3). Furthermore, the EHNA inhibition was long lasting, because no activity could be detected after passage in culture medium 1 and 6 h after the EHNA treatment (Table 1). The low ADA activity detected after 24 h (0.27 ± 0.05 nmol NH3 min−1 mg−1 protein) was probably due to new trophozoites grown after the incubation in the culture medium. We have evaluated the interaction of EHNA-treated T. vaginalis on NO production by human neutrophils stimulated with T. vaginalis. Figure 4 shows that neutrophils alone produced low levels of NO (1.98 ± 0.35 μM); however, when stimulated
with lipopolysaccharide (positive control), the concentration increased 35 times (70.26 ± 14.69 μM). When the trichomonad-culture supernatants from EHNA-treated Saracatinib nmr trichomonads and the T. vaginalis lysate were incubated with neutrophils, both conditions inhibited the NO production. On the other hand, and expectedly, the co-culture with intact T. vaginalis trophozoites produced a high CP 690550 amount of NO. However, when incubated in the presence of 1 h EHNA-treated parasites, the NO production effect was reverted. The same effect was observed with adenosine and inosine. In order to identify the ADA-related sequences on T. vaginalis genome, we performed a phylogenetic analysis. NCBI blast searches
of GenBank yielded two complete T. vaginalis ADA-related sequences (XP_001317231 and XP_001325125). Semi-quantitative RT-PCR experiments were performed and the relative abundance of ADA-related genes ada(125) and ada(231) mRNA vs. α-tubulin was determined by densitometry. As shown in Fig. 5a and b, both genes were expressed, although ada(231) in higher quantity when compared with the ada(125) : α-tubulin ratio. The phylogenetic tree was constructed using the neighbor-joining method and proportional (p) distance
(Fig. 5c). Four well-resolved terminal clades supported by high bootstrap values were identified, confirming the presence of two ADA orthologues for T. vaginalis. The first clade grouped consistently ADA1 vertebrate sequences and ADA-related sequence from T. spiralis. The second clade was formed BCKDHA by E. histolytica, D. discoideum and T. vaginalis sequences. The third clade grouped the ADAL sequences, whereas the fourth clade was formed by ADA2 sequences. Plasmodium falciparum and L. major ADA-related sequences were placed independently between the four clades mentioned. Trypanosoma brucei and E. coli were the most divergent sequences. The tree topology strongly suggests homologous functions on the T. vaginalis genome. In order to screen freshly isolated clinical isolates besides TV-VP60, we have determined ADA activity in five other T. vaginalis isolates.