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PubMed1979

The role of heme in the regulation of the late program of Friend cell erythroid differentiation.

The addition of a chemical inducer, such as dimethylsulfoxide (DMSO), to cultures of mouse Friend erythroleukemic cells results in the induction of a number of late erythroid events, including the accumulation of globin mRNA, the inducation of hemoglobin synthesis, the appearance of erythrocyte membrane antigens (EMA), and the cessation of cell division. The experiments presented in this study demonstrate that heme is necessary but not sufficient for the loss of proliferative capacity associated with DMSO-induced Friend cell differentiation, whereas the accumulation of globin mRNA and EMA can occur in the absence of heme synthesis or heme itself. These conclusions were reached by selectively inhibiting heme synthesis in DMSO-treated cells in two independent ways: (i) Inducible cells were treated with 3-amino-1,2,4-triazole (AT), a drug which inhibits the induction of heme synthesis in Friend cells in a dose-dependent manner. Treatment of inducible Friend cells with 1.5% DMSO for five days caused the plating efficiency in methyl cellulose to decrease to 1% of that in untreated cultures. However, treatment of the cells with DMSO plus AT almost totally prevented this decrease in plating efficiency. The addition of exogenous hemin, which alone had no significant effect on plating efficiency, largely reversed the effect of AT in DMSO-treated cells, reducing the plating efficiency to below 5%. In contrast to the marked effects of AT on the proliferative capacity of differentiating Friend cells, the levels of globin mRNA and EMA were only partially decreased in cells treated with DMSO plus AT, compared to cells treated with DMSO alone. (ii) The relationship between heme synthesis, terminal cell division, and the induction of globin mRNA was investigated further through the use of non-inducible Friend cell variant clones. One such non-inducible clone, M18, appears to be a phenotypic analog of inducible cells treated with DMSO plus AT. Clone M18 did not accumulate heme or hemoglobin, as detected by benzidine staining, nor lose its proliferative capacity in response to DMSO. However, globin mRNA was induced by DMSO in this clone. Treatment of clone M18 with DMSO plus hemin overcame the block in hemoglobin accumulation suggesting that M18 has a defect in the induction of heme biosynthesis. In addition, exposure of M18 cells to DMSO plus hemin caused a gradual decrease in plating efficiency which was not due to non-specific toxicity. Prior incubation of M18 cells in DMSO for three to five days was necessary before hemin caused a rapid loss of proliferative capacity. Thus, these results, in agreement with the AT studies on inducible Friend cells and previous studies on the induction of EMA in clone M18, indicate that there may be both heme-dependent and heme-independent events in the program of Friend cell differentiation.

Amitrole↗
PubMed1994

Reactive oxygen metabolite-induced toxicity to cultured bovine endothelial cells: status of cellular iron in mediating injury.

We aimed to determine the status of iron in mediating oxidant-induced damage to cultured bovine aortic endothelial cells. Chromium-51-labeled cells were exposed to reaction mixtures of xanthine oxidase/hypoxanthine and glucose oxidase/glucose; these produce superoxide and hydrogen peroxide, or hydrogen peroxide, respectively. Xanthine oxidase caused a dose dependent increase of 51Cr release. Damage was prevented by allopurinol, oxypurinol, and extracellular catalase, but not by superoxide dismutase. Prevention of xanthine oxidase-induced damage by catalase was blocked by an inhibitor of catalase, aminotriazole. Glucose oxidase also caused a dose-dependent increase of 51Ci release. Glucose oxidase-induced injury, which was catalase-inhibitable, was not prevented by extracellular superoxide dismutase. Both addition of and pretreatment with deferoxamine (a chelator of Fe3+) prevented glucose oxidase-induced injury. The presence of phenanthroline (a chelator of divalent Fe2+) prevented glucose oxidase-induced 51Cr release, whereas pretreatment with the agent did not. Apotransferrin (a membrane impermeable iron binding protein) failed to influence damage. Neither deferoxamine nor phenanthroline influenced cellular antioxidant defenses, or inhibited lysis by non-oxidant toxic agents. Treatment with allopurinol and oxypurinol, which inhibited cellular xanthine oxidase, failed to prevent glucose oxidase injury. We conclude that (1) among the oxygen species extracellularly generated by xanthine oxidase/hypoxanthine, hydrogen peroxide induces damage via a reaction on cellular iron; (2) deferoxamine and phenanthroline protect cells by chelating Fe3+ and Fe2+, respectively; and (3) reduction of cellular stored iron (Fe3+) to Fe2+ may be prerequisite for mediation of oxidant-induced injury, but this occurs independently of extracellular superoxide or cellular xanthine oxidase-derived superoxide.

