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1.
Parasitol Res ; 120(2): 705-713, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33415404

RESUMO

Leishmaniasis is one of the most neglected parasitic infections of the world and current therapeutic options show several limitations. In the search for more effective drugs, plant compounds represent a powerful natural source. Artemisinin is a sesquiterpene lactone extracted from Artemisia annua L. leaves, from which dihydroartemisinin (DQHS) and artesunic acid (AA)/artesunate are examples of active derivatives. These lactones have been applied successfully on malaria therapy for decades. Herein, we investigated the sensitivity of Leishmania braziliensis, one of the most prevalent Leishmania species that cause cutaneous manifestations in the New World, to artemisinin, DQHS, and AA. L. braziliensis promastigotes and the stage that is targeted for therapy, intracelular amastigotes, were more sensitive to DQHS, showing EC50 of 62.3 ± 1.8 and 8.9 ± 0.9 µM, respectively. Cytotoxicity assays showed that 50% of bone marrow-derived macrophages cultures were inhibited with 292.8 ± 3.8 µM of artemisinin, 236.2 ± 4.0 µM of DQHS, and 396.8 ± 6.7 µM of AA. The control of intracellular infection may not be essentially attributed to the production of nitric oxide. However, direct effects on mitochondrial bioenergetics and H2O2 production appear to be associated with the leishmanicidal effect of DQHS. Our data provide support for further studies of artemisinin and derivatives repositioning for experimental leishmaniasis.


Assuntos
Antiprotozoários/farmacologia , Artemisininas/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Metabolismo Energético/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Leishmania braziliensis/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Mitocôndrias/metabolismo , Succinatos/farmacologia
2.
Acta Trop ; 215: 105803, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33373585

RESUMO

Chagas disease is a neglected tropical disease strongly associated with low socioeconomic status, affecting nearly 8 million people - mainly Latin Americans. The current infection risk is based on acute case reports, most of which are typically associated with oral transmissions. In the semi-arid region of Northeastern Brazil, serious outbreaks of this transmission type have surged in the last years. One of those occurred in 2016 in the state of Rio Grande do Norte. Rural residents of four municipalities surrounding Marcelino Vieira ingested sugar cane juice - which was probably ground with Trypanosoma cruzi-infected insects. Eighteen cases of Chagas disease were confirmed serologically, with two deaths reported. Socioeconomic information, schooling of residents and the structure of peridomestic and domestic environments in the rural area of Marcelino Vieira, along with entomological indicators, were investigated to understand better the factors related to the outbreaks in this region. We found triatomines (mainly Triatoma brasiliensis) in 35% (24/67) of domiciliary units and all rocky outcrops inspected (n = 7). Overall, 25% (91/357) of examined T. brasiliensis were infected by T. cruzi in artificial ecotopes, with almost the same prevalence in the sylvatic environment (22%; 35/154). Among all ecotopes investigated, wood/tile/brick piles were the ones linked to high insect infestations and triatomine T. cruzi infection prevalence. Ninety-five percent of people interviewed recognized the triatomines and knew the classic route of transmission of disease - triatomine bite-dependent. However, only 7.5% admitted knowledge that Chagas disease can also be acquired orally - which poses a risk this transmission route currently recognized. Here, we highlight the physical proximity between humans and triatomine populations with high T. cruzi infection prevalence as an additional risk factor to oral/vector contaminations. In sum, residents have low income, low level of education, and/or a willful disregard for the routes of Chagas disease transmission (specifically oral transmission), a combination of factors that may have favored the Chagas disease outbreak. We here provide recommendations to avoid further outbreaks.


Assuntos
Doença de Chagas/transmissão , Surtos de Doenças , Animais , Brasil/epidemiologia , Doença de Chagas/epidemiologia , Entomologia , Humanos , Insetos Vetores , Prevalência , Fatores de Risco , Fatores Socioeconômicos
3.
Pathog Dis ; 78(6)2020 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-32926094

