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J W Tracy

Publications and source records attributed to J W Tracy.

At least 19 recordsLinked to original sources
PubMed2000

SmMAK16, the Schistosoma mansoni homologue of MAK16 from yeast, targets protein transport to the nucleolus.

The SmMAK16 gene from Schistosoma mansoni was cloned by chance when an adult worm cDNA library was probed with antiserum to affinity-purified S. mansoni GSH S-transferases. SmMAK16 encodes a hydrophilic protein of 259 amino acids with a molecular mass of 31 kDa. The protein shares 43% sequence identity and 66% similarity to the nuclear protein MAK16 of Saccharomyces cerevisiae that has been implicated both in cell cycle progression and biogenesis of 60S ribosomal subunits. Both proteins display a similar degree of sequence similar to the hypothetical protein CeMAK16 from Caenorhabditis elegans. These proteins share a number of apparent protein motifs, including two nuclear localization signals (NLS), multiple sites for phosphorylation by protein kinase CK2 and four conserved cysteine residues that resemble a zinc binding domain. SmMAK16 mRNA is more highly expressed in adult female worm than males. Recombinant SmMAK16 was phosphorylated by human protein kinase CK2. When chimeric constructs containing SmMAK16 fused the green fluorescent protein (GFP) were transiently transfected into COS-7s cells, the reporter was localized not in nuclei, but exclusively in nucleoli. The yeast and nematode homologues were likewise able to direct nucleolar accumulation of the fluorescent reporter. The high degree of sequence conservation together with the ability to direct nucleolar protein transport supports the hypothesis that MAK16 proteins play a key role in the biogenesis of 60S subunits.

Amino Acid Sequence↗
PubMed1998

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.

Animals↗
PubMed1997

Schistosoma japonicum GSH S-transferase Sj26 is not the molecular target of praziquantel action.

It has been suggested that Sj26, a Schistosoma japonicum GSH S-transferase, is the molecular target of the antischistosomal drug praziquantel (McTigue et al., 1995, J. Mol. Biol. 246, 21-27). We tested this hypothesis by asking two questions: (1) does praziquantel inhibit Sj26 activity with a variety of model substrates; and (2) does praziquantel prevent the binding to Sj26 of physiologically relevant nonsubstrate ligands? High concentrations of praziquantel (up to 500 microM) did not inhibit Sj26 activity using the model substrates 1-chloro-2,4-dinitrobenzene, 3,4-dichloronitrobenzene, or ethacrynic acid. Sj26 had no measurable activity with two higher molecular weight GSH S-transferase substrates: 5-androsten-3,17-dione and sulfobromophthalein. We also assessed the ability of praziquantel to prevent the inhibition of Sj26 by a series of S-alkyl-GSH conjugates. The half-maximal inhibitory concentrations of S-hexyl-GSH, S-octyl-GSH, and S-decyl-GSH (10, 10, and 5 microM, respectively) for Sj26 were not affected by up to 500 microM praziquantel. This suggests that praziquantel does not compete with GSH for Sj26 binding. In order to determine if praziquantel disrupts binding of nonsubstrate ligands to Sj26, we tested praziquantel for its ability to prevent the inhibition of Sj26 by both bilirubin and hematin. Praziquantel (100 or 500 microM) did not alter inhibition of Sj26 by 3 microM bilirubin, but partially protected Sj26 against inhibition by hematin (0.1 to 2.0 microM). Interestingly, in a similar reaction, 100 microM S-methyl-GSH protected Sj26 from inhibition equally as well as praziquantel. Bovine serum albumin (5 microM) completely protected against inhibition by 1 microM hematin. These results indicate that although praziquantel partially protects Sj26 from hematin inhibition, this protection is neither specific to praziquantel nor physiologically relevant. Our results do not support the hypothesis that the mechanism of praziquantel action involves competitive inhibition of Sj26 catalytic activity or blocking binding of nonsubstrate ligands. We can, therefore, find no evidence that Sj26 is the molecular target of the antischistosomal activity of praziquantel.

Animals↗
PubMed1993

Induction of Schistosoma mansoni glutathione S-transferase by xenobiotics.

