Fragility and glassy dynamics of 2Ca(NO(3))(2)center dot 3KNO(3) under pressure: Molecular dynamics simulations

Molecular dynamics simulations of the glass-forming liquid 2Ca(NO(3))(2)center dot 3KNO(3) (CKN) were performed from high temperature liquid states down to low temperature glassy states at six different pressures from 10(-4) to 5.0 GPa. The temperature dependence of the structural relaxation time indicates that the fragility of liquid CKN changes with pressure. In line with recent proposal [Scopigno , Science 302, 849 (2003)], the change on liquid fragility is followed by a proportional change of the nonergodicity factor of the corresponding glass at low temperature. (c) 2008 American Institute of Physics.

Validation of dot blot hybridization and denaturing high performance liquid chromatography as reliable methods for TP53 codon 72 genotyping in molecular epidemiologic studies

Background: Mutations in TP53 are common events during carcinogenesis. In addition to gene mutations, several reports have focused on TP53 polymorphisms as risk factors for malignant disease. Many studies have highlighted that the status of the TP53 codon 72 polymorphism could influence cancer susceptibility. However, the results have been inconsistent and various methodological features can contribute to departures from Hardy-Weinberg equilibrium, a condition that may influence the disease risk estimates. The most widely accepted method of detecting genotyping error is to confirm genotypes by sequencing and/or via a separate method. Results: We developed two new genotyping methods for TP53 codon 72 polymorphism detection: Denaturing High Performance Liquid Chromatography (DHPLC) and Dot Blot hybridization. These methods were compared with Restriction Fragment Length Polymorphism (RFLP) using two different restriction enzymes. We observed high agreement among all methodologies assayed. Dot-blot hybridization and DHPLC results were more highly concordant with each other than when either of these methods was compared with RFLP. Conclusions: Although variations may occur, our results indicate that DHPLC and Dot Blot hybridization can be used as reliable screening methods for TP53 codon 72 polymorphism detection, especially in molecular epidemiologic studies, where high throughput methodologies are required.

Molecular and morphological characterization of Amathia distans Busk and Amathia brasiliensis Busk (Bryozoa: Ctenostomata) from the tropical and subtropical Western Atlantic

Morphological and molecular analyses have proven to be complementary tools of taxonomic information for the redescription of the ctenostome bryozoans Amathia brasiliensis Busk, 1886 and Amathia distans Busk, 1886. The two species, originally described from material collected by the `Challenger` expedition but synonymized by later authors, now have their status fixed by means of the selection of lectotypes, morphological observations and analyses of DNA sequences described here. The morphological characters allowing the identification of living and/or preserved specimens are (1) A. brasiliensis: whitish-pale pigment spots in the frontal surface of stolons and zooids, and a wide stolon with biserial zooid clusters growing in clockwise and anti-clockwise spirals along it, the spirality direction being maintained from maternal to daughter stolons; and (2) A. distans: bright yellow pigment spots in stolonal and zooidal surfaces including lophophores, and a slender stolon, thickly cuticularized, with biserial zooid clusters growing in clockwise and anti-clockwise spirals along it and the spirality direction not maintained from maternal to daughter stolons. Pairwise comparisons of DNA sequences of the mitochondrial genes cytochrome c oxidase subunit I and large ribosomal RNA subunit revealed deep genetic divergence between A. brasiliensis and A. distans. Finally, analyses of those sequences within a Bayesian phylogenetic context recovered their genealogical species status.

Neutron Activation Analysis: A Primary (Ratio) Method to Determine SI-Traceable Values of Element Content in Complex Samples

The metrological principles of neutron activation analysis are discussed. It has been demonstrated that this method can provide elemental amount of substance with values fully traceable to the SI. The method has been used by several laboratories worldwide in a number of CCQM key comparisons - interlaboratory comparison tests at the highest metrological level - supplying results equivalent to values from other methods for elemental or isotopic analysis in complex samples without the need to perform chemical destruction and dissolution of these samples. The CCOM accepted therefore in April 2007 the claim that neutron activation analysis should have the similar status as the methods originally listed by the CCOM as `primary methods of measurement`. Analytical characteristics and scope of application are given.

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process. Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays. The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3. Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.

