Expression of mRNA for sodium-glucose transporter 1 and fatty acid translocase in the ruminant gastrointestinal tract before and after weaning

The mRNA expression of sodium‐glucose transporter 1 (SGLT1) and fatty acid translocase (CD36) in the gastrointestinal tract of Holstein cattle and Saanen goats before and after weaning was investigated. Before weaning, the expression of both SGLT1 and CD36 was highest in the jejunum, relative to the other parts of the gastrointestinal tract in both species. The expression of SGLT1 and CD36 in the duodenum was second highest in the goats. After weaning, SGLT1 and CD36 expression in the small intestine significantly decreased in both species. The expression of both types of transporters was also detected in the forestomach. From these results, it was concluded that the jejunum is probably the major absorption site for glucose and long‐chain fatty acids before weaning, and that the expression of both types of transporters decreases after weaning in cattle and goats.     

Differential Expression of Monocarboxylate Transporter 1 and Ancillary Protein CD147 in Red Blood Cells of Show Jumping Horses

We compare the expression levels of the lactate transporter complex consisting of the lactate transporter, monocarboxylate transporter 1 (MCT1), and its ancillary protein, cluster of differentiation 147 (CD147), in the membranes of red blood cells (RBCs) from two breeds of jumping horses and associate the expression levels of these proteins with their jumping ability. The expression levels of MCT1 and CD147 proteins on the membranes of RBCs collected from 30 show jumping horses of two different breeds were quantified: the Brazilian Sport Horses (n = 17) and the European Warmbloods (n = 13). The levels of MCT1 and CD147 in the RBC membranes were measured by western blot using horse-specific antibodies. Statistical analyses included unpaired Student t test and chi-squared test. According to the expression levels of MCT1 and CD147 proteins, 88% of the Brazilian Sport Horses were categorized as high lactate transporters (HTs) and the remaining 12% as low lactate transporters (LTs). The opposite was found for the European Warmbloods, where most animals (77%) were classified as LTs and the remaining animals (23%) were classified as HTs. Brazilian Sport Horses express statistically significantly higher levels of CD147 and MCT1 than European Warmbloods. The classification of horses considering the expression of proteins involved in the ability to transport lactate through the complex MCT1-CD147 seems to be breed dependent, with horses that are able to jump higher obstacles showing lower expression of the MCT1-CD147 complex in their RBCs.     

Recovery of intact IgG in the gastrointestinal tract of the growing rat following ingestion of an ovine serum immunoglobulin

The aim of this study was to determine whether orally ingested ovine serum IgG partly resists digestion in the growing rat. Fifteen Sprague‐Dawley male rats were allocated to one of three diets for a 3‐week study: a control diet (CON) and two test diets containing either freeze‐dried ovine serum immunoglobulin (FDOI) or inactivated ovine serum immunoglobulin (IOI). Samples of stomach chyme and intestinal digesta from the ad libitum‐fed rats were subjected to ELISA and Western blot analysis. Amounts of intact ovine IgG for the FDOI diet were found to be 13.9, 20.0, 34.1, 13.0 and 36.9 μg in the total wet digesta from the stomach chyme, duodenal, jejunal, ileal and colonic digesta respectively. Qualitative detection by Western blot revealed the presence of intact ovine serum IgG with a ~150 kDa MW. This was detected in all of the gut segments (stomach chyme, duodenal, jejunal, ileal and colonic digesta) for growing rats fed the FDOI diet. No ovine IgG was detected in the chyme or digesta from rats fed the CON or the IOI diets. Ovine serum IgG partly resisted digestion in the growing rat fed the FDOI diet and was found throughout the digestive tract. These results provide a basis to explain the reported biological effects of orally administered immunoglobulin.     

布莱凯特黑牛CB1基因克隆测序及表达分析

本试验旨在研究布莱凯特黑牛CB1基因CDS区序列和表达情况,探讨CB1基因与脂肪沉积的关系.运用RT-PCR技术克隆CB1基因CDS区序列,并进行生物学分析;运用qRT-PCR技术检测不同组织的mRNA相对表达量;以及运用免疫组化技术对蛋白进行定位分析,运用Western blot技术检测CB1蛋白在不同组织的表达量....     

