Spermine action on mitochondrial H(+)-ATPase activity and proliferation rate of Trypanosoma cruzi.

Activity of H(+)-ATPase of Trypanosoma cruzi (RA strain) submitochondrial particles is increased in parasites which have a low spermine content caused by growing in a polyamine free medium or in a medium containing inhibitors of polyamine synthesis. Under these conditions, the proliferation rate is markedly decreased. Kinetics of the enzyme inhibition by spermine indicates a non-competitive inhibition. Spermine, through its action on the H(+)-ATPase hydrophobic environment, affects the enzyme activity. Together with the ATPase protein inhibitor, this could be a mechanism regulating ATP levels needed for the parasite proliferation.

Polyamines in Trypanosoma cruzi.

Growth and polyamine content of epimastigotes of Trypanosoma cruzi were studied in the presence of precursors or inhibitors of putrescine synthesis. Arginine and agmatine turned out to be better precursors than ornithine. alpha-D-difluoromethylarginine, an inhibitor of arginine decarboxylase, inhibited cellular proliferation and decreased putrescine, spermidine and spermine contents while that of cadaverine remained unchanged. These effects were reversed both by agmatine and putrescine. alpha-D-difluoromethylornithine, an inhibitor of ornithine decarboxylase did not inhibit growth of parasites grown in a polyamine free medium. These results suggest that epimastigotes need polyamines to grow, and that the parasite are able to synthesize them mainly through the arginine decarboxylase pathway.

[Growth and DNA synthesis inhibition and superoxide anion formation by iminoquinones in Trypanosoma cruzi]. / Inhibición del crecimiento y la síntesis de DNA y formación de anión superóxido por quinona-iminas en Trypanosoma cruzi.

Despite its epidemiological importance, chemotherapy of Chagas' disease is still an unsolved problem. Many drugs have been assayed for their trypanocidal action on T. cruzi, but, so far, naftoquinone-imines have not been included in drug screenings. Fernández A.E. et al., prepared a series of 4-(aminomethylisoxazolyl)-1,2-naftoquinones. Some of these, namely, IIIA (2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naftoquinone-4- imine), IIIC hydroxy-N-3,5-dimethyl-4-isoxazolyl)-1,4-naftoquinone-4-i min e, IIIE (2-hydroxy-N-(3-methyl-5-isoxazolyl)-1,4-naftoquinone-4-i min e), and IVD (5-methyl-3-isoxazolyl)-1,2-naftoquinone-4-imine) were assayed on T. cruzi epimastigotes. Parasite growth and DNA synthesis were inhibited 12-36% and 14-49%, respectively, by 18-19 microM quinones or 44-83% and 37-73%, respectively, by 60-78 microM quinones (Table 1). The assayed compounds (37-100 microM concentration) stimulated oxygen uptake (Figs. 2 and 3) and superoxide anion generation by epimastigotes to 0.20-0.70 nmol/min/mg of cell protein (O2- measured by the adrenochrome method, Table 2). In the presence of quinones, T. cruzi mitochondrial fraction supplemented with 0.42 mM NADH produced O2- at a rate of 1.10-1.45 nmol/min/mg of protein (31 and 75 microM quinone, respectively; Table 3) whereas, under the same experimental conditions, the microsomal fraction supplemented with NADPH produced O2- at rate of 0.85 and 1.03, respectively (same units). The kinetics of O2- generation by the mitochondrial fraction supplemented with quinone IIIC and NADH revealed two interacting sites (Fig. 5). The corresponding Km(s) were 5.2 and 17 microM and the Vm, 13 and 17 nmoles O2-/min/mg of protein, respectively. The quinone interaction with the mitochondrial membranes involved a "quinone reductase" located on the substrate site of the antimycin site, as shown by the 3.6-fold increase of the oxygen uptake rate induced by quinone IIIE in Fig. 2. Apparently, the quinone excess inhibited the reductase since by varying the quinone concentration in the 0-500 microM range, the oxygen uptake versus quinone concentration plots showed a maximum at 100-150 microM quinone (Fig. 3). O2- generation by quinones depended on the latter redox-cycling, as shown by a) the decrease of quinone absorption at 480-500 nm after incubation with T. cruzi epimastigotes for the time necessary to exhaust the reaction mixture oxygen, and b) the reappearance of the quinone absorption band after oxygenation of the anaerobic reaction mixture (Fig. 6).

