Different true-protein sources do not modify the metabolism of crossbred Bos taurus x Bos indicus growing heifers

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

Effects of protein supplementation frequency on physiological responses associated with reproduction in beef cows

The objective of this experiment was to determine if frequency of protein supplementation impacts physiological responses associated with reproduction in beef cows. Fourteen nonpregnant, nonlactating beef cows were ranked by age and BW and allocated to 3 groups. Groups were assigned to a 3 x 3 Latin square design, containing 3 periods of 21 d and the following treatments: 1) soybean meal supplementation daily (D), 2) soybean meal supplementation 3 times/week (3WK), and 3) soybean meal supplementation once/week (1WK). Within each period, cows were assigned to an estrus synchronization protocol: 100 mu g of GnRH + controlled internal drug release device (CIDR) containing 1.38 g of progesterone (P-4) on d 1, 25 mg of PGF(2 alpha) on d 8, and CIDR removal + 100 mu g of GnRH on d 11. Grass-seed straw was offered for ad libitum consumption. Soybean meal was individually supplemented at a daily rate of 1 kg/cow (as-fed basis). Moreover, 3WK was supplemented on d 0, 2, 4, 7, 9, 11, 14, 16, and 18 whereas 1WK was supplemented on d 4, 11, and 18. Blood samples were collected from 0 (before) to 72 h after supplementation on d 11 and 18 and analyzed for plasma urea-N (PUN). Samples collected from 0 to 12 h were also analyzed for plasma glucose, insulin, and P-4 (d 18 only). Uterine flushing fluid was collected concurrently with blood sampling at 28 h for pH evaluation. Liver biopsies were performed concurrently with blood sampling at 0, 4, and 28 h and analyzed for mRNA expression of carbamoyl phosphate synthetase I (CPS-I; h 28) and CYP2C19 and CYP3A4 (h 0 and 4 on d 18). Plasma urea-N concentrations were greater (P < 0.01) for 1WK vs. 3WK from 20 to 72 h and greater (P < 0.01) for 1WK vs. D from 16 to 48 h and at 72 h after supplementation (treatment x hour interaction, P < 0.01). Moreover, PUN concentrations peaked at 28 h after supplementation for 3WK and 1WK (P < 0.01) and were greater (P < 0.01) at this time for 1WK vs. 3WK and D and for 3WK vs. D. Expression of CPS-I was greater (P < 0.01) for 1WK vs. D and 3WK. Uterine flushing pH tended (P <= 0.10) to be greater for 1WK vs. 3WK and D. No treatment effects were detected (P >= 0.15) on expression of CYP2C19 and CYP3A4, plasma glucose, and P-4 concentrations, whereas plasma insulin concentrations were greater (P <= 0.03) in D and 3WK vs. 1WK. Hence, decreasing frequency of protein supplementation did not reduce uterine flushing pH or plasma P-4 concentrations, which are known to impact reproduction in beef cows.

Metabolism and ruminal parameters of Holstein × Gir heifers fed sugarcane and increasing levels of crude protein

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

The ability of Nile tilapia to regulate protein and energy intake evaluated by carbon relative enrichement

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Protein Hydrolysates Are Avoided by Herbivores but Not by Omnivores in Two-Choice Preference Tests

Background: The negative sensory properties of casein hydrolysates (HC) often limit their usage in products intended for human consumption, despite HC being nutritious and having many functional benefits. Recent, but taxonomically limited, evidence suggests that other animals also avoid consuming HC when alternatives exist. Methodology/Principal Findings: We evaluated ingestive responses of five herbivorous species (guinea pig, mountain beaver, gopher, vole, and rabbit) and five omnivorous species (rat, coyote, house mouse, white-footed mouse, and deer mouse; N = 16–18/species) using solid foods containing 20% HC in a series of two-choice preference tests that used a nonprotein, cellulose-based alternative. Individuals were also tested with collagen hydrolysate (gelatin; GE) to determine whether it would induce similar ingestive responses to those induced by HC. Despite HC and GE having very different nutritional and sensory qualities, both hydrolysates produced similar preference score patterns. We found that the herbivores generally avoided the hydrolysates while the omnivores consumed them at similar levels to the cellulose diet or, more rarely, preferred them (HC by the white-footed mouse; GE by the rat). Follow-up preference tests pairing HC and the nutritionally equivalent intact casein (C) were performed on the three mouse species and the guinea pigs. For the mice, mean HC preference scores were lower in the HC v C compared to the HC v Cel tests, indicating that HC’s sensory qualities negatively affected its consumption. However, responses were species-specific. For the guinea pigs, repeated exposure to HC or C (4.7-h sessions; N = 10) were found to increase subsequent HC preference scores in an HC v C preference test, which was interpreted in the light of conservative foraging strategies thought to typify herbivores. Conclusions/Significance: This is the first empirical study of dietary niche-related taxonomic differences in ingestive responses to protein hydrolysates using multiple species under comparable conditions. Our results provide a basis for future work in sensory, physiological, and behavioral mechanisms of hydrolysate avoidance and on the potential use of hydrolysates for pest management.

