Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI- 1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell- bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.     

Function and expression of somatostatin receptors of the endocrine pancreas

Somatostatin (SST) regulates multiple biological processes via five genetically distinct, G-protein coupled receptors. Clinical interest in therapy for neuroendocrine and metabolic disorders has resulted in the development of new tools for exploring the function of somatostatin receptors (SSTRs). The development of highly SSTR-selective agonists and antagonists, animal models with the deletion of individual SSTRs, as well as SSTR-specific antibodies have all been utilized in delineating SSTR functions. In the pancreas, SST is a potent regulator of insulin and glucagon secretion. Indeed, the inappropriate regulation of pancreatic A- and B-cell function in metabolic diseases provides an impetus to evaluate the SSTRs as therapeutic targets. By combining the results obtained from molecular biology, pharmacology and immunochemical studies the current review provides a summary of important recent developments which have extended our knowledge of SST actions in the endocrine pancreas.     

Orexin-A inhibits glucagon secretion and gene expression through a Foxo1-dependent pathway

Orexin-A (OXA) regulates food intake and energy homeostasis. It increases insulin secretion in vivo and in vitro, although controversial effects of OXA on plasma glucagon are reported. We characterized the effects of OXA on glucagon secretion and identify intracellular target molecules in glucagon-producing cells. Glucagon secretion from in situ perfused rat pancreas, isolated rat pancreatic islets, and clonal pancreatic A-cells (InR1-G9) were measured by RIA. The expression of orexin receptor 1 (OXR1) was detected by Western blot and immunofluorescence. The effects of OXA on cAMP, adenylate-cyclase-kinase (AKT), phosphoinositide-dependent kinase (PDK)-1, forkhead box O-1 (Foxo1), and cAMP response element-binding protein were measured by ELISA and Western blot. Intracellular calcium (Ca(2+)(i)) concentration was detected by fura-2and glucagon expression by real-time PCR. Foxo1 was silenced in InR1-G9 cells by transfecting cells with short interfering RNA. OXR1 was expressed on pancreatic A and InR1-G9 cells. OXA reduced glucagon secretion from perfused rat pancreas, isolated rat pancreatic islets, and InR1-G9 cells. OXA inhibited proglucagon gene expression via the phosphatidylinositol 3-kinase-dependent pathway. OXA decreased cAMP and Ca(2+)(i) concentration and increased AKT, PDK-1, and Foxo1 phosphorylation. Silencing of Foxo1 caused a reversal of the inhibitory effect of OXA on proglucagon gene expression. Our study provides the first in vitro evidence for the interaction of OXA with pancreatic A cells. OXA inhibits glucagon secretion and reduces intracellular cAMP and Ca(2+)(i) concentration. OXA increases AKT/PDK-1 phosphorylation and inhibits proglucagon expression via phosphatidylinositol 3-kinase- and Foxo-1-dependent pathways. As a physiological inhibitor of glucagon secretion, OXA may have a therapeutic potential to reduce hyperglucagonemia in type 2 diabetes.     

人成釉蛋白抗体的制备及组织表达特异性

目的:制备抗人成釉蛋白抗体,观察成釉蛋白在各组织中的表达。方法:实验于2002-03在解放军第四军医大学基础部生物化学与分子生物学教研室完成。以重组、纯化的成釉蛋白C端肽为抗原,混入完全/不完全弗氏佐剂,免疫新西兰大白兔,经5次免疫后,颈动脉取血,分离血清并用饱和硫酸铵纯化,制备兔抗人成釉蛋白多克隆抗体,用双向免疫扩散试验和ELISA检测抗体的效价。用WesternBlot检测人成釉蛋白的组织表达特异性。结果:①ELISA检测结果:表明兔抗人成釉蛋白多克隆抗体效价达到1∶10000。②WesternBlot显示:成釉蛋白在人牙胚组织总蛋白中有特异性表达,相对分子质量约为65000,在脑、心、肝、脾、肺、肾、胰腺、胸腺、骨骼肌等组织中未见表达条带。结论:制备了抗人成釉蛋白抗体,为研究成釉蛋白在人牙胚中的组织表达以及利用抗体纯化蛋白提供了基础,从蛋白水平证实成釉蛋白为牙胚组织特异性蛋白,并证实人牙胚组织中的成釉蛋白相对分子质量约为65000。     

延边朝鲜族新生儿血红蛋白Gγ/(Gγ+Aγ)比值测定

目的:血红蛋白γ链包括Gγ和Aγ,基因图谱分析发现新生儿常有γ链基因的异常。实验测定延边朝鲜族新生儿血红蛋白Gγ/(Gγ Aγ)比值,对于研究人类遗传变异和基因调节具有重要意义。方法:实验于2005-12/2007-04在延边大学医学部基础医学院生物化学与分子生物学教研室完成。①材料来源:97例朝鲜族新生儿脐带血由延边大学医院妇产科产室提供,产妇均签署知情同意书。②实验方法:新生儿脐带血取样后立即冷冻于液氮之中,EDTA-Na2抗凝,进行血红蛋白解链,每500μL脐带血与0.9%的NaCl溶液1000μL相混合稀释,离心去上清,分离血红蛋白。用酸性聚丙烯酰胺以凝胶电泳法分离血红蛋白中的Gγ和Aγ肽链,电压200V,电泳50min,电泳方向为正极到负极。电泳后凝胶用丽春红2R染色,乙酸脱色致本底无色。用UVP扫描凝胶成像分析系统对凝胶板上的Gγ和Aγ肽链进行扫描定量,测定血红蛋白中Gγ/(Gγ Aγ)比值。结果:朝鲜族新生儿脐带血中的血红蛋白Gγ/(Gγ Aγ)比值分布情况:1例处于30%～48%低Gγ区,占总例数的1.03%,平均值27.60%;96例处于50%～79%中间区,占总例数的98.97%,平均值(68.40±2.90)%,明显低于相关文献报道的新疆维吾尔族、广西壮族、北京市汉族、西藏藏族新生儿Gγ/(Gγ Aγ)的整体均值(75.4±2.50)%,差异有显著性意义(P<0.05);未发现>80%高Gγ区者。结论:延边朝鲜族新生儿血红蛋白Gγ/(Gγ Aγ)比值多分布于50%～79%中间区,其平均值与其他民族比较相对较低。