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PubMed2006

Effect of ascorbate oxidase over-expression on ascorbate recycling gene expression in response to agents imposing oxidative stress.

Ascorbate oxidase (AO) is a cell wall-localized enzyme that uses oxygen to catalyse the oxidation of ascorbate (AA) to the unstable radical monodehydroascorbate (MDHA) which rapidly disproportionates to yield dehydroascorbate (DHA) and AA, and thus contributes to the regulation of the AA redox state. Here, it is reported that in vivo lowering of the apoplast AA redox state, through increased AO expression in transgenic tobacco (Nicotiana tabacum L. cv. Xanthi), exerts no effects on the expression levels of genes involved in AA recycling under normal growth conditions, but plants display enhanced sensitivity to various oxidative stress-promoting agents. RNA blot analyses suggest that this response correlates with a general suppression of the plant's antioxidative metabolism as demonstrated by lower expression levels of AA recycling genes. Furthermore, studies using Botrytis cinerea reveal that transgenic plants exhibit increased sensitivity to fungal infection, although the response is not accompanied by a similar suppression of AA recycling gene expression. Our current findings, combined with previous studies which showed the contribution of AO in the regulation of AA redox state, suggest that the reduction in the AA redox state in the leaf apoplast of these transgenic plants results in shifts in their capacity to withstand oxidative stress imposed by agents imposing oxidative stress.

Ascorbate Oxidase↗
PubMed1991

A unique function for ascorbate.

Vitamin C is a reducing substance, an electron donor. When vitamin C donates its two high-energy electrons to scavenge free radicals, much of the resulting dehydroascorbate is re-reduced to vitamin C and therefore used repeatedly. Conventional wisdom is correct in that only small amounts of vitamin C are necessary for this function because of its repeated use. The point missed is that the limiting part in nonenzymatic free radical scavenging is the rate at which extra high-energy electrons are provided through NADH to re-reduce the vitamin C and other free radical scavengers. When ill, free radicals are formed at a rate faster than the high-energy electrons are made available. Doses of vitamin C as large as 1-10 g per 24 h do only limited good. However, when ascorbate is used in massive amounts, such as 30-200+ g per 24 h, these amounts directly provide the electrons necessary to quench the free radicals of almost any inflammation. Additionally, in high concentrations ascorbate reduces NAD(P)H and therefore can provide the high-energy electrons necessary to reduce the molecular oxygen used in the respiratory burst of phagocytes. In these functions, the ascorbate part is mostly wasted but the necessary high-energy electrons are provided in large amounts.

Ascorbic Acid↗
PubMed2000

The effects of ethylene, depressed oxygen and elevated carbon dioxide on antioxidant profiles of senescing spinach leaves.

It has been suggested that antioxidants play a role in regulating or modulating senescence dynamics of plant tissues. Ethylene has been shown to promote early plant senescence while controlled atmospheres (CA; reduced O2 levels and elevated CO2 levels) can delay its onset and/or severity. In order to examine the possible importance of various antioxidants in the regulation of senescence, detached spinach (Spinacia oleracea L.) leaves were stored for 35 d at 10 degrees C in one of three different atmospheres: (1) ambient air (0.3% CO2, 21.5% O2, 78.5% N2), (2) ambient air + 10 ppm ethylene to promote senescence, or (3) CA (10% CO2, 0.8% O2 and 89.2% N2) to delay senescence. At weekly intervals, material was assessed for activities of the antioxidant enzymes ascorbate peroxidase (ASPX; EC 1.11.1.11), catalase (CAT; EC 1.11.1.6), dehydroascorbate reductase (DHAR; EC 1.8.5.4), glutathione reductase (GR; EC 1.6.4.2), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), and superoxide dismutase (SOD; EC 1.15.1.1), and concentrations of the water-soluble antioxidant compounds ascorbate and glutathione. Indicators of the rate and severity of senescence (lipid peroxidation, chlorophyll, and soluble protein levels) were also determined. Results indicated that the rate and severity of senescence was similar between the leaves stored in ambient air or CA until day 35, at which point the ambient air-stored leaves exhibited a sharp increase in lipid peroxidation. Tissues under both storage regimes demonstrated significant declines only in levels of ASPX, CAT, and ascorbate. Glutathione content in the CA-stored tissue also significantly dropped, but only on day 35. In contrast, spinach leaves stored in ambient air + ethylene experienced a rapid decrease in levels of all the antioxidants assessed except SOD. Declines in levels of ASPX, CAT, and ascorbate over the 35 d storage period regardless of the composition of the storage atmosphere suggests that regulation of H2O2 levels plays an important role in both the dynamics and severity of post-harvest senescence of spinach.

