bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Cell Lines Carrying Self-Replicating Hepatitis C Virus RNAs
Authors: Pietschmann Thomas , Lohmann Volker , Rutter Gabriel , Kurpanek Katharina , Bartenschlager Ralf
Published: July 2002

Subgenomic selectable RNAs of the hepatitis C virus (HCV) have recently been shown to self-replicate to high levels in the human hepatoma cell line Huh-7 (V. Lohmann, F. Körner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110–113, 1999). Taking advantage of this cell culture system that allows analyses of the interplay between HCV replication and the host cell, in this study we characterized two replicon-harboring cell lines that have been cultivated for more than 1 year. During this time, we observed no signs of cytopathogenicity such as reduction of growth rates or ultrastructural changes. High levels of HCV RNAs were preserved in cells passaged under continuous selection. When selective pressure was omitted replicon levels dropped, but depending on culture conditions the RNAs persisted for more than 10 months. A tight coupling of the amounts of HCV RNA and proteins to host cell growth was observed. Highest levels were found in exponentially growing cells, followed by a sharp decline in resting cells, suggesting that cellular factors required for RNA replication and/or translation vary in abundance and become limiting in resting cells. Studies of polyprotein processing revealed rapid cleavages at the NS3/4A and NS5A/B sites resulting in a rather stable NS4AB5A precursor that was processed slowly into individual products. Half-lives (
t
1/2
s) of mature proteins ranged from 10 to 16 h, with the exception of the hyperphosphorylated form of NS5A, which was less stable (t
1/2
, ∼7 h). Results of immunoelectron microscopy revealed an association of the majority of viral proteins with membranes of the endoplasmic reticulum, suggesting that this is the site of RNA replication. In summary, replicon-bearing cells are a good model for viral persistence, and they allow the study of various aspects of the HCV life cycle.
Hepatitis C Virus NS5A Physically Associates with p53 and Regulates p21/waf1 Gene Expression in a p53-Dependent Manner
Authors: Majumder Mainak , Ghosh Asish K. , Steele Robert , Ray Ranjit , Ray Ratna B.
Published: July 2002

We have previously demonstrated that hepatitis C virus (HCV) NS5A protein promotes cell growth and transcriptionally regulates the p21/waf1 promoter, a downstream effector gene of p53. In this study, we investigated the molecular mechanism of NS5A-mediated transcriptional repression of p21/waf1. We observed that transcriptional repression of the p21/waf1 gene by NS5A is p53 dependent by using p53 wild-type (+/+) and null (−/−) cells. Interestingly, p53-mediated transcriptional activation from a synthetic promoter containing multiple p53 binding sites (PG13-LUC) was abrogated following expression of HCV NS5A. Additional studies using pull-down experiments, in vivo coimmunoprecipitation, and mammalian two-hybrid assays demonstrated that NS5A physically associates with p53. Confocal microscopy revealed sequestration of p53 in the perinuclear membrane and colocalization with NS5A in transfected HepG2 and Saos-2 cells. Together these results suggest that an association of NS5A and p53 allows transcriptional modulation of the p21/waf1 gene and may contribute to HCV-mediated pathogenesis.
Macrophage Tropism of Human Immunodeficiency Virus Type 1 Facilitates In Vivo Escape from Cytotoxic T-Lymphocyte Pressure
Authors: Schutten M. , van Baalen C. A. , Guillon C. , Huisman R. C. , Boers P. H. M. , Sintnicolaas K. , Gruters R. A. , Osterhaus A. D. M. E.
Published: July 2002

Early after seroconversion, macrophage-tropic human immunodeficiency virus type 1 (HIV-1) variants are predominantly found, even when a mixture of macrophage-tropic and non-macrophage-tropic variants was transmitted. For virus contracted by sexual transmission, this is presently explained by selection at the port of entry, where macrophages are infected and T cells are relatively rare. Here we explore an additional mechanism to explain the selection of macrophage-tropic variants in cases where the mucosa is bypassed during transmission, such as blood transfusion, needle-stick accidents, or intravenous drug abuse. With molecularly cloned primary isolates of HIV-1 in irradiated mice that had been reconstituted with a high dose of human peripheral blood mononuclear cells, we found that a macrophage-tropic HIV-1 clone escaped more efficiently from specific cytotoxic T-lymphocyte (CTL) pressure than its non-macrophage-tropic counterpart. We propose that CTLs favor the selective outgrowth of macrophage-tropic HIV-1 variants because infected macrophages are less susceptible to CTL activity than infected T cells.
Vaccinia Virus Blocks Gamma Interferon Signal Transduction: Viral VH1 Phosphatase Reverses Stat1 Activation
Authors: Najarro Pilar , Traktman Paula , Lewis John A.
Published: July 2002

We have analyzed the effects of vaccinia virus (VV) on gamma interferon (IFN-γ) signal transduction. Infection of cells with VV 1 to 2 h prior to treatment with IFN-γ inhibits phosphorylation and nuclear translocation of Stat1 and consequently blocks accumulation of mRNAs normally induced by IFN-γ. While phosphorylation of other proteins in the IFN-γ pathway was not affected, activation of Stat1 by other ligand-receptor systems was also blocked by VV. This block of Stat1 activation was dose dependent, and although viral protein synthesis was not required, entry and uncoating of viral cores appear to be needed to block the accumulation of phosphorylated Stat1. These results suggest that a virion component is responsible for the effect. VV virions contain a phosphatase (VH1) that is sensitive to the phosphatase inhibitor Na3VO4but not to okadaic acid. Addition of Na3VO4but not okadaic acid restored normal Stat1 phosphorylation levels in VV-infected cells. Moreover, virions containing reduced levels of VH1 were unable to block the IFN-γ signaling pathway. In vitro studies show that the phosphatase can bind and dephosphorylate Stat1, indicating that this transcription factor can be a substrate for VH1. Our results reveal a novel mechanism by which VV interferes with the onset of host immune responses by blocking the IFN-γ signal cascade through the dephosphorylating activity of the viral phosphatase VH1.
Close but Distinct Regions of Human Herpesvirus 8 Latency-Associated Nuclear Antigen 1 Are Responsible for Nuclear Targeting and Binding to Human Mitotic Chromosomes
Authors: Piolot Tristan , Tramier Marc , Coppey Maité , Nicolas Jean-Claude , Marechal Vincent
Published: July 2002

