP53 region in the various cell lines\nAccording to Knudson's ‘two hit’ model of tumor suppressor gene functional inactivation, the mutation of one allele is supposed to be associated with a deletion of the second allele. To assess this genetic mechanism, we performed FISH experiments to search for deletions of one copy of the TP53 region, especially in cell lines with a mutated TP53 gene. For this purpose, metaphase preparations of the studied cell lines were cohybridized with a p53 DNA probe labeled in red (BAC clone RP11-199F11) as described previously (25) and a chromosome 17-specific centromeric probe labeled in green. The three cell lines shown previously to express a wt p53 protein (LAN5, IMR32 and SH-SY5Y), displayed as expected two signals with each probe, confirming the presence of both TP53 alleles in these cells (Figure 8 and Table 3). Conversely, IGR-N-91 and SK-N-AS cell lines displayed only one fluorescent signal for each probe, suggesting a whole chromosome 17 lost, or at least losses of the 17p arm and the centromeric region. The SK-N-BE(2) cell line has been described as containing only one chromosome 17 and one TP53 signal (10). In our analysis, only 10% of the cells displayed this characteristic, whilst most of the cells had two copies of both (Figure 8 and Table 3). p53 sequencing, however, confirmed the previously described mutation, and the absence of a normal allele, suggesting that the cells used in our study had acquired, during culture, an uniparental disomy for the TP53-mutated chromosome 17. Finally, the other two cell lines (LAN-1 and IGR-NB8) displayed highly variable genetic heterogeneity from one cell to the next (Table 3). Surprisingly, although p53 transcripts are extremely faintly expressed in LAN-1 cell line, all cells showed several FISH signals with the 167 kb BAC probe used here. To understand this apparent contradiction, an array-CGH experiment was performed on an oligo-array Agilent, which indicated a 133 kb interstitial deletion corresponding to the p53 coding region and the 97 kb upstream region. Accordingly, the fluorescent spots observed in FISH experiments on LAN-1 cells should be related to the hybridization of the 57 kb region downstream of p53 gene present in the BAC probe. IGRN-B8 cell line displayed a number of signals of both colors ranging from 0 to 4, with 87% of cells displaying a loss for one TP53 allele. Despite this genomic variability, analysis of the p53 protein showed a single shortened form in IGR-N-B8 cell line (Figure 1). Consequently, and as suggested for SK-N-BE(2) cell line, IGR-N-B8 cell line should contain a variable number of copies of chromosome 17 with mutated p53.\n\n\nDISCUSSION\nThe p53 gene, the ‘genome guardian’, is mutated in over 50% of human cancers, with the most common mutations being missense mutations (>2/3 of mutations) (26). In human neuroblastoma tumors, p53 mutations are rarely present at the time of diagnosis (5,27); however, oncogenic p53 mutations can be found in advanced neuroblastomas that often relapse following high-dose chemotherapy (10). In contrast, in breast cancers, it has been reported that p53 mutations might improve response to high-dose chemotherapy including therapy with epirubicin and cyclophosphamide (28).\nAn investigation into the p53 genomic status and functions of eight human NB lines revealed that all five of the mutated cell lines had distinct genetic characteristics as is schematically represented in Figure 9: SK-N-BE(2) with a single missense mutation in the p53 gene, encoding a highly stable full-length protein. SK-N-AS and IGR-NB8 proteins, although they have intact transactivation and DBDs, were truncated at the C-terminus generating 341 and 326 amino acids respectively; they therefore lack the tetramerization domain that is essential for an active conformation. Very recently, Bourdon et al. (22) reported the putative occurrence of β and γ isoforms from different tissues due to alternate splicing that indicates the similarity to those of p73 and p63 as identified previously by Daniel Caput and co-workers (29). In the p53 isoforms scheme proposed by Bourdon et al. (22), the SK-N-AS cell line that elicits p53i9 protein expression is consistent with the p53β isoform. Genomic analysis reveals that the only occurrence of the p53β isoform in SK-N-AS results from a deletion spanning the intron 9/exon 10 junction. Similar to the p53β isoform in SK-N-AS, the p53 in IGR-NB8 that lacks 67 amino acids at C-terminus was, alone, unable to induce p21/WAF1 promoter activation except with endogenous wt-p53 on SH-SY5Y cells where transfection with IGR-NB8 significantly augmented the transcriptional activation of the p21/Waf-1 promoter (Figure 5A). Studies by other authors have reported the interaction between the C-terminal domain and another region that impedes the active conformation of p53, suggesting an allosteric model for p53 activity regulation (30). Such events have been demonstrated for the 342-stop mutant, generated by mutagenesis, which can modulate transactivation, growth and apoptosis (31). Moreover, Harms and Chen (32) reported that the C-terminal basic domain inhibits induction of the proapoptotic target gene insulin-like growth factor binding protein 3, suggesting that IGR-NB8 might induce this gene. IGR-N-91 had an abnormally high molecular weight protein due to the duplication of wild-type exons 7-8-9, thus affecting the DBD and OD; and LAN-1, with a mutation at codon 182 (Cys→stop) concurred with an earlier report showing extremely low levels of mRNA and undetectable protein expression (9).\nNotably, all the p53 variants, including SK-N-AS (β isoform) and IGR-NB8 (C-terminal truncated p53), elicited a total lack of p21 promoter activation. In particular, the p53β isoform was unable to induce endogenous p21 expression in SK-N-AS (Figure 6), concurring with data obtained from in vitro transfection experiments in H1299 cells by Bourdon et al. (22). For the IGR-N-91 cells, although p53 was mutated and unable to transactivate the p21/WAF1 promoter, the FASAY global test was not conclusive since ∼80% of colonies were white and nearly 20% (see also Table 2), though not enough, were red. Moreover, in this particular line, standard sequencing on cDNA using primers located within each exon as used for routine tumor analysis was unable to detect any anomalies in p53 genetic status (data not shown). These results enlighten the limit of the conventional tests to detect a transcription inactivation of p53 brought by duplication within the DBD.\nAnalysis of p53 genomic status was explored by FISH experiments, in search for a potential biallelic inactivation of p53, with a mutation of one allele and a deletion of the second one. This situation was indeed clearly observed in IGR-N-91 and SK-N-AS cell lines, with an unambiguous loss of one chromosome 17p arm in all cells of both. SK-N-BE(2), LAN-1 and IGR-NB-8 cell lines showed a more complex genomic situation which should be relevant of variable copy numbers of chromosome 17 bearing in most cases (LAN-1) or in all cases [SKN-BE(2), IGR-NB8] the mutated characteristic p53 allele. Our data therefore clearly demonstrate that each technique has a role and a combination of techniques is required in order to correctly define the p53 phenotype and genotype in tumor and particularly in NB cells.\nOur data enlighten a high frequency of the C-terminal abnormalities (3/5 mutated) in NB cell lines. For SK-N-AS and IGR-NB8, a part of the oligomerization domain was lost and IGR-N-91 gained an extra oligomerization domain. According to FASAY assay the p53 expressed in IGR-N-91 still specifically bind DNA but not the p53 expressed in SK-N-AS and IGR-NB8 in agreement with previous published data obtained by electrophoretic mobility shift assay (33).\nWith regards to biological relevance, different mutants within the DBD vary in their oncogenicity. They are classified into two types depending of the location of the mutation, mutations of class I occur in the DNA contact areas, while class II mutations occur in areas important for the conformational stability of p53 protein (30). Although both class I and class II mutants have loss its ability to specifically bind DNA, class II mutations have been shown to be more oncogenic than class I. However, to our knowledge the oncogenicity of mutant affecting the C-terminal domain have not been studied. The biological role of the C-terminal mutants needs now to be thoroughly investigated in NB tumors.\n\n\n" ], "offsets": [ [ 0, 31245 ] ] } ]

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[ { "id": "pmcA1521150__text", "type": "Article", "text": [ "Peculiarities of carcinogenesis under simultaneous oral administration of benzo(a)pyrene and o-cresol in mice.\nAbstract\nA modifying influence of ortho-cresol (o-cresol) on the carcinogenic effect of benzo(a)pyrene (BaP) with combined oral administration to CC57Br mice had been found. During simultaneous administration of o-cresol (1 mg) and BaP (1 mg), the incidence of tumors, the multiplicity of tumors, and the degree of malignancy all increased, but the latency was shortened. When o-cresol was administered before or after BaP (in identical doses), the carcinogenic effect was weakened. When o-cresol (10 mg) and BaP (5 mg) were administered simultaneously, the incidence of malignant tumors was similar to controls receiving BaP only (13.8%), indicating inhibition of carcinogenesis.\n\n\n\n\n Environnmental Health Perspectives Supplements Vol. 101 (Suppl. 3): 341-344 (1993) \n\n Peculiarities of Carcinogenesis under Simultaneous Oral Administration of \n\n Benzo(a)Pyrene and o-Cresol in Mice \n\n by N. Ya. Yanysheva,' N. V. Balenko, 1. A. Chernichenko,1 and V. F. Babiy' \n\n A modifying influence of ortho-cresol (o-cresol) on the carcinogenic effect of benzo(a)pyrene (BaP) with combined oral administration to CC57Br mice had been found. During simultaneous administration of o-cresol (1 mg) and BaP (1 mg), the incidence of tumors, the multiplicity of tumors, and the degree of malignancy all increased, but the latency was shortened. When o-cresol was administered before or after BaP (in identical doses), the carcinogenic effect was weakened. When o-cresol (10 mg) and BaP (5 mg) were administered simultaneously, the incidence of malignant tumors was similar to controls receiving BaP only (13.8%), indicating inhibition of carcinogenesis. \n\n Introduction \n\n Numerous experiments have shown a modifying influence of different chemicals on benzo(a)pyrene (BaP)induced carcinogenic effects. Both enhancing or weakening effects have been seen (1-12). In some experiments ubiquitious environmental carcinogenic and toxic chemical pollutants were used (13-17). \n\n The stimulating effect of phenol, nitrogen oxides, and sulfur dioxide upon BaP's blastogenic action on the respiratory tract and phenol on the digestive tract (forestomach) have been reported (12-18). We have established the relationship between the doses of carcinogenic and toxic agents and their modifying effects (17,18). The maximal enhancing effect was observed after BaP (2.5 mg) and NO2 (0.87 mg/m3) were exposed to rat respiratory tract. The effect weakened with decreasing dose. At concentrations at the MPC level (MPC BaP, 1 ng/m3, MPC NO2, 0.04 mg/m3) no effect was seen. Phenol at 1 mg orally to CC57Br mice enhanced the BaP (1 mg) effect, but no effect was seen at 0.02 mg. Phenolic compounds showed either enhancing or inhibiting effects on carcinogenesis depending on their chemical structure (16,18-25). \n\n This report presents the results of combined oral administration of BaP and artho-cresol (o-cresol) in mice, \n\n 'Ukrainian State Medical Center for Environmental Health, Kiev, Ukraine. \n\n Address reprint requests to N. Y. Yanysheva, Ukrainian State Medical Center for Environmental Health, Popudrenko St. 50, 253660 Kiev-94, Ukraine. \n\n chemicals commonly found in ambient water because of their industrial use (coke chemistry, oil processing, shale processing, and other industries) (27-30). \n\n Materials and Methods \n\n The experiment was conducted on CC57Br female mice weighing 12-14 g. Animals were divided in 15 groups. This experiment included three types of sequential combinations for the introduction of compounds: a) simultaneous BaP and o-cresol administration; b) BaP after o-cresol (stage 1); and c) BaP before o-cresol (stage 2). Appropriate controls were included (Table 1). \n\n The chemicals were administered twice per week (for a total of 10, 20, and 40 doses) using a syringe and a needle with a soldered olive on its tip. BaP (1 or 5 mg) or o-cresol (0.02, 1, or 10 mg) was placed in triethylene glycol (TEG) and administered as 0.1-mL water solutions. The evaluation of o-cresol's modifying effect on the incidence of tumors, especially malignant tumors, the tumor latency period (tl), and the average time (ta) of the appearance of tumors, as well as the multiplicity index, M (the average number of tumors per animal for tumor-bearing animals) was recorded. Because the M could be identified at the initial stage of carcinogenesis by the third month after the beginning of the trial when neoplasms began to emerge, moribund animals were killed by ether inhalation if they did not die spontaneously. \n\n The experiments with large chemical doses (BaP, 5 mg; o-cresol, 10 mg) lasted for 30 weeks. Some animals were killed after the 1st, 3rd, 5th, and 10th procedures for the \n\n YANYSHEVA ET AL. \n\n evaluation of initial stages of carcinogenesis; others were killed at 26 and 30 weeks. The stomachs were distended with formalin solution and the mucosa examined macroscopically. All tumors > 1 mm in diameter were recorded. Organs and tissues were fised in 10% neutral formalin solution, embedded in paraffin and routine histological slides prepared. Microscopic data were processed according to Mostkovoy (30). \n\n Results \n\n The data from this study demonstrate the modifying influence of o-cresol on BaP-induced carcinogenesis with combined oral exposure. The combined exposure and BaP alone caused benign and malignant epithelial tumors of the forestomach. The benign neoplasms were papillomas, and the malignant neoplasms were invasive carcinomas. \n\n The combined administration of BaP and o-cresol showed different results depending on doses and sequential combinations of both chemicals. As shown in Table 2, the simultaneous administration of BaP (1 mg) and o-cresol (1 mg) affected all parameters of carcinogenesis that were measured. This included a significant increase in the incidence of tumors, shortening of the time to the appearance of the first tumor, and the mean time of tumor \n\n development as compared to animals that received 1 mg of BaP alone and had tumors. One hundred percent of the mice that received 5 mg of BaP alone had tumors. \n\n Shortening of the time to appearance of malignant tumors was also observed: the mice with forestomach cancer died between 23 and 26.2 weeks after the beginning of experiment, but the controls survived to 58.5 weeks. Muliple small metastases in the lungs and mediastinum were found in 42.8% of mice, which signifies a high degree of malignancy. \n\n o-Cresol at 0.02 mg did not modify carcinogenesis in comparison to the control. o-Cresol administration before or after BaP did not modify tumor incidence or the multiplicity index as compared to control, but the latency period was longer (Table 2). When o-cresol was administered after BaP (stage 2), there was an absence of malignant tumors. \n\n Besides the quantitative aspects, other peculiarities of carcinogenesis in mice simultaneously administered BaP and o-cresol should be noted. The combined chemical administration resulted in diffuse verrucosa vegetations over the forestomach surface, especially near the greater curvature. Highly aggressive malignant neoplasms developed earlier with more metastases as compared to control. \n\n Many mice were emaciated, and the tumors could be \n\n Table 1. Scheme of BaP and o-cresol combined action under different regimes of oral administration. \n\n Theatmenta \n\n No. of mice Stage 1 Stage 2 \n\n Groups of in each No. of No. of \n\n animals group Substances Dose, mg applications Substances Dose, mg applications \n\n 1 55 BaP, o-cresol 5 +10 10 - - 2 45 BaP 5 10 - - 3 40 TEG la 10 - - 4 45 TEG, o-cresol 1b + 10 10 - - 5 30 BaP, o-cresol 1+1 20 - - 6 30 BaP, o-cresol 1 + 0.02 20 - - 7 30 o-Cresol 1 20 BaP 1 20 8 30 BaP 1 20 o-Cresol 1 20 9 30 BaP 1 20 - 10 30 - - - BaP 1 20 11 30 o-Cresol 1 20 - 12 30 - - - o-Cresol 1 20 13 30 o-Cresol + TEG 1+2' 20 - 14 30 o-Cresol + TEG 0.02 +2b 20 \n\n 15 30 TEG 2b 20 - - Abbreviations: BaP, benzo[a]pyrene; TEG, triethylene glycol. \n\n Table 2. Occurrence of forestomach tumors in CC57Br mice after combined oral administration of BaP and o-cresol. \n\n Number of animals with forestomach tumors \n\n Order of administration of substances Total Benign Malignant \n\n (dose, mg) Absolute % % M t, ta % t, ta BaP (5), o-cresol (10) 29 100.0 86.2 ND ND ND 13.8 ND ND BaP (1), o-cresol (1) 19 95.0 60.0 9.6 10.7 16.3 35.0 23.0 25.2 BaP (1), o-cresol (0.02) 7 35.0 30.0 1.6 13.5 19.8 5.0 56.8 56.8 BaP (1), stage 1, o-cresol (1), stage 2 7 31.8 31.8 1.4 16.2 31.1 0 0 0 \n\n BaP (1), stage 2 6 35.3 29.4 2.8 15.8 41.2 5.9 24.8 24.8 BaP (5), stage 1 18 100.0 50.0 ND ND ND 50.0 ND ND BaP (1), stage 1 8 33.3 28.7 1.4 14.0 21.3 4.6 58.5 58.5 BaP (1), stage 2 7 36.8 31.6 2.8 10.0 13.3 5.2 55.7 55.7 \n\n Abbreviations: BaP, benzo[a]pyrene; 0, not observed; ND, not determined; t1, time of the first tumor appearance in weeks; ta, mean time of tumor development in weeks; M, multiplicity. \n\n 342 \n\n CARCINOGENESIS OF BaP AND o-CRESOL 343 \n\n palpated through the abdominal wall. At autopsy, enlargement of the stomach with tuberculous white superficial vegetations were observed. The forestomach and glandular part of the stomach was often obliterated by tumor masses. \n\n The stomach was often adhered to the pancreas, liver, and mesentery. Hemmorrhage and inflammation were found in tumors foci. When o-cresol was introduced before BaP, the tumors were more frequently found closer to the small curvature of the stomach between the forestomach and esophageal entrance. Over the large curvature less prominent mucosal folds were observed. Microscopically, a decrease in mucosa papillae, epithelial atrophy (one to two cullular layers), decreased keratonization, and nuclear pycnosis were observed. With the simultaneous administration of large doses of o-cresol and BaP, the final carcinogenic effect (30 weeks after the first dose) was similar to BaP alone but differences were observed at the earlier stages (after the 1st, 3rd, 5th, and 10th exposures). \n\n In the BaP control, multiple forestomach epithelial proliferative and hyperplastic changes were found after the third dose. Multiple papillomas occurred (9-15 in each mouse). The stomach's mucosal folds appeared thickened diffusely, then papillomas appeared and finally merged together. \n\n Approximately half of the tumors were malignant. The neoplasms filled almost the whole forestomach cavity and infiltrated the wall with tuberculous vegetations, which were visible on the serosal surface. Thmor infiltration in the liver, pancreas, and wide dissemination of peritoneum were also observed. In mice simultaneously administered BaP and o-cresol, the proliferative alterations of forestomach epithelium were seen after the third dose. However, they occurred as the single foci at damaged mucosa and even in the later stages were not diffuse. Adjacent to the hyperplastic foci, the mucosa was atrophied with decreased keratonization. The epithelial cells also showed cytoplasm coagulation and nuclear pycnosis. Edema, inflammation of the mucosa, submucosa microabscesses, and erosion were seen. There were fewer papillomas per mouse (four to eight in each mouse) than in the control group. Even in the late stages the papillomas were isolated and elevated on the atrophied mucosal folds. \n\n In the final experiment the papillomas prevailed as compared to the previous experiments. Only 4 out of 29 mice (13.8%) developed malignant tumors. Neoplasms were smaller and appeared as single verrucosa vegetations 5-10 mm in diameter. Thus, the toxic dose of o-cresol inhibits the carcinogenic process of induced forestomach tumors by decreasing multiplicity, frequency, and percentage of malignant tumors. \n\n Discussion \n\n Our results and the literature suggest a hypothesis on modifying carcinogenesis mechanisms. The primary effect of the toxic agents, including carcinogenic agents, may relate to cellular membrane damage with the subsequent increased permeability (31-35), which may be the mechanism of the o-cresol. \n\n With simultaneous introduction of o-cresol at low toxic doses and BaP there may be increased carcinogen penetration to the target cells. In addition, membrane damage may alter other cellular systems responsible for energy and xenobiotic detoxication. Damage of these processes may activate free-radical reactions, glycolysis, or alter carcinogenic metabolism, which promotes the oncogenic effect. The o-cresol effect on these systems was confirmed by our previous investigations on cytochrome P450, ferrosulfuric nonhemic proteins, and semiquinon radical content affected by the combined action of BaP and o-cresol (36). \n\n Another effect was obtained with BaP exposure after o-cresol. The atrophic alterations in the stomach induced by 2.5 months of o-cresol administration might decrease the natural conditions of retention (muscosal folds, frontier torn), and thus shorten the contact of BaP with the forestomach. In our opinion, this explains the decreased carcinogenic effect seen after o-cresol exposure. \n\n Considering the toxic effect of o-cresol at low toxic doses (1 mg) after BaP and the effect of a super toxic dose (10 mg) simultaneously with BaP administration, it is possible that the inhibition of carcinogenesis in both trials was related to the toxic effects of o-cresol. Cytotoxicity may not only hamper tumor growth, but also promote the regression of inducible and spontaneous neoplasms (37-39). There is also a possibility that BaP damages metabolic systems and decreases resistance to carcinogenesis. \n\n Conclusions \n\n The data obtained demonstrate that simultaneous administration of BaP modifies the induced carcinogenesis depending on the dose and the sequence of administration. Administered with BaP (1 mg), a low toxic o-cresol dose (about minimally effective) produces an enhanced cocarcinogenic effect reflected in the incidence, frequency, multiplicity, and degree of malignancy of forestomach tumors. \n\n o-Cresol administration at low toxic doses before or after BaP at the same dose level (1 mg) and its administration at super toxic doses (10 mg) with the BaP optimal dose (5 mg) may inhibit carcinogenesis. Simultaneous introduction of a noneffective o-cresol dose (0.02 mg) with the BaP (1 mg) does not change its carcinogenic activity. Controlling both chemicals in the environment is the most effective measure of preventing potential risk and is undoubtedly of paramount significance. \n\n REFERENCES \n\n 1. Bingham, E., and Falk, H. L. Environmental carcinogenesis. The \n\n modifying effect of cocarcinogens on the threshold response. Arch. Environ. Health 19: 779-783 (1969). \n\n 2. Goldschmidt, B. M., Katz, C., and Van Duuren, B. L. The cocar\n\n cinogenic activity of non-carcinogenic aromatic hydrocarbons. Proc. Am. Assoc. Cancer Res. 17: 84 (1973). \n\n 3. Kaufman, D. C., and Madison, R. M. Synergistic effects of \n\n benzo(a)pyrene and N-methyl-N-nitrosourea respirators carcinogenesis in Syrian golden hamsters. In: Proceedings of the Sym\n\n 344 YANYSHEVA ET AL. \n\n posium on Epithelial Respiratory Carcinogenesis and Bioassays. Batelle Seattle Research Center, Seattle, WA, 1974, p. 17. \n\n 4. Montesano, R., Saffiotti, U, Ferrero, A, and Kaufman, D. 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Ilnitsky, Eds.), Budapest, 1979, pp. 111-113. \n\n 28. Djatlovitskaya, F. G., and Maktas, E. D. Separate determination of fly \n\n phenols in water using thin-layer chromatography [in Russian]. Gig. Sanit. 6: 60-63 (1965). \n\n 29. Kostovetsky, Ya. I., and Zholdakova, Z. I. About hygienic rate setting \n\n of phenol in the water of waterpools [in Russian]. Gig. Sanit. 7: 7-10 (1971). \n\n 30. Mostkovoy, M. I. Practicum on variational-statistical processing of \n\n clinical material [in Russian]. Ashkhabad, 1954, p. 132. \n\n 31. Weinstein, I. B. The scientific basis for carcinogen detection and \n\n primary cancer prevention. Cancer 47: 1133-1141 (1981). \n\n 32. Merkulov, A. I, and Skvortsova, R. I. About toxic effect of phenol [in \n\n Russian]. Gig. Sanit. 1: 79-80 (1984). \n\n 33. Weinstein, I. B., Mufson, R. A., Lee, L. S., et al. Membrane and other \n\n biochemical effects of the phorbol esters and their relevance to tumor promotion. In: Carcinogenesis. Fundamental Mechanisms and Environmental Effects. London, 1980, pp. 543-563. \n\n 34. Yamasaki, H., and Weinstein, I. B. Cellular and molecular mecha\n\n nisms of tumor promotion and their implications for risk assessment. In: Methods for Estimating Risk of Chemical Injury: Human and Non-human Biota and Ecosystems (V. B. Vouk, G. C. Butler, D. G. Hoel, and D. B. Peakall, Eds.), SCOPE, 1985, pp. 155-180. \n\n 35. Karu, T, Kirso, U. E., and Andrianov, L. A. Dynamics of resorption of \n\n 3,4-benzpyrene with phenols from mouse skin [in Russian]. Voprosy Onkol. 5: 80-84 (1973). \n\n 36. Yanysheva, N. Ya., Yurkovskaya, T. N., Beregovskaya, N. N., et al. \n\n Metalloenzymic complexes of energetic and detoxicating systems of cell during benz(a)pyrene-induced carcinogenesis. In: Proceedings of the 4th National Congress of Oncology with International Participation. Sofia, 1985, p. 102. \n\n 37. Mizell, M. Anuran (Lucke) tumor breakdown in regenerating anuran \n\n tadpole tails. Anat. Res. 137: 382-383 (1960). \n\n 38. Sheremetieva, E. N. Spontaneous melanoma in regenerating tails of \n\n axolotis. J. Exp. Zool. 158: 101-122 (1965). \n\n 39. Tsonis, P. A, and Eguchi, G. Carcinogens on regeneration. Effects on \n\n N-methyl-N-nitro-N-nitrosoguanidine and 4-nitrogninoline-1-oxide on limb regeneration in adult newts. Differentiation 20: 52-60 (1981). " ], "offsets": [ [ 0, 24527 ] ] } ]

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[ { "id": "pmcA2632666__text", "type": "Article", "text": [ "Protein subfamily assignment using the Conserved Domain Database\nAbstract\nBackground\nDomains, evolutionarily conserved units of proteins, are widely used to classify protein sequences and infer protein function. Often, two or more overlapping domain models match a region of a protein sequence. Therefore, procedures are required to choose appropriate domain annotations for the protein. Here, we propose a method for assigning NCBI-curated domains from the Curated Domain Database (CDD) that takes into account the organization of the domains into hierarchies of homologous domain models.\n\nFindings\nOur analysis of alignment scores from NCBI-curated domain assignments suggests that identifying the correct model among closely related models is more difficult than choosing between non-overlapping domain models. We find that simple heuristics based on sorting scores and domain-specific thresholds are effective at reducing classification error. In fact, in our test set, the heuristics result in almost 90% of current misclassifications due to missing domain subfamilies being replaced by more generic domain assignments, thereby eliminating a significant amount of error within the database.\n\nConclusion\nOur proposed domain subfamily assignment rule has been incorporated into the CD-Search software for assigning CDD domains to query protein sequences and has significantly improved pre-calculated domain annotations on protein sequences in NCBI's Entrez resource.\n\n\n\nBackground\nA major goal in the post-genomic world is to infer protein function from sequence information. One popular approach is to classify protein families or domains by grouping homologous sequences and annotating the groups with properties such as general function, intracellular location, three-dimensional structure, conserved sequence patterns or motifs, evolutionary origin, and binding and active sites. Novel proteins can be characterized quickly by assigning a group via profile search methods. However, more than one family or subfamily may exhibit similarity to overlapping sequence intervals and to a degree that seems convincing (Figure 1). Assigning the protein to the correct group not only yields the correct annotations, but may also help to avoid propagating annotation errors and alleviate current issues with mislabelling in protein sequence databases [1,2]. Here, we examine the problem of making correct domain assignments from the Conserved Domain Database [3,4].\nDomains are evolutionarily conserved units in proteins and frequently correspond to recurrent structural and functional units. The particular function of a protein depends on its combination of domains; two-thirds of prokaryotic proteins and 80% of eukaryotic proteins have more than one domain. To create new protein functions, novel domain architectures arise through domain rearrangement and recombination, frequently through gene duplication and fission or fusion events [5,6]. A domain may be represented as a multiple sequence alignment (MSA) of homologous sequence fragments. To identify the domains in a query protein sequence, the MSAs are converted into scoring models such as hidden Markov model or position-specific scoring matrix for use with database search algorithms such HMMER [7] and RPS-BLAST [8].\nTo refine protein annotation, domains models may be subdivided to represent more specific functions or conserved features. CDD curators apply phylogenetic and structural analysis to construct hierarchies of homologous domain models, related by common descent, to reflect aspects of their evolutionary histories [3,4]. Curation follows an iterative procedure to split domain models into subfamilies that redistributes sequences into more narrowly defined models. In the hierarchy tree structure, the leaf domains represent highly conserved and often orthologous protein subgroups. Their precursor (internal) domains, on the other hand, reflect ancient gene duplication events, as CDD aims to categorize ancient conserved domain families.\nIt may seem natural that once the profiles have been defined, the most significant match to a query sequence is the correct one. Indeed, domains from Pfam [9,10] and SMART [11] are assigned following the lowest alignment E-value that exceeds a family-specific cutoff [12,13]. This straightforward approach works well when the candidate domains are disjoint. Domain subfamilies may be obtained through automated methods such as the SCI-PHY algorithm for identifying functional subtypes of known domain families [14,15] or by mirroring other hierarchical domain classifications such as SCOP [16] and CATH [17]. However, subfamily assignment methods generally attempt to classify a member of a family at the subfamily level given that the family is known, as in a statistical pairwise/profile method proposed for SUPERFAMILY [18-20].\nThe systematic arrangement of CDD domains requires identifying the most suitable level of resolution among domain models that offer more or less fine-grained descriptions of a protein. We take the viewpoint that if a protein cannot be associated unambiguously with a specific subgroup or may be a member of a subgroup that has not been defined, the protein can be assigned a more generic domain model or the superfamily in general. Consequently the ideal domain assignment to a query sequence will be the most specific domain, within a candidate hierarchy, with a strong match to the sequence. Here, we analyze a set of correct domain assignments from CDD to establish an improved method for assigning domains to query sequences. The effectiveness of a traditional alignment score and domain-specific threshold is of particular interest, as this method is efficient and makes use of alignment information that is already computed for CDD.\nConstructing a benchmark set of correct domain assignments\nTo benchmark domain assignment heuristics, a reference set of domain assignments is constructed from the NCBI-curated portion of CDD v. 2.12. This set contains every sequence fragment present among the MSAs and its domain assignment. The NCBI-curated domains have undergone rigorous testing to optimize the MSAs and distributions of representative sequence fragments.\nThe correct or most specific domain for each sequence in a hierarchy is defined as the domain having no descendant that contains an overlapping sequence interval. Two sequence intervals from one protein are said to overlap if one sequence interval contains at least 30% of the positions of the other. While each sequence has been placed in the most specific domain model that characterizes it, this step is required as parent and child domains share overlapping sequences (Figure 1). Sequences with overlapping regions from more than one hierarchy are counted once for each hierarchy. Non-overlapping regions of a protein are treated independently. Alignments between all NCBI-curated domains and proteins present in the public Entrez protein set at time of analysis (September 2007) [21] have been pre-computed using RPS-BLAST. In this analysis, the alignment score refers to the bitscore, a normalized version of the raw alignment score between the query sequence and the PSSM, which allows alignments from different searches to be compared. The bitscore corresponds roughly to the alignment E-value and is used instead to avoid real value rounding issues.\nA significant PSSM-sequence alignment is called a hit, for brevity. We call a match between a sequence region and its correct domain a self hit to distinguish it from other hits to overlapping sequence regions. Other hits to the sequences in the reference dataset serve as examples of incorrect domain assignments.\nCDD v 2.12 contains 3078 NCBI-curated domains in 495 hierarchies, including 298 single-domain \"hierarchies\" and 197 trees with 2357 leaf and 423 internal domains. Many sequence fragments used to construct the NCBI-curated domain profiles come from proteins that have been replaced with newer versions or declared obsolete. Among the 109186 representative sequences in NCBI-curated domain hierarchies, over 21% have no hits and more than 90% of those sequences are no longer present in Entrez. This analysis excludes the 149 curated domains without corresponding live data in Entrez, leaving 2929 domains.\n\nPerformance of a simple high-score assignment method\nWe begin by assessing the performance of the previous method for assigning NCBI-curated domains from CDD. The NCBI CD-Search tool [22] has historically relied on alignment E-value and properties such as the source domain database to highlight one or a few most likely domain assignments, without claiming to pinpoint the correct domain assignment. Analysis of all hits to the sequences in the benchmark set reveals that assigning domains by high alignment score alone achieves 96% accuracy over all sequences and 100% accuracy over the representative sequences for 91.5% of domain models. Further, categorizing non-self hits by their hierarchical relationships to the correct domain reveals that assigning to a subclass of the correct domain is the most common type of error when a sequence matches the correct domain and other domains (Table 1). For simplicity, all non-self hits are labelled as incorrect hits in the tables although some child/descendant and parent/ancestor assignments may not be regarded as actual classification errors. Child/descendant domains score higher than the self hit for 21.8% of sequences with both types of hits. These higher scores may reflect computational bias from longer profiles, overly cautious assignment of a sequence to a more generic domain, or missing subfamilies. In contrast, higher scores from parent/ancestor domains or domains from other branches of a hierarchy are rarely observed. For additional data and discussion of all analyses described in this document, see [Additional file 1].\nWe define a score threshold for each domain to be the lowest self-hit score to that domain among all of its sequences in the benchmark set. This additional heuristic, in particular, reduces incorrect assignments to subclasses as only 9.1% of hits to subclasses score above the thresholds for those subclasses (Table 2). The threshold definition works around the issue of small data size–over 60% of domains have 20 or fewer self hits–and addresses variances in scores between domains due to properties such as length and residue composition, or practical issues such as incomplete local alignments, which are not considered by simple high-score heuristics. The definition is more restrictive than its Pfam counterpart, the minimum alignment score among all sequences in the automated \"full alignment\", as NCBI-curated hierarchies in CDD tend to present a finer-grained classification of a protein domain family.\n\nProposed rule for specific domain assignment\nWe propose to label a single domain as correct or specific for a protein sequence region if its alignment score is highest among all domains that align to overlapping regions of the protein sequence and the score exceeds a pre-calculated threshold for the domain, defined as the minimum alignment score among confirmed members of the domain. Sequence intervals that are difficult to group with a specific subclass with high confidence following this rule may receive only generic domain assignments. Assuming that the set of overlapping domains represents an ancient domain superfamily, such a generic assignment would be characterized as membership with the respective superfamily.\n\nReducing misclassifications and errors due to missing subfamilies\nA more concrete picture of the effect of the proposed rule may be gleaned by quantifying misclassifications, defined to be either descendants of the correct domain or domains that lie in other branches of the correct hierarchy. Averaged over domains in multi-domain hierarchies and counting only sequences with self hits, the misclassification rate using high scores only is 2.6%. Incorporating score thresholds to eliminate low-scoring best hits reduces the misclassification rate to 0.85%. Misclassifications may also be used to estimate error due to missing subfamilies. Not all subclasses in a domain hierarchy may have been identified as the available sequence databases only provide a terse snapshot of protein domain diversity. We simulate a cross-validation experiment to ask, if an existing domain model were missing from a hierarchy, what fraction of its sequence intervals have best hits to other models in the hierarchy that are not ancestors of the correct model? Averaged over leaf domains, 50.9% of domain assignments made from high alignment score alone are misclassifications, compared to 6.0% of domain assignments after thresholds are used to screen hits.\n\nFunction and classification through specific domain assignments: Glycyl radical enzymes\nTo illustrate the effect of our proposed method, we examine domain assignments from the glycyl radical enzymes (RNR_PFL hierarchy). Its subgroups have distinct and important functions, including ribonucleotide reductases (RNRs), which synthesize deoxyribonucleotides, and pyruvate-formate lysases (PFLs), a family of catabolic enzymes. The proposed method places the sequence [Entrez:CAA42118] into RNR class 1 and places [Entrez: AAZ61477] into RNR class-1-like domain. The functions of these proteins are inferred by their subclass. Other proteins receive generic assignments to this family. For example, PFL2 (cd01677) is the best match to [Entrez:ABX41552] and [Entrez:EDQ26237] with alignment scores that fall short of the PFL2 threshold. The first alignment includes a long insertion (gap), and the latter exhibits weak sequence similarity; in both of these scenarios the transfer of functional annotation may not be straightforward.\nDomain assignments also help to make biological insights. RNRs fall into classes that use different mechanisms and/or cofactors. Class 1 is oxygen dependent and class 3 is used by strictly or facultative anaerobic organisms. RNR_1_like has a similar active site to class 1 and at the time of curation, no specific literature was available about this subclass. We observed that the strictly anaerobic organism Chlorobium limicola DSM 24 has RNR_3 proteins (e.g. [Entrez:ZP_00512827]) as well as an enzyme ([Entrez:ZP_00512727]) that matches RNR_1_like, suggesting that RNR_1_like, a subfamily lacking experimental characterization, may contain non-oxygen dependent versions of RNR_1.\n\n\nDiscussion\nWhile many sequences can be classified by sequence similarity, profiles of protein domain families make it possible to quickly classify more distant homologs [23] and can better handle multi-domain proteins. An important step in transferring annotations from known protein families is identifying the subclass that provides the best characterization for the protein. Here, we conducted the first focused analysis of domain assignments from CDD in order to assess existing methods for domain and domain subfamily assignment and identify ways to improve the quality of assignments. We find that best-scoring hits are sometimes too specific, causing a sequence to be mislabelled by a subfamily of the correct domain. We propose a subclass assignment procedure that enables concrete assignments, computed quickly using existing data, and demonstrate that this procedure largely avoids over-predictions or false positive assignments and is robust enough to deal with situations such as incomplete hierarchies in which not all subfamilies have been identified. We elected to not employ standard jack-knife or cross-validation testing for a sequence against its correct domain, as the task is to classify sequence fragments that are very similar to a subfamily, where the subfamily model is also constructed from very similar sequences.\nAlthough the sequence and domain databases evolve rapidly, we expect our findings to provide an accurate snapshot for some time. A version of our proposed method has been incorporated into the current version of the CD-Search program and the pre-calculated annotation of proteins with domains in NCBI's Entrez system. Domain assignments to specific orthologous subfamilies or ancient subfamilies are distinguished from non-specific assignments to a domain superfamily. High-confidence annotation of functional sites is also provided following these results. We hope the improved ability to quickly and accurately classify proteins will be a valuable step toward simplifying protein sequence analysis and the computational annotation of genomes.\n\nCompeting interests\nThe authors declare that they have no competing interests.\n\nAuthors' contributions\nJF carried out the experiments and drafted the manuscript. AMB conceived the study, participated in its design, and helped to draft the manuscript.\n\nSupplementary Material\n\n\n" ], "offsets": [ [ 0, 16845 ] ] } ]

[ { "id": "pmcA2632666__T0", "type": "species", "text": [ "Chlorobium limicola" ], "offsets": [ [ 14205, 14224 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "1092" } ] } ]

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[ { "id": "pmcA1838407__text", "type": "Article", "text": [ "Video analysis of the escape flight of Pileated Woodpecker Dryocopus pileatus: does the Ivory-billed Woodpecker Campephilus principalis persist in continental North America?\nAbstract\nBackground\nThe apparent rediscovery of the Ivory-billed Woodpecker Campephilus principalis in Arkansas, USA, previously feared extinct, was supported by video evidence of a single bird in flight (Fitzpatrick et al, Science 2005, 308:1460–1462). Plumage patterns and wingbeat frequency of the putative Ivory-billed Woodpecker were said to be incompatible with the only possible confusion species native to the area, the Pileated Woodpecker Dryocopus pileatus.\n\nResults\nNew video analysis of Pileated Woodpeckers in escape flights comparable to that of the putative Ivory-billed Woodpecker filmed in Arkansas shows that Pileated Woodpeckers can display a wingbeat frequency equivalent to that of the Arkansas bird during escape flight. The critical frames from the Arkansas video that were used to identify the bird as an Ivory-billed Woodpecker are shown to be equally, or more, compatible with the Pileated Woodpecker.\n\nConclusion\nThe identification of the bird filmed in Arkansas in April 2004 as an Ivory-billed Woodpecker is best regarded as unsafe. The similarities between the Arkansas bird and known Pileated Woodpeckers suggest that it was most likely a Pileated Woodpecker.\n\n\n\nBackground\nThe reported rediscovery of the Ivory-billed Woodpecker in 2004–5 in the Big Woods of Arkansas gave new impetus to efforts to conserve the mature bottomland woodlands of the south-eastern USA. Several sightings have been reported without photographic evidence being obtained [1]. Unless sightings are, however, independently verifiable on the basis of photographic or other recorded evidence, the possibility that mistakes have been made cannot be eliminated. Crucial to the scientific case for the persistence of the Ivory-billed Woodpecker was a 4 s video of a large woodpecker in flight recorded by M.D. Luneau on 25 April 2004 (henceforth referred to as the 'Luneau video') and published in 2005 [1], which was claimed to be inconsistent with the plumage patterns of the superficially similar Pileated Woodpecker (a common resident bird of the area). Both species are large, black-and-white woodpeckers [2]. The upperwing of the Ivory-billed Woodpecker is black, with white secondary feathers and white on some inner primary feathers. Pileated Woodpeckers have a largely black upperwing, with white restricted to the 'wrist' due to white bases to the primary feathers. The underwing of Pileated Woodpecker has all-white underwing coverts, giving an appearance of a white underwing with a broad black outline (the black flight feathers). These plumage differences result in the Ivory-billed Woodpecker having a white trailing edge to the wings (upper and lower sides), whereas the Pileated Woodpecker has a black trailing edge to the wings. Both species have black wing-tips. These and other plumage characteristics are shown in [1,2]. The wingbeat frequency of the bird in the Luneau video was measured at 8.6 beats s-1, similar to that inferred from archival sound recording of a single Ivory-billed Woodpecker, but claimed to be outside the range of Pileated Woodpeckers (which generally have slower wingbeats) [1,3].\nSibley et al [4] questioned the video evidence, in particular providing alternative explanations for the plumage patterns of the Luneau bird in flight and at rest. They pointed out individual frames of the Luneau video that appear to show three features that are each inconsistent with Ivory-billed Woodpecker: (1) apparently black secondary feathers on the upper surface of the left wing, (2) particularly bright white primary bases, and (3) a black band curving smoothly round the wing tip (see Figure 3 in [4]). They hypothesized that flexing of a Pileated Woodpecker's wings during flight could produce the appearance of white trailing edges on both wings in low-quality videos [4]. They offered, however, no direct evidence to show that this could cause a video of a Pileated Woodpecker to look like the bird in the Luneau video. Fitzpatrick et al [5] in turn rebutted some aspects of the hypothesis of Sibley et al [4], publishing video stills of Pileated Woodpeckers, and a model of a Pileated Woodpecker, that appeared to show a black trailing edge to the wings inconsistent with Ivory-billed Woodpecker and the Luneau video. Fitzpatrick et al [5] neither rebutted nor discussed the three key inconsistencies described above. Without further evidence, this became largely a theoretical debate over interpretation of field characters that were barely visible in the very small images originally obtained. On one hand, as pointed out in Sibley et al [4], some of the frames of the bird in the Luneau video do appear to be inconsistent with Ivory-billed Woodpecker. On the other hand, the flight pattern of the bird in the Luneau video is asserted to be atypical for Pileated Woodpecker (but matching anecdotal descriptions of Ivory-billed Woodpecker). Furthermore, the general impression of the bird in the Luneau video was that there is far too much white in the wings for it to be a Pileated Woodpecker, and that if it was a Pileated, then it must be an aberrant one with abnormally extensive white plumage. Such birds occasionally occur, and have been observed in the Arkansas study area [6].\nThis study was undertaken to determine whether the flight and plumage of the bird in the Luneau video really was inconsistent with either a normal or partial albino Pileated Woodpecker. Independent analyses of the plumage patterns and wingbeat frequencies observable in Pileated Woodpeckers are presented, and it is concluded that the identification of the bird in the Luneau video as definite Ivory-billed Woodpecker is probably unsafe.\n\nResults\nOn January 28 and February 5, 2006, David Nolin (DN) video-recorded Pileated Woodpeckers Dryocopus pileatus at a bird-feeder in Dayton, Ohio, USA. A Hi-8 Sony Handycam was used, hand-held, at approximately 5 m from the feeder. Birds on the tree trunk were alarmed by movement, and their escape flights recorded. Four escape flights were captured that approximate to that recorded for the putative Ivory-billed Woodpecker Campephilus principalis by Luneau in April 2004 and published in Fitzpatrick et al [1]. The videos are not directly equivalent because the Pileated Woodpeckers made only short escape flights to nearby trees, whereas the putative Ivory-billed Woodpecker in the Luneau video showed little sign of coming to rest before being lost from view. Nevertheless, interesting comparisons can be made.\nWingbeat frequency of Pileated Woodpecker\nThe woodpecker in the Luneau video maintains a steady rapid wingbeat rate of 8.6 beats s-1 for at least 8 wingbeats [1], a figure that was confirmed by independent analysis during preparation of this paper. The Pileated woodpeckers in DN's video do not do this – after initial rapid flapping immediately after take-off, they settle into a more relaxed level flight. As shown in Tables 1 and 2, although the mean wingbeat frequencies of the Pileated Woodpeckers in DN's video are slower than the 8.6 s-1 recorded for the bird in the Luneau video [1,3,5] the first four wingbeats, the initial escape response, are faster than those claimed for Pileated Woodpeckers in the literature [1,3,5]. For the four escape flights, the mean frequency values for the first four wingbeats are 7.1, 6.7, 8.6, and 8.0 s-1, respectively. The 8.6 beats s-1 of the bird identified in the Luneau video, while consistent with the limited data (n = 1; see Discussion) for Ivory-billed Woodpecker, is equally consistent with Pileated Woodpecker in its initial escape flight. The bird in the Luneau video maintains a frequency of 8.6 s-1 for the next four wingbeats too, whereas the Pileated Woodpeckers recorded here all slowed their flight as they prepared to land in nearby trees. There are no data to suggest whether Pileated Woodpeckers can maintain a wingbeat frequency approaching 8.6 s-1 for eight or more wingbeats, like the bird in the Luneau video. It remains possible that the flight pattern of the bird in the Luneau video is unusual for Pileated Woodpecker, but a frequency of 8.6 s-1 is consistent with a Pileated Woodpecker gaining initial speed and height in escape flight, and by itself cannot be taken as strong evidence that the Luneau video bird was an Ivory-billed Woodpecker. This is discussed further below.\n\nPlumage pattern of Pileated Woodpeckers in flight\nThe video of Pileated Woodpeckers in flight was obtained in avi format, decompiled and examined frame by frame. Comparisons of Pileated Woodpecker images with key images of Luneau video are shown in Figures 1 and 2, and suggest a genuine resemblance between the bird in the Luneau video and a Pileated Woodpecker. Analysis is complicated by the different digital processing of the two videos, and in the case of the Nolin videos it is important to concentrate only on those frames or part-frames where apparent plumage features are not an artifact of blurred images. Thirty-six frames from the fourth example of Pileated escape flight, which most resembled the flight path of the Luneau video bird, were analysed systematically frame by frame. They represent seven complete wingbeats (1.20 s from the middle of the second wingbeat to middle of wingbeat 9) and were directly compared frame-by-frame with the equivalent fields (middle wingbeat 2 – middle wingbeat 9) of the Luneau video. This comparison is shown in Figure 3. The images of the birds are not identical, but in every frame of the 36 frames available, there are sufficient similarities to suggest that the bird in the Luneau video is consistent with the known Pileated Woodpecker. Further comparisons of the Luneau bird with the other three Pileated escape flights recorded are presented in the supplementary material (see Additional file 1).\nKey findings of the video analysis are:\n1. Pileated Woodpeckers flying near-horizontally away from the observer show much more white in poor-quality video than would be expected from their general plumage pattern. They present an appearance of a black-bodied bird with largely white wings and black wingtips, very similar to the bird in the Luneau video; compare in particular Figure 1B, frame 758, with Figure 1A, frame 283.3. The expected appearance of the upperwing of Pileated Woodpecker – mostly black with a small white patch at the base of the primaries – is often not seen, and is only clearly resolvable when birds are flying near-vertically before landing on a tree trunk; something the bird in the Luneau video did not do.\n2. The black trailing edge to the underwing of Pileated Woodpecker is often very inconspicuous and may disappear completely. Due to motion and flexion of the wing, the black trailing edge is much more obvious towards the wingtips. This produces an apparent plumage pattern that matches the patterns shown by the Luneau video bird (compare Figure 1B, frames 175 and 457 with Figure 1A, frames 300 and 416.7). In many frames of Pileated Woodpecker, a black trailing edge to the wing is discernable (though due to bleeding of white as a video artifact, it appears narrower than it really is). However, analysis of the bird in the Luneau video in light of images of known Pileated Woodpeckers confirms that a similar black trailing edge to the wing is discernable in some frames of the Luneau video (compare Figure 1B, frame 775 with Figure 1A, frame 366.7: the apparent plumage patterns are similar, and inconsistent with Ivory-billed Woodpecker). It is argued here that the hypothesis put forward in Sibley et al [4] is correct, and that the black trailing edge of the underwing of Pileated Woodpecker can indeed, due to flexion of the wings during the downstroke, be misinterpreted as the black leading edge and wingtips of the upperwing of an Ivory-billed Woodpecker.\n3. Figure 3 shows that the plumage patterns shown by the Luneau bird, throughout several wingbeat cycles, are compatible with Pileated Woodpecker. The three plumage features described in Sibley et al [4] that are incompatible with Ivory-billed Woodpecker (black secondary feathers on upper surface of left wing, brighter white primary bases, and a black band curling round the wing tip) are seen consistently in the Luneau video and are recapitulated throughout the video of Pileated Woodpecker.\n\n\nDiscussion\nEvidence is presented here to show that the distinctive plumage features of Pileated Woodpecker are surprisingly difficult to resolve in poor-quality video of birds in escape flight away from the camera, and that they can show apparent plumage patterns that might more readily be associated with Ivory-billed Woodpecker. Irrespective of the identity of the bird in the Luneau video, this knowledge will be critical to assessment of further claims of Ivory-billed Woodpeckers during the current intensive search effort. It is, however, suggested here that critical frames used for identification of the Luneau video woodpecker as an Ivory-billed Woodpecker are also consistent with Pileated Woodpecker. The wingbeat frequency of the bird in the Luneau video is also perhaps consistent with Pileated Woodpecker, at least for short periods of flight.\nAnalysis of the videos of Pileated Woodpecker has supported the hypothesised interpretations of key frames of the Luneau video by Sibley et al [4]. Although the rebuttal of that comment in Fitzpatrick et al [5] asserted that flexion and motion of wings of Pileated Woodpeckers could not produce the images seen in the Luneau video, it has been shown here that they can.\nThe Luneau video as presented in Fitzpatrick et al [1], shows features that are consistent with Pileated Woodpecker, and inconsistent with Ivory-billed Woodpecker. It is argued in this paper that, in fact, the black trailing edge of the wing of a Pileated Woodpecker is seen clearly in the Luneau video, during the downstroke of the wingbeat cycle, but that it has been misinterpreted as black wingtips (Figure 1, 2, 3).\nA fuller analysis of the Luneau video by the Cornell University team is presented online [7]. Although it is not peer-reviewed, the points this article makes should be taken into account. The authors summarise nine diagnostic traits from their analysis of the Luneau video that identify the bird as Ivory-billed Woodpecker. These are listed and discussed point-by point below.\n1. 'The underwing pattern in flight consistently appears largely white, giving the appearance of having black wingtips but lacking any black along the rear, or trailing edge.'\nData presented in this paper show that this statement is not wholly supported, and in any case the underwing of Pileated Woodpeckers can present the same appearance.\n2. 'The upperwing pattern in flight consistently shows a broad, white trailing edge, with no frames demonstrating the conspicuous dark rear border to be expected of normal Pileated Woodpeckers.'\nNotwithstanding that certain frames of the Luneau video (e.g. frame 350) do appear to show a black trailing edge to the upperwing, data presented in this paper shows that, at this angle of view and resolution of video, Pileated Woodpeckers also may fail to show this feature. This analysis has shown that the hypothesis presented in Sibley et al [4] is plausible, i.e. that some of the frames interpreted by [1] to show the upperwing of an Ivory-billed Woodpecker may in fact show large amounts of white and the black trailing edge from the underwing of a Pileated Woodpecker.\n3. 'The wings are longer relative to the body diameter than in Pileated Woodpecker and consistent with the wing shape of Ivory-billed Woodpecker.'\nFitzpatrick et al [5] agreed that accurate measurements were not possible from the video images presented in their original paper [1], and it seems unlikely that much confidence can be placed in the wing-length measurements of the bird in the Luneau video. Comparison of, for example, Figure 1A, frame 283.3 with Figure 1B, frame 578 suggests that any differences will be very difficult to prove.\n4. 'Reenactment of the scene using life-sized, realistically painted, dynamically flapping models produced images remarkably similar to those of the Luneau video using the Ivory-billed Woodpecker model, and images clearly identifiable as Pileated Woodpecker using a model of that species.'\nInterpretation of model re-enactments is hampered by the fact that the stiff, flat-winged models cannot reflect the wing flexion and curvature of real birds. Reenactment of the scene using real Pileated Woodpeckers has produced images remarkably similar to the Luneau video.\n5. 'The wingbeat frequency is 8.6 beats per second, which is almost identical to that recorded for Ivory-billed Woodpecker (as documented by one acoustic record from 1935). The wing-beat frequencies of Pileated Woodpecker are not known to exceed 7.5 beats per second, and more typically range between 3 and 6 beats per second.'\nThe fact that in only four recorded escape flights of Pileated Woodpecker, two were recorded for which the initial escape flight wingbeat frequency (8.0 s-1 and 8.6 s-1) exceeded that previously recorded for this species shows that previous datasets were too limited to make this conclusion. Birds flap more rapidly at take off to gain altitude and speed than they do in sustained level flight: Pileated Woodpecker flight data in the literature [1,4,5] was derived from the work of Tobalske [8], which explicitly excluded the initial take-off period, and therefore cannot be used to support the elimination of Pileated Woodpecker in the Luneau video. Furthermore, the bird in the Luneau video is consistently gaining height from a low position above water and, whatever its species, might be expected to flap more rapidly than if it were in level flight. Tanner [9] noted that Pileated Woodpeckers can maintain extended fast direct flight. He was of the opinion that flight pattern was not a useful character for separating the two species in the field, and that Pileated Woodpeckers frequently fly in a manner that was in no way different to Ivory-billed Woodpeckers.\nThe figure of 8.6 wingbeats per second for the Luneau bird (data reanalysed here) is taken as consistent with Ivory-billed Woodpecker on the basis of analysis of a single archival audio recording [3]. The Ivory-billed Woodpecker in that audio tape is clearly flapping its wings, but without accompanying visual confirmation it is not clear that it is in flight. In general, larger birds are expected to flap their wings more slowly than smaller birds of comparable wing morphology. Tobalske [8] showed that, across species, smaller woodpeckers tend to flap more quickly than larger ones, and that there was considerable intraspecific variation. The assertion that Ivory-billed Woodpeckers flap their wings more quickly than Pileated Woodpeckers is therefore counterintuitive. Further comment is conjecture: while the flight pattern and wing posture of the bird in the Luneau video may be unusual, it has not been shown that it is outside the range of variability of Pileated Woodpecker, and cannot therefore be used to eliminate the possibility that it was the commoner species.\n6. 'White plumage on the back is visible on the retreating bird as it begins to gain altitude. Ivory-billed Woodpecker has white on the back; Pileated Woodpecker has entirely black back.'\nThis was discussed by Sibley et al [4], who argued that the images thought to show white on the dorsum were too small to be accepted uncritically. In all the frames of the Luneau video that appear to show white on the dorsum, the bird is distant (dorsal white is not visible on the higher resolution images earlier in the video) and partially obscured, making it difficult to distinguish dorsum from wing. Spurious areas of white pixels appear as artifacts in both videos. Nevertheless, this remains the best evidence that the Luneau bird was not a standard Pileated Woodpecker.\n7. 'The dorsal view of the right wing as it begins to unfold shows a triangle of white that matches in size and position the white on the folded wing of an Ivory-billed Woodpecker beginning to launch into flight.'\nNo further comment is provided here. An alternative explanation was offered by Sibley et al [4] and rebutted by Fitzpatrick et al [5]. The statement requires a degree of certainty about the position of the wing.\n(8) 'The distance between the wrist area and the tip of the tail (32–36 cm, as measured when the bird begins to take flight) is comparable to known measurements of Ivory-billed Woodpecker and considerably larger than even the largest Pileated Woodpecker we measured.'\nAs stated under (3), above, there is general agreement that accurate measurements are not possible from the Luneau video because too many uncontrolled variables are involved [4,5].\n9. 'Only 20 seconds before the woodpecker flees, a bird with the size and color pattern of an Ivory-billed Woodpecker was perched within 3 m of the site from which the woodpecker took flight.'\nThis would be a strong argument if it could be shown that the object in question was a bird and not, as is now apparently thought likely, a section of branch or tree stump [10]. The Luneau video reveals several white triangular patches apparently visible on or around tree trunks, most or all of which must therefore be images of tree topography or video artifacts. This was discussed in the literature (see [4,5]).\nCentral to the identification of the flying bird seen in the Luneau video was the evidence that plumage and flight patterns were inconsistent with Pileated Woodpecker. A very basic video analysis presented here has suggested that this may not be the case, and that further research is needed. Any identification of the bird in the Luneau video as an Ivory-bill must take into account the data presented here and in Sibley et al [4], which shows it is largely consistent with Pileated Woodpecker and points out apparent inconsistencies with Ivory-billed Woodpecker. This does not of course necessarily imply that the Ivory-billed Woodpecker is extinct, nor indeed entirely rule out the possibility that the bird in the Luneau video was one. There appears to be no reason to question the anecdotal sight records of Ivory-billed Woodpecker presented in Fitzpatrick et al [1] (or in many online sources), because some of them appear credible, albeit brief. Audio evidence has since been published [11] although this too is far from conclusive. However, to regard the Luneau video by itself as presenting anything other than an unidentified woodpecker falls below the standards of proof normally required for scientific publication: the images are not good enough. The Ivory-billed Woodpecker may persist in continental North America, and there is enough anecdotal evidence to make this a possibility, but the Luneau video does not support the case. The balance of evidence would suggest that the bird in the Luneau video is more likely to have been a Pileated Woodpecker, but the search for Ivory-billed Woodpecker should continue.\nWhile this paper was under review, a report of sight records and sound recordings of Ivory-billed Woodpeckers was published from a location in Florida [12]. This very exciting claim is strengthened by reports of sighting of the white dorsal stripes on one bird in flight. Unfortunately, several sightings were made without optical aids and cannot be considered proven. The 'kent' calls recorded from the Florida location are spectrographically similar to the 'bleat' calls of young White-tailed Deer, as described in Richardson et al [13]. A clear photograph will be required from this location too before the presence of Ivory-billed Woodpeckers can be considered confirmed. It is hoped that this paper will help with assessment of any further low quality photographs or videos.\n\nConclusion\nFlight and plumage patterns of the putative Ivory-billed Woodpecker recorded in Arkansas in 2005 are recapitulated by confirmed Pileated Woodpeckers. The bird in the Arkansas video is best regarded as not fully identified, and is probably a Pileated Woodpecker.\n\nMethods\nVideo recording\nPileated Woodpeckers were attracted to a bird feeder containing suet at Grants Trail, Dayton, OH 45459. The suet feeder was placed approximately 2.1 m high on a tree trunk, and the distance to the suet feeder from the observation point was approximately 5 m. Birds on the feeder were startled by movement of window blinds on January 28 and February 5, 2006, and their escape flights were filmed using a Sony Hi-8 SteadyShot video camera at 29.97 frames s-1. At least two birds feature in the videos, male and female.\nAnalogue tape was converted to digital by connecting the Hi-8 camera directly to a Sony DCR-HC30 digital video camera and recording onto that camera's mini dv cassette. The resulting images were converted to an avi file using Windows Movie Maker on a Windows XP PC. The video is freely available in wmv format [14] and in avi format from the author or David Nolin (via the author). The video was decompiled using Blaze Media Pro (Mystik Media, Hampstead, NC, USA) for a detailed analysis. Import into Avid® Xpress Pro HD for deinterlacing did not reduce the wing flicker seen in the images, and further professional processing could not improve the resolution, so the original decompiled file was used for analysis. Hence some frames contain two overlaid images, which may lower the resolution in some cases. The decompiled file was examined frame by frame and compared to the decompiled images of the putative Ivory-billed Woodpecker presented in Fitzpatrick et al [1].\nWingbeat frequencies were calculated by noting the frame number of the midpoint of the downstroke of each wingbeat (e.g. Figure 1B, frame 758) and calculating the length of time taken per wingbeat as (number of frames between downstroke midpoints)/29.97.\n\n\nAuthors' contributions\nJMC performed the data analysis and drafted the manuscript.\n\nSupplementary Material\n\n\n" ], "offsets": [ [ 0, 26106 ] ] } ]

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"text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 16817, 16840 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T97", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 16920, 16939 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T98", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 17100, 17119 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T99", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 17441, 17460 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T100", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 17656, 17675 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T101", "type": "species", "text": [ "Pileated Woodpeckers" ], "offsets": [ [ 17923, 17943 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T102", "type": "species", "text": [ "Pileated Woodpeckers" ], "offsets": [ [ 18109, 18129 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T103", "type": "species", "text": [ "Ivory-billed Woodpeckers" ], "offsets": [ [ 18189, 18213 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T104", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 18325, 18348 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T105", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 18420, 18443 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T106", "type": "species", "text": [ "Ivory-billed Woodpeckers" ], "offsets": [ [ 18879, 18903 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T107", "type": "species", "text": [ "Pileated Woodpeckers" ], "offsets": [ [ 18939, 18959 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T108", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 19181, 19200 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T109", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 19389, 19412 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T110", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 19436, 19455 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T111", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 20040, 20059 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T112", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 20217, 20240 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T113", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 20651, 20674 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T114", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 20721, 20740 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T115", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 21030, 21053 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T116", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 21692, 21711 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T117", "type": "species", "text": [ "Ivory-bill" ], "offsets": [ [ 21895, 21905 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T118", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 22020, 22039 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T119", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 22085, 22108 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T120", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 22161, 22184 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T121", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 22358, 22381 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T122", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 22809, 22832 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T123", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 23092, 23111 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T124", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 23132, 23155 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T125", "type": "species", "text": [ "Ivory-billed Woodpeckers" ], "offsets": [ [ 23258, 23282 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T126", "type": "species", "text": [ "White-tailed Deer" ], "offsets": [ [ 23655, 23672 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9874" } ] }, { "id": "pmcA1838407__T127", "type": "species", "text": [ "Ivory-billed Woodpeckers" ], "offsets": [ [ 23795, 23819 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T128", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 24009, 24032 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] }, { "id": "pmcA1838407__T129", "type": "species", "text": [ "Pileated Woodpeckers" ], "offsets": [ [ 24093, 24113 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T130", "type": "species", "text": [ "Pileated Woodpecker" ], "offsets": [ [ 24206, 24225 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T131", "type": "species", "text": [ "Pileated Woodpeckers" ], "offsets": [ [ 24252, 24272 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "51359" } ] }, { "id": "pmcA1838407__T132", "type": "species", "text": [ "Ivory-billed Woodpecker" ], "offsets": [ [ 25680, 25703 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "386521" } ] } ]

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[ { "id": "pmcA334876__text", "type": "Article", "text": [ "The c1 genes of P1 and P7.\nAbstract\nThe c1 genes of the heteroimmune phages P1 and P7 were sequenced and their products were compared. P7c1 expression was correlated with the translation in vitro of a protein whose predicted molecular weight (33,000 daltons) is indistinguishable from that of the P1c1 repressor. The c1 regions from both P1 and P7 were found to contain open reading frames capable of coding for a 283-amino acid protein whose predicted secondary structure lacks the helix-turn-helix motif commonly associated with repressor proteins. Two P1c1 amber mutations were localized to the 283-amino acid open reading frame. The P1c1 and P7c1 sequences were found to differ at only 18 positions, all but two of which alter the third position of the affected codon and do not alter the amino acid sequence of the protein. Plasmids expressing the c1 gene from either phage cause the repression of transcription from a cloned promoter situated upstream of P1c1.Images\n\n\n\n\n\n\n Volme 7 Nmbe 19198 Nulei Acds eserc \n\n The cl genes of P1 and P7 \n\n Francis A.Osborne, Sonja R.Stovall and Barbara R.Baumstark* \n\n Department of Biology, Georgia State University, Atlanta, GA 30303, USA \n\n Received July 11, 1989; Revised and Accepted August 29, 1989 EMBL accession nos X16005, X16006 \n\n ABSTRACT \n\n The cl genes of the heteroimmune phages P1 and P7 were sequenced and their products were compared. P7cl expression was correlated with the translation in vitro of a protein whose predicted molecular weight (33,000 daltons) is indistinguishable from that of the Plcl repressor. The cl regions from both P1 and P7 were found to contain open reading frames capable of coding for a 283-amino acid protein whose predicted secondary structure lacks the helix-tum-helix motif commonly associated with repressor proteins. Two Plcl amber mutations were localized to the 283-amino acid open reading frame. The Plcl and P7cl sequences were found to differ at only 18 positions, all but two of which alter the third position of the affected codon and do not alter the amino acid sequence of the protein. Plasmids expressing the cI gene from either phage cause the repression of transcription from a cloned promoter situated upstream of Plcl. \n\n INTRODUCTION \n\n The cl genes of the plasmid prophages P1 and P7 code for repressor proteins that are required for the establishment and maintenance of lysogeny (reviewed in 1). A protein corresponding to the Plcl repressor has been isolated and shown to be a sequence-specific DNA binding protein that recognizes several widely dispersed sites on the phage DNA (2-7). The consensus DNA sequence recognized by the Plcl repressor (ATTTATTAGAGCA[A/T]T) contains no discernable bilateral symmetry, a feature that is highly unusual among prokaryotic operator sites. \n\n P1 and P7 are heteroimmune; that is, each phage is able to establish a lytic infection on a lysogen of the other phage. In this sense, their relationship is analogous to that of phage X and 434, which differ in the DNA specificity of their cI repressor proteins (8). However, genetic studies indicate that Plcl and P7cl can be crossed into the heterologous phage without affecting the immunity specificity of the recipient (9). The basis for P1/P7 heteroimmunity has been localized to a second regulatory gene, c4, that is unlinked to cl. The c4 gene products prevent the expression of antireb, a closely linked gene that interferes with cl-mediated repression (10, 11). According to current models, P1/P7 heteroimmunity results from the inability of the c4 repressor of one phage to prevent antireb expression from the heteroimmune phage genome (10, 11). \n\n Because the cl genes of P1 and P7 are genetically interchangeable, it is anticipated that the two gene products carry out similar or identical regulatory functions. The studies presented in this paper were undertaken to investigate the biochemical basis for the apparent genetic identity of the two cl genes. In this paper, we present the DNA sequence of the cl genes of P1 and P7 and the predicted amino acid sequence of the cl repressor proteins. We report that Plcl and P7cl code for proteins of identical size (283 amino acids) and \n\n Nucleic Acids Research \n\n Volume 17 Number 19 1989 \n\n 767 1 \n\n (r IRL Press \n\n Nucleic Acids Research \n\n nearly identical sequence. We report further that both repressors prevent the expression of a promoter located immediately upstream of the Plc 1 open reading frame, an observation that confirms their functional similarity and suggests an autoregulatory role for the two proteins. Analysis of the secondary structure predicted by the open reading frames does not reveal the characteristic helix-turn-helix (12) or other motifs commonly associated with DNA binding proteins. \n\n MATERIALS AND METHODS Bacterial and phage strains. \n\n E. coli K336 is a SuO derivative of K140 (13). E. coli CB454 is a recA-, lacZderivative of K-12 (14). P1 + is described by Scott (13). P7+ is the strain of Smith (15), as described by Scott (16). P7cl. 1 contains a missense mutation in the cl gene (17). The P7 phage strains and the cl amber mutant phage strains PIci .245Cm, Plc1. 169 and P Ic .55 (11) were generously provided by June Scott. Enzymes and reagents. \n\n Restriction enzymes, T4 DNA ligase and polymerase, and the Klenow fragment of E. coli DNA polymerase were purchased from Boehringer Biochemicals or New England Biolabs and reactions were carried out according to the manufacturers' instructions. DNA sequencing kits and in vitro transcription-translation kits were purchased from Bethesda Research Laboratories and Amersham Corporation, respectively. Synthetic oligonucleotides to be used as sequencing primers were prepared on an Applied Biosystems DNA synthesizer. Plasmid construction. \n\n pBRB7.2. pBRB7.2 (2) contains a 3.2 kb EcoRI/PvuII fragment from the cl region of P1 (Figure 1) inserted into the 2.3 kb EcoRIlPvuII fragment of pBR322 that contains the origin of replication and the f3-lactamase gene. \n\n pFA02. P7 plasmid DNA was digested with PvuII, ligated to similarly digested pBR322, and transformed into E. coli K336. Ampicillin-resistant colonies were screened for cl activity by cross-streak complementation analysis against P7cl.1 (18). pFAO2 contains a 3.5 kb insert of P7 DNA. The fragment was localized to the cl region of the P7 genome by Southern hybridization against P1 and P7 DNA that had been digested with BamHI and BglII (data not shown). \n\n pBRBJ69. 1 and pBRB55. 1. The PI cI open reading frame was previously localized to a 2.6 kb EcoRlIBamHI fragment derived from PlEcoRI-7 (2). This fragment also contains the wildtype allele for the conditional lethal mutation am43 (19, 20). To clone the cl reading frame from the amber mutant phage Plc1.169 and P Ic .55, we digested phage DNA with EcoRI and BamHI, ligated the digestion products into similarly digested pBR322, and transformed the ligation mixture into E. coli K336. Ampicillin-resistant, tetracyclinesensitive colonies were screened by cross-streak complementation analysis for their ability to support the growth of Plam43. Plasmid DNA isolated from complementation-positive cells was shown by agarose gel electrophoresis to carry plasmids containing the 2.6 kb EcoRIlBamHI fragment from the cl region. The cl mutant open reading frames were placed under the control of normal regulatory signals present in the cI region by digesting the cl.55 and cl. 169-containing plasmids with BamHI and PvuII and inserting a 601 bp BamHI/PvuH fragment containing the cl promoter region (2). The resulting plasmids, pBRB55.1 and pBRB169.1, respectively, contain the 3.2 kb EcoRllPvuIl fragment analogous to that present in pBRB7.2 (Figure 1). \n\n pBCB2. 13-2.18. To identify cl-repressible promoters, we introduced selected fragments \n\n 7672 \n\n Nucleic Acids Research \n\n 4 f - - + \n\n I 4 P1 4- 4- 4 \n\n ECAR H C B R P \n\n 4 4.4,~ 4, 1 1 4. I 4, I 11 \n\n t t t t t E G R N E B R P \n\n 4 4 - \n\n 4\n\n 1 ' '110z I I I I I I 1 \n\n 3.2 1.5 1.0 0.5 0 \n\n P7 \n\n pBRB7.2 \n\n -------------------cl1-------------\n\n r ~ / >' pFAO2 \n\n *-lacZ pBCB2.13 \n\n <-lacZ Z pBCB2.16 <-lacZX pBCB2.18 \n\n Fig. 1. The cl regions of P1 and P7. A restriction map is indicated by the solid line in the upper part of the figure. Sites for EcoRI (E), Pvul (P), NnrI (N), Bgll (G), BamHI (B), and EcoRV (R) are shown. The sequencing strategy is indicated by the horizontal arrows. Letters and arrows above and below the map refer to sites and sequence analysis for P1 and P7, respectively. The size of this region (in kilobase pairs) is indicated below the map. The DNA fragments present in selected plasmids are illustrated by boxes at the bottom of the figure. The dashed line reveals the approximate position of the cl gene (2). The sites of the -yb mutations introduced into pFA02 are indicated by asterisks. pFA02.16 and pFA02.26 contain insertions located 0.9 kb and 1.4 kb, respectively, from the PvuIl site at the left side of the map. The direction of the lacZ open reading frame in pBCB2.13-18 is indicated by the adjacent arrow. \n\n from the cI region of P1 into pCB192, a promoter-probe vector containing promoterless copies of lacZ and galK extending in opposite directions from a multiple cloning site (21). The source of P1 DNA for these constructions was pZHA3, a derivative of pBRB7.2 that contains a HindIlI linker at the single EcoRV site located about 200 bps upstream of the cl open reading frame (Figure 1). pBCB2.13 contains a 460 bp fragment of P1 DNA extending from the EcoRV site to a Bgll site within the cI open reading frame. pBCB2. 16 contains a 130 bp fragment extending from the EcoRV site to a BamHI site located about 100 bps upstream of the cl open reading frame, while pBCB2.18 contains the region extending from this BamHI site to the Bglll site within the open reading frame (Figure 1). The orientation of the P1 DNA fragments within these plasmids was confirmed by restriction mapping and DNA sequencing. \n\n To test for the regulation of promoter expression by cl, we transformed pBCB2.13 and its derivatives into CB454(pBRB7.152) and CB454(pFAO2.152), two strains that express P7cl and Pll, respectively, from the pCB192-compatible kanamycin-resistant vector pDPT152 (22). Cells harboring both plasmids were selected by their resistance to both ampicillin and kanamycin. lacZ expression was measured by the procedure of Miller (23). pBRB7.152 was generated by introducing PlEcoRI-7 into pDPT152. pBRB7.152 has sustained a spontaneous deletion within the EcoRI-7 fragment that results in the loss of 2.5 kb of P1 DNA from the far left side of the P1 genetic map, but retains the 3.2 kb PvuHlEcoRI fragment required for cl expression that is present in pBRB7.2 (Figure 1). \n\n 7673 \n\n z \n\n I 011Z I \n\n e., \n\n Nucleic Acids Research \n\n Table 1. Complementation of PIci .245 by plasmids that contain cI genes. \n\n Phenotype Efficiency Relative \n\n of of Efficiency of Plasmid cI gene Lysogeny Lysogeny pBR322 1.5x 10-6 6.0x 10-6 pBRB7.2 Plcl+ 2.5 x 10-' 1.0 \n\n pBRB55.1 Plcl-am 1.4 x 10-6 5.6 x 10-6 pBRB169.1 PlCl-am 2.1 x 10-6 8.4 x 10-6 pFAO2 P7cl+ 8.7 x 10-2 0.35 \n\n pFAO2.16 P7c I-, 4.7 x 10-6 1.9 X 10-5 pFAO2.26 P7cI -1 3.1 x 10-6 1.2 x 10-5 \n\n Complementation for lysogeny was carried out as described by Devlin et al. (26). The plasmids were carried by E. coli K336. Cells were grown to mid-log phase at 370 in LB containing 50 tig/m1 sodium ampicillin and infected with Plcl.245 at a multiplicity of infection of 5 in the presence of 50 mM CaC12. After 10 minutes, non-absorbed phage were removed by centrifugation and the infection was allowed to proceed for 2 hours at 370. The infected cells were plated on LB plates containing 50 pLg/ml sodium ampicillin, 50 pLg/ml chloramphenicol, and 40 mM sodium citrate. The efficiency of lysogeny is defined as the number of ApRCmR cells at the end of infection divided by the number of ApR cells present at the start of the infection. \n\n To construct pFA02.152, we introduced a 2.8 kb EcoRV fragment of P7 DNA containing the cl gene (Figure 1) into the single EcoRI site of pDPT152 after it had been rendered blunt-ended by extension with T4 DNA polymerase. cl expression by cells harboring either pBRB7.152 or pFA02.152 was confirmed by measuring their ability to form chloramphenicol-resistant lysogens when infected with Plcl.245Cm. ,yb insertional mutagenesis. \n\n Insertional mutagenesis of P7cl was carried out using the 'yb transposon of F (24) as described by Devlin et al. (25). E. coli W1485(pFA02) was mated with the F- strain MX648 and subsequently plated on ampicillin (to select for the plasmid) and streptomycin sulfate (to select for the recipient strain). Transconjugants which could support only lytic growth upon infection by P7cl .1 (as scored by cross-streak analysis; 18) were assumed to have lost cl-complementing activity and were characterized further. The positions of two cl - insertional mutations, carried by pFA02.16 and pFA02.26, were identified by restriction mapping (Figure 1). DNA sequencing. \n\n DNA sequence analysis was carried out using the M13-dideoxy technique of Sanger et al. (26). Selected DNA fragments containing the cl wildtype or mutant genes were introduced into M13 mp8 or mp9. 18-nucleotide oligomers complementary to defined sequences within the cl gene were extended using the Klenow fragment of DNA polymerase in the presence of dideoxynucleotide triphosphates and analyzed by polyacrylamide-urea gel electrophoresis. The sequencing strategy is shown in Figure 1. RESULTS \n\n Localization of the P7cJ gene. \n\n Initial localization of the P7cl gene was undertaken by subjecting pFA02 to 'yb mutagenesis and determining the map position of inserts which destroy the ability of the plasmids to complement a P7cl - mutation (as determined by cross-streak analysis). pFA02 and the -yb insertion mutants were tested further by comparing their ability to complement a PIcI amber mutation with the complementation activity of plasmids containing cl genes isolated \n\n 7674 \n\n Nucleic Acids Research \n\n B C D E F G \n\n *.4 \n\n 68\n\n 43\n\n 29- _ - =I = Am~ W _ \n\n 18\n\n 14 p \n\n Fig. 2. In vitro transcription-translation of plasmids carrying the cl region of P1 and P7. Proteins encoded by selected plasmids were labeled with 35S methionine according to the procedure of DeVries and Zubay (27), using a commercial in vitro transcription/tramnslation kit from Amersham Corporation. The reaction mixtures were subjected to electrophoresis on a 12.5% SDS-polyacrylamide gel and the labeled proteins were visualized by autoradiography. The migration of 14C-labeled protein molecular weight standards (Bethesda Research Laboratories) is indicated at the left side of the figure. Plasmids present in each lane are: A. pBRB55.1; B. pBRB169.1; C. pBRB7.2; D. pFAO2; E. pFAO2.16; F. pFAO2.26; G. pBR322. \n\n from P1 wildtype and amber mutants. Lysogeny by cells infected with Plcl.245Cm was scored as the growth of infected cells on ampicillin (to select for the resident plasmid) and chloramphenicol (to select for the phage genome). The values observed for the two plasmids containing Plcl and P7cl (pBRB7.2 and pFA02, respectively) are very similar and significantly higher than those obtained for pBR322 or for any of the plasmids carrying \n\n Table 2. Assay for lacZ expression from plasmids containing P1 DNA fragments. \n\n ,3-galactosidase activity (units) \n\n minus cI plus Pici plus P7cl relative activity \n\n (pDPT152) (pBB7. 152) (pFA02.152) +PICJ +P7cJ \n\n pCB192 0.58 0.55 0.54 0.95 0.93 pBCB2.13 154 15.2 13.6 0.10 0.09 pBCB2.16 1.1 1.2 1.1 1.1 1.0 pBCB2.18 4.0 3.4 3.9 0.9 1.0 \n\n Cells containing derivatives of the ApR promoter-probe plasmid pCB192 and the compatible KnR plasmid pDPT152 were grown in LB at 37?. When they reached mid-log phase, the cells were chilled, lysed, and assayed for (3-galactosidase activity according to the procedure of Miller (23). Plasmids derived from pCB192 are indicated at the left side of the Table. Plasmids derived from pDPT152 are indicated in parentheses across the top of the Table. The values reported are the average of two independent experiments. Relative activity is defined as the f3-galactosidase activity measured in cells harboring plasmids expressing cl divided by the activity measured in cells carrying only pDPT152. \n\n 7675 \n\n Nucleic Acids Research \n\n GATATCCAATCAGGAGTACC GCATCACCCAAGACGACCTG GATGATCTCACTGACACAAT CGAATATCTCATGGCCACTA ACCAGCCAGACTCACAATAAATGCA 105 v-- \n\n TtgAca TATAATG \n\n CTAATAAATCTATTATTTTC GTTGGATCCTTCTATAATGG TGGCCAACAACTCCCAGTGT AATCCGCTGTGAGTTGTTGG CCATGTCAATTCTGGAGGAGGATCA 210 \n\n b----- I I GGAGGtG \n\n ATG ATA AAT TAT GTC TAC GGC GAA CAA CTG TAC CAG GAG TTC GTC AGC TTC AGG GAT CTC TTT CTA AAA AAA GCT GTT GCA CGC GCC CAA 300 MET lIe Asn Tyr Vat Tyr Gly Glu Gin Leu Tyr Gin Gtu Phe Vat Ser Phe Arg Asp Leu Phe Leu Lys Lys Ala Val Ala Arg Ala Gtn \n\n tag(cl .55) \n\n CAC GTT GAT GCC GCC AGC GAC GGT CGT CCT GTT CGC CCG GTT GTC GTT CTG CCG TTC AM GM ACG GAC AGC ATT CAG GCT GMA ATT GAT 390 His Val Asp Ala Ala Ser Asp Gly Arg Pro Vat Arg Pro Vat Vat Vat Leu Pro Phe Lys Glu Thr Asp Ser lIe Gin Ala Glu lie Asp \n\n T A C A G \n\n AAA TGG ACA TTA ATG GCG CGG GAA CTG GAG CAG TAC CCA GAT CTC MT ATC CCA MG ACT ATT TTA TAT CCT GTA CCT AAC ATC CTT CGC 480 \n\n 9\n\n Lys Trp Thr Leu MET Ala Arg Glu Leu Gtu Gin Tyr Pro Asp Leu Asn lIe Pro Lys Thr lie Leu Tyr Pro Vat Pro Asn lIe Leu Arg \n\n A T C \n\n GGT GTG CGT AAG GTT ACG ACT TAT CAG ACA GAA GCA GTG MC AGC GTC AAT ATG ACC GCT GGC CGC ATT ATT CAT CTG ATT GAT AAG GAC 570 Gly Vat Arg Lys Vat Thr Thr Tyr Gin Thr Glu Ala Vat Asn Ser Vat Asn MET Thr Ala Gly Arg lIe lIe His Leu lIe Asp Lys Asp \n\n G \n\n ATT CGC ATC CAA AM AGC GCG GGG ATC MT GAG CAC AGT GCG AAA TAC ATA GAG MC CTG GAA GCA ACA AM GAG CTA ATG AAG CAG TAC 660 lle Arg lIe Gin Lys Ser Ala Gly lIe Asn Gtu His Ser Ala Lys Tyr lIe Gtu Asn Leu Gtu Ala Thr Lys Gtu Leu MET Lys Gin Tyr \n\n T T \n\n CCG GAG GAT GAA AAA TTC CGT ATG CGC GTA CAC GGC TTT AGC GAA ACA ATG CTG CGC GTC CAT TAC ATT TCC AGT AGC CCT AAC TAC AAT 750 Pro Glu Asp Glu Lys Phe Arg MET Arg Vat His Gly Phe Ser Gtu Thr MET Leu Arg Vat His Tyr lie Ser Ser Ser Pro Asn Tyr Asn \n\n Phe \n\n T C G T T \n\n I ~~~I I II \n\n GAT GGC MA TCA GTT AGT TAC CAT GTG CTG CTA TGT GGC GTG TTT ATC TGC GAT GM ACT CTC CGA GAT GGA ATC ATC ATC AAC GGT GAA 840 \n\n e.. Asp Gly Lys Ser Vat Ser Tyr His Vat Leu Leu Cys Gly Vat Phe lie Cys Asp Glu Thr Leu Arg Asp Gly lIe lie lIe Asn Gly Gtu \n\n Pro \n\n C tag(cl .169) \n\n TTT GAG AM GCA AAA TTT AGC CTT TAT GAC TCT ATA GM CCG ATC ATC TGC GAC CGC TGG CCG CAG GCA AM ATA TAT CGC CTG GCA GAT 930 Phe Gtu Lys Ala Lys Phe Ser Leu Tyr Asp Ser lIe Glu Pro lie lie Cys Asp Arg Trp Pro Gin Ala Lys lIe Tyr Arg Leu Ala Asp \n\n T \n\n ATT GM MT GTA AM AM CM ATT GCC ATC ACT CGC GM GAG AAA G GTC AM TCA GCC GCA TCA GTT ACG CGC AGC CGC AAA ACT AAG 1020 \n\n n-----\n\n lie Glu Asn Vat Lys Lys Gin lIe Ala lie Thr Arg Glu Glu Lys Lys Vat Lys Ser Ala Ala Ser Vat Thr Arg Ser Arg Lys Thr Lys \n\n AAG GGG CAG CCA GTA AAC GAC MC CCC GAA AGC GCG CM TAG Lys Gly Gin Pro Val Asn Asp Asn Pro Glu Ser Ala Gin ter \n\n Fig. 3. DNA sequence of PIcI and P7cl. The DNA sequence of PIcI is indicated. Positions where the sequence of P7cl differs from that of Plcl are indicated above the P1 sequence. The amino acid sequence predicted by the open reading frame is given below the sequence. The two amino acid substitutions present in P7cl are shown below the open reading frame. The locations of the amber mutant codons in cl.55 and ci. 169 are indicated by small letters above the sequence. Sites for selected restriction enzymes (EcoRV [v]; BamHI [b]; Bgll [g]; EcoRP [e]; and NruI [n]) are illustrated by dashed lines beneath the sequence. The cl repressor binding site is underlined. Inverted arrows beneath the sequence illustrate the inverted repeat sequence upstream of the open reading frame. Predicted promoter ribosome binding sites are indicated by the presence of the consensus sequences above and below the line, respectively. The DNA sequences of Plcl from bp 1-134 and bp 1-434 were reported previously (2,5). \n\n mutant cl genes from either P1 or P7 (Table 1). The efficiency with which a cloned P7cl gene complements a PlcI mutation confirms previous genetic studies indicating that these two genes are functionally interchangeable (9). The location of the 'y6 mutations that destroy cl-complementing activity suggests that the P7cl open reading frame occupies a map position similar to that of the P1 open reading frame (Figure 1). 7676 \n\n Nucleic Acids Research \n\n Proteins produced by fragments containing Plcl. \n\n As an initial step in the comparison of the P1 and P7 repressors, we analyzed the gene products expressed from the cloned cl regions. In an in vitro transcription-translation reaction, plasmids coding for the wildtpe alleles of either PIcI or P7cl direct the production of a protein with an estimated molecular weight of 33,000 daltons (Figure 2, Lanes C and D), a size that agrees closely with the predicted molecular weight of the PIcI repressor reported previously (3,28). The loss of the 33,000 dalton protein in the cl - 'ya-induced P7 mutant plasmids (Figure 2, Lanes E and F) is consistent with its designation as the P7cl repressor. As expected, the 33,000 dalton protein is not observed when reaction mixtures contain DNA from Plcl amber mutants (Figure 2, Lanes A and B). DNA sequence analysis of the cl genes. \n\n To make a direct comparison between the Plcl and P7cl DNA sequences and to predict the amino acid sequences of the repressor proteins, we carried out M 13-dideoxy sequence analysis of cloned fragments carrying the cl genes. The sequences of about 1 kb of P1 and P7 DNA were determined starting from a common EcoRV site predicted to lie approximately 200 bps upstream of the cl genes. The P1 and P7 sequences (Figure 3) both contain an ATG initiation codon preceded by a putative ribosome binding sequence (29) situated 211 bps downstream of the EcoRV site. In each case, the initiation codon is followed by an open reading reading frame extending for 283 codons. The P1 and P7 open reading frames code for proteins with predicted molecular weights (32,515 and 32,499 daltons, respectively) that agree closely with the values of the proteins expressed from the cloned DNA fragments (Figure 2) and with results predicted independently for the purified PIcI repressor (3-4). The localization of two PIcI amber mutations to the P1 open reading frame confirms its identification as the cI coding sequence. cI. 169 contains an amber mutation that would result in a protein fragment of 26,680 daltons, a value that agrees well with the size of a protein fragment observed under the in vitro transcription/translation reaction conditions (Figure 2, Lane B). The cl.55 amber mutation lies close to the N-terminal region of the protein, resulting in the production of a fragment of 55 amino acids that is apparently too small to resolve under the electrophoretic conditions used for separation of the proteins. Over 60% of the amino acid sequence predicted for the PIcI open reading frame has been verified by amino acid sequence analysis of peptide fragments isolated from the purified repressor protein (see accompanying paper, reference 3). \n\n The DNA sequences of P1 and P7 are identical for a 399-bp region that extends from the EcoRV site at the 5' side of the cl gene to a point 188 bps within the open reading frame. The sequences within the Plcl and the P7cl open reading frames differ at only 18 positions, all but two of which occur in the wobble position of the predicted codon. From these results, we conclude that the functional identity of the P1 and P7 cl genes is a consequence of their nearly identical amino acid sequence. Analysis of promoters upstream of the cl open reading frame. \n\n Expression of Plcl was shown previously to require sequences on the distal side of a BamHI site (2, 5) located about 100 bps upstream of the open reading frame (Figure 3). A binding site for the cl repressor has also been shown to exist close to this BamHI site (2, 5, 6). To determine whether this region contains a promoter that is detectable in vivo and, further, to determine whether this promoter can be regulated by cl repressor proteins from either P1 or P7, we introduced several DNA fragments from this region into the promoter probe vector pCB192, screened for promoter activity (as monitored by lacZ expression) and checked for repression of this activity in the presence of a compatible \n\n 7677 \n\n Nucleic Acids Research \n\n plasmid expressing Plcl or P7cl. Cells harboring pBCB2.13 (a plasmid which carries a 460 bp fragment of P1 DNA that extends across the BamHI site upstream of cl into the open reading frame) are dark blue in the presence of Xgal and produce significant levels of 3-galactosidase (Table 2). In contrast, pBCB2.16 and pBCB2.18 (which each contain DNA from only one side of the BamHI site located in pBCB2. 13) do not confer a blue color on their host cell in the presence of X-Gal and express negligible amounts of 3-galactosidase (Table 2). These observations suggest that expression from the promoter identified here requires sequences that span the BamHI site upstream of cl. Expression of cl from a compatible plasmid in the presence of pBCB2. 13 results in a 90% reduction in promoter strength (Table 2). This reduction is seen in the presence of either Plcl or P7cl, indicating that the two repressor proteins are both capable of repressing expression from this promoter. DISCUSSION. \n\n The DNA sequences of Plcl and P7cl differ at only 18 sites, all but two of which occur at the third position of the affected codon. This observation provides biochemical confirmation of the functional identity predicted on the basis of previous genetic analysis (9). A number of DNA binding proteins exhibit a common structural motif in which two helices are separated by a glycine residue (12). This motif is not observed in the predicted secondary structures (30) of the Plcl and P7cl amino acid sequences. A sequence with some similarity to the XCro helix-turn-helix region was previously reported near the Nterminus of the PIcI protein (5); however, it was noted that the potential for helix formation is disrupted by the presence of several prolines within the region. The secondary structure predicted for the Plcl and P7cl repressor proteins (30) does not reveal other structural characteristics (e.g., Zn fingers (31), leucine zippers (32), or helix-loop-helix motifs (33)) that have been associated with DNA binding activity in other systems. A search of the GenBank and EMBL databases does not reveal any other known regulatory proteins with significant amino acid similarity to the Plcl or the P7cl repressor sequences. Since the Plcl repressor differs from most other repressors in DNA binding specificity (i.e., in its recognition of an asymmetric operator sequence), it is not unexpected to find that the protein does not exhibit common structural motifs at the amino acid level. \n\n The cl-repressible promoter described in this report is located in a region just upstream of the cl open reading frame and is oriented in the direction of cl. Because the promoter is present on a multicopy plasmid, it is not possible to make a direct calculation of promoter strength; however, the values observed are about five-fold lower than the levels produced by a derivative of pCB192 that contains the plac promoter from pUC19 (34). Because sequences on both sides of the BamHI site located upstream of cl are required for promoter activity (Table 2), we suggest that the promoter spans this site. Less than 10 bps downstream of this BamHI site is a heptanucleotide sequence (TATAATG) that is identical to the -10 consensus sequence for RNA polymerase (35). If this sequence does indeed correspond to the -10 region of the promoter, the -35 region would be predicted to lie on the other side of the BamHI site in a region that overlaps a known cI repressor binding site (2-5). Analysis of this region does not reveal any sequences with significant similarity to the -35 consensus sequence. The best fit is the sequence TCTATT (Figure 3), which matches only two positions of the -35 consensus (TTGACA). The lack of a strong -35 region is often observed with genes that require an activator. Although a pentanucleotide sequence corresponding to the conserved portion of the CRP protein consensus binding site (36) \n\n 7678 \n\n Nucleic Acids Research \n\n is located just upstream of the predicted -35 region (at position 91; Figure 3), a role for CRP-mediated activation in cl expression has not previously been described. The orientation of the promoter and its cl-repressible character raise the possibility that cl expression is autoregulatory. If this is so, one potential activator would be the cl repressor itself. Expression cannot be absolutely dependent on cl-mediated activation, however, because the cloned promoter exhibits significant activity in the absence of the cl gene (Table 2). Under the conditions reported here, the presence of the cl gene results in a decrease rather than an increase in lacZ expression; however, these observations do not rule out a potential activator role for the cl protein, since the ratios of repressor and operator provided by the multicopy plasmids may not be optimal for activation. Physiologically, the role of additional repressor binding sites in regulating cl expression also cannot be discounted. Three potential operator sites have been identified several hundred bps upstream of the cI open reading frame (2-5); one or more of these could be involved (possibly through a DNA looping mechanism; 37) in the activation or repression of cl expression during phage growth. \n\n A cl-repressible promoter oriented in the direction of cl was previously reported (38) to be located entirely within PlBamHI-9, a fragment located upstream of cl which is bracketed by the BamHI site within pBCB2. 13. Because sequences on both sides of this BamHI site are required for the activity of the promoter in pBCB2.13, we suggest that the previously identified promoter is distinct from the one reported here. The promoter from BamHI-9 could correspond to a consensus promoter sequence that is situated about 500 bps upstream of cl and overlaps a cl repressor binding site (2). If so, cl expression is likely to be controlled by more than one promoter. Located between this promoter sequence and the promoter encoded on pBCB2. 13 is an open reading frame whose product (termed coi, or c-one inactivator) has been implicated in the establishment of lytic growth (1, 39; B.R. Baumstark, unpublished results). It has been suggested (2) that the decision to enter lytic or lysogenic growth is influenced by the level of transcription initiated from the distal promoter (which would transcribe coi prior to the transcription of cl) relative to that of the promoter located immediately upstream of the cl gene (which would transcribe only ci). \n\n A 32-nucleotide hyphenated inverted repeat sequence is located just upstream of the cl open reading frame (positions 146-188; Figure 3). It is not currently known whether this sequence has any regulatory effect on cl expression. Conceivably, the sequence could serve as a recognition site for an as-yet-unidentified regulatory protein. Alternatively, it may affect the secondary structure of the messenger RNA. A transcript extending from a promoter located upstream of the putative coi open reading frame would be capable of forming a stable stem-loop structure containing 16 bps with a single bp mismatch (AG = -33.6 Kcal) of this inverted repeat sequence. Such a structure could potentially serve as a recognition site for a regulatory factor or, alternatively, could mask such a site. On the other hand, transcription originating from the promoter spanning the BamHI site just upstream of cl would start at a site within the inverted repeat sequence, forming a comparatively less stable stem-loop structure of about 8 bps. The role of the inverted repeat region in the regulation of cl expression is currently under investigation. \n\n ACKNOWLEDGEMENTS \n\n We thank Heinz Schuster for his review of the manuscript. This work was supported by National Science Foundation grant DMB-8704146. \n\n 7679 \n\n Nucleic Acids Research \n\n Abbreviations: bp, basepairs; kb, kilobase pairs; X-Gal, 5-Bromo-4-Chloro-3-indolylbeta-D-galactopyranoside. \n\n *To whom correspondence should be addressed \n\n REFERENCES \n\n 1. Yarmolinsky, M.B., and Steinberg, N. (1988). In Calendar, R., (ed.), The Bacteriophages, Plenum Publishing \n\n Corp., NY, Vol. 1, pp. 291-438. \n\n 2. Baumstark, B.R., Stovall, S.R., and Ashkar, S. (1987). Virology 156, 404-413. \n\n 3. Dreiseikelmann, B., Velleman, M., and Schuster, H. (1988). J. Biol. Chem. 263, 1391-1397. \n\n 4. Heinrich, J., Riedel, H.-D., Baumstark, B.R., Kimura, M., and Schuster, H. (1989). Nucleic Acids Res, \n\n this volume. \n\n 5. Eliason, J.L., and Stemnberg, N. (1987). J. Mol. Biol. 198, 281-293. \n\n 6. Velleman, M., Dreiseikelmann, B., and Schuster, H. (1987). Proc. Natl. Acad. Sci. USA 84, 5570-5574. 7. Citron, M., Velleman, M., and Schuster, H. (1988). J. Biol. Chem. 264, 3611-3617. \n\n 8. Chadwick, P., Pirotta, V., Steinberg, R., Hopkins, N., and Ptashne, M. (1970). Cold Spring Harbor Symp. \n\n Quant. Biol. 35, 283-294. \n\n 9. Chesney, R.H., and Scott, J.R. (1975). Virology 67, 375-384. \n\n 10. Wandersman, C., and Yarmolinsky, M. (1977). Virology 78, 267-276. \n\n 11. Scott, J.R., West, B.W., and Laping, J.L. (1978). Virology 85, 587-600. 12. Pabo, C.O., and Sauer, R. A. (1984). Ann. Rev. Biochem. 53, 293-321. 13. Scott, J.R. (1974). Virology 62, 344-349. \n\n 14. Schneider, K., and Beck, C.F. (1987). Methods in Enzymol. 153, 452-461. 15. Smith, H.W. (1972). Nature New Biol. 238, 205-206. 16. Scott, J.R. (1975). Virology 65, 173-178. \n\n 17. Scott, J.R., Kropf, M.M., and Mendelson, L. (1977). Virology 76, 39-46. 18. Scott, J.R. (1968). Virology 36, 564-574. \n\n 19. Walker, D.H., Jr., and Walker, J.T. (1976). J. Virol. 20, 177-187. 20. Stemnberg, N. (1979). Virology 96, 129-142. \n\n 21. Schneider, K., and Beck, C.F. (1986). Gene 42, 37-48. \n\n 22. Taylor, D.P., and Cohen, S.N. (1979). J. Bacteriol. 137, 92-104. \n\n 23. Miller, J.H. (1972). In Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring \n\n Harbor, N.Y. 352-355. \n\n 24. Guyer, R.S. (1978). J. Mol. Biol. 126, 347-365. \n\n 25. Devlin, B.H., Baumstark, B.R., and Scott, J.R. (1982). Virology 120, 360-375. \n\n 26. Sanger, F., Nicklen, S., and Coulson, A.R. (1977). Proc. Natl. Acad. Sci. USA 74, 5463-5467. 27. DeVries, J.K., and Zubay, G. (1967). Proc. Natl. Acad. Sci. USA 57, 1010-1012. \n\n 28. Heilmann, H., Reeve, J.R., and Puhler, A. (1980). Mol. Gen. Genet. 178, 149-154. 29. Shine, J., and Dalgarno, L. (1974). Proc. Natl. Acad. Sci. USA 71, 1342-1346. 30. Chou, P.Y., and Fasman, G.D. (1978). Adv. Enzymol. 47, 45-148. 31. Berg, J.M. (1986). Nature 319, 264-265. \n\n 32. Landschultz, W.H., Johnson, P.F., and McKnight, S.L. (1988). Science 240, 1759-1764. 33. Murre, C., McCaw, P., and Baltimore, D. Cell 56, 777-783. \n\n 34. Anderson, B.E., Baumstark, B.R., and Bellini, W.J. (1988). J. Bacteriol. 170, 4493-4500. 35. Rosenberg, M., and Court, D. (1979). Annu. Rev. Genet. 13, 319-353. \n\n 36. Ebright, R.H., Cossart, P., Gicquel-Sanzey, B., and Beckwith, J. (1984). Nature 311, 232-235. 37. Ptashne, M. (1986). Nature 322, 697-701. \n\n 38. Stemnberg, N., and Hoess, R. (1983). Annu. Rev. Genet. 17, 123-154. 39. Scott, J.R. (1980). Curr. Top. Microbiol. Immunol. 90, 49-65. \n\n 7680 \n\n This article, submitted on disc, has been automatically \n\n converted into this typeset format by the publisher. " ], "offsets": [ [ 0, 37490 ] ] } ]

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[ { "id": "pmcA1779460__text", "type": "Article", "text": [ "MUC1 alters oncogenic events and transcription in human breast cancer cells\nAbstract\nIntroduction\nMUC1 is an oncoprotein whose overexpression correlates with aggressiveness of tumors and poor survival of cancer patients. Many of the oncogenic effects of MUC1 are believed to occur through interaction of its cytoplasmic tail with signaling molecules. As expected for a protein with oncogenic functions, MUC1 is linked to regulation of proliferation, apoptosis, invasion, and transcription.\n\nMethods\nTo clarify the role of MUC1 in cancer, we transfected two breast cancer cell lines (MDA-MB-468 and BT-20) with small interfering (si)RNA directed against MUC1 and analyzed transcriptional responses and oncogenic events (proliferation, apoptosis and invasion).\n\nResults\nTranscription of several genes was altered after transfection of MUC1 siRNA, including decreased MAP2K1 (MEK1), JUN, PDGFA, CDC25A, VEGF and ITGAV (integrin αv), and increased TNF, RAF1, and MMP2. Additional changes were seen at the protein level, such as increased expression of c-Myc, heightened phosphorylation of AKT, and decreased activation of MEK1/2 and ERK1/2. These were correlated with cellular events, as MUC1 siRNA in the MDA-MB-468 line decreased proliferation and invasion, and increased stress-induced apoptosis. Intriguingly, BT-20 cells displayed similar levels of apoptosis regardless of siRNA, and actually increased proliferation after MUC1 siRNA.\n\nConclusion\nThese results further the growing knowledge of the role of MUC1 in transcription, and suggest that the regulation of MUC1 in breast cancer may be more complex than previously appreciated. The differences between these two cell lines emphasize the importance of understanding the context of cell-specific signaling events when analyzing the oncogenic functions of MUC1, and caution against generalizing the results of individual cell lines without adequate confirmation in intact biological systems.\n\n\n\nIntroduction\nMUC1 is the founding member of the mucin family: proteins characterized by heavy O-glycosylation centering around a variable number of tandem repeats that are rich in serine and threonine residues [1,2]. MUC1 is a transmembrane heterodimer with one subunit solely extracellular (MUC1-EX), and the other subunit composed of a short extracellular stem, a single transmembrane domain, and the cytoplasmic tail (together called the MUC1-CT). MUC1 possesses both pro- and anti-adhesive capacities, as the MUC1-EX provides binding sites for a variety of adhesion proteins, while its large size and extended structure prevents cell-cell contact [3-5].\nInitially described as a tumor antigen overexpressed in >90% of breast cancers, MUC1 is now known to be an oncogene with roles in both tumor formation and progression [1,6]. Mouse studies have been integral to the current understanding of MUC1 in cancer. Muc1 knockout mice (Muc1-/-; MUC1 is human; Muc1 is mouse) show a reduction in tumorigenic phenotype when crossed onto mice overexpressing the Wnt-1 [7] or polyomavirus middle T antigen [8] oncogenes in the mammary gland. In contrast, MUC1 overexpression in the mammary gland drives tumor formation [9], indicating that MUC1 is a true oncogene.\nMany of the oncogenic effects of MUC1 stem from its cytoplasmic tail, which binds to several proteins implicated in cancer, including c-Src [10,11] and the epidermal growth factor receptor (EGFR) family [12,13]. MUC1 stimulates mitogen activated protein kinase (MAPK) signaling through the extracellular signal regulated kinases (ERK1/2) [12,14]; this can occur through MUC1 association with Grb2 and son of sevenless to activate Ras [15]. ERK1/2 signaling is commonly stimulated by the Ras-Raf-MEK (MAPK and ERK kinase) cascade downstream of mitogens such as EGFR [16], and regulates transcription via factors like the activator protein-1 complex. Loss of MUC1 can reduce EGFR expression [17], providing another means of affecting MAPK signaling. Our results describe a novel mechanism by which MUC1 regulates the ERK1/2 pathway, through modulating transcription of the genes encoding MEK1, Raf-1, and c-Jun.\nMUC1 expression correlates with increased survival in response to cytotoxic or oxidative agents [18-21], and can activate the phosphoinositol-3 kinase-AKT pathway as part of an anti-apoptotic response [18]. MUC1 has also recently been linked to transcription, as the MUC1-CT localizes to the nucleus [22] and affects transcription by β-catenin [22,23], FOXO3a [21], p53 [24], and estrogen receptor α [25]. However, there are indications that the role of MUC1 in oncogenesis is regulated by cell type and signaling context. For example, MUC1 can stimulate Fas-mediated apoptosis [26], while Muc1 is specifically down-regulated in c-neu-induced mammary tumors [27]. This report emphasizes the complexity of MUC1 signaling in breast cancer by contrasting results from two established breast cancer cell lines.\nTo understand MUC1 function in cells with high endogenous expression, that is, cells likely to have evolved with active MUC1 signaling, we used small interfering RNA (siRNA) to knock down MUC1 in MDA-MB-468 and BT-20 cells. We then analyzed transcription of 84 genes involved in cancer, as well as the effects upon cellular events linked to oncogenesis, such as apoptosis and proliferation. Though the cell lines show some similarity in transcriptional alterations after transfection with MUC1 siRNA, their phenotypes are quite dissimilar: MDA-MB-468 increases apoptosis and reduces proliferation and invasion, while BT-20 proliferates more rapidly after loss of MUC1. This last may reflect the striking amount of active AKT in BT-20; AKT activity is increased in both cell lines after MUC1 siRNA, which agrees with a previous study of MUC1 siRNA [21], but disagrees with results from 3Y1 fibroblasts [18]. Recent studies have emphasized the complex and context-specific regulation of even such classic oncogenes as AKT [28]. The differences between the two breast cancer cell lines in this study suggest that MUC1 oncogenic functions are also subject to cell-specific regulation, and stress the need for understanding the cellular signaling context when interpreting results.\n\nMaterials and methods\nCell culture and siRNA transfection\nMDA-MB-468 and BT-20 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) plus 10% fetal calf serum, 1% Glutamax (Invitrogen) and 1% penicillin/streptomycin. Stable cell lines (468.Neo and 468.MUC1Δ8) were selected with 0.5 mg/ml G418. For epidermal growth factor (EGF) stimulation, MDA-MB-468 cells were serum-starved overnight and treated for 10 minutes at 37°C with 100 ng/ml EGF. Transient siRNA transfection was performed with Lipofectamine2000 (Invitrogen) and 100 nM siRNA oligonucleotides. The commercially available siRNA constructs (all from Dharmacon, Lafayette, CO, USA) were scrambled (siCONTROL non-targeting siRNA #1), or directed against firefly luciferase (siCONTROL non-targeting siRNA #2) or MUC1 (siGENOME smartpool). The independent oligonucleotides designed in our laboratory target sequences beginning at MUC1 codons 882 and 956, and have been described previously [29]. The scrambled siRNA construct was used only in BT-20 cells as it causes a non-specific knockdown of MUC1 in the MDA-MB-468 line.\n\nCloning of MUC1 WT vector and stable transfection\nTwo silent mutations (G891A and T894C) were introduced into the MUC1 cDNA (called MUC1Δ8) to make it resistant to the 882 siRNA that targets that region of the mRNA. The mutant cDNA was cloned into the pLNCX.1 vector with neomycin resistance (gift of Joseph Loftus, Mayo Clinic, Arizona, USA). Stable transfection was performed with Lipofectamine2000; cells were selected beginning 24 hours post-transfection and maintained as a polyclonal population.\n\nWestern blots and antibodies\nCells were lysed in buffer (20 mM HEPES pH 8.0, 150 mM sodium chloride, 1% Triton X-100, 2 mM EDTA) with commercial protease (Complete inhibitor cocktail, Roche, Pleasanton, CA, USA) and phosphatase inhibitors (10 mM sodium fluoride, 2 mM sodium vanadate, 50 μM ammonium molybdate). Protein concentration was determined by BCA (Pierce, Rockford, IL, USA); 50 μg of lysate were loaded on SDS-PAGE gels for each experiment, except for the pMEK1/2 blot in MDA-MB-468, where 150 μg were used. Non-commercial antibodies used were: BC2, a mouse monoclonal to the MUC1-EX (gift of Dr McGuckin, Queensland University, Queensland, Australia), and CT2, an Armenian hamster monoclonal to the MUC1-CT developed in our lab [12]. Antibodies to pMEK1/2, MEK1/2, ERK1/2, Myc, pAKT, AKT, β-tubulin (all Cell Signaling, Danvers, MA, USA), β-actin and dpERK1/2 (both Sigma, St. Louis, MO, USA) were used according to manufacturers' recommendations. All antibodies except β-actin (1:2,500) and dpERK1/2 (1:10,000) were used at 1:1,000 dilution for western blots. Flow cytometric analysis of MUC1 was done with HMPV-FITC, which recognizes the core peptide of the MUC1-EX tandem repeats (Pharmingen, San Diego, CA, USA). Bromodeoxyuridine (BrdU) staining was performed with a fluorescently conjugated antibody to BrdU (BrdU-PE, BD Biosciences, San Diego, CA, USA) as described below. Densitometry was performed using the public domain ImageJ program (developed at the NIH and available at [30]. Each band was measured in three places; the results were averaged and normalized to tubulin to control for loading.\n\nTranswell invasion assays\nCells were serum-starved beginning 24 hours post-siRNA transfection. Cells were re-plated in serum-free medium 48 hours post-transfection at 50,000 cells per insert (sized for 24-well plates), with serum-containing medium in the bottom of the growth well as an attractant. Transwell inserts (BD Biosciences) pre-coated with laminin, fibronectin, collagen IV, or control (no matrix) were used, and cells were permitted to invade for 48 hours. At this point (96 hours post-transfection), visual inspection of the growth wells confirmed that negligible numbers of cells went through to the bottom of the plate. Non-invaded cells were swabbed from the tops of half of the inserts ('samples', containing only invaded cells), and retained in the others ('controls', all cells). Inserts were stained for 10 minutes with crystal violet (0.5% in 20% methanol) and washed with water. Membranes were cut out and destained for 10 minutes in 10% acetic acid in a 96-well plate; membranes were removed and absorbance was read at 570 nm. Percent invasion is defined as (absorbance of samples/absorbance of controls) × 100.\n\n[3H]Thymidine incorporation assays\nCells were re-plated in quadruplicate 24 hours post-siRNA transfection at 15,000 cells/well (96-well plate) with [3H]thymidine (1 μCi/well), then incubated in normal conditions for 24 hours. At this time (48 hours post-siRNA transfection) excess radioactivity was washed off and the cells were harvested and read on a TopCount plate reader. Statistical analysis was performed using JMP 5.1.2 software (SAS Institute, Inc., Cary, NC, USA); the student's t test was used to determine p values and significance was confirmed with Wilcoxon rank sum and Pearson chi squared analyses.\n\nBrdU incorporation\nBrdU (50 μM) was given to cells 48 hours post-siRNA transfection and permitted to incorporate for 1.5 hours. Cells were then washed with PBS, trypsinized, and washed again. BrdU staining was performed according to an adaptation of the manufacturer's protocol: cells were re-suspended in PBS, mixed 1:1 with -20°C neat ethanol, and incubated 1 hour at -20°C to fix. Fixed cells were then washed gently and denatured in 2 M HCl for 20 minutes at room temperature. Following washing and 2 minute's incubation with 0.1 M Tris to neutralize the acid, cells were re-suspended in FACS buffer (0.5% fetal calf serum in PBS) and stained with Phycoerythrin (PE)-conjugated anti-BrdU according to the manufacturer's protocol for flow cytometry analysis on a FACScan instrument.\n\nApoptosis and trypan blue staining\nApoptosis was measured using a kit (BD Biosciences) containing propidium iodide (PI) and FITC-conjugated annexin V. Cells were stained according to the manufacturer's protocol and the level of apoptosis determined by flow cytometry. Quadrants are: early apoptosis (annexin V+/PI-, lower right) late apoptosis (annexin V+/PI+, upper right) and non-apoptotic cell death (annexin V-/PI+, upper left). Treatments for the stress panel were: no treatment (control); DMSO as a control for celecoxib; 20 mM celecoxib, brand name Celebrex™ (dissolved in DMSO) [31]; 0.2 mM H2O2 [32]; or 1 mg/mL G418 (Pfizer, New York, NY, USA).\n\nReal-time PCR arrays\nTranscriptional analysis using Cancer PathwayFinder RT2 profiler arrays (SuperArray, Frederick, MD, USA) was performed according to the manufacturer's protocol. Briefly, total RNA was isolated using an RNeasy extraction kit (Qiagen, Valencia, CA, USA); 1 μg of RNA was reverse transcribed with the cDNA synthesis kit (SuperArray) and cDNA was subjected to real-time PCR using SYBR green to detect product. Arrays were performed independently at least twice for each cell line; all PCR products were checked on agarose gels. Values were obtained for the threshold cycle (Ct) for each gene and normalized using the average of four housekeeping genes on the same array (HPRT1, RPL13A, GAPD, ACTB). Ct values for housekeeping genes and a dilution series of ACTB were monitored for consistency between arrays. Change (ΔCt) between MUC1 siRNA and control siRNA was found by:\nΔCt = Ct(MUC1 siRNA) - Ct(control siRNA)\nand fold change by:\nFold change = 2(-ΔCt)\nValues are given as fold change; only genes showing two-fold or greater change were considered. Both luciferase and scrambled siRNA controls were used in BT-20; only genes showing consistent alteration with both controls were included in the results reported here. The scrambled siRNA could be not used in MDA-MB-468 as these cells decrease MUC1 expression in response to this construct.\n\n\nResults\nsiRNA transfection decreases MUC1 expression in breast cancer cell lines\nTwo human breast cancer cell lines, MDA-MB-468 and BT-20, were transiently transfected with a pool of four siRNA oligonucleotides directed against the MUC1 mRNA (468.siMUC1 and BT.siMUC1), or a control oligonucleotide directed against luciferase (468.siLuc and BT.siLuc). Both cell lines express high levels of MUC1, making them promising targets for this analysis. Western blots (Figure 1a) show successful knockdown of both the extracellular domain and cytoplasmic tail fragments of MUC1; luciferase siRNA does not substantially change the level of MUC1 compared to parental cells. 468.siMUC1 show a substantial decrease in the amount of MUC1-CT, while BT.siMUC1 show slightly less knockdown of MUC1-CT. Both MDA-MB-468 and BT-20 display a less dramatic decrease of MUC1 extracellular domain compared to MUC1-CT (Figure 1a); this likely represents protein synthesized prior to transfection, and may reflect differences in the turnover rates of the two subunits.\nAnalysis of the MUC1 extracellular domain by flow cytometry confirms that both cell lines substantially decrease MUC1 expression after siRNA (Figure 1b). By flow cytometry, 468.siMUC1 averaged 75% knockdown of MUC1 compared to 468.siLuc; and BT.siMUC1 averaged 50% knockdown relative to BT.siLuc. These effects could be titrated with the concentration of siRNA, were seen as early as 24 hours post-transfection (data not shown) and lasted to at least 96 h post-transfection (Figure 1b). All experiments were conducted within 48 to 96 hours after siRNA transfection. Similar results were obtained using two independent oligonucleotides designed in our lab (data not shown), designated '882' and '956' for the initial codon recognized by each.\n\nTranscriptional changes are seen after MUC1 siRNA\nRecent work indicates that MUC1 may affect transcription both directly via interaction with transcription factors and indirectly (for example, through modulating signaling). To study the effects of MUC1 knockdown in breast cancer cell lines, real-time PCR arrays were used to analyze transcription of 84 genes implicated in cancer. Only genes with greater than two-fold change were considered. Three genes (MAP2K1, VEGF, PDGFA) were altered two-fold or more after MUC1 siRNA in both MDA-MB-468 and BT-20 cells (Figure 2); two genes (ITGAV, MMP2) changed only in 468.siMUC1; and five genes (TIMP3, RAF1, JUN, TNF, CDC25A) only in BT.siMUC1. This list represents all genes affected greater than two-fold after MUC1 siRNA, rather than a select group. Three genes whose transcription was changed by less than two-fold are shown, two of which (PDGFB and ITGB1) are listed because they relate closely to genes altered by two-fold (PDGFA and ITGAV). The third, MYC, is included because western blots confirmed a substantial change at the protein level (Figure 3a) that may reflect both transcriptional and post-transcriptional regulation.\nInterestingly, transcription of MAP2K1 was decreased in both cell lines after MUC1 siRNA. This gene encodes MEK1, one of the primary regulators of the ERK1/2 MAPK pathway [33], a network that has been linked several times to MUC1 [12,34-36]. We examined MEK1 and MEK2 levels by western blot to confirm decreased protein in MUC1 siRNA-treated cells (Figure 3a), and found that not only were total MEK1/2 levels lower in 468.siMUC1 and BT.siMUC1 compared to controls (0.48 and 0.68 relative to siLuc, respectively), but so were the basal amounts of active (phosphorylated) MEK1/2 (pMEK1/2; 0.12 and 0.42 relative to siLuc, respectively). Both 468.siMUC1 and BT.siMUC1 also showed reduced activation of ERK1/2 (dpERK1/2; 0.21 and 0.27 relative to siLuc, respectively), as would be expected with diminished signaling through MEK1/2; total ERK1/2 levels remain unchanged.\nAs both lines have high levels of EGFR and thus activate the MEK-ERK cascade intensely when stimulated with EGF [37], siRNA-transfected cells were treated with EGF. Notably, MUC1 siRNA impairs this important oncogenic pathway in MDA-MB-468 cells, as 468.siMUC1 display less pMEK1/2 in response to EGF than do 468.siLuc (Figure 3b). Interestingly, EGF treatment of BT-20 cells results in slightly higher pMEK1/2 levels in BT.siMUC1 compared to BT.siLuc. Though this result seems paradoxical in light of decreased MAP2K1 transcription in BT.siMUC1, it likely results from differential functions of Raf isoforms in combination with the increased RAF1 transcription (Figure 2) and protein level (Figure 3a) in these cells. Specifically, B-Raf is thought to be the main activator of MEK under normal conditions; Raf-1 activates MEK in response to stimulus [38]. Thus, it appears that basal pMEK1/2 levels are not greatly affected by Raf-1 overexpression in BT.siMUC1 cells, likely because MEK is regulated primarily by B-Raf under normal growth conditions. In contrast, when the cells are stimulated (EGF), increased Raf-1 levels in BT.siMUC1 leads to heightened pMEK1/2 (Figure 3b).\n\nMUC1 siRNA increases apoptosis in MDA-MB-468 but not BT-20\nWe next examined whether MUC1 knockdown and its associated transcriptional alterations would affect overall cellular events. As several of the genes shown in Figure 2 are important in regulating proliferation and survival, and because of the recently described role of MUC1 in modulating apoptosis in response to cellular stresses [20,21,24], we first analyzed whether MUC1 siRNA would alter apoptosis in these lines. Although there was no change in basal apoptosis in either line (Figure 4a), we observed that the cell lines responded differently when trypsinized for re-plating 24 hours after transfection (Figure 4b). Interestingly, 468.siMUC1 cells show greater apoptosis after trypsinization than do 468.siLuc (49.8% versus 34.0%, respectively), while BT-20 cells from both siRNA treatments display similar levels of apoptosis (around 22%).\nTo examine whether this phenomenon is specific to trypsin treatment or part of a general stress response involving MUC1, we subjected cells to a panel of stresses and measured cell death. In agreement with the patterns seen with trypsinization, BT.siLuc and BT.siMUC1 respond similarly to all treatments (data not shown), while 468.siMUC1 die more readily than 468.siLuc in response to trypsin, G418, hydrogen peroxide, or celecoxib, a chemotherapeutic that targets the cyclooxygenase-2 (COX-2) pathway (Figure 4c); these data were confirmed with two independent siRNA constructs (data not shown).\nLike the MAPK pathway, AKT signaling has been linked to MUC1 in cancer. Although transcription of AKT was not altered in MUC1 siRNA-treated cells, the results of our apoptosis studies prompted us to investigate levels of AKT further. As expected, the total AKT protein level is not greatly changed after MUC1 siRNA in either cell line, though the active form (pAKT) is increased in both 468.siMUC1 and BT.siMUC1 compared to controls (Figure 3a). This result disagrees with MUC1 activation of the AKT pathway in rat 3Y1 cells [18], and may reflect regulation more appropriate to breast cancer cells; this is supported by activation of AKT in response to MUC1 siRNA in other lines [21]. In addition, there is a striking difference in the relative amounts of AKT and pAKT in the two cell lines (Figure 4d). When lysates from both lines are exposed to film for the same length of time (overexposure masks the differences between BT.siLuc and BT.siMUC1 that are apparent in Figure 3a), it is clear that pAKT levels are much higher in BT-20 than in MDA-MB-468, despite lower total AKT expression. This difference in AKT activation between MDA-MB-468 and BT-20 likely contributes to the disparity in their sensitivity to the increased apoptosis expected with loss of MUC1.\n\nMUC1 siRNA alters proliferation and invasion\nAs MUC1 is involved in apoptosis, we next analyzed its effects on proliferation. BrdU and [3H]thymidine incorporation were used to analyze proliferation after MUC1 siRNA. 468.siMUC1 cells show a significant decrease in [3H]thymidine incorporation compared to 468.siLuc, while intriguingly, BT.siMUC1 cells show a significant increase in proliferation (Figure 5a). Growth curves mirror these results, as do experiments with the two independent MUC1 siRNA oligonucleotides (data not shown). Note that these assays require trypsinizing cells 24 hours post-transfection; therefore, the results in the MDA-MB-468 line could stem from the changes in apoptosis described in the previous section, rather than a true effect on proliferation. To control for this, we incubated non-trypsinized, siRNA-transfected cells at similar confluence with BrdU to measure incorporation. The 'clumped' profile of cells (contrast to Figure 4b) is likely a result of the acid denaturation (recommended by the antibody manufacturer), as it occurs uniformly in these experiments. BrdU incorporation (Figure 5b) confirms that the [3H]thymidine results are not solely due to alterations in apoptosis, as 468.siMUC1 cells incorporate less BrdU than 468.siLuc; once again, BT.siMUC1 cells show increased proliferation over BT.siLuc.\nGiven the role of MUC1 in adhesion, we examined whether MUC1 siRNA affects cellular invasion. In transwell assays, BT-20 cells invaded poorly, regardless of the siRNA used (data not shown). However, MDA-MB-468 cells invade more readily, and were analyzed on a panel of three different extracellular matrix proteins. Interestingly, 468.siMUC1 cells display somewhat decreased invasion on collagen IV, laminin, and fibronectin matrices, and on a no-matrix control (Figure 5c), which is in agreement with the trend towards decreased metastasis observed in Muc1-/- × MMTV-PyV MT mice [8].\n\nTransfection of MUC1 rescues the 468.siMUC1 phenotype\nTo determine if the above effects are specific to MUC1, we created stable transfectants of the MDA-MB-468 line using empty vector (468.Neo) or a full-length MUC1 construct (468.MUC1Δ8) that is resistant to one of the independent MUC1-directed oligonucleotides ('882'). These cells were maintained in G418-containing medium to retain transgene selection. As expected, 468.MUC1Δ8 cells show higher levels of both the MUC1 extracellular domain and the MUC1-CT than do 468.Neo (Figure 6a). Note that 468.Neo have MUC1 expression comparable to parental MDA-MB-468; the exposures in Figure 6a are lighter than those in Figure 1a, in order to clearly show the relative levels of MUC1 in the stable transfectants. After MUC1 siRNA, 468.MUC1Δ8 lose some MUC1 (likely endogenous protein, which is not siRNA-resistant) but retain high-level expression, while 468.Neo show a decrease in MUC1 levels similar to parental 468.siMUC1 cells (Figures 6a,b). The difference in the amount of MUC1 knockdown between 468.Neo and 468.MUC1Δ8 is highlighted by the purple shading in Figure 6b.\nBrdU incorporation (Figure 6c) indicates that 468.Neo show decreased nucleotide incorporation after MUC1 siRNA compared to control (3.3% versus 25.0%, respectively); this is not seen in 468.MUC1Δ8 cells, which show similar levels of BrdU incorporation regardless of the siRNA used (21.5% for luciferase, 23.9% for MUC1). 468.Neo cells display a more dramatic decrease in BrdU incorporation after MUC1 siRNA than what is seen in parental 468.siMUC1 cells, which may reflect the additional stress of being maintained in G418-containing medium. Similarly, analysis of apoptosis in trypsinized cells indicates that the increased apoptosis seen in parental 468.siMUC1 cells is also present in the 468.Neo line after MUC1 siRNA (Figure 6d; 43.6% in control versus 59.6% in MUC1 siRNA). However, in 468.MUC1Δ8 cells, the level of apoptosis after luciferase siRNA (34.1%) is lower than that in 468.Neo cells; MUC1 siRNA increases the amount of apoptosis slightly (42.8%), restoring it to a level similar to that seen in luciferase siRNA-treated 468.Neo cells. Together, these studies suggest that the above-described results are specific to MUC1, as stable transfection of an siRNA-resistant MUC1 rescues the phenotype seen in 468.siMUC1 cells.\n\n\nDiscussion\nThis report describes both the transcriptional alterations seen after transfection with MUC1 siRNA in human breast cancer cells and the effects on events such as apoptosis and proliferation. The two cell lines used (MDA-MB-468 and BT-20) were chosen for high expression of MUC1 and a substantial (50% to 75%), consistent decrease in MUC1 expression after siRNA. Both lines have epithelial morphology, form tumors slowly in nude mice [37], have mutant p53 [39,40], express EGFR [37], and lack estrogen receptor α [41]. One striking difference between these lines, however, is their response to MUC1 siRNA. MDA-MB-468 cells behave as expected for loss of an oncogene: MUC1 siRNA correlates with increased apoptosis in response to stress, decreased proliferation, and reduced invasion. In contrast, BT.siMUC1 cells proliferate more rapidly than BT.siLuc cells with little effect on apoptosis.\nMuch of the phenotype of these cells can be understood in light of protein levels and transcriptional activity after MUC1 siRNA. As mentioned, both MDA-MB-468 and BT-20 display increased pAKT after MUC1 siRNA, but the ratio of active to total AKT is considerably higher in the BT-20 line, which may help these cells resist the increased apoptosis expected with loss of MUC1. Myc levels are also higher in both cell lines after MUC1 siRNA, although the ability of Myc to promote proliferation and apoptosis in different cellular contexts [42] complicates the interpretation of this finding.\nBoth cell lines show reduced transcription of VEGF, PDGFA, PDGFB, and MAP2K1 (MEK1) after MUC1 siRNA. The genes encoding vascular endothelial growth factor (VEGF) and the A and B chains of platelet-derived growth factor (PDGF-A and PDGF-B) are interesting as these proteins have been heavily implicated in angiogenesis, suggesting a novel function for MUC1 in regulating this process. Vascular endothelial growth factor expression in cancer is linked to tumor growth and metastasis [43,44]; platelet-derived growth factor is also angiogenic, but has an additional role in stimulating desmoplasia [45]. Reduced transcription of these genes after MUC1 siRNA suggests that MUC1 may foster angiogenesis and stromal proliferation, although this must be confirmed in a more appropriate model system.\nDecreased MAP2K1 (MEK1) transcription after MUC1 siRNA provides a novel mechanism by which MUC1 can affect the ERK1/2 MAPK pathway. MUC1 has often been linked to the Ras-Raf-MEK-ERK cascade [12,34-36,46], and at least two mechanisms by which MUC1 can alter MAPK signaling have been described: MUC1 interaction with and phosphorylation by the EGFR family [12,13], and MUC1 binding to the Grb2/Sos complex that activates Ras [46]. Reduction of MEK1 levels after MUC1 siRNA agrees with the role of MUC1 in strengthening MAPK signaling, and indicates that MUC1 can regulate both the transcription and activity of members of this pathway.\nTwo additional MAPK pathway members are altered specifically in BT.siMUC1, with no corresponding change in 468.siMUC1 cells. These genes are RAF1 and JUN which are increased and decreased, respectively, after MUC1 siRNA. Raf-1 and c-Jun both function outside of the ERK1/2 MAPK pathway, which may explain the seeming paradox of increased RAF1 transcription with simultaneous decreases in MAP2K1 and JUN. Specifically, Raf-1 can inhibit ASK1 (apoptosis signal-regulated kinase 1) upstream of p38 and JNK (Jun N-terminal kinase) [38]. ASK1 phosphorylates JNK in response to stress, resulting in activation of c-Jun and stimulation of apoptosis [47], indicating that the coordinate up-regulation of RAF1 and down-regulation of JUN may provide a potent anti-apoptotic effect in BT.siMUC1.\nRegulation of life and death is also a hallmark of the CDC25A and TNF gene products. CDC25A is a phosphatase that stimulates cell cycle progression [48], thus the effects of its decrease in BT.siMUC1 are unclear in light of the increased proliferation of these cells. However, the CDC25 proteins (A, B, and C) were recently shown to have greater functional overlap than was previously thought [49], suggesting that the other two isoforms may compensate for reduced CDC25A levels. TNF encodes tumor necrosis factor (TNF)α, known for its potent, cell type-specific control of life and death. In tumor cells, TNFα expression can promote proliferation and inhibit apoptosis [50], suggesting that increased TNF transcription in BT.siMUC1 could contribute to the increased proliferation seen in these cells.\nInterestingly, the increase in TNF is accompanied by decreased transcription of TIMP3, encoding tissue inhibitor of metalloproteinases (TIMP)3. The TIMP family disrupts the function of matrix metalloproteinases (MMPs), generally resulting in decreased invasion [51]. TIMP3 is unique in that it can also inhibit TNFα converting enzyme (TACE), which activates TNFα by cleaving it from the cell surface [50]. Reduced expression of TIMP3 would, therefore, foster signaling through TNFα by releasing inhibition of TNFα converting enzyme. In agreement with this, TIMP3 can promote apoptosis [52]; thus, its down-regulation in BT.siMUC1 provides another mechanism by which these cells are able to resist the increased apoptosis expected with loss of MUC1.\nAnother TIMP target responds to MUC1 siRNA, as 468.siMUC1 cells show significantly increased expression of MMP2 (encoding MMP-2/gelatinase A), the product of which degrades type IV collagen [52]. In breast cancer, the ratio of active to latent MMP-2 increases with tumor progression; MMP-2 may facilitate both angiogenesis and metastasis [52]. Its increase after loss of MUC1 is, therefore, unexpected, but at least two factors may clarify this result. First, though MMP-2 levels are increased in mouse mammary tumors, its expression is confined to the stroma [53], suggesting that increased MMP2 transcription after loss of the epithelium-specific MUC1 might reflect a shift towards a more mesenchymal phenotype. Second, MMP-2 levels are increased by overexpression of erbB2 [52]; previous studies have shown that erbB2 and Muc1 expression are mutually exclusive in mammary tumors [27], implying that MMP2 might be part of a transcriptional profile linked to low MUC1 levels.\nIt is intriguing that, despite increased MMP2 transcription, invasion is decreased in 468.siMUC1 cells, even on collagen IV. This may reflect insufficient activation of MMP-2, as the precursor protein must be cleaved for enzymatic function [52]. Alternatively, the slowed invasion of these cells may relate to impaired adhesion resulting from decreased transcription of ITGAV and ITGB1 (αv and β1 integrins, respectively). Integrin signaling is tied to life-or-death decisions in epithelial cells, and integrin expression is vital for processes from wound healing to metastasis [54]. Integrin αvβ3 is implicated in facilitating metastasis of breast cancer cells to bone [55]; decreased transcription of ITGAV after MUC1 siRNA may, therefore, suggest that MUC1 is involved in this lethal process as well.\nThe MUC1 oncogene has been linked to apoptosis [18,20,26], proliferation [17], and transcription [21,23-25] in cancer. However, the two cell lines chosen for our study display very different responses to MUC1 siRNA, indicating that regulation of MUC1 in breast cancer is likely quite complex and cautioning against over-generalization of results from individual cell lines. Previous reports suggest that, though most studies outline a clearly oncogenic role for MUC1 in breast cancer, the exact details may vary depending on factors such as cell type and signaling context. For example, MUC1 stimulates Fas-mediated apoptosis in CHO cells [26], quite unlike the inhibition of apoptosis seen in other cell lines. Similarly, though MUC1 drives mammary oncogenesis in its own right [9] and facilitates tumorigenesis driven by other oncogenes [7,8], Muc1 is selectively down-regulated in c-neu-induced mouse mammary tumors [27], indicating that the context of oncogenic signaling is vital to understanding the function of MUC1.\nThus, it is important to consider the relative levels of knockdown of MUC1 in the two cell lines: BT-20 cells reduce MUC1 expression after siRNA less strongly than do MDA-MB-468 (50% versus 75% knockdown, respectively). As MUC1 serves as a scaffold [11], overexpression of MUC1 relative to its associated signaling proteins might create a dilution effect, sequestering signal transducers away from each other; this would be relieved by MUC1 siRNA. Thus, enough MUC1 may be retained in BT.siMUC1 cells for its oncogenic effects, while signaling complex formation would be enhanced by lowering the amount of MUC1 relative to other signaling proteins.\n\nConclusion\nThe contrast between the MDA-MB-468 and BT-20 lines in response to MUC1 siRNA serves as a reminder that simplified models such as cell lines fail to encompass the complexity of intact biological systems. This report describes transcriptional alterations seen after MUC1 knockdown: decreased transcription of MAP2K1, VEGF, PDGFA, ITGAV, TIMP3, CDC25A, and JUN, and increased transcription of MMP2, TNF, and RAF1. The alterations in MAP2K1, RAF1, and JUN represent a novel means by which MUC1 can affect ERK1/2 signaling: transcriptional regulation of MAPK pathway members. Oncogenic events are also altered in both cell lines after MUC1 siRNA. These results strengthen the growing ties linking MUC1 and transcriptional regulation, and suggest that the role of MUC1 in breast cancer may be more complex than a direct correlation between MUC1 level and oncogenic function.\n\nAbbreviations\nBrdU = bromodeoxyuridine; EGF = epidermal growth factor; EGFR = epidermal growth factor receptor; ERK = extracellular signal regulated kinase; MAPK = mitogen activated protein kinase; MEK = MAPK and ERK kinase; MMP = matrix metalloproteinases; MUC1-CT = MUC1 cytoplasmic tail; MUC1-EX = MUC1 extracellular subunit; PBS = phosphate-buffered saline; PI = propidium iodide; siRNA = small interfering RNA; TIMP = tissue inhibitor of metalloproteinases; TNF = tumor necrosis factor.\n\nCompeting interests\nThe authors declare that they have no competing interests.\n\nAuthors' contributions\nCLH performed all studies and composed the manuscript. SJG participated in the design and coordination of the studies and contributed strongly to the revision of the manuscript. Both authors have read and approved the manuscript.\n\n\n" ], "offsets": [ [ 0, 36538 ] ] } ]

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[ { "id": "pmcA147033__text", "type": "Article", "text": [ "Molecular cloning of four novel murine ribonuclease genes: unusual expansion within the ribonuclease A gene family.\nAbstract\nWe have characterized four novel murine ribonuclease genes that, together with the murine eosinophil-associated ribonucleases 1 and 2, form a distinct and unusual cluster within the RNase A gene superfamily. Three of these genes (mR-3, mR-4, mR-5) include complete open reading frames, encoding ribonucleases with eight cysteines and appropriately spaced histidines (His11 and His124) and lysine (Lys35) that are characteristic of this enlarging protein family; the fourth sequence encodes a non-functional pseudogene (mR-6P). Although the amino acid sequence similarities among these murine ribonucleases varies from 60 to 94%, they form a unique cluster, as each sequence is found to be more closely related to another of this group than to either murine angiogenin or to murine pancreatic ribonuclease. Interestingly, the relationship between the six genes in this 'mR cluster' and the defined lineages of the RNase A gene family could not be determined by amino acid sequence homology, suggesting the possibility that there are one or more additional ribonuclease lineages that have yet to be defined. Although the nature of the evolutionary constraints promoting this unusual expansion and diversification remain unclear, the implications with respect to function are intriguing.\n� 1997 Oxford University Press \n\n Nucleic Acids Research, 1997, Vol. 25, No. 21 \n\n 4235�4239 \n\n Molecular cloning of four novel murine ribonuclease genes: unusual expansion within the Ribonuclease A gene family Dean Batten+, Kimberly D. Dyer, Joseph B. Domachowske� and Helene F. Rosenberg* The Laboratory of Host Defenses, Building 10, Room 11N104, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA Received August 4, 1997; Revised and Accepted September 17, 1997 DDBJ/EMBL/GenBank accession numbers: U72031, U72032, AF017258�AF017261 \n\n ABSTRACT We have characterized four novel murine ribonuclease genes that, together with the murine eosinophilassociated ribonucleases 1 and 2, form a distinct and unusual cluster within the RNase A gene superfamily. Three of these genes (mR-3, mR-4, mR-5) include complete open reading frames, encoding ribonucleases with eight cysteines and appropriately spaced histidines (His11 and His124) and lysine (Lys35) that are characteristic of this enlarging protein family; the fourth sequence encodes a non-functional pseudogene (mR-6P). Although the amino acid sequence similarities among these murine ribonucleases varies from 60 to 94%, they form a unique cluster, as each sequence is found to be more closely related to another of this group than to either murine angiogenin or to murine pancreatic ribonuclease. Interestingly, the relationship between the six genes in this `mR cluster' and the defined lineages of the RNase A gene family could not be determined by amino acid sequence homology, suggesting the possibility that there are one or more additional ribonuclease lineages that have yet to be defined. Although the nature of the evolutionary constraints promoting this unusual expansion and diversification remain unclear, the implications with respect to function are intriguing. INTRODUCTION Ribonuclease A (RNase A, bovine pancreatic ribonuclease) is the prototype of an enlarging family of proteins defined by distinctive elements of conserved primary structure and enzymatic activity (1,2). The features shared by all members of the RNase A family include six to eight cysteines and specific histidine and lysine residues that form the ribonuclease active site. To date, several distinct lineages within the RNase A superfamily have been identified. Pancreatic ribonucleases (RNases 1) have been isolated from an extensive array of mammalian species (3,4), and mRNA encoding human pancreatic ribonuclease has been detected in \n\n numerous somatic tissues in addition to pancreas (5). RNases 2 and 3, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), respectively, have been characterized primarily with respect to eosinophil function (6,7), although expression of EDN [also known as RNase Us (8) or human liver ribonuclease (9)] is also widespread (5). Angiogenin (RNase 5), a structurally atypical member of this family, was originally identified as an agent promoting blood vessel growth and development (10,11). RNase 4 (12,13) and RNase k6 (14) are also members of the RNase A superfamily, although their functions are currently obscure. Recently, Larson and colleagues (15) described cDNAs encoding two highly homologous murine ribonucleases, the murine eosinophil-associated ribonucleases (mEAR) 1 and 2, which were cloned and identified via tryptic peptides isolated from murine eosinophil proteins. Although Southern analysis demonstrated that multiple copies of sequence homologous to mEAR-1 and mEAR-2 were present in murine genomic DNA, the precise nature of these copies--as pseudogenes, polymorphisms, or distinct functional genes--was unclear. In this work, we have identified four of these homologous sequences, three encoding novel ribonucleases, and one encoding a pseudogene. Together with mEAR-1 and mEAR-2, these six ribonuclease genes form an `mR cluster,' whose members are more closely related to one another than they are to other murine ribonucleases, yet whose position with respect to the defined RNase A lineages remains unclear. MATERIALS AND METHODS Isolation of genomic fragments encoding murine ribonucleases by polymerase chain reaction (PCR) Genomic DNA was isolated from cells of the murine 3T3 fibroblast cell line. The mR-3, mR-5 and mR-6P sequences were amplified using oligonucleotide primers derived from the published coding sequence of mEAR-1 as follows: 5-CAA ACC CTT TCC CAG AAG TTT GCC-3 (amino acids 28�33); 5-AAA TGT CCC ATC CAA GTG AAC TGG ACC-3 (amino acids 156�148) (15). The PCR reactions were performed as described previously (14), and the multiple products present in the single 400 bp product were identified by dideoxy\n\n *To whom correspondence should be addressed. Tel: +1 301 402 9131; Fax: +1 301 402 4369; Email: hr2k@nih.gov Present addresses: +Duke University School of Medicine, Durham, NC 27701, USA and �Department of Pediatrics, State University of New York-Health Sciences Center at Syracuse, Syracuse, NY 13210, USA \n\n \f4236 Nucleic Acids Research, 1997, Vol. 25, No. 21 \n\n Figure 1. (A) Alignment of amino acid sequences encoded by six related murine ribonuclease genes. Regions of amino acid sequence identity among the five functional genes are enclosed in boxes; the boxes enclosing the active site residues (His11, Lys35, His124) and eight cysteines conserved in all members of the RNase A superfamily are shaded. (B) Percent similarities between amino acid sequences determined via the BESTFIT algorithm of the Wisconsin Genetics Computer Group (WGCG) program on-line at the National Institutes of Health. GenBank accession numbers: mEAR (murine eosinophil-associated ribonuclease)-1, U72032; mEAR-2, U72031; mR (murine ribonuclease)-3, AF017258; mR-4, AF017259; mR-5, AF017260; mR-6P (pseudogene), AF017261. \n\n sequencing of individual plasmids after subcloning into the pCR II TA cloning vector (Invitrogen, San Diego, CA). The complete open reading frames were obtained by extension in both 5 and 3 directions by uni-directional PCR (Genome Walker kit, Clontech, Palo Alto, CA), involving two amplifications with nested primers and Tth polymerase as described previously (14). The nested gene-specific primer pairs were as follows: for 5 extension of mR-3, 5-CTG GAA CCA CTG GAT ACG TGG GAC TGT CCT-3 and 5-ACG TGG GAC TGT CCT GTG GAG TTC TGG GTT-3; for 3 extension of mR-3, 5-ATG CTG TTG GTG TGT GTG GAA ATC CAA GTG-3 and 5-GCT TGT GCA GTG ACA ATA TAA GTC AAA ACT-3; for 5 extension of mR-5, 5-GGT GGG ACG GTC CTT TGG AGT TCT GGG GTT ACA-3 and 5-GGG GTT ACA GGC AAC TGT GTA GGA CTT CTT TCC-3; for 3 extension of mR-5, 5-GAT GTT GTC CGT GTG TGT CAC AAT CCA CCC-3 and 5-AAG ACT TGC AAA GAC GGG ACA AGT CCA AAT-3; for 5 extension of mR-6P, 5-TTG TTG TTT TGC ATC TGC ATT GTG CAT AAC TGC-3 and 5-TTC TGC ATA ACT GCT TGC TGA ACT TGT GAG TGA-3; for 3 extension of mR-6P, 5-ATG TGA TAA TGC AAT GCT GTC TCT TAG CAG TTA-3 and 5-TAC AAG \n\n AGT ATG TAA GCC ATT GAA TCA TTT TCT-3. The products of these reactions were likewise subcloned into the PCR II TA vector and evaluated by dideoxy-sequencing. The mR-4 sequence was amplified with a 36 bp primer derived from sequence encoding the amino-terminal signal sequence of mEAR-1; 5-ATG GGT CCG AAG CTG CTT GAG TCC CGA CTT TGT CTC-3 with the carboxy-primer described above. \n\n Northern analysis The murine multi-tissue Northern blot was purchased from Clontech, and pre-hybridized and hybridized as per manufacturer's instructions with radiolabelled oligonucleotide probes. The membrane was washed with 5� SSPE with 0.1% SDS for 1 h at 37_C and the autoradiogram developed after exposure to film at �80_C. The mEAR-1/mEAR-2/mR-3 probe: 5-CTC TTG TCA CTG CAC AAG CCA CTT GGA TTT CC-3, the mR-5 probe: 5-GTC CCG TCT TTG CAA GTC TTG GGT GGA TTG TG-3, and the actin probe: 5-GCA CAT GCC GGA GCC GTT GTC GAC GAC GAG CGC GGC GAT ATC ATC ATC-3 (16). \n\n \f4237 Nucleic Acids Research, 1997, Vol. 25, No. Nucleic Acids Research, 1994, Vol. 22, No. 121 4237 \n\n Figure 2. (A) Dendrogram depicting relationships among the eight characterized murine ribonucleases as determined by a modified UPGMA method (17). Abbreviations are as in Figure 1A, also mPR (murine pancreatic ribonuclease, sw:rnp_mouse), mANG (murine angiogenin, U72672), hEDN (human eosinophil-derived neurotoxin, M24157), hECP (human eosinophil cationic protein, X15161), hRK6 (human ribonuclease k6, U64998), pRK6 (porcine kidney ribonuclease (24), and bRK6 (bovine kidney ribonuclease, sw:rnkd_bovin). (B) Calculated isoelectric points from amino acid sequences in (A) as determined via the PEPTIDESORT algorithm of WGCG. Table 1. Amino acid sequence comparisons of murine and human ribonucleases \n\n Sequence analysis All DNA sequence analysis and comparisons were performed with the assistance of the Wisconsin Genetics Computer Group and Sequencher (Gene Codes Corporation) programs available at the National Institutes of Health. The dendrogram in Figure 2A was constructed by a modified version of the unweighted pair group method with arithmetic mean (UPGMA) (17), constructed using four initial pairings, mEAR-2 to mR-3, mR-5 to mR-6P, hEDN to hECP and bRK6 to pRK6. RESULTS Isolation of genomic fragments encoding novel murine ribonucleases The alignment in Figure 1A displays the amino acid sequences encoded by the six related ribonuclease genes. Murine eosinophil-associated ribonucleases-1 and -2 (mEAR-1 and mEAR-2) are the predicted amino acid sequences from two genes described previously by Larson and colleagues (15); murine ribonucleases 3, 4, 5 and 6-pseudogene (mR-3, mR-4, mR-5 and mR-6P), are the predicted amino acid sequences encoded by novel DNA \n\n hEDN mEAR-1 mEAR-2 mR-3 mR-4 mR-5 mANG mPR 56 56 54 60 60 43 53 \n\n hECP 59 59 57 59 58 49 52 \n\n hRK6 58 59 59 53 56 49 59 \n\n hPR 47 48 49 48 57 55 79 \n\n hR4 50 53 51 52 54 57 66 \n\n hANG 49 48 51 47 47 80 60 \n\n Values are expressed as percent similarity between pairs of amino acid sequences as determined by the BESTFIT algorithm of the Wisconsin Genetics Computer Group program on-line at the National Institutes of Health. Value representing the highest degree of sequence homology in each row is indicated in boldface. Abbreviations are as defined in Figures 1 and 2, and also include hR4 (human ribonuclease 4, sw:rnl4_human), hANG (human angiogenin, M11567), and hPR (human pancreatic ribonuclease, X62946). \n\n \f4238 Nucleic Acids Research, 1997, Vol. 25, No. 21 fragments amplified from murine genomic DNA. All five functional genes encode amino acid sequences with eight cysteines and appropriately spaced catalytic histidines and lysine that are conserved among the members of the RNase A superfamily. The predicted coding sequence of the non-functional pseudogene (mR-6P) includes two aberrant stop codons (positions 19 and 100) as well as a point mutation resulting in the destruction of the cysteine at position 78. Each genomic fragment also encodes a 28 residue amino terminal signal sequence (not shown). The amino acid sequences are displayed in order of decreasing similarity to mEAR-1 (Fig. 1B). Genomic fragments encoding mEAR-1 and mEAR-2 were also detected. Relationships among the murine ribonucleases A dendrogram depicting the relationships among several of the murine and human ribonucleases is shown in Figure 2A. The mEAR-1, mEAR-2, and mR-3 sequences form a closely related sub-group, with only nine amino acid sequence differences noted between mEAR-2 and mR-3, and 12 and 17 between these two and mEAR-1, respectively. The mR-4, mR-5, and mR-6P sequences diverge from this subgroup and from one another as well. These six sequences are more closely related to one another than they are to either murine pancreatic ribonuclease (mPR) or to murine angiogenin (mANG) or to any of the three human ribonucleases shown (hEDN, hECP or hRK6). Isoelectric points The isoelectric points of all six related ribonucleases range from 8�11, as is typical for members of the RNase A superfamily (Fig. 2B). Interestingly, the isoelectric points within the mEAR-1/mEAR-2/mR-3 subgroup vary by a full 1.0 unit (mEAR-1 at 9.2 to mEAR-2 at 10.2) in response to only 12 discrepancies in amino acid sequence. As increased charge has been associated with increased toxicity in other branches of the RNase A gene family (18), this finding has potential consequences with respect to the function of these proteins (see Discussion). Northern analysis Tissue-specific expression of mRNA encoding these murine ribonucleases is shown in Figure 3. The probe complementary to all three sequences of the mEAR-1/mEAR-2/mR-3 subgroup hybridizes to an 1.4 kb mRNA isolated from renal tissue (Fig. 3A). As the probe cannot distinguish among the three genes, it is not possible to conclude whether the band detected represents mRNA encoding one, two, or all three of the ribonucleases of this subgroup. In contrast, the probe complementary to the mR-5 genomic fragment hybridizes to two prominent mRNA species in hepatic tissue, one at 1.4 kb, and a second at 2.0 kb. A prominent mRNA of 1.8 kb was also detected in mRNA isolated from murine testicular tissue; fainter hybridizing mRNAs of varying size were detected in lung and skeletal muscle. The size variation may represent differential splicing of the mR-5-encoding mRNA, or may represent mRNAs encoding one or more as yet undiscovered murine ribonucleases that are more closely related to mR-5 than those whose sequences are reported here. \n\n Figure 3. (A) Murine RNA probed with a 32 bp oligonucleotide complementary to mEAR-1, mEAR-2 and mR-3 coding sequences. Tissue source of each RNA is indicated above each lane. (B) Same as in (A), probed with a 32 bp oligonucleotide complementary to mR-5. (C) Same as in (A), probed with a 48 bp oligonucleotide complementary to human actin (16), demonstrating relative loading of each lane. \n\n Relationship to human ribonucleases The data in Table 1 denote the amino acid sequence similarity between pairs of murine and human ribonucleases of the RNase A superfamily. Overall, the similarities within the identified cluster range from 47 to 60%, with remarkably little variation among the pairs. Although the sequences of the mEAR-1/mEAR-2/mR-3 subgroup are slightly more similar to ECP and RNase k6, and mR-4 and mR-5, to EDN and ECP, the orthologous relationships of these murine ribonucleases cannot be discerned from these data. This stands in direct contrast to mPR and mANG, whose human orthologs can be clearly distinguished on the basis of their amino acid sequence homologies. \n\n DISCUSSION In this work, we have identified four novel murine ribonuclease genes that, together with mEAR-1 and mEAR-2 defined by Larson and colleagues (15), form an unusual cluster within the RNase A gene superfamily. The six genes within this cluster have varying amino acid sequence homologies to one another, but are clearly more closely related to one another than to either murine pancreatic ribonuclease (mPR) or angiogenin (mANG), or to any of the human ribonucleases of the RNase A family. These results suggest that the `mR cluster' emerged via multiple duplications of a gene that had already diverged from those encoding the other murine ribonucleases. Although this type of expansion is unusual in this gene family, it is actually not unique; similar expansion has occurred in bovine species, with gene duplication resulting in the three closely-related bovine pancreatic, bovine brain (19,20) and bovine seminal (21) ribonucleases. One interesting feature of the mR cluster is that its relationship to any of to the six known human RNase A-type genes cannot be \n\n \f4239 Nucleic Acids Research, 1997, Vol. 25, No. Nucleic Acids Research, 1994, Vol. 22, No. 121 determined from their respective amino acid sequence homologies. Although the amino acid sequences encoded by mEAR-1 and mEAR-2 match those of tryptic peptides derived from murine eosinophil proteins (15), the homology data do not stand in overwhelming support of a unique relationship between any of the mR cluster ribonucleases and the human eosinophil ribonucleases EDN and ECP. The existence of an additional, as yet unidentified human ribonuclease (or ribonucleases) more closely related to those of the mR cluster cannot be ruled out. Perhaps the most important issue raised by this work is the question of why so much evolutionary energy has been devoted to enlarging and diversifying the RNase A gene superfamily. To date, this superfamily now includes several distinct lineages, two species-limited clusters, and the two most rapidly evolving functional coding sequences known among primates (22). The question of evolutionary energy takes on particular significance here, where five functional ribonucleases have emerged in what appears to be a remarkably short period of evolutionary time (15). It is conceivable that each of these ribonucleases might serve a unique but related function. On this point, a number of studies have suggested a distinct role for the human ribonuclease ECP in eosinophil-mediated host defense (6,7); in addition, we have recently shown that eosinophil ribonucleases can inhibit retroviral transduction of human target cells (23). The possibility that certain ribonucleases have diverged to promote distinct and specific host defense-related activities remains an intriguing hypothesis. ACKNOWLEDGEMENTS We would like to thank Dr Jianzhi Zhang for helpful discussions, and Drs Harry L. Malech and John I. Gallin for their ongoing suppport of the work in our laboratory. D.B. is a Howard Hughes Medical Institute-National Institutes of Health Research Scholar REFERENCES 1 D'Alessio, G. and Riordan, J.F. (1997) In D'Alessio, G. and Riordan, J.F. (eds) Ribonucleases: structure and function. Academic Press, Inc., New York, NY. 2 Sorrentino, S. and Libonati, M. 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[ { "id": "pmcA1634875__text", "type": "Article", "text": [ "Molecular polymorphism, differentiation and introgression in the period gene between Lutzomyia intermedia and Lutzomyia whitmani\nAbstract\nBackground\nLutzomyia intermedia and Lutzomyia whitmani (Diptera: Psychodidae) are important and very closely related vector species of cutaneous leishmaniasis in Brazil, which are distinguishable by a few morphological differences. There is evidence of mitochondrial introgression between the two species but it is not clear whether gene flow also occurs in nuclear genes.\n\nResults\nWe analyzed the molecular variation within the clock gene period (per) of these two species in five different localities in Eastern Brazil. AMOVA and Fst estimates showed no evidence for geographical differentiation within species. On the other hand, the values were highly significant for both analyses between species. The two species show no fixed differences and a higher number of shared polymorphisms compared to exclusive mutations. In addition, some haplotypes that are \"typical\" of one species were found in some individuals of the other species suggesting either the persistence of old polymorphisms or the occurrence of introgression. Two tests of gene flow, one based on linkage disequilibrium and a MCMC analysis based on coalescence, suggest that the two species might be exchanging alleles at the per locus.\n\nConclusion\nIntrogression might be occurring between L. intermedia and L. whitmani in period, a gene controlling behavioral rhythms in Drosophila. This result raises the question of whether similar phenomena are occurring at other loci controlling important aspects of behavior and vectorial capacity.\n\n\n\nBackground\nThe Phlebotominae sand flies Lutzomyia intermedia Lutz & Neiva 1912 and Lutzomyia whitmani Antunes & Coutinho 1912 are vectors of cutaneous leishmaniasis in Brazil. These are closely related species that can be only distinguished by a few morphological differences [1] and both show high anthropophily and reported natural infections with Leishmania in different regions of Brazil [2].\nDespite their importance as vectors, only a handful of studies have been carried out in these two species using molecular techniques [3-6]. One of the most important findings from an epidemiological perspective is the evidence obtained for introgression between the two species using mitochondrial DNA [4]. This was particularly interesting because apparently, only lineages of L. whitmani sympatric with L. intermedia have been involved in cutaneous leishmaniasis transmission in the peridomestic environment [4], which suggests that genes controlling aspects of vectorial capacity could be passing from one species to the other. In fact, mitochondrial introgression has been reported in other sand fly species [7,8] suggesting that might be a common phenomenon in these insect vectors. However, because mitochondrial genes can introgress relatively easily between closely related species [9], it becomes important to examine whether introgression can occur with nuclear genes.\nThe Drosophila period (per) gene homologue was isolated in sand flies by Peixoto et al. [10]. This circadian clock gene was originally identified using mutagenesis by Konopka and Benzer [11], but is also known to control the differences in the \"lovesong\" rhythms between D. melanogaster and D. simulans [12], that are important to the sexual isolation between these two species [13-15]. In addition, per was implicated in the control of species-specific circadian mating rhythms in Drosophila and Bractocera, which might also constitute a reproductive isolation mechanism [16-18]. Thus per may possibly represent an example of a Drosophila speciation gene [19], and in fact it has been used as a molecular marker in a number of speciation and evolutionary studies, not only in Drosophila (reviewed in [20]) but also in other insects (e.g. [21]) including sand flies [22-24].\nBecause per controls the circadian clock in different insects [25], it is almost certainly involved in the rhythms of activity and biting of sand flies [26], which are very important to leishmaniasis transmission. In addition, per might be involved in reproductive isolation in sand flies, via mating rhythms, or via their \"lovesongs\" [2,27]. per is thus a particularly interesting marker, among the few available, for an introgression analysis in L. intermedia and L. whitmani. Evidence for introgression in per might suggest that gene flow between these two vector species is occurring at other genes controlling important aspects of behavior and vectorial capacity. It might also suggest that per does not have a strong role in their reproductive isolation. In the current study, we analyzed the molecular variation within the per gene of L. intermedia and L. whitmani in five different localities in Eastern Brazil.\n\nResults\nPolymorphism and divergence between L. intermedia and L. whitmani\nA total of 68 sequences from L. intermedia and 53 from L. whitmani homologue to a fragment of the period gene were analyzed from populations of five localities in Eastern Brazil (Fig 1). The alignment of 72 variable sites is shown in Fig 2. Although most of the changes are either synonymous or occur within the 58 bp intron, non-synonymous substitutions are observed causing 9 amino acid differences among the sequences (Fig 2).\nTable 1 shows the number of sequences of each population of the two species, the number of polymorphic sites (S) and the estimates of molecular polymorphism θ (based on the total number of mutations) and π. Table 1 also shows the Tajima's [28] and Fu & Li's [29] statistics. Within each species, all populations present similar levels of polymorphism with the exception of L. whitmani from Ilhéus, which seems to be less polymorphic than the others. This population was also the only one presenting a significant value in the Fu & Li test but only at the 5% level. Finally, the last column of Table 1 presents the recombination estimator γ [30] indicating that both species show evidence of intragenic recombination in the per gene.\nTo investigate the level of intra and interspecific differences, initially an AMOVA was carried out as shown in Table 2. The results show a non-significant within species and a significant between species molecular variation at the per locus. Table 3 shows a more detailed analysis of the intraspecific differentiation among populations of L. intermedia and L. whitmani. None of the pairwise and overall fixation indexes (Fst) are significant in the case of L. intermedia and only one (Posse × Ilhéus) has a borderline significant value in L. whitmani. The results therefore show that no significant geographical heterogeneity was detected among the populations of the two species. The estimated number of migrants per generation, based on the overall Fst values, is 20.683 for L. intermedia and 23.125 for L. whitmani.\nTable 4 shows measures for DNA divergence between species (Dxy and Da), as well as the Fst and Nm values considering each species as a unique population. Dxy is the average number of nucleotide substitutions per site between alleles from two different populations and Da is the number of net nucleotide substitutions between two populations. Table 4 also shows the number of polymorphisms exclusive for each species (Sint and Swhit), the number of shared polymorphisms (Ss) and the number of fixed differences (Sf) between species. As one can note, there is a high number of shared polymorphisms between species, and no fixed differences between them suggesting either the persistence of ancestral polymorphisms or the occurrence of introgression. In fact, there is one shared haplotype between the two species (IPO13, WPO10 and WPO19) and three L. whitmani sequences (WAC02, WPO13 and WPO14) which show only one nucleotide difference to \"typical\" L. intermedia haplotypes (see also below).\n\nGenealogy of period sequences\nA phylogenetic analysis of the period gene sequences from L. intermedia and L. whitmani was carried out with the Minimum Evolution method using the Kimura 2-parameter distance (Fig 3). A sequence from L. umbratilis, a related species from the same subgenus Nyssomyia, was used as outgroup [24]. The tree shows L. intermedia and L. whitmani as non-monophyletic. However, despite the low bootstrap values, which are below 50% in most cases, there is a large group that contains most L. intermedia sequences and a second large group with most L. whitmani sequences. A few other sequences are clustered outside these two main groups. It is interesting to note that there are three L. whitmani alleles (WAC2, WPO13 and WPO14) inside L. intermedia main group, as well as one L. intermedia allele (ICP16) inside the L. whitmani main group. In addition, a second L. intermedia allele (IPO13) is a shared haplotype between the two species as mentioned above. Again, the results suggest either the persistence of ancestral polymorphisms or the occurrence of introgression between the two species. Very similar results were obtained using the maximum likelihood algorithm as implemented in PAUP 4.0b10 software [31] (data not shown).\nAs mentioned before, there is evidence of intragenic recombination in the per gene fragment of both species (see Table 1) and for that reason the bifurcating tree shown in Fig 3 has to be viewed with caution, as different regions of the gene might have different phylogenetic histories [32]. Therefore, we constructed Minimum Evolution trees with the two most polymorphic non-recombining blocks of the per gene fragment identified using the Hudson and Kaplan [33] method available in the DNAsp 4.1 program [34]. We did not observed major changes in the genealogy of the L. intermedia and L. whitmani per sequences, especially regarding the five haplotypes (ICP16, IPO13, WAC2, WPO13 and WPO14) that clearly cluster with sequences of the other species (data not shown).\nFinally, a haplotype network was estimated from per sequences using statistical parsimony, as described by Templeton et al. [35] and implemented in the TCS1.21 software [36] (Fig 4). A small number of ambiguities were resolved as suggested by Crandall and Templeton [37]. The haplotype network shows connections between sequences from each species, separating most of the sequences of L. intermedia and L. whitmani in two groups. No intraspecific geographical structuring was found. Once again, some of the L. whitmani sequences (WAC2, WAC10, WPO13 and WPO14) appear more closely related to L. intermedia haplotypes. In addition, one L. intermedia allele (ICP16) is connected by a small number of mutations to some of the main L. whitmani haplotypes and IPO13 is a shared haplotype between the two species. These results confirm the same putative introgressed sequences indicated by the phylogenetic reconstructions.\n\nLD test of introgression\nWe tested the hypothesis of gene flow between L. intermedia and L. whitmani using a method based on linkage disequilibrium (LD) developed by Machado et al. [38]. In this test, x is the difference between the average LD found among all pairs of shared polymorphisms (DSS) between the two species and the average LD among all pairs of sites for which one member is a shared polymorphism and the other is an exclusive polymorphism (DSX). In case of gene flow x should tend to be positive [see [38] for more details].\nBecause of limitations on the total number of sequences that could be handled by the WH program we could not perform the simulations with all sequences. Therefore, we carried out the LD test of introgression between each pair of sympatric populations of L. intermedia and L. whitmani from the localities of Posse, Afonso Claudio and Corte de Pedra. The input files were prepared using the values of recombination and linkage disequilibrium calculated by the SITES program [30] for each population (data not shown). Although no significant values were found for the smaller samples of Afonso Claudio and Corte de Pedra, the results (Table 5) present evidence for introgression in the period gene in both directions (from L. intermedia to L. whitmani and vice-versa) in the locality of Posse.\n\nIsolation with Migration model\nTo further examine the gene flow between L. intermedia and L. whitmani we used the IM software [39]. The Isolation with Migration model has six demographic parameters that include two migration rates, one for each population. The IM software estimates the posterior probability for each of the model parameters, fitting the Isolation with Migration model to the data. One of the assumptions of this model is that the loci studied do not have internal recombination. Therefore, we identified four different non-recombining blocks of our fragment of per, which were then treated as different loci in the analysis. The four-gametes test [33] implemented in DnaSP4.1 was used for the identification of possible recombination events. Since the program estimates parameters for a pair of closely related populations or species, all sequences of each species were used in the analysis as a single population. We performed MCMC runs using the IM software with different seed numbers, in order to guarantee convergence of the sample.\nMaximum likelihood estimates of migration parameters revealed a non-zero value for both species, m1 = 1.398 and m2 = 1.014 (m1 – from L. whitmani towards L. intermedia; m2 – from L. whitmani towards L. intermedia). Fig 5 shows the posterior distributions for migration rates and reveals a null probability for the absence of migration from L. whitmani towards L. intermedia. In addition, the absence of migration in the opposite direction is not included in the 95% confidence interval (values range from 0.222 to 8.898), thus supporting the presence of migration in both directions. The conversion of the migration rate estimate to population migration rate per generation (m1 and m2) is not accurate when the population size is based on a single locus. However, the average of the migrant number per generation for both species was very close to the Nm estimate based on Fst values (Nm ~0.49 in Table 4, m1 ~0.52 and m2 ~0.34).\n\n\nDiscussion\nThere is some evidence that L. intermedia and L. whitmani might represent sibling-species complexes in Brazil. Lutzomyia neivai Pinto 1926, a sibling of L. intermedia is found in parts of Southern and Western Brazil and some other countries of South America [40]. The present study did not include populations of this species. In the case of L. whitmani, mitochondrial data [3,6] indicates three main lineages in Brazil: an Amazonian group, a North-South group and a Northeast group. We did not find strong evidence of a geographical differentiation in the period gene among populations of L. whitmani although one of the pairwise Fst comparisons (Posse × Ilhéus) was significant at the 5% level.\nWhen we compare L. intermedia and L. whitmani, we find a highly significant Fst value (0.3373), which is however smaller than that observed for the period gene between sympatric siblings of Lutzomyia longipalpis (Fst = 0.3952) [23], a complex of cryptic species that are vectors of American visceral leishmaniasis. Therefore, despite the presence of diagnostic morphological characters to identify L. intermedia and L. whitmani [1] the level of molecular divergence in period is not as high as the cryptic L. longipalpis siblings.\nEven though it is hard to distinguish introgression from the persistence of ancestral polymorphisms, a test of gene flow based on the signature introgression leaves on the patterns of linkage disequilibrium [38] as well as simulations that fit the \"Isolation with Migration\" model to the data suggest that L. intermedia and L. whitmani might be exchanging alleles at the per locus. This is further supported by the presence of shared haplotypes between the two species in Posse and very similar sequences in all sympatric populations. There is mounting evidence that introgression plays a major role in the evolution of closely related insect vector species. Introgression among vectors may have important epidemiological consequences. Gene flow in loci that affect vectorial capacity, such as those controlling host preference and susceptibility to parasite infection, can change the transmission patterns and consequently make the disease control a harder task. Introgression of genes that control adaptation to particular types of environment can also have a major impact on the spread of vector-borne diseases as was proposed for the major African malaria vector Anopheles gambiae [41]. The same can be said about genes controlling insecticide resistance. For example, Weill et al. [42] found a kdr mutation responsible for pyrethroid resistance in the Mopti form of Anopheles gambiae, a normally susceptible taxon of this species complex. Sequence analysis reveals that this resistant allele probably originates through introgression from the Savanna form.\nAlthough L. intermedia and L. whitmani are closely related and only distinguished by a few morphological differences, they do show differentiation in some other important traits. For example, in Posse, one of the localities we studied, the two species show differences in abundance during the year. L. intermedia is more abundant in the summer while L. whitmani is more frequent in the winter months [2]. They also show differences in microhabitat preferences, L. intermedia being more common in the peridomestic area while L. whitmani is found mainly in the surrounding forest [2]. In addition, the two species show marked differences in their tendencies to bite humans in the early morning, with L. whitmani showing higher feeding rates than L. intermedia [26]. Therefore, despite the evidence of introgression in the period gene in this locality, there are important ecological and behavioral differences between the two species in Posse suggesting that gene flow is probably rather limited in loci controlling these traits. Hence, it is yet not clear whether introgression has played an important role in the evolution of L. intermedia and L. whitmani. Further work with other genes might help clarify the issue.\n\nConclusion\nEvidence for introgression between L. intermedia and L. whitmani obtained using mitochondrial DNA [4] seems to be corroborated by our data on the period gene, a nuclear marker. Nevertheless, considering that period is potentially involved in reproductive isolation and might be, therefore, less prone to introgression than the \"average\" gene [43], it is possible that much higher levels of gene flow between the two species occur at other genes. It might, on the other hand, suggest that this behavioral gene, or at least the fragment we analyzed, did not play a role in speciation between L. intermedia and L. whitmani. In fact the same has been suggested for some Drosophila species [44] despite per's role controlling lovesong and mating rhythm differences between D. melanogaster and D. simulans [13-16].\nAlthough the evidence for introgression in the per gene between L. intermedia and L. whitmani is not overwhelming, it does indicate the need to extend this analysis to other loci in the future. We are currently isolating new molecular markers in the two species to carry out a multi-locus approach [39] that might help determining how much variation in gene flow and differentiation there is across the genome of these two very important leishmaniasis vectors.\n\nMethods\nSand fly samples\nSand fly samples used in this work were all the F1 generation from wild collected females from the Brazilian localities of Posse (Petrópolis, Rio de Janeiro State, 22°30'S 43°10'W), Jacarepaguá (Rio de Janeiro, Rio de Janeiro State, 22°55'S 43°21'W), Afonso Claudio (Espírito Santo State, 20°04'S 41°07'W), Corte de Pedra (Presidente Tancredo Neves, Bahia State, 13°27'S 39°25'W) and Ilhéus (Bahia State, 14°50'S 39°06'W). L. intermedia and L. whitmani were identified according to Young and Duncan [1]. The progeny of each wild caught female was raised separately according to Souza et al. [45] and only one F1 male of each female was used for the molecular analysis, which included 68 individuals of L. intermedia (12 from Afonso Claudio, 18 from Posse, 20 from Corte de Pedra and 18 from Jacarepaguá) and 51 individuals of L. whitmani (12 from Afonso Claudio, 17 from Posse, 3 from Corte de Pedra and 19 from Ilhéus). Note that, although the distribution of the two species shows considerable overlap in Eastern Brazil, in many localities only one species is found or is far more abundant than the other. There are also seasonal and microhabitat differences in abundance between them in areas of sympatry [2].\n\nDNA methods\nGenomic DNA was prepared according to Jowett [46] with slight modifications and the PCR was carried out for 30 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, using Abgene, Amersham Biosciences or Biotools reagents according to manufacturers directions. The per primer sequences are: 5llper2: 5'-AGCATCCTTTTGTAGCAAAC-3' (forward) and 3llper2: 5'-TCAGATGAACTCTTGCTGTC-3' (reverse). These primers amplify a 486 bp fragment of the sand fly per gene homologue that includes part of the PAS/CLD domain, an intron (58 bp) and the beginning of the perS domain [24]. The amplified fragments were cloned using the pMOSBlue blunt ended cloning kit (Amersham Biosciences) and plasmid DNA preparation was carried out using the \"Flexiprep\" Kit (Amersham Biosciences). Cloned PCR fragments were sequenced at Fundação Oswaldo Cruz and at University of Leicester using ABI 377 sequencers. With the exception of two L. whitmani individuals from Corte de Pedra (see below), only one sequence of each sand fly (representing one of the two possible alleles) was used in the analysis but an average of three sequences per individual were obtained in order to check possible PCR induced mutations. In addition, PCR fragments were also sequenced directly in some cases for the same reason. In the case of the two L. whitmani mentioned above 6 and 9 clones were sequenced respectively from specimens WCP01 and WCP03 to determine both alleles simply to increase the size of this small sample.\nNegative controls were performed for all amplification reactions. In addition, PCR, cloning and sequencing were repeated for two individuals to confirm putative introgressed sequences and to exclude the possibility that they were the result of PCR contamination. Finally, for at least two individuals with putative introgressed sequences, we could define the other allele from additional clones (not included in the analysis), which showed to be typical of the species, indicating no identification problems.\nThe sequences were submitted to GenBank (accession numbers AY927062 to AY927182).\n\nSequence analyses\nThe preliminary sequence editing was carried out using the Wisconsin Package Version 9.1, Genetics Computer Group (GCG), Madison, and ClustalX [47] was used to perform the multiple alignment. Analyses of population polymorphisms and differentiation between populations were carried out using DNAsp4.1 [34] and ProSeq [48] softwares, while Arlequin v. 2.0 [49] was used for an analysis of molecular variance (AMOVA) between populations. The Minimum Evolution phylogenetic tree was constructed using MEGA 3.1 software [50]. The haplotype network was estimated using TCS1.21 [36]. Recombination and linkage disequilibrium analyses were performed using the DNAsp4.1 and SITES program [30]. Linkage disequilibrium simulations were carried out by the WH program [51,52] and Markov Chain Monte Carlo (MCMC) simulations of the isolation with migration model were performed using the algorithm implemented in the IM program [39].\n\n\nAuthors' contributions\nCJM generate and analyzed all the data and drafted the manuscript. NAS and CAC collected and maintained sand fly samples. CPK helped to write the manuscript and supervised CJM during her stay in Leicester. AAP is the principal investigator, participated in its design and coordination, and helped to write the manuscript. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 24096 ] ] } ]

