Gopinadhan Paliyath

Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana.

Earlier studies with Arabidopsis thaliana exposed to ultraviolet B (UV-B) and ozone (O3) have indicated the differential responses of superoxide dismutase and glutathione reductase. In this study, we have investigated whether A. thaliana genotype Landsberg erecta and its flavonoid-deficient mutant transparent testa (tt5) is capable of metabolizing UV-B- and O3-induced activated oxygen species by invoking similar antioxidant enzymes. UV-B exposure preferentially enhanced guaiacol-peroxidases, ascorbate peroxidase, and peroxidases specific to coniferyl alcohol and modified the substrate affinity of ascorbate peroxidase. O3 exposure enhanced superoxide dismutase, peroxidases, glutathione reductase, and ascorbate peroxidase to a similar degree and modified the substrate affinity of both glutathione reductase and ascorbate peroxidase. Both UV-B and O3 exposure enhanced similar Cu,Zn-superoxide dismutase isoforms. New isoforms of peroxidases and ascorbate peroxidase were synthesized in tt5 plants irradiated with UV-B. UV-B radiation, in contrast to O3, enhanced the activated oxygen species by increasing membrane-localized NADPH-oxidase activity and decreasing catalase activities. These results collectively suggest that (a) UV-B exposure preferentially induces peroxidase-related enzymes, whereas O3 exposure invokes the enzymes of superoxide dismutase/ascorbate-glutathione cycle, and (b) in contrast to O3, UV-B exposure generated activated oxygen species by increasing NADPH-oxidase activity.

Influence of Salicylic Acid on H2O2 Production, Oxidative Stress, and H2O2-Metabolizing Enzymes (Salicylic Acid-Mediated Oxidative Damage Requires H2O2)

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.

Changes in Activities of Antioxidant Enzymes and Their Relationship to Genetic and Paclobutrazol-Induced Chilling Tolerance of Maize Seedlings

The potential role of antioxidant enzymes in protecting maize (Zea mays L.) seedlings from chilling injury was examined by analyzing enzyme activities and isozyme profiles of chilling-susceptible (CO 316) and chilling-tolerant (CO 328) inbreds. Leaf superoxide dismutase (SOD) activity in CO 316 was nearly one-half that of CO 328, in which the high activity was maintained during the chilling and postchilling periods. Activity of glutathione reductase (GR) was much higher in roots than in leaves. CO 328 also possessed a new GR isozyme that was absent in roots of CO 316. Ascorbate peroxidase (APX) activity was considerably lower in leaves of CO 328 than in CO 316, and nearly similar in roots. Paclobutrazol treatment of CO 316 induced several changes in the antioxidant enzyme profiles and enhanced their activities, especially those of SOD and APX, along with the induction of chilling tolerance. These results suggest that increased activities of SOD in leaves and GR in roots of CO 328, as well as SOD and APX in leaves and roots of paclobutrazol-treated CO 316, contribute to their enhanced chilling tolerance.

Selective Cytotoxicity of a Red Grape Wine Flavonoid Fraction Against MCF-7 Cells

Red wine is a rich source of polyphenolic components such as anthocyanins and flavonoids. The inhibitory effects of red wine polyphenolics on human breast cancer cells have been demonstrated earlier, but their effects on normal cells have not been fully established. Red wine (Merlot) was fractionated by hydrophobic interaction chromatography and different flavonoid fractions with increasing hydrophobicity were obtained. These fractions were tested for their inhibitory effect on human breast cancer cells (MCF-7), normal human mammary epithelial cells (HMEC), and a non-tumorigenic MCF-10A cell line. By contrast to the authentic flavonoids such as quercetin, naringenin and catechin which inhibited the growth of HMEC much more than that of MCF-7 cancer cells, a red wine fraction, that was comprised mainly of the flavonoid aglycones, showed maximal inhibition of the growth of breast cancer cells, with relatively low cytotoxicity towards HMEC and MCF-10A cells. In the presence of this flavonoid fraction, the normal cells grew normally, whereas the breast cancer cells underwent a change in morphology into spherical forms. Cytotoxicity analyses suggested that these cells had become apoptotic. The efficiency of inhibition of cell proliferation by various flavonoid fractions appeared to be related to their inhibition of calcium and calmodulin-promoted phosphodiesterase activity, suggesting that flavonoids may interfere with calcium second messenger function. The results suggest that certain grape wine ingredients have anticancer properties and these ingredients may be helpful for developing designer functional foods with cancer-preventive properties.

