bluuebunny/crossref_metadata_2025 huggingface dataset:
High Prevalence of TT Virus (TTV) and TTV-Like Minivirus in Cervical Swabs
Authors: Fornai Claudia , Maggi Fabrizio , Vatteroni Maria Linda , Pistello Mauro , Bendinelli Mauro
Published: July 2002

Genomes of TT virus (TTV) and TTV-like minivirus DNA were detected in 80% and 61% of cervical swabs from healthy women, respectively, regardless of concurrent human papillomavirus infection. These results show that the potential exists for sexual transmission of TTV and related viruses.
Relevance of the Number of Positive Bottles in Determining Clinical Significance of Coagulase-Negative Staphylococci in Blood Cultures
Authors: Mirrett Stanley , Weinstein Melvin P. , Reimer Larry G. , Wilson Michael L. , Reller L. Barth
Published: July 2002

Coagulase-negative staphylococci (CNS) are the most commonly isolated contaminants from blood cultures, yet they frequently cause true infections. Determining the clinical significance of CNS is difficult, and clinicians often consider the number of positive bottles within a set of blood culture bottles in their assessment. Therefore, in three separate studies, we counted the number of positive bottles within blood culture sets comprising two, three, or four bottles in order to predict whether or not CNS were clinically significant isolates (CSI) in adult patients with suspected sepsis. Each culture was evaluated by independent, published clinical criteria to determine its clinical importance. Of 486 positive sets that included two adequately filled bottles, 127 (26%) CNS were CSI, 329 (67%) were contaminants, and 30 (6%) were indeterminate as a cause of sepsis. Among CSI, 39 and 61% were isolated from one and two bottles, respectively. The positive predictive value for sepsis was 18% when one bottle was positive and 37% when both bottles were positive. Of 235 positive sets that included three adequately filled bottles, 81 (34%) were CSI, 109 (46%) were contaminants, and 45 (19%) were indeterminate as a cause of sepsis. Of CSI, 43, 38, and 19% were found in one, two, and three bottles, respectively. The positive predictive value for sepsis was 28, 52, and 30% when one, two and three bottles were positive. Of 303 positive blood culture sets that included four adequately filled bottles, 64 (21%) were considered CSI, 197 (65%) were contaminants, and 42 (14%) were indeterminate as a cause of sepsis. Of CSI, 27, 28, 19, and 27% were found in one, two, three, and four bottles, respectively. The positive predictive value for sepsis was 11, 30, 34, and 37% when one, two, three, and four bottles were positive. We conclude that the number of culture bottles positive in a given culture set cannot reliably predict the clinical significance of the CNS isolated and, therefore, should not be used as a criterion for determining whether or not an isolate represents true infection or contamination.
Molecular Characterization of Group A Streptococcus Strains Isolated during a Scarlet Fever Outbreak
Authors: Perea-Mejía Luis M. , Inzunza-Montiel Alma E. , Cravioto Alejandro
Published: July 2002

Forty group A streptococcus (GAS) isolates, recovered during a scarlet fever outbreak, were grouped based on their
Dde
I restriction profiles from
emm
amplicons. Twenty-seven isolates were identified by sequencing as
emm2
. The
emm2
isolates showed the
speA1
,
speB1
, and
speC1
alleles. Isolation of this GAS type from scarlet fever outbreaks is uncommon.
The VP5 Domain of VP4 Can Mediate Attachment of Rotaviruses to Cells
Authors: Zárate Selene , Espinosa Rafaela , Romero Pedro , Méndez Ernesto , Arias Carlos F. , López Susana
Published: July 2002

Some animal rotaviruses require the presence of sialic acid (SA) on the cell surface to infect the cell. We have isolated variants of rhesus rotavirus (RRV) whose infectivity no longer depends on SA. Both the SA-dependent and -independent interactions of these viruses with the cell are mediated by the virus spike protein VP4, which is cleaved by trypsin into two domains, VP5 and VP8. In this work we have compared the binding characteristics of wild-type RRV and its variant nar3 to MA104 cells. In a direct nonradioactive binding assay, both viruses bound to the cells in a saturable and specific manner. When neutralizing monoclonal antibodies directed to both the VP8 and VP5 domains of VP4 were used to block virus binding, antibodies to VP8 blocked the cell attachment of wild-type RRV but not that of the variant nar3. Conversely, an antibody to VP5 inhibited the binding of nar3 but not that of RRV. These results suggest that while RRV binds to the cell through VP8, the variant does so through the VP5 domain of VP4. This observation was further sustained by the fact that recombinant VP8 and VP5 proteins, produced in bacteria as fusion products with glutathione
S
-transferase, were found to bind to MA104 cells in a specific and saturable manner and, when preincubated with the cell, were capable of inhibiting the binding of wild-type and variant viruses, respectively. In addition, the VP5 and VP8 recombinant proteins inhibited the infectivity of nar3 and RRV, respectively, confirming the results obtained in the binding assays. Interestingly, when the infectivity assay was performed on neuraminidase-treated cells, the VP5 fusion protein was also found to inhibit the infectivity of RRV, suggesting that RRV could bind to the cell through two sequential steps mediated by the interaction of VP8 and VP5 with SA-containing and SA-independent cell surface receptors, respectively.
A Recent Outbreak of Human Immunodeficiency Virus Type 1 Infection in Southern China Was Initiated by Two Highly Homogeneous, Geographically Separated Strains, Circulating Recombinant Form AE and a Novel BC Recombinant
Authors: Piyasirisilp Sucheep , McCutchan Francine E. , Carr Jean K. , Sanders-Buell Eric , Liu Wei , Chen Jie , Wagner Ralf , Wolf Hans , Shao Yiming , Lai Shenghan , Beyrer Chris , Yu Xiao-Fang
Published: July 2002

