GeLC-MS-based proteomics of Chromobacterium violaceum: comparison of proteome changes elicited by hydrogen peroxide.

Chromobacterium violaceum is a free-living bacillus with several genes that enables it survival under different harsh environments such as oxidative and temperature stresses. Here we performed a label-free quantitative proteomic study to unravel the molecular mechanisms that enable C. violaceum to survive oxidative stress. To achieve this, total proteins extracted from control and C. violaceum cultures exposed during two hours with 8 mM hydrogen peroxide were analyzed using GeLC-MS proteomics. Analysis revealed that under the stress condition, the bacterium expressed proteins that protected it from the damage caused by reactive oxygen condition and decreasing the abundance of proteins responsible for bacterial growth and catabolism. GeLC-MS proteomics analysis provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress ultimately aggregating knowledge of the response of this organism to environmental stress. This study identified approximately 1500 proteins, generating the largest proteomic coverage of C. violaceum so far. We also detected proteins with unknown function that we hypothesize to be part of new mechanisms related to oxidative stress defense. Finally, we identified the mechanism of clustered regularly interspaced short palindromic repeats (CRISPR), which has not yet been reported for this organism.

Feed efficiency indexes and their relationships with carcass, non-carcass and meat quality traits in Nellore steers.

Five hundred and seventy-five Nellore steers were evaluated for residual feed intake and residual feed intake and gain and their relationships between carcass, non-carcass and meat quality traits. RFI was measured by the difference between observed and predicted dry matter intake and RIG was obtained by the sum of -1*RFI and residual gain. Efficient and inefficient animals were classified adopting ±0.5 standard deviations from RFI and RIG mean. A mixed model was used including RFI or RIG and contemporary group as fixed effects, initial age as covariate and sire and experimental period as random effects, testing the significance of the regression slope for each evaluated trait. RIG was positively related to longissimus muscle area. Efficient-RFI animals had lower liver and internal fat proportions compared to inefficient-RFI animals. Efficient-RFI and efficient-RIG animals had 11.8% and 11.2% lower extracted intramuscular fat, compared to inefficient-RFI and inefficient-RIG animals, respectively. Efficient-RFI animals had tougher meat compared to inefficient-RFI animals.

Identification and evaluation of polymorphisms in FABP3 and FABP4 in beef cattle.

Single nucleotide polymorphisms (SNPs) were screened in FABP3 and FABP4 by automatic sequencing of pools of DNA from crossbred animals whose phenotypes belonged to the upper and lower extremes for back fat and marbling, as well as of a pool of DNA from sires used for crossbreeding. Five SNPs were identified in FABP3 and another nine SNPs were identified in FAPB4. Of these, only one SNP had no previous registry in the SNAP database (dbSNP). Three polymorphisms were selected for further evaluation of their association with production traits using restriction fragment length polymorphism-PCR (RFLP-PCR) or real-time PCR genotyping. All 3 markers were in Hardy-Weinberg equilibrium at the 5% significance level for all 7 genetic groups analyzed. Significant association was observed between FABP3-G/A with rib eye area (P = 0.035) and the rib eye area/hot carcass weight ratio (P = 0.025) and between FABP4/TasI with marbling (P = 0.052) and meat texture (P = 0.053). No significant association was observed between the FABP4-G/C polymorphism and any of the observed traits. Previous association studies with allelic variants in these genes have shown mixed results, probably because of the small effect of the genes for these traits, which suggests that results should be replicated in other populations.

Identification, characterisation and molecular modelling of two AP endonucleases from base excision repair pathway in sugarcane provide insights on the early evolution of green plants.

Unlike bacteria and mammals, plant DNA repair pathways are not well characterised, especially in monocots. The understanding of these processes in the plant cell is of major importance, since they may be directly involved in plant acclimation and adaptation to stressful environments. Hence, two sugarcane ESTs were identified as homologues of AP endonuclease from the base-excision repair pathway: ScARP1 and ScARP3. In order to understand their probable function and evolutionary origin, structural and phylogenetic studies were performed using bioinformatics approaches. The two predicted proteins present a considerable amino acid sequence similarity, and molecular modelling procedures indicate that both are functional, since the main structural motifs remain conserved. However, inspection of the sort signal regions on the full-length cDNAs indicated that these proteins have a distinct organelle target. Furthermore, variances in their promoter cis-element motifs were also found. Although the mRNA expression pattern was similar, there were significant differences in their expression levels. Taken together, these data raise the hypothesis that the ScARP is an example of a probable gene duplication event that occurred before monocotyledon/dicotyledon segregation, followed by a sub-functionalisation event in the Poaceae, leading to new intracellular targeting and different expression levels.

