bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Analysis of a Novel Heparin-Binding Glycoprotein Encoded by Human Herpesvirus 7
Authors: Skrincosky David , Hocknell Peter , Whetter Linda , Secchiero Paola , Chandran Bala , Dewhurst Stephen
Published: July 2002

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related betaherpesviruses that encode a number of genes with no known counterparts in other herpesviruses. The product of one such gene is the HHV-6 glycoprotein gp82-105, which is a major virion component and a target for neutralizing antibodies. A 1.7-kb cDNA clone from HHV-7 was identified which contains a large open reading frame capable of encoding a predicted primary translational product of 468 amino acids (54 kDa) with 13 cysteine residues and 9 potential N-linked glycosylation sites. This putative protein, which we have termed gp65, was homologous to HHV-6 gp105 (30% identity) and contained a single potential membrane-spanning domain located near its amino terminus. Comparison of the cDNA sequence with that of the viral genome revealed that the gene encoding gp65 contains eight exons, spanning almost 6 kb of the viral genome at the right (3′) end of the HHV-7 genome. Northern (RNA) blot analysis with poly(A)+RNA from HHV-7-infected cells revealed that the cDNA insert hybridized to a single major RNA species of 1.7 kb. Antiserum raised against a purified, recombinant form of gp65 recognized a protein of roughly 65 kDa in sucrose density gradient-purified HHV-7 preparations; treatment with PNGase F reduced this glycoprotein to a putative precursor of approximately 50 kDa. Gp65-specific antiserum also neutralized the infectivity of HHV-7, while matched preimmune serum did not do so. Finally, analysis of the biochemical properties of recombinant gp65 revealed a specific interaction with heparin and heparan sulfate proteoglycans and not with closely related molecules such asN-acetylheparin and de-N-sulfated heparin. At least two domains of the protein were found to contribute to heparin binding. Taken together, these findings suggest that HHV-7 gp65 may contribute to viral attachment to cell surface proteoglycans.
Directional Transneuronal Infection by Pseudorabies Virus Is Dependent on an Acidic Internalization Motif in the Us9 Cytoplasmic Tail
Authors: Brideau Amy D. , Eldridge Marlies G. , Enquist Lynn W.
Published: July 2002

The Us9 gene is conserved among most alphaherpesviruses. In pseudorabies virus (PRV), the Us9 protein is a 98-amino-acid, type II membrane protein found in the virion envelope. It localizes to the
trans
-Golgi network (TGN) region in infected and transfected cells and is maintained in this compartment by endocytosis from the plasma membrane. Viruses with Us9 deleted have no observable defects in tissue culture yet have reduced virulence and restricted spread to retinorecipient neurons in the rodent brain. In this report, we demonstrate that Us9-promoted transneuronal spread in vivo is dependent on a conserved acidic motif previously shown to be essential for the maintenance of Us9 in the TGN region and recycling from the plasma membrane. Mutant viruses with the acidic motif deleted have an anterograde spread defect indistinguishable from that of Us9 null viruses. Transneuronal spread, however, is not dependent on a dileucine endocytosis motif in the Us9 cytoplasmic tail. Through alanine scanning mutagenesis of the acidic motif, we have identified two conserved tyrosine residues that are essential for Us9-mediated spread as well as two serine residues, comprising putative consensus casein kinase II sites, that modulate the rate of PRV transneuronal spread in vivo.
Identification of a
cis
-Acting Replication Element within the Poliovirus Coding Region
Authors: Goodfellow Ian , Chaudhry Yasmin , Richardson Andrew , Meredith Janet , Almond Jeffrey W. , Barclay Wendy , Evans David J.
Published: July 2002

The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the
Enterovirus
genus of the
Picornaviridae
. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus
cis
-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.
Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins
Authors: Zhang Jie , Pekosz Andrew , Lamb Robert A.
Published: July 2002

Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt− or NAt−) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M1) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt− or NAt− virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M2protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt− and NAt− is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt− and NAt− viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition.
The Epithelial Integrin αvβ6 Is a Receptor for Foot-and-Mouth Disease Virus
Authors: Jackson Terry , Sheppard Dean , Denyer Michael , Blakemore Wendy , King Andrew M. Q.
Published: July 2002

