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Biomedical subjects

C G Clark

Publications and source records attributed to C G Clark.

At least 37 records · Page 2Linked to original sources
PubMed1997

Extensive genetic diversity in Blastocystis hominis.

Blastocystis homonis is a common human parasite of uncertain role in human disease. Approximately equal numbers of reports implicate it and exonerate it as a pathogen. Genetic diversity in B. hominis was investigated using riboprinting to study sequence variation in the small subunit ribosomal RNA genes of 30 randomly selected isolates. Extensive sequence variation was discovered in B. hominis ribosomal RNA genes and this species consists of at least seven morphologically identical but genetically quite distinct organisms. If only a subset of the B. hominis variants have the potential to cause disease in humans this might explain the disparate findings reported. Future clinical studies must take the heterogeneity of B. hominis into account.

Animals↗
PubMed1997

Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar.

The electrophoretic patterns of hexokinase and phosphoglucomutase have been widely used to distinguish Entamoeba histolytica from Entamoeba dispar isolates. Although E. histolytica and E. dispar, previously called pathogenic and nonpathogenic Entamoeba histolytica, differ clearly in sequences of many homologous genes, a conversion between the two has been reported by several laboratories, in each case showing the conversion of hexokinase (ATP, D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzyme patterns. An apparent mobility shift of this enzyme may either be due to posttranslational modification or processing, or to the appearance of a new isoform encoded by a second gene. In this study we observed that the four observed bands in the isoenzyme patterns of pathogenic and nonpathogenic forms of Entamoeba were correlated with four different cDNAs, and that the four recombinant hexokinases produced in Escherichia coli comigrated with their natural counterparts. Polymerase chain reaction (PCR) experiments did not reveal hidden genes which might be responsible for conversion phenomena. These results strongly support the redefinition of pathogenic and nonpathogenic Entamoeba histolytica as two closely related species Entamoeba histolytica and Entamoeba dispar.

Amino Acid Sequence↗
PubMed1996

A possible mitochondrial gene in the early-branching amitochondriate protist Trichomonas vaginalis.

Trichomonads are anaerobic flagellated protists that, based on analyses of ribosomal RNA sequences, represent one of the earliest branching lineages among the eukaryotes. The absence of mitochondria in these organisms coupled with their deep phylogenetic position has prompted several authors to suggest that trichomonads, along with other deeply-branching amitochondriate protist groups, diverged from the main eukaryotic lineage prior to the endosymbiotic origin of mitochondria. In this report we describe the presence of a gene in Trichomonas vaginalis specifically related to mitochondrial chaperonin 60 (cpn60). A recent study indicates that a protein immunologically related to cpn60 is located in trichomonad hydrogenosomes. Together, these data provide evidence that ancestors of trichomonads perhaps harbored the endosymbiotic progenitors of mitochondria, but that these evolved into hydrogenosomes early in trichomonad evolution.

Animals↗
PubMed1996

Salmonella typhimurium InvA expression probed with a monoclonal antibody to the C-terminal peptide of InvA.

The Salmonella typhimurium InvA protein is a component of a sec-independent secretion apparatus necessary for full virulence of the bacteria. We generated a monoclonal antibody to the C-terminal portion of the InvA protein that recognized proteins in S. typhimurium and weakly in Y. enterocolitica, but not in several other species of bacteria, including S. flexneri. S. typhimurium grown without agitation produced relatively constant amounts of membrane InvA throughout the growth cycle, whereas bacteria grown with agitation had a sharp increase in the amount of membrane InvA at late exponential phase. Levels of InvA present in Salmonella membranes under some growth conditions do not appear to correlate with levels of invasion under the same conditions.

Antibodies, Monoclonal↗
PubMed1995

Direct evidence for secondary loss of mitochondria in Entamoeba histolytica.

Archezoan protists are though to represent lineages that diverged from other eukaryotes before acquisition of the mitochondrion and other organelles. The parasite Entamoeba histolytica was originally included in this group. Ribosomal RNA based phylogenies, however, place E. histolytica on a comparatively recent branch of the eukaryotic tree, implying that its ancestors had these structures. In this study, direct evidence for secondary loss of mitochondrial function was obtained by isolating two E. histolytica genes encoding proteins that in other eukaryotes are localized in the mitochondrion: the enzyme pyridine nucleotide transhydrogenase and the chaperonin cpn60. Phylogenetic analysis of the E. histolytica homolog of cpn60 confirmed that it is specifically related to the mitochondrial lineage. The data suggest that a mitochondrial relic may persist in this organism. Similar studies are needed in archezoan protists to ascertain which, if any, eukaryotic lineages primitively lack mitochondria.

Amino Acid Sequence↗
PubMed1995

Further characterisation of a monoclonal antibody reactive with Escherichia coli O157:H7.

Monoclonal antibody (MAb) 4E8C12 has been previously reported to recognise low mol. wt proteins from enterohaemorrhagic Escherichia coli (EHEC) serotypes O157:H7 and O26:H11. Crude lipopolysaccharide (LPS) preparations from proteinase K-digested bacterial suspensions reacted in Western blots with MAb 4E8C12, as did highly purified LPS from O157:H7 strains. The material recognised by this antibody was, therefore, LPS. The LPS epitope was identified by a whole-cell ELISA in several EHEC, verotoxin producing E. coli (VTEC) and verotoxin-negative strains in addition to E. coli serotypes O157:H7 and O26:H11. Acriflavine and bile salts enhanced the production or availability of the epitope at the cell surface and in culture supernates. These data indicate that the presence of the epitope did not correlate with the virulence of these organisms.

