bluuebunny/crossref_metadata_2025 huggingface dataset:
Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells
Authors: Holtkamp Silke , Kreiter Sebastian , Selmi Abderraouf , Simon Petra , Koslowski Michael , Huber Christoph , Türeci Özlem , Sahin Ugur
Published: August 2006

Adoptive transfer of dendritic cells (DCs) transfected with in vitro–transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacokinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3′ of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription–polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tail measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tail with a free 3′ end rather than one extended with unrelated nucleotides, and 2 sequential β-globin 3′ untranslated regions cloned head to tail between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4+ and CD8+ T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations.
Polymerization of fibrin: direct observation and quantification of individual B:b knob-hole interactions
Authors: Litvinov Rustem I. , Gorkun Oleg V. , Galanakis Dennis K. , Yakovlev Sergiy , Medved Leonid , Shuman Henry , Weisel John W.
Published: August 2006

The polymerization of fibrin occurs primarily through interactions between N-terminal A- and B-knobs, which are exposed by the cleavage of fibrinopeptides A and B, respectively, and between corresponding a- and b-holes in the γ- and β-modules. Of the potential knob-hole interactions—A:a, B:b, A:b, and B:a—the first has been shown to be critical for fibrin formation, but the roles of the others have remained elusive. Using laser tweezers–based force spectroscopy, we observed and quantified individual B:b and A:b interactions. Both desA-fibrin with exposed A-knobs and desB-fibrin bearing B-knobs interacted with fragment D from the γD364H fibrinogen containing b-holes but no functional a-holes. The strength of single B:b interactions was found to be 15 to 20 pN, approximately 6-fold weaker than A:a interactions. B:b binding was abrogated by B-knob mimetic peptide, the (β15-66)2 fragment containing 2 B-knobs, and a monoclonal antibody against the β15-21 sequence. The interaction of desB-fibrin with fragment D containing a- and b-holes produced the same forces that were insensitive to A-knob mimetic peptide, suggesting that B:a interactions were absent. These results directly demonstrate for the first time B:b binding mediated by natural B-knobs exposed in a fibrin monomer.
Wnt expression is not correlated with β-catenin dysregulation in Dupuytren's Disease
Authors: O'Gorman David B , Wu Yan , Seney Shannon , Zhu Rebecca D , Gan Bing Siang
Published: August 2006

Background

Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in finger flexion contractures. Increased cellular β-catenin levels have been identified as characteristic of this disease. As Wnts are the most widely recognized upstream regulators of cellular β-catenin accumulation, we have examined Wnt gene expression in surgical specimens and in DD-derived primary cell cultures grown in two-dimensional monolayer culture or in three-dimensional FPCL collagen lattice cultures.

Results

The Wnt expression profile of patient-matched DD and unaffected control palmar fascia tissue was determined by a variety of complimentary methods; Affymetrix Microarray analysis, specific Wnt and degenerative primer-based Reverse Transcriptase (RT)-PCR, and Real Time PCR. Microarray analysis identified 13 Wnts associated with DD and control tissues. Degenerate Wnt RT-PCR analysis identified Wnts 10b and 11, and to a lesser extent 5a and 9a, as the major Wnt family members expressed in our patient samples. Competitive RT-PCR analysis identified significant differences between the levels of expression of Wnts 9a, 10b and 11 in tissue samples and in primary cell cultures grown as monolayer or in FPCL, where the mRNA levels in tissue > FPCL cultures > monolayer cultures. Real Time PCR data confirmed the down-regulation of Wnt 11 mRNA in DD while Wnt 10b, the most frequently isolated Wnt in DD and control palmar fascia, displayed widely variable expression between the methods of analysis.

Conclusion

These data indicate that changes in Wnt expressionper seare unlikely to be the cause of the observed dysregulation of β-catenin expression in DD.
Microarray data on gene modulation by HIV-1 in immune cells: 2000–2006
Authors: Giri Malavika S , Nebozhyn Michael , Showe Louise , Montaner Luis J
Published: August 2006

Here, we review 34 HIV microarray studies in human immune cells over the period of 2000–March 2006 with emphasis on analytical approaches used and conceptual advances on HIV modulation of target cells (CD4 T cell, macrophage) and nontargets such as NK cell, B cell, and dendritic cell subsets. Results to date address advances on gene modulation associated with immune dysregulation, susceptibility to apoptosis, virus replication, and viral persistence following in vitro or in vivo infection/exposure to HIV-1 virus or HIV-1 accessory proteins. In addition to gene modulation associated with known functional correlates of HIV infection and replication (e.g., T cell apoptosis), microarray data have yielded novel, potential mechanisms of HIV-mediated pathogenesis such as modulation of cholesterol biosynthetic genes in CD4 T cells (relevant to virus replication and infectivity) and modulation of proteasomes and histone deacetylases in chronically infected cell lines (relevant to virus latency). Intrinsic challenges in summarizing gene modulation studies remain in development of sound approaches for comparing data obtained using different platforms and analytical tools, deriving unifying concepts to distil the large volumes of data collected, and the necessity to impose a focus for validation on a small fraction of genes. Notwithstanding these challenges, the field overall continues to demonstrate progress in expanding the pool of target genes validated to date in in vitro and in vivo datasets and understanding the functional correlates of gene modulation to HIV-1 pathogenesis in vivo.
A dominant role for FcγRII in antibody-enhanced dengue virus infection of human mast cells and associated CCL5 release
Authors: Brown Michael G , King Christine A , Sherren Christine , Marshall Jean S , Anderson Robert
Published: August 2006

