RESUMO
Trichomonas vaginalis infection is associated with important problems of public health, including the spreading of other sexual transmitted infections. The existence of clinical resistant isolates metronidazole and tinidazole, the drugs approved for the treatment of trichomoniasis, points to the necessity of continue searching for trichomonicidal substances. Here we optimize a colorimetric assay with MTT to assess trichomonas viability. The absence of ascorbic acid and cysteine in the culture medium was indispensable to perform the assay, as these compounds spontaneously reduce the MTT. Linearity of absorbance was verified versus trichomonas counts. Medium inhibitory concentration of metronidazole was determined using the sigmoidal Emax model, by comparing the absorbance of test cultures with controls. The obtained value was in the range of published data. The test would be used for the evaluation of trichomonicidal activity of chemical compounds and natural products.
Assuntos
Antitricômonas/farmacologia , Colorimetria/métodos , Metronidazol/farmacologia , Trichomonas vaginalis/efeitos dos fármacos , Meios de Cultura/química , Feminino , Humanos , Testes de Sensibilidade Parasitária , Sais de Tetrazólio/química , Tiazóis/química , Tricomoníase/tratamento farmacológico , Tricomoníase/parasitologia , Trichomonas vaginalis/isolamento & purificaçãoRESUMO
The ACT-DHOD gene in the kinetoplastid Bodo saliens encodes aspartate carbamoyltransferase and dihydroorotate dehydrogenase, the second and fourth enzymes of pyrimidine biosynthesis. Although the single mRNA species yielded a 70-kDa ACT-DHOD protein, Western blotting with anti-DHOD-peptide antibody showed a major band of 35-kDa and minor bands. In-gel digestion and liquid chromatography-tandem mass (MS/MS) spectrometry showed that the 35-kDa band contained DHOD-specific polypeptides and an ACT-specific polypeptide, suggesting the occurrence of independent DHOD and ACT. Immunoprecipitation and MS/MS analysis identified a 70-kDa ACT-DHOD and a 35-kDa DHOD independently, and the N-terminal amino acid of 35-kDa DHOD was blocked. In vitro processing assay showed that recombinant ACT-DHOD was decreased by the B. saliens lysate, accompanying the appearance of 35-kDa DHOD and 35-kDa ACT. These results indicate that fused ACT-DHOD is the precursor to mature DHOD. Large amount of 35-kDa DHOD in B. saliens is discussed from a viewpoint of its physiological roles.
Assuntos
Aspartato Carbamoiltransferase/metabolismo , Fusão Gênica , Genes de Protozoários , Kinetoplastida/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Sequência de Aminoácidos , Animais , Aspartato Carbamoiltransferase/genética , Di-Hidro-Orotato Desidrogenase , Kinetoplastida/genética , Dados de Sequência Molecular , Família Multigênica , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Peptídeos/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em TandemRESUMO
Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses designated T. vaginalis virus (TVV), which may have important implications for trichomonal virulence and disease pathogenesis. We tested for TVV in 40 fresh T. vaginalis isolates from Cuban patients by total extraction of nucleic acids (DNA and RNA). TVV was detected in 22 (55 percent) of the 40 T. vaginalis isolates. This gives an estimate of the infection rate of Cuban T. vaginalis isolates by the dsRNA virus. Future research should focus on the association between trichomonosis symptoms and the presence of TVV.