Inhibición del crecimiento y la síntesis de DNA y formación de anión superóxido por quinona-iminas en Trypanosoma cruzi. / [Growth and DNA synthesis inhibition and superoxide anion formation by iminoquinones in Trypanosoma cruzi]

Despite its epidemiological importance, chemotherapy of Chagas disease is still an unsolved problem. Many drugs have been assayed for their trypanocidal action on T. cruzi, but, so far, naftoquinone-imines have not been included in drug screenings. Fernández A.E. et al., prepared a series of 4-(aminomethylisoxazolyl)-1,2-naftoquinones. Some of these, namely, IIIA (2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naftoquinone-4- imine), IIIC hydroxy-N-3,5-dimethyl-4-isoxazolyl)-1,4-naftoquinone-4-i min e, IIIE (2-hydroxy-N-(3-methyl-5-isoxazolyl)-1,4-naftoquinone-4-i min e), and IVD (5-methyl-3-isoxazolyl)-1,2-naftoquinone-4-imine) were assayed on T. cruzi epimastigotes. Parasite growth and DNA synthesis were inhibited 12-36

and 14-49

, respectively, by 18-19 microM quinones or 44-83

and 37-73

, respectively, by 60-78 microM quinones (Table 1). The assayed compounds (37-100 microM concentration) stimulated oxygen uptake (Figs. 2 and 3) and superoxide anion generation by epimastigotes to 0.20-0.70 nmol/min/mg of cell protein (O2- measured by the adrenochrome method, Table 2). In the presence of quinones, T. cruzi mitochondrial fraction supplemented with 0.42 mM NADH produced O2- at a rate of 1.10-1.45 nmol/min/mg of protein (31 and 75 microM quinone, respectively; Table 3) whereas, under the same experimental conditions, the microsomal fraction supplemented with NADPH produced O2- at rate of 0.85 and 1.03, respectively (same units). The kinetics of O2- generation by the mitochondrial fraction supplemented with quinone IIIC and NADH revealed two interacting sites (Fig. 5). The corresponding Km(s) were 5.2 and 17 microM and the Vm, 13 and 17 nmoles O2-/min/mg of protein, respectively. The quinone interaction with the mitochondrial membranes involved a [quot ]quinone reductase[quot ] located on the substrate site of the antimycin site, as shown by the 3.6-fold increase of the oxygen uptake rate induced by quinone IIIE in Fig. 2. Apparently, the quinone excess inhibited the reductase since by varying the quinone concentration in the 0-500 microM range, the oxygen uptake versus quinone concentration plots showed a maximum at 100-150 microM quinone (Fig. 3). O2- generation by quinones depended on the latter redox-cycling, as shown by a) the decrease of quinone absorption at 480-500 nm after incubation with T. cruzi epimastigotes for the time necessary to exhaust the reaction mixture oxygen, and b) the reappearance of the quinone absorption band after oxygenation of the anaerobic reaction mixture (Fig. 6).

Terminal oxidases in the trypanosomatid Trypanosoma cruzi.

Titration of Trypanosoma cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. The inhibitions obtained at low cyanide concentrations (0-300 microM), taken together with cyanide effects on cytochrome aa3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa3 as T. cruzi main terminal oxidase. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was active in both kinetoplastic and dyskinetoplastic (cytochrome aa3-deficient) epimastigotes, and azide- and antimycin-insensitive. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa3, b, c558, o and possibly d, as components of the parasite electron transport system. Fractionation studies demonstrated that both o and d were bound to the mitochondrial membrane. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.

[Cytochromes in different stages, strains, and populations of Trypanosoma cruzi]. / Citocromos en diferentes estadios, cepas y poblaciones de Trypanosoma cruzi.