Human herpesvirus 8 is associated with all forms of Kaposi's sarcoma, AIDS-associated body cavity-based lymphomas, and some forms of multicentric Castleman's disease. Herpesvirus 8, like other gammaherpesviruses, can establish a latent infection in which viral genomes are stably maintained as multiple episomes. The latent nuclear antigen (LANA or LNAI) may play an essential role in the stable maintenance of latent episomes, notably by interacting concomitantly with the viral genomes and the metaphase chromosomes, thus ensuring an efficient transmission of the neoduplicated episomes to the daughter cells. To identify the regions responsible for its nuclear and subnuclear localization in interphase and mitotic cells, LNAI and various truncated forms were fused to a variant of green fluorescent protein. This enabled their localization and chromosome binding activity to be studied by low-light-level fluorescence microscopy in living HeLa cells. The results demonstrate that nuclear localization of LNAI is due to a unique signal, which maps between amino acids 24 and 30. Interestingly, this nuclear localization signal closely resembles those identified in EBNA1 from Epstein-Barr virus and herpesvirus papio. A region encompassing amino acids 5 to 22 was further proved to mediate the specific interaction of LNA1 with chromatin during interphase and the chromosomes during mitosis. The presence of putative phosphorylation sites in the chromosome binding sites of LNA1 and EBNA1 suggests that their activity may be regulated by specific cellular kinases.
Human Immunodeficiency Virus Type 1 Subtype C Molecular Phylogeny: Consensus Sequence for an AIDS Vaccine Design?
Authors: Novitsky V. , Smith U. R. , Gilbert P. , McLane M. F. , Chigwedere P. , Williamson C. , Ndung'u T. , Klein I. , Chang S. Y. , Peter T. , Thior I. , Foley B. T. , Gaolekwe S. , Rybak N. , Gaseitsiwe S. , Vannberg F. , Marlink R. , Lee T. H. , Essex M.
Published: July 2002

An evolving dominance of human immunodeficiency virus type 1 subtype C (HIV-1C) in the AIDS epidemic has been associated with a high prevalence of HIV-1C infection in the southern African countries and with an expanding epidemic in India and China. Understanding the molecular phylogeny and genetic diversity of HIV-1C viruses may be important for the design and evaluation of an HIV vaccine for ultimate use in the developing world. In this study we analyzed the phylogenetic relationships (i) between 73 nonrecombinant HIV-1C near-full-length genome sequences, including 51 isolates from Botswana; (ii) between HIV-1C consensus sequences that represent different geographic subsets; and (iii) between specific isolates and consensus sequences. Based on the phylogenetic analyses of 73 near-full-length genomes, 16 “lineages” (a term that is used hereafter for discussion purposes and does not imply taxonomic standing) were identified within HIV-1C. The lineages were supported by high bootstrap values in maximum-parsimony and neighbor-joining analyses and were confirmed by the maximum-likelihood method. The nucleotide diversity between the 73 HIV-1C isolates (mean value of 8.93%; range, 2.9 to 11.7%) was significantly higher than the diversity of the samples to the consensus sequence (mean value of 4.86%; range, 3.3 to 7.2%,
P
< 0.0001). The translated amino acid distances to the consensus sequence were significantly lower than distances between samples within all HIV-1C proteins. The consensus sequences of HIV-1C proteins accompanied by amino acid frequencies were presented (that of Gag is presented in this work; those of Pol, Vif, Vpr, Tat, Rev, Vpu, Env, and Nef are presented elsewhere [
http://www.aids.harvard.edu/lab_research/concensus_sequence.htm
]). Additionally, in the promoter region three NF-κB sites (GGGRNNYYCC) were identified within the consensus sequences of the entire set or any subset of HIV-1C isolates. This study suggests that the consensus sequence approach could overcome the high genetic diversity of HIV-1C and facilitate an AIDS vaccine design, particularly if the assumption that an HIV-1C antigen with a more extensive match to the circulating viruses is likely to be more efficacious is proven in efficacy trials.
Detection of CD4
+
- and CD8
+
-T-Cell Responses to Human Papillomavirus Type 1 Antigens Expressed at Various Stages of the Virus Life Cycle by Using an Enzyme-Linked Immunospot Assay of Gamma Interferon Release
Authors: Steele Jane C. , Roberts Sally , Rookes Susan M. , Gallimore Phillip H.
Published: July 2002