Dengue virus is a major mosquito-borne human pathogen with four known serotypes. The presence of antidengue virus antibodies in the serum of individuals prior to dengue virus infection is believed to be an important risk factor for severe dengue virus disease as a result of the phenomenon of antibody-dependent enhancement operating on Fc receptor (FcR)-bearing cells. In addition to blood monocytes, mast cells are susceptible to antibody-enhanced dengue virus infection, producing a number of inflammatory mediators including IL-1, IL-6, and CCL5. Using the human mast cell-like lines KU812 and HMC-1 as well as primary cultures of human cord blood-derived mast cells (CBMC), we aimed to identify the participating FcRs in antibody-enhanced mast cell dengue virus infection, as FcRs represent a potential site for therapeutic intervention. CBMC expressed significant levels of FcγRI, FcγRII, and FcγRIII, and mast cell-like HMC-1 and KU812 cells expressed predominantly FcγRII. All four serotypes of dengue virus showed antibody-enhanced binding to KU812 cells. Specific FcγRII blockade with mAb IV.3 was found to significantly abrogate dengue virus binding to KU812 cells and CBMC in the presence of dengue-specific antibody. Dengue virus infection and the production of CCL5 by KU812 cells were also inhibited by FcγRII blockade.
Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the signals induced by the NACHT-LRR (NLR) pattern recognition receptors
Authors: Netea Mihai G , Azam Tania , Ferwerda Gerben , Girardin Stephen E , Kim Soo-Hyun , Dinarello Charles A
Published: August 2006

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a member of a new family of myeloid receptors, encoded by a gene cluster linked to the MHC. Engagement of TREM-1 stimulates intracellular signals, resulting in activation of phagocytosis, neutrophil degranulation, and amplification of cytokine production induced by TLRs. In the present study, a novel property following engagement of TREM-1 is described, namely the amplification of cytokine production induced by the second major class of pattern recognition receptors, the NAIP, CIITA, HET-E, TP-1-leucine-rich repeat (NACHT-LRR; NLR) receptors, which recognize intracellular microorganisms through sensing their muropeptide components of peptidoglycan. The TREM-1/NLR synergism was observed for the production of TNF-α, IL-1β, and IL-6, leading to an increase in cytokine production up to tenfold greater than the additive value of TREM-1 or muropeptide stimulation alone. Several putative mechanisms are proposed to be involved in the synergism between NLRs and TREM-1, including the increase in TREM-1 expression by NLR ligands, and of the expression of nucleotide oligomerization domain-2 receptor by TREM-1 engagement. In contrast, although caspase-1 modulates IL-1β and IL-6 production after stimulation with anti-TREM-1 antibodies or NLR ligands, it does not appear to be responsible for the synergism between these two pathways. These findings demonstrate that TREM-1 acts on both major recognition pathways of bacterial structures: the extracellular TLR receptors, and the intracellular NLR molecules. This latter finding supports the concept that TREM-1 provides optimal amplification of cytokine-induced inflammation during the initiation of host defense.
Type 1 Collagen Marker of Bone Turnover, Insulin-Like Growth Factor, and Leptin in Dichorionic Twins with Discordant Birth Weight
Authors: Bajoria Rekha , Sooranna Suren R. , Chatterjee Ratna
Published: August 2006

Objective: We investigated the relationship between IGF-I-IGF binding protein (IGFBP)-1 and leptin levels with type 1 collagen markers of bone turnover in dichorionic twins with or without discordant birth weight of 20% or greater.

Methods: Maternal and cord bloods were collected from gestational age-matched dichorionic twins with (n = 16) or without (n = 16) discordant birth weight. The samples were assayed for cross-linked carboxyl terminal telopeptide (ICTP, a marker of bone resorption) and propeptide (PICP, a marker of bone formation) of type I collagen, leptin, IGF-I, and IGFBP-1 by RIA.

Results: The intrauterine growth-restricted (IUGR) twins of the discordant group had higher fetal ICTP (P < 0.001) and IGFBP-1 (P < 0.001) levels, whereas PICP (P < 0.001), IGF-I (P < 0.001), and leptin (P < 0.001) were lower than the cotwins with normal weight (AGA). In contrast, concentrations of IGF-I, IGFBP-1, ICTP, PICP, and leptin were comparable between concordant twin pairs. Leptin levels were positively correlated with PICP (r = 0.61; P < 0.001) and negatively with ICTP (r = −0.57; P < 0.001) in concordant and AGA twins but not in IUGR twins. In IUGR twins, IGF-I had positive association with PICP (r = 0.76; P < 0.001) and negative association with ICTP (r= −0.76; P < 0.001), whereas IGFBP-1 was negatively correlated with PICP levels (r = −0.65; P < 0.01). No such association was found in concordant and AGA twins.

