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Biomedical subjects

J D Wetherall

Publications and source records attributed to J D Wetherall.

At least 19 recordsLinked to original sources
PubMed1992

Typing of methicillin-resistant Staphylococcus aureus with an M13 repeat probe.

A bacteriophage M13 tandem repeat has been used to probe EcoRI digested genomic DNA of methicillin-resistant Staphylococcus aureus (MRSA). The patterns generated were found to be useful in typing MRSA and generally confirmed the relationships that had previously been recognized in other studies based on antimicrobial resistance and plasmid profiles. The epidemic MRSA of London hospitals (EMRSA) and the majority of the epidemic MRSA of eastern Australian hospitals (EA MRSA) gave the same pattern. However, two isolates previously classified as EA MRSA gave a different pattern and a third another pattern. One isolate from Dublin, two isolates from Nuneaton and two isolates from Singapore gave the same pattern as the two EA MRSA. With the exception of the early or classic MRSA all the other isolates examined gave their own distinctive patterns. With one exception the classic MRSA belonged to a separate group. The exception was of particular interest because it gave the same pattern as the majority of the EA MRSA. This suggests that there may be an evolutionary relationship between some of the classic MRSA and the EMRSA of London and the EA MRSA of Australia.

Bacterial Typing Techniques↗
PubMed1991

Particle-enhanced turbidimetric immunoassay of sex-hormone-binding globulin in serum.

A particle-enhanced turbidimetric immunoassay (PETIA) for human sex-hormone-binding globulin (SHBG) is described. The method involves use of antibody covalently coupled to latex particles and is almost fully automated, with sample processing being complete in less than 20 min. The working reagents are stable for at least three months, and full calibration of the assay each day is not essential. A particular advantage is that pretreatment of samples is rarely required because the working range of the assay is from 2.0 to 320 nmol/L for nondiluted serum. Intra- and interassay CVs were less than 4.5% and 8.5%, respectively, and mean analytical recovery was 101.5%. SHBG concentrations of 129 serum samples determined by this method and by a commercially available immunoradiometric assay correlated highly.

Female↗
PubMed1988

Purification and characterisation of ovine C4: evidence for two molecular forms in ovine plasma.

A relatively rapid procedure is described for the isolation of the fourth component of complement (C4) from ovine plasma. The method, which recovers approximately 30% C4, is based upon DEAE Sephacel anion exchange chromatography of PEG precipitated plasminogen depleted plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration. SDS-PAGE of purified ovine C4 under reducing conditions revealed a complex pattern of bands which was interpreted on the basis of a three polypeptide chain structure for each of two distinct species, or isotypes, of C4 molecule herein termed C4A and C4B. Each isotype differs in the mol. wt of the alpha chain--108 and 95 K respectively. Nucleophilic substitution of immunoprecipitated ovine C4 with radiolabelled methylamine revealed that both C4 species contained a reactive thiol ester site and that each could be cleaved into an activated form (presumably C4b) characterised by a truncated alpha' chain some 8 K lower in mol. wt. A comparison of the isotype composition of purified C4 with that of immunoprecipitated C4 from the same animal indicated that the purification procedure favoured isolation of the C4B isotype. The mol. wts of both the alpha and beta chains were lowered following digestion of ovine C4 with neuraminidase.

Animals↗
PubMed1987

Class I major histocompatibility complex antigens and C4 concentrations in sheep plasma.

Data are presented demonstrating that high concentrations of complement protein C4 in sheep plasma are associated with a particular class I OLA specificity. By way of contrast, a similar association could not be demonstrated between C3 plasma concentrations and OLA specificities. These data support the hypothesis that gene(s) determining C4 plasma concentrations are linked to the ovine MHC.

Animals↗
PubMed1987

Purification and characterisation of the fourth component of bovine complement.

