bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Wide-Range Quantitative Nested Real-Time PCR Assay for
Mycobacterium tuberculosis
DNA: Development and Methodology
Authors: Takahashi Teruyuki , Tamura Masato , Asami Yukihiro , Kitamura Eiko , Saito Kosuke , Suzuki Tsukasa , Takahashi Sachiko Nonaka , Matsumoto Koichi , Sawada Shigemasa , Yokoyama Eise , Takasu Toshiaki
Published: March 2008

Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for
Mycobacterium tuberculosis
DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control “calibrator” to measure the copy number of
M. tuberculosis
DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (
F
= 1.086,
P
= 0.774) against a range (1 to 10
5
) of copy numbers of mimic
M. tuberculosis
DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.
RNase P RNA Gene (rnpB) Phylogeny of Hemoplasmas and OtherMycoplasmaSpecies
Authors: Peters Iain R. , Helps Chris R. , McAuliffe Laura , Neimark Harold , Lappin Michael R. , Gruffydd-Jones Timothy J. , Day Michael J. , Hoelzle Ludwig E. , Willi Barbara , Meli Marina , Hofmann-Lehmann Regina , Tasker Séverine
Published: March 2008

Partial sequences of the RNase P RNA gene (rnpB) were obtained from a number of hemoplasmas and otherMycoplasmaspecies. Phylogenetic analysis of these sequences showed that all hemoplasmas were present within a single clade and were most closely related toMycoplasma fastidiosum, similar to the results found with 16S rRNA gene phylogeny.
New and Sensitive Assay for Determining
Pseudomonas aeruginosa
Metallo-Beta-Lactamase Resistance to Imipenem
Authors: Tan Jingyuan , Pitout Johann D. D. , Guttman David S.
Published: March 2008

We present an imipenem lysate metallo-beta-lactamase (MBL) functional assay. This assay eliminates false-positive results due to the bactericidal effects of EDTA, can be performed with inexpensive reagents available in most laboratories, and is as accurate as the MBL Etest. It is appropriate for both high-accuracy screens and laboratories in developing countries with limited resources.
Stability of
Trichomonas vaginalis
DNA in Urine Specimens
Authors: Ingersoll Jessica , Bythwood Tameka , Abdul-Ali Deborah , Wingood Gina M. , Diclemente Ralph J. , Caliendo Angela M.
Published: March 2008

Trichomonas vaginalis
is an important pathogen in both men and women. Culture is considered the diagnostic gold standard, although studies have shown that PCR is more sensitive than either culture or wet mount for the diagnosis of
T. vaginalis
infections. We sought to identify a simple method for stabilizing
T. vaginalis
DNA in urine samples that could be easily applied to molecular testing. The stability of
T. vaginalis
DNA in 40 urine samples was assessed by storage for various times at either 4°C or room temperature with or without the Becton Dickinson urine preservative transport (UPT) kit. Overall, there was better stability of
T. vaginalis
DNA when specimens were stored at 4°C than when they were stored at 20 to 22°C and when the UPT system was used.
T. vaginalis
DNA was stable in specimens stored without using the UPT at 4°C for about 3 days and at room temperature for only 1 day. For specimens placed in the UPT within 24 h (times of 1, 6, and 24 h) of collection, the DNA was stable for up to 30 days when stored at 4°C. For specimens stored at room temperature, the urine should be added to the UPT ideally within 1 hour of collection, and in this case the DNA remained stable for up to 30 days. When storing specimens at room temperature, a delay of 24 h prior to adding to UPT led to an unacceptably high loss of assay sensitivity.
Histone Hyperacetylation Occurs on Promoters of Lytic Cycle Regulatory Genes in Epstein-Barr Virus-Infected Cell Lines Which Are Refractory to Disruption of Latency by Histone Deacetylase Inhibitors
Authors: Countryman Jill K. , Gradoville Lyndle , Miller George
Published: March 2008

Activation of the Epstein-Barr virus (EBV) lytic cycle is mediated through the combined actions of ZEBRA and Rta, the products of the viral BZLF1 and BRLF1 genes. During latency, these two genes are tightly repressed. Histone deacetylase inhibitors (HDACi) can activate viral lytic gene expression. Therefore, a widely held hypothesis is that Zp and Rp, the promoters for BZLF1 and BRLF1, are repressed by chromatin and that hyperacetylation of histone tails, by allowing the access of positively acting factors, leads to transcription of BZLF1 and BRLF1. To investigate this hypothesis, we used chromatin immunoprecipitation (ChIP) to examine the acetylation and phosphorylation states of histones H3 and H4 on Zp and Rp in three cell lines, Raji, B95-8, and HH514-16, which differ in their response to EBV lytic induction by HDACi. We studied the effects of three HDACi, sodium butyrate (NaB), trichostatin A (TSA), and valproic acid (VPA). We also examined the effects of tetradecanoyl phorbol acetate (TPA) and 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor, on histone modification. In Raji cells, TPA and NaB act synergistically to activate the EBV lytic cycle and promote an increase in histone H3 and H4 acetylation and phosphorylation at Zp and Rp. Surprisingly, however, when Raji cells were treated with NaB or TSA, neither of which is sufficient to activate the lytic cycle, an increase of comparable magnitude of hyperacetylated and phosphorylated histone H3 at Zp and Rp was observed. In B95-8 cells, NaB inhibited lytic induction by TPA, yet NaB promoted hyperacetylation of H3 and H4. In HH514-16 cells, NaB and TSA strongly activated the EBV lytic cycle and caused hyperacetylation of histone H3 on Zp and Rp. However, when HH514-16 cells were treated with VPA, lytic cycle mRNAs or proteins were not induced, although histone H3 was hyperacetylated as measured by immunoblotting or by ChIP on Zp and Rp. Taken together, our data suggest that open chromatin at EBV BZLF1 and BRLF1 promoters is not sufficient to activate EBV lytic cycle gene expression.
Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in
Cyprinus carpio koi
Authors: Costes B. , Fournier G. , Michel B. , Delforge C. , Raj V. Stalin , Dewals B. , Gillet L. , Drion P. , Body A. , Schynts F. , Lieffrig F. , Vanderplasschen A.
Published: March 2008

