mdl-201053 and 4-(2-aminoethyl)benzenesulfonylfluoride

Third-stage larvae (L3) of Ascaris suum develop and molt to fourth-stage larvae (L4) during in vitro cultivation; consistently greater than 80% of the larvae develop to L4 during 7 days in culture (DIC). To assess the role of proteases in this process, the effect of protease class-specific inhibitors was studied. The presence of either a serine protease inhibitor (AEBSF, 100 microM) or an aspartic protease inhibitor (pepstatin A, 100 microM) had no effect on the percentage of L4 after 7 DIC. However, the presence of either a cysteine protease inhibitor (Z-Phe-Ala-FMK, 100 microM) or an aminopeptidase inhibitor (amastatin, 100 microM) resulted in 77% and 34% reductions, respectively, in the percentage of L4 compared to untreated cultures; viability of the larvae was not affected. The effect of Z-Phe-Ala-FMK on molting was time and dose dependent. In contrast to Z-Phe-Ala-FMK, E-64, another specific inhibitor of cysteine proteases, had no effect on molting. The data support a role for an aminopeptidase and suggest a role for a cysteine protease in the development of the L3 to L4 stage of A. suum.

Topics: Animals; Anti-Bacterial Agents; Ascaris suum; Coloring Agents; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Ketones; Larva; Movement; Pepstatins; Peptides; Protease Inhibitors; Sulfones; Swine; Tetrazolium Salts; Thiazoles; Trypsin Inhibitors

The incorporation of radioactivity from [3H]leucine-labeled hemoglobin (Hb) into adult Haemonchus contortus proteins was investigated. Further, the role of previously described cysteine proteases present in intestinal tissue and excretory/secretory products of H. contortus was assessed in the breakdown of Hb. A cell lysate preparation (predominantly Hb) was obtained from reticulocytes metabolically labeled, in vitro, with [3H]leucine. Following 24-h incubation in the presence of [3H]Hb, adult H. contortus incorporated radioactivity. The presence of the protein synthesis inhibitor puromycin (200 micrograms ml-1) reduced incorporation by 72%, indicating that this process was dependent on protein synthesis. The specific cysteine protease inhibitor Z-phe-ala-FMK (PAF) at 0.1 mM had no effect on incorporation of radioactivity; however, the breakdown of Hbg in the culture medium was reduced by 50%. In contrast, PAF at 1.0 mM caused a 78% reduction in incorporated radioactivity. Parasite viability was also decreased by 1.0 mM PAF, and thus the reduction of incorporation of radioactivity may not be due to specific enzyme inhibition. The serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) at 1.0 mM caused a 40% reduction in incorporation of radioactivity; the aspartic protease inhibitor pepstatin (1 mM) was without effect. Adult H. contortus also incorporated radioactivity from [3H]leucine-labeled intact reticulocytes. This incorporation was inhibited-by 1.0 mM PAF and AEBSF in a manner similar to that for the cell lysate preparation. These data indicate that adult H. contortus degrade Hbg and incorporate the radioactivity into their macromolecules. The specific action of the endogenous cysteine protease in the digestion of Hbg could not be demonstrated unequivocally. However, the hypothesis that the secreted cysteine protease functions in extracorporeal digestion was supported.

Topics: Animals; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; Haemonchus; Hemoglobins; Ketones; Pepstatins; Protease Inhibitors; Puromycin; Sulfones