New outbreaks of human immunodeficiency virus type 1 (HIV-1) among injecting drug users (IDUs) are spreading in China along heroin trafficking routes. Recently, two separate HIV-1 epidemics among IDUs were reported in Guangxi, Southern China, where partial sequencing of the
env
gene showed subtype C and circulating recombinant form (CRF) AE. We evaluated five virtually full-length HIV-1 genome sequences from IDUs in Guangxi to determine the genetic diversity and the presence of intersubtype recombinants. Sequence analysis showed two geographically separated, highly homogeneous HIV-1 strains. B/C intersubtype recombinants were found in three IDUs from Baise City, in a mountainous region near the Yunnan-Guangxi border. These were mostly subtype C, with portions of the capsid and reverse transcriptase (RT) genes from subtype B. The subtype B portion of the capsid was located in the N-terminal domain, which has been shown to influence virus core maturation, virus infectivity, and binding to cyclophilin A, whereas the subtype B portion of RT was located in the palm subdomain, which is the active site of the enzyme. These BC recombinants differed from a BC recombinant found in Xinjiang Province in northwestern China. CRF AE strains were found in IDUs from Nanning, the capital of Guangxi, and in IDUs from Pingxiang City near the China-Vietnam border. The AE and BC recombinants were both remarkable for their low interpatient diversity, less than 1% for the full genome. Rapid spread of HIV-1 among IDUs may foster the emergence of highly homogeneous strains, including novel recombinants in regions with multiple subtypes.
Mutations in Herpes Simplex Virus Glycoprotein D Distinguish Entry of Free Virus from Cell-Cell Spread
Authors: Rauch Daniel A. , Rodriguez Nilda , Roller Richard J.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) is an essential component of the entry apparatus that is responsible for viral penetration and subsequent cell-cell spread. To test the hypothesis that gD may serve distinguishable functions in entry of free virus and cell-cell spread, mutants were selected for growth on U
S
11cl19.3 cells, which are resistant to both processes due to the lack of a functional gD receptor, and then tested for their ability to enter as free virus and to spread from cell to cell. Unlike their wild-type parent, HSV-1(F), the variants that emerged from this selection, which were named SP mutants, are all capable of forming macroscopic plaques on the resistant cells. This ability is caused by a marked increase in cell-cell spread without a concomitant increase in efficiency of entry of free virus. gD substitutions that arose within these mutants are sufficient to mediate cell-cell spread in U
S
11cl19.3 cells but are insufficient to overcome the restriction to entry of free virions. These results suggest that mutations in gD (i) are sufficient but not necessary to overcome the block to cell-cell spread exhibited by U
S
11cl19.3 cells and (ii) are insufficient to mediate entry of free virus in the same cells.
Species-Specific TT Viruses and Cross-Species Infection in Nonhuman Primates
Authors: Okamoto Hiroaki , Fukuda Masako , Tawara Akio , Nishizawa Tsutomu , Itoh Yukio , Hayasaka Ikuo , Tsuda Fumio , Tanaka Takeshi , Miyakawa Yuzo , Mayumi Makoto
Published: July 2002

