bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Membrane Fusion by Murine Leukemia Viruses Is Controlled in
cis
or in
trans
by Interactions between the Receptor-Binding Domain and a Conserved Disulfide Loop of the Carboxy Terminus of the Surface Glycoprotein
Authors: Lavillette Dimitri , Boson Bertrand , Russell Stephen J. , Cosset François-Loı̈c
Published: July 2002

Cell entry of retroviruses is initiated by the recognition of cellular receptors and the subsequent membrane fusion between viral and cellular membranes. These two steps are mediated by the surface (SU) and transmembrane (TM) subunits of the retroviral envelope glycoprotein (Env), respectively. Determinants regulating membrane fusion have been described throughout SU and TM, but the processes coupling receptor recognition to fusion are still elusive. Here we establish that a critical interaction is formed between the receptor-binding domain (RBD) and the major disulfide loop of the carboxy-terminal domain (C domain) of the murine leukemia virus SU. Receptor binding causes an alteration of this interaction and, in turn, promotes further events of Env fusion activation. We characterize mutations which, by lowering this interaction and reducing the compatibility between the RBD and C domains of Env glycoprotein chimeras, affect both Env fusogenicity and sensitivity to receptor interference. Additionally, we demonstrate that suboptimal interactions in such mutant Env proteins can be compensated in
trans
by soluble RBDs in a manner that depends on their compatibility with the C domain. Our results therefore indicate that RBD/C domain interactions may occur in
cis
, via the proper RBD of the viral Env itself, or in
trans
, via a distinct RBD expressed by virion-free Env glycoproteins expressed endogenously by the infected cells or provided by neighboring Env trimers.
Requirements for Assembly of Poliovirus Replication Complexes and Negative-Strand RNA Synthesis
Authors: Teterina Natalya L. , Egger Denise , Bienz Kurt , Brown David M. , Semler Bert L. , Ehrenfeld Ellie
Published: July 2002

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5′ noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5′-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3D
pol
coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5′-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3′ end.
In Vivo Accumulation of Cyclin A and Cellular Replication Factors in Autonomous Parvovirus Minute Virus of Mice-Associated Replication Bodies
Authors: Bashir Tarig , Rommelaere Jean , Cziepluch Celina
Published: July 2002

Autonomous parvovirus minute virus of mice (MVM) DNA replication is strictly dependent on cellular factors expressed during the S phase of the cell cycle. Here we report that MVM DNA replication proceeds in specific nuclear structures termed autonomous parvovirus-associated replication bodies, where components of the basic cellular replication machinery accumulate. The presence of DNA polymerases α and δ in these bodies suggests that MVM utilizes partially preformed cellular replication complexes for its replication. The recruitment of cyclin A points to a role for this cell cycle factor in MVM DNA replication beyond its involvement in activating the conversion of virion single-stranded DNA to the duplex replicative form.
Infection of Cells with Human Cytomegalovirus during S Phase Results in a Blockade to Immediate-Early Gene Expression That Can Be Overcome by Inhibition of the Proteasome
Authors: Fortunato Elizabeth A. , Sanchez Veronica , Yen Judy Y. , Spector Deborah H.
Published: July 2002

Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G
0
using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE
+
in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE
+
cells that gradually accumulated within the S and G
2
/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU
+
cells were IE
−
and had progressed into G
2
/M or back to G
1
. The majority of the IE
+
cells in S and G
2
/M were BrdU
−
. Only a few cells were IE
+
BrdU
+
, and they resided in G
2
/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.
Preexisting Immunity to Adenovirus in Rhesus Monkeys Fails To Prevent Vector-Induced Toxicity
Authors: Varnavski Andrei N. , Zhang Yi , Schnell Michael , Tazelaar John , Louboutin Jean-Pierre , Yu Qian-Chun , Bagg Adam , Gao Guang-ping , Wilson James M.
Published: July 2002

In an earlier study we evaluated innate immune responses to a first-generation adenoviral vector infused into the portal vein of rhesus monkeys who had never been exposed to adenovirus previously. In these animals, the systemic administration of E1/E3-deleted adenoviral vectors resulted in immediate activation of innate immunity and serious toxicity caused by targeting of vector to antigen-presenting cells and systemic inflammation. We analyze here how these responses are affected by vector-specific preexisting immunity that was induced by intramuscular immunization 6 months prior to evaluation. Our results show that preexposure to the vector substantially diminishes the transgene expression in most tissues but has little effect on gene transfer. Significantly, preimmunization does not eliminate systemic vector-induced toxicity. These conclusions are based on the presence of clinical features of coagulopathy and elevated levels of proinflammatory cytokine interleukin-6 in the serum of animals treated with vector after intramuscular immunization. Furthermore, preexisting immunity appears to induce a vector-specific inhibitory effect on erythroid progenitor development in the bone marrow that is not found when naive animals are challenged with vector.
Virus-Like Particles of a Fish Nodavirus Display a Capsid Subunit Domain Organization Different from That of Insect Nodaviruses
Authors: Tang Liang , Lin Chan-Shing , Krishna Neel K. , Yeager Mark , Schneemann Anette , Johnson John E.
Published: July 2002