Allopurinol↗
PubMed1994

Histochemical demonstration of hydrogen peroxide production by leukocytes in fixed-frozen tissue sections of inflammatory lesions.

The production of H2O2 by cells in cold paraformaldehyde-fixed frozen sections of inflammatory lesions was histochemically demonstrated by incubating them with diaminobenzidine (DAB) for 2 to 6 h. Catalase (150 micrograms/ml, about 1400 U/ml) inhibited the reaction, indicating that H2O2 was required to produce the chromogenic DAB product. Granulocytes (PMNs and eosinophils) were the main types of cells stained by the DAB reaction. Positive staining of macrophages was less frequent. The H2O2 was produced by metabolic enzymes that were still active after cell death and mild fixation. An atmosphere of 95 to 100% oxygen enhanced the specific DAB reaction, and an atmosphere of 100% nitrogen eliminated it. The DAB histochemical reaction to detect H2O2 requires the presence of peroxidases to produce the colored reaction product. Within our tissue sections, such peroxidases were evidently present in excess, because addition of low concentrations of H2O2 significantly increased the reaction product. Although some of the H2O2 produced by the granulocytes may have been derived from the dismutation of superoxide (O2-), the NADPH oxidase pathway for O2- formation did not seem to be involved: NADPH oxidase, a rather labile enzyme, should not be active after mild fixation, and diphenyleneiodonium (100 microM), an inhibitor of flavine-requiring NADPH oxidase, did not inhibit the reaction. Reactive nitrogen intermediates were also not involved, because NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester, inhibitors of nitric oxide synthetase, did not appreciably inhibit the reaction. We conclude that stable, non-flavine-requiring oxidases, possibly cyclooxygenases or lipoxygenases, produced the H2O2 measured histochemically by our DAB reaction. These studies were made on tissue sections of acute dermal inflammatory lesions produced in rabbits by the topical application of 1% sulfur mustard [bis(2-chloroethyl) sulfide] in methylene chloride. Both intact PMNs and disintegrating PMNs in the base of the crust produced H2O2. Despite the production of H2O2 and the presence of peroxidase activity, no tissue damage was seen microscopically near the H2O2-producing cells, which indicates that the tissues are well protected by the antioxidants present in this self-limiting inflammatory reaction.

Amitrole↗
PubMed1990

Influence of oxidative stress on the age-linked alterations of the cerebral glutathione system.

The glutathione system (reduced and oxidized glutathione; redox index) was studied in the forebrain of male Wistar rats of 5, 15, and 25 months of age following the administration for 2 months in drinking water of chemicals that induce oxidative stress: paraquat and diethyldithiocarbamate (DDC) to increase superoxide radical formation, aminotriazole and hydrogen peroxide to increase hydroxyl radical generation, as well as diamide and ferrous chloride to decrease the glutathione cycle activity. Chronic oral administration of phosphatidylcholine for 2 months was evaluated in 25-month-old rats. Aging accentuated the changes produced by chemicals that induce oxidative stress; i.e., the changes in the glutathione redox index were most pronounced in the forebrains of the older paraquat-, DDC-, H2O2-, and diamide-treated rats. Markedly different adaptative changes occurred within the various drug groups. The reduced glutathione was increased (by paraquat, DDC and aminotrazole), decreased (by H2O2) or unchanged (by iron and diamide). Furthermore, in older rats, paraquat and DDC increased the glutathione redox index, whereas H2O2 and diamide decreased the glutathione redox index or were ineffective (i.e., aminotriazole, iron). The glutathione redox index altered by chronic drug administration was modified by the concomitant administration of phosphatidylcholine.

