bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production and Application of New Monoclonal Antibodies Specific for a FecalHelicobacter pyloriAntigen
Authors: Suzuki Nobuyuki , Wakasugi Masahiko , Nakaya Seigo , Okada Keiko , Mochida Ritsuko , Sato Masami , Kajiyama Hirofumi , Takahashi Ryoki , Hirata Haruhisa , Ezure Yohji , Koga Yasuhiro , Fukuda Yoshihiro , Shimoyama Takashi 
Published: July 2002

ABSTRACTThe aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecalHelicobacter pyloriantigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity forH. pyloriantigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form ofH. pylori, and fecal samples fromH. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41H. pyloriclinical isolates and in fecal samples from sevenH. pylori-positive subjects. Several kinds ofHelicobacterspecies (Helicobacter felis,Helicobacter hepaticus,Helicobacter mustelae, andHelicobacter cinaedi) exceptH. pylori, major bacteria in feces (Campylobacter jejuni,Bacteroides vulgatus,Bifidobacterium breve,Bifidobacterium infantis, andEscherichia coli), and fecal samples from sixH. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis ofH. pyloriinfection.
Immunization with Alpha-Toxin Toxoid Protects the Cornea against Tissue Damage during Experimental
Staphylococcus aureus
Keratitis
Authors: Hume Emma B. H. , Dajcs Joseph J. , Moreau Judy M. , O'Callaghan Richard J. 
Published: July 2002

ABSTRACT

Alpha-toxin is a major virulence factor in
Staphylococcus aureus
keratitis. Active or passive immunization with alpha-toxin toxoid could protect against corneal damage. Results show that either form of immunization did not kill bacteria but did significantly protect against corneal pathology, especially epithelial erosion.
Cloning and DNA Sequence Analysis of an Immunogenic Glucose-Galactose MglB Lipoprotein Homologue fromBrachyspira pilosicoli, the Agent of Colonic Spirochetosis
Authors: Zhang P. , Cheng X. , Duhamel G. E. 
Published: July 2002

ABSTRACTColonic spirochetosis (CS) is a newly emerging infectious disease of humans and animals caused by the pathogenic spirocheteBrachyspira(formerlySerpulina)pilosicoli. The purpose of this study was to characterize an antigen that was recognized by antibodies present in sera of challenge-exposed pigs. The gene encoding the antigen was identified by screening a plasmid library of humanB. pilosicolistrain SP16 (ATCC 49776) genomic DNA with hyperimmune and convalescent swine sera. The predicted amino acid sequence encoded by the clonedB. pilosicoligene had a high degree of similarity and identity to glucose-galactose MglB lipoprotein. Located 106 bp downstream of the putativemglBgene was a 3′-truncated open reading frame with 73.8% similarity and 66.3% identity tomglAofEscherichia coli, suggesting a gene arrangement within an operon which is similar to those of other bacteria. A single copy of the gene was present inB. pilosicoli, and homologous sequences were widely conserved among porcine intestinal spirochetesSerpulina intermedia,Brachyspira innocens,Brachyspira murdochii, and the avianBrachyspira alvinipulli, but not in porcineBrachyspira hyodysenteriae, humanBrachyspira aalborgi, and porcineTreponema succinifaciens. The deduced molecular weight of the mature MglB lipoprotein was consistent with expression by the cloned gene of a polypeptide with an apparent molecular weight of 36,000, as determined by Western blot analysis and [3H]palmitate labeling. Because mucin is the principal constituent of the colonic mucus gel and consists of glycoproteins that can serve as the substrate for growth and chemotaxis ofB. pilosicoliin vitro, a role for MglB in mucosal localization of the spirochete appears consistent with the pathogenesis of CS. However, the presence of homologous sequences in closely related but nonpathogenic commensal spirochetes suggests that other virulence determinants may be required for pathogenesis.
Attenuated
Shigella flexneri
2a Vaccine Strain CVD 1204 Expressing Colonization Factor Antigen I and Mutant Heat-Labile Enterotoxin of Enterotoxigenic
Escherichia coli
Authors: Koprowski Hilary , Levine Myron M. , Anderson Richard J. , Losonsky Genevieve , Pizza Mariagrazia , Barry Eileen M. 
Published: July 2002

ABSTRACT

A multivalent live oral vaccine against both
Shigella
spp. and enterotoxigenic
Escherichia coli
(ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution—serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into
S. flexneri
2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM
1
binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-
S. flexneri
2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type
S. flexneri
2a. This prototype
Shigella
-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated
Shigella
vaccine strain and engendering immune responses against both the heterologous antigens and vector strain.
Analysis of the Cellular Localization of Bdr Paralogs in
Borrelia burgdorferi
, a Causative Agent of Lyme Disease: Evidence for Functional Diversity
Authors: Roberts David M. , Theisen Michael , Marconi Richard T. 
Published: July 2002