RESUMO

Screenings of natural products have significantly contributed to the discovery of novel leishmanicidal agents. In this study, three known cruzioseptins-antibacterial peptides from Cruziohyla calcarifer skin-were synthesized and evaluated against promastigotes and amastigotes stages of Leishmania (L.) amazonensis and L. (V.) braziliensis. EC50 ranged from 9.17 to 74.82 µM, being cruzioseptin-1 the most active and selective compound, with selectivity index > 10 for both promastigotes and amastigotes of L. (V.) braziliensis. In vitro infections incubated with cruzioseptins at 50 µM showed up to ∼86% reduction in the amastigote number. Cruzioseptins were able to destabilize the parasite's cell membrane, allowing the incorporation of a DNA-fluorescent dye. Our data also demonstrated that hydrophobicity and charge appear to be advantageous features for enhancing parasiticidal activity. Antimicrobial cruzioseptins are suitable candidates and alternative molecules that deserve further in vivo investigation focusing on the development of novel antileishmanial therapies.


Assuntos
Antiprotozoários/química , Antiprotozoários/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Anfíbios/metabolismo , Animais , Humanos , Leishmaniose/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Pele/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-31454702

RESUMO

Phospholipase A2 toxins present in snake venoms interact with biological membranes and serve as structural models for the design of small peptides with anticancer, antibacterial and antiparasitic properties. Oligoarginine peptides are capable of increasing cell membrane permeability (cell penetrating peptides), and for this reason are interesting delivery systems for compounds of pharmacological interest. Inspired by these two families of bioactive molecules, we have synthesized two 13-mer peptides as potential antileishmanial leads gaining insights into structural features useful for the future design of more potent peptides. The peptides included p-Acl, reproducing a natural segment of a Lys49 PLA2 from Agkistrodon contortrix laticinctus snake venom, and its p-AclR7 analogue where all seven lysine residues were replaced by arginines. Both peptides were active against promastigote and amastigote forms of Leishmania (L.) amazonensis and L. (L.) infantum, while displaying low cytotoxicity for primary murine macrophages. Spectrofluorimetric studies suggest that permeabilization of the parasite's cell membrane is the probable mechanism of action of these biomolecules. Relevantly, the engineered peptide p-AclR7 was more active in both life stages of Leishmania and induced higher rates of ethidium bromide incorporation than its native template p-Acl. Taken together, the results suggest that short peptides based on phospholipase toxins are potential scaffolds for development of antileishmanial candidates. Moreover, specific amino acid substitutions, such those herein employed, may enhance the antiparasitic action of these cationic peptides, encouraging their future biomedical applications.


Assuntos
Venenos de Crotalídeos/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Peptídeos/farmacologia , Fosfolipases A2/farmacologia , Agkistrodon/metabolismo , Animais , Células Cultivadas , Venenos de Crotalídeos/síntese química , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/síntese química
5.
Oxid Med Cell Longev ; 2019: 5080798, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30728886

RESUMO

Prostate cancer development has been associated with changes in mitochondrial activity and reactive oxygen species (ROS) production. Melatonin (MLT) and docosahexaenoic acid (DHA) have properties to modulate both, but their protective role, mainly at early stages of prostate cancer, remains unclear. In this study, the effects of MLT and DHA, combined or not, on PNT1A cells with regard to mitochondria bioenergetics, ROS production, and proliferation-related pathways were examined. Based on dose response and lipid accumulation assays, DHA at 100 µM and MLT at 1 µM for 48 h were chosen. DHA doubled and MLT reduced (40%) superoxide anion production, but coincubation (DM) did not normalize to control. Hydrogen peroxide production decreased after MLT incubation only (p < 0.01). These alterations affected the area and perimeter of mitochondria, since DHA increased whereas MLT decreased, but such hormone has no effect on coincubation. DHA isolated did not change the oxidative phosphorylation rate (OXPHOS), but decreased (p < 0.001) the mitochondrial bioenergetic reserve capacity (MBRC) which is closely related to cell responsiveness to stress conditions. MLT, regardless of DHA, ameliorated OXPHOS and recovered MBRC after coincubation. All incubations decreased AKT phosphorylation; however, only MLT alone inhibited p-mTOR. MLT increased p-ERK1/2 and, when combined to DHA, increased GSTP1 expression (p < 0.01). DHA did not change the testosterone levels in the medium, whereas MLT alone or coincubated decreased by about 20%; however, any incubation affected AR expression. Moreover, incubation with luzindole revealed that MLT effects were MTR1/2-independent. In conclusion, DHA increased ROS production and impaired mitochondrial function which was probably related to AKT inactivation; MLT improved OXPHOS and decreased ROS which was related to AKT/mTOR dephosphorylation, and when coincubated, the antiproliferative action was related to mitochondrial bioenergetic modulation associated to AKT and ERK1/2 regulation. Together, these findings point to the potential application of DHA and MLT towards the prevention of proliferative prostate diseases.


Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Metabolismo Energético/fisiologia , Melatonina/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Ácidos Docosa-Hexaenoicos/farmacologia , Humanos , Masculino , Melatonina/farmacologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Espécies Reativas de Oxigênio , Transdução de Sinais
6.
Int J Parasitol Drugs Drug Resist ; 8(3): 430-439, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30293058

RESUMO

Tritryps diseases are devastating parasitic neglected infections caused by Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei subspecies. Together, these parasites affect more than 30 million people worldwide and cause high mortality and morbidity. Leishmaniasis comprises a complex group of diseases with clinical manifestation ranging from cutaneous lesions to systemic visceral damage. Antimonials, the first-choice drugs used to treat leishmaniasis, lead to high toxicity and carry significant contraindications limiting its use. Drug-resistant parasite strains are also a matter for increasing concern, especially in areas with very limited resources. The current scenario calls for novel and/or improvement of existing therapeutics as key research priorities in the field. Although several studies have shown advances in drug discovery towards leishmaniasis in recent years, key knowledge gaps in drug discovery pipelines still need to be addressed. In this review we discuss not only scientific and non-scientific bottlenecks in drug development, but also the central role of public-private partnerships for a successful campaign for novel treatment options against this devastating disease.


Assuntos
Descoberta de Drogas/métodos , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Animais , Antiprotozoários/efeitos adversos , Antiprotozoários/uso terapêutico , Antiprotozoários/toxicidade , Doença de Chagas/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/estatística & dados numéricos , Descoberta de Drogas/legislação & jurisprudência , Descoberta de Drogas/estatística & dados numéricos , Descoberta de Drogas/tendências , Humanos , Doenças Negligenciadas/tratamento farmacológico , Doenças Negligenciadas/parasitologia , Parcerias Público-Privadas , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Trypanosomatina/efeitos dos fármacos
7.
J Nat Prod ; 81(1): 188-202, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29297684

RESUMO

The isolation and identification of a series of new pseudoceratidine (1) derivatives from the sponge Tedania brasiliensis enabled the evaluation of their antiparasitic activity against Plasmodium falciparum, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) infantum, and Trypanosoma cruzi, the causative agents of malaria, cutaneous leishmaniasis, visceral leishmaniasis, and Chagas disease, respectively. The new 3-debromopseudoceratidine (4), 20-debromopseudoceratidine (5), 4-bromopseudoceratidine (6), 19-bromopseudoceratidine (7), and 4,19-dibromopseudoceratidine (8) are reported. New tedamides A-D (9-12), with an unprecedented 4-bromo-4-methoxy-5-oxo-4,5-dihydro-1H-pyrrole-2-carboxamide moiety, are also described. Compounds 4 and 5, 6 and 7, 9 and 10, and 11 and 12 have been isolated as pairs of inseparable structural isomers differing in their sites of bromination or oxidation. Tedamides 9+10 and 11+12 were obtained as optically active pairs, indicating an enzymatic formation rather than an artifactual origin. N12-Acetylpseudoceratidine (2) and N12-formylpseudoceratidine (3) were obtained by derivatization of pseudoceratidine (1). The antiparasitic activity of pseudoceratidine (1) led us to synthesize 23 derivatives (16, 17, 20, 21, 23, 25, 27-29, 31, 33, 35, 38, 39, 42, 43, 46, 47, 50, and 51) with variations in the polyamine chain and aromatic moiety in sufficient amounts for biological evaluation in antiparasitic assays. The measured antimalarial activity of pseudoceratidine (1) and derivatives 4, 5, 16, 23, 25, 31, and 50 provided an initial SAR evaluation of these compounds as potential leads for antiparasitics against Leishmania amastigotes and against P. falciparum. The results obtained indicate that pseudoceratidine represents a promising scaffold for the development of new antimalarial drugs.


