Effects of dietary urea levels on milk protein fractions of Holstein cows

The aim of this study was to evaluate the effects of substituting soybean meal for urea on milk protein fractions (casein, whey protein and non-protein nitrogen) of dairy cows in three dietary levels. Nine mid-lactation Holstein cows were used in a 3 x 3 Latin square arrangement, composed of 3 treatments, 3 periods of 21 days each, and 3 squares. The treatments consisted of three different diets fed to lactating cows, which were randomly assigned to three groups of three animals: (A) no urea inclusion, providing 100% of crude protein (CP), rumen undegradable protein (RUP) and rumen degradable protein (RDP) requirements, using soybean meal and sugarcane as roughage; (B) urea inclusion at 7.5 g/kg DM in partial substitution of soybean meal CP equivalent; (C) urea inclusion at 15 g/kg DM in partial substitution of soybean meal CP equivalent. Rations were isoenergetic and isonitrogenous-1 60 g/kg DM of crude protein and 6.40 MJ/kg DM of net energy for lactation. When the data were analyzed by simple polynomial regression, no differences were observed among treatments in relation to milk CP content, true protein, casein, whey protein, non-casein and non-protein nitrogen, or urea. The milk true protein:crude protein and casein:true protein ratios were not influenced by substituting soybean meal for urea in the diet. Based on the results it can be concluded that the addition of urea up to 15 g/kg of diet dry matter in substitution of soybean meal did not alter milk protein concentration casein, whey protein and its non-protein fractions, when fed to lactating dairy cows. (c) 2007 Elsevier B.V. All rights reserved.

The non-coding RNA BC1 is down-regulated in the hippocampus of Wistar Audiogenic Rat (WAR) strain after audiogenic kindling

The aim of this study was to identify molecular pathways involved in audiogenic seizures in the epilepsy-prone Wistar Audiogenic Rat (WAR). For this, we used a suppression-subtractive hybridization (SSH) library from the hippocampus of WARs coupled to microarray comparative gene expression analysis, followed by Northern blot validation of individual genes. We discovered that the levels of the non-protein coding (npc) RNA BC1 were significantly reduced in the hippocampus of WARs submitted to repeated audiogenic seizures (audiogenic kindling) when compared to Wistar resistant rats and to both naive WARs and Wistars. By quantitative in situ hybridization, we verified lower levels of BC1 RNA in the GD-hilus and significant signal ratio reduction in the stratum radiatum and stratum pyramidale of hippocampal CA3 subfield of audiogenic kindled animals. Functional results recently obtained in a BC1-/- mouse model and our current data are supportive of a potential disruption in signaling pathways, upstream of BC1, associated with the seizure susceptibility of WARs. (C) 2010 Elsevier B.V. All rights reserved.

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAD genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1 Delta) of this gene. The gat 1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gal is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans. (C) 2010 Elsevier Inc. All rights reserved.

Effects of kinetin and nitrogen on growth rates, pigment and protein contents in wild and phycoerythrin-deficient strains of Hypnea musciformis (Rhodophyta)

Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. is the main source for carrageenan production in Brazil and strains with selected characteristics could improve the production of raw material. The effects of kinetin on growth rates, morphology, protein content, and concentrations of pigments (chlorophyll a, phycoerythrin, phycocyanin, and allophycocyanin) were assessed in the wild strain (brown phenotype) and in the phycoerythrin-deficient strain (green phenotype) of H. musciformis. Concentrations of kinetin ranging from 0 to 50 mu M were tested in ASP 12-NTA synthetic medium with 10 mu M nitrate (N-limited) and 100 mu M nitrate (N-saturated). In N-limited condition, kinetin stimulated growth rates of the phycoerythrin-deficient strain and formation of lateral branches in both colour strains. Kinetin stimulated protein biosynthesis in both strains. However, differences between both nitrogen conditions were significant only in the phycoerythrin-deficient strain. In the wild strain, effects of kinetin on concentrations of phycobiliproteins were not significant in both nitrogen conditions, except for chlorophyll content. However, the phycoerythrin-deficient strain showed an opposite response, and kinetin stimulated the phycobiliprotein biosynthesis, with the highest concentrations of phycoerythrin in N-saturated medium, while the highest concentrations of allophycocyanin and phycocyanin were observed in N-limited medium. These results indicate that the effects of kinetin on growth, morphology, protein and phycobiliprotein contents are influenced by nitrogen availability, and the main nitrogen storage pools in phycoerythrin-deficient strain of H. musciformis submitted to N-limited conditions were phycocyanin and allophycocianin, the biosynthesis of which was enhanced by kinetin.

Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp. nov.

A Gram-negative, rod-shaped, non-spore-forming and nitrogen-fixing bacterium, designated ICB 89(T), was isolated from stems of a Brazilian sugar cane variety widely used in organic farming. 16S rRNA gene sequence analysis revealed that strain ICB 89(T) belonged to the genus Stenotrophomonas and was most closely related to Stenotrophomonas maltophilia LMG 958(T), Stenotrophomonas rhizophila LMG 22075(T), Stenotrophomonas nitritireducens L2(T), [Pseudomonas] geniculata ATCC 19374(T), [Pseudomonas] hibiscicola ATCC 19867(T) and [Pseudomonas] beteli ATCC 19861(T). DNA-DNA hybridization together with chemotaxonomic data and biochemical characteristics allowed the differentiation of strain ICB 89(T) from its nearest phylogenetic neighbours. Therefore, strain ICB 89(T) represents a novel species, for which the name Stenotrophomonas pavanii sp. nov. is proposed. The type strain is ICB 89(T) (=CBMAI 564(T) =LMG 25348(T)).

Identification of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS

The nutritional value of maize seed is limited due to its high content of storage proteins (zeins), which are deficient in essential amino acids such as lysine and tryptophan. In a previous paper, we showed that protein bodies obtained from BR473 maize variety, developed by Embrapa (Brazilian Agricultural Research Corporation), were mainly constituted by Z27 and a smaller quantity of Z50 gamma-zeins. Besides zein proteins, other not identified protein band in the SDS/PAGE was also observed, which could indicate the presence of non-zein proteins additionally to gamma-zeins. In the present paper, we have demonstrated the presence of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS and database searching. This fact could be related to the excellent energetic value and higher protein quality of BR473 maize grains, since high lysine concentration in some maize varieties has been related to the presence of cytoskeleton proteins that are non-zeins. We have identified the following proteins: Brittle-1 protein (chloroplast precursor), Legumin-1, glyceroldehyde-3-phosphate dehydrogenase, and elongation factor 1-alpha.

Recovery and upgrading bovine rumen protein by extrusion: Effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9% or 3.8%) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein Solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9% residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8% in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end. (C) 2009 Elsevier Ltd. All rights reserved.

Amaranth protein presents cholesterol-lowering effect

This study describes amaranth`s protein cholesterol-lowering effect and investigates its mechanisms hypercholesterolaemia was induced in male hamsters through diet rich in casein (300 g/kg diet) containing regular levels of cholesterol (0.5 kg/g) fed during 3 weeks. Animals were divided into three groups and fed ad libitum diets for 4 weeks containing as the sole source of protein: casein (control), amaranth protein isolate or, casein + amaranth protein isolate. Plasma concentrations of cholesterol and triacylglycerols were measured at four different points: at the beginning of the study. after hypercholesterolaemia was induced, in the first week and then at the end of the experimental diet period. The reduction of the total plasma cholesterol concentration at the end of experimental period for animals fed on diets containing amaranth protein isolate pure and with casein were 27% (P < 0.05) and 48% (P < 0.05). respectively, being the non-HDL fractions the most affected. Digestibility of protein as well as excretion of cholesterol and bile acid, were investigated as the possible mechanisms for this significant hypocholesterolaemic effect. Cholesterol excretion was related to the hypocholesterolaemia but could not explain all the observed reduction. Our findings suggest that amaranth protein has a metabolic effect on endogenous cholesterol metabolism. (C) 2009 Elsevier Ltd. All rights reserved.

Cholesterol-Lowering Properties of Whole Cowpea Seed and Its Protein Isolate in Hamsters

Hypercholesterolemic hamsters were fed for 4 wk on diets rich in saturated fatty acids and cholesterol, differing only in protein source (20%): casein (control group, HC), whole cowpea seed (HWS), and cowpea protein isolate (HPI). Hamsters fed on HWS and HPI presented significant reductions in plasma total cholesterol and non-HDL cholesterol. HPI and HC presented similar protein digestibility, which were significantly higher than that of HWS. Animals fed on HWS presented significantly higher levels of bile acids and cholesterol in feces than did the animals fed on casein or HPI diets. Histological analyses of the liver showed that HC diet resulted in steatosis widely distributed throughout the hepatic lobule, while HWS and HPI diets promoted reductions in liver steatosis. The effectiveness of HWS for modulating lipid metabolism was greater than that of HPI, as measured by plasma cholesterol reduction and liver steatosis.

