Localisation and expression of TRPV6 in all intestinal segments and kidney of laying hens

1. The aim of this study was to investigate the localisation and expression of the epithelial Ca2+ channel TRPV6 (transient receptor potential vanilloid channel type 6) in different intestinal segments and kidney of laying hens during peak lay. 2. Immunohistochemical analysis of the intestine indicated that TRPV6 was localised to the brush-border membranes of the duodenum, jejunum, ileum, caecum, and rectum. Expression was weaker in the rectum, and little or no expression was found in crypt and goblet cells. In addition, TRPV6 mRNA was quantified amongst different intestinal segments, and expression was highest in the duodenum and jejunum. Furthermore, Western blotting indicated that the duodenum expressed the greatest amount of TRPV6 and the rectum the least with the other segments expressing intermediate levels. 3. In the kidney, distinct immunopositive staining for TRPV6 was detected at the apical domain of the distal convoluted tubules (DCT) and medullary connecting tubules (CNT). Interestingly, distribution of TRPV6 extended to the proximal convoluted tubules (PCT). Furthermore, the kidney expressed lower TRPV6 mRNA and protein levels compared with that in the duodenum. 4. In conclusion, the epithelial Ca2+ channel TRPV6 is strongly expressed in the apical cells of the entire intestine and the renal tubules, suggesting that active Ca2+ transcellular transport plays a crucial role in dietary calcium (re)absorption in laying hens.     

The effects of restriction of movement on the reliability of heart rate variability measurements in the horse (Equus caballus)

Analysis of heart rate variability (HRV) is a noninvasive approach for investigating the sympathovagal balance of the autonomic nervous system. In recent years, HRV has been increasingly evaluated in animal research. In horses, it has been suggested that basal resting conditions can be achieved by restraining them. The aim of this study was to verify how restriction of movement influences HRV i2n horses. Ten healthy standardbred mares were used to measure the electrocardiographic signal under 2 conditions: free to move in the stall and restrained in the stock. Results indicate that the restriction of movement is associated with increased nervous system sympathetic activity not consistent with resting conditions.     

Assessment of Sperm DNA Fragmentation in Stallion (Equus caballus) and Donkey (Equus asinus) Using the Sperm Chromatin Dispersion Test

Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.     

Diversity of the infracommunities of strongylid nematodes in the ventral colon of Equus caballus from Rio de Janeiro state, Brazil

Nematodes from the ventral colon of 31 adult horses, 24 males and 9 females, in the metropolitan region of the state of Rio de Janeiro were analysed. There were 53,444 (86.4%) adults of the total recovered strongylid nematodes. They belonged to 21 species of Cyathostominae and seven of Strongylinae. Larval forms made up 13.6% (8407) of the total recovered, and 49% of the strongylid nematodes were observed in ventral colon. The most prevalent and abundant species were Cyathostomum tetracanthum, Cylicocyclus nassatus, Cylicostephanus minutus, Cylicostephanus longibursatus, Cylicostephanus leptostomus, Cylicostephanus calicatus and Cylicostephanus goldi, corresponding to 93.2% of the total adult population; these species were classified as central species, except C. goldi; and other species were classified as secondary (n = 8) and satellite (n = 14). The low Green's aggregation index, presented to central species, indicated high dispersion because they were found in a greater number of infracommunities and were proven by the greater prevalence. Only 12.1% of the horses were uninfected or infected by a single species; the other infections were multiple, ranging from 3 to 23 species.     

蛋鸡TRPV6基因的原核表达、纯化及抗体制备

为制备蛋鸡TRPV6蛋白多克隆抗体,根据其基因序列,设计1对特异性引物,以卵巢组织中提取的总RNA为模板,扩增蛋鸡TRPV6基因1 801～2 176nt的375bp序列,构建原核表达质粒pET-32a（＋）-TRPV6;将重组质粒转化BL21（DE3）,经IPTG诱导表达TRPV6融合蛋白,通过镍离子螯合柱纯化后免疫新西兰大白兔,获得兔抗鸡TRPV6多克隆抗体,分别通过酶联免疫吸附试验（ELISA）法和Western blot检测抗体的效价和抗体特异性。结果表明,试验成功构建原核表达载体pET-32a（＋）-TRPV6,SDS-PAGE蛋白电泳检测发现目的蛋白大小约35 000;Western blot分析显示,表达的TRPV6融合蛋白具有良好的免疫原性,其抗体可与大肠杆菌表达的产物特异性结合;ELISA显示其抗体效价达1∶100 000。获得纯化的融合蛋白和多克隆抗体对研究TRPV6钙离子通道在蛋鸡髓质骨形成的作用机理具有重要作用。     

Cloning,tissue distribution and functional characterization of the donkey (Equus asinus) oligopeptide transporter 2

