RESUMEN
OBJECTIVE: MiR-551b has been reported to display tumor-suppressive and oncogenic potential in several cancers, but there has been no study on the roles of miR-551b in colorectal cancer (CRC). In this work, we aimed to explore the potential functions and mechanisms of miR-551b in the modulation of the CRC progression. PATIENTS AND METHODS: The expressions of miR-551b were examined in 122 pairs of CRC cancer tissues and adjacent non-tumor samples by Real Time-Polymerase Chain Reaction (RT-PCR). The clinical significance of miR-551b in CRC patients was explored using a Chi-square test, Kaplan-Meier assays, and multivariate analysis. MiR-551b mimics and inhibitors were used to establish miR-551b upregulation and downregulation models in CRC cells to examine the functions of miR-551b on cells proliferation, migration, invasion, and apoptosis. Dual-luciferase reporter assays were conducted for the validation of the possible modulation of a putative target of miR-551b. RESULTS: We showed that miR-551b was significantly down-regulated in CRC tissues and cell lines. It was observed that miR-551b expressions were correlated with lymph nodes metastasis, TNM stage, and poor prognosis. Multivariate analysis identifies low level of miR-551b expressions as an independent predictor for a shorter overall survival. The functional assessment suggested that forced miR-551b expression distinctly suppressed CRC cells growth, invasion, and migration, while the suppression of miR-551b displayed the opposite trend. Mechanistic studies confirmed that PTP4A3 was identified as a direct target of miR-551b in CRC. Interesting observations revealed that the cells capacities were higher in miR-551b +PTP4A3 group, when compared with those in miR-551b group, indicating that up-regulation of PTP4A3 rescued the repressive functions of miR-551b overexpression on CRC cells growth and migration. CONCLUSIONS: The findings of our study first showed that miR-551b, a potential tumor suppressor, may be used as a promising prognostic predictor and therapeutic target for CRC.
Asunto(s)
Neoplasias Colorrectales/metabolismo , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Apoptosis , Carcinogénesis , Movimiento Celular , Proliferación Celular , Células Cultivadas , Distribución de Chi-Cuadrado , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , Femenino , Humanos , Estimación de Kaplan-Meier , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Masculino , MicroARNs/genética , Persona de Mediana Edad , Análisis Multivariante , Proteínas de Neoplasias/genética , Pronóstico , Proteínas Tirosina Fosfatasas/genéticaRESUMEN
The nicotinic acetylcholine receptor (AChR) from the electric organ of T. californica is highly phosphorylated on tyrosine residues in vivo. In contrast, tyrosine phosphorylation of the AChR in rat myotube cultures is barely detectable. To determine whether this low level of tyrosine phosphorylation of the AChR in muscle cell cultures is due to a lack of neuronal innervation, we examined tyrosine phosphorylation of the AChR in rat diaphragm in vivo. Immunofluorescent double labeling of cryostat sections of rat diaphragm using antibodies specific for phosphotyrosine or the AChR showed a direct colocalization of phosphotyrosine with the AChR at the neuromuscular junction. Using anti-phosphotyrosine antibodies, immunoblots of AChR partially purified from rat diaphragm demonstrated that the rat AChR contains high levels of phosphotyrosine. Denervation of rat diaphragm induced a time-dependent decrease in tyrosine phosphorylation of the AChR, as measured by immunocytochemical and immunoblot techniques. Tyrosine phosphorylation of the AChR occurred late in the development of the neuromuscular junction, between postnatal days 7 and 14. These studies suggest that muscle innervation regulates tyrosine phosphorylation of the AChR and that tyrosine phosphorylation may play an important role in the developmental regulation of the AChR.