Viruses resembling human TT virus (TTV) were searched for in sera from nonhuman primates by PCR with primers deduced from well-conserved areas in the untranslated region. TTV DNA was detected in 102 (98%) of 104 chimpanzees, 9 (90%) of 10 Japanese macaques, 4 (100%) of 4 red-bellied tamarins, 5 (83%) of 6 cotton-top tamarins, and 5 (100%) of 5 douroucoulis tested. Analysis of the amplification products of 90 to 106 nucleotides revealed TTV DNA sequences specific for each species, with a decreasing similarity to human TTV in the order of chimpanzee, Japanese macaque, and tamarin/douroucouli TTVs. Full-length viral sequences were amplified by PCR with inverted nested primers deduced from the untranslated region of TTV DNA from each species. All animal TTVs were found to be circular with a genomic length at 3.5 to 3.8 kb, which was comparable to or slightly shorter than human TTV. Sequences closely similar to human TTV were determined by PCR with primers deduced from a coding region (N22 region) and were detected in 49 (47%) of the 104 chimpanzees; they were not found in any animals of the other species. Sequence analysis of the N22 region (222 to 225 nucleotides) of chimpanzee TTV DNAs disclosed four genetic groups that differed by 36.1 to 50.2% from one another; they were 35.0 to 52.8% divergent from any of the 16 genotypes of human TTV. Of the 104 chimpanzees, only 1 was viremic with human TTV of genotype 1a. It was among the 53 chimpanzees which had been used in transmission experiments with human hepatitis viruses. Antibody to TTV of genotype 1a was detected significantly more frequently in the chimpanzees that had been used in transmission experiments than in those that had not (8 of 28 [29%] and 3 of 35 [9%], respectively;
P = 0.038).
These results indicate that species-specific TTVs are prevalent in nonhuman primates and that human TTV can cross-infect chimpanzees.
Evolutionary Rate and Genetic Drift of Hepatitis C Virus Are Not Correlated with the Host Immune Response: Studies of Infected Donor-Recipient Clusters
Authors: Allain Jean-Pierre , Dong Yan , Vandamme Anne-Mieke , Moulton Vincent , Salemi Marco
Published: July 2002

Six donor-recipient clusters of hepatitis C virus (HCV)-infected individuals were studied. For five clusters the period of infection of the donor could be estimated, and for all six clusters the time of infection of the recipients from the donor via blood transfusion was also precisely known. Detailed phylogenetic analyses were carried out to investigate the genomic evolution of the viral quasispecies within infected individuals in each cluster. The molecular clock analysis showed that HCV quasispecies within a patient are evolving at the same rate and that donors that have been infected for longer time tend to have a lower evolutionary rate. Phylogenetic analysis based on the split decomposition method revealed different evolutionary patterns in different donor-recipient clusters. Reactivity of antibody against the first hypervariable region (HVR1) of HCV in donor and recipient sera was evaluated and correlated to the calculated evolutionary rate. Results indicate that anti-HVR1 reactivity was related more to the overall level of humoral immune response of the host than to the HVR1 sequence itself, suggesting that the particular sequence of the HVR1 peptides is not the determinant of reactivity. Moreover, no correlation was found between the evolutionary rate or the heterogeneity of the viral quasispecies in the patients and the strength of the immune response to HVR1 epitopes. Rather, the results seem to imply that genetic drift is less dependent on immune pressure than on the rate of evolution and that the genetic drift of HCV is independent of the host immune pressure.
Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit
Authors: Werner Susanne , Wöhrl Birgitta M.
Published: July 2002

The genes encoding the α (63-kDa) and β (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV αβ and αPol RTs within which one subunit was selectively mutated. When αβ heterodimers contained the Asp 181→Asn mutation in their β subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their α subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in αβ heterodimers mutated in the β subunit but was lost when the mutation was present in the α subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the α subunit of the heterodimeric RSV RT αβ.
Molecular Analysis of the Abnormal Prion Protein during Coinfection of Mice by Bovine Spongiform Encephalopathy and a Scrapie Agent
Authors: Baron Thierry G. M. , Biacabe Anne-Gaelle
Published: July 2002

Molecular features of the proteinase K-resistant prion protein (PrP res) may discriminate among prion strains, and a specific signature could be found during infection by the infectious agent causing bovine spongiform encephalopathy (BSE). To investigate the molecular basis of BSE adaptation and selection, we established a model of coinfection of mice by both BSE and a sheep scrapie strain (C506M3). We now show that the PrP res features in these mice, characterized by glycoform ratios and electrophoretic mobilities, may be undistinguishable from those found in mice infected with scrapie only, including when mice were inoculated by both strains at the same time and by the same intracerebral inoculation route. Western blot analysis using different antibodies against sequences near the putative N-terminal end of PrP res also demonstrated differences in the main proteinase K cleavage sites between mice showing either the BSE or scrapie PrP res profile. These results, which may be linked to higher levels of PrP res associated with infection by scrapie, were similar following a challenge by a higher dose of the BSE agent during coinfection by both strains intracerebrally. Whereas PrP res extraction methods used allowed us to distinguish type 1 and type 2 PrP res, differing, like BSE and scrapie, by their electrophoretic mobilities, in the same brain region of some patients with Creutzfeldt-Jakob disease, analysis of in vitro mixtures of BSE and scrapie brain homogenates did not allow us to distinguish BSE and scrapie PrP res. These results suggest that the BSE agent, the origin of which remains unknown so far but which may have arisen from a sheep scrapie agent, may be hidden by a scrapie strain during attempts to identify it by molecular studies and following transmission of the disease in mice.
Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase
Authors: Boyer Paul L. , Sarafianos Stefan G. , Arnold Edward , Hughes Stephen H.
Published: July 2002

Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. The AZT resistance mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient. So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. Steric constraints involving the azido group cause the end of an AZT 5′-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.
Identification of Specific Cucumber Necrosis Virus Coat Protein Amino Acids Affecting Fungus Transmission and Zoospore Attachment
Authors: Kakani Kishore , Sgro Jean-Yves , Rochon D'Ann
Published: July 2002

