Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats.

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.     

Supplemental ascorbate or alpha-tocopherol induces cell death in Cu-deficient HL-60 cells

Cytochrome c oxidase (CCO) is the Cu-dependent, terminal respiratory complex of the mitochondrial electron transport chain. Inhibition of CCO can promote oxidative stress by increasing mitochondrial production of reactive oxygen species (ROS). Because mitochondria have an important role in apoptosis as both a target and source for ROS, enhanced ROS production resulting from inhibition of CCO by Cu deficiency may trigger apoptosis. The present study focuses on the mitochondrial effects of N,N'-bis(2-aminoethyl)-1,3-propanedi-amine (TET), which inhibits CCO by causing cellular Cu deficiency, and the antioxidants ascorbate and alpha-tocopherol in a human promyelocytic leukemia cell line (HL-60). The following effects were observed: (i) TET reduced both cell growth and viability only in the presence of ascorbate or alpha-tocopherol; (ii) TET reduced CCO activity and increased mitochondrial ROS production as indicated by increased expression of Mn super-oxide dismutase, but the induction of Mn superoxide dismutase was not affected by ascorbate or alpha-tocopherol; (iii) TET acted independently of ascorbate or alpha-tocopherol in disrupting mitochondrial membrane potential; (iv) TET did not increase caspase-8 activity in the absence of ascorbate or alpha-tocopherol; and (v) TET did not increase transfer of cytochrome c from mitochondria to the cytosol unless alpha-tocopherol was present. These findings indicate that reduction in CCO activity by TET-induced Cu deficiency increased oxidative stress in HL-60 cells sufficiently to disrupt the electrochemical gradient of the inner mitochondrial membrane but did not trigger cell death. Also, ascorbate and alpha-tocopherol did not alleviate oxidative stress but may have become pro-oxidants, adding to the oxidant burden sufficiently to trigger cell death in TET-treated cells.     

Effect of the combined action of flavonoids, ascorbate and alpha-tocopherol on peroxidation of phospholipid liposomes induced by Fe2+ ions

The effect of alpha-tocopherol, ascorbate, rutin and dihydroquercetin on chemiluminescence (CL) accompanying the Fe2+-induced peroxidation of unsaturated fatty acids in phospholipid liposomes has been investigated. The amplitude of CL decreased and the latent period increased in the presence of alpha-tocopherol, rutin and dihydroquercetin which is typical of peroxide radical traps. Ascorbate also reduced the CL amplitude but only at small concentrations up to about 4 microM. A further increase of ascorbate concentration had a negligible effect on the amplitude. At the same time, the latent period in CL development increased with the growth of ascorbate concentration, apparently, as a result of recycling of divalent iron oxidized in the course of lipid peroxidation. The effects of rutin and dihydroquercetin on the liposomal CL in the presence of alpha-tocopherol and ascorbate in all experiments were almost the same as when these compounds were added individually. The antioxidant effects were merely summed up without any mutual enhancement or inhibition of each other's action.     

Stress-induced changes of plasma antioxidants in aquacultured sea bass,Dicentrarchus labrax

Antioxidant plasma activities of ascorbate, alpha-tocopherol and glutathione peroxidase were analysed in adult male sea bass, Dicentrarchus labrax, in normal conditions and after hypoxia-recovery. In addition, tank measurements of temperature, pH, salinity and chlorine changes were carried out. Ascorbate and alpha-tocopherol were measured using a high-pressure liquid chromatography method and glutathione peroxidase activity enzymatically. Ascorbate and alpha-tocopherol showed a relationship with the velocity of body growing in normal and hypoxia-recovery conditions. In sea bass exposed to hypoxia, only ascorbate and alpha-tocopherol levels were significantly lower compared with the control group. Slope study and expression percent of antioxidants reduction after stress conditions revealed a predominant role of plasma alpha-tocopherol. Sea bass subjected to variations of salinity and chlorine showed a significant decrease in plasma alpha-tocopherol. A relationship could be suggested between antioxidant defence and fish response in aquaculture.     

