bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of
rpoB
Gene Analysis for Detection and Identification of
Bartonella
Species
Authors: Renesto Patricia , Gouvernet Joanny , Drancourt Michel , Roux Veronique , Raoult Didier 
Published: July 2002

ABSTRACT

Identification of
Bartonella
species is of increasing importance as the number of infections in which these bacteria are involved increases. To date, these gram-negative bacilli have been identified by various serological, biochemical, and genotypic methods. However, the development of alternative tools is required, principally to circumvent a major risk of contamination during sample manipulation. The aim of our study was to investigate the possible identification of various
Bartonella
species by comparison of RNA polymerase beta-subunit gene (
rpoB
) sequences. This approach has previously been shown to be useful for the identification of members of the family
Enterobacteriaceae
(C. M. Mollet, M. Drancourt, and D. Raoult, Mol. Microbiol. 26:1005–1011, 1997). Following PCR amplification with specific oligonucleotides, a 825-bp region of the
rpoB
gene was sequenced from 13 distinct
Bartonella
strains. Analysis of these sequences allowed selection of three restriction enzymes (
Apo
I,
Alu
I, and
Afl
III) useful for discerning the different strains by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. To confirm the potential value of such an approach for identification of
Bartonella
, the
rpoB
PCR was then applied to 94 clinical samples, and the results obtained were identical to those obtained by our reference PCR method. Twenty-four isolates were also adequately identified by PCR-RFLP analysis. In all cases, our results were in accordance with those of the reference method. Moreover, conserved regions of DNA were chosen as suitable primer targets for PCR amplification of a 439-bp fragment which can be easily sequenced.
Simultaneous Detection of Measles Virus, Rubella Virus, and Parvovirus B19 by Using Multiplex PCR
Authors: del Mar Mosquera María , de Ory Fernando , Moreno Mónica , Echevarría Juan E. 
Published: July 2002

ABSTRACT

We describe here a multiplex reverse transcription-PCR (RTMNPCR) assay designed to detect and differentiate measles virus, rubella virus, and parvovirus B19. Serial dilution experiments with vaccine strains that compared cell culture isolation of measles in B95 cells and rubella in RK13 cells showed sensitivity rates of 0.004 50% tissue culture infective dose (TCID
50
) for measles virus and 0.04 TCID
50
for rubella virus. This RTMNPCR can detect as few as 10 molecules for measles virus and rubella virus and one molecule for parvovirus B19 in dilution experiments with plasmids containing inserts of the primary reaction amplification products. Five pharyngeal exudates from measles patients and 2 of 15 cerebrospinal fluid samples from measles-related encephalitis were found to be positive for measles virus by this RTMNPCR. A total of 3 of 27 pharyngeal exudates from vaccinated children and 2 pharyngeal exudates, plus one urine sample from a case of congenital rubella syndrome, were found to be positive for rubella virus by RTMNPCR, whereas 16 of 19 sera from patients with erythema infectiosum were determined to be positive for parvovirus B19 by RTMNPCR. In view of these results, we can assess that this method is a useful tool in the diagnosis of these three viruses and could be used as an effective surveillance tool in measles eradication programs.
Assessment of Clarithromycin-Resistant
Helicobacter pylori
among Patients in Shanghai and Guangzhou, China, by Primer-Mismatch PCR
Authors: Pan Zhi-Jun , Su Wan-Wen , Tytgat Guido N. J. , Dankert Jacob , van der Ende Arie 
Published: July 2002

ABSTRACT

Of 96
Helicobacter pylori
isolates from patients in Shanghai and Guangzhou, China, 5 had the A2143G 23S rRNA mutation as determined by primer-mismatch PCR and were resistant to clarithromycin by the E-test. The remaining isolates were primer-mismatch PCR negative and susceptible to clarithromycin. The conclusion is that the prevalence of clarithromycin-resistant
H. pylori
isolates among these Chinese patients is 5%.
Trypsin-Induced Structural Transformation in Aquareovirus
Authors: Nason Emma L. , Samal Siba K. , Venkataram Prasad B. V. 
Published: July 2002

