Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Physiological implications of the regulation of vacuolar H+-ATPase by chloride ions

Vacuolar H+-ATPase is a large multi-subunit protein that mediates ATP-driven vectorial H+ transport across the membranes. It is widely distributed and present in virtually all eukaryotic cells in intracellular membranes or in the plasma membrane of specialized cells. In subcellular organelles, ATPase is responsible for the acidification of the vesicular interior, which requires an intraorganellar acidic pH to maintain optimal enzyme activity. Control of vacuolar H+-ATPase depends on the potential difference across the membrane in which the proton ATPase is inserted. Since the transport performed by H+-ATPase is electrogenic, translocation of H+-ions across the membranes by the pump creates a lumen-positive voltage in the absence of a neutralizing current, generating an electrochemical potential gradient that limits the activity of H+-ATPase. In many intracellular organelles and cell plasma membranes, this potential difference established by the ATPase gradient is normally dissipated by a parallel and passive Cl- movement, which provides an electric shunt compensating for the positive charge transferred by the pump. The underlying mechanisms for the differences in the requirement for chloride by different tissues have not yet been adequately identified, and there is still some controversy as to the molecular identity of the associated Cl--conducting proteins. Several candidates have been identified: the ClC family members, which may or may not mediate nCl-/H+ exchange, and the cystic fibrosis transmembrane conductance regulator. In this review, we discuss some tissues where the association between H+-ATPase and chloride channels has been demonstrated and plays a relevant physiologic role.

Participation of the NO/cGMP/K+ATP pathway in the antinociception induced by Walker tumor bearing in rats

Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K+ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K+ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E2- (PGE2, 400 ng/paw; 50 µL; intraplantar injection) and carrageenan-induced hypernociception (500 µg/paw; 100 µL; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE2) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE2) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE2; 8 µg/paw) and the ATP-sensitive K+ channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE2; 160 µg/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels.

Spectrum of K ras mutations in Pakistani colorectal cancer patients

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

Estudo químico de alimentos formulados à base de palmito Bactris gasipaes H.B.K. (pupunha) desidratado

O coração da palmeira Bactris gasipaes H.B.K. (pupunha) foi utilizado, sob forma desidratada, na formulação de uma sopa-creme para merenda escolar. Análises químicas do palmito e do coração da palmeira (açúcares redutores, lipídeos, proteínas totais, aminograma, fibras, calorias e minerais) foram feitas tanto na forma in natura como na forma processada, bem como na de sopa-creme. Não foram encontradas diferenças relevantes em relação a essas determinações, podendo-se ressaltar a boa qualidade aminoacídica do coração da palmeira.

Características sensoriais de compotas de pêssego light elaboradas com sucralose e acesulfame-K

O trabalho objetivou realizar uma caracterização sensorial de compotas de pêssego com reduzidas calorias. As compotas foram elaboradas pela substituição parcial de açúcar pelos edulcorantes sucralose, acesulfame-K e sucralose+acesulfame-K. Além desta caracterização, investigou-se a vida-de-prateleira destes produtos durante um período de 90 dias. Para isto, foram avaliados os atributos de aparência geral, cor, brilho, maciez, sabor geral e acidez, logo após o processamento, aos 30, 60 e 90 dias de estocagem. Ao término do período de estocagem executou-se, ainda, um teste de preferência. De acordo com a avaliação sensorial dos diferentes atributos, detectou-se diferenças significativas entre as quatro formulações de compota, especialmente em relação aos atributos de sabor e acidez. As formulações com acesulfame-K e sucralose+acesulfame-K mostraram valores inferiores às demais. Durante o período de armazenamento não se constatou expressivas alterações nas características sensorias das compotas. Os resultados do teste de preferência mostraram que não houve distinção na preferência dos consumidores por qualquer das formulações.