Cucumber necrosis virus (CNV) is naturally transmitted in the soil by zoospores of the fungal vector
Olpidium bornovanus
. Successful transmission requires that virus particles attach to the surface of zoospores prior to zoospore encystment on host roots. Mechanically passaged CNV was screened for mutants deficient in fungus transmission. We found six such mutants, exhibiting transmission efficiencies ranging from approximately 14 to 76% of that of wild-type (WT) CNV. Results of in vitro virus-zoospore binding assays show that each mutant binds to zoospores less efficiently than WT CNV (21 to 68%), suggesting that defects in transmission for these mutants are at least partially due to inefficient zoospore binding. Analysis of the structure of the CNV coat protein subunit and trimer indicates that affected amino acids in all of the mutants are located in the shell or protruding domain and that five of six of them are potentially exposed on the surface of the virus particle. In addition, several of the mutated sites, along with a previously identified site in a region of subunit-subunit interaction in the coat protein shell domain (M. A. Robbins, R. D. Reade, and D. M. Rochon, Virology 234:138–146, 1997), are located on the particle quasi-threefold axis, suggesting that this region of the capsid may be important in recognition of a putative zoospore receptor. The individual sites may directly affect attachment to a receptor or could indirectly affect attachment via changes in virion conformation.
Transmembrane Topology of PiT-2, a Phosphate Transporter-Retrovirus Receptor
Authors: Salaün Christine , Rodrigues Pierre , Heard Jean Michel
Published: July 2002

PiT-1 and PiT-2 are related multiple transmembrane proteins which function as sodium-dependent phosphate transporters and as the cell receptors of several oncoretroviruses. Two copies of a homology domain that is found in distantly related species assign these proteins to a large family of phosphate transporters. A current membrane topology model of PiT-1 and PiT-2 predicts 10 transmembrane domains. However, the validity of this model has not been addressed experimentally. We addressed this issue by a comprehensive study of human PiT-2. Evidence was obtained for glycosylation of asparagine 81. Epitope tagging showed that the N- and C-terminal extremities are extracellular. The orientation of C-terminal-truncation mutants expressed in cell-free translation assays and incorporated into microsomal membranes was examined by immunoprecipitation. Data were interpreted with respect to previous knowledge about retrovirus binding sites, to the existence of repeated homology domains, and to predictions made in family members. A model in which PiT-2 has 12 transmembrane domains and extracellular N- and C-terminal extremities is proposed. This model, which differs significantly from previous predictions about PiT-2 topology, may be useful for further investigations of PiT-2 interactions with other proteins and for the understanding of PiT-2 transporter and virus receptor functions.
Displacement of YY1 by Differentiation-Specific Transcription Factor hSkn-1a Activates the P
670
Promoter of Human Papillomavirus Type 16
Authors: Kukimoto Iwao , Kanda Tadahito
Published: July 2002

Transcription from human papillomavirus type 16 (HPV16) P
670
, a promoter in the E7 open reading frame, is repressed in undifferentiated keratinocytes but becomes activated upon differentiation. We showed that the transient luciferase expression driven by P
670
was markedly enhanced in HeLa cells cotransfected with an expression plasmid for human Skn-1a (hSkn-1a), a transcription factor specific to differentiating keratinocytes. The hSkn-1a POU domain alone, which mediates sequence-specific DNA binding, was sufficient to activate the expression of luciferase. Electrophoretic mobility shift assay revealed the presence of two binding sites, sites 1 and 2, upstream of P
670
, which were shared by hSkn-1a and YY1. Site 1 bound more strongly to hSkn-1a than site 2 did. YY1 complexing with a short DNA fragment having site 1 was displaced by hSkn-1a, indicating that hSkn-1a's affinity with site 1 was stronger than YY1's. Disrupting the binding sites by nucleotide substitutions raised the basal expression level of luciferase and decreased the enhancing effect of hSkn-1a. In HeLa cells transfected with circular HPV16 DNA along with the expression plasmid for hSkn-1a, the transcript from P
670
was detectable, which indicates that the results obtained with the reporter plasmids are likely to have mimicked the regulation of P
670
in authentic HPV16 DNA. The data strongly suggest that the transcription from P
670
is repressed primarily by YY1 binding to the two sites, and the displacement of YY1 by hSkn-1a releases P
670
from the repression.
Unique Monoclonal Antibody Recognizing the Third Extracellular Loop of CXCR4 Induces Lymphocyte Agglutination and Enhances Human Immunodeficiency Virus Type 1-Mediated Syncytium Formation and Productive Infection
Authors: Tanaka Reiko , Yoshida Atsushi , Murakami Tsutomu , Baba Eishi , Lichtenfeld Julliane , Omori Takeru , Kimura Tohru , Tsurutani Naomi , Fujii Nobutaka , Wang Zi-Xuan , Peiper Stephen C. , Yamamoto Naoki , Tanaka Yuetsu
Published: July 2002

