Phosphamidon, methylparathion and dichlorvos impact on tissue oxidative metabolism in penaeid prawn, Metapenaeus monoceros

Changes in oxidative metabolism of hepatopancreas and muscle tissues of penaeid prawn, Metapenaeus monoceros was studied, following its exposure to selected organophosphorous insecticides phosphamidon, dichlorovos and methylparathion. The OPI are found to inhibit the activity levels of acetylcholinesterase, succinate dehydrogenase, isocitrate dehydrogenase, pyruvate dehydrogenase, lactate dehydrogenase and cytochrome-c-oxidase and cause accumulation of acetylcholine in the hepatopancreas and muscle tissues. These changes in the activity levels of selected oxidative enzymes during insecticide exposure in these tissues of prawn indicates the shift in the metabolic emphasis from aerobic to anaerobic conditions and is interpreted as a functional adaptation to insecticide induced metabolic stress. These observed changes at cellular level pave way for successful survival of prawns in insecticide polluted environ.     

The effects of beta-mercaptoethanol and sodium dodecyl sulfate on the Humicola insolens beta-glucosidase

Hydrolysis of p-nitrophenyl-beta-D-glucoside by the beta-glucosidase of a thermophilic and cellulolytic fungus, Humicola insolens was stimulated by two-fold in the presence of high concentrations of beta-mercaptoethanol. This enzyme did not have any free sulfhydryl groups and high concentrations of beta-mercaptoethanol (5% v/v) reduced all of the three disulfide bonds present in the enzyme. In contrast, the hydrolysis of cellobiose and cellulose polymers was inhibited by 50% under the same conditions. Sodium dodecyl sulfate (1% w/v) even in combination with beta-mercaptoethanol did not show any significant effects on this enzyme. These unusual properties suggest that this enzyme may be of significant importance for understanding the structure of the enzyme.     

Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development.

Two general mechanisms mediate glucose transport, one is a sodium-coupled glucose transporter found in the apical border of intestinal and kidney epithelia, while the other is a sodium-independent transport system. Of the latter, several facilitated transporters have been identified, including GLUT1 (erythrocyte/brain), GLUT2 (liver) and GLUT4 (adipose/muscle) isoforms. In this study, we used Western-blot analysis and high resolution immunoelectron microscopy (IEM) to investigate the stage-related expression and cellular localization of GLUT1, 2 and 4. The Western blot results demonstrate that GLUT1 is detectable in the oocyte and throughout preimplantation development. GLUT2 isoforms were not detectable until the blastocyst stage, while the GLUT4 isoform was undetectable in the oocyte through blastocyst stages. The present findings confirm previous studies at the molecular level which demonstrated that mRNAs encoding the same GLUT isoforms are detectable at corresponding developmental stages. GLUT1 and GLUT2 display different cellular distributions at the blastocyst stage as shown by IEM studies. GLUT1 has a widespread distribution in both trophectoderm and inner cell mass cells, while GLUT2 is located on trophectoderm membranes facing the blastocyst cavity. This observation suggests a different functional significance for these isoforms during mouse preimplantation development.     

Variable alpha-tocopherol stimulation and protection of glutathione peroxidase activity in established and malignant fibroblasts.

Studies on glutathione (GSH) metabolism in an established baby hamster kidney fibroblast cell line (BHK-21/C13) and in its polyoma virus-transformed counterpart (BHK-21/PyY) have revealed a significant stimulation of intracellular GSH peroxidase (GSHpx) activity (selenium-independent plus selenium-dependent) by alpha-tocopherol supplementation (14 microM). This stimulation was found to be much greater in the transformed cells. Other GSH-requiring enzyme activities (i.e. GSH reductase and GSH S-transferase) were unaltered by alpha-tocopherol treatment, suggesting a degree of specificity in its action on GSHpx. In unsupplemented growth media, the GSHpx activity in both cell lines was significantly decreased by oxidative stress. However, the same stress applied to the alpha-tocopherol-supplemented cells had no effect on the stimulated GSHpx activity, suggesting that some protection was afforded by the alpha-tocopherol.     