Prediction of culture performance of juvenile Litopenaeus vannamei by in vitro (pH-stat) degree of feed protein hydrolysis with species-specific enzymes

Rapid in vitro methods for measuring digestibility may be useful in analysing aqua feeds if the extent and limits of their application are clearly defined. The pH-stat protein digestibility routine with shrimp hepatopancreas enzymes was previously related to apparent protein digestibility with juvenile Litopenaeus vannamei fed diets containing different protein ingredients. The potential of the method to predict culture performance of shrimp fed six commercial feeds (T3, T4, T5, T6, T7 and T8) with 350 g kg(-1) declared crude-protein content was assessed. The consistency of results obtained using hepatopancreas enzyme extracts from either pond or clear water-raised shrimp was further verified in terms of reproducibility and possible diet history effects upon in vitro outputs. Shrimps were previously acclimated and then maintained over 56 days (initial mean weight 3.28 g) on each diet in 500-L tanks at 114 ind m(-2), clear water closed system with continuous renewal and mechanical filtering (50 mu m), with four replicates per treatment. Feeds were offered four times daily (six days a week) delivered in trays at feeding rates ranging from 4.0% to 7.0% of stocked shrimp biomass. Feed was accessible to shrimp 4 h daily for 1-h feeding period after which uneaten feed was recovered. Growth and survival were determined every 14 days from a sample of 16 individuals per tank. Water quality was monitored daily (pH, temperature and salinity) and managed by water back flushing filter cleaning every 7-10 days. Feeds were analysed for crude protein, gross energy, amino acids and pepsin digestibility. In vitro pH-stat degree of protein hydrolysis (DH%) was determined for each feed using hepatopancreas enzyme extracts from experimental (clear water) or pond-raised shrimp. Feeds resulted in significant differences in shrimp performance (P < 0.05) as seen by the differences in growth rates (0.56-0.98 g week(-1)), final weight and feed conversion ratio (FCR). Shrimp performance and in vitro DH% with pond-raised shrimp enzymes showed significant correlation (P < 0.05) for yield (R-2 = 0.72), growth rates (R-2 = 0.72-0.80) and FCR (R-2 = -0.67). Other feed attributes (protein : energy ratio, amino acids, true protein, non-protein nitrogen contents and in vitro pepsin digestibility) showed none or limited correlation with shrimp culture performance. Additional correlations were found between growth rates and methionine (R-2 = 0.73), FCR and histidine (R-2 = -0.60), and DH% and methionine or methionine+cystine feed contents (R-2 = 0.67-0.92). pH-stat assays with shrimp enzymes generated reproducible DH% results with either pond (CV <= 6.5%) or clear water (CV <= 8.5%) hepatopancreas enzyme sources. Moreover, correlations between shrimp growth rates and feed DH% were significant regardless of the enzyme origin (pond or clear water-raised shrimp) and showed consistent R-2 values. Results suggest the feasibility of using standardized hepatopancreas enzyme extracts for in vitro protein digestibility.

Maternal Moderate Physical Training during Pregnancy Attenuates the Effects of a Low-Protein Diet on the Impaired Secretion of Insulin in Rats: Potential Role for Compensation of Insulin Resistance and Preventing Gestational Diabetes Mellitus

The effects of pregestational and gestational low-to-moderate physical training on insulin secretion in undernourished mothers were evaluated. Virgin female Wistar rats were divided into four groups as follows: control (C, n = 5); trained (T, n = 5); low-protein diet (LP, n = 5); trained with a low-protein diet (T + LP, n = 5). Trained rats ran on a treadmill over a period of 4 weeks before mate (5 days week(-1) and 60 min day(-1), at 65% of VO2max). At pregnancy, the intensity and duration of the exercise were reduced. Low-protein groups were provided with an 8% casein diet, and controls were provided with a 17% casein diet. At third day after delivery, mothers and pups were killed and islets were isolated by collagenase digestion of pancreas and incubated for a further 1 h with medium containing 5.6 or 16.7 mM glucose. T mothers showed increased insulin secretion by isolated islets incubated with 16.7 mM glucose, whereas LP group showed reduced secretion of insulin by isolated islets when compared with both C and LP + T groups. Physical training before and during pregnancy attenuated the effects of a low-protein diet on the secretion of insulin, suggesting a potential role for compensation of insulin resistance and preventing gestational diabetes mellitus.

Protein diets promote the maturation of oocytes and spawning of Piaractus mesopotamicus kept in cages

The objective of this study was to evaluate the effects of balanced diets on the maturation of oocytes and the reproductive performance of P. mesopotamicus in cages. A completely random design with 224 fish in 16 cages measuring 5 m(3) was employed for this purpose. The treatments consisted of diets containing 18, 24, 30, and 36% crude protein (CP) provided ad libitum. The external and internal morphological characteristics of the specimens were examined, as well as: the position of the germinal vesicle, the distribution of oocyte diameters, the fertilization and hatching rates, the number of oocytes released, the total number of oocytes, the remaining weight and total weight of the ovaries, the gonadosomatic index, the condition factor (K), and the histology of the oocytes and ovaries post-spawning and during ovarian regression. The diameters of the oocytes collected before the first hormonal application displayed a unimodal distribution for the lowest protein content and a polymodal distribution for the other treatments. A similar situation was seen during spawning. The lowest fertilization and hatching rates were found as a consequence of the treatment with 30% CP (P < 0.05). The greatest hatching rate occurred in the females fed 18% CP. The greatest total oocyte weight was found in the specimens that received between 30 and 36% CP. The lowest K index was found in the females fed 36% CP. In conclusion, a diet containing 18% CP satisfies the reproductive requirements of females adapted to this system.