The structure of recombinant virus-like particles of malabaricus grouper nervous necrosis virus (MGNNV), a fish nodavirus isolated from the grouper
Epinephelus malabaricus
, was determined by electron cryomicroscopy (cryoEM) and three-dimensional reconstruction at 23-Å resolution. The cryoEM structure, sequence comparison, and protein fold recognition analysis indicate that the coat protein of MGNNV has two domains resembling those of tomato bushy stunt virus and Norwalk virus, rather than the expected single-domain coat protein of insect nodaviruses. The analysis implies that residues 83 to 216 fold as a β-sandwich which forms the inner shell of the T=3 capsid and residues 217 to 308 form the trimeric surface protrusions observed in the cryoEM map. The structural similarities between fish nodaviruses and members of the tombusvirus and calicivirus groups provide significant new data for understanding the evolution of the nodavirus family.
γδ T Cells Promote a Th1 Response during Coxsackievirus B3 Infection In Vivo: Role of Fas and Fas Ligand
Authors: Huber Sally , Shi Cuixia , Budd Ralph C.
Published: July 2002

Fas/Fas ligand (FasL) interactions regulate disease outcome in coxsackievirus B3 (CVB3)-induced myocarditis. MRL+/+mice infected with CVB3 develop severe myocarditis, a dominant CD4+Th1 (gamma interferon [IFN-γ+]) response to the virus, and a predominance of γδ T cells in the myocardial infiltrates. MRLlpr/lprand MRLgld/gldmice, which lack normal expression of Fas and express a mutated FasL, respectively, have minimal myocarditis and show a dominant CD4+Th2 (interleukin-4 [IL-4+]) phenotype to CVB3. Spleen cells from virus-infected wild-type,lpr, andgldanimals proliferate equally to virus in vitro. Adoptive transfer of γδ T cells from hearts of CVB3-infected MRL+/+mice (FasL+) into infected MRLgld/gldrecipients (FasL−/Fas+) restores both disease susceptibility and Th1 cell phenotype. However, transfer of these cells into MRLlpr/lprrecipients (FasL+/Fas−) did not promote myocarditis and the viral response remained Th2 biased. This paralleled the expression of very high surface levels of FasL by myocardial γδ T cells, as well as their propensity to selectively lyse Th2 virus-specific CD4+T cells. These results demonstrate that Fas/FasL interactions conferred by γδ Τ cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.
Vaccinia Virus G4L Glutaredoxin Is an Essential Intermediate of a Cytoplasmic Disulfide Bond Pathway Required for Virion Assembly
Authors: White Christine L. , Senkevich Tatiana G. , Moss Bernard
Published: July 2002

Our previous studies provided evidence that E10R, a vaccinia virus protein belonging to the ERV1/ALR family, has a redox function and is required for virion assembly. Repression of E10R prevented the formation of intramolecular disulfide bonds of the G4L glutaredoxin, the L1R membrane protein, and the structurally related F9L protein. Here, we demonstrate an oxidation pathway (E10R
SS
→ G4L
SS
→ L1R
SS
, F9L
SS
) in which G4L occupies an intermediate position. By reacting free thiols with 4-acetamido-4′-malemideylstilbene-2,2′-disulfonic acid, alkylated and nonalkylated disulfide-bonded forms of G4L could be resolved from each other by polyacrylamide gel electrophoresis. The cysteines of intracellular G4L were in both disulfide and reduced forms, whereas those of E10R, L1R, and F9L and virion-associated G4L were mostly disulfide bonded. Repression of G4L expression prevented the formation of disulfide bonds in both L1R and F9L but not E10R. Both cysteines of G4L were required for L1R and F9L disulfide bond formation or for
trans
-complementation of virus infectivity when G4L expression was repressed. No role in the E10R-G4L redox pathway was found for O2L, a nonessential glutaredoxin encoded by vaccinia virus. We suggest that cytoplasmic G4L is a redox shuttle between membrane-associated E10R and L1R or F9L.
Paired Charge-to-Alanine Mutagenesis of Dengue Virus Type 4 NS5 Generates Mutants with Temperature-Sensitive, Host Range, and Mouse Attenuation Phenotypes
Authors: Hanley Kathryn A. , Lee Jay J. , Blaney Joseph E. , Murphy Brian R. , Whitehead Stephen S.
Published: July 2002

