Higher Ascorbate Research Articles

Ascorbate transport by primary cultured neurons and its role in neuronal function and protection against excitotoxicity

Neurons maintain relatively high intracellular concentrations of ascorbic acid, which is achieved primarily by the activity of the sodium-dependent vitamin C transporter SVCT2. In this work, we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate as well as whether this system contributes to maturation of neuronal function and cellular defense against oxidative stress and excitotoxic injury. We found that the SVCT2 helps to maintain high intracellular ascorbate levels, normal ascorbate transport kinetics, and activity-dependent ascorbate recycling. Immunocytochemistry studies revealed that SVCT2 is expressed primarily in the axons of mature hippocampal neurons in culture. In the absence of SVCT2, hippocampal neurons exhibited stunted neurite outgrowth, less glutamate receptor clustering, and reduced spontaneous neuronal activity. Finally, hippocampal cultures from SVCT2-deficient mice showed increased susceptibility to oxidative damage and N-methyl-D-aspartate-induced excitotoxicity. Our results revealed that maintenance of intracellular ascorbate as a result of SVCT2 activity is crucial for neuronal development, functional maturation, and antioxidant responses.

Ascorbate deficiency results in impaired neutrophil apoptosis and clearance and is associated with up-regulation of hypoxia-inducible factor 1α.

Some cells, including neutrophils, accumulate high intracellular ascorbate concentrations, which suggests that they have an important function in these cells. In this study we have used L-gulono-γ-lactone oxidase (Gulo)-/- mice, which are unable to synthesize ascorbate, to generate ascorbate-deficient neutrophils and have used these to investigate the effect of ascorbate on neutrophil function. Peritoneal neutrophils from ascorbate-deficient animals had normal morphology and respiratory burst activity but failed to undergo spontaneous apoptosis, determined by morphology and the surface expression of phosphatidylserine. Initially, there was increased cell survival, but death eventually occurred by necrosis within 48 h. Neutrophils persisted in thioglycollate-induced inflammation in Gulo-/Ȓ mice with the later appearance of necrotic cells, suggesting that apoptosis was also affected in vivo. Also, ascorbate-deficient neutrophils were not recognized by macrophages in an in vitro assay for phagocytosis, providing further evidence for defective apoptosis and clearance. Neutrophils from Gulo-/- mice had elevated levels of hypoxia-inducible factor (HIF)-1α, a transcription factor regulated by Fe2+ -dependent hydroxylases which require ascorbate for optimal activity. HIF-1α has been shown previously to inhibit neutrophil apoptosis under hypoxic conditions. Our results suggest that in ascorbate deficiency, up-regulation of HIF-1α blocks neutrophil apoptosis under normoxic conditions and that this represents a novel and important function for vitamin C in inflammatory cells.

Biosynthesis of ascorbic acid in legume root nodules.

Ascorbic acid (vitamin C) is a major antioxidant and redox buffer, but is also involved in other critical processes of plants. Recently, the hypothesis has been proposed that legume nodules are unable to synthesize ascorbate and have to import it from the shoot or root, thus providing a means by which the plant regulates nodule senescence. The last step of ascorbate biosynthesis in plants is catalyzed by L-galactono-1,4-lactone dehydrogenase (GalLDH). The mRNAs encoding GalLDH and three other enzymes involved in ascorbate biosynthesis are clearly detectable in nodules. Furthermore, an active membrane-bound GalLDH enzyme is present in nodule mitochondria. Biochemical assays on dissected nodules reveal that GalLDH activity and ascorbate are correlated in nodule tissues and predominantly localized in the infected zone, with lower levels of both parameters (relative to the infected tissues) in the apex (87%) and senescent region (43%) of indeterminate nodules and in the peripheral tissues (65%) of determinate nodules. In situ RNA hybridization showed that the GalLDH mRNA is particularly abundant in the infected zone of indeterminate and determinate nodules. Thus, our results refute the hypothesis that ascorbate is not synthesized in nodules and lend support to a previous conclusion that ascorbate in the infected zone is primarily involved in the protection of host cells against peroxide damage. Likewise, the high ascorbate and GalLDH activity levels found in the apex of indeterminate nodules strongly suggest a participation of ascorbate in additional functions during symbiosis, possibly related to cell growth and division and to molecular signaling.