Administration, Oral↗
PubMed1990

Changes on cerebral antioxidant enzymes, peroxidation, and the glutathione system of frogs after aging and catalase inhibition.

The five major antioxidant enzymes, glutathione, and in vivo or in vitro stimulated (Fe(++)-ascorbate) peroxidation were similar in old and young Rana perezi frogs. Long-term (2.5 months) treatment with aminotriazole strongly decreased cerebral catalase (CAT) activity and increased in vivo but not in vitro peroxidation in the brain. This suggests that the increase in endogenous brain peroxidation after CAT inhibition is due to an increased free-radical attack on cerebral membranes, and not to a possible increase in their sensitivity to peroxidative damage. The increase of in vivo peroxidation is especially remarkable taking into account the low levels of CAT present in the vertebrate brain. On the other hand, these changes were not accompanied by any effect on the survival of the animals. Comparison of these results with those obtained in other species suggests the possibility that O2-free radicals be of minor importance in relation to brain aging in animals with low rates of oxygen consumption.

Aging↗
PubMed2006

Acyl-CoA oxidase activity from Beauveria bassiana, an entomopathogenic fungus.

Beauveria bassiana produces acyl-Co oxidase (ACO) in the P(20000 g) fraction of glucose and alkane-grown cultures that catalyze the oxidation of acyl-CoAs of different chain length. The activity was measured indirectly over the formation of H2O2 via the oxidative-coupled assay system. ACO activity was assessed spectrophotometrically in the P(20000 g) fraction of glucose-grown (FS0) and n-alkane grown cultures (FS(alk)), employing acyl-CoAs of 16 to 24 carbons as substrates. A significant increment in the activity was observed in FS(alk) as compared to that of controls (FS0) in all conditions tested. Tetracosane-grown cultures showed the highest activity with lignoceroyl-CoA. The reaction conditions were optimized employing lignoceroyl-CoA as substrate. A variable lag phase was observed when the activity was measured as a function of time. In the presence of 3-amino-1,2,4-triazole (AT) to prevent H2O2 consumption by endogenous catalase, the lag phase became shorter and disappeared when AT concentrations were raised from 40 to 200 mM, thus enhancing acyl-CoA oxidation. Enzyme activity reached its maximal value in the presence of 240 microg peroxidase, 0.08% Triton X-100 and 36 microM bovine serum albumin. The apparent Km using lignoceroyl as substrate was estimated 2.5 microM. ACO showed high activity and stability between 30 and 40 degrees C, as well as between 7.0 and 9.0 pH, for 120 min, being 7.0 the optimum pH.

Acyl-CoA Oxidase↗
PubMed1990

Occurrence of the general control of amino acid biosynthesis in yeasts.

The response of three amino acid biosynthetic enzymes, threonine dehydratase, tyrosine aminotransferase and saccharopine dehydrogenase, to conditions of histidine, tryptophan or lysine limitation was investigated in 15 yeast species. The activities of all these enzymes increased about two- to fourfold as a result of action of the general control of amino acid biosynthesis in Brettanomyces anomalus, Candida maltosa, Hansenula polymorpha, Rhodosporidium toruloides, Saccharomyces cerevisiae and Yarrowia lipolytica. No evidence for the existence of the general control was found in Candida brumptii, Candida utilis, Hansenula anomala, Hansenula henricii, Kluyveromyces marxianus, Pichia guilliermondii, Saccharomycopsis capsularis, Trichosporon adeninovorans and Trigonopsis variabilis.

Amino Acids↗
PubMed2001

Measurement of inhibin A: a modification to an enzyme-linked immunosorbent assay.

