James M. Smith

Schistosome Satellite DNA Encodes Active Hammerhead Ribozymes

ABSTRACT Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Smα repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in bothcis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Smα ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum orHeterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.

Antioxidant systems in Schistosoma mansoni: correlation between susceptibility to oxidant killing and the levels of scavengers of hydrogen peroxide and oxygen free radicals.

Abstract The relative susceptibility of Schistosoma mansoni schistosomula and adults to cell-free oxidant mediated killing has been studied and correlated with levels of oxidant scavengers in the parasite stages. Over 95% of the schistosomula were killed in H 2 O 2 (0.25 m m )) or H 2 O 2 generated by glucose (5.5 m m )-glucose oxidase (5 mU ml −1 ); and in superoxide, H 2 O 2 , and possibly hydroxyl radical and singlet oxygen, generated by xanthine (0.5 m m )- or acetaldehyde (0.5 m m )-xanthine oxidase (10 mU ml −1 ). Under similar conditions less than 2% of the adults were killed. The oxidant scavenger enzymes superoxide dismutase, cytochrome c peroxidase, glutathione peroxidase, glutathione reductase, and the tripeptide glutathione were present in the parasite, but not the enzyme catalase. Enzyme activities and glutathione levels were significantly lower in the schistosomula than adult worms. Juvenile and adult S. mansoni show differences in susceptibility to in vitro oxidant killing that correlate with differences in their levels of endogenous antioxidants.

The aging process of the nematode Caenorhabditis elegans in bacterial and axenic culture

Abstract While much is known of the morphological and some physiological changes which occur during the aging of Caenorhabditis elegans, little attempt has been made to measure the changes in behaviour. Wild type C. elegans (var. Bristol) were cultured axenically, individually observed each day for 15 minutes and their behavioural actions recorded on a multi-channel event recorder or on a video tape recorder of a closed circuit TV- Particular attention was paid to the rate of backwardly directed somatic waves, pharyngeal bulb pulsations, the interval between defecations and oviposition. C. elegans lived significantly longer in axenic culture than in bacteria. A gradual linear decline occurred in the rate of backward waves between maturation (day 4) and death (day 20) for those worms in axenic culture. In striking contrast, the mean maximum rate of pharyngeal bulb pulsations maintained a plateau from day 4 to 18, while the mean interval between defecations doubled from 60 sec (days 4 to 8) to 120 sec (days...

Schistosoma mansoni: levels of antioxidants and resistance to oxidants increase during development.

The effects of cell-free generated oxidants on migrating and developing stages of Schistosoma mansoni were investigated and the levels of antioxidant enzymes and of glutathione were determined for each stage. Schistosomula and 2-week-old parasites recovered from the livers of infected mice showed similar susceptibility to killing by added hydrogen peroxide and t-butylhydroperoxide. However, when glucose (0.5 mM)-glucose oxidase (2.5 mU ml-1) and xanthine (0.5 mM) or hypoxanthine (0.5 mM)-xanthine oxidase (5.0 mU ml-1) systems were used to generate hydrogen peroxide and oxygen free-radicals, schistosomula were more susceptible to oxidative killing than the 2-week-old parasites. The 4- and 8-week-old worms were more resistant to oxidants than all of the younger stages. High levels of superoxide dismutase (16.2-24.8 U mg-1 protein) were present in all stages. Catalase was not detected. Glutathione peroxidase activity with cumene hydroperoxide as substrate was not detectable in the schistosomula but the activity was present in the 2-week-old parasites. However, hydrogen peroxide-sensitive glutathione peroxidase activity was present in all the stages with a threefold difference in activity between schistosomula and the adult stages. Glutathione-s-transferase activity was significantly lower in the schistosomula, lung stages, and the 2-week-old parasites than in the older stages. Progressive increases in the levels of glutathione reductase and glutathione were also observed with development. The differences in the levels of antioxidants between different stages of development may partly explain the increase in resistance to oxidant-mediated damage as the parasite develops.

Antioxidant systems in Schistosoma mansoni: Evidence for their role in protection of the adult worms against oxidant killing

Abstract In contrast to schistosomula, adult S. mansoni are relatively resistant to oxidant killing in cell free systems, and possess significantly higher levels of oxidant scavengers. In this study, we observed that the adult S. mansoni had a significantly greater capacity to remove hydrogen peroxide than the schistosomula. In addition, they protected a significant proportion of the schistosomula against oxidant killing in vitro . The susceptibility of schistosomula to oxidant killing was inversely dependent on the weight of schistosomules used per assay. However, when the weight of schistosomules and that of adults were equivalent, the adults afforded greater protection from the lethal effects of the oxidants to the schistosomules than the schistosomules could themselves. The protein content of the adults was significantly greater than that of schistosomula of equal wet weight. A significant proportion of the adult worms was rendered susceptible to killing by hydrogen peroxide, when they were exposed to inhibitors of the enzyme cytochrome c peroxidase or glutathione depleting agents. These findings indicate that adult S. mansoni have a greater capacity for oxidant detoxification than the schistosomula, and suggest that cytochrome c peroxidase and glutathione may be important for hydrogen peroxide detoxification in the parasite.