Ascorbate Transport and Recycling by SH-SY5Y Neuroblastoma Cells: Response to Glutamate Toxicity

Neurons maintain relatively high intracellular concentrations of vitamin C, or ascorbic acid. In this work we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate, as well as how this system responds to oxidant stress induced by glutamate. Cultured SH-SY5Y neuroblastoma cells took up ascorbate, achieving steady-state intracellular concentrations of 6 mM and higher at extracellular concentrations of 200 microM and greater. This gradient was generated by relatively high affinity sodium-dependent ascorbate transport (Km of 113 microM). Ascorbate was also recycled from dehydroascorbate, the reduction of which was dependent on GSH, but not on D-glucose. Glutamate in concentrations up to 2 mM caused an acute concentration-dependent efflux of ascorbate from the cells, which was prevented by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Intracellular ascorbate did not affect radiolabeled glutamate uptake, showing absence of heteroexchange.

Effects of a natural extract from Mangifera indica L, and its active compound, mangiferin, on energy state and lipid peroxidation of red blood cells

Following oxidative stress, modifications of several biologically important macromolecules have been demonstrated. In this study we investigated the effect of a natural extract from Mangifera indica L (Vimang), its main ingredient mangiferin and epigallocatechin gallate (EGCG) on energy metabolism, energy state and malondialdehyde (MDA) production in a red blood cell system. Analysis of MDA, high energy phosphates and ascorbate was carried out by high performance liquid chromatography (HPLC). Under the experimental conditions, concentrations of MDA and ATP catabolites were affected in a dose-dependent way by H 2O 2. Incubation with Vimang (0.1, 1, 10, 50 and 100 μg/mL), mangiferin (1, 10, 100 μg/mL) and EGCG (0.01, 0.1, 1, 10 μM) significantly enhances erythrocyte resistance to H 2O 2-induced reactive oxygen species production. In particular, we demonstrate the protective activity of these compounds on ATP, GTP and total nucleotides (NT) depletion after H 2O 2-induced damage and a reduction of NAD and ADP, which both increase because of the energy consumption following H 2O 2 addition. Energy charge potential, decreased in H 2O 2-treated erythrocytes, was also restored in a dose-dependent way by these substances. Their protective effects might be related to the strong free radical scavenging ability described for polyphenols.

Influence of ascorbate and pH on the activity of the diatom xanthophyll cycle‐enzyme diadinoxanthin de‐epoxidase

In this study, the enzyme activity of partially purified diadinoxanthin de‐epoxidase (DDE) from the diatom Cyclotella meneghiniana was investigated at different ascorbate concentrations and pH values. In comparison with spinach violaxanthin de‐epoxidase (VDE), we found a much higher affinity of the enzyme for the co‐substrate ascorbate. The Km value of DDE at pH 5 (0.7 mM) was significantly lower than that observed for VDE (2.3 mM). The pH‐optimum of DDE activity was found at pH 5 at low ascorbate concentrations. At high ascorbate concentrations, we observed a strong shift of the pH optimum towards higher pH values, and significant DDE activity was still present at almost neutral pH values. This is in contrast to VDE, where despite a slight shift towards higher pH values, enzyme activity was never observed above pH 6.5. The pH optimum of VDE was always found in a narrow range between pH 5 and 5.2, irrespective of the presence of high or low ascorbate concentrations. The high affinity of DDE for ascorbate indicates that, even at a limited availability of reduced ascorbate, high enzyme activity is possible at low pH values. At high ascorbate concentrations, on the other hand, DDE activity can be shifted towards neutral pH values, thereby facilitating a very fast and strong response to small pH changes in the thylakoid lumen. The importance of the high ascorbate affinity of DDE for the physiology of intact diatom cells is discussed.