Expression of KA1 kainate receptor subunit in the substantia gelatinosa of the trigeminal subnucleus caudalis in mice

The KA1 kainate receptor (KAR) subunit in the substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been implicated in the processing of nociceptive information from the orofacial region. This study compared the expression of the KA1 KAR subunit in the SG of the Vc in juvenile, prepubescent and adult mice. RT-PCR, Western blot and immunohistochemistry analyses were used to examine the expression level in SG area. The expression levels of the KA1 KAR subunit mRNA and protein were higher in juvenile mice than in prepubescent or adult mice. Quantitative data revealed that the KA1 KAR subunit mRNA and protein were expressed at levels approximately two and three times higher, respectively, in juvenile mice than in adult mice. A similar expression pattern of the KA1 KAR subunit was observed in an immunohistochemical study that showed higher expression in the juvenile (59%) than those of adult (35%) mice. These results show that the KA1 KAR subunits are expressed in the SG of the Vc in mice and that the expression level of the KA1 KAR subunit decreases gradually with postnatal development. These findings suggest that age-dependent KA1 KAR subunit expression can be a potential mechanism of age-dependent pain perception.     

GHSR—1a在奶山羊胃肠道的分布与表达

试验采用免疫组织化学、Real—timePCR和Western blotting方法测定ghrelin的功能性受体GHSR-1a（Growth hormone seeretagogue receptor-1a,GHSR-1a）在奶山羊胃肠道的分布和表达。免疫组织化学结果显示,GHSR—1a免疫阳性细胞广泛分布于奶山羊胃肠道。在皱胃主要定位于黏膜层和肌层;瘤胃、网胃和瓣胃黏膜层及肌层中也可见GHSR-1a免疫阳性细胞;在小肠主要位于十二指肠、空肠和回肠的黏膜层、黏膜下层和肌层;在结肠、盲肠和直肠GHSR—1a免疫阳性细胞也有广泛分布;GHSR—1a主要表达于内在神经丛神经细胞、胃底腺上皮细胞、肠腺上皮细胞、复层鳞状上皮细胞、平滑肌细胞中。real—timePCR和Westernblotting结果显示,皱胃、十二指肠、空肠、回肠、盲肠、结肠和直肠GHSR—1a的表达水平相对较高,显著高于瘤胃、网胃和瓣胃的表达（P〈0．05）。结果表明,ghrelin可能通过GHsR-1a对奶山羊胃肠功能具有重要的调节作用。     

A 57‐bp deletion in the ovine KAP6‐1 gene affects wool fibre diameter

High glycine–tyrosine keratin‐associated proteins (HGT‐KAPs) are predominantly present in the orthocortex of wool fibres. They vary in abundance in different wools and have been implicated in regulating wool fibre properties, but little is known about the functional roles of these proteins in the fibre matrix. In this study, we used polymerase chain reaction – single‐strand conformational polymorphism (PCR‐SSCP) analysis to screen for variation in a gene encoding the ovine HGT‐KAP6‐1 protein. We identified three gene variants (A, B and C). Variants A and B were similar to each other, with only three nucleotide differences occurring downstream of the coding sequence. However, variant C had a 57‐bp deletion that would notionally result in a loss of 19 amino acids in the protein. The presence of C was found to be associated with an increase in mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVFD) and prickle factor (percentage of fibres over 30 microns; PF). Sheep of genotype BC produced wool of greater MFD, FDSD and PF than sheep of genotypes AA, AB and BB. The CVFD was greater in the BC sheep than the AB sheep. The results suggest that variation in ovine KRTAP6‐1 affects wool fibre diameter‐associated traits and that the 57‐bp deletion in this gene would lead to coarser wool with greater FDSD, CVFD and PF.     

Seroprevalences for ovine enzootic abortion in Switzerland

Our aim was to assess the seroprevalence of Chlamydophila (Cd) abortus (Chlamydia psittaci serotype 1), denoted ovine enzootic abortion (OEA), in the Swiss sheep population. A competitive enzyme-linked immunosorbent assay (cELISA) was adapted for the investigation of pooled serum samples (pool approach) and receiver-operator characteristic (ROC) analysis was applied to define the cut-off of the pool approach. At a cut-off value of 30% inhibition, the flock-level pooled sensitivity and specificity were 92.9% and 97.6% when compared to classifying the flock based on individual-animal samples.

