Identification of a cDNA/Protein Leading to an Increased P i -uptake in Xenopus laevis Oocytes

In a previous report we documented an increased Na⁺-dependent transport of inorganic phosphate (P _i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P _i -uptake stimulator) lead to a 3-4-fold stimulation of Na⁺-dependent P _i -uptake (10ng cRNA injected, 3–5 days of expression). Na⁺-independent uptake of P _i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K _m -values for the induced Na⁺-dependent uptake were 0.26 ± 0.04 mm for P _i and 14.8 ± 3.0 mm for Na⁺. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P _i -uptake into Xenopus laevis oocytes, but which is not a P _i -transporter itself. Received: 31 July 1996/Revised: 16 October 1996     

鸡传染性喉气管炎病毒gB基因的克隆及其在耻垢分枝杆菌中的表达

以ILTV基因组为模板 ,利用PCR特异扩增出gB基因 ,定向克隆到中间质粒载体pY_α ,构建了中间质粒pY_α_gB。然后以中间质粒pY_α_gB为模板 ,扩增出含有人结核分枝杆菌启动子hsp70基因和堪萨斯分枝杆菌α信号肽基因的hsp_α_gB片段 ,回收补平后与穿梭表达载体pRR3平端连接 ,从而构建大肠杆菌_分枝杆菌穿梭表达质粒pR_α_gB。再将其电转化至耻垢分枝杆菌M .smegmatismc2 15 5 ,ELISA检测表明重组菌株M .smegmatismc2 15 5 (pR_α_gB)的表达产物具有很好的反应原性。Westernblot检测说明gB基因在分枝杆菌中获得了表达并具有良好的免疫原性。鸡胚中和试验结果表明该重组菌株可以中和 1个剂量EID50 的ILTV强毒 ,能够保护SPF鸡胚抵抗强毒攻击     

mRNA差别显示技术及其在生命科学中的应用

ｍＲＮＡ表达水平的变化决定细胞的功能状态,个体发育、细胞增殖分化与凋谢、生理刺激和药物治疗等过程,都会出现ｍＲＮＡ 表达水平的变化。阐明这种变化有助于揭示细胞生理过程的分子机制。该技术无需更多的背景资料,可快速有效地分离表达水平出现差别的基础,这些基因编剧编码的功能多肽在细胞生理过程中扮演着重要角色。     

Diversity of glutamate transporter expression and function in the mammalian retina

Summary. Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. Between photoreceptors and second order neurons, increases in light intensity are signaled by decreases in the concentration of glutamate within the synaptic cleft. In such a system the precise control of glutamate in the synaptic cleft is thus essential and glutamate transporters are thought to contribute to this process. As demonstrated here, all neuronal and macroglial cells of the retina appear to express high-affinity glutamate transporters. GLAST1, GLT1, EAAC1 and EAAT5 are expressed in the retina and exhibit unique localisation and functional properties. In the present study we summarize retinal glutamate transporter expression, identify the major glutamate uptake site in the mammalian retina and discuss the possible functional roles of different glutamate transporter subtypes in glutamatergic neurotranmission in the retina. Received August 31, 1999 Accepted September 20, 1999     

Apolipoprotein D modulates arachidonic acid signaling in cultured cells: implications for psychiatric disorders

Deficiencies in arachidonic acid (AA) parameters have been reported in schizophrenic patients. AA is a primary binding ligand for apolipoprotein D (apoD), which is increased in response to antipsychotic drug treatment and elevated in subjects with schizophrenia and bipolar disorder. In this study, we investigated whether apoD might modulate AA signaling/mobilization in cultured embryonic kidney (HEK) 293T cells. Immunofluorescent labeling revealed both cytosolic and membrane-bound expression of apoD protein in apoD-transfected cells. In cells expressing apoD, phorbal 12-myristate 13-acetate-induced AA release was inhibited compared to controls and membrane levels of AA were elevated, as indicated by the amount of AA maximally incorporated into membrane phospholipids. In addition, exogenous apoD added directly to the incubation media prevented cellular uptake of free [3H]AA. These results suggest that apoD acts to stabilize membrane-associated AA by preventing release and sequestering free AA in the cell. These actions of apoD may be beneficial to psychiatric patients.