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use the RGD-dependent integrin αvβ3 as a cellular receptor on cultured cells. However, several other RGD-dependent integrins may have the potential to act as receptors for FMDV in vivo. Of these, αvβ6 is a likely candidate for use as a receptor by FMDV as it is expressed on epithelial cells, which correlates with the tissue tropism of the virus. In this report, we show that human colon carcinoma cells (SW480) that are normally nonpermissive for FMDV become susceptible to infection as a result of transfection with the integrin β6 subunit and expression of αvβ6 at the cell surface. Integrin αvβ6 is the major site for virus attachment on the β6-transfected cells, and binding to αvβ6 serves to increase the rate of virus entry into these cells. In addition, we show that virus binding and infection of the β6-transfected cells is mediated through an RGD-dependent interaction that is specifically inhibited by a monoclonal antibody (10D5) that recognizes αvβ6. These studies establish a role for αvβ6 as a cellular receptor for FMDV.
Synergistic Action of GA-Binding Protein and Glucocorticoid Receptor in Transcription from the Mouse Mammary Tumor Virus Promoter
Authors: Aurrekoetxea-Hernández Koldo , Buetti Elena
Published: July 2002

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position −139 to −146 from the cap site) and fp2 (at −157 to −164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.
Host Protein Interactions with the 3′ End of Bovine Coronavirus RNA and the Requirement of the Poly(A) Tail for Coronavirus Defective Genome Replication
Authors: Spagnolo Jeannie F. , Hogue Brenda G.
Published: July 2002

RNA viruses have 5′ and 3′ untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5′ end and has a 3′ UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus (MHV). Specific host protein interactions with the BCV 3′ UTR [287 nt plus poly(A) tail] were identified using gel mobility shift assays. Competition with the MHV 3′ UTR [301 nt plus poly(A) tail] suggests that the interactions are conserved for the two viruses. Proteins with molecular masses of 99, 95, and 73 kDa were detected in UV cross-linking experiments. Less heavily labeled proteins were also detected in the ranges of 40 to 50 and 30 kDa. The poly(A) tail was required for binding of the 73-kDa protein. Immunoprecipitation of UV-cross-linked proteins identified the 73-kDa protein as the cytoplasmic poly(A)-binding protein (PABP). Replication of the defective genomes BCV Drep and MHV MIDI-C, along with several mutants, was used to determine the importance of the poly(A) tail. Defective genomes with shortened poly(A) tails consisting of 5 or 10 A residues were replicated after transfection into helper virus-infected cells. BCV Drep RNA that lacked a poly(A) tail did not replicate, whereas replication of MHV MIDI-C RNA with a deleted tail was detected after several virus passages. All mutants exhibited delayed kinetics of replication. Detectable extension or addition of the poly(A) tail to the mutants correlated with the appearance of these RNAs in the replication assay. RNAs with shortened poly(A) tails exhibited less in vitro PABP binding, suggesting that decreased interactions with the protein may affect RNA replication. The data strongly indicate that the poly(A) tail is an important
cis
-acting signal for coronavirus replication.
Cell Fusion Activity of Hepatitis C Virus Envelope Proteins
Authors: Takikawa Shingo , Ishii Koji , Aizaki Hideki , Suzuki Tetsuro , Asakura Hitoshi , Matsuura Yoshiharu , Miyamura Tatsuo
Published: July 2002

To examine the cell fusion activity of hepatitis C virus (HCV) envelope proteins (E1 and E2), we have established a sensitive cell fusion assay based on the activation of a reporter gene as described previously (O. Nussbaum, C. C. Broder, and E. A. Berger, J. Virol. 68:5411–5422, 1994). The chimeric HCV E1 and E2 proteins, each consisting of the ectodomain of the E1 and E2 envelope protein and the transmembrane and cytoplasmic domains of the vesicular stomatitis virus G glycoprotein, were expressed on the cell surface. Cells expressing the chimeric envelope proteins and T7 RNA polymerase were cocultured with the various target cell lines transfected with a reporter plasmid encoding the luciferase gene under the control of the T7 promoter. After cocultivation, the cell fusion activity was determined by the expression of luciferase in the cocultured cells. The induction of cell fusion requires both the chimeric E1 and E2 proteins and occurs in a low-pH-dependent manner. Although it has been shown that HCV E2 protein binds human CD81 (P. Pileri, Y. Uematsu, S. Campagnoli, G. Galli, F. Falugi, R. Petracca, A. J. Weiner, M. Houghton, D. Rosa, G. Grandi, and S. Abrignani, Science 282:938–941, 1998), the expression of human CD81 alone is not sufficient to confer susceptibility to cell fusion in the mouse cell line. Treatment of the target cells with pronase, heparinase, or heparitinase reduced the cell fusion activity induced by the chimeric envelope proteins. These results suggest (i) that both HCV E1 and E2 proteins are responsible for fusion with the endosomal membrane after endocytosis and (ii) that certain protein molecules other than human CD81 and some glycosaminoglycans on the cell surface are also involved in the cell fusion induced by HCV.
A Single Amino Acid Change in the Newcastle Disease Virus Fusion Protein Alters the Requirement for HN Protein in Fusion
Authors: Sergel Theresa A. , McGinnes Lori W. , Morrison Trudy G.
Published: July 2002