To increase insight into the structural basis of CXCR4 utilization in human immunodeficiency virus type 1 (HIV-1) infection, a new generation of three monoclonal antibodies (MAbs) was developed in WKA rats. The A80 MAb, which binds an epitope in the third extracellular loop (ECL3) of CXCR4, has unique biologic properties that provide novel insights into CXCR4 function. This agent enhanced syncytium formation in activated human peripheral blood mononuclear cells (PBMC) infected with X4 or R5 and CEM cells infected with X4 HIV-1 strains. Exposure to A80 increased the productive infection of activated CD4+T cells and CEM cells with R5 and X4 viruses, respectively. This antibody uniquely induced agglutination of PBMC and CEM cells but did not activate calcium mobilization. Agglutination induced by A80 was inhibited by stromal cell-derived factor 1, T22, and phorbol 12-myristate 13-acetate but was not significantly altered by pretreatment of cells with pertussis toxin, wortmannin, or MAbs to LFA-1, ICAM-1, ICAM-2, and ICAM-3. The binding of the A145 and A120 MAbs was mapped to the N-terminal extracellular domain and a conformational epitope involving ECL1 and ECL2, respectively. Both of these MAbs inhibited HIV-1 infection and lacked the novel properties of A80. These results suggest a new role for CXCR4 in homologous lymphocyte adhesion that is ligand independent and in HIV-1 infection.
Vesicular Stomatitis Virus G-Pseudotyped Lentivirus Vectors Mediate Efficient Apical Transduction of Polarized Quiescent Primary Alveolar Epithelial Cells
Authors: Borok Zea , Harboe-Schmidt Jens Erik , Brody Steven L. , You Yingjian , Zhou Beiyun , Li Xian , Cannon Paula M. , Kim Kwang-Jin , Crandall Edward D. , Kasahara Noriyuki
Published: July 2002

We investigated the use of lentivirus vectors for gene transfer to quiescent alveolar epithelial cells. Primary rat alveolar epithelial cells (AEC) grown on plastic or as polarized monolayers on tissue culture-treated polycarbonate semipermeable supports were transduced with a replication-defective human immunodeficiency virus-based lentivirus vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein and encoding an enhanced green fluorescent protein reporter gene. Transduction efficiency, evaluated by confocal microscopy and quantified by fluorescence-activated cell sorting, was dependent on the dose of vector, ranging from 4% at a multiplicity of infection (MOI) of 0.1 to 99% at an MOI of 50 for AEC grown on plastic. At a comparable titer and MOI, transduction of these cells by a similarly pseudotyped murine leukemia virus vector was ∼30-fold less than by the lentivirus vector. Importantly, comparison of lentivirus-mediated gene transfer from the apical or basolateral surface of confluent AEC monolayers (R
t
> 2 kΩ · cm
2
; MOI = 10) revealed efficient transduction only when VSV-G-pseudotyped lentivirus was applied apically. Furthermore, treatment with EGTA to increase access to the basolateral surface did not increase transduction of apically applied virus, indicating that transduction was primarily via the apical membrane domain. In contrast, differentiated tracheal epithelial cells were transduced by apically applied lentivirus only in the presence of EGTA and at a much lower overall efficiency (∼15-fold) than was observed for AEC. Efficient transduction of AEC from the apical cell surface supports the feasibility of using VSV-G-pseudotyped lentivirus vectors for gene transfer to the alveolar epithelium and suggests that differences exist between upper and lower airways in the polarity of available receptors for the VSV-G protein.
Abnormal Priming of CD4+T Cells by Dendritic Cells Expressing Hepatitis C Virus Core and E1 Proteins
Authors: Sarobe Pablo , Lasarte Juan José , Casares Noelia , López-Díaz de Cerio Ascensión , Baixeras Elena , Labarga Pablo , García Nicolás , Borrás-Cuesta Francisco , Prieto Jesús
Published: July 2002

Patients infected with hepatitis C virus (HCV) have an impaired response against HCV antigens while keeping immune competence for other antigens. We hypothesized that expression of HCV proteins in infected dendritic cells (DC) might impair their antigen-presenting function, leading to a defective anti-HCV T-cell immunity. To test this hypothesis, DC from normal donors were transduced with an adenovirus coding for HCV core and E1 proteins and these cells (DC-CE1) were used to stimulate T lymphocytes. DC-CE1 were poor stimulators of allogeneic reactions and of autologous primary and secondary proliferative responses. Autologous T cells stimulated with DC-CE1 exhibited a pattern of incomplete activation characterized by enhanced CD25 expression but reduced interleukin 2 production. The same pattern of incomplete lymphocyte activation was observed in CD4+T cells responding to HCV core in patients with chronic HCV infection. However, CD4+response to HCV core was normal in patients who cleared HCV after alpha interferon therapy. Moreover, a normal CD4+response to tetanus toxoid was found in both chronic HCV carriers and patients who had eliminated the infection. Our results suggest that expression of HCV structural antigens in infected DC disturbs their antigen-presenting function, leading to incomplete activation of anti-HCV-specific T cells and chronicity of infection. However, presentation of unrelated antigens by noninfected DC would allow normal T-cell immunity to other pathogens.
The Pseudorabies Virus VP22 Homologue (UL49) Is Dispensable for Virus Growth In Vitro and Has No Effect on Virulence and Neuronal Spread in Rodents
Authors: del Rio T. , Werner H. C. , Enquist L. W.
Published: July 2002

