bluuebunny/crossref_metadata_2025 huggingface dataset:
STAT5A interacts with and is phosphorylated upon activation of the µ‐opioid receptor
Authors: Mazarakou Georgia , Georgoussi Zafiroula
Published: April 2005

Signal Transducers and Activators of Transcription (STATs) are transcription factors shown to be activated by G protein‐coupled receptors. In the present study, we demonstrate that acute morphine or [D‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin (DAMGO) exposure of COS‐7 cells transiently transfected with the µ‐opioid receptor and STAT5A, leads to receptor‐dependent tyrosine phosphorylation of STAT5A. Activation of HEK293 cells, stably expressing the µ‐opioid receptor with µ‐opioid agonists results in the transcriptional activation of a STAT‐responsive reporter gene. Pertussis toxin has no effect on the level of STAT5A phosphorylation, while the Src inhibitor PP1 abolishes opioid‐dependent STAT5A phosphorylation. All three opioid receptor subtypes –µ, δ and κ– share the conserved motif YXXL (amino‐acids 336–339 for the µ‐opioid receptor), known to be critical for STAT5A/5B binding. Co‐immunoprecipitation and pull‐down experiments using a GST‐carboxyl‐terminal tail of the µ‐opioid receptor and rat brain, or COS‐7 cell cytosolic extracts, demonstrate the direct binding of STAT5A to this region. Mutation of the Y336 to alanine does not prevent STAT5A binding, whereas deletion of the entire putative STAT5A binding site YXXL abolishes STAT5A interaction to the carboxyl‐terminal tail of the µ‐opioid receptor. Collectively, our results demonstrate the association of STAT5A with the µ‐opioid receptor and reveal novel signalling pathways in the regulation of transcription by the µ‐opioid receptor.
Role of oxidative stress in paraquat‐induced dopaminergic cell degeneration
Authors: McCormack Alison L. , Atienza Jennifer G. , Johnston Louisa C. , Andersen Julie K. , Vu Susan , Di Monte Donato A.
Published: April 2005

Systemic treatment of mice with the herbicide paraquat causes the selective loss of nigrostriatal dopaminergic neurons, reproducing the primary neurodegenerative feature of Parkinson's disease. To elucidate the role of oxidative damage in paraquat neurotoxicity, the time‐course of neurodegeneration was correlated to changes in 4‐hydroxy‐2‐nonenal (4‐HNE), a lipid peroxidation marker. When mice were exposed to three weekly injections of paraquat, no nigral dopaminergic cell loss was observed after the first administration, whereas a significant reduction of neurons followed the second exposure. Changes in the number of nigral 4‐HNE‐positive neurons suggest a relationship between lipid peroxidation and neuronal death, since a dramatic increase in this number coincided with the onset and development of neurodegeneration after the second toxicant injection. Interestingly, the third paraquat administration did not cause any increase in 4‐HNE‐immunoreactive cells, nor did it produce any additional dopaminergic cell loss. Further evidence of paraquat‐induced oxidative injury derives from the observation of nitrotyrosine immunoreactivity in the substantia nigra of paraquat‐treated animals and from experiments with ferritin transgenic mice. These mice, which are characterized by a decreased susceptibility to oxidative stress, were completely resistant to the increase in 4‐HNE‐positive neurons and the cell death caused by paraquat. Thus, paraquat exposure yields a model that emphasizes the susceptibility of dopaminergic neurons to oxidative damage.
Presenilin function and γ‐secretase activity
Authors: Brunkan A. L. , Goate A. M.
Published: April 2005

Alzheimer's disease (AD) is the most common form of dementia and is characterized pathologically by the accumulation of β‐amyloid (Aβ) plaques and neurofibrillary tangles in the brain. Genetic studies of AD first highlighted the importance of the presenilins (PS). Subsequent functional studies have demonstrated that PS form the catalytic subunit of the γ‐secretase complex that produces the Aβ peptide, confirming the central role of PS in AD biology. Here, we review the studies that have characterized PS function in the γ‐secretase complex in Caenorhabditis elegans, mice and in in vitro cell culture systems, including studies of PS structure, PS interactions with substrates and other γ‐secretase complex members, and the evidence supporting the hypothesis that PS are aspartyl proteases that are active in intramembranous proteolysis. A thorough knowledge of the mechanism of PS cleavage in the context of the γ‐secretase complex will further our understanding of the molecular mechanisms that cause AD, and may allow the development of therapeutics that can alter Aβ production and modify the risk for AD.
SALARIES—A FRAMEWORK FOR THE STUDY OF TRENDS
Authors: Berghoefer Fred G.
Published: April 2005

This paper proposes that salary structures and the development of salaries over time be considered within the framework of the distribution of income over time. In particular, it examines certain salary scales in the U.S. and the progressions of typical individuals’ salaries during the period 1948–1969, in comparison with the percentile distributions of household income in the same period, as reflected by the Current Population Survey of the Bureau of the Census.
SOCIAL MATRICES AND INDICATORS IN THE TRADITIONAL AFRICAN ECONOMY
Authors: O'Loughlin Carleen , Ewusi Kodwo
Published: April 2005

This paper discusses the relevance of the conventional national accounts systems to the traditional African economy and concludes that they contribute little because they omit certain economic activities and fail to recognise the reciprocity between social and economic activities. Social accounting is thus more relevant. Lack of statistical data may make it necessary to conduct special surveys and in some cases a tribe or village or economic region may be a more useful accounting unit than a nation. A modified system of accounts is suggested, based on the frame work of the four consolidated accounts of the SNA. It provides linkages to many more nonmonetised activities. Other linkages would be provided through supporting tables emphasising social activities and transfers. A system of transactor accounts in matrix form is also suggested. In the case of communities smaller than the nation several external transactor sectors could be included. It is recognised that the problem of evaluation of social activities and a number of economic activities remains to be solved and it is concluded that “time spent” may be the only common unit or value to equate such activities.

