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J W Tracy

Publications and source records attributed to J W Tracy.

At least 37 records · Page 2Linked to original sources
PubMed1988

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.

Animals↗
PubMed1985

Evaluation of a purified Schistosoma japonicum glycoprotein egg antigen for the immunodiagnosis of infection in man.

A purified glycoprotein, glycoprotein-2, which was isolated from Schistosoma japonicum soluble egg antigen, was tested in an enzyme-linked immunoadsorbent assay for its potential as an immunodiagnostic reagent. Using empirically-derived criteria of endpoint titer, 39/40 sera from S. japonicum-infected individuals were scored correctly as positive in a blind test. Of these, 26/40 were from individuals who excreted fewer than 100 eggs per gram of stool. No cross reactions were observed with sera from uninfected individuals (0/20) or from individuals infected with S. mansoni (0/22), Trichinella spiralis (0/6) or Brugia malayi (0/4). Cross reactions were observed with 3/24 individuals infected with S. haematobium who excreted greater than 2,000 eggs per 10 ml urine. Glycoprotein-2 appears to satisfy the requisite criteria of specificity and sensitivity to warrant further evaluation.

Animals↗
PubMed1984

1-thiocarbamoyl-2-imidazolidinone, a metabolite of niridazole in Schistosoma mansoni.

Niridazole, a nitro heterocyclic antischistosomal drug, is extensively metabolized to unknown metabolites by Schistosoma mansoni. We report that 1-thiocarbamoyl-2-imidazolidinone was isolated by high pressure liquid chromatography and identified by high resolution electron impact mass spectroscopy as a niridazole metabolite in schistosomes. After a 20-h in vitro incubation in 30 ml of medium containing 10 micrograms ml-1 [14C]niridazole (5.2 Ci mol-1), 100 S. mansoni worm pairs contained approximately 275 ng of 1-thiocarbamoyl-2-imidazolidinone. This amount represented 4% of the total metabolized fraction of niridazole in the parasite. Incubation of schistosomes with 1-thiocarbamoyl-2-[2 14C]imidazolidinone (2.7 Ci mol-1) indicated that this metabolite was not taken up. However, schistosomes released an average of 44 ng ml-1 or 1% of the total 1-thiocarbamoyl-2-imidazolidinone found in the worm back into 1 ml of medium during incubation. No host oxidative metabolites of niridazole were found in the parasites.

Animals↗
PubMed1983

Reductive metabolism of niridazole by adult Schistosoma mansoni. Correlation with covalent drug binding to parasite macromolecules.

Niridazole, an antischistosomal nitrothiazole derivative, is metabolized by adult Schistosoma mansoni to one or more reactive intermediates, as evidenced by extensive covalent binding of [14C]niridazole to parasite macromolecules. When worm pairs were incubated for 16 hr in culture medium containing 70 microM [14C]niridazole, 26-34% of the total parasite-associated radioactivity was irreversibly bound to trichloroacetic acid-precipitable material. Drug binding was both time- and [14C]niridazole concentration-dependent. Of the bound drug fraction, 85-90% was associated with parasite proteins, 3-5% with RNA and 4-7% with DNA. When schistosomes were recovered from infected mice, treated with periodic doses of [14C]niridazole, over 40% of the total parasite-associated radioactivity was bound to macromolecules. Niridazole caused up to a 40% decrease in the concentration of total nonprotein thiols in intact schistosomes incubated with the drug over an 8-hr period. Under strictly anaerobic conditions, cell-free schistosome preparations catalyzed a reduced pyridine nucleotide-dependent reduction of niridazole's essential nitro group, as evidenced by disappearance of absorption at 400 nm. Net nitroreduction did not occur under aerobic conditions, although the drug did stimulate oxidation of the pyridine nucleotide cofactor. Covalent binding of [14C]niridazole also took place in this cell-free system, with requirements identical with those needed for enzymatic nitroreduction. Covalent drug binding, but not nitroreduction, was inhibited up to 80-85% by 2 mM L-cysteine, N-acetyl-L-cysteine, or glutathione; S-carboxymethyl-L-cysteine, which has no free sulfhydryl group, was not inhibitory. [14C]4'-Methylniridazole, a nonschistosomicidal analogue of niridazole, was taken up by intact schistosomes in vitro, but was not metabolized and did not bind covalently to parasite macromolecules. Furthermore, 4'-methylniridazole did not affect the concentration of nonprotein thiols in intact parasites and did not serve as a substrate for schistosomal nitroreductase in vitro. These results indicate a positive correlation between proximal metabolic activation of niridazole within these facultative anaerobic organisms and its antiparasitic activity.