The tegument of herpesvirus virions is a distinctive structure whose assembly and function are not well understood. The herpes simplex virus type 1 VP22 tegument protein encoded by the UL49 gene is conserved among the alphaherpesviruses. Using cell biology and viral genetics, we provide an initial characterization of the pseudorabies virus (PRV) VP22 homologue. We identified three isoforms of VP22 present in PRV-infected cells that can be resolved by polyacrylamide gel electrophoresis. The predominant form is not phosphorylated and is present in virions, while the other two species are phosphorylated and excluded from virions. VP22 localized to the nucleus by 6 h postinfection, as determined by immunofluorescence and cell fractionation. VP22 immunofluorescence in the nucleus was both diffuse and in punctate structures. The punctate nuclear localization was the most pronounced form of staining and did not localize exclusively to sites of viral DNA replication. Unexpectedly, a VP22 null mutant had no obvious phenotypes during tissue culture infections and was similar to the wild type in all respects. Moreover, the VP22 null mutant was as virulent and neuroinvasive as the wild-type virus after infection of the rodent eye and spread to the brain using both anterograde and retrograde neuronal circuits.
The Maturation Process of pVP2 Requires Assembly of Infectious Bursal Disease Virus Capsids
Authors: Chevalier Christophe , Lepault Jean , Erk Inge , Da Costa Bruno , Delmas Bernard
Published: July 2002

Infectious bursal disease virus (IBDV) is a nonenveloped avian virus with a two-segment double-stranded RNA genome. Its T=13 icosahedral capsid is most probably assembled with 780 subunits of VP2 and 600 copies of VP3 and has a diameter of about 60 nm. VP1, the RNA-dependent RNA polymerase, resides inside the viral particle. Using a baculovirus expression system, we first observed that expression of the pVP2-VP4-VP3 polyprotein encoded by the genomic segment IBDA results mainly in the formation of tubules with a diameter of about 50 nm and composed of pVP2, the precursor of VP2. Very few virus-like particles (VLPs) and VP4 tubules with a diameter of about 25 nm were also identified. The inefficiency of VLP assembly was further investigated by expression of additional IBDA-derived constructs. Expression of pVP2 without any other polyprotein components results in the formation of isometric particles with a diameter of about 30 nm. VLPs were observed mainly when a large exogeneous polypeptide sequence (the green fluorescent protein sequence) was fused to the VP3 C-terminal domain. Large numbers of VLPs were visualized by electron microscopy, and single particles were shown to be fluorescent by standard and confocal microscopy analysis. Moreover, the final maturation process converting pVP2 into the VP2 mature form was observed on generated VLPs. We therefore conclude that the correct scaffolding of the VP3 can be artificially induced to promote the formation of VLPs and that the final processing of pVP2 to VP2 is controlled by this particular assembly. To our knowledge, this is the first report of the engineering of a morphogenesis switch to control a particular type of capsid protein assembly.
Equine Infectious Anemia Virus and the Ubiquitin-Proteasome System
Authors: Ott David E. , Coren Lori V. , Sowder Raymond C. , Adams Julian , Nagashima Kunio , Schubert Ulrich
Published: July 2002

Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9
Gag
proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors.
Solid-Phase Proteoliposomes Containing Human Immunodeficiency Virus Envelope Glycoproteins
Authors: Grundner Christoph , Mirzabekov Tajib , Sodroski Joseph , Wyatt Richard
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein gp120 mediates receptor binding and is the major target for neutralizing antibodies. A broadly neutralizing antibody response is likely to be a critical component of the immune response against HIV-1. Although antibodies against monomeric gp120 are readily elicited in immunized individuals, these antibodies are inefficient in neutralizing primary HIV-1 isolates. As a chronic pathogen, HIV-1 has evolved to avoid an optimal host response by a number of immune escape mechanisms. Monomeric gp120 that has dissociated from the functional trimer presents irrelevant epitopes that are not accessible on functional trimeric envelope glycoproteins. The resulting low level of antigenic cross-reactivity between monomeric gp120 and the functional spike may contribute to the inability of monomeric gp120 to elicit broadly neutralizing antibodies. Attempts to generate native, trimeric envelope glycoproteins as immunogens have been frustrated by both the lability of the gp120-gp41 interaction and the weak association between gp120 subunits. Here, we present solid-phase HIV-1 gp160ΔCT (cytoplasmic tail-deleted) proteoliposomes (PLs) containing native, trimeric envelope glycoproteins in a physiologic membrane setting. We present data that indicate that the gp160ΔCT glycoproteins on PLs are trimers and are recognized by several relevant conformational ligands in a manner similar to that for gp160ΔCT oligomers expressed on the cell surface. The PLs represent a significant advance over present envelope glycoprotein formulations as candidate immunogens for HIV vaccine design and development.
Apical Budding of a Recombinant Influenza A Virus Expressing a Hemagglutinin Protein with a Basolateral Localization Signal
Authors: Mora Rosalia , Rodriguez-Boulan Enrique , Palese Peter , García-Sastre Adolfo
Published: July 2002

Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.
Expression of Simian Immunodeficiency Virusnefin Immune Cells of Transgenic Mice Leads to a Severe AIDS-Like Disease
Authors: Simard Marie-Chantal , Chrobak Pavel , Kay Denis G. , Hanna Zaher , Jothy Serge , Jolicoeur Paul
Published: July 2002

In order to study the functions of simian immunodeficiency virus (SIV) Nef in vivo in a small-animal model, we constructed transgenic (Tg) mice expressing the SIVmac239nefgene in the natural target cells of the virus under the control of the human CD4 gene promoter (CD4C). These CD4C/SHIV-nefSIVTg mice develop a severe AIDS-like disease, with manifestations including premature death, failure to thrive or weight loss, wasting, thymic atrophy, an especially low number of peripheral CD8+T cells as well as a low number of peripheral CD4+T cells, diarrhea, splenomegaly, and kidney (interstitial nephritis, segmental glomerulosclerosis), lung (lymphocytic interstitial pneumonitis), and heart disease. In addition, these Tg mice fail to mount a class-switched antibody response after immunization with ovalbumin, they produce anti-DNA autoantibodies, and some of them developPneumocystis cariniilung infections. All these results suggest a generalized Nef-induced immunodeficiency. The low numbers of peripheral CD8+and CD4+T cells are likely to reflect a thymic defect and may be similar to the DiGeorge-like “thymic defect” immunophenotype described for a subgroup of human immunodeficiency virus type 1-infected children. Therefore, it appears that SIV Nef alone expressed in mice, in appropriate cell types and at sufficient levels, can elicit many of the phenotypes of simian and human AIDS. These Tg mice should be instrumental in studying the pathogenesis of SIV Nef-induced phenotypes.
Mass Transfer in Liquid in Apparatus with Mobile Packing. Application of Model of Cascade of Ideal Mixers
Authors: Palatý Zdeněk
Published: July 2002

The paper deals with the determination of parameters of a cascade of ideal mixers (i.e. their number and mean residence time of liquid in each member) in modelling the liquid flow on a plate with mobile packing. The number of cascade members has been found practically independent of the gas and liquid velocities and the static bed heights in the gas velocity range from 1.0 to 3.5 m s-1, liquid velocity range from 5.36 . 10-3 to 12.5 . 10-3 m s-1, and the range of static bed height from 21 . 10-3 to 47 . 10-3 m. The mean residence time is increased with increasing static bed height and is decreased with increasing velocities of both gas and liquid. The calculated parameters of the model of flow of liquid and the experimental data on desorption of CO2 from water by a stream of air have been used to calculate the values of mass transfer coefficient in liquid which have been compared with those obtained from the dispersion model. The both approaches have been found to give practically identical results.
Short Glass Micro-Packed Columns for Gas Chromatography
Authors: Komárek Karel
Published: July 2002

For a quick analysis of simple mixtures of lower chlorinated hydrocarbons a possible application has been studied of short glass micro-packed columns packed with commercial sorbents for extraction by solid phase. For analyses of lower alcohols, carboxylic acids, and other oxygen-containing substances the columns were packed with the organic porous sorbent, a copolymer of divinylbenzene and 1,4-di(methacryloyloxymethyl)naphthalene. The chosen sorbents were placed into glass capillary columns of 0.4 - 0.7 m length and 0.7 mm i.d. As the sorbent amount in the column is small, the columns are cheap. Whenever necessary, they can be prepared in any laboratory. The analyses of simple mixtures of above-mentioned classes of compounds lasted from 90 to 270 s.
Determination of Limiting Flux and Threshold Pressure in Stirred Ultrafiltration of Colloidal Suspensions
Authors: Vladisavljevic Goran T. , Pavasovic Vladeta Lj.
Published: July 2002

The stationary permeate flux during ultrafiltration of colloidal suspensions reaches a limiting value, Jl, at high pressure differences. According to the gel polarization model employed here, the existence of this limiting flux is a consequence of gelation of the solution at the membrane-solution interface. The limiting permeate flux, Jl, can easily be determined from experimental data by plotting ∆p/Js against ∆p. Furthermore, the membrane resistance at zero pressure difference, Rm0, and the pressure difference necessary to reach 95 per cent of the limiting flux, i.e. the threshold pressure, ∆pt, may be calculated, too. The agreement between the model and experimental data is satisfactory. The laboratory-made silica sol consisting of uniform spherical particles with a surface average diameter of 18 nm was selected as a working fluid throughout this study.
On the Transition from Homogeneous to Bubbling Fluidization
Authors: Hartman Miloslav , Beran Zdeněk , Veselý Václav , Svoboda Karel
Published: July 2002

