James W. Tracy

Purification of three cytosolic glutathione S-transferases from adult Schistosoma mansoni☆

The cytosolic fraction of adult Schistosoma mansoni contains glutathione S-transferase (EC 2.5.1.18) activity, determined with the prototype substrate 1-chloro-2,4-dinitrobenzene, that is 5- to 50-fold greater than that found in other metazoan parasites. A survey of several model substrates revealed that enzymes in male and female schistosomes have distinct but overlapping substrate specificities. Four forms of glutathione S-transferase were detected, three of which, SmGST-1, SmGST-2, and SmGST-3, were purified to apparent homogeneity by glutathione affinity chromatography and HPLC chromatofocusing. The purified enzymes displayed very similar catalytic and physicochemical properties. They could be distinguished by differences in activity with ethacrynic acid and trans-4-phenyl-3-buten-2-one, but not with aryl halide substrates. The isoelectric points of SmGST-1, SmGST-2, and SmGST-3 were estimated to be 7.2, 7.1, 6.9, respectively. A polyclonal antiserum to SmGST-3 cross-reacted with the other two forms, but not with other soluble schistosome proteins. Each of the purified enzymes displayed an apparent subunit molecular weight of 28,500 by polyacrylamide gel electrophoresis under denaturing conditions. Gel filtration chromatography yielded a molecular weight of 30,800 for the catalytically active form of the enzyme. Unlike all known glutathione S-transferases, the three enzyme forms purified from S. mansoni appear to be catalytically active monomeric proteins.

Quantitative analysis of hemolymph monophenol oxidase activity in immune reactive Aedes aegypti

Abstract A hemocyte-mediated melanotic encapsulation reaction is elicited in adult Aedes aegypti in response to intrathoracically inoculated microfilariae of Dirofilaria immitis. The activity of monophenol oxidase in cell-free hemolymph collected from uninoculated, microfilariae-inoculated and saline-inoculated control mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of l -[3,5-3H]tyrosine to dopa. Enzyme activity in immune reactive hosts examined 2 days postinoculation was aproximately twice as high (96–206 nmol/min per mg protein) as in uninoculated or saline inoculated insects (34–80 nmol/min per mg protein). The augmented activity of the enzyme coincides in time with the early development of melanotic capsules around the microfilariae. The possible involvement of hemocytes in the activation and/or generation of monophenol oxidase in response to infection, and the metabolism of catecholamines in relation to insect immune responses are discussed.

Effects of juglone (5-hydroxy-1,4-naphthoquinone) on midgut morphology and glutathione status in Saturniid moth larvae.

Actias luna and Callosamia promethea larvae were fed birch foliage supplemented with juglone (5-hydroxy-1,4-naphthoquinone) to determine whether juglone causes oxidative stress in midguts of these species. Juglone is a substituent of walnut foliage. A. luna, but not C. promethea, thrives on walnut foliage, as well as birch foliage supplemented with juglone. After 2 and 3 days on juglone-containing diets, midgut samples from these animals were compared histologically and were analyzed for GSH and GSSG content. C. promethea, but not A. luna, midguts revealed partial loss of epithelial structure. In contrast, GSH and GSSG did not change significantly in either species. In a separate experiment, live midgut explants from each species were cultured for 4 h in 0, 0.05, and 0.25% juglone. In juglone-treated explants, GSSG increased 2.1 and 5.6-fold, respectively, for A. luna, and 1.6 and 2.7-fold, respectively, for C. promethea. There was also a small dose-dependent decrease in GSH in C. promethea, but not A. luna. Although histology indicates that the midgut is a target of juglone toxicity in C. promethea, GSH analyses from either species do not support the expectation that changes in GSH/GSSG explain differences in susceptibility to juglone toxicity.

Phenol oxidase activity in hemolymph compartments of Aedes aegypti during melanotic encapsulation reactions against microfilariae

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity was assessed in hemocytes, cell-free plasma and complete hemolymph collected from Aedes aegypti Liverpool strain, intrathoracically inoculated with saline alone, immune activated by the inoculation of Dirofilaria immitis microfilariae (mff), and uninoculated. Enzyme activities between groups were compared using a radiometric hydroxylation assay (MPO) and a high pressure liquid chromatography with electrochemical detection assay (DPO). There were no significant differences in enzyme activity in hemocytes, cell-free plasma, and complete hemolymph between uninoculated and saline-inoculated controls. Both MPO and DPO activity of mosquito hemocytes and complete hemolymph from immune-activated mosquitoes were significantly increased at 12 and 24 h postinoculation as compared with the enzyme activity from saline-inoculated mosquitoes, but no significant increase in enzyme activity was detected in cell-free plasma from immune-activated mosquitoes. Increases of MPO and DPO activity in hemocytes and hemolymph following immune activation were proportional, thereby suggesting that a single enzyme might react with both monophenols and o-diphenols within the hemolymph of A. aegypti. Results also suggest that augmented phenol oxidase activity associated with melanotic encapsulation reactions is associated primarily with hemocytes.