The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F
1
portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation.
Mov34 Protein from Mouse Brain Interacts with the 3′ Noncoding Region of Japanese Encephalitis Virus
Authors: Ta Malancha , Vrati Sudhanshu
Published: July 2002

The plus-sense RNA genome of Japanese encephalitis virus (JEV) contains noncoding regions (NCRs) of 95 and 585 bases at its 5′ and 3′ ends, respectively. The last 83 nucleotides of the 3′-NCR are predicted to form stable stem-loop (SL) structures. The shape of this 3′-SL structure is highly conserved among divergent flaviviruses even though only small stretches of nucleotide sequence contained within these structures are conserved. These SL structures have been predicted to function as
cis
-acting signals for RNA replication and as such may bind to viral and cellular proteins that may be involved in viral replication. We have studied the interaction of the JEV 3′-NCR RNA with host proteins using gel retardation assays. We show that the JEV 3′-SL structure RNA forms three complexes with proteins from the S100 cytoplasmic extract prepared from the neonatal mouse brain. These complexes could be obtained in the presence of 200 mM KCl, indicating that the RNA-protein interaction may be physiologically relevant. UV-induced cross-linking and Northwestern blotting analyses detected three proteins with apparent molecular masses of 32, 35, and 50 kDa that bound to the JEV 3′-SL structure RNA. Screening of the neonatal mouse brain cDNA library with the JEV 3′-SL structure RNA identified a 36-kDa Mov34 protein interacting with it. Competition experiments using the RNA extracted from JEV virions established that the 36-kDa Mov34 protein indeed bound to the JEV genome. Murine Mov34 belongs to a family of proteins whose members have been shown to be involved in RNA transcription and translation. It is, therefore, likely that the murine Mov34 interaction with JEV 3′-NCR has a role in RNA replication.
Mutational Analysis of the Rubella Virus Nonstructural Polyprotein and Its Cleavage Products in Virus Replication and RNA Synthesis
Authors: Liang Yuying , Gillam Shirley
Published: July 2002

Rubella virus nonstructural proteins, translated from input genomic RNA as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. To examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral RNA synthesis, we introduced into a rubella virus infectious cDNA clone a panel of mutations that had variable defective effects on p200 processing. The virus yield and viral RNA synthesis of these mutants were examined. Mutations that completely abolished (C1152S and G1301S) or largely abolished (G1301A) cleavage of p200 resulted in noninfectious virus. Mutations that partially impaired cleavage of p200 (R1299A and G1300A) decreased virus replication. An RNase protection assay revealed that all of the mutants synthesized negative-strand RNA as efficiently as the wild type does but produced lower levels of positive-strand RNA. Our results demonstrated that processing of rubella virus nonstructural protein is crucial for virus replication and that uncleaved p200 could function in negative-strand RNA synthesis, whereas the cleavage products p150 and p90 are required for efficient positive-strand RNA synthesis.
Influenza A Viruses Lacking Sialidase Activity Can Undergo Multiple Cycles of Replication in Cell Culture, Eggs, or Mice
Authors: Hughes Mark T. , Matrosovich Mikhail , Rodgers M. Elizabeth , McGregor Martha , Kawaoka Yoshihiro
Published: July 2002

Influenza A viruses possess both hemagglutinin (HA), which is responsible for binding to the terminal sialic acid of sialyloligosaccharides on the cell surface, and neuraminidase (NA), which contains sialidase activity that removes sialic acid from sialyloligosaccharides. Interplay between HA receptor-binding and NA receptor-destroying sialidase activity appears to be important for replication of the virus. Previous studies by others have shown that influenza A viruses lacking sialidase activity can undergo multiple cycles of replication if sialidase activity is provided exogenously. To investigate the sialidase requirement of influenza viruses further, we generated a series of sialidase-deficient mutants. Although their growth was less efficient than that of the parental NA-dependent virus, these viruses underwent multiple cycles of replication in cell culture, eggs, and mice. To understand the molecular basis of this viral growth adaptation in the absence of sialidase activity, we investigated changes in the HA receptor-binding affinity of the sialidase-deficient mutants. The results show that mutations around the HA receptor-binding pocket reduce the virus's affinity for cellular receptors, compensating for the loss of sialidase. Thus, sialidase activity is not absolutely required in the influenza A virus life cycle but appears to be necessary for efficient virus replication.
Chimeric Retroviral Helper Virus and Picornavirus IRES Sequence To Eliminate DNA Methylation for Improved Retroviral Packaging Cells
Authors: Young Won-Bin , Link Charles J.
Published: July 2002

