bluuebunny/crossref_metadata_2025_split huggingface dataset:
Murine Coronavirus Nonstructural Protein p28 Arrests Cell Cycle in G0/G1Phase
Authors: Chen Chun-Jen , Sugiyama Kazuo , Kubo Hideyuki , Huang Cheng , Makino Shinji 
Published: September 2004

ABSTRACTMurine coronavirus mouse hepatitis virus (MHV) gene 1 encodes several nonstructural proteins. The functions are unknown for most of these nonstructural proteins, including p28, which is encoded at the 5′ end of the MHV genome. Transient expression of cloned p28 in several different cultured cells inhibited cell growth, indicating that p28 expression suppressed cell proliferation. Expressed p28 was exclusively localized in the cytoplasm. Cell cycle analysis by flow cytometry demonstrated that p28 expression induced G0/G1cell cycle arrest. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that p28 expression resulted in an accumulation of hypophosphorylated retinoblastoma protein (pRb), tumor suppressor p53, and cyclin-dependent kinase (Cdk) inhibitor p21Cip1. Expression of p28 did not alter the amount of p53 transcripts yet increased the amount of p21Cip1transcripts, suggesting that p28 expression increased p53 stability and that p21Cip1was transcriptionally activated in a p53-dependent manner. Our present data suggest the following model of p28-induced G0/G1cell cycle arrest. Expressed cytoplasmic p28 induces the stabilization of p53, and accumulated p53 causes transcriptional upregulation of p21Cip1. The increased amount of p21Cip1suppresses cyclin E/Cdk2 activity, resulting in the inhibition of pRb hyperphosphorylation. Accumulation of hypophosphorylated pRb thus prevents cell cycle progression from G0/G1to S phase.
Induction of Antiviral Immunity by Double-Stranded RNA in a Marine Invertebrate
Authors: Robalino Javier , Browdy Craig L. , Prior Sarah , Metz Adrienne , Parnell Pamela , Gross Paul , Warr Gregory 
Published: September 2004

ABSTRACTVertebrates mount a strong innate immune response against viruses, largely by activating the interferon system. Double-stranded RNA (dsRNA), a common intermediate formed during the life cycle of many viruses, is a potent trigger of this response. In contrast, no general inducible antiviral defense mechanism has been reported in any invertebrate. Here we show that dsRNA induces antiviral protection in the marine crustaceanLitopenaeus vannamei. When treated with dsRNA, shrimp showed increased resistance to infection by two unrelated viruses, white spot syndrome virus and Taura syndrome virus. Induction of this antiviral state is independent of the sequence of the dsRNA used and therefore distinct from the sequence-specific dsRNA-mediated genetic interference phenomenon. This demonstrates for the first time that an invertebrate immune system, like its vertebrate counterparts, can recognize dsRNA as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response.
Human Immunodeficiency Virus Type 1 (HIV-1) Antigen Secretion by Latently Infected Resting CD4
+
T Lymphocytes from HIV-1-Infected Individuals
Authors: Fondere Jean-Michel , Petitjean Gael , Huguet Marie-France , Salhi Sharon Lynn , Baillat Vincent , Macura-Biegum Anna , Becquart Pierre , Reynes Jacques , Vendrell Jean-Pierre 
Published: September 2004

ABSTRACT

In resting CD4
+
T lymphocytes harboring human immunodeficiency virus type 1 (HIV-1), replication-competent virus persists in patients responding to highly active antiretroviral therapy (HAART). This small latent reservoir represents between 10
3
and 10
7
cells per patient. However, the efficiency of HIV-1 DNA-positive resting CD4
+
T cells in converting to HIV-1-antigen-secreting cells (HIV-1-Ag-SCs) after in vitro CD4
+
-T-cell polyclonal stimulation has not been satisfactorily evaluated. By using an HIV-1-antigen enzyme-linked immunospot assay, 8 HIV-1-Ag-SCs per 10
6
CD4
+
resting T cells were quantified in 25 patients with a plasma viral load of <20 copies/ml, whereas 379 were enumerated in 10 viremic patients. In parallel, 369 and 1,238 copies of HIV-1 DNA per 10
6
CD4
+
T cells were enumerated in the two groups of patients, respectively. Only a minority of latently HIV-1 DNA-infected CD4
+
T cells could be stimulated in vitro to become HIV-1-Ag-SCs, particularly in aviremic patients. The difference between the number of HIV-1 immunospots in viremic versus aviremic patients could be explained by HIV-1 unintegrated viral DNA that gave additional HIV-1-Ag-SCs after in vitro CD4
+
-T-cell polyclonal stimulation. The ELISPOT approach to targeting the HIV-1-Ag-SCs could be a useful method for identifying latently HIV-1-infected CD4
+
T cells carrying replication-competent HIV-1 in patients responding to HAART.
Infectious Entry of West Nile Virus Occurs through a Clathrin-Mediated Endocytic Pathway
Authors: Chu J. J. H. , Ng M. L. 
Published: September 2004

