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Biomedical subjects

R Parton

Publications and source records attributed to R Parton.

72 records · Page 4Linked to original sources
PubMed1987

Acellular pertussis vaccine prepared by a simple extraction and toxoiding procedure.

An extract containing predominantly pertussis toxin (PT) and filamentous haemagglutinin (FHa) was obtained from culture supernates of Bordetella pertussis by a single-step procedure using dye-ligand chromatography. The pathophysiological activities associated with the pertussis toxin component were removed by treatment with a water-soluble carbodiimide. The product was stable at 4 degrees C, was non-toxic for mice, induced high levels of IgG antibodies to both PT and FHa in vaccinated animals as judged by ELISA, and protected mice from intracerebral and intranasal challenge with B. pertussis.

Animals↗
PubMed1986

R-plasmid-mediated chromosome mobilization in Bordetella pertussis.

Antibiotic-resistant and auxotrophic mutants of Bordetella pertussis were isolated. These were used as recipients for the uptake from Escherichia coli of broad-host-range R plasmids R68.45, RP1, and RP1 and RP4 carrying transposons Tn501 and Tn7 respectively. B. pertussis transconjugants from these crosses were used as donors to mobilize StrR, NalR, thr+ and gly+ chromosomal markers to B. pertussis or to B. parapertussis recipient strains. The frequency of plasmid transfer varied and depended on the donor and recipient strains used. Differences in chromosome mobilization frequencies of individual markers were observed and appeared to depend on the presence or absence of transposons Tn501 and Tn7 on the plasmid. Linkage was detected between the gly+ and NalR markers.

Bordetella pertussis↗
PubMed1986

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector.

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.

Anthranilate Synthase↗
PubMed1986

Effect of anti-inflammatory agents on the haemorrhagic response of mouse skin to Bordetella pertussis heat-labile toxin.

Bordetella pertussis heat-labile toxin (HLT) was assayed by the haemorrhagic response produced by subcutaneous injection into weaned mice. Young mice, 3-5 weeks old of either sex, were highly responsive but they became resistant to HLT as they matured. Two anti-inflammatory agents, prednisolone and meclofenamate, inhibited the skin reactions in young mice. When given intraperitoneally, prednisolone was most inhibitory if it was injected just before, or at the same time as, HLT challenge. Prednisolone given 3 h after challenge, when the skin reactions had started to develop, did not significantly attenuate the final response. Both drugs were even more effective when mixed with the HLT challenge and injected subcutaneously. These findings are discussed in relation to the possible mechanism of action of HLT and to the reported beneficial effects of corticosteroids in the treatment of whooping cough.

Animals↗
PubMed1985

The effect of growth conditions on adenylate cyclase activity and virulence-related properties of Bordetella pertussis.

Growth of Bordetella pertussis in Stainer & Scholte medium in which the NaCl had been replaced by one of several inorganic or organic salts resulted in a large decrease in adenylate cyclase activity, histamine-sensitizing activity and in the amounts of two cell-envelope polypeptides of Mr 28000 and 30000. Although some variation between strains was observed, there was never a case where one of these properties was lost independently of the others. Cultures in which these properties were lost had decreased amounts of extracellular cAMP when compared to NaCl-grown cultures. Adenylate cyclase activity was detected in three locations of B. pertussis cultures (extracellular, extracytoplasmic but cell-associated, and cytoplasmic). After growth in medium containing high concentrations of MgSO4, enzyme activity was decreased to a similar extent in all three locations.

Adenylyl Cyclases↗
PubMed1985

Adenylate cyclase activity during phenotypic variation of Bordetella pertussis.