Animals↗
PubMed1982

Suppression of cell-mediated immune responses in vivo and in vitro by 1-thiocarbamoyl-2-imidazolidinone.

Both in vivo and in vitro, the niridazole metabolite, 1-thiocarbamoyl-2-imidazolidinone (TCI), was found to suppress various manifestations of cell-mediated immunity. In vivo, doses of 10(-6) g/kg every other day delayed rejection of BALB/cJ (H--2d) skin allografts by C57BL/6J (H--2b) mice. Much lower single doses (approximately 10(-11) g/kg) markedly reduced both pulmonary granuloma formation around. Schistosoma mansoni eggs in C57BL/bJ mice and delayed footpad swelling in sensitized CF1 mice responding to S. mansoni egg antigen (SEA). Reduction of footpad swelling occurred when TCI was given 24 h before, but not 4 hr after, antigenic challenge. In vitro. SEA-dependent production of eosinophil stimulation promoter (ESP) activity by sensitized mouse spleen cells was decreased greater than 90% by 10(-10)g TCI/ml. TCI failed, however, to inhibit the action of preformed ESP lymphokine on eosinophils. The blastogenic response of sensitized lymph node cells to SEA was reduced by greater than 70% by 10(-11) to -10 (-8)g TCI/ml. The drug produced no effect on either antigen-dependent lymphokine production or blastogenesis if it was added 5 min after, instead of before, stimulating antigen.

Animals↗
PubMed1982

Opsonic requirements for phagocytosis of Borrelia hermsii by human polymorphonuclear leukocytes.

Opsonic requirements for phagocytosis by human polymorphonuclear leukocytes (PMNLs) of a laboratory strain of Borrelia hermsii were examined. Intracellular localization of spirochetes was confirmed by electron microscopy and cinephotomicroscopy. Phagocytosis of 3H-labeled spirochetes was increased with higher concentrations of pooled human serum or greater ratios of spirochetes to PMNLs and was unaffected by diminished classical complement pathway activity. Immune rabbit serum markedly increased phagocytosis of spirochetes beyond levels observed with preimmune rabbit serum. Immune rabbit serum also demonstrated direct activity against B. hermsii, which decreased the motility and viability and increased agglutination of the spirochetes independently of complement. Both the direct activity against B. hermsii and the opsonic activity of immune rabbit serum were eliminated by immunoabsorption of immunoglobulins. These observations suggest a role for the alternative complement pathway and for immunoglobulins in the opsonization of spirochetes for phagocytosis by PMNLs. Immunoglobulins may also play a role in the direct clearance of spirochetes.

Animals↗
PubMed1982

Direct and indirect effects of soluble extracts of Schistosoma mansoni eggs on fibroblast proliferation in vitro.

The possibility that soluble products of Schistosoma mansoni eggs might participate in the pathogenesis of hepatic fibrosis in schistosomiasis was investigated. Both crude saline extracts of eggs (soluble egg antigen [SEA]) and a partially purified SEA fraction contained activity which stimulated guinea pig and human dermal fibroblasts to proliferate in vitro, as measured by uptake of [3H]thymidine. Maximum activity was present in fractions which eluted from Sephacryl S-200 with an apparent molecular weight of less than or equal to 12,500 and in fractions which had an estimated pI 8, as determined by preparative isoelectric focusing of partially purified SEA. Activity in crude SEA was not removed by chromatography on concanavalin A-Sepharose 4B. When concanavalin A-binding glycoproteins lacking intrinsic fibroblast-stimulating activity were incubated with spleen cells from infected or uninfected mice, fibroblasts-stimulating activity was detected in the culture supernatants. Thus, SEA contains two functionally distinct molecular species. One of these directly stimulates fibroblasts, whereas the other induces the release of a fibroblast-stimulating activity from lymphocytes or macrophages or both. Since these fibroblast-stimulating factors might be elaborated in the livers of infected individuals, these observations suggest a potential role of soluble schistome products in the pathogenesis of hepatic fibrosis in schistosomiasis.

Animals↗
PubMed1982

Isolation of Schistosoma japonicum egg glycoprotein antigens which sensitize mice to lung granuloma formation and elicit an immediate hypersensitivity response.

