Cloning and characterisation of a peroxiredoxin from the swine roundworm Ascaris suum

Antioxidant enzymes in parasites play an important role in protection against the oxygen radicals by generating during aerobic metabolism, as well as in defence against host immune cell assault. Here we report the cloning and characterisation of a cDNA encoding peroxiredoxin from Ascaris suum (AsPrx). AsPrx is 776bp long and contains the nematode 22bp splice leader sequence at the 5' end and polyadenylation signal followed by poly(A) tail at the 3' end. AsPrx codes a full-length protein with a predicted molecular mass of 22. 6kDa, and possesses two cysteine residues at amino acid 49 and 168 that are conserved among Prx proteins. GenBank() analysis showed that the deduced amino acid sequence had significant similarity to parasite and mammalian Prx at the amino acid level. DNA nicking revealed that Escherichia coli-expressed recombinant AsPrx (rAsPrx) is enzymatically inhibited to form oxidative-nicking of supercoiled plasmid DNA. Two-dimensional immunoblot analysis with mouse anti-rAsPrx serum reacted two major constituent protein spots in extracts of adult female worms, suggesting that the native AsPrx might be function as a major antioxidant enzyme in Ascaris suum.     

Alternative migration routes of Ascaris suum in the pig

Experiments were conducted to investigate possible alternative routes of extraintestinal migration of Ascaris suum larvae in the pig. Pigs were infected with A. suum via injection of newly hatched larvae into cecal veins (i.v.), into cecal lymph nodes (LN), or intraperitoneally (i.p.), and control animals were inoculated orally with infective eggs (p.o.). Two pigs per inoculation route were necropsied on days 1, 4, and 13 postinoculation. The numbers of liver lesions and the percentage of larvae recovered was considerably greater in pigs inoculated i.v. or p.o. on each necropsy day. However, irrespective of inoculation route, at least a proportion of larvae passed through the livers and were able to complete migration to the small intestine by day 13. The results indicate that larval penetration of the intestinal wall is not necessary for liver-lung migration and that passage through the liver may be favorable for migrating A. suum larvae, although a delayed arrival in the small intestine cannot be ruled out for larvae following alternative routes.     

Opiate alkaloids in Ascaris suum

The parasitic worm Ascaris suum contains the opiate alkaloids morphine and morphine-6-glucuronide as determined by HPLC coupled to electrochemical detection and by gas chromatography/mass spectrometry. The level of morphine in muscle tissue of female and male is 252 +/- 32.68, 1168 +/- 278 and 180 +/- 23.47 (ng/g of wet tissue), respectively. The level of M6G in muscle tissue of female and male is 167 +/- 28.37 and 92 +/- 11.45 (ng/g of wet tissue), respectively. Furthermore, Ascaris maintained for 5 days contained a significant amount of morphine, as did their medium, demonstrating their ability to synthesize the opiate alkaloid. The anatomic distribution of morphine was examined by indirect immunofluorescent staining and HPLC of various tissues dissected from male and female adult worms. Immunofluorescence revealed morphine in the subcuticle layers, in the animals' nerve chords and in the female reproductive organs. Morphine was found to be most prevalent in the muscle tissue and there is significantly more morphine in females than males, probably due to the large amounts in the female uterus. Morphine (10(-9) M) and morphine-6-glucuronide (10(-9) M) stimulated the release of NO from Ascaris muscle tissue. Naloxone (10(-7) M), and L-NAME (10(-6) M) blocked (P < 0.005) morphine-stimulated NO release from A. suum muscle. CTOP (10(-7) M) did not block morphine's NO release. However, naloxone could not block M6G stimulated NO release by muscle tissue, whereas CTOP (10(-7) M) blocked its release. These findings were in seeming contradiction to our inability to isolate a mu opiate receptor messenger RNA by RT-PCR using a human mu primer. This suggests that a novel mu opiate receptor was present and selective toward M6G.     