Basic carboxyl groups of hemoglobin S: Influence of oxy-deoxy conformation on the chemical reactivity of Glu-43(β)

The -carboxyl groups of Glu-43() and Glu-22() of hemoglobin-S (HbS), two intermolecular contact residues of deoxy protein, are activated by carbodiimide atp H 6.0. The selectivity of the modification by the two nucleophiles, glycine ethyl ester (GEE) and glucosamine, is distinct. Influence ofN-hydroxysulfosuccinimide, a reagent that rescues carbodiimide-activated carboxyl (O-acyl isourea) as sulfo-NHS ester, on the overall selectivity and efficiency of the coupling of Glu-22() and Glu-43() with nucleophiles has been investigated. Sulfo-NHS increases the extent of coupling of nucleophiles to HbS. The rescuing efficiency of sulfo-NHS(increase in modification) with GEE and galactosamine as nucleophiles is 2.0 and 2.8, respectively. In the presence of sulfo-NHS, the extent of modification of a carboxyl group is a direct reflection of the extent to which it is activated (i.e., the protonation state of the carboxyl group). The modification reaction exhibits very high selectivity for Glu-43() with GEE and galactosamine (GA) in the presence of sulfo-NHS. From the studies of the kinetics of amidation of oxy-HbS at its Glu-43() (i.e., chemical reactivity) as a function of thepH in the region of 5.5–7.5, the apparentpKa of its -carboxyl group has been calculated to be 6.35. Deoxygenation of HbS, nearly doubles the chemical reactivity of Glu-43() of HbS atpH 7.0. It is suggested that the increased hydrophobicity of the microenvironment of Glu-43(), which occurs on deoxygenation of the protein, is reflected as the increased chemical reactivity of the -carboxyl group and could be one of the crucial preludes to the polymerization process.     

Production of triple-stranded recombination intermediates by RecA protein, in vitro

During the directional strand exchange that is promoted by RecA protein between linear duplex DNA and circular single-stranded DNA, a triple-stranded DNA intermediate was formed and persisted even after the completion of strand transfer followed by deproteinization. In the deproteinized three-stranded DNA complexes, the sequestered linear third strand resisted digestion by E coli exonuclease I. In relation to polarity of strand exchange which defines the proximal and distal ends of the duplex DNA, when homology was restricted to the distal region of duplex substrate, the joints formed efficiently and were stable even upon complete deproteinization. Enzymatic probing of deproteinized distal joints with nuclease P1 revealed that the joints consist of long three-stranded structures that at neutral pH lack significant single-stranded character in any of the three strands. Instead of circular single-stranded DNA, when a linear single strand is recombined with partially homologous duplex DNA, in the presence of SSB, the formation of homologous joints by RecA protein, is significantly more efficient at distal end than at the proximal. Taken together, these observations suggest that with any single-stranded DNA (circular or linear), RecA protein efficiently promotes the formation of distal joints, from which, however, authentic strand exchange may not occur. Moreover, these joints might represent an intermediate which is trapped into a stable triple stranded state.     

Redox cycling and sulphydryl arylation; their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes

Quinones are believed to be toxic by a mechanism involving redox cycling and oxidative stress. In this study, we have used 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ), which redox cycles to the same degree as menadione, but does not react with free thiol groups, to distinguish between the importance of redox cycling and arylation of free thiol groups in the causation of toxicity to isolated hepatocytes. Menadione was significantly more toxic to isolated hepatocytes than 2,3-diOMe-1,4-NQ. Both menadione and 2,3-diOMe-1,4-NQ caused an extensive GSH depletion accompanied by GSSG formation, preceding loss of viability. Both compounds stimulated a similar increase in oxygen uptake in isolated hepatocytes and NADPH oxidation in microsomes suggesting they both redox cycle to similar extents. Further evidence for the redox cycling in intact hepatocytes was the detection of the semiquinone anion radicals with electron spin resonance spectroscopy. In addition we have, using the spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide), demonstrated for the first time the formation of superoxide anion radicals by intact hepatocytes. These radicals result from oxidation of the semiquinone by oxygen and further prove that both these quinones redox cycle in intact hepatocytes. We conclude that while oxidative processes may cause toxicity, the arylation of intracellular thiols or nucleophiles also contributes significantly to the cytotoxicity of compounds such as menadione.     

Characterization of a glutathione conjugate of the 1,4-benzosemiquinone-free radical formed in rat hepatocytes

Rat hepatocytes treated with 1,4-benzoquinone formed 1,4-benzosemiquinone and 2-S-glutathionyl-1,4-benzosemiquinone radicals as detected by ESR spectroscopy. The 2-S-glutathionyl-1,4-benzosemiquinone radical was first obtained from the reaction of 1,4-benzoquinone with glutathione. Glutathione both reduced benzoquinone to form benzosemiquinone and conjugated benzoquinone to form 2-S-glutathionyl-1,4-benzosemiquinone radical. The ratio of these two radicals depended upon the ratio of 1,4-benzoquinone to glutathione. At near equimolar ratios, the 2-S-glutathionyl-1,4-benzosemiquinone radical was predominantly formed. This radical was characterized by computer simulation of the experimental spectra and identified by comparison of its hyperfine coupling constants with those of chemical analogues. The 2-S-glutathionyl-1,4-benzosemiquinone radicals formed inside hepatocytes, and then crossed the plasma membrane into the media.