bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of theChlamydomonasCell Cycle by a Stable, Chromatin-Associated Retinoblastoma Tumor Suppressor Complex
Authors: Olson Bradley J.S.C. , Oberholzer Michael , Li Yubing , Zones James M. , Kohli Harjivan S. , Bisova Katerina , Fang Su-Chiung , Meisenhelder Jill , Hunter Tony , Umen James G.
Published: October 2010

We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit—RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle–regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.
Assessment of liver fibrosis before and after antiviral therapy by different serum marker panels in patients with chronic hepatitis C
Authors: Martinez S. M. , Fernández‐Varo G. , González P. , Sampson E. , Bruguera M. , Navasa M. , Jiménez W. , Sánchez‐Tapias J. M. , Forns X.
Published: October 2010

Aliment Pharmacol Ther 2011; 33: 138–148

Background  Liver biopsy is the reference standard to assess liver fibrosis in chronic hepatitis C.

Aim  To validate and compare the diagnostic performance of non‐invasive tests for prediction of liver fibrosis severity and assessed changes in extracellular matrix markers after antiviral treatment.

Methods  The performances of Forns’ score, AST to platelet ratio index (APRI), FIB‐4 index and Enhanced Liver Fibrosis (ELF) score were validated in 340 patients who underwent antiviral therapy. These scores were determined 24 weeks after treatment in 161 patients.

Results  Forns’ score, APRI, FIB‐4 and ELF score showed comparable diagnostic accuracies for significant fibrosis [area under the receiver operating characteristic curve (AUROC) 0.83, 0.83, 0.85 and 0.81, respectively]. To identify cirrhosis, FIB‐4 index showed a significantly better performance over APRI and ELF score (AUROC 0.89 vs. 0.83 and 0.82, respectively). ELF score decreased significantly in patients with sustained virological response (SVR) (P < 0.0001) but remained unchanged in nonresponders. Non‐1 hepatitis C virus (HCV) genotype, baseline lower HCV RNA, glucose, hyaluronic acid and higher cholesterol levels were independently associated with SVR.

Conclusions  Simple panel markers and ELF score are accurate at identifying significant fibrosis and cirrhosis in chronic hepatitis C. A decrease in ELF score after antiviral treatment reflects the impact of viral clearance in hepatic extracellular matrix and probably in the improvement of liver fibrosis.
Parenting, family functioning and lifestyle in a new culture: the case of African migrants in Melbourne, Victoria, Australia
Authors: Renzaho André M. N. , Green Julie , Mellor David , Swinburn Boyd
Published: October 2010

This study documented the parenting styles among African migrants now living in Melbourne, Victoria, Australia, and assessed how intergenerational issues related to parenting in a new culture impact on family functioning and the modification of lifestyles. A total of 10 focus group discussions (five with parents and five with 13–17‐year‐old children; N = 85 participants) of 1.5–2 hours duration were conducted with Sudanese, Somali and Ethiopian migrant families. The analysis identified three discrete themes: (i) parenting‐related issues; (ii) family functioning and family relations; and (iii) lifestyle changes and health. African migrant parents were restrictive in their parenting; controlled children's behaviours and social development through strict boundary‐setting and close monitoring of interests, activities, and friends; and adopted a hierarchical approach to decision‐making while discouraging autonomy among their offspring. Programmes seeking to improve the health and welfare of African migrants in their host countries need to accommodate the cultural and social dimensions that shape their lives. Such programmes may need to be so broad as to apply an acculturation lens to planning, and to assist young people, parents and families in addressing intergenerational issues related to raising children and growing up in a different social and cultural milieu.
The prevalence and phenomenology of self‐injurious and aggressive behaviour in genetic syndromes
Authors: Arron K. , Oliver C. , Moss J. , Berg K. , Burbidge C.
Published: October 2010

Background Self‐injurious and aggressive behaviours are reported as components of some behavioural phenotypes but there are few studies comparing across syndrome groups. In this study we examined the prevalence of these behaviours and the associated person characteristics in seven genetic syndromes.

Methods Questionnaire data on self‐injury and aggression, mood, hyperactivity, autism spectrum disorder and repetitive behaviour were collected on Angelman (AS,n = 104), Cornelia de Lange (CdLS, 101), Cri du Chat (CdCS, 58), Fragile X (FXS, 191), Lowe (LS, 56), Prader–Willi (PWS, 189) and Smith–Magenis (SMS, 42) syndromes.