Estabilidade de ácido ascórbico e antocianinas em néctar de camu-camu (Myrciaria dubia (H. B. K.) McVaugh)

O camu-camu é um fruto com notável potencial nutricional, pelo seu alto conteúdo em ácido ascórbico. Sendo assim, objetivou-se avaliar a estabilidade do ácido ascórbico e pigmentos presentes no néctar de camu-camu, armazenados sob diferentes condições de luminosidade e temperatura. Os frutos foram despolpados e avaliados quanto às características físico-químicas. O néctar, obtido a partir da polpa, foi acondicionado em garrafas PET e armazenado em temperatura ambiente e sob refrigeração na presença e ausência de luz, sendo avaliado por 120 dias quanto à estabilidade de ácido ascórbico e antocianinas. O teor de ácido ascórbico nos néctares armazenados sob luz não diferiu estatisticamente dos armazenados protegidos da luz (343,25 e 340,48 mg.100 g -1), respectivamente, nos armazenados sob refrigeração, e (330,48 e 333,56 mg.100 g -1) nos armazenados em temperatura ambiente. Constatou-se que esta vitamina em néctares armazenados por 120 dias em temperatura de refrigeração apresentou boa estabilidade, com perda de apenas 12 a 14%. Quanto às antocianinas, a temperatura ambiente contribuiu negativamente, ocasionando uma degradação mais acelerada, no entanto, a exposição à luz não teve efeito. Nestas condições experimentais, conclui-se que o fator luminosidade tem pouca influência sobre o ácido ascórbico e antocianinas no néctar de camu-camu, e que a temperatura ambiente de armazenamento é fator negativo na estabilidade destes pigmentos.

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Propriedades funcionais das proteínas de amêndoas da faveleira (Cnidosculus phyllacanthus (Mart.) Pax. et K. Hoffm.) com e sem espinhos

A faveleira possui amêndoas com potencial em óleos e proteínas alimentares e é encontrada em todos os estados do nordeste brasileiro, principalmente nas regiões do sertão e caatinga. Objetivando a sua aplicação em formulações alimentícias, as propriedades funcionais das proteínas de amêndoas da faveleira com espinhos (FCE), e sem espinhos (FSE) foram estudadas. As amêndoas apresentaram, elevado conteúdo de lipídio (40,56 e 40,21%) e proteína (33,00 e 35,77%) e depois de desengordurada, apresentou 57,55 e 63,00% respectivamente. Na obtenção dos isolados proteicos, os índices em proteínas extraídas foram de 83,51% para a FCE e 80,57% para a FSE em pH 10,5. Os índices de proteínas recuperadas através da precipitação isoelétrica foram de 72,84 e 72,11% em pH 4,5. Os índices de proteínas solúveis e as propriedades de emulsificação mostraram-se dependentes do pH, com valores mínimos no ponto isoelétrico e mais elevados em pH ácido e básico ao pI. O isolado proteico da FSE apresentou melhor capacidade de absorção de água e de óleo em relação à FCE. Quando avaliadas através da atividade e da estabilidade de emulsão os isolados apresentaram performance apropriada tanto na região ácida quanto alcalina do pI.

Association between aflatoxin and aflatoxigenic fungi in Brazil nut (Bertholletia excelsa H.B.K.)

Brazil nut has a high nutritional content and is a very important trade commodity to some Latin American countries. In order to evaluate its safety, 120 samples from different stages of the productive chain were analyzed in terms of: moisture content (mc), aflatoxigenic fungi and aflatoxin (LOQ = 1.95 μg.kg-1 total aflatoxin) using TLC. Among all samples, 4 (6.7%) from the receiving area, and 5 (16.7%), from retail presented aflatoxins above the LOQ, but the amount of aflatoxins was below the LOQ after the samples were dried in the plant. The positive samples were above the limit of total aflatoxin permitted by the European Union (4.0 μg.kg-1) and Brazil (30 μg.kg-1). The mc mean was 22.43% in the receiving area, which is higher than that in the other stages samples. All the A. flavus strains were aflatoxigenic, and there was statistic association between the presence of aflatoxin and flavus strains. The aflatoxigenic fungi strains associated to the aflatoxins levels in the samples show that an effective control is necessary for the food safety in the Brazil nut production chain.