ABSTRACT

Aquareovirus
, a member of the family
Reoviridae
, is a large virus with multiple capsid layers surrounding a genome composed of 11 segments of double-stranded RNA. Biochemical studies have shown that treatment with the proteolytic agent trypsin significantly alters the infectivity of the virus. The most infectious stage of the virus is produced by a 5-min treatment with trypsin. However, prolonged trypsin treatment almost completely abolishes the infectivity. We have used three-dimensional electron cryomicroscopy to gain insight into the structural basis of protease-induced alterations in infectivity by examining the structural changes in the virion at various time intervals of trypsin treatment. Our data show that after 5 min of trypsinization, projection-like spikes made of VP7 (35 kDa), associated with the underlying trimeric subunits, are completely removed. Concurrent with the removal of VP7, conformational changes are observed in the trimeric subunit composed of putative VP5 (71 kDa). The removal of VP7 and the accompanied structural changes may expose regions in the putative VP5 important for cell entry processes. Prolonged trypsinization not only entirely removes the outer capsid layer, producing the poorly infectious core particle, but also causes significant conformational changes in the turret protein. These changes result in shortening of the turret and narrowing of its central channel. The turret, as in orthoreoviruses, is likely to play a major role in the capping and translocation of mRNA during transcription, and the observed conformational flexibility in the turret protein may have implications in rendering the particle transcriptionally active or inactive.
Sendai Virus C Proteins Must Interact Directly with Cellular Components To Interfere with Interferon Action
Authors: Garcin Dominique , Curran Joseph , Kolakofsky Daniel 
Published: July 2002

ABSTRACTSendai virus (SeV) infection of interferon (IFN)-competent cells is one of the most efficient ways of inducing IFN production. Virus replication is nevertheless largely unaffected, since SeV infection also interfers with IFN action, a prerequisite for the establishment of an antiviral state. This property has been mapped by reverse genetics to the viral C gene, which is also known to act as a promoter-specific inhibitor of viral RNA synthesis. Using luciferase reporter plasmids containing IFN-responsive promoters, we have found that all four C proteins effectively interdict IFN signaling when expressed independently of SeV infection. The C proteins must therefore interact directly with cellular components to carry this out. The C gene in the context of an SeV infection was also found to induce STAT1 instability in some cells, whereas in other cells it apparently acts to prevent the synthesis of STAT1 in response to the virus infection or IFN treatment. The SeV C proteins appear to act in at least two ways to counteract the IFN induced by SeV infection.
Human Monoclonal Antibodies That Inhibit Binding of Hepatitis C Virus E2 Protein to CD81 and Recognize Conserved Conformational Epitopes
Authors: Hadlock Kenneth G. , Lanford Robert E. , Perkins Susan , Rowe Judy , Yang Qing , Levy Shoshana , Pileri Piero , Abrignani Sergio , Foung Steven K. H. 
Published: July 2002

ABSTRACT
The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.
Structural and Functional Dissection of Human Cytomegalovirus US3 in Binding Major Histocompatibility Complex Class I Molecules
Authors: Lee Sungwook , Yoon Juhan , Park Boyoun , Jun Youngsoo , Jin Mirim , Sung Ha Chin , Kim Ik-Hwan , Kang Seongman , Choi Eui-Ju , Ahn Byung Yoon , Ahn Kwangseog 
Published: July 2002

