Adipocytes Are the Only Site of Glutamine Synthetase Expression Within the Lactating Mouse Mammary Gland.

Background: Glutamine in milk is believed to play an important role in neonatal intestinal maturation and immune function. For lactating mothers, glutamine utilization is increased to meet the demands of the enlarged intestine and milk production. However, the source of such glutamine during lactation has not been studied. Objectives: We aimed to assess the effects of lactation on the expression of glutamine synthetase (GS) in the mammary gland and other tissues of lactating mice. Methods: Mouse tissues were sampled at 4 time points: 8-wk-old (virgin, control), post-delivery day 5 (PD5, early lactation), PD15 (peak lactation), and involution (4 days after weaning at PD21). We examined the gene expression and protein concentrations of GS and the first 2 enzymes of branched-chain amino acid catabolism: branched-chain aminotransferase 2 (BCAT2) and branched-chain ketoacid dehydrogenase subunit E1α (BCKDHA). Results: The messenger RNA (mRNA) expression and protein concentrations of GS in mammary glands were significantly lower at PD5 and PD15 compared with the control but were restored at involution. Within the mammary gland, GS protein was only detected in adipocytes with no evidence of presence in mammary epithelial cells. Compared with the control, mRNA and protein concentrations of BCAT2 and BCKDHA in mammary glands significantly decreased during lactation and involution. No changes in GS protein concentrations during lactation were found in the liver, skeletal muscle, and lung. In non-mammary adipose tissue, GS protein abundance was higher during lactation compared with the virgin. Conclusions: This work shows that, within the mouse mammary gland, GS is only expressed in adipocytes and that the relative GS abundance in mammary gland sections is lower during lactation. This suggests that mammary adipocytes may be a site of glutamine synthesis in the lactating mouse. Identifying the sources of glutamine production during lactation is important for optimizing milk glutamine concentration to enhance neonatal and maternal health.

The Welfare of Horses Competing in Three-Barrel Race Events Is Shown to Be Not Inhibited by Short Intervals between Starts.

Equestrian sports require precise animal welfare and health evaluations. To test the hypothesis that horses maintain their welfare when subjected to two three-barrel (3TB) races with 2 min intervals, an experiment was designed to evaluate their surface temperature using infrared thermography (IRT) in regions of interest (barrel, flank, neck, jaw, corner of the mouth, and ocular caruncle) and also measure blood biomarkers (hemogram, total plasma protein, fibrinogen, urea, creatinine, GGT, CK, cortisol, IL-6, and IL-1ß). Ten Quarter Horses were monitored through thermography (pre-race, +1, +4, and +24 h post-race) and blood sampling (pre-race, +1, +4, and +24 h post-race). ANOVA and Tukey test at 5% were used. IRT in six regions of interest (Left/Right-barrel, flank, neck muscles) increased at +, with no differences between values recorded at +1 and +4 when compared to those measured pre-race (p > 0.05). Plasma protein, RBC count, hemoglobin, hematocrit, WBC count, neutrophils, and lymphocytes (p < 0.05) increased immediately after the races, with recovery at +1 h. Other biomarkers did not change, including cortisol, IL-6, and IL-1ß (p > 0.05). Results indicate that well-conditioned 3TB horses subjected to two races at short intervals do not show changes that could be related to impaired health or welfare.

Effects of the Ingestion of Ripe Mangoes on the Squamous Gastric Region in the Horse.