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[ { "id": "pmcA150219__text", "type": "Article", "text": [ "Rap1p and other transcriptional regulators can function in defining distinct domains of gene expression\nAbstract\nBarrier elements that are able to block the propagation of transcriptional silencing in yeast are functionally similar to chromatin boundary/insulator elements in metazoans that delimit functional chromosomal domains. We show that the upstream activating sequences of many highly expressed ribosome protein genes and glycolytic genes exhibit barrier activity. Analyses of these barriers indicate that binding sites for transcriptional regulators Rap1p, Abf1p, Reb1p, Adr1p and Gcn4p may participate in barrier function. We also present evidence suggesting that Rap1p is directly involved in barrier activity, and its barrier function correlates with local changes in chromatin structure. We further demonstrate that tethering the transcriptional activation domain of Rap1p to DNA is sufficient to recapitulate barrier activity. Moreover, targeting the activation domain of Adr1p or Gcn4p also establishes a barrier to silencing. These results support the notion that transcriptional regulators could also participate in delimiting functional domains in the genome.\n\nINTRODUCTION\nThe eukaryotic genome is organized into discrete functional domains in which gene expression is either permitted or repressed. Domains permissive for gene expression are usually defined as euchromatin or active chromatin, and domains that repress gene expression heterochromatin or silent chromatin. The HML and HMR loci in Saccharomyces cerevisiae are silent chromatin domains (1). Compared to active chromatin, silent chromatin is more compact and its histone components are hypoacetylated (2). The SIR complex consisting of Sir2p through Sir4p is an integral part of yeast silent chromatin. Sir3p and Sir4p interact with the N-terminal tails of histones H3 and H4 providing the structural basis of silent chromatin (1). Sir2p was recently found to be an NAD-dependent histone deacetylase and was suggested to be responsible for histone hypoacetylation in silent chromatin (3). Silent chromatin is initiated at small cis-acting DNA elements known as the E and I silencers flanking each of the HM loci (4).\nIncreasing evidence indicates that histone deacetylation is key to the propagation of silent chromatin (4). In vertebrates and fission yeast, spread of silent chromatin involves a chain of events of histone H3 deacetylation → H3 methylation → binding of methylated H3 by HP1 (heterochromatin protein 1) or swi6 (4). In S.cerevisiae, there is evidence that Sir3p (hence the SIR complex) has much higher affinity to unacetylated histone H4 than acetylated H4 (5). Based on this and the fact that Sir2p is a histone deacetylase, a refined model for the propagation of silent chromatin can be proposed. In this model, Sir2p recruited to the silencer deacetylates histones in an adjacent nucleosome enabling it to bind another SIR complex with high affinity. The nucleosome-bound SIR complex in turn deacetylates the neighboring nucleosome allowing it to recruit a new SIR complex. In this manner, the SIR complex promotes its own propagation along an array of nucleosomes (4).\nThe fact that transcriptionally silent chromatin can encroach upon active chromatin poses the question of how interspersed domains of silent and active chromatin are demarcated. Studies of the Drosophila and vertebrate genomes demonstrated that some domains are delimited by boundary or insulator elements (6). These elements are specialized DNA sequences that act as barriers to enhancers and/or silent chromatin. One of the best characterized insulators is the chicken HS4 insulator at the β-globin locus. Histones surrounding this insulator are hyperacetylated indicating the presence of histone acetyltransferase (HAT) activity at the insulator (6). Recently, sequences that could block the spread of silent chromatin have been discovered in S.cerevisiae (7,8). These sequences (referred to as silent chromatin barriers) include sub-telomeric anti-silencing regions (STARs), the right boundary of HMR, and the upstream activating sequence (UAS) of the highly expressed TEF2 gene (9–11). The mechanisms underlying barrier functions in yeast are not known.\nTEF2-UAS contains three tandem repressor activator protein 1 (Rap1p)-binding sites that are necessary and sufficient for its barrier function (11). Rap1p is an abundant sequence-specific DNA-binding protein (12). Variants of the 13 bp consensus sequence for Rap1p binding (ACACC CRYACAYM) (13) lie not only in the UASs of numerous genes but also in the silencers of the HM loci and in telomeric C1–3A repeats. Accordingly, Rap1p functions as a global regulator of transcriptional activation and repression, silencing, as well as telomere length (12). Rap1p performs these diverse functions by interacting with different factors in respective contexts. As an activator, Rap1p binds to the promoters of 362 ORFs (13) and can function via at least three mechanisms. First, Rap1p binding can ‘open up’ local chromatin structure at a promoter to help another activator to bind (14). Secondly, Rap1p bound to DNA can help Gcr1p bind to an adjacent site in the promoter through physical interaction (15). Thirdly, Rap1p is involved in the recruitment of the NuA4 HAT complex and TFIID to ribosome protein genes (RPGs) (16,17). As a silencing factor, Rap1p binds to silencers or telomeric repeats and recruits Sir3p/Sir4p through direct interactions (18). Rap1p also interacts with telomere-specific proteins in executing its role in regulating telomere length (19). It is puzzling that a protein that helps establish silent chromatin could also act as a barrier to its propagation.\nIn this study, we found that in addition to TEF2-UAS, UASs of many yeast genes exhibit barrier activity. Analyses of these UASs indicate that binding sites for transcriptional regulators Abf1p, Reb1p, Adr1p, Gcn4p, as well as Rap1p, may participate in barrier function. These UASs are dispersed across the genome and might play a role in defining functional chromosomal domains. Moreover, we demonstrated that targeting the activation domain of Rap1p, Adr1p or Gcn4p alone, led to barrier function. Since the activation domain of an activator was usually involved in interacting with co-activators and/or components of the transcriptional machinery, these data indicated that other factors might also be involved in barrier function.\n\nMATERIALS AND METHODS\nPlasmids and strains\nPlasmids 1–53 were used to make strains 1–53, respectively (see Figs 1–6). Plasmid 1 was previously named pMB22-a that contained a HindIII–BamHI fragment of chromosome III (coordinates 14838–16263) with a URA3 gene inserted at its EcoRV site (20). Plasmid 2 was derived from 1 by inserting TEF2-UAS (–511 to –407 bp relative to the translation start codon) at the SnaBI site. Plasmids 3–38 were identical to 2 except that different fragments from various promoters (see Figs 1 and 2) were inserted at the SnaBI site. Plasmid 29 had a sequence of chromosome III (295327–295713) containing the HMR-tRNA gene (21) inserted at the SnaBI site of plasmid 1. Plasmids 39 and 40 were previously named pYXB26 and pYXB59, respectively (11). Plasmid 41 was identical to pYXB59 except bearing mutations illustrated in Figure 3A. Plasmids 42–48 were previously described as pYXB29, 48, 28, 27, 59, 31 and 37, respectively (11). Plasmid 50 was derived from plasmid 1 by inserting two copies of a sequence bearing the consensus binding sequence of LexA (bold) (22), GGGGTACGTACTGTATGTACATACAGGATATCGGGG, at the SnaBI site. Plasmid 51 was identical to 50 except having only one copy of the LexA-binding sequence. Plasmid 52 was derived from pYXB26 (11) by inserting a SpeI-ADE2-AvrII sequence at the SpeI site. Plasmid 53 was derived from plasmid 52 by inserting TEF2-UAS (–511 to –407 bp relative to the translation start codon) at the SpeI site.\nPlasmid L1 (see Fig. 5A) was made by inserting LEU2 into pBTM116 (23) carrying the LexA gene flanked by the promoter and terminator of ADH1. L2 through L8 were derived from L1 by fusing various sequences to LexA (see Fig. 5A and B). Plasmid pRS425 (24) carried the 2 µm origin and LEU2.\nStrain YXB76 was MATa ura3-52 leu2-3,112 ade2-1 lys1-1 his5-2 can1-100, E-HMLα-Iinverted (20). Y2047b was MATa HMRa HMLα EΔ79–113::SUP4-o IΔ242 LEU2-GAL10-FLP1 ura3-52 ade2-1 lys1-1 his5-1 can1-100 [cir0] (25). Strain 1 was made by transforming YXB76 to Ura+ with HindIII + BamHI digested plasmid 1. Strains 2–38, 50 and 51 were similarly made with corresponding plasmids, respectively (e.g. strain 2 was made with plasmid 2). Strains 39, 40 and 42–48 were previously described as YXB26, 48, 29, 48, 28, 27, 59, 31 and 37, respectively (11). Strains 41, 52 and 53 (see Figs 3C and 6) were made by transforming Y2047b to canavanine-resistant by BamHI-digested plasmids 41, 52 and 53, respectively. Strains 40′–48′ were sir3::URA3 derivatives of strains 40–48, respectively. The relevant genotypes of all the strains were confirmed by Southern blotting.\n\nQuantitative mating assay\nQuantitative mating was performed as described (11).\n\nElectrophoresis mobility shift assay (EMSA)\nRap1p was expressed in Escherichia coli BL21 (DE3) from pL3S5, a pET vector carrying the RAP1 gene downstream of a T7 promoter (26). Crude protein extracts were prepared from 40 ml cultures harvested after 3 h of IPTG induction and resuspended in 1 ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). A control extract was prepared from a culture without IPTG induction. Western blotting revealed that Rap1p protein was present in the induced extract but not the uninduced one (data not shown). DNA fragments of 26 bp in length (see Fig. 3A) were end-labeled with 32P-phosphate. Fifteen microliter binding reactions were prepared, each consisting of 20 000 c.p.m. radiolabeled DNA probe, 5 µg of crude protein extract, non-specific competitor DNAs (0.3 µg of yeast tRNA, 0.3 µg of salmon sperm DNA and 0.5 µg of E.coli DNA), 10 µg of BSA, 10 mM Tris–HCl (pH 8.0), 10 mM MgCl2 and 8% glycerol. The reactions were incubated at 25°C for 20 min and loaded onto non-denaturing polyacrylamide gels (4–5%). Gels were run at 40 mA for 1 h in 0.5× TBE buffer.\n\nAnalysis of the supercoiling of DNA circles\nCells were grown in YPR (yeast extract + bacto peptone + 2% raffinose) to early log phase. Galactose (2%) was then added to the culture to induce the expression of FLP1. After 2.5 h of incubation, nucleic acid was isolated using the glass bead method and fractionated on agarose gels with 30 µg/ml chloroquine. DNA circles were detected by Southern blotting.\n\n\nRESULTS\nUASs of many highly expressed genes can function as barriers to the spread of silencing\nWe have previously shown that TEF2-UAS was able to block the spread of silencing in a silencer-blocking assay (11). Such an assay tested if a sequence had the ability to prevent a silencer from silencing a reporter gene when it was positioned between the silencer and the reporter. In this report we used a new silencer-blocking assay to identify more barrier elements. This assay employed the URA3 gene and an inverted HML-I that could silence sequences to the right of HML (20) (Fig. 1, strain 1). URA3 expression makes cells sensitive to 5-fluoroorotic acid (FOA) thus URA3 silencing could be measured by cell growth on FOA medium (Fig. 1, strain 1). As expected, TEF2-UAS and another known barrier element, HMR-tRNA gene (21) exhibited barrier activity in this assay as indicated by the lack of silencing of URA3 in strains 2 and 29 (Fig. 1).\nTEF2 encoding translation elongation factor 1α is one of the most highly expressed genes in yeast (27). Other highly expressed genes include ribosome protein genes (RPGs) and genes coding for glycolytic enzymes (27). The coordinated regulation of most of these genes requires the so-called general regulatory factors Rap1p, Abf1p and Reb1p (28,29). Using the above silencer-blocking assay, we tested if the UASs of other highly expressed genes could also block URA3 silencing (Fig. 1). Seventeen new barrier elements were identified from a total of 26 UASs tested. These 17 elements were UASs of eight RPGs (see Fig. 1, strains 3–10), eight glycolytic genes (strains 18–25) and the HIS3 gene (strain 26). All 17 elements except ADH2-UAS and HIS3-UAS exhibited very strong barrier activity as evidenced by the absence of URA3-silencing in strains 3–10 and 18–24 (Fig. 1).\nSeven of the eight RPG-UASs that had barrier activity, contained a single or a pair of tandem Rap1p-binding sites (Fig. 1, strains 3–9). Three of them also contained an Abf1p or Reb1p site (strains 6, 8 and 9). One barrier RPG-UAS (strain 10) had only a predicted Abf1p site. Of the seven RPG-UASs that had no barrier activity (Fig. 1, 11–17), only two bore no site for Rap1p, Abf1p or Reb1p (11 and 12). The remaining five each contained one to three Rap1p sites (strains 13–17). RPL15A-UAS (17) also contained an Abf1p site. For most of the 15 RPG-UASs tested (3–17), the presence or absence of predicted Rap1p sites correlated with the presence or absence of Rap1p association in vivo as previously examined by chromatin immunoprecipitation assays (13). Exceptions were the UASs of RPS24B, RPS28A and RPL15A (13).\nOf the eight UASs of glycolytic genes tested (Fig. 1, strains 18–25), seven exhibited strong barrier activity (18–24) and one had weaker but detectable barrier activity (strain 25). Glycolytic genes are among the most highly expressed genes and their high expression depends on the functions of Rap1p, Abf1p or Reb1p as well as Gcr1p (29,30). The seven strong glycolytic barriers all consist of one or two Rap1p sites plus one or two Reb1p or Abf1p sites (Fig. 1, strains 18–24). In addition, one or more Gcr1p-binding sites are found adjacent to each Rap1p site. It was proposed that the function of Rap1p was to facilitate the binding of Gcr1p to the UAS (15). Abf1p or Reb1p was shown to play a role similar to that of Rap1p in activating glycolytic genes (31–33).\nAlthough the above data reinforced the notion that certain Rap1p-binding UASs could serve as barriers to silencing, they raised new questions about the essential components of a barrier element. For example, some of the UASs that contained only a single Rap1p site had barrier activity (e.g. RPL19B-UAS, strain 3) whereas others that contained two or three sites had no activity (e.g. RPL39, strain 16). One explanation was that sequences flanking the Rap1p site(s) in a particular UAS could positively or negatively influence barrier function. Moreover, the presence of Abf1p or Reb1p sites in 11 of the 17 new barriers made us wonder if they also participated in barrier function. We began to address these issues by analyzing three new barrier elements (Fig. 2). RPS10A-UAS containing an Abf1p-binding site and a pair of Rap1p sites was divided into three fragments that were tested in a silencer-blocking assay (Fig. 2, strains 30–32). It was clear that only the fragment containing Rap1p sites functioned as a barrier (strain 31). TPI1-UAS bearing a Reb1p site and a Rap1p site was divided into two parts with one containing the Rap1p site (Fig. 2, strain 34) and the other Reb1p site (strain 33). Neither of these fragments alone retained barrier activity indicating that concerted action of Reb1p and Rap1p was required for barrier activity. Duplicating the Reb1p-bearing part of TPI1-UAS didn’t restore barrier activity (strain 35). However, triplicating the Rap1p-containing part did re-create barrier function (strain 36). RPS28A-UAS bore, in addition to a Rap1p site, an Abf1p site and an adjacent T-rich region that are required for efficient transcription of RPS28A (34). A sequence of RPS28A-UAS deleted for a fragment bearing the Rap1p site lost the barrier activity (Fig. 2, strain 37). However, duplicating this sequence restored barrier activity (strain 38). The above results indicated again that Rap1p sites could form barriers (strains 31 and 36). On the other hand, they also suggested that synergistic actions of Abf1p sites (strain 38), Reb1p site + Rap1p site (strain 18), or Abf1p site + Rap1p site (strain 16) could all lead to barrier function. This was not very surprising since Rap1p, Abf1p and Reb1p could perform similar and sometimes interchangeable functions in gene regulation (16,35,36). Note, however, we haven’t ruled out the possibility that other factors (e.g. Gcr1p) or structure features of DNA (e.g. T-tracks) were also involved in barrier function.\nIt was interesting that ADH2-UAS and HIS3-UAS bearing no site for Rap1p, Abf1p or Reb1p exhibited detectable barrier activity (Fig. 1, strains 25 and 26). However, binding sites for other transcriptional activators existed in these UASs. The major regulator that binds to ADH2-UAS is Adr1p (37). On the other hand, Gcn4p binds to multiple sites in HIS3-UAS and activates HIS3 transcription (38). The possible involvement of Adr1p or Gcn4p in barrier function was examined below. Note Adr1p function was subject to glucose repression (37), which might explain why ADH2-UAS only exhibited limited barrier activity since glucose was used in the media used in our assay.\n\nEffect of mutating Rap1p-binding sites in TEF2-UAS on barrier function\nAlthough we had shown that the three Rap1p-binding sites in TEF2-UAS were necessary and sufficient for its barrier activity (11), we had not shown if binding of Rap1p to these sites was required. To address this we performed mutational analysis of TEF2-UAS. As shown in Figure 3A and B, the three Rap1p-binding sites in TEF2-UAS (designated as R1, R2 and R3) were all able to bind Rap1p in an EMSA (Fig. 3B). Single or double C→A mutations were introduced into R1, R2 and R3 resulting in m1, m2 and m3, respectively (Fig. 3A). The m2 and m3 sites had both lost the ability to bind Rap1p, whereas m1 still bound Rap1p (Fig. 3B), which could be predicted from the specific mutations in each site (Fig. 3A, compare m1, m2 and m3 to the consensus sequence). We then tested a mutated TEF2-UAS containing m1, m2 and m3 in a silencer-blocking assay using the HML-E silencer and the HMLα genes (Fig. 3C). In this assay, the HML-I silencer was deleted but the HML-E silencer was sufficient to silence the HMLα genes (Fig. 3C, strain 39) (11). Silencing in strain 39 (a-type) was measured by its ability to mate with an α-type strain (Fig. 3C). As predicted, the wild-type TEF2-UAS blocked HMLα silencing (11) (Fig. 3C, strain 40). On the other hand, the mutated TEF2-UAS containing m1, m2 and m3 had lost barrier activity (Fig. 3C, compare the mating efficiencies of strains 41 and 40). Therefore, mutations that prevented Rap1p from binding two of the three sites in TEF2-UAS abolished its barrier activity, indicating that association of Rap1p with TEF2-UAS at more than one site was required for barrier function.\n\nThe barrier TEF2-UAS alters local chromatin structure\nIn eukaryotic cells, the topology of local DNA reflects the chromatin structure in which it resides. We had previously developed a DNA topology-based assay to examine chromatin structure in vivo, which involved excising a chromosomal region of interest from its genomic location as a circle using site-specific recombination (39). Using this strategy, we demonstrated that DNA in silenced chromatin was more negatively supercoiled than that in active chromatin (39). In this report we used the topology assay to address if barrier elements altered local chromatin structure as Drosophila or chicken insulators did (40,41).\nVarious sequences (designated X) from UASs of TEF2, AgTEF (TEF gene from Ashbya gossypii) and ADE2 were inserted between HML-E and HMLα at HMLΔI (HML locus deleted for the I silencer) flanked by the FRT sites for the Flp1p site-specific recombinase (Fig. 4A and B). The FLP1 gene was under the control of the inducible GAL10 promoter (39). The mating efficiency of each strain measured silencing of HMLα (Fig. 4C). In strains 40, 42, 44 and 47, silencing was restricted to the left of the X insertions and HMLα was derepressed (Fig. 4C). Consistently, the proportion of silent chromatin in the region flanked by the FRT sites in each of these strains was decreased, as indicated by the reduced negative supercoiling of the excised circle (11) (data not shown). Note that the above effect of a barrier on HML chromatin reflected both change in the scope of silencing caused by the barrier activity and alteration (if any) in chromatin structure at the barrier independent of the silencing state of the locus. To exclusively examine the latter, we analyzed the topology of HML DNA in a silencing-deficient background. The SIR3 gene in strains described in Figure 4B was disrupted rendering them defective in silencing (strains 40′–48′ in Fig. 4D). Recombination mediated by Flp1p in these strains led to the excision of a group of chromosomal circles that differed only in the X insertion. The supercoiling of these circles was analyzed as a function of the length of the insertion (Fig. 4D). If an insertion simply lengthened a circle without causing abnormal changes in nucleosomal structure and/or density, the topoisomers of this circle would migrate slower relative to those of a similar circle with a smaller size. Otherwise, migration of the topoisomers would deviate from that predicted based on the size of the circle. As shown in Figure 4D, the circle from strain 40′ bearing a 104 bp barrier element migrated faster rather than slower than the circle from strain 46′ bearing a 91 bp non-barrier element. Circles bearing other barrier elements also exhibited unexpected faster migration (compare strains 44′ to 45′, 47′ to 43′, 42′ to 48′, respectively). Therefore, these data indicated that barrier function correlated with altered topology of local DNA. This was further demonstrated by that mutations in TEF2-UAS that abolished its barrier activity also abolished its effect on DNA topology (Fig. 4D, compare 41′ to 40′; note that circles from both strains had the same size). Since in our gel assay more negatively supercoiled DNA migrate slower, we concluded that every fragment with barrier activity caused a reduction in negative supercoiling of approximately one to two turns in HMLΔI DNA (Fig. 4D, compare the centers of distributions of topoisomers in pairs of lanes, e.g. 40′ and 41′).\nThe topology of eukaryotic DNA reflects mainly the wrapping of DNA into nucleosomes (approximately one negative superhelical turn is associated with each nucleosome) (42). Therefore, removal of one nucleosome would eliminate approximately one turn. On the other hand, histone acetylation can reduce negative supercoiling associated with a nucleosome by approximately 0.2 turns (43). Therefore, acetylating five nucleosomes would reduce negative supercoiling by approximately one turn. Consequently, the linking number change of one to two brought about by TEF2-UAS can be accounted for by either the loss of one to two nucleosomes or acetylation of five to 10 nucleosomes. Further experiments are under way to clarify the nature of change in chromatin induced by a barrier.\n\nTargeting the transcription activation domain of Rap1p recapitulates its barrier activity\nThe multifunctional Rap1p can be divided into at least four functional domains (Fig. 5A) (12). We were interested in defining which of these domains might be involved in the barrier activity of Rap1p. To this end, we fused various parts of Rap1p to the bacterial LexA protein and tested if targeting any of the fusion proteins to LexA-binding sites could re-create a barrier to silencing (Fig. 5A). The LexA-RAP1 fusion genes were carried on 2 µm based plasmids marked by LEU2 (designated L1–L6, Fig. 5A). When introduced into strain 50 in which two LexA-binding sites had been inserted between the inverted HML-I and URA3, LexA alone had no effect on URA3 silencing (Fig. 5A, plasmid L1). Targeting the DNA binding domain, DNA bending domain, silencing domain, or the activating + silencing domain of Rap1p also did not affect URA3 silencing (L2–L5). On the other hand, targeting the activation domain alone recapitulated the barrier activity of Rap1p (L6). These results indicated that the activation domain of Rap1p was involved in barrier activity. The inability of the Rap1p-silencing domain to block URA3 silencing in our assay did not necessarily mean that this domain had no barrier function, since such a function might be suppressed by much stronger silencing activity associated with the silencing domain.\nWas the lack of barrier function of fusion constructs L2–L5 due to a lack of expression of these proteins? To address this possibility, we examined the levels of L1–L6 proteins as well as L7 and L8 proteins described in Figure 5B by western blotting using an antibody against LexA. As shown in Figure 5C, L5 was expressed at a level comparable to L6, and L4 was expressed at a higher level than L6. Therefore, the lack of barrier function of L4 and L6 was not the result of their lack of expression. On the other hand, the level of L2 or L3 was significantly lower than that of L6, hence the lack of barrier function of L2 and L3 might be due to their insufficient expression.\nAs an important control for the above targeting experiments, we showed that plasmids L1–L6 (Fig. 5A) as well as L7 and L8 (Fig. 5B) bearing LexA-fusion genes had no effect on URA3 silencing in strain 1 (Fig. 1) in which there was no binding site for LexA present between HML-I and URA3 (data not shown). Hence, the effect of the fusion proteins on URA3 silencing was the result of their targeting to specific loci, not the consequence of non-specific, indirect functions (if any) of these proteins.\n\nTargeted activation domain of Adr1p or Gcn4p can also act as a barrier factor\nWe have shown above that ADH2-UAS bearing Adr1p-binding sites and HIS3-UAS bearing Gcn4p sites both had detectable barrier activity (Fig. 1, strains 25 and 26). Given that tethering the activation domain of Rap1p is sufficient for barrier function, we tested if targeting the activation domain of Adr1p or Gcn4p could also establish a barrier. Adr1p contains four separate activation domains (TADI–TADIV) that can contact Ada2p, Gcn5p and/or TFIIB (45). TAD III (415–467) interacts with only Gcn5p but not Ada2p or TFIIB. We fused this domain of Adr1p and the activation domain of Gcn4p (102–149) to LexA, respectively (Fig. 5B, L7 and L8). Targeting these fusion proteins to two LexA-binding sites between HML-I and URA3 in strain 50 dramatically reduced URA3 silencing (Fig. 5B, compare L7 and L8 to L1). Therefore, like LexA-Rap1p (627–695), both LexA-Adr1p (415–467) and LexA-Gcn4p (102–149) also had barrier activity. Barrier function of these fusion proteins was significantly decreased in strain 51 in which only one LexA site was inserted between HML-I and URA3 (compare strains 50 and 51 carrying plasmid L7), indicating that the potency of such barriers was dependent on the amount of tethered fusion proteins.\n\n\nDISCUSSION\nWe have shown in this report that UASs of many highly expressed genes could function as barriers to the spread of transcriptional silencing. These newly identified barrier elements together with previously described barrier elements (STARs, HMR-tRNA gene, TEF2-UAS and CHA1-UAS) (9–11,21) are scattered across the yeast genome and may play a role in dividing the genome into functional domains. Analyses of the barrier UASs suggest that binding sites for transcriptional regulators Rap1p, Abf1p, Reb1p, Adr1p and Gcn4p can participate in barrier function. Barrier activity often requires concerted actions of more than one factor-binding site. Our mutational analysis of TEF2-UAS suggests a direct involvement of Rap1p in its barrier function. It is noteworthy that not all UASs of highly expressed genes have barrier activity. Many non-barrier UASs also consist of multiple regulator binding sites, therefore other unknown factors might determine whether a UAS could act as a barrier.\nHow Rap1p and the other transcriptional regulators act to block silencing is not clear. Since they can all act as transcriptional activators, one natural explanation is that they may overcome the silencing effect of a silencer and directly activate the reporter gene. However, it has been well documented that Rap1p and the other transcriptional regulators usually act at relatively short distances (less than ∼600 bp upstream of the translation start codon) (28,29,46), but the binding sites for these regulators were >800 bp from the translation start codon in most of the silencing-blocking tests in this report (Figs 1 and 2). In the test shown in Figure 6, TEF2-UAS blocked ADE2 silencing when it was ∼2 kb downstream from the translation start codon of the gene. Moreover, Fourel et al. (44) demonstrated that the barrier function (or anti-silencing function) of an activator was independent of its role in activating transcription. Therefore, it is unlikely that the ability of a UAS to prevent gene silencing is due to its direct activation of the gene. In light of this, our results are in agreement with the notion that transcriptional regulators can also participate in defining the boundaries of functional chromosomal domains.\nA few models have been proposed for the barrier functions of transcriptional regulators. One model proposed that binding of a regulator to a barrier led to the formation of a nucleosome-free region thereby disrupting a continuous nucleosome array (11). Since the SIR complexes spread by self-interaction and interacting with adjacent nucleosmes in a stepwise fashion (4), a nucleosome-free gap could present a barrier to the spreading SIR complex. This ‘nucleosome gap’ hypothesis is consistent with that certain barrier factors such as Rap1p and Reb1p could indeed prevent nucleosome formation around their binding sites (14) and the barrier function of TEF2-UAS correlated with its ability to alter local chromatin structure (this report). In fact, many insulator/boundary elements in higher organisms have also been shown to alter local chromatin structure. For instance, the chicken β-globin insulator was linked to changes in nucleosome positioning, and the Drosophila scs and scs′ boundary elements are associated with unusual chromatin structures that are hypersensitive to nucleases (40,41). However, it is not clear whether any chromatin change associated with barrier function in yeast or insulator function in higher cells is the cause or effect of the function.\nAnother model for barrier action proposed by R. Kamakaka and colleagues (21) proposed that barrier factors recruit chromatin modifying and/or remodeling complexes to counteract the more compact, hypoacetylated silent chromatin. In agreement with this hypothesis, targeted HATs SAS2, GCN5 and ESA1 all had barrier activity (21; Y.-H. Chiu, Q. Yu and X. Bi, unpublished results). Interestingly, Rap1p, Adr1p, Gcn4p and Abf1p share the ability to recruit HAT complexes NuA4 and/or SAGA to target genes (16,45,47–49). Therefore, these factors that are capable of binding to the newly identified barriers may function as barrier factors by recruiting HAT complexes. In fact, recruitment of HAT activity has also been suggested to be the mechanism underlying the function of the chicken β-globin insulator (6), although it was not known what HAT might be involved. Our demonstration that tethering the activation domain of Rap1p, Adr1p or Gcn4p alone recapitulates barrier activity is consistent with the above HAT model for barrier function, since at least the activation domain of Adr1p has been shown to directly interact with HAT complexes (45). However, we haven’t ruled out the possibility that the barrier function of the targeted activation domain of Rap1p, Adr1p or Gcn4p is not related to the barrier function of the native protein. Note that the ‘nucleosome gap’ model and the HAT model are not mutually exclusive since the nucleosome gap could result from destabilization of nucelosomes caused by HAT function. A recent intriguing study showed that targeting proteins of the nuclear pore complex (NPC) could also establish a boundary for silent chromatin, and that tethering to NPC was required for boundary activity (50). This led to a revisit to the looping model for the function of boundary/insulator elements (51). However, an alternative explanation was that the NPC compartment could simply be rich in transcriptional activating factors or poor in silencing factors like the SIR proteins (50).\nIn summary, results from this and other studies indicate that factors previously identified as positive and/or negative regulators of gene activation could also function in demarcating distinct domains of gene activity. How these factors carry out their boundary function is not resolved but recruitment of other factors (e.g. chromatin modifying complexes) is likely to be involved.\n\nNOTE ADDED IN PROOF\nAfter this work was completed and submitted for publication the first time, Fourel et al. (52) reported that targeted activation domains of Rap1p and Abf1p had insulating capacity.\n\n\n" ], "offsets": [ [ 0, 33131 ] ] } ]

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[ { "id": "pmcA2562362__text", "type": "Article", "text": [ "Host immunity in the protective response to vaccination with heat-killed Burkholderia mallei\nAbstract\nBackground\nWe performed initial cell, cytokine and complement depletion studies to investigate the possible role of these effectors in response to vaccination with heat-killed Burkholderia mallei in a susceptible BALB/c mouse model of infection.\n\nResults\nWhile protection with heat-killed bacilli did not result in sterilizing immunity, limited protection was afforded against an otherwise lethal infection and provided insight into potential host protective mechanisms. Our results demonstrated that mice depleted of either B cells, TNF-α or IFN-γ exhibited decreased survival rates, indicating a role for these effectors in obtaining partial protection from a lethal challenge by the intraperitoneal route. Additionally, complement depletion had no effect on immunoglobulin production when compared to non-complement depleted controls infected intranasally.\n\nConclusion\nThe data provide a basis for future studies of protection via vaccination using either subunit or whole-organism vaccine preparations from lethal infection in the experimental BALB/c mouse model. The results of this study demonstrate participation of B220+ cells and pro-inflammatory cytokines IFN-γ and TNF-α in protection following HK vaccination.\n\n\n\nBackground\nBurkholderia mallei, the etiologic agent of glanders, is a gram-negative, capsulated, non-motile, facultative intracelluar bacterium. Most known members of the Burkholderiaceae are resident in the soil; however, B. mallei is thought to be an obligate mammalian pathogen. Horses are highly susceptible to infection and considered the natural reservoir for infection, although mules and donkeys are also susceptible [1]. Clinically, glanders in solipeds can present as either a chronic (horses) or acute (mules and donkeys) form. Naturally acquired human infection with B. mallei, although not seen in the United States since 1945, has occurred rarely and sporadically among laboratory workers and those in direct contact with infected animals [2]. However, glanders is endemic among domestic animals in Africa, Asia, the Middle East, and Central and South America. The course of infection is dependent on the route of exposure. Direct contact with the skin can lead to a systemic infection. Inhalation of aerosol or dust containing B. mallei can lead to septicemic, pulmonary, or chronic infections of the muscle, liver and spleen. The disease has a 95% case fatality rate for untreated septicemic infections and a 50% case fatality rate in antibiotic-treated individuals [3].\nThere is no human or animal vaccine available for glanders, and development of a partial or fully protective adaptive host response to the organism has not been well-defined. Previous studies with B. mallei and the host response have shown that a mixed immune response consisting of both Th1 and Th2-associated cytokines with a predominant IgG1 subclass does not correlate with protection [4]. Additional studies with passive transfer of monoclonal antibodies specific for B. mallei have correlated with early protection from infection [5]. Recent studies have also shown the Th1 cytokine IL-12 to mediate partial protection to non-viable B. mallei-vaccinated mice [6]. Thus, full correlates of protection mediated by the adaptive immune system against B. mallei remain to be fully elucidated.\nIn this series of studies, we sought to address the impact of depletion of the major effector lymphoid cell populations (B220+ B cells, CD4+ or CD8+ T cells) and key pro-inflammatory/Type 1 cytokines (IFN-γ or TNF-α) on survival in BALB/c mice vaccinated with heat killed (HK) bacilli followed by an intraperitoneal (i.p.) challenge with live organism. In addition, studies investigating the effect of complement on opsonization of organism and antibody production were assessed. Heat killed bacteria were used as a model of vaccination to allow evaluation of B. mallei specific immune responses. The results of this study demonstrate participation of B220+ cells and pro-inflammatory cytokines IFN-γ and TNF-α in protection following HK vaccination.\n\nResults\nHeat-killed B. mallei vaccination mediates partial protection from lethal challenge\nTo begin to address this issue in an animal model of acute infection, we established that immunologically naive BALB/c mice challenged i.p. with 2 × 107 CFU resulted in death by day 4–6, while i.p. immunization with 1 × 105 heat killed (HK) bacteria provided partial protection against a subsequent challenge. Two independent experiments resulted in similar findings of 40% survival for HK-vaccinated mice with a mean survival time (MST) of 8 days versus 4 days in naïve mice (Fig. 1). The administration of vaccines for B. mallei during an outbreak would mandate relatively rapid onset of protection for human or veterinary use. Based on non-routine use and vaccine implementation in the course of an outbreak, a 14 day window was chosen for assessment of protection. Our results indicate that HK vaccination can afford partial protection to an otherwise lethal challenge of B. mallei by the i.p. route.\n\nEffects of cell depletion on HK-vaccinated survival\nTo dissect the cellular basis for protection mediated by HK vaccination, 13 days after immunization with HK bacteria (day -1), and at day of challenge, mice were dosed with antibodies to deplete CD4+, CD8+ or B220+ cells. Antibody depletion of CD4+, CD8+, or B220+ cells in these mice was confirmed by flow cytometric analysis with depletion efficiencies for CD4, CD8, and B220 populations at 99.7%, 96%, and 95%, respectively, relative to mice treated with isotype control monoclonal antibodies (data not shown). Our results demonstrated decreased survival rates in B220 (p = 0.3418), CD4+ (p = 0.5417) and CD8+ (p = 0.4684) antibody depleted mice, compared to isotype control antibody, a finding that indicated a possible role for vaccine induced antibody production. When challenged with 2 × 107 CFU/mouse by the i.p. route, loss of T cells resulted in reduced survival (50%) relative to the non-specific isotype control (Fig. 2). In contrast to the loss of T cells, depletion of B220+ cells resulted in 100% mortality relative to the non-specific isotype control (Fig. 2). To further evaluate the necessity of these effector cells in providing protection following HK vaccination, relatively resistant C57BL/6 mice, deficient in mature B-cells (μMT), CD4 T-cells (CD4-/-) or CD8 T-cells (CD8-/-) were subjected to an identical HK vaccination and challenge regimen. Mature, B-cell-deficient mice demonstrated a 50% decreased survival (p = 0.0888) compared to the wild-type mice with an MST of 35.5 days (Fig. 3). CD4-/- and CD8-/- mice exhibited a 60% (p = 0.1343) and 0% reduced survival, respectively (Fig. 3).\n\nEffects of cytokine depletion on HK vaccination\nSimilar studies were performed to determine the role of IFN-γ or TNF-α in acute infection in BALB/c mice immunized with HK bacteria. Six hours before challenge, mice were dosed with antibodies that neutralize IFN-γ or TNF-α. Individual depletion of either TNF-α (p = 0.0145) or IFN-γ (p = 0.0446) resulted in 100% mortality with an MST of 3 and 2 days, respectively, compared to the HK-vaccinated isotype control mice (Fig. 4). In contrast, 40% of HK-vaccinated, isotype control mice survived to at least 12 days post-challenge (Fig 4). To further evaluate the host TNF-α response during an established B. mallei chronic infection, we infected 12 BALB/c mice by the i.p. route with 1 × 106 CFU B. mallei. One animal was terminally ill on day 37 post-infection. On day 42 post-infection, the remaining 11 mice were dosed with either anti-TNF-α (n = 6), or control mAb (AFRC Mac 49) (n = 5). No further deaths were observed in the control mAb-treated mice. Rapid mortality was observed in the anti-TNF-α-treated group, with all mice dying within 7 days of treatment (p = 0.0023) relative to the isotype-treated controls (Fig. 5).\n\nJ774A.1 uptake of serum treated B. mallei\nComplement mediated uptake assays were performed to evaluate opsonization. Results indicated enhanced bacterial uptake in J774A.1 phagocytes inoculated with serum treated B. mallei (p = .0082), compared to B. mallei alone, while heat-inactivated serum produced uptake percentages similar to those prior to serum addition (Fig. 6). Taken together, these results imply an active role for complement components in the uptake of organism by macrophages.\n\nImmunoglobulin production in HK vaccinated BALB/c mice\nWe further characterized the ability of HK vaccination to induce a predominant IgG isotype by determining IgG2a/IgG1 ratios in i.p. and i.n. vaccinated BALB/c mice. Pre (day 14 post vaccination) and post (day 2 post infection) exposure serum samples were obtained and evaluated for IgG isotype concentrations (Table 1). No appreciable differences in IgG pre-exposure levels were seen when comparing i.n. to i.p. vaccination. In addition, cobra venom factor-treated animals showed no significant differences to non-cobra venom factor-treated animals in IgG pre-exposure (challenge) levels. Conversely, isotype switching in the cobra venom factor treated animals was enhanced in post-exposure serum IgG2a (Table 1).\n\n\nDiscussion\nRecent studies have shown a key role in protection from lethal challenge for IFN-γ in non-vaccinated mice from either NK and/or NKT cells following experimental exposure to B. mallei and B. pseudomallei [7,8]. A similar protective role in the innate response to infection has been demonstrated for TNF-α in B. pseudomallei infection [8]. The studies presented here are consistent with the essential role of these factors in the relative levels of protection conferred by vaccination with heat-killed B. pseudomallei and would appear to be viable early markers for protection from lethal acute infection [9]. Currently, there are no fully protective vaccines against B. mallei or B. pseudomallei in a murine model, particularly for the sensitive BALB/c versus C57BL6 models. Previous studies have also demonstrated that both the humoral and cell-mediated arms are essential for protection from B. pseudomallei infection [10]. Thus, loss or reduction of TNF-α and IFN-γ levels result in significantly reduced survival rates, substantiating previous reports of the role of these factors in protection against B. mallei [7]. Moreover, we demonstrate a role for sustained TNF-α production in the maintenance of host survival throughout the course of B. mallei infection. Mice with an established B. mallei chronic infection rapidly lost the ability to control the growth of the bacillus upon neutralization of TNF-α. This would suggest a potential role for TNF-α in the maintenance of productive granulomas which may limit the spread of bacteria in chronically infected hosts, or, alternatively, in direct or indirect microbicidal or bacteriostatic activities at the sites of infection. Additional studies are underway to determine more precisely the role of TNF-α in host protection to B. mallei.\nMultiple innate and adaptive cell types may contribute to the production of IFN-γ in response to infection with B. mallei following vaccination. Our results with individual depletion of CD4+ and CD8+ T cells suggests that both cell types may compensate for the functional loss of the other effector cell type in the production of this key cytokine. The effector role for IFN-γ in mediating protection against B. mallei may include both immunoregulatory and non-regulatory functions. Regardless, the requirement of IFN-γ, as demonstrated by administration of neutralizing antibody prior to infection, indicates that stimulation of IFN-γ response is a desirable goal for a B. mallei vaccine.\nSimilarly, B220-positive cells appear to play a role in protection following vaccination with heat-killed B. mallei. Interestingly, this protective immunity, occurring in other intracellular pathogens, is not exclusively dependent on B cells [11]. Passive protection has been demonstrated against acute Burkholderia infection by monoclonal antibodies [5,12]. Protection against B. pseudomallei infection by anti-LPS, capsular polysaccharide and proteins has been short-lived, suggesting that antibody production offers limited protection in the initial stages of infection by an as-yet-undefined mechanism [12]. We have shown that following depletion of B220+ cells, survival rates decreased as much as 100% relative to non-depleted controls and individual CD4/CD8-depleted mice via the intraperitoneal route. Results from C57BL/6 mice deficient in mature B-cells (μMT), CD4 T-cells (CD4-/-) or CD8 T-cells (CD8-/-) substantiate the requirement for B-cell involvement by evidence of μMT and CD4-/- decreased survival. The lack of an effective CTL response to vaccination did not appear to alter survival in what would appear to be a CD4/B-cell (humoral)-driven response. In CD4-deficient mice, we have the additional potential variable that a CD4-dependent antibody response might also be inhibited during the vaccination phase relative to mice treated with antibody immediately prior to and during the early phases of infection. Although not statistically significant, we did observe a decrease in survival in μMT (mature B cell) deficient mice as early as day 9 post challenge, whereas CD4-deficient mice produced similar results at day 32 post challenge, indicating a role for B cells independent of CD4 T cell help, perhaps through a T-independent mechanism of antibody production. Although CD8-/- C57BL/6 demonstrated no decreased survival in our HK-vaccinated model, a lack of potential endogenous protein production by HK B. mallei may have contributed to limited MHC-I presentation.\nComplement associated studies revealed increased J774A.1 uptake of serum-treated B. mallei. Complement-mediated uptake studies of B. pseudomallei by polymorphonuclear leukocytes (PMNs) suggest that capsule production contributes to resistance of phagocytosis by reducing C3b bacterial deposition [13]. Previous studies have demonstrated that a polysaccharide capsule is present in B. mallei, [14,15] although in the present study enhanced uptake with serum-treated B. mallei was observed. Intracellular survival assays of complement mediated uptake of organisms were not performed in the present study, thus, the role of complement opsonization on intracellular survival is not fully resolved. Previous reports have demonstrated the ability of B. mallei to survive within macrophage without the aid of serum coating organisms [16]. Conversely, the idea of antibody mediated opsonization to facilitate macrophage activation and clearance of intracellular organisms may offer support to the role of B cells in an effective immune response. A possible protective mechanism may include HK vaccination induced production of opsonizing antibodies which may aid in complement mediated uptake, thereby limiting the initial bacterial threshold below a lethal level.\nImmunoglobulin responses to HK vaccination resulted in modest levels of IgG1 following 2 weeks post vaccination, while post-exposure levels were indicative of efficient class switching to a favorable IgG2a isotype. Importantly, cobra venom factor treatment of animals at time of vaccination did not alter their ability to produce immunoglobulin. In fact, cobra venom factor treated animals resulted in higher IgG2a levels when compared to non-treated. Complement activation can modulate both the primary and secondary immune responses and has been shown to enhance secondary immune responses to vaccination [17]. The current results suggest that cobra venom factor treatment may affect the modulation of the immune response to B. mallei infection through B cell activation and/or memory B cell generation.\n\nConclusion\nIn summary, our results provide a basis for future studies of protection via vaccination using either subunit or whole-organism vaccine preparations from lethal infection in the experimental BALB/c mouse model. Understanding and defining the role of B cells in adaptive B. mallei immunity will likely be fundamental to the design of an efficacious vaccine and important goals of future research.\n\nMethods\nBacterial strain and mice\nB. mallei strain ATCC 23344 (China 7) was cultured on Luria-Bertani agar supplemented with 4% glycerol (LB+4%G) agar plates for 48 h at 37°C. Isolated colonies were sub-cultured to LB+4%G broth, and cultures were incubated at 37°C until optical density readings at 600 nm (OD600) reached an exponential phase of growth. Bacteria were pelleted by centrifugation, washed and re-suspended in sterile 1× phosphate-buffered saline (PBS, pH 7.4) to obtain the desired CFU/ml. To obtain HK inoculums, bacterial suspensions were incubated at 85°C for 3 h and stored at 4°C until use. The absence of live B. mallei organisms in the HK preparations was confirmed after plating 10% of the total inoculums (v/v) and incubating these at 37°C for 48 h. All procedures were performed under a class II biosafety cabinet in a biosafety level 3 laboratory. Female, 6- to 8-week-old, BALB/c mice (n = 5–7) were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, Indiana). Female, 6- to 8-week-old, C57BL/6 mice deficient in mature B-cells (μMT), CD4 T-cells (CD4-/-) and CD8 T-cells (CD8-/-) and wild-type mice were obtained from The Jackson Laboratory (Bar Harbor, Maine).\n\nVaccination and challenge\nBALB/c and C57BL/6 mice were grouped and vaccinated with 0.5 μg of HK B. mallei (without adjuvant) by i.p. injection using a 25-gauge syringe. Two weeks post HK vaccination mice were injected i.p. with 2 × 107 CFU/100 μl of live B. mallei (~20 LD50) [18]. Complement depleted animals were challenged with 2.5 × 104 CFU/50 μl (~0.25 LD50) by intranasal (i.n.) route. Aliquots from the inoculums were plated to confirm the infecting dose. All procedures and animal protocols used in this study were approved by the Biosafety and IACUC committees at UTMB and conducted in either BSL-3 or ABSL-3 laboratories.\n\nCell and cytokine depletions\nAcute in vivo cell/cytokine depletion was performed with monoclonal rat anti-mouse CD4 (GK1.5), CD8α (53-6.7) or B220 (RA3-6B2) obtained from R&D Systems, Inc. (Minneapolis, MN) by methods similar to those we have previously described [19]. Functional grade purified rat anti-mouse interferon-gamma (IFN-γ, AN-18) was obtained from eBioscience (San Diego, CA) and purified anti-mouse tumor necrosis factor (TNF-α, MP6-XT3) from BD Pharmingen (San Diego, CA). IFN-γ and TNF-α antibodies were injected i.p. 6 h prior to challenge, 200 μg per mouse in 200 μl PBS or at later time points as indicated. Rat IgG isotype control was obtained from Southern Biotech (Birmingham, AL) and administered i.p. on day of challenge, 200 μg/mouse. Rat anti-mouse CD4, CD8α and B220 were injected i.p. twice, 1 day prior to challenge and on day of challenge, with an equivalent dosage sufficient to deplete T or B cells from 6 × 108 bone marrow cells per injection. The efficiency of depletion at time of infection for CD4+, CD8+, and B220+ cells was confirmed by flow cytometry analysis immediately prior to infection.\n\nComplement depletion with cobra venom factor\nMice, six to seven per group, were vaccinated i.p. with 1 × 105 CFU of nonviable B. mallei cell preparation in a total volume of 0.1 ml. Two weeks later, 24 h and 1 h before challenge, complement depleted mice were treated i.p. with 12.5 units total cobra venom factor (Quidel Corporation Speciality Products, San Diego, CA) in 0.1 ml of PBS. Complement depletion was confirmed prior to challenge by micro-titer hemolytic complement activity (CH50) assay as previously described [20].\n\nB. mallei J774A.1 uptake assays\nJ774A.1 cells were seeded (5 × 105) onto Corning costar 24 well plates (Corning, NY) with DMEM and incubated overnight at 37°C with 5% CO2. Bacterial suspensions were incubated at 37°C for 45 minutes supplemented with 2% mouse serum from Sigma-Aldrich (St. Louis, MO.), heat inactivated mouse serum (56°C 30 minutes), or bacteria alone and then added at an MOI of 10:1 to J774A.1 cells in triplicate. Inoculated wells were centrifuged at 800 g for 2 minutes and incubated for 2 hours at 37°C with 5% CO2 followed by a PBS wash (×2) and 2 hour incubation with 250 μg/ml kanamycin. Wells were washed twice with PBS and lysed with 0.1% Triton X-100, followed by serial 10-fold dilutions plated on LBG plates and incubated at 37°C for 2 days. Colony forming units were enumerated and uptake expressed as a percentage of initial inoculating dose ± SEM.\n\nAntibodies and flow cytometry\nFlow cytometric analysis was performed on 0.1-ml blood samples transferred to micro centrifuge tubes containing 90 μl of acid citrate dextrose (ACD) solution. Red blood cells were lysed using ACK-lysing buffer (Biosource International, Inc., Camarillo, CA) according to the manufacturer's instruction. Antibodies used for analysis of surface markers included: FITC-conjugated rat anti-mouse CD45R/B220 (RA3-6B2, BD Pharmingen San Diego, CA) for B cells; FITC-conjugated rat anti-mouse CD8α (53-6.7) and CD4 (GK1.5, BD Pharmingen, San Diego, CA) for CD8+ or CD4+ cells, respectively. Samples evaluated for CD4+ and CD8α+ cells were also incubated with biotin-conjugated hamster anti-mouse CD3e (145-2C11) monoclonal antibody (BD Pharmingen, San Diego, CA) and subsequently with streptavidin APC Cy7. Isotype-matched, non-specific controls were assayed in parallel (BD Pharmingen, San Diego, CA). Surface staining was performed according to previously published protocols [21]. Following cell staining, the samples were fixed with 2% buffered paraformaldehyde overnight prior to analysis by flow cytometry. Samples were analyzed using a FACSCalibur flow cytometer with BD CellQuest Pro software.\n\nAntibody assays\nImmunoglobulin subclass IgG1 and IgG2a titers in mice were determined by a whole bacterial cell ELISA performed in 96-well, Immulon 2 HB, round-bottom plates (Dynex Technologies). B. mallei antigen was diluted in 0.1 M carbonate buffer (pH 9.5) and 50 μl of diluted cells placed into wells. Plates were stored overnight at 4°C. The plates were washed with washing solution (1× PBS, 0.05% Tween 20), and incubated with 100 μl of blocking solution (1× PBS, 1% bovine serum albumin, 0.05% Tween 20) for 1 h at 37°C. Dilutions of mouse sera were made with blocking solution in duplicate and plates were incubated for 1 h at 37°C. Following incubation, plates were washed and 50 μl of anti-Ig-horseradish peroxidase subclass conjugate, diluted accordingly to manufacturer's instructions (Southern Biotechnology Associates, Inc. Birmingham, Ala.), was added to each well and incubated for 1 h at 37°C. After washing, 50 μl of 2,2'-azino-di-(3-ethylbenzthizoline)-6-sulfonate (ABTS) peroxidase substrate (KPL, Inc., Gaithersburg, Maryland) was added to each well and plates incubated for 25 min at room temperature. The amount of bound antibody was determined colorimetrically by absorbance at 405 nm.\n\nStatistical analysis\nSurvival curves were calculated by Kaplan Meier survival analysis with log-rank tests between groups using GraphPad Prism (V.4.03 for windows). Statistical analysis was generally performed with the paired Student's t-test. P value ≤ 0.05 was considered significant.\n\n\nAbbreviations\nHK: Heat-killed; i.p.: intraperitoneal; i.n.: intranasal.\n\nAuthors' contributions\nGCW designed and conducted experiments and drafted the manuscript. BMJ carried out the immunoassays and animal work. SP provided analysis of data and contributed to design and animal work. RAL participated in the generation and analysis of chronic TNF-α data. DME conceived the study, and participated in its design and coordination and helped to draft the manuscript. AGT participated in the bacterial work and drafting of the manuscript. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 23914 ] ] } ]

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[ { "id": "pmcA328328__text", "type": "Article", "text": [ "Mapping irradiation hybrids to cosmid and yeast artificial chromosome libraries by direct hybridization of Alu-PCR products.\nAbstract\nA direct hybridization protocol is described for screening cosmid and yeast artificial chromosome libraries with pools of Alu-PCR products from somatic cell or irradiation hybrids. This method eliminates purification, cloning and analysis of each individual Alu-PCR product before library screening. A series of human X chromosome irradiation hybrids were mapped by this method, using a cosmid reference library for comparisons between overlapping hybrids to identify interesting clones for further analysis.Images\n\n\n\n\n\n\n Nucleic Acids Research, Vol. 19, No. 12 3315 \n\n Mapping irradiation hybrids to cosmid and yeast artificial chromosome libraries by direct hybridization of Alu-PCR products \n\n Anthony P.Monaco*, Veronica M.S.Lam2, Gunther Zehetner, Gregory G.Lennon, Christal Douglas, Dean Nizetic, Peter N.Goodfellow1 and Hans Lehrach \n\n Genome Analysis Laboratory and 'Molecular Human Genetics Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK and 2Department of Biochemistry, Li Shu Fan Building, Sassoon Road, University of Hong Kong, Hong Kong \n\n Received March 12, 1991; Revised and Accepted May 16, 1991 \n\n ABSTRACT \n\n A direct hybridization protocol is described for screening cosmid and yeast artificial chromosome libraries with pools of Alu-PCR products from somatic cell or irradiation hybrids. This method eliminates purification, cloning and analysis of each individual AluPCR product before library screening. A series of human X chromosome irradiation hybrids were mapped by this method, using a cosmid reference library for comparisons between overlapping hybrids to identify interesting clones for further analysis. \n\n INTRODUCTION \n\n The generation of human DNA probes specific for individual chromosomes and subregions of chromosomes has been advanced with Alu-sequence primed polymerase chain reaction amplification (Alu-PCR, 1-3). This method specifically amplifies sequences between Alu repeats from human DNA in somatic cell hybrids and yeast artificial chromosomes (YACs, 4). Individual Alu-PCR products can be purified from agarose gels or ligated into plasmid vectors to screen for single copy sequences. Unique Alu-PCR products are then localized to certain chromosome regions using DNA blots of somatic cell hybrid panels. Once localized, Alu-PCR fragments can be screened against genomic libraries to isolate longer DNA fragments from the region of interest. As an alternative to this multistep process we have developed a hybridization protocol for screening of cosmid and YAC libraries directly with pools of Alu-PCR products. \n\n Two new human specific Alu primers were used to generate DNA probes from a series of irradiation-reduced hybrids containing multiple human X chromosome fragments of 1-2000 kb on a hamster chromosome background (5; P.N.G., unpublished). The Alu-PCR products were hybridized as a pool of probes to X-specific cosmid and YAC libraries, after \n\n competitive reassociation with an excess of human DNA to both the library filters and radioactively labelled Alu-PCR products. Comparisons were made between clones identified by overlapping irradiation hybrids and single copy DNA probes hybridized to the cosmid and YAC libraries. \n\n METHODS \n\n Two human Alu sequence primers were generated which were shown to be human specific; 3144 from the 3' end of Alu: 5'-GAGCGAGACTCCGTCTCAAA-3' and 2729 from the 5' end of Alu: 5'-GTGGATCACCTGAGGTCAGGAGTTC-3'. All PCR reactions were carried out with 100 ng of hybrid DNA and 0.7 ,^g of a single Alu primer in 100 d41 of 0.01 M Tris-HCl pH 8.3, 0.0015 M MgCl2, 0.05 M KCI, 200 AtM each of dNTPs, 10% dimethlysulfoxide, and 2.5 units of Cetus Taq polymerase. Reactions were 30 cycles of 94?C for 2 min, 57?C for 2 min, and 74?C for 4 min followed by a final extension time at 74?C for 9 min. Reactions products were analyzed on 1 % agarose gels and shown to contain between five and twenty fragments, with sizes ranging from 0.1 to 2.0 kb. Chinese hamster DNA and no DNA PCR reactions were done to control for non-human products (data not shown). \n\n Alu-PCR products were separated from Alu oligomers over Qiagen columns, and approximately 50-100 ng were labelled by random hexamer priming (6). The radioactively labelled pool of fragments was prehybridized with 37.5 ,^g of total human DNA and 12.5 tsg of hamster DNA immobilized on a cellulose support matrix, prepared as previously described (7). Reactions were at 65?C in 1 ml of 0.75M NaCl, 0.05M sodium phosphate pH 7.2, 0.005M EDTA, 0.1 % sodium dodecyl sulphate (SDS), 0.5 mg/ml heparin, and 100 jig/ml denatured salmon sperm DNA. The cellulose was pelleted and the supernatant boiled for 2 min every 12-16 hours (three times in two days). \n\n Cosmid and YAC library filters (Hybond N+, Amersham) were prehybridized at 42?C for 16 hours with 100 jig/ml \n\n * To whom correspondence should be addressed at Human Genetics Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK \n\n 1991 Oxford University Press \n\n 3316 Nucleic Acids Research, Vol. 19, No. 12 \n\n Chromosome X \n\n .,.., ., < \n\n _ ., . < ., \n\n *2.' 1- 7-_ \n\n _-_ 1. J \n\n ,.1 .. - 1 , _ i - _ 1 . 1 \n\n 1 ' 4 - -1 + \n\n 1 1 L L . i \n\n . 1 . _ 1 \n\n 1 1 s. 1 i \n\n 1 i ;. --r' F 1 -\n\n 1 -1-l \n\n j i-_1. 1 \n\n - _ 1 .. \n\n s- S . i n -. \n\n .. : ,1' ,. -1 _ E I i ? \n\n ,. . _ _ _ \n\n G-\n\n . \n\n *.-.,., z \n\n _ \n\n _ , \n\n z ;- [X1 \n\n 21 , In \n\n 27 38 45 48 54 74 86 \n\n I 1 I \n\n I \n\n I \n\n 107 \n\n MD \n\n I \n\n :I I \n\n I I \n\n I I \n\n Figur 2a and 2b: Hybridization of Alu-PCR products generated with Alu primer 3144 from irradiation hybrid 48 to duplicate copies of 22 x22cm filters containing 9216 human X chromosome cosmids (8). 2c: Hybridization of Alu-PCR products generated with Alu primer 3144 from an independent hybrid (54) to a third identical cosmid filter. \n\n Figure 1: A schematic diagram of the human X chromosome alongside the approximate cytogenetic location of fragments identified in nine irradiation hybrids (numbers across the top). The human X fragments were identified by hybridization of 27 known DNA markers (indicated by a black line; P.N.G, unpublished) or by cosmids in common with unique X chromosome probes in the reference library database (open boxes and Table 1). The size of the lines and boxes relate to the best cytogenetic location of the probes used according to Human Gene Mapping 10.5 (14) and does not indicate the physical extent of the irradiation hybrid fragments. \n\n denatured and sheared total human DNA in 50% formamide, 4XSSC, 0.05 M sodium phosphate pH 7.2, 0.001 M EDTA, 10% dextran sulphate, 1.0% SDS, 50 isg/ml denatured salmon sperm DNA and OxDenhardt's solution. The radioactively labelled Alu-PCR products were denatured and added to fresh \n\n hybridization solution without human DNA competitor at 1 x 106 \n\n cpm/ml and hybridized at 42?C for 16 hours. Filters were washed in 0.1 xSSC and 1.0% SDS, twice at room temperature and twice at 65?C for 30 min each and exposed to Kodak X-OMAT film for 2-3 days at -70?C with an intensifying screen. \n\n For each hybridization, two sets of duplicate cosmid filters were used from the ICRF reference library system (8), each containing 9216 flow-sorted human X chromosome cosmid clones or approximately 2 chromosome equivalents on a 22 x 22 cm filter (9). The coordinates of signals positive on duplicate cosmid filters were entered into the reference library database (G. Z, unpublished) using a digitizing tablet. For the X chromosome specific YAC library (A.P.M. and H.L., unpublished), about 420 YAC colonies were spotted manually onto filters from 96 well microtiter dishes using a 96 prong device. After growth on selective media for 3 days, YAC filters were processed for hybridization as previously described (10). \n\n RESULTS \n\n A panel of 195 X chromosome irradiation hybrids was constructed (50,000 rads) and characterized by DNA hybridization using 27 X chromosome markers and flourescence in situ hybridization using total human DNA as probe (Benham et al., 1989; P.N.G., unpublished). This analysis indicated that the hybrids contained multiple small fragments (4-10 fragments of 1000-5000 kb each) with a preferential retaining of centromere sequences (90%). From this panel, nine irradiation hybrids were chosen that contained less than five different regions by DNA probe hybridizations, mostly from the short arm of the X chromosome (Fig 1). All nine hybrids were used in PCR reactions with 3'-Alu primer 3144 and two were used with 5'-Alu \n\n Figure 3: Hybridization of Alu-PCR products generated with Alu primer 3144 from irradiation hybrid 54 to a filter containing 420 YAC clones from the human X chromosome. The positive YAC was also identified in a separate hybridization with the DMD probe P20 (12). \n\n primer 2729. Example hybridizations to a human X chromosome cosmid filter in Fig 2 shows the intensity and reliability of positive clones identified on duplicate filters with Alu-PCR products from the same irradiation hybrid (48) and the independence of clones identified with Alu-PCR products from a different hybrid (54). Fig 3 shows a single positive YAC clone after hybridization of Alu-PCR products from irradiation hybrid 54 to a filter containing about 420 YAC clones specific for the human X chromosome. \n\n The total number of cosmids identified with each pool of AluPCR products for each hybrid is shown in Table 1. From the average number of cosmids identified (24) in four chromosome equivalents screened and the estimated average DNA content in each hybrid (3000-15000 kb), the Alu-PCR products generated by a single primer were calculated on average to be 300-1500 kb apart, similar to published estimates for this method (1,2). Only 3-4 cosmids were identified in common using Alu-PCR products generated with either 3' or 5' Alu primers (3144 or 2729) from two hybrids (38 and 45). This shows that separate products were amplified with the two Alu primers since they prime synthesis from opposite ends of the Alu consensus sequence and Alu sequences are oriented in the genome either head to head, \n\n A \n\n B \n\n C \n\n El \n\n I0 \n\n Nucleic Acids Research, Vol. 19, No. 12 3317 \n\n Table 1. Cosmids identified by hybrids and unique probes \n\n hybrids 21 27 38 38 45 45 48 54 74 86 107 unique \n\n primer 3144 3144 3144 2729 3144 2729 3144 3144 3144 33144 3144 probes \n\n 21 3144 59 1 27 3144 7 14 2 38 3144 3 1 28 2 38 2729 0 0 4 25 0 45 3144 13 2 2 0 32 3 45 2729 1 0 0 0 3 31 0 48 3144 10 2 4 0 2 0 28 3 54 3144 11 3 0 0 5 0 5 30 2 74 3144 5 0 0 0 4 0 5 12 20 3 86 3144 3 1 0 0 4 1 3 4 3 17 0 107 3144 1 0 1 1 0 0 3 0 0 0 13 0 \n\n tail to tail or head to tail relative to each other. Therefore, by using the 3'- and 5'-Alu primers in separate PCR reactions with the same hybrid DNA, the total number of products and cosmid clones identified was almost doubled. \n\n Table 1 also indicates how many cosmids were identified by Alu-PCR products from other hybrids, and 16 cosmids previously identified with unique DNA probes in the reference library database. As can be seen in Fig 1 and previous irradiation hybrid analysis, there is a preferential retention of centromere sequences (2,11). However, there were no cosmids identified in common from all the hybrids positive with centromere sequences. This is probably due to a paucity of Alu repeats in the correct orientation in alphoid centromere sequences and thus few or no Alu-PCR products would be amplified from the centromere. \n\n Cosmids identified in common by several irradiation hybrids (Table 1) were most likely from regions of overlap outside the centromere area as shown by the previous DNA probe characterization (Fig 1). The overlap regions between hybrids were also seen by 16 cosmids (Table 1, Fig 1) that were hybridization targets of unique DNA markers in the reference library database that mapped in independent experiments to the overlap region. At least for several cosmids this showed that the Alu-PCR products identified target cosmids that were definitely from the expected region contained in the hybrids. For example, hybrids 21 and 54 were both shown to contain part of the Duchenne muscular dystrophy (DMD) locus (Fig 1; P.N.G., unpublished) and had 11 cosmids in common, including one identified by the probe P20 from the deletion hotspot region of the DMD gene (12). In Fig 3 the hybridization of Alu-PCR products from hybrid 54 identified a YAC clone which was also positive for the DMD probe P20 (data not shown). This method also identified fragments in the hybrids that were not seen in the initial DNA characterization (Fig 1 and Table 1). Since the 27 DNA probes were not close enough to each other along the chromosome to detect all possible hybrid fragments (1000-5000 kb), many regions would have been untested. For example, AluPCR products from hybrids 45 and 48 identified several cosmids, also seen by the cDNA for chronic granulomatous disease gene (CYBB, 13) in Xp2 1.1, although this region was not tested in the original hybrid characterization. \n\n DISCUSSION \n\n The direct hybridization of Alu-PCR products from somatic cell hybrids to genomic libraries can bypass gel purification or ligation of fragments into plasmid vectors and individual analysis for single copy sequences. Hybridization of Alu-PCR products as \n\n a pool to ordered array libraries such as the flow-sorted X chromosome cosmid library (9), allows the direct comparison of overlapping hybrids to pinpoint cosmids most likely to be from the region of interest. In conjunction with the reference library database (G.Z, unpublished) with 183 X chromosome probe hybridization entries, cosmids identified with both Alu-PCR products and uniquely mapped X probes immediately map them to the region of interest and proove that the method has worked. Similar hybridization experiments using Alu-PCR products from four overlapping irradiation hybrids identified four cosmids in common that mapped to the region of overlap by independent experiments (F.Muscatelli, A.P.M., P.N.G., H.L. and M.Fontes, in preparation). Since only 27 probes from the X chromosome were used to initially characterize the hybrids and the length of individual human fragments in the irradiation hybrids is about 1000-5000 kb, many regions could have been undetected in the original analysis. The direct hybridization of Alu-PCR products to the cosmid reference library detected such fragments since they identified cosmids in common with uniquely mapped probes in regions not tested originally (Table 1 and Fig 1). This should prove to be a sensitive and efficient method to determine content and overlap of irradiation hybrids in conjunction with DNA blot hybridization. However, since the exact length of the human DNA fragments for each hybrid and the spacing of Alu-PCR products along the chromosome is not known, it is difficult to directly correlate the number of target cosmids to the DNA content of the hybrids. \n\n The direct hybridization of Alu-PCR products from irradiation or somatic cell hybrids to total genomic YAC libraries will be especially useful to construct long range YAC contigs from specific subregions of chromosomes. The dissection of a total genomic YAC library by this method may be more efficient than generating chromosome specific YAC libraries from somatic cell hybrids (usually a haploid human chromosome on a diploid or greater rodent background) or flow-sorted chromosomes, because of the low transformation efficiency of yeast. \n\n ACKNOWLEDGEMENTS \n\n We would like to thank Gert-Jan Van Ommen for the probe P20 and Stuart Orkin for the CYBB cDNA probe. A.P.M is supported in part by a research fellowship from the Muscular Dystrophy Association of America. V.M.S.L. is supported in part by the Medical Research Grant of the University of Hong Kong. Reference library filters and cosmids identified by Alu-PCR products can be requested from the Reference Library Database, ICRF, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K. \n\n 3318 Nucleic Acids Research, Vol. 19, No. 12 \n\n REFERENCES \n\n 1. 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(eds.), Genome Analysis Volume 1: Genetic and Physical Mapping. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 39-81. \n\n 9. Nizetic, D., Zehetner, G., Monaco, A.P., Gellen, L., Young, B.D., and \n\n Lehrach, H. (1991) Proc. Natl. Acad. Sci. USA, 88:3233-3237. \n\n 10. Larin, Z. and Lehrach, H. (1990) Genet. Res. Camb., 56:203-208. \n\n 11. Cox, D,R., Pritchard, C.A., Uglum, E., Casher, D., Koborl. J. and Myers, \n\n R.M. (1989) Genomics, 4:397-407. \n\n 12. Wapenaar, M.C., Kievits, T., Hart, K.A., Abbs S., Blonden, L.A.J., \n\n denDunnen, J.T., Grootscholten, P.M., Bakker, E., Verellen-Dumoulin, C., Bobrow, M., vanOmmen, G.J.B., and Pearson, P.L. (1988) Genomics, 2:101-108. \n\n 13. Royer-Pokora, B., Kunkel, L.M., Monaco, A.P., Goff, S.C., Newburger, \n\n P.E., Baehner, R.L., Cole F.S., Curnette J.T., and Orkin, S.A. (1986) Nature, 322: 32-38. \n\n 14. Davies, K.E., Mandel, J.L., Monaco, A.P., Nussbaum, R.L. and Willard, \n\n H.F. (1990) Cytogenet. Cell Genet. 55: 254-313. " ], "offsets": [ [ 0, 18947 ] ] } ]

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[ { "id": "pmcA2553092__text", "type": "Article", "text": [ "TOPS++FATCAT: Fast flexible structural alignment using constraints derived from TOPS+ Strings Model\nAbstract\nBackground\nProtein structure analysis and comparison are major challenges in structural bioinformatics. Despite the existence of many tools and algorithms, very few of them have managed to capture the intuitive understanding of protein structures developed in structural biology, especially in the context of rapid database searches. Such intuitions could help speed up similarity searches and make it easier to understand the results of such analyses.\n\nResults\nWe developed a TOPS++FATCAT algorithm that uses an intuitive description of the proteins' structures as captured in the popular TOPS diagrams to limit the search space of the aligned fragment pairs (AFPs) in the flexible alignment of protein structures performed by the FATCAT algorithm. The TOPS++FATCAT algorithm is faster than FATCAT by more than an order of magnitude with a minimal cost in classification and alignment accuracy. For beta-rich proteins its accuracy is better than FATCAT, because the TOPS+ strings models contains important information of the parallel and anti-parallel hydrogen-bond patterns between the beta-strand SSEs (Secondary Structural Elements). We show that the TOPS++FATCAT errors, rare as they are, can be clearly linked to oversimplifications of the TOPS diagrams and can be corrected by the development of more precise secondary structure element definitions.\n\nSoftware Availability\nThe benchmark analysis results and the compressed archive of the TOPS++FATCAT program for Linux platform can be downloaded from the following web site: \n\nConclusion\nTOPS++FATCAT provides FATCAT accuracy and insights into protein structural changes at a speed comparable to sequence alignments, opening up a possibility of interactive protein structure similarity searches.\n\n\n\nBackground\nStructural biology is one of the most successful fields of modern biology. Over 50,000 solved protein structures illustrate details of many specific biological processes. The same data also provide us with information about the global features of protein structure space and can be studied to discover the evolutionary, physical, and mathematical rules governing them. How many fundamentally different protein shapes (folds) are there? How do protein structures evolve? How do new structural features appear, and if they are coupled with changes in function, how does this process occur? Such questions can be studied by classifying, comparing and analyzing known protein structures. Two different, but synergistic strategies are typically used for this purpose. In classification systems such as SCOP [1] or CATH [2], human intuition is used to simplify the description of protein structures to a manageable size, and a human eye, sometimes supported by automated analysis, can recognize patterns and types of structures. In the second approach, specialized comparison algorithms, such as DALI [3], CE [4], or FATCAT [5] can be used to calculate a distance-like metric in the protein structure space. This in turn can be used to cluster proteins into groups. Many such algorithms have been developed over the past few decades and have been mostly used for the classification of protein structures into families.\nAn exact solution of an alignment between two structures is formally equivalent to a threading problem and is therefore NP-complete [6]. However, a practical solution can be obtained by heuristics reducing the problem to a manageable size [7]. In human classification systems, the protein is usually reduced to a set of several structural elements, which obviously involve many arbitrary thresholds. Automated algorithms have the same problem and also suffer from inconsistencies between different numerical measures of protein structure similarity [8]. Interestingly, despite these problems, results of different approaches are broadly similar. They all identify approximately a few hundred general classes of protein structures, usually called folds [1] or topologies [2], distinguished by how the main chain of the protein folds around itself in the three-dimensional space. At the same time, the comparison of different approaches, both between and within the two classes, shows that fold/topologies (or cluster) definitions are somewhat fuzzy, with some proteins being occasionally difficult to classify and joining different groups depending on various assumptions. This lead some to question the concept of the fold [9], but practical application of protein structure comparison leaves little doubt that protein structure space has some natural granularity that overlaps well with the traditional fold classification.\nComparison and classification of protein structures is significantly simplified by the fact that proteins have naturally modular structures, being mostly composed of locally regular structures: alpha helices and beta strands. These two types of secondary structures constitute a little over 50% of an average protein's length. With the average length of a secondary structure being around 10 amino acids, this makes it possible to describe protein structure as an arrangement of a much smaller number of elements. Protein structures are often visualized in a simplified form, with the so-called ribbon diagram with secondary structures shown as helices and arrows being the most popular (see Figure 1). This picture can be simplified further by showing individual secondary structure elements as simple symbols (circles or boxes/triangles). These depictions, called fold diagrams, originally proposed in the 70s [10-12] are best captured by a TOPS (Topology of Protein Structures) algorithm, which attempts to automate the process of creation of the topology cartoon [13]. While useful in protein classification, such simplified descriptions are not used in the most popular automated protein structure comparison algorithms such as DALI [3] or CE [4]. Kleywegt and Jones developed a method for finding similar motifs based on comparing distance matrices that are constructed by representing protein as a set of SSEs with their directional vectors and angle between those vectors [14]. Programs that used SSEs either for structure comparison based on hierarchical superposition of both SSEs and atomic representation [15] or for finding common substructures in the comparison process based on subgraph isomorphism, such as [16,17] and recent applications of the TOPS diagram [18,19], usually struggle with translating the comparison results from the secondary structure to the individual residue level. Although the SSM method uses graph-matching procedures at the SSE level followed by an interactive 3D alignment of the protein C-alpha atom [20], it lacks the topological relationships between the SSEs, which are essential features in identifying common scaffolds in distantly related proteins. A TOPS pattern was used to guide the sequence alignment, for instance, to build multiple structural alignments of the distantly related family of beta-rich protein domains [21]. The Multiple Sequence Alignment Tool (MSAT) automates this approach, merging it with a popular ClustalW program [22]. DALI [3], CE [4] or FATCAT [5] introduce their own methods of decomposing the protein structure into smaller units, such as 7 × 7 dense distance map fragments (DALIs) or aligned fragment pairs (AFPs) (CE and FATCAT). The large number of such fragments and the combinations of the fragments that need to be evaluated by structure comparison programs is the main reason for the significant computational requirements of such algorithms. However, more importantly, TOPS+ method is used here to enable a structural comparison that takes into account flexibility in protein structures and not only classifies the differences, but also can recognize such rearrangements – which is a first such application using the SSEs language. In this contribution, we explore the question of whether it would be possible to combine insights provided by topology diagrams into automated protein structure alignment algorithms, focusing on the FATCAT program developed previously in our group.\n\nMethods\nFlexible structure alignment method FATCAT\nFATCAT [5] is a unique structure alignment method that allows for flexibility in the structures being compared. It builds the alignment by chaining aligned fragment pairs (AFPs) [23] together using a unified scoring function where AFP extensions, gaps, and twists each have their specific scores (Figure 2). Introducing a twist into the alignment is penalized, but this penalty may be compensated for by the gain in the score of the resulting alignment (i.e., longer alignment and/or better RMSD). Rigid alignment can be treated as a special case, in which no twist is allowed in chaining AFPs. FATCAT program provides alignment in both, \"rigid\" mode and \"flexible\" (default) mode.\nFATCAT, as well as most other protein structure comparison programs, is very slow when compared to sequence alignments. The computing time of FATCAT is determined by the size of the collection of AFPs detected between the two structures being compared. FATCAT is available from a server with an option to search in SCOP or PDB databases for similar structures. This search typically takes between 8 to 16 hours of CPU time, and this is the main obstacle to broader use of this option. FATCAT has been used to construct a Flexible Structure Neighborhood (FSN) database that contains pre-computed results of structure similarity searches and it takes several weeks of CPU time to update the FSN database. Other protein structure comparison resources, such as DALI or CE have very similar problems.\n\nTOPS cartoons and TOPS graph models\nAs discussed in the Background, TOPS cartoons capture the simplified, fold-level description of protein structure and at the same time can be automated [24]. The TOPS algorithm uses structural features such as hydrogen bonds and chirality of the beta strands to provide a scoring function to optimize the cartoon (see Figure 1(b)). In TOPS, the secondary structural elements (SSEs) are derived from the DSSP program [25]. Based on TOPS cartoons, a formal graph model and graph-based definitions of protein topology and pattern discovery and comparison methods were developed [26,27]. The TOPS database and comparison, pattern discovery and matching programs are accessible from .\n\nNovel TOPS+ and TOPS+ strings models\nThe TOPS model was further enhanced to incorporate features such as protein-ligand interaction information and more detailed secondary structural segment information. This enhanced model is called TOPS+ model (see Figure 3a). This TOPS+ model can be described formally in a TOPS+ strings language (Figure 3b) at a reduced linear level. The enhanced TOPS+ strings models can be used in fast string-based structure matching and comparison, at the same time avoiding issues of NP-completeness associated with graph alignments.\nIn detail, each node (SSE segment) of the TOPS+ strings is described by its type, orientation, PDB start number, segment length, total number of incoming (InArc) and outgoing (OutArc) arcs (edges), total number of ArcTypes, and total number of ligand arcs (LigArc). The type of the segment (SSEType) could be one of [E, e, H, h, U, u], where, \"E\" and \"e\" represent the \"up\"- and \"down\"-oriented beta strands; \"H\" and \"h\" indicate the \"up\"- and \"down\"-oriented alpha helices; and \"U\" and \"u\" represent ligand-bound and ligand-free loops. The InArcType can be classified as an/a [R, L, P, A], where \"R\" and \"L\" represent right and left chiralities; and \"P\" and \"A\" represent parallel and anti-parallel hydrogen bonds, respectively. The OutArcType is represented in a similar manner by [R', L', P', A']. Ligand arcs are indicated by LT = AA, where LT is the ligand type and AA is the PDB number. For example, Figure 3(a) and 3(b) contain visual representations of TOPS+ and TOPS+ strings models, respectively, for the protein domain d1fnb_1. Here the triangles represent the beta strands; the red curve represents the alpha helix; gray ellipsoids indicate loops; and green arcs indicate hydrogen bonds between two beta strands, called anti-parallel beta sheets. The length of a TOPS+ strings model is defined by number of SSEs; thus, the length of d1fnb_1 is 19. For further details, see [28].\n\nTOPS+ strings comparison method\nTOPS+ is a comparison method that computes a distance between TOPS+ strings models of two proteins based on a dynamic programming approach and identifies the longest common subsequence (LCS), consisting of the list of the topologically equivalent SSEs between two proteins. For example, Figure 3(c) shows the TOPS+ strings alignment between Dihydropteridine reductase proteins from rat (1dhr) and human (1hdr). The TOPS+ strings models for 1dhr and 1hdr are represented by a linear string-model, where a yellow triangle and red curves indicate the beta strands and alpha helices in their \"up\" or \"down\" orientations, respectively. The grey line and purple stubs represent the loop regions and the NAD ligand interactions, respectively. Note that the ligand-interaction information is optional and in this work we have not used it. The incoming and outgoing arcs are depicted in the SSEs (top and bottom of the beta strands), where red and green arcs represent the parallel and anti-parallel hydrogen-bond interactions that show beta-sheet information, while yellow and blue arcs indicate the right and left chirality relationships between the SSEs. A pink arrow between the TOPS+ strings elements indicates the conserved SSE. The dotted arrows indicate the conserved alpha helices and beta strands, while the plain arrows indicate the conserved loop regions.\n\nTOPS++FATCAT method\nIn this work, we want to test the general idea of pruning the search space of the FATCAT comparison process using topological constraints derived from the TOPS+ strings alignment. Many of the AFPs considered in the FATCAT alignment could be easily eliminated from the comparison by constraining the alignment region. Here we explore constraints obtained from the TOPS+ strings alignment, which identifies topologically equivalent secondary structure elements (alpha helices, beta strands, and loops) for this purpose. Such equivalences define blocks that restrict the alignment region; AFPs that fall outside these regions are simply not considered (see Figure 4(b)). We introduce a parameter r to control the strictness of constraints by TOPS+ strings alignments; r equals 0 if the alignment region is strictly restrained by TOPS+ strings alignment, and r is set to 1 by default in our program to allow certain flexibility to the constrained alignment region (Figure 4(c)). We then can speed up the FATCAT alignment by considering only the AFPs within the constrained alignment area (Figure 4(d)). The rigid structural alignment can be treated as a special case of TOPS++FATCAT, in which no twist is allowed in chaining AFPs. However, the TOPS++FATCAT program provides alignment in both, \"rigid\" mode and \"flexible\" mode (default).\n\nBenchmarking\nFor benchmarking and comparison, we have used the PDB40 dataset of 1,901 protein domain pairs (DP) corresponding to SCOP version 1.61 from the ASTRAL database [29]. Table 1 provides the SCOP superfamily level homolog versus non-homolog statistics for the four main SCOP classes i.e., all-alpha, all-beta, alpha/beta, alpha+beta, and all proteins regardless of their structural classes.\n\nEvaluation Analyses\nWe performed the Receiver Operating Characteristics (ROC) curve and the AUC (Area Under the ROC Curve) analyses to compare the performance of the TOPS++FATCAT method with the original FATCAT method, using SCOP classification at the superfamily level as a standard of comparison [30].\n\n\nResults\nROC and AUC Analyses\nWe have compared the performance of the TOPS++FATCAT method against the original FATCAT method using the SCOP classification information at the superfamily level. We have plotted the ROC curves based on P-values obtained from the FATCAT and the TOPS++FATCAT methods. We have plotted the ROC curves separately for the main SCOP classes, i.e., all-alpha, all-beta, alpha/beta, alpha+beta, and all proteins regardless of the class they belong to (see Figure 5(a) to 5(e)). In the graph, the x- and y-axes represent the false positive and true positive rates of the performance of the comparison methods respectively. In the legend, rF-pvalue and fF-pvalue indicate results from the rigid and flexible FATCAT methods, respectively; similarly, rT2F-pvalue and fT2F-pvalue represent the rigid and flexible TOPS++FATCAT methods, respectively. We have calculated the AUC values for all the SCOP classes based on ROC curves obtained from the FATCAT and TOPS++FATCAT methods with the flexible/rigid options (see Table 2).\nFor all protein classes, the rigid FATCAT performs best, usually followed by the flexible FATCAT, the rigid TOPS++FATCAT, and the flexible TOPS++FATCAT. The performance of all four methods is best for all alpha and all beta proteins, and all four perform markedly worse (but similar to each other) for alpha/beta proteins. Only alpha+beta proteins show a clear difference between the FATCAT and TOPS++FATCAT methods. It is important to note that the TOPS+ strings models consider the parallel and anti-parallel properties of the beta-sheet information in the form of total number of incoming and outgoing arcs with their ArcTypes. Thus, the TOPS++FATCAT method discriminates the protein domain pairs more efficiently compared to the original FATCAT method. For example, in the all-beta protein domain pairs, both the flexible and the rigid TOPS++FATCAT methods perform well. The flexible TOPS++FATCAT method covers nearly 84% of protein domains with 0% false positives, but the flexible and rigid FATCAT methods cover only 76% and 49% of the true positives, respectively, with 0% false positives. The zoomed-in version of the ROC curves with up to 10% false positives for all-beta rich protein families is shown in Figure 5(f); where both the rigid TOPS++FATCAT (green) and flexible (red) TOPS++FATCAT methods have coverage rates of 82% and 84% true positives respectively with 0% false positives. The overall results for all protein classes show that TOPS++FATCAT performance is only slightly lower (3%–7% AUC value difference (see Table 2)) as compared to FATCAT while providing a significant, more than 10-fold speedup (see next section).\n\nAFP and Runtime Analyses\nWe tested both the FATCAT and TOPS++FATCAT methods using the Mac OS X version 10.4.10 computer system with a 2 × 2.66-GHz Dual-Core Intel Xeon processor and 1-GB 667 MHz memory. We have performed runtime analysis on 1,901 protein domain pairs and counted the total number of AFPs and the corresponding runtime from both the FATCAT and the TOPS++FATCAT methods. The results show an exponential increase in AFPs (Figure 6(b)) and corresponding runtime (Figure 6(a)) for the FATCAT method as compared to the TOPS++FATCAT method (see Table 3) For example, the average number of AFPs for the TOPS++FATCAT method is 530, but the average number of AFPs for the FATCAT method is 15,019. This represents the number of average AFPs used by the FATCAT method is increased by a factor of 28 (see Table 3). This result leads to the conclusion that TOPS++FATCAT is 22 times faster compared to the FATCAT because this method must take into account more number of AFPs in the comparison process (see Table 3).\n\nCase Studies\nWhile the overall accuracy of both rigid and flexible FATCAT methods is better than their TOPS++FATCAT equivalents, an interesting example where the opposite is true lies in the comparison of two proteins, d2trxa_ (108 aa) from Escherichia coli and d1kte__ (105 aa) from Sus scrofa (pig) from the thioredoxin-like superfamily. For this pair, the flexible_TOPS++FATCAT method provides an alignment with 88 equivalent positions with 1.67 Å chain RMSD and 3.06 Å of optimal RMSD without any twist, giving the alignment with 10% sequence identity (see Table 4). On the other hand, the flexible_FATCAT method provides an alignment with 86 aligned positions using a twist in the C-terminal region; it has a higher chain RMSD of 5.14 Å, and its optimal RMSD is 3.48 Å. For more information regarding the chain and optimal RMSDs refer [5]. The flexible_FATCAT method uses the twist to align a helix in the C-terminal region, which is positioned incorrectly with a beta-sheet core (see Table 4). Figure 7(a) shows the superposition of d2trxa_ (gray) and d1kte__ (orange) domains from the flexible_FATCAT method, where the blue color indicates the d1kte__ protein domain from the flexible_TOPS++FATCAT method. The incorrect alignment of the C-terminal domain alpha helix of the d1kte__ domain (orange) is visible in the core of the beta-sheet region. Figure 7(b) and 7(c) shows the AFPs from the flexible_FATCAT and flexible_TOPS++FATCAT methods, respectively. The hinge region provides a twist in the flexible_FATCAT method indicated by an arrow and the AFPs represented by a different color (see Figure 7(b)). In this case, the alignment constraints from the TOPS+ strings alignment allow the TOPS++FATCAT method to avoid a spurious alignment.\nThe Erythrocruorin protein domain d1eca__ (136 aa) from Chironomus thummi and the Phycocyanin alpha subunit protein domain d1cpca_ (162 aa) from Fremyella diplosiphon (Cyanobacterium) belong to the Globin-like superfamily. For these protein domain pairs, the FATCAT method provides a better alignment with 120 and 118 aligned positions with the chain RMSD of 4.02 Å based on the flexible and rigid options, respectively. The flexible_TOPS++FATCAT method gives an alignment of 63 aligned positions with the 3.23 Å optimal RMSD and the 6.28 Å chain RMSD. In this case, the flexible_TOPS++FATCAT method misses the N-terminal region helix and misaligns some helices. For example, Figure 8(a) shows the superposition of d1eca__ (gray) and d1cpca_ (orange) domains from the flexible_FATCAT method, while d1cpca_ (blue) domain is from the flexible_TOPS++FATCAT method. The AFP chaining alignment and the actual alignment from FATCAT are shown in Figure 8(b) and 8(e), respectively. Figure 8(c) shows the AFP alignment from TOPS++FATCAT, in which this method misses the N-terminal region and incorrectly aligns some of the C-terminal regions (see Figure 8(d)). However, the rigid_TOPS++FATCAT method produces an alignment of 108 aligned positions with optimal and chain RMSDs of 3.22 Å and 6.28 Å respectively. In general, TOPS comparison does not work well for alpha-rich proteins due to the lack of hydrogen bonds between SSEs [26]. The same is true for TOPS+ strings comparison to some extent; however, this method takes advantage of ligand-interaction information to compare protein domains more efficiently; for example the DNA binding motifs such as helix-turn-helix and helix-loop-helix can be easily recognized [28]. However, we have not explored that ligand pattern discovery option within the TOPS+ strings comparison in this paper. In addition, the TOPS+ strings alignment provides only a basic alignment; the scoring function to find the best alignment has not been optimized. These problems can be addressed in future development by considering the advanced TOPS+ and TOPS+ strings models based on helix-helix packing relationships and SSE-ligand interaction properties together with the right and left chiralities. Furthermore, the TOPS+ strings comparison can be optimized in both the comparison process as well as in the alignment process in order to take into account indels (insertion/deletion) of SSEs which exist in nature across the different members of the protein superfamilies [31].\n\n\nDiscussion and conclusion\nThe overall results for all protein classes show that TOPS++FATCAT performance is only slightly lower (3%–7% AUC value difference) as compared to FATCAT while providing a significant, more than 10-fold speedup. The main reason for the discrepancies is that TOPS+ strings alignments occasionally misalign the secondary structure elements and subsequent FATCAT alignment, constrained by the TOPS+ strings alignment, cannot overcome the earlier errors. There is a clear trade-off between the runtime and the accuracy; limiting the pool of fragments being compared speeds up the algorithm but results in (slightly) lower accuracy. At the same time, these results offer clear suggestions for future development. Using a more advanced version of the TOPS+ strings comparison method would remove some of the false positives might be at a cost of significantly slowing the total performance of the TOPS++FATCAT method.\n\nAuthors' contributions\nMV developed the TOPS++FATCAT algorithm, performed the calculations and prepared the figures, YY provided advice and oversight in the project, verified the code and provided FATCAT results for comparison, AG contributed to the original idea and to writing of the manuscript.\n\n\n" ], "offsets": [ [ 0, 25028 ] ] } ]

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[ { "id": "pmcA1913570__text", "type": "Article", "text": [ "Elevated Blood Lead Levels of Children in Guiyu, an Electronic Waste Recycling Town in China\nAbstract\nBackground\nElectronic waste (e-waste) recycling has remained primitive in Guiyu, China, and thus may contribute to the elevation of blood lead levels (BLLs) in children living in the local environment.\n\nObjectives\nWe compared the BLLs in children living in the e-waste recycling town of Guiyu with those living in the neighboring town of Chendian.\n\nMethods\nWe observed the processing of e-waste recycling in Guiyu and studied BLLs in a cluster sample of 226 children < 6 years of age who lived in Guiyu and Chendian. BLLs were determined with atomic absorption spectrophotometry. Hemoglobin (Hgb) and physical indexes (height and weight, head and chest circumferences) were also measured.\n\nResults\nBLLs in 165 children of Guiyu ranged from 4.40 to 32.67 μg/dL with a mean of 15.3 μg/dL, whereas BLLs in 61 children of Chendian were from 4.09 to 23.10 μg/dL with a mean of 9.94 μg/dL. Statistical analyses showed that children living in Guiyu had significantly higher BLLs compared with those living in Chendian (p < 0.01). Of children in Guiyu, 81.8% (135 of 165) had BLLs > 10 μg/dL, compared with 37.7% of children (23 of 61) in Chendian (p < 0.01). In addition, we observed a significant increasing trend in BLLs with increasing age in Guiyu (p < 0.01). It appeared that there was correlation between the BLLs in children and numbers of e-waste workshops. However, no significant difference in Hgb level or physical indexes was found between the two towns.\n\nConclusions\nThe primitive e-waste recycling activities may contribute to the elevated BLLs in children living in Guiyu.\n\n\n\nDisposal of electronic waste, or e-waste, is an emerging global environmental issue, as these wastes have become the most rapidly growing segment of the municipal waste stream in the world [Dahl 2002; Halluite et al. 2005; Jang and Townsend 2003; Schmidt 2002; Silicon Valley Toxics Coalition (SVTC) 2001]. It is reported that approximately 500 million computers became obsolete between 1997 and 2007 in the United States (National Safety Council 1999). Up to 80% of e-waste from the United States has seeped into Asia and Africa (Johnson 2006; Puckett et al. 2002; Schmidt 2002, 2006; SVTC 2001). It is noteworthy that the United States is the only developed country today that has not ratified the United Nations Basel Convention, which bans the export of hazardous wastes to developing countries (United Nations Environment Programme 1992, 2006; USA Today 2002).\nTogether with New Delhi in India, Guiyu in Shantou, Guangdong Province, China (Figure 1), is one of the popular destinations of e-waste (Brigden et al. 2005; Puckett et al. 2002). Within a total area of 52 km2 and local population of 132,000 (in 2003), Guiyu has accommodated millions of tons of e-waste from overseas and domestic a year. Nearly 60–80% of families in the town have engaged in e-waste recycling operations conducted by small scale family-run workshops, with approximately 100,000 migrant workers employed in processing e-waste. Because the implementation of a clean and safe high-tech recovery process was very expensive (Allsopp et al. 2006), the processes and techniques used during the recycling activities in Guiyu were very primitive. The result was that many tons of e-waste material and process residues were dumped in workshops, yards, roadsides, open fields, irrigation canals, riverbanks, ponds, and rivers. Hazardous chemicals can be released from e-wastes through disposal or recycling processes, threatening the health of local residents. Several studies have reported the soaring levels of toxic heavy metals and organic contaminants in samples of dust, soil, river sediment, surface water, and groundwater of Guiyu (Brigden et al. 2005; Puckett et al. 2002; Wang and Guo 2006; Wang et al. 2005; Wong et al. 2006; Yu et al. 2006). Previously, we have shown that the residents in Guiyu had high incidence of skin damage, headaches, vertigo, nausea, chronic gastritis, and gastric and duodenal ulcers, all of which may be caused by the primitive recycling processing of e-waste (Qiu et al. 2004).\nOf many toxic heavy metals, lead is the most widely used in electronic devices for various purposes, resulting in a variety of health hazards due to environmental contamination (Jang and Townsend 2003; Musson et al. 2006; Vann et al. 2006). Lead enters biological systems via food, water, air, and soil. Children are particularly vulnerable to lead poisoning—more so than adults because they absorb more lead from their environments (Baghurst et al. 1992; Grigg 2004; Guilarte et al. 2003; Jain and Hu 2006; Needleman 2004; Safi et al. 2006; Wasserman et al. 1998). The U.S. Centers for Disease Control and Prevention (CDC) defined elevated blood lead levels (BLLs) as those ≥ 10 μg/dL in children ≤ 6 years of age (CDC 1991). Nevertheless, studies have increasingly shown that low blood lead concentrations, even < 10 μg/dL, were inversely associated with children’s IQ scores and academic skills (Canfield et al. 2003; Lanphear et al. 2000, 2005; Nevin 2000; Schnaas et al. 2006). Therefore, no safety margin at existing exposures has been identified (Chiodo et al. 2004; Koller et al. 2004).\nConsidering the potential heavy metal contamination in the local living environment of Guiyu, we hypothesized that children living in Guiyu may have elevated BLLs and thus their physical and mental development may have been affected. In this study, we evaluated the mean BLLs in children 1–6 years of age living in Guiyu and compared them with those living in the neighboring town of Chendian, where no e-waste processing was taken.\nMaterials and Methods\nGeographic location and site description\nThere are 28 villages with a total area of 52 km2 and a resident population of 132,000 and around 100,000 migrant workers in Guiyu (Figure 1). We chose four villages for their differences in the scale and type of e-waste processing. Beilin village has dense e-waste workshops mainly involved in equipment dismantling, circuit board baking, and acid baths; Dutou village specializes in plastics sorting, including manually stripping plastic materials from electronic products and then crudely classifying them; Huamei village had workshops similar to those of Beilin, but they are fewer and scattered; and Longgang village was involved in plastic reprocessing in which plastics collected from Dutou and other villages were washed and smashed into tiny pieces of recycled plastic. We used the neighboring town of Chendian as a control because the local residents work mainly in the textiles industry, not in e-waste processing. The population, traffic density, lifestyle, and socioeconomic status were very similar to those of Guiyu.\n\nStudy population\nThe study population was composed of children ≤ 6 years of age. No children involved in the study had any occupational exposure to e-waste. A cluster sample of 165 children with a median age of 5.0 years lived in the four villages of Guiyu (Figure 1). Sixty-one children with a median age of 4.0 years resided in Chendian were included in the study for comparison. After written informed consent was obtained from the parents or guardians, blood samples were collected from the children at village kindergartens. To facilitate the counseling process, advice on dietary and eating habits to minimize lead exposure were provided to the local residents. All children found to have high BLLs were advised to get further hospital treatment. The study was approved by the Human Ethics Committee of Shantou University Medical College.\n\nMeasurement of BLLs and hemoglobin\nVenipuncture blood samples were obtained from each volunteer at the kindergarten, and collected in lead-free tubes by trained nurses. Lead in total blood was analyzed by graphite furnace atomic absorption spectrometry (GFAAS), which consisted of a Shimadzu AA-660 AAS and GFA-4B graphite furnace atomizer and an ACS-60G autosampler (Shimadzu Corporation, Kyoto, Japan). The main parameters used for the determination were a wavelength of 283.3 nm, current of 8 mA, a slit width of 1.00 nm, drying at 150°C, ashing at 325°C, and atomization at 1,400°C. The accuracy of the method was controlled by recoveries between 95% and 107% from the spiked blood samples. Repeated analyses of standard solutions confirmed the method’s precision. The BLLs were expressed in micrograms per deciliter (1 μg/dL = 0.0484 μmol/L). Meanwhile, we assessed hemoglobin (Hgb) levels by hemoglobin cyanide method with hemoglobinometer (XK-2, JiangSu, China).\n\nEvaluation of physical developmental indexes\nChildren’s physical growth and development, such as body height, weight, and head and chest circumferences were measured when blood samples were collected. Weight and height were measured using a weighing and height scale (TZ120; Yuyao Balance Instrument Factory, Yuyao, China) with maximum weight of 120 kg (minimum scale, 50 g) and minimum height of 70 cm (minimum scale, 0.5 cm). Head and chest circumferences were measured using graduated anthropometric tapes.\n\nStatistical analyses\nWe performed statistical analyses using SPSS version 10.0 software (SPSS, Chicago, IL, USA). We used independent sample t-tests or covariance analyses for comparisons of mean, chi-square analyses for test of frequency data, and linear regression analysis for the association between BLLs and age. Differences were considered significant with a p-value < 0.05.\n\n\nResults\nObservation of e-waste processing\nThe primitive e-waste recycling procedures in Guiyu were mainly as follows: a) Old electronic equipment was dismantled (Figure 2) with electric drill, cutter, hammer, and screwdriver into component parts such as monitor, hard drive, CD driver, wires, cables, circuit boards, transformer, charger, battery, and plastic or metal frame that are sold for reuse or to other workshops for further recycling. b) Circuit boards (Figure 3) of computers and other large appliances were heated over coal fires to melt the solder to release valuable electronic components, such as diodes, resistors, and microchips. c) Circuit boards of cell phones and other hand-held devices were taken apart by a electrothermal machine (Figure 4), which was a particular environmental and human health concern in the processing of e-waste in Guiyu. d) In acid baths (Figure 5), some microchips and computer parts were soaked to extract precious gold and palladium, from which the waste acids were discharged into nearby fields and streams. e) Wires and cables were stripped or simply burnt in open air to recover metals. f) Printer cartridges were ripped apart for their toner and recyclable aluminum, steel, and plastic parts. g) Plastic [e.g., polyvinyl chloride (PVC), acrylonitrile butadiene styrene copolymer (ABS), high-density polyethylene (HDPE)] was sorted by workers according to rigidity, color, and luster. Plastic scraps that cannot be sorted visually must be burned and classified by burning odor. Another way to sort different plastics was gravitational separation into ceramic jugs with brine (Figure 6), after which the pieces were spread on the sidewalk to dry; h) For reprocessing, after sorting plastic scraps were fed into grinders that spit out tiny pieces of plastic. i) For metals sorting and reprocessing, transformers, chargers, batteries, and cathode-ray tubes were separated and hammered open for recycling metals such as copper, steel, silver, aluminum, which were then reprocessed to raw material.\nAlthough the methods for processing e-waste were primitive, the coordination of e-waste recycling in Guiyu was very well organized into specific tasks. Workshops specializing in dismantled equipment would not conduct circuit board baking or plastics and metals reprocessing. The chain of recycling components from each type of e-waste was well established in the town.\n\nBLLs in children\nWe collected blood from 165 children in Guiyu and 61 children in Chendian and measured the BLLs in these children. Table 1 shows that the BLLs corresponded to the children’s age, sex, and town of residence. As expected, BLLs among Guiyu children were much higher than those in the children of Chendian (p < 0.01). Among Guiyu children, 135 (81.8%) had BLLs > 10 μg/dL, whereas 23 (37.7%) in Chendian (p < 0.01) had high levels. Among 135 (81.8%) Guiyu children with elevated BLLs, 61.8% and 20% had BLLs > 10 μg/dL and 20 μg/dL respectively, but lead levels > 45 μg/dL were not found. And BLLs of Guiyu increased somewhat with age (p < 0.01); older children tended to have higher BLLs than younger ones. We found no evidence for the association in lead concentrations or prevalence of elevated BLLs differentiated by sex (both p > 0.05).\nTable 2 presents BLLs for 165 exposed children in the four villages. The findings showed that BLLs from different villages were in the following descending order: Beilin, 19.34 μg/dL > Dutou, 17.86 μg/dL > Huamei, 14.23 μg/dL > Longgang, 13.13 μg/dL (Table 2). Children living in Beilin, where the number of e-waste workshops specializing in equipment dismantling, circuit board baking, and acid baths, had the highest BLLs. Dutou, which had many workshops specializing in plastics sorting, including strip plastic materials from e-waste, had the second highest BLLs in children. Huamei had e-waste workshops similar to those of Beilin, but fewer and less centralized; the BLLs of Huamei children were much lower than those of Beilin and Dutou. Longgang, a village specializing in reprocessing plastics collected from other villages that had no workshops directly processing e-waste, had the lowest BLLs. There was a significant difference in BLLs among the children of the four villages (p < 0.01). In Beilin and Dutou, 88.8% and 100% children had elevated BLLs > 10 μg/dL, respectively.\nAs far as physical indexes and Hgb levels were concerned, there was no significant difference between Guiyu and Chendian (p > 0.05, Table 3).\n\n\nDiscussion\nIn this study, we observed that the processing of e-waste in Guiyu was very primitive and the recycling industry depended mainly on manual processing methods. Despite the fact that the coordination of the e-waste recycling is well organized in family-based small business units, the manual processing methods and the deposition of the e-waste have contributed to the contamination by heavy metals in the living environment. Examination of the possible impact of the e-waste industry on the BLLs of children living in Guiyu revealed that Guiyu children had significantly higher BLLs than Chendian children. Of children tested in Guiyu, 81.8% had BLLs > 10 μg/dL, indicating a correlation between the BLLs in children and the numbers of e-waste workshops. We speculated that the elevated BLLs in Guiyu children may be directly caused by the contamination of the lead during e-waste recycling. However, further study should be conducted to determine the relationship between BLLs in Children and the actual lead contamination in the environment.\nLead is considered one of the major heavy metal contaminants during the process of e-waste recycling. A cathode ray tube inside a television set or a computer monitor contains an average of 4–8 lb lead; monitor glass contains about 20% lead by weight; a typical battery weighs 36 lb and contains about 18 lb of lead. For decades, lead as a major component of solders has been used to attach electronic components to printed circuit boards. Lead compounds have also been used as stabilizers in some PVC cables and other products. Our study demonstrated in Guiyu a significant increasing trend in BLLs with increasing age; older children tended to have higher BLLs than younger ones. This might be the result of increasing exposure risk because older children might have more outdoor activities. In addition, it may also be attributed to the fact that the heaviest lead-contaminated zone in air after the burning of the e-waste was 75–100 cm above the ground (Wang and Zhang 2006), which was the height range for normal Chinese children 5–6 years of age.\nIn China, the mean BLL of children was 9.29 μg/dL, and 33.8% of the subjects had BLLs > 10 μg/dL; boys’ mean BLL was 9.64 μg/dL, significantly higher than the girls’ mean BLL of 8.94 μg/dL (p < 0.001) (Wang and Zhang 2006). Generally in China, BLLs of children living in industrial and urban areas were significantly higher than those of children in suburbs and rural areas (Wang and Zhang 2006). In Guiyu, the BLLs of children were higher than the mean level in China, and there were no significant different between boys and girls. Although Guiyu is rural, the children’s BLLs were nearly double those of a nearby urban area, Shantou City (7.9 μg/dL; Luo et al. 2003). Compared with results from studies conducted in some other part of Guangdong province, such as Zhongshan City (7.45 μg/dL; Huang et al. 2003) and Shenzhen City (9.06 μg/dL; Wang et al. 2003), we observed higher BLLs not only in Guiyu children, but also in Chendian children (9.94 μg/dL). The lead contamination may have spread from Guiyu to nearby Chendian by dust, river, and air and contributed to the elevation of Chendian children’s BLLs.\nIn conclusion, elevated BLLs in Guiyu children are common as a result of exposure to lead contamination caused by primitive e-waste recycling activities. Lead contamination from e-waste processing appears to have reached the level considered to be a serious threat to children’s health around the e-waste recycling area. Based on these threats, it is necessary to increase public awareness about the effects of exposure to lead from e-waste and arouse local governments’ interest in public health and safety, so that an infrastructure for safe management of e-waste can be established. More important, responsible management strategies should be undertaken to minimize e-waste production and make e-waste components more easily recycled and reused.\n\nCorrection\nIn the Abstract and Discussion, the percentage of Guiyu children with BLLs > 10 μg/dL has been corrected from 88% in the original manuscript published online to 81.8%.\n\n\n" ], "offsets": [ [ 0, 18166 ] ] } ]

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[ { "id": "pmcA1551914__text", "type": "Article", "text": [ "Travel-Related Venous Thrombosis: Results from a Large Population-Based Case Control Study (MEGA Study)\nAbstract\nBackground\nRecent studies have indicated an increased risk of venous thrombosis after air travel. Nevertheless, questions on the magnitude of risk, the underlying mechanism, and modifying factors remain unanswered.\n\nMethods and Findings\nWe studied the effect of various modes and duration of travel on the risk of venous thrombosis in a large ongoing case-control study on risk factors for venous thrombosis in an unselected population (MEGA study). We also assessed the combined effect of travel and prothrombotic mutations, body mass index, height, and oral contraceptive use.\nSince March 1999, consecutive patients younger than 70 y with a first venous thrombosis have been invited to participate in the study, with their partners serving as matched control individuals. Information has been collected on acquired and genetic risk factors for venous thrombosis. Of 1,906 patients, 233 had traveled for more than 4 h in the 8 wk preceding the event. Traveling in general was found to increase the risk of venous thrombosis 2-fold (odds ratio [OR] 2.1; 95% confidence interval [CI] 1.5–3.0). The risk of flying was similar to the risks of traveling by car, bus, or train. The risk was highest in the first week after traveling. Travel by car, bus, or train led to a high relative risk of thrombosis in individuals with factor V Leiden (OR 8.1; 95% CI 2.7–24.7), in those who had a body mass index of more than 30 kg/m2 (OR 9.9; 95% CI 3.6–27.6), in those who were more than 1.90 m tall (OR 4.7; 95% CI 1.4–15.4), and in those who used oral contraceptives (estimated OR > 20). For air travel these synergistic findings were more apparent, while people shorter than 1.60 m had an increased risk of thrombosis after air travel (OR 4.9; 95% CI 0.9–25.6) as well.\n\nConclusions\nThe risk of venous thrombosis after travel is moderately increased for all modes of travel. Subgroups exist in which the risk is highly increased.\n\n\nBackground.\nRecently there has been increasing concern that blood clots (thromboses) in the leg or lungs occur with greater frequency after air travel. Several theories have been put forward to explain why this increase might happen, including the fact that air passengers tend to not move around much, or possibly that reduced amounts of oxygen in the blood make the blood more likely to clot. Understanding what causes such clots is important as it would help us come up with suggestions of ways to prevent them.\n\nWhy Was This Study Done?\nIt is not possible to test in a controlled trial whether travel causes an increase in blood clots, so the next best way of studying this problem is to do a case-control study, in which people with blood clots (cases) are compared with similar people who don't have a blood clot (controls—in this case, the partners of the cases), and the differences in a number of contributing factors are assessed.\n\nWhat Did the Researchers Do and Find?\nSince 1999, the MEGA (Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis) study has aimed to identify all people in an area of the Netherlands who develop a blood clot for the first time, by seeking out people who receive treatment for blood clots. At the time of this report, 1,906 people with clots had been found; of these, 233 had traveled for more than four hours in the eight weeks preceding the event. Traveling in general was found to increase the risk of clots two-fold, and the risk was highest in the week after traveling. The risk of flying was similar to the risk of traveling by car, bus, or train, and was highest in the first week after traveling. Certain other factors increased the risk of a blood clot even more, such as having a particular mutation (known as factor V Leiden) in a gene involved in blood clotting, having a body mass index of more than 30 kg/m2 (over 30 kg/m2 is defined as being obese), being more than 1.90 meters tall, and using oral contraceptives. All these factors made the risk of clots especially after air travel worse; in addition, people shorter than 1.60 meters also had an increased risk of thrombosis after air travel. However, it should be borne in mind that the number of cases in each of these various groups was quite small, and the overall risk of getting a thrombosis is still low.\n\nWhat Do These Findings Mean?\nSince the risks of thrombosis are increased for all types of long travel, it seems that the main factor causing the thrombosis is immobility. However, since the risk is even higher for air travel, the relative lack of oxygen may also play a part. One interesting aspect of this study is that the researchers used partners as controls; in order to be sure that doing this did not make the results invalid, the researchers had to carefully adjust for differences between the cases and controls, such as the fact that partners were generally of the opposite sex. In a related Perspective (DOI: 10.1371/journal.pmed.0030300), Kenneth Rothman discusses the study further.\n\nAdditional Information.\nPlease access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030307.\n\n\n\n\nIntroduction\nInterest in the role of air travel in the pathogenesis of venous thrombosis has heightened in the past 5 y [1–5]. Venous thrombosis was first linked to air travel in 1954 [6], and as air travel has become more and more common, many case reports and case series have been published since. Several clinical studies have shown an association between air travel and the risk of venous thrombosis. In a series of individuals who died suddenly at Heathrow Airport, death occurred far more often in the arrival than in the departure area [7]. Two similar studies described a “dose-response” relation: the risk of pulmonary embolism in air travelers increased with the distance traveled [5,8]. A number of case-control studies, however, have shown conflicting results [9–11]. More recently, a 2-fold increased risk in patients who had traveled by air was described in a case-control study among 210 patients and 210 controls [3]. A case-crossover study based on record linking in Australia described a 4-fold increased risk of venous thrombosis in the first 2 wk after a long-haul flight [1]. In terms of absolute risk, two studies found similar results: one performed in New Zealand found a frequency of 1% of venous thrombosis in 878 individuals who had traveled by air for at least 10 h [2], and a German study found venous thrombotic events in 2.8% of 964 individuals who had traveled for more than 8 h in an airplane, as compared to 1% in 1,213 controls [4]. The events in both studies were mostly asymptomatic.\nThe available evidence suggests that the overall risk of venous thrombosis is moderately increased after air travel. Nevertheless, many questions remain unanswered: the exact underlying mechanism is still unknown, and, related to this, it is not clear whether the risk is increased after air travel only or after long-distance travel in general. Furthermore, the effect of the combination of other risk factors for venous thrombosis and travel has not yet been systematically studied, with the exception of a study by Martinelli et al., who found an additionally increased risk in patients with thrombophilia and patients who used oral contraceptives [3].\nThe Multiple Environmental and Genetic Assessment (MEGA) study of risk factors for venous thrombosis is a large ongoing case-control study aimed at assessing the combined effect of genetic and acquired risk factors for venous thrombosis. Cases and control individuals are questioned about—among many other items—travel that occurred shortly before the event. This provides an opportunity to assess the effect of travel on the risk of thrombosis in an unselected population, as well as the effect of the combination of travel with several other risk factors for thrombosis.\n\nMethods\nStudy Design\nSince March 1999, consecutive patients younger than 70 y with a first deep-vein thrombosis (DVT) or pulmonary embolism (PE) have been identified at six regional anticoagulation clinics in the Netherlands. Anticoagulant clinics monitor the anticoagulant therapy of all patients in a well-defined geographical area, allowing us to identify consecutive and unselected patients with thrombosis. Patients who were unable to fill in the questionnaire (because of language or severe psychiatric problems), as well as those who died soon after the venous thrombosis or who were in the end stage of a disease and for that reason did not participate, were not included. All others were considered eligible. Partners of these patients were invited as control individuals, and the same exclusion criteria were applied.\nAll participants filled in a detailed standardized questionnaire on general demographic and anthropomorphic characteristics, as well as risk factors for venous thrombosis. The questionnaire was sent to all participants within a few weeks after the event and covered the period of 1 y prior to the date of the thrombotic event (index date). When the participant was unable to fill in the questionnaire we asked questions by phone, using a standardized mini-questionnaire. Three months after the patients had discontinued their oral anticoagulant therapy, they were invited with their partners to the anticoagulation clinic for a blood sample. In those patients who continued to take oral anticoagulant therapy for more than 1 y after the event, blood was drawn during therapy. If participants were unable to come to the clinic, a buccal swab was sent by mail to replace the blood sample for DNA extraction.\nThe study protocol was approved by the Ethics Committee of the Leiden University Medical Center. Written informed consent was obtained from all participants [12].\n\nValidation Study of Thrombosis Diagnosis\nDischarge letters or diagnostic reports of the venous thrombotic event were obtained for a sample of 742 patients who had their first thrombosis between March 1999 and March 2000. The diagnostic management of the patients was compared to the diagnostic procedure as described in the Dutch consensus [13]. Diagnosis of clinically suspected DVT of the leg is based on a clinical score, serial compression ultrasonography, and D-dimer assay. Objective testing of clinically suspected pulmonary embolism is based on perfusion and ventilation scintigraphy, ultrasonography of the leg veins, pulmonary angiography, or helical computed tomography. Out of 395 patients with DVT of the leg, 384 (97%) were objectively diagnosed, while out of 347 patients with PE, 271 (78%) were confirmed with objective testing as certainly having PE. Since the diagnosis appears to be made by objective methods in virtually all cases of DVT, while being more ambiguous for PE, we also analyzed these two manifestations of venous thrombosis separately.\n\nCurrent Analysis\nFor the current analysis we were interested in the effects of travel, and its combined effect with other common risk factors for venous thrombosis. Patients with a solitary arm thrombosis were excluded from this analysis. Of 3,902 eligible cases, diagnosed up to May 2002, 656 did not participate for various reasons (such as not willing or not reachable), leading to a response of 83%. A further 3% responded only to the mini-questionnaire, taken by phone, which did not contain questions about travel. Of the remaining 3,111 cases, 78% had a partner, 77% of whom were willing to participate, which left 1,867 couples. Additionally, 229 partners were identified for whom the corresponding patient originally participated but was later found not to be eligible (aged over 70 y, or not a first thrombotic event). These control individuals were matched on sex and 5-y age groups to one of the 557 patients whose partner did not want to participate, so an extra 229 pairs were included, making a total of 4,192 participants (2,096 pairs). As part of the general questionnaire, questions had been asked about whether or not respondents had traveled for more than 4 h in the 3 mo before the index date, about the travel date, and about mode and duration of travel. We assessed the occurrence of thrombosis in relation to the period of time that had passed since traveling. Travel was defined in the analysis as at least one journey with a duration of at least four uninterrupted hours during the 8-wk period before the event. During the analysis it appeared that some individuals had provided dates of travel after the event instead of before. As there was only one opportunity to fill in such a date, we had no information about the period before the event. This was the case in 88 cases and 146 controls. We excluded these individuals and their partners, which left 3,812 participants (1,906 pairs) for the analysis.\nBecause we selected the partners of the cases as control individuals, and because it turned out, as expected, that couples tend to travel together, we performed a conditional logistic regression analysis to calculate odds ratios (ORs) for the relation between travel and venous thrombosis. This method fully takes this matching into account, and leads to unbiased estimates, with adjustment for all factors in which cases and controls tend to be similar, e.g., socioeconomic class [14]. Details of this method can be found in Protocol S1. The 95% confidence intervals (CIs) were derived from the model.\nWe assessed the combined effect of traveling and the following risk factors for thrombosis: factor V Leiden mutation, prothrombin G20210A mutation, body mass index (BMI, as kg/m2), and height. We were also interested in the combined effect of oral contraceptive use and travel. However, as the control individuals were nearly always of the opposite sex (partners of the cases were recruited as controls), it was not possible to perform a matched analysis for the combination of oral contraceptive use and travel. Therefore, we performed a case-only analysis [15]. This method allows one to examine the association between two exposures among case individuals only. ORs are interpreted as a synergy index (SI) on a multiplicative scale, with independence assumed between the exposures. As this analysis depends only on cases, it was possible to perform it in all consecutive cases, therefore also including those without a partner.\n\nLaboratory Measurements\nBlood was collected from the antecubital vein into vacuum tubes containing 0.106 mol/l trisodium citrate. High molecular weight DNA was isolated from leukocytes using a standard salting-out procedure [16] and stored at −20 °C. When a blood sample was not available, DNA was extracted from buccal swabs. Three large cotton swabs in a total of 6 ml of SDS–proteinase K solution (100 mM NaCl, 10 mM EDTA, 10 mM Tris-HCl [pH 8.0], 0.5% SDS, 0.1 mg/ml proteinase K) were obtained. Upon arrival, the proteinase K concentration was raised to 0.2 mg/ml, and the sample was incubated for 2 h at 65 °C. Subsequently, the solute was recovered by centrifugation. Potassium acetate was added to the supernatant to a final concentration of 1.6 M. After 15 min incubation on ice, proteins were removed using chloroform/isomylalcohol (24:1) treatment. The DNA in the water phase was subsequently ethanol precipitated. After centrifugation, the pellet was resuspended in 200 μl of 10 mM Tris-HCl and 10 mM EDTA (pH 8.0), and frozen at −20 °C until further analysis. The factor V Leiden mutation (G1691A) and the prothrombin mutation (G20210A) were simultaneously detected by duplex polymerase chain reaction [17,18]. The technician was blinded concerning the origin of the sample, i.e., whether it was from a patient or from a control individual.\n\n\nResults\nVenous Thrombosis in Relation to Travel\nTable 1 shows general characteristics of the 1,906 patients. They ranged in age from 18 to 69 y (median 50.4 y); 51% were men. Diagnosis was DVT in 57% of the cases, PE in 32%, and both in 11%. As partners of the cases were included as control individuals, the sex distribution of the control individuals was the opposite; the age distribution differed only trivially.\nOf the patients, 233 individuals (12%) had traveled for at least 4 h by air, bus, car, or train within the 8 wk preceding the index date, as compared to 182 of the control individuals (9.5%). As the cases and control individuals were selected as couples, many pairs (135) had traveled together and were uninformative: as a consequence, 145 pairs in which either the patient (98) or the control (47) had traveled could be used for the matched analysis (Table 2). This analysis showed a 2-fold increased risk of venous thrombosis for all modes of travel combined (OR 2.1; 95% CI 1.5–3.0) compared to not traveling. For air travel alone, 49 individuals (31 cases and 18 controls) had traveled without their partner, and the analysis yielded an OR of 1.7 (95% CI 1.0–3.1). For the other modes of travel (car, bus, and train) the relative risks were essentially similar to each other and to that of air travel (Table 2).\nThe risk of venous thrombosis was not clearly related to increased duration of travel (Table 2). Of the 233 events that occurred within 8 wk after traveling, 68 (29%) were diagnosed in the first week, after which the incidence gradually decreased (Figure 1).\n\nThe Effect of Other Risk Factors Combined with Travel\nProthrombotic mutations.\nInformation on the factor V Leiden mutation and prothrombin G20210A genotype was available for 1,713 patients (90%) and for 1,629 of the control individuals (85%). Factor V Leiden was present in 259 cases (14%) and 84 control individuals (4%) (OR 3.1; 95% CI 2.4–4.1).\nThe risk of venous thrombosis was 8-fold increased in people with factor V Leiden who had traveled by bus, car, or train (modes combined) as compared to noncarriers who did not travel (OR 8.1; 95% CI 2.7–24.7). For the combined effect of air travel and factor V Leiden, the risk seemed even slightly higher (OR 13.6; 95% CI 2.9–64.2).\nThe prothrombin G20210A mutation was found in 83 cases (4%) and in 29 control individuals (2%) (OR 2.7; 95% CI 1.7–4.2). The risk in individuals with this mutation who had traveled was difficult to interpret because of the small numbers but appeared not to increase more than additively (Table 3).\n\nBMI.\nThe effect of BMI was studied by dividing individuals into three categories with the following BMI values: <25, 25–30, and >30 kg/m2 [19]. A BMI of 25–30 kg/m2 was associated with an increased risk of venous thrombosis (OR 1.4; 95% CI 1.2–1.7), and the risk was slightly higher in patients with a BMI of 30 kg/m2 or more (OR 1.7; 95% CI 1.4–2.1).\nThe combined effect of a higher BMI and travel was the sum of the individual risks (Table 3), with the exception of people with a BMI of more than 30 kg/m2 who traveled by car, bus, or train, for whom the risk was 10-fold increased (OR 9.9; 95% CI 3.6–27.6). This increase in risk was not found in people who traveled by air.\n\nHeight.\nParticularly short or tall people may be subjected during travel to even more unnatural sitting positions than individuals with average height. Therefore, we assessed the effect of extremes of heights in combination with travel on the risk of venous thrombosis by comparing short (less than 1.60 m) and tall individuals (more than 1.90 m) with people of average height (1.60–1.90 m). Compared to people of average height, the risk of venous thrombosis was lower for short people (OR 0.7; 95% CI 0.5–0.9) and did not differ for very tall individuals (OR 0.9; 95% CI 0.7–1.1). The risk was found to be increased in people of more than 1.90 m who traveled (OR 4.7; 95% CI 1.4–15.4 for travel by car, bus, or train; OR 6.8; 95% CI 0.8–60.6 for air travel) compared to non-traveling people of average height. Interestingly, the risk of venous thrombosis was also increased in short people but only after air travel (OR 4.9; 95% CI 0.9–25.6), not after other modes of travel (OR 1.0; 95% CI 0.3–2.8, all relative to non-traveling people of average height).\n\nOral contraception.\nTo study the association between oral contraceptive use, travel, and the risk of venous thrombosis, we performed a case-only analysis in all female patients who were less than 50 y of age. As we needed only cases, it was also possible to include women without a partner for this analysis, which led to a total of 1,025 women aged under 50. Non-users who did not travel were used as the reference group. The case-only estimate of the SI for women who traveled by car, bus, or train was 2.4 (95% CI 1.5–3.7). This indicates that the OR for the combination of travel and oral contraceptive use is 2.4 times the product of the separate ORs. As oral contraceptive use generally increases the risk of venous thrombosis about 4-fold [20], the combination with travel by car, bus, or train would lead to an estimated OR of about 20 (4 × 2 × 2.4). A clearly stronger interaction of travel by air with oral contraceptive use was found: the case-only estimate of the SI was 4.9 (95% CI 2.1– 11.4), which would result in an OR of about 40 (4 × 2 × 4.9).\n\n\nEffect of Risk Factors in DVT Patients Only\nOf the 1,906 cases, 1,082 were diagnosed with DVT. As the diagnosis was more unambiguous in these patients (97% objectively diagnosed as compared to 78% of the PE patients), we repeated the analysis in these patients only.\nIn this analysis, the overall effect of travel on the risk of DVT was equal to the effect on all venous thrombosis (DVT and PE combined). However, here we found a stronger risk for travel by air (OR 3.0; 95% CI 1.3–7.1) then for travel by car, bus, or train (OR 1.9; 95% CI 1.1–3.2) (Table 4). Also, the analysis of the combination of other risk factors with travel resulted in more clear-cut effects, despite the smaller number of cases: the risk of DVT was still clearly synergistically increased in patients with factor V Leiden who traveled, whereas the prothrombin G20210A mutation did not further increase the risk of travel (Table 4). Furthermore, a BMI of more than 30 kg/m2 in combination with travel yielded high ORs for DVT both in people who traveled by car, bus, or train and in those who flew. Being more than 1.90 m tall in combination with travel resulted in higher ORs for DVT; the risk for short people was more increased after travel by air (OR 6.8; 95% CI 1.1–43.5) (Table 4). The effect of oral contraceptive use in combination with travel by car, bus, or train on the risk of DVT was studied in 589 women and was somewhat lower than the effect on the risk of all venous thrombosis (SI 1.9; 95% CI 0.9–4.2). In those who traveled by air it was also a bit lower (SI 3.4; 95% CI 1.3–8.8), but still indicative of a strong synergistic effect.\n\n\nDiscussion\nIn this population-based case-control study, long-distance traveling increased the risk of venous thrombosis 2-fold. Travel by air increased the risk to the same extent as travel by car, bus, or train. The risk was highest in the first week after traveling. As venous thrombosis is a disease in which many factors (genetic and acquired) interact [21], we identified groups with additional risk factors in which the risk was further increased. This was the case for individuals with factor V Leiden, obese people (BMI > 30 kg/m2), and short (only for travel by air) and tall people, as well as for women using oral contraceptives. Some of these synergistic effects were more apparent for air travel.\nAlthough the studies that have been published so far have not yielded entirely consistent results, those that did report an increased risk of venous thrombosis in air travelers showed similar risk estimates of a 2- to 3-fold increased risk (even in one with asymptomatic events only [4]). The occurrence of venous thrombosis was highest in the first week after travel, and slowly declined afterwards, a pattern that was also described in a recent record-linking study from Australia [1], supporting a causal relation.\nAs a possible mechanism for an extra risk in travelers who fly, an effect of hypobaric hypoxia on the coagulation system was postulated, which has already been studied a number of times, mainly in hypobaric chambers, with unclear results so far. Our study showed an increased risk in all types of travel, which suggests that the increased risk of flying is caused mainly by immobilization. Additionally, the risk is further increased in short and tall people, who are likely to experience more immobilization and venous compression than other travelers. However, as some of our findings were more pronounced for air travel, we cannot exclude an additional effect of hypobaric hypoxia, possibly in risk groups only. This possibility is supported by a recent study of our group [22] in which we found that thrombin generation occurred in some healthy volunteers after flying for 8 h but happened to a far lesser extent after being immobilized for 8 h in a cinema. The high response in the fliers was associated with the presence of risk factors for thrombosis, i.e., oral contraceptive use, the factor V Leiden mutation, and the combination of the two. This finding indicates an effect of an additional factor in an airplane, such as hypobaric hypoxia, to which mainly individuals with risk factors respond.\nNone of the studies published so far have systematically studied the effect of traveling in combination with other risk factors, with the exception of the study by Martinelli et al. [3]. In an analysis of 210 patients, they found a 16-fold increased risk for patients who traveled by air and had some form of thrombophilia, as well as a 14-fold increased risk in women who flew and used oral contraceptives, findings that confirm both the results of the present study and our finding of activated coagulation in individuals with risk factors after flying [22].\nThe finding that taller and shorter people had an increased risk of venous thrombosis after traveling should be interpreted with some caution, as the numbers were small in these strata. On the other hand, it is biologically plausible: very tall people are subjected to even more cramped seating than average-height individuals, and very short people's feet may not touch the floor, which would lead to extra compression of the popliteal veins. Interestingly, the increased risk for short people was only found in people who traveled by air. This may have to do with the fact that seats in cars are generally lower, and more individually adjustable, than those in airplanes.\nAs the diagnosis of DVT is usually more unambiguous than that of PE [23], as was the case in our study population as well, we repeated the analysis using only DVT as the outcome of interest (97% objectively diagnosed). In this analysis, despite using smaller numbers, most findings were either similar or appeared more evident, and inconsistencies that were found when using both DVT and PE as endpoints disappeared.\nTo our knowledge, this is the first large population-based case-control study in which the effect of travel on the risk of venous thrombosis has been studied. Because the control individuals were closely matched, being partners of the cases, and couples tend to travel together, only the cases and control individuals who had not traveled together could be used for the analysis. Also because of this design, the effect of sex and age could not be studied. It has to be noted, however, that for all other research questions on the effect of genetic and acquired risk factors on the risk of venous thrombosis, this design has no limitations and the close matching of cases and controls renders confounding by, for instance, lifestyle and socioeconomic class less likely than in previous unmatched studies (see also Protocol S1). Another advantage of this approach is the minimization of recall bias, as the cases and controls would generally fill in the questionnaire together.\nMany questions are still left unanswered that necessitate more research. First of all, our study results apply only to people younger than 70 y of age. Furthermore, it is likely that other characteristics exist that also increase the risk—person-specific (e.g., other drug use), behavioral (e.g., use of sleeping pills or alcohol consumption), and flight-specific (e.g., class or seating)—that need to be identified. These further variables are part of our ongoing study as part of the World Health Organization Research Initiative into the Global Hazards of Travel (WRIGHT study). For those who have an increased risk, such as oral contraceptive users and individuals with factor V Leiden, prevention may be warranted. Prevention may vary from simple measures, such as exercises during the flight, to measures that carry a risk themselves, such as anticoagulants. Specific studies are needed to assess the efficacy of these measures and their risk–benefit ratio.\nIt can be concluded that the risk of venous thrombosis is 2-fold increased for all travelers and to the same extent for all modes of travel. In individuals who use oral contraceptives, are carriers of the factor V Leiden mutation, or are particularly tall, short, or obese, this risk is considerably higher, to such an extent that studies into the efficacy of prophylactic measures are required.\n\nSupporting Information\n\n\n" ], "offsets": [ [ 0, 29331 ] ] } ]