ABSTRACTThe human cytomegalovirus US3, an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, forms a complex with major histocompatibility complex (MHC) class I molecules and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes. To identify which parts of US3 confine the protein to the ER and which parts are responsible for the association with MHC class I molecules, we constructed truncated mutant and chimeric forms in which US3 domains were exchanged with corresponding domains of CD4 and analyzed them for their intracellular localization and the ability to associate with MHC class I molecules. All of the truncated mutant and chimeric proteins containing the luminal domain of US3 were retained in the ER, while replacement of the US3 luminal domain with that of CD4 led to cell surface expression of the chimera. Thus, the luminal domain of US3 was sufficient for ER retention. Immunolocalization of the US3 glycoprotein after nocodazole treatment and the observation that the carbohydrate moiety of the US3 glycoprotein was not modified by Golgi enzymes indicated that the ER localization of US3 involved true retention, without recycling through the Golgi. Unlike the ER retention signal, the ability to associate with MHC class I molecules required the transmembrane domain in addition to the luminal domain of US3. Direct interaction between US3 and MHC class I molecules could be demonstrated after in vitro translation by coimmunoprecipitation. Together, the present data indicate that the properties that allow US3 to be localized in the ER and bind MHC class I molecules are located in different parts of the molecule.
Bcl-2 Blocks a Caspase-Dependent Pathway of Apoptosis Activated by Herpes Simplex Virus 1 Infection in HEp-2 Cells
Authors: Galvan Veronica , Brandimarti Renato , Munger Joshua , Roizman Bernard 
Published: July 2002

ABSTRACT

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type virus blocks the execution of the cell death program triggered by expression of viral genes, by the Fas and tumor necrosis factor pathways, or by nonspecific stress agents. In particular, an earlier report from this laboratory showed that the mutant virus
d
120 lacking the genes encoding infected cell protein 4 (ICP4), the major regulatory protein of the virus, induces a caspase-3-independent pathway of apoptosis in human SK-N-SH cells. Here we report that the pathway of apoptosis induced by the
d
120 mutant in human HEp-2 cells is caspase dependent. Specifically, in HEp-2 cells infected with
d
120, (i) a broad-range inhibitor of caspase activity, z-vad-FMK, efficiently blocked DNA fragmentation, (ii) cytochrome
c
was released into the cytoplasm, (iii) caspase-3 was activated inasmuch as poly(ADP-ribose) polymerase was cleaved, and (iv) chromatin condensation and fragmentation of cellular DNA were observed. In parallel studies, HEp-2 cells were transfected with a plasmid encoding human Bcl-2 and a clone (VAX-3) expressing high levels of Bcl-2 was selected. This report shows that Bcl-2 blocked all of the manifestations associated with programmed cell death caused by infection with the
d
120 mutant. Consistent with their resistance to programmed cell death, VAX-3 cells overproduced infected cell protein 0 (ICP0). An unexpected observation was that ICP0 encoded by the
d
120 mutant accumulated late in infection in small, quasi-uniform vesicle-like structures in all cell lines tested. Immunofluorescence-based colocalization studies indicated that these structures were not mitochondria or components of the endoplasmic reticulum or the late endosomal compartment. These studies affirm the conclusion that HSV can induce programmed cell death at multiple steps in the course of its replication, that the
d
120 mutant can induce both caspase-dependent and -independent pathways of programmed cell death, and that virus-induced stimuli of programmed cell death may differ with respect to the pathway that they activate.
Adaptive Mutations in Sindbis Virus E2 and Ross River Virus E1 That Allow Efficient Budding of Chimeric Viruses
Authors: Kim Kyongmin Hwang , Strauss Ellen G. , Strauss James H. 
Published: July 2002

