Evidence for catabolic pathway of propionate metabolism in CNS: expression pattern of methylmalonyl-CoA mutase and propionyl-CoA carboxylase alpha-subunit in developing and adult rat brain.

Methylmalonyl-CoA mutase (MCM) and propionyl-CoA carboxylase (PCC) are the key enzymes of the catabolic pathway of propionate metabolism and are mainly expressed in liver, kidney and heart. Deficiency of these enzymes leads to two classical organic acidurias: methylmalonic and propionic aciduria. Patients with these diseases suffer from a whole spectrum of neurological manifestations that are limiting their quality of life. Current treatment does not seem to effectively prevent neurological deterioration and pathophysiological mechanisms are poorly understood. In this article we show evidence for the expression of the catabolic pathway of propionate metabolism in the developing and adult rat CNS. Both, MCM and PCC enzymes are co-expressed in neurons and found in all regions of the CNS. Disease-specific metabolites such as methylmalonate, propionyl-CoA and 2-methylcitrate could thus be formed autonomously in the CNS and contribute to the pathophysiological mechanisms of neurotoxicity. In rat embryos (E15.5 and E18.5), MCM and PCC show a much higher expression level in the entire CNS than in the liver, suggesting a different, but important function of this pathway during brain development.

Reticulocyte dynamic and hemoglobin variability in hemodialysis patients treated with Darbepoetin alfa and C.E.R.A.: a randomized controlled trial.

BACKGROUND: In a simulation based on a pharmacokinetic model we demonstrated that increasing the erythropoiesis stimulating agents (ESAs) half-life or shortening their administration interval decreases hemoglobin variability. The benefit of reducing the administration interval was however lessened by the variability induced by more frequent dosage adjustments. The purpose of this study was to analyze the reticulocyte and hemoglobin kinetics and variability under different ESAs and administration intervals in a collective of chronic hemodialysis patients. METHODS: The study was designed as an open-label, randomized, four-period cross-over investigation, including 30 patients under chronic hemodialysis at the regional hospital of Locarno (Switzerland) in February 2010 and lasting 2 years. Four subcutaneous treatment strategies (C.E.R.A. every 4 weeks Q4W and every 2 weeks Q2W, Darbepoetin alfa Q4W and Q2W) were compared with each other. The mean square successive difference of hemoglobin, reticulocyte count and ESAs dose was used to quantify variability. We distinguished a short- and a long-term variability based respectively on the weekly and monthly successive difference. RESULTS: No difference was found in the mean values of biological parameters (hemoglobin, reticulocytes, and ferritin) between the 4 strategies. ESAs type did not affect hemoglobin and reticulocyte variability, but C.E.R.A induced a more sustained reticulocytes response over time and increased the risk of hemoglobin overshooting (OR 2.7, p = 0.01). Shortening the administration interval lessened the amplitude of reticulocyte count fluctuations but resulted in more frequent ESAs dose adjustments and in amplified reticulocyte and hemoglobin variability. Q2W administration interval was however more favorable in terms of ESAs dose, allowing a 38% C.E.R.A. dose reduction, and no increase of Darbepoetin alfa. CONCLUSIONS: The reticulocyte dynamic was a more sensitive marker of time instability of the hemoglobin response under ESAs therapy. The ESAs administration interval had a greater impact on hemoglobin variability than the ESAs type. The more protracted reticulocyte response induced by C.E.R.A. could explain both, the observed higher risk of overshoot and the significant increase in efficacy when shortening its administration interval.Trial registrationClinicalTrials.gov NCT01666301.

Comportamiento de glicinina, beta-conglicinina y alfa-amilasa en semillas de soja deterioradas y no deterioradas

El objetivo del trabajo fue estudiar el comportamiento de glicinina y beta-conglicinina y la actividad de alfa-amilasa en semillas deterioradas y no deterioradas de 10 cultivares de soja [Glycine max (L.) Merr.]. Las semillas se sometieron a dos tratamientos: deterioradas por envejecimiento acelerado y no deterioradas. Se determinó la presencia de las proteínas de reserva a partir de semillas con 0, 3 y 8 días de germinadas por electroforesis en geles de poliacrilamida (SDS-PAGE). La actividad de la alfa-amilasa se determinó bioquímicamente en semillas con 0, 3, 8 y 12 días de germinadas. No hubo diferencias en la presencia de bandas de glicinina y beta-conglicinina en semillas no deterioradas hasta los 8 días de germinadas. Las semillas deterioradas se comportaron en forma similar a las no deterioradas. La actividad de la alfa-amilasa aumentó en semillas germinadas hasta 8 días y disminuyó a los 12 días. En semillas deterioradas la actividad enzimática disminuyó con respecto a las no deterioradas. El deterioro artificial no afectó la presencia de glicinina y beta-conglicinina pero alteró la actividad de la alfa-amilasa hasta los 12 días de germinación. Los cultivares estudiados mostraron comportamiento diferencial frente a la actividad de esta enzima.

