Antioxidant activities of seabuckthorn (Hippophae rhamnoides) during hypoxia induced oxidative stress in glial cells

The present study reports the cytoprotective and antioxidant properties of alcoholic leaf extract of seabuckthorn (SBT) against hypoxia induced oxidative stress in C-6 glioma cells. Exposure of cells to hypoxia for 12 h resulted in a significant increase in cytotoxicity and decrease in mitochondrial transmembrane potential compared to the controls. Further an appreciable increase in nitric oxide and reactive oxygen species (ROS) production was noted which in turn was responsible for fall in intracellular antioxidant levels and GSH/GSSG ratio. There was a significant increase in DNA damage during hypoxia as revealed by comet assay. Pretreatment of cells with alcoholic leaf extract of SBT at 200 μg/ml significantly inhibited cytotoxicity, ROS production and maintained antioxidant levels similar to that of control cells. Further, the leaf extract restored the mitochondrial integrity and prevented the DNA damage induced by hypoxia. These results indicate that the leaf extract of SBT has strong antioxidant and cytoprotective activity against hypoxia induced oxidative injury. (Mol Cell Biochem 278: 9–14, 2005)     

Pre-steady-state kinetic analysis of enzyme-monitored turnover during cystathionine β-synthase-catalyzed H(2)S generation

Cystathionine β-synthase (CBS) catalyzes the first step in the transsulfuration pathway in mammals, i.e., the condensation of serine and homocysteine to produce cystathionine and water. Recently, we have reported a steady-state kinetic analysis of the three hydrogen sulfide (H(2)S)-generating reactions that are catalyzed by human and yeast CBS [Singh, S., et al. (2009) J. Biol. Chem. 284, 22457-22466]. In the study presented here, we report a pre-steady-state kinetic analysis of intermediates in the H(2)S-generating reactions catalyzed by yeast CBS (yCBS). Because yCBS does not have a heme cofactor, in contrast to human CBS, it is easier to observe reaction intermediates with yCBS. The most efficient route for H(2)S generation by yCBS is the β-replacement of the cysteine thiol with homocysteine. In this reaction, yCBS first reacts with cysteine to release H(2)S and forms an aminoacrylate intermediate (k(obs) of 1.61 ± 0.04 mM(-1) s(-1) at low cysteine concentrations and 2.8 ± 0.1 mM(-1) s(-1) at high cysteine concentrations, at 20 °C), which has an absorption maximum at 465 nm. Homocysteine binds to the E·aminoacrylate intermediate with a bimolecular rate constant of 142 mM(-1) s(-1) and rapidly condenses to form the enzyme-bound external aldimine of cystathionine. The reactions could be partially rate limited by release of the products, cystathionine and H(2)S.     

Impeded electron transfer from a pathogenic FMN domain mutant of methionine synthase reductase and its responsiveness to flavin supplementation

Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metabolism and is characterized by elevated levels of plasma homocysteine. In this study, we have examined the molecular basis of MSR dysfunction associated with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione reduction is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochemical penalty associated with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy.     

Characterization of a succinyl-CoA radical-cob(II)alamin spin triplet intermediate in the reaction catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase

The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co(2+) in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co(2+). Labeling of the substrate with (13)C and (2)H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2'-(13)C]methylmalonyl-CoA and [2-(13)C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon alpha to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme.     

Assembly and protection of the radical enzyme, methylmalonyl-CoA mutase, by its chaperone

MeaB is a recently described P-loop GTPase that plays an auxiliary role in the reaction catalyzed by the radical B12 enzyme, methylmalonyl-CoA mutase. Defects in the human homologue of MeaB result in methylmalonic aciduria, but the role of this protein in coenzyme B12 assimilation and/or utilization is not known. Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA that uses reactive radical intermediates that are susceptible to oxidative inactivation. In this study, we have examined the influence of MeaB on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase and on the thermodynamics of cofactor binding. MeaB alone has a modest effect on the affinity of the mutase for the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor, increasing it 2-fold from 404 +/- 71 to 210 +/- 22 nM. However, in the presence of GDP, the affinity for the cofactor decreases 5-fold to 1.89 +/- 0.33 microM, while in the presence of guanosine 5'(beta-gamma imino)triphosphate, a nonhydrolyzable analogue of GTP, the binding of AdoCbl to the mutase is not detected. Protection against oxidative inactivation of the mutase by MeaB is dependent upon the presence of nucleotides with the MeaB/GDP and MeaB/GTP complexes decelerating the rate of formation of oxidized cofactor by 3- and 15-fold, respectively. This study suggests that MeaB functions in the GTP-dependent assembly of holomethylmalonyl-CoA mutase and subsequent protection of radical intermediates during catalysis.     

Response of cyanobacteria to arsenic toxicity

Arsenic content of cyanobacterial biomass, soil and water samples from arsenic-contaminated area of eastern India were estimated. It was found that arsenic content in cyanobacterial biomass (276.9 μg g⁻¹) was more than soil (19.01 μg g⁻¹) or water sample (244.13 μg L⁻¹). Shallow tube well water showed more arsenic (244.13 μg L⁻¹) than deep tube well water (146.13 μg L⁻¹). Arsenic resistant genera recorded from the contaminated area were Oscillatoria princeps, Oscillatoria limosa, Anabaena sp. and Phormidium laminosum. Among these, P. laminosum was isolated and exposed to different concentration of Arsenic in vitro (0.1–100 ppm) to study the toxicity level of arsenic. Modulation in stress enzymes and stress-related compounds were studied in relation to lipid peroxidase, catalase, super oxide dismutase (SOD), ascorbate peroxidase (APX), reduced glutathione and carotenoids in arsenic exposed biomass to understand the resistance mechanism of the genus both in laboratory condition as well as in natural condition. Arsenic content of cyanobacterial biomass from contaminated area was more (276.9 μg g⁻¹) than laboratory exposed sample (37.17 μg g⁻¹), indicating bioconcentration of arsenic in long-term-exposed natural biomass. Overall, more activity of catalase was recorded in cyanobacterial biomass of natural condition whereas SOD and APX were at higher level in laboratory culture.     