Genome scan for meat quality traits in Nelore beef cattle.

Meat quality traits are economically important because they affect consumers' acceptance, which, in turn, influences the demand for beef. However, selection to improve meat quality is limited by the small numbers of animals on which meat tenderness can be evaluated due to the cost of performing shear force analysis and the resultant damage to the carcass. Genome wide-association studies for Warner-Bratzler shear force measured at different times of meat aging, backfat thickness, ribeye muscle area, scanning parameters [lightness, redness (a*), and yellowness] to ascertain color characteristics of meat and fat, water-holding capacity, cooking loss (CL), and muscle pH were conducted using genotype data from the Illumina BovineHD BeadChip array to identify quantitative trait loci (QTL) in all phenotyped Nelore cattle. Phenotype count for these animals ranged from 430 to 536 across traits. Meat quality traits in Nelore are controlled by numerous QTL of small effect, except for a small number of large-effect QTL identified for a*fat, CL, and pH. Genomic regions harboring these QTL and the pathways in which the genes from these regions act appear to differ from those identified in taurine cattle for meat quality traits. These results will guide future QTL mapping studies and the development of models for the prediction of genetic merit to implement genomic selection for meat quality in Nelore cattle.

Intake and Performance of Yearling Steers Grazing Guineagrass (Panicum maximum cv. Tanzânia) Pasture Supplemented with Different Energy Sources.

The aim of this study was to evaluate the efficiency of supplements containing different energy sources in relation to mineral supplementation of steers grazing guineagrass (Panicum maximum cv Tanzânia) pasture, during the dry season. The experimental design was a randomized block with three treatments and four replications. The treatments consisted of a mineral supplementation and two other supplements, one based on corn seed and the other based on soybean hulls, and provided at 0.8% of body weight. Forty-eight, 12 month-old crossbred steers with an average initial body weight of 267 kg, were assigned to twelve paddocks (1,125 ha) of guineagrass. The animals that were fed with soybean hulls and corn seed presented a greater average daily gain (0.982 and 0.937) when compared with the mineral supplementation. Soybean hulls can be used as a satisfactory food source, replacing corn as an energy source in the supplementation of beef cattle without compromising animal performance.

Candidate genes for production traits in Nelore beef cattle.

Meat quality is an important trait for the beef industry. Backfat thickness, ribeye area, and shear force are traits measured late in life, and the investigation of molecular markers associated with these traits can help breeding programs. In cattle, some polymorphisms have been related to production traits. Thus, the purpose of this study was to assess the presence of polymorphisms in the candidate genes insulin-like growth factor 1 (IGF1), fatty acid-binding protein 4 (FABP4), and peroxisome proliferative active receptor gamma coactivator 1 A (PPARGC1A) and associate them with production traits in reference families of Nelore cattle. We used 270 steers descendent from 20 sires that were chosen to represent variability in this breed. The investigation of marker effects on the traits was performed using a mixed model under the restricted maximum likelihood method. A significant allele substitution effect was found for IGF1 and yearling weight (P ≤ 0.017). The mean allele substitution effect was 6.9 kg, with the 229 allele associated with reduced yearling weight in this Nelore population.

A methodological approach to estimate the lactation curve and net energy and protein requirements of beef cows using nonlinear mixed-effects modeling.