ABSTRACT

The
bdr
(
Borrelia
direct repeat) gene family of the genus
Borrelia
encodes a polymorphic group of proteins that carry a central repeat motif region containing putative phosphorylation sites and a hydrophobic carboxyl-terminal domain. It has been postulated that the Bdr proteins may anchor to the inner membrane via the C-terminal domain. In this study, we used cellular fractionation methodologies, salt and detergent treatments, and immunoblot analyses to assess the association of the Bdr proteins with the cellular infrastructure in both
Borrelia burgdorferi
(a Lyme disease spirochete) and
B. turicatae
(a relapsing fever spirochete). Triton X-114 extraction and partitioning experiments demonstrated that most Bdr paralogs are associated with the inner membrane-peptidoglycan complex. Analyses of cells treated with the highly chaotropic bile salt detergent deoxycholic acid demonstrated that some Bdr paralogs may also interact with the peptidoglycan, as evidenced by their tight association with the insoluble cellular matrix. In addition, immunoprecipitation (IP) experiments revealed an enhanced IP of all Bdr paralogs when the cell lysates were boiled prior to addition of the precipitating antibody. Furthermore, some Bdr paralogs were accessible to antibody in the IP experiments only in the boiled cell lysates. These observations suggest that different Bdr paralogs may carry out different structural-functional roles. Demonstration of the inner membrane localization of the Bdr proteins and of the differences in nature of the interaction of individual Bdr paralogs with the cell infrastructure is an important step toward defining the functional role of this unique protein family in the genus
Borrelia
.
The Molecular Weight Distribution of Succinoglycan Produced by
Sinorhizobium meliloti
Is Influenced by Specific Tyrosine Phosphorylation and ATPase Activity of the Cytoplasmic Domain of the ExoP Protein
Authors: Niemeyer Dagmar , Becker Anke 
Published: July 2002

ABSTRACT

It is thought that in the gram-negative soil bacterium
Sinorhizobium meliloti
the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I). The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed. The cytoplasmic domain of the ExoP protein was shown to have ATPase activity. Mutations in the highly conserved Walker A ATP-binding motif prevented ATPase activity of the ExoP protein. Phenotypically, these mutations resulted in much lower levels of succinoglycan which consisted only of monomers of the octasaccharide repeating unit. The ExoP protein has similarities to proteins with autophosphorylating protein tyrosine kinase activity. We found that ExoP was phosphorylated on tyrosine and that site-directed mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP resulted in an altered ratio of low-molecular-weight succinoglycan to high-molecular-weight succinoglycan.
Identification, Characterization, and Functional Analysis of a Gene Encoding the Ferric Uptake Regulation Protein in
Bartonella
Species
Authors: Park Sarah Y. , Kelminson Karen L. , Lee Anthea K. , Zhang Peng , Warner Rachel E. , Rehkopf David H. , Calderwood Stephen B. , Koehler Jane E. 
Published: July 2002

ABSTRACT

Environmental iron concentrations coordinately regulate transcription of genes involved in iron acquisition and virulence via the ferric uptake regulation (
fur
) system. We identified and sequenced the
fur
gene and flanking regions of three
Bartonella
species. The most notable difference between
Bartonella
Fur and other Fur proteins was a substantially higher predicted isoelectric point. No promoter activity or Fur autoregulation was detected using a
gfp
reporter gene fused to the 204 nucleotides immediately upstream of the
Bartonella fur
gene.
Bartonella henselae fur
gene expression complemented a
Vibrio cholerae fur
mutant.
Bacillus subtilisfunctional genomics: global characterization of the stringent response by proteome and transcriptome analysis
Authors: Eymann Christine , Homuth Georg , Scharf Christian , Hecker Michael 
Published: July 2002

ABSTRACTThe stringent response inBacillus subtiliswas characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type andrelAmutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response todl-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in therelAmutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknowny-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.
Infection of Central Nervous System by Motile
Enterococcus
: First Case Report
Authors: Kurup Asok , Tee Wen Sim Nancy , Loo Liat Hui , Lin Raymond 
Published: July 2002

ABSTRACT

A 66-year-old man with four indwelling ventriculoperitoneal shunts for multiloculated hydrocephalus from a complicated case of meningitis a year before developed shunt infection based on a syndrome of fever, drowsiness, and cerebrospinal fluid neutrophil pleocytosis in the background of repeated surgical manipulation to relieve successive shunt blockages. The cerebrospinal fluid culture, which yielded a motile
Enterococcus
species, was believed to originate from the gut. This isolate was lost in storage and could not be characterized further. The patient improved with vancomycin and high-dose ampicillin therapy. He relapsed a month later with
Enterococcus gallinarum
shunt infection, which responded to high-dose ampicillin and gentamicin therapy. This is probably the first case report of motile
Enterococcus
infection of the central nervous system.
Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease
Authors: Liu Xin , Yang Jenny , Ghazi A. Mohamad , Frey Teryl K. 
Published: July 2002