Inhibin A is a useful prenatal marker of Down syndrome. Currently, the available enzyme-linked immunosorbent assays (ELISAs) for inhibin A are based upon the same paired monoclonal antibodies. In the present study we have confirmed for one of those ELISAs that short-term sample storage as whole blood leads to a significant decline in detectable inhibin A and that this is most likely due to erythrocyte catalase interference with a critical oxidation step in the assay. While this interference can be eliminated by heating the samples pre-assay, this process is labour intensive. In the present study we have demonstrated that the addition of 3-amino-1,2,4-triazole (AT), a catalase 'suicide' inhibitor, also prevents the decline of inhibin A levels in samples stored as whole blood. We suggest that the addition of AT to samples prior to assay is a simple modification to the inhibin A ELISA that affords optimum performance.

Amitrole↗
PubMed1993

Cloning and sequence of ADP-ribosylation factor 1 (ARF1) from Schizosaccharomyces pombe.

A gene encoding a homologue of the ADP-ribosylation factor (ARF) family of small GTP binding proteins was cloned from a Schizosaccharomyces pombe cDNA library by a functional screen of suppressors of sensitivity to 3-aminotriazole in a gcn3 null strain of Saccharomyces cerevisiae. Two independent isolates each contained the full coding region of the ARF1 gene. The encoded SpARF1 protein has a predicted molecular weight of 20,618 and is 88% and 79% identical to human and S. cerevisiae ARF1 proteins, respectively. As independent isolates were obtained, this effect of the SpARF1 appears to be a real phenomenon, but cannot currently be easily understood within the context of the evidence for a role(s) for ARF proteins in the protein secretory pathway.

ADP-Ribosylation Factor 1↗
PubMed1995

Characterization of Schizosaccharomyces pombe his1 and his5 cDNAs.

We have isolated Schizosaccharomyces pombe cDNAs corresponding to the genes his1+ and his5+. The his1 cDNA was isolated by functional complementation of the His- phenotype in a his1-29 gcn3 Saccharomyces cerevisiae strain, while the his5 cDNA was isolated as a suppressor of the 3-amino-1,2, 4-triazole (3-AT) sensitivity in a gcn3 S. cerevisiae strain. his1 and his5 are each present in single copy in haploid S. pombe. As is the case with S. cerevisiae, we have found that the growth of wild-type strains of S. pombe is sensitive to 3-AT, an inhibitor of imidazoleglycerol-phosphate dehydratase. This enzyme is encoded by the HIS3 gene in S. cerevisiae and the his5+ gene in S. pombe. Treatment of S. pombe cells with 3-AT leads to a small increase in the level of the his5 transcript, but no effect is seen on the level of the his1 transcript. This is in contrast to larger increases in transcription of amino acid biosynthetic genes, regulated by the general amino acid control, seen previously in similarly treated cultures of S. cerevisiae. These results suggest that there are likely to be some differences in the regulation of amino acid biosynthesis between these two yeasts.

Amino Acid Sequence↗
PubMed1993

Evidence that catalase is a major pathway of ethanol oxidation in vivo: dose-response studies in deer mice using methanol as a selective substrate.

