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G H Coombs

Publications and source records attributed to G H Coombs.

At least 127 records · Page 7Linked to original sources
PubMed1985

Subcellular localisation of purine-metabolising enzymes in Leishmania mexicana mexicana.

Leishmania mexicana mexicana cultured promastigotes were fractionated by isopycnic centrifugation on linear sucrose gradients. Guanine, hypoxanthine and xanthine phosphoribosyltransferase activities were found to be associated with glycosomes, whereas adenine phosphoribosyltransferase was cytosolic. 3'- and 5'-nucleotidases and IMP dehydrogenase were shown to be particulate, the former two possibly being associated with the plasma membrane, IMP dehydrogenase with the endoplasmic reticulum. Nucleosidases and deaminases were found to be cytosolic. The results demonstrate that intracellular separation of enzymes could play a part in the regulation of the parasite's purine metabolism.

Animals↗
PubMed1985

Purine phosphoribosyltransferases of Leishmania mexicana mexicana and other flagellate protozoa.

Amastigotes and cultured promastigotes of Leishmania mexicana mexicana and L. m. amazonensis, cultured promastigotes of L. donovani and L. tarentolae, and the culture forms of Crithidia fasciculata, Herpetomonas muscarum muscarum and H. m. ingenoplastis all possessed four phosphoribosyltransferase (PRTase) activities: adenine PRTase, hypoxanthine PRTase, guanine PRTase and xanthine PRTase. The enzymes of L. m. mexicana required divalent cations for activity; Mn2+ or Co2+ produced maximal activity in most cases. Hypoxanthine PRTase, guanine PRTase and xanthine PRTase from all organisms were sedimentable in part, suggesting that they may occur within glycosomes. The enzymes of L. m. mexicana cultured promastigotes were inhibited by a range of purine analogues.

Adenine Phosphoribosyltransferase↗
PubMed1985

Amine production by Leishmania mexicana.

Growing promastigotes of Leishmania mexicana mexicana excreted large amounts of urea and ammonia into the culture medium. Both promastigotes and amastigotes in short-term, high-density cultures also produced urea and ammonia; the excretion rate was higher in promastigotes. Putrescine was excreted by growing promastigotes but spermine and spermidine excretion apparently did not occur. Both promastigote and amastigote cell extracts contained putrescine and spermidine. Both polyamines, but especially putrescine, were present at higher concentrations in promastigotes than amastigotes. Trace amounts of spermine were found in the promastigote extracts but none was detected in the amastigotes.

Amines↗
PubMed1984

Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum: comparative proteolytic activity.

At least four proteolytic activities were detected in the lysates of each of Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum. These were HPAase, a dithiothreitol-dependent activity on hide powder azure; AZCase, a dithiothreitol-dependent activity on azocasein; and two distinct activities towards peptide nitroanilide derivatives--one was optimally active at pH 7 and stimulated by dithiothreitol; the other had no dithiothreitol requirement and was highly active at pH 5. HPAase and AZCase were active over a broad pH range. Overall, with respect to these four activities, T. batrachorum and T. vaginalis were quite similar. In contrast, T. vaginalis and T. foetus differed from one another in several respects, notably the level of HPAase activity and the properties of the dithiothreitol-independent activity. Multiple bands of proteinase activity were demonstrated with each species after electrophoresis of parasite extracts on polyacrylamide gels containing denatured haemoglobin. They appeared optimally at acid pH and in the presence of dithiothreitol. The proteinase band patterns of T. foetus were similarly complex (at least six bands), whereas T. batrachorum gave a much simpler pattern (three bands). The sensitivities to proteinase inhibitors suggested that all the activities were due to cysteine proteinases. The results show that there are some similarities in the proteolytic activities of all three trichomonad species, and that the two parasites of the urinogenital tracts of mammals possess additional features in common.

Animals↗
PubMed1984

Inhibition of Leishmania amastigote growth by antipain and leupeptin.

Antipain and leupeptin were found to be potent inhibitors of the growth of Leishmania mexicana mexicana amastigotes in explanted mouse unstimulated peritoneal macrophages. Antipain at 100 mg l-1 reduced eight-fold the percentage of macrophages infected and to 5% of the control the number of amastigotes present after seven days incubation. At 1 mg l-1, antipain and leupeptin were as effective antileishmanial agents as pentamidine isethionate, all three stopping parasite multiplication over the seven-day incubation.

