RESUMO
Evolutionary relationships among parasites of the subfamily Leishmaniinae, which comprises pathogen agents of leishmaniasis, were inferred based on differential protein expression profiles from mass spectrometry-based quantitative data using the PhyloQuant method. Evolutionary distances following identification and quantification of protein and peptide abundances using Proteome Discoverer and MaxQuant software were estimated for 11 species from six Leishmaniinae genera. Results clustered all dixenous species of the genus Leishmania, subgenera L. (Leishmania), L. (Viannia), and L. (Mundinia), sister to the dixenous species of genera Endotrypanum and Porcisia. Placed basal to the assemblage formed by all these parasites were the species of genera Zelonia, Crithidia, and Leptomonas, so far described as monoxenous of insects although eventually reported from humans. Inferences based on protein expression profiles were congruent with currently established phylogeny using DNA sequences. Our results reinforce PhyloQuant as a valuable approach to infer evolutionary relationships within Leishmaniinae, which is comprised of very tightly related trypanosomatids that are just beginning to be phylogenetically unraveled. In addition to evolutionary history, mapping of species-specific protein expression is paramount to understand differences in infection processes, tissue tropisms, potential to jump from insects to vertebrates including humans, and targets for species-specific diagnostic and drug development.
Assuntos
Leishmania , Filogenia , Trypanosomatina , Leishmania/genética , Leishmania/metabolismo , Leishmania/classificação , Trypanosomatina/genética , Trypanosomatina/metabolismo , Trypanosomatina/classificação , Evolução Molecular , Animais , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteômica/métodos , Proteoma/genética , Proteoma/análise , Proteoma/metabolismo , Crithidia/genética , Crithidia/metabolismoRESUMO
Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.
Assuntos
Crithidia/efeitos dos fármacos , Crithidia/microbiologia , Fosforilcolina/análogos & derivados , Simbiose , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Crithidia/metabolismo , Crithidia/ultraestrutura , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Fosfatidilcolinas/biossíntese , Isótopos de Fósforo/metabolismo , Fosforilcolina/farmacologiaRESUMO
Crithidia deanei, a monoxenic trypanosomatid, presents an endosymbiotic bacterium in its cytoplasm. Both the protozoan and the bacterium maintain intensive metabolic exchange, resulting in an interesting model to study the coevolution of metabolisms. The relevance of l-proline for the growth of C. deanei and its transport into these cells was studied. Both the endosymbiont-containing (wild) and the endosymbiont-free protozoa (aposymbiont or cured) strains, when grown in medium supplemented with l-proline, reached higher cell densities than those grown in unsupplemented media. We biochemically characterized the uptake of l-proline in both the wild (K(m)=0.153+/-0.022 mM, V(max)=0.239+/-0.011 nmol min(-1) per 4 x 10(7) cells) and the aposymbiont strains (K(m)=0.177+/-0.049 mM, V(max)=0.132+/-0.012 nmol min(-1) per 4 x 10(7) cells). These data suggest a single type of proline transporter whose activity is upregulated by the presence of the symbiotic bacterium. Proline transport was further characterized and was found to be insensitive to the extracellular concentration of Na+, but sensitive to K+ and pH. The abolition of proline uptake by respiratory chain inhibitors and valinomycin indicates that the proline transport in C. deanei is dependent on the plasma membrane K+ gradient.