The final section deals with investment in human resources and proposes a balance sheet approach to indicative planning. This exercise would be related to demographic projections in several variants. Other factors to be analysed dynamically would be education and health status, public finance and, ultimately, distribution of income and wealth since it is noted that the process of monetisation is having an impact which may have important welfare implications.
Intracranial EEG Substrates of Scalp EEG Interictal Spikes
Authors: Tao James X. , Ray Amit , Hawes‐Ebersole Susan , Ebersole John S.
Published: April 2005

Summary:  Purpose: To determine the area of cortical generators of scalp EEG interictal spikes, such as those in the temporal lobe epilepsy.

Methods: We recorded simultaneously 26 channels of scalp EEG with subtemporal supplementary electrodes and 46 to 98 channels of intracranial EEG in 16 surgery candidates with temporal lobe epilepsy. Cerebral discharges with and without scalp EEG correlates were identified, and the area of cortical sources was estimated from the number of electrode contacts demonstrating concurrent depolarization.

Results: We reviewed ∼600 interictal spikes recorded with intracranial EEG. Only a very few of these cortical spikes were associated with scalp recognizable potentials; 90% of cortical spikes with a source area of >10 cm2 produced scalp EEG spikes, whereas only 10% of cortical spikes having <10 cm2 of source area produced scalp potentials. Intracranial spikes with <6 cm2 of area were never associated with scalp EEG spikes.

Conclusions: Cerebral sources of scalp EEG spikes are larger than commonly thought. Synchronous or at least temporally overlapping activation of 10–20 cm2 of gyral cortex is common. The attenuating property of the skull may actually serve a useful role in filtering out all but the most significant interictal discharges that can recruit substantial surrounding cortex.
Occipital Lobe Epilepsy: Clinical Characteristics, Surgical Outcome, and Role of Diagnostic Modalities
Authors: Kun Lee Sang , Young Lee Seo , Kim Dong‐Wuk , Soo Lee Dong , Chung Chun‐Kee
Published: April 2005

Summary:  Purpose: To assess the role of various diagnostic modalities, to identify surgical prognostic factors and concordances with presurgical evaluations, and to characterize the clinical features of occipital lobe epilepsy (OLE), we studied 26 patients who were diagnosed as having OLE and underwent epilepsy surgery.

Methods: Diagnoses were established by standard presurgical evaluations, which included magnetic resonance imaging (MRI), fluorodeoxyglucose–positron emission tomography (FDG‐PET), ictal single‐photon emission computed tomography (SPECT), scalp video‐EEG monitoring, and intracranial EEG monitoring. After epilepsy surgery, patients were followed up for >2 years.

Results: Sixteen (61.5%) of the 26 became seizure free after surgery, and another eight patients had a favorable outcome. Sixteen of the 26 patients experienced a type of visual aura (i.e., visual hallucination, visual illusion, blindness, or a field defect). Nine patients had both automotor seizures and secondary generalized tonic–clonic seizures at different times. Interictal EEG showed correctly localizing spikes in 10 of the 16 patients who became seizure free, and in three of the 10 non–seizure‐free patients. MRI correctly localized the lesion in seven of these 16 seizure‐free patients, and in three of the 10 non–seizure‐free patients. FDG‐PET correctly localized the lesion in eight of the 16 seizure‐free patients, and in three of nine non–seizure‐free patients. Ictal SPECT was performed in 19 patients and correctly localized the lesion in only three of 12 seizure‐free patients, and in four of seven non–seizure‐free patients. Ictal EEG correctly localized the lesion in 13 of the 16 seizure‐free patients, and in five of the 10 non–seizure‐free patients. No significant relation was found between the diagnostic accuracy of any modality and surgical outcome. The localizations of epileptogenic zones by these different diagnostic methods were complementary. The concordance of three or more modalities was significantly observed in seizure‐free patients (p = 0.042). However, no definite relation was observed between the presence of lateralizing clinical seizure manifestation and surgical outcome (p = 0.108).

Conclusions
: Some specific auras indicated an occipital epilepsy onset. Various diagnostic methods can be useful to diagnose OLE, and a greater concordance between presurgical evaluation modalities indicates a better surgical outcome.
High-Level Expression of Marek's Disease Virus Glycoprotein C Is Detrimental to Virus Growth In Vitro
Authors: Tischer B. Karsten , Schumacher Daniel , Chabanne-Vautherot Danièlle , Zelnik Vladimir , Vautherot Jean-François , Osterrieder Nikolaus
Published: April 2005

Expression levels of Marek's disease virus (MDV) glycoprotein C (gC) are significantly reduced after serial virus passage in cell culture. Reduced gC expression coincides with enhanced MDV growth in vitro and attenuation. To analyze this phenomenon in detail, a full-length infectious MDV clone was modified by Red-based and shuttle mutagenesis in
Escherichia coli
. Besides a gC-negative deletion mutant harboring a kanamycin resistance gene, a markerless mutant with the