Asunto(s)
Unión Neuromuscular/metabolismo , Receptores Nicotínicos/metabolismo , Tirosina , Tirosina/análogos & derivados , Animales , Diafragma/metabolismo , Órgano Eléctrico/metabolismo , Músculos/metabolismo , Fosforilación , Fosfotirosina , Ratas , Receptores Nicotínicos/aislamiento & purificación , Torpedo , Tirosina/análisisRESUMEN
Reduction of extracellular ferricyanide by intact cells reflects the activity of an as yet unidentified trans-plasma membrane oxidoreductase. In human erythrocytes, this activity was found to be limited by the ability of the cells to recycle intracellular ascorbic acid, its primary trans-membrane electron donor. Ascorbate-dependent ferricyanide reduction by erythrocytes was partially inhibited by reaction of one or more cell-surface sulfhydryls with p-chloromercuribenzene sulfonic acid, an effect that persisted in resealed ghosts prepared from such treated cells. However, treatment of intact cells with the sulfhydryl reagent had no effect on NADH-dependent ferricyanide or ferricytochrome c reductase activities of open ghosts prepared from treated cells. When cytosol-free ghosts were resealed to contain trypsin or pronase, ascorbate-dependent reduction of extravesicular ferricyanide was doubled, whereas NADH-dependent ferricyanide and ferricytochrome c reduction were decreased by proteolytic digestion. The trans-membrane ascorbate-dependent activity was also found to be inhibited by reaction of sulfhydryls on its cytoplasmic face. These results show that the trans-membrane ferricyanide oxidoreductase is limited by the ability of erythrocytes to recycle intracellular ascorbate, that it does not involve the endofacial NADH-dependent cytochrome b(5) reductase system, and that it is a trans-membrane protein that contains sensitive sulfhydryl groups on both membrane faces.
Asunto(s)
Ácido Ascórbico/metabolismo , Membrana Eritrocítica/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Ácido Ascórbico/análisis , Transporte de Electrón , Membrana Eritrocítica/enzimología , Eritrocitos/metabolismo , Ferricianuros/metabolismo , Glutatión/análisis , Humanos , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/metabolismo , Oxidación-Reducción , Oxidorreductasas/metabolismo , Pronasa , Reactivos de Sulfhidrilo/farmacología , TripsinaRESUMEN
Ascorbic acid is an important antioxidant in human blood. Erythrocytes contribute to the antioxidant capacity of blood by regenerating ascorbate and possibly by exporting ascorbate-derived reducing equivalents through a transmembrane oxidoreductase. The role of ascorbate as an electron donor to the latter enzyme was tested in human erythrocytes and ghosts using nitroblue tetrazolium as an electron acceptor. Although nitroblue tetrazolium was not directly reduced by ascorbate, erythrocyte ghosts facilitated reduction of nitroblue tetrazolium in the presence of ascorbate and ascorbate derivatives containing a reducing double bond. The resulting blue monoformazan product was deposited directly in ghost membranes. Ascorbate-induced monoformazan deposition showed several features of an enzyme-mediated process, including hyperbolic dependence on substrate and acceptor concentrations, as well as sensitivity to enzyme proteolysis, detergent solubilization, and sulfhydryl reagents. Incubation of intact erythrocytes with nitroblue tetrazolium caused deposition of the monoformazan in ghost membranes prepared from the cells. This deposition reflected the intracellular ascorbate content and was inhibited by extracellular ferricyanide, a known electron acceptor for the transmembrane oxidoreductase. Although nitroblue tetrazolium did not cross the cell membrane, like the cell-impermeant ferricyanide, it oxidized intracellular [14C]ascorbate to [14C]dehydroascorbate, which then exited the cells. In resealed ghosts, both monoformazan deposition and ferricyanide reduction were proportional to the intravesicular ascorbate concentration. NADH was only about half as effective as a donor for the enzyme as ascorbate in both open and resealed ghosts. These results suggest that not only can ascorbate donate electrons to a transmembrane oxidoreductase, but that it may be the major donor in intact erythrocytes.
