qBrn-2, a POU III gene in quail: distinct developmental expression revealed by a specific antibody

We examined qBrn-2 protein expression in quail from its onset to final profile with a specific antibody we prepared. qBrn-2 expression employed onset-widespread-restriction pattern, and precisely concurred with formation and differentiation of neural tube. qBrn-2 protein was also located outside neural tube. Obvious differences in expression were observed compared with that of Brn-2.     

Developmental expression of the POU transcription factor qBrn-2 during somitic myogenesis in quail

We prepared a specific antiserum to the qBrn-2 protein and examined the developmental distribution of this protein during quail somitic myogenesis. In contrast to its mammalian homolog N-Oct-3, qBrn-2 exhibited an impressive spatio-temporal profile in somitic myogenesis, in addition to the orthodox expression observed in the developing neural tube. In somites, qBrn-2 was expressed in the outer epithelial cells, but not in the core cells. During the somite differentiation, qBrn-2 expression was enhanced and restricted to myotome. The location of qBrn-2 expression seemed to overlap with that of myf5 and myoD in myotome. However, in cells that just began to express myf5 or myoD, qBrn-2 expression was not obvious. As embryonic development proceeded, qBrn-2 positive cells in myotome migrated dorsally and ventrally, and qBrn-2 expression was still observed at dorsal and ventral muscle masses in the forelimb. On the basis of our observations, it seems that qBrn-2 may play important roles in the determination, differentiation and migration of muscle precursor cells, in addition to its known roles in neurogenesis.     

大肠埃希菌Mn-SOD基因的克隆、表达及多克隆抗体制备

目的实现Mn-SOD基因在大肠埃希菌中的高可溶性表达,制备Mn-SOD的多克隆抗体。方法用PCR方法从一株野生型大肠埃希菌（E.coli）基因组中扩增Mn-SOD基因编码区．将它克隆到原核表达载体上进行大量表达和纯化,再用纯化的蛋白对新西兰大白兔进行背部多点注射,40d后取其血清,用Western-blot印迹实验测定抗体效果。结果SDS-PAGE分析表明SOD的表达量约为细菌总蛋白的50％;黄嘌呤氧化酶法测定表达蛋白活性,结果表明每毫克菌体可溶性总蛋白中表达产物酶比活为3921．77U／mg,是对照BL21的276．77倍;并制备了高效价的多克隆抗体。结论该研究成功地构建了大肠埃希菌Mn-SOD基因高效原核表达系统,所表达的Mn-SOD具有良好的免疫原性和免疫反应性。     

大鼠脑红蛋白(NGB)的原核表达、抗体制备及其细胞分布

脑红蛋白 (NGB)是新发现的与脑内氧供应密切相关的分子 .为了检测细胞内脑红蛋白的表达、亚细胞分布从而对该分子进行深入的功能研究 ,成功地将大鼠脑红蛋白基因编码区构建于原核表达载体pGEX 4T 2 ,转化大肠杆菌BL2 1(DE3) ,获得融合表达产物 .对含有融合蛋白的包含体进行溶解和复性 ,用谷胱甘肽S 转移酶 (GST)亲和层析柱纯化 ,通过免疫家兔获得了兔源性抗NGB多克隆抗体 .采用Western印迹分析技术 ,用该抗体检测NGB基因的真核表达产物 ,证明该抗体有较好的针对NGB蛋白的专一性 ,可用于对NGB的结构和功能研究 .同时 ,用该抗体进行免疫组化分析发现 ,正常成年大鼠神经系统中有较多的NGB免疫反应阳性细胞分布 ,提示NGB是与神经系统功能密切相关的重要分子     

A monoclonal antibody recognizing a common antigen on neurons and fibroblasts in chicken and quail