Animals↗
PubMed1984

The in-vitro activity of metronidazole against strains of Escherichia coli with impaired DNA repair systems.

The minimal inhibitory concentration (MIC) of metronidazole was lower for strains of Escherichia coli with lesions in their DNA repair systems than for their parent strains. The viable numbers of all strains of E. coli tested were considerably reduced by metronidazole, even at sub-MIC levels, and the effect was greater when DNA repair lesions were more severe. In addition, metronidazole caused cell elongation of E. coli and slowly disappeared from the medium upon incubation with the bacterium. The drug-induced decrease in viable numbers of E. coli was followed by an increase. Most cells in this 'recovery' population were found to be anaerogenic and were relatively unaffected by subsequent exposure to metronidazole. The results show that E. coli consumes and is susceptible to metronidazole, under anaerobic conditions. DNA damage is involved in the antibacterial action of the drug, and E. coli resistant to the drug can be obtained readily in in-vitro incubations.

DNA Repair↗
PubMed1984

Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes.

Leishmania mexicana mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, two main proteinase activity peaks were separated with both methods. These accounted for approximately 10% and 90% of the total activity. Characterization of the two activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed one polypeptide with a molecular weight in the region of 31 000. In contrast, the activity of the minor peak eluted from the columns was of higher molecular weight (67 000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only one activity peak from both column techniques. This activity (mol. wt 67 000) corresponded to the high molecular weight proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivatives of various small peptides. The high molecular weight proteinases of both amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. In contrast, the low molecular weight amastigote proteinases were particularly active against two of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. These results indicate that a highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.

Animals↗
PubMed1983

A comparative study of the proteolytic enzymes of Trypanosoma brucei, T. equiperdum, T. evansi, T. vivax, Leishmania tarentolae and Crithidia fasciculata.

Four types of proteolytic activity were detected in the bloodstream form of each of the four Trypanosoma species: (i) HPAase, active on hide powder azure and detected on polyacrylamide gels containing denatured haemoglobin; (ii) AZCase, active on azocasein; (iii) type 1, active on the chromogenic peptide N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine p-nitroanilide in the presence of dithiothreitol, and (iv) type 2, active against several nitroanilide derivatives in the absence of dithiothreitol. Studies of the pH optimum, dithiothreitol requirement and inhibitor sensitivities of the proteolytic activities suggested that: (a) HPAase and type 1 activities could be due to the same enzymes, probably a family of cysteine proteinases; (b) AZCase had some characteristics of a cysteine proteinase, but was not identical to HPAase, and (c) type 2 activity could be due to a serine proteinase. Procyclic T. brucei contained relatively low cysteine proteinase activities (HPAase, AZCase and type 1) but high type 2 activity. Their proteolytic enzymes thus were apparently more similar to those in Crithidia fasciculata and Leishmania tarentolae promastigotes than those in T. brucei bloodstream forms.

Anilides↗
PubMed1983

An analysis of the proteinases of Trichomonas vaginalis by polyacrylamide gel electrophoresis.

The proteinases of Trichomonas vaginalis have been analysed by electrophoresis in polyacrylamide gels containing denatured haemoglobin. Seven bands of activity were detected indicating multiple proteinases. All of the enzymes were stimulated by 1 mM dithiothreitol and had inhibitor sensitivities characteristic of cysteine proteinases. The enzymes differed significantly, however, with respect to pH optima and relative sensitivities to inhibitors.

Animals↗
PubMed1983

Leishmania mexicana: drug sensitivities of promastigotes and transforming amastigotes.

A comparison has been made of the effects of a range of antiprotozoal drugs and other metabolic inhibitors upon growing promastigotes and transforming amastigotes of Leishmania mexicana mexicana. Amastigotes transforming to promastigotes in vitro were highly susceptible to 2-mercaptoacetate, 4-pentenoate, alpha-difluoromethylornithine, ethidium bromide, acridine orange, pentamidine isethionate, allopurinol, amphotericin B, menoctone and pentostam. Promastigotes were in most cases less sensitive to these inhibitors. The results reiterate that the biochemical differences between the two developmental forms are reflected in their sensitivities to inhibitors. The transformation in vitro of Leishmania amastigotes to promastigotes may be a useful model for use as a primary screen for antileishmanial agents.