Most retroviral packaging cell lines were established by a helper virus plasmid cotransfected with a separate plasmid encoding a selection marker. Since this selection marker coexisted in
trans
with the helper virus sequence, helper virus gene expression could be inactivated by host DNA methylation despite selection for the cotransfected selection marker. We have reported that DNA methylation could occur in the long terminal repeat (LTR) region of helper virus in vector producer cells (VPC) in up to 2% of the population per day (W. B. Young, G. L. Lindberg, and C. J. Link, Jr., J. Virol. 74:3177–3187, 2000). To overcome host cell DNA methylation that suppresses viral gene expression, we constructed a chimeric retroviral helper virus, pAM3-IRES-Zeo, that contains Moloney murine leukemia virus as a helper virus and a picornavirus internal ribosome entry site (IRES) sequence followed by a Zeocin selection marker at the 3′ end of the
env
sequence. This pAM3-IRES-Zeo permitted selection for intact and functional helper virus in transfected cells without subcloning. By selection with Zeocin, a mixed population of pAM3-IRES-Zeo-transfected NIH3T3 cells (AMIZ cells) was maintained with little or no DNA methylation of the helper virus 5′ LTR. The high level of pAM3-IRES-Zeo gene expression resulted in no detectable vector superinfection and in high vector titers (2 × 10
6
to 1.5 × 10
7
CFU/ml) after introduction of a retroviral vector. When Zeocin selection was withdrawn from AMIZ cells, methylation of the 5′ LTR increased from 17 to 36% of the population during 67 days of continuous culture and the cells became susceptible to superinfection. During this period, gene expression of pAM3-IRES-Zeo decreased and vector titer production was reduced to 2 × 10
4
CFU/ml. These data demonstrate an important role of DNA methylation in the genetic instability of VPC. The chimeric helper virus allows the establishment of a mixed population of packaging cells capable of high-level and sustained vector production without cloning procedures.
HLA-B57-Restricted Cytotoxic T-Lymphocyte Activity in a Single Infected Subject toward Two Optimal Epitopes, One of Which Is Entirely Contained within the Other
Authors: Goulder Philip J. R. , Tang Yanhua , Pelton Stephen I. , Walker Bruce D.
Published: July 2002

Viral peptides are recognized by cytotoxic T lymphocytes (CTL) as a complex with major histocompatibility complex (MHC) class I molecules, but the extent to which a single HLA allele can accommodate epitope peptides of different length and sequence is not well characterized. Here we report the identification of clonal CTL responses from the same donor that independently recognize one of two HLA-B57-restricted epitopes, KAFSPEVIPMF (KF11; p24Gagresidues 30 to 40) and KAFSPEVI (KF8; p24Gagresidues 30 to 37). Although lysis studies indicated that the KF11 peptide stabilized the HLA-B57-peptide complex more efficiently than the KI8 peptide, strong clonal responses were directed at each epitope. In samples from a second donor, the same phenomenon was observed, in which distinct CTL clones recognized peptide epitopes presented by the same HLA class I allele (in this case, HLA-A3) which were entirely overlapping. These data are relevant to the accurate characterization of CTL responses, which is fundamental to a detailed understanding of MHC class I-restricted immunity. In addition, these studies demonstrate marked differences in the length of peptides presented by HLA-B57, an allele which is associated with nonprogressive human immunodeficiency virus infection.
Mutation of a Conserved Residue (D123) Required for Oligomerization of Human Immunodeficiency Virus Type 1 Nef Protein Abolishes Interaction with Human Thioesterase and Results in Impairment of Nef Biological Functions
Authors: Liu Lang Xia , Heveker Nikolaus , Fackler Oliver T. , Arold Stefan , Le Gall Sylvie , Janvier Katy , Peterlin B. Matija , Dumas Christian , Schwartz Olivier , Benichou Serge , Benarous Richard
Published: July 2002

Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.
Mutational Definition of Functional Domains within the Rev Homolog Encoded by Human Endogenous Retrovirus K
Authors: Bogerd Hal P. , Wiegand Heather L. , Yang Jin , Cullen Bryan R.
Published: July 2002

Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a
cis
-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the ∼25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.
Determination of Essential Amino Acids Involved in the CD4-Independent Tropism of the X4 Human Immunodeficiency Virus Type 1 m7NDK Isolate: Role of Potential N Glycosylations in the C2 and V3 Regions of gp120
Authors: Dumonceaux Julie , Goujon Caroline , Joliot Veronique , Briand Pascale , Hazan Uriel
Published: July 2002