Crude Schistosoma japonicum soluble egg antigen (SEA) could be resolved into at least 25-30 Coomassie blue-reactive polypeptides when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Two of these polypeptides also gave a strong reaction for carbohydrate with the periodic acid-Schiff (PAS) reagent. Although only four antigens could be demonstrated by double immunodiffusion in agar against the sera of mice with a 9-week S. japonicum infection, double antibody precipitation of 125I-labeled SEA, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, revealed the presence of at least 20-22 antigenic species. Crude SEA was fractionated by Concanavalin A-Sepharose 4B chromatography. The resulting fractions, together with crude SEA, were tested for their ability to subcutaneously sensitize mice to lung granuloma formation around S. japonicum eggs subsequently injected intravenously. Strikingly, only the egg glycoprotein antigen fraction, which bound to the lectin column and was eluted with alpha-methyl-D-mannoside, possessed demonstrable sensitizing activity. Likewise, only this glycoprotein fraction elicited a significant immediate (15-min) footpad response, when injected into the footpads of S. japonicum-infected mice. The biologically active glycoproteins were further purified by repetitive gel filtration chromatography. Two of the isolated glycoproteins (designated gp-1 and gp-2) were shown to be greater than or equal to 90% pure and to be PAS-reactive; whereas the third (gp-3) was found to be still heterogenous. When the individual purified glycoproteins were tested in the lung granuloma model, all three displayed significant sensitizing activity; however, gp-2 appeared to be the most active. Similarly, the same glycoprotein antigen elicited a significantly greater immediate footpad reaction in infected mice than did the others.

Animals↗
PubMed1982

Adoptive transfer of modulation of granuloma formation and hepatosplenic disease in murine schistosomiasis japonica by serum from chronically infected animals.

The course of disease due to Schistosoma japonicum in C57BL/6J mice was examined. Animals infected with 25 cercariae of the Philippine strain of S. japonicum for 9 wk develop a peak portal hypertension of 13 +/- 0.6 cm H2O and mean hepatic granuloma area of 459 X 10(3) micrometers 2. By 30 wk, disease manifestations were reduced; mean portal pressure was 7 +/- 0.5 cm H2O and mean hepatic granuloma area was 12 X 10(3) micrometers 2. Adoptive transfer of serum but not 1 X 10(8) spleen or 4 X 10(7) lymph node cells from chronically infected mice resulted in lowering portal pressure and decreasing granuloma size in recipients. Granuloma formation in the sensitized lung model was also suppressed by serum from chronically infected mice. The immunosuppressive component of this serum was localized in the IgG1 fraction.

Acute Disease↗
PubMed1981

Activation of antigen-specific suppressor cells in human schistosomiasis mansoni by fractions of soluble egg antigens nonadherent to Con A sepharose.

The nature of the mononuclear suppressor cell and the fraction of crude soluble egg antigen (SEA) capable of activating antigen-specific suppressor cells were studied in 32 Egyptian children infected with schistosomiasis mansoni. Crude SEA was fractionated on Con A Sepharose, and the unbound and bound material (eluted with alpha-methyl-D-mannoside) was recovered for use in the generation of suppressor cells by the use of a co-culture technique. The glycoprotein fraction was further purified by DEAE cellulose chromatography to isolate the major serologic antigen (MSA-1). We found that both the crude SEA and the "protein" fraction (nonadherent to Con A Sepharose) of SEA was capable of inducing antigen-specific suppressor cell activity in a dose-dependent manner. The glycoprotein fraction of SEA (including MSA-1) was responsible for inducing nonspecific suppression. The antigen-specific suppressor cell was characterized in terms of its adherence properties of plastic and the presence of a receptor for sheep erythrocytes (E). The nonadherent, E-rosette-positive mononuclear cells were found to be activated by the crude SEA, and the "protein" fraction of SEA was found to exert an antigen-specific suppressor influence. Clinically, suppressor cell activity was found to be statistically greater in patients with a high intensity of infection compared to those with a low intensity of infection, but it was not different in patients with regard to the presence or absence of organomegaly. These results provide further information on one of the immunoregulatory mechanisms operative in human S. mansoni infection and on the nature of the antigen(s) that activates this response during the course of infection.

Antigens↗
PubMed1981

The formation of 1-thiocarbamoyl-2-imidazolidinone from niridazole in mouse intestine.