The possible involvement of peroxidase in defense of yellow lupine embryo axes against Fusarium oxysporum

Peroxidase activity (EC 1.11.1.7) towards phenolic substrates, i.e. pyrogallol, syringaldazine and guaiacol, and ascorbate peroxidase activity (EC 1.11.1.11) were analyzed in embryo axes of Lupinus luteus L. cv. Polo cultured on Heller medium for 96h after inoculation with the necrotrophic fungus Fusarium oxysporum f.sp. Schlecht lupini. Four variants were compared: inoculated embryo axes cultured with 60mM sucrose (+Si) or without it (-Si), and non-inoculated embryo axes cultured with 60mM sucrose (+Sn) or without it (-Sn). Between 0 and 96h of culture, peroxidase activity towards the phenolic substrates increased in all variants except -Si, where a decrease was noted in peroxidase activity towards syringaldazine and guaiacol, but not towards pyrogallol. In +Si tissues, a considerable increase in enzyme activity towards these substrates was recorded starting from 72h of culture. Lignin content of +Si tissues increased already at the first stage of infection, i.e. 24h after inoculation. Additionally, in +Sn tissues, high ascorbate peroxidase activity was observed during the culture. Its activity increased in +Si tissues, beginning at 72h after inoculation. However, this was lower than in +Sn tissues. At 72h after inoculation, a considerably stronger development of the infection was observed in -Si than in +Si tissues during our earlier research [Morkunas, I. et al., 2005. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum. Plant Physiol Biochem 2005; 43: 363-73]. Both peroxidases assayed towards phenolic substrates and ascorbate peroxidase was less active in -Si tissues than in -Sn tissues. Hydrogen peroxide concentration was much higher in -Si than in +Si tissues. These results indicate that peroxidases may be some of the elements of the defense system that are stimulated by sucrose in yellow lupine embryo axes in response to infection caused by F. oxysporum.

Effect of iron and ascorbate on uroporphyria in ascorbate-requiring mice as a model for porphyria cutanea tarda

Excess hepatic iron is known to enhance both porphyria cutanea tarda (PCT) and experimental uroporphyria. Since previous studies have suggested a role for ascorbate (AA) in suppressing uroporphyria in AA-requiring rats (in the absence of excess iron), the present study investigated whether AA could suppress uroporphyria produced by excess hepatic iron. Hepatic URO accumulation was produced in AA-requiring Gulo(-/-) mice by treatment with 3,3',4,4',5-pentachlorbiphenyl, an inducer of CYP1A2, and 5-aminolevulinic acid. Mice were administered either sufficient AA (1000 ppm) in the drinking water to maintain near normal hepatic AA levels or a lower intake (75 ppm) that resulted in 70 % lower hepatic AA levels. The higher AA intake suppressed hepatic URO accumulation in the absence of administered iron, but not when iron dextran (300-500 mg Fe/kg) was administered. This effect of iron was not due to hepatic AA depletion since hepatic AA content was not decreased. The effect of iron to prevent AA suppression of hepatic URO accumulation was not observed until a high hepatic iron threshold was exceeded. At both low and high AA intakes, hepatic malondialdehyde (MDA), an indicator of oxidative stress, was increased three-fold by high doses of iron dextran. MDA was considerably increased even at low iron dextran doses, but without any increase in URO accumulation. The level of hepatic CYP1A2 was unaffected by either AA intake. In this mouse model of PCT, AA suppresses hepatic URO accumulation at low, but not high hepatic iron levels. These results may have implications for the management of PCT.

Sepsis inhibits recycling and glutamate-stimulated export of ascorbate by astrocytes

Sepsis causes brain dysfunction. Because neurotransmission requires high ascorbate and low dehydroascorbic acid (DHAA) concentrations in brain extracellular fluid, the effect of septic insult on ascorbate recycling (i.e., uptake and reduction of DHAA) and export was investigated in primary rat and mouse astrocytes. DHAA raised intracellular ascorbate to physiological levels but extracellular ascorbate only slightly. Septic insult by lipopolysaccharide and interferon-γ increased ascorbate recycling in astrocytes permeabilized with saponin but decreased it in those with intact plasma membrane. The decrease was due to inhibition of the glucose transporter (GLUT1) that translocates DHAA because septic insult slowed uptake of the nonmetabolizable GLUT1 substrate 3- O-methylglucose. Septic insult also abolished stimulation by glutamate of ascorbate export. Specific nitric oxide synthase (NOS) inhibitors and nNOS and iNOS deficiency failed to alter the effects of septic insult. Inhibitors of NADPH oxidase generally did not protect against septic insult, because only one of those tested (diphenylene iodonium) increased GLUT1 activity and ascorbate recycling. We conclude that astrocytes take up DHAA and use it to synthesize ascorbate that is exported in response to glutamate. This mechanism may provide the antioxidant on demand to neurons under normal conditions, but it is attenuated after septic insult.