U
L

44
gene deleted was constructed. On the basis of this deletion mutant, the original or a modified

U
L

44
gene with a mutated start codon (AUG→ACG) was reinserted into the authentic locus. Similarly, mutants expressing authentic gC or the start codon mutation under the control of a strong constitutive promoter were generated. In vitro studies demonstrated that gC deletion mutants induced twofold-larger plaques than the parental virus did, whereas constitutive overexpression of the glycoprotein resulted in a more than twofold reduction in plaque size. In addition, plaque sizes of the gC deletion mutant were reduced when virus was grown using supernatants from cells infected with parental virus, but supernatants obtained from cells infected with the gC deletion mutant had no measurable effect on plaque size. The results indicated that (i) expression of MDV gC, albeit at low levels in a highly passaged virus, had a significant negative impact on the cell-to-cell spread capabilities of the virus, which was alleviated in its absence and exacerbated by its overexpression, and that (ii) this activity was mediated by the secreted form of MDV gC.
Evaluation of Human Monoclonal Antibody 80R for Immunoprophylaxis of Severe Acute Respiratory Syndrome by an Animal Study, Epitope Mapping, and Analysis of Spike Variants
Authors: Sui Jianhua , Li Wenhui , Roberts Anjeanette , Matthews Leslie J. , Murakami Akikazu , Vogel Leatrice , Wong Swee Kee , Subbarao Kanta , Farzan Michael , Marasco Wayne A.
Published: April 2005

In this report, the antiviral activity of 80R immunoglobulin G1 (IgG1), a human monoclonal antibody against severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein that acts as a viral entry inhibitor in vitro, was investigated in vivo in a mouse model. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced by more than 4 orders of magnitude to below assay limits. The essential core region of S protein required for 80R binding was identified as a conformationally sensitive fragment (residues 324 to 503) that overlaps the receptor ACE2-binding domain. Amino acids critical for 80R binding were identified. In addition, the effects of various 80R-binding domain amino acid substitutions which occur in SARS-like-CoV from civet cats, and which evolved during the 2002/2003 outbreak and in a 2003/2004 Guangdong index patient, were analyzed. The results demonstrated that the vast majority of SARS-CoVs are sensitive to 80R. We propose that by establishing the susceptibility and resistance profiles of newly emerging SARS-CoVs through early S1 genotyping of the core 180-amino-acid neutralizing epitope of 80R, an effective immunoprophylaxis strategy with 80R should be possible in an outbreak setting. Our study also cautions that for any prophylaxis strategy based on neutralizing antibody responses, whether by passive or active immunization, a genotyping monitor will be necessary for effective use.
Amino Acid 36 in the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Controls Fusogenic Activity: Implications for the Molecular Mechanism of Viral Escape from a Fusion Inhibitor
Authors: Kinomoto Masanobu , Yokoyama Masaru , Sato Hironori , Kojima Asato , Kurata Takeshi , Ikuta Kazuyoshi , Sata Tetsutaro , Tokunaga Kenzo
Published: April 2005

We have previously described a human immunodeficiency virus type 1 (HIV-1) proviral clone, pL2, derived from defective viral particles with higher fusogenicity than the prototypic NL4-3 virus. In this study, we attempted to determine the region that confers the enhanced fusion activity by creating envelope recombinants between pL2 and pNL4-3, as well as point mutants based on pNL4-3. The results indicate that amino acid 36 of gp41 is key for the fusogenic activity and infectivity enhancement and that glycine 36 (36G) of gp41 in pL2 is conserved in nearly all HIV-1 isolates except for pNL4-3. The mutation 36G→D in a primary-isolate-derived Env decreased syncytium-forming activity and infectivity. The assays for cell-cell fusion and viral binding suggested that the enhanced fusion mediated by the 36D→G mutation is not due to increased binding efficiency but is directly due to actual enhancement of viral fusion activity. Interestingly, this amino acid position is exactly equivalent to that at which the mutation of HIV-1 isolates that have escaped from a fusion inhibitor, enfuvirtide (T-20), has been frequently observed. The correlation between these previous findings and our findings was suggested by structural analysis. Our finding, therefore, has implications for a molecular basis of the viral escape from this drug.
Functional Mimicry of a Human Immunodeficiency Virus Type 1 Coreceptor by a Neutralizing Monoclonal Antibody
Authors: Xiang Shi-Hua , Farzan Michael , Si Zhihai , Madani Navid , Wang Liping , Rosenberg Eric , Robinson James , Sodroski Joseph
Published: April 2005

Interaction of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein with the primary receptor, CD4, promotes binding to a chemokine receptor, either CCR5 or CXCR4. The chemokine receptor-binding site on gp120 elicits CD4-induced (CD4i) antibodies in some HIV-1-infected individuals. Like CCR5 itself, the CD4i antibody 412d exhibits a preference for CCR5-using HIV-1 strains and utilizes sulfated tyrosines to achieve binding to gp120. Here, we show that 412d binding requires the gp120 β19 strand and the base of the V3 loop, elements that are important for the binding of the CCR5 N terminus. Two gp120 residues in the V3 loop base determined 412d preference for CCR5-using HIV-1 strains. A chimeric molecule in which the 412d heavy-chain third complementarity-determining loop sequence replaces the CCR5 N terminus functioned as an efficient second receptor, selectively supporting the entry of CCR5-using HIV-1 strains. Sulfation of N-terminal tyrosines contributed to the function of this chimeric receptor. These results emphasize the close mimicry of the CCR5 N terminus by the gp120-interactive region of a naturally elicited CD4i antibody.
Nuclear Localization of the Nipah Virus W Protein Allows for Inhibition of both Virus- and Toll-Like Receptor 3-Triggered Signaling Pathways
Authors: Shaw Megan L. , Cardenas Washington B. , Zamarin Dmitriy , Palese Peter , Basler Christopher F.
Published: April 2005

