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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two monoclonal antibodies against filamentous haemagglutinin (F-HA) from Bordetella have been produced. In immunoblotting of a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of high ionic strength extracts of Bordetella pertussis, the two monoclonal antibodies both stained several high molecular weight bands. The two monoclonal antibodies were used for immuno-affinity column one-step purification of F-HA from high-ionic-strength extracts of Bordetella pertussis. In gradient SDS-PAGE, the eluted F-HA is present as a major double band with a molecular weight of approximately 240,000 daltons and several minor bands with molecular weights down to 90,000 daltons. The monoclonal antibodies were used in a monoclonal antibody catching enzyme linked immunosorbent assay (ELISA) for sensitive and specific detection of F-HA. This ELISA system proved valuable in optimizing the elution conditions for the monoclonal affinity columns.
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PMID:Purification and partial characterization of filamentous haemagglutinin from Bordetella pertussis using monoclonal antibodies. 609 92

Bordetella pertussis microorganisms were treated with several extracting agents followed by ultracentrifugation to remove particulate matter. Analysis of the resulting supernatants by SDS gel electrophoresis showed one major component after simple salt extraction, and much more complex, although consistent pattern following detergent treatment. The yield of the solubilized protein in detergent extracts exceeded by far the values recorded for salt extracts. In order to prevent irreversible precipitation of the solubilized proteins upon removal of the denaturing agent, a novel procedure was developed. After extraction with urea-salt, the solubilized material was absorbed on a mineral carrier prior to the separation of the denaturing agent. The resulting absorbed vaccine was highly potent in the mouse-protection test, whereas the toxic reactions, elicited upon injection into experimental animals, were reduced in the comparison to the starting material. This diminished reactogenic potential was accompanied by the partial loss of the leukocytosis-promiting factor, whose activity was greatly diminished by urea-salt at alkaline pH-values. The procedure described may be applied to large-scale processing of Bordetella persussis microorganisms. Clinical trials now in progress should confirm or rebut the thesis that increased tolerability of the product, inferred from animal experiments, is reflected by fewer adverse reactions in humans. In the former case, the detergent extract vaccine may constitute a realistic alternative to conventional whole-cell vaccines against whooping-cough.
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PMID:Extracted protective antigen of Bordetella pertussis. I. Preparation and properties of the solubilized surface of components. 626 98

The filamentous haemagglutinin of Bordetella pertussis has been purified from static, liquid culture supernatants and from extracts of cells grown on a solid medium. SDS-PAGE of the purified protein has shown multiple polypeptides with molecular weights ranging from 220 000 to about 58 000. By transferring the SDS-dissociated polypeptides to nitrocellulose paper and reacting with several monoclonal antibodies, it has been shown that many of the polypeptides are probably fragments of the polypeptide of highest molecular weight.
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PMID:Heterogeneity of the filamentous haemagglutinin of Bordetella pertussis studied with monoclonal antibodies. 631 62

The isolated polysaccharide chain, PS-1, of the Bordetella pertussis endotoxin was examined by isoelectric focusing, SDS-polyacrylamide gel electrophoresis and gel filtration for heterogeneity and for possible contamination by the parent endotoxin. This polysaccharide, previously found to be a very potent, macrophage-dependent, polyclonal B-cell activator and to mediate the specific binding of the endotoxin to macrophages, stimulated the interleukin 1 (IL 1) secretion by human monocytes; its potency was similar to that measured for the endotoxin. It was concluded that endotoxin-induced IL 1 production may be initiated by the interaction of the polysaccharide chain of the B. pertussis endotoxin and a specific structure present on macrophages.
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PMID:Interleukin 1 secretion by human monocytes stimulated by the isolated polysaccharide region of the Bordetella pertussis endotoxin. 633 May 37