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[ { "id": "pmcA1075922__text", "type": "Article", "text": [ "Tissue-dependent isoforms of mammalian Fox-1 homologs are associated with tissue-specific splicing activities\nAbstract\nAn intronic hexanucleotide UGCAUG has been shown to play a critical role in the regulation of tissue-specific alternative splicing of pre-mRNAs in a wide range of tissues. Vertebrate Fox-1 has been shown to bind to this element, in a highly sequence-specific manner, through its RNA recognition motif (RRM). In mammals, there are at least two Fox-1-related genes, ataxin-2 binding protein 1 (A2BP1)/Fox-1 and Fxh/Rbm9, which encode an identical RRM. Here, we demonstrate that both mouse Fxh and A2BP1 transcripts undergo tissue-specific alternative splicing, generating protein isoforms specific to brain and muscle. These tissue-specific isoforms are characterized for their abilities to regulate neural cell-specific alternative splicing of a cassette exon, N30, in the non-muscle myosin heavy chain II-B pre-mRNA, previously shown to be regulated through an intronic distal downstream enhancer (IDDE). All Fxh and A2BP1 isoforms with the RRM are capable of binding to the IDDE in vitro through the UGCAUG elements. Each isoform, however, shows quantitative differences in splicing activity and nuclear distribution in transfected cells. All Fxh isoforms and a brain isoform of A2BP1 show a predominant nuclear localization. Brain isoforms of both Fxh and A2BP1 promote N30 splicing much more efficiently than do the muscle-specific isoforms. Skeletal muscles express additional isoforms that lack a part of the RRM. These isoforms are incapable of activating neural cell-specific splicing and, moreover, can inhibit UGCAUG-dependent N30 splicing. These findings suggest that tissue-specific isoforms of Fxh and A2BP1 play an important role in determining tissue specificity of UGCAUG-mediated alternative splicing.\n\nINTRODUCTION\nAlternative splicing of pre-mRNA is one of the fundamental mechanisms for the regulation of gene expression in higher eukaryotes (1,2). Developmentally regulated, cell type- or tissue-specific, and signal-induced alternative splicing of pre-mRNAs takes place in multicellular organisms throughout their lifetimes. Misregulation or abnormalities in pre-mRNA splicing, in some instances, leads to cellular dysfunctions found in human and animal diseases (3–5). Using various model systems of regulated alternative splicing, a number of pre-mRNA features that influence alternative splice site selection have been defined (1,2). These include enhancer and repressor RNA sequences located in exons and introns. Identification of RNA-binding proteins targeting these cis-regulatory elements is currently in progress. In vertebrates, participation of the SR family proteins and hnRNP proteins, such as PTB and hnRNPA1, in alternative splicing regulation via binding to the cis-regulatory elements have been shown in many tissue-specific splicing models (6,7). Although these RNA-binding proteins are ubiquitously expressed, their different abundance in different cells, differences in their post-translational modifications in different cellular contexts and their different abilities to assemble multiprotein complexes in different pre-mRNA contexts are thought to contribute to the determination of cell type-specific patterns of alternative splicing. For the last few years, tissue-specific and tissue-enriched RNA-binding proteins have begun to be identified as splicing regulators. These include brain-specific (or enriched) Nova-1, nPTB (brPTB) and some of the CELF family proteins (8–12). Discovery of these proteins has had a great impact on studies aimed at understanding the molecular mechanisms of alternative splicing regulation.\nOne of the intronic cis-elements, which are involved in tissue-specific or differentiation stage-dependent regulation of alternative splicing, is the hexanucleotide UGCAUG. The importance of this element was originally recognized in fibronectin pre-mRNA by Huh and Hynes (13). Since then, alternative splicing specific to a variety of tissues or cell types, including neural cells, muscles, epithelial cells and erythrocytes, has been shown to be modulated via this element (14–21). Recently, Jin et al. (21) discovered that a zebrafish homolog of Caenorhabditis elegans Fox-1 (22) could bind specifically to the pentanucleotide GCAUG by in vitro selection from randomized RNA sequences. This pentanucleotide is almost identical to the hexanucleotide UGCAUG except for the first U. Moreover, the zebrafish Fox-1 homolog, as well as the mouse Fox-1 homolog, are capable of repressing the inclusion of an alternative cassette exon of the ATP synthase F1γ pre-mRNA via binding to GCAUG, which mimics muscle-specific exclusion of this exon. This mouse homolog is identical to the ataxin-2 binding protein 1 (A2BP1), which has been previously cloned in humans and mice as the cDNA encoding a protein, which interacts with ataxin-2, the product of the causative gene for spinocerebellar ataxia type 2 (23,24). In addition to A2BP1/Fox-1, another mouse homolog of C.elegans Fox-1, Fxh, has been independently cloned as a cDNA, which is induced by androgen in motor neurons (25). Of note is that A2BP1/Fox-1 and Fxh share an identical RNA recognition motif (RRM) at the amino acid level. Therefore, two genes in the mouse genome encode homologs of nematode Fox-1. According to the names given by the first cDNA cloning, we used the nomenclature of A2BP1 and Fxh in this report. A2BP1 and Fxh have been named A2BP1 and Rbm9, respectively, in the human and mouse genomes.\nWe have been studying regulatory mechanisms of neural cell-specific alternative splicing using the non-muscle myosin heavy chain II-B (NMHC-B) gene as a model system (14,26). NMHC-B mRNA is expressed ubiquitously. However, an alternative exon, N30, which encodes a 30 nt coding sequence, is included in the mRNAs from some neural cells, but is skipped in those from all other cells in mammals and birds (27,28). In cultured cells, a switch in N30 splicing from exclusion to inclusion can be seen in neural retinoblastoma Y79 cells during the post-mitotic and differentiated stages triggered by butyrate treatment. We have previously defined an intronic distal downstream enhancer (IDDE), which confers neural cell specificity on N30 inclusion, using this cell line (14). The IDDE includes two copies of UGCAUG. Mutation of these hexanucleotides results in N30 skipping in post-mitotic differentiated Y79 cells.\nIn this study, we investigated the possible involvement of A2BP1 and Fxh in the regulation of N30 splicing. To this end, we have isolated cDNA clones for A2BP1 and Fxh from brain and muscles. cDNA cloning revealed the existence of tissue-specific (enriched) isoforms of both A2BP1 and Fxh. Of importance, different isoforms of A2BP1 and Fxh show different activities with respect to N30 splicing as well as different subcellular localizations.\n\nMATERIALS AND METHODS\nDatabase disposition\nThe sequences reported in this paper have been deposited in the GenBank database with accession numbers AY659951 (F011), AY659952 (F411), AY659953 (F402), AY659954 (A016), AY659955 (A030), AY659956 (A713), AY659957 (A715) and AY659958 (A704).\n\nRNA preparation and RT–PCR\nTotal RNAs were isolated from mouse tissues and cultured cells using an RNA isolation kit (Stratagene) or an RNeasy mini kit (Qiagen). To obtain the full-length coding regions of cDNAs for Fxh and A2BP1, RT–PCRs were performed using Superscript II RNase H− reverse transcriptase (Invitrogen) and Pfu Turbo DNA polymerase (Stratagene). The PCR primers used to obtain all Fxh cDNAs were 5′-ctcaggcctccactagttATGGAGAAAAAGAAAATGGTAACTC-3′ and 5′-ctcaggcctcctctagaaGTAGGGGGCAAATCGGCTGTA-3′. The upstream primers for the brain (A016 and A030) and muscle (A713, A715 and A704) A2BP1 cDNAs were 5′-ctcaggcctccactagtgATGAATTGTGAAAGAGAGCAGCT-3′ and 5′-ctcaggcctccactagtcATGTTGGCGTCGCAAGGAGTCC-3′, respectively, and the downstream primer for all A2BP1 cDNAs was 5′-ctcaggcctcctctagagATATGGAGCAAAACGGTTGTATCC-3′. Lower case letters represent adapter sequences including restriction enzyme sites. 5′ Rapid amplification of cDNA ends (RACE) was performed using Marathon-Ready cDNA (BD Biosciences Clontech). For the analysis of minigene and NMHC-B mRNAs, RT–PCRs were performed as described previously (14,26). Sequences of primers P1–P9 shown in Figures 1, 4 and 6 are as follows: P1, 5′-AATTCACCCAGCAACCAGAAT-3′; P2, 5′-TAGAGGGATGTAAGTGTTGATGCC-3′; P3, 5′-CAGAGGGCGGACAGTGTATGGT-3′; P4, 5′-GGCGGCAGGGGCGAGGGCAT-3′; P5, 5′-CCGTGGTCGCACCGTGTACAAC-3′; P6, 5′-CAGCGGCAGTGGCAGGGGTG-3′; P7, 5′-AGGAAGAAAGGACCATAATATTCC-3′; P8, 5′-CCTCCACCCAGCTCCAGTTGT-3′; and P9, 5′-CCTGTAGTTATTAAATCCTTCAAG-3′.\n\nPreparation of expression constructs and minigenes\nThe cDNAs of Fxh and A2BP1 were introduced into a plasmid pCS3 + MT, which contains a myc-epitope, and its modified version pCS3 + MT + NLS, which in addition contains the nuclear localization signal (NLS) of the SV40 large T antigen (26). Minigenes G, J without the IDDE, and H with the wild-type IDDE are the same as minigenes W, D4 and Cm0, respectively in ref. (14). The 201 nt IDDEs with mutations were generated by recombinant PCR using the appropriate primers, which included mutated sequences. The hexanucleotide TGCATG sequences at the 5′ and 3′ sides were changed to GTTACT and ACCTAC, respectively.\n\nElectrophoresis mobility shift assay\nTemplate DNAs for in vitro RNA transcription were prepared by PCR using the wild-type and mutant IDDEs in the minigenes as templates and an upstream primer that included the T7 promoter sequence at the 5′ end. The probe and competitor RNAs were transcribed by T7 RNA polymerase in the presence of [α-32P]UTP and a trace amount of [35S]UTPαS, respectively, using a MAXIscript kit (Ambion). Mole concentrations of synthesized RNAs were estimated by radioactivities. Fxh and A2BP1 proteins with a myc tag were synthesized in vitro by using a TNT quick-coupled transcription/translation system (Promega) from pCS3 + MT constructs, which include the SP6 promoter. Binding reactions were carried out in a 10 μl mixture that contains 10 mM HEPES (pH 7.9), 2 mM MgCl2, 50 mM KCl, 5% glycerol, 0.5 mM DTT, 5 μg tRNA, 1 μl reticulocyte lysate reaction mixture and 15 fmol of probe, on ice for 20–30 min. An aliquot of 5 μg of heparin was added to the reaction 10 min before gel electrophoresis. The reaction mixtures were analyzed by electrophoresis in a 6% polyacrylamide gel using a 0.5× TBE buffer (Invitrogen).\n\nCell culture and transfection\nThe human retinoblastoma cell line Y79 was cultured and transfected with DNAs as described previously (14,26). Total amounts of transfected DNAs were adjusted to be constant by addition of the empty vector. Either Lipofectin (Invitrogen) or Effectene transfection reagent (Qiagen) was used for the transfection. For stable transfection, the pCS3+MT expression constructs were co-transfected with a plasmid carrying a neomycin resistant gene and selected by 0.2 mM geneticin (Invitrogen). For differentiation of Y79 cells, cells were plated on the poly-d-lysine-coated plates and then treated with 2.0–2.5 mM sodium butyrate for 4–5 days. HeLa cells were cultured as described and transfected with DNA using Effectene reagent (14,26).\n\nImmunoblot analysis\nSamples that required both protein and mRNA analysis were split upon harvesting. Total cell proteins were subjected to SDS–PAGE and blotted as described previously (26). The primary antibodies used are monoclonal antibodies to a myc-epitope (Invitrogen) and green fluorescent protein (GFP) (Clontech). Binding of antibodies was detected with the SuperSignal System (Pierce) or ECL (Amersham).\n\nImmunofluorescent microscopy\nHeLa or Y79 cells grown in a four-chamber glass slide were transfected as described above. Cells were fixed with 10% formaldehyde 24–48 h after transfection, and permealized with 0.5% Triton X-100 then blocked with 5% goat serum. Primary antibodies used were mouse anti-myc (Invitrogen), rabbit anti-NMHC-B (29). Secondary antibodies were Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes). DAPI was used for DNA staining. Specimens were mounted in ProLong antifed kit (Molecular Probe). The images were collected using Leica SP confocal microscopy (Leica).\n\n\nRESULTS\nTissue-dependent isoforms of Fxh and A2BP1 and their subcellular distribution\nThe A2BP1 mRNAs are detected almost exclusively in brain and striated muscles (heart and skeletal muscles) in adult mice, whereas the Fxh mRNAs are expressed in a wide variety of tissues with the highest expression in brain and heart [(21,25) and S. Kawamoto, unpublished data]. These expression profiles prompted us to characterize the mRNAs of A2BP1 and Fxh in brain and muscles. We have cloned cDNAs for two gene transcripts from brain, heart and skeletal muscles by RT–PCR and 5′ RACE. The isolated cDNAs are schematically presented with genomic structures in Figure 1 (for amino acid sequences see also Figure 8). Both gene transcripts are found to undergo tissue-specific alternative splicing. In both cases, brain and striated muscles express unique splice variants generated by mutually exclusive splicing of exons B40 and M43, respectively, which provide different coding sequences in the middle of the carboxyl half of the molecules. Southern blot analysis of the RT–PCR products of the Fxh mRNAs from the different tissues probed by oligonucleotides corresponding to B40 and M43 shows that M43 is exclusively used in heart and skeletal muscles, while B40 is predominantly used in brain (Figure 2A). Digestion of the RT–PCR products of the A2BP1 mRNAs with restriction enzymes unique to B40 and M43 demonstrates that B40 and M43 are used almost exclusively in brain and muscles, respectively (Figure 2B). A2BP1 contains an additional cassette type alternative exon A53, consisting of 53 nt. Inclusion and exclusion of exon A53 results in two different amino acid sequences at the C-terminal region owing to a frame shift. In the case of the A2BP1 gene, moreover, it appears that brain and striated muscle utilize alternative promoters, resulting in different amino acid sequences at the N-terminus. The 5′ RACE of skeletal muscle mRNAs yielded essentially a single species of sequence with 29 unique N-terminal amino acids. The 5′ RACE using brain mRNAs; however, yielded multiple products with multiple deduced amino acid sequences, all of which differ from the muscle amino acid sequence at the very N-terminus. Here, we focus our analysis on a clone containing nine unique amino acids at the N-terminus, which was obtained most frequently. Exon–intron organization of Fxh and A2BP1 shows remarkable similarities and a RRM is encoded in four exons (Figure 1B). Of note is that the significant amounts of Fxh and A2BP1 mRNAs from skeletal muscles are missing a part of the RRM by exon skipping. Typically, as shown in Figure 2C, they lack the 93 nt exon that encodes RNP1, one of the two most critical motifs of the RRM (30). Some of them lack almost the entire RRM (e.g. F402 in Figure 1A).\nTo examine the subcellular distribution of each isoform of Fxh and A2BP1, myc-tagged proteins were transiently expressed in cultured cells and immunostained with an anti-myc antibody. Initially, HeLa cells were used to investigate subcellular localization, since these cells are more suited for these studies. Representative confocal images are shown in Figure 3A. DAPI and anti-NMHC-B antibodies serve as markers for nuclei and cytoplasm, respectively. As noted, the ratio of protein distributed between nuclei and cytoplasm differs among the proteins. All isoforms of Fxh have a predominant nuclear localization. The brain isoform of A2BP1 without the A53 exon (A016) localizes to both nuclei and cytoplasm, whereas the other brain isoform with the A53 exon (A030) localizes predominantly to cytoplasm with only a minimum being in the nuclei. The relative amounts of both muscle isoforms of A2BP1 (A713 and A715) are somewhat between the amounts of the two brain isoforms. These data are summarized in Figure 1A. The subcellular distribution of representative isoforms (F011, A016 and A030) was also examined in retinoblastoma Y79 cells, which were used as host cells for the transfection experiments in order to characterize splicing activities of Fxh and A2BP proteins (see below). Although Y79 cells have a spherical shape and have only thin cytoplasm, exogenously expressed isoforms of Fxh and A2BP1 show a similar subcellular distribution as in HeLa cells (Figure 3B). F011 localizes predominantly to nuclei, A016 to both nuclei and cytoplasm and A030 predominantly to cytoplasm.\n\nSpecific interaction of Fxh and A2BP1 with IDDE via a hexanucleotide UGCAUG\nFxh and A2BP1 share an identical RRM and A2BP1 has been reported to bind specifically to the pentanucleotide GCAUG through this RRM (21). We have previously reported that the IDDE of the NMHC-B transcript, which is indispensable for the regulation of neural cell-specific cassette type exon N30 splicing, has two copies of GCAUG (14). Therefore, we investigated whether Fxh and/or A2BP1 bound to the IDDE. Electrophoretic mobility shift assays (EMSAs) were carried out using labeled IDDE and in vitro transcribed and translated Fxh and A2BP1, which include a myc-epitope. Since all Fxh and A2BP1 isoforms, except F402 and A704, contain the identical RRM, representative isoforms were analyzed. The expression of F011 in reticulocyte lysate causes the formation of a RNA–protein complex whose migration shift distinguishes it from those of the control reticulocyte lysate (C in Figure 4B, lanes 3 and 10). The unlabeled wild-type IDDE competes with the probe efficiently for the formation of the specific complex C (Figure 4B, lane 4). On the other hand, the mutant mc (Figure 4A), which has a mutation in both copies of UGCAUG, does not (Figure 4B, lane 7). The mutant ma, which has a mutation in the hexanucleotide at the 5′ side, shows less efficient competition, compared with mb, which has a mutation in the 3′ side of the hexanucleotide (Figure 4B, lanes 5 and 6), indicating that the nucleotides at the 5′ side are more important than those at the 3′ side. The presence of an anti-myc antibody, but not a non-specific antibody, inhibits the formation of the specific complex (Figure 4B, lane 8). Synthesis of the full-length F011 protein using a reticulocyte lysate and specificity of the myc antibody for the expressed protein are verified by immunoblot analysis (Figure 4B, lanes 11 and 12). A016 and A030 show essentially identical results to those with F011 (data not shown). These results indicate that Fxh and A2BP1 can bind to the IDDE and that the hexanucleotide UGCAUG is required for their binding.\n\nFxh and A2BP1 enhance N30 inclusion in a UGCAUG-dependent manner\nTo study whether Fxh and A2BP1 regulate neural cell-specific splicing via binding to UGCAUG, the Fxh and A2BP1 expression constructs were co-transfected into retinoblastoma Y79 cells with a number of the reporter minigene constructs, which include the wild-type or a mutant version of UGCAUG in the IDDE (Figure 4A). Minigenes H and J consist of the exons E5, N30 and E6 and their flanking introns with some deletions in the introns. The IDDE with or without mutations in the hexanucleotide is included or excluded between N30 and E6. As described above, each isoform of Fxh and A2BP1 enters the nucleus to a different extent. To see the effects of different proteins on N30 splicing itself, independent of their differential properties in nuclear localization, an exogenous NLS was added to the expressed proteins. Since Fxh and A2BP1 proteins contain the identical RRM, and F011, A016 and A030 all show the same UGCAUG-dependent binding to the IDDE in vitro, F011 and A030 were used for these experiments. Host cells Y79 at the proliferating stage exclude the N30 exon in ∼90% of the endogenous NMHC-B mRNAs (e.g. see Figure 6A, lane 1).\nAs shown in Figure 4C, the mRNAs derived from minigene J exclude N30 without exogenous expression of Fxh or A2BP1 in Y79 cells, similar to the endogenous NMHC-B mRNAs (Figure 4C, upper panel, lanes 1–5). However, in the presence of exogenous expression of F011 and A030, the N30 inclusion is increased (Figure 4C, upper panel, lanes 7 and 12). The N30 inclusion is absolutely dependent on the presence of the IDDE (Figure 4C, upper panel, lanes 6 and 11). Moreover, mutation of either one of the two copies of UGCAUG (ma, mb) abolishes the inclusion of N30 (Figure 4C, upper panel, lanes 8–10, 13–15). In the context of minigene H, which contains shorter introns, the larger extent of N30 inclusion is induced by either F011 or A030 overexpression (Figure 4C, middle panel, lanes 7 and 12). The N30 inclusion of the minigene H mRNAs also depends on the presence of the IDDE (Figure 4C, middle panel, lanes 6 and 11). Mutation of both copies of UGCAUG (mc) results in a complete loss of N30 inclusion (Figure 4C, middle panel, lanes 10 and 15). The mutant ma shows stronger inhibition of N30 inclusion compared with mb (Figure 4C, middle panel, lanes 8 and 9). This observation is consistent with the competition experiments of the EMSA shown in Figure 4B, indicating that the 5′ hexanucleotide is more important than the 3′ hexanucleotide for binding of Fxh or A2BP1 to the IDDE as well as an activation of N30 splicing. Comparable amounts of F011 and A030 are expressed in each transfection as verified by immunoblots using an anti-myc antibody (Figure 4C, lower panel, lanes 6–17).\nSince minigenes J and H lack a portion of the intron between exons N30 and E6 and, therefore, the IDDE is located ∼100 nt downstream of N30, instead of 1.5 kb as in the native gene, we also analyzed the effects of Fxh and A2BP1 on the N30 splicing of the wild-type minigene G, which includes full-length introns among E5, N30 and E6 (Figure 4A). As shown in Figure 4C (right panel), both F011 and A030 are capable of promoting N30 inclusion, with F011 showing a higher activity (Figure 4C, lanes 16 and 17). Although A030 can promote N30 inclusion as efficiently as F011 in the contexts of the minigene J and H transcripts, it can do so less efficiently than F011 in the context of the minigene G transcript. Interpretation of this observation will be discussed below.\nTaken together, Fxh and A2BP1 can activate N30 inclusion in an IDDE-dependent manner. The hexanucleotide motif UGCAUG is indispensable for this activation.\n\nDifferential activities of alternatively spliced isoforms of Fxh and A2BP1 in promoting N30 inclusion\nBoth Fxh and A2BP1 mRNAs are expressed in brain and A2BP1 is also expressed in striated muscles and Fxh is expressed in an even wider variety of tissues. As demonstrated above, however, both Fxh and A2BP1 transcripts undergo tissue-dependent alternative splicing, producing muscle-specific and brain-enriched isoforms. Therefore, the relative activity of individual isoform of Fxh and A2BP1 in promoting N30 inclusion was compared using minigene G.\nFirst, in order to evaluate the relative specific activity of each isoform in the splicing reaction separately from its ability to localize to nuclei, an exogenous NLS was included in the expressed protein to equalize the nuclear concentration of the expressed protein in these experiments. Essentially, all of the expressed proteins with the exogenous NLS are localized to the nucleus (data not shown). Therefore, the relative nuclear concentrations of the expressed proteins can be estimated easily by immunoblots. As shown in immunoblots in Figure 5A, similar quantities of proteins are expressed in a dose-dependent manner. Expression of the brain isoform F011 causes a dose-dependent increase in N30 inclusion and the extent of N30 inclusion reaches a plateau with ∼85% of the mRNAs including N30. In contrast, inclusion of N30 promoted by F411, the predominant isoform from skeletal muscles, reaches a plateau with only 40% of the mRNAs. With respect to A2BP1, the brain isoform A030 shows higher activity in N30 inclusion than the muscle isoform A715, which shows almost no activation, as shown in Figure 5A. Including additional isoforms, the activities of individual isoforms with an exogenous NLS in promoting N30 inclusion are shown in Figure 5B (lanes 8–13) and are also summarized in Figure 1A (nls). Special care was taken to ensure that a similar quantity of each protein was expressed and, if not, different amounts were tested to obtain comparable expression. In the presence of the exogenous NLS (nls), F011 and A016 show the highest activities among all isoforms tested. A30 shows a considerably lower activity than A016. Each of the muscle isoforms (F411, A713 and A715) has a lower activity than that of their brain counterparts (F011, A016 and A030, respectively). As expected, isoforms lacking a part or all of the RRM (F402 and A704) have no activity for N30 inclusion.\nNext, the splicing activities of the wild-type proteins without an exogenous NLS were examined. Representative data are shown in Figure 5B (lanes 2–7) and the relative activities are summarized in Figure 1A (wt). The protein amounts detected by immunoblots in these experiments represent the total amounts of the proteins distributed to both the nuclei and the cytoplasm. The splicing activities of the wild-type proteins are consistent with the activities that combine the splicing activities of the proteins with the exogenous NLS and the activities of the native proteins to localize to nuclei. In the absence of the exogenous NLS (wt), the brain isoforms F011, and A016 to a lesser extent, are still capable of activating N30 inclusion efficiently. The muscle isoforms for both Fxh and A2BP1 (F411, A713 and A715), as well as the brain isoform A030, show only minimal activities. Therefore, F011 and A016 appear to have the most physiological relevance to N30 splicing activation.\n\nOverexpression of Fxh and A2BP1 activates N30 inclusion of endogenous NMHC-B mRNAs\nThe human NMHC-B gene consists of 41 constitutive exons and 3 alternative exons. Its pre-mRNA is ∼156 kb in length and it is much more complex than the pre-mRNA from the minigenes. In addition, the minigene pre-mRNAs are driven by a heterologous promoter. Therefore, we next examined if Fxh and A2BP1 were capable of promoting N30 inclusion of the endogenous transcript. Y79 cells were stably transfected with the expression construct for F011 or A016. Both F011 and A016 are enriched in the brain and show higher activation of N30 inclusion in the minigene transcripts. mRNAs encoding endogenous NMHC-B were analyzed by using RT–PCR. As shown in Figure 6, the inclusion of exon N30 with and without another alternative exon, R18, in the endogenous mRNAs is markedly increased in the clones which were stably transfected with the construct for F011 or A016 containing an exogenous NLS (Figure 6, lanes 4 and 5). Although to a lesser extent, transfection of the wild-type constructs without an exogenous NLS also results in a significant increase in N30 inclusion (Figure 6, lanes 2 and 3). Thus, exogenously expressed Fxh and A2BP1 are capable of activating N30 inclusion not only in the transcripts from the minigenes, but also in those from the native NMHC-B gene in Y79 cells.\n\nFxh may cooperate with other factor(s) to promote N30 inclusion\nTo address the role of endogenous Fxh and its potential interaction with other proteins in promoting N30 inclusion, we made use of an isoform of Fxh, F402, which lacks the RRM. The mutant proteins lacking an RRM for other RNA-binding proteins have previously been reported to function in a dominant-negative fashion and inhibit the activities of the wild-type proteins (26,31). Therefore, the effects of F402 on the N30 inclusion of minigene H mRNAs were examined in the context of the Y79 cells treated with butyrate. Upon butyrate treatment, as reported previously, Y79 cells enter in a post-mitotic and differentiated stage, and importantly, the endogenous as well as the minigene mRNAs in those cells include N30 to a large extent, unlike those in the untreated cells that predominantly exclude N30 (14). As shown in Figure 7, in the absence of exogenous expression of F402, large quantities of the mRNAs derived from minigene H-wt, which contains the wild-type IDDE, include N30 (Figure 7, upper panel, lane 4). In contrast, only small quantities of N30 inclusion are detected in the mRNAs from minigene H-mc, which has mutations in both copies of UGCAUG in the IDDE (Figure 7, upper panel, lane 8). Therefore, the butyrate-treated Y79 cells contain factor(s), which are capable of activating the UGCAUG-dependent N30 inclusion. Co-transfection of the wild-type minigene H-wt with the F402 expression construct causes a dose-dependent inhibition of N30 inclusion (Figure 7, lanes 1–3), indicating that F402 has an antagonistic effect on the endogenous factors with respect to N30 inclusion. Notably, the UGCAUG-independent N30 inclusion seen in the mutant minigene H-mc is not affected by the co-expression of F402 (Figure 7, lanes 5–7), indicating that the inhibitory activity of F402 depends on the UGCAUG element. The parallel experiment with the F011 expression construct does not show a significant effect on N30 splicing in either the wild-type or mutant minigene (Figure 7, lanes 9–16). This may be due to the fact that N30 inclusion of the wild-type minigene mRNAs has already reached a maximal level and is consistent with the idea that butyrate-treated Y79 cells contain sufficient amounts of protein(s) functionally equivalent to F011, which can promote N30 inclusion in a UGCAUG-dependent manner. Since F402 does not bind to the UGCAUG element, and has a UGCAUG-dependent antagonistic effect on N30 splicing, it most probably disrupts protein–protein interactions of the endogenous proteins that are required for the UGCAUG-dependent activation of N30 splicing. Endogenous Fxh (and/or A2BP1) would be a good candidate whose function could be antagonized by exogenously expressed F402. Thus, this observation suggests that the endogenous Fxh has an effect on the activation of N30 splicing and that other proteins cooperate with Fxh for N30 activation. Since muscle cells express the RRM-defective isoforms to a significant extent (Figure 2C) and the wild-type F402 localize to nuclei efficiently, this also raises the possibility that the RRM-defective isoforms may have an inhibitory function on the N30 splicing in muscle cells.\n\n\nDISCUSSION\nTwo major findings are described in this report. First, Fxh and A2BP1 facilitate neural cell-specific inclusion of the cassette-type exon via binding to the specific intronic sequence UGCAUG. In addition to a minigene model system, Fxh and A2BP1 are capable of facilitating N30 inclusion of the endogenous pre-mRNA. This result provides an important demonstration of physiological relevance and supports the notion that the NMHC-B pre-mRNA is likely to be the true target for Fxh or A2BP1-mediated regulation. However, whether the endogenous Fxh or A2BP1 regulates endogenous NMHC-B pre-mRNA splicing needs to be determined in a future study. In vertebrates, small interfering RNAs and gene targeting strategies have recently been used successfully to address the roles of endogenous splicing regulators in alternative splicing of endogenous target pre-mRNAs (8,32–36). A second and more novel finding is the identification of tissue-specific isoforms of Fxh and A2BP1 with different splicing activities as well as different subcellular localizations. This finding raises the possibility that the products of the Fxh and A2BP1 genes can contribute to a mechanism as to how tissue specificity of alternative splicing is achieved.\nMany splicing factors are detected not only in the nuclei, but also in the cytoplasm (37). They are shuttling between the nucleus and the cytoplasm and, in some instances, extracellular stimuli trigger changes in subcellular distribution of these proteins. Such translocations have been reported for hnRNPA1 and PTB (38,39). Moreover, a number of RNA-binding proteins have been demonstrated to play a role in multiple steps during gene expression in different subcellular compartments, such as pre-mRNA processing in nuclei, mRNA export from nuclei to cytoplasm and mRNA localization, stability and translation in cytoplasm (37,40). Therefore, not surprisingly, Fxh and A2BP1 isoforms were found to be distributed in both the nuclei and the cytoplasm in HeLa and Y79 cells. However, the relative ratios of proteins distributed between the two subcellular compartments at steady-state differ among the isoforms. In agreement with Jin et al. (21), substantial amounts of the brain isoform A016 are detected in nuclei. Other A2BP1 isoforms, the brain isoform A030 and the muscle isoforms A713 and A715, are only poorly detected in nuclei. This observation is consistent with the reports where endogenous A2BP1 in cerebellar Purkinje cells, hippocampus neurons and cardiac myocytes were shown to be localized essentially to the cytoplasm (23,24). Thus, inclusion and exclusion of A53 and differences in the very N-terminal sequences results in A2BP1 isoforms with a distinct subcellular localization. It is likely that A2BP1 proteins have multiple roles, involving both nuclear and cytoplasmic events. In contrast, all three Fxh isoforms predominantly localized to the nuclei. Therefore, in terms of their localization, Fxh proteins are better candidates for regulators of the pre-mRNA splicing that takes place in nuclei. Of note, however, our preliminary results of 5′ RACE, as well as the EST database, detect multiple 5′ end sequences for both Fxh and A2BP1 mRNAs, which are presumably generated by alternative promoters and alternative splicing. The diversity of the 5′ end cDNA sequences leads to the generation of a number of unique N-terminal amino acid sequences. Therefore, this study does not exclude the possible existence of other isoforms with different subcellular localizations for both Fxh and A2BP1. Our study also does not exclude the possibility that some of the isoforms translocate between the nucleus and the cytoplasm following stimuli.\nThe main aim of this study is to determine the relative activities of tissue-dependent isoforms of Fxh and A2BP1 in neural cell-specific and UGCAUG element-dependent alternative splicing. To obtain an indication of the relative specific activity of each isoform in transfected cells, the same amounts of the expressed proteins should be available for the splicing reaction in the nuclei. For this reason, an exogenous NLS was included in the expressed proteins. Essentially, all of the expressed proteins with the exogenous NLS localized to nuclei. Thus, the amounts of the expressed proteins determined by immunoblots represent the nuclear concentrations. The analysis using the proteins expressed with the exogenous NLS allowed us to compare directly the splicing activities of these proteins. Furthermore, this analysis also allowed us to define the critical regions of the proteins for splicing activation.\nAs shown in Figure 5, the splicing activities of the various isoforms of Fxh and A2BP1 are intrinsically different, regardless of the subcellular localization properties of the wild-type proteins. Among the isoforms tested in this study, F011 and A016, which include B40, are found to have higher activities in promoting N30 inclusion. When the primary amino acid sequences, outside of the RRM, of these two proteins are compared, the C-terminal regions (amino acids 190–377 of F011) show a higher homology with 71% identity, whereas the N-terminal regions (amino acids 1–112 of F011) show only 53% identity. The C-terminal region includes four subregions of nearly identical stretches of amino acids (Figure 8, I–IV). One subregion (II) includes 13 amino acids encoded by exon B40. Substitution of this subregion with exon M43 in Fxh causes substantial changes in amino acid sequences resulting in only a 21% identity in this region between F011 and F411. Another subregion (IV) is located at the C-terminal end and A030 lacks this homologous region by the inclusion of exon A53, which results in a frame shift. Since F411 and A030 show poor splicing activation compared with F011 and A016, respectively, these two subregions of F011 and A016 appear to serve as activation domains, presumably by interacting with other proteins. This notion is supported by the finding that the RRM-lacking isoform F402, which includes the same subregions II and IV as F011, functions apparently as a dominant-negative mutant to the wild-type Fxh, consistent with the interpretation that the mutant and wild-type are competing to interact with other protein(s). To date, Fyn tyrosine kinase and estrogen receptor-α have been reported to interact with Fxh, and ataxin-2 with A2BP1 (23,41,42). Whether these proteins participate in the regulation of pre-mRNA splicing is currently unknown. Of interest, A030 with the exogenous NLS activates N30 splicing as efficiently as F011 in the pre-mRNAs derived from minigenes J and H, which contain the shorter intron, whereas this isoform poorly activates N30 splicing in the pre-mRNA from minigene G, which contains the full-length intron. This observation implies that the interactions of A030 with different factors are required in the different pre-mRNA contexts. Therefore, the isoforms of Fxh and A2BP1 described here may have different effects on other UGCAUG-regulated alternative splicing.\nThe involvement of the hexanucleotide UGCAUG in regulated alternative splicing has been experimentally demonstrated in a number of neural cell-specific, as well as other tissue-specific, model systems (13–21). This hexanucleotide element plays a role, in most cases, as an enhancer in regulating alternative splicing of cassette-type exons as well as mutually exclusive exons. Furthermore, computational analysis has revealed that UGCAUG is over-represented in the introns in which splicing is regulated, compared with the constitutively spliced introns (43). This analysis also pointed to the UGCAUG element as playing a role in the regulation of tissue-specific alternative splicing in a wide range of tissues, but not in specific tissues.\nTo date, KH-type splicing regulatory protein (KSRP) (44), A2BP1 (21) and Fxh (this study) are known to be capable of binding to UGCAUG. KSRP is expressed ubiquitously and tissue-specific variants of this gene have not been described so far. In this study, we have described the existence of tissue-dependent isoforms of Fxh and A2BP1, which, while not identical in some areas of the molecule, may contain the same RRM. The physiological relevance of these isoforms is that they have different splicing activities and different subcellular localizations. The brain isoforms promote N30 inclusion more efficiently than the muscle isoforms of both Fxh and A2BP1. The isoforms lacking the RRM are normally expressed to a significant extent in skeletal muscles. This isoform is incapable of activating N30 splicing and, moreover, can inhibit N30 inclusion. The properties of these isoforms are consistent with Fxh and A2BP1 acting as regulators for N30 splicing, since N30 is included in neuronal cells, but excluded in muscles. Therefore, despite the tissue-independent occurrence of UGCAUG as a regulatory element, given the tissue-dependent isoforms of the UGCAUG-binding proteins (Fxh and A2BP1) with different activities, the hexanucleotide UGCAUG could confer tissue specificity on regulated splicing. One of the major problems in understanding the mechanisms responsible for alternative pre-mRNA splicing is the manner in which tissue specificity is determined. In vertebrates, to date, only a few tissue-specific proteins have been identified as splicing regulators (8–12). Here, we have shown that the tissue-dependent isoforms of the sequence-specific RNA-binding proteins, which themselves are generated by alternative splicing, have different activities in tissue-specific alternative splicing of target pre-mRNA. Therefore, these isoforms play a role in the determination of tissue specificity of target pre-mRNA splicing. The discovery of these tissue-dependent isoforms of the UGCAUG-binding proteins with different splicing activities adds an important new dimension to the molecular mechanisms responsible for regulating tissue-dependent alternative splicing mediated via UGCAUG.\n\n\n" ], "offsets": [ [ 0, 40476 ] ] } ]

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[ { "id": "pmcA1079799__text", "type": "Article", "text": [ "Mutations of PIK3CA in gastric adenocarcinoma\nAbstract\nBackground\nActivation of the phosphatidylinositol 3-kinase (PI3K) through mutational inactivation of PTEN tumour suppressor gene is common in diverse cancer types, but rarely reported in gastric cancer. Recently, mutations in PIK3CA, which encodes the p110α catalytic subunit of PI3K, have been identified in various human cancers, including 3 of 12 gastric cancers. Eighty percent of these reported mutations clustered within 2 regions involving the helical and kinase domains. In vitro study on one of the \"hot-spot\" mutants has demonstrated it as an activating mutation.\n\nMethods\nBased on these data, we initiated PIK3CA mutation screening in 94 human gastric cancers by direct sequencing of the gene regions in which 80% of all the known PIK3CA mutations were found. We also examined PIK3CA expression level by extracting data from the previous large-scale gene expression profiling study. Using Significance Analysis of Microarrays (SAM), we further searched for genes that show correlating expression with PIK3CA.\n\nResults\nWe have identified PIK3CA mutations in 4 cases (4.3%), all involving the previously reported hotspots. Among these 4 cases, 3 tumours demonstrated microsatellite instability and 2 tumours harboured concurrent KRAS mutation. Data extracted from microarray studies showed an increased expression of PIK3CA in gastric cancers when compared with the non-neoplastic gastric mucosae (p < 0.001). SAM further identified 2910 genes whose expression levels were positively associated with that of PIK3CA.\n\nConclusion\nOur data suggested that activation of the PI3K signalling pathway in gastric cancer may be achieved through up-regulation or mutation of PIK3CA, in which the latter may be a consequence of mismatch repair deficiency.\n\n\n\nBackground\nThe phosphatidylinositol 3-kinase (PI3K)-AKT signalling pathway is involved in the regulation of diverse cellular processes, including cell growth, survival and motility. Abnormal activation of this pathway is frequently observed in various cancer types, leading to aberrant cell cycle progression, altered adhesion and motility, inhibition of apoptosis and induction of angiogenesis [1]. It has been previously reported that genetic alterations involving various members along this signalling pathway could lead to its activation in cancer. These include mutation, allelic loss or promoter methylation of the negative regulator PTEN [2]; or alternatively, chromosomal amplification or over-expression of the positive regulators PIK3CA [3-5] and the various AKT kinases [6,7]. Furthermore, changes in other related pathways that are commonly altered in cancer, such as those involved in growth factor stimulation via the G-protein-coupled receptors or through direct interaction with the activated form of small GTPase RAS, can also lead to PI3K-AKT pathway activation [8]. Activation of this pathway results in the phosphorylation of AKT at Thr-308/309 and Ser-473/474. These phosphorylated forms of AKT proteins have been detected by Western blot or immunohistochemistry in various cancer types, suggesting the frequent activation of PI3K-AKT pathway in the carcinogenic process [7,9].\nAlthough genetic changes along the PI3K-AKT pathway have been repeatedly documented in brain, ovarian, endometrial, breast, prostate and thyroid cancers [1,2], reports on its mechanism of activation in gastric cancer are limited. Gastric cancer is the second most common cancer worldwide but its molecular basis of tumourigenesis is still poorly understood. Previous immunohistochemical study has demonstrated the presence of the phosphorylated form of AKT in 78% of gastric cancer [10], suggesting that activation of this pathway may also be common in gastric cancer. Though loss of heterozygosity (LOH) involving the PTEN locus has been demonstrated in 47% of gastric cancer in a recent study, mutation or promoter methylation was absent even in cases with LOH [11]. Thus data from this study could not support the two-hit inactivation of PTEN in gastric cancer, while the biological significance of PTEN haploinsufficiency remains controversial. Alternatively, amplification of AKT1 has been reported in a single case of gastric cancer [12], and amplification of PIK3CA associated with elevated mRNA levels has been found in 36% of gastric cancer [11]. More recently, Samuels et al. screened a diverse spectrum of human cancers for mutation in 16 PI3K or PI3K-like genes and found a high frequency of somatic mutation in PIK3CA, which encodes the p110α catalytic subunit. Major screening in colorectal cancer (CRC) identified PIK3CA mutations in 74 out of 234 (32%) cases, while mutations were also noted in 3 out of 12 (25%) gastric cancers. Reported mutations were mostly of missense type, and clustered within 2 regions in the helical and kinase domains. Expression of a \"hot-spot\" mutant, H1047R, conferred a significant up-regulation of lipid kinase activity of PIK3CA, suggesting it as an activating mutation [13]. In this study, we have examined a series of 94 human gastric adenocarcinomas for PIK3CA mutation. We have also examined PIK3CA expression level by extracting data from a large-scale gene expression profiling study previously performed for these cases [14,15]. Using SAM, genes with significant correlating expression with PIK3CA have also been identified.\n\nMethods\nPatient samples preparation\nDNA samples used for sequencing were prepared from frozen tumour and non-tumour gastric mucosae from 94 gastric cancer patients who underwent gastrectomy in the Department of Surgery, Queen Mary Hospital, The University of Hong Kong, as previously described [16]. Majority of the frozen samples (n = 81) showed tumour component of over 70%, whereas in 13 cases a lower proportion between 50 to 70% was accepted due to the tumours' inherent diffuse infiltrative nature with entrapment of non-neoplastic components. Analysis for microsatellite instability (MSI), BRAF and KRAS mutation have been performed and reported previously [16]. RNA preparation and gene expression profiling using a cDNA microarray containing 44,500 cDNA clones, representing around 30,300 unique genes, has been performed and reported in 90 of these tumours in comparison to 22 non-tumour gastric mucosae [14,15]. This study was approved by the Ethics Committee of the University of Hong Kong.\n\nMutational screening\nMutation screening of PIK3CA was performed for exons 9 and 20, covering the mutational hotspots; and for exon 18, from which a mutation was found in a gastric cancer. Mutations in these 3 exons constituted 80% of all PIK3CA mutations detected in the previous study [13]. PIK3CA intron-specific external amplification primers and internal sequencing primers were designed according to the previous study [13] with some modifications [see Additional file 1]. In particular, primers for exon 9 have been modified to avoid amplification of homologous sequences located in other chromosomes. PCR products were generated using the external primers and directly sequenced using the internal primers with the DYEnamic™ ET Terminator Cycle Sequencing Kit (Amersham Biosciences, Freiburg, Germany) according to the manufacturer's instruction. Electrophoresis was performed in the ABI Prism® 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). For each exon, PCR products were generated from 2 independent PCR reactions for sequencing of the forward and reverse strands. For exon 9, 2 independent PCR followed by sequencing of the forward strand were performed. Analysis of the chromatograms was performed using the mutation analysis software Mutation Explorer™ (SoftGenetics, State College, PA, USA).\n\nExtraction of expression data and statistical analysis\nGene expression data were extracted from the microarray database containing 126 samples (90 gastric cancers, 14 lymph node metastasis and 22 non-tumour gastric mucosae) based on a 3-fold signal above background ratio for either channel and with 80% good data [14]. Gene expression data from 20,336 cDNA clones satisfied this selection criteria and were extracted, which included a cDNA clone corresponding to PIK3CA (IMAGE clone number 345430, GenBank accession no. W72473). Expression data for PIK3CA was extracted and the differences in expression levels between tumour and non-tumour tissues were examined using the Student's t-Test. SAM was performed to identify genes with significant correlating expression with PIK3CA [17]. The missing values in the dataset were estimated by a K-nearest neighbours impute algorithm using 10 nearest neighbour [18] followed by 5000 permutations in the SAM analysis.\n\n\nResults\nAmong the 94 gastric adenocarcinoma analysed, we have detected PIK3CA mutation in 4 cases. Two cases harboured the mutation A3140G (H1047R) in exon 20, and the other 2 cases with mutations G1624A (E542K) and G1633A (E545K) in exon 9. Representative sequence chromatograms are shown in figure 1. All four mutations were absent in the corresponding non-neoplastic mucosae and thus were confirmed as somatic mutations. Though the overall mutation frequency (4.3%) was lower than that of the previous study, the nature of the 4 mutations found were consistent with those identified at the reported hotspots. In particular, the H1047R mutation has been reported in 2 gastric cancers and 15 colorectal cancers [13]. While the E542K and the E545K mutations were not found in gastric cancer in the previous series, a large number of colorectal tumours did harbour these 2 mutations.\nPIK3CA mutation spectrum and their corresponding clinico-pathological features were listed in Table 1. We noted a higher tendency of high-level MSI in gastric cancers with PIK3CA mutations (3 in 4, 75%) than in those without (18 in 90, 20%). Moreover, though the overall incidence of KRAS mutation in the studied population was low (8 in 94), 2 of the 4 gastric cancers with PIK3CA mutation also harboured a KRAS mutation.\nSince over-expression of PIK3CA has been reported in gastric cancer [11], we have also extracted PIK3CA expression data from our previous cDNA microarray study of these cases [14,15]. We have confirmed that expression level of PIK3CA was significantly higher in gastric cancers (n = 87, mean = 0.099, SD = 0.428) when compared with non-neoplastic gastric mucosae (n = 22, mean = -0.418, SD = 0.426; Student's t-Test, p < 0.001). Using PIK3CA expression level as a continuous variable for SAM analysis [17], we found 2910 cDNA clones (corresponding to about 2546 unique genes) whose expression associated positively with PIK3CA expression (median number of false significant = 0.372, Delta = 1.107) [see Additional file 2]. Interestingly, no gene was found to be negatively associated with PIK3CA expression.\n\nDiscussion\nIn this study, we have reported the presence of PIK3CA gene mutation in 4.3% of gastric cancer. A high tendency (3 in 4) of mismatch repair deficiency was noted in cases harbouring PIK3CA mutation. Though the small number of PIK3CA mutations in our study may not justify statistical claim of significance; suggestion of such, despite of its not being mentioned by the authors, can be found from a previous study in CRC by Samuels et al.. From their study of 33 MSI and 201 microsatellite stable (MSS) CRC cases, PIK3CA mutation was present in 48% of the MSI tumours, but only in 29% of the MSS tumours. A significant association would have been revealed if statistical analysis had been applied (Fisher's exact test, p = 0.014) [13]. Gastrointestinal tract cancers with MSI are known to have a different molecular pathway of tumour evolution compared with their MSS counterparts [19,20]. This can be attributed to their propensity for frameshift mutations in repeat sequences, resulting in selective disruption of genes with such sequences within their coding regions. With 2 poly-adenine tracts within its coding region, PTEN can be inactivated through frameshift mutations in MSI CRC, resulting in the selective targeting of the PI3K-AKT signalling pathway [21,22]. It is also known that mismatch repair deficiency would lead to an elevated rate of missense mutation due to impaired single nucleotide mismatch repair [23]. Thus, the observed higher incidence of PIK3CA missense mutation in MSI colorectal and gastric cancers suggests yet another mechanism for the activation of the PI3K-AKT signalling pathway through mismatch repair deficiency.\nOur data also showed a higher tendency of KRAS mutation in cases with PIK3CA mutations (2 in 4) than in those without (6 in 90). Yet again due to the low incidence of both mutations in our samples, statistical significance may not be claimed. In the study by Samuels et al., some of the colorectal tumours with PIK3CA mutation also harboured KRAS or BRAF mutation [13]. The PI3K-AKT pathway is known to have a close association with the RAS-MEKK signalling pathway [8]. Constitutively active RAS can interact with the catalytic subunit of PI3K and lead to its activation. Ras-dependent PI3K activation contributes to the transforming phenotype by mediating anchorage-independent growth, cytoskeletal reorganisation and apoptosis evasion. It has been observed that genes involved in the same signalling pathway may manifest mutations in cancer cells in a mutually exclusive manner, presumably due to the lack of selective growth advantage in having a second hit in the already altered pathway. A prominent example is the mutually exclusive occurrence of the BRAF hotspot mutation (V600E) and KRAS mutations in colorectal cancer [24,25]. However, there exist other examples of alterations in multiple components of the same signalling pathway that may lead to a multi-level modulation of its activity. For example, non-V600E BRAF mutations tend to occur together with KRAS mutations [26], and inactivation of the secreted frizzled-related proteins (antagonists of WNT) by promoter methylation frequently coincides with mutations in the Adenomatous Polyposis Coli gene to achieve multi-level activation of the WNT signalling pathway in colorectal cancers [27]. Whether PIK3CA functions independently from RAS, or acts synergistically with RAS to produce additive effects on the activation of the same pathway awaits further clarification.\nBy extracting data from microarray, we have confirmed the up-regulation of PIK3CA expression in gastric cancer tissues compared with the non-neoplastic gastric mucosae and identified a large number of genes that showed a significant positive correlation in expression level with PIK3CA. These genes participate in diverse cellular processes with 177 as putative cell cycle-regulated genes [28] and 126 mapped to genes with known functions in cell cycle regulation, cell proliferation or DNA replication [see Additional file 2]. While some of these genes maybe induced by PIK3CA, others maybe co-ordinately regulated by common upstream signals. Expression data set at one point was limited in differentiating the above cause and consequence, yet it certainly revealed the complexity of the carcinogenic process and the intricate relationship of PIK3CA signalling with other cellular processes.\nContrary to our expectation, the incidence of PIK3CA mutation found in the current study (4%) is much lower compared with that observed by Samuel et al. (25%) [13]. The reason for discrepancy may simply be a result of sample bias as the previous study involved only a small number of gastric cancers (n = 12). However, ethnic differences can also be another possibility. The diverse pathological spectrum and aetiological factors of gastric cancers in different geographical locations may be paralleled by differences in molecular pathway of tumour development. Since our current study is only based on a Chinese population with an intermediate gastric cancer incidence, further studies involving patients from different ethnic groups will be able to address this possibility.\n\nConclusion\nLarge-scale screening of gastric adenocarcinomas for PIK3CA mutations revealed a mutation incidence of 4.3%. Increased PIK3CA expression level was observed in gastric tumours compared with non-neoplastic mucosae. This increase in PIK3CA level was associated with the elevated expression of a large number of genes, which may constitute the upstream regulators or downstream targets of PIK3CA along the PI3K signalling pathway.\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\nAuthors' contributions\nVSWL carried out the molecular analysis, performed data analysis and drafted the manuscript. CWW, TLC, WZ assisted in the molecular analysis. KMC provided the clinical data. ASWC assisted in data analysis and edited the manuscript. SS and XC participated in the microarray study and data analysis. STY and SYL conceived of the study, participated in its design, coordination and data analysis, and edited the manuscript. All authors read and approved the final manuscript.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\nSupplementary Material\n\n\n" ], "offsets": [ [ 0, 17097 ] ] } ]

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[ { "id": "pmcA2575403__text", "type": "Article", "text": [ "Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence\nAbstract\nTuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.\n\nIntroduction\nTuberculosis caused by Mycobacterium tuberculosis (M.tb) remains one of the leading causes of mortality in the world, with around 2 million deaths each year [1]. Most individuals remain asymptomatic after the primary infection with only 10% at risk of developing an active disease during their life [2]. In asymptomatic individuals, the bacilli are not cleared but rather persist in a dormant state, from which they may reactivate and induce clinical disease at later stages [3].\nThe prognosis of the disease depends on the host's efficiency to constrain the bacilli at the site of infection. When inhaled M.tb reach the lungs, they are internalized by lung macrophages. The latter trigger the accumulation at the infectious site of macrophages, lymphocytes and dendritic cells, to form a granuloma, which is a major histo-pathological feature of TB. Within granulomas, macrophages differentiate into epithelioïd cells (differentiated macrophages), and/or fuse to form multinucleated giant cells (MGC). Macrophages with large numbers of lipid-free vacuoles, as well as macrophages filled with lipid-containing bodies, also called foamy macrophages (FM) are also found within granulomatous structures in both experimental animal models and human disease [4],[5]. The above cells are surrounded by a rim of lymphocytes, and at later stages, a tight coat of fibroblasts encloses the structure [6]. Although the structure and cell composition of granulomas are well known, the biology of these inflammatory structures and, more specifically, the role of granuloma-specific cell types, remain largely unknown.\nWe have previously developed an in vitro model of human tuberculous granulomas to gain insight into the survival strategies of the tubercle bacillus within its human host. This model now enables the characterization of granuloma-specific cell types, and their modulation by M.tb [7]. The main advantage of this model over in vivo animal models or ex vivo human biopsy samples, is the availability of live granuloma cells which facilitates analysis of their cell biology. Using this model we have recently shown that, within granulomas, large multinucleated giant cells, also known as Langhans giant cells, result from the induction of granuloma macrophage fusion by M.tb glycolipids [8]. We have shown that these cells have lost the ability to mediate bacterial uptake upon maturation, but have conserved their ability to mediate antigen presentation [9].\nThe differentiation of macrophages into FMs has been particularly well described in individuals developing a postprimary, also known as secondary or adult, TB. These postprimary infections are considered to be the result of re-infection or reactivation of a primary TB [10]. FMs have been described in leprosy patients or M. avium-infected AIDS (Acquired ImmunoDeficiency Syndrome) patients, and in chronic stages of M.tb infection in mice [4],[11],[12]. The foamy aspect of these macrophages is the result of intracellular lipid accumulation within lipid bodies, also called lipid droplets or lipid vacuoles [13],[14],[15]. In an experimental model of leukocyte infection, it was recently suggested that BCG (Bacille Calmette Guerin) infection can induce, in a TLR2-dependent fashion, the rapid formation of lipid bodies carrying out part of the eicosanoïd biosynthesis that usually accompanies the infection, thus pointing to an active role for lipid bodies during the course of infection [16]. However, the mechanisms regulating this lipid accumulation during mycobacterial infection and their significance in the physiopathology of tuberculosis are not understood. Most of the studies on TB granulomas have focused on the contribution of host components, but very little is known about the role played by bacterial constituents in terms of granuloma formation and progression.\nThe present work was aimed at deciphering the role of FMs in M.tb survival within human granulomas. To test our working hypothesis according to which FMs constitute a nutrient-rich reservoir for M.tb persistence, we used our in vitro model of human granulomas to analyze the formation of FMs and their role during M.tb infection. We showed that only highly virulent mycobacteria (M.tb, M. avium) and not saprophytic ones (M. smegmatis) could induce the formation of FMs in mature granulomas. Moreover, we demonstrated that oxygenated mycolic acids specifically produced by the above pathogenic species were responsible for FMs formation. Once differentiated, FMs were unable to mediate phagocytosis of new bacilli and their microbicidal activity was reduced. M.tb was not killed in FMs but instead persisted in a non-replicating state, and over-expressed dormancy genes. Noteworthy, in foamy macrophages, M.tb-containing phagosomes were shown to migrate towards lipid bodies which they progressively surrounded and engulfed. As a result, bacteria were freed into lipid bodies, thus favoring the bacilli's access to nutrients. From these data, we propose that FMs could form a secure reservoir for the tubercle bacilli.\n\nMethods\nHuman samples\nHuman blood samples, purchased from the French National Blood provider of Toulouse, were collected from fully anonymized non-tuberculous control donors, an ethical committee approval was, therefore, not necessary. This study was conducted according to the principles expressed in the Helsinki Declaration, with informed consent obtained from each donor.\nWe chose to work on lymph node samples rather than lung biopsies, which are usually only paraffin-embedded, because staining for lipids can only be performed on frozen samples. Lymph node biopsies were taken for diagnosis purposes, in ten non-HIV patients. For each biopsy, a fragment was sent to the microbiology laboratory, another was frozen in liquid nitrogen and the main part was fixed in formalin and paraffin-embedded for histological examination. M.tb was identified in 9 lymph node biopsies and from the lung aspiration in the last patient. This latter case showed no signs of necrosis, and no FMs were found in the lymph node biopsy. This study complies with the guidelines of the declaration of Helsinki.\n\nBacterial strains and culture conditions\nWild-type M. smegmatis and M. smegmatis/hma strains were previously described [17], M. tuberculosis-GFP were a kind gift from Dr. C. Guilhot (CNRS-IPBS, Toulouse France). Bacilli were grown in Middlebrook 7H9 medium (Difco) supplemented with 10% albumin–dextrose–catalase (Difco). Fluorescent M. smegmatis and M. smegmatis/hma were obtained by FITC labelling as described in [18].\n\nIsolation of RNA from intraphagosomal M.tb\nSix and 12 days post-infection (MOI 10), macrophages and FMs (5×106) were washed twice with PBS, scraped off the cell dishes and recovered by centrifugation. The cell pellets were lysed with lysis buffer (RNEasy mini kit, Quiagen) and transferred to 2 ml Eppendorf-tubes containing a 0.5 ml suspension of 0.1 mm-diameter glass beads (Biospec). Mycobacteria were disrupted using a bead beater (Retsch) followed by a 5 min centrifugation at 14 000g. RNA contained in the supernatant was then column-purified according to the manufacturer's conditions using the RNEasy mini kit (Qiagen) and quantified.\n\nQuantitative Real-Time RT-PCR\nIn RNA samples DNA contamination was excluded by DNAse I treatment (Ambion). 1 µg total RNA was reverse-transcribed using random hexamer primers (Ambion) and Superscript III reverse transcriptase (Invitrogen). Real-time PCR was performed on cDNA using the SYBR green essay (Applied Biosystems). Reverse and forward primers used are listed below in Table 1. Fluorescence was measured by ABIPrism 7300 (Applied Biosystems). The calculated threshold cycle (Ct) value for each gene of interest was normalized to the Ct value for 16S and the fold expression was calculated using the formula: fold change = 2−Δ.(ΔCt) [19]. Real-time PCR conditions include initial activation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing and extension at 65°C for 1 min. The gene induction ratios were obtained by comparing gene expression levels in intracellular bacilli with those of log-phase in vitro-grown bacilli. RNAs were isolated from two independent macrophage infections.\n\nIn vitro human granuloma formation\nIn vitro granulomas were obtained as previously described [7]. Briefly, 1×106 freshly isolated Peripheral Blood Mononuclear Cells (PBMCs) were incubated with 1×104 viable M.tb, or 1×103 viable M. smegmatis or M. smegmatis/hma. The culture medium was RPMI-1640+Glutamax (Difco), containing 7.5% human AB serum (Sigma-Aldrich).\n\nMacrophage differentiation\n2.5×106 PBMCs prepared in RPMI-1640+Glutamax (Difco) were plated over coverslips in 24-well plates. After 2 h culture at 37°C, cells were washed 3 times with PBS and then refed with RPMI-1640+Glutamax (Difco), containing 7.5% human AB serum. After 6 days of culturing, macrophages were differentiated.\n\nRespiratory burst assay with Nitroblue tetrazolium (NBT)\nHuman monocyte derived macrophages were stimulated with M. smegmatis/hma for 2 h at 37°C, washed and reincubated in mycobacterium-free medium. After 2 days of differentiation into FMs, macrophages were co-stained with NBT (2 mg/ml Sigma-Aldrich) and Nile red (Sigma-Aldrich) for 30 min at 37°C. Stained cells were fixed and then analysed with an inverted microscope (Nikon TE 300).\n\nPhagocytosis and Survival test\nFor phagocytosis assays, differentiated macrophages were incubated with M. smegmatis/hma mycolic acids for 2 days and then infected with 1×108 labeled mycobacteria per well for 90 min, washed 3 times with PBS and chased for 3 h in fresh culture medium.\nFor survival experiments, macrophages were infected with M. tuberculosis-GFP (10 bacteria/cell), washed 3 times with PBS (Gibco) and re-incubated in fresh culture medium. At selected time points thereafter (1, 3, 6, 10 and 14 days) cells were labeled with Nile red (Sigma-Aldrich), fixed and observed under a confocal microscope. The amount of mycobacteria per cells was evaluated. (100 cells were analyzed for each time point).\n\nLipid body staining and immunostaining\nGranuloma cells were collected and plated onto glass coverslips with a cytospin (Thermo Shandon) fixed for 30 min in PBS-PFA 4% and stained with Oil red-O (Sigma-Aldrich) as described [20]. The slides were then counterstained with haematoxylin (Dako Cytomation) and observed under an inverted microscope (Nikon TE 300).\nFor fluorescence analysis, granuloma cells or macrophages were collected in PBS, lipid bodies were stained with Nile red (Sigma-Aldrich, 0.1 µg/ml, from a stock solution in methanol) for 15 min washed with PBS, fixed for 30 min in PBS-PFA 4%, mounted with the fluorescent mounting medium (Dako Cytomation) and observed under a confocal microscope.\nIn order to distinguish the lipids contained within lipid bodies, from those of the cell membrane, we used the fluorescent emission spectrum properties of Nile red which depend upon the kind of lipid associated with Nile red, i.e. for triacylglycerol: λmax em = 590 nm, for phospholipids: λmax em = 640 nm (Molecular Probes handbook). On confocal microscopy pictures, the phospholipid background of both macrophages and FM appears in red and the triacylglycerol-rich lipid bodies appear in white. Cells were considered to be positive for Nile red staining when more than 50% of the cell surface was stained (see Figure S2).\n\nMycolic acid isolation\nBacterial residues obtained after lipid extraction with organic solvents [17] were saponified with a mixture of 40% KOH aqueous solution and methoxyethanol (1∶7, v/v) at 110°C for 3 hours in a screw-capped tube. After acidification, fatty acids were extracted with diethylether, derivatised into methyl esters with diazomethane and analyzed by analytical thin-layer chromatography on silica Gel 60 (Silica Gel 60 Macherey-Nagel) using either dicholoromethane or petroleum ether/diethylether (9∶1, v/v, five runs). Visualization of lipid spots was performed by spraying the plates with molybdophosphoric acid (10% in ethanol), followed by charring.\n\nProcessing for electron microscopy\nGranulomas were fixed for 1 hour at room temperature with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, containing 0.1 M sucrose, 5 mM CaCl2 and 5 mM MgCl2. After two successive 15-min washes with the same buffer, the granulomas were postfixed for 1 hour at RT with 1% osmium tetroxide (Electron Microscopy Science) in the same buffer devoid of sucrose. The granulomas were scraped off the culture dishes with a rubber policeman and concentrated in 1% agarose in the same buffer. After a one hour treatment at room temperature with 1% uranyl acetate in Veronal buffer, the samples were dehydrated in a graded series of ethanol and embedded in Spurr resin. Thin sections were stained with uranyl acetate and lead citrate.\n\nImage acquisition in confocal microscopy\nThe images were obtained using a Leica confocal fluorescence microscope (SP2) equipped with a Plan Apo 40×1.4 Ph 6 objective (Olympus Optical) and CoolSNAP-Pro CF digital camera in conjunction with Image-Pro Plus version 4.5.1.3 software (Media Cybernetics). The images were edited using Adobe Photoshop CS2 9 software (Adobe Systems).\n\n\nResults\nFoamy macrophages are strongly associated with necrotic lesions and often contain M. tuberculosis\nWe analyzed lymph node biopsies from 10 tuberculous patients as a first step for evaluating the role of FMs within tuberculous granulomas. A section through a representative biopsy is shown in Figure 1. Well-circumscribed and -differentiated granulomatous structures were observed in all the samples (Figure 1A). Classically, lesions display a necrotic center (N), an interface area between the necrotic center and the histiocytes (I), and some peripheral granulomas (G). Only seven out of ten patients presented lesions displaying central necrosis (Table 2). Staining of the histology samples with Oil red-O, a classic lipid stain, confirmed the presence of FMs within the granulomatous structures in six out of seven samples presenting necrosis (Figure 1B), whereas no FMs were found in the three non-necrotic lesions (Table 2). Noteworthy, in samples with necrotic areas, FMs were always found in the interface region flanking the central necrosis (Figure 1B). These observations firstly confirmed the presence of FMs in most TB patients' lesions thereby suggesting that these cells play an important role in the formation/maintenance of such lesions. Second, FMs seem to be associated with necrosis, which is a hallmark of TB lesions, since they were observed only in lesions with a necrotic center and preferentially located around the necrotic area.\nInterestingly, staining, in parallel, of serial thin sections from a patient's lesion biopsy with Oil red-O (Figure 1C) and Ziehl Nielsen (Figure 1D), showed that most of the bacilli (arrow) were located in the same area as FMs, thus suggesting a strong association between the persisting tubercle bacilli and FMs within granulomas.\n\nM. tuberculosis induces the formation of FMs within in vitro human tuberculous granulomas\nTo further characterize the role of FMs in the granulomatous response, we assessed whether FM formation in granulomatous structures was triggered only by pathogenic mycobacterial species (M.tb), or by low virulent ones (M. smegmatis) as well. PBMCs from non-tuberculous control individuals were infected with M.tb or M. smegmatis, following the procedure previously described for the induction of granulomatous structures ([7],[9] and Figure S1). Granuloma cells collected at days 3 and 11 were stained with Oil red-O to visualize the lipid droplets within FMs under the light microscope (Figure 2). At day 3, several M.tb-induced granuloma cells already showed lipid bodies (Figure 2A). In contrast, the cells collected from M. smegmatis-induced granulomas were seldom (5%) positively stained (Figure 2B). Interestingly, M. avium induced FM formation in a similar way to M.tb (not shown). By day 11, the amount of positively stained cells had increased in M.tb-induced granulomas, but not in M. smegmatis-induced ones. In addition, the number of lipid bodies per cell increased dramatically with time, as depited in the enlarged views (Figures 2A, C). The quantitative evaluation of the percentage of FMs within granulomas induced by both strains confirmed the differences observed under the light microscope, and showed a seven-fold difference (44% vs 6% respectively) between M.tb and M. smegmatis in terms of their ability to induce FM formation (Figure 2D). Our results therefore show that virulent species such as M.tb and M. avium, contrary to poorly or avirulent ones such as M. smegmatis, are able to induce the formation of FMs within our experimental model.\n\nOxygenated mycolic acids induce the maturation of macrophages into FMs\nMycolic acids from M.tb incorporated into liposomes were recently shown to trigger the differentiation of mice peritoneal macrophages into foamy-like cells [21]. Interestingly, both M.tb and M. avium, which induce FM formation, express a family of oxygenated mycolic acids, especially ketomycolic acids, which are not produced by M. smegmatis (Figure 3A). In this context, inactivation of the M.tb hma gene (mmaA4-Rv0642c) was shown to abolish the synthesis of oxygenated keto- and hydroxyl-mycolic acid in the mutant strain [17]. Conversely, transforming M. smegmatis with the hma gene induced the production of both keto- and hydroxyl-mycolic acids [22], (Figures 3B, C).\nIn the light of both data, we anticipated that oxygenated mycolic acids specifically produced by M.tb and M. avium, under the control of hma, are responsible for FM formation within human granulomas. To test this hypothesis, we compared FM formation after infection of PBMCs with either the wild-type, or the hma-expressing M. smegmatis strain (M. smegmatis/hma). Granulomas cells were collected 3 days later and stained with Oil red-O to visualize lipid bodies. Wild-type M. smegmatis-induced granulomas displayed only 5.5% of FM, whereas the hma gene-expressing strain induced granulomas bearing a majority (67%) of brightly stained Oil red-O positive cells (Figures 4A, B, C). Induction of FM formation was even greater if isolated macrophages were directly infected with either strain. After only 4 hours of infection, M. smegmatis/hma had already transformed 64% of the infected macrophages into lipid body-positive cells (see Figure S2 for Nile red positive cells), whereas only 9% of the cells contained lipid bodies after infection with the wild-type strain (Figures 4D, E, F). To confirm the specific role of hma-dependent oxygenated mycolic acids in FM formation, and to rule out a possible combined effect of oxygenated mycolic-acids with other mycobacterial components, mycolic acids isolated from wild-type M. smegmatis or M. smegmatis/hma were incubated with isolated macrophages. With mycolic acids isolated from the wild-type strain, only 13% of the macrophages were transformed into FM whereas 66% of the macrophages incubated with mycolic acids isolated from M. smegmatis/hma were strongly stained for lipid bodies (Figures 4G, H, I).\nThese results therefore indicate that oxygenated mycolic acids play a leading role in M.tb-induced FM formation.\n\nThe phagocytic and bactericidal activities are arrested in FMs\nTo assess the function of granuloma FM, we first evaluated the ability of such cells to mediate phagocytosis. For this purpose, macrophages isolated from PBMCs were exposed to M. smegmatis/hma-derived mycolic acids to induce FM formation. Two days later, the cell population contained a mixture of FM (50–70%) and macrophages (30–50%), as assessed by Nile red staining (not shown). The mixed cell population was infected with FITC-labelled M. smegmatis. Intracellular bacilli were found only within Nile red-negative macrophages thereby indicating that FMs are unable to ingest bacteria (Figure 5A). This result was reproduced using other mycobacterial strains, such as M.tb and M. bovis BCG, for infection (not shown). These results further suggest that the bacilli found in granuloma FMs were internalized by macrophages prior to their transformation into FMs.\nTo assess whether FMs are able to develop a respiratory burst, which is a major intracellular bactericidal activity, the ability of Nile red positive cells (i.e. FMs) to mediate NBT reduction was determined. As shown in Figure 5D, only 8% of the NBT-positive cells (Figure 5B) were Nile red positive FMs (Figure 5C). This strongly suggests that once macrophages have differentiated into FMs, they lose the ability to mediate intracellular bactericidal activity. We postulate that FMs could, therefore, form a secure reservoir for the tubercle bacilli.\n\nM. tuberculosis persists in a dormant non-replicative state in FMs\nTo evaluate the validity of the above hypothesis, we analyzed the ability of M.tb to replicate within FMs. For this purpose, isolated macrophages were infected with M.tb. At selected intervals post-infection, the amount of bacilli per cell was compared in both non-differentiated macrophages and FMs (Figure 5E). Until day 6, M.tb replicated in a similar fashion in both cell types. In contrast, after day 6 post-infection, the amount of bacilli remained stationary in FMs, whereas it continued to increase in macrophages. It is interesting to note that arrest of bacterial replication coincided with completion of macrophage differentiation into FM, i.e. starting from day 3 post-infection. Our data suggest, that the bacilli found in granuloma FMs were internalized by macrophages prior to their differentiation into FMs and also that bacilli can terminate their replication cycle while macrophages are being transformed into FM, but that replication comes to a halt as soon as the maturation process is complete.\nWe next determined whether the non-replicative bacilli observed in FMs were still alive. For this purpose, we analyzed the expression of a series of genes known to be up-regulated when bacilli are in a persistent non-replicating state [23],[24]. RNA was, therefore, prepared from both in vitro-grown M.tb and intracellular bacilli at day 6 and 12 post-infection. The respective amounts of RNA corresponding to isocitrate lyase, α-cristallin, a very hypothetical 7.6 kDa protein, CHP and DosR proteins were then quantified by RT-PCR. As shown in Table 3, the dormancy genes were all strongly up-regulated in intracellular bacilli at day 12 post-infection. These results further demonstrate that the bacilli are not killed in FMs, but rather persist in a dormant, and therefore non-replicative stage [25]. Interestingly, the dormancy genes were not as strongly expressed at day 6, time at which bacteria were still able to replicate in macrophages undergoing differentiation into FMs.\n\nCharacterization of M.tb survival within FMs and interaction with lipid bodies\nTo gain further insight into the morphological appearance of M.tb within FM and into the interactions between FM lipid bodies (LB) and M.tb-containing phagosomes, granulomas were fixed and processed for conventional electron microscopy at days 3 and 11 post-infection.\nWhatever the time point at which granuloma cells were observed, bacteria were all enclosed in phagosomes, most of which contained a single bacterium. None of them (over a thousand which were examined under the electron microscope) were free in the cytoplasm and only one was enclosed in a classical autophagic vacuole. At day 3 post-infection, FMs profiles (thin sections) were scarce, representing at most 9% of the total population of macrophage profiles observed under the electron microscope (Figure 6A). In addition, 86% of the FM profiles displayed at most 5 small LBs (Figure 6B). At this stage of granuloma formation, bacteria were infrequent in FM, but were found in other types of macrophages. One of these displayed large numbers of vacuoles containing flocculent material and often one or two LBs. Over 95% of the bacteria located in the different granuloma macrophages were morphologically intact, and therefore alive [26]. Intact bacteria present no breaks in the cell wall or cytoplasmic membrane and their cytoplasm has preserved its ultrastructural organization and electron opacity. Furthermore, they display no electron translucent intracytoplasmic lipid inclusions (ILI). Bacteria were also observed in between cells, probably as a result of cell lysis within granulomas. These bacteria were also morphologically intact and devoid of ILI (not shown).\nAt day 11 post-infection, M.tb-containing macrophages displaying large numbers of vacuoles with flocculent material were less frequently observed. Interestingly, the amount of such vacuoles had strongly decreased in most of these cells while the number of LBs had increased. The percentage of FM had increased to reach 41% of the total population of macrophage profiles within the granulomas (Figure 6A). From these observations, it is tempting to assume that the highly vesiculated macrophages give rise to FMs. Within FMs, the size and amount of LBs had also increased with time since 48% of the FM profiles now displayed more than 5 LB per FM thin section (Figure 6B), randomly distributed within the cells (Figure 6C). About 30% of the FM profiles displayed between 1 and 20 bacteria, which were morphologically intact and enclosed within phagosomes (Figure 6C, enlarged view).\nThe interaction between these bacteria and the cellular LBs was next examined at day 11. Sixty percent of the bacilli were scattered throughout the FMs and displayed no obvious signs of interaction with the cellular LBs. A small fraction of the bacteria (21%), however, were observed in the close vicinity of cellular LBs. The membrane of the phagosomes in which they were enclosed clearly interacted with cellular LBs (Figures 6C, 7A, 7B, arrows) and became tightly apposed to an increasingly larger surface area of the LB. As a result, the phagosomes started to surround LBs in a zippering fashion (Figures 7E, F). Ultimately, bacilli (19%) were translocated to cellular LBs (Figure 7C). From these observations, it is tempting to assume that M.tb-containing phagosomes engulf cellular LBs rather than fusing with them. This process, which is reminiscent of autophagy, resulted in the transfer of free bacteria into the lumen of cellular LBs (Figure 7C), some of which displayed up to 21 bacteria (Figure 7G). Interestingly, only altered M.tb found within FM lipid bodies exhibited electron translucent ILIs (Figures 7D, G), thereby suggesting that they are able to accumulate host cell lipids. In previous work, the term altered bacteria had been used to define live bacteria that had acquired ILIs [26]. Since the presence of ILIs within the cytoplasm of M.tb is typical for non-replicating bacteria in a state of dormancy [27], this further confirms that these bacteria are dormant.\n\n\nDiscussion\nStudies carried out several decades ago suggested that postprimary tuberculosis starts as a lipid pneumonia [28],[29]. Indeed, following a first inflammatory process leading to exudates of mononuclear cells within alveolar spaces, early pathologists observed an accumulation of lipid droplets in alveolar macrophages of TB patients. Tubercle bacilli were shown to reside in these lipid-rich macrophages which were named foamy macrophages (FMs) [30]. Recently, histo-pathological analysis of biopsies from patients with untreated tuberculosis confirmed the century-old histological descriptions of postprimary tuberculosis [10]. In this study, Hunter et al showed that postprimary tuberculosis begins as a lipid pneumonia with the accumulation of large amounts of lipid-rich FMs, accompanied by bronchial obstruction. It was also shown that in alveolar foamy macrophages, the bacilli were mainly found within lipid droplets. All these observations underline the important, yet often neglected, role of lipid accumulation, and more precisely FM formation, at the infectious site in the physiopathology of TB.\nIn murine experimental models, FMs accumulate within the outermost layer of granulomatous structures occupying the alveolar spaces during the chronic phase of infection. This strongly suggests that FMs could be involved in lesion cleaning via phagocytic uptake of cellular debris generated by the local inflammatory response. Once filled with debris, FMs would leave the parenchyma through the alveolar spaces up to the superior bronchial tree, to be finally swallowed and digested in the stomach [31]. In fact, this process is very well known, and is a crucial factor for TB diagnosis in children. As infants do not usually generate cavitary lesions, and because it is difficult to detect bacilli in the sputum, the diagnosis is linked to the detection of bacilli in the gastrointestinal lavage [32].\nWe show that M.tb-induced the transformation of in vitro-grown human granuloma macrophages into FMs within 6 days, and even more rapidly (3–4 hours) in cultured macrophages. Although this event occurs more quickly than in vivo, or in animal models, our data are consistent with the above in vivo observations. Within FMs, bacilli and LBs were often tightly linked, to the point that a non-negligible amount of bacteria were ultimately observed within LBs. Interestingly, some of the bacilli transferred into lipid bodies displayed their own intracytoplasmic lipid inclusions, which are considered to be one of the hallmarks of non-replicating (dormant) M.tb [27]. The recent observation of persistent ILI-containing tubercle bacilli within adipocyte LBs [33] is in good agreement with our observations. Since bacilli residing in phagosomes that do not interact with cellular lipid bodies do not display ILIs, it is tempting to assume that lipids within ILIs are of cellular origin. The accumulation of lipids within bacilli [47], via interaction with FM lipid bodies could, therefore, be crucial to M.tb persistence. It is indeed known that M.tb accumulates lipids, and more precisely triacylglycerols, during dormancy [34],[35] from which it derives both carbon and energy for its own metabolism. Intracellular persistence of M.tb is also critically linked to the acquisition of host cholesterol through the Mce4 transporter system [36].\nThe question that arises is how do bacteria gain access to lipids from LB? Direct fusion of phagosomes with FM lipid bodies seems unlikely as the membranes of both structures are quite different from one another. Our observations suggest instead that once M.tb-containing phagosomes have established close contact with a lipid body, they surround and engulf the latter by a process that remains to be deciphered. This phenomenon is somewhat reminiscent of autophagy, as observed under conditions of cholesterol depletion in macrophages infected with M. avium [37]. After degradation of the resulting inner membrane, bacteria would be freed within the lipids of the engulfed LB, and therefore be in direct contact with cellular lipids. How bacilli translocate the cellular lipids to their own cytoplasm remains to be established.\nAnother important phenomenon underlined by our study is the strong correlation between the presence of FMs in the granulomatous structures and the development of necrosis within the lesion, as suggested by Pagel over 80 years ago [29]. Interestingly, FMs were systematically located at the interface region between the histiocytes and the central necrosis area of the biopsied lesions. Although necrosis formation could depend on an indirect effect of the global immune response, our data indicate that the formation of FMs is an important factor favoring the appearance of necrosis. Analysis of larger series of biopsy samples are, however, needed to definitely demonstrate our actual hypothesis according to which FMs play a direct and unique role in necrosis formation. Consistent with this hypothesis, we observed that FMs induced from M.tb-infected macrophages displayed permanent TNF-α secretion, a potent pro-necrotic factor, whereas M. smegmatis-infected macrophages were poor producers of TNF-α. At day 4 post-infection, TNF-α secretion was indeed twofold higher in macrophages (of which 70% had differentiated into FMs) infected with M.tb than in those infected with M. smegmatis, as measured both by ELISA and RNA quantification (Peyron, unpublished observations). However, one must keep in mind that the association of FMs and necrosis may be the consequence of the FM cleaning process of lipoproteins released into the necrotic tissue, as observed in atherosclerosis lesions [38]. It is thus tempting to propose that M.tb mycolic acids may be responsible for the development of necrotic lesions, due to their ability to induce TNF-α production by FMs. Whether mycolic acids are directly involved in TNF-α production, or only indirectly by inducing FM formation, remains a matter of debate currently under study.\nOur observations are strikingly similar to the phenomena described for postprimary tuberculosis, that seems to begin as localized foci of pneumonia followed by massive necrosis leading to the formation of pulmonary cavities [10]. If this proves to be the case, then the traditionally admitted phenomenon of cavitation arising from the erosion of caseating granulomatous structures from bronchi can be ruled out [39]. In our study, we successfully induced FM formation from isolated macrophages infected with M.tb, i.e. outside a granulomatous structure, which is consistent with Hunter's recent proposal.\nUntil now, mycolic acids have been considered to be indirectly involved in virulence mechanisms as being part of complex molecules of the mycobacterial envelope. The most widely studied mycolic acid-containing mycobacterial compound trehalose 6,6′ dimycolate (TDM), has been extensively analyzed for its role in virulence since the mid-fifties [40]. Recently, it was shown to interfere with the host granulomatous response [41]. Overall, TDM was mainly shown to mediate macrophage activation and a Th1-type response to M.tb infection (for review, see [25]).\nOur results demonstrate a direct role of oxygenated mycolic acids for FM formation, independently from the appearance and stage of the disease. M.tb-specific mycolic acids indeed trigger the transformation of both isolated and granuloma macrophages, into FM. Given the absence of FM formation in M. smegmatis-induced granulomas, despite the induction of a comparative inflammatory response, ascertained by the similar induction of granulomas, this phenomenon clearly depends upon a direct contact with the bacilli, and not to the inflammatory response. Mycolic acids are major and hallmark components of the mycobacterial cell wall. They constitute 40–60% of dry weight of the envelope [42]. All members of the complex (e.g. M.tb, Mycobacterium africanum, Mycobacterium bovis and Mycobacterium microti) are able to synthesize the same combination of mycolic acids, i.e. cyclopropanated α-mycolic acids, ketomycolic and methoxymycolic acids [17], which are not synthesized by non-pathogenic mycobacterial species [43]. These structural specificities probably account for part of the pathogenicity of these species, as shown by the impaired virulence of mutant strains deprived of keto and methoxyl groups in experimental infections [17],[44],[45].\nOur study, therefore, gives the first proof of a direct role of isolated mycolic acids in the interplay between M.tb and host cells. Interestingly, this effect is expressed both by whole bacilli and isolated lipids, suggesting that oxygenated mycolic acids are either secreted by the bacilli, or exposed at the cell wall surface in a manner enabling their bioactivity. According to our results, mycolic acids trigger the formation, within granulomas, of FMs in which bacilli can hide and survive. Oxygenated mycolic acids, either free, as constituents of TDM [44], or linked to the cell wall arabinogalactan [46],[47], should, therefore, be considered as major virulence factors enabling M.tb survival for long periods of time in a persistent state. Being an inducer of host lipid accumulation, and FM formation at the site of infection, these oxygenated mycolic acids could, therefore, also be responsible for the induction of necrosis within lesions, thus favoring M.tb dissemination.\nInterestingly, deletion of the mmaA4 (Rv0642c) gene also drastically decreased the ability of M.tb to induce the differentiation of macrophages into FMs (data not shown). However, the residual ability of this mutant to induce FMs suggests that other mycobacterial factors might partially trigger the formation of FM. With regard to the mycolic acid methyltransferases, given that (i) mmaA2 (Rv0644c) and mmaA3 (Rv0643c) are pseudogenes in M. leprae and (ii) mmaA4 KO present no trans cyclopropanation [45], thus excluding the involvement of the cmaA2 (Rv0503c) gene, we expect that at least pcaA (Rv0470c), which introduces cis-cyclopropane, may play the same role. Consistent with this hypothesis, a pcaA null mutant is unable to persist within infected mice [46], thus demonstrating the role of a mycolic acid methyltransferase in the chronic stage of infection.\nOverall, our study has shed light on a previously uncharacterized cell population participating in human tuberculous granulomas, namely foamy macrophages. We propose that the specific induction of FM by M.tb would create a favourable environment for persistent bacteria. In our opinion, FMs could be a safe shelter because they preserve bacilli from a direct contact with granuloma lymphocytes and histiocytes, they lose one of the major macrophage bactericidal activities and they constitute an important source of nutrients for the bacilli thanks to the fatty acids accumulated in their lipid granules.\n\nSupporting Information\n\n\n" ], "offsets": [ [ 0, 39660 ] ] } ]

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[ { "id": "pmcA1131934__text", "type": "Article", "text": [ "HP1 modulates the transcription of cell-cycle regulators in Drosophila melanogaster\nAbstract\nHeterochromatin protein 1 (HP1) was originally described as a non-histone chromosomal protein and is required for transcriptional gene silencing and the formation of heterochromatin. Although it is localized primarily at pericentric heterochromatin, a scattered distribution over a large number of euchromatic loci is also evident. Here, we provide evidence that Drosophila HP1 is essential for the maintenance of active transcription of euchromatic genes functionally involved in cell-cycle progression, including those required for DNA replication and mitosis. Depletion of HP1 in proliferating embryonic cells caused aberrant progression of the cell cycle at S phase and G2/M phase, linked to aberrant chromosome segregation, cytokinesis, and an increase in apoptosis. The chromosomal distribution of Aurora B, and the level of phosphorylation of histone H3 serine 10 were also altered in the absence of HP1. Using chromatin immunoprecipitation analysis, we further demonstrate that the promoters of a number of cell-cycle regulator genes are bound to HP1, supporting a direct role for HP1 in their active transcription. Overall, our data suggest that HP1 is essential for the maintenance of cell-cycle progression and the transcription of cell-cycle regulatory genes. The results also support the view that HP1 is a positive regulator of transcription in euchromatin.\n\nINTRODUCTION\nChromatin in higher eukaryotes is subdivided into different functional compartments termed heterochromatin and euchromatin (1). Heterochromatin differs from euchromatin in its DNA composition, replication timing, condensation throughout the cell cycle, and its ability to silence euchromatic genes placed adjacent to or within its territory, often described as position-effect-variegation (PEV) (2).\nHeterochromatin protein 1 (HP1) was the first protein identified in Drosophila melanogaster as a heterochromatin-associated protein (3); the corresponding gene has been cloned from a number of organisms and is highly conserved from yeast to human (4). Polytene chromosome staining showed that, in Drosophila, HP1 is distributed mainly in pericentric heterochromatin, telomeric heterochromatin, the banded small fourth chromosome (5–8), as well as ∼200 individual loci scattered throughout the euchromatic chromosomal arms (5). The gene encoding HP1 in D.melanogaster, Su(var)2-5, was isolated as a suppressor of PEV (9–11). The protein contains a highly conserved motif, the chromo (chromatin organization modifier) domain, similar to Polycomb (Pc), a repressor of homeotic genes (12). The association between HP1 and pericentric heterochromatin is believed to occur via the chromo domain of HP1 and the N-terminal tail of histone H3 methylated at lysine 9 (13,14), generated by histone methyltransferase-Su(var)3-9, a partner of HP1 in pericentric heterochromatin (15). The C-terminal chromo ‘shadow’ domain of HP1 interacts with other silencing complexes to suppress local transcriptional activity (15–18). However, studies of HP1 chromosomal distribution also showed that HP1 does not always co-localize with lysine 9 methylated histone H3 or Su(var)3-9, especially in euchromatic regions (19–21); in some cases, HP1 is found directly bound to DNA (22,23). All these features argue for distinct roles for HP1 in chromatin and in epigenetic gene regulation.\nHP1 is believed to be an essential structural protein protecting the integrity of chromosomes during cell division (8,24). Swi6, the homolog of HP1 in fission yeast, is dispensable for survival, but its deletion results in lagging chromosomes during anaphase, and a high rate of chromosome loss (25,26). Mutations of HP1 in D.melanogaster result in late larval lethality, chromosome breakages/loss, telomere fusion and a high frequency of cells with abnormal anaphase (8,27). Null alleles of the HP1 functional partner in mice (SUVAR39) also showed various chromosomal defects (28), supporting a conserved role for heterochromatin proteins in the regulation of chromosome dynamics during cell-cycle progression. However, the mechanism(s) involved remains to be understood.\nIn this study, we utilized Drosophila embryonic Kc cells and an RNA interference (RNAi)-based approach to demonstrate that HP1 plays an important role at S phase and G2/M phases during the cell cycle. We further show that nearly one-third of known/predicted cell-cycle regulators require HP1 to maintain their active transcription. These genes include MCMs, Orc4, CDC45L, INCENP, Aurora B, CAF1, Bub1, Bub3 and a few other cell-cycle regulators. ChIP analysis suggests that HP1 plays a direct role in their transcription. Therefore, the results of this study provide an alternative explanation for the specific role of HP1 in the regulation of chromatin dynamics and in cell-cycle progression.\n\nMATERIALS AND METHODS\nRNAi in Kc cells\nDrosophila Kc cells were routinely cultured at 25°C in Schneider Drosophila medium (GIBCO) supplemented with 10% fetal calf serum, 160 μg/ml penicillin, 250 μg/ml streptomycin, and 4 mM l-glutamine. Double-stranded RNA (dsRNA) of HP1 was generated by incubation of single-stranded RNA in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 3 min at 95°C and then placed in a beaker with water at 75°C and allowed to cool slowly to room temperature. The detailed procedure of RNAi was carried out according to the established protocols (). Briefly, Kc cells were seeded in a six-well dish using serum-free medium at 1 × 106 cells/ml. HP1 dsRNA (5 μg/ml) was added to the cultured Kc cells. After 60 min at room temperature, 2 ml of medium containing 10% serum was added to each well and the plates transferred to 25°C for up to 8 days. Western blotting and RT–PCR were carried out using the extract/total RNA isolated from control and dsRNA-treated cells on days 2, 6 and 8.\n\nCell-cycle and apoptosis analysis\nThe procedure for flow cytometric analysis of Kc cells followed that in the manual provided with the BrdU flow kit (BD PharMingen). The cells were fed with BrdU for 4 h, then scraped and collected. Fluorescence was measured using a FACSCalibur (Becton Dickinson). Data collection and analysis were performed using CellQuest software.\n\nElectrophoresis and immunoblotting\nCell extracts (15 μg) were fractionated by 10% SDS–PAGE, then transferred to Hybond-P PVDF membranes (Amersham) and probed with primary antibodies (CIA9), and secondary antibodies (anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG), obtained from Jackson Immunoresearch Laboratories. Enhanced chemiluminescence reagents (Amersham Pharmacia Biotech) were used for signal detection.\nFor the analysis of H3 ser10 phosphorylation, we used whole-cell extracts from 700 000 Kc cells (control and RNAi at day 8). Western blotting was performed using polyclonal antibodies against ser10-phosphorylated histone H3 at a dilution of 1:1000 (Upstate). Kc control cells arrested in mitosis by incubation in 25 µM colchicine (Sigma) for 24 h were also analyzed for comparison.\n\nImmunofluorescence\nKc cells were seeded onto polylysine slides, fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 5 min. The incubation with primary antibodies was carried out in blocking solution for 1 h.\nFor staining of mitotic cells, the cells were permeabilized using PBST (PBS containing 0.3% Triton X-100) and stained with polyclonal antibody against Drosophila Aurora B at 1:200 dilution and monoclonal mouse at anti-β-tubulin 1:300 dilution (Chemicon International) as primary antibodies. Secondary antibodies were anti-rabbit coupled with Alexa 488 (1:500) and anti-mouse coupled to Alexa 546 (1:500) (Molecular Probes, Eugene, Oregon). Images were acquired using a confocal LSM510 META microscope (Zeiss). Stacks of images were analyzed using the IMARIS 4.0 program (Media cybernetics, Carlsbad, CA).\n\nAntibodies\nAffinity-purified polyclonal antibodies of HP1 (rabbit #192 and #187, 5 μg) and 5 μg of polyclonal anti-HA antibodies (Sigma) were used in each ChIP reaction. The specificity of the HP1 polyclonal antibodies was determined using various approaches, including western blotting assay, immunofluorescence staining and immunoprecipitation to pull down HP1 (data not shown). The monoclonal antibody HP1–CIA9 (5) was used at a dilution of 1:20 in immunoblotting assays.\n\nMicroarray analysis and RT–PCR\nTotal RNA was isolated from control and HP1-depleted Kc cells at day 8 using an RNeasy kit (Qiagen). RNA labeling and microarray data analysis followed the standard protocol from Affymetrix. We used ANOVA (P < 0.001) to assess the expression confidence for each gene.\nFor RT–PCR analysis, poly(A)+ mRNA was purified with the Oligotex Direct mRNA kit (Qiagen) according to the manufacturer's instructions. The purified poly(A)+ RNA was reverse transcribed using the Thermoscript kit (Invitrogen). The cDNA was then used for PCR amplification for 35 cycles with gene-specific primers. PCR products were scanned after electrophoretic separation with a Typhoon Scanner, quantified using ImageQuant software (Amersham Biosciences) and normalized for amplification of the Actin5c transcript. The sequence of primers used for RT–PCR and ChIP analysis are provided in the Supplementary Material.\n\nChIP\nChIP was performed according to Orlando et al. (29) and the protocol provided by Upstate () with some modifications. In brief, 1–2 × 108 Kc cells were prepared and fixed in 1% formaldehyde. Nuclei were isolated according to a standard procedure in Current Protocols (), then resuspended in 1.7 ml of lysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitors) and sonicated using a Branson sonifier 250. Chromatin fractions in the size range 0.2–0.8 kb were used to perform immunoprecipitation experiments. We used 5 μg affinity-purified polyclonal antibodies (#192 and #182 for HP1; HA antibody for control) and 1 ml of salmon sperm DNA/protein-A-agarose (Upstate) pre-cleared chromatin lysate in each reaction. The mixture was then rotated at 4°C overnight and the recovered beads were washed twice with 1 ml of Low salt buffer (Upstate), once with High salt buffer (Upstate), once with LiCl buffer (Upstate) and twice with TE at 4°C for 8 min. ChIP DNA was extracted according to the standard procedures (29).\n\n\nRESULTS\nDepletion of HP1 in Drosophila Kc cells\nVarious chromosomal defects in the cell cycle have been observed in embryos or larval tissues of Drosophila HP1 mutants (8,27). However, the presence of maternally loaded HP1 in embryos and the lethality of HP1 mutants at late larval stages have so far precluded a systematic study of the role of HP1 in cell-cycle regulation. Therefore, we used Drosophila Kc cells, a cell line derived from Drosophila embryos, as a model system to address this problem. HP1 transcripts were depleted using an RNAi-based approach (see Materials and Methods). The reduction in HP1 expression was measured both by RT–PCR and by western blotting analysis (Figure 1A). A significant reduction in the HP1 expression was already evident after 2 days treatment with HP1 dsRNA. Cells at day 8 showed a reduction in HP1 of ∼90% (Figure 1A) and were therefore used in all subsequent experiments.\n\nCell-cycle progression at S and G2/M phase is altered in the absence of HP1\nThe impact of HP1 loss on the cell cycle of Kc cells was determined using cell-cycle profile analysis of HP1-depleted and control cells. The percentage of cells in S phase was determined by BrdU incorporation, and total DNA content by 7-amino-actinomycin (7-AAD). The results showed that the depletion of HP1 (day 8) caused a decrease in S-phase cells of at least 4-fold, and a 2-fold decrease in G2/M-phase cells (Figure 1B), although no significant effect was found at the G1 phase. In addition, depletion of HP1 caused a greater than 7-fold increase in the number of apoptotic cells. These results, therefore, confirm that HP1 is an important regulator during the cell cycle, especially at the S and G2/M phases.\n\nCell-cycle regulators require HP1 to maintain their active transcription\nTo ask whether the cell-cycle defects were due to changes in the transcription of genes functionally involved in S phase and the G2/M phase, we next assessed global changes in gene transcription following depletion of HP1. Expression profile analysis was performed using total RNA isolated from both HP1-depleted Kc cells and control Kc cells, and an Affymetrix Drosophila chip. For each experiment, we used total RNA isolated from two independent HP1-depleted and control samples, and at least two independent experiments were performed.\nThe microarray analysis showed that loss of HP1 function in Kc cells resulted in alterations in transcription of >500 genes: ∼400 genes were down-regulated and ∼120 genes were up-regulated (>1.5-fold, ANOVA). The function of these genes ranged from cellular enzymes, signal transduction molecules, and membrane and cell structural proteins, to nucleic acid-binding proteins and cell-cycle regulators (Figure 2A). At the chromosomal level, the genes targeted by HP1 appeared to be distributed along all euchromatic chromosomal arms (data not shown), supporting a global role of HP1 in euchromatic gene regulation (20).\nAmong 60 known/predicted genes associated with DNA replication function, 15 were down-regulated in the absence of HP1 (Figure 2B). These included McM2, McM5, McM6 and CDC45L, which are required for processive DNA replication and correct chromosome condensation (30–32). Other genes involved in DNA replication, such as components of the origin recognition complex (Orc)—Orc4, Caf1, Gnf1, Dref1, DNA polymerase-γ and Tam—were also down-regulated (Figure 2B). Aurora B and inner centromere protein (INCENP), known to be required for kinetochore assembly, chromosome condensation and bipolar chromosome attachment during mitosis (33), also showed a reduction in transcription. A similar loss of transcription was observed in Bub1 and Bub3 (Figure 2B), encoding mitotic checkpoint control proteins (34,35). Loss function of Bub1 has been shown to cause chromatin bridges to extend between the two separating groups of chromosomes, and extensive chromosome fragmentation in anaphase cells (35).\nWe confirmed the changes in the transcription of cell-cycle regulators using semi-quantitative RT–PCR, which gave results consistent with the microarray analysis. In addition, cell-cycle regulator genes, such as McM3, McM7 and Asp (abnormal spindle), were also confirmed to be down-regulated (Figure 2C). Collectively, these results demonstrate that HP1 is indeed involved in the regulation of transcription of cell-cycle regulators.\n\nHP1 is required for Aurora B distribution and histone H3 phosphorylation\nINCENP is localized to the centromeric region of chromosomes at metaphase and the spindle midzone at anaphase, which then targets Aurora B, a kinase essential for histone H3 ser10 phosphorylation, to these sites (36). Loss of function of both these ‘chromosomal passenger’ proteins causes abnormal chromosomal segregation at metaphase, as well as certain cytokinesis defects (36,37). The loss of transcription of both INCENP and Aurora B after depletion of HP1, therefore, raised the possibility that localization of Aurora B (Figure 3 and data not shown) may be altered. Staining of HP1-depleted Kc cells with anti-Aurora B antibodies indeed revealed an altered localization of Aurora B and, in a number of cases, a complete loss of Aurora B (Figure 3A). Consistent with the loss function of Aurora B, the spindles in the metaphase cells were also disorganized, with a large number of cells showing an altered prometaphase chromosome alignment (Figure 3A). Some showed extensive chromosome fragmentation (Figure 3B), or the presence of a third spindle pole-like structure as indicated by beta-tubulin (Figure 3A). At telophase, we observed defective separating cells with an extra cell envelope-like structure without nuclei (Figure 3A). Chromatin bridges or lagging chromatids at telophase were also evident in some cells (Figure 3C); however, in some cases, localization of Aurora B appeared not to be affected, arguing that other pathways are possibly involved.\nWe next analyzed changes in histone H3 serine 10 phosphorylation, since the loss of transcription of INCENP is known to affect localization of Aurora B (33), which is essential in the regulation of histone H3 phosphorylation (36). Total cell extracts from HP1-depleted Kc cells were analyzed by western blotting (Figure 3D). The results indeed showed a severalfold reduction in H3 ser10 phosphorylation after depletion of HP1, consistent with the functional disruption of INCENP and Aurora B in the absence of HP1.\n\nHP1 directly targets genes encoding cell-cycle regulators in euchromatin\nTo test whether the loss of transcription of genes involved in DNA replication and mitosis was a direct effect of the loss of HP1, we performed a ChIP analysis to determine whether HP1 is physically associated with these genes. Chromatin lysates from formaldehyde-fixed Kc cells were sonicated into small chromatin fragments (0.2–0.8 kb) and immunoprecipitated with polyclonal antibodies against Drosophila HP1. As a control, we used a mock precipitation (beads only) and polyclonal antibodies against HA. Our ChIP results showed that known transposable elements distributed in heterochromatin, such as F-element, TART and 1360 (7,38), were all enriched in HP1 binding (Figure 4), which is also consistent with a previous study (20).\nUsing the same ChIP DNA material, we then attempted to determine whether HP1 was enriched in genes involved in DNA replication. Primers were designed to cover the promoter regions of selected genes. The results showed that McM3, McM5 and Tam were all enriched in HP1 binding (Figure 4). However, McM7 appeared to be HP1-negative, although its transcription was also affected by the loss of HP1 function. Genes essential for mitosis, such as Aurora B, were also HP1-positive (Figure 4). These results demonstrate that these cell-cycle regulator genes are directly targeted by HP1 in their promoter regions.\n\n\nDISCUSSION\nIn this study, we used microarray and RT–PCR techniques to demonstrate that transcription of cell-cycle regulators is misregulated in the absence of HP1. Certain defects in S phase may be a direct consequence of the loss of transcription of DNA replication genes such as McM2, McM5, McM6, CDC45L, Orc4 and others, since these genes have been functionally implicated in the initiation of DNA replication and/or the progression of replication forks (39). Depletion or mutation of these genes has been shown to result in DNA damage (32), the blockage of replication forks (39), increased chromosome loss/genome instability, and defective condensation (30).\nThe reduction in the number of cells in G2/M phase may be a consequence of the reduction in transcription or functional disruption of INCENP, Aurora B, Bub1, and Bub3 (34,36). Chromosome segregation defects, such as chromosome fragmentation and chromatin bridges in anaphase/telophase cells, and certain cytokinesis defects in HP1-depleted cells, mimic the phenotype of cells with loss function of INCENP, Aurora B or Bub1 (35–37). The mislocalization of Aurora B in the absence of HP1 is also consistent with the loss of transcription and functional disruption of INCENP (37), and the reduction in Aurora B transcription may be partially responsible for the observed chromosomal defects, including loss of histone H3 phosphorylation at serine 10.\nHP1 is also known to physically interact with certain components of replication complexes such as ORCs and MCMs (30,40,41), with the inner centromere protein INCENP (42) and the chromatin assembly factor CAF1 (43) promoting delivery of HP1 to heterochromatin sites (44). Loss of HP1 is, therefore, expected to cause disruption to such HP1-associated complexes, and will partially contribute to the chromatin/chromosomal defects in HP1 mutants (8,27) and HP1-depleted Kc cells. It is therefore well possible that the loss of transcription of these cell-cycle regulator genes, and consequent disruption of HP1 functional complexes or heterochromatin structure, all contributed to the cell-cycle defects observed.\nThe ChIP assay supports the hypothesis that the loss of transcription of cell-cycle regulator genes is a direct effect of the lack of HP1. Aurora B, McM3, and McM5 were all bound by HP1 at their promoter regions, although other cell-cycle regulators, such as McM7, were HP1-negative, implying that the altered transcription in these genes might be a secondary effect of the loss of HP1.\nA previous study in Drosophila Kc cells (20) employed an approach based on the ectopic expression of a fusion protein of HP1 with a prokaryotic DNA adenine methyltransferase and identified a number of methylated targets in the genome. In this study, MCM3 and MCM5 were not found to be methylated, indicating lack of association with HP1. On the other hand, heterochromatin repeats, such F-element and 1360, were consistently found to be HP1-enriched both here and in the previous study. It remains to be determined whether these discrepancies are due to the different experimental systems used. However, we note that the previous study was performed using a cDNA array, while we observe binding of endogenous HP1 at the promoter of these genes. Similarly, another study using chromatin immunoprecipitation in larvae also showed few HP1-positive genes that were not detected in Kc cells by the Dam ID approach (21).\nA large number of genes affected by the loss of D.melanogaster HP1 in larval tissues (21) seem to be different from that in embryonic Kc cells. The change in the transcription of Aurora B and few cell-cycle regulators reported in this study is also not found among the HP1-affected genes at larval stage (21). This may be due to specific role(s) of HP1 in different stages of development. Alternatively, it is also possible that the impact of HP1 in the transcription of cell-cycle regulators in proliferating cells is underestimated when performing the analyses on larval tissues, and thus on mixed populations of both proliferating and differentiating/differentiated cells.\nHP1 is generally known as a transcriptional repressor, as supported by several lines of evidence: silencing of a euchromatic reporter gene in heterochromatin requires HP1 (10,11), tethering of HP1 next to a euchromatic reporter gene causes silencing (45), and the repression of genes within euchromatic region 31 bound by HP1 is relieved in the absence of HP1 (46). In contrast, genes in heterochromatin, known as heterochromatic genes, such as light and rolled, seem to require HP1 to maintain their active transcription (47,48). The level of transcription of heterochromatic genes was dramatically reduced in a mutated HP1 background (47–49). It was therefore proposed that HP1 may function as a positive regulator of transcription of these genes (50,51), although the exact regulation mechanism remains unclear. A study of heat-shock genes found that HP1 is associated with RNA transcripts in the coding region, and is also a positive regulator of their transcription (52,53). The chromatin association of HP1 at the promoter region of active euchromatic genes demonstrated from this work and others, and its independence from histone H3K9 methylation (21), all suggest that mechanism whereby HP1 modulates transcription of euchromatic genes is potentially distinct from its role in heterochromatin formation.\nCollectively, the results of this study demonstrate that HP1 plays an essential role in cell-cycle progression, and support the view that HP1, in addition to its role in heterochromatin, can act as a positive transcriptional regulator of euchromatic genes.\n\nSUPPLEMENTARY MATERIAL\nSupplementary Material is available at NAR Online.\n\nSupplementary Material\n\n\n" ], "offsets": [ [ 0, 24037 ] ] } ]

[ { "id": "pmcA1131934__T0", "type": "species", "text": [ "Drosophila melanogaster" ], "offsets": [ [ 60, 83 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T1", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 456, 466 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T2", "type": "species", "text": [ "Drosophila melanogaster" ], "offsets": [ [ 1947, 1970 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T3", "type": "species", "text": [ "yeast" ], "offsets": [ [ 2111, 2116 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4932" } ] }, { "id": "pmcA1131934__T4", "type": "species", "text": [ "human" ], "offsets": [ [ 2120, 2125 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] }, { "id": "pmcA1131934__T5", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 2176, 2186 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T6", "type": "species", "text": [ "D.melanogaster" ], "offsets": [ [ 2431, 2445 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T7", "type": "species", "text": [ "fission yeast" ], "offsets": [ [ 3590, 3603 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4896" } ] }, { "id": "pmcA1131934__T8", "type": "species", "text": [ "D.melanogaster" ], "offsets": [ [ 3763, 3777 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T9", "type": "species", "text": [ "mice" ], "offsets": [ [ 3961, 3965 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "10090" } ] }, { "id": "pmcA1131934__T10", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 4239, 4249 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T11", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 4946, 4956 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T12", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 5011, 5021 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T13", "type": "species", "text": [ "rabbit" ], "offsets": [ [ 6556, 6562 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9986" } ] }, { "id": "pmcA1131934__T14", "type": "species", "text": [ "mouse" ], "offsets": [ [ 6571, 6576 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "10090" } ] }, { "id": "pmcA1131934__T15", "type": "species", "text": [ "horseradish" ], "offsets": [ [ 6577, 6588 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "3704" } ] }, { "id": "pmcA1131934__T16", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 7538, 7548 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T17", "type": "species", "text": [ "mouse" ], "offsets": [ [ 7591, 7596 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "10090" } ] }, { "id": "pmcA1131934__T18", "type": "species", "text": [ "rabbit" ], "offsets": [ [ 7709, 7715 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9986" } ] }, { "id": "pmcA1131934__T19", "type": "species", "text": [ "mouse" ], "offsets": [ [ 7756, 7761 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "10090" } ] }, { "id": "pmcA1131934__T20", "type": "species", "text": [ "rabbit" ], "offsets": [ [ 8052, 8058 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9986" } ] }, { "id": "pmcA1131934__T21", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 10455, 10465 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T22", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 10572, 10582 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T23", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 10821, 10831 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T24", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 10867, 10877 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T25", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 12574, 12584 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T26", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 17318, 17328 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T27", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 20795, 20805 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1131934__T28", "type": "species", "text": [ "D.melanogaster" ], "offsets": [ [ 21738, 21752 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] } ]

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[ { "id": "pmcA1851977__text", "type": "Article", "text": [ "Molecular Basis for a Lack of Correlation between Viral Fitness and Cell Killing Capacity\nAbstract\nThe relationship between parasite fitness and virulence has been the object of experimental and theoretical studies often with conflicting conclusions. Here, we provide direct experimental evidence that viral fitness and virulence, both measured in the same biological environment provided by host cells in culture, can be two unrelated traits. A biological clone of foot-and-mouth disease virus acquired high fitness and virulence (cell killing capacity) upon large population passages in cell culture. However, subsequent plaque-to-plaque transfers resulted in profound fitness loss, but only a minimal decrease of virulence. While fitness-decreasing mutations have been mapped throughout the genome, virulence determinants—studied here with mutant and chimeric viruses—were multigenic, but concentrated on some genomic regions. Therefore, we propose a model in which viral virulence is more robust to mutation than viral fitness. As a consequence, depending on the passage regime, viral fitness and virulence can follow different evolutionary trajectories. This lack of correlation is relevant to current models of attenuation and virulence in that virus de-adaptation need not entail a decrease of virulence.\nVirulence expresses the harm that parasites inflict upon their hosts. Many studies have addressed the basis of virulence and its effect on host and parasite survival. It has generally been accepted that one of the components of parasite virulence is fitness, or the capacity of the parasite to multiply in its host. Some models have equated virulence with fitness. In the present study, we use foot-and-mouth disease virus (FMDV) to document that virulence and fitness—measured in the same biological environment provided by cells in culture—can be unrelated traits. This has been achieved by multiplying the virus in a manner that mutations accumulated in its genome. Mutations decreased fitness dramatically, but not virulence. Chimeric and mutant viruses were constructed to show that virulence is influenced by only some of the FMDV genes, while fitness is influenced by the entire genome. For this reason, virulence is more robust (“resistant”) than fitness to the effects of deleterious mutations. The fact that virulence can be unrelated to fitness has implications for the design of anti-viral vaccines because it suggests that it may be possible to design high fitness, low virulence strains to stimulate the host immune response. Furthermore, in modelling studies it cannot be assumed that virulence is equal to fitness.\n\n\n\nIntroduction\nThe relationship between fitness and virulence is an unsettled question, and sometimes fitness is considered a component of the virulence phenotype of parasites. RNA viruses are ideal systems to address this important question because of their high mutability and fecundity, which result in a potential for rapid evolution, and also because of the availability of quantitative fitness and virulence assays.\nRNA viruses replicate as complex and dynamic mutant spectra, termed viral quasispecies. Key to quasispecies dynamics are mutation rates in the range of 10−3 to 10−5 substitutions per nucleotide copied, and competition among continuously arising variant genomes [1–4], which prompt rapid movements in sequence space, with corresponding changes of position in the fitness landscape [5]. Indeed, large population passages of RNA viruses in cell culture permit competitive optimization of mutant distributions that generally result in fitness gain [6,7], while repeated bottleneck events (experimentally realized as plaque-to-plaque transfers) lead to random accumulation of deleterious mutations (operation of Muller's ratchet [8]) and result in average fitness decreases [9–13]. Fitness recovery of low fitness foot-and-mouth disease virus (FMDV) clones occurs mainly with introduction of mutations along the genome, with very few true reversions.\nAn understanding of the consequences of fitness variation for viral virulence is a key question for viral pathogenesis and evolution. Here, we approach this issue with FMDV, an important viral pathogen in veterinary medicine [14], and one that fully participates of quasispecies dynamics. Our laboratory has characterized multiple FMDV variants that derive from one original biological clone, and that occupy widely different fitness levels when replicating in a defined environment in cell culture. We define fitness as the replication capacity of a mutant FMDV, relative to a reference FMDV, in direct growth-competition upon coinfection of baby hamster kidney 21 (BHK-21) cells [15–17]. Fitness of FMDV in BHK-21 cells is a multigenic trait [7].\nIn the present study, we define virulence of FMDV as the capacity of the virus to kill BHK-21 cells under a standard set of cell culture conditions [18]. Thus, the FMDV–BHK-21 system offered a means to investigate in a direct and comparative fashion the relationship between fitness and virulence of a virus, measured in the same biological environment provided by BHK-21 cells. We describe the behavior of an FMDV clone (\n\t\t\t\t), which has a history of repeated serial plaque-to-plaque transfers in BHK-21 cells [11], that attained a very low fitness value relative to its parental reference virus (C-S8c1), and yet, its virulence for BHK-21 cells was significantly higher than that of C-S8c1. A comparative study of the capacity to kill BHK-21 cells of chimeric FMDVs constructed with cDNA copies of the two parental FMDVs indicates that the enhanced virulence for BHK-21 cells of the low fitness clone is a polygenic trait, with participation of the regions encoding capsid proteins and non-structural proteins 2A, 2B, and 2C as virulence determinants. Three specific amino acid replacements in 2C have been identified as redundant virulence determinants of FMDV for BHK-21 cells. Thus, while large population passages of the virus resulted in a gain of both fitness and virulence, subsequent bottleneck passages resulted in a decrease of fitness but not of virulence.\n\t\t\t\nThe results suggest that fitness is very vulnerable to mutation in any genomic region. In contrast, because of the involvement of several (but not all) viral genes in virulence, and the redundant effect of three 2C substitutions, virulence is a more robust phenotypic trait than fitness, and less vulnerable to accumulation of mutations. Therefore, we provide direct evidence that viral fitness and capacity to kill cells can (in some cases) be unrelated traits. Furthermore, the relationship between fitness and virulence, of being either linked or unrelated traits, depends on the evolutionary history of the virus. This observation has implications for viral pathogenesis, and sheds light on models of virulence proposed on the basis of theoretical and experimental studies with cellular organisms.\n\nResults\nInability of FMDV \n\t\t\t\t\t to Establish a Persistent Infection in BHK-21 Cells\n\t\t\t\t\nSeveral biological clones and populations were obtained by passaging FMDV biological clone C-S8c1 [19–22] in BHK-21 cells, either as large population passages or plaque-to-plaque transfers (Figure 1). The biological clones and populations differed up to 236-fold in relative fitness (Table 1). The fitness differences found are expected from previous results on fitness gain upon large population passages of RNA viruses [6,7] and fitness decrease upon plaque-to-plaque (bottleneck) transfers [9–13]. The initial experiment was aimed at testing whether \n\t\t\t\t\t, because of its low fitness (0.11 times that of its parental C-S8c1 [12,23,24] [Table 1]), had an advantage in establishing a persistent non-cytopathic infection in BHK-21 cells as compared with its parental clone, C-S8c1 (Figure 1). A persistent FMDV infection is established by growing the cells that survive a standard cytolytic infection with FMDV [25]. Confluent monolayers of BHK-21 cells were infected either with C-S8c1 or with \n\t\t\t\t\t at a multiplicity of infection (MOI) of 0.02–0.1 plaque-forming units (PFU)/cell (2 × 106 cells infected with 4 × 104 −2 × 105 PFU). Unexpectedly, at 24 h postinfection, the cells infected with \n\t\t\t\t\t showed extensive cytopathology, and at 48 h postinfection, no surviving cells were observed. The frequency of surviving cells in parallel infections with C-S8c1 was 5 × 10−3–9 × 10−3, which is consistent with previous determinations [25]. No persistently infected BHK-21 cell cultures could be established with \n\t\t\t\t\t, despite several attempts. Thus, C-S8c1, which displays a 9-fold higher relative fitness than \n\t\t\t\t\t in BHK-21 cells, showed a capacity to kill BHK-21 cells that was at least 103-fold lower than the killing capacity of \n\t\t\t\t\t in the infectivity assay intended to establish a persistent FMDV infection.\n\t\t\t\t\n\nFMDV Fitness and Capacity to Kill BHK-21 Cells May Not Be Positively Correlated\nThe capacity of \n\t\t\t\t\t to kill BHK-21 cells despite its low fitness in BHK-21 cells led us to quantitatively examine the relationship between fitness of FMDV and its capacity to kill BHK-21 cells. To this aim, FMDV clones or populations were compared in a cell killing assay, consisting in determining the time required to kill 104 BHK-21 cells as a function of the PFU added (described in Materials and Methods). The results (Figure 2A) indicate that over the time range of 12 h to 48 h postinfection, the number of PFUs needed to kill 104 BHK-21 cells varied logarithmically as a function of time. Similar quantifications of relative virulence were obtained by measuring the PFU needed to kill 104 cells in 24 h, and then by extrapolating the PFU values to 0 h postinfection (Tables 1 and S1). Virulence of \n\t\t\t\t\t was 29 to 35 times higher than virulence of C-S8c1, despite the latter displaying a 9-fold higher fitness (Tables 1 and S1). The high virulence of \n\t\t\t\t\t was not due to the plaque-to-plaque transfers, since a high virulence was also quantitated for its parental clone, \n\t\t\t\t\t, and for population C-S8p113 (Figure 2B; Tables 1, 2, and S1). \n\t\t\t\t\t deviated from a line that correlated relative fitness of FMDV and the logarithm of cell killing capacity, as reflected in the decrease of the regression coefficient (R2) (inset in Figure 2A). Probably, this deviation is due to the fact that \n\t\t\t\t\t lost fitness due to plaque-to-plaque transfers, and the other viruses were not subjected to plaque-to-plaque transfers. On the other hand, virulence determinants were acquired during the large population passages done between C-S8c1 and C-S8c1p113. The 29- to 35-fold higher virulence of \n\t\t\t\t\t with respect to C-S8c1 (Tables 1 and S1), despite its low fitness, indicates that viral fitness and virulence can be two unrelated traits.\n\t\t\t\t\n\nMapping Virulence Determinants in the \n\t\t\t\t\t Genome\n\t\t\t\t\nThe comparison of the consensus nucleotide sequence of the \n\t\t\t\t\t genome with that of C-S8c1 revealed a total of 47 mutations (Table S2), leading to 21 amino acid replacements affecting structural and non-structural proteins (Figure 3). To identify the genomic regions associated with the increased virulence of \n\t\t\t\t\t with respect to C-S8c1, we measured the BHK-21 cell killing capacity of nine chimeric viruses rescued from constructs obtained by introducing fragments of cDNA of the \n\t\t\t\t\t genome into plasmid pMT28, which encodes infectious C-S8c1 RNA [21] (Figure 4). The results (Figure 5; Tables 2 and S1) show that several genomic regions contribute to the virulence of \n\t\t\t\t\t for BHK-21 cells, and that the major contributors map within genomic positions 2046 to 3760 (residues encoding part of VP2, VP3, and part of VP1, Figure 5A) and 3760 to 5839 (residues encoding 2A, 2B, 2C, and 3A, Figure 5B). The results exclude the internal ribosome entry site and the 3C- and 3D-coding regions as significant virulence determinants of \n\t\t\t\t\t for BHK-21 cells (virulence of the relevant chimeric viruses ≤ 2.5, relative to C-S8c1; Tables 2 and S1). Infectious progeny production by each chimeric virus was intermediate between the production of the parental viruses pMT28 and \n\t\t\t\t\t, with no significant differences that could be correlated with virulence (Table 2).\n\t\t\t\t\n\nNon-Structural Protein 2C Is a Determinant of the Virulence of FMDV for BHK-21 Cells\nAmino acid substitutions in human rhinovirus protein 2C promoted cytopathology for mouse L cells [26]. Remarkably, \n\t\t\t\t\t shares with other FMDV clones and populations, notably, MARLS and C-S8p260p3d (the two viruses showing the highest virulence for BHK-21 cells; Figure 2A; Tables 1 and S1), three amino acid substitutions in 2C: S80N, T256A, and Q263H. In addition, MARLS and CS8p260p3d include replacement M283V in 2C, relative to C-S8c1 [27,28]. To test whether any (or a combination) of the three shared amino acid substitutions in 2C contributed to the increased virulence of FMDV, each of the mutations was introduced individually into plasmid pMT28 by site-directed mutagenesis, as described in Materials and Methods. Transcripts of the three mutants, termed pMT28 (SN), pMT28 (TA), and pMT28 (QH) (Figure 4), were used to transfect BHK-21 cells, and the viruses obtained were tested with the BHK-21 cell killing assay. Viruses having any of the substitutions in 2C have a virulence intermediate between that of C-S8c1 and \n\t\t\t\t\t (Figure 6A). To test whether the combination of the three substitutions in 2C could produce an additional increase of virulence, the three mutations were introduced in pMT28 to rescue the triple mutant pMT28 (SN, TA, QH) (Figure 4). The results (Figure 6B) show that the virulence of the triple 2C mutant is similar to the virulence of the individual 2C mutants. The 2C mutations did not significantly affect the infectious progeny production (Table 2). A testable prediction of this result is that the introduction of the wild-type 2C-3A-coding region in the genetic background of \n\t\t\t\t\t should produce a virus with lower virulence than \n\t\t\t\t\t. Indeed, the results with such a chimeric virus (Figure 5D) indicate that the presence of the 2C- and 3A-coding region as the only genetic region of the pMT28 in the genetic background of \n\t\t\t\t\t resulted in an FMDV with a 2.4- to 4.8-fold lower virulence than \n\t\t\t\t\t. We conclude that mutations in 2C contribute to virulence of FMDV for BHK-21 cells.\n\t\t\t\t\nThus, a virus that evolves towards low fitness levels due to the operation of Muller's ratchet may nevertheless maintain its capacity to kill the same cells in which it displays low fitness. In FMDV, the enhanced capacity to kill BHK-21 cells was multigenic, including participation of non-structural protein 2C with three amino acid substitutions acting in a redundant fashion. In conclusion, the results provide a molecular interpretation of why fitness and virulence of an animal virus can follow disparate evolutionary trajectories, culminating in two unrelated traits.\n\n\nDiscussion\nThe capacity of a virus to kill cells is probably influenced by several steps in the virus life cycle, including receptor affinity (which may trigger signalling pathways and alter cell functions) and intracellular viral replication that may lead to metabolic alterations such as transcriptional or translational shut-off [29]. A parallel increase of virulence and fitness as a virus improves its adaptation to a host cell type is expected, since a key parameter that should contribute to the fitness level of a cytophatic virus is the accumulation of infectious particles and its release from cells, which are events often associated with cell killing. This expectation was fulfilled in our experiments (increase of both fitness and cell killing following large population passages of C-S8c1 [Figure 2]), and also in other virus–host systems. In a comparison of two genetically divergent isolates of the whispovirus white spot syndrome virus, whose virulence for the shrimp host Penaeus monodon was measured by in vivo cumulative mortality rates, virulence correlated with competitive fitness in vivo [30]. The onset of type 1 diabetes by coxsackievirus B strains was linked to the viral replication rate and to the infectious dose [31]. In engineered alphavirus replicons, a direct correlation between the level of RNA replication and cytopathogenicity was observed [32]. At an epidemiological level, a greater replicative fitness of historical versus current human immunodeficiency virus type 1 (HIV-1) isolates was taken as evidence of HIV-1 attenuation over time, assuming a direct connection between fitness and virulence [33]. In vivo, viral fitness may vary among specific organs, and virulence may be affected only when fitness for some specific target tissues is affected [34].\nReplicative fitness is, however, but one of several factors which influence the progression of a viral infection in vivo. In a comparative analysis, R5-tropic and X4-tropic clones of HIV-1 showed similar replication capacity in mitogen-activated T cells. However, X4 clones were transferred more efficiently than R5 clones from dendritic cells to CD4(+) T cells, a fact that can contribute to the competitive advantage of X4 viruses in AIDS patients [35]. Simian immunodeficiency virus SIVmac239 infects both the sooty mangabey and the rhesus macaque, reaching high viral loads in both hosts, yet it is only virulent for the rhesus macaque [36]. Deviations of a positive correlation between viral fitness and virulence were observed also in the plant viruses cucumber mosaic virus [37] and barley stripe mosaic virus [38]. A study of the effect of lysis timing on bacteriophage fitness revealed that a strain with an intermediate lysis time had the highest fitness [39]. The time of transmission may also affect virulence. Nuclear polyhedrosis virus transmitted early to its host, the moth Lymantria dispar, was more virulent than virus transmitted late, although the latter was more productive because the virus could use more host tissue for replication [40]. In a study of the susceptibility of North American and non–North American breeds of Lymantria to several isolates of the fungus Entomaphaga maimaiga, mortality was scored in all cases. However, virulence of the fungus, quantitated by the time of death of Lymantria, was, in some cases, inversely proportional to fitness, quantitated by fungal reproduction in the moth [41]. In all these cases, the molecular basis of the lack of positive correlation between fitness and virulence is not understood.\nModel for a Lack of Correlation between Viral Fitness and Virulence\nThe results with FMDV clones H5 have documented that both fitness-enhancing and virulence-enhancing mutations can be incorporated in the viral genome in such a fashion that subsequent fitness-decreasing mutations associated with bottleneck (plaque-to-plaque) transfers produce only minimal effects on virulence (Figure 2). The dissection of accompanying molecular events, achieved through quantification of virulence of recombinant and mutant genomes (Tables 2 and S1), provides an interpretation of the lack of positive correlation between virulence and fitness. Multiple fitness-decreasing mutations occur in the course of plaque-to-plaque transfers, distributed throughout the FMDV genome [11]. In contrast, determinants of virulence for BHK-21 cells are multigenic, but concentrated mainly in some FMDV genomic regions. Similar multigenic but discrete virulence determinants have been described also in other virus–host systems [42,43]. To decrease virulence, mutations occurring randomly in the course of plaque-to-plaque transfers should affect specific genomic sites, and this will occur with a lower probability than fitness-decreasing mutations, which can hit any of the multifunctional picornaviral proteins and regulatory regions [11]. This model is reinforced by the observation that three amino acid substitutions in 2C (S80N, T256A, and Q263H) had a similar effect in enhancing FMDV virulence, and the three mutations in the same genome had an effect comparable to each mutation individually (Figure 6; Tables 2 and S1). It is not clear what the basis of the contribution of 2C to virulence for BHK-21 could be. 2C is involved in RNA synthesis and contains a nucleotide-binding domain, although none of the substitutions found in \n\t\t\t\t\t and \n\t\t\t\t\t lie within such a domain. An unlikely triple reversion would be required to eliminate the virulence-enhancing effect of the three mutations in 2C. We propose that a higher robustness of the FMDV genome with regard to virulence for BHK-21 cells, rather than to replicative fitness in the same cells, underlies the different trajectories followed by fitness and virulence upon subjecting the virus to repeated bottleneck transfers. Obviously, we cannot exclude that parameters of the virus life cycle, other than fitness as measured in our experiments, could correlate with virulence for BHK-21.\n\t\t\t\t\nThe comparative analysis of FMDV clones and populations shows that shifts in virulence can occur even through the evolution of a single viral clone (C-S8c1), with its restricted genetic diversity prompted by different replication regimes in the same host cells, which also have a clonal origin (see Materials and Methods). We conjecture that the demonstration that fitness and virulence can follow different evolutionary courses has been possible thanks to the consequences of the extreme passage regimes to which the viral populations were subjected: competitive evolution of an ample mutant spectra during repeated large population passages, and accumulation of deleterious (with regard to fitness, but not with regard to virulence) mutations upon plaque-to-plaque transfers (predominance of genetic drift and operation of Muller's ratchet) [12,15].\n\nImplications for Models of Virulence\nIt must be emphasized that fitness and virulence are relative values that pertain to a defined physical and biological environment. Virulence determinants of FMDV, identified here for BHK-21 cells, need not apply to virulence for the natural animal hosts of FMDV [44]. However, the observation of a lack of correlation between fitness and virulence in a FMDV clone is relevant to current models of attenuation and virulence, since it shows that more virulent forms of a virus need not have a reproductive advantage, and that viral virulence is not necessarily a byproduct of viral fitness. Even if virulence is regarded as an unavoidable consequence of parasite adaptation [45], virus de-adaptation (fitness loss) need not entail a decrease of virulence.\nMost current definitions of virulence include both the ability of the pathogen to multiply and to cause harm to its host; some authors, however, assume a direct relationship between fitness and capacity to produce disease [46–48]. In relating the results with FMDV to general models of virulence in host–parasite systems, it must be considered that in the FMDV system, evolution of the host BHK-21 cells could not influence FMDV evolution, because clonal cell populations with a controlled passage history were supplied in constant numbers at each infection event (see Materials and Methods). Therefore, changes in host density, or mobility, as well as pathogen survival in the external environment, all of which are relevant parameters in virulence models [48,49], cannot play a role in our system. A consistent finding in serial passage experiments is that virulence of a parasite increases with passage number in a new host [50]. The results with FMDV infecting BHK-21 cells cytolytically imply that the increase of virulence can be conditioned to the history of passage regimes undergone by a virus.\nThe invariance of BHK-21 cells in the course of serial cytolytic passages of FMDV is in contrast with the parallel system consisting of BHK-21 cells persistently infected with FMDV C-S8c1 [25], in which the cells are passaged and coevolve with the resident virus [51]. Host–virus coevolution has generally favored a decrease of viral virulence in the field, a classical example being myxoma virus and myxomatosis in rabbits [52].\nOur comparison of FMDV clones did not provide evidence of clones with high fitness and low virulence, which, with regard to natural hosts, is an aim of biomedicine to obtain vaccine strains. Yet, the existence of specific mutations that differentially affect fitness and virulence opens the way to engineer candidate vaccine strains unable to kill the host, while maintaining replicative competence. Virulence is, however, a feature of the host–parasite relationship [46], and the mutations needed to impair virulence are expected to be host-dependent [53,54].\n\n\nMaterials and Methods\nCells, viruses, and infections.\nThe BHK-21 cells used in the present study were cloned by end-point dilution, followed by preparation of a cell stock from a single cell; they were passaged a maximum of 30 times before being used for FMDV infection [25,51]. Procedures for cell growth, infection of BHK-21 cell monolayers with FMDV in liquid medium, and plaque assays in semi-solid agar medium were carried out as previously described [11,19,25,27]. Mock-infected cells were handled in parallel in all infectivity and plaque assays to monitor absence of viral contamination. The FMDVs used in the present study (Figure 1) are (i) the reference clone C-S8c1, which has been assigned a relative fitness of 1.0 [11]. (ii) MARLS, a monoclonal antibody escape mutant isolated from population C-S8c1p213 [55]; MARLS has a fitness of 25 relative to C-S8c1 [24]. (iii) C-S8p260p3d, a standard FMDV virus rescued by low MOI passage of C-S8p260. The latter is a virus that evolved by passage of C-S8c1 at a high MOI, which resulted in dominance of two defective FMDV genomes (both including internal deletions) that were infectious by complementation, in the absence of standard virus [22,24,28]; C-S8p260p3d has a relative fitness of 20 [24]. (iv) REDpt60, obtained after 60 successive plaque-to-plaque transfers of RED (a monoclonal antibody escape mutant isolated from population C-S8c1p100) [20]; REDpt60 has a fitness of 1.9 relative to C-S8c1. (v) C-S8c1p113, a viral population obtained after 113 serial cytolytic passages of C-S8c1 at a high MOI in BHK-21 cells (2 × 106 BHK-21 cells infected with the virus contained in 200 μl of the supernatant from the previous infection). (vi) Clone \n\t\t\t\t\t, a biological clone isolated from population C-S8c1p113 [11]; its relative fitness is 26 (unpublished data). (vii) Clone \n\t\t\t\t\t, obtained after 95 successive plaque-to-plaque transfers of \n\t\t\t\t\t [11]; its relative fitness is 0.11 [23].\n\t\t\t\t\n\nCell killing assay.\nThe capacity of FMDV to kill BHK-21 cells was measured as previously described [18,22]. The assay consists in determining the minimum number of PFU required to kill 104 BHK-21 cells after variable times of infection. The assay was performed in M96 multiwell plates with monolayers of 104 BHK-21 cells per well infected with serial dilutions of virus. At different times postinfection, cells were fixed with 2% formaldehyde and stained with 2% crystal violet in 2% formaldehyde. Results are expressed as the logarithm of the number of PFUs needed for complete cell killing (as judged by cell staining with crystal violet, with series of control wells with known numbers of cells) as a function of time postinfection [18,22].\n\nDetermination of relative fitness of \n\t\t\t\t\t.\n\t\t\t\t\nThe relative fitness of FMDV \n\t\t\t\t\t was determined by growth competition in BHK-21 cells as previously described [7,10,11,24,56]. FMDV \n\t\t\t\t\t was mixed with appropriate proportions of \n\t\t\t\t\t, which was used as reference virus. This virus has a fitness 8.5-fold higher than that of the reference clone of C-S8c1 in BHK-21 cells [7,10,11,24,56]. Four serial infections were carried out at MOI of 0.1 PFU/cell. The proportion of the two competing genomes at different passages was determined by real-time reverse transcription (RT)–PCR, employing primers 5531wtnew and \n\t\t\t\t\t, which are able to discriminate FMDV \n\t\t\t\t\t RNA from \n\t\t\t\t\t RNA. The nucleotide sequences of the primers will be provided upon request. The fitness vector obtained for \n\t\t\t\t\t corresponded to the equation y = 0,0206e1,1074x; R2 = 0.9507. The antilogarithm (base e) of the vector slope is the fitness of the assayed virus relative to the reference virus [56].\n\t\t\t\t\n\ncDNA synthesis, molecular cloning, and nucleotide sequencing.\nViral RNA was extracted by treatment with Trizol as previously described [57]. Reverse transcription of FMDV RNA was carried out with avian myeloblastosis virus reverse transcriptase (Promega, http://www.promega.com) or Transcriptor reverse transcriptase (Roche, http://www.roche.com), and PCR amplification was performed by using either Ampli-Taq polymerase (PerkinElmer, http://las.perkinelmer.com) or an Expand High Fidelity polymerase system (Roche), as instructed by the manufacturers. The FMDV genome-specific oligonucleotide primers used have been previously described [22,58]. In all RT-PCR amplifications, negative amplification controls, including all reaction components except template RNA, were run in parallel to monitor absence of contamination.\nChimeric viruses containing selected regions of \n\t\t\t\t\t in the genetic background of C-S8c1 (Figure 4) were obtained by replacing the corresponding DNA fragment of pMT28 by a cDNA copy of \n\t\t\t\t\t RNA, using specific restriction sites. To obtain pMT28/\n\t\t\t\t\t (436-2046), a chimera that included nucleotides 436 to 2046 of \n\t\t\t\t\t (the residue numbering of the FMDV genome is as in [11]), \n\t\t\t\t\t RNA was amplified by RT-PCR using primers NR2 and JH2, and the cDNA was digested with Hpa I (position 436) and Xba I (2046), and ligated into pMT28 DNA digested with the same enzymes. To obtain pMT28/\n\t\t\t\t\t (2046–3760), \n\t\t\t\t\t RNA was amplified by RT-PCR using primers 2R1New and pU, and then the cDNA was digested with Xba I (2046) and Avr II (3760). To obtain pMT28/\n\t\t\t\t\t (3760–5839), \n\t\t\t\t\t RNA was amplified by RT-PCR using primers 3R2New and 3CD1, and then the cDNA was digested with Avr II (3760) and Rsr II (5839). To obtain pMT28/\n\t\t\t\t\t (5839–7427), \n\t\t\t\t\t RNA was amplified by RT-PCR using primers 5531 wt new and C-Not-Pol, and then the cDNA was digested with Rsr II (5839) and Bam HI (7427). To obtain pMT28/\n\t\t\t\t\t (436-3760), \n\t\t\t\t\t RNA was amplified by RT-PCR using primers NR2 and JH2, and then the cDNA was digested with Hpa I (position 436) and Xba I (2046), and ligated into pMT28/\n\t\t\t\t\t (2046–3760) DNA digested with the same enzymes. To obtain pMT28/\n\t\t\t\t\t (3760–7427), \n\t\t\t\t\t RNA was amplified by RT-PCR using primers 3R2New and 3CD1, and then the cDNA was digested with Avr II (3760) and Rsr II (5839), and ligated into pMT28/\n\t\t\t\t\t (5839–7427) DNA digested with the same enzymes. To obtain pMT28/\n\t\t\t\t\t (2046–7427), \n\t\t\t\t\t RNA was amplified by RT-PCR using primers 2R1New and pU, and then the cDNA was digested with Xba I (2046) and Avr II (3760), and ligated into pMT28/\n\t\t\t\t\t (3760–7427) DNA digested with the same enzymes. To obtain pMT28/\n\t\t\t\t\t (436-7427), \n\t\t\t\t\t RNA was amplified by RT-PCR using primers NR2 and JH2; the cDNA was digested with Hpa I (position 436) and Xba I (2046), and ligated into pMT28/\n\t\t\t\t\t (2046–7427) DNA digested with the same enzymes. To obtain \n\t\t\t\t\t/2C-3A(pMT28), pMT28 was digested with Bgl II (4201) and Rsr II (5839), and a DNA fragment including wild-type 2C-3A-coding region was inserted into pMT28/\n\t\t\t\t\t (436-7427) DNA digested with the same enzymes. DNA ligation, transformation of Escherichia coli DH5α, isolation of DNA from bacterial colonies, and characterization of DNA by restriction enzyme digestion were performed by standard procedures [59]. The primers used for molecular cloning and site-directed mutagenesis are described in Table S3.\n\t\t\t\t\nTo obtain FMDV C-S8c1 containing the mutations found in gene 2C of \n\t\t\t\t\t, plasmid pMT28 was subjected to site-directed mutagenesis using an oligonucleotide including the required nucleotide replacement, and 3R2New or 3CD1 as external oligonucleotide primer (Table S3; Figure 4). A DNA fragment termed A was obtained by subjecting plasmid pMT28 to site-directed mutagenesis using primers (reverse) mutSNu, mutTAu, and mutQHu (to introduce mutations S80N, T256A, and Q263H, respectively) and an external oligonucleotide primer (3R2New, forward). A DNA fragment termed B was obtained amplifying pMT28 with primers (forward) mutSNd, mutTAd, and mutQHd (to introduce mutations S80N, T256A, and Q263H, respectively) and an external oligonucleotide primer (3CD1, reverse). DNA fragments A and B, including the desired mutations, were recombined by shuffling PCR using equimolar amounts of DNA fragments and two external primers (3R2New and 3CD1). The DNA with the desired mutation(s) in the 2C gene was digested with Avr II (genomic position 3760) and Rsr II (position 5839), and cloned into pMT28 to generate pMT28 (SN), pMT28 (TA), and pMT28 (QH). To obtain pMT28 (SN, TA, QH), plasmid pMT28 (SN) was subjected to site-directed mutagenesis to introduce mutation T256A in a similar way as described above, and then, plasmid pMT28 (SN, TA) was subjected to site-directed mutagenesis to introduce mutation Q263H. All chimeric viruses and mutants were analyzed by nucleotide sequencing using Big Dye Terminator Cycle Sequencing kit (Abi Prism; PerkinElmer) and sequencer ABI373 as previously described [58]. Sequences were analyzed using DNASTAR 4.0 (http://www.dnastar.com), GeneDoc, and GCC (University of Wisconsin). Each sequence was determined at least twice, with products obtained using different oligonucleotide primers.\n\t\t\t\t\nDNA from pMT28 or its recombinant and mutant derivatives was linearized with Nde I and transcribed with SP6 RNA polymerase as previously described [22,27]. Transcript RNA integrity and concentration were estimated by agarose gel electrophoresis, in parallel runs with known amounts of standard C-S8c1 RNA. BHK-21 cell monolayers (70% confluent, about 1 × 106 cells) were transfected with RNA transcripts (1 μg RNA) using lipofectin as previously described [59]. Virus was collected from the culture supernatant at 72 h post-transfection. The virus obtained by transfection was passaged twice before using it in biological studies. RNA was extracted and sequenced to ascertain that the virus maintained the genomic structure and mutations of the initial transcript.\nConsensus genomic nucleotide sequences of FMDV clones were obtained by RT-PCR amplification of virion RNA using specific primers [7,22,28].\n\n\nSupporting Information\nAccession Numbers\nThe GenBank accession numbers for the C-S8c1, \n\t\t\t\t\t, \n\t\t\t\t\t, CS8p260p3d, and MARLS genomic sequences are AJ133357, AM409190, AM409325, DQ409185, and AF274010, respectively. Nucleotide and amino acid sequences for picornaviruses can be found at http://www.iah.bbsrc.ac.uk/virus/picornaviridae/SequenceDatabase/3Ddatabase/3D.HTM.\n\t\t\t\t\n\n\n\n" ], "offsets": [ [ 0, 34882 ] ] } ]

[ { "id": "pmcA1851977__T0", "type": "species", "text": [ "foot-and-mouth disease virus" ], "offsets": [ [ 466, 494 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T1", "type": "species", "text": [ "foot-and-mouth disease virus" ], "offsets": [ [ 1706, 1734 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T2", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 1736, 1740 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T3", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 2144, 2148 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T4", "type": "species", "text": [ "foot-and-mouth disease virus" ], "offsets": [ [ 3875, 3903 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T5", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 3905, 3909 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T6", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 4180, 4184 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T7", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 4343, 4347 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T8", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 4570, 4574 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T9", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 4600, 4604 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T10", "type": "species", "text": [ "hamster" ], "offsets": [ [ 4660, 4667 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "10026" } ] }, { "id": "pmcA1851977__T11", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 4713, 4717 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T12", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 4806, 4810 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T13", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 4925, 4929 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T14", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 5171, 5175 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T15", "type": "species", "text": [ "FMDVs" ], "offsets": [ [ 5524, 5529 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T16", "type": "species", "text": [ "FMDVs" ], "offsets": [ [ 5579, 5584 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T17", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 5921, 5925 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T18", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 6960, 6964 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T19", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 7098, 7102 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T20", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 7836, 7840 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T21", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 7936, 7940 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T22", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 8837, 8841 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T23", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 8859, 8863 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T24", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 9092, 9096 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T25", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 9149, 9153 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T26", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 10158, 10162 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T27", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 12284, 12288 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "12110" } ] }, { "id": "pmcA1851977__T28", "type": "species", "text": [ "human rhinovirus" ], "offsets": [ [ 12334, 12350 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "169066" } ] }, { "id": "pmcA1851977__T29", "type": "species", "text": [ "mouse" ], "offsets": [ [ 12389, 12394 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "10090" } ] }, { "id": "pmcA1851977__T30", "type": "species", "text": [ "FMDV" ], "offsets": [ [ 12446, 12450 ] ], "normalized": [ { 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[ { "id": "pmcA2365769__text", "type": "Article", "text": [ "IL-5 drives eosinophils from bone marrow to blood and tissues in a guinea-pig model of visceral larva migrans syndrome\nAbstract\nThis study was undertaken to evaluate the role of IL-5 in eosinophil migration and in the maintenance of eosinophilia in a guinea-pig model of visceral larva migrans syndrome. The results show that the infection of animals with Toxocara canis induced an early increase in serum IL-5 levels that might be essential for eosinophil differentiation and proliferation and for the development of eosinophilia. When infected guinea-pigs were treated with mAb anti-IL-5 (TRFK-5) given at the same time or 1 or 3 days after infection, there was a high percentage of reduction of eosinophil counts 18 days after infection. However, when the mAb was administered during the peak of eosinophilia, there was high inhibition in blood, no inhibition in bronchoalveolar lavage fluid (BALF) or peritoneum and an increase in eosinophil numbers in bone marrow. Thus, a basic level of IL-5 may be essential to drive eosinophils from bone marrow to blood and tissues, and for the maintenance of eosinophilia in infected animals. We may also conclude that when eosinophils have already migrated to the lungs, TRFK-5 has no power to inhibit eosinophilia, which is also under control of local lung cells producing IL-5. In this way, only one later TRFK-5 treatment may not be sufficient to modify the lung parenchyma microenvironment, since T. canis antigens had already stimulated some cell populations to produce IL-5.\nResearch Paper Mediators of Inflammation 5, 24-31 (1996) \n\n Tins study was undertaken to evaluate the role of 11-5 in eosinophil migration and in the maintenance of eosinophilia in a guinea-pig model of visceral larva migrans syndrome. The results show that the infection of animals with Toxocara canis induced an early increase in serum 11-5 levels that might be essential for eosi\n\n IL-5 drives eosinophils from bone marrow to blood and tissues in a guinea-pig model of visceral larva migrans syndrome L. H. Faccioli, 1\"cA V. F. Mokwa, C. L. Silva, G. M. Rocha, J. I. Araujo, M. A. Nahori 2 and B. B. Vargaftig 2 \n\n nophil differentiation and proliferation and for the development of eosinophilia. When infected guinea-pigs were treated with mAb anti-11-5 (TRFK-5) given at the same time or 1 or 3 days after infection, there was a high percentage of reduction of eosinophil counts 18 days after infection. However, when the mAb was administered during the peak of eosinophilia, there was high inhibition in blood, no inhibition in bronchoalveolar lavage fluid (BALF) or peritoneum and an increase in eosinophil numbers in bone marrow. Thus, a basic level of 11-5 may be essential to drive eosinophils from bone marrow to blood and tissues, and for the maintenance of eosinophilia in infected animals. We may also conclude that when eosinophils have already migrated to the lungs, TRFK-5 has no power to inhibit eosinophilia, which is also under control of local lung cells producing 11-5. In this way, only one later TRFK-5 treatment may not be sufficient to modify the lung parenchyma microenvironment, since T. canis antigens had already stimulated some cell populations to produce 11-5. \n\n 1Department of Parasitology, Microbiology and Immunology, School of Medicine of Ribeir,5o Preto, 14049-900, Ribeiro Preto, SP, Brazil. Fax: (+55) 16 633 6631 2Unit de Pharmacologie Cellulaire, Unit Associe Institut Pasteur/INSERM n. 285, Paris, \n\n France. \n\n CACorresponding Author \n\n Key words: Eosinophil, Eosinophilia by Toxocara canis, IL-5 in eosinophilia, Toxocara canis \n\n Introduction \n\n treatment in mice infected with \n\n Eosinophilia has been associated with parasitic diseases, particularly when the parasites invade the tissues or injure the mucosal surfaces. Toxocara canis is an intestinal parasite of dogs, and is the most common aetiologic agent of visceral larva migrans syndrome (VLMS). In humans, VLMS results from the ingestion of embryonated eggs of T. canis, that eclode in the small intesfine. The infective larvae invade the mucosa, move into the liver via the portal circulation, and from there to the lungs. 2 Beaver et al., 3 who were the first to describe this syndrome, noted the intense eosinophilia which reaches more than 90% of total leucocyte counts. However, there are few studies regarding the mechanisms involved in the blood and tissue eosinophilia obseeeed in VLMS. Several investigators have suggested a direct correlation between eosinophilia and interleukin5 (IL-5) in human helminth infections4'5 and in experimental animal models. 67 Inhibition of eosinophilia has been demonstrated by anti-IL-5 24 Mediators of Inflammation Vol 5 \n\n brasiliensis, 8 $chistosoma mansoni, Toxocara canis7 and Heligmosomoidespolygyrus. 1 IL-5 has also been shown to support the terminal differentiation, proliferation of eosinophil precursors 11'12 and eosinophil activation. 13 Although IL-5 does not demonstrate eosinophil chemotactic activity in vivo14 there is some evidence suggesting that this cytokine may modulate a selective eosinophil accumulation at the site of inflammation. Moreover, Sehmi et a/.15 reported that IL-5 has a selective priming effect on eosinophil migratory response to nonselective chemoattractant mediators in vitro. Also, Moser et a/. 16 have demonstrated that in order to acquire the ability to transmigrate, eosinophils must be primed with IL-5, IL-3 and GM-CSF. Thus, the involvement of IL-5 in eosinophilia is not fully understood. In the present study we have used a guinea-pig model of VLMS to investigate the involvement of IL-5 in eosinophil migration and in the maintenance of eosinophilia in blood, bone marrow, lung and peritoneal cavity. (C) 1996 Rapid Science Publishers \n\n Nipostrongylus \n\n 1996 \n\n \fIL-5 drives eosinophils in guinea-pig \n\n Materials and Methods Animals: Outbred albino weighing 300-400g at the ments were obtained from the School of Medicine of versity of So Paulo, Brazil. \n\n female guinea-pigs start of the experi\n\n were routinely processed, embedded in paraffin, sectioned at 4-61.tm, and stained with Chromothope 2R and haematoxylin, for examination by light microscopy. Determination oflL-5 in serum: The IL-5 level in the serum of guinea-pigs was measured using an enzyme-linked immunosorbent assay (ELISA). Briefly, ELISA plates (96-well Immunoplate MaxiSorp, Nunc, Roskilde, Denmark) were coated with IL-5-specific monoclonal antibody (TRFK-5, 5 lag/ml in phosphate buffered saline, pH 7.4, PBS, 100 l.tl/well). After 2 h of incubation at 37C, the wells were washed four times with PBS containing 0.1% Tween 20 (PBS-T). Then, 100 btl of samples or recombinant murine IL-5 standards (0.15-200ng/ml) in PBS-T and 2% BSA (PBSTBSA) were added to each well. After incubation for l h at 37C, the wells were washed three times and 100 l.tl of biotinylated rat anti-murine IL-5 (biotinylated-TRFK-5, 1 lag/ml in PBS-TBSA) was added. After incubation for 1 h at 37C, the wells were washed again three times and 100 of peroxidase-labelled streptavidin (1/1000, Kirkegaard & Perry Laboratories Inc., Maryland, USA) were added to each well. Following incubation for l h at 37C and further washing, the enzyme was developed using the TMB substrate peroxidase for 5 min. The reaction was stopped by adding 501.tl of 2.0 N HCl, and the optical densities were read at 490nm using an automated plate reader. The sensitivity of the assay was 0.15 ng/ml and the upper limit 100ng/ml. \n\n the animal house of Ribeiro Preto, Uni\n\n Infection of animals: T. canis eggs were obtained by the methods of Olson and Schulz, 17 with minor modifications. Briefly, gravid female worms were recovered from dogs, and the eggs were rescued from the uterus, washed and allowed to develop to the infective stage in shallow dishes containing 0.5% formalin at 37C. Under light ether anaesthesia, the animals were infected with I ml saline containing 500 T. canis eggs, by gastric intubation using a metal cannula. Blood cell counts: Guinea-pigs were anaesthetized with sodium pentobarbitone (30mg/kg, i.v.) and blood samples were collected by cardiac puncture with 10% EDTA. Total cell counts were carried out using diluting fluid in a Neubauer chamber. Differential countings were obtained using Rosenfeld-stained cytocentrifuge preparations, \n\n Bronchoalveolar lavage fluid. The guinea-pigs were killed by an overdose of sodium pentobarbitone and 5ml of phosphate-buffered saline (PBS) containing 0.5% sodium citrate (PBS/SC), at room temperature, were instilled through a polyethylene cannula introduced into the trachea. The cells present in the bronchoalveolar lavage fluid (BALF) were recovered immediately. The procedure was repeated once. The leucocyte counts in the BAI_ were determined as described above, Peritoneal cells: The cells from the peritoneal cavities were harvested by injection of 10ml of PBS/SC into the peritoneum. Only 5-8 ml of the \n\n fluid was withdrawn for cell counts, as described above, \n\n Monoclonal antibodies: The rat monoclonal antibody TRFK-5 was a generous gift from Dr P. Minoprio, Institut Pasteur, Paris. The neutralizing antibody was purified by precipitation with ammonium sulfate (45%) from ascites prepared in CD1 nude mice (Charles River, St Aubin les Elbeuf, France) inoculated 1 week before the injection of hybridoma cells, with I ml of pristane (Sigma). After precipitation and dialysis of the ascite fluid overnight against PBS, the dialysate was further purified on a Protein G1 column (HiTrapTM, Pharmacia Upsala, Sweden). \n\n Bone marrow cells: Bone marrow cells were collected by flushing the contents of the guinea-pig femur with 10 ml of PBS/SC. Total cell numbers were determined as above. In the differential cell counts the cell populations were divided into mature neutrophils, mature eosinophils and others (mainly precursors and mononuclear \n\n cells), Histopathological studies: Tissues were removed from guinea-pigs at various times post-infection and immediately fixed in 10% formalin. Tissues \n\n Eosinophil and cytokine depletion: Guinea-pigs were injected i.p. with TRFK-5 or with the irrelevant antibody (rat IgG against total anti-human IgG) once, 2mg/animal, at the time of infection or at different intervals (1, 3, 12 or 17 days) thereafter. The animals in this group were sacririced 18 days after infection. liver: One lobule of was used to determine the larval each liver counts from infected guinea-pigs. Larval recovery \n\n Recovery \n\n of larvae from \n\n Mediators of Inflammation Vol 5 \n\n 1996 \n\n 25 \n\n \fL. H. Faccioli et al. \n\n was evaluated as described by Kayes and Oaks, 18 with minor modifications. Briefly, the tissue was chopped and digested with pepsin-HC1 (pH 1.5\n\n 1.8) for 2h at 37C. Larval counts for each sample were performed after centrifugation and examination of three 100-l.tl samples under the light microscope. Statistical analysis: Data are presented as the mean _+ S.E.M. and were analysed statistically using the Mann-Whitney test for unpaired data. A p < 0.05 value was considered to be statistically \n\n significant. \n\n Results \n\n of eosinophil counts in blood bone marrow, BALF and peritoneum: Guinea-pigs infected with T. canis eggs showed a timedependent blood, bone-marrow, BAUV and peritoneal eosinophilia (Fig. 1). The results represent the mean of nine animals obtained in three different experiments. The eosinophil number Kinetics 16\n\n increased significantly from 0.55 +_ 0.37 x 105 at the beginning of experiment to 6.0 +__ 1.03 x 105 at 6 days post-infection, peaked at day 18 (12.0 _+ 2.31 x 105), and decreased by day 24 (8.11 2.85 x 105) (Fig. 1A). A rise in the percentage of mature eosinophils in bone marrow\" was observed 12 days after infection (ranging from 6 2% to 14 2%) and peaked at 18 days (17 2%) (Fig. 1B). As in blood, the number of eosinophils in BALF increased significantly from 0.14 0.06 x 105 to 1.37 _+ 0.35 x 105 at 6 days after infection, reaching a peak at 18 days (10.23 2.62 x 105) with an increase in relative number of as much as 90% in eosinophil counts in relation to controls, and was still elevated at day 24 (9.07___ 3.47 x 10 s) (Fig. 1C). The remaining cells in the BALF were alveolar macrophages, lymphocytes, mast cells and ciliated cells. In contrast to blood and BALF, the number of eosinophils in the peritoneal cavity increased significantl only at day 12 post-infection (onset, 2.06 1.04 x 105; day 6, 3.68 _+ 0.82 x 105; day 12, 5.77 +_ 1.12 x 10>; and increased progres\n\n __ _ _ _. (B) \n\n (AI Blood \n\n 24 20\" \n\n Bone Marrow \n\n 1612 8 \n\n 4 O0\n\n 3 \n\n 6 \n\n 9 \n\n 12 \n\n 15 \n\n 111 \n\n 21 \n\n 24 \n\n 16\n\n (c) BALF \n\n 16\n\n ID) Peritoneum \n\n 12\n\n __o \n\n 12\n\n 8 \n\n 4 \n\n 0 3 \n\n O6 9 \n\n 12 \n\n 15 \n\n 16 \n\n 21 \n\n 24 \n\n Days postinfoction \n\n FIG. 1. Number of eosinophils in blood, BALF and peritoneal cavity, and percentage, of eosinophils in bone marrow of T. canisqnfected guinea-pigs. Values are the mean -t-S.E.M. (n=8 to 9). Asterisks indicate a significant difference between infected and noninfected animals (n 5 6). *p < 0.05 and **p < 0.01. 26 Mediators of Inflammation Vol 5 \n\n 1996 \n\n \fIL-5 drives eosinophils in guinea-pig \n\n sively until day 24, 12.44 4- 2.72 x 105) (Fig. 1D). The percentage of eosinophils in some animals reached 55% at the peak of infection. No increase in the number of mononuclear cells was seen in any compartment analysed. Larval counts: The percentage of inoculated T. canis larvae recovered by peptic digestion of the liver of experimental animals 4 h and 1, 2, 3, 4, 9, 12 and 18 days after inoculation of 500 eggs per animal is shown in Fig. 2. Most of the larvae were recovered 2 to 4 days after infection and 10% recovery was also observed on day 18 in the liver of the animals. \n\n 150 \n\n 120 \n\n IL-5 level in serum of infected animals: IL-5 was measured in the serum of infected and normal guinea-pigs. Each time point in Fig. 3 represents the mean of results from three to five infected animals, and from six controls. Two peaks of IL5 were present in the serum of infected guineapigs 1 day after infection (102 __+ 22 pg/ml), and 18 days later (59 7 pg/ml). The level of IL-5 in the controls was 31 4- 4 pg/ml. Eosinophil numbers in infected animals treated with TRFK-5: When guinea-pigs received an i.p. injection of TRFK-5, the monoclonal antibody against IL-5, at the time of egg administration or 1 day later, the number of eosinophils in blood, BALF, peritoneal cavity and bone marrow was \n\n _ \n\n 30\n\n 0 \n\n 0 \n\n 3 \n\n 6 \n\n 12 \n\n 18 \n\n Days postinfection \n\n FIG. 3. IL-5 concentration in serum of T. caniinfected guineapigs (n 3-5). Basal IL-5 concentrations were of 31 4 pg/ml (n= 12). \n\n _ \n\n 24 \n\n 60\n\n 50\n\n 40\n\n 0 \n\n \"T\" \n\n 1\" \n\n 0 \n\n 3 \n\n 6 \n\n 9 \n\n 12 \n\n 15 \n\n 18 \n\n 21 \n\n 24 \n\n Days postinfection FIG. 2. Percentage of T. canis larvae recovered from liver of guinea-pigs studied at various times post-infection. Data obtained from five animals. \n\n drastically reduced, even when determined 18 days after infection (Table 1). No inhibition of eosinophil counts was observed when the animals were inoculated with the irrelevant antibody at the time of infection (Table 1). Fig. 4 shows the comparative results of eosinophilia obtained when the antibody was given 3 days or 17 days after egg inoculation. The antibody given at 3 days after infection induced a high percentage of inhibition in eosinophil counts in all the compartments analysed 18 days after infection (Fig. 4A). However, when TRFK-5 was administered to the infected animals on day 17 post-infection (thus 1 day before sacrifice), a significant inhibition in number and percentage of eosinophils was observed only in the blood (p=0.030) (Fig. 4B). A small non-significant decrease was seen in BALF (p 0.790) and peritoneum (p= 0.222). Moreover, the number of mature eosinophils in bone marrow increased by 140% (p 0.038). As demonstrated in Fig. 4B, the behaviour of eosinotShilia in BALF was completely different from that observed in blood. Thus, to better understand the eosinophilia in the lungs of infected animals, we monitored eosinophil numbers in BALF after administration of TRFK-5 at the same time, or 1, 3, 12 or 17 days after infection. The animals were sacrificed 18 days after infection. In another group, TRFK-5 was administered 18 days post-infection and the animals were sacrificed 6 days later. When the mAb was administered at the same time or 1 or 3 days postinfection there was a significant inhibition in the number of eosinophils (Fig. 5). These data show Mediators of Inflammation Vol 5 \n\n 1996 \n\n 27 \n\n \fL. H. Faccioli et al. Table 1. Eosinophils in T. caniinfected guinea-pigs treated or untreated with TRFK-5 Compartment \n\n Time of sacrifice \n\n (days) \n\n Non18 24 18 24 18 24 18 24 \n\n treated \n\n Blood \n\n 12.18 5.28 10.08 -t- 2.85 16.50 4.42 6.57 +__ 1.68 \n\n BALF Peritoneal cavity \n\n 12.31 -I- 2.35 12.44 2.33 9 6 \n\n Bone marrow \n\n _ _ _ _ _ ___ _ __ __ _ __ Days of treatment with TRFK-5 after egg administration \n\n Irrelevant Ab at the time of infection \n\n 0 \n\n 3 \n\n 12 \n\n 17 \n\n 18 \n\n (n 6) \n\n (n=4) \n\n (n=4) \n\n (n 5) \n\n (n 5) \n\n (n=4) \n\n 0.14 -t- 0.14\" \n\n 0.26 0.79 \n\n 0.26* 0.24* \n\n 0.17 _-t- 0.17\" \n\n 0.15 \n\n 0.15\" \n\n 2.59 \n\n 0.80* 4.36 2.21 \n\n 13.32 \n\n 4.54 \n\n O. 15 \n\n 0.15* \n\n 0.36 \n\n 0.10\" \n\n 1.22 -t- 0.50* \n\n 7.07 -t- 2.39 \n\n 15.24 \n\n 29.31 _+ 15.90 16.09 \n\n 10.33 _+ 4.10 3.79 \n\n 1.47 -t- 0.76* 3 \n\n 0.48 \n\n _+ 0.35* \n\n 0.46 2.6 \n\n 0.27* 1.3\" \n\n 2.89 \n\n 0.94* \n\n 10.42 \n\n 4.00 \n\n 1.58\" \n\n -+ \n\n 1\" \n\n 1.25 -I- 0.25* \n\n + \n\n 5 \n\n 15 -t- 2 \n\n 9_+1 \n\n 2 \n\n 0.7* \n\n In blood BALF and peritoneal cavity the values represent mean -t-_ S.E.M. x 10 eosinophils. *p < 0.05. \n\n ml-1 \n\n and in bone marrow mean -t-_ S.E.M. of the percentage of mature \n\n that the inhibition of the first peak of IL-5 which appeared at 1 to 3 days after infection as shown in Fig. 3, is also very important for the establishment of eosinophilia in the lungs. However, 24- (A) \n\n when the mAb was administered 12, 17 or 18 days after infection there was no significant inhibition in the numbers of eosinophils in BALF (Fig. 5), showing that once established, the eosi18\n\n 15x \n\n 16\n\n 12\n\n ._ . 0 \n\n 12 \n\n o \n\n BIo o d \n\n BALF \n\n P e rit oneum \n\n Bone marrow \n\n 24- (Is) \n\n 1815\n\n E x \n\n 20\n\n 16\n\n o \n\n 12///. \n\n o = o \n\n 12\n\n 0 Blood \n\n O\" \n\n BALF \n\n Peritoneum \n\n Bone marrow FIG. 4. Number of eosinophils in blood, BALF and peritoneum and percentage of eosinophils in bone marrow of T. caniinfected guinea-pigs submitted or not to treatment with TRFK-5. (A) 2 mg/animal at 3 days post-infection; (B) 2 mg/animal 17 days post-infection. The treated and control animals were sacrificed 18 days after infection. Asterisks indicate a significant difference from infected controls (n=5-6) and from animals treated with TRFK-5 (n=4- 5). *p < 0.05 and **p < 0.01. 28 Mediators of Inflammation Vol 5 \n\n 1996 \n\n \fIL-5 drives eosinophils in guinea-pig 24\n\n E \n\n 20\n\n heart; data not shown) and muscle, as reported by other investigators, 8 were infiltrated. The factors responsible for in vivo eosinophil accumulation at inflammatory sites have been \n\n 0 \n\n TRFK-5 i.p. \n\n 0 \n\n 3 \n\n 12 17 \n\n A \n\n B \n\n FIG. 5. Number of eosinophils in BALF of T. caniinfected guinea-pigs submitted or not to treatment with TRFK-5. (A) animals were sacrificed 18 days post-infection and (B) 24 days after infection. Asterisks indicate a significant difference from infected control and from animals treated with TRFK-5 (p< 0.01). \n\n nophilia persists in lungs, probably by the secretion of IL-5 from cells localized in the lung microenvironment, \n\n Histopathological analysis: The treatment of T. canis-infected animals with irrelevant antibody showed a widespread eosinophilic infiltration as in untreated animals (Fig. 6A, B). However, the treatment of animals with TRFK-5 at the same time of infection, or 1 day or 3 days later ted to a complete inhibition of eosinophil infiltration in the lung parenchyma (Fig. 6C). By contrast, the mononuclear cell infiltration in the lungs was not modified. When the infected guinea-pigs received TRFK-5 1 day before sacrifice (or 17 days post-infection), eosinophil infiltration in the lung parenchyma was also inhibited (Fig. 6D) but not to the same extent as observed in the group receiving TRFK-5 given at the time of infection or 3 days later. Thus, the histological determination of eosinophil infiltration in these lungs corroborates a reduction but not a sizeable inhibition of eosinophil numbers as observed in the BALF of the same infected animals, \n\n poorly defined, although T lymphocytes and mast cells appear to be involved in eosinophilia. 9'2 IL-5, a T cell-derived factor that regulates B cell functions, is an eosinophil differentiation factor11 as well as a stimulating and survival-prolonging factor specific for eosinophils in vitro. 2 Also, several investigators have demonstrated that sTstemic eosinophilia in mice infected with parasites is mediated by IL-5 produced in response to the infection. 2'22 In the present study, the i.p. administration of the TRFK-5 antibody markedly inhibited the widespread eosinophilia observed in T. canis-infected guinea-pigs, indicating that IL5 participated in a guinea-pig model of VLMS eosinophJlia. Most of the T. canis larvae which penetrated the intestinal wall had migrated into the liver within 72h after inoculation as demonstrated here and elsewhere. 2 It is apparently during this interval that the worm provides the signals to cytokine-producing cells, which in turn trigger increased serum levels of specific cytokine as demonstrated here for IL-5, 24 to 72 h after infection. The signals may be provided directly by the invading parasite or by cells in response to the parasite. The cytokine pattern that develops at this early stage, probably induced by a T-cell independent pathway, may also influence the pattern of T cell differentiation into a Th2 type, which may be responsible for the second peak of IL-5 observed in our experimental model (Fig. 3), although a second cycle of larval invasion (Fig. 2) with a rapid peak of IL-5 liberation cannot be ruled out. \n\n Discussion The results of the present study show that in our experimental model widespread eosinophilia follows the infection of guinea-pigs with second stage eggs from T. canis, as also noted in humans and in other experimental animals. 7'7 T. canis is a potent stimulus for systemic eosinophilia, since blood, BALF, peritoneum and all tissues examined (kidney, eyes, spleen, thymus, \n\n Thus, our results suggest that the eosinophilia against helminth larvae may be initiated by the release of IL-5 when the parasites migrate from the intestine to the liver by stimulation of specific cell populations. Then, an early release of IL-5 quickly induces eosinophil recruitment, probably first from the stored mature eosinophil pool from vascular endothelium or by the mobilization of eosinophils from extravascular sites to the blood. This fact could explain why we found increased eosinophils first in blood and later in other compartments. The early IL-5 release may also serve as a signal for eosinophil differentiation and maturation in bone marrow. The time \n\n inteeeal observed between the first peak of IL-5 release and the increase of eosinophils in blood coincides with that reported to be necessary for eosinophil differentiation and maturation in vitro. 12Increased eosinophil production and liberation into blood and other tissues occurs Mediators of Inflammation Vol 5 \n\n 1996 \n\n 29 \n\n \fL. H. Faccioli et al. \n\n FIG. 6. (A) photomicrographs of lung parenchyma from guinea-pigs infected for 18 days with T. cani,, (B) infected animals which were treated with irrelevant antibody at the time of infection; (C) infected animals which were treated with the mAb TRFK-5 at 3 days after infection; (D) mAb administration 17 days after infection. The animals were sacrificed 18 days after infection. Note the intense eosinophil infiltration into the lung in A and B, the inhibition of eosinophils in C and the reduction of eosinophils in D. \n\n thereafter. Thus, early and later IL-5 release provides a necessary level of this cytokine, which is involved in the maintenance of eosinophilia. We may assume that the inhibition of the first peak of IL-5 release by TRFK-5 does not permit the subsequent T cell stimulation and differentiation. This may explain the long-lasting effect of TRFK5 treatment observed here and also reported by others. 8 In agreement with our results, there is an important observation of Svetic et al. 24 showing that a specific and highly reproducible IL-5 gene expression pattern is detectable in Peyer's patches by 6 to 12h after Heligmosomoides polygyrus infection. The early increase in IL-5 gene expression after infection was probably T cell-independent, inasmuch as it was obseeeed in Peyer's patches of congenitally athymic mice and of conventional mice treated with anti-CD4 30 Mediators of Inflammation Vol 5 \n\n and anti-CD8 mAb. Moreover, Kusama eta/. 25 have observed two peaks of eosinophilia in normal and athymic mice, and suggested that IL-5 observed in the first peak was produced by cells other than CD4 T cells, since anti-CD4 and anti-CD3 mAb reduced only the second peak of eosinophilia in normal mice and slightly reduced the first peak of eosinophilia in both normal and nu/nu mice. The local lung cells producing IL-5 may also help us to explain the reason why 12, 17 or 18 days post-infection TRFK-5 treatment only partially inhibits, or does not inhibit eosinophil infiltration into the lungs, as demonstrated in Figs 5 and 6. We may suggest that when eosinophils have already migrated to the lungs, TRFK-5 has no power to inhibit eosinophilia, which is also under control of local lung cells producing IL-5. In this way, only one later TRFK\n\n 1996 \n\n \fIL-5 drives eosinophils in guinea-pig \n\n 5 treatment may not be sufficient to modify the lung parenchyma microenvironment, since T. canis antigens have already stimulated some cell populations to produce IL-5, as demonstrated by Kusama et aL25These results suggest that eosinophilia in lungs is under the control of different factors when compared to that observed in blood and the peritoneal cavity. One of the most important results obtained here was the inhibition of circulating eosinophil numbers by the different mAb treatments, even when the antibody was given at the peak of blood eosinophilia, which was accompanied by an increase of mature eosinophils in bone marrow. This suggests that IL-5, apart from being required for the terminal differentiation of eosinophils in bone marrow, 26 is also likely to drive eosinophils from the bone marrow to the blood and then to the tissues, probably by upregulating VLA-4 expression in eosinophils. Moser et aL have demonstrated that in order to acquire the ability to transmigrate, eosinophils must be primed with cytokines such as IL-5, IL-3 or GM-CSF for expression of adhesion molecules such as VI-4. Recently, Pretolani et al. 27 have indeed shown that an anti-VLA-4 antibody suppresses eosinophil recruitment to lung in the guinea-pig and, as a consequence, inhibits the accompanying bronchopulmonary hyperresponsiveness. \n\n tant in protective immunity to a gastrointestinal nematode infection in mice. Proc Natl Acad Sci USA 1991; 88= 5513-5517. 11. Yamaguchi A, Suda T, Suda J, et al. Purified interleukin (IL-5) supports the terminal differentiation and proliferation of murine eosinophilic \n\n precursors. J Exp Med 1988; 16'7: 43-56. 12. Yamaguchi Y, Hayashi Y, Sugama Y, et al. Highly purified murine interleukin (IL-5) stimulates eosinophil function and prolongs in vitro survival. IL-5 as an eosinophil chemotactic factor. J Exp Med 1988; 16'7: 17371752. 13. Rothenberg ME, Petersen J, Stevens RL, Silberstein DS, McKenzie DT, Austen KF, Owen WF. IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity, and sustained antibodydependent cytotoxicity. J Immuno11989; 143; 2311-2316. 14. Collins PD, Weg VB, Faccioli LH, Watson ML, Moqbel R, Williams TJ. Eosinophil accumulation induced by human interleukin-8 in the guinea pig in vivo. Immunology 1993; '7}; 312-318. 15. Sehmi R, Wardlavo AJ, Cromwell O, Kurihara K, Waltmann P, Kay AB. Interleukin-5 selectively enhances the chemotactic response of eosinophils obtained from normal but not eosinophilic subjects. Blood 1992; '79; 2952-2959. 16. Moser R, Fehr J, Bruijnzeel PLB. IL-4 controls the selective endotheliumdriven transmigration of eosinophils from allergic individuals. J Immunol 1992; 149: 1432-1438. 17. Olson LJ, Schulz CW. Nematode induced hypersensitivity reactions in guinea pigs: onset of eosinophilia and positive Schultz-Dale reactions following graded infection with Toxocara canis. Ann N Y Acad Sci 1963; 113; 440-455. 18. Kayes SG, Oaks \n\n JA. Development \n\n of the granulomatous response in \n\n murine toxocariasis. I. Initial events. Am J Patho11978; ,}3; \n\n 277-294. \n\n 19. Basten A, Beeson PB. Mechanisms of eosinophilia. II. Role of the lymphocyte. J Exp Med 1970; 131; 1288-1305. 20. Plaut M, Pierce JH, Watson CJ, Hanley-Hyde J, Nordan RP, Paul WE. Mast cell lines produce lymphokines in response to cross-linkage of Fc epsilon RI or to calcium ionophoras. Nature 1989; 339: 64-67. is 21. Sher A, Coffman RL, Hieny S, Scott P, Cheever AW. Interleukin required for the blood and tissue eosinophilia but not granuloma formation induced by infection with Schistosoma mansoni. Proc Natl Acad Sci USA 1990; 8'7; 61-65. 22. Herndon FJ, Kayes SG. Depletion of eosinophils by anti-IL-5 monoclonal antibody treatment of mice infected with Trichinella spiralis does not alter parasite burden or immunologic resistance to reinfection. J Immuno11992; 149: 3642-3647. 23. Oshima T. Standardization of techniques for infecting mice with Tox24. ocara canis and observations on the normal migration routes of the larvae. J Parasito11961; 4'7= 652. Svetic A, Madden KB, Zhou XD, et al. A primary intestinal helminthic infection rapidly induces a gut-associated elevation of Th2-associated cytokines and IL-3. J Immuno11993; 150: 3434-3441. Kusama Y, Takamoto M, Kasahara T, Takatsu K, Nariuchi H, Sugane K. Mechanisms of eosinophilia in BALB/c-nu/+ and congenitally athymic BALB/c-nu/nu mice infected with Toxocara canis. Immunology 1995; 84; 461-468. Rennick DM, Thompson-Snipes L, Coffman RL, Seymour BWP, Jackson JD, Hudak S. In vivo administration of antibody to interleukin-5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice. Blood 1990; '76: 312-316. Petrolani M, Ruffle C, Lapa e Silva JR, Joseph D, Lobb RR, Boris Vargaftig B. Antibody to very late activation antigen 4 prevents antigen-induced bronchial hyperreactivity and cellular infiltration in guinea pig airways. J Exp Med 1994; 180= 795-805. \n\n References 25. 1. Nutman \n\n 2. \n\n 3. 4. 5. \n\n 6. 7. \n\n 8. \n\n 9. \n\n TB, Ottesen EA, Cohen SG. The eosinophil, eosinophilia, and eosinophil-related disorders. Allergy Proc 1989; 10; 47-62. Glickman LT, Schantz PM. Epidemiology and pathogenesis of zoonotic toxocariasis. Epidem Rev 1981; 3= 230-250. Beaver P, Snyder H, Carrera G, Dent J, Lafferty J. Chronic eosinophilia due to visceral larva migrans. Pediatrics 1952; 9: 7-19. Limaye AP, Abrams JS, Silver JE, Ottesen EA, Nutman TB. Regulation of parasite-induced eosinophilia: selectively increased interleukin production in helminth-infected patients. J Exp Med 1990; l'7:a; 399-402. Steel C, Nutman TB. Regulation of IL-5 in onchocerciasis: a critical role for IL-2. J Immuno11993; 15{}; 5511-5518. Yamaguchi Y, Matsui T, Kasahara T, et al. In vivo changes of hemapoietic progenitors and the expression of the interleukin gene in eosinophilic mice infected with Toxocara canis. Exp Hemato11990; 18; 1152-1157. Parsons JC, Coffman RL, Grieve RB. Antibody to interleukin prevents blood and tissue eosinophilia but not liver trapping in murine larval toxocariasis. Parasite Immuno11993; 15: 501-508. Coffman RL, Seymour BWP, Hudak S, Jackson J, Rennick D. Antibody to interleukin-5 inhibits helminth-induced eosinophilia in mice. Science 1989; 245; 308-310. Sher A, Coffman RL, Hieny S, Cheever AW. Ablation of eosinophil and IgE responses with anti-IL-5 or anti-IL-4 antibodies fails to affect immunity against Schistosoma mansoni in the mouse. J Immuno11990; 145: 3911\n\n 26. \n\n 27. \n\n ACKNOWLEDGEMENTS. This work was funded by grant 300652/85-2 from Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) and Grant 92/5105-7 from Fundago de Amparo ft Pesquisa do Estado de So Paulo (FAPESP). We wish to thank Mrs M.A. Fernandes for technical assistance, Mrs M.M.O. Rossi for the histological sections, M. Costa Gongalves for the photography artwork, and Dr P. Minoprio, Institut Pasteur, for kindly providing TRFK-5. \n\n 3916. 10. Urban \n\n Jr JF, \n\n Katona IM, Paul WE, Finkelman FD. Interleukin 4 is impor\n\n Received 12 October 1995; accepted 17 November 1995 \n\n Mediators of Inflammation Vol 5 \n\n 1996 \n\n 31 \n\n \f " ], "offsets": [ [ 0, 33074 ] ] } ]