Human papillomavirus (HPV) antigens are expressed in epithelial cells at different stages of differentiation, and this may affect how they are handled by the immune system. We assessed the relative immunogenicities of four different HPV type 1 proteins: E6 and E7, which are made early in basal or parabasal cells; E4, which is made suprabasally in differentiating cells; and L1, a late protein which appears in the highly differentiated upper spinous layers. Pools of 15-mer peptides covering the primary sequences of all four proteins were used to screen 15 normal donors in enzyme-linked immunospot assays of gamma interferon release for both CD4
+
- and CD8
+
-T-cell reactivities. CD8
+
-T-cell responses were detected to the L1 protein in 7 of the 15 samples examined. No responses to E6, E7, or E4 were detected. CD4
+
-T-cell reactivities were again detected in 7 of the 15 donors. A broader spectrum of responses to E6 (three of seven), E4 (six of seven), and L1 (three of seven) was apparent, but there was no reactivity to E7. The predominant CD4
+
response was to E4. Reactivities were seen in some cases to corresponding regions on other common HPV types but were probably due to a multiple infection rather than to a cross-reaction. Antibodies to HPV1 virus-like particles were detected in 12 of the 15 (80%) donors, but antibody status did not correlate with T-cell reactivity. The differences in the relative immunogenicities of the four proteins revealed in this study are discussed in relation to how they may be processed and presented to the immune system by differentiating epithelial cells.
Antibodies to Rotavirus Outer Capsid Glycoprotein VP7 Neutralize Infectivity by Inhibiting Virion Decapsidation
Authors: Ludert Juan Ernesto , Ruiz Marie Christine , Hidalgo Carlos , Liprandi Ferdinando
Published: July 2002

The rotavirus capsid is composed of three concentric protein layers. Proteins VP4 and VP7 comprise the outer layer. VP4 forms spikes, is the viral attachment protein, and is cleaved by trypsin into VP8* and VP5*. VP7 is a glycoprotein and the major constituent of the outer protein layer. Both VP4 and VP7 induce neutralizing and protective antibodies. To gain insight into the virus neutralization mechanisms, the effects of neutralizing monoclonal antibodies (MAbs) directed against VP8*, VP5*, and VP7 on the decapsidation process of purified OSU and RRV virions were studied. Changes in virion size were followed in real time by 90° light scattering. The transition from triple-layered particles to double-layered particles induced by controlled low calcium concentrations was completely inhibited by anti-VP7 MAbs but not by anti-VP8* or anti-VP5* MAbs. The inhibitory effect of the MAb directed against VP7 was concentration dependent and was abolished by papain digestion of virus-bound antibody under conditions that generated Fab fragments but not under conditions that generated F(ab′)
2
fragments. Electron microscopy showed that RRV virions reacted with an anti-VP7 MAb stayed as triple-layered particles in the presence of excess EDTA. Furthermore, the infectivity of rotavirus neutralized via VP8*, but not that of rotavirus neutralized via VP7, could be recovered by lipofection of neutralized particles into MA-104 cells. These data are consistent with the notion that antibodies directed at VP8* neutralize by inhibiting binding of virus to the cell. They also indicate that antibodies directed at VP7 neutralize by inhibiting virus decapsidation, in a manner that is dependent on the bivalent binding of the antibody.
Segregation of CD4 and CXCR4 into Distinct Lipid Microdomains in T Lymphocytes Suggests a Mechanism for Membrane Destabilization by Human Immunodeficiency Virus
Authors: Kozak Susan L. , Heard Jean Michel , Kabat David
Published: July 2002

Recent evidence has suggested that plasma membrane sphingolipids and cholesterol spontaneously coalesce into raft-like microdomains and that specific proteins, including CD4 and some other T-cell signaling molecules, sequester into these rafts. In agreement with these results, we found that CD4 and the associated Lck tyrosine kinase of peripheral blood mononuclear cells and H9 leukemic T cells were selectively and highly enriched in a low-density lipid fraction that was resistant at 0°C to the neutral detergent Triton X-100 but was disrupted by extraction of cholesterol with filipin or methyl-β-cyclodextrin. In contrast, the CXCR4 chemokine receptor, a coreceptor for X4 strains of human immunodeficiency virus type 1 (HIV-1), was almost completely excluded from the detergent-resistant raft fraction. Accordingly, as determined by immunofluorescence with confocal microscopy, CD4 and CXCR4 did not coaggregate into antibody-induced cell surface patches or into patches of CXCR4 that formed naturally at the ruffled edges of adherent cells. The CXCR4 fluorescent patches were extracted with cold 1% Triton X-100, whereas the CD4 patches were resistant. In stringent support of these data, CD4 colocalized with patches of cholera toxin bound to the raft-associated sphingoglycolipid GM1, whereas CXCR4 did not. Addition of the CXCR4-activating chemokine SDF-1α did not induce CXCR4 movement into rafts. Moreover, binding of purified monomeric gp120 envelope glycoproteins from strains of HIV-1 that use this coreceptor did not stimulate detectable redistributions of CD4 or CXCR4 between their separate membrane domains. However, adsorption of multivalent gp120-containing HIV-1 virion particles appeared to destabilize the local CD4-containing rafts. Indeed, adsorbed HIV-1 virions were detected by immunofluorescence microscopy and were almost all situated in nonraft regions of the cell surface. We conclude that HIV-1 initially binds to CD4 in a raft domain and that its secondary associations with CXCR4 require shifts of proteins and associated lipids away from their preferred lipid microenvironments. Our evidence suggests that these changes in protein-lipid interactions destabilize the plasma membrane microenvironment underlying the virus by at least several kilocalories per mole, and we propose that this makes an important contribution to fusion of the viral and cellular membranes during infection. Thus, binding of HIV-1 may be favored by the presence of CD4 in rafts, but the rafts may then disperse prior to the membrane fusion reaction.
Single Mutation in the Flavivirus Envelope Protein Hinge Region Increases Neurovirulence for Mice and Monkeys but Decreases Viscerotropism for Monkeys: Relevance to Development and Safety Testing of Live, Attenuated Vaccines
Authors: Monath Thomas P. , Arroyo Juan , Levenbook Inessa , Zhang Zhen-Xi , Catalan John , Draper Ken , Guirakhoo Farshad
Published: July 2002

