Digitonin Permeabilization Does Not Affect Mitochondrial Function And Allows The Determination Of The Mitochondrial Membrane Potential Of Trypanosoma Cruzi In Situ

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Digitonin can be used to permeabilize selectively the plasma membrane of Trypanoaoma cruzi epimastigotes without significantly affecting the functional integrity of mitochondria. Addition of digitonin at concentrations close to 64 μM caused decrease in the rate of basal respiration of epimastigotes similar to that caused by oligomycin. A further addition of carbonyl cyanide p-trifluorophenylhydrazone (FCCP) brought respiration to the same rate observed prior to the inclusion of digitonin or oligomycin. This suggests that like oligomycin, digitonin is shifting respiration to a nonphosphorylating state probably by depleting the cells from adenine nucleotides due to permeabilization of the plasma membrane. The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of these cells in situ using safranine O. The response of epimastigotes mitochondrial membrane potential to phosphate, FCCP, valinomycin, nigericin, ADP, and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In addition, T. cruzi mitochondria are able to build up and retain a membrane potential of a value comparable to that of mammalian mitochondria. The trypanocidal drug crystal violet, as well as other cationic drugs such as dequalinium, induced a rapid dose-related collapse of the inner mitochondrial membrane potential.266221443114434CAPES; São Paulo Research Foundation; CNPq; São Paulo Research Foundation; FAPESP; São Paulo Research FoundationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Ca2+ Transport In Digitonin-permeabilized Trypanosomatids

The use of digitonin to permeabilize Leishmania mexicana mexicana, Leishmania agamae, and Crithidia fasciculata plasma membranes enabled us to study Ca2+ transport in situ. The present results show that the mitochondria of these trypanosomatids are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by inorganic phosphate. Large amounts of Ca2+ were retained by C. fasciculata mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butylhydroperoxide and diamide. In contrast, Ca2+ was not retained in the matrix of Leishmania sp. mitochondria for long periods of time. 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Role Of Trypanosoma Cruzi Peroxiredoxins In Mitochondrial Bioenergetics

