New anti-protozoal agents.
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Antiprotozoal Agents↗
Biomedical subjects
W E Gutteridge
Publications and source records attributed to W E Gutteridge.
Purine base uptake in Trypanosoma cruzi: adaptations and effects of inhibitors.
1. The pattern of purine base uptake in culture epimastigotes of Trypanosoma cruzi can be predicted from cell growth rate and cell concentration, with the late log phase showing the greatest variability. 2. Uptake rates are unexpectedly low in the reproductive tissue amastigotes and high in the non-reproductive blood trypomastigotes. It is suggested that blood trypomastigotes metabolize and accumulate reserves of purine metabolites, whereas amastigotes depend on degradation of host cell RNA and nucleotides as purine sources. 3. All parasite forms salvage hypoxanthine and guanine in preference to adenine. Nifurtimox, dipyridamole and cytochalasin have no effect on uptake, whereas amphotericin B, allopurinol, xanthine and urate inhibit it. The alterations caused by urate are complex, apparently involving inhibition of the monooxypurine phosphoribosyltransferase and induction of permeation of purines into the cells.
Adenine↗
The regulation of phosphofructokinase in epimastigote Trypanosoma cruzi.
Glycosomal (microbody)-enriched fractions prepared from epimastigote Trypanosoma cruzi were used as a partially purified source of phosphofructokinase. D-Fructose 6-phosphate showed sigmoidal kinetics at pH 7.0, but hyperbolic kinetics at pH 8.0. Various adenosine nucleotides were positive effectors; 5'-AMP was the most powerful. ATP showed hyperbolic kinetics under all conditions tested. Several described inhibitors and activators of mammalian phosphofructokinase were without significant effect on the trypanosomal enzyme; the absence of effect of D-fructose 2,6-bisphosphate is of particular note.
Adenosine Diphosphate↗
The occurrence and distribution of alpha-hydroxy-acid dehydrogenase in some members of the order Kinetoplastida.
LDH-X is the isoenzyme of lactate dehydrogenase found in mammalian spermatozoa, occurring in cytosolic and mitochondrial locations. Gossypol strongly inhibits it, and the spermicidal action of this compound is attributed to the disruption of a reducing shuttle. The flagellated protozoan, Trypanosoma cruzi, contains an enzyme activity similar to LDH-X, called alpha-hydroxy-acid dehydrogenase, which is here shown to possess cytosolic and glycosomal components. The glycosome is a microbody-like organelle containing the early glycolytic enzymes. We postulate that the inhibition of replication of T. cruzi by gossypol derives from interference with glycosomal reducing shuttles. T. lewisi resembles T. cruzi in this respect.
Alcohol Oxidoreductases↗
A strategy for the prevention of the transmission of Chagas' disease during blood transfusion.
Our strategy for preventing the transmission of Chagas' disease during blood transfusion is discussed. In addition, the possibility that the Peru, Sonya, Tulahuen and Y strains of Trypanosoma cruzi show varying sensitivities to a series of amphiphilic cationic drugs in vitro at 4 degrees C was investigated using a microscope lysis test. All 21 drugs tested at a concentration of 10(-3) M lysed Sonya bloodstream trypomastigotes, but Peru, Tulahuen and Y strains were affected by 17, 17 and 11 drugs, respectively. All four strains were most sensitive to the acridines; acranil, aminacrine and mepacrine. Although some variation was seen in their responses to certain drugs, no one strain was particularly insensitive to the series as a whole. The effects of gentian violet, maprotiline and mepacrine on the infectivity of Sonya trypomastigotes following incubation at 4 degrees C for 24 h were evaluated. Mepacrine, at a concentration of 2.5 X 10(-4) M greatly decreased the viability of trypomastigotes, while 10(-3) M concentrations of both maprotiline, mepacrine, and gentian violet (at low parasite densities only) apparently abolished all infectivity. Although the compounds we tested did not show a significant improvement over gentian violet, the compound currently used in some blood banks, other existing amphiphilic cationic drugs could be of use in preventing the transmission of Chagas' disease during blood transfusion.
Chagas Disease↗
Uptake of purine bases by Trypanosoma cruzi culture epimastigotes.