Mechanisms of vitamin C stabilization by K562 erythroleukemic cells

Summary K562 cells display several possibilities to keep ascorbic acid in the surrounding medium in the reduced state and prevent its loss by degradation of the oxidized form, dehydroascorbic acid: (1) A semidehydroascorbic acid reductase with high affinity for the ascorbate radical scavenges this before it disproportionates into the two parent forms of vitamin C (ascorbate and dehydroascorbic acid). (2) Dehydroascorbic acid in the extracellular medium is slowly converted to ascorbate by a different mechanism with low affinity which may or may not involve uptake of the oxidized and release of the reduced form. (3) Ascorbate remains relatively stable in the cell culture medium in presence, but also in absence of the cells after their removal, This is most probably due to the presence of released peptides in the cell-conditioned medium which can chelate transition metal ions and thus prevent catalytic autoxidation of ascorbate.     

Ascorbate free radical and its role in growth control

Summary Ascorbate and its free radical potentiates proliferation of HL-60 cells in serum-limiting media. Dehydroascorbate does not affect growth. This stimulation of growth is due to a general shortening of the cell cycle. The incubation of HL-60 cells with ascorbate free radical produces a significant change of the redox potential of cells. The presence of cells in culture media avoids the total oxidation of ascorbate, and also HL-60 cells induce the short-term stabilization of ascorbate. Ascorbate free radical potentiates also the onset of DNA synthesis in CCL39 cells induced by fetal calf serum, although itself does not affect quiescense to proliferation transition. This transition induced by fetal calf serum also potentiates the capacity of CCL39 cells to stabilize ascorbate. We discuss here the role of ascorbate free radical on growth control by its reduction by the plasma membrane redox system and its meaning for cell physioslogy.     

Osmotic Swelling Stimulates Ascorbate Efflux from Cerebral Astrocytes

Abstract: Ascorbate (reduced vitamin C) is an important enzyme cofactor, neuromodulator, and antioxidant that is stored at millimolar concentrations in the cytosol of cerebral astrocytes. Because these cells swell during hyponatremia, cerebral ischemia, and trauma, we investigated the effects of osmotic stress on astrocytic transport of ascorbate. Ascorbate efflux from primary cultures of rat astrocytes was rapidly (within 1 min) increased by incubation in hypotonic medium. Efflux also increased when astrocytes, which had been adapted to a hypertonic environment, were swollen by transfer to isotonic medium. Swelling-induced ascorbate efflux was inhibited by the anion-transport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). The pathway that mediates ascorbate efflux was found to be selective because a larger anion, 2',7'-bis(carboxyethyl)-5-(or -6)-carboxyfluorescein (BCECF), was retained in the swollen astrocytes. Na⁺-dependent ascorbate uptake into astrocytes was inhibited slightly during the first minute of hypotonic stress, indicating that the sodium ascorbate cotransporter does not mediate swelling-induced efflux. Cell concentration of authentic ascorbate was measured by HPLC with electrochemical detection. When astrocytes were incubated in ascorbate-free medium, hypotonicity decreased cell ascorbate concentration by 50% within 3 min. When astrocytes were incubated in ascorbate-supplemented hypotonic medium, intracellular ascorbate concentration was restored within 10 min because uptake remained effective. Many pathological conditions cause brain cell swelling and formation of reactive oxygen species. Ascorbate release during astrocytic swelling may contribute to cellular osmoregulation in the short-term and the scavenging of reactive oxygen species.     

BOTANICAL BRIEFING: The Function and Metabolism of Ascorbic Acid in Plants

Ascorbate is a major metabolite in plants. It is an antioxidantand, in association with other components of the antioxidantsystem, protects plants against oxidative damage resulting fromaerobic metabolism, photosynthesis and a range of pollutants.Recent approaches, using mutants and transgenic plants, areproviding evidence for a key role for the ascorbate–glutathionecycle in protecting plants against oxidative stress. Ascorbateis also a cofactor for some hydroxylase enzymes (e.g. prolylhydroxylase) and violaxanthin de-epoxidase. The latter enzymelinks ascorbate to the photoprotective xanthophyll cycle. Arole in regulating photosynthetic electron transport has beenproposed. The biosynthetic pathway of ascorbate in plants hasnot been identified and evidence for the proposed pathways isreviewed. Ascorbate occurs in the cell wall where it is a firstline of defence against ozone. Cell wall ascorbate and cellwall-localized ascorbate oxidase (AO) have been implicated incontrol of growth. High AO activity is associated with rapidlyexpanding cells and a model which links wall ascorbate and ascorbateoxidase to cell wall extensibility is presented. Ascorbate hasalso been implicated in regulation of cell division by influencingprogression from G1 to S phase of the cell cycle. There is aneed to increase our understanding of this enigmatic moleculesince it could be involved in a wide range of important functionsfrom antioxidant defence and photosynthesis to growth regulation. Ascorbic acid; ascorbate oxidase; cell division; cell wall; growth; oxidative stress; photosynthesis; ozone; vitamin C     

