GLUT1-mediated effective anti-miRNA21 pompon for cancer therapy

Oncogenic microRNAs are essential components in regulating the gene expression of cancer cells. Especially miR21, which is a major player involved of tumor initiation, progression, invasion and metastasis in several cancers. The delivery of anti-miR21 sequences has significant potential for cancer treatment. Nevertheless, since anti-miR21 sequences are extremely unstable and they need to obtain certain concentration to function, it is intensely difficult to build an effective delivery system for them. The purpose of this work is to construct a self-assembled glutathione (GSH)-responsive system with tumor accumulation capacity for effective anti-miR21 delivery and cancer therapy. A novel drug delivery nanosphere carrying millions of anti-miR21 sequences was developed through the rolling circle transcription (RCT) method. GSH-responsive cationic polymer polyethyleneimine (pOEI) was synthesized to protect the nanosphere from degradation by Dicer or other RNase in normal cells and optimize the pompon-like nanoparticle to suitable size. Dehydroascorbic acid (DHA), a targeting molecule, which is a substrate of glucose transporter 1 (GLUT 1) and highly expressed on malignant tumor cells, was connected to pOEI through PEG, and then the polymer was used for contracting a RNA nanospheres into nanopompons. The anti-miR21 nanopompons showed its potential for effective cancer therapy.

HPLC-UV method for the simultaneous determinations of ascorbic acid and dehydroascorbic acid in human plasma

A high performance liquid chromatography (HPLC) paired with UV-vis detection method to determine ascorbic acid and its oxidation product, dehydroascorbic acid, in human plasma was developed. Ascorbic acid in human plasma was extracted and stabilized using 10% metaphosphoric acid, and was analyzed by a Symmetry C18 column with 5 mM Hexadecyltrimethylammonium bromide and 50 mM KH2PO4 solution as the mobile phase (1.0 mL/min flow rate). Isoascorbic acid served as the internal standard and ultraviolet detector wavelength was 254 nm and 265 nm. Dehydroascorbic acid concentration was calculated from the differences in ascorbic acid concentration before and after reduction by dithiothreitol reagent. Quantification for ascorbic acid in human plasma was linear from 1–100 µg/mL. The inter- and intra-day precisions and accuracy were determined and the results were found to be within ±15%. This method was successfully applied to a human pharmacokinetic study of ascorbic acid as well as dehydroascorbic acid after oral administration of 4,000 mg vitamin C tablets to healthy Korean volunteers.

Dehydroascorbic Acid Attenuates Ischemic Brain Edema and Neurotoxicity in Cerebral Ischemia: An in vivo Study

Ischemic stroke results in the diverse phathophysiologies including blood brain barrier (BBB) disruption, brain edema, neuronal cell death, and synaptic loss in brain. Vitamin C has known as the potent anti-oxidant having multiple functions in various organs, as well as in brain. Dehydroascorbic acid (DHA) as the oxidized form of ascorbic acid (AA) acts as a cellular protector against oxidative stress and easily enters into the brain compared to AA. To determine the role of DHA on edema formation, neuronal cell death, and synaptic dysfunction following cerebral ischemia, we investigated the infarct size of ischemic brain tissue and measured the expression of aquaporin 1 (AQP-1) as the water channel protein. We also examined the expression of claudin 5 for confirming the BBB breakdown, and the expression of bcl 2 associated X protein (Bax), caspase-3, inducible nitric oxide synthase (iNOS) for checking the effect of DHA on the neurotoxicity. Finally, we examined postsynaptic density protein-95 (PSD-95) expression to confirm the effect of DHA on synaptic dysfunction following ischemic stroke. Based on our findings, we propose that DHA might alleviate the pathogenesis of ischemic brain injury by attenuating edema, neuronal loss, and by improving synaptic connection.