Assuntos
Alcaloides/química , Alcaloides/farmacologia , Antiparasitários/química , Antiparasitários/farmacologia , Poríferos/química , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antiprotozoários/química , Antiprotozoários/farmacologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Relação Estrutura-Atividade , Trypanosoma cruzi/efeitos dos fármacos
8.
Biol Trace Elem Res ; 168(1): 133-40, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25850543

RESUMO

Cadmium is a heavy metal of increasing environmental concern that has long been associated to several human pathological processes. Recent population surveys have correlated cadmium non-occupational exposure to widespread idiopathic pathologies. Food and tobacco are reported to be the main exposure sources of cadmium to the general population, as phosphate fertilizers are rich in such a metal, thus contaminating the crops. Although its mechanisms of toxicity are not a consensus in the literature, it is well established that reactive oxygen species play a key role in this process, leading to the oxidation of several biological molecules. We have therefore assessed whether three environmentally realistic doses of cadmium alter the oxidative status of Wistar rat testis and eventually result in histological damages. Our results show that even the lowest environmental dose of cadmium was able to disturb the endogenous antioxidant system in Wistar testis, although an increase in lipid peroxidation was observed only within the group exposed to the highest environmental dose. Despite that no remarkable morphological changes were observed in any group, significant alterations in blood vessel lumen were reported for some cadmium-exposed animals, suggesting that endothelium is one of the primary targets involved in cadmium toxicity.


Assuntos
Antioxidantes/metabolismo , Intoxicação por Cádmio/complicações , Cádmio/análise , Exposição Ambiental/efeitos adversos , Animais , Intoxicação por Cádmio/epidemiologia , Intoxicação por Cádmio/patologia , Endotélio Vascular/efeitos dos fármacos , Fertilizantes/análise , Glutationa/metabolismo , Células Intersticiais do Testículo/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Testículo/metabolismo , Testículo/patologia , Ganho de Peso/efeitos dos fármacos
9.
J Eukaryot Microbiol ; 61(4): 381-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24801399

RESUMO

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 Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -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 (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 µM Ca(2+) 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 Ca(2+) this lectin, at 50 µg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm 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 Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.


Assuntos
Ciclosporina/farmacologia , Fabaceae/química , Lectinas/farmacologia , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Sementes/química , Animais , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo
10.
PLoS Negl Trop Dis ; 7(6): e2279, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23785540

RESUMO

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.


Assuntos
Dano ao DNA , Guanina/análogos & derivados , Estresse Oxidativo , Trypanosoma cruzi/fisiologia , Animais , Sobrevivência Celular , Células Cultivadas , Doença de Chagas/parasitologia , Doença de Chagas/patologia , Modelos Animais de Doenças , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Fibroblastos/parasitologia , Expressão Gênica , Guanina/metabolismo , Peróxido de Hidrogênio/toxicidade , Macrófagos/parasitologia , Camundongos , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Parasitemia/parasitologia , Parasitemia/patologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento
11.
Free Radic Biol Med ; 63: 65-77, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23665397

RESUMO

Tryparedoxins (TXNs) are multipurpose oxidoreductases from trypanosomatids that transfer reducing equivalents from trypanothione to various thiol proteins. In Trypanosoma cruzi, two genes coding for TXN-like proteins have been identified: TXNI, previously characterized as a cytoplasmic protein, and TXNII, a putative tail-anchored membrane protein. In this work, we performed a comparative functional characterization of T. cruzi TXNs. Particularly, we cloned the gene region coding for the soluble version of TXNII for its heterologous expression. The truncated recombinant protein (without its 22 C-terminal transmembrane amino acids) showed TXN activity. It was also able to transfer reducing equivalents from trypanothione, glutathione, or dihydrolipoamide to various acceptors, including methionine sulfoxide reductases and peroxiredoxins. The results support the occurrence and functionality of a second tryparedoxin, which appears as a new component in the redox scenario for T. cruzi.


