Solution structure of the human signaling protein RACK1

Background: The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results: In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 +/- 0.2) x 10(6) M(-1) and resulted in a dissociation constant (KD) of (0.7 +/- 0.1) x 10(-6) M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion: Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.

Seed phytate and protein content in beans depending on the application of basalt powder

Seed phytate and protein content in beans depending on the application of basalt powder. The content of phytate in the grains is correlated with the supply of phosphorus to the plant, but there is a lack of knowledge as to possible effect of slower availability of nutrients in the soil. The objectives of this study were to assess the effect of rock powder, alone or combined with cattle manure, on the productivity, levels of phosphorus, protein and phytate content in beans. The experiment was carried out in a randomized blocks design, with four replications. The treatments were control (limestone, granite and natural phosphate); conventional fertilization; powder basalt (2.5, 5.0, 10.0 and 20.0 ton. ha(-1)); cattle manure, and doses of powder basalt with cattle manure. In the treatment with conventional fertilizer, the total phosphorus content in grain was higher than the control, but the application of powder of basalt did not show a difference significant. Increase in the doses of basalt powder increased the phosphorus content, but phytate content remained constant. Basalt powder proved to be an alternative to maintain low levels of phosphorus in the form of phytate in the grains.

Fatty acids decrease catalase activity in human leukaemia cell lines

Fatty acid (FA) may disturb the redox state of the cells not only by an increase in reactive oxygen species (ROS) generation but also due to a reduction in antioxidant enzyme activities. The effect of various FAs (palmitic, stearic, oleic, linoleic, gamma-linolenic and eicosapentaenoic acids (EPAs)) on Jurkat and Raji cells, (human T and B leukaemic cell lines was investigated). The following measurements were carried out: FA composition of the cells, cell proliferation and activities of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). The protective effect of alpha-tocopherol on cell death was also investigated. Each cell line presented a specific FA composition. All the tested ENS reduced catalase activity. The toxic effect of FA was abolished by the pre-incubation with physiological concentrations of alpha-tocopherol. The findings support the proposition that the increase in oxidative stress induced by FA partially occurs due to a reduction in catalase activity. In spite of the decrease in the enzyme activity, catalase protein and mRNA levels were not changed, suggesting a post-translational regulation. Copyright (C) 2007 John Wiley & Sons, Ltd.

Aerobic exercise training improves skeletal muscle function and Ca(2+) handling-related protein expression in sympathetic hyperactivity-induced heart failure

Bueno CR Jr, Ferreira JC, Pereira MG, Bacurau AV, Brum PC. Aerobic exercise training improves skeletal muscle function and Ca(2+) handling-related protein expression in sympathetic hyperactivity-induced heart failure. J Appl Physiol 109: 702-709, 2010. First published July 1, 2010; doi: 10.1152/japplphysiol.00281.2010.-The cellular mechanisms of positive effects associated with aerobic exercise training on overall intrinsic skeletal muscle changes in heart failure (HF) remain unclear. We investigated potential Ca(2+) abnormalities in skeletal muscles comprising different fiber compositions and investigated whether aerobic exercise training would improve muscle function in a genetic model of sympathetic hyperactivity-induced HF. A cohort of male 5-mo-old wild-type (WT) and congenic alpha(2A)/alpha(2C) adrenoceptor knockout (ARKO) mice in a C57BL/6J genetic background were randomly assigned into untrained and trained groups. Exercise training consisted of a 8-wk running session of 60 min, 5 days/wk (from 5 to 7 mo of age). After completion of the exercise training protocol, exercise tolerance was determined by graded treadmill exercise test, muscle function test by Rotarod, ambulation and resistance to inclination tests, cardiac function by echocardiography, and Ca(2+) handling-related protein expression by Western blot. alpha(2A)/alpha(2C)ARKO mice displayed decreased ventricular function, exercise intolerance, and muscle weakness paralleled by decreased expression of sarcoplasmic Ca(2+) release-related proteins [alpha(1)-, alpha(2)-, and beta(1)-subunits of dihydropyridine receptor (DHPR) and ryanodine receptor (RyR)] and Ca(2+) reuptake-related proteins [sarco(endo) plasmic reticulum Ca(2+)-ATPase (SERCA) 1/2 and Na(+)/Ca(2+) exchanger (NCX)] in soleus and plantaris. Aerobic exercise training significantly improved exercise tolerance and muscle function and reestablished the expression of proteins involved in sarcoplasmic Ca(2+) handling toward WT levels. We provide evidence that Ca(2+) handling-related protein expression is decreased in this HF model and that exercise training improves skeletal muscle function associated with changes in the net balance of skeletal muscle Ca(2+) handling proteins.

