Fasciola hepatica: site of resistance to reinfection in cattle

The effective site of resistance to reinfection of cattle with Fasciola hepatica was examined by recovery of challenge flukes from either the liver or body cavity. Calves infected 18 or 26 weeks previously with F. hepatica showed levels of resistance to reinfection of 56 and 94%, respectively, as assessed by recovery of flukes from the liver 15-16 weeks after challenge. Plasma glutamate dehydrogenase (EC 1.4.1.3; GLDH) enzyme activity estimations revealed only a marginal increase in these latter resistant calves compared with previously naive controls, indicating minimal liver damage as a result of migrating flukes. By comparison, when immature challenge flukes were recovered from the body cavity 4 or 14 days after infection of corresponding previously infected or naive calves, there was no significant difference in numbers. It appears, therefore, that, in cattle, resistance mechanisms are effective against challenge flukes at or soon after penetration of the liver.     

Fasciola hepatica: motility response to fasciolicides in vitro

The effects of a wide range of fasciolicides on the in vitro motility of Fasciola hepatica have been determined by means of an isometric transducer system. Carbon tetrachloride and diamphenethide do not affect movement at concentrations up to 500 and 100 micrograms/ml, respectively; at 1000 micrograms/ml, however, carbon tetrachloride induces a rapid tonic paralysis. Brotianide and the deacetylated metabolite of diamphenethide cause a rapid flaccid paralysis of the fluke at concentrations of 1.0 micrograms/ml and above. In contrast, the effect of MK-401 is a long-term one, a flaccid paralysis occurring after 20 hr only at 200 micrograms/ml. Praziquantel also produces a flaccid paralysis of the fluke, but this follows an initial increase, then decrease in muscle tone. The effect is rapid at 500 micrograms/ml, but long-term at 100 and 200 micrograms/ml; at these lower concentrations there is also a stimulation of activity. Oxyclozanide , rafoxanide, niclofolan , bithionol, and hexacholorophene induce a rapid spastic paralysis of the fluke at concentrations of 1.0 micrograms/ml and above. Both phasic and tonic components are evident in the response at concentrations of 1.0 micrograms/ml and below; the phasic component disappears at higher concentrations. Nitroxynil produces a similar effect, evident at higher concentrations. Among the benzimidazoles, mebendazole, oxfendazole, and albendazole sulphoxide cause a suppression of motility, whilst thiabendazole and albendazole produce a stimulation of movement. The effects are not rapid, however, for only mebendazole at 500 micrograms/ml causes total inactivity of the fluke within a 12-hr period. Possible explanations for these effects on fluke motility are discussed.     

Fasciola hepatica: effect of 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide on glycolysis in vitro

In vitro, 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide, a potent fasciolicide, causes a potent concentration-dependent inhibition of glucose uptake by mature Fasciola hepatica. In F. hepatica treated with the disulfonamide and then fed [U-¹⁴C]glucose, there was a 60% inhibition of glucose utilization and a corresponding inhibition of acetate and propionate formation. Treated fluke parasites possessed much lower levels of adenosine triphosphate, phosphoenolpyruvate, glucose 6-phosphate, and fructose 6-phosphate than untreated parasites and contained higher levels of glycerol and the free sugars fructose and mannose. Direct measurement of the effect of the disulfonamide on the glycolytic enzymes of F. hepatica demonstrated that 3-phosphoglycerate kinase (EC 2.7.2.3) and phosphoglyceromutase (EC 2.7.5.3) were inhibited. It is therefore suggested that the fasciolicidal activity of 4-amino-6-trichloroethenyl-1, 3-benzenedisulfonamide is due to inhibition of the enzymes 3-phosphoglycerate kinase and phosphoglyceromutase which effectively blocks the Embden-Myerhof glycolytic pathway.     

Fasciola hepatica: the presence of particle-associated and soluble nonspecific acid phosphatases.