Subsequently, sera from 775 randomly selected flocks out of 11 cantons of Switzerland were investigated using the pool approach. The cantons included in the study represented 72% of the Swiss sheep flocks and 76% of Swiss sheep population. Antibodies against Cd. abortus were found in almost 19% (144) of the 775 examined sheep flocks. Test prevalences were adjusted for the imperfect test characteristics using the Rogan–Gladen estimator and Bayesian inference. Seroprevalence was highest (43%) in the canton Graubünden. In the remaining 10 cantons the seroprevalence ranged from 2 to 29%. The cELISA in combination with testing pooled sera and statistical methods for true prevalence estimation provided a good survey tool at lower costs and time when compared to other approaches.     

Ⅰ型鸭肝炎病毒VP1基因串联表达载体的构建

为了构建Ⅰ型鸭肝炎病毒双拷贝VP1基因的原核表达质粒,试验采用RT-PCR技术扩增鸭肝炎病毒VP1基因,将其连接到克隆载体pGEM-T Easy中得到重组克隆质粒pGEM-T-VP1,阳性克隆质粒经鉴定正确后,分别用BamHⅠ、XholⅠ、BglⅡ进行酶切,重组后得到pGEM-T-2VP1,然后将双拷贝VP1基因亚克隆入原核表达载体pET-30a(+)中,得到重组质粒pET-30a(+)-2VP1。结果表明:得到了含有目的基因的阳性克隆。说明Ⅰ型鸭肝炎病毒双拷贝VP1基因原核表达质粒构建成功。     

Molecular Monitoring of the Main Changes in Bacterial Floral Diversity in the Gastrointestinal Tract of a Thoroughbred Foal with Catarrhal Enteritis by Using PCR-DGGE

In this study, the main changes in bacterial floral diversity in the gastrointestinal tract of a Thoroughbred foal were monitored by using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). The foal died of catarrhal enteritis of the cecum and large colon. Diarrheal feces and gastrointestinal contents were compared with normal feces. The closest relatives of the bacterium in the samples were Lactobacillus johnsonii (100% similarity), uncultured Bacteroides sp. (92.5% similarity), Bacteroides fragilis (96.3% similarity), and Enterococcus faecium/Enterococcus durans (100% similarity); these were detected by PCR-DGGE using a universal primer set. Monitoring revealed that the numbers of Escherichia coli/Shigella sonnei (97.9% similarity) were significantly higher in the diarrheal feces. Thus, PCR-DGGE is a useful tool for monitoring the main changes in bacterial floral diversity occurring in the gastrointestinal tracts of Thoroughbreds.     

Changes in the gene expression of adiponectin and glucose transporter 12 (GLUT12) in lactating and non-lactating cows

Glucose delivery and uptake by the mammary gland is a rate‐limiting step in milk synthesis. Insulin resistance is believed to increase throughout the body following the onset of lactation. To study glucose metabolism in peak‐, late‐, and non‐lactating cows we analyzed the expression of an adipokine, namely, adiponectin, decreased insulin resistance, leptin, and a novel insulin‐responsive glucose transporter (GLUT12) in the adipose tissue and mammary gland by using real‐time polymerase chain reaction. Our results demonstrated that the mRNA level of adiponectin in the adipose tissue was greater in non‐lactating cows than in peak‐lactating cows. In the adipose tissue, there were no significant differences in the abundance of GLUT12 mRNA between the peak‐, late‐, and non‐lactating cows. In contrast, in the mammary gland, the mRNA level of GLUT12 was greater in non‐lactating cows than in peak‐ and late‐lactating cows. In the adipose tissue, the mRNA level of leptin and peroxisome proliferator‐activated receptor gamma 2 (PPARγ2) was greater in non‐lactating cows than in peak‐lactating cows. The results of the present study suggest that in lactating cows adiponectin plays an important role in insulin resistance in the adipose tissue; in the mammary gland, GLUT12 expression is believed to be an important factor for insulin‐dependent glucose metabolism.     