Results A significantly higher prevalence of self‐injury was evident in CdCS, CdLS, FXS, PWS, LS and SMS. The prevalence of aggression was significantly heightened in AS and SMS. Self‐injury was associated with repetitive and impulsive behaviour in CdLS, FXS, PWS and LS. Impulsivity and overactivity were significantly higher in those showing aggression across all syndrome groups.

Conclusions These data quantify the risk for self‐injury and aggression in the syndromes studied with implications for early intervention. The associations between these behaviours and person characteristics both within and between syndromes warrant further research.
Electronically measured adherence to immunosuppressive medications and kidney function after deceased donor kidney transplantation
Authors: Israni Ajay K. , Weng Francis L. , Cen Ye‐Ying , Joffe Marshall , Kamoun Malek , Feldman Harold I.
Published: October 2010

Israni AK, Weng FL, Cen Y‐Y, Joffe M, Kamoun M, Feldman HI. Electronically measured adherence to immunosuppressive medications and kidney function after deceased donor kidney transplantation.  Clin Transplant 2011: 25: E124–E131. © 2010 John Wiley & Sons A/S.

Abstract:  Background:  Non‐adherence with immunosuppressive medications can result in allograft rejection and eventually allograft loss.

Methods:  In a racially diverse population, we utilized microelectronic cap monitors to determine the association of adherence with a single immunosuppressive medication and kidney allograft outcomes post‐transplantation. This prospective cohort study enrolled 243 patients from eight transplant centers to provide adherence and kidney allograft outcomes data. To determine the association of adherence with change in estimated glomerular filtration rate (eGFR), we fit mixed effects models with the outcome being change in eGFR over time. We also fit Cox proportional hazards models to determine the association of adherence with time to persistent 25% and 50% decline in eGFR.

Results:  The distribution of adherence post‐transplant was as follows: 164 (68%), 49 (20%), and 30 (12%) had >85–100%, 50–85%, and <50% adherence, respectively. Seventy‐nine (33%) and 36 (15%) of the subjects experienced a persistent 25% decline in eGFR or allograft loss and 50% decline in eGFR or allograft loss during follow‐up. Adherence was not associated with acute rejection or 25% decline or 50% decline in eGFR. In the adjusted and unadjusted model, adherence and black race were not associated with change in eGFR over time.

Conclusions:  Non‐adherence with a single immunosuppressive medication was not associated with kidney allograft outcomes.
Rapidly progressive hepatic alveolar echinococcosis in an ABO‐incompatible renal transplant recipient
Authors: Geyer M. , Wilpert J. , Wiech T. , Theilacker C. , Stubanus M. , Kramer‐Zucker A. , Fischer K.‐G. , Drognitz O. , Frydrychowicz A. , Kern W. , Walz G. , Pisarski P.
Published: October 2010

M. Geyer, J. Wilpert, T. Wiech, C. Theilacker, M. Stubanus, A. Kramer‐Zucker, K.‐G. Fischer, O. Drognitz, A. Frydrychowicz, W. Kern, G. Walz, P. Pisarski. Rapidly progressive hepatic alveolar echinococcosis in an ABO‐incompatible renal transplant recipient. Transpl Infect Dis 2011: 13: 278–284. All rights reserved

Abstract: We report on the case of an ABO‐incompatible renal re‐transplant recipient maintained on an intensified immunosuppressive regimen for recurrent cellular rejection episodes and transplant glomerulopathy who presented with rapidly growing hepatic tumors, radiologically suggestive of hemangiosarcoma. Upon resection and pathological work‐up, the lesions revealed alveolar echinococcosis, a rare but potentially life‐threatening parasitosis. Usually infection with Echinococcus multilocularis remains asymptomatic for extended periods of time and can go unrecognized for years. In the case presented, we observed an atypically rapid growth pattern of E. multilocularis that might have been due to the extent of the immunosuppressive regimen, which included repetitive anti‐CD20 treatments. Retrospectively performed serological studies with enzyme‐linked immunosorbent assays known to provide high sensitivity and specificity for the detection of echinococcosis in the general population, yielded ambiguous results in our immunocompromised host, which could be, in part, explained by B‐cell depletion and its effects on antibody production and indirect actions on cellular immunity. In conclusion, this is the first report to our knowledge of hepatic alveolar echinococcosis in a renal transplant recipient. This case documents an altered clinical course of the parasitosis and the challenge of serological diagnostic tools under an intensified regimen of immunosuppressive agents, including rituximab.
PEGylation prevents bacteria‐induced platelet activation and biofilm formation in platelet concentrates
Authors: Greco C. A. , Maurer‐Spurej E. , Scott M. D. , Kalab M. , Nakane N. , Ramírez‐Arcos S. M.
Published: October 2010

