Effect of styrene on testicular enzymes of growing rat.

Effect of styrene (100 or 200 mg/kg body wt/day) for 60 days was observed on testicular enzymes of postnatally maturing rats. A significant decrease in epididymal spermatozoa count was observed only at 200 mg/kg body weight dose. Activities of testicular sorbitol dehydrogenase and acid phosphatase decreased while activities of lactate dehydrogenase, beta-glucuronidase, glucose-6-phosphate dehydrogenase, and gamma-glutamyl transpeptidase significantly increased only in animals exposed to styrene at a dose of 200 mg/kg body weight. The results suggest that exposure to high dose of styrene during developmental period alters the activities of enzymes associated with specific cell type of testis.     

Effect of malathion and radiation separately and jointly upon rat enzymes in vivo.

The effect of malathion (50 mg/kg) and radiation (900R) separately and jointly upon phosphatases and succinic dehydrogenase has been studied in female albino rats. Malathion and radiation when given separately, at the dose levels used in this study, appear to have no effect on acid and alkaline phosphatase of kidney, heart and brain. In preirradiated rats given malathion a significant decrease in phosphatases was observed in kidney and brain tissue after as little as 2 h and the values persisted for up to 24 h. No significant change was observed in heart tissue. At the dose level of 50 mg/kg of malathion, no significant change in succinic dehydrogenase was observed; however, radiation caused a significant increase in succinic dehydrogenase of brain and kidney tissue. In preirradiated rats given malathion the effect appeared to be enhanced in time to a certain degree. One could therefore speculate that simple non-specific stress caused by malathion adds to a similar effort of radiation by reducing phosphatases and enhancing the succinic dehydrogenase enzyme.     

Testicular toxicity of di-n-butyl phthalate in adult rats: effect on marker enzymes of spermatogenesis

Di-n-butyl phthalate (DBP) was administered to adult male rats by gavage at the doses of 250, 500 and 1000 mg/kg body weight/day for 15 days. A significant decrease in epididymal spermatozoa counts was observed at 500 and 1000 mg/kg doses of DBP. The activity of sorbitol dehydrogenase was found to be significantly decreased while that of lactate dehydrogenase, gamma-glutamyl transpeptidase, beta-glucuronidase, and glucose-6-phosphate dehydrogenase, significantly increased in the animals exposed to 500 and 1000 mg/kg of DBP. Decrease in the activity of acid phosphatase was also observed at all dose levels. Histopathological studies revealed marked degeneration of seminiferous tubules, further confirming testicular toxicity of DBP. The results suggest that testicular atrophy caused by DBP is associated with an alteration in the activities of enzymes related with specific events of spermatogenesis.     

Changes of blood enzymes in brook trout induced by infection with Aeromonas salmonicida

Furunculosis was induced in brook trout, Salvelinus fontinalis , by experimental inoculation with Aeromonas salmonidda. The levels of blood enzymes were determined and the most significant modifications were observed for aldolase, creatine phosphokinase and ornithine carbamyl transferase. Alkaline phosphatase, glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase showed minor modification. There was no significant change of activity for acid phosphatase, cholinesterase, lactic dehydrogenase, phosphohexose iso-merase, isocitric dehydrogenase and aminopeptidase.     

Investigation of the possible protective role of gallic acid on paraoxanase and arylesterase activities in livers of rats with acute alcohol intoxication

