碱性成纤维细胞生长因子受体基因在牛眼晶体上皮细胞内的表达

目的探讨碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ，ｂＦＧＦ）对晶体上皮细胞促增殖作用的机理。方法从培养的第二代牛眼晶体上皮细胞提取ＲＮＡ，经逆转录反应合成ｃＤＮＡ。借助从人体胎盘纤维细胞ｂＦＧＦ受体序列合成的寡核苷酸引物，聚合酶链反应体外扩增ｃＤＮＡ。扩增的ｃＤＮＡ片段克隆后，Ｓａｎｇｅｒ双脱氧链终止法测定其序列。结果探测出牛眼晶体上皮细胞ｂＦＧＦ受体的ｍＲＮＡ，测序后发现其氨基酸序列片段中仅３个氨基酸与人体不同。结论晶体上皮细胞存在ｂＦＧＦ受体，当ｂＦＧＦ与其受体结合后，对促进晶体上皮细胞的增殖以及白内障术后后囊混浊具有重要作用。     

人自然杀伤细胞抑制性受体P58.2基因克隆与鉴定

目的：P58．2基因克隆与鉴定。方法：采用RT—PCR技术，从正常人外周血单个核细胞中扩增自然杀伤细胞抑制性受体P58．2基因全长cDNA，经酶切后构建重组克隆载体，双酶切和测序鉴定。结果：RT—PCR获得了预期的扩增产物P58．2全长cDNA，成功构建了pSPORT1-P58．2重组克隆载体，酶切、酶谱分析与预期结果相符。DNA测序结果与GenBank登记的人P58．2cDNA全长碱基序列一致。结论：pSPORT1-P58．2重组克隆载体构建成功；P58．2基因序列与文献报道一致，为下一步构建表达载体和基因转染等工作打下良好的基础。     

Excitatory amino acid receptor regulation after subthalamic nucleus lesions in the rat

The subthalamic nucleus plays a pivotal role in the regulation of basal ganglia output. Recent electrophysiologic, lesion and immunocytochemical studies suggest that the subthalamic nucleus uses an excitatory amino acid as a neurotransmitter. After complete ablation of the subthalamic nucleus, we have examined the NMDA, AMPA, kainate and metabotropic subtypes of excitatory amino acid receptors in two major subthalamic projection areas (globus pallidus and substantia nigra pars reticulata) with quantitative autoradiography. Two weeks after ablation, binding sites for [³H]AMPA and [³H]kainate increased in substantia nigra pars reticulata ipsilateral to the lesion. In globus pallidus on the lesioned side, [³H]glutamate binding to the NMDA recognition site decreased. The results suggest that glutamate receptors regulate after interruption of subthalamic nucleus output.     

cAMP对胰岛素刺激的NIH3T3细胞丝裂原激活蛋白激酶活性的抑制作用

目的 探讨cAMP-PKA信号途径对胰岛素激活的Ras-丝裂原激活蛋白激酶(MAPK)信号途径的影响。方法 以3-异丁基-1-甲基黄嘌呤(IBMX)提高细胞内环腺苷酸(cAMP)浓度，藉四唑蓝比色法(MTT法)和蛋白免疫印迹试验分别观察cAMP对胰岛素刺激的小鼠成纤维细胞(NIH3T3细胞)的生长增殖和胞内丝裂原激活蛋白激酶(MAPK)活性的影响。结果 IBMX(0．5mmol/L)抑制静息的和胰岛素(10mU／mL)刺激的NIH3T3细胞的增殖，作用呈时间依赖性。在胰岛素(10mU／mL)刺激的NIH3T3细胞，MAPK出现酪氨酸磷酸化；而以IBMX(0．5mmol/L)预处理细胞30min后，再以胰岛素作用10min，胰岛素刺激的MAPK的酪氨酸磷酸化受抑制。结论：cAMP可通过抑制MAPK磷酸化活化对NIH3T3细胞增殖起抑制作用。     

内皮素-1及受体基因在良性前列腺增生组织中的表达及意义

目的　探讨内皮素 1(ET 1)及受体A、B(ETAR、ETBR)在良性前列腺增生 (BPH)组织中的表达及意义。　方法　应用免疫组化和RT PCR方法检测前列腺组织ET 1及ETAR、ETBRmRNA的表达 ,并与患者临床参数行相关分析。　结果　ET 1、ETARmRNA和ETBRmRNA在BPH组织中的表达 (吸光度A值分别为 0 .94± 0 .0 8、0 .6 4± 0 .0 8、0 .97± 0 .0 8)与正常前列腺组织(0 .5 7± 0 .0 6、0 .37± 0 .0 5、0 .5 1± 0 .0 4 )相比 ,差别均有显著性意义 ,P均 <0 .0 5 ,其中ET 1、ETARmRNA与IPSS、前列腺体积、前列腺尿道长度、前列腺尿道压、最大尿道压呈正相关 ,与最大尿流率、平均尿流率呈负相关。　结论　ET 1和ETAR在BPH中的表达量与膀胱出口梗阻严重程度密切相关。     

p38丝裂原活化蛋白激酶在糖尿病大鼠神经病理性痛中的作用

目的探讨p38丝裂原活化蛋白激酶（p38MAPK）在链脲菌素诱导的糖尿病大鼠神经病理性痛中的作用。方法雌性Wistar大鼠31只，3月龄，体重180～220g，随机分为3组：对照组（C组，n=10）、糖尿病神经病理性痛组（D组，n=11）和p38MAPK抑制剂组（Ⅰ组，n=10）。D组、Ⅰ组单次腹腔注射链脲菌素65mg／kg制备糖尿病模型。糖尿病模型制备成功后，Ⅰ组尾静脉注射p38MAPK抑制剂SB203580 0．5mg／kg，1次／周，连续4周；C组和D组尾静脉注射等体积的生理盐水。给药4周后，测定机械缩足反应阈值（MWT）、左侧坐骨神经传导速率（NCV）、背根神经节（DRG）和脊髓的磷酸化p38MAPK水平。结果与C组比较，D组、Ⅰ组MWT下降，NCV减慢，伴有脱髓鞘现象，DRG和脊髓的磷酸化p38MAPK水平升高；与D组比较，Ⅰ组MWT升高，NCV增快，脱髓鞘程度减轻，DRG和脊髓的磷酸化p38MAPK水平下降。结论p38MAPK信号转导通路参与了糖尿病大鼠神经病理性痛的形成。     