Animals↗
PubMed1983

Antitrichomonal activity of compounds that affect DNA and its repair.

The trichomonacidal activity of dimethyl sulphate (DMS) was increased a 100-fold by administration in combination with benzamide and aminobenzamides, compounds thought to interfere with DNA repair in mammalian cells. The same benzamides did not enhance the trichomonacidal activities of metronidazole or a thiosemicarbazone. The results suggest that Trichomonas vaginalis is able to repair damaged DNA and that neither metronidazole nor the thiosemicarbazone affect trichomonal DNA in the same way as DMS. They do not rule out the possibility that the drugs have some other action on DNA.

Animals↗
PubMed1983

The antagonistic effects of acetate and lactate upon the trichomonacidal activity of metronidazole.

Lactate and acetate at 5 mM were found to be antagonistic to the killing action of metronidazole on Trichomonas vaginalis under aerobic but not anaerobic conditions. These compounds had no effect on the growth of the parasite, or the rate of metronidazole reduction by, and the activities of NADH oxidase and NADPH oxidase in, parasite homogenates. The phenomenon reported could be one reason why metronidazole is not always effective in the treatment of trichomoniasis.

Acetates↗
PubMed1982

Proteinase inhibitors as antileishmanial agents.

Leishmania mexicana mexicana amastigote proteinase activity was largely inhibited by low concentrations of leupeptin, antipain and two epoxysuccinates, compounds known to affect cysteine proteinases. Of these inhibitors, only two had leishmanicidal activity. trans-Dicyclohexylepoxysuccinate at 10 microgram/ml inhibited the in vitro transformation of L. m. mexicana amastigotes to promastigotes by greater than 50%. Antipain was a potent antileishmanial agent, which inhibited promastigote growth over seven days by 50% at 0 . 5 microgram/ml. The number of amastigotes that transformed in vitro to promastigotes was reduced 78% by antipain at 0 . 1 microgram/ml. Each of the three diamidines investigated (pentamidine isothionate, amicarbilide and M and B 4596) exhibited marked antileishmanial activity, but only M and B 4596 had any significant effect (36% inhibition at 33 microgram/ml) on L. m. mexicana amastigote proteinase activity.

Animals↗
PubMed1982

A comparative study of Leishmania mexicana amastigotes and promastigotes. Enzyme activities and subcellular locations.

Leishmania mexicana mexicana amastigotes have been shown to contain greater activities than promastigotes of the enzymes that catalyse the beta-oxidation of fatty acids, but lower activities of several glycolytic enzymes, with the activity of pyruvate kinase being especially low. The results suggest the beta-oxidation of fatty acids is relatively more important to Leishmania amastigotes than promastigotes, whereas the reverse is true for glycolysis. Succinic dehydrogenase and peptidase activities were much higher in promastigotes than amastigotes. The activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, acid phosphatase and glucose-6-phosphate dehydrogenase varied less, although in each case the activity was significantly lower in the mammalian stage. A method for lysing and fractionating L. m. mexicana promastigotes has been developed. Using this procedure it has been established that many of the glycolytic and functionally related enzymes are located in cell organelles, that hexokinase is intimately connected with the particulate part of the parasite, and that the microsomal fraction of L. m. mexicana is very different in composition from the microsomes of mammalian liver cells.

Coenzyme A Ligases↗
PubMed1982

Proteinases of Leishmania mexicana and other flagellate protozoa.

The amastigote form of the human pathogen Leishmania mexicana contains high proteinase activity, some 20 times greater than that in the promastigote form and macrophages and appreciably higher than the activity in other flagellate protozoa. The main amastigote enzymes are soluble, whereas those of the promastigote are particulate, and have inhibitor sensitivities characteristic of cysteine proteinases. The very high soluble proteinase activity of L. mexicana amastigotes may be a primary factor in the survival and growth of this mammalian stage in its potentially degradative intracellular habitat.

Animals↗