Animals↗
PubMed1994

Host-parasite relationships in amebiasis: conference report.

Entamoeba histolytica has been redescribed as the separate species E. histolytica and Entamoeba dispar. E. dispar is apparently never invasive in humans, while E. histolytica is the etiologic agent of amebic colitis and liver abscess. Virulence factors that may enable E. histolytica to invade include a galactose-specific adhesin, secreted proteases, extracellular matrix receptors, and a cell surface lipophosphoglycan. Progress in vaccine development includes the identification of the cysteine-rich domain of the adhesin and the serine-rich surface protein as protective antigens.

Amebiasis↗
PubMed1994

The 70-kilodalton pertussis toxin-binding protein in Jurkat cells.

125I-ASD photoaffinity-labeling derivatives of pertussis toxin (125I-ASD-PT) or lipopolysaccharide (125I-ASD-LPS) labeled similar 70-kDa proteins in Jurkat cells, a cell line derived from human CD4+ T lymphocytes. Labeling of this 70-kDa protein by 125I-ASD-PT was inhibited by underivatized PT but not by underivatized LPS. However, an immunoglobulin M monoclonal antibody with specificity for the p73 LPS receptor in murine splenocytes (S. W. Bright, T.-Y. Chen, L. M. Flebbe, M.-G. Lei, and D. C. Morrison, J. Immunol. 145:1-7, 1990) inhibited 125I-ASD-PT labeling of the 70-kDa species in Jurkat cells. Our results suggested that PT may bind to the same 70-kDa protein as LPS does in Jurkat cells but that PT and LPS bind to different sites on this receptor candidate. 125I-ASD-PT photoaffinity labeling of the 70-kDa protein was also inhibited by underivatized glycoproteins to which PT has been shown to bind, and this inhibition correlated with the relative binding affinities of the glycoproteins for PT. 125I-ASD derivatives of two sialic acid-specific plant lectins, Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin, with oligosaccharide binding specificities similar to those of PT also labeled a 70-kDa protein in Jurkat cells. This suggests that the 70-kDa PT receptor candidate in Jurkat cells likely contains sialooligosaccharide sequences to which PT, M. amurensis leukoagglutinin, and S. nigra agglutinin bind. The cross-reacting epitope recognized by monoclonal antibody 5D3 in this 70-kDa species might overlap the PT- and LPS-binding sites.

CD4-Positive T-Lymphocytes↗
PubMed1993

Hydrophobic binding of pertussis toxin is enhanced by oligosaccharide receptors.

Pertussis toxin is one of several virulence factors produced by Bordetella pertussis, the etiologic agent of whooping cough. Pertussis toxin is an oligomeric A-B class toxin composed of an ADP-ribosyltransferase S1 (A) subunit and a B oligomer containing lectin-like binding domains. The carbohydrate binding specificity of the B oligomer is for sialooligosaccharide sequences expressed on target cell receptors and asparagine-linked glycans found in many serum glycoproteins. Pertussis toxin also has the ability to bind to the inert surfaces of culture tubes. In this report we present data showing that pertussis toxin binding to polypropylene microcentrifuge tubes was enhanced in a time- and concentration-dependent manner by the addition of soluble glycoprotein or oligosaccharide receptor analogs. Evidence obtained using the hydrophilic and hydrophobic surfaces of Gel Bond electrophoresis casting film indicated that receptor-enhanced binding was likely due to hydrophobic interactions. Hydrophobic binding of the isolated B oligomer of pertussis toxin was enhanced only in the presence of high concentrations of glycoproteins. Therefore, the S1 (A) subunit of pertussis holotoxin appears to play a role in receptor-enhanced hydrophobic binding. We propose, therefore, that pertussis toxin binding to its receptors may expose or preferentially orient hydrophobic residues that may contribute to the functional association of the toxin with host cell plasma membranes and delivery of the S1 subunit to its intracellular target.

Cell Membrane↗
PubMed1993

Entamoeba histolytica: a method for isolate identification.

The ability to identify individual isolates of Entamoeba histolytica Schaudinn 1903 (Emend. Walker 1911) is necessary before several important epidemiological questions can be answered. We have developed such a method based on our discovery of extensive polymorphism in two E. histolytica genes--the serine-rich antigen gene and the "strain specific gene"--each of which has an internal tandemly repeated structure. Using the polymerase chain reaction we detected both size and restriction site polymorphisms in the repetitive regions. When the two genes were used in combination we obtained 16 distinct DNA patterns out of 18 isolates examined. Moreover, these patterns proved to be stable under a variety of conditions--long-term culture, axenization, cell cloning, and animal passage.

Animals↗
PubMed1993

Entamoeba histolytica: an explanation for the reported conversion of "nonpathogenic" amebae to the "pathogenic" form.

The reported conversion of "nonpathogenic" Entamoeba histolytica isolates to the "pathogenic" form during attempted axenization of the amebae is highly controversial. After failing to obtain conversions ourselves we concluded that the simplest explanation for the published observations would be contamination of nonpathogenic cultures with pathogenic amebae. To address this possibility we used a method based on analysis of stable DNA polymorphisms that allows the positive identification of individual pathogenic isolates. The DNA patterns obtained using the "converted" amebae proved to be identical to those of reference isolates present in the laboratories at the time of conversion. We also found that very few cells need be transferred for a pathogenic contaminant to become established in a nonpathogenic culture. Cross-contamination fully explains the conversion phenomenon and thus recognition of nonpathogenic and pathogenic amebae as the distinct species Entamoeba dispar Brumpt 1925 and E. histolytica Schaudinn 1903 (Emend. Walker 1911), respectively, is upheld.

Animals↗