Crystallization and preliminary crystallographic analysis of a new class of glutathione transferase from nematodes.

Mouse and Heligmosomoides polygyrus constitute a readily manipulated small-animal laboratory model for investigating host-nematode interactions. Two major forms of glutathione transferase (GST) are expressed in H. polygyrus adult worms following primary infection. One of these forms belongs to a new class of GST which has only been found in the nematode phylum and therefore presents a possible target for nematode control. In this study, crystals were obtained of a recombinant representative of this new GST class from H. polygyrus. These crystals belong to the triclinic space group P1, with unit-cell parameters a = 72.7, b = 74.0, c = 88.6 A, alpha = 79.1, beta = 80.1, gamma = 81.5 degrees, and are likely to contain four homodimers in the asymmetric unit. X-ray diffraction data were collected to 1.8 A resolution on station A1 at the Cornell High-Energy Synchrotron Source (CHESS).

Acid phosphatase activity in excretion/secretion products from Heligmosomoides polygyrus adults: an indicator of the physiological status of the worms.

Acid phosphatase (AP) activity was detected in 24 h culture media from adult Heligmosomoides polygyrus. Female and male excretion/secretion products showed similar specific activity. For both, the AP had a pH optimum of 4.0 and was inhibited by sodium fluoride, tartaric acid, and sodium orthovanadate. The release of AP by adult worms was significantly inhibited by adverse incubation conditions (temperatures of 20 degrees C and 4 degrees C), known physiological perturbers ( t-butylhydroperoxide and sodium azide), and broad spectrum anthelmintics (albendazole, levamisole, morantel, and ivermectin). These results indicate that the AP activity level in the culture medium may be an indicator of the physiological status of the worms.

The in vitro secretion of acetylcholinesterase by adult stages of Heligmosomoides polygyrus: the effects of broad-spectrum anthelmintics.

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 +/- 0.07 mumol min-1 l-1 mg-1) than for female (0.56 +/- 0.04 mumol min-1 mg-1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug-free medium (RPMI medium supplemented with 0.5% BSA) for 24 h or continuously exposed to the drugs in supplement-free medium (24 h), the concentration- and time-dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50% (IC50) relative to drug-free controls were estimated. The IC50 values ranged from 0.012 microM (IVM) to 2.96 microM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad-spectrum anti-nematodal activity is discussed.

Differential expression of glutathione S-transferase (GST) by adult Heligmosomoides polygyrus during primary infection in fast and slow responding hosts.

Glutathione S-transferase (GST) specific enzymatic activity, assayed with the model substrate 1-chloro-2,4-dinitrobenzene, was 45% higher in adult Heligmosomoides polygyrus passaged through a slow responder mouse strain, C57/BL10 compared to worms passaged through a fast-responder strain (SWR x SJL) F1. Western analysis using polyclonal antisera raised to purified H. polygyrus GSTs did not appear to positively correlate the expression of GST protein with functional enzymatic activity. However, western blotting did indicate a sex-linked expression pattern of GST protein, with male worms expressing a higher ratio of the 24 kDa to the 23 kDa GST family than female worms.

Glutathione S-transferases from the gastrointestinal nematode Heligmosomoides polygyrus and mammalian liver compared.

Glutathione S-transferases have been partially characterised from the gastrointestinal nematode Heligmosomoides polygyrus. Two major subunit families were purified (24 and 23 kDa) with N-terminal homology to the mammalian Alpha family. Four dimeric forms of GST were purified from the nematode by glutathione-affinity chromatography, two major enzymes (pI 8.1, 5.0) and two minor forms (pI 5.8, 5.3). The purified GST pool could neutralize model and lipid peroxides via peroxidase activity but not peroxidation derived reactive carbonyls via glutathione transferase activity. Antisera raised to the pooled nematode GSTs appeared to recognize other Strongylida GSTs more strongly on Western blotting compared to mammalian GSTs.

A PCR strategy for the isolation of glutathione S-transferases (GSTs) from nematodes.

Anchor based PCR technology has been used to isolate a GST sequence from the gastro-intestinal nematode Heligmosomoides polygyrus. A 800 base pair product was amplified from first-strand cDNA using primers based on the N-terminal sequence of purified H. polygyrus GST (upstream primer) and a non-specific polyadenylate tail with an anchor sequence (downstream primer). The product was cloned into pUC18 and sequenced. A reading frame of 648 bases in the sequence encoded a protein which has 30% homology with the alpha family of mammalian glutathione S-transferases.

The effect of levamisole on acetylcholinesterase secretion by male and female Heligmosomoides polygyrus during ageing.

The inhibition of acetylcholinesterase secretion by male and female Heligmosomoides polygyrus was tested on worms taken from experimentally infected mice and maintained for 3 days in vitro in levamisole. The dose inhibiting 50% of enzyme secretion (ID 50) of male worms was twice the ID 50 for female worms. A similar difference was observed in vivo between the dose of levamisole removing 50% (LD50) of male and female worms from the mouse. Acetylcholinesterase secretion by worms and ID 50 were tested in vitro at 3-weekly intervals from 3 to 21 weeks post infection (WPI). Acetylcholinesterase secretion was always significantly higher for male than for female worms. A decrease of ID 50, correlated with the age of the worms was observed: from 1.5 to 0.5 micrograms/ml for males and from 0.7 to 0.1 micrograms/ml for females. These results were confirmed in vivo by a higher efficacy of the anthelminthic at 21 than at 4 WPI.