Theoretical study on the molecular and electronic properties of some substances used for diabetes mellitus treatment

Diabetes mellitus (DM) is a disease that affects a large number of people, and the number of problems associated with the disease has been increasing in the past few decades. These problems include cardiovascular disorders, blindness and the eventual need to amputate limbs. Therefore, the quality of life for people living with DM is less than it is for healthy people. In several cases, metabolic syndrome (MS), which can be considered a disturbance of the lipid metabolism, is associated with DM. In this work, two drugs used to treat DM, pioglitazone and rosiglitazone, were studied using theoretical methods, and their molecular properties were related to the biological activity of these drugs. From the results, it was possible to correlate the properties of each substance-particularly electronic properties-with the biological interactions that are linked to their pharmacological effects. These results suggest that there are future prospects for designing or developing new drugs based on the correlation between theoretical and experimental properties.

Exercise training and caloric restriction prevent reduction in cardiac Ca(2+)-handling protein profile in obese rats

Previous studies show that exercise training and caloric restriction improve cardiac function in obesity. However, the molecular mechanisms underlying this effect on cardiac function remain unknown. Thus, we studied the effect of exercise training and/or caloric restriction on cardiac function and Ca(2+) handling protein expression in obese rats. To accomplish this goal, male rats fed with a high-fat and sucrose diet for 25 weeks were randomly assigned into 4 groups: high-fat and sucrose diet, high-fat and sucrose diet and exercise training, caloric restriction, and exercise training and caloric restriction. An additional lean group was studied. The study was conducted for 10 weeks. Cardiac function was evaluated by echocardiography and Ca(2+) handling protein expression by Western blotting. Our results showed that visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels were higher in rats on the high-fat and sucrose diet compared with the lean rats. Cardiac nitrate levels, reduced/oxidized glutathione, left ventricular fractional shortening, and protein expression of phosphorylated Ser(2808)-ryanodine receptor and Thr(17-)phospholamban were lower in rats on the high-fat and sucrose diet compared with lean rats. Exercise training and/or caloric restriction prevented increases in visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels and prevented reduction in cardiac nitrate levels and reduced: oxidized glutathione ratio. Exercise training and/or caloric restriction prevented reduction in left ventricular fractional shortening and in phosphorylation of the Ser(2808)-ryanodine receptor and Thr(17)-phospholamban. These findings show that exercise training and/or caloric restriction prevent cardiac dysfunction in high-fat and sucrose diet rats, which seems to be attributed to decreased circulating neurohormone levels. In addition, this nonpharmacological paradigm prevents a reduction in the Ser(2808)-ryanodine receptor and Thr(17-)phospholamban phosphorylation and redox status. (Hypertension. 2010;56:629-635.)

Intracellular mechanisms of specific beta-adrenoceptor antagonists involved in improved cardiac function and survival in a genetic model of heart failure

beta-blockers, as class, improve cardiac function and survival in heart failure (HF). However, the molecular mechanisms underlying these beneficial effects remain elusive. In the present study, metoprolol and carvedilol were used in doses that display comparable heart rate reduction to assess their beneficial effects in a genetic model of sympathetic hyperactivity-induced HF (alpha(2A)/alpha(2C)-ARKO mice). Five month-old HF mice were randomly assigned to receive either saline, metoprolol or carvedilol for 8 weeks and age-matched wild-type mice (WT) were used as controls. HF mice displayed baseline tachycardia, systolic dysfunction evaluated by echocardiography, 50% mortality rate, increased cardiac myocyte width (50%) and ventricular fibrosis (3-fold) compared with WT. All these responses were significantly improved by both treatments. Cardiomyocytes from HF mice showed reduced peak [Ca(2+)](i) transient (13%) using confocal microscopy imaging. Interestingly, while metoprolol improved [Ca(2+)](i) transient, carvedilol had no effect on peak [Ca(2+)](i) transient but also increased [Ca(2+)] transient decay dynamics. We then examined the influence of carvedilol in cardiac oxidative stress as an alternative target to explain its beneficial effects. Indeed, HF mice showed 10-fold decrease in cardiac reduced/oxidized glutathione ratio compared with WT, which was significantly improved only by carvedilol treatment. Taken together, we provide direct evidence that the beneficial effects of metoprolol were mainly associated with improved cardiac Ca(2+) transients and the net balance of cardiac Ca(2+) handling proteins while carvedilol preferentially improved cardiac redox state. (C) 2008 Elsevier Inc. All rights reserved.

