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.     

Replication of neurovirulent equine herpesvirus type 1 (EHV-1) in CD172a+ monocytic cells

Equine herpesvirus type 1 (EHV-1) is responsible for respiratory disorders, abortion and myeloencephalopathy (EHM) in horses. Two pathotypes of EHV-1 strains are circulating in the field: neurovirulent (N) and non-neurovirulent (NN). For both strains, CD172a⁺ monocytic cells are one of the main carrier cells of EHV-1 during primary infection, allowing the virus to invade the horse’s body. Recently, we showed that EHV-1 NN strains showed a restricted and delayed replication in CD172a⁺ cells. Here we characterize the in vitro replication kinetics of two EHV-1 N strains in CD172a⁺ cells and investigate if the replication of these strains is similarly silenced as shown for EHV-1 NN strains. We found that EHV-1 N replication was restricted to 7–8% in CD172a⁺ cells compared to 100% in control RK-13 cells. EHV-1 N replication was not delayed in CD172a⁺ cells but virus production was significant lower (10^3.0 TCID₅₀/10⁵ inoculated cells) than in RK-13 cells (10^8.5 TCID₅₀/10⁵ inoculated cells). Approximately 0.04% of CD172a⁺ cells produced and transmitted infectious EHV-1 to neighbour cells compared to 65% of RK-13 cells. Unlike what we observed for the NN strain, pretreatment of CD172a⁺ cells with histone deacetylases inhibitors (HDACi) did not influence the replication of EHV-1 N strains in these cells. Overall, these results show that the EHV-1 replication of N strains in CD172a⁺ cells differs from that observed for NN strains, which may contribute to their different pathogeneses in vivo.     

THE EFFECT OF A COMBINATION OF MEDETOMIDINE-BUTORPHANOL AND MEDETOMIDINE, BUTORPHANOL, ATROPINE ON GLOMERULAR FILTRATION RATE IN DOGS

The effects of intramuscularly administered medetomidine and butorphanol (MB), and medetomidine, butorphanol, atropine (MBA) on glomerular filtration rate (GFR) were determined in six dogs as measured by 99m-Tc-labeled diethylenetriaminepentaacetic acid (99mTc-DTPA) nuclear scintigraphy. Direct systolic, diastolic, and mean arterial blood pressures and heart rate were measured at regular time intervals before, during, and after GFR calculations. The mean GFR measurement following MB was significantly greater (4.44 ml/min/kg) than following MBA (3.82 ml/min/kg) or saline treatment (3.41 ml/min/kg). There was no significant difference between the mean GFR measurements following MBA injection and following saline injection. Diastolic and mean arterial pressures following MBA injection were significantly higher than the values recorded after either MB or saline alone. Heart rate following MB administration was significantly lower than that recorded for dogs receiving MBA or saline alone. The results of this study indicate that the administration of medetomidine in combination with butorphanol significantly increases total GFR in healthy dogs, while the administration of the combination of medetomidine, butorphanol, and atropine does not.     

影响体细胞核移植效率的因素

哺乳动物体细胞核移植 (克隆 )是近年来迅速发展起来的一项新技术 ,对濒危动物保护、优良种畜快速扩群以及核质关系的研究、药物试验等诸多领域都具有重大意义。但体细胞核移植是一项程序繁杂的工作 ,每一步操作所采用的试剂、材料和方案等都会影响到核移植效率 ,因而其影响因素十分众多。文章根据目前已有资料 ,论述了卵母细胞来源、卵母细胞与供体细胞的胞质容量、卵母细胞的去核方案、体细胞供体动物的年龄、供体细胞的组织来源与传代次数及供体细胞的冷藏、冷冻和所处的细胞周期阶段对核移植效率的影响。另外 ,对重组胚的融合 -激活方案、核移植方案及重构胚的培养方案对核移植效率的影响也作了简要论述。     