Conclusion: These data suggest that IUGR twins had high bone turnover, which is independent of maternal factors and perhaps may be due to altered IGF axis.
Copeptin and Arginine Vasopressin Concentrations in Critically Ill Patients
Authors: Jochberger Stefan , Morgenthaler Nils G. , Mayr Viktoria D. , Luckner Günter , Wenzel Volker , Ulmer Hanno , Schwarz Siegfried , Hasibeder Walter R. , Friesenecker Barbara E. , Dünser Martin W.
Published: August 2006

Context: Determination of arginine vasopressin (AVP) concentrations may be helpful to guide therapy in critically ill patients. A new assay analyzing copeptin, a stable peptide derived from the AVP precursor, has been introduced.

Objective: Our objective was to determine plasma copeptin concentrations.

Design: We conducted a post hoc analysis of plasma samples and data from a prospective study.

Setting: The setting was a 12-bed general and surgical intensive care unit (ICU) in a tertiary university teaching hospital.

Patients: Our subjects were 70 healthy volunteers and 157 ICU patients with sepsis, with systemic inflammatory response syndrome (SIRS), and after cardiac surgery.

Interventions: There were no interventions.

Main Outcome Measures: Copeptin plasma concentrations, demographic data, AVP plasma concentrations, and a multiple organ dysfunction syndrome score were documented 24 h after ICU admission.

Results: AVP (P < 0.001) and copeptin (P < 0.001) concentrations were significantly higher in ICU patients than in controls. Patients after cardiac surgery had higher AVP (P = 0.003) and copeptin (P = 0.003) concentrations than patients with sepsis or SIRS. Independent of critical illness, copeptin and AVP correlated highly significantly with each other. Critically ill patients with sepsis and SIRS exhibited a significantly higher ratio of copeptin/AVP plasma concentrations than patients after cardiac surgery (P = 0.012). The American Society of Anesthesiologists’ classification (P = 0.046) and C-reactive protein concentrations (P = 0.006) were significantly correlated with the copeptin/AVP ratio.

Conclusions: Plasma concentrations of copeptin and AVP in healthy volunteers and critically ill patients correlate significantly with each other. The ratio of copeptin/AVP plasma concentrations is increased in patients with sepsis and SIRS, suggesting that copeptin may overestimate AVP plasma concentrations in these patients.
Circulating Bioactive Androgens in Midlife Women
Authors: Chen Jiangang , Sowers MaryFran R. , Moran Francisco M. , McConnell Daniel S. , Gee Nancy A. , Greendale Gail A. , Whitehead Cheryl , Kasim-Karakas Sidika E. , Lasley Bill L.
Published: August 2006

Context: It is important to characterize the biological activity of circulating androgenic steroid hormones during the menopausal transition because these appear to impact the metabolic and cardiovascular health risk factors of women.

Objective: The objective of the study was to develop and characterize a cell-based bioassay that measures the androgen receptor-mediated signal transduction in serum.

Design: This was a clinically relevant experimental study nested in a sample population of a longitudinal cohort study.

Setting: The study was conducted at a university laboratory.

Methods: A receptor-mediated luciferase expression bioassay based on HEK 293 cells that were stably cotransfected with plasmids containing the human androgen receptor and luciferase gene was developed. In 49 samples from menstruating women aged 42–52 yr, total testosterone (T) and SHBG concentrations were measured by immunoassay; free T concentrations were calculated from the total T and SHBG concentrations.

Results: Mean total T concentration of the sample was 1.15 nm (sd 0.46, range 0.57–3.86 nm). The mean bioactive androgen detected was 1.00 nm (sd 0.24, range 0.53–1.60 nm). Calculated free T (mean 0.0156 nm) was significantly lower than the levels of bioactive androgens measured by receptor-mediated bioassay. There was significant positive correlation between bioactive androgen levels and total T values in young women and polycystic ovarian disorder patients, whereas no correlation was found between the two values in middle-aged women.

Conclusions: An androgen receptor-mediated bioassay can provide additional information in the evaluation of total bioactive androgens in midlife women. Our data suggest that levels of circulating SHBG may have a significant impact on the levels of total circulating bioavailable androgens.
Serum Aldosterone Concentration and Cardiovascular Risk in Women with Polycystic Ovarian Syndrome
Authors: Cascella Teresa , Palomba Stefano , Tauchmanovà Libuse , Manguso Francesco , Di Biase Sebastiano , Labella Donato , Giallauria Francesco , Vigorito Carlo , Colao Annamaria , Lombardi Gaetano , Orio Francesco
Published: August 2006

Context: Polycystic ovary syndrome (PCOS) is associated with early impairment of vascular structure and a low-grade chronic inflammation. Aldosterone is a well-recognized cardiovascular risk (CVR) factor and is related to inflammatory processes.

Objective: Our objective was to investigate serum aldosterone levels in PCOS and correlate them to some CVR factors and early atherosclerotic markers.

Design and Setting: A prospective baseline-controlled clinical study was conducted at the University “Federico II” of Naples School of Medicine (Naples, Italy).

Patients: Fifty PCOS women age- and body mass index-matched with 50 healthy women were enrolled.

Mean Outcome Measures: Anthropometric, hormonal, and metabolic patterns, including plasma aldosterone, renin, and C-reactive protein, were measured in each subject. Intima-media thickness was also evaluated in each patient and control.