A monoclonal antibody, FiN1, obtained by immunization of a mouse with homogenates of embryonic quail nodose ganglia, was found to react with a surface antigenic determinant, both in quail and chick, present on practically all neurons of the spinal cord and of the peripheral nervous system and on a subpopulation of fibroblasts. An ontogenetic study performed on tissue sections, cell suspensions and cultures showed that FiN1 defines a differentiation marker which appears relatively late in development, during the second half of embryonic life, and persists after hatching. The onset and evolution of its expression during development varies in a tissue-specific manner.     

hSef基因在不同人组织和细胞系的表达分布

将人源Sef蛋白胞内区编码序列与GST融合构建原核表达质粒进行重组蛋白的表达与纯化并制备多抗 .在COS 7细胞中转染表达hSef显示 ,其分子量分别为 80kD ,10 0kD ,比体外翻译的分子量偏大 ,提示可能有糖基化存在 .Northern印迹的结果表明 ,hSefmRNA主要分布在人肾和睾丸组织 .RT PCR检测到hSefmRNA在众多细胞系有广泛存在 .免疫组化的结果显示 ,hSef蛋白在人肾和睾丸及相应癌组织表达水平较高 .     

The role of gicerin, a novel cell adhesion molecule, in development, regeneration and neoplasia

Neurite outgrowth factor (NOF) is an extracellular matrix (ECM) protein in the laminin family and its ligand, gicerin, is a novel cell adhesion molecule in the immunoglobulin superfamily. Gicerin has a homophilic adhesive activity as well as a heterotypic manner to NOF. In the nervous systems, gicerin is expressed during developmental stage when neurons migrate or extend neurites to form a neural network. Gicerin promotes neurite extension and migration of embryonic neurons in vitro by its homophilic and heterophilic adhesion activities. Introduction of antigicerin antibody into early developing eyes perturbs the layer formation of neural retina. These data suggest that gicerin participates in the formation of neural tissues. Gicerin is also expressed in other non-neural tissues; in epithelia of trachea, kidney and oviduct, gicerin expression is restricted in the developmental period. In contrast, muscular tissues and endothelial cells express gicerin continuously even after maturation. Interestingly, gicerin re-appears strongly in the regenerating epithelia of trachea, kidney and oviduct, and also anti-gicerin antibody disrupts the healing process of trachea. Furthermore, gicerin and NOF are overexpressed in the chicken nephroblastomas (Wilm's tumor) and oviductal adenocarcinomas. In vitro analyses show that gicerin adhesive activities can promote binding among tumor cells and adhesion of tumor cells to NOF. A polyclonal antibody against gicerin also perturbs the re-attachment of cancer cells onto metastasizing sites. It is clear from these studies that gicerin is a potential effector for pathological tissue formation as well as for normal development.     

Galectin-1 plays essential roles in adult mammalian nervous tissues. Roles of oxidized galectin-1

Previous data have suggested that galectin-1 is expressed widely in nervous tissues at embryonic stages but becomes restricted mainly to peripheral nervous tissues with maturation. Though the expression is intense in adult mammalian peripheral neurons, there had been no report about functions of galectin-1 there. Recently we discovered a factor that enhanced peripheral axonal regeneration. The factor was identified as oxidized galectin-1 with three intramolecular disulfide bonds and showed no lectin activity. Oxidized recombinant human galectin-1 (rhGAL-1/Ox) showed the same nerve growth promoting activity at very low concentrations (pg/ml). rhGAL-1/Ox at similarly low concentration was also effective in in vivo experiments of axonal regeneration. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited the axonal regeneration in vivo as well as in vitro. Since galectin-1 is expressed in the regenerating sciatic nerves as well as in both sensory neurons and motor neurons, these results suggest that galectin-1 is secreted into the extracellular space to be oxidized, and then, in its oxidized form, to regulate initial repair after axotomy. The administration of oxidized galectin-1 effectively promoted functional recovery after sciatic nerve injury in vivo. Oxidized galectin-1, hence, appears to play an important role in promoting axonal regeneration, working as a kind of cytokine, not as a lectin. Recent reports indicated additional roles of cytosolic galectin-1 in neural diseases, such as ALS. Furthermore galectin-1 has been proved to be a downstream target of DeltaFosB. In hippocampus of rat brain, expression of DeltaFosB is induced immediately after ischemia-reperfusion, suggesting that galectin-1 may also play important roles in central nervous system after injury.     