Erosions and gastric ulcers may be present in horses at any age and under different conditions of rearing and handling. In tropical regions, horses can feed on fruits rich in soluble carbohydrates, such as mangoes, but little is known about how these foods interact with their digestive systems. To test the hypothesis that the ingestion of ripe mangoes with peels could cause disturbances in the digestive processes of horses, an experiment was developed to monitor animals that had free access to ripe mangoes in their pasture areas. Horses (purebred Arabians, n = 5; ~340 kg, ~13 years) were evaluated by video gastroscopy and blood analysis. A controlled postprandial glucose curve for mango intake was also performed. Gastroscopies were performed at intervals of 15 days, starting in December, just before the beginning of the harvest, until the beginning of February, and days after the end of the harvest. Blood collection was performed on the same day between November and February for blood analysis. The results were submitted to ANOVA and Tukey's test, with a significance level of p < 0.05. Gastroscopies indicated that four out of five horses had erosions and ulcers in the squamous region between 15 and 30 days after the start of the season. Biochemical tests indicated a reduction in plasma proteins during the harvest period, and the postprandial glucose curve showed concentrations above 200 mg/dL between 30 and 180 min after ingestion of 5.37 kg mangoes. The animals were not treated and recovered after 15 days of harvest and without ripe mangoes on the ground. It is concluded that the indiscriminate ingestion of mangoes favors the appearance of lesions in the gastric squamous region, to varying degrees, and that animals recover naturally after an average of 15 days from the end of the season when the animals return to their regular feeding with hay and grass pasture.

Zinc, manganese and copper amino acid complexed in laying hens' diets affect performance, blood parameters and reproductive organs development.

In the intestinal lumen, excess of oxides and sulfates interfere with the absorption of minerals due to competition from the same absorption site. Amino acids-mineral complexed (AACM) is intended to minimize these problems, which might be absorbed by different absorption sites. Then, a study including Zinc (Zn), Manganese (Mn) and Copper (Cu) from different sources was carried out to evaluate the performance, blood parameters and reproductive organs development of Brown Laying Hens. A total of 800 Lohmann Brown Lite were fed, from one-day-old to 182-days-old, Zn, Mn and Cu from different sources. Measurements were made from 105 to 182-days-old. The laying hens were distributed according to a completely randomized design with 20 replicates and 20 birds per experimental unit. The treatments consisted of a diet supplemented with 70, 70 and 8 mg/kg of Zn, Mn and Cu; respectively, from inorganic sources (IM). The second treatment contained 40, 40 and 2.75 mg/kg of Zn, Mn and Cu, respectively from IM plus 30, 30 and 5.25 mg/kg of Zn, Mn and Cu; respectively, from AACM sources. Performance and reproductive organs development (oviduct and ovary weight), tibia weight, liver weight, egg output and body weight, and blood variables were evaluated. Data were compared by Student's t-test (P < 0.05). Laying hens fed AACM reached 35% of egg output two days earlier and presented heavier tibia bone than the IM group. Those hens also presented greater oviduct weight, greater hematocrit and greater serum concentration of total leukocytes, erythrocytes, eosinophils, monocytes and the hormones T4 and FSH, than the hens fed IM. The supplementation of AACM in laying hens' diets since one-day-old improves the productive performance from the beginning of egg output to peak production, which is justified by better development of bones and oviduct, hormone production and immune system support.

Behavioral and physiological evaluation of sows raised in outdoors systems in the Brazilian semiarid region.

The objective of this study was to evaluate the biochemical, behavioral, and physiological parameters in pregnant sows of native and improved crossbreeds reared within a thermally stressful outdoors system. Twenty pregnant sows of two different genetic groups (native and improved lineage) were used in this research, all animals were distributed in a completely randomized design. The behavioral evaluation was organized in subdivided plots, the sub-plots were the four periods of the day (early morning, morning, afternoon, and night), and the evaluation of physiological parameters were in three periods of the day (9:00 A.M., 12:00 A.M. and 3:00 P.M.), which was conducted for three consecutive days. Blood collection was performed by retro-orbital sinus puncture 1 day before the start of behavior analysis. Glucose levels, total protein, and creatinine showed differences between improved crossbreeds and native animals. It was observed that creatinine and total protein presented larger values for the group of improved crossbreeds, while glucose levels were higher for native animals. While behavioral variables showed behavior indicative of heat stress, as more time was spent by pregnant sows of improved genotypes getting wet, searching for water and staying in outdoors, while native animals showed much more movement behavior during morning and afternoon periods. It was observed differences were observed of time for respiratory rates, while no differences were found for rectal temperature. This indicates that in a region with hot climate, it would be more possible to raise native pregnant sows, due to their rusticity and ability to acclimate to a local condition, according to behavioral evaluation and physiological parameters. Pregnant sows of improved breeding genotypes presented higher difficulties of adaptation than native sows.