Ringo: Discordance between the molecular and clinical manifestation in a Golden Retriever Muscular Dystrophy dog

Of the various genetic homologues to Duchenne Muscular Dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog, which presents a variable but usually severe and progressive muscle weakness, has the closest relevance to DMD in both clinical severity and histopathological change. Among 77 GRMD dogs born in our colony in Brazil, we have identified a very mildly affected dog, Ringo, born July 2003. Among his descendants, at least one male, Suflair, is also showing a mild course. In an attempt to better characterize these two dogs, we studied the pattern of muscle proteins expression in Ringo and Suflair, as compared to severely affected and normal control dogs. Dystrophin was absent in both and utrophin was overexpressed in a pattern similar to the observed in severely affected dogs. Understanding the mechanism that is protecting Ringo and Suflair from the deleterious effect of the dystrophin gene mutation is of utmost interest, In addition it points out that the clinical impact of therapeutic trials should be interpreted with caution. (C) 2009 Elsevier B.V. All rights reserved.

MicroRNAs 29 are involved in the improvement of ventricular compliance promoted by aerobic exercise training in rats

Soci UPR, Fernandes T, Hashimoto NY, Mota GF, Amadeu MA, Rosa KT, Irigoyen MC, Phillips MI, Oliveira EM. MicroRNAs 29 are involved in the improvement of ventricular compliance promoted by aerobic exercise training in rats. Physiol Genomics 43: 665-673, 2011. First published March 29, 2011; doi:10.1152/physiolgenomics.00145.2010.-MiRNAs regulate cardiac development, hypertrophy, and angiogenesis, but their role in cardiac hypertrophy (CH) induced by aerobic training has not previously been studied. Aerobic training promotes physiological CH preserving cardiac function. This study assessed involvement of miRNAs-29 in CH of trained rats. Female Wistar rats (n = 7/group) were randomized into three groups: sedentary (S), training 1 (T1), training 2 (T2). T1: swimming sessions of 60 min/5 days/wk/10 wk. T2: similar to T1 until 8th wk. On the 9th wk rats swam 2x/day, and on the 10th wk 3x/day. MiRNAs analysis was performed by miRNA microarray and confirmed by real-time PCR. We assessed: markers of training, CH by ratio of left ventricle (LV) weight/body wt and cardiomyocytes diameter, pathological markers of CH (ANF, skeletal alpha-actin, alpha/beta-MHC), collagen I and III (COLIAI and COLIIIAI) by real-time PCR, protein collagen by hydroxyproline (OH-proline) concentration, CF and CH by echocardiography. Training improved aerobic capacity and induced CH. MiRNAs-1, 133a, and 133b were downregulated as observed in pathological CH, however, without pathological markers. MiRNA-29c expression increased in T1 (52%) and T2 (123%), correlated with a decrease in COLIAI and COLIIIAI expression in T1 (27%, 38%) and T2 (33%, 48%), respectively. MiRNA-29c was inversely correlated to OH-proline concentration (r = 0.61, P = 0.05). The E/A ratio increased in T2, indicating improved LV compliance. Thus, these results show that aerobic training increase miR-29 expression and decreased collagen gene expression and concentration in the heart, which is relevant to the improved LV compliance and beneficial cardiac effects, associated with aerobic high performance training.

Fermentation kinetics for xylitol production by a Pichia stipitis D-Xylulokinase mutant previously grown in spent sulfite liquor

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3 Delta) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h(-1)). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 g(xylose)/g(cel) h) and xylitol production (0.059 g(xylitol)/g(cel) h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

The Influence of Initial Xylose Concentration, Agitation, and Aeration on Ethanol Production by Pichia stipitis from Rice Straw Hemicellulosic Hydrolysate