The Nipah virus V and W proteins, which are encoded by the P gene via RNA editing, have a common N-terminal domain but unique C-terminal domains. They localize to the cytoplasm and nucleus, respectively, and have both been shown to function as inhibitors of JAK/STAT signaling. Here we report that V and W proteins also block virus activation of the beta interferon (IFN-β) promoter and the IFN regulatory factor 3 (IRF3)-responsive IFN-stimulated gene 54 promoter. Surprisingly, only W protein shows strong inhibition of promoter activation in response to stimulation of Toll-like receptor 3 (TLR3) by extracellular double-stranded RNA. This activity is dependent on the nuclear localization of W protein. Within the unique C-terminal domain of W protein, we have identified a nuclear localization signal (NLS) that requires basic residues at positions 439, 440, and 442. This NLS is responsible for mediating the preferential interaction of W protein with karyopherin-α 3 and karyopherin-α 4. Nuclear localization of W protein therefore enables it to target both virus and TLR3 pathways, whereas the cytoplasmic V protein is restricted to inhibiting the virus pathway. We propose that this discrepancy is in part due to the V protein being less able to block signaling in response to the kinase, TBK-1, whereas both V and W can prevent promoter activation in response to IKKε. We demonstrate that, when the TLR3 pathway is stimulated, the levels of phosphorylated IRF3 are reduced in the presence of W protein but not V protein, confirming the differential effects of these proteins and illustrating that W protein-mediated inhibition is due to a loss of active IRF3.
Comparative Host Gene Transcription by Microarray Analysis Early after Infection of the Huh7 Cell Line by Severe Acute Respiratory Syndrome Coronavirus and Human Coronavirus 229E
Authors: Tang Bone S. F. , Chan Kwok-hung , Cheng Vincent C. C. , Woo Patrick C. Y. , Lau Susanna K. P. , Lam Clarence C. K. , Chan Tsun-leung , Wu Alan K. L. , Hung Ivan F. N. , Leung Suet-yi , Yuen Kwok-yung
Published: April 2005

The pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) at the cellular level is unclear. No human cell line was previously known to be susceptible to both SARS-CoV and other human coronaviruses. Huh7 cells were found to be susceptible to both SARS-CoV, associated with SARS, and human coronavirus 229E (HCoV-229E), usually associated with the common cold. Highly lytic and productive rates of infections within 48 h of inoculation were reproducible with both viruses. The early transcriptional profiles of host cell response to both types of infection at 2 and 4 h postinoculation were determined by using the Affymetrix HG-U133A microarray (about 22,000 genes). Much more perturbation of cellular gene transcription was observed after infection by SARS-CoV than after infection by HCoV-229E. Besides the upregulation of genes associated with apoptosis, which was exactly opposite to the previously reported effect of SARS-CoV in a colonic carcinoma cell line, genes related to inflammation, stress response, and procoagulation were also upregulated. These findings were confirmed by semiquantitative reverse transcription-PCR, reverse transcription-quantitative PCR for mRNA of genes, and immunoassays for some encoded proteins. These transcriptomal changes are compatible with the histological changes of pulmonary vasculitis and microvascular thrombosis in addition to the diffuse alveolar damage involving the pneumocytes.
Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein
Authors: Dawe Sandra , Corcoran Jennifer A. , Clancy Eileen K. , Salsman Jayme , Duncan Roy
Published: April 2005

Select members of the
Reoviridae
are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusion-inducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N
exoplasmic
/C
cytoplasmic
topology. This topology results in the translocation of the smallest ectodomain (∼20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of protein-mediated membrane fusion.
Biochemical Characterization of Rous Sarcoma Virus MA Protein Interaction with Membranes
Authors: Dalton Amanda K. , Murray Paul S. , Murray Diana , Vogt Volker M.
Published: April 2005

The MA domain of retroviral Gag proteins mediates association with the host cell membrane during assembly. The biochemical nature of this interaction is not well understood. We have used an in vitro flotation assay to directly measure Rous sarcoma virus (RSV) MA-membrane interaction in the absence of host cell factors. The association of purified MA and MA-containing proteins with liposomes of defined composition was electrostatic in nature and depended upon the presence of a biologically relevant concentration of negatively charged lipids. A mutant MA protein known to be unable to promote Gag membrane association and budding in vivo failed to bind to liposomes. These results were supported by computational modeling. The intrinsic affinity of RSV MA for negatively charged membranes appears insufficient to promote efficient plasma membrane binding during assembly. However, an artificially dimerized form of MA bound to liposomes by at least an order of magnitude more tightly than monomeric MA. This result suggests that the clustering of MA domains, via Gag-Gag interactions during virus assembly, drives membrane association in vivo.
Impact of Viral Factors on Very Early In Vivo Replication Profiles in Simian Immunodeficiency Virus SIVagm-Infected African Green Monkeys
Authors: Pandrea Ivona , Kornfeld Christopher , Ploquin Mickael J.-Y. , Apetrei Cristian , Faye Abdourahmane , Rouquet Pierre , Roques Pierre , Simon François , Barré-Sinoussi Françoise , Müller-Trutwin Michaela C. , Diop Ousmane M.
Published: April 2005