A chimeric yellow fever (YF) virus/Japanese encephalitis (JE) virus vaccine (ChimeriVax-JE) was constructed by insertion of the prM-E genes from the attenuated JE virus SA14-14-2 vaccine strain into a full-length cDNA clone of YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to the emergence of a small-plaque virus containing a single Met→Lys amino acid mutation at E279, reverting this residue from the SA14-14-2 to the wild-type amino acid. A similar virus was also constructed by site-directed mutagenesis (J. Arroyo, F. Guirakhoo, S. Fenner, Z.-X. Zhang, T. P. Monath, and T. J. Chambers, J. Virol. 75:934-942, 2001). The E279 mutation is located in a beta-sheet in the hinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of the infected cell. In independent transfection-passage studies with FRhL or Vero cells, mutations appeared most frequently in hinge 4 (bounded by amino acids E266 to E284), reflecting genomic instability in this functionally important region. The E279 reversion caused a significant increase in neurovirulence as determined by the 50% lethal dose and survival distribution in suckling mice and by histopathology in rhesus monkeys. Based on sensitivity and comparability of results with those for monkeys, the suckling mouse is an appropriate host for safety testing of flavivirus vaccine candidates for neurotropism. After intracerebral inoculation, the E279 Lys virus was restricted with respect to extraneural replication in monkeys, as viremia and antibody levels (markers of viscerotropism) were significantly reduced compared to those for the E279 Met virus. These results are consistent with the observation that empirically derived vaccines developed by mouse brain passage of dengue and YF viruses have increased neurovirulence for mice but reduced viscerotropism for humans.
Analysis of an
Autographa californica
Nucleopolyhedrovirus
lef-11
Knockout: LEF-11 Is Essential for Viral DNA Replication
Authors: Lin Guangyun , Blissard Gary W.
Published: July 2002

The
Autographa californica
nucleopolyhedrovirus (Ac
M
NPV)
lef-11
gene was previously identified by transient late expression assays as a gene important for viral late gene expression. The
lef-11
gene was not previously identified as necessary for DNA replication in transient origin-dependent plasmid DNA replication assays. To examine the role of
lef-11
in the context of the infection cycle, we generated a deletion of the
lef-11
gene by recombination in an Ac
M
NPV genome propagated as a BACmid in
Escherichia coli
. The resulting Ac
M
NPV
lef-11-
null BACmid (vAc
lef11KO
) was unable to propagate in cell culture, although a “repair” Ac
M
NPV BACmid (vAc
lef11KO-REP
), which was generated by transposition of the
lef-11
gene into the
polyhedrin
locus of the vAc
lef11KO
BACmid, was able to replicate in a manner similar to wild-type or control Ac
M
NPV viruses. Thus, the
lef-11
gene is essential for viral replication in Sf9 cells. The vAc
lef11KO
BACmid was examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in late transcription. The
lef-11-
null BACmid and control BACmids were transfected into Sf9 cells, and viral DNA replication was monitored. The viral DNA genome of the
lef-11-
null BACmid (vAc
lef11KO
) was not amplified, whereas replication and amplification of the genomes of the repair BACmid (vAc
lef11KO-REP
), wild-type Ac
M
NPV, and a nonpropagating
gp64-
null control BACmid (vAc
GUSgp64KO
) were readily detected. Northern blot analysis of transcripts from selected early, late, and very late genes showed that late and very late transcription was absent in cells transfected with the
lef-11-
null BACmid. Thus, in contrast to prior studies using transient replication and late expression assays, studies of a
lef-11-
null BACmid indicate that LEF-11 is required for viral DNA replication during the infection cycle.
The Gene 10 (UL49.5) Product of Equine Herpesvirus 1 Is Necessary and Sufficient for Functional Processing of Glycoprotein M
Authors: Rudolph Jens , Seyboldt Christian , Granzow Harald , Osterrieder Nikolaus
Published: July 2002

The functional cooperation of equine herpesvirus 1 (EHV-1) glycoprotein M (gM) and the gene 10 (UL49.5) product was analyzed. Transient-transfection experiments using gM and UL49.5 expression plasmids as well as RK13 cell lines constitutively expressing UL49.5 (RK49.5) or gM (RKgM) demonstrated that the endo-β-
N
-acetylglucosaminidase H (endo H)-resistant mature form of gM was detectable only after coexpression of the two proteins. Deletion of the EHV-1 UL49.5-homologous gene 10 in strain KyA resulted in a small-plaque phenotype and up to 190-fold-reduced virus titers. The growth defects of the mutant KyAΔ49.5 virus, which were very similar to those of a gM-negative KyA virus, could be completely compensated for by growth of the mutant virus on RK49.5 cells or by repairing the deletion of gene 10 in the revertant virus KyAΔ49.5R. Analysis of cells infected with the UL49.5-negative EHV-1 demonstrated that gM was not transported to the trans-Golgi network in the absence of the UL49.5 product. In contrast, gM was efficiently transported and processed to the endo H-resistant mature form in KyAΔ49.5-infected RK49.5 cells. Furthermore, radioimmunoprecipitation experiments demonstrated that gM maturation was observed only if a 10,000-
M
r
protein was coprecipitated with gM in KyA- or KyAΔ49.5R-infected cells or virions. This protein was absent in cells infected with KyΔ49.5 or KyAΔgM, suggesting that it was the EHV-1 UL49.5 product. Taken together, our results demonstrate that the expression of the EHV-1 UL49.5 product is necessary and sufficient for gM processing and that it is required for efficient virus replication.
Herpes Simplex Virus Vectors Elicit Durable Immune Responses in the Presence of Preexisting Host Immunity
Authors: Brockman Mark A. , Knipe David M.
Published: July 2002

