RESUMO
In the present work we characterized the secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This housefly parasite hydrolyzed p-nitrophenylphosphate at a rate of 10.26 nmol Pi/mg protein/min. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate promoted a decrease in this phosphatase activity. When the parasites were assayed in the presence of sodium tartrate, an inhibitor of Leishmania spp-secreted acid phosphatases, this activity was drastically diminished. Cytochemical analysis showed the localization of this enzyme on the external surface and in the flagellar pocket of these parasites. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor (PAF) inhibited the phosphatase activity determined in the supernatant of living H. m. muscarum.
Assuntos
Fosfatase Ácida/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Moscas Domésticas/parasitologia , Fator de Ativação de Plaquetas/farmacologia , Trypanosomatina/enzimologia , Animais , Meios de Cultura , Trypanosomatina/crescimento & desenvolvimentoRESUMO
The effects of platelet-activating factor (PAF) on the ecto-phosphatase activity of Trypanosoma cruzi were investigated. Living parasites hydrolyzed p-nitrophenyl phosphate (p-NPP) at a rate of 5.71 +/- 0.37 nmol P(i) mg(-1) min(-1). This ecto-phosphatase activity increased to 8.70 +/- 1.12 nmol P(i) mg(-1) min(-1) when the cells were grown in the presence of 10(-9) M PAF. This effect was probably due to stimulation of the release of the ecto-phosphatase and/or the secretion of an intracellular phosphatase to the extracellular medium, as suggested by cytochemical analysis. Modulation of the ecto-phosphatase activity was also observed when PAF was added during the time course of the reaction. WEB 2086, a competitive PAF antagonist, was able to revert PAF effects when both were used at the same concentration. When PAF was added to a membrane enriched fraction preparation of T. cruzi, no alteration on the phosphatase activity was observed. This result suggests an involvement of intracellular signaling, as PAF was only effective on intact cells. Sphingosine and phorbol-12-myristate-13-acetate (PMA) were then used to investigate a possible involvement of protein kinase C (PKC) with PAF-induced phosphatase secretion. Sphingosine by itself stimulated the secretion of a phosphatase but did not significantly interfere with PAF effects on this enzyme. On the other hand, PMA was able to abrogate PAF-induced release of this phosphatase. These data are highly suggestive of a putative involvement of signal transduction mediated by a ligand of mammalian origin (PAF), through PKC and a specific receptor located on the cell surface of the human parasite Trypanosoma cruzi.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Trypanosoma cruzi/efeitos dos fármacos , Animais , Azepinas/farmacologia , Meios de Cultivo Condicionados/química , Fosfoproteínas Fosfatases/análise , Fator de Ativação de Plaquetas/antagonistas & inibidores , Inibidores da Agregação Plaquetária/farmacologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Triazóis/farmacologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
In the present work ecto-phosphatase activity in Herpetomonas muscarum muscarum has been characterized using live parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 4.27 nmol Pi/mg of protein.min. A pH curve was generated, in which these intact flagellates showed the highest phosphatase activity at pH 6.5. Classical inhibitors for acid phosphatase, such as sodium orthovanadate, sodium tartrate, and ammonium molybdate, were used in the experiments and showed different patterns of inhibition. Lithium fluoride, aluminum chloride, and fluoroaluminate complexes were also tested. Although lithium fluoride and fluoroaluminate complexes were capable of inhibiting the phosphatase activity, aluminum chloride stimulated this enzyme. Cytochemical analysis showed the localization of this enzyme on the parasite surface. This ecto-phosphatase activity was also significantly diminished when the parasites were treated with 10(-6) M platelet-activating factor (PAF), a potent phospholipid mediator that promoted cellular differentiation in this parasite.
Assuntos
4-Nitrofenilfosfatase/antagonistas & inibidores , 4-Nitrofenilfosfatase/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Trypanosomatina/enzimologia , Animais , Diferenciação Celular/efeitos dos fármacos , Sistema Livre de Células , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hidrólise , Cinética , Trypanosomatina/citologia , Trypanosomatina/efeitos dos fármacosRESUMO
The effects of platelet-activating factor (PAF), at doses ranging from 10(-6) M to 10(-10) M, on cell growth and on cell differentiation of Herpetomonas muscarum muscarum were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF did not interfere with the protozoan growth. However, parasites grown in the presence of PAF (10(-6) M) were significantly more differentiated than those grown in the absence of PAF, since the first day of culture. On the first two days of culture, PAF doses ranging from 10(-10) M to 10(-7) M, did not significantly interfere with the differentiation of these parasites, although after the third day of culture, all PAF doses used significantly increased the protozoan differentiation. Specific PAF receptor antagonists totally abrogated (WEB 2086 and WEB 2170) or significantly decreased (BN 52021) PAF effect on cell differentiation. These findings indicate PAF triggers the process of cell differentiation in Herpetomonas muscarum muscarum and suggest these parasites have receptors for PAF.
Assuntos
Fator de Ativação de Plaquetas/farmacologia , Trypanosomatina/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Trypanosomatina/citologiaRESUMO
ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).
Assuntos
Adenosina Trifosfatases/metabolismo , Leishmania tropica/enzimologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , 4-Nitrofenilfosfatase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Leishmania tropica/metabolismo , Magnésio/farmacologia , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Especificidade por SubstratoRESUMO
The effects of platelet-activating factor (PAF), at (10)-6M and (10)-9M, on cell growth and cell differentiation of Trypanosoma cruzi were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF slightly interfered with the protozoan growth. However, parasites growth in the presence of PAF were significantly more differentiated than those grown in the absence of PAF, beginning on the fourth day of culture. A specific PAF receptor antagonist (WEB 2086) totally abrogated PAF effect on cell differentiation. These findings indicate that PAF triggers the process of cell differentiation in T. cruzi and suggest that these parasites have receptors for PAF.
Assuntos
Fator de Ativação de Plaquetas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Azepinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Cinética , Microscopia Eletrônica , Inibidores da Agregação Plaquetária/farmacologia , Triazóis/farmacologia , Trypanosoma cruzi/ultraestruturaRESUMO
PIP: The probabilities of survival of patients carrying the AIDS virus are presented in the city of Santos, Brazil, where it is estimated that 1056 cases were reported in 1988, 1989, and 1990. Records of epidemiological investigations delivered by outpatient services or obtained by means of active search realized routinely in city hospitals were analyzed. Information on deaths were derived from the offices of the civil registry of Santos, Sao Vincente, and Guaruja, and from the notifications received. Losses (deaths registered in offices of other regions) were estimated at less than 2%, the proportion of incidence in the general population according to data of the Foundation SEADE. The patients were grouped by sex and stage of progression of the disease according to classification of cases (IV B, IV C, IV D, and IV E conform to the classification of the Centers for Disease control) [CDC, suspected cases (class IV A of CDC), and asymptomatic seropositive cases (classes I, II, and II of CDC).^ieng