Bacterial contamination of platelet concentrates represents the greatest post‐transfusion infectious risk. Biofilm formation in this environment resulting from platelet–bacteria interactions can lead to non‐uniform contaminant distribution and thus missed detection. As formation of platelet–bacteria aggregates is largely based on receptor–ligand interactions, we examined whether shielding these events would result in reduced biofilm formation by contaminant bacteria. We introduced methoxypoly(ethylene glycol) to covalently modify the platelet surface using a process termed ‘PEGylation’. In the first study of its kind, we demonstrate that PEGylated platelet concentrates inoculated with Staphylococcus epidermidis display a significant reduction in bacterial binding and biofilm formation.
5‐α‐Reductase inhibitors for prostate cancer chemoprevention: an updated Cochrane systematic review
Authors: Wilt Timothy J. , MacDonald Roderick , Hagerty Karen , Schellhammer Paul , Tacklind James , Somerfield Mark R. , Kramer Barnett S.
Published: October 2010

OBJECTIVE

To estimate the benefits and harms of 5‐α‐reductase inhibitors (5‐α‐RIs) in preventing prostate cancer.

MATERIALS AND METHODS

We searched MEDLINE and the Cochrane Collaboration Library through June 2010 to identify randomized trials.

We included articles if they examined 5‐α‐RI vs control, were ≥1 year in duration and provided clinical outcomes.

Our primary outcome was prostate cancer period‐prevalence ‘for‐cause’.

RESULTS

Eight studies met inclusion criteria but only the Prostate Cancer Prevention Trial and the Reduction by Dutasteride of Prostate Cancer Events were designed to assess the impact of 5‐α‐RIs on prostate cancer period‐prevalence. The mean age of enrolees was 64 years, 92% were White, and mean PSA level was 3.1 ng/mL. For‐cause prostate cancers comprised 54% of all cancers detected in placebo‐controlled studies.

Compared with placebo, 5‐α‐RI resulted in a 25% relative risk (RR) reduction in prostate cancers detected for‐cause [RR 0.75, 95% confidence interval (CI) 0.67–0.83; 1.4% absolute risk reduction (3.5% vs 4.9%)]. One BPH trial reported that the risk of prostate cancers detected for‐cause was significantly reduced with dutasteride and combined dutasteride plus tamsulosin compared with tamsulosin monotherapy.

Six trials vs placebo assessed prostate cancers detected overall. There was a 26% RR reduction favouring 5‐α‐RI [RR 0.74, 95% CI 0.55–1.00; 2.9% absolute risk reduction (6.3% vs 9.2%)]. There were reductions across categories of age, race and family history of prostate cancer.

One placebo‐controlled trial of men that investigators considered at greater risk for prostate cancer (based on age, elevated PSA level and having a previous suspicion of prostate cancer leading to a prostate biopsy) reported that dutasteride did not reduce prostate cancers detected for‐cause based on needle‐biopsy but did reduce risk of overall incident prostate cancer detected by biopsy by 23%[RR 0.77, 95% CI 0.70–0.85; absolute reduction 16.1% vs 20.8%]. There were reductions across age, family history of prostate cancer, PSA level, and prostate volume subgroups.

Incidences of erectile dysfunction, ejaculate volume, decreased libido, and gynaecomastia were greater with 5‐α‐RI vs placebo.

CONCLUSIONS

5‐α‐RIs reduce the risk of being diagnosed with prostate cancer among men who are screened regularly for prostate cancer.

Information is inadequate to assess the effect of 5‐α‐RIs on prostate cancer or all‐cause mortality.

5‐α‐RIs increase sexual and erectile dysfunction.
The Learning Region between Pedagogy and Economy
Authors: PIAZZA ROBERTA
Published: October 2010

Economic growth is stimulated through learning. In ‘the learning economies’ of those European regions that chose to develop their human and intellectual capital wisely, benefits have been visible. But this is a one‐dimensional outlook in a multi‐dimensional world. A ‘Learning Region’ is an entirely different entity, pooling and mobilising its resources from the community, its institutions, culture and heritage, and industry, to mention just a few, for the common, social, economic and ecological good. This article examines and critiques recent ideas and perceptions behind the concept of the learning region and suggests why, in the Italian context, they have not been successful so far in entering the consciousness of regional leaders. It argues that greater individualisation, privatisation and the ascendancy of the market approach to learning are, perhaps paradoxically, barriers to establishing the regional structures that would implement lifelong learning for all and enhance economic and social progress. Cooperation, partnership, sharing and the integration of stakeholders in respecting a common goal are far more difficult to achieve in today's climate.
Pictures in Wittgenstein's Later Philosophy
Authors: Egan David
Published: October 2010

