Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)

The MNK (Menkes disease protein; ATP7A) is a major copper- transporting P-type ATPase involved in the delivery of copper to cuproenzymes in the secretory pathway and the efflux of excess copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in Menkes disease, a fatal neurodegenerative copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic detergent DDM (n-dodecyl b-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 µM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6 µM; h=5.0). The purified MNK demonstrated active ATP-dependent vectorial 64Cu transport when reconstituted into soya-bean asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian copper-transporting P-type ATPase associated with a human disease.

The relationship betwen NaCl concentration and taste perception of saltiness in bread

Background – Sodium (Na+ ) is present in food in the form of sodium chloride (NaCl). There is strong evidence that high dietary Na+ intakes increase the risk of developing various adverse health conditions. Many international organisations encourage Na+ reduction in both the diet and the food supply. One of the major dietary sources of NaCl is bread, where NaCl has the essential function of imparting flavour. At present, no literature has been published examining taste interactions that may play a role in limiting the maximum saltiness perception in bread.
Objective – To determine the extent the physical structure of bread inhibits salty taste perception. Additionally, to determine whether common commercial bread additives suppress saltiness of bread.
Design – Subjects (n=14, 12 females) tasted and rated samples with varying NaCl concentrations in water (0 – 1724 mg NaCl/100 g) and bread (125 – 1550 mg NaCl/100 g) using the general Labelled Magnitude Scale. Psychophysical curves plotting NaCl concentration against NaCl intensity were constructed for water and bread. Breads of fixed NaCl concentration (1125 mg NaCl/100 g) and various common additives (sucrose, soya flour, canola oil, gluten) were also rated to assess perceived saltiness.
Outcomes – There was a significant difference between Na+ psychophysical curves in water and bread (P<0.05) with the bread matrix suppressing maximum possible saltiness by 25% to 70%. Suppression of saltiness was observed after the addition of sucrose (55% decrease) or soya flour (60% decrease) during bread production compared to prototypical bread (both P<0.05).
Conclusions – The physical structure of bread and some common additives have a major influence on perceptual saltiness of bread. The removal of additives that suppress saltiness combined with strategies to modify the texture of bread could lead to significant reduction in dietary Na+, whilst maintaining optimal salty taste.

Effect of diet, sex and age on fatty acid metabolism in broiler chickens : SFA and MUFA

The present study was conducted to evaluate the effect of different dietary lipid sources, age and sex on the SFA and MUFA metabolism in broiler chickens using a whole body fatty acid balance method. Four dietary lipid sources (palm fat, Palm; soyabean oil, Soya; linseed oil, Lin; and fish oil, Fish) were added at 3% to a basal diet containing 5% Palm. Diets were fed to female and male chickens from day 1 to either day 21 or day 42 of age. The accumulation (percentage of net intake and ex novo production) of SFA and MUFA was significantly lower in broilers fed on Palm than in broilers fed on the other diets (85·7 v. 97·4 %). Conversely, β-oxidation was significantly higher in Palm-fed birds than the average of the other dietary treatments (14·3 v. 2·6 %). On average, 33·1% of total SFA and MUFA accumulated in the body were elongated, and 13·8% were Δ-9 desaturated to longer chain or more unsaturated metabolites, with lower proportions being elongated and desaturated for the Palm and Fish diets than for the Soya and Lin diets. Total in vivo apparent elongase activity decreased exponentially in relation to the net intake of SFA and MUFA, while it increased with age. Total in vivo apparent Δ-9 desaturase activity was not significantly affected by dietary treatment or age. Total ex novo production and β-oxidation of SFA and MUFA showed a negative and positive curvilinear relationship with net intake of SFA and MUFA, respectively. Sex had no effect on SFA and MUFA metabolism.

