RESUMO
This paper presents unequivocal results about the presence of trypanothione and its precursor glutathionespermidine from the opportunistic human pathogen Acanthamoeba polyphaga. They were isolated by RP-HPLC as thiolbimane derivatives and characterized using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF). Additionally RP-HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were also present in A. polyphaga. Besides trypanothione, we want to report four new peptides in trophozoites, a tetrapeptide, a hexapeptide, a heptapeptide and a nonapeptide. Trypanothione and two of the thiol peptides, the hexapeptide and heptapeptide, are oxidized since the reduced forms increase in amount when the normal extract is treated by DTT or by electrolytic reduction that convert the oxidized forms to reduced ones. On the other hand, they disappear when the amoeba extract is treated with NEM or when the amoeba culture is treated with various inhibitors of NADPH-dependent disulfidereducing enzymes. Comparison of the thiol peptides, including trypanothione from A. polyphaga with extracts from human lymphocytes showed that they are not present in the latter. Therefore, some of the peptides here reported could be used as antigens for rapid detection of these parasites. In regard to the presence of the enzymes that synthesize and reduce trypanothione in A. polyphaga we suggest that they can be used as drug targets.
Assuntos
Acanthamoeba/química , Acanthamoeba/metabolismo , Glutationa/análogos & derivados , Peptídeos/química , Proteínas de Protozoários/química , Espermidina/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Glutationa/química , Glutationa/isolamento & purificação , Glutationa/metabolismo , Humanos , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espermidina/química , Espermidina/isolamento & purificação , Espermidina/metabolismoRESUMO
Trypanosoma cruzi is exposed to sudden temperature changes during its life cycle. Adaptation to these variations is crucial for parasite survival, reproduction, and transmission. Some of these conditions may change the pattern of genetic expression of proteins involved in homeostasis in the course of stress treatment. In the present study, the proteome of T. cruzi epimastigotes subjected to heat shock and epimastigotes grow normally was compared by two-dimensional gel electrophoresis followed by mass spectrometry for protein identification. Twenty-four spots differing in abundance were identified. Of the twenty-four changed spots, nineteen showed a greater intensity and five a lower intensity relative to the control. Several functional categories of the identified proteins were determined: metabolism, cell defense, hypothetical proteins, protein fate, protein synthesis, cellular transport, and cell cycle. Proteins involved in the interaction with the cellular environment were also identified, and the implications of these changes are discussed.
Assuntos
Proteínas/metabolismo , Proteoma/metabolismo , Trypanosoma cruzi/metabolismo , Adaptação Fisiológica , Eletroforese em Gel Bidimensional/métodos , Resposta ao Choque Térmico/fisiologia , Humanos , Espectrometria de Massas/métodos , Proteínas/análise , Proteínas/classificação , Proteoma/análiseRESUMO
Tyrianthins A (1) and B (2), two new partially acylated glycolipid ester-type heterodimers were isolated from Ipomoea tyrianthina. Scammonic acid A was determined as the glycosidic acid in both monomeric units. Tyrianthin A (1) (IC(50) 0.24+/-0.09 microM and E(max) 81.80+/-0.98%), and tyrianthin B (2) (IC(50) 0.14+/-0.08 microM and E(max) 87.68+/-0.72%) showed significant in vitro relaxant effect on aortic rat rings, in endothelium- and concentration-dependent manners. Also, these compounds were able to increase the release of GABA and glutamic acid in brain cortex, and displayed weak antimycobacterial activity.
Assuntos
Glicolipídeos/química , Ipomoea/química , Vasodilatadores/química , Animais , Antituberculosos/química , Antituberculosos/isolamento & purificação , Antituberculosos/farmacologia , Dimerização , Ácido Glutâmico/metabolismo , Glicolipídeos/isolamento & purificação , Glicolipídeos/farmacologia , Camundongos , Raízes de Plantas/química , Ratos , Vasodilatadores/isolamento & purificação , Vasodilatadores/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
In this paper, we present definitive data to show, from ESI (electrospray ionization) studies, that the thiol-bimane compound isolated and purified from Entamoeba histolytica trophozoites, corresponds unequivocally to the structure of trypanothione. Trypanothione disulphide was shown to have a molecular ion of m/z 722. It was further demonstrated by MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) MS that this thiol compound also corresponds to the characteristic monoprotonated ion of trypanothione-(bimane)(2), which has a molecular ion of m/z 1103.95. The ion pattern of the thiol-bimane compound prepared from the commercial trypanothione standard is identical with the E. histolytica thiol-bimane compound. After HPLC separation, chemical amino acid analysis by dabsylation and dansylation of the thiol bimane compound from Entamoeba showed the presence of the following trypanothione components: glutamic acid, cysteic acid, glycine and spermidine. We can conclude from these highly reliable MS experiments and chemical analyses that E. histolytica contains the thiol compound trypanothione, which was previously thought to occur only in trypanosomatids.