Dinucleotide repeat polymorphism adjacent to sheep complement factor B.
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Biomedical subjects
Publications and source records attributed to D M Groth.
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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.
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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.
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.
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.
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.
Antibodies to the membrane antigens of human T-cell lymphotropic virus-I (anti-HTLV-MA) have been detected in patients with the acquired immune deficiency syndrome (AIDS) and in patients with hemophilia. The authors examined sera from 71 AIDS patients and 46 hemophiliac children for the presence of anti-HTLV-MA using an indirect membrane immunofluorescence assay with flow cytometry analysis. Thirty-seven of the 71 (52%) AIDS patients and 7 of the 46 (15%) hemophiliac patients had high titered anti-HTLV-MA, using a T-lymphoid cell line infected with the leukemia virus. None of the 78 control subjects had high titered antibody. All seven hemophiliac patients with elevated anti-HTLV-MA used Factor VIII concentrates, and all had inverted T-lymphocyte helper-suppressor (T4 [Leu-3]/T8 [Leu 2]) ratios. No correlations were found between inverted T4/T8 ratios and antibody to cytomegalovirus, Toxoplasma gondii, or hepatitis B. This work supports contentions that HTLV-like organisms cause AIDS and that these organisms are transmitted by blood products such as Factor VIII concentrate.
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.