To better understand which factors govern the levels of viral loads in early lentiviral infections of primates, we developed a model that allows distinguishing between the influences of host and viral factors on viremia. Herein we report that two species of African green monkeys (Chlorocebus sabaeusandC. pygerythrus) infected with their respective wild-type simian immunodeficiency virus SIVagm viruses (SIVagm.sab92018 and SIVagm.ver644) consistently showed reproducible differences in viremia during primary infection but not at later stages of infection. Cross-infections of SIVagm.sab92018 and SIVagm.ver644 into, respectively,C. pygerythrusandC. sabaeusrevealed that the dynamics of viral replication during primary infection were dependent on the viral strain used for the infection but not on the host. Hence, the kinetics of SIVagm.sab92018 and SIVagm.ver644 were similar in both sabaeus and vervet animals, indicating that the difference in viremia levels between the two groups during the early phase of infection was not associated with the host. Coreceptor usage for these two strains showed a larger coreceptor repertoire for SIVagm.sab92018, which is able to efficiently use CXCR4 in addition to CCR5, than for SIVagm.ver644, which showed a classical CCR5 coreceptor usage pattern. These differences could not be explained by different charges of the V3 loop for SIVagm.sab92018 and for SIVagm.ver644. In conclusion, our study showed that the extent of virus replication during the primary infection is primarily dependent on viral determinants.
New Antiviral Pathway That Mediates Hepatitis C Virus Replicon Interferon Sensitivity through ADAR1
Authors: Taylor Deborah R. , Puig Montserrat , Darnell Miriam E. R. , Mihalik Kathleen , Feinstone Stephen M.
Published: April 2005

While many clinical hepatitis C virus (HCV) infections are resistant to alpha interferon (IFN-α) therapy, subgenomic in vitro self-replicating HCV RNAs (HCV replicons) are characterized by marked IFN-α sensitivity. IFN-α treatment of replicon-containing cells results in a rapid loss of viral RNA via translation inhibition through double-stranded RNA-activated protein kinase (PKR) and also through a new pathway involving RNA editing by an adenosine deaminase that acts on double-stranded RNA (ADAR1). More than 200 genes are induced by IFN-α, and yet only a few are attributed with an antiviral role. We show that inhibition of both PKR and ADAR1 by the addition of adenovirus-associated RNA stimulates replicon expression and reduces the amount of inosine recovered from RNA in replicon cells. Small inhibitory RNA, specific for ADAR1, stimulated the replicon 40-fold, indicating that ADAR1 has a role in limiting replication of the viral RNA. This is the first report of ADAR's involvement in a potent antiviral pathway and its action to specifically eliminate HCV RNA through adenosine to inosine editing. These results may explain successful HCV replicon clearance by IFN-α in vitro and may provide a promising new therapeutic strategy for HCV as well as other viral infections.
Compartmentalization of Hepatitis C Virus Genotypes between Plasma and Peripheral Blood Mononuclear Cells
Authors: Roque-Afonso Anne-Marie , Ducoulombier Delphine , Di Liberto Gaëtana , Kara Rachid , Gigou Michele , Dussaix Elisabeth , Samuel Didier , Féray Cyrille
Published: April 2005

Differences in hepatitis C virus (HCV) variants of the highly conserved 5′ untranslated region (UTR) have been observed between plasma and peripheral blood mononuclear cells (PBMC). The prevalence and the mechanisms of this compartmentalization are unknown. Plasma and PBMC HCV variants were compared by single-strand conformation polymorphism (SSCP) and by cloning or by genotyping with a line probe assay (LiPA) in 116 chronically infected patients, including 44 liver transplant recipients. SSCP patterns differed between compartments in 43/109 analyzable patients (39%). Differences were significantly more frequent in patients with transplants (21/38 [55%] versus 22/71 [31%];
P
< 0.01) and in those who acquired HCV through multiple transfusions before 1991 (15/20; 75%) or through drug injection (16/31; 52%) than in those infected through an unknown route (7/29; 24%) or through a single transfusion (5/29; 17%;
P
< 0.001). Cloning of the 5′ UTR, LiPA analysis, and nonstructural region 5B sequencing revealed different genotypes in the two compartments from 10 patients (9%). In nine patients, the genotype detected in PBMC was not detected in plasma and was weak or undetectable in the liver in three cases. This genotypic compartmentalization persisted for years in three patients and after liver transplantation in two. The present study shows that a significant proportion of HCV-infected subjects harbor in their PBMC highly divergent variants which were likely acquired through superinfections.
Stimulation of Poliovirus Synthesis in a HeLa Cell-Free In Vitro Translation-RNA Replication System by Viral Protein 3CD
pro
Authors: Franco David , Pathak Harsh B. , Cameron Craig E. , Rombaut Bart , Wimmer Eckard , Paul Aniko V.
Published: April 2005

The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CD
pro
, is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CD
pro
in virus synthesis. We observed that purified 3CD
pro
protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CD
pro
is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CD
pro
, 3C
pro
R84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3D
pol
domain of 3CD
pro
. Surprisingly, virus synthesis is strongly inhibited by the addition of both 3C
pro
and 3CD
pro
at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3D
pol
, and 3C
pro
or with cellular protein PCBP2. These results suggest that 3CD
pro
has to be present in the reaction at the time the replication complexes are assembled and that both the 3C
pro
and 3D
pol
domains of the protein are required for its activity that stimulates virus production.
Comparative Genomics of Foot-and-Mouth Disease Virus
Authors: Carrillo C. , Tulman E. R. , Delhon G. , Lu Z. , Carreno A. , Vagnozzi A. , Kutish G. F. , Rock D. L.
Published: April 2005