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[ { "id": "pmcA1885853__text", "type": "Article", "text": [ "A global gene evolution analysis on Vibrionaceae family using phylogenetic profile\nAbstract\nBackground\nVibrionaceae represent a significant portion of the cultivable heterotrophic sea bacteria; they strongly affect nutrient cycling and some species are devastating pathogens.\nIn this work we propose an improved phylogenetic profile analysis on 14 Vibrionaceae genomes, to study the evolution of this family on the basis of gene content.\nThe phylogenetic profile is based on the observation that genes involved in the same process (e.g. metabolic pathway or structural complex) tend to be concurrently present or absent within different genomes. This allows the prediction of hypothetical functions on the basis of a shared phylogenetic profiles. Moreover this approach is useful to identify putative laterally transferred elements on the basis of their presence on distantly phylogenetically related bacteria.\n\nResults\nVibrionaceae ORFs were aligned against all the available bacterial proteomes. Phylogenetic profile is defined as an array of distances, based on aminoacid substitution matrixes, from single genes to all their orthologues. Final phylogenetic profiles, derived from non-redundant list of all ORFs, was defined as the median of all the profiles belonging to the cluster. The resulting phylogenetic profiles matrix contains gene clusters on the rows and organisms on the columns.\nCluster analysis identified groups of \"core genes\" with a widespread high similarity across all the organisms and several clusters that contain genes homologous only to a limited set of organisms. On each of these clusters, COG class enrichment has been calculated. The analysis reveals that clusters of core genes have the highest number of enriched classes, while the others are enriched just for few of them like DNA replication, recombination and repair.\n\nConclusion\nWe found that mobile elements have heterogeneous profiles not only across the entire set of organisms, but also within Vibrionaceae; this confirms their great influence on bacteria evolution even inside the same family. Furthermore, several hypothetical proteins highly correlate with mobile elements profiles suggesting a possible horizontal transfer mechanism for the evolution of these genes. Finally, we suggested the putative role of some ORFs having an unknown function on the basis of their phylogenetic profile similarity to well characterized genes.\n\n\n\nBackground\nOver the past ten years, a great number of microbial genomes have been sequenced covering a wide representation of prokaryots as well as multiple strains of some species. The study of these genomes both by computational and experimental approaches has highly improved our understanding on physiology, phylogenetic relationship and pathogenicity of many organisms. Furthermore, it has provided new knowledge on microbial genome evolution, revealing a gene core shared by the great majority of bacteria, genes characteristic of particular groups and \"novel\" genes that possibly originated by lateral gene transfer from some unknown source.\nAnalysis performed on closely related genomes revealed that a substantial fraction of genes in any genome seem to be strain specific. These genes might sometime arise by gene duplication followed by a rapid divergence, or by lineage-specific loss of genes in one strain, resulting in a unique gene in other strains. However, there are several lines of evidence indicating that lateral gene transfer may be the main mechanism to acquire novel genes. Indeed, this could be one of the main forces driving bacterial adaptation and evolution. Phage DNA is thought to be one of the main vectors for lateral gene transfer among bacteria [1] and many virulence factors from bacterial pathogen are phage encoded [2]. For example, the genes for CT, the most important virulence factor of V. cholerae, are encoded in the genome of phage CTXφ, integrated in the bacterial chromosome 1.\nSince lateral gene transfer plays a relevant role in bacterial evolution, the reconstruction of phylogeny is very complex and phylogenetic trees built by standard sequence analysis may not lead to a reliable picture of the evolutionary history. In fact, alternative trees can be obtained when different proteins are considered.\nFor many aspects the classification of bacteria on the basis of their global gene content may give a better description of their evolutionary history. This may be particularly important when bacteria of the same group are compared, since newly acquired genes could be relevant to confer peculiar features that allows the exploitation of different ecological niches.\nIn this study we propose a bioinformatic procedure to investigate bacterial genome evolution, taking into account the global gene content, as well as sequence similarity. We based our analysis on modified phylogenetic profiles [3]; however, we do not consider only the presence/absence of orthologue genes, but also their distance, based on a substitution matrix.\nA phylogenetic profile is a non-sequence-homology-based method developed to infer a possible functional relationship between genes. It is based on the idea that proteins that are involved in the same metabolic pathway or structural complex are likely to evolve in a correlate fashion and during evolution appear phylogenetically linked, showing a tendency to be either preserved or eliminated as a whole. Therefore, genes showing similar phylogenetic profiles are likely to be functionally related. We extended the use of phylogenetic profiles to produce an evolutionary tree based on a hierarchical clusterization of organisms with similar phylogenetic profiles.\nFor this study we took the whole gene dataset of 320 prokaryotic genomes, however, we limited the analysis to the orthologous groups that are present in at least one of the 14 considered species of the Vibrionaceae family. These bacteria belong to the Gammaproteobacteria group and are highly abundant in aquatic environment, they strongly influence nutrient cycling and various species are also devastating pathogens. Since we focused our analysis on this particular group, the aim of this study is not the construction of a global evolutionary tree, but rather a Vibrionaceae perspective of bacterial diversity, based on phylogenetic profiles.\n\nResults and discussion\nPhylogenetic matrix\nThe analysis was performed on 14 bacteria belonging to the Vibrionaceae family (Table 1). The redundant list of Vibrionaceae ORFs was clustered to reduce the number of proteins to analyze and the phylogenetic profile for each cluster was calculated as described in the Method section.\nMany authors proposed and successfully applied different measure methods to calculate the phylogenetic profile values.\nPellegrini et al. [3] firstly proposed a phylogenetic profile described as a string of bits, each bit representing the absence or presence of an homologous gene in a given genome. This method lacks a weighting procedure, giving the same weight (value 1) to all the sequences that are considered homologous given a similarity threshold. Enault and colleagues proposed an improved phylogenetic profile based on a normalized Blastp bit score [4]. This method, compared to the approach implemented by Pellegrini, allows weighting each point of the profile proportionally to the length and the quality of the alignment. Jingchun and colleagues optimized the phylogenetic profiles method by integrating phylogenetic relationships among reference organisms and sequence homology information, based on E-value score, to improve prediction accuracy [5].\nThe measure index I proposed in this work is similar to the others described above, taking into account both the quality and the length of the alignment using a substitution matrix. Moreover our approach considers also the total length of the sequences, penalizing good alignments occurring between ORFs having different lengths and taking into consideration that ORFs could differentiate mainly for the presence of functional domains.\nThe final phylogenetic profile for each cluster was defined as the median of all the profiles belonging to the cluster, named \"meta-profile\", which describes the profile of conserved ORFs belonging to an entire family.\n\nHierarchical cluster analysis\nA hierarchical cluster analysis was performed on the entire phylogenetic profile matrix and it was calculated a statistical support based on bootstrap method for the nodes of the columns tree (Fig 1). The branch tree colors represent the bootstrap percentage support. This constitutes a phylogenetic tree based on gene content using Vibrionaceae ORFs as a reference. Genomes belonging to the same taxonomic group tend to cluster together and the Vibrionaceae species are closely related. As expected, according to the Vibrionaceae branch lengths it is evident that variability within this group is higher compared to the other groups. The dataset used for phylogenetic matrix calculation is indeed composed by Vibrionaceae ORFs. This implies that the similarity measures between these ORFs and the corresponding orthologues will be nearly zero in most of the other species and significantly higher in the Vibrionaceae family, increasing the variability into this group. Moreover the average percentage of clusters shared by the Vibrionaceae members is only 47.5% (average number of shared clusters divided by the total number of clusters) that again indicates a high variability inside this family. It is also interesting to note that organisms belonging to the same or closely related taxa split into different subgroups. This highlights the existence of a high variability among lineages, due to genetic and evolutionary processes such as lateral gene transfer, concerted evolution and gene duplication [6]. In terms of gene content, the organisms more related to the Vibrionaceae belong to the gamma and beta proteobacteria. In particular Altermonadales, Enterobacteriales and Burkholderiales are closely related to Vibrionaceae, and share the higher number of cluster of genes (average percentage of 20%). As expected, Archea are the most distant group sharing just 3.8% of clusters.\nClusters and genes distribution, as shown in Fig 2, reveals that the number of clusters and genes shared by the organisms decreases as the number of organisms considered increases. The analysis was performed considering for each cluster profile the number of organisms sharing the same numbers of clusters (and genes). The majority of gene cluster groups no more than 21 species on a total of 320. The highest blue spike corresponds to the higher number of genes shared by 105 groups of 14 organisms. Among these groups, as expected, Vibrionaceae are highly represented. Other species represented are Colwellia psychrerythraea 34H and Shewanella oneidensis, that belong to the Alteromonadales family.\nThe cluster analysis performed on genes is shown in Fig. 3. From now on, to avoid confusing interpretation between clusters derived from the cluster analysis and cluster derived from the ORFs clustering we will use the term \"gene\" in place of cluster of ORFs.\nThe different gradient of color, from bright to dark red, represents decreasing similarity values. The cluster analysis allows the detection of three main groups of genes. The first one (Fig 3, panel B) contains the most conserved and established genes shared almost by all the organisms. These core genes can be defined as the set of all genes shared as orthologous by all members of an evolutionary coherent group. In our analysis we identify four clusters, for a total of 145 genes, shared by all the 320 organisms.\nThe ORFs belonging to these clusters are predicted to codify for the ATP binding subunit of ABC transporters (annotated as ABC-type polar amino acid transport system, ABC-type antimicrobial peptide transport system, ABC-type histidine transport system and ABC-type transport system involved in lysophospholipase L1 biosynthesis). This finding is surprising since this is the first report where these ORFs are assigned to the core genes. Anyway two different explanations can be traced. First, it is known that the ABC transporters represent an essential transport system in the prokaryotes and that their ATP binding subunits are apparently overrepresented compared to the other two subunits (ligand binding and permease subunit) in all genomes sequenced thus far [7]. Second, one organism, Buchnera aphidicola, presents these genes with a similarity just below the cut-off used for the analysis, but they have been considered since it is well known that in this mutualistic endosymbiont the accelerated evolution and AT bias affect all its genes, including the 16S rRNA [8,9].\nThe dataset used for the analysis includes genomes in draft quality (Vibrio cholerae 0395, Vibrio cholerae MO10, Vibrio cholerae RC385, Vibrio cholerae V51, Vibrio cholerae V52, Photobacterium profundum 3TCK, Vibrio MED222, Vibrio splendidus12B01). Wrong ORFs prediction or missing genes due to incomplete genome sequences can explain the low number of core genes identified. To avoid such problems we repeated the analysis excluding the draft genomes and thus considering 312 genomes. The results, reported in Table 2, show an increased number of the core genes and in particular ribosomal proteins and tRNA synthetase, as reported by Charlesbois and Doolittle [10]. This could be considered as a sort of \"minimal genome\" containing the group of genes that are necessary to maintain a free-living organism.\nThe low number of genes shared by all the organisms can be due to many factors. First of all we used the Vibrionaceae ORFs as a reference, limiting the number of genes we were able to identify. It was further demonstrated that the core gene size decreases as more genome sequences are analyzed [10].\nGenes that are considered to belong to the core set when close organisms are compared, are classified as flexible genes when distantly related genomes are analyzed [6]. Finally, genes within core genomes might be transferred or replaced, introducing new versions of existing genes into genomes. Such transfers can replace even highly conserved genes by non-homologous counterparts but the advantages provided are difficult to explain. It is also to take into consideration that many symbiotic and parasitic bacteria undergo a reduction of their genomes, loosing many genes required by free-living cell.\nThe second group (Fig 3, panel C) represents genes shared mainly among Vibrionaceae and other gamma proteobacteria (as Altermonadales, Burkholderiales and Enterobactidiales).\nFinally, the third group (Fig. 3, panel D) is composed by genes that are mainly specific to the Vibrionaceae.\n\nk-mean cluster analysis and cluster enrichment\nWe performed a k-means cluster analysis, setting the k value to 14. As shown in Fig. 4, the clusters 3, 4, 11, 13 and 14 contain the higher percentage of genes, accounting for more that 50% of the total genes, while clusters 9 and 10 contain the lower number of ORFs (3% of genes). The variance in each k-means cluster is very low (Fig. 4), meaning that the clusters contain genes with compact and similar profiles. As described in Fig. 4, the majority of the clusters (1, 2, 3, 4, 5, 6, 7, 9, 12, 13) contains genes with a similar profile, with the average values (red line) near zero, except for the presence of some spikes correspondent to an increasing similarity with some isolated organisms. As shown in Table 3, clusters 1, 3, 4, 5, and 9 contains genes that have a high similarity in a small subset of organisms. The majority of these ORFs are annotated as hypothetical proteins or phage related proteins. Clusters 8, 10 and 14 present genes shared among almost all the organisms. In particular cluster 10 is composed by the core genes described before having an high value of similarity widespread among all the organisms; cluster 8 contains genes shared mainly by gamma proteobacteria and cluster 14 is composed of genes in common between Vibrionaceae and Enterobacteriaceae.\nA functional annotation has been performed on each gene cluster using COG (Cluster of Orthologous Genes), KEGG pathway map and GO databases. For each k-mean cluster the enrichment probability with respect to the total number of clusters has been obtained with the hypergeometric distribution.\nFig. 5 shows COG enrichment results for each cluster. As expected clusters represented by conserved genes (cluster 8, 10 and 14) have the higher number of enriched COG codes, while cluster specific of few organisms are characterized by a small number of enriched COGs. The majority of clusters presents COG codes enrichment for S (function unknown), R (poorly characterized) and – (absence of COG code) categories. This is due to the large abundance of unknown and hypothetical proteins presents in the Vibrionaceae proteomes.\nIt is worth noting that cluster 3, mainly represented by Photobacterium profundum SS9 ORFs, is enriched only by C (Energy production and conversion), L (DNA replication, recombination and repair) and M (Cell envelope biogenesis, outer membrane). Probably the L class overrepresentation is determined by the high number of transposons that are present in the SS9 genome [11]. The role played by these transposable elements in the survival of this deep-sea bacterium it is still a matter of debate [12].\nIn addition V. vulnificus YJ016 and V. vulnificus CMCP6 (cluster 13) seem to share genes belonging to the enriched COG classes K (Transcription), L and T (Signal transduction mechanisms). It was previously reported an enrichment in genes belonging to the transcription class in the genome of V. vulnificus respect to the V. cholerae genome [13]. This class is clearly related to the T class and seems to indicate that this organism is able to receive and translate in a transcriptional response specific environmental signals. Despite this, the large majority of the genes in clusters 3 and 13 lacks COG annotation.\nCluster 7, as shown in Table 3, accounts organisms with large genome size (see Table 1). This can explain the fact the this cluster contains almost all the COG class enriched and suggests a more complex and flexible life-style of these organisms compared to the other Vibrionaceae members.\nKEGG annotation is limited to metabolic or structural complex network and so a reduced number of genes have a KEGG entry. This causes the presence of clusters without enriched map (cluster 2–5, 7, 13, see Fig 5). Also in this case, the clusters presenting the higher number of significant KEGG map are those containing the conserved genes. The most enriched KEGG clusters are cluster 14, 10 and 8 accounting for the majority of the metabolic pathways. Cluster 1 is enriched for map3080 (type IV secretion system). In fact V. fischeri genome contains 10 separate pilus gene clusters, including eight type-IV pilus loci. The presence of multiple pilus gene clusters suggests that different pili may be expressed in different environments or during multiple stages of its development as a symbiont [14].\nCluster 11 is enriched for map3090 (type II secretion system). The type II pathway is conserved among gram-negative bacteria, including many pathogens, and secretes a variety of virulence factors and degradative enzymes [15].\nCluster 9 is enriched for map 00860 (Porphyrin and chlorophyll metabolism). These genes are involved in the cobalamin (coenzyme B12) biosynthetic pathway [16]. Some organisms, such as Salmonella typhimurium and Klebsiella pneumoniae, can synthesize cobalamin de novo [17], while E. coli and large part of the Vibrionaceae perform cobalamin biosynthesis only when provided with cobinamide. It is interesting to observe that the genes belonging to the de novo pathway are only shared by Archea, some other organisms like Salmonella, Pseudomonas and Vibrio MED222.\nFinally cluster 6 is enriched by map2010 (ABC transporter), map2020 (two-component system), map2030 and map2031 (bacterial chemotaxis), map2040 (Flagellar assembly) and map3090 (type II secretion system). This cluster contains genes shared with a high similarity by all Vibrio and with a lower similarity with Photobacterium profundum species. Among the Vibrio species the organisms showing the highest similarity (Tab. 3) are V. cholerae strains.\n\nVibrionaceae specific genes\nWe identify 1940 clusters specific to the Vibrionaceae. All the Vibrionaceae considered in the analysis share 108 clusters. Among these genes we identify ToxR and ToxS genes. ToxR gene encodes a transmembrane regulatory protein firstly identified in V. cholerae, in which it co-ordinates many virulence factors in response to several environmental parameters [18]. V. cholerae ToxR activity is enhanced by a second transmembrane protein, ToxS, encoded downstream toxR [19]. This family of proteins is involved in response to temperature, pH, osmolarity and in Photobacterium profundum SS9, a piezophilic bacterium, to hydrostatic pressure [20]. The widespread presence of these genes among the Vibrionaceae suggests their importance in regulatory mechanisms.\nWe identify two other noteworthy groups of genes composed by 257 and 160 genes respectively shared just by two strains, mainly annotated as \"hypothetical protein\". The first group of genes is shared between Photobacterium profundum SS9 and Photobacterium profundum 3TCK, while the second is shared between V. vulnificus CMCP6 and YJ016. These strains are closely related and this explains the high number of shared genes; while, inside the Vibrionaceae family, the number of specific shared genes highly decreases, showing a high inter-species variability (Fig. 6)\n\nProphages and transposases\nProphages recover different biological roles both as quantitatively important genetic elements of the bacterial chromosome, and as vectors of lateral gene transfer among bacteria, due to their characters of mobile DNA elements. Indeed, numerous virulence factors from bacterial pathogens are phage encoded. It was postulated that this role of prophages is not limited to pathogenic bacteria but some adaptations of nonpathogenic strains to their ecological niche might also be mediated by prophages acquisition [21].\nTo better understand the importance of mobile elements within Vibrionaceae family, we performed a hierarchical cluster analysis using gene profiles annotated as \"phage protein\" and \"transposase\", for a total of 172 clusters of genes (Fig 7). We found that a high inter-strain genetic variability exists and phages and transposases are both shared by almost all Vibrionaceae, and specific to just some organisms. We identified five major clusters of mobile elements that are specific to a single organism. A group composed by 26 clusters containing both transposase and phage proteins seem to be unique to V. splendiduds 12B01 (Fig 7). Another one composed by 16 clusters is specific of V. vulnificus CMCP6 (Fig 7) while V. parahaemoliticus has a cluster of 11 genes (Fig 7). Moreover there is another group of transposases and phage genes shared mainly by V. cholerae 0395, Shewanella oneidensis and V. cholerae V51 (Fig 7). Finally a big cluster of almost 30 genes, all predicted to codify for transposases, was found in P. profundum SS9 genome (Fig. 7). The high presence of transposases in this bacterium seems to correlate with its deep-sea habitat, a feature presumably shared with other deep-sea microorganisms [12]. As shown in Fig 7, many of the clusters well conserved in an organism, are partially shared with a low similarity by other organisms. This agrees with the idea that prophages are not maintained in the genome over a long period of time and part of their genes may be deleted from the chromosome. Moreover, microarray analysis and PCR scanning demonstrated that prophages are frequently strain specific within a given bacterial species [22-24]. According to the modular theory of phage evolution, phage genomes are mosaics of modules, groups of genes functionally related, that are free to recombine in genetic exchanges between distinct phages infecting the same cell [21]. This can result in the occurrences of different part of phage distributed in far related genomes. Phylogenetic profile of some transposases is similar to the phage ones, suggesting a possible transfer mechanism phage-mediated for such mobile elements.\n\n\nConclusion\nIn this work we propose an improved phylogenetic profile analysis on 14 Vibrionaceae genomes, to study this family on the basis of gene content. Using a phylogenetic profile for each cluster of genes defined as the median of all the profiles belonging to the cluster (meta-profile) we investigate the evolution of groups of ORFs belonging to the entire family. A two-way cluster analysis allows us to identify similarity structures on the entire phylogenetic matrix composed by 8,239 clusters of genes and 320 organisms.\nThe phylogenetic tree obtained with the cluster analysis does not reflect the global evolutionary tree because of the Vibrionaceae ORFs dataset used for the analysis, but rather can be considered as the Vibrionaceae perspective of bacterial diversity. The phylogenetic tree reflects the evolutionary processes that shape genomes, as lateral gene transfer, genes genesis and loss. In this context, the tree allows to group together genomes on the base of their global gene content.\nWe found that genomes belonging to the same taxonomic group tend to cluster together and that Vibrionaceae species are closely related. Moreover organisms belonging to the same or closely related taxa split into different subgroups, confirming the existence of a high variability among lineages, due to genetic and evolutionary process such as lateral gene transfer, concerted evolution and genes duplication.\nOn the other hand several groups of genes characterised by different homogeneous profiles have been identified. In particular we found, 1) a set of conserved genes (with a high similarity values across all organisms) that reflects the \"minimal genome\" composition defined in other previous works; 2) a set of genes mainly shared by Vibrionaceae and other Gamma proteobacteria and 3) genes specific to different sets of Vibrionaceae.\nFinally a further analysis on prophage and transposase has confirmed the high inter-strain genetic variability even among closely related species.\nThe increasing number of genomes included in this type of analysis surely add new sorces of variability and noise, anyway we think that the use of meta-profiles can be useful for complexity reduction and data analysis to study global gene evolution.\n\nMethods\nDatasets\nThe Vibrionaceae species used in this analysis were selected among the freely available complete and draft genome sequences. The proteomes of V. cholerae N16961, V. parahaemolyticus, V. vulnificus YJ016, V. vulnificus CMCP6, V. fischeri ES114, Photobacterium profundum SS9 were downloaded from the NCBI ftp site [25]. Protein sequences of Vibrio cholerae MO10, Vibrio cholerae 0395, Vibrio cholerae RC385, Vibrio cholerae V51, Vibrio cholerae V52 were downloaded from the NCBI genome database, while sequences of Vibrio MED222, Vibrio splendidus 12B01, Photobacterium profundum 3TCK from the J. Craig Venter Institute web site.\nThe 320 complete genomes update at 03/06 were downloaded from the NCBI ftp site.\n\nSimilarity search and phylogenetic profile construction\nAll the Vibrionaceae ORFs were merged generating a redundant list of 59,669 proteins and were compared to all open reading frame from 320 bacterial and archeal genomes using Blastp. To determine the presence of an orthologous we used a combination of three different thresholds; a similarity value equal to or higher than 30%, an alignment length equal or higher than 70% and an Evalue score lower than or equal to e-6. After determining the presence of an orthologous gene, we computed a similarity index I for each pair of orthologous (a point of the phylogenetic profile) as follow:\nI=SqsSqq⋅min⁡(lq,ls)max⁡(lq,ls)\n MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqWGjbqscqGH9aqpdaWcaaqaaiabdofatnaaBaaaleaacqWGXbqCcqWGZbWCaeqaaaGcbaGaem4uam1aaSbaaSqaaiabdghaXjabdghaXbqabaaaaOGaeyyXIC9aaSaaaeaacyGGTbqBcqGGPbqAcqGGUbGBcqGGOaakcqWGSbaBdaWgaaWcbaGaemyCaehabeaakiabcYcaSiabdYgaSnaaBaaaleaacqWGZbWCaeqaaOGaeiykaKcabaGagiyBa0MaeiyyaeMaeiiEaGNaeiikaGIaemiBaW2aaSbaaSqaaiabdghaXbqabaGccqGGSaalcqWGSbaBdaWgaaWcbaGaem4CamhabeaakiabcMcaPaaaaaa@532D@\nwhere lq and ls are the query and subject length sequence respectively and Sqs is the similarity score between the query and the subject sequence. Sqs is defined as follow:\nSqs=∑i=1MαAqi,Asi+GP\n MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqWGtbWudaWgaaWcbaGaemyCaeNaem4Camhabeaakiabg2da9maaqahabaacciGae8xSde2aaSbaaSqaaiabdgeabjabdghaXjabdMgaPjabcYcaSiabdgeabjabdohaZjabdMgaPbqabaaabaGaemyAaKMaeyypa0JaeGymaedabaGaemyta0eaniabggHiLdGccqGHRaWkcqWGhbWrcqWGqbauaaa@4620@\nwhere M is the match length between the query and subject sequence; Aqi and Asi respectively the query and subject amino acid in position i; α the BLOSUM substitution matrix value for amino acid pair Aqi, Asi and GP the gap penalty. GP is defined as follow:\nGP = GOP+GEP(k-1)\nwhere GOP is the Gap Open Penalty set to -11, GEP the Gap Extension Penalty set to -1 and k the gap length. Sqq represents the score of the self-aligned query sequence.\nSqs is always smaller than Sqq and the score S range between 0 and 1. In order to take into account also the different sequence lengths, we multiplied the score S by the ratio between the minimum length between query and subject and the maximum length between query and subjects. In this way the total score is weighted on the base of the length, resulting in a lower similarity value if the lengths of the sequences are different.\nThe phylogenetic profile for each ORF is an array of index I with length equal to the number of genomes considered (320).\n\nORFs clustering\nThe redundant list of 59,669 Vibrionaceae ORFs contained multiple copies of the same genes due to the presence of conserved genes in the considered genomes. In order to reduce the redundancy, we clustered proteins using a two-step approach. The first step is based on COG (Cluster of Othologous Genes) annotation. COG classifies conserved genes according to their homologous relationships. All the Vibrionaceae ORFs were annotated using COG clusters and proteins sharing the same COG code were considered belonging to the same cluster. In particular, the annotation process consists of a similarity search of all the ORFs against the COG proteins using blast and considering the best hit for each protein. 43,024 ORFs presented a similarity with a COG entry, producing 2,463 different clusters. In the second step, the remaining 16,645 ORFs without similarity with any COG entry were clustered using CD-HIT software [26]. CD-HIT program clusters protein sequence database at high sequence identity threshold and efficiently removes high sequence redundancy. This last clustering process produced 9,613 different groups of similar proteins.\nFinally from the 12,076 total clusters obtained by this methodology, those composed by ORFs that do not have any ortologous genes (with a phylogenetic profile composed by an array with all zero values except for one position match with itself) were eliminated, resulting in a dataset composed by 8,239 distinct clusters.\nThe final phylogenetic profile for each cluster (meta-profile) was defined as the median of all the profiles belonging to the cluster. At the end of these procedures the final phylogenetic matrix was composed by 8,239 rows (cluster of genes) and 320 columns (organisms). In each cell the median of the index in the cluster was reported.\n\nCluster analysis\nSeveral clustering techniques have been used to identify the similarity structure underneath our data. A k-means and a two-way hierarchical cluster analysis with Euclidean distance and complete linkage were performed on the phylogenetic matrix.\nThe goal of a cluster analysis is to partition the elements into subsets without any constrains or a priori information, so that two criteria are satisfied: homogeneity, elements inside a cluster are highly similar to each other; and separation, elements from different clusters have low similarity to each other.\nThe Figure of Merit (FOM) is a measure of fit of the expression patterns for the clusters produced by a particular algorithm that estimates the predictive power of a clustering algorithm. It is computed by removing each sample in turn from the data set, clustering genes based on the remaining data, and calculating the fit of the withheld sample to the clustering pattern obtained from the other samples. On our data FOM analysis identified the best number of k-means clusters between 10 and 15. We decided to set k (in the k-means analysis) equal to 14. In each of these 14 clusters subsequent hierarchical cluster analysis was performed with bootstrap cluster assessment. All the previous analyses were performed with TMEV software [27], freely available at [28].\n\nEnrichment categories\nEach cluster of genes has been annotated according to COG code, GO terms and KEGG pathway maps. Class enrichment (with respect to the entire matrix) has been calculated according to the hypergeometric distribution that was used to obtain the chance probability of observing the number of genes annotated with a particular COG, GO and KEGG category among the selected cluster. The probability P of observing at least k genes of a functional category within a group of n genes is given by:\nP=∑i=kn(fi)(g−fn−i)(gn)\n MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqWGqbaucqGH9aqpdaaeWbqaamaalaaabaWaaeWaaeaafaqabeGabaaabaGaemOzaygabaGaemyAaKgaaaGaayjkaiaawMcaamaabmaabaqbaeqabiqaaaqaaiabdEgaNjabgkHiTiabdAgaMbqaaiabd6gaUjabgkHiTiabdMgaPbaaaiaawIcacaGLPaaaaeaadaqadaqaauaabeqaceaaaeaacqWGNbWzaeaacqWGUbGBaaaacaGLOaGaayzkaaaaaaWcbaGaemyAaKMaeyypa0Jaem4AaSgabaGaemOBa4ganiabggHiLdaaaa@47C6@\nwhere f is the total number of genes with the same category (in the matrix) and g is the total number of genes in our matrix.\n\n\n\n" ], "offsets": [ [ 0, 34715 ] ] } ]

[ { "id": "pmcA1885853__T0", "type": "species", "text": [ "V. cholerae" ], "offsets": [ [ 3856, 3867 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "666" } ] }, { "id": "pmcA1885853__T1", "type": "species", "text": [ "Colwellia psychrerythraea 34H" ], "offsets": [ [ 10788, 10817 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "167879" } ] }, { "id": "pmcA1885853__T2", "type": "species", "text": [ "Shewanella oneidensis" ], "offsets": [ [ 10822, 10843 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "70863" } ] }, { "id": "pmcA1885853__T3", "type": "species", "text": [ "Buchnera aphidicola" ], "offsets": [ [ 12458, 12477 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9" } ] }, { "id": "pmcA1885853__T4", "type": "species", "text": [ "Vibrio cholerae 0395" ], "offsets": [ [ 12814, 12834 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345073" } ] }, { "id": "pmcA1885853__T5", "type": "species", "text": [ "Vibrio cholerae MO10" ], "offsets": [ [ 12836, 12856 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345072" } ] }, { "id": "pmcA1885853__T6", "type": "species", "text": [ "Vibrio cholerae RC385" ], "offsets": [ [ 12858, 12879 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345074" } ] }, { "id": "pmcA1885853__T7", "type": "species", "text": [ "Vibrio cholerae V51" ], "offsets": [ [ 12881, 12900 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345075" } ] }, { "id": "pmcA1885853__T8", "type": "species", "text": [ "Vibrio cholerae V52" ], "offsets": [ [ 12902, 12921 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345076" } ] }, { "id": "pmcA1885853__T9", "type": "species", "text": [ "Photobacterium profundum 3TCK" ], "offsets": [ [ 12923, 12952 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "314280" } ] }, { "id": "pmcA1885853__T10", "type": "species", "text": [ "Vibrio MED222" ], "offsets": [ [ 12954, 12967 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "314290" } ] }, { "id": "pmcA1885853__T11", "type": "species", "text": [ "Vibrio splendidus12B01" ], "offsets": [ [ 12969, 12991 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "314291" } ] }, { "id": "pmcA1885853__T12", "type": "species", "text": [ "Photobacterium profundum SS9" ], "offsets": [ [ 16952, 16980 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "298386" } ] }, { "id": "pmcA1885853__T13", "type": "species", "text": [ "V. vulnificus YJ016" ], "offsets": [ [ 17409, 17428 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "196600" } ] }, { "id": "pmcA1885853__T14", "type": "species", "text": [ "V. vulnificus CMCP6" ], "offsets": [ [ 17433, 17452 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "216895" } ] }, { "id": "pmcA1885853__T15", "type": "species", "text": [ "V. vulnificus" ], "offsets": [ [ 17689, 17702 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "672" } ] }, { "id": "pmcA1885853__T16", "type": "species", "text": [ "V. cholerae" ], "offsets": [ [ 17718, 17729 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "666" } ] }, { "id": "pmcA1885853__T17", "type": "species", "text": [ "V. fischeri" ], "offsets": [ [ 18825, 18836 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "668" } ] }, { "id": "pmcA1885853__T18", "type": "species", "text": [ "Salmonella typhimurium" ], "offsets": [ [ 19514, 19536 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "90371" } ] }, { "id": "pmcA1885853__T19", "type": "species", "text": [ "Klebsiella pneumoniae" ], "offsets": [ [ 19541, 19562 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "573" } ] }, { "id": "pmcA1885853__T20", "type": "species", "text": [ "E. coli" ], "offsets": [ [ 19609, 19616 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "562" } ] }, { "id": "pmcA1885853__T21", "type": "species", "text": [ "Vibrio MED222" ], "offsets": [ [ 19877, 19890 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "314290" } ] }, { "id": "pmcA1885853__T22", "type": "species", "text": [ "Photobacterium profundum" ], "offsets": [ [ 20202, 20226 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "74109" } ] }, { "id": "pmcA1885853__T23", "type": "species", "text": [ "V. cholerae" ], "offsets": [ [ 20319, 20330 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "666" } ] }, { "id": "pmcA1885853__T24", "type": "species", "text": [ "V. cholerae" ], "offsets": [ [ 20619, 20630 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "666" } ] }, { "id": "pmcA1885853__T25", "type": "species", "text": [ "V. cholerae" ], "offsets": [ [ 20734, 20745 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "666" } ] }, { "id": "pmcA1885853__T26", "type": "species", "text": [ "Photobacterium profundum SS9" ], "offsets": [ [ 20929, 20957 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "298386" } ] }, { "id": "pmcA1885853__T27", "type": "species", "text": [ "Photobacterium profundum SS9" ], "offsets": [ [ 21335, 21363 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "298386" } ] }, { "id": "pmcA1885853__T28", "type": "species", "text": [ "Photobacterium profundum 3TCK" ], "offsets": [ [ 21368, 21397 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "314280" } ] }, { "id": "pmcA1885853__T29", "type": "species", "text": [ "V. vulnificus CMCP6" ], "offsets": [ [ 21434, 21453 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "216895" } ] }, { "id": "pmcA1885853__T30", "type": "species", "text": [ "YJ016" ], "offsets": [ [ 21458, 21463 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "196600" } ] }, { "id": "pmcA1885853__T31", "type": "species", "text": [ "V. splendiduds 12B01" ], "offsets": [ [ 22843, 22863 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "314291" } ] }, { "id": "pmcA1885853__T32", "type": "species", "text": [ "V. vulnificus CMCP6" ], "offsets": [ [ 22924, 22943 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "216895" } ] }, { "id": "pmcA1885853__T33", "type": "species", "text": [ "V. parahaemoliticus" ], "offsets": [ [ 22958, 22977 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "670" } ] }, { "id": "pmcA1885853__T34", "type": "species", "text": [ "V. cholerae" ], "offsets": [ [ 23094, 23105 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "666" } ] }, { "id": "pmcA1885853__T35", "type": "species", "text": [ "Shewanella oneidensis" ], "offsets": [ [ 23112, 23133 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "70863" } ] }, { "id": "pmcA1885853__T36", "type": "species", "text": [ "V. cholerae" ], "offsets": [ [ 23138, 23149 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "666" } ] }, { "id": "pmcA1885853__T37", "type": "species", "text": [ "P. profundum SS9" ], "offsets": [ [ 23260, 23276 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "298386" } ] }, { "id": "pmcA1885853__T38", "type": "species", "text": [ "V. cholerae N16961" ], "offsets": [ [ 26801, 26819 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "243277" } ] }, { "id": "pmcA1885853__T39", "type": "species", "text": [ "V. parahaemolyticus" ], "offsets": [ [ 26821, 26840 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "670" } ] }, { "id": "pmcA1885853__T40", "type": "species", "text": [ "V. vulnificus YJ016" ], "offsets": [ [ 26842, 26861 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "196600" } ] }, { "id": "pmcA1885853__T41", "type": "species", "text": [ "V. vulnificus CMCP6" ], "offsets": [ [ 26863, 26882 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "216895" } ] }, { "id": "pmcA1885853__T42", "type": "species", "text": [ "V. fischeri ES114" ], "offsets": [ [ 26884, 26901 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "312309" } ] }, { "id": "pmcA1885853__T43", "type": "species", "text": [ "Photobacterium profundum SS9" ], "offsets": [ [ 26903, 26931 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "298386" } ] }, { "id": "pmcA1885853__T44", "type": "species", "text": [ "Vibrio cholerae MO10" ], "offsets": [ [ 26998, 27018 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345072" } ] }, { "id": "pmcA1885853__T45", "type": "species", "text": [ "Vibrio cholerae 0395" ], "offsets": [ [ 27020, 27040 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345073" } ] }, { "id": "pmcA1885853__T46", "type": "species", "text": [ "Vibrio cholerae RC385" ], "offsets": [ [ 27042, 27063 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345074" } ] }, { "id": "pmcA1885853__T47", "type": "species", "text": [ "Vibrio cholerae V51" ], "offsets": [ [ 27065, 27084 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345075" } ] }, { "id": "pmcA1885853__T48", "type": "species", "text": [ "Vibrio cholerae V52" ], "offsets": [ [ 27086, 27105 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "345076" } ] }, { "id": "pmcA1885853__T49", "type": "species", "text": [ "Vibrio MED222" ], "offsets": [ [ 27172, 27185 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "314290" } ] }, { "id": "pmcA1885853__T50", "type": "species", "text": [ "Vibrio splendidus 12B01" ], "offsets": [ [ 27187, 27210 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "314291" } ] }, { "id": "pmcA1885853__T51", "type": "species", "text": [ "Photobacterium profundum 3TCK" ], "offsets": [ [ 27212, 27241 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "314280" } ] } ]

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[ { "id": "pmcA2531092__text", "type": "Article", "text": [ "A case of demand ischemia from phendimetrazine\nAbstract\nIntroduction\nPhendimetrazine is a medication currently being used to help patients with weight loss. It shares a chemical structure with amphetamines. As such, it shares some of the same toxicities, which can include cardiac toxicity. This case highlights this principle.\n\nCase presentation\na 54 year old Caucasian female presented to our urgent care facility with complaints of chest pains and other symptoms suggestive of acute coronary syndrome. Ultimately, she was transferred to the emergency room. After evaluation there, it appeared she was having demand ischemia from prescription diet pills\n\nConclusion\nThis case report demonstrates the potential dangers of amphetamine based diet pills. There have been other cases of cardiomyopathies related to phendimetrazine, but it is something that is rarely recognized in an outpatient setting. A case such as this demonstrates the importance of obtaining a careful medication history in all patients and in recognizing diet pills with an amphetamine base can cause cardiac toxicity.\n\n\n\nCase presentation\nA 54 year-old Caucasian female presented to our urgent care facility complaining of nausea and vomiting, sense of impending doom and vague chest pain radiating toward her left side for about five hours. She never had similar symptoms in the past. She also denied anything that could have precipitated these symptoms. Her only past medical history was significant for spina bifida. Her medications included occasional Fiorinal (unknown dose), Xanax 0.5 mg as needed, and Phendimetrazine (unclear dose). Her social history was significant for smoking 1/2 pack per day cigarette use. She denied alcohol use. Family history was non contributory. She worked from home. Her physical exam showed a tachycardia of around 100 beats per minute, respiratory rate of 16, temperature of 98.1, and O2 saturation of 100% on room air. She was approximately 5'7\" and 145 pounds. In general, she was an anxious appearing, diaphoretic woman in moderate distress, she had no elevated JVD at 30 degrees, her heart was tachycardic, but otherwise without murmur, gallops, or rubs, her lungs were clear, abdomen soft, and she had no peripheral edema. An EKG was checked which appears below (figure 1). After examination, there was concern for acute coronary syndrome (ACS). She was given nitroglycerin with relief of her chest discomfort. She was also given aspirin to chew. EMS was called and she was transferred to a local emergency room. She was hospitalized there for three days and after her discharge, we got permission from her to request records. While hospitalized, she was ruled out for ACS with negative troponins. She was also given beta blockade which resolved her tachycardia and her T wave changes on EKG. The next morning, she had an adenosine stress test which revealed normal uptake with no areas of ischemia and an ejection fraction of 55%. She was monitored for one more day and then discharged with instructions to discontinue her diet pills.\n\nDiscussion\nPhendimetrazine is a medication currently being used for weight loss, with potential for illicit use. It has a similar chemical composition of amphetamines, which is thought to account for its clinical actions [1]. Amphetamines are well recognized as an etiology of cardiac ischemia, however phendimetrazine is more rarely described in the literature as causing cardiac events. [2,3]. Acute effects include hyperpyrexia, mydriasis, chest pain, arrhytmias, delirium, and, rhabdomylosis, among others [2]. Long term use has been associated with dilated cardiomyopathies, some of which have resolved with discontinuation of the medication [3]. In this particular case, it appears she may have developed a demand ischemia from the medication. It is not known how much of the drug she was taking. Initially, she was resistant to accepting that phendimetrazine could induce side effects, and there was suspicion that she could have been taking more of the drug that recommended. In addition, she was not prescribed the medication and would not admit to where she obtained it. As the public seems to have more focus on using medications to induce weight loss, this may be a more recognized complication and heart conditions should likely be monitored prior to starting amphetamine based weight loss pills.\n\nConclusion\nDue to potentially detrimental effects of this medication, phendimetrazine should be used cautiously in many situations. As it shares its chemical structure with amphetamines, it also shares many of the side effects and the potential for abuse/addiction. There have been other reports in literature describing adverse outcomes from phendimetrazine as well as other weight loss medications. Therefore, cautious use is warranted.\n\nAbbreviations\nACS: Acute Coronary Syndrome.\n\nCompeting interests\nThe authors declare that they have no competing interests.\n\nAuthors' contributions\nDL, JJ, GG have all been involved in and approve of the writing of this case presentation.\n\nConsent\nWritten informed consent was obtained from the patient for publication purposes. A copy can be obtained if requested by the Editor in Chief of this journal.\n\n\n" ], "offsets": [ [ 0, 5210 ] ] } ]

[ { "id": "pmcA2531092__T0", "type": "species", "text": [ "patients" ], "offsets": [ [ 130, 138 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] }, { "id": "pmcA2531092__T1", "type": "species", "text": [ "patients" ], "offsets": [ [ 998, 1006 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] }, { "id": "pmcA2531092__T2", "type": "species", "text": [ "woman" ], "offsets": [ [ 2027, 2032 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] } ]

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[ { "id": "pmcA1885664__text", "type": "Article", "text": [ "The recombination-associated protein RdgC adopts a novel toroidal architecture for DNA binding\nAbstract\nRecA plays a central role in the nonmutagenic repair of stalled replication forks in bacteria. RdgC, a recombination-associated DNA-binding protein, is a potential negative regulator of RecA function. Here, we have determined the crystal structure of RdgC from Pseudomonas aeruginosa. The J-shaped monomer has a unique fold and can be divided into three structural domains: tip domain, center domain and base domain. Two such monomers dimerize to form a ring-shaped molecule of approximate 2-fold symmetry. Of the two inter-subunit interfaces within the dimer, one interface (‘interface A’) between tip/center domains is more nonpolar than the other (‘interface B’) between base domains. The structure allows us to propose that the RdgC dimer binds dsDNA through the central hole of ∼30 Å diameter. The proposed model is supported by our DNA-binding assays coupled with mutagenesis, which indicate that the conserved positively charged residues on the protein surface around the central hole play important roles in DNA binding. The novel ring-shaped architecture of the RdgC dimer has significant implications for its role in homologous recombination.\n\nINTRODUCTION\nHomologous recombination systems contribute to the maintenance of genome integrity and are essential for the repair of stalled replication forks (1). Genetic studies of the recombination-dependent growth C (rdgC) gene in bacterial systems have suggested that it is involved in DNA replication and recombination (2,3). In Neisseria gonorrhoeae (the gonococcus), RdgC is required for efficient pilin antigenic variation and plays some role in cell growth (4,5). The Escherichia coli rdgC gene encodes a DNA-binding protein of 34 kDa, whose expression level was at its highest during exponential phase, reaching its maximum at ∼1000 dimers per cell (2). Its level decreased sharply to ∼50 dimers per cell in stationary phase. This profile suggests that RdgC might function during the period of DNA replication (2).\nThe RecA proteins and their homologs play a key role in recombinational DNA repair in bacteria and eukaryotes. Their aberrant reactions could result in gross chromosomal rearrangements that lead to human diseases, including cancer. Therefore, their functions must be tightly regulated, and understanding how the RecA family of recombinases is regulated is of utmost importance (1). The E. coli RdgC protein is a potential negative regulator of the function of RecA (1). It inhibits RecA-promoted DNA strand exchange, RecA-mediated ATPase activity and RecA-dependent LexA cleavage (1). Sedimentation equilibrium data indicated that E. coli RdgC exists in solution as a mixture of oligomeric states in equilibrium, most likely monomers, dimers and tetramers (1). This concentration-dependent change in the oligomeric state appears to affect its mode of binding to DNA and its capacity to inhibit RecA (1). The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA-binding sites, especially on double-stranded DNA (dsDNA) (1).\nDeletion of the E. coli rdgC gene alone causes no obvious phenotype but is highly deleterious in strains lacking certain enzymes involved in recombination and replication restart (2,3). RdgC is essential for the growth of an E. coli strain lacking PriA, indicating that it might affect replication fork progression or fork rescue (2). PriA provides a means to load the DnaB replicative helicase at DNA replication fork and D loop structures (2). RdgC is, therefore, a key factor in the rescue of stalled or broken forks and subsequent replication restart. dnaC suppressors of PriA can overcome this inviability, especially when RecF, RecO or RecR is inactivated, which indicates that RdgC avoids or counters the toxic effect of these proteins by limiting inappropriate RecA loading on SSB-coated ssDNA (2).\nThe DNA-binding ability of the RdgC protein has been demonstrated clearly using electrophoretic mobility shift assays (2). Escherichia coli RdgC binds non-specifically to dsDNA and with higher affinity than ssDNA (2). RdgC from N. meningitidis also binds DNA with little specificity for sequence or structure, like the E. coli protein (4). RdgC exhibits no preference for DNA replication or recombination intermediates (2). However, no detectable domains or nuclease activity could be identified for the E. coli RdgC protein (2,6). The complexes of E. coli RdgC with both linear and supercoiled circular plasmid DNA were imaged using atomic force microscopy (7). RdgC was found to have an increased affinity to DNA ends and to promote bending of DNA (7). RdgC has the effect on DNA superstructure; the promotion of DNA condensation was observed at high protein concentrations (7). Recombination is largely enhanced by close contacts of distant regions along the DNA strands through condensation (7).\nPseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that belongs to the γ subdivision of the Proteobacteria. Orthologs of the rdgC gene are found only in β and γ subdivisions of the Proteobacteria (2). The amino acid sequence of P. aeruginosa RdgC, a 306-residue protein, shows 46 and 36% identities against those of E. coli and N. meningitidis, respectively. Despite the importance of DNA-binding ability of RdgC in its biological roles, the structural details of RdgC and the mechanism of its action remain unclear. Here, we report the crystal structure of P. aeruginosa RdgC as determined by the multiwavelength anomalous diffraction (MAD) method of X-ray crystallography. It reveals that RdgC dimer is ring-shaped, with a central hole of ∼30 Å diameter. The inside surface around the central hole is rich in conserved, positively charged residues, which are implicated in DNA-binding by mutagenesis. We propose that dsDNA binds to RdgC through this central hole. Our structure provides a solid structural framework for a better understanding of the role of RdgC in homologous recombination.\n\nMATERIALS AND METHODS\nProtein expression and purification\nThe rdgC gene (PA3263) was amplified by the polymerase chain reaction (PCR) using the genomic DNA of P. aeruginosa strain PAO1 as a template. The oligonucleotide primers designed using the published genome sequence (8) were 5′-G GAA TTC CAT ATG TGG TTC CGC AAT CTG CTC G-3′ (forward) and 5′-CCG CCG CTC GAG GAC GCC CTG GGG GAT TTC TTC-3′ (reverse). The bases in bold denote the NdeI and XhoI cleavage sites, respectively. The PCR product was digested with NdeI and XhoI, and was then inserted into the NdeI/XhoI-digested expression vector pET-21a(+) (Novagen). This vector construction adds an eight-residue tag (LEHHHHHH) to the carboxyl terminus of the gene product to facilitate protein purification.\nThe protein was expressed in E. coli C41 (DE3) cells (9). The cells were grown at 37°C up to OD600 of 0.6 in Luria–Bertani medium that contained 50 μg ml−1 ampicillin, and the protein expression was induced by 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The cells were allowed to grow at 20°C for 22 h after IPTG induction and were harvested by centrifugation at 4200 g (6000 rev min−1; Sorvall GSA rotor) for 10 min at 4°C. The cell pellet was resuspended in an ice-cold lysis buffer [20 mM Tris-HCl (pH 7.9), 500 mM sodium chloride, 5 mM imidazole] and was then homogenized with an ultrasonic processor. The crude cell extract was centrifuged at 36 000 g (18 000 rev min−1; Hanil Supra 21 K rotor) for 60 min at 4°C, and the recombinant protein in the supernatant fraction was purified in three chromatographic steps.\nThe first purification step utilized the C-terminal hexahistidine-tag by Ni2+-chelated HiTrap chelating column (GE Healthcare). The eluent was diluted 3-fold with 50 mM Tris-HCl at pH 7.4. The diluted solution was applied to a 20-ml heparin-Sepharose column (GE Healthcare), which was previously equilibrated with a buffer of 50 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol and 1 mM EDTA. The protein was eluted with a linear gradient of 0–2.0 M sodium chloride in the same buffer. The first peak corresponded to high molecular aggregates of RdgC and the second peak to RdgC dimers, which was also consistent with our dynamic light scattering measurements. When the aggregates were not separated from the dimers, the protein readily aggregated within a day. The dimer fractions were concentrated to ∼6 mg ml−1 concentration using an YM10 ultrafiltration membrane (Millipore-Amicon) and further purified by gel filtration on a HiLoad XK 16 Superdex 200 prep-grade column (GE Healthcare), which was previously equilibrated with a buffer of 50 mM Tris-HCl (pH 7.4), 200 mM sodium chloride, 1 mM dithiothreitol and 1 mM EDTA. The first peak corresponding to the high molecular aggregates of RdgC was only a minor component, and the second peak corresponding to RdgC dimers was stable for several weeks when frozen at −70°C. The procedure for purifying the SeMet-substituted protein was the same, except for the presence of 10 mM dithiothreitol in all buffers used during purification steps. Dynamic light scattering experiments were performed at 24°C, with the protein (at 2 mg ml−1 concentration) dissolved in a buffer consisting of 50 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol, 1 mM EDTA and 200 mM sodium chloride on a Model DynaPro-801 instrument (Wyatt, Santa Barbara, CA, USA). The protein concentration was estimated by measuring the absorbance at 280 nm, employing the calculated extinction coefficient of 20 910 M−1 cm−1 (SWISS-PROT; http://www.expasy.ch/).\n\nMutagenesis and electrophoretic mobility shift assay\nPoint mutants were prepared using the QuikChange Site-Directed Mutagenesis kit (Stratagene), and the mutations were confirmed by DNA sequencing. The seven mutants R4A, F120A, K146A, R198A, R208A, Q212A and R252A were well expressed and soluble. The three mutants K70A, R81A and R211A were not expressed, while R122A mutant was expressed but was not soluble. Therefore, we mutated Lys70, Arg81, Arg122 and Arg211 into aspartate. The K70D, R81D, R122D and R211D mutants were well expressed and soluble. The soluble mutants were expressed and purified under the conditions identical to those for the wild type, except that the heparin-Sepharose column step was omitted. All of the soluble mutants had similar elution profiles as the wild type upon gel filtration, and the dimer fractions were used for DNA-binding assays. We mixed 5 μl of RdgC (1.1 μg μl−1) and 5 μl of a linear dsDNA with 414 bp (63 ng μl−1), corresponding to the molar ratio of 64:1 for RdgC dimer to dsDNA. The reaction mixtures were incubated for 10 min at room temperature. RdgC–DNA complexes were analyzed by 1.5% (w/v) agarose gel electrophoresis in 1× TAE buffer (45 mM Tris-acetate at pH 8.3, 1 mM EDTA). DNA bands were visualized by ethidium bromide staining. For DNA-binding assays, the protein concentration was measured with a Bio-Rad protein assay kit.\n\nCrystallization and X-ray data collection\nCrystals were grown by the hanging-drop vapor diffusion method at 24°C by mixing equal volumes (2 μl each) of the protein solution (at 19 mg ml−1 concentration in a buffer consisting of 50 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol, 1 mM EDTA and 200 mM sodium chloride) and the reservoir solution. To grow the native crystals, we used a reservoir solution consisting of 0.1 M sodium HEPES (pH 7.5), 1.0 M tri-sodium citrate and 1% (w/v) Anapoe®35 as the detergent. Native crystals grew to approximate dimensions of 0.2 mm × 0.1 mm × 0.1 mm within a few days. The SeMet-substituted protein was crystallized under conditions identical to those for the native crystals except for the presence of 10 mM dithiothreitol in the protein solution.\nA crystal of the SeMet-substituted protein was dipped into a cryoprotectant solution for a few seconds and was flash-cooled in the cold nitrogen gas stream at 100 K. The cryoprotectant solution consisted of 0.1 M sodium HEPES (pH 7.5), 1.0 M tri-sodium citrate, 1% (w/v) Anapoe®35 as the detergent and 10% (v/v) glycerol. A set of Se MAD data was collected at 100 K at three different wavelengths using an Area Detector System Corporation Quantum 210 charge-coupled device detector at the beamline NW12A of Photon Factory, Tsukuba, Japan. The crystal was rotated through a total of 180° with a 1° oscillation range per frame. The raw data were processed and scaled using the program suit HKL2000 (10). The SeMet-substituted crystal belongs to the space group P21212, with unit cell parameters of a = 87.04 Å, b = 115.60 Å and c = 76.07 Å.\nThe native crystals were soaked for 10 min in 10 mM ethyl mercury thiosalicylate (EMTS) to collect a set of Hg MAD data at four different wavelengths. X-ray diffraction data from the EMTS-derivatized crystal, as well as a native crystal, were collected on a Bruker charge-coupled device detector at the beamline BL-6B of Pohang Light Source, Pohang, Korea. The EMTS-soaked crystal belongs to the space group P21212, with unit cell parameters of a = 87.16 Å, b = 116.02 Å and c = 74.97 Å. The native crystal belongs to the space group P21212, with unit cell parameters of a = 87.49 Å, b = 116.04 Å and c = 76.03 Å. If the presence of a dimeric molecule in the asymmetric unit is assumed, the calculated crystal volume per protein mass (VM) is 2.75 Å3 Da−1 and the solvent content is 55.3%. Supplementary Table 1 summarizes the statistics of X-ray diffraction data collection.\n\nStructure solution and refinement\nHeavy atom sites were located with the program SOLVE (11). Four Se sites from the SeMet-substituted crystal and two Hg sites from the EMTS-soaked crystal were located. Two sets of the initial Se and Hg MAD phases were separately improved using the program RESOLVE (12). Two electron density maps were independently interpreted, and the fragments of the two models were combined into a single model, since the two maps were complementary to each other. Non-crystallographic symmetry (NCS) matrices were found from a partial model built from the initial electron density map, and phases were further improved by the 2-fold NCS averaging, solvent flattening and histogram matching with the program DM (13). The model was built with O (14). The model was refined with the program CNS (15), including the bulk solvent correction. 10% of the data were randomly set aside as the test data for the calculation of Rfree (16). Several rounds of model building, simulated annealing, positional refinement and individual B-factor refinement were performed. Subsequently, this model was used to refine the structure of the native protein. The model has excellent stereochemistry, as evaluated by the program PROCHECK (17). Refinement statistics are summarized in Supplementary Table 1.\n\n\nRESULTS AND DISCUSSION\nModel quality and monomer structure\nThe structure of P. aeruginosa RdgC was solved using two sets of MAD data collected from a crystal of the selenomethionine (SeMet)-substituted protein and from a mercury derivative crystal of the native protein (Supplementary Table 1). The model has subsequently been refined using the 20.0–2.50 Å data from a native crystal to crystallographic Rwork and Rfree values of 23.4 and 27.9%, respectively, with no sigma cut-off. The refined model contains 612 residues of the two monomers in the asymmetric unit of the crystal (residues 1–306 for both chains A and B) and 87 water molecules. The C-terminal eight-residue fusion tag has no electron density in both subunits and has not been modeled. Conformations of the two monomers in the asymmetric unit are similar. The root-mean-square (r.m.s.) difference between the two monomers is 0.69 Å for 306 Cα atom pairs, with a maximum deviation of 8.8 Å for the Cα atom of Gly305. The residues giving r.m.s. differences larger than 2.0 Å are 14–16, 94–96, 201–202 and 303–306, which correspond to loops connecting secondary structure elements or the C-terminus. They are located on the surface of the dimer. Subunit A has a better map quality than subunit B, and thus has a lower mean B-factor than subunit B (40.3 Å2 versus. 56.1 Å2 for all 4788 atoms) (Supplementary Figure 1). Therefore, subunit A is chosen for discussions, unless otherwise stated.\nThe P. aeruginosa RdgC monomer is J-shaped and has approximate dimensions of 72 Å × 60 Å × 40 Å (Figure 1). It can be divided into three structural domains: center domain, tip domain and base domain. Center domain (residues 1–73 and 121–167) includes a six-stranded anti-parallel β-sheet with two flanking α helices (α1 and α4). The helix α1 runs nearly parallel to the two outermost strands β2 and β3, which in turn lie adjacent to β4. Strands β2 and β3 are separated by a loop. The helix α4 is nearly orthogonal to the β-strands. Tip domain (residues 74–120) contains two α helices (α2 and α3) and is inserted between strands β4 and β5 of the center domain. It sticks out from the center domain. The two connections between the center domain and the tip domain are rich in conserved residues, hinting at an important functional role of these joints. They could act as a hinge for a possible conformational change. The C-terminal region of the polypeptide chain is folded into the base domain (residues 168–306), which consists of a five-stranded anti-parallel β-sheet with four α-helices, three of which (α5, α6 and α8) cover one side of the β-sheet. The long helix α8 is slightly bent.\n\n\nStructural similarity searches\nWhen we searched for structural similarity against the Protein Data Bank database using the program DALI (18), the P. aeruginosa RdgC monomer showed no significant similarity with a Z score above 5. Therefore, we conclude that the overall fold of the RdgC polypeptide chain is unique. When we elaborated the structural comparisons with individual domains of P. aeruginosa RdgC, only the center domain gave a Z score above 5.\nUsing the center domain (residues 1–73 and 121–167) alone, the highest structural similarity is found with the human β2-adaptin appendage domain (PDB code 1E42, Z score = 7.1, r.m.s. deviation = 2.5 Å for 85 structurally aligned residues, residues 1–12, 14–30, 44–47, 58–72, 122–133 and 135–160 of P. aeruginosa RdgC chain A and residues 838–841, 848–880, 885–906 and 912–967 of the human β2-adaptin appendage domain chain A) (19). There does not appear to be any functional relatedness between these two proteins. The next highest similarity is found with the yeast TATA-box-binding protein (PDB code 1YTB, Z score = 5.7, r.m.s. deviation = 2.6 Å for 79 structurally aligned residues, residues 4–12, 14–19, 21–27, 56–70, 124–132 and 134–166 of P. aeruginosa RdgC chain A and residues 69–96, 100–126 and 132–155 of TATA-box-binding protein, N-terminal half of chain A) (20). The TATA-box-binding protein is composed of a ten-stranded antiparallel β-sheet with four flanking α-helices on the convex side of the β-sheet (20). It has an internal quasi 2-fold symmetry, and its β-strands are involved in DNA binding. There is, however, no structural similarity of functional significance, because the two center domains of the RdgC dimer are well separated and do not associate into a single unit (Figure 1C and D).\nUsing the tip domain (residues 74–120) alone, the ATPase domain of bovine Hsc70 chaperone shows highest structural similarity (PDB code 1BA1, Z score = 4.8, r.m.s. deviation = 1.7 Å for 45 structurally aligned residues, residues 76–120 of P. aeruginosa RdgC chain A and residues 230–253 and 256–276 of the Hsc70 ATPase domain) (21). There does not seem to be any significant functional relationship between these two proteins. The α-helix-loop-α-helix structure of the tip domain is reminiscent of the helix–hairpin–helix (HhH) DNA-binding motif that is present in a number of DNA repair enzymes (22–25). The HhH motifs are characterized by the conserved sequence motif hxxhxGhGxxxAxxxhh, where h is any hydrophobic residue (VILMWFYA) and x any residue (23). Two conserved glycines allow a tight turn between two α-helices. However, the tip domain of P. aeruginosa RdgC protein does not have such a sequence motif, and the loop in the RdgC tip domain is much more extended than that of the HhH motifs. Therefore, the RdgC tip domain is uniquely folded.\nWith the base domain (residues 168–306) alone, the highest similarity is found with P. aeruginosa isochorismate-pyruvate lyase (unpublished deposition; PDB code 2H9C, Z score = 4.0, r.m.s. deviation = 4.8 Å for 51 structurally aligned residues, residues 186–191, 237–240, 257–272 and 274–298 of P. aeruginosa RdgC chain A and residues 36–41, 50–53, 55–66 and 68–96 of P. aeruginosa isochorismate-pyruvate lyase chain A). There does not appear to be any functional relationship between the two proteins, and the base domain of RdgC is uniquely folded. The 101-residue chain of P. aeruginosa isochorismate-pyruvate lyase is folded into three α-helices, two of which overlap with two α-helices (α2 and α3) of the base domain of RdgC.\n\nRing-shaped dimer structure and inter-subunit interface\nOur structure reveals that P. aeruginosa RdgC forms a ring-shaped dimer of approximate 2-fold symmetry in the crystal (Figure 1). This observation is consistent with the results of previous gel filtration and sedimentation equilibrium experiments, which suggested the presence of dimeric species of E. coli RdgC in solution (1,2). Depending on the concentration, RdgC could also exist in solution as monomers, tetramers and higher oligomers (1). In the crystal lattice, no monomeric, trimeric or tetrameric species exist. Our structure unequivocally rules out the monomer–trimer model, which fits the sedimentation equilibrium data approximately, as well as the monomer–dimer–tetramer model (1). The RdgC dimer has approximate dimensions of 80 Å × 60 Å × 40 Å, with a central hole of ∼30 Å diameter.\nOnly a moderate amount of surface area is buried upon the formation of the RdgC dimer. The monomer–monomer interface buries a solvent-accessible surface area of ∼1360 Å2 per monomer, corresponding to ∼8% of the total surface area of the monomer (Protein–Protein Interaction Server at http://www.biochem.ucl.ac.uk/bsm/PP/server/). Polar and nonpolar atoms in interface are 44 and 56%, respectively. The interface can be divided into two kinds: ‘interface A’ involving both the tip domains and center domains, and ‘interface B’ involving the base domains (Figure 2A). These two interfaces have significantly different characters.\n\nThe interface A is formed largely by the crossing over of the two tip domains in the dimer (Figure 1). This interface covers a surface area of ∼565 Å2 and is largely nonpolar, with the polar and nonpolar atoms in the interface being 37.5 and 2.5%, respectively. Phe120 contributes considerably to the nonpolar character of the interface between the tip domains, accounting for ∼150 Å2 of the interface area. Phe120 is part of a highly conserved sequence segment and lies at the boundary between the tip domain and the center domain. The aromatic ring of Phe120′ inserts into a hydrophobic pocket, which is formed by the hydrophobic residues Ile74, Leu75, Pro76, Val79, Leu115, Ala119 and Phe120 of the neighboring monomer (Figure 2B). The primed residue belongs to the adjacent subunit. These residues are well conserved in the RdgC family (indicated by green triangles below the aligned sequences in Figure 3). As these hydrophobic residues are located near the molecular 2-fold axis, the two clusters of hydrophobic side chains are merged into a single, large cluster in the dimer (Figure 2B). Of the 16 hydrogen bonds that exist between the two subunits, only one is made in this interface between Arg118 and Arg118′.\n\nThe interface B is contributed to exclusively by the two base domains. This interface covers a surface area of ∼795 Å2. The polar and nonpolar atoms in this interface are 50% each. The two β-sheets of the two base domains merge into a single ten-stranded antiparallel β-sheet in this interface (Figure 2A). Thus, 15 of 16 inter-subunit hydrogen bonds are clustered in this interface. The hydrogen bond networks can be divided into two types. The first type involves the main chain atoms of Val206, Arg208 and Lys210 in strand β9; this strand associates with the equivalent strand from the other subunit in an antiparallel manner (Figure 2C). The other type involves both the side chain and main chain atoms. The side chain of Arg211′ interacts with the backbone of Gly204, while the side chain of Lys227 hydrogen bonds to those of both Gln212′ and Glu218 (Figure 2D). In addition, the side chain of Gln212′ interacts with that of Asp199 and the backbone of Arg211′ (Figure 2D). Arg211, Gln212, Glu218 and Lys227 are highly conserved among the RdgC family members. The residues participating in the hydrogen bond networks are indicated by red circles below the aligned sequences in Figure 3. The observation that conserved residues play important roles in forming the dimer implies that dimerization of RdgC is critical for its function.\nIf one or both of interfaces A and B are to be broken for functional reasons such as for allowing dsDNA to enter into the central hole of the RdgC dimer, we can conceive of three scenarios. The first possibility would be a simultaneous breakage of the two interfaces A and B. The second would be the sequential disruption of the interfaces A and B. The third would be the disruption of only one of the two interfaces A and B. Sedimentation equilibrium indicated the presence of monomers in solution (1), which is consistent with the first two scenarios. If the third scenario holds, RdgC dimers could possibly exist in two conformations in solution, i.e. a closed form as observed in the present crystal structure, and an open form, in which one of the two interfaces A and B is broken.\n\nA model for DNA binding by RdgC and DNA-binding assay\nThe net charge of P. aeruginosa RdgC is highly negative, since each monomer contains 12 aspartate, 13 glutamate, 10 lysine, 9 arginine and 2 histidine residues. The net charge of a dimer would be −12 (or −8), if we assume none (or all) of histidines to be positively charged. The electrostatic potential at the molecular surface of RdgC dimer shows that the charge distribution on the protein surface is highly nonuniform (Figure 4A). Since the inner surface of the RdgC dimer is rich in conserved, positively charged residues and the diameter of the central hole (∼30 Å) is approximately similar to that of other toroidal DNA-binding proteins (26), it appears likely that RdgC dimers bind dsDNA through the central hole. Despite extensive co-crystallization and soaking trials with linear dsDNAs of different lengths (14-mer, 16-mer and 18-mer), we could not locate the bound DNA experimentally. We attribute this difficulty to the nature of sequence-nonspecific DNA binding by RdgC. Therefore, we built a crude model of a DNA complex of the RdgC dimer by fitting dsDNA into the central hole (Figure 4B). This model is supported by the DNA-binding assays coupled with mutagenesis, as described below. It is also consistent with the suggested binding site size of ∼16 nt for the E. coli RdgC dimer (1) and the approximate thickness (∼40 Å) of the P. aeruginosa RdgC dimer.\n\nWhen we performed electrophoretic mobility shift assay for dsDNA binding, P. aeruginosa RdgC did not require Mg2+ ions (Supplementary Figure 2), unlike Deinococcus radiodurans RecR, which required high concentrations of Mg2+ ions (27). ATP was not required for dsDNA binding by P. aeruginosa RdgC, either (Supplementary Figure 2). This is consistent with an apparent lack of an ATP-binding pocket in the RdgC dimer structure.\nIn order to identify the residues that are important for dsDNA binding, we carried out mutational analyses. The residues for mutagenesis were selected on the basis of the modeled complex between RdgC dimer and dsDNA, together with sequence conservation (Figure 4C). We prepared eleven point mutants (R4A, K70D, R81D, F120A, R122D, K146A, R198A, R208A, R211D, Q212A and R252A). These eleven mutated residues are well conserved among RdgC proteins. Eight mutants (R4A, K70D, R81D, R122D, K146A, R198A, R208A and R252A) had a lower affinity for dsDNA than the wild-type RdgC (Figure 4D). These eight mutated residues are indicated by blue squares below the aligned sequences in Figure 3. This result indicates that dsDNA binds to the inside surface around the central hole of the RdgC dimer, since the mutated residues are located on the inner surface of the RdgC dimer. It is worth mentioning that a single mutation introduces two alterations per RdgC dimer.\nThe three mutants (F120A, R211D and Q212A) were prepared to investigate whether dimerization of RdgC is crucial for dsDNA binding. Phe120 is a key residue located in the inter-subunit interface A (Figure 2B), while Arg211 makes hydrogen bonds in the interface B (Figure 2D) and Gln212 is involved in both intra- and inter-subunit interactions (Figure 2D). dsDNA binding by the F120A mutant is considerably weakened, compared to either the R211D or Q212A mutant (Figure 4D). This result suggests that destabilization of the ring-shaped architecture of the RdgC dimer is deleterious for tight dsDNA binding and that interface A is probably more important than interface B for dsDNA binding. dsDNA-binding affinity of the Q212A mutant was lower than the wild type (Figure 4D), whereas that of the R211D mutant was comparable to the wild type (Figure 4D). This result suggests that disruption of the inter-subunit interface B is also detrimental for dsDNA binding but to a smaller extent than disruption of the interface A. It further suggests that the side chain of Arg211 is not involved in recognizing dsDNA. It is interesting that the DNA complex for wild type forms a distinct band (lane 3 in Figure 4D), as opposed to a more diffuse band as one would expect if the complexes contained different numbers of RdgC dimers. Assuming a binding site size of ∼16 nt (1), the 414-bp duplex DNA would bind ∼25 RdgC dimers if it is fully saturated.\n\nRdgC is unique among ring-shaped DNA-binding proteins\nPseudomonas aeruginosa RdgC adds a unique member to the repertoire of toroidal DNA-binding proteins (26) such as D. radiodurans RecR (27) and DNA polymerase processivity factors, including the β subunit of E. coli DNA polymerase III (28), gp45 of T4 DNA polymerase (29) and PCNA of eukaryotic DNA polymerase δ (30,31) (Figure 5). These proteins do not have an overall sequence similarity among themselves, and their quaternary structures also vary. P. aeruginosa RdgC and E. coli β clamp are homodimers, T4 gp45 and eukaryotic PCNAs are homotrimers and D. radiodurans RecR is a homotetramer.\n\nIn P. aeruginosa RdgC, the inner surface of the ring-shaped dimer is largely formed by β-strands, while the outer surface of the ring is mostly formed by α-helices (Figure 5). The predicted DNA-binding site of P. aeruginosa RdgC dimer is primarily formed by β-strands and loops. In contrast, in other ring-shaped DNA-binding proteins such as the β subunit of E. coli DNA polymerase III (28), gp45 of T4 DNA polymerase (29), PCNA of eukaryotic DNA polymerases δ (30,31) and D. radiodurans RecR (27), α-helices are located toward the center of the ring and are surrounded by β-strands in the outermost side of the ring (Figure 5). Despite a superficial resemblance of the overall ring-shaped architecture of the RdgC dimer with those of other toroidal DNA-binding proteins, the internal arrangement of the secondary structure elements in the RdgC dimer is strikingly different from those of other toroidal DNA-binding proteins. Thus, RdgC is unique among ring-shaped DNA-binding proteins.\nA number of other proteins involved in DNA metabolism also adopt a ring-shaped structure despite diversity in the molecular functions (26). They include NAD+-dependent DNA ligase (25), λ exonuclease (32), hexameric helicases, topoisomerases, the trp RNA-binding attenuation protein, the bacteriophage head-to-tail connector and translin (26). There are also many examples of the proteins in DNA recombination pathways that assemble into multi-subunit toroids containing a central channel (26). Electron microscopy showed that the E. coli RuvB protein, in the presence of ATP, forms a dodecamer on double-stranded DNA in which two stacked hexameric rings encircle the DNA and are oriented in opposite directions with D6 symmetry (33). The human RAD52 protein was found to assemble into heptameric rings with a large funnel-shaped channel, 40–60 Å in diameter (34). The human DMC1 protein has so far been detected only as an octameric ring (35).\n\nInhibition mechanism of RecA function by RdgC\nRdgC inhibits RecA-promoted DNA strand exchange, RecA-mediated ATPase activity and RecA-dependent LexA cleavage (1). It was observed that the effects of RdgC on RecA-promoted DNA strand exchange were much more pronounced than the effects of RdgC on other RecA functions, when RdgC was added to the reaction late (1). By examining the effects of the DinI protein on RdgC-mediated inhibition of RecA activities, it was concluded that the potent effect of RdgC in the inhibition of strand exchange did not primarily reflect a displacement of RecA in the nucleoprotein filament by RdgC, but instead a binding of RdgC to the dsDNA substrate so as to make it unavailable to the filaments for strand exchange (1).\nOur work contributes to a better understanding of the inhibition mechanism of RecA function by RdgC. The ring-shaped architecture with a central hole that is suitable for the binding of dsDNA lends RdgC with a capacity to hold dsDNA very tightly. Therefore, we suggest that RdgC dimers probably function as a tight dsDNA gripper and physically block access to the target dsDNA by RecA filaments, because tight encircling of dsDNA by RdgC dimers, possibly as a linear aggregate, would prevent the two strands from dissociating. However, it remains unclear how the closed-ring structure of the RdgC dimer might allow binding of a large number of RdgC dimers on the duplex DNA. It may be conceivable that each RdgC dimer can open temporarily at one or both of its two inter-subunit interfaces so that dsDNA can enter the central hole.\nRdgC was at its highest level during exponential phase, reaching its maximum of ∼1000 dimers per E. coli cell. Its level decreased sharply to ∼50 dimers per cell in stationary phase (2). Under normal cellular conditions, RecA is maintained at ∼1000 molecules per cell (http://www.els.net/). Following LexA repressor cleavage, the level of RecA protein in the cell increases by as much as 20-fold. RdgC inhibition of the RecA function by a simple competition mechanism at the cellular concentration of RdgC clearly requires a much higher affinity for DNA by RdgC than RecA. Possible protein–protein interactions between RdgC dimers might have a profound effect on the affinity for DNA, when a large number of RdgC dimers are bound on a duplex DNA. If only one of the two inter-subunit interfaces can open up and the 2-fold symmetry axes of the RdgC dimers are misaligned sufficiently, the escape of DNA from a linear array of interacting RdgC dimers would be extremely difficult. Alternatively, both inter-subunit interfaces of each dimer may open up simultaneously. A line of indirect evidence for the possible protein–protein interactions between RdgC dimers is provided by the cooperative binding of RdgC to DNA (7). Cooperativity of RdgC binding may be caused by local and distant protein–protein interactions (7). A kind of side-by-side interaction between two adjacent molecules of RdgC dimer is present in the crystal, burying an accessible surface area of ∼750 Å2 at the interface. The interaction involves mostly the less polar side surface of the dimer (left side of Figure 4A), with 2-fold symmetry axes of the two interacting RdgC molecules making an angle of ∼90° when viewed down the central holes and ∼15° when viewed perpendicular to the holes. This may explain the observed DNA bending by RdgC, which was speculated to be a necessary step in the working mechanism of RdgC, if one can assume that the strong protein–protein interactions in the crystal were relevant for protein–protein interactions in solution (7).\nRdgC protein was shown to have a potent effect on RecA protein-promoted DNA strand exchange in E. coli (1). In a series of experiments, RdgC was added after RecA filaments had formed on the ssDNA and 5 min after the linear dsDNA was added. With 3 μM RecA, a sharp reduction in DNA strand exchange products was seen with 0.4 μM RdgC, and the generation of products was abolished at 0.8 μM RdgC (1). This suggests that the binding affinity of RdgC to dsDNA is considerably higher than that of RecA. The high affinity of RdgC could be explained by its ring shape and the suggested mode of DNA binding. Tetrameric species of E. coli RdgC, which started to form at around 2 µM concentrations, were speculated to be the species with the most potent inhibitory effect, possibly by having a higher affinity for DNA (1). Higher affinity for DNA of the tetrameric species could be understood, if the formation of a tetramer in solution involves a protein–protein interaction similar to the side-by-side interaction between two adjacent RdgC dimers in the crystal, although there is no experimental evidence. Obviously, there are still many open questions that need to be addressed in future biochemical experiments.\n\n\nSUMMARY\nThe crystal structure of RdgC, a recombination-associated DNA-binding protein, determined in this study reveals that the polypeptide chain is uniquely folded and two monomers associate to form a ring-shaped dimer with a central hole of ∼30 Å diameter. The inner surface around the central hole is rich in positively charged residues. These structural features suggest that dsDNA runs through the central hole when it binds to RdgC dimers. This idea is supported by our mutational studies, which indicate that the conserved, positively charged residues around the central hole are important for dsDNA binding. The toroidal architecture of the RdgC dimer provides a sound framework for a better understanding of its role in homologous recombination.\nCoordinates\nAtomic coordinates and the structure factor data have been deposited in the Protein Data Bank (accession code 2OWY).\n\n\nSUPPLEMENTARY DATA\nSupplementary Data are available at NAR Online.\n\nSupplementary Material\n\n\n" ], "offsets": [ [ 0, 38472 ] ] } ]

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[ { "id": "pmcA553979__text", "type": "Article", "text": [ "Medicinal herb use among asthmatic patients attending a specialty care facility in Trinidad\nAbstract\nBackground\nThere is an increasing prevalence of asthma in the Caribbean and patients remain non-compliant to therapy despite the development of guidelines for management and prevention. Some patients may self-medicate with medicinal herbs for symptomatic relief, as there is a long tradition of use for a variety of ailments. The study assessed the prevalence of use and the factors affecting the decision to use herbs in asthmatic patients attending a public specialty care clinic in Trinidad.\n\nMethods\nA descriptive, cross-sectional study was conducted at the Chest Clinic in Trinidad using a de novo, pilot-tested, researcher-administered questionnaire between June and July 2003.\n\nResults\nFifty-eight out of 191 patients (30.4%) reported using herbal remedies for symptomatic relief. Gender, age, ethnicity, and asthma severity did not influence the decision to use herbs; however, 62.5% of patients with tertiary level schooling used herbs, p = 0.025. Thirty-four of these 58 patients (58.6%) obtained herbs from their backyards or the supermarket; only 14 patients (24.1%) obtained herbs from an herbalist, herbal shop or pharmacy. Relatives and friends were the sole source of information for most patients (70.7%), and only 10.3% consulted an herbalist. Ginger, garlic, aloes, shandileer, wild onion, pepper and black sage were the most commonly used herbs.\n\nConclusions\nAmong patients attending the Chest Clinic in Trinidad the use of herbal remedies in asthma is relatively common on the advice of relatives and friends. It is therefore becoming imperative for healthcare providers to become more knowledgeable on this modality and to keep abreast with the latest developments.\n\n\n\nBackground\nRecent reports from the Caribbean suggest that the incidence of asthma is following the global trend of increasing prevalence. In Jamaica, a prevalence of 20.8% for exercise-induced asthma was estimated in a cross-sectional study in schoolchildren [1]. About one in ten patients attending an Accident and Emergency Department in Trinidad were treated for acute severe asthma [2] and over 15,000 patients attended four A&E departments throughout the island over a 12-month period [3].\nInhaled corticosteroids as prophylaxis and 'as required' bronchodilator for symptomatic relief are established modalities for asthma management and prevention and the Commonwealth Caribbean Medical Research Council/Global Initiative for Asthma guidelines were adopted in the Caribbean in 1997 [4]. It has been noted that inefficient management predisposes patients to frequent hospitalization and reduced quality of life. In Trinidad, non-compliance and inadequate inhaler technique negatively impact on effective disease management [5,6]. The frequent unavailability of medication at public health facilities and the prohibitive cost at private pharmacies are significantly associated with non-compliance and consequently poor disease control. In these studies, some patients indicated their use of herbal remedies as an alternative to conventional medicines.\nOver the last few decades, a global resurgence in the use of herbal remedies has fuelled the growing multi-billion dollar international trade of botanical products. Many patients, dissatisfied with conventional medicines because they expect permanent cures, believe that herbal remedies are 'natural' and sometimes self-medicate without informing their attending physician.\nAlthough there is a long history of traditional use of medicinal herbs throughout the Caribbean [7,8] few studies were done to assess the prevalence of use. Surveys in Jamaica reported an almost 100% use of herbal teas and remedies by respondents throughout the island [9] and 71% in paediatrics inpatients at the University Hospital [10]. These studies, however, assessed only the lifetime use of medicinal herbs and did not identify their use for any particular disease. In Trinidad and Tobago, the use of 'bush medicine' in diabetic patients attending primary healthcare facilities throughout the island was assessed and although 42% reportedly used herbs, only 24% used this healthcare modality for self-management of diabetes [11]. Another survey conducted at an outpatient surgical facility in Trinidad indicated a lifetime prevalence of 86% among patients [12] for any healthcare issue.\nThis study was undertaken to assess the extent of use of herbal remedies by asthmatic patients attending a specialty chest clinic in Trinidad for symptomatic relief and to determine the factors influencing the patient's decision to use herbs.\n\nMethods\nThe study was approved by the Ethics Committee of the Faculty of Medical Sciences, University of the West Indies, St. Augustine campus and permission to interview patients was granted by the Director of the Chest Clinic of the Ministry of Health, Trinidad and Tobago. The study was conducted over the two-month period June to July 2003.\nSample and setting\nThe Chest Clinic was chosen as the source of subjects as this is the only national tertiary level health facility specializing in the management of respiratory diseases. Patients entering the study were physician-diagnosed asthmatics based on self-reporting symptoms of wheezing, chest tightness and nocturnal coughing in the previous year. Patients were recruited by consecutive sampling and the nature and purpose of the study were explained on an individual basis. Those confirming their willingness to participate signed their informed consent and were interviewed using a de novo, pilot-tested, researcher-administered questionnaire.\n\nInterview instrument\nThe questionnaire assessed demographic data such as age, gender, ethnicity, residential district, education, employment and socioeconomic status. Subjects reported their disease severity as intermittent, moderate or severe as determined by the Global Initiative for Asthma (GINA) guidelines with respect to symptom frequency [4]. Patients also reported their use of herbal remedies, identified the herbs used, the frequency of use, source of herbal medicines and the reasons for the use of herbs.\n\nStatistical analysis\nThe sample size was calculated as 185 patients assuming a prevalence of 86% [13] with a confidence level of 95%. Since all variables were categorical, χ2 tests were performed to determine whether there were statistically significant associations between the use of herbs and these variables. The p value was set at <0.05 for statistical significance. The data was analyzed using SPSS for Windows (Version 9.0, Chicago, IL).\n\n\nResults\nDemography\nDuring the study period one hundred and ninety one patients consented to participate. The demographic details of the sample are given in Table 1. Patients between 35 and 64 years of age formed the largest portion of the sample (62.3%). There was a significant gender difference with females outnumbering males by a 2:1 ratio, p < 0.01. Most patients were of Asian Indian origin (58.1%) and resided in suburban areas (60.2%). There was a high level of unemployment (30.4%); this could be correlated to primary schooling (seven or less years of formal education) being the highest educational level attained in 52.9% and no formal schooling in 5.2% of the sample population. Income was low, with 42.9% of the sample population earning below US$4,000 per year.\n\nAntiasthmatic drug use\nThe GINA guidelines were recently adopted in the Caribbean and asthmatic patients are currently treated according to their symptom severity. In our sample population, particularly in patients with moderate and severe symptoms, corticosteroids (controllers) and β2-agonists (relievers) were prescribed at very high rates, Table 2. Almost 90% of all patients with moderate symptoms were prescribed drugs in these classes. Almost all patients with severe symptoms were prescribed β2-agonists. This high level of prescription and use of β2-agonists suggest a lack of symptomatic control in our sample population. Theophylline and anticholinergics were prescribed in both categories of patients, but to a lesser extent.\n\nFactors influencing the use of herbal remedies\nGender, age, ethnicity, residential district, employment status, income and asthma severity had no statistically significant effect on the use of herbal remedies within the sample population, Table 3. However, almost two-thirds (62.5%) of patients with tertiary education used herbal remedies for asthma, p = 0.025.\n\nCharacteristics of patients using herbal remedies\nMost patients (70.7%) using herbs were advised by a relative or friend and only 10.3% sought the advice of an herbalist, Table 4. A cultural/traditional basis was the reason for herbal remedy usage in twenty-one (36.2%) patients and another twelve (20.7%) patients used herbs because they felt that were either 'natural' or 'healthy'. Twelve (20.7%) patients used herbs because they believed that their physician-prescribed allopathic medicines were not working.\nMost patients (58.6%) obtained their herbs or medicinal plants from either their backyards or the supermarket. Only fourteen (24.1%) obtained their herbal supplies from an herbalist, herbal shop or pharmacy. Seventeen (29.3%) of these patients reported using herbs within the last week and most these patients (60.3%) used herbs within the last six months.\nMany of these patients were using both physician-prescribed antiasthmatic drugs and herbal remedies, Table 5. No patient with either moderate or severe symptoms indicated that herbal remedies alone were sufficient to relieve symptomatic episodes. It is interesting to note that most patients with moderate symptoms (57.1%) believed that concurrent use of conventional medications and herbs gave better symptomatic relieve. One the other hand, most patients with severe symptoms (53.8%) believed that physician-prescribed medications worked better than herbal remedies, while 23.1% believed that neither relieved their symptoms.\n\nHerbs used in asthma\nMost patients in the sample used more than one medicinal herb simultaneously, which were usually prepared and administered as mixtures in teas. Almost one in four patients using medicinal herbs (22.5%) used either garlic (Allium sativum) or ginger (Zingiber officinale) for symptomatic relief of asthma, Table 6. Aloes (Aloe vera) shandileer (Leonotis nepetifolia), wild onion (Hymenocallis tubiflora), pepper (Capsicum spp.) tulsi (Ocimum gratissimum), black sage (Cordia curassavica), shadon beni (Eryngium foetidium), lemongrass (Cymbopogon citratus) and nutmeg (Myristica fragrans) were the more popular traditional indigenous West Indian medicinal plants used. Two patients reported using marijuana (leaves and roots). Herbs of European and North American origin, identified as Echinacea (Echinacea purpurea), Golden Seal (Hydrastis canadensis) and Chamomile (Matricaria chamomilla) were less frequently used. Five patients reported using trade name imported tablets for asthma.\n\nEffect of income and education on the use of herbs\nPatients using easily accessible herbs such as ginger (Zingiber officinale) and aloes (Aloe vera), and traditional indigenous medicinal herbs such as shandileer (Leonotis nepetifolia) and tulsi (Ocimum gratissimum) were more likely to be earning less than US$12,000, Table 7. Herbs of European or North American origin (Echinacea purpurea and Matricaria chamomilla) were more likely to be used by patients earning in excess of US$12,000 per annum. Income did not affect the use of either garlic or cocoa onion.\nAloes (Aloe vera), tulsi (Ocimum gratissimum) and golden seal were preferred in patients with at least twelve years of formal education, Table 7. Garlic and Echinacea were the preferred herbal medicines in patients with more than twelve years formal education. Educational level did not affect the patients' decision to use shandileer (Leonotis nepetifolia), wild onion (Hymenocallis tubiflora) or ginger (Zingibe officinale).\n\n\nDiscussion\nThis is the first study of its kind in the Caribbean to assess the use of medicinal herbs by asthmatic patients attending a specialty care clinic. The findings of this study are instructive as the use of medicinal herbs for self-medication in disease management has far reaching implications on the quality of healthcare delivery [14]. We report a prevalence of 30.4% in our patient sample, which is significantly higher than that in the UK, Denmark, Singapore and in the US [15-18].\nMost patients using medicinal herbs relied on the advice of relatives and friends as their sole source of information, as were caregivers of children in a US study [19]. We suggest that this information on the use of medicinal plants could have come from traditional/cultural knowledge, anecdotal evidence or from the greater public awareness through information networks such as the internet on the potential medicinal benefits of herbs. Asthma is an emerging chronic disease in the Caribbean and we suggest that the traditional knowledge in this area may be relatively 'new' and exist in relation to other diseases affecting the respiratory tract, such as cough, the common cold and the flu. This may be one of the reasons for the low prevalence of use of herbs in elderly asthmatic patients, as a strong traditional knowledge may not have existed.\nWe expected a higher prevalence of herbal use in individuals living in rural areas as these districts are depots for traditional knowledge as was reported in Jamaica where rural respondents used a larger variety of herbs than those living in urban areas [10]. As suggested earlier, we suspect that due to the recent emergence of asthma as a chronic disease in the Caribbean it is reasonable to expect that traditional knowledge in the management of this disease is not strong and our results are indicative of this.\nWe suspected that employment status could have predicted the use of herbs, however, this was not the case in our study sample. Unemployed patients did not improvise more in their use of herbal remedies than those in other income groups, even though most of the herbs used were relatively common, readily available and cheap. The low socioeconomic status of the majority of the sample may have prohibited both consultation with qualified herbalists and the purchase of imported, processed herbs that would have incurred additional out-of-pocket expense to the patient. What we noted was that there was no difference in the use of herbs across the income ranges and that in fact, patients earning relatively modest annual incomes between $US12,000 and $US19,999 were most likely to use herbs, although this did not reach statistical significance.\nAttaining a higher education positively influence the decision to use herbs. We suggest that in the absence of traditional knowledge regarding the medicinal use of herbs for asthma, a higher educational level may predispose an individual to greater access to general knowledge, especially with greater exposure to the internet and other sources of information, and this could be a factor in positively influencing the individual's decision to use medicinal herbs. The availability of scientific evidence-based information on the efficacy of herbs for diverse healthcare problems may be particularly significant in patients with the resources to avail themselves to such information, particularly those with higher educational and income levels. This is particularly true for garlic and Echinacea, which have been extensively researched and furthermore patients with higher educational and income levels would be more likely be at an advantage to access information via literature or on the world wide web regarding the use of these medicinal plants.\nPatients using imported, processed, and obviously more expensive herbal medications were on the higher end of the socioeconomic scale and were more likely to afford these medications. It was also observed that garlic and Echinacea were the herbs of choice in patients with higher educational levels. These herbs have a long tradition of use and are widely researched in Europe and North America. The traditional use and strong scientific evidence to support their therapeutic efficacy could be important factors influencing the patient's decision. It has been suggested elsewhere that patients with higher educational levels also tend be more involved in the management of their health; they tend to self-medicate or even suggest to their physicians the course of therapy.\nAlthough one in five patients using medicinal herbs stated that \"conventional medicines were not working\" as the reason for using this alternative healthcare modality, we noted that asthma severity does not affect the decision to use herbs. In previous studies, poor management was associated with non-compliance with prescribed pharmacotherapy and poor inhaler technique [5,6].\nThe backyard and home garden were major sources of readily available herbs such as aloes, shadon beni and lemongrass. Wild growing 'weeds' such as shandileer, tulsi, cocoa onion and black sage were also identified. The supermarket was a major source of inexpensive common medicinal herbs such as garlic, ginger and nutmeg. The identification of these medicinal herbs provides an opportunity to investigate West Indian plants used to treat asthma to determine whether they possess pharmacological properties. Scientific investigations have shown that some of these herbs possess pharmacological and anti-inflammatory properties, and these may be useful in suppressing the characteristic exaggerated immune response in asthma [20-24]. Pepper and bayleaf have also been shown to exhibit anti-inflammatory properties [[25,26]27]. There is an imperative to commence scientific investigations on traditional West Indian medicinal plants to determine their therapeutic efficacy and safety.\nThe survey instrument specifically asked questions on the use of medicinal herbs in asthma and did not inquire about the use of herbs as customary teas or tonics. We therefore did not determine lifetime prevalence for the use of herbs in our patient sample, but we suppose that had this been included that there might have been a prevalence similar to those reported in the Jamaica [10,11] and Trinidad [13] surveys. The survey was also limited in that by electing to conduct the study at a public health facility we obviously had a bias towards patients at the lower rung of the socioeconomic ladder, with lower income and educational status. As a consequence, the results reflected patients from this demographic background. We may have expected a different outcome in asthmatic patients attending private institutions, where their characteristics would have been slightly different, as we noted that even in our sample the small number of persons with higher income and educational status tended to use more medicinal herbs for symptomatic relief.\nWe did not assess whether patients informed their attending physician at the clinic about their use of herbs or determined whether the knowledge or attitudes of these physicians regarding the use of herbs influenced the patients' decision to use herbs. The study was also limited in that we did not ascertain the out-of-pocket expense for herbal remedies by patients, although most stated that herbal medicines (which we supposed were processed, imported products) were more expensive than conventional medicines. We assumed that an additional expense would have only been incurred by those patients purchasing processed, imported herbs obtained from a herbalist, herbal shop or pharmacy (24.1%) and who actually consulted a herbalist (10.3%). We also reasoned that since all the other herbs used were inexpensive and available from either the backyard garden or supermarket (58.6%) that the cost to patients selecting these remedies was minimal.\n\nConclusions\nThe findings of this study are important in that local medicinal plants in Trinidad have been identified in the self-management of asthma in a significant number of patients attending the specialty clinic. These identified herbs can now be targeted for scientific investigation to determine whether their pharmacological efficacy will assist in the development of viable healthcare alternatives in a developing country. These findings are also important for policymakers in the health sector who are given the mandate to regulate issues pertaining to the public's health. We are also becoming more aware of the potential for critical interplay between herbs and drugs when taken concomitantly to produce life-threatening interactions. Since herbs are here to stay and patients will continue to self-medicate with increasing frequency, it is imperative that healthcare providers become more knowledgeable on this modality and keep abreast with the latest developments in herbal therapy.\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\nAuthors' contributions\nYNC was the P.I. in this study. He was responsible for the study concept, development of methodology, coordinating the research activities, analyzing the data, and writing the manuscript. AFW was responsible for data input and analysis. DA was involved in methodological development, data collection, data input and analysis and presentation at regional conference. RC was involved in methodological development, data collection, data input and analysis. NW was involved in methodological development, data collection and input. RM was involved in methodological development, data collection and input. OS was involved in methodological development, data collection and input. DW was involved in methodological development, data collection and input. All authors read and approved the final manuscript.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\n\n" ], "offsets": [ [ 0, 21898 ] ] } ]

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[ { "id": "pmcA2646695__text", "type": "Article", "text": [ "A life threatening uterine inversion and massive post partum hemorrhage caused by placenta accrete during Caesarean section in a primigravida: a case report\nAbstract\nBackground\nA 32-year-old Caucasian primigravida was admitted for elective Caesarean Section at 36 weeks and 6 days with the diagnosis of preeclampsia.\n\nCase presentation\nTraction of the umbilical cord after delivery of a healthy baby resulted in uterine inversion. The placenta was found to be densely adherent to the posterior uterine wall. Piecemeal excision of the placenta as close as possible to the uterine lining was then performed.\n\nConclusion\nIn this way we were able to control a massive post partum hemorrhage and preserve the fertility of the patient.\n\n\n\nBackground\nPlacenta accreta is defined as abnormal adherence, either in whole or in part of the afterbirth to the underlying uterine wall. Placenta accreta and other pathological placentations (such as increta, percreta) are rare complications of pregnancy with potential life threatening and fertility threatening consequences. The incidence of placenta accreta has increased ten times over the last fifteen years, which reflects the increase in the rate of Caesarean Sections (CS) [1]. Placenta accreta has become the most important cause of peripartum hysterectomy. A life threatening acute uterine inversion and massive PPH can be caused by placenta accreta during CS but seldom in a primigravida (Figure 1).\n\nCase presentation\nA 32 year old, primigravida, was admitted in a District General Hospital for elective Caesarean section at 36 weeks and 6 days with the diagnosis of preeclampsia. She had two antenatal ultrasound examinations showing a healthy fetus and posterior fundal placenta.\nThe patient had lower segment CS of a healthy male infant under spinal anesthesia. The placenta was found to be densely adherent to the posterior uterine wall. Traction of the umbilical cord was applied and subsequently resulted in uterine inversion.\nThe placenta was removed by 'piecemeal' excising as close as possible to the uterine lining. About 80% of the placental tissue was removed until the uterine inversion was corrected. The uterus was closed in two layers. Two intra-abdominal drains were sited. The estimated blood loss was 2.5 litres and five units of blood were transfused together with 2 units of FFP during intra-operative and post-operative period. In addition, the patient was treated with intravenous oxytocin infusion, pr misoprostol and antibiotics.\nOn the second post partum day, vaginal Doppler ultrasound scan showed significant amount of placental tissues with increased vascularity measuring 2.7 × 6.6 × 6.8 cms within the endometrial cavity (Figure 2).\nThe patient was discharged on the fifth post-operative day with a conservative management. A follow up Ultrasound scan after two weeks showed reduction of placental mass (Figure 3). In addition, there was significant decrease in serum beta HCG levels from 2300 u/L on day 1 to 13 u/L at four weeks post operatively. The patient remained with minimal vaginal bleeding without abdominal pain. She had two normal periods after stopping breastfeeding and was feeling well. She was discharged from the early pregnancy unit.\n\nDiscussion\nA life-threatening uterine inversion can be rarely caused by placenta accreta [2]. Placenta accreta classically presents with retained placenta and hemorrhage. The association between uterine inversion and placenta accreta is unclear, however, strong traction on the umbilical cord with fundal placenta, excessive fundal pressure, relaxed uterus, short umbilical cord, uterine anomalies and antepartum use of magnesium sulphate are known associated factors [2]. Uterine inversion and retain placenta accreta can both be fatal complications [3].\nIn the case described the placenta accreta was complicated by uterine inversion and subsequent massive post partum hemorrhage, significantly increasing the risk of maternal mortality. Massive post partum hemorrhage is a major cause of maternal mortality in the United Kingdom (why women die latest report) [4].\nIn this case, the placenta was clamped as close to the uterine cavity as possible and cut. The base of the placenta was overrun with haemostatic sutures and this was repeated until as much of the placenta as possible was removed (Figure 4). Placental removal enabled correction of uterine inversion.\nDespite the many conditions associated with uterine inversion risk assessment is often lacking making the condition usually unexpected at the time of presentation. The association between abnormal placentation such as placenta accreta and uterine inversion is well supported [2]. Therefore, we advocate antenatal evaluation and risk assessment for placenta accreta [5]. Prenatal Ultrasound reported sensitivity of 94% and specificity of 79% for placenta accreta, but offer no more than provisional diagnostic probability statement [6]. Moreover, because 45% of placenta accreta cases were not detected by ultrasound, it is important to consider avoiding manual removal of placenta if there were intraoperative signs of accreta [6]. If clinically or sonographically the patient is suspected antenatally to be at risk of placenta accreta, appropriate management options should be considered, such as attempted conservative management or hysterectomy and counseling provided about potential sequelae [6]. The traditional management is abdominal hysterectomy, but this operation terminates fertility and may have devastating psychological effects. However, in correct circumstances, a conservative approach may be suitable. Conservative management of abnormally invasive placentation can be effective and fertility can be preserved. It should be only considered in highly selected cases when blood loss is minimal and there is wish for fertility preservation [7].\nFor women who want to preserve their fertility the placenta should be left intact if possible after caesarean delivery as this approach lowers the risk of subsequent hysterectomy from 85% to 15%. For women who have completed their family, hysterectomy with placenta left in situ is preferable to lower the maternal morbidity rates [6].\nThis case report involved conservative management. Peripartum hysterectomy was avoided and the aim was to preserve fertility. Prophylactic antibiotics, post partum oxytocics and the use of misoprostol post operatively helped to prevent further post partum hemorrhage.\nWhen a patient isinitially opted for conservative management, the possibility of recurrence should be discussed [8]. Furthermore, placentation should be carefully monitored for recurrence in any subsequent pregnancy, particularly if the placenta is located at the same site as the previous placenta accreta.\nConservative treatment for placenta accreta may be an alternative procedure in some selected cases.\n\nConclusion\nWe suggest an alternative approach for managing uterine inversion caused by placenta accreta that involved conservative management. This way hysterectomy was avoided and fertility was preserved.\n\nAbbreviations\nCS: Caesarean Section; FFP: Fresh Frozen Plasma\n\nConsent\nWritten informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor – in Chief of this journal.\n\nCompeting interests\nThe authors declare that they have no competing interests.\n\nAuthors' contributions\nAll authors have made substantial contribution to concept this case report.\n\n\n" ], "offsets": [ [ 0, 7543 ] ] } ]

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[ { "id": "pmcA2538543__text", "type": "Article", "text": [ "Personal and environmental correlates of active travel and physical activity in a deprived urban population\nAbstract\nBackground\nEnvironmental characteristics may be associated with patterns of physical activity in general or with particular types of physical activity such as active travel (walking or cycling for transport). However, most studies in this field have been conducted in North America and Australia, and hypotheses about putative correlates should be tested in a wider range of sociospatial contexts. We therefore examined the contribution of putative personal and environmental correlates of active travel and overall physical activity in deprived urban neighbourhoods in Glasgow, Scotland as part of the baseline for a longitudinal study of the effects of opening a new urban motorway (freeway).\n\nMethods\nWe conducted a postal survey of a random sample of residents (n = 1322), collecting data on socioeconomic status, perceptions of the local environment, travel behaviour, physical activity and general health and wellbeing using a new 14-item neighbourhood rating scale, a travel diary, the short form of the International Physical Activity Questionnaire (IPAQ) and the SF-8. We analysed the correlates of active travel and overall physical activity using multivariate logistic regression, first building models using personal (individual and household) explanatory variables and then adding environmental variables.\n\nResults\nActive travel was associated with being younger, living in owner-occupied accommodation, not having to travel a long distance to work and not having access to a car, whereas overall physical activity was associated with living in social rented accommodation and not being overweight. After adjusting for personal characteristics, neither perceptions of the local environment nor the objective proximity of respondents' homes to motorway or major road infrastructure explained much of the variance in active travel or overall physical activity, although we did identify a significant positive association between active travel and perceived proximity to shops.\n\nConclusion\nApart from access to local amenities, environmental characteristics may have limited influence on active travel in deprived urban populations characterised by a low level of car ownership, in which people may have less capacity for making discretionary travel choices than the populations studied in most published research on the environmental correlates of physical activity.\n\n\n\nBackground\nUntil recently, research on correlates of physical activity was dominated by studies of individual demographic and psychosocial characteristics [1]. This reflected an emphasis on promoting sport, recreation or health-directed exercise using techniques to encourage individual behaviour change [2]. However, there is little evidence that such approaches are effective in increasing physical activity in the medium-to-long term [3]. If habitual patterns of behaviour are environmentally cued, sustained change is likely to require a supportive environment in which people can be active [4,5]. There is therefore increasing interest in the influence of the social and physical environment on physical activity.\nWith respect to the physical (natural or built) environment, a growing body of evidence suggests that certain environmental characteristics may be associated with patterns of physical activity in general or with particular types of physical activity such as walking or cycling as modes of transport [4-10]. Among the correlates most frequently identified in such reviews – some ascertained using 'objective' measures, and others in terms of people's perceptions – are the aesthetic quality of the surroundings, the presence of pavements (sidewalks), the convenience of facilities for being active, the availability of green space, access to amenities (destinations) within walking or cycling distance, safety from traffic and personal attack, and the lack of heavy traffic. Some of these local characteristics reflect higher-order aspects of urban design and spatial policy such as population density, connectivity and mixed land use [6,8]. Importantly, different characteristics may be associated with different types of physical activity; for example, Owen and colleagues found that the aesthetic quality of the surroundings was associated with walking for exercise or recreation and with walking in general, but not with walking for transport, whereas perceptions of traffic were associated with walking for transport and walking in general, but not with walking for exercise or recreation [5].\nDespite the growing volume of published studies in this field, many authors remain circumspect in their interpretation of the available evidence. Giles-Corti and Donovan have described access to a supportive physical environment as a necessary, but insufficient, condition for an increase in physical activity in the population [11], while Handy found 'convincing' evidence of an association between physical activity and the built environment in general but 'less convincing' evidence as to which specific environmental characteristics were most strongly associated [7]. One limitation of the available evidence is that most research has been conducted in North America and Australia [9,12], and it is not clear whether associations observed in those countries are generalisable to other settings with different aggregate socioeconomic characteristics (e.g. wealth or access to private cars) or environmental characteristics (e.g. climate, patterns of land use, or availability of public transport). For example, North American researchers are often interested in the presence or absence of pavements (sidewalks), but it is unusual for streets in the United Kingdom (UK) not to have a pavement or footpath beside them. Hypotheses about putative environmental correlates of physical activity therefore need to be tested in a wider range of settings.\nA more profound limitation of the available evidence is that identifying a relationship between, for example, urban form and walking for transport is not the same thing as showing that changing the built environment will lead to a change in behaviour [13]. Few researchers have taken up the opportunity (or challenge) presented by 'natural experiments' to investigate the effects of environmental interventions on physical activity [14]. We therefore established a longitudinal study to examine changes associated with the opening of a new urban section of the M74 motorway (freeway) currently under construction in Glasgow, Scotland. The rationale and design for this study have been described previously [15]. It is claimed that the new motorway, which will mostly pass through or close to densely-populated urban neighbourhoods, will contribute to the regeneration of a region which includes some of the most deprived and least healthy working-class communities in Europe [16]. It is also claimed that the new motorway will divert traffic from local streets, reduce traffic noise and bring new local employment opportunities, thereby improving characteristics of the local environment held to be associated with active travel. Others claim that the new motorway will encourage car use, degrade the aesthetic quality of the surroundings and reduce the safety and attractiveness of routes for pedestrians and cyclists across the line of the motorway – all changes which may be expected to discourage active travel [15]. The eventual aim of the M74 study will be to assess the effects of this major modification to the urban built environment and transport infrastructure on perceptions of the local environment and on population health and health-related behaviour, the primary outcome of interest being a change in the quantity of 'active travel' (walking and cycling for transport).\nIn this paper, we report findings from the cross-sectional (baseline) phase of the study which contribute evidence on the environmental correlates of physical activity in this comparatively deprived urban population. We focus on two specific hypotheses: first, that levels of active travel and overall physical activity vary with demographic and socioeconomic characteristics, but not necessarily in the same way; second, that these relationships may be partly explained by the perceived characteristics of the local environment in which people live and by their objectively-assessed proximity to motorway and major road infrastructure.\n\nMethods\nDelineation of study areas\nWe used spatially referenced census and transport infrastructure data held and analysed in a geographical information system (GIS), combined with field visits, to delineate three study areas in Glasgow with similar aggregate socioeconomic characteristics and broadly similar topographical characteristics apart from their proximity to urban motorway infrastructure (Table 1, Figure 1). All three study areas extended from inner mixed-use districts close to the city centre to residential suburbs, contained major arterial roads other than motorways, and contained a mixture of housing stock including traditional high-density tenements, high-rise flats and new housing developments (Figure 2).\n\nSampling and survey administration\nWe used the Royal Mail Postcode Address File (PAF) (version 2005.3) to identify all residential addresses whose unit postcode (zip code) was within one of the study areas (total n = 35601) and drew a random sample of 3000 households from each area. Unit postcodes (e.g. G12 8RZ) are the smallest available unit of postal geography in the UK; residential unit postcodes cover about 15 addresses on average. We sent the survey to all households (total n = 9000) between 28 September and 4 October 2005 and resent the survey to all non-responding households between 26 and 31 October 2005. We alerted households to the survey by means of a postcard sent a few days in advance, used coloured paper for some of the survey materials, and posted survey packs in white envelopes printed with the university crest; these techniques have been shown in a meta-analysis to be associated with increased response rates to postal surveys [17]. We asked householders to ensure that the questionnaire was completed by a resident aged 16 or over; if more than one resident was eligible, we asked householders to select the person with the most recent birthday. Respondents who consented to follow-up were entered into a prize draw to win a £50 (€63; US$92) gift voucher. Responses received more than three months after the first mailing wave were disregarded in analysis.\n\nData collection\nThe questionnaire included items on demographic and socioeconomic characteristics, health and wellbeing (including the the SF-8 scale), perceptions of the local environment, travel behaviour and the short form of the International Physical Activity Questionnaire (IPAQ) (Additional file 1). We developed a new 'neighbourhood scale' to assess perceptions of relevant characteristics of the local environment (aesthetics, green space, access to amenities, convenience of routes, traffic, road safety and personal safety). The development, principal components analysis and reliability of the items in this scale and the derivation and reliability of summary variables are reported in an accompanying paper [18].\n\nData cleaning and derivation of variables\nDemographic and socioeconomic characteristics\nWe excluded from analysis all respondents who failed to enter their age or sex. We then examined the distributions of all raw variables and carried out range and consistency checks to identify any anomalous values or variables with a high proportion of missing responses. As a consequence, we collapsed responses on distance to place of work or study, housing tenure, car access and working situation into fewer categories by merging categories with small numbers of responses; we also disregarded household composition and working situation of spouse or partner in analysis because of the large numbers of missing values for these variables.\n\nHealth and wellbeing\nWe calculated body mass index (BMI) by converting, where necessary, self-reported heights and weights from imperial to metric units and dividing the height in metres by the square of the weight in kilograms; we also categorised respondents into quintiles of BMI. We calculated physical (PCS-8) and mental (MCS-8) health summary scores from the SF-8 data and scaled these to population norms using the method and coefficients given in the SF-8 manual [19].\n\nObjective environmental characteristics\nWe linked each record to the unit postcode of residence. We then constructed concentric buffers at 100-metre intervals up to 500 metres around the routes and access points of existing and planned motorways and around the network of other major (A- and B- class) roads, and assigned each respondent to a category of proximity to each type of road infrastructure (within 100 metres, 101–200 metres, etc.) based on the location of the centroid of their unit postcode.\n\nTravel behaviour\nFor travel time analysis we included travel diaries which recorded no travel at all, but we disregarded travel data from respondents who had not been at home on the day of the travel diary, whose questionnaire had been misprinted such that the travel diary pages were unusable, who had recorded journeys without reporting valid quantitative data on the durations of those journeys, or whose completed travel diary appeared implausible. We also disregarded journeys whose purpose was not stated or was beyond the scope of the travel diary (Additional file 1, page 8). We summed the reported travel time for each mode of transport, calculated a total travel time by active modes (walking plus cycling) and by all modes combined, and calculated the proportion of total travel time contributed by each mode of transport.\n\nPhysical activity\nWe cleaned and analysed IPAQ data in accordance with the IPAQ scoring protocol . We therefore disregarded physical activity data from respondents who had reported more than 16 hours of physical activity per day or who had missing or internally inconsistent data on the frequency or duration of any of the three categories of physical activity (walking, moderate-intensity activity or vigorous activity). We also recoded reported durations of activity of less than ten minutes to zero, and of greater than 180 minutes to 180 minutes. We calculated the estimated total physical activity energy expenditure for each respondent (MET-min/week) and used a combination of frequency, duration and total energy expenditure to assign each respondent to a 'high', 'moderate' or 'low' category of overall physical activity in accordance with the prescribed IPAQ algorithm. The 'high' category corresponds to a sufficient level of physical activity to meet current public health recommendations for adults [20].\n\n\nAnalysis\nWe considered it unlikely that the statistical assumptions required for linear regression could be met because the distributions of time spent walking and cycling and of estimated total physical activity energy expenditure were both strongly positively skewed and dominated by a large number of zero values which meant that the data were not amenable to log-transformation. We therefore modelled the correlates of active travel and physical activity using multivariate logistic regression. We defined 'active travel' as a binary condition achieved by any respondent who had reported at least 30 minutes of travel by walking, cycling or both in their travel diary, reflecting the current recommendation that adults should accumulate at least 30 minutes of moderate-intensity physical activity on most days of the week [20], and we defined 'physical activity' as a binary condition achieved by any respondent whose overall physical activity was categorised as 'high' using IPAQ. We then built separate multivariate models for active travel and physical activity following the method of Hosmer and Lemeshow [21], first including only 'personal' (individual or household) variables and then adding 'environmental' variables (Additional file 2).\n\n\nResults\nResponse\nWe received 1345 completed questionnaires. After subtracting from the numerator 23 completed questionnaires with missing critical demographic data (age or sex), and after subtracting from the denominator 676 addresses from which survey packs were returned as undeliverable, this left 1322 valid responses to be entered into analysis – a response rate of 1322/(9000-676) = 15.9%.\n\nCharacteristics of study participants\nDemographic and socioeconomic characteristics\nRespondents were aged between 16 and 89 years (median age 48 years). 804 (61%) were women. Only 136 (26%) of the men and 145 (18%) of the women reported having access to a bicycle. For those who usually travelled to a place of work or study, the median reported distance was 3.5 miles (about 5.5 kilometres). Other characteristics of study participants are summarised in Table 2.\n\nHealth and wellbeing\n25% of respondents reported difficulty walking for a quarter of a mile, 39% reported a long-term health problem or disability, and 50% were overweight (median BMI 25.1 kg/m2). The median mental health summary score (MCS-8) was significantly lower (i.e. poorer) than the population norm (median 47.3, 95% CI 46.4 to 48.1); the median physical health summary score (PCS-8) was not significantly different from the population norm (median 50.9, 95% CI 49.6 to 51.7).\n\n\nDescriptive data on travel behaviour and physical activity\nTravel behaviour\n1099 travel diaries were suitable for travel time analysis. Men and women were equally likely to have returned usable travel time data, but respondents who were older, retired, or living in social rented accommodation or who did not have access to a car were less likely to have returned usable data. On average, respondents recorded about an hour's travel per day (mean 61.5 minutes, median 50.0 minutes), of which a minority was spent using active modes of transport (walking or cycling: mean 20.0 minutes, median 10.0 minutes) (Table 3). 304 respondents (28%) recorded at least 30 minutes of active travel, of whom 294 (97%) recorded at least 30 minutes of walking.\n\nPhysical activity\n833 respondents returned complete physical activity data suitable for analysis. Women and respondents who were older, retired, or living in social rented accommodation or who did not have access to a car were less likely to have returned usable data. Respondents reported a mean of 318 minutes' walking per week and a mean estimated total physical activity energy expenditure of 3000 MET-minutes per week (Table 4). Only 316 respondents (38%) were categorised as having achieved a 'high' (i.e. sufficient) level of physical activity.\n\n\nCorrelates of active travel\nActive travel was significantly associated with being younger, living in owner-occupied accommodation, not having to travel more than four miles to work, having access to a bicycle, not having access to a car, and the absence of any difficulty walking. The final best model of the 'personal' correlates of active travel provided satisfactory goodness-of-fit (Hosmer and Lemeshow test: χ2 = 13.04, df = 8; P = 0.11) and explained nearly one-fifth of the total variance in active travel (Nagelkerke's R2 = 18.7%) (Table 5). Adding 'environmental' variables to the model showed an additional significant positive association between active travel and perceived proximity to shops, and an additional significant negative association between active travel and perceived road safety for cyclists. The final best model of the personal and environmental correlates of active travel also provided satisfactory goodness-of-fit (Hosmer and Lemeshow test: χ2 = 10.61, df = 8; P = 0.23) and explained slightly more of the total variance in active travel than did the personal model alone (Nagelkerke's R2 = 20.1%) (Figure 3).\nIn order to aid interpretation, we also partitioned the dataset into two strata ('No car available' and 'Car available') and refitted the final model separately to each stratum of the dataset (Table 6). This showed that the subset of respondents with no access to a car accounted for the significant overall relationship between active travel and access to a bicycle, whereas those with access to a car accounted for the significant overall relationships with distance to place of work or study and perceptions of the local environment. The relationship with difficulty walking was also stronger in this group than in those without access to a car.\n\nCorrelates of physical activity\nPhysical activity was significantly associated with living in social-rented accommodation, not being overweight, and the absence of any difficulty walking. The final best model of the 'personal' correlates of physical activity provided satisfactory goodness-of-fit (Hosmer and Lemeshow test: χ2 = 3.89, df = 7; P = 0.89) and explained about one-sixth of the total variance in physical activity (Nagelkerke's R2 = 15.9%) (Table 7). Adding 'environmental' variables to the model showed an additional significant negative association between physical activity and perception of traffic volume (i.e. respondents who perceived there to be a higher volume of traffic were more likely to report physical activity). The final best model of the personal and environmental correlates of physical activity also provided satisfactory goodness-of-fit (Hosmer and Lemeshow test: χ2 = 3.86, df = 8; P = 0.87) and explained slightly more of the total variance in physical activity than did the personal model alone (Nagelkerke's 16.6%) (Figure 3).\n\n\nDiscussion\nPrincipal findings\nIn this deprived urban population, the likelihood of reporting active travel was associated with being younger, living in owner-occupied accommodation, not having to travel a long distance to work and not having access to a car, whereas overall physical activity was associated with living in social-rented accommodation and not being overweight. After adjusting for individual and household characteristics, neither perceptions of the local environment nor the objective proximity of respondents' homes to motorway or major road infrastructure appeared to explain much of the variance in active travel or overall physical activity, although we did find a significant positive association between active travel and perceived proximity to shops.\n\nRepresentativeness and completeness of survey data\nOur difficulty in obtaining a representative sample of the resident population is not unique to our study. Although our final response rate was low, it was almost identical to that achieved in a recent population-based intervention study elsewhere in Glasgow [22]. Some of the challenges of recruiting research participants in areas of deprivation have been described elsewhere [23]; these are superimposed on a downward trend in participation in even the best-resourced national population surveys [24] and an upward (and socially biased) trend in opt-outs from the main alternative sampling frame, the edited electoral register [25]. Although our achieved sample contained a higher proportion of respondents from owner-occupied and car-owning households than predicted from 2001 census data for the same census output areas, these differences may be partly accounted for by an upward background trend in owner occupation and car access between 2001 and 2005. Our achieved sample is still clearly disadvantaged overall, in terms of socioeconomic and health status, compared with the country as a whole. It also contains sufficient heterogeneity to enable us to examine, in time, how the effects of the intervention are distributed between socioeconomic groups. We therefore consider our achieved sample fit for purpose.\nWe had to disregard a substantial proportion of cases in analysis because respondents had returned unusable travel time data or had returned physical activity data that were incomplete, internally inconsistent or included a 'Don't know' response and were therefore unacceptable according to the IPAQ scoring protocol. Most published studies using the same, short form of IPAQ have either not reported the distribution of the continuous summary measures or have not reported data for the UK separately from those for other countries where higher levels of physical activity are reported. Despite the high proportion of missing physical activity data in our dataset, however, the aggregate continuous data we obtained were broadly comparable to those reported in Rütten and colleagues' study of a random sample of UK adults [26]. We could have included more cases in physical activity analysis by, for example, imputing missing values, but the results would not have been comparable with others' owing to the substantial deviations from the scoring protocol which would have been required. The frequency of unusable responses was not reported in the international multi-centre study which originally established the validity and reliability of IPAQ [27]. It is possible that offering a 'Don't know' option in the self-completed IPAQ questionnaire encourages respondents to select this rather than to enter what may be a reasonably precise estimate of the actual time spent in physical activity; the respondent has no way of knowing that a single 'Don't know' response will result in all of their physical activity data being disregarded in analysis. This should be considered in any future revision of the IPAQ questionnaire and scoring protocol.\n\nContribution of active travel to overall physical activity\nThe explanatory variables that were significantly associated with active travel but not with physical activity (distance to place of work or study, access to a bicycle, access to a car, perceived proximity to shops, and perceived road safety for cyclists) all have an obvious intuitive relationship with the use of walking or cycling as modes of transport. That they were not significantly associated with overall physical activity suggests either that active travel contributes only a minority of respondents' overall physical activity or that other factors not measured in this study are more important correlates of overall physical activity than those which determine active travel. A crude comparision of the quantity of active travel reported in the one-day travel diaries with the quantities of physical activity reported using IPAQ suggests that on average, active travel may indeed make only a small (~15%) contribution to overall physical activity in this study population. However, the real contribution may be substantially greater than this if, as has been shown previously, respondents tend to over-report their physical activity using IPAQ [28]. There can be little doubt that active travel makes a substantial contribution to the total quantity of walking reported in this study population. Irrespective of the true contribution of active travel to overall physical activity, however, it remains likely that other unmeasured personal and social factors beyond the scope of this study may be more important correlates of overall physical activity.\n\nSocio-spatial patterning of active travel and overall physical activity\nRespondents living in owner-occupied households were more likely to report active travel than those living in social-rented accommodation, but less likely to report sufficient overall physical activity. Since neither working situation nor perceived financial situation emerged as significantly associated with active travel or overall physical activity, housing tenure and car access are the remaining explanatory variables in this dataset which can be interpreted as markers of socioeconomic status. Although having access to a car clearly reflects the possession of a material asset, it has been argued that this is a less direct marker of socioeconomic status than some other markers because, in Scotland at least, access to a car is a more-or-less essential requirement for living in many rural areas, whereas it is possible to live in a dense urban settlement such as Glasgow without using a car. In the final models in this study, therefore, housing tenure may be regarded as the primary marker of socioeconomic status. The findings consequently suggest conflicting socioeconomic gradients in prevalence: more advantaged respondents were more likely to report active travel, but more disadvantaged respondents were more likely to report sufficient overall physical activity. The higher prevalence of sufficient overall physical activity among the more disadvantaged despite their lower propensity for active travel is likely to reflect higher quantities of physical activity in other domains, particularly occupational and domestic activities, since leisure-time physical activity tends to be higher among more advantaged groups [29].\n\nEnvironmental characteristics: paradoxical, unmeasured, or irrelevant?\nThe two environmental variables that emerged as significantly associated with active travel, particularly among those without access to a car, were perceived proximity to shops and perceived road safety for cyclists. The positive association with perceived proximity to shops suggests that for active travel to be undertaken in this population, it may be more important that people live close to the amenities they need than that they live in an environment with more favourable subjective or discretionary considerations such as attractiveness or noise. This would be consistent with an understanding that walking as a mode of transport is primarily a way of undertaking journeys which have to be made anyway, as opposed to more discretionary (recreational) forms of walking which may be more susceptible to the influence of less-structural characteristics.\nAlthough the negative association with perceived road safety for cyclists appears counter-intuitive, similar 'paradoxical inverse relationships' have been reported elsewhere, for example by Titze and colleagues in a study of the correlates of cycling among students [30] and by Humpel and colleagues in a study of correlates of walking for pleasure [31]. Titze and colleagues suggest that respondents who cycle regularly are more likely to be aware of, and report, the danger posed by traffic than non-cyclists or infrequent cyclists. A similar phenomenon could explain the negative association between physical activity and perception of traffic volume.\nOverall, the influence of the putative environmental characteristics examined in this study on active travel and physical activity appeared small compared with that of the personal characteristics found to be significant, and including environmental characteristics in the models did not substantially modify the influence of personal characteristics.\nOn the one hand, this could reflect an artefact of the research methods (a false negative error), which could have arisen in various ways. In particular, the 'wrong' environmental exposure may have been measured, in that the environmental characteristics examined were those of the immediate surroundings of respondents' homes, whereas the propensity to choose active modes of transport may be more strongly influenced by the characteristics of the environment elsewhere on their routes [30], for example the perceived danger of cycling in the city centre – an association which may be absent, or at least diluted, when the 'exposure' examined is limited to the residential environment. It could also be argued that the apparently weak influence of environmental characteristics in this study reflects a reliance on respondents' perceptions which have not been objectively verified and may therefore be a weak proxy for the 'true' objectively-measured characteristics of their surroundings. However, as recent reviews have pointed out, the current weight of evidence for objective environmental correlates of walking is no greater than that for subjective environmental correlates [5] and it is entirely plausible that people's perceptions of their environment may be at least as important as their objective conditions in influencing their behaviour [6].\nOn the other hand, we may have demonstrated a real absence of any major association. Although at first sight this appears at odds with the growing body of review-level evidence for environmental correlates of physical activity, Wendel-Vos and colleagues noted that of all the environmental factors examined in all the studies included in their review, analysis showed a 'null association' in 76% of cases [9], and our finding that personal factors account for a much larger proportion of the variance in active travel or physical activity than is accounted for by environmental factors is consistent with those of some other European studies [32,33]. In the particular context of this study, residents may simply have adapted to adverse conditions in their local environment in the ways identified by Hedges in a qualitative study of people living close to new roads built in the UK in the 1970s [34] – particularly by attitudinal adaptation, which Hedges characterises as developing an attitude that it is futile to resist. One can imagine that in the most deprived areas of Glasgow, people may have become resigned to the nature of their surroundings, seeing them as inevitable and not amenable to change either through environmental improvement or through their moving to another area.\n\n\nConclusion\nAfter demographic and socioeconomic characteristics were taken into account, neither perceptions of the local environment nor objective proximity to major road infrastructure appeared to explain much of the variance in active travel or overall physical activity in this study. Our study population may be both objectively constrained by their socioeconomic circumstances (including comparatively limited access to private cars) and adapted to living in conditions which others would consider to pose a barrier to active travel. Under these circumstances, environmental characteristics which have been found to influence discretionary active travel in studies in other, more affluent populations may simply be irrelevant in a population which is more captive in its travel choices. Environmental correlates of active travel should not be assumed to be generalisable between populations; researchers should continue to test hypotheses about putative environmental correlates in different settings, and policymakers should recognise that the effects of interventions to change the environment are likely to vary between populations and between socioeconomic groups within populations.\n\nCompeting interests\nThis paper is based on material contained in the first author's PhD thesis.\n\nAuthors' contributions\nDO had the original idea for the study, designed the study and the survey materials, applied for ethical approval, cleaned and coded the survey data, carried out all the geographical and statistical analyses and wrote the paper. MP was DO's PhD supervisor. RM, NM, MP and SP constituted the steering group for the study, contributed to and advised on the design of the study and the interpretation of the emerging findings, and contributed to the critical revision of the paper. All authors read and approved the final manuscript.\n\nSupplementary Material\n\n\n" ], "offsets": [ [ 0, 35350 ] ] } ]

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[ { "id": "pmcA2409186__text", "type": "Article", "text": [ "Comparative Genome Analysis of Filamentous Fungi Reveals Gene Family Expansions Associated with Fungal Pathogenesis\nAbstract\nFungi and oomycetes are the causal agents of many of the most serious diseases of plants. Here we report a detailed comparative analysis of the genome sequences of thirty-six species of fungi and oomycetes, including seven plant pathogenic species, that aims to explore the common genetic features associated with plant disease-causing species. The predicted translational products of each genome have been clustered into groups of potential orthologues using Markov Chain Clustering and the data integrated into the e-Fungi object-oriented data warehouse (http://www.e-fungi.org.uk/). Analysis of the species distribution of members of these clusters has identified proteins that are specific to filamentous fungal species and a group of proteins found only in plant pathogens. By comparing the gene inventories of filamentous, ascomycetous phytopathogenic and free-living species of fungi, we have identified a set of gene families that appear to have expanded during the evolution of phytopathogens and may therefore serve important roles in plant disease. We have also characterised the predicted set of secreted proteins encoded by each genome and identified a set of protein families which are significantly over-represented in the secretomes of plant pathogenic fungi, including putative effector proteins that might perturb host cell biology during plant infection. The results demonstrate the potential of comparative genome analysis for exploring the evolution of eukaryotic microbial pathogenesis.\n\nIntroduction\nFungi and oomycetes are responsible for many of the world's most devastating plant diseases including late blight disease of potato, caused by the oomycete pathogen Phytophthora infestans and rice blast disease caused by the ascomycete fungus Magnaporthe grisea, both of which are responsible for very significant harvest losses each year. The enormous diversity of crop diseases caused by these eukaryotic micro-organisms poses a difficult challenge to the development of durable disease control strategies. Identifying common underlying molecular mechanisms necessary for pathogenesis in a wide range of pathogenic species is therefore a major goal of current research. Approximately 100,000 species of fungi have so far been described, but only a very small proportion of these are pathogenic [1]. Phylogenetic studies have, meanwhile, shown that disease-causing pathogens are not necessarily closely-related to each other, and in fact are spread throughout all taxonomic groups of fungi, often showing a close evolutionary relationship to non-pathogenic species [2], [3]. It therefore seems likely that phytopathogenicity has evolved as a trait many times during fungal and oomycete evolution [1] and in some groups may be ancestral to the more recent emergence of saprotrophic species. A significant effort has gone into the identification of pathogenicity determinants– individual genes that are essential for a pathogen to invade a host plant successfully, but which are dispensable for saprophytic growth [4], [5]. However, far from being novel proteins encoded only by the genomes of pathogenic fungi, many of the genes identified so far encode components of conserved signalling pathways that are found in all species of fungi, such as the mitogen activated protein (MAP) kinases [6], adenylate cyclase [7] and G-protein subunits [8]. The MAP kinase pathways, for example, have been studied extensively in the budding yeast Saccharomyces cerevisiae and trigger morphological and biochemical changes in response to external stimuli such as starvation stress or hyperosmotic conditions [9]. In pathogenic fungi, components of these pathways have evolved instead to regulate the morphological changes associated with plant infection. For example, appressorium formation in the rice blast fungus Magnaporthe grisea, stimulated by hard, hydrophobic surfaces is regulated by a MAP kinase cascade [10]. This pathway deploys novel classes of G-protein coupled receptors not found in the genome of S. cerevisiae [11], but the inductive signal is transmitted via a MAP kinase, Pmk1, that is a functional homologue of the yeast Fus3 MAP kinase where it serves a role in pheromone signalling [10]. Similarly, conserved metabolic pathways such as the glyoxylate cycle and amino acid biosynthesis are also important for pathogenesis [12]–[14]. This may in some cases reflect the nutritional environment the pathogen encounters when growing in the host plant tissue, and in others shows the importance of simple metabolites for pathogenic processes, such as the role of glycerol as a compatible solute for generating turgor pressure in the appressorium of M. grisea [15]. It is undoubtedly the case, however, that identification of such genes has also been a consequence of the manner in which these studies have been carried out, often using yeast as a model organism to test hypotheses concerning the developmental biology and biochemistry of plant pathogenic species.\nOther pathogenicity factors identified to date have been shown to be involved in functions associated with host infection, such as plant cell wall degradation, toxin biosynthesis and protection against plant defences [reviewed in 5]. Identification of a pathogenicity factor generally involves making a mutant fungal strain with a non-functioning version of the gene by targeted gene deletion and assaying the ability of the mutant to cause disease. Therefore, most pathogenicity factors identified so far, have been validated in only a small number of genetically tractable pathogenic fungi, such as M. grisea and the corn smut Ustilago maydis and many of the advances in understanding the developmental biology of plant infection have occurred in these model pathogens [16], [17]. However, there are severe limitations to studying pathogenicity by mutating one gene at a time and working predominantly with a hypothesis-driven, reverse genetics approach. Many virulence-associated processes, for instance, such as the development of infection structures and haustoria, are likely to involve a large number of gene products and so there is likely to be redundancy in gene function. One example of this is cutinase, a type of methyl esterase that hydrolyses the protective cutin layer present on the outside of the plant epidermis. Cutinase was excluded as a pathogencity factor for M. grisea on the basis that a mutant strain containing a non-functional cutinase-encoding gene was still able to cause rice blast disease [18]. However, sequencing of the M. grisea genome has shown the presence of eight potential cutinase-encoding genes implicated in virulence [19]. Additionally, targeted gene deletion is not feasible in many important pathogens and the normal definition of fungal pathogenicity cannot be applied in the case of obligate biotrophs, such as the powdery mildew fungus Blumeria graminis, which cannot be cultured away from living host plants. Therefore, new approaches are needed to identify genes that are vital for the process of pathogenicity. These include high-throughput methods such as microarray analysis, serial analysis of gene expression (SAGE), insertional mutagenesis, proteomics and metabolomics [19], [20] and are dependent on the availability of genome sequence information.\nAfter the initial release of the genome of the budding yeast S. cerevisiae in 1996 [21], the number of publicly available sequenced fungal genomes has recently risen very quickly. A large number of fungal genome sequences are now publicly available, including those from several phytopathogenic fungi, including M. grisea [22], Ustilago maydis [23], Gibberella zeae [24] (the causal agent of head blight of wheat and barley), Stagonospora nodorum [25] (the causal agent of glume blotch of wheat), the grey mould fungus Botrytis cinerea and the white mould fungus Sclerotinia sclerotiorum [reviewed in 19]. Comparison of gene inventories of pathogenic and non-pathogenic organisms offers the most direct means of providing new information concerning the mechanisms involved in fungal and oomycete pathogenicity. In this report, we have developed and utilized the e-Fungi object-oriented data warehouse [26], which contains data from 36 species of fungi and oomycetes and deploys a range of querying tools to allow interrogation of a significant amount of genome data in unparalleled detail. We report the identification of new gene families that are over represented in the genomes of filamentous ascomycete phytopathogens and define gene sets that are specific to diverse fungal pathogen species. We also report the putatively secreted protein sets which are produced by plant pathogenic fungi and which may play significant roles in plant infection.\n\nResults\nIdentification of orthologous gene sets from fungal and oomcyete genomes\nGenome sequences and sets of predicted proteins were analysed from 34 species of fungi and 2 species of oomycete (Table 1). In order to compare such a large number of genomes, an object-oriented data warehouse has been constructed known as e-Fungi [26] which integrates genomic data with a variety of functional data and has a powerful set of queries that enables sophisticated, whole-genome comparisons to be performed. To compare genome inventories, the entire set of predicted proteins from the 36 species (348,787 proteins) were clustered using Markov Chain Clustering [27] as described previously [28], [29]. A total of 282,061 predicted proteins were grouped into 23,724 clusters, each cluster representing a group of putative orthologues. The remaining 66,934 sequences were singletons, the products of unique genes. A total of 165 clusters contained proteins from all 36 species used in this study (Table S1). Not surprisingly, they included many proteins involved in basic cellular processes, such as ribosomal proteins, components of transcription, translation and DNA replication apparatus, cytoskeletal proteins, histones, proteins involved in the secretory pathway, protein folding, protein sorting and ubiquitin-mediated proteolysis and enzymes involved in primary metabolism. Only 16 clusters contained proteins that were found in all 34 species of fungi, but which were absent from the two species of oomycete (Table S2). This number of fungal-specific clusters is surprisingly low considering the phylogenetic distance between the oomycetes and fungi [30]. The list however, is consistent with the fundamental differences in biology between fungi and oomycetes and included proteins involved in fungal septation, glycosylation, transcriptional regulation, cell signalling, as well as two amino-acyl tRNA synthetases. The obligate mammalian pathogen Encephalitozoon cuniculi, a microsporidian fungus, has a reduced genome that codes only for 1,997 proteins and lacks genes encoding enzymes of many primary metabolic pathways such as the tricarboxylic acid cycle, fatty acid β-oxidation, biosynthetic enzymes of the vast majority of amino acids, fatty acids and nucleotides, as well as components of the respiratory electron transport chain and F1-F0 ATP synthase. It also lacks mitochondria and peroxisomes [31]. Therefore, we reasoned that the inclusion of this species in the analysis of MCL clusters is likely to result in underestimation of the number of groups of conserved proteins. By discarding E. cuniculi, there are 377 clusters that contained proteins from 35 species of fungi and oomycetes (Table S3). This relatively small number of fungal-conserved clusters reflects the large evolutionary distance between members of the fungal kingdom, as well as complex patterns of gene gains and losses during the evolution of fungi. Basidiomycetes and ascomycetes are thought to have diverged nearly 1,000 million years ago [32] and the Saccharomycotina alone are more evolutionarily diverged than the Chordate phylum of the animal kingdom [33]. Since the divergence of Saccharomycotina (hemiascomycetes) and Pezizomycotina (euascomycetes), the genomes of the latter have greatly increased in size, partly due to the appearance of novel genes related to the filamentous lifestyle. Lineage-specific gene losses have also been shown in a number of hemiascomycete species [34]. As well as the groups of proteins mentioned above (Table S1), the fungal-conserved clusters included those containing enzymes from primary metabolic pathways not present in E. cuniculi, such as the tricarboxylic acid cycle, amino acid metabolism, fatty acid biosynthesis, cholesterol biosynthesis and nucleotide metabolism, as well as components of the respiratory electron transport chain and F1-F0 ATP synthase. The conserved protein clusters also include a number of transporters (including mitochondrial transporters), enzymes involved in haem biosynthesis, autophagy-related proteins, those involved in protein targeting to the peroxisome and vacuole and additional groups of proteins involved in signal transduction that are not present in E. cuniculi (including those involved in inosine triphosphate and leukotriene metabolism). The analysis also showed there were 105 clusters that contained proteins from 33 species of fungi (excluding E. cuniculi), but not from the two species of oomycete (see Table S4). As well as those mentioned previously (Table S2), the group includes a number of clusters of transporters that are conserved in fungi but not found in oomycetes, as well as proteins involved in fungal cell wall synthesis, and lipid metabolism. It may be the case that the genomes of oomycete species do not possess orthologues of the fungal genes in these clusters, or alternatively, the large evolutionary distance between the oomycetes and fungi mean that the corresponding orthologues from each Kingdom cluster separately.\n\nComparative analysis of yeasts and filamentous fungi\nOne striking difference in the morphology of species of fungi is between those that have a filamentous, multi-cellular growth habit and those that grow as single yeast cells. There is some overlap between these two groups; because some fungi are dimorphic or even pleiomorphic, switching between different growth forms depending on environmental conditions or the stage of their life cycle. For example, the corn-smut fungus Ustilago maydis can exist saprophytically as haploid yeast-like cells, but needs to form a dikaryotic filamentous growth form in order to infect the host plant [23]. Generally the genomes of the filamentous fungi contain more protein-encoding genes (9,000–17,000) than those from unicellular yeasts (5,000–7,000), perhaps reflecting their greater morphological complexity and secondary metabolic capacity. U. maydis, however, has 6,522 protein encoding genes, perhaps reflecting its lack of extensive secondary metabolic pathways and its potential usefulness in defining the minimal gene sets associated with biotrophic growth [23]. The increase in proteome size in filamentous ascomycetes may be due to the expansion of certain gene families or the presence of novel genes that are essential for the filamentous lifestyle.\nFor the purposes of this study, the filamentous fungi were defined as the filamentous ascomycetes (subphylum Pezizomycotina), basidiomycetes and zygomycetes and the unicellular fungi were defined as the budding yeasts (order Saccharomycetales), the archiascomycete Schizosaccharomyces pombe and the microsporidian fungus Encephalitozoon cuniculi. A total of 37 MCL clusters contained proteins from all species of filamentous fungi, but no species of unicellular fungi (Table 2). Interestingly, eight of these clusters also contained proteins from both species of oomycete represented in e-Fungi. The filamentous-fungal specific clusters included a number of proteins that are involved in cytoskeletal rearrangements (dedicator of cytokinesis protein, integrin beta-1-binding protein, dynactin p62 family, dynein light intermediate chain 2), it seems likely that these are required for the complex morphological changes that filamentous fungi undergo during their lifecycle and the production of differentiated cells, such as spores, fruiting bodies and infection structures. The results also suggest that filamentous fungal species make a greater use of lipids as signalling molecules than yeast species. For example, the occurrence of filamentous fungal-specific clusters representing two groups of lysophospholipases, as well as ceramidases that are involved in sphingolipid signalling [35] and linoleate diol synthases that can catalyse the formation of leukotrienes [36]. Interestingly, one of the products of linoleate diol synthase has been shown to be a sporulation hormone in Aspergillus nidulans [37]. There is also a cluster that represents homologues of a novel human gene (LRP16) that acts downstream of a steroid receptor and promotes cell proliferation [38]. Two clusters of filamentous fungal-specific proteins represent enzymes involved in molypterin biosynthesis (MCL2420, MCL2581). Molypterin is a molybdenum-containing co-factor for nitrate reductase, an enzyme that is known to be absent from the species of yeast used in this study [39]. Both these clusters are also found in oomycetes. There are other clusters representing proteins important for activities specific to filamentous fungi, such as homologues of Pro11 (striatin) which regulates fruiting body formation in Sordaria macrospora [40], the vegetatible incompatibility protein HET-E-1, which prevents the formation of heterokaryons between incompatible fungal strains in Podospora anserina [41], anucleate primary sterigmata protein A from Aspergillus nidulans, which is essential for nuclear migration and conidiophore development [42] and cytochrome P450 and polyketide synthase-encoding genes, both of which are involved in a number of secondary metabolic pathways including toxin biosynthesis [43].\n\nPathogenicity-associated gene functions in fungi\nAs the selected set of fungi includes both saprotrophic and pathogenic species, this allows us to compare the gene inventories of phytopathogenic and closely related non-pathogenic fungi to look for genes that are unique to phytopathogens. Analysis of MCL clusters showed that there were no clusters that contained proteins from all species of fungal phytopathogen in e-Fungi (namely B. cinerea, Eremothecium gossypii, G. zeae, M. grisea, S. sclerotiorum, S. nodorum and U. maydis) but did not contain proteins from non-pathogenic species. There were, however, four clusters that were exclusive to filamentous ascomycete phytopathogens (namely B. cinerea, G. zeae, M. grisea, S. sclerotiorum, S. nodorum as shown in Table 3). Significantly, none of the members of these clusters had homology to any known proteins or contained motifs from the Pfam database [44], so we were unable to predict their function, although two of the clusters (MCL4854 and MCL8229) consisted entirely of proteins that were predicted to be secreted. Taken together, the observations indicate that a battery of completely novel secreted proteins may be associated with ascomycete fungal pathogens.\nPathogenicity factors have been defined as genes that are essential for successful completion of the pathogen lifecycle but dispensable for saprophytic growth [4]. This is an experimental definition based on whether null mutations of a given gene reduce the virulence of the pathogen on its host. We wished to ascertain whether homologues of previously characterised and experimentally-validated pathogenicity factors were limited to the genomes of pathogenic species. A search was therefore made for pathogenicity factors that have been identified experimentally for the species of phytopathogens represented in e-Fungi using PHI-base, the plant-host interaction database [45]. The matching locus was identified for each pathogenicity factor in the corresponding genome sequence by comparing a published protein sequence with sets of predicted proteins for each genome using BLASTP. This produced a list of 105 pathogenicity factors, although corresponding loci could not be found in genome sequences for all the published genes (see Table S5). MCL clusters containing these proteins were identified (76 unique clusters) and the species distribution of members of these clusters analysed. In total, 29 of the MCL clusters contained pathogenicity factors with members from at least 34 of the 36 species represented in e-Fungi (Table 4). Not surprisingly, many of these clusters contain conserved components of signalling pathways such as protein kinases, adenylate cyclases, G-proteins and cell cycle regulators. Cellular morphogenesis is known to be important for infection of the host plant by many phytopathogens, for example, in appressorium formation in Magnaporthe grisea [46] or the switch in the growth form of Ustilago maydis from yeast-like growth to filamentous invasive growth [47]. Links between successful plant infection and cell cycle control have also been demonstrated [48]. It seems likely that conserved signalling pathways that control activities, such as mating and morphogenesis in all fungi, have evolved to control processes essential for pathogencity in phytopathogens. Other conserved pathogenicity factors encode enzymes of metabolic pathways that are present in nearly all fungi, but seem to be important for the life cycle of particular pathogenic species, for example, enzymes involved in beta-oxidation of fatty acids, the glyoxylate shunt, amino acid metabolism and the utilisation of stored sugars. When considered together, this may indicate that nutritional conditions which fungi encounter when invading host plant tissue require mobilisation of stored lipids prior to nutrition being extracted from the host plant. Seventeen of the MCL clusters containing pathogenicity factors were specific to filamentous ascomycetes (Table 5). These include a number of enzymes involved in secondary metabolism, such as those involved in the synthesis of the fungal toxin trichothecene in G. zeae [43] and those involved in melanin biosynthesis [49], as well as structural proteins, some of which are components of differentiated cell types not seen in yeasts, for example, hydrophobins which are components of aerial structures such as fruiting bodies [50] but are also involved in pathogenicity [16]. There also seems to be a number of filamentous ascomycete specific receptor proteins (transducin beta-subunit, G-protein coupled receptor, tetraspanins) that have evolved in pathogens to be used in sensing environmental cues that are essential for successful infection of the host [51]. The Woronin body is a structure found only in filamentous ascomycetes, and has been shown to be essential for pathogenicity in M. grisea [52]. A major constituent of the woronin body, encoded by MVP1, is a pathogenicity factor for M. grisea, but also has homologues in nearly all species of filamentous ascomycetes. Two proteins that were initially discovered as being highly expressed in the appressoria of M. grisea and essential for pathogenicity (Mas1 and Mas3) [53] also have homologues in a number of species of filamentous fungi (Table 5). Thus, many innovations that have allowed filamentous ascomycetes to have a more complex morphology than unicellular yeasts have also evolved to be essential for plant infection by phytopathogenic species. Interestingly, none of the MCL clusters containing known pathogenicity factors contained members only from phytopathogenic fungi, apart from those that were restricted to just one species. These are therefore likely to represent highly-specialised proteins that have evolved for the specific lifecycle of just one species of phytopathogen, for example the Pwl proteins involved in determining host range of different strains of M. grisea [54]. Two of the proteins specific to M. grisea, the metallothionein Mmt1 [55] and the hydrophobin Mpg1 [56] are small polypeptides and are members of highly divergent gene families, other members of which do not cluster together using BLASTP.\n\nComparative analysis of plant-pathogenic and saprotrophic filamentous ascomycetes\nBased on the analysis reported, it is likely that in general there are a large number of differences in gene inventories between filamentous and yeast-like fungi. Therefore, in order to compare the genomes of phytopathogens and saprotrophs, we focused on filamentous ascomycetes in order to resolve in greater detail the distinct differences in gene sets between these two ecologically separate groups of fungi. In this way differences due to phylogeny between the species would be minimised. We compared the gene inventories of the phytopathogens B. cinerea, G. zeae, M. grisea, S. sclerotiorum, S. nodorum with the non-pathogens Aspergillus nidulans, Chaetomium globosum, Neurospora crassa and Trichoderma reesei. Phylogenetic analysis suggests that the phytopathogenic species do not form a separate clade from the pathogenic species (Figure 1), [3] and we assumed that differences in gene inventory should therefore reflect lifestyle rather than evolutionary distance. In order for such a comparison to be considered valid, the completeness and quality of the fungal genome sequences used should, however, also be comparable. Table S6 summarises the available data about genome sequence coverage, genome size and the number of predicted proteins for each species. This shows that the genome coverage is greater than 5x and the number of predicted proteins in the range of 10,000–16,000 for all genomes used, suggesting a high level of equivalence between species with regard to sequence quality. From our work it seems unlikely that there are pathogenicity factors conserved in, and specific to, all species of phytopathogen. It may, for instance, be the case that differences in the gene inventories are due to the expansion of certain gene families in the genomes of phytopathogenic species associated with functions necessary for pathogenesis. To define protein families, we used the Pfam database which contains protein family models based on Hidden Markov Models [44], [57]. Sets of predicted proteins for each fungal species in e-Fungi were analysed for the occurrence of Pfam motifs and the number of proteins containing each domain across fungal species ascertained. The sets of predicted protein sequences used in this study have been automatically predicted as part of each individual genome project and are likely to contain a number of artefactual sequences. The use of Pfam motifs to define gene families in this study reduces the likelihood of such sequences affecting the data, since Pfam motifs are based on multiple sequence alignments of well-studied proteins.\nA small number of Pfam motifs were not found in the proteomes of the filamentous ascomycete non-pathogens, but were found in the proteomes of at least three species of filamentous ascomycete phytopathogens (Table 6). These include the Cas1p-like motif (PF07779), found in 4 species of phytopathogen, including five copies in G. zeae, and the Yeast cell wall synthesis protein KRE9/KNH1 motif (PF05390), which was found in three species of phytopathogen. Cas1p is a membrane protein necessary for the O-acetylation of the capsular polysaccharide of the basidiomycete animal pathogen Cryptococcus neoformans [58]. KRE9 and KNH1 are involved in the synthesis of cell surface polysaccharides in S. cerevisiae [59]. Taken together this suggests that synthesis of cell surface polysaccharides is important for phytopathogens, perhaps helping to shroud the fungus from plant defences. The function of the YDG/SRA domain motif (PF02182) is unknown, but is found in a novel mouse cell proliferation protein Np95, in which the domain is important both for the interaction with histones and for chromatin binding in vivo [60]. As well as domains of unknown function, the list of phytopathogen-specific Pfam motifs includes Allophanate hydrolase (PF02682) which is found in an enzyme involved in the ATP-dependent urea degradation pathway [61], a peptidase motif, an opioid growth receptor motif (PF04664) and Mnd1 (PF03962), which is involved in recombination and meiotic nuclear division [62].\nTo detect potential gene family expansion, we decided to identify Pfam motifs that were present in both phytopathogenic and non-pathogenic species of filamentous ascomycetes, but that were more common in the genomes of the former. The Pfam motifs were ranked on the ratio of the mean number of proteins containing each motif in phytopathogens, when compared to non-pathogens (Table 7). The tables only show ratios of greater than or equal to 2.5. Pfam motifs that were more common in the proteomes of pathogens, include some found in enzymes involved in secondary metabolic pathways. These include novel enzymes that have only previously been studied in non-fungal species, such as the chalcone synthases; type III polyketide synthases involved in the biosynthesis of flavonoids in plants [63] and lipoxygenases; components of metabolic pathways resulting in the synthesis of physiologically-active compounds such as eicosanoids in mammals [64] and jasmonic acid in plants [65] as well as antibiotic synthesis monooxygenases. It seems likely that secondary metabolism is essential in phytopathogenic species for the synthesis of mycotoxins, antibiotics, siderophores and pigments [66], but it may also offer fungal pathogens a distinct alternative means of perturbing host metabolism, cell signalling or plant defence, in contrast to bacterial pathogens that rely on protein secretion to achieve this. There also seems to be number of protease and peptidase domains that are more common in the genomes of phytopathogens as well as domains from two classes of cell-wall degrading enzymes: namely cutinase (PF01083) and Glycosyl hydrolase family 53 (PF07745) which is found in arabinogalactan endo-1,4-beta-galactosidases that hydrolyze the galactan side chains that form part of the complex carbohydrate structure of pectin [67]. Two other domains found in enzymes involved in pectin degradation, pectinesterase (PF01095) and Glycosyl hydrolases family 28 (PF00295) are both more than twice as common in the genomes of phytopathogens than saprotrophs. In contrast, domains found in cellulases have fairly equal distribution between the proteomes of phytopathogens and non-pathogens (data not shown). Therefore, for phytopathogens the most essential enzymes for pathogenesis may well be those that allow the fungus to penetrate the protective cutin layer of the plant epidermis and disrupt the pectin matrix of the plant cell wall in which cellulose fibrils are embedded. Pectin-degrading enzymes have already been shown to be pathogenicity factors in a number of fungi [68]. NPP1 motifs are characteristic of a group of proteins called NLPs (Nep1-like proteins) that trigger defence responses, necrosis and cell death in plants and may act as virulence factors [69]. The NLPs are more common in the genomes of phytopathogenic, when compared to non-pathogenic ascomycetes, but are even more numerous in the proteomes of the oomycetes (64 proteins in Phytophthora ramorum and 75 in Phytophthora sojae). Proteins containing the Chitin recognition protein domain (PF00187) are also very common in the proteomes of phytopathogens (18 in M. grisea and 16 in S. nodorum). A role for chitin-binding proteins has been proposed in protecting the fungal cell wall from chitinases produced by host plants [70]. There are also two other Pfam motifs, which are more common in the proteomes of phytopathogens, that are found in enzymes involved in the catabolism of toxic compounds, namely arylesterase (PF01731) and EthD protein (PF07110) which breakdown organophosphorus esters [71] and ethyl tert-butyl ether [72], respectively.\n\nComparative secretome analysis of phytopathogenic and saprotrophic filamentous ascomycetes\nStudies in bacterial pathogens and oomycetes have shown that a range of secreted proteins known as effectors are important for establishing infection of the host plant [73], [74]. These secreted proteins may disable plant defences and subvert cellular processes to suit the needs of invading pathogens. Therefore, we decided also to compare gene family size in the secretomes of phytopathogens and non-pathogens. There are a number of programs available that predict whether a protein is likely to be secreted, although the predictions they give significantly differ from each other. Therefore we defined the secretome of each fungal species based on those proteins that are predicted to be secreted by two different programs: SignalP 3.0 [75] and WoLFPSORT [76]. The size of each secretome is summarised in Figure 2. Even when using two programs, the sizes of predicted secretomes can vary greatly. For example, a similar analysis for M. grisea using SignalP and ProtComp (www.Softberry.com) predicted only 739 secreted proteins (out of a proteome of 11,109) compared to our prediction of 1,546 secreted proteins (out of a proteome of 12,841) [22]. The size of the secretomes for each species varied from 5%–12% of the total proteome. Overall, the size of the secretomes from phytopathogens did not differ greatly from that of non-pathogens.\nTable 8 shows a list of Pfam motifs, not found in the secretomes of non-pathogenic filamentous ascomycetes, that were present in at least three phytopathogenic fungal species. The Isochorismatase motif (PF00857) was found in the secretomes of all five species of phytopathogen. Isochorismatase catalyses the conversion of isochorismate to 2,3-dihydroxybenzoate and pyruvate. It has been implicated in the synthesis of the anti-microbial compound phenazine by Pseudomonas aeruginosa [77] and the siderophore, enterobactin, by Escherichia coli [78]. The isochorismatase motif is also found in a number of hydrolases, such as nicotinamidase that converts nicotinamide to nicotinic acid [79]. Members of this family are found in all filamentous ascomycetes, but interestingly they are only secreted in phytopathogens. Salicylic acid is synthesised in plants in response to pathogen attack and mediates plant defences. As isochorismate is a precursor of salicyclic acid [80], it may be worth speculating that isochorismatases secreted by fungi could act to reduce salicylic acid accumulation in response to pathogen attack and thus inhibit plant defence responses. The secreted isochorismatases (apart from one of the proteins from S. nodorum) all show sequence similarity to ycaC from E. coli, an octameric hydrolase of unknown function [81]. Pfam motifs found in the secretomes of at least three species of phytopathogens, but not in any of the non-pathogens also include those found in enzymes potentially involved in detoxification, such as arylesterase and amidohydrolase, and also beta-ketoacyl synthase, which catalyses the condensation of malonyl-ACP with a growing fatty acid chain and is found as a component of a number of enzyme systems, including fatty acid synthases and polyketide synthases [82], [83].\nTable 9 shows a list of Pfam motifs that are more common in the secretomes of phytopathogens as compared to saprotrophs. These include a number of secreted proteases, transcription factors and components of signal transduction pathways. The Kelch domain (PF01344) shows the most striking difference in distribution between phytopathogenic and non-pathogenic genomes. This 50-residue domain is found in a number of actin-binding proteins [84], as well as enzymes such as galactose oxidase and neuraminidase. The putative function of each secreted Kelch domain-containing protein was ascertained by performing a BLAST search against the NCBI non-redundant protein database (Table 10). A number of these seem to be galactose oxidases, enzymes which catalyse the oxidation of a range of primary alcohols, including galactose, to the corresponding aldehyde with the concomitant reduction of oxygen to hydrogen peroxide (H2O2) [85]. Galactose oxidase shares a copper radical oxidase motif with the hydrogen peroxide-generating glyoxal oxidases involved in lignin-degradation in Phanerochaete chrysosporium [86]. H2O2-producing copper oxidases have been shown to have roles in morphogenesis, in the corn-smut fungus Ustilago maydis for example, a glyoxal oxidase is required for filamentous growth and pathogenicity [87] and a galactose oxidase is involved in fruiting body formation in the gram-negative bacterium Stigmatella aurantiaca [88]. Interestingly, the list of Pfam motifs more common in the secretomes of phytopathogens also includes those found in copper amine oxidases, H2O2-generating enzymes that catalyse the oxidative deamination of primary amines to the corresponding aldehydes [89] and peroxidases, haem-containing enzymes that use hydrogen peroxide as the electron acceptor to catalyse a number of oxidative reactions. Secreted fungal peroxidases include enzymes involved in lignin breakdown by the white rot fungus Phanerochaete chrysosporium [90], but in plants they generate reactive oxygen species and are involved in defence responses and growth induction [91]. A number of other secreted Kelch domain-containing proteins have similarity to proteins of unknown function from species of the bacterial phytopathogen Xanthomonas. Many Kelch domain-containing proteins are involved in cytoskeletal rearrangement and cell morphology [92], [93]. It may be worth speculating that secreted Kelch domain-containing proteins could act as effectors, causing changes in the arrangement of the cytoskeleton of infected plants to aid the proliferation of fungal hyphae. It has recently been shown, for example, that M. grisea co-opts plasmodesmata to move from cell to cell in infected rice leaves [94] and would therefore need to peturb cytoskeletal organisation in rice epidermal cells. There are other Pfam domains that are more common in the secretomes of phytopathogens that may potentially be found in effectors such as the PAN domain (PF00024), that mediates protein-protein and protein-carbohydrate interactions [95] and the F5/8 type C domain (PF00754), found in the discoidin family of proteins involved in cell-adhesion or developmental processes [96].\n\n\nDiscussion\nOne of the most fundamental aims in plant pathology research is to define precisely the difference between pathogenic and non-pathogenic microorganisms. The answer cannot be one of simple phylogeny, because phytopathogenic species are found in all taxonomic divisions of fungi and are often closely related to non-pathogenic species [3]. Before the availability of genomic sequences and high throughput approaches to study gene function [20], research was concentrated on the search for single pathogenicity factors; genes that are dispensable for saprophytic growth but essential for successful infection of the host plant [4], [97]. However, rather than encoding novel proteins found only in phytopathogens, the majority of pathogenicity factors discovered in this way have been found to be involved in signalling cascades and metabolic pathways and hence are conserved in most species of fungi [5]. Components of signalling cascades that in the budding yeast S. cerevisiae are responsible for responses to pheromones, nutritional starvation and osmotic stress [9] have in many cases evolved different roles in the life cycle of pathogens, such as controlling appressorium formation, dimorphism and growth [10]. Although the central components of signalling are conserved between phytopathogens and S. cerevisiae, the receptors are often different, reflecting the different environmental cues to which the pathogen needs to respond [11], [98].\nAnalysis of all available genome sequences from a wider range of fungal species has for the first time allowed us to address the differences between phytopathogens and non-pathogens at a whole genome level. For this purpose, the e-Fungi data warehouse provides a means to interrogate the vast amounts of genomic and functional data available in a simple integrated manner [26]. Previous research, in which EST datasets were compared with genomic sequences, suggested that the expressed gene inventories of phytopathogenic species were not significantly more similar to one another than to those of saprotrophic filamentous fungi [99]. We clustered sets of predicted proteins from 36 different species of fungi and oomycetes into groups of potential orthologues and the species distribution of members of each cluster was ascertained. There were no clusters that were completely specific to phytopathogenic species across both fungi and oomycetes, suggesting that the presence of novel, universal pathogenicity factors in the genomes of phytopathogens is unlikely. This was confirmed by looking at clusters containing empirically defined pathogenicity factors, where homologues of many of these were found in all species studied and none were conserved in the genomes only of phytopathogens. A small number were only found in a single species of fungus and probably represented proteins that are highly specialised for a particular role in a specific pathogenic species, for example in host-plant recognition [54]. Previous research also suggested that the gene inventories of filamentous fungi were more similar to each other than to those of unicellular yeasts [99]. Analysis of the clusters of similar proteins show some clusters that are found in all species of filamentous fungi (including ascomycetes, basidiomycetes and zygomycetes) but are not present in the genomes of yeasts, consistent with the original conclusion. These contain a number of proteins that are likely to be involved in morphological changes associated with the more complex filamentous lifestyle, as well those involved in secondary metabolism and signalling cascades that are not found in yeasts. In particular, our results suggest that filamentous fungi use a wider variety of lipid molecules for the purpose of signalling. Some of these may act as pheromones, or hormones– chemical messengers diffusing from one cell to another to elicit a physiological or developmental response [37]. A number of these innovations to the filamentous lifestyle may serve important roles in pathogenesis as well, because homologues of a number of pathogenicity factors are found only in filamentous ascomycetes. The distribution of filamentous fungi-specific proteins, such as involved in those cytoskeletal rearrangements and fruiting body formation, throughout the fungal kingdom (and in some cases in oomycetes as well), suggests that the last common ancestral fungus may well have been multi-cellular and the evolution of uni-cellular fungi was likely associated with massive gene loss. For example, it has been shown that early in ascomycete evolution there was a proliferation of subtilase-type protease-encoding genes that have been retained in some filamentous ascomycete lineages, but lost in the yeast lineage [100].\nIt has previously been speculated that the evolution of phytopathogenesis was associated with the expansion of certain gene families [1]. Duplication of an ancestral gene, followed by mutation allows members of the family to take on new functions [101]. For example, genomes of the filamentous ascomycetes studied here have between 40 and 140 cytochrome P450-encoding genes (data not shown) that are involved in toxin biosynthesis, lipid metabolism, alkane assimilation and detoxification [102] and which probably arose via gene duplication and functional diversification. In contrast, the genome of the budding yeast S. cerevisiae has only three cytochrome P450-encoding enzymes. We have shown here that there are likely to be large differences in the gene inventories of filamentous fungi compared to unicellular yeasts.\nTo study the differences between phytopathogenic and saprophytic fungi, we concentrated on the filamentous ascomycetes where there are a number of phytopathogenic species genomes have been sequenced along with closely related non-pathogens. Protein families were defined using Pfam motifs [57] and the predicted protein sets for each species analysed in order to identify domains that were specific to or more common in the genomes of phytopathogens. Not surprisingly, many of the protein families we identified are likely to be associated with pathogenic processes such as plant cell wall degradation, toxin biosynthesis, formation of reactive oxygen species and detoxification [5]. Studies of bacterial phytopathogens have shown the importance of effectors, secreted proteins that disable plant defences and subvert metabolic and morphological processes for the benefit of the invading pathogen and which require delivery via a type III secretion system that are often deployed during pathogenesis [73]. Bacterial type III secreted effectors (T3SEs) have been shown to target salicyclic acid and abscisic acid-dependent defences, host vesicle trafficking, transcription and RNA metabolism, and several components of the plant defence signalling networks [103]. Very recently, potential effector-encoding genes have been identified in the genomes of several species of oomycete pathogens and are defined by the presence of a conserved RXLR-EER motif downstream of the signal peptide sequence [74]. The RXLR-EER motif is necessary for delivery of effector proteins into host plant cells and is therefore critical to their biological activity [74].\nTo identify potential fungal effectors, we compared Pfam motif frequency between the secretomes of phytopathogens and non-pathogens. This analysis identified potential effector-encoding genes, including secreted proteases, transcription factors and proteins that may be involved in cytoskeletal rearrangements (such as Kelch-domain containing proteins) and protein-protein interactions, as well as a group of pathogen-specific secreted isochorisimatases that potentially could suppress salicyclic acid-dependent host plant defences. Bacterial T3SEs are injected directly into the host cytoplasm via the type III secretion injection apparatus [73]. In contrast, the potential fungal effectors identified in this study appear to be secreted by the normal cellular secretory pathway via the endoplasmic reticulum and the mechanism by which fungal effectors might be taken up by plant cells and enter into the host cytoplasm is currently unknown.\nAlthough the evolution of phytopathogenicity is likely to have happened several times and the lifestyles of these fungi are diverse, a comparison of gene inventories of a number of species using a powerful resource, such as e-Fungi, has allowed us to pinpoint new gene families that may serve important roles in the virulence of phytopathogens, allowing their selection for gene functional studies, that are currently in progress. The analyses deployed here may also offer a blueprint for the types of larger, more comprehensive studies that will be necessary to interpret the large flow of genetic data that will result from next generation DNA sequence analysis utilizing both a much wider variety of fungal pathogen species and also large sets of individual isolates of existing species.\n\nMaterials and Methods\nClustering of sequences\nSets of predicted proteins were downloaded for each of the 36 genomes from respective sequencing project websites (Table 1). Proteins less than 40 amino acids in length were not included in this analysis. Proteins were clustered using “all against all” BLASTP [104] followed by Markov Chain Clustering (MCL) [27] with 2.5 as a moderate inflation value and 10−10 as an E-value cut-off, as described previously [28], [29]. Clusters were annotated based on best hit against Swiss-Prot protein database [105] of members of that cluster (e-value <10−20 using BLASTP), or Pfam motifs contained in proteins from the cluster in the absence of Swiss-Prot hits.\n\nIdentification of Pfam motifs\nThe Pfam-A library from release 18.0 of the Pfam database was downloaded from the Pfam website (http://www.sanger.ac.uk/Software/Pfam/). This library contains 7973 protein models constructed from manually curated multiple alignments and covers 75% of proteins in UniProt [44], [57]. This library was used to analyse the sequences of predicted proteins for all 36 fungal genomes to identify the Pfam motifs that each protein contains. The analysis was performed using the “pfam_scan” perl script (version 0.5) downloaded from the Pfam website and HMMER software (downloaded from http://hmmer.wustl.edu/). Default thresholds were used, which are hand-curated for every family and designed to minimise false positives [44].\n\nIdentification of secreted proteins\nThe N-terminal sequence of each predicted protein from the 36 fungal genomes used in this study was analysed for the presence of a signal peptide using SignalP 3.0 [75] and sub-cellular localisation was predicted using WoLF PSORT [76]. Both these programs were installed locally. SignalP 3.0 uses two different algorithms to identify signal sequences. The secretome for each fungal species was defined as containing those proteins that were predicted have a signal peptide by both prediction algorithms from SignalP 3.0 and also predicted to be extracellular by WoLF PSORT.\n\nData analysis\nAll the data produced, as described above, was stored in the e-Fungi data warehouse [26] from which it can be accessed via a web-interface (http://www.e-fungi.org.uk/). Analyses described in this study were performed using the e-Fungi database.\n\n\nSupporting Information\n\n\n" ], "offsets": [ [ 0, 49634 ] ] } ]

