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Biomedical subjects

C J Bacchi

Publications and source records attributed to C J Bacchi.

108 records · Page 6Linked to original sources
PubMed1977

Polyamines in trypanosomatids.

Polyamines were determined by n-butanol extraction and thin-layer chromatography in four trypanosomatids: Trypanosoma brucei (rat infection) and cultures of Crithidia fasciculata, Leptomonas sp., and Trypanosoma mega. All had putrescine and spermidine but no detectable spermine. Putrescine and spermidine levels were quantitated for extracts of leptomonas during the normal growth cycle. Spermidine values peaked 18 h before peak cell populations. Spermidine-putrescine ratios for all organisms were related to the presumed phylogeny of the group.

Animals↗
PubMed1975

Drug-resistant Leptomonas: cross-resistance in trypanocide-resistant clones.

A Leptomonas of insect origin was highly susceptible to several standard trypanocides and leishmanicides in vitro. Resistance was induced to some of these drugs; clones were isolated from each strain. Cross-resistance patterns of the clones were derived for diamidines, quinapyramine (Antrycide), acriflavin, phenanthridines, and other drugs active against trypanosomes and leishmanias. Clones tested included two each that were resistant to acriflavin, Antrycide, diminazene aceturate (Berenil), and pentamidine and one that was resistant to stilbamidine. Appreciable cross-resistance was evident for all clones. Differences were observed between clones from the same parent strain. Collateral susceptibility towards isometamidium and oxophenarsine was detected in most clone-derived populations. In clones passaged without drug to test for drug fastness, acriflavin and pentamidine clones lost resistance within 10 transfers, whereas Berenil and Antrycide clones retained considerable resistance after 20 to 30 subcultures without drug. Considerations of differences in life cycles suggest that the clone collection may be useful in screening for agents effective against leishmanias and stercorarian trypanosomes rather than against salivary trypanosomes.

Clone Cells↗
PubMed1970

Biochemical properties of trypanosomatid lactate dehydrogenases.

Lactate dehydrogenase (LDH, E.C.1.1.1.27) was found in supernatant (cytoplasmic enzyme) fractions of the trypanosomatid flagellates Trypanosoma conorhini and Crithidia fasciculata if 10 mm cysteine was present in the homogenizing medium. The T. conorhini LDH activity with pyruvate as substrate was increased 35% if 5 mm cysteine was also included in reaction mixtures. K(m) values for the T. conorhini enzyme were 3.3 x 10(-4)m with pyruvate, and 1.6 x 10(-4)m with alpha-ketobutyrate. Cysteine inhibited alpha-ketobutyrate reduction. Comparison of trypanosomatid and human serum LDH enzymes with respect to K(m), substrate activity and inhibition, pH optima, and K(i) values for oxalate and oxamate indicated that the trypanosomatid isoenzymes differed significantly from serum LDH. C. fasciculata LDH was extremely labile, since 59% of the activity was lost 90 min after isolation. The role of LDH enzymes in trypanosomatid metabolism is discussed, and the results are related to other trypanosomatid LDH enzymes. The comparison of homologous enzymes in host and parasite is discussed with regard to metabolic function and a possible model system for chemotherapy.

Animals↗
PubMed1969

Effects of some antitumor agents on growth and glycolytic enzymes of the flagellate Crithidia.