Gallic acid, a polyphenyl class natural product from gallnut and green tea, is known to be antioxidant, anti‐inflammatory and radical scavenger. In this study, we aimed to investigate the possible protective effects of gallic acid on paraoxonase and arylesterase activities in liver exposed to acute alcohol intoxication. Paraoxonase and arylesterase activities in liver tissue and serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase levels were measured. Histological investigations were also made. In our study, we observed a significant increase of serum alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase activities, which are indicators of liver damage after acute ethanol consumption. Gallic acid therapy has significantly reduced the increase in these biomarkers, indicating a possible hepatoprotective effect of gallic acid. Ethanol consumption caused a significant decrease in liver paraoxonase activity (P < 0.001). Gallic acid treatment partly restored this decreased paraoxonase activity, which resulted from ethanol administration. A gallic acid dose of 100 mg/kg was observed as highest restoring effect for paraoxonase activity (P < 0.05). The activity of arylesterase was decreased in the ethanol group as compared with the control group, but this was not significant. However, 50 mg/kg of gallic acid treatment restored the loss of this activity due to ethanol exposure (P < 0.001). We observed that gallic acid ameliorates the liver damage caused by excessive alcohol consumption in a dose‐dependent way. Our results in this study showed that gallic acid might have a protective effect against alcoholic liver disease. Copyright © 2012 John Wiley & Sons, Ltd.     

Activity of several erythrocyte enzymes in chick embryos and chicks after gamma-irradiation

After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.     

Bile acid induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridium limosum

When grown in the presence of bile acids, two strains of Clostridium limosum were found to contain significant amounts of NADP-dependent 7 alpha/7 beta-hydroxysteroid dehydrogenase and NAD-dependent 7 alpha-hydroxysteroid dehydrogenase which were active against conjugated and unconjugated bile acids. No measurable activity could be found when deoxycholic acid (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid) was used as substrate. No 7 beta-hydroxysteroid dehydrogenase activity and only a trace of 7 alpha-hydroxysteroid dehydrogenase activity could be demonstrated when bile acid was deleted from the growth medium. If bile acid was added after the time of inoculation, the amounts of 7 alpha/7 beta-hydroxysteroid dehydrogenase were greatly reduced. Enzyme enhancement was blocked by addition of rifampicin. The 7 alpha/7 beta-hydroxysteroid dehydrogenase components had pH optima of approximately 10.5. Both the 7 alpha/7 beta-hydroxysteroid dehydrogenase activities were heat-labile, with the 7 beta-component being the more stable of the two. When ranked according to the level of enzymes induced, the order in increasing bile acid induction power on an equimolar scale (0.4 mM) was: 7-ketodeoxycholic acid, cholic acid, chenodeoxycholic acid, and deoxycholic acid. Both 7-ketolithocholic acid and ursodeoxycholic acid were ineffective as enzyme inducers. Optimal induction was achieved with high concentrations of cholic acid (5 mM) and a harvest time of 24 hr. Addition of ursodeoxycholic acid to medium containing optimal concentrations of deoxycholic acid suppressed enzyme induction.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of methanol on citric acid production from galactose by Aspergillus niger

Summary The effect of methanol on the ability of several strains of Aspergillus to produce citric acid from galactose has been investigated. In the absence of methanol, very little production (less than 1 g/l) was observed. In the presence of methanol (final concentration 1% v/v), however, citric acid production and yeilds were increased considerably. Strong relationships were observed between citric acid production and the activities of the enzymes 2-oxoglutarate dehydrogenase and pyruvate carboxylase in cell-free extracts. During citric acid production, in the presence of methanol, the activity of 2-oxoglutarate dehydrogenase was low and that of pyruvate carboxylase high. In the absence of methanol, where little citric acid was produced, the reverse was true. It is suggested that the presence of methanol may increase the permeability of the cell to citrate, and the cell responds to the diminished intracellular level by increasing production via repression of 2-oxoglutarate dehydrogenase.     

Retinoic acid synthesis by cytosol from the alcohol dehydrogenase negative deermouse

