Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Infection with Campylobacter pyloridis has been strongly associated with gastritis in humans although its etiologic significance is currently undefined. We examined the structure and antigenicity of whole-cell, outer-membrane, acid-extractable surface protein, and proteinase K-treated whole cell lysate preparations from eight C. pyloridis strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with homologous and heterologous immune rabbit serum. Whole-cell and outer-membrane profiles observed in all strains of C. pyloridis were nearly identical; none were similar to those of C. jejuni and C. fetus. Major whole-cell bands migrated at 26,000, 29,000, 56,000, and 62,000 molecular weights. The acid-extracted protein profiles of all C. pyloridis strains also were similar to one another and showed similarities with acid-extracted proteins from C. jejuni, with major bands migrating at 29,000, 48,000 to 53,000, and 62,000. All proteinase K-treated lysates showed different lipopolysaccharide (LPS) profiles, ranging from rough to smooth with multiple repeating side chains. Immunoblots of whole-cell and proteinase K-treated preparations of the C. pyloridis strains showed that there was antigenic cross-reactivity of proteins migrating at 62,000 and 56,000, but not in other regions, and cross-reactivity between LPS core regions but not side chains. These results suggest that C. pyloridis has both protein and core LPS group antigens and strain-specific protein and LPS side chain antigens.
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PMID:Conservation and diversity of Campylobacter pyloridis major antigens. 355 97

The outer membrane proteins of a series of fluorescent, root-colonizing, plant-growth-stimulating Pseudomonas spp. having been characterized (L. A. de Weger et al., J. Bacteriol. 165:585-594, 1986), the lipopolysaccharides (LPSs) of these strains were examined. The chemical composition of the LPSs of the three best-studied plant-growth-stimulating Pseudomonas strains WCS358, WCS361, and WCS374 and of P. aeruginosa PAO1 as a reference strain was determined and appeared to differ from strain to strain. The 2,6-dideoxy-2-aminosugar quinovasamine was the most abundant compound in the LPS of strain WCS358. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified LPS and of proteinase K-treated cell envelopes revealed ladderlike patterns for most of these strains. These patterns were not substantially influenced by differences in culture conditions. Analysis of proteinase K-treated cell envelopes of 24 root-colonizing Pseudomonas spp. revealed a unique band pattern for each strain, suggesting a great variety in the LPS structures present in these root colonizers. Therefore, electrophoretic analysis of LPS can be used for characterization and identification of the fluorescent root-colonizing Pseudomonas strains.
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PMID:Lipopolysaccharides of Pseudomonas spp. that stimulate plant growth: composition and use for strain identification. 355 19

Human gingival fibroblasts were exposed in culture to cell extracts of different black-pigmented Bacteroides species, and their growth was monitored by determining thymidine uptake and counting cells. Of the Bacteroides species tested (B. gingivalis, B. asaccharolyticus, and B. intermedius), B. gingivalis gave the extract with the strongest inhibitory effect on fibroblast thymidine uptake. Linear inhibition reaching 80% of the control level was obtained with a dose of 100 micrograms of B. gingivalis extract protein per ml. The effect of B. asaccharolyticus resembled that of B. gingivalis, but even at the highest dose tested B. intermedius had only a slight inhibitory effect. When fibroblasts were counted after 2- and 4-day exposures to B. gingivalis extracts, a clear depression in the number of fibroblasts was found. The effects of extracts obtained from early and late growth phases of B. gingivalis cultures were similar. A fraction of B. gingivalis consisting essentially of lipopolysaccharides (LPSs) was obtained by degrading the extract proteins with proteinase K. Silver staining of polyacrylamide gels revealed a LPS pattern with a molecular mass ranging from 37 to 60 kilodaltons. This LPS-rich fraction caused inhibition of thymidine uptake by gingival fibroblasts similar to that caused by the native extract alone. Thus, the inhibition of gingival fibroblast growth by B. gingivalis appeared to be LPS mediated. This inhibitory effect of B. gingivalis on oral fibroblast growth may be a virulence factor of this bacterium.
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PMID:Inhibition of gingival fibroblast growth by Bacteroides gingivalis. 379 30

A serotyping system for nontypable Haemophilus influenzae (NTHI) was developed by using isolated outer membrane protein (OMP) preparations and rabbit antisera. OMPs of 23 strains were isolated by molecular sieve chromatography of outer membranes in 1.5% sodium deoxycholate buffer. These OMP preparations were relatively free of lipopolysaccharide as determined by silver staining of sodium dodecyl sulfate gels and by dot assay with a monoclonal antibody which is specific for the lipid A of H. influenzae. Three antisera raised to whole organisms were used to serotype 21 of 23 strains with a kinetic enzyme-linked immunosorbent assay. Digestion of OMP preparations with proteinase K removed greater than 90% of the antigenic reactivity, indicating that the system is based on OMP antigens. Marked antigenic heterogeneity of OMPs exists among strains of NTHI. By determining the pattern of serological reactivity of OMPs with the three antisera, isolates were divided into groups based on antigenic differences. Six serotypes were identified. This OMP serotyping system is based on multiple antigenic determinants. Future studies will focus on identifying serotype-specific epitopes to further refine this serological classification scheme for NTHI.
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PMID:Antigenic heterogeneity of outer membrane proteins of nontypable Haemophilus influenzae is a basis for a serotyping system. 387 83