Biomarcadores sanguíneos de caprinos Saanen com diferentes faixas etárias / Blood biomarkers of saanen goats with different ages

Objetivou-se estabelecer o perfil aminoacídico, bioquímico e hematológico de caprinos sadios da raça Saanen de diferentes faixas etárias criados em regime intensivo no Nordeste do Brasil. A partir de amostras de sangue de cada animal foi realizada a análise de aminoácidos, perfil hematológico e bioquímico. Os dados foram submetidos a ANOVA e ao Teste de Tukey (P<0,05). A concentração de glutamato [GLU] e a de glutamina e glutamato [GLN+GLU] apresentaram variações (P<0,05) enquanto que a [GLN] não variou (P>0,05). A [GLU] e de [GLN+GLU] foram 91% e 43% superiores nos animais do grupo cria quando comparados com os demais. Ocorreram também valores significativos nas concentrações de proteínas plasmáticas totais [PPT], ureia [URE], creatinina [CREAT], ácido úrico [AcU], alanina aminotransferase [ALT], creatina quinase [CK], glicose [GLIC], triglicérides [TRIG] e colesterol total [COLES-T] (P<0,05), diferentemente das albumina [ALB] e aspartato aminotransferase [AST]. Nos índices hematológicos houve diferenças para volume corpuscular médio (VCM), concentração de hemoglobina corpuscular média (CHCM), coeficiente de variação de amplitude de distribuição dos eritrócitos (RDW-CV) (P<0,05), mas não ocorreram variações significativas de resultados nas células brancas do sangue [CGB], hemácias [HEM], hemoglobina [Hb] e na percentagem do hematócrito (P>0,05). Destaca-se que este conhecimento possibilite melhor entendimento dos processos metabólicos nos animais hígidos e enfermos, levando em consideração as condições alimentar e do manejo da região. Contribuindo assim para aumentar a produtividade do rebanho na região tropical dada a grande importância da caprinocultura no Nordeste do Brasil.

The aim of this study was to establish biochemical, hematological and amino acid profiles from healthy Saanen goats, from different ages bred on intensive system, in Northeastern Brazil. It was used blood Samples to analyse these parameters. Data was submitted to ANOVA and Tukey test (P<0.05). The glutamate [GLU] and glutamine plus glutamate [GLN+GLU] showed differences (P<0.05), while [GLN] didn't (P>0.05). The [GLU] and [GLN+GLU] was 91% and 43% higher on post-weaning group. Also it was seen significance on total protein plasmatic [TPP], urea [URE], creatinine [CREAT], uric acid [UAC], alanine aminotransferase [ALT], creatine kinase [CK], glucose [GLU], total triglyceride [TG] and total cholesterol [CHOL]. Unlike albumin [ALB] and aspartate aminotransferase [AST]. Haematological indices were no differences of mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), coefficient of variation of amplitude red cell distribution (RDW-CV) (P <0.05), but no significant changes in results in white blood cell [WBC], [HEM] red blood cells, hemoglobin [Hb] and the percentage of hematocrit (P> 0.05). Metabolic processes on healthy and sick animals, should be better understood by this study, considering nutritional and environmental condition in this region. This contribution may increase herd productivity in this region by the great importance of goat breeding in northeastern Brazil

Trypanosoma brucei histone H1 inhibits RNA polymerase I transcription and is important for parasite fitness in vivo.

Trypanosoma brucei is a unicellular parasite that causes sleeping sickness in humans. Most of its transcription is constitutive and driven by RNA polymerase II. RNA polymerase I (Pol I) transcribes not only ribosomal RNA genes, but also protein-encoding genes, including variant surface glycoproteins (VSGs) and procyclins. In T. brucei, histone H1 (H1) is required for VSG silencing and chromatin condensation. However, whether H1 has a genome-wide role in transcription is unknown. Here, using RNA sequencing we show that H1 depletion changes the expression of a specific cohort of genes. Interestingly, the predominant effect is partial loss of silencing of Pol I loci, such as VSG and procyclin genes. Labelling of nascent transcripts with 4-thiouridine showed that H1 depletion does not alter the level of labelled Pol II transcripts. In contrast, the levels of 4sU-labelled Pol I transcripts were increased by two- to sixfold, suggesting that H1 preferentially blocks transcription at Pol I loci. Finally, we observed that parasites depleted of H1 grow almost normally in culture but they have a reduced fitness in mice, suggesting that H1 is important for host-pathogen interactions.