Seven mutations in the C2, V3, and C3 regions of gp120 are implicated in the tropism of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. Site-directed mutagenesis revealed that three amino acids are essential to maintain this tropism, one in the C2 region and two in the V3 loop. Two mutations implied N glycosylation modifications.
Structural Flexibility and Functional Valence of CD4-IgG2 (PRO 542): Potential for Cross-Linking Human Immunodeficiency Virus Type 1 Envelope Spikes
Authors: Zhu Ping , Olson William C. , Roux Kenneth H.
Published: July 2002

CD4-immunoglobulin G2 (CD4-IgG2) incorporates four copies of the D1D2 domains of CD4 into an antibody-like molecule that potently neutralizes primary human immunodeficiency virus type 1. Here electron microscopy was used to explore the structure and functional valence of CD4-IgG2 in complex with gp120. CD4-γ2, a divalent CD4-immunoglobulin fusion protein, was evaluated in parallel. Whereas CD4-γ2–gp120 complexes adopted a simple Y-shaped structure, CD4-IgG2–gp120 complexes consisted of four gp120s arrayed about a central CD4-IgG2 molecule, a structure more reminiscent of complement C1q. Molecular modeling corroborated the electron microscopy data and further indicated that CD4-IgG2 but not CD4-γ2 has significant potential to cross-link gp120-gp41 trimers on the virion surface, suggesting a mechanism for the heightened antiviral activity of CD4-IgG2.
Protection against Woodchuck Hepatitis Virus (WHV) Infection by Gene Gun Coimmunization with WHV Core and Interleukin-12
Authors: Garcı́a-Navarro R. , Blanco-Urgoiti B. , Berraondo P. , Sánchez de la Rosa R. , Vales A. , Hervás-Stubbs S. , Lasarte J. J. , Borrás F. , Ruiz J. , Prieto J.
Published: July 2002

Woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) are closely similar with respect to genomic organization, host antiviral responses, and pathobiology of the infection. T-cell immunity against viral nucleocapsid (HBcAg or WHcAg) has been shown to play a critical role in viral clearance and protection against infection. Here we show that vaccination of healthy woodchucks by gene gun bombardment with a plasmid coding for WHcAg (pCw) stimulates proliferation of WHcAg-specific T cells but that these cells do not produce significant levels of gamma interferon (IFN-γ) upon antigen stimulation. In addition, animals vaccinated with pCw alone were not protected against WHV inoculation. In order to induce a Th1 cytokine response, another group of woodchucks was immunized with pCw together with another plasmid coding for woodchuck interleukin-12 (IL-12). These animals exhibited WHcAg-specific T-cell proliferation with high IFN-γ production and were protected against challenge with WHV, showing no viremia or low-level transient viremia after WHV inoculation. In conclusion, gene gun immunization with WHV core generates a non-Th1 type of response which does not protect against experimental infection. However, steering the immune response to a Th1 cytokine profile by IL-12 coadministration achieves protective immunity. These data demonstrate a crucial role of Th1 responses in the control of hepadnavirus replication and suggest new approaches to inducing protection against HBV infection.
Application of Chelating Ion Exchanger Ostsorb DTTA to Determination of Lead and Cadmium in Soils, Plants, and Fertilizers
Authors: Svoboda Ladislav , Tomek Miloslav , Tolar Petr
Published: July 2002

The paper investigates a possible application of chelating cellulose ion exchanger with the functional groups of diethylenetriaminetetraacetic acid to concentrating lead and cadmium in extracts from soils and industrial fertilizers or mineralizates of plant materials. A favourable recovery of the metals needs the knowledge of the approximate content of the complex forming cations in the solutions and the suppression of formation of iron and aluminium hydroxides during modification of acidity of extracts to the value of pH 4 which is the optimum for the formation of chelates. The degree of concentrating reached the values of fE = 10 - 50, the recovery of lead and cadmium depended on the nature of sample and was in the range of R = 60 - 100%.
119Sn and 13C NMR Spectral Study of Some Vinyltin(IV) Compounds Involving the Sn-S Bond
Authors: Handlíř Karel , Lyčka Antonín , Holeček Jaroslav , Nádvorník Milan , Pejchal Vladimír , Sebald Angelika
Published: July 2002