A relatively rapid method for the isolation of complement protein C4 from bovine plasma is described. The method consists of DEAE Sephacel anion exchange chromatography of plasminogen-depleted bovine plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration on a TSK G3000 SW column. A yield of approximately 20% was obtained. Conventional SDS-PAGE of purified bovine C4 showed the presence of alpha, beta and gamma polypeptide chains, the molecular weights of which were determined from Ferguson plots to be 95,000 +/- 2,500, 80,500 +/- 2,000 and 30,000 +/- 500 daltons, respectively. SDS-PAGE of C4 immunoprecipitated from the plasma of individual cattle in gels with a reduced proportion of crosslinker showed size polymorphism of the alpha chain. The presence of dual alpha chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine alpha chains is approximately 1,800 daltons. On activation of bovine C4 both alpha chains were cleaved into alpha' chains (87,000 and 85,000 daltons) characteristic of C4b.

Animals↗
PubMed1987

Analysis of C3 and C4 in ovine plasma.

The distributions of plasma concentrations of complement proteins C3 and C4 were studied in sample populations of merino and Suffolk sheep. No differences between the breeds or the sexes were observed. The distribution for ovine C4 was polymodal and very disperse relative to that for C3. It was found, however, that C3 concentrations were elevated in specimens from 20 merino sheep bred as high responders to a Trichostrongylus vaccine. Significantly decreased plasma C4 concentrations were observed in representatives of both merino and merino X Border Leicester cross-bred sheep affected with congenital progressive ovine muscular dystrophy. Agarose gel electrophoretic variants of ovine C3 were not detected. Evidence for electrophoretic variants of ovine C4 in agarose gels was found although individual allotypes could not be reliably identified. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) did not reveal size heterogeneity for the alpha and beta chains of immunoprecipitated ovine C3. Analysis of reduced immunoprecipitated ovine C4 by SDS-PAGE revealed considerable size heterogeneity in the alpha chain consistent with isotypic and/or allotypic variability. The data presented strongly suggest the presence of two C4 loci in sheep, each of which exhibits polymorphism.

Animals↗
PubMed1987

Sex hormone-binding globulin secretion by human hepatocarcinoma cells is increased by both estrogens and androgens.

The secretion of sex hormone-binding globulin (SHBG) by the human hepatocarcinoma cell line Hep G2 was increased significantly not only by estradiol (E2) but also by testosterone (T), dihydrotestosterone, and the synthetic androgen danazol in the presence, but not the absence, of fetal calf serum. The secretion of SHBG also was stimulated by the antiestrogen tamoxifen, although this required the use of longer incubation periods and higher concentrations than were required for the steroids. The antiandrogen cyproterone acetate and cortisol (5 microM) decreased SHBG secretion. Pregnanediol (5 microM) had no discernible effect. These changes were considered specific as none of the compounds altered either the secretion of total protein by the cells or their total protein content. Cells incubated with a mixture of E2 (0.5 microM) and T (0.5 microM) secreted significantly more SHBG than did cells incubated with E2 (1.0 microM), indicating these steroids exert their effects through different mechanisms. The increases with E2 and T were reduced significantly by tamoxifen and cyproterone acetate, respectively, suggesting receptor mediation of the steroid effects. The E2-related increase was abolished by cycloheximide, indicating that the changes were due to alterations in the synthesis of SHBG rather than to alterations in the release of previously synthesized protein. These findings suggest the T-related decrease in plasma SHBG levels may be due to causes other than a decrease in the hepatic synthesis of the protein. Additionally, they indicate that Hep G2 cells may prove suitable for examining the regulation of the SHBG gene by a variety of compounds.

Carcinoma, Hepatocellular↗
PubMed1987

Breed differences in the frequency of bovine lymphocyte antigens.

Lymphocytes from 1,564 cattle of 18 breeds and cross-bred groups in Australia were tested for major histocompatibility system class 1 antigens. Gene frequencies were calculated for the Angus, Belmont Red, Brahman, Hereford and Holstein-Friesian breeds. There were substantial differences among these breeds in antigen and gene frequency. There were striking differences among all 18 breeds in the presence or absence of certain antigens. Two antigens, CA13 and CA36, were strongly associated in Hereford cattle but occurred independently of each other in the other breeds.

Animals↗
PubMed1985

Distribution of complement protein C4 concentrations in bovine plasma.