Efecto de antioxidantes y señalizadores en plantas de papa (Solanum tuberosum L.) infectadas con Candidatus Liberibacter solanacearum bajo condiciones de invernadero / Effect of antioxidants and sings in potato plants (Solanum tuberosum L.) infected with Candidatus Liberibacter solanacearum under greenhouse conditions

En México, las pérdidas ocasionadas en el cultivo de la papa por el síndrome punta morada (PMP), son del orden del 30 al 95 %. Este síndrome ha sido asociado principalmente a la bacteria Candidatus Liberibacter solaneacearum, la cual, ocasiona reducción en el rendimiento y calidad de la cosecha ya que los tubérculos presentan pardeamiento interno, lo cual, no es deseable ni para el consumo en fresco ni para la industria. Con base en lo anterior, el objetivo de este trabajo fue evaluar la eficiencia de tres compuestos que actúan como antioxidantes y/o señalizadores para reducir el daño causado en la papa por Ca, L. solanacearum bajo condiciones de invernadero. Se utilizaron plantas producidas in vitro, infectadas con Ca. L. solaneacearum y sin infección, Los compuestos evaluados fueron: ácido dehidroascórbico (600 ppm), ácido ascórbico (600 ppm) y peróxido de hidrógeno (1 mM), y se aplicaron en plantas infectadas y no infectadas dos veces por semana. Los controles fueron plantas infectadas y no infectadas sin aplicación de antioxidantes. Para evitar el error experimental en la aplicación de los compuestos evaluados el diseño experimental utilizado fue el de bloques completos al azar. Se observó reducción en el daño ocasionado por Ca. L. solaneacearum y se obtuvieron diferencias significativas entre tratamientos en la producción de tubérculos. El mayor número de tubérculos en los tratamientos que incluyeron plantas no infectadas fue obtenido por la aplicación del peróxido de hidrógeno, superando en un 33 %, 48 % y 59 % a las plantas tratadas con ácido dehidroascórbico, con ácido ascórbico y a las plantas controles respectivamente. De igual manera, el mayor número de tubérculos en tratamientos que incluyeron plantas infectadas se obtuvo con la aplicación de peróxido de hidrógeno, superando en un 33 %, 17 % y 67 % a los tratamientos con ácido dehidroascórbico, con ácido ascórbico y a plantas control respectivamente. Estos resultados demuestran un efecto potencial de los compuestos evaluados en la tolerancia de las plantas de papa contra Ca. L. solanacearum, aunque su función no es disminuir el crecimiento o desarrollo de la bacteria.

In Mexico, the losses caused by the purple top syndrome in potato range from 30 to 95%. This syndrome has been greatly associated with the bacterium Candidatus Liberibacter solaneacearum, which produces reduction in yield and in crop quality, the tubers have internal browning, which is not desirable either for fresh consumption or for the industry. Present study was conducted to evaluate the efficiency of three products that act as antioxidants and / or signs to reduce damage on potato caused by Ca. L. solanacearum under greenhouse conditions. Plants uninfected and infected with the bacterium, produced in vitro were used. The products evaluated were: dehydroascorbic acid (600 ppm), ascorbic acid (600 ppm) and hydrogen peroxide (1 mM), and were applied to infected and uninfected plants twice a week. Infected and uninfected plants without application of antioxidants were used as control treatments. To avoid experimental error in the application of the products evaluated the experimental design was a randomized complete block. A reduction of potato damage by Ca. L solaneacearum was registered, and there were significant differences among treatments in the tubers production. The largest tuber number in treatments that included uninfected plants was obtained in the hydrogen peroxide application, which exceeded a 33 %, 48 % and 59 % to plants treated with dehydroascorbic acid, with ascorbic acid and the controls respectively. Similarly, the largest tuber number in treatments included infected plants was obtained by hydrogen peroxide, and it exceeded a 33%, 17 % and 67 % to treatments with dehydroascorbic acid, with ascorbic acid and the controls respectively. These results show a potential effect of the products evaluated to protect potato plants against Ca. L. solanacearum, although its function is not to diminish the growth or development of bacteria.

Vitamina C, carotenoides, fenólicos totais e atividade antioxidante de goiaba, manga e mamão procedentes da ceasa do estado de Minas Gerais / Vitamin C, carotenoids, phenolic compounds and antioxidant activity of guava, mango and papaya from ceasa of Minas Gerais state