The kinetic and physical properties of acid phosphatases in the lysosomal and microsomal fractions of F. hepatica were found to be similar, indicating that they are one and the same enzyme. In contrast, the biochemical properties of the soluble acid phosphatase (EC 3.1.3.2) were quite different from those of the lysosomal and microsomal fractions. This indicated the presence of two distinct forms of the enzyme one particle associated and the other soluble. Electrophoretic heterogeneity of these two types of acid phosphomonoesterase was seen. Two bands of activity were observed in both lysosomal and microsomal fractions and three bands in the soluble fraction.     

Fasciola hepatica: immunisation of rats by intraperitoneal injection of adult fluke antigen in Freund's adjuvant

Intraperitoneal injections of rats with freeze-dried, adult Fasciola hepatica material, which had been resuspended in phosphate-buffered saline and emulsified in Freund's incomplete adjuvant, reduced fluke burdens by 48 to 81% following oral infection. The addition of Bordetella pertussis to the adjuvant antigen emulsion enhanced the protection slightly (but not to a statistically significant degree); fluke antigens with B. pertussiss alone induced no protection.     

Control of glycogen synthase and phosphorylase by insulin in rat adipocytes which is unrelated to the concentration of cyclic AMP

Incubation of adipocytes in glucose-free medium with adrenocorticotrophic hormone, epinephrine, isoproterenol, or norepinephrine increased the concentration of cyclic AMP and the percentage of phosphorylase a activity, and decreased the percentage of glycogen synthase I activity. Glucose was essentially without effect on glycogen synthase or phosphorylase in either the presence or absence of epinephrine. Although glucose potentiated the action of insulin to activate glycogen synthase, the hexose did not enhance the effectiveness of insulin in the presence of epinephrine. Likewise, glucose did not increase the ability of insulin to oppose the activation of phosphorylase by epinephrine.The activation of glycogen synthase by insulin was not associated with a decrease in the concentration of cyclic AMP. Insulin partially blocked the rise in cyclic AMP due to isoproterenol, adrenocorticotrophic hormone, and norepinephrine. The maximum effects of isoproterenol on glycogen synthase and phosphorylase were observed when the concentration of cyclic AMP was increased twofold. However, insulin clearly opposed the changes in enzyme activity produced by isoproterenol (and also adrenocorticotrophic hormone, epinephrine and norepinephrine) even though concentrations of cyclic AMP were still increased three- to fourfold. Nicotinic acid opposed the increases in cyclic AMP due to adrenocorticotrophic hormone, isoproterenol and norepinephrine to the same extent as insulin; however, nicotinic acid was ineffective in opposing the activation of phosphorylase and inactivation of glycogen synthase produced by these agents. Thus, it is unlikely that the effects of insulin on glycogen synthase and phosphorylase result from an action of the hormone to decrease the concentration of cyclic AMP.     

Fasciola hepatica: role of developmental stages in the rat's resistance to challenge

Rats were sensitized by subcutaneous implantation of either metacercariae, 4 week-old juveniles, adult worms, or eggs of Fasciola hepatica and then challenged with 30 metacercariae 2 weeks later. Worm burdens were determined 8 weeks after challenge. Apart from adult worms, all implanted stages conferred a significant degree of protection on the recipients. The effectiveness of adult worm implants was not improved by using worms from different sources (sheep and cattle rather than rats) nor by extending the period of sensitization prior to challenge.     

Theileria annulata and Babesia ovis: ultracytochemical lactic dehydrogenase activity of sporozoites in salivary glands of female ticks, Hyalomma anatolicum excavatum and Rhipicephalus bursa

The occurrence of a marker enzyme of glycolysis, lactic dehydrogenase (LDH) (EC 1.1.1.27.), was studied ultracytochemically in sporozoites of Babesia ovis in the tick Rhipicephalus bursa and in sporozoites of Theileria annulata in the tick Hyalomma anatolicum excavatum. Female ticks infected transstadially were fed on rabbits and dissected 3–5 days post infestationem. The salivary glands were removed and incubated in the cytochemical medium unfixed or after fixation in buffered paraformaldehyde solution. A modified ferricyanide medium adjusted to pH 6.5 was used for incubation. Controls were performed by preincubating specimens in 10^?3 M iodine or by omitting NAD⁺ or lactate in the incubation medium. Following incubation, the specimens were fixed in buffered solutions of paraformaldehyde or glutaraldehyde, postfixed in osmium tetroxide, and embedded in Durcupan ACM. Mature “schizonts” consisting of an abundant number of sporozoites were examined in both piroplasmean species. In sporozoites of B. ovis the final enzymic product was deposited within the nucleus. No cytoplasmic reaction was observed. However, the membrane delimiting the schizont from the adjacent glandular cells was distinctly reactive. In T. annulata reactivity was usually confined to the cytoplasm. Sometimes, a reaction within mitochondria could be observed. The reaction product had formed aggregations which often appeared to be attached to micronemes. There was no nuclear reactivity in this species.The results suggest the existence of a glycolytic metabolic pathway with different subcellular localizations in sporozoites of the two piroplasmean species.     