Activation of a Ca2+-dependent K+ current by the oncogenic receptor protein tyrosine kinase v-Fms in mouse fibroblasts

We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K⁺ current that is absent in control cells. The activation of the K⁺ current was Ca²⁺-dependent, voltage-independent, and was completely blocked by the K⁺ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC₅₀ values of 3nM, 18 nM and 76nM, respectively. To identify signalling components that mediate the activation of this K⁺ current, NIH3T3 cells that express different mutants of the wildtype v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K⁺ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K⁺ current. Treatment of wild-type v-Fms cells with Clostridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K⁺ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K⁺ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K⁺ channel activation by the oncogenic RTK v-Fms. Received: 19 November 1997 / Accepted: 21 January 1998     

Lack of effect of opioid peptides,morphine and naloxone on superoxide formation in human neutrophils and HL-60 leukemic cells

Summary There are controversial reports in the literature concerning the effects of opioids on superoxide (O ₂ ^– ) formation in phagocytes, these agents being either inhibitory or stimulatory. We re-examined this issue and compared the effects of the Chemotactic peptide, N-formyl-l,-methionyl-l-leucyl-l-phenylalanine (fMet-Leu-Phe), phorbol myristate acetate (PMA), ATP, platelet activating factor (PAF), cytochalasin B (CB) and prostaglandin E₁ (PGE₁) with those of various opioids on O ₂ ^– formation in human neutrophils and HL-60 leukemic cells under defined experimental conditions. In the presence of CB, fMet-Leu-Phe and PAF concentration-dependently activated O ₂ ^– formation in neutrophils with EC₅₀ values of 20 nM and 100 nM, respectively. In the absence of CB, fMet-Leu-Phe and PAF were much less effective. PAF synergistically enhanced O ₂ ^– formation induced by fMet-Leu-Phe. ATP at a concentration of 100 M and the opioids, methionine enkephalin, -endorphin, dynorphin, [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin, [d-Ala²-d-Leu⁵]-enkephalin and morphine at concentrations between 10 pM to 1 M did not activate O ₂ ^– formation. ATP but not \-endorphin potentiated fMet-Leu-Phe-induced O ₂ ^– formation. O ₂ ^– formation induced by a maximally stimulatory concentration of PMA (100 ng/ml) was enhanced by fMet-Leu-Phe but was unaffected by methionine enkephalin or PGE₁. PMA at a non-stimulatory concentration (2 ng/ml) potentiated the effect of fMet-Leu-Phe but did not induce responsiveness to PAF, ATP or -endorphin. PGE₁ strongly inhibited fMet-Leu-Phe-induced O ₂ ^– formation, whereas morphine, methionine enkephalin and the opioid antagonist, naloxone, were without effect. In HL-60 cells differentiated with dibutyryl cAMP, fMet-Leu-Phe, PAF and ATP but not -endorphin activated O ₂ ^– formation. Our results show that O ₂ ^– formation is differentially regulated by various classes of intercellular signal molecules and that opioids do not play a role in the regulation of O ₂ ^– formation. The precise definition of the experimental conditions and control experiments with established modulators of O ₂ ^– formation are essential to evaluate the role of opioids in the regulation of this effector system.Send offprint requests to R. Seifert at the above address     

LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus

Cellular attachment factors like the C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) can augment viral infection and might promote viral dissemination in and between hosts. The lectin LSECtin is encoded in the same chromosomal locus as DC-SIGN/R and is coexpressed with DC-SIGNR on sinusoidal endothelial cells in liver and lymphnodes. Here, we show that LSECtin enhances infection driven by filovirus glycoproteins (GP) and the S protein of SARS coronavirus, but does not interact with human immunodeficiency virus type-1 and hepatitis C virus envelope proteins. Ligand binding to LSECtin was inhibited by EGTA but not by mannan, suggesting that LSECtin unlike DC-SIGN/R does not recognize high-mannose glycans on viral GPs. Finally, we demonstrate that LSECtin is N-linked glycosylated and that glycosylation is required for cell surface expression. In summary, we identified LSECtin as an attachment factor that in conjunction with DC-SIGNR might concentrate viral pathogens in liver and lymph nodes.     

PD98059对人骨髓间质干细胞分化为成骨细胞的影响

目的:探讨胞外信号调节激酶(ERK)信号传导途径对骨髓间质干细胞(MSC)分化为成骨细胞的影响。方法:采用Ficoll-Paque淋巴细胞分离液分离成人MSC,体外扩增,应用地塞米松、β-甘油磷酸钠、vitaminC定向诱导MSC分化为成骨细胞。在成骨诱导液中加入不同剂量的PD98059,观察其对成骨细胞形成的影响。结果:MSC体外扩增15代可获得(3-4)×10¹²个细胞。在成骨诱导液作用下,MSC可在体外定向分化为成骨细胞。不同剂量的PD98059均可抑制MSC分化为成骨细胞,并有剂量依赖关系;同时促使部分细胞转化为脂肪细胞。结论:ERK信号传导途径可能在MSC分化为成骨细胞和脂肪细胞过程中起关键作用。