Cytosolic alcohol dehydrogenase in the deermouse is coded by a single genetic locus and a strain of the deermouse which is alcohol dehydrogenase negative exists. These two strains of the deermouse were used to extend insight into the role of cytosolic alcohol dehydrogenases in the conversion of retinol into retinoic acid. Retinoic acid synthesis from physiological concentrations of retinol (7.5 microM) with cytosol from the alcohol dehydrogenase negative deermouse was 13% (liver), 14% (kidney), 60% (testes), 78% (lung), and 100% (small intestinal mucosa) of that observed with cytosol from the positive deermouse. The rates in the negative strain ranged from 0.3 to 0.7 nmol/h/mg protein: sufficient to fulfill cellular needs for retinoic acid. Ten millimolar 4-methylpyrazole inhibited retinoic acid synthesis 92, 94, 26, and 30% in kidney, liver, lung, and testes of the positive deermouse, respectively, but only 50, 30, 0, and 0% in the same tissues from the negative deermouse. Ethanol (300 mM) did not inhibit retinoic acid synthesis in kidney cytosol from the negative strain. Therefore multiple cytosolic dehydrogenases, including alcohol dehydrogenases, contribute to retinol metabolism in vitro. The only enzyme(s) likely to be physiologically significant to retinoic acid synthesis in vivo, however, is the class of dehydrogenase, distinct from ethanol dehydrogenase, that is common to both the positive and the negative deermouse. This conclusion is supported by the data described above, the kinetics of retinoic acid synthesis and retinal reduction in kidney cytosol from the negative deermouse, and the very existence of the alcohol dehydrogenase negative deermouse. This work also shows that microsomes inhibit the cytosolic conversion of retinol into retinoic acid and that the synthesis of retinal, a retinoid that has no known function outside of the eye, does not reflect the ability or capacity of a sample to synthesize retinoic acid.     

Plant Leaf and Stem Proteins. II. Isozymes and Environmental Cabbage

The activity of 10 enzymes separated by acrylamide disc gel electrophoresis of leaf and stem extracts from Dianthus grown under summer and winter conditions was studied. While banding was constant and highly reproducible under each environment, differences between the 3 cultivars and between the tissues were evident. No significant differences in the isozyme patterns of glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and catalase were observed between the 2 environments. Loss of activity was observed under winter conditions with amylase and lactate dehydrogenase and loss of certain isozymic components was evident with acid phosphatase and esterase. Prominent changes were observed in peroxidase isozymes, the hardy cultivars developing additional isozymic components under winter conditions. Only minor changes in the total protein banding were seen. The enzymes showed considerable stability in those tissues killed by the freezing conditions.     

Dihydroorotic acid dehydrogenase activity in actinomycin-D-treated and normal chick embryos

The effect of actinomycin D on chick embryos cultivated in vitro by New's culturing method was studied. Exposure of chick embryos to actinomycin D (0.05 micrograms/ml) at the primitive streak stage (stage 4; Hamburger and Hamilton) for 6 h showed interference in orotic acid formation. The assay of the enzyme dihydroorotic acid dehydrogenase was carried out in both treated and control embryos. No enzymic activity was observed in actinomycin-D-treated embryos in contrast to the considerable activity in the controls. These observations suggest an interference by actinomycin D in the biogenesis of the enzyme dihydroorotic acid dehydrogenase.     

Beneficial effects of DL-alpha-lipoic acid on cyclophosphamide-induced oxidative stress in mitochondrial fractions of rat testis

The present study investigated the protective efficacy of dl-alpha-lipoic acid on the peroxidative damage and abnormal antioxidant levels in the mitochondrial fraction of testis in cyclophosphamide (CP) administered rats. Male Wistar rats of 140+/-20 g were categorized into four groups. Two groups were administered CP (15 mg/kg body weight once a week for 10 weeks by oral gavage) to induce testicular toxicity; one of these groups received lipoic acid treatment (35 mg/kg body weight intraperitoneally once a week for 10 weeks, 24 h prior to CP administration). A vehicle-treated control group and a lipoic acid drug control group were also included. The mitochondrial fraction of untreated CP-exposed testis showed 1.84-fold increase in lipid peroxidation, along with a significant (P<0.001) increase in hydrogen peroxide levels. In CP-exposed rats, we observed abnormal changes in the activities/levels of mitochondrial enzymic (superoxide dismutase, glutathione peroxidase and glutathione reductase) and non-enzymic (reduced glutathione, ascorbate and alpha-tocopherol) antioxidants. CP-treated rats also showed decline in the activities of mitochondrial enzymes such as succinate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase. In contrast, rats pretreated with lipoic acid showed normal lipid peroxidation and antioxidant defenses, thereby showing the protection rendered by lipoic acid.     

