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R Parton

Publications and source records attributed to R Parton.

At least 37 records · Page 2Linked to original sources
PubMed1996

Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro.

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation reaction has not been determined. We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli. Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells. A single-step purification was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vitro with palmitic acid, as reported previously for activation of the homologous E. coli haemolysin (HlyA). The in vitro activation system may be used to investigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target cell specificity and toxic activity of CyaA.

Adenylate Cyclase Toxin↗
PubMed1994

Construction of conjugative shuttle and suicide vectors for Pasteurella haemolytica and P. multocida.

A shuttle cloning vector, pAKA16, and suicide derivatives pAKA19 and pAKA22 have been developed for gene transfer to Pasteurella haemolytica and P. multocida. pAKA16 was constructed by insertion of the lacZ alpha-peptide-encoding region and a multiple cloning site into a plasmid which was originally isolated from P. haemolytica serotype A1. The vector encodes ampicillin resistance, and contains at least 14 unique restriction sites and the property of phenotypic identification of recombinant clones in Escherichia coli by insertional inactivation of beta-galactosidase activity. It can be transferred by conjugation to P. haemolytica or P. multocida and is stably maintained in both species. The type-II chloramphenicol acetyltransferase-encoding gene (cat), cloned into pAKA16, was stably expressed in both P. haemolytica and P. multocida. Plasmids pAKA19 and pAKA22 were constructed by replacement of the origin of DNA replication (ori) of pAKA16 with a ColE1-type ori from pBR322 or an ori of plasmid R6K (oriR6K) from pJM703.1, respectively. These derivatives replicate in E. coli, but not in either P. haemolytica or P. multocida, and are suitable for use as suicide vectors for these Pasteurella species.

Chloramphenicol O-Acetyltransferase↗
PubMed1994

Evaluation of different methods for the detection of outer membrane proteins and lipopolysaccharides of Pasteurella haemolytica by immunoblotting.

The optimal conditions for the detection of outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of Pasteurella haemolytica by immunoblotting were evaluated. The variables examined included the equilibration time of the gels before transfer, composition of the transfer buffer, type of blotting membrane, blocking agent, effect of the zwitterionic detergent Empigen BB on protein renaturation, and the development reagent. The composition of the transfer buffer and time of gel equilibration significantly affected the efficiency of transfer of both OMPs and LPS. However, the optimal conditions for the transfer of OMPs were not the same as those for LPS. Thus, optimal transfer of OMPs occurred in Tris-glycine buffer, with prior equilibration of the gels to allow for expansion, whereas optimal transfer of LPS was achieved in Tris-glycine-methanol buffer with no equilibration of the gels. In Tris-glycine-methanol buffer, gel equilibration resulted in a significantly reduced transfer of both OMPs and LPS, probably due to the removal of SDS from these components. The use of Zeta-Probe blotting membrane which, unlike nitrocellulose, does not require methanol for optimal protein binding, did not result in improved binding of OMPs or LPS in the absence of methanol and, even after prolonged blocking (> 2 h), gave higher background staining than did nitrocellulose. Effective blocking of nitrocellulose was achieved with 3% (w/v) gelatin, 2.5% (w/v) skimmed milk or 0.3% (v/v) Tween 20, whereas increased background staining occurred with 1% (w/v) bovine serum albumin or 1% (w/v) ovalbumin. The incorporation of Empigen BB in the primary antibody buffer did not improve antibody recognition of proteins as a result of their renaturation. For the horseradish-peroxidase enzyme development system, the substrate 3,3'-diaminobenzidine tetrahydrochloride was more sensitive, and developed more quickly, than 4-chloro-1-naphthol, but faded more rapidly after drying of the membrane. 4-chloro-1-naphthol was more suitable for identifying OMPs because less background staining occurred, whereas 3,3'-diaminobenzidine tetrahydrochloride was more suitable for the detection of LPS due to its greater sensitivity.

Bacterial Outer Membrane Proteins↗
PubMed1994

Development of an intraperitoneal implant chamber for the study of in vivo-grown Pasteurella haemolytica in cattle.