Effect of ascorbate on oxygen uptake and growth of Escherichia coli B

The addition of ascorbate to aerobically growing cultures of Escherichia coli B caused only a short pause in growth and no subsequent change in the rate or extent of growth. The effect of ascorbate on oxygen uptake varied from inhibition in minimal medium to stimulation in rich medium. Cyanide-resistant growth and oxygen uptake were stimulated by ascorbate. Both the rate and extent of anaerobic growth were stimulated in proportion to the amount of ascorbate added when fumarate was the terminal electron acceptor. Ascorbate had no effect on any aspect of anaerobic growth in the absence of a terminal electron acceptor or in the presence of nitrate.     

Formation of Ascorbate Oxidase in Cultured Pumpkin Cells

Ascorbate oxidase activity rapidly increased during callus formationfrom pumpkin fruit tissue. The activity reached a maximum at5 days after transfer and then declined. In callus which hadbeen subcultured at about 4-week intervals for more than oneyear, the activity also increased after transfer to fresh mediumand reached a maximum in the early logarithmic phase of growth.Light had little effect on the appearance of ascorbate oxidaseactivity in pumpkin callus. In the callus grown in the presenceof 10µM CuSO₄, the activity was about 10 times that inthe presence of 0.1 µM CuSO₄, suggesting that the formatonof ascorbate oxidase in pumpkin callus is stimulated by copper,a prosthetic metal of the enzyme. From 45 to 75% of the totalascorbate oxidase activity in pumpkin cell suspension cultureswas found in the medium. Ascorbate oxidase activity in the medium,as well as that in the cells, increased soon after transferto fresh medium, and reached a maximum at about 5 days. (Received July 2, 1987; Accepted November 21, 1987)     

Deleterious synergistic effects of ascorbate and copper on the development of Plasmodium falciparum: an in vitro study in normal and in G6PD-deficient erythrocytes

The effects of ascorbate and copper on the development of Plasmodium falciparum were studied in two modes: pretreatment of uninfected erythrocytes followed by infection by P. falciparum and treatment of parasitized erythrocytes. Pretreatment of G6PD(+) cells with ascorbate caused a slight enhancement in parasite development, while in G6PD(-) cells a suppressive effect on the plasmodia was demonstrated. Copper alone interfered with parasite growth in both cell types. The combination of copper and ascorbate arrested parasite maturation, an effect which was more pronounced in G6PD(-) cells. Synergism between copper and ascorbate was better demonstrated following the treatment of infected erythrocytes: while ascorbate alone supported parasite development and copper alone had only a marginal suppressive effect, the combination of copper and ascorbate yielded a marked inhibition of parasite growth. Ascorbate proved destructive to the parasites in the presence of adventitious copper, or on the second day of the parasite life cycle. In these cases it acted as a pro-oxidant, while in other systems, in particular in the presence of a chelator, ascorbate acted as an antioxidant and promoted parasite growth. The understanding of the role of transition metals and free radicals in parasite development and injury could shed light on novel approaches to fight malaria.     

In vitro lipid peroxidation of human serum catalyzed by cupric ion: antioxidant rather than prooxidant role of ascorbate.

