Molecular characterization of the mouse Tem1/endosialin gene regulated by cell density in vitro and expressed in normal tissues in vivo

Human tumor endothelial marker 1/endosialin (TEM1/endosialin) was recently identified as a novel tumor endothelial cell surface marker potentially involved in angiogenesis, although no specific function for this novel gene has been assigned so far. It was reported to be expressed in tumor endothelium but not in normal endothelium with the exception of perhaps the corpus luteum. Here we describe the cDNA and genomic sequences for the mouse Tem1/endosialin homolog, the identification and characterization of its promoter region, and an extensive characterization of its expression pattern in murine and human tissues and murine cell lines in vitro. The single copy gene that was mapped to chromosome 19 is intronless and encodes a 92-kDa protein that has 77.5% overall homology to the human protein. The remarkable findings are 1) this gene is ubiquitously expressed in normal human and mouse somatic tissues and during development, and 2) its expression at the mRNA level is density-dependent and up-regulated in serum-starved cells. In vitro, its expression is limited to cells of embryonic, endothelial, and preadipocyte origin, suggesting that the wide distribution of its expression in vivo is due to the presence of vascular endothelial cells in all the tissues. The ubiquitous expression in vivo is in contrast to previously reported expression limited to corpus luteum and highly angiogenic tissues such as tumors and wound tissue.     

The lipophilicity of phorbol esters as a critical factor in determining the pattern of translocation of protein kinase C delta fused to green fluorescent protein

Our previous study showed differential subcellular localization of protein kinase C (PKC) delta by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC delta. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13-dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13-dibutyrate and phorbol 12,13-dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nM to 10 microM). The highly lipophilic derivatives, phorbol 12,13-didecanoate and phorbol 12, 13-diundecanoate, at 1 microM caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC delta localization and thereby potentially to their different biological activities.     

小鼠淋巴细胞抗原CTLA-4的胞外肽段表达及纯化

目的: 真核细胞表达小鼠淋巴细胞抗原CTLA-4胞外段肽,研究表达肽段与抗原呈递细胞B7分子结合后减轻小鼠淋巴细胞刺激后的增殖抑制,从而启动T淋巴细胞进一步增殖。方法:从小鼠脾脏淋巴细胞获得总RNA,通过逆转录PCR扩增出CTLA-4全长基因,克隆并测序。依据胞外段序列和真核表达载体pcDNA3.1序列,合成引物扩增胞外片段,两者经内切核酸酶处理、连接构建重组表达pcDNA3.1载体,重组质粒经测序验证后,采用lipofectamine 2000转染入小鼠肝癌细胞Hepa1-6,经G418筛选获得稳定表达细胞株。结果:获得小鼠CTLA-4胞外段真核表达载体和小鼠肝癌细胞Hepa1-6稳定表达转染细胞株,制备了CTLA-4胞外肽段,经His标签抗体和小鼠CTLA-4抗体Western blot检测表达蛋白带均呈阳性。结论:获得CTLA-4胞外段肽,为进一步研究该肽的作用打下基础。     

博尔纳病病毒对BALB/c小鼠感染性检测

目的分析博尔纳病病毒（Borna disease virus,BDV）H1766株对BALB/c小鼠的感染性。方法选择病毒滴度为2.0×107FFU/ml的BDV病毒液分别对新生和成年BALB/c小鼠进行脑内接种,并用相同病毒液对原代培养的新生BALB/c小鼠脑细胞进行接种。经过一定时间的病毒作用后分别提取总RNA,采用巢式RT-PCR方法检测BDV-p40基因,并通过免疫组化方法检测脑内接种脑组织中BDV-P40蛋白。结果脑内接种病毒的小鼠脑组织中可以检测到BDV-p40基因和BDV-P40蛋白,培养的小鼠脑细胞中可以检测到BDV-p40基因。结论BDVH1766株可以感染新生和成年的BALB/c小鼠。