Charge-to-alanine mutagenesis of dengue virus type 4 (DEN4) NS5 gene generated a collection of attenuating mutations for potential use in a recombinant live attenuated DEN vaccine. Codons for 80 contiguous pairs of charged amino acids in NS5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant DEN4 cDNA constructs. These mutant viruses were tested for temperature-sensitive (ts) replication in both Vero cells and HuH-7 human hepatoma cells. Of the 32 mutants, 13 were temperature sensitive (ts) in both cell lines, 11 were not ts in either cell line, and 8 exhibited a host range (
tshr
) phenotype. One
tshr
mutant was ts only in Vero cells, and seven were ts only in HuH-7 cells. Nineteen of the 32 mutants were 10-fold or more restricted in replication in the brains of suckling mice compared to that of wild-type DEN4, and three mutants were approximately 10,000-fold restricted in replication. The level of temperature sensitivity of replication in vitro did not correlate with attenuation in vivo. A virus bearing two pairs of charge-to-alanine mutations was constructed and demonstrated increased temperature sensitivity and attenuation relative to either parent virus. This large set of charge-to-alanine mutations specifying a wide range of attenuation for mouse brain should prove useful in fine-tuning recombinant live attenuated DEN vaccines.
Human Papillomavirus Type 16 E7 Maintains Elevated Levels of the cdc25A Tyrosine Phosphatase during Deregulation of Cell Cycle Arrest
Authors: Nguyen Don X. , Westbrook Thomas F. , McCance Dennis J.
Published: July 2002

Essential to the oncogenic properties of human papillomavirus type 16 (HPV-16) are the activities encoded by the early gene product E7. HPV-16 E7 (E7.16) binds to cellular factors involved in cell cycle regulation and differentiation. These include the retinoblastoma tumor suppressor protein (Rb) and histone deacetylase (HDAC) complexes. While the biological significance of these interactions remains unclear, E7 is believed to help maintain cells in a proliferative state, thus establishing an environment that is conducive to viral replication. Most pathways that govern cell growth converge on downstream effectors. Among these is the cdc25A tyrosine phosphatase. cdc25A is required for G
1
/S transition, and its deregulation is associated with carcinogenesis. Considering the importance of cdc25A in cell cycle progression, it represents a relevant target for viral oncoproteins. Accordingly, the present study focuses on the putative deregulation of cdc25A by E7.16. Our results indicate that E7.16 can impede growth arrest induced during serum starvation and keratinocyte differentiation. Importantly, these E7-specific phenotypes correlate with elevated cdc25A steady-state levels. Reporter assays performed with NIH 3T3 cell lines and human keratinocytes indicate that E7 can transactivate the
cdc25A
promoter. In addition, transcriptional activation by E7.16 requires the distal E2F site within the
cdc25A
promoter. We further demonstrate that the ability of E7 to abrogate cell cycle arrest, activate
cdc25A
transcription, and increase cdc25A protein levels requires intact Rb and HDAC-1 binding domains. Finally, by using the cdk inhibitor roscovitine, we reveal that E7 activates the
cdc25A
promoter independently of cell cycle progression and cdk activity. Consequently, we propose that E7.16 can directly target
cdc25A
transcription and maintains
cdc25A
gene expression by disrupting Rb/E2F/HDAC-1 repressor complexes.
Intracellular Distribution of Human T-Cell Leukemia Virus Type 1 Gag Proteins Is Independent of Interaction with Intracellular Membranes
Authors: Le Blanc Isabelle , Blot Vincent , Bouchaert Isabelle , Salamero Jean , Goud Bruno , Rosenberg Arielle R. , Dokhélar Marie-Christine
Published: July 2002

Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system.
CDP Binding to Multiple Sites in the Mouse Mammary Tumor Virus Long Terminal Repeat Suppresses Basal and Glucocorticoid-Induced Transcription
Authors: Zhu Quan , Dudley Jaquelin P.
Published: July 2002

Mouse mammary tumor virus (MMTV) is transcribed at high levels in the lactating mammary gland to ensure transmission of virus from the milk of infected female mice to susceptible offspring. We previously have shown that the transcription factor CCAAT displacement protein (CDP) is expressed in high amounts in virgin mammary gland, yet DNA-binding activity for the MMTV long terminal repeat (LTR) disappears as mammary tissue differentiates during lactation. CDP is a repressor of MMTV expression and, therefore, MMTV expression is suppressed during early mammary gland development. In this study, we have shown using DNase I footprinting and electrophoretic mobility shift assays that there are at least five CDP-binding sites in the MMTV LTR upstream of those previously described in the promoter-proximal negative regulatory element (NRE). Single mutations in two of these upstream sites (+691 or +692 and +735 relative to the first base of the LTR) reduced CDP binding to the cognate sites and elevated reporter gene expression from the full-length MMTV LTR. Combination of a mutation in the promoter-distal NRE with a mutation in the proximal NRE gave approximately additive increases in LTR-reporter gene activity, suggesting that these binding sites act independently. Mutations in several different CDP-binding sites allowed elevation of reporter gene activity from the MMTV promoter in the absence and presence of glucocorticoids, hormones that contribute to high levels of MMTV transcription during lactation by activation of hormone receptor binding to the LTR. In addition, overexpression of CDP in transient-transfection assays suppressed both basal and glucocorticoid-induced LTR-mediated transcription in a dose-dependent manner. These data suggest that multiple CDP-binding sites contribute independently to regulate binding of positive factors, including glucocorticoid receptor, to the MMTV LTR during mammary gland development.
Role of an Intact Splenic Microarchitecture in Early Lymphocytic Choriomeningitis Virus Production
Authors: Müller Stefan , Hunziker Lukas , Enzler Susanne , Bühler-Jungo Myriam , Di Santo James P. , Zinkernagel Rolf M. , Mueller C.
Published: July 2002