The word “picture” occurs pervasively in Wittgenstein's later philosophy. Not only does Wittgenstein often use literal pictures or the notion of mental pictures in his investigations, but he also frequently uses “picture” to speak about a way of conceiving of a matter (e.g. “A picture held us captive” at Philosophical Investigations§115). I argue that “picture” used in this conceptual sense is not a shorthand for an assumption or a set of propositions but is rather an expression of conceptual bedrock on the model of an organising myth. This reading builds primarily on work by Gordon Baker and Stanley Cavell.
Cartilage Thickness Distribution Affects Computational Model Predictions of Cervical Spine Facet Contact Parameters
Authors: Womack Wesley , Ayturk Ugur M. , Puttlitz Christian M.
Published: October 2010

With motion-sparing disk replacement implants gaining popularity as an alternative to anterior cervical discectomy and fusion (ACDF) for the treatment of certain spinal degenerative disorders, recent laboratory investigations have studied the effects of disk replacement and implant design on spinal kinematics and kinetics. Particularly relevant to cervical disk replacement implant design are any postoperative changes in solid stresses or contact conditions in the articular cartilage of the posterior facets, which are hypothesized to lead to adjacent-level degeneration. Such changes are commonly investigated using finite element methods, but significant simplification of the articular geometry is generally employed. The impact of such geometric representations has not been thoroughly investigated. In order to assess the effects of different models of cartilage geometry on load transfer and contact pressures in the lower cervical spine, a finite element model was generated using cadaver-based computed tomography imagery. Mesh resolution was varied in order to establish model convergence, and cadaveric testing was undertaken to validate model predictions. The validated model was altered to include four different geometric representations of the articular cartilage. Model predictions indicate that the two most common representations of articular cartilage geometry result in significant reductions in the predictive accuracy of the models. The two anatomically based geometric models exhibited less computational artifact, and relatively minor differences between them indicate that contact condition predictions of spatially varying thickness models are robust to anatomic variations in cartilage thickness and articular curvature. The results of this work indicate that finite element modeling efforts in the lower cervical spine should include anatomically based and spatially varying articular cartilage thickness models. Failure to do so may result in loss of fidelity of model predictions relevant to investigations of physiological import.
The Relationship Between Sperm Velocity and Pressures Applied to the Zona Pellucida During Early Sperm-Oocyte Penetration
Authors: Gefen Amit
Published: October 2010

Sperm velocity is long known to be an important indicator of sperm quality but without much biophysical theory explaining it. Contact mechanics based modeling was developed here to determine the effects that sperm velocity (vs) and sperm head density (ρs)—which is an indicator of the stiffness of the head, have on the peak sperm-zona pellucida contact pressure (pm) during the early phase of sperm penetration. The modeling identified vs as being more influential on pm compared with the influence that ρs has, which means that for spermatozoa competing on the same oocyte, greater vs is a more important advantage than higher ρs. Specifically, pm was more sensitive by a power of 2 to changes in vs than to changes in ρs. It was further demonstrated that each 0.1 g/cc increase in ρs (within the physiologically relevant range of 1.3–1.7 g/cc) would be equivalent to just ∼3% rise in vs, indicating again that faster swimming is a better strategy for spermatozoa compared with head stiffening. The modeling hence provided some useful insights regarding sperm biomechanics, which theoretically elucidate the well-recognized importance of sperm velocity measurements as being indicative of sperm quality.
The Inducible Costimulator (ICOS) Is Critical for the Development of Human T
H
17 Cells
Authors: Paulos Chrystal M. , Carpenito Carmine , Plesa Gabriela , Suhoski Megan M. , Varela-Rohena Angel , Golovina Tatiana N. , Carroll Richard G. , Riley James L. , June Carl H.
Published: October 2010

The inducible costimulator ICOS is critical for the differentiation and expansion of human T
H
17 cells and promotes the antitumor capacity of these cells.
A Flexible Analysis of Gene-Environment Interactions
Authors: Carlson Nichole
Published: October 2010

Bayesian analysis of gene-environment interplay offers a way to better balance case-only and case-control strategies.
Presence of Host ICAM-1 in Laboratory and Clinical Strains of Human Immunodeficiency Virus Type 1 Increases Virus Infectivity and CD4+-T-Cell Depletion in Human Lymphoid Tissue, a Major Site of Replication In Vivo
Authors: Bounou Salim , Leclerc Jacques E. , Tremblay Michel J.
Published: October 2010