Here we present complete genome sequences, including a comparative analysis, of 103 isolates of foot-and-mouth disease virus (FMDV) representing all seven serotypes and including the first complete sequences of the SAT1 and SAT3 genomes. The data reveal novel highly conserved genomic regions, indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Previously undescribed invariant motifs were identified in the 5′ and 3′ untranslated regions (UTR), as was tolerance for insertions/deletions in the 5′ UTR. Fifty-eight percent of the amino acids encoded by FMDV isolates are invariant, suggesting that these residues are critical for virus biology. Novel, conserved sequence motifs with likely functional significance were identified within proteins L
pro
, 1B, 1D, and 3C. An analysis of the complete FMDV genomes indicated phylogenetic incongruities between different genomic regions which were suggestive of interserotypic recombination. Additionally, a novel SAT virus lineage containing nonstructural protein-encoding regions distinct from other SAT and Euroasiatic lineages was identified. Insights into viral RNA sequence conservation and variability and genetic diversity in nature will likely impact our understanding of FMDV infections, host range, and transmission.
Human Circovirus TT Virus Genotype 6 Expresses Six Proteins following Transfection of a Full-Length Clone
Authors: Qiu Jianming , Kakkola Laura , Cheng Fang , Ye Chaoyang , Söderlund-Venermo Maria , Hedman Klaus , Pintel David J.
Published: April 2005

The expression profile of the circovirus TTV has not yet been fully characterized. In this paper, we show that following transfection of a full-length viral clone of TTV genotype 6, each of the three virally encoded mRNAs is translated from two initiating AUGs, and therefore, the TTV genome generates at least six proteins. Localization studies of hemagglutinin-tagged versions of these proteins in fixed cells, and green fluorescent protein-tagged versions of these proteins in living cells, expressed following transfection, demonstrated that two were primarily nuclear, two were primarily cytoplasmic, and two were found throughout the cell.
Replication-Defective Adenovirus Serotype 5 Vectors Elicit Durable Cellular and Humoral Immune Responses in Nonhuman Primates
Authors: Santra Sampa , Seaman Michael S. , Xu Ling , Barouch Dan H. , Lord Carol I. , Lifton Michelle A. , Gorgone Darci A. , Beaudry Kristin R. , Svehla Krisha , Welcher Brent , Chakrabarti Bimal K. , Huang Yue , Yang Zhi-Yong , Mascola John R. , Nabel Gary J. , Letvin Norman L.
Published: April 2005

The magnitude and durability of immune responses induced by replication-defective adenovirus serotype 5 (ADV5) vector-based vaccines were evaluated in the simian-human immunodeficiency virus/rhesus monkey model. A single inoculation of recombinant ADV5 vector constructs induced cellular and humoral immunity, but the rapid generation of neutralizing anti-Ad5 antibodies limited the immunity induced by repeated vector administration. The magnitude and durability of the immune responses elicited by these vaccines were greater when they were delivered as boosting immunogens in plasmid DNA-primed monkeys than when they were used as single-modality immunogens. Therefore, administration of ADV5-based vectors in DNA-primed subjects may be a preferred use of this vaccine modality for generating long-term immune protection.
Selection for Human Immunodeficiency Virus Type 1 Envelope Glycosylation Variants with Shorter V1-V2 Loop Sequences Occurs during Transmission of Certain Genetic Subtypes and May Impact Viral RNA Levels
Authors: Chohan Bhavna , Lang Dorothy , Sagar Manish , Korber Bette , Lavreys Ludo , Richardson Barbra , Overbaugh Julie
Published: April 2005

Designing an effective human immunodeficiency virus type 1 (HIV-1) vaccine will rely on understanding which variants, from among the myriad of circulating HIV-1 strains, are most commonly transmitted and determining whether such variants have an Achilles heel. Here we show that heterosexually acquired subtype A HIV-1 envelopes have signature sequences that include shorter V1-V2 loop sequences and fewer predicted N-linked glycosylation sites relative to the overall population of circulating variants. In contrast, recently transmitted subtype B variants did not, and this was true for cases where the major risk factor was homosexual contact, as well as for cases where it was heterosexual contact. This suggests that selection during HIV-1 transmission may vary depending on the infecting subtype. There was evidence from 23 subtype A-infected women for whom there was longitudinal data that those who were infected with viruses with fewer potential N-linked glycosylation sites in V1-V2 had lower viral set point levels. Thus, our study also suggests that the extent of glycosylation in the infecting virus could impact disease progression.
Structural Elements of the tRNA TΨC Loop Critical for Nucleocytoplasmic Transport Are Important for Human Immunodeficiency Virus Type 1 Primer Selection
Authors: Kelly Nathan J. , Morrow Casey D.
Published: April 2005