The objective of this study was to evaluate methods to predict the secretion of milk and net energy and protein requirements of beef cows (Bos indicus and B. taurus) after approximately 1 mo postpartum using nonlinear mixed-effect modeling (NLME). Twenty Caracu × Nellore (CN) and 10 Nellore (NL) cows were inseminated to Red Angus bulls, and 10 Angus × Nellore (AN) were bred to Canchim bulls. Cows were evaluated from just after calving (25 ± 11 d) to weaning (220 d). Milk yield was estimated by weighing calves before and after suckling (WSW) and by machine milking (MM) methods at 25, 52, 80, 109, 136, 164, 193, and 220 ± 11 d of lactation. Brody and simple linear equations were consecutively fitted to the data and compared using information criteria. For the Brody equation, a NLME model was used to estimate all lactation profiles incorporating different sources of variation (calf sex and breed of cow, cow as a nested random effect, and within-cow auto-correlation). The CV for the MM method (29%) was less than WSW (45%). Consequently, the WSW method was responsible for reducing the variance about 1.5 times among individuals, which minimized the ability to detect differences among cows. As a result, only milk yield MM data were used in the NLME models. The Brody equation provided the best fit to this dataset, and inclusion of a continuous autoregressive process improved fit (P < 0.01). Milk, energy and protein yield at the beginning of lactation were affected by cow genotype and calf sex (P < 0.001). The exponential decay of the lactation curves was affected only by genotype (P < 0.001). Angus × Nellore cows produced 15 and 48% more milk than CN and NL during the trial, respectively (P < 0.05). Caracu × Nellore cows produced 29% more milk than NL (P < 0.05). The net energy and net protein requirements for milk yield followed a similar ranking. Male calves stimulated their dams to produce 11.7, 11.4, and 11.9% more milk, energy and protein, respectively (P < 0.05). The MM method is better than the WSW technique to detect genetic or environmental differences in milk yield among beef cows. The data obtained by the MM method and analyzed by NLME models allows the inclusion of fixed effects, random effects and an auto-regressive process in lactation equations to describe lactation curves and net energy and protein requirements. The NLME is a powerful tool to describe differences in the secretion of milk due to heterosis and cell mammary external stimulus in beef cows.

A SNP in ASAP1 gene is associated with meat quality and production traits in Nelore breed.

The ASAP1 gene is located in a QTL region for meat production traits and to access the role of the ASAP1 gene, the association between a SNP in this gene and production traits in beef cattle was studied. For this, about 270 steers of reference families of Nelore breed were used. The investigation of marker effects on the traits was performed using a mixed model under the restricted maximum likelihood method. Novel association of a SNP in the ASAP1 gene and shear force measured at 24 h post mortem (P≤0.0083) was described in this population of Nelore cattle. This polymorphism accounted for 1.13% of the total additive variance and 17.51% of total phenotypic variance of the trait, suggesting that this marker could be used in marker assisted selection.

Effects of dietary supplementation of rumen-protected conjugated linoleic acid to grazing cows in early lactation.

Conjugated linoleic acids (CLA) are potent anticarcinogens in animal and in vitro models as well as inhibitors of fatty acid synthesis in mammary gland, liver, and adipose tissue. Our objective was to evaluate long-term CLA supplementation of lactating dairy cows in tropical pasture on milk production and composition and residual effects posttreatment. Thirty crossbred cows grazing stargrass (Cynodon nlemfuensis Vanderyst var. nlemfüensis) were blocked by parity and received 150 g/d of a dietary fat supplement of either Ca-salts of palm oil fatty acids (control) or a mixture of Ca-salts of CLA (CLA treatment). Supplements of fatty acids were mixed with 4 kg/d of concentrate. Grazing plus supplements were estimated to provide 115% of the estimated metabolizable protein requirements from 28 to 84 d in milk (treatment period). The CLA supplement provided 15 g/d of cis-9,trans-11 and 22g of cis-10,trans-12. Residual effects were evaluated from 85 to 112 d in milk (residual period) when cows were fed an 18% crude protein concentrate without added fat. The CLA treatment increased milk production but reduced milk fat concentration from 2.90 to 2.14% and fat production from 437 to 348 g/d. Milk protein concentration increased by 11.5% (2.79 to 3.11%) and production by 19% (422 to 504 g/d) in the cows fed CLA. The CLA treatment decreased milk energy concentration and increased milk volume, resulting in unchanged energy output. Milk production and protein concentration and production were also greater during the residual period for the CLA-treated cows. The CLA treatment reduced production of fatty acids (FA) of all chain lengths, but the larger effect was on short-chain FA, causing a shift toward a greater content of longer chain FA. The CLA treatment increased total milk CLA content by 30% and content of the trans-10,cis-12 CLA isomer by 88%. The CLA treatment tended to decrease the number of days open, suggesting a possible effect on reproduction. Under tropical grazing conditions, in a nutritionally challenging environment, CLA-treated cows decreased milk fat content and secreted the same amount of milk energy by increasing milk volume and milk protein production.

