Immunolocalization of arginine kinase (AK) in Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides.

Arginine kinase (AK) is a member of the phosphagen kinase family. AK plays a major role in cellular energy metabolism in invertebrates including nematodes. In the present study, we performed the direct immunofluorescence test to determine the immunolocalization of AK in different stages of the life cycle (eggs, larvae, and adult worms) of Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides. Our results indicated variable levels of expression of AK in different stages. Moreover, strong fluorescence was observed in cleaving eggs than in dormant eggs. The highest activity of the enzyme was observed in the fully developed eggs. This may be due to high expression of AK in embryonic development, which is associated with increased energy demand due to cleavage and cellular differentiation. Surprisingly, expression of AK is significantly higher in the middle part and posterior end compared to anterior end of the larvae. In addition, AK is highly concentrated in cellular and metabolically active parts of the body such as hypodermis, muscle, intestine, ovaries, oviducts, and uterus, while it is absent in noncellular areas like cuticle. The present study revealed the presence of AK in T. canis, A. lumbricoides, and T. vitulorum and that it plays a major role in energy metabolism of these nematodes. Interestingly, antiserum was prepared against the recombinant T. canis AK and reacts with the native AKs of T. canis, A. lumbricoides, and T. vitulorum. AK levels could vary in relation to maximum potential rates of ATP turnover, oxidative capacity, and energy output. Further studies on subcellular localization of AK in these important helminths provide new information for researchers to develop effective anthelmintics against the parasites of veterinary and of public health importance.

An alternate technique for isolation of Toxocara canis excretory-secretory antigens / Uma técnica alternativa para o isolamento de Toxocara canis excreção e secreção de antígenos

The aim of the present study was to test the effectiveness of a sausage-casing membrane for dialysis of Toxocara excretory-secretory antigens (TES). The protein concentrated by the tested membrane was compared with that obtained using a Sigma commercial membrane, as were the protein fractions found by polyacrylamide gel electrophoresis. Standard positive and negative serum samples were evaluated in an ELISA immunoassay, and equivalent data were obtained in all steps, indicating that the sausage-casing membrane is efficient, besides being less expensive to process.

O objetivo do presente estudo foi testar a eficácia de uma membrana utilizada para o preparo de embutidos, na obtenção do antígeno de excreção e secreção de Toxocara (TES). A concentração protéica foi comparada com a obtida com a membrana Sigma tanto quanto as frações protéicas separadas por eletroforese em gel de poliacrilamida. Amostras de soros padrão positivo e negativo foram avaliadas no teste imunoenzimático ELISA. Dados equivalentes foram observados em todas as etapas, sugerindo que a membrana possa ser utilizada para diálise por ser eficiente e de menor custo no preparo do antígeno.

Kinetics of the reduction of oxaloacetate catalyzed by mitochondrial malate dehydrogenase of Toxocara canis muscle.

1. The reaction of reduction of oxaloacetate to L-malate in the presence of NADH catalyzed by mitochondrial malate dehydrogenase (EC 1.1.1.37) of Toxocara canis muscle has been studied. 2. The data obtained in initial velocity experiments as well as those involving product inhibition suggest that the reaction mechanism is of the sequential type with a kinetically significant ternary complex and in which the coenzymes bind to the free enzyme. 3. The kinetic parameters, including the inhibition constant for NADH were estimated by non-linear regression analysis using the appropriate rate equations.

Effects of temperature on the mitochondrial malate dehydrogenase of adult muscle of Toxocara canis.

Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.

Toxocara canis: proteolytic enzymes secreted by the infective larvae in vitro.

Second-stage larvae of the dog nematode Toxocara canis are infective to man and cause the syndromes of visceral larva migrans and ocular toxocariasis. Larvae cultured in vitro secrete proteases which degrade components of a model of extracellular matrix and basement membranes. These enzymes have been characterized using a variety of techniques. Multiple enzyme activities were demonstrated by substrate gel electrophoresis, associated with proteins of molecular weights of 120 and 32 kDa. The enzyme activity was inhibited both in substrate gels and in a radiogelatin microplate assay by phenylmethylsulfonyl fluoride. Optimal activity occurred at pH 9, with minor activities apparent at pH 5 and 7; the relationship between these proteolytic activities is currently under investigation.

The secreted and somatic antigens of the third stage larva of Anisakis simplex, and antigenic relationship with Ascaris suum, Ascaris lumbricoides, and Toxocara canis.

The in vitro-released 'excretory/secretory' (ES) and somatic antigens of the third stage (infective) larva of Anisakis simplex were characterised by radioiodination, immunoprecipitation, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Oral infection with the parasite elicited a substantial IgG antibody response to ES in infected rabbits and humans, with a minimal response to somatic materials. Serial serum sampling in experimental infection showed that there was a sequential recognition of distinct ES components. In contrast to oral infection, intraperitoneal exposure of rats with living parasites induced a strong response to both ES and somatic antigen preparations. Sequential recognition of ES antigens, and differential responses to somatic components, might, therefore, have application in the estimation of the age and degree of penetration by the nematodes in human infection. Extensive antigenic relationships were found between A. simplex and three other species of ascaridoid nematodes, namely Ascaris lumbricoides, Ascaris suum, and Toxocara canis, but none with a panel of non-ascaridoid nematodes. Evidence is presented that a Mr 14,000 component of A. simplex has a homologue in all of the ascaridoids examined, but does not elicit an antibody response in anisakiasis. Finally, the ES of A. simplex is shown to contain two proteinase activities, of approximately Mr 23,400 and 46,100, as revealed by separation on gelatin substrate gels, although the antigenicity of the enzymes remains to be established.

Superoxide dismutase activity in nematodes.