Glutathione-Associated Regulation of Plant Growth and Stress Responses

A reduced form of glutathione (GSH) is considered to protect the cell from oxidative damage, based on its redox buffering action and abundance in the cell. However, in plants, the high redox potential molecule ascorbate exists at comparable or higher concentrations and is used for scavenging hydrogen peroxide as an electron donor. Recently, examples that cannot be explained simply by the antioxidant activity of GSH have been increasing in number. This article summarizes the recent findings on the glutathione-associated events in plants, in particular, growth and development including cell differentiation, cell death and senescence, pathogen resistance, and enzymatic regulation.

Effects of heat acclimation pretreatment on changes of membrane lipid peroxidation, antioxidant metabolites, and ultrastructure of chloroplasts in two cool-season turfgrass species under heat stress

The membrane lipid peroxidation, antioxidant metabolites, as well as ultrastructure of chloroplasts of leaves were investigated in two cool-season turfgrass species, perennial ryegrass (heat sensitive) and tall fescue (moderate), exposed to three heat stress levels (38, 42, 46 °C) for 14 h, respectively, with a heat acclimation pretreatment at 30 °C for 3 days in a growth chamber. Relative water content (RWC), cell membrane thermostability, malondialdehyde (MDA) content, accumulation of hydrogen peroxide (H 2O 2) and superoxide (O 2 −), ascorbate (AsA) and glutathione (GSH) contents were measured in leaves. RWC decreased with increase in heat stress levels in tall fescue and perennial ryegrass leaves. However, leaves with heat acclimation pretreatment kept lower decrease in RWC than those without heat acclimation pretreatment at three heat stress levels. Under heat stresses, leaves heat-acclimated pretreatment in two turfgrass species maintained higher membrane thermostability and lower membrane lipid peroxidation product (MDA) than those in no heat-acclimated leaves; however, perennial ryegrass leaves showed greater membrane injury than tall fescue leaves regardless of heat acclimation pretreatment, especially at 46 °C. Significant accumulation of H 2O 2 and O 2 − was observed in two turfgrass species leaves regardless of heat-acclimated pretreatment at all stress levels; however, their degree of accumulation kept lower level in heat-acclimated leaves than in no heat-acclimated leaves. AsA and GSH are two important metabolites in antioxidant system in plants. Our results showed the contents of AsA and GSH in heat-acclimated leaves were higher than those in control at same heat stress level though the contents of AsA and GSH showed the trend of significant decrease with increase in heat stress levels. After heat acclimation pretreatment, ultrastructure of chloroplasts had lower damage in two turfgrass species leaves under heat stress than those without heat acclimation pretreatment, which may contribute to the decrease in membrane lipid peroxidation, increase in AsA and GSH contents at same heat stress level. However, it was insufficient to protect the chloroplast from damage with increase in heat stress level, even if with high AsA and GSH contents to remove reactive oxygen species (ROS).

Reactivity of Peroxynitrite and Nitric Oxide with LDL

Low density lipoprotein (LDL) oxidation by peroxynitrite is a complex process, finely modulated by control of peroxynitrite formation, LDL availability and free-radical scavenging by nitric oxide (*NO), ascorbate and alpha-tocopherol (alpha -TOH). In the presence of CO2, lipid targets are spared at the expense of surface constituents. Since surface damage may lead to oxidation-induced LDL aggregation and particle recognition by scavenger receptors, CO2 cannot be considered an inhibitor of peroxynitrite-dependent LDL modifications. Chromanols, urate and ascorbate cannot scavenge peroxynitrite in the vasculature, although intermediates of urate oxidation and high ascorbate concentrations may do soin vitro. Most if not all of the protection against peroxynitrite-induced LDL oxidation afforded by urate, ascorbate, chromanols and also*NO should be considered to depend on their free radical scavenging abilities, including inactivation of lipid peroxyl radicals (LOO),*NO2, and CO3*-; as well as their capacity to reduce high oxidation states of metal centers. Peroxynitrite direct interception by reduced manganese (II) porphyrins is possibly the most powerful although unspecific strategy to inhibit peroxynitrite reactions. In light of the recent demonstration of nitrated bioactive lipids in vivo, renewed interest in the mechanisms of peroxynitrite- and nitric oxide-mediated lipid nitration and nitrosation is guaranteed.