Results: Aldosterone levels were significantly increased (P < 0.001) in PCOS compared with healthy women (10.5 ± 3.2 vs. 5.7 ± 2.5 ng/dl). In PCOS, a significant (P < 0.001) direct correlation between plasma aldosterone and homeostasis model assessment, C-reactive protein, intima-media thickness, and mean blood pressure was found. On the other hand, high-density lipoprotein cholesterol and potassium were inversely (P < 0.001) related to serum aldosterone. Multiple linear regression analysis showed that the area under the curve for insulin and homeostasis model assessment was linearly related to aldosterone in PCOS.

Conclusion: PCOS women show an insulin resistance related increase in serum aldosterone levels.
Novel HESX1 Mutations Associated with a Life-Threatening Neonatal Phenotype, Pituitary Aplasia, but Normally Located Posterior Pituitary and No Optic Nerve Abnormalities
Authors: Sobrier Marie-Laure , Maghnie Mohamad , Vié-Luton Marie-Pierre , Secco Andrea , di Iorgi Natascia , Lorini Renata , Amselem Serge
Published: August 2006

Context: Hesx1 is one of the earliest homeodomain transcription factors expressed during pituitary development. Very few HESX1 mutations have been identified in humans; although in those cases the disease phenotype shows considerable variability, all but one of the patients display an ectopic posterior pituitary and/or optic nerve abnormalities.

Objective: The objectives of the study were to describe the complex phenotype associated with the panhypopituitarism of two unrelated Italian patients who, at birth, presented with hypoglycemic seizures and respiratory distress complicated by shock, in a familial context of neonatal death in one family and spontaneous miscarriage in both families and to identify the molecular basis of this unusual syndrome.

Main Outcome Measures: Magnetic resonance imaging of the pituitary region, study of HESX1 gene and transcripts, and assessment of the ability of mutated HESX1 proteins to repress transcription were measured.

Results: Magnetic resonance imaging examination showed an anterior pituitary aplasia in a flat sella turcica and a normally located posterior pituitary in both patients. A constellation of extrapituitary developmental defects were found in the two patients, but without any optic nerve abnormalities. Sequencing of HESX1 exons and their flanking intronic regions revealed two different homozygous mutations. A frameshift (c.449_450delAC) was identified in one case, whereas the other patient carried a splice defect (c.357 + 2T>C) confirmed by the study of HESX1 transcripts. If translated, these mutations would lead to the synthesis of truncated proteins partly or entirely lacking the homeodomain, with no transcriptional repression, as shown by their inability to inhibit PROP1 activity.

Conclusions: These observations reveal two novel HESX1 mutations in a so-far-undescribed disease phenotype characterized by a life-threatening neonatal condition associated with anterior pituitary aplasia, in the absence of ectopic posterior pituitary and optic nerve abnormalities, two features classically associated with HESX1 defects.
Baseline Characteristics and Effects of Growth Hormone Therapy over Two Years in Younger and Elderly Adults with Adult Onset GH Deficiency
Authors: Franco Celina , Johannsson Gudmundur , Bengtsson Bengt-Åke , Svensson Johan
Published: August 2006

Context: The effects of GH replacement in elderly GH-deficient (GHD) adults are not well known.

Objective/Design/Patients: In this prospective, single-center, open-label study, baseline characteristics and the effects of 2-yr GH replacement were determined in 24 GHD adults above 65 yr of age and in 24 younger GHD patients (mean age, 37 yr; range, 27–46 yr). All patients had adult onset disease, and both groups were comparable in terms of the number of pituitary hormonal deficiencies, gender, body mass index, and waist/hip ratio. Duration of hypopituitarism was, however, longer in the elderly patients.

Results: The mean maintenance dose of GH was 0.31 (sem, 0.03) mg/d in the elderly GHD patients and 0.44 (0.04) mg/d in the younger patients. The less marked response in IGF-I sd score, total body fat, and extracellular water in the elderly patients lost significance when the dose of GH was accounted for in the statistical analyses. Despite the lower dose in the elderly GHD group, these patients had a more marked reduction in waist/hip ratio and serum low-density lipoprotein-cholesterol level, and these differences remained also after correction for duration of hypopituitarism. There was no difference at baseline or in responsiveness in lean mass, bone mineral density, and glucose homeostasis.

Conclusions: This study identifies elderly GHD adults as a GH-sensitive group in whom a low dose of GH can improve body composition and serum lipid profile without any significant impairment of glucose metabolism. GH replacement should therefore be considered in elderly GHD adults.
Circulating Estradiol Is an Independent Predictor of Progression of Carotid Artery Intima-Media Thickness in Middle-Aged Men
Authors: Tivesten Åsa , Hulthe Johannes , Wallenfeldt Karin , Wikstrand John , Ohlsson Claes , Fagerberg Björn
Published: August 2006

Context: Estrogen treatment of men with prostate cancer is associated with increased cardiovascular morbidity and mortality; however, the role of endogenous estrogen levels for atherosclerotic disease in men is unknown.

Objective: The objective of the study was to determine whether endogenous serum estradiol (E2) levels predict the progression of carotid artery intima-media thickness in men.