This study was designed to identify the site of formation of 1-thiocarbamoyl-2-imidazolidinone (TCI), a potent immunoactive metabolite of the antihelminthic drug, niridazole. When niridazole was administered intragastrically to C57Bl/6J mice, a 4-hr delay was observed before TCI was detected in the serum. By contrast, 4-hydroxyniridazole, a marker of hepatic niridazole metabolism, appeared in the serum within 30 min. Changing the route of niridazole administration from intragastric to intracaecal abolished the lag period in the rise of serum TCI concentrations relative to the 4-hydroxyniridazole marker. Pretreatment of mice with neomycin sulfate reduced the amount of TCI excreted in the urine by about 90% over a 24-hr period, but did not affect the amount of 4-hydroxyniridazole excreted. Injection of niridazole into isolated segments of mouse intestine resulted in TCI production, with the greatest conversion noted in the caecum. Subsequent incubation of niridazole with suspensions of mouse caecum contents in vitro also resulted in the formation of TCI, but not 4-hydroxyniridazole. Attempts to demonstrate TCI formation in vitro with various fractions of mouse liver were unsuccessful. These results indicate a dissociation of TCI formation from the major hepatic pathway of niridazole metabolism and support the view that TCI is formed from niridazole in the gastrointestinal tract as a result of the action of intestinal microflora.

Animals↗
PubMed1975

Reversible, coenzyme-A-mediated inactivation of biosynthetic condensing enzymes in yeast: a possible regulatory mechanism.

alpha-Isopropylmalate synthase [3-hydroxy-4-methyl-3-carboxyvalerate 2-oxo-3-methylbutyrate-lyase (CoA-acetylating); EC 4.1.3.12], the enzyme catalyzing the first committed step in leucine biosynthesis, and homocitrate synthase [3-hydroxy-3-carboxyadipate 2-oxoglutarate-lyase (CoA-acetylating); EC 4.1.3.21], the first enzyme in lysine biosynthesis in yeast, are rapidly inactivated in the presence of low concentrations of coenzyme A, a product of both reactions. Closely related compounds like 3-dephospho-coenzyme A or oxidized coenzyme A are almost without effect, as are other sulfhydryl compounds. Citrate (si)-synthase [citrate oxaloacetate-lyase (pro-3S-CH2-COO-minus leads to acetyl-CoA); EC 4.1.3.7] appears to be completely resistant against inactivation by coenzyme A. Inactivated alpha-isopropylmalate and homocitrate synthases can be reactivated by dialysis, but not by adding excess substrate. Protection against coenzyme-A-mediated inactivation is provided by relatively high concentrations of the alpha-ketoacid substrate or the specific end product inhibitor of each of the two enzymes. The coenzyme-A-mediated inactivation of alpha-isopropylmalate synthase has been more closely investigated. It requires the presence of divalent metal ions, with Zn++being most effective. The inactivation does not require molecular oxygen. It occurs in the presence of low concentrations of substrates and is observed in toluene-treated cells. These results, together with evidence that alpha-isopropylmalate synthase and homocitrate synthase are located in the mitochondria, suggest a mechanism by which increasing intra-mitochondrial coenzyme A concentrations might serve as a signal of decreasing acetyl-coenzyme A levels, triggering a temporary inactivation of biosynthetic acetyl-coenzyme A-consuming reactions in order to channel the available acetyl-coenzyme A into the citrate cycle.

2-Isopropylmalate Synthase↗
PubMed1973

Subcellular localization of the leucine biosynthetic enzymes in yeast.

When baker's yeast spheroplasts were lysed by mild osmotic shock, practically all of the isopropylmalate isomerase and the beta-isopropylmalate dehydrogenase was released into the 30,000 x g supernatant fraction, as was the cytosol marker enzyme, glucose-6-phosphate dehydrogenase. alpha-Isopropylmalate synthase, however, was not detected in the initial supernatant, but could be progressively solubilized by homogenization, appearing more slowly than citrate synthase but faster than cytochrome oxidase. Of the total glutamate-alpha-ketoisocaproate transaminase activity, approximately 20% was in the initial soluble fraction, whereas solubilization of the remainder again required homogenization of the spheroplast lysate. Results from sucrose density gradient centrifugation of a cell-free particulate fraction and comparison with marker enzymes suggested that alpha-isopropylmalate synthase was located in the mitochondria. It thus appears that, in yeast, the first specific enzyme in the leucine biosynthetic pathway (alpha-isopropylmalate synthase) is particulate, whereas the next two enzymes in the pathway (isopropylmalate isomerase and beta-isopropylmalate dehydrogenase) are "soluble," with glutamate-alpha-ketoisocaproate transaminase activity being located in both the cytosol and particulate cell fractions.

Acyltransferases↗
PubMedDate not supplied

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.

Aedes↗