Vitamin D deficiency and a CYP27B1-1260 promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy.

BACKGROUND & AIMS: Vitamin D is an important immune modulator and preliminary data indicated an association between vitamin D deficiency and sustained virologic response (SVR) rates in hepatitis C virus (HCV) genotype 1 patients. We, therefore, performed a comprehensive analysis on the impact of vitamin D serum levels and of genetic polymorphisms with functional relevance within the vitamin D cascade on chronic hepatitis C and its treatment. METHODS: Vitamin D serum levels, genetic polymorphisms within the vitamin D receptor and 1α-hydroxylase were determined in a cohort of 468 HCV genotype 1, 2, and 3 infected patients who were treated with interferon-alfa based regimens. RESULTS: Chronic hepatitis C was associated with a high incidence of severe vitamin D deficiency compared to controls (25(OH)D(3)<10 ng/ml in 25% versus 12%, p<0.00001). 25(OH)D(3) deficiency correlated with SVR in HCV genotype 2 and 3 patients (50% and 81% SVR for patients with and without severe vitamin D deficiency, respectively, p<0.0001). In addition, the CYP27B1-1260 promoter polymorphism rs10877012 had substantial impact on 1,25-dihydroxyvitamin D serum levels (72, 61, and 60 pmol/ml for rs10877012 AA, AC, and CC, respectively, p=0.04) and on SVR rates in HCV genotype 1, 2, and 3 infected patients (77% and 65% versus 42% for rs10877012 AA, AC, and CC, respectively, p=0.02). CONCLUSIONS: Chronic hepatitis C virus infection is associated with vitamin D deficiency. Reduced 25-hydroxyvitamin D levels and CYP27B1-1260 promoter polymorphism leading to reduced 1,25-dihydroxyvitamin D levels are associated with failure to achieve SVR in HCV genotype 1, 2, and 3 infected patients.

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis.

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.

Funcionalidade da oleoresina de páprica microencapsulada em goma-arábica e amido de arroz/gelatina

O objetivo deste trabalho foi avaliar a funcionalidade da oleoresina de páprica microencapsulada em goma-arábica e amido de arroz/gelatina, incorporada nos sistemas-modelo bolo e gel de gelatina. Avaliou-se a cor por meio de medida instrumental e aceitação sensorial por atributos de aparência e aceitação global. O pigmento encapsulado solubilizou-se, tingiu e se distribuiu uniformemente nos bolos, os quais apresentaram boa aceitação global. Nos géis, a goma apresentou desempenho superior ao amido/gelatina quanto à aparência, porém, todos os tratamentos obtiveram baixa aceitação global. A presença da oleoresina microencapsulada não interferiu negativamente no sabor, aroma ou textura dos sistemas analisados.

Surface-functionalized ultrasmall superparamagnetic nanoparticles as magnetic delivery vectors for camptothecin.

Drug-nanoparticle conjugates: The anticancer drug camptothecin (CPT) was covalently linked at the surface of ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) via a linker, allowing drug release by cellular esterases. Nanoparticles were hierarchically built to achieve magnetically-enhanced drug delivery to human cancer cells and antiproliferative activity.The linking of therapeutic drugs to ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) allowing intracellular release of the active drug via cell-specific mechanisms would achieve tumor-selective magnetically-enhanced drug delivery. To validate this concept, we covalently attached the anticancer drug camptothecin (CPT) to biocompatible USPIOs (iron oxide core, 9-10 nm; hydrodynamic diameter, 52 nm) coated with polyvinylalcohol/polyvinylamine (PVA/aminoPVA). A bifunctional, end-differentiated dicarboxylic acid linker allowed the attachment of CPT to the aminoPVA as a biologically labile ester substrate for cellular esterases at one end, and as an amide at the other end. These CPT-USPIO conjugates exhibited antiproliferative activity in vitro against human melanoma cells. The intracellular localization of CPT-USPIOs was confirmed by transmission electron microscopy (iron oxide core), suggesting localization in lipid vesicles, and by fluorescence microscopy (CPT). An external static magnetic field applied during exposure increased melanoma cell uptake of the CPT-USPIOs.