Design, Setting and Participants: This was a population-based, prospective cohort study (the Atherosclerosis and Insulin Resistance study) conducted in Göteborg, Sweden, among 313 Caucasian men without cardiovascular or other clinically overt diseases. Carotid artery intima-media thickness, an index of preclinical atherosclerosis, was measured by ultrasound at baseline (58 yr of age) and after 3 yr of follow-up. Serum sex hormone levels and cardiovascular risk factors (body mass index, waist to hip ratio, systolic blood pressure, serum triglycerides, plasma c-peptide, and smoking status) were assessed at study entry.

Intervention: There was no intervention.

Main Outcome Measures: Association between baseline total and free E2 levels and progression of carotid intima-media thickness over 3 yr with adjustments for cardiovascular risk factors was measured.

Results: In univariate analyses, both total and free E2 levels at baseline were positively associated with the annual change in intima-media thickness. In linear regression models including E2 and cardiovascular risk factors, low-density lipoprotein and high-density lipoprotein cholesterol and E2 were identified as independent predictors of progression of carotid artery intima-media thickness (total E2 beta = 0.187, P = 0.001; and free E2 beta = 0.183, P = 0.003).

Conclusions: Circulating E2 is a predictor of progression of carotid artery intima-media thickness in middle-aged men. Further studies are needed to investigate the role of endogenous E2 for incident cardiovascular disease events.
The Effects of Growth Hormone Treatment on Bone Mineral Density and Body Composition in Girls with Turner Syndrome
Authors: Ari Mim , Bakalov Vladimir K. , Hill Suvimol , Bondy Carolyn A.
Published: August 2006

Background: Many girls with Turner syndrome (TS) are treated with GH to increase adult height. In addition to promoting longitudinal bone growth, GH has effects on bone and body composition.

Objective: The objective was to determine how GH treatment affects bone mineral density (BMD) and body composition in girls with TS.

Method: In a cross-sectional study, we compared measures of body composition and BMD by dual energy x-ray absorptiometry, and phalangeal cortical thickness by hand radiography in 28 girls with TS who had never received GH and 39 girls who were treated with GH for at least 1 yr. All girls were participants in a National Institutes of Health (NIH) Clinical Research Center (CRC) protocol between 2001 and 2006.

Results: The two groups were similar in age (12.3 yr, sd 2.9), bone age (11.5 yr, sd 2.6), and weight (42.8 kg, sd 16.6); but the GH-treated group was taller (134 vs. 137 cm, P = 0.001). The average duration of GH treatment was 4.2 (sd 3.2) yr (range 1–14 yr). After adjustment for size and bone age, there were no significant differences in BMD at L1–L4, 1/3 radius or cortical bone thickness measured at the second metacarpal. However, lean body mass percent was higher (P < 0.001), whereas body fat percent was lower (P < 0.001) in the GH-treated group. These effects were independent of estrogen exposure and were still apparent in girls that had finished GH treatment at least 1 yr previously.

Conclusions: Although GH treatment has little effect on cortical or trabecular BMD in girls with TS, it is associated with increased lean body mass and reduced adiposity.
Homologous gene sequences mediate transcription-domain formation
Authors: Binnie Alexandra , Castelo-Branco Pedro , Monks Joan , Proudfoot Nicholas J.
Published: August 2006

The organisation of transcription in the mammalian nucleus is a topic of particular interest because of its relevance to gene regulation. RNA polymerase II transcription occurs at hundreds of sites throughout the nucleoplasm. Recent data indicate that coordinately regulated genes can localise to shared transcription sites. Other transcribed sequences have also been shown to cluster in the nucleus. The ribosomal RNA genes cluster in the nucleoli. Similarly, transiently transfected plasmids and dsDNA viruses form transcription domains (TDs) containing multiple templates. Intriguingly, plasmids expressing β-globin gene sequences recruit the endogenous β-globin loci to their TDs. In light of this observation, we have investigated plasmid TDs as a model for gene recruitment. We find that TD formation is dependent on the presence of homologous gene sequences. Plasmids containing non-homologous gene sequences form separate TDs, independent of homology in the backbone or promoter sequences. TD formation is also favoured by low plasmid concentrations. This effect is sequence-specific and high concentrations of one plasmid do not disrupt domain formation by non-homologous plasmids in the same cell. We conclude that recruitment into TDs is an active process that is driven by homologies between transcribed sequences and becomes saturated at high copy numbers.
Adenoviral vector saturates Akt pro-survival signaling and blocks insulin-mediated rescue of tumor-necrosis-factor-induced apoptosis
Authors: Miller-Jensen Kathryn , Janes Kevin A. , Wong Yun-Ling , Griffith Linda G. , Lauffenburger Douglas A.
Published: August 2006

