Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
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Lipopolysaccharides (LPS) isolated from Bordetella pertussis (Bp), B. parapertussis (Bpp), and B. bronchiseptica (Bbs) were analysed for their chemical composition, molecular heterogeneity, and immunological and biological properties. All LPS contained heptose, KDO, GlcN, uronic acid, phosphate, and fatty acids. The fatty acids C14:0, C16:0 and 3-OHC14:0 were common to all LPS preparations. By SDS-PAGE, Bp-LPS had two bands of low molecular mass, and Bpp- and Bbs-LPS showed a low molecular mass band together with ladder bands of high molecular mass. Immunological assays demonstrated that Bp-LPS reacted with antisera prepared from Bp and Bpp; Bpp-LPS reacted with antisera against Bpp and Bbs, and Bbs-LPS reacted with antisera against any of the three species. Bp-LPS showed biological activities comparable to those of E. coli LPS in terms of lethal toxicity, pyrogenicity, mitogenicity, macrophage activation, and induction of tumor necrosis factor. All activities of Bpp-LPS, except mitogenicity, were lower than those of E. coli LPS. Biological activities stronger or comparable to those of E. coli LPS were observed for Bbs-LPS.
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PMID:Structural and biological comparison of lipopolysaccharides (LPS) from Bordetella species. 177 13

An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca2+ to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca2+ and this interaction may be important for its invasion into animal cells. Finally, the technique for purifying the catalytic subunit by SDS-polyacrylamide gel electrophoresis may prove useful in studying the interaction of the adenylyl cyclase with other components produced by the bacteria, as well as the interaction of the enzyme with eukaryotic target cells.
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PMID:Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis. 185 26

The outer membrane of Wolinella recta ATCC 33238 was isolated by French pressure cell disruption and differential centrifugation. Outer membrane proteins (OMPs) were solubilized by Zwittergent 3.14 extraction and separated by DEAE-Sephacel ion-exchange chromatography. The major OMPs that were found in W. recta ATCC 33238 and in several other Wolinella spp. consisted of proteins with apparent molecular masses of 51, 45, and 43 kDa. These three conserved proteins were purified to essential homogeneity by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized chemically. Heating at between 75 and 100 degrees C revealed both the 43- and 51-kDa proteins to be heat modified from apparent molecular masses of 32 and 38 kDa, respectively. The 45-kDa protein was unmodified at all temperatures tested. Two-dimensional isoelectric focusing-SDS-PAGE revealed the 51-kDa protein to be composed of multiple pIs between a pH of 5.0 and greater than 8.0 while the 43- and 45-kDa proteins had a pI of approximately 6.0. N'-terminal amino acid sequence analysis of the first 30 to 40 amino acids and search of the Protein Identification Resource data base for similar proteins only revealed the 43-kDa protein to be similar to the P.69 OMP of Bordetella pertussis; however, the homology was weak (33%). Amino acid analysis revealed the 43-kDa protein to be noncharged and the 45- and 51-kDa proteins to be hydrophilic, containing between 38 to 42% polar residues but no cysteine. This study reports the purification and partial characterization of three conserved proteins in W. recta ATCC 33238.
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PMID:Extraction, purification, and characterization of major outer membrane proteins from Wolinella recta ATCC 33238. 189 72

In comparison with haemagglutinin (HA)-active strains of Bordetella bronchiseptica, the HA-deficient strains lacked a 150 kDa protein band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Adsorption of partially purified HA with bovine erythrocytes showed the loss of the 150 kDa band in the supernatant. This 150 kDa protein was purified by high-performance liquid chromatography using gel filtration columns. Electron microscopic examination revealed that the purified HA possessed a fine filamentous structure with dimensions of approximately 2 x 150 nm, which was considered to represent the filamentous haemagglutinin of B. bronchiseptica. Both the cell-free antigen obtained from the culture supernatant of phase-I strain of the bacteria and the bovine erythrocytes, which combined with crude HA showed an excellent protective activity of the challenge by virulent strain of B. bronchiseptica in mice.
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PMID:Characterization of haemagglutinin from Bordetella bronchiseptica. 195 98

The viability of four strains of Bordetella bronchiseptica, two strains of B. pertussis and one strain of B. parapertussis exposed to hyperimmune and pre-colostrum porcine serum was examined. Viable cell numbers (cfu/ml) of the B. pertussis strains and a rough strain of B. bronchiseptica (CSU-P-1) decreased by 99% and 99.99%, respectively, after exposure for 1 h to porcine hyperimmune serum. In contrast, smooth B. bronchiseptica strains and the B. parapertussis strain showed no significant decrease in viable cell numbers after the same treatment. B. bronchiseptica strain CSU-P-1 also showed a 99% decrease in viable cell numbers after exposure to pre-colostrum porcine serum for 1 h whereas the other strains tested showed no decrease in viable numbers under the same conditions. Heating the hyperimmune and pre-colostrum serum at 56 degrees C for 30 min resulted in the loss of bactericidal activity suggesting the involvement of complement in both systems. Analysis of silver-stained SDS-PAGE profiles of lipopolysaccharide (LPS) extracted from the bacterial cells indicated that the smooth strains of B. bronchiseptica and the B. parapertussis strain possessed high mol. wt O-side chain-like material, whereas the B. pertussis strains and B. bronchiseptica strain CSU-P-1 did not. Gel filtration of acid-hydrolysed LPS samples indicated two distinct carbohydrate peaks for the strains with high mol. wt O-side chain-like material, whereas the other strains each yielded one distinct peak. Western-blot analysis indicated a positive reaction for anti-B. bronchiseptica antibodies to the high mol. wt O-side chain-like material of all serum-resistant strains used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Serum sensitivity and lipopolysaccharide characteristics in Bordetella bronchiseptica, B. pertussis and B. parapertussis. 201 Sep 7