Effects of different levels of protein intake and physical training on growth and nutritional status of young rats

This study aimed to investigate the effects of physical training, and different levels of protein intake in the diet, on the growth and nutritional status of growing rats. Newly-weaned Wistar rats (n=48) were distributed into six experimental groups: three of them were subjected to physical swim training (1 h per day. 5 d per week, for 4 wk, after 2 wk of familiarization) and the other three were considered as controls (non-trained). Each pair of groups, trained and non-trained, received diets with a different level of protein in their composition: 14%. 21% or 28%. The animals were euthanized at the end of the training period and the following analyses were performed: proteoglycan synthesis as a biomarker of bone and cartilage growth, IGF-I (insulin-like growth factor-I) assay as a biomarker of growth and nutritional status. total RNA and protein concentration and protein synthesis measured in vivo using a large-dose phenylalanine method. As a main finding, increased dietary protein, combined with physical training, was able to improve neither tissue protein synthesis nor muscle growth. In addition, cartilage and bone growth seem to be deteriorated by the lower and the higher levels of protein intake. Our data allow us to conclude that protein enhancement in the diet, combined with physical exercise, does not stimulate tissue protein synthesis or muscle mass growth. Furthermore, physical training, combined with low protein intake, was not favorable to bone development in growing animals.

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.

Ethanol-induced vasoconstriction is mediated via redox-sensitive cyclo-oxygenase-dependent mechanisms

The present study investigated the role of ROS (reactive oxygen species) and COX (cyclooxygenase) in ethanol-induced contraction and elevation of [Ca(2+)](i) (intracellular [Ca(2+)]). Vascular reactivity experiments, using standard muscle bath procedures, showed that ethanol (1-800 mmol/l) induced contraction in endothelium-intact (EC(50): 306 +/- 34 mmol/l) and endothelium-denuded (EC(50): 180 +/- 40 mmol/l) rat aortic rings. Endothelial removal enhanced ethanol-induced contraction. Preincubation of intact rings with L-NAME [N(G)-nitro-L-arginine methyl ester; non-selective NOS (NO synthase) inhibitor, 100 mu mol/l], 7-nitroindazole [selective nNOS (neuronal NOS) inhibitor, 100 mu mol/l], oxyhaemoglobin (NO scavenger, 10 mu mol/l) and ODQ (selective inhibitor of guanylate cyclase enzyme, 1 mu mol/l) increased ethanol-induced contraction. Tiron [O(2)(-) (superoxide anion) scavenger, 1 mmol/l] and catalase (H(2)O(2) scavenger, 300 units/ml) reduced ethanol-induced contraction to a similar extent in both endothelium-intact and denuded rings. Similarly, indomethacin (non-selective COX inhibitor, 10 mu mol/l), SC560 (selective COX- I inhibitor, 1 mu mol/l), AH6809 [PGF(2 alpha) (prostaglandin F(2 alpha))] receptor antagonist, 10 mu mol/l] or SQ29584 [PGH(2)(prostaglandin H(2))/TXA(2) (thromboxane A(2)) receptor antagonist, 3 mu mol/l] inhibited ethanol-induced contraction in aortic rings with and without intact endothelium. In cultured aortic VSMCs (vascular smooth muscle cells), ethanol stimulated generation of O(2)(-) and H(2)O(2). Ethanol induced a transient increase in [Ca(2+)](i), which was significantly inhibited in VSMCs pre-exposed to tiron or indomethacin. Our data suggest that ethanol induces vasoconstriction via redox-sensitive and COX-dependent pathways, probably through direct effects on ROS production and Ca(2+) signalling. These findings identify putative molecular mechanisms whereby ethanol, at high concentrations, influences vascular reactivity. Whether similar phenomena occur in vivo at lower concentrations of ethanol remains unclear.