An intraperitoneal implant chamber was developed for the study of the in vivo growth of Pasteurella haemolytica in calves. The chamber had a volume of approximately 100 ml and featured an external sampling port which allowed multiple and sequential sampling of the chamber contents. A single polycarbonate diffusion membrane with a pore size of 0.22 micron allowed host peritoneal fluid to diffuse into the chamber and maintained the bacterial population free of white blood cells. Chambers were implanted into the peritoneal cavities of four five-month-old dairy-cross calves, demonstrated to be sero-negative by indirect haemagglutination assay. Three days later, four different P. haemolytica isolates, of serotypes A1 or A2, were inoculated into the chambers. In all cases, there was a slow decline in the viable bacterial numbers within the chambers. Western blot analysis of the antibody content of the chamber fluids revealed IgG antibodies to P. haemolytica OMPs in the fluid prior to inoculation and both 9 and 15 days after inoculation. Furthermore, there was no significant change in the IgG antibody content of the chamber fluid, either quantitatively or qualitatively, during the course of the experiment. Analysis of the bactericidal activity of pre-inoculation chamber fluid against the corresponding bacterial isolate suggested that an antibody-dependent complement-mediated process was not responsible for the decline in bacterial numbers. Overall, the chamber design was demonstrated to be extremely effective for in vivo studies of P. haemolytica in calves, allowing easy and regular sampling of the chamber contents and maintaining bacteria free of white blood cells. Although there was a slow decline in bacterial numbers over time, sufficient numbers of cells could be obtained for analysis of cell-surface antigens.

Animals↗
PubMed1994

Cough production, leucocytosis and serology of rats infected intrabronchially with Bordetella pertussis.

Adult Sprague-Dawley rats infected intrabronchially with Bordetella pertussis strain 18-323 encased in agarose beads (BP-beads), developed a paroxysmal cough and leucocytosis, both of which peaked at around day 10. When animals were exposed to ether for 2 min after delivery of the beads, there was an enhancement of the number of subsequent coughing episodes. Inclusion of carrageenan in the beads also enhanced coughing. Control rats, given sterile beads or left untreated, showed only a low level of coughing or no coughing, depending upon their source. Rats challenged by the same route with heat-killed B. pertussis in beads, or with live organisms in suspension (without beads) showed no cough induction or leucocytosis. However, intranasal delivery of B. pertussis suspension gave rise to a moderate amount of coughing and leucocytosis. Serum IgG responses to B. pertussis antigens were greatest in rats infected with BP-beads and antibodies against both pertussis toxin and filamentous haemagglutinin were detected. Since the rat is the only conveniently accessible laboratory animal species in which B. pertussis induces an intermittent paroxysmal cough, as in man, it merits further study for determining the mechanisms of pathogenesis and immunity in pertussis.

Animals↗
PubMed1994

Responses to Bordetella pertussis mutant strains and to vaccination in the coughing rat model of pertussis.

Phase I strains 18-323, Tohama and L-84 of Bordetella pertussis produced paroxysmal coughing when encased in agarose beads and administered intrabronchially to adult Sprague-Dawley rats. In contrast, the Phase IV variant of strain L-84 was inactive in cough induction, as was strain BP 357, a transposon-insertion mutant which is deficient only in pertussis toxin (PT). Strain BPM 1809, which lacks only the heat-labile toxin, was similar to the unmodified Phase I strains for cough induction, indicating that this toxin is not needed to induce coughing. B. parapertussis also was inactive as a cough inducer. These results indicate that PT, present in Phase I strains of B. pertussis, and absent from Phase IV strains, strain BP 357 and B. parapertussis, is essential for the induction of paroxysmal coughing in this rat model of whooping cough. Prior injection of DTP (whole-cell) vaccine greatly reduced the incidence of coughing in rats challenged subsequently with Phase I B. pertussis. Serological responses were monitored after intrabronchial infection with the various bacterial strains and after vaccination and challenge. The PT-positive or -negative status of the strains in vivo was confirmed by the appropriate presence or absence of anti-PT IgG in the convalescent sera.

Animals↗
PubMed1994

Comparison of outer-membrane proteins of Pasteurella haemolytica expressed in vitro and in vivo in cattle.

