2 M glycine solution, pH 10.7, and the optical density determined at 405 nm in an ELISA reader (Labsystems Multiskan Ex). The extent of secretion was expressed as the see more net percentage of the total β-hexosaminidase activity in the supernatant of unstimulated cells. The results represent the mean of quadruplicate tests ± standard deviation (SD). Medium 199 was used for the cultivation of promastigotes

of Leishmania major (MHOM/SU/73/5ASKH). Promastigotes were cultured in the medium [supplemented with heat-inactivated (56 °C for 30 min) fetal bovine serum (10%)] at 27 °C, in a 5% CO2 atmosphere in an incubator ( Takahashi et al., 2004). The leishmanicidal effects of the peptides were assessed using the improved 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrasodium bromide (MTT) method as follows. Cultured promastigotes were seeded at 4 × 105/50 mL of the medium per well in Nutlin 3a 96-well microplates, and then 50 mL of different concentrations of test compounds dissolved in a mixture of DMSO and the medium were added to each well. Each concentration was tested in triplicate. The microplate was incubated

at 27 °C in 5% CO2 for 48 h. TetraColor ONE (10 mL) a mixture of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt and 1-methoxy-5-methylphenazinium methosulfate was added to each well and the plates were incubated at 27 °C for 6 h. Optical density values (test wavelength 450 nm; reference wavelength 630 nm) were measured using a microplate reader (Thermo BioAnalysis Japan Co., Ltd., Kanagawa, Japan). The values of 50% inhibitory concentration of the peptides were estimated from the dose–response curve. The venom extracts of E. rubrofemoratus were subjected to reversed-phase HPLC, and the purity and complexity of each fraction was triclocarban examined by MALDI-TOF MS. The HPLC profile was rather simple, having only several intense peaks ( Fig. 1A). The two major fractions eluted at 26.1 and 27.6 min showed a high purity with protonated molecular ion peaks at m/z 1623.9 and 1474.9 (MH+, monoisotopic), respectively. The molecular weight and chromatographic behavior

suggested these components to be peptides, which we named eumenitin-R and eumenine mastoparan-ER (EMP-ER), respectively. The sequence of the peptides was analyzed first by MALDI-TOF/TOF MS. Eumenitin-R had a sequence of 15 amino acids as I/L-N-I/L-K/Q-G-I/L-I/L-K/Q-K/Q-V-A-S-I/L-I/L-N, which was consistent with the observed molecular mass. However, contrary to expectation, there was no d or w ions observed, and therefore, no information about the I/L and K/Q differentiation. Accordingly, the sequence was determined by Edman degradation using an automated sequencer, giving whole sequence as L-N-L-K-G-L-I-K-K-V-A-S-L-L-N. The solid-phase synthesis of this peptide and the HPLC comparison of the synthetic specimen with the natural peptide finally corroborated the sequence.