Glutamine and glutamate supplementation raise milk glutamine concentrations in lactating gilts.

Glutamine is the most abundant amino acid in milk, and lactation is associated with increased glutamine utilization both for milk synthesis and as a fuel for the enlarged small intestine. A number of recent studies have indicated that lactation is accompanied by a mild catabolic state in which skeletal muscle proteins are degraded to provide amino acids that are used to synthesize additional glutamine. In this study we tested the hypothesis that supplemental L-glutamine or the commercially available glutamine supplement Aminogut (2.5% by weight mixed into daily feed) provided to gilts from 30 days prior to parturition until 21 days post-parturition would prevent a decrease in skeletal muscle glutamine while increasing the glutamine content of the milk. Muscle glutamine content decreased (P < 0.05) in control animals during lactation but this was prevented by supplementation with either L-glutamine or Aminogut. In this study, neither lactation nor supplementation had any effect on plasma glutamine or glutamate content. Free glutamine, and the total glutamine plus glutamate concentrations in milk from the control and the Aminogut group rose (P < 0.05) during the first 7 days of lactation, with milk concentrations in the L-glutamine supplemented group showing a similar trend (P = 0.053). Milk glutamate remained constant between day 7 and 21 of lactation in the control and L-glutamine supplemented groups, but by day 21 of lactation the free glutamine, glutamate, and glutamine plus glutamate concentrations in milk from Aminogut-treated gilts were higher than those of control gilts. Thus dietary glutamine supplementation can alleviate the fall in intramuscular glutamine content during lactation in gilts, and may alleviate some of the catabolic effects of lactation. Furthermore, the increased milk glutamine content in the supplemented gilts may provide optimum nutrition for piglet development.

TTC7B emerges as a novel risk factor for ischemic stroke through the convergence of several genome-wide approaches.

We hereby propose a novel approach to the identification of ischemic stroke (IS) susceptibility genes that involves converging data from several unbiased genetic and genomic tools. We tested the association between IS and genes differentially expressed between cases and controls, then determined which data mapped to previously reported linkage peaks and were nominally associated with stroke in published genome-wide association studies. We first performed gene expression profiling in peripheral blood mononuclear cells of 20 IS cases and 20 controls. Sixteen differentially expressed genes mapped to reported whole-genome linkage peaks, including the TTC7B gene, which has been associated with major cardiovascular disease. At the TTC7B locus, 46 tagging polymorphisms were tested for association in 565 Portuguese IS cases and 520 controls. Markers nominally associated in at least one test and defining associated haplotypes were then examined in 570 IS Spanish cases and 390 controls. Several polymorphisms and haplotypes in the intron 5-intron 6 region of TTC7B were also associated with IS risk in the Spanish and combined data sets. Multiple independent lines of evidence therefore support the role of TTC7B in stroke susceptibility, but further work is warranted to identify the exact risk variant and its pathogenic potential.

Variants within the nitric oxide synthase 1 gene are associated with stroke susceptibility.

OBJECTIVE: Animal studies have allowed important insights into the role of the nitric oxide synthase (NOS) enzymes in atherosclerosis and hypertension, as well as in stroke. In this study we tested the hypothesis that the NOS1 and NOS3 genes, respectively encoding neuronal NOS (nNOS) and endothelial NOS (eNOS), influence stroke susceptibility and outcome after a stroke event. METHODS: We conducted a case-control association study in 551 ischemic stroke patients and 530 controls to assess the role of NOS1 and NOS3 variants in stroke susceptibility. The same genes were tested for association with stroke outcome in a subset of 431 patients. RESULTS: Four NOS1 single nucleotide polymorphisms (SNPs) (rs2293050, rs2139733, rs7308402 and rs1483757) and four haplotypes were significantly associated with stroke susceptibility after adjusting for demographic, clinical and life-style risk factors, and correcting for multiple testing using the false discovery rate (FDR) method (SNPs: 0.004<(uncorrected)P<0.007 and 0.036

Variants in the inflammatory IL6 and MPO genes modulate stroke susceptibility through main effects and gene-gene interactions.