Outer-membrane protein (OMP) profiles of two serotype A1 isolates of Pasteurella haemolytica were compared by SDS-PAGE and Western blotting with bovine convalescent serum after growth (a) in vitro under iron-sufficient and -deficient conditions, (b) in vivo in the lungs of experimentally infected calves and (c) in vivo in diffusion chambers implanted into the peritoneal cavities of calves. Lung-grown bacteria differed from iron-sufficient in vitro-grown bacteria in having enhanced expression of the previously recognized 71, 77 and 100 kDa iron-regulated proteins, reduced expression of 18, 31, 39.5 and 50 kDa proteins, and expression of a 19 kDa protein. Differences were also apparent in the Western blot profiles of OMPs of in vitro- and lung-grown bacteria. These included the apparent lack of recognition of the 100 kDa protein in the lung-grown bacteria, but not in the in vitro-grown bacteria, and more intense staining of a 47 kDa protein in in vitro-grown bacteria, but not in lung-grown bacteria. The OMP profiles of the chamber-grown bacteria resembled those of the lung-grown bacteria in that expression of the 18, 19, 31 and 39.5 kDa proteins was similar. These similarities demonstrated that the chamber-grown bacteria had adapted to the in vivo environment, and that growth conditions within the chambers resembled, but not perfectly, those within the lungs. For example, expression of the three iron-regulated OMPs was very low in the chamber-grown bacteria compared to the lung-grown bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A new assay for the invasive adenylate cyclase toxin of Bordetella pertussis based on its morphological effects on the fibronectin-stimulated spreading of BHK21 cells.

When baby hamster kidney (BHK) cells are allowed to spread on fibronectin-coated substrata in the absence of serum and the presence of agents which elevate intracellular 3':5'-cyclic AMP (cAMP) levels they adopt an abnormal, stellated morphology. To determine whether the invasive adenylate cyclase (AC) toxin of Bordetella pertussis induced the same response, cell extracts were prepared from several B. pertussis strains. They were characterized for AC toxin production by enzymic assay and by immunoblotting with an AC-toxin-specific monoclonal antibody. Extracts of strains producing AC toxin induced elevated levels of intracellular cAMP in BHK cells and promoted a stellation response during cell spreading. Extracts prepared from strains defective in AC toxin production showed no effect. Using image analysis to quantify the morphological change, we have demonstrated that the effect of AC toxin on cell spreading is dose dependent. This technique is a rapid and sensitive assay for the invasive AC toxin.

Adenylate Cyclase Toxin↗
PubMed1994

Adjuvant effect of pertussis toxin on the production of anti-ovalbumin IgE in mice and lack of direct correlation between PCA and ELISA.

In studies designed to optimize the production and detection of anti-ovalbumin (anti-Oa) IgE in Ham/ICR mice, a range of doses of both Oa and pertussis toxin as the IgE adjuvant was explored. As determined by 48-hour passive cutaneous anaphylaxis (PCA) tests, the highest titre of anti-Oa IgE was obtained in a three-injection protocol with 0.1 micrograms Oa and 1 microgram pertussis toxin for the priming dose, followed by two further doses of 0.1 microgram Oa alone. In single-dose immunizations, the highest PCA responses were obtained in sera from mice given 20 micrograms Oa and 1 microgram pertussis toxin. These data confirm that murine IgE production to Oa depends on particular combinations of immunization variables. There was no direct correlation between the PCA and anti-IgE enzyme-linked immunosorbent assay (ELISA) titres of the PCA-positive sera; indeed there was a significant negative correlation. This relationship was not due to interference by IgG1, as there was no correlation between the anti-Oa IgG1 and anti-Oa IgE ELISA titres of the sera. These results highlight the need for caution in assuming that serum IgE levels as measured by ELISA will necessarily correlate positively with IgE biological activity as measured by allergen challenge in vivo.

Adjuvants, Immunologic↗
PubMed1994

Haemolytic assay for the detection of adenylate cyclase toxin of Bordetella pertussis.

The aim of this study was to device a simple, sensitive and convenient assay for the detection of toxicity of the Adenylate cyclase toxin (ACT), a newly recognized toxin of Bordetella pertussis. Haemolytic activity of ACT was analysed as an assay and sheep RBCs were found to be suitable to detect the toxic effect down to the dose of 6.25 nmol cAMP/min/ml enzymic activity applied. The haemolytic activity was found to be dose-dependent.

Journal Article↗
PubMed1993

Chromosomal DNA from both flagellate and non-flagellate Bordetella species contains sequences homologous to the Salmonella H1 flagellin gene.

