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Five Bordetella pertussis strains of phase I were grown in conventional casamino-acid medium and in media modified by adding high concentrations of MgSO4 or nicotinic acid. Cells grown in high-magnesium media (in the C-mode) had only about 4% of the protective antigen (PA) and 6% of the histamine-sensitising factor (HSF) of cells from the normal medium. Envelopes from C-mode organisms when examined by SDS-PAGE showed a loss of 28K and 30K polypeptide bands. Similar parallel losses of PA, HSF and 28K and 30K bands were found with cells from the high-nicotinic-acid medium. A medium with a high concentration of nicotinamide gave cells with normal amounts of PA, HSF and 28K and 30K bands. Growth in high concentrations of Na2SO4 caused partial losses of PA, HSF and 28K and 30K bands, while a high-succinate medium gave cells with somewhat diminished PA and HSF but without appreciable attenuation of the 28K and 30K bands. Because of the close correlation between the presence or absence of PA, HSF and 28K and 30K envelope polypeptides, it is suggested that the latter may represent or be closely associated with the components responsible for PA and HSF activities.
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PMID:Loss of protective antigen, histamine-sensitising factor and envelope polypeptides in cultural variants of Bordetella pertussis. 5 40

Cell-envelope polypeptides of eight phase-I and five phase-IV strains of Bordetella pertussis were compared by SDS-polyacrylamide gel electrophoresis. All phase-I strains gave a strikingly similar but complex pattern of protein bands, which did not appear to vary with known differences in heat-labile agglutinogens. Phase-IV strains gave the same pattern as phase-I strains, except that one band was missing and another was either much reduced or absent. Envelopes from phase-I strains grown in Hornibrook medium rich in Mg-2+ ions to produce "antigenically-modulated" C-mode cells gave a pattern of bands indistinguishable from phase-IV strains. A phase-IV strain grown in the high-Mg-2+ medium gave the same pattern of bands as when grown in unmodified Hornibrook medium. We suggest that the two polypeptide bands that show changes may be responsible for one or more of the immunological or physiopathological activities that are lost during phase variation and antigenic modulation in B. pertussis.
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PMID:Cell-envelope proteins of Bordetella pertussis. 16 97

Differential centrifugation was used to prepare fractions from broken cells of Bordetella pertussis strain 114. Whole cells and several fractions were then assayed for potency and for safety. Crude ribosomal fractions were uniformly protective. However, ribosomes purified by washing in high salt solution and recentrifugation were at least 40 fold less potent. Protective antigen was found in the wash fluid. Wash fluid was subjected to SDS-polyacrylamide gel electrophoresis. No specific protein or carbohydrate has yet been identified as a protective immunogen. It is clear that ribosomes are not protective, but copurify with protective antigen. SDS-polyacrylamide gel analysis of soluble material purified from ribosomes may be of value in experimental studies on pertussis vaccine. If the protective immunogen can be identified, this procedure may also be of value in vaccine standardization.
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PMID:Subcellular fractions for immunizing against pertussis. 22 10

In active anaphylactic shock of rats pretreated with Bordetella pertussis vaccine, both plasma thrombin clotting time and the amount of antigenically active fibrinogen degradation products in the serum were increased. The formation of clottable fibrinogen fragments was shown by SDS polyacrylamide gel electrophoresis of thrombin-induced clots. When plasma of rats pretreated with 125I rat fibrinogen and then subjected to anaphylaxis was submitted to SDS polyacrylamide gel electrophoresis, fibrinogen-split products were also detected. Fibrinogen degradation results from the proteolytic effect of an activated fibrinolytic enzyme.
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PMID:Evidence of fibrinogen degradation in rat anaphylaxis. 115 26

A method is described for the separation and purification of proteins from complex mixtures of foreign antigens in a form suitable for stimulating T cells in vitro. The technique involves electrophoretic separation of proteins followed by elution, concentration and adsorption of the polypeptide subunits to latex microspheres. Alternatively, where a specific antibody is available, proteins may be affinity-purified from a heterogeneous mixture of antigens, using antibody-coated latex microspheres. Nanogram quantities of protein coupled to latex were shown to be highly efficient stimulators of antigen-specific T cells as tested by in vitro proliferation and cytokine release assays. The utility of this technique was demonstrated using poliovirus capsid proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and coupled to latex microspheres for specificity analysis of T cell clones. Antigen reactivity of the T cell clones was confirmed using recombinant baculoviruses expressing individual poliovirus proteins. Furthermore, recombinant proteins coupled to latex microspheres were used for efficient stimulation and in vitro propagation of T cell clones specific for the simian immunodeficiency virus (SIV) envelope (env) protein. Although the technique is illustrated in this report using viral antigens, it has also proved to be an efficient method for the separation of bacterial antigens in studies of polyclonal T cell responses to Bordetella pertussis antigens.
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PMID:Preparative separation of foreign antigens for highly efficient presentation to T cells in vitro. 133 64