Hemocyte monophenol oxidase activity in mosquitoes exposed to microfilariae of Dirofilaria immitis.

Monophenol oxidase (MPO) activity in hemocytes collected from Aedes aegypti Liverpool strain and Aedes trivittatus intrathoracically inoculated with saline alone, inoculated with Dirofilaria immitis microfilariae (mff), or from uninoculated mosquitoes was compared using a radiometric tyrosine hydroxylation assay. Hemocyte MPO activity in mff-inoculated (= immune-activated) mosquitoes was significantly increased at 24 hr postinoculation (PI) in A. aegypti and at 6, 12, and 24 hr PI in A. trivittatus as compared with saline-inoculated controls. Baseline and immune-activated levels of hemocyte MPO activity in A. trivittatus were significantly higher compared with those seen in A. aegypti. Baseline hemocyte population levels were similar in both species, but immune activation did not elicit increases in total hemocyte populations in A. trivittatus as has been demonstrated for A. aegypti. Likewise, immune activation by the inoculation of mff did not significantly alter plasma MPO activity in A. trivittatus as compared with uninoculated or saline-inoculated mosquitoes. Plasma MPO activity in A. aegypti, however, appears to constitute a major component of the immune response. The importance of phenol oxidase(s) in the immune response of mosquitoes against mff and the relationship of observed differences in MPO activity to differences in immunological capability between A. aegypti and A. trivittatus are assessed.

IMMUNOCYTOCHEMICAL LOCALIZATION OF THE MAJOR GLUTATHIONE S-TRANSFERASES IN ADULT SCHISTOSOMA MANSONI

Indirect immunofluorescence was used to investigate the tissue distribution of the major isoenzymes of Schistosoma mansoni glutathione S-transferase (GSH S-transferase). When polyclonal rabbit antisera against GSH S-transferase isoenzymes SmGST-1, -02, and -3 were applied to cryostat or plastic-embedded sections of fixed adult worms, a punctate pattern of enzyme distribution was observed that was restricted to the parenchyma. Labeling was much more pronounced in males than females, consistent with the biochemically determined distribution of these enzymes between the sexes. Intense immunolabeling was noted within the subectocytoplasmic core tissue of the tubercles of the male that appeared to be connected to deep parenchymal cells by immunoreactive cell processes. Immunofluorescence could be blocked completely by prior incubation of antisera with affinity-purified enzyme. Although schistosome GSH S-transferases have been reported to be protective antigens, no immunoreactivity was detected within or on the tegument, including the dorsal spines of the male. The lack of tegumental immunoreactivity was confirmed by immunoblotting of tegumental membrane preparations following SDS-PAGE. Muscle fibers, vitelline cells, and cecal epithelium also failed to react. The fact that the GSH S-transferases were not uniformly distributed among all parenchymal cells suggests the existence of subpopulations of parenchymal cells that are preferentially involved in the conjugation of electrophiles with glutathione.

Schistosoma mansoni : single-step purification and characterization of glutathione S-transferase isoenzyme 4

A soluble glutathione S-transferase isoenzyme, designated SmGST-4 was purified to apparent homogeneity in a single step from the cytosol of adult Schistosoma mansoni by selective elution of the enzyme from a glutathione-agarose affinity column using glutathione disulfide. SmGST-4, which comprised about 5% of the bound glutathione S-transferase activity, could be distinguished from the previously characterized glutathione S-transferase isoenzyme family (SmGST-1/2/3), by its unique chromatographic behavior, lower subunit M(r) (26,000), differences in substrate specificity and inhibitor sensitivity, and a lack of reactivity with antiserum to SmGST-3. The purified isoenzyme catalyzed the conjugation of several model xenobiotics including 1-chloro-2,4-dinitrobenzene, ethacrynic acid, and trans-4-phenyl-3-buten-2-one. Like the SmGST-1/2/3 isoenzyme family, SmGST-4 failed to catalyze the conjugation of a model epoxide substrate, 1,2-epoxy-3-(p-nitrophenoxy)propane. Because glutathione S-transferases from other organisms play a role in protecting cells against the toxic products of lipid peroxidation, SmGST-4 and the members of the SmGST-1/2/3 isoenzyme family were tested for their capacity to reduce cumene hydroperoxide and to catalyze the conjugation of 4-hydroxyalk-2-enals. Although all four isoenzymes catalyzed both reactions, the specific activity of SmGST-1, SmGST-2, and SmGST-3 toward cumene hydroperoxide was at least 10-fold greater than that of SmGST-4. In contrast, the latter more effectively conjugated a homologous series of 4-hydroxyalk-2-enal isomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Schistosoma mansoni : glutathione S-transferase-catalyzed detoxication of dichlorvos