Most of the isolates of Bordetella bronchiseptica obtained by this laboratory possessed a characteristic colonial morphology when grown on Bordet- Gengou agar (BGA) at 37 degrees C. The colonies appeared domed (Dom+) with a smooth colonial surface (Scs+) and a clear zone of hemolysis ( Hly +). From these Dom+ Scs+ Hly + BGA colony types arose flat (Dom-), smooth colonial surface (Scs+) and nonhemolytic ( Hly -) variants at frequencies of 10(-2) to 10(-3). Isogenic pairs of Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA phenotype variants (BGA-PVs) were picked from 11 strains of B. bronchiseptica, and their whole cell lysates were compared with each other by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Characteristic SDS-PAGE profiles were observed for each of the Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA-PVs with regard to (i) surface-exposed proteins, based on autoradiographs of 125I- Iodogen -labeled organisms, (ii) polypeptide differences, based on gels stained with Coomassie brilliant blue R-250, and (iii) lipopolysaccharide differences based on gels stained with silver after oxidation with periodic acid. SDS-PAGE profiles were then used to monitor the phenotypes expressed by Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA-PVs transferred and grown on brucella agar, Trypticase soy agar, and nutrient agar. When grown on non-BGA media, the Dom+ Scs+ Hly + BGA-PVs from six of eight strains showed SDS-PAGE profiles identical to those of Dom- Scs+ Hly - BGA-PVs. This phenotypic change was reversible even after 15 subcultures on the non-BGA media, since Dom+ Scs+ Hly + organisms passed back onto BGA expressed both Dom+ Scs+ Hly + colonial morphology and Dom+ Scs+ Hly + SDS-PAGE profiles. The influence of cultural conditions on maintenance of virulence is discussed.
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PMID:Phenotypic variation and modulation in Bordetella bronchiseptica. 637 14

We report here the identification of a virulence-associated factor, Tcf, (tracheal colonization factor), produced by strains of Bordetella pertussis but not Bordetella parapertussis or Bordetella bronchiseptica. This protein is encoded by the tcfA gene. When a strain of B. pertussis 18323 lacking this protein is used to infect mice with an aerosol challenge, the number of bacteria isolated from the tracheas is decreased 10-fold when compared with the parent 18323. The derived amino acid sequence of tcfA predicts a 68 kDa RGD-containing, proline-rich protein, which after cleavage of a typical prokaryotic signal sequence would be 64 kDa. Amino acid sequence analysis demonstrates that the C-terminal 30 kDa of this protein shows 50% identity to the 30 kDa C-terminus of another Bordetella protein, the pertactin precursor. The N-terminal 34 kDa region contains the three amino-acid motif RGD and is 16.5% proline. Coupled in vitro transcription and translation analysis indicates that the tcfA gene product migrates as two bands of approximately 90 kDa. A fusion protein of the N-terminal, 34 kDa portion of Tcf to maltose-binding protein migrates, on SDS-PAGE, 30 kDa higher than expected from the combined molecular weights. Polyclonal antisera raised against the unique N-terminal portion of Tcf recognizes 90 kDa and 60 kDa bands in immunoblots of whole-cell lysates of strains of B. pertussis; it does not recognize any protein in whole-cell lysates of B. bronchiseptica or B. parapertussis. Supernatants of cultures of B. pertussis 18323 contain the 60 kDa form of the protein. Southern blot analysis of chromosomal DNA from strains of B. bronchiseptica and B. parapertussis, using a probe derived from tcfA, shows strong hybridization only to B. pertussis DNA. Thus, Tcf appears to be a unique virulence factor of B. pertussis.
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PMID:Tracheal colonization factor: a Bordetella pertussis secreted virulence determinant. 747 58

We studied the biochemical mechanism of morphological changes in cells treated with Bordetella dermonecrotizing toxin (DNT). DNT caused the morphological changes of serum-starved MC3T3-E1 cells from flat shapes to reflactile ones. These changes were accompanied by the assembly of actin stress fibers and focal adhesions, which is known to be regulated by the small GTP-binding protein rho. Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and inactivates rho protein, 'rounded' the cells within 2 hours after addition to the extracellular fluid and their rounded shapes were maintained for at least 10 hours. However, when the cells were co-treated with C3 exoenzyme and DNT, they were rounded at 2 hours but recovered an apparently intact morphology after 3-8 hours of incubation. rho proteins in lysates from DNT-treated cells and untreated cells were radiolabeled by [32P]ADP-ribosylation with C3 exoenzyme and analyzed by SDS-polyacrylamide gel electrophoresis. Whereas the lysate from untreated cells showed a single band of [32P]ADP-ribosylated rho protein, the lysate from DNT-treated cells showed an additional two bands as well as the band identical to that of the lysate from untreated cells. Recombinant rhoA protein treated with DNT in vitro also showed a mobility shift in SDS-polyacrylamide gel electrophoresis. These results indicate that DNT causes the assembly of actin stress fibers and focal adhesions by directly modifying rho protein.
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PMID:Bordetella bronchiseptica dermonecrotizing toxin stimulates assembly of actin stress fibers and focal adhesions by modifying the small GTP-binding protein rho. 759 85