Recently, it was demonstrated that 4-methylpyrazole was not only an inhibitor of alcohol dehydrogenase but also caused competitive inhibition of fatty acyl-CoA synthetase, the enzyme which activates fatty acids (B. U. Bradford, D. T. Forman, and R. G. Thurman, 1993, Mol. Pharmacol. 43, 115-119). Rates of catalase-dependent alcohol metabolism were decreased in alcohol dehydrogenase-negative (ADH-) deer mice because the H2O2 supply for catalase via peroxisomal fatty acid oxidation was inhibited due to substrate limitation. In light of these findings it became necessary to reevaluate the role of catalase and alcohol dehydrogenase in alcohol metabolism. In this study, methanol, a selective substrate for catalase in rodents, was compared with ethanol. Rates of ethanol and methanol metabolism were studied in vivo at blood alcohol levels ranging from 50 to 500 mg/dl. In the ADH- deer mouse, rates of methanol and ethanol metabolism increased when alcohol was elevated from 0 to 100 mg/dl and were maximal at values around 6-8 mmol/kg/h (half-maximal rates were observed at blood alcohol levels around 50 mg/dl). In the ADH+ deer mouse, rates of ethanol metabolism increased to values around 9 mmol/kg/h at 100 mg/dl and remained constant at blood levels up to 500 mg/dl. In contrast, rates of methanol metabolism increased to values of only 5 mmol/kg/h at levels of 100 mg/dl (the half-maximal rate was about 2.5 mmol/kg/h at 50 mg/dl) followed by a steady increase to 9 mmol/kg/h as the blood level was increased from 100 to 500 mg/dl (the half-maximal rate for this second component was around 6 mmol/kg/h at 300 mg/dl). Rates of methanol uptake were 50 +/- 4 nmol/min/mg protein in 10,000g pellets from ADH- deer mouse livers; however, methanol was not metabolized by isolated microsomes. The catalase inhibitor aminotriazole decreased ethanol and methanol metabolism 75% in ADH- deer mice. Further, olive oil, which is rich in oleate, increased methanol metabolism from 7 to 11.5 mmol/kg/h. This stimulation was blocked by fructose, which diminishes ATP and decreases H2O2 supply. In the ADH+ deer mouse, fructose (2 g/kg) stimulated ethanol metabolism as expected; however, inhibition of both ethanol and methanol metabolism was observed with higher doses of fructose (10 g/kg). Taken together, these data support the hypothesis that catalase is the predominant pathway for alcohol metabolism in the ADH- deer mouse. The contribution of catalase was about 50% in the ADH+ mutant at low doses of ethanol and approached 100% as the alcohol concentration was elevated.

Alcohol Dehydrogenase↗
PubMed1994

The role of glucose in cellular defences against cytotoxicity of hydrogen peroxide in Chinese hamster ovary cells.

The presence of glucose in the cellular environment influences the response of cells to hydrogen peroxide. This study examines the effect of glucose on clonogenic cell survival of Chinese hamster ovary cells exposed to micromolar concentrations of exogenous hydrogen peroxide. Exposure to hydrogen peroxide (20 mumol/10(7) cells) resulted in considerable cytotoxicity that was unaffected by the presence or absence of glucose. However, glucose protected the cells from killing induced by milder exposure (1 mumol/10(7) cells) to the oxidant causing a shift in the dose-response curve. This effect was considered in terms of glucose metabolism via the pentose phosphate cycle. A low nontoxic concentration of hydrogen peroxide (0.1 mumol/10(7) cells) markedly increased pentose phosphate cycle activity in normal cells. Clonogenic survival and activity of the cycle for cells depleted of total glutathione to about 8.6% of its initial value and/or with catalase activity reduced to about 10% of control levels were also determined. Neither of these modifications alone completely abolished the protective effect of glucose. Efficacy of glucose protection against cytotoxicity of hydrogen peroxide diminished in cells depleted of glutathione, and this was not accompanied by any detectable increase in pentose phosphate cycle activity above the control level. Cells depleted of catalase alone had a profile of survival and pentose phosphate cycle activity similar to that of control cells when exposed to hydrogen peroxide. Cells depleted of both glutathione and catalase were almost as sensitive to hydrogen peroxide as the cells incubated without glucose. They also did not express any detectable increase in pentose phosphate cycle activity. Survival of those cells, when exposed to hydrogen peroxide, was almost the same regardless if glucose was present or not. These results demonstrate an important role for the glutathione redox cycle, catalase, and the pentose phosphate cycle in protection against hydrogen peroxide in Chinese hamster ovary cells. They confirm the essential role of glucose and pentose phosphate cycle activity for the detoxification of hydrogen peroxide via the glutathione redox cycle. The data suggest that the ability of catalase to metabolise peroxide may also depend on metabolism of glucose via the pentose phosphate cycle. A clear understanding of the protective mechanisms in cells against hydrogen peroxide has many applications since this common reactive oxygen species is implicated in several pathophysiologies and in the action of certain chemotherapeutic drugs.

Amitrole↗
PubMed1996

Correlation between glutathione and stimulation of the pentose phosphate cycle in situ in Chinese hamster ovary cells exposed to hydrogen peroxide.