An acute infection with lymphocytic choriomeningitis virus (LCMV) is efficiently controlled by the cytotoxic-T-cell (CTL) response of the host, and LCMV titers in the spleen and peripheral solid organs usually fall sharply after day 4 to 6 postinfection. Surprisingly, infection of immunodeficient recombination-activating gene 2-deficient (RAG2−/−) mice with 5 × 102PFU of LCMV-WE causes about 80-fold-lower LCMV titers in the spleen on day 4 postinfection compared with C57BL/6 control mice. This could not be attributed to NK cell activity, since common gamma-chain-deficient RAG2−/−mice lacking NK cells show low LCMV titers comparable to those for RAG2−/−mice. Furthermore, the reduced early LCMV production in spleens could not be explained by an enhanced gamma interferon production in RAG2−/−mice. Analysis of mutant mice exhibiting various defects in the splenic microarchitecture, including (i) tumor necrosis factor alpha-negative (TNF-α−/−), lymphotoxin alpha-negative (LTα−/−), B-cell-deficient μMT mice, (ii) immunoglobulin M-negative mice, and (iii) RAG−/−mice reconstituted with wild-type versus TNF-α−/−LTα−/−B cells, revealed a clear correlation between an intact splenic marginal zone, rapid early replication of LCMV in the spleen, and efficient CTL induction. These results suggest that by the preferential infection of the highly organized splenic microarchitecture, LCMV seems to successfully exploit one of the key elements in the chain of the adaptive immune system. Not only does the early tropism of LCMV for the splenic marginal zone trigger a potent immune response, but at the same time the marginal zone may also become a target of early CTL-mediated immunopathology that impairs immune responsiveness.
The Fusion Peptide of Semliki Forest Virus Associates with Sterol-Rich Membrane Domains
Authors: Ahn Anna , Gibbons Don L. , Kielian Margaret
Published: July 2002

Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl β-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.
The Tat Protein of Human Immunodeficiency Virus Type 1 (HIV-1) Can Promote Placement of tRNA Primer onto Viral RNA and Suppress Later DNA Polymerization in HIV-1 Reverse Transcription
Authors: Kameoka Masanori , Morgan Max , Binette Marc , Russell Rodney S. , Rong Liwei , Guo Xiaofeng , Mouland Andrew , Kleiman Lawrence , Liang Chen , Wainberg Mark A.
Published: July 2002

Human immunodeficiency virus type-1 Tat has been proposed to play a role in the regulation of reverse transcription. We previously demonstrated that wild-type Tat can augment viral infectivity by suppressing the reverse transcriptase (RT) reaction at late stages of the viral life cycle in order to prevent the premature synthesis of potentially deleterious viral DNA products. Here we have performed a detailed analysis of the cell-free reverse transcription reaction to elucidate the mechanism(s) whereby Tat can affect this process. Our results show that Tat can suppress nonspecific DNA elongation while moderately affecting the specific initiation stage of reverse transcription. In addition, Tat has an RNA-annealing activity and can promote the placement of tRNA onto viral RNA. This points to a functional homology between Tat and the viral nucleocapsid (NC) protein that is known to be directly involved in this process. Experiments using a series of mutant Tat proteins revealed that the cysteine-rich and core domains of Tat are responsible for suppression of DNA elongation, while each of the cysteine-rich, core, and basic domains, as well as a glutamine-rich region in the C-terminal domain, are important for the placement of tRNA onto the viral RNA genome. These results suggest that Tat can play at least two different roles in the RT reaction, i.e., suppression of DNA polymerization and placement of tRNA onto viral RNA. We believe that the first of these activities of Tat may contribute to the overall efficiency of reverse transcription of the viral genome during a new round of infection as well as to enhanced production of infectious viral particles. We hypothesize that the second activity, illustrating functional homology between Tat and NC, suggests a potential role for NC in the displacement of Tat during viral maturation.
Selection of RNA Aptamers That Are Specific and High-Affinity Ligands of the Hepatitis C Virus RNA-Dependent RNA Polymerase
Authors: Biroccio Antonino , Hamm Jörg , Incitti Ilario , De Francesco Raffaele , Tomei Licia
Published: July 2002