Differential reduced minus oxidized (Red-Ox) or reduced. CO minus reduced (Red. CO-Red) spectra of Trypanosoma cruzi metacyclic trypomastigotes (Tulahuen strain), revealed the presence of cytochromes aa3, b, c5 5 8, o and possibly d (Fig. 1). Mitochondrial membranes from the epimastigote form of the parasite showed similar cytochrome spectra (Fig. 2A). Extraction of the mitochondrial membranes with guanidine and cholate, and spectroscopy of the cytochrome o -cyanide derivative proved that this cytochrome is an integral constituent of the mitochondrial membranes (Figs. 2B and 4, and Table 1). Contamination of the investigated samples with hemoglobin, peroxidase, catalase, cytochrome P-420, cytochrome P-450 or culture medium hemoproteins was ruled out by filtering the cells and mitochondrial membranes on Sephadex G-50, or by differential spectroscopy of pigments, or by differential centrifugations of samples. Investigation of pyridine-hemochromes revealed hemes A, B, C and D (Fig. 3), thus confirming the presence of the postulated cytochromes. Comparative spectroscopy of a series of T. cruzi stocks (including Tulahuen and Y strains), many of them obtained from acute or chronic forms of Chagas disease, revealed significant variability in the cytochrome content (Table 2). Taking cytochromes o and b as standard for comparison, the epimastigotes samples could be grouped as follows (in parenthesis number of passages through the culture medium): 1) stocks with a relatively high content of cytochromes b and o, prevailing the former (stocks Y (116), RA (114), AF, FN, TN and MG (14 y 16); 2) stocks with a relatively low content of both cytochromes: Y (119), AWP and UP; 3) stocks with a low content of cytochrome b, without cytochrome o: CA-I and CA-I (V); 4) stocks without cytochromes: Y(117 and 118) and RA(113). In some strains (e.g. Y and RA), significant variation of cytochrome content in different stocks of the same isolate was observed (Table 2), but the Tulahuen strain proved to be less variable. Comparison of cytochrome distribution and other properties of parasites, namely, lethality for mice and morphology, did not allow to establish positive correlations.

Citocromos en diferentes estadíos, cepas y poblaciones de Trypanosoma cruzi / Cytochromes in different stages, strains and populations of Trypanosoma cruzi

Los espectros diferenciales de muestras "reducida menos oxidada" (Red-Ox) y "reducida. CO menos reducida" (Re. CO-Red) de tripomastigotes metacíclicos del Trypanosoma cruzi (cepa Tulahuen) revelaron la presencia de los citocrómos aa3, b, c555 y o. Espectros concordantes se obtuvieron con la fracción mitocondrial de la forma epimastigote de la misma cepa, después de filtrar las membranas por Sephadex G-50 para excluir hemoproteínas espúreas provenientes del medio de cultivo o hemoproteínas solubles (hemoglobina, peroxidasa, catalasa, etc.), o citocrómos P-420 y P-450. El tratamiento de la fracción mitocondrial cruda con guanidina y colato de Na y la formación de complejos con cianuro, revelaron que el citocrómo o es un constituyente integral de esas membranas. El análisis de los piridinahemocrómos provenientes de los citocrómos mitocondriales demostró la presencia de los hemos A, B y C. Una banda espectral a 625 nm en los espectros de los tripomastigotes, en las membranas mitocondriales, y en los piridina-hemocrómos indicó la presencia de un citocrómo d. Un examen comparativo del contenido en citocrómos o y b de una serie de poblaciones de T. cruzi, provenientes originariamente en su mayoría de pacientes con formas agudas o crónicas de la enfermedad de Chagas, demostró variabilidad en el contenido en citocrómos y falta de correlación de este parámetro con las propiedades biológicas del parásito, en particular con la letalidad para el ratón

Citocromos en diferentes estadios, cepas y poblaciones de Trypanosoma cruzi. / [Cytochromes in different stages, strains, and populations of Trypanosoma cruzi]

Differential reduced minus oxidized (Red-Ox) or reduced. CO minus reduced (Red. CO-Red) spectra of Trypanosoma cruzi metacyclic trypomastigotes (Tulahuen strain), revealed the presence of cytochromes aa3, b, c5 5 8, o and possibly d (Fig. 1). Mitochondrial membranes from the epimastigote form of the parasite showed similar cytochrome spectra (Fig. 2A). Extraction of the mitochondrial membranes with guanidine and cholate, and spectroscopy of the cytochrome o -cyanide derivative proved that this cytochrome is an integral constituent of the mitochondrial membranes (Figs. 2B and 4, and Table 1). Contamination of the investigated samples with hemoglobin, peroxidase, catalase, cytochrome P-420, cytochrome P-450 or culture medium hemoproteins was ruled out by filtering the cells and mitochondrial membranes on Sephadex G-50, or by differential spectroscopy of pigments, or by differential centrifugations of samples. Investigation of pyridine-hemochromes revealed hemes A, B, C and D (Fig. 3), thus confirming the presence of the postulated cytochromes. Comparative spectroscopy of a series of T. cruzi stocks (including Tulahuen and Y strains), many of them obtained from acute or chronic forms of Chagas disease, revealed significant variability in the cytochrome content (Table 2). Taking cytochromes o and b as standard for comparison, the epimastigotes samples could be grouped as follows (in parenthesis number of passages through the culture medium): 1) stocks with a relatively high content of cytochromes b and o, prevailing the former (stocks Y (116), RA (114), AF, FN, TN and MG (14 y 16); 2) stocks with a relatively low content of both cytochromes: Y (119), AWP and UP; 3) stocks with a low content of cytochrome b, without cytochrome o: CA-I and CA-I (V); 4) stocks without cytochromes: Y(117 and 118) and RA(113). In some strains (e.g. Y and RA), significant variation of cytochrome content in different stocks of the same isolate was observed (Table 2), but the Tulahuen strain proved to be less variable. Comparison of cytochrome distribution and other properties of parasites, namely, lethality for mice and morphology, did not allow to establish positive correlations.