鸡粒细胞巨噬细胞集落刺激因子-猪干扰素α1融合基因在大肠埃希菌中的可溶性表达及其生物学活性研究

为可溶性表达重组鸡粒细胞巨噬细胞集落刺激因子(ChGM-CSF)与猪干扰素α1(PoIFNα1)融合蛋白,并研究其生物学活性,本研究分别提取鸡、猪肝脏细胞总RNA,采用RT-PCR方法扩增ChGM-CSF和PoIFNα1基因,经linker连接上述两种基因后将其克隆于pET-32a原核表达载体,转化E.coliBL21(DE3)菌株进行诱导表达,SDS-PAGE和western blot检测融合蛋白表达产物。在ST细胞/VSV病毒测定系统以细胞病变抑制法滴定该融合蛋白的抗病毒活性;同时用MTT法检测其对鸡淋巴细胞增殖活性的促进作用。结果显示,PCR扩增并融合后的ChGM-CSF和PoIFNα1融合基因约为1000bp,构建重组表达载体后,诱导表达的rChGM-CSF-PoIFNα1融合蛋白分子量约55ku,主要存在于破碎菌体的上清中,表达量较高。Westernblot检测结果显示,该融合蛋白分别能够与ChGM-CSF多抗和PoIFNα单克隆抗体特异性结合,其抗病毒比活性约为1.1×10^6IU/mg,并且具有促进鸡淋巴细胞增殖的活性。本研究为rChGM-CSF-PoIFNα1重组融合蛋白的研制及其相关活性的测定提供实验依据。     

梅花猪胃肠道5-羟色胺、胃饥饿素和胰高血糖素样肽-1免疫阳性细胞分布特征研究

为了解中国优良地方猪种梅花猪胃肠道内分泌细胞的分布与形态特征,本试验选取6头30日龄断奶健康梅花仔猪,在相同条件下饲养至65日龄,所有仔猪饲喂相同日粮,自由采食饮水。试验结束时,收集仔猪胃肠道各段组织样品,应用免疫组织化学方法观察3种胃肠道内分泌激素5-羟色胺（5-HT）、胃饥饿素（ghrelin）和胰高血糖素样肽-1（GLP-1）免疫阳性细胞的分布及形态特征,并对阳性细胞密度进行分析。结果显示,梅花猪的肠道不同部位（十二指肠、空肠、回肠和结肠）均存在5-HT阳性细胞,主要位于肠上皮组织中,呈锥形、圆形、梭形等形态,可分为开放型和闭合型,其中十二指阳性细胞肠密度显著高于其他肠段（P<0.05）;梅花猪的胃肠道不同部位均有Ghrelin阳性细胞,阳性细胞密度也是随胃肠道走向递减,胃腺体部密度最高;梅花猪的肠道不同部位均有GLP-1阳性细胞,阳性细胞密度沿肠道走向呈抛物线分布,其中回肠数量最多。本试验结果为进一步探究梅花猪胃肠道生理奠定了基础。     

Distribution of 5-hydroxytryptamine,Ghrelin or Glucagon-like Peptide-1 Immunopositive Cells in the Gastrointestinal Tract of Meihua Pig

In order to analyze the distributions and densities of endocrine cells in the gastrointestinal tract of Meihua pig (an elite native breed of Guangdong province, China), 6 healthy piglets weaned at 30 days of age were fed with the same diet till 65 days of age. At 65 days of age, different sections of the gastrointestinal tract of each piglet were collected, and the features and densities of 5-hydroxytryptamine (5-HT), ghrelin and glucagon-like peptide-1 (GLP-1) immunopositive cells were detected by immunohistochemistry. The results showed that the 5-HT positive cells distributed at the epithelial layer throughout the intestinal tract (duodenum, jejunum, ileum and colon) with a variety of features (e.g. cone, round, spindle), and they could be classified into either open-shape or closed-shape type, the density of 5-HT positive cells in duodenum was highest; The ghrelin positive cells were located along the gastrointestinal tract (gastric glands, pylorus, duodenum and jejunum) with both open-shape and closed shape, and its density decreased along the tract with most cells in gastric glands; The GLP-1 positive cells were observed along the intestinal tract, but its density increased from duodenum to ileum and then decreased in colon. This study might provide supporting data for further study of the gastrointestinal physiology of Meihua pig.     

C型口蹄疫病毒VP1基因的原核表达及重组蛋白的纯化

利用PCR技术扩增获得C型口蹄疫病毒VP1基因,将其克隆到原核表达载体pET-30α(+)上,构建重组表达质粒pET-VP1。质粒pET-VP1转化大肠杆菌BL21(DE3)感受态细胞,用IPTG诱导VP1基因的表达,收集不同时间的菌液进行SDS-PAGE、Western blot分析,结果得到分子量约为33 ku的目的条带,表达产物占菌体总蛋白的35%,且该目的条带能被C型FMDV阳性血清识别,说明VP1基因在大肠杆菌中得到高效表达。融合表达蛋白经镍柱纯化,重组蛋白纯度达90%以上。     

Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0 = day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17β (E₂) were detected in day 25 conceptuses. Concentrations of E₂ were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E₂ and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1α and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E₂ and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.