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a
loxP
-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.
The Thiazolobenzimidazole TBZE-029 Inhibits Enterovirus Replication by Targeting a Short Region Immediately Downstream from Motif C in the Nonstructural Protein 2C
Authors: De Palma Armando M. , Heggermont Ward , Lanke Kjerstin , Coutard Bruno , Bergmann Mirko , Monforte Anna-Maria , Canard Bruno , De Clercq Erik , Chimirri Alba , Pürstinger Gerhard , Rohayem Jacques , van Kuppeveld Frank , Neyts Johan
Published: March 2008

TBZE-029 {1-(2,6-difluorophenyl)-6-trifluoromethyl-1
H
,3
H
-thiazolo[3,4-a]benzimidazole} is a novel selective inhibitor of the replication of several enteroviruses. We show that TBZE-029 exerts its antiviral activity through inhibition of viral RNA replication, without affecting polyprotein processing. To identify the viral target of TBZE-029, drug-resistant coxsackievirus B3 (CVB3) was selected. Genotyping of resistant clones led to the identification of three amino acid mutations in nonstructural protein 2C, clustered at amino acid positions 224, 227, and 229, immediately downstream of NTPase/helicase motif C. The mutations were reintroduced, either alone or combined, into an infectious full-length CVB3 clone. In particular the mutations at positions 227 and 229 proved essential for the altered sensitivity of CVB3 to TBZE-029. Resistant virus exhibited cross-resistance to the earlier-reported antienterovirus agents targeting 2C, namely, guanidine hydrochloride, HBB [2-(alpha-hydroxybenzyl)-benzimidazole], and MRL-1237 {1-(4-fluorophenyl)-2-[(4-imino-1,4-dihydropyridin-1-yl)methyl]benzimidazole hydrochloride}. The ATPase activity of 2C, however, remained unaltered in the presence of TBZE-029.
Generation of Antiviral Major Histocompatibility Complex Class I-Restricted T Cells in the Absence of CD8 Coreceptors
Authors: Andrews Nicolas P. , Pack Christopher D. , Lukacher Aron E.
Published: March 2008

The CD8 coreceptor is important for positive selection of major histocompatibility complex I (MHC-I)-restricted thymocytes and in the generation of pathogen-specific T cells. However, the requirement for CD8 in these processes may not be essential. We previously showed that mice lacking β2-microglobulin are highly susceptible to tumors induced by mouse polyoma virus (PyV), but CD8-deficient mice are resistant to these tumors. In this study, we show that CD8-deficient mice also control persistent PyV infection as efficiently as wild-type mice and generate a substantial virus-specific, MHC-I-restricted, T-cell response. Infection with vesicular stomatitis virus (VSV), which is acutely cleared, also recruited antigen-specific, MHC-I-restricted T cells in CD8-deficient mice. Yet, unlike in VSV infection, the antiviral MHC-I-restricted T-cell response to PyV has a prolonged expansion phase, indicating a requirement for persistent infection in driving T-cell inflation in CD8-deficient mice. Finally, we show that the PyV-specific, MHC-I-restricted T cells in CD8-deficient mice, while maintained long term at near-wild-type levels, are short lived in vivo and have extremely narrow T-cell receptor repertoires. These findings provide a possible explanation for the resistance of CD8-deficient mice to PyV-induced tumors and have implications for the maintenance of virus-specific MHC-I-restricted T cells during persistent infection.
MAXIMAL NON-SYMMETRIC ENTROPY LEADS NATURALLY TO ZIPF'S LAW
Authors: LIU CHENG-SHI
Published: March 2008

As the most fundamental empirical law, Zipf's law has been studied from many aspects. However, its meaning is still an open problem. Some models have been constructed to explain Zipf's law. In the letter, a concept of auxiliary information of an event is introduced, and a new entropy named non-symmetric entropy is defined. We prove that maximizing non-symmetric entropy leads naturally to Zipf's law under the special auxiliary information parameters. At the same time, if we take other auxiliary parameters we will get other distribution laws.
Role of serotonergic input to the ventrolateral medulla in expression of the 10-Hz sympathetic nerve rhythm
Authors: Orer Hakan S. , Gebber Gerard L. , Barman Susan M.
Published: March 2008

