Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The monokine eosinophil cytotoxicity enhancing factor (ECEF) increases antibody-dependent cytotoxicity of eosinophils towards helminth larvae. A monokine biochemically indistinguishable from ECEF increases the release of leukotriene C4 and other arachidonic acid metabolites by eosinophils. We have developed monoclonal antibodies (mAb) to these monokines by immunizing mice with ECEF made by the U-937 histiocytic lymphoma cell line. mAb 81.10.C9 (IgG2b) and 9A6G (IgG1) inhibit the effect of the monokine on release of AA products. Both mAb bind ECEF, which appears after affinity chromatography purification as a major 13-14-kDa and a minor 62-kDa component (13-14 kDa and 52 kDa after reduction) in silver-stained gels. An additional component of 30 kDa is detectable after radioiodination of the immunopurified material. The specificity of both mAb was studied in several ways. In immunoprecipitation, both recognize the 13-14-kDa and the 30-kDa components, while the 62-(52)-kDa protein is not significantly precipitated. Both mAb react in enzyme-linked immunosorbent assay with products secreted by peripheral blood mononuclear cells and monocytes, as well as with those secreted by phorbol 12-myristate 13-acetate and lipopolysaccharide-stimulated U-937 cells and with the immunopurified proteins. These were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroeluted and assayed for ECEF activity. Activity was associated with the 13-14-kDa and the 30-kDa fractions, as seen by increased eosinophil antibody-dependent adherence to schistosomula and cytotoxicity. Granulocyte-monocyte-colony-stimulating factor and interleukin 1, but not tumor necrosis factor, could be detected in crude U-937 supernatants. However, active immunopurified ECEF has no activity in assays for granulocyte-monocyte-colony-stimulating factor, interleukin 1 or tumor necrosis factor. Immunocytochemical localization of ECEF employing the mAb shows strong surface staining of viable monocytes and U-937 cells, suggesting that ECEF is associated to the cell surface. These properties distinguish ECEF from other monokines previously reported to activate eosinophils.
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PMID:Eosinophil cytotoxicity enhancing factor: purification, characterization and immunocytochemical localization on the monocyte surface. 219 4

Smooth (S)-lipopolysaccharide (LPS) preparations from reference and field strains of several biovars of Brucella abortus, B. melitensis, and B. suis were prepared by (i) the hot phenol-water method, (ii) hot sodium dodecyl sulfate extraction and proteinase K digestion, or (iii) dimethyl sulfoxide extraction. These S-LPS-enriched fractions were further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining after periodate oxidation. Immunoblots were developed by using either monoclonal antibodies specific for Brucella A or M antigens or polyclonal polyspecific or monospecific sera from rabbits, cattle, and goats. The specificity of monoclonal antibodies reactive with Brucella unique (A or M) epitopes was demonstrated by enzyme-linked immunosorbent assay, LPS latex agglutination, or agglutination inhibition. The most-represented subunits of S-LPS ranged in Mr from 30,000 to 70,000 relative to marker proteins. According to A or M immunodominance, two sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns were clearly distinguished among biovars, whatever the fraction tested: a close succession of regularly spaced narrow bands for A greater than M strains and regularly spaced triplets of bands including either (i) a first thin band followed by two thick bands for B. abortus M greater than A strains or (ii) one thick band between two thin bands for B. melitensis or B. suis M greater than A strains. Moreover, A and M specificities were reaffirmed by sandwich enzyme immunoassay and latex agglutination inhibition with monoclonal antibodies and polyclonal sera.
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PMID:Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of smooth-lipopolysaccharide heterogeneity among Brucella biovars related to A and M specificities. 222 39

The protein and lipopolysaccharide (LPS) compositions of 10 autoagglutinating Aeromonas hydrophila and Aeromonas sobria strains were studied; one group consisted of five serogroup O:11 strains that contained an S layer, while a second group was composed of diverse serogroups that were S layer negative by transmission electron microscopy. All serogroup O:11 strains were found to contain a predominant 52,000- to 54,000-molecular-weight protein that was present on both whole-cell and outer membrane protein profiles; this protein was found to be glycine extractable under low-pH (pH 4) conditions and was identified as the surface array protein. LPS analysis revealed that all O:11 strains exhibited homogeneous-length O-polysaccharide side chains characterized primarily by two or three major bands. In contrast, S-layer-negative autoagglutinating strains of other serogroups lacked this predominant surface array protein, and silver stain analysis of LPS indicated that such profiles mainly consisted of core antigens and were deficient in or devoid of O-polysaccharide side chains. These collective results offer potential explanations for observed differences between these two groups in virulence, disease spectrum, and pathogenic properties.
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PMID:Electrophoretic analysis of the surface components of autoagglutinating surface array protein-positive and surface array protein-negative Aeromonas hydrophila and Aeromonas sobria. 222 47