Vitamin C status and collagen cross-link ratios in Gambian children

Vitamin C (ascorbate) is essential for hydroxylation of prolyl and lysyl residues in nascent collagen, the failure of which leads to connective tissue lesions of scurvy. Of the pyridinium-type cross-links in mature collagen, pyridinoline requires more hydroxylysyl residues than does deoxypyridinoline. Our study tested the hypothesis that pyridinoline:deoxypyridinoline ratios in urinary degradation products may vary with ascorbate status in man. These ratios were compared between British and Gambian prepubertal boys, mean age 8.3 years, and in Gambian boys between two seasons with contrasting ascorbate availability. The mean cross-links ratio in 216 British boys was 4.36 (SD 0.71), significantly greater (P<0.0001) than in sixty-two Gambian boys: 3.83 (SD 0.52). In the Gambians the cross-links ratio was significantly higher in the dry season (with high ascorbate intake and status) than in the rains (with low intake and status). A 7-week controlled intervention was carried out in Gambian boys during the rainy season (the 'hungry' season, when vitamin C-containing foods are virtually unavailable): 100 mg ascorbate/d was given to one group of thirty-two Gambian boys and placebo to another group. The intervention did not, however, significantly alter the cross-link ratio, possibly because the response time and/or intervention-response delay is >7 weeks. If confirmed, the putative association between ascorbate and collagen cross-link ratios in man could become the basis for a functional test for adequacy of ascorbate status.

Duodenal ascorbate and ferric reductase in human iron deficiency1–3

The first step in iron absorption requires the reduction of ferric iron to ferrous iron, a change that is catalyzed by duodenal ferric reductase. Iron deficiency is associated with high iron absorption, high ferric reductase activity, and high duodenal ascorbate concentrations in experimental animals, but it is not known whether a relation between reductase and ascorbate is evident in humans. The objective of the study was to assess the relation between ferric reductase activity in human duodenal biopsy specimens and ascorbate concentrations in iron-replete and iron-deficient subjects. Patients and control subjects were overnight-fasted adults presenting sequentially for upper gastrointestinal endoscopic investigation. Ferric reductase activity in duodenal biopsy specimens was assayed by using nitroblue tetrazolium. Ascorbate was assayed in duodenal biopsy specimens and plasma. Iron-deficient patients had significantly higher reductase activity (n = 6-9; P < 0.05) and duodenal (n = 20; P < 0.001) and plasma (n = 6; P < 0.001) ascorbate concentrations than did control subjects. Incubation of biopsy specimens with dehydroascorbate (to boost cellular ascorbate) increased reductase activity in the tissues that initially had normal activity (n = 9; P < 0.01) but inhibited reductase activity in the tissues that already had high reductase activity (n = 13; P < 0.001). Iron deficiency in humans is associated with increased duodenal ascorbate concentrations. This finding suggests that increased reductase activity is partly due to an increase in this substrate for duodenal cytochrome b reductase 1.

Protective role of corneal epithelium against ultraviolet radiation damage

It is known that the corneal epithelium strongly absorbs ultraviolet radiation (UVR). The aim of the present study was to examine the protective role of corneal epithelium against UVR damage by comparing the biological effect of UVR exposure on whole corneas with that on de-epithelialized corneas. Six New Zealand albino rabbit corneas were exposed to UVR centred around 280 nm at a dose that causes biomicroscopically significant keratitis (012 J/cm(2)). Three corneas underwent manual de-epithelialization prior to UVR exposure. A control group of three rabbits underwent only manual de-epithelialization. The animals were killed 76 hours after treatment. The corneas were stained with haematoxylin and evaluated by light microscopy. Corneas that underwent only the exposure to UVR showed a loss of epithelial cells in the treated area. No damage to keratocytes or the stroma was detected. Corneas that underwent manual de-epithelialization showed a loss of epithelial cells, and also keratocytes in the anterior quarter of the corneal stroma. However, corneas that were exposed to UVR after manual de-epithelialization showed very deep stromal damage. The keratocytes disappeared through the entire thickness of the stroma in the UVR-exposed area. Exposure to UVR at 280 nm alone does not result in any deep damage to the corneal stroma and keratocytes. Manual de-epithelialization causes the disappearance of anterior keratocytes. However, the stromal damage caused by UVR in the de-epithelialized corneas was very deep. The corneal epithelium serves to protect the deeper corneal structures against UVR damage, probably by absorbing a substantial amount of the UVR energy applied to the eye.