During MgSO4-induced modulation of Bordetella pertussis, adenylate cyclase activity, histamine-sensitizing activity (HSA) and the major cell-envelope polypeptides with Mr 28000 and 30000 (X polypeptides) were lost synchronously at a rate which could be accounted for by a simple growth-dilution effect. MgSO4 and other compounds which induced the above phenotypic change caused little inhibition of adenylate cyclase activity. Nicotinic acid was the sole exception and at 4.1 mM-caused 60% inhibition of activity. Lysates of modulated cells, mixed with lysates of unmodulated cells, had no effect on either adenylate cyclase activity or HSA. Protein synthesis was a prerequisite for MgSO4-induced modulation and also for the reversal of this process. Exogenous cAMP and dibutyryl cAMP (5 mM) had no counteracting effect on MgSO4- or nicotinic acid-induced modulation. The concentration of MgSO4 required to induce loss of the X polypeptides (10 to 11 mM) was not altered by promoting adenylate cyclase activity by including an activator in the growth medium. In one culture containing 10 mM-MgSO4 and activator, partial loss of the X polypeptides occurred and yet the extracellular cAMP concentration was twice that of cultures without activator and where full expression of the X polypeptides occurred. [3H]cAMP-binding activity was detected in cell extracts of several strains of B. pertussis, but antiserum against purified Escherichia coli catabolite repressor protein gave no reaction with B. pertussis cell extracts. Respiration rates with amino acids were similar for modulated and unmodulated variants and an avirulent strain of B. pertussis. These results are discussed in relation to a possible causal role for adenylate cyclase in modulation of B. pertussis.

Adenylyl Cyclases↗
PubMed1985

Effect of prednisolone on the toxicity of Bordetella pertussis for mice.

Prednisolone, given orally or intraperitoneally before challenge, protected mice against the lethal effect of a crude cell extract of Bordetella pertussis containing heat-labile toxin (HLT) as the major toxic component. Prednisolone did not diminish the lethal toxicity of heated B. pertussis cell suspensions containing pertussis toxin and endotoxin but devoid of HLT. This suggests that the protective effect of the steroid was directed against the HLT. When live bacteria were injected intraperitoneally, prednisolone showed a protective effect against the initial toxaemia. By day 7, however, the protection was no longer evident and the steroid promoted the survival of the organisms within the peritoneal cavity. These findings are discussed in the light of reports of the beneficial effects of corticosteroids in the treatment of whooping cough and in relation to a possible role for HLT in the pathogenesis of the disease.

Animals↗
PubMed1984

Purification and partial characterization of filamentous haemagglutinin from Bordetella pertussis using monoclonal antibodies.

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.

Antibodies, Monoclonal↗
PubMed1981

Effect of the growth environment on cell-envelope components of Escherichia coli in relation to sensitivity to human serum.

Three smooth strains of urinary Escherichia coli were grown in a chemostat under carbon-limited (C-lim) and magnesium-limited (Mg-lim) conditions over a range of dilution rates (D). Strain LP1674 was resistant to human serum under C-lim but became sensitive when grown under Mg-lim, the degree of sensitivity increasing as D increased. The transition to serum sensitivity was accompanied by loss of ability to produce extractable K1 antigen and a reduction in the amount of a 46k envelope polypeptide. C-lim cells of strain LP729 exhibited a delayed sensitive response to serum, the degree of lag in serum killing becoming less pronounced with increasing values of D; Mg-lim cells were more sensitive with little or no lag in serum killing. The degree of lag appeared to be directly related to the amount of the O side-chain sugar mannose associated with the lipopolysaccharide. C-lim and Mg-lim cultures of E. coli strain LP1395 were resistant to serum except when growing at near maximal rates. Although C-lim cultures contained more acidic polysaccharide than Mg-lim cells, transition to serum sensitivity did not appear to be related to exopolysaccharide production. Rapidly growing cells of strain LP1395 did, however, have lower lipopolysaccharide 0 side-chain sugar:core-sugar ratios than more slowly growing cells. With all three strains, changes in dilution rate and in the nature of the limiting nutrient were accompanied by changes in envelope protein composition. This study demonstrates that many cell-surface changes occur in response to alterations in the growth environment and some of these may be correlated with changes in sensitivity to serum.

Bacterial Proteins↗
PubMed1981

Rapidity of antigenic modulation of Bordetella pertussis in modified Hornibrook medium.

Modulation of Bordetella pertussis was induced by growth in Hornibrook medium with a high content of magnesium sulphate (C-medium); four pathophysiological activities in the cells or in the whole culture were measured at intervals. Modulation, shown by the extensive loss of protective antigen, histamine-sensitising factor, leuko-cytosis-promoting factor, heat-labile toxin and X-mode specific envelope proteins, occurred during the first 10 h of incubation of X-mode cells in C-medium at 35 degrees C and before the onset of the logarithmic phase of growth. The rapidity of these losses was greater than could be explained by a simple growth-dilution effect and did not appear to be due to release of activity into the culture fluid. It seemed, therefore, that there was selective destruction of pathophysiological activities as well as cessation of synthesis. The activities appeared to be lost at different rates. Mouse-protective activity was lost more rapidly than histamine-sensitising and leukocytosis-promoting activities; heat-labile toxicity was lost more slowly or less completely. Modulation was shown to be easily reversed.