Catalase activity during the development of the parasitic nematode, Ascaris suum

1. Catalase activity was partially purified from body wall muscle of the parasitic nematode, Ascaris suum, and was similar to catalases isolated from mammalian tissues. It exhibited a broad pH optimum and was unaffected by 2 mM ethylenediaminetetra-acetate. In contrast, it was inhibited reversibly by 1 mM cyanide and irreversibly by prior incubation in 40 mM 3-amino-1:2:4-triazole for 1 hr or heating at 80 degrees C for 15 min. 2. Catalase activity was highest in the unembryonated "egg" and decreased dramatically as development proceeded. 3. Catalase activity in adult body wall muscle was similar to that in rat skeletal muscle, but dramatically lower than that in rat liver. Catalase activity was barely detectable in A. suum testis. 4. Cytochrome-c peroxidase activity did not appear to be present in adult A. suum muscle mitochondria.     

Chitin synthesis in zygotes of Ascaris suum

In Ascaris suum chitin is formed in the zygote immediately after oocyte fertilization, and its synthesis is completed in the eggs from the distal half of the uterus. Incorporation of radiocarbon [14C] glucose into chitin of the eggshell was 40-fold higher than incorporation of [14C] glucosamine. The same rank order also holds for the incorporation of label from these isotopes into the glycogen of the ovaries. A large part of the radiolabel was incorporated first into oocyte glycogen and only after fertilization was it incorporated into eggshell chitin. Actinomycin D inhibited chitin synthesis in the eggs from the distal half of the uterus and it significantly reduced incorporation of radiocarbon from glucose into chitin.     

Ascaris suum: immunoglobulin responses in mice

The immune response in mice to infection with Ascaris suum was characterized by determining (1) changes in serum immunoglobulin levels and (2) changes in the relative proportions of immunoglobulin-containing cells in major lymphoid tissues and sites of possible local immunoglobulin production.     

Polymorphism of malate dehydrogenase in Ascaris suum

Starch gel electrophoresis of homogenates prepared from adult Ascaris suum revealed polymorphism for the number, staining intensity, and electrophoretic mobility of the cytoplasmic isozymes of malate dehydrogenase (MDH). Five different variant isozymic patterns were found among the 2160 worms surveyed. The most acceptable formulation for the molecular basis of the variant patterns supports the hypothesis that the synthesis of supernatant MDH in Ascaris suum is under the control of two separate genetic loci, MDH_A and MDH_B.This work was supported by National Institutes of Health Grant HD-00994.     

Poly(ADP-ribosylation) in Ascaris suum

Poly(ADP-ribosylation) was demonstrated in the intestinal parasite Ascaris suum, especially in the reproductive tissues. The activity of the ADP-ribosyltransferase was found to depend on divalent cations and to be stimulated by deoxyribonuclease I about 5-fold. The reaction rate was optimal at a temperature of 30 degrees C and at pH about 8.4. The apparent Km value for NAD was estimated to be 0.2mM. The enzyme activity was effectively inhibited by nicotinamide (Ki = 65 microM) benzamide (6 microM), 3-aminobenzamide (10 microM), theophylline (35 microM) and thymidine (50 microM). The type of inhibition by these compounds was found to be competitive with respect to NAD.     

Patch-clamp and chloride channels in Ascaris suum

Single-channel electrophysiology is an invaluable tool fo the study of ion channels. However, it is a technique that has failed to attract widespread use by parasitologists. Here, Diane Dixon and Richard Martin outline the principles undelrying single channel recording and highlight its uses in the discovery of a new and unusual chloride channel in the musculature of Ascaris suum.     

Proteinase activity in Ascaris suum eggs, hatching fluid, and excretions-secretions.

Hatching fluid and the excretions and secretions (E.S.) of hatched larvae of Ascaris suum revealed proteinase activity when assayed by 2 different procedures employing collagen or casein as substrates. Both assays apparently detected similar levels of proteinase activity in hatching fluid and E.S. of hatched larvae. The Anson (casein substrate) assay worked best in 0.05 M phosphate buffer, pH 8.0. The Azocoll (collagen substrate) assay worked best in 0.05 M borate buffer at pH 8.8. Azocoll assays done at temperatures ranging from 25 to 65 C revealed maximal proteinase activity at 55 C. Analysis of hatching fluid from 18-, 21-, and 28-day-old embryos and of extracts from sonicated 0- to 28-day-old developmental stages showed that proteinase activity increased markedly 18 days after embryonation had begun. Prior to the 18th day of embryonation proteinase levels were relatively low.