The effects of 3-methylcholanthrene, butylated hydroxyanisole, and phenobarbital on the expression of glutathione S-transferase (GST, EC 2.5.1.18) were examined in the human parasite Schistosoma mansoni. GST specific activity toward 1-chloro-2,4-dinitrobenzene increased by 170% in parasites recovered from mice injected with 3-methylcholanthrene and 230% in parasites recovered from mice maintained on a diet containing butylated hydroxyanisole. These increases in specific enzyme activity were paralleled by accumulation of mRNA hybridizing to pGT16.4, a cDNA clone that encodes the most abundant GST subunit, SmGST-3. Northern hybridization analysis showed a 5-fold increase in mRNA hybridizing to pGT16.4 72 h after exposure to 3-methylcholanthrene, a 10-fold increase after 12 days exposure to butylated hydroxyanisole, and a 6-fold increase 16 h after treatment with phenobarbital. In contrast, no accumulation of mRNA hybridizing to either of two other cDNA clones that encode the SmGST-4 and SmGST-6 subunits was detected. Hybrid select translation using pGT16.4 combined with reverse-phase high-pressure liquid chromatographic analysis demonstrated that in addition to SmGST-3 mRNA, the clone also hybridized to mRNA species encoding the SmGST-1 subunit, a member of the same isoenzyme family. High-pressure liquid chromatographic analysis of GST affinity purified from butylated hydroxyanisole-exposed parasites revealed a 2.5-fold increase in the concentration of SmGST-1 and SmGST-3 present compared with an equivalent amount of tissue from control organisms. There was no change, however, in the SmGST-1 to SmGST-3 ratio (1:6), indicating that both subunits were induced to the same extent by this agent. The results of these studies suggest that alterations in GST expression may influence the parasite's survival within the host environment.

Animals↗
PubMed1992

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)

Animals↗
PubMed1992

Relationship of hemolymph phenol oxidase and mosquito age in Aedes aegypti.

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity in hemocytes and cell-free plasma perfused from 7-, 14-, 21-, and 28-day-old Aedes aegypti mosquitoes were compared. A progressive decrease of enzyme activity was detected as mosquito age increased, and this decrease was significant in both hemocytes and cell-free plasma when mosquitoes were 28 days old as compared with that found in 7-day-old mosquitoes. There was no significant difference in total hemolymph protein as mosquito age increased. Although this decreased MPO and DPO activity might be partially responsible for the reduced immune response against filarial worms previously reported for older mosquitoes, other factors undoubtedly play a significant role.

Aedes↗
PubMed1992

External gamma-ray counting of selected tissues from a Thorotrast patient.

Results of gamma-ray measurements of selected tissues from a patient who was injected with Thorotrast almost 36 y ago are reported. The purposes of this study were: 1) to determine the relative tissue distribution and activities of specific radionuclides in the 232Th decay chain, specifically 228Ra (as measured by 228Ac), 212Pb, and 224Ra (measured directly and as measured by 212Pb), and 2) to evaluate the level of radioactive disequilibrium among the daughter products. The spleen and liver had the highest concentrations of radioactivity. Bone also appears to be a long-term sink for 232Th daughter products based on estimates from a small portion of one rib. Larynx and esophagus contained measurable activity, which may have been due to their proximity to the "Thorotrastoma." Radioactivity in the remaining measured tissues were low, as expected. Secular equilibrium could be demonstrated in bone, pancreas, larynx, esophagus, and breast. Significant disequilibrium was observed for spleen, liver, kidney, and red blood cells. Radioactivity measurements reported here will be useful in estimating radiation doses to selected tissues. Such dose estimates are valuable in refining current risk estimates (e.g., liver) and in identifying tissues at risk for further epidemiologic studies. These results, while consistent with other published studies, should be interpreted with caution since measurements were made on only one patient.

Aged↗
PubMed1991

Analysis of glutathione S-transferase-catalyzed S-alkylglutathione formation by high-performance liquid chromatography.