An alternative high-throughput staining method for detection of neutral lipids in green microalgae for biodiesel applications

A simple and high-throughput method for determining in situ intracellular neutral lipid accumulation in Chlorella ellipsoidea and Chlorococcum infusionum with flow cytometry and confocal microscopy was established by employing different solvents and a lipophilic dye, Nile red. Seven different organic solvents, acetic acid, dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, n-hexane, and chloroform at different concentrations ranging from 0 to 80% (v/v) were tested. The fluorescence signal for neutral lipids was collected with a 586/42 emission filter (PE-A) and the maximum fluorescence intensity (% grandparent) was measured as 74.01 ± 4.82% for Chlorella and 70.1 ± 5.52% for Chlorococcum at 30% acetic acid (v/v). The statistical analysis of Nile red-stained cells showed a high coefficient of variation (CV), standard deviation (SD), mean, and median values in the acetic acid-based staining method, followed by DMSO, n-hexane and chloroform. Confocal microscopy revealed a high rate of accumulation of cytosolic neutral lipids when stained with Nile red and other organic solvents. Higher lipid accumulation in Fesupplemented conditions was also detected and a maximum lipid content of 57.36 ± 0.41% (4-fold) in Chlorella and 48.20 ± 0.43% (4-fold) in Chlorococcum were measured at 0.001 g/L of ferrous sulfate (FeSO₄). High fluorescence intensity (75.16 ± 0.24% in Chlorella and 72.24 ± 1.07% in Chlorococcum) in Fe-treated cells confirmed the efficiency of the staining procedure.     

Microbial Activity in High-Sulfur Reclaimed Coal Mine Overburden Sites

The high-sulfur containing coalmine overburden (OB) dumping ground of the Tirap colliery under the northeastern coalfields in Assam, India, was reclaimed by phytoremediation techniques. Native plant species were planted with line and field methods and by amending with cow dung and unmined soil. In this study, the changes in microbial activity of the reclaimed mine OB wasteland of the Tirap colliery are examined. The enzyme activities of urease, dehydrogenase, and phosphatase were compared over time. Improvements in soil pH, bulk density, and water-holding capacity in the mine OB were found. After four years of reclamation, 1.01% of particles were soil sized (=0.255 mm grain size) with the maximum degradation in the mine OB grain sizes of ?20 mm to +12 mm and of +25 mm. A 1,000-fold increase in microbial colony forming units (CFU) in the four years at the remediated site along with an increase in total DNA were evident for the mine OB reclaimed sites. The increase of microbial CFUs in the mine OB was related to a 100- to 2,000-fold increase in enzyme activity for the reclaimed areas. From this investigation, it can be concluded that the improvement of enzyme activities and the increase in microbial populations of reclaimed mine OB will yield information pertaining to the reclamation index of mine OB.     

PLP-dependent H(2)S biogenesis

The role of endogenously produced H(2)S in mediating varied physiological effects in mammals has spurred enormous recent interest in understanding its biology and in exploiting its pharmacological potential. In these early days in the field of H(2)S signaling, large gaps exist in our understanding of its biological targets, its mechanisms of action and the regulation of its biogenesis and its clearance. Two branches within the sulfur metabolic pathway contribute to H(2)S production: (i) the reverse transsulfuration pathway in which two pyridoxal 5'-phosphate-dependent (PLP) enzymes, cystathionine β-synthase and cystathionine γ-lyase convert homocysteine successively to cystathionine and cysteine and (ii) a branch of the cysteine catabolic pathway which converts cysteine to mercaptopyruvate via a PLP-dependent cysteine aminotransferase and subsequently, to mercaptopyruvate sulfur transferase-bound persulfide from which H(2)S can be liberated. In this review, we present an overview of the kinetics of the H(2)S-generating reactions, compare the structures of the PLP-enzymes involved in its biogenesis and discuss strategies for their regulation. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

Brugia malayi: establishment in inbred and outbred strains of mice

Immunocompetent mouse model for human filarial parasite Brugia malayi is urgently required in view of the paucity of commercial reagents for other susceptible rodent viz. mastomys and gerbil. Genes within the major histocompatibility complex have been reported to influence the susceptibility of mouse to helminth parasites. Attempts have therefore been made in the present investigation to experimentally infect various inbred strains of mice viz. NZB/BINJ, BALB/c, AKR, C(3)H, and SJL/J with H-2 haplotype (H-2: d, d, k, k, s, respectively) and outbred strains of mice viz. Parks and Swiss. Findings indicate that susceptibility of mice to B. malayi is strain associated. This is the first report on the successful completion of full developmental cycle of subperiodic B. malayi in NZB/BINJ, an immunocompetent mouse strain. In some of the other strains, partial development or low degree of establishment of worms was observed.