ABSTRACT

The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products. A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X. Liu, S. L. Ropp, R. J. Jackson, and T. K. Frey, J. Virol. 72:4463–4466, 1998). To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF. When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn. Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn
65
binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding. All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV–CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein. Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril. In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.
Nitric Oxide Synthesis Enhances Human Immunodeficiency Virus Replication in Primary Human Macrophages
Authors: Blond Donatienne , Raoul Hervé , Le Grand Roger , Dormont Dominique 
Published: July 2002

ABSTRACTMacrophages are suspected to play a major role in human immunodeficiency virus (HIV) infection pathogenesis, not only by their contribution to virus dissemination and persistence in the host but also through the dysregulation of immune functions. The production of NO, a highly reactive free radical, is thought to act as an important component of the host immune response in several viral infections. The aim of this study was to evaluate the effects of HIV type 1 (HIV-1) Ba-L replication on inducible nitric oxide synthase (iNOS) mRNA expression in primary cultures of human monocyte-derived macrophages (MDM) and then examine the effects of NO production on the level of HIV-1 replication. Significant induction of the iNOS gene was observed in cultured MDM concomitantly with the peak of virus replication. However, this induction was not accompanied by a measurable production of NO, suggesting a weak synthesis of NO. Surprisingly, exposure to low concentrations of a NO-generating compound (sodium nitroprusside) andl-arginine, the natural substrate of iNOS, results in a significant increase in HIV replication. Accordingly, reduction ofl-arginine bioavailability after addition of arginase to the medium significantly reduced HIV replication. The specific involvement of NO was further demonstrated by a dose-dependent inhibition of viral replication that was observed in infected macrophages exposed toNG-monomethyll-arginine andNG-nitro-l-arginine methyl ester (l-NAME), two inhibitors of the iNOS. Moreover, an excess ofl-arginine reversed the addition of L-NAME, confirming that an arginine-dependent mechanism is involved. Finally, inhibitory effects of hemoglobin which can trap free NO in culture supernatants and in biological fluids in vivo confirmed that endogenously produced NO could interfere with HIV replication in human macrophages.
Low Plasma Human Immunodeficiency Virus Type 2 Viral Load Is Independent of Proviral Load: Low Virus Production In Vivo
Authors: Popper Stephen J. , Sarr Abdoulaye Dieng , Guèye-Ndiaye Aïssatou , Mboup Souleymane , Essex Myron E. , Kanki Phyllis J. 
Published: July 2002

ABSTRACTLevels of virus in the plasma are closely related to the pathogenicity of human immunodeficiency virus type 1 (HIV-1). HIV-2 is much less pathogenic than HIV-1, and infection with HIV-2 leads to significantly lower plasma viral load. To identify the source of this difference, we measured both viral RNA and proviral DNA in matched samples from 34 HIV-2-infected individuals. Nearly half had undetectable viral RNA loads (<100 copies/ml), but levels of proviral DNA were relatively high and confirmed that quantities of provirus in HIV-1 and HIV-2 infection were similar. Overall, HIV-2 proviral DNA load did not correlate with viral RNA load, and higher viral RNA load was associated with increased production of plasma virus from the proviral template. These results suggest that low viral load in HIV-2 infection is due to decreased rates of viral production, rather than differences in target cell infectivity.
A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2A
pro
: Effects on Cellular and Viral Gene Expression
Authors: Barco Angel , Feduchi Elena , Carrasco Luis 
Published: July 2002

ABSTRACT

A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2A
pro
) under the control of tetracycline has been obtained. Synthesis of 2A
pro
induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2A
pro
cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2A
pro
, prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2A
pro
still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2A
pro
. Moreover, synthesis of 2A
pro
in 2A7d cells complements the translational defect of a poliovirus 2A
pro
-defective variant. These results show that poliovirus 2A
pro
expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2A
pro
functions, to complement poliovirus 2A
pro
mutants, and to test antiviral compounds.
Frameshift Signal Transplantation and the Unambiguous Analysis of Mutations in the Yeast Retrotransposon Ty1 Gag-Pol Overlap Region
Authors: Lawler Joseph F. , Merkulov Gennady V. , Boeke Jef D. 
Published: July 2002

ABSTRACT

The yeast retrotransposon Ty1 encodes a 7-nucleotide RNA sequence that directs a programmed, +1 ribosomal frameshifting event required for Gag-Pol translation and retrotransposition. We report mutations that block frameshifting, which can be suppressed in
cis
by “transplanting” the frameshift signal to a position upstream of its native location. These “frameshift transplant” mutants transpose with only a modest decrease in efficiency, suggesting that the location of the frameshift signal in a functional Ty1 element may vary. The genomic architecture of Ty1 is such that Gag, Ty1 PR (PR), and the Gag-derived p4 peptide share a common sequence. The functional independence of the movement of the frameshift signal to a new location within the Ty1 element is used to unambiguously attribute the effect of mutations deleterious to transposition in this region of overlapping coding sequences to effects on the Ty1 (PR). This work defines the amino terminus of the Ty1 PR and introduces a new technique for studying viral genome organization.