Assuntos
Glutationa/metabolismo , Tiorredoxinas/genética , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Expressão Gênica , Glutationa/análogos & derivados , Oxirredução , Proteína Dissulfeto Redutase (Glutationa) , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Espermidina/análogos & derivados , Espermidina/metabolismo , Tiorredoxinas/metabolismo
12.
Arch Biochem Biophys ; 520(2): 117-22, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22425758

RESUMO

Trypanosoma cruzi's antioxidant system is unique and relevant to the parasite. In this study, quantitative assays were performed to determine cytosolic and mitochondrial tryparedoxin peroxidases and superoxide dismutases expression (TcCPx, TcMPx, SODB and SODA) in correlation to H(2)O(2) release and O(2)(-) production. Differences were observed regarding H(2)O(2) release and O(2)(-) production between strains and along the growth curve. All of the enzymes studied exhibited varied expression as a function of time in culture. Although at lower levels, the Y strain exhibited the same pattern of Tulahuen 2 enzyme expression for all of the proteins studied, except SODA. In the stationary phase, the degree of expression of all of the enzymes in the Y strain returned to similar levels as those detected in the log phase with the exception of TcCPx and SODA. In Tulahuen 2, a higher expression of TcMPx, SODA and SODB was detected in the early stationary phase, and a slight decrease was observed in the late stationary phase for each enzyme, excluding TcMPx, which exhibited a marked decrease, and TcCPx, which increased its level. Because of the significance of ROS in redox signaling, these differences in enzyme expression underscore the importance of these parameters for epimastigote proliferation.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Trypanosoma cruzi/fisiologia
13.
J Bioenerg Biomembr ; 43(6): 651-61, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22081211

RESUMO

Trypanosoma cruzi is a hemoflagellate protozoan that causes Chagas' disease. The life cycle of T. cruzi is complex and involves different evolutive forms that have to encounter different environmental conditions provided by the host. Herein, we performed a functional assessment of mitochondrial metabolism in the following two distinct evolutive forms of T. cruzi: the insect stage epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate the entry of electrons into the electron transport chain by increasing complex II-III activity. Interestingly, cytochrome c oxidase (CCO) activity and the expression of CCO subunit IV were reduced in bloodstream forms, creating an "electron bottleneck" that favored an increase in electron leakage and H(2)O(2) formation. We propose that the oxidative preconditioning provided by this mechanism confers protection to bloodstream trypomastigotes against the host immune system. In this scenario, mitochondrial remodeling during the T. cruzi life cycle may represent a key metabolic adaptation for parasite survival in different hosts.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Estágios do Ciclo de Vida/fisiologia , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Adaptação Fisiológica/fisiologia , Animais , Doença de Chagas/imunologia , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Complexo II de Transporte de Elétrons/imunologia , Complexo III da Cadeia de Transporte de Elétrons/imunologia , Humanos , Peróxido de Hidrogênio/imunologia , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/imunologia , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/imunologia
14.
J Bioenerg Biomembr ; 43(4): 409-17, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21732174

RESUMO

Understanding the energy-transduction pathways employed by Trypanosoma cruzi, the etiological agent of Chagas disease, may lead to the identification of new targets for development of a more effective therapy. Herein, the contribution of different substrates for O(2) consumption rates along T. cruzi epimastigotes (Tulahuen 2 and Y strains) growth curve was evaluated. O(2) consumption rates were higher at the late stationary phase not due to an increase on succinate-dehydrogenase activity. Antimycin A and cyanide did not totally inhibit the mitochondrial respiratory chain (MRC). Malonate at 10 or 25 mM was not a potent inhibitor of complex II. Comparing complex II and III, the former appears to be the primary site of H(2)O(2) release. An update on T. cruzi MRC is presented that together with our results bring important data towards the understanding of the parasite's MRC. The findings mainly at the stationary phase could be relevant for epimastigotes transformation into the metacyclic form, and in this sense deserves further attention.