Asunto(s)
Ácido Ascórbico/metabolismo , Membrana Eritrocítica/enzimología , Oxidorreductasas/metabolismo , Antioxidantes/metabolismo , Transporte de Electrón , Humanos , NAD/química , Nitroazul de Tetrazolio/químicaRESUMEN
Arginine vasopressin (AVP), oxytocin (OT), and angiotensin-II (AII) elicit a biphasic ACTH secretory response by perifused anterior pituitary cells consisting of an initial transient (less than 3-min) spike phase and a subsequent sustained plateau phase. In contrast, CRF produces a monophasic sustained plateau type of ACTH secretory response. We have previously demonstrated that 1) influx of extracellular Ca2+ (Cae2+) via L-type voltage-sensitive Ca2+ channels is involved in both the response to CRF and the sustained phase of the response to AVP and OT; 2) release of intracellular Ca2+ (Cai2+) is involved in the spike phase of the response to AVP, OT, and AII; and 3) activation of protein kinase-C is required for the sustained phase, but not for the spike phase, of the response to AVP. CRF action is mediated by activation of protein kinase-A. In this study we further examined the role of Cai2+ by exploiting the fact that a low concentration (1 microM) of ionomycin, a potent Ca2+ ionophore, releases Cai2+ from nonmitochondrial inositol-1,4,5-trisphosphate (IP3)-sensitive Cai2+ stores without causing Cae2+ influx. Pretreatment with ionomycin for 10 min decreased the spike phase of the response to 100 nM AVP, OT, and AII, but had no effect on the response to 10 nM CRF or the sustained phase of the responses to the other agonists. The combination of CRF plus AVP induced a biphasic and synergistic release of ACTH. Ionomycin pretreatment reduced the spike phase, especially the first 1 min, without any effect on the sustained phase. These results indicate that Cai2+ release, but not Cae2+ influx, is involved in the spike phase of the response to AVP, OT, and AII and that Cai2+ is not involved in the synergistic effect of the combination of CRF plus AVP. Having established these relationships, we examined the effect of 2-h perifusion with 100 nM dexamethasone (DEX) on stimulated ACTH release. DEX pretreatment reduced the total response to CRF, the sustained phase of the responses to AVP and OT, and the sustained phase of the synergistic response to CRF plus AVP. However, DEX had no effect on the spike phase of the responses to AVP, OT, or AII or the spike phase of the response to CRF plus AVP. These results indicate that DEX inhibits ACTH release mediated by activation of either protein kinase-A or protein kinase-C, but does not affect inositol-1,4,5-trisphosphate/Cai2(+)-mediated ACTH release.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , Calcio/deficiencia , Glucocorticoides/farmacología , Membranas Intracelulares/metabolismo , Adenohipófisis/metabolismo , Angiotensina II/farmacología , Animales , Arginina Vasopresina/farmacología , Hormona Liberadora de Corticotropina/farmacología , Dexametasona/farmacología , Ionomicina/farmacología , Oxitocina/farmacología , Perfusión/métodos , Adenohipófisis/citología , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Human erythrocytes efficiently reduce dehydroascorbic acid (DHA) to ascorbate, which helps to maintain the ascorbate content of blood. Whereas erythrocyte DHA reduction is thought to occur primarily through a direct chemical reaction with GSH, this work addresses the role of enzyme-mediated DHA reduction by these cells. The ability of intact erythrocytes to recycle DHA to ascorbate, estimated as DHA-dependent ferricyanide reduction, was decreased in parallel with GSH depletion by glutathione-S-transferase substrates. In contrast, the sulfhydryl reagent phenylarsine oxide inhibited DHA reduction to a much greater extent than it decreased GSH in intact cells. DHA reduction in excess of that due to a direct chemical reaction with GSH was also observed in freshly prepared hemolysates. Hemolysates likewise showed NADPH-dependent reduction of DHA that appeared due to thioredoxin reductase, because this activity was inhibited 68% by 10 microM aurothioglucose, doubled by 5 microM E. coli thioredoxin, and had an apparent Km for DHA (1.5 mM) similar to that of purified thioredoxin reductase. Additionally, aurothioglucose-sensitive, NADPH-dependent DHA reductase activity was decreased 80% in hemolysates prepared from phenylarsine oxide-treated cells. GSH-dependent DHA reduction in hemolysates was more than 10-fold that of NADPH-dependent reduction. Nonetheless, the ability of phenylarsine oxide to decrease DHA reduction in intact cells with little effect on GSH suggests that enzymes, such as thioredoxin reductase, may contribute more to this activity than previously considered.
