An increase of acidic isoform of catalase in red blood cells from HIV(+) population

A systemic oxidative stress of HIV (+) individuals has been recognized from a low glutathione level and a high level of inflammatory cytokines such as TNF. Previously, we demonstrated that the catalase enzyme activity in HIV (+) population is significantly altered depending on the cell types; the level was significantly high in red blood cells while the enzymes in white blood cells were remarkably low (Res Commun Subs Abuse 16: 161–176, 1995). In this study, we further characterized the difference in RBC catalase molecules between HIV (+) and control population. We have found that RBC from HIV (+) population, whether they were asymptomatic or symptomatic, contained a significantly elevated catalase protein accompanied by the enzyme activities, and that the majority of the elevated protein were acidic pl of the molecules with an identical subunit mass of approximately 60 KDa. These results suggest that catalase is induced prior to and/or during erythroid differentiation lineage in HIV (+) population as a somatic defense to respond and compensate for a systemic oxidative stress and for an anemic condition. (Mol Cell Biochem 165: 77–81, 1996)     

Distribution and activity of microsomal NADPH-dependent monooxygenases and amino acid decarboxylases in cruciferous and non-cruciferous plants, and their relationship to foliar glucosinolate content

The NADPH-dependent conversion of amino acids to their aldoximes is an initial step in glucosinolate biosynthesis. A number of microsomal aldoxime-forming monooxygenase activities were detected in leaves from a variety of glucosi-nolate-containing species, whereas barley, bean and tobacco leaves did not contain any such activities. The substrates for these monooxygenases in each species largely correlated with the spectrum of glucosinolates found in that species. No activity was detected that metabolized homomethionine (supposed precursor of 2-propenylglucosinolate [sinigrin]), even in species where sinigrin was the major glucosinolate. In Sinapis species containing hydroxybenzylglucosinolate (sinalbin), activity with L-Tyr was detected, whereas Brassica species containing sinalbin had no such activity. However, these Brassicas did contain an L-Phe monooxygenase activity. Partial characterization of the monooxygenases indicated that in Brassica species, Nasturtium officinalis and Raphanus sativus these resembled the flavin-linked monooxygenases previously found in oilseed rape (Brassica napus) and Chinese cabbage (Brassica campestris). The L-Tyr-dependent activity in Sinapis species, and the L-Phe-dependent activity in Tropacolum majus, had characteristics of cytochrome P450-type enzymes. No similarity was found with any other known amino acid metabolizing enzymes (including decarboxylases, amino acid oxidases and diamine/polyamine oxidases).     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, fromArabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the -glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.     

Antioxidative protection in the inducible CAM plant Sedum album L. following the imposition of severe water stress and recovery

The antioxidative protection during the C₃-CAM shift induced by water stress was investigated in the temperate succulent Sedum album L. The C₃-CAM shift was characterized in terms of CO₂ exchange, titratable acidity and phosphoenolpyruvate carboxylase activity. Well-watered plants displayed C₃-like patterns of gas exchange and exhibited a mild day-night acid fluctuation indicating that those plants were performing CAM-cycling metabolism. Imposed drought highly stimulated CAM cycling, decreasing the net CO₂ uptake during the day, eliminating net CO₂ efflux at night and stimulating tissue acid fluctuations. As water deficit developed, chlorophyll fluorescence measurements showed a decrease in the Fv/Fm ratio, indicating that photoinhibition could follow after severe drought. Protection might be performed by the increased activity of enzymes involved in the destruction of free radicals and oxidants, but their response depended on the water status of the plant. Ascorbate peroxidase and superoxide dismutase activities increased in plants subjected to mild stress but declined during severe water stress. Catalase activity, however, was quite stable under mild water stress and was clearly inhibited under severe water stress. At this stage, glutathione reductase and monodehydroascorbate reductase seemed to be very important in the protection against oxidants, both increasing considerably their activities under severe water stress. Even if recycling has been shown to alleviate photoinhibition, our results clearly demonstrate that antioxidative enzymes play an important role in the protection of plants from oxidants during the C₃-CAM shift induced by water stress.     

Chilling,oxidative stress and antioxidant responses inArabidopsis thaliana callus

Chilling ofArabidopsis thaliana (L.) Heynh. callus tissue to 4 °C led to conditions of oxidative stress, as indicated by increased levels of the products of peroxidative damage to cell membranes. Cellular H₂O₂ was also observed to increase initially upon chilling but by day 8 cellular levels had declined to below control levels. Although levels of catalase activity remained similar to those in unchilled tissue, activity of ascorbate peroxidase increased between days 4 and 8 of chilling to 4 °C. In callus held at 23 °C, levels of reduced glutathione remained static whereas they rose in callus held at 4 °C. Levels of oxidised glutathione were initially low but increased significantly by day 4 in the chilled callus. At 23 °C, however, levels of oxidised glutathione remained low. Between days 1 and 3 at 4 °C, levels of glutathione reductase activity increased but by day 8 glutathione reductase activity was similar to that in cells held at 23 °C. Exposure of callus to abscisic acid at 23 °C also led to increased activities of ascorbate peroxidase and glutathione reductase.Abbreviations ABA abscisic acid - GSH reduced glutathione - GSSG oxidised glutathione - TTC 2,35-triphenyltetrazolium chloride This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.     