Human immunodeficiency virus type 1 (HIV-1) incorporates several host proteins. Earlier studies have indicated that such foreign constituents can modulate the virus life cycle, although the potential roles that these proteins might play in the viral pathology in vivo remain unclear. In an attempt to shed light on this issue, we first exposed explants of human lymphoid tissue to isogenic viruses except for the presence or absence of host-derived ICAM-1. Incorporation of ICAM-1 alone increased HIV-1 infectivity for human tonsillar tissue cultured ex vivo. This observation was made for viruses bearing distinct coreceptor utilization profiles. Conversion of LFA-1 to a high-affinity-high-avidity state for ICAM-1 further augmented the susceptibility of human tonsillar histocultures to infection by ICAM-1-bearing virions. A more massive depletion of CD4+T lymphocytes was seen with X4 ICAM-1/POS viruses than with isogenic ICAM-1/NEG virions. Exposure of X4 and R5 primary isolates of HIV-1 to a blocking anti-ICAM-1 antibody resulted in a decrease of virus infection. Finally, X4 and R5 virions derived from a natural human lymphoid tissue microenvironment incorporated high levels of ICAM-1. Altogether, these results indicate that the incorporation of host ICAM-1 can significantly modulate the biology of HIV-1 in a cellular milieu recognized as the major site of replication in vivo and suggest that host proteins found in HIV-1 particles may participate in the pathogenesis of this disease.
The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles
Authors: Müller Barbara , Patschinsky Tilo , Kräusslich Hans-Georg
Published: October 2010

The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [
ortho-
32
P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.
Conserved Region CR2 of Epstein-Barr Virus Nuclear Antigen Leader Protein Is a Multifunctional Domain That Mediates Self-Association as well as Nuclear Localization and Nuclear Matrix Association
Authors: Tanaka Michiko , Yokoyama Akihiko , Igarashi Mie , Matsuda Go , Kato Kentaro , Kanamori Mikiko , Hirai Kanji , Kawaguchi Yasushi , Yamanashi Yuji
Published: October 2010

Self-association of viral proteins is important for many of their functions, including enzymatic, transcriptional, and transformational activities. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) contains various numbers of W1W2 repeats and a unique carboxyl-terminal Y1Y2 domain. It was reported that EBNA-LP associates with a variety of cellular proteins and plays a critical role in EBV-induced transformation. We report here that EBNA-LP self-associates in vivo and the domain responsible for the homotypic association is a multifunctional domain mediating nuclear localization, nuclear matrix association, and EBNA-2-dependent coactivator function of the protein. Our conclusions are based on the following observations. (i) EBNA-LP interacts with itself or its derivatives in the yeast two-hybrid system. (ii) A purified chimeric protein consisting of glutathione
S
-transferase fused to EBNA-LP specifically formed complexes with EBNA-LP transiently expressed in COS-7 cells. (iii) When Flag epitope-tagged EBNA-LP with either one or two W1W2 repeats and EBNA-LP containing four W1W2 repeats were coexpressed in COS-7 cells, the latter was specifically coimmunoprecipitated with the former. (iv) Mutational analyses of EBNA-LP with deletion mutants revealed that the region between codons 19 and 39 (relative to the first amino acid residue of the W2 domain) is essential for self-association of the protein. The mapped region almost completely overlaps with CR2 and CR3, regions conserved among a subset of primate γ-herpesviruses and critical for EBNA-2-dependent coactivator function. Amino acid substitutions in CR2 alone abolished the ability of the protein to self-interact. This laboratory previously reported that CR2 is also responsible for nuclear localization and nuclear matrix association (A. Yokoyama, Y. Kawaguchi, I. Kitabayashi, M. Ohki, and K. Hirai, Virology 279:401–413, 2001). (v) Sucrose gradient sedimentation showed that amino acid substitutions in CR2 reduced the ability of the protein to form protein complexes in B cells. These results suggest that self-association of EBNA-LP may be important for its various functions and interactions of the protein with multiple cellular proteins.
Adeno-Associated Virus Type 2 Rep78 Inhibition of PKA and PRKX: Fine Mapping and Analysis of Mechanism
Authors: Schmidt Michael , Chiorini John A. , Afione Sandra , Kotin Robert
Published: October 2010