Asunto(s)
Ácido Ascórbico/metabolismo , Enzimas/metabolismo , Eritrocitos/enzimología , Eritrocitos/metabolismo , Reductasa de Tiorredoxina-Disulfuro/fisiología , Arsenicales/farmacología , Aurotioglucosa/farmacología , Membrana Celular/química , Sistema Libre de Células/química , Sistema Libre de Células/efectos de los fármacos , Ácido Deshidroascórbico/metabolismo , Dinitroclorobenceno/farmacología , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Ferricianuros/metabolismo , Glutatión/efectos de los fármacos , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Hemólisis , Humanos , Cetonas/farmacología , NADP/farmacología , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Vitamina E/análisisRESUMEN
Ascorbic acid is an important antioxidant in human plasma, but requires efficient recycling from its oxidized forms to avoid irreversible loss. Human erythrocytes prevented oxidation of ascorbate in autologous plasma, an effect that required recycling of ascorbate within the cells. Erythrocytes had a high capacity to take up dehydroascorbate, the two-electron oxidized product of ascorbate, and to reduce it to ascorbate. Uptake and conversion of dehydroascorbate to ascorbate was saturable, was half-maximal at 400 microM dehydroascorbate, and achieved a maximal intracellular ascorbate concentration of 1.5 mM. In the presence of 100 microM dehydroascorbate, erythrocytes had the capacity to regenerate a 35 microM ascorbate concentration in blood every 3 min. Ascorbate recycling from DHA required intracellular GSH. Depletion of erythrocyte GSH by more than 50% with diamide did not acutely affect the cellular ascorbate content, but did impair the subsequent ability of GSH-depleted cells to recycle dehydroascorbate to ascorbate. Whereas erythrocyte ascorbate recycling was coupled to GSH, an overwhelming extracellular oxidant stress depleted both ascorbate and alpha-tocopherol before the GSH content of cells fell appreciably. Recycled ascorbate was released from cells into plasma, but at a rate less than one tenth that of dehydroascorbate uptake and conversion to ascorbate. Nonetheless, ascorbate released from cells protected endogenous alpha-tocopherol in human LDL from oxidation by a water soluble free radical initiator. These results suggests that recycling of ascorbate in erythrocytes helps to maintain the antioxidant reserve of whole blood.
Asunto(s)
Antioxidantes/metabolismo , Ácido Ascórbico/sangre , Eritrocitos/metabolismo , Glutatión/sangre , Estrés Oxidativo/fisiología , Vitamina E/sangre , Transporte Biológico/fisiología , Radicales Libres , Humanos , Lipoproteínas LDL/sangre , Valores de ReferenciaRESUMEN
Lipophilic derivatives of ascorbic acid may protect lipid bilayers and micelles against lipid peroxidation. In this work the binding, accessibility, and reducing capacity of ascorbate 6-palmitate (A6P) were studied in human erythrocyte membranes. In contrast to less lipophilic carbon-6-modified ascorbate derivatives, A6P bound to erythrocyte membranes in a concentration-dependent manner. This binding was preserved following centrifugation washes, but was largely reversed by extraction with bovine serum albumin. Most of the ascorbyl groups of membrane-bound A6P were readily accessible to oxidation by water-soluble oxidants. Ferricyanide quantitatively oxidized membrane-bound A6P, but the latter spared endogenous tocopherols from destruction. In EPR studies, A6P was much more effective than ascorbate in reducing nitroxide spin labels positioned at either carbon-5 or carbon-16 of membrane-bound stearic acid in both intact cells and in membranes. A6P, thus, appears to intercalate into the erythrocyte membrane with the ascorbyl group located superficially, but with access to the hydrophobic membrane interior, and with the ability to recycle endogenous alpha-tocopherol during oxidant stress.
Asunto(s)
Ácido Ascórbico/análogos & derivados , Membrana Eritrocítica/metabolismo , Antimutagênicos , Ácido Ascórbico/sangre , Ácido Ascórbico/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Ferricianuros , Radicales Libres , Humanos , Cinética , Liposomas , Oxidación-Reducción , Albúmina Sérica Bovina , Marcadores de SpinRESUMEN
The uptake, recycling, and function of ascorbic acid was evaluated in cultured U-937 monocytic cells. Dehydroascorbic acid, the two-electron oxidized form of the vitamin, was taken up on the glucose transporter and reduced to ascorbate to a much greater extent than ascorbate itself was accumulated by the cells. In contrast to dehydroascorbic acid, ascorbate entered the cells on a sodium- and energy-dependent transporter. Intracellular ascorbate enhanced the transfer of electrons across the cell membrane to extracellular ferricyanide. Rates of ascorbate-dependent ferricyanide reduction were saturable, fivefold greater than basal rates, and facilitated by intracellular recycling of ascorbate. Whereas reduction of dehydroascorbic acid concentrations above 400 microM consumed reduced glutathione (GSH), even severe GSH depletion by 1-chloro-2,4-dinitrobenzene was without effect on the ability of the cells to reduce concentrations of dehydroascorbic acid likely to be in the physiologic range (< 200 microM). Dialyzed cytosolic fractions from U-937 cells reduced dehydroascorbic acid to ascorbate in an NADPH-dependent manner that appeared due to thioredoxin reductase. However, thioredoxin reductase did not account for the bulk of dehydroascorbic acid reduction, since its activity was also decreased by treatment of intact cells with 1-chloro-2,4-dinitrobenzene. Thus, U-937 cells loaded with dehydroascorbic acid accumulate ascorbate against a concentration gradient via a mechanism that is not dependent on GSH or NADPH, and this ascorbate can serve as the major source of electrons for transfer across the plasma membrane to extracellular ferricyanide.