There is substantial evidence that inflammation within the central nervous system contributes to stroke risk and recovery. Inflammatory conditions increase stroke risk, and the inflammatory response is of major importance in recovery and healing processes after stroke. We investigated the role of inflammatory genes IL1B, IL6, MPO, and TNF in stroke susceptibility and recovery in a population sample of 672 patients and 530 controls, adjusting for demographic, clinical and lifestyle risk factors, and stroke severity parameters. We also considered the likely complexity of inflammatory mechanisms in stroke, by assessing the combined effects of multiple genes. Two interleukin 6 (IL6) and one myeloperoxidase (MPO) single-nucleotide polymorphisms were significantly associated with stroke risk (0.022<(corrected)P<0.042), highlighting gene variants of low to moderate effect in stroke risk. An epistatic interaction between the IL6 and MPO genes was also identified in association with stroke susceptibility (P=0.031 after 1,000 permutations). In a subset of 546 patients, one IL6 haplotype was associated with stroke outcome at 3 months ((corrected)P=0.024), an intriguing finding warranting further validation. Our findings support the association of the IL6 gene and present novel evidence for the involvement of MPO in stroke susceptibility, suggesting a modulation of stroke risk by main gene effects, clinical and lifestyle factors, and gene-gene interactions.

Association of a genetic variant in the ALOX5AP with higher risk of ischemic stroke: a case-control, meta-analysis and functional study.

BACKGROUND: Variants in the 5-lipoxygenase-activating protein (ALOX5AP) and phosphodiesterase 4D (PDE4D) genes have first been associated with ischemic stroke (IS) through whole-genome linkage screens. However, association studies obtained conflicting results. We aimed to investigate the contribution of selected single nucleotide polymorphisms (SNPs) in these genes for the first time in a large Iberian population. METHODS: A case-control design was used to analyze one SNP in ALOX5AP and five SNPs in PDE4D in a total of 1,092 IS patients and 781 healthy controls of two different subsets from Spain and Portugal. The analysis was adjusted for confounding variables and the results were integrated in a meta-analysis of all case-control studies. In addition, ALOX5AP gene expression levels were determined in controls and IS cases. RESULTS: A first meta-analysis of both subsets showed that the T allele of the SG13S114 SNP in ALOX5AP was a risk factor for IS after Bonferroni correction [OR = 1.22 (1.06-1.40); p = 0.006]. A second meta-analysis of white populations confirmed these results [OR = 1.18 (1.07-1.31); p = 0.001]. ALOX5AP gene expression analysis in a subset of controls and cases revealed that the SG13S114 genotypes modulate mRNA levels of ALOX5AP (p = 0.001) and mRNA levels were higher in IS cases (2.8 +/- 2.4%) than in controls (1.4 +/- 1.3%; p = 0.003). No association of the variants in PDE4D with IS was observed in our study. CONCLUSIONS: The ALOX5AP SG13S114 variant is an independent risk factor for IS in the Iberian population and is associated with ALOX5AP expression levels. The role of this gene in stroke merits further investigation.

Variants of the Matrix Metalloproteinase-2 but not the Matrix Metalloproteinase-9 genes significantly influence functional outcome after stroke.