The uptake of adenine, guanine and hypoxanthine by Trypanosoma cruzi culture epimastigotes was studied over short time periods at 22 degrees C. The uptake process is concentrative, driven by the purine phosphoribosyltransferases and saturable at about 10 microM hypoxanthine and 1 microM adenine or guanine. Each purine base is apparently taken up by a separate route and the oxypurines are regulated in parallel. Adenine inhibits oxypurine uptake. When the material was incubated with guanine or hypoxanthine for more than 2 min, a decrease in the nucleotide/free base ratio was observed, indicating inhibition of the guanine and hypoxanthine phosphoribosyltransferases. The uptake rates during the ascending phase of uptake and of culture growth rates were of the order of 50 for hypoxanthine and 20 for guanine, relative to that of adenine. These rates were within the physiological range for cell growth in these cultures. During the phase of descending growth and decreased purine uptake activity rates, uptake was depressed to 20% of the rates required for the growth observed in the cultures. It is proposed that the decline in growth rate leads to an increase of the nucleotide pools in the cells which inhibit uptake. This depression may be a cause of the mitotic blockade which occurs during the stationary phase.
Adenine↗
Unusual RNA polymerase content of Trypanosoma brucei nuclei.
Nuclei were isolated from bloodstream forms of Trypanosoma brucei by nitrogen cavitation and sedimentation through percoll density gradients. Transcription studies with these nuclei in vitro demonstrated features not seen with other eukaryotes: RNA synthesis was much greater in the presence of Mn2+ than with Mg2+ and was sensitive to high concentrations (10-100 micrograms/ml) of alpha-amanitin at all salt concentrations tested (25-300 mM ammonium sulphate). RNA polymerase extracted from nuclei by sonication at high ionic strength chromatographed as a single peak, sensitive to high alpha-amanitin concentrations, on DEAE-sephadex under conditions which resolved the classic three RNA polymerase forms when rat liver nuclear extracts were used.
Amanitins↗
Trypanosoma cruzi: possible control of parasite transmission by blood transfusion using amphiphilic cationic drugs.
About 200 clinically used amphiphilic cationic drugs have been shown to be active in vitro against Trypanosoma cruzi at concentrations of less than or equal to 1 mM. Activity against epimastigote and trypomastigote forms was similar, and in both cases the most potent drugs were litracene, maprotiline, thioproperazine, and the acridines: acranil, aminacrine, and mepacrine. Fluorescence microscopy demonstrated that epimastigotes rapidly accumulate acridines initially in discrete subcellular organelles. The amount of drug incorporated during 15 min of incubation was sufficient to produce subsequent lysis of both trypomastigotes and epimastigotes within 24 hr at 4 C. Trypanocidal activity was dependent on the extracellular pH (optimum greater than or equal to 8) and drug exposure time, but was independent of red blood cell density, serum dilution, and temperature (4 to 37 C). Despite their trypanocidal activity, amphiphilic cationic drugs appear to have no significant effect on the energy state of red blood cells at a concentration of 1 mM. These drugs have a possible role in the prevention of Chagas' disease by blood transfusion.
Aminoacridines↗
Existing chemotherapy and its limitations.
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Chagas Disease↗
Pyrimidine metabolism in Trichomonas vaginalis.
Pyrimidine metabolism in Trichomonas vaginalis was investigated using washed cell suspensions of the organism with radiolabelled pyrimidine ring precursors and preformed pyrimidines. The precursors [14C]orotate, [14C]bicarbonate and [14C]aspartate were not incorporated into the pyrimidine bases of trichomonal nucleic acids, indicating that the protozoan is unable to synthesise the pyrimidine ring and is dependent on the salvage of exogenous pyrimidines. [3H]uracil, [3H]uridine, [3H]cytidine, deoxy[3H]cytidine and [3H]thymidine were all efficiently salvaged, and interconversion between cytosine and uracil nucleotides was detected. Thymidylate synthase activity was not detected, suggesting that T. vaginalis is dependent upon an exogenous supply of thymidine for TMP synthesis.
Animals↗
A comparison of the interaction of anthelmintic benzimidazoles with tubulin isolated from mammalian tissue and the parasitic nematode Ascaridia galli.