The morphological heterogeneity of lipopolysaccharides (LPSs) among salmonella mutants with different LPS chemotypes was analyzed in silver-stained polyacrylamide gels. The biochemical differences in the LPS chemotypes were reflected in the unique profiles of the purified LPSs. The LPS profiles in the whole-cell lysates were also unique for each chemotype. (Whole-cell lysates were assessed by a method which preferentially silver stains LPS and by a proteinase K digest of whole-cell lysates. The silver-stained LPS profiles of proteinase K-digested lysates were similar to the homologous purified LPS and could be used to preliminarily characterize the LPS chemotype before purification.) In summary, biochemical variation in LPS composition can be detected in silver-stained polyacrylamide gels.
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PMID:Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. 618 29

The lysogenization of Pseudomonas aeruginosa PAO by phage D3 results in derivatives which are resistant to superinfection by phage D3c by virtue of the fact that homologous phage cannot adsorb to these cells. The serologically and morphologically unrelated phage E79 showed a markedly decreased adsorption rate to the lysogen PAO(D3). Since both of these phages are lipopolysaccharide specific, these results suggested lysogenic conversion of the phage receptor. The lipopolysaccharide was extracted from strain PAO by the hot phenol-water technique, but this procedure was ineffective with PAO(D3). We developed a technique involving cold trichloroacetic acid extraction, followed by ultracentrifugation, digestion of the high-speed pellet with proteinase K, and ultimate purification on CsCl step gradients. The lipopolysaccharide from the wild type had inactivating activity against D3 and E79, whereas that from PAO(D3) inactivated neither. Chromatographic analysis indicated that the convertant lipopolysaccharide was smooth, and quantitative chemical analyses of the two preparations showed no differences in the level of the major fatty acids, amino compounds, or neutral sugars. On the other hand, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the side chains had a decreased migration rate through the gel matrix. The application of 1H and 13C nuclear magnetic resonance spectroscopic analysis revealed that the PAO side chain is chemically identical to that of serotype O:2a,d, containing 2,3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, and 2-acetamido-2,6-dideoxy-D-galactose (D-fucosamine). The molecular basis of the conversion event was (i) the introduction of an acetyl group into position 4 of the fucosamine residue and a change in the bonding between trisaccharide repeating units from alpha 1 leads to 4 to beta 1 leads to 4.
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PMID:O-antigen conversion in Pseudomonas aeruginosa PAO1 by bacteriophage D3. 619 Jul 94

The techniques of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining, and immunoblotting were used to analyze the lipopolysaccharide (LPS) structure of 20 strains of Campylobacter jejuni and 4 strains of Campylobacter coli belonging to more than 22 thermostable serotypes. The LPSs of all strains examined were shown to be of a low-molecular-weight type, and these low-molecular-weight LPSs conferred heat-stable serospecificity. High-molecular-weight banding observed with both in vivo LPS in proteinase K digests of whole cell lysates and purified LPS was shown to be due to the ready ability of Campylobacter lipopolysaccharide to form aggregates rather than to the presence of O polysaccharide chains. Purified LPSs from two strains of C. jejuni were also subjected to gross chemical analysis. The high-lipid A to low-neutral sugar ratio of both LPSs was typical of LPSs lacking O polysaccharide chains.
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PMID:Structural and antigenic heterogeneity of lipopolysaccharides of Campylobacter jejuni and Campylobacter coli. 620 38