In order to find small RNA molecules that are specific and high-affinity ligands of nonstructural 5B (NS5B) polymerase, we screened by SELEX (systematic evolution of ligands by exponential amplification) a structurally constrained RNA library with an NS5BΔC55 enzyme carrying a C-terminal biotinylation sequence. Among the selected clones, two aptamers appeared to be high-affinity ligands of NS5B, with apparent dissociation constants in the low nanomolar range. They share a sequence that can assume a stem-loop structure. By mutation analysis, this structure has been shown to correspond to the RNA motif responsible for the tight interaction with NS5B. The aptamers appeared to be highly specific for the hepatitis C virus (HCV) polymerase since interaction with the GB virus B (GBV-B) NS5B protein cannot be observed. This is consistent with the observation that the activity of the HCV NS5B polymerase is efficiently inhibited by the selected aptamers, while neither GBV-B nor poliovirus 3D polymerases are affected. The mechanism of inhibition of the NS5B activity turned out to be noncompetitive with respect to template RNA, suggesting that aptamers and template RNA do not bind to the same site. As a matter of fact, mutations introduced in a basic exposed surface of the thumb domain severely impaired both the binding of and activity inhibition by the RNA aptamers.
Variability in the Human Immunodeficiency Virus Type 1 gp120 Env Protein Linked to Phenotype-Associated Changes in the V3 Loop
Authors: Hoffman Noah G. , Seillier-Moiseiwitsch Francoise , Ahn JaeHyung , Walker Jason M. , Swanstrom Ronald
Published: July 2002

Isolates of human immunodeficiency virus type 1 (HIV-1) are classified according to the chemokine receptor (coreceptor) used in conjunction with CD4 to target and enter cells: viruses using CCR5 and CXCR4 are classified as R5 and X4, respectively. The major determinant of entry-related HIV-1 phenotypes is known to reside in the third variable region of gp120 (V3). It is clear, however, that positions outside of V3 play some role in influencing phenotype, although marked context dependence and extensive variability among HIV-1 isolates have made the identification of these positions difficult. We used the presence of previously described substitutions in V3 to classify a large set of HIV-1 subtype B gp120 sequences available in public databases as X4-like or R5-like. Using these classifications, we searched for positions outside of V3 where either amino acid composition or variability differed significantly among sequences of different inferred phenotypes. Our approach took the epidemiological relationships among sequences into account. A cluster of positions linked to changes in V3 was identified between amino acids 190 and 204 of gp120, immediately C-terminal of V2; changes at position 440 in C4 were also linked to inferred phenotype. Structural data place these positions at the coreceptor-binding face of gp120 in a surface-exposed location. We also noted a significant increase in net positive charge in a highly variable region of V2. This study both confirms previous observations and predicts specific positions that contribute to a functional relationship between V3, V2, and C4.
Avian
bic
, a Gene Isolated from a Common Retroviral Site in Avian Leukosis Virus-Induced Lymphomas That Encodes a Noncoding RNA, Cooperates with c-
myc
in Lymphomagenesis and Erythroleukemogenesis
Authors: Tam Wayne , Hughes Stephen H. , Hayward William S. , Besmer Peter
Published: July 2002

bic
is a novel gene identified at a common retroviral integration site in avian leukosis virus-induced lymphomas and has been implicated as a collaborator with c-
myc
in B lymphomagenesis. It lacks an extensive open reading frame and is believed to function as an untranslated RNA (W. Tam, Gene 274:157-167, 2001; W. Tam, D. Ben-Yehuda, and W. S. Hayward, Mol. Cell. Biol. 17:1490-1502, 1997). The oncogenic potential of
bic
, particularly its ability to cooperate with c-
myc
in oncogenesis, was tested directly by expressing c-
myc
and
bic
, either singly or in pairwise combination, in cultured chicken embryo fibroblasts (CEFs) and in chickens using replication-competent retrovirus vectors. Coexpression of c-
myc
and
bic
in CEFs caused growth enhancement of cells. Most importantly, chick oncogenicity assays demonstrated that
bic
can cooperate with c-
myc
in lymphomagenesis and erythroleukemogenesis. The present study provides direct evidence for the involvement of untranslated RNAs in oncogenesis and provides further support for the role of noncoding RNAs as riboregulators.
Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction
Authors: Cuconati Andrea , Degenhardt Kurt , Sundararajan Ramya , Anschel Alan , White Eileen
Published: July 2002

Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.
Stabilization of a Full-Length Infectious cDNA Clone of Transmissible Gastroenteritis Coronavirus by Insertion of an Intron
Authors: González José M. , Pénzes Zoltan , Almazán Fernando , Calvo Enrique , Enjuanes Luis
Published: July 2002

The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in
Escherichia coli
cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs.
Separation and Determination of Some Aromatic Sulfones by Reversed-Phase High-Performance Liquid Chromatography
Authors: Královský Josef , Kalhousová Marta , Šlosar Petr
Published: July 2002

