Biomedical subjects

H M Ward

Publications and source records attributed to H M Ward.

10 recordsLinked to original sources

PubMed1998

Identification of the second heparin-binding domain in human complement factor H.

Complement factor H (fH) regulates activation of the alternative pathway of C, reducing the amount of C3b deposited on sialic acid-rich surfaces. Heparin binding has been used as a model for examining the sialic acid-binding characteristics of fH. We have previously shown that of the 20 short consensus repeat (SCR) modules of fH, SCR 7 contains an important heparin binding site, but other SCRs also play a role in heparin binding. To localize the other sites, we prepared recombinant truncated and SCR deletion mutants of fH and tested them by heparin-agarose affinity chromatography. The 5 C-terminal SCRs were found to contain a heparin binding site as an SCR 7 deletion mutant of the N terminal 15 SCRs did not bind heparin, but a construct consisting of SCRs 16-20 was shown to bind heparin. Double deletion of SCRs 7 and 20 from fH abrogated binding to heparin, indicating that SCR 20 contains a heparin binding site. This finding was confirmed with the observation that attachment of SCR 20 to a group of nonbinding SCRs produced a heparin-binding protein. A protein consisting of SCRs 19 and 20 did not bind heparin, whereas SCRs 18-20 did, indicating that, although SCR 20 contains a heparin binding site, at least two nonspecific adjacent SCRs are required. fH-related protein-3 (FHR-3) possesses an SCR homologous to SCR 7 of fH and bound heparin, whereas FHR-4, which lacks such an SCR, did not. Thus, fH contains two separate heparin binding sites, which are located in SCRs 7 and 20.

Apolipoproteins↗

PubMed1998

Lipopolysaccharide O-antigen expression and the effect of its absence on virulence in rfb mutants of Vibrio cholerae O1.

Using defined rfb mutants, defective in the biosynthesis of the O-antigen of the lipopolysaccharide (LPS), and monoclonal antibodies (MAbs) to the A, B and C LPS antigens, we have examined the distribution of the antigens and the effects of their loss. By immunogold electron microscopy, it has been possible to determine the relative amounts of the A, B and C antigens on Inaba and Ogawa cells, confirming previous studies based upon bacterial agglutination and hemagglutination inhibitions. These antigens are absent from rfb::Tn mutants selected as resistant to phages which have been shown to use the O-antigen as their receptor. These mutants were severely attenuated as measured by both LD50 and their ability to compete with the wild-type parents when analyzed in the infant mouse cholera model. These mutants were unchanged in the export of cholera toxin or other secreted proteins but revealed an altered outer membrane protein profile. The competition defect suggested an effect on TCP (toxin-coregulated pilus). An analysis of the rfb::Tn mutants revealed that they were unable to assemble TCP on their surface, but the major subunit, TcpA, could be found as an intracellular pool. These mutants could be complemented back to wild-type using the cloned rfb region, implying that functional TCP assembly is dependent upon an intact LPS.

Animals↗

PubMed1998

M protein of the group A Streptococcus binds to the seventh short consensus repeat of human complement factor H.

Streptococcus pyogenes evades complement by binding the complement-regulatory protein factor H (fH) via the central conserved C-repeat region of M protein. However, the corresponding binding region within fH has not previously been precisely localized. fH is composed of 20 conserved modules called short consensus repeats (SCRs), each of which contains approximately 60 amino acids. A series of fH truncated and deletion mutants were prepared, and their interaction with M6 protein was examined. The M protein binding site was initially localized to SCRs 6 to 15 as demonstrated by ligand dot blotting, chemical cross-linking, and enzyme-linked immunosorbent assay. SCR 7 was then shown to contain the M protein binding site, as a construct consisting of the first seven SCRs bound M protein but a construct containing the first six SCRs did not bind. In addition, deletion of SCR 7 from full-length fH abolished binding to M protein. SCR 7 is known to contain a heparin binding domain, and binding of fH to M6 protein was almost totally inhibited in the presence of 400 U of heparin per ml. These results localize the M6 protein binding site of fH to SCR 7 and indicate that it is in close proximity to the heparin binding site.

Animals↗

PubMed1997

Cloning and analysis of the human complement factor H gene promoter.

The 5' flanking region of human factor H was cloned using nested polymerase chain reaction (PCR) and the promoter finder method. A total of 1.2 kb has been sequenced and a number of putative regulatory elements identified including glucocorticoid response elements cAMP responsive element, HTF-1, and acute phase signal sequences. A 717 b.p. fragment was cloned into a CAT reporter vector and transfected into HeLa cells. A series of truncations from the 5' end of this fragment were also cloned into the CAT vector. Analysis of CAT activity of the cell lysates showed that the region from -699 to +18 is likely to contain promoter elements for the factor H gene as it was able to drive transcription of the CAT gene.