Recombinant adenoviruses are used extensively as delivery vectors in clinical gene therapy and in molecular biology, but little is known about how the viral carrier itself contributes to cellular responses. Here we show that infection with an E1/E3-deleted adenoviral vector (Adv) sensitizes human epithelial cells to tumor necrosis factor (TNF)-induced apoptosis. To explore the mechanism of Adv-mediated sensitization, we measured activity time courses for three protein kinases (MK2, IKK and JNK1) centrally involved in the TNF-receptor signaling network, as well as two kinases (Akt and ERK) activated by growth factors. Both the pro-apoptotic signal MK2 and the anti-apoptotic signal Akt were upregulated when Adv-infected cells were stimulated with TNF, and MK2 and Akt each contributed significantly to TNF-induced cell fate. Surprisingly, further activation of Akt in Adv-infected cells via insulin treatment did not significantly reduce apoptosis or MK2 activity. We show that the ineffectiveness of insulin-mediated anti-apoptotic signaling through Akt is due to saturation of Akt-effector substrate phosphorylation in Adv-infected cells. Normalizing Akt signaling relative to its Adv-induced baseline activity identified a global dose-response curve that relates Akt signaling to cellular survival. Thus, the background Akt activity induced by Adv limits the transmission of anti-apoptotic signals in response to further cytokine or growth-factor stimulation. The phenotypic and intracellular synergy between Adv and TNF may have implications for interpreting cellular responses in gene-therapy and laboratory applications.
Helix-1 of the cAMP-specific phosphodiesterase PDE4A1 regulates its phospholipase-D-dependent redistribution in response to release of Ca2+
Authors: Huston Elaine , Gall Irene , Houslay Thomas M. , Houslay Miles D.
Published: August 2006

The unique N-terminal regions of PDE4 cAMP-specific phosphodiesterases confer interaction with distinct signalling and scaffolding proteins. The PDE4A1 isoform is unique in being entirely membrane associated. Its N-terminal region is formed from two helices separated by a mobile hinge, where helix-2 contains a TAPAS1 domain that inserts into the lipid bilayer in a Ca2+-triggered fashion. Here we show that helix-1 is important for intracellular targeting of PDE4A1 in living cells, facilitating membrane association, targeting to the trans-Golgi stack and conferring Ca2+-stimulated intracellular redistribution in a manner that is dependent on the phospholipase-D-mediated generation of phosphatidic acid. The LxDFF motif within helix-1 is pivotal to this, where Leu4-Phe6-Phe7 forms a compact hydrophobic pocket on one side of helix-1 whereas Asp5, located on the opposite face of helix-1, provides the Ca2+-regulation site. Mutation of Asp5 to Ala or the release of Ca2+ from intracellular stores de-restricts trans-Golgi localisation of PDE4A1 allowing it to redistribute in cells in a phosphatidic-acid-dependent manner. This study provides the first evidence for Ca2+-triggered relocalisation of a cAMP phosphodiesterase and indicates a potential means for allowing cross-talk between the cAMP, phospholipase D and Ca2+-signalling pathways.
TFIIH trafficking and its nuclear assembly during earlyDrosophilaembryo development
Authors: Aguilar-Fuentes Javier , Valadez-Graham Viviana , Reynaud Enrique , Zurita Mario
Published: August 2006

We present the first analysis of the dynamics of the transcription DNA-repair factor TFIIH at the onset of transcription in early Drosophila development. TFIIH is composed of ten polypeptides that are part of two complexes - the core and the CAK. We found that the TFIIH core is initially located in the cytoplasm of syncytial blastoderm embryos, and that after mitotic division ten and until the cellular blastoderm stage, the core moves from the cytoplasm to the nucleus. By contrast, the CAK complex is mostly cytoplasmic during cellularization and during gastrulation. However, both components are positioned at promoters of genes that are activated at transcription onset. Later in development, the CAK complex becomes mostly nuclear and co-localizes in most chromosomal regions with the TFIIH core, but not in all sites, suggesting that the CAK complex could have a TFIIH-independent role in transcription of some loci. We also demonstrate that even though the CAK and the core coexist in the early embryo cytoplasm, they do not interact until they are in the nucleus and suggest that the complete assembly of the ten subunits of TFIIH occurs in the nucleus at the mid-blastula transition. In addition, we present evidence that suggests that DNA helicase subunits XPB and XPD are assembled in the core when they are transported into the nucleus and are required for the onset of transcription.
TheArabidopsis SKP1homologASK1controls meiotic chromosome remodeling and release of chromatin from the nuclear membrane and nucleolus
Authors: Yang Xiaohui , Timofejeva Ljudmilla , Ma Hong , Makaroff Christopher A.
Published: August 2006

During early stages of meiotic prophase I the nucleus undergoes considerable reorganization, including the clustering of telomeres, the release of contacts between chromosomes and the nuclear membrane, the reorganization of the nucleolus, and chromatin remodeling. Using a light squashing technique for the analysis of meiotic chromosomes along with fluorescent in situ hybridization, transmission electron microscopy and immunolocalization studies with antibodies to modified histones, we demonstrate that ASK1 is essential for early nuclear reorganization events. A relatively large number of meiotic alterations have been identified in ask1-1 plants. We show that many of these defects, including alterations in homolog pairing, nucleolus migration and the missegregation of chromosomes, may arise from alterations in chromatin structure and the inability of chromosomes to resolve and release properly from the nuclear membrane and nucleolus during leptotene. These results raise the interesting possibility that ASK1 controls chromatin structure by targeting of either an early regulator of meiotic progression or possibly matrix attachment proteins for destruction.
Epilysin (MMP-28) induces TGF-β mediated epithelial to mesenchymal transition in lung carcinoma cells
Authors: Illman Sara A. , Lehti Kaisa , Keski-Oja Jorma , Lohi Jouko
Published: August 2006