The reversed-phase high-performance liquid chromatography of some selected, industrially important aromatic sulfones has been investigated. The chromatographic behaviour of three groups of aromatic sulfones has been studied. The optimum conditions of separation and UV spectra of the sulfones and some of their hydroxy and benzyloxy derivatives are presented. The dependences of capacity factors vs methanol content in mobile phase are mentioned. The results obtained have been applied to the quantitative analysis of different technical-grade samples and isomer mixtures. For all the separation methods mentioned the concentration ranges of linear calibration curves have been determined.
Thermal Cyclocondensation of Ethyl (1-Methyl-5- and 6-Benzimidazolyl/benzotriazolyl)aminomethylenepropanedioates
Authors: Milata Viktor , Ilavský Dušan , Goljer Igor , Zalibera Lubomír
Published: July 2002

Thermal cyclocondensation of starting ethyl (1-methyl-5- and 6-benzimidazolyl/benzotriazolyl)aminomethylenepropanedioates (Ia - Ie) in aprotic medium gives angularly condensed ethyl azolo[4,5-f]quinolonecarboxylates IIa, IIc, IIe and azolo[5,4-f]quinolonecarboxylates IIb, IId, respectively, which are hydrolyzed to the corresponding carboxylic acid III in acid medium. The structure of reaction products has been confirmed by 1H and 13C NMR, IR, and UV spectroscopy.
Improvement of Properties of Catalyst for Industrial Production of Acrolein from Propylene
Authors: Machek Jaroslav , Tichý Josef , Martinec Alexander , Švachula Jiří
Published: July 2002

The catalytic gas-phase oxidation of propylene on a multicomponent Mo-Co-Fe-Bi-K-O catalyst gives up to 85% yield of acrolein and acrylic acid along with undesirable side products formaldehyde and acetaldehyde. Effects of four factors have now been studied with the aim of suppressing of formation of the by-products viz. the effect of potassium content in the catalyst, that of final annealing temperature of the catalyst, and those of addition of other components (tungsten, nickel). It has been found that a change in potassium content in catalyst affects distinctly its activity and also selectivity for products of total oxidation of propylene but does not practically affect the yields of formaldehyde and acetaldehyde. The yields of the said side products decreased with increasing annealing temperature, however, with concomitant considerable decrease in the catalyst activity. Whereas a partial substitution of molybdenum atoms in the catalyst by tungsten atoms is accompanied by an increase in production of formaldehyde and acetaldehyde, some catalysts with nickel gave acrolein as the only aldehyde.
Prodrugs of Analogs of Nucleic Acid Components
Authors: Alexander Petr , Holý Antonín
Published: July 2002

This review regards various attitudes to the rational design of prodrugs derived of nucleoside and nucleotide analogues with pronounced biological (antiviral, anticancer) activity. Particular attention is focused on the recent development of prodrugs of approved therapeutic agents with the above structural association.
Radical Addition of Triethylamine and 1-Methylpiperidine to Tetrafluoroethene
Authors: Hampl František , Liška František
Published: July 2002

The radical addition of triethylamine (I) and 1-methylpiperidine (II) to tetrafluoroethene (III) initiated by UV and γ-60Co radiation produces tertiary amines containing 1,1,2,2-tetrafluoroethyl group at α-position (1 : 1 adducts) and/or at α,α'-positions (1 : 2 adducts). 1 : 2 Telomers, 1 : 3 adducts, and products formed by recombination of 1 : 1 adduct radicals represent minor products.
Two Crystalline Forms of Sesquiterpene Lactone Helenalin - X-Ray and NMR Study
Authors: Rychłewska Urszula , Buděšínský Miloš , Szczepanska Beata , Błoszyk Ełżbieta , Holub Miroslav
Published: July 2002

Helenalin, a naturally occurring sesquiterpene lactone of the pseudoguaianolide type, has been reported to exist in two polymorphic forms, both crystallizing in the monoclinic space group P21. In this paper we report the X-ray structure of the third room temperature polymorph, crystallizing in the orthorhombic space group P212121. The isolated molecules in all three polymorphs exhibit nearly the same molecular conformation, so the three crystalline modifications differ mainly in the mode the molecules pack in the solid state. Two main hydrogen bond acceptors, the oxygen atom of the cyclopentenone ring and the oxygen atom of the lactone carbonyl group, are competing in the hydrogen bond formation with the secondary hydroxyl group. Having an access to two polymorphs which differ in the hydrogen bond acceptors, we have performed the solid state 13C NMR measurements using a cross-polarization magic angle spinning technique. By comparison of the spectra taken in non-polar solvent with the spectra taken in the solid state we were able to demonstrate the shielding effects resulting from the intermolecular hydrogen bonding in crystals. The fast conformational equilibrium between two conformers TC(10) and TC(7) was suggested from NMR study of helenalin in solution.
Generalized Transient Leveque Problem
Authors: Wein Ondřej
Published: July 2002