The genus Bordetella contains four species: two are non-motile, the human pathogens B. pertussis and B. parapertussis; and two are motile, the broad host-range mammalian pathogen B. bronchiseptica, and the avian pathogen B. avium. The motility of the latter two species is due to peritrichous flagella. Here we show that strains of all four species contain DNA sequences homologous to flagellin genes. Two types of gene probe were hybridised to Bordetella chromosomal DNa in Southern blots: the structural gene for H1 flagellin of Salmonella typhimurium and an oligonucleotide derived from the conserved N-terminal amino acid sequences of various flagellin proteins. ClaI-digested DNa from all four Bordetella species hybridised with both probes in Southern blots, although each species gave a characteristic pattern of hybridisation. This indicates that the non-motile B. pertussis and B. parapertussis species contain non-expressed flagellin genes.

Base Sequence↗
PubMed1993

Internalization and recycling of activated thrombin receptors.

Shortly after activation by either thrombin or the tethered ligand domain peptide SFLLRN, thrombin receptors undergo homologous desensitization, temporarily losing their ability to respond to both agonists. We have examined the role of receptor internalization and recycling in this process using receptor-directed antibodies as probes. The results show within 1 min of activation > 85% of the approximately 200,000 thrombin receptors on megakaryoblastic human erythroleukemia (HEL) and CHRF-288 cells are sequestered into endosomes via coated pits, after which the majority are transferred to lysosomes. This process does not require proteolysis of the receptor and occurs with sufficient speed to play a major role in the regulation of thrombin receptor function. Although most of the internalized receptors are ultimately degraded, approximately 25% return to the cell surface. These recycled receptors are in a state in which they can respond to SFLLRN but not thrombin; nor do they self-activate despite the apparent continued presence of the tethered ligand. In contrast to other G protein-coupled receptors, which are internalized and then recycled in an activatable state, recovery of the thrombin response occurs only after the expression on the cell surface of adequate numbers of newly synthesized receptors.

Amino Acid Sequence↗
PubMed1993

Identification of Bordetella pertussis in nasopharyngeal swabs by PCR amplification of a region of the adenylate cyclase gene.

The polymerase chain reaction (PCR) was used to amplify a 522-bp region of the adenylate cyclase toxin (cyaA) gene of Bordetella pertussis. As few as 100 cfu from a suspension of B. pertussis could be detected by this procedure when the amplified PCR product was detected by ethidium bromide staining of agarose gels. However, simulated clinical specimens, prepared from swabs impregnated with known numbers of B. pertussis cells, only yielded a positive reaction with > or = 10(4) cfu. Hybridisation of a Southern blot of the PCR products from the swab samples with a cya-specific probe gave a positive reaction with as few as 8 cfu, but the hybridisation signal was uniformly weak with fewer than 10(4) cfu. Nevertheless, three of 13 nasopharyngeal swabs, taken from suspected clinically defined cases of whooping cough and stored frozen for up to 18 months, gave a positive PCR reaction.

Adenylate Cyclase Toxin↗
PubMed1992

Interactions of rab5 with cytosolic proteins.

Rab proteins, one of the subfamilies of ras-like small GTP-binding proteins, are attached to cellular compartments or transport vesicles and may determine the specificity of fusion between these compartments and vesicles. It has been proposed that they alternate between a membrane-bound and a cytosolic state during their functional cycle. We have used a photo-crosslinking approach to identify their cytosolic interaction partners. In vitro synthesized rab5 was cross-linked in the presence of ATP mainly to three cytosolic proteins of 52, 65, and 85 kDa. Sucrose density gradient centrifugation of the cross-linked products suggested that they were part of a 10-14 S complex. Furthermore, rab5 was cross-linked to these and additional cytosolic proteins of 42, 48, and 160 kDa in the absence of ATP. Unexpectedly, upon ATP depletion of the cytosol cross-linked and noncross-linked rab5 was found in a sedimentable high molecular weight structure. Other members of the rab subfamily, but not N-ras, also sedimented under these conditions. Electrophoretic and electron microscopic analysis of the pelleted material revealed that it contained actin filament bundles and intermediate filaments. Our data suggest that cytosolic rab proteins interact with several proteins in a 10-14 S complex, and that the rab proteins may interact directly or indirectly via this complex with the cytoskeleton.

Actins↗
PubMed1992

Lipopolysaccharide heterogeneity in Pasteurella haemolytica isolates from cattle and sheep.

