Sodium-dependent transport of ascorbic acid in U937 cell mitochondria.

U937 cells exposed to physiological concentrations of ascorbic acid (AA) accumulate the reduced form of the vitamin in the cytosol and even further in their mitochondria. In both circumstances, uptake was dependent on Na(+) -AA-cotransport, with hardly any contribution of hexose transporters, which might be recruited to transport the oxidized form of the vitamin. There was an identical linear relationship between the mitochondrial accumulation of the vitamin and the extramitochondrial AA concentration, regardless of whether detected in experiments using intact cells or isolated mitochondria. Western blot experiments revealed expression of both SVCT1 and 2 in plasma membranes, whereas SVCT2 was the only form of the transporter expressed at appreciable amounts in mitochondria. These results therefore provide the novel demonstration of SVCT2-dependent mitochondrial transport of AA and hence challenge the present view that mitochondria only take up the oxidized form of the vitamin

Temporal correlation of morphological and biochemical changes with the recruitment of different mechanisms of reactive oxygen species formation during human SW872 cell adipogenic differentiation

none8noHuman SW872 preadipocyte conversion to mature adipocytes is associated with time-dependent changes in differentiation markers' expression and with morphological changes accompanied by the accumulation of lipid droplets (LDs) as well as by increased mitochondriogenesis and mitochondrial membrane potential. Under identical conditions, the formation of reactive oxygen species (ROS) revealed with a general probe was significant at days 3 and 10 of differentiation and bearly detectable at day 6. NADPH oxidase (NOX)-2 activity determined with an immunocytochemical approach followed a very similar pattern. There was no evidence of mitochondrial ROS (mROS), as detected with a selective fluorescence probe, at days 3 and 6, possibly due to the triggering of the Nrf-2 antioxidant response. mROS were instead clearly detected at day 10, concomitantly with the accumulation of very large LDs, oxidation of both cardiolipin and thioredoxin 2, and decreased mitochondrial glutathione. In conclusion, the morphological and biochemical changes of differentiating SW872 cells are accompanied by the discontinuous formation of ROS derived from NOX-2, increasingly implicated in adipogenesis and adipose tissue dysfunction. In addition, mROS formation was significant only in the late phase of differentiation and was associated with mitochondrial dysfunction.openFiorani, Mara; De Matteis, Rita; Canonico, Barbara; Blandino, Giulia; Mazzoli, Alessandro; Montanari, Mariele; Guidarelli, Andrea; Cantoni, OrazioFiorani, Mara; De Matteis, Rita; Canonico, Barbara; Blandino, Giulia; Mazzoli, Alessandro; Montanari, Mariele; Guidarelli, Andrea; Cantoni, Orazi

Modelo de estrategia de sustentabilidad, propuesta de aplicación para emprendimientos regionales en Misiones

Este proyecto surge en el contexto del Proyecto de investigación “Estrategias innovadoras de desarrollo regional: Emprendimientos basados en el diseño y la sustentabilidad”, de la Universidad Nacional de Misiones-Facultad de Arte y Diseño. Se busca generar modelos de estrategias proyectuales innovadoras dentro de la práctica del diseño basadas en la sustentabilidad y la refuncionalización de objetos y materiales. Desde una perspectiva global, el desarrollo sustentable de productos y servicios se ha posicionado rápidamente y es una ventaja competitiva para emprendimientos muy diversos. En nuestro país, y especialmente en Misiones, no se ha percibido aún con precisión la potencia que puede tener en la economía local la implementación de unidades económicas basadas en estrategias de esta índole. Se parte del análisis de casos preexistentes nacionales e internacionales que desarrollan estrategias de producción sustentable, especialmente aquellos que incorporan a la refuncionalización. La investigación está enfocada en casos de empresas industriales tales como: empresas que integran el sector foresto maderero (caso 1), que es uno de los de mayor potencial, tanto desde la escala industrial como desde la posibilidad de generar valor (e identidad). Y una empresa de producción de envases de papel y laminados (caso 2), se incluye porque contienen elementos diversos, además la identidad se constata en que esta empresa es la principal proveedora de material para envases de las yerbateras locales. En mencionadas empresas existen materiales de descarte, tales como: Caso 1: Despuntes de pino en diferentes estados de proceso, multilaminados y placas de terciados, descartes por problemas de producción, despuntes de los palos, y aserrín. Caso 2: Films de PP, envases de yerba, tubos de cartón, postizos plásticos de PE, tablas de madera aglomerada, pallets, entre otros. Los mencionados descartes aún no son aprovechados. Se pretende promover el desarrollo sustentable de la región y potenciar la gestión como herramienta de diseño, que ayude a articular los distintos actores que intervienen en una cadena de valor, fortaleciendo el capital humano, su cultura e identidad. El objetivo es la incubación de propuestas que sirvan como modelo para emprendedores regionales, a fin de promover y sensibilizar acerca del uso de estrategias de sustentabilidad tanto en emprendedores, como empresas e instituciones de la región, comunicando el valor económico, ambiental y social de la incorporación de procesos sustentables como variable de innovación en dichas unidades productivas.publishedVersio