Epilysin (MMP-28) is the newest member of the matrix metalloproteinase (MMP) family. Although it is expressed in a number of tissues, no biological substrates or functions for this enzyme have been identified yet. We have expressed recombinant epilysin in A549 lung adenocarcinoma cells and found that this resulted in stable and irreversible epithelial to mesenchymal transition (EMT) accompanied by loss of cell surface E-cadherin, proteolytic processing of latent TGF-β-complexes and increased levels of active TGF-β. The cascade of events leading to the onset of EMT is prevented by the MMP inhibitor GM6001 or antibodies neutralizing the activity of TGF-β. Once EMT had occurred the cell phenotype could, however, not be reversed by the MMP-inhibitor. Importantly, the expression of epilysin also resulted in upregulation of MT1-MMP and gelatinase-B (MMP-9) and in the collagen invasive activity of A549 cells. Further, we found that epilysin and the recombinant hemopexin domain were targeted to the surface of epithelial cells. This cell surface interaction was sensitive to the proteolytic activity of MT1-MMP, and was lost after EMT. Current results indicate that epilysin can induce EMT and cell invasion through a TGF-β-dependent mechanism suggesting novel biological roles for this enzyme in the regulation of epithelial cell function and in the induction of carcinogenesis.
Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and endosomes
Authors: Young Andrew R. J. , Chan Edmond Y. W. , Hu Xiao Wen , Köchl Robert , Crawshaw Samuel G. , High Stephen , Hailey Dale W. , Lippincott-Schwartz Jennifer , Tooze Sharon A.
Published: August 2006

Autophagy, fundamentally a lysosomal degradation pathway, functions in cells during normal growth and certain pathological conditions, including starvation, to maintain homeostasis. Autophagosomes are formed through a mechanism that is not well understood, despite the identification of many genes required for autophagy. We have studied the mammalian homologue of Atg9p, a multi-spanning transmembrane protein essential in yeast for autophagy, to gain a better understanding of the function of this ubiquitious protein. We show that both the N- and C-termini of mammalian Atg9 (mAtg9) are cytosolic, and predict that mAtg9 spans the membrane six times. We find that mAtg9 is located in the trans-Golgi network and late endosomes and colocalizes with TGN46, the cation-independent mannose-6-phosphate receptor, Rab7 and Rab9. Amino acid starvation or rapamycin treatment, which upregulates autophagy, causes a redistribution of mAtg9 from the TGN to peripheral, endosomal membranes, which are positive for the autophagosomal marker GFP-LC3. siRNA-mediated depletion of the putative mammalian homologue of Atg1p, ULK1, inhibits this starvation-induced redistribution. The redistribution of mAtg9 also requires PI 3-kinase activity, and is reversed after restoration of amino acids. We speculate that starvation-induced autophagy, which requires mAtg9, may rely on an alteration of the steady-state trafficking of mAtg9, in a Atg1-dependent manner.
Energetic influence on gull flight strategy selection
Authors: Shamoun-Baranes Judy , van Loon Emiel
Published: August 2006

During non-migratory flight, gulls (Larids) use a wide variety of flight strategies. We investigate the extent to which the energy balance of a bird explains flight strategy selection. We develop a model based on optimal foraging and aerodynamic theories, to calculate the ground speeds and airspeeds at which a gull is expected to flap or soar during foraging flight. The model results are compared with observed flight speeds, directions, and flight strategies of two species of gulls, the black-headed gull Larus ridibundus and the lesser black-backed gull Larus fuscus. The observations were made using a tracking radar over land in The Netherlands.

The model suggests that, especially at combinations of low ground speed(∼5-10 m s-1), high air speed (∼20-25 m s-1) and low ground and air speed, gulls should favor soaring flight. At intermediate ground and air speeds the predicted net energy gain is similar for soaring and flapping. Hence the ratio of flapping to soaring may be higher than for other air and ground speed combinations. This range of speeds is broadest for black-headed gulls. The model results are supported by the observations. For example, flapping is more prevalent at speeds where the predicted net energy gain is similar for both strategies. Interestingly, combinations of air speed and flight speed that, according to the model, would result in a loss of net energy gain, were not observed. Additional factors that may influence flight strategy selection are also briefly discussed.
A reaction-diffusion analysis of energetics in large muscle fibers secondarily evolved for aerobic locomotor function
Authors: Hardy Kristin M. , Locke Bruce R. , Da Silva Marilia , Kinsey Stephen T.
Published: August 2006