Asunto(s)
Ácido Ascórbico/metabolismo , Monocitos/metabolismo , Ácido Deshidroascórbico/metabolismo , Transporte de Electrón , Glutatión/metabolismo , Humanos , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Células U937RESUMEN
Human erythrocytes regenerate ascorbate from its oxidized product, dehydroascorbate. The extent to which such ascorbate recycling occurs by a GSH-dependent mechanism was investigated. In the presence of glucose, erythrocytes took up over 90% of extracellular [14C]dehydroascorbate and rapidly converted it to [14C]ascorbate, which was trapped within the cells. Dehydroascorbate uptake and reduction was not associated with generation of a monoascorbyl free radical intermediate. Uptake and reduction of dehydroascorbate by glucose-depleted erythrocytes coordinately decreased GSH and raised GSSG concentrations in erythrocytes. This effect was reversed by D-glucose, but not by L-lactate. Conversely, depletion of cellular GSH decreased the ability of cells to recycle dehydroascorbate to ascorbate, as reflected in the extent to which cells were able to reduce extracellular ferricyanide. Monoascorbyl free radical was formed during the reduction of extracellular ferricyanide, indicating that one electron transfer steps were involved in this process. In GSH-depleted cells, addition of L-lactate as an energy source for glycolysis-dependent NADH regeneration did cause a partial recovery of the ability of cells to reduce ferricyanide. However, in resealed erythrocyte ghosts containing either 4 mM GSH or 400 mu M NADH, only the GSH-containing ghosts supported regeneration of ascorbate from added dehydroascorbate. These results suggest that in human erythrocytes ascorbate regeneration from dehydroascorbate is largely GSH dependent, and that it occurs through either enzymatic or nonenzymatic reactions not involving the monoascorbyl free radical.
Asunto(s)
Ácido Ascórbico/sangre , Ácido Deshidroascórbico/sangre , Eritrocitos/fisiología , Glutatión/fisiología , Ferricianuros/metabolismo , Humanos , Oxidación-ReducciónRESUMEN
Lipid-soluble antioxidants, such as alpha-tocopherol, protect cell membranes from oxidant damage. In this work we sought to determine whether the amphipathic derivative of ascorbate, ascorbate 6-palmitate, is retained in the cell membrane of intact erythrocytes, and whether it helps to protect the cells against peroxidative damage. We found that ascorbate 6-palmitate binding to erythrocytes was dose-dependent, and that the derivative was retained during the multiple wash steps required for preparation of ghost membranes. Ascorbate 6-palmitate remained on the extracellular surface of the cells, because it was susceptible to oxidation or removal by several cell-impermeant agents. When bound to the surface of erythrocytes, ascorbate 6-palmitate reduced ferricyanide, an effect that was associated with generation of an ascorbyl free radical signal on EPR spectroscopy. Erythrocyte-bound ascorbate 6-palmitate protected membrane alpha-tocopherol from oxidation by both ferricyanide and a water-soluble free radical initiator, suggesting that the derivative either reacted directly with the exogenously added oxidant, or that it was able to recycle the alpha-tocopheroxyl radical to alpha-tocopherol in the cell membrane. Ascorbate 6-palmitate also partially protected cis-parinaric acid from oxidation when this fluorescent fatty acid was intercalated into the membrane of intact cells. These results show that an amphipathic ascorbate derivative is retained on the exterior cell surface of human erythrocytes, where it helps to protect the membrane from oxidant damage originating outside the cells.