The dual role of ascorbate as an antioxidant and a prooxidant has been clearly documented in the literature. Ascorbate acts as an antioxidant by protecting human serum from lipid peroxidation induced by azo dye-generated free radicals. On the other hand, ascorbate is readily oxidized in the presence of transition metal ions, (especially cupric ion) and accelerates lipid peroxidation in tissue homogenates by producing free radicals. Interestingly, we observed an antioxidant rather than an expected prooxidant role of ascorbate when human serum supplemented with 1.2mmol/L ascorbate underwent lipid peroxidations initiated by 2mmol/L copper sulfate. The antioxidant role of ascorbate was confirmed by studying the conventional thiobarbituric acid reactive substances (TBARS) as well as by observing the protective effect of ascorbate on the copper-induced peroxidation of unsaturated and polyunsaturated fatty acids. The antioxidation protection provided by ascorbate was comparable to that of equimolar alpha-tocopherol when incubated for 24h. However, lipid peroxidation products were lower in serum supplemented with alpha-tocopherol after 48h of incubation. This effect may be attributed to the binding of copper by plpha-tocopherol after serum proteins, thus preventing direct interaction between cupric ions and ascorbate. This proposed mechanism is based on the observation that the concentration of ascorbate decreased more slowly in serum than in phosphate buffer at physiological pH. Our results also indicate an outstanding anti-oxidant property of human serum due to the chelation of transition metal ions (even at high concentrations) by various serum proteins.     

Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells

The biological function of ascorbate oxidase (AAO) was not yet clarified, although it was suggested that AAO may be involved in cell growth. We investigated AAO expression and ascorbate metabolism during non-synchronous, synchronous, and elongation cultures of tobacco BY-2 cells. In non-synchronous culture, AAO mRNA was abundant in logarithmic growth phase. Ascorbate content greatly increased during the growth, whereas dehydroascorbate content was slightly increased. In synchronous division culture, AAO mRNA was detected in all phases, but the levels were quite low in G1 phase. Ascorbate content was high in all phases, whereas dehydroascorbate content was low, especially in G1 phase. In elongation culture, the levels of AAO mRNA increased during elongation of the cells. AAO activity in the culture medium, as well as ascorbate and dehydroascorbate contents in the cells, also increased during the elongation. We propose that AAO expression and production of dehydroascorbate are under the control of the cell cycle and that AAO may function apoplastically as an ascorbate oxidizer in the process of cell elongation.     

Regulation of ascorbate oxidase expression in pumpkin by auxin and copper

Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation.     

马头山羊成纤维细胞系的建立与生物学特性分析

采用组织块贴壁培养法对马头山羊耳缘组织进行培养, 成功构建了马头山羊耳缘组织成纤维细胞系, 并对其形态学、生长动力学、细胞活力测定、中期染色体、微生物污染等特性进行了研究。结果表明: 培养细胞形态为典型的成纤维细胞, 细胞群体倍增时间(PDT)约为36 h。细胞冻存复苏后的活率为96.7%, 传代后生长状况与冻存前一致。细胞中期染色体二倍体(2n=60)占主体约为96%。微生物检测细菌、真菌、病毒支原体检测结果为阴性。细胞系各项指标均达到美国典型培养中心(ATCC)标准。此细胞库的建立在细胞水平上对马头山羊的遗传资源进行了保存, 也为今后的生物学研究以及体细胞克隆保种等研究提供了理想的实验材料。     

A novel cold-inducible gene from Arabidopsis,RCI3, encodes a peroxidase that constitutes a component for stress tolerance

Myrothamnus flabellifolia, a short woody shrub from southern Africa, can survive severe desiccation of its vegetative organs. We studied mechanisms protecting this plant from oxidative damage during desiccation for 2 weeks, 4 and 8 months, and also during subsequent rehydration. This plant retains high concentrations of chlorophyll during desiccation, and these chlorophyll molecules are probably a source for potentially harmful singlet oxygen production. Desiccation triggered substantial increases in zeaxanthin and redox shifts of the antioxidants glutathione and ascorbate towards their oxidised forms. Simultaneously, the concentrations of violaxanthin, beta-carotene, ascorbate, alpha-tocopherol, and glutathione reductase activity progressively decreased. Antheraxanthin, gamma-tocopherol, lutein, neoxanthin and glucose-6-phosphate dehydrogenase displayed less pronounced changes in response to desiccation. Even after 4 months of desiccation, Myrothamnus flabellifolia recovered rapidly upon rehydration. Re-watering induced formation of ascorbate and glutathione, simultaneous reduction of their oxidised forms, and rapid production of alpha-tocopherol and of various carotenoids. Only after 8 months of desiccation did the antioxidant system of M. flabellifolia break down; 3 weeks after the onset of rehydration, these plants abscised their leaves, but even then they were still able to recover and develop new ones. Ascorbate, beta-carotene and alpha-tocopherol were totally depleted after 8 months of desiccation and did not recover upon rehydration; glutathione was partly maintained, but only in the oxidised form. We present a model demonstrating which parts of antioxidant pathways break down as oxidative stress becomes detrimental and we discuss some potential implications of our results for the genetic modification of crop plants to improve their drought tolerance.     