Fasciola hepatica: cytochrome c oxidoreductases and effects of oxygen tension and inhibitors

Studies were made on the mechanism of respiration in Fasciola hepatica (Trematoda). Respiration was found to be dependent on the oxygen tension. The respiratory enzyme systems, NADH-cytochrome c oxidoreductase (EC 1.6.2.1), succinate-cytochrome c oxidoreductase (EC 1.3.99.1) NADH oxidase and cytochrome c-oxygen oxidoreductase (EC 1.9.3.1) were detected in a mitochondrial preparation, the NADH oxidase activity being markedly stimulated by addition of mammalian cytochrome c. Amytal and rotenone inhibited NADH oxidase activity. Antimycin A inhibited succinoxidase activity only at relatively high concentrations. Azide was inhibitory at high concentrations. However, cyanide was found to stimulate respiration. Hydrogen peroxide was found to be an end product of respiration in F. hepatica.     

The binding of beta- and gamma-cyclodextrins to glycogen phosphorylase b: kinetic and crystallographic studies

A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, alpha-, beta-, and gamma-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with K(i) values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with beta- and gamma-cyclodextrins at 1.94 A and 2.3 A resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that beta- and gamma-cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of alpha-cyclodextrin and octakis (2,3,6-tri-O-methyl)-gamma-cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction.     

Schistosoma mansoni: Inhibition of glucosephosphate isomerase and glycolysis by sugar phosphates

Glucosephosphate isomerase (EC 5.3.1.9) of Schistosoma mansoni is inhibited competitively by a number of tetrose, pentose, and hexose phosphates with inhibitor constant (K_i) values in the range of 0.5 to 400 μM. The most potent inhibitor is 5-phospho-d-arabinonate which resembles the cis-enediolate transition state intermediate of the reaction. These analogs were also found to be effective inhibitors of the production of lactate from glucose by suitably supplemented worm homogenates. The rank order of potency of inhibition of glycolysis was inversely related to the magnitudes of the K_i values for glucosephosphate isomerase. These K_i values were similar to those previously reported for mammalian glucosephosphate isomerase, suggesting similarities in the steric and electronic characteristics of the active sites of these isofunctional enzymes. This conclusion was further supported by the observed pH dependence of the inhibition by 5-phospho-d-arabinonate. Although glucosephosphate isomerase is not a rate-limiting enzyme of glycolysis, in the conventional sense, its selective inhibition could be of chemotherapeutic importance, in part because of the accumulation in glycolyzing systems of glucose 6-phosphate which is a potent feedback inhibitor of hexokinase.     

Fasciola hepatica: effects on the antioxidative properties and lipid peroxidation of rat serum

Fasciola hepatica infection is accompanied by increased formation of reactive oxygen species. The aim of this study was to analyze antioxidative properties of rat serum in the course of fasciolosis. Wistar rats were infected per os with 30 metacercariae of F. hepatica. Activities of antioxidant enzymes and concentrations of non-enzymatic antioxidants in serum were determined at 4, 7, and 10 weeks post-infection (wpi). Activity of superoxide dismutase (Cu, Zn-SOD) significantly decreased (by 35% during the migratory phase, by 40 and 23% at 7 and 10 wpi, respectively), while glutathione reductase activity significantly increased (by 62, 65, and 41%, at 4, 7, and 10 wpi, respectively). No significant changes were found in the activity of glutathione peroxidase. Significant decreases in concentrations of reduced glutathione, vitamins C, E, and A were observed, particularly during the migratory phase of fasciolosis (at 4 wpi). These changes were accompanied by enhancement of lipid peroxidation processes as evidenced by increased levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Concentrations of MDA and 4-HNE at 4 wpi increased by 38% and by 59%. MDA increased by 51% at 7 wpi and by 79% at 10 wpi, while 4-HNE increased by 87 and 118%, respectively. The results indicate that fasciolosis is associated with enhanced oxidative reactions and reduced antioxidant defense capability of rat serum.     

