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T K Blackmore

Publications and source records attributed to T K Blackmore.

12 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

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↗
PubMed1996

Emerging resistance in Enterococcus spp.

Enterococcus spp. are becoming increasingly important nosocomial pathogens. They are intrinsically resistant to most antibiotics, and effective therapy depends primarily on the penicillins, vancomycin and the aminoglycosides. Under antibiotic selection pressure they have developed high level resistance to these agents, and the first vancomycin-resistant enterococcal infection in Australia was described recently. The vancomycin-resistance genes are of particular concern because of their potential to transfer to other gram-positive organisms. The prevention and control of resistant enterococci is a major challenge that is best met by a combination of active infection control measures and restriction of broad-spectrum antibiotic use.

Anti-Bacterial Agents↗
PubMed1995

Identification of complement regulatory domains in human factor H.

Factor H, a regulator of complement activation, contains 20 short consensus repeat (SCR) domains common among the family of C3b/C4b-binding proteins. Chinese hamster ovary cells transfected with cDNA corresponding to the N-terminal tryptic fragment of factor H (containing SCR 1-5 and part of SCR 6) secreted protein with cofactor activity for factor I-dependent cleavage of C3b. A series of deletion mutants, each lacking one of the first five SCR, were constructed, and the supernatants of transfected Chinese hamster ovary cells were tested for cofactor activity. Supernatants of Chinese hamster ovary cells transfected with SCR 1, SCR 4, and SCR 5 deletion mutants retained cofactor function, although the SCR 1 deletion had reduced cofactor activity. Deletion of SCR 2 or 3 totally abolished cofactor activity. Expression and functional analysis of SCR units 1-3, 2-3, and 2-4 demonstrated that the SCR 1-3 unit is sufficient for cofactor activity, but SCR 1-4 is required for full activity. For assays involving cell protection, a construct linking SCR 1-5 to the glycosyl-phosphatidylinositol anchor of decay-accelerating factor was prepared, and stable transfectants were obtained. These cells were protected against complement-mediated cytotoxicity, similarly to decay-accelerating factor- and membrane cofactor protein-transfected cells. These studies define the complement regulatory domains in factor H and suggest that the general complement functional unit for C3 convertase regulation involves three or four consecutive SCR units.

Animals↗
PubMed1995

Comparison of nasopharyngeal aspirates and throat swab specimens in a polymerase chain reaction-based test for Mycoplasma pneumoniae.

Nasopharyngeal aspirates and throat swab specimens were compared in a polymerase chain reaction (PCR)-based test for Mycoplasma pneumoniae. The pathogen was detected in 50% and 45% of throat swab specimens and aspirates, respectively. However, in specimens negative for Mycoplasma pneumoniae by PCR, amplification inhibitors were detected in 0% and 36% of throat specimens and aspirates, respectively. Further investigations confirmed that no throat specimens, but one-quarter of aspirates, are likely to be rejected for containing inadequate respiratory material or excess amplification inhibitors. Because rejection of most of the unsuitable specimens is possible only after PCR, the use of aspirates is less cost-effective. This, and the reluctance to subject patients to aspiration, make the aspirate an inferior specimen for detection of Mycoplasma pneumoniae by PCR.

Adult↗
PubMed1995

Clinical utility of the polymerase chain reaction to diagnose Mycoplasma pneumoniae infection.

The diagnosis of Mycoplasma pneumoniae infection currently relies on serological methods which may be slow to produce diagnostic results and may be inconvenient for both the clinician and the patient. This study was designed to assess whether or not the polymerase chain reaction (PCR) is a useful additional diagnostic method. Comparison was therefore made with serology as it is routinely practiced. PCR was used to examine for the presence of M. pneumoniae DNA in throat swab specimens obtained from 99 hospitalized patients investigated for a range of respiratory pathogens including M. pneumoniae. PCR detected M. pneumoniae DNA in 24 adults and 25 children, which is significantly more than the 32 patients found to be antibody positive by the particle agglutination test (p = 0.001). M. pneumoniae DNA was not detected in any of the throat swabs from 32 apparently healthy volunteers. PCR inhibitors were not detected in any of the samples tested. Significantly more children (88%) than adults (38%) were found to be anti-mycoplasma antibody-positive (p < 0.0001). Routine clinical practice was reflected in the fact that 56 patients (57%) had indeterminate serological results because only single sera were obtained. The sensitivity and specificity of PCR were assessed to be 92% and 98% respectively, using a combination of serological and clinical data as the benchmark. PCR appears to have advantages over serological testing, both with respect to accuracy and convenience of single specimen testing. The poor performance of serological tests in adults makes PCR especially useful in this age group.

Adult↗
PubMed1994

Cystic pulmonary lesions in Pneumocystis carinii infection.

Thick-walled pulmonary cavities are an uncommon manifestation of Pneumocystis carinii pneumonia (PCP). A case of PCP in a man with Acquired Immunodeficiency Syndrome (AIDS) presenting with a thick-walled pulmonary cavity and multiple smaller pulmonary air cysts is described. All of these lesions showed dramatic healing with therapy. A brief review of the recent literature is also presented.

AIDS-Related Opportunistic Infections↗
PubMed1992

Prevalence of antibodies to hepatitis C virus in patients receiving renal replacement therapy, and in the staff caring for them.

Two hundred and forty-three patients receiving renal replacement therapy (RRT) and 20 renal unit staff were tested for antibodies to hepatitis C (HCV). Three patients (1.2%) were positive by the first generation test kit, the lowest rate in patients receiving RRT reported in the literature to date. These three, and eight other patients tested positive by the second generation kit, a prevalence rate of 4.5%. Anti-HCV antibody positivity was associated with higher mean serum alanine aminotransferase (p = 0.0003) and aspartate aminotransferase (p = 0.018) levels. However, only one of the 11 anti-HCV positive patients had liver transaminase levels more than twice the upper limit of the laboratory reference range. Anti-HCV positivity was associated with a higher mean number of units of blood transfused (p = 0.035). None of 20 staff were anti-HCV positive. Twenty-five of 212 (11.7%) patients reported a history of liver disease; none of these were anti-HCV positive. Hepatitis B surface antigen was detected in eight of 215 (3.7%) patients, of which three were e antigen positive. There was evidence of past hepatitis B infection in 53 of 215 (24.7%) patients, more frequently in Maoris (p = 0.001). Overall, significantly raised liver transaminases were present in three of 198 (1.5%) patients and in no staff. This unit has a remarkably low prevalence of antibodies to HCV, an observation supported by the low rate of abnormal serum liver enzymes.

Adolescent↗