Citocromos en diferentes estadíos, cepas y poblaciones de Trypanosoma cruzi / Cytochromes in different stages, strains and populations of Trypanosoma cruzi

Los espectros diferenciales de muestras "reducida menos oxidada" (Red-Ox) y "reducida. CO menos reducida" (Re. CO-Red) de tripomastigotes metacíclicos del Trypanosoma cruzi (cepa Tulahuen) revelaron la presencia de los citocrómos aa3, b, c555 y o. Espectros concordantes se obtuvieron con la fracción mitocondrial de la forma epimastigote de la misma cepa, después de filtrar las membranas por Sephadex G-50 para excluir hemoproteínas espúreas provenientes del medio de cultivo o hemoproteínas solubles (hemoglobina, peroxidasa, catalasa, etc.), o citocrómos P-420 y P-450. El tratamiento de la fracción mitocondrial cruda con guanidina y colato de Na y la formación de complejos con cianuro, revelaron que el citocrómo o es un constituyente integral de esas membranas. El análisis de los piridinahemocrómos provenientes de los citocrómos mitocondriales demostró la presencia de los hemos A, B y C. Una banda espectral a 625 nm en los espectros de los tripomastigotes, en las membranas mitocondriales, y en los piridina-hemocrómos indicó la presencia de un citocrómo d. Un examen comparativo del contenido en citocrómos o y b de una serie de poblaciones de T. cruzi, provenientes originariamente en su mayoría de pacientes con formas agudas o crónicas de la enfermedad de Chagas, demostró variabilidad en el contenido en citocrómos y falta de correlación de este parámetro con las propiedades biológicas del parásito, en particular con la letalidad para el ratón (AU)

Disminucion de los citocromos y la biosintesis proteica mitocondrial en musculo esqueletico de ratas diabeticas. / Decrease cytochromes and mitochondrial protein biosynthesis in skeletal muscle of diabetic rats

Se determino el contenido en citocromos aa3, b, c y c1 en mitocondrias de musculo esqueletico, higado y corazon de ratas diabeticas por inyeccion de estreptozotocina (65 mg/kg, dosis unica, intravenosa), 30 dias despues de la iniciacion de la diabetes. Se aislaron las mitocondrias y se determino su contenido en citocromos. Solamente en las mitocondrias de musculo esqueletico se comprobo disminucion significativa (p < 0.001) de los citocromos, especialmente aa3 y b (en las mitocondrias de higado hubo aumento de los citocromos aa3).La variacion de los citocromos musculares concordo con la disminucion de la sintesis de proteinas, comprobada utilizando el procedimiento de Favalukes y col. para medir la incorporacion de aminoacidos en proteinas mitocondriales

Disminucion de los citocromos y la biosintesis proteica mitocondrial en musculo esqueletico de ratas diabeticas. / Decrease cytochromes and mitochondrial protein biosynthesis in skeletal muscle of diabetic rats

Se determino el contenido en citocromos aa3, b, c y c1 en mitocondrias de musculo esqueletico, higado y corazon de ratas diabeticas por inyeccion de estreptozotocina (65 mg/kg, dosis unica, intravenosa), 30 dias despues de la iniciacion de la diabetes. Se aislaron las mitocondrias y se determino su contenido en citocromos. Solamente en las mitocondrias de musculo esqueletico se comprobo disminucion significativa (p < 0.001) de los citocromos, especialmente aa3 y b (en las mitocondrias de higado hubo aumento de los citocromos aa3).La variacion de los citocromos musculares concordo con la disminucion de la sintesis de proteinas, comprobada utilizando el procedimiento de Favalukes y col. para medir la incorporacion de aminoacidos en proteinas mitocondriales