In a previous work we demonstrated that Trypanosoma cruzi exoantigens of pI 4.5 (Ea 4.5), whose most important epitopes are glucidic, are able to induce a partially protective immune response in mice. To ascertain the involvement of antibody isotypes in this protection, we immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The analysis of immune response by skin test revealed the occurrence of specific immediate type hypersensitivity on Day 15 after the last immunization. By ELISA and using Ea 4.5 as antigen, specific IgG1 antibody was detected. When formaldehyde-fixed epimastigotes were used as antigen, binding of IgG1 and IgG2 was observed. Trypomastigotes incubated for 1 hr at 33 degrees C with the immune sera and then injected in normal syngeneic mice produced a significantly lower parasitemia than trypomastigotes incubated with the control sera. This capacity of anti-Ea 4.5 sera was resistant to 56 degrees C for 2 hr and was diminished after the absorption of immune sera with the carbohydrate moiety of Ea 4.5. The assay with the immune IgG1 and IgG2, separated through protein A-Sepharose affinity chromatography, showed that IgG1 retains most of this capacity. Purified immune IgG1 revealed two antigenic bands of molecular weight between 50 and 55 kDa in SDS-PAGE of Ea 4.5.
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PMID:Trypanosoma cruzi: involvement of IgG isotypes in the parasitemia control of mice immunized with parasite exoantigens of isoelectric point 4.5. 163 59

Pertussis toxin (PT), an oligomeric exotoxin of Bordetella pertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid single-step procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (S1), 70% (S2), greater than 90% (S3), greater than 90% (S4), and 50% (S5), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes.
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PMID:Purification and immunological characterization of HPLC-purified pertussis toxin subunits. 165 40

We obtained highly purified fimbrae from Bordetella pertussis cells which gave a single band in SDS-polyacrylamide gel electrophoresis. Using the purified fimbriae, we prepared anti-fimbriae rabbit or goat IgGs by fimbriae-Sepharose affinity chromatography, and developed ELISA for its determination. The concentration of fimbriae in our former type of acellular vaccine (80 micrograms per ml) was determined to be 20 ng per ml, and that in our component vaccine was at an almost negligible level. To estimate the protective effects of the small levels of fimbriae, we evaluated the protective activities of three types of vaccines; the former type acellular vaccine (pertussis toxin (PT): filamentous hemagglutinin (FHA): fimbriae = 15:61:0.02 (microgram per ml)), the two-component vaccine (16:64:0.0001), and the three-component vaccine (16:63:0.8). The three types of vaccines showed no significant differences in protectivity against the experimental aerosol infection suggesting that these levels of fimbriae are not effective against the experimental aerosol infection of mice with Bordetella pertussis.
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PMID:Comparison of the protective effects of the pertussis acellular vaccine with the component vaccine, which have different amounts of fimbriae, against the experimental aerosol infection of mice with Bordetella pertussis. 168 13

Spleen cells from mice immunized with a Bordetella pertussis N-lauroyl sarcosine membrane extract (SME) were used to generate hybridoma cells lines producing monoclonal antibodies (mAbs). Seven mAbs were shown to be specific to B. pertussis lipo-oligosaccharide (LOS) by immunoblotting of SME or purified LOS following SDS-PAGE. All mAbs reacted with the B. pertussis Tohama I strain of the LOS AB phenotype, and did not react with the atypical variant strain 134 of the LOS B phenotype. The immune reactivity of the mAbs was retained after treatment of SME with proteinase K and was lost after sodium periodate treatment. No cross-reactivity was observed with the mAbs when tested against B. parapertussis and other Gram-negative bacteria. However, all mAbs reacted with B. bronchiseptica. Binding assays with live B. pertussis cells demonstrated that mAbs strongly reacted with cell surface exposed antigenic determinants. High bacterial cell lytic capability was observed for five of these mAbs. Concentrations between 0.22 and 2.2 micrograms mAb ml-1 (0.1 and 1 microgram per 450 microliter assay) purified by protein A were required to kill at least 50% of the bacteria. Competition immunoassays with biotinylated antibodies showed that the bacteriolytic and non-bacteriolytic mAbs were directed to different epitopes of the B. pertussis LOS A.
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PMID:Characterization and comparative bactericidal activity of monoclonal antibodies to Bordetella pertussis lipo-oligosaccharide A. 171 58

The heterohybridoma cell line HBp2 secreting human monoclonal antibody (hMAb) directed against Bordetella pertussis was generated by fusing SP2/HPT heteromyeloma cells with human spleen lymphocytes, after in vitro stimulation for 6 days. The hybridoma was maintained in culture for more than 1 year with continuous antibody secretion. The hMAb HBp2, an IgM, reacted with untreated and proteinase K-treated B. pertussis outer membrane antigens, whereas the reactivity was lost when the antigen was treated with sodium periodate. Human MAb HBp2 was shown to be specific to B. pertussis LOS by immunoblotting of whole cell extracts after SDS-PAGE. In a dot enzyme immunoassay, HBp2 reacted with all B. pertussis strains and clinical isolates tested except for four atypical variant strains of the LOS B phenotype. Human MAb HBp2 also reacted with a clinical isolate of B. bronchiseptica. No reaction was observed against B. parapertussis and other gram-negative species. Together these studies suggested that HBp2 is reactive with carbohydrate epitopes present on the LOS A. Binding assays with live bacteria demonstrated that hMAb HBp2 reacted with cell surface exposed epitopes on B. pertussis but the antibody did not bind significantly to the surface on intact B. bronchiseptica cells. When examined for bactericidal activity in the presence of complement, hMAb HBp2 showed high lytic capability against B. pertussis while no killing was obtained against B. bronchiseptica. These experiments established that LOS A is a target for human bactericidal antibodies. This antigen merits further investigation as a potentially important component in human immunity to B. pertussis infection.
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PMID:Biological activity of a human monoclonal antibody to Bordetella pertussis lipooligosaccharide. 172 52


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