The effect of glutathione on stimulation of pentose phosphate cycle activity during oxidative challenge was evaluated in intact Chinese hamster ovary cells in situ. Glutathione was depleted to varying levels with L-buthionine-[S,R] sulfoximine. The level of stimulation of pentose phosphate cycle activity by exogenous H2O2 (4 mumol/10(7) cells) was dependent on the time of pretreatment with L-buthionine-[S,R] sulfoximine and was proportional to the total glutathione concentration. This was not related to the amount of GSSG, since its level was exceedingly low under conditions where H2O2 stimulated pentose phosphate cycle activity. The amount of GSSG in cells increased after exposure to 10-fold higher concentrations of H2O2 under conditions where there was no stimulation of pentose phosphate cycle activity above the basal level. Paraquat caused stimulation of pentose phosphate cycle activity which was independent of L-buthionine-[S,R] sulfoximine pretreatment and of the glutathione content of cells. The stimulatory effects of both oxidants on pentose phosphate cycle activity appeared to be independent of glutathione reductase activity since they were unaffected in cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea. The inhibitory effect of L-buthionine-[S,R] sulfoximine on stimulation of pentose phosphate cycle activity by H2O2 did not appear to be due to the inhibitor itself, but rather to the overall level of glutathione. Glutathione could have a role in maintaining activity of the pentose phosphate cycle at a level which is appropriate for the severity of the oxidative challenge as well as for the capacity of the cellular antioxidant defenses.

Amitrole↗
PubMed1999

Enhancement of cytotoxicity of hydrogen peroxide by hyperthermia in chinese hamster ovary cells: role of antioxidant defenses.

Regional hyperthermia has potential for human cancer treatment, particularly in combination with systemic chemotherapy or radiotherapy. The mechanisms involved in heat-induced cell killing are currently unknown. Hyperthermia may increase oxidative stress in cells, and thus, oxidative stress could have a role in the mechanism of cell death. We use hydrogen peroxide as a model oxidant to improve understanding of interactions between heat and oxidative stress. Heat increased cytotoxicity of hydrogen peroxide in Chinese hamster ovary cells. Altered levels of cellular antioxidants should create an imbalance between prooxidant and antioxidant systems, thus modifying cytotoxic responses to heat and to oxidants. We determine the involvement of the two cellular antioxidant defenses against peroxides, catalase and the glutathione redox cycle, in cellular sensitivity to heat, to hydrogen peroxide, and to heat combined with the oxidant. Defense systems were either inhibited or increased. For inhibition studies, intracellular glutathione was diminished to less than 15% of its initial level by treatment with L-buthionine sulfoximine (1 mM, 24 h). Inhibition of catalase was achieved with 3-amino-1,2,4-triazole (20 mM, 2 h), which caused a 80% decrease in endogenous enzyme activity. To increase antioxidants, cells were pretreated with the thiol-containing reducing agents, N-acetyl-L-cysteine, 2-oxo-4-thiazolidine carboxylate, and 2-mercaptoethane sulfonate. These compounds increased intracellular glutathione levels by 30%. Catalase activity was increased by addition of exogenous enzyme to cells. We show that levels of glutathione and catalase affect cellular cytotoxic responses to heat and hydrogen peroxide, either used separately or in combination. These findings are relevant to mechanisms of cell killing at elevated temperatures and suggest the involvement of oxidative stress.

Acetylcysteine↗
PubMed1997

Purification and characterization of a homodimeric catalase-peroxidase from the cyanobacterium Anacystis nidulans.

