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The presence of cholinesterases (ChE) is reported in T. pseudospiralis excretion-secretion products (ESP) by spectrophotometry method, using acetylthiocholine (ATCI) and butyrilthiocholine (BTCI) as substrates. By inhibition assays, we found that T. pseudospiralis release both acetyl- and butiryl-cholinesterases (AchE and BchE, respectively). The sedimentation coefficientes of these enzymes were determined by sucrose density gradient. We studied the in vivo ChE secretion by immunoblot assays using AchE from Electrophorus (electric eel) and sera from normal or infected mice with T. pseudospiralis or T. spiralis. The presence of anti-AchE antibodies was only demonstrated in the sera from T. pseudospiralis infected mice. Moreover the in vivo secretion was corroborated by the high difference determinate between the ChE activity of the immuno complexes from T. pseudospiralis infected sera and the immunocomplexes from T. spiralis infected sera as well as normal sera. Finally, we analyzed the effect of the organophosphate NeguvónR (metrifonate) on the ChE activity from the J. pseudospiralis ESP. The drug inhibits in part this activity. Moreover NeguvónR (metrifonate) showed a high activity against the T. pseudospiralis viability.

Cholinesterases were detected in protoscolices of Echinococcus granulosus spectrophotometrically and electrophoretically. To characterize these activities as acetylcholinesterases or pseudocholinesterases, BW284C51 and the organophosphate anthelmintic Neguvón® were assayed as specific inhibitors of acetylcholinesterases, while Iso-OMPA was employed as specific inhibitor of pseudocholinesterases. We concluded that these Cholinesterase (ChE) activities would be considered as possible targets in chemotherapy.

A preliminary purification has been carried out by continuous elution electrophoresis of a 49.5 kDa protease of crude extracts from Dicrocoelium dendriticum eggs. The enzyme showed a high capacity to degrade the collagen derivative azocoll at acidic pH. Although it is necessary to carry out further experiments to confirm any physiological role, this protease could be implicated in penetration mechanisms.

In the present study we report the presence of acetylcholinesterase activity and gamma-aminobutyric acid binding sites in crude extracts of Dicrocoelium dendriticum. This indirectly demonstrates the presence of acetylcholine and GABA. The presence of these neurotransmitters could indicate the existence of two systems implicated in the neurotransmission of the Digenea.

In the present report we study the proteolytic activity of the excretion-secretion and crude extracts of different stages of Trichinella spiralis (Owen, 1835) Railliet, 1895, (muscle-stage larvae, adult worms before and after mating, and newborn larvae) using natural substrates (structural and hematic mammalian proteins). The analysis of the results allow us to set up a certain stage-specificity, as well as an important relationship between the protease patterns throughout the parasite life cycle and how the parasite may overcome both mechanical and humoral barriers within the host. Muscle-stage larvae present a great activity against structural proteins (collagen), while newborn larvae and adult worms degrade principally hematic proteins (hemoglobin, fibrinogen and immunoglobulin G).

A protease from excretion-secretion products of Trichinella spiralis muscle-stage larvae was purified by continuous elution electrophoresis. The state of purification was analyzed electrophoretically using one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme was shown to be a single polypeptide with an estimated molecular mass of 35 kDa and isoelectric point of 6.2. Following purification, the enzyme activity was measured by hydrolysis of gelatin, azocoll, azoalbumin, azocasein and collagen as substrates. Maximal azocollytic activity was at pH 5 and a temperature of 37 degrees C. Finally, the proteolytic activity was partially inhibited by N-alpha-p-tosyl-lysine chloromethyl ketone, chymostatin and E-64.

In the present study we analyzed the humoral response of Trichinella spiralis-infected mice to a 35-kDa protease (purified from the excretory-secretory products of T. spiralis muscle larvae) by a Western-blot procedure and an enzyme-linked immunosorbent assay (ELISA) technique using a panel of postinfection mouse anti-Trichinella sera. The results demonstrated that this response was time-dependent and that infected mice could be distinguished from controls. In addition, inhibition assays demonstrated that these antisera were capable of abolishing the proteinase activity of the 35-kDa protease in vitro. The occurrence of proteases seems to be a very common feature in parasite crude extracts and excretory-secretory products (McKerrow 1989). It is also known that these enzymes are implicated in important host-parasite interactions, and for this reason, recent reports have proposed the use of parasite proteases both as alternative targets for an induced immune response and as a rich source of antigenic material for diagnostic testing (Hotez et al. 1985; Yamasaki et al. 1989; Song et al. 1990; Frank and Grieve 1991; Britton et al. 1992; Song and Chappell 1993). We have recently purified a protease (mol. wt., 35 kDa) from the excretory-secretory (ES) products of Trichinella spiralis (GM-1 strain) muscle larvae and established some of the biochemical properties of this protease (Armas-Serra et al. 1994).(ABSTRACT TRUNCATED AT 250 WORDS)