目的研究突触小体相关蛋白(snaptosome associated protein of 23 KD,Snap23)与Munc 18C蛋白的相互作用。方法从人肌肉组织中钓取人Snap23基因并克隆入pEYFP-N1载体中,构建pEYFP-Snap23质粒,瞬时转染入CHO细胞后荧光显微镜观察并拍照,...目的研究突触小体相关蛋白(snaptosome associated protein of 23 KD,Snap23)与Munc 18C蛋白的相互作用。方法从人肌肉组织中钓取人Snap23基因并克隆入pEYFP-N1载体中,构建pEYFP-Snap23质粒,瞬时转染入CHO细胞后荧光显微镜观察并拍照,免疫蛋白印迹检测EYFP-Snap23蛋白的表达。共转染pEYFP-Snap23和PECFP-Munc 18c质粒,利用荧光共振能量转移的方法检测二者是否有相互作用,同时共转染pEYFP-Snap23和pECFP-N1作为对照组。结果成功构建pEYFP-Snap23载体,荧光共振能量转移实验证明Snap23与Munc 18C蛋白有相互作用。结论突触小体相关蛋白Snap23可能与Munc 18C相互作用。展开更多
AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ducta...AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS: One and 6 μmol/L carbachol evoked a dose- dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.展开更多
Objective Munc18 is considered as an intracellular protein that plays an important role in exocytosis of neurotransmitters.Previous studies have demonstrated the presence of autoantibodies against Munc18 in a subgroup...Objective Munc18 is considered as an intracellular protein that plays an important role in exocytosis of neurotransmitters.Previous studies have demonstrated the presence of autoantibodies against Munc18 in a subgroup of Rasmussen’s encephalitis patients.However,the machinery of Munc18 autoimmunity is still elusive.The present study was aimed to investigate Munc18 release from primary cultured neurons,Munc18 distribution on the outer plasma membrane of neurons,and the neurotoxicity of Munc18 antibody.Methods The cerebral cortical neurons from embryonic day 17 SpragueDawley rats were prepared and cultured in neurobasal medium.The proteins in culture medium were precipitated with 10% trichloroacetic acid,and analyzed by immunoblotting.The proteins on neuronal surface were biotinylated with EZ-Link-sulfoNHS-LC-Biotin,and collected with avidin-conjugated agarose beads followed by immunoblotting analysis.For cell surface immunofluorescent staining,the living neurons were labeled with anti-Munc18 antibody at 4 °C.Neuronal injury was assessed by lactate dehydrogenase（LDH） release.Results Munc18 was detected in culture medium by immunoblotting analysis.After treatment with 50 μmol/L glutamate for 1 h,Munc18 content in medium was increased.Meanwhile,β-actin and syntaxin1 were not detected in culture medium,and LDH release was not significantly increased.Moreover,glutamate enhanced Munc18 distribution on outer plasma membrane.Living neuron staining also demonstrated the localization of Munc18 on neuronal surface after glutamate treatment,especially at contacting regions between neurons.Glutamate-induced increase of surface Munc18 distribution was suppressed by NMDA receptor antagonist MK801,but not by AMPA receptor antagonist NBQX.Moreover,compared with c-Fos antibody,Munc18 antibody could induce neuronal injury,when culture medium contained the components of serum.Conclusion A portion of Munc18 can be released from neurons or distributed on neuronal surface,which can be enhanced by glutamate treatment via activation of NMDA receptors.Besides,Munc18 antibody-induced neuronal injury depends on the serum components.展开更多
Objective Muncl8-1 has an important role in neurotransmitter release, and controls every step in the exocy- totic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change ...Objective Muncl8-1 has an important role in neurotransmitter release, and controls every step in the exocy- totic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change of Muncl8 localization in neuronal nuclei was analyzed. Methods Epilepsy models were established by injection of kainic acid （KA） solution into hippocampus of Sprague-Dawley （SD） rats or intraperitoneal injection of KA in Kunming mice. The hippocampal neurons were prepared from embryonic day 18 SD rats, and cultured in neurobasal medium, followed by treatment with glutamate for 3 h. Neuronal and glial nuclei of hippocampus were separated by sucrose density gradient centrifugation. The nucleus-enriched fractions were stained with 0.1% Cresyl Violet for morphological assay. Immuno- chemistry and immunoelectron microscopy with anti-Muncl 8-1 antibody were used to determine the nuclear locatization of Munc 18-1. Immunoblotting was used to detect the protein level of Munc 18-1. Results The localization of Munc 18-1 in nucleus of rat hippocampal neuron was confirmed by immunochemistry, immunoelectron microscopy, and immunob- lotting detection of neuronal nucleus fraction. In animals receiving intrahippocampal or intraperitoneal injection of KA, immunostaining revealed that the expression of Muncl 8-1 decreased in pyramidal cell layer of CA regions, as well as in hilus and granular cell layer of dentate gyrus in hippocampus. Moreover, immunoblotting analysis showed that the expres- sion level of Muncl 8-1 in nucleus fraction of hippocampus significantly decreased in KA-treated animals. The relation- ship between the change of Muncl8-1 expression in neuronal nuclei and neuronal over-activation was also tested in pri- mary cultured neurons. After treatment with 50 ~tmol/L glutamate acid for 3 h, Muncl8-1 level was decreased in nucleus fraction and increased in cytoplasmic fraction of primary cultured neurons. Conclusion These results suggest that excit- atory stimulation can induce the distribution change of Munc 18-1 in neuron, which may subsequently modulate neuronal functions in brain.展开更多
文摘目的研究突触小体相关蛋白(snaptosome associated protein of 23 KD,Snap23)与Munc 18C蛋白的相互作用。方法从人肌肉组织中钓取人Snap23基因并克隆入pEYFP-N1载体中,构建pEYFP-Snap23质粒,瞬时转染入CHO细胞后荧光显微镜观察并拍照,免疫蛋白印迹检测EYFP-Snap23蛋白的表达。共转染pEYFP-Snap23和PECFP-Munc 18c质粒,利用荧光共振能量转移的方法检测二者是否有相互作用,同时共转染pEYFP-Snap23和pECFP-N1作为对照组。结果成功构建pEYFP-Snap23载体,荧光共振能量转移实验证明Snap23与Munc 18C蛋白有相互作用。结论突触小体相关蛋白Snap23可能与Munc 18C相互作用。
基金Grants to H.Y.G. from the U.S. National Institute of Health, R21 AA015579-01A1 and to S.V. form the Canadian Institute of Health Research
文摘AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS: One and 6 μmol/L carbachol evoked a dose- dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.