BACKGROUND: Multiple lines of evidence suggest that genetic factors contribute to stroke recovery. The matrix metalloproteinases -2 (MMP-2) and -9 (MMP-9) are modulators of extracellular matrix components, with important regulatory functions in the Central Nervous System (CNS). Shortly after stroke, MMP-2 and MMP-9 have mainly damaging effects for brain tissue. However, MMPs also have a beneficial activity in angiogenesis and neurovascular remodelling during the delayed neuroinflammatory response phase, thus possibly contributing to stroke functional recovery. METHODS: In the present study, the role of MMP-2 and MMP-9 genetic variants in stroke recovery was investigated in 546 stroke patients. Functional outcome was assessed three months after a stroke episode using the modified Rankin Scale (mRS), and patients were classified in two groups: good recovery (mRS 1). Haplotype tagging single nucleotide polymorphisms (SNPs) in the MMP-2 (N = 21) and MMP-9 (N = 4) genes were genotyped and tested for association with stroke outcome, adjusting for significant non-genetic clinical variables. RESULTS: Six SNPs in the MMP-2 gene were significantly associated with stroke outcome (0.0018

Kalirin: a novel genetic risk factor for ischemic stroke.

Cerebrovascular and cardiovascular diseases are the leading causes of death and disability worldwide. They are complex disorders resulting from the interplay of genetic and environmental factors, and may share several susceptibility genes. Several recent studies have implicated variants of the Kalirin (KALRN) gene with susceptibility to cardiovascular and metabolic phenotypes, but no studies have yet been performed in stroke patients. KALRN is involved, among others, in the inhibition of inducible nitric oxide synthase, in the regulation of ischemic signal transduction, and in neuronal morphogenesis, plasticity, and stability. The goal of the present study was to determine whether SNPs in the KALRN region on 3q13, which includes the Ropporin gene (ROPN1), predispose to ischemic stroke (IS) in a cohort of Portuguese patients and controls. We genotyped 34 tagging SNPs in the KALRN and ROPN1 chromosomal region on 565 IS patients and 517 unrelated controls, and performed genotype imputation for 405 markers on chromosome 3. We tested the single-marker association of these SNPs with IS. One SNP (rs4499545) in the ROPN1-KALRN intergenic region and two SNPs in KALRN (rs17286604 and rs11712619) showed significant (P < 0.05) allelic and genotypic (unadjusted and adjusted for hypertension, diabetes, and ever smoking) association with IS risk. Thirty-two imputed SNPs also showed an association at P < 0.05, and actual genotyping of three of these polymorphisms (rs7620580, rs6438833, and rs11712039) validated their association. Furthermore, rs11712039 was associated with IS (0.001 < P < 0.01) in a recent well-powered genomewide association study (Ikram et al. 2009). These studies suggest that variants in the KALRN gene region constitute risk factors for stroke and that KALRN may represent a common risk factor for vascular diseases.

Mitochondrial haplogroup H1 is protective for ischemic stroke in Portuguese patients.

BACKGROUND: The genetic contribution to stroke is well established but it has proven difficult to identify the genes and the disease-associated alleles mediating this effect, possibly because only nuclear genes have been intensely investigated so far. Mitochondrial DNA (mtDNA) has been implicated in several disorders having stroke as one of its clinical manifestations. The aim of this case-control study was to assess the contribution of mtDNA polymorphisms and haplogroups to ischemic stroke risk. METHODS: We genotyped 19 mtDNA single nucleotide polymorphisms (SNPs) defining the major European haplogroups in 534 ischemic stroke patients and 499 controls collected in Portugal, and tested their allelic and haplogroup association with ischemic stroke risk. RESULTS: Haplogroup H1 was found to be significantly less frequent in stroke patients than in controls (OR = 0.61, 95% CI = 0.45-0.83, p = 0.001), when comparing each clade against all other haplogroups pooled together. Conversely, the pre-HV/HV and U mtDNA lineages emerge as potential genetic factors conferring risk for stroke (OR = 3.14, 95% CI = 1.41-7.01, p = 0.003, and OR = 2.87, 95% CI = 1.13-7.28, p = 0.021, respectively). SNPs m.3010G>A, m.7028C>T and m.11719G>A strongly influence ischemic stroke risk, their allelic state in haplogroup H1 corroborating its protective effect. CONCLUSION: Our data suggests that mitochondrial haplogroup H1 has an impact on ischemic stroke risk in a Portuguese sample.