Herpes simplex virus (HSV) recombinants are being developed as vaccine vectors for the expression of heterologous antigens. There is concern, however, that preexisting HSV immunity may decrease their effectiveness. We have addressed this issue in an animal model. Immunized mice were inoculated with a replication-defective HSV-1 vector that expressed theEscherichia coliβ-galactosidase protein as a model antigen. We assessed vector efficacy by analyzing the immunoglobulin G (IgG) antibody response and cellular proliferative response directed against β-galactosidase. We report that the ability of the vector to induce antibody or proliferative responses was not diminished by preexisting immunity to HSV. Of further note, the anti-HSV and anti-β-galactosidase IgG responses following vector administration were extremely durable in both immunized and naive mice. These results indicate that the ability of a replication-defective HSV-derived vaccine vector to elicit long-lived immune responses in mice is not impaired by prior HSV exposure.
Parameters of Human Hepatitis Delta Virus Genome Replication: the Quantity, Quality, and Intracellular Distribution of Viral Proteins and RNA
Authors: Gudima Severin , Chang Jinhong , Moraleda Gloria , Azvolinsky Anna , Taylor John
Published: July 2002

Assembly of hepatitis delta virus (HDV) in infected human hepatocytes involves association of the 1,679- nucleotide single-stranded genomic RNA (δRNA) with multiple copies of both small and large forms of the delta protein (δAg) to form a ribonucleoprotein particle which in turn interacts with envelope proteins of the natural helper virus, hepatitis B virus. Subsequently, for initiation of a new round of replication, the amount of small δAg within the assembled HDV particle is both necessary and sufficient. Quantitative assays were used in order to better understand just how much δAg is needed. The molar ratio of δAg species to genomic δRNA in assembled HDV particles was approximately 200. Next, this ratio was determined for cells under several different experimental situations in which HDV genome replication was occurring. These included replication in woodchuck liver and also in mouse liver and skeletal muscle, as well as replication in stably and transiently transfected cultured human hepatoblastoma cells. Surprisingly, in almost all these situations the molar ratios were comparable to that observed for HDV particles. This was true for different times after the initiation of replication and was independent of whether or not virus assembly was occurring. Cell fractionation combined with quantitative assays was used to test whether the majority of δAg and δRNA were colocalized during HDV replication in transfected cells. The cytoplasmic fraction contained the majority of δAg and genomic δRNA. Finally, the quality of δAg and δRNA, especially at relatively late times after the initiation of replication, was examined by using reverse transcription-PCR, cloning, and sequencing through the entire δAg open reading frame. When virus assembly and spread were not possible, 20% or less of the predicted δAg would have been able to support HDV replication. In summary, an examination of the quantity, quality and intracellular distribution of δAg and δRNA in several different experimental systems has provided a better understanding of the parameters associated with the initiation, maintenance, and ultimate decline of HDV genome replication.
Transcriptional Targeting of Lentiviral Vectors by Long Terminal Repeat Enhancer Replacement
Authors: Lotti Francesco , Menguzzato Emilio , Rossi Claudia , Naldini Luigi , Ailles Laurie , Mavilio Fulvio , Ferrari Giuliana
Published: July 2002

Gene therapy of many genetic diseases requires permanent gene transfer into self-renewing stem cells and restriction of transgene expression to specific progenies. Human immunodeficiency virus (HIV)-derived lentiviral vectors are very effective in transducing rare, nondividing stem cell populations (e.g., hematopoietic stem cells) without altering their long-term repopulation and differentiation capacities. We developed a strategy for transcriptional targeting of lentiviral vectors based on replacing the viral long terminal repeat (LTR) enhancer with cell lineage-specific, genomic control elements. An upstream enhancer (HS2) of the erythroid-specific GATA-1 gene was used to replace most of the U3 region of the LTR, immediately upstream of the HIV type 1 (HIV-1) promoter. The modified LTR was used to drive the expression of a reporter gene (the green fluorescent protein [GFP] gene), while a second gene (a truncated form of the p75 nerve growth factor receptor [ΔLNGFR]) was placed under the control of an internal constitutive promoter to monitor cell transduction, or to immunoselect transduced cells, independently from the expression of the targeted promoter. The transcriptionally targeted vectors were used to transduce cell lines, human CD34
+
hematopoietic stem-progenitor cells, and murine bone marrow (BM)-repopulating stem cells. Gene expression was analyzed in the stem cell progeny in vitro and in vivo after xenotransplantation into nonobese diabetic-SCID mice or BM transplantation in coisogenic mice. The modified LTR directed high levels of transgene expression specifically in mature erythroblasts, in a TAT-independent fashion and with no alteration in titer, infectivity, and genomic stability of the lentiviral vector. Expression from the modified LTR was higher, better restricted, and showed less position-effect variegation than that obtained by the same combination of enhancer-promoter elements placed in a conventional, internal position. Cloning of the woodchuck hepatitis virus posttranscriptional regulatory element at a defined position in the targeted vector allowed selective accumulation of the genomic transcripts with respect to the internal RNA transcript, with no loss of cell-type restriction. A critical advantage of this targeting strategy is the use of a spliced, major viral transcript to express a therapeutic gene and that of an internal, independently regulated promoter to express an additional gene for either cell marking or in vivo selection purposes.
Benzazolylmethylheteroarylamines
Authors: Milata Viktor , Kada Rudolf
Published: July 2002