The activities of the enzymes superoxide dismutase (E.C.:1.15.1.1) and catalase (E.C.:1.11.1.6) were studied in purified extracts of four nematodes: Ascaris suum, Toxascaris leonina, Toxocara canis and T. cati adult males and females. No catalase activity was found in any of the extracts. The results reveal that the SOD activities of the four parasites presented species differences and also sexual differences within each species. Polyacrylamide gel electrophoresis pattern analysis confirmed that the mobilities, widths and band intensities varied according to the species and sex of the parasite from which the enzyme was obtained.

Biochemical and immunological systematics of some ascaridoid nematodes: genetic divergence between congeners.

Vertical starch gel electrophoresis and trefoil immunodiffusion were used to study the systematics of some ascaridoid nematodes. Within the Ascarididae, the time scale of divergence was too great for intergeneric electrophoretic comparisons. Congeneric electrophoretic comparisons of Baylisascaris procyonis (host--raccoon) versus Baylisascaris transfuga (host--black bear), and Toxocara canis (host--domestic dog) versus Toxocara cati (host--domestic cat) yielded Nei genetic distance coefficients of 1.21 and 1.55, respectively. Estimates of times of divergence made from 1 electrophoretic clock calibration suggest that the Baylisascaris species have not shared a common ancestor for 25 million years (Myr), and that the Toxocara species diverged 33 Myr ago. The Baylisascaris divergence estimate corresponds to host-family divergence estimates based on immunological and paleontological evidence, which suggests that cospeciation has occurred. In contrast to this, Ascaris suum (host--pig) and Ascaris lumbricoides (host--human) have a distance coefficient of 0.09. This indicates that these species diverged comparatively recently and may represent a case of host range expansion. Trefoil immunodiffusion comparisons of ascaridoid albumins yielded reactions of identity for A. suum, A. lumbricoides, Parascaris equorum, B. procyonis, B. transfuga, T. canis, and T. cati. This confirms that these taxa are members of a monophyletic group.

Biochemical polymorphism in Parascaris equorum, Toxocara canis and Toxocara cati.

Vertical starch gel electrophoresis was used to resolve proteins encoded by 18 gene loci in ascaridoid nematodes. Estimates of genetic variability were made from population samples of the dog ascarid (Toxocara canis), cat ascarid (Toxocara cati), and the horse ascarid (Parascaris equorum). Levels of polymorphism and mean heterozygosity were high, which is not consistent with the hypothesis that the intestinal environment selects for monomorphism among endoparasites. Most observed allele frequencies conformed to Hardy-Weinberg equilibrium expectations as tested by chi2 goodness-of-fit, suggesting that the proteins evaluated are inherited in a Mendelian fashion and that these nematodes are mating at random. Subunit structures of the following enzymes, deduced from electrophoretic phenotypes of heterozygotes, corresponded to those of vertebrates: lactate dehydrogenase; malate dehydrogenase; 6-phosphogluconate dehydrogenase; phosphoglucomutase; esterase D; peptidase B; peptidase D; and mannose-6-phosphate isomerase. This observation substantiates the conservative nature of polypeptide subunit number across phylogenetically diverse groups of organisms.

Purification and properties of mitochondrial malate dehydrogenase of Toxocara canis muscle.

Mitochondrial malate dehydrogenase was purified from muscle extracts of Toxocara canis by means of Sephadex G-100 gel filtration, DEAE-Sephadex ion-exchange chromatography and 5'AMP-Sepharose 4B affinity chromatography. The purified enzyme showed an optimum pH for the reduction of oxaloacetate of 7.3 in Tris-HCl buffer and of pH 7.5-7.8 in phosphate buffer. The m-MDH showed values of 3.2 kcal/mol and 10.5 kcal/mol for the energy of activation, calculated from the Arrhenius equation. The mitochondrial enzyme was found to be more susceptible to thermal inactivation as compared with the cytosolic isoenzyme. Kinetic experiments showed that the m-MDH of Toxocara canis is inhibited by excess oxaloacetate but not by excess NADH. The apparent Km for oxaloacetate reduction was 53 microM and 0.54 mM for L-malate oxidation.

Cytosolic malate dehydrogenase in muscle extracts of Toxocara canis.

1. Malate dehydrogenase (L-malic acid:NAD+ oxydoreductase, EC 1.1.1.37) was partially purified from muscle extracts of Toxocara canis by means of gel chromatography in Sephadex G-150 and affinity chromatography in Sepharose-4B-Blue dextran. 2. The purified enzyme was very active in reducing oxalacetate and less active in oxidizing L-malate. It was inhibited by excess oxalacetate but not by L-malate. 3. The kinetic parameters of the enzyme were obtained and these included: pH and temperature optima and apparent Michaelis constants for the substrates. 4. The results suggest that the enzyme from Toxocara canis behaves like the enzyme of the model helminth Ascaris lumbricoides.

A comparison of the sites of acid phosphatase activity in an adult filaria, Setaria sp. and in some gastro-intestinal nematodes.

The histochemical localization of acid phosphatase in an adult filaria, Setaria sp. obtained from the peritoneal cavity of a cow was closely examined and compared with that of adult nematodes parasitic in the host alimentary canal; special attention was paid to the intestine and body wall of the parasites. Setaria sp. was found to show high acid phosphatase activity in the interchordal hypodermis of the body wall and uterine microfilariae, and similar activity is suspected to occur in the cuticle. The intestine of this nematode exhibited very low, if any, activity. In contrast, nematodes parasitic on the alimentary canal, such as Toxocara cati, T. canis, Physaloptera sp. and Ancylostoma caninum, showed no activity in the body wall and very high activity in the luminal surface of their intestine. The possible function of the abundant acid phosphatase in the body wall of this filaria is discussed.