Hormones and neurotransmitters utilize cyclic AMP (cAMP) as a second messenger in signal transduction pathways to regulate cell growth and division, differentiation, gene expression, and metabolism. Adeno-associated virus type 2 (AAV-2) nonstructural protein Rep78 inhibits members of the cAMP signal transduction pathway, the protein kinases PKA and PRKX. We mapped the kinase binding and inhibition domain of Rep78 for PRKX to amino acids (aa) 526 to 561 and that for PKA to aa 526 to 621. These polypeptides were as potent as full-length Rep78 in kinase inhibition, which suggests that the kinase-inhibitory domain is entirely contained in these Rep peptides. Steady-state kinetic analysis of Rep78-mediated inhibition of PKA and PRKX showed that Rep78 appears to increase the
K

m

value of the peptide kinase substrate, while the maximal velocity of the reaction was unaffected. This indicates that Rep78 acts as a competitive inhibitor with respect to the peptide kinase substrate. We detected homology between a cellular pseudosubstrate inhibitor of PKA, the protein kinase inhibitor PKI, and the PRKX and PKA inhibition domains of Rep78. Due to this homology and the competitive inhibition mechanism of Rep78, we propose that Rep78 inhibits PKA and PRKX kinase activity by pseudosubstrate inhibition.
Thermostability of Reovirus Disassembly Intermediates (ISVPs) Correlates with Genetic, Biochemical, and Thermodynamic Properties of Major Surface Protein μ1
Authors: Middleton Jason K. , Severson Tonya F. , Chandran Kartik , Gillian Anne Lynn , Yin John , Nibert Max L.
Published: October 2010

Kinetic analyses of infectivity loss during thermal inactivation of reovirus particles revealed substantial differences between virions and infectious subvirion particles (ISVPs), as well as between the ISVPs of reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The difference in thermal inactivation of T1L and T3D ISVPs was attributed to the major surface protein μ1 by genetic analyses with reassortant viruses and recoated cores. Irreversible conformational changes in ISVP-bound μ1 were shown to accompany thermal inactivation. The thermal inactivation of ISVPs approximated first-order kinetics over a range of temperatures, permitting the use of Arrhenius plots to estimate activation enthalpies and entropies that account for the different behaviors of T1L and T3D. An effect similar to enthalpy-entropy compensation was additionally noted for the ISVPs of these two isolates. Kinetic analyses with other ISVP-like particles, including ISVPs of a previously reported thermostable mutant, provided further insights into the role of μ1 as a determinant of thermostability. Intact virions, which contain ς3 bound to μ1 as their major surface proteins, exhibited greater thermostability than ISVPs and underwent thermal inactivation with kinetics that deviated from first order, suggesting a role for ς3 in both these properties. The distinct inactivation behaviors of ISVPs are consistent with their role as an essential intermediate in reovirus entry.
Replication of Subgenomic Hepatitis A Virus RNAs Expressing Firefly Luciferase Is Enhanced by Mutations Associated with Adaptation of Virus to Growth in Cultured Cells
Authors: Yi MinKyung , Lemon Stanley M.
Published: October 2010

Replication of hepatitis A virus (HAV) in cultured cells is inefficient and difficult to study due to its protracted and generally noncytopathic cycle. To gain a better understanding of the mechanisms involved, we constructed a subgenomic HAV replicon by replacing most of the P1 capsid-coding sequence from an infectious cDNA copy of the cell culture-adapted HM175/18f virus genome with sequence encoding firefly luciferase. Replication of this RNA in transfected Huh-7 cells (derived from a human hepatocellular carcinoma) led to increased expression of luciferase relative to that in cells transfected with similar RNA transcripts containing a lethal premature termination mutation in 3D
pol
(RNA polymerase). However, replication could not be confirmed in either FrhK4 cells or BSC-1 cells, cells that are typically used for propagation of HAV. Replication was substantially slower than that observed with replicons derived from other picornaviruses, as the basal luciferase activity produced by translation of input RNA did not begin to increase until 24 to 48 h after transfection. Replication of the RNA was reversibly inhibited by guanidine. The inclusion of VP4 sequence downstream of the viral internal ribosomal entry site had no effect on the basal level of luciferase or subsequent increases in luciferase related to its amplification. Thus, in this system this sequence does not contribute to viral translation or replication, as suggested previously. Amplification of the replicon RNA was profoundly enhanced by the inclusion of P2 (but not 5′ noncoding sequence or P3) segment mutations associated with adaptation of wild-type virus to growth in cell culture. These results provide a simple reporter system for monitoring the translation and replication of HAV RNA and show that critical mutations that enhance the growth of virus in cultured cells do so by promoting replication of viral RNA in the absence of encapsidation, packaging, and cellular export of the viral genome.
Binding of Hepatitis C Virus-Like Particles Derived from Infectious Clone H77C to Defined Human Cell Lines
Authors: Wellnitz Sabine , Klumpp Bettina , Barth Heidi , Ito Susumu , Depla Erik , Dubuisson Jean , Blum Hubert E. , Baumert Thomas F.
Published: October 2010

