RESUMO
We investigated the effect of dehydroepiandrosterone sulfate (DHEAS) and 17beta-estradiol on NTPDase activity in fresh clinical (VP60) and long-term-grown (30236 ATCC) isolates of Trichomonas vaginalis followed by NTPDase gene transcriptional analysis. ATP hydrolysis was activated in vitro by 17beta-estradiol (0.01-1.0microM) in the VP60 isolate. Treatment for 2h with 17beta-estradiol (0.01-1microM) promoted an inhibition in nucleotide hydrolysis in the 30236 isolate whereas the 12h-treatment promoted an activation of nucleotide hydrolysis in both isolates. ADP hydrolysis was inhibited in vitro by 1.0-5.0microM DHEAS in the ATCC isolate. The treatment with DHEAS (0.01-1.0microM) for 2h inhibited ATP and ADP hydrolysis in VP60; however, during a 12h-treatment with DHEAS, nucleotide hydrolysis was inhibited in both isolates. Two NTPDase orthologous (NTPDaseA and NTPDaseB) were identified and the treatment with DHEAS for 12h was able to inhibit mRNA NTPDaseA transcript levels from the VP60. These findings demonstrate that NTPDase activity and gene expression pattern are modulated by exposure to steroids in T. vaginalis.
Assuntos
Sulfato de Desidroepiandrosterona/farmacologia , Estradiol/farmacologia , Estrogênios/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Nucleosídeo-Trifosfatase/metabolismo , Trichomonas vaginalis/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Análise de Variância , Animais , Humanos , Hidrólise/efeitos dos fármacos , Cinética , Camundongos , Dados de Sequência Molecular , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/efeitos dos fármacos , Nucleosídeo-Trifosfatase/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/genética , Trichomonas vaginalis/crescimento & desenvolvimentoRESUMO
Trichomonas vaginalis infection may be influenced by the vaginal concentrations of estrogens. We have investigated the effects of 17beta-estradiol and dehydroepiandrosterone sulfate (DHEAS) on the ecto-5'-nucleotidase activity in fresh clinical (VP60) and in long-term-grown (30236 ATCC) isolates of T. vaginalis. In vitro exposure to DHEAS and 17beta-estradiol did not induce any changes in adenosine monophosphate (AMP) hydrolysis in these isolates. The treatment of parasites in the presence of DHEAS (0.01-1.0 microM) for 2 h inhibited AMP hydrolysis in VP60 isolate, whereas there were no significant changes in nucleotide hydrolysis in the presence of 17beta-estradiol. DHEAS and 17beta-estradiol (0.01-1.0 microM) for 2 h inhibited AMP hydrolysis in 30236 isolate. The 12 treatment with 0.1 microM DHEAS inhibited AMP hydrolysis, whereas 17beta-estradiol did not alter the nucleotide hydrolysis in VP60 isolate. Our findings have shown that the complex effect of steroid hormones and their receptors on T. vaginalis may promote changes in ecto-5'-nucleotidase activity during exposure to these hormones.
Assuntos
Monofosfato de Adenosina/metabolismo , Hormônios/metabolismo , Nucleotidases/antagonistas & inibidores , Esteroides/metabolismo , Trichomonas vaginalis/efeitos dos fármacos , Trofozoítos/efeitos dos fármacos , Trofozoítos/metabolismo , Animais , Sulfato de Desidroepiandrosterona/metabolismo , Estradiol/metabolismo , Hidrólise , Trichomonas vaginalis/metabolismoRESUMO
Monocercomonas sp. is a flagellate protozoan found in the large intestine of snakes and in insects. Light microscopy revealed the measurements of morphological features of the trophozoites. Scanning electron microscopy showed in detail the emergence of the three anterior flagella, the recurrent flagellum, the axostyle, and the absence of undulating membrane. In addition, we described spherical forms which are probably pseudocysts. The investigation on the occurrence of this process was carried out through the incubation of Monocercomonas sp. trophozoites in several stressful conditions, such as pH change, nutrient depletion and different temperatures. Results revealed high pseudocyst formation at acidic pH values (4.0, 5.0, and 6.0), in absence of serum and in incubation at 37 degrees C. The occurrence of these pseudocystic forms in trichomonads life cycle is under investigation. This study describes the external structure of Monocercomonas sp., as demonstrated by light and scanning electron microscopy. Moreover, to our knowledge, this is the first time that formation of probable pseudocysts is shown in Monocercomonas sp., contributing to the research field on termite protozoa biology.
Assuntos
Trichomonadida/crescimento & desenvolvimento , Trichomonadida/ultraestrutura , Animais , Flagelos/ultraestrutura , Resposta ao Choque Térmico , Concentração de Íons de Hidrogênio , Estágios do Ciclo de Vida , Microscopia , Microscopia Eletrônica de Varredura , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/ultraestruturaRESUMO
This study describes the enzymatic properties of an ecto-5'-nucleotidase in Trichomonas gallinae. The enzyme hydrolyzes nucleoside monophosphates at pH 7.2 and is activated by divalent cations, such as magnesium. Ecto-5'-nucleotidase activity was insensitive to levamisole, tetramisole (alkaline phosphatase inhibitors), and AMPCP (adenosine 5'-[alpha,beta-methylene]diphosphate), an ecto-5'-nucleotidase inhibitor, whereas 0.1mM ammonium molybdate (considered a potent inhibitor of 5'-nucleotidase activity) completely inhibited the enzyme activity. The apparent K(M) (Michaelis constant) and Vmax (maximum velocity) values for Mg2+-AMP were 466+/-57 microM and 3.7+/-0.59 nmolPi/min/10(6) trichomonads, respectively. Considering that trichomonads lack the ability to synthesize purines and pyrimidines de novo, the presence of an ecto-5'-nucleotidase in intact trophozoites of T. gallinae could be important in regulating the extracellular nucleotide levels and generating adenosine, essential for the survival strategies of the parasite.