Effect of monensin on performance in growing ruminants reared under different environmental temperatures

To evaluate the effect of monensin on the performance of growing cattle under different environmental temperatures, 24 male calves (81.9 +/- 7.7 kg mean weight and 100 days old) were distributed in a 2 x 2 factorial arrangement, contrasting 0 or 85 mg monensin/animal per day at 24.3 or 33.2 degrees C (environmental temperatures). Monensin supplementation increased weight gain (P=0.036), improved feed efficiency (P=0.040), increased ruminal concentrations of volatile fatty acids (VFA; P=0.003) and decreased the molar proportion of butyrate (P=0.034); all effects irrespective of environmental temperatures. A temperature-dependent monensin effect was detected on nitrogen retention (P=0.018) and N retained:N absorbed ratio (P=0.012). Animals fed monensin retained higher N amounts than those of the non-supplemented ones when the environmental temperature was 33.2 degrees C. Environmental temperature and monensin supplementation showed an interaction effect on urine N concentration (P=0.003). Temperature did not affect N excretion in monensin-fed animals, but increased N excretion in the non-supplemented ones. Monensin increased the crude protein (CP) digestibility (P=0.094) for

Production and properties of casein hydrolysate microencapsulated by spray drying with soybean protein isolate

The aim of this work was to encapsulate casein hydrolysate by spray drying with soybean protein isolate (SPI) as wall material to attenuate the bitter taste of that product. Two treatments were prepared: both with 12 g/100 g solids and containing either two proportions of SPI: hydrolysate (70:30 and 80:20), called M1 and M2, respectively. The samples were evaluated for morphological characteristics (SEM), particle size, hygroscopicity, solubility, hydrophobicity, thermal behavior and bitter taste with a trained sensory panel using a paired-comparison test (non-encapsulated samples vs. encapsulated samples). Microcapsules had a continuous wall, many concavities, and no porosity. Treatments M1 and M2 presented average particle sizes of 11.32 and 9.18 mu m, respectively. The wall material and/or the microencapsulation raised the hygroscopicity of the hydrolysate since the free hydrolysate had hygroscopicity of 53 g of water/100 g of solids and M1 and M2 had 106.99 and 102.19 g of water/100 g of solids, respectively. However, the hydrophobicity decreases, the absence of a peak in encapsulated hydrolysates, and the results of the panel sensory test considering the encapsulated samples less bitter (p < 0.05) than the non-encapsulated, showed that spray drying with SPI was an efficient method for microencapsulation and attenuation of the bitter taste of the casein hydrolysate. (c) 2008 Elsevier Ltd. All rights reserved.

Rapid and slow modulation of nitrate reductase activity in the red macroalga Gracilaria chilensis (Gracilariales, Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.

Prolonged exposure to palmitate impairs fatty acid oxidation despite activation of AMP-activated protein kinase in skeletal muscle cells

The aim of this study was to investigate the chronic effects of palmitate on fatty acid (FA) oxidation, AMPK/ACC phosphorylation/activation, intracellular lipid accumulation, and the molecular Mechanisms involved in these processes in skeletal muscle cells. Exposure of L6 myotubes for 8 h to 200, 400, 600, and 800 mu M of palmitate did rot affect cel viability but significantly reduced FA oxidation by similar to 26.5%, similar to 43.5%, similar to 50%, and similar to 47%, respectively. Interestingly, this occurred despite significant increases in AMPK (similar to 2.5-fold) and ACC (similar to 3-fold) phosphorylation and in malonyl-CoA decarboxylase activity (similar to 38-60%). Low concentrations of palmitate (50-100 mu M) caused an increase (similar to 30%) in CPT-I activity. However, as the concentration of palmitate increased, CPT-I activity decreased by similar to 32% after exposure for 8 h to 800 mu M of palmitate. Although FA uptake was reduced (similar to 35%) in cells exposed to increasing, palmitate concentrations, intracellular lipid accumulation increased in a dose-dependent manner, reaching values similar to 2.3-, similar to 3-, and 4-fold higher than control in muscle cells exposed to 400, 600, and 800 mu M palmitate, respectively. Interestingly, myotubes exposed to 400 mu M of palmitate for 1h increased basal glucose uptake and glycogen synthesis by similar to 40%. However, as time of incubation in the presence of palmitate progressed from 1 to 8h, these increases were abolished and a time-dependent inhibition of insulin-stimulated glucose uptake (similar to 65%) and glycogen synthesis (30%) was observed in myotubes. These findings may help explain the dysfunctional adaptations that occur in glucose and FA Metabolism in skeletal muscle under conditions of chronically elevated circulating levels of non-esterified FAs. Such as in obesity and Type 2 Diabetes.