Rhizobium leguminosarum B556 and 8002 differ only with respect to carrying symbiotic plasmids with specificity for Pisum or Phaseolus hosts, respectively. Protease-treated samples derived from free-living cultures of both strains revealed a ladder of lipopolysaccharide (LPS-1) bands after periodate-silver staining of sodium dodecyl sulfate-polyacrylamide gels. These bands were arranged as doublets. After Western (immuno-) blotting, all LPS-1 bands reacted with monoclonal antibody JIM 21, whereas monoclonal antibody MAC 57 reacted only with the upper (slower-migrating) band and monoclonal antibody MAC 114 reacted only with the lower band of each doublet pair. Preparations obtained from bacteroids of Pisum or Phaseolus nodules showed significant differences in the size distribution and antigenicity of LPS. In bacteroids from Phaseolus sp., JIM 21 and MAC 57 each stained a ladder of LPS-1 bands on sodium dodecyl sulfate-polyacrylamide gels which corresponded in mobility to the upper band of each doublet pair seen in free-living cultures. MAC 114 did not react with the LPS from Phaseolus sp.-derived bacteroids. In bacteroids from Pisum sp., only fast-migrating (lower-molecular-weight) forms of LPS-1 could be visualized on gels, but both upper and lower bands of each doublet were still present and could be stained by the appropriate monoclonal antibody, MAC 57 or MAC 114, respectively. Similarly, bacteroids from R. leguminosarum 3841, which nodulates Pisum species, differed with respect to the structure and antigenicity of their LPS-1 from bacteroids of a related strain, B625, which nodulates Phaseolus species. Physiological factors were investigated that could account for these differences between the structures of LPS-1 from free-living cultures of B556 and 8002 and that from bacteroids. The following modifications in growth conditions each tended to reduce the expression of MAC 114 antigen and enhance the expression of MAC 57 antigen: succinate rather than glucose as the carbon source; microaerobic (2.5%, vol/vol) oxygen concentrations; and acidic (pH 5 to 6) culture medium. When all three of these conditions were combined, the LPS-1 that resulted was very similar to that in bacteroids from Pisum nodules. However, it was not possible to reproduce the LPS-1 pattern observed for bacteroids from Phaseolus nodules, which maintained a ladder of LPS bands reacting with MAC 57 antibody.
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PMID:Immunochemical analysis of lipopolysaccharides from free-living and endosymbiotic forms of Rhizobium leguminosarum. 231 3

A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been shown to have no effect on the recognition of this epitope by the monoclonal antibody. On the contrary, the inhibition of the antigen reactivity to the monoclonal antibody after a mild periodate oxidation suggests the involvement of a carbohydrate moiety in the epitope. Moreover, the SDS-PAGE analysis of phenol-water extracts has shown an additional compound, detected by silver staining but not recognized by the monoclonal antibody.
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PMID:Demonstration of a surface antigen of Clostridium tyrobutyricum by use of immunoblotting with a monoclonal antibody. 232 79

A simple physical-chemical method for estimating the lipopolysaccharide (LPS, i.e. endotoxin) content of meningococcal polysaccharide vaccines is described. The low molecular weight LPS was first separated from the high molecular weight polysaccharide by SDS-polyacrylamide gel electrophoresis. The LPS was then selectively visualized by a highly sensitive silver stain which can detect 5 ng of meningococcal LPS. The method can detect concentrations of LPS in meningococcal polysaccharides as low as 0.01%, and was used to measure semiquantitatively the LPS in bulk powders of meningococcal polysaccharides and in vaccines in final containers. The LPS in the bulk powders and in the vaccines was also assayed by the gelation of Limulus polyphemus ameobocyte lysate and the results of the two methods were in agreement.
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PMID:The analysis of lipopolysaccharide (endotoxin) in meningococcal polysaccharide vaccines by silver staining following SDS-polyacrylamide gel electrophoresis. 242 Aug 3