We studied the changes in inferior cardiac sympathetic nerve discharge (SND) produced by unilateral microinjections of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists into the ventrolateral medulla (VLM) of urethane-anesthetized, baroreceptor-denervated cats. Microinjection of the 5-HT2 receptor antagonist LY-53857 (10 mM) into either the rostral or caudal VLM significantly reduced ( P ≤ 0.05) the 10-Hz rhythmic component of basal SND without affecting its lower-frequency, aperiodic component. The selective depression of 10-Hz power was accompanied by a statistically significant decrease in mean arterial pressure (MAP). Microinjection of LY-53857 into the VLM also attenuated the increase in 10-Hz power that followed tetanic stimulation of depressor sites in the caudal medullary raphé nuclei. Microinjection of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)2-amino-propane (DOI; 10 μM) into the VLM selectively enhanced 10-Hz SND, and intravenous DOI (1 mg/kg) partially reversed the reduction in 10-Hz SND produced by 5-HT2 receptor blockade in the VLM. Microinjection of the 5-HT1A receptor agonist, 8-hydroxy-2-(di- n-propylamino)tetralin (8-OHDPAT; 10 mM), into either the rostral or caudal VLM also selectively attenuated 10-Hz SND and significantly reduced MAP. The reduction in 10-Hz SND produced by 8-OHDPAT was partially reversed by intravenous WAY-100635 (1 mg/kg), which selectively blocks 5-HT1A receptors. These results support the view that serotonergic inputs to the VLM play an important role in expression of the 10-Hz rhythm in SND.
Involvement of apical P2Y2receptor-regulated CFTR activity in muscarinic stimulation of Cl−reabsorption in rat submandibular gland
Authors: Ishibashi Kazunari , Okamura Kazuhiko , Yamazaki Jun
Published: March 2008

Previously, we presented in vivo evidence for a physiological significance of cAMP-regulated CFTR Cl−channels in Ca2+-activated Cl−reabsorption in the ductal system of the rat submandibular gland. Here, we address the mechanism by which basal CFTR activation contributes to the transepithelial Cl−movement evoked by muscarinic stimulation. The Cl−concentration ([Cl−]) was increased in the final saliva from rat submandibular gland during pilocarpine stimulation when a small interfering RNA for CFTR or a specific CFTR inhibitor, CFTRinh-172, was injected retrogradely into the gland's own duct, indicating that basal CFTR activation is involved in Cl−reabsorption. Systemically administered propranolol failed to alter the [Cl−], suggesting little involvement of a β-adrenergic pathway in the Cl−movement that occurs through basal CFTR activation. Intraductal injection of suramin (a nonspecific P2-receptor antagonist) increased the salivary [Cl−], indicating the existence of endogenous purinergic activation. Upon separate intraductal injection, ATP and a P2Y2-receptor agonist, UTP, decreased the salivary [Cl−] almost equipotently. CFTRinh-172 and suramin each prevented these effects, whereas 2′,3′- O-(4-benzoylbenzoyl)-ATP (Bz-ATP), a P2X7agonist, had no specific effect. Pilocarpine stimulation evoked ATP secretion into the salivary fluid. Immunohistochemistry revealed the partial coexistence of CFTR and P2Y2receptors on the luminal surface of epithelial cells in the striated ducts of this gland. These results raise the possibility that muscarinic stimulation-induced Cl−reabsorption occurs through basal CFTR activity and that this is regulated by P2Y2receptors in the ductal epithelium via stimulation by ATP secreted into the salivary fluid.
Volume expansion stimulates monoubiquitination and endocytosis of surface-expressed skate anion-exchanger isoform
Authors: Musch Mark W. , Puffer Amanda B. , Goldstein Leon
Published: March 2008

In hyposmotic medium, skate erythrocytes swell and then lose taurine and other solutes with obligate water to achieve a regulatory volume decrease (RVD) over a 90-min period. The skate erythrocyte anion-exchanger isoform 1 (skAE1) participates in the RVD, and increased surface expression after hyposmolality-induced volume expansion occurs within 5 min but decreases to baseline within 120 min. The subsequent fate of skAE1 is the focus of these studies. SkAE1 sent to the surface becomes monoubiquitinated, a modification that is present while skAE1 is associated with clathrin and Rab5 but is removed before skAE1 is passed to the Rab4 compartment. Endocytosis of skAE1 involves clathrin-mediated internalization. Surface plasma membrane skAE1 forms tetramers and demonstrates increased tyrosine phosphorylation, and both of these processes decrease before skAE1 appears in the Rab5 compartment. Volume expansion-stimulated surface skAE1 comes from an intracellular pool in a buoyant membrane fraction resistant to nonionic detergent extraction (DRM), and the amount of skAE1 increases in this buoyant DRM fraction on the surface. Clathrin heavy chain is found largely in the erythrocyte DRM, but in dense, rather than buoyant, fractions. Rab5- and Rab4-containing membranes are largely detergent soluble, suggesting that as skAE1 is passed to clathrin and then to Rab5 compartments, the membrane microdomain composition changes. The present studies demonstrate that skAE1, which appears on the surface after hyposmolality-induced volume expansion, is monoubiquitinated, a modification that may serve as a signal for removal of skAE1 from the surface. This modification is eliminated after clathrin-mediated removal of skAE1 in a membrane domain containing Rab5, potentially permitting recycling and reuse of skAE1.
Deleterious effects of endogenous and exogenous testosterone on mesenchymal stem cell VEGF production
Authors: Ray Rinki , Herring Christine M. , Markel Troy A. , Crisostomo Paul R. , Wang Meijing , Weil Brent , Lahm Tim , Meldrum Daniel R.
Published: March 2008

