A. Jiménez-González

Proteolytic enzymes from Trichinella spiralis larvae

Trichinella spiralis larvae infect their hosts by the penetration of small intestine enterocytes. The exact mechanism of penetration is unknown, but the presence of proteolytic enzymes is suspected. In this study, whole worm extracts and excretory-secretory (ES) components were obtained and their proteolytic enzymes examined. Enzymes from worm extracts were capable of hydrolysing azocoll, a general protease substrate in a wide range of pH (2-8), with maximal activity at pH 5. Trichinella spiralis larval enzymes were sensitive to metalloprotease and serine protease inhibitors. Three proteases were identified in worm extracts at molecular weight (MW) 48, 54 and 62 kDa by incorporating a gelatine substrate into a standard or a modified sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) set-up, in which we used low SDS concentration in gel and electrophoresis buffer (0.01%). Intact larvae incubated in a medium containing azocoll showed azocollytic activity. Subsequent analysis of ES products by modified SDS-PAGE in gels containing gelatine demonstrated the presence of three protease of apparent MW 33, 62 and 230 kDa.

Purification and preliminary characterization of a protease from the excretion—secretion products of Trichinella spiralis muscle-stage larvae

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.

Interaction of ivermectin with γ-aminobutyric acid receptors in Trichinella spiralis muscle larvae

Abstract The value of the γ-aminobutyric acid (GABA) receptor of nematodes as a target for ivermectins mode of action remains unclear. Using binding assays, we examined extracts from Trichinella spiralis muscle larvae for the presence of [3H]-ivermectin and [3H]-GABA binding sites. Tissue preparations displayed affinity binding sites for [3H]-ivermectin with a dissociation constant (Kd) of 83 nM and a receptor density (Bmax) of 145 fmol/mg protein. We also identified a specific [3H]-GABA binding activity with a Kd of 1.2 μM and a Bmax of 4.78 pmol/mg protein. In competition studies, ivermectin was found to be a competitive inhibitor of specific [3H]-GABA binding activity with an inhibition constant (Ki) of 3.39 nM, suggesting that GABA receptors could be implicated in the mechanism of action of ivermectin in nematodes.

Preliminary characterization and interaction of tubulin from Trichinella spiralis larvae with benzimidazole derivatives.

Tubulin was estimated to account for 0.3% of the total soluble protein in Trichinella spiralis cytosolic fractions. Tubulin from T. spiralis was partially purified by precipitation with either taxol or vinblastine sulphate. Immunoblotting with alpha- and beta-tubulin monoclonal antibodies revealed the presence of tubulin in T. spiralis partially purified preparations. Electrophoretic mobility of T. spiralis tubulin in sodium dodecyl sulphate-polyacrylamide gels was very similar to that shown by pig brain tubulin. Further studies with colchicine binding assays indicated that T. spiralis tubulin has binding features similar to that of tubulin from other nematodes: colchicine association constant = 8.1 x 10(-4) M and competitive inhibition of colchicine binding by podophyllotoxin, with an inhibition constant of 1.3 x 10(-6) M. Finally, inhibition of colchicine binding by several benzimidazoles (mebendazole, fenbendazole, oxibendazole and albendazole) was investigated. All the benzimidazoles inhibited colchicine binding in a competitive manner, with inhibition constant values ranging from 1.4 x 10(-7) M (mebendazole) to 3.9 x 10(-6) M (fenbendazole).

Biochemical effects of luxabendazole on Trichinella spiralis

Biochemical changes produced by luxabendazole in muscle-stageTrichinella spiralis larvae consisted of a decrease in free glucose and glycogen levels (46.71% and 35.66%, respectively) after in vivo treatment, slight in vitro inhibition of fumarate reductase activity (24.15%) and, finally, inhibition of [3H]-colchicine-tubulin binding, which was found to be of a competitive nature, with an inhibition constant (Ki) of 0.9×10−7M. In a parallel study, luxabendazole did not appear to be inhibitory to [3H]-colchicine binding to pig-brain tubulin.

A comparative study of the succinate dehydrogenase–fumarate reductase complex in the genus Trichinella

Succinate dehydrogenase and fumarate reductase activities from a participate fraction of 4 isolates of the supposed ‘species’ of Trichinella, T. spiralis, T. pseudospiralis, T. nativa and T. nelsoni have been determined using spectrophotometric methods. The ‘ in vitro ’ effect of the anthelmintics thiabendazole, mebendazole and ivermectin on succinate dehydrogenase and fumarate reductase activities from the above isolates is also described. The significance of the results is discussed in the context of the controversy that surrounds speciation of the genus Trichinella .

Levamisole binding sites in Haemonchus contortus

Larval and adult extracts from isolates of Haemonchus contortus were assayed for specific [3H]levamisole binding activity. All of the tissue preparations displayed [3H]levamisole binding sites. The sensitive isolate SE and resistant isolate RJ showed no differences in larval and adult binding data. Larval SE extracts had higher receptor density (Bmax = 648 fmol mg-1) and dissociation constant (Kd = 1.28 microM) for [3H]levamisole than larval extracts of the American isolate RUSA (Bmax = 87 fmol mg-1 and Kd = 0.15 microM). Extracts of adult SE and RUSA isolates contain as much as 327 fmol mg-1 of protein and 205 fmol mg-1 of protein, respectively, and similar dissociation constants (Kd = 0.77 microM and Kd = 0.81 microM, respectively). There was a good correlation between specific binding activity of larval and adult extracts in both SE and RUSA isolates. The nicotinic cholinergic antagonist alpha-bungarotoxin had no effects in either isolate on [3H]levamisole binding activity. The results confirm that levamisole acts at a cholinergic receptor in H. contortus, and suggest that target site modification could be involved in the development of levamisole resistance.

Actin isoforms in the parasitic nematodeHaemonchus contortus

Actin proteins in the parasitic nematodeHaemonchus contortus were partially purified by a new method based on precipitation with podophyllotoxin and dimethylsulfoxide. Two-dimensional gel electrophoresis of partially purified actin proteins revealed differences in isoform composition between adults and third-stage larvae. The significance of this finding is possibly related to the existence of differential functional or developmental characteristics between these two life-cycle stages.

Characterization of levamisole binding sites in Trichinella spiralis

Characterization of the levamisole receptor was performed with total extracts of Trichinella spiralis muscle larvae using binding assays with tritiated levamisole ([3H]LEV, 291 GBq/mmol). We detected a specific [3H]LEV binding activity with a dissociation constant (Kd) of 4.76 μM and a receptor density (Bmax) of 2.14 pmol/mg of protein. In inhibition studies, only dimethylphenylpiperazinium iodide (DMPP) and hexamethonium were found to be competitive inhibitors of the [3H]LEV binding with an inhibition constant (Ki) of 31.04 and 4.43 μM, respectively, whereas d-tubocurarine and α-bungarotoxine had no effect on [3H]LEV binding activity, and procaine and atropine potentiated the [3H]LEV-receptor binding. All these data support the idea that levamisole acts as a cholinergic agonist in T. spiralis.