To determine whether cysteine residues have a contribution to the mechanism of color silver staining, we silver stained sodium dodecylsulfate polyacrylamide gel electrophoresis separations of proteins which have few or no cysteines. Proteins without cysteine stained negatively (yellow against a yellow background) with silver. Proteins with one or more cysteines stained orange, red, brown, or green/gray depending on the mole percentage of cysteine and whether they contained covalently attached lipids. The colors could not be correlated with the mole percentages of cysteine of these proteins indicating that some components other than cysteine affect the staining color of cysteine-containing proteins. Silver staining of amino acids, sugars, nucleotide bases, or lipopolysaccharide dot-blotted onto nitrocellulose paper implicated adenine, lipids, the basic amino acids, and glutamine, but not sugars or other amino acids in silver/protein complexes.
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PMID:Requirement for cysteine in the color silver staining of proteins in polyacrylamide gels. 242 85

Rough mutants from Salmonella typhimurium and Salmonella minnesota were transformed with a plasmid containing a 6.5-kilobase insert of DNA from Chlamydia trachomatis assumed to encode a glycosyltransferase. Transformation resulted in the expression of a genus-specific chlamydial epitope on the lipopolysaccharide (LPS) of the recombinant strains. Proteinase K-digested whole-cell lysates of the recombinants and of controls were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining or Western blot analysis. Two LPS populations were detected in the recombinants, the parent LPS and a faster-migrating component. The latter stained with monoclonal antibody against the genus-specific chlamydial epitope and was not seen in the controls. LPS was extracted and purified from recombinants of S. minnesota R595 and R4 and characterized by the passive hemolysis and passive hemolysis inhibition assays and by hydrolysis kinetics. Different antigenic determinants could be distinguished from each other by the passive hemolysis inhibition test with monospecific antigen-antibody reactions. Rabbits were immunized with heat-killed recombinant bacteria to study the immunogenic properties of the recombinants. In all animals, antibodies were raised against the parent core specificity and against the chlamydia-specific epitope. The data show that the recombinant bacteria are useful as immunogens to prepare polyclonal antisera against chlamydiae and that LPS isolated from them exhibits the same antigenic determinants as chlamydial LPS and may thus be used as a substitute for chlamydial LPS in serological assays.
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PMID:Antigenic and immunogenic properties of recombinants from Salmonella typhimurium and Salmonella minnesota rough mutants expressing in their lipopolysaccharide a genus-specific chlamydial epitope. 243 22

Some strains of Campylobacter jejuni react with more than one reference antiserum from the serotyping scheme based on heat-stable lipopolysaccharide antigens. To investigate the molecular basis of these cross-reactions, lipopolysaccharides from the reference strains for serotypes 4, 13, 16, 43, and 50 and isolates recovered during two different outbreaks of C. jejuni enteritis were analyzed by passive haemagglutination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis coupled with silver staining or immunoblotting. The results showed that lipopolysaccharides from the reference strains and the isolates reacted with antisera prepared against heterologous strains in various combinations and that both silver-stainable, low Mr and non-silver-stainable, high Mr lipopolysaccharide components provided the antigenic determinants associated with the cross-reactions. There were strain-to-strain differences in the structural and antigenic properties of these macromolecules and shared antigenic determinants were not always provided by a common structure. Analysis of the silver-stained lipopolysaccharide profiles, outer membrane protein patterns, and chromosomal DNA restriction patterns indicated that strains with the same lipopolysaccharide profile could have the same outer membrane protein pattern and the same DNA restriction pattern. These results provided evidence for the presence of clones within this antigenic complex and implicated antigenic variation in some strains as the phenomenon responsible for the multiplicity of cross-reactions.
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PMID:Characterization of cross-reacting serotypes of Campylobacter jejuni. 247 59

The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM1330 expressed O-antigen in E. coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex. Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E. coli K-12. Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was close relationship among the O101 ETEC isolates.
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PMID:Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E. coli B41 (O101:K99/F41) and the genetic relationship to other O101 rfb loci. 247 71


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