Modulating the paracrine effects of bone marrow mesenchymal stem cells (BMSCs) may be important for the treatment of ischemic myocardial tissue. In this regard, endogenous estrogen may enhance BMSC vascular endothelial growth factor (VEGF) production. However, little information exists regarding the effect of testosterone on stem cell function. We hypothesized that 1) endogenous or exogenous estrogen will enhance stem cell production of VEGF and 2) endogenous or exogenous testosterone will inhibit BMSC VEGF production. BMSCs were collected from adult male, female, castrated male, and ovariectomized female rats. One hundred thousand cells were incubated with testosterone (1, 10, or 100 nM) or estrogen (0.15, 1.5, or 15 nM) for 48 h. Cell supernatants were collected, and VEGF was measured by ELISA. BMSCs harvested from castrated males, normal females, and ovariectomized females produced more VEGF compared with normal males. Castration was associated with the highest level (1,018 ± 98.26 pg/ml) of VEGF production by BMSCs, which was significantly more than that produced by BMSCs harvested from normal male and normal female animals. Exogenous testosterone significantly reduced VEGF production in BMSCs harvested from ovariectomized females in a dose-dependent manner. Exogenous estrogen did not alter BMSC VEGF production. These findings suggest that testosterone may work on BMSCs to decrease protective growth factor production and that effective removal of testosterone's deleterious effects via castration may prove to be beneficial in terms of protective factor production. By manipulating the mechanisms that BMSCs use to produce growth factors, we may be able to engineer stem cells to produce maximum growth factors during therapeutic use.
MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells
Authors: Elberg Gerard , Chen Lijuan , Elberg Dorit , Chan Michael D. , Logan Charlotte J. , Turman Martin A.
Published: March 2008

Transforming growth factor-β1 (TGF-β1) is known to induce epithelial-mesenchymal transition in the kidney, a process involved in tubulointerstitial fibrosis. We hypothesized that a coactivator of the serum response factor (SRF), megakaryoblastic leukemia factor-1 (MKL1), stimulates α-smooth muscle actin (α-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-β1. Herein, we study the effect of MKL1 expression on α-SMA in these cells. We demonstrate that TGF-β1 stimulation of α-SMA transcription is mediated through CC(A/T)6-rich GG elements known to bind to SRF. These elements also mediate the MKL1 effect that dramatically activates α-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces α-SMA expression regardless of treatment with TGF-β1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufficient to induce α-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-β1 stimulation of α-SMA expression. Therefore, MKL1 is also absolutely required for TGF-β1 stimulation of α-SMA expression. Western blot and immunofluorescence analysis show that overexpressed and endogenous MKL1 are located in the nucleus in non-stimulated RTC. Chromatin immunoprecipitation assay demonstrates that TGF-β1 induces binding of endogenous SRF and MKL1 to the α-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating α-SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myofibroblast formation and function in the kidney.
Modulation of Trigeminal Spinal Subnucleus Caudalis Neuronal Activity Following Regeneration of Transected Inferior Alveolar Nerve in Rats
Authors: Saito Kimiko , Hitomi Suzuro , Suzuki Ikuko , Masuda Yuji , Kitagawa Junichi , Tsuboi Yoshiyuki , Kondo Masahiro , Sessle Barry J. , Iwata Koichi
Published: March 2008

Modulation of trigeminal spinal subnucleus caudalis neuronal activity following regeneration of transected inferior alveolar nerve in rats. To clarify the neuronal mechanisms of abnormal pain in the face innervated by the regenerated inferior alveolar nerve (IAN), nocifensive behavior, trigeminal ganglion neuronal labeling following Fluorogold (FG) injection into the mental skin, and trigeminal spinal subnucleus caudalis (Vc) neuronal properties were examined in rats with IAN transection. The mechanical escape threshold was significantly higher at 3 days and lower at 14 days after IAN transection, whereas head withdrawal latency to heat was significantly longer at 3, 14, and 60 days after IAN transection. The number of FG-labeled ganglion neurons was significantly reduced at 3 days after IAN transection but increased at 14 and 60 days. The number of wide dynamic range (WDR) neurons with background (BG) activity was significantly higher at 14 and 60 days after IAN transection compared with naïve rats, and the number of WDR and low-threshold mechanoreceptive (LTM) neurons with irregularly bursting BG activity was increased at these two time points. Mechanically evoked responses were significantly larger in WDR and LTM neurons 14 days after IAN transection compared with naïve rats. Heat- and cold-evoked responses in WDR neurons were significantly lower at 14 days after transection compared with naïve rats. Mechanoreceptive fields were also significantly larger in WDR and LTM neurons at 14 and 60 days after IAN transection. These findings suggest that these alterations may be involved in the development of mechanical allodynia in the cutaneous region innervated by the regenerated IAN.
Intercostal and Abdominal Respiratory Motoneurons in the Neonatal Rat Spinal Cord: Spatiotemporal Organization and Responses to Limb Afferent Stimulation
Authors: Giraudin Aurore , Cabirol-Pol Marie-Jeanne , Simmers John , Morin Didier
Published: March 2008