Intracellular location of enzymes involved in citrate production by Aspergillus niger.

The intracellular distribution and maximal activities of nine enzymes involved in the biosynthesis and degradation of citric acid in Aspergillus niger were determined under conditions of growth and of citric acid production. Under these conditions the intracellular location of the enzymes in most cases resembled that described for other filamentous fungi. Pyruvate carboxylase was found predominantly or exclusively in the cytosol. A single isoenzyme of NADP-isocitrate dehydrogenase was present, which appeared to be localised in the mitochondrion. No significant differences in maximal enzyme activities were observed except for NADP-isocitrate dehydrogenase, which showed decreased activity in production-phase mycelia. The results obtained support the scheme proposed by C.P. Kubicek for the intracellular organisation of citric acid formation but provide little evidence that this process is controlled at the level of the biosynthesis of any of the enzymes examined here.     

Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis.

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.     

The effect of progesterone on prolactin stimulation of fatty acid synthesis, glycerolipid synthesis and lipogenic-enzyme activities in mammary glands of pseudopregnant rabbits, after explant culture or intraductal injection.

The activities of lipogenic enzymes, such as acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase, and glycerolipid synthesis increased significantly in mammary explants of 11-day-pseudopregnant rabbits in response to prolactin, in the presence of near-physiological concentrations of insulin and corticosterone in culture. Increasing the concentration of progesterone in culture resulted in suppression of glycerolipid synthesis and activities of acetyl-CoA carboxylase and fatty acid synthetase, but not the pentose phosphate dehydrogenases. However, at near-physiological concentration of progesterone, only acetyl-CoA carboxylase activity was decreased. Injection of prolactin intraductally into 11-day-pseudopregnant rabbits stimulated glycerolipid synthesis, fatty acid synthesis and enzymes involved in fatty acid synthesis, after 3 days. Intraductal injection of progesterone separately or together with prolactin had no significant effect on basal or stimulated lipogenesis in mammary glands. Intramuscular injection of progesterone at 10 mg/day did not suppress fatty acid synthesis stimulated when prolactin was injected intraductally, but a significant inhibition was observed at a higher dose (80 mg/day).     

Ferulic acid with ascorbic acid synergistically extenuates the mitochondrial dysfunction during beta-adrenergic catecholamine induced cardiotoxicity in rats

Disruption of mitochondria and free radical mediated tissue injury have been reported during cardiotoxicity induced by isoproterenol (ISO), a beta-adrenergic catecholamine. The present study was designed to investigate the effect of the combination of ferulic acid (FA) and ascorbic acid (AA) on the mitochondrial damage in ISO induced cardiotoxicity. Induction of rats with ISO (150 mg/kg b.wt., i.p.) for 2 days resulted in a significant decrease in the activities of respiratory chain enzymes (NADH dehydrogenase and cytochrome c-oxidase), tricarboxylic acid cycle enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, alpha-ketoglutarate dehydrogenase), mitochondrial antioxidants (GPx, GST, SOD, CAT, GSH), cytochromes (b, c, c1, aa3) and in the level of mitochondrial phospholipids. A marked elevation in mitochondrial lipid peroxidation, mitochondrial levels of cholesterol, triglycerides and free fatty acids were also observed in ISO intoxicated rats. Pre-co-treatment with the combination of FA (20 mg/kg b.wt.) and AA (80 mg/kg b.wt.) orally for 6 days significantly enhanced the attenuation of these functional abnormalities and restored normal mitochondrial function when compared to individual drug treated groups. Mitigation of ISO induced biochemical and morphological changes in mitochondria were more pronounced with a combination of FA and AA rather than the individual drug treated groups. Transmission electron microscopic observations also correlated with these biochemical parameters. Hence, these findings demonstrate the synergistic ameliorative potential of FA and AA on mitochondrial function during beta-adrenergic catecholamine induced cardiotoxicity and associated oxidative stress in rats.     