ABSTRACT
The pathway of West Nile flavivirus early internalization events was mapped in detail in this study. Overexpression of dominant-negative mutants of Eps15 strongly inhibits West Nile virus (WNV) internalization, and pharmacological drugs that blocks clathrin also caused a marked reduction in virus entry but not caveola-dependent endocytosis inhibitory agent, filipin. Using immunocryoelectron microscopy, WNV particles were seen within clathrin-coated pits after 2 min postinfection. Double-labeling immunofluorescence assays and immunoelectron microscopy performed with anti-WNV envelope or capsid proteins and cellular markers (EEA1 and LAMP1) revealed the trafficking pathway of internalized virus particles from early endosomes to lysosomes and finally the uncoating of the virus particles. Disruption of host cell cytoskeleton (actin filaments and microtubules) with cytochalasin D and nocodazole showed significant reduction in virus infectivity. Actin filaments are shown to be essential during the initial penetration of the virus across the plasma membrane, whereas microtubules are involved in the trafficking of internalized virus from early endosomes to lysosomes for uncoating. Cells treated with lysosomotropic agents were largely resistant to infection, indicating that a low-pH-dependent step is required for WNV infection. In situ hybridization of DNA probes specific for viral RNA demonstrated the trafficking of uncoated viral RNA genomes to the endoplasmic reticulum.
Ring Finger Protein ZIN Interacts with Human Immunodeficiency Virus Type 1 Vif
Authors: Feng Feng , Davis Adam , Lake Julie-Anne , Carr Jill , Xia Wei , Burrell Christopher , Li Peng 
Published: September 2004

ABSTRACT
Virion infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary human CD4 T lymphocytes and macrophages. Vif overcomes the HIV-inhibitory effects of cellular factor APOBEC3G, which has cytidine deaminase activity. We previously reported the isolation of a Vif-interacting ring finger protein, Triad 3, from a human leukocyte cDNA library, using the yeast two-hybrid system. The full-length cellular protein homologue of Triad 3 has been recently identified as the zinc finger protein inhibiting NF-κB (ZIN). Sequence analysis indicates that Triad 3 protein contains all four major ring-like motifs of ZIN. We report here that ZIN binds to purified Vif in vitro and that Triad 3/ZIN interacts with HIV-1 Vif in transfected human 293T cells, as demonstrated by coimmunoprecipitation. To test the biological relevance of this interaction, we produced infectious HIV-1 NL4.3 in the presence or absence of cotransfected ZIN. HIV-1 NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were twofold less infectious in a single-cycle infectivity assay than virus produced in the absence of exogenous ZIN. It was further shown that cells infected with HIV NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were impaired in viral DNA synthesis by twofold. The impairment in viral reverse transcription and the reduction in single-cycle viral infectivity were both shown to be dependent on the presence of Vif in the virus producer cells. The possible mechanisms by which ZIN interferes with the early events of HIV-1 replication are discussed.
Lysines Close to the Rous Sarcoma Virus Late Domain Critical for Budding
Authors: Spidel Jared L. , Craven Rebecca C. , Wilson Carol B. , Patnaik Akash , Wang Huating , Mansky Louis M. , Wills John W. 
Published: September 2004

