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Biomedical subjects

C G Clark

Publications and source records attributed to C G Clark.

At least 19 recordsLinked to original sources
PubMed1998

A mitochondrial-like chaperonin 60 gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria.

Diplomonads, parabasalids, as represented by trichomonads, and microsporidia are three protist lineages lacking mitochondria that branch earlier than all other eukaryotes in small subunit rRNA and elongation factor phylogenies. The absence of mitochondria and plastids in these organisms suggested that they diverged before the origin of these organelles. However, recent discoveries of mitochondrial-like heat shock protein 70 and/or chaperonin 60 (cpn60) genes in trichomonads and microsporidia imply that the ancestors of these two groups once harbored mitochondria or their endosymbiotic progenitors. In this report, we describe a mitochondrial-like cpn60 homolog from the diplomonad parasite Giardia lamblia. Northern and Western blots reveal that the expression of cpn60 is independent of cellular stress and, except during excystation, occurs throughout the G. lamblia life cycle. Phylogenetic analyses position the G. lamblia cpn60 in a clade that includes mitochondrial and hydrogenosomal cpn60 proteins. The most parsimonious interpretation of these data is that the cpn60 gene was transferred from the endosymbiotic ancestors of mitochondria to the nucleus early in eukaryotic evolution, before the divergence of the diplomonads and trichomonads from other extant eukaryotic lineages. A more complicated explanation requires that these genes originated from distinct alpha-proteobacterial endosymbioses that formed transiently within these protist lineages.

Amino Acid Sequence↗
PubMed1998

Microbiological and epidemiological investigation of cholera epidemic in Ukraine during 1994 and 1995.

The Ukraine cholera epidemic of 1994 and 1995 was caused by Vibrio cholerae O1, serotype Ogawa, biotype El Tor. This epidemic was centred in the area around Respublika Krim (Crimea) and Mykolajiv, and spread to include parts of southern Ukraine. Cases of cholera occurred between September and November of 1994 and between June and October of 1995. The 32 fatalities among 1370 recorded cases (case fatality ratio, 2.3%) occurred throughout the course of the epidemic. V. cholerae from patients with cholera produced cholera toxin and were resistant to multiple antibiotics, though no resistance plasmids were found. Conjugation experiments suggested that resistance to multiple antibiotics may be present on a self-transmissible genetic element. Environmental sources of V. cholerae O1 El Tor included sewage, sea and surface water, and fresh water and marine fish. All but one of the environmental V. cholerae isolated during the epidemic were very similar to selected isolates from patients at the same time, supporting the role of these environmental sources in the spread of disease.

Cholera↗
PubMed1998

Investigation of the 1994-5 Ukrainian Vibrio cholerae epidemic using molecular methods.

Thirty-seven Vibrio cholerae and four non-cholera Vibrio isolates from Ukraine, including strains from the epidemic of 1994-5, were analysed by molecular methods. Results from PFGE and ribotyping indicated that all Ukrainian toxigenic V. cholerae were closely related to each other and to an isolate from a patient from Pakistan. A non-toxigenic river water strain obtained during the height of the epidemic was more distantly related to these V. cholerae strains, while the Vibrio parahaemolyticus isolates and Vibrio alginolyticus isolate were not closely related to V. cholerae or each other. ERIC- and REP-PCR allowed the differentiation of strains identical by other methods. The results obtained confirm that the epidemic Ukrainian strains are most closely related to seventh pandemic strains from Asia and support a hypothesis that the Ukrainian epidemic of 1994-5 was caused by toxigenic environmental strains surviving since the time of the 1991 Ukrainian epidemic or before.

Cholera↗
PubMed1998

Detection of microsporidia spore-specific antigens by monoclonal antibodies.

Microsporidia (phylum Microspora) are unicellular parasites commonly found in invertebrates, fish, and laboratory animals; however, microsporidiosis is an emerging problem in patients with the acquired immunodeficiency syndrome (AIDS). The infective stage of these parasites is the spore, which possesses a rigid cell wall that protects the parasite outside its host. Little is known about their antigenic composition. Sensitive, reliable, and easily performed methods for identification and speciation are generally not available. Here, we report the production of 21 MAbs specific to spore antigens of several species of Microsporidia. MAbs were generated to purified spores of Encephalitozoon intestinalis and Encephalitozoon hellem, and their reactivities were tested against spores and intracellular developing forms of E. intestinalis, E. hellem, Encephalitozoon cuniculi, and Vittaforma corneae. Both species-specific and broad-reactivity MAbs were produced. Five MAbs reacted against the spores of all four species tested: 7 with 3 species, 6 with 2 species, 1 with E. intestinalis, and 4 with the polar tube of all species. Immunoelectron microscopy confirmed the reactivity of specific MAbs to the spore wall or the polar tube. These MAbs reacted to a few antigens as determined by Western blot, and none of the epitopes were periodate-sensitive. These MAbs may be useful in the diagnosis and speciation of Microsporidia as well in the purification, cloning, and detection of these antigens.

Animals↗
PubMed1998

Trypanosoma cruzi infection of free-ranging lion-tailed macaques (Macaca silenus) and ring-tailed lemurs (Lemur catta) on St. Catherine's Island, Georgia, USA.

Free-ranging Old World primates released on St. Catherine's Island, Georgia (USA), were tested for infection with Trypanosoma cruzi as part of a study of the epizootiology of sylvatic T. cruzi in the southeastern USA. The parasite was observed in liver infusion tryptose medium cultures of blood from seven of 11 lion-tailed macaques (Macaca silenus) and one of 19 ring-tailed lemurs (Lemur catta). Cultures of blood from 10 black and white ruffed lemurs (Varecia variegata) were all negative. Analysis of small subunit ribosomal RNA gene polymorphisms detected using polymerase chain reaction techniques indicates that the parasites isolated from both the lion-tailed macaques and the ring-tailed lemur are probably the same as T. cruzi parasites isolated from raccoons (Procyon lotor) trapped on St. Catherine's Island and other locations in the southeastern USA. Foraging lion-tailed macaques were observed to handle and partially consume specimens of Triatoma sanguisuga, the triatomine bug thought to be a vector of T. cruzi in the southeastern USA. Oral transmission of the parasite may have occurred as a result of this behavior.

Animals↗
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↗