Asunto(s)
Antioxidantes/farmacología , Ácido Ascórbico/análogos & derivados , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Antioxidantes/metabolismo , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Membrana Eritrocítica/metabolismo , Ácidos Grasos Insaturados/metabolismo , Compuestos Férricos/metabolismo , Ferricianuros/metabolismo , Humanos , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Vitamina E/metabolismoRESUMEN
The antidiabetic drug troglitazone contains the active chromanol ring of alpha-tocopherol, which should give it antioxidant properties within cells. In these studies, the antioxidant effects of troglitazone were tested in human erythrocytes and in their ghosts. Troglitazone bound to erythrocyte ghosts in a linear manner and was retained even after centrifugation washes. In response to an oxidant stress generated by a water-soluble free radical initiator, troglitazone that was bound to erythrocyte ghosts was oxidized, but induced a lag-phase in the disappearance of endogenous alpha-tocopherol and in the appearance of lipid hydroperoxides. Troglitazone also delayed loss of endogenous alpha-tocopherol and hemolysis in washed intact erythrocytes in response to free radical-induced extracellular oxidant stress. To mimic exposure of erythrocytes to lipid hydroperoxides in vivo, erythrocytes were incubated with phospholipid liposomes that contained small amounts of preformed lipid hydroperoxides. This induced an oxidant stress in both the liposomes and cells. Troglitazone in concentrations above 4 microM almost completely prevented further appearance of lipid hydroperoxides in the liposomes, and also completely preserved alpha-tocopherol in the erythrocytes. The present results suggest that troglitazone will help to prevent peroxidative damage to erythrocytes in areas of excessive oxidant stress in the vascular bed.
Asunto(s)
Antioxidantes/uso terapéutico , Cromanos/uso terapéutico , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Tiazoles/uso terapéutico , Tiazolidinedionas , Células Cultivadas , Cromanos/química , Relación Dosis-Respuesta a Droga , Membrana Eritrocítica/metabolismo , Radicales Libres/metabolismo , Humanos , Peróxidos Lipídicos/metabolismo , Liposomas/metabolismo , Estrés Oxidativo , Tiazoles/química , Factores de Tiempo , Troglitazona , Vitamina E/metabolismoRESUMEN
Liver is the site of ascorbic acid synthesis in most mammals. As human liver cannot synthesize ascorbate de novo, it may differ from liver of other species in the capacity or mechanism for ascorbate recycling from its oxidized forms. Therefore, we compared the ability of cultured liver-derived cells from humans (HepG2 cells) and rats (H4IIE cells) to take up and reduce dehydroascorbic acid (DHA) to ascorbate. Neither cell type contained appreciable amounts of ascorbate in culture, but both rapidly took up and reduced DHA to ascorbate. Intracellular ascorbate accumulated to concentrations of 10-20 mM following loading with DHA. The capacity of HepG2 cells to take up and reduce DHA to ascorbate was more than twice that of H4IIE cells. In both cell types, DHA reduction lowered glutathione (GSH) concentrations and was inhibited by prior depletion of GSH with diethyl maleate, buthionine sulfoximine, and phenylarsine oxide. NADPH-dependent DHA reduction due to thioredoxin reductase occurred in overnight-dialyzed extracts of both cell types. These results show that cells derived from rat liver synthesize little ascorbate in culture, that cultured human-derived liver cells have a greater capacity for DHA reduction than do rat-derived liver cells, but that both cell types rely largely on GSH- or NADPH-dependent mechanisms for ascorbate recycling from DHA.
Asunto(s)
Ácido Ascórbico/metabolismo , Glutatión/metabolismo , Glutatión/fisiología , Hígado/citología , Hígado/efectos de los fármacos , Animales , Arsenicales/farmacología , Butionina Sulfoximina/farmacología , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Maleatos/farmacología , NADP/metabolismo , Ratas , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Reductasa de Tiorredoxina-Disulfuro/farmacología , Factores de Tiempo , Agua/químicaRESUMEN
The role of ascorbic acid in transferrin-independent ferric iron reduction and uptake was evaluated in cultured U-937 monocytic cells. Uptake of 55Fe by U-937 cells was doubled by 100 microM extracellular ascorbate, and by pre-incubation of cells with 100 microM dehydroascorbic acid, the two-electron-oxidized form of ascorbate. Reduction of extracellular ferric citrate also was enhanced by loading the cells with dehydroascorbic acid. Dehydroascorbic acid was taken up rapidly by the cells and reduced to ascorbate, such that the latter reached intracellular concentrations as high as 6 mM. However, some ascorbate did escape the cells and could be detected at concentrations of up to 1 microM in the incubation medium. Further, addition of ascorbate oxidase almost reversed the effects of dehydroascorbic acid on both 55Fe uptake and ferric citrate reduction. Thus, it is likely that extracellular ascorbate reduced ferric to ferrous iron, which was then taken up by the cells. This hypothesis also was supported by the finding that during loading with ferric citrate, only extracellular ascorbate increased the pool of intracellular ferrous iron that could be chelated with cell-penetrant ferrous iron chelators. In contrast to its inhibition of ascorbate-dependent ferric iron reduction, ascorbate oxidase was without effect on ascorbate-dependent reduction of extracellular ferricyanide. This indicates that the cells use different mechanisms for reduction of ferric iron and ferricyanide. Therefore, extracellular ascorbate derived from cells can enhance transferrin-independent iron uptake by reducing ferric to ferrous iron, but intracellular ascorbate neither contributes to this reduction nor modifies the redox status of intracellular free iron.