Influence of Redox Environments on the Solubility of Arsenic in Soils

Arsenic (As) is a semimetallic element that is notorious for its toxicity and carcinogenicity. Arsenic can be removed by some ferns. The objectives of this study were to investigate the ability of Pteris vittata L. (Pteridophyta) and Phlebodium aureum (L.) J. Sm. (Polypodiaceae) to absorb inorganic As, in the form of arsenate and arsenite. The removal of As by ferns was observed at varying anion concentrations and As solubility in the absorbing plant. Results obtained with ferns on As-contaminated soil indicate that redox potential and iron (Fe) presence affected the solubility of As and the absorption capacity of ferns. Upon reduction to -200mV, the soluble As content increased to 400mV. The results indicate that Fe oxides and the influence of redox potential strongly affect As absorption. Under nonreducing conditions, Phlebodium aureum did not remove As as well as Pteris vittata. Under more reducing conditions (-200 to 0mV) and under similar soil conditions, the results show that the both ferns remove As.

MULTIVARIATE STATISTICAL METHOD IN OPTIMIZATION OF PROTEIN EXTRACTION FROM SOYBEAN FLOUR WITH DAIRY WHEY

The aim objective of this project was to evaluate the protein extraction of soybean flour in dairy whey, by the multivariate statistical method with 2(3) experiments. Influence of three variables were considered: temperature, pH and percentage of sodium chloride against the process specific variable ( percentage of protein extraction). It was observed that, during the protein extraction against time and temperature, the treatments at 80 degrees C for 2h presented great values of total protein (5.99%). The increasing for the percentage of protein extraction was major according to the heating time. Therefore, the maximum point from the function that represents the protein extraction was analysed by factorial experiment 2(3). By the results, it was noted that all the variables were important to extraction. After the statistical analyses, was observed that the parameters as pH, temperature, and percentage of sodium chloride, did not sufficient for the extraction process, since did not possible to obtain the inflection point from mathematical function, however, by the other hand, the mathematical model was significant, as well as, predictive.

Penetration of hydrolysed soy protein-added brine and its effect on yield and pH of beef steaks from the biceps femoris muscle

This study was aimed to evaluate the penetration behaviour of different brines with tumbled beef steaks from the biceps femoris muscle, specifically their interactions with pH and effects on yield. Six muscles from different animals, divided into origin (OP) and insertion (IP) portions, were cut into 60 steaks of 2.5 cm thickness and tumbled for 30 or 60 min. The steaks were tumbled with two brines, with (WTB/HSP) or without (WTB) hydrolysed soy protein (HSP), and steaks that were not tumbled with brine or water were used as controls. Brine penetration was verified by measuring the amount of dye-containing brine (absorbance at 627 nm) recovered from homogenates of four thin (2 mm) slices from the surface of the beef steaks after tumbling. The WTB/HSP steaks exhibited greater (P < 0.05) brine penetration when tumbled for 60 min than for 30 min. The OP steaks showed greater yield and lower pH (P < 0.05) than IP steaks. HSP-added brine increased the water absorption and retention in the first slices of the steaks, and its efficiency was increased with a longer tumbling time. The portion of the biceps femoris muscle used influenced brine absorption and retention, impacting meat yield. (C) 2010 Elsevier Ltd. All rights reserved.

Identification of protein coding regions using the modified Gabor-wavelet transform

An important topic in genomic sequence analysis is the identification of protein coding regions. In this context, several coding DNA model-independent methods based on the occurrence of specific patterns of nucleotides at coding regions have been proposed. Nonetheless, these methods have not been completely suitable due to their dependence on an empirically predefined window length required for a local analysis of a DNA region. We introduce a method based on a modified Gabor-wavelet transform (MGWT) for the identification of protein coding regions. This novel transform is tuned to analyze periodic signal components and presents the advantage of being independent of the window length. We compared the performance of the MGWT with other methods by using eukaryote data sets. The results show that MGWT outperforms all assessed model-independent methods with respect to identification accuracy. These results indicate that the source of at least part of the identification errors produced by the previous methods is the fixed working scale. The new method not only avoids this source of errors but also makes a tool available for detailed exploration of the nucleotide occurrence.