Inhibition of phospholipase D

Phospholipases A2 are involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Inhibitors of these enzymes are proving useful in determining the biological roles of phospholipases A2 in complex cellular processes and may also have therapeutic potential. Inhibition of these lipolytic enzymes is more difficult to characterize as the enzymatic reaction occurs at a lipid/water interface. This review focuses on the description of a number of classes of rationally designed phospholipase A2 inhibitors. The development of a theoretical framework for the proper analysis of inhibitors is presented. Structural studies of phospholipase A2‐inhibitor complexes suggest how the lipolysis reaction is catalyzed. Finally, some recent results on the use of phospholipase A2 inhibitors in living cells and tissues are revealed.— Gelb, M. H., Jain, M. K., Berg, O. G. Inhibition of phospholipase A2. FASEB J. 8: 916‐924; 1994.

Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion

Wild blueberries are rich in polyphenols and have several potential health benefits. Understanding the factors that affect the bioaccessibility and bioavailability of polyphenols is important for evaluating their biological significance and efficacy as functional food ingredients. Since the bioavailability of polyphenols such as anthocyanins is generally low, it has been proposed that metabolites resulting during colonic fermentation may be the components that exert health benefits. In this study, an in vitro gastrointestinal model comprising sequential chemostat fermentation steps that simulate digestive conditions in the stomach, small intestine and colon was used to investigate the breakdown of blueberry polyphenols. The catabolic products were isolated and biological effects tested using a normal human colonic epithelial cell line (CRL 1790) and a human colorectal cancer cell line (HT 29). The results showed a high stability of total polyphenols and anthocyanins during simulated gastric digestion step with approximately 93% and 99% of recovery, respectively. Intestinal digestion decreased polyphenol- and anthocyanin- contents by 49% and 15%, respectively, by comparison to the non-digested samples. During chemostat fermentation that simulates colonic digestion, the complex polyphenol mixture was degraded to a limited number of phenolic compounds such as syringic, cinnamic, caffeic, and protocatechuic acids. Only acetylated anthocyanins were detected in low amounts after chemostat fermentation. The catabolites showed lowered antioxidant activity and cell growth inhibition potential. Results suggest that colonic fermentation may alter the biological activity of blueberry polyphenols.

Modulation of phospholipase D and lipoxygenase activities during chilling. Relation to chilling tolerance of maize seedlings

Phospholipase D (phosphatidylcholine choline hydrolase, EC 3.1.4.4) and lipoxygenase activities (linoleate: oxygen oxidoreductase, EC 1.13.11.12) were analysed in chilling-susceptible and chilling-tolerant maize (Zea mays L.) seedlings in relation to their chilling tolerance. Before chilling, phospholipase D activity in the leaf microsomal fraction was 2-fold higher in the chilling-susceptible CO 316 when compared to that of paclobutrazol-treated, chilling-tolerant CO 316. Phospholipase D activity in the heavy membrane fraction comprising chloroplast and mitochondria showed a 3 to 4-fold increase during chilling and post-chilling periods, albeit with temporal variations. An increase in cytosolic phospholipase D activity was observed only in CO 316 and CO 328. Also, phospholipase D activity in the heavy membrane and cytosolic fractions of roots increased in CO 316 during chilling and post-chilling periods. Lipoxygenase activity was low in both leaves and roots of CO 328. Paclobutrazol treatment of CO 316 did not appear to affect the level of cytosolic lipoxygenase activity in roots during the early part of chilling and post-chilling, but increased the activity in leaves. The results suggest that during chilling and post-chilling periods, temporal variations in phospholipase D and lipoxygenase activities in both leaves and roots of CO 316, CO 316P and CO 328 could result in differential metabolism of phospholipids. Enhanced degradation coupled with low levels of turnover could lead to development of chilling injury in the tissue.

