To investigate if incubation with heme or H2O2 affects cell viability, we used the highest concentration of the compounds tested and evaluated the cell integrity by the MTT assay

Soon after this concentration, there is a pronounced drop in H2O2 2-Pyrrolidinecarboxamide, N-[(2S)-2-hydroxy-2-phenylethyl]-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-, (2S,4E)- generation (Fig two).Fig 1. Hydrogen peroxide production for the duration of L. amazonensis proliferation days. The graph exhibits the hydrogen peroxide generation (shut circles) and the amount of cells current in the society medium on every of the times (open up circles). The values symbolize the indicate normal mistake of at least a few unbiased experiments. Statistically important when compared to day one (P < 0.05).We have shown that heme activates Na+/K+ ATPase in L. amazonensis [25]. Incubating intact promastigotes with 2.5 M heme, a concentration at which higher H2O2 levels were achieved, we confirmed the increase in Na+/K+ ATPase activity (Fig 3A). To investigate if H2O2 would be able to activate Na+/K+ ATPase, living promastigotes were incubated in the presence of increased concentrations of H2O2 and the Na+/K+ ATPase activity was assayed (Fig 3B). There is a sharp increase in Na+/K+ ATPase activity in response to low increased amounts of H2O2, with a peak of 1.16 0.06 NMOL PI X H-1 x mg protein-1 when the cells were incubated with 0.1 M H2O2 (Fig 3B). Concentrations of H2O2 above this threshold caused a decrease in the activation of Na+/K+ ATPase activity, indicating a small window where H2O2 could work as a signaling molecule in the enzyme activity.Fig 2. Effect of increasing concentrations of heme on the production of hydrogen peroxide by L. amazonensis. Living parasites were incubated for 20 min in the reaction medium with the addition of increasing concentrations of heme, as indicated on the abscissa. The values represent the mean standard error of at least three independent experiments. Statistically significant when compared to control (P < 0.05).Fig 3. Effect of heme and hydrogen peroxide on the Na+/K+ ATPase activity in L. amazonensis. The intact cells were incubated with (white bar) or without (black bar) 2.5 M heme (A) or with increasing concentrations of hydrogen peroxide (B) for 20 min. The values represent the mean standard error of at least three independent experiments. Statistically significant when compared to control (P < 0.05). CTRL, control.22991416To investigate if incubation with heme or H2O2 affects cell viability, we used the highest concentration of the compounds tested and evaluated the cell integrity by the MTT assay. Neither 50 M heme nor 2.5 M H2O2 were able to affect cell viability when compared to control (Fig 4). Triton X-100 was used as a positive control for non-viable cells.The generation of H2O2 is higher in the log phase than the stationary phase (Fig 1).