The activation of ADR is arginine concentration-dependent.

A. The minimal arginine concentration required for activation of ADR in axenic promastigotes. The promastigotes were cultured either in M199 medium containing 0.45 mM arginine or in arginine deprived M199 medium containing different concentrations of arginine (0.5 μM, 5 μM and 50 μM). The promastigotes were cultured in the respective medium for two hours, the total RNA was isolated and Northern blot analysis was carried out. Changes in the LdAAP3 and LdPT expression levels were determined by gene specific probes as described in Materials and Methods. Data are representative of three independent experiments. The two asterisks (**) in the figures refer to the two transcripts (5-and 7-kb) synthesized from the LdBPK_310910.1 gene. B and C. The minimum concentration of arginine required for ADR activation in intracellular amastigotes. THP-1 cells were infected with L. donovani in RPMI medium containing 0 (actual arginine concentration in RPMI medium is 0.008 mM), 0.1 mM, 0.5 mM or 1.5 mM arginine for 48 h. The total RNA was then extracted, and the resulting cDNA was subjected to real-time PCR analysis using primers specific for LdAAP3 (B) and LdPT (C). The results are expressed as fold-change of control (2 h infected cells). Values are mean ± S.E.M. The results are representative of three independent experiments performed in triplicates. **, p < 0.001–0.01. The real time PCR primers do not amplify any products (LdAAP3 or LdPT) in uninfected macrophages. D and E. Time-course of ADR. THP-1 cells were infected with L. donovani in RPMI medium containing 0.1 mM arginine for 2 h, 24 h and 48 h. The total RNA was then extracted, and the resulting cDNA was subjected to real-time PCR analysis using primers specific for LdAAP3 (D) and LdPT (E). The results are expressed as fold-change of control (2 h infected cells). Values are mean ± S.E.M. The results are representative of three independent experiments performed in triplicates. *, p < 0.01–0.05, **, p < 0.001–0.01, ***, p < 0.0001–0.001. The real time PCR primers did not amplify any products (LdAAP3 or LdPT) in uninfected macrophages.