Fontes protéicas e de amido com diferentes degradabilidades ruminais para alimentar vacas leiteiras

O objetivo deste trabalho foi avaliar fontes de amido (milho moído fino x milho processado como pipoca) e fontes protéicas (farelo de soja x uréia x farinha de peixe) com degradabilidades ruminais diferentes para alimentar vacas leiteiras. Foram utilizadas 56 vacas Holandesas com 112 dias em lactação. Os tratamentos foram: MFS (milho moído fino + farelo de soja); PFS (pipoca + farelo de soja); PFP (pipoca + farelo de soja + farinha de peixe); PU (pipoca + farelo de soja + uréia). O tratamento PFS não afetou a produção de leite, mas diminuiu o teor de gordura, a produção de gordura e o teor de proteína do leite. A produção de leite foi maior no tratamento PFS do que nos tratamentos PFP e PU. O tratamento PFP diminuiu o teor de gordura, a produção de gordura e de leite corrigida para 3,5% de gordura, mas aumentou o teor de proteína no leite.

Mudanças relacionadas ao amaciamento da graviola durante a maturação pós-colheita

Este trabalho teve por objetivo avaliar mudanças físicas, químicas e bioquímicas relacionadas ao amaciamento de graviola (Annona muricata L.) Crioula durante a maturação. Os frutos foram colhidos no estádio de maturidade fisiológica, em plantas cultivadas na Estação Experimental da Embrapa Agroindústria Tropical, em Pacajus, Ceará, armazenados a 26,3±0,6 ºC e 88±12% de umidade relativa, durante 1, 2, 3, 4 e 5 dias. Utilizou-se o delineamento experimental inteiramente casualizado, com quatro repetições. As variáveis analisadas foram: perda de massa, amido, pectina total e solúvel, e atividades das enzimas amilase, pectinametilesterase, poligalacturonase, alfa-galactosidases e beta-galactosidases. Não houve variação significativa no conteúdo de pectina solúvel. Após cinco dias, a perda de massa atingiu 5%, mas não promoveu murcha dos frutos. As reduções nos conteúdos de amido e de pectina total coincidiram com período de expressiva atividade, respectivamente, das enzimas amilase e da poligalacturonase e beta-galactosidase de parede celular. As modificações mais acentuadas no conteúdo de amido, de pectina total, na solubilização de pectinas e na atividade das enzimas amilase, pectinametilesterase, poligalacturonase e beta-galactosidase de parede celular ocorreram no período do segundo ao quarto dia após a colheita.

Desempenho de cordeiros e estimativa da digestibilidade do amido de dietas com diferentes fontes protéicas

O objetivo deste trabalho foi avaliar a influência de fontes protéicas sobre desempenho, características da carcaça e da carne de cordeiros confinados, e estimar a digestibilidade do amido de rações com alta proporção de grãos. Foram distribuídos 28 cordeiros Santa Inês, em blocos completos ao acaso, de acordo com o peso vivo e a idade, no início do experimento. As fontes protéicas foram os farelos de: soja, amendoim, canola e algodão, em dietas isonitrogenadas com 90% de concentrado e 10% de volumoso (feno de coast-cross). Na determinação da digestibilidade, foram utilizados quatro borregos em delineamento experimental em quadrado latino 4x4, e a digestibilidade do amido foi estimada a partir do teor de amido fecal. Não houve diferenças (p>0,05) quanto ao consumo de matéria seca, ganho de peso vivo médio, conversão alimentar, características da carcaça e da carne, entre as fontes protéicas avaliadas. A digestibilidade do amido apresentou coeficiente de determinação de 93%. Independentemente da fonte protéica utilizada, o teor de amido nas fezes é um indicador eficiente na estimativa da digestibilidade do amido de dietas com alta proporção de concentrado para cordeiros.

Orthogonal protection of peptides and peptoids for cyclization by the thiol-ene reaction and conjugation

Cyclic peptides and peptoids were prepared using the thiolene Michael-type reaction. The linear precursors were provided with additional functional groups allowing for subsequent conjugation: an orthogonally protected thiol, a protected maleimide, or an alkyne. The functional group for conjugation was placed either within the cycle or in an external position. The click reactions employed for conjugation with suitably derivatized nucleoside or oligonucleotides were either cycloadditions (Diels-Alder, Cu(I)-catalyzed azide-alkyne) or the same Michael-type reaction as for cyclization.