Colchicine and a range of anthelmintic benzimidazoles inhibited the in vitro polymerization of tubulin purified from the parasitic nematode Ascaridia galli. In most cases, this inhibition was more pronounced than that detected when these drugs were incubated with tubulin purified from mammalian tissue. In particular, oxfendazole and thiabendazole had virtually no effect on mammalian tubulin assembly whereas they were both good inhibitors of nematode tubulin polymerization. Electron microscopic examinations revealed no morphological differences between microtubules from either nematode or mammalian tissues polymerized in the presence or absence of drug, though the length and number of microtubules was reduced in the drug-incubated samples. These results show that the benzimidazole group of anthelmintics interacts specifically with nematode tubulin and that their selectivity, at least in part, is a direct consequence of such interaction.
Animals↗
A novel series of chemical structures active in vitro against the trypomastigote form of Trypanosoma cruzi.
A rapid in vitro test system has shown that many drugs which possess a product licensed for use in man are also active at a concentration of less than 1mM against the blood forms of Trypanosoma cruzi. 62 of these are structurally related amphiphilic cationic drugs which completely lyse the trypomastigotes at 4 degrees C within 24 hr, yet most leave the erythrocytes intact. Three polyene and two anthracycline antibiotics were also found to be selectively trypanocidal under the same conditions.
Animals↗
Purine and pyrimidine metabolism in the Trypanosomatidae.
The pathways leading to purine and pyrimidine nucleotide production in members of the family Trypanosomatidae are discussed with special emphasis on data relating to pathogenic species published from 1974 to 1983 inclusive. Trypanosomes and leishmania in general lack a de novo purine biosynthetic pathway, but have a multiplicity of possible routes for purine salvage. In contrast, pyrimidine nucleotides can be produced by either de novo or salvage pathways. The properties of these pathways in trypanosomatids are compared and contrasted with those of their hosts.
Animals↗
Characterization of satellite DNA in Trypanosoma brucei and Trypanosoma cruzi.
We have determined the properties of the simple-sequence satellite DNAs from two protozoa, Trypanosoma brucei and Trypanosoma cruzi. The T. brucei satellite DNA contains 29 mol% guanine plus cytosine and is made up of long tandem arrays of a 177 base-pair repeat. Sequence heterogeneity in these repeats is limited and restricted to certain positions as shown by sequence analysis, restriction enzyme digestion and two-dimensional analysis of nucleotides bordering the AluI and HhaI recognition sites in the repeat. The repeat contains two copies of a 19 base-pair sequence differing by a single base-pair substitution and several additional copies of part of this sequence. Sequence variants of the repeat are clustered in the DNA. Satellite DNA is not detectably linked to other DNA and no transcripts of this DNA are found in T. brucei. The T. cruzi satellite DNA repeat is 196 base-pairs long and contains 53 mol% guanine plus cytosine. Direct repetitions longer than eight base-pairs were not observed in the nucleotide sequence of this repeat. The nucleotide sequences of the satellites of T. brucei and T. cruzi are not related. In cell fractionation experiments, the T. brucei and T. cruzi satellite DNAs were recovered from the nuclear fraction. Micrococcal nuclease digestion of nuclear fractions yielded 193 and 197 base-pair nucleosomal oligomers in T. brucei and T. cruzi, respectively; these oligomers contained satellite DNA but not the extranuclear kinetoplast DNA. The 193 base-pair nucleosomal repeat of T. brucei is significantly different from the 177 base-pair satellite repeat. Satellite and nucleosomal repeats are, therefore, not in phase in T. brucei. These satellite DNAs are the first to be observed in protozoa, and we conclude that their properties are similar to those of satellites from animals or plants.
Animals↗
Studies on the glycosomal orotate phosphoribosyl transferase of Trypanosoma cruzi.
The orotate phosphoribosyltransferase of the epimastigote form of Trypanosoma cruzi was studied in its particulate state in preparations derived from glycosomes. Maximum activity was observed at pH 9. There was little activity in the absence of Mg2+; optimum [Mg2+] was related to [5'-phosphoribosyl-alpha-1-pyrophosphate]; Mn2+ could partially substitute for it. Kinetic analyses ruled out a substituted mechanism and suggested instead that the mechanism may be sequential. The apparent Km orotate was 2 microM; that for 5'-phosphoribosyl-alpha-1-pyrophosphate was 8 microM. The enzyme could not use uracil as substrate and was apparently not regulated by naturally-occurring nucleotides. It was, however, sensitive to inhibition by a wide range of pyrimidine analogues, the most active of which was 5-fluoroorotate. These inhibitors were as effective against the enzyme activity of intact glycosomes as broken preparations. This observation, when considered with an apparent lack of latency, suggests that the enzyme is located on the outside of the glycosome. The product of its reaction, orotidine 5'-phosphate, did not exchange readily with exogenous orotidine 5'-phosphate, suggesting that it is channeled directly to orotidine 5'-phosphate decarboxylase, the next enzyme in the pathway.