Oligopeptide transporter 2 (PepT2) is an important transporter of oligopeptides. In the present study, we describe the molecular cloning, tissue distribution and functional characterization of a donkey (Equus asinus) PepT2. The cloned cDNA sequence was 2202 bp at full length, encoding a 733 amino acid peptide with a molecular weight of 81.9 kDa and a theoretical pI of 8.92. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of PepT2. The expression of PepT2 in the kidney and lung was significantly higher than that observed in the ileum, duodenum, jejunum, spleen, liver, heart and stomach. Functional characterization by heterologous expression in Chinese hamster ovary cells showed that the uptake of β-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA) by donkey PepT2-Chinese hamster ovary cells was dependent on time, pH and substrate concentration, with a low Km value of 91.51 ± 14.14 μM and a maximum velocity of 41.37 ± 2.193 pmol/min/mg protein. In the present study, for the first time, the expression and functional characteristics of donkey PepT2 were evaluated, the results of which provide new insights and a better understanding of its crucial role in oligopeptide transport in donkeys.     

Sequences and polymorphisms of the canine monoamine transporter genes SLC6A2, SLC6A3, and SLC6A4 among five dog breeds

Monoamine transporters have roles to regulate the monoamine concentrations in synaptic clefts in the central nervous system and are the targets of antidepressants and psychostimulants. They include transporters for norepinephrine, dopamine and serotonin, which are encoded by the SLC6A2, SLC6A3, and SLC6A4 genes, respectivily. We sequenced the full lengths of the coding regions of these genes for dogs and identified four single nucleotide polymorphisms (SNPs) in SLC6A2 and four in SLC6A3. One SLC6A3 SNP was non-synonymous and caused an amino acid substitution from threonine to serine. The genotype frequencies of these polymorphisms differed significantly among five breeds with different behavioral traits, suggesting that novel SLC6A2 and SLC6A3 SNPs would provide additional useful information for behavioral genetic studies in dogs.     

猪繁殖与呼吸综合征病毒结构蛋白基因ORF3、ORF5和ORF6在真核细胞中表达

用PCR方法从重组质粒pOKCH-1a扩增出猪繁殖与呼吸综合征病毒结构蛋白基因ORF3、ORF5、ORF6,将它们分别定向克隆到含有鸡β-actin启动子的高效真核表达载体pCAGGS的多克隆位点,经酶切、PCR及测序分析,筛选鉴定出含有ORF3、ORF5、ORF6基因的重组质粒,分别命名为pCAGGS-ORF3、pCAGGS- ORF5、DCAGGS-ORF6。将重组质粒纯化后转染293T细胞,用RT-PCR扩增出了特征性的基因片段,并采用特异性单抗进行间接免疫荧光检测,结果在胞浆内有亮绿色荧光,而在细胞膜上没有见到荧光,这说明表达的蛋白在胞浆内而不在细胞膜上。通过Westerm blot检测确定表达的GP3、GP5和M蛋白的分子量分别为42 Ku、25 Ku和19 Ku。本试验结果为进一步研究PRRSV膜蛋白伪病毒粒子及DNA疫苗奠定了基础。     

Up-regulation of Endothelin-1 and Endothelin type A receptor genes expression in the heart of broiler chickens versus layer chickens

To compare Endothelin (ET) production and genes expression of ET-1 and ET_A receptor (ET_AR) between broiler and layer chickens during rearing, semi-quantitative RT-PCR and enzyme immunometric assay were performed in the heart ventricles and serum. There were gradual elevations of ET-1 and ET_AR mRNAs in the left ventricle of broiler and layer chicken groups that were mainly significant (P < 0.05) at 28, 35 and 42 days of age with compared to previous days whereas were not significant between two groups.These gradual elevations of ET-1 and ET_AR mRNAs were also observed in the right ventricle that were significant (P < 0.05) at 28, 35 and 42 days of age in broilers and 42 days of age in layers with compared to previous days. Increasing of these mRNAs in the right ventricle of broiler chickens were significantly (P < 0.05) more than layer chickens at 28, 35 and 42 days. Serum ET in broilers was significantly (P < 0.05) higher than layer chickens at 28 and 42 days of age. It is concluded that circulating ET and cardiac ET-1, ET_AR genes expression is higher in broiler chickens than in layer chickens particularly after 21 days of age. It is probably that these breed differences make broiler chickens to be more susceptible to Endothelin related-cardiomyopathies such as congestive heart failure and ascites.     

Reference gene screening for analyzing gene expression in the heart,liver, spleen,lung and kidney of forest musk deer