[ { "id": "pmcA2409186__T0", "type": "species", "text": [ "potato" ], "offsets": [ [ 1773, 1779 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4113" } ] }, { "id": "pmcA2409186__T1", "type": "species", "text": [ "Phytophthora infestans" ], "offsets": [ [ 1813, 1835 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4787" } ] }, { "id": "pmcA2409186__T2", "type": "species", "text": [ "rice" ], "offsets": [ [ 1840, 1844 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4530" } ] }, { "id": "pmcA2409186__T3", "type": "species", "text": [ "Magnaporthe grisea" ], "offsets": [ [ 1891, 1909 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "148305" } ] }, { "id": "pmcA2409186__T4", "type": "species", "text": [ "yeast" ], "offsets": [ [ 3576, 3581 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4932" } ] }, { "id": "pmcA2409186__T5", "type": "species", "text": [ "Saccharomyces cerevisiae" ], "offsets": [ [ 3582, 3606 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4932" } ] }, { "id": "pmcA2409186__T6", "type": "species", "text": [ "rice blast fungus" ], "offsets": [ [ 3932, 3949 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "148305" } ] }, { "id": "pmcA2409186__T7", "type": "species", "text": [ "Magnaporthe grisea" ], "offsets": [ [ 3950, 3968 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "148305" } ] }, { "id": "pmcA2409186__T8", "type": "species", "text": [ "S. cerevisiae" ], "offsets": [ [ 4147, 4160 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4932" } ] }, { "id": "pmcA2409186__T9", "type": "species", "text": [ "yeast" ], "offsets": [ [ 4269, 4274 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4932" } ] }, { "id": "pmcA2409186__T10", "type": "species", "text": [ "M. grisea" ], "offsets": [ [ 4799, 4808 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "148305" } ] }, { "id": "pmcA2409186__T11", "type": "species", "text": [ "yeast" ], "offsets": [ [ 4986, 4991 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4932" } ] }, { "id": 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"id": "pmcA2409186__T30", "type": "species", "text": [ "Encephalitozoon cuniculi" ], "offsets": [ [ 10820, 10844 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "6035" } ] }, { "id": "pmcA2409186__T31", "type": "species", "text": [ "E. cuniculi" ], "offsets": [ [ 11473, 11484 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "6035" } ] }, { "id": "pmcA2409186__T32", "type": "species", "text": [ "E. cuniculi" ], "offsets": [ [ 12521, 12532 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "6035" } ] }, { "id": "pmcA2409186__T33", "type": "species", "text": [ "E. cuniculi" ], "offsets": [ [ 13094, 13105 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "6035" } ] }, { "id": "pmcA2409186__T34", "type": "species", "text": [ "E. cuniculi" ], "offsets": [ [ 13294, 13305 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "6035" } ] }, { "id": "pmcA2409186__T35", "type": "species", "text": [ "yeast" ], "offsets": [ [ 14107, 14112 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4932" 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"db_name": "ncbi", "db_id": "5145" } ] }, { "id": "pmcA2409186__T48", "type": "species", "text": [ "Aspergillus nidulans" ], "offsets": [ [ 17716, 17736 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "162425" } ] }, { "id": "pmcA2409186__T49", "type": "species", "text": [ "B. cinerea" ], "offsets": [ [ 18413, 18423 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "40559" } ] }, { "id": "pmcA2409186__T50", "type": "species", "text": [ "Eremothecium gossypii" ], "offsets": [ [ 18425, 18446 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "33169" } ] }, { "id": "pmcA2409186__T51", "type": "species", "text": [ "G. zeae" ], "offsets": [ [ 18448, 18455 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "5518" } ] }, { "id": "pmcA2409186__T52", "type": "species", "text": [ "M. grisea" ], "offsets": [ [ 18457, 18466 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "148305" } ] }, { "id": "pmcA2409186__T53", "type": "species", "text": [ "S. sclerotiorum" ], "offsets": [ [ 18468, 18483 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[ { "id": "pmcA509286__text", "type": "Article", "text": [ "How can Health Behavior Theory be made more useful for intervention research?\nAbstract\nBackground\nThe present paper expresses the author's views about the practical utility of Health Behavior Theory for health behavior intervention research. The views are skeptical and perhaps even a bit exaggerated. They are, however, also based on 20-plus years of in-the-trenches research focused on improving health behavior practice through research.\n\nDiscussion\nThe author's research has been theoretically driven and has involved measurement of varying variables considered to be important theoretical mediators and moderators of health behavior. Regretfully, much of this work has found these variables wanting in basic scientific merit. Health Behavior Theory as we have known it over the last 25 years or so has been dominated by conceptualizations of behavior change processes that highlight cognitive decision-making. Although much of health behavior practice targets what people do rather than what they think, the logic of focusing on thoughts is that what people think about is the key to what they will do in the future, and that interventions that can measure and harness those processes will succeed to a greater extent than those that do not. Unfortunately, in the author's experience, the premise of cognitive theories has fallen short empirically in a number of ways. The cognitive schemata favored by most health behavior theories are difficult to measure, they do not predict behavioral outcomes very well, there is little evidence that they cause behavior, and they are hard to change directly.\n\nSummary\nIt is suggested that health behavior researchers reconsider their use of these theories in favor of models whose variables are more accessible to observation and experimental manipulation and that most importantly have strong empirical support.\n\n\n\nBackground\nThe author has been conducting research on behavioral treatment of obesity for about 25 years. During that time, the dominant conceptual models guiding intervention development have been cognitive behavior models that have their origin in psychological theory. Those most often cited include the Health Belief Model [1], Protection Motivation Theory [2], Subjective Expected Utility Theory [3], the Theory of Reasoned Action [4], Social Cognitive Theory [5], and the Transtheoretical Model [6]. All of these theories are concerned with how people make behavioral choices and the general idea is that people decide what to do based on the extent to which they expect that their choices will produce results that they value. Much of the content of the theories is concerned with factors that may affect value/expectancy calculations. As summarized by Weinstein in a comparative review of four social psychological theories [7], variables thought to influence value/expectancy judgments include such factors as perceived rewards of current behavior, self-efficacy, normative beliefs, motivation, and the perceived consequences of not changing behavior.\nWeinstein's summary is illustrative of the fact that Health Behavior Theory has tended to be particularly interested in understanding people's motivation to change behavior rather than ability to change. Moreover, motivation is thought to be the result of a relatively complex, but logical, interpretation of large quantities of information about self and environment. The theories that Weinstein reviewed deal almost exclusively with behavioral decision processes in people's minds. They have few if any terms relating to how information gets into peoples minds or how subsets of it receive more or less attention. Broader health behavior theories such as Social Cognitive Theory or the Transtheoretical model have addressed issues and variables outside the person to a greater extent, but the fundamental interest in and belief in psychological variables as the key force in determining health behavior remains.\nThe implications of the focus of health behavior theory on psychological determinants of behavioral decision-making for my own research area of interest, obesity treatment, are several. One is the inclusion of measures of psychological characteristics in most research protocols (e.g., assessment of behavioral intentions, self-efficacy, perception of barriers to change, perception of social support, and outcome expectations). A second is the inclusion of treatment elements that specifically target psychological perceptions and processes independent of the diet and physical activity behaviors that actually produce weight change (e.g., how to deal with emotional eating, how to deal with the frustration of lapses and relapses, and how to talk to yourself to increase self-motivation). A third is the belief that psychological reactions to treatment experiences themselves are very important and deserve independent attention. Common behavioral prescriptions for weight-loss goals and frequency of self-weighing are exemplary (i.e., recommending infrequent weighing to prevent discouraging feedback about progress and encouraging smaller and thus \"more attainable\" behavior and weight-loss goals in the belief that they will be more motivating).\nThe problem with the emphasis on cognitive variables in weight-control research is that they have so far failed to meet fundamental scientific criteria for empirical verification. Thus, they also have not led to a better understanding of the weight-loss process, have not improved our ability to predict weight-loss outcomes, and have not led to improvement in treatment methods. In some cases it is even arguable that they have made treatment worse. I will illustrate these problems with results from my own research.\n\nDiscussion\nLike most behavioral researchers in the obesity area, I have attempted to measure elements of health behavior theory in every obesity intervention project I have ever conducted. I have assessed weight-loss goals, behavioral and weight-loss self-efficacy, psychological well-being, perceived barriers to diet and physical activity change, stages-of-change, and perceived social support. How well have empirical examinations of these factors fared as predictors of success in weight control?\nSelf-efficacy\nWe have examined the predictive value of self-efficacy assessments in several of our studies and describe the results from three of these here in more detail [8-10]. In the first study, self-efficacy was assessed at baseline, posttreatment, and one year later in 85 men participating in a 15-week weight-loss program [8]. The self-efficacy instrument had subscales for emotional states (e.g., anxiety) and situations (e.g., eating away from home). Higher baseline self-efficacy on both subscales was associated with greater weight loss in treatment and at 1- and 2-year follow-up. Emotional self-efficacy at posttreatment did not predict weight loss at 1- or 2-year follow-up. Situational self-efficacy at posttreatment predicted weight loss at 1-year but not 2-year follow-up.\nThe second study examined mood and situational self-efficacy in 55 men and 58 women before and after a 16-week weight-loss treatment with a 1-year follow-up [9]. Women had lower pretreatment self-efficacy than men. Self-efficacy was predictive of weight loss and maintenance in men but not in women. Change in self-efficacy over time was positively related to weight change in women but not in men.\nThe third study examined predictors of weight change over a 2-year period in 460 men and 1172 women who received a low-intensity weight-loss intervention delivered through their HMO [10]. The self-efficacy measure was the WEL questionnaire. Men again were found to have higher baseline self-efficacy than women. Self-efficacy did not predict weight change in men but was positively, though weakly, related to weight change at 6 months only in women.\nOur overall conclusion from the analyses described above, as well as others not pursued in as great detail, is that self-efficacy is a weak predictor of weight loss and is inconsistent across study populations and gender. It tends to increase with weight loss. However, treatment-induced increases in efficacy are not predictive of longer-term weight-loss success.\n\nBarriers to Adherence\nWe have also attempted to measure barriers to adherence to weight-control behaviors in many of our studies [11-14]. The instruments used for this have typically been formatted similarly to efficacy questionnaires in that people are asked to indicate how difficult they find situational, knowledge, and motivational challenges to achieving diet and exercise changes. The findings in these studies have been quite consistent. Baseline assessments of perceived barriers to behavior change are not predictive of weight change. Weight loss is associated with reported decreases in perceived barriers. Treatment-induced change in perceived barriers are not predictive of future weight change. In other words, barrier perceptions as we have measured them do not appear to have pragmatic significance.\n\nWeight Goals\nGoal-setting has long been of interest to health behavior theory and in recent years has attracted attention in weight-loss research when it was realized that most people who enter weight-loss treatments want to lose a lot more weight than is realistic given the potency of current weight-loss methodologies [15]. When asked to describe weight losses they deem to represent \"dream, happy, acceptable, and disappointing,\" many individuals in treatment fail to reach even \"disappointing\" weight losses even though in objective medical terms the results are positive. Based on the argument that failure to reach gratifying weight-loss goals leads to psychological distress that lowers weight self-efficacy and undermines weight-loss efforts, it has become popular to recommend counseling in weight-loss treatments specifically targeting the lowering of weight-loss goals. The theoretical argument is that excessive outcome expectations undermine behavioral efforts. We have now completed three sets of formal analyses examining whether weight goals are predictive of weight-loss success. In one of these analyses the relationship between weight-loss goals, weight-loss goal attainment, and long-term (30 months) weight-loss attainment and psychological well-being were assessed in 69 men and 61 women participating in an intensive behavioral treatment program [16]. Results indicated that weight-loss goals were unrealistically high on average and that lower goals were more likely to be reached. Nevertheless, weight-loss goals did not predict either short- or long-term weight losses and were not associated with elevated psychological distress. Two more recent analyses we have conducted looking at weight-loss goals as predictors of success have produced similar results [Linde JA, Jeffery RW, Levy RL, Pronk NP and Boyle RG, unpublished data [17]]. Weight-loss goals either did not predict weight loss at all or were slightly positively related to weight-loss success.\n\nPerceived Social Support\nPerceived social support is another psychological factor thought to influence health behavior decision-making. We have measured social support in a variety of ways in our studies, ranging from single-item questions to multipaged assessments attempting to differentiate among informational, instrumental, and emotional support. The results, unfortunately, have closely paralleled those we have seen with other assessments of barriers to adherence. Assessments of social support prior to treatment do not predict weight loss. Average reports of social support tend to parallel weight loss itself. When people lose weight they report more social support. When they regain, they report less. In other words, perceptions of social support are not predictive of success in weight-loss treatments.\n\nFrequency Weight Self-monitoring\nSelf-monitoring of health behavior is incorporated into many health behavior theories, usually as part of a person's assessment of achieved outcomes. Although self-monitoring is usually considered a positive element in the adoption of health behavior, in obesity treatment frequent self-monitoring of weight has tended to be down-played or even discouraged on the grounds that disappointing results (i.e., less than desired weight change) may undermine motivation. This is another example in which health behavior theory may have indirectly led to incorrect treatment recommendations. In weight-loss treatments, active discouragement of frequent self-observation of weight has become popular based on the premise that more frequent weighting will cause psychological stress and lower self-efficacy. Recently, we have examined the relationship between frequency of self-weighing and body weight in both clinical and population samples and have found, somewhat to our surprise, that frequency of self-weighing is one of the strongest single predictors of body weight cross-sectionally, and change in the frequency of self-weighing is one of the strongest predictors of weight change [Linde JA, Jeffery RW and French SA, unpublished data]. The direction of predictions, however, is opposite that derived from theory. People who weigh themselves more weigh less and are more successful in losing weight.\n\nStage-of-Change\nA final failure of current health behavior theory to prove useful in weight-control research is a recent examination of the relationship between a stage-of-change measure adopted from Prochaska and short- and long-term weight loss [18]. Categories of precontemplation, contemplation, preparation, and action were defined based on questions about weight-loss intentions and recent weight-loss attempts. Despite a large sample size, excellent follow-up rates, and well-measured objective outcomes, we were unable to demonstrate that staging algorithms recommended by proponents of the Transtheoretical Model could predict weight-loss outcomes.\n\nExperimental Modification of Expectations\nOur most recent effort to utilize health behavior theory in obesity intervention research is a study that attempted to examine the effectiveness of experimentally-induced outcome expectancies on weight loss [Finch EA, Linde JA, Jeffery RW, Rothman AJ and King CM, unpublished data]. Obese men and women participated in an 8-week weight-loss program with 18-month follow-up in which they were assigned to one of two expectancy groups. The optimistic group was told that focusing exclusively on the positive benefits of weight loss would be valuable in ensuring that they remained motivated in their weight-loss efforts and was given assignments during weekly group sessions and homework between sessions to reinforce this optimistic mindset. A \"balanced\" expectancy group received the instructions that focusing on both the positive and negative aspects of weight loss, a balanced approach, would be most conducive to maintaining weight-loss motivation. This group also received assignments to reinforce their message. Results of this study indicated that the expectation induction was successful initially but difficult to maintain in the face of real weight-loss experience. We were also unable to show that experimentally-induced expectations influenced weight-loss success.\n\n\nSummary and Conclusion\nTo summarize the findings described above, I have had considerable difficulty over the last 25 years in confirming that the psychosocial variables favored by health behavior theory are of much value for obesity intervention research. They do not predict weight loss well, either as mediators or moderators. There is little evidence to support the idea that targeting them for intervention improves weight-loss outcomes. It is, of course, arguable that the weak findings relating to health behavior theory variables are due in large part to methodological weaknesses, either in measurement tools and/or their frequency of measurement. I would argue, however, that 25 years is long enough to wait for improved methods and that it is time to look elsewhere for variables that better predict weight-change outcomes and that, therefore, may form a better basis for improving future treatments.\nImplication for Weight-Loss Treatment\nGiven the lack of success finding support for cognitive mediators of behavior change in weight loss, one might surmise that progress in improving weight-loss interventions over the last 20 years must have been dreary indeed. Somewhat surprisingly, however, that is not the case. In fact, the short-term (6 to 12 months) success of weight-loss treatments has approximately doubled over that time and several variables have been identified that reliably enhance treatment outcomes. It has been clearly shown experimentally that increasing treatment length [19], prescribing low-energy intakes [20], prescribing high-energy expenditure [21], using a deposit contract and group-based reward systems [22], and simplifying adherence to diet through meal substitutes [23] and exercise by providing exercise equipment [24] all improve initial weight loss. From a theoretical perspective, however, one thing is noteworthy about these successful innovations. Although not incompatible with health behavior theory, none of them are specifically derived from cognitive decision-making models. Indeed, health behavior theory does not include variables like these in its models.\n\nWhere Do We Go From Here?\nThe argument above about the practical limitations of many popular theories of health behavior is not meant to be a call to abandon theory. Behavior scientists have amassed much useful information about the principles underlying human behavior that should be valuable for health behavior interventions. Much is known about human perception, learning, motivation, and responsiveness to environmental opportunities and contingencies. Health behavior intervention lies at the interface between people and their environment. Interventionists change aspects of the environment (cues, information, behavioral contingencies) with the intention of producing changes in how people behave. What is needed to advance health behavior intervention is theory that addresses relationships between modifiable aspects of the environment and behavior. There is no doubt that cognitive processes are involved in these relationships. However, the extent to which current theories capture this is questionable. Data now available suggest that easily obtainable information about people's cognitive processes adds little to our ability to predict the results of interventions. Thus, it may be wise to pay more attention to applied theories like classical behavior theory [25], communications theory [26], and learning theory [27] than to those coming out of the social cognitive traditions.\n\n\nCompeting interests\nNone declared.\n\n\n" ], "offsets": [ [ 0, 18798 ] ] } ]

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[ { "id": "pmcA2430230__text", "type": "Article", "text": [ "Emergence of Delayed Methylmercury Toxicity after Perinatal Exposure in Metallothionein-Null and Wild-Type C57BL Mice\nAbstract\nBackground\nAlthough a long latency period of toxicity after exposure to methylmercury (MeHg) is known to exist in humans, few animal studies have addressed this issue. Substantiation of delayed MeHg toxicity in animals would affect the risk evaluation of MeHg.\n\nObjectives\nOur goal in this study was to demonstrate the existence of a latency period in a rodent model in which the toxicity of perinatal MeHg exposure becomes apparent only later in life. Our study included metallothionein (MT) knockout mice because studies have suggested the potential susceptibility of this strain to the neurodevelopmental toxicity of MeHg.\n\nMethods\nPregnant MT-null and wild-type C57Bl/6J mice were exposed to MeHg through their diet containing 5 μg Hg/g during gestation and early lactation. We examined behavioral functions of the offspring using frequently used paradigms, including open field behavior (OPF), passive avoidance (PA), and the Morris water maze (MM), at ages of 12–13 and 52–53 weeks.\n\nResults\nAt 12 weeks of age, behavioral effects of MeHg were not detected, except for OPF performance in MeHg-exposed MT-null females. At 52 weeks of age, the MeHg-exposed groups showed poorer performance both in PA and MM, and their OPF activity differed from controls. These effects of MeHg appeared exaggerated in the MT-null strain. The brain Hg concentration had leveled off by 13 weeks of age.\n\nConclusions\nThe results suggest the existence of a long latency period after perinatal exposure to low-level MeHg, in which the behavioral effects emerged long after the leveling-off of brain Hg levels. Hence, the initial toxicologic event responsible for the late effects should have occurred before this leveling-off of brain Hg.\n\n\n\nMethylmercury (MeHg) poses serious and practical concerns for human populations regarding perinatal exposure. Fish, especially large predator (carnivore) fish species, accumulate high concentrations of MeHg through the marine food chain, and exposure of pregnant women to MeHg through the consumption of fish has evoked widespread concern due to potential effects on offspring. Two large-scale cohort studies in fish-eating populations of Seychelles and Faroe islanders are being conducted; although the former has not found consistent adverse developmental effects of MeHg (Myers et al. 2007), the latter has reported adverse effects (Debes et al. 2006). Fundamental reasons for this discrepancy have not been completely elucidated, and many questions remain regarding the neurotoxicity of MeHg, despite extensive study.\nAmong the unanswered questions is whether there is a long latency period for behavioral manifestations after exposure to MeHg (Clarkson and Magos 2006; Landrigan et al. 2005; Rice 1996; Weiss et al. 2005a, 2005b). Typical examples of latent toxicity in humans, including both acute and chronic MeHg exposures, have been described in detail elsewhere (Weiss et al. 2005a). Davidson et al. (2006) recently suggested that effects of perinatal exposure to MeHg may emerge 9 years after birth in the Seychelles cohort. Consequently, risk assessments of MeHg exposure could be inaccurate because studies (human or animal) usually do not focus on later stages of life and therefore could miss delayed effects. The possibility of delayed toxicity is exemplified by the expanded Barker hypothesis, which posits that the origin of some neurodegenerative diseases such as Parkinson and Alzheimer diseases lies in early exposure to environmental chemicals (Landrigan et al. 2005). Although epidemiologic evidence would be ideal for exploring the possibility of delayed toxicity (and, indeed, data from epidemiologic studies form the basis of current risk assessment for developmental toxicity of MeHg), considering the complex effects of numerous potential confounders and the existence of multiple exposures in human populations, animal models would likely make important contributions to this field.\nAlthough numerous animal studies have described the developmental neurotoxicity of MeHg (Watanabe and Satoh, 1996), few have addressed the latency issue. Few studies have evaluated the neurobehavioral effects in rodents longitudinally beyond 6 months after perinatal exposure. Spyker (1975) addressed this issue in her pioneering work, reporting the late development of behavioral toxicity in mice prenatally exposed to MeHg; it appeared, however, that the substantial mortality and retarded growth among the exposed mice were apparent before weaning, indicating that the doses used (even though some lower dose levels were included) exerted severe toxicity. Using a relatively complex schedule-controlled operant behavior method, rats whose parents were exposed to MeHg (0.5 or 6.4 mg/L) from 4 weeks before mating and continuing to postnatal day (PND) 16 were shown to be less sensitive to a change in the reinforcement schedule than were their nonexposed counterparts at 28–32 months of age (Newland et al. 2004). Mice that were perinatally exposed to 1 or 3 mg/L MeHg in drinking water did not show significant deviation from controls in behavioral performance (motor performance, memory, and learning) at 5, 15, or 26 months of age, but the lifetime-exposed groups did show a significant deviation (Weiss et al. 2005c). The existence of a latency period (i.e., the absence of effects earlier in life followed by the emergence of effects at a later stage of life) has not been demonstrated in any rodent study. In nonhuman primates, delayed emergence of the signs of neurotoxicity was observed several years after the cessation of a 7-year postnatal exposure (Rice 1996).\nMetallothionein (MT) protects against the toxicities of a variety of metals. We examined the neurotoxicity and developmental toxicity of metallic Hg in MT I/II-knockout mice (Yoshida et al. 2004) and demonstrated the susceptibility of this genetically manipulated strain to the toxicity of metallic Hg. In contrast to metallic Hg, MeHg does not induce MT, and MT would not substantially influence the kinetics of MeHg (Yasutake et al. 1998). Several reports, however, have demonstrated protective effects of MT against MeHg toxicity, which was ascribed to the radical scavenging effect of MT (Yao et al. 2000). We also showed that perinatal exposure to MeHg results in altered metabolism of thyroid hormones in neonates that was more distinct in MT-null strains than their wild-type counterparts (Mori et al. 2006). The vulnerability of the MT-null strain suggests that delayed neurobehavioral toxicity due to MeHg, if it does exist, might be more distinctive in this strain.\nBy utilizing the MT-null strain, we aimed to answer the following two questions: First, could we generate a model in which the toxicity of MeHg would emerge or at least become exaggerated later in life as opposed to earlier in life (i.e., at 3–6 months, which was the timing for most of the earlier studies that used behavioral evaluations)? Second, would the MT-null strain be affected more than its parent C57BL strain? Answering either of these questions not only could influence the risk evaluation of MeHg, but it could also lead to a better understanding of the mechanism of toxicity for perinatal MeHg exposure. To address these issues, we used three behavioral paradigms, the open field (OPF), passive avoidance (PA), and Morris (water) maze (MM) tests, which are often used in this field and which we used in our previous studies on the effects of Hg vapor (Yoshida et al. 2004, 2006). Performances in the MM and PA are said to be the most sensitive to aging (Gower and Lamberty 1993).\nWe used a dose of 5 μg MeHg/g in the diet, which resulted in a brain Hg level relevant to human risk assessment. We evaluated the behavioral end points twice, once around 3 months of age and the other time around 1 year; the latter time roughly corresponds to the period when many behavioral performances, including OPF (Acevedoa et al. 2006; Carrie et al. 1999; Gower and Lamberty 1993), PA (Gower and Lamberty 1993), and MM (Bach et al. 1999; Carrie et al. 1999), show alterations in this mouse strain.\nMaterials and Methods\nAnimals and MeHg exposure\nOLA129/C57BL/6J strain mice (wild type) and MT I/II-knockout mice (MT-null) of this strain were provided by K.H. Choo of the Murdoch Institute, Parkville, Australia (Michalska and Choo 1993) and were of a mixed genetic background of 129/Ola and C57BL/6 strains. F1 hybrid mice were mated with C57BL/6 mice for six generations at the National Institute for Environmental Studies (Tsukuba, Japan). At 10 weeks of age, single male and female mice were allowed to cohabit; every female mouse was checked each morning for the presence of a vaginal plug. When a plug was confirmed, the day was designated as day 0 of gestation (GD0).\nThe diet, NIH-07PLD formula (CLEA Japan, Inc., Tokyo, Japan), contained vitamins and trace elements as follows (per kilogram diet): 3.2 mg CuSO4, 88 mg FeSO4, 149 mg MnSO4, 25 mg ZnCO3, 1.6 mg Ca(IO3)2, 11 mg vitamin B1, 4.7 mg vitamin B2, 1.9 mg vitamin B6, 44 mg vitamin E, in addition to 5 μg MeHg/g. This diet was fed to the pregnant mice starting from GD0 through 10 days after delivery (i.e., PND10). Thereafter, we switched mice to a diet that did not contain MeHg. We chose GD0 as the beginning of exposure because exposures that started before conception often resulted in fairly high Hg concentrations in fetal/neonatal brains (Kakita et al. 2003), and we chose PND10 to cover the early neonatal period, in which considerable brain growth occurs. In our experimental setting, the neonatal mice started to eat from the diet bucket and drink from the water bottle from PND10 onward. Control mice were kept on the same diet but without MeHg (< 0.01 μg Hg/g). On PND1, to avoid the confounding effects due to different litter size, we reduced each litter to six pups (three males and three females when possible), and on PND10, two males and two females from each litter were killed for chemical analyses.\nThe remaining male and female offspring per litter were weaned on PND28 and used for subsequent behavioral analyses (either at 12–13 weeks or 52–53 weeks, depending on the litter) as described below. We measured body weights of the weaned mice every 2 weeks. Thus, four experimental groups were used (with or without MeHg exposure for two strains), and each group consisted of 12–13 litters. For half of the litters, the behavioral analyses were conducted at 12–13 weeks of age, and upon completion of the behavioral analyses, the animals were killed under ether anesthesia. We then dissected the organs (brain, liver, and kidneys) for Hg analyses. For the remaining half of the litters, we conducted the behavioral tests at 52–53 weeks of age. The mice were treated humanely and with regard to alleviation of suffering according to the National Institute for Environmental Studies’ Guidelines for Animal Welfare and the guidelines of St. Marianna University.\n\nBehavioral evaluations\nThe details of each behavioral procedure have been described elsewhere (Yoshida et al. 2006). Brief descriptions follow.\nFor the OPF test, we used an OPF apparatus (Ohara Co., Ltd., Tokyo, Japan) with a 60 × 60-cm floor surrounded by walls 60 cm high. The experimental room light was turned off, and a dim light of 80 lux was lit during the experiment. We placed a mouse in the center of the floor and monitored its behavior for 10 min using a CCD camera connected to a computer. The position of the center of gravity was calculated by image-analyzing software, which was used to calculate the total distance traveled by the mouse as well as the positional preference (either center or peripheral, where peripheral was defined as the area within 10 cm from the wall). We cleaned the OPF apparatus with 70% ethanol between trials.\nThe apparatus for the PA test (Ohara Co. Ltd.) consisted of a light compartment that illuminated by a 400-lux light and a dark compartment with black opaque walls and lids. The two compartments were separated by a mobile guillotine door. On the first day (training trial), we placed each mouse in the light compartment facing away from the guillotine door, which was closed. After 30 sec of introduction, the door was opened; when the mouse entered the dark compartment, a brief electric shock (4 mA for 2 sec) was delivered through the metal grid floor. This would force the mouse back to the light component. The interval between the opening of the door and the entry to the dark room (in seconds) was recorded and defined as the latency. On the next day, the same procedure was repeated, but without the electric shock (retaining trail). In this PA paradigm, aversive learning was assumed to be established in the training trial, and we used its retention in the retaining trail as the index of learning. Between each individual trial, we cleaned the apparatus with ethanol. The cutoff time of the retention session was 300 sec.\nThe MM test apparatus (Ohara Co., Ltd.) was a round-shaped water pool with a diameter of 120 cm. A small platform was submerged in the water, which provided a place for mice to escape from the water (i.e., an aversive stimulus, water temperature = 23 ± 1°C). The water was made opaque by adding white paint so that the mouse could not see the submerged platform. In each trial, we released a mouse into the pool from a determined position along the wall, and the performance of the mouse was monitored by a CCD camera/image analyzer. The time required to reach the platform was recorded. If a mouse could not find the platform within 60 sec after release, it was led to the platform and placed on it for 20 sec before being removed. In these cases, a latency of 60 sec was recorded. We conducted the trial once a day up to the fifth day for each mouse, and the order of each mouse was counterbalanced across the day. On the sixth day, a transfer test (or probe test), which is a trial without the platform, was conducted; in this procedure, we counted the number of times that the mouse crossed the position where the platform had been.\n\nTissue Hg concentration\nThe tissue samples were homogenized (10% weight/volume) in distilled water using a Polytron homogenizer (Kinematica GmbH, Littau, Switzerland). We determined Hg levels in the homogenates by the oxygen combustion–gold amalgamation method (Ohkawa et al. 1977) using an atomic absorption Hg detector MD-1 (Nippon Instruments, Co. Ltd., Osaka, Japan). To ensure the accuracy of the measurement, we included reference material from a dogfish (DORM-2; National Research Council of Canada, Ottawa, Ontario, Canada) with a certified value of 4.64 ± 0.26 μg/g in the analyses; the observed values fell within the certified range. The detection limit of the measurement was 0.1 ng Hg.\n\nStatistics\nWe analyzed behavioral data for OPF and PA by analysis of variance (ANOVA), taking sex, strain, and MeHg exposure as the factors. All the interactions among these factors were put into the model. When any of the interactions was highly significant, we analyzed the data separately in an appropriate way; for example, if sex × Hg was significant, the data for males and females were separately analyzed for the effects of strain and Hg. Whenever appropriate, ANOVAs were followed by Mann-Whitney U or Student’s t-tests, depending on the nature and distribution of the variables. We analyzed data for the MM by repeated-measures ANOVA, taking the exposure as between-group and trials as within-group variables. The test was performed for each of the four sex and strain combinations separately. The significance level was set at p < 0.05.\n\n\nResults\nBody weight\nUp to 20 weeks of age, the body weight values of the control and MeHg-exposed groups were not different, regardless of strain or sex. After 28 weeks, except for the wild-type female groups, the MeHg-exposed groups weighed significantly less than the controls (Figure 1).\n\nOPF\nAt 12 weeks of age, a three-way ANOVA of the locomotion distance revealed that only strain was a significant factor (p < 0.001), reflecting the longer distance traveled by the MT-null mice (Figure 2A). Strain was also a significant factor for the proportion of the central-area locomotion (Figure 3A), and Hg exposure marginally affected this outcome. In MT-null females, the proportion of central-area locomotion was higher in MeHg-exposed mice than in the controls; this difference was not observed in any other strain–sex combination. At 52 weeks of age, the strain × Hg interaction was highly significant (p < 0.001) in an ANOVA of locomotion distance (Figure 2B); MeHg exposure was associated with decreased locomotion distance in wild-type mice and with increased distance in MT-null mice. A strain-wise two-way ANOVA (with sex and Hg as the factors) revealed that only Hg was significant in both strains (p < 0.01). Regarding the proportion of the central-area locomotion, the effects of MeHg appeared to depend on sex [i.e., sex × Hg was highly significant (p < 0.001) in the three-way ANOVA; Figure 3B]. Indeed, a sex-wise two-way ANOVA showed significant effects of Hg only in females (p < 0.001).\n\nPA\nAt 12 weeks of age, all the groups showed prolonged latency in the second (retention) trial, and no consistent effect of MeHg was recognized regardless of strain or sex (Figure 4A).\nAt 52 weeks of age, a three-way ANOVA revealed a significant interaction between strain and Hg (p < 0.05); strain-wise two-way ANOVAs revealed a significant effect of Hg on learning in MT-null mice of both sexes; these groups of mice showed significantly shorter (less than half) latency times compared to control mice (Figure 4B). A notable difference between the results at 52 weeks of age and those at 12 weeks was that many of the tested mice exceeded the cutoff time in the retention trials at 52 weeks, except for the MT-null groups.\n\nMM\nAt 13 weeks of age, repeated-measures ANOVA did not indicate any effects of MeHg (Figure 5A). At 52 weeks of age (Figure 5B), wild-type males and MT-null females shared the same statistical results; Hg as well as the Hg × trial interaction were statistically significant. Thus, in both cases, the MeHg groups showed a longer latency, hampering learning performance.\n\nTissue Hg concentration\nAt PND10, which was immediately after the exposure, brain Hg concentrations of the neonatal mice were approximately ≤ 0.5 μg/g (Table 1). Although the MT-null mice and females showed slightly higher brain Hg concentrations than the corresponding wild-type group and males, respectively, neither of these differences was significant. At 13 weeks, when the behavioral tests were completed, the brain Hg concentration was comparable to the control (nonexposed) level (approximately 5 ng/g in both the exposed and control brains). Interestingly, MT-null mice had a significantly lower brain Hg concentration than the corresponding wild-type groups.\n\n\nDiscussion\nResults of the present study demonstrate the delayed emergence of neurobehavioral toxicity due to perinatal MeHg exposure, which presumably developed after brain MeHg concentrations had leveled off. This emergent toxicity was exaggerated in MT-null mice and was more distinct in females. To our knowledge, our findings show the first clear-cut demonstrations of a long latency period of MeHg neurobehavioral toxicity in rodents and possible genetic susceptibility for the emergent toxicity.\nThe exposure level should be considered before discussing the end points. On PND10, immediately after the cessation of MeHg exposure, the brain Hg concentration was approximately 0.5 μg/g, regardless of the strain or sex. In a previous study, the brain Hg concentration in mice perinatally exposed to 6 mg MeHg/L (via water) peaked between PND0 and PND4 and was approximately three times higher than on PND21 (Goulet et al. 2003). Therefore, the peak brain Hg concentration, which is presumably observed around birth, can be estimated as about 3-fold higher than that on PND10 and would be approximately 1.5 μg/g (0.5 μg/g × 3), which is one of the lowest levels among recent rodent studies. As shown by Sakamoto et al. (2002) in their Figure 2, prenatal exposure of rats to MeHg showed a peak brain Hg concentration around PND1 that was four to five times greater than the level on PND10. Also, Newland and Rasmussen (2000) reported a slight alteration of a complex operant behavior in rats at ages < 2 years at brain Hg concentrations as low as 0.5 μg/g at birth, although statistical significance of this particular effect was not clear. It should be noted that rats have different Hg kinetics (Hirayama and Yasutake 1986; Yasutake and Hirayama 1990) due to the high affinity of rat hemoglobin for MeHg (Clarkson and Magos 2006).\nThe most important observation of the present study was that the effects of low-level MeHg exposure were detected only at later stages in the lives of the mice. Except for the central-area occupancy in OPF in MT-null females, no statistically significant effects of MeHg were observed in any of the three behavioral tests at 12 weeks of age. In contrast, significant effects were observed in all three tests at 52 weeks of age. The brain Hg concentration of the exposed groups had leveled off and was not distinguishable from the non-exposed group at 13 weeks of age, immediately after the first phase of the behavioral testing. Therefore, in the present study, there was a latency period in which the dose and effects could not be detected, although effects were observed 9 months later. Another notable observation was that the emergent manifestation of toxicity was also recognized in the suppression of body weight (except for wild-type females), which only became apparent on or after 28 weeks of age.\nThe existence of a latency period of as long as several years after chronic (7 years from birth), low-level exposure to MeHg has been described in nonhuman primates (Rice 1996). In that case, however, the Hg concentration in the brain remained elevated, presumably as a result of the long exposure. Indeed, Rice (1996) argued that the minute amount of residual brain Hg could have caused the delayed toxicity. This was clearly not the case in the present study because the Hg concentration leveled off around the time of the first phase of the behavioral study. The absence of behavioral effects at 12 weeks of age ruled out the possibility that the residual behavioral effects were due to elevated Hg early in life. Therefore, the behavioral toxicity that surfaced at 52 weeks of age must have had its origin before the brain Hg concentration leveled off (at or before 13 weeks of age), although the redistribution of Hg to the brain from other sites of deposition, such as the liver, cannot be completely excluded. The long silent period before the manifestations of toxicity emerged suggests that a slow process plays a role in this latent toxicity.\nAlthough an example of a slow process is aging, 52 weeks of age might not be sufficiently old for a mouse to be considered aged in the physiologic sense because C57BL/6 mice have a relatively long life span among mouse strains [median survival of 27–31 months (Gower and Lamberty 1993)] and a survival rate at 18–19 months as high as 90% (Institute on Aging HP) (National Institute on Aging 2008). Nevertheless, various behavioral examinations have shown age-related changes in the performance of mice at approximately 1 year of age in the OPF (Acevedoa et al. 2006; Carrie et al. 1999), MM (Bach et al. 1999; Carrie et al. 1999), and PA (Gower and Lamberty 1993) tests. The observed effects of MeHg, including the deterioration in the PA and MM and suppression in the OPF (in wild-type mice), were consistent with these reported effects of aging on behavioral function (in the sense described above), except for the increased OPF activity in the MT-null mice.\nRegardless of its neural basis, the basis of neurobehavioral toxicity should be sought in early life stages when the brain Hg concentration is highly elevated (approximately 1.5 μg/g at its peak). Some in vivo experiments have demonstrated several candidate mechanisms of perinatal exposure to MeHg, including abnormal migration of neurons and/or glias (Kakita et al. 2003; Rodier et al. 1984), but at higher Hg concentrations. Using exactly the same exposure protocol as the present study, we found suppressed activity of type III iodo-thyronine deiodinase, a thyroid hormone-metabolizing enzyme, in the brains of PND10 mouse neonates (Mori et al. 2006), consistent with our previous study of higher MeHg doses (Watanabe et al. 1999). This perturbation could be one of the candidate mechanisms responsible for the later anomalous behaviors because even a transient change in thyroid hormones during the critical period of perinatal life exerts long-term consequences (Auso et al. 2004).\nThe effects of MeHg at 52 weeks of age were influenced by two potential modifying factors, sex and strain. In the OPF, while the locomotion was affected in both strains (although the direction was opposite), center occupancy was significantly increased only in the MT-null mice. In addition, the effects on PA were significant only in MT-null mice, whereas MeHg at a higher dose was reported to worsen PA performance in rats (6–8 weeks of age; Sakamoto et al. 2002). In addition, body weight gain was suppressed in both male and female MT-null mice, whereas in wild-type mice the suppression was observed only in males. Taken together, the MT-null strain appeared to be slightly more susceptible to the late-emergent effects of MeHg. Several lines of evidence have shown that MT-I,II is protective against the toxicity of MeHg (Leiva-Presa et al. 2004; Yao et al. 2000), and the present results were basically consistent with these reports. We have also reported the susceptibility of the MT-null strain to the neurotoxic effects of metallic Hg (Yoshida et al. 2004, 2006).\nThe difference in the susceptibility to MeHg between sexes is still debated (Clarkson and Magos 2006; National Research Council 2000; Vahter 2007). In the present study, some responses to MeHg were different between the sexes, including OPF performance at 12 and 52 weeks of age and MM performance at 52 weeks. The fact that the MT-null female group was the only group affected by MeHg at 12 weeks may suggest the particular susceptibility of females in this strain. This point needs to be clarified in further experiments.\nThe question remains of why MT-null mice are susceptible to the delayed neurotoxicity of perinatal MeHg. Apparently kinetics play only a minor role because the strain did not show distinct effects on the brain Hg concentration at PND10. The significantly lower brain Hg concentration in MT-null mice compared with corresponding wild-type mice at 13 weeks of age indicated that MT-I,II might play a significant role in the retention of Hg (or MeHg). This is consistent with the results of studies of metallic Hg exposure (Yoshida et al. 2004, 2005); a lower brain Hg concentration may not guarantee lower toxicity, supporting the protective role of the protein. Earlier studies suggest that brain MT-I,II has an important role both in the response to oxidative injury (Potter et al. 2007) and in the process of aging (Kojima et al. 1999). Therefore, the lack of MT can exaggerate the toxicity of MeHg by enhancing the initial effects due to oxygen radicals and/or by accelerating functional aging. Apart from this, an intriguing possibility is that the brain-specific isoform, MT-III, might contribute to the results we obtained because the expression of MT-III, together with MT-I, is increased in the brain of old rats, resulting in the low availability of free zinc for synapses (Mocchegiani et al. 2004). The age-dependent expressions of MT isoforms might be modified in MT-null mice.\nResults of the present study might allow the possibility of alternative interpretations due to some potentially confounding factors. For example, except for the wild-type females, we observed significant differences in body weight between MeHg-exposed and non-exposed groups. Because these differences only became clear later in life, they might be associated with the toxicity that also emerged later in life. Manipulation of body weight in rodents alters activity levels, although the reported results are not always consistent with each other (Harrison and Archer 1987; Samuelsson et al. 2008). Also, the differential performance in PA could be related with the potential effects of MeHg on (electric) shock sensitivity, which we did not examine. At least one high-dose study with adult rats showed reduced electric sensitivity due to mercury exposure (Wu et al. 1985). These possibilities need to be addressed in future studies.\nTo summarize, the present results suggest that an initial (or triggering) toxicologic event occurs before the brain Hg concentration stabilizes and that the nature of this event should be either an acceleration of the aging process or interaction with the aging process. Thus, by identifying the physiologic events associated with the functional aging of the examined behavioral tasks, the fundamental toxicologic scar might be revealed.\n\n\n" ], "offsets": [ [ 0, 29313 ] ] } ]

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[ { "id": "pmcA1347483__text", "type": "Article", "text": [ "EndoNet: an information resource about endocrine networks\nAbstract\nEndoNet is a new database that provides information about the components of endocrine networks and their relations. It focuses on the endocrine cell-to-cell communication and enables the analysis of intercellular regulatory pathways in humans. In the EndoNet data model, two classes of components span a bipartite directed graph. One class represents the hormones (in the broadest sense) secreted by defined donor cells. The other class consists of the acceptor or target cells expressing the corresponding hormone receptors. The identity and anatomical environment of cell types, tissues and organs is defined through references to the CYTOMER® ontology. With the EndoNet user interface, it is possible to query the database for hormones, receptors or tissues and to combine several items from different search rounds in one complex result set, from which a network can be reconstructed and visualized. For each entity, a detailed characteristics page is available. Some well-established endocrine pathways are offered as showcases in the form of predefined result sets. These sets can be used as a starting point for a more complex query or for obtaining a quick overview. The EndoNet database is accessible at .\n\nINTRODUCTION\nTheoretical analyses in the post-sequencing era, in particular in the context of systems biology approaches, increasingly investigate the properties of all kinds of pathways and networks such as metabolic and signaling pathways. It is commonly accepted that we need formal descriptions of these networks to make systematic use of the overwhelming body of facts gathered over decades of laboratory work both in narrow or global scale.\nCorresponding databases have been created and are available now for metabolic networks (KEGG) (1,2), protein interaction networks (BIND and DIP) (3,4) and signaling pathways (CSNDB, Patika and TRANSPATH®) (5–8), just to name a few. So far, however, their main focus is on intracellular processes. Intercellular signaling is addressed only insofar as usually the pathways modeled start with extracellular ligands, and as there exist catalogs and databases about secreted proteins, or the ‘secretome’, of certain systems (9–12).\nThis shortcoming is part of the more comprehensive problem, the genotype–phenotype gap: from a certain genotype, we are able to infer a ‘molecular phenotype’, but for correlating it with a more complex phenotype such as a biological process, or even a certain disease and its clinical appearance, we still depend largely on the mere description of an observed correlation. There is no way to infer such a phenotype through all the different layers of increasing complexity between genomic DNA sequences and the physiological function of whole organs and their interplay within an organism.\nThere may be principal barriers preventing such an inference across different complexity levels, but even to explore these limits, we have to make attempts to bridge the genotype–phenotype gap. We have to do the next step towards modeling intercellular networks that are inextricably linked to the physiology of multicellular organisms (13). Being one of the most complex constructs in the body, the endocrine system comprises numerous cells and tissues that secrete hormones which pass through the body, activate specific receptors of target cells and initiate there multiple intracellular signaling pathways.\nHere, we present a new database, EndoNet, which provides information about the components of endocrine networks and their relations, and enables the analysis of intercellular regulatory pathways in humans. The EndoNet database is accessible at .\n\nRESULTS\nThe biological schema of endocrine actions\nDevelopment and function of different organs as well as the response of a whole multicellular organism to its environment is coordinated through a complex communication system between specialized cells that are part of its organs. This communication is mostly mediated by hormones. In a broader sense, this functional class of biomolecules also comprises growth factors, cytokines, chemokines and other signal transmitters. This generic view is supported, for instance, by the definition given for ‘hormone activity’ by Gene Ontology (GO) (14): ‘Any substance formed in very small amounts in one specialized organ or group of cells and carried (sometimes in the bloodstream) to another organ or group of cells, in the same organism, upon which it has a specific regulatory action’. This definition is also broad enough to include modes of hormonal actions as diverse as endocrine, paracrine and autocrine effects. Accordingly, with the term ‘hormone’ one might refer to any extracellular substance that induces specific responses in target cell and helps to coordinate growth, differentiation, gene expression and metabolic activities of various cells, tissues and organs in multicellular organisms (15).\nHormones can be classified based on their chemical nature, solubility, the distance over which the signal acts and so on (15,16). From the viewpoint of genome–phenotype relations, it is reasonable to distinguish between polypeptide, thus, genome-encoded hormones, on one hand, and those low-molecular weight hormones such as steroids, with only the machinery of their synthesis being genome-encoded, on the other hand. Another classification of hormones, which seems to be overlapping with the previous one refers to the intracellular location of their receptors and, thus, how the subsequent signal is further transduced: membrane-bound receptors usually trigger more or less complex signaling cascades towards the nucleus, whereas nuclear receptors, mainly bound by low-molecular weight hormones, have a very short signaling pathway downstream since they act as transcription factors themselves (15,16).\nIn intercellular communication, we can basically differentiate between two kinds of cells: donor cells which synthesize and secrete a hormone, and acceptor cells which express a hormone receptor (Figure 1a). Donor cells become active under the influence of an external, mostly environmental, stimulus. In the acceptor cell, binding of the hormone to a receptor triggers an intracellular signal transduction cascade with different kinds of end nodes and effects: transcription factors affecting the gene expression program of the acceptor cell, metabolic enzymes controlling the cell's metabolism, structural components which define the acceptor's morphological features, or components of the secretory apparatus regulating the release of other extracellular molecules. If synthesis and secretion of another hormone is among the effects exerted by receptor activation, the acceptor is turned into a donor cell, thus becoming an internal node of the organism's endocrine network. Acceptor cells which do not become producers of another hormone are called ‘terminal target cells’ of the endocrine network, but finally constitute the overall physiological effect of the respective hormonal pathway, or simply the phenotype (Figure 1a).\n\nThe EndoNet data model\nIn the EndoNet data model, two classes of entities—hormones (in the broadest sense) and their acceptor or target cells expressing the corresponding receptors—span a bipartite directed graph. Since one and the same hormone may be secreted by multiple cell types (donor cells), each such secretion event is represented by a hormone node on its own. Similarly, each cell type known to express a hormone receptor (acceptor cell) leads to an individual node. The graph's edges represent hormone transport and binding to a receptor (intercellular edges), on one hand, and triggering or inhibition of hormone secretion by a receptor activated by hormone binding (intracellular edges), on the other hand. Optionally, an edge representing the transport of a hormone can be subdivided by introducing the transport medium (usually blood) as an additional, intermediary node.\nThus, in the conceptual schema of the EndoNet database (Figure 1b), the links between cells/organs and hormone define donor cells (‘D’), those between cells/organs and receptors acceptor cells (‘A’). If an acceptor cell synthesizes another hormone in response to an incoming signal, it becomes an internal node in the emerging hormonal network.\nIn EndoNet, the pathway between a hormone receptor expressed in an acceptor cell and a hormone synthesized in the same cell (intracellular edge) is handled as a black box. In case of genome-encoded peptide hormones, cross-references to entries in the TRANSPATH® database, which describe the signaling cascade starting from the hormone's receptor and ending at the hormone's gene, are provided, if available. Datasets on non-peptide hormones will in future be enriched by a specific metabolic add-on which will include references to the databases KEGG (1) and BRENDA (17), allowing for further characterization of the steps performed during the hormone's synthesis and the regulation of both activity and expression of the enzymes involved.\nBy now, the EndoNet structure already allows for including descriptions of the physiological effects induced by hormone binding (see below, Future Developments).\n\nThe contents of EndoNet\nIn the present version of EndoNet, and as a first approach, we consider the endocrine (hormonal) network of the human body. For each molecule (hormone or receptor), a primary name and synonyms are given. In case of peptide hormones, the sequence of the processed polypeptide, rather than that of the protein precursor, is specified. For a multimeric protein hormone, the subunit composition as well as the sequences of all subunits are stored; the same holds true for hormone receptors.\nAdditionally, all peptide hormone and receptor datasets have links to HumanPSD™ (18) and to the Swiss-Prot database, The structures of non-peptide hormones can be accessed through the corresponding hyperlinks to the KEGG COMPOUND section. Finally, all molecules may have links to the TRANSPATH® database.\nAs described, EndoNet utilizes data about the tissues from which hormones are secreted and in which receptors are expressed to define donor and acceptor cells, respectively. The identity and anatomical environment of cell types, tissues and organs is defined through references to the CYTOMER® ontology (19,20); in the numerous cases where the receptors are ubiquitously expressed, just the root term ‘human body’ is linked.\nData on whether a hormone's synthesis is triggered or inhibited by another hormone through its respective receptor in a particular cell was obtained by manual selection from textbooks [e.g. (16), ], monographies (21), original literature, the EST library information and the linked databases (TRANSPATH®, HumanPSD™ and Swiss-Prot). The contents of EndoNet are summarized in Table 1.\n\nWeb interface, queries and visualization\nEndoNet can be accessed through the WWW via a JSP-based web interface. Hormones, receptors and tissues can be queried for their names, and detailed information on all identified components is available through individual characteristics pages. Each hormone's individual entry page displays its source and target tissues (donor and acceptor cells) as well as its receptors, along with some molecular data. Similarly, each receptor entry exhibits the tissues in which the receptor is expressed, and the hormones it interacts with. Finally, each tissue entry lists the hormone receptors that are found in, as well as the hormones that are synthesized and secreted by the tissue. It is also indicated whether the corresponding tissue exerts gender-specific properties; additional information based on the CYTOMER® ontology is available through the corresponding link on the tissue detail page.\nInstead of searching for a name of a hormone, one can also browse the hierarchical hormone classification featured by EndoNet (available at the ‘Search’ page).\nEach query result can be used as starting point for an extended query. Different items of interest can be selected and added to a common result set. Since several search results can be combined, it is possible to create sets with multiple search parameters.\nAt any step of this incremental retrieval process, the sets of hormones, receptors and tissues obtained so far can be used as starting points for reconstructing a network by a depth-first graph traversal algorithm. The maximum number of steps can be selected separately for the upstream and downstream part of the reconstruction process. Subsequently, the graph will be displayed using a Graphviz-based visualization method [(22), ]. In the resulting image, hormones and receptors are represented as nodes grouped together into subgraphs representing the tissues (cells/organs) they are secreted from or expressed in, respectively (Figure 2). Autocrine loops (donor and acceptor cell being identical) are treated specially for visualization. On demand, the hormones' transport media can be included in the visualization of intercellular edges, enabling the user to choose between different complexities of output. Intracellular edges, which connect a receptor to a hormone, represent the influence of a receptor's activation on the secretion of a hormone from the same cell and are displayed differently, depending on whether this influence is triggering or inhibitory in nature.\nThe graph is displayed as a clickable image map, linking each entity to its detailed characteristics page, thus making the database entries accessible from the graphical overview of a network, too. The graph is available in two different formats: PNG and SVG. While virtually every browser supports PNG (a ‘pixel’ or ‘bitmap’ format), only a few of them provide a zoom functionality for bitmap pictures. Scalable vector graphics, (SVG) provides more functionality (including perfect image quality throughout all zoom factors), but until now most browsers do not support SVG natively and require an SVG plugin () for displaying this vector-based format.\nSome well-established endocrine pathways are offered as showcases in the form of predefined result sets. For instance, sets representing the hypothalamic–hypophyseal axis with a focus on either thyroid hormones, adrenal hormones, growth hormones or prolactin are provided. These predefined sets can be used for obtaining a quick overview or as starting points for more complex queries.\n\n\nFUTURE DEVELOPMENTS\nAmong the important improvements of EndoNet which we are currently working on is the possibility to represent intercellular communication at different levels of the hierarchical organization of organs, tissues and cells in the organism, as well as to distinguish between such communications in male and female organisms. These options will be introduced by a tighter integration with the CYTOMER-based ontology (19,20).\nThe next step will be to expand the contents of EndoNet towards the details of the processes occurring in the transport medium, usually the blood. Since not all hormones are transported as free molecules and some hydrophobic hormones (e.g. steroids and thyroids) need to be bound to specific carrier proteins, proper description of such transporters and their interaction with the corresponding hormones will be required. It is planned to involve quantitative data about the regular or pathological levels of the hormones, the overall kinetics of each hormone in the blood (monotonous decay, oscillating concentrations, increase in response to certain stimuli, etc.), its turnover and metabolic products, etc. That will allow utilization of EndoNet contents for diagnostic purposes. In future, the EndoNet data model will be extended in order to incorporate external stimuli (e.g. light) and physiological states (stress, age, etc.) in a formalized manner, allowing to determine whether or not and in which quantity a hormone will be secreted under the given circumstances. EndoNet will then link the physiological effects of hormones with the intracellular molecular processes leading to its synthesis and secretion in the donor cells, and to the effects on its acceptor cells. At many places of these intracellular and intercellular networks, genetically determined aberrations may cause specific, sometimes pathological phenotypes. Thus, EndoNet will enable to bridge the gap between known genotypes and their molecular and clinical phenotypes in this area of medical research and its applications.\n\nDISCUSSION\nAt its present state, EndoNet provides a high coverage of molecules that are conventionally considered as hormones as well as other molecules that are involved in intercellular communication, such as growth factors, lymphokines and chemokines and their known receptors. The aim of the database is to provide a useful resource for studying the principal features of hormonal networks in a comprehensive way, as it was done more exemplarily in the past for these kinds of networks (23), but was done globally for many other intracellular network types, such as metabolic, protein interaction and transcription networks [reviewed in (24,25)]. EndoNet database certainly is not yet complete but will grow rapidly.\n\n\n" ], "offsets": [ [ 0, 17196 ] ] } ]

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[ { "id": "pmcA2367462__text", "type": "Article", "text": [ "Comparison of strategies for identification of regulatory quantitative trait loci of transcript expression traits\nAbstract\nIn order to identify regulatory genes, we determined the heritability of gene transcripts, performed linkage analysis to identify quantitative trait loci (QTLs), and evaluated the evidence for shared genetic effects among transcripts with co-localized QTLs in non-diseased participants from 14 CEPH (Centre d'Etude du Polymorphisme Humain) Utah families. Seventy-six percent of transcripts had a significant heritability and 54% of them had LOD score ≥ 1.8. Bivariate genetic analysis of 15 transcripts that had co-localized QTLs on 4q28.2-q31.1 identified significant genetic correlation among some transcripts although no improvement in the magnitude of LOD scores in this region was noted. Similar results were found in analysis of 12 transcripts, that had co-localized QTLs in the 13q34 region. Principal-component analyses did not improve the ability to identify chromosomal regions of co-localized gene expressions.\n\nBackground\nThere is a breadth of information being generated by the Human Genome Project and the interpretation of these data has been a major area of research. For simple Mendelian disorders, the identification of genetic effects is fairly straightforward due to understanding the biology that drives these disorders. However, for complex oligogenic or polygenic disorders, understanding all the interconnections between genes influencing a trait is a difficult task because the understanding of the biology of many of these disorders is still evolving. Multiple gene × gene and gene × environment interactions can influence the expression of phenotypes. Genes can interact by modifying the expression of other genes and therefore function as regulatory genes [1].\nIn an effort to dissect some of these complexities, we performed linkage analysis of gene expression transcripts of members of Centre d'Etude du Polymorphisme Humain (CEPH) Utah families to determine the heritability of transcripts and the evidence for regulatory quantitative trait loci (QTLs) and we performed pairwise bivariate linkage analysis and principal-component analysis (PCA) for data-reduction to evaluate the evidence for shared genetic effects. The ability to assess gene expression traits simultaneously and to link them to QTLs offers the possibility of identifying previously unknown underlying molecular processes for future investigation.\n\nMethods\nPopulation and phenotypes\nWe used the Genetic Analysis Workshop 15 (GAW 15) Problem 1 microarray gene expression profiles for the analyses. Data were available for 14 three-generation CEPH Utah families. Expression levels of genes were obtained from lymphoblastoid cells using the Affymetrix Human Focus Arrays [2]. We were provided with data on 3554 transcripts that showed greater variation between individuals than within the same individual.\nFamily members were genotyped for 2882 autosomal and X-linked single-nucleotide polymorphisms (SNP) generated by the SNP Consortium . Genetic map positions were obtained using the SNP Mapping web application developed by the University College Dublin Conway Institute of Biomolecular and Biomedical Research , which uses the Rutgers Combined Linkage-Physical Map of the Human Genome and data taken from the NCBI dbSNP Build 123 (in Kosambi centimorgans). This information was used to calculate multipoint identity by descent matrices (MIBDs) with Merlin and Minx [3], after removal of Mendelian inconsistencies and double recombinants with Preswalk (based on Simwalk mistyping probabilities) [4]. MIBDs were used for linkage analyses.\nTranscript distributions were normalized using an inverse normalization transformation of z-scores of individual transcripts regressed on the mean individual transcript level. We further adjusted for the effects of age, age2, sex, age × sex and age2 × sex interaction using predictive linear regression models in SAS 9.1 (Cary, NC). We generated these residuals as part of our processing of the transcripts for linkage analyses.\n\nHeritability estimation and linkage analysis\nHeritability was estimated using maximum likelihood variance decomposition methods in SOLAR [5,6]. Genome scans were performed using multipoint variance-components models that test for linkage between traits and genetic variants by partitioning the variance of the expression level into its additive genetic and environmental variance components [7]. For transcripts with co-localized QTLs, we performed bivariate linkage analysis to identify shared genetic effects. The bivariate polygenic model estimates correlations caused by residual additive genetic effects (ρG) and correlations caused by random environmental effects (ρE) [8]. To test for additive genetic correlation among pairs of traits, the log likelihood of a model in which ρG is constrained to 0 (null hypothesis, no correlation) or ρG = 1 (null hypothesis, complete shared genetic effect) is compared to that of a model in which ρG is estimated for the traits. Significant differences among the models (ρG ≠ 0) suggest that some of the same genes influence both traits. We also performed linkage analysis using the factors obtained from the PCA in a sample of transcripts with co-localized QTLs.\n\nPrincipal-component analysis\nPCA was used to reduce the number of expression profiles into statistically meaningful groups while retaining the original total variance using all the expression profiles [9,10]. We selected two different chromosomal regions of a length of 10 to 12 MB in which the QTLs of at least 10 transcripts were co-localized. Only transcripts of genes that were not located in these selected chromosomal regions were included in the analyses (trans-regulatory genes). Because of the small number of individuals in the study and concerns of overfitting the model, a maximum sample of 50 transcript values were considered at one time [11]. The number of factors was determined using the eigenvalue-one requirement [11]. Factors are interpreted by examining the varimax-rotated factor loadings, which are the correlations between each phenotype and the factor in question. Factor loadings greater than or equal to 0.40 in absolute value were used to interpret factors and to characterize the factor structures; this criterion ensures that the individual factor variables share at least 15% of their variance with the given factor [9]. The principle components were obtained by calculating the eigenvalues of the sample covariance matrix, which represent the amount of variance contributed by each factor. Only factors with eigenvalues higher than 1 were considered for linkage analysis.\n\nIntegrating data from linkage analysis for gene co-expression\nLinkage signals of individual transcript expressions were recorded and the location of QTLs was compared to the location of the transcript gene in order to identify trans-regulatory sites. In addition, the location and LOD scores of QTLs identified in single individual transcript analysis (univariate analysis) were compared with the location of the QTLs identified using bivariate analysis or factors of the PCA. This allowed a determination of whether the bivariate analysis or PCA data reduction analysis improved our evidence for linkage, and if so, a more in-depth examination of the transcripts included in the principal components needs to be examined for biologic interactions on complex disorders.\n\n\nResults\nAmong 194 individuals from 14 families, 17 individuals with missing information on age were excluded. Seventy-six percent (n = 2688) of the transcripts had significant heritability (p < 0.05) and were considered for the linkage analysis. Of this, 1448 (54%) transcripts displayed suggestive evidence of linkage (had a maximum LOD score ≥ 1.8 [12]). The QTLs of 1661 transcripts (759 of which with LOD ≥ 1.8) were localized in a different region than the gene transcript (trans-regulatory sites). We used two different chromosomal regions with co-localized transcript QTLs, chromosomes 4q28.2-q31.1 and 13q34, for more in-depth analyses.\nChromosome 4q28.2-q31.1 region\nTable 1 reports the results for the chromosome 4q28.2-q31.1 region. Fifteen transcripts co-localized in this region in the univariate linkage analysis, and the LOD scores ranged from 1.17 to 3.72. The strongest linkage signals were observed for the transcripts of the MX2, NUCB2, and SNX4 genes. Using PCA, we obtained five factors from the 15 transcripts with eigenvalues greater than 1. Only one factor, with a high positive loading for the MX2 gene transcript, had a significant heritability and a LOD score ≥ 1.8. The linkage analysis using this factor identified the chromosome region for the MX2 gene, but the LOD score was lower than the one obtained by single linkage analysis of the MX2 transcript.\nWe then performed bivariate analysis of all pairwise co-localized transcripts on 4q28.2-q31.1 and found evidence for genetic correlation of co-localized genes, although without much increase in the magnitude of the LOD score (Figure 1). This analysis identified two networks of gene expressions (Figure 1). We obtained two factors using PCA of the first network (Group 1, SNX4, YWHAQ, ASS, MX2, and ISGF3G gene transcripts). Both factors had significant heritability; however, only Factor 1, loading heavily on the MX2 gene, localized to the 4q28.2-q31.1 region (Table 1), and the magnitude of the LOD score was lower than that of the univariate MX2 gene transcript analysis (LOD = 2.28). The heritability of one factor obtained using PCA for Group 2 transcripts was not significant and further analysis was not performed.\n\nChromosome 13q34 region\nWe performed analysis in an additional chromosome region of co-localized transcripts, 13q34 region, and noted similar results. Using univariate analysis, 12 transcripts co-localized in this region; and bivariate analysis revealed an intricate network of correlated traits (Table 2 and Figure 2). Using PCA, we obtained five factors, three of them with significant heritability. Similar to our previous findings on chomosome 4, PCA factors did not improve the magnitude of the LOD scores when compared to univariate analysis.\n\n\nDiscussion\nIn this study, we identified co-localized QTLs of individual transcripts and compared the univariate and bivariate linkage results using single transcripts to those using factors obtained from PCA. By using factors that accounted for the variance of multiple transcripts with co-localized QTLs, we attempted to reduce the number of linkage analyses performed as well as possibly identifying previously unknown patterns of associated gene expression profiles. The PCA did in fact reduce the number of linkage analyses performed, but it did not improve the magnitude of signals in the target QTLs as compared with univariate or bivariate analyses. In fact, in at least one case, PCA was unable to detect a linkage signal for the main gene transcript loading in the factor (Table 1, Group 1, Factor 2).\nWe also performed pairwise bivariate genetic analysis on those transcripts that co-localized to the same genomic region, presumably because this area of the genome harbored genes involved in the regulation of these transcripts [2]. We detected significant genetic correlation of these co-localized transcripts, indicating potential gene networks operating in these regions. However, in most cases, bivariate linkage analysis did not improve the magnitude of the LOD score compared to univariate analysis. Most traits were highly correlated (ρG > 0.60), and therefore they may provide redundant information that may reduce the power for detection of the bivariate signal [8]. In addition, because ρG is a test of the overall additive genetic correlation among two traits and not the QTL-specific pleiotropy, it is possible that the co-localized linkage signals are not in fact genetically correlated. Further analysis is required to address these issues.\nThe chromosome regions selected for detailed analyses were arbitrarily chosen as we identified multiple other regions with co-localized linkage of gene expressions. The results from our univariate genome scan differ markedly from those reported by Morley et al. [2] because we included a smaller sample of individuals so that adjustment for covariate effects of age could be made. Our analysis strategy also adjusted for the effects of age and sex, which could also add to the observed differences [13]. Finally, our definition of genome window size for co-localized gene expressions was twice larger than the one described in the study of Morley et al.\n\nConclusion\nWe identified several chromosomal regions of co-localized trans-regulatory genes with significant heritability. Some of these regulatory genes displayed strong additive genetic correlations, and may be part of genetic networks. However, when compared to univariate analysis, linkage analysis of bivariate phenotypes and factor scores obtained from PCA did not improve the ability to identify chromosomal regions of co-localized gene expressions.\n\nList of Abbreviations\nCEPH: Centre d'Etude du Polymorphisme Humain\nGAW: Genetic Analysis Workshop\nH2: heritability\nLOD: logarithm of the odds\nMIBD: multipoint identity-by-descent matrices\nN/A: not apply\nNCBI: National Center for Biotechnology Information\nPCA: principal-component analysis\nQTL: quantitative trait loci\nSE: standard error of the mean\nSNP: single-nucleotide polymorphism\nSOLAR: Sequential Oligogenic Linkage Analysis Routines\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\n\n" ], "offsets": [ [ 0, 13636 ] ] } ]

[ { "id": "pmcA2367462__T0", "type": "species", "text": [ "participants" ], "offsets": [ [ 396, 408 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] }, { "id": "pmcA2367462__T1", "type": "species", "text": [ "Human" ], "offsets": [ [ 1114, 1119 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] }, { "id": "pmcA2367462__T2", "type": "species", "text": [ "Human" ], "offsets": [ [ 2771, 2776 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] }, { "id": "pmcA2367462__T3", "type": "species", "text": [ "Human" ], "offsets": [ [ 3295, 3300 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] } ]

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[ { "id": "pmcA140144__text", "type": "Article", "text": [ "Gender differences in factors influencing insulin resistance in elderly hyperlipemic non-diabetic subjects\nAbstract\nBackground\nThe increase in the prevalence of insulin resistance-related metabolic syndrome, a disorder that greatly increases the risk of diabetes, heart attack and stroke, is alarming. One of the most frequent and early symptoms of metabolic syndrome is hypertriglyceridemia. We examined the gender differences between various metabolic factors related to insulin resistance in elderly non-diabetic men and postmenopausal women of comparable age suffering from hypertriglyceridemia, and compared them with healthy subjects of equal age.\n\nResults\nThe indexes of insulin resistance HOMA IR and QUICKI were significantly higher in both hyperlipemic men and women than in controls; 95% confidence limits of hyperlipemic subjects did not overlap with controls. In both normolipemic and hyperlipemic men and women serum leptin correlated significantly with insulin resistance, while HDL-cholesterol correlated inversely with HOMA-IR only in women (both normo- and hyperlipemic), and serum tumor necrosis factor α (TNFα) only in hyperlipemic women. According to results of multiple regression analysis with HOMA-IR as a dependent variable, leptin played a significant role in determining insulin resistance in both genders, but – aside from leptin – triglycerides, TNFα and decreased HDL-cholesterol were significant determinants in women, while body mass index and decreased HDL-cholesterol were significant determinants in men. The coefficient of determination (R2) of HOMA IR by above mentioned metabolic variables was in women above 60%, in men only about 40%.\n\nConclusion\nThe significant role of serum leptin in determination of insulin resistance in both elderly men and postmenopausal women of equal age was confirmed. However, the study also revealed significant gender differences : in women a strong influence of triglycerides, TNFα and decreased HDL-cholesterol, in men only a mild role of BMI and decreased HDL-cholesterol.\n\n\n\nBackground\nIn association with pandemic obesity the prevalence of the insulin resistance-related metabolic syndrome is constantly growing [1]. As a consequence of this fact, type 2 diabetes mellitus and cardiovascular mortality occurs in much younger age groups [2]. A typical hyperlipemia, consisting of an increase of serum triglycerides and a decrease of serum HDL-cholesterol, is a characteristic and an early symptom of this syndrome [3].\nWith increasing age, body mass index (BMI) and adiposity, insulin sensitivity declines and the number of cardiovascular risk factors increases in both genders [4-6]. It was repeatedly demonstrated that plasma concentration of leptin – a hormone produced mainly by adipose tissue – is substantially higher in all age groups of women than in men [7-10]. This may be caused by different size and/or distribution of fat tissue compartments influenced by hormones: estrogens stimulate, whereas testosterone inhibits leptin secretion. In women subcutaneous fat mass prevails – and during augmentation of overweight it increases – while in men intra-abdominal fat mass prevails [11-13]. Subcutaneous fat in particular serves as a substantial source of tumor necrosis factor α (TNFα), which represents one of the factors that interfere with insulin signal transduction into the cells [14-16]. Leptin, TNFα and some other factors are abundantly expressed in adipose tissue and contribute to the insulin resistance that accompanies overweight and obesity. Leptin correlates positively with hyperinsulinemia, BMI, fat mass and hypertriglyceridemia, respectively, and correlates inversely with HDL-cholesterol and lean body mass [17-25].\nThe incidence and mortality of ischemic heart disease and of other consequences of atherosclerosis increases with age in both genders, especially after the age of sixty. In premenopausal women, however, the incidence of these disorders is considerably less frequent than in men of appropriate age. After the menopause the prevalence of metabolic syndrome and cardiovascular mortality in women gradually increases, attaining values comparable to men at about the age of 70 [2,26]. Paradoxically, it takes place at the time when serum leptin concentration in women has relatively decreased [27,28].\nThe aim of this study was to analyze the interrelations between several metabolic variables and factors related to insulin resistance in groups of both normal and hyperlipemic postmenopausal women and men of appropriate age, and to attempt to elucidate the gender differences and some pathophysiologic mechanisms of these differences. We compared homeostatic indexes of insulin resistance HOMA IR and QUICKI, serum lipid and insulin parameters, uric acid, leptin and TNFα between groups of subjects without apparent symptoms of metabolic syndrome, and groups showing mild hypertriglyceridemia with decreased HDL-cholesterol. In addition, serum concentration of the heart fraction of fatty acid binding protein (hFATP) was explored as a factor that might reflect the regulative role of PPAR gamma in lipid homeostasis [29,30], and serum IgG anticardiolipin (ACL-IgG) was investigated as an indirect indicator of oxidized lipid fractions related to atherosclerotic complications [31,32].\n\nMethods\nSubjects\nThe study was carried out on 70 out-patients of the Metabolic Center at the hospital in Sternberk, Czech Republic. From these, 40 patients (20 men and 20 women) were selected with mild hyperlipidemia, i.e. with plasma triglyceride concentration exceeding 2.0 mmol/l, total cholesterol exceeding 6.0 mmol/l, LDL cholesterol exceeding 4.0 mmol/l, and with HDL cholesterol concentration in men under 1.0 mmol/l, and in women under 1.2 mmol/l. These groups were denominated as \"hyperlipemic\". Two other groups (10 men and 20 women) with approximately normal serum values of these variables were taken as \"controls\". The average age in men was 59.1 ± 10.6 y, and in women 59.4 ± 10.1 y, respectively. The differences between lipid parameters of hyperlipemic and control groups were highly statistically significant, while the age differences were insignificant (see Table 1). None of the patients had clinically apparent diabetes mellitus, but some of the hyperlipemic patients exerted impaired glucose tolerance or impaired fasting glucose (values between 6.1 and 7.0 mmol/l, or between 6.1 and 7.8 mmol/l, respectively). None of the patients was treated with insulin, peroral antidiabetics or antihyperlipemic drugs; some of them were treated with antihypertensive therapy. No signs of major clinical or laboratory symptoms of other diseases were present in any group of the explored patients. Blood samples were obtained in the morning via a venipuncture after overnight fasting. After clotting the serum was separated and stored at -20° until used. An informed consent was obtained from all probands.\nBody mass indexes (BMI), defined as weight in kilograms divided by the square of height in meters, were calculated.\n\nBiochemical methods\nSerum leptin concentrations were measured by a sandwich ELISA test kit (Human Leptin ELISA, BioVendor Laboratory Medicine, Inc, Czech Republic). Its sensitivity limit was 0.2 ng/ml, intraassay CV 6.1% at the level of 7.5 ng/m, inter-assay CV 8.5% at the level of 4.8 ng/ml. Tetramethylbenzidine was used as a substrate; quality controls were human based. Several other hormones and peptides were estimated by routine immunochemical tests: insulin, C-peptide, TNFα (IMMULITE, Diagnostic Products Corporation, Los Angeles, CA, U.S.A.), proinsulin intact (DAKO, Denmark), IgG anticardiolipin (ACL-IgG, IMMCO Diagnostics, Buffalo, NY, U.S.A.) and heart fatty acid binding protein (hFABP, Hbt HUMAN H-FABP, HyCult Biotechnology, Uden, the Netherlands). Serum concentration of glucose, total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, Apoprotein B and uric acid were measured on a ILAB-600 biochemical analyzer (Instrumentation Laboratory, Lexington, Ma, U.S.A.) using BioVendor sets. All samples were processed and examined according to principles of good laboratory practice and under constant intralaboratory and external quality control.\nThe homeostatic indexes of insulin resistance (HOMA IR and QUICKI) were calculated according to the homeostasis model of assessment [33-35] as follows:\nHOMA IR = fasting insulin (μU/ml) * fasting glucose (mmol/l) / 22.5;\nQUICKI = 1 / [log fasting insulin (μU/ml) + log fasting glucose (mg/100 ml)].\n\nStatistics\nStatistical analysis was performed using the Version 6 SAS/STAT software (SAS Institute, Inc., Cary, NC, U.S.A.). The Shapiro-Wilks tests were used in testing the normality of distribution. Some of the data obtained were not normally distributed. The statistical significance of differences between the means in the hyperlipemic and control groups were evaluated using the unpaired Student's T-test in the case of normal distribution of data sets, and using the Kolmogorov-Smirnov test when at least in one of the data sets the normal distribution was excluded. Spearman's rank-order correlation was used for correlation analysis. Multiple regression analysis was performed using HOMA IR indexes of insulin resistance as dependent variables, and other metabolic and hormonal factors (lipid parameters, BMI, leptin, TNFα, hFABP, ACL-IgG) as independent variables. The so-called step-down regression model was used to select dominant independent variables. Various four-member groups of independent (explanatory) variables were used for the analysis and the non-zero intercept was taken into account. The independent variables were then dropped, one at a time; at each stage one variable making the least contribution to the dependent variable (i.e. that showed the least p-value in the test of the regression coefficient being zero) was excluded. The coefficient of determination R2, which can be viewed as a percentage explaining the total variance, was simultaneously monitored. A great drop in R2 after excluding some independent variable enabled selection of those independent variables that could be thought to be the most important determinants of the dependent variable.\n\n\nResults\nTable 1 demonstrates mean parameters in individual groups of subjects matched according to sex, lipid parameters and age. While the age of all four groups did not differ substantially, the concentrations of total serum cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol differ very significantly in both male and female hyperlipemic groups as compared with controls. In addition, the concentration of triglycerides in control women was significantly higher than in control men, the concentration of triglycerides in hyperlipemic women was lower than in hyperlipemic men, and the concentration of HDL-cholesterol in hyperlipemic women was very significantly higher when compared with hyperlipemic men.\nTable 2 shows the values of other metabolic and insulin parameters, factors related to insulin resistance and indexes of insulin resistance, respectively. Body mass indexes and uric acid concentration were significantly higher in hyperlipemic men as compared to controls, but not in hyperlipemic women. Uric acid concentration was substantially lower in hyperlipemic women than in hyperlipemic men. Plasma concentrations of glycemia, insulin and intact proinsulin were significantly higher in both hyperlipemic men and women as compared with controls of identical gender, while the concentration of leptin increased only in hyperlipemic men. However, serum leptin concentrations of both control and hyperlipemic women were significantly higher than in corresponding groups of men. Serum concentrations of TNFα, hFABP and ACL-IgG in hyperlipemic groups of both men and women were not significantly different from control groups. On the other hand, the indexes of insulin resistance HOMA IR and QUICKI differed very significantly in hyperlipemic groups of both men and women as compared with corresponding control groups, more distinctly in women.\nFrom Fig. 1, presenting 95% confidence limits of insulin resistance indexes HOMA IR and QUICKI, we concluded that in groups of hyperlipemic patients of both genders the insulin resistance was substantially higher than in control groups; the groups did not overlap each other.\nIn Table 3 the results of Spearman's correlations between insulin resistance index HOMA IR and various metabolic parameters are presented. In the control group of men, positive significant correlation between HOMA IR and serum leptin concentration, and inverse significant correlation between HOMA IR and ACL IgG, respectively, were found. In the control group of women, the significance of Spearman's correlation between HOMA IR and leptin was more expressive; inverse correlation between HOMA IR and HDL-cholesterol was also present.\nIn the hyperlipemic group of men, the significance of the correlation between HOMA IR was more expressive in relation to the control group, and no significant correlation between HOMA IR and ACL IgG was found. In the hyperlipemic group of women, however, the significance of Spearman's correlation between HOMA IR and serum leptin concentration weakened, the inverse correlation between HOMA IR and HDL-cholesterol remained approximately unchanged, and a positive correlation between HOMA IR and serum concentration of TNFα appeared.\nTable 4 shows results of multiple regression analysis, when data from both control and hyperlipemic groups of each gender were judged together. HOMA IR was considered as a dependent variable and differently changed constellations of metabolic and other factors were taken as independent variables.\nIn men, BMI and leptin seemed to play a main role in influencing the insulin resistance index HOMA IR, while TGL, ACL IgG and LDL-cholesterol didn't play any significant role (see left columns of Table 4). The decreasing of HDL-cholesterol concentration may also have some influence (see a significant drop of R2 after exclusion of this factor in Table 4A, 4B). But in the presence of leptin in the group of independent factors, the drop of R2 after exclusion of HDL-cholesterol from these factors was minimal (see Table 4C). On the other hand, after the exclusion of TNFα from the group of independent variables (see Table 4B, 4D) the value of R2 has unexpectedly risen, which could reflect the interference of TNFα with factors increasing the insulin resistance.\nIn women (see right columns of Table 4), the maximal values of R2 were achieved with combination of independent variables containing TGL, leptin and HDL-cholesterol (about 60% influence on HOMA IR! – see Table 4A, 4B, 4C). TNFα seemed to play quite a different role than in men: after exclusion of this factor from the group of independent factors R2significantly decreased (see Table 4B, 4D). In contrast to men, the role of BMI seemed to be minimal. As in men, the role of ACL IgG and LDL-cholesterol in influencing HOMA IR was negligible, but in contrast to men, hFABP might play a certain role in this process (see Table 4D).\nGenerally, the insulin resistance (represented by HOMA IR) was in men much less influenced by metabolic variables than in women; while in women in some combinations of dependent variables R2 reached 64 %, in men the maximal value of R2 was only 39 %.\n\nDiscussion\nIn our previous paper [36], the mean value of HOMA IR in healthy subjects of both genders and of age comparable with our controls was 1.57 ± 0.87, and the mean value of index QUICKI 0.366 ± 0.029, respectively. These values, as well as the 95 % confidence limits, correspond to values found in controls in this study.\nIn accordance with many previous papers, serum concentrations of leptin in women (both control and hyperlipemic) were substantially higher than in men. In the control group of women the correlation between leptin and HOMA IR was highly significant. However, in hyperlipemic women the significance of this correlation lessened, because HOMA IR increased considerably (and significantly) but serum concentration of leptin only slightly (insignificantly). In men the significance of correlations between serum leptin and HOMA IR was high and approximately the same in both the control and hyperlipemic groups, because the values of HOMA IR as well as serum leptin have nearly doubled in hyperlipemic in relation to control groups. In non-hyperlipemic postmenopausal women the high concentration of serum leptin was not associated with higher insulin resistance: HOMA IR did not differ substantially from men. A significant increase of insulin resistance in hyperlipemic women was associated by only slight and insignificant increase of leptin concentration. According to Spearman's correlations, an increase of serum TNFα and/or a decrease of HDL-cholesterol might also play a distinct role in this respect. (see Table 3). In contrast to women, in hyperlipemic men the increase of insulin resistance index was approximately proportional with the increase of leptin concentration.\nMultiple regression analysis affirmed the importance of leptin serum in increasing of insulin resistance in both genders. In men, only BMI and HDL-cholesterol from other factors studied seemed to play a certain role, but the maximal values of influencing HOMA IR reached only 39%, with leptin and BMI being the more important factors. On the other hand, in women the maximal determination of HOMA IR as high as 60% was registered in combination of serum leptin, TGL and decreased HDL-cholesterol as independent factors; the role of BMI was insignificant.\nIt is not known how leptin is regulated. A strong correlation between plasma leptin and fasting insulin undoubtedly exists, but hyperleptinemia in both obese and lean humans is not likely the result of hyperinsulinemia [37]. A relationship between leptin and insulin dependent on sex or BMI was reported, but relationship between triglyceride concentrations and leptin was independent of sex, BMI, and insulin [18,24]. Hyperleptinemia, as an early sign of obesity, was closely linked to subcutaneous fat mass [39,40]. Percentage of body fat has been shown to be the strongest predictor of leptin levels even in lean women [41]. Leptin was highly correlated with percentage of body fat and with fat mass in adults irrespective of gender and age; however, the mean determinant of leptin plasma concentration in men and postmenopausal women was BMI, while in premenopausal women it was only the fat mass [42]. These findings contrast with our results showing minimal influence of BMI on HOMA IR in postmenopausal women.\nAll factors mentioned are connected with fat tissue: leptin and TNFα are directly produced chiefly by adipocytes, BMI growth is obviously accompanied by fat mass increase, and the typical hypertriglyceridemia associated with a decrease of HDL-cholesterol goes along with obesity and fat mass growth. The gender differences in circulating leptin were best explained by percentage of body fat and – inversely – by lean body mass [25]. In both genders the intra-abdominal fat correlated with insulin resistance, while the subcutaneous fat correlated with circulating leptin [11,12]. In men obesity led to a prevalent increase of intra-abdominal fat, while in women of subcutaneous fat [13]. Influences of different compartments of adipose tissues could elucidate the variability of correlations between insulin resistance and high leptin concentrations in lean and obese subjects of both genders [43]. In our non-hyperlipemic postmenopausal women the content of subcutaneous fat mass might be higher than in non-hyperlipemic men of appropriate age, which indicated a higher serum concentration of leptin. However, the insulin resistance – related to intra-abdominal fat mass – did not differ from men. The significant increase of insulin resistance and leptin concentration in hyperlipemic men might reflect the growing content of both subcutaneous and intra-abdominal fat mass (see the significant increase of BMI). In hyperlipemic women the significant increase of insulin resistance accompanying only minimal insignificant increase of leptin could be caused by prevalent growing of intra-abdominal fat mass.\nIn elderly postmenopausal women, an association between leptin and plasma lipoprotein concentration was found which depended on adiposity [17], and inverse correlations between serum leptin and HDL-cholesterol were described [44]. In our study, insulin resistance in women seemed to be more notably than in men influenced by lipid disorders, i.e. positively by serum triglycerides and inversely by HDL-cholesterol. These findings might be important in considering the concept of treatment of insulin resistance-related disorders in postmenopausal women.\nThe significant role of TNFα in insulin resistance, caused by inhibiting the transduction of insulin signaling and by down-regulation of glucose transporter GLUT-4 and insulin receptor substrate-1, has been repeatedly confirmed [45-48]. Our results supported these findings unambiguously only in women, while in men TNFα seemed paradoxically to interfere with other factors – mainly BMI and leptin – in influencing insulin resistance, thus playing a quite different role. Previously it was found [46] that correlation between serum TNFα on the one side, and insulin, HOMA IR, serum triglycerids, respectively, on the other side, was substantially more significant in women than in men. Serum concentration of TNFα in patients with type 2 diabetes of both genders correlated only with the quantity of intra-abdominal fat compartment [50]. Visceral obesity correlated with plasmatic aldosterone and with insulin resistance only in premenopausal women, but not in men [51].\nFrom all these data we might support our above mentioned conclusion – that rising of insulin resistance in hyperlipemic women was associated with an increase of intra-abdominal fat, because this fat mass in particular is a source of TNFα, which interfered with insulin sensitivity only in women. We came to this conclusion irrespective of the finding that the increase of serum TNFα in hyperlipemic women was statistically insignificant; results of Spearman's correlation (Table 3) and multiple regression analysis confirm a distinct role of this factor. In hyperlipemic men not only the serum concentration of TNFα has decreased instead of increasing, but according to multiple regression analysis it played a quite different role in influencing insulin sensitivity, interfering with factors that determined insulin resistance (leptin and BMI).\nIn the control group of men IgG anticardiolipin was inversely correlated to HOMA IR. The significance of this finding is not clear. These antibodies indicate vascular and thrombotic complications and oxidative modification of lipoproteins [52,53] and may represent an increased risk of atherogenic and inflammatory complications. In this case, however, their growing might be connected with an increase in insulin sensitivity. Anyway, ACL IgG evidently did not participate significantly in influencing the increase of insulin resistance associated with hyperlipidemia, although other anti-cardiolipin correlations could be masked by the relatively large inter-individual variations in this parameter.\nNeither serum concentration of hFABP, a factor ensuring transmembrane transport and oxidative metabolisation of long-chain fatty acids [54,55], was significantly changed in hyperlipemic and insulin resistant subjects of both genders. This factor was very weakly associated only with HOMA IR in women (see Table 4D), indicating that enhanced metabolisation of fatty acids in cells might to some degree contribute to insulin resistance.\n\nConclusions\nIn postmenopausal women as well as in men of approximately equal age serum leptin plays a significant role as an important determinant of insulin resistance. In addition to this factor, in women the grade of insulin resistance is very considerably influenced by serum triglycerides, tumor necrosis factor alpha, and by decreased concentration of HDL-cholesterol, while in men only a mild influence of BMI and decreased HDL-cholesterol is observed. These findings are explained as a consequence of gender-related differences in adipose tissue composition and/or distribution in both normal-weight and over-weight subjects and should be taken into account in treatment of patients with metabolic risk factors of cardiovascular diseases.\n\nList of abbreviations\nHDL-Cholesterol = high-density cholesterol\nLDL-cholesterol = low-density cholesterol\nHOMA IR = Homeostasis Assessment of Insulin Resistance\n= fasting insulin (μU/ml) * fasting glucose (mmol/l) / 22,5\nQUICKI = 1 / [log fasting insulin (μU/ml) + log fasting glucose (mg/100 ml) ]\nTNFα = tumor necrosis factor alpha\nBMI = body mass index\nR2 = coefficient of determination\nhFABP = heart fatty acid binding protein\nACL-IgG = IgG fraction of anticardiolipin\nTGL = triglycerides\nGLUT-4 = glucose transporter-4\nPPARγ = Peroxisome Proliferator-Associated Receptor gamma\nCV = coefficient of variation\n\nAuthors' contributions\nDr. Radka Lichnovská collected the clinical material, performed analysis of biochemical values and edited the manuscript.\nDr. Simona Gwozdziewiczová performed analysis of clinical and biochemical data and edited the manuscript.\nProf. Jirí Hrebícek initiated the study, participated in its design and coordination, and wrote and edited the manuscript.\n\n\n" ], "offsets": [ [ 0, 25493 ] ] } ]

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[ { "id": "pmcA2636784__text", "type": "Article", "text": [ "Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct\nAbstract\nBackground\nIn both vertebrates and invertebrates, the oviduct is an epithelial tube surrounded by visceral muscles that serves as a conduit for gamete transport between the ovary and uterus. While Drosophila is a model system for tubular organ development, few studies have addressed the development of the fly's oviduct. Recent studies in Drosophila have identified mating-responsive genes and proteins whose levels in the oviduct are altered by mating. Since many of these molecules (e.g. Muscle LIM protein 84B, Coracle, Neuroglian) have known roles in the differentiation of muscle and epithelia of other organs, mating may trigger similar differentiation events in the oviduct. This led us to hypothesize that mating mediates the last stages of oviduct differentiation in which organ-specific specializations arise.\n\nResults\nUsing electron- and confocal-microscopy we identified tissue-wide post-mating changes in the oviduct including differentiation of cellular junctions, remodeling of extracellular matrix, increased myofibril formation, and increased innervation. Analysis of once- and twice-mated females reveals that some mating-responsive proteins respond only to the first mating, while others respond to both matings.\n\nConclusion\nWe uncovered ultrastructural changes in the mated oviduct that are consistent with the roles that mating-responsive proteins play in muscle and epithelial differentiation elsewhere. This suggests that mating triggers the late differentiation of the oviduct. Furthermore, we suggest that mating-responsive proteins that respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Taken together, our results establish the oviduct as an attractive system to address mechanisms that regulate the late stages of differentiation and maintenance of a tubular organ.\n\n\n\nBackground\nMost internal organs, including the vascular and respiratory systems and the gastro-intestinal and urinary-genital tracts are comprised of a single epithelial tube or a network of tubes. Tubular organs serve as conduits for the transport of gases, liquids, or solutes, and serve as barriers between biological compartments. To create tubes with specific flow and barrier properties, the morphology of the tube must be precisely specified during development and modulated by physiology. To accommodate specific physiological roles, tissue-specific programs for differentiation are employed at the last stages of development. While much is known about the molecular and cellular basis of tube formation [1-6], little is known about the mechanisms that regulate the late stages of differentiation in which organ-specific specializations arise.\nThe conservation of genes and similarity in tubular organ design across taxa make Drosophila an excellent model for understanding organogenesis in higher animals. In Drosophila, the best understood tubular organs from a developmental point of view are the trachea and salivary gland. Studies of these organs reveal a general program for tubular organ development, in which combinatorial expression of global patterning genes specifies positions within the embryo for the subsequent activation of tissue-specific early genes and transcription factors. This program results in the activation of downstream genes involved in terminal differentiation of organ-specific specializations such as the cuticle that lines the tracheal lumen [1,2,4,7-9].\nThe Drosophila female reproductive tract is another tubular system, consisting of the uterus and a common oviduct (the main tube) that branches into two lateral oviducts. Regional differences in function are observed along the length of the tract, with egg activation occurring largely at the proximal end, in the lateral oviducts, and fertilization occurring at the distal end, in the uterus [10,11]. Unlike other tubular organs, little is known about the development of the female reproductive tract. However, regional differences in function suggest the presence of region-specific differentiation programs within the female reproductive tract.\nIn Drosophila, mating induces changes in female behavior and physiology via molecules transmitted in the seminal fluid. These changes are rapid and lead to a mated female state which is profoundly different from the unmated female state. While an unmated female lays few eggs and readily accepts the courtship efforts of a male, a mated female exhibits increased egg-laying and actively rejects males [12-19]. Microarray studies of whole flies reveal that the changes in egg-laying rate are accompanied by a change in gene expression. Within three hours of mating there is an increase in expression of a small number of genes [20,21]. Rapid changes in gene expression, as well as protein abundance, have also been observed in the female reproductive tract [22,23].\nIn the upper reproductive tract (lateral and common oviducts, hereafter, oviduct), mating induces an increase in immune related transcripts and down regulates transcription factors involved in cell growth and differentiation. At the protein level mating induces increased abundance of proteins associated with muscle assembly and function and cytoskeletal proteins associated with epithelial morphogenesis [23]. Since many of these mating-responsive proteins act in late differentiation pathways of muscle and epithelia elsewhere (e.g. Bent, Muscle LIM protein 84B(Mlp84B), Neuroglian (Nrg), Coracle (Cora)), we hypothesize that mating triggers similar differentiation in the oviduct. To test our hypothesis we characterized the ultrastructure of oviduct epithelia and muscle, as well as the pattern of innervation before and after mating. We then examined the effect of different mating regimes on oviduct mating-responsive cytoskeletal proteins and on female reproductive output. Our results suggest that active tissue remodeling takes place in the oviduct epithelia and musculature in response to mating. Furthermore, we found a striking increase in innervation of the oviduct after mating.\nOur results show that the reproductive tract is an attractive system to address mechanisms that regulate the late stages of tissue differentiation in a tubular organ. Unlike other tubular organs, the last differentiation stage of the oviduct is triggered by an extrinsic cue (mating). This makes it possible to experimentally control the onset of differentiation, with an opportunity to independently examine the effects of mating and age. In addition, it allows us to examine processes essential for reproduction.\n\nResults\nMating induces changes in oviduct lumen\nOur previous molecular profiling showed that mating promotes changes in actin-based cytoskeletal molecules and suggests that mating triggers molecular changes and tissue remodeling in the female reproductive tract that mediate its progression to a mature functional stage [23]. To gain insight into the mechanisms that underlie this progression, we used light and electron microscopy to determine the morphological status of the oviduct in unmated and mated 3-day old females.\nIn nearly all mated reproductive tracts processed for microscopy (8/9), an egg was located in one of the lateral oviducts, whereas an egg was never observed in the oviduct of unmated reproductive tracts (5/5) (Figure 1). This observation is consistent with previous studies that report increased ovulation and egg-laying at 6 h post-mating [24]. In all unmated reproductive tracts examined, the region between the lateral oviducts and the middle of the common oviduct was either tapered or constricted, whereas this region appeared relaxed in the mated reproductive tract (Figure 1A and 1D). These observations raise the possibility that the lumen is narrow in the unmated oviduct and larger in the mated oviduct. To address this possibility, we collected serial 1 μm longitudinal sections through the reproductive tracts of unmated and mated females and stained these sections with toluidine blue to survey the appearance of the lumen along the entire length of the oviduct (Figure 1B and 1E). Our examination reveals that, in unmated reproductive tracts, the lateral oviduct lumen has an irregular shape, while the common oviduct lumen appears straight. Moreover, in all the unmated reproductive tracts sectioned, we detected patches of darkly stained material in the lumen of the lower common oviduct. In the mated reproductive tract, the lumen of the upper oviduct (defined as the lateral oviduct and upper part of the common oviduct) has an irregular shape, and the lumen of the lower oviduct (defined as the lower part of common oviduct) appears straight. In addition, the lumen of the lower oviduct appears wider in mated than unmated reproductive tracts (Figure 1C and 1F). Interestingly, darkly stained material was not detected in the oviduct lumen of mated females. This observation appears to be consistent with the description made by Mahowald et al. [25], who reported that the oviduct lumen of unmated females is nearly filled with an intima-like matrix and that this matrix is reduced after mating.\nBecause 3-day-old mated females lay eggs, it is unclear whether the lack of lumenal material and increase in lumen size in mated females occurred before or after the passage of eggs. We suggest that mating directly or indirectly induces morphological changes in the oviduct that facilitate egg passage through the duct. Taken together, our observations lead us to propose that the oviduct lumen is closed and/or obstructed in the unmated reproductive tract, and that mating induces changes in the epithelia and/or muscle that \"open\" the oviduct lumen.\n\nInitial formation of cell-cell junctions in oviduct epithelia is mating-independent\nTo determine whether mating induces specific morphological changes in the oviduct epithelia post-mating, we next examined the ultrastructure of the oviduct epithelia in unmated and mated reproductive tracts. Since molecular profiling demonstrates that proteins associated with cellular junctions such as α- and β-Spectrin (Spec), Cora, and Nrg [23] increase post-mating, we first determined the status of the cellular junctions in the oviduct epithelia of unmated females, and whether these junctions change post-mating. In Drosophila, most ectodermally derived epithelia (such as the epidermis and trachea), with a few exceptions, are joined apically by a belt-like adherens junction called the zonal adherens junction (ZA) followed basally by a septate junction (SJ) [26]. Our analysis reveals that the oviduct is lined, along its entire length, by a monolayered epithelium comprised of squamous-type cells. Although region-specific differences in morphology were observed, all oviduct epithelia examined, in both unmated and mated females, are joined along their lateral membranes by an extensive SJ and lack an apical ZA (Figure 2). SJs and ZAs form complete belts that surround the epithelial cell, thus making these junctions easily visible in transverse sections through the epithelium. Because ZAs were not detected in our transverse sections through the oviduct, this implies that ZAs never formed, or developed earlier and were lost (Figure 2D). Interestingly, we did not detect any ultrastructural differences in the SJs at 6 h post-mating, but we did uncover differences in SJ ultrastructure in different regions of the oviduct.\nBased on their ultrastructure, two types of SJs, smooth and pleated, can be distinguished in Drosophila [26]. Smooth SJs are distinguished by the lack of visible septae and the appearance of electron dense material in the intercellular space, while pleated SJs are distinguished by the ladder-like appearance of septae. In the lateral oviducts and upper common oviduct, septa were not detected in the SJ, thus these SJs represent smooth SJs or an immature stage of pleated SJ (Figure 2A, 2A', 2A\"). In contrast, a ladder-like arrangement of septae was often visible in the SJs of the lower common oviduct (Figure 2C'), thus these SJs can be classified as pleated. Unlike the smooth-like SJs of the upper oviduct, the pleated SJs of the lower oviduct are followed basally by spot type adherens junction (spot AJs) (Figure 2B, 2B'; additional file 1). Further analysis is necessary to determine if the SJs of the upper and lower oviduct represent different types or different developmental stages. Our findings demonstrate that the initial formation of SJs, as well as spot AJs in the lower oviduct, are mating-independent. This raises an interesting question. Why are SJ proteins such as Cora and Nrg up-regulated post-mating if SJs are formed prior to mating? It is possible that the increased expression of SJ proteins is associated with functional changes in polarized secretion post-mating. Recent studies have shown that SJs play an unexpected role in regulating the apical secretion of specialized extracellular matrix molecules in the trachea [27,28], and that these molecules are important regulators of lumen size.\n\nMating modulates apical secretory activity in the oviduct\nGiven the presence of extensive SJs in the oviduct and the role of SJs in regulating apical secretion of extracellular matrix molecules in other epithelia (e.g. trachea), we asked if mating modulates apical secretion in the oviduct epithelia. Our ultrastructural analysis reveals that different regions of the oviduct display different apical membrane morphology (i.e microvilli or pleats) (see Figures 2 and additional file 2A and 2A), but all epithelia are covered by an electron dense apical extracellular matrix (AECM) and a thin layer of cuticle. We found that mating induces ultrastructural changes in the AECM and cuticle in both the upper and lower oviduct. In the upper oviduct of the unmated female, the AECM varies in thickness along the apical surface (Figure 3A). Some areas have little AECM, while other areas are covered by a distinct layer of AECM (~1–2 μm in thickness; Figure 3B). However, the AECM of mated females is more evenly distributed along the apical surface, (~2 μm in thickness; Figure 3E). Strikingly, the AECM and cuticle of mated females have a ruffled appearance, suggesting that the AECM and cuticle have increased in surface area (Figure 3F). Electron dense granules up to ~1.5 μm in diameter were occasionally observed in both the AECM and cell cytoplasm (Figure 3F). Although further analysis is needed to determine the role of these granules in the oviduct epithelia, it is possible that these granules participate in the secretion and deposition of the AECM. Taken together, our findings suggest that polarized secretion via the AECM, while ongoing in the upper oviduct of the unmated female is enhanced and/or modulated post-mating.\nPost-mating changes in AECM ultrastructure are also observed in the lower common oviduct. However, unlike the AECM of the upper oviduct, the AECM of the lower oviduct is well developed prior to mating. In the lower oviduct of the unmated female, the AECM consists of an amorphous electron dense material and is unevenly distributed, forming a thick, bulbous layer above the plasma membrane in some regions (additional file 2). Matrix-like material is also observed in the lumen, but this material is more electron dense than the AECM (additional file 2A and 2A). In the mated female, the AECM is flattened against the plasma membrane and is uniformly distributed along the apical surface (additional file 2B and 2B). Matrix-like material was not observed in the center of the lumen, but small pools of very electron dense material were detected in the spaces between the epithelial folds (additional file 2C and 2C). This may explain why lumenal matrix was not detected at the light microscopic level in the mated female oviduct (see Figure 1E and 1F). Taken together, our observations suggest that the lower common oviduct is a site of active apical secretion in both mated and unmated females, and that matrix secretion, particularly in the lumen, is reduced post-mating. These findings raise the intriguing possibility that the AECM and lumenal matrix function as a plug in the lower oviduct, and that mating induces the breakdown of this plug.\n\nMating induces changes in hemi-adherens junctions in upper oviduct\nIn addition to modulating secretion at the apical membrane, mating induces changes at the basolateral membrane. In many epithelia, one of the last steps of differentiation is the development of a layer of extracellular matrix (ECM) called the basal lamina that covers the apical and/or basal membranes and the concomitant development of hemi-adherens junctions (HAJs). HAJs connect the cell cytoskeleton with the ECM and are formed at virtually all cell surfaces that contact an ECM. HAJs can be distinguished at the ultrastructural level as a patch-like, electron dense undercoat of the plasma membrane that opposes the basal lamina ([26]; Figure 3G, 3H). In the Drosophila embryo, the HAJs and basal lamina are formed at the same time [26]. Because the basal lamina is established at a time when the majority of extracellular matrix molecules are actively secreted [26,29], this suggests that the formation of HAJs is tightly coordinated with the secretion of the ECM.\nOne of the most striking post-mating changes observed in the oviduct epithelia was the appearance of numerous HAJs along the basolateral membrane in the upper oviduct (Figure 3). The importance of HAJs, particularly in the upper oviduct, is underscored by the extensive infolding of the basolateral membrane that is observed in both unmated and mated females (Figure 3A and 3E). The infolded membrane gives rise to a highly branched intercellular space that is filled with an ECM (Figure 3C and 3G). This ECM is contiguous with the basal lamina that surrounds the epithelia. Few HAJs were observed in the upper oviduct of the unmated female, and these were largely restricted to the basal membrane, and not observed along the basolateral infolding (Figure 3C). In contrast, numerous HAJs appear along the basolateral infolding post-mating in this region of the oviduct (Figure 3F–3H). In addition, the intercellular space appears wider post-mating (Figure 3C–3D and 3G–3H), suggesting that mating induces increased secretion and/or deposition of the ECM in this cellular compartment and brings the ECM to a threshold concentration that can support the development of HAJs. HAJs were also detected along the basal membrane, but they were not detected along the apical membrane even though this membrane was covered by an ECM. Interestingly, while the basolateral membrane forms very shallow folds in the lower oviduct (see additional file 2), HAJs were observed along this membrane in unmated reproductive tracts (data not shown), thus suggesting that the epithelia is more differentiated in this region of the oviduct, and that the differentiation of the upper and lower oviduct may be under different control.\n\nMuscle differentiation is enhanced post-mating\nWhile we uncovered post-mating changes in the oviduct epithelia that might facilitate its transition to a high egg-laying state, this transition may also be mediated by changes in oviduct muscle properties and/or activity. The oviduct is lined by circular muscle fibers with supercontractile characteristics [30]. Our previous studies showed that mef2 and mlp84B genes that regulate muscle differentiation, are expressed and increased post-mating in the oviduct, as well as in the sperm storage regions of the reproductive tract [22,23]. This suggests that mating induces muscle differentiation in the reproductive tract. Muscle differentiation is characterized by the assembly of myofilaments into bundles called myofibrils. As muscles differentiate, myofibrils and z-bodies appear simultaneously, and increase in number until the cytoplasm is filled with myofibrils [31]. Like epithelia, one of the last steps of muscle differentiation is the secretion of a basal lamina that surrounds the muscle fiber. To determine if mating induces structural changes in muscles (such as increased myofibrils) we examined the ultrastructure of muscle fibers in the upper and lower parts of the oviduct. Our analysis revealed that, in both unmated and mated reproductive tracts, the muscles of the lower oviduct are highly differentiated as evidenced by the high density of myofibrils, well developed and aligned z-bodies, and secretion of a thick, electron dense basal lamina (Figure 4E and 4F). In contrast, muscle fibers in the lateral oviducts and upper common oviduct appear less differentiated than muscles in the lower common oviduct, as evidenced by the lesser density of myofibrils and z-bodies, and little or no basal lamina (Figure 4A and 4B). Moreover, the muscles of the upper oviduct appear more differentiated in the mated than in unmated reproductive tracts (Figure 4A–4D). Interestingly, we observed neighboring muscle fibers in different states of differentiation in the lateral oviducts in both unmated and mated reproductive tracts, but not elsewhere in the oviduct (Figure 4B). These results suggest that mating enhances the rate of muscle differentiation in the upper oviduct, and that muscle differentiation is delayed in the upper as compared to the lower oviduct. The increased muscle differentiation in the upper oviduct is not dramatic and likely reflects the short post-mating period examined in this study. The delayed differentiation of the upper oviduct muscles resembles the delay in the onset of development between the adult thoracic muscles and abdominal muscles during metamorphosis [32]. Since the ovaries and the other parts of the reproductive tract are known to have different segmental origins [33], we hypothesize that different parts of the oviduct develop at different rates or begin development at different times.\n\nIncreased innervation in the oviduct post-mating\nNerve-muscle interactions play an important role in regulating adult muscle development and refining the final pattern of innervation [34,35]. Given that oviduct muscle differentiation is enhanced post-mating, we predicted that mating either directly or indirectly induces changes in innervation. To address this prediction, we quantified the number of nerve terminals or boutons innervating the lateral oviducts and common oviduct in unmated and mated reproductive tracts. Studies of oviduct innervation in Drosophila reveal that the fly's oviduct receives aminergic, peptidergic and glutamatergic input [30,36-39]. In both larval and adult Drosophila, different types of boutons are formed by neurons that express different neurotransmitters and modulators [40-42]. By similarity to the boutons described at the larval and adult neuromuscular junction, Middleton et al. [30] report that the fly's oviduct is innervated by glutamatergic type I boutons and tyraminergic/octopaminergic type II boutons. Rodgriguez-Valentin et al. [43] further report that the oviduct type II boutons co-express octopamine and glutamate. The neurons that give rise to the type I innervation have not been identified. However, it is well established that type II innervation arises from octopaminergic neurons located in the abdominal ganglion [30,43]. In addition, it has been shown that some or all of these neurons express a GAL4 insertion line for the bullwinkle (bwk) gene [43]. Bwk encodes a HMG-box containing putative transcription factor [44]. To determine if mating induces any changes in the number of type I and II boutons innervating the oviduct muscles, we used the pan-neural marker, anti-HRP to label all oviduct boutons in unmated and mated females. To distinguish between type I and II boutons we used an antibody against the Disc Large (DLG) protein [45]. Type I boutons were identified by their DLG postsynaptic staining and large size (> 8 μm in diameter) (Figure 4G), while type II boutons were distinguished by their absence of DLG staining and smaller size (< 2 μm) (Figure 4H). We find that type I and II boutons innervate the lateral oviducts and common oviduct, and that the type I innervation is restricted to a few axons that run parallel to the length of the oviduct, while the type II innervation is more widespread. We quantified the number of boutons in the lateral oviducts and common oviduct and observed a 74% increase in bouton number in the lateral oviduct and a 66% increase in the common oviduct post-mating (Figure 4I). More over, we observed no significant change in the number of type I boutons in the lateral oviduct and common oviduct. However, we detected a 1.5-fold increase in the number of type II boutons in the lateral oviduct and a 1.8-fold increase in type II innervation in the common oviduct. Dramatic increases in bouton growth are also observed during development. For example, a ten-fold increase in bouton number is observed at the neuromuscular junction during the larval period [46]. To determine if the increase in type II innervation was specific to mating or reflected normal growth in 3 day-old females, we quantified type I and II innervation in the oviducts of 5 day-old unmated females. We found no significant difference in type I and II innervation in unmated 3 day-old and 5-day-old females, indicating that mating, either directly or indirectly, induces a dramatic increase in type II innervation (Figure 4I). To determine if the post-mating increase in innervation is unique to the oviduct, we asked if mating induces a global change in innervation. We quantified bouton number in the adult ventral midline muscles of the 5th abdominal segment. These muscles are innervated by boutons that increase in number during metamorphosis [47]. No significant difference in bouton number was detected at these muscles in unmated and mated females (additional file 3). Though further analysis is needed, this suggests that the post-mating increase in innervation is oviduct-specific. Because the type II boutons are octopaminergic, the increased type II innervation may result in increased octopamine (OA) release in the oviduct. In support of this possibility, we have preliminary evidence that OA is released in the oviduct post-mating (Heifetz and Wolfner, in preparation). Studies in locust and Drosophila demonstrate that OA inhibits oviduct contraction, while glutamate activates oviduct contraction [30,43,48]. In Drosophila, electrical stimulation of the posterior abdominal nerve gives rise to a series of muscle contractions in the oviduct followed by a period of muscle fatigue or relaxation [43]. This pattern of muscle contraction and relaxation may facilitate the proper movement of the egg through the oviduct. In their study of bwk expressing neurons that innervate the oviduct, Rodriguez-Valentin et al. [43] show that OA and glutamate interact to produce the pattern of oviduct contraction and relaxation described above. It is therefore possible that the post-mating increase in type II innervation in the oviduct plays an important role in the increased rate of ovulation and egg-laying observed post-mating.\n\nFemale mating history affects the enrichment of cytosekeletal proteins in the oviduct\nTo gain insights into the role of cytoskeletal protein enrichment ([23]; additional file 4) in mediating the morphological changes detected in this study, we examined the effect of different mating regimes on cytoskeletal protein abundance. We focused on a subset of mating-responsive cytoskeletal proteins with well established roles in the differentiation of muscle and epithelia. These include: (i) Mlp84B which regulates the late differentiation pathway of muscle [49]; (ii) Cora and Nrg which are required for the formation of septate junctions in epithelia [50], and (iii) Hu-li tai shao (Hts), also known as adducin-like protein, which functions in assembly of the cytoskeletal network. Na+ pump α subunit (ATPα), another protein associated with septate junctions in epithelia, is not a mating-responsive protein and was used as a control. Using western blots, we first determined the abundance of the cytoskeletal proteins in oviducts of 3-day-old unmated and mated females at 6 hrs post-mating. We confirmed the proteomic results of Kalpenikov et al. [23] and found that mating increases the abundance of all proteins, except ATPα in mated oviducts relative to their abundance in unmated oviducts (Figure 5A).\nTo determine whether the increased abundance of mating-responsive proteins persists for longer times post-mating, we examined oviducts of 10-day-old females that mated once at 3 days of age, and calculated the abundance of the mating-responsive proteins relative to their level in oviducts of 3-day-old unmated females. We found no change or a slight increase in the relative abundance of all cytoskeletal proteins except Mlp84B at 7 days post-mating (Figure 5A). Strikingly, the level of Mlp84B declines by 7 days post-mating to the level observed prior to mating. Thus Mlp84B levels rise and fall after mating, while the epithelial-related proteins rapidly rise and are maintained at a high level after mating. This raises the possibility that a second mating might trigger an increase in Mlp84B protein expression as observed in 3-day-old females at 6 h post-mating. To test this possibility, females were mated twice (once at day 3, and once on day 10 of age), and their oviducts were examined at 6 hrs after the second mating. We calculated the abundance of the cytoskeletal proteins in the twice mated oviducts relative to their abundance in oviducts of 3-day-old unmated females. Our results show that a second mating has little or no effect on Mlp84B abundance. Thus, Mlp84B may represent a class of mating-responsive proteins that is only needed after the first mating. Interestingly, the effect of the second mating on the epithelial-related mating-responsive proteins appears to be different for each protein. While Cora levels drop to the level observed in 3-day-old unmated females, Nrg and Hts are maintained at a high level.\nTo determine if the changes in cytoskeletal protein abundance are mating-dependent we measured their abundance in the oviducts of unmated 5- and 10-day-old females. We calculated their abundance relative to their level in oviducts of unmated 3-day-old females (Figure 5B). We rationalized that if the change in cytoskeletal protein abundance is mating-dependent we will not see similar changes in unmated females. We observed a slow increase in the relative abundance of all mating-responsive proteins with time post-eclosion (Figure 5B). Because unmated females lay more eggs as they age (see additional file 5C) one possible interpretation of the increased level of cytoskeletal proteins in unmated females is that these proteins are associated with an intrinsic program for oviduct maturation and that mating accelerates this process to maximize egg-laying efficacy. Alternatively, it is possible that the slow increase in protein abundance observed in unmated females is due to the passage of eggs through the oviduct.\nTaken together, our results suggest that mating is essential to fine-tune the levels of the mating-responsive proteins examined in this study. Because the changes in cytoskeletal protein abundance are different in unmated and mated females, this suggests that the post-mating changes are mating-dependent. Furthermore, we suggest that these post-mating changes are linked to changes in oviduct function.\n\nEarly or prior mating increases fecundity\nIn Drosophila, female fecundity decreases with age [51-54]. It has been proposed that this decrease is due, in part, to the loss of germline and somatic stem cells [55]. Since the expression of the oviduct cytoskeletal proteins examined in this study change with age and mating experience, the state of the oviduct may also play a role in fecundity. To separate the effects of age and mating, we measured the fecundity of females that mated twice, first at 3 days post-eclosion and again at 10 days, and compared that to the fecundity of females that mated once at 3 days and females that mated once at 10 days. Fecundity was measured as the number of eggs laid per day per female during the first three days after mating. Once-mated 3-day-old females laid nearly twice as many eggs as once-mated 10-day-old females during the three days examined (24.5 ± 0.7 versus 13.3 ± 0.9, p < 0.0001). Twice-mated 10-day-old females also laid about 50% more eggs than once-mated females of the same age (19.3 ± 0.9 versus 13.3 ± 0.9, p < 0. 0001), but about 20% fewer than laid by once-mated 3-day-old females during the three days examined (19.3 ± 0.9 versus 24.5 ± 0.7, p < 0.0001) (Figure 6A, see also additional file 5A and 5B). Thus, the difference in fecundity between once-mated 10-day-old and once-mated 3-day-old females is not a result of age alone. Rather the main determinant of fecundity at 10 days is whether there had been a prior mating at 3 days. We also calculated the fertility (number of adults eclosed) of once- and twice-mated females. Once-mated 3-day-old females are more fertile than once-mated 10-day-old females (69.5 ± 1.6% versus 56.1 ± 3.0%, p < 0.0001) and slightly more fertile than twice-mated 10-day-old females (69.5 ± 1.6% versus 62.7 ± 2.4%, p < 0.015) (Figure 6B, see also additional file 5D). Because there is no significant difference in fertility between once-mated and twice-mated 10-day-old females, this suggests that (1) a prior mating has no significant effect on the fertility of 10-day-old mated females and (2) fertility decreases with age. One intriguing interpretation of these results is that an early mating increases fecundity which partially compensates for the age-related decrease in fertility. Thus, cytoskeletal mating-responsive protein changes may be associated with structural changes in the oviduct that result in increased fecundity. This may counteract the effects of decreased fertility on the reproductive output.\n\n\nDiscussion\nDespite the use of Drosophila as a model system for organ-level biology and the emerging parallels between mammalian and Drosophila reproductive biology [56], this is the first integrative tissue-wide study of post-mating changes in the Drosophila oviduct. Our results provide several lines of evidence at the molecular, morphological and physiological levels suggesting that mating induces tissue-wide differentiation in the oviduct. Moreover, we identify ultrastructural changes in the mated oviduct that are consistent with the roles that some of the mating-responsive proteins examined in this study (e.g. Mlp84B, Cora, Nrg) are reported to play in muscle and epithelial differentiation elsewhere. For example, the increased abundance of Mlp84B, a major regulator of the late differentiation pathway of muscle [49] is consistent with the increased muscle differentiation in the upper oviduct post-mating (Figure 4A–4F). Similarly, the increased abundance of Cora and Nrg, molecules that are essential for SJ development and function [50] is consistent with the observation that SJs in the upper oviduct are immature and/or that mating induces changes in the apical extracellular matrix whose secretion may be regulated, in part, by SJs as occurs in the trachea [27,28]. Other post-mating changes that indicate that mating induces tissue-wide differentiation include increased HAJs along the basolateral membrane and increased innervation.\nAnalysis of protein abundance following different mating regimes (unmated, once-mated, twice-mated) gave us further insights into the possible roles that mating-responsive proteins play in the oviduct. For example, Mlp84B is only responsive to the first mating, while the epithelial proteins examined (Cora, Nrg and Hts) are responsive to the first and second mating. Furthermore, the response to the second mating is different from the response to the first mating. Taken together, these results suggest that Mlp84B is required for the final maturation of the oviduct, while the epithelial proteins examined are required for both the maturation and maintenance of the oviduct at a high functional state. Moreover, the post-mating pattern of Mlp84B supports the idea that the first mating induces the final maturation of the oviduct. The rise and fall of Mlp84B abundance after the first mating (Figure 5) parallels the expression pattern of Mlp84B during development where peaks in Mlp84B transcription occur during periods of embryogenesis and metamorphosis when muscle is differentiating [49].\nUsing different mating regimes we tested whether the first/early mating is essential for maintenance of high reproductive output in the second mating. Our results suggest that mating at an early age is essential to achieve maximum reproductive output (i.e. high fecundity and fertility). Since the first mating increases reproductive output (evidence from our mating regime experiments), it is likely that the ultrastructural changes detected in mated 3-day-old females lead to a highly functional oviduct.\nWe suggest that the final maturation of the oviduct includes a mating-dependent stage. We propose that during the first few days post-eclosion, the oviduct undergoes the first phase of differentiation, after which the oviduct is developmentally poised for a rapid response to an extrinsic cue (mating). Mating then triggers the second phase of maturation (tissue remodeling and modulation) which is essential for proper oviduct function (Figure 7). We further propose that the second phase of maturation consists of processes that are mating-independent and -dependent, and that both of these pathways are essential to produce a functional oviduct. For example, initial formation of SJs occurs prior to mating (mating-independent) while the increased apical secretion and development of HAJs are mating-dependent. The oviduct musculature is an example where both mating-independent and -dependent processes play a role. Muscles are highly differentiated in the lower oviduct prior to mating, while muscle differentiation is ongoing in the upper oviduct and increases after mating. Although the onset of muscle differentiation in both regions is mating-independent, the further differentiation of muscle in the upper oviduct is mating-dependent. Another possible interpretation of the role of mating on oviduct maturation is that mating accelerates and synchronizes processes that are essential for the functional maturation of the oviduct. It will therefore be interesting to examine the oviducts of older females to determine the status of oviduct maturation.\nWhat is the benefit of mating-induced differentiation of the oviduct tissues? Unmated females are capable of laying eggs, albeit at a reduced rate as compared to mated females of the same age. One possible interpretation is that reproduction is energetically costly, thus delaying oviduct maturation until sperm is available is advantageous to the female. This may reflect the evolution of a mechanism to optimize reproductive capacity in early adulthood in short-lived animals.\nIn summary, we have identified events at the cellular, molecular and physiological levels that are part of an efficient and specific program for reproduction. Drosophila affords us the opportunity to uncover the signaling pathways that coordinate these events to produce a physiologically functional organ.\n\nMethods\nFlies\nWild-type Canton-S flies were used for the fecundity/fertility experiments and confocal analysis. Wild-type Canton-S5 [57] flies were used for electron microscopy. All flies were kept in a 12 hrs light/dark cycle at 23 ± 2°C. Upon eclosion, females and males were collected on ice and held separately until 3 (females and males) or 10 (females) days of age.\n\nSample preparation\nFor all assays (unless described differently) unmated females were placed with 3-day-old unmated males and observed until mating initiated. At the end of mating, females were aspirated into fresh vials and held for 6 hrs. At 6 hrs after the start of mating, females were placed on ice for dissection. Previous molecular studies indicate changes in protein abundance at 3 hrs post-mating. We hypothesize that these molecular changes will translate into morphological changes in the next few hours. We chose to analyze the morphology of mated females at 6 h post-mating as opposed to later times post-mating because the changes observed at later times may be due, in part, to the high rate of eggs passing through the oviduct.\n\nElectron microscopy\nReproductive tracts were dissected in Schneider's Drosophila medium (Sigma) on ice and processed for electron microscopy as described in [42]. Tracts were flat-embedded between two sheets of Aclar (Electron Microscopy Sciences), which allowed us to image the entire tract at the light microscopic level prior to sectioning. Sections were cut on a Reichart Ultracut microtome. One-μm thick sections were stained with 1% toluidine blue, and viewed with a Zeiss Axoplan microscope. Ultrathin sections (~100 nm) were mounted on formvar grids, stained with lead citrate, and viewed with a Philips/FEI Morgagni 268 TEM at 80 kV. Our analysis is based on 4 unmated samples and 3 mated samples. Two unmated samples were cut in the longitudinal plane and two additional unmated samples were cut in the transverse plane, while one mated sample was cut in the longitudinal plane and two additional mated samples were cut in the transverse plane. For longitudinal sections, the entire tract was re-embedded and cut. For samples cut in the transverse plane, the flat-embedded reproductive tract was divided into three regions: (1) lateral oviducts and upper common oviducts, (2) middle common oviduct, and (3) lower common oviduct. Each region was re-embedded and sectioned. It is beyond the scope of this paper to describe all three regions, and our analysis focuses on the uppermost and lowermost regions.\n\nImmunocytochemistry\nReproductive tracts were dissected in Yamamoto's Ringer (10 mM MOPS; 80 mM NaCl; 10 mM KCL; 0.2 mM MgCl2; 0.1 mM CaCl2) with 5% (w/v) sucrose on ice, fixed in 4% paraphormaldehyde in PBS (phosphate-buffered saline; 0.85% NaCl, 1.4 mM KH2 PO4, 8 mM Na2 HPO4, pH 7.4) for 45 min and then washed in PBS. The reproductive tracts were then incubated in blocking solution (0.5% Triton x-100, 3% NGS, 0.1% BSA) for 2 hrs at room temperature. The following primary antibodies, reagents, and dilutions were used: Cy3-conjugated goat anti-HRP, 1:200 (Jackson Immunochemicals, West Grove, PA); mouse anti-Disc Large (DLG), 1:1000 (Developmental Hybridoma Bank), Alexa Fluor 488-phalloidin, 1:200 (Invitrogen, Molecular Probes, Scotland). Secondary antibodies were Alexa Flour 488-conjugated Goat anti-mouse, 1:200 and Alexa Flour 546-conjugated Goat anti-rabbit, 1:200 (Invitrogen, Molecular Probes, Scotland). Reproductive tracts were incubated with the different primary antibodies (diluted in PBS + 0.2% Triton x-100) for 2 hr at room temperature, washed with PBST, incubated with secondary antibodies for 2 hrs at room temperature and washed with PBS. Reproductive tracts of the different treatments were mounted with Antifade media [58] on a multi-well glass slide (Hendley-Essex, UK). For each treatment (unmated, mated) and antibody/reagent (HRP, DLG, phalloidin) a minimum of ten reproductive tracts from at least two independent biological replicates were prepared.\n\nConfocal microscopy\nReproductive tracts were viewed with a Zeiss 510 laser scanning confocal microscope using 20× and 60× objective with additional zooming. Optical sections from different focal plans of each reproductive tract region (lateral oviducts, common oviduct, uterus) were collected and projected as a reconstructed three-dimensional image using LSM image browser (version 3,5,0,376) software. Image collections were identical for each of the different reproductive tract regions analyzed.\n\nQuantitation of bouton number\nTo quantify the number of boutons in the lateral and common oviducts we used ImageJ software (1.37b, National Institutes of Health) to analyze confocal images of anti-HRP and anti-DLG labeled boutons in the oviduct. The number of boutons per unit area was quantified with the Particle Analysis Tool. Briefly, to differentiate between the boutons, the particle analysis tool requires the image to be a \"binary\" image (i.e., black or white), thus we first converted the images to gray scale. We then set a \"threshold\" range so that pixels in the image whose value lies in this range are converted to black; pixels with values outside this range are converted to white. We next defined a region of interest (ROI) within the oviduct to count particles (i.e. count boutons). This ROI was saved and served to measure the number of boutons per unit area in each treatment. For each oviduct we counted the number of anti-HRP and anti-DLG labeled boutons in two ROIs within the lateral oviducts and two ROIs in the common oviduct. One-way ANOVA (SPSS 15.0) was used to measure the difference in bouton number per unit area in different regions of the oviducts, in both unmated and mated females.\n\nQuantitation of cytoskeleton proteins\nSample preparation\nTo evaluate the effect of mating on the abundance of the cytoskeleton proteins tested, females were: (i) aged for 3 days, mated with 3-day-old unmated males and their oviducts were dissected after 6 hrs post-mating (Once3); (ii) aged for 3 days, mated with 3-day-old unmated males and their oviducts were dissected after 7 days (Once3 day10); (iii) aged for 3 days, mated first with 3-day-old unmated males and held singly for 7 days. At 10 days of age, female were mated again with 3-day-old unmated males. Oviducts were dissected at 6 hrs post-second mating (Twice3&10). We also examined 5-day-old and 10-day-old unmated females (UM5, UM10 respectively).\n\nSDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting\nFor each mating regime, sixty oviducts were pooled and 30 μl of SDS-PAGE sample buffer was added as described in [59]. Samples were boiled, and then frozen at -20°C until loading. SDS-PAGE was performed on 12% polyacrylamide gels and western blotted as in [60]. Proteins were cross-linked to the filter. The following primary antibodies and dilutions were used: mouse anti-Neuroglian (kindly provided by M. Hortsch) 1:250; Guinea pig anti-Coracle (kindly provided by R.G. Fehon) 1:2500; rabbit anti-Mlp84B (kindly provided by M. Beckerle) 1:1000; mouse anti-hts (1B1, Developmental Studies Hybridoma Bank, DSHB) 1:75; mouse anti-Na, K-ATPase (α5, DSHB) 1:100. Secondary antibodies included: anti-Guinea pig IgG (peroxidase conjugated), anti-Rabbit IgG and anti-Mouse IgG (developed in goat, Sigma, Israel) 1:10,000. Proteins were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham Piscataway, NJ).\n\nAnalysis\nThe developed film was scanned and the signal intensity (protein abundance) of each band was determined using ImageJ software (1.37d, National Institutes of Health). We evaluated protein abundance by measuring the mean gray value of a specific band and the background. The mean gray value of the background was then subtracted from that of the measured band. Relative protein abundance in mated oviduct vs. 3-day-old unmated oviduct was then calculated. Four independent biological replicates were prepared for each mating status. The reported abundance (see Figure 5) is the relative ratio (mated/unmated or unmated/unmated) of at least three replicates that showed the same trend.\n\n\nExamination of female reproductive output\nMating regimes\nTo evaluate the effect of mating on reproductive output females were treated as follows: (i) aged for 3 days and mated with 3-day-old unmated males (Once3); (ii) aged for 10 days and mated with 3-day-old unmated males (Once10); (iii) aged for 3 days, mated first with 3-day-old unmated males, held for 7 days and mated again with 3-day-old unmated males (Twice3&10). In all cases male and female pairs were observed to record mating initiation and termination.\n\nAnalysis\nFollowing mating, females were aspirated into fresh vials, held singly and allowed to lay eggs for 6 hrs, then transferred daily (each 24 hrs) to fresh vials. The number of eggs laid and the number of eclosed adults were counted from vials created at 6 hrs, 1, 2 and 3 days post-mating. To ascertain the baseline of female egg-laying, we also included in our experiment unmated females that were kept in the same conditions as mated females. The number of eggs laid by unmated females was counted from vials created at 6 hrs, 1, 2 and 3 days after placing the females in the holding vials. In addition, we also recorded the pattern of unmated female egg-laying for 10 days.\nTo determine the effect of different mating regimes on female reproductive output (i.e. fecundity and fertility), we used One-way ANOVA (SPSS 15.0).\n\n\n\nAbbreviations\nNrg: Neuroglian; Cora: Coracle; Spec: α- and β-Spectrin; SJ: septate junction; SSJ: smooth septate junction; PSJ: pleated septate junction; ZA: zonal adherens junction; AJ: adherens junction; AECM: apical extracellular matrix; ECM: extracellular matrix; HAJ: hemi-adherens junction; SAJ: spot adherens junction; OA: Octopamine; Hts: Hu-li tai shao; ATP α: Na+ pump α subunit; Mlp84B: Muscle LIM protein at 84B; DLG: Disc Large; HRP: horseradish peroxidas; UM: unmated; M: mated.\n\nAuthors' contributions\nAK and PKR contributed equally to this manuscript. AK, PKR and YH conceived and designed the project and analyzed the data. AK performed the confocal, Western blots and fertility assays. PKR and AK performed the light microscopy. PKR conducted the electron microscopy. AK, PKR and YH wrote the manuscript. RRH contributed to design of the study and revision of the manuscript. All authors participated in the discussion and approval of the final manuscript.\n\nSupplementary Material\n\n\n" ], "offsets": [ [ 0, 50056 ] ] } ]

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[ { "id": "pmcA1564398__text", "type": "Article", "text": [ "The role of the muscarinic system in regulating estradiol secretion varies during the estrous cycle: the hemiovariectomized rat model\nAbstract\nThere is evidence that one gonad has functional predominance. The present study analyzed the acute effects of unilateral ovariectomy (ULO) and blocking the cholinergic system, by injecting atropine sulfate (ATR), on estradiol (E2) serum concentrations during the estrous cycle. The results indicate that ULO effects on E2 concentrations are asymmetric, vary during the estrous cycle, and partially depend on the cholinergic innervation.\n\nBackground\nEstradiol secretion is regulated by pituitary [follicle stimulating hormone (FSH) and luteinizing hormones (LH), prolactin, and adrenocorticotropin (ACTH)]. The effects of these hormones are modulated by neurotransmitters released by the intrinsic ovarian innervation near the follicular wall. Acetylcholine produced by the follicle may be one of the neurotransmitters participating in modulating the effects of pituitary hormones on the follicle [1-3].\nEvidence suggesting that one gonad has functional predominance in mammals and birds have been published [1,4-8]. In previous studies we have shown that unilateral ovariectomy (ULO) modifies progesterone and/or testosterone serum concentrations, and that the effects of ULO depend on both, the stage of the estrous cycle when ULO was performed and the ovary (left or right) remaining in situ [9-11].\nAsymmetry in ovarian functions has been explained by differences in the ovarian innervation participating in modulating the effects of gonadotropin on the ovarian follicles [1,6]. Kawakami et al [12] showed that electrical stimulation of the medial basal pre-chiasmatic area, the ventro-medial hypothalamus, and the areas in the mesencephalon of hypophysectomized and adrenalectomized female rats resulted in a significant increase of estradiol (E2) and progesterone (P4) plasma concentrations in the contra-lateral ovarian venous blood. In turn, stimulating the dorsal hippocampus, the lateral amygdala, and the mesencephalic areas resulted in lower E2 and P4 concentrations. Ovarian denervation of rats in proestrus stage blocks E2 secretion induced by stimulating the medial basal pre-chiasmatic area. In addition, the electrochemical stimulation in proestrus day of the medial basal pre-chiasmatic area of untreated rats increased E2 and P4 concentrations in serum. This effect was not observed when stimulation was applied to the pre-optic supra-chiasmatic area. According to the authors' interpretation of the results, the efferent neural system connecting the brain and the ovaries is supplementary to the brain-pituitary-ovarian hormonal mechanisms regulating ovarian steroid secretion, and the system may be required for adjusting ovarian responsiveness and sensitivity to gonadotropins [12,13].\nGerendai et al. [14] described a multi-synaptic neural pathway between the central nervous system and the ovaries, with the vagus nerve being one of the main neural pathways. In ULO treated rats, bi-lateral sectioning the vagus nerve (ventral or dorsal) results in lower compensatory ovarian hypertrophy. The effects of sectioning the left vagus nerve depend on the remaining ovary in situ: rats with the left ovary in situ had a larger proportion of ovulating animals, compensatory ovarian hypertrophy and number of ova shed. In turn, rats with the right ovary in situ showed a decrease in all parameters studied [15].\nBased on available information, the present study aims to analyze if changes in E2 secretion by the left and right ovaries vary during the estrous cycle, using the unilateral ovariectomized animal as a model of study.\nWe also investigated if, throughout estrous cycle diestrus 1 (D1), diestrus 2 (D2) and proestrus (P), the cholinergic system modulates E2 secretion in an asymmetric way. For this purpose, we analyzed the effects of injecting ATR at 13.00 h to rats on D1, D2 or P with or without unilateral or bilateral ovariectomy.\n\nMaterials and methods\nThe study was performed with virgin adult female rats (195–225-g body weight) of the CIIZ-V strain from our own stock. Animals were kept under controlled lighting conditions (lights on from 05:00 to 19:00 h), with free access to food (Purina S.A., Mexico) and tap water; following NIH Guide parameters for the care of laboratory animals. The Committee of the Facultad de Estudios Superiores Zaragoza approved the experimental protocols.\nEstrous cycles were monitored by daily vaginal smears. Only rats showing at least two consecutive 4-day cycles were used in the experiment. All surgeries were performed under ether anesthesia, between 13:00–13:15 hours. Rats were sacrificed by decapitation one hour after treatment.\nExperimental groups\nRats were randomly allotted to one of the experimental groups described below. Animals from different experimental groups were treated simultaneously and sacrificed one hour after surgery. The number of animals used in each experimental group is presented in Tables 1, 2 and 3.\nControl group (N = 48). Non-treated cyclic rats sacrificed at 14:00 h on D1 (17 rats), D2 (19 rats) and P (12 rats).\nEther anesthesia (N = 24): Groups of rats, on specific stages (D1, D2 or P) of the estrous cycle, were anesthetized for 10 min and sacrificed one hour later.\nUnilateral peritoneal perforation (sham operation) (N = 53): A unilateral incision was performed 2-cm below the last rib; affecting skin, muscle, and peritoneum. The ovaries were not injured or manipulated. After surgical procedures the wound was sealed.\nBilateral peritoneal perforation (sham operation 2) (N = 27). A bilateral incision below the last rib, including skin and muscle, was performed. The ovaries were not injured or manipulated. After surgical procedures the wound was sealed.\nUnilateral ovariectomy (ULO) (N = 50): A unilateral incision below the last rib, including skin and muscle was performed, and the right or left ovary was extirpated. The wound was subsequently sealed.\nBilateral ovariectomy (N = 23): A bilateral incision below the last rib, including skin and muscle was performed, and the ovaries removed. The wound was subsequently sealed.\n\nBlocking the cholinergic system\nTo analyze the effects of blocking the cholinergic system, groups of animals were injected with atropine sulfate (ATR, Sigma Chem. Co. St. Louis, Mo.). ATR was injected one hour before surgery at doses known to block ovulation: in D1, 100 mg/kg body weight (b.w.); in D2, 300 mg/kg b.w.; and in P, 700 mg/kg b.w. [16].\nOne hour after ATR treatment, rats were randomly allotted to one of the following treatments: unilateral peritoneal perforation, bilateral peritoneal perforation, ULO, or bilateral ovariectomy. All animals were sacrificed one hour after surgery. For control purposes, untreated rats, on D1, D2 or P, were injected with ATR in the same dose as in their corresponding treatment group. The animals were sacrificed two hours after treatment.\n\nAutopsy procedures\nAnimals were sacrificed by decapitation. The blood of the trunk was collected in a test tube, allowed to clot at room temperature for 30 minutes and centrifuged at 3,000 rpm for 15 minutes. Serum was stored at -20°C, until E2 concentrations were measured.\n\nHormone assay\nConcentrations of E2 in serum were measured by Radio-Immuno-Assay (RIA); using kits purchased from Diagnostic Products (Los Angeles, CA). Results are expressed in pg/ml. The Intra- and inter-assay variation coefficients were 6.9% and 10.8 %, respectively.\n\nStatistics\nData on hormonal concentrations in serum were analyzed using multivariate analysis of variance (MANOVA) followed by Tukey's test. Differences in serum hormone concentrations between two groups were analyzed by Student's t-test. A probability value of less than 5% was considered significant.\n\n\nResults\nEffects of ether anesthesia and unilateral or bilateral perforation of the peritoneum\nIn the control group, animals sacrificed on P showed significantly higher E2 serum concentration than animals sacrificed on D1 or D2 (D1: 55.3 ± 8.0; D2: 59.1 ± 7.9; P: 158.4 ± 1.8). Compared to the control group, ether anesthesia treatment did not modify E2 serum concentrations (D1: 62.9 ± 8.4; D2: 69.5 ± 12.0; P: 164.1 ± 17.6). Since ether anesthesia did not modify E2 serum concentrations, treatment results are compared to their respective control group.\nThe effects on E2 serum concentrations of unilaterally or bilaterally perforating the peritoneum depended on the side of the peritoneum and the stage of the estrous cycle when perforation surgery was performed. Perforating the left peritoneum on D1 resulted in lower E2 serum concentrations (55%), while bilateral perforation, or perforating the right side of the peritoneum, had no apparent effects (Table 1).\nPerforating the right side of the peritoneum on D2 day resulted in E2 concentration increases (184%), while perforating the left side resulted in a decrease (51%) of E2 serum concentrations. Bilateral perforation had no apparent effects on hormone concentrations. Perforating the peritoneum on P phase (left, right or bilateral) resulted in hormone serum concentration decreases (Left 30%; Right 50%; Bilateral 41%). Results are summarized in Table 1.\n\nEffects of unilateral or bilateral ovariectomy\nWhen surgery was performed on D1, no significant differences in E2 serum concentrations were observed between rats with ULO (animals with intact left or right ovary in situ) or perforation of the peritoneum (Figure 1). Animals with the left intact ovary in situ showed significantly higher E2 serum concentrations than animals with the right intact ovary in situ (61.5 ± 9.4 vs. 17.3 ± 4.3, p < 0.05 Student's t test).\nCompared to animals with unilateral perforation of the peritoneum, animals with right ULO (left ovary in situ) performed on D2 had lower E2 serum concentrations (55%). Such differences were not observed in rats with left ULO (Figure 1). As in rats treated on D1, E2 serum concentrations were significantly higher in animals treated on D2 with the left ovary in situ (right ULO) than in animals with the right ovary in situ (49.5 ± 10.8 vs. 26.0 ± 6.9).\nIn animals treated on P, right ULO (left ovary in situ) resulted in higher E2 (180%) serum concentrations than in animals with unilateral peritoneum perforation. ULO performed on the left side (right ovary in situ), resulted in significantly lower (45%) E2 serum concentrations compared to rats with a unilateral perforation of the peritoneum (Figure 1). As observed in rats treated on D1 or D2, when the intact left ovary remains in situ, estradiol serum concentrations were significantly higher than in animals with the intact right ovary in situ (142.0 ± 14.1 vs. 61.5 ± 6.0). Compared to animals with a bilateral perforation of the peritoneum, bilateral ovariectomy resulted in significantly lower E2 serum concentrations, regardless of the stage of the estrous cycle surgery performed (D1 74%; D2 73%; P 84%). Results are summarized in Table 2.\n\nEffects of blocking the cholinergic system\nInjecting ATR on D1 or P resulted in E2 serum concentrations decreases (84% and 67%, respectively), and had no apparent effects on E2 serum concentrations when injected on D2 (Table 3).\nFigure 2 shows that the effects of blocking the cholinergic system of rats with unilateral perforation of the peritoneum depended on both, the side (left or right) and the phase of the estrous when surgery was performed. Injecting ATR on D1 or D2 resulted in a significant drop in E2 serum concentrations in animals with sham treatment on the right side. Blocking the cholinergic system of rats with left side peritoneum perforation on D1 or P resulted in a drop in E2 serum concentrations (52%; 47%, respectively), while the same treatment performed on D2 resulted in a significant E2 concentrations increase (157%).\nFigure 3 shows the effects of blocking the cholinergic system of rats with ULO. ATR treatment on D1 or P stages performed on rats with the left ovary in situ resulted in a significant drop of E2 serum concentrations (65%; 62% respectively). Such effects were not observed in rats treated on D2. When ATR treatment was performed on rats with the right ovary in situ on D1, E2 serum concentrations were lower (48%) than in ULO animals. Blocking the cholinergic system on D2 resulted in E2 serum concentrations increase (159%). When the treatment was performed on P, no significant differences in E2 serum concentrations were observed.\nCompared to bilateral treatment, perforation of the peritoneum or bilateral ovariectomy, ATR treatment on D1 resulted in significant E2 serum concentrations decreases, 90% in bilateral peritoneal perforation and 60% in bilateral ovariectomized animals.\nBlocking the cholinergic system on D2, to rats with bilateral perforation of the peritoneum or bilateral ovariectomy resulted in E2 serum concentrations increases (159% and 253% respectively), while injecting ATR to animals treated on P had no apparent effects (Figure 4).\n\n\nDiscussion\nThe results obtained in the present study suggest that the ability to compensate the secretion of E2 by the missing ovary is different between the right and left ovaries and varies during the estrous cycle. Similarly, our results suggest that the cholinergic system participates in regulating E2 secretion by the ovary, and that such participation varies depending on the ovary remaining in situ and the stage of the estrous cycle when the surgical procedure was performed.\nPreviously, we suggested the existence of a neural pathway arising from the peritoneum that participates in regulating E2 [9], P4 [10] and testosterone secretion [11]. In the rat, the sensory information arising from the peritoneum is sent to the nucleus tractus solitarius and stimulates neurokinine-B receptors [17]. Since perforating the peritoneum unilaterally on each day of the estrous cycle changed E2 serum concentrations, we think that each side of the peritoneum sends different neural information through the superior ovarian nerve (SON) to the ovary and the central nervous system, perhaps reaching nuclei related to the vagus nerve.\nA study analyzing the distribution of sensory neurons innervating the peritoneum showed that when tracer was placed on the area where the peritoneum covers the abdominal wall, labeled neurons were observed only in the ipsilateral dorsal root ganglia [18]. The authors suggest that most of the parietal peritoneum receives sensory nerves from dorsal root ganglia, and visceral peritoneum from both, the spinal and vagus nerves.\nAccording to Stener-Victorin et al. (19) repeated electro-acupunture treatments in rats with polycystic ovary syndrome (PCO), induced by a single injection of estradiol valerate, resulted in lower nerve growth factor (NGF) concentrations at the ovarian level than in non-electro-acupunture treated PCO rats. In our experiments, perforating the peritoneum affected the same somatic segments employed by Stener-Victorin et. al. [19].\nWe presume that peritoneum surgery resulted in an increase of NGF concentrations at the ovarian level, which in turn induced hyper-androgenism, as observed in women with PCO [20]. Previously, we showed that the unilateral perforation of the peritoneum results in a significant increase in testosterone serum concentrations [11]. Because E2 serum concentrations did not increase after left or bilateral perforation of the peritoneum, we suppose that the neural information originating from the peritoneum inhibits the mechanisms regulating aromatase activity within the follicle.\nOne of the ovaries' sources of catecholamines arrives through the SON. In the ovary, the SON fibers are mainly distributed in the peri-follicular theca layer, and in close relation with the cells of the theca interna [21,22]. Sectioning the SON of rats in P results in a sudden drop of P4 and E2 concentrations in the ovarian vein effluent [23], while the same procedure on estrus did not modify E2 concentrations [24]. Therefore, it is possible that perforating the peritoneum modifies the type and/or rate of information arriving to the ovary via the SON. Another possibility is that perforating the left side of the peritoneum results in an increase release of ovarian gamma amino butyric acid (GABA), and a subsequent increase of E2 concentrations.\nAccording to Erdö, et. al. [25] and Laszlo, et. al. [26], injecting GABA into pseudo-pregnant rats increases E2 concentration in the blood. Present results indicate that injecting ATR, before unilateral or bilateral perforation of the peritoneum, to rats in D1 or P, results in lower E2serum concentrations; leading us to think that some of the neural fibers present in the peritoneum are muscarinic. It is also possible that blocking the cholinergic innervation, by ATR treatment, results in lower adrenaline and norepinephrine release by the adrenal medulla.\nOur results, and those of others, suggest that stimulating on D2 the sensory receptors located on the left side of the peritoneum triggers an E2 secretion inhibitory mechanism, that the sensory pathway arising from the right side has a stimulatory effect, and that both are mediated by the cholinergic muscarinic system.\nPreviously, we showed that injecting ATR to rats in D2 results in increases of P4 serum concentrations originating from the adrenals [8], without having apparent effects on testosterone serum concentrations [11]. Since P4 and androgens are precursors in the synthesis of E2, we presume that this mechanism may explain the increase in E2 serum concentrations observed in rats with peritoneum perforation previously injected with ATR.\nAnother possibility explaining the differences on E2 secretion regulation during the estrous cycle is that the effects of the cholinergic system take place through changes at the celiac ganglion level. According to Aguado and Ojeda [23], acetylcholine inhibits P4 secretion in the celiac ganglion-SON-ovary preparation obtained from rats in D1 or D2, while the preparation obtained from rats in P resulted in only a moderate stimulation. Since there is evidence that fibers from the vagus nerve innervate neurons in the celiac ganglion [27], we presume that the cholinergic system modulates the sympathetic post-ganglionar activity and the secretory ability of the ovaries through the SON.\n\nConclusion\nBased on the differences in E2 serum concentrations in rats with ULO, present results suggest that the capacity to release E2 by the left and right ovaries varies during the estrous cycle. We presume that the left ovary releases more E2 than the right one. As previously proposed, another possibility is that neural communication between the ovaries modulates E2 secretion.\n\n\n" ], "offsets": [ [ 0, 18743 ] ] } ]

