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Moderately controlled transport of ascorbate into aortic endothelial cells against slowdown of the cell cycle, decreasing of the concentration or increasing of coexistent glucose as compared with dehydroascorbate.

Uptake of L-[1-(14)C]ascorbic acid (Asc) of 12.5-200 microM for 1 h into bovine aortic endothelial BAE-2 cells grown to confluence was as low as 43-64% (per cell) of uptake into the cells grown to nearly one-fourth confluence. [14C]Asc undergoing transmembrane uptake was concentrated and accumulated in the cell less efficiently ([Asc]in/ex = 8-13) at confluence than at subconfluence ([Asc]in/ex = 15-24). The declined Asc uptake at confluence is attributable to slowdown of the cell cycle, because a similar decrease in [Asc]in/ex was shown by subconfluent cells precultured in serum-insufficient medium, resulting in an increase in G1 phase and concurrent decreases in S and G2 + M phase distributions as determined by flow cytometry. [1-(14)C]Dehydroascorbic acid (DehAsc) was taken up and accumulated as Asc, after metabolic reduction, without detectable DehAsc. The [Asc]in/ex values for DehAsc at confluence were as low as 15-69% of those at subconfluence in contrast to the values as retentive as 62-75% for Asc, suggesting the moderate control of Asc uptake against slowdown of the cell cycle. At either confluence or subconfluence, dose-dependence for DehAsc uptake was more marked than for Asc uptake as shown by an uphill slope in a curve of doses versus [Asc]in/ex for DehAsc in contrast to a downhill slope for Asc, suggesting the moderate control for Asc uptake against fluctuation of the dose. Increasing of coexistent glucose of 5 mM to 20-40 mM, plasma concentrations in diabetic patients, declined DehAsc uptake to 46-48%, which was less moderately controlled than Asc uptake retained to 59-73%. Asc uptake did not compete with DehAsc uptake, suggesting different transporter proteins for Asc and DehAsc. Thus, Asc uptake into the aortic endothelial cells is more moderately controlled against slowdown of the cell cycle, decreasing of the extracellular concentrations or increasing of coexistent glucose than DehAsc uptake, suggesting a homeostatic advantage of Asc over DehAsc in terms of retention of intracellular Asc contents within a definite range.

Animals↗

Activity staining on polyacrylamide gels of trypsin inhibitors from leaves of sweet potato (Ipomoea batatas L. Lam) varieties.

The failure of activity staining of trypsin inhibitors in crude leaf extracts of sweet potato varieties including Tainong 27, Tainong 34, and Tainong 57 on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel was prevented by dipping the gels in solutions containing 10-40 mM hydrogen peroxide, 10 mM Tris buffer (pH 7.9) for 30 min before staining.

Acrylic Resins↗

Dielectric resonator-based flow and stopped-flow EPR with rapid field scanning: A methodology for increasing kinetic information.

We report methodology which combines recently developed dielectric resonator-based, rapid-mix, stopped-flow EPR (appropriate for small, aqueous, lossy samples) with rapid scanning of the external (Zeeman) magnetic field where the scanning is preprogrammed to occur at selected times after the start of flow. This methodology gave spectroscopic information complementary to that obtained by stopped-flow EPR at single fields, and with low reactant usage, it yielded more graphic insight into the time evolution of radical and spin-labeled species. We first used the ascorbyl radical as a test system where rapid scans triggered after flow was stopped provided "snapshots" of simultaneously evolving and interacting radical species. We monitored ascorbyl radical populations either as brought on by biologically damaging peroxynitrite oxidant or as chemically and kinetically interacting with a spectroscopically overlapping nitroxide radical. In a different biophysical application, where a spin-label lineshape reflected rapidly changing molecular dynamics of folding spin-labeled protein, rapid scan spectra were taken during flow with different flow rates and correspondingly different times after the mixing-induced inception of protein folding. This flow/rapid scan method is a means for monitoring early immobilization of the spin probe in the course of the folding process.

Antioxidants↗

Oxyradical scavenging effects of dehydroascorbate.

Dehydroascorbate (DHA) has been shown to possess vitamin C like activities as well as to protect the lens against oxidative stress and cataract formation. The results presented here suggest that some of the beneficial effects of DHA can be attributed to its property of undergoing peroxidative decarboxylation and of O2-. scavenging. Incubation of 1-14C-DHA with peroxide at physiological pH has been found to liberate 14CO2 in quantitative yields, with recovery of 79-94%. The recovery increases with the increase in the amount of DHA used. Its O2-. scavenging activity was apparent by inhibition of O2-. dependent reduction of ferricytochrome c and nitroblue tetrazolium. The authenticity of the latter mechanism was proved by inhibition of the O2-. dependent reactions also by superoxide dismutase.