ABSTRACT

Alphavirus glycoproteins E2 and E1 form a heterodimer that is required for virus assembly. We have studied adaptive mutations in E2 of Sindbis virus (SIN) and E1 of Ross River virus (RR) that allow these two glycoproteins to interact more efficiently in a chimeric virus that has SIN E2 but RR E1. These mutations include K129E, K131E, and V237F in SIN E2 and S310F and C433R in RR E1. Although RR E1 and SIN E2 will form a chimeric heterodimer, the chimeric virus is almost nonviable, producing about 10
−7
as much virus as SIN at 24 h and 10
−5
as much after 48 h. Chimeras containing one adaptive change produced 3 to 20 times more virus than did the parental chimera, whereas chimeras with two changes produced 10 to 100 times more virus and chimeras containing three mutations produced yields that were 180 to 250 times better. None of the mutations had significant effects upon the parental wild-type viruses, however. Passage of the triple variants eight or nine times resulted in variants that produced virus rapidly and were capable of producing >10
8
PFU/ml of culture fluid within 24 h. These further-adapted variants possessed one or two additional mutations, including E2-V116K, E2-S110N, or E1-T65S. The RR E1-C433R mutation was studied in more detail. This Cys is located in the putative transmembrane domain of E1 and was shown to be palmitoylated. Mutation to Arg-433 resulted in loss of palmitoylation of E1. The positively charged arginine residue within the putative transmembrane domain of E1 would be expected to alter the conformation of this domain. These results suggest that interactions within the transmembrane region are important for the assembly of the E1/E2 heterodimer, as are regions of the ectodomains possibly identified by the locations of adaptive mutations in these regions. Further, the finding that four or five changes in the chimera allow virus production that approaches the levels seen with the parental SIN and exceeds that of the parental RR illustrates that the structure and function of SIN and RR E1s have been conserved during the 50% divergence in sequence that has occurred.
The Conserved Serine 177 in the Delta Antigen of Hepatitis Delta Virus Is One Putative Phosphorylation Site and Is Required for Efficient Viral RNA Replication
Authors: Mu Jung-Jung , Chen Ding-Shinn , Chen Pei-Jer 
Published: July 2002

ABSTRACT
Hepatitis delta virus (HDV) small delta antigen (S-HDAg) plays a critical role in virus replication. We previously demonstrated that the S-HDAg phosphorylation occurs on both serine and threonine residues. However, their biological significance and the exact phosphorylation sites of S-HDAg are still unknown. In this study, phosphorylated S-HDAg was detected only in the intracellular compartment, not in viral particles. In addition, the number of phosphorylated isoforms of S-HDAg significantly increased with the extent of viral replication in transfection system. Site-directed mutagenesis showed that alanine replacement of serine 177, which is conserved among all the known HDV strains, resulted in reduced phosphorylation of S-HDAg, while the mutation of the other two conserved serine residues (2 and 123) had little effect. The S177A mutant dramatically decreased its capability in assisting HDV RNA replication, with a preferential and profound impairment of the antigenomic RNA replication. Furthermore, the viral RNA editing, a step relying upon antigenomic RNA replication, was also abolished by this mutation. These results suggested that phosphorylation of S-HDAg, with serine 177 as a presumable site, plays a critical role in viral RNA replication, especially in augmenting the replication of antigenomic RNA.
Activated Peripheral CD8 Lymphocytes Express CD4 In Vivo and Are Targets for Infection by Human Immunodeficiency Virus Type 1
Authors: Imlach S. , McBreen S. , Shirafuji T. , Leen C. , Bell J. E. , Simmonds P. 
Published: July 2002