Antioxidants↗
PubMed2007

Exogenous proline and glycinebetaine increase NaCl-induced ascorbate-glutathione cycle enzyme activities, and proline improves salt tolerance more than glycinebetaine in tobacco Bright Yellow-2 suspension-cultured cells.

Up-regulation of the antioxidant system provides protection against NaCl-induced oxidative damage in plants. Antioxidants and activity of enzymes involved in the ascorbate-glutathione (ASC-GSH) cycle in tobacco Bright Yellow-2 (BY-2) were investigated to assess the antioxidant protection offered by exogenous proline and glycinebetaine (betaine from now on) against salt stress using cells grown in suspension culture. Reduced ascorbate (ASC) was detected in BY-2 cells but dehydroascorbate (DHA) was not. Large quantities of a reduced form of glutathione (GSH) and smaller quantities of an oxidized form of glutathione (GSSG) were detected in BY-2 cells. Salt stress significantly reduced the contents of ASC and GSH as well as activities of ASC-GSH cycle enzymes such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR). Exogenous proline or betaine increased the activities of all enzymes except MDHAR involved in NaCl-induced ASC-GSH cycle. Levels of ASC and GSH in BY-2 cells under salt stress were lower in the presence of proline or betaine than in the absence of proline or betaine whereas there was no difference in redox status. Proline proved more effective than betaine in maintaining the activity of enzymes involved in NaCl-induced ASC-GSH cycle. Neither proline nor betaine had any direct protective effect on NaCl-induced enzyme activity involved in the antioxidant system; however, both improved salt tolerance by increasing enzyme activity. The present study, together with our earlier findings [Hoque MA, Okuma E, Banu MNA, Nakamura Y, Shimoishi Y, Murata Y. Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities. J Plant Physiol 2006;164:553-61.], suggests that proline offered greater protection against salt stress than betaine did because proline was more effective in increasing the activity of enzymes involved in the antioxidant system.

Antioxidants↗
PubMed2004

Ascorbate as a "redox sensor" and protector against irradiation-induced oxidative stress in 32D CL 3 hematopoietic cells and subclones overexpressing human manganese superoxide dismutase.

PURPOSE: To determine whether increased expression of manganese superoxide dismutase (MnSOD) protects cells from irradiation by preventing the production of reactive oxygen species (ROS), a new approach to detecting free radical intermediates using ascorbate as an endogenous spin trap was used. MATERIALS AND METHODS: Cells from the 32D cl 3 hematopoietic cell line or a subclone overexpressing MnSOD (2C6) were incubated with dehydroascorbate for 30 min and irradiated to doses from 0 to 50 Gy. Radical intermediates reacting with spin traps or ascorbate were measured by electron spin resonance spectroscopy. Results were compared to irradiation-induced changes in thiol levels, irradiation survival curves, and accumulation of 8-OHdG as a measurement of DNA oxidative damage. RESULTS: Manganese superoxide dismutase-overexpressing 2C6 cells maintained higher levels of ascorbate (5.4 +/- 0.5 and 2.6 +/- 0.5 nmol/10(6) cells, respectively) and thiols (14.0 +/- 0.1 and 11.1 +/- 0.2 nmol/10(6) cells) compared to 32D cl 3 parent cells. Cells overexpressing MnSOD produced lower levels of ROS than did the parental 32D cl 3 cells, as evidenced by lower expenditure of ascorbate and GSH after irradiation. Increased ascorbate levels protected both 32D cl 3 and 2C6 cells from irradiation killing, as demonstrated by an increased shoulder on survival curves and decreased DNA 8-OHdG accumulation. CONCLUSIONS: Manganese superoxide dismutase overexpression protects 2C6 cells from irradiation damage by scavenging ROS that readily interact with major endogenous antioxidants--ascorbate and GSH--in nontransfected hematopoietic 32D cl 3 cells.

Animals↗
PubMed2000

Correlation of antioxidant capacities to oxygen radical scavenging enzyme activities in blackberry.