Este estudo teve como objetivo analisar o conteúdo de compostos antioxidantes (ácido ascórbico - AA, ácido desidroascórbico - ADA, vitamina C total, licopeno, β-caroteno, β-criptoxantina e estimativa de compostos fenólicos) e avaliar a atividade antioxidante, em goiaba, manga e mamão. A análise de carotenoides e vitamina C foi realizada por Cromatografia Líquida de Alta Eficiência (CLAE). O teor de fenólicos totais foi determinado utilizando o reagente de Folin-Ciocalteu e leitura espectrofotométrica. A atividade antioxidante foi avaliada pelo Teste do 2,2-diphenil-2-picril-hidrazil (DPPH•) e do Poder Redutor. A Anova (α= 0,05) foi utilizada para a análise dos dados. Os teores dos constituintes antioxidantes diferiram entre as três frutas, mas a goiaba foi a fruta que apresentou teores mais elevados de compostos fenólicos, vitamina C total, ADA e licopeno, além dos maiores valores para atividade antioxidante. Foi constatada forte correlação entre os testes que avaliaram a atividade antioxidante e o teor de fenólicos totais, demonstrando serem estes os principais compostos antioxidantes a contribuírem para a atividade antioxidante das frutas analisadas, em ambos os testes. É importante incentivar a utilização das frutas avaliadas neste estudo, tanto em nível doméstico quanto em estabelecimentos de alimentação coletiva para aumentar o consumo de antioxidantes naturais pela população.

This study aimed to analyze the content of antioxidant compounds (ascorbic acid - AA, dehydroascorbic acid - DHA, total vitamin C, lycopene, β-carotene, β-cryptoxanthin and phenolic compounds) and to evaluate the antioxidant activity in guava, mango and papaya. The analysis of carotenoids and vitamin C was performed by high performance liquid chromatography (HPLC). The content of phenolic compounds was determined using the Folin-Ciocalteu reagent and spectrophotometric reading. Antioxidant activity was evaluated by testing the 2.2-diphenyl-2-picryl-hydrazil (DPPH•) and reducing power. ANOVA was used for data analysis (α= 0.05). The levels of antioxidant constituents differed among the three fruits; guava was the fruit that had the highest levels of phenolic compounds, total vitamin C, lycopene and DHA, and the highest values for antioxidant activity. There was a strong correlation between tests that evaluated antioxidant activity and the content of phenolic compounds, demonstrating that these are the main antioxidant compounds to contribute to antioxidant activity of fruits examined in both tests. It is important to encourage the use of fruits evaluated in this study, both at home and in food service establishments, in order to increase the intake of natural antioxidants by the population.

Espinaca fresca, supercongelada y en conserva: contenido de vitamina C pre y post cocción / Fresh, frozen and canned spinach: vitamin C content before and after cooking

Vitamin C stability, new variables of cultivation, management and post harvest conservation of vegetables and culinary treatments before consumption can produce modifications in the content of this vitamin. To obtain actualized information of vitamin C content in fresh and processed food, this nutrient was determined in spinach in the fresh state and after subjected to current methods of conservation. Vitamin C determination by HPLC included ascorbic and dehydroascorbic acid. Mean values of vitamin C (mean +SD) in spinach were: fresh, 44.0 +/- 8,74 mg/100g; quick frozen, 24,2+14.81 mg/100g; and canned, 25.0 +/-1.9 mg/100g. This variability depends of genotypic factors and conservation conditions. Microwave and steam cooking generated the most retention of the nutrient, while boiling generated great losses of both vitamin forms.

La labilidad de la vitamina C, las nuevas variables de cultivo, manejo y conservación postcosecha de los vegetales y los tratamientos culinarios previos a su consumo pueden provocar modificaciones en el contenido de este nutriente. Para contar con información actualizada y propia del contenido de vitamina C en alimentos frescos y procesados, se determinó este nutriente en espinacas frescas y sometidas a los métodos de conservación actuales considerando también, la influencia de los sitios de expendio y la aplicación de los tratamientos culinarios más comunes. La determinación de vitamina C, constituida por ácido ascórbico y dehidroascórbico, se realizó empleando HPLC. Los valores medios de vitamina en espinacas sin procesar y su desvío estándar son 44.0 y 8,74 mg/100g, en productos supercongelados 24,2 y 14,81 mg/100g y en conservas de 25.0 y 1.9 mg/100g. Esta variabilidad depende de factores genotípicos y condiciones de conservación. La cocción por microondas y vapor generaron las mayores retenciones del nutriente, mientras que el hervor provocó grandes pérdidas de ambas formas vitamínicas.