Respiration requires the coordinated rhythmic contractions of diverse muscles to produce ventilatory movements adapted to organismal requirements. During fast locomotion, locomotory and respiratory movements are coordinated to reduce mechanical conflict between these functions. Using semi-isolated and isolated in vitro brain stem-spinal cord preparations from neonatal rats, we have characterized for the first time the respiratory patterns of all spinal intercostal and abdominal motoneurons and explored their functional relationship with limb sensory inputs. Neuroanatomical and electrophysiological procedures were initially used to locate intercostal and abdominal motoneurons in the cord. Intercostal motoneuron somata are distributed rostrocaudally from C7–T13segments. Abdominal motoneuron somata lie between T8and L2. In accordance with their soma distributions, inspiratory intercostal motoneurons are recruited in a rostrocaudal sequence during each respiratory cycle. Abdominal motoneurons express expiratory-related discharge that alternates with inspiration. Lesioning experiments confirmed the pontine origin of this expiratory activity, which was abolished by a brain stem transection at the rostral boundary of the VII nucleus, a critical area for respiratory rhythmogenesis. Entrainment of fictive respiratory rhythmicity in intercostal and abdominal motoneurons was elicited by periodic low-threshold dorsal root stimulation at lumbar (L2) or cervical (C7) levels. These effects are mediated by direct ascending fibers to the respiratory centers and a combination of long-projection and polysynaptic descending pathways. Therefore the isolated brain stem-spinal cord in vitro generates a complex pattern of respiratory activity in which alternating inspiratory and expiratory discharge occurs in functionally identified spinal motoneuron pools that are in turn targeted by both forelimb and hindlimb somatic afferents to promote locomotor-respiratory coupling.
The Brain Stem Saccadic Burst Generator Encodes Gaze in Three-Dimensional Space
Authors: Van Horn Marion R. , Sylvestre Pierre A. , Cullen Kathleen E.
Published: March 2008

When we look between objects located at different depths the horizontal movement of each eye is different from that of the other, yet temporally synchronized. Traditionally, a vergence-specific neuronal subsystem, independent from other oculomotor subsystems, has been thought to generate all eye movements in depth. However, recent studies have challenged this view by unmasking interactions between vergence and saccadic eye movements during disconjugate saccades. Here, we combined experimental and modeling approaches to address whether the premotor command to generate disconjugate saccades originates exclusively in “vergence centers.” We found that the brain stem burst generator, which is commonly assumed to drive only the conjugate component of eye movements, carries substantial vergence-related information during disconjugate saccades. Notably, facilitated vergence velocities during disconjugate saccades were synchronized with the burst onset of excitatory and inhibitory brain stem saccadic burst neurons (SBNs). Furthermore, the time-varying discharge properties of the majority of SBNs (>70%) preferentially encoded the dynamics of an individual eye during disconjugate saccades. When these experimental results were implemented into a computer-based simulation, to further evaluate the contribution of the saccadic burst generator in generating disconjugate saccades, we found that it carries all the vergence drive that is necessary to shape the activity of the abducens motoneurons to which it projects. Taken together, our results provide evidence that the premotor commands from the brain stem saccadic circuitry, to the target motoneurons, are sufficient to ensure the accurate control shifts of gaze in three dimensions.
CX3CR1 Is Expressed by Prostate Epithelial Cells and Androgens Regulate the Levels of CX3CL1/Fractalkine in the Bone Marrow: Potential Role in Prostate Cancer Bone Tropism
Authors: Jamieson Whitney L. , Shimizu Saori , D'Ambrosio Julia A. , Meucci Olimpia , Fatatis Alessandro
Published: March 2008

We have previously shown that the chemokine fractalkine promotes the adhesion of human prostate cancer cells to bone marrow endothelial cells as well as their migration toward human osteoblasts in vitro. Thus, the interaction of fractalkine with its receptor CX3CR1 could play a crucial role in vivo by directing circulating prostate cancer cells to the bone. We found that although CX3CR1 is minimally detectable in epithelial cells of normal prostate glands, it is overexpressed upon malignant transformation. Interestingly, osteoblasts, stromal and mesenchymal cells derived from human bone marrow aspirates express the cell-bound form of fractalkine, whereas the soluble form of the chemokine is detected in bone marrow supernatants. To investigate the mechanisms regulating the levels of soluble fractalkine in the bone marrow, we focused on androgens, which play a critical role in both prostate cancer progression and skeletal metastasis. Here, we show that dihydrotestosterone dramatically increases the cleavage of fractalkine from the plasma membrane of bone cells and its action is reversed by nilutamide—an antagonist of the androgen receptor—as well as the wide-spectrum inhibitor of matrix metalloproteases, GM6001. However, dihydrotestosterone was unable to induce fractalkine-cleavage from human bone marrow endothelial cells. Thus, androgens could promote the extravasation of CX3CR1-bearing cancer cells on a fractalkine concentration gradient, while leaving unaltered their ability to adhere to the bone marrow endothelium. In conclusion, our results indicate that CX3CR1, fractalkine, and the enzymes responsible for its cleavage might represent suitable targets for therapies aiming to counteract skeletal secondary tumors from prostate adenocarcinoma. [Cancer Res 2008;68(6):1715–22]
Rad6B Is a Positive Regulator of β-Catenin Stabilization
Authors: Shekhar Malathy P.V. , Gerard Brigitte , Pauley Robert J. , Williams Bart O. , Tait Larry
Published: March 2008

