Purification and characterization of nitroreductase from red alkaliphilic bacterium Aquiflexum sp. DL6.

Nitroaromatic compounds are toxic to living organisms. Most of them exhibit human mutagenic and carcinogenic potential. Biotransformation and bioremediation processes can convert these compounds into non-toxic compounds. Acclimatization of bacterial strain Aquiflexum sp. DL6 with nitro-aromatics resulted in significant induction of nitroreductase (EC 1.5.1.34). The enzyme was purified by the combination of DEAE-cellulose and Sephadex G-100 column chromatography with 80-fold purification and 22% yield. Molecular weight of purified nitroreductase was estimated to be 29 kDa by SDS-PAGE. The enzyme characteristics were explored by varying the pH and temperatures, and the optimum activity was found at pH 9.5 and 40 degrees C. It was revealed that the substrate specificity of nitroreductase of Aquiflexum sp. DL6 was wide for the most of the tested nitro-aromatic compounds. The kinetic parameters like Michaelis constant and velocity maxima were determined with o-nitrophenol and NADH as substrates.

Chromate reduction by Burkholderia cepacia MCMB-821, isolated from the pristine habitat of alkaline crater lake.

The Cr(VI)-reducing bacterial strain MCMB-821 was isolated from the alkaline crater lake of Lonar and was identified as Burkholderia cepacia. MCMB-821 was resistant to 1,000-ppm Cr(VI) and reduced 98% of the 75 ppm Cr(VI) within 36 h at pH 9.0 in the presence of 2% salt and lactose as the electron donor. The chromate-reducing efficiency of MCMB-821 was comparable under both aerobic as well as anaerobic conditions. Electron paramagnetic resonance spectroscopy data suggested that MCMB-821 reduced Cr(VI) to Cr(III) via the formation of transient Cr(V) intermediate. The chromate-reducing ability of MCMB-821 was suppressed in the presence of membrane inhibitors and enhanced in the presence of 2,4-dinitrophenol, suggesting the involvement of electron transport chain in the Cr(VI) bioreduction.

Age-related reductions in the activities of antioxidant enzymes in the rat inferior colliculus.

The inferior colliculus (IC) is a major relay and processing center of auditory signals in the midbrain and receives inputs from most other auditory nuclei. A number of studies have indicated age-related declines in the GABAergic and excitatory amino acid systems in the IC, including losses in both GABA immunoreactive (+) and GABA immunonegative (-) synapses. The goal of this project was to identify potential biochemical and morphological changes in the IC that may contribute to deficits in the functions of these neurotransmitters, using three age groups of Fischer-344 rats. Homogenates obtained from the IC showed age-dependent reductions in activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), with a concomitant increase in lipid peroxidation. Dephosphorylation of IC homogenates with alkaline phosphatase reduced the activities of SOD and CAT in all age groups, which could be restored by protein kinase C (PKC)-dependent phosphorylation. Restoration of enzyme activity was specific to the PKC-alpha isozyme, but not to the beta1, beta2, delta or gamma forms. No age-dependent change in the levels of PKC isoforms (alpha, beta1, beta2 and gamma) was detectable in IC homogenates. Morphological analyses indicate decreases in mitochondrial density in the somata of both GABA+ and GABA- IC neurons in 19- and 28-month-old rats when compared to 3-month-olds, along with significantly higher matricial abnormalities. These data indicate age-related increases in oxidative stress in the IC, which could be partially restored by PKC. The progressive increase in oxidative stress with age may underlie changes in neuronal morphology and function of the IC.

Comparative assessment of the effect of aflatoxin B1 on hepatic dysfunction in some mammalian and avian species.

1. Aflatoxin B1 (1.5 mg/kg body weight, i.p.) was administered to rats, mice, quail and chickens to examine the comparative effect on hepatic microsomal drug-metabolizing enzymes, cytosolic glutathione S-transferase and serum enzymes. 2. Administration of aflatoxin B1 to rats resulted in a significant decrease in microsomal cytochrome P-450, NADPH-cytochrome c reductase, activities of aminopyrine N-demethylase, aniline hydroxylase, cytosolic glutathione S-transferase and liver glutathione content. However, no significant changes in these parameters were seen in mice. 3. Quail showed a significant decrease in the content of cytochrome P-450 and the activities of aminopyrine N-demethylase, aniline hydroxylase and cytosolic glutathione S-transferase. A similar treatment did not affect these biotransformation enzymes in chickens. 4. The activities of serum enzymes, sorbitol dehydrogenase, alanine aminotransferase and aspartate aminotransferase were increased significantly in rats and quail. Mice exhibited a significant increase in the activities of sorbitol dehydrogenase and aspartate aminotransferase, while chickens showed a significant increase only in alanine aminotransferase.

Lack of in vitro and in vitro effects of fenbendazole on phase I and phase II biotransformation enzymes in rats, mice and chickens.

Intraperitoneal administration of 10 mg fenbendazole/kg bw daily for 5 d caused no significant alterations in the activities of hepatic microsomal drug-metabolizing enzymes viz aminopyrine N-demethylase, aniline hydroxylase and cytosolic glutathione S-transferase in rats, mice and chickens. Similarly no significant difference in the amount of microsomal cytochrome P-450 and NADPH-cytochrome c reductase was found between control and treated animals. In vitro incubation of fenbendazole with rat, mouse and chicken microsomes suggests that the drug neither binds to microsomal protein cytochrome P-450 nor inhibits the activities of aminopyrine N-demethylase and aniline hydroxylase. Similarly in vitro addition of fenbendazole to cytosolic glutathione S-transferase from the above species did not alter the activity of this enzyme. The results indicate that fenbendazole does not alter the activity of hepatic microsomal monooxygenase system significantly in rats, mice and chickens at a dosage level of 10 mg/kg body weight. In vitro studies also indicate that fenbendazole does not interact with the hepatic microsomal monooxygenase system, indicating it is not a substrate for cytochrome P-450-dependent monooxygenase system.

Purification and characterization of glutathione-S-transferase from liver cytosol of phenobarbital-treated rabbits.

Glutathione-S-transferase (GST) from the liver cytosol of phenobarbital (PB)-treated rabbits was purified by DEAE-cellulose, CM-cellulose and hydroxylapatite column chromatography. Four species of GST were obtained by eluting the CM-cellulose column with a linear KCl gradient, and the major protein investigated. The purified enzyme from PB-treated and untreated rabbit had specific activities of 125.16 units/mg and 72.8 units/mg of protein, respectively, and the apparent Km was 0.6 X 10(-3) M for GSH and 1.6 X 10(-3) M for 1-chloro-2,4-dinitrobenzene. The optimum pH value was 8.7 and the enzyme was able to conjugate with 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(p-nitrophenoxy)propane and p-nitrobenzyl chloride.