Effects of Ascorbic and Dehydroascorbic Acids on Apoptotic Cell Death in Hippocampal Slice Culture

Ascorbic acid (AA) and dehydroascorbic acid (DHA) are known to have protective effects in experimental central nerve system disorder models such as stroke, ischemia, and epileptic seizures. The present study was conducted to examine the protective effect of AA and DHA on kainic acid (KA) neurotoxicity using organotypic hippocampal slice cultures (OHSC). Protective effects of AA and DHA on KA-induced cell death were evaluated by analyzing caspase-3. In addition, to determine if the prooxidant effect of AA is related to iron, the effect of AA on cell death was examined using desferrioxamine (DFO), an iron chelator. After 12h-KA treatment, significant delayed neuronal death was detected in CA3 region, but not in CA1. The AA (500 micrometer) and DHA (100 and 500 micrometer) pretreatments significantly prevented cell death by inhibiting caspase-3 activation in CA3 region. In the concentration of 1,000 micrometer, however, AA pretreatment might have prooxidant effect, but AA-induced oxidative reaction is mainly not related to transition metal ions. These data showed that the pretreatments of intermediate-dose AA and DHA protected KA-induced neuronal damage in OHSCs and co-pretreatment of AA and DFO did not affect cell death except for a few cases. These data suggest that both AA and DHA pretreatment have antioxidant or prooxidant effect depending on doses treated on KA-induced neuronal injury and the possible prooxidant effect of AA may not depend on the Fenton reaction.

Effect of dehydroascorbate on reactive oxygen species in mesangial cell induced by high glucose / 中华肾脏病杂志

Objective To investigate the effect of dehydroascorbate on reactive oxygen species (ROS) in mesangial cell induced by high glucose. Methods Mesangial cells were cultured in RPMI-1640 medium containing 10% newborn calf serum. Intracellular AA and DHA contents were measured with vitamin C assay system. The intracellular formation of ROS was detected with the fluorescent probe CM-H2DCFDA by using confocal microscopy. Activity of AP-1 was detected by EMSA. Results AA entry into cells was not significantly different from background noise. At a DHA concentration of 1 mmol/L, increasing concentrations of glucose competitively inhibited DHA entry into the cells such that the accumulation of DHA was smaller than half maximal at about 22 mmol/L glucose. Cytochalasin B,a kind of hexose transporter inhibitor,inhibited DHA entry into the cells. At a glucose concentration of 25 mmol/L, DHA inhibited intracellular ROS formation in a dose-dependent manner when DHA level was smaller than 4 mmol/L. In addition, the inhibitory effect of DHA on ROS generation was accompanied by lowering AP-1 activity in mesangial cell incubated by high glucose. Conclusions Mesangial cells are DHA dependent. VitC exclusion from mesangial cells through competition of glucose and DHA for common transport mechanism will deprive the cells of the central antioxidant and can lead to ROS accumulation. Proper doses of DHA will protect mesangial cell from injury of high glucose by inhibition on ROS formation and AP-1 activation.

Rapid uptake of oxidized ascorbate induces loss of cellular glutathione and oxidative stress in liver slices

The observation that ascorbate known to retain pro-oxidant properties induces cell death in a number of immortal cell lines, led us to examine its mechanism and whether it is involved in oxidative stress injury in such asocorbate-enriched tissue cells as hepatocytes. In rat liver homogenates, higher concentrations (1 and 3 mM) of ascorbate suppressed lipid peroxide productions but lower concentrations (0.1 and 0.3 mM) did not. In contrast to the homogenate, ascorbate increased lipid peroxide production in liver slices in a concentration dependant manner. Iso-ascorbate, the epimer of ascorbate did not cause an increase the oxidative stress in liver slices. This differential effect between homogenates and liver slices implies that cellular integrity is required for ascorbate to induce oxidative stress. Wortmannin, an inhibitor of the GLUT (glucose transporter) thought to transport dehydroascorbate into cells, inhibited [14C]- ascorbate uptake and suppressed oxidative stress in liver slices. Wortmannin suppressed that [14C]- ascorbate uptake by GLUT following oxidation to [14C]dehydroascorbate. Taken together, these observations support our hypothesis that ascorbate is oxidized to dehydroascorbate by molecular oxygen in solution (i.e., plasma and culture medium) which is then carried into hepatocytes (via a GLUT) where it is reduced back to ascorbate causing oxidative stress.