Mutations in β-catenin or other Wnt pathway components that cause β-catenin accumulation occur rarely in breast cancer. However, there is some evidence of β-catenin protein accumulation in a subset of breast tumors. We have recently shown that Rad6B, an ubiquitin-conjugating enzyme, is a transcriptional target of β-catenin/TCF. Here, we show that forced Rad6B overexpression in MCF10A breast cells induces β-catenin accumulation, which despite being ubiquitinated is stable and transcriptionally active. A similar relationship between Rad6B, β-catenin ubiquitination, and transcriptional activity was found in WS-15 and MDA-MB-231 breast cancer cells, and mouse mammary tumor virus–Wnt-1 mammary tumor—derived cells, implicating Rad6B in physiologic regulation of β-catenin stability and activity. Ubiquitinated β-catenin was detectable in chromatin immunoprecipitations performed with β-catenin antibody in MDA-MB-231 but not MCF10A cells. Rad6B silencing caused suppression of β-catenin monoubiquitination and polyubiquitination, and transcriptional activity. These effects were accompanied by a reduction in intracellular β-catenin but with minimal effects on cell membrane–associated β-catenin. Measurement of β-catenin protein stability by cycloheximide treatment showed that Rad6B silencing specifically decreases the stability of high molecular β-catenin with minimal effect upon the 90-kDa nascent form. In vitro ubiquitination assays confirmed that Rad6B mediates β-catenin polyubiquitination, and ubiquitin chain extensions involve lysine 63 residues that are insensitive to 26S proteasome. These findings, combined with our previous data that Rad6B is a transcriptional target of β-catenin, reveal a positive regulatory feedback loop between Rad6B and β-catenin and a novel mechanism of β-catenin stabilization/activation in breast cancer cells. [Cancer Res 2008;68(6):1741–50]
Regulation of CXCR4 by the Notch Ligand Delta-like 4 in Endothelial Cells
Authors: Williams Cassin Kimmel , Segarra Marta , De La Luz Sierra Maria , Sainson Richard C.A. , Tosato Giovanna , Harris Adrian L.
Published: March 2008

Gene-targeting studies have shown that Delta-like 4 (Dll4) is required for normal embryonic vascular remodeling, but the mechanisms underlying Dll4 regulatory functions are not well defined. We generated primary human umbilical vascular endothelial cells that express Dll4 protein to study Dll4 function and previously showed that Dll4 down-regulates vascular endothelial growth factor (VEGF) receptor 2 and NRP1 expression and inhibits VEGF function. We now report that expression of Dll4 in endothelial cells inhibited attachment and migration to stromal-derived growth factor 1 (SDF1) chemokine. Cell surface, total protein, and mRNA levels of CXCR4, principal signaling receptor for SDF1, were significantly decreased in Dll4-transduced endothelial cells, attributable to a significant reduction of CXCR4 promoter activity. An immobilized recombinant extracellular portion of Dll4 (rhDLL4) was sufficient to down-regulate CXCR4 mRNA and protein, whereas protein levels of SDF1, VEGF, and RDC1 were unchanged. The γ-secretase inhibitor L-685,458 significantly reconstituted CXCR4 mRNA in rhDLL4-stimulated endothelial cells. CXCR4 mRNA levels were significantly reduced in mouse xenografts of Dll4-transduced human gliomas compared with control gliomas, and vascular CXCR4 was not detected by immunohistochemistry in the enlarged vessels within the Dll4 gliomas. Thus, Dll4 may contribute to vascular differentiation and inhibition of the angiogenic response by regulating multiple receptor pathways. [Cancer Res 2008;68(6):1889–95]
AXL Is a Potential Target for Therapeutic Intervention in Breast Cancer Progression
Authors: Zhang Yi-Xiang , Knyazev Peter G. , Cheburkin Yuri V. , Sharma Kirti , Knyazev Yuri P. , Őrfi László , Szabadkai István , Daub Henrik , Kéri György , Ullrich Axel
Published: March 2008

Protein kinases play important roles in tumor development and progression. A variety of members of this family of signal transduction enzymes serve as targets for therapeutic intervention in cancer. We have identified the receptor tyrosine kinase (RTK) AXL as a potential mediator of motility and invasivity of breast cancer cells. AXL is expressed in most highly invasive breast cancer cells, but not in breast cancer cells of low invasivity. Ectopic expression of AXL was sufficient to confer a highly invasive phenotype to weakly invasive MCF7 breast cancer cells. Experimental inhibition of AXL signaling by a dominant-negative AXL mutant, an antibody against the extracellular domain of AXL, or short hairpin RNA knockdown of AXL decreased motility and invasivity of highly invasive breast cancer cells. To selectively interfere with cancer cell properties defining the rate of disease progression, we identified 3-quinolinecarbonitrile compounds, which displayed potent inhibitory activity against AXL and showed strong interference with motility and invasivity of breast cancer cells. Our findings validated the RTK AXL as a critical element in the signaling network that governs motility and invasivity of breast cancer cells, and allowed the identification of experimental anti-AXL small molecular inhibitors that represent lead substances for the development of antimetastatic breast cancer therapy. [Cancer Res 2008;68(6):1905–15]
Kaposi's Sarcoma–Associated Herpesvirus Viral IFN Regulatory Factor 3 Stabilizes Hypoxia-Inducible Factor-1α to Induce Vascular Endothelial Growth Factor Expression
Authors: Shin Young C. , Joo Chul-Hyun , Gack Michaela U. , Lee Hye-Ra , Jung Jae U.
Published: March 2008

