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
Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides.
Assuntos
Inibidores Enzimáticos/farmacologia , Penicilinas/farmacologia , Pirofosfatases/metabolismo , Estreptomicina/farmacologia , Trichomonas/efeitos dos fármacos , Trichomonas/enzimologia , Animais , Cálcio/farmacologia , Ativadores de Enzimas/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Magnésio/farmacologia , Nucleosídeos de Purina/metabolismo , Nucleosídeos de Pirimidina/metabolismo , Azida Sódica/farmacologia , Especificidade por Substrato , Trofozoítos/efeitos dos fármacos , Trofozoítos/enzimologiaRESUMO
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
A doença de Chagas é a infecção tecidual e hematológica causada pelo protozoário flagelado Trypanosoma cruzi, sendo importante causa de mortalidade de adultos jovens. O diagnóstico da doença de Chagas depende comumente do resultado de provas sorológicas, sendo utilizados testes usando antígenos T. cruzi: hemaglutinação passiva (HA), imunofluorescência indireta (IFI) e enzimaimunoensaio(ELISA). O objetivo deste estudo foi verificar a soroprevalência da doença de Chagas em 8228 doadores de sangue do Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul de Porto Alegre (RS), bem como averiguar a qualidade técnica dos testes utilizados e sua concordância. A prevalência de amostras positivas para T. cruzi foi de 0,4% na comunidade analisada, sendo que o teste de ELISA apresenta sensibilidade de 100%, especificidade de 99,9%, eficiência de 99,9% e valor preditivo negativo de 100%. O coeficiente Kappa achado entre os testes de ELISA e HA foi de 99%, mostrando uma concordância quase perfeita. Neste estudo mostra-se a grande qualidade do teste de ELISA, que se apresenta como um ensaio muito adequado para a sorologiaanti-T. cruzi, já que a correta identificação dos portadores da doença de Chagas é de importância ímpar no que tange à saúde pública e sobretudo à triagem de doadores em bancos de sangue.
Chagas disease is a tissue and hematological infection caused by flagellate protozoan Trypanosoma cruzi, an important cause of mortality in young adults. The diagnosis of Chagas disease depends of the results of serological tests, being utilized tests using antigens T. cruzi: indirect hemaglutination (HA), indirect immunofluorescence (IFI) and enzyme-linked immunosorbent assay (ELISA). The aim of this study was verify the seroprevalence of Chagas disease in 8228 blood donors of São Lucas Hospital of Pontifícia Universidade Católica of Rio Grande do Sul in Porto Alegre (RS), as well like ascertain the technical quality of the tests utilized and its agreement. The prevalence of positive samples for T. cruzi was of 0,4% in the community analyzed, being that the test of ELISA presents sensibility of 100%, specificity of 99,9%, efficiency of 99,9% and negative predictive value of 100%. The coefficient Kappa found between the tests of ELISA and HA was of 99%, showing a barely perfect agreement. In our study we show to big quality of the test of ELISA, which is presented like a very adequate assay for the serologic investigation anti-T. cruzi, since to correct identification of the porters of the Chagas disease is of unequal importance in what plays the public health and to the selection of givers in banks of blood.
Assuntos
Humanos , Bancos de Sangue , Doença de Chagas , Ensaio de Imunoadsorção Enzimática , Prevalência , Estudos Soroepidemiológicos , Trypanosoma cruziRESUMO
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.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/metabolismo , Antitricômonas/farmacologia , Inibidores Enzimáticos/farmacologia , Tricomoníase/veterinária , Trichomonas/enzimologia , Monofosfato de Adenosina/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Animais , Cálcio/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Levamisol/farmacologia , Magnésio/metabolismo , Tetramizol/farmacologia , Tricomoníase/parasitologiaRESUMO
Here we report the effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis was 5- to 7-fold higher for the fresh clinical strain, when compared with the ATCC (American Type Culture Collection) strain. ATP hydrolysis was activated in the presence of metronidazole in the ATCC strain, whilst it was inhibited 33% by 50 microM tinidazole in a fresh clinical isolate. The treatment of cells in the presence of metronidazole for 2 h inhibited ATP and ADP hydrolysis, whilst treatment with tinidazole inhibited ATP and ADP hydrolysis only in the fresh clinical isolate. The drugs did not change the ecto-5'-nucleotidase activity for both strains. Our results suggest that the modulation of extracellular ATP and ADP levels during treatment with these drugs could be a parasitic defence strategy as a survival mechanism in an adverse environment.