Genotoxicity assessment in aquatic environment impacted by the presence of heavy metals.

The aim of this study was to access the genotoxic potential of Extremoz Lake waters in Northeastern Brazilian coast, using the Allium cepa system, piscine micronucleus test and comet assay. In addition, heavy metal levels were quantified by atomic absorption flame spectrometry. The results of the A. cepa system showed significant changes in the frequency of chromosome aberrations and in the mitotic index compared to negative control. No significant changes were observed in micronuclei frequency in the erythrocytes of Oreochromis niloticus. The comet assay showed a statistically significant alteration in the level of DNA breaks of O. niloticus. Chemical analysis detected an increase in heavy metal levels in different sampling periods. These results point out a state of deterioration of water quality at Extremoz Lake, caused by heavy metal contamination and genotoxic activity. It is recommended to establish a monitoring program for the presence of genotoxic agents in this water lake.

Functional interactions between the estrogen receptor and the transcription activator Sp1 regulate the estrogen-dependent transcriptional activity of the vitellogenin A1 io promoter.

Two distinct, TATA box-containing promoters regulate the transcriptional activity of the Xenopus vitellogenin A1 gene. These two promoters are of different strength and are separated by 1.8 kilobase pairs of untranslated sequence. Estrogen receptor (ER) and its ligand, 17beta-estradiol, induce the activity of both promoters. The estrogen response elements (EREs) are located proximal to the downstream i promoter while no ERE-like sequences have been identified in the vicinity of the upstream io promoter. We show here, that transcriptional activity of the upstream io promoter is Sp1-dependent. Moreover, we demonstrate that estrogen inducibility of the io promoter results from functional interactions between the io bound Sp1 and the ER bound at the proximity of i. Functional interactions between Sp1 and ER do not require the presence of a TATA box for transcriptional activation, as is demonstrated using the acyl-CoA oxidase promoter. The relative positions that ER and Sp1 occupy with respect to the initiation site determines whether these two transcription activators can synergize for transcription initiation.

Identification of two steroid-responsive promoters of different strength controlled by the same estrogen-responsive element in the 5'-end region of the Xenopus laevis vitellogenin gene A1.

A structural and functional analysis of the 5'-end region of the Xenopus laevis vitellogenin gene A1 revealed two transcription initiation sites located 1.8 kilobases apart. A RNA polymerase II binding assay indicates that both promoters form initiation complexes efficiently. In vitro, using a transcription assay derived from a HeLa whole-cell extract, the upstream promoter is more than 10-fold stronger than the downstream one. In contrast, both promoters have a similar strength in a HeLa nuclear extract. In vivo, that is in estrogen-stimulated hepatocytes, it is the downstream promoter homologous to the one used by the other members of the vitellogenin gene family, which is 50-fold stronger than the upstream promoter. Thus, if functional vitellogenin mRNA results from this latter activity, it would contribute less than 1% to the synthesis of vitellogenin by fully induced Xenopus hepatocytes expressing the four vitellogenin genes. In contrast, both gene A1 promoters are silent in uninduced hepatocytes. Transfection experiments using the Xenopus cell line B3.2 in which estrogen-responsiveness has been introduced reveal that the strong downstream promoter is controlled by an estrogen responsive element (ERE) located 330 bp upstream of it. The upstream promoter can also be controlled by the same ERE. Since the region comprising the upstream promoter is flanked by a 200 base pair long inverted repeat with stretches of homology to other regions of the X. laevis genome, we speculate that it might have been inserted upstream of the vitellogenin gene A1 by a recombination event and consequently brought under control of the ERE lying 1.5 kilobases downstream.