Trypanosoma cruzi cytosolic (TcCPx) and mitochondrial tryparedoxin peroxidase (TcMPx) play a fundamental role in H 2O 2 detoxification. Herein, mitochondrial bioenergetics was evaluated in cells that overexpressed TcCPx (CPx) and TcMPx (MPx) and in pTEX. In MPx, a higher expression was observed for TcCPx, and the same correlation was true for CPx. Differences in H 2O 2 release among the overexpressing cells were detected when the mitochondrial respiratory chain was inhibited using antimycin A or thenoyltrifluoroacetone. MPx had higher O 2 consumption rates than pTEX and CPx, especially in the presence of oligomycin. In all of the cells, the mitochondrial membrane potential and the ATP levels were similar. Because of the mild uncoupling that was observed in MPx, the presence or induction of a proton transporter in the mitochondrial membrane is suggested when TcMPx is expressed at higher levels. Our results show a possible interplay between the cytosolic and mitochondrial antioxidant systems in a trypanosomatid. © Springer Science+Business Media, LLC 2011.434419424Barros, M.H., Bandy, B., Tahara, E.B., Kowaltowski, A.J., (2004) J Biol Chem, 279, pp. 49883-49888Boveris, A., Stoppani, A.O.M., (1977) Experientia, 33, pp. 1306-1308Boveris, A., Sies, H., Martino, E.E., Docampo, R., Turrens, J.F., Stoppani, A.O., (1980) Biochem J, 188, pp. 643-648Cadenas, E., (2004) Mol Asp Med, 25, pp. 17-26Castellani, O., Ribeiro, L., Fernandes, F., (1967) J Protozool, 14, pp. 447-451Castro, H., Sousa, C., Santosm, N., Budde, H., Cordeiro-Da-Silva, A., Flohé, L., Tomás, A.M., (2004) Mol Biochem Parasitol, 36, pp. 137-147Castro, H., Romao, S., Carvalho, S., Teixeira, F., Sousa, C., Tomás, A.M., (2010) PLoS One, 5 (E1), p. 2607Jms, D., Lowry, C.V., Kja, D., (1995) Arch Biochem Biophys, 317, pp. 1-6Facundo, H.T., De Paula, J.G., Kowaltowski, A.J., (2007) Free Radical Biol Med, 42, pp. 1039-1048Finzi, J.K., Chiavegatto, C.W., Corat, K.F., Lopez, J.A., Cabrera, O.G., Mielniczki-Pereira, A.A., Colli, W., Gadelha, F.R., (2004) Mol Biochem Parasitol, 133, pp. 37-43Hernandez, F., Turrens, J., (1998) Mol Biochem Parasitol, 93, pp. 135-137Jo, S.H., Son, M.K., Koh, H.J., Lee, S.M., Song, I.H., Kim, Y.O., Lee, Y.S., Huh, T.L., (2001) J Biol Chem, 276, pp. 16168-16176Kowaltowski, A.J., Costa, A.D., Vercesi, A.E., (1998) FEBS Lett, 425, pp. 213-216Kowaltowski, A., Souza-Pinto, N., Castilho, R., Vercesi, A., (2009) Free Radical Biol Med, 47, pp. 333-343Mehta, A., Shaha, C., (2004) J Biol Chem, 279, pp. 117798-117813Meziane-Cherif, D., Kmi, A., Sergheraert, C., Tartar, A., Dubremetz, J.F., Ouaissi, M.A., (1994) Exp Parasitol, 79, pp. 536-541Mielniczki-Pereira, A., Chiavegatto, C., Lopez, J., Colli, W., Alves, M.J., Gadelha, F.R., (2007) Acta Trop, 101, pp. 54-60Monteiro, G., Kowaltowski, A.J., Barros, M.H., Netto, L.E., (2004) Arch Biochem Biophys, 425, pp. 14-24Nogueira, F.B., Krieger, M.A., Nirdé, P., Goldenberg, S., Romanha, A.J., Smf, M., (2006) Acta Trop, 100, pp. 119-132Piacenza, L., Irigoin, F., Alvarez, M.N., Peluffo, G., Taylor, M., Kelly, J., Wilkinson, S.R., Radi, R., (2007) Biochem J, 403, pp. 323-334Piacenza, L., Peluffo, G., Alvarez, M.N., Kelly, J.M., Wilkinson, S.R., Radi, R., (2008) Biochem J, 410, pp. 359-368Piacenza, L., Zago, M.P., Peluffo, G., Alvarez, M.N., Basombrio, M.A., Radi, R., (2009) Int J Parasitol, 39, pp. 1455-1464Piñeyro, M.D., Parodi-Talice, A., Arcari, T., Robello, C., (2008) Gene, 408, pp. 45-50Prathalingham, S.R., Wilkinson, S.R., Horn, D., Kelly, J.M., (2007) Antimicrob Agents Chemother, 51, pp. 755-758Tian, W., Brausntein, L.D., Pang, J., Stuhlmeier, K.M., Xi, Q., Tian, X., Stanton, R.C., (1998) J Biol Chem, 273, pp. 10609-10617Vercesi, A.E., Bernardes, C.F., Hoffmann, M.E., Gadelha, F.R., Docampo, R., (1991) J Biol Chem, 266, pp. 14431-14434Vercesi, A.E., Hoffmann, M.E., Bernardes, C.F., Docampo, R., (1993) J Med Braz Biol Res, 26, pp. 355-363Vercesi, A.E., Kowaltowski, A.J., Oliveira, H.C., Castilho, R.F., (2006) Front Biosci, 11, pp. 2554-2564Wiese, A.G., Pacifici, R.E., Davies, K.J., (1995) Arch Biochem Biophys, 318, pp. 231-240Wilkinson, S.R., Temperton, N.J., Mondragon, A., Kelly, J.M., (2000) J Biol Chem, 275, pp. 8220-8225Wilkinson, S.R., Meyer, D.J., Taylor, M.C., Bromley, E.V., Miles, M.A., Kelly, J.M., (2002) J Biol Chem, 277, pp. 17062-17071Wilkinson, S.R., Horn, D., Prathalingam, S.R., Kelly, J.M., (2003) J Biol Chem, 278, pp. 31640-31646Wilkinson, S.R., Taylor, M.C., Horn, D., Kelly, J.M., Cheeseman, I., (2008) PNAS, 105, pp. 5022-502

Release of the cytosolic tryparedoxin peroxidase into the incubation medium and a different profile of cytosolic and mitochondrial peroxiredoxin expression in H2O2-treated Trypanosoma cruzi tissue culture-derived trypomastigotes

AbstractTrypanosoma cruzi antioxidant enzymes are among the factors that guarantee parasite survival and maintain infection, enabling T. cruzi to cope with oxidative stress. Herein, the expression of cytosolic (TcCPx) and mitochondrial (TcMPx) tryparedoxin peroxidases was evaluated in tissue culture-derived trypomastigotes upon incubation with different concentrations of H2O2. TcCPx expression slightly increased (5.4%) in cells submitted to 10μM H2O2 treatment when compared to the control, but decreased when higher H2O2 concentrations (20–50μM) were employed. Under these conditions, TcMPx expression increased (∼53%) with 10μM-treatment compared to the control, followed by a reduction that reached ∼46% of the control when using the highest concentration tested. Interestingly, in the supernatant of the incubations, TcCPx, but not TcMPx, was detected, and its levels increased concomitantly with its decreased expression in the intracellular compartment. Our data show that peroxiredoxins in the tissue culture-derived trypomastigote can be modulated under oxidative stress and are present in higher amounts when compared to the epimastigote stage of T. cruzi. Additionally, due to the different expression patterns observed upon H2O2-treatment, each peroxiredoxin may play a distinct role in protecting the parasite under oxidative stress conditions