Effect of diet, sex and age on fatty acid metabolism in broiler chickens : n-3 and n-6 PUFA

The PUFA metabolism in broiler chicken was studied through the whole body fatty acid balance method. Four dietary lipid sources (palm fat, Palm; soyabean oil, Soya; linseed oil, Lin; fish oil, Fish) were added at 3% to a basal diet containing 5% palm fat. Diets were fed to female and male birds from day 1 to either day 21 or day 42 of age. Birds fed the Lin diet showed a significantly higher 18 : 2n-6 accumulation compared with the other diets (85·2 v. 73·6% of net intake), whereas diet did not affect 18 : 3n-3 accumulation (mean 63% of net intake). Bioconversion of 18 : 2n-6 significantly decreased in the order Palm.> Lin > Soya > Fish (4·7, 3·9, 3·4 and 1% of net intake, respectively). The 18 : 3n-3 bioconversion on the Palm and Soya diets was similar and significantly higher than in broilers on the Lin diet (9·1 v. 5·8% of net intake). The β-oxidation of 18 : 2n-6 was significantly lower on the Lin diet than on the other diets (10·8 v. 23·3% of net intake), whereasβ-oxidation of 18 : 3n-3 was significantly higher on the Fish diet than on the other diets (41·5 v. 27·3% of net intake). Feeding fish oil suppressed apparent elongase and desaturase activity, whereas a higher dietary supply of 18 : 3n-3 and 18 : 2n-6 enhanced apparent elongation and desaturation activity on the PUFA involved in the n-3 and n-6 pathway, respectively. Accumulation of 18 : 2n-6 and 18 : 3n-3 increased andβ -oxidation decreased with age. Sex had a marginal effect on the PUFA metabolism.

Feeding of Spodoptera eridania (Lepidoptera: Noctuidae) on soybean genotypes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of a probiotic soy product and physical exercise on formation of pre-neoplastic lesions in rat colons in a short-term model of carcinogenic

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Piroxicam encapsulated in liposomes: Characterization and in vivo evaluation of topical anti-inflammatory effect

Liposomes of soya phosphatidylcholine, cholesterol, and stearylamine (molar ratio 6/3/1) and 0.1% alpha-tocopherol were prepared by the extrusion of multilamellar vesicles through 0.2-mu m polycarbonate membrane. Liposomes were characterized by electron transmission microscopy, and the mean structure diameter was 278 nm. The encapsulation efficiency obtained was 12.73%. The topical anti-inflammatory effect was evaluated in vivo by the cotton pellet granuloma method. We analyzed free piroxicam at 4 mg/kg, piroxicam encapsulated in liposomes added to 1.5% hydroxyethylcellulose (HEC) gel at 1.6 mg/kg, and piroxicam encapsulated in liposomes added to HEC gel at 4 mg/kg; the inhibition of inflammation obtained was 21.1%, 32.8%, and 47.4%, respectively. These results showed that the encapsulation of piroxicam produced an increase of topical anti-inflammatory effect, suggesting that the inhibition of inflammation can be obtained with lower drug concentrations.

Study of the diclofenac/phospholipid interactions with liposomes and monolayers

The interaction of diclofenae sodium (SD) with soya phosphatidylcholine (SPC) has been studied with floating Langmuir monolayers and liposomes. SD was either introduced into the subphase of SPC monolayers or co-spread with SPC on an aqueous subphase. In both cases, SD caused the surface pressure isotherm to become more expanded, thus demonstrating the affinity between SD and SPC. The incorporation of SD caused SPC liposomes to have a decreased diameter according to light scattering experiments. When SPC liposomes were injected into an aqueous subphase, their destruction yielding surface-active monomers could be monitored by changes in surface pressure. SD-loaded liposomes displayed a much faster kinetics when the surface density of surface-active monomers was plotted against time, with rate constants increasing significantly with the SD concentration. The kinetic profile can be quantitatively analyzed by plotting In[1 - (Gamma/Gamma(infinity))] versus t(1/2) (C) 2004 Elsevier B.V. All rights reserved.