The lipopolysaccharide (LPS) of Neisseria gonorrhoeae whole-cell lysates and proteinase K-digested lysates was examined and compared with purified homologous LPS by a method which preferentially stains LPS in polyacrylamide gels. The silver-stained profile of gonococcal LPS in the proteinase K-digested lysate was similar to that of homologous purified LPS; however, the LPS profile in whole-cell lysates was much smaller than that of digested lysates or purified LPS. Conditions of solubilization did not affect these differences. Since it is known that LPS migrates in a unique fashion in second-dimension electrophoresis, the location of LPS in the whole-cell lysates was probed by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a variety of stains and radiolabels. Results from these experiments indicated a stable and reproducible association of LPS with proteins ranging between 23,000 to 36,000 in Mr, in particular major outer membrane protein I. In addition to staining with the silver method, which preferentially stains LPS, the putative LPS was resistant to digestion by proteinase K, did not stain with Coomassie brilliant blue, and was not labeled extrinsically with 125I (Iodogen method) or intrinsically with [35S]methionine. Analysis of two-dimensional gels by immunoblotting with rabbit antisera prepared from protein I bands removed from a polyacrylamide gel revealed the presence of antigens in the same area of the gel (below proteins that were 23,000 to 36,000 in Mr). Antibodies to constituents which migrated below the diagonal were essentially removed by adsorption of antisera with purified LPS, as were antibodies to homologous LPS and LPS in proteinase K-digested whole-cell lysates. Immunoblotting with a monoclonal antibody specific for LPS demonstrated reactivity of the antibody with LPS and with the protein I band. On the basis of these data, we conclude that protein I and perhaps other proteins in the whole-cell lysate are stably associated with LPS; this complex is resistant to dissociation in sodium dodecyl sulfate at high temperature (approximately 100 degrees C) but does, for unknown reasons, dissociate with electrophoresis in the second dimension. The association of LPS with protein antigens in sodium dodecyl sulfate-polyacrylamide gels adds another dimension of complexity to analysis of these antigens by immunoelectroblotting. Furthermore, the tight association of LPS with the major outer membrane protein I may alter the nature of the immune response generated by "purified" protein I vaccine antigens. The possible role of protein-LPS complexes in the pathogenesis of gonorrhea is discussed.
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PMID:Analyses of gonococcal lipopolysaccharide in whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: stable association of lipopolysaccharide with the major outer membrane protein (protein I) of Neisseria gonorrhoeae. 620 9

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the lipopolysaccharides of typical and atypical strains of the fish pathogen Aeromonas salmonicida. 32P intrinsically radiolabeled lipopolysaccharide in sarcosinate-extracted outer membrane preparations, lipopolysaccharide stained by silver in proteinase K-digested outer membrane preparations and whole cell lysates, as well as purified lipopolysaccharide, displayed O-polysaccharide chains which were unusually homogeneous with respect to chain length. Chemical analysis further revealed that the sugar composition of the smooth lipopolysaccharide purified from three typical strains was very similar. Immunoblotting and immunofluorescent staining with both polyclonal and monoclonal antibody showed that the O-polysaccharide chains were strongly immunogenic and were antigenically cross-reactive on typical and atypical strains from diverse origins. Immunofluorescence analysis and phage binding studies demonstrated that a number of these O-polysaccharide chains traversed the surface protein array of virulent strains of A. salmonicida and were exposed on the cell surface.
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PMID:Structural and immunochemical homogeneity of Aeromonas salmonicida lipopolysaccharide. 637 Sep 55

Monoclonal antibodies were prepared by the fusion of murine myeloma NS1 cells with spleen cells of BALB/c mice immunized with Formalin-killed elementary bodies of the Chlamydia trachomatis L2 serovar. The specificity of these monoclonal antibodies was determined with a solid-phase immunoassay in which HeLa 229 cells infected with C. trachomatis serovars D, G, H, I, L2 and the Chlamydia psittaci meningopneumonitis strain Cal-10 were used. An immunoglobulin G3 monoclonal antibody (L2I-6) was identified that reacted with both C. trachomatis- and C. psittaci-infected HeLa cells. The immunoreactivity of the genus-specific epitope was heat resistant (100 degrees C, 10 min) but was destroyed by sodium metaperiodate treatment. Further characterization of the chlamydial specificity of monoclonal antibody L2I-6 by microimmunofluorescence showed that it was reactive with all 15 C. trachomatis serovars and seven C. psittaci strains isolated from five different animal species. We undertook studies to identify the biochemical nature of the chlamydial component on which the genus-specific epitope was located. The immunoreactive component was isolated by hot phenol-water extraction of dithiothreitol-reduced chlamydial elementary bodies. The component was positive in the Limulus amoebocyte lysate test (results of Limulus amoebocyte lysate assay were identical with those of Salmonella typhimurium LT2 SAI 377 Re lipopolysaccharide [LPS]), contained 8.8% 2-keto-3-deoxyoctulosonic acid, was resistant to proteinase K, and possessed electrophoretic mobility and silver-staining characteristics in sodium dodecyl sulfate-polyacrylamide gel electrophoresis consistent with a rough LPS or glycolipid. On the basis of these findings, we conclude that the genus-specific epitope recognized by monoclonal L2I-6 is located on chlamydial LPS. We further characterized the antigenic properties of the chlamydial LPS epitope by examining the immunoreactivity of monoclonal antibody L2I-6 by immunoblotting analyses against isolated LPSs extracted from Neisseria gonorrhoeae, S. typhimurium, and Escherichia coli. Monoclonal antibody L2I-6 did not bind LPS of these organisms, demonstrating that the chlamydial genus-specific LPS epitope is apparently not shared by these gram-negative bacteria. We were able, however, to show that the chlamydial LPS does share antigenic determinants with LPS of gram-negative organisms. Polyclonal rabbit antisera raised against S. typhimurium Re LPS or lipid A showed intense immunological cross-reactivity with chlamydial LPS by immunoblotting.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Monoclonal antibody against a genus-specific antigen of Chlamydia species: location of the epitope on chlamydial lipopolysaccharide. 642 19


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