Assuntos
Crithidia/metabolismo , Crithidia/microbiologia , Prolina/metabolismo , Simbiose , Animais , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Bactérias/metabolismo , Meios de Cultura , DNA Bacteriano/análise , Depressão Química , Concentração de Íons de Hidrogênio , Monensin/farmacologia , Potássio/metabolismo , RNA Ribossômico 16S/análise , Rotenona/farmacologia , Sódio/metabolismo , Temperatura , Fatores de Tempo , Regulação para Cima , Valinomicina/farmacologiaRESUMO
In this study, the role of phospholipid biosynthetic pathways was investigated in the establishment of the mutualistic relationship between the trypanosomatid protozoan Crithidia deanei and its symbiotic bacterium. Although the endosymbiont displays two unit membranes, it lacks a typical Gram-negative cell wall. As in other intracellular bacteria, phosphatidylcholine is a major component of the symbiont envelope. Here, it was shown that symbiont-bearing C. deanei incorporates more than two-fold (32)Pi into phospholipids as compared with the aposymbiotic strain. The major phospholipid synthesized by both strains was phosphatidylcholine, followed by phosphatidylethanolamine and phosphatidylinositol. Cellular fractioning indicated that (32)Pi-phosphatidylcholine is the major phospholipid component of the isolated symbionts, as well as of mitochondria. Although the data indicated that isolated symbionts synthesized phospholipids independently of the trypanosomatid host, a key finding was that the isolated bacteria synthesized mostly phosphatidylethanolamine, rather than phosphatidylcholine. These results indicate that phosphatidylcholine production by the symbiont depends on metabolic exchanges with the host protozoan. Insight about the mechanisms underlying lipid biosynthesis in symbiont-bearing C. deanei might help to understand how the prokaryote/trypanosomatid relation has evolved in the establishment of symbiosis.
Assuntos
Bactérias/metabolismo , Crithidia/microbiologia , Fosfatidilcolinas/metabolismo , Simbiose , Animais , Membrana Celular/metabolismo , Crithidia/crescimento & desenvolvimento , Crithidia/metabolismo , Meios de Cultura , Mitocôndrias/metabolismo , Fosfolipídeos/metabolismo , Radioisótopos de Fósforo/metabolismoRESUMO
Blastocrithidia culicis and Crithidia deanei are trypanosomatid protozoa of insects that normally contain intracellular symbiotic bacteria. The protozoa can be rid of their endosymbionts by antibiotics, producing a cured cell line. Here, we analyzed the glycoconjugate profiles of endosymbiont-harboring and cured strains of B. culicis and C. deanei by Western blotting and flow cytometry analyses using lectins that recognize specifically sialic acid and mannose-like residues. The absence of the endosymbiont increased the intensity of the lectins binding on both trypanosomatids. In addition, wild and cured strain-specific glycoconjugate bands were identified. The role of the surface saccharide residues on the interaction with explanted guts from Aedes aegypti gut was assessed. The aposymbiotic strains of B. culicis and C. deanei presented interaction rates 3.3- and 2.3-fold lower with the insect gut, respectively, when compared with the endosymbiont-bearing strains. The interaction rate of sialidase-treated cells of the wild and cured strains of B. culicis and C. deanei was reduced in at least 90% in relation to the control. The interaction of B. culicis (wild strain) with explanted guts was inhibited in the presence of mucin (56%), fetuin (62%), sialyllactose (64%) and alpha-methyl-D-mannoside (80%), while in C. deanei (wild strain) the inhibition was 53%, 56%, 79% and 34%, respectively. Collectively, our results suggest a possible involvement of sialomolecules and mannose-rich glycoconjugates in the interaction between insect trypanosomatids and the invertebrate host.
Assuntos
Aedes/parasitologia , Fenômenos Fisiológicos Bacterianos , Crithidia/metabolismo , Crithidia/microbiologia , Glicoconjugados/análise , Trypanosomatina/metabolismo , Trypanosomatina/microbiologia , Animais , Western Blotting , Sistema Digestório/parasitologia , Citometria de Fluxo , Glicoconjugados/biossíntese , Glicoconjugados/fisiologia , Lectinas/metabolismo , Manose/fisiologia , Ácido N-Acetilneuramínico/fisiologia , Coloração e Rotulagem , SimbioseRESUMO
In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41+/-0.13 nmol P(i) h(-1) 10(7) cells(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg(2+)-dependent ecto-ATPase activity was 4.05+/-0.17 nmol P(i) h(-1) 10(7) cells(-1). Mg(2+)-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93+/-0.26 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.26+/-0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg(2+)-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg(2+)-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.