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[ { "id": "pmcA1779435__text", "type": "Article", "text": [ "Presence of antibodies against cyclic citrullinated peptides in patients with 'rhupus': a cross-sectional study\nAbstract\n'Rhupus' is a rare condition sharing features of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). If rhupus is a distinctive entity, an overlap between RA and SLE or a subset of SLE is currently debated. This study was performed to explore the prevalence of antibodies against cyclic citrullinated peptides (anti-CCP antibodies) in rhupus. Patients meeting American College of Rheumatology criteria for RA, SLE, or both were included. Clinical and radiographic features were recorded and sera were searched for anti-CCP antibodies, rheumatoid factor, antinuclear antibodies, anti-extractable nuclear antigens, and antibodies against double-stranded DNA (anti-dsDNA antibodies). Seven patients for each group were included. Clinical and serological features for RA or SLE were similar between rhupus and RA patients, and between rhupus and SLE patients, respectively. Values for anti-CCP antibodies obtained were significantly (p < 0.05) higher in RA (6/7) and rhupus (4/7) than in SLE patients (0/7) and healthy subjects (0/7). Our data support the possibility that rhupus is an overlap between RA and SLE, because highly specific autoantibodies for RA (anti-CCP) and for SLE (anti-dsDNA and anti-Sm) are detected in coexistence.\n\nIntroduction\nThe clinical coexistence of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) was first described in 1969 by Kantor and was termed 'rhupus syndrome' by Schur (both cited in [1]). Since then, fewer than 100 cases of rhupus have been published [1-3]. In an epidemiological study including about 7,000 new patients, the prevalence of RA was 15% and for SLE it was 8.9%. The expected coincidence of RA and SLE by chance would therefore be 1.2%. However, the observed prevalence of rhupus was 0.09%, less than one-tenth of that expected [1].\nPrevious reports have shown that the patients with rhupus display an array of autoantibodies including anti-double-stranded DNA (anti-dsDNA), anti-Sm (both highly specific for SLE), anti-SSA, anti-SSB, anti-ribonucleoprotein, antinuclear antibodies (ANA), anti-cardiolipins, and rheumatoid factor (RF) [1,2]. However, no study has yet been performed to investigate the presence of antibodies against cyclic citrullinated peptides (anti-CCP antibodies), which have a specificity for RA of 96 to 98% (for second-generation assays (anti-CCP2)) [4,5]. Recent data have confirmed that these antibodies are rarely if ever present in other autoimmune diseases such as SLE, Sjögren's syndrome (SS), scleroderma and myositis [6]. Nowadays, it is a matter of debate whether rhupus is a clinically and immunologically distinctive entity [2], a true overlap between SLE and RA [7], or a subgroup of patients with lupus [8].\nThis descriptive, cross-sectional study was performed to investigate the frequency of anti-CCP antibodies in a cohort of patients with rhupus.\n\nMaterials and methods\nWe included all patients fulfilling American College of Rheumatology (ACR) classification criteria for both RA [9] and SLE [10] who belonged to our cohorts of patients with RA and with SLE. Comparisons were made with age- and gender-matched patients with RA and with SLE, and healthy subjects. The study was approved by the local ethics committee, and informed consent was obtained. Serum samples were obtained and stored at -75°C until use. Sera were analyzed for anti-CCP2 antibodies by ELISA (Inova Diagnostics, San Diego, CA, USA) with a cutoff value of 60 U/ml. Fine antinuclear reactivities (ELISA; Inova Diagnostics), RF (nephelometry), ANA (indirect immunofluorescence on HEp-2 slides), and anti-dsDNA (indirect immunofluorescence on Crithidia luciliae substrate) antibodies were also determined. Except for healthy individuals, standard radiographs of hands were available. For statistical analysis, ANOVA and the Mann–Whitney U test were performed as appropriate with GraphPad Prism 4.0 software (GraphPad Inc, San Diego, CA, USA).\n\nResults\nSeven female patients with a median age of 44 years (range 25 to 64) met our inclusion criteria. The major clinical and laboratory findings are presented in Table 1. Healthy individuals and all patients belonged to cohorts from the same ethnic group (Hispanic mestizo). No differences in demographic data were found between groups.\nMean ACR criteria for SLE were 5.57 (range 4 to 8) in the SLE group, and 5.57 (4 to 8) in the rhupus group. In the same way, mean ACR criteria for RA were 6 (4 to 7) in the RA group, and 5.14 (4 to 6) for the patients with rhupus. In all patients with rhupus, RA was presented as the initial disease, as has been described previously [2]. In accordance with another report, in two patients the disease started during their childhood as juvenile chronic arthritis [1].\nAnti-CCP antibodies were found in four of seven (57%) patients with rhupus, and in six of seven (86%) patients with RA, whereas neither patients with SLE nor healthy individuals showed reactivity. When the concentrations in each group were compared, a statistical significant difference between groups was found (ANOVA, p < 0.05). The mean concentration of anti-CCP antibodies was 584 U/ml (range 0 to 1,237) in patients with rhupus (Figure 1), 875 U/ml (0 to 1,178) in the RA group (not significant compared with rhupus), 1 U/ml (0 to 10) for SLE individuals (p < 0.05 compared with rhupus), and 0 U/ml (0 to 2) for healthy controls (p < 0.05 compared with rhupus). Two of three patients with rhupus who were negative for anti-CCP antibodies were also negative for anti-dsDNA antibodies, RF and anti-extractable nuclear antigen antibodies, although both patients met RA and SLE classification criteria, including ANA and erosive arthritis.\nDifferences in ANA, anti-dsDNA and anti-extractable nuclear antigen autoantibodies between patients with rhupus and those with SLE were not found. We also found no difference in the prevalence of RF between patients with rhupus and those with RA. Surprisingly, one healthy subject was positive for RF, ANA and anti-SSA antibodies, although she was asymptomatic and no features of any disease were found.\n\nDiscussion\nThe close association between different type II human leukocyte antigen (HLA) molecules and the risk of RA is well established. These major histocompatibility complex (MHC) class II molecules share the same amino acid sequence (QKRAA or QRRAA) in positions 69 to 74 of the β-chain, namely the 'shared epitope'. Recent works have demonstrated that this 'shared epitope' preferentially binds peptides containing the non-standard amino acid citrulline (deiminated arginine) [11]. In addition, an abnormally increased function of the enzyme peptidylarginine deiminase 4 (PAD4; responsible for the deimination of arginine) and an elevated anti-CCP autoantibody production in patients with RA have been demonstrated [12]. These facts have built the first bridge between cellular and humoral autoimmunity in a major rheumatic disease, supporting a pathogenetic role for an abnormal metabolism of citrulline in the development of RA [13,14].\nPatients with SLE are often part of the control group when determining the specificity of anti-CCP antibodies for RA [15], although some studies have been performed specifically on patients with SLE. These studies contribute some clues to the role of anti-CCP antibodies in rhupus. Mediwake and colleagues [16], in a study exploring the predictive value of anti-CCP antibodies to distinguish erosive arthritis in SLE, found ten patients (out of 231) with erosive arthritis, two of whom had anti-CCP antibodies. In concord with this, Hoffman and colleagues [15] demonstrate that three patients with erosive arthritis, included in a cohort of 235 patients with SLE, were positive for anti-CCP antibodies. These authors suggest that the presence of anti-CCP antibodies can predispose for a chronic RA-like arthritis in patients with SLE. Additionally Weissman and colleagues [17] demonstrated that patients with SLE can display radiographic abnormalities similar to those of RA, although the presence of marginal erosions is a rare finding.\nIn the present study we demonstrate that the patients with rhupus show a very similar arthritis pattern (including erosive disease) and similar autoantibody production (RF and anti-CCP antibodies) to those in patients with RA. In addition, patients with rhupus display a clinical and serological profile indistinguishable from patients with SLE. Moreover, the presence of other coexistent autoimmune diseases was similar in all groups of patients (two patients with rhupus, three patients with RA, and three patients with SLE also had SS).\nWe found high titers of anti-CCP antibodies in four of seven (57%) patients with rhupus, a frequency similar to that reported for RA [4]. This finding, together with the clinical similarity, supports the contention that rhupus belongs to the RA spectrum. The high prevalence of anti-CCP antibodies in RA found in our study could be explained by a selection bias because only patients with RA with an aggressive disease (namely erosive arthritis and RF+) were included. In contrast, the mean ACR criterion for SLE was similar between patients with rhupus and those with SLE, including the 'robust' features of SLE such as renal and neurological involvement, and anti-dsDNA and anti-Sm antibodies. These clinical and serological features shared between patients with rhupus and those with SLE also place rhupus in the SLE spectrum.\nTitration of anti-CCP antibodies in the rhupus group clearly shows a bimodal distribution, suggesting the existence of two different subpopulations. Because of the small number of patients, we are unable to define the differential features underlying each subset. However, two of three patients negative for anti-CCP antibodies were also negative for both RF and anti-dsDNA antibodies.\n\nConclusion\nOn the basis of the presence of shared clinical features of RA (mainly erosive arthritis) and SLE (including renal and neurological involvement) along with the presence of anti-dsDNA and anti-CCP autoantibodies in our patients with rhupus, our findings strongly support the contention that rhupus is a true overlap between RA and SLE, not merely a part of the clinical spectrum of the articular involvement seen in SLE. Moreover, on the basis of the mean ACR criteria for both diseases, we have confirmed that patients with rhupus have more RA-associated and less SLE-associated damage, an issue that has been suggested previously [2].\nTo our knowledge, this is the first report exploring the prevalence of anti-CCP antibodies specifically in patients with rhupus. More studies are needed to expand the pathogenetic knowledge of this overlap syndrome.\n\nAbbreviations\nANA = antinuclear antibodies; anti-CCP antibodies = antibodies against cyclic citrullinated peptides; anti-dsDNA antibodies = antibodies against double-stranded DNA; ELISA = enzyme-linked immunosorbent assay; RA = rheumatoid arthritis; RF = rheumatoid factor; SLE = systemic lupus erythematosus; SS = Sjögren's syndrome.\n\nCompeting interests\nThe authors declare that they have no competing interests.\n\nAuthors' contributions\nLA-G participated in the conception and design of the experiments, in the acquisition, analysis and interpretation of data, and was involved in drafting the manuscript. RS performed the immunoassays. RM-V participated in the analysis and interpretation of data and performed the statistical analysis. LG-G participated in the analysis and interpretation of data. AV participated in the recruitment of patients and the acquisition of data. RB participated in the interpretation of data, revising the manuscript for intellectual content and giving the final approval of the version to be published. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 11903 ] ] } ]

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[ { "id": "pmcA1891629__text", "type": "Article", "text": [ "A secondary structural common core in the ribosomal ITS2 (internal transcribed spacer) of\n Culexspecies from diverse geographical locations\nAbstract\nIn the present study, sequence and structural analysis of ITS2 region (the spacer segment between 5.8S and 28S rRNA of \n mature rRNA sequences) of 7 Culex species belonging to 5 different geographical locations was carried out. Alignment of the \n ITS2 sequence from the 7 species revealed 8 homologous domains. Four species namely C. vishnui, C. annulus, C. pipiens, \n C. quiquefasciatusshowed high sequence (98­100%) and RNA secondary structure similarity. The ITS2 similarity among different \n species is high despite their varying geographical locations. Several common features of secondary structure are shared among \n these species, with some of them supported by compensatory changes, suggesting the significant role by ITS2 as an RNA domain \n during ribosome biogenesis.\n\nBackground\n\n\t\t\t\tCulex mosquito species have been described from a wide range of environments and are involved in pathogen transmission from \n human to reservoirs and vice-versa. [1] Sequence similarity have been \n reported for ITS2 (the spacer segments between 5.8S and 28S rRNA of mature rRNA sequences) from 5 Culex species of diverse \n geographic locations. [3–4–\n 5–9–\n 20] The ITS spacers are versatile as genetic markers and have been used for the determination of taxonomic \n classification. [17] Recent functional analyses performed on yeast ribosomal \n RNA genes clearly show that the structural integrity of the transcribed spacer regions is an essential prerequisite for the correct \n processing of mature rRNA and for the biogenesis of active ribosomal subunits. [2–\n 3] The derivation of reliable secondary structure models for each transcribed spacer \n region would represent a major step towards a detailed understanding of their biological roles.\nComparative sequence analysis provides a powerful tool for identifying biologically relevant folding patterns in RNA molecules. \n [4] This involves collection of sequences exhibiting substantial nucleotide \n differences while retaining unequivocal sequence similarity. However, a high degree of sequence variation in the transcribed \n spacers is known even among closely related species. [5–6–7] Here, we analyze ITS2 from 7 \n Culex species characteristic of 5 different geographical locations to assemble common RNA structural features.\n\nMethodology\nDataset\nITS2 nucleotide sequences from 7 Culex mosquitoes characteristic of 5 geographical locations (Italy, China, Africa, \n America, and Japan) were downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank\n ). The GenBank accession numbers for ITS2 sequences \n from C. pipens (North America), C. tritaeniorhynchus (China), C. quinquefasciatus\n (Africa), C. tarsalis (Italy), C. annulus (China), C. pseudovishnui \n (China), and C. vishnui (Japan) were X75817, AF305558, Z48468, U33031, AF453488, AF453498 and AF165900, \n respectively.\n\nSequence alignments\nMultiple sequence alignments were performed using CLUSTALW with a gap opening penalty of 15 and gap extension penalty of 6.66. \n [8]\n\nSecondary structure prediction\nThe RNA secondary structures for ITS2 were predicted using RNADRAW. [21]\n RNADRAW predicts RNA structures by identifying suboptimal structures using the free energy optimization methodology at a default \n temperature of 370°C. In the current study, ITS2 and 5.8S regions (the first 170 nucleotides) were used for RNA structure \n prediction. The minimum energy structure prediction algorithm in RNADRAW was ported from the RNAFOLD program included in the \n Vienna RNA package. [24] The dynamic programming algorithm employed in \n RNADRAW was based on the work of Zuker and Stiegler [25] and uses energy \n parameters taken from Turner [26], Freier [27] and Jaeger. [28]\n\nPhylogenetic analysis\nThe phylogenetic Genebee service was used for phylogenetic tree construction. [22\n ]\n\nRNA fold\nThe Sribo program in Sfold (Statistical Folding and Rational Design of Nucleic Acids) was \n used to predict the probable target accessibility sites (loops) for trans-cleaving ribozymes in ITS2. [24] The prediction of accessibility is based on a statistical sample of the Boltzmann \n ensemble for secondary structures. Here, we assessed the likelihood of unpaired sites for potential ribozyme target. Each mRNA \n exists as a population of different structures. Hence, stochastic approach to the evaluation of accessible sites was found \n appropriate. [29] The probability profiling approach by Ding and Lawrence \n [30] reveals target sites that are commonly accessible for \n a large number of statistically representative structures in the target RNA. This novel approach bypasses the long-standing \n difficulty in accessibility evaluation due to limited representation of probable structures due to high statistical confidence \n in predictions. The probability profile for individual bases (W = 1) is produced for the region that \n includes a triplet and two flanking sequences of 15 bases each in every site of the selected cleavage triplet (e.g., GUC).\n \n\n\nResults and Discussion\nITS2 sequences showed a taxonomic trend similar to that in phylogeny construction (Figure 1A\n\t\t\t). A multiple sequence alignment \n of 5.8S rDNA and ITS2 showed in Figure 1B indicated that more distantly related species have \n lower sequence similarity in the \n ITS2 region. The predicted features of ITS2 using RNADRAW are given in Table 1. The stems \n (double stranded paired regions) \n stabilize RNA secondary structures and the number of stems present in each ITS2 is given (Table 1). ITS2 RNA structures from \n C. pipiens and C. quinquefasciatus have the highest negative free energy (-149.38 Kcal and \n -148.23 Kcal) followed by C. tarsalis (-129.2), C. annulus and then by C. vishnui\n (-115.3), C. pseudovishnui (-105.66) and C. tritaeniorhyncus (- 82.57). Visual comparison shows \n that this is related to the trend in the cladogram given in Figure 1A. A high degree of sequence similarity is observed at the \n 5' end compared to the 3' end (Figure 1A). This is due to factors such as genetic drift, the relative number and size of repeats, \n rates of unequal crossover, gene conversion, immigration and the number of the loci influencing the length. [10] Furthermore, a high level of sequence similarity is found between C. \n annulus and C. vishnui as well as C. quinquefasciatus and C. pipiens.\n The simple tandem repeats shown in Figure 1B as bolded regions are found to be similar. \n This similarity is seen in corresponding \n RNA structures and computed energies.\nThe Culex species considered in this study were then grouped into three classes based on the similarity of \n RNA stems and loops. Despite their different geographic locations with varying eco-climatic conditions, class I \n (C. annulus, C. pseudovishnui and C. vishnui) isolates (China and Japan) showed high similarity \n in secondary structural features. Similar observations were seen in class II (C. pipiens, C. quinquefasciatus and \n C. tarsalis) isolates from North America and Africa. [19] \n A high degree of similarity in the 5.8S region unlike the ITS2 is shown for different isolates due to relative evolutionary \n selection. [9] The loops 11 and 12 having sequences UGUCG and CUUCGGUG, \n respectively are highly conserved in all classes.\nFigure 1C shows the distribution of different types of loops (hairpin, bulge, multi branched, interior and exterior) among \n different isolates. The segments of the ITS2 having score ≥ 50 are further probed carefully for target site to assess the \n likelihood of unpaired segments. Interestingly, the observed phylogenetic trend was identified with respect to the target \n accessibility sites for the seven Culex isolates. The order of preference is interior loop, bulge loop, multiple \n branched loop, hairpin loop and exterior loops in all the isolates.\nSeveral homologous domains were observed in the ITS regions of Aedes mosquito species by Wesson \n\t\t\tet al\t [11] and was shown that these domains base pair to \n\t\t\tform a core region central to \n several stem features. This suggested that conserved core region of rDNA is more important for a functional rRNA folding \n pattern. Barker found that ITS2 is unique in the 16 populations of Boophilus microplus, Boophilus decoloratus, \n Rhipicephalus appendiculatus, Rhipicephalus zambesiensis, Rhipicephalus evertsi, Rhipicephalus sanguineus, Rhipicephalus \n turanicus, Rhipicephalus pumilio and Rhipicephalus camicasi from different geographical locations. \n [12] These results suggest that the differences and similarity observed \n in the ITS2 of different species are not simply accumulated due to random mutation and have evolved for functional selection \n in ribosome biogenesis. However, it was shown in three related mosquito genera (Aedes, Psorophora and Haemogogus)\n that the intra-spacer variable regions appear to co-evolve and that ITS2 variation is constrained by its secondary structure. \n [11] Further studies have demonstrated that the ITS2 is essential \n for the correct and efficient processing and maturation of 26S rRNA ribosomal units. [\n 13] Furthermore, the information required for the efficient removal of ITS2 from its RNA precursor is not \n localized but dispersed throughout the ITS2 region. Thus, insertions and deletions (indels) that affect secondary structures \n alter rRNA processing. Critical changes in the rRNA folding pattern due to evolutionary sequence variation in the ITS spacer \n regions may thus have an important role on the kinetics of precursor rRNA formation for the efficient functioning of rDNA clusters.\n \n\nConclusion\nComparison of ITS2 sequences from different isolates of Culex show similarity and variations. Surprisingly, species displaying \n sequence similarity belong to different geographical locations with diverse climatic and ecological conditions. This implies that \n the ITS2 regions have less selective pressure than the ribosomal regions. Several common structural folds were shared among the \n selected mosquitoes for maintaining functional equivalents. Construction of an evolutionary tree using more isolates of \n Culex will provide an understanding for their functional selection.\n\n\n" ], "offsets": [ [ 0, 10736 ] ] } ]

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"type": "species", "text": [ "C. pipens" ], "offsets": [ [ 2856, 2865 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7175" } ] }, { "id": "pmcA1891629__T7", "type": "species", "text": [ "C. tritaeniorhynchus" ], "offsets": [ [ 2883, 2903 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7178" } ] }, { "id": "pmcA1891629__T8", "type": "species", "text": [ "C. quinquefasciatus" ], "offsets": [ [ 2913, 2932 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7176" } ] }, { "id": "pmcA1891629__T9", "type": "species", "text": [ "C. tarsalis" ], "offsets": [ [ 2950, 2961 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7177" } ] }, { "id": "pmcA1891629__T10", "type": "species", "text": [ "C. annulus" ], "offsets": [ [ 2971, 2981 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "182211" } ] }, { "id": "pmcA1891629__T11", "type": "species", "text": [ "C. pseudovishnui" ], "offsets": [ [ 2991, 3007 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "100677" } ] }, { "id": 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"db_id": "6941" } ] }, { "id": "pmcA1891629__T30", "type": "species", "text": [ "Boophilus decoloratus" ], "offsets": [ [ 8711, 8732 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "60189" } ] }, { "id": "pmcA1891629__T31", "type": "species", "text": [ "Rhipicephalus appendiculatus" ], "offsets": [ [ 8741, 8769 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "34631" } ] }, { "id": "pmcA1891629__T32", "type": "species", "text": [ "Rhipicephalus zambesiensis" ], "offsets": [ [ 8771, 8797 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "60191" } ] }, { "id": "pmcA1891629__T33", "type": "species", "text": [ "Rhipicephalus evertsi" ], "offsets": [ [ 8799, 8820 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "60190" } ] }, { "id": "pmcA1891629__T34", "type": "species", "text": [ "Rhipicephalus sanguineus" ], "offsets": [ [ 8822, 8846 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "34632" } ] }, { "id": "pmcA1891629__T35", "type": "species", "text": [ "Rhipicephalus pumilio" ], "offsets": [ [ 8880, 8901 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "127007" } ] }, { "id": "pmcA1891629__T36", "type": "species", "text": [ "Rhipicephalus camicasi" ], "offsets": [ [ 8906, 8928 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "0" } ] }, { "id": "pmcA1891629__T37", "type": "species", "text": [ "Haemogogus" ], "offsets": [ [ 9288, 9298 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "0" } ] } ]

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[ { "id": "pmcA1526545__text", "type": "Article", "text": [ "B lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease activity correlates better with blood leukocyte BLyS mRNA levels than with plasma BLyS protein levels\nAbstract\nConsiderable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and ΔBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and ΔBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and ΔBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and ΔBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.\n\nIntroduction\nB lymphocyte stimulator (BLyS; a trademark of Human Genome Sciences, Inc., Rockville, MD, USA) is a 285-amino-acid member of the tumor necrosis factor ligand superfamily [1-3]. A causal relation between constitutive overproduction of BLyS and development of systemic lupus erythematosus (SLE)-like illness has incontrovertibly been established in mice. BLyS-transgenic mice often develop SLE-like features as they age [3-5], and SLE-prone (NZB × NZW)F1 (BWF1) and MRL-lpr/lpr mice respond clinically to treatment with BLyS antagonists (decreased disease progression and improved survival) [3,6].\nConsiderable inferential evidence points to a role for BLyS overproduction in human SLE as well. Cross-sectional studies have demonstrated elevated circulating levels of BLyS in 20–30% of human SLE patients tested at a single point in time [7,8]. Moreover, a 12-month longitudinal study documented persistently elevated serum BLyS levels in about 25% of SLE patients and intermittently elevated serum BLyS levels in an additional 25% of patients [9]. Remarkably, circulating BLyS levels did not correlate with disease activity (measured using the SLE Disease Activity Index [SLEDAI]) in these cross-sectional or longitudinal studies [7-9]. Although a statistically significant correlation between circulating BLyS levels and SLEDAI has been appreciated in a more recent 24-month longitudinal study of 245 SLE patients (with >1,700 plasma samples analyzed) [10], the correlation remains weak.\nThe limited correlation between circulating BLyS protein levels and disease activity in these studies may have exposed an inadequacy of the former to reflect faithfully endogenous BLyS overproduction. In addition to the rate of BLyS protein production, several other factors (for example, utilization and excretion) can affect circulating BLyS protein levels. Although there are no practicable means of directly measuring in vivo BLyS production per se in humans, the level of BLyS mRNA may serve as a better surrogate marker of in vivo BLyS production than does the level of BLyS protein. Candidate BLyS mRNA isoforms include the full-length BLyS mRNA isoform, which encodes the full-length protein, and the alternatively spliced ΔBLyS mRNA isoform, which encodes a protein with a small peptide deletion [11]. (ΔBLyS does not bind to cells expressing BLyS receptors, and therefore it has no agonistic activity. Moreover, ΔBLyS can form heterotrimers with full-length BLyS, thereby actually functioning as a dominant-negative antagonist of BLyS activity.)\nIn this report we demonstrate that peripheral blood leukocytes from SLE patients express elevated mRNA levels of both full-length BLyS and ΔBLyS relative to those levels expressed by patients with rheumatoid arthritis (RA) or by normal control individuals. In the SLE patients, both full-length BLyS and ΔBLyS mRNA levels are more closely associated with disease activity (SLEDAI) than are BLyS protein levels. Accordingly, BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.\n\nMaterials and methods\nGeneral details\nThis study was approved by the institutional review boards of the University of Southern California and the Scripps Research Institute. All participants gave their written informed consent before participation in this study.\n\nParticipants\nPatients receiving outpatient medical care at the rheumatology clinics of the Los Angeles County + University of Southern California Medical Center were recruited into the study. Diagnoses of SLE (n = 60) or RA (n = 60) were based on established clinical criteria [12]. Healthy control individuals (n = 30) were recruited from Los Angeles County + University of Southern California Medical Center and University of Southern California Keck School of Medicine personnel. No exclusions were made on any basis other than an inability to give informed consent. Each patient's sex, race, age, and medications at the time of the phlebotomy were recorded (Table 1).\nBased solely on the patient's willingness to donate a second blood sample, repeat blood samples were collected from 37 of the SLE patients 147–511 days (median 371 days) after collection of the first samples. These patients were not selected on the basis of any demographic, clinical, or laboratory feature.\nClinical disease activity for the SLE patients was assessed using the SLEDAI [13] and using a modified SLEDAI that excludes the contribution of anti-double-stranded DNA (anti-dsDNA) antibodies from the total score. Each patient's medical chart was reviewed for results of standard clinical laboratory tests within the previous or subsequent 1-month period.\n\nPlasma BLyS determination\nWhole venous blood was centrifuged to yield plasma and a buffy coat. The plasma was harvested, stored at -70°C, and assayed for BLyS levels by ELISA [8,14] using Fab fragments of the capture antibody rather than the whole antibody to reduce assay interference by rheumatoid factor. The lower limit of detection in this assay is 0.3 ng/ml. For statistical purposes, plasma samples with BLyS concentrations below the lower limit of detection were assigned a value of 0.25 ng/ml.\n\nBlood BLyS mRNA determination\nThe buffy coat from centrifuged whole blood was harvested, added to RNAlater™ (Ambion, Austin, TX, USA) at a 1:4 vol/vol ratio for RNA stabilization, stored at -70°C, and assayed for full-length BLyS and ΔBLyS mRNA levels by real-time PCR. Total RNA was purified from buffy coat samples using RNAeasy miniprep kits (Qiagen, Valencia, CA, USA), and contaminating genomic DNA was removed by DNAse-I digestion. One-tenth volume of total RNA was used as template in the first-strand cDNA reaction using oligo-dT and the Superscript III first-strand synthesis system (Invitrogen, Carlsbad, CA, USA). Duplicate samples of cDNA were amplified with primers against β-actin, full-length BLyS, or ΔBLyS: β-actin sense 5'-CGAGAAGATGACCCAGATCATGT-3'; β-actin anti-sense 5'-GGCATACCCCTCGTAGATGG-3'; full-length BLyS sense 5'-GCAGACAGTGAAACACCAACTATAC-3'; ΔBLyS sense 5'-CAGAAGAAACAGGATCTTACACAT-3'; and full-length BLyS/ΔBLyS anti-sense 5'-TGCCAGCTGAATAGCAGGAATTAT-3'.\nA 165 bp amplicon for β-actin was PCR-amplified using the 7900 HT ABI Prism machine (Qiagen) with annealing at 65°C. A 296 bp amplicon for full-length BLyS was PCR-amplified, with annealing at 64°C. A 270 bp amplicon for ΔBLyS was PCR-amplified with annealing at 61°C. The annealing conditions for full-length BLyS and ΔBLyS were determined so that each primer set remained specific to the respective BLyS isoform and yielded a PCR efficiency similar to those of cloned cDNA standards. Melting curve analysis revealed a single peak for each gene amplified. The threshold cycle (Ct) values for each reaction were determined using Sequence Detection System software (Applied Biosystems, Foster City, CA, USA). Results are presented as ratios of full-length BLyS or ΔBLyS mRNA to β-actin mRNA, which were calculated using the following formulae:\n2 exp(Ctβ-actin - Ctfull-length BLyS)\n2 exp(Ctβ-actin - CtΔBLyS)\n\nDetermination of anti-BLyS autoantibodies\nBLyS was bound to microtiter plates by first coating the plates with streptavidin and then adding biotinylated recombinant BLyS. Using these plates as the capture reagent, plasma samples were incubated, and horseradish peroxidase-conjugated anti-human IgA/IgM/IgG (Southern Biotechnology Associates, Birmingham, AL, USA; 1:20,000 final dilution) or horseradish peroxidase-conjugated anti-human IgG (Southern Biotechnology; 1:10,000 final dilution) were used as the detector reagents.\n\nStatistical analysis\nAll analyses were performed using SigmaStat software (SPSS, Chicago, IL, USA). Results that did not follow a normal distribution were log-transformed to achieve normality. Parametric testing between two matched or unmatched groups was performed using the paired or unpaired t test, respectively. Parametric testing among three or more groups was performed using one-way analysis of variance. When log-transformation failed to generate normally distributed data or the equal variance test was not satisfied, nonparametric testing was performed using the Mann–Whitney rank sum test between two groups and by Kruskal–Wallis one-way analysis of variance on ranks among three or more groups. Correlations were determined using Pearson product moment correlation for interval data and using Spearman rank order correlation for ordinal data or for interval data that did not follow a normal distribution. Nominal data were analyzed using χ2 analysis-of-contingency tables.\n\n\nResults\nElevated plasma BLyS levels and blood levels of full-length BLyS and ΔBLyS mRNA isoforms in systemic lupus erythematosus patients\nPrevious reports of elevated circulating BLyS levels in SLE patients were based on a BLyS ELISA that utilized a whole (unfragmented) capture anti-BLyS monoclonal antibody [7-9]. Since the publication of these reports, it has been recognized that the presence of rheumatoid factor can potentially interfere with the assay and lead to spurious overestimation of the true circulating BLyS levels (Human Genome Sciences, Inc.; unpublished observations). To mitigate potential interference from rheumatoid factor, the BLyS ELISA has been modified and the capture anti-BLyS monoclonal antibody is now utilized as a Fab fragment. Despite the changes in the ELISA format, our findings are entirely consistent with those of the previous reports. Plasma BLyS levels were significantly greater in the SLE group than in either RA or normal control group (P < 0.001; Figure 1a). Arbitrary assignment of the 95th percentile value among the normal control individuals as the upper limit of 'normal' revealed that two of the 30 normal control individuals, 15 of the 60 RA patients, and 29 of the 60 SLE patients harbored elevated plasma BLyS levels (P < 0.001).\nOverexpression of BLyS in SLE patients was also established by measuring BLyS mRNA levels normalized to β-actin mRNA levels in peripheral blood leukocytes (buffy coats). The geometric mean full-length BLyS mRNA and ΔBLyS mRNA levels among the SLE patients were each significantly greater than those among the RA patients and normal control individuals, respectively (P < 0.001 for each; Figure 1b,c). Arbitrary assignment of the 95th percentile values for full-length BLyS and ΔBLyS mRNA levels among the normal control individuals as the upper limits of 'normal' revealed that two of the 30 normal control individuals, four of the 60 RA patients, and 20 of the 60 SLE patients had elevated full-length BLyS mRNA levels (P < 0.001), and that two of the 30 normal control individuals, three of the 60 RA patients, and 19 of the 60 SLE patients had elevated ΔBLyS mRNA levels (P < 0.001). Levels of full-length BLyS and ΔBLyS mRNA strongly correlated with each other (r = 0.703; P < 0.001) in the SLE cohort, and plasma BLyS levels also correlated significantly with levels of each BLyS isoform (r = 0.429, P < 0.001; and r = 0.290, P = 0.024, respectively). Among these SLE patients, none of the measured BLyS parameters correlated with patient age, sex, race, or daily dose of corticosteroids (data not shown). Because the racial composition of the normal cohort was not as predominantly Hispanic as were those of the RA and SLE cohorts, we assessed the BLyS parameters in the respective Hispanic subpopulations. As for the entire populations, values for SLE were significantly greater than those for either RA or normal controls (P ≤ 0.004; data not shown).\n\nCorrelations between BLyS parameters and plasma immunoglobulin levels\nBLyS is a potent B cell survival factor [15-21], and administration of exogenous BLyS to mice leads to B cell expansion and hypergammaglobulinemia [1]. Previous studies with numbers of SLE patients greater than were included in the present study documented a modest but significant correlation between serum levels of BLyS and IgG [8,10]. In our SLE cohort of limited size, plasma BLyS levels failed to show significant correlations with plasma levels of total immunoglobulin, IgG, or IgA. In contrast, full-length BLyS and ΔBLyS mRNA levels correlated significantly with each (Figure 2). (None of the BLyS parameters correlated with plasma IgM levels.) The absence of significant correlation between plasma BLyS levels and the immunoglobulin parameters also persisted when just the 53 patients with detectable plasma BLyS levels were considered (r = -0.133, P = 0.346 for total immunoglobulin; r = -0.048, P = 0.734 for IgG; and r = 0.033, P = 0.817 for IgA).\n\nCorrelations between BLyS parameters and disease activity\nPrevious studies either have failed to demonstrate a significant correlation between disease activity and circulating BLyS levels [7-9] or have detected only a weak correlation between the two [10]. Consonant with those studies, we identified no significant correlation between plasma BLyS levels and SLEDAI in our cohort of 60 SLE patients (Figure 3a). The failure to demonstrate a significant correlation cannot be attributed to a skewing of the results by the patients in whom plasma BLyS levels were below the limit of detection, because no significant correlation was detected among the 53 SLE patients in whom plasma BLyS levels were in the detectable range (r = 0.185, P = 0.183). In contrast, a significant correlation between SLEDAI and full-length BLyS mRNA levels was readily discernible (Figure 3b). A trend toward a correlation between SLEDAI and ΔBLyS mRNA levels was also observed, although it did not achieve statistical significance (Figure 3c).\nA component of the SLEDAI is the presence of circulating anti-dsDNA antibodies. Because circulating BLyS levels may affect the presence and/or titers of circulating anti-dsDNA antibodies [7-10], we assessed correlations between the individual BLyS parameters and a modified SLEDAI that excludes any consideration of anti-dsDNA antibodies. As with the unmodified SLEDAI, the modified SLEDAI did not correlate with plasma BLyS levels (Figure 3d) either among the SLE cohort overall or among the 53 patients in whom plasma BLyS levels were in the detectable range (r = 0.160, P = 0.252), but it significantly correlated with full-length BLyS mRNA levels (Figure 3e) and exhibited a trend toward correlation with ΔBLyS mRNA levels (Figure 3f). Thus, the stronger correlations between BLyS mRNA levels and disease activity cannot solely be explained by any effects that BLyS may have on anti-dsDNA antibodies per se.\nMoreover, among the 37 SLE patients who were evaluated on two separate occasions, trends toward correlation were appreciated between changes in the unmodified or modified SLEDAI and changes in full-length BLyS or ΔBLyS mRNA levels but not changes in plasma BLyS levels (Figure 4). These results cannot be ascribed to changes in medications taken by the patients, because changes in neither disease activity nor in any of the BLyS parameters correlated with changes in the doses of corticosteroids or cytotoxics taken by the patients (data not shown). The failure to demonstrate a meaningful association between changes in SLEDAI score and changes in plasma BLyS protein levels cannot be attributed to a skewing of the results by the patients in whom plasma BLyS levels were below the limit of detection, because the absence of association between the two persisted among the 27 SLE patients in whom plasma BLyS levels were in the detectable range in both samples (r = -0.069, P = 0.727 for plasma BLyS versus unmodified SLEDAI; r = -0.020, P = 0.919 for plasma BLyS versus modified SLEDAI).\n\nLack of correlation between levels of BLyS mRNA isoforms and percentages of individual leukocyte cell types\nAmong cells in peripheral blood, BLyS is predominantly expressed by cells of the myeloid lineage (monocytes and neutrophils) [1,14,22,23]. Accordingly, a shift in the differential leukocyte count away from lymphocytes to monocytes and/or neutrophils could substantially alter BLyS mRNA results. Because of the limited amount of blood we were permitted to obtain from the SLE patients (consequent to the high prevalence of anemia among these patients), we were unable to purify the individual leukocyte populations for BLyS mRNA analysis. Nevertheless, to demonstrate that the elevated BLyS mRNA levels in SLE did not simply reflect a shift in differential leukocyte count, we assessed the correlations between the individual BLyS parameters on the one hand and the percentages of blood neutrophils, monocytes, and lymphocytes on the other. No correlations were appreciated (Figure 5).\n\nPresence of anti-BLyS autoantibodies in patients with systemic lupus erythematosus\nThe poorer correlation between plasma BLyS protein levels and disease activity compared with that between BLyS mRNA levels and disease activity was striking. Patients with SLE frequently develop autoantibodies against self-antigens, and so some of the SLE patients might have harbored autoantibodies to BLyS. Such autoantibodies could have complexed with BLyS and enhanced its clearance, thereby masking BLyS overproduction. Alternatively, such autoantibodies might have sterically blocked the epitopes recognized by the detecting antibodies in the in vitro ELISA. In this case, measured BLyS levels would have been spuriously reduced, again masking BLyS overproduction.\nIn our cohort, IgA/IgM/IgG anti-BLyS antibodies were detected in six out of the 60 SLE patients. Such autoantibodies were also detected in two out of 60 RA patients and in one out of 30 normal control individuals, demonstrating that anti-BLyS autoantibodies are not restricted to SLE patients. IgG anti-BLyS autoantibodies were detected in 3 SLE patients but in no RA patients or normal control individuals.\n\n\nDiscussion\nElevated blood levels of BLyS protein and mRNA are well described features of human SLE [7-9]. We confirmed these observations in our study and extended them by documenting increases not just in levels of full-length BLyS mRNA but also in levels of ΔBLyS mRNA (Figure 1). Of note, BLyS mRNA levels were elevated in SLE but not in RA, raising the possibility that BLyS overproduction in SLE is systemic whereas BLyS overproduction in RA may be more focused to the affected arthritic joints [24]. The modestly elevated plasma BLyS protein levels in RA patients may reflect, at least in part, release of locally overproduced BLyS into the circulation.\nThe relationship between circulating BLyS protein levels and disease activity was addressed in several previous studies, but significant correlations between the two measures did not emerge [7-9]. In the largest study to date, a 2-year longitudinal study of 245 patients in which more than 1,700 plasma samples were analyzed, a significant but weak correlation between the two was appreciated [10]. In the present study, a significant correlation between plasma BLyS protein levels and disease activity was again not realized (Figure 3a,d).\nThe weak, at best, correlation between circulating BLyS levels and disease activity is seemingly rather surprising. There is a clear-cut association in BlyS transgenic mice between BLyS overexpression and development of SLE-like features [3-5], and treatment of SLE-prone mice with BLyS antagonists retards the progression of disease and improves survival [3,6]. Moreover, development of precocious glomerular pathology in autoimmune-prone mice correlates strongly with circulating BLyS levels [25].\nThe likely explanation for the weak correlation between circulating BLyS levels and disease activity in human SLE is not that disease activity in SLE patients is insensitive to the degree of BLyS overproduction. Rather, a more tenable explanation is that circulating BLyS levels in human SLE do not always accurately reflect excessive endogenous BLyS production. We can identify at least three nonmutually exclusive mechanisms to explain a dissociation between the two.\nFirst, SLE patients frequently develop autoantibodies to a myriad of self-targets (for example, erythrocytes, lymphocytes). Indeed, we detected circulating IgA/IgM/IgG anti-BLyS autoantibodies in 10% (6/60) of the tested SLE patients, and we detected circulating IgG anti-BLyS autoantibodies in 5% (3/60). These percentages may be underestimates of the true prevalence of anti-BLyS autoantibodies, because some of these autoantibodies may be saturated in vivo with circulating BLyS, rendering them incapable of binding to BLyS in the in vitro detection assay. We do not yet know whether the anti-BLyS autoantibodies are functionally neutralizing but, regardless, such autoantibodies could enhance the clearance of BLyS and/or interfere with in vitro detection of BLyS, thereby masking endogenous BLyS overproduction.\nSecond, increased urinary excretion of BLyS has been reported in SLE patients, especially among those with clinically overt renal involvement [26]. At least four of the patients we studied manifested nephrotic-range proteinuria (≥3 g/24 hours), and so urinary loss of BLyS was probably substantial in at least these patients. A validated assay for urinary BLyS detection has not yet been developed so we were unable to quantify urinary BLyS levels. Once an assay for urinary BLyS levels is validated, we should be able to assess the effect of urinary BLyS excretion on circulating BLyS levels.\nThird, BLyS promotes in vivo expansion of B cells [1]. Freshly isolated SLE B cells, despite intact surface expression of BLyS receptors, bind less biotinylated BLyS ex vivo than do freshly isolated normal B cells [27]. Although other interpretations are possible, the most likely explanation is that BLyS receptors on B cells in SLE patients are occupied in vivo by soluble BLyS. Accordingly, it is likely that BLyS receptors expressed by the expanded B cell population do bind BLyS and remove it from the circulation, resulting in a homeostatic pathway that modulates the effects of BLyS overproduction on circulating BLyS levels. Indeed, circulating levels of BLyS rise with peripheral blood B cell depletion and fall with re-emergence of peripheral blood B cells in rituximab-treated RA or SLE patients [28,29], highlighting this inverse relationship between circulating BLyS levels and B cell load. Moreover, one of the hallmarks of active disease in human SLE is the increased percentages of activated B cells and plasma cells in peripheral blood [30-34], probably reflecting increased systemic numbers of activated B cells and plasma cells. Although not yet formally tested, differential BLyS receptor expression by these cells compared with expression by nonactivated B cells may result in increased peripheral BLyS utilization, further dampening the effects of BLyS overproduction on circulating protein levels.\nTo circumvent these confounding processes, we used BLyS mRNA levels as a surrogate marker of endogenous BLyS production. Overall, the correlations between disease activity and either full-length BLyS or ΔBLyS mRNA levels were much stronger than that between disease activity and BLyS protein levels (Figures 3 and 4). This was the case regardless of whether we used the standard SLEDAI or the modified SLEDAI as a measure of disease activity. These correlations were not spurious ones consequent to shifts in percentages of leukocyte subpopulations in peripheral blood, because BLyS mRNA levels did not correlate with percentages of blood neutrophils, monocytes, or lymphocytes (Figure 5).\nA similar pattern was observed between plasma immunoglobulin levels and the BLyS parameters, with plasma levels of total immunoglobulin, IgG, and IgA correlating significantly with full-length BLyS and ΔBLyS mRNA levels but not with plasma BLyS levels (Figure 2). These significant correlations between full-length BLyS or ΔBLyS mRNA levels and plasma immunoglobulin levels again highlight the greater ability of BLyS mRNA levels, compared with plasma BLyS protein levels, to reflect ongoing BLyS overproduction.\nAt present, it is not known whether soluble ΔBLyS protein is present in the circulation of SLE patients or of normal individuals. Although full-length BLyS protein is readily cleaved and released from cells transfected with a vector containing murine full-length BLyS, ΔBLyS protein is not cleaved or released from murine ΔBLyS transfectants [11]. Given the strong similarities between murine and human full-length BLyS and ΔBLyS, it is likely that human soluble ΔBLyS protein is also not cleaved from the cell surface and released into the circulation. Moreover, soluble ΔBLyS protein is not released from cells transfected with a vector containing just the extracellular domain of human ΔBLyS (which encodes the soluble protein; A.L. Gavin, unpublished observations). Whether this reflects rapid intracellular degradation of soluble ΔBLyS or some other impediment to its release remains unknown. Regardless, if the inability to release soluble ΔBLyS in vitro faithfully recapitulates in vivo biology, then the stronger associations between SLE disease activity and full-length BLyS or ΔBLyS mRNA levels compared with that between SLE disease activity and BLyS protein levels could not be attributable to interference by biologically inactive (inhibitory) ΔBLyS protein in the BLyS protein detection ELISA. Importantly, even if soluble ΔBLyS protein is present in the circulation and is detected by the BLyS protein detection ELISA, then the stronger correlations between SLE disease activity and full-length BLyS or ΔBLyS mRNA levels than that between disease activity and total BLyS (including ΔBLyS) protein levels suggest that full-length BLyS and/or ΔBLyS mRNA levels may operationally serve as useful biomarkers of disease activity in SLE. Although the complexity and labor intensiveness associated with quantitative real-time PCR may render measurement of BLyS mRNA levels impracticable for routine clinical practice, such measurement could readily be incorporated into clinical trials and yield valuable information.\nLongitudinal observations in large numbers of SLE patients will be necessary to establish or refute the utility of full-length BLyS and/or ΔBLyS mRNA to subserve this clinically vital function.\nAlthough expression of the two major BLyS isoforms was highly coordinate among SLE patients, there were several patients in whom ΔBLyS mRNA levels were markedly greater than or less than the expected values based on full-length BLyS mRNA levels (data not shown). This raises the possibility that dysregulation of ΔBLyS may contribute to overall BLyS dysregulation in at least some SLE patients. It is known that interferon-γ, interleukin-10, interferon-α, and CD154 can upregulate full-length BLyS mRNA levels [14,22,35], but it is not known what effects these or other cytokines/cell-surface structures have on ΔBLyS expression. Further investigation of the regulation of ΔBLyS and the differential expression of BLyS isoforms is certainly warranted.\nAlthough the associations between full-length BLyS and/or ΔBLyS mRNA levels and disease activity in SLE were usually strong when the SLE cohort was analyzed in aggregate, there were several SLE patients in whom BLyS mRNA levels were quite high despite little objective ongoing disease activity, and there were several SLE patients in whom BLyS mRNA levels were low despite considerable ongoing disease activity. One must recognize that the bulk of the pathogenic autoimmune responses probably takes place in the spleen and lymph nodes, rather than in the peripheral blood, where myeloid lineage cells (for example, dendritic cells) produce BLyS and support B cell survival and expansion [36]. Local BLyS production in the secondary lymphoid tissues will be more important to the development and maintenance of an autoimmune response than will remote BLyS levels in the circulation. Because at least some autoreactive B cells may be more sensitive to BLyS-mediated survival signals than non-autoreactive B cells [37,38], local increases in BLyS production could preferentially promote expansion of autoreactive B cells. These cells, in turn, could activate autoreactive T cells by presenting autoantigen to them, and some of the autoreactive B cells would respond to T cell derived signals and mature into (pathogenic) autoantibody secreting plasma cells. In contrast to murine studies, in which investigators can readily harvest and analyze lymphoid and myeloid lineage cells from any site (for example, spleen, bone marrow), such is not the case for human studies. Peripheral blood is the only site readily accessible for human studies, and it is possible that, at least in some patients, BLyS mRNA levels in circulating leukocytes do not reflect local BLyS production in the secondary lymphoid tissues.\nOne must also recognize that disease activity in SLE is not solely driven by B cells. Systemic inflammation and SLE flares can be triggered via B cell independent means. Not all SLE patients treated with a B cell depleting course of rituximab experience clinical remission [39], strongly pointing to the importance of non-B cells in disease pathogenesis/maintenance. Conversely, not all pathogenic B cells necessarily require high levels of BLyS to effect their pathogenicity. Murine studies have unequivocally documented B cell subpopulations that do not depend upon BLyS for their survival [40-42]. Although mice completely devoid of BLyS have reduced numbers of mature B cells and harbor reduced levels of immunoglobulin, these reductions are incomplete. Thus, it is possible that some SLE patients harbor pathogenic B cells that are relatively insensitive to BLyS and could drive considerable disease activity even in the context of low endogenous BLyS production. Conversely, patients with high BLyS mRNA levels may be those patients whose disease is strongly driven by BLyS and may be especially helped by BLyS antagonist therapy. Future clinical trials should be able to establish whether the BLyS mRNA levels are good predictors of response to such agents.\n\nConclusion\nPlasma total immunoglobulin, IgG, and IgA levels and disease activity (as measured by SLEDAI) in SLE patients correlate more closely with peripheral blood leukocyte levels of BLyS mRNA than with plasma levels of BLyS protein. These findings suggest that BLyS mRNA levels better reflect in vivo BLyS production than do circulating BLyS protein levels, and may be a useful biomarker in the clinical monitoring of SLE patients. These findings also support the premise that BLyS overexpression not only promotes development of disease but also actively contributes to the ongoing maintenance of disease in SLE patients. This reinforces the rationale underlying clinical trials with BLyS antagonists in SLE.\n\nAbbreviations\nanti-dsDNA = anti-double-stranded DNA; BLyS = B lymphocyte stimulator; bp = base pairs; Ct = threshold cycle; ELISA = enzyme-linked immunosorbent assay; PCR = polymerase chain reaction; RA = rheumatoid arthritis; SLE = systemic lupus erythematosus; SLEDAI = SLE Disease Activity Index.\n\nCompeting interests\nTSM and DMH were employees of Human Genome Sciences (HGS) at the time the investigation was conducted. (DMH has since left the company.) WS has received research support from HGS and has served as a consultant to HGS (<$10,000). CEC, ALG, and DN declare that they have no competing interests.\n\nAuthors' contributions\nCEC identified and recruited all participants; collected all the blood samples and reviewed all the medical charts; and wrote the initial working draft of this manuscript. ALG developed and performed all the real-time PCR assays and assisted in the interpretation of the results and in writing the final version of the manuscript. TSM performed the plasma BLyS protein and anti-BLyS assays and assisted in the interpretation of the results and in writing the final version of the manuscript. DMH assisted in the design in the study, in the interpretation of the results, and in writing the final version of the manuscript. DN assisted in the design in the study, in the interpretation of the results, and in writing the final version of the manuscript. WS conceived the study, supervised the recruitment of participants, performed the statistical analyses, assisted in the interpretation of the results, and supervised the editing of the manuscript to its final form. All authors read and approved the final manuscript version.\n\n\n" ], "offsets": [ [ 0, 34453 ] ] } ]

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[ { "id": "pmcA280693__text", "type": "Article", "text": [ "p8 inhibits the growth of human pancreatic cancer cells and its expression is induced through pathways involved in growth inhibition and repressed by factors promoting cell growth\nAbstract\nBackground\np8 is a stress-induced protein with multiple functions and biochemically related to the architectural factor HMG-I/Y. We analyzed the expression and function of p8 in pancreatic cancer-derived cells.\n\nMethods\nExpression of p8 was silenced in the human pancreatic cancer cell lines Panc-1 and BxPc-3 by infection with a retrovirus expressing p8 RNA in the antisense orientation. Cell growth was measured in control and p8-silenced cells. Influence on p8 expression of the induction of intracellular pathways promoting cellular growth or growth arrest was monitored.\n\nResults\np8-silenced cells grew more rapidly than control cells transfected with the empty retrovirus. Activation of the Ras→Raf→MEK→ERK and JNK intracellular pathways down-regulated p8 expression. In addition, the MEK1/2 inhibitor U0126 and the JNK inhibitor SP600125 up-regulates expression of p8. Conversely, p38 or TGFβ-1 induced p8 expression whereas the specific p38 inhibitor SB203580 down-regulated p8 expression. Finally, TGFβ-1 induction was in part mediated through p38.\n\nConclusions\np8 inhibits the growth of human pancreatic cancer cells. p8 expression is induced through pathways involved in growth inhibition and repressed by factors that promote cell growth. These results suggest that p8 belongs to a pathway regulating the growth of pancreatic cancer cells.\n\n\n\nBackground\nWhile studying the molecular response of the injured pancreas, we identified a new gene, called p8, whose expression is strongly induced during the acute phase of pancreatitis [1]. Further experiments have shown that p8 mRNA is activated in almost all cells in response to several stresses [2], including minimal stresses such as after routine change of the culture medium in the absence of any added substance [3], indicating that p8 is a ubiquitous protein induced by cellular stress. The p8 gene was cloned in human, rat, mouse, and Xenopus laevis [1,4-6], conceptually translated from the Drosophila melanogaster genome or deduced from EST libraries (Bos taurus, Xenopus tropicalis, Zebrafish, Orzzias latipes, Bombyx mori and Paralichthys olivaceous). The overall degree of homology with human p8 ranged from 81 to 40%. Secondary structure prediction methods indicated that within the homologous region of the eleven proteins, there is a basic Helix-Loop-Helix secondary structure motif, characteristic of some classes of transcription factors [1]. Even though a small protein such as p8 would not need a nuclear localization signal (NLS) to be transported to the nucleus, a clear NLS can be predicted for the eleven proteins comprising a bipartite domain of positively charged aminoacids. In addition, a nuclear/cytoplasmic location has been demonstrated for human p8 upon overexpression of the recombinant protein and immunohistochemistry [4], and for recombinant Xenopus laevis p8 fused to green fluorescent protein [6]. Homology searching in databases did not reveal significant similarity of p8 with other proteins of known function. However, biochemical properties of the mammalian p8 proteins are shared by some high mobility group proteins (HMG) [7], particularly by the HMG-I/Y family. The overall identity of human p8 with human HMG-I/Y is only about 35%, but the molecular mass, isoelectric point, hydrophilicity plot, the resistance to denaturation after heating at 100°C and the charge separation are very similar [8]. The p8 protein seems to bind DNA weakly, as shown by electrophoretic mobility shift assay, without preference for DNA sequences. Finally, human p8 has also been shown to be a substrate for protein kinase A in vitro and phosphorylated p8 has a higher content of secondary structure and binding to DNA is highly increased [8]. An architectural role in transcription has been proposed for this protein, in analogy with the HMG-I/Y proteins, and a recent work seems to confirm this hypothesis [9].\nFunctions of p8 appear to be multiple and complex. For example, p8 mRNA expression was strongly induced in 3T3 cells upon TGFβ-1 treatment which in turn enhances the Smad-transactivating function responsible for TGFβ-1 activity [10]. We also found that p8 is involved in cell cycle regulation since p8-deficient embryonic fibroblasts grew more rapidly and incorporated more [3H] thymidine and BrdU than p8-expressing cells [11]. Moreover, expression of p8 in breast cancer-derived cells seems to mediate the inhibition of cell growth induced by 1,25-Dihydroxyvitamin D3 [12]. On the contrary, we also reported that p8 may promote cell growth when overexpressed in Cos-7, AR42J and HeLa cells [1,4]. In addition, p8 seems to be involved in other intracellular functions such as apoptosis since p8-expressing fibroblasts are more sensitive than p8-deficient fibroblasts to the apoptosis induced by DNA damage. Also, p8 is required for endothelin-induced mesangial cell hypertrophy in diabetic kidney, in a mechanism involving ERK, JNK and PI3 kinase [13]. p8 seems to play a functional role in the initiation of LHβ gene expression during embryonic cell differentiation [14]. Moreover, the Drosophila melanogaster p8 homologue is involved in response to starvation and might be activated to stop cell growth in case of nutrient deprivation [15]. Finally, a particularly attractive role in tumour progression was recently proposed for p8 [16]. Fibroblasts obtained from p8-expressing or p8-deficient animals were transformed with a retroviral vector expressing both the rasV12 mutated protein and the E1A adenoviral oncogene. In soft-agar assays, transformed p8-expressing cells formed colonies at high frequency, as expected, but p8-deficient transformed fibroblasts were unable to form colonies. Similarly, transformed p8-expressing cells produced tumours in all athymic nude mice when injected subcutaneously or intraperitoneally, whereas transformed p8-deficient fibroblasts did not. On the other hand, studies by another laboratory revealed that expression of the Com1 protein [17], which is identical to human p8, mediates the growth of tumour cells after metastatic establishment in a secondary organ, indicating that activated expression of Com1/p8 in metastatic cells is required for tumour progression. These results strongly suggest that p8 is involved in the cellular pathway(s) required for tumour progression and metastasis.\nOur aim is to check the relevance of p8 to cancer progression in human. As a first step, we investigated in the present study the function of p8 in two cell lines derived from human pancreatic cancer. We observed that inhibition of p8 expression increased the cells growth rate. In addition, activations of the Ras→Raf→MEK→ERK and JNK intracellular pathways, which promote the growth of pancreatic cells, down-regulated p8 expression, whereas activation of p38 or TGFβ-1, which inhibit cell growth, induced its expression. It was concluded that i/ p8 inhibits the growth of human pancreatic cancer cell lines, ii/ p8 expression is induced through pathways involved in growth inhibition and, conversely, repressed by factors that promote cell growth.\n\nResults\np8 is silenced in pancreatic cancer cells by infection with a retrovirus expressing p8 RNA in the antisense orientation\nPanc-1 and BxPc-3 pancreatic cells were chosen for this study because, on the one hand, both cells express higher level of p8 (Figure 1) and, on the other hand, because Panc-1 is wild-type for Smad4/DPC4 and mutated for K-ras, while BxPc-3 is Smad4/DPC4 mutated and K-ras wild-type [18,19], therefore representing different mechanisms of transformation and different genetic backgrounds. K-ras and Smad4/DPC4 mutations are the major mechanisms involved in pancreatic cancer development. We inhibited p8 expression in both Panc-1 and BxPc-3 pancreatic cells by infecting cells with a retrovirus expressing the p8 asRNA (antisense RNA) and carrying the puromycin resistance. The antibiotic-selected cells were analyzed by Western blotting to evaluate the intracellular amounts of p8 protein. As shown in Figure 2, the p8 protein was clearly visible in both Panc-1 and BxPc-3 pancreatic cells infected with the empty retrovirus but almost undetectable in cells infected with the retrovirus encoding the p8 asRNA showed, indicating that our anti-sense strategy is efficient to silence p8 gene expression in pancreatic cancer cells. Preliminary studies had been conducted to select the best strategy to inhibit p8 expression. We compared the efficacy of the stable transfection of a siRNA, using a retroviral expression vector to the asRNA strategy described above. In our hands, the antisense strategy worked best, as judged from Western blot assessment of p8 protein expression (data not shown).\n\np8-silenced pancreatic cells grow more rapidly\nWe compared in the two cell lines the influence on growth parameters of blocking p8 expression with the p8 asRNA. Figure 3 shows that both Panc-1 and BxPc-3 cells in which p8 has been silenced grew more rapidly than cells infected with the empty vector suggesting that inhibition by p8 of pancreatic cancer cell growth is independent from the mechanism of transformation and genetic background.\n\nSerum-stimulated cellular growth down-regulates p8 expression\nFetal calf serum, which contains a complex mix of growth factors, can be used as inductor of cell growth. As shown in Figure 4 expression of p8 mRNA was down-regulated in both Panc-1 and BxPc-3 when the cells were shifted from culture media containing 0.1% fetal calf serum to media containing 10% FCS. p8 protein showed a similar behavior. These results show that p8 expression is down-regulated in growing pancreatic cells.\n\nThe Ras→Raf→MEK→ERK pathway down-regulates p8 expression in pancreatic cancer cells\nMost human pancreatic cancers harbor mutations in the K-ras oncogene, which happens relatively early in pancreatic tumorigenesis [20]. The oncogenic mutation of the K-ras gene stabilizes the Ras protein in a GTP-bound form, which is constitutively active and make the cells grow more rapidly. Contrary to the activated Ras protein, p8 inhibits cell growth (Figure 3). We looked whether the Ras→Raf→MEK→ERK pathway was also involved in the regulation of p8 expression, and which step(s) were critical. Figure 5 shown that expression of a mutated form of the Ras protein (rasV12) in BxPc-3 cells, which are wild-type for ras, resulted in decreased p8 mRNA concentration and protein level suggesting that the activated ras inhibits p8 expression. Figure 6 shows that overexpression of Raf, but not of Raf301 (a negative mutant of Raf), and of ERK also inhibited the expression of the p8-CAT construct in Panc-1 as well as in BxPc-3. Finally, the MEK1/2 specific inhibitor U0126 [21] activated p8 mRNA expression in pancreatic cells whether they carry mutated ras (Panc-1) or wild-type (BxPc-3). Similar results were observed when expression of the p8 protein was monitored by Western blotting (Figure 7).\n\nActivation of the JNK pathway down-regulates p8 expression in pancreatic cancer cells\nc-Jun NH2-terminal kinase (JNK) is another major MAPK pathway which converts extracellular signals into expression of specific target genes through phosphorylation and activation of transcription factors. JNK activation has been implicated in various, often opposite cellular responses, such as cell proliferation, transformation and apoptosis. As shown in Figure 8, overexpression of JNK down-regulates the gene reporter activity of the p8-CAT construct in Panc-1 cells. Similar results were found in BxPc-3 cells. Treatment of these cells with the JNK specific inhibitor SP600125 [22] up-regulates expression of the p8 mRNA and p8 protein (Figure 9). These results show that the JNK pathway is involved in the regulation of p8 expression.\n\nThe p38 pathway up-regulates p8 expression in pancreatic cancer cells\nThe p38 signal transduction pathway also plays an essential role in regulating several cell functions including growth, response to inflammation, differentiation and apoptosis. In fact, in pancreatic cancer cells, p38 is a strong inhibitor of proliferation [23] contrary to the Ras→Raf→MEK→ERK and JNK pathways. We therefore analyzed the putative role of the p38 pathway in regulating p8 expression in pancreatic cancer cells. Figure 10 shows that over-expression of the plasmid encoding p38 significantly increases p8-CAT activity in Panc-1 as well as in BxPc-3 cells. Then, cells were treated with SB203580, a specific inhibitor of p38 [24], and p8 expression was measured. p8 mRNA as well as the encoded protein were down-regulated after inhibition of the p38 activity (Figure 11). These results indicate that the p38 pathway is a positive regulator of p8 expression in pancreatic cancer cells.\n\nTGFβ-1 up-regulates p8 expression in pancreatic cancer cells\nThe most prominent biological activity of TGFβ-1 is its potent inhibition of cell growth in a wide variety of cell types including pancreatic cells. TGFβ-1 signals are sent through two types of transmembrane serine/threonine kinase receptors. In fact, TGFβ-1 binds and brings together the type I and type II receptors. In the resulting complex, the constitutively active TGFβ-1 type II receptor phosphorylates the type I receptor, which then plays a major role in transducing the signal to downstream components to affect gene expression through phosphorylation of SMAD proteins. Phosphorylated receptor-regulated SMADs then form heteromeric complexes with the common partner SMAD4. These heteromeric complexes then move to the nucleus, where SMAD4 will bind DNA and contribute to transcriptional activation. In general, pancreatic cancer cells present with defects in TGFβ-1 signaling and are resistant to TGFβ-1-mediated growth suppression. Since TGFβ-1 and p8 are inhibitors of pancreatic cell growth we analyzed whether p8 could mediate, at least in part, the effect of TGFβ-1. First, we found that treatment of Panc-1 cells with TGFβ-1 increased p8 mRNA levels and p8 protein as judged by Western blot (Figure 12). Then, to confirm that overexpression is regulated at the transcriptional level, we analyzed the effect of some constructs expressing constitutively activated type I TGFβ receptor, dominant negative type II TGFβ receptor, a dominant negative of Smad4 and the wild-type Smad4 on the p8-CAT activity. As expected, the constitutively activated type I TGFβ receptor but not the dominant negative type II TGFβ receptor increased CAT activity. Also, expression of the Smad4, contrary to that of the negative mutant, induced p8 transcription (Figure 13). Together, these results indicate that p8 is positively regulated by TGFβ-1.\nBeside the Smad proteins, TGFβ-1 also activates the p38 MAPK pathway in pancreas-derived cells, which may play an important role in TGFβ-1 induced genes [25]. Therefore, we analyzed the p38-dependent effect of TGFβ-1 on p8 transcription. As shown in Figure 13, inhibition of p38 activity with the SB203580 specific inhibitor decreased about 40% the activity of TGFβ-1 on the p8 promoter indicating that the effect of TGFβ-1 on p8 promoter is mediated by both p38-dependent and p38-independent pathways.\n\n\nDiscussion\nPancreatic adenocarcinoma is the fourth leading cause of death from malignant diseases [26]. The aggressive nature of the neoplasia, the lack of early detection, and the limited response to treatments such as chemotherapy and radiotherapy contribute to the high mortality rate of the disease. Therefore, a better understanding of the molecular mechanism leading to pancreatic cancer remains a major goal because it may help proposing strategies for earlier diagnosis and better treatment. The most commonly altered genes in pancreatic adenocarcinoma are K-ras (75 to 100%), p16INK4a (95%), p53 (50 to 75%) and DPC4 (50%) [27-31]. Whereas K-ras is a proto-oncogene all the others are tumour suppressor genes. Additional genes have been found altered at lower frequency. Panc-1 and BxPc-3 pancreatic cells were chosen for this study because they both express high levels of p8 (Figure 1) and because they present with different mechanisms of transformation and genetic backgrounds, Panc-1 being wild-type for Smad4/DPC4 but mutated for K-ras and BxPc-3 mutated for Smad4/DPC4 and wild-type for K-ras [18,19]. This work presents evidences that p8 inhibits the growth rate of pancreatic cancer-derived cells and that the intracellular pathways promoting cell growth down-regulate p8 expression whereas those promoting growth arrest up-regulate its expression. Together, these results suggest that p8 is downstream of some cell growth regulators and therefore regulation of p8 expression or its activity could be used as a target for treating pancreatic cancer.\nSilencing p8 expression was able to strongly promote cell growth in both cell types, Panc-1 and BxPc-3, suggesting that p8 may act downstream of the ras- or Smad4/DPC4-dependent ways. Also, we found that stimulating cell growth by the complex combination of growth factors contained in fetal calf serum down-regulated expression of p8 whereas, on the contrary, treating the cells with TGFβ-1, which promotes cell cycle arrest, stimulates p8 expression. Therefore, p8 gene expression seems to be regulated in opposite directions by mechanisms promoting cell growth or cell cycle arrest. It is interesting to note that while p8 expression is under the control of cell growth regulatory pathways such as Ras→Raf→MEK→ERK, JNK, p38 and TGFβ-1, p8 can affect cell cycle progression, suggesting that p8 is a target for factors regulating pancreatic cell growth.\nA mechanism by which p8 could regulate cell cycle progression in embryonic fibroblasts was previously proposed [11]. In fact, p8 seems to take action upstream from cyclin-dependent kinases because the intracellular levels and activities of Cdk2 and Cdk4 are decreased when p8 is expressed. Concomitantly, the cyclin-dependent kinase inhibitor p27 is expressed at a low level in p8-deficient cells which may explain the increased activity of Cdk2 and Cdk4. The mechanism by which p8 regulates the intracellular level of those proteins remains to be determined. However, because p8 is a transcriptional cofactor, it is possible that regulation of expression of these molecules takes place, at least in part, at the transcription level.\nInterestingly, expression of p8 mRNA seems to be regulated in a cell type- and stimulus-specific manner since, for example, p38 can induce p8 expression in response to stress in fibroblasts [3] but not in renal mesangial cells treated with endothelin [13]. In pancreatic cancer-derived cells p38 seems to play a major role since it is involved in p8 activation as judged by transient transfection assays and using a specific p38 inhibitor (Figures 10 and 11). In addition, p38 is also involved in TGFβ-1-induced p8 expression because about 40% of the TGFβ-1 effect was abolished when p38 activity was specifically blocked (Figure 13). On the other hand, ERK and JNK are inducers of p8 expression in mesangial cells treated with endothelin, but not involved in the activation of p8 in response to stress in fibroblasts [3], and even repressors in pancreatic cells (Figures 5, 6, 7, 8 and 9). Finally, PI3 kinase is an inducer of p8 expression in both endothelin-mediated p8 activation in mesangial cells [13] and pancreatic cells (data not shown).\nBased on these observations, overexpression of p8 could be considered a possible goal for treating pancreatic tumours, in order to limit their growth. However, we previously reported that p8 repression would prevent rasV12/E1A transformed fibroblasts from evolving as tumours in nude mice [16]. This apparent contradiction needs to be resolved before considering p8 as a target for treating cancer progression.\n\nConclusions\nIn conclusion (see Figure 14), we report in this paper that inhibition of p8 expression by an anti-sense strategy increases the growth rate of both Panc-1 and BxPc-3 pancreatic cancer-derived cells. Moreover, ERK- and JNK-mediated pathways down-regulate p8 expression, whereas p38 and TGFβ-1 pathways induce p8 expression. Also, cell growth triggered by expression of a RAS mutated protein or by 10% fetal calf serum induces down-regulation of p8 expression. Together, these results indicate that p8 is an intracellular cell growth inhibitor and that it is oppositely regulated by growth-promoting or growth-inhibiting factors in pancreatic cancer-derived cells.\n\nMaterial and Methods\nCell lines and cell culture conditions\nThe human pancreatic cancer cell lines Panc-1 and BxPc-3 were a kind gift of Dr C. Susini (INSERM U.531, Toulouse) and A. Hajri (IRCAD, Strasbourg) respectively. Panc-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicilin G and 100 μg/ml streptomycin. BxPc-3 were cultivated in RPMI 1640 medium in the presence of 2 mM L-glutamine, 4.5 g/L glucose, 10 mM Hepes, 1.0 mM sodium pyruvate, 10% fetal calf serum and 100 IU/ml penicilin G and 100 μg/ml streptomycin. Human recombinant TGFβ-1 was obtained from Sigma, and specific SB203580, U0126 and SP600125 inhibitors were from Calbiochem and utilized at 10 μM.\n\nExpression plasmids\nExpression plamids encoding p38 (pCEFL HA p38), Erk2 (pcDNAIII HA ERK2), JNK (pcDNAIIIB HA JNK), the wild-type Raf (pcDNA RAF BXB) and the Raf dominant negative (pcDNA RAF 301 K375W) were obtained from O Coso (University of Buenos Aires). Plamids encoding the constitutively activated type I TGFβ receptor (RI ACT), the dominant negative type II TGFβ receptor (RII DN) and the Smad4 dominant negative (DPC4 1–514 a.a.) were obtained from R Urrutia (Mayo Clinic, Rochester) and the wild type Smad4 was from C Heldin (Ludwig Institute, Uppsala).\n\nPancreatic p8-deficient cells\nTo silence p8 expression in pancreatic cells, we infected these cells with a retrovirus expressing human p8 in the antisense orientation. The retroviral vector was constructed as follows: human p8 cDNA was subcloned in HindIII and XhoI restriction sites of the pLPC plasmid (obtained from S. Lowe) in the antisense orientation. Amphotrope human p8 expressing retrovirus was then generated by transient transfection using Phoenix amphotrope packaging cells. Viral supernatant was used to infect Panc-1 and BxPc-3 pancreatic cells and the population of p8-silenced cells was isolated by selection in presence of puromycin (1 μg/ml). As control, cells were infected with the pLPC empty vector.\n\np8 expression in arrested and growing cells\nOne million of Panc-1 or BxPc-3 cells were cultivated on 10-cm Petri plates in standard conditions (with 10% FCS). After 48 h, culture media were changed for fresh media with FCS restricted to 0.1%, in order to stop growth. After 24 hours of growth arrest, culture medium was replaced either by medium with 10% fetal calf serum to resume cell growth or, as control, by medium with 0.1% fetal calf serum. Twenty four hours later cells were recovered and RNA and protein extracted.\n\np8 expression in TGFβ-1-treated Panc-1 cells\nOne million of Panc-1 cells were cultivated in 10-cm culture dishes for 48 hours under standard conditions before TGFβ-1 treatment. Human recombinant TGFβ-1 (5 ng/ml) was added to cells, without changing the culture medium, and cells were collected 12 hours later for RNA and protein preparation.\n\nBxPc-3 rasV12-expressing cells\npLPC-rasV12 and pLPC plasmids were obtained from S. Lowe. Phoenix amphotrope packaging cells (106) were plated in a 6-well plate, incubated for 24 hours, then transfected with PEI with 5 μg of retroviral plasmid. After 48 hours, the medium containing virus was filtered (0.45 μm filter, Millipore) to obtain the viral supernatant. Target BxPc-3 were plated at 2 × 105 cells per 35-mm dish and incubated overnight. For infections, the culture medium was replaced by an appropriate mix of the viral supernatant and culture medium (V/V), supplemented with 4 μg/ml polybrene (Sigma), and cells were incubated at 37°C. BxPc-3 rasV12-expressing cells were selected with puromycin (1 μg/ml). Cells infected with the pLPC empty vector were used as control.\n\nWestern-blot analyses\nOne hundred μg of total protein extracted from cells was separated with standard procedures on 15.0% SDS-PAGE using the Mini Protean System (Bio-Rad) and transferred to a nitrocellulose membrane (Sigma). The intracellular level of p8 was estimated by Western blot using a polyclonal antibody (1:1000) raised against human p8 [4].\n\nGrowth curves\nOne hundred thousand cells per well were plated in a series of 35-mm culture dishes. The cell number was estimated daily in triplicate, during 1 to 5 days, in a haemocytometer. Within experiments, each point was determined at least two times.\n\nCell transfection and gene reporter assays\nPanc-1 and BxPc-3 (105) were cultivated in 30 mm diameter culture dishes for 24 hours then transiently transfected with 0.5 μg of p8-CAT reporter plasmid and 0.5 μg of pCMV/βgal plasmid (to control transfection efficiency) using the Fugene reagent in accordance with the manufacturer's protocol (Roche Molecular Biochemicals). The p8-CAT plasmid is the previously reported p-1471/+37p8-CAT promoter construct [5]. Reporter activities were measured as previously described [5]. Briefly, cell extracts were prepared with the reporter lysis buffer (Promega) 24 hours after transfection and CAT activity was determined by the phase extraction procedure [32] and β-galactosidase assay was performed essentially as described in Sambrook et al. [33]. CAT activity was normalized to β-galactosidase activity. Experiments were carried out in triplicate and repeated at least two times. Expression plasmids (0.5 μg) were co-transfected with p8-CAT and pCMV/βgal plasmids as indicated.\n\nRT-PCR analysis\nRNA was extracted using the Trizol (Life Technologies) procedure. Total RNA (1 μg) was analyzed by RT-PCR with the SuperScript™ One-step RT-PCR System and the Platinum Taq kit (Life Technologies). RT-PCR was performed using different numbers of cycles to verify that the conditions chosen were within the linear range. The mRNA coding for p8 was specifically amplified with sense (5' GAAGAGAGGCAGGGAAGACA 3') and antisense (5' CTGCCGTGCGTGTCTATTTA 3') primers, in positions 72 and 643 of the cDNA (accession # NM_012385), respectively. As control, the transcript coding for the ribosomal protein RL3 was specifically amplified for 22 cycles with sense (5'GAAAGAAGTCGTGGAGGCTG3') and antisense (5'ATCTCATCCTGCCCAAACAC3') primers, in positions 216 and 637 of the cDNA, respectively.\n\n\nAuthor's contributions\nCM prepared cells and retrovirus, carried out RNA purification, RT-PCR, Western blots, and cell growth experiments, NL carried out CAT assays, SV participated in the design of the study and analysis of data, JLI participated in the analysis of data and wrote the manuscript. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 27085 ] ] } ]

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[ { "id": "pmcA2567314__text", "type": "Article", "text": [ "Syndrome of arachnomelia in Simmental cattle\nAbstract\nBackground\nThe syndrome of arachnomelia is an inherited malformation mainly of limbs, back and head in cattle. At present the arachnomelia syndrome has been well known mainly in Brown Swiss cattle. Nevertheless, the arachnomelia syndrome had been observed in the Hessian Simmental population during the decade 1964–1974. Recently, stillborn Simmental calves were observed having a morphology similar to the arachnomelia syndrome. The goal of this work was the characterization of the morphology and genealogy of the syndrome in Simmental to establish the basis for an effective management of the disease.\n\nResults\nThe first pathologically confirmed arachnomelia syndrome-cases in the current Simmental population appeared in the year 2005. By 2007, an additional 140 calves with the arachnomelia syndrome were identified. The major pathological findings were malformed bones affecting the head, long bones of the legs and the vertebral column. It could be shown that, with the exception of two cases that were considered as phenocopies, all of the paternal and about two-third of the maternal pedigrees of the affected calves could be traced back to one common founder. Together with the data from experimental matings, the pedigree data support an autosomal recessive mutation being the etiology of the arachnomelia syndrome. The frequency of the mutation in the current population was estimated to be 3.32%.\n\nConclusion\nWe describe the repeated occurrence of the arachnomelia syndrome in Simmental calves. It resembles completely the same defect occurring in the Brown Swiss breed. The mutation became relatively widespread amongst the current population. Therefore, a control system has to be established and it is highly desirable to map the disease and develop a genetic test system.\n\n\n\nBackground\nIn the year 2006 a syndrome was described in the German and Austrian Simmental (Fleckvieh, as it is locally called, is the main dual-purpose breed in Germany, in short called Simmental in the further text) population, that was pathologically similar to the arachnomelia syndrome in Brown Swiss cattle [1]. The congenital arachnomelia syndrome (AS, OMIA Phene ID 139, Group 000059) is mainly a malformation of the skeletal system in cattle that was initially described by Rieck and Schade [2] in Holstein Friesian, Red Holstein and Simmental.\nThe main pathological changes are skeletal malformations of the legs, the spinal column and the skull. The legs are thinner and appear longer than normal (dolichostenomelia, arachnomelia) since the diameter of the diaphyses is reduced. These long bones are more fragile and, in combination with stiffened joints, they tend to fracture during calving. The fetlock joints are deformed, often stiffened and show hyperextension. The malformation of the spinal column leads to kyphosis and scoliosis. The skull malformations are characterized by a shortened lower jaw (brachygnathia inferior), convex rounding of the frontal bone leading to a marked stop (\"pointer head\") and rotation of the anterior cranium. In some cases, additional malformations like hydrocephalus externus develop [2-5].\nSince the report of Rieck and Schade [2] no further cases were reported in Simmental cattle, but in the 1980s the syndrome was dispersed in another breed, the European Brown Swiss cattle, by the use of American Brown Swiss sires [4,6]. In Brown Swiss an autosomal recessive mode of inheritance was supposed and a control program based on the identification of carriers by pedigree analyses was established [5]. Recently, four cases of arachnomelia syndrome were reported in Italy [3].\nIn this study, we present the data of 152 pathologically confirmed cases of arachnomelia syndrome in Simmental that were collected from October 2005 to March 2007. We describe the pathological findings, the familial occurrence and an estimate of the frequency of the diseases allele in Simmental cattle. Additional support for the mode of inheritance and the genetic basis of the arachnomelia syndrome is given by the result of experimental matings of obligate carriers.\n\nResults and discussion\nAS has not been reported again in Simmental since its first description in the 1970s, more than thirteen years ago. In autumn 2004 a number of stillborn calves with similar malformations of the legs and head were recorded within the monitoring system of anomalies in Simmental. Some of these calves were sent to the veterinary service laboratory for examination, and in December 2005 the first 15 cases of AS were pathologically confirmed. Subsequently, farmers and veterinarians had been encouraged to report cases by an information leaflet and various articles in local trade journals. An increasing number of suspected cases was reported and an additional 136 affected calves were identified by pathological examination by June 2007.\nFamilial occurrence and case presentation of the syndrome of arachnomelia\nThe geographical origins of the cases were the southern part of Germany and Austria, reflecting the regional distribution of the Simmental breed. Both sexes were equally represented in the 152 (80 male, 72 female, χ2 = 0.21, p = 0.64) affected calves. The largest number of cases was registered in 2006 (Figure 1). In retrospect, it could be shown that the main reason for the rapid increase of cases in the years 2005 and 2006 was the high popularity of certain sires carrying the AS mutation (ROMEL, ISO-Nr. 276000911043667, born in 1995; EGEL, 276000915512806, 1985; REXON, 276000913008210, 1989). The latter two sires represent the key-nodes of the pedigree pathways of the mutation from the founder into the current population (Figure 2). ROMEL, for example, sired more than 40,000 cows 4 to 6 years ago. Furthermore, 115 sons of ROMEL born from 2001 to 2005 are registered and listed in the breeding database [7]. These progeny were now mated to ROMEL and sons or grandsons of EGEL and REXON resulting in a high probability for the occurrence of affected calves. Increasing awareness of the disease and abandoning of selling the semen from carriers led to a sharp drop of cases in 2007. The disease was successfully managed by efficient collaboration of the Institute for Animal Breeding of the Bavarian State Research Centre for Agriculture (LfL), the Landeskuratorium der Erzeugerringe für tierische Veredelung in Bayern e.V (LKV), the Bavarian Animal Health Service (TGD) and breeding organizations.\n\nPathological findings\nCalves under suspicion of the arachnomelia syndrome were sent to the pathology department of the TGD for macroscopic examination. The observed major pathological findings were (1) facial deformation, including brachygnathia inferior and concave rounding of the maxilla forming a dent ('pointer-head'); (2) abnormally thin diaphyses of the long bones (the outer diameter of the diaphyses is diminished, whereas the width of the substantia compacta is normal) leading to frequent fractures of the metacarpus and metatarsus in the course of forced birth assistance ('spider-legs', dolichostenomelia). The deformations of other bones of the legs were less apparent and the scapula was usually unaffected; (3) angular deformations of the distal parts of the legs characterized by bilateral stiff and hyperextended fetlocks with the extremity of the toe forward and parallel to the trunk of the body; and (4) defects of the vertebral column (kyphosis and scoliosis), but not of the ribs (Figure 3A–C). Additionally, inconsistent pathological findings included cerebral herniation combined with a malformed foramen magnum, microphthalmia, and external and internal hydrocephalus. The latter seem to develop secondarily, due to the enlarged foramen magnum.\nHistological examination of selected cases revealed the presence of hemorrhages at the osteochondral junction of the epiphysis and an abrupt transmission from chondral to osteogenic tissue.\nCases never showed isolated malformations, e.g. of the head or legs, but usually a combination of all pathological findings that are characteristic for the syndrome. Nevertheless, the degree of the lesions ranged from obvious spider-leg cases to moderate or mild changes, making a definite diagnosis difficult. The latter cases (3) were excluded from the initial pedigree analyses. Meanwhile, an indirect gene test is available that has been developed at the Institute for Animal Breeding of the Bavarian State Research Centre for Agriculture (ITZ) and it could be shown that these cases are most probably not genetically affected (Buitkamp et al., in preparation).\n\nCarrier identification\nTwo criteria were used for carrier identification. The first was the presence of a calf that was diagnosed by pathological investigation. In many cases more than one affected calf per sire was identified [8]. Some sires had only one affected calf, but a large number of risk-matings. In these cases a second criterion, the statistical evaluation of risk-matings, was used to identify potential phenocopies. For this purpose, the probability of observing only a single affected calf among a certain number of risk-matings of the sire in question was calculated. Risk-matings were defined as matings with direct progenies of identified AS carriers. The probability of observing an affected calf depends also on the probability that such a calf is reported to the LKV. We assumed this probability to be 50%. Under these conditions, the probability of observing only one affected calf is lower than 1.0 percent, if at least 104 risk-matings are given for a single sire. In this case it is very likely that the single affected calf is a phenocopy. In 2006 and 2007 this was the case for two sires used for artificial insemination that had no pedigree connection to SEMPER (see below).\n\nExperimental matings of obligate carriers\nFour out of seven cows that were known AS carriers brought to the facilities of the ITZ were used for embryo transfer (Table 1). 33 of the 60 recipients (55%) were confirmed pregnant on day 35. Four of the 33 pregnant heifers (12%) aborted between days 36 and 49 of pregnancy. Of the remaining 29 recipients, 6 were slaughtered on day 150, 6 on day 200, and 17 animals on day 225 of pregnancy (Table 1). Four fetuses (three male and one female) out of 29 (14%) showed the typical pathological changes of the arachnomelia syndrome as described above (Figure 4C,D). All other fetuses showed no signs of AS (Figure 4A,B).\nMale fetuses represented 76% (22 of 29) of pregnancies (χ2 = 3.123, p = 0.077, Yates corrected for sample size <30). Female weight (Table 2), crown-rump length at day 225 and chest circumference at day 225 of normal fetuses were lower than that of male fetuses. Fetuses that were affected by the arachnomelia syndrome showed lower weight than normal fetuses. The affected and the normal fetuses had similar crown-rump length, but the chest circumference of affected fetuses was higher than that of normal fetuses (Table 2). Due to the small number of affected female fetuses, a comparison with unaffected animals for sex was not possible. To compare unaffected and affected animals in total, the data were analyzed for statistical differences by the non parametric Mann-Whitney-Test. The only trait that was significantly different between unaffected and affected fetuses was the chest circumference (p = 0.004, Table 2). The body weight of AS affected calves was tendentially lower than that of normal calves. Since affected calves did not have different crown-rump-length and their chest circumference was even higher, this can best be explained by a reduced bone mass.\n\nPedigree Analysis and mode of inheritance\nEight-generation pedigrees of all cases were extracted from the joint German and Austria pedigree data and screened for common ancestors. The pedigree of the majority of affected calves (paternal line 150, maternal line 106 out of 152, Table 3) could be traced back to one founder, SEMPER (ISO-Nr. 27000979299305), a sire born in 1964, 6–9 generations before the affected calves were born (Figure 2). Most of the affected calves inherited the AS mutation via REXON or EGEL (Table 3, Figure 2). In 44 cases the maternal paths were not linked to the common pedigree (Table 3). One explanation would be the existence of additional, hereto unknown origins of the mutation. This could happen if the AS mutation is much more ancient and additional pedigree paths exist or if an independent mutation event happened leading to the same phenotype. An alternative, more plausible explanation could be the occurrence of misparentages. It is well known that in the pre parentage-test era, the frequency of false paternity, especially of the cows, was reasonable high (up to 23% [9]). Therefore, there is a good chance of a false registry within 6–9 generations.\nThere is strong support for the assumption that the AS is regulated by a single autosomal locus acting in a recessive manner. First of all, the pedigree structure of the affected calves in Simmental can best be explained by a recessive mode of inheritance. The paternal branch of the pedigree could be traced back to one sire, SEMPER, for all affected calves, the maternal branch in the majority of the cases. Inbreeding loops over a few generations are present in several pedigrees of affected calves, e.g. cases P3364 and P1787 (Figure 2). Sex-dependent inheritance can obviously be excluded and a dominant mode with reduced penetrance seems to be unlikely. Secondly, the experimental matings resulted in 4 affected and 25 unaffected fetuses, a result that most closely resembles the expectation of a recessive mode of inheritance. Thirdly, the occurrence of cases corresponded well with the numbers expected under the assumption of a recessively acting mutation. We tested this on the progeny of ROMEL, the largest dataset available from one carrier. We analyzed the period from the beginning of the recording system for malformations to May 2007. In that period 44,170 calves were born that were sired by ROMEL. From these, 662 were considered as risk pairings, i.e. the mother had a risk of 0.5 to be a carrier (i.e. one of the grandparents was an obligate carrier) and 35 calves out of these were diagnosed as affected. Since it is expected that about 1/2 to 1/3 of the affected calves were recorded, this result is very close to the expected 1:7 ratio of affected to unaffected calves. Moreover, these findings are concordant with the historical description of the arachnomelia syndrome in Simmental [2] and the analyses of cases in Brown Swiss [5]. Finally, when applying linkage analyses using microsatellite markers, evaluations with a model assuming recessive autosomal inheritance gave the highest lod scores (Buitkamp et al., in preparation).\n\nAllelic frequency of carriers in the present Simmental cow population\nSince the arachnomelia syndrome-allele was passed to the current population through two parental lines (REXON and EGEL) and the main carriers are known, it is possible to estimate the frequency of the disease allele by an allele-counting method [10]. The allelic frequency was calculated for all cows from the breeding population who were alive in June 2007. In 10.4 percent of the pedigrees of 540,725 cows an identified carrier was found and the probability that individual cows were carrier of the arachnomelia syndrome-allele was calculated. E.g. in 14,740 and 41,032 cases a known carrier appeared as sire and grandsire, respectively. In these cases the probability of transmitting the allele is 50 and 25 percent, respectively, if no further carrier is present in the two generation pedigree. The averaged rate of the arachnomelia syndrome carriers based on known carriers over all cows alive in Bavarian Simmental was 3.32 percent.\nUsing this approach, the frequency of carriers was calculated for each year (always based on the actual datasets from August 2008) from 2003 to 2008 (Figure 1). The calculations were done twice, considering all known carriers together, and also by using only ROMEL as a carrier to show the numeric contribution of his progeny (Figure 1). For these analyses, the sires that are designated to be non-carriers by the number of risk pairings without having a case or the indirect gene test are set as non-carrier. Therefore, these frequencies are slightly lower than the initial frequency estimate of 3.32 percent.\n\n\nConclusion\nThe cases of malformed Simmental calves presented here showed the same morphology described in the arachnomelia syndrome in Brown Swiss [e.g. [3]], even though there is a certain morphological variation from mild to severe malformations. The main findings, brachygnathia inferior and convex frontal bone of the face, deformation of vertebrae, and dysplasia of the limbs, namely the diaphyses of metatarsus and -carpus and the fetlocks, can best be explained by irregularly developed bone structure at the corresponding locations.\nWithout pathological examination it is difficult to distinguish the arachnomelia syndrome from other malformations of the limbs. Therefore, low numbers of cases in Simmental probably passed unrecognized before 2005. In that year the allelic frequency of the disease in the cow population increased sharply because some sires that had been carriers of the mutation had become very popular 2–4 years before.\nThe identification of a common ancestor, the results from the experimental matings and the analyses of numbers of cases from risk matings strongly support the hypothesis of an autosomal recessively inherited disease. Furthermore, this assumption is concordant with the historical description of the syndrome in Simmental and Brown Swiss. The allelic frequency of the arachnomelia syndrome in the current population is well above 3 percent and a substantial number of progeny from known carriers with superior genetic merit shall be used as sires during the next years. Therefore, a control system has to be established and the arachnomelia syndrome-gene should be mapped as a prerequisite for the development of an indirect gene test for carrier identification. The availability of pathologically well characterized cases from the field and from the ET-generated full-sib families will be an excellent material for a genetic mapping procedure.\n\nMethods\nRecording system for congenital malformations\nA system for monitoring inherited congenital malformations in Bavarian cattle populations was established by the Institute for Animal Breeding of the Bavarian State Research Centre for Agriculture (ITZ) in cooperation with the Bavarian milk recording organization (LKV) [11]. In short, a questionnaire was developed for detailed recording of malformed calves. The malformation was described according to its location (e.g. head, legs) and its characteristics (e.g. hernia). The standardized data were stored in a database at the LKV, that is evaluated monthly for a potential genetic background of malformations.\nSires that fit into the pedigree (progeny of REXON or EGEL) with at least one affected calf with confirmed paternity were defined as obligate carriers and marked in the breeding information system [7]. In cases without connection to the pedigree and only one recorded calf the number of \"risk pairings\" (matings to cows where at least one parent is a known carrier, enabling the calculation of the probability for the occurrence of cases) was calculated. When the probability that a case occurs was above 99% for the sire in question the case was considered to be a phenocopy. The number of calves affected by the arachnomelia syndrome and their parentage is routinely published [8].\n\nPathological examinations\nPathological examinations followed standard procedures. Calves were photographed and size and weight measurements were recorded. Tissue specimens from the condyle (epiphysis) and from the diaphysis of the femur were collected for histological examination. Specimens were fixed in 10% formalin and kept in a decalcifying solution (Ossafixonafor) for 24 hours. Thereafter, specimens were processed in an automated embedding system, sectioned at 4–6 microns and finally stained with haematoxyline and eosin.\n\nExperimental matings and embryo transfer\nKnown carriers of the arachnomelia syndrome (seven cows that had produced at least one affected calf) were brought to the facilities of the ITZ for embryo transfer (Table 1). Late morulae and blastocysts collected on day 7 (day 0 = estrus) from superovulated donor cows were nonsurgically transferred to heifers [12].\n\nMode of inheritance and allele frequency\nThe pedigree of all cases was constructed from the pedigree that is used for the joint breeding evaluation of Germany and Austria. The graphical presentation of the pedigree was performed with the Pedigraph TM software [13]. The allelic frequency of the AS mutation in the current cow population was estimated from ancestors with known genotypes following the allele-counting method [10]. For this reason two generation pedigrees of herd book cows in Bavarian Simmental were analyzed for obligate carriers. We considered all cows that were alive in June 2007 and included in the herd book. All animals were bred by the use of artificial insemination.\n\nStatistical analyses\nThe non parametric Mann-Whitney-Test was performed using SPSS Version 14.0, the Chi-square test was performed using R 2.4.0 [14].\n\n\nAuthors' contributions\nJB drafted the manuscript and analyzed the pedigrees. BL conceived the monitoring system for inherited diseases. RE extracted the data from the database and estimated the allelic frequencies of the arachnomelia syndrome. HR and MW performed the embryo collection, transfer and recorded the morpho-metrical data of the experimental matings. BS examined the calves pathologically. NM and KG participated in study design and coordination and critically revised the manuscript.\nAll authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 21958 ] ] } ]

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[ { "id": "pmcA2610030__text", "type": "Article", "text": [ "Human genetic selection on the MTHFR 677C>T polymorphism\nAbstract\nBackground\nThe prevalence of genotypes of the 677C>T polymorphism for the MTHFR gene varies among humans. In previous studies, we found changes in the genotypic frequencies of this polymorphism in populations of different ages, suggesting that this could be caused by an increase in the intake of folate and multivitamins by women during the periconceptional period. The aim was to analyze changes in the allelic frequencies of this polymorphism in a Spanish population, including samples from spontaneous abortions (SA).\n\nMethods\nA total of 1305 subjects born in the 20th century were genotyped for the 677C>T polymorphism using allele specific real-time PCR with Taqman® probes. A section of our population (n = 276) born in 1980–1989 was compared with fetal samples (n = 344) from SA of unknown etiology from the same period.\n\nResults\nAn increase in the frequency of the T allele (0.38 vs 0.47; p < 0.001) and of the TT genotype (0.14 vs 0.24; p < 0.001) in subjects born in the last quarter of the century was observed. In the 1980–1989 period, the results show that the frequency of the wild type genotype (CC) is about tenfold lower in the SA samples than in the controls (0.03 vs 0.33; p < 0.001) and that the frequency of the TT genotype increases in the controls (0.19 to 0.27) and in the SA samples (0.20 to 0.33 (p < 0.01)); r = 0.98.\n\nConclusion\nSelection in favor of the T allele has been detected. This selection could be due to the increased fetal viability in early stages of embryonic development, as is deduced by the increase of mutants in both living and SA populations.\n\n\n\nBackground\nThe methylenetetrahydrofolate reductase enzyme (MTHFR) catalyzes a reaction that produces 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for homocysteine in the synthesis of methionine. The 677C>T mutation of the MTHFR gene has been associated with a thermolabile enzyme with decreased activity that may cause an increase in plasma homocysteine concentrations [1] when folate status is poor. This polymorphism is one of the most widely studied clinically relevant polymorphisms in humans, as it is related to cardiovascular disease [2] and neural tube defects (NTD; 601634) [3].\nA large number of studies have provided a broad overview of the prevalence of the 677C>T polymorphism in different human populations, showing that the distribution of frequencies is diverse [4]. These differences have been also observed between groups of different ages in the same Spanish population (older and younger than 24 years) [5] and in a Swiss population (older and younger than 60 years) [6], as well as in a Japanese population [7].\nIn some populations, such the Toscanians in Italy [8] and Mexicans [9], the homozygous mutated genotype (TT) has reached frequencies greater than 30%. On the other hand, in Africans the frequency of the TT genotype is very low (less than 1%) [10,11], but, in African-Americans, it has already reached 2% [12]. Studies based on the distribution of genotypic and allelic frequencies of the 677C>T polymorphism and the 1298A>C polymorphism in the MTHFR gene in Israeli, Japanese and Ghanaian Africans populations [13] concluded that the 677T mutation in the MTHFR gene emerged as a founder haplotype with some selective advantage. Recently, preliminary evidence of genetic selection of this polymorphism related to folate intake has been reported [14].\nThe aim of the present study is to analyze the changes in frequencies of the 677C>T polymorphism during the 20th century and particularly the evolution of the frequencies during the decade of 1980–1989, by comparing the genotype frequencies between living subjects born in this period versus samples of spontaneous abortions (SA) that occurred during in the same time period.\n\nMethods\nSubjects\nThis study was approved by the Ethics Committee at the University Hospital \"Virgen de la Victoria\" (Málaga). One of the study groups consisted of 344 fetal tissue samples from SA, obtained from the Department of Pathology of the University Hospital Carlos Haya (Málaga). These samples were selected after checking the clinical history and by the inclusion criteria of containing histologically confirmed fetal tissue collected in the 1980s from SA at less than 3 months (11 ± 1.70 week) and of unknown etiology. These fetal samples were compared with a control population of 276 subjects born in the 1980s with an average age of 22 ± 4.58.\nAnother population of subjects born in the south of Spain in the 20th century were genotyped (1305 subjects, 697 women and 608 men) and divided into four groups according to birth date: 1900 to 1925 (n = 206); 1926 to 1950 (n = 320), 1951 to 1975 (n = 408), 1976 to 2000 (n = 371). Individuals were selected randomly from different areas of the province of Malaga, in southern Spain, and from different social statuses to avoid a selection bias. All the selected individuals were Caucasian and residents of the study area. The parents of all subjects included in the study were also Caucasian and born in Spain. The possibility of a founder effect or genetic drift was investigated and rejected. All the selected individuals were also genotyped for an insertion/deletion polymorphism in the angiotensin converting enzyme (ACE) gene and/or the 2756A>G polymorphism in the methionine synthase gene (MTR), in order to determine whether or not our adult and young populations were genetically homogeneous. No significant differences were observed in allelic or genotypic frequencies for these genes between the different groups.\nThe population studied was randomly selected according to age. Subjects 0–12 years old were selected from dried blood spots from neonatal screening papers; subjects 10–24 years old were recruited from students in primary and secondary schools and in university; subjects 25–50 years old and >51 years old were recruited using their Andalusia Health Service identity cards. After approval by the University Hospital Ethical Committee, all the subjects were contacted, and, from those whose written consent was obtained, 10 ml of blood was taken. The investigation in this study conforms to the principles outlined in the Declaration of Helsinki.\n\nGenetic analysis\nThe fetal samples were extracted from the archived formalin-fixed, paraffin-embedded tissue sections. Genomic DNA was extracted from fetal tissue using the method described by Coombs et al. (1999) [15]. Genomic DNA of the second and third groups was extracted from peripheral leukocytes using the AquaPure Genomic DNA Blood Kit (Bio-Rad).\nGenotyping was performed using Real Time PCR with allele specific Taqman® probes and primers described by Ulvik et al. (2001) [16] and the following optimized protocol for 45 cycles: 10 s – 94°C, 40 s – 54°C, 15 s – 72°C. The PCR mix (25 μl total volume) consisted of 5 μl of genomic DNA, 0.5 μl of sense primer, 0.62 μl of anti-sense primer, 0.85 μl Taqman® probe FAM, 0.43 μl Taqman® probe TET, 20 μl PCR-buffer iQ-SupermixTM (Bio-Rad) (containing 100 mM KCl, 40 mM Tris-HCl, (pH 8.4) 1.6 mM dNTP (dATP, dCTP, dGTP and dTTP), iTaq® polymerase (50 units/mL) and 6 mM MgCl2) and 17.75 μl H2O.\n\nStatistical and mathematical analysis\nAll samples were genotyped, and the allelic and genotypic frequencies were compared. Differences were analyzed statistically using the chi-square test or Fisher's exact test. Correlations are expressed using Pearson's coefficient (r).\nCompliance of genotype distributions with Hardy-Weinberg (HW) equilibrium was evaluated by chi-square analysis. For all tests, a p-value < 0.05 was considered to be statistically significant. Values are expressed as the mean ± SD.\nThe genetic selection model was calculated for the evolution of the 677C>T genotypes. The genetic selection could be classified as codominant or incompletely dominant and directional with the heterozygous genotype having an intermediate fitness. For this kind of selection, the most appropriate mathematical model is dq = [spq(2hp+q-h)]/[p2 + 2pq(1-hs) + q2 × (1-s)], where dq is the change of frequency of the allele with lower fitness, s is the fraction of that genotype lost to selection, h is the degree of dominance (between 0, for no dominance and 1, for complete dominance), and p is the frequency of the allele with higher fitness.\n\n\nResults\nWe analyzed the genotype frequencies of the 677C>T polymorphism in a population born during the 20th century. A total of 1305 subjects were divided into four groups of 25 years according to birth date. The genotype frequencies were compared between the four quarters of the century and showed very significant changes (p < 0.001) in the group born in the last quarter of the 20th century (1976–2000), when compared to any of the other groups. The changes show a decrease of the CC genotype and an increase of the TT genotype in the last 25 years of the 20th century. (Table 1)\nConsidering that each 25 year period corresponds to a generation, allelic frequencies did not change during the first 75 years of the century (HW equilibrium). However, we found that allelic and genotypic frequencies for the 677C>T polymorphism in the last quarter of the century are significantly different compared to the previous generation (1951–1975). The genotype frequencies in the last quarter of the century are not the expected by a HW calculation using the allelic frequencies of the previous generation. This could be described as a consequence of genetic selection found in this population, in the absence of other causes. Applying the mathematical model described above to our population, the calculated fitness (s) is 0.5, and it can be predicted that both alleles will be approximately at a frequency of 50% in the next generation and allele T will be at 90% after seven generations (Figure 1A). Another possibility is that a scenario could be predicted in which both alleles will have frequencies of about 50% in the next generation and that they will maintain this stability while conditions remain unchanged. (Figure 1B)\nThe comparison of the genotype frequencies between a group of fetal samples from SA that occurred during the 1980–1989 decade and living subjects born in the same decade showed significant differences in genotype frequencies (p < 0.001). CC genotypes were almost absent in abortion samples compared to living subjects (0.03 vs 0.33), while CT and TT genotypes were overrepresented in the same group. When 3-year periods are studied in the decade, we detected a significant increase of the mutated subjects during the decade (CT p < 0.05; TT p < 0.01). Allele frequencies showed the same pattern (p < 0.05). Controls showed the same tendency but without statistical significance. (Table 2)\nThe evolution of genotype frequencies during the 1980–1989 decade of the TT genotypes correlates well in both living populations as well as fetal samples r = 0.98 (p = 0.11).\n\nDiscussion\nDifferent reports show that the prevalence of the 677C>T polymorphism of the MTHFR gene differs dramatically among human populations. Evidence of this dynamism can be observed in many reports: frequency variations between populations that are geographically very close, even in the same country [8]; changes found in the same race or ethnic group such as Africans [10,11] and African-Americans [12]; the high prevalence of the 677C>T poymorphism in populations with special nutritional features such as Mexicans [9] and Japanese [13]; and changes in frequencies between generations of the same population, as has been observed in Spain [5], Switzerland [6] and Japan [7].\nThere are numerous interpretations of this great diversity, and most tend to be related to adaptation to external conditions such as climate or nutritional status. Dependence of folate degradation on skin pigmentation [17], nutritional habits or human intervention periconceptional periods could explain this genetic variation. Definitely, external factors in combination with different levels of MTHFR enzyme activity, conditioned by polymorphisms, could influence the fetal viability of certain genotypes.\nIn 1998, we suggested the possibility of genetic selection in Spain in favor of the mutants of the 677C>T polymorphism in the MTHFR gene based on the fact that treatment with vitamins and folates during pregnancy increased the viability of fetuses with the TT homozygous genotype. This hypothesis was based on the increase in the number of mutated individuals found in our population since the mid-1970s [5] and the coincident increased intake of vitamins and folate by pregnant women in Spain [18,19]. In 2002, a new study found changes in genotype frequencies for the 677C>T and 1298A>C polymorphisms in different age groups. Total homocysteine (tHcy) levels in plasma were also analyzed according to the different genotype interactions [20]. That study hypothesized about fetal viability and about a genetic selection model on the basis of non-linkage disequilibrium between both polymorphisms. Recently, a study with fetal and control populations showed the strong influence of these polymorphisms, though mainly of the 677C>T polymorphism, on spontaneous early abortion [21]. In the present study, significant changes in allelic and genotypic frequencies are detected, as is Hardy-Weinberg disequilibrium, at the 677C>T polymorphism. We hypothesize that there is a dynamic process of genetic selection that favors the T allele. This process of selection started during the last quarter of the 20th century, during which the frequency for mutant homozygous (TT) rose significantly from 14% to 24%. We propose that this increase in mutants is due to the inclusion of an external factor that enhances mutant fetal viability.\nIf we apply the mathematical model for dynamic selection developed for diploid organisms with sexual reproduction, the T allele could reach to 90% in seven generations in our population (Figure 1A). However, this model assumes selection in a constant environment that applies to all individuals in the population studied. In our case, we suggest that the external factor is related to an increase in folate and vitamin intake in women in periconceptional period and does not affect to all individuals [18,19]. We assume that prediction of a classic selection model in this case is only theoretical.\nOn the basis of a competition between alleles in which an environmental factor favors one allele versus the other, the final result would be that predicted by the previous mathematical model. However in this case, the environment is not selecting against the wild type allele but rather allowing the survival of more mutated alleles. Therefore, the expected result would be not a systematic increase of the mutated allele but the creation of an allelic balance dependent on vitamin and folate abundance conditions. In this case, the mutation would have a lower influence on fetal viability (Figure 1B).\nThe results showed an increase in mutated genotypes (CT and TT) and a strong protection against abortion by the wild type genotype (CC), which is practically non-existent in the SA group. The frequency of the CC genotype shows no change over the decade studied (1980–1989), which indicates that folate does not exert a visible effect on this genotype. However, the frequency of the mutated allele increases during this decade, especially in fetuses from abortions, and this increase correlated with the increase of the T allele in the control population. This finding suggests that the effect of folate is crucial to viability during the early stages of embryonic development, but, even with folate, not all embryos will survive until birth.\nIn this population, the mutant allele with lower enzymatic activity has higher fitness than the wild type. In the folate cycle, it can be observed that 5,10-methyleneTHF availability may be important. 5,10-methyleneTHF is the substrate for several reactions in the cycle, but two of them (5-methylTHF and thymidilate synthesis) might be essential for embryo development in folate deficiency conditions.\nIn both cases, complete or limited MTHFR activity will produce higher or lower 5,10-methyleneTHF availability, which might be an essential factor for embryo development, such that a greater folate levels can compensate the lower enzymatic activity of the mutant.\nThe implications of this polymorphism in nucleotide synthesis have not yet been determined, but certain data, such as high levels of uric acid found in mutated subjects [22,23], suggest that there are different turnover rates associated with different polymorphisms.\n\nConclusion\nWe suggest that there is genetic selection in our population for the T allele of the MTHFR – 677C>T polymorphism, whose origin could be an increase in fetal viability during the early stages of embryonic development because of an increase in folate and vitamin intake by women in the periconceptional period that began to be established in Spain in the last quarter of the 20th century [18,19]. Higher frequencies for the T allele and TT genotype in our population are observed in the living and SA populations.\n\nCompeting interests\nThe authors declare that they have no competing interests.\n\nAuthors' contributions\nAMO performed the statistical analysis, helped to draft the manuscript and revised it for publication. GC is the corresponding author, participated in the acquisition of samples and carried out the genotyping. ARP carried out the bibliographic search and helped to draft the manuscript. AJJ participated in the selection and the processing of samples. MJG coordinated the laboratory work and selected the genotyping method. AR selected the control subjects and designed the consent form. MR helped in the interpretation of data and tables performance. ARE conceived the study and is the guarantor of this work and the general coordinator. All authors read and approved the final manuscript.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\n\n" ], "offsets": [ [ 0, 18053 ] ] } ]

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[ { "id": "pmcA2526157__text", "type": "Article", "text": [ "Identification and Analysis of Co-Occurrence Networks with NetCutter\nAbstract\nBackground\nCo-occurrence analysis is a technique often applied in text mining, comparative genomics, and promoter analysis. The methodologies and statistical models used to evaluate the significance of association between co-occurring entities are quite diverse, however.\n\nMethodology/Principal Findings\nWe present a general framework for co-occurrence analysis based on a bipartite graph representation of the data, a novel co-occurrence statistic, and software performing co-occurrence analysis as well as generation and analysis of co-occurrence networks. We show that the overall stringency of co-occurrence analysis depends critically on the choice of the null-model used to evaluate the significance of co-occurrence and find that random sampling from a complete permutation set of the bipartite graph permits co-occurrence analysis with optimal stringency. We show that the Poisson-binomial distribution is the most natural co-occurrence probability distribution when vertex degrees of the bipartite graph are variable, which is usually the case. Calculation of Poisson-binomial P-values is difficult, however. Therefore, we propose a fast bi-binomial approximation for calculation of P-values and show that this statistic is superior to other measures of association such as the Jaccard coefficient and the uncertainty coefficient. Furthermore, co-occurrence analysis of more than two entities can be performed using the same statistical model, which leads to increased signal-to-noise ratios, robustness towards noise, and the identification of implicit relationships between co-occurring entities. Using NetCutter, we identify a novel protein biosynthesis related set of genes that are frequently coordinately deregulated in human cancer related gene expression studies. NetCutter is available at http://bio.ifom-ieo-campus.it/NetCutter/).\n\nConclusion\nOur approach can be applied to any set of categorical data where co-occurrence analysis might reveal functional relationships such as clinical parameters associated with cancer subtypes or SNPs associated with disease phenotypes. The stringency of our approach is expected to offer an advantage in a variety of applications.\n\n\n\nIntroduction\nBiological research has experienced a paradigm shift in the last decade catalyzed by the availability of genome sequences and the resulting development of high-throughput technologies. The large data volumes produced by these novel technologies are often published as supplementary material and/or stored in extensive data repositories [1]. Functional interpretation of these data is an ongoing challenge. Co-occurrence analysis, based on the hypothesis that co-occurring entities are functionally linked, is a technique that has been used in three main areas of biological research:\nCo-occurrence of genes in sequenced genomes relies on the fact that proteins do not function in isolation and are dependent on other proteins, either as direct binding partners, or as catalysts of substrates. Thus, when two proteins significantly co-occur in a large number of genomes or can be observed as fusion proteins in a subset of genomes, they are likely to be binding partners or enzymes needed for a specific metabolic pathway. Examples of those studies have been reported by [2]–[7].\nText mining is a quickly evolving field that aims at developing technologies helping to cope with the functional interpretation of large volumes of publications. Co-occurrence of gene names in publication abstracts, entire publications, or other gene-related databases has been used to derive co-occurrence networks with clear evidence that edges in those networks are reflecting functionally relevant relationships [8]–[11]. Gene names have also been analyzed for co-occurrence with other entities such as mutations [12], chemical compounds [13], and disease related keywords [14]. From the resulting networks, hypotheses about candidate genes involved in inherited diseases and drug targets can be derived. Clustering of gene related publications using keywords has been applied to enhance the quality of gene expression clusters [15], [16]. More general (non gene-centric) approaches try to organize the literature into functional areas based on co-occurrence of MeSH terms, keywords, diseases, phenotypes, chemicals, and similar objects of biomedical research interest [17]–[21].\nCo-occurrence analysis of transcription factor binding motifs has been carried out in a variety of slightly differing ways in a wide range of organisms, including humans. [22]–[33]. The underlying hypothesis is that co-regulated genes, identified usually by gene expression studies, should contain specific combinations of transcription factor binding motifs in their upstream regulatory regions, the identification of which would allow the reverse-engineering of transcription regulatory networks [34].\nWe have recently applied co-occurrence analysis to studying published gene expression signatures and showed that co-occurrence patterns of genes reflect cancer signaling pathways [35]. Although co-occurrence analysis has a respectable history, the methodologies used in the studies mentioned above could not be easily applied to studying gene expression signatures. There are three main reasons that dictated the use of a different approach. First, gene expression signatures can vary in size by orders of magnitude. Obviously, the larger a signature the more likely it is to find two or more genes co-occurring in that signature. Thus, the significance of co-occurrences must be evaluated in the presence of considerable heterogeneity of co-occurrence probabilities among gene lists. As a consequence, the statistics used to evaluate the significance of co-occurrence events must reflect this heterogeneity. In particular, it must be based on list-specific co-occurrence probabilities. Second, in the vast majority of previous studies, co-occurrence is analyzed for pair-wise combinations of co-occurring entities. We found that the resulting stringency of this approach is not adequate for the analysis of published gene expression signatures [35]. Third, the null-model against which the significance of co-occurrences is tested does not work well for gene expression signatures. A common procedure is to use generic randomization of the entire data set under analysis or to select subsets of data entries randomly for comparison purposes. However, gene expression signatures are composed of distinct gene sets and the null-model must maintain this property, which is not guaranteed using these approaches. Furthermore, the list-specific nature of co-occurrence probabilities cannot be dealt with properly.\nNetCutter was developed to address these challenges and to provide a generic tool for generating and analyzing co-occurrence networks. Although NetCutter has been developed for the analysis of gene expression signatures, it is based on abstract concepts that make it applicable to a wide variety of problems. The input is represented by a bipartite graph that is composed of list-entry pairs, which are stored in tab-separated text format. Co-occurrence of entries in lists is analyzed using pair-wise or higher order combinations of entries. The significance of co-occurrence is tested using a novel bi-binomial approximation of Poisson-binomial statistics (which is a binomial distribution with trial specific probabilities) that handles list-length-heterogeneity properly and provides a novel measure of association that is found to be superior to the Jaccard and the uncertainty coefficients. Occurrence probabilities are obtained from an edge-swapping procedure that maintains vertex degrees in the underlying bipartite graph and distinct sets of entries per list. As we shall see below, this procedure has a number of advantages over other possible null-models and permits co-occurrence analysis with near maximum stringency. Last but not least, NetCutter is equipped with a number of algorithms for the identification of network communities, vertex ranking, and convenience tools needed in the analysis of co-occurrence networks, or any undirected graph. We illustrate the utility of NetCutter in the identification of corresponding clusters of genes and publications from the PubLiME data set. PubLiME (Published Lists of Microarray Experiments) is a repository of published cancer related gene expression signatures (http://bio.ifom-ieo-campus.it/Publime). The concept of cluster correspondence follows from the bipartite graph representation of the data. Reversing the list-entry order in the bipartite graph permits identifying communities of entries as well as communities of lists. We show that communities of publications corresponding to communities of genes in the PubLiME data set can be used to generate hypotheses about the putative function of gene communities.\n\nResults\nThe bipartite graph model of co-occurrence analysis\nCo-occurrence analysis using NetCutter is based on the abstraction of list-entry pairs. Any entity that co-occurs with some other entity must be confined to some sort of container where co-occurrence is observed. For example, in the case of gene name co-occurrence in PubMed abstracts, the abstract is the container and the gene names are the co-occurring entities. Similarly, co-occurrence of transcription factor binding motifs is observed in gene promoters. The promoters are the containers where motif entities co-occur. The containers generally host more than one entity (otherwise co-occurrence would be impossible) and can be conveniently interpreted as lists. The co-occurring entities are the list entries. Lists and entries form a bipartite graph with one part of the graph representing lists and the other part representing entries. The presence of a given entry in a given list is indicated by an edge between the corresponding list and entry vertices. It is required that each entry can be linked to the same list only once. Without loss of generality, let's consider genes as entries and PubMedID_listIDs as lists in the following, unless otherwise specified (Fig. 1A). This interpretation of lists and entries has been applied in the co-occurrence analysis of published gene expression signatures [35].\n\nOccurrence probabilities and null-models\nA prerequisite for co-occurrence analysis is the availability of occurrence probabilities of genes per list. The occurrence probabilities can be derived from randomizing the bipartite graph and are dependent on the choice of the null-model. A null-model creates an occurrence probability matrix where the occurrence probability for each list–gene pair is listed. As a general property of this matrix, the sum of all matrix elements must equal the number of edges in the bipartite graph. This is because each edge is linked to either side of the bipartite graph with certainty and therefore the sum of occurrence probabilities over all lists (which can be calculated as the row sum if genes are listed vertically or as the column sum if genes are listed horizontally) followed by summing the results over all genes must be 1 for every edge. The number of matrix elements is given by #genes*#lists and therefore the average occurrence probability for any null-model must be #edges/(#genes*#lists). As a consequence, different null-models will only be distinguished by the way they attribute occurrence probabilities to vertices with different vertex degrees but not by the average occurrence probability.\nWe consider six different strategies to randomize the bipartite graph. First, we could reconnect all edges of the graph randomly. The probability of being connected by an edge for a given list-gene pair is given by (1/#genes)*(1/#lists). Since there are #edges edges to be reconnected, the occurrence probability for a single list-gene pair is #edges/(#genes*#lists), i.e. equal to the average occurrence probability. Thus, this model provides equal occurrence probabilities for all gene-list pairs and does not consider vertex degrees. We call this model the generic randomization (GR) model in the following.\nSecond, we could disconnect the edges on only the list side of the bipartite graph and reconnect them randomly. The occurrence probability of a gene vertex would be given by (gene vertex degree)/#lists. The sum of these probabilities over all lists is equal to the gene vertex degree and the sum of all gene vertex degrees is equal to the total number of edges. Thus, the sum of all matrix elements is equal to the number of edges, as required. Since this model considers gene vertex degrees, we call it the gene vertex degree (GVD) model.\nThird, we disconnect the edges on the gene side of the bipartite graph and reconnect them randomly. The probability of a list vertex being connected to a gene would be given by (list vertex degree)/#genes. The sum of these probabilities over all genes is equal to the list vertex degree and the sum of all list vertex degrees is equal to the total number of edges. Again, the sum of all matrix elements is equal to the total number of edges. Since this model considers list vertex degrees, we call it the list vertex degree (LVD) model.\nIn model four and five, we reconnect edges considering both gene and list vertex degrees and allow multiple edges between list-gene pairs. The occurrence probabilities in model four are calculated according to the binomial distribution. We calculate the probability of a list-gene pair for being connected as the cumulative binomial probability of the list-gene pair being chosen at least once in the process of randomly reconnecting the edges. This can be achieved by setting the number of trials equal to the gene vertex degree, the probability of success equal to the list vertex degree divided by the total number of edges, and the number of successes equal to 0. The occurrence probability of a list-gene pair is then given by the complement of this probability. This model is called the binomial (BN) model. In model five, we calculate occurrence probabilities according to the hypergeometric distribution. The number of successes in the sample is equal to 0, the sample size is equal to the gene vertex degree, the number of successes in the population is set to the list vertex degree, and the population size is the total number of edges. Again, the occurrence probability of a list-gene pair is obtained as the complement of this probability. We call this model the hypergeometric (HG) model. Calculating occurrence probabilities in this manner does not guarantee that the matrix elements add up to the total number of edges. Therefore, the matrices are normalized such that this condition is satisfied by multiplying each matrix element with the factor #edges/(observed matrix sum), which is generally quite close to 1, however.\nIn model six, we again consider vertex degrees, but we require that each list is composed of distinct sets of genes. Thus, multiple edges are forbidden. This condition is satisfied by applying an edge-swapping procedure during graph randomization. Edge-swapping works by randomly choosing two list-gene pairs from the bipartite graph and prior to performing the edge-swap, a test is performed to ensure that the two genes are not already linked to the respective target lists. This procedure is performed a large number of times. To ensure complete randomization of the graph, the number of swaps performed should be significantly larger than the number of edges. After performing R randomizations of the graph and counting the number of times a gene has been linked to a particular list, division of this number by R gives the occurrence probability of a gene in a given list. As will be shown below, edge-swapping produces occurrence probabilities that closely approximate occurrence probabilities obtained by generating a complete permutation set of the bipartite graph, counting the number of times a gene is found part of a list, and dividing this number by the total number of permutations. In the permutation model, the sum of occurrence probabilities of a gene over all lists equals the gene vertex degree (see below) and thus the sum of all matrix elements is the number of edges. Since permutation sets of bipartite graphs are difficult to calculate, we use the edge-swapping procedure as a close approximation and call this model the edge-swapping (ES) model.\nFig. 1 shows the occurrence probabilities of the different null-models for the bipartite graph shown in Fig. 1A. The GR model yields identical occurrence probabilities for all list-gene pairs, which is equal to the average occurrence probability in all models. In the other models, the occurrence probabilities deviate to varying extent from the average occurrence probability as a function of vertex degrees. In the GVD model, the deviations are a function of gene vertex degree and in the LVD model the deviations are dependent on list vertex degrees. In the remaining models, the deviations are functions of both the gene and the list vertex degrees. In all cases, larger than average occurrence probabilities are obtained for larger vertex degrees at the expense of smaller than average occurrence probabilities for smaller vertex degrees. From these data, it is difficult to choose the most effective null-model. A hint can be gleaned from gene1, however. Gene1 is present in all lists. Therefore, the co-occurrence probability of gene1 with other genes, which is calculated by multiplying the occurrence probabilities of gene1 and geneX for every list under study, should depend only on the occurrence probability of this other gene. In other words, the occurrence probability of gene1 in all lists should be 1.0. Only two models satisfy this constraint: The GVD and the ES models. Since the GVD model does not consider list vertex degrees, it seems that the ES model is the preferred null-model.\n\nExpected number of co-occurrences\nAs a general criterion for comparing the effectiveness of different null-models, we have to compare them for the number of expected co-occurrences. The most effective null-model will be the one that maximizes the expected number of co-occurrences. If the expected number of co-occurrences is larger, an observed number of co-occurrences in a real bipartite graph will be less significant and thus such a null-model permits co-occurrence analysis with higher stringency. The expected number of co-occurrences depends in an obvious fashion on the list vertex degree. If pair-wise co-occurrences are considered, the number of co-occurrences in a list of vertex degree N is given by the binomial coefficient N over 2. Larger lists will give rise to more co-occurrences and the number increases quickly with list vertex degree. The dependency of the expected number of co-occurrences on the gene vertex degree is less obvious and depends strongly on the null-model. A gene that is part of a list with vertex degree N will give rise to N-1 co-occurrences in that list. The null-model permits calculating the probability to find this gene in a given list. Thus, the expected number of co-occurrences of a gene is given by the sum of expected co-occurrences in all lists where for a single list the expected co-occurrences are given by (Nl−1)*pl. Nl is the list vertex degree and pl is the occurrence probability of the gene in that list as determined by the null-model.\nWe used the PubLiME data set [35] to calculate the expected number of co-occurrences with different null-models. The results are shown in Fig 2A. The expected number of co-occurrences was calculated for all genes in all lists using all null-models and the sum of expected co-occurrences per gene is shown as a scatter plot with the gene vertex degree on the x-axis and the expected number of co-occurrences on the y-axis. The results in Fig. 2A suggest the following ranking of null-models: GR<GVD<LVD<BN = HG<ES. The BN and the HG models perform in an essentially identical way. However, the ES model is the model that yields the largest estimates of expected co-occurrences. The results are also in line with the intuitive expectation that genes with higher vertex degree give rise to more co-occurrences. However, it can be seen that this is not true for all null-models. In particular, it is not true for the GR and the LVD models, which do not consider gene vertex degrees.\nAs outlined above, it is expected that the null-model that yields the highest estimates of expected co-occurrences should permit co-occurrence analysis with the highest stringency. In Fig. 2B, this hypothesis is tested directly again using the PubLiME data set [35]. For all null-models, co-occurrence analysis was carried out using module size 3 and support 5 (co-occurrence modules must be present in at least five publications). The choice of these parameters has been discussed in [35]. The number of co-occurrence modules was then determined that have a Z-score higher or equal than the cut-off shown in Fig. 2B. The Z-score is calculated from the mean and variance of the Poisson-binomial distribution as shown in the Materials and Methods section and published in [35]. More details on the probability distribution will be provided below. The GR and GVD models perform very poorly and identify large numbers of modules with high Z-scores. The LVD model performs a little better and approximates the BN and HG models at higher Z-score cut-offs. The BN and HG models give essentially identical results. However, the ES model is the model that yields the fewest number of significant co-occurrence modules and is thus the most stringent. The increased stringency of the ES model over the BN and HG models is also reflected in a higher signal-to-noise ratio calculated as the number of significant co-occurrence modules in the real bipartite graph divided by the number of modules found in a randomized bipartite graph (Fig. 2C).\nThe reason for the superior stringency of the ES model over all other models can be explained by examining the average occurrence probability per gene and list vertex degree. Fig. 2D and E show the average occurrence probability of genes with the same gene vertex degree as a function of the gene vertex degree. It can be seen that the ES model yields higher occurrence probability estimates for genes with higher vertex degrees as compared to the BN and HG models. In GR and LVD models, gene vertex degrees are ignored and occurrence probabilities for genes with large vertex degree are very small, which is compensated by larger occurrence probabilities for genes with small vertex degree. The GVD model is identical to the ES model in this setting. Fig. 2E shows the average occurrence probability of all lists with the same vertex degree as a function of list vertex degree. It can be seen that the ES model provides higher occurrence probability estimates for large lists as compared to the BN and HG models. In this setting, the LVD model performs like the ES model while the GR and GVD models yield small occurrence probabilities for large lists. Since it has been shown above that long lists and genes with high vertex degree are responsible for a large part of the total number of co-occurrences for the most stringent null-models, the null-model that provides larger occurrence probability estimates for genes and lists with high vertex degree at the expense of lower estimates for smaller degrees will be the most stringent because large occurrence probabilities make co-occurrence more likely and thus less significant. By these criteria, the ES model is the most stringent of all models tested.\n\nThe ES model as an approximation of the permutation null-model\nThe data shown above have revealed that the ES model is the best of the models tested. One may wonder, however, whether yet more effective null-models can be found. An obvious choice would be the permutation model. In the permutation model, a complete permutation set of the bipartite graph is created such that each list is composed of distinct sets of genes. The number of graphs where a gene is present in a given list divided by the total number of permutations then provides the occurrence probability estimate. The permutation model is the ideal null-model because it is exhaustive. The problem is that a complete permutation set of bipartite graphs of some complexity is very time consuming to calculate. For example, the simple bipartite graph from Fig. 1A is part of a permutation set of 455 graphs. The number of permutations is increasing quickly as the numbers of genes and lists grow. However, since edge-swapping ensures that gene lists are composed of distinct sets of genes, each edge-swap produces a graph that is part of the permutation set of the bipartite graph. Edge-swapping can thus be viewed as a random sampling procedure from the permutation set of the bipartite graph. Therefore, occurrence probability estimates derived by edge-swapping should approximate those obtained from the permutation model.\nWe generated a complete permutation set of the graph shown in Fig. 1A to verify this hypothesis. The results are shown in Fig. 3. Fig. 3A shows how the number of possible permutations can be calculated. Gene1 is present in all lists and does not have an impact on the total number of permutations. Gene2, having vertex degree two, is present in two out of three lists in one out of three possible ways. The remaining genes have vertex degree 1 and can be freely chosen to fill the empty slots. We can now count exactly how many times a gene is linked to a list and divide these counts by 455, the size of the permutation set, to obtain exact occurrence probabilities. These numbers are shown in graphical form in Fig. 3B and in numerical form in Fig. 3C. Fig. 3B also shows the occurrence probability estimates obtained by edge-swapping side-by-side to the exact occurrence probabilities. The graph in Fig. 1A was subjected to edge-swapping 1000 times and the number of times a gene was found present in a list was divided by 1000 to obtain the occurrence probability. At each run, 100 random edge swaps were performed to ensure complete randomization of the graph. This procedure was repeated 10 times and the mean and standard deviation of occurrence probability estimates for each gene in each list are shown. In all cases, the mean differs from the real probability by less than two standard deviations, in most cases by less than one standard deviation. Thus, edge-swapping provides reliable estimates of exact occurrence probabilities as determined from a complete permutation set.\nAs an interesting observation, we provide evidence that occurrence probabilities are non-linear functions of vertex degrees in the edge-swapping model. This is illustrated in Fig. 3C. Individual and average occurrence probabilities are shown as a function of gene and list vertex degrees. Non-linearity of individual occurrence probabilities can be verified from the counts table underneath the plots. However, the average occurrence probability is found to depend on vertex degrees in a linear fashion instead. This is a consequence of the fact that occurrence probabilities of a gene over all lists add up to the gene vertex degree and that the occurrence probabilities of all genes for a given list add up to the list vertex degree. At the same time, since the most stringent permutation based null-model predicts non-linear dependencies of individual occurrence probabilities on vertex degrees, assuming such linearity in statistical models of co-occurrence will be linked to loss of stringency.\nWe conclude that the ES null-model is the null-model that permits co-occurrence analysis with the highest stringency among the models tested and that it closely approximates occurrence probabilities derived from an ideal permutation model. The increased stringency of the ES model over other models is a consequence of higher occurrence probabilities for genes and list with high vertex degrees, which are giving rise to a large part of all co-occurrences in the bipartite graph. Since large occurrence probabilities make co-occurrence more likely, the analysis becomes more stringent.\n\nCo-occurrence probabilities\nCo-occurrence analysis can be thought of as a Bernoulli experiment with a binomial outcome (a given combination of entries is either present or not present in a given list). Thus, the Binomial distribution (BD) is a natural choice for judging the significance of the number of co-occurrences. However, the BD is defined for a probability of success which is equal in all trials. The list-specific nature of occurrence probabilities is not compatible with this condition (analysis of each list represents one trial), which means that co-occurrence analysis in the presence of list-length-heterogeneity is better described as a series of Poisson trials, where the probability of success varies from trial to trial. Therefore, the significance of co-occurrences must be evaluated using a binomial distribution with trial-specific probabilities, i.e. the Poisson-binomial distribution (PBD). The probability of success in a single Poisson trial can be calculated by multiplying the list-specific occurrence probabilities for the combination of genes under study. The number of occurrence probabilities that need to be multiplied is equal to the module size, i.e. the number of genes whose combination is studied. An observed number of co-occurrences for a combination of genes can then be evaluated using the PBD, which is given by the formula [36]:(18)\nThe structure of this formula is very similar to the structure of the formula used to calculate the binomial distribution, except that multiplication with a binomial coefficient is replaced by summation over individual terms, which makes calculation of P-values using (18) inefficient (note that equation numbering starts in the Materials and Methods section). Here, Ak denotes the kth set of indices of the i lists where genes are co-occurring (“success”). There are possible sets and summation is carried out accordingly. denotes the set of indices of N−i lists where genes are not co-occurring (“failure”). [36] have reported two fast procedures for calculating exact PBD P-values. However, both procedures work with probability ratios and suffer from numerical overflow/underflow problems for large numbers of trials. NetCutter uses two workarounds to circumvent this problem. One is based on using Poisson-binomial Z-scores, which can be calculated very easily instead (see below). The other relies on a fast approximation procedure for calculating Poisson-binomial P-values, which we call bi-binomial approximation (BBA) or bi-binomial distribution (BBD).\n\nZ-scores and P-values of BBD\nGiven the mean μ (1) and variance σ2 (2) of PBD (see Materials and Methods), the Z-score associated with a given number of co-occurrences x is obtained as:(19)\nConsidering the structure of formulae (1) and (2) (Materials and Methods section), PBD Z-scores can be calculated very easily and provide a simple estimate of the significance of co-occurrence modules. However, in contrast to normally distributed Z-scores, binomial and Poisson-binomial Z-scores do not correspond to the same P-value for different sets of probabilities of success. To see this, calculate for example the probability of success in a series of 100 Bernoulli trials with success probability 0.1 and 0.9 for the expectation of 10 and 90 successes, respectively. The Z-score will be 0 in both cases but the corresponding cumulative P-values are 0.5832 and 0.5487. Therefore, exact levels of significance cannot be derived from Z-scores alone. Thus, a fast and reliable procedure for calculating Poisson-binomial P-values is needed. The BBD approximation was developed to solve this problem.\nThe BBD approximation of PBD P-values follows from the relationship between the variance of PBD and the population variance of trial-specific probabilities of success. This relationship is shown in Materials and Methods to be described by (4):(4)\nThis equation shows that there is an inverse linear relationship between the population variance S2 of the N trial probabilities and the variance of PBD σ2, which means that PBD becomes increasingly narrow as the variance of trial probabilities grows. It also shows that, for constant mean μ and number of trials N, the shape of PBD depends only on the variance of trial probabilities. Therefore, relationship (4) suggests an easy way to approximate PBD P-values. The P-value can be obtained by constructing a set of trial probabilities with equal variance as the original set of trial probabilities, which, however, are not all different. In other words, the series of Poisson trials can be replaced by two sets of Bernoulli trials with trial probabilities p1 and p2 constructed such that the variance is equal to the original set of trial probabilities. This strategy is illustrated in Fig. 4 and explains why this approximation is called bi-binomial. The details on how to obtain the values of the two sets of Bernoulli trial probabilities and the number of trials with p1 and p2 as probabilities of success are provided in the Materials and Methods section. The precision of the BBD approximation is discussed in supplementary material Simulation S1.\nIn order to evaluate whether BBD P-values as a significance measure of co-occurrence offer an advantage over other measures such as the Jaccard coefficient or the uncertainty coefficient, pair-wise co-occurrence of two genes in 200 lists with and without list-length-heterogeneity was studied (Fig. 5). Each gene is assumed to occur in 100 lists. Therefore, the occurrence probabilities of both genes over all 200 lists must add up to 100, regardless of list-length-heterogeneity. For simplicity, occurrence probabilities of both genes are assumed to be equal in any particular list. The co-occurrence probability in a list is then given by the square of the occurrence probability in that list. For all possible co-occurrences from 0 to 100, the Jaccard and uncertainty coefficients were calculated as detailed in the Materials and Methods section. In addition, cumulative BBD P-values were calculated using the co-occurrence probabilities as trial probabilities. To illustrate the advantage of BBD over BD as co-occurrence probability distribution, cumulative BD P-values of a BD with the same mean as BBD but constant trial probabilities is shown. These trial probabilities can be obtained by dividing the mean of BBD by the number of lists.\nThree different cases of list-length-heterogeneity are considered in Fig. 5: No heterogeneity (standard deviation 0), heterogeneity with standard deviation 0.283 and heterogeneity with standard deviation 0.401 in the occurrence probabilities. The Jaccard and uncertainty coefficients are by definition insensitive to list-length-heterogeneity because differences in co-occurrence probabilities in a given list cannot be considered in their calculation. This is because both coefficients are defined by the counts of the four list classes: both genes absent, both genes present, first gene absent second gene present, and first gene present second gene absent, i.e. by the corresponding contingency table, which does not change with different list-length-heterogeneity. In the absence of list-length-heterogeneity, the cumulative P-values of BD and BBD (which are perfectly overlapping as expected) assume 0.5 at 50 co-occurrences, which corresponds to the expected number of co-occurrences calculated as (50 = 100 occurrences per gene/200 lists) ˆ2*200 lists. The uncertainty coefficient is found to be 0 and the Jaccard coefficient is 0.33333 at that point. When there is modest list-length-heterogeneity (standard deviation 0.283), the mean of BBD is shifting to the right. This is because the sum of squares of varying occurrence probabilities (i.e. the sum of co-occurrence probabilities used as trial probabilities, which is equal to the mean of BBD) is always larger than the sum of squares of constant occurrence probabilities with the same average occurrence probability (0.5). The corresponding BD in the presence of list-length-heterogeneity is obtained by dividing the expected number of co-occurrences by the total number of lists, which means assuming equal co-occurrences in all lists. This visualization is shown to illustrate how BBD (which is narrower than the corresponding BD) gives rise to a steeper cumulative distribution of P-values and as a consequence to more significant P-values for numbers of co-occurrence that are far from the expectation. As the level of list-length-heterogeneity grows (standard deviation of occurrence probabilities 0.401), the mean of BBD is shifted even further to the right and BBD P-values are distributed in a still steeper fashion as compared to corresponding BD P-values and the interval of non-significant co-occurrences is shrinking further. With modest list-length-heterogeneity, the expected number of co-occurrences is 66, which is associated with a Jaccard coefficient of 0.49 and an uncertainty coefficient of 0.075. In the case of large list-length-heterogeneity, the expected number of co-occurrences is 82 with J = 0.69 and UC = 0.32.\nTaken together, these data show that the expected number of co-occurrences varies strongly with the level of list-length-heterogeneity and that the expected number of co-occurrences is associated with different values of UC and J. To complicate matters further, 66 co-occurrences (J = 0.49, UC = 0.075) represent significant positive association (PBBD = 0.996) with equal list lengths, no significant association with modest differences in list length (PBBD = 0.536) and strongly negative association (meaning one gene excludes the other) with strong list-length-heterogeneity (PBBD = 0.00016). Thus, the same J and UC association measure is obtained for positive, negative, and absence of association. Therefore, the meaning of these measures cannot be interpreted properly in the absence of knowledge about the occurrence probabilities of the co-occurring entities. Furthermore, the data in Fig. 5 also show that neither J nor UC can distinguish between positive and negative association while this is easy with cumulative BBD P-values: Large P-values mean positive association and low P-values mean negative association. In summary, we conclude that BBD provides a novel association measure that offers a number of advantages over the existing contingency table based association measures Jaccard coefficient and uncertainty coefficient. The results in Fig. 5 also show that significance of association depends critically on the specific distribution of co-occurring entities over lists of varying length (because this distribution determines the occurrence probabilities) and that contingency table based methods (which cannot capture this distribution) should be avoided in the presence of significant list-length-heterogeneity.\n\nGeneration of co-occurrence networks and the identification of communities\nThe procedures outlined above allow the identification of significant co-occurrence modules in any type of bipartite graph. Three user defined parameters have an impact on the stringency of co-occurrence analysis: The module size, the support, and the Z-score/P-value cutoff. The module size determines how many entries will be tested for co-occurrence, the support sets a lower boundary on the required number of co-occurrences, and the Z-score/P-value cutoff sets the significance threshold. In general, higher module size leads to more stringent co-occurrence analysis at the cost of computational complexity. The support parameter allows limiting this complexity by filtering out co-occurrence modules which co-occur less frequently than required by the support. The significance cutoff permits adjusting the signal-to-noise ratio, which is calculated as the number of co-occurrence modules observed in the real versus a randomized bipartite graph. The impact of these parameters on the stringency of co-occurrence analysis has been reported previously for the PubLiME data set [35] and is illustrated in a simulation study provided as supplementary material Simulation S1. From the set of significant co-occurrence modules, a co-occurrence network is generated by considering each entry a vertex and drawing an edge between any two vertices, which have been part of the same significant co-occurrence module [35].\nAn important question in the analysis of co-occurrence networks regards the presence of network communities. Communities can be understood as groups of vertices with the property that the number of edges running within groups is larger than expected by chance and that the number of edges running between groups is lower than expected by chance [37]. This problem of partitioning a graph is often referred to as the graph-cut problem (hence the name NetCutter). NetCutter is built on the Java Universal Network and Graph framework (JUNG) software package (http://jung.sourceforge.net), which provides algorithms for solving this problem. In particular, NetCutter implements the Bicomponent clustering algorithm [38], the Edge-Betweenness clustering algorithm [39], and the Exact Flow Community algorithm [40]. Furthermore, there is a clustering tool that is not part of the JUNG package, namely an algorithm identifying communities using eigenvectors of the modularity matrix [37]. The code for this algorithm was kindly provided by Mark Newman in C++ and ported to Java. In addition to these tools, NetCutter provides a number of convenience functions for the analysis of co-occurrence networks, such as testing the significance of lists reporting a set of entries making up a network community, ranking of vertices, random graph generators for topological analysis of co-occurrence networks, and others. Details on all functions are provided in the NetCutter software documentation.\nOne of the possible applications of NetCutter is illustrated below. This application is tightly linked to the bipartite graph representation of the data. Namely, NetCutter can be used to perform co-occurrence analysis of genes or list derived from the same bipartite graph. The network communities identified in each both reflect the same underlying structure of the bipartite graph. In the case of gene expression signatures stored in PubLiME, clusters of genes correspond to clusters of publications, which can reveal possible functions of gene clusters.\n\nCluster correspondence and association studies\nThe co-occurrence analysis of the PubLiME data set published previously [35] identified 5 major network communities of genes with consistent functional annotations that are deregulated in cancer related gene expression signatures. This analysis was performed by considering all genes mentioned in a particular publication as a single signature, even though they might have been part of different tables and cluster analyses. Here we present an advanced analysis of the PubLiME data set where each table and/or cluster identified in a given publication is considered as a separate signature. This brings the total number of signatures to be analyzed to 1015 comprising a total of 7358 differentially regulated genes derived from 233 publications reporting cancer related signatures derived from human samples. We use this analysis to illustrate three major points: First, the set of communities reported previously is reproduced by this more fine-grained analysis. Second, the set of gene communities corresponds to a set of publication communities. Third, associations between publications and gene communities can be calculated with higher stringency using the edge-swapping null-model in conjunction with bi-binomial P-values as compared to binomial or hypergeometric statistics.\nThe bipartite graph to be analyzed is composed of PubMedID_listID-gene pairs (see supplementary material Table S1). Co-occurrence analysis was carried out in two ways: First, gene co-occurrence was analyzed and communities of co-occurring genes were defined by edge-betweenness clustering as described in Materials and Methods. Second, co-occurrence of PubMedID_listIDs was analyzed. To this end, the order of PubMedID_listID-gene pairs was reversed to form GENE-PUBMEDID_LISTID pairs. Thus, the lists in the resulting bipartite graph are formed by genes and the entries are the PubMedID_listIDs where the genes are reported as differentially regulated. Occurrence probabilities for the reversed bipartite graph can be obtained by transposing the occurrence probability matrix of the original bipartite graph. Since the gene communities identified in gene co-occurrence analysis reflect the structure inherent in the bipartite graph (which is not affected by reversing the list-entry order), co-occurrence analysis of the reversed bipartite graph will result in PubMedID_listID communities that reflect the same underlying structure in the bipartite graph. In other words, PubMedID_listID communities correspond to gene communities. In less abstract terms, the PubMedID_listID communities should correspond to sets of publications that report similar sets of genes as differentially regulated. The identification of communities of publications can help the researcher to easily identify publications studying genes in a gene community that is of interest to the researcher.\nThe results of both types of co-occurrence analysis are displayed in Fig. 6. Fig. 6A shows the gene clusters identified. The clusters are named after significant enrichments of gene categories as determined by functional category enrichment using DAVID [41]. The P-values shown are Benjamini corrected for multiple testing as reported by DAVID. The clusters are very similar to the clusters published previously [35]. There is one new cluster that is strongly enriched for ribosomal proteins (“protein biosynthesis” cluster), which has not reached significance in our previous analysis. The “surface antigen” cluster contains many genes that had been reported as part of the “signal transduction” cluster. Altogether, however, these results strongly support the notion that the gene clusters in the PubLiME data set can be reproduced by the more fine-grained analysis that considers sublists in each publication as separate signatures.\nThe corresponding clusters of PubMedID_listIDs are shown in Fig. 6B. There are five clusters, which have been named after their corresponding gene cluster. Only one cluster (the “extracellular matrix-immune response cluster”) cannot be separated by edge-betweenness clustering at the point of maximal graph modularity. To see that this naming is indeed justified, we needed to investigate how strongly a given PubMedID_listID is associated with a given gene cluster, i.e. how significant is the overlap of the genes reported in a gene cluster and the genes reported in a PubMedID_listID. Binomial or hypergemetric statistics are generally used to calculate this significance. However, the bipartite graph model in conjunction with the edge-swapping null-model offers a more fine-grained approach based on bi-binomial statistics.\nThe edge-swapping null-model determines occurrence probabilities in such a way that the number of genes in a given PubMedID_listID is associated with insignificant P-values in the context of the complete bipartite graph. However, when a subset of genes is analyzed, e.g. all the genes that are reported in a particular list, the P-value associated with the number of genes contained in this list will likely be highly significant according to how unlikely it is to obtain all the genes contained in a given list in a random draw from all genes present in the bipartite graph. Thus, PubMedID_listID association with a set of genes in the bipartite graph model can be calculated in the following way: The set of genes that is used to analyze association is used to extract a subgraph from the original bipartite graph where occurrence probabilities for each gene-PubMedID_listID pair are identical to those in the original bipartite graph (i.e. they are not recalculated by edge-swapping). The vertex degree of the PubMedID_listID vertices in the subgraph indicates the number of genes contained in each PubMedID_listID overlapping with the set of genes used to extract the subgraph. From the occurrence probabilities of the genes in a given PubMedID_listID, the bi-binomial P-value can then be calculated for every list vertex degree observed in the subgraph. In Fig. 7, the significance of association of the PubMedID_listIDs (see Fig. 6B) with the cell cycle cluster of genes (Fig. 6A) is calculated. For comparison, binomial and hypergeometric P-values are also shown. It can be seen that the bi-binomial P-value is larger than the binomial and hypergeometric P-values, which means that the strength of association is evaluated in a more stringent manner using BBD statistics (see Discussion for an explanation of this observation).\nThe analysis of significant associations between PubMedID_listIDs and gene clusters now permit answering the question whether there is correspondence between gene clusters and PubMedID_listID clusters. The naming of PubMedID_listID clusters shown in Fig. 6B is based on the number of PubMedID_listID that are significantly associated with gene clusters shown in Fig. 6A. First, for each gene cluster, all the PubMedID_listIDs that are associated with that cluster with more than 95% confidence (i.e. cumulative bi-binomial P-values> = 0.95) were identified. Second, the number of significant PubMedID_listIDs in each PubMedID_listID cluster was counted for every gene cluster. The significance of this number was then calculated using binomial statistics. The results of this analysis are shown in Table 1. Negative decadic logarithms of the binomial P-value are displayed. It is apparent that each PubMedID_listID cluster is strongly associated with at least one gene cluster, except for the “extracellular matrix-immune response” cluster, which is associated with two gene clusters. The strength of these associations suggests that the PubMedID_listID clusters are indeed corresponding to the gene clusters and that both the gene and the PubMedID_listID clusters reflect the structure of the bipartite graph representing the PubLiME data set.\nDetails about all the lists analyzed are attached as supplementary material Table S2. Looking at these lists, some general conclusions about the gene clusters can be drawn. Cell cycle cluster genes have been found deregulated in a wide variety of tumor types such as colon cancer, breast cancer, in biliary tract cancer, pancreatic cancer, gastric cancer, prostate cancer, T-cell leukemia, glioma, acute lymphoblastic and myeloblastic leukemias, soft tissue sarcoma, neuroblastoma, as well as in a number of cellular model systems in response to different stimuli. Thus, the cell cycle cluster seems to consist of genes with a general role in oncogenesis. The surface antigen cluster instead seems to be derived preferentially from studies on leukemia. The interferon cluster genes are found deregulated in virus induced pathologies such as papilloma virus induced cervical cancer, and viral hepatitis. Immune response cluster genes were reported as differentially regulated in inflammatory conditions such as ulcerative colitis, Crohn's disease, and Helicobacter pylori infections. Genes of the extracellular matrix cluster seem to be associated with cancer progression studies and metastatic potential. For the protein biosynthesis cluster, there are 15 signatures that are significantly enriched for those genes. The cancers studied comprise medulloblastoma, glioblastoma, pancreatic cancer, soft tissue sarcoma, lung carcinoma, breast carcinoma, prostate carcinoma, multiple myeloma, and lymphocytic leukemia. The genes are also found deregulated in response to DNA damage. Although the number of signatures is limited, the variation in conditions where the genes are deregulated is compatible with the hypothesis that protein biosynthesis genes, as cell cycle genes, are deregulated in many cancer types, which might reflect the general property of cancer cells to divide and grow in an uncontrolled fashion.\n\n\nDiscussion\nHere we have investigated basic aspects of co-occurrence analysis and present a software tool, NetCutter, which can be used to identify and analyze generic co-occurrence networks. In NetCutter, a co-occurrence data set is represented as a bipartite graph with one part representing lists and the other part list entries whose co-occurrence patterns are studied. The bipartite graph representation of co-occurrence data sets allows the efficacy of different null-models to be tested systematically. We have shown that an edge-swapping procedure used to randomize the bipartite graph generates a null-model that allows co-occurrence analysis with the highest stringency. The other null-models tested here tend to underestimate occurrence probabilities of entries per list for lists and genes with high vertex degrees, i.e. for lists and genes where most co-occurrences are observed. As a result, co-occurrences are judged more significant than they really are.\nCo-occurrence data sets with exactly equal lists lengths are likely to be the exception from the rule. It can be assumed that some list-length-heterogeneity will be present in most circumstances. An important consequence of list-length-heterogeneity regards the co-occurrence probability distribution used to evaluate the significance of the observed number of co-occurrences. Co-occurrence analysis in the presence of list-length-heterogeneity is best performed using the Poisson-binomial distribution (a binomial distribution with trial specific probabilities). However, calculating Poisson-binomial P-values for large numbers of lists is difficult using existing procedures [36]. We have presented an approximation to the Poisson-binomial distribution, called bi-binomial distribution, which is based on replacing the set of Poisson trials by two sets of Bernoulli trials. The resulting distribution reproduces the Poisson-binomial distribution nearly exactly and its P-values can be calculated with ease even for thousands of lists (see also supplementary material Simulation S1 for details on the precision of BBD). Importantly, BBD provides a novel measure of association, which is shown to be superior to existing measures such as the Jaccard coefficient and the uncertainty coefficient, whose values cannot be interpreted properly in the absence of knowledge about the occurrence probabilities of co-occurring entities.\nIt is worth noting that Poisson-binomial Z-scores are distinguished from Gaussian Z-scores by the fact that they do not correspond to the same P-value for different PBDs, BBDs, and even BDs. This is because the Z-score is an explicit part of the function defining the normal probability density while it is not part of the definitions of BD, PBD, and BBD densities. As a consequence, the simple Poisson-binomial Z-score based approach to evaluating significance of co-occurrence must be complemented with the BBD to approximate Poisson-binomial P-values in order to enable multiple testing corrections and to allow calculation of confidence levels in association studies precisely. However, NetCutter is equipped with a bipartite graph randomization tool that permits measuring the number of false positives due to multiple testing directly by comparing the number of significant co-occurrence modules in the real bipartite graph to the corresponding number in a randomized version thereof. Randomization is performed by edge-swapping in order to preserve vertex degrees. The resulting signal-to-noise ratios that are plotted for each Z-score/P-value cutoff provide a highly reliable and visually intuitive defense mechanism against false positives (see also supplementary material Simulation S1).\nIn the vast majority of co-occurrence studies, pair-wise co-occurrences have been analyzed using different statistical models. We have observed that the stringency of pair-wise co-occurrence analysis is far below the stringency of co-occurrence analysis using higher order combinations of co-occurring entities [35]. In NetCutter, co-occurrence analysis is preceded by occurrence analysis, i.e. the occurrence probability of each entry in each list is determined. Starting from occurrence probabilities, co-occurrence probabilities for any size of co-occurrence modules under study can be obtained by multiplying the respective list-specific occurrence probabilities. Given the list-specific co-occurrence probabilities, bi-binomial P-values are then calculated in exactly the same way for any module size. As a consequence, NetCutter can perform co-occurrence analysis for higher order combinations of co-occurring entries (i.e. larger module sizes) using the same statistical model. One of the benefits of using higher module sizes is robustness of the analyses in the presence of noise. This is because each edge in the resulting co-occurrence network is evaluated many times since every pair of co-occurring entries can be part of many higher order co-occurrence modules [35]. Another advantage is that implicit relationships between entries, which have never occurred together [18], can be derived as a natural by-product of using module sizes larger than 2. As shown in a simulation study (supplementary material Simulation S1), the result is a dramatic reduction of misclassifications at higher module sizes.\nNetCutter can be used to calculate the strength of association between a subset of entries and lists reporting those entries. In this case, the analysis is performed on a subgraph of the original bipartite graph. The subgraph can correspond to communities of entries in the co-occurrence network, or any set of entries of interest. NetCutter will then calculate the significance of observing a given number of occurrences of an entry in the user defined subset of lists using bi-binomal statistics. This analysis mode corresponds to association studies with the advantage that the structure of the underlying bipartite graph (i.e. list length heterogeneity) is considered and handled appropriately using the bi-binomial distribution. As a consequence, association studies can be performed with higher stringency.\nThis result can be understood by examining the occurrence probability matrix that is implicitly assumed in performing binomial or hypergeometric tests for the significance of overlaps. In both tests, a gene is assumed to have an equal opportunity to be present in a list. Therefore, the probability of success for a gene to be part of a list is given by the list vertex degree divided by the total number of genes. In other words, both tests are implicitly based on the list vertex degree model, which has been shown previously to underestimate the occurrence probability and the expected number of co-occurrences for genes with high vertex degree (see Fig. 2A). Since the BBD P-values are calculated from the ES-model, which assigns higher occurrence probabilities to genes with higher vertex degree and more expected co-occurrences, the observed number of overlaps between a set of genes of interest and the content of a given list (which can be viewed as co-occurrence of the overlapping genes in that list) will be judged less significant when the overlapping genes are of high vertex degree (and vice versa when the overlapping genes are of low vertex degree) as compared to binomial or hypergeometric tests. Since the BBD P-values are derived from the most stringent ES null-model, BBD P-values provide a more reliable estimate for the significance of overlap.\nCo-occurrence analysis of data represented as bipartite graphs permits visualizing the structure of the bipartite graph either as communities of list entries (genes) or as communities of lists (PubMedID_ListID) in co-occurrence networks. We have analyzed the PubLiME data set for the presence of corresponding gene and list clusters. In addition to previously published clusters of genes, we describe a novel gene cluster that is composed of protein biosynthesis associated genes [35]. We found that the corresponding clusters of PubMedID_ListID (gene expression signatures) are in general strongly enriched for genes reported in the corresponding gene cluster and that interrogation of corresponding clusters can be used to deduct hypotheses about the putative function of gene clusters.\nIn addition to co-occurrence analysis, NetCutter offers a number of tools for the analysis of co-occurrence networks, or any undirected graph. In particular, community identification is supported by four different community identification algorithms. NetCutter also offers a range of convenience functions that are of help in network analysis. Worthy of mentioning are the random graph generators that can provide control graphs for topological studies. The complete set of options is described in the software documentation.\nIn summary, we present a general framework for co-occurrence analysis with many potential applications. We illustrate a number of advantages of using the bipartite graph representation of data and the associated statistics. In particular, the identification of corresponding clusters permits the identification of functional subunits such as gene clusters on the one hand, and the generation of hypotheses about the function of those units by analyzing the corresponding list clusters on the other hand. Future developments will be directed towards the analysis of data sets that are considerably larger than the data sets analyzed so far. For example, co-occurrence analysis might be of interest for the analysis of single nucleotide polymorphism (SNP) data sets and association studies of genome variability with disease. Each patient is characterized by a specific range of SNPs. Co-occurrence patterns of patients according to their SNPs could be compared to clinical parameters with the aim of identifying genomic regions associated with disease. The increased stringency of association studies offered by NetCutter may be of use in the analysis of polygenic diseases where conventional methods fail. For being useful in this setting, NetCutter must be capable of analyzing bipartite graphs with millions instead of thousands of vertices.\n\nMaterials and Methods\nImplementation of NetCutter\nNetCutter is written in Java using NetBeans6 software (http://www.netbeans.info/) and tested on the Java Runtime environment 1.6.0.0. on a Windows XP Professional computer. The Java Runtime environment, which can be downloaded from http://java.sun.com/, must be installed on a computer that is intended to run NetCutter. NetCutter is provided as a single jar file and should run by double clicking the jar file, provided that the Java runtime environment is properly installed. NetCutter makes use of the following software packages and classes: JUNG version 1.3 (http://jung.sourceforge.net/download.html), Apache Jakarta Commons Collections 3.1 (http://jakarta.apache.org/commons/collections/), Cern Colt Scientific Library 1.2.0 (http://dsd.lbl.gov/hoschek/colt/), Xerces (http://xerces.apache.org/xerces2-j/index.html), Jama (http://math.nist.gov/javanumerics/jama/), Netlib Java LAPACK (http://www.netlib.org/lapack/), JFreeChart (http://www.jfree.org/jfreechart/), partition.java (http://astro.u-strasbg.fr/fmurtagh/mda-sw/java/partition.java).\n\nBi-binomial approximation of Poisson-binomial distribution\nThe Poisson-binomial distribution (binomial distribution with trial specific probabilities) has recently been proposed as a statistic that properly handles largely differing sizes of gene expression signatures in meta-analysis of gene expression data [35]. Z-scores have been used to estimate the significance of co-occurrence because P-value calculation is cumbersome and error prone. Two methods reported by [36] suffer from numerical overflow/underflow problems when large numbers of Poisson trials with probabilities deviating significantly from 0.5 are being analyzed. Therefore, we propose a fast approximation of P-values based on a bi-binomial distribution. The bi-binomial distribution is a special case of the Poisson-binomial distribution where the probability of success can assume only two values. In order to achieve a good approximation of the underlying Poisson-binomial distribution, the values of these two probabilities and the number of trials where they are assumed must be determined carefully. As is shown in the following, the values of the two trial probabilities and their number of occurrences follow from the formula used to calculate the variance of the Poisson-binomial distribution and from the formula yielding the population variance of trial probabilities of the Poisson-binomial distribution to be approximated.\nThe mean μ and the variance σ2 of the Poisson-binomial distribution are given by equation (1) and (2), respectively.(1)(2)pi is the trial-specific probability of success and N is the total number of trials. For the sake of completeness, a formal proof of equation (1) is reported as supplementary material Proof S1 and the proof of equation (2) can be obtained in an analogous fashion.\nThe population variance S2 of trial probabilities pi is given by equation (3).(3)\nRearranging equation (3) considering (1) and (2) leads to (4) and (5), where pa denotes the average trial probability of success and qa its complement.(4)(5)Now let's define two trial probabilities p1 and p2, which are used N1 and N2 times during the Poisson trials, respectively. Thus, N1 and N2 add up to N.(6)\nConsidering (1), the average trial probability pa can then be obtained from (7).(7)\nUsing (7), p1 can thus be calculated as (8).(8)\nSimilarly, considering (2), the variance σ2 is given by (9).(9)\nSubstituting p1 in (9) using (8) followed by substituting σ2 in (5) by (9) leads to a quadratic equation for p2 as a function of pa, N, and S2, as shown in equation (10).(10)\nThe solution to (10) is given by (11).(11)\nSetting p2 to(12)p1 can be obtained from (8) and shown to be given by formula (13):(13)Choosing p2 as(12a)leads to p1(13a)\nComparing (13a) to (12) and (12a) to (13), it can be seen that the formulae are identical except for the fact that N1 and N2 are reversed. Since the assignment of which set of trials is called N1 and which set of trials is called N2 is completely arbitrary, we can limit the remaining analysis on (12) and (13) without loss of generality.\nNote that (12) and (13) do not guarantee that p1 and p2 are always confined between 0 and 1 for any combination of N1 and N2. While probabilities smaller than 0 or bigger than 1 would still result in a distribution with the same overall variance as the original distribution, P-value calculation will be imprecise because the tails of the distribution will deviate significantly from the original distribution. Thus, we need to define the values N1 and N2 in such a way that p2< = 1 and p1> = 0. This can be achieved by evaluating (12) and (13).\nEvaluating (12) for the condition that p2< = 1, solving the resulting inequality for N2, and considering (5), which relates S2 and σ2, we obtain (14).(14)\nSimilarly, evaluating (13) for the condition p1> = 0, solving the resulting inequality for N2, considering (5), which relates S2 and σ2, and defining μf the expected number of failures as N * (1−pa) (15),(15)we obtain (16)(16)\nThe meaning of these boundaries is best illustrated by considering a Poisson-binomial distribution whose variance is 0, i.e. that assumes 1 at X = μ and 0 otherwise. In this case (14) requires N2< = μ while (16) requires N2> = μ. These conditions can only be fulfilled contemporaneously when N2 is set to μ. Intuitively, this means that there are μ trials with probability of success 1 and N−μ trials with probability of 0, resulting in a Poisson-binomial distribution with variance σ2 = 0 and mean μ. When σ2 is larger than 0, the choice of N1 and N2 is more flexible. However, since the choice of N2 = μ is valid for all possible values of σ2, this is how NetCutter determines N1 and N2. When μ is not an integer, N2 is set to the integer closest to μ.\nHaving determined p2 (12) and p1 (13) as well a N1 and N2 (14, 16, 6), we can now calculate the bi-binomial approximation of the Poisson-binomial distribution in a fashion that is very similar to calculate the binomial P-value. With q1 = 1−p1 and q2 = 1−p2 we obtain:(17)\nThe summation is necessary because i successes can be obtained from any combination of j p1 and i−j p2 trials, where j can assume any value from 0 to i.\n\nCalculating Jaccard and uncertainty coefficients\nFor the purpose of comparing the efficacy of the bi-binomial distribution as a significance measure of co-occurrence, Jaccard and uncertainty coefficients (which are also called measures of association) were calculated using the formulae:\nThe Jaccard coefficient J is calculated as the number of times A and B occur together divided by the number of times A occurs without B plus the number of times B occurs without A plus the number of times A and B occur together [42].\nThe uncertainty coefficient [42] is calculated as:H is the entropy associated with A, B, and AB. For A, the entropy is calculated from the probabilities of A occurring in n1 out of N lists (n1/N) and A not occurring in n2 out of N lists (n2/N). Analogous calculations lead to the entropy associated with B. For H(A,B), the probabilities of A occurring without B, B occurring without A, A and B occurring together, and neither A nor B occurring in the lists are used.\n\nCo-occurrence analysis of the PubLiME data set\nThe bipartite graph to be analyzed is composed of 27619 PubMedID_listID-gene pairs (see supplementary material Table S1). Edge-swapping (1000 simulations, see above) was used to determine occurrence probabilities and gene co-occurrence was analyzed using module size 3 (co-occurrence of three genes), bi-binomial Z-score cutoff 6, bi-binomial P-value cutoff 1.0E-5, and support 5. Supplementary material Simulation S1 provides details on why module size 3 is chosen. The support parameter ensures that each 3-gene co-occurrence module is present in at least 5 signatures. We identified 1654 significant modules in the test data compared to 5 modules in a randomized bipartite graph, corresponding to a signal-to-noise ratio of 331. The co-occurrence network was generated from the significant co-occurrence modules by drawing an edge between each pair wise combination of genes that are part of the same co-occurrence module. Gene communities were identified in this network by edge-betweenness clustering removing 4 edges, which resulted in a maximal network modularity of 0.63. Modularity is calculated as described by [43].\nFor the identification of PubMedID_listID clusters, the PubMedID_listID-gene pairs in the original bipartite graph were reversed to form gene-PubMedID_listID pairs. Occurrence probabilities were obtained by transposing the original occurrence probability matrix determined by edge-swapping as described above. PubMedID_listID co-occurrence was analyzed using module size 5, Z-score cutoff 6, bi-binomial P-value cutoff 1.0E-5, and support 3. Please note that the choice of these parameters is dictated by the parameters used in gene co-occurrence analysis. The reversal of the bipartite graph necessitates the support parameter used in gene co-occurrence analysis (5) to be used as module size in PubMedID_listID co-occurrence analysis and the module size used in gene co-occurrence analysis (3) to be used as the support parameter in PubMedID_listID co-occurrence analysis if the scope of the analysis is the identification of PubMedID_listID clusters that correspond to gene clusters identified before. The significance cutoffs remain unchanged. PubMedID_listID co-occurrence analysis revealed 448 significant co-occurrence modules in the real bipartite graph and 6 significant co-occurrence modules in the randomized bipartite graph with a signal-to-noise ratio of 75. Communities in the resulting co-occurrence network were identified by edge-betweenness clustering removing 130 edges. The resulting maximal network modularity was found to be 0.47.\n\n\nSupporting Information\n\n\n" ], "offsets": [ [ 0, 72440 ] ] } ]

