Evaluation by blue native polyacrylamide electrophoresis colorimetric staining of the effects of physical exercise on the activities of mitochondrial complexes in rat muscle

Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 ± 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 ± 0.65, P < 0.05) and soleus (9.8 ± 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.

Angiotensin II stimulates MCP-1 production in rat glomerular endothelial cells via NAD(P)H oxidase-dependent nuclear factor-kappa B signaling

Angiotensin II (Ang II) plays a crucial role in the pathogenesis of renal diseases. The objective of the present study was to investigate the possible inflammatory effect of Ang II on glomerular endothelial cells and the underlying mechanism. We isolated and characterized primary cultures of rat glomerular endothelial cells (GECs) and observed that Ang II induced the synthesis of monocyte chemoattractant protein-1 (MCP-1) in GECs as demonstrated by Western blot. Ang II stimulation, at concentrations ranging from 0.1 to 10 µm, of rat GECs induced a rapid increase in the generation of reactive oxygen species as indicated by laser fluoroscopy. The level of p47phox protein, an NAD(P)H oxidase subunit, was also increased by Ang II treatment. These effects of Ang II on GECs were all reduced by diphenyleneiodonium (1.0 µm), an NAD(P)H oxidase inhibitor. Ang II stimulation also promoted the activation of nuclear factor-kappa B (NF-κB). Telmisartan (1.0 µm), an AT1 receptor blocker, blocked all the effects of Ang II on rat GECs. These data suggest that the inhibition of NAD(P)H oxidase-dependent NF-κB signaling reduces the increase in MCP-1 production by GECs induced by Ang II. This may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with chronic renal disease.

Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Caracterização imunoquímica da ACC (ácido 1-carboxílico-1-aminociclopropano) oxidase em frutos climatéricos

Com o objetivo de caracterizar, por via imunoquímica, a enzima ACC (ácido 1-carboxílico-1-aminociclopropano) oxidase em frutos climatéricos, foram preparados anticorpos policlonais específicos para esta proteína. Utilizou-se, como antígeno, uma proteína recombinante, produzida em Escherichia coli K38/pGP1,2, contendo o vetor de expressão pT7-7A4 no qual foi inserido um clone de DNA da ACC oxidase. A especificidade dos anticorpos foi demonstrada pela técnica de "Western blot", a partir de extratos protéicos de maçãs e tomates em diferentes estágios de maturação. Verificou-se que o aumento da produção de etileno, quando os frutos passaram do estágio pré-climatérico para o climatérico, está diretamente correlacionada com o aumento da síntese da ACC oxidase. Em estágios de maturação mais avançados houve uma redução da produção de etileno e da atividade ACC oxidase, mas esta proteína continuava presente. Quando o "Western blot" foi realizado com tomates transgênicos, onde a produção de etileno e a síntese da ACC oxidase foram inibidos em mais de 95%, nenhuma reação imunoquímica foi detectada. O conjunto de resultados obtidos indica que os anticorpos detectam especificamente ACC oxidase.

TEOR DE VITAMINA C, ATIVIDADE DE ASCORBATO OXIDASE E PERFIL SENSORIAL DE MANGA (Mangífera índica L.) VAR. HADEN, DURANTE O AMADURECIMENTO

A composição química e bioquímica da manga, varia de acordo com as condições da cultura, variedade e estágio de maturação, geralmente contendo alto conteúdo de ácido ascórbico. Com o objetivo de estabelecer o papel da ascorbato oxidase &#091;E.C.1.10.3.3&#093;, sobre os níveis de ácido ascórbico durante o processo de amadurecimento de manga (Mangífera índica L.) var. Haden, foram analisadas amostras da fruta correspondentes aos estágios verde maturo (zero) e armazenadas por 2, 4, 6, 8, 10, 12 e 14 dias a 20 ± 2oC. As amostras foram obtidas das polpas cortadas em pequenos cubos de aproximadamente 8 cm3 de 8 mangas com textura sem diferença significativa entre elas, medidas com auxílio de um penetrômetro Magness-Taylor. Em cada amostra foi determinada atividade de ascorbato oxidase para verificar sua participação em possíveis quedas de ácido ascórbico durante o amadurecimento das frutas. Também foram determinados periodicamente o teor de ácido ascórbico e o perfil sensorial durante o período de amadurecimento. A atividade enzimática foi determinada espectrofotometricamente a 245 nm 30oC, o ácido ascórbico foi analisado de acordo com a metodologia da AOAC modificada e a análise sensorial através de análise descritiva quantitativa. Os dados da análise sensorial foram analisados através de análise de variância (ANOVA), testes de médias de Tukey, análise de componentes principais e análise discriminante por passos. Durante o amadurecimento, a atividade da ascorbato oxidase aumentou e o teor de ácido ascórbico diminuiu, havendo significativa (p£0,05) correlação linear negativa (r=-0,98). Os termos descritores para a manga foram: sabor característico, aroma característico, acidez, adstringência, coloração amarela da polpa, doçura e suculência. O perfil sensorial apresentou significativa melhora com o amadurecimento. Todos os atributos sensoriais aumentaram significativamente (p£0,05) durante o amadurecimento das mangas, exceto acidez e adstringência.