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Ácido Ascórbico/metabolismo , Transferrina/metabolismo , Quelantes/farmacología , Ácido Deshidroascórbico/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Hierro/metabolismo , Oxidación-Reducción , Fenantrolinas/farmacología , Células U937RESUMEN
This report describes isolation of virus particles from plants of rice, maize, wheat and sorghum with symptoms of dwarfing collected from two provinces of China, purification of double-stranded RNA (dsRNA) from the virus particles, and synthesis of full-length cDNAs of genome segments 9 (S9) and 10 (S10) by reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis showed that the S9 sequences of the Chinese isolates and a Japanese rice black-streaked disease virus (RBSDV) isolate were very similar (89.1-89.6% homology at nucleotide level and 92.3-92.9% and 95.8-98.6% homology at amino acid level for ORF1 and ORF2, respectively). Analogical similarity was found also for the S10 sequences of the isolates under comparison: 93.0-95.4% homology at nucleotide level and 96.2-97.0% homology at amino acid level. However, there was a relatively lower similarity for S9 and S10 segments ofthe Chinese isolates and an Italian maize rough dwarf virus (MRDV) isolate. The phylogenetic analysis indicated that the Chinese isolates that infect rice, maize, wheat and sorghum and cause similar symptoms could represent the same virus species, RBSDV.
Asunto(s)
Grano Comestible/virología , Enfermedades de las Plantas/virología , ARN Bicatenario/análisis , Reoviridae/aislamiento & purificación , Secuencia de Aminoácidos , China , Grano Comestible/clasificación , Grano Comestible/crecimiento & desarrollo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , ARN Bicatenario/genética , ARN Viral/análisis , ARN Viral/genética , Reoviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
Several peptides that could bind specifically to the outer coat protein encoded by the S10 gene of Rice black streaked virus (RBSDV) were isolated from a phage-display random 12-mer peptide library. The sequence analysis showed that the amino acid motif (K)K**(*)P, the asterisk denoting any amino acid, might be the core sequence by which the peptides bind to the target protein. The peptide 1 that had a high affinity to RBSDV outer coat protein was synthesized by a chemical method and its fusion protein with glutathione-S-transferase (GST) was produced in an Escherichia coli expression system. The dot and Western blot analyses indicated that RBSDV could be detected with a high sensitivity in crude extracts of diseased plant leaves using a purified GST fusion protein. The circular dichroism (CD) spectroscopy revealed that the synthesized binding peptide but not a nonbinding peptide could bring about a marked change in the conformation of outer coat RBSDV protein. Since the protein functions only when it has correct conformation, the peptides binding specifically to it could possibly disturb the function of the virus outer coat protein and might be used to block the transmission pathway of the virus. Summing up, as these peptides showed a high specificity and sensitivity and diagnostic potential for RBSDV, they may represent the basis of a novel strategy for development of resistance to RBSDV.
Asunto(s)
Bacteriófagos/genética , Proteínas de la Cápside/metabolismo , Biblioteca de Péptidos , Virus de Plantas/genética , Reoviridae/genética , Fusión Artificial Génica , Secuencia de Bases , Western Blotting , Proteínas de la Cápside/aislamiento & purificación , Expresión Génica , Glutatión Transferasa/metabolismo , Datos de Secuencia Molecular , Oryza/virología , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Virus de Plantas/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Reoviridae/metabolismo , Sensibilidad y EspecificidadRESUMEN
Phages with high affinity to the P20 protein of rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of phage display screening. Nine different peptides from the enriched library were selected by enzyme-linked immunosorbent assay (ELISA). The P20 protein from raw extracts of rice leaves infected with RSV could be detected by those 9 peptides displayed on the phage, which suggested that a peptide could be an effective tool for diagnosis of RSV in rice and planthopper. Circular dichroism (CD) spectra of P20 fusion proteins with the binding phages and non-binding phages showed that the conformation of P20 protein was changed after binding to each of the 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of the P20 protein. Thereafter, those peptides might be used to develop plant resistance and disrupt virus transmission. Three of the 12-mer peptide genes were fused with the glutathione-S-transferase (GST) gene in the vector pGEX 3X. The fusion proteins were obtained from an Escherichia coli expression system and purified. The fusion proteins might have a potential to develop a plant peptide-based resistance to its pathogens and virus diagnosis. It also provided a tool (i) to confirm the inhibition of the function of P20 protein by the fusion peptides in vivo, and (ii) to detect the function of P20 protein and the interaction between the virus and its vector.