Decarboxylation↗

Copper-induced changes in the growth, oxidative metabolism, and saponin production in suspension culture roots of Panax ginseng in bioreactors.

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 microM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 microM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 muM but decreased at 50 microM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O2*-) accumulation, and H2O2 content at 5 and 10 microM Cu-treated roots were not increased but strongly increased at 50 microM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 microM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 microM Cu, but both G-POD and CAT activities were inhibited at 50 microM Cu. Glutathione metabolism enzymes such as gamma-glutamylcysteine synthetase (gamma-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 microM Cu but were strongly inhibited at 50 microM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 microM Cu was associated to the strong induction of gamma-glutamyltranspeptidase (gamma-GGT) activity. These results indicate that plant could grow under Cu stress (5-25 microM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.

Bioreactors↗

Oxidation of NADH by vanadium: kinetics, effects of ligands and role of H2O2 or O2.

The mechanism of oxidation of NADH by either vanadium(V) or vanadium(IV) was examined in the presence of reducing agents, complexing agents, and hydrogen peroxide. Reducing agents that stimulate the oxidation of NADH by V(V) include: a variety of cysteine analogues, glutathione, beta-mercaptoethanol, dithiothreitol, and ascorbate. Complexing agents which stimulate NADH oxidation by V(V) include cystine, glutathione disulfide, and dehydroascorbate. Vanadium(IV)-dependent systems which oxidize NADH include combinations of V(IV) with cysteine or air alone. Combination of either V(V) or V(IV) with hydrogen peroxide leads to NADH oxidation. Based on kinetic analysis and the use of the diagnostic inhibitors--superoxide dismutase, catalase, albumin, mannitol, ethanol, and anaerobic conditions--we have assigned two major mechanisms of NADH oxidation. One is the previously reported mechanism which involves V(V)-superoxide as the NADH oxidant. This reaction is inhibited by superoxide dismutase and anaerobic conditions but not by catalase or ethanol. This reaction is observed for V(V) in the presence of reducing agents and complexing agents. The second reaction mechanism operates when V(IV) comes in contact with hydrogen peroxide and involves V(III)-superoxide as the NADH oxidant. This reaction is inhibited by catalase (if unligated hydrogen peroxide is an intermediate) and superoxide dismutase but not anaerobic conditions or ethanol. This mechanism is observed for reactions of V(IV) with air or hydrogen peroxide.

Albumins↗

Enzymatic recycling of oxidized ascorbate in pig heart: one-electron vs two-electron pathway.

Enzymatic systems able to reduce either dehydroascorbate or ascorbyl radical back to ascorbate by "recycling" vitamin C may contribute to lowering the nutritional requirement of it and to increase tissue antioxidant capacity. The activities of two enzymatic activities, GSH-dehydroascorbate reductase (two-electron reduction pathway) and NADH-semidehydroascorbate reductase (one-electron reduction pathway) in pig tissues, have been investigated. The activity of glutathione-dependent reduction of dehydroascorbate, although measurable, appeared negligible taking into consideration the low physiological substrate concentration. On the other hand, the one-electron reduction of ascorbyl radical resulted fast enough to slow down the consumption of the antioxidant vitamin.

Animals↗

Human cell dehydroascorbate reductase. Kinetic and functional properties.

Dehydroascorbate reductase (glutathione: dehydroascorbate oxidoreductase, EC 1.8.5.1) activity was examined in crude cytosol extracts of human cells. In blood neutrophil and lymphocyte extracts, the Km at pH 6.85 for dehydroascorbate was 1.3 mM, and for reduced glutathione 3.8 mM. Rates of dehydroascorbate uptake by intact human neutrophils, monocytes, lymphocytes and cultured fibroblasts were proportional to cytosol dehydroascorbate reductase activities. Dehydroascorbate reduction during dehydroascorbate uptake by these cells may be entirely enzymatic.

Cells, Cultured↗

Reduction of extracellular potassium ferricyanide by transmembrane NADH: (acceptor) oxidoreductase of human erythrocytes.