ABSTRACT
The release of retroviruses from the plasma membrane requires host factors that are believed to be recruited to the site of budding by the late (L) domain of the virus-encoded Gag protein. The L domain of Rous sarcoma virus (RSV) has been shown to interact with a ubiquitin (Ub) ligase, and budding of this virus is dependent on Ub. RSV is similar to other retroviruses in that it contains ∼100 molecules of Ub, but it is unique in that none of these molecules has been found to be conjugated to Gag. If transient ubiquitination of RSV Gag is required for budding, then replacement of the target lysine(s) with arginine should prevent the addition of Ub and reduce budding. Based on known sites of ubiquitination in other viruses, the important lysines would likely reside near the L domain. In RSV, there are five lysines located just upstream of the L domain in a region of the matrix (MA) protein that is dispensable for membrane binding, and replacement of these with arginine (mutant 1-5KR) reduced budding 80 to 90%. The block to budding was found to be on the plasma membrane; however, the few virions that were released had normal size, morphology, and infectivity. Budding was restored when any one of the residues was changed back to lysine or when lysines were inserted in novel positions, either within this region of MA or within the downstream p10 sequence. Moreover, the 1-5KR mutant could be rescued into particles by coexpression of budding-competent Gag molecules. These data argue that the phenotype of mutant 1-5KR is not due to a conformational defect. Consistent with the idea that efficient budding requires a specific role for lysines, human T-cell leukemia virus type 1, which does not bud well compared to RSV and lacks lysines close to its L domain, was found to be released at a higher level upon introduction of lysines near its L domain. This report strongly supports the hypothesis that ubiquitination of the RSV Gag protein (and perhaps those of other retroviruses) is needed for efficient budding.
Retroviruses Pseudotyped with the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Efficiently Infect Cells Expressing Angiotensin-Converting Enzyme 2
Authors: Moore Michael J. , Dorfman Tatyana , Li Wenhui , Wong Swee Kee , Li Yanhan , Kuhn Jens H. , Coderre James , Vasilieva Natalya , Han Zhongchao , Greenough Thomas C. , Farzan Michael , Choe Hyeryun 
Published: September 2004

ABSTRACT
Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2). Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-protein-pseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS.
Cell Fusion Activities of Hantaan Virus Envelope Glycoproteins
Authors: Ogino Michiko , Yoshimatsu Kumiko , Ebihara Hideki , Araki Koichi , Lee Byoung-Hee , Okumura Megumi , Arikawa Jiro 
Published: September 2004

ABSTRACT
Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.
Variation in alternative splicing across human tissues
Authors: Yeo Gene , Holste Dirk , Kreiman Gabriel , Burge Christopher B 
Published: September 2004

Abstract
Background
Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues.

Results
Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues.

Conclusions
This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells.
Clinical pharmacology and determinates of response to gefitinib in non-small cell lung cancer
Authors: Palmer Matt , Geurink Justin , Hahn Kristine , Kolesar Jill 
Published: September 2004

Objective. Provide a review and update of gefitinib (Iressa®), including recent data on mutations in the epidermal growth factor receptor (EGFR) and possible implications in non-small cell lung cancer patients. Data sources. Primary literature and review articles were researched using Pub-Med. Articles through June 2004 were considered for preparation of the review. All clinical trials with published results and articles reporting EGFR mutations were considered for inclusion by the authors. Conclusions. Gefitinib is a novel EGFR tyrosine kinase inhibitor approved for treatment of chemo-resistant NSCLC. Response rates of 10-19% have been observed in clinical trials with no improvement when combined with chemotherapy. Currently, gefitinib is used as a single agent for advanced NSCLC that is resistant to chemotherapy. Mutations in EGFR correlating with response to gefitinib have recently been described. Based on these early results, genetic testing of NSCLC patients upon diagnosis could predict response to gefitinib. Early treatment of NSCLC patients positive for EGFR mutations with gefitinib remains to be tested.
Estimating the direct aerosol radiative perturbation: Impact of ocean surface representation and aerosol non‐sphericity
Authors: Bellouin N. , Boucher O. , Vesperini M. , Tanré D. 
Published: September 2004

AbstractAtmospheric aerosols are now actively studied, in particular because of their radiative and climate impacts. Estimations of the direct aerosol radiative perturbation, caused by extinction of incident solar radiation, usually rely on radiative transfer codes and involve simplifying hypotheses. This paper addresses two approximations which are widely used for the sake of simplicity and limiting the computational cost of the calculations. Firstly, it is shown that using a Lambertian albedo instead of the more rigorous bidirectional reflectance distribution function (BRDF) to model the ocean surface radiative properties leads to large relative errors in the instantaneous aerosol radiative perturbation. When averaging over the day, these errors cancel out to acceptable levels of less than 3% (except in the northern hemisphere winter). The other scope of this study is to address aerosol non‐sphericity effects. Comparing an experimental phase function with an equivalent Mie‐calculated phase function, we found acceptable relative errors if the aerosol radiative perturbation calculated for a given optical thickness is daily averaged. However, retrieval of the optical thickness of non‐spherical aerosols assuming spherical particles can lead to significant errors. This is due to significant differences between the spherical and non‐spherical phase functions. Discrepancies in aerosol radiative perturbation between the spherical and non‐spherical cases are sometimes reduced and sometimes enhanced if the aerosol optical thickness for the spherical case is adjusted to fit the simulated radiance of the non‐spherical case. Copyright © 2004 Royal Meteorological Society
Properties and first application of an error‐statistics tuning method in variational assimilation
Authors: Chapnik Bernard , Desroziers Gerald , Rabier Florence , Talagrand Olivier 
Published: September 2004