Reproducing the organic matter model of anthropogenic dark earth of Amazonia and testing the ecotoxicity of functionalized charcoal compounds

The objective of this work was to obtain organic compounds similar to the ones found in the organic matter of anthropogenic dark earth of Amazonia (ADE) using a chemical functionalization procedure on activated charcoal, as well as to determine their ecotoxicity. Based on the study of the organic matter from ADE, an organic model was proposed and an attempt to reproduce it was described. Activated charcoal was oxidized with the use of sodium hypochlorite at different concentrations. Nuclear magnetic resonance was performed to verify if the spectra of the obtained products were similar to the ones of humic acids from ADE. The similarity between spectra indicated that the obtained products were polycondensed aromatic structures with carboxyl groups: a soil amendment that can contribute to soil fertility and to its sustainable use. An ecotoxicological test with Daphnia similis was performed on the more soluble fraction (fulvic acids) of the produced soil amendment. Aryl chloride was formed during the synthesis of the organic compounds from activated charcoal functionalization and partially removed through a purification process. However, it is probable that some aryl chloride remained in the final product, since the ecotoxicological test indicated that the chemical functionalized soil amendment is moderately toxic.

Oxidation proteomics: the role of thiol modifications

Identification of thiol modifications has gained significant importance. It is increasingly recognized that cysteines play an important role in protein function under both physiological and patho-physiological conditions. Here we reviewed different approaches that are used to identify oxidized proteins and discuss different fluorescent labeling techniques, differential two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization - time of flight identification, in short MALDI-TOF. We illuminate processes that depend on protein oxidation of cysteines and we look into consequences of thiol oxidation during aging and in a variety of diseases, with a special reference to Alzheimer's disease. There is an urgent need for methods that detect specifically oxidized proteins and are able to distinguish different oxidation types.

Intracellular thiol-mediated modulation of epithelial sodium channel activity.

The epithelial sodium channel ENaC is physiologically important in the kidney for the regulation of the extracellular fluid volume, and in the lungs for the maintenance of the appropriate airway surface liquid volume that lines the pulmonary epithelium. Besides the regulation of ENaC by hormones, intracellular factors such as Na(+) ions, pH, or Ca(2+) are responsible for fast adaptive responses of ENaC activity to changes in the intracellular milieu. In this study, we show that ENaC is rapidly and reversibly inhibited by internal sulfhydryl-reactive molecules such as methanethiosulfonate derivatives of different sizes, the metal cations Cd(2+) and Zn(2+), or copper(II) phenanthroline, a mild oxidizing agent that promotes the formation of disulfide bonds. At the single channel level, these agents applied intracellularly induce the appearance of long channel closures, suggesting an effect on ENaC gating. The intracellular reducing agent dithiothreitol fully reverses the rundown of ENaC activity in inside-out patches. Our observations suggest that changes in intracellular redox potential modulate ENaC activity and may regulate ENaC-mediated Na(+) transport in epithelia. Finally, substitution experiments reveal that multiple cysteine residues in the amino and carboxyl termini of ENaC subunits are responsible for this thiol-mediated inhibition of ENaC.

Espalhamento e secagem de filme de amido-glicerol-fibra preparado por "tape-casting"

O objetivo deste trabalho foi avaliar processos de espalhamento e secagem de filme de amido-glicerol-fibras de celulose, preparado por "tape-casting". O espalhamento da suspensão foi avaliado a 50, 150 e 250 cm min-1, seguido de secagem em estufa com circulação forçada de ar, a 40 ou 70ºC; avaliou-se também o espalhamento da suspensão a 150 cm min-1, seguido da secagem do filme sobre suporte de "tape-casting" a 22ºC e 60% de umidade relativa, com velocidades do ar de 4, 6 e 8 m s-1 no túnel de secagem. Ensaios reológicos mostraram que o espalhamento das suspensões a temperaturas inferiores a 50ºC apresentou módulo de elasticidade superior ao módulo viscoso, em todo o espectro de frequências de oscilação. As microscopias mostraram filmes secos a 40 e 70ºC sem defeitos de formação, e cujas propriedades mecânicas não diferiam. A velocidade de espalhamento e a do ar não modificam o tempo de secagem nem as propriedades dos filmes. O tempo de secagem pode ser reduzido para aproximadamente duas horas, o que é importante para a produção dos filmes em larga escala.