vaginalis, the aim of this study was to characterize ADA activity, an enzyme involved in nucleoside metabolism, and to evaluate the relative mRNA expression of ADA-related genes in this

mucosal parasite. Trichomonas vaginalis clinical isolate TV-VP60 (Michel et al., 2006) was used throughout this enzyme characterization study. The other five isolates were TV-30236 (from the American Type Culture Collection, ATCC) and the clinical isolates TV-LACM1, TV-LACM2, TV-LACH1 and TV-LACH2 from our Clinical Laboratory surveys (Universidade Federal do Rio Grande do Sul, Brazil). Trichomonads were cultured axenically in vitro and maintained in trypticase–yeast extract–maltose (TYM) medium (Diamond, 1957), pH 6.0, supplemented with 10% (v/v) inactivated bovine serum at 37 °C. Organisms from the logarithmic phase were evaluated before and after assays based on motility and viability using trypan blue (0.2%) exclusion. The parasites were then harvested by centrifugation PI3K inhibitor and washed three times with phosphate-buffered saline (PBS) added with 2.0 mM EDTA and 2.0 mM EGTA. The final pellet was resuspended and used for the subsequent assays.

Trichomonas vaginalis lysates were obtained in liquid nitrogen, at 0.1 mg−1 protein−1 mL−1, in the presence of 1.0 mM protease inhibitor cocktail. An aliquot from the parasite suspension was added to the reaction mixture containing 50 mM sodium phosphate buffer (pH 7.5) to maintain the protein concentration (50–150 μg mL−1) in the final volume of 200 μL. The samples were then preincubated for 10 min at 37 °C. The selleck screening library reaction was initiated with the addition of the substrate adenosine (3.0 mM) and stopped, after a determined time (10–40 min), by adding the samples on 500 μL of phenol-nitroprusside reagent (50.4 mg of phenol and 0.4 mg of sodium nitroprusside mL−1). Controls with the addition of the enzyme preparation after the termination of reaction were used to correct nonenzymatic deamination of the substrate. The reaction mixtures were mixed with 500 μL of alkaline-hypochlorite reagent (sodium hypochlorite to 0.125% available chlorine, in 0.6 M PtdIns(3,4)P2 NaOH). Samples were incubated at 37 °C for 15 min. The colorimetric

assay was carried out at 635 nm (Giusti, 1974) to measure the ammonia produced by the enzymatic reaction and the ADA activity was expressed as nmol NH3 min−1 mg−1 protein. In all assays, at least three different experiments were performed in triplicate. The protein quantification was performed in triplicate for the parasite suspensions (Bradford, 1976) using bovine serum albumin as a standard. After the standardization of incubation time and the protein concentration in order to maintain the linearity of the enzymatic reaction, assays to determine the optimum pH were performed using 50 mM sodium phosphate buffer (mixture: 0.2 M disodium phosphate and 0.2 M sodium phosphate, pH 6.5–7.5) and sodium carbonate bicarbonate buffer (mixture: 0.2 M sodium carbonate and 0.