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C G Clark

Publications and source records attributed to C G Clark.

251 records · Page 14Linked to original sources
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The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica.

Formalin fixation is the most common storage, transportation and preservation method for stool samples. However, fixation dramatically reduces our ability to extract from stool samples DNA that is a suitable template for polymerase chain reaction (PCR)-based diagnostic tests. In this study we evaluated the effects of formalin concentration and of the time stored in fixative on the success of PCR amplification. We found that the deleterious effects of formalin are both time and concentration dependent and may result from fragmentation of fixed DNA during its purification.

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Genome structure and evolution of Naegleria and its relatives.

In the 4 yr since the molecular biology of DNA in Naegleria was last reviewed several major advances have been made, and these are reviewed here: isolation and characterization of mitochondrial and ribosomal DNAs; enumeration of chromosomal DNAs by pulsed field gel electrophoresis; sequence analysis of differentially expressed genes; phylogenetic placement of the genus Naegleria among the eukaryotes and Naegleria species within the genus.

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Electrophoretic karyotype and linkage groups of the amoeboflagellate Naegleria gruberi.

We have constructed a molecular karyotype for two strains of Naegleria gruberi using pulsed field gel electrophoresis. Each strain has about 23 chromosomes, considerably more than any previous estimate. These chromosomes range in size from 400 kilobasepairs to over 2,000 kilobasepairs. In Naegleria, construction of the DNA karyotype depends on assessment of the anomalous electrophoretic mobility of the circular ribosomal RNA genes. We have determined the chromosomal locations of an identified unique gene (flagellar calmodulin) and four identified multigene families (alpha- and beta-tubulin, actin, ubiquitin), as well as three differentially expressed genes of unknown functions. The ca. 12 actin genes are dispersed over at least seven chromosomes, whereas the majority of the more than eight alpha-tubulin genes are confined to a single chromosome. The ubiquitin genes are found on five chromosomes in one strain and seven in the other and the beta-tubulin genes are on three or four. Our observations provide a foundation for molecular genetic studies in this organism.

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A redescription of Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911) separating it from Entamoeba dispar Brumpt, 1925.

Explaining the low incidence of invasive disease (10%) in humans infected with Entamoeba histolytica has occupied the attention of generations of both clinical and nonclinical investigators. One possible explanation would be the existence of two morphologically identical species-one an invasive pathogen, the other noninvasive. This was first proposed by Brumpt in 1925, but his explanation was virtually ignored until 1978 when the first of several publications appeared suggesting that E. histolytica did indeed consist of two species. We have reexamined Brumpt's claim in light of recent biochemical, immunological and genetic studies and conclude that the data derived from these investigations provide unequivocal evidence supporting his hypothesis. With this in mind, we redescribe the invasive parasite retaining the name Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911), and set it apart from the noninvasive parasite described by Brumpt, Entamoeba dispar Brumpt, 1925.

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Phylogenetic relationships among anuran trypanosomes as revealed by riboprinting.

Twenty trypanosome isolates from Anura (frogs and toads) assigned to several species were characterized by riboprinting-restriction enzyme digestion of polymerase chain reaction amplified small subunit ribosomal RNA genes. Restriction site polymorphisms allowed distinction of all the recognized species and no intraspecific variation in riboprint patterns was detected. Phylogenetic reconstruction using parsimony and distance estimates based on restriction fragment comigration showed Trypanosoma chattoni to be only distantly related to the other species, while T. ranarum and T. fallisi appear to be sister taxa despite showing non-overlapping host specificities.

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YI-S, a casein-free medium for axenic cultivation of Entamoeba histolytica, related Entamoeba, Giardia intestinalis and Trichomonas vaginalis.

Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa, especially Entamoeba, Giardia, and trichomonads. The digest used almost exclusively in the development of these media, Medo-Peptone (Trypticase BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo-Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica. In response to this problem we have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein-dependent medium TYI-S-33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen-dwellers.

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Axenic cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879.

Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria.

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Riboprinting: a tool for the study of genetic diversity in microorganisms.

Classical morphology-based methods of taxonomic and phylogenetic analysis are inadequate in many groups of structurally simple eukaryotes. Molecular methods can generate data independently of the complexity of the organisms' morphology. Riboprinting is one such technique, and involves restriction enzyme analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes. The utility of the method is illustrated with examples from several genera of intestinal and bloodstream parasites. Among the applications of riboprinting are the detection of cryptic genetic variation within species, organism misidentifications and culture mix-ups, independent verification of DNA sequences, and the rapid generation of data useful in phylogenetic analyses.

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Intraspecific variation and phylogenetic relationships in the genus Entamoeba as revealed by riboprinting.

Eighty-seven isolates of amebae assigned to the genus Entamoeba have been studied by riboprinting (restriction enzyme polymorphism analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes). Twenty-four distinct patterns were obtained, most of which corresponded to previously described species. In three species (Entamoeba coli, Entamoeba gingivalis and Entamoeba moshkovskii) intraspecific variation was detected that led to the grouping of isolates into 'ribodemes' (populations of amebae that share the same riboprint pattern). The riboprint data were used to estimate genetic distances among and within species for the construction of phylogenetic trees based on parsimony and distance analyses. The trees obtained with the two methods are largely congruent. In some cases the estimated distances between species were greater than the upper limit recommended for the fragment comigration method of analysis indicating unusually deep branches within this genus. However, it appears that those species producing cysts with eight nuclei, those producing cysts with one nucleus, and those producing cysts with four nuclei form morphologically based groups that are supported by the riboprint data. The oral parasite Entamoeba gingivalis, which does not encyst, clusters with the third group indicating secondary loss of this ability.

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Monoclonal antibodies reacting with colorectal tumours.

25 mouse monoclonal antibodies were screened for cell surface reactivity with colorectal tumours using the indirect immunoperoxidase method. Five antibodies of the IgG subclasses were selected. LICR-LON-M8 was raised against human milk fat globule membrane (HMFGM). The remaining four antibodies 48-1, 3-48-2, 77-1, and 8-30-3 were raised against human bladder metastases. M8, 77-1, and 8-30-3 which showed epithelial membrane antigen (EMA) - like reactivity exhibited greater selectivity for colorectal cancer compared with normal colon. M8 and 77-1 are being evaluated for immunoscintigraphy of colorectal carcinoma.

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