Reduction of extracellular ferricyanide by intact erythrocytes proceeds by a membrane bound, NADH-dependent reaction. It is depressed by a glycolysis inhibitor and a non penetrable sulfhydryl reagent, and activated by dehydroascorbate. Dehydroascorbate activation cannot be accounted for by release of reducing equivalents from the cells. It is concluded that the observed reaction is brought about by transmembrane NADH-acceptor oxidoreductase with donor binding at the inner and acceptor binding at the outer cell surface.

Cytochrome Reductases↗

alpha-Phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion.

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (.OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systemically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (alpha N = 15.4 G and alpha beta H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of alpha-phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF), significantly (p < 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.

Animals↗

The influence of oxygenated water on the immune status, liver enzymes, and the generation of oxygen radicals: a prospective, randomised, blinded clinical study.

BACKGROUND & AIMS: Oxygenated water with an oxygen concentration of 30-120 mg/l water is believed to improve the immune status, without any toxicological effects. The purpose of this clinical study was to assess the effects of long-term drinking of oxygenated water on the immune status. METHODS: In this prospective, double-blinded, randomised study 24 volunteers of either sex (age 18-63 years) drank daily 3 times 500 ml either oxygenated (Verum-group: n=12) or normal mineral water (Placebo-group: n=12) for 28 days. On day 1 and day 28 standard laboratory tests, IgG, IgA and IgM, lymphocyte subpopulations and functional analysis of T-cells by flow cytometry, were done. Furthermore, the oxygen radicals were determined by the detection of the ascorbyl radicals. RESULTS: Drinking of normal or oxygenated water had no effect on whole blood count or the liver enzymes. Interestingly the volunteers in the Verum-group showed a significant increase in ascorbyl radicals after drinking oxygenated water for 14 and 21 days. CD4+ and CD4+CD45RA+ lymphocytes as well as lymphocyte activation marker (CD69) and soluble IL-2 receptor increased in both groups, in contrast T-helper2 cells and IgG decreased during the study. The only differences between the two groups were a significant decrease of NK-cells form 13.42%+ or -5.04 to 10.83%+ or -4.82 (P<.002) and an increase of the Th1/Th2-ratio from 2.77%+ or -1.07 to 6.68%+ or -5.33 (P<.03) in the Verum-group. CONCLUSION: Long-term consumption of oxygenated water has no apparent harmful effect on the liver, blood and the immune system. Moreover it leads to a transient moderate increase of oxygen radicals in the blood. An interesting observation is the increase of the Th1/Th2-ratio in the Verum group, whereas in both groups T-cell activation after mitogen stimulation, the soluble IL-2 receptor, the CD4+ and the naive CD4+CD45RA+ cells increased.

Adolescent↗

Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR.

Methylviologen (MV) induces oxidative damages in leaves. In order to understand its mechanism we studied initial biochemical events under light in MV-fed spinach leaves. When isolated chloroplasts were illuminated in the presence of MV, both stromal and thylakoid-bound ascorbate peroxidases (APX) were inactivated rapidly at the same rates, and their inactivation was retarded by ascorbate (AsA) at higher concentrations. Since MV accelerates the photoproduction of O2- in Photosystem (PS) I and simultaneously inhibits the photoreduction of monodehydroascorbate (MDA) to AsA, the inactivation of APX was attributed to the loss of AsA and accumulation of H2O2 in the stroma. Following APX, superoxide dismutase and NADP(+)-glyceraldehyde 3-phosphate dehydrogenase, both of which are vulnerable to H2O2, were inactivated by MV plus light. Dehydroascorbate reductase, monodehydroascorbate reductase, PS II, PS I and ferredoxin-NADP(+) reductase were far less sensitive to the treatment. In the treated leaves, cytosolic APX and guaiacol-specific peroxidase were also inactivated, but slower than chloroplastic APXs were. Catalase was not inactivated. Thus the MV-induced photooxidative damages of leaves are initiated with the inactivation of chloroplastic APXs and develop via the inactivation of other H2O2-sensitive targets. The decay half-life of the MDA signal after a short illumination in the leaves, as determined by in vivo electron spin resonance spectrometry (ESR), was prolonged when the H2O2-scavenging capacity of the leaf cells was abolished by the inactivation of chloroplastic and cytosolic APXs. The measurement of MDA in leaves by ESR, therefore, allows to estimate in vivo cellular capacity to scavenge the photoproduced H2O2.

Ascorbate Peroxidases↗