Animals↗
Purification and characterisation of tubulin from the parasitic nematode, Ascaridia galli.
We have developed a method for the purification of tubulin from a parasitic nematode using DEAE-Sephadex column chromatography and temperature-dependent assembly. The resulting microtubules were morphologically similar to those obtained from mammalian brain. The nematode tubulin showed similar properties to mammalian tubulin on one and two dimensional polyacrylamide gels, although certain electrophoretic conditions revealed a slight difference in the alpha-tubulins from mammals and nematodes. This was confirmed by limited proteolytic peptide mapping. The beta subunit of nematode tubulin appeared almost identical to that of mammals. Peptide maps of these tubulins were also compared with those of eukaryotic micro-organisms and these results interpreted in terms of the evolution of the tubulin polypeptides and the sensitivity of helminths to antimicrotubular agents.
Animals↗
The enzymes of purine salvage in Trypanosoma cruzi, Trypanosoma brucei and Leishmania mexicana.
We have previously shown the presence of various purine salvage enzymes in Trypanosoma cruzi, including phosphoribosyltransferase, aminohydrolase, kinase, phosphorylase and hydrolase activities. We now report that a similar situation occurs in Leishmania mexicana amazonensis and Trypanosoma brucei brucei. In all three organisms we found higher levels of activity for the phosphoribosyltransferase enzymes than for the nucleoside kinases, suggesting a preference for the salvage of purine bases rather than nucleosides. Similarly, absence of inosine phosphorylase activity suggests that only one route for the salvage of hypoxanthine is available to the three organisms. The most striking difference was that whereas T. cruzi and T. brucei possessed adenosine aminohydrolase activity, this was not detected in L. mexicana; instead adenine aminohydrolase activity was found. The overall similarity, as judged by the distribution of enzyme activities, of purine salvage in these three members of the kinetoplastida suggest a broad spectrum of activity for any inhibitor acting in this area; the plethora of alternative salvage pathways, however, suggests that in no case would such inhibition be cidal.
Aminohydrolases↗
UMP synthesis in the kinetoplastida.
All six enzymes of pyrimidine biosynthesis de novo have been detected in homogenates of the culture promastigote form of Leishmania mexicana amazonensis, the blood trypomastigote form of Trypanosoma brucei and the culture epimastigote, blood trypomastigote and intracellular amastigote forms of Trypanosoma cruzi. Dihydroorotate dehydrogenase is mitochondrial in mammals, but the isofunctional enzyme, dihydroorotate oxidase was found to be cytoplasmic, whereas orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase, which are cytoplasmic in mammals, were found to be particulate. Analysis by isopycnic sedimentation in sucrose showed that both particulate enzymes co-sedimented with glycosomal-(microbody-)marker enzymes such as hexokinase. Electron microscopy indicated that fractions containing these activities consisted essentially only of microbodies. It is concluded therefore that these enzymes are associated with glycosomes. Kinetic studies with intact glycosomal preparations suggested that there was no membrane barrier between 5-phosphoribose 1-pyrophosphate (P-Rib-PP) and orotate phosphoribosyltransferase, indicating either that the active site of this enzyme is probably on the outside of the glycosome or that the glycosome may have an efficient transport site for P-Rib-PP. Not all the UMP salvage enzymes assayed were detected. No uridine kinase activity was found in any of the species investigated, suggesting that uridine salvage might be routed via a uridine phosphorylase and uracil phosphoribosyltransferase. In agreement with this suggestion, these latter activities were detected in all organisms tested except the intracellular amastigote form of T. cruzi, where uracil phosphoribosyltransferase appeared absent. All the UMP salvage enzymes investigated occurred in cytoplamic fractions.
Animals↗