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[ { "id": "pmcA2602716__text", "type": "Article", "text": [ "A Case-Control Study to Assess the Relationship between Poverty and Visual Impairment from Cataract in Kenya, the Philippines, and Bangladesh\nAbstract\nBackground\nThe link between poverty and health is central to the Millennium Development Goals (MDGs). Poverty can be both a cause and consequence of poor health, but there are few epidemiological studies exploring this complex relationship. The aim of this study was to examine the association between visual impairment from cataract and poverty in adults in Kenya, Bangladesh, and the Philippines.\n\nMethods and Findings\nA population-based case–control study was conducted in three countries during 2005–2006. Cases were persons aged 50 y or older and visually impaired due to cataract (visual acuity < 6/24 in the better eye). Controls were persons age- and sex-matched to the case participants with normal vision selected from the same cluster. Household expenditure was assessed through the collection of detailed consumption data, and asset ownership and self-rated wealth were also measured. In total, 596 cases and 535 controls were included in these analyses (Kenya 142 cases, 75 controls; Bangladesh 216 cases, 279 controls; Philippines 238 cases, 180 controls). Case participants were more likely to be in the lowest quartile of per capita expenditure (PCE) compared to controls in Kenya (odds ratio = 2.3, 95% confidence interval 0.9–5.5), Bangladesh (1.9, 1.1–3.2), and the Philippines (3.1, 1.7–5.7), and there was significant dose–response relationship across quartiles of PCE. These associations persisted after adjustment for self-rated health and social support indicators. A similar pattern was observed for the relationship between cataract visual impairment with asset ownership and self-rated wealth. There was no consistent pattern of association between PCE and level of visual impairment due to cataract, sex, or age among the three countries.\n\nConclusions\nOur data show that people with visual impairment due to cataract were poorer than those with normal sight in all three low-income countries studied. The MDGs are committed to the eradication of extreme poverty and provision of health care to poor people, and this study highlights the need for increased provision of cataract surgery to poor people, as they are particularly vulnerable to visual impairment from cataract.\n\nBackground.\nGlobally, about 45 million people are blind. As with many other conditions, avoidable blindness (preventable or curable blindness) is a particular problem for people in developing countries—90% of blind people live in poor regions of the world. Although various infections and disorders can cause blindness, cataract is the most common cause. In cataract, which is responsible for half of all cases of blindness in the world, the lens of the eye gradually becomes cloudy. Because the lens focuses light to produce clear, sharp images, as cataract develops, vision becomes increasingly foggy or fuzzy, colors become less intense, and the ability to see shapes against a background declines. Eventually, vision may be lost completely. Cataract can be treated with an inexpensive, simple operation in which the cloudy lens is surgically removed and an artificial lens is inserted into the eye to restore vision. In developed countries, this operation is common and easily accessible but many poor countries lack the resources to provide the operation to everyone who needs it. In addition, blind people often cannot afford to travel to the hospitals where the operation, which also may come with a fee, is done.\n\nWhy Was This Study Done?\nBecause blindness may reduce earning potential, many experts believe that poverty and blindness (and, more generally, poor health) are inextricably linked. People become ill more often in poor countries than in wealthy countries because they have insufficient food, live in substandard housing, and have limited access to health care, education, water, and sanitation. Once they are ill, their ability to earn money may be reduced, which increases their personal poverty and slows the economic development of the whole country. Because of this potential link between health and poverty, improvements in health are at the heart of the United Nations Millennium Development Goals, a set of eight goals established in 2000 with the primary aim of reducing world poverty. However, few studies have actually investigated the complex relationship between poverty and health. Here, the researchers investigate the association between visual impairment from cataract and poverty among adults living in three low-income countries.\n\nWhat Did the Researchers Do and Find?\nThe researchers identified nearly 600 people aged 50 y or more with severe cataract-induced visual impairment (“cases”) primarily through a survey of the population in Kenya, Bangladesh, and the Philippines. They matched each case to a normally sighted (“control”) person of similar age and sex living nearby. They then assessed a proxy for the income level, measured as “per capita expenditure” (PCE), of all the study participants (people with cataracts and controls) by collecting information about what their households consumed. The participants' housing conditions and other assets and their self-rated wealth were also measured. In all three countries, cases were more likely to be in the lowest quarter (quartile) of the range of PCEs for that country than controls. In the Philippines, for example, people with cataract-affected vision were three times more likely than normally sighted controls to have a PCE in the lowest quartile than in the highest quartile. The risk of cataract-related visual impairment increased as PCE decreased in all three countries. Similarly, severe cataract-induced visual impairment was more common in those who owned fewer assets and those with lower self-rated wealth. However, there was no consistent association between PCE and the level of cataract-induced visual impairment.\n\nWhat Do These Findings Mean?\nThese findings show that there is an association between visual impairment caused by cataract and poverty in Kenya, Bangladesh, and the Philippines. However, because the financial circumstances of the people in this study were assessed after cataracts had impaired their sight, this study does not prove that poverty is a cause of visual impairment. A causal connection between poverty and cataract can only be shown by determining the PCEs of normally sighted people and following them for several years to see who develops cataract. Nevertheless, by confirming an association between poverty and blindness, these findings highlight the need for increased provision of cataract surgery to poor people, particularly since cataract surgery has the potential to improve the quality of life for many people in developing countries at a relatively low cost.\n\nAdditional Information.\nPlease access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050244.\n\n\n\nIntroduction\nImprovements in health are at the heart of the Millennium Development Goals, with the recognition that better health is central to the primary aim of reducing poverty as well as important in its own right. Empirical data are needed to back up this claim. Unravelling the relationship between blindness and poverty therefore has important implications, and may also be informative for the association between poverty and other disabilities.\nBlindness is a common condition globally, affecting approximately 45 million people, and more than a third of blindness is caused by cataract [1,2]. Globally, the prevalence of blindness is five-fold higher in poor than rich countries [2]. Limited data show that within countries the poor are also more likely to be blind [3,4]. It is frequently asserted that blindness is both a cause and consequence of poverty, but there are few empirical data to support this claim. Poverty may cause cataract blindness, because access to cataract surgery is limited in low-income countries [5]. Furthermore, within poor countries some evidence suggests that lack of money is a major barrier to uptake of cataract surgery by individuals [6–8]. Blindness may also cause poverty, as the blind individual, or the household members who care for them, have a reduced earning potential [4,9]. This complex problem could have serious implications; estimates from The Gambia suggest that there is a substantial economic burden from lost productivity among blind people [10]. Therefore, blindness prevention may ultimately be cost saving [11]. Extrapolations on a global level indicate that a successful eye care programme could prevent more than 100 million cases of blindness between 2000 and 2020, and consequently save at least US$102 billion, which would otherwise be lost to reductions in productivity associated with blindness [12]. However, these estimates are based on extrapolations from limited data and were not based on individual-level data. It is also difficult to identify the component of productivity loss that is due to blindness, as this condition mainly affects older people, who may suffer from other comorbidities that restrict their employment opportunities or make them dependent on the care of others.\nThe Cataract Impact Study was undertaken to assess the relationship between cataract visual impairment and “economic poverty” and quality of life, and to estimate the impact of cataract surgery on these factors in three low-income countries. The aim of the current paper is to assess the association at baseline between visual impairment from cataract and household poverty (measured through consumption, asset ownership, and self-rated wealth) in a population-based case–control study in Kenya, the Philippines, and Bangladesh.\n\nMethods\nSetting\nCase and control participants were recruited from Nakuru district, Kenya (January–February, 2005); Negros island (May–June, 2005) and Antique district (April–May, 2006), Philippines; and Satkhira district, Bangladesh (November–December, 2005).\n\nSelection of Cases and Controls\nPersons with cataract visual impairment (cases) and persons without (controls) were primarily recruited through a population-based survey of adults aged ≥ 50 y [6–8]. Clusters of 50 people (regardless of visual impairment) aged ≥ 50 y were selected through probability-proportionate to size sampling, using either the census (Philippines and Bangladesh) or electoral role (Kenya) as the sampling frame. Households within clusters were selected through a modification of compact segment sampling, whereby a map was drawn of the enumeration area that was divided into segments, each including approximately 50 people aged ≥ 50 y, and one segment was chosen at random [13]. Households in the segment were included sequentially until 50 people aged ≥ 50 y were identified. The surveys included 3,503 (93% response rate) people aged ≥ 50 y in Kenya, 4,868 (92%) in Bangladesh, 2,774 (76%) in Negros, and 3,177 (83%) in Antique.\nAll people in the survey aged ≥ 50 y underwent visual acuity (VA) testing and ophthalmic examination. VA was measured in full daylight with available spectacle correction with a Snellen tumbling “E” chart using optotype size 6/18 (20/60) on one side and size 6/60 (20/200) on the other side at 6 or 3 metres. If the VA was <6/18 in either eye then pinhole vision was also measured. Participants with pinhole vision <6/18 but >6/60 in the better eye due to age-related cataract were given a second VA test using an “E” of size 6/24. The ophthalmologist examined all eyes with a presenting VA <6/18 with a torch (i.e., flashlight), direct ophthalmoscope, and/or portable slit lamp. The principal cause of blindness or visual impairment was recorded, according to the WHO convention in which the major cause is assigned to the primary disorder or, if there are two existing primary disorders, to the one that is easiest to treat [14].\nSurvey participants were eligible for inclusion as cases if they were aged ≥ 50 y with best corrected visual acuity <6/24 in the better eye due to cataract, as diagnosed by an ophthalmologist. All eligible cases identified from these surveys were invited to participate in the study. Participants were eligible to be controls if they were aged ≥ 50 y, did not have VA <6/24 in the better eye due to cataract and did not live in the same household as a case. During the survey a list was maintained of all eligible controls, by age group (50–54, 55–59, 60–64, 65–69, and >70) and sex. Whenever a case was identified, one age- and sex-matched control was randomly selected from the list for inclusion (or up to two controls in Bangladesh). If no matching eligible controls had been identified in that cluster at that stage of the survey, then the next eligible control in the cluster was recruited.\nBecause of logistical and time constraints, additional cases were also included through community-based case detection. In Kenya and Negros (Philippines), clusters were randomly selected through probability proportionate to size using the same cluster sampling procedure after completion of the population-based survey. Clusters were visited in advance and asked that all people ≥ 50 y with vision problems come to a central point on a specified day, and that a list be made of people unable to attend (e.g., due to blindness or other physical disability). After examining patients at the central point, the survey team then visited those unable to leave their houses. Any identified eligible cases that agreed to be part of the study were interviewed in their homes. In Bangladesh and Antique (Philippines), community case detection was carried out simultaneously with the survey by two of the four teams, so that controls were included for these cases. Within each cluster from the survey, one interviewer was asked to be taken to two community members aged ≥ 50 y with eye problems, living within the cluster boundaries but not from the segments selected for the survey. If VA was <6/24 with pinhole in the better eye, the ophthalmologist was called to carry out the full eye examination, and eligible cases were included in the study.\nFor the purposes of the present analyses, control individuals with any visual impairment (VA <6/18 in the better eye) were excluded (n = 14 in Kenya, n = 53 in Bangladesh, n = 24 in the Philippines). Case and control participants who were significantly communication impaired (e.g. deafness, dementia, or psychiatric disease) were excluded (fewer than five per country), and one case was excluded in the Philippines because of missing age data. One household had two eligible cases (Kenya), and one of these participants was excluded for the poverty analyses as poverty was assessed through household level indicators (see below).\nIn total, 147 cases (82 from the survey and 65 from case detection) and 79 controls were included in Kenya; 217 cases (162 from survey and 55 from case detection) and 280 controls in Bangladesh; and 238 cases (146 survey and 92 case detection) and 180 controls in the Philippines.\n\nData Collection\nAll case and control participants were interviewed in their homes by trained interviewers in the local language. Each interview lasted approximately 1 h.\nMeasures of poverty.\nPoverty was measured through (a) monthly per capita expenditure (PCE) to indicate consumption, (b) asset ownership, and (c) self-rated wealth. The economic part of the questionnaires was adapted through interviews, focus group discussions, and pilot testing in each country to ensure local relevance.\nThe person primarily responsible for household finances (which may have been the case/control or another household member) was interviewed to assess PCE and assets. PCE was measured using methods based on the World Bank's Living Standards Measurement Study [15]. Items were included on food (42–52 items per country), education (three items), health (five items), household expenses (nine items), and personal expenses (21 or 22 items). In total, 85 items were included in the questionnaire in Kenya, 90 in the Philippines, and 79 in Bangladesh. The informant was asked to recall the monetary value of food that was purchased, consumed from home production, or received as payment in kind or as gifts. Consumption was assessed over a 1-wk period for frequently consumed items, and this was scaled up to estimate monthly consumption. The amount consumed monthly was assessed for items that were consumed more rarely. Monthly rent was recorded among households who rented, and households who owned their property were asked to estimate the amount that they could charge in rent per month. The consumption on all items was summed to calculate total monthly household consumption, and this was converted to United States dollars (US$) at the 2005 exchange rate ($1 = 76 Kenya shillings, 64 Bangladesh taka, 55 Philippine pesos). Total monthly household consumption was divided by the number of household members to calculate monthly PCE for the household.\nThe household informant was also asked about the number and type of context-specific assets owned by the household, including different types of furniture, electrical equipment, cattle, and vehicles. Information was collected on household characteristics, including the building material of the floor, roof, and walls; type of toilet; and the number of rooms.\nSelf-rated wealth was assessed by asking the household informant to rank the household's wealth relative to others in the community on a scale from 1 (poorest) to 10 (richest).\n\nCovariates.\nCase and control individuals were interviewed about standard sociodemographic indicators, including household composition, education, and employment. Information was collected on vision-related quality of life using the World Health Organization Prevention of Blindness and Deafness 20-item Visual Functioning Questionnaire [16,17], and health-related quality of life was assessed using items from the European Quality of Life Questionnaire [18]. Detailed time-use data were collected using methods based on the World Bank's Living Standards Measurement Study [15].\n\n\nTraining and Fieldwork\nInterviewers were trained for 1 wk, including 2 d of pilot testing. Attempts were made to minimise measurement bias by emphasising the need for consistency in data collection among cases and controls. The questionnaires were translated into the local languages (three in Kenya, three in the Philippines, and one in Bangladesh) and back-translated by independent translators (one for each language) who were also asked to comment on appropriateness of language used for the target population. A review was held to discuss differences in translation and modify accordingly. The questionnaire was piloted in each setting and small modifications to wording of some items were made, where appropriate, to ensure local understanding. Teams were accompanied by a field supervisor at least 1 d per wk to ensure that high quality was maintained and interviews were observed randomly throughout the study.\n\nStatistical Analysis\nMicrosoft Access was used for data entry, and all data were double entered and validated. Analyses were undertaken in SAS version 8.2.\nThe mean and range of each expenditure item was calculated to assess whether answers were plausible, and to identify and exclude gross outliers (none identified). Rental equivalents were imputed based on household characteristics and non-rent expenditure for households where these estimates were missing or unreasonably low (< $1 per mo) (four in Kenya, three in Bangladesh, 18 in the Philippines). Total monthly household consumption was divided by the number of household members to calculate per capita household expenditure. Per capita household expenditure was divided into quartiles, separately for each country, based on the distribution of the data for the case and control participants combined. Households with incomplete expenditure data were excluded from analyses (five cases and four controls in Kenya; one case and one control in Bangladesh).\nA relative index of household assets was derived using principal components analysis (PCA) to determine weights for a list of assets and wealth indicators [19]. Variables entered into the PCA included building materials of the house, ownership of ten household assets, animal ownership, and education of the head of the household. The derived index was divided into quartiles from poorest (lowest socioeconomic status [SES] index) to least poor (highest SES index). PCA analyses were undertaken separately for each country. The means of the poverty variables were first compared for cases recruited through the two different methods, and then from cases and controls using t-tests for continuous variables (e.g., PCE and assets). For categorical variables (e.g., household rank) we used the Mann-Whitney test and presented medians and interquartile ranges. PCE was highly skewed and therefore was log transformed for the t-tests. The two-way correlations were calculated between PCE, assets, and household rank, in turn.\nLogistic regression analyses were undertaken separately for each country, assessing the association between case/control status and sociodemographic and poverty variables. Conditional logistic regression was not undertaken, since the matching was incomplete, so all analyses were adjusted for the matching variables (age, sex, and rural/urban location). Likelihood ratio tests were undertaken to assess the significance of adding covariates with more than two levels (e.g., age groups, self-rated health groups) to the model. Tests for trend were undertaken across quartiles of the poverty variables and assessed using the p-value for trend. Analyses were also conducted adjusting for the logistic regression analyses for poverty by social support indicators (marital status and household size) and self-rated health, since these variables may confound the association between cataract visual impairment and poverty. Analyses from the Philippines were also adjusted for study site, since data were obtained from two settings (Negros and Antique). An attempt was made to disentangle the relationship between poverty and cataract by stratifying the analyses by age, sex, and level of visual impairment among the cases.\n\nEthical Approval\nInformed signed or thumb-printed consent was obtained from all cases and controls. In Kenya and Bangladesh all cases were offered free cataract surgery at the local hospital, with free transport. In the Philippines, patients were referred for surgery, which was subsidised for patients who could not afford the fee. Ethical approval for this study was obtained from the ethics committees of the London School of Hygiene & Tropical Medicine, the Kenya Medical Research Institute, the Bangladesh Medical Research Council, and the University of St. La Salle, Bacolod, Philippines. This study complied with the guidelines of the Declaration of Helsinki.\n\n\nResults\nSociodemographic Characteristics of Cases and Controls\nCase and control participants were matched reasonably closely by sex and location. However, within the age category ≥ 70 y, cases tended to be older than the controls, so that cases were over-represented in the oldest age groups (75–79 and ≥ 80 y) compared to controls (Table 1). Cases were less likely to be married than controls, in Kenya (OR 0.6, 95% CI 0.3–1.1), Bangladesh (0.6, 0.4–1.0), and the Philippines (0.7, 0.4–1.0), although this only reached statistical significance in Bangladesh (p = 0.03). There was a strong protective effect of literacy and education on cataract in Bangladesh and Kenya that was not evident in the Philippines. Cases were substantially less likely to have a job other than working in the field compared to controls in all three countries. Cases reported significantly poorer self-rated health than controls—this pattern was particularly evident in the Philippines (OR for lowest versus highest quartile of self-rated health = 5.7, 95% CI 3.0–10.7) but also apparent in Kenya (2.6, 1.1–6.2) and Bangladesh (3.3, 2.1–5.3).\n\nSummary Wealth Measures\nAll three settings were poor. The mean PCE was less than US$1 per person per day in all three settings: US$26.4 (standard deviation [SD] = US$34.9) in Kenya, US$21.7 (US$48.0) in Bangladesh and US$26.1 (US$23.5) in the Philippines. The biggest expense was food in all three settings, making up 55% of PCE in Kenya, 47% in Bangladesh, and 64% in the Philippines, followed by household expenses including rent (21% in Kenya, 28% Bangladesh, and 22% Philippines) (Figure 1). The majority of food consumption was from direct purchase (70% in Kenya, 75% in Bangladesh, and 77% in the Philippines) or home-grown production (24% in Kenya, 22% in Bangladesh, and 17% in the Philippines), and little was from gifts or payments.\nAn asset score was created through PCA in the three settings. The first principal component explained 22% of the variability in asset variables in Kenya, 25% in Bangladesh, and 24% in the Philippines. Self-perceived wealth of the household clustered around the average with a large proportion of households in Kenya (48%), Bangladesh (43%), and the Philippines (64%); households stating that they were ranked between 4 and 6, on a scale from 1 to 10, in terms of wealth in their community. The three measures of poverty were highly correlated, each showing significant correlation (p < 0.001) with the other measure.\n\nEconomic and Household Characteristics of Cases and Controls\nThere were no significant differences in PCE, assets, or household rank between cases recruited through the population-based survey and those recruited through case detection, with the exception that the case-detection cases had lower household rank in Kenya (mean = 3.7 versus 3.1, p = 0.02). Consequently, cases recruited through the two methods were combined in the subsequent analyses.\nCases were poorer than controls, in all three settings according to all three poverty measurements (Table 2). The mean PCE was 20%–28% lower for members of households with a case than for control households, and this difference was highly significant in Bangladesh and the Philippines; for Kenya it was lower but did not reach significance (p = 0.07). The PCA score for assets was significantly lower among cases than controls in Kenya and Bangladesh, and it was lower in the Philippines although it did not reach significance (p = 0.06). Self-perceived wealth was significantly lower for households with a case compared to control households in Kenya (3.4 versus 4.5) and Bangladesh (3.9 versus 4.6), though not in the Philippines (4.1 versus 4.3).\nThere was no difference in the size of the households of cases and controls in any of the three settings. The ratio of dependents (i.e., household member aged <15 or ≥ 50 y) to independents (i.e., household member aged 15–50 y) was similar between cases and controls in Bangladesh (1.4 versus 1.4), but the dependency ratio was higher for controls than cases in Kenya (2.1 versus 1.6) and the Philippines (1.7 versus 1.3), due to the smaller number of people of working age.\n\nPatterns of Expenditure in Cases and Controls\nFigure 1 shows the total PCE and the allocation of expenditure within quartiles of PCE for cases and controls. Monthly PCE was similar for cases and controls within each of the quartiles of expenditure. There was a gradual increase in PCE between the first three quartiles, and then a rapid increase between the third and the richest quartile. Within the first three quartiles of PCE the majority of expenditure was on food. Substantial expenditure on non-food items was observed only in the highest quartile of expenditure, where about half of expenditure was on non-food items. Similar patterns of PCE were observed for cases and controls in Kenya, Bangladesh, and the Philippines within each quartile of expenditure. These results demonstrate that cataract visual impairment was related to reduced PCE, but not allocation of expenditure.\n\nMultivariate Analyses of Poverty and Cataract Visual Impairment\nMultivariate analyses showed that case participants were consistently poorer than controls in Kenya, Bangladesh, and the Philippines, using three different measures of poverty (Table 3). Cases were more likely than controls to be in the lowest quartile of PCE rather than the highest quartile in Kenya (OR 2.3, 95% CI 0.9–5.5), Bangladesh (1.9, 1.1–3.2) and the Philippines (3.1, 1.7–5.7). In all three settings these associations showed significant dose–response as assessed by the p-value for trend across the quartiles, with decreasing PCE related to case status and these relationships persisted after adjustment for self-rated health and social support indicators. A similar pattern was observed for the relationship between case–control status and asset ownership. Cases were significantly more likely to be in the lowest quartile of asset ownership rather than the highest quartile compared to controls in Kenya (3.7, 1.4–9.6), Bangladesh (2.6, 1.5–4.4), and the Philippines (2.1, 1.1–3.8). Cases were also significantly more likely to be in the lowest quartile of household rank rather than the highest, compared to controls in Kenya (3.5, 1.5–8.0), Bangladesh (2.7, 1.6–4.7) and the Philippines (2.3, 1.1–4.8). The associations with assets and household rank also showed a significant dose–response relationship, and the associations were largely unchanged after adjustment for self-rated health and social support indicators. In Kenya and Bangladesh the relationship between PCE and case status was somewhat weaker than for the other measures of poverty, while the reverse was true in the Philippines.\nStratifying the association between PCE and cataract visual impairment by level of visual impairment showed an inconsistent pattern (Table 4). In Kenya, the association with low PCE was somewhat stronger comparing cataract blind cases to controls (OR 3.1, 95% CI 0.9–10.8) than comparing moderate visually impaired cases to controls (1.8, 0.6–5.4), while this pattern was reversed in Bangladesh (blind cases versus controls: 1.8, 1.0–3.4; moderately visually impaired cases versus controls: 3.1, 1.3–7.2). In the Philippines the association with low PCE was strongest comparing severely visually impaired cases to controls (5.9, 2.0–17.6). The association between cataract visual impairment and PCE was stronger among men than women in Bangladesh and the Philippines, while the reverse was true in Kenya (Table 5). In Kenya and the Philippines the strongest association between cataract and PCE was among people aged 70–79 y, while in Bangladesh the strongest effect was in people aged over 80 y. Stratifying the association between assets and household rank with cataract by level of visual impairment, sex, or age broadly repeated these findings, and generally supported the lack of consistent pattern (unpublished data).\n\n\nDiscussion\nThis large, multicentre population-based case–control study provides evidence that people with visual impairment from cataract are poorer than control participants with normal vision matched for age and sex. This pattern was evident whether poverty was measured in terms of PCE, assets, or self-rated wealth. Marital status seemed to be protective for cataract visual impairment, possibly indicating the role of social support in health-seeking behaviour. Reduced self-rated health was also strongly related to cataract visual impairment. This demonstrates the impact of poor vision on overall assessments of health and supports our previous finding of a relationship between cataract and quality of life [17].\nAdjustment for marital status and self-rated health did not entirely explain the association between poverty and cataract visual impairment, suggesting that it operated through other pathways. Visual impairment could cause poverty through reduced employment opportunities. We might therefore expect to see a stronger relationship between cataract and poverty among the blind case participants who may have fewer employment opportunities than among those less impaired (i.e., moderate visual impairment). Poverty may also cause visual impairment through restricted access to cataract surgery. In this case we would expect to see a stronger relationship between poverty and less severely affected cases (i.e., moderate visual impairment), as poor families may allocate money for surgery on members who are blind from cataract, so that poverty mainly restricts access to surgery among people who are moderately visually impaired. The relationships that we observed between level of visual impairment and cataract were inconsistent across the three settings. Perhaps this shows that both pathways were operating or that the dynamics of the relationship between poverty and blindness vary in different settings. Levels of literacy and education were lower among cases than controls. These long-term indicators of disadvantage are unlikely to have changed after the onset of cataract. This observation provides some evidence that poverty preceded blindness in our study participants.\nIt is frequently asserted that blindness is both a cause and consequence of poverty, but there are few empirical data to support this claim. Globally, the prevalence of blindness is five-fold higher in poor than rich countries [2], and data from Pakistan and India suggest that within countries the poor are more likely to be blind [3, 4]. Some blinding conditions are a direct consequence of poverty, notably trachoma, which thrives in poor areas lacking water and sanitation [20]. Other blinding diseases clearly contribute to poverty, such as onchocerciasis, which results in the abandonment of the fertile areas near to the rivers where the disease vector thrives [9]. A larger literature shows that poor people are more likely to be ill or disabled than their richer compatriots, ranging from general disability in India, Bulgaria, and Ghana [21]; common mental disorders in Brazil, Chile, India, and Zimbabwe [22]; deafness in Brazil [23]; and tuberculosis in China [24]. There are also some exceptions such as a case-control study in Rwanda which failed to show an association between PCE and musculoskeletal impairment, perhaps because the population was almost universally poor [25].\nPoverty may increase the incidence of disease, particularly preventable diseases such as tuberculosis. Poverty may also restrict access to appropriate health care and so prolong the duration of disease. A study in rural Tanzania showed that care-seeking behaviour for childhood illness is worse among poorer families than among the relatively rich families [26]. Another Tanzanian study found that people with higher levels of asset ownership were more likely to obtain antimalarials even though they were less likely to be parasitaemic [27]. With respect to cataract, there is little evidence that prevention is possible, and so the main pathway from poverty to blindness is likely to be through reduced access to cataract surgical services. High health care costs may also exacerbate poverty. A study in rural China showed that ill health increases medical expenditure significantly, which detracts from expenditure on food, education, investment in farming, and participation in social activities [28]. Inability to afford cataract surgery is cited as the major barrier to the uptake of surgery in the surveys conducted in Kenya, the Philippines, and Bangladesh [6–8]. This indicates that the cost of surgery is perceived as substantial by many households, notwithstanding the problems of assessing the complex issue of barriers in the absence of in-depth qualitative interviews. Consequently, there are lower rates of cataract surgery among the poor [3].\nPoverty may also limit the employment opportunities of the person with disability or their household members. This pattern has been demonstrated for people with HIV in South Africa [29], tuberculosis in China [24], or disability in Sri Lanka [22]. An impact of blindness on reduced employment or income has been observed in Guinea [9] and India [4]. A belief that blindness reduces the employment opportunities of household members is widespread, but so far there is limited supportive evidence. There is a further complication to investigations of the relationship between cataract and poverty, as the individuals with cataract are likely to be elderly and facing multiple disabilities. Our study took account of the potential impact of multiple disabilities, as we adjusted for self-rated health, which is closely related to overall health, and this adjustment had no overall impact on our results [30].\nStudy Strengths\nThis was a large population-based case–control study, conducted in three countries, allowing international comparisons. This was the first study, to our knowledge, to relate PCE to visual impairment. We also measured assets, which reflects long-term access to resources, and self-rated wealth. We used expenditure as a proxy for income, which has aided both academic and nonacademic investigations. As one example, the notorious Chicago gangster Al Capone managed to escape prosecution for smuggling, gambling, bootlegging, and murder for years, but was eventually convicted of tax evasion, because the jury was convinced that his exorbitant expenses on clothes, furnishing, foods, and gifts were inconsistent with his claim that he had no income. Expenditure often provides a better measure of poverty than income for a number of reasons. Income may be variable by season, whereas households attempt to smooth expenditure over the year. People are more comfortable sharing information about expenditure than income, and it may be a more meaningful measure than income in an agrarian society as it reflects what the household is able to command based on its current income, borrowing ability, or household savings [31]. PCE also has advantages over assets, as it may be more responsive to change, which will be important for the follow-up analyses of the study participants after they have undergone cataract surgery.\n\nStudy Limitations\nThere are a number of limitations relating to the measurement of poverty in this study. Our analyses focus on monetary indicators of poverty, while we acknowledge that health, education, and housing are also important. We concede that it is difficult to measure expenditure accurately [32,33], but this also true for the measurement of diet and other variables, which is standard practise in many epidemiological studies. Furthermore, a large number of items were included in our measure of expenditure so that the measure was comprehensive [33]. Expenditure data were not validated through diaries or other means, although assets and self-rated wealth correlated highly with PCE. Other recent estimates of expenditure are not available from surveys conducted in these countries to allow comparison. The per capita estimates of monthly gross national income from the World Development Indicators database show somewhat higher estimates in Kenya (US$48) and Bangladesh (US$40) than our PCE derived estimates, and far higher estimates for the Philippines (US$108). This discrepancy may be reasonable, as the World Development Indicators reflect national averages, while we sampled the households with elderly people in poor regions of the country, many of whom were visually impaired from cataract. PCE was calculated simply by dividing the total household expenditure by the number of household members, without inclusion of economies of scale or equivalence scales. There is no widely accepted alternative to the simple equal-sharing convention, and the majority of expenditure was on food which does not allow for economies of scale. Furthermore, there were slightly fewer people of working age in the control households in Kenya and the Philippines, so adjustment for equivalence scores would be unlikely to explain the higher poverty among cases. The case and control households were of similar sizes in the three settings, so economies of scales are unlikely to have explained the differences.\nThere were a number of limitations relating to study design. Unfortunately, we did not record the exact numbers of cases and controls who refused to participate or were unable to communicate (believed to be fewer than five in each country), so the response rate is unknown, but was believed to be high. A variety of methods were used for case recruitment, as we were not able to obtain enough cases through the survey alone. However, cases recruited through the population-based survey and through case detection had similar poverty characteristics.\n\nConclusions\nOur data show that people with visual impairment due to cataract were poorer than controls in three low income countries, Bangladesh, Kenya, and the Philippines. The Millennium Development Goals are committed to the eradication of extreme poverty and provision of health care to poor people. This study confirms an association between poverty and blindness and highlights the need for increased provision of cataract surgery to poor people, particularly since cataract surgery is a highly cost-effective intervention in these settings [34].\n\n\n\n" ], "offsets": [ [ 0, 41272 ] ] } ]

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[ { "id": "pmcA1808052__text", "type": "Article", "text": [ "A disease-specific measure of health-related quality of life for use in adults with immune thrombocytopenic purpura: Its development and validation\nAbstract\nBackground\nNo validated disease-specific measures are available to assess health-related quality of life (HRQoL) in adult subjects with immune thrombocytopenic purpura (ITP). Therefore, we sought to develop and validate the ITP-Patient Assessment Questionnaire (ITP-PAQ) for adult subjects with ITP.\n\nMethods\nInformation from literature reviews, focus groups with subjects, and clinicians were used to develop 50 ITP-PAQ items. Factor analyses were conducted to develop the scale structure and reduce the number of items. The final 44-item ITP-PAQ, which includes ten scales [Symptoms (S), Bother-Physical Health (B), Fatigue/Sleep (FT), Activity (A), Fear (FR), Psychological Health (PH), Work (W), Social Activity (SA), Women's Reproductive Health (RH), and Overall (QoL)], was self-administered to adult ITP subjects at baseline and 7–10 days later. Test-retest reliability, internal consistency reliability, construct and known groups validity of the final ITP-PAQ were evaluated.\n\nResults\nSeventy-three subjects with ITP completed the questionnaire twice. Test-retest reliability, as measured by the intra-class correlation, ranged from 0.52–0.90. Internal consistency reliability was demonstrated with Cronbach's alpha for all scales above the acceptable level of 0.70 (range: 0.71–0.92), except for RH (0.66). Construct validity, assessed by correlating ITP-PAQ scales with established measures (Short Form-36 v.1, SF-36 and Center for Epidemiologic Studies Depression Scale, CES-D), was demonstrated through moderate correlations between the ITP-PAQ SA and SF-36 Social Function scales (r = 0.67), and between ITP-PAQ PH and SF-36 Mental Health Scales (r = 0.63). Moderate to strong inter-scale correlations were reported between ITP-PAQ scales and the CES-D, except for the RH scale. Known groups validity was evaluated by comparing mean scores for groups that differed clinically. Statistically significant differences (p < 0.01) were observed when subjects were categorized by treatment status [S, FT, B, A, PH, and QoL, perceived effectiveness of ITP treatment [S], and time elapsed since ITP diagnosis [PH].\n\nConclusion\nResults provide preliminary evidence of the reliability and validity of the ITP-PAQ in adult subjects with ITP. Further work should be conducted to assess the responsiveness and to estimate the minimal clinical important difference of the ITP-PAQ to more fully understand the impact of ITP and its treatments on HRQoL.\n\n\n\nBackground\nImmune thrombocytopenic purpura (ITP) is a disorder characterized by autoimmune-mediated platelet destruction and suboptimal platelet production [1-3] that results in a decrease in the number of circulating platelets and increases the risk of bleeding events. The estimated prevalence rate for ITP in the United States is 9.5/100,000 [4]. Adult women are disproportionately affected by the disorder, with a female to male ratio of nearly two to one [5]. The disorder rarely remits spontaneously in adult subjects [1]. The mortality rate is relatively low (< 1%) [6] in adults less than 65 years of age. Morbidity increases above age 65, primarily as a result of an increase in age-related major bleeding events [7].\nInitial therapy for ITP consists of some combination of glucocorticoids, intravenous immune globulin (IVIg) or anti-D [8]. Splenectomy is often considered if these therapies fail. Approximately two-thirds of patients treated with splenectomy achieve a sustained remission [1,5,9]. Patients who fail splenectomy are treated with a wide variety of agents including corticosteroids, danazol, and chemotherapeutic agents. Morbidity and mortality in these refractory patients are substantial [1,6,8].\nPatient-reported outcomes (PRO), including health-related quality of life (HRQoL) measures, are critical components for evaluating and understanding treatment effects from the subject's perspective. The Food and Drug Administration (FDA) indicates that PRO measures are important to assess because they may: 1) detect treatment effects known only to the subject; 2) understand the subject's perspective regarding treatment effect; or 3) provide information not included in a clinician's subject notes [10]. Furthermore, the Committee for Medicinal Products for Human Use of the European Medicines Agency defines HRQoL as \"the subject's subjective perception of the impact of his disease and its treatment(s) on his daily life, physical, psychological and social functioning and well-being\" [11].\nCurrently, limited data are available on the assessment of the impact of symptoms in adult subjects with ITP. Symptoms of ITP, such as spontaneous bruising, menorrhagia, mucosal bleeding and prolonged bleeding with injury, may significantly affect HRQoL in ITP subjects [12]. Treatments for chronic ITP can also be associated with substantial side effects [5,8]. In addition, subjects who are resistant to current therapies are likely to experience an even greater decrement to their HRQoL than responders to treatment. Thus, restoring and/or maintaining quality of life should be an important goal of treatment. While the primary markers for ITP include hematologic measures such as platelet counts, clinical measures typically do not assess a subject's functioning and well-being. Therefore, subjects and physicians may want to weigh the impact of ITP therapies on HRQoL endpoints when making treatment decisions.\nPreviously, no validated disease-specific measures were available to evaluate quality of life in adult ITP subjects; however, two disease-specific HRQoL questionnaires have been developed for use in children with ITP [13,14]. Thus, we sought to develop a questionnaire that would be appropriate to assess the impact of ITP symptoms and its treatments on HRQoL in adult subjects. The objective of this current manuscript is to describe the development and initial validation of a newly developed HRQoL questionnaire for use in adult subjects with ITP.\n\nMethods\nOverview\nA newly developed questionnaire, which assesses issues of importance to ITP subjects, was developed based on available published literature, existing questionnaires, expert clinical opinion, and input from subjects with ITP.\n\nSubjects and Procedures\nTo develop the questionnaire, three focus groups with ITP subjects were conducted in geographically diverse locations (San Diego, CA, New York, NY, and Oklahoma City, OK). Each of the three sites recruited a convenience sample of five to eight ITP subjects who were being treated on an outpatient basis. To be eligible, subjects were required to have active disease and be ≥ 18 years of age. Although platelet count data were not required for participation in the focus groups, clinicians at each site considered the subjects to have active disease, usually an indication that platelet levels have dropped below 120 × 109L and the subject requires treatment and/or more frequent monitoring. In total, 23 ITP subjects participated in the focus groups after providing their informed consent.\nTo validate the questionnaire, a convenience sample of subjects was recruited from the same three clinical sites in New York, NY, Oklahoma City, OK, and La Jolla, CA. Subjects were eligible if they were ≥ 18 years of age, had active disease, and were willing to complete a self-administered questionnaire at two time points. Target enrollment was roughly 72 subjects (24 subjects from each site).\nThe study protocol was approved by a local institutional review board at each site and was carried out in accordance with Good Clinical Practice and International Conference on Harmonization guidelines and the Declaration of Helsinki. Written informed consent was obtained from each subject prior to enrollment.\n\nCreation of Questionnaire/Item Selection\nA trained moderator led all focus groups using a detailed discussion guide. Subjects discussed their ITP history, treatment, ITP symptoms, and the impact of ITP on daily activities. Each focus group lasted approximately three hours, and subjects were provided with an honorarium for their time. Following the focus group session, all subjects completed a questionnaire which included the SF-36 v1 [15] and ITP-specific questions. The ITP-specific items were developed based on clinical input [12,16-18]. The ITP-specific items assessed the impact of ITP on the subject's overall quality of life, relationships, ability to sleep, menstruation/gynecological history, and sexual activity. The ITP-specific questions also assessed the subject's response to ITP treatments and any side effects. Transcripts of the focus groups were summarized and reviewed by the study team. The initial draft of the ITP Subject Assessment Questionnaire (ITP-PAQ) was developed after reviewing information from the literature searches, existing questionnaires, expert opinion, focus group transcripts, and the questionnaire responses from the focus group subjects.\nThe initial ITP-PAQ consisted of 50 items that assesses the impact of ITP in the areas of physical health, mental health, work, social activity, reproductive health (relevant for women only), and overall quality of life. Factor analyses were conducted which yielded six unique domains. The impact of ITP on physical health was measured by four scales that evaluated ITP-related symptoms, Fatigue/Sleep, Bother-Physical Health, and Activity. Its impact on mental health was measured by two scales that evaluated psychological distress and fear. A copy of the questionnaire can be obtained by contacting Janet L. Nichol and sample items are included Table 2.\n\nStatistical Analyses\nA validation study was conducted to evaluate the psychometric properties of the newly developed ITP-PAQ questionnaire so that it could be used to measure the impact of ITP in adult subjects in future studies. Standard psychometric methods were used to evaluate the reliability and validity of the questionnaire [19,20].\nEligible subjects completed the baseline questionnaire at the site or by mail after providing telephone consent. An informed consent form and baseline questionnaire was mailed to those subjects who gave their initial consent via telephone. These completed documents were returned by mail to the investigators. At follow up, each subject was mailed the same questionnaire and asked to complete it a second time (for evaluating test-retest reliability) approximately two weeks later. Additionally, subjects completed the SF-36 and the CES-D[21] for validation purposes at both assessments. Demographic and clinical characteristics were also solicited in order to more fully describe the study population. Each study subject received an honorarium for completing the questionnaires.\n\nScale creation and confirmatory factor analysis\nConfirmatory factor analyses were conducted to test the hypothesized structure of the scales. Two models using LISREL version 8 were tested [22]. The first model consisted of all 50 HRQoL items and 10 factors, whereas the second model consisted of a subset of the 50-item correlation matrix. Only women respond to the six items comprising the Reproductive Health scale, so the items were not included in the second LISREL model to avoid estimation biases. The remaining 44 items were analyzed. Model fit was evaluated using the goodness-of-fit (GFI), the normed fit index (NFI), the non-normed fit index (NNFI), the comparative fit index (CIF), and the root mean square error of approximation (RMSEA). For the confirmatory models, index values greater than 0.95 indicate better fit, and RMSEA values less than 0.05 are considered evidence of adequate fit [23].\n\nReliability and stability\nTwo forms of reliability were assessed: test-retest reliability and internal consistency reliability. Test-retest reliability, a measure of the degree to which the questionnaire yields stable scores over a short period of time (assuming there is no underlying change), was measured by the intra-class correlation coefficient (ICC) [24,25]. An ICC of ≥ 0.70 was considered acceptable [26].\nInternal consistency reliability, the extent to which items within each scale correlate with each other to form a multi-item scale, was assessed using Cronbach's alpha [25,27]. Data from both assessments were used to evaluate internal consistency reliability. An alpha coefficient of ≥ 0.70 was considered acceptable, which is the commonly accepted minimal standard for reliability coefficients endorsed by the Scientific Advisory Committee of the Medical Outcomes Trust [26].\n\nConstruct Validity\nConstruct validity was assessed by examining the inter-scale correlations between the ITP-PAQ and the CES-D and the ITP-PAQ with the SF-36 and by examining the strength of the within ITP-PAQ scale correlations [25,28]. For both inter-scale and intra-scale correlations, we made a priori hypotheses about the directionality and magnitude of the correlation and observed the extent to which hypothesized relationships held. For example, we hypothesized that the scales of the ITP-PAQ would be negatively correlated with the CES-D and positively correlated with those of the SF-36. We expected the Pearson correlations to be moderate in size.\n\nKnown Groups\nKnown groups validity evaluates the ability of the measure to discriminate between groups known to be clinically different [28]. We only collected patient-reported information using the questionnaire and did not collect clinical information such as platelet counts. Therefore, the following four criteria were identified as proxies for severity:\n• Currently on treatment\n• Splenectomy status\n• Subjects' self-perception of the effectiveness of current medication\n• Length of time since diagnosis\nIt was hypothesized that subjects not being treated, who did not have a splenectomy, who perceived their medication to be more effective, and who had been diagnosed with ITP for a longer time would be healthier and therefore report higher HRQoL scores. In contrast, subjects on any treatment, who had received a splenectomy, who perceived their medication to be less effective, and who were diagnosed more recently would report worse HRQoL. In addition, subjects were also categorized by gender. Subjects were categorized into two groups for each of the analyses: female vs. male, intact spleen vs. removed spleen, currently on ITP treatment vs. not currently on ITP treatment, subject's perception of the effectiveness of their current ITP medication (extremely/moderately effective vs. not at all effective), and ITP diagnosis less than one year ago vs. ITP diagnosis more than one year ago.\n\n\nResults\nDemographics and clinical characteristics\nTable 1 describes the demographic and clinical characteristics of the 73 subjects included in the validation analyses. The majority were female (77%) and Caucasian (84%). The mean age was 45 years (SD = 15.7), and most of the subjects had been diagnosed with ITP for at least five years (57%). Fifty-two percent of the subjects reported that they were currently taking medications for their ITP. Furthermore, 58% indicated that they had a splenectomy. Among the 42 subjects who had a splenectomy, 55% reported that the removal of their spleen did not cure their ITP. With one exception, the remaining subjects did not provide a response.\n\nConfirmatory factor analysis\nThe first confirmatory factor analysis of the 50-item and ten factors model converged in 28 iterations. However, neither the inter-item correlation matrix nor the inter-factor correlation matrix was positive-definite, which suggests that the proposed model is wrong for the data or the data are inadequate for the model[22]. The chi-square value of the model was 316.64 with 1129 degrees of freedom (p = 1.0), which does not support the hypothesized scale structure of the initial ITP-PAQ. The confirmatory analysis of this LISREL model indicate that computing domain scores for the Physical Health and Mental Health domains is not appropriate for the ITP-PAQ.\nThe second LISREL model was analyzed to confirm the scale structure, excluding the Reproductive Health scale. For this model, 126 parameters were estimated: 46 factor loadings, 44 error terms, and 36 inter-factor correlations. The model converged in 39 iterations, with a chi-square value of 1043.10 with 864 degrees of freedom (p < 0.01). The Goodness of Fit Index (GFI), Normed Fit Index (NFI), Non-normed Fit Index (NNFI), and Comparative Fit Index (CFI) was 0.60, 0.63, 0.91, and 0.92, respectively, and the RMSEA was 0.05 [90% CI, 0.04–0.065]. Furthermore, the inter-factor correlations ranged form 0.33 between the Symptoms and Work scales to 0.96 between the Bother-Physical Health and Overall QoL scales.\nIn addition to the confirmatory factor analyses, Cronbach's alphas and item-to-total correlations were used for item reduction. Items with low factor loadings and item-to-total correlations that reduced the internal consistency were eliminated. Although initial factor analyses identified six domains for future use, the final version of the ITP-PAQ contained 44 items that included the following ten scales: Symptoms, Bother-Physical Health, Fatigue/Sleep, Activity, Fear, Psychological Health, Work, Social Activity, Women's Reproductive Health, and Overall QoL. Table 2 provides information on the number of items, item variability and sample items from each scale of the questionnaire. Each scale is scored from 0 to 100, with higher scores representing better quality of life.\n\nTest-retest reliability\nOf the 73 subjects who completed the first administration of the questionnaire, most of the subjects completed the second questionnaire within a 15-day period (75%), during which subjects were expected to remain clinically stable. However, 20% of the 73 subjects completed the questionnaire within three weeks following the first administration. The remaining 5% of subjects completed it between four and nine weeks after the first \"test.\" ICC's were computed for the entire sample (n = 73) and for a sub-sample of respondents who completed the second questionnaire within three weeks (n = 69). With the exception of the Bother-Physical Health and Activity scales, all scales had acceptable test-retest reliability (ICC ≥ 0.70) as measured by the ICC (Table 3). For the entire sample, ICC values ranged from 0.52–0.90, while ICC values for the sub-sample ranged from 0.56–0.89.\n\nInternal consistency reliability\nInternal consistency reliability, measured by Cronbach's alpha, ranged from 0.66 to 0.92 (Table 3). With the exception of the Reproductive Health scale, Cronbach's alpha coefficients exceeded the acceptable level of 0.70. Cronbach's alpha for the Symptoms, Bother-Physical Health, Fatigue/Sleep, and Activity scales ranged from 0.71–0.89, while Cronbach's alpha for the Psychological Health and Fear scales ranged from 0.87–0.92. Additionally, Cronbach's alphas for the Social Activity, Work, Reproductive Health, and Overall QoL scales were 0.72, 0.86, 0.66, and 0.89, respectively.\n\nConstruct validity\nTable 4 displays the results of inter-scale Pearson correlation coefficients for the initial test administration of the ITP-PAQ. As expected, the Symptoms, Bother-Physical Health, Fatigue/Sleep, and Activity scales were moderately to strongly inter-correlated based on the data from the initial administration (correlation coefficients ranged from 0.56–0.75; p < 0.05). The Overall QoL scale was moderately to strongly correlated with the other ITP-PAQ scales, with the exception of the Reproductive Health scale.\nIn addition to examining the correlations within the ITP-PAQ scales, construct validity was also assessed by comparing the ITP-PAQ scale scores with those of the CES-D and the SF-36. The CES-D was negatively correlated with all ITP-PAQ scales, except for the Reproductive Health scale. Other than the Reproductive Health scale, Pearson correlations ranged from -0.37 to -0.70 (p < 0.05) (data not shown). Most of the ITP-PAQ scales were moderately correlated with the SF-36 scales; however, the Reproductive Health scale was not significantly correlated with any of the SF-36 scales.\nThe mean SF-36 scores of the subjects with ITP were compared to those of the general U.S. population norms [15]. Results from t-tests indicate that there were statistically significant differences (p < 0.05) in SF-36 mean scores between subjects with ITP (range, 43.04–72.86) and the general U.S. population (range, 60.86–84.15). Subjects with ITP reported lower scores on each SF-36 scale compared to the US norm (data not shown).\n\nKnown groups validity\nSubjects were categorized into two groups according to gender, splenectomy status, current ITP treatment status, subject's perception of the effectiveness of ITP treatment, and time elapsed since ITP diagnosis. When subjects were grouped according to gender or splenectomy status, no statistically significant differences were observed for any of the ITP-PAQ scales (data not shown). Subjects who were currently receiving treatment for ITP reported lower scores on all ITP-PAQ scales compared to subjects who were not currently receiving treatment. Statistically significant differences (p < 0.01) were reported for the following ITP-PAQ scales when subjects were categorized by treatment status: Symptoms, Fatigue/Sleep, Bother-Physical Health, Activity, Psychological Health, and Overall QoL (Figure 1). When subjects were categorized by effectiveness of ITP treatment, statistically significant differences (p < 0.05) were observed for the Symptoms and Activity scales (Figure 1), while statistically significant differences were only found for the Psychological Health scale when subjects were categorized according to time elapsed since ITP diagnosis (data not shown). Subjects who had been diagnosed with ITP for < 1 year had a lower mean score on the Psychological Health scale compared to subjects who had been diagnosed with ITP for at least one year (50.38 vs. 66.46, respectively; p = 0.02) (data not shown).\n\n\nDiscussion\nThe goal of this study was to develop and undertake initial validation analyses of the ITP-PAQ as a tool for measuring HRQoL specifically related to adult subjects with ITP. The results of this study provide preliminary evidence of the reliability and validity of the ITP-PAQ in this population.\nThe results indicate that, with the exception of the Reproductive Health scale, the questionnaire has good internal consistency. The Reproductive Health scale may not have reached an acceptable level because the items could in fact be measuring slightly different concepts. For example, the Reproductive Health scale includes items that assess symptom bother related to menstruation in addition to items that ask how ITP impacts reproductive choices, such as becoming pregnant, giving birth, and adopting children. Perhaps, the symptom bother items in this scale may fit more appropriately with the Bother-Physical Health scale, and the reproductive choice items could comprise a separate scale.\nMost of the ITP-PAQ scales also demonstrated acceptable test-retest reliability, even though the time interval between test and retest administrations of the questionnaire exceeded the targeted time interval of seven to ten days. However, two scales, the Bother-Physical Health and Activity scales, reported ICC values below the acceptable value of 0.70. In addition to the lag between the two administrations of the questionnaire, subjects may have experienced an increase in bother and/or a decrease in activity due to ITP during the extended time interval. Additionally, the comparatively low ICC values of the Bother-Physical Health and Activity scales may be due in part to the relatively low number of items contained in each of these scales (four and two items, respectively) compared to the Symptoms scale which contains six items.\nIn general, the construct validity of the questionnaire was supported by inter-scale correlations. As expected, the Bother-Physical Health, Symptoms, Fatigue/Sleep, and Activity scales were more strongly correlated to one another than with other scales. However, the Reproductive Health scale had a lower internal consistency reliability and it was weakly correlated with the ITP-PAQ scales, the SF-36, and the CES-D, possibly due to the differing concepts measured by the items within this scale or the all-female sample.\nMost of the ITP-PAQ scales were moderately correlated with the SF-36 scales and the CES-D; however, correlations between some of the scales were < 0.40 (e.g., Fear and SF-36 Mental Health, 0.30; p < 0.05). This low correlation could be due to the ITP-PAQ assessing fear associated with ITP (e.g., fear of having a bleeding episode), while the SF-36 provides a more general assessment of mental health issues (e.g., felt downhearted and blue).\nThe known-groups validity results indicate that some of the ITP-PAQ scales (Symptoms, Fatigue/Sleep, Bother-Physical Health, Activity, Psychological Health, and Overall QoL scales) were able to differentiate ITP subjects who were currently receiving ITP treatment from those who were not receiving treatment for ITP, providing preliminary evidence of the ITP-PAQ's ability to distinguish between groups known to be different. However, the ITP-PAQ scales were generally unable to distinguish between subjects when they were grouped by gender, splenectomy status, perceived effectiveness of treatment and length of time since ITP diagnosis. The ITP-PAQ may not be able to differentiate between female and male subjects because the disorder may affect females and males similarly. Additionally, significant differences may not have been observed between subjects who have undergone splenectomy and subjects who have not because 55% of subjects who had a splenectomy indicated that it did not cure their ITP. Specifically, the known-groups could be defined as 'subjects without a splenectomy' versus 'subjects with a failed splenectomy' (for whom QoL likely worsened) versus 'subjects with a successful splenectomy' (for whom QoL may have improved). In the future, to assess whether the ITP-PAQ scales can differentiate between groups of subjects, it may be worthwhile to categorize subjects by a more clinically relevant measure, such as platelet count.\nSeveral limitations should be considered when interpreting our findings. Subjects were drawn from a convenience sample. The study population was fairly homogeneous, comprised primarily of Caucasian female subjects. The data was validated using only patient-reported data collected via questionnaire. The lack of clinical data in this initial validation study will be addressed in on-going pivotal trials that will collect clinical data such as platelet counts and platelet response. In addition, the time interval between the initial and retest administration of the questionnaire may have been too lengthy to properly evaluate the test-retest reliability. Because 25% of subjects did not complete the questionnaire within the targeted fifteen day interval, those subjects may have undergone clinical changes that may have affected their responses. In future validation studies platelet counts or type of platelet response should be used to identify a stable cohort for the test-retest analyses. Furthermore, the criteria used to categorize the subjects for the known groups validity evaluation may not have been sufficient to allow for the ITP-PAQ scales to detect differences between groups. Grouping the subjects by a different criterion, such as a relevant clinical measure, may bolster the findings for its known groups validity.\n\nConclusion\nThe primary goal of this manuscript was to describe the development of a new ITP-specific HRQoL questionnaire for adults with ITP and to present our initial findings on the psychometric properties of this questionnaire. The results of this initial validation study indicate that the questionnaire generally has acceptable reliability and validity. We plan to conduct additional analyses using more objective clinical measures such as platelet counts as a criterion for known groups validity. Further validation work should also be conducted to assess its responsiveness and to estimate its minimal clinical important difference value so that it can become a more widely used HRQoL measure in the ITP population.\n\nCompeting interests\nThe validation study design, analysis, interpretation of results, and the writing of the manuscript represent the joint collaboration of all authors of this study, which was funded solely by Amgen, Inc, Thousand Oaks, California, USA. Ovation Research Group provided no additional funding for this study. The decision to submit this manuscript for publication was subject to the approval of Amgen, Inc. and all authors.\nGary Okano and Janet Nichol are employees of Amgen, Inc. James Bussel is an employee of Weill Cornell Medical Center. James George is employed by the University of Oklahoma Health Sciences Center. Robert McMillan is a Professor Emeritus of the Scripps Research Institute. Susan Mathias is an employee of Ovation Research Group.\n\nAuthors' contributions\nSDM supervised the interpretation of the results from the validation study, and drafted the manuscript. JBB, JNG, RM, and JLN provided clinical expertise in the development of the questionnaire, and participated in the design and execution of the study. GJO assisted in interpreting the results and drafting the manuscript. All authors read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 29578 ] ] } ]

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[ { "id": "pmcA328326__text", "type": "Article", "text": [ "Enzymatic multiplex DNA sequencing.\nAbstract\nThe problem of reading DNA sequence films has been reformulated using an easily implemented, multiplex version of enzymatic DNA sequencing. By utilizing a uniquely tagged primer for each base-specific sequencing reaction, the four reactions can be pooled and electrophoresed in a single lane. This approach has been previously proposed for use with fluorescently labelled probes (1), and is analogous to the principle used in four-dye fluorescence sequencing except that the signals are resolved following electrophoresis (2). After transfer to a nylon membrane, images are obtained separately for each of the four reactions by hybridization using oligonucleotide probes. The images can then be superimposed to reconstitute a complete sequence pattern. In this way the correction of gel distortion effects and accurate band registration are considerably simplified, as each of the four base-specific ladders require very similar corrections. The methods therefore provide the basis for a second generation of more accurate and reliable film reading programs, as well as being useful for conventional multiplex sequencing. Unlike the original multiplex protocol (3), the approach described is suitable for small projects, as multiple cloning vectors are not used. Although more than one vector can be utilized, only a library of fragments cloned into any single phage, phagemid or plasmid vector is actually required, together with a set of tagged oligonucleotide primers.Images\n\n\n\n\n\n\n Nucleic Acids Research, Vol. 19, No. 12 3301 \n\n Enzymatic multiplex DNA sequencing \n\n Mark Chee \n\n Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 20H, UK \n\n Received March 15, 1991; Revised and Accepted May 2, 1991 \n\n ABSTRACT \n\n The problem of reading DNA sequence films has been reformulated using an easily implemented, multiplex version of enzymatic DNA sequencing. By utilizing a uniquely tagged primer for each base-specific sequencing reaction, the four reactions can be pooled and electrophoresed in a single lane. This approach has been previously proposed for use with fluorescently labelled probes (1), and is analogous to the principle used in four-dye fluorescence sequencing except that the signals are resolved following electrophoresis (2). After transfer to a nylon membrane, images are obtained separately for each of the four reactions by hybridization using oligonucleotide probes. The images can then be superimposed to reconstitute a complete sequence pattern. In this way the correction of gel distortion effects and accurate band registration are considerably simplified, as each of the four basespecific ladders require very similar corrections. The methods therefore provide the basis for a second generation of more accurate and reliable film reading programs, as well as being useful for conventional multiplex sequencing. Unlike the original multiplex protocol (3), the approach described is suitable for small projects, as multiple cloning vectors are not used. Although more than one vector can be utilized, only a library of fragments cloned into any single phage. phagemid or plasmid vector is actually required, together with a set of tagged oligonucleotide primers. \n\n INTRODUCTION \n\n The community of biologists is undertaking the sequencing of representative genomes of various free-living organisms, ranging in size from Mycoplasma (800kb) to mammals (3 Gb) (4). However, the largest contiguous DNA sequences which have been determined so far are the genomes of several dsDNA eukaryotic viruses (5, 6, 7, 8, 9) and plant chloroplasts (10, 11, 12). The largest of these is the 229kb genome of human cytomegalovirus (8). The difficulty in sequencing millions of base pairs of DNA is that several steps in the methods are relatively labour intensive, although the sequencing reactions themselves are rapid and easily performed. Two limiting steps in conventional procedures are the size fractionation of sequencing reaction products by gel electrophoresis and the subsequent reading of sequence ladders. The former problem can be overcome by multiplexing, which theoretically allows an enormous amount of \n\n data to be obtained from a single gel by processing clones as mixtures rather than individually (3). Each sequence in the mixture is labelled by a unique short oligonucleotide 'tag' sequence. This allows the mixture to be resolved following electrophoresis: the superimposed sequence ladders are blotted from the gel to a nylon membrane, and detected one at a time by hybridization using tag-specific oligonucleotide probes. In practice, at least 50 sets of sequences can be obtained from a single gel (3). \n\n Unfortunately, a bottleneck in the multiplex procedure is the reading of sequence films. In previous large-scale sequencing projects this task has been performed with the aid of a sonic digitizer (13, 14). Although film reading programs have been under development for some time (15), and some programs are commercially available, their error rates are presently more variable and unpredictable than that of a skilled person and the accurate interpretation of film-imaged sequence ladders by computer programs is difficult to achieve in routine practice. Programs specifically designed to read multiplex films have an advantage. This is because a sequence image can be used as an 'internal standard' to help interpret other images derived from the same membrane (3). However, the original implementation of the multiplex strategy used chemical sequencing (16), which yields a more complex sequence ladder than the enzymatic dideoxynucleotide chain-termination method (17). Most successful large scale sequencing projects have used the chaintermination method and bacteriophage M13 vectors, which allows the routine production of clean and easily interpretable sequences (18). It was therefore decided to adapt the original multiplex protocol for use with enzymatic sequencing, using tagged primers. \n\n MATERIALS AND METHODS \n\n Eight oligonucleotide sequencing primers were synthesized, each 37 nucleotides in length. The 3' end of each primer consists of the 17 nucleotide M13 universal priming sequence [GTAAAACGACGGCCAGT3']. The 5' ends of the primers bear different 20mer tag sequences (Figure 1). In four of the primers, UEO1C, UPOIC, UE02C and UP02C, these tags are complementary to the EO1, PO1, E02 and P02 probe sequences respectively (copied from the original 'plex' vectors (3)). A second set of four primers, UJOL14C, UJOL15C, UJOL16C and UJOL17C, have the following tag sequences: 5' CAAGTTTGAAGGTACTCATT, TATCAATTAAATTGTllTGAC, GTGTTGCTACCCAAGAAGCA, and TGTCACTAGAGCTGTCACTT, respectively. The \n\n ?=) 1991 Oxford University Press \n\n 3302 Nucleic Acids Research, Vol. 19, No. 12 \n\n oligonucleotides were gel-purified (19) and used to sequence ssDNA templates prepared by phenol extraction (20) or SDS denaturation (21). Conventional sequencing reactions were performed as previously described (20). \n\n For hybridization experiments, radioactively labelled nucleotides were omitted from the sequencing reactions. Instead, the 21d of each nucleotide mix added to the reaction mixture consisted of the following: 'A' mix: 6.25MtM dATP, 62.5lM ddATP; 'C' mix: 6.25MM dCTP, 40MtM ddCTP; 'G' mix: 6.25MtM dGTP, 80MtM ddGTP; 'T' mix: 6.25MM dTTP, 250yM ddTTP; as well as 125MM of each of the three other dNTPs in each mix. Apart from the use of these modified mixes, no changes were made to the conventional sequencing procedure (20). \n\n Sequencing reactions were pooled and ethanol precipitated as appropriate. Precipitation in microtitre trays was carried out as follows: a mixture of 3.2M1 3M NaAc pH 5.0 and 112Mi1 EtOH was dispensed to individual wells of a microtitre plate (Falcon 3911 or Corning 25855) using an 8-channel pipettor. Each set of four reactions was added to the EtOH/NaAc mixture, and the tray sealed using a Falcon 3073 plate sealer. The samples were mixed by inversion and stored at -20?C for 30 minutes. The DNA was collected by a 20 minute centrifugation at 4 000 rpm in an IEC Centra 3C centrifuge. The sealer was removed, and the plate inverted to discard the supernatant. After blotting the tray on tissue paper, 200MI of 95 % EtOH was added to each well. The plate was covered with a plastic lid and recentrifuged for 2 minutes. The EtOH was discarded and the plate inverted for several minutes on tissue paper, then left for 20 minutes to air dry. Precipitated samples were resuspended in 6M1 deionized water by vortexing on an SMI multi-tube vortexer for 1 minute. Samples were denatured and electrophoresed on 6 % polyacrylamide buffer gradient gels as previously described (20). \n\n Following electrophoresis, the gel was transferred to a dry piece of Whatman 3MM blotting paper, and placed on a second sheet of blotting paper supported on a glass plate and saturated in 4 x SSC (SSC: 150mM NaCl, l5mM trisodium citrate). This sheet was wicked in a tray containing 1 litre of 4 x SSC. The DNA was transferred to a nylon membrane (Amersham Hybond N) by capillary blotting overnight (22). DNA was fixed to the membranes by U.V. crosslinking (23). \n\n Plex oligonucleotide probes were a kind gift of Dr.George Church. Probes were tailed at their 3' ends using [a-32p] dCTP as previously described (3). For the preparation of digoxigenin (DIG) labelled probes, identical tailing reactions were carried out substituting I0pmols of DIG-II dUTP (Boehringer Mannheim) for [a-32P] dCTP. Membranes were prehybridized for at least 10 minutes in 4 x SSC, 5 x Denhardts' (0.1 % (w/v) each of BSA (heated at 80?C for 30 minutes to inactivate any alkaline phosphatase activity), Ficoll (Pharmacia) and polyvinylpyrrolidone), 0.5% (w/v) SDS, 5mM NaHPO4 (23). Hybridization was carried out in 25-50M1 of prehybridization buffer per cm2 of membrane. The probe concentration was approximately lnM. After lh at 42?C, unbound probe was removed by five 1 minute washes at room temperature in 1 x SSC, 0.5% SDS (200MI/cm2 membrane). Radioactive blots were covered in Saran wrap and exposed to film immediately. Detection of DIG labelled probes used an anti-DIG antibodyalkaline phosphatase conjugate (Boehringer Mannheim) according to the manufacturer's instructions, except that all volumes were reduced by 70% and the conjugate was used at a 1:10 000 dilution. Blots were developed in 25M1 of 100mM Tris.Cl pH9.5, \n\n mantane4-methoxy4(3 \"-phosphoryloxy)phenyl-1 ,2-dioxetane); Tropix)/cm2 for 30 minutes at 37?C, prior to exposure to film. Probes and dioxetane were stripped from the membranes by two 10 minute washes at 700C with 0.2% SDS, 2mM EDTA (200,ul/cm2 membrane). \n\n The hybridization and washing procedures were carried out in plastic bags. However, washing steps have also been performed with gentle agitation in a perspex tub (43 x 27 x 15cm) mounted on a reciprocal shaker, with equivalent results. In the latter case a minimum wash volume of 500mls was used. The use of a tub is more convenient for batch processing and should be straightforward to automate. \n\n RESULTS \n\n Autoradiograms revealed no difference in sequence quality when tagged primers were used instead of the 17mer universal primer in conventional [a-35S] dATP labelled sequencing reactions and in multiplex hybridization experiments using [a-32P] dCTP-tailed probes (results not shown). Experiments were then conducted to determine the feasibility of pooling the four base reactions for each clone and fractionating them in a single lane to obtain a superimposed but interpretable set of sequence ladders. The question addressed was whether or not difficulties in band registration might arise as a result of mobility differences between the different primer sequences and/or distortion of the membrane between probings. It is relevant that an automated film reader employing an internal standard requires that the nylon membrane does not undergo significant distortions between probings (George Church, personal communication). Clones were sequenced using the four tagged primers UEOlC, UPOIC, UE02C, and UP02C, one for each base reaction (Figure 1). The A, C, G and T reactions for each clone were pooled, and processed as described above. A complete set of sequence autoradiograms was obtained from four consecutive rounds of probing with [a-32P] dCTP-labelled oligonucleotides. Alignment of the films showed that sequence-specific mobility effects and distortion of the membrane between probings were sufficiently minor to allow accurate registration of the bands, and hence accurate reading of the sequence. At least 200 nucleotides of sequence could be read accurately from a single clone by simply tracing the four sets of bands using different colours, overlaying the tracings, and reading the bands sequentially. In order to assess the practicality of reading the sequences by machine, the images were scanned to provide optical density profiles (Figure 2). These profiles were overlaid, and were found to be sufficiently in register to allow accurate interpretation of the sequence for at least 300 nucleotides. This was essentially the limit of resolution of the gel for accurate manual reading. \n\n In order to ensure that the relatively minor mobility differences observed between the four primers were not coincidental to the oligonucleotides used, a second set of four tagged M13 universal primers was synthesised, this time incorporating 20mer sequences derived from the genome of murine herpesvirus-68 (UJOL14C, 15C, 16C, 17C). Sequencing reactions were performed using [cx-35S] dATP to label the DNA directly. Various templates were sequenced, and in all cases correctly ordered sequence ladders were obtained following conventional electrophoresis in which the four reactions were run side-by-side (results not shown). \n\n Initial hybridization experiments were conducted using [f -32p] \n\n dCTP tailed oligonucleotide probes. However, the use of \n\n lOOmM NaCl, 5OmM MgC12, 0.15mM AMPPD ([3-(2'-ada\n\n B \n\n C \n\n 'Ordinary' Plex' 4-CJ3 4-rn + \n\n Primeir Primer \n\n \"All \"C\" \"G\" \" T\" \n\n 'Plex' 'Ordinary' Pool and \n\n Vector Vector fractionate \n\n Resolve by sequential \n\n hybridizations \n\n Sequencing reactions \n\n SequencingreactIo product \n\n Sequencing reaction product \n\n - n; \n\n = = \n\n - = \n\n - n: = \n\n Figure 1. Approaches to enzymatic multiplex DNA sequencing. a) A set of sequence-tagged vectors can be used. The tag site is shown in red, and the insert to be sequenced in blue. However, the original plex vectors (3) are plasmids, and therefore amenable only to dsDNA sequencing. Sets of bacteriophage M13 vectors have been constructed bearing either one (32) or two [Chee, unpublished] of the plex tag sites flanking the polylinker, which can be used for this approach. b) The strategy used in this paper. In this case the tag site is carried on the primer. c) If tagged primers are used, there is no practical impediment to performing each base reaction using a different primer, as depicted. The reactions can then be pooled in any combination desired. The configuration shown, in which the four reactions are electrophoresed in a single lane, is designed to facilitate accurate band registration and reading by an automatic film reader. In order to read the sequence manually, base reactions would be run side-by-side. The logistics of processing the reactions are essentially the same with either configuration; the same number of probings are required. \n\n Figure 2. Four overlaid one-dimensional optical density profiles for a single clone shown in two overlapping sections. The optical density profiles are unprocessed, except for a simple transform to correct for the relative displacement (translation and rotation) of the four images from which they are extracted. The profiles read 5' to 3' from right to left. Nucleotides positions 66 to 214 from the start of the universal priming site are shown. The sequence is that of Bluescribe M13+ (template DNA obtained by rescue with M13K07 helper phage (30)), and was determined using the primers UEOIC, UPOIC, UE02C, and UP02C for the T, C, G and A \n\n specific reactions respectively. Detection was by autoradiography following hybridization with [a- 32p] dCTP tailed plex probes. \n\n A \n\n Nucleic Acids Research, Vol. 19, No. 12 3303 \n\n I \n\n 3304 Nucleic Acids Research, Vol. 19, No. 12 \n\n a) \n\n 175-... \n\n -w\n\n .m; \n\n b) \n\n 182\n\n a :...... \n\n F.VW \n\n an \n\n - w \n\n -1 \n\n S - K~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~i \n\n 'F C C; A I1 'CG A \n\n Figure 3. Four separate base-specific reactions imaged from a single lane using chemiluminescent detection. The clones sequenced are: a) Bluescribe M13 + (obtained by rescue with M13K07 helper phage (30)) and b) an M13 recombinant clone prepared in a microtitre tray (21) (a kind gift of Victoria Smith). Nucleotide positions shown on the figure are numbered from the start of the universal priming site. The clones were sequenced using UEOlC, UPOIC, UE02C, and UP02C for the T, C, G and A specific reactions respectively. The blot was probed with corresponding DIG-11-dUTP labelled oligonucleotides. \n\n radioactivity on the scale envisioned for a large sequencing project is undesirable for reasons of safety. The relatively long exposure times required (6 to 24 hours) and the short half lives of the probes might also be inconvenient. It has been shown that a biotin/streptavidin/alkaline phosphatase based detection system used in conjunction with a chemiluminescent dioxetane substrate overcomes these disadvantages (24, 25, 26). We utilized a different bridging system with similar results. Digoxigenin (DIG) labelled oligonucleotide probes were detected using anti-DIG antibody-alkaline phosphatase conjugates and a chemiluminescent dioxetane substrate. Exposure times of 10 to 15 minutes were typically required, following a one hour preincubation period (Figure 3). In our hands the DIG bridging system was similar in sensitivity to the streptavidin based system (24), and the practical lower limit of template ssDNA required in order to obtain an easily interpretable sequencing ladder was estimated to be in the range of 20 to 50fmols per reaction. However, the sensitivity of detection was limited only by enzymaticallytriggered background luminescence, and not by the level of signal obtained. The nonradioactive methods described have been used successfully in an 8-plex system. \n\n DISCUSSION \n\n Although the original multiplex protocol was based on a set of tagged vectors (3), tagged primers have also been used or proposed for various forms of multiplex DNA sequencing (George Church, personal communication; 2, 27). For example, a proposal was recently put forward for multiplex sequencing using sequence-labelled primers and fluorophor-labelled probes \n\n (1), similar in principle to the methods used here. However, we use tagged primers and the superposition of the four sequencing reactions to address the problem of reading DNA sequence films; a part of this solution is to utilize M13 dideoxynucleotide sequencing, thereby improving the quality of the data to be analyzed. In addition, the proposal for fluorophor-labelled probes does not take into consideration any of the practical sequencing problems addressed here, and, in the version described, remains a promising but unproven scheme for large scale DNA sequencing. \n\n There are several advantages to tagging primers instead of vectors. Firstly, there is no need to prepare multiple libraries of clones in special vectors. This means that workers can use vector/host combinations that yield good results in their hands, and an increased depth of multiplexing can easily be accomodated by synthesizing more primers. This should make multiplexing more accessible to workers undertaking smaller projects. A theoretical disadvantage of tagged primers is that the procedure can only be multiplexed following primer annealing (1), or following the sequencing reactions (this paper; in practice, pooling immediately after the annealing step might lead to increased backgrounds if one or more primers were present in excess over their template DNAs). This is a relatively late stage. In the original procedure (3), clones were pooled prior to amplification by growth, an early step. However, we do not believe the sacrifice to be of practical importance when using phage vectors. In our experience, recombinant M13 phage have variable growth rates and the effects of competition are likely to severely limit the number of clones that can usefully be pooled for growth. In contrast, by growing clones individually, the depth of multiplexing is only really limited by probe sensitivity. We have not investigated the factors influencing variability in phage growth rates. \n\n It is worth noting that reliable protocols have been developed for growing large numbers of individual M13 clones and preparing high quality ssDNA templates in microtitre trays (28, 21). It is relatively simple to prepare manually two microtitre trays of ssDNA templates (192 clones) in a day. Sufficient clones can be prepared in a week to sequence a 20kb fragment to a redundancy of 10 (Victoria Smith, personal communication). In this laboratory, ssDNA is now prepared with the aid of a commercially available robotic workstation (21). As sequencing reactions are also carried out in microtitre trays, manually or robotically (20, 29), the entire M13-based dideoxynucleotide sequencing procedure is amenable to automation (29). For these reasons we see little practical advantage in pooling clones early. Finally, by not pooling clones early, the ability to easily retrieve individual clones is retained, which may facilitate directed sequencing later in a project should this become necessary. \n\n Multiplex DNA sequencing is currently limited by the lack of a robust computer program which can correct for the large variety of gel and sequencing artefacts that are normally encountered. The foundation of a film reading program is the ability to bring into register precisely vertical arrays of base-specific bands. This requires the ability to track lanes, correct for distortions, and order bands based on their relative spacing. A method of sequencing which has successfully overcome the problem of sequence reading uses real-time detection of fluorescently labelled DNA samples migrating through the gel (2). This system also utilizes the principle of running the four base reactions down the same lane (2). However, bands are detected at a fixed location \n\n in space, and their detection is separated in time. Hence the \n\n Nucleic Acids Research, Vol. 19, No. 12 3305 \n\n problem of gel distortion is essentially avoided, although corrections for the different mobilities of the four dyes must be carried out. In contrast, we utilize the advantages of single lane electrophoresis to address the problem of superimposing four relatively large and complex two-dimensional images. Furthermore, by using sequence-tagged oligonucleotides which are detected by hybridization, a much greater depth of multiplexing can realistically be achieved than by real-time detection. \n\n The use of two-dimensional colour traces to depict the processed output of a film reader is consistent with the method of displaying fluorescence traces, and should facilitate the checking and editing of sequence databases in which both kinds of data have been entered. The sequence compilation programs used in this laboratory, which are already capable of handling large shotgun databases (8, 31), have recently undergone extensive improvements (Rodger Staden, personal communication). There is now an interactive database editor which allows the graphical display of fluorescence traces, and it is envisaged that this feature could be extended to allow the handling of data from a film reader when a suitable machine is developed. \n\n ACKNOWLEDGEMENTS \n\n I am particularly grateful to George Church for thought-provoking discussions and gifts of vectors and oligonucleotide probes and to John Sulston for advice. I also thank Victoria Smith for the gift of DNA samples, Bart Barrell for long-term support, Tom O'Keefe of Milligen/Biosearch for lessons in multiplexing and Amersham International for the optical density overlays shown in Figure 2. M.C. is supported by a fellowship from Applied Biosystems. \n\n 12. Hiratsuka, J., Shimada, H., Whittier, R., Ishibashi, T., Sakamoto, M., Mori, \n\n M., Kondo, C., Honju, Y., Sun, C. -R, Meng, B. -Y, Li, Y. -Q, Kanno, A., Nisizawa, Y., Hirai, A., Shinozaki, K. and Sugiura, M. (1989) Molecular and General Genetics, 217, 185-194. \n\n 13. Komaromy, M. and Govan, H. (1984) Nucleic Acids Research, 12, 675-678. 14. Staden, R. (1984) Nucleic Acids Research, 12, 499-503. \n\n 15. Elder, J. K., Green, D. K. and Southern, E. M. (1986) Nucleic Acids \n\n Research, 14, 417-424. \n\n 16. Maxam, A. M. and Gilbert, W. (1977) Proceedings of the National Academy \n\n of Sciences, U.S.A., 74, 560-564. \n\n 17. Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Proceedings of the National \n\n Academy of Sciences, U.S.A., 74, 5463-5467. \n\n 18. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H. and Roe, B. \n\n A. (1980) Journal of Molecular Biology, 143, 161-178. 19. Applied Biosystems User Bulletin (1987) 13, 11-16. \n\n 20. Bankier, A. T., Weston, K. M. and Barrell, B. G. (1987) Methods in \n\n Enzymology, 155, 51-93. \n\n 21. Smith, V., Brown, C. M., Bankier, A. T. and Barrell, B. G. (1990) DNA \n\n Sequence, 1, 73-78. \n\n 22. Southern, E. M. (1975) Journal of Molecular Biology, 98, 503-517. \n\n 23. Church, G. M. and Gilbert, W. (1984) Proceedings of the National Academy \n\n of Sciences, U.S.A., 81, 1991-1995. \n\n 24. Beck, S., O'Keefe, T., Coull, J. M. and Koster, H. (1989) Nucleic Acids \n\n Research, 17, 5115-5123. \n\n 25. Tizard, R., Cate, R. L., Ramachandran, K. L., Wysk, M., Voyta, J. C., \n\n Murphy, 0. J. and Bronstein, I. (1990) Proceedings of the National Academy of Sciences, U.S.A., 87, 4514-4518. \n\n 26. Beck, S. and Koster, H. (1990) Analytical Chemistry, 62, 2558-2570. \n\n 27. Jacobson, K. B., Arlinghaus, H. F., Schmitt, H. W., Sachleben, R. A., \n\n Brown, G. M., Thonnard, N., Sloop, F. V., Foote, R. S., Larimer, F. W., Woychik, R. P., England, M. W., Burchett, K. L. and Jacobson, D. A. (1991) Genomics, 9, 51-59. \n\n 28. Eperon, I. C. (1986) Analytical Biochemistry, 56, 406-412. \n\n 29. Bankier, A. T. and Barrell, B. G. (1989) In Howe, C. J. and Ward, E. \n\n S. (ed), Nucleic acids sequencing: a practical approach. IRL Press, Oxford, Vol. 1, pp. 37-78. \n\n 30. Vieira, J. and Messing, J. (1987) Methods in Enzymology, 153, 3-11. 31. Davison, A. DNA Sequence, in press. \n\n 32. Heller, C., Radley, E., Khurshid, F. A. and Beck, S. Gene, in press. \n\n REFERENCES \n\n 1. Yang, M. M. and Youvan, D. C. (1989) Biotechnology, 7, 576-580. \n\n 2. Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connell, \n\n C. R., Heiner, C., Kent, S. B. H. and Hood, L. E. (1986) Nature, 321, 674-679. \n\n 3. Church, G. M. and Kieffer-Higgins, S. (1988) Science, 240, 185-188. 4. Watson, J. D. (1990) Science, 248, 44-49. \n\n 5. Baer, R., Bankier, A. T., Biggin, M. D., Deininger, P. L., Farrell, P. J., \n\n Gibson, T. J., Hatfull, G., Hudson, G. S., Satchwell, S. C., Seguin, C., Tuffnell, P. S. and Barrell, B. G. (1984) Nature, 310, 207-211. \n\n 6. Davison, A. J. and Scott, J. E. (1986) Journal of General Virology, 67, \n\n 1759-1816. \n\n 7. McGeoch, D. J., Dalrymple, M. A., Davison, A. J., Dolan, A., Frame, \n\n M. C., McNab, D., Perry, L. J., Scott, J. E. and Taylor, P. (1988) Journal of General Virology, 69, 1531-1574. \n\n 8. Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, \n\n R., Horsnell, T., Hutchison III, C. A., Kouzarides, T., Martignetti, J. A., Satchwell, S. C., Tomlinson, P., Weston, K. M. and Barrell, B. G. (1990) Current Topics in Microbiology and Immunology, 154, 125-169. \n\n 9. Goebel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., Winslow, J. \n\n P. and Paoletti, E. (1990) Virology, 179, 247-266. \n\n 10. Ohyama, K., Fukuzawa, H., Kohchi, T., Shirai, H., Sano, T., Sano, S., \n\n Umesono, K., Shiki, Y., Takeuchi, M., Chang, Z., Aota, S. -I, Inokuchi, H. and Ozeki, H. (1986) Nature, 322, 572-574. \n\n 11. Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., \n\n Matsubayashi, T., Zaita, N., Chunwongse, J., Obokata, J., YamaguchiShinozaki, K., Ohto, C., Torazawa, K., Meng, B. Y., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J., Takaiwa, F., Kato, A., Tohdoh, N., Shimada, H. and Sugiura, M. (1986) EMBO Journal, 5, 2043-2049. " ], "offsets": [ [ 0, 28881 ] ] } ]

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[ { "id": "pmcA1352346__text", "type": "Article", "text": [ "Plasma D-dimer concentration in patients with systemic sclerosis\nAbstract\nBackground\nSystemic sclerosis (SSc) is an autoimmune disorder of the connective tissue characterized by widespread vascular lesions and fibrosis. Little is known so far on the activation of the hemostatic and fibrinolytic systems in SSc, and most preliminary evidences are discordant.\n\nMethods\nTo verify whether SSc patients might display a prothrombotic condition, plasma D-dimer was assessed in 28 consecutive SSc patients and in 33 control subjects, matched for age, sex and environmental habit.\n\nResults and discussion\nWhen compared to healthy controls, geometric mean and 95% confidence interval (IC95%) of plasma D-dimer were significantly increased in SSc patients (362 ng/mL, IC 95%: 361–363 ng/mL vs 229 ng/mL, IC95%: 228–231 ng/mL, p = 0.005). After stratifying SSc patients according to disease subset, no significant differences were observed between those with limited cutaneous pattern and controls, whereas patients with diffuse cutaneous pattern displayed substantially increased values. No correlation was found between plasma D-dimer concentration and age, sex, autoantibody pattern, serum creatinine, erythrosedimentation rate, nailfold videocapillaroscopic pattern and pulmonary involvement.\n\nConclusion\nWe demonstrated that SSc patients with diffuse subset are characterized by increased plasma D-dimer values, reflecting a potential activation of both the hemostatic and fibrinolytic cascades, which might finally predispose these patients to thrombotic complications.\n\n\n\nBackground\nSystemic sclerosis (SSc) is an autoimmune disorder of the connective tissue characterized by widespread vascular lesions and fibrosis. In SSc, vasospasm causes frequent episodes of reperfusion injury and free radical-mediated endothelial dysfunction, which might finally influence the onset of local thrombotic complications. The characteristic vascular involvement affects primarily small arteries and capillaries, causing reduced blood flow and tissue ischemia and supporting the typical clinical manifestations of this unique autoimmune disorder [1]. However, mechanisms involved in the endothelial injury are as yet elusive and most biochemical evidences are often inconclusive or controversial. Some earlier investigations suggested that SSc patients might be characterized by a procoagulant state, reporting depressed basal and stimulated fibrinolytic activity, while others studies have reported normal plasma fibrinolytic activity and normal skin and plasma tissue plasminogen activator (tPA) levels [2-4]. It has been also reported that the lack of a consistent and homogenous increase of some fibrinolytic markers, in the presence of normal levels of antithrombin, might indirectly highlight an impairment of the heparan sulphate-antithrombin system, which would finally promote thrombin generation [3]. Conversely, Cerinic and colleagues provided evidence that fibrinolysis might be impaired in SSc, as shown by reduced D-dimer and decreased levels of plasminogen activator inhibitor [4]. In synthesis, there are no conclusive evidences on the activity of the hemostatic and fibrinolytic pathways in SSc so far.\nD-dimer, a breakdown product of cross-linked fibrin, was proven useful for the diagnostic evaluation of several thrombotic disorders. Moreover, an increased D-dimer value in plasma is a reliable marker of a systemic prothrombotic state, likely superior to alternative fibrinolytic markers, and its measurement might be helpful in predicting or preventing thrombotic events in the single patient [5]. Therefore, to investigate whether SSc patients might be characterized by a potential prothrombotic condition, plasma D-dimer vales were measured in a subset of SSc patients, compared with those of a healthy matched control population and further associated with SSc disease subset.\n\nMethods\nPlasma D-dimer was measured in 28 consecutive SSc patients (2 males and 26 females; mean age 50 ± 15 years, 17 with limited and 11 with diffuse disease patterns), who fulfilled the American Rheumatism Association's criteria for the diagnosis of SSc [6] and in 33 control subjects, matched for age (48 ± 13 years), sex (3 males, 30 females) and environmental habit, recruited among healthy hospital personnel. Samples were collected in the morning; all subjects were in a fasted state. The research was carried out according to the principles of the Declaration of Helsinki and an informed consent for testing was received from all individuals recruited to the study. Blood was collected after an overnight fast into siliconized vacuum tubes, containing 0.105 mol/l sodium citrate (Becton-Dickinson, Oxford, UK). Samples were gently mixed and centrifuged for 10 min at 15°C at 1500 × g; plasma was separated and stored in aliquots at -70°C until measurement. Plasma D-dimer was measured employing Vidas DD, a rapid and quantitative automated enzyme linked immunosorbent assay with fluorescent detection, on the Mini Vidas immunoanalyzer (bioMerieux, Marcy l'Etoile, France). Analytical imprecision, expressed in terms of mean inter-assay coefficient of variation (CV), was quoted by the manufacturer as being lower than 5%. Significance of differences between samples was assessed, following logarithmic conversion of data, by parametric tests (Student's t-test, ANOVA test, Pearson's correlation); the level of statistical significance was set at p < 0.05.\n\nResults and discussion\nWhen compared to healthy controls, geometric mean and 95% confidence interval (IC95%) of plasma D-dimer concentration appeared significantly increased in SSc patients (362 ng/mL, IC 95%: 361–363 ng/mL vs 229 ng/mL, IC95%: 228–231 ng/mL, p = 0.005). After stratifying SSc patients according to disease subset, no significant differences were observed between those with limited cutaneous pattern (lcSSc) and controls (geometric mean plasma D-dimer: 283 ng/mL, IC95%: 282–285 ng/mL; p = 0.61), whereas patients with diffuse cutaneous pattern (dcSSc) displayed substantially increased values (geometric mean plasma D-dimer: 538 ng/mL, IC95%: 536–539 ng/mL; p < 0.001). Additionally, patients with active disease, as evaluated according to the European Scleroderma Study Group criteria [7], displayed higher D-dimer levels as compared to patients with inactive disease (p = 0.027). As further shown in table 1, D-dimer concentration correlated significantly with the modified Rodnan total skin score (TSS) and the forced vital capacity (FVC). No correlation was observed between plasma D-dimer concentration and age, sex, autoantibody pattern, serum creatinine, erythrosedimentation rate, nailfold videocapillaroscopic pattern and pulmonary involvement, ascertained according to the score proposed by Medsger et al [8].\nThe pathogenesis of the endothelial injury in SSc is as yet elusive and most biochemical evidences are often inconclusive or controversial. Although endothelial cell apoptosis and impaired angiogenesis have received major attention among the mechanisms involved in the characteristic vascular dysfunction, recent studies provided clear evidence of a significant activation of the coagulation cascade, resulting in a procoagulant state that might finally raise the relative risk of thrombotic events in these patients. In SSc, the peculiar vascular lesions and fibrosis were claimed to impair endothelial function, as suggested by impairment of fibrinolysis and activation of the coagulation pathway. The following loss of the balance between fibrinolysis and coagulation might finally contribute to vessel engulfment with fibrin and breakdown of vessel patency, symptomatic of a tendency to the development of thrombotic complications in this particular autoimmune disorder [4]. D-dimer is a heterogeneous class of end-stage degradation products that directly reflect the level of lysed cross-linked fibrin, occurring in vivo with a wide range of molecular weights. Therefore, D-dimer is a well-recognized marker of a systemic prothrombotic state [5,9] and appears a strong, consistent predictor of cardiovascular events in the general population, in patients with cardiovascular disease and in other pathologies characterized by an increased risk of thrombosis [10-12]. Accordingly, D-dimer measurement could be reliably used as an initial screening test in patients with clinically suspected thrombosis, as its high negative predictive value enables to validly rule out ongoing thrombotic complications [12]. Little is known on the thrombotic tendency of SSc patients so far [13]. At variance with previous investigations [2-4], we demonstrated that SSc patients with diffuse subset are characterized by increased plasma D-dimer values, reflecting a potential activation of both the coagulation and fibrinolytic pathways.\n\nConclusion\nAlthough increased D-dimer values in SSc patients were occasionally observed in earlier studies, the association between plasma D-dimer and disease subset is likely an original and innovative issue. The significant correlation observed with disease activity, cutaneous involvement and forced vital capacity, further suggests that SSc patients, especially those with diffuse subset, display a hypercoagulable state, which might finally predispose this peculiar subset of patients to the development of thrombotic complications.\n\nAuthors' contributions\nGL: conceived of the study, participated in its design and coordination and drafted the manuscript; AV: participated in the design of the study, performed the statistical analysis and helped to draft the manuscript; PC: participated in the design and coordination of the study; GLS: participated in the design of the study; MM: participated in the design and coordination of the study and performed the measurement; GCG: participated in the design and coordination of the study. All authors read and approved the final manuscript. The authors declare that they have no competing interests.\n\n\n" ], "offsets": [ [ 0, 9969 ] ] } ]

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[ { "id": "pmcA1937013__text", "type": "Article", "text": [ "Dynein Modifiers in C. elegans: Light Chains Suppress Conditional Heavy Chain Mutants\nAbstract\nCytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis.\nMicrotubules and microtubule-dependent motor proteins segregate chromosomes during mitosis and also promote cellular organization in nondividing cells. An essential motor protein complex called cytoplasmic dynein powers many aspects of microtubule-dependent transport, but it is currently unclear how dynein is regulated such that it can execute different processes. We have performed a genome-wide screen to isolate genes that are involved in dynein-dependent processes. We determined that 20 of the 49 genes we identified specifically influenced the viability of dynein mutant strains but not the viability of other C. elegans mutants. Many of the proteins that specifically influence dynein localized to subcellular sites where the dynein heavy chain has been reported by others to be found. Additionally, we identified four dynein components that appear to negatively regulate the force-generating dynein heavy chain. The identification and initial characterization of this group of genes represents a route to identify genes that are not themselves essential but do participate in essential processes.\n\n\n\nIntroduction\nThe microtubule motor called cytoplasmic dynein has roles in diverse cellular processes including meiotic and mitotic spindle assembly and function, neuronal transport, and organelle positioning [1]. Cytoplasmic dynein is composed of a dimer of heavy chains (HCs), along with several accessory chains (ACs: intermediate, light intermediate, and light chains). Other dynein-interacting proteins, such as dynactin and LIS1, are likely present at substoichiometric levels and further modulate dynein function. The HCs contain both ATPase and microtubule binding activities and are sufficient for microtubule-based motility in vitro, moving toward the minus, or slow-growing, end of microtubules [2]. The dynein ACs provide cargo docking sites and often are encoded by multigene families in any one species [reviewed in 1,3]. In C. elegans, a single gene called dhc-1 encodes a cytoplasmic dynein 1 HC, while 11 other genes encode five classes of predicted dynein ACs [3,4].\nThe early C. elegans embryo is an excellent system for investigating gene contributions for essential cellular processes, including cytoskeletal functions [5]. The C. elegans dynein HC DHC-1 is essential and required for multiple microtubule-dependent events during early embryogenesis [6–9]. Depletion of DHC-1 by RNA interference (RNAi) in early C. elegans embryos produces defects in female meiotic divisions, migration of the oocyte and sperm pronuclei after fertilization, and centrosome separation during mitotic spindle assembly [6]. Analysis of fast-acting dhc-1 temperature-sensitive (ts) mutants has further revealed that dynein is required for chromosome congression to the metaphase plate during mitosis, as well as for mitotic spindle positioning [10].\nWhile many requirements for cytoplasmic dynein are known, our knowledge of the molecular mechanisms that target and regulate dynein remains limited. Clearly, the multiple ACs can couple the dynein HC to particular substrates [11], including vesicles, nuclei, viruses, kinetochores, and rhodopsin [see table in 1]. However, reducing the function of only four of the eleven dynein ACs in C. elegans produces lethal phenotypes [12]. Thus, it remains unclear how ACs influence the different essential requirements for dynein. Another potential route for dynein regulation involves the phosphorylation state of the different dynein chains, which in some cases confers distinctive functional properties to the motor. While many examples of dynein phosphorylation exist, and cell cycle dependent changes in phosphorylation have been described [13–15], few if any studies have demonstrated a requirement for such modification during mitosis. Large-scale forward genetic screens have identified genes with requirements similar to those for dynein, but many of these encode core components of the microtubule cytoskeleton and few are known to directly influence dynein itself [12,16]. Genes that do influence dynein function might also have other essential roles, leading to pleiotropic mutant phenotypes that obscure their relationship to dynein [17–19]. Moreover, redundancy within the multigene dynein subunit families, and also perhaps between the different ACs, may complicate the identification of single gene requirements that are important for dynein function. Thus far, reducing the function of individual genes has not provided substantial insight into the mechanisms that regulate and mediate the many different requirements for cytoplasmic dynein during mitosis.\nTo identify potential regulators of cytoplasmic dynein, we have used a sensitized genetic background to conduct a genome-wide screen for modifiers of dynein function in C. elegans. Other groups have successfully used RNAi modifier screens to identify genes that function in particular pathways [20–23]; we have used RNAi to screen for genes that, when reduced in function, suppress the embryonic lethality associated with a temperature-sensitive (ts) allele of the dhc-1 dynein HC gene. Using the dhc-1ts genetic background, we found 49 genes that, when depleted, suppress the partial loss of HC function. Twenty of these genes suppress conditional dynein HC mutants but not other conditional mutants with unrelated defects. Finally, we show that some of these dynein-specific suppressors encode proteins that may overlap with the dynein HC in subcellular localization.\n\nResults\nTo identify dynein suppressors, we used three different conditional and recessive dhc-1 mutants that were identified previously [7]. These ts alleles of dhc-1 (or195, or283, and or352) produce similar defects at the restrictive temperature of 26 °C, including incomplete mitotic spindle assembly in one-cell embryos and embryonic lethality [7]. We sequenced the dhc-1 locus in the three mutants. The independently isolated or195 and or283 alleles each change a conserved serine to leucine at codon 3200, within the coiled-coil region of the microtubule-binding stalk domain (Figure 1A and 1C). The or352 allele replaces a conserved glycine with aspartic acid at codon 2158, in the ATP-binding walker A motif of the second AAA ATPase domain (Figure 1B and 1C). As both missense mutations affect conserved residues, they may prove useful for engineering ts alleles in other organisms. The temperature versus viability curves of the dynein ts mutants feature a steep central transition zone ideal for modifier screening because subtle changes in temperature produce large changes in embryonic viability (Figure 2A).\nTo identify genes that, when reduced in function, can suppress conditional dhc-1 mutants, we developed a high-throughput RNAi-based screen (Figure 2B). To reduce gene function we used a library of 16,757 bacterial strains that each express a dsRNA corresponding to exon-rich gene sequences [17,19]. We then tested over 99% of the bacterial strains in this library for RNAi-mediated suppression of dhc-1(or195) embryonic lethality at 23 °C, after raising synchronized L1 larvae to adulthood on dsRNA-expressing bacterial lawns in 48-well agar plates. This screening procedure should work to identify nonessential and essential suppressor genes, because RNAi does not always fully reduce gene function [24,25], and even if RNAi does produce lethality, cosuppression could restore viability. Nevertheless, essential genes may be missed due to earlier requirements that produce strong larval arrest, sterile, or embryonic lethal phenotypes.\nUsing this screening procedure, we identified 49 bacterial clones that consistently increased embryonic viability at the semipermissive temperature. The dsRNA-producing plasmids were then sequenced to verify gene identity. Quantification of embryonic viability using dhc-1(or195) animals showed that the RNAi-mediated depletion of suppressor gene function increased viability to 5%–100%, compared to less than 2% in unsuppressed controls (Figure 3A; Table S1). The proteins encoded by the suppressor genes we identified are summarized in Figure 4.\nAs a more direct assay for dynein activity in the suppressed dhc-1 embryos, we measured spindle length and cytokinesis success: dhc-1 mutant embryos have severe spindle assembly defects and subsequent cytokinesis failures [6,7,10]. We shifted dhc-1 adult hermaphrodites from 23 °C to the fully restrictive temperature of 26 °C for 3–5 hours and made time-lapse video micrographs using Nomarski optics to monitor the first embryonic cell division. This procedure results in dhc-1(or195) embryos with P0 spindles 30% the length of wild-type spindles (Figure S1). In the suppressed dhc-1(or195) backgrounds, spindle lengths ranged from 30%–83% of wild-type lengths (Figure S1). Similarly, cytokinesis failed in unsuppressed dhc-1(or195) embryos 89% of the time, but most of the suppressors rescued this phenotype (Figure S1). These results indicate that most of the suppressors influence dynein-dependent cellular processes, as expected given their ability to restore viability when reduced in function.\nSpecificity of Suppression\nBecause RNAi can reduce the function of unintended targets (so-called “off-target effects” [26,27]), we also used available mutations in some of the suppressor genes we identified to reduce their function. We constructed double mutant strains using dhc-1(or195) and viable deletion alleles for two suppressor genes, dylt-1(ok417) and ufd-2(tm1380), and examined embryonic viability (Figure 3B). The deletion alleles of dylt-1 (encoding a Tctex1-type dynein light chain), and ufd-2 (encoding a ubiquitin conjugating enzyme) both recapitulated the suppression produced by RNAi knockdown (Figure 3B). The dpy-3(e27) and dpy-10(e128) point mutation alleles [28] also suppressed embryonic lethality in double mutants (Figure 3B). Based on this small sampling, and because RNAi in C. elegans appears to be highly gene specific in the absence of close paralogs [12,19], we conclude that many of the suppressors we have identified will prove to be suppressor locus specific. The dsRNA-expressing bacterial clones we used to deplete two of the dynein suppressors (tag-300 and ZK1127.10) probably also knock down expression of one close paralog for each locus [29].\nWe next asked whether the suppressors are specific for dynein function or if their depletion more generally stabilizes ts proteins. We tested for specificity using two conditional mutants with cell fate patterning defects unrelated to dynein function, lit-1(or131) and spn-4(or191). The lit-1 gene encodes a MAP kinase-related protein [30], while spn-4 encodes a protein with an RNA binding motif [31]. We found that ten of the dhc-1-interacting genes significantly increased embryonic viability in both lit-1 and spn-4 mutants, while 18 others suppressed one or the other of these two conditional mutants when reduced in function using the same RNAi protocol as that used for dhc-1ts mutants (Figure 4, right two columns; Table S1). Therefore, about half of the suppressors appear to act nonspecifically on multiple ts mutants to restore embryonic viability. From here on, we will refer to the suppressors that only acted on dhc-1, and not on lit-1 and spn-4 ts mutants, as dynein-specific suppressors.\nBecause many ts mutations exert their effect via protein assembly or unfolding mechanisms [32], suppressor genes reduced in function by RNAi might not be expected to exhibit allele specificity with most ts mutations. To determine if the dynein suppressors are either allele or strain specific, we tested the two other conditional dhc-1 strains (containing the or283 and or352 alleles). Although the or283 allele is identical to or195, it provides a useful control for the presence of background mutations because the two strains were isolated independently. In most cases, depletion of the dynein-specific suppressors also restored viability to the other two ts dhc-1 alleles. Y40B1B.5, a putative translation initiation factor, suppressed only one conditional dhc-1 strain, and we consider this as an example of a nonspecific interaction (Figure 4, left three columns; Table S1). Two dsRNAs that do not suppress lit-1 or spn-4 mutants produced suppression in the dhc-1(or195) and dhc-1(or283) strains, but not in the dhc-1(or352) strain, perhaps indicating allele specificity or variability in the RNAi treatments. We conclude that strain background differences are relatively rare, and that the majority of the suppressors are allele-independent. To summarize, we have identified 20 genes that when reduced in function specifically suppress multiple dynein ts strains but not unrelated ts loci.\n\nSurvey of Putative Dynein Accessory Chains\nWe were surprised to discover that depleting two predicted dynein ACs specifically suppressed the partial loss of HC function, because most dynein accessory subunits are presumed to promote dynein function by aiding dynein complex formation or mediating cargo attachment [3,11,33]. Depletion of either dylt-1 (encoding a Tctex1-type light chain) or dyrb-1 (encoding a roadblock-type light chain) suppressed embryonic lethality in all three ts dynein HC mutant strains (Figure 4). To extend this observation, we surveyed all genes encoding predicted dynein components for suppression of the three ts dhc-1 mutants (Figure 5A; Table S2). We reasoned that some dynein subunit genes could have been missed in the primary screening and several dynein AC genes were not represented in the E. coli RNAi library. After using RNAi to reduce their function, we found that one of three Tctex1 homologs (dylt-1), one of four LC8 homologs (dlc-1), one of two light intermediate chains (dli-1), as well as the sole roadblock homolog (dyrb-1) each strongly suppressed the three conditional dynein mutants. Lower-level suppression was also seen for the second light intermediate chain, xbx-1, when its function was reduced. Thus, one gene of each of four subunit classes restores viability to the three dhc-1 mutant strains when depleted by RNAi.\nThe only subunit class not found to suppress was the intermediate chain, encoded by a single gene in C. elegans, dyci-1. When reduced in function by RNAi, dyci-1 produces a larval arrest phenotype like that observed for dhc-1(RNAi); this phenotype precludes any suppression of the conditional embryonic lethality (shown as “la” in Figure 5A). In contrast, knockdown of either dlc-1 or dli-1 suppresses embryonic lethality in the dhc-1ts mutants, even though reducing their function in otherwise wild-type embryos produces dhc-1-like defects, including embryonic lethality [12,34] (see Figure 5B and Discussion). The suppressing cytoplasmic dynein subunits and DYCI-1 are shown in a putative complex in Figure 5E.\nWe performed several genetic assays to better understand how the suppressor genes may be operating. First, suppression of dhc-1 lethality by reducing AC function may indicate that our dhc-1 alleles express a neomorphic and toxic DHC-1 protein: if the suppressor dynein AC subunits positively function in dynein processes, depleting them might suppress any neomorphic effects. This explanation is perhaps unlikely, because the dhc-1ts alleles are all recessive, but remained a possibility in dhc-1 homozygotes. We therefore reduced dynein function using RNAi in animals that had passed through the larval arrest points for dhc-1(RNAi) and dyci-1(RNAi). Specifically, we transferred dhc-1(or195) L4 hermaphrodites to plates with bacteria expressing dhc-1 or dyci-1 dsRNA. As control we performed dylt-1(RNAi) using the same procedure. We observed substantial suppression with dylt-1 in this assay, but we saw no suppression with the heavy or intermediate chains (Figure 5C). This suggests that the DHC-1ts protein is not toxic and that dyci-1 acts more like dhc-1 than the other suppressing ACs because it does not suppress the heavy chain mutant.\nTo further examine the nature of the AC suppression, we asked if depletion of the suppressor chains could bypass the requirement for dhc-1. We transferred wild-type L4 larvae to plates with bacteria expressing dsRNA corresponding to both the suppressor ACs and dhc-1. We did not observe any suppression in these double RNAi assays (Figure 5D), suggesting that dhc-1ts suppression requires the residual activity of the defective DHC-1 protein. We conclude that the dynein AC suppressors inhibit or somehow oppose the function of the DHC-1ts protein, and that the dhc-1(or195ts) mutation does not produce a toxic gene product but simply reduces DHC-1 activity to a low, but non-null, level.\n\nLocalization of the Dynein Suppressor Proteins\nTo further explore how the suppressor proteins function, we examined the subcellular localization of nine of them as stably expressed N-terminal GFP::S fusions. We chose to first focus on the suppressor genes that were conserved but poorly characterized in any system, or were conserved but uncharacterized during early C. elegans embryogenesis. Prior dynein immunocytochemistry-based localization studies serve as a comparison [6,10,35]. As in other species, C. elegans DHC-1 is associated with mitotic spindles, centrosomes, the nuclear envelope, the cell cortex, the midbody, and throughout the cytoplasm. Most of the suppressor proteins we examined localized to sites where DHC-1 is known to act or localize (Figure 6). However, the nearly ubiquitous distribution of DHC-1 in early embryonic cells makes colocalization likely but not necessarily meaningful, and biochemical studies are needed to conclusively address any direct or indirect physical associations.\nFour suppressor GFP fusion proteins localized to nuclear membranes and to spindle poles or pericentrosomal regions. The DYLT-1 and DYRB-1 dynein light chains were associated with nuclear envelopes and centrosomes, as well as meiotic and mitotic spindle poles (Figure 6A–6H; Videos S1 and S2). The potential coiled-coil protein K04F10.3 was present on the nuclear envelope and in a pericentrosomal position during mitosis, similar to endoplasmic reticulum proteins [36] (Figure 6I–6L; Video S3). K04F10.3 was also highly enriched at meiotic spindle poles (Figure 6I), which has been observed for other endoplasmic reticulum proteins [36]. The NPP-22 transmembrane nucleoporin was found at nuclear envelopes (Figure 6M–6P; Video S4), as previously reported for later stage embryos [37], and it also surrounded centrosomes during mitosis. Two splice isoforms of the pleckstrin homology domain–containing EFA-6/Y55D9A.1, an ARF guanine nucleotide exchange factor, were enriched cortically both in the anterior portion of the one-cell zygote and at the blastomere boundary in two-cell embryos (Figure 6Q–6T; Videos S5 and S6). The conserved Mo25 homolog MOP-25.2/Y53C12A.4 was found enriched in a single spot after cytokinesis that appears to correspond to the midbody (Figure 6U–6X; Video S7). F10E7.8, a highly conserved ortholog of S. cerevisiae Far11, appears nuclear (Figure 6Y–6B′ and Video S8). Finally, the nonspecific suppressor protein STAR-2, a predicted RNA binding protein, appears to be associated with P-granules (like its homolog GLD-1), where dynein is neither localized nor known to function (Figure 6C′–6F′).\n\nDLYT-1 and DYRB-1: Dynein Light Chain Localization\nThe C. elegans dynein HC protein weakly localizes to spindle poles during early embryonic cell cycles [6,10], and so did DYRB-1 and DYLT-1 (Videos S9 and S10). However, ts mutant forms of the DHC-1 protein (including DHC-1 encoded by the or195 allele) strongly localize to centrosomes when shifted to the non-permissive temperature [10]. The mechanism underlying this enhanced localization is not known, but it may represent trapping of the defective protein at a normally transient location. We exploited this behavior of the mutant DHC-1 protein to determine whether redistribution of the putative DYRB-1 and DYLT-1 dynein light chains also occurred in the dhc-1(or195) background.\nWe found that the cellular distributions of DYRB-1 and DYLT-1 were dramatically altered in dhc-1(or195) mutant embryos. After shifting the parental worms to the restrictive temperature for 3–5 h prior to collecting embryos, these two proteins were prominently localized to centrosomes and to spindle poles that did not separate in one-cell stage embryos (Figure 7; Videos S11 and S12). The spindle pole to cytoplasmic fluorescence ratio during late anaphase was 5-fold higher in both of the dhc-1 homozygous mutant strains when compared to wild-type embryos expressing the GFP fusions. We also assayed localization of the two putative dynein light chains after short temperature shifts to the nonpermissive temperature in the dhc-1(or195) mutant background, which yields mitotic spindles with an overall wild-type appearance and function. These short temperature shifts also resulted in robust localization of these two dynein light chains to centrosomes (unpublished data). Finally, we examined the localization of GFP::DYRB-1 and GFP::DYLT-1 in embryos from dhc-1(or195) −/+ worms grown at the dhc-1(or195) permissive temperature of 15 °C. Even though embryos from mothers heterozygous for this recessive mutation are viable and develop normally, even at 26 °C [7], we observed a substantial increase in both GFP fusion proteins at the mitotic spindle poles in early embryos (Figure 7; Videos S13 and S14). Importantly, localization of DYLT-1 and DYRB-1 to centrosomes does not occur in embryos depleted for DHC-1 with RNAi (our unpublished results), indicating that these proteins require the mutant DHC-1 polypeptide for centrosomal targeting in the dhc-1(or195) embryos. In summary, the DYRB-1 and DYLT-1 proteins localize to sites where the DHC-1 HC is also found in wild-type embryos, and all three proteins respond similarly to mutational alterations in DHC-1.\n\nGenetic Characterization of the DYLT-1 and DYRB-1 Dynein Light Chains\nWe obtained putative null alleles to determine if dylt-1 and dyrb-1 function in dynein-dependent processes. DYLT-1 is 40% identical to human DYNLT3 and 38% identical to Drosophila Dlc90F (see alignment in Figure 8A). Two other C. elegans genes, dylt-2 and dylt-3, encode more divergent members of this protein family. DYRB-1 is 49% identical to both human DYNLRB1 and Drosophila Robl (see alignment in Figure 8A). There do not appear to be other Roadblock genes in the C. elegans genome [3]. Deletion alleles for both dylt-1 and dyrb-1 have been isolated (Figure 8B). The dylt-1(ok417) deletion removes the entire DYLT-1 open reading frame and does not affect adjacent coding regions. The dyrb-1(tm2645) deletion removes 69% of the dyrb-1 coding region, leaving 29 predicted N-terminal amino acids, and does not affect adjacent coding regions.\nBoth deletions are currently annotated as homozygous viable [29]. However, we found that the dyrb-1(tm2645) strain was in fact heterozygous for the deletion and that most embryos produced by dyrb-1(tm2645) homozygous animals failed to hatch (Figure 8C). Homozygous dyrb-1(tm2645) worms also showed an egg-laying defect and produced small broods (unpublished data). To determine if the dyrb-1 deletion was responsible for the embryonic lethality, we crossed the GFP::dyrb-1 transgene into the deletion background. The presence of the transgene fully rescued the embryonic lethality (Figure 8C), but not the egg-laying defect: the transgene is driven from a germline-specific promoter and so would not be expected to rescue zygotic phenotypes. The embryonic lethality exhibited by dyrb-1(tm2645) mutants is consistent with RNAi studies performed by injection or soaking [12,38]. In contrast, homozygous dylt-1 deletion mutants did not exhibit any larval or embryonic lethality (Figure 8C).\nTo determine if these dynein light chain mutants exhibit dynein HC-like phenotypes, we observed the completion of meiotic polar body extrusion and the first two mitotic cell divisions in mutant embryos (Figure 8D). The dylt-1 embryos appeared wild type for completion of meiosis, pronuclear migration, and spindle assembly and function. However, the dyrb-1 embryos occasionally contained extra female pronuclei (observed in four of 12 recordings, Figure 8D), suggesting that polar body extrusion was defective, and pronuclear migration was often slow compared to wild-type embryos. Once formed, spindles appeared functional using Nomarski optics, although they were frequently positioned improperly and had large spindle poles, as has also been observed after RNAi knockdown [12]. Thus, these two genes are not strictly essential, but the DYRB-1 protein clearly is required for dynein-dependent processes.\n\n\nDiscussion\nBy using the suppressor screening method outlined in Figure 2B, we have isolated and characterized 49 genes that when reduced in function can suppress a partial loss of dynein HC function. This screening procedure takes advantage of sensitized genetic backgrounds using conditional mutants, can be completed for one mutant in less than 5 wk, and is scalable so that many mutants can be screened in parallel. In fact, we have performed 15 such screens in different sensitized backgrounds (unpublished data). By using three dhc-1ts mutant strains, we found that strain background differences and allele specificity are minimal because most of these genes suppress all three dynein mutants when reduced in function using RNAi. Furthermore, by using two unrelated ts mutants to assay for specificity, we found that 57% of the suppressor genes suppress multiple unrelated mutant loci. Thus, it is clear that assaying the specificity of suppression is critical for evaluating the functional significance of these RNAi interactions. Eliminating the analysis of these nonspecific genes in future screens will save time and resources. Most of the specific suppressor proteins we examined appear to overlap in subcellular localization with the dynein HC, based on previous studies of DHC-1, while one nonspecific suppressor protein, STAR-2, localized to germline P-granules, where dynein is not known to function.\nMany of the 20 genes that specifically suppress multiple dhc-1ts alleles are nonessential in C. elegans but well conserved nonetheless. Six of eight deletion alleles available for the 20 specific suppressor genes are homozygous viable, and six additional specific genes do not display lethal phenotypes when reduced in function by RNAi in wild-type worms [29]. Thus, our genetic screening has identified roles in an essential process for at least 12 apparently nonessential genes. Fourteen of the specific dynein suppressor genes have human orthologs as determined by best reciprocal BLAST hits (Table 1), while mop-25.2 has a conserved human homolog but also a paralog in C. elegans. Eleven of these conserved genes are nonessential in C. elegans. Interestingly, eight of the conserved genes in Table 1 have been implicated in human disease etiology, with three of them identified as the causative gene [39–41]. Thus, using sensitized genetic backgrounds for genome-wide modifier screens can identify roles for nonessential but conserved genes and thereby provide insights into human disease.\nNonspecific Suppression of Conditional Mutants\nWe examined the predicted molecular functions of the suppressor proteins to better understand the basis for the nonspecific suppression phenomenon. Strikingly, many of the nonspecific suppressor genes encode proteins with predicted roles in mitochondrial, ribosomal, and collagen function (18 of 29 genes, or 62%), while only two such genes appeared to specifically suppress dhc-1 (2 of 20 genes or 10%). It is possible that stress produced by RNAi knockdown of these suppressor genes triggers the activity of molecular chaperones that can generally restore function to ts proteins. Indeed, mutation of dpy-10 is known to suppress three other ts mutants: glp-1, emb-5, and mup-1 [42–44]. Furthermore, RNAi reduction of dpy-10, star-2, osr-1, or C50D2.1 (all suppressors of dhc-1, lit-1, and spn-4 ts alleles) induces the glycerol biosynthetic gene gpdh-2, while dpy-10 and osr-1 mutants exhibit increased glycerol levels, a condition that promotes protein stability [45]. We suggest that partial loss of central metabolic processes can invoke stress responses that nonspecifically alleviate protein-folding problems in ts proteins. Filtering out these nonspecific interactions by testing unrelated conditional mutants increases the likelihood that the remaining suppressor genes are more directly involved with dynein function. However, ts mutants likely differ in their susceptibility to nonspecific suppression mechanisms, and some apparently unrelated ts mutants might share common cofactors such that both mutants are suppressed by depletion of the same cofactor. Nevertheless, we expect that more extensive testing for specificity will prove very useful for judging the significance of modifier interactions.\n\nPossible Relevance of Suppressor Proteins to Dynein Function\nWe examined the localization of a number of GFP fusions to suppressor proteins to gain insight into their functional relationship to dynein. In several cases, the subcellular distribution of the suppressor proteins overlapped in different ways with the known and nearly ubiquitous distribution of cytoplasmic dynein in the early C. elegans embryo. In fact, the only specific suppressor that did not display dynein-like localization was F10E7.8, a homolog of yeast Far11 of unknown function [46], which was nuclear. The one nonspecific suppressor protein we examined did not show any dynein-like localization patterns. The subcellular localizations of the GFP::suppressor protein fusions are intriguing. However, given the nearly ubiquitous distribution of dynein in early embryonic cells, biochemical tests for direct association are needed to address the significance of any colocalization detected using light microscopy.\nWe are particularly interested in suppressor proteins that localize to mitotic spindle poles: the association of the DYLT-1 and DYRB-1 predicted dynein light chains with centrosomes and spindle poles suggests that they may be components of cytoplasmic dynein in C. elegans. Localization of cytoplasmic dynein to centrosomes and spindle poles is well established [47,48], and the inhibition of dynein function prevents centrosome separation, centrosome attachment to nuclei, and the formation of bipolar spindles [6,10,49,50]. Moreover, the centrosomal localization of DHC-1, DYLT-1, and DYRB-1 are all greatly enhanced in dhc-1ts mutant embryos: this dependence of the light chain distribution on the HC further suggests they reside in the same motor complex (Figure 7 and [10]). Furthermore, roadblock light chains are well-established components of dynein, and all of the roadblock protein in mammalian liver extracts is dynein associated [51,52]. Finally, a DYLT-1 homolog in vertebrates is a stoichiometric subunit of cytoplasmic dynein [53]. The presence of these two light chains in a dynein complex is consistent with them having either positive or negative roles in the regulation of HC function (see below).\nCytoplasmic dynein is found on the nuclear envelope where it is thought to regulate nuclear membrane breakdown during mitosis [54], and dynein plays roles during the trafficking of endoplasmic reticulum components [55,56]. Therefore, the nuclear envelope/endoplasmic reticulum proteins NPP-22 and K04F10.3 could couple dynein activity to either of these structures. The anc-1 gene was also isolated in our screening and ANC-1 is localized to the nuclear envelope where it maintains nuclear positioning in postembryonic cells [57]. Reducing the function of these three genes may suppress partial loss of dynein HC mutants by reducing the need for dynein during nuclear envelope breakdown, through constitutive partial destabilization of the nuclear envelope.\nThe distribution of the cytoplasmic dynein HC includes sites other than spindle poles and nuclear envelopes in C. elegans, for example, at the cell cortex and at the cell division remnant called the midbody [6,10]. The MOP-25.2 protein was found at the midbody and faintly at spindle poles. The MOP-25.2 ortholog in S. pombe, Pmo25, is also present at the cell division site and on spindle poles [58]. Mammalian MOP-25.2 homologs stimulate the kinase activity of the LKB1 tumor suppressor (the C. elegans ortholog is PAR-4), which in turn activates MARK microtubule-destabilizing kinases [59,60]. The C. elegans MARK ortholog, PAR-1, controls cell polarity during embryogenesis, and orthologs have been implicated in regulation of microtubule dynamics from yeast to humans [61–64].\nLastly, the two splice isoforms of EFA-6 were associated with the anterior cell cortex in late one-cell embryos. Cortically localized dynein may have important roles in applying forces to astral microtubules that influence mitotic spindle positioning and chromosome separation during anaphase [10,65,66]. EFA6 ARF guanine nucleotide exchange factors require their pleckstrin homology domain for cortical targeting, and are known to regulate cortical actin dynamics in vertebrate cells by promoting guanine nucleotide exchange on ARF6 [67,68]. Our results identifying efa-6 as a dynein HC suppressor suggest a functional linkage of the actin and microtubule cytoskeletons at the cell cortex. Interestingly, two yeast pleckstrin homology domain proteins, Num1 and mcp5+, localize to the cell cortex and direct astral microtubule and dynein function, although they do not contain a Sec7 domain like EFA-6 does [69–71].\n\nFunction of Dynein Intermediate, Light Intermediate, and Light Chains\nThe dynein chains in C. elegans exhibit strikingly different functional requirements. The DYRB-1 roadblock light chain is required for completion of meiosis and pronuclear migration, but an at least partially functional mitotic spindle forms in the absence of DYRB-1 (Figure 8). The DLI-1 light intermediate chain is required for multiple dynein-dependent functions: pronuclear migration, centrosome separation, and meiotic and mitotic spindle function [12,34]. DLI-1 may promote nuclear envelope targeting of both centrosomes and DHC-1 by interacting with the nuclear envelope protein ZYG-12 [35]. The second worm light intermediate chain gene, xbx-1, is required for cilia function but not early embryonic development [12,72]. RNAi knockdown of DLC-1, one of three LC8 proteins in C. elegans, produces defects similar to dli-1 but knockdown of the other two LC8-related genes does not result in any phenotypes [12]. RNAi depletion of dyci-1 results in severe meiotic, pronuclear migration, and mitotic spindle assembly defects [12] and in our feeding RNAi regimen dyci-1(RNAi) produces a larval arrest phenotype similar to that observed for dhc-1. Finally, the three Tctex1 proteins in C. elegans, DYLT-1, 2, and 3, are not essential for dynein-related functions [12]. As the dyrb-1, dlc-1, and dli-1 dynein AC genes display some dhc-1-like requirements, they positively influence dynein function. However, because reducing their function suppresses dhc-1ts mutants, they may also exert negative regulation (along with dylt-1) on the heavy chain.\n\nNegative Regulation of Dynein HC by Light Chain Subunits\nFinding that reducing the function of light and light intermediate dynein chains suppressed the partial loss of HC function was a striking result. One member of each of four subunit classes can suppress the embryonic lethality associated with three dhc-1 ts mutants (Figure 5). We have considered two different models to explain how RNAi-mediated depletion of these dynein ACs can suppress reduced HC function. First, these dynein subunits could be in functional complexes with, and exert negative regulation on, the DHC-1 HC (Figure 5B). The suppression mechanism in this case proposes that removal of the suppressing ACs increases residual mutant DHC-1 activity. The other, nonsuppressing, accessory subunits might then function in nonmitotic cellular processes such as neuronal transport or organelle positioning. In support of this view, physical removal of the intermediate chains of rat cytoplasmic dynein increased HC ATPase activity by about 4-fold (light chains were not monitored in this study but were likely removed as well) [73]. Thus, at least with respect to ATPase activity, some dynein ACs do act as biochemical negative regulators of HC function.\n\nAn Assortment of Essential and Nonessential Dynein Complexes\nAlternatively, an assortment of dynein complexes (with different ACs) could coexist within early embryonic cells, with only a subset required for the essential mitotic functions that require DHC-1. In this case, suppression might result from the release of DHC-1 HCs from less essential motor complexes, allowing more of the functionally compromised HCs to participate in the essential process of mitosis. We currently disfavor this hypothesis because two of the suppressing light chains (DYLT-1 and DYRB-1) can indeed localize to meiotic and mitotic spindles (Figures 6 and 7), sites where DHC-1 has been shown by others to localize and function. Furthermore, the distribution of DYRB-1 and DYLT-1 closely resembles the distribution of the HC in dhc-1(or195) embryos, suggesting that these two light chains associate with the HC during mitosis (Figure 7 and [10]). Finally, dhc-1-like phenotypes result from mutation or RNAi knockdown of three suppressing ACs in otherwise wild-type worms, indicating that they share at least some common and essential requirements. Regardless of the suppression mechanism, our identification of ACs that genetically interact with the DHC-1 HC provides a basis for functionally classifying the paralogs of these dynein subunit gene families, and for further investigation of dynein composition and function.\n\nNonessential Dynein Subunits and Negative Regulation of the HC\nSome ACs are nonessential, supporting the view that some cytoplasmic dynein subunits could function by exerting negative regulation on the HC, rather than positively influencing essential HC function. For example, DYRB-1 is not absolutely required for viability because worms lacking this protein can be propagated, although they are extremely sick and do exhibit two dhc-1-like phenotypes (Figure 8). Also, homozygous dylt-1 deletion mutants appear fully viable (Figure 8). The two additional Tctex1 C. elegans genes could be functionally redundant with DYLT-1, but simultaneously reducing the function of DYLT-2 and DYLT-3 by RNAi in the dylt-1 deletion strain did not cause lethality (unpublished data). Because RNAi does not always completely reduce function, the question of redundancy in the Tctex1 C. elegans gene family remains unresolved. However, Drosophila contains only a single Tctex1 gene, Dlc90F [74,75]. A Dlc90F null allele that deletes 80% of the open reading frame is essential only for sperm production but not for viability of male or female flies, despite the wild-type protein being incorporated into dynein motors and expressed in various Drosophila tissues [74]. Thus, at least in Drosophila, the Tctex1 dynein light chain family is not required for cell division processes like the HC is. Interestingly, budding yeast does not possess genes for the Tctex1 or roadblock ACs, indicating that functional cytoplasmic dynein does not require these subunits that are conserved in many other organisms. The AC genes that yeast does posses are not required for HC motility in vitro because dynein purified from yeast with mutations in these genes remains fully active [2]. Thus, dynein function in several contexts does not require AC subunits, and we suggest that in some cases they may have negative regulatory roles. Negative regulation of cytoplasmic dynein may be redundant with other modes of HC regulation or only required during special circumstances. Further studies of subunit localization, and in vitro studies of C. elegans dynein motility, may provide further insight into the modes of AC regulation and function.\n\n\nMaterials and Methods\nC. elegans strains and culture.\nStrains were cultured according to standard procedures [28]. ts mutants were maintained at 15 °C and GFP-expressing strains in a wild-type background were maintained in a 23 °C incubator. dhc-1(or195) was outcrossed six times to the N2 Bristol wild-type strain and the or283 and or352 dhc-1 mutants were each outcrossed four times with N2. For sequencing mutant dhc-1 loci, genomic DNA was amplified as overlapping ∼1-kb fragments from the start codon to the stop codon and sequenced at the University of Oregon DNA sequencing laboratory. For double mutant constructions, the dhc-1(or195) mutation was monitored by sequencing or by assaying a restriction fragment length polymorphism caused by the mutation with Hpy188I (New England Biolabs, http://www.neb.com), following PCR amplification of the mutated region. The dylt-1(ok417) and ufd-2(tm1380) alleles were monitored by PCR amplification of genomic sequence encompassing the deletions and assaying product size by agarose gel electrophoresis.\n\nGFP imaging.\nVisualization of GFP fusion protein localization was accomplished by mounting embryos on M9 + 3% agarose pads on microscope slides covered with a coverslip. Time-lapse videos were obtained on a spinning disk Nikon Eclipse TE2000-U microscope (Nikon Instruments, http://www.nikon.com) fitted with an ORCA-ER digital camera (Hamamatsu Photonics, http://www.hamamatsu.com) using a Nikon 60×, 1.4 NA Plan Apo oil objective lens. Videos were adjusted for contrast in ImageJ (National Institutes of Health, http://rsb.info.nih.gov/ij/) [76], images were adjusted for levels in Adobe Photoshop (http://www.adobe.com/).\n\nRNAi screening.\nAfter obtaining the E. coli RNAi library from the MRC Geneservice (Cambridge, UK) [17,19], we rearrayed it into a 48-well microplate format using a liquid-handling Qiagen BioRobot 8000 (http://www.qiagen.com). E. coli strains were thawed from −80 °C storage and inoculated into 1 ml of LB + 100 mg/ml ampicillin-containing 96-well growth plates (Whatman, http://www.whatman.com) and covered with microporous sealing film (USA Scientific, http://www.usascientific.com). Only 48 wells of the 96-well growth plates were filled with media, corresponding to the rearrayed E. coli library. After overnight shaking incubation at 37 °C, 20 μl of the cultures were dispensed with a 24-channel electronic repeating pipette (Rainin, http://www.rainin.com) onto 48-well plates (Nunc, http://www.nuncbrand.com/) containing NGM agar, 100 μg/ml ampicillin, and 1 mM IPTG and allowed to dry and induce dsRNA at 37 °C overnight. The 48-well agar plates were filled with a Wheaton Unispense peristaltic pump (http://www.wheaton.com) equipped with a custom-made adaptor (University of Oregon Technical Science Administration) that allowed simultaneous filling of eight wells with the agar solution. Approximately 15 hypochlorite-synchronized L1 mutant larvae were pipetted into each well of the 48-well plates with a multichannel pipette and allowed to produce broods. Screening for F1 viability was performed by visual examination with a dissecting microscope. Phenotypes were recorded on an Excel spreadsheet (http://www.microsoft.com) and organized in a FileMaker Pro (http://www.filemaker.com) database. We qualitatively identified 295 initial positive suppressing E. coli strains, for which we repeated the assay on 60-mm plates with E. coli again thawed from the library (not streak purified). If the observed phenotypes reproduced, the assay was performed with three streak-purified E. coli colonies, and a single isolate that again displayed the interaction was kept for further analysis.\n\nEmbryonic viability quantitation methods.\nTo quantitate embryonic viability we used the following procedure. Cultures of dsRNA-producing bacteria were grown overnight in LB + 100 μg/ml ampicillin. Cultures (0.2 ml) were seeded onto 60-mm NGM agar plates containing 100 μg/ml ampicillin and 1 mM IPTG and allowed to induce dsRNAs overnight at room temperature. The L4440 control vector-containing strain was used as the bacterial lawn for the experiments shown in Figures 2A and 3B. Approximately 80 synchronized L1 larvae (obtained from hypochlorite-treated worms) were pipetted onto the plates and allowed to grow to young adulthood. Five gravid worms were transferred to prepared NGM agar plates supplemented with 100 μg/ml ampicillin and 1 mM IPTG containing a small RNAi bacterial lawn produced from ∼5 μl of overnight E. coli culture. After producing broods, the adult worms were removed and the embryos were allowed to develop for at least 24 h. Embryos and larvae were then counted immediately or after storage at 4 °C. We considered only suppressor dsRNAs that increased embryonic viability greater than 3-fold above the background viability observed with the L4440 control vector (in the dhc-1(or195)) screen to be significant enough for continued study.\n\nMolecular biology.\nWe introduced a polylinker site containing six unique restriction enzyme recognition sites into the pIC26 GFP-S protein plasmid by using phosphorylated and PAGE-purified synthetic oligonucleotides [77]. Following ligation, the new plasmid was sequence verified. The modified vector, pSO26, allows the use of additional restriction enzymes and directional cloning for inserting genes of interest (Figure S2). The SpeI site was recreated at the 5′ end of the polylinker but not at the 3′ end. We amplified N2 genomic DNA or cDNA (Invitrogen, http://www.invitrogen.com) with Pfu Turbo polymerase (Stratagene, http://www.stratagene.com), and cloned A-tailed PCR products into either pGEM-T or pGEM-T-easy shuttle vectors (Promega, http://www.promega.com). Inserted genes were sequence verified at the University of Oregon DNA sequencing laboratory prior to cleavage and ligation to pSO26 (see Table S3 for restriction sites and primer sequences used). All of the constructs used in this study were cloned as SpeI-AsiSI or AscI-AsiSI fragments, except for the STAR-2 gene, which was amplified as a SpeI fragment and cloned into pIC26.\nTo construct dynein subunit dsRNA-expressing plasmids not available in the RNAi library, gene fragments were amplified from N2 genomic DNA with the following primers: F41G4.1: 5′-AAGATATCACCCAAAATGGTCCAAAACAAAG-3′ and 5′-CGGATATCTCGACTGAAGCTGGTTCTGA-3′, xbx-1: 5′-AAGATATCTACGACGATGGAAGTTTGAAG-3′ and 5′-CGGATATCCGTGCCTCTGCAGC-3′, dlc-3: 5′-AAGATATCAATTTCAGGTGGACACTGGC-3′ and 5′-CGGATATCAGCACACTTGCATCATCTGAA-3′. The PCR products were cut with EcoRV, ligated to EcoRV-digested L4440, and sequence verified.\n\nIsolation of transgenic worms.\nGFP fusion plasmids were bombarded into unc-119(ed3) worms as previously described except with the following two changes [78]. Three milligrams of gold particles were used per hepta adaptor bombardment. Also, we briefly sonicated the gold particles (prior to DNA coating and while suspended in 50% glycerol) with a Branson sonifier 450 (http://www.sonifier.com/) fitted with a small tip set to power level 1, to disrupt gold aggregates. Non-Unc worms were picked to new plates and allowed to produce broods, which were assayed for GFP fluorescence with a Zeiss axioskop microscope (http://www.zeiss.com/) fitted with an X-Cite 120 illumination system (EXFO life sciences, Mississauga, Ontario, Canada). For each fluorescent line, 12 GFP-positive worms were singled to new plates to determine if the constructs were integrated or were carried as extrachromosomal arrays.\n\n\nSupporting Information\nAccession Numbers\nThe National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi?itool=toolbar) accession numbers for the dhc-1 homologs discussed in this paper are C. elegans, NP_491363; Dictyostelium discoideum, XP_643185; Drosophila melanogaster, AAA60323; Homo sapiens, NP_001367; Mus musculus, NP_084514; Saccharomyces cerevisiae, NP_012980; and Schizosaccharomyces pombe, NP_001018285.\nThe NCBI accession numbers for the Drosophila and human DYLT-1 and DYRB-1 protein homologues, respectively, are Dlc90F, NP_477356; DYNLT3, NP_006511; DYNLRB1, NP_054902; and robl, NP_523771.\n\n\n\n" ], "offsets": [ [ 0, 50169 ] ] } ]

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} ] }, { "id": "pmcA1937013__T79", "type": "species", "text": [ "Mus musculus" ], "offsets": [ [ 49868, 49880 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "10090" } ] }, { "id": "pmcA1937013__T80", "type": "species", "text": [ "Saccharomyces cerevisiae" ], "offsets": [ [ 49893, 49917 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4932" } ] }, { "id": "pmcA1937013__T81", "type": "species", "text": [ "Schizosaccharomyces pombe" ], "offsets": [ [ 49934, 49959 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "4896" } ] }, { "id": "pmcA1937013__T82", "type": "species", "text": [ "Drosophila" ], "offsets": [ [ 50010, 50020 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "7227" } ] }, { "id": "pmcA1937013__T83", "type": "species", "text": [ "human" ], "offsets": [ [ 50025, 50030 ] ], "normalized": [ { "db_name": "ncbi", "db_id": "9606" } ] } ]

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[ { "id": "pmcA2636797__text", "type": "Article", "text": [ "Efficacy of intra-articular hyaluronan (Synvisc®) for the treatment of osteoarthritis affecting the first metatarsophalangeal joint of the foot (hallux limitus): study protocol for a randomised placebo controlled trial\nAbstract\nBackground\nOsteoarthritis of the first metatarsophalangeal joint (MPJ) of the foot, termed hallux limitus, is common and painful. Numerous non-surgical interventions have been proposed for this disorder, however there is limited evidence for their efficacy. Intra-articular injections of hyaluronan have shown beneficial effects in case-series and clinical trials for the treatment of osteoarthritis of the first metatarsophalangeal joint. However, no study has evaluated the efficacy of this form of treatment using a randomised placebo controlled trial. This article describes the design of a randomised placebo controlled trial to evaluate the efficacy of intra-articular hyaluronan (Synvisc®) to reduce pain and improve function in people with hallux limitus.\n\nMethods\nOne hundred and fifty community-dwelling men and women aged 18 years and over with hallux limitus (who satisfy inclusion and exclusion criteria) will be recruited.\nParticipants will be randomised, using a computer-generated random number sequence, to receive a single intra-articular injection of up to 1 ml hyaluronan (Synvisc®) or sterile saline (placebo) into the first MPJ. The injections will be performed by an interventional radiologist using fluoroscopy to ensure accurate deposition of the hyaluronan in the joint. Participants will be given the option of a second and final intra-articular injection (of Synvisc® or sterile saline according to the treatment group they are in) either 1 or 3 months post-treatment if there is no improvement in pain and the participant has not experienced severe adverse effects after the first injection. The primary outcome measures will be the pain and function subscales of the Foot Health Status Questionnaire. The secondary outcome measures will be pain at the first MPJ (during walking and at rest), stiffness at the first MPJ, passive non-weightbearing dorsiflexion of the first MPJ, plantar flexion strength of the toe-flexors of the hallux, global satisfaction with the treatment, health-related quality of life (assessed using the Short-Form-36 version two questionnaire), magnitude of symptom change, use of pain-relieving medication and changes in dynamic plantar pressure distribution (maximum force and peak pressure) during walking. Data will be collected at baseline, then 1, 3 and 6 months post-treatment. Data will be analysed using the intention to treat principle.\n\nDiscussion\nThis study is the first randomised placebo controlled trial to evaluate the efficacy of intra-articular hyaluronan (Synvisc®) for the treatment of osteoarthritis of the first MPJ (hallux limitus). The study has been pragmatically designed to ensure that the study findings can be implemented into clinical practice if this form of treatment is found to be an effective treatment strategy.\n\nTrial registration\nAustralian New Zealand Clinical Trials Registry: ACTRN12607000654459\n\n\n\nBackground\nOsteoarthritis (OA) is a degenerative joint disease that commonly presents within the first metatarsophalangeal joint (MPJ) of the foot. The terms hallux limitus and hallux rigidus have frequently been used interchangeably to describe differing severities of pain and limitation of motion associated with OA at the first MPJ [1]. Hallux limitus is a progressive osteoarthritic condition of the first MPJ that may advance to an end-stage presentation of hallux rigidus where the joint fuses and there is a complete restriction of motion [1]. First MPJ OA is the second most common disorder affecting the foot after hallux valgus [2]. The prevalence of the condition increases with age, and it has been reported that radiographic changes in the first MPJ affect are evident in approximately 46% of women and 32% of men at 60 years of age [3]. Osteoarthritis at the first MPJ is characterised by the symptoms of pain and stiffness at the joint [1]. Secondary painful symptoms relate to compensations during gait that may occur due to the reduced motion of the first MPJ [1]. The presence of pain associated with first MPJ OA impacts on normal walking and quality of life [4].\nTreatment of hallux limitus involves conservative measures (such as physical therapy, foot orthoses, footwear modification, joint manipulation and injection with corticosteroid) [5], or surgical intervention (either joint-salvage or joint-destructive procedures) [6]. Pharmacological treatment is also often undertaken as an adjunct for pain relief in the management of hallux limitus [6]. However, although non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 inhibitors have been found to be effective in the management of various forms of OA, gastrointestinal complications remain a concern [7]. In light of these limitations with existing treatments, an alternative treatment termed 'viscosupplementation' – the intra-articular injection of hyaluronan into arthritic joints with the aim of restoring the viscoelasticity of the synovial fluid [8] – has been proposed and has attracted considerable attention in the medical literature as a treatment for OA [9]. In particular, both the American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) recommend hyaluronan in the management of OA of the knee [10,11]. Although the results of systematic reviews investigating the effectiveness of this type of treatment for knee OA are controversial, the most recent update of the Cochrane systematic review evaluating viscosupplementation for the treatment of knee OA concluded that viscosupplementation was both safe and effective for the treatment of OA and was superior or equivalent to any form of systemic intervention or intra-articular corticosteroids [9,12].\nDespite there being a large number of studies investigating the effectiveness of hyaluronan for knee OA, few studies have investigated the effects of this form of treatment for OA at the first MPJ [13]. In a case-series retrospective study, 14 patients with radiographically confirmed OA at the first MPJ that received up to 3 intra-articular injections of 1 ml hyaluronan (Ostenil® Mini) (sodium hyaluronate) reported a statistically significant reduction in pain (reported using a visual analogue scale) after 6 months [14]. The treatment was well tolerated, with 3/14 (21%) participants reporting mild adverse reactions at the injection site. In another study, Pons et al[13] compared a single intra-articular injection of 1 ml Ostenil® Mini (sodium hyaluronate) with 1 ml Trigon depot® (triamcinolone acetonide, a corticosteroid) for the treatment of painful, grade 1 hallux limitus (Karasick and Wapner [15] scale) in 37 participants (40 feet) [13]. Both treatment groups showed statistically significant reductions in pain at rest or on palpation for up to 12 weeks post-injection. However, hyaluronan treatment resulted in a statistically significant greater reduction in pain during walking and greater improvement in the American Orthopaedic Foot and Ankle Society (AOFAS) hallux MPJ score compared to treatment with triamcinolone acetonide. The treatment with hyaluronan was well tolerated, with 2/20 (10%) participants reporting mild adverse reactions at the injection site.\nAlthough both of these studies suggest that intra-articular hyaluronan is safe and effective for the treatment of hallux limitus, neither used a placebo control group [13,14]. This limitation is significant as a placebo effect can account for 79% of the efficacy of intra-articular hyaluronan treatment [16]. Further, both studies are limited in that neither of the studies used blinding of both the participants and assessors in their protocols. It is therefore possible that the positive effects of hyaluronan may have been overestimated. Accordingly, the aims of this project are to conduct a double blind randomised controlled trial to determine the effectiveness of intra-articular hyaluronan (Synvisc®) on (i) foot pain and function; (ii) the range of motion of the first MPJ; (iii) the strength of the plantarflexor muscles of the first MPJ; (iv) the health related quality of life; and (v) the use of pain-relieving medications in people with hallux limitus. The study protocol is presented in this paper, consistent with the recommendations of Editorial Board of BioMed Central [17].\n\nMethods\nDesign\nThis study is a parallel group, participant and assessor blinded, randomised controlled trial with a 6 month follow-up (Figure 1). It has been developed using the principles described by Osteoarthritis Research Society International (OARSI) Clinical Trials Task Force guidelines [18]. Participants will be randomised to receive a single intra-articular injection of up to 1 ml hyaluronan (Synvisc®) or sterile saline (placebo) into the first MPJ. Allocation to either the Synvisc® or placebo groups will be achieved using a computer-generated random number sequence. The allocation sequence will be generated and held by an external person not directly involved in the trial. Concealment of the allocation sequence will be ensured as each participant's allocation will be contained in a sealed opaque envelope. Envelopes will be made opaque by using a sheet of aluminium foil inside the envelope. In addition, a system using carbon paper will be employed so the details (name and date of recruitment) are transferred from the outside of the envelope to the paper inside the envelope containing the allocation prior to opening the seal. Assessors and participants will be blinded to group allocation. Participants will be given the option of a second and final intra-articular injection (of Synvisc® or sterile saline according to the treatment group they are in) on days 30 or 90 if there is no improvement in pain and the participant has not experienced severe adverse effects after the first injection).\n\nParticipants\nThe Human Studies Ethics Committee at La Trobe University (Human Ethics Committee Application No. 07-45) and the Radiation Advisory Committee of the Victorian Department of Human Services have given approval for the study. Written informed consent will be obtained from all participants prior to their participation. People with hallux limitus will be recruited from a number of sources:\n(i) advertisements in relevant Melbourne (Australia) newspapers;\n(ii) mail-out advertisements to health-care practitioners in Melbourne;\n(iii) advertisements using relevant internet web-sites (including );\n(iv) posters displayed in local retirement villages, community centres and universities located in Melbourne.\nRespondents will initially be screened by telephone interview to ensure they are suitable for the study. Suitable individuals will then be invited to participate in the study and attend an initial assessment.\nTo be included in the study, participants must meet the following inclusion criteria:\n(i) be aged at least 18 years;\n(ii) report having symptoms of pain, during walking or rest, in the first MPJ for at least 3 months;\n(iii) report having pain rated at least 20 mm on a 100 mm visual analogue pain scale (VAPS);\n(iv) have pain upon palpation of the dorsal aspect of the first MPJ;\n(v) radiographic evidence of OA (score 1 or 2 for either osteophytes or joint space narrowing using a previously published radiographic classification) [19] at the first MPJ.\n(vi) able to walk household distances (>50 meters) without the aid of a walker, crutches or cane;\n(vii) be willing to attend the La Trobe University Medical Centre (Melbourne, Australia) for treatment with either Synvisc® or placebo (single intra-articular injection) and attend the Health Sciences Clinic at La Trobe University (Melbourne, Australia) for the initial assessment and the outcome measurements (at baseline and 1, 3 and 6 months post-treatment);\n(viii) not receive other intra-articular injections into the first MPJ during the course of the study, apart from those dictated by the study;\n(ix) be willing to discontinue taking all pain-relieving medications (analgesics and non-steroidal anti-inflammatory medications (NSAIDs), except paracetamol up to 4 g/day, taken by mouth or applied topically):\n- for at least 14 days prior to the baseline assessment;\n- during the study period (6 months after the final treatment with Synvisc®).\nParticipants who do take paracetamol need to discontinue its use at least 24 hours prior to the baseline assessment and follow-up assessments at 1, 3 and 6 months after the treatment;\n(x) be willing to not receive any physical therapy on the involved MPJ or trial of shoe modifications or foot orthoses during the study period.\nExclusion criteria for participants in this study will be:\n(i) Severe radiographic evidence of OA (score 3 for either osteophytes or joint space narrowing) at the first MPJ using a previously published radiographic classification [19];\n(ii) previous surgery on the first MPJ;\n(iii) intra-articular steroid, or any other intra-articular injection at the first MPJ in the previous 6 months;\n(iv) treatment with systemic steroid (excluding inhalation or topical steroids), immunosuppressives or anticoagulants (except for acetylsalicylic acid at dosages of up to 325 mg/day);\n(v) presence of joint infection(s) of the foot;\n(vi) significant deformity of the first MPJ including hallux abducto valgus (grade of 3 or 4 scored using the Manchester Scale [20];\n(vii) presence of peripheral vascular disease. Peripheral vascular disease will be considered to be present if any of the following are present [21];\n▪ past history of, vascular surgery, Raynaud's phenomenon, vasculitis associated with connective tissue diseases, Buerger's disease, arterial emboli, deep vein thrombosis or lower limb ulcers;\n▪ history of intermittent claudication or rest pain;\n▪ presence of atrophy, ulcers or significant oedema;\n▪ inability to palpate at least one pedal pulse;\n▪ Ankle Brachial Pressure Index <0.9;\n(viii) presence of one or more conditions that can confound pain and functional assessments of the first MPJ, such as metatarsalgia, plantar fasciitis, pre-dislocation syndrome, sprains of the foot, Achilles tendinopathy, degenerative joint disease of the foot (other than the first MPJ) or painful corns and callus;\n(ix) planning to undergo any surgical procedure or receive any injections, apart from those dictated by the study, at the involved first MPJ during the study period;\n(x) presence of systemic inflammatory condition or infection, such as inflammatory arthritis, diagnosed with rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, septic arthritis, acute pseudogout, or any other connective tissue disease;\n(xi) evidence of gout or other musculoskeletal disease other than OA within the feet. Gout will be screened for using clinical history and physical assessment (painful joint, abrupt onset, swelling), radiographic assessment (asymmetrical joint swelling, subcortical cysts without erosion and tophi) as well as serum uric acid levels (hyperuricaemia = serum uric acid > mean + 2 SD from normal population) [22];\n(xii) active skin disease or infection in the area of the injection site;\n(xiii) any medical condition that, in the opinion of the investigators, makes the participant unsuitable for inclusion (e.g., severe progressive chronic disease, malignancy, bleeding disorder, clinically important pain in a part of the musculoskeletal system other than the first MPJ, or fibromyalgia);\n(xiv) pregnant or lactating women, or women who are of child bearing age or have not undergone menopause (Synvisc® has not been tested in pregnant women or women who are nursing);\n(xv) cognitive impairment (defined as a score of < 7 on the Short Portable Mental Status Questionnaire) [23];\n(xvi) known hypersensitivity (allergy) to hyaluronan preparations, or to avian proteins, feathers or egg products;\n(xvii) involvement in any clinical research study in the previous 3 months that could be considered to affect the results of this study.\n\nIntra-articular injections for the treatment groups\nParticipants will be randomised to receive a single intra-articular injection of up to 1 ml of hyaluronan (Synvisc®; Genzyme Biosurgery, Genzyme Corporation, NJ, USA) or sterile saline (placebo) into the first MPJ. Each 2 ml ampoule of Synvisc® contains 16 mg of hylan G-F 20 (cross-linked hylan polymers; hylan A and B), 17 mg sodium chloride, 0.32 mg disodium hydrogen phosphate, 0.08 mg sodium dihydrogen phosphate monohydrate. The hyaluronan is extracted from chicken combs and the purified material has an average molecular weight of 6,000 kDa.\nThe injections will be performed by the same experienced interventional radiologist (AEZ) using fluoroscopic imaging to ensure accurate deposition of the hyaluronan within the joint. As the Synvisc® is provided in ampoules that are labelled with the product name, it will not be possible to blind the injector, however this person is not involved in generation of the allocation order, recruitment, assessment or data analysis. The intra-articular injection will be performed using a 21 gauge (0.80 × 19 mm) Surflo® (Terumo® Corp., Tokyo, Japan) winged infusion set under aseptic procedures. Either a dorso-lateral or dorso-medial approach for injection will be used at the discretion of the injector (depending on which approach provides minimum interference from the osteophytes at the first MPJ joint margins). No anaesthetic will be used. If the participant has bilateral painful first MPJs, only one side (the most painful side) will be treated and used for data collection. The injector will record the volume of the agent that is injected.\nParticipants will be given the option of a second and final intra-articular injection (of Synvisc® or sterile saline according to the treatment group they are in) on days 30 or 90 if there is no improvement in pain (assessed using the VAPS for pain during walking or at rest) and the participant has not experienced severe adverse effects after the first injection).\n\nAssessments\nInitial assessments\nAn initial assessment will be performed to determine the eligibility of participants for this study. Demographic data will be collected including the age, gender, height and weight of participants. Data will also be obtained concerning the presentation of symptoms (foot affected, duration of symptoms). If the participant has bilateral painful first MPJs, the most painful side will be used for data collection and subsequent treatment. To establish eligibility for the study, participants will undergo a clinical assessment, have one set of dorso-plantar and lateral weight-bearing x-rays taken of their feet to grade the severity of first MPJ OA as well as undergo a blood test to assess serum uric acid levels (to exclude gout).\nWeightbearing dorso-plantar and lateral radiographic views will be obtained from both feet with the participant standing in a relaxed bipedal stance position. All x-rays will be taken by the same medical imaging department using a Shimadzu UD150LRII 50 kw/30 kHz Generator and 0.6/1.2 P18DE-80S high speed x-ray tube from a ceiling suspended tube mount. AGFA MD40 CR digital phosphor plates in a 24 cm × 30 cm cassette will be used. For dorso-plantar projections, the x-ray tube will be angled 15° cephalad and centered at the base of the third metatarsal. For lateral projections, the tube will be angled 90° and centered at the base of the third metatarsal. The film focus distance will be set at 100 cm [19].\n\nBaseline assessments and outcome measures\nParticipants who are eligible for the study will be invited to attend a baseline assessment. During the baseline assessment, participants will undergo primary and secondary outcome measurements prior to receiving their injection. The outcome measurements have been developed in accordance of the recommendations of the OARSI Clinical Trials Task Force guidelines [18].\n\nPrimary outcome measures\nOutcome measurements (primary and secondary) will occur at four time-points at baseline, 1, 3 and 6 months post-treatment (after the intra-articular injection of Synvisc® or placebo). The assessor performing the measurements will be blinded as to which treatment group participants have been allocated to. Participants who receive a second treatment at day 30 or 90 will be followed for a further 30 days or 90 days respectively and undergo outcome measurements at 7 or 9 months respectively.\nThe primary outcome measures will be the Pain and Function subscales of the Foot Health Status Questionnaire (FHSQ) [24]. The FHSQ includes 13 questions that assess four domains of foot health, Foot pain, Foot function, Footwear and General foot health. The FHSQ has been subjected to an extensive validation (content, criterion and construct validity) process. It has a high test-retest reliability (intraclass correlation coefficients ranging from 0.74 to 0.92) and a high degree of internal consistency (Cronbach's α ranging from 0.85 to 0.88) [24]. Rigorous reviews have rated it as one of the highest quality foot health status measures currently available [25-27].\n\nSecondary outcome measures\nThe secondary outcome measures will be:\n(i) Severity of pain\nSeverity of pain at the first MPJ during walking, and during rest, over the past week will be assessed using a 100 mm visual analogue pain scale. The left side of the scale (0 mm) will be labelled \"no pain\" and the right side of the scale (100 mm) will be labelled \"worst pain possible\" for each question [25,28].\n\n(ii) Severity and duration of stiffness at the first metatarsophalangeal joint\nThe severity of stiffness at the first MPJ during walking over the past week will be assessed using a 100 mm visual analogue scale. The left side of the scale (0 mm) will be labelled \"not stiff at all\" and the right side of the scale (100 mm) will be labelled \"most stiff possible\". The average duration of stiffness at the first MPJ over the past week will be assessed using a four category scale response. The responses are: \"none\", \"1–15 minutes\", \"16–30 minutes\" and \"greater than 30 minutes\" [29].\n\n(iii) Passive, non-weightbearing dorsiflexion range of motion of the first metatarsophalangeal joint\nFirst MPJ dorsiflexion range of motion will be measured using a goniometer as the maximum angle at which the hallux cannot be passively moved into further extension in a non-weightbearing position (Figure 2) [30]. The test will be performed two times and the average will be used for analysis. This measurement technique shows high intra-reliability (ICC = 0.95, standard error of mean = 1.3°) [30].\n\n(iv) Plantar flexion strength of the toe-flexors of the hallux\nPlantar flexion strength of the toe-flexors of the hallux will be measured using the Mat Scan® plantar pressure measurement device [31]. Participants will be seated with the hip, knee, and ankle at 90 degrees and their foot placed over the Mat Scan® plantar pressure measurement device (Tekscan, Boston, MA, USA) (Figure 3a). This system consists of a 5-mm thick floor mat (432 × 368 mm) incorporating 2288 resistive sensors (1.4 sensors/cm2) sampling at a rate of 40 Hz. The mat will be calibrated for each participant using his or her own bodyweight before each testing session. Participants will be instructed to use their toe-flexor muscles to maximally push their hallux down on the MatScan® device and forces under the hallux will be recorded (Figure 3b). The test will be performed three times for the hallux and the maximal force will be used for analysis. The test-retest reliability of this measurement technique has previously been shown to be high, with intraclass correlation coefficients (ICCs) = 0.88 (95% CI 0.81 – 0.93) [31].\n\n(vi) Plantar pressure measurement\nPlantar pressures will be recorded during level barefoot walking using the MatScan® system (Tekscan®, Boston, MA, USA). The two-step gait initiation protocol will be used to obtain foot pressure data, as it requires fewer trials than the mid-gait protocol and has similar re-test reliability [32]. Three trials will be recorded, which has been found to be sufficient to ensure adequate reliability of pressure data [32,33]. Following data collection, the Research Foot® software (version 5.24) will be used to construct individual \"masks\" to determine maximum force (kg) and peak pressure (kg/cm2) under seven regions of the foot: hallux, lesser toes, 1st MPJ, 2nd MPJ, 3rd to 5th MPJs, midfoot and heel (Figure 4a). For each region, the median of the three trials will be used for analysis. Typical plantar pressure recordings from a participant are shown in Figure 4b.\n\n(vi) Global satisfaction with the treatment\nGlobal satisfaction with the treatment will be assessed using a 5-point Likert scale, as well as a dichotomous (yes/no) scale. The five point-Likert scale will ask \"How satisfied are you with the treatment you received for your big-toe joint pain?\", and will have the following five responses: \"Dissatisfied\", \"Only moderately satisfied\", \"Fairly satisfied\", \"Clearly satisfied\" and \"Very satisfied\". The dichotomous scale of satisfaction will be answered as \"Yes\"' or \"No\" in response to the question: \"Would you recommend the treatment that you received to someone else with big-toe joint pain\".\n\n(vii) Health related quality of life\nThe Short-Form-36 (version two) (SF-36) questionnaire will be used to assess health related quality of life. The SF-36 is a 36 question survey that measures eight health concepts most affected by disease and treatment. The eight health concepts can then be used to form two summary measures: Physical health and Mental health. The Short Form-36 (SF-36) has been extensively validated and is one of the most widely used instruments to measure health status. The SF-36 shows content, concurrent, criterion, construct, and predictive evidence of validity. The reliability of the eight concepts and two summary measures has been assessed using both internal consistency and test-retest methods. Reliability statistics have exceeded 0.80 [34-37].\n\n(viii) Self-reported magnitude of symptom change\nSelf-reported magnitude of symptom change will be measured using a 15-point Likert scale. The scale will ask participants \"how much have your symptoms in your big-toe joint have changed from the beginning of the study to now?\". The fifteen responses will range from \"A very great deal better\" to \"A very great deal worse\".\n\n(ix) Use of rescue medications to relieve pain at the first metatarsophalangeal joint\nThe number of participants who consumed rescue medication (e.g., paracetamol) and mean consumption of rescue medication to relieve pain at the first MPJ (mean grams of paracetamol/participant/month] will be assessed using a medications diary that participants will self-complete [38,39]. The diary will be returned to the assessor at monthly intervals for analysis.\n\n(x) Frequency and severity of adverse events as safety variables\nThe frequency (number of participants affected and number of cases) and types of adverse events (including adverse drug reactions) in each treatment group during the trial will be recorded using a questionnaire that participants will complete during the follow-up appointments at 1, 3 and 6 months post-treatment [40]. To classify the 'type' of adverse event, a blinded assessor will classify the adverse event as being serious or non-serious [40]. Any serious adverse events, defined as adverse events leading to serious disability, hospital admission, or prolongation of hospitalisation, life-threatening events; or death) will be further classified using the International Classification of Diseases (ICD) codes [41]. Non-serious adverse events will include both local (pain, effusion and heat, with each classified as mild, moderate, severe) and systemic adverse events. An open-response type format will also be available for participant responses.\n\n\n\nSample size\nThe sample size for the study has been pre-specified using an a priori power analysis using the primary outcome measure of the pain domain of the FHSQ [42]. One hundred and forty two participants (i.e. 71 per group) will provide power of 90% to detect a minimally important difference in the pain domain of the FHSQ (i.e. 14 points on the FHSQ questionnaire) with the significance level set at p < 0.05. A difference of 14 points was determined to be a clinically significant difference worth detecting [43] and a standard deviation of 25 was derived from a previous report [44]. This calculation included a 5% drop-out rate [13]. However, we will aim to recruit 150 participants (~75 participants per intervention group). Further, we have conservatively ignored the extra precision provided by covariate analysis when estimating the sample size.\n\nStatistical analysis\nStatistical analysis will be undertaken using SPSS version 14.0 (SPSS Corp, Chicago, Ill, USA) and STATA 8 (Stata Corp, College Station, Tex., USA) statistical software. All analyses will be conducted on an intention-to-treat principle using all randomised participants [45-47]. Missing data will be replaced with the last score carried forward [48]. Standard tests for normal distribution will be used and transformation carried out if required.\nDemographic characteristics (gender, age, weight, height, body mass index) will be determined for the baseline visit for each treatment group. Summary statistics will be calculated for duration of symptoms, side affected (left, right, bilateral), grade of OA at the first MPJ [19] as well as all primary and secondary outcome measurements for each treatment group.\nAnalyses will be conducted on 1, 3 and 6 month outcome measures. The continuously-scored outcome measures at 1, 3 and 6 months will be compared using analysis of covariance with baseline scores and intervention group entered as independent variables [49,50]. The exception to this will be the plantar pressure measurements which will be analysed at baseline, 1, 3 and 6 months using two-way repeated measures analysis of variance statistics. Post-hoc comparisons will be performed using Bonferroni-adjusted t-tests. Nominal and ordinal scaled data will be compared at 1, 3 and 6 months using Mann-Whitney U-tests and chi-square analyses (or Fisher's exact test where appropriate) respectively. Effect sizes will be determined using Cohen's d (continuous scaled data) or odds ratios (nominal scaled data and ordinal scaled data) as appropriate.\nThe outcome measurements obtained at 7 or 9 months for participants that receive a second and final intra-articular injection (of Synvisc® or sterile saline according to the treatment group they are in) on days 30 or 90 respectively, will also be analysed as described above. These analyses will be classified as secondary outcomes.\n\n\nDiscussion\nThis study is a randomised placebo controlled trial designed to investigate the efficacy of intra-articular hyaluronan (Synvisc®) to reduce pain and improve function in people with OA of the first MPJ (hallux limitus). Two studies have previously investigated the efficacy of intra-articular hyaluronan for the treatment of first MPJ OA [13,14]. However, neither of these studies used a placebo control group. To our knowledge, this is the first randomised controlled trial using intra-articular hyaluronan for OA of the first MPJ.\nThe use of a placebo control group is essential for studies evaluating the effects of intra-articular therapies as there is likely to be a large placebo response related to the injection procedure and this may inflate the results in uncontrolled evaluations [51]. Indeed, a recent meta-analysis of hyaluronan for knee OA concluded that a placebo effect accounted for 79% of the efficacy of intra-articular hyaluronan [16].\nThe study protocol and outcome measures have been developed in accordance of the recommendations of the OARSI Clinical Trials Task Force guidelines [18]. The outcome measures are pain and function subscales of the FHSQ, pain and stiffness at the first MPJ, range of motion (dorsiflexion) of the first MPJ, plantar flexion strength of muscles of the first MPJ, generic health related quality of life (SF-36), patient satisfaction with treatment, consumption of rescue medication as well as frequency and nature of adverse effects. These outcomes will be measured at baseline then at 1, 3 and 6 months after treatment. Previous research suggests that the effects of intra-articular hyaluronan persist for up to 12 months following treatment [9,38]. Thus, the use of follow-up assessments at 6 month post-treatment will allow us to determine if the effects, if any, of intra-articular hyaluronan persist in the longer term.\nParticipants will be given the option of a second and final intra-articular injection (of Synvisc® or sterile saline according to the treatment group they are in) on days 30 or 90 if there is no improvement in their symptoms. Although this has the potential to complicate the interpretation of the results of the study, this protocol was included as it is likely to be more reflective of clinical practice [14], and this is in keeping with the pragmatic nature of this trial.\nIn summary, this project is the first randomised controlled trial to be conducted to evaluate the efficacy of intra-articular hyaluronan for reducing pain and improving function in people with hallux limitus. The study protocol, including interventions, have been pragmatically designed to ensure that the study findings are generaliseable to clinical practice. Recruitment for the study will commence in June 2008, and we expect final results to be available in mid-2010.\n\nCompeting interests\nHBM and KBL are Editor-in-Chief and Deputy Editor-in-Chief, respectively, of Journal of Foot and Ankle Research. It is journal policy that editors are removed from the peer review and editorial decision making processes for papers they have co-authored.\n\nAuthors' contributions\nSEM, HBM, KBL and CJH conceived the idea and obtained funding for the study. SEM, HBM, KBL, AEZ and JDL designed the trial protocol. SEM, HBM, KBL and GVZ drafted the manuscript. All authors have read and approved the final manuscript.\n\n\n" ], "offsets": [ [ 0, 34260 ] ] } ]