Dietary flavonoids as intracellular substrates for an erythrocyte trans-plasma membrane oxidoreductase activity

The plasma membrane oxidoreductase (PMOR) activity, which mainly utilises ascorbate as intracellular electron donor, represents a major mechanism for cell-dependent reduction of extracellular oxidants and might be an important process used by the erythrocytes to keep a reduced plasma environment. We previously reported that in human erythrocytes, myricetin and quercetin act as intracellular substrates of a PMOR showing a novel mechanism whereby these flavonoids could exert beneficial effects under oxidative stress conditions. Here, we evaluated the ability of different flavonoids (quercetin, myricetin, morin, kaempferol, fisetin, catechin, luteolin, apigenin, acacetin, rutin, taxifolin, naringenin, genistein) and of two in vivo O-methylated metabolites of quercetin (isorhamnetin and tamarixetin) to be substrates of PMOR, by comparing their antioxidant capacity (i.e. direct interaction with the oxidant ferricyanide or with the free radical 1,1-diphenyl-2-picryl-hydrazil) with their ability to penetrate the erythrocytes and donate electrons to the PMOR. The results obtained indicate that, although most of the flavonoids display significant antioxidant activities, only those (quercetin, myricetin, fisetin) that combine the cathecol structure of the B ring (responsible for the reducing activity) with the 2,3 double bond and 4-oxo function of the C ring (responsible for the uptake by erythrocytes) can act as intracellular substrates for PMOR. It is of note that the metabolites of quercetin enter erythrocytes and donate electrons to the PMOR as the parent compound. The present data show a relationship between the flavonoid structures and their ability to provide electrons to the PMOR, suggesting an additional mechanism whereby dietary flavonoids may exert beneficial effects in man

Mitochondrial reactive oxygen species: the effects of mitochondrial ascorbic acid vs untargeted and mitochondria-targeted antioxidants

Premise: Mitochondria represent critical sites for reactive oxygen species (ROS) production, which dependent on concentration is responsible for the regulation of both physiological and pathological processes.Purpose: Antioxidants in mitochondria regulate the redox balance, prevent mitochondrial damage and dysfunction and maintain a physiological ROS-dependent signaling. The aim of the present review is to provide critical elements for addressing this issue in the context of various pharmacological approaches using antioxidants targeted or non-targeted to mitochondria. Furthermore, this review focuses on the mitochondrial antioxidant effects of ascorbic acid (AA), providing clues on the complexities associated with the cellular uptake and subcellular distribution of the vitamin.Conclusions: Antioxidants that are not specifically targeted to mitochondria fail to accumulate in significant amounts in critical sites of mitochondrial ROS production and may eventually interfere with the ensuing physiological signaling. Mitochondria-targeted antioxidants are more effective, but are expected to interfere with the mitochondrial ROS-dependent physiologic signaling. AA promotes multiple beneficial effects in mitochondria. The complex regulation of vitamin C uptake in these organelles likely contributes to its versatile antioxidant response, thereby providing a central role to the vitamin for adequate control of mitochondrial dysfunction associated with increased mitochondrial ROS production