Membrane deterioration during postharvest senescence of broccoli florets: modulation by temperature and controlled atmosphere storage

Abstract When freshly cut heads of broccoli ( Brassica oleracea L.) are stored in air at 23°C or 10°C, the florets rapidly senesce. Chlorophyll levels decline by 80–90% within 4 days at 23°C and within 10 days at 10°C, and under the same storage conditions microsomal phospholipid phosphate and fatty acids in the florets decline by 70–90%. The breakdown of membrane phospholipids is accompanied by a large decrease (… 35%) in bulk lipid fluidity of the membranes. These changes are temperature-dependent inasmuch as they are completely inhibited during storage for comparable periods in air at 5°C. Also, the potential of microsomal membranes isolated from florets to degrade exogenous, radiolabelled phosphatidylcholine increases during storage in air at 23°C or 10°C, but not at 5°C. Storage at 5°C or 10°C in a controlled atmosphere (5% CO 2 , 3% O 2 , 92% N 2 ) at … 80% relative humidity strongly inhibits loss of chlorophyll and the change in microsomal bulk lipid fluidity reflecting phospholipid breakdown. Thus reduced integrity of thylakoids and membrane phospholipid degradation appear to be inherent features of broccoli floret senescence that are sensitive, directly or indirectly, to ambient CO 2 and O 2 concentrations. These observations confirm the potential of using controlled atmospheres to enhance the storability of broccoli and identify physiological and biochemical parameters of floret senescence that can be used to establish optimum controlled atmosphere conditions.

Biochemical Basis for Functional Ingredient Design from Fruits

Functional food ingredients (nutraceuticals) in fruits range from small molecular components, such as the secondary plant products, to macromolecular entities, e.g., pectin and cellulose, that provide several health benefits. In fruits, the most visible functional ingredients are the color components anthocyanins and carotenoids. In addition, several other secondary plant products, including terpenes, show health beneficial activities. A common feature of several functional ingredients is their antioxidant function. For example, reactive oxygen species (ROS) can be oxidized and stabilized by flavonoid components, and the flavonoid radical can undergo electron rearrangement stabilizing the flavonoid radical. Compounds that possess an orthodihydroxy or quinone structure can interact with cellular proteins in the Keap1/Nrf2/ARE pathway to activate the gene transcription of antioxidant enzymes. Carotenoids and flavonoids can also exert their action by modulating the signal transduction and gene expression within the cell. Recent results suggest that these activities are primarily responsible for the health benefits associated with the consumption of fruits and vegetables.

Grape and wine polyphenols down-regulate the expression of signal transduction genes and inhibit the growth of estrogen receptor-negative MDA-MB231 tumors in nu/nu mouse xenografts.

The antitumor properties of the Merlot grape (and Merlot wine) polyphenols were evaluated in relation to their ability to modulate gene expression in developing tumors using an athymic nude mouse model transplanted with the estrogen receptor-negative MDA-MB231 cells. Groups of mice were fed a modified AIN 93G diet (Research Diets Inc, New Brunswick, NJ) with the experimental groups receiving 100 mg/kg body weight equivalent of polyphenols by gavage 3 times per week. After 1 week of acclimation and another week of polyphenol supplementation, MDA-MB231 cells were transplanted and the growth patterns of the tumors monitored. After 33 days of tumor growth, the animals were euthanized, the tumors isolated, and gene expression profiles analyzed using signal transduction and cell cycle arrays. The development of tumors was almost totally arrested in grape polyphenol-treated mice. Total polyphenols isolated from the wine were more effective in reducing tumor growth as compared with a hydrophobic polyphenol fraction isolated from the wine, showing a 50% and 60% reduction in tumor growth on day 33, respectively. Analysis of gene expression showed that genes such as CDK2, FAS, LEF1, PRKCE, and PTGS2, belonging to the NFkappaB, phospholipase C, and calcium signaling pathways, were down-regulated in tumors that developed in grape polyphenol-treated mice. Several genes related to cell cycle regulation, such as CDK5RAP1, RBBP8, and SERTAD1, were up-regulated in these tumors. Changes in the expression of these genes were less pronounced in tumors of wine polyphenol-treated mice. The study highlights the potential influences of dietary polyphenolic components on gene expression in estrogen receptor-negative tumors and its relation to inhibition of tumor growth.