Asunto(s)
Tenuivirus/genética , Proteínas Virales/química , Electroforesis en Gel de Poliacrilamida , Biblioteca de Péptidos , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Tenuivirus/químicaRESUMEN
The NS3 and NC protein genes encoded by RNA3 of RStV, the NCP and NSvc4 protein genes encoded by RNA4 were subcloned into the E. coli expression vector pGEX3X to express four groups of fusion protein under IPTG induction. These fusion proteins were used to immunize rabbits to raise antisera. The antisera against the E. coli-expressed proteins were available for probing the presence of the viral gene products in both rice plant and insect hosts. The expected gene products can be probed only in diseased rice plant with NCP antiserum and the corresponding products detected in both plant and RStV particle preparation with NC antiserum. The viral gene products probed by NS3 and NSvc4 antisera were different from the expected ones in size.
Asunto(s)
Oryza/virología , Virus de Plantas/química , Proteínas Recombinantes de Fusión/análisis , Proteínas Virales/análisis , Animales , Western Blotting , Escherichia coli/genética , Insectos , ConejosRESUMEN
The anterior pituitary (AP) tissues were removed from adult male Sprague-Dawley rats by decapitation. The dispersed cells with high viability (greater than or equal to 95%) were prepared using trypsin digestion and mechanical dissociation, and then mixed with Bio-Gel P2, packed into the columns, and perfused continuously with M199 medium for more than 24 hours. Different doses of LHRH were administered by 6-min pulses at one hour intervals. A steady and detectable basal LH secretion was present in all columns during the experiment. LHRH stimulation pulse could induce LH secretion rapidly, and repeated LHRH pulses of same dose produced statistically equal LH pulses. The dose-response curve of LH secretion was linear within the range of 1 X 10(-10) to 1 X 10(-7) mol/L LHRH. These results indicate that continuous perfusion system of dispersed AP cells which offers significant advantages over other methodologies provides a very useful in vitro model for studying the mechanisms on LHRH regulation of LH secretion.
Asunto(s)
Hormona Liberadora de Gonadotropina/fisiología , Hormona Luteinizante/metabolismo , Adenohipófisis/citología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hormona Liberadora de Gonadotropina/farmacología , Cinética , Masculino , Perfusión , Radioinmunoensayo , Ratas , Ratas EndogámicasRESUMEN
In the present study, a perfusion system of dispersed cells was used to investigate the effects of LHRH pulse amplitude and frequency, and LHRH continuous stimulation on LH secretion by anterior pituitary cells of adult male rats. The results have shown that, in the range of LHRH concentrations from 1 X 10(-10) to 1 X 10(-6) mol/L, the dose-response curve of LH secretion was linear. LHRH pulse frequency generated a biphasic LH response: increasing LHRH pulse frequency increased the basal LH secretion and decreased LH/pulse. When 1 X 10(-9) mol/L or greater LHRH was given at frequencies of 3 pulses/h or higher, it was observed that a maximal LH peak was induced and then the LH release declined progressively to its LH basal level, i.e. LHRH self-priming effect and LH desensitization occurred. Enhancement of amplitude of LHRH pulses could reduce pulse frequency required for priming. Increases in frequency of LHRH pulses with high amplitude would provoke the priming effect more quickly. In addition, continuous perfusion of LHRH with different concentrations could also elicit the LHRH self-priming effect and lH desensitization. LHRH with low concentration (1 X 10(-10) mol/L) would take much longer to evoke a self-priming effect. These results indicate that the LH secretion pattern is dependent on LHRH pulsatile amplitude and frequency, and will help to clarify the kinetics mechanisms by which LH pulses fluctuate in vivo.