| pubmed-article:8500582 | pubmed:abstractText | D. immitis third-stage larvae (L3) were cultured with fluoromethyl ketone cysteine protease inhibitors. By Day 5 in culture, none of the larvae cultured with 0.1, 0.2, 0.6, or 1.0 mM benzyloxycarbonyl-Phe-Ala-CH2F (Z-Phe-Ala-CH2F) has molted, while 63.2% of larvae in media without inhibitor had molted. At the two lower concentrations of inhibitor more larvae had initiated, but not completed, the molt. In addition to Z-Phe-Ala-CH2F, four other fluoromethyl ketone derivatives, Z-Phe-Arg-CH2F, amorpholine urea-(Mu)-Leu-Phe-CH2F, Mu-Tyr-Phe-CH2F, and Mu-Phe-Phe-CH2F, were tested to determine their effects on L3 in culture. All fluoromethyl ketones tested except Z-Phe-Arg-CH2F inhibited molting. Larvae cultured in inhibitors were determined to be alive as judged qualitatively by motility and quantitatively by reduction of 3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium. Electron microscopy demonstrated that L3 which were unable to molt after being cultured in a fluoromethyl ketone derivative had synthesized the new fourth-stage (L4) cuticle but had not shed the L3 cuticle. The same fluoromethyl ketone derivative that did not inhibit molting, Z-Phe-Arg-CH2F, was a slightly less effective inhibitor of larval extract-initiated hydrolysis of the synthetic peptide substrate, Z-Val-Leu-Arg-7-amino-4-methyl coumarin. L3 were also cultured through the molt in media containing the synthetic peptide substrate Z-Val-Leu-Arg-4- methoxy-B-naphthylamide to examine cysteine protease activity in situ. Fluorescence as seen on Days 0-4 during the molting process was first observed on the anterior tip of the larvae, and subsequently in the pharynx, with progression down the L4 as it shed the L3 cuticle. | lld:pubmed |