The onset of the aggregative mode of liquid-solid fluidization was explored. The experimental findings were interpreted by means of the dynamic (elastic) wave velocity and the voidage propagation (continuity) wave velocity. For widely different systems, the mapping of regimes has been presented in terms of the Archimedes number, the Froude number and the fluid-solid density ratio. The proposed diagram also depicts the typical Geldart's Group A particles fluidized with air.
Dimeric Structures of Cp'TiCl2 Compounds with Bulky Substituents at the Cyclopentadienyl Rings
Authors: Schmid Günter , Thewalt Ulf , Sedmera Petr , Hanuš Vladimír , Mach Karel
Published: July 2002

The complexes [(η5-C5Me4Ph)TiCl(μ-Cl)]2 (1) and [(η5-C5Bn5)TiCl(μ-Cl)]2 (2) (Bn = benzyl) were obtained by the reaction of TiCl3 and the corresponding alkali metal cyclopentadienides in a THF-toluene mixture. The X-ray diffraction single-crystal analysis revealed that both compounds form centrosymmetric dimers bridged via a pair of chloride ligands. Compound 1 crystallizes in the monoclinic space group P21/n (No. 14; a = 11.387(3) Å, b = 16.496(2) Å, c = 8.383(2) Å, β = 110.786(15)°, V = 1 472.2(6) Å3, Z = 2), and compound 2 in the monoclinic space group P21/c (No. 14; a = 18.967(5) Å, b = 10.7190(10) Å, c = 17.996(5) Å, β = 116.747(15)°, V = 3 267.2(13) Å3, Z = 2). Common features of the structures differ only negligibly. The Ti-Ti distance (in 1 3.3345(11) Å, in 2 3.374(2) Å) is considerably shorter than in paramagnetic dimeric titanocene monochlorides (3.91-3.95 Å). The compounds are diamagnetic, probably due to spin-paired d1 electrons.
Adsorption of Iodide Ion from Aqueous NaI-NaClO4- Butan-1-ol at the Mercury-Electrolyte Interface
Authors: Saba Jadwiga , Sykut Kazimierz , Nieszporek Jolanta , Szaran Jan
Published: July 2002

Properties of the adsorption layers I--ClO4--H2O and I--ClO4--H2O-butan-1-ol on the mercury electrode were described using adsorption and kinetic parameters for the Zn(II) ion reduction. The systems were characterized by the measurements of differential capacity, zero charge potential and surface tension at this potential. The data were analysed to obtain the surface pressure and the charge due to specific adsorption of I- ions as a function of charge and concentration of I- ions. The standard Gibbs energy of adsorption ∆G0ad and parameter B obtained from the virial isotherm were compared. The rate constants of Zn(II) ion reduction as an ion affecting the adsorption equilibrium were determined.
Electrocatalysis of Hydrogen Evolution by Transition Metal Complexes
Authors: Heyrovský Michael
Published: July 2002

Voltammetric reduction of monothiocyanate complexes of the iron group metals is accompanied by catalytic evolution of hydrogen in two potential regions. Around -1.3 V vs SCE, hydrogen is evolved at the surface of the electrodeposited metal, around -1.6 V at the surface of mercury. The catalyst is the protonated reduced, zero-valent form of the complex which has limited stability. Minor differences in behaviour of the complexes of the three metals are due to different values of their complexation constant.
Diastasis of the pubic symphysis peculiar to horse riders: modern aspects of pelvic pommel injuries
Authors: Mulhall K J , Khan Y , Ahmed A , O'Farrell D , Burke T E , Moloney M
Published: July 2002

Diastasis of the pubic symphysis is a well documented injury typically associated with high energy trauma. Three cases in horse riders are here described, emphasising the appropriate modern investigation, including computed tomography, and orthopaedic and urological management.
A checklist to facilitate cultural awareness and sensitivity
Authors: Seibert P S , Stridh-Igo P , Zimmerman C G
Published: July 2002

United States of America demographic profiles illustrate a nation rich in cultural and racial diversity. Approximately 29% of the population are minorities and demographic projections indicate an increase to 50% by the year 2050. This creates a highly mobile and constantly changing environment, revealing the need for new levels of cultural awareness and sensitivity. These issues are particularly critical in the medical community where medical professionals must understand the impact cultural differences and barriers can have on evaluation, treatment, and rehabilitation. During times of stress, such as when injury strikes, problems associated with lack of cultural sensitivity are intensified. Cultural diversity is of particular concern when standard measures for diagnosis and prognosis are derived from established norms for responding, because culture defines norms. This paper details a ten point checklist designed to facilitate cultural awareness and sensitivity in medical settings to ensure maximum successful recovery and outcomes for all patients.