The muscles that power swimming in the blue crab, Callinectes sapidus, grow hypertrophically, such that in juvenile crabs the cell diameters are <60 μm, whereas fibers of the adult crabs often exceed 600μm. Thus, as these animals grow, their muscle fibers greatly exceed the surface area to volume ratio and intracellular diffusion distance limits of most cells. Previous studies have shown that arginine phosphate (AP) recovery in the anaerobic (light) fibers, which demonstrate a fiber size dependence on anaerobic processes following contraction, is too slow to be restricted by intracellular metabolite diffusive flux, in spite of the fiber's large size. By contrast, the aerobic (dark) fibers have evolved an intricate network of intracellular subdivisions that maintain an effectively small `metabolic diameter' throughout development. In the present study, we examined the impact of intracellular metabolite diffusive flux on the rate of post-contractile AP resynthesis in the dark muscle, which has a much higher aerobic capacity than the light muscle. AP recovery was measured for 60 min in adults and 15 min in juveniles following burst contractile activity in dark fibers, and a mathematical reaction-diffusion model was used to test whether the observed aerobic rates of AP resynthesis were fast enough to be limited by intracellular metabolite diffusion. Despite the short diffusion distances and high mitochondrial density, the AP recovery rates were relatively slow and we found no evidence of diffusion limitation. However, during simulation of steady-state contraction, which is an activity more typical of the dark fibers, there were substantial intracellular metabolite gradients, indicative of diffusion limitation. This suggests that high ATP turnover rates may lead to diffusion limitation in muscle even when diffusion distances are short, as in the subdivided dark fibers.
PTHrP regulation and calcium balance in sea bream (Sparus auratusL.) under calcium constraint
Authors: Abbink Wout , Bevelander Gideon S. , Hang Xiaoming , Lu Weiqun , Guerreiro Pedro M. , Spanings Tom , Canario Adelino V. M. , Flik Gert
Published: August 2006

Juvenile gilthead sea bream were exposed to diluted seawater (2.5‰salinity; DSW) for 3 h or, in a second experiment, acclimated to DSW and fed a control or calcium-deficient diet for 30 days. Branchial Ca2+influx, drinking rate and plasma calcium levels were assessed. Sea bream plasma parathyroid hormone related protein (sPTHrP) was measured, and mRNAs of pthrp, its main receptor, pth1r, and the calcium-sensing receptor (casr) were quantified in osmoregulatory tissues and the pituitary gland. When calcium is limited in water or diet, sea bream maintain calcium balance; however, both plasma Ca2+ and plasma sPTHrP concentrations were lower when calcium was restricted in both water and diet. Positive correlations between plasma sPTHrP and plasma Ca2+(R2=0.30, N=39, P<0.05), and plasma sPTHrP and body mass of the fish (R2=0.37, N=148, P<0.001) were found. Immunoreactive sPTHrP was demonstrated in pituitary gland pars intermedia cells that border the pars nervosa and co-localises with somatolactin. In the pituitary gland, pthrp, pth1r and casr mRNAs were downregulated after both short-and long-term exposure to DSW. A correlation between pituitary gland pthrp mRNA expression and plasma Ca2+(R2=0.71, N=7, P<0.01) was observed. In gill tissue, pthrp and pth1r mRNAs were significantly upregulated after 30 days exposure to DSW, whereas no effect was found for casr mRNA expression. We conclude that in water of low salinity,declining pituitary gland pthrp mRNA expression accompanied by constant plasma sPTHrP levels points to a reduced sPTHrP turnover and that sPTHrP, through paracrine interaction, is involved in the regulation of branchial calcium handling, independently of endocrine pituitary gland sPTHrP.
Bi-directional route learning in wood ants
Authors: Graham Paul , Collett Thomas S.
Published: August 2006

Some ants and bees readily learn visually guided routes between their nests and feeding sites. They can learn the appearance of visual landmarks for the food-bound or homeward segment of the route when these landmarks are only present during that particular segment of their round trip. We show here that wood ants can also acquire landmark information for guiding their homeward path while running their food-bound path, and that this information may be picked up, when ants briefly reverse direction and retrace their steps for a short distance. These short periods of looking back tend to occur early in route acquisition and are more frequent on homeward than on food-bound segments.
Actions of kinin peptides in the stomatogastric ganglion of the crabCancer borealis
Authors: Saideman Shari R. , Christie Andrew E. , Torfs Pieter , Huybrechts Jurgen , Schoofs Liliane , Nusbaum Michael P.
Published: August 2006

To fully understand neuronal network operation, the influence of all inputs onto that network must be characterized. As in most systems, many neuronal and hormonal pathways influence the multifunctional motor circuits of the crustacean stomatogastric ganglion (STG), but the actions of only some of them are known. Therefore, we characterized the influence of the kinin peptide family on the gastric mill (chewing) and pyloric (filtering of chewed food)motor circuits in the STG of the crab Cancer borealis. The kinins are myoactive in arthropods and they occur within the arthropod central nervous system (CNS), but their CNS actions are not well characterized in any species. The pevkinins were first identified in the shrimp Penaeus vannamei,but they have yet to be studied in the STG of any species. We identified kinin-like immunolabeling (KLI) in the pericardial organs (POs) in C. borealis, but there was no KLI within the STG. The POs are a major source of hormonal influence on the STG. Pevkinin peptides activated the pyloric circuit and they caused a modest increase in the speed of ongoing pyloric rhythms. This modest influence on cycle speed resulted in part from pevkinin excitation of the lateral pyloric neuron, whose strengthened inhibitory synapse onto the pyloric pacemaker neurons limited the pevkinin-mediated increase in cycle speed. The pevkinin excitation of the pyloric rhythm was not strong enough to interfere with the previously documented, gastric mill rhythm-mediated weakening of the pyloric rhythm. Pevkinin also had little influence on the gastric mill rhythm. These results indicate that the kinin peptides have distinct and selective modulatory actions on the pyloric rhythm.