Recently, Katritzky and coworkers in a series of papers described preparation of various 1-(1-aryl/alkylaminoalkyl)benzotriazoles by reaction of benzotriazole, an aliphatic aldehyde (or the product of their reaction - 1-(1-hydroxyalkyl)benzotriazole) and an amine in ethanol. In some cases the formation of 1-bis(1-benzotriazolylalkyl)aryl/alkylamines was also observed. The aim of our study was to compare the reactivity of 2-, 3- and 4-aminopyridine in the reaction with 1-hydroxymethylbenzotriazole, and to find out whether and under which conditions also 1-hydroxymethylbenzimidazole enters an analogous reaction. We further studied the reaction of these two compounds with 5-amino-1-arylbenzazoles and the structure of the products.
Regularized Positive Exponential Sum (REPES) Program - A Way of Inverting Laplace Transform Data Obtained by Dynamic Light Scattering
Authors: Jakeš Jaromír
Published: July 2002

Computer program REPES, which has been used in treating dynamic light scattering data for eight years but never published, and the algorithm used within it are described in detail. A list of papers quoting REPES and a brief discussion of suitability and performance of regularizing methods in inverting Laplace transform are given.
Reactivity of Nitrogen Oxides Towards Sodium Carbonate and Hydrogencarbonate Based Solid Sorbents
Authors: Lippert Erich , Bach Pavel , Stejskalová Květoslava , Erdös Emerich , Mocek Karel
Published: July 2002

The reactivity of nitrogen oxides towards sodium carbonate and hydrogencarbonate based solids was measured within a range of variables which is of technological interest. The reactivity is significant at lower temperatures only and is not affected by the presence of carbon dioxide or oxygen. Water vapor acts as a catalyst. Nitrogen dioxide is more reactive than nitrogen monoxide. The reactivity of the solid depends substantially on its origin and way of preparation. As compared with SO2 or HCl, nitrogen oxides exhibit a lower reactivity, presumably because the solid is blocked by the reaction products.
Long-Range Effect of 17-Substituents in 3-Oxo Steroids on 4,5-Double Bond Hydrogenation
Authors: Šídová Romana , Stránský Karel , Kasal Alexander , Slavíková Barbora , Kohout Ladislav
Published: July 2002

The long-range effect of substituents in the 17-position on the hydrogenation of double bond of the steroidal ∆4-3-ketones in acetic acid on a platinum catalyst is described in a series of testosterone (1) and epitestosterone (5) esters with carboxylic acids of varying alkyl chain length. The ratio 5α- to 5β-products is affected by the nature of substituents in the position 17.
Autoxidation vs Hydrolysis in 16α-Acyloxy Steroids
Authors: Slavíková Barbora , Kasal Alexander , Buděšínský Miloš
Published: July 2002

Some enolizable α-hydroxy ketones are extremely susceptible to oxidation with traces of air in a reaction vessel. Autoxidation can be used in synthesis of oxo acids or diacids and their derivatives. Yet alkaline hydrolysis of the substrate is possible though under strictly air-free conditions.
32P-Postlabelling Analysis of DNA Adducts with 1-(Phenylazo)-2-naphthol (Sudan I, Solvent Yellow 14) Formed in vivo in Fisher 344 Rats
Authors: Stiborová Marie , Schmeiser Heinz H. , Breuer Andrea , Frei Eva
Published: July 2002

We report the analysis of DNA adducts with 1-(phenylazo)-2-naphthol in the liver and urinary bladder of Fisher 344 rats treated orally with this dye. DNA adducts were detected and quantitated using the nuclease P1-enhanced version of the 32P-postlabelling assay. Two variations of multidirectional chromatographic systems were used to resolve either bulky and/or smaller (polar) 32P-labelled adducts by TLC. In the present study, a double oral administration of the dye (500 mg/kg) for one day yielded negative results in 32P-postlabelling assay of liver DNA (24 h after dosing). However, three DNA adducts in the urinary bladder were detected under the same conditions of treatment. Chromatography experiments indicated that the two principal DNA adducts detected in the urinary bladder of Fisher 344 rats were the same as those detected in DNA modified by 1-(phenylazo)-2-naphthol and its metabolite 1-(phenylazo)naphthalene-2,6-diol after their activation with peroxidase in vitro. The results presented here strongly suggest that peroxidase itself or in a combination with cytochrome P450 participates in the initiation phase of 1-(phenylazo)-2-naphthol carcinogenesis in the urinary bladder.
Synthesis and Analgesic Activity of Some 1-Benzofurans, 1-Benzothiophenes and Indoles
Authors: Rádl Stanislav , Hezký Petr , Urbánková Jitka , Váchal Petr , Krejčí Ivan
Published: July 2002

3-Unsubstituted 1-benzofurans 1e and 1f, 3-methyl-1-benzofurans 1a-1d, and 3-amino-1-benzofurans 2a-2l, as well as 3-amino-1-benzothiophenes 3a, 3b and 3-aminoindoles 4a-4f, 11a, and 11b were prepared and tested as analgesics. The 3-amino-1-benzofurans 2 were prepared from the corresponding 2-hydroxybenzonitriles 5 and phenacyl bromides 6 via intermediates 7. Similar treatment of 2-sulfanylbenzonitrile (8) provided 3-amino-1-benzothiophenes 3. Appropriately substituted 2-aminobenzonitriles 9 then provided N-substituted 3-aminoindoles 4. 1-(Ethoxycarbonyl)indoles 4e and 4f were successfully deprotected giving indoles 11a and 11b, respectively.
Preconcentration and Separation of Gold on Modified Silica Gel
Authors: Hájková Jana , Kanický Viktor , Otruba Vítězslav
Published: July 2002