Human immunodeficiency virus type 1 (HIV-1) selects a host cell tRNA as the primer for the initiation of reverse transcription. In a previous study, transport of the intact tRNA from the nucleus to the cytoplasm during tRNA biogenesis was shown to be a requirement for the selection of the tRNA primer by HIV-1. To further examine the importance of tRNA structure for transport and the selection of the primer, yeast tRNA
Phe
mutants were designed such that the native tRNA structure would be disrupted to various extents. The capacity of the mutant tRNA
Phe
to complement a defective HIV-1 provirus that relies on the expression of yeast tRNA
Phe
for infectivity was determined. We found a direct relationship between intact tRNA conformation and the capacity to be selected by HIV-1 for use in reverse transcription. tRNA
Phe
mutants that retained the capacity for nucleocytoplasmic transport, indicative of overall intact conformation, complemented the defective provirus. The mutant tRNAs were not aminoacylated, and the levels of complementation were lower than that for wild-type tRNA
Phe
, which did undergo transport and aminoacylation. Taken together, these results demonstrate that HIV-1 primer selection is most dependent on a tRNA structure necessary for nucleocytoplasmic transport, consistent with primer selection occurring in the cytoplasm at or near the site of protein synthesis.
Identification of a Peach Latent Mosaic Viroid Hairpin Able To Act as a Dicer-Like Substrate
Authors: Landry Patricia , Perreault Jean-Pierre
Published: April 2005

The ability of several viroids to induce posttranscriptional gene silencing has been demonstrated; however, the structure recognized by the Dicer enzyme(s) responsible for the initiation of this mechanism remains a mystery. Here, we show that the hairpin known to be implicated in the replication of peach latent mosaic viroid has the ability to trigger the Dicer enzyme(s). This domain, which is composed of a succession of several small stems separated by symmetrical bulges, is reminiscent of the precursor micro-RNAs.
Melanocytic dysplastic naevi occupy the middle ground between benign melanocytic naevi and cutaneous malignant melanomas: emerging clues
Authors: Hussein M R
Published: April 2005

Although several studies have confirmed the aetiological importance of melanocytic dysplastic naevi (MDN) in the development of cutaneous malignant melanoma (CMM), the analysis of these lesions was directed mostly towards the study of melanomas. The underlying reasons include the relatively large size of CMMs, their direct lethal outcome, and the feasibility of establishing melanoma cell lines. In contrast, because of their relatively small size, questionable malignant potential, and the difficulty in establishing in vitro cultures, MDN have been studied less extensively. Hypothetically, transformed melanocytes can give rise to any lesion in the hierarchy of melanocytic tumours. Based on this hypothetical perspective, and on the epidemiological, morphological, immunohistochemical, and genetic characteristics of MDN, it is not surprising that these lesions occupy an intermediate position in the hierarchy of melanocytic lesions, and may be precursors of CMM. Although this argument appears to be straightforward, it is still controversial. This review explores the components of this argument and provides supporting evidence for this hypothesis.
Cyclooxygenase 2 expression in nasopharyngeal carcinoma: immunohistochemical findings and potential implications
Authors: Tan K-B , Putti T C
Published: April 2005

Background:Cyclooxygenase 2 (COX-2), an inducible prostaglandin synthase, participates in inflammatory and neoplastic processes. It is expressed by various tumours and contributes to carcinogenesis. Notably, COX-2 inhibitors appear to have tumour suppressor effects and are being evaluated in clinical trials.

Aims:To investigate COX-2 expression in nasopharyngeal carcinoma (NPC), a common tumour in parts of Asia, and to discuss potential implications.

Methods:Eighty five cases of NPC were reviewed. COX-2 immunohistochemistry and semiquantitative assessment of expression in nasopharyngeal biopsies were performed. Because COX-2 is proangiogenic, tumour microvessel density was also assessed with the use of CD31 immunohistochemistry.

Results:Histologically, 78 NPCs were undifferentiated, six were non-keratinising, and one was keratinising. Thirty nine NPCs had adjacent dysplastic epithelium. COX-2 expression was noted in 60 NPCs, 14 of 39 samples of dysplastic epithelium, and only one of 25 samples of normal epithelium (p < 0.01). Microvessel density was not significantly different between COX-2 positive and COX-2 negative tumours (p  =  0.774). Tumour COX-2 positivity was not associated with higher tumour stage (p  =  0.423).

Conclusion:COX-2 expression is more frequently seen as nasopharyngeal epithelium progresses from normal to dysplastic to carcinoma. This suggests that COX-2 contributes to the multistep process of NPC carcinogenesis. COX-2 represents a therapeutic target for COX-2 inhibitors, and there is thus a basis for the further investigation of this adjuvant treatment modality for NPC. COX-2 inhibitors are known to potentiate the antitumour effects of radiotherapy, which is the primary treatment for NPC.
High expression of Mcl-1 in ALK positive and negative anaplastic large cell lymphoma
Authors: Rust R , Harms G , Blokzijl T , Boot M , Diepstra A , Kluiver J , Visser L , Peh S-C , Lim M , Kamps W A , Poppema S , van den Berg A
Published: April 2005

Aim:To gain more insight into the genes involved in the aetiology and pathogenesis of anaplastic large cell lymphoma (ALCL).

Methods:Serial analysis of gene expression (SAGE) was undertaken on the CD4+ALK+ (anaplastic lymphoma kinase positive) ALCL derived cell line Karpas299 and as comparison on CD4+ T cells. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed on five ALCL derived cell lines and 32 tissue samples to confirm the SAGE data.