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.
A Gene Capable of Blocking Apoptosis Can Substitute for the Herpes Simplex Virus Type 1 Latency-Associated Transcript Gene and Restore Wild-Type Reactivation Levels
Authors: Perng Guey-Chuen , Maguen Barak , Jin Ling , Mott Kevin R. , Osorio Nelson , Slanina Susan M. , Yukht Ada , Ghiasi Homayon , Nesburn Anthony B. , Inman Melissa , Henderson Gail , Jones Clinton , Wechsler Steven L.
Published: October 2010

After ocular herpes simplex virus type 1 (HSV-1) infection, the virus travels up axons and establishes a lifelong latent infection in neurons of the trigeminal ganglia. LAT (latency-associated transcript), the only known viral gene abundantly transcribed during HSV-1 neuronal latency, is required for high levels of reactivation. The LAT function responsible for this reactivation phenotype is not known. Recently, we showed that LAT can block programmed cell death (apoptosis) in neurons of the trigeminal ganglion in vivo and in tissue culture cells in vitro (G.-C. Perng et al., Science 287:1500–1503, 2000; M. Inman et al., J. Virol. 75:3636–3646, 2001). Consequently, we proposed that this antiapoptosis function may be a key to the mechanism by which LAT enhances reactivation. To study this further, we constructed a recombinant HSV-1 virus in which the HSV-1 LAT gene was replaced by an alternate antiapoptosis gene. We used the bovine herpes virus 1 (BHV-1) latency-related (LR) gene, which was previously shown to have antiapoptosis activity, for this purpose. The resulting chimeric virus, designated CJLAT, contains two complete copies of the BHV-1 LR gene (one in each viral long repeat) in place of the normal two copies of the HSV-1 LAT, on an otherwise wild-type HSV-1 strain McKrae genomic background. We report here that in both rabbits and mice reactivation of CJLAT was significantly greater than the LAT null mutant dLAT2903 (
P
< 0.0004 and
P
= 0.001, respectively) and was at least as efficient as wild-type McKrae. This strongly suggests that a BHV-1 LR gene function was able to efficiently substitute for an HSV-1 LAT gene function involved in reactivation. Although replication of CJLAT in rabbits and mice was similar to that of wild-type McKrae, CJLAT killed more mice during acute infection and caused more corneal scarring in latently infected rabbits. This suggested that the BHV-1 LR gene and the HSV-1 LAT gene are not functionally identical. However, LR and LAT both have antiapoptosis activity. These studies therefore strongly support the hypothesis that replacing LAT with an antiapoptosis gene restores the wild-type reactivation phenotype to a LAT null mutant of HSV-1 McKrae.
The Cytoplasmic Tail of Infectious Bronchitis Virus E Protein Directs Golgi Targeting
Authors: Corse Emily , Machamer Carolyn E.
Published: October 2010

We have previously shown that the E protein of the coronavirus infectious bronchitis virus (IBV) is localized to the Golgi complex when expressed exogenously from cDNA. Here, we report that neither the transmembrane domain nor the short lumenal domain of IBV E is required for Golgi targeting. However, an N-terminal truncation containing only the cytoplasmic domain (CTE) was efficiently localized to the Golgi complex, and this domain could retain a reporter protein in the Golgi. Thus, the cytoplasmic tail of the E protein is necessary and sufficient for Golgi targeting. The IBV E protein is palmitoylated on one or two cysteine residues adjacent to its transmembrane domain, but palmitoylation was not required for proper Golgi targeting. Using C-terminal truncations, we determined that the IBV E Golgi targeting information is present between tail amino acids 13 and 63. Upon treatment with brefeldin A, both the E and CTE proteins redistributed to punctate structures that colocalized with the Golgi matrix proteins GM130 and p115 instead of being localized to the endoplasmic reticulum like Golgi glycosylation enzymes. This suggests that IBV E is associated with the Golgi matrix through interactions of its cytoplasmic tail and may have interesting implications for coronavirus assembly in early Golgi compartments.
Potent Immunosuppressive Activities of Cytomegalovirus- Encoded Interleukin-10
Authors: Spencer Juliet V. , Lockridge Kristen M. , Barry Peter A. , Lin Gaofeng , Tsang Monica , Penfold Mark E. T. , Schall Thomas J.
Published: October 2010