The specificity of the myelin basic protein gene promoter studied in transgenic mice.

The myelin basic proteins (MBPs) are a family of polypeptides that are predominantly expressed in the nervous system where they play a major role in myelination. We have generated four lines of transgenic mice carrying a transgene in which 1.34 kb of the 5'-flanking sequence of the mouse MBP gene was fused upstream of the coding region of the Escherichia coli lac Z gene in order to investigate developmental and tissue-specific expression of the MBP gene. Expression of both the lacZ transgene and the endogenous MBP gene followed a common developmental pattern in mouse brain. Transgene expression was detected in primary oligodendrocytes, but not in type 2 astrocytes. In addition, the lacZ gene product was expressed in epithelial cells of certain nonneural tissues, namely kidney, epididymis, ureter, and seminal vesicles. The ectopic expression of the transgene was associated with the development of DNase I hypersensitive sites at the site of insertion which was found to be within the intron 1 region of the endogenous MBP gene. The results reported here strongly suggest that the 1.34-kb 5'-flanking region of the MBP gene contains cis-regulatory elements that confer developmental regulation of the MBP gene, although this region appears to lack elements that restrict its expression to the nervous system.     

The Sodium-Dependent Inorganic Phosphate Transporter SLC34A1 (NaPi-IIa) Is Not Localized in the Mouse Brain: A Case of Tissue-Specific Antigenic Cross-Reactivity

The sodium-dependent inorganic phosphate transporter NaPi-IIa is expressed in the kidney. Here, the authors used a polyclonal antiserum raised against NaPi-IIa- and NaPi-IIa-deficient mice to characterize its expression in nervous tissue. Western blots showed that a NaPi-IIa immunoreactive band (~90 kDa) was only present in wild-type kidney membranes and not in kidney knockout or wild-type brain membranes. In the water-soluble fraction of wild-type and knockout brains, another band (~50 kDa) was observed; this band was not detected in the kidney. Light and electron microscopic immunohistochemistry using the NaPi-IIa antibodies showed immunolabeling of kidney tubules in wild-type but not knockout mice. In the brain, labeling of presynaptic nerve terminals was present also in NaPi-IIa-deficient mice. This labeling pattern was also produced by the NaPi-IIa preimmune serum. The authors conclude that the polyclonal antiserum is specific toward NaPi-IIa in the kidney, but in the brain, immunolabeling is caused by a cross-reaction of the antiserum with an unknown cytosolic protein that is not present in the kidney. This tissue-specific cross-reactivity highlights a potential pitfall when validating antibody specificity using knockout mouse-derived tissue other than the specific tissue of interest and underlines the utility of specificity testing using preimmune sera.     

一个麻疯树RING型锌指蛋白基因JcRFP1的克隆及表达

首次从麻疯树胚乳cDNA丈库中克隆得到一个RJNG型锌指蛋白基（GenBank登录号为JF920726）,命名为JcRFP1。该cDNA长度为728bp,包含编码JcRFP1蛋白的完整开放阅读框（516bp）。脚,基因在麻疯树各器官中均检测到表达且表达量依次为：叶〉茎〉花〉果实〉种子〉根。将克隆到的JcRFP1基因的cDNA序列连接到表达载体pET32a（＋）上,导入BL21（DE3）pLysS菌株,成功诱导表达相对分子质量为33．2kDa的可溶性融合蛋白。该融合蛋白免疫新西兰大白兔,得到效价为1：6500的抗血清。研究表明,JcRFP1蛋白具有体外泛素连接酶E3活性,在麻疯树体内可能参与油菜素甾醇信号转导途径。