Plasma ascorbate deficiency is associated with impaired reduction of sulfamethoxazole-nitroso in HIV infection.

The objective of these studies was to determine the role of ascorbate deficiency in HIV infection in the defective detoxification of sulfamethoxazole-nitroso, the metabolite thought to mediate sulfonamide hypersensitivity reactions. Fifty-one HIV-infected patients and 26 healthy volunteers were evaluated. Vitamin supplementation histories were obtained, and blood samples were collected for determination of plasma ascorbate, dehydroascorbate, and cysteine concentrations, erythrocyte glutathione concentrations, and plasma reduction of sulfamethoxazole-nitroso in vitro. Plasma ascorbate concentrations were significantly lower in HIV-positive patients not taking vitamin supplements (29.5 +/- 22.3 microM) than in healthy subjects (54.8 +/- 22.3 microM; P = 0.0005) and patients taking 500-1000 mg of ascorbate daily (82.5 +/- 26.3 microM; P < 0.0001). Plasma ascorbate deficiency was strongly correlated with impaired reduction of sulfamethoxazole-nitroso to its hydroxylamine (r = 0.60, P < 0.0001), and during in vitro reduction, the loss of plasma ascorbate was strongly associated with the amount of nitroso reduced (r = 0.70, P < 0.0001). Ascorbate added ex vivo normalized this reduction pathway. Erythrocyte glutathione concentrations were significantly lower in HIV-positive patients (0.98+/-0.32 mM) than in healthy subjects (1.45+/-0.49 mM; P = 0.001), but this finding was unrelated to ascorbate supplementation. There was trend toward lower plasma cysteine concentrations in patients (8.4+/-3.9 microM) than in controls (10.3+/-4.3 microM), but this trend was similarly unrelated to ascorbate supplementation. Dehydroascorbate concentrations were not significantly higher in HIV-positive patients (7.4+/-10.5%) than in healthy controls (4.0+/-6.2%), even in the subset of patients taking ascorbate (8.4+/-9.4%). Ascorbate deficiency is common in HIV-positive patients and is associated with impaired detoxification of sulfamethoxazole-nitroso, the suspected proximate toxin in sulfonamide hypersensitivity. Patients taking daily ascorbate supplements (500-1000 mg) achieved high plasma ascorbate concentrations and did not show this detoxification defect. Ascorbate deficiency (or supplementation) was not associated with changes in glutathione or cysteine concentrations. These data suggest that ascorbate deficiency, independent of thiol status, may be an important determinant of impaired drug detoxification in HIV infection.

Drought‐induced spikelet sterility is associated with an inefficient antioxidant defence in rice panicles

Water stress‐induced spikelet sterility limits rice production under upland conditions. The causes of spikelet sterility under drought stress are poorly understood. In this study the role of antioxidant defence management in drought‐induced spikelet sterility was investigated in two rice (Oryza sativa) genotypes differing in drought resistance. Drought‐resistant N22 genotype showed less water stress‐induced spikelet sterility when compared to the susceptible N118 genotype under upland conditions. The N22 panicles maintained higher RWC and turgor potential and lower H2O2 levels across the developmental stages under water stress than that of N118 panicles. Drought‐induced enhancement in superoxide dismutase (SOD, EC 1.15.1.1) activity coupled with higher ascorbate (AsA), glutathione (GSH) content and enhanced ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities resulted in lower H2O2 levels in N22 panicles. In contrast, insufficient enhancement in SOD, APX and GR activities resulted in relatively higher H2O2 levels under water stress in N118 panicles. The N22 panicles exhibited a higher number of SOD and APX isozymes in comparison with N118 panicles that might provide better reactive oxygen species scavenging. Hence it is concluded that well‐equipped antioxidant defence plays an important role in minimizing water stress‐induced spikelet sterility in upland rice.