Assuntos
Adenosina Trifosfatases/metabolismo , Crithidia/enzimologia , Pirofosfatases/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Adenosina/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Cátions/classificação , Cátions/metabolismo , Células Cultivadas , Crithidia/metabolismo , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Pirofosfatases/análise , Especificidade por Substrato , Suramina/antagonistas & inibidores , Suramina/metabolismo , Fatores de TempoRESUMO
High mannose-type, N-linked oligosaccharides devoid of glucose units may be glucosylated directly from UDP-Glc in mammalian, plant, fungal and protozoan cells. The glucosylated compounds thus formed (protein-linked Glc1Man5-9GlcNAc2, depending on the organisms) are immediately deglucosylated by glucosidase II, an enzyme located, the same as the glucosylating activity, in the endoplasmic reticulum. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated in the trypanosomatid Crithidia fasciculata (a microorganism transferring Man7GlcNAc2 in protein N-glycosylation) cells of the parasite were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and/or castanospermine that were several hundred-fold higher than those required to inhibit 50% of the activity of the protozoan enzyme. The inhibitors did not affect the cell growth rate and, although glucose analogs, did not interfere with the entry of glucose into the cells. About 40-43% of total N-linked oligosaccharides appeared to be glucosylated. As on the average there are several N-linked oligosaccharides per glycoprotein, more than 40-43% (but probably not all of them) are transiently glucosylated in the endoplasmic reticulum.
Assuntos
Crithidia/metabolismo , Glucose/metabolismo , Glicoproteínas/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , 1-Desoxinojirimicina , Acetilglucosaminidase/metabolismo , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Eletroforese em Papel , Glucosamina/análogos & derivados , Glucosamina/farmacologia , Glucosidases/metabolismo , Glicosilação , Indolizinas/farmacologia , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Microssomos/metabolismo , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Trissacarídeos/metabolismoRESUMO
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. In addition to the mitochondrial Ca2+ uptake, a vanadate-sensitive Ca2(+)-transporting system was also detectable in these trypanosomatids.
Assuntos
Cálcio/metabolismo , Digitonina/farmacologia , Trypanosoma/metabolismo , Animais , Transporte Biológico , Crithidia/efeitos dos fármacos , Crithidia/metabolismo , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/metabolismo , Potenciais da Membrana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Trypanosoma/efeitos dos fármacosRESUMO
Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis in Trypanosoma cruzi and stimulated O2 uptake and O2-. generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and O2-. generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H2O2 production were obtained with Crithidia fasciculata despite the presence of catalase in this organism.
Assuntos
Antiprotozoários , Crithidia/efeitos dos fármacos , Iminas/farmacologia , Isoxazóis/farmacologia , Naftoquinonas/farmacologia , Oxazóis/farmacologia , Oxigênio/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Animais , Crithidia/crescimento & desenvolvimento , Crithidia/metabolismo , DNA/biossíntese , Radicais Livres , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