Chronic low frequency/low volume resistance training reduces pro-inflammatory cytokine protein levels and TLR4 mRNA in rat skeletal muscle

Skeletal muscle is the source of pro- and anti-inflammatory cytokines, and recently, it has been recognized as an important source of interleukin 6 (IL-6), a cytokine that exerts inhibitory effects on several pro-inflammatory cytokines. Although dynamic chronic resistance training has been shown to produce the known ""repeated bout effect"", which abolishes the acute muscle damage, performing of high-intensity resistance training has been regarded highly advisable, at least from the hypertrophy perspective. On the other hand, a more therapeutic, ""non-damaging"" resistance training program, mainly composed of concentric forces, low frequency/low volume of training, and the same exercise, could theoretically benefit the muscle when the main issue is to avoid muscle inflammation (as in the treatment of several ""low-grade"" inflammatory diseases) because the acute effect of each resistance exercise session could be diminished/avoided, at the same time that the muscle is still being overloaded in a concentric manner. However, the benefits of such ""less demanding"" resistance training schedule on the muscle inflammatory profile have never been investigated. Therefore, we assessed the protein expression of IL-6, TNF-alpha, IL-10, IL-10/TNF-alpha ratio, and HSP70 levels and mRNA expression of SCF(beta-TrCP), IL-15, and TLR-4 in the skeletal muscle of rats submitted to resistance training. Briefly, animals were randomly assigned to either a control group (S, n = 8) or a resistance-trained group (T, n = 7). Trained rats were exercised over a duration of 12 weeks (two times per day, two times per week). Detection of IL-6, TNF-alpha, IL-10, and HSP70 protein expression was carried out by western blotting and SCF(beta-TrCP) (SKP Cullin F-Box Protein Ligases), a class of enzymes involved in the ubiquitination of protein substrates to proteasomal degradation, IL-15, and TLR-4 by RT-PCR. Our results show a decreased expression of TNF-alpha and TLR4 mRNA (40 and 60%, respectively; p < 0.05) in the plantar muscle from trained, when compared with control rats. In conclusion, exercise training induced decreased TNF-alpha and TLR-4 expressions, resulting in a modified IL-10/TNF-alpha ratio in the skeletal muscle. These data show that, in healthy rats, 12-week resistance training, predominantly composed of concentric stimuli and low frequency/low volume schedule, down regulates skeletal muscle production of cytokines involved in the onset, maintenance, and regulation of inXammation.

Enalapril treatment corrects the reduced response to bradykinin in diabetes increasing the B2 protein expression

Considering the growing importance of the interaction between components of kallikreinkinin and renin-angiotensin systems in physiological and pathological processes, particularly in diabetes mellitus, the aim of the present study was to investigate the effect of enalapril on the reduced response of bradykinin and on the interaction between angiotensin-(1-7) (Ang-(1-7)) and bradykinin (BK), important components of these systems, in an insulin-resistance model of diabetes. For the above purpose, the response of mesenteric arterioles of anesthetized neonatal streptozotocin-induced (n-STZ) diabetic and control rats was evaluated using intravital microscopy. In n-STZ diabetic rats, enalapril treatment restored the reduced response to BK but not the potentiation of BK by Ang-(1-7) present in non-diabetic rats. The restorative effect of enalapril was observed at a dose that did not correct the altered parameters induced by diabetes such as hyperglycernia, glicosuria, insulin resistance but did reduce the high blood pressure levels of n-SZT diabetic rats. There was no difference in mRNA and protein expressions of B1 and B2 kinin receptor subtypes between n-STZ diabetic and control rats. Enalapril treatment increased the B2 kinin receptor expression. From our data, we conclude that in diabetes enalapril corrects the impaired BK response probably by increasing the expression of B2 receptors. The lack of potentiation of BK by Ang-(1-7) is not corrected by this agent. (c) 2008 Elsevier Inc. All rights reserved.