The screening of reference genes for real-time quantitative PCR (qPCR) in forest musk deer (FMD) tissue is of great significance to the basic research on FMD. However, there are few reports on the stability analysis of FMD reference genes so far. In this study, We used qPCR to detect the expression levels of 11 reference gene candidates (18S rRNA, beta-actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box-binding protein [TBP], hypoxanthine phosphoribosyltransferase 1 [HPRT1], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide [YWHAZ], hydroxymethylbilane synthase [HMBS], eukaryotic translation elongation factor 1 alpha 1 [EEF1A1], succinate dehydrogenase complex flavoprotein subunit A [SDHA], peptidylprolyl isomerase B [PPIB], and ubiquitin C [UBC]) in heart, liver, spleen, lung and kidney of FMD. After removing 18S rRNA on account of its high expression level, geNorm, NormFinder, BestKeeper and ΔCt algorithms were used to evaluate the expression stability of the remaining genes in the five organs, and further comprehensive ranking was calculated by RefFinder. According to the results, the selected reference genes with the most stable expression in the heart of FMD are SDHA and YWHAZ, while in the liver are ACTB and SDHA; in the spleen and lung are YWHAZ and HPRT1; in the kidney are YWHAZ and PPIB. The use of common reference genes in all five organs is not recommended. The analyses showed that tissue is an important variability factor in genes expression stability. Meanwhile, the result can be used as a reference for the selection of reference genes for qPCR in further study.     

Heat shock protein 60 expression in heart, liver and kidney of broilers exposed to high temperature

The objective of this study was to investigate the expression and localization of HSP60 in the heart, liver, and kidney of acutely heat-stressed broilers at various stressing times. The plasma creatine kinase (CK) and glutamic pyruvic transaminase (GPT) concentrations statistic increased following heat stress. After 2 h of heat stress, the tissues showed histopathological changes. Hsp60 expressed mainly in the cytoplasm of parenchyma cells heat stress. The intensity of the cytoplasmic staining varied and exhibited an organ-specific distribution pattern. Hsp60 levels in the hearts of heat-stressed chickens gradually increased at 1 h (p < 0.05) and peaked (p < 0.05) at 5 h; Hsp60 levels in the liver gradually decreased at 3 h (p < 0.05); Hsp60 levels in the kidney had no fluctuation. It is suggested that Hsp60 expression is tissue-specific and this may be linked to tissue damage in response to heat stress. The Hsp60 level is distinct in diverse tissues, indicating that Hsp60 may exert its protective effect by a tissue- and time-specific mechanism.     

猪GPX5,FUT1和PRLR基因的遗传多态性及遗传效应分析

为了分析谷胱甘肽过氧化物酶5基因(Glutathion Peroxidase 5,GPX5),岩藻糖转移酶1基因(α1-Fucosytransferase,FUT1)和催乳素受体基因(Prolactin receptor,PRLR)在野猪与大白猪杂交的F1代群体中的遗传多态性及遗传效应。采用PCR-RFLP方法检测了GPX5,FUT1和PRLR基因的多态性,分析其对初生重(LiB)和30日龄体重(Li30)的遗传效应。结果表明,除了PRLR基因外,其余2个基因的基因频率与大白猪已有研究结果基本一致;PRLR基因的AA型个体在Li30上比AB和BB型个体可显著增重1.42～1.47 kg(P0.05);GPX5和FUT1基因对这2个性状的效应均不显著。结果提示,野猪血统的引入没有显著改变现有猪群的遗传基础,PRLR基因可在生产中考虑应用。     

Variation in the expression of Hsp27, αB-crystallin mRNA and protein in heart and liver of pigs exposed to different transport times

Twenty pigs were randomly divided into four groups of five pigs each (not transported – control, 1, 2 and 4 h of transportation). A significant increase of ALT, AST and CK in the blood serum and acute parenchyma cell lesions were observed and those were characterized by acute degenerations in the heart and liver. Hsp27 expression levels increased significantly in the heart after 2 h and in the liver after 4 h of transportation, accompanying with the hsp27 mRNA increasing significantly in the heart and liver after 1 h of transportation. αB-crystallin expression levels were fluctuant (not significantly) in the heart and liver during transporting, however, αB-crystallin mRNA increase notably in the heart after 1 h and decrease significantly in the liver at 1 and 2 h of transportation, respectively. In conclusion, the cellular damage to the heart and liver is highest after 1 h of transportation, Hsp27 and αB-crystallin play dissimilar roles and show tissue-specific response in different tissues during transportation.     

Sequencing of canine 5-hydroxytriptamine receptor (5-HTR) 1B, 2A, 2C genes and identification of polymorphisms in the 5-HTR1B gene

Polymorphisms of human genes encoding 5-hydroxytriptamine (serotonin) receptors (5-HTRs) are thought to be associated with psychiatric disorders and behavioral traits. In the present study, we searched for corresponding polymorphisms in the dog and compared allelic frequencies for the canine 5-HTR1B, 5-HTR2A, and 5-HTR2C genes among five canine breeds. The canine genes consisted of the following: 5-HTR1B, 1170 bp; 5-HTR2A, 1413 bp; and 5-HTR2C, 1377 bp. All of these genes were highly homologous with the human genes. We found six single nucleotide polymorphisms (SNPs) in the 5-HTR1B gene (G57A, A157C, G246A, C660G, T955C, and G1146C). Genotyping of the respective SNPs revealed that there were inter-breed variations in the genotypes and allelic frequencies for four out of the six identified SNPs, suggesting that further analyses of the polymorphisms of the 5-HTR1B gene would be useful in order to gain an understanding of the genetic background underlying the diversified behavioral traits among canine species.