Metabolism of alkyl halides and some organophosphorous compounds by glutathione S-transferases (EC 2.5.1.18) leads to formation of an S-alkylglutathione as a common product. We have developed an HPLC assay for formation of S-methylglutathione and S-ethylglutathione that is applicable to measuring enzyme activity toward a variety of xenobiotic substrates. The conjugates are derivatized with 1-fluoro-2,4-dinitrobenzene to form the corresponding N-2,4-dinitrophenyl derivatives, which are then separated by reverse-phase HPLC with gradient elution. The utility of the method is illustrated by the use of partially purified preparations of rat liver glutathione S-transferases and several prototypic substrates including iodomethane, iodoethane, dichlorvos, and methyl parathion. The limit of detection is about 50 pmol of N-(2,4-dinitrophenyl)-S-alkylglutathione. Advantages of the method over other assays of S-alkyl transferase activity are discussed.

2,4-Dinitrophenol↗
PubMed1991

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.

Animals↗
PubMed1990

Electrochemical determination of diphenol oxidase activity using high-pressure liquid chromatography.

A quantitative assay for the diphenol oxidase activity of tyrosinase (EC 1.14.18.1) using high-pressure liquid chromatography with electrochemical detection is described. The assay is based on the observation (M. Sugumaran, 1986, Biochemistry 25, 4489-4492) that tyrosinase catalyzes the oxidative decarboxylation of 3,4-dihydroxymandelic acid to 3,4-dihydroxybenzaldehyde. The substrate and product were readily separated on a reverse-phase column equilibrated with 0.1 M citrate buffer, pH 3.2, containing 0.5 mM Na2 EDTA, and 5% (v/v) acetonitrile. The reaction of DHMA with mushroom tyrosinase was linear with time and proportional to the amount of enzyme present. The specific activity of mushroom tyrosinase using the method was about fourfold greater than that obtained using a spectrophotometric assay for diphenol oxidase following dopachrome formation from L-3,4-dihydroxyphenylalanine. The applicability of the high-pressure liquid chromatographic assay to determination of diphenol oxidase activity in small biological sample sizes was demonstrated by using microgram quantities of crude, cell-free hemolymph from Aedes aegypti mosquitoes.

Aedes↗
PubMed1990

High-pressure liquid chromatographic analysis of hemolymph plasma catecholamines in immune-reactive Aedes aegypti.

Tyrosine and catecholamines have been implicated as substrates for the encapsulation reactions involved in the immune response of mosquitoes to microfilariae (mff). Identification and quantitation of tyrosine and catecholamines present in Aedes aegypti hemolymph plasma were accomplished by ion-pair high-pressure liquid chromatography with electrochemical detection at either +650 or +850 mV vs Ag/AgCl. Tyrosine, dopamine, and N-beta-alanyldopamine were detected in the hemolymph plasma of naive A. aegypti. Although no differences in these compounds were observed in hemolymph plasma from A. aegypti inoculated with Dirofilaria immitis mff, the chromatogram showed a single major peak (PI) (65 microM, expressed as dopamine equivalents) that was not present in naive hemolymph plasma. Saline-inoculated controls contained only 5% of the PI in immune reactive hemolymph plasma. A high concentration of PI (127 +/- 39 microM) was also detected after treatment of hemolymph plasma with mild alkaline conditions (pH 9.0), indicating that it is normally present as an electrochemically inert form in naive mosquitoes. High concentrations of PI were also detected in the naive hemolymph plasma from three other mosquito species, but no PI was found in A. trivittatus under any conditions. PI did not cochromatograph with any of the catecholamines commonly thought to be involved in immune responses of dipterans against metazoan parasites, suggesting that it may be a unique substrate for these reactions. The biological relevance of PI was evidenced by its appearance in the hemolymph plasma of two strains of D. immitis-inoculated A. aegypti.

Aedes↗
PubMed1989

Studies of mechanisms of niridazole-elicited embryotoxicity: evidence against a major role for covalent binding.