The 13C and 119Sn NMR spectra were examined for two series of vinyltin(IV) compounds involving the Sn-S bond, of general formulas (CH2=CH)nSn(SR)4 - n (n = 0 - 4) and (CH2=CH)nSn(Cl)(SR)3 - n (n = 0 - 3) where R = 1-C4H9, C(S)N(C2H5)2 or 8-quinolinyl C9H6N, or SR = 1/2 (SCH2CH2S). Deuteriochloroform and hexadeuteriodimethyl sulfoxide served as non-coordinating and coordinating solvents, respectively. The 13C and 119Sn NMR data provided a basis for discussion of the nature and strength of the Sn-S bonding, coordination of the central tin atom including the shape of the coordination polyhedron, and the ability of the compounds to form donor acceptor complexes.
Fluorescence Reagents for Labelling of Biomolecules. Part III. Study of the Reactions of 2- and 4-Substituted 9-Isothiocyanatoacridines with Glycine
Authors: Sabolová Danica , Mazagová Dana , Kristian Pavol , Antalík Marián , Podhradský Dušan , Imrich Ján
Published: July 2002

Kinetics of nucleophilic addition reaction of 2- and 4-substituted 9-isothiocyanatoacridines I - VII with glycine in buffered aqueous dimethylformamide has been studied. The addition products, N-(9-acridinylthiocarbamoyl)glycines VIII - XIV, were characterized by IR, UV, 1H NMR, mass and fluorescence spectra. Derivatives VIII, X and XII exhibited higher fluorescence intensity than the starting isothiocyanates; the highest fluorescence was found for the unsubstituted compound X. The reaction mechanism is discussed on the basis of properties of the reaction products and kinetic characteristics.
4-(2',4'-Difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic Acid and Its Derivatives
Authors: Kuchař Miroslav , Poppová Marie , Zůnová Hana , Knězová Eva , Vosátka Václav , Příhoda Marek
Published: July 2002

Friedel Crafts reaction of itaconic anhydride (III) with 2,4-difluorobiphenyl (II) afforded unsaturated acid IV which was hydrogenated to give 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid (I). A series of functional derivatives and salts of the acid I has been prepared. The antiinflammatory effect of these compounds was compared with that of acid I using selected experimental inflammation models. The analgesic activity in the intraperitoneal irritation test was also evaluated. In the case of (R)-(+)-1-phenylethylamide Vf the ratio of the diastereoisomers was determined by HPLC.
Free Level Effect on the Impeller Power Input in Baffled Tanks
Authors: Wichterle Kamil
Published: July 2002

Analysis of extended data on turbine impeller power input in geometrically similar agitated baffled tanks shows that the power number Po is a function of Reynolds number Po = Po*(Re) until the emergence of surface aeration. Though it is usually anticipated that Po* = const in high Reynolds number region, some, whatever weak, function should be taken into consideration in more detailed analysis of the power data even here. In practice, disturbances of level and gas captured in the impeller region play also a significant role, namely in smaller tanks at higher impeller speeds. Decrease of power input can be explained by decrease of gas-liquid mixture density, or in other words by increase of efficient gas holdup eE just in the impeller region. The value eE defined by the relation Po = Po*(Re)/(1 + eE) was determined from the available data. Like other effects of the surface aeration it depends mainly on the dimensionless number Nc = (We Fr)1/4. A simple correlation eE (Nc) is suggested as a correction factor for prediction of impeller power in presence of gas capture.
Approximate Solution of Momentum Transfer in System Generalized Newtonian Fluid-Fluidized Bed of Spherical Particles Using Modified Rabinowitsch-Mooney Equation
Authors: Dolejš Václav , Mikulášek Petr , Doleček Petr
Published: July 2002

The modified Rabinowitsch-Mooney equation together with the corresponding relations for consistency variables has been adopted for approximate solution of momentum transfer between generalized Newtonian fluid with laminar flow and surface of fluidized bed of spherical particles inclusive of wall surface. The solution has been concretized for a fluid characterized by power-law and Ellis flow models in the creeping flow region. The range of values of ratios of particle diameter to column diameter and that of porosity values e in which the suggested relation satisfactorily agrees with experimental results for pseudoplastic fluids have been delimitated experimentally.
New Method for Preparation of 1-Azabicyclo[2.2.2]octane-4-carbonitrile and 1-Azabicyclo[3.2.2]nonane-5-carbonitrile
Authors: Svoboda Jiří , Paleček Jaroslav
Published: July 2002

The title compounds were prepared from pyridine-4-carboxamide. Its alkylation with 2-chloroethanol or 3-chloropropanol, followed by hydrogenation gave 1-substituted piperidine-4-carboxamides IIIa and IIIb, respectively, which on treatment with thionyl chloride were converted into the respective 1-substituted halogenoalkylpiperidine-4-carbonitriles IVa and IVb. On cyclization, compounds IVa and IVb afforded 1-azabicyclo[2.2.2]octane-4-carbonitrile and 1-azabicyclo[3.2.2]nonane-5-carbonitrile, respectively.
Jatropham 5-O-β-D-Glucopyranoside from Lilium candidum L.
Authors: Mučaji Pavel , Haladová Mária , Eisenreichová Eva , Buděšínský Miloš , Ubik Karel
Published: July 2002