Cytosolic extracts of the cyanobacterium Anacystis nidulans exhibit both catalase and o-dianisidine peroxidase activity. Native polyacrylamide gel electrophoresis demonstrates one distinct enzyme, which has been purified to essential homogeneity and found to be composed of two identical subunits of equal size (80.5 kDa). The isoelectric point is at pH 4.7. It is a very efficient catalase with a broad pH optimum between 6.5 and 7.5 and a Km for H2O2 of 4.3 mM, a calculated turnover number of 7200 s(-1), and an overall-rate constant of 3.5 x 10(6) M(-1) s(-1). The behaviour of this protoheme-enzyme is typical of the class of prokaryotic catalase-peroxidases, which is sensitive to cyanide (Ki = 27.2 microM) and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole. The enzyme accepts electrons from o-dianisidine, but not from ascorbate, glutathione, and NADH. With hydrogen peroxide in steady-state conditions the enzyme is mainly in the ferric state indicating that Compound I is much faster reduced by H2O2 than it is formed. The native enzyme is in the high-spin state, which is transformed to low-spin upon addition of cyanide. With peroxoacetic acid Compound I is formed at a rate of 5.9 x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C with about 50% hypochromicity, a Soret-maximum at 405 nm and isosbestic points at 354 and 427 nm.

Amitrole↗
PubMed1998

The protective effect of 17 beta-estradiol on vasomotor responses of aorta from cholesterol-fed rabbit is reduced by inhibitors of superoxide dismutase and catalase.

The involvement of endogenous antioxidant enzymes in the protective effect of 17 beta-estradiol against hypercholesterolemia was evaluated on aorta from cholesterol-fed rabbits treated with estradiol. 17 beta-Estradiol, more potent than alpha-tocopherol, restored the acetylcholine-induced relaxation, which was impaired by cholesterol chow, and enhanced the vasorelaxation induced by sodium nitroprusside. Diethyldithiocarbamate (5 mM), a superoxide dismutase (SOD) inhibitor, reduced relaxation to acetylcholine down to the level obtained in cholesterol-fed rabbits not treated with estrogen. This inhibition was dose-dependently reversed by addition of exogenous SOD. Aminotriazole (5 mM), a catalase inhibitor, reduced slightly this response, which was not reversed by exogenous catalase. A similar concentration of diethyldithiocarbamate prevented the potentiation of response to sodium nitroprusside induced by estrogen, whereas aminotriazole had no effect. These results suggest that, on aorta from cholesterol-fed rabbit, superoxide dismutase and catalase are involved in the protective effect of estrogen on the vasomotor response to acetylcholine, but only superoxide dismutase participates in response to sodium nitroprusside.

Acetylcholine↗
PubMed1999

The early phase of apoptosis in human neuroblastoma CHP100 cells is characterized by lipoxygenase-dependent ultraweak light emission.

Human neuroblastoma CHP100 cells were forced into apoptosis (programmed cell death, PCD) or necrosis by treatment with calcium chloride or sodium nitroprusside (a nitric oxide donor), respectively. Cellular luminescence, a marker of membrane lipid peroxidation, was increased by calcium but not by nitroprusside, and reached a maximum of 4-fold the control value 2 hours after treatment. The increase in luminescence was paralleled by increased 5-lipoxygenase (up to 250% of the control value) and decreased catalase (down to 50%) activity within the same time window. Consistently, incubation of CHP100 cells with inhibitors of 5-lipoxygenase (5,8,11,14-eicosatetraynoic acid and MK886) reduced light emission and PCD, whereas inhibition of catalase by 3-amino-1, 2,4-triazole enhanced both processes. Treatment of CHP100 cells with retinoic acid or cisplatin, unrelated PCD inducers reported to activate the lipoxygenase pathway, also gave enhanced light emission parallel to PCD increase. Altogether, these results suggest that cellular luminescence is an early marker of apoptotic, but not necrotic, program(s) involving generation of hydrogen peroxide and activation of 5-lipoxygenase.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1995

A GCN-like response in Candida albicans.

The control of amino acid and purine biosynthesis in the yeast Saccharomyces cerevisiae is mediated by the transcriptional activator GCN4. We previously identified the presence of two putative GCN4 responsive elements (GCREs) in the promoter sequence of the Candida albicans ARO3 gene, which encodes an enzyme in the aromatic amino acid pathway. We now show that amino acid deprivation results in a dramatic rise in the steady-state level of ARO3-specific mRNA, indicative of a GCN-like pathway in C. albicans.

Amino Acids↗