The activities of the oxygen radical scavenging enzymes [glutathione-peroxidase (GSH-POD), superoxide dismutase (SOD), and guaiacol peroxidase (G-POD)], hydrogen peroxide scavenging enzymes in the ascorbate-glutathione cycle [ascorbate peroxidase (AsA-POD), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR)], the nonenzyme components [ascorbate (AsA), dehydroascorbate (DHAsA), glutathione (GSH), and oxidized glutathione (GSSG)], and their antioxidant capacity [oxygen radical absorbance capacity (ORAC)] were measured in the juice of six different thornless blackberry (Rubus sp.) cultivars. The 'Hull Thornless' cultivar contained the highest levels, whereas 'Black Satin' consistently had the lowest activities for all the enzymes tested in this study. ORAC values were also the highest in 'Hull Thornless' and lowest in 'Black Satin'. The highest levels of AsA and DHAsA were in the juice of 'Hull Thornless' blackberries with 1. 09 and 0.15 micromol/g fresh wt, respectively. 'Hull Thornless' also had the highest ratio of AsA/DHAsA among the six blackberry cultivars studied. The 'Smoothstem' cultivar contained the lowest amounts of AsA and DHAsA. 'Hull Thornless' had the highest GSH content with 78.7 nmol/g fresh wt, while 'Chester Thornless' contained the largest amount of GSSG. The highest GSH/GSSG ratio was 4.90 which was seen in the 'Hull Thornless' cultivar. The correlation coefficient between ORAC values and AsA/DHAsA ratios was as high as 0.972. A correlation (r = 0.901) was also detected between ORAC values and GSH content. The antioxidant activity in blackberry juice was positively correlated to the activities of most antioxidant enzymes (r = 0.902 with SOD; r = 0.858 with GSH-POD; r = 0.896 with ASA-POD; and r = 0.862 with GR).

Antioxidants↗
PubMed2001

Taurine counteracts oxidative stress and nerve growth factor deficit in early experimental diabetic neuropathy.

Oxidative stress has a key role in the pathogenesis of diabetic complications. We have previously reported that taurine (T), which is known to counteract oxidative stress in tissues (lens, kidney, retina) of diabetic rats, attenuates nerve blood flow and conduction deficits in early experimental diabetic neuropathy (EDN). The purpose of this study was to evaluate whether dietary T supplementation counteracts oxidative stress and the nerve growth factor (NGF) deficit in the diabetic peripheral nerve. The experiments were performed in control rats and streptozotocin-diabetic rats fed standard or 1% T-supplemented diets for 6 weeks. All measurements were performed in the sciatic nerve. Malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HA) were quantified with N-methyl-2-phenylindole. GSH, GSSG, dehydroascorbate (DHAA), and ascorbate (AA) were assayed spectrofluorometrically, T by reverse-phase HPLC, and NGF by ELISA. MDA plus 4-HA concentration (mean +/- SEM) was increased in diabetic rats (0.127 +/- 0.006 vs 0.053 +/- 0.003 micromol/g in controls, P < 0.01), and this increase was partially prevented by T (0.096 +/- 0.004, P < 0.01 vs untreated diabetic group). GSH levels were similarly decreased in diabetic rats treated with or without taurine vs controls. GSSG levels were similar in control and diabetic rats but were lower in diabetic rats treated with T (P < 0.05 vs controls). AA levels were decreased in diabetic rats (0.133 +/- 0.015 vs 0.219 +/- 0.023 micromol/g in controls, P < 0.05), and this deficit was prevented by T. DHAA/AA ratio was increased in diabetic rats vs controls (P < 0.05), and this increase was prevented by T. T levels were decreased in diabetic rats (2.7 +/- 0.16 vs 3.8 +/- 0.1 micromol/g in controls, P < 0.05) and were repleted by T supplementation (4.2 +/- 0.3). NGF levels were decreased in diabetic rats (2.35 +/- 0.20 vs 3.57 +/- 0.20 ng/g in controls, P < 0.01), and this decrease was attenuated by T treatment (3.16 +/- 0.28, P < 0.05 vs diabetic group). In conclusion, T counteracts oxidative stress and the NGF deficit in early EDN. Antioxidant effects of T in peripheral nerve are, at least in part, mediated through the ascorbate system of antioxidative defense. The findings are consistent with the important role for oxidative stress in impaired neurotrophic support in EDN.

Aldehydes↗