Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.     

鸡源株与人源株H9N2流感病毒血凝素受体结合位点氨基酸比较与分析

H19N2病毒可以感染多种禽类和哺乳动物，包括人类。流感病毒血凝素受体结合位点的氨基酸可以影响受体结合特性。为了解鸡源株与人源株H19N2病毒受体结合部氨基酸的差异，作者对二者进行了比较与分析，结果表明，鸡源株与人源株血凝素受体结合部位氨基酸的第137、183、190、226位点存在差异，鸡源株在这些位点分别为K、N、AorVorT、LorQ而人源株则分别为R、H、E、L。虽然尚不清楚这些位点的改变对病毒与细胞亲和力及宿主范围的影响，但由于H9N2在我国养鸡业的普遍存在，加强H9N2病毒的分子流行病学监测有重要意义。     

棉花黄萎病菌毒素结合位点初探

以纯化的棉花黄萎病菌毒素(PLPC)免疫新西兰白兔制备了PLPC特异性抗血清。将PLPC(15μg·ml 1)用不同浓度的抗体(1～20μg·ml 1IgG)吸附后处理泗棉3号的切根苗,毒素所引起的症状都有不同程度的减轻。表明所制备的抗体在与毒素发生特异性免疫学反应的同时,可部分封闭毒素分子上与毒素受体结合的位点。利用竞争ELISA测定了泗棉3号幼苗子叶的质膜制剂与PLPC的结合活性。结果表明,质膜制剂与毒素结合后能部分阻断毒素与其抗体的免疫学反应,即质膜制剂中含有毒素的结合位点。分别用胰蛋白酶和煮沸处理质膜制剂后,质膜制剂对毒素与其抗体的反应的抑制作用消失,初步表明质膜制剂中与毒素结合的是蛋白质。     

Adrenomedullin (AM) and adrenomedullin binding protein (AM-BP) in the bovine mammary gland and milk: Effects of stage of lactation and experimental intramammary E. coli infection

Adrenomedullin (AM) has been characterized as an endogenous tissue survival factor and modulator of many inflammatory processes. Because of the increased susceptibility of the mammary gland to infection during the time surrounding parturition in the cow, we investigated how milk and tissue content of AM and its binding protein (AM-BP) might be affected by the stage of lactation and the udder health status. Milk and mammary biopsy samples were obtained from Holstein cows 21 days prior to and at various times after calving to represent the dry period and early and mid-stages of lactation. Additional cows received an intramammary challenge with Escherichia coli for immunohistochemical characterization of AM and AM-BP. Milk AM concentrations were relatively constant across the stages of lactation while AM-BP increased two-fold (P<0.04) between early and mid-lactation. Milk AM (P<0.04) and AM-BP (P<0.03) increased as somatic cell counts (SCCs) increased within a given stage of lactation. Tissue content of both (AM and AM-BP) were significantly affected by stage of lactation, lowest in the dry period and progressively increasing to peak at mid-lactation as well as increasing in association with higher levels of SCCs. Following E. coli challenge, AM increased in epithelial cells surrounding mammary alveoli presenting high levels of SCCs. The data suggest that AM and AM-BP are cooperatively regulated in the mammary gland during lactation; changes in localized tissue AM and AM-BP content reflect a dynamic regulation of these tissue factors in the bovine mammary gland consistent with their protective effects within inflamed tissue.     

Comparative pharmacokinetics of marbofloxacin in healthy and Mannheimia haemolytica infected calves

The pharmacokinetics of marbofloxacin were investigated in healthy (n=8) and Mannheimia haemolytica naturally infected (n=8) Simmental ruminant calves following intravenous (i.v.) and intramuscular (i.m.) administration of 2 mg kg(-1) body weight. The concentration of marbofloxacin in plasma was measured using high performance liquid chromatography with ultraviolet detection. Following i.v. administration of the drug, the elimination half-life (t(1/2 beta)) and mean residence time (MRT) were significantly longer in diseased calves (8.2h; 11.13 h) than in healthy ones (4.6 h; 6.1 h), respectively. The value of total body clearance (CL(B)) was larger in healthy calves (3 ml min(-1) kg(-1)) than in diseased ones (1.3 ml min(-1) kg(-1)). After single intramuscular (i.m.) administration of the drug, the elimination half-life, mean residence time (MRT) and maximum plasma concentration (C(max)) were higher in diseased calves (8.0, 12 h, 2.32 microg ml(-1)) than in healthy ones (4.7, 7.4 h, 1.4 microg ml(-1)), respectively. The plasma concentrations and AUC following administration of the drug by both routes were significantly higher in diseased calves than in healthy ones. Protein binding of Marbofloxacin was not significantly different in healthy and diseased calves. The mean value for MIC of marbofloxacin for M. haemolytica was 0.1+/-0.06 microg ml(-1). The C(max)/MIC and AUC(24)/MIC ratios were significantly higher in diseased calves (13.0-64.4 and 125-618 h) than in healthy calves (8-38.33 and 66.34-328 h). The obtained results for surrogate markers of antimicrobial activity (C(max)/MIC, AUC/MIC and T > or = MIC) indicate the excellent pharmacodynamic characteristics of the drug in diseased calves with M. haemolytica, which can be expected to optimize the clinical efficacy and minimize the development of resistance.     