Jatropham 5-O-β-D-glucopyranoside [(3-methyl-2-oxo-3-pyrroline-5-yl)-β-D-glucopyranoside] was isolated from the ethanolic extract of the petals of Lilium candidum L.
Effect of the Solvent Basicity and Additives on the Electroreduction of Picric Acid and Tetramethylammonium Picrate in Aprotic Media
Authors: Janderka Pavel , Fischer Oldřich , Fischerová Eva
Published: July 2002

The electroreduction of picric acid and tetramethylammonium picrate in acetonitrile, acetone, N,N-dimethylformamide, dimethyl sulfoxide and hexamethylphosphoramide has been studied by normal pulse polarography, cyclic voltammetry and by coulometric analysis. The reduction of undissociated acid starts in all aprotic media with consumption of one electron. The reduction potential is shifted to more negative potentials with rising basicity of the solvent, i.e. with rising dissociation constant of picric acid. The formation of anion radicals is followed by the sequence of acido-basic selfprotonation reactions as well as by the disproportionation leading to a nitroso intermediate. The reduction mechanism does not change by addition of proton acceptor (basic alumina) or by amfiprotic species (anthranilic acid). In the presence of a strong acid, picric acid can mediate the electroreduction of added protons. Proposed mechanisms were supported by comparison with electrochemical behaviour of related nitrophenols in the same aprotic media.
Influence of C-Terminal Modifications of Bradykinin Antagonists on Their Activity
Authors: Prahl Adam , Wierzba Tomasz , Winklewski Pawel , Wszedybyl Magdalena , Cherek Maciej , Juzwa Witold , Lammek Bernard
Published: July 2002

In the present study we have described the synthesis and some pharmacological properties of four new analogues of bradykinin (BK). Two peptides were designed by substitution of positions 7 and 8 of known B2 antagonists with N-methyl-L-phenylalanine [Phe(Me)]. The next two analogues were obtained by replacement of D-Phe residue in position 7 of known B2 antagonist with 1-naphthyl-D-alanine or 2-naphthyl-D-alanine. The antagonistic potency of peptides was assessed by their ability to inhibit vasodepressor response to exogenous bradykinin in conscious rats. Although our studies demonstrated disadvantegous influence of Phe(Me)7,8 modification for B2 antagonism, we showed that D-amino acid residue in position 7 of BK antagonists may be replaced by suitable L-amino acid residue. As regards (D-Nal)7 substitution, we found strikingly different antagonistic potencies of analogues which differ only in the presence of D-1-Nal and D-2-Nal. We assume that it is due to different conformations of these peptides, proving the importance of the shape of the C-terminal part of B2 antagonists for their activity.
Influence of pH on Phosphate Intercalation in Zinc-Aluminum Layered Double Hydroxide
Authors: Badreddine Mohamed , Legrouri Ahmed , Barroug Allal , De Roy André , Besse Jean-Pierre
Published: July 2002

The exchange of chloride ion by phosphate ions in [Zn-Al-Cl] layered double hydroxide was investigated using X-ray diffraction and infrared spectroscopy. The effects of the pH of the solution containing the phosphate ions on the ion exchange was studied. The best sample in terms of crystallinity, was obtained at pH 8. This sample was further characterized by scanning electron microscopy and microanalysis.
Comparative Study of Polymerization of 2-, 3- and 4-Iodophenylacetylenes with Rh-, Mo- and W-Based Catalysts
Authors: Vohlídal Jiří , Sedláček Jan , Patev Nikolay , Pacovská Marta , Lavastre Olivier , Cabioch Sandrine , Dixneuf Pierre H. , Blechta Vratislav , Matějka Pavel , Balcar Hynek
Published: July 2002