Human red blood cells as a natural flavonoid reservoir

Quercetin is rapidly and avidly taken up by human red blood cells (RBC) via a passive diffusion mechanism, driven by flavonoid binding to haemoglobin and resulting in an almost quantitative accumulation of the flavonoid. Heamoglobin-free resealed ghosts accumulated quercetin exclusively in the membrane fraction. Cell-associated quercetin was biological active and could be quantitatively utilised to support the reduction of extracellular oxidants mediated by a transplasma-membrane oxido-reductase. Additional experimental evidence revealed that quercetin uptake declined in the presence of albumin and that, under these conditions, the amount of cell-associated quercetin is enhanced by increasing the RBC number. Quercetin release from flavonoid-preloaded RBC was observed only in the presence of albumin (or in human plasma) and this response was progressively inhibited upon incubation in solutions containing albumin previously exposed to increasing concentrations of quercetin and cleared of the unbound fraction of the flavonoid. Furthermore, exposure to quercetin pre-saturated albumin promoted accumulation of the flavonoid in fresh RBC and this response was a direct function of the extent of albumin saturation. These results, indicating a flow of quercetin from albumin to haemoglobin, and vice versa, are therefore consistent with the possibility that human RBC play a pivotal role in the distribution and bioavailability of circulating flavonoids

Low Concentrations of Arsenite Target the Intraluminal Inositol 1, 4, 5-Trisphosphate Receptor/Ryanodine Receptor Crosstalk to Significantly Elevate Intracellular Ca2

Arsenite is an established human carcinogen inducing cyto- and genotoxic effects through poorly defined mechanisms involving the formation of reactive oxygen species (ROS) and deregulated Ca2+ homeostasis. We used variants of the U937 cell line to address the central issue of the mechanism whereby arsenite affects Ca2+ homeostasis. We found that a 6 h exposure to the metalloid (2.5 μM), while not associated to an immediate or delayed toxicity, causes a significant increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism characterized by the following components: i) it was not affected by ROS produced under the same conditions; ii) a small amount of Ca2+ was mobilized from the inositol-1,4,5-trisphosphate receptor (IP3R). This response was not further augmented by greater concentrations of the metalloid; iii) large amounts of Ca2+ were instead dose-dependently mobilized from the ryanodine receptor (RyR) in response to IP3R stimulation; iv) the cells maintained an intact responsiveness to agonist-stimulated Ca2+ mobilization from both channels; v) arsenite, even at 5-10 μM, failed to directly mobilize Ca2+ from the RyR; vi) arsenite failed to enhance Ca2+ release from the RyR under conditions in which the [Ca2+]i was increased by either RyR agonists or ionophore-stimulated Ca2+ uptake. We therefore conclude that arsenite elevates the [Ca2+]i by directly targeting the IP3R and its intraluminal crosstalk with the RyR. This mechanism likely mediates mitochondrial superoxide formation, downstream damage on various biomolecules, including genomic DNA, and mitochondrial dysfunction/apoptosis eventually occurring after longer incubation to, or exposure to greater concentrations of, arsenite

Mitochondrial Uptake and Accumulation of Vitamin C: What Can We Learn From Cell Cultures Studies?

The mitochondrial fraction of L-ascorbic acid (AA) is of critical importance for the regulation of the redox status of these organelles and for cell survival. Recent Advances: Most cell types take up AA by high affinity sodium-dependent vitamin C transporter 2 (SVCT2), sensitive to inhibition by dehydroascorbic acid (DHA). DHA can also be taken up by glucose transporters (GLUTs) and then reduced back to AA. DHA concentrations, normally very low in biological fluids, may only become significant next to superoxide releasing cells. Very little is known on the mechanisms mediating the mitochondrial transport of the vitamin