Differential secretion of acetylcholinesterase and proteases during the development of Heligmosomoides polygyrus.

The development of the gastrointestinal nematode Heligmosomoides polygyrus was studied in the mouse. Levels of production of acetylcholinesterase and proteases were measured in excretory/secretory products of various stages of the parasite. The production of acetylcholinesterase was found to be maximal between days 4 and 6 post-infection, corresponding to the fourth larval stage of the parasite's life-cycle. Analysis of proteolytic activity revealed both quantitative and qualitative differences between the stages. Quantitative examination showed a maximal concentration of proteolytic enzymes in the early third larval stage (L3). Qualitative analysis revealed L3-associated molecules at 96, 15 and 8 kDa, L4-associated molecules at 58 and 33 kDa and adult-associated molecules at 116, 102, 39 and 25 kDa. A number appeared to be shared by all stages (18, 16 and 13 kDa), whilst others (76 and 42 kDa) appeared to be associated with the late L4/early adult parasite. The biological and immunological implications of variation in the production of proteases and acetylcholinesterase during the development of H. polygyrus are discussed.

The possible absence of cytochrome P-450 linked xenobiotic metabolism in helminths.

The distribution of cytochrome P-450, b5 and associated oxidations, aniline hydroxylase, 7-ethoxycoumarin O-deethylase and 4-nitroanisole O-demethylase were studied in the parasitic nematode Heligmosomoides polygyrus, its mammalian host, Mus musculus and the free-living nematode Panagrellus redivivus. Cytochrome P-450, its associated oxidations and cytochrome b5 could not be detected in whole homogenates or subcellular fractions (mitochondrial, microsomal and soluble fractions) of either nematode under a variety of assay conditions which included attempted induction with sodium phenobarbital. P. redivivus was able to reduce 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene and azobenzene, which is predominantly microsomal. The implications of these results in terms of chemotherapy are discussed.

Proteolytic enzymes in excretory-secretory products from adult Nematospiroides dubius.

Excretory-secretory products (ES), collected from in vitro cultures of adult Nematospiroides dubius, were examined for proteolytic enzyme activity. ES enzymes had a pH optimum of 8.0 and their activity was sensitive to serine-proteinase inhibitors. Three SDS-resistant proteases were identified in ES at molecular weight (mol. wt) 200,000, 105,000 and 48,000 by incorporating substrates into the matrices of sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels.

In vitro acetylcholinesterase secretion by male or female Heligmosomoides polygyrus.

Acetylcholinesterase (AChE) secreted in vitro by adult male and female Heligmosomoides polygyrus were assayed over a 4-day period using a micro-method. The rate of AChE secretion by 1, 2 or 4 worms was approximately linear, and three times higher for male worms than for female worms.

Glutamine and asparagine synthesis in the nematodes Heligmosomoides polygyrus and Panagrellus redivivus.

Low levels of glutamine synthetase were demonstrated in Panagrellus redivivus and Heligmosomoides polygyrus. Asparagine synthetase was detected in P. redivivus but activity was too low to be detected in H. polygyrus.

Heligmosomoides polygyrus: peroxidase activity.

Peroxidase activity in Heligmosomoides polygyrus was located primarily in the mitochondrion. The enzyme was active with a range of organic and inorganic electron donors and, in addition to hydrogen peroxide, it could utilize cumene peroxide, but the highest activity was obtained with linoleic acid peroxide. The effects of electron chain substrates and inhibitors on H. polygyrus mitochondrial peroxidase activity was consistent with the enzyme being linked functionally to cytochrome c, although in vivo, this may not be the only electron donor. The interaction of the peroxidase with electron transport is discussed.

Amino acid catabolism in the nematodes Heligmosomoides polygyrus and Panagrellus redivivus. 1. Removal of the amino group.

The major transaminase in Heligmosomoides polygyrus, Panagrellus redivivus and rat liver was the 2-oxoglutarate-glutamate system, with relatively few amino acids acting as donors for the pyruvate-alanine and oxaloacetate-aspartate systems. The relative effectiveness of the different amino acid donors in the three transaminase systems was similar in all three tissues. Both H. polygyrus and P. redivivus can oxidatively deaminate a range of L-amino acids, although D-amino acid oxidase activity was low. Serine and threonine dehydratase activity and histidase activity were present in H. polygyrus and P. redivivus and both nematodes were also able to deaminate glutamine, asparagine and arginine. When NAD(H) was the cofactor the glutamate dehydrogenases of H. polygyrus and P. redivivus showed similar regulatory properties to the mammalian enzyme. However, with NADP(H) the results were anomalous. The capacity of both nematodes to transaminate and oxidatively deaminate amino acids was broadly similar and comparable to mammalian tissue. Glutamate dehydrogenase is probably the major route for deamination in these nematodes. A complete sequence of urea cycle enzymes could not be demonstrated in either P. redivivus or H. polygyrus.

Amino acid catabolism in the nematodes Heligmosomoides polygyrus and Panagrellus redivivus. 2. Metabolism of the carbon skeleton.

All of the enzymes of proline catabolism were present in Heligmosomoides polygyrus and Panagrellus redivivus and the activities were, in general, similar to those found in rat liver. Both nematodes were also shown to be able to catabolize the branched-chain amino acids leucine, isoleucine and valine, by pathways similar to those found in mammalian liver. There were no significant differences in amino acid catabolism between the animal-parasitic and free-living species of nematode.