Assuntos
Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Antimicina A/farmacologia , Cianetos/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos
15.
Mol Biochem Parasitol ; 176(1): 8-16, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21073906

RESUMO

Components of the DNA mismatch repair (MMR) pathway are major players in processes known to generate genetic diversity, such as mutagenesis and DNA recombination. Trypanosoma cruzi, the protozoan parasite that causes Chagas disease has a highly heterogeneous population, composed of a pool of strains with distinct characteristics. Studies with a number of molecular markers identified up to six groups in the T. cruzi population, which showed distinct levels of genetic variability. To investigate the molecular basis for such differences, we analyzed the T. cruzi MSH2 gene, which encodes a key component of MMR, and showed the existence of distinct isoforms of this protein. Here we compared cell survival rates after exposure to genotoxic agents and levels of oxidative stress-induced DNA in different parasite strains. Analyses of msh2 mutants in both T. cruzi and T. brucei were also used to investigate the role of Tcmsh2 in the response to various DNA damaging agents. The results suggest that the distinct MSH2 isoforms have differences in their activity. More importantly, they also indicate that, in addition to its role in MMR, TcMSH2 acts in the parasite response to oxidative stress through a novel mitochondrial function that may be conserved in T. brucei.


Assuntos
Proteína 2 Homóloga a MutS/metabolismo , Estresse Oxidativo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Adenosina Trifosfatases/metabolismo , Cisplatino/farmacologia , Reagentes para Ligações Cruzadas/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA , DNA Mitocondrial/genética , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Peróxido de Hidrogênio/farmacologia , Dados de Sequência Molecular , Proteína 2 Homóloga a MutS/genética , Mutação , Oxidantes/farmacologia , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/efeitos dos fármacos
16.
J Bioenerg Biomembr ; 41(3): 299-308, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19618257

RESUMO

In trypanosomatids the involvement of mitochondrial complex I in NADH oxidation has long been debated. Here, we took advantage of natural Trypanosoma cruzi mutants which present conspicuous deletions in ND4, ND5 and ND7 genes coding for complex I subunits to further investigate its functionality. Mitochondrial bioenergetics of wild type and complex I mutants showed no significant differences in oxygen consumption or respiratory control ratios in the presence of NADH-linked substrates or FADH(2)-generating succinate. No correlation could be established between mitochondrial membrane potentials and ND deletions. Since release of reactive oxygen species occurs at complex I, we measured mitochondrial H(2)O(2) formation induced by different substrates. Significant differences not associated to ND deletions were observed among the parasite isolates, demonstrating that these mutations are not important for the control of oxidant production. Our data support the notion that complex I has a limited function in T. cruzi.


Assuntos
Complexo I de Transporte de Elétrons/genética , Metabolismo Energético/fisiologia , Mitocôndrias/fisiologia , Trypanosoma cruzi/fisiologia , Animais , Respiração Celular/fisiologia , Metabolismo Energético/genética , Peróxido de Hidrogênio/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Oxirredução , Consumo de Oxigênio/fisiologia , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética
17.
Biochem J ; 418(3): 595-604, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19053945

RESUMO

The epimastigote stage of Trypanosoma cruzi undergoes PCD (programmed cell death) when exposed to FHS (fresh human serum). Although it has been known for over 30 years that complement is responsible for FHS-induced death, the link between complement activation and triggering of PCD has not been established. We have previously shown that the mitochondrion participates in the orchestration of PCD in this model. Several changes in mitochondrial function were described, and in particular it was shown that mitochondrion-derived O(2)(*-) (superoxide radical) is necessary for PCD. In the present study, we establish mitochondrial Ca(2+) overload as the link between complement deposition and the observed changes in mitochondrial physiology and the triggering of PCD. We show that complement activation ends with the assembly of the MAC (membrane attack complex), which allows influx of Ca(2+) and release of respiratory substrates to the medium. Direct consequences of these events are accumulation of Ca(2+) in the mitochondrion and decrease in cell respiration. Mitochondrial Ca(2+) causes partial dissipation of the inner membrane potential and consequent mitochondrial uncoupling. Moreover, we provide evidence that mitochondrial Ca(2+) overload is responsible for the increased O(2)(*-) production, and that if cytosolic Ca(2+) rise is not accompanied by the accumulation of the cation in the mitochondrion and consequent production of O(2)(*-), epimastigotes die by necrosis instead of PCD. Thus our results suggest a model in which MAC assembly on the parasite surface allows Ca(2+) entry and its accumulation in the mitochondrion, leading to O(2)(*-) production, which in turn constitutes a PCD signal.


Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Proteínas do Sistema Complemento/fisiologia , Mitocôndrias/fisiologia , Trypanosoma cruzi/citologia , Animais , Ativação do Complemento/efeitos dos fármacos , Digitonina/farmacologia , Humanos , Ionomicina/farmacologia , Mitocôndrias/efeitos dos fármacos , Soro/fisiologia , Superóxidos/metabolismo , Trypanosoma cruzi/metabolismo
18.
Chem Biol Interact ; 176(2-3): 143-50, 2008 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-18771661

RESUMO

Goniothalamin is a styryllactone synthesized by plants of the genus Goniothalamus. The biological activities of this molecule, particularly its anti-protozoan, anti-fungal, and larvicidal properties, have received considerable attention. In this work, we investigated the action of the natural and synthetic enantiomers (R)-goniothalamin (1) and (S)-goniothalamin (ent-1) on cell viability, nitric oxide synthase (NOS) expression and activity, and the expression of selected proteins involved in apoptosis and autophagy in renal cancer cells. Both compounds were cytotoxic and decreased the mitochondrial function of renal cancer cells. However, the enantiomers differentially affected the expression/activity profiles of some signaling pathway mediators. Ent-1 (4 nM) was more potent than 1 (6.4 microM) in inhibiting constitutive NOS activity (54% and 59% inhibition, respectively), and both enantiomers decreased the protein expression of neuronal and endothelial NOS, as assessed by western blotting. Ent-1 and 1 caused down-regulation of Ras and TNFR1 and inhibition of protein serine/threonine phosphatase 2A (PP2A). Compound 1 markedly down-regulated Bcl2, an anti-apoptotic protein, and also induced PARP cleavage. Despite inducing an expressive down-regulation of Bax, ent-1 was also able to induce PARP cleavage. These results suggest that these compounds caused apoptosis in renal cancer cells. Interestingly, ent-1 enhanced the expression of LC3, a typical marker of autophagy. NFkappaB was down-regulated in 1-treated cells. Overall, these results indicate that the anti-proliferative activity of the two enantiomers on renal cancer cells involved distinct signaling pathways, apoptosis and autophagy as dominant responses towards 1 and ent-1, respectively.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Óxido Nítrico/metabolismo , Pironas/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Conformação Molecular , Óxido Nítrico Sintase/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Proteína Fosfatase 2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pironas/síntese química , Pironas/química , Receptores Tipo I de Fatores de Necrose Tumoral/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Estereoisomerismo , Células Tumorais Cultivadas , Proteínas ras/efeitos dos fármacos , Proteínas ras/metabolismo
19.
Exp Parasitol ; 120(4): 421-3, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18809403

RESUMO

Within the mitochondrion of Leishmania infantum, hydroperoxide metabolism relies on the activity of tryparedoxin-dependent peroxidases (TXNPxs). Tryparedoxins (TXNs) are thioredoxin-related oxidoreductases, which in vitro are reduced by the trypanothione reductase/trypanothione [TR/T(SH)(2)] redox couple. Still, there is no evidence that this actually occurs in the mitochondrion. This communication addresses the question of how the mitochondrial TXN/TXNPx system is reduced. First, using a digitonin fractionation assay, we show that TR activity is absent from the L. infantum mitochondrion. The possibility that this organelle possesses alternative electron sources for TXN/TXNPx is then investigated. Biochemical assays performed with purified recombinant enzymes, revealed that TR and T(SH)(2) can be replaced, albeit less efficiently, by the dihydrolipoamide dehydrogenase/lipoamide redox system as TXN/TXNPx electron donor. This result challenges the classical view that T(SH)(2) is the only reductant for TXNs and add new prospects regarding the involvement of 2-oxo acid dehydrogenase complexes in L. infantum mitochondrial hydroperoxide metabolism.


Assuntos
Leishmania infantum/metabolismo , Mitocôndrias/metabolismo , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismo , Tiorredoxinas/metabolismo , Animais , Digitonina , Indicadores e Reagentes , Leishmania infantum/enzimologia , Leishmania infantum/ultraestrutura , Mitocôndrias/enzimologia , Oxirredução , Peróxidos/metabolismo
20.
Apoptosis ; 11(10): 1761-71, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16927017

RESUMO

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 disease.


Assuntos
Morte Celular/efeitos dos fármacos , Leucemia/patologia , Riboflavina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Luz , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Riboflavina/efeitos da radiação , Riboflavina/uso terapêutico
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