ABSTRACTThere is increasing evidence that CD8 lymphocytes may represent targets for infection by human immunodeficiency virus type 1 (HIV-1) in vivo whose destruction may contribute to the loss of immune function underlying AIDS. HIV-1 may infect thymic precursor cells destined to become CD4 and CD8 lymphocytes and contribute to the numerical decline in both subsets on disease progression. There is also evidence for the induction of CD4 expression and susceptibility to infection by HIV-1 of CD8 lymphocytes activated in vitro. To investigate the relationship between CD8 activation and infection by HIV-1 in vivo, activated subsets of CD8 lymphocytes in peripheral blood mononuclear cells (PBMCs) of HIV-seropositive individuals were investigated for CD4 expression and HIV infection. Activated CD8 lymphocytes were identified by expression of CD69, CD71, and the human leukocyte antigen (HLA) class II, the β-chain of CD8, and the RO isoform of CD45. CD4+and CD4−CD8 lymphocytes, CD4 lymphocytes, other T cells, and non-T cells were purified using paramagnetic beads, and proviral sequences were quantified by PCR using primers from the long terminal repeat region. Frequencies of activated CD8 lymphocytes were higher in HIV-infected study subjects than in seronegative controls, and they frequently coexpressed CD4 (mean frequencies on CD69+, CD71+, and HLA class II+cells of 23, 37, and 8%, respectively, compared with 1 to 2% for nonactivated CD8 lymphocytes). The level of CD4 expression of the double-positive population approached that of mature CD4 lymphocytes. That CD4 expression renders CD8 cell susceptible to infection was indicated by their high frequency of infection in vivo; infected CD4+CD8 lymphocytes accounted for between 3 and 72% of the total proviral load in PBMCs from five of the eight study subjects investigated, despite these cells representing a small component of the PBMC population (<3%). Combined, these findings provide evidence that antigenic stimulation of CD8 lymphocytes in vivo induces CD4 expression that renders them susceptible to HIV infection and destruction. The specific targeting of responding CD8 lymphocytes may provide a functional explanation for the previously observed impairment of cytotoxic T-lymphocyte (CTL) function disproportionate to their numerical decline in AIDS and for the deletion of specific clones of CTLs responding to HIV antigens.
STICKY SPHERES IN QUANTUM MECHANICS
Authors: PENROSE M. D. , PENROSE O. , STELL G. 
Published: July 2002

For a 3-dimensional system of hard spheres of diameter D and mass m with an added attractive square-well two-body interaction of width a and depth ε, let BD, a denote the quantum second virial coefficient. Let BD denote the quantum second virial coefficient for hard spheres of diameter D without the added attractive interaction. We show that in the limit a → 0 at constant α: = ℰma2/(2ħ2) with α < π2/8, [Formula: see text] The result is true equally for Boltzmann, Bose and Fermi statistics. The method of proof uses the mathematics of Brownian motion. For α > π2/8, we argue that the gaseous phase disappears in the limit a → 0, so that the second virial coefficient becomes irrelevant.
ON THE CENTER PROBLEM FOR DEGENERATE SINGULAR POINTS OF PLANAR VECTOR FIELDS
Authors: MAÑOSA VÍCTOR 
Published: July 2002

The center problem for degenerate singular points of planar systems (the degenerate-center problem) is a poorly-understood problem in the qualitative theory of ordinary differential equations. It may be broken down into two problems: the monodromy problem, to decide if the singular point is of focus-center type, and the stability problem, to decide whether it is a focus or a center. We present an outline on the status of the center problem for degenerate singular points, explaining the main techniques and obstructions arising in the study of the problem. We also present some new results. Our new results are the characterization of a family of vector fields having a degenerate monodromic singular point at the origin, and the computation of the generalized first focal value for this family V1. This gives the solution of the stability problem in the monodromic case, except when V1 = 1. Our approach relies on the use of the blow-up technique and the study of the blow-up geometry of singular points. The knowledge of the blow-up geometry is used to generate a bifurcation of a limit cycle.
EXTENSIBLE INFORMATION BROKERS
Authors: LU JIANGUO , MYLOPOULOS JOHN 
Published: July 2002

The number and size of information services available on the internet has been growing exponentially over the past few years. This growth has created an urgent need for information agents that act as brokers in the sense that they can autonomously search, gather, and integrate information on behalf of a user. To remain useful, such brokers will have to evolve throughout their lifetime to keep up with evolving and ever-changing information services. This paper proposes a framework named XIB (eXtensible Information Brokers) for building and evolving information brokers. The XIB takes as input a description of required information services and supports the interactive generation of an integrated query interface. It also generates wrappers for each information service dynamically. Once the query interface and wrappers are in place, the user can specify a query and get back a result which integrates data from all wrapped information sources. The XIB depends heavily on XML-related techniques. More specifically, we use DTDs to model the input and output of each service, and XML to represent both input and output values. Based on such representations, the paper investigates service integration in the form DTD integration, and studies query decomposition in the form of XML element decomposition. Within the proposed framework, it is easy to add or remove information services to a broker, thereby facilitating maintenance, evolution and customization of information brokers.