Pertussis toxin (PT), preactivated with 20 mm dithiothreitol (DTT), was incubated with different serum dilutions (1/10-1/200) before addition to the reaction mixture. Final concentrations of the reagents were: PT (50 ng/ml), dithiothreitol (DTT)(less than or equal to 0.3 mM), bovine transducin (2 micrograms/ml), ATP (1 mM). GTP (1 mM), lysophosphatidylcholine (LTC) (0.1 mg/ml), sodium acetate (NaAc) (0.1 M), Tris-HCl, pH 7.1 (0.06 M) and 32P-NAD+ (10 microCi 28 Ci/mM). The reaction was stopped by precipitation with 10% TCA (w/v), the pellet was collected and the samples were submitted to SDS-PAGE followed by autoradiography. ADP-ribosylation was detected as the radiolabelling of a protein band (m.w. approximately 40 kD) corresponding to the alpha-subunit of transducin. 32P-ADP incorporation was a linear function of PT concentration. By this assay quantitative differences in PT neutralizing antibodies were found between sera which were not revealed by measuring PT neutralizing antibodies by a Chinese hamster ovary (CHO) cell culture test or by an enzyme-linked immunosorbent assay (ELISA). The conditions of the ADP-ribosylation of bovine transducin have been optimized to permit detection of the enzymic activity of as low amounts of PT as 0.5 ng. This opens the possibility of the study of the presence and avidity of neutralizing antibodies to PT in post-vaccination sera without preceding purification and concentration of the antibodies and thus may provide a useful tool for evaluation of whooping cough vaccines.
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PMID:A sensitive method for measuring neutralizing antibodies to Bordetella pertussis toxin: optimized ADP-ribosylation of transducin. 204 77

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.
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PMID:Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis. 206 Jul 61

Lipopolysaccharides (LPS) isolated from Bordetella pertussis, B. parapertussis and B. bronchiseptica were analysed for their chemical composition, molecular heterogeneity and immunological properties. All the LPS preparations contained heptose, 3-deoxy-D-manno-2-octulosonic acid, glucosamine, uronic acid, phosphate and fatty acids. The fatty acids C14:0, C16:0 and beta OHC14:0 were common to all the LPS preparations. LPS from B. pertussis strains additionally contained isoC16:0, those from B. parapertussis contained isoC14:0 and isoC16:0, and those from B. bronchiseptica contained C16:1. By SDS-PAGE, LPS from B. pertussis had two bands of low molecular mass, and the LPS from B. parapertussis and B. bronchiseptica showed low molecular mass bands together with a ladder arrangement of high molecular mass bands. Immunodiffusion, quantitative agglutination and ELISA demonstrated that the LPS from B. pertussis strains reacted with antisera prepared against whole cells of B. pertussis and B. bronchiseptica; LPS from B. parapertussis reacted with antisera to B. parapertussis and B. bronchiseptica, and LPS from B. bronchiseptica reacted with anti-whole cell serum raised against any of the three species. From these results, it is concluded that LPS from B. bronchiseptica has structures in common with LPS from B. pertussis and B. parapertussis, while the LPS from B. pertussis and B. parapertussis are serologically entirely different from each other.
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PMID:Biochemical and immunological comparison of lipopolysaccharides from Bordetella species. 211 65

In order to investigate arthritis-triggering, serologically cross-reactive antigens, sera from patients infected with arthritis-associated microbes, Salmonellae, Chlamydia trachomatis, and Sindbis-like alphavirus, were reacted against SDS-PAGE separated and immunoblotted Yersinia enterocolitica 0:3 antigens. These sera reacted with Yersinia to the same extent as did the control sera taken from patients with streptococcal, staphylococcal and Bordetella pertussis infections or from healthy blood donors. Moreover, the various sera produced different reactivity patterns, directed against several different antigens. Although sera from test subjects, as well as from controls including healthy individuals, recognized some Yersinia antigens, these patterns differed markedly from those recognized by sera taken from patients with Yersinia infection. Significantly, analysis of the reactivities against the different molecular weight antigen components of Yersinia revealed no dominant band or pattern which could thus have been defined as arthritis-associated.
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PMID:Serological cross-reactions against Yersinia enterocolitica in patients infected with other arthritis-associated microbes. 216 7

The quantitation of pertussis toxin (PT) in two sandwich ELISAs was tested for specificity. The detection of the captured PT was obtained by using either polyspecific rabbit anti Bordetella pertussis serum (RaBp-ELISA) or a monoclonal anti-PT antibody (McaPT-ELISA). No major differences in the estimation of PT in highly purified preparations were noted using either ELISA variants. In contrast, the quantitation of PT in crude extracts of B. pertussis cultures by the RaBp-ELISA was found to be over-estimated and showed greater variability when compared to the McaPT-ELISA. Comparison of the distribution of PT in the eluate fractions following partial purification by hydroxylapatite chromatography revealed that the results of the McaPT-ELISA were more specific as judged by SDS-PAGE analysis.
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PMID:Quantitation of pertussis toxin in an enzyme linked immunosorbent assay with improved specificity. 237 59


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