AbstractThe method for tuning observational or background error statistics is presented and some of its properties are exposed using theoretical considerations and experiments carried out in a simplified framework. In particular, the method is shown to be equivalent to a maximum‐likelihood evaluation and its efficiency is seen to depend on the number of observations. The results of several experiments carried out with the variational assimilation system of the French numerical weather‐prediction system ARPEGE, both with simulated and actual datasets involving satellite radiances, are also presented. The temporal stability of the results and their consistency with the known quality of the measurements are shown. Copyright © 2004 Royal Meteorological Society
High-Sensitivity Detection of the A3243G Mutation of Mitochondrial DNA by a Combination of Allele-Specific PCR and Peptide Nucleic Acid-Directed PCR Clamping
Authors: Urata Michiyo , Wada Yui , Kim Sang Ho , Chumpia Worawan , Kayamori Yuzo , Hamasaki Naotaka , Kang Dongchon 
Published: September 2004

Abstract
Background: The A3243G mutation of mitochondrial DNA (mtDNA) is involved in many common diseases, including diabetes mellitus and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). For detection of this mutation, allele-specific PCR is highly sensitive but requires strict control of PCR conditions; it thus is not adequate for a routine clinical test. We aimed to develop a routinely available PCR method for quantitative detection of low-level heteroplasmy of the A3243G mutation.
Methods: Quantitative allele-specific PCR for the A3243G mutation was performed in the presence of peptide nucleic acid (PNA), in which PNA is complementary to the wild-type mtDNA, with one primer having a 3′ end matched to nucleotide position 3243 of the mutant.
Results: With our method, amplification of wild-type mtDNA was suppressed 7000-fold compared with amplification of the mutant mtDNA under a broad range of conditions: DNA, 5–100 ng; annealing temperature, 61–66 °C; and PNA, 1.5–3.5 μmol/L. Hence, 0.1% heteroplasmy of the A3243G mutation can be reliably quantified by this method. Blood samples form 40 healthy volunteers showed &lt;0.06% heteroplasmy, suggesting that 0.1% is diagnostically significant.
Conclusions: PNA maintains the specificity of allele-specific PCR over a wide range of conditions, which is important for routine clinical testing.
Survivin Is an Independent Prognostic Marker for Risk Stratification of Breast Cancer Patients
Authors: Span Paul N , Sweep Fred C G J , Wiegerinck Erwin T G , Tjan-Heijnen Vivianne C G , Manders Peggy , Beex Louk V A M , de Kok Jaques B 
Published: September 2004

Abstract
Background: Results in previous qualitative studies of the association of the apoptosis inhibitor survivin with prognosis of breast cancer patients have been contradictory.
Methods: Survivin mRNA was measured by quantitative TaqMan reverse transcription-PCR in 275 breast cancer tissues from patients with operable tumors and was correlated with established clinicopathologic factors, relapse-free survival [(RFS); 102 events], and overall survival [(OS); 81 events].
Results: High survivin mRNA concentrations were found mainly in tissues from younger patients and in high-grade cancer tissues. High survivin concentrations were most strongly associated with estrogen receptor- or progesterone receptor-negative tumors. In univariate Cox regression analysis for RFS, survivin concentrations were significantly associated with poor prognosis with a hazard ratio (HR) of 1.99 (95% confidence interval, 1.31–3.02; P = 0.001) for every 10-fold increase in expression. For OS, a significant contribution of survivin to poor prognosis was found with a HR of 2.76 (1.67–4.55; P &lt;0.001). Multivariate analyses were performed including established clinicopathologic factors. For RFS, age (P = 0.027), nodal category (P &lt;0.001), and survivin [HR = 1.78 (1.18–2.68); P = 0.006] contributed significantly to the model. For OS, only nodal category (P &lt;0.001) and survivin [HR = 3.05 (1.83–5.10); P &lt;0.001] were significant.
Conclusion: Survivin demonstrates a strong, independent, association with poor prognosis. Survivin might be used as a new marker to stratify breast cancer patients for more optimal treatment modalities, or it could be a promising new target for therapy.