Selective preconcentration of gold in the form of ion associates of Au(III) chloro complex or Au(I) cyano complex with quaternary bases was studied using modified silica gel. Solutions containing gold in the presence of the complexing anion and a quaternary base were pumped through a Separon™ SGX C18 column. After elution with 96 vol.% ethanol, the eluate was evaporated in the presence a complexing agent (chloride or cyanide). The resulting aqueous solutions were analyzed with inductively coupled plasma atomic emission spectrometry. The influence of concentration of onium salts, chlorides, nitrates, and the influence of the sample volume on the sorption were studied. The sorption flow rate and the elution volume were optimized, and the separation from a geological sample matrix was verified. The best recovery (100%) of preconcentration of Au(III) chloro complex was obtained with [1-(ethoxycarbonyl)pentadecyl]trimethylammonium bromide and 1-hexadecylpyridinium chloride at concentrations of 0.006 and 0.004 mol dm-3, respectively. The sorption flow rate of a sample may vary in the range 0.4-7 cm3 min-1, the elution volume was between 5 and 6 cm3. The recovery decreased with increasing sample volume above 250 cm3 and was diminished by 10% in the presence of nitrates above 0.4 mol dm-3. Major accompanying elements Fe and Mn, which considerably interfere at the Au I 242.795 nm line, were quantitatively separated from gold. A Czech certified reference material 1001 Mokrsko was analyzed using the developed preconcentration technique and inductively coupled plasma atomic emission spectrometry, inductively coupled plasma mass spectrometry, and atomic absorption spectrometry. Sorption of ion associates of Au(I) cyano complex was also efficient.
Characterisation of breathing and associated central autonomic dysfunction in the Rett disorder
Authors: Julu P O O , Kerr A M , Apartopoulos F , Al-Rawas S , Engerström I Witt , Engerström L , Jamal G A , Hansen S
Published: July 2002

AIM

To investigate breathing rhythm and brain stem autonomic control in patients with Rett disorder.

SETTING

Two university teaching hospitals in the United Kingdom and the Rett Centre, Sweden.

PATIENTS

56 female patients with Rett disorder, aged 2–35 years; 11 controls aged 5–28 years.

DESIGN

One hour recordings of breathing movement, blood pressure, ECG R-R interval, heart rate, transcutaneous blood gases, cardiac vagal tone, and cardiac sensitivity to baroreflex measured on-line with synchronous EEG and video. Breathing rhythms were analysed in 47 cases.

RESULTS

Respiratory rhythm was normal during sleep and abnormal in the waking state. Forced and apneustic breathing were prominent among 5–10 year olds, and Valsalva breathing in the over 18 year olds, who were also most likely to breathe normally. Inadequate breathing peaked among 10–18 year olds. Inadequate and exaggerated breathing was associated with vacant spells. Resting cardiac vagal tone and cardiac sensitivity to baroreflex were reduced.

CONCLUSIONS

Labile respiratory rhythms and poor integrative inhibition in Rett disorder suggest brain immaturity. Linking this to an early monoaminergic defect suggests possible targets for the MECP2 gene in clinical intervention. Exaggerated and inadequate autonomic responses may contribute to sudden death.
Physiological and biomechanical adaptations to the cycle to run transition in Olympic triathlon: review and practical recommendations for training
Authors: Millet Gregoire P , Vleck Veronica E
Published: July 2002

Current knowledge of the physiological, biomechanical, and sensory effects of the cycle to run transition in the Olympic triathlon (1.5 km, 10 km, 40 km) is reviewed and implications for the training of junior and elite triathletes are discussed. Triathlon running elicits hyperventilation, increased heart rate, decreased pulmonary compliance, and exercise induced hypoxaemia. This may be due to exercise intensity, ventilatory muscle fatigue, dehydration, muscle fibre damage, a shift in metabolism towards fat oxidation, and depleted glycogen stores after a 40 km cycle. The energy cost (CR) of running during the cycle to run transition is also increased over that of control running. The increase in CRvaries from 1.6% to 11.6% and is a reflection of triathlete ability level. This increase may be partly related to kinematic alterations, but research suggests that most biomechanical parameters are unchanged. A more forward leaning trunk inclination is the most significant observation reported. Running pattern, and thus running economy, could also be influenced by sensorimotor perturbations related to the change in posture. Technical skill in the transition area is obviously very important. The conditions under which the preceding cycling section is performed—that is, steady state or stochastic power output, drafting or non-drafting—are likely to influence the speed of adjustment to transition. The extent to which a decrease in the average 10 km running speed occurs during competition must be investigated further. It is clear that the higher the athlete is placed in the field at the end of the bike section, the greater the importance to their finishing position of both a quick transition area time and optimal adjustment to the physiological demands of the cycle to run transition. The need for, and current methods of, training to prepare junior and elite triathletes for a better transition are critically reviewed in light of the effects of sequential cycle to run exercise.
Short term power output in relation to growth and maturation
Authors: Armstrong N , Welsman J R , Chia M Y H
Published: July 2002

Objective—To examine short term power output during growth and maturation using a multilevel modelling approach.