Cytomegalovirus (CMV) has highly evolved mechanisms for avoiding detection by the host immune system. Recently, in the genomes of human and primate CMV, a novel gene comprising segments of noncontiguous open reading frames was identified and found to have limited predicted homology to endogenous cellular interleukin-10 (IL-10). Here we investigate the biological activities of the CMV IL-10-like gene product and show it to possess potent immunosuppressive properties. Both purified bacterium-derived recombinant CMV IL-10 and CMV IL-10 expressed in supernatants of human cells were found to inhibit proliferation of mitogen-stimulated peripheral blood mononuclear cells (PBMCs), with specific activity comparable to that of recombinant human IL-10. In addition, CMV IL-10 expressed from human cells inhibited cytokine synthesis, as treatment of stimulated PBMCs and monocytes with CMV IL-10 led to a marked decrease in production of proinflammatory cytokines. Finally, CMV IL-10 was observed to decrease cell surface expression of both major histocompatibility complex (MHC) class I and class II molecules, while conversely increasing expression of the nonclassical MHC allele HLA-G. These results demonstrate for the first time that CMV has a biologically active IL-10 homolog that may contribute to immune evasion during virus infection.
Influenza A Virus M2Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture
Authors: Takeda Makoto , Pekosz Andrew , Shuck Kevin , Pinto Lawrence H. , Lamb Robert A.
Published: October 2010

The amantadine-sensitive ion channel activity of influenza A virus M2protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2protein-mediated equilibration of the intralumenal pH of thetransGolgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656–5662, 2001). An M2protein containing a deletion in the transmembrane (TM) domain (M2-del29–31) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del29–31virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2residue N31is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del29–31virus. We found that the M2-del29–31virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del29–31virus in 4 days after cocultivation of a 100:1 ratio of M2-del29–31virus to wt virus, respectively. We conclude that the M2ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.
The g5R (D250) Gene of African Swine Fever Virus Encodes a Nudix Hydrolase That Preferentially Degrades Diphosphoinositol Polyphosphates
Authors: Cartwright Jared L. , Safrany Stephen T. , Dixon Linda K. , Darzynkiewicz Edward , Stepinski Janusz , Burke Richard , McLennan Alexander G.
Published: October 2010

The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap
4
A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap
4
A hydrolases, having highest activity with GTP, followed by adenosine 5′-pentaphosphate (p
5
A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp
3
A. In common with eukaryotic diadenosine hexaphosphate (Ap
6
A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP
5
and [PP]
2
-InsP
4
. A comparison of the kinetics of substrate utilization showed that the
k
cat
/
K

m

ratio for PP-InsP
5
is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-β-phosphate from both PP-InsP
5
and [PP]
2
-InsP
4
. ASFV infection led to a reduction in the levels of PP-InsP
5
, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective
K

m

values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.
Oncoretrovirus and Lentivirus Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus Glycoprotein: Generation, Concentration, and Broad Host Range
Authors: Beyer Winfried R. , Westphal Manfred , Ostertag Wolfram , von Laer Dorothee
Published: October 2010

Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.
Adaptation of Wild-Type Measles Virus to Tissue Culture
Authors: Waku Kouomou Diane , Wild T. Fabian
Published: October 2010

Measles has a host range restricted to humans and monkeys in captivity. Fresh measles virus (MV) isolates replicate readily in several human and simian B-cell lines but need a period of adaptation to other types of cells. The identification of CD46 and CD150 (SLAM) as cellular receptors for MV has helped to clarify certain aspects of the immunobiology of MV infections. We have examined the properties of an MV wild-type strain grown in the epithelial cell line Vero. After adaptation, this virus expressed high levels of both the viral glycoproteins (hemagglutinin and fusion protein) but did not induce fusion (syncytia). No changes in the amino acid sequence were found in either of the viral glycoproteins. Using several approaches, the Vero-adapted virus could not be shown to interact with CD46 either in the initiation or during the course of infection. The presence of human SLAM expressed in the Vero cells rapidly gave rise to fusion and lower yields of infectious virus.
Sequence and Genetic Arrangement of the USRegion of the Monkey B Virus (Cercopithecine Herpesvirus 1) Genome and Comparison with the USRegions of Other Primate Herpesviruses
Authors: Ohsawa Kazutaka , Black Darla H. , Sato Hiroshi , Eberle R.
Published: October 2010

The sequence of the unique short (US) region of monkey B virus (BV) was determined. The 13 genes identified are arranged in the same order and orientation as in herpes simplex virus (HSV). These results demonstrate that the BV USregion is entirely colinear with that of HSV type 1 (HSV-1), HSV-2, and simian agent 8 virus.