The Cysteine-Rich Protein Thimet Oligopeptidase as a Model of the Structural Requirements for S-glutathiolation and Oxidative Oligomerization

Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10-50 mu M) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.

Botanical Origin of Protein Sources Used by Honeybees (Apis mellifera) in an Atlantic Forest

Productive and reproductive traits of beehives are influenced by climate and food availability in the region where the bees are reared or maintained, thus honey and pollen storage, egg-laying conditions of the queen as well as comb occupation are subject to seasonal variations. The present study was conducted in the apiary of the Department of Entomology and Acarology, College of Agriculture Luiz de Queiroz, ESALQ/USP, in the municipality of Piracicaba, in an area containing fruit trees, ornamental plants and a fragment of a native forest. The objective was to identify protein sources used by honeybees (Apis mellifera) over a whole year (2010-2011) in remnants of the Atlantic forest, information that can be used in the conservation and restoration of degraded areas. For sample preparation, the acetolysis method was adopted (Eredtman 1952) and the quantitative analysis was performed by counting successive samples of 900 grains per sample which were grouped by botanical species and/or pollen types. The results show that the bees used various plant types in the area, including ruderal species, to maintain their colonies. Apis mellifera seeks food sources in all plants in the surroundings of the apiary, including herbaceous, shrubs, trees, native or introduced. Eucalyptus sp. played an important role as a food source in all seasons due to its wide availability around the apiary and its high flower production. The most frequent pollen types (greater than 10% of the sample) were Anadenanthera sp., Acacia sp, Miconia sp. and Eucalyptus sp. in winter; Philodendron sp., Mikania cordifolia, Parthenium and Eucalyptus sp. in spring; Alternanthera ficoidea, Chamissoa altissima and Eucalyptus sp. in summer; Philodendron sp., Raphanus sp. and Eucalyptus sp. in autumn.

Functional properties of gelatin-based films containing Yucca schidigera extract produced via casting, extrusion and blown extrusion processes: A preliminary study

Gelatin-based films containing both Yucca schidigera extract and low concentrations of glycerol (0.25-8.75 g per 100 g protein) were produced by extrusion (EF) and characterized in relation to their mechanical properties and moisture content. The formulations that resulted in either larger or smaller elongation values were used to produce films via both blown extrusion (EBF) and casting (CF) and were characterized with respect to their mechanical properties, water vapor permeability, moisture content, solubility, morphology and infrared spectroscopy. The elongation of the EF films was significantly higher than that of the CF and EBF films. The transversal section possessed a compact, homogeneous structure for all of the films studied. The solubility of the films (36-40%) did not differ significantly between the different processes evaluated. The EBF films demonstrated lower water vapor permeability (0.12 g mm m-(2) h(-1) kPa(-1)) than the CF and EF films. The infrared spectra did not indicate any strong interactions between the added compounds. Thermoplastic processing of the gelatin films can significantly increase their elongation; however, a more detailed assessment and optimization of the extrusion conditions is necessary, along with the addition of partially hydrophobic compounds, such as surfactants. (C) 2012 Elsevier Ltd. All rights reserved.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

PLGA microspheres containing bee venom proteins for preventive immunotherapy

Bee venom (BV) allergy is potentially dangerous for allergic individuals because a single bee sting may induce an anaphylactic reaction, eventually leading to death. Currently, venom immunotherapy (VIT) is the only treatment with long-lasting effect for this kind of allergy and its efficiency has been recognized worldwide. This therapy consists of subcutaneous injections of gradually increasing doses of the allergen. This causes patient lack of compliance due to a long time of treatment with a total of 30-80 injections administered over years. In this article we deal with the characterization of different MS-PLGA formulations containing BV proteins for VIT. The PLGA microspheres containing BV represent a strategy to replace the multiple injections, because they can control the solute release. Physical and biochemical methods were used to analyze and characterize their preparation. Microspheres with encapsulation efficiencies of 49-75% were obtained with a BV triphasic release profile. Among them, the MS-PLGA 34 kDa-COOH showed to be best for VIT because they presented a low initial burst (20%) and a slow BV release during lag phase. Furthermore, few conformational changes were observed in the released BV. Above all, the BV remained immunologically recognizable, which means that they could continuously stimulate the immune system. Those microspheres containing BV could replace sequential injections of traditional VIT with the remarkable advantage of reduced number of injections. (C) 2011 Elsevier B.V. All rights reserved.

Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

Abstract Background All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. Results The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. Conclusion Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.