Ammonia assimilation in Bacillus polymyxa. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha,gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed.     

Pentalenolactone biosynthesis: Molecular cloning and assignment of biochemical function to PtlF, a short-chain dehydrogenase from Streptomyces avermitilis, and identification of a new biosynthetic intermediate

Pentalenolactone (1) is an antibiotic that has been isolated from many species of Streptomyces. The putative dehydrogenase encoded by the ptlF gene (SAV2993) found within the Streptomyces avermitilis pentalenolactone gene cluster was cloned and overexpressed in Escherichia coli. PtlF, which belongs to the short-chain dehydrogenase/oxidoreductase superfamily, was shown to catalyze the oxidation of 1-deoxy-11beta-hydroxypentalenic acid (9) to 1-deoxy-11-oxopentalenic acid (10), a new intermediate of the pentalenolactone biosynthetic pathway. The methyl ester of 10 was characterized by NMR, GC-MS and high resolution mass spectrometry. PtlF exhibited a 150-fold preference for beta-NAD(+) over beta-NADP(+). PtlF had a pH optimum of 8.0 in the physiological pH range, while a significant activity enhancement was observed from pH 9.0 to 11.3. At pH 8.0, PtlF had a k(cat) of 0.65+/-0.03 s(-1), with a K(m) for 9 of 6.5+/-1.5 microM and K(m) for NAD(+) of 25+/-3 microM.     

A synthetic peptide-directed antibody as a probe of the phosphorylation site of pyruvate dehydrogenase

A synthetic peptide, corresponding to the 14-amino acid tryptic fragment containing phosphorylation sites one and two of bovine mitochondrial pyruvate dehydrogenase, was coupled to Limulus polyphemus hemocyanin and used to produce rabbit polyclonal antibodies. A positive signal was observed when Western blots of bovine, porcine, or yeast mitochondrial pyruvate dehydrogenase complexes were probed with the antibodies but not with blots of bacterial, cellular slime mold, plant mitochondrial, or plant plastid pyruvate dehydrogenase complexes. The antibodies also gave a positive signal when used to probe blots of the bovine kidney branched chain 2-oxo acid dehydrogenase complex. The ATP-dependent phosphorylation/inactivation of rat liver mitochondrial pyruvate dehydrogenase complex could be inhibited by prior incubation with the anti-peptide antibodies.     

One-step synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid using a multienzymatic system

12-ketoursodeoxycholic acid (12-keto-UDCA) is a key intermediate for the synthesis of ursodeoxycholic acid (UDCA), an important therapeutic agent for non-surgical treatment of human cholesterol gallstones and various liver diseases. The goal of this study is to develop a new enzymatic route for the synthesis 12-keto-UDCA based on a combination of NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH, EC 1.1.1.201) and NADH-dependent 3α-hydroxysteroid dehydrogenase (3α-HSDH, EC 1.1.1.50). In the presence of NADPH and NADH, the combination of these enzymes has the capacity to reduce the 3-carbonyl- and 7-carbonyl-groups of dehydrocholic acid (DHCA), forming 12-keto-UDCA in a single step. For cofactor regeneration, an engineered formate dehydrogenase, which is able to regenerate NADPH and NADH simultaneously, was used. All three enzymes were overexpressed in an engineered expression host Escherichia coli BL21(DE3)Δ7α-HSDH devoid of 7α-hydroxysteroid dehydrogenase, an enzyme indigenous to E. coli, in order to avoid formation of the undesired by-product 12-chenodeoxycholic acid in the reaction mixture. The stability of enzymes and reaction conditions such as pH value and substrate concentration were evaluated. No significant loss of activity was observed after 5 days under reaction condition. Under the optimal condition (10 mM of DHCA and pH 6), 99 % formation of 12-keto-UDCA with 91 % yield was observed.