Trichomonas gallinae: charaterization and regulatory properties of lactic dehydrogenase.

The lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1.1.1.27, LDH)of Trichomonas gallinae was characterized and some of its regulatory properties studied. Electrophoretic analysis, with specific enzymatic staining of crude and dialyzed cell-free extracts and dialyzed ammonium sulfate fractions, all revealed a single band of enzymatic activity suggesting only one molecular form of the enzyme. The pH optima were found to be the following: 7.0 in the pyruvate to lactate direction and 9.0 in the reverse direction. Thermal inactivation studies showed a narrow temperature optimum peaking at 35 C. The K_m values for all four reaction components were determined and found to be: NADH, 70 μm; pyruvate, 88 μm; NAD, 65 μm; and l-lactate, 4.6 mM. T. gallinae LDH was absolutely specific for NAD, NADH, l-lactate, and pyruvate. The enzyme exhibited negative cooperativity, with both NADH and l-lactate, as evidenced by curvilinear Lineweaver-Burk kinetics and Hill coefficients of less than one. Several glycolytic intermediates lowered the K_m of NADH with variable effects on the K_m of pyruvate. The regulation of LDH by glycolytic intermediates is discussed.     

Leishmania donovani: Surface membrane acid phosphatase activity of promastigotes

Subcellular fractionation of Leishmania donovani promastigotes yielded plasma membranes, which were enriched in acid phosphatase (E.C.3.1.3.2.) activity. Cytochemically, the enzyme displayed a uniform distribution over the surface of intact protozoa. The enzyme was also visualized on the external face of the isolated plasma membranes, as indicated by the distribution of subpellicular microtubules. Various parameters of the membrane-bound enzyme were also determined including pH and temperature optima and substrate specificity. The results suggest that these organisms are adapted for existence in a hydrolytic environment.     

Leishmania donovani: action of excreted factor on hydrolytic enzyme activity of macrophages from mice with genetically different resistance to infection

The effect of purified excreted factor from promastigotes of Leishmania donovani upon the activity of four enzymes from lysed peritoneal exudate cells of mice (C3H and C57BL) was determined. There was no demonstrable effect on acid phosphatase (EC3.1.3.2), β-glucuronidase (EC3.2.1.21), and N-acetyl-β-glucosaminidase (EC3.2.1.29), but β-galactosidase (EC3.2.1.23) was inhibited up to 72% after 3 hr of incubation at 37 C. Inhibition of C57BL mouse enzymes was not significantly different from that of C3H mice. Protamine sulfate combined with the highly negatively charged excreted factor of L. donovani to migrate as a single positively charged band on immunoelectrophoresis. Protamine sulfate also reversed the β-galactosidase inhibition, though this was without direct effect on the enzyme. The excreted factor did not change or lose its charge or antigenicity with regard to precipitating antibody, when incubated with extracts of mouse peritoneal exudate cells, splenocytes, or liver homogenate—irregardless of whether the mice had been infected with leishmaniasis for 1 or 2 weeks or were uninfected.     

The turnover of skeletal muscle glycogen phosphorylase studied using the cofactor, pyridoxal phosphate, as a specific label

The turnover of glycogen phosphorylase has been measured using the cofactor, pyridoxal phosphate, as a label specific for this enzyme in skeletal muscle. Radiolabelled pyridoxine administered in vivo is incorporated into a protein-bound fraction in skeletal muscle, shown by several criteria to be equivalent to glycogen phosphorylase. This pool of radiolabel disapears slowly with a half-life of 11.9 days, taken to be a good estimate of the intracellular half-life of the enzyme. The use of the cofactor in this fashion minimises overestimation of half-life that results from reincorporation of the label. Further, premature dissociation of the cofactor from native enzyme, which would lead to underestimation of half-life, is unlikely. At the level of sensitivity given by this method there was little evidence for the appearance of pyridoxal phosphate-labelled degradation intermediates of the enzyme.     