Complement component C4 concentrations were measured in 40 pure bred Hereford cattle and 40 cattle from a mixed breed herd. Significant differences were not observed between the two groups studied nor between bulls and cows. However, the distribution of C4 concentrations was relatively disperse and appeared polymodal suggesting the presence of two isotypes of C4. Polyacrylamide gel electrophoresis of immunoprecipitated bovine C4 showed many samples to have two C4 alpha chains differing in relative molecular mass by about 1800. Isoelectric focusing of bovine plasma in agarose gels followed by immunofixation with specific anti-C4 antisera revealed two populations of native C4 differing in pI by about 0.3 pH unit. An association between the type of C4 alpha chain present and the pI of the native C4 molecule was observed. Collectively these findings indicate the presence of two structural C4 genetic loci in cattle.

Animals↗
PubMed1985

Plasma sex hormone binding globulin concentration and binding capacity in children before and during puberty.

The steroid binding capacity and concentration of plasma sex hormone binding globulin have been compared in 116 children aged between 2 and 14 years. Concentration was measured by electroimmunodiffusion standardised with reference to the mass of the pure protein and binding capacity by quantitating the binding of radiolabelled 5 alpha-dihydrotestosterone. Binding capacity correlated highly with concentration in all subjects and neither differed significantly between the sexes before or during puberty. However, both were significantly lower in pubertal than in pre-pubertal children. These findings suggest the metabolism of the protein is similar in boys and girls and that the fall in its steroid binding capacity at puberty in fact is due to a fall in its concentration rather than to changes in its physicochemical properties.

Adolescent↗
PubMed1983

A chromatographic method for the purification of K99 pili from enterotoxigenic Escherichia coli.

Details of a relatively inexpensive method for the purification of K99 pili in their native conformation are reported. The method involved sequential precipitation of K99 pili with (NH4)2SO4, followed by precipitation in 12% (w/v) mannose or sorbitol solution. The crude pili preparation was adsorbed with Bio-Gel A-5m and subjected to sequential gel filtration on Bio-Gel A-5m equilibrated with Tris/EDTA/NaN3/NaCl buffer and KSCN/KCl solution, respectively. The K99 containing peak was subjected to sequential ion-exchange chromatography on DEAE-Bio-Gel A equilibrated with 0.02 M-Tris/HCl, pH 8.6 containing 0.05 M-NaCl and DEAE-Sephadex A-50 equilibrated with 0.05 M-phosphate buffer, pH 7.2. The purified pili yielded a single band on SDS-PAGE with an estimated molecular weight of 13000. Attempts to purify pili by other methods evaluated, viz. MgCl2 precipitation and chromatofocusing were unsuccessful. While the amino acid composition of purified K99 pili was similar to that reported previously the N-terminal amino acid was apparently blocked.

Amino Acids↗
PubMed1982

A rapid procedure for the isolation of human sex steroid binding protein.

A three stage procedure is described which provides a rapid, simple, and reliable means to isolate sex steroid binding protein from human serum. Purification is achieved by the sequential use of affinity chromatography, isoelectric focusing in granulated gels and ion exchange chromatography. Between 3--4 mg of protein can be recovered from one litre of pregnancy serum without four days. A molecular radius of 2.98 nm and an apparent molecular mass of 91 000 were obtained for the purified native protein by polyacrylamide gel electrophoresis. A value of 50 000 was obtained for the molecular mass of the reduced protein by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The purified protein was used to prepare a monospecific rabbit antiserum and a murine monoclonal antibody. Partial characterisation of the latter has been achieved.

Animals↗
PubMed1979

HLA antigens and the response to influenza A virus.

An attempt was made to confirm a previously reported association between HLA type B16 and resistance to infection with live attenuated influenza A virus vaccine. No differences were found either in the ability of influenza virus to attach to fractionated white blood cells from HLB B16 subjects compared to cells from subjects of other HLA types, nor in the incidence of antibodies to three consecutive epidemic strains of influenza in sera from B16 subjects and control subjects. The association between HLA B16 and resistance to influenza could not be confirmed in a retrospective analysis of a clinical trial of a live attenuated influenza vaccine.

Adsorption↗