LOCALIZATION OF P-GLYCOPROTEIN mRNA IN THE TISSUES OF HAEMONCHUS CONTORTUS ADULT WORMS AND ITS RELATIVE ABUNDANCE IN DRUG-SELECTED AND SUSCEPTIBLE STRAINS

Membrane transporter P-glycoproteins (Pgps) are present in a number of nematode species, including Haemonchus contortus. Allelic variation in some Pgp genes has been found to be associated with resistance to the anthelmintic ivermectin (IVM), although functional verification of a role for Pgps in IVM resistance has yet to be demonstrated. By in situ hybridization, the distribution of Pgp mRNA was visualized in transverse cryosections of adult H. contortus, using a digoxigenin-labeled cDNA probe encoding the ATP-binding region of an H. contortus Pgp. The probe sequence targeted a conserved ATP-binding region of Pgp-A (97.9% identity). It also shared 49.7–71.1% identity with 11 other Pgp sequences previously identified in H. contortus and may hybridize these sequences to give an overall measure of the total P-glycoprotein mRNA. Staining was predominately localized along the intestinal tract of the worms, with the most intense staining localized in the pharynx and anterior intestine. In the mid- and posterior intestinal regions, staining was restricted to the luminal side of the intestine. Some staining was also associated with the vas deferens and the lateral hypodermal chords anterior to the nerve ring. Using densitometry, the levels of Pgp mRNA in the pharynges of unselected and IVM- and moxidectin (MOX)-selected strains of male and female H. contortus were compared. No differences were detected between the levels of expression of Pgp in the susceptible strain versus the IVM- or MOX-selected strains. Evidence in the literature suggests that not all Pgp homologues are linked to chemical resistance phenotypes. It is thus possible that expression of 1 of the H. contortus Pgps is altered in IVM-resistant strains but that this phenomenon was undetectable in our experiments.

Glutathione redox state, lipid peroxide levels, and activities of glutathione enzymes in oltipraz-treated adult Schistosoma mansoni

A decrease in reduced glutathione (GSH) levels in adult Schistosoma mansoni exposed in vitro to the antischistosomal drug oltipraz (OPZ) (20-60 nM) was accompanied by a significant increase in oxidized glutathione (GSSG) levels. The total glutathione (GSH + GSSG) levels also diminished in drug-treated parasites. The activities of the parasite glutathione peroxidase (GPO), utilizing cumene hydroperoxide as a substrate, and glutathione S-transferase (GST), measured 18 hr after in vitro incubation with the drug, were elevated significantly, but there were no significant alterations in the activities of the GPO, utilizing H2O2, or glutathione reductase (GR). Drug-treated worms showed increased lipid peroxidation. In vivo, the proportion of the worms recovered from infected mice given OPZ (100 mg/kg body wt) gradually declined with time, to about 30% of that recovered from infected untreated control mice by day 14 after drug administration, and consisted predominantly of male worms. Accompanying this significant decline in the proportion of worms recovered were significant decreases in the activities of the enzymes GR and GST in drug-exposed worms. On the other hand, a slight initial increase in the GPO activity with cumene hydroperoxide was followed by a return to control values, and the GPO activity with H2O2 was decreased only slightly with time. Interestingly, the 4-hydroxyalk-2-enal aldehydes, known products of lipid peroxidation, inhibited the GST reaction with 1-chloro-2,4-dinitrobenzene (CDNB). The OPZ-induced changes in S. mansoni could increase parasite susceptibility to oxidative attack by host phagocytes, and are probably linked with the antischistosomal action of the drug in vivo.

Differential effects of oltipraz and its oxy-analogue on the viability of Schistosoma mansoni and the activity of glutathione S-transferase.

Adult worms of Schistosoma mansoni recovered from mice treated with oltipraz (OPZ) showed a significant diminution in their ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan, a measure of parasite viability. Incubation of glutathione S-transferase (GST) from S. mansoni with OPZ resulted in a time- and concentration-dependent inhibition of enzyme activity. RP 36,642 (an inactive oxy-derivative of OPZ) had a minimal effect on the viability of the worms and no effect on GST activity. The structural integrity of OPZ, particularly the thione sulphur, appears to be necessary for expression of the antischistosomal effects of the drug. OPZ-induced inhibition of GST was non-competitive with either reduced glutathione (GSH) or 1-chloro-2,4-dinitrobenzene (CDNB), indicating that the drug is not a substrate for GST-catalysed conjugation reactions. In addition, the inhibition of GST could not be reversed by dialysis or repurification of the enzyme via a GSH-agarose affinity column. The effects of OPZ on GST activity could render the parasite vulnerable to damage by host-derived reactive oxygen species and aldehydic products of lipid peroxidation. The effects of OPZ on GST activity may play a role in the antischistosomal action of OPZ.

Primates as a Source of Entamoeba histolytica, Their Zymodeme Status and Zoonotic Potential

A survey of 17 species of non-human captive primates in Quebec, Canada, revealed that 40% of the animals were infected with the protozoan Entamoeba histolytica. Isoenzyme analysis of lysates prepared from cultured amebic isolates showed them to belong to either a zymodeme III or VIII, and therefore, characteristic of non-pathogenic strains of E. histolytica from humans. These isolates were also of low virulence in gerbil ceca. Serum samples from infected and uninfected primates had negative titres for E. histolytica and all the animals appeared to be healthy.

Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.