Base Sequence↗

PubMed1996

Identification of a heparin binding domain in the seventh short consensus repeat of complement factor H.

Surface polyanions such as sialic acid and heparin are thought to enhance the binding of complement factor H (fH) to C3b deposited on particles and cell surfaces, thereby reducing complement activation. fH contains 20 short consensus repeat (SCR) domains, and it has been proposed that SCR 13 contains a heparin binding site. We used recombinant proteins to determine the heparin binding site on fH. Full-length fH (H20) and truncated and SCR deletion mutant proteins were cloned and expressed in Chinese hamster ovary cells. Supernatants were applied to heparin-agarose affinity columns to determine their binding and elution profiles. Deletion of SCR 13 from H20 did not prevent heparin binding nor alter its salt elution profile, indicating that SCR 13 does not contain an essential heparin binding site. We found that SCR 7 contains a heparin binding site, as SCRs 1 through 7 were the smallest truncated proteins to bind heparin (89 +/- 3%). Furthermore, deletion of SCR 7 from a protein containing SCRs 1 through 9 reduced heparin binding, whereas deletion of SCR 6 did not (17 +/- 13 vs 81 +/- 13%; p = 0.02). It is likely that other heparin binding sites exist within SCRs 10 through 20; an SCR 7 deletion mutant of H20 eluted earlier than H20, but still showed >99% binding to immobilized heparin. SCR 13 does not contain such a site because a double deletion of SCRs 7 and 13 from H20 showed >97% heparin binding and had an elution profile smilar to that of a single deletion of SCR 7.

Animals↗

PubMed1995

A gene-targeting suicide vector for Streptococcus bovis.

A gene-targeting suicide vector for Streptococcus bovis has been constructed using the Escherichia coli/Streptococcus shuttle plasmid, pMU1328, and a region derived from the broad host-range, Gram-positive transposon, Tn916. This suicide plasmid replicates autonomously in E. coli, but not in Strep. bovis or Strep. bovis Tn916. Under positive selection, the plasmid was shown to integrate into Strep. bovis Tn916 chromosomal DNA at a frequency of 3 x 10(-8) cell-1 and was stably maintained for at least 100 generations in the absence of selection. This is the first report of a recombination system in ruminal bacteria. The ability to target genes, knock out specific functions or introduce novel genes into these micro-organisms will allow ruminal species to be manipulated and may eventually lead to improved animal production.

Bacterial Proteins↗

PubMed1992

A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania.

A Trypanosoma cruzi expression vector has been constructed using sequences derived from the flanking regions of the glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. The neomycin phosphotransferase (neor) gene was incorporated as a selectable marker. Using electroporation we have introduced this vector into both T. cruzi and Leishmania cells and conferred G418 resistance. Transformation is mediated by large extrachromosomal circular elements composed of head-to-tail tandem repeats of the vector. The transformed phenotype is stable for at least 6 months in the absence of G418 and can be maintained during passage through the T. cruzi life-cycle. Foreign genes inserted into an expression site within the vector (pTEX) can be expressed at high levels in transformed cells. To our knowledge this paper describes the first trypanosome shuttle vector and the first vector which functions in both trypanosomes and Leishmania.

Animals↗

PubMed1989

Mapping of chromosomal loci associated with lipopolysaccharide synthesis and serotype specificity in Vibrio cholerae 01 by transposon mutagenesis using Tn5 and Tn2680.

Vibrio cholerae strains of the 01 serotype have been classified into three subclasses, Ogawa, Inaba and Hikojima, which are associated with the O-antigen of the lipopolysaccharide (LPS). The DNA encoding the biosynthesis of the O-antigen, the rfb locus, has been cloned and analysed (Manning et al. 1986; Ward et al. 1987). Transposon mutagenesis of the Inaba and Ogawa strains of V. cholerae, using Tn5 or Tn2680 allowed the isolation of a series of independent mutants in each of these serotypes. Some of the insertions were mapped to the rfb region by Southern hybridization using the cloned rfb DNA as a probe, confirming this location to be responsible for both O-antigen production and serotype specificity. The other insertions allowed a second region to be identified which is involved in V. cholerae LPS biosynthesis.

Bacteriophages↗

PubMed1987

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae O1.

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.

Antigens, Bacterial↗

PubMedDate not supplied

Emergency: a case history.

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Dental Care for Persons with Disabilities↗