Response of an electrodiffusion friction sensor to a finite step of the wall shear rate is studied by numerically solving the relevant mass-transfer problem. The resulting numerical data on transient currents are treated further to provide reasonably accurate analytical representations. Existing approximations to the general response operator are checked by using the obtained exact solution.
Calculation of the Primary Current Distribution in Cells with Curved Electrodes Using the Finite Difference, Conservative Scheme, and Finite Element Methods
Authors: Zoric Josip , Roušar Ivo
Published: July 2002

The primary current distribution was calculated in cells with a curvilinear shape of the electrodes by the finite difference (FDM), the conservative scheme (CS), and the finite element methods (FEM). These methods were used for the solutions of the Laplace equation (LE) for a 2D cross-section of a cell consisting of two concentric cylinders (tubes) as electrodes and the inter-electrode space filled with electrolyte. For this cell the analytical solution of LE is known. The local current density on the approximated shape of the electrodes was calculated. The error in the normalized local current density relative to the mean was 5.2%, 52% or 0.2% with FDM using a 64 o 64 mesh, CS using 64 o 64 mesh or FEM using 969 nodes, respectively. Also the boundary element method (BEM) has been used. With 199 elements at the electrode the error in the normalized current density was 0.2%. Taking into account the simplicity of programming and the possibility of using previously developed modules in other calculations, FEM and BEM showed the best performance.
Gas Chromatography and Mass Spectrometry of Isomeric Cyclohexylnonadecanes and 1-Cyclopentylicosane
Authors: Stránský Karel , Valterová Irena
Published: July 2002

Gas-chromatographic behaviour of 1-, 2-, 3-, 4-, 5-, 6-, 7-, and 10-cyclohexylnonadecanes (1-8) and 1-cyclopentylicosane (9) was studied. Kovats retention indices (I) on capillary fused silica columns DB-1 and DB-WAX were calculated and complete mass spectra of all the nine isomeric hydrocarbons are described.
Activities and Stabilities of Redox Molecular Sieve Catalysts in Liquid Phase Oxidations. A Review
Authors: Sheldon Roger A. , Arends Isabel W. C. E. , Lempers Hans E. B.
Published: July 2002

The use of redox molecular sieves as heterogeneous catalysts for liquid phase oxidations with H2O2 and alkyl hydroperoxides is reviewed. Emphasis is placed on titanium- and chromium-substituted molecular sieves as examples of catalysts operating via peroxometal and oxometal pathways, respectively. Various titanium-substituted molecular sieves were shown to be stable, heterogeneous catalysts for, e.g., olefin epoxidation, but the remarkable activity of the progenitor of this family of catalysts, titanium-silicalite (TS-1), has not been equalled. The catalytic activity of chromium-substituted molecular sieves was shown to be entirely due to small amounts of homogeneous chromium species leached from the catalyst during reaction. The catalytic activities and stabilities of other materials, including zirconium-substituted hexagonal mesoporous silicas and a vanadium bipyridyl complex encapsulated in zeolite Y, were also tested. They were either unstable (vanadium) towards leaching or showed low activity (zirconium). Attempts to incorporate molybdenum in silicalite were unsuccessful and previously reported activity was shown to be due to the blank reaction. A review with 64 references.
Chaperone-Like Activity of Human Haptoglobin: Similarity with α-Crystallin
Authors: Pavlíček Zdeněk , Ettrich Rüdiger
Published: July 2002

Human haptoglobin (Hp) has been shown to have chaperone-like activity in preventing thermally induced aggregation of catalase and γ-crystallin. No differences in the chaperone- like behaviour of genetic types Hp 1-1 and a mixture of types Hp 2-1 and Hp 2-2 (i.e. Hp II) were found. Haptoglobin not only suppresses heat-induced aggregation of proteins but also prevents γ-crystallin from aggregation by oxidative stress. In addition, haptoglobin also provides protection against glycation-induced inactivation of catalase by glyceraldehyde. Chaperone-like activity of haptoglobin decreases in the course of its glycation. Refolding studies have shown that Hp exhibits its chaperone-like activity predominantly on the unfolding and not on the refolding pathway. Although Hp and α-crystallin have no sequence similarities, it seems that their chaperone-like activities are of the same type.
Phosphorylation of Purine (Phosphonomethoxy)alkyl Derivatives by Mitochondrial AMP Kinase (AK2 Type) from L1210 Cells
Authors: Krejčová Romana , Horská Květoslava , Votruba Ivan , Holý Antonín
Published: July 2002