基金supported by the National Natural Science Foundation of China(No.30470536,30670654,90919004)
文摘Objective Munc18 is considered as an intracellular protein that plays an important role in exocytosis of neurotransmitters.Previous studies have demonstrated the presence of autoantibodies against Munc18 in a subgroup of Rasmussen’s encephalitis patients.However,the machinery of Munc18 autoimmunity is still elusive.The present study was aimed to investigate Munc18 release from primary cultured neurons,Munc18 distribution on the outer plasma membrane of neurons,and the neurotoxicity of Munc18 antibody.Methods The cerebral cortical neurons from embryonic day 17 SpragueDawley rats were prepared and cultured in neurobasal medium.The proteins in culture medium were precipitated with 10% trichloroacetic acid,and analyzed by immunoblotting.The proteins on neuronal surface were biotinylated with EZ-Link-sulfoNHS-LC-Biotin,and collected with avidin-conjugated agarose beads followed by immunoblotting analysis.For cell surface immunofluorescent staining,the living neurons were labeled with anti-Munc18 antibody at 4 °C.Neuronal injury was assessed by lactate dehydrogenase（LDH） release.Results Munc18 was detected in culture medium by immunoblotting analysis.After treatment with 50 μmol/L glutamate for 1 h,Munc18 content in medium was increased.Meanwhile,β-actin and syntaxin1 were not detected in culture medium,and LDH release was not significantly increased.Moreover,glutamate enhanced Munc18 distribution on outer plasma membrane.Living neuron staining also demonstrated the localization of Munc18 on neuronal surface after glutamate treatment,especially at contacting regions between neurons.Glutamate-induced increase of surface Munc18 distribution was suppressed by NMDA receptor antagonist MK801,but not by AMPA receptor antagonist NBQX.Moreover,compared with c-Fos antibody,Munc18 antibody could induce neuronal injury,when culture medium contained the components of serum.Conclusion A portion of Munc18 can be released from neurons or distributed on neuronal surface,which can be enhanced by glutamate treatment via activation of NMDA receptors.Besides,Munc18 antibody-induced neuronal injury depends on the serum components.
基金supported by grants from the National Natural Science Foundation of China (No. 81071017, 30470536, 90919004)
文摘Objective Muncl8-1 has an important role in neurotransmitter release, and controls every step in the exocy- totic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change of Muncl8 localization in neuronal nuclei was analyzed. Methods Epilepsy models were established by injection of kainic acid （KA） solution into hippocampus of Sprague-Dawley （SD） rats or intraperitoneal injection of KA in Kunming mice. The hippocampal neurons were prepared from embryonic day 18 SD rats, and cultured in neurobasal medium, followed by treatment with glutamate for 3 h. Neuronal and glial nuclei of hippocampus were separated by sucrose density gradient centrifugation. The nucleus-enriched fractions were stained with 0.1% Cresyl Violet for morphological assay. Immuno- chemistry and immunoelectron microscopy with anti-Muncl 8-1 antibody were used to determine the nuclear locatization of Munc 18-1. Immunoblotting was used to detect the protein level of Munc 18-1. Results The localization of Munc 18-1 in nucleus of rat hippocampal neuron was confirmed by immunochemistry, immunoelectron microscopy, and immunob- lotting detection of neuronal nucleus fraction. In animals receiving intrahippocampal or intraperitoneal injection of KA, immunostaining revealed that the expression of Muncl 8-1 decreased in pyramidal cell layer of CA regions, as well as in hilus and granular cell layer of dentate gyrus in hippocampus. Moreover, immunoblotting analysis showed that the expres- sion level of Muncl 8-1 in nucleus fraction of hippocampus significantly decreased in KA-treated animals. The relation- ship between the change of Muncl8-1 expression in neuronal nuclei and neuronal over-activation was also tested in pri- mary cultured neurons. After treatment with 50 ~tmol/L glutamate acid for 3 h, Muncl8-1 level was decreased in nucleus fraction and increased in cytoplasmic fraction of primary cultured neurons. Conclusion These results suggest that excit- atory stimulation can induce the distribution change of Munc 18-1 in neuron, which may subsequently modulate neuronal functions in brain.