Kaposi's sarcoma–associated herpesvirus (KSHV) is the etiologic agent associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of both developmental and pathologic angiogenesis, composed of an oxygen-sensitive α-subunit and a constitutively expressed β-subunit. HIF-1 activity in tumors depends on the availability of the HIF-1α subunit, the levels of which are increased under hypoxic conditions. Recent studies have shown that HIF-1 plays an important role in KSHV reactivation from latency and pathogenesis. Here, we report a novel mechanism by which KSHV activates HIF-1 activity. Specific interaction between KSHV viral IFN regulatory factor 3 (vIRF3) and the HIF-1α subunit led to the HIF-1α stabilization and transcriptional activation, which induced vascular endothelial growth factor expression and ultimately facilitated endothelial tube formation. Remarkably, the central domain of vIRF3, containing double α-helix motifs, was sufficient not only for binding to HIF-1α but also for blocking its degradation in normoxic conditions. This indicates that KSHV has developed a unique mechanism to enhance HIF-1α protein stability and transcriptional activity by incorporating a viral homologue of cellular IRF gene into its genome, which may contribute to viral pathogenesis. [Cancer Res 2008;68(6):1751–9]
14-3-3ζ Down-regulates p53 in Mammary Epithelial Cells and Confers Luminal Filling
Authors: Danes Christopher G. , Wyszomierski Shannon L. , Lu Jing , Neal Christopher L. , Yang Wentao , Yu Dihua
Published: March 2008

Recent progress in diagnostic tools allows many breast cancers to be detected at an early preinvasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. Previously, we discovered that 14-3-3ζ is overexpressed in >40% of advanced breast cancers, and this overexpression predicts poor patient survival. Here, we examined at what stage of breast disease 14-3-3ζ overexpression occurs, and we found that increased expression of 14-3-3ζ begins at atypical ductal hyperplasia, an early stage of breast disease. To determine whether 14-3-3ζ overexpression is a decisive early event in breast cancer, we overexpressed 14-3-3ζ in MCF10A cells and examined its effect in a three-dimensional culture model. We discovered that 14-3-3ζ overexpression severely disrupted the acini architecture resulting in luminal filling. Proper lumen formation is a result of anoikis, apoptosis due to detachment from the basement membrane. We found that 14-3-3ζ overexpression conferred resistance to anoikis. Additionally, 14-3-3ζ overexpression in MCF10A cells and in mammary epithelial cells (MEC) from 14-3-3ζ transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3ζ induced hyperactivation of the phosphoinositide 3-kinase/Akt pathway which led to phosphorylation and translocation of the MDM2 E3 ligase resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3ζ–overexpressing MCF10A acini in three-dimensional cultures. These data suggest that 14-3-3ζ overexpression is a critical event in early breast disease, and down-regulation of p53 is one of the mechanisms by which 14-3-3ζ alters MEC acini structure and increases the risk of breast cancer. [Cancer Res 2008;68(6):1760–7]
Mammalian Target of Rapamycin Repression by 3,3′-Diindolylmethane Inhibits Invasion and Angiogenesis in Platelet-Derived Growth Factor-D–Overexpressing PC3 Cells
Authors: Kong Dejuan , Banerjee Sanjeev , Huang Wei , Li Yiwei , Wang Zhiwei , Kim Hyeong-Reh Choi , Sarkar Fazlul H.
Published: March 2008

Platelet-derived growth factor-D (PDGF-D) is a newly recognized growth factor known to regulate many cellular processes, including cell proliferation, transformation, invasion, and angiogenesis. Recent studies have shown that PDGF-D and its cognate receptor PDGFR-β are expressed in prostate tumor tissues, suggesting that PDGF-D might play an important role in the development and progression of prostate cancer. However, the biological role of PDGF-D in tumorigenesis remains elusive. In this study, we found that PDGF-D–overexpressing PC3 cells (PC3 cells stably transfected with PDGF-D cDNA and referred to as PC3 PDGF-D) exhibited a rapid growth rate and enhanced cell invasion that was associated with the activation of mammalian target of rapamycin (mTOR) and reduced Akt activity. Rapamycin repressed mTOR activity and concomitantly resulted in the activation of Akt, which could attenuate the therapeutic effects of mTOR inhibitors. In contrast, B-DIM (BR-DIM from Bioresponse, Inc.; a chemopreventive agent) significantly inhibited both mTOR and Akt in PC3 PDGF-D cells, which were correlated with decreased cell proliferation and invasion. Moreover, conditioned medium from PC3 PDGF-D cells significantly increased the tube formation of human umbilical vein endothelial cells, which was inhibited by B-DIM treatment concomitant with reduced full-length and active form of PDGF-D. Our results suggest that B-DIM could serve as a novel and efficient chemopreventive and/or therapeutic agent by inactivation of both mTOR and Akt activity in PDGF-D–overexpressing prostate cancer. [Cancer Res 2008;68(6):1927–34]
Cancer-Associated Transforming Growth Factor β Type II Receptor Gene Mutant Causes Activation of Bone Morphogenic Protein-Smads and Invasive Phenotype
Authors: Bharathy Savita , Xie Wen , Yingling Jonathan M. , Reiss Michael
Published: March 2008