2-, 3- and 4-Iodophenylacetylenes (IPA) have been polymerized with [Rh(cod)(OCH3)]2 complex in THF (system Rh), MoCl5 in benzene (Mo), WOCl4 in benzene (WB) and WOCl4/Ph4Sn in benzene-dioxane 1 : 1 (WD). All the systems provide poly(iodophenylacetylene)s (PIPA) and diverse amounts of oligomers (mostly cyclic trimers and tetramers) but the system 2-IPA/Rh that provides mostly dimers only. 2-IPA has also been polymerized with MoOCl4/Bu4Sn/EtOH in toluene (Masuda's catalyst system), however, neither living nor pseudo-living polymerization has been observed. The ring-iodine steric effects control the polymerization activity of the Rh and WB systems and the oligomerization activity of the Mo system. The ring-iodine electronic effects are important for the oligomerization activity of the Rh and WB systems and both oligomerization and polymerization activity of the WD system. Both types of the ring-iodine effect are important for polymerization activity of the Mo system. Rh catalyst provides high-cis-transoid, head-to-tail (HT) 3-PIPA that slowly isomerizes in CDCl3 solution to an insoluble trans-polymer. In the case of 4-PIPA, the isomerization is extremely fast upon polymer entering into contact with methanol. On the basis of spectral changes accompanying this isomerization, the Raman band at 1 338 cm-1 is assigned to the presence of HT sequences of both cis-transoid and trans-cisoid type. The other catalysts provide irregular PIPAs with content of both cis units and HT sequences decreasing in order Mo > WD > WB. Solubility of PIPA samples is shown as a result of complex interplay of the ring-iodine position and the polymer microstructure and molecular weight.
Intercalation of Ketones in Vanadyl Phosphate and Isostructural Hosts
Authors: Melánová Klára , Beneš Ludvík , Zima Vítězslav , Čapková Pavla , Trchová Miroslava
Published: July 2002

The intercalation of acetone and butan-2-one in vanadyl and niobyl phosphates and niobyl arsenate is reported. All intercalates prepared are tetragonal and their basal spacings are close to each other. The intercalates contain one molecule of the ketone per formula unit. The existence of keto-enol tautomerism of guest molecules in interlayer space is assumed.
Floor vibration control using semi-active tuned mass dampers
Authors: Setareh Mehdi
Published: July 2002

This paper discusses the application of a new class of semi-active tuned mass dampers, called ground-hook tuned mass dampers (GHTMD), for the reduction of floor vibrations due to human movements. The TMD introduced uses a continuously variable semi-active damper (ground-hook damper) to achieve reduction in the floor acceleration. Here, the GHTMD is applied to a single degree of freedom system representative of building floors. The GHTMD design parameters are defined in terms of non-dimensional values. The optimum values of these parameters are found based on the minimization of the acceleration response of the floor for different GHTMD mass ratios and floor damping ratios. The performance of the GHTMD is compared to that of the equivalent passive TMD. In addition, the effects of off-tuning due to variations in the mass ratios and frequency ratios of the TMD and GHTMD are studied. Comparison of the results demonstrates the efficiency and robustness of GHTMD with respect to equivalent TMD. Finally, a guide for the design of GHTMDs is presented.Key words: floor vibrations, semi-active tuned mass dampers, tuned vibration absorbers, vibration control, ground-hook dampers, human-induced vibrations, annoying vibrations, optimum design parameters.
Studies on the rearrangement of ortho-nitrobenzylidenemalonates and their Analogues to 2-aminobenzoate derivatives
Authors: Lewandowska Elzbieta , Kinastowski Stefan , Wnuk Stanislaw F
Published: July 2002

Reaction of the diethyl 2-nitro-4-(trifluoromethyl)benzylidenemalonate with diethylamine in alcohols resulted in the reduction of the nitro group and the oxidation of the vinylic carbon attached to the phenyl ring. Simultaneous migration of the malonic fragment gave the appropriate 2-amino-4-(trifluoromethyl)benzoate esters. The presence of at least two nitro groups, or one nitro group and trifluoromethyl group on the phenyl ring, attached to the α-carbon and strongly electron withdrawing substituents at the β-carbon (CO2Et, CN) in ortho-nitrobenzylidene systems is necessary for this reductiveoxidative rearrangement to proceed. Reaction of nitrocinnamates with thiols in the presence of triethylamine in tetrahydrofuran gave Michael addition products with different regioselectivity of addition. Ethyl 2-nitrocinnamate undergoes standard β-addition of thiols to a carboncarbon double bond. However, 2,4-dinitro- and 2,4,6-trinitrocinnamates undergo α-addition of thiols, indicating that the presence of two nitro groups on the phenyl ring can reverse polarity of the carboncarbon double bond in cinnamate acceptors.Key words: abnormal Michael reactions, aromatic nitro compounds, benzylidene compounds, rearrangements.
A NEW ALGEBRA FROM THE REPRESENTATION THEORY OF FINITE GROUPS
Authors: JUYUMAYA J.
Published: July 2002

In this work we define a new algebra. The definition of our algebra arises naturally in the study of certain generators (non standard) for Yokonuma–Hecke algebra [8]. This algebra is linked to the Knot theory via the Vassiliev algebra defined by J. Baez [2].