1. Dirofilaria immitis: effect of fluoromethyl ketone cysteine protease inhibitors on the third- to fourth-stage molt
J K Richer, W G Hunt, J A Sakanari, R B Grieve Exp Parasitol. 1993 May;76(3):221-31. doi: 10.1006/expr.1993.1027.
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.
2. Partial purification and some properties of tryptophan decarboxylase from a Bacillus strain
K G Büki, D Q Vinh, I Horváth Acta Microbiol Hung. 1985;32(1):65-73.
Bacteria of different origin were screened for tryptophan decarboxylase activity. The best producer belonged to an unidentified taxonomic entity of the genus Bacillus. In complete medium it produced tryptamine from tryptophan. The decarboxylase could partially be purified from the cells by sonication and DEAE-cellulose chromatography. The enzyme had an Mr of 150 000 and a pH optimum of about 7, was stable up to 37 degrees C, and its Km was about 0.3 mM for tryptophan. The enzyme needed pyridoxal phosphate for maximum activity.
3. Metabolic labeling of Dirofilaria immitis third- and fourth-stage larvae and their excretory-secretory products
G R Frank, R B Grieve J Parasitol. 1991 Dec;77(6):950-6.
Infective third-stage larvae of Dirofilaria immitis were collected from Aedes aegypti and cultured in vitro to the fourth stage. Larval proteins were labeled metabolically using [35S]cysteine and methionine in different media and for different lengths of time. Labeled proteins in the excretory-secretory component and the larval homogenates were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and by 2-dimensional gel electrophoresis. Numerous proteins ranging from 14 to greater than 200 kDa were identified from both the excretory-secretory components and the larval homogenates. Both fractions demonstrated shared and unique proteins. Using timed labeling, age- and stage-specific proteins were identified; at least 2 proteins of approximately 20.5 and 22 kDa were associated in time with the molt from the third to fourth stage. Two proteins of the same molecular weight were specifically recognized by immune dog sera, but not by sera of their infected nonimmune cohorts.