Bordetella (B.) bronchiseptica isolates from the respiratory tract of rat and pig in their virulent phase-I and their spontaneously developed avirulent phase-III were investigated. The strains were cultured on Bordet-Gengou agar, on nutrition agar with 10% horse blood, and with 5% sheep blood, on yeast agar, on minimal nutrition agar (MM) and Simmon's citrate agar with glycerol, starch, and nicotinic acid. Antigenic modulation was induced by MgSO4 on Bordet-Gengou agar. The B. bronchiseptica strains were cultivated at 37 degrees C for 48 h. The influence of different MgSO4 concentrations after five passages (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100 mM/ml and low (20 degrees C) and high (42 degrees C) temperatures on antigenic modulation was investigated on Bordet-Gengou (B-G) agar. B. bronchiseptica colonies were characterized in relation to morphology, haemolysis, slide agglutination with B. bronchiseptica phase-I polyclonal antibodies, haemagglutination with horse and calf erythrocytes and polypeptide and LPS patterns in SDS-PAGE. Cultivation of B. bronchiseptica phase-I on B-G agar and ACMM agar at 37 degrees C for 48 h resulted in strong phase-I antigenic patterns, and, on peptone-rich media, in antigenic modulation. The morphology of B. bronchiseptica phase-I-strain colonies on peptone media was different from that of B-G and ACMM agar. The LPS pattern of both strains resembled that of phase-III strains. MgSO4 concentrations of 1 mM/ml (strain 1636 I) and 3 mM/ml (Ratte I) were able to induce LPS-pattern-like phase III.
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PMID:Influence of culture conditions on expression of antigens in Bordetella bronchiseptica. 759 57

Antiserum to canine serum amyloid A (SAA) was prepared in rabbits by immunization with crude SAA which was prepared from high-density lipoprotein 3 (HDL3) obtained from canine acute-phase serum. The antiserum was absorbed for contaminating antibodies by affinity chromatography using Sepharose 4B coupled with normal canine serum proteins. The rabbit anti-canine SAA serum reacted with a protein and formed a single precipitin line at the position of the alpha 1-region of the immunoelectrophoresis of canine acute-phase serum but did not react with the normal canine serum on immunoelectrophoresis. The antibody to canine SAA was also confirmed by Western blotting analysis. Canine SAA was purified as a low molecular weight protein component from crude SAA by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after gel filtration chromatography. Purified canine SAA had a molecular weight of 15,000 as estimated by SDS-PAGE. This SAA level was found by enzyme-linked immunosorbent assay (ELISA) to increase 1 day after inoculation with Bordetella bronchiseptica to 9.0-20.1 times the preinoculation value.
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PMID:Preparation of anti-canine serum amyloid A (SAA) serum and purification of SAA from canine high-density lipoprotein. 806 95

The R1.1 mouse thymoma cell line expresses a high-affinity kappa opioid binding site. Opioid binding to this site is inhibited by guanine nucleotides, suggesting that the receptor is coupled to a guanine nucleotide-binding protein. Here, we present evidence that the kappa opioid binding site on R1.1 cell membranes is negatively coupled to adenylyl cyclase. The kappa-selective agonists (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide methane-sulfonate hydrate [(-)-U50,488], (5 alpha,7 alpha, 8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxas- piro(4,5)dec-8-yl)benzeneacetamide (U69,593) and several dynorphin peptides inhibited basal and forskolin-stimulated cyclic AMP production by up to 40% in R1.1 cell membranes. The order of potency for the inhibition of adenylyl cyclase activity by opioid agonists correlated with their Ki values for the inhibition of [3H]U69,593 binding. Opioid-mediated inhibition of adenylyl cyclase activity was stereoselective, as (-)-U50,488 was more potent than the (+) isomer, and the inhibition was blocked by the kappa-selective antagonist nor-binaltorphimine. The opioid-mediated inhibition of adenylyl cyclase activity was also completely blocked by incubating R1.1 cells with Bordetella pertussis toxin (PTX). Incubation of R1.1 cell membranes with PTX and [adenylate-32P]NAD+ resulted in the exclusive labeling of a 41-kDa protein, as determined by separating the membrane proteins under reducing conditions on a SDS polyacrylamide gel, followed by autoradiography. These results suggest that a PTX-sensitive inhibitory guanine nucleotide-binding protein mediates the link between the thymoma kappa opioid receptor and adenylyl cyclase.
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PMID:The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line is coupled to adenylyl cyclase through a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein. 810


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