Se ensayaron varias aminoisoxazolil-1,2-naftoquinomas sobre la formación de anión superóxido (O2) y peróxido de hidrógeno (H2O2) por Crithidia fasciculata y Leptomonas seymouri. Los compuestos IVD (N-(5-metil-3-isoxazolil)-4 amino-1,2-naftoquinona; IIID (2-hidroxi-N-(5-metil-3-isoxazoli)-1,4-naftoquinona-4-imina) y IIIE (2-hidroxi-N-(3-metil-5-isoxazolil)-1,4-naftoquinona-4-imina) estimularon la produción de H2O2 mientras que IIIE, IIIC (2-hidroxi-N-(3,5-dimetil-4-isoxazoli)-1,4-naftoquinona-4-imina) y IIIA (2-hidroxi-N-3,4-dimetil-5-isoxazolil)-1,4-naftoquinona-4-imina), pero no IVD, estimularon la formación de O2 en los organismos estudiados. El ciclo redox de las quinona-iminas se verificó por a) la variación de su absorbancia en función de la concentración de oxígeno en el medio de suspensión de las células; b) su reducción anaeróbica, relativamente rápida, con velocidad máxima para IVD; c) la oxidación de los quinoles correspondientes, obtenidos por reducción con borohidruro. C. fasciculata y L. seymouri contenían superóxido dismutasa, enzima esencial para la formación de peróxidos como consecuencia del ciclo redox de las quinonas, y también catalasa, cuya actividad fue seis veces mayor en C. fasciculata. La presencia de catalasa no impidió la formación de H2O2, como producto metabólico de las quinona-iminas. No se pudo demostrar actividad de ascorbato peroxidasa, benzidina peroxidasa o guayacol peroxidasa en los organismos estudiados
Assuntos
Animais , Peróxido de Hidrogênio/metabolismo , Naftoquinonas/farmacologia , Trypanosomatina/efeitos dos fármacos , Crithidia/efeitos dos fármacos , Crithidia/metabolismo , Iminas/farmacologia , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismo , Trypanosomatina/metabolismoRESUMO
Se ensayaron varias aminoisoxazolil-1,2-naftoquinomas sobre la formación de anión superóxido (O2) y peróxido de hidrógeno (H2O2) por Crithidia fasciculata y Leptomonas seymouri. Los compuestos IVD (N-(5-metil-3-isoxazolil)-4 amino-1,2-naftoquinona; IIID (2-hidroxi-N-(5-metil-3-isoxazoli)-1,4-naftoquinona-4-imina) y IIIE (2-hidroxi-N-(3-metil-5-isoxazolil)-1,4-naftoquinona-4-imina) estimularon la produción de H2O2 mientras que IIIE, IIIC (2-hidroxi-N-(3,5-dimetil-4-isoxazoli)-1,4-naftoquinona-4-imina) y IIIA (2-hidroxi-N-3,4-dimetil-5-isoxazolil)-1,4-naftoquinona-4-imina), pero no IVD, estimularon la formación de O2 en los organismos estudiados. El ciclo redox de las quinona-iminas se verificó por a) la variación de su absorbancia en función de la concentración de oxígeno en el medio de suspensión de las células; b) su reducción anaeróbica, relativamente rápida, con velocidad máxima para IVD; c) la oxidación de los quinoles correspondientes, obtenidos por reducción con borohidruro. C. fasciculata y L. seymouri contenían superóxido dismutasa, enzima esencial para la formación de peróxidos como consecuencia del ciclo redox de las quinonas, y también catalasa, cuya actividad fue seis veces mayor en C. fasciculata. La presencia de catalasa no impidió la formación de H2O2, como producto metabólico de las quinona-iminas. No se pudo demostrar actividad de ascorbato peroxidasa, benzidina peroxidasa o guayacol peroxidasa en los organismos estudiados (AU)
Assuntos
Animais , Naftoquinonas/farmacologia , Trypanosomatina/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Crithidia/efeitos dos fármacos , Crithidia/metabolismo , Iminas/farmacologia , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismo , Trypanosomatina/metabolismoRESUMO
An assay for UDP-Glc:glycoprotein glucosyltransferase was developed. Incubation of rat liver microsomes with UDP-[14C]Glc led to the formation of hot trichloroacetic acid insoluble material identified as protein-linked Glc1Man7-9GlcNAc2. Addition of 8 M urea-denatured thyroglobulin to the incubation mixtures stimulated up to 10-12-fold the formation of the same compounds but only in the presence of detergents. Native thyroglobulin was ineffective. Several experiments indicated that the stimulation was due to the transfer of glucose residues from UDP-Glc to high-mannose oligosaccharides in urea-denatured thyroglobulin and that this transfer reaction did not involve dolichol mono- or diphosphate derivatives as intermediates. The glycoprotein glucosylating activity was mainly located in the endoplasmic reticulum and could glucosylate glycopeptides derived from the digestion of thyroglobulin with an unspecific protease. Glucosylation of oligosaccharides in those glycopeptides occurred, however, at a rate at least 2 orders of magnitude slower than that of the same compounds in urea-denatured thyroglobulin. Tryptic