Effect of NaCI on nitrate reductase,glutamate dehydrogenase and glutamate synthase inVigna radiata calli

The effect of NaCI stress on the activities of nitrate reductase (NR), glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT) in callus lines ofVigna radiata which differ in salt resistance, was studied at weekly intervals upto 28 d of growth. After 28 d, the NaCI resistant callus (selected at 300 mM NaCI) at NaCI concentrations higher than 200 mM maintained higher NR activity than non-selected line. NaCI stress also affects aminating and deaminating activities of GDH. The NADH-GDH activity in the presence of NaCI was higher in the resistant than non-selected line. On the other hand, NAD-GDH activity in both the lines was completely inhibited after 7 d of growth. The increased activity of NADH-GDH in resistant calli may play a vital role in protecting the cells from toxic effect of increased endogenous level of ammonia which probably accumulates due to efficient NO₃ ⁻ reduction. NADH-GOGAT activity was found to decrease under salt stress in both the callus lines. Nitrogen assimilation in salt-resistant calli under salt stress was found to be characterized by high NR and NADH-GDH activities, concomitantly with low GOGAT activity. The authors are grateful to DST and CSIR for financial assistance.     

The growth form of the inducing microorganism and chitin addition affect mycolytic enzyme production byTrichoderma harzianum

The effect of the growth form of the inducing microorganism on specificTrichoderma harzianum mycolytic enzyme production was studied. The pelleted form ofRhizopus nigricans gave a better product concerning protoplast formation ability. The maximum yield of protoplasts from the target fungusCochliobolus lunatus was 1×10⁸ ml^–1. Analysis of individual specific enzyme activities inTrichoderma mycolytic enzyme preparations confirms the importance of high chitinase and low protease activity for high protoplast yields. Supplementation of the production medium with chitin increased the chitinase activity in theTrichoderma exoenzyme mixture.     

Inhibition of rabbit sperm acrosomal enzymes by gossypol

The effect of gossypol on the activities of 10 acrosomal enzymes of the rabbit sperm was evaluated. Acrosin, Azocoll proteinase, neuraminidase, and arylsulfatase were significantly inhibited or completely inactivated by 12–76 μM gossypol. Hyaluronidase, β-glucuronidase, and acid phosphatase were inhibited only at a higher concentration of gossypol (380 μM). Phospholipase C, alkaline phosphatase, and β-N-Acetyl glucosaminidase were not inhibited even at 380 μM gossypol. Gossypol was found to be a noncompetitive inhibitor of arylsulfatase with a Ki of 120 μM. The inhibition was reversible and dose-dependent. As the acrosomal enzymes were more sensitive to the inhibition by gossypol compared to sperm enzymes involved in glycolysis or energy production, these assays may serve as a more reliable indicator for monitoring the occurence of gossypol-induced sterility. © 1995 Wiley-Liss, Inc.     

The Origin of the Oxidative Burst in Plants

A large number of publications recently have drawn strong analogies between the production of active oxygen species in plant cells and the “oxidative burst” of the phagocyte, even to the point of constructing elaborate models involving receptor mediated G-protein activation of a plasmalemma NADPH oxidase in plant cells. However there are potentially other active oxygen species generating systems at the plant cell surface. The present work examines these alternatives with particular emphasis on the rapid production of active oxygen species, in common with a number of other systems, by suspension-cultured cells of French bean on exposure to an elicitor preparation from the fungal pathogen Colletotrichum lindemuthianum. The cells show a rapid increase in oxygen uptake which is followed shortly afterwards by the appearance of a burst of these active oxygen species, as measured by a luminescence assay, which is probably all accounted for by hydrogen peroxide. An essential factor in this production of H₂O₂ appears to be a transient alkalinization of the apoplast where the pH rises to 7.0-7.2. Dissipation of this pH change with a number of treatments, including ionophores and strong buffers, substantially inhibits the oxidative burst. Little evidence was found for enhanced activation of a membrane-bound NADPH oxidase. However the production of H₂O₂ under alkaline conditions can be modelled in vitro with a number of peroxidases, one of which, an M_r 46,000 wall-bound cationic peroxidase, is able to sustain H₂O₂ production at neutral pH unlike the other peroxidases which only show low levels of this reaction under such conditions and have pH optima at values greater than 8.0. On the basis of such comparative pH profiles between the cells and the purified peroxidase and further inhibition studies a direct production of H₂O₂ from the wall peroxidase in French bean cells is proposed. These experiments may mimic some of the responses to plant pathogens, particularly the hypersensitive response, which is an important feature of resistance. A cell wall peroxidase-origin for the oxidative burst is clearly different from a model consisting of receptor activation of a plasmaiemma-localised NADPH oxidase generating superoxide. An alternative simple and rapid mechanism thus exists for the generation of H₂O₂ which does not require such multiple proteinaceous components.