Polyphenol-enriched azuki bean (Vina angularis) extract reduces the oxidative stress and prevents DNA oxidation in the hearts of streptozotocin-induced early diabetic rats.

We examined the changes in the heart of rats at the early stages of streptozotocin (STZ)-induced diabetes, and whether azuki bean extract (ABE) could influence these changes. The experimental diabetic rats received 0 or 40 mg/kg of ABE orally for 4 weeks, whereas the control group rats received distilled water. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) and expression of proteins associated with peroxisomal FA ß-oxidation as well as oxidative stress markers were examined. The levels of peroxisomal ACOX1 and catalase of the diabetic groups were significantly higher than those in the control group. The levels of p62, phosphorylated-p62 (p-p62) and HO-1 in the STZ group were significantly higher than those in the control group, and the levels of p-p62, HO-1, and 8-OHdG were significantly lower by ABE administration. The STZ-induced early diabetes increases the levels of proteins related to peroxisomal FA ß-oxidation and oxidative stress markers in hearts. ABE protects diabetic hearts from oxidative damage.

Efecto de dos antioxidantes en la supervivencia, las actividades neurológicas y la función mitocondrial de ratones senescentes / Effect of two antioxidants on survival, neurological activities and mitochondrial function in senescent mice

Objetivos: valorar la influencia de tratamientos crónicos con los antioxidantes vitamina E y tioprolina en: a) la supervivencia de ratones; b) la actividad neurológica de los animales envejecidos, y c) la disminución de actividades enzimáticas mitocondriales y el daño oxidativo mitocondrial asociados al proceso de envejecimiento. Material y método: los ratones recibieron desde la semana 28 de vida, y durante toda su vida, una suplementación en la dieta de vitamina E (5 g de acetato de dl-*-tocoferol/kg de comida) o de tioprolina (2 g de l-4-ácido tiazolidín carboxílico/kg de comida). Para evaluar la actividad neurológica los ratones se sometieron cada 2 semanas a 2 pruebas de comportamiento. En mitocondrias aisladas de cerebro se determinó el daño oxidativo, medido como proteínas o lípidos oxidados, así como por disminución de las actividades enzimáticas NADH-citocromo c reductasa, succinato-citocromo c reductasa, citocromo oxidasa y óxido nítrico sintasa mitocondrial (mtNOS). Resultados: la expectativa de vida de los ratones macho aumentó después de la suplementación con vitamina E en un 34-34% (vida media y longevidad máxima, respectivamente), y después de la suplementación con tioprolina en un 33-24%. La vitamina E y la tioprolina fueron efectivas en la disminución de los marcadores mitocondriales de daño oxidativo (TBARS y carbonilos proteínicos), y en el retardo de la disminución de las actividades enzimáticas y neurológicas asociadas al envejecimiento. Conclusiones: las actividades enzimáticas de mtNOS, NADH deshidrogenasa y citocromo oxidasa pueden usarse como indicadores de tratamientos efectivos del déficit neurológico asociado al envejecimiento

Aims: the aim of this study was to evaluate the influence of chronic treatments with the antioxidants vitamin E and thioproline on: 1) survival in mice; 2) the neurological activities of aged animals; and 3) the decreased mitochondrial enzymatic activities and oxidative damage associated with the ageing process. Material and method: mice received food supplemented with vitamin E (5 g dl-*-tocopherol acetate/kg of food) or with thioproline l-4-thioproline/kg (2 g l-4-thiazolidine carboxylic acid/kg of food) from 28 weeks of age and during their entire lifespan. To evaluate neurological activity the animals underwent two behavioural tests every 15 days from weeks 28 to 76 of age. Oxidative damage to isolated brain mitochondria was evaluated by determining protein and lipid oxidation products and mitochondrial enzyme activities: NADH-cytochrome c reductase, succinate-cytochrome c reductase, cytochrome oxidase, and mitochondrial nitric oxide synthase (mtNOS). Results: lifespan was increased in male mice by 34-34% (mean and maximal lifespan, respectively) after supplementation with 5 g vitamin E/kg food and by 33-24% (mean and maximal lifespan, respectively) after supplementation with 2 g thioproline/kg food. Vitamin E and thioproline were effective in decreasing the level of markers of oxidative damage (TBARS and protein carbonyls) in isolated mitochondria and in delaying the decreases in mitochondrial enzyme activities and the loss of neurological function associated with ageing. Conclusions: the activities of mtNOS, NADH dehydrogenase and cytochrome oxidase can be used as indicators of the effectiveness of treatments for age-dependent neurological impairment

Dual subcellular distribution of cytochrome b5 in plant, cauliflower, cells.

Subfractionation studies showed that cytochrome b(5) (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A-insensitive NADH-cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.

Evidence for an association of ndh B, ndh J gene products and ferredoxin-NADP-reductase as components of a chloroplastic NAD(P)H dehydrogenase complex.

Using non-denaturing gel electrophoresis and staining with nitro-blue tetrazolium, we reveal the presence of two NAD(P)H oxidoreductase activity bands within thylakoids membranes of Solanum tuberosum L. Second dimension SDS-PAGE and Western analysis show that one of the activity bands contains several polypeptides, two of them being recognized by antibodies directed against peptides corresponding to conserved domains of chloroplastic genes products NDH B and NDH J (at 32 and 18 kDa, respectively). Both activity bands also contain a polypeptide (around 36 kDa) recognized by an antibody directed against ferredoxin-NADP(+)-reductase (FNR). We conclude from these results that both chloroplastic ndh B and ndh J gene products are components of a thylakoid NAD(P)H dehydrogenase complex. The association with FNR is suggested to allow the complex to use NADPH instead of NADH as a preferential substrate.

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

Analysis of NADH dehydrogenases from plant [mung bean (Phaseolus aureus)] mitochondrial membranes on non-denaturing polyacrylamide gels and purification of complex I by band excision.

The present paper describes the analysis of plant mitochondrial NADH dehydrogenases using a recently developed non-dissociating gradient polyacrylamide-gel-electrophoresis technique [Kuonen, Roberts & Cottingham (1986) Anal. Biochem. 153, 221-226]. Solubilized mung-bean (Phaseolus aureus) submitochondrial particles were analysed on 3-22% (w/v) gradient polyacrylamide gels containing 0.1% Triton X-100 and stained for multiple NADH dehydrogenase activities. A rotenone-sensitive NADH dehydrogenase (Complex I) was identified on the basis of co-migration with the purified mammalian enzyme. The polypeptide composition of the plant enzyme was further analysed by band excision and SDS/polyacrylamide-gel electrophoresis.