Transforming growth factor β (TGFβ) plays a key role in maintaining tissue homeostasis by inducing cell cycle arrest, differentiation and apoptosis, and ensuring genomic integrity. Furthermore, TGFβ orchestrates the response to tissue injury and mediates repair by inducing epithelial to mesenchymal transition and by stimulating cell motility and invasiveness. Although loss of the homeostatic activity of TGFβ occurs early on in tumor development, many advanced cancers have coopted the tissue repair function to enhance their metastatic phenotype. How these two functions of TGFβ become uncoupled during cancer development remains poorly understood. Here, we show that, in human keratinocytes, TGFβ induces phosphorylation of Smad2 and Smad3 as well as Smad1 and Smad5 and that both pathways are dependent on the kinase activities of the type I and II TGFβ receptors (TβR). Moreover, cancer-associated missense mutations of the TβRII gene (TGFBR2) are associated with at least two different phenotypes. One type of mutant (TGFBR2E526Q) is associated with loss of kinase activity and all signaling functions. In contrast, a second mutant (TGFBR2R537P) is associated with high intrinsic kinase activity, loss of Smad2/3 activation, and constitutive activation of Smad1/5. Furthermore, this TGFBR2 mutant endows the carcinoma cells with a highly motile and invasive fibroblastoid phenotype. This activated phenotype is TβRI (Alk-5) independent and can be reversed by the action of a dual TβRI and TβRII kinase inhibitor. Thus, identification of such activated TβRII receptor mutations in tumors may have direct implications for appropriately targeting these cancers with selective therapeutic agents. [Cancer Res 2008;68(6):1656–66]
Cannabinoids Inhibit Glioma Cell Invasion by Down-regulating Matrix Metalloproteinase-2 Expression
Authors: Blázquez Cristina , Salazar María , Carracedo Arkaitz , Lorente Mar , Egia Ainara , González-Feria Luis , Haro Amador , Velasco Guillermo , Guzmán Manuel
Published: March 2008

Cannabinoids, the active components of Cannabis sativa L. and their derivatives, inhibit tumor growth in laboratory animals by inducing apoptosis of tumor cells and impairing tumor angiogenesis. It has also been reported that these compounds inhibit tumor cell spreading, but the molecular targets of this cannabinoid action remain elusive. Here, we evaluated the effect of cannabinoids on matrix metalloproteinase (MMP) expression and its effect on tumor cell invasion. Local administration of Δ9-tetrahydrocannabinol (THC), the major active ingredient of cannabis, down-regulated MMP-2 expression in gliomas generated in mice, as determined by Western blot, immunofluorescence, and real-time quantitative PCR analyses. This cannabinoid-induced inhibition of MMP-2 expression in gliomas (a) was MMP-2–selective, as levels of other MMP family members were unaffected; (b) was mimicked by JWH-133, a CB2 cannabinoid receptor–selective agonist that is devoid of psychoactive side effects; (c) was abrogated by fumonisin B1, a selective inhibitor of ceramide biosynthesis; and (d) was also evident in two patients with recurrent glioblastoma multiforme. THC inhibited MMP-2 expression and cell invasion in cultured glioma cells. Manipulation of MMP-2 expression by RNA interference and cDNA overexpression experiments proved that down-regulation of this MMP plays a critical role in THC-mediated inhibition of cell invasion. Cannabinoid-induced inhibition of MMP-2 expression and cell invasion was prevented by blocking ceramide biosynthesis and by knocking-down the expression of the stress protein p8. As MMP-2 up-regulation is associated with high progression and poor prognosis of gliomas and many other tumors, MMP-2 down-regulation constitutes a new hallmark of cannabinoid antitumoral activity. [Cancer Res 2008;68(6):1945–52]
Prediction of Cancer Driver Mutations in Protein Kinases
Authors: Torkamani Ali , Schork Nicholas J.
Published: March 2008

A large number of somatic mutations accumulate during the process of tumorigenesis. A subset of these mutations contribute to tumor progression (known as “driver” mutations) whereas the majority of these mutations are effectively neutral (known as “passenger” mutations). The ability to differentiate between drivers and passengers will be critical to the success of upcoming large-scale cancer DNA resequencing projects. Here we show a method capable of discriminating between drivers and passengers in the most frequently cancer-associated protein family, protein kinases. We apply this method to multiple cancer data sets, validating its accuracy by showing that it is capable of identifying known drivers, has excellent agreement with previous statistical estimates of the frequency of drivers, and provides strong evidence that predicted drivers are under positive selection by various sequence and structural analyses. Furthermore, we identify particular positions in protein kinases that seem to play a role in oncogenesis. Finally, we provide a ranked list of candidate driver mutations. [Cancer Res 2008;68(6):1675–82]