Trypanosoma cruzi tryparedoxin II interacts with different peroxiredoxins under physiological andoxidative stress conditions.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has to cope with reactive oxygen and nitrogen species during its life cycle in order to ensure its survival and infection. The parasite detoxifies these species through a series of pathways centered on trypanothione that depend on glutathione or low molecular mass dithiol proteins such as tryparedoxins. These proteins transfer reducing equivalents to peroxidases, including mitochondrial and cytosolic peroxiredoxins, TcMPx and TcCPx, respectively. In T. cruzi two tryparedoxins have been identified, TXNI and TXNII with different intracellular locations. TXNI is a cytosolic protein while TXNII due to a C-terminal hydrophobic tail is anchored in the outer membrane of the mitochondrion, endoplasmic reticulum and glycosomes. TXNs have been suggested to be involved in a majority of biological processes ranging from redox mechanisms to protein translation. Herein, a comparison of the TXNII interactomes under physiological and oxidative stress conditions was examined. Under physiological conditions, apart from the proteins with unknown biological process annotation, the majority of the identified proteins are related to cell redox homeostasis and biosynthetic processes, while under oxidative stress conditions, are involved in stress response, cell redox homeostasis, arginine biosynthesis and microtubule based process. Interestingly, although TXNII interacts with both peroxiredoxins under physiological conditions, upon oxidative stress, TcMPx interaction prevails. The relevance of the interactions is discussed opening a new perspective of TXNII functions.Fil: Dias, L.. Universidade Estadual de Campinas; BrasilFil: Peloso, E.F.. Universidade Estadual de Campinas; BrasilFil: Leme, A.F.P.. Associaçáo Brasileira de Informática;Fil: Carnielli, C.M.. Associaçáo Brasileira de Informática;Fil: Pereira, C.N.. Universidade Estadual de Campinas; BrasilFil: Werneck, C.C.. Universidade Estadual de Campinas; BrasilFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Gadelha, F.R.. Universidade Estadual de Campinas; Brasi

A promising action of riboflavin as a mediator of leukaemia cell death

Besides having a pivotal biological function as a component of coenzymes, riboflavin appears a promissing antitumoral agent, but the underlying molecular mechanism remains unclear. In this work, we demonstrate that irradiated riboflavin, when applied at microM concentrations, induces an orderly sequence of signaling events finally leading to leukemia cell death. The molecular mechanism involved is dependent on the activation of caspase 8 caused by overexpression of Fas and FasL and also on mitochondrial amplification mechanisms, involving the stimulation of ceramide production by sphingomyelinase and ceramide synthase. The activation of this cascade led to an inhibition of mitogen activated protein kinases: JNK, MEK and ERK and survival mediators (PKB and IAP1), upregulation of the proapoptotic Bcl2 member Bax and downregulation of cell cycle progression regulators. Importantly, induction of apoptosis by irradiated riboflavin was leukaemia cell specific, as normal human lymphocytes did not respond to the compound with cell death. Our data indicate that riboflavin selectively activates Fas cascade and also constitutes a death receptor-engaged drug without harmful side effects in normal cells, bolstering the case for using this compound as a novel avenue for combating cancerous diseas

The Cratylia Mollis Seed Lectin Induces Membrane Permeability Transition In Isolated Rat Liver Mitochondria And A Cyclosporine A-insensitive Permeability Transition In Trypanosoma Cruzi Mitochondria

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca2+ and mitochondrial dysfunction due to matrix Ca2+ overload. In order to investigate the mechanism of Ca2+-induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (ΔΨm) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca2+ was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca2+ this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ΔΨm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca2+ dependent mechanisms in both mitochondria. 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Characterization Of The Intracellular Ca2+ Pools Involved In The Calcium Homeostasis In Herpetomonas Sp. Promastigotes

Trypanosomatids of the genus Herpetomonas comprises monoxenic parasites of insects that present proand opisthomastigotes forms in their life cycles. In this study, we investigated the Ca2+ transport and the mitochondrial bioenergetic of digitonin-permeabilized Herpetomonas sp. promastigotes. The response of promastigotes mitochondrial membrane potential to ADP, oligomycin, Ca2+, and antimycin A indicates that these mitochondria behave similarly to vertebrate and Trypanosoma cruzi mitochondria regarding the properties of their electrochemical proton gradient. Ca2+ transport by permeabilized cells appears to be performed mainly by the mitochondria. Unlike T. cruzi, it was not possible to observe Ca2+ release from Herpetomonas sp. mitochondria, probably due to the simultaneous Ca2+ uptake by the endoplasmic reticulum. In addition, a vanadate-sensitive Ca2+ transport system, attributed to the endoplasmic reticulum, was also detected. 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