Studies reported here were designed to examine the hypothesis that covalent binding of reactive intermediates to macromolecules of the conceptus represents a major mechanism for the embryotoxicity of niridazole (NDZ). The roles of embryonic thiol content and oxygenation on: 1) malformation incidence; 2) reductive metabolism; and 3) covalent binding to embryonic macromolecules of metabolites resulting from reductive biotransformation of NDZ were studied. Results were compared with those from studies with the nondysmorphogenic analog of NDZ, 4'-methylniridazole (MNDZ). Day 10 rat embryos were pretreated for 5 hours in vitro with either L-buthionine-S, R-sulfoximine (BSO) or N-acetylcysteine (NAC) to modulate their glutathione (GSH) content. BSO reduced GSH levels, but NAC was ineffective. Following pretreatment, embryos were cultured for an additional 15 hours in the presence of [14C]NDZ or [14C]MNDZ with an initial oxygen concentration of 5%. At the end of the culture period (day 11, AM), those embryos with active heartbeat and vitelline circulation were examined for asymmetric malformations. Drug metabolites were subjected to multiple extractions from the culture medium and subjected to quantitative high-performance liquid chromatography (HPLC) analysis. Homogenates of the embryos were extracted with trichloroacetic acid (TCA) to estimate the covalent binding of radiolabeled parent compound/metabolites. Autoradiographic analyses were performed on other embryos. BSO pretreatment, which reduces embryonic GSH tissue levels, dramatically increased both the conversion of NDZ to 1-thiocarbamoyl-2-imidazolidinone (TCI) (generated via reductive metabolism of NDZ) and covalently bound label but failed to increase embryotoxicity. NAC, by contrast, did not significantly affect embryonic GSH levels, TCI generation, or covalent binding. Because both rates of metabolism of NDZ to TCI and covalent binding could vary independently of malformation incidence, we concluded that they do not represent critical mechanistic factors for the embryotoxicity of NDZ and related nitroheterocycles.

Animals↗
PubMed1989

Schistosoma mansoni: influence of the female parasite on glutathione biosynthesis in the male.

The glutathione (GSH) content of male Schistosoma mansoni increases in the absence of the female. This phenomenon, originally observed in vitro, also occurs within the host. At the time of recovery from mice, the GSH content of males from single-sex infections was 1.7-fold higher than that of paired males from mixed sex infections (P less than 0.01). The effect of mating status on male GSH biosynthetic and turnover rates was examined to determine the basis for increased GSH content in unpaired males. GSH turnover rates, measured when GSH biosynthesis was inhibited by greater than 95% with 5.0 mM DL-buthionine-SR-sulfoximine, were indistinguishable between unpaired and paired males with a first-order rate constant of 0.018 hr-1. In contrast, incorporation of L-[35S]cysteine into GSH revealed that GSH biosynthesis was 5-fold higher in unpaired than in paired males. Transport of L-cystine into male schistosomes, the presumed rate-limiting step in GSH biosynthesis, was unaffected by mating status. The GSH content increased when males were incubated in medium that had previously contained females or when separated from females by a microporous membrane. Males paired to 50% ethanol-fixed females had unchanged GSH content in vitro. It appears that male GSH biosynthesis may be regulated by a response stimulated by the female's physical presence in the gynechophoral canal and not by a soluble factor released from the female.

Animals↗
PubMed1989

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.

Aedes↗
PubMed1989

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.

Animals↗
PubMed1988

Niridazole metabolism by rat embryos in vitro.

We report the results of studies on the reductive activation of the schistosomicidal agent, niridazole (NDZ). Intact rat embryos in vitro reduced this compound, generating a stable metabolite in the presence of 5% O2. By contrast, embryo and yolk sac homogenates or liver microsomes appeared to require anaerobiasis. Malformation incidence--specifically, axial asymmetry--showed a strong correlation with nitroreductase activity rates when the latter were modulated by oxygen tension. Data presented here suggest that when embryos are exposed to NDZ under conditions of low oxygen in vitro, redox cycling ensues with molecular oxygen serving to oxidize early reduction products. This process continues, regenerating the parent compound until oxygen is depleted locally. The basis of this localized depletion is unknown, but inability of the immature supply system to replete oxygen or demand by precociously aerobic tissues may be involved. Once local anaerobiasis is attained, further reduction could generate toxic metabolites capable of covalently binding cellular macromolecules. Localized hypoxia represents another potential mechanism of dysmorphogenesis.

Abnormalities, Drug-Induced↗
PubMed1988

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.

Animals↗