Methods—Body mass, stature, and triceps and subscapular skinfold thicknesses of boys and girls, aged 12.2 (0.4) years (mean (SD)) at the onset of the study, were measured at age 12, 13, and 17 years. Sexual maturation, classified according to Tanner's stage of pubic hair development, was assessed on the first two occasions and assumed to be stage 5 at 17 years. Peak power (PP) and mean power (MP) were assessed on each occasion using the Wingate anaerobic test.

Results—Initial models, founded on 417 determinations of short term power output, identified body mass, stature, and age as significant explanatory variables of both PP and MP. The values for girls were significantly lower than those for boys, and a significant age by sex interaction described a progressive divergence in the MP of boys and girls. The introduction of sum of two skinfold thicknesses produced a model with an improvement in fit as indicated by a significant change in log likelihood. The stature term was negated and the body mass term increased. The age and sex terms were reduced but remained significant. The age by sex interaction term remained a significant explanatory variable for MP. Maturity effects were non-significant additional explanatory variables in all models of power output.

Conclusion—The values of PP and MP for boys are higher than those for girls, and, for MP, sex differences increase with age. Body mass and skinfold thicknesses are significant influences on both PP and MP, but age exerts a positive but non-linear effect on power output independent of body size and fatness.
Effect of warming up on knee proprioception before sporting activity
Authors: Bartlett M J , Warren P J
Published: July 2002

Background: It is now generally accepted that the ligamentous structures of the knee not only act as mechanical restraints but also have a neurophysiological role in joint function and protection. A report that knee joint laxity increases with exercise raised the question as to whether there is any compensatory change in joint position appreciation.

Objective: To test whether there is a compensatory mechanism for increased ligamentous laxity during normal levels of activity.

Methods: Joint position appreciation was measured, using a previously reported technique, in the knees of sportsmen at rest and after warm up.

Results: Joint position appreciation was found to be significantly more sensitive after warm up (p = 0.005).

Conclusions: These findings indicate that joint position appreciation within the knee accommodates physiological changes within the ligaments and muscles after exercise.
Effects of exercise on soluble transferrin receptor and other variables of the iron status
Authors: Schumacher Y O , Schmid A , König D , Berg A
Published: July 2002

Background:Soluble transferrin receptor (sTfr) is a new marker of iron status and erythropoietic activity. It has been included in multivariable blood testing models for the detection of performance enhancing erythropoietin misuse in sport.

Objective:To evaluate the effect of different types and volumes of physical activity on sTfr concentration, variables of iron status (ferritin, transferrin, iron, and protein), and haematological indices.

Methods:Thirty nine subjects were divided into three groups: 1, untrained (n = 12); 2, moderately trained (n = 14); 3, highly trained (n = 13, seven men, six women). Groups 1 and 2 carried out two exercise tests: an incremental running test until exhaustion (test A) and a 45 minute constant speed running test at 70% Vo2max(test B). Group 3 performed three days (women) or four days (men) of prolonged aerobic cycling exercise. The above variables together with haemoglobin and packed cell volume were analysed in venous blood samples before and after exercise. Changes in blood and plasma volume were estimated.

Results:sTfr levels were slightly increased in trained and untrained subjects immediately after test A. Test B and aerobic exercise had no significant effect on sTfr. Ferritin levels were increased after the laboratory tests for trained and untrained subjects and after prolonged aerobic exercise in male cyclists. Transferrin was increased significantly in trained and untrained subjects after both laboratory tests, but remained unchanged after prolonged exercise. Plasma and blood volumes were decreased after the laboratory tests but increased after aerobic exercise. No differences in the variables were observed between trained and untrained subjects with respect to response to exercise.

Conclusion:The changes in sTfr and the variables of iron status can be mainly attributed to exercise induced changes in volume. Taking these limitations into account, sTfr can be recommended as a marker of iron deficiency in athletes.
Low frequency of HLA-DRB1*11 in hepatitis C virus induced end stage liver disease
Authors: Tillmann H L , Chen D-F , Trautwein C , Kliem V , Grundey A , Berning-Haag A , Böker K , Kubicka S , Pastucha L , Stangel W , Manns M P
Published: July 2002

Hepatitis C virus (HCV) infection becomes chronic in more than 70% of patients, leading to end stage liver disease in about 20–30% of these patients. Apart from the virus itself, host factors that modulate the immune response are likely to be involved in determining the outcome of HCV infection. Studies on the association of human leucocyte antigens (HLAs) and HCV infection have shown inconsistent results. Selection of patient subgroups may be crucial. However, any association relevant to HCV disease progression will become evident, especially in those patients with end stage liver disease. Therefore, we analysed the phenotype frequencies of HLA antigens in two groups of 69 and 39 patients with HCV induced liver cirrhosis who had received a transplant or were awaiting liver transplantation. The first group was typed serologically and compared with 331 blood and liver donors. The second group, prospectively HLA typed by a polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) procedure for HLA-DRB and DQB alleles, was compared with another 170 PCR-SSO typed and randomly selected blood donors. Decreased frequencies for HLA-DR5 and HLA-DQ3 were found in one group of patients with HCV induced liver cirrhosis compared with the control groups. In the second analysis comparing 39 patients with end stage liver cirrhosis with blood donors, we confirmed the significant decrease in HLA-DRB1*11 and HLA-DQB1*03, which corresponded to serological HLA-DR5 and HLA-DQ3 antigens, respectively. Our results show that the presence of HLA-DRB1*11 and HLA-DQB1*03 alleles is associated with a reduced risk for the development of HCV induced end stage liver disease.