Inibição da síntese da ACC (ácido 1-carboxílico-1-aminociclopropano) oxidase em maçãs frigoconservadas em atmosfera controlada

Foi estudada a expressão da ACC oxidase em maçãs, cv. Jonagold, colhidas no estádio pré-climatérico e armazenadas sob refrigeração em atmosfera normal (0ºC, 95% UR - AN) e controlada (0ºC, 95% UR, 1,5% O2 e 2,5% CO2 - AC), durante 180 dias. Na instalação do experimento, aos 90 e aos 120 dias, foram coletadas amostras para a determinação da firmeza de polpa, da acidez total titulável, dos sólidos solúveis totais, da produção de etileno, da atividade ACC oxidase e para a detecção imunoquímica das isoformas desta enzima. A dosagem da atividade ACC oxidase foi realizada por cromatografia gasosa a partir de extrato protéico solúvel acrescido de 250µM de ACC, 10µL de sulfato ferroso e 30µL de ascorbato de sódio. Para a detecção imunoquímica utilizou-se a técnica "western blot", com anticorpos policlonais anti-ACC oxidase de maçã, após separação das proteínas em eletroforese e isoeletrofocalização. Não foi detectada ACC oxidase em maçãs pré-climatéricas. Porém, após 120 horas em condições ambientais, houve a síntese dessa enzima e um incremento na produção de etileno. A refrigeração não exerceu controle na síntese da ACC oxidase e produção de etileno, resultando em significativas perdas físico-químicas nas frutas armazenadas em AN. Já a utilização de AC permitiu controlar a via de biossíntese do etileno, pela inibição da síntese da ACC oxidase, mantendo o material com boa qualidade para o consumo in natura. A adição de ACC e dos cofatores aumentou a atividade ACC oxidase e alterou o pI da ACC oxidase.

Effect of polyphenol oxidase (PPO) and air treatments on total phenol and tannin content of cocoa nibs

Cocoa flavour is greatly influenced by polyphenols. These compounds undergo a series of transformations during cocoa processing leading to the characteristic cocoa flavour. The use of exogenous polyphenol oxidase (PPO) proved to be useful to reduce polyphenol content in cocoa nibs. The effect of a PPO associated or not with air over total phenol and tannin content was evaluated. Cocoa nibs were autoclaved and treated with a PPO or water in the absence or presence of an air flow for 0.5, 1, 2 and 3 hours. Total phenol content was reduced in PPO or water treatments, but when associated with air there was an increase in phenol content. Tannin content was reduced only by the treatment with water and air.

Characterization of polyphenol oxidase in two cocoa (Theobroma cacao L.) cultivars produced in the south of Bahia, Brazil

Abstract The reactions leading to the formation of precursors of chocolate flavor are performed by endogenous enzymes present in the cocoa seed. Polyphenol oxidase (PPO) presence and activity during fermentation of cocoa beans is responsible for the development of flavor precursors and is also implicated in the reduction of bitterness and astringency. However, the reliability of cocoa enzyme activities is complicated due to variations in different genotypes, geographical origins and methods of fermentation. In addition, there is still a lack of systematic studies comparing different cocoa cultivars. So, the present study was designed to characterize the activity of PPO in the pulp and seeds of two cocoa cultivars, PH 16 and TSH 1188. The PPO activity was determined spectrophotometrically and characterized as the optimal substrate concentration, pH and temperature and the results were correlated with the conditions during the fermentation process. The results showed the specificity and differences between the two cocoa cultivars and between the pulp and seeds of each cultivar. It is suggested that specific criteria must be adopted for each cultivar, based on the optimal PPO parameters, to prolong the period of maximum PPO activity during fermentation, contributing to the improvement of the quality of cocoa beans.

Extraction optimization of polyphenols, antioxidant and xanthine oxidase inhibitory activities from Prunus salicina Lindl.

Abstract Optimization of polyphenols extraction from plum (Prunus salicina Lindl.) was evaluated using response surface methodology. The Box-Behnken experimental results showed the optimal conditions involved an extraction temperature of 59 °C, a sonication time of 47 min, and an ethanol concentration of 61% respectively. The maximum extraction yield of total polyphenols was 44.74 mg gallic acid equivalents per gram of dried plum at optimal conditions. Polyphenol extracts exhibited stronger antioxidant activities than Vc by evaluating of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activity. Furthermore, polyphenol extracts (IC50 = 179 g/mL) showed obvious inhibitory effects on xanthine oxidase. These findings suggest that polyphenol extracts from P. salicina can be potentially used as natural antioxidant and xanthine oxidase inhibitory agents.