肌原纤维蛋白浓度对风味物质吸附能力的影响

为探究肌原纤维蛋白浓度对风味物质的作用机制,本研究建立了肌原纤维蛋白和风味物质作用的复合体系,研究了不同浓度肌原纤维蛋白对17种典型风味化合物吸附能力的影响。结果表明,风味化合物的种类对肌原纤维蛋白的吸附能力有显著影响,一定浓度的蛋白对醛、酮、醇、酯的吸附作用强弱依次为:醛类酯类酮类醇类。蛋白浓度由2 mg·m L~(-1)增加至4 mg·m L~(-1)时,对所有风味化合物吸附作用显著增强;由4 mg·m L~(-1)增加至6 mg·m L~(-1)时,蛋白对2-甲基丁醛、戊醛、己醛、丁酸乙酯吸附作用明显增强,而当蛋白浓度由6 mg·m L~(-1)增加至8 mg·m L~(-1)时,肌原纤维蛋白对所有风味化合物的吸附作用都明显减弱。蛋白对风味化合物的吸附能力的增加可能是由于较高的蛋白质浓度改变了风味化合物在气、液两相间的分配系数;然而过高浓度的蛋白对风味物质吸附能力的削弱则是蛋白质之间相互作用增强或表面张力降低的结果。本研究结果为肉类风味感知研究过程中确定蛋白浓度、挥发性风味化合物种类等关键参数和改善蛋白类食品的风味提供了参考。     

小麦TaFKBP62c-2B基因克隆、组织表达及亚细胞定位

FK506结合蛋白(FK506 binding protein,FKBP)具有肽基-脯氨酰顺反异构酶(peptidyl-proly cistrans isomerase,PPIase)活性,可以作为一种分子伴侣在蛋白折叠中发挥作用.本研究采用同源基因克隆获得小麦(Triticum aestivum)高温胁迫应答基因TaFKBP62c-2B,利用qRT-PCR和绿色荧光蛋白(green fluorescent protein,GFP)示踪技术明确了TaFKBP62c-2B的表达模式和亚细胞定位信息.序列分析结果表明,小麦TaFKBP62c-2B基因含有37 bp的5'UTR区、1 926 bp的完整CDS以及176 bp的3'UTR区,共编码642个氨基酸(GenBank登录号:KU350629).结构域分析发现,TaFKBP62c-2B是由3个FK506结合保守域(FKBP_C)和3个三十四肽重复序列(tetratricopeptide repeat,TPR)构成的多域FKBP.qRT-PCR结果显不,TaFKBP62c-2B强烈响应高温胁迫;组织表达分析表明,TaFKBP62c-2B主要在小麦的茎和小穗等植株地上组织中表达,在根部的表达量较低.亚细胞定位表明,TaFKBP62c-2B位于内质网上.本研究获得了小麦TaFKBP62c-2B基因全长、明确了其表达规律以及亚细胞定位信息,为进一步了解TaFKBP62c-2B的生物学功能提供了基础资料.     

opaque-2玉米近等基因系的构建与赖氨酸含量快速检测

我国高赖氨酸玉米种质资源狭窄,opaque-2(o2)突变基因能大幅提高玉米赖氨酸含量,通过分子标记辅助选择构建o2玉米近等基因系并检测其赖氨酸含量具有重要意义。其中,要解决的两个关键问题是如何准确地将不同供体的o2突变基因导入受体系和如何快速地检测导入系的赖氨酸含量。本研究利用O2基因内紧密连锁的SSR共显性标记引物phi057检测玉米供体和受体自交系的多态性,利用其特异性和共显性构建o2近等基因系;参考已有研究,改进染料结合(DBL)法,测定18组构建成功的o2近等基因系的赖氨酸含量。结果表明,不仅在不同供体(CA339和山东2548)之间存在多态性,而且在不同受体系间也存在多态性,利用phi057能够成功地将不同供体的o2突变基因导入受体系,构建o2近等基因系;改进的DBL法分析表明,不同受体系赖氨酸含量变化较大,不同背景的受体系导入o2突变基因后赖氨酸含量增加的幅度差异也较大;普通玉米自交系间赖氨酸含量为0.223%~0.368%,构建成功的不同o2近等基因系间,赖氨酸含量为0.373%~0.527%,与受体亲本相比,赖氨酸增加幅度最低为13%,最高为74%。分析表明,phi057能准确筛选导入o2突变基因的受体系,结合改进的DBL法能快速地选择赖氨酸含量高的玉米。     

Salmonid fish viruses and cell interactions at early steps of the infective cycle

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.