Dialyzed cytosol of adult Schistosoma mansoni worm pairs catalyzed the glutathione-dependent O-demethylation of dichlorvos (2,2-dichlorovinyl dimethylphosphate), the active form of the antischistosomal drug metrifonate, to form a thioether conjugate, S-methylglutathione, and desmethyl dichlorvos. The reaction rate was dependent on both time and protein concentration, and no product was formed when either dichlorvos or glutathione was omitted from the reaction mixture. Female worm cytosols were about 2.5-fold more active per milligram of protein that those of males. Partial purification of glutathione S-transferases from male worms by affinity chromatography on glutathione-agarose showed that the reaction could be catalyzed by a preparation containing the three major isoenzymes, but that the unbound fraction, which contains at least one additional form of the enzyme that is particularly active with epoxide substrates, was 16-fold more active toward dichlorvos than the bound fraction. S-Methylglutathione also was formed by S. mansoni worm pairs incubated in the presence but not in the absence of dichlorvos. Because GSH S-transferase-catalyzed metabolism of dichlorvos results in the formation of desmethyldichlorvos, which unlike the parent compound is not an effective acetylcholinesterase inhibitor, the reaction represents a pathway of detoxication in schistosomes. It is the first example of a clinically used schistosomicide shown to be detoxicated by a conjugation pathway. These results raise the possibility that dichlorvos detoxication by S. mansoni may help explain why this species is normally refractory to metrifonate.

Effect of pairing in vitro on the glutathione level of male Schistosoma mansoni.

The effect of in vitro incubation on the level of the intracellular nucleophile, glutathione (GSH), in adult Schistosoma mansoni was investigated. The GSH levels of freshly collected adult male and female parasites were 8.5 +/- 2.5 and 2.7 +/- 0.7 nmol/10 worms, respectively, as determined by an enzymatic assay. Twenty-four-hour incubation of unpaired males in RPMI-1640 medium at 37 C resulted in a 1.7-fold increase (P less than 0.001) in GSH level that remained elevated for at least 7 days. The increase was dependent on exogenous L-cystine, suggesting that it was due to biosynthesis of GSH. Biosynthesis in male S. mansoni was confirmed by isolating [3H] GSH from parasites incubated in medium containing L-[3H] cystine or [3H] glycine. In contrast to unpaired males, the GSH level of paired males as well as that of unpaired or paired females did not increase after 24 hr in vitro. When males that had been incubated unpaired for 24 hr were allowed to couple in vitro with freshly collected females, their GSH level fell to that of continuously paired males. These observations provide evidence that in vitro female schistosomes can influence the physiology of the male.

Differential toxicity of juglone (5-hydroxy-1,4-naphthoquinone) and related naphthoquinones to saturniid moths.

The preferred hosts of the saturniid mothActias luna include members of the Juglandaceae, whose foliage contain the toxin juglone (5-hydroxy-1,4-naphthoquinone). The performance ofActias luna andCallosamia promethea was compared when fourth-instar larvae of each were fed birch foliage, a mutually acceptable food plant, or birth supplemented with 0.05% (w/w) juglone.A. luna fed juglone exhibited no changes in developmental time or mortality compared to a diet without juglone. In contrast, juglone-supplemented diets, when fed toC. promethea, caused negative growth rate, and a 3.6-fold decrease in consumption rate. The performance ofA. luna also was compared on birch and walnut; larvae developed and grew more rapidly on an all-walnut vs. an all-birch diet. To examine the effect of 1,4-naphthoquinone structure onA. luna survival, first instars were fed on birch supplemented with varying concentrations of juglone (J), menadione (M), plumbagin (P), or lawsone (L). In diets supplemented at 0.05% (w/w), none of the compounds produced effects significantly different from controls. In diets supplemented at 0.5% (w/w), the treatments produced significant toxic effects in the order P>M=L>J for mortality, and P>L>M=J for increased developmental time. Late-instarA. luna are clearly resistant to juglone compared toC. promethea, and early-instarA. luna are resistant to several related 1,4-naphthoquinones. These results suggest a chemical basis for host choice among saturniids. In addition, the luna-walnut system may be a valuable model for studying quinone detoxication.