Structure and viscoelastic behavior of pharmaceutical biocompatible anionic microemulsions containing the antitumoral drug compound doxorubicin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural Properties Induced by the Composition of Biocompatible Phospholipid-Based Microemulsion and Amphotericin B Association

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dieta hiperlipídica com farinha de soja como fonte proteica: utilização na seleção de ratos propensos e resistentes à obesidade

OBJETIVO: Desenvolver uma dieta hiperlipídica de baixo custo, tendo farinha de soja como fonte proteica, que seja eficiente na seleção de ratos propensos e resistentes à obesidade e que permita alcançar fenótipo obeso nos animais propensos. Além desses requisitos, a dieta deve ser palatável e não rejeitada a curto prazo pelo animal. MÉTODOS: A dieta proposta foi obtida misturando-se leite condensado (15,5%), amendoim (18,5%), farinha de soja (20,0%), óleo de milho (6,0%), ração Bio Tec (30,0%) e bolacha wafer de chocolate (10,0%). A mistura foi peletizada e submetida à análise bromatológica. A dieta foi ofertada a ratos Wistar durante uma semana; posteriormente, os animais foram divididos em três grupos, de acordo com o ganho de peso. O terço superior foi considerado propenso à obesidade e o terço inferior, resistente à obesidade. Após 80 dias de oferta da dieta, os animais foram sacrificados e foram quantificados o peso corpóreo, consumo alimentar, gorduras retroperitoneal, periepididimal, de carcaça e gorduras totais. RESULTADOS: Verificou-se que a dieta apresentava 5,31kcal/g, com a seguinte composição: 22,3% de gordura, 22,2% de proteína, 15,9% de fibra, estimando-se 35,7% de carboidrato. Ratos propensos à obesidade, alimentados por 87 dias com a dieta hipercalórica, apresentaram peso corpóreo, gorduras retroperitoneal, periepididimal e totais significativamente maiores do que animais resistentes à obesidade (p<0,05). O consumo de alimentos também foi maior em animais propensos (p<0,05). Verificou-se também que a substituição da caseína pela farinha de soja, como componente proteico da ração, levou à diminuição de 96,0% no custo do estudo. CONCLUSÃO: A dieta formulada com farinha de soja apresentou custo reduzido e foi capaz de desenvolver o fenótipo obeso em ratos propensos, à semelhança do observado na literatura com outras dietas.

Oxidative Stability and alpha-Tocopherol Retention in Soybean Oil with Lemon Seed Extract (Citrus Limon) under Thermoxidation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Relationship between structural features and in vitro release of doxorubicin from biocompatible anionic microemulsion

In this work structural features of anionic microemulsions, containing the pharmaceutical biocompatible components soya phosphatidylcholine (SPC), eumulgin HRE 40 (EU) and sodium oleate (SO) as surfactant, cholesterol (CHO) as oil phase and aqueous buffer were studied. Microemulsions were formulated with and without the antitumor drug doxorubicin (DOX). The various microstructures characterized in the pseudo-temary phase diagram were analyzed by polarized light microscopy, small-angle X-ray scattering (SAXS) and X-ray diffraction (XRD) as well as by their ability to incorporate and release DOX. The experimental results demonstrated a correlation between the composition, the structural features and drug delivery. It was found that at higher cholesterol contents, the crystallization of CHO polymorph phases changed the mobility of DOX molecules. Droplets were formed with short-range spatial correlation from a microemulsion (ME) with a low surfactant:oil ratio. More ordered structures with lamellar arrangements formed by the increasing of the CHO proportions in the formulation may be due to CHO crystallization. The in vitro release of DOX showed that the presence of a high content of crystalline CHO prolongs the release of DOX from ME. The retention of DOX in the internal oil phase of the ME may modulate the drug release for a prolonged time. These results clearly demonstrate the potential of ME as a drug-delivery system. (c) 2007 Elsevier B.V. All rights reserved.

Oil-in-water lecithin-based microemulsions as a potential delivery system for amphotericin B

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Desenvolvimento de snacks extrusados a partir de misturas de farinha de soja, fécula e farelo de mandioca

Neste trabalho, objetivou-se avaliar o efeito das condições de extrusão sobre as propriedades físicas de produtos extrusados. Foi utilizado o delineamento central composto rotacional com quatro variáveis independentes (2(4)) e a metodologia de superfície de resposta para avaliar os resultados de índice de expansão, volume específico, índice de absorção de água, índice de solubilidade em água e cor, de acordo com as variações de temperatura de extrusão, rotação da rosca, porcentagem de farelo de mandioca e porcentagem de farinha de soja. Os resultados indicam que é possível produzir novos produtos extrusados com boas propriedades físicas a partir de misturas de farinha de soja, fécula e farelo de mandioca.