Results:High expression of Mcl-1 was seen in the Karpas299 cell line, whereas the two other antiapoptotic Bcl-2 family members, Bcl-2 and Bcl-XL, were not detected in the SAGE library. Quantitative RT-PCR confirmed the high expression of Mcl-1 mRNA and low expression of Bcl-2 and Bcl-XLin Karpas299 and in four other ALCL cell lines. To expand on these initial observations, primary tissue samples were analysed for Mcl-1, Bcl-XL, and Bcl-2 by immunohistochemistry. All 23 ALK+ and nine ALK− ALCL cases were positive for Mcl-1. Bcl-2 and Bcl-XLwere expressed infrequently in ALK+ ALCL cases, but were present in a higher proportion of ALK− ALCL cases.

Conclusion:The consistent high expression of Mcl-1 in ALK+ and ALK− ALCL suggests that Mcl-1 is the main antiapoptotic protein in this disease. The high frequency of Mcl-1, Bcl-2, and Bcl-XLpositive ALCL cases in the ALK− group compared with the ALK+ group indicates that ALK induced STAT3 activation is not the main regulatory pathway in ALCL.
Sections of the nipple and quadrants in mastectomy specimens for carcinoma are of limited value: Table 1
Authors: Sikand K , Lee A H S , Pinder S E , Elston C W , Ellis I O
Published: April 2005

Aim:To assess the value of nipple and quadrant sections in mastectomy specimens for carcinoma in detecting Paget’s disease and multifocal carcinoma.

Methods:Two hundred and forty eight consecutive mastectomies performed for carcinoma were reviewed. The presence of Paget’s disease of the nipple and mode of identification of any multifocal carcinoma was recorded.

Results:Nipple sections showed Paget’s disease in eight specimens: in five the diagnosis had been made on previous biopsy and in three (1%) this was a new diagnosis. In the 220 specimens in which all four quadrants were sampled, multifocal disease was identified more often in specimens with invasive carcinoma (39 of 186; 21%) than in those with only ductal carcinoma in situ (0 of 34). In specimens with invasive carcinoma, multifocality was identified macroscopically in 20: on microscopy of tumour sections in four, on microscopic examination of quadrant sections in 11, in the nipple in three, and in both quadrant and nipple sections in one. Overall, multifocality was found on microscopic examination of quadrant or nipple sections in 15 of 220 specimens (7%).

Conclusions:The low frequency of detection of multifocality or Paget’s disease in nipple and quadrant sections from mastectomy specimens, combined with the fact that such findings do not affect patient management, suggest that nipple and quadrant sections should only be taken if resources permit.
Synthesis of tungsten complexes that contain hexaisopropylterphenyl-substituted triamidoamine ligands, and reactions relevant to the reduction of dinitrogen to ammonia
Authors: Yandulov Dmitry V , Schrock Richard R
Published: April 2005

[HIPTN3N]WCl (WCl) can be synthesized readily by adding H3[HIPTN3N] to WCl4(DME) followed by LiN(SiMe3)2 ([HIPTN3N]3 = [(HIPTNCH2CH2)3N]3 where HIPT = 3,5-(2,4,6-i-Pr3C6H2)2C6H3 = HexaIsoPropylTerphenyl). Reduction of WCl with KC8 in benzene under N2 yields WN=NK. WN=NK is readily oxidized in THF by ZnCl2 to yield zinc metal and WN2. Reduction of WN2 to [WN2] is reversible at 2.27 V vs. FeCp2+/0 in 0.1 mol/L [Bu4N][BAr′4]/PhF electrolyte (Ar′ = 3,5-(CF3)2C6H3), while oxidation of WN2 to [WN2]+ is also reversible at 0.66 V. Protonation of WN=NK by [Et3NH][OTf] in benzene yields WN=NH essentially quantitatively. Protonation of WN=NH at Nβ with [H(OEt)2][BAr′4] in ether affords [W=NNH2][BAr′4] quantitatively. Electrochemical reduction of [W=NNH2][BAr′4] in 0.1 mol/L [Bu4N][BAr′4]/PhF is irreversible at scan rates of up to 1 V/s. Addition of NaBAr′4 and NH3 to WCl in PhF yields [W(NH3)][BAr′4]. Electrochemical reduction of [W(NH3)][BAr′4] in 0.1 mol/L [Bu4N][BAr′4]/PhF is irreversible at 2.06 V vs. FeCp2+/0 at a scan rate of 0.5 V/s. Treatment of [W(NH3)][BAr′4] with triethylamine and [FeCp2][PF6] in C6D6, followed by LiN(SiMe3)2, yielded W≡N. Treatment of [W(NH3)][BAr′4] with LiBHEt3 (1 mol/L in THF) results in formation of WH, which is converted to WH3 upon exposure to an atmosphere of H2. Attempts to prepare WN=NH by treating WN2 with [2,6-LutH][BAr′4] and CoCp2 yielded only [W=NNH2][BAr′4]. [W=NNH2][BAr′4] is reduced to W=NNH2 by CoCp*2, but this species disproportionates to yield WN=NH, W≡N, and ammonia. Reduction of [W(NH3)][BAr′4] with CoCp*2 does not yield any observable W(NH3). Attempted catalytic reduction of dinitrogen using WN2 as the catalyst under conditions identical or similar to those employed for catalytic reduction of dinitrogen by MoN2 and related Mo complexes failed. Single crystal X-ray studies were carried out on W-N=NK, WN2, W-N=NH, [W=NNH2][BAr′4], and [W(NH3)][BAr′4].Key words: dinitrogen, reduction, tungsten, ammonia.