Ascorbate-induced changes in gangliosides of calf aortic smooth muscle cells in culture: Possible influence of extracellular matrix

Summary The ganglioside composition of calf aortic smooth muscle cells, cultured in the presence and absence of ascorbate, was analyzed. Previous work has shown that ascorbate supplementation leads to the formation of an extracellular matrix consisting primarily of collagen and that this matrix influences the biosynthetic capabilities of the cell. Cell cultures were supplemented with ascorbate for 3 wk and labeled with [¹⁴C]glucosamine for 3 d before harvesting. Ascorbate supplementation resulted in increased ganglioside sialic acid levels and a change in chromatographic profile involving both absolute and relative increases in GD1a. The latter, along with polysialo species, showed increased incorporation of [¹⁴C]glucosamine. These findings are interpreted in relation to the proposed role of gangliosides as mediators in the interaction of various cells with extracellular matrix. This work was supported by grants 1-P01-AG05554 and 2-R01-NS-04834 from the Public Health Service, Washington, DC, as well as a Presidential Junior Faculty Development Award (JAS) from the Purchase College Foundation.     

Ascorbic acid blunts oxidant stress due to menadione in endothelial cells

Endothelial cells are exposed to potentially damaging reactive oxygen species generated both within the cells and in the bloodstream and underlying vessel wall. In this work, we studied the ability of ascorbic acid to protect cultured human-derived endothelial cells (EA.hy926) from oxidant stress generated by the redox cycling agent menadione. Menadione caused intracellular oxidation of dihydrofluorescein, which required the presence of D-glucose in the incubation medium, and was inhibited by intracellular ascorbate and desferrioxamine. At concentrations of 100 microM and higher, menadione depleted the cells of both GSH and ascorbate, and ascorbate loading partially prevented the decrease in GSH due to menadione. Menadione increased L-arginine uptake by the cells, but inhibited endothelial nitric oxide synthase, an effect that was prevented by acute loading with ascorbate. Ascorbate blunts menadione-induced oxidant stress in EA.hy926 cells, which may help to preserve nitric oxide synthase activity under conditions of excessive oxidant stress.     

Cardiac differentiation at an initial low density of human-induced pluripotent stem cells

A high density of human-induced pluripotent stem cells (hiPSCs) improves the efficiency of cardiac differentiation, suggesting the existence of indispensable cell-cell interaction signals. The complexity of interactions among cells at high density hinders the understanding of the roles of cell signals. In this study, we determined the minimum cell density that can initiate differentiation to facilitate cell-cell interaction studies. First, we co-induced cardiac differentiation in the presence of the glycogen synthase kinase-3β inhibitor CHIR99021 and activin A at various cell densities. At an initial low density, cells died within a few days in RPMI-based medium. We then investigated the culture conditions required to maintain cell viability. We used a basal medium excluding important components for the maintenance of hiPSC pluripotency, including activin A, basic fibroblast growth factor, and insulin. Supplementation of the basal medium with Rho-associated protein kinase inhibitor and insulin improved cell viability. Interestingly, addition of basic fibroblast growth factor enabled the expression of cardiac markers at the mRNA level but not the protein level. After further modification of the culture conditions, 10% of the cells expressed the cardiac troponin T protein, which is associated with cell contraction. The novel protocol for cardiac differentiation at an initial low cell density can also be used to evaluate high cell density conditions. The findings will facilitate the identification of cell signals required for cardiomyocyte formation.