Fasciola hepatica: inhibition of phosphoenolpyruvate carboxykinase, and end-product formation by quinolinic acid and 3-mercaptopicolinic acid

Quinolinic acid and 3-mercaptopicolinic acid act as inhibitors of Fasciola hepatica phosphoenolpyruvate carboxykinase. Low concentrations of these compounds (0.1 mM quinolinate and 0.01 mM 3-mercaptopicolinate) resulted in noncompetitive inhibition, which became mixed inhibition at higher concentrations (1.5 and 0.15 mM, respectively). 3-mercaptopicolinic acid proved to be a much more potent effector than quinolinic acid. Both quinolinic acid and 3-mercaptopicolinic acid caused a significant reduction in the total amount of end product excreted, again 3-mercaptopicolinate being more effective than quinolinate. When glucose was present in the medium, both propionate and acetate levels fell significantly with both inhibitors; however, only 3-mercaptopicolinic acid caused an effect in the absence of glucose.     

Fasciola hepatica and Fasciola gigantica: comparison of cellular response to experimental infection in sheep

Cellular responses to Fasciola gigantica and to Fasciola hepatica infection in sheep were compared. Eosinophil numbers increased more quickly and strongly in F. gigantica-infected sheep than in F. hepatica-infected sheep. In both groups, peripheral blood mononuclear cell (PBMC) proliferation in response to the parasitic excretory-secretory products (ESP) showed similar kinetics. Interferon-gamma (IFN-gamma) production by ESP-stimulated PBMC was early and showed similar kinetics in both groups. Interleukin-10 (IL-10) production by FhESP-stimulated PBMC was very high throughout infection even at 0 weeks post-infection (WPI) in F. hepatica-infected sheep, while in F. gigantica-infected sheep, IL-10 production by FgESP-stimulated PBMC increased between 1 and 4 WPI. IL-10 production in F. gigantica-infected sheep was significantly lower than in F. hepatica-infected sheep during infection. The lower susceptibility to F. gigantica infection in sheep could be explained by the more intense cellular response induced by the parasite and the weaker capacity of F. gigantica to evade the immune response.     

Effect of the acclimation to high environmental temperature on the activity of hepatic glycogen phosphorylase (a+b and a), liver glycogen content and blood glucose level in rats

(1) Changes in the activity of hepatic glycogen phosphorylase a+b and a (GPh-ase a+b and a), liver glycogen content and blood glucose level during acclimation to moderate high environmental temperature (35±1 °C) were studied. (2) Experiments were carried out on adult fed Wistar rats of both sexes, previously given either short-term (1, 4 and 7 days) or long-term (14, 21, 30 and 60 days) exposure to high environmental temperature. The controls were continuously kept at room temperature (20±2 °C). (3) The results obtained showed that in the period of short-term exposure the liver glycogen content was decreased significantly (after the first and fourth days in male rats and after first day in female rats) and the GPh-ase a activity increased (after first day in male rats and after first, fourth and seventh day in female rats). Long-term exposure caused significant increased liver glycogen content (beginning from the 14th day in male rats and the 21st day in female rats) until the end of the acclimation period (60 days). The elevated activity of GPh-ase a persists after 14th day of exposure only in female rats while there are no significant changes over the rest of the acclimation period in both sexes. There were no significant changes in total GPh-ase activity during the whole period of exposure. Blood glucose level was significantly decreased throughout the whole period of acclimation to high environmental temperature, in both sexes (except in the 1 day exposed groups). (4) The increased activity of hepatic GPh-ase a and decreased glycogen content suggested that the short-term exposure to heat stimulates the glycogenolytical processes. Decreased blood glucose level, and elevated liver glycogen content (r=-0.7467 in male and r=-0.6548 in female rats) suggested that prolonged exposure to high environmental temperature stimulated glycogenogenesis, without changes in the GPh-ase activity.