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[ { "id": "pmcA2453154__text", "type": "Article", "text": [ "A Novel Role of the NRF2 Transcription Factor in the Regulation of Arsenite-Mediated Keratin 16 Gene Expression in Human Keratinocytes\nAbstract\nBackground\nInorganic sodium arsenite (iAs) is a ubiquitous environmental contaminant and is associated with an increased risk of skin hyperkeratosis and cancer.\n\nObjectives\nWe investigated the molecular mechanisms underlying the regulation of the keratin 16 (K16) gene by iAs in the human keratinocyte cell line HaCaT.\n\nMethods\nWe performed reverse transcriptase polymerase chain reaction, luciferase assays, Western blots, and electrophoretic mobility shift assays to determine the transcriptional regulation of the K16 gene by iAs. We used gene overexpression approaches to elucidate the nuclear factor erythroid-derived 2 related factor 2 (NRF2) involved in the K16 induction.\n\nResults\niAs induced the mRNA and protein expression of K16. We also found that the expression of K16 was transcriptionally induced by iAs through activator protein-1–like sites and an antioxidant response element (ARE) in its gene promoter region. Treatment with iAs also enhanced the production and translocation of the NRF2 transcription factor, an ARE-binding protein, into the nucleus without modification of its mRNA expression. In addition, iAs elongated the half-life of the NRF2 protein. When overexpressed in HaCaT cells, NRF2 was also directly involved in not only the up-regulation of the detoxification gene thioredoxin but also K16 gene expression.\n\nConclusions\nOur data clearly indicate that the K16 gene is a novel target of NRF2. Furthermore, our findings also suggest that NRF2 has opposing roles in the cell—in the activation of detoxification pathways and in promoting the development of skin disorders.\n\n\n\nInorganic sodium arsenite (iAs), a ubiquitous element, is one of the most toxic metals present in the natural environment (Bagla and Kaiser 1996). Arsenicals are found as naturally occurring constituents of soil, food, and drinking water (Wu et al. 1989; Yoshida et al. 2004), and exposure to iAs has been associated with a variety of disease outcomes, including disorders of the skin, urinary bladder, liver, and lung (Tchounwou et al. 2004). In particular, skin hyperkeratosis is a characteristic dermatologic lesion associated with ingestion of arsenic from contaminated groundwater (McLellan 2002; Yoshida et al. 2004). There is also a significant association between hyperkeratosis, nonmelanoma skin cancer (e.g., basal cell carcinoma and squamous cell carcinoma), and Bowen disease (Col et al. 1999; Rossman et al. 2004). Furthermore, the pathologic features associated with arsenic-induced hyperkeratosis present as typical acanthotic types of psoriasis-like keratosis, characterized by the aberrant proliferation and terminal differentiation of epidermal keratinocytes (Lee et al. 2006). Many epidemiologic studies have shown that hyperkeratoses are the most frequent precursor lesions of some skin cancers (Bagla and Kaiser 1996; Col et al. 1999).\nThe keratins are the most prominent cytoskeletal proteins in keratinocytes and comprise a large family of proteins that form intermediate filament networks in all epithelial cell types (Moll et al. 1982). Keratin 16 (K16 ) and keratin 6 (K6) genes are constitutively expressed in a number of stratified epithelial levels, including the palmar and plantar epidermis (Moll et al. 1982). In skin diseases characterized by aberrant proliferation and differentiation, such as psoriasis and cancer, K16 is detectable at higher levels compared with normal tissue (Haider et al. 2006). Furthermore, the tissue-specific overexpression of wild-type K16 in the epidermis of transgenic mice results in the hyperproliferation of keratinocytes and aberrant keratinization of cornified layers, leading to hyperkeratosis of the skin (Takahashi et al. 1994).\nNuclear factor erythoid-derived 2 related factor 2 (NRF2), a “cap ’n’ collar” basic leucine zipper transcription factor, regulates a transcriptional program that maintains cellular redox homeostasis and protects cells from oxidative stress and xenobiotic agents (Ishii et al. 2000; Moi et al. 1994). Several detoxifying and antioxidant genes, including glutathione-S-transferases (GSTs), heme oxygenase-1 (HMOX1), and thioredoxin (TXN), are regulated by NRF2 through the antioxidant responsive element (ARE) in the respective promoter regions of these genes (McMahon et al. 2001; Wakabayashi et al. 2004). NRF2 is held in the cytoplasm by a cytoskeletal-associated specific inhibitory protein (kelch-like ECH-associated protein 1; KEAP1) under normal cellular redox state conditions, where it is continuously targeted by the proteasomal degradation pathway (McMahon et al. 2003). Upon exposure of the cell to oxidative stress or electrophiles, NRF2 can escape this KEAP1-mediated repression, translocate to the nucleus, and activate the expression of its target genes (Dinkova-Kostova et al. 2002; McMahon et al. 2003).\nRecently, studies of Keap1–/–mice have shown that NRF2 accumulates in the nucleus and constitutively activates the transcription of its target genes, even in the absence of stress signals (Wakabayashi et al. 2003). Most interestingly, however, the skin, esophagus, and forestomach of Keap1-deficient mice show cornified layer and hyperkeratosis phenotypes. In addition, previous studies have also shown that the expression of NRF2 and ARE-controlled genes is induced by iAs in some cell types (Pi et al. 2003; Sakurai et al. 2005). Furthermore, histochemical analyses have indicated that the expression of K16 is increased in Bowen disease, basal cell carcinoma, and squamous cell carcinoma induced by arsenicals (Yu et al. 1993). However, it remains to be determined whether NRF2 can regulate the transcriptional activation of K16 upon iAs exposure in human keratinocytes. Hence, these findings prompted us to investigate the molecular mechanisms underlying the regulation of the K16 gene by iAs-induced NRF2 mediation.\nMaterials and Methods\nChemicals and reagents\nA purified preparation of inorganic sodium arsenite (iAs; NaAsO2; Merck, Darmstadt, Germany) was dissolved in phosphate-buffered saline (PBS) and added directly to the culture medium. A fresh iAs solution was prepared for each new experiment. Cycloheximide (CHX), dimethyl-sulfoxide (DMSO), and a protease inhibitor cocktail were purchased from Sigma (St. Louis, MO, USA). CHX was dissolved in DMSO and stored –20°C until use.\n\nCells and culture conditions\nThe human keratinocyte HaCaT cell line was obtained from N.E. Fusenig (German Cancer Research Center, Heidelberg, Germany). Cells were maintained in monolayer cultures in 95% air and 5% CO2 at 37°C in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin and 50 mg/mL streptomycin and nonessential amino acids (Gibco BRL, Paisley, UK).\n\nRNA preparation and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis\nWe determined RNA expression levels by semiquantitative RT-PCR analysis as described previously (Sugioka et al. 2004). Total RNA was isolated from HaCaT cells using the GeneElute Mammalian Total RNA Kit (Sigma). The specific primers used for first-strand cDNA synthesis and PCR were as follows: K16 [forward, 5′-GAT GCT TGC TCT GAG AGG TC-3′, and reverse, 5′-CCA GCA AGA TCT GGT ACT CC-3′; Gene Bank accession no. NM_005557 (National Center for Biotechnology Information 2007)]; c-Jun (forward, 5′-CCT GTT GCG GCC CCG AAA CT-3′, and reverse, 5′-ACC ATG CCT GCC CCG TTG AC-3′; NM_002228); c-Fos (forward, 5′-TTT GCC TAA CCG CCA CGA TGA T-3′, and reverse, 5′-TTG CCG CTT TCT GCC ACC TC-3′; NM_005252); NRF2 (forward, 5′-AGA TTC ACA GGC CTT TCT CG-3′, and reverse, 5′-CAG CTC TCC CTA CCG TTG GA-3′; AF323119); KEAP1 (forward, 5′-CAG AGG TGG TGG TGT TGC TTA T-3′, and reverse, 5′-AGC TCG TTC ATG ATG CCA AAG-3′; NM_012289); TXN (forward, 5′-CAG GGG AAT GAA AGA AAG G-3′, and reverse, 5′-CAA GGT GAA GCA GAT CG-3′; NM_003329), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control (forward, 5′-ACC ACA GTC CAT GCC ATC AC-3′, and reverse, 5′-TCC ACC ACC CTG TTG CTG TA-3′, NM_002046). PCR products were separated on a 1.8% agarose gel and stained with ethidium bromide.\n\nWestern blot analysis\nWe performed Western blot analysis as described previously (Sugioka et al. 2004). Briefly, nuclear and cytoplasmic proteins were extracted using the NE-PER nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. For protein extraction, the cells were lysed in a buffer containing complete protease inhibitor cocktail. We measured protein concentrations using the DC Protein Assay Kit (Bio-Rad, Richmond, CA, USA). Equal amounts of protein were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Amersham Biosciences, Bucks, UK). Immunoblotting was carried out with specific antibodies in Tris-buffered saline with 0.05% Tween 20. The primary antibodies were as follows: K16 (Neomarkers, Fremont, CA, USA), NRF2 and KEAP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-Jun (Cell Signaling, Beverly, MA, USA), and β-actin (Sigma). After washing, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies and developed by chemiluminescence using the ECL Plus Detection Kit (Amersham Biosciences).\n\nPlasmids, transfections, and luciferase assays\nHuman K16 promoter regions of varying lengths (pXK-1, 3, 4, 5-1, and 5-2) were provided by Y-N Wang (National Cheng Kung University, Taiwan). These DNA fragments were prepared from HaCaT cells and were ligated into the pXP-1 luciferase vector (Wang and Chang 2003). The p3xARE/Luc vector, harboring three tandem repeats of ARE, was donated by X.L. Chen (Discovery Research, AtheroGenics Inc., Alpharetta, GA, USA) (Chen et al. 2003). The wild-type NRF2 expression vector (WT-NRF2) was a gift from H.S. So (Wonkwang University School of Medicine, Korea) (So et al. 2006). NRF2 cDNA was subcloned into a pcDNA3.1(+) vector (Invitrogen, San Diego, CA, USA). For the transfection of reporter plasmids, we seeded HaCaT cells into six-well plates at a density of 80% the previous day. Cells were then transfected with a total of each luciferase reporter construct (2.5 μg) using LipofectAMINE plus (Invitrogen). To control for the efficiency of transfection, Renilla luciferase gene expression was monitored using either the pRL-CMV or pRL-TK vectors (Promega, Madison, WI, USA). For overexpression of WT-NRF2, we normalized the total plasmid concentration using the pcDNA3.1(+) empty vector. Thirty-six hours after transfection, the medium was replaced with fresh medium containing either vehicle (PBS) or iAs for 6 hr. After iAs exposure, we harvested cells and analyzed them for luciferase activity using a Dual-Luciferase Reporter Assay System (Promega).\nFor the investigation of the role of NRF2 in regulating K16 gene expression, transfection of an NRF2 expression plasmid into HaCaT cells was carried out using LipofectAMINE 2000 (Invitrogen). Cells were cultured in 100-mm plates 24 hr before transfection. The expression plasmid WT-NRF2 (15 μg) was then transfected into the cells for 48–60 hr. As a negative control, we used 15 μg of the pcDNA3.1(+) empty vector.\n\nElectrophoretic mobility-shift assays (EMSA)\nWe extracted and measured nuclear proteins as described above. Nuclear protein/ DNA complexes were subjected to electrophoresis in nondenaturing 5% polyacrylamide gels containing 2% glycerol in 0.25% Tris-borate/EDTA buffer and transferred to Hybond-N+ nylon transfer membranes (Amersham Biosciences) for detection using the Light-Shift EMSA kit (Pierce) according to the manufacturer’s protocol, with minor modifications. We incubated 10-μg aliquots of nuclear extract with the DNA probe in a binding reaction buffer containing 10 mM Tris/HCl (pH 7.6), 50 mM KCl, 0.5 mM dithiothreitol, 0.25 mM EDTA, 5% glycerol, 2.5 mM MgCl2, 0.05% NP-40 detergent, and 2 μg of poly(dI-dC)·poly(dI-dC) for 30 min at room temperature. For supershift assays, 2 μg of either a polyclonal anti-NRF2 or an anti-c-Jun antibody (Santa Cruz Biotechnology) was added with the nuclear protein for 2 hr at 4°C before the labeled oligonucleotide probe was added. Biotin-labeled, double-stranded oligonucleotides WT-K16ARE (–157/–132, 5′-GGGGAACCTGGAGTCAGCAGT-TAGGA-3′), containing an ARE site (–148/ –140, underlined) in the human K16 promoter region, and Mut-K16ARE (5′-GGGGAA-CCTGGAGTCAaaAGTTAGGA-3′, mutated GC box in the ARE) were prepared by Fasmac (Kanagawa, Japan). A consensus ARE probe was purchased from Panomics, Inc. (Redwood City, CA, USA). For competition binding of the K16 ARE-complexes, we used an unlabeled AP-1 consensus oligonucleotide (5′-TATC-GATAAGCTATGAGTCATCCGGG-3′). The binding specificity was confirmed in each case by the addition of a 100-fold molar excess of unlabeled oligonucleotide.\n\nCHX chase experiment\nWe investigated the posttranscriptional regulation of both the steady-state levels and half-life of the NRF2 protein by CHX chase analysis. Cells were incubated in serum-free medium in the absence or presence of iAs for 6 hr. The culture medium was then replaced with serum-free medium containing CHX (100 μg/mL). We prepared cell lysates at 0, 10, 30, 60, 120, and 240 min after iAs treatment. Whole-cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against NRF2.\n\nStatistics\nAll the data generated from at least three independent experiments and expressed as the mean ± SD were analyzed by the Student’s t-test. Statistical comparisons were made by logarithmic transformation of the normalized values. We considered p-values < 0.01 to be statistically significant.\n\n\nResults\nK16 expression is induced by iAs in HaCaT cells\nWe wanted to determine whether the K16 mRNA is transcriptionally regulated by iAs, and treated HaCaT cells with this compound for various time periods over a range of doses. After treatment of HaCaT cells with 1–20 μM iAs, the expression of K16 mRNA was increased compared with the control at 6 hr (Figure 1A) but had declined to basal levels at 24 hr. The increase in the K16 protein levels after 6 hr of iAs exposure was just detectable at 10–20 μM, but a dose-dependent increase was more evident at 10 hr (Figure 1B). This enhancement of K16 expression had declined to basal levels at 24 hr.\n\nIdentification of the iAs responsive region in the K16 gene promoter\nTo investigate the mechanisms underlying the transactivation of the K16 gene by iAs, we first examined the response of the K16 regulatory region to this compound using a luciferase reporter gene assay. The dose-dependent activation of K16 transcription after iAs treatment was observed with a construct containing a 515-bp fragment of the K16 promoter (Figure 2A). To further elucidate the region containing the iAs responsive element, we examined a series of deletions of the 5′-flanking region of K16 gene. The ARE sequence in the pXK-5–1 vector contains an activator protein-1 (AP-1)–like element followed by a GC box. As shown in Figure 2B, an enhancement in the reporter activity levels was observed for the promoter constructs, pXK-1, 3, 4, and 5-1, in response to 20 μM iAs. A decline in reporter activity, however, depended on the number of AP-1–like sites, and the results for the pXK-5-1 construct show also that ARE is activated by iAs. In contrast, no significant activation was observed using a pXK-5-2 construct in response to 20 μM iAs.\n\nExpression of AP-1 transcriptions factor and c-Jun production following iAs treatment\nWe examined AP-1 transcription factors c-Jun and c-Fos expression in iAs-treated HaCaT cells by semiquantitative RT-PCR. iAs-induced c-Jun expression was observed during the first 3 hr after treatment (data not shown). An appreciable induction of c-Jun was also confirmed after 6 hr, but this was down-regulated by 24 hr after iAs treatment (Figure 3A). In contrast, the expression of c-Fos was only transiently detectable at 3 hr (data not shown) but was not observed during the 6–24 hr period of this experiment. As shown in Figure 3B, iAs-enhanced c-Jun production can be observed in a dose-dependent manner at 6 hr, but it declines from 10 to 24 hr.\n\niAs potently induces the translocation of NRF2 and activates the ARE of the K16 promoter\nThe results of our reporter assays suggested that iAs stimulates not only the AP-1–like sites but also the ARE site within the K16 gene promoter in HaCaT cells (Figure 3). In addition, several oxidative stress agents and toxic chemicals, including iAs, have been reported to induce the expression of ARE-dependent genes in several cell types (Pi et al. 2003; Sakurai et al. 2005). On the basis of our observations and some recent reports, we thus hypothesized that iAs would have the ability to activate the ARE of the K16 gene promoter directly, resulting in the induction of K16 expression in HaCaT cells. To confirm that the K16 ARE indeed functions as an iAs-responsive transcriptional control element, we performed transient transfections of HaCaT cells with a p3xARE/Luc construct and then subjected these cells to iAs for 6 hr. As shown in Figure 4A, treatment of HaCaT cells with iAs results in a dramatic increase in ARE-driven promoter activity. Likewise, EMSA using a consensus ARE probe show that iAs-induced ARE-binding complexes increase markedly, in a dose-dependent fashion (Figure 4B). These results indicate that iAs has the ability to activate the ARE-driven genes. We performed further EMSA experiments using an ARE probe specific to the K16 proximal promoter region (WT-K16ARE) and found that K16ARE–nuclear protein complexes formation is augmented by iAs in a dose-dependent manner (Figure 4C). Moreover, the formation of these complexes is specifically inhibited by the addition of excess unlabeled oligonucleotide competitor (Figure 4B,C), whereas an excess of an unlabeled AP-1 probe competes only marginally for K16ARE binding (Figure 4C).\nThe NRF2 transcription factor has been shown to bind to AREs upon translocation into the nucleus, resulting in the induction of ARE-mediated genes (Wakabayashi et al. 2004). To examine whether iAs induces and translocates NRF2 into the nucleus in HaCaT cells, we treated these cells with iAs for either 3 or 6 hr. As shown in Figure 4D, a dose-dependent accumulation of NRF2 protein was observed in the nucleus upon treatment with iAs for 6 hr. This was not observed in the parallel experiment performed over the 3-hr time course.\nSupershift EMSA analysis using an NRF2 antibody showed that the iAs-induced and iAs-translocated NRF2 protein binds to the WT-K16ARE probe containing the ARE sequence of the K16 proximal promoter region (5′-GGAGTCAGC-3′) that comprises an AP-1–like site and a GC box, whereas the supershift of c-Jun was not observed (Figure 4E). To identify whether the GC box is dispensable for the iAs-stimulated binding activity of NRF2, we next performed EMSA analyses with either WT- or a Mut-K16ARE probe containing an intact AP-1–like element but a mutated GC box. As shown in Figure 4F, the K16ARE–nuclear protein complexes and supershifted bands that were enhanced by iAs treatment were largely abolished by the addition of the Mut-K16ARE probe.\n\niAs stabilizes the NRF2 protein\nWe examined the effects of iAs treatment on the function of KEAP1 in HaCaT cells. Treatment with iAs did not alter the expression levels of KEAP1 mRNA or protein over either a 3 or 6 hr time course (Figure 5A). Next, we examined the effects of iAs on the expression of NRF2 mRNA in HaCaT cells. Exposure to iAs did not significantly alter the steady-state levels of NRF2 mRNA (data not shown). Production of NRF2 protein, however, was observed to increase in both a dose-and time-dependent manner (Figure 4D). To further examine the stabilization of NRF2 protein by iAs, we monitored the decay of basal and iAs-induced NRF2 proteins after inhibition of protein synthesis by CHX (Figure 5B). The results of this analysis revealed that the NRF2 protein levels decrease by approximately 50% within 30 min of treatment with CHX in cells that had not been exposed to iAs. Only trace amounts of NRF2 are then detectable after 60 min of exposure to CHX in these cells. The HaCaT cells were then pretreated with iAs for 6 hr before their exposure to CHX in a similar timecourse experiment. The levels of NRF2 in these iAs-treated cells were again found to decrease by about 50%, but only after 120 min of CHX exposure.\n\nNRF2 plays a crucial role in the regulation of K16 gene expression in HaCaT cells\nTo confirm the functional role of NRF2 in the induction of K16 gene expression by iAs, we investigated whether the expression of K16 mRNA is induced by the overexpression of NRF2 (WT-NRF2) in HaCaT cells. We also investigated the expression of the detoxification gene TXN, which is highly induced by a variety of oxidative stimuli through NRF2-mediated ARE transactivation (Kim et al. 2001). The expression of TXN gene in untransfected cells after treatment with iAs was stronger than that of the control cells (Figure 6A). When the cells were transfected with WT-NRF2 and then treated with or without iAs, the expression of TXN mRNA was augmented markedly compared with the empty-vector control. Similarly, the expression of K16 mRNA was also induced in cells transfected with WT-NRF2 in the absence or presence of iAs. We next performed a transient transfection of HaCaT cells with the pXK-5–1 luciferase vector together with the WT-NRF2 vector. The overexpression of NRF2 in increasing concentrations resulted in significant enhancement of the ARE-mediated K16 promoter activation (Figure 6B).\n\n\nDiscussion\nIn the present study, we showed for the first time that iAs induces the transcriptional activation of K16 in the human keratinocyte cell line, HaCaT, through the ARE present in its gene promoter. It has been reported previously that treatment with iAs enhances the production and translocation of NRF2 into the nucleus in several cell types. However, until now it has remained uncertain whether the induction of NRF2 by iAs mediates the transcriptional activation of the K16 gene in keratinocytes. In our current experiments, we demonstrated that iAs elongates the half-life of the NRF2 protein, which results in its increased expression levels. Furthermore, this iAs-induced NRF2 protein was shown to bind to the ARE sequences in the promoter region of the K16 gene. Finally, by overexpressing NRF2, we have clarified that its induction is involved in not only the gene expression of the detoxification gene TXN, but also in the upregulation of K16 expression in HaCaT cells through the ARE in the K16 gene promoter. These experiments indicate an important and novel function for NRF2 in the regulation of K16 in keratinocytes and also help to further explain the molecular mechanisms underlying arsenic-mediated epidermal hyperkeratosis.\nIn our present experiments, the expression levels of K16 mRNA and protein were indeed found to be enhanced by iAs in a dose-dependent manner (Figure 1). In addition, luciferase assays of the K16 promoter revealed that iAs enhances its activity in a dose-dependent fashion, which is stimulated by AP-1–like sites and an ARE (Figure 2). The promoter of the human K16 gene was recently cloned and sequenced, and several AP-1–like sites were found within the –515-bp region of the gene (Wang and Chang 2003). AP-1 transcription factor can be formed by the dimerization of either Jun or Jun/Fos family members (Eferl and Wagner 2003). In the present study, the increased expression of c-Jun, but not c-Fos was evident in the nuclei of HaCaT cells after iAs treatment (Figure 3). Our findings thus suggest that the activation of c-Jun/AP-1 is one of the essential steps in the regulation of K16 gene expression by iAs exposure in HaCaT cells.\nIt has been well documented that the ARE core sequence includes an AP-1–like binding site (TGAC/GTCA), followed by a GC box (Rushmore et al. 1991; Xie et al. 1995). We have found in our current analyses that the AP-1–like site within the K16 promoter region from –148 to –140 bp (5′-GGAGTCAGC-3′) resembles a consensus ARE sequence. Recent studies have shown that AREs can be specifically bound by complexes of several basic-leucine zipper transcription factors, including NRF2 (Ishii et al. 2000; Moi et al. 1994). NRF2 heterodimerizes with either AP-1 or small MAF (MAFG, MAFK, and MAFF) proteins (MAF, v-maf musculoaponeurotic fibrosarcoma oncogene homolog) and binds to the ARE to induce the transcription of ARE-mediated genes (Motohashi et al. 2002). In the present investigation, EMSA and supershift assays showed that the NRF2 proteins in the nuclei bind to the ARE sequences of K16 promoter region after iAs exposure. iAs-induced c-Jun, however, does not bind to this ARE (Figure 4E). c-Jun may thus act on other AP-1 sites within the K16 promoter region. These results also suggest that other heterodimer partners of NRF2 are involved in the ARE regulation of K16 promoter region underlying iAs-mediated the K16 gene expression. Gel shifts with an K16Mut-ARE probe (harboring a mutation in the ARE GC box) clearly show that the ARE sequence in the K16 promoter, particularly the terminal GC dinucleotide, is essential for mediating iAs-induced K16 transactivation and NRF2 binding (Figure 4F). Several investigations have suggested that the GC nucleotides within the ARE are essential for both the basal and oxidative stress–induced activities of the ARE-related genes, NAD(P)H dehydrogenase quinone 1 (NQO1) and glutamate-cysteine ligase catalytic subunit (GCLC) (Wasserman and Fahl 1997; Wild et al. 1998). Our current results are consistent with these earlier studies in showing that the formation of the iAs-responsive NRF2/ARE complexes is reduced by a mutation in the GC box. Collectively, our present observations reveal a new molecular mechanism in which iAs-induced K16 gene expression is also regulated by activation NRF2/ARE pathways.\nIt has been widely accepted that oxidative stress disrupts sequestration of NRF2 by KEAP1, permits NRF2 translocation to the nucleus, and transactivates the expression of various NRF2-mediated genes (Dinkova-Kostova et al. 2002; McMahon et al. 2003). Our present study showed that iAs elongates the half-life of the NRF2 protein but has no effects upon KEAP1 expression (Figure 5). Other studies have also demonstrated that the production of NRF2 is increased by various inducers via posttranscriptional control (Nguyen et al. 2003; Stewart et al. 2003). Several earlier reports also indicated that either oxidative stress or antioxidant substances stabilize the expression of the NRF2 protein, either by directly modifying the cysteine residues on KEAP1 to disrupt the NRF2/KEAP1 complex (Dinkova-Kostova et al. 2002) or by facilitating the release of NRF2 through the phosphorylation of the NRF2/KEAP1 complex (Bloom and Jaiswal 2003). These findings are largely consistent with our present finding that iAs stabilizes the expression of NRF2 in HaCaT cells by elongating the protein half-life.\nRecently, Wakabayashi et al. (2003) demonstrated that NRF2 accumulates in the nucleus at constitutively high levels and produces various cytoprotective genes in embryonic fibroblast- and liver-derived Keap1-null mice. Surprisingly, these Keap1-deficient mice also show a thicker stratum corneum epidermis, abnormal keratinization, and cornification in the esophagus and forestomach (hyperkeratosis). K6 was found to be strongly expressed in the esophageal epithelium of these mice. These results indicate that K6 is also a target gene of NRF2. In addition, the promoter of the K6 gene bears a remarkable sequence similarity to the K16 promoter (Jiang et al. 1993). Therefore, we examined whether K16 gene expression is also regulated by NRF2. In the present study, the gene expression and transactivation of K16 were dramatically induced by transfection with WT-NRF2 via in HaCaT cells, clearly demonstrating that NRF2 acts as a direct transcriptional regulator of the K16 gene (Figure 6). In addition, we also showed that transfection of HaCaT cells with WT-NRF2 induces the expression of detoxification gene TXN (Figure 6A). NRF2 may thus have a major role to play in the development of hyperkeratosis, whereas the expression and induction of NRF2 is implicated in cell protection against a variety of genotoxic and cytotoxic effects. Hence, based on these results and on the findings from studies of Keap1 knockout mice, iAs may both cause hyperkeratosis and induce detoxification enzymes via the modification of NRF2. Given that there are both beneficial and adverse effects of NRF2 activity, caution will therefore be needed when using antioxidants for prevention and therapy. Although further investigations are needed, we believe that our findings provide important clues for the design of future therapies for arsenic-mediated hyperkeratosis and for treatments involving the molecular targeting of NRF2.\n\nConclusion\nOur findings clearly demonstrate that the induction of the K16 gene in human keratinocytes by iAs depends on NRF2 activation. Our results thus represent a valuable initial effort to elucidate the relationship between the K16 gene and the NRF2 transcription factor, which may be responsible for hyperkeratosis.\n\n\n" ], "offsets": [ [ 0, 29472 ] ] } ]

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[ { "id": "pmcA2518116__text", "type": "Article", "text": [ "CHEK2 Mutations Affecting Kinase Activity Together With Mutations in TP53 Indicate a Functional Pathway Associated with Resistance to Epirubicin in Primary Breast Cancer\nAbstract\nBackground\nChemoresistance is the main obstacle to cure in most malignant diseases. Anthracyclines are among the main drugs used for breast cancer therapy and in many other malignant conditions. Single parameter analysis or global gene expression profiles have failed to identify mechanisms causing in vivo resistance to anthracyclines. While we previously found TP53 mutations in the L2/L3 domains to be associated with drug resistance, some tumors harboring wild-type TP53 were also therapy resistant. The aim of this study was; 1) To explore alterations in the TP53 gene with respect to resistance to a regular dose epirubicin regimen (90 mg/m2 every 3 week) in patients with primary, locally advanced breast cancer; 2) Identify critical mechanisms activating p53 in response to DNA damage in breast cancer; 3) Evaluate in vitro function of Chk2 and p14 proteins corresponding to identified mutations in the CHEK2 and p14(ARF) genes; and 4) Explore potential CHEK2 or p14(ARF) germline mutations with respect to family cancer incidence.\n\nMethods and Findings\nSnap-frozen biopsies from 109 patients collected prior to epirubicin (as preoperative therapy were investigated for TP53, CHEK2 and p14(ARF) mutations by sequencing the coding region and p14(ARF) promoter methylations. TP53 mutastions were associated with chemoresistance, defined as progressive disease on therapy (p = 0.0358; p = 0.0136 for mutations affecting p53 loop domains L2/L3). Germline CHEK2 mutations (n = 3) were associated with therapy resistance (p = 0.0226). Combined, mutations affecting either CHEK2 or TP53 strongly predicted therapy resistance (p = 0.0101; TP53 mutations restricted to the L2/L3 domains: p = 0.0032). Two patients progressing on therapy harbored the CHEK2 mutation, Arg95Ter, completely abrogating Chk2 protein dimerization and kinase activity. One patient (Epi132) revealed family cancer occurrence resembling families harboring CHEK2 mutations in general, the other patient (epi203) was non-conclusive. No mutation or promoter hypermethylation in p14(ARF) were detected.\n\nConclusion\nThis study is the first reporting an association between CHEK2 mutations and therapy resistance in human cancers and to document mutations in two genes acting direct up/down-stream to each other to cause therapy failure, emphasizing the need to investigate functional cascades in future studies.\n\n\n\nIntroduction\nChemoresistance is the main obstacle to cure in most malignancies, including breast cancer. While adjuvant chemotherapy may reduce the hazard rate of relapse by about one third in breast cancer patients [1], the majority among patients harboring micro- metastases are not cured by today's standards. Considering patients harboring distant metastases, resistance and therapy failure inevitably occurs, in general over a time period of less than one year for each individual regimen [2].\nDespite extensive experimental research [3], little data are available considering chemoresistance in vivo. For anthracycline therapy in breast cancer, topoisomerase-II amplifications have been associated with a dose-responsiveness different from what is observed in non-amplified tumors 4, 5. Several studies have tried to generate “prediction profiles” based on gene expression microarrays [6], [7], [8], however, none of the different profiles generated expressed a sensitivity suitable for clinical applications, or have been successfully reproduced by others (see references to original works in [9] and [10]).\np53 (the protein encoded by the TP53 gene) plays a key role in executing DNA-damage induced apoptosis and growth arrest [11]. Previously, our group reported mutations in the zink-binding domains L2 (codons 163–195) and L3 (codons 236–251) of p53 critical to DNA binding [12] to be associated with but not fully predictive for resistance to chemotherapy with a low-dose weekly anthracycline [13] or a mitomycin plus 5-fluoro-uracil containing [14] regimen. Similar findings were reported by another group [15]. In contrast, others reported TP53 mutations to predict sensitivity to a dose-dense epirubicin-cyclophosphamide regimen [16].\nThe finding that some tumors harboring wild-type TP53 may be resistant to anthracycline therapy lead us to postulate that other genes involved in the p53 pathway could be mutated in these tumors [3]. p53 is activated by post-translational modifications, and the protein is phosphorylated at multiple amino acids [17]. Phosphorylation at Ser 20 (Ser 23 in mice) by the Chk2 protein (coded by the CHEK2 gene) in response to DNA damage activates p53 by inhibiting binding to, and deactivation by, the MDM2 (Mouse Minute 2 homolog; HDM2) protein [18], [19], [20]. While experimental studies have suggested a critical role of Chk2 in activating p53 apoptotic response to genotoxic stress [21], [22], others claim Chk2 to be dispensable for p53 activation with respect to apoptosis as well as growth arrest [23]. Following an initial report of a CHEK2 germline mutation in a family filling the characteristics of a Li-Fraumeni syndrome (LFS) [24], recent papers have suggested germline mutations in CHEK2 to be associated with a moderately increased risk of breast and colon cancers (see references in [25]). Recently, we discovered a somatic, nonsense CHEK2 mutation in a single patient expressing resistance to doxorubicin low dose therapy [26].\nA second mechanism of p53 activation is through p14(ARF) (p19 in mice) function. p14(ARF) does not phosphorylate p53, but inhibits MDM2 dependent p53 degradation through direct MDM2 binding. While p14(ARF)-mediated p53 activation has been linked to oncogene-induced p53 activation and, in general, considered not involved in response to DNA damage (see references in [27]), p14(ARF) may be activated through the E2F1/retinoblastoma pathway [28]. Importantly, two recent studies revealed lack of p19 (mouse homologue of human p14(ARF)) function in mice to inhibit p53 tumor suppressor function in response to ionizing radiation as well as DNA damaging agents [29], [30].\nThe aim of this study was 1) to explore alterations in the TP53 gene with respect to resistance to a regular dose epirubicin regimen (90 mg/m2 body surface every 3 week) in patient with primary, locally advanced, breast cancer; 2) To explore defects in potential mechanisms activating p53 in response to DNA damage in breast cancer as a cause of drug resistance in wild-type tumors. To do so, we sequenced the complete coding regions for the CHEK2 and p14(ARF) genes and analyzed for p14(ARF) promoter hypermetylations; 3) Evaluate in vitro function of potential Chk2 and p14(ARF) protein translates corresponding to identified mutations in the CHEK2 and p14(ARF) genes; 4) Identify potential TP53, CHEK2 and p14(ARF) mutations to be germline, explore the incidence of different cancers among affected relatives with respect to specific mutations. By comparing in vitro characteristics of specific mutations to drug sensitivity and family cancer risk syndromes, this may add to our understanding of the importance of these gene cascades executing response to DNA damage versus tumor suppression activity.\nAnalyzing tumor samples from a total of 109 primary locally advanced breast cancer patients treated with epirubicin 90mg/3 weekly, we found TP53 mutations affecting the L2/L3 domains or protein dimerization, as well as non-functional CHEK2 mutations abrogating dimerization and phosphorylation, to be associated with therapy resistance; no mutation or promoter hypermethylations of the p14(ARF) gene was discovered. Our findings suggest a critical role for Chk2 with respect to DNA-damage-dependent p53 activation and resistance to anthracycline therapy in human breast cancer.\n\nMaterials and Methods\nPatients\nA total of 223 patients with locally advanced non-inflammatory breast cancer (T3-4 and/or N2) were randomly allocated to primary treatment either with epirubicin 90 mg/m2 or paclitaxel 200 mg/m2. The primary aim of the study was identification of markers predicting drug resistance to the regimens. Thus, the reason for randomizing patients was not for effect comparison, but to achieve similar patient cohorts in the two arms. Based on the findings of a clinical lack of cross-resistance between anthracyclines and taxane therapies in breast cancer [31], we hypothesized the mechanisms of resistance to be different between the two compounds. While the analysis of tumor samples from the paclitaxel is ongoing, we here report our findings from the patients allocated to the epirubicin arm.\nThe epirubicin arm included a total of 109 patients (age 28 to 70 years, median 51 years). Two patients were analyzed for gene mutations but omitted from statistical analysis as protocol violators; histopathological examination revealed one patient (Epi089) to harbor a sarcomatoid tumor, while one patient Epi232 was erroneously enrolled with stage II disease.\nThe study protocol was approved by the Regional Ethical Committee (Norwegian Health Region III), including formal Biobank registration in accordance to Norwegian law. The study and protocol is registered under the Norwegian Social Science Data services ((www.nsd/uib/personvern/database/), University of Bergen project no 16297 and Helse Bergen project no 13025). Each patient gave written informed consent.\n\nTissue Sampling\nBefore commencing chemotherapy, each patient had an incisional tumor biopsy as described previously [14]. All tissue samples were snap-frozen immediately on removal in the theatre.\n\nTreatment Regime and Staging\nPrimary treatment consisted of epirubicin (90 mg/m2) administered as a 3-weekly schedule. Treatment was scheduled for four cycles unless progression occurred at an earlier stage. Clinical response was assessed before each treatment cycle, and the final response evaluated 3 weeks after the 4th cycle for overall response classification. Because the protocol was implemented by October 1997 with patients enrolled between November 1997 and December 2003, responses were consistently graded by the UICC system [32] and not the more recently implemented “RECIST” criteria [33]. Thus, responses were classified as CR (Complete Response, complete disappearance of all tumor lesions), PR (Partial Response, reduction ≥50% in the sum of all tumor lesions, calculated for each as the product of the largest diameter and the one perpendicular to it), PD (Progressive Disease, increase in the diameter product of any individual tumor lesion by ≥25%), and SD (Stable Disease, anything between PR and PD). To analyze for the predictive value of the different parameters, similar to our previous studies [13], [14] we compared PD tumors (non responders) with the combined group of tumors classified as SD/PR/CR (responders); the reason for this approach is discussed in detail elsewhere [34]. Median follow-up time was defined from patient inclusion in the study up to October 31, 2006. Deaths attributable to causes other than breast cancer were treated as censored observations.\nAll patient records were subject to central audit for response classification (by E.L., B.Ø. and P.E.L.). Response classifications were completed and approved without any knowledge about result from laboratory analysis.\n\nRNA Purification\nTotal RNA was purified by Trizol (Life Technologies, Inc.) extraction from snap-frozen tissue samples according to manufacturer's instructions. After extraction, the RNA was dissolved in 100 µl of DEPC treated ddH2O. cDNA was synthesized by reverse transcription using Transcriptor reverse transcriptase (Roche), according to the manufacturer's protocol.\n\nDNA Purification\nGenomic DNA from tumor biopsies and blood lymphocytes was isolated using QIAamp DNA Mini kit (Qiagen, Chatsworth, CA) according to the manufacturer's protocol.\n\nMutation Analysis\nAll mutational analysis was performed blinded to clinical data. Mutations in TP53, CHEK2 and p14(ARF) genes were analyzed by PCR (or nested PCR) amplification and sequencing of PCR product, or by cloning of PCR products and sequencing of the resulting plasmids (all primers described in Table 1). Cloning was performed using the TOPO TA Cloning kit (Invitrogen). Sequencing of clones was performed until at least 10 different sequences covered all parts of the CHEK2 coding sequence. DNA sequencing was carried out directly on 1 µl PCR product or plasmid using Big Dye terminator mix (Applied Biosystems). Capillary gel electrophoresis, data collection, and sequence analysis were done on an automated DNA sequencer (ABI 3700). When a mutation was detected, the relevant exon was amplified by PCR from genomic tumor DNA and DNA from blood lymphocytes and sequenced for verification and germline detection. (Primers described in Table 1).\n\nLoss of Heterozygosity (LOH)\nLoss of heterozygosity (LOH) in tumors with mutations in CHEK2 was assessed using the microsatellite marker, D22S275, which maps to intron 4 of CHEK2. LOH in tumors with mutation in TP53 was assessed using two markers, one variable number tandem repeat in intron 1 [35] and a CA repeat close to the TP53 gene [36]. Fluorescently end-labeled primers were used in the PCR, and the PCR products were analyzed on an ABI 3700. LOH was evaluated by comparing the allele peak-height ratios from blood DNA and tumor DNA. A sample was scored as having AI (Allelic Imbalance) when a reduction in peak height of one allele in tumor sample was at least 18% compared with that of blood DNA from the same patient [37].\n\nAnalysis of p14(ARF) promoter methylation\nGenomic DNA was subjected to bisulphate conversion using the CpGenome DNA Modification Kit (Intergen) according to the manufacturer's protocol. Both the unmethylated- and methylated-specific PCRs were performed in 50 µl reaction mixes containing 2.5 U AmpliTaq Gold DNA Polymerase (Applied Biosystems), 1× PCR buffer, 1.5 mM MgCl2, 0.1 mM of each deoxynucleotide triphosphate, 0.2 µM of each primer (Table 1) and 2 µl of modified genomic DNA. Thermocycling conditions for both the unmethylated- and methylated-specific PCRs were an initial step of 5 minutes at 95°C followed by 35 cycles of 30 sec. at 94°C, 30 sec. at 60.5°C and 60 sec. at 72°C before a final elongation step at 72°C for 7 min.\n\nChk2 Dimerisation\nChk2 mutant's ability to form dimers with the wild-type protein was investigated by immunoprecipitation. U-2-OS cells were co-transfected with expression vectors expressing wild-type Chk2 with N-terminal Xpr-tag (pcDNA4/HisMax, Invitrogen) and mutated Chk2 forms with C-terminal V5-tag (pcDNA3.1/V5-His, Invitrogen). Transfection was performed using FuGene 6.0 transfection reagent (Roche) according to the manufacturer's instructions. Cells were harvested in lysisbuffer (50 mM TrisHCl pH 8.0, 150 mM NaCl, 0.5% NP40, 5 mM EDTA pH 8.0) 48 hours after transfection. An aliquote of the cell lysate was harvested for subsequent Chk2-mutant-V5 transfection verification. Samples were further incubated with A/G Pluss Agarose beads (Santa Cruz Biotechnology) at 4°C for 25 minutes before the beads were removed by centrifugation at 5000g for 4 minutes and the samples were incubated with 1.5 µg anti-V5 (Invitrogen) at 4°C for 90 minutes. Fresh A/G Pluss Agarose beads were added and the samples were incubated for another 90 minutes at 4°C. The beads were washed three times with 1×PBS, before being separated on a 10% polyacrylamide gel and blotted on to a nitrocellulose membrane. Chk2-wild-type-Xpr co-precipitated with Chk2-mutant-V5 was detected through incubations with anti-Xpr antibody (Invitrogen), HRP-conjugated secondary antibody and ECL detection reagent (GE Healthcare).\n\nKinase Activity\nChk2 mutant's ability to function as kinases was investigated through an in vitro kinase assay. The V5 expression vectors used for the dimerisation study were also used to express Chk2 mutants in the kinase assay. U-2-OS cells were transfected using the FuGene 6.0 transfection reagent (Roche) according to the manufacturer's instructions. Cells were then incubated at 37°C in 5% CO2 and humidified atmosphere. After 24 hours doxorubicin (Nycomed Pharma) was added to the media to a final concentration of 50ng/ml and the cells were further incubated for 24 hours before harvest. 75 cm2 of 90% confluent cells were harvested in 500 µl lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 2 mM MgCl2, 5 mM EDTA), and the cytosol was incubated for 90 minutes at 4°C with 50 µl 50% Glutathione Sepharose beads (Amersham Biosciences) linked to anti-V5 antibody (Invitrogen). The beads were then washed twice with lysisbuffer containing 500 mM NaCl and twice with kinase assay buffer (50 mM HEPES, 10 mM MgCl2, 5 mM MnCl2, 2.5 mM EGTA). The beads received 30 µl kinase assay buffer with 7.5 µM cold ATP, 10 µCi 32P-gamma-ATP (GE Healthcare) and 2 µg isolated Cdc25C peptide, and was incubated at 30°C for 30 minutes. Samples were separated on a 12.5% polyacrylamide gel and blotted on to a nitrocellulose membrane. A radiosensitive imaging plate was exposed to the membrane and the plate was read in a FLA200 imager (Fuji).\nThe kinase assay described above was also used to determine the Chk2 mutants' kinase activity after co-transfection of each Chk2 mutant and wild-type Chk2 in equal amounts.\n\nStatistical Analysis\nStatistical analysis was performed using the Primer of Biostatistics system, version 5.0 [38]. The differences in the distribution of TP53 and CHEK2 mutations among patients revealing a PD and the responders were analyzed with use of Fisher's exact test. P-values are reported as accumulated two-sided. Because of the limited time of the follow-up, no formal statistical assessment of overall survival was performed. Relapse-free survival was analyzed by the log-rank test. Details regarding outcome in individual patients with mutations are shown in Table 2 and 3 to make them available to the reader.\n\n\nResults\nTP53 Mutations and Response to Therapy\nThe TP53 mutations identified in the tumors of the patients treated with epirubicin together with the clinical response to therapy and follow-up data are presented in Table 2. Somatic TP53 mutations were identified in 23 (21.5%) of the patients. Normal tissue (WBC) was available from 18 of these for germline characterization, revealing none of the mutations identified to be germline alterations. Of the 23 mutations detected, 20 were missense and 3 were nonsense. One mutation (del483CAT) has not been reported previously either in breast cancer or in any other tumor type (IARC database: http://www.iarc.fr/p53/). Twelve of the mutations directly or indirectly affected the L2/L3 domains of the p53 protein (Table 2) previous found to predict a poor prognosis [39] and drug resistance [14], [40]. For statistical comparison, mutation Gly325Ter (patient Epi215) located to the tetramerization domain is grouped together with the mutations affecting the L2/L3 domain, since this mutation leads to truncation of the protein and with loss of tetramerization and functional defects similar to L2/L3 mutations [41].\nThere was a statistical significant correlation between TP53 mutation status and lack of treatment response (PD) (Table 4; p = 0.0358; Fisher exact test). When tumors harboring TP53 mutations affecting the p53 L2/L3 DNA-binding domains were compared to those with wild-type TP53 or TP53 mutations outside the L2/L3 domains, this correlation was further strengthened (p = 0.0136).\nThe previously described TP53 polymorphism, Arg72Pro [42] was detected in 31 (29%) of our patients. No correlation was found between this polymorphism and lack of treatment response (p = 0.2750; Fisher exact test) or TP53 mutational status (p = 0.2024).\n\nCHEK2 Mutations and Response to Therapy\nTable 3 presents the patients with detected CHEK2 mutations together with a description of the clinical response and follow up-data. CHEK2 mutations were identified in three out of the 109 patients (2.8%). Notably, each of the CHEK2 mutations identified was also present in patient lymphocyte DNA, confirming a germline origin. The Arg95Ter (C283T) mutation is novel. This mutation was present in two patients (Epi132 and Epi203) living in different parts of Norway with no known family relationship. However, linkage analysis using microsatellite markers (D22S275, D22S272, D22S1172 and D22S423) suggested a common founder mutation (data not shown). The C283T transition generates a novel stop codon in exon 1 of CHEK2, leading to truncation of the Chk2 protein. LOH analysis indicated loss of the wild-type CHEK2 allele in the both tumors from the two patients harboring this mutation (Epi132 and Epi203). Both these tumors were non-responsive to epirubicin therapy (PD). In contrast, the third patient with a germline CHEK2 mutation (patient Epi151; point mutation at T1091C, Ile364Thr) had a partial response to epirubicin therapy. This tumor was non-informative with respect to LOH. Taking all CHEK2 mutations together, they predicted resistance to epirubicin (p = 0.0226).\nThe previously described silent Glu84Glu (A252G) polymorphism [24], [43] in exon 1 was detected in two (1.9%) patients. No association between this polymorphism and treatment response was recorded.\nOne of the tumors (Epi203) harboring the C283T substitution (Arg95Ter) also harbored a somatic TP53 mutation in codon 175, Arg175His, located in the L2 domain of p53 (Table 2). This mutation was detected in another four of our patients treated with epirubicin (Table 2). In addition, TP53 Arg175His mutation was recorded in one patient of our previous study evaluating response to doxorubicin [13]. The fact that none of the Arg175His patients presented here or in our previous study revealed resistance to therapy (PD) suggests this mutation may not cause resistance to anthracyclines in breast cancers in vivo. Omitting the tumor harboring both a CHEK2 and a TP53 mutation (patient Epi203) from statistical analysis, Chk2 mutations (n = 2) were non-significantly associated with therapy resistance (p = 0.1633). In a previous study [26], however, we analyzed for CHEK2 mutation status in relation to therapy outcome in a cohort of patients from doxorubicin study [13]. In that study [26], we detected the previously identified mutation Ile157Thr. In addition, we detected a novel nonsense somatic mutation (1368InsA). This mutation was associated with lack of function in vitro; moreover, it was associated with drug resistance in vivo. Analyzing our material and this cohort [26] together, (n = 160), CHEK2 mutations (n = 5 in total) predicted for resistance to doxorubicin and epirubicin therapy (p = 0.0123). Even though, excluding patient Epi203 (harboring TP53 Arg175His and Arg95Ter CHEK2 mutation) as well as other patients harboring TP53 L2/L3 mutations (n = 129), CHEK2 mutations (n = 4 in total) predicted for resistance to doxorubicin and epirubicin therapy (p = 0.030).\n\nTP53 and CHEK2 Mutations Combined and Response to Therapy\nAssuming that TP53 and CHEK2 mutations may substitute for each other, we analyzed for the predictive effect of mutations in both genes. The occurrence of a mutation affecting either CHEK2 or TP53 strongly predicted therapy resistance (p = 0.0101; Fisher exact test). When tumors harboring TP53-L2/L3 mutations and CHEK2 mutations were compared with those wild-type or TP53 mutations outside the L2/L3 domain, the correlation was further strengthened (p = 0.0032; Fisher exact test). The significance was preserved when comparing patients with a PD to objective responders (CR and PR) excluding patients with stable disease (SD) from the statistical analysis (Table 4).\n\np14(ARF) Mutations and Promoter Methylations\nNeither mutations nor polymorphisms in the coding region of p14(ARF) were observed among the 107 patients analyzed. Likewise, no promoter methylations were detected.\n\nInfluence of CHEK2 and TP53 Mutation Status on Relapse-Free Survival\nBecause of the limited time of the follow-up, no formal statistical assessment of overall survival was performed. Details regarding outcome for individual patients with mutations are described in Table 2 and 3 to make these data available to the reader. Relapse-free survival is depicted in (Figure 1). Figure 1A shows relapse-free survival for the patients with TP53 and CHEK2 mutations (all mutations found) compared to patients without any TP53 or CHEK2 mutations, no difference in relapse-free survival was observed. Similar, no difference was seen when grouping TP53 mutations outside L2/L3 and CHEK2 mutation not affecting kinase function (Ile364Thr) as wild-type (Figure 1B). Grouping tumors harboring a mutation in L2/L3 together with CHEK2 mutations affecting kinase domain (Arg95Ter) in one group, mutations outside TP53 L2/L3 and Ile364Thr as one group and tumors without any found mutations in TP53 and CHEK2 separately, again no noticeably difference in relapse-free survival were seen (Figure 1C). Notably, in addition to a short median follow-up time, a total of 35 patients with a sub-optimal response to epirubicin received subsequent treatment with paclitaxel, which may have influenced the outcome.\n\nCHEK2 Mutant's Capability to Form Dimers\nTo investigate whether the identified CHEK2 mutations affect the ability of the Chk2 protein to form dimers, co-transfection and immunopresipitation of V5-tagged mutants and Xpress-tagged wild-type Chk2 were performed using CHEK2 low-expressing U-2-OS cells. As we identified the previously characterized CHEK2 germline mutants variants Arg117His (n = 2 and Ile157Thr (n = 1) among patients allocated to primary treatment with paclitaxel in our ongoing study, these mutants were evaluated together with Arg95Ter and Ile364Thr. The results presented in Figure 2 show that all Chk2 variants carrying a point mutation were able to form dimers with wild-type Chk2, whereas the Arg95Ter variant was not.\n\nKinase Activity of CHEK2 Mutants\nTo investigate whether the identified CHEK2 mutants retained the wild-type kinase activity, an in vitro Chk2 kinase assay with respect to Chk2 autophosphorylation and Cdc25 substrate phosphorylation was performed. The U-2-OS cells were preferred for this assay because they were previously found to express only low levels of endogenous Chk2 [44]. This was confirmed by us using an antibody recognizing endogenous protein (data not shown). These cells have previously been used by other investigators to study Chk2 kinase activity [44], [45], [46].\nThe two mutants Arg117Gly and Ile157Thr were previously tested for in vitro kinase activity [47], but were both included here, together with wild-type CHEK2 as controls. Compared to wild-type Chk2, the Ile157Thr mutant retained wild-type kinase activity. The mutant Ile364Thr showed partially reduced kinase activity both in term of Cdc25-phosphorylation and autophosphorylation (Figure 3). In contrast, the mutant Arg117Gly showed strongly reduced kinase activity while the Arg95Ter mutant was totally devoid of any Chk2 kinase activity. The activity recorded for Ile157Thr and Arg117Gly was consistent with previously reported results for these two mutants [47]. Notably, there was an internal consistency with respect to percentage activity reduction comparing individual mutants with respect to autophosphorylation and phosphorylation of Cdc25 (Figure 3).\nSince enzymatically active Chk2 exists as dimers, it was important to determine the effect of Chk2 mutants on wild-type/mutant heterodimer kinase activity. The effect on Chk2 kinase activities (Chk2 autophosphorylation and Cdc25 substrate phosphorylation) of the individual mutants were therefore determined after co-transfection with wild-type Chk2 as described in Materials and Methods. The results from this co-transfection-kinase assay (Figure 4) were similar to those of the single-transfection assay (Figure 3) except in the case of the Arg117Gly mutant, which expressed a substantial kinase activity when complexed with wild-type Chk2. This is consistent with previous data indicating that the Arg117Gly mutant has neglectable kinase activity itself but dimerizes efficiently to Chk2 wild-type without strongly affecting the wild-type Chk2 activity. Hence, the activity detected is probably caused by the co-transfected and co-precipitated wild-type protein.\nTo rule out the possibility that endogenously expressed wild-type Chk2 contributed to observed Arg117Gly kinase activity shown in Figure 4, we compared the Arg117Gly variant activity in the presence or absence of co-transfected wild-type Chk2 to the activities of Arg95Ter under the same conditions. The Arg95Ter variant does not form dimers with wild-type Chk2. As seen in Figure 5, Arg117Gly, which forms dimers with Chk2 wild-type, allows increased activity when co-transfected with wild-type as compared to the corresponding activity for the Arg95Ter mutant. The fact that Arg117Gly, when transfected alone, displays very similar activity as Arg95Ter or negative control (background levels), strongly indicates that the contribution of endogenous Chk2, which, similarly to exogenously expressed wild-type Chk2 co-precipitate with Arg117Gly is non-significant.\n\nFamily Cancer Incidence in Relation to CHEK2 Germline Mutations\nFollowing an initial report of a family with a CHEK2 germline mutation expressing an increased cancer incidence resembling the Li-Fraumeni syndrome [24], recent studies have revealed the more common CHEK2 mutations to be associated with a moderately increased risk of breast and colorectal cancers. We hypothesized that CHEK2 mutations having a detrimental effect on drug sensitivity could be associated with a more aggressive, Li-Fraumeni or a Li-Fraumeni-like (LFL) cancer syndrome [48]. Except from the patient harboring the Ile364Thr mutation who did not have any known congestion of cancer disease in the family, a detailed assessment of family cancer history was performed for each patient harboring a germline CHEK2 mutation. The family cancer pedigrees are depicted in Figure 6.\nWhile patients harboring CHEK2 germline mutations revealed different types of cancers (mainly breast and tumors of the gastrointestinal area) in their family, surprisingly, no distinct pattern discriminating families harboring the Arg95Ter mutation from the other CHEK2 mutated families could be identified. One of them (Epi203), who inherited the mutation from her father's side of the family, had no accumulation of either breast or colorectal cancer on that side. It should be noted, however, that two brothers of her fathers mother had prostate cancer, and two siblings of his father having hepatocellular carcinoma and bladder cancer, respectively), while the other expressed a disease pattern resembling what has been seen with the more common CHEK2 mutations, like del1100C [25].\n\n\nDiscussion\nTP53 plays a key role as a tumor suppressor gene. Its protein product activates processes such as growth arrest, DNA repair, apoptosis and/or senescence in response to genotoxic damage as well as oncogene activity [49], [50]. Despite being extensively studied, critical issues regarding regulation of the p53 protein remain poorly understood, and conflicting evidence obtained in different experimental systems make the clinical relevance of experimental data questionable.\nChemoresistance is the main obstacle to cancer cure in most malignancies, including breast cancer. Previously, we found TP53 mutations affecting the L2/L3 DNA binding domain to be associated with lack of responsiveness to doxorubicin monotherapy [13] as well as mitomycin and 5-fluoro-uracil in concert [14]. However, some tumors revealed therapy resistance despite harboring wild-type TP53. Postulating that these tumors may harbor genetic disturbances in genes playing a key role in the p53 pathway, we here sequenced TP53 along with CHEK2 and p14(ARF), the latter two known to play a critical role as p53 activators, in tumors from 109 patients treated with epirubicin monotherapy. Our results confirm TP53 mutations, in particular those affecting the L2/L3 domains, to be associated with drug resistance. Most importantly, we also found CHEK2 mutations generating a non-functional protein in our in vitro assays to be associated with drug resistance. In contrast, none of our tumors harbored either mutations or expressed promoter hypermethylations affecting the p14.\nBased on in vitro assays, we were able to classify the different Chk2 mutants with respect to dimerization capability as well as kinase activity (Chk2 autophosphorylation and Cdc25 substrate phosphorylation). In addition, the kinase activities of the Chk2 wild-type/mutant complexes were monitored in co-transfection experiments. Notably, each point mutation (except for Arg117Gly) revealed similar relative kinase efficacy whether co-transfected with wild-type Chk2 or not (Figure 3 and 4). Cells co-transfected with Arg117Gly and wild-type Chk2 revealed kinase activity, probably due to the contribution of the wild type protein in Chk2 mutant – wild-type heterodimers. In contrast, cells transfected with Arg95Ter revealed no kinase activity whether co-transfected with wild-type Chk2 or not, clearly distinguishing this mutation from the others (Figure 3 and 5).\nAll in vitro assays were based on transfection of the U-2-OS cell line, a cell line known to express wild-type Chk2 at low levels, and previously used by other investigators to study Chk2 activity [44], [45], [46]. Since we were not able to obtain satisfactory technical quality of the kinase assay in cell lines negative for Chk2 (HCT 15 and HCT 116), we assessed potential background kinase activity due to endogenous Chk2 by performing western blot analysis revealing the endogenous levels of Chk2 in U-2-OS cells to be non-significant compared to the exogenously expressed Chk2 levels (data not shown). We also performed a separate kinase assay, directly comparing the effect of binding partners for the dimerizing Arg117Gly and the non-dimerizing Arg95Ter. This assay also revealed the contribution of endogenous Chk2 to be non-significant (Figure 5).\nTaking our in vitro findings together with in vivo observations, our present data confirm that the functionally defective CHEK2 Arg95Ter mutation, together with LOH, is associated with resistance to anthracycline therapy. In contrast, the patient harboring the Ile364Thr mutation, moderately reducing phosphorylation activity, responded well to therapy. The other missense mutations; Arg117Gly and Ile157Thr were observed among patients receiving paclitaxel therapy only; thus, their influence on anthracycline sensitivity in vivo could not be addressed. Yet, based on the finding that the Arg117Gly mutant expressed no intrinsic activity, but readily dimerized to the wild-type protein without abolishing its activity, we hypothesize that this mutation and, probably, other yet unidentified CHEK2 mutations with a similar lack of intrinsic kinase activity, may cause resistance to anthracycline therapy if combined with LOH in breast cancer.\nOur present findings have two major implications. First, we confirm that mutations in genes encoding proteins located within the same functional pathway may substitute for each other with respect to drug sensitivity, revealing for the first time a functional pathway critical to chemotherapy response in vivo. Second, the identification of mutations in the CHEK2 but not in the p14(ARF) gene in resistant tumors suggests that Chk2 mediated phosphorylation of p53 is a critical event in executing anti-tumor effect as a response to DNA damaging agents in breast cancer. This adds to our understanding not only of the function of p53 but Chk2 as well. p53 undergoes phosphorylation at multiple sites by different kinases, including Chk2 [51]. While activation of the ATM leading to direct (Ser 15) and Chk2-mediated (Ser 20) phosphorylation of p53 is considered an important mechanism for triggering p53 activation in response to DNA damage [52], some reports suggest ATM [53] and even Chk2 [23] to be redundant to this function. Importantly, Chk2 has been shown capable of inducing ATM-independent apoptosis in vitro [21]. While Chk2 phosphorylates p53 at Ser 20, thereby stabilizing p53 by preventing MDM2 binding [19], Chk2 also phosphorylates p53 at six additional sites, including Ser 313 and Ser 314 located in the nuclear localization signal domain of p53 [51]. In addition, Chk2 phosphorylates other important targets like BRCA1, Cdc25A and Cdc25C involved in DNA repair, G1 and G2 arrest, respectively [54]. Despite the wide range of known Chk2 substrates relevant for DNA repair and cell cycle control, our present findings that CHEK2 mutations leading to non-functional Chk2 protein may substitute for p53 mutations strongly advocate a role for Chk2 with respect to drug sensitivity executed through p53 activation.\nNotably, one of the tumors (Epi203) with the Arg95Ter CHEK2 mutation in addition harbored a somatic TP53 mutation, Arg175His, with allelic imbalance for the TP53 gene (Table 2). Importantly, among another four patients in this study (Epi063, Epi071, Epi087, Epi153) and one patient from our previous doxorubicin protocol [13] harboring the Arg175His mutation together with allelic imbalance for TP53, all five of these patients responded to anthracycline therapy either with a partial response or stable disease. In contrast, Epi132 and the only patient for whom we previously identified a non-functional CHEK2 mutation (1368InsA; coding for a non-functional protein translate with cytoplasmic location [26]) expressed resistance to epirubicin and doxorubicin, respectively. Arg175His is a p53 “hot-spot” structural mutation reported to have defects with respect to transcriptional activation and also to negatively interact with wild-type p53 [55]. While this mutation has been shown to enhance chemoresistance upon transfection into p53 null Saos-2 cells [56], these osteosarcoma-derived cells may not necessarily be representative for breast cancers in vivo. Recent evidence strongly support p53 to be involved also in non-transcriptional mediated apoptosis by interacting with the Bcl-2/Bax system [57], and transcription-defect structural p53 mutants have been shown to execute non-transcriptional apoptosis in experimental systems [58]. Concomitant inactivation of Chk2 and p53 in breast cancer has been recorded by others [59], and the finding that a somatic mutation may generate a “growth advantage” in tumor cells already harboring a germline CHEK2 mutation may not implicate an effect on drug sensitivity in tumors not yet exposed to cytotoxic compounds. Rather, it may indicate a growth advantage, probably related to loss of p21 function. Notably, in a previous study we found the p21 polymorphism G251A to be associated with an increased risk of developing large breast cancers but to have no effect on drug sensitivity [60], indicating that growth rate and drug resistance may be regulated independently. Taken together, we believe our findings advocate a role for Chk2 in executing cellular response to anthracycline-induced DNA damage.\nAs mentioned above, removing TP53 mutated tumors including the double-mutated Epi203 from statistical analysis, CHEK2 mutation status still predicted for resistance to anthracycline therapy. In addition, removing the tumors harboring the Arg175His mutation from the p53 “L2/L3” group strengthened the correlation to lack of treatment response to epirubicin (p = 0.0005).\nComparing the effects of mutations in the CHEK2 gene to TP53 mutations indirectly underlines the importance of the role of Chk2 to chemoresistance. Our present findings as well as results from our previous studies [13], [14] revealed that about 50% of the patients with tumors harboring TP53 L2/L3 mutations to be non-responders to primary therapy. In contrast, all our three patients harboring a non-functional CHEK2 mutation (the two Arg95Ter mutated patients here and our previous patient harboring the 1368InsA) expressed primary resistance to therapy. We previously hypothesized that therapy response in tumors harboring TP53 L2/L3 mutations could be due to redundant pathways acting in concert [3]. Although no definite conclusion should be drawn from a limited number of observation, the fact that Chk2 not only phosphorylates p53 but also phosphorylates other substrates such as Cdc25A and Cdc25C [54] and E2F1 in response to etoposide-induced DNA damage [61] may indicate that inactivation of redundant pathways could take place in parallel.\nThe literature remains inconsistent with respect to whether the border amino acids 163, 195, 236 and 251 should be included in the p53 L2 and L3 domains [12]. Taking a conservative approach, we classified patient Epi56, harboring a mutation in codon 163, as a L2/L3 mutant. The patient harboring this mutation responded to therapy (PR). If this mutation was classified as outside the L2 domain, our p-value had been strengthened from p = 0.0136 to p = 0.0096.\nGermline mutations in TP53 cause the Li-Fraumeni and Li-Fraumeni-like cancer disposition syndromes. However, while the germline and somatic mutations associated with these syndromes reveal a preference for the same codons [48], TP53 mutations affecting the DNA-binding domains seem associated with a poor prognosis [62], [63], [64] and, in particular, drug resistance [14], [40] in breast cancer. Thus, tumor suppression and tumor cell response to chemotherapeutics may involve different parts of p53 protein function. Following an initial report identifying a CHEK2 mutation in a family expressing characteristics of the Li-Fraumeni syndrome [65], recent evidence has linked CHEK2 founder mutations to a moderately increased risk of breast- and colorectal cancers with some additional disposition for other malignancies as well [66]. However, cancer incidence and phenotypes did not reveal an aggressive Li-Fraumeni or Li-Fraumeni-like tumor pattern. Similar to the two patients in our paclitaxel treatment arm harboring the rare but previously characterized mutation Arg117Gly and the patient with the Ile157Thr mutation, they expressed a moderately increased risk of breast and gastrointestinal cancers (Fig. 6). Thus, CHEK2 resembles TP53 in as much as there seems to be no direct correlation between effects of individual mutations with respect to tumor suppression and drug resistance.\nOur finding that TP53 mutations located to the DNA-binding domains predicts drug resistance may indicate transcriptional mechanisms to be involved in drug-induced cell death. p53 induced apoptosis has been associated with transcriptional induction of genes including Puma and Noxa as well as Bax in experimental systems [55], [67], [68]. Yet, recent evidence has revealed p53 to induce apoptosis through non-transcriptional mechanisms by direct protein interactions with members of the Bcl-2/Bax system and mitochondrial release of cytochrom c [57], [69]. In deed, there is evidence that the DNA-binding domains, in particular the L3 part of the protein, may be critical also to transcriptional-independent apoptosis [70]. Of particular note is the finding that Chk2 may regulate transcriptional-independent p53-mediated apoptosis in response to DNA-damage created through ionizing irradiation [71]. Interestingly, Krajewski et al [72] reported low expression of Bax assessed by immunostaining to be associated with a low response to chemotherapy in metastatic breast cancer. Although no conclusion should be drawn at this stage, together these findings are consistent with the challenging hypothesis that transcription-independent activation of Bax following Chk2-phosphorylation may represent a key pathway in p53 dependent cell death in breast cancer in vivo.\np14 acts by releasing p53 from MDM2 binding, and has been related to oncogene-induced p53 activation [73]. Recently, p14 was shown to affect p53 by additional mechanisms, including acetylations [74], response to ionizing radiation in human fibroblasts [75], and tumor-suppression following ionizing radiation in mice [76], [77]. These findings further links the retinoblastoma and p53 pathways [28]. As such, we believe the negative finding with respect to its role in chemoresistance adds important information.\nContrasting earlier findings by us and others [15], a recent study revealed TP53 mutations to be associated with increased likelihood of having a complete response to chemotherapy [16]. These results may not necessarily be at conflict. In the latter study, patients received treatment with a “dose-dense” chemotherapy regimen; if confirmed, the combined data may outline a therapeutic indication for aggressive dose-dense therapy based on tumor TP53/CHEK2 status.\nSo far attempts to identify single markers and, more recently, gene expression arrays predicting chemoresistance have not proved successful (see refs in [9], [10]). The findings presented here reveal for the first time defects in a functional gene cascade to be associated with drug resistance in a human cancer in vivo. Moreover, the findings are made in breast cancer, the most frequent malignant disease among women in the industrialized world, and relate to resistance to anthracyclines, the type of cytotoxic compounds most frequently employed for this malignancy.\nWhile the only study we are aware of comparing TP53 mutation status in primaries and their distant metastases suggested an increasing fraction of tumors to express mutated TP53 during progression [78], we do not know the potential contribution of either TP53 or CHEK2 mutations to drug resistance in micrometastases or in metastatic disease. Yet the finding that one of our non-functional CHEK2 mutations associated with chemoresistance (1368InsA) occurred as a somatic, not germline mutation, suggest such mutations may be selected for during tumor progression. We propose the findings presented here provide important beacons identifying a functional pathway [3] likely to be disturbed through different mechanisms in relation to therapy resistance in advanced disease.\nIn conclusion, we believe our findings here that mutations in the TP53 and CHEK2 genes each may cause resistance to anthracycline therapy in primary tumors to have wide implications to future research in this area. While results from experimental systems are mandatory generating hypotheses, conflicting data from in vitro studies underlines the pivotal role of identifying defects associated with therapy resistance in vivo. Either through mutations of the genes themselves, or inactivation of this functional cascade through co-factors, we believe identification of the Chk2 – p53 axis as critical to anthracycline therapy response provides a functional clue for further investigations in this area.\n\n\n" ], "offsets": [ [ 0, 46897 ] ] } ]

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[ { "id": "pmcA1524813__text", "type": "Article", "text": [ "Comparison of age-specific cataract prevalence in two population-based surveys 6 years apart\nAbstract\nBackground\nIn this study, we aimed to compare age-specific cortical, nuclear and posterior subcapsular (PSC) cataract prevalence in two surveys 6 years apart.\n\nMethods\nThe Blue Mountains Eye Study examined 3654 participants (82.4% of those eligible) in cross-section I (1992–4) and 3509 participants (75.1% of survivors and 85.2% of newly eligible) in cross-section II (1997–2000, 66.5% overlap with cross-section I). Cataract was assessed from lens photographs following the Wisconsin Cataract Grading System. Cortical cataract was defined if cortical opacity comprised ≥ 5% of lens area. Nuclear cataract was defined if nuclear opacity ≥ Wisconsin standard 4. PSC was defined if any present. Any cataract was defined to include persons who had previous cataract surgery. Weighted kappa for inter-grader reliability was 0.82, 0.55 and 0.82 for cortical, nuclear and PSC cataract, respectively. We assessed age-specific prevalence using an interval of 5 years, so that participants within each age group were independent between the two surveys.\n\nResults\nAge and gender distributions were similar between the two populations. The age-specific prevalence of cortical (23.8% in 1st, 23.7% in 2nd) and PSC cataract (6.3%, 6.0%) was similar. The prevalence of nuclear cataract increased slightly from 18.7% to 23.9%. After age standardization, the similar prevalence of cortical (23.8%, 23.5%) and PSC cataract (6.3%, 5.9%), and the increased prevalence of nuclear cataract (18.7%, 24.2%) remained.\n\nConclusion\nIn two surveys of two population-based samples with similar age and gender distributions, we found a relatively stable cortical and PSC cataract prevalence over a 6-year period. The increased prevalence of nuclear cataract deserves further study.\n\n\n\nBackground\nAge-related cataract is the leading cause of reversible visual impairment in older persons [1-6]. In Australia, it is estimated that by the year 2021, the number of people affected by cataract will increase by 63%, due to population aging [7]. Surgical intervention is an effective treatment for cataract and normal vision (> 20/40) can usually be restored with intraocular lens (IOL) implantation.\nCataract surgery with IOL implantation is currently the most commonly performed, and is, arguably, the most cost effective surgical procedure worldwide. Performance of this surgical procedure has been continuously increasing in the last two decades. Data from the Australian Health Insurance Commission has shown a steady increase in Medicare claims for cataract surgery [8]. A 2.6-fold increase in the total number of cataract procedures from 1985 to 1994 has been documented in Australia [9]. The rate of cataract surgery per thousand persons aged 65 years or older has doubled in the last 20 years [8,9]. In the Blue Mountains Eye Study population, we observed a one-third increase in cataract surgery prevalence over a mean 6-year interval, from 6% to nearly 8% in two cross-sectional population-based samples with a similar age range [10]. Further increases in cataract surgery performance would be expected as a result of improved surgical skills and technique, together with extending cataract surgical benefits to a greater number of older people and an increased number of persons with surgery performed on both eyes.\nBoth the prevalence and incidence of age-related cataract link directly to the demand for, and the outcome of, cataract surgery and eye health care provision. This report aimed to assess temporal changes in the prevalence of cortical and nuclear cataract and posterior subcapsular cataract (PSC) in two cross-sectional population-based surveys 6 years apart.\n\nMethods\nThe Blue Mountains Eye Study (BMES) is a population-based cohort study of common eye diseases and other health outcomes. The study involved eligible permanent residents aged 49 years and older, living in two postcode areas in the Blue Mountains, west of Sydney, Australia. Participants were identified through a census and were invited to participate. The study was approved at each stage of the data collection by the Human Ethics Committees of the University of Sydney and the Western Sydney Area Health Service and adhered to the recommendations of the Declaration of Helsinki. Written informed consent was obtained from each participant.\nDetails of the methods used in this study have been described previously [11]. The baseline examinations (BMES cross-section I) were conducted during 1992–1994 and included 3654 (82.4%) of 4433 eligible residents. Follow-up examinations (BMES IIA) were conducted during 1997–1999, with 2335 (75.0% of BMES cross section I survivors) participating. A repeat census of the same area was performed in 1999 and identified 1378 newly eligible residents who moved into the area or the eligible age group. During 1999–2000, 1174 (85.2%) of this group participated in an extension study (BMES IIB). BMES cross-section II thus includes BMES IIA (66.5%) and BMES IIB (33.5%) participants (n = 3509).\nSimilar procedures were used for all stages of data collection at both surveys. A questionnaire was administered including demographic, family and medical history. A detailed eye examination included subjective refraction, slit-lamp (Topcon SL-7e camera, Topcon Optical Co, Tokyo, Japan) and retroillumination (Neitz CT-R camera, Neitz Instrument Co, Tokyo, Japan) photography of the lens. Grading of lens photographs in the BMES has been previously described [12]. Briefly, masked grading was performed on the lens photographs using the Wisconsin Cataract Grading System [13]. Cortical cataract and PSC were assessed from the retroillumination photographs by estimating the percentage of the circular grid involved. Cortical cataract was defined when cortical opacity involved at least 5% of the total lens area. PSC was defined when opacity comprised at least 1% of the total lens area. Slit-lamp photographs were used to assess nuclear cataract using the Wisconsin standard set of four lens photographs [13]. Nuclear cataract was defined when nuclear opacity was at least as great as the standard 4 photograph. Any cataract was defined to include persons who had previous cataract surgery as well as those with any of three cataract types. Inter-grader reliability was high, with weighted kappa 0.82 for cortical cataract, 0.55 (simple kappa 0.75) for nuclear cataract and 0.82 for PSC grading. The intra-grader reliability for nuclear cataract was assessed with simple kappa 0.83 for the senior grader who graded nuclear cataract at both surveys. All PSC cases were confirmed by an ophthalmologist (PM).\nIn cross-section I, 219 persons (6.0%) had missing or ungradable Neitz photographs, leaving 3435 with photographs available for cortical cataract and PSC assessment, while 1153 (31.6%) had randomly missing or ungradable Topcon photographs due to a camera malfunction, leaving 2501 with photographs available for nuclear cataract assessment. Comparison of characteristics between participants with and without Neitz or Topcon photographs in cross-section I showed no statistically significant differences between the two groups, as reported previously [12]. In cross-section II, 441 persons (12.5%) had missing or ungradable Neitz photographs, leaving 3068 for cortical cataract and PSC assessment, and 648 (18.5%) had missing or ungradable Topcon photographs, leaving 2860 for nuclear cataract assessment.\nData analysis was performed using the Statistical Analysis System (SAS, SAS Institute, Cary, NC, USA). Age-adjusted prevalence was calculated using direct standardization of the cross-section II population to the cross-section I population. We assessed age-specific prevalence using an interval of 5 years, so that participants within each age group were independent between the two cross-sectional surveys.\n\nResults\nCharacteristics of the two survey populations have been previously compared [14] and showed that age and sex distributions were similar. Table 1 compares participant characteristics between the two cross-sections. Cross-section II participants generally had higher rates of diabetes, hypertension, myopia and more users of inhaled steroids.\nCataract prevalence rates in cross-sections I and II are shown in Figure 1. The overall prevalence of cortical cataract was 23.8% and 23.7% in cross-sections I and II, respectively (age-sex adjusted P = 0.81). Corresponding prevalence of PSC was 6.3% and 6.0% for the two cross-sections (age-sex adjusted P = 0.60). There was an increased prevalence of nuclear cataract, from 18.7% in cross-section I to 23.9% in cross-section II over the 6-year period (age-sex adjusted P < 0.001). Prevalence of any cataract (including persons who had cataract surgery), however, was relatively stable (46.9% and 46.8% in cross-sections I and II, respectively).\nAfter age-standardization, these prevalence rates remained stable for cortical cataract (23.8% and 23.5% in the two surveys) and PSC (6.3% and 5.9%). The slightly increased prevalence of nuclear cataract (from 18.7% to 24.2%) was not altered.\nTable 2 shows the age-specific prevalence rates for cortical cataract, PSC and nuclear cataract in cross-sections I and II. A similar trend of increasing cataract prevalence with increasing age was evident for all three types of cataract in both surveys. Comparing the age-specific prevalence between the two surveys, a reduction in PSC prevalence in cross-section II was observed in the older age groups (≥ 75 years). In contrast, increased nuclear cataract prevalence in cross-section II was observed in the older age groups (≥ 70 years). Age-specific cortical cataract prevalence was relatively consistent between the two surveys, except for a reduction in prevalence observed in the 80–84 age group and an increasing prevalence in the older age groups (≥ 85 years).\nSimilar gender differences in cataract prevalence were observed in both surveys (Table 3). Higher prevalence of cortical and nuclear cataract in women than men was evident but the difference was only significant for cortical cataract (age-adjusted odds ratio, OR, for women 1.3, 95% confidence intervals, CI, 1.1–1.5 in cross-section I and OR 1.4, 95% CI 1.1–1.6 in cross-section II). In contrast, men had slightly higher PSC prevalence than women in both cross-sections but the difference was not significant (OR 1.1, 95% CI 0.8–1.4 for men in cross-section I and OR 1.2, 95% 0.9–1.6 in cross-section II).\n\nDiscussion\nFindings from two surveys of BMES cross-sectional populations with similar age and gender distribution showed that the prevalence of cortical cataract and PSC remained stable, while the prevalence of nuclear cataract appeared to have increased. Comparison of age-specific prevalence, with totally independent samples within each age group, confirmed the robustness of our findings from the two survey samples. Although lens photographs taken from the two surveys were graded for nuclear cataract by the same graders, who documented a high inter- and intra-grader reliability, we cannot exclude the possibility that variations in photography, performed by different photographers, may have contributed to the observed difference in nuclear cataract prevalence. However, the overall prevalence of any cataract (including cataract surgery) was relatively stable over the 6-year period.\nAlthough different population-based studies used different grading systems to assess cataract [15], the overall prevalence of the three cataract types were similar across different study populations [12,16-23]. Most studies have suggested that nuclear cataract is the most prevalent type of cataract, followed by cortical cataract [16-20]. Ours and other studies reported that cortical cataract was the most prevalent type [12,21-23].\nOur age-specific prevalence data show a reduction of 15.9% in cortical cataract prevalence for the 80–84 year age group, concordant with an increase in cataract surgery prevalence by 9% in those aged 80+ years observed in the same study population [10]. Although cortical cataract is thought to be the least likely cataract type leading to a cataract surgery, this may not be the case in all older persons.\nA relatively stable cortical cataract and PSC prevalence over the 6-year period is expected. We cannot offer a definitive explanation for the increase in nuclear cataract prevalence. A possible explanation could be that a moderate level of nuclear cataract causes less visual disturbance than the other two types of cataract, thus for the oldest age groups, persons with nuclear cataract could have been less likely to have surgery unless it is very dense or co-existing with cortical cataract or PSC. Previous studies have shown that functional vision and reading performance were high in patients undergoing cataract surgery who had nuclear cataract only compared to those with mixed type of cataract (nuclear and cortical) or PSC [24,25]. In addition, the overall prevalence of any cataract (including cataract surgery) was similar in the two cross-sections, which appears to support our speculation that in the oldest age group, nuclear cataract may have been less likely to be operated than the other two types of cataract. This could have resulted in an increased nuclear cataract prevalence (due to less being operated), compensated by the decreased prevalence of cortical cataract and PSC (due to these being more likely to be operated), leading to stable overall prevalence of any cataract.\nPossible selection bias arising from selective survival among persons without cataract could have led to underestimation of cataract prevalence in both surveys. We assume that such an underestimation occurred equally in both surveys, and thus should not have influenced our assessment of temporal changes.\nMeasurement error could also have partially contributed to the observed difference in nuclear cataract prevalence. Assessment of nuclear cataract from photographs is a potentially subjective process that can be influenced by variations in photography (light exposure, focus and the slit-lamp angle when the photograph was taken) and grading. Although we used the same Topcon slit-lamp camera and the same two graders who graded photos from both surveys, we are still not able to exclude the possibility of a partial influence from photographic variation on this result.\nA similar gender difference (women having a higher rate than men) in cortical cataract prevalence was observed in both surveys. Our findings are in keeping with observations from the Beaver Dam Eye Study [18], the Barbados Eye Study [22] and the Lens Opacities Case-Control Group [26]. It has been suggested that the difference could be related to hormonal factors [18,22]. A previous study on biochemical factors and cataract showed that a lower level of iron was associated with an increased risk of cortical cataract [27]. No interaction between sex and biochemical factors were detected and no gender difference was assessed in this study [27]. The gender difference seen in cortical cataract could be related to relatively low iron levels and low hemoglobin concentration usually seen in women [28]. Diabetes is a known risk factor for cortical cataract but in this particular population diabetes is more prevalent in men than women in all age groups [29]. Differential exposures to cataract risk factors or different dietary or lifestyle patterns between men and women may also be related to these observations and warrant further study.\n\nConclusion\nIn summary, in two population-based surveys 6 years apart, we have documented a relatively stable prevalence of cortical cataract and PSC over the period. The observed overall increased nuclear cataract prevalence by 5% over a 6-year period needs confirmation by future studies, and reasons for such an increase deserve further study.\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\nAuthors' contributions\nAGT graded the photographs, performed literature search and wrote the first draft of the manuscript. JJW graded the photographs, critically reviewed and modified the manuscript. ER performed the statistical analysis and critically reviewed the manuscript. PM designed and directed the study, adjudicated cataract cases and critically reviewed and modified the manuscript. All authors read and approved the final manuscript.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\n\n" ], "offsets": [ [ 0, 16562 ] ] } ]

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[ { "id": "pmcA1621059__text", "type": "Article", "text": [ "Case report: rapidly fatal bowel ischaemia on clozapine treatment\nAbstract\nBackground\nThere have been previous reported deaths due to clozapine-induced constipation. In all these cases patients have experienced prior abdominal symptoms over a period of weeks or months.\n\nCase presentation\nWe report the sudden death due to constipation of a healthy young male patient on clozapine without any known history of prior abdominal symptoms.\n\nConclusion\nPsychiatrists need to be alert to the medical emergencies which can occur in the context of clozapine treatment and also need to make other clinicians who may have contact with their patients aware of these.\n\n\n\nBackground\nThere have been six previously published cases of death secondary to clozapine-induced constipation [1-3]. Of these, two patients died from faecal peritonitis, two from aspiration of faeculent vomitus as a result of bowel obstruction and two from bowel necrosis. In all these cases there had been prior complaints of constipation and/or other abdominal symptoms for weeks to months before the fatal event. Here we describe a case of constipation, presumably clozapine-induced, where death from bowel ischaemia occured within 2 days from the first complaint of constipation and without any prior reported abdominal symptoms which might have provided a warning to the clinicians involved.\n\nCase presentation\nA 20-year-old male with a year long history of schizophrenia which had been unresponsive to trials of two atypical antipsychotic drugs was commenced on clozapine. The dose was titrated over the next year to 900 mg daily. Due to persisting negative symptoms amisulpiride 400 mg twice daily was added with good response after one month. The patient was reviewed regularly over the next year, continued to improve and did not report any side effects to members of the multidisciplinary mental health team working to support him in the community. He appeared to be fit and healthy. Although he usually lived in supported accommodation he was staying temporarily with his family and from their account he complained of having constipation for 2 days before presenting to his GP with severe abdominal pain. He was prescribed medication and returned home but his condition deteriorated further and a few hours later an ambulance was called. He collapsed and died before reaching hospital. Post mortem examination revealed that he had impacted faeces which had pressed against the bowel wall causing ischaemia. This had led to infarction of this part of the bowel.\n\nConclusion\nThis case demonstrates that death can occur over a very short time course from constipation, in this case presumably induced by clozapine. Death from constipation and subsequent bowel infarction is relatively common in elderly patients and infarction causes a far more rapid and dangerous deterioration than does intestinal obstruction. In the present case this meant that this patient did not have any contact with psychiatric services between the onset of his symptoms and his rapid demise, in spite of regular follow-up. Although the risk of neutropenia is relatively well-known, it should be borne in mind that clozapine is reported to be associated with a number of other syndromes which may be rapidly fatal including not only constipation and obstruction but also cardiovascular collapse, seizures and ketoacidosis. Psychiatrists working with such patients should not only themselves be vigilant regarding such complications but should take steps to see that other clinicians to whom the patient may present are also aware of them.\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\nAuthors' contributions\nBoth authors were equally involved in the preparation of this manuscript.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\n\n" ], "offsets": [ [ 0, 3857 ] ] } ]

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[ { "id": "pmcA2151861__text", "type": "Article", "text": [ "Recombinant activated protein C in sepsis: endothelium protection or endothelium therapy?\nAbstract\nEndothelium dysfunction is one of the hallmarks of sepsis. Looney and Mattay, in the previous issue of Critical Care, highlight the role of activated protein C (APC) as a protective endothelial drug in septic situations. Nevertheless, the results of in vivo studies are less explicit and it remains uncertain whether these properties are relevant in human septic shock. Before considering recombinant APC (rAPC) as a therapeutic drug for the endothelium, we have to demonstrate its efficiency to protect or to reduce endothelium injury when infused a long time after the septic challenge. Nevertheless, if rAPC is efficient when infused in the early phase of septic challenge, we thus need to treat our patients earlier. At the least, genetically engineered variants have been designed with greater anti-apoptotic activity and reduced anticoagulant activity relative to wild-type APC. Further studies are needed to demonstrate the usefulness of these variants in septic shock therapy.\n\n\nThe use of recombinant activated protein C (rAPC) is one of the hottest topics in septic shock therapy. The pivotal phase 3 placebo-controlled Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) clinical trial demonstrated a 19.4% relative risk reduction in 28-day mortality (6.1% absolute risk reduction) with an increased risk (3.5% versus 2.0%) of serious bleeding events compared with placebo.\nTwo recent and important articles have highlighted the role of APC as a protective endothelial drug [1] and as a cyto-protective drug [2]. Beneficial effects of rAPC in the PROWESS study were thought to be related to a reduction in coagulation and, to a lesser extent, to a reduction in inflammatory response to sepsis [3]. Post-PROWESS investigations have been associated with a myriad of cellular or animal studies demonstrating that rAPC, through reactions mediated by endothelial protein C receptor and the effector receptor, protease activated receptor-1, acts directly on cells to exert multiple cytoprotective effects including: down regulation of pro-inflammatory gene expression [4]; anti-inflammatory activities [5]; anti-apoptotic activity [6]; and protection of endothelial barrier function [1,2].\nEndothelium dysfunction is one of the hallmarks of sepsis [7]. Sepsis, per se, may induce phenotypic modulations of the endothelium through direct or indirect interaction of the endothelial layer with components of the bacterial wall, inducing a myriad of host-derived factors from endothelial cells. Phenotypic modifications include changes in pro-coagulant and proadhesive properties, increased endothelial permeability, endothelial cell apoptosis and changes in vasomotor properties; the last of these is crucial since vasoplegia is directly related to septic shock mortality. Recent animal and human data have suggested that rAPC may improve both vascular and myocardial dysfunction and vascular reactivity to catecholamine during endotoxin and/or septic challenge [8,9].\n\nFrom bench to bedside\nExperimental evidence supports a role of APC in maintaining the integrity of the endothelium through both direct and indirect mechanisms. Nevertheless, the results of in vivo studies are less explicit. In a retrospective study of septic shock in humans, Monnet and colleagues [9] demonstrated that APC infusion was associated with a decrease in the amount of delivered norepinephrine. Wiel and colleagues [10] demonstrated in a rabbit model of endotoxin induced shock that APC decreased aorta endothelial injury. By contrast, in a lung model of endotoxin induced inflammation, Robriquet and colleagues [11] demonstrated a trend to an increased vascular permeability using high doses of human APC. This last result was in sharp contrast with the results obtained by Nick and colleagues [12] in a human model of pulmonary endotoxin administration. APC appears to improve mortality in septic shock with a high APACHE 2 score and is potentially detrimental in severe sepsis. In rats, APC markedly decreased tumour necrosis factor concentrations whereas they remained unchanged in either human septic shock or endotoxemia. The question arises, therefore, as to whether it is truly possible to reconcile all these discrepancies? Moreover, can these stirring laboratory data be translated into clinical practice?\n\nLimitations of experimental studies: endothelium protection versus endothelium therapy\nClearly, in cellular and animal models, rAPC has been given either as a pre-treatment or concurrent with septic challenge. This mode of administration favours the anti-inflammatory effects of rAPC, which are particularly efficient in murine models in protecting the endothelium from cytokine-mediated apoptosis or upregulation of endothelial adhesion molecules. Thus, studies using post-injury treatment are needed in models that mimic septic shock, such as experimental pneumonia or peritonitis treated by antibiotics and volume resuscitation, and where the effects of rAPC would be investigated 16 to 24 hours after septic challenge. If we can demonstrate the efficiency of rAPC to protect or to reduce endothelium injury in these conditions, we can ultimately postulate that rAPC is also a therapeutic drug for the endothelium.\n\nThe earlier the better\nIf rAPC is efficient when infused in the early phase of septic challenge, we thus need to treat our patients earlier. At least two studies suggest that treatment with rAPC within 24 hours may carry a larger survival advantage for patients with severe sepsis, compared with those treated more than 24 hours after organ dysfunction [13]. Interventions directed at specific endpoints, when initiated early in the 'golden hours' of a patient's condition, seem to be promising [14]. The beneficial effects of earlier administration of rAPC to appropriate patients may fit into this paradigm.\n\nThe future\nExtensive in vivo and in vitro studies have focused on the cytoprotective effects of APC and most authors agree that its anticoagulant and cytoprotective effects are mediated by distinct APC structural features. Positively charged residues in surface loops in the APC protease domain have been identified as participating in the anticoagulant activity but not in cellular effects. Hence, variants have been designed with greater anti-apoptotic activity and reduced anticoagulant activity relative to wild-type APC [2]. Whether these genetically engineered variants actually provide superior pharmacological properties remains to be elucidated in vivo. Such investigations may allow the design of therapeutic APC variants with decreased anticoagulant activity to reduce the risk of bleeding on the one hand, but also with normal cytoprotective properties in order to retain full beneficial effects on sepsis outcome.\n\nAbbreviations\nPROWESS = Protein C Worldwide Evaluation in Severe Sepsis; rAPC = recombinant activated protein C.\n\nCompeting interests\nBL has received reimbursements and funding from Eli Lilly, France.\n\n\n" ], "offsets": [ [ 0, 7067 ] ] } ]

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[ { "id": "pmcA1590010__text", "type": "Article", "text": [ "Drug information resources used by nurse practitioners and collaborating physicians at the point of care in Nova Scotia, Canada: a survey and review of the literature\nAbstract\nBackground\nKeeping current with drug therapy information is challenging for health care practitioners. Technologies are often implemented to facilitate access to current and credible drug information sources. In the Canadian province of Nova Scotia, legislation was passed in 2002 to allow nurse practitioners (NPs) to practice collaboratively with physician partners. The purpose of this study was to determine the current utilization patterns of information technologies by these groups of practitioners.\n\nMethods\nNurse practitioners and their collaborating physician partners in Nova Scotia were sent a survey in February 2005 to determine the frequency of use, usefulness, accessibility, credibility, and current/timeliness of personal digital assistant (PDA), computer, and print drug information resources. Two surveys were developed (one for PDA users and one for computer users) and revised based on a literature search, stakeholder consultation, and pilot-testing results. A second distribution to nonresponders occurred two weeks following the first. Data were entered and analysed with SPSS.\n\nResults\nTwenty-seven (14 NPs and 13 physicians) of 36 (75%) recipients responded. 22% (6) returned personal digital assistant (PDA) surveys. Respondents reported print, health professionals, and online/electronic resources as the most to least preferred means to access drug information, respectively. 37% and 35% of respondents reported using \"both print and electronic but print more than electronic\" and \"print only\", respectively, to search monograph-related drug information queries whereas 4% reported using \"PDA only\". Analysis of respondent ratings for all resources in the categories print, health professionals and other, and online/electronic resources, indicated that the Compendium of Pharmaceuticals and Specialties and pharmacists ranked highly for frequency of use, usefulness, accessibility, credibility, and current/timeliness by both groups of practitioners. Respondents' preferences and resource ratings were consistent with self-reported methods for conducting drug information queries. Few differences existed between NP and physician rankings of resources.\n\nConclusion\nThe use of computers and PDAs remains limited, which is also consistent with preferred and frequent use of print resources. Education for these practitioners regarding available electronic drug information resources may facilitate future computer and PDA use. Further research is needed to determine methods to increase computer and PDA use and whether these technologies affect prescribing and patient outcomes.\n\n\n\nBackground\nChallenges with knowledge management for health care professionals\nIn 1986, Haynes et al. published a series of 6 articles entitled \"how to keep up with the medical literature\" in an effort to help clinicians with information management, but this challenge has not decreased in last two decades [1-6]. Alper et al. suggest that maintaining currency with relevant literature in primary care would \"require 627.5 hours per month, or about 29 hours per weekday, or 3.6 full-time equivalents of physician effort\" [7]. The volume of information associated with keeping up to date is frequently cited as a barrier [8]. It is estimated that annually there are approximately 10,000 new randomized trials in MEDLINE and over 450,000 clinical trials identified by the Cochrane Collaboration [9,10]. Keeping up to date has been described with several analogies including clinicians attempting to drink water from a fire hose and swimming in rivers of clinical research with unprecedented depth, velocity, and turbulence [11,12].\nDifficulties with dissemination of research evidence and keeping up to date on pharmacotherapeutic interventions are reported despite the development of tools such as clinical practice guidelines and systematic reviews that are intended to reduce the need for practitioners to evaluate original research [13]. To complicate matters further, there are often issues of credibility, timeliness, and volume of clinical practice guidelines and reviews. Many guidelines are criticized for their methodological development. Shaneyfelt et al. reviewed 279 guidelines for methodological standards from peer reviewed medical literature [14]. These authors found that only 51%, 33.6%, and 46% adhered to standards on guideline development and format, evidence identification and summary, and formulation of recommendations, respectively [14]. A Canadian review on drug therapy guidelines found significant variation in quality depending on the developer [13]. Approximately 25% of guidelines were not recommended for use in practice by the appraisers' criteria [13]. As an example of the volume of clinical practice guidelines available, eleven recent guidelines on community acquired pneumonia exist [15]. To add to the complexities involved with keeping current with pharmacotherapeutic management strategies, as of 2000, there were over 22,000 drug products approved for sale in Canada for human use [16].\nThere is also considerable debate regarding what constitutes \"evidence\" in practice, which contributes to confusion for clinicians [17,18]. Sim et al. succinctly describe the gap between evidence and action as difficulties with obtaining, systematically reviewing, applying in context, and measuring the outcome following application of evidence [19].\n\nMaintaining competence – nurse practitioners as a new group of prescribers\nCompetencies for nurse practitioners (NPs) on a local and international level include critically appraising and applying literature and research findings in practice [20-23]. The Canadian Nurses Association (CNA) has developed the Canadian Nurse Practitioner Core Competency Framework that describes the knowledge, skills, judgment, and attributes required for practice. Evidence based practice is integral to pharmacotherapeutic interventions and prescribing competencies [23]. The National Prescribing Centre, an organization of the National Health Service in the UK, describes several competencies around information needs relevant to prescribing and emphasis is placed on using relevant and up to date information in various formats (e.g. print, electronic, verbal). Several related competencies include understanding advantages and disadvantages of information sources and the currency of resources [21]. Researchers in the US developed NP informatics competencies for integration into advanced nursing practice curricula [24]. Competencies related to informatics knowledge include critical analysis of data and information for use in evidence based practice, evaluating and applying relevant information, synthesizing best evidence, and using optimal search strategies to locate clinically sound and useful studies from information resources [24]. Achieving and maintaining competence in these domains as well as a solid foundation in pharmacology is necessary to support NPs in their relatively new role as a prescriber [25-27].\n\nKnowledge management and information seeking behaviours among nurse practitioners and physicians\nInformation seeking behaviours of physicians are better documented than NPs [11]. Information related to diagnosis is important to both groups but drug therapy queries may occur more frequently with NPs [28-33]. Research on nurses' behaviours related to information seeking is available from the hospital setting [33-35] but the generalizability of these behaviours to NPs with a prescribing role is unclear. Differences in nursing roles, responsibilities, and legislation, including prescriptive authority, exist depending on the country of practice.\n\nNurse practitioners and their collaborating physician partners in Nova Scotia\nNova Scotia is a Canadian province with a population of approximately 942,000 [36]. The province is divided into six health zones that include nine district health authorities, one of which includes the provincial capital and is considered to be urban [37,38]. Health care service delivery is challenging due to many factors including the rural nature of the province, which is estimated to be 60% of population [37,39].\nStarting in 1998, the Nova Scotia Department of Health led an initiative to explore different methods of delivering, managing, and funding primary care services. The Strengthening Primary Care in Nova Scotia Communities Initiative (SPCI) was established with the selection of four primary care demonstration sites where a primary health care NP was hired to practice collaboratively with one or more family/general physicians and other members of an interdisciplinary team. Each demonstration site adopted alternative (non fee-for-service) physician payment mechanisms and used electronic patient records (EPRs) to support service delivery [41]. Demonstration sites participated in project evaluation components that included, but were not limited to, NP roles, alternative fee structures, consumer satisfaction, and implementation and integration of EPRs [41,42].\nLegislation to allow NPs to practice collaboratively with physicians in Nova Scotia was passed in 2002, part way through the SPCI project [39]. Prescriptive authority granted through legislation authorizes NPs to prescribe from a schedule of drugs [43,44]. At the time of conducting this research project, 16 primary health care NPs were in active practice [43].\nThe EPR component of the SPCI project evaluation provided information on the use of technologies in the community context. Results from the implementation process indicated that considerable attention is required for technology literacy, time for training, and selection of software for EPRs [41]. Although the majority of community-based, non-institutional clinical practice settings in Nova Scotia primarily operate with paper-based charting systems, there is a movement toward integrating electronic technologies, including the EPR, in practice among health care providers, administrators, and the provincial government. In addition to recording patient visit information, a component of the EPR package serves to provide drug information resources.\nDrug therapy information resources for NPs and nurse prescribers have frequently been described as essential in supporting practice [25,28,29]. The role of NPs is relatively new in Canada [39] and there is limited information available to indicate the type of resources (e.g. print, electronic, EPR based) these prescribers use for drug and therapeutic information queries at the point of care. It is unknown as to whether differences exist regarding types of resources used, drug information needs, and utilization patterns among NPs and collaborating physician partners. Some research has suggested that the degree of multidisciplinary team functioning relates to the adoption of technology or innovations in practice but more research is required to determine the extent of these relationships [45,46].\nThe use of EPR technology is increasing in Nova Scotia but little information is available regarding the readiness of practitioners for use of specific features such as drug information resources. Based on the EPR related results of the SPCI evaluation, use of these functions could be challenging without proper facilitation. The purpose of the survey for this research was to describe drug information resources used by NPs and their collaborating physician partners at the point of care. The results of the survey will be used to guide further technology implementation strategies and stimulate further discussion around drug information resource usage at the point of care.\n\n\nMethods\nSurvey development\nSurvey development involved three stages including identification of important content areas, development of draft questions, and survey refinement.\nIdentifying important content areas for inclusion in the survey involved conducting a comprehensive English language literature search, consultation with relevant stakeholders (e.g. members of the Nova Scotia Department of Health), and input from subject matter experts at Dalhousie University. The literature review was conducted using the following bibliographic databases: PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), International Pharmaceutical Abstracts (IPA), and Web of Science Citation Databases. Hand and electronic searching of relevant journals was also conducted. Broad search terms were used without limits on publication date or place as nurse practitioner titles, roles and scopes of practice, and terminology regarding technology vary nationally and internationally. Some examples of terms used included nurse practitioner, nurse prescriber, nurse clinicians, district nurse, health visitor, drug information resources, drug information services, information needs, and information technology.\nThe draft survey was reviewed by the research team to reduce the number of items and improve clarity. The layout of the questionnaire was carefully examined to ensure that it was easy to follow and complete. Research results from a previous investigation of Nova Scotian physicians' behaviours regarding drug information were also used to further revise the survey [47]. This draft questionnaire was pilot tested by two out of province NPs and one physician. The results of the pilot were used to make final revisions to the survey. Based on pilot-testing feedback and investigator consensus, the final survey was divided into 2 versions, one for personal digital assistant (PDA) users and one for computer users.\nThe 10 page surveys for PDA and computer users had 5 or 6 sections, respectively, and 37 questions, many with multiple parts. The survey content included demographics, computer or PDA use and experience, drug and therapeutic resource use and preferences, PDA future use, perceived barriers and facilitators to PDA use, and technology training preferences.\nSection one contained demographic questions such as gender, age, job title, volume of patients, and EPR availability in the practice setting. Section two was designed to determine PDA or computer use and experience in the practice setting with questions regarding length of use, costs, and work versus home usage. This section also addressed usage and rating of different drug information resources. Resource ratings were based on the frequency of usage, usefulness, accessibility, credibility, and current/timeliness. Resources were grouped as print (i.e. books, journals, and clinical practice guidelines), online/electronic resources, and health professionals and other. Respondents used 5-point Likert scales (strongly agree to strongly disagree) for rating opinions related to resources. A rating of 6 (not applicable, I do not use this resource) was also included for respondents who did not use a particular resource. Frequency of searching for specific information was rated on a 3-point Likert scale (frequently to never). The final sections of the survey included categorical, open-ended, and Likert scale questions regarding preferred resources, technology barriers, PDA future use, and technology training preferences. Copies of the surveys are attached as an appendix in PDF format [see additional file 1 and 2] or can also be accessed from the Initiative for Medication Management, Policy Analysis, Research & Training (IMPART) website [48].\nEthics approval for the survey was granted through Dalhousie University Research Ethics Board on February 3, 2005.\n\nSurvey population\nLicensed, actively practicing, primary health care NPs (n = 16) and their collaborating physician partners (n = 21) were eligible to participate.\n\nSurvey procedures\nThe survey recruitment procedures were based on the methods of Dillman [49] and Salant and Dillman [50]. Survey packages contained a cover letter, separate surveys for PDA and computer users, and a return self-addressed stamped envelope. The covering letter instructed respondents to self-select the appropriate survey (either PDA or computer) based on their drug information seeking behaviours. Participants who had used a PDA at any time were instructed to complete the PDA version of the survey. Those who had never used a PDA for drug information were instructed to complete the computer version of the survey. Several strategies were used to optimize response rate and included: personalized cover letters, coloured paper for surveys, stamped return envelopes, follow-up mailing, and a priority post mailing [51]. The covering letter included coloured logos of Dalhousie University and the Nova Scotia Department of Health representing the investigator affiliations and endorsement of the project.\nA master mailing list with names and addresses of NPs and their collaborating physician partners was created. To maintain confidentiality of respondents, a number placed on the bottom right corner of each survey corresponded to a name on the confidential master mailing sheet. The postage paid return envelopes were addressed to the research coordinator at the School of Nursing, Dalhousie University, who matched respondents to the mailing list from the first distribution. The cross-referenced mailing list was not accessible to those entering or analysing data. The research coordinator sent the second distribution to those who had not initially responded. A fluorescent coloured page was included in the second mailing to notify recipients of the second and final mailing status. The second mailing followed 2 weeks after the initial mailing (February 2005). The surveys were sent via Xpresspost™ through Canada Post.\n\nData analyses\nQuantitative\nData were entered and analysed in Statistical Package for Social Sciences (SPSS) (version 11.5 for Windows). Five surveys were randomly selected as a check for accuracy of data entry. Descriptive statistics were used to describe resource usage by practitioners. Chi Square (Fisher's Exact when cell count less than 5) analyses were used to determine differences in computer or PDA use based on predetermined variables (e.g. high speed Internet connection, number of patients per day). Mann Whitney U tests were used to compare physician and NPs Likert scale ratings (1 = strongly agree to 5 = strongly disagree) of resource use. Physician and NP rankings of all resources (print, online/electronic, and health professionals and other) were determined from means of Likert scale ratings (1 = strongly agree, 5 = strongly disagree) for each of the pre-specified characteristics (e.g. frequency of use, accessibility, etc.) and the frequency of use of the resources. The best rankings were assigned for the lowest mean scores and the largest number of the sample using a resource. These rankings (ranks based on mean and ranks based on sample) were then entered into a formula to calculate an overall rank. The formula includes: rank = [(rank according to % of sample using the resource + rank based on mean score) ÷ 2]. This formula was used to account for mean scores based on small samples as these numbers could potentially over or underestimate the value of a resource. Ratings of 6 (i.e. not applicable, I do not use this resource) were excluded from the analyses.\n\nQualitative\nComments were entered in a word-processing program and organized by type of respondent (PDA versus computer) and question number. The coded survey number and respondent type (NP or physician) were also included next to comments. Investigators determined themes and categorized comments based on previous experience, knowledge, and familiarity with the topic.\n\n\n\nResults\nSurveys were completed and returned by 75% of eligible participants (27 of 36). One physician survey was undeliverable. The response rates from within the NP and physician samples were 88% and 65%, respectively. Complete demographic information is available in Table 1.\nMethods for accessing resources and self-reported resource use\nResource use was similar amongst practitioners. Respondents indicated that print resources (mean 4.56, SD 0.80), health professionals (mean 3.26, SD 0.90), and online/electronic resources (mean 2.70, SD 1.20) were the preferred method (1 = least preferred to 5 = most preferred) for accessing drug information. Thirty-seven percent of respondents reported that searching for specific questions related to drug information (e.g. usual dosage, duration of therapy) was conducted using both print and electronic resources (but print use greater than electronic) (Table 2). The preferred means (i.e. print) to access resources was consistent with the most common means of conducting searches for specific drug information queries.\nRespondents' ratings for pre-specified print, online/electronic, and professional resources and other, based on means from Likert scales and number of respondents using the resources, are presented in Tables 3, 4, and 5. Of all resources within the print, online/electronic, and health professionals or other categories, NPs and physicians rated the Compendium of Pharmaceuticals and Specialties (CPS) [52] and pharmacists as the top two most frequently used resources for providing drug and therapeutic information. Physicians rated other physicians as the third most frequently used resource. The book Therapeutic Choices [53] ranked third for NPs. Based on written feedback, physicians and NPs consulted pharmacists and other physicians most frequently. The CPS and pharmacists were also ranked as the top two resources overall in terms of usefulness, accessibility, credibility, and current/timeliness for physicians. Rankings by NPs were similar for usefulness, accessibility, and credibility. NPs ranked pharmacists, Therapeutic Choices, and academic detailing first and the CPS as second for current/timeliness.\nWithin the online/electronic category, electronic clinical practice guidelines (eCPGs) were rated the highest for all characteristics (e.g. usefulness, credibility). Although eCPGs were highly ranked, approximately 30% of the sample reported not using this resource. Other resources in this category were infrequently used based on respondents' self-reports.\nPharmaceutical industry representatives were used as a source of drug information by 85% and 86% of physicians and NPs, respectively (Table 5). This was higher than regional drug information services (used by 23% of physicians and 50% of NPs). After exclusion of traditional health professionals (i.e. physicians, nurses, pharmacists, allied health) in the health professionals and other category, pharmaceutical industry representatives received rankings for second or third for frequency of use, usefulness, accessibility, credibility, and current/timeliness, based on means and number of respondents using this resource (data not shown).\n\nDifferences between nurse practitioners and physicians\nA series of Mann Whitney U tests were used to compare the responses of NPs and physicians on their use of print, online/electronic, and health professional resources. In total 95 statistical tests were conducted. The large number of tests increases the likelihood of a type I error as five significant differences would be expected by chance alone at an alpha threshold of 0.05. It is therefore important to treat these results with caution. A limited number of statistically significant (p < 0.05) differences were identified between physicians and NPs and are reported in Table 6. Therapeutic Choices differed significantly for frequency of use with more NPs making use of this resource. Allied health professionals significantly differed between NPs and physicians for accessibility and current/timeliness while NPs were more in agreement with these characteristics of the resource. Nurse colleague credibility and current/timeliness was rated significantly higher by NPs versus physicians.\n\nFactors influencing electronic technology use at the point of care\nFactors such as gender, age, practitioner type (NP vs physician), accessibility, technical support, Internet connection speed, patient volume, presence of an EPR, and home computer use were examined to determine if they were associated with the use of a work computer to search for drug information at the point of care. No statistically significant associations were found (Fisher's Exact).\n\nAdditional resources from respondent comments\nRespondents indicated other resources and programs, such as clinical calculators, that they would like to access from their computer or PDA. The top three resources that were desired included Canadian clinical practice guidelines, patient education information, and ability to track clinical activities/statistics. Further comments from two NP computer survey respondents revealed that a resource on drug interactions and dosages would be desired. One other NP also indicated \"up to date info [sic] on drugs to treat various illnesses ie doseage [sic], length of use etc.\"\n\nComputer or personal digital assistant use in practice\nApproximately 50% of computer survey respondents reported using their work computers for searching drug or therapeutic information related to patient care. Of those respondents, just over half (54%) also reported using their home computer for this purpose. Sixty-seven and 17% of PDA survey respondents reported using their PDA for searching drug or therapeutic information related to patient care at work and home, respectively.\n\nSearching on a weekly basis for specific information related to drugs\nOf the 24 specified categories of drug information included in the survey, the majority were reported as infrequently searched and a smaller percentage as never searched by respondents (data not shown). The top three categories rated as frequently searched were side effects, adult or usual drug dosage, and most appropriate drug for an indication. (Table 7)\n\nIssues related to personal digital assistants\nRespondents reported their level of agreement with statements related to how PDAs may influence their practice. The statements included aspects of workload (organization and paper work), convenience, and improving quality of care and patient outcomes. (Table 8) Respondents agreed that PDAs are a convenient resource but indicated that PDAs would not decrease paperwork or improve patient health outcomes.\n\nBarriers and facilitators to personal digital assistants: themes from written comments\nPeer support from colleagues, convenience, standardized usage, and financial and technical support were the main perceived facilitators to PDA use reported by respondents. The main perceived barrier to PDA use reported by respondents (n = 10) included cost. Other factors such as technology literacy, time, lack of peer support, no high speed internet for downloads, lack of needed resources, keeping up to date on resources, and searching speed were also reported.\n\nFuture use of personal digital assistants\nFifty-two percent, including current PDA users, reported that they would use a PDA in the future. Twenty two percent were uncertain and 19% reported that they would not use a PDA in the future. Two people did not respond.\n\nConfidentiality\nFifty two percent of respondents indicated that patient confidentiality with PDAs was no more concerning compared to use of other technologies. Forty-four percent did not know if they had a policy on patient confidentiality with regard to technologies.\n\nTechnology training and reimbursement\nRespondents rated (1 = least preferred to 5 = most preferred) one on one instruction and group learning led by an expert facilitator as the most preferred (mean 4.32, SD 0.99) means by which to receive instruction on a new technology. Least preferred methods included online discussions/chatrooms (mean 1.52, SD 1.04), internet videos (live: mean 1.70, SD 1.10, or static: mean 1.87, SD 1.14), video cassettes (mean 2.30, SD 1.55), trial and error learning (mean 2.32, SD 1.28), and written manuals (mean 2.92, SD 1.44). Paid leave for attendance at technology training sessions was the preferred means (mean 1.77, SD 0.86; 1 = strongly agree to 5 = strongly disagree) of remuneration for respondents. Respondents also indicated that if financial remuneration was to occur, it should correspond to the amount of time for training that is required (versus a flat rate) (mean 1.96, SD 1.08). Continuing education credits were not viewed as an incentive (mean 2.69, SD 1.44).\n\n\nDiscussion\nPreferred resources\nIn our study, printed materials (e.g. compendia, journals, textbook resources) and professionals (e.g. pharmacists) were the most preferred and frequently used means to access information. Physician reliance on text and compendia relative to online/electronic resources has been frequently reported [11]. In a study examining family doctors' use of information sources to answer clinical questions, human resources (e.g. doctor, pharmacist), non-prescribing print information (e.g. textbooks and journal articles), and prescribing texts were used 36%, 32%, and 25% of the time, respectively [54]. Books from the workplace were reported by approximately 79% of UK primary care nurses as a commonly used source of knowledge and information used to support practice [55]. Fewer than one-third (31%) reported using electronic resources (e.g. Internet, electronic journals) for this purpose [55]. Results of a postal questionnaire to NPs demonstrated that 61% and 51% of respondents reported using drug reference manuals and textbooks, respectively, a few times a week or more [29]. These frequencies were second and third only to consulting with their physician supervisor (63%). Data from structured interviews of a sample of 22 community nurse prescribers reported by Hall et al. revealed that the majority relied on print materials to access information, namely the British National Formulary [32]. A survey of a primary care practice-based research network in the US that included physicians, physician assistants, and nurse practitioners, revealed that interpersonal and rapidly accessed print resources were preferred. Sixty-one and 58% of respondents reported using drug reference sources such as the Physician's Desk Reference (PDR) and medical textbooks, respectively, a few times a day or daily [56].\nThe clinicians in our sample perceived the Canadian compendium, the CPS, to be useful, accessible, credible, and current/timely. The CPS, is described as \"the Canadian drug reference for health professionals\" and is intended to provide a central source of drug information on drug products available in Canada [52]. It is available in print (English and French) and became available online in June 2004. The CPS includes drug monographs for commonly used products approved for use in Canada, but it does not include all drugs available on the Canadian market [57]. The majority of these product monographs are based on monographs submitted by pharmaceutical manufacturers and approved by Health Canada. Some of the monographs are written by the Canadian Pharmacists Association and are described as being evidence-based [52]. The CPS also includes more than 100 pages of clinical tools [52]. The CPS has been criticized for including pharmaceutical company advertising and requiring manufacturer payment for inclusion of product monographs [58]. The accuracy of particular components of CPS monographs has also been investigated. A review of overdose management in 119 monographs from the 2001 CPS revealed considerable variability in the utility of information with 50% of the monographs containing misleading or dangerous advice [59]. Since 2004, the CPS has included an alert box in the overdose section of monographs notifying users to contact Poison Control Centres for overdose management information. Some authors have criticized references that are similar to the CPS as being inadequate with regard to inclusion of evidence based information [60].\nThe NPs in our sample also rated Therapeutic Choices highly for all characteristics. This finding is most likely attributable to the fact that it is a recommended resource for coursework associated with the Dalhousie NP university program curriculum. Therapeutic Choices is a concise therapeutics reference text published by the Canadian Pharmacists Association. The text contains approximately 120 extensively referenced chapters with a disease management approach including easy to use algorithms and tables. An editorial board is responsible for extensively reviewing the content to ensure unbiased and objective information is presented [53].\n\nHealth professionals\nReliance on other health professionals, especially pharmacists and physicians, as a resource for information was evident from our study and concurs with the findings of others [28,32,55,61]. Nurse practitioners have reported that collaborative relationships with pharmacists increase NP role satisfaction [61]. NPs frequently consult with allied health care professionals in their primary health care provider role and this is supported by written feedback from our sample regarding frequently consulted health professionals. Nursing colleagues are also likely to be rated highly by NPs due to their affiliation with peers from the same profession.\nSome investigators have shown that non-human references (e.g. textbook) are sought for more technical aspects of prescribing (e.g. dose), whereas guidance regarding selection of agents (i.e. right drug for an indication) is sought from human resources (e.g. pharmacists or physicians) [62]. We were unable to determine what kinds of resources were used for specific purposes from our study.\n\nOnline and electronic resources, computers, and personal digital assistants\nFrom our study, computer survey respondents ranked online/electronic resources third in preference following print and health professionals. Various barriers and facilitators to accessing information online/electronically or via the Internet have been described in the literature [55,63-66]. Variables that have been described by others as barriers such as accessibility, high speed internet access, patient volume, age, practitioner type, and technology support did not appear to influence computer searching for information on drugs or therapeutics related to patient care in our results. Some qualitative feedback does however support this notion. As an example, in response to a request for a rationale for not using computers one physician commented: \"Retro tech [sic]/old fashion. I still like to use my mind and have always been a fan of pen and paper\". Barriers that were identified with our sample regarding the use of handheld technologies such as PDAs included cost, time, and issues related to technology literacy. Several people questioned the value of PDAs. One GP stated when referring to a PDA: \"So far I have not discovered a use for one\". Other respondents reinforced their preferences for other resources (e.g. books) and resistance to technology. When responding to barriers for the use of PDAs, one NP commented, \"My huge dislike for machinery that frequently requires updating and patience\". A physician responded, \"as stated, I like to use my own mind, and can get all the info I need from books relatively quickly\". Facilitators to the use of PDAs mainly included convenience factors such as having resources all in one place, faster means to get information, and portability. Our sample was not in agreement with some convenience factors in that they did not feel that PDAs would decrease paperwork. Practitioners from our sample felt relatively neutral about PDAs improving patient's health outcomes with 41% responding in this manner. Results from a sample of primary care practitioners in the US revealed that 76% agreed that the use of handheld devices for electronic prescribing would substantially reduce medical errors and improve the quality of health care [67].\nOur study also suggests that resources such as the Cochrane Library and its Database of Systematic Reviews were not frequently used. This finding is similar to that of other investigators [30,35,64]. Despite the desire of some clinicians to use these resources, lack of confidence and ability to use them appropriately has been found [30,64,68,69]. Our study suggests that although this resource is perceived as credible, current/timely, and useful, it is also perceived to be somewhat inaccessible. The Cochrane Library is available to the health professionals (e.g. nurses, physicians, pharmacists, occupational therapists, physiotherapists, etc.) in our sample through professional bodies via the Atlantic Health Knowledge Partnership [70].\n\nTechnology training: preferences and incentives\nWith regard to receiving training for a new technology, our study demonstrates that in person conferences or one on one training sessions are the preferred means to receive continuing education. Person to person interaction has been reported as the preferred and most frequently used means to access continuing education or training by other investigators [55,71].\nOur study also indicates that this group of practitioners may benefit from accessing resources [72-80] that provide guidance on useful drug information resources available for devices such as PDAs. This is exemplified by one respondent's statement \"knowledge regarding good software programs\" as a barrier to the use of PDAs.\n\nPharmaceutical industry\nThe influence of the pharmaceutical industry on physician prescribing and research outcomes has been documented [81,82]. Although NP use of industry representatives as a source of pharmacological information has been documented, the influence on prescribing is largely uninvestigated [32,61,83-85]. The CNA competency framework includes a statement regarding prescribing and industry relations [23]. In our study, the physician and NP rankings of industry representatives were similar. Within the health professionals and other category, pharmaceutical representatives were used as a resource by more of the sample than regional drug information services and comparably to academic detailing services. Academic detailing is a form of continuing medical education where a trained health professional visits prescribers for a fifteen to twenty minute session to provide objective information regarding a therapeutic topic based on best available evidence [86,87]. Following academic detailing, physician and NP rankings of pharmaceutical industry representatives were second or third for frequency of use, usefulness, accessibility, credibility, and current/timeliness.\n\nLimitations\nWe do not have demographics or information regarding the reasons why survey recipients did not respond. As per ethical requirements to maintain confidentiality of respondents, we were not able to match respondents from their respective place of practice and therefore cannot conclude whether the practitioners within a practice setting influenced the others' responses. The sample size of the survey is small although it includes 88% response from community based NPs in Nova Scotia. The generalizability of the results is limited due to the variations in NP scopes of practice nationally and internationally. It is unknown whether the findings are generalizable to nonresponding physicians within Nova Scotia collaborating with NPs or to physicians not in collaborative practices with NPs as they were not included as a part of the sample. Due to multiple statistical comparisons (Mann Whitney U), the results comparing NP and physician ratings of results should be interpreted with caution.\n\n\nConclusion\nRespondent ratings of resources and preferences for resource use were consistent with self-reported means of conducting searches for specific drug information queries. The use of computers and PDAs remains limited and also matches preferences and resource ratings. Education to this group of practitioners regarding available drug information resources may facilitate use of computer and PDA resources. Further research is needed to determine methods to increase the use of computers and PDAs and if use of these technologies affects prescribing and patient outcomes.\n\nCompeting interests\nIngrid Sketris holds a Chair from Canadian Institutes of Health Research (CIHR), Canadian Health Services Research Foundation (CHSRF) co-sponsored by the Nova Scotia Health Research Foundation (NSHRF). Andrea Murphy received salary support through this Chair as a research fellow at the time of conducting this research. The survey was performed in fulfillment of the requirements for the Drug Use Management and Policy Residency that Murphy participated in as a part of her fellowship. The residency was conducted with a decision making partner from the Nova Scotia Department of Health.\nThe opinions expressed in this paper are those of the authors and do not represent the opinions of the Nova Scotia Department of Health, CIHR/CHSRF or NSHRF.\nMF, MM, RMM, and DG have no competing interests to declare.\n\nAuthors' contributions\nAM conceptualized the design and composed the survey instruments, carried out the study, entered and analyzed the data, drafted the original manuscript, and modified subsequent drafts based on authors' and reviewers' feedback. MF, RMM, IS, MM, and DG reviewed and suggested revisions to the survey tools, covering letters, overall study design, and contributed to feedback on the analysis and manuscript revisions.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\nSupplementary Material\n\n\n" ], "offsets": [ [ 0, 41661 ] ] } ]

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[ { "id": "pmcA2367482__text", "type": "Article", "text": [ "Modeling the effect of PTPN22 in rheumatoid arthritis\nAbstract\nIn order to model the effect of PTPN22 on rheumatoid arthritis (RA), we determined the combination of single-nucleotide-polymorphisms (SNPs) showing the strongest association with RA. Three SNPs (rs2476601-rs12730735-rs11102685) were selected for which we estimated the genotypic relative risks (GRRs) of the corresponding genotypes. On the basis of these GRRs we defined four at-risk genotypic classes. Relative to the class of reference risk, individuals had a risk approximately multiplied by two, three, or four. This classification was confirmed by the excess of identity-by-descent (IBD) sharing (IBD = 2) for the sibs of an index in the high-risk class and by excess of non-IBD sharing (IBD = 0) when the index belonged to the low-risk class. The observed data could not be explained by the role of a single variant but were compatible either with a joint effect of the three typed SNPs of PTPN22 on RA or with the role of two untyped variants.\n\nBackground\nThe single-nucleotide polymorphism (SNP) R620W, also denoted rs2476601, is located within the hematopoietic-specific protein tyrosine phosphatase gene, PTPN22. This SNP (C/T) codes for an amino-acid change and the frequency of its minor allele T has been recently and repeatedly shown to be increased in patients with rheumatoid arthritis (RA) [1]. The allele T confers 1.7- to 1.9-fold increased risk to heterozygote and higher risks to homozygote carriers [2] compared to the non-carrier individuals. This variant is also well known to be associated with several other autoimmune diseases [3], such as systemic lupus erythematosus and type 1 diabetes. Recently, Carlton et al. [2] studied the PTPN22 genetic variations in the North American Rheumatoid Arthritis Consortium (NARAC) data. Using the information on several SNPs typed in PTPN22, they compared the haplotype distributions in NARAC patients and controls. They demonstrated that SNP R620W does not fully explain the association between PTPN22 and RA and suggested the effect of at least one additional variant in the PTPN22 gene.\nWe propose here to reanalyze the NARAC data using both association and linkage information for modeling the role of PTPN22 in RA.\n\nMethods\nData\nWe selected from the NARAC data the 511 families with affected sib pairs typed for 14 SNPs of PTPN22, and 1404 unrelated controls also typed for all these SNPs. For each affected sib pair we considered the proband as an index RA patient. The R620W SNP is one of the 14 SNPs in PTPN22. It is located at the ninth position, so it will be subsequently denoted as SNP 9. A preliminary study of linkage disequilibrium (LD) among the 14 SNPs was examined in the 1404 controls. The LD analysis lead us to exclude 3 SNPs (SNP 2, SNP 12, SNP 13), which are in complete LD with one (or more) other SNP(s).\n\nSelection of associated SNPs\nThe combination test [4] was used on the 11 remaining SNPs to select the subset of SNPs showing a significant difference in the genotypic distribution between RA index patients and controls. Its principle consists in testing all possible combinations of SNPs within a gene. Here, there are (211-1) possible combinations. Such a systematic testing of all SNPs and all SNP combinations raises the problem of multiple and non-independent tests. This problem is generally solved by the implementation of a permutation procedure that allows estimation of corrected p-values. Here, associated combinations are very significant and the number of permutations necessary to discriminate them would be extremely high and almost unreachable. Nevertheless, the chi-square values of the genotypic association test are so high that even the conservative Bonferroni correction can be used. We selected the most associated and parsimonious subset of SNPs by nested chi-square tests (NCST) in a forward procedure. The NCST compares the strength of association between nested significant subsets.\n\nGenotypic relative-risk estimation\nFor the selected subset of SNPs, we used the marker association segregation chi-square (MASC) method [5] to compute the genotypic relative risk (GRR) of each genotype. The genotype distributions of index and controls was conditional on the fact that the index has an affected sib.\n\nStratified sib pair IBD estimation\nConditional on each marker genotype of the index cases, the number of parental alleles identical by descent (IBD) shared by the index case and one affected sib were estimated on PTPN22 with the MERLIN software [6]. MERLIN is able to take into account LD between SNPs during the IBD computation. So the estimated IBD distributions are computed on the overall set of SNPs even if they are in LD. The fit of a model to the IBD distributions stratified on index marker genotypes [7] may then be tested by the MASC method.\n\nModeling PTPN22 effect\nWe applied the MASC method [5] to find the most parsimonious model explaining the overall observations, i.e., the genotype and the stratified sib-pair IBD distributions. To do this, MASC requires the haplotype frequencies in the general population, which were estimated on the unrelated controls by the MERLIN software.\nThe MASC method computes the expected genotype marker distribution and the expected sib-pair IBD distributions stratified on marker genotypes for a given genetic model. Here, the computation of the genotypic distribution is conditioned on the fact that index cases have an affected sib. The global expected likelihood of the genetic model given the observed data is then computed as the product of the likelihoods of each expected distribution, and is maximized on the model parameters. The fit of the model to the observed data is tested by a likelihood ratio test (LRT) between global expected likelihood and the likelihood of the saturated model.\n\n\nResults\nSelection of associated SNPs\nMany subsets of SNPs show significant associations. Table 1 presents a selection of the most associated combinations of one, two, and three SNPs. When considering only the effect of a single SNP, the only significant associated one after correction for multiple testing is SNP 9. The combination of SNPs 9–10 is the one which, among the combination of two SNPs, best improves the association shown by the SNP 9 alone (p = 0.017). The subset SNPs 9-10-11 (rs2476601-rs12730735-rs11102685) is the only one that improves significantly the association shown by the SNPs 9–10 (p = 0.038). Adding another SNP to this subset does not significantly improve the association. Consequently, all the subsequent analyses have been done considering SNPs 9-10-11 and their ten corresponding genotypes.\n\nGRR estimation\nTable 2 displays the genotypes and the corresponding GRRs for SNP 9 taken alone (columns 1 and 2) and for the set of the three SNPs 9-10-11 (columns 3 and 4). The GRRs vary from 1 to 2.7 when considering only SNP 9, whereas the variation ranges from 1 to 4.7 when the information on the three SNPs is taken into account. Interestingly, the CC genotype of the SNP 9 can be subdivided in several genotypes when taking into account the genotypes for SNPs 10 and 11 (rows 1 to 6) with GRRs ranging from 1 (CC-GG-AA) to 3.6 (CC-AA-GG). This observation demonstrates the importance of using the additional information on SNPs 10–11.\n\nSib pair IBD estimation\nThe proportion of RA sibs sharing 0, 1, or 2 parental alleles for PTPN22 is 0.26 (181 pairs), 0.51 (362 pairs), and 0.23 (167 pairs), respectively, and does not differ from the IBD distribution 0.25; 0.5; 0.25 expected under no linkage. However, if our GRRs correctly reflect the differential risk of RA, we expect to see differences in the IBD vectors stratified on the genotypes of the subset of SNPs 9-10-11 [7]. To avoid cells with small numbers of individuals we pooled sib pairs with the index genotypes (SNP 9-10-11) that have similar risk. We thus defined four arbitrary at risk genotypic classes: the low risk class (L; GRR = 1; 19 pairs), the intermediate risk class 1 (I1; 1 < GRR ≤ 2; 295 pairs), the intermediate risk class 2 (I2; 2 < GRR ≤ 3; 157 pairs), and the high risk class (H; GRR > 3; 34 pairs).\nTable 3 shows that the proportion of IBD = 0 decreases from 0.47 to 0.09 according to the fact that the index belongs to class L or class H and conversely, the proportion of IBD = 2 increases from 0.11 to 0.26. These stratified IBD distributions are consistent with the risk genotypic classes. In contrast, the IBD sharing distributions stratified only on SNP 9 genotypes are not consistent with the GRR estimates on this SNP (Table 4).\n\nModeling PTPN22 effect\nWe apply the MASC method in using the genotype distribution only on the SNP 9 and the IBD stratified on the SNP 9 genotypes. In that case, the single and causal effect of the SNP 9 is not rejected (p = 0.29). Then, we model the effect of PTPN22 using the four genotypic groups of risk defined on the genotypes of the combination of the SNPs 9-10-11 and the IBD information stratified on them. In this case, we reject the direct effect of SNP 9 (p = 0.005). We also reject the effect of a single untyped SNP (p = 0.04). However, we do not reject the interactive effect of the 3 SNPs (p = 0.53) or the interactive effect of two untyped SNPs.\n\n\nDiscussion\nThe involvement of PTPN22 and HLA in RA susceptibility is no longer disputed. However, as shown by Carlton et al. and confirmed in this study, the role of PTPN22 cannot be explained only by the R620W SNP.\nA correct modeling of PTPN22 is important and shows that the genotypic risk varies much more (1 to 4.7) than reported in the literature (1 to 2.7) [4]. In this study we proposed, for the first time, a model for the effect of PTPN22, taking into account both association and linkage information.\nAnother method, called LAMP [8] was recently proposed for joint modeling of linkage and association [8]. The linkage information used by the LAMP method is the global IBD sharing of affected sib pairs. However, it is very important to note that the power of model discrimination strongly depends on the association and linkage information that is used. As shown here, the information on SNP 9 alone and on the global IBD is very poor as compared with that of the three SNPs 9-10-11 and to the stratified IBD distributions on the four at-risk genotype groups.\nIn conclusion, we applied a four-step strategy to model the effect of a candidate gene covered by several SNPs: 1) to select the most associated set of SNPs; 2) to group the corresponding genotypes according their GRRs; 3) to stratify IBD sharing information on the at-risk genotype groups; 4) to model the effect of the candidate gene while taking into account both linkage and association information.\nThis strategy allowed better modeling of the effect of PTPN22 in RA susceptibility. Recently, du Montcel et al. [9] refined the modeling of HLA in RA susceptibility. A next step will be to use simultaneously the PTPN22 and HLA information to evaluate their joint effects while taking into account important covariables such as age and gender.\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\n\n" ], "offsets": [ [ 0, 11148 ] ] } ]

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[ { "id": "pmcA1459157__text", "type": "Article", "text": [ "Piezoelectric osteotomy in hand surgery: first experiences with a new technique\nAbstract\nBackground\nIn hand and spinal surgery nerve lesions are feared complications with the use of standard oscillating saws. Oral surgeons have started using a newly developed ultrasound bone scalpel when performing precise osteotomies. By using a frequency of 25–29 kHz only mineralized tissue is cut, sparing the soft tissue. This reduces the risk of nerve lesions. As there is a lack of experience with this technique in the field of orthopaedic bone surgery, we performed the first ultrasound osteotomy in hand surgery.\n\nMethod\nWhile performing a correctional osteotomy of the 5th metacarpal bone we used the Piezosurgery® Device from Mectron [Italy] instead of the usual oscillating saw. We will report on our experience with one case, with a follow up time of one year.\n\nResults\nThe cut was highly precise and there were no vibrations of the bone. The time needed for the operation was slightly longer than the time needed while using the usual saw. Bone healing was good and at no point were there any neurovascular disturbances.\n\nConclusion\nThe Piezosurgery® Device is useful for small long bone osteotomies. Using the fine tip enables curved cutting and provides an opportunity for new osteotomy techniques. As the device selectively cuts bone we feel that this device has great potential in the field of hand- and spinal surgery.\n\n\n\nBackground\nFor osteotomies of the hand oscillating saws are usually used [1]. Even though they are varied in size, they are not very precise for use in the vicinity of nerves and arteries. They also pose problems while being used in conjunction with magnification, as one's range of sight and focus is restricted when wearing magnifying glasses. For that reason oral surgeons have moved to using the newly developed piezoelectrical bone scalpel when operating in the near vicinity of nerves or arteries. The tip of this instrument oscillates in the frequency of ultrasound [2]. The mechanism of this device is based on the so called Piezo – Effect. French Physicists Jean and Marie Curie first mentioned the direct Piezo-Effect 1880, whereby certain crystals produce electrical current while under mechanical pressure. The reciprocal effect, by which the crystals are deformed when under electrical current, was then discovered a while later. This is the effect being used by the Piezosurgery Device®. In this device, the electrical field is located in the handle of the saw [3]. Due to the deformation caused by the electrical current, a cutting – hammering movement is produced at the tip of the instrument. These micro movements are in the frequency range of 25 to 29 kHz and, depending on the insert, with an amplitude of 60 to 210 μm. This way only mineralized tissue is selectively cut. Neurovascular tissue and other soft tissue would only be cut by a frequency of above 50 kHz [3-5]. Depending on the strength of the bone and the blade geometry, the efficiency of the cutting can be regulated by the frequency modulator and the power level. For cooling there is an integrated pump with five different working levels. This pump automatically washes physiological solution to the area being cut. The cost of the device is about 7.000 USD. Additional costs per operation are for the cooling liquid and are in the range of a few dollars. We have used the Piezosurgery Device® by Mectron [Italy] [3] for the first time in osteotomies of the long bone in the field of hand surgery. We will report on our experience with one case, with a follow up time of one year.\n\nMethod\nThe correctional osteotomy was performed on a 23 year old worker who suffered a malunited metacarpal bone fracture of the fifth finger on his dominant hand. The X-ray revealed a 45 degree angular deformity of the fifth metacarpal neck with internal rotation. (Figure 1). The operation was performed under regional anesthesia. A longitudinal incision was made over the fifth metacarpal. The tendon of the extensor digiti minimi was found and on its radial side the periosteum of metacarpal five was reached. The periosteum was opened longitudinally over the defect as usual. For the correction of the defect of 45 degrees, a bone wedge was excised. Instead of using the traditional oscillating saw, the Piezosurgery Device® [3] was used (Figure 2). We used a sharp hardened saw coated with titannitrid (Figure 3). For most of the surgery the highest power level, the boosted burst c, was used. We set the automatic cooling of the area with water to its highest level. The angulation and rotation was corrected and fixed with a 1.5 mm titanium five-hole plate and four screws. Closure of the wound was done in layers. Mobilization was started on the 10th postoperative day. The overall time of observation was one year.\n\nResults and discussion\nThe Piezosurgery® Device is ideally sized for hand surgery. The cutting was very precise. The edges of the osteotomy were all sharp to the edge, there was no need to split the bone with a chisel, nor was there the danger of a break out. During the osteotomy there were no disturbing vibrations in the area of operation. This absence of vibration is very practical for operations using a magnifier. Vercellotti mentions that to overcome any problems during surgery, instead of increasing pressure on the hand piece, as in traditional techniques, it is necessary to find the correct pressure to achieve the desired result. With piezoelectric surgery, increasing the working pressure above a certain limit impedes the vibrations of the insert [4]. We have also experienced this in our study. The instrument can be moved in all directions comparable to a pen. The tip of the instrument is exchangeable. Using the fine tip enables multiplanar as well as curved cutting. Because of the automatic water cooling during the whole procedure, there is always a clear view onto the object. This is something oral surgeons found especially useful [6]. The authors mention that the downside of the device is the relative slow sawing process. We needed about 30 seconds for one cut of the relatively small bone. This is about 20 seconds longer than the time needed for cutting with the usual saw. Although the power can be regulated with the power box and the use of different scalpels, we agree with other authors that the optimal use of this device is in surgeries of small bones where precise and soft tissue friendly cutting is required [7]. As other literature has shown, the device selectively cuts bone while sparing nerves and other soft tissue [2,3]. This allows for minimal invasive surgeries with limited retraction of soft tissue and minimal stripping of the periosteum, saves time and might have a positive effect on the healing process. Our aim of the first time use of the Piezosurgery® Device in hand surgery was to check its usability in osteotomies of tubular bones. The preparation of the bone was done in the usual manner as is done when cutting with an oscillating saw. The reason for this was to fully visualize the cutting process using this new device, although in the future, it should be possible to minimize the bony exposure. In our patient the postoperative healing of the wound and the bone consolidation (Figure 4) were smooth. The duration of postoperative sick leave was four weeks which is more rapid than the usual recovery period. The patient regained full use of his finger according to the state before the fracture. At no point was there any loss of sensitivity. The patient as well as the surgeons were fully satisfied with the result.\n\nConclusion\nThe Piezosurgery® Device is a useful device for small long bone osteotomies. We feel that this device has great potential in the field of hand- and spinal surgery. As the device selectively cuts bone, considerable nerve lesions can be avoided and minimal invasive surgeries are possible. Using the fine tip enables curved cutting and provides an opportunity for new osteotomy techniques.\n\nCompeting interests\nThe author(s) declare that they have no competing interests.\n\nAuthors' contributions\nDJH initiated and coordinated the new application of Piezosurgery® device and wrote the publication.\nStSt lead the osteotomy as he was experienced with this tool from oral surgery. He played a major part in writing the technical aspects.\nOVK was the treating surgeon, performed the operation and evaluated the new tool.\nSS performed a literature review and wrote part of the publication.\nPH was the treating chief surgeon, evaluated the new tool and lead the treatment in all aspects.\n\nConsent\nWe obtained oral consent from the patient but could not obtain written consent.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\n\n" ], "offsets": [ [ 0, 8778 ] ] } ]

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[ { "id": "pmcA331414__text", "type": "Article", "text": [ "Primitive Neuroectodermal Tumor (PNET) of the kidney: a case report\nAbstract\nBackground\nA case of Primitive Neuroectodermal Tumor (PNET) of the kidney in a 27-year-old woman is presented. Few cases are reported in the literature with a variable, nonspecific presentation and an aggressive behaviour. In our case, a radical nephrectomy with lymphadenectomy was performed and there was no residual or recurrent tumour at 24-month follow-up.\n\nMethods\nThe surgical specimens were formalin-fixed and paraffin embedded. The sections were stained with routinary H&E. Immunohistochemistry was performed.\n\nResults\nThe immunohistochemical evaluation revealed a diffuse CD99 positivity in the cytoplasm of the neoplastic cells. Pankeratin, cytokeratin AE1/AE3, vimentin, desmin, S100, cromogranin were negative. The clinical presentation and the macroscopic aspect, together with the histological pattern, the cytological characteristic and the cellular immunophenotype addressed the diagnosis towards primary PNET of kidney.\n\nConclusions\nSince sometimes it is difficult to discriminate between PNET and Ewing's tumour, we reviewed the difficulties in differential diagnosis. These tumors have a common precursor but the stage of differentiation in which it is blocked is probably different. This could also explain their different biological behaviour and prognosis.\n\n\n\nBackground\nThe peripheral Primitive Neuroectodermal Tumor (PNET), firstly recognized by Arthur Purdy Stout in 1918, is a member of the family of \"small round-cell tumors\". Primitive renal localization is very rare. There are almost 50 cases reported in the literature, although it is difficult to estimate the exact number since often it has not been differentiated from Ewing's Sarcoma [1-13]. Renal PNET is more aggressive than in the other sites. It frequently arises during childhood or adolescence, having an aggressive clinical course towards metastatic disease and death. It often recurs locally and metastasises early to regional lymph nodes, lungs, liver, bone and bone marrow, resulting in a poor prognosis. The 5-year disease-free survival rate, for patients presenting well confined extra-skeletal PNET, is around 45–55% and cases with advanced disease at presentation have a median relapse-free survival of only 2 years [1].\n\nCase presentation\nA 27-year-old woman was referred because of a mild left flank pain and haematuria. Ultrasonography identified a left renal mass homogeneously hyperechogenic in comparison with renal parenchyma. CT scan showed a 11 mm × 8 mm × 6 mm tumor replacing the upper half of the left kidney with extension into the renal vein. Chest x-ray was negative. Pathological stage after radical nephrectomy was T3aN0Mx.\nThe surgical specimens were formalin-fixed and paraffin embedded. The sections were stained with routinary H&E. Immunohistochemistry was performed using avidin biotin complex technique and diaminobenzidine as chromogen. The antibodies used included CD99 (Dako, M3601), pankeratin (Dako, M0821), cytokeratin AE1/AE3 (Dako, M3515), vimentin (Dako, M7010), desmin (Dako, M0760), S100 (Dako, Z0311), and chromogranin A (Dako, M0869), at suggested dilution. We performed also appropriate routinely positive and negative controls.\nThe tumor was multilobular, grey, glistening, focally hemorrhagic, surrounded by a capsule and with a sharp demarcation from the uninvolved kidney. Histologically, the tumor consisted of small round cells with round nuclei and scant cytoplasm. It presented different patterns, with cohesive lobules or rosettes and perivascular pseudo-rosettes or, in some areas, spindle cellular elements (fig. 1).\nThe immunohistochemical evaluation revealed a diffuse CD99 positivity in the cytoplasm of the neoplastic cells (fig. 2); tumoral cells were also visible in the vascular lumens (fig. 3). By contrast, pankeratin, cytokeratin AE1/AE3, vimentin, desmin, S100, cromogranin were negative.\nThe clinical presentation and the macroscopic aspect, together with the histological pattern, the cytological characteristic and the cellular immunophenotype addressed the diagnosis towards primary PNET of kidney. A bone scan did not reveal positive areas. Eight cycles of chemotherapy with Vincristine, Ifosfamide and Adriamycin, four cycles of Ifosfamide and VP16 and eight sittings of local radiotherapy were sequentially performed. Follow-up examinations with CT and bone scan failed to show residual or recurrent tumor after 24 months.\n\nConclusions\nPrimitive Neuroectodermal Tumor of the kidney is a rare entity. The few cases reported revealed a variable presentation and an aggressive behaviour. The distinction from other primary malignancies of the kidneys is crucial for prognosis. The differential diagnosis includes extra-osseous Ewing's sarcoma, rhabdomyosarcoma, Wilm's tumor, carcinoid, neuroblastoma, clear cell sarcoma of the kidney, lymphoma, the small cell variant of osteosarcoma, desmoplastic small round cell tumor and nephroblastoma [5].\nThe Homer-Wright type rosettes, commonly scarce of number or less defined in extra skeletal Ewing's sarcoma (ES), are a typical histological feature for PNET and can address the diagnosis although they can be found also in neuroblastoma [5]. To better address the diagnosis, an immunohistochemical analysis is necessary. In our case the presence of MIC-2 gene products, known also as CD99, 12E7, E2, 013 and HBA71, suggested a PNET diagnosis. Primitive neuroectodermal tumors only immunorreactive to CD99, even if uncommon, are reported in the literature [13]. The reactivity to vimentin, NSE and S-100 may facilitate the diagnosis but is not patognomonic, while CD 99 positivity is nowadays a clue for the diagnosis. Moreover cytogenetic studies (not performed in our case) demonstrated that PNET and Ewing's sarcoma can both be associated to a translocation of the long arms of chromosome 11 and 22, t(11;22)(q22;q12) [5]. Despite their genetic and antigenic similarity, many authors currently recognize PNET and extra-skeletal Ewing's sarcoma of the kidney as separate entities. It is also important to keep separate renal PNET and malignant rhabdomyosarcoma tumor (MRT). Weeks et al reported 8 cases suggestive for PNET but mimicking MRT [14]. Although renal PNET and MRT show similar clinico-pathological features, the latter usually occurs in very young children, having a more aggressive prognosis.\nRodriguez et al postulated that these two renal neoplasms share a common undifferentiated precursor to explain their similarity and we agree with these Authors [12]. Indeed, the hypothesis that tumors arise from stem cells (SCs) as a consequence of a maturative arrest is now growing [15]. SCs are present in almost all tissues and may originate different cellular lineages by the multi-step process named \"differentiation\". The role of SCs in tumorigenesis was clearly demonstrated in a number of carcinogenic models showing that solid and haematopoietic cancers could arise from tissue-specific SCs [16-19]. In agreement with Sell and Pierce, we retain that the degree of malignancy of a carcinoma depends by the stage in which SCs differentiation stopped during carcinogenesis [19]. In particular, since PNET, Ewing's tumour and MRT have a similar morphology, our hypothesis is that the mesenchimal stem precursor of these tumors is the same, but the stage of differentiation in which it is blocked is different. This could explain why sometimes it is difficult to discriminate between these tumors, notwithstanding they present a different biological behaviour.\n\nCompeting interests\nNone declared.\n\nAuthors' contribution\nAll authors contributed.\n\nPre-publication history\nThe pre-publication history for this paper can be accessed here:\n\n\n\n" ], "offsets": [ [ 0, 7735 ] ] } ]

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