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. (C) 2010 Elsevier Inc. All rights reserved.

Growth performance and body composition of pacu Piaractus mesopotamicus (Holmberg 1887) in response to dietary protein and energy levels

Improper dietary protein and energy levels and their ratio will lead to increased fish production cost. This work evaluated effects of dietary protein : energy ratio on growth and body composition of pacu, Piaractus mesopotamicus. Fingerling pacu (15.5 +/- 0.4 g) were fed twice a day for 10 weeks until apparent satiation with diets containing 220, 260, 300, 340 or 380 g kg-1 crude protein (CP) and 10.9, 11.7, 12.6, 13.4 or 14.2 MJ kg-1 digestible energy (DE) in a totally randomized experimental design, 5 x 5 factorial scheme (n = 3). Weight gain, specific growth rate increased and feed conversion ratio (FCR) decreased significantly (P < 0.05) when CP increased from 220 to 271, 268 and 281 g kg-1 respectively. Pacu was able to adjust feed consumption in a wide range of dietary DE concentration. Fish fed 260 CP diets showed best (P < 0.05) protein efficiency ratio and FCR with 11.7-12.6 MJ kg-1; but for the 380 CP-diets group, significant differences were observed only at 14.2 MJ kg-1 dietary energy level, suggesting that pacu favours protein as energy source. DE was the chief influence on whole body chemical composition. Minimum dietary protein requirement of pacu is 270 g kg-1, with an optimum CP : DE of 22.2 g MJ-1.

Growth and haematology of pacu, Piaractus mesopotamicus, fed diets with varying protein to energy ratio

Haematopoiesis and blood cells` functions can be influenced by dietary concentration of nutrients. This paper studied the effects of dietary protein:energy ratio on the growth and haematology of pacu, Piaractus mesopotamicus. Fingerling pacu (15.5 +/- 0.4 g) were fed twice a day for 10 weeks until apparent saciety with diets containing 220, 260, 300, 340 or 380 g kg(-1) crude protein (CP) and 10.88, 11.72, 12.55, 13.39, 14.22 MJ kg(-1) digestible energy (DE) in a totally randomized experimental design, 5 x 5 factorial scheme (n=3). Weight gain and specific growth rate were affected (P < 0.05) by protein level only. Protein efficiency ratio decreased (P < 0.05) with increasing dietary protein at all levels of dietary energy. Daily feed intake decreased (P < 0.05) with increasing dietary energy. Mean corpuscular haemoglobin concentration was affected (P < 0.05) by DE and interaction between dietary CP and DE. Total plasma protein increased (P < 0.05) with dietary protein and energy levels. Plasma glucose decreased (P < 0.05) with increasing dietary protein. The CP requirement and optimum protein:energy ratio for weight gain of pacu fingerlings, determined using broken-line model, were 271 g kg(-1) and 22.18 g CP MJ(-1) DE respectively. All dietary CP and DE levels studied did not pose damages to fish health.

Digestibility of plant protein-based diets by largemouth bass Micropterus salmoides

Commercial farming of carnivorous fish demands the reduction of environmental impact of feeds; that requires minimal use of dietary animal protein. This study investigated the digestibility of diets formulated exclusively out of plant protein, added feed attractants, by the carnivore largemouth bass, Micropterus salmoides. Juvenile largemouth bass (14.0 +/- 1.0 cm) conditioned to accept artificial, dry feed were confined in polypropylene cages and fed ad libitum in three daily meals, seven experimental diets containing varying levels of vegetable and animal protein sources, added of different feed stimulants. After last daily meal, cages were transferred to cylindrical-conical-bottomed, 200-L aquaria, where faeces were collected by sedimentation into refrigerated containers, preserved and later analysed for chemical composition. Soybean meal can be used as partial substitute of animal protein in diets for largemouth bass; the poultry by-product meal shows as a good option as animal protein source in these rations. Control treatment - 50PP : 50AP - yielded best performances; the need for the use of fish meal in the formulation for carnivorous diets is, at least, questionable. Results of the digestibility trials demonstrated the importance of determining the diet digestibility, if precision in the formulation of least-cost feeds for carnivorous fish is the ultimate goal.