Rice straw hemicellulosic hydrolysate was used as fermentation medium for ethanol production by Pichia stipitis NRRL Y-7124. Shaking bath experiments were initially performed aiming to establish the best initial xylose concentration to be used in this bioconversion process. In the sequence, assays were carried out under different agitation (100 to 200 rpm) and aeration ((V) under bar (flask)/V(medium) ratio varying from 2.5 to 5.0) conditions, and the influence of these variables on the fermentative parameters values (ethanol yield factor, Y(P/S); cell yield factor, Y(X/S); and ethanol volumetric productivity, Q(P)) was investigated through a 2(2) full-factorial design. Initial xylose concentration of about 50 g/l was the most suitable for the development of this process, since the yeast was able to convert substrate in product with high efficiency. The factorial design assays showed a strong influence of both process variables in all the evaluated responses. The agitation and aeration increase caused a deviation in the yeast metabolism from ethanol to biomass production. The best results (Y(P/S) = 0.37 g/g and Q(P) = 0.39 g/l. h) were found when the lowest aeration (2.5 V(flask)/V(medium) ratio) and highest agitation (200 rpm) levels were employed. Under this condition, a process efficiency of 72.5% was achieved. These results demonstrated that the establishment of adequate conditions of aeration is of great relevance to improve the ethanol production from xylose by Pichia stipitis, using rice straw hemicellulosic hydrolysate as fermentation medium.

Interesterification of milkfat and soybean oil blends catalyzed by immobilized Rhizopus oryzae lipase

Milkfat (MF)/soybean oil (SBO) blends ranging from 50% to 100% of milkfat (w/w) were enzymatically interesterified with a sn-1,3 specific lipase from Rhizopus oryzae immobilized on polysiloxane-polyvinyl alcohol matrix, in a solvent free medium. Interesterification progress was monitored by following the changes in the relative proportions of 50-carbon triacylglycerols (TAGS) to 44-carbon TAGs (50/44 ratio) in the reaction. The starting materials and products were also analyzed in terms of consistency measured in a texturometer. All reactions gave interesterified (IE) products with lower consistency than non-interesterified (NIE) MF:SBO blends and interesterification degree varied from 0.54 to 2.60 in 48 h reaction. The highest interesterification degree was achieved for 65:35 MF:SBO blends, which gave 76% reduction in the consistency. These results showed the potential of the immobilized lipase to change the TAGs profile of the MF:SBO blend allowing to obtain cold-spreadable milkfat. (C) 2010 Elsevier B.V. All rights reserved.

Packed-Bed Reactor Running on Babassu Oil and Glycerol to Produce Monoglycerides by Enzymatic Route using Immobilized Burkholderia cepacia Lipase

The aim of this study was the glycerolysis of babassu oil catalyzed by immobilized lipase from Burkholderia cepacia, in a continuous packed-bed reactor. The best reaction conditions were previously established in batchwise via response surface methodology as a function of glycerol-to-oil molar ratio and reaction temperature. The reactor operated continuously for 22 days at 50 A degrees C, and during the first 6 days, no significant decrease on the initial lipase activity was observed. Monoglycerides concentration was in the range from 25 to 33 wt.%. Subsequently, a progressive decrease in the activity was detected, and an inactivation profile described by Arrhenius model estimated values of 50 days and 1.37 x 10(-2) h(-1), for the half-life and deactivation coefficient, respectively.

Biomimetic oxidative treatment of spruce wood studied by pyrolysis-molecular beam mass spectrometry coupled with multivariate analysis and (13)C-labeled tetramethylammonium hydroxide thermochemolysis: implications for fungal degradation of wood

In this work, pyrolysis-molecular beam mass spectrometry analysis coupled with principal components analysis and (13)C-labeled tetramethylammonium hydroxide thermochemolysis were used to study lignin oxidation, depolymerization, and demethylation of spruce wood treated by biomimetic oxidative systems. Neat Fenton and chelator-mediated Fenton reaction (CMFR) systems as well as cellulosic enzyme treatments were used to mimic the nonenzymatic process involved in wood brown-rot biodegradation. The results suggest that compared with enzymatic processes, Fenton-based treatment more readily opens the structure of the lignocellulosic matrix, freeing cellulose fibrils from the matrix. The results demonstrate that, under the current treatment conditions, Fenton and CMFR treatment cause limited demethoxylation of lignin in the insoluble wood residue. However, analysis of a water-extractable fraction revealed considerable soluble lignin residue structures that had undergone side chain oxidation as well as demethoxylation upon CMFR treatment. This research has implications for our understanding of nonenzymatic degradation of wood and the diffusion of CMFR agents in the wood cell wall during fungal degradation processes.