Adjuvants, Immunologic↗
PubMed1979

Degradation of bacterial lipopolysaccharide by gut juice os the snail Helix pomatia.

Lipopolysaccharides from several bacteria were selectively degarded by gut juice of the snail Helix pomatia with extensive loss of anticomplementary activity and changes in the electrophoretic pattern in polyacrylamide gels. The gut juice had little effect on ketodeoxyoctonate content or immunodominant sugars. The lipid A moiety of the lipopolysaccharide appeared to be the main site of attack.

Animals↗
PubMed1979

Loss of adjuvanticity in rats for the hyperacute form of allergic encephalomyelitis and for reaginic antibody production in mice of a phenotypic variant of Bordetella pertussis.

The adjuvanticity of a phenotypic (C-mode) variant of B. pertussis, known to be deficient in certain immunological and physiopathological properties, was compared to that of the normal (X-mode) strain. The X-mode vaccine was a potent adjuvant for induction of hyperacute experimental allergic encephalomyelitis to guinea-pig spinal cord in Lewis rats whereas C-mode vaccine was inactive. X-mode vaccine was also highly active in the induction of reaginic (both IgE and IgGl) antibodies to ovalbumin in mice while C-mode vaccine caused only a transitory increase in the IgE level. These data support the view that an adjuvant component of B. pertussis, which is probably identical with the histamine-sensitizing and leukocytosis promoting factor, is much diminished in C-mode cells while the lipopolysaccharide adjuvant remains unchanged.

Adjuvants, Immunologic↗
PubMed1977

A protein factor associated with serum resistance in Escherichia coli.

Immunogel-diffusion studies showed that 60 degree C LiCl extracts of the smooth serum-resistant mutant Escherichia coli strain 17 contained greater amounts of a protein antigen than did extracts of the parent strain LP729. An extract of strain 17 was fractionated on Sepharose 4B and the protein antigen was found as the only detectable antigen in a number of fractions; sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that these fractions contained one major polypeptide band with a molecular weight of 46 000 daltons. We suggest that this protein antigen may be partly responsible for the serum resistance of strain 17 though its presence in other serum-sensitive strains suggests that additional factors are essential for full serum resistance.

Bacterial Proteins↗
PubMed1976

Loss of protective antigen, histamine-sensitising factor and envelope polypeptides in cultural variants of Bordetella pertussis.

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.

Animals↗
PubMed1976

Radiolabelling of Bordetella pertussis envelope proteins by the 125 I-lactoperoxidase method.

Bordetella pertussis strain number 18334 was grown in media which yielded cells with either a normal complement of surface antigens (X-model), or cells which were phenotypically altered (C-model). Neither X- nor C-mode bacteria incorporated more than traces of radioactivity when exposed to Na 125 I, lactoperoxidase and a source of H2O2 under conditions which gave substantial labelling of BSA and other soluble proteins. In contrast, envelope preparations were readily labelled. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that several polypeptides had incorporated 125 I but not in amounts proportional to their abundance in the envelopes. Envelopes from C-mode cells gave a labelling pattern similar to those of X-mode except in one region. Control experiments suggested that the failure of intact cells to become labelled may be due to bacterial inhibition of the reagents.

Autoradiography↗
PubMed1975

Envelope proteins in Salmonella minnesota mutants.

Envelope proteins of one smooth (S) strain and seven rough (R) mutants of Salmonella minnesota were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All strains gave similar band patterns although some consistent differences were detected. A major polypeptide band at 54k, which coincided with the flagellar component, was more prominent in S, Ra and Rb than in the Rc, Rd and Re chemotypes. The latter strains, however, showed more prominent bands at 48, 19 and 18k. The stage of growth at which the cultures were harvested was also found to affect the band patterns, particularly in the 54 and 40k regions. A closer examination of S, Ra and Re strains suggested that the levels of the major 40 and 37k bands were slightly reduced in Re. It is concluded that the progressive loss of lipopolysaccharide components which occurs from the S chemotype through various degrees of roughness to Re is accompanied by a change in the envelope protein composition, apparently between Rb and Rc.

Bacterial Proteins↗
PubMed1975

Cell-envelope proteins of Bordetella pertussis.

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.

Allergens↗