digestion of urea-denatured thyroglobulin did not affect its glucosylation rate. The structure of Glc1Man9GlcNAc2 linked to urea-denatured thyroglobulin was identical with that of Glc1Man9GlcNAc2-P-P-dolichol. The assay of UDP-Glc:glycoprotein glucosyltransferase allowed detection of the activity in microsomal membranes in which endogenous acceptors appeared to be absent or almost absent, such as those derived from mung bean, Mucor rouxii, Crithidia fasciculata, and Trypanosoma cruzi cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glicoproteínas/metabolismo , Hexosiltransferases , Proteínas de Membrana , Microssomos/metabolismo , Plantas/metabolismo , Streptomyces griseus/metabolismo , Trypanosoma cruzi/metabolismo , Acetilglucosaminidase/metabolismo , Animais , Crithidia/metabolismo , Glucose/metabolismo , Glucosiltransferases/metabolismo , Glicosilação , Masculino , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Microssomos Hepáticos/metabolismo , Mucor/metabolismo , Peptídeo Hidrolases/metabolismo , Desnaturação Proteica , Ratos , Saccharomyces cerevisiae/metabolismo , Frações Subcelulares/metabolismo , Transferases/metabolismoRESUMO
It has been synthesized a series of 4-aminoisoxazol-1,2-naftoquinones, which inhibit growth and DNA synthesis in Trypanosoma cruzi. This effect was related to quinone induction of oxyradical generation. N-(5-methyl-3-isoxazolyl)-4-amino-1,2-naftoquinone and 2-hydroxy-N-(3,5-dimethyl-1-4-isoxazolyl)-1,4-naftoquinone-4 -imine inhibited also growth of Crithidia fasciculata and Leptomonas symouri, two organisms that may prove useful for the assay of trypanocidal drugs. In order to establish the role of oxyradicals for quinone-imine toxicity, the latter redox-cycling was demonstrated by a) reversible change in their spectra; b) reduction under anaerobic conditions, and c) quinol oxidation by oxygen. Production of H2O2, measured by the microperoxidase method, was maximal with N-(5-methyl-3-isoxazolyl)-4-amine-1,2-naftoquinone (IVD) (0.29-0.26 nmol/min/mg of cell protein), either with C. fasciculata or L. seymouri. Lesser, though significant activities were obtained with 2-hydroxy-N-(5-methyl-3-isoxazolyl)-1,4-naftoquinone-4-imine (IIID) and 2-hydroxy-N-(3-methyl-5-isoxazolyl)-1,4-naftoquinone-4-imine (IIIE) whereas IIIA and IIIC were inactive. O2-. generation, measured by the adrenochrome method, was induced by naftoquinone-imines with a hydroxyl group at C-2 (IIIE, 2-hydroxy-N-(3,5-dimethyl-4-isoxazolyl)-1,4-naftoquinone-4-i mine (IIIC) and 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naftoquinone-4-i mine (IIIA), but no by IVD, with a carbonyl group at C-2. Measurement of catalase and superoxide dismutase in both organisms yielded significant activities, but ascorbate peroxidase, guaiacol peroxidase and bencidine peroxidase were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Peróxido de Hidrogênio/metabolismo , Naftoquinonas/farmacologia , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/metabolismo , Animais , Crithidia/efeitos dos fármacos , Crithidia/metabolismo , Iminas/farmacologia , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
1. Addition of nifurtimox or benznidazole to the NADH-containing mitochondrial fraction of Crithidia fasciculata led to the appearance of the characteristic ESR spectra corresponding to their nitro anion radicals, suggesting that the nitro anion radical is a necessary intermediate in the reduction of both nitro compounds. 2. Nifurtimox anion radical generation by the mitochondrial fraction was insensitive to rotenone and antimycin A but was enhanced by KCN. 3. The nifurtimox anion radical reacted with oxygen under aerobic conditions leading to an increase in the cyanide-insensitive respiration of the intact cells and in the rate of O2- and H2O2 production by the C. fasciculata mitochondrial fraction. 4. In contrast, generation of O2- and H2O2 was not stimulated with pharmacological concentrations of benznidazole. Furthermore, benznidazole inhibited the cyanide-insensitive respiration of the intact cells.