Substrate activity of purine (phosphonomethoxy)alkyl derivatives towards mitochondrial AMP kinase (AK2 type) from L1210 cells was studied. The native AMP kinase, purified nearly to homogeneity, is a monomer with molecular weight 26 kDa. The purified AMP kinase is specific for natural adenine nucleotides (AMP and dAMP) as phosphate acceptors but has a broad specificity to nucleoside 5'-triphosphates as phosphate donors. In addition to adenine acyclic nucleotide analogues, the enzyme is capable of phosphorylating also analogous derivatives containing 2,6-diaminopurine moiety. Kinetic data show that the substrate activity of these acyclic nucleoside phosphonates towards AK2 isoenzyme decreases in the order (S)-HPMPA > (R)-PMPA > PMEA > PMEDAP > (S)-PMPDAP > (R)-PMPDAP >> (S)-PMPA. Acyclic nucleotide analogues do not exhibit any inhibitory activity towards AK2 isoenzyme.
Identification and Characterization of Redox Sites in Supramolecular Systems and Their Relevance for the Design of Photoactive Devices. Ru(II)/C60-Based Donor-Acceptor Dyads
Authors: Carano Maurizio , Ceroni Paola , Maggini Michele , Marcaccio Massimo , Menna Enzo , Paolucci Francesco , Roffia Sergio , Scorrano Gianfranco
Published: July 2002

Two dyads consisting of a mononuclear or a dinuclear ruthenium complex covalently linked to a fullerenopyrrolidine through a rigid androstane spacer, have been prepared through azomethine ylide cycloaddition to C60. Electrochemical and photophysical studies revealed that ground-state electronic interactions between the dinuclear ruthenium chromophore and the fullerenopyrrolidine moiety are small. The redox series observed for the dyads correspond to the superimposition of the reduction patterns of the fullerene core and of the Ru(II)-bipyridine moieties. The results of the electrochemical investigation allow us to rationalize the photophysical behaviour of the two species by identifying the thermodynamically allowed and forbidden routes for the deactivation of the Ru-based metal-to-ligand charge transfer (MLCT) excited state.
Cytostatic 6-Arylpurine Nucleosides IV. Synthesis of 2-Substituted 6-Phenylpurine Ribonucleosides
Authors: Hocek Michal , Holý Antonín , Dvořáková Hana
Published: July 2002

A series of 2-X-substituted-6-phenyl-9-(β-D-ribofuranosyl)purines (X = Cl, Br, I, CH3, CF3and Ph) was prepared by halo-deaminations of protected 2-amino-6-phenylpurine ribonucleoside, by regioselective Suzuki-Miyaura reactions of 2,6-dihalopurines with phenylboronic acid or by cross-coupling reactions of the corresponding 2-halo-6-phenylpurines followed by deprotection. None of the title nucleosides exhibited any considerable cytostatic activity.
Hydrolytic Processing of Rice Husks in Aqueous Media: A Kinetic Assessment
Authors: Vila Carlos , Garrote Gil , Domínguez Herminia , Parajó Juan Carlos
Published: July 2002

The kinetics of hemicellulose decomposition in the non-isothermal rice husk autohydrolysis was experimentally assessed. Experimental data on the time course of the concentrations of hemicellulosic polymers (xylan, araban and acetyl groups) and their autohydrolysis products (including sugar oligomers, monosaccharides, furfural and acetic acid) were fitted to several kinetic models based on multiple pseudohomogeneous first-order reactions. Sugar oligomers were the major reaction products. The hydrolytic conversion of xylan, araban and acetyl groups was well interpreted by kinetic models based on consecutive and parallel reactions.
An evidence and consensus based guideline for acute diarrhoea management
Authors: Armon K , Stephenson T , MacFaul R , Eccleston P , Werneke U
Published: July 2002

OBJECTIVE

To develop an evidence and consensus based guideline for the management of the child who presents to hospital with diarrhoea (with or without vomiting), a common problem representing 16% of all paediatric medical attenders at an accident and emergency department. Clinical assessment, investigations (biochemistry and stool culture in particular), admission, and treatment are addressed. The guideline aims to aid junior doctors in recognising children who need admission for observation and treatment and those who may safely go home.

EVIDENCE

A systematic review of the literature was performed. Selected articles were appraised, graded, and synthesised qualitatively. Statements on recommendation were generated.

CONSENSUS

An anonymous, postal Delphi consensus process was used. A panel of 39 selected medical and nursing staff were asked to grade their agreement with the generated statements. They were sent the papers, appraisals, and literature review. On the second and third rounds they were asked to re-grade their agreement in the light of other panellists' responses. Consensus was predefined as 83% of panellists agreeing with the statement.

RECOMMENDATIONS

Clinical signs useful in assessment of level of dehydration were agreed. Admission to a paediatric facility is advised for children who show signs of dehydration. For those with mild to moderate dehydration, estimated deficit is replaced over four hours with oral rehydration solution (glucose based, 200–250 mOsm/l) given “little and often”. A nasogastric tube should be used if fluid is refused and normal feeds started following rehydration. Children at high risk of dehydration should be observed to ensure at least maintenance fluid is tolerated. Management of more severe dehydration is detailed. Antidiarrhoeal medication is not indicated.

VALIDATION

The guideline has been successfully implemented and evaluated in a paediatric accident and emergency department.