Protective effect of ascorbate against oxidative stress in the mouse lens.

The purpose of this study was to determine if high ascorbate of the human aqueous protects the lens against oxidative stress. Previous studies with the rat lens have been inconclusive because of its fortification with aldose reductase (AR), an important antioxidant. The human lens is deficient in this activity. These studies were hence done with the mouse lens, a species deficient in this enzyme. The reactive oxygen species (ROS)-induced physiological damage to the tissue was assessed in organ culture, by measuring its ability to actively transport 86Rb(+) ions, in the absence and presence of ascorbate. In addition, the status of tissue metabolism and its antioxidant reserve were assessed by quantitating ATP and glutathione (GSH). As expected, ROS decreased the membrane transport activity as well as the levels of ATP and GSH. Ascorbate minimized these toxic effects substantially. The presence of high ascorbate, therefore, appears highly beneficial in protecting the lens against oxidative damage and cataract formation, despite a deficiency of AR. The findings therefore appear to be significant from the point of view of using this nutrient for delaying the onset of cataract development in human beings, therapeutically as well as nutritionally.

THE EFFECT OF HOT AIR TREATMENTS IN AIR OR IN LOW O2 ATMOSPHERE ON THE QUALITY AND ANTIOXIDANTS OF TOMATO FRUIT

Tomato fruit is an excellent source of antioxidants. However, the fruit is sensitive to chilling injury (CI). We have determined the effect of forced hot air treatments on the quality, CI, and the contents of some antioxidants. ‘Rapsody’ tomato fruit was exposed to 34oC for 24 h in air, to 38oC for 24 h in air, or to 38oC for 24 h in 5% O2, all at low RH, and then stored in air at 4oC or 10oC for up to 30 d. Fruit exposed to 38oC at 5% O2 were injured the most, while fruit that were not heated, and those heated in air at 34oC for 24 h were the least injured. Fruit that were maintained at 10oC and were previously either not heated, or heated at 34oC developed the best color when ripened, having the least chlorophyll and the highest lycopene contents. Color developed slowly at 4oC than at 10oC. -carotene increased in fruit that were not heated and to a lesser extent in those that were heated in air at 34oC or 38oC. Fruit that were exposed to 38oC in 5% O2 had lower –tocopherol content and higher peroxidase, ascorbate peroxidase, superoxide dismutase, glutathione reductase, and glutathione S-transferase activity than fruit of other treatments. We conclude that heating of tomato fruit at 34oC for 24 h caused the least injury and fruit color developed adequately.

Effects of leaf ascorbate content on defense and photosynthesis gene expression in Arabidopsis thaliana.

Ascorbate deficiency in the Arabidopsis thaliana vtc1 mutant had no effect on photosynthesis, but modified defense pathways. The ascorbate content of vtc1 leaves was increased 14-fold after 10 mM ascorbate was supplied, without a concomitant change in redox state. High ascorbate modified the abundance of 495 transcripts. Transcripts encoding dehydroascorbate reductase, pathogenesis-related protein 1, and a peroxiredoxin were decreased, whereas those encoding salicylate induction-deficient protein 1, Cu,Zn superoxide dismutase, iron superoxide dismutase, metallothionein, and glutathione transferases were increased. Catalase transcripts were unaffected, but ascorbate peroxidase isoforms APX1 and tAPX were slightly decreased and sAPX transcripts increased. A number of nuclear encoded transcripts for photosynthetic electron transport components were repressed as a result of ascorbate accumulation, whereas those that were chloroplast-encoded were increased. High ascorbate caused decreases in mRNAs encoding chloroplast enzymes such as fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase that are activated by reduced thioredoxin. In contrast, others, such as glucose 6-phosphate dehydrogenase, whose activity is inactivated by reduced thioredoxin, were repressed. Together, these results show that ascorbate is involved in metabolic cross-talk between redox-regulated pathways. The abundance of this antioxidant provides information on redox buffering capacity that coordinates redox processes associated with the regulation of photosynthesis and plant defense.