Lipopolysaccharide (LPS) from 40 isolates of Pasteurella haemolytica, comprising 23 serotype A1, seven serotype A2, one serotype T4, one serotype T10 and eight untypable isolates, obtained from diseased and healthy cattle or sheep, was characterized by SDS-PAGE and Western blotting. Ten different SDS-PAGE LPS profiles, five smooth and five rough, were identified among the biotype A and untypable isolates and designated LPS types 1-10. LPS types 1 and 2 were smooth, had similar O-antigen banding-patterns but differed in the low-molecular-mass or core-oligosaccharide regions; type 3 LPS was rough but had a core-oligosaccharide region similar to that of LPS type 1. No similarities were observed between these LPS types and types 6, 7 and 9, which were smooth, and types 4, 5, 8 and 10, which were rough. Most serotype A1 isolates (19/23) were of LPS type 1, whereas two isolates each had LPS of types 2 and 3. The majority (5/7) of serotype A2 isolates possessed type 3 LPS, whereas the remaining two isolates each had LPS of types 4 and 5. There was much greater heterogeneity within the untypable group of isolates, which comprised LPS of types 1 and 9 (two isolates each), and 6, 7, 8 or 10 (one isolate each). Western blotting analysis demonstrated that LPS types 1 and 2 had immunologically identical O-antigen side-chains but differed in their core-oligosaccharide regions, whereas the core-oligosaccharide region of rough LPS type 3 was immunologically very similar to that of LPS type 1. The other LPS types were immunologically unrelated to these three LPS types. The majority (20/23) of serotype A1 isolates originated from cattle and possessed LPS types 1 or 2, different from most (5/7) of the serotype A2 isolates which originated from sheep and possessed LPS of types 3 or 4. However, two of the three ovine serotype A1 isolates had the same type 3 LPS as occurred in most of the ovine serotype A2 isolates, suggesting a possible correlation between LPS type and host specificity. This study has demonstrated that LPS diversity within different serotypes of P. haemolytica is greater than was previously thought and that certain LPS types might be host-specific.

Animals↗
PubMed1992

Outer-membrane protein and lipopolysaccharide variation in Pasteurella haemolytica serotype A1 under different growth conditions.

Growth characteristics, as well as outer-membrane protein (OMP) and lipopolysaccharide (LPS) profiles in SDS-polyacrylamide gels, of two serotype A1 isolates of Pasteurella haemolytica were examined under different in vitro growth conditions. The two isolates were chosen as representatives of disease (S/C 82/1) and non-disease (W/D 83/4) isolates, respectively. The growth rates and final cell densities of both isolates increased as the degree of aeration increased. In particular, the final cell densities varied significantly according to the degree of aeration. Under anaerobic conditions, however, both the growth rate and final cell density were significantly reduced. There was reduced expression of a 40.5 kDa protein under anaerobic conditions in both isolates, whereas in S/C 82/1 expression of the 71, 77 and 100 kDa iron-regulated proteins increased as aeration decreased. There were also differences in low-molecular-mass components of LPS between cells grown anaerobically and those grown aerobically. Growth in the presence of 5% CO2 did not significantly alter the growth rate and had little, if any, affect on OMPs or LPS. Differences in the expression of certain proteins occurred as growth progressed from the exponential to the stationary phase. Growth in the presence of the iron chelators 2,2'-dipyridyl, ethylenediamine-dihydroxyphenylacetic acid (EDDA), desferrioxamine mesylate (desferal), ovotransferrin (conalbumin) and bovine transferrin was inhibited within a very narrow concentration range. In the presence of 2,2'-dipyridyl, EDDA or desferal, 71 and 100 kDa iron-regulated OMPs increased in both isolates whereas a 77 kDa protein increased in isolate S/C 82/1 only. In the presence of ovotransferrin or bovine transferrin there was, in both isolates, increased expression of the 71 kDa protein, a slight increase in expression of the 100 kDa protein but no expression of the 77 kDa protein; there was also increased production of the 40.5 kDa protein, and synthesis of two additional proteins of 23 and 26 kDa. Other differences occurred after growth in foetal and newborn calf sera. In foetal calf serum there was enhanced expression of the 71 but not of the 100 kDa protein. In newborn calf serum there was no enhanced expression of the 71, 77 or 100 kDa proteins, but expression of novel proteins of 97 and 98 kDa as well as a high-molecular-mass protein occurred. There was also slight quantitative differences in the LPS profiles of cells grown in foetal or newborn calf sera compared to those of cells grown in other media.(ABSTRACT TRUNCATED AT 400 WORDS)

Aerobiosis↗