Some antitumor agents known to specifically inhibit certain tumor cell enzymes were examined for activity against glycolytic enzymes and growth of the insect trypanosomatid, Crithidia fasciculata. The cytoplasmic enzymes hexokinase, alpha-glycerophosphate dehydrogenase, malic dehydrogenase, and glucose-6-phosphate dehydrogenase were tested. Agaricic acid (2-hydroxy-1,2,3-nonadecane tricarboxylic acid) was highly inhibitory (50 to 100%) to malic and alpha-glycerophosphate dehydrogenases at approximately 3 x 10(-5)m; 2-(p-hydroxyphenyl)-2-phenylpropane (2 x 10(-4)m), and 5,6-dichloro-2-benzoxazolinone (5 x 10(-4)m) were less effective (50% inhibition) against them. The antiprotozoal agents primaquine (4 x 10(-4)m) and Melarsoprol (8 x 10(-4)m) were 30 to 40% inhibitory. Agaricic acid, 2-(p-hydroxyphenyl)-2-phenylpropane, and 5,6-dichloro-2-benzoxazolinone inhibited growth of Crithidia at less than 10(-4)m. Eight other test compounds from the Cancer Chemotherapy National Service Center (CCNSC) were not toxic to cell growth, although two (4-biphenylcarboxylic acid and 1-[p-chlorobenzyl]-2-ethyl-5-methyl-indole-3-acetic acid) inhibited Crithidia alpha-glycerophosphate dehydrogenase below 1 mm. All of the compounds used specifically inhibited cancer cell alpha-glycerophosphate dehydrogenase. The corresponding enzyme in pathogenic African trypanosomes is important in their terminal respiration. C. fasciculata may be useful in preliminary evaluation of chemotherapeutic agents as potential trypanocides.

Acetates↗
PubMedDate not supplied

Subcellular localization of the enzymes of the arginine dihydrolase pathway in Trichomonas vaginalis and Tritrichomonas foetus.

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56-80%) in the non sedimentable fraction of both species. A large proportion (35-40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non-ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.

Animals↗
PubMedDate not supplied

Rapid methylation of cell proteins and lipids in Trypanosoma brucei.

The fate of the [methyl-14C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-14C]methionine, [U-14C]methionine, S-[methyl-14C]AdoMet or [33S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-14C] and [U-14C]methionine to [35S]methionine. After 4-h incubation with [U-14C]methionine, [methyl-14C]methionine or [35S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-14C] or [methyl-14C]methionine in the presence of cycloheximide (50 micrograms/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [35S]methionine and cycloheximide. [Methyl-14C] and [U-14C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-14C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-14C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-14C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.

Animals↗
PubMedDate not supplied

Effects of carboxylmethylation and polyamine synthesis inhibitors on methylation of Trypanosoma brucei cellular proteins and lipids.

The fate of methionine in eukaryotic cells is divided between protein synthesis and the branched pathway encompassing polyamine synthesis, methylation of proteins and lipids, and transsulphuration reactions. Aside from protein synthesis, the first step to all other uses of methionine is conversion to S-adenosylmethionine. Blockade of polyamine synthesis in African trypanosomes by the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (Ornidyl, DFMO) the AdoMet decarboxylase inhibitor 5'-[[(Z)-4-amino-2-butenyl]-methylamino]-5'-deoxyadenosine or the protein methylase inhibitor sinefungin induces dramatic increases in intracellular AdoMet. In a previous study, distribution and pool sizes of [35S] or [U-14C]methionine were followed in bloodform trypanosomes as incorporation into the total TCA precipitable fractions. In the present study, the effects of pretreatment with DFMO (1 mM), MDL 73811 (1 microM) and sinefugin (2 nM) on [35S] and [U-14C]methionine incorporation were studied in blood forms. DFMO or MDL 73811 pretreatment increased protein methylation 1.5-fold through incorporation of [U14C]methionine, while sinefungin caused a 40% reduction of incorporation. The increases in incorporation of [U-14C]methionine due to DFMO and MDL 73811 were reduced 40% to 70% by including cold AdoMet (1 mM) in the incubation medium, an indication of AdoMet transport by bloodform trypanosomes and the utilization of [U-14C]methionine as AdoMet. Exogenous AdoMet had no effect on [35S]methionine incorporation. The agents studied are curative for African trypanosomiasis infections, either clinically (DFMO) or in model infections (MDL 73811, sinefungin) and thus highlight interference with AdoMet metabolism and methylation reactions as biochemical consequences of these agents.

Adenosine↗
PubMedDate not supplied

Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi.

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.

Acetyltransferases↗