人血栓调节蛋白基因的表达及转基因小鼠的建立

用PCR方法从人的基因组DNA中扩增了人血栓调节蛋白（hTM)基因，将其克隆到带有人EF－1α启动子的pEF-neo哺乳动物表达载体上，得到表达质粒pEF-TM。在转染pEF-TM的COS－1细胞中，hTM得到了瞬时表达，并且正确地定位到细胞膜上。用pEF-TM转染猪内皮细胞（PEC），经G418筛先得到稳定转染的克隆。凝血活性测定结果显示，表达hTM的PEC凝血时间明显延长，表明其抗凝血能力增强。通过显微注射方法，得到了1只F0代hTM转基因小鼠。Southern杂交结果表明，该转基因小鼠整合了9个拷贝的pEF-TM DNA,并能将外源DNA遗传给后代。     

牛皮肤成纤维细胞的体外培养与冻存

利用牛皮肤组织块直接培养法，得到牛皮肤细胞的原代培养物，再用酶消化法和反复贴壁法处理，能够纯化成纤维细胞。成纤维细胞的冻存是通过选用6种分别含有二甲基亚砜（DMSO）、甘油（GL）及乙二醇（EG）的保护液，以相同的冻前处理方法，对牛皮肤成纤维细胞进行缓慢冷冻，冰箱预冷平衡1－2h，逐步投入液氮（－196℃）中保存，再经37℃水浴解冻，Hanks液脱保护剂，以贴壁率评价冻存效果。结果表明，20％DMSO保护液对牛皮肤成纤维细胞表现出较好且稳定的冷冻保护效果，其平均贴壁率达87．9％。     

BmNPV半胱氨酸蛋白酶基因的核苷酸序列研究

对家蚕核型多角体病毒苏州株（BmNPVsu）光胱氨酸蛋白酶基因（CP）的序列分析表明,该基因读码框为972个核苷酸,编码323个氨基酸。同源性分析表明,BmNPVsu的CP与首蓿银纹夜蛾核型多角体病毒（AcNPV）、美国白蛾核型多角体病毒（HcNPV）、云杉卷叶蛾核型多角体病毒（CfNPV）、黄杉毒蛾核型多角体病毒（OpNPV）、舞毒蛾核型多角体病毒（Ld-NPV）在DNA水平上的同源性分别为96.5％、76.2％、74.9％、72.7％、62.9％;在氨基酸水平上的同源性分别为96.9％、77.1％、79.3％、77.1％、65.6％。BmNPV CP的氨基酸序列与不同来源的木瓜蛋白酶超家族的CP也具有较高的同源性,特别与 Trpanosoma brucei的CP具有较高的一致性,达32％。在组织蛋白酶B、H、L、S以及木瓜蛋白酶的36个保守氨基酸残基中有31种出现在BmNPVsu的CP中,BmNPVsu CP同其它杆状病毒CP一样,可看作木瓜蛋白酶超家族成员。     

转基因植物对蜜蜂的安全性（下）

转基因植物已在全球范围内大面积推广应用 ,全球 5大转基因植物中的油菜、棉花、玉米均是主要的蜜粉源植物 ,对养蜂生产有重要意义 ,其对蜜蜂的安全性问题值得关注。 2种主要的抗虫 (Bt毒蛋白、蛋白酶抑制剂 )基因或抗真菌基因的产物只有在高浓度时才可能对工蜂的寿命、消化酶活性、行为等有不良影响。从目前的研究来看 ,转基因植物对蜜蜂是安全的     

Feline Ubiquitin Fusion Protein Genes

Using cDNA from a CRFK cell line as a template, PCR amplification was performed with the Ub1S and poly(dT) primers to isolate feline ubiquitin genes. Sequencing of the 495 bp PCR fragment revealed that the putative amino acids induced by this fragment gave a fusion protein consisting of a ubiquitin polypeptide (76 amino acids) and an extension protein of ribosomal proteins L40 (52 amino acids). The putative amino acid sequence of ubiquitin was identical to those of humans, rats and pigs.The recombinant glutathione S-transferase (GST)–feline ubiquitin fusion proteins were produced in Escherichia coli and purified. The fusion proteins had a molecular weight of about 42 kDa and were detected by immunoblot assay with rabbit anti-ubiquitin antiserum.The mRNAs from heat-shocked and non-heat-shocked cells were subjected to RT-PCR (Ub1S and poly(dT) primers) analysis. The molecular weights of the ubiquitinated proteins in heat-shocked CFRK cells were between 18 kDa and 24 kDa by immunoblot assay.These results suggested that there were more ubiquinated proteins in the heat-shocked CRFK cells than in the pre-heat-shocked cells.