Alcoholism and acquired immune deficiency syndrome are associated with severe muscle wasting.This impairment in nitrogen balance arises from increased protein degradation and a decreased rate of protein synthesis.The ...Alcoholism and acquired immune deficiency syndrome are associated with severe muscle wasting.This impairment in nitrogen balance arises from increased protein degradation and a decreased rate of protein synthesis.The regulation of protein synthesis is a complex process involving alterations in the phosphorylation state and protein-protein interaction of various components of the translation machinery and mammalian target of rapamycin(mTOR) complexes.This review describes mechanisms that regulate protein synthesis in cultured C2C12 myocytes following exposure to either alcohol or human immunodeficiency virus antiretroviral drugs.Particular attention is given to the upstream regulators of mTOR complexes and the downstream targets which play an important role in translation.Gaining a better understanding of these molecular mechanisms could have important implications for preventing changes in lean body mass in patients with catabolic conditions or illnesses.展开更多
目的研究srGAP2(Slit-Robo GTPase activating Protein 2)、γ-氨基丁酸以及单核细胞趋化蛋白-1(MCP-1)在难治性癫痫患儿脑组织中的表达情况及意义。方法选取11例患儿的颞叶脑组织和同期11例正常标本的颞叶脑组织,使用免疫组化和免疫印...目的研究srGAP2(Slit-Robo GTPase activating Protein 2)、γ-氨基丁酸以及单核细胞趋化蛋白-1(MCP-1)在难治性癫痫患儿脑组织中的表达情况及意义。方法选取11例患儿的颞叶脑组织和同期11例正常标本的颞叶脑组织,使用免疫组化和免疫印迹等方法检测srGAP2、γ-氨基丁酸以及MCP-1的表达情况。结果难治性癫痫患儿脑组织中,srGAP2的阳性表达率显著高于正常脑组织。难治性癫痫患儿脑组织和正常脑组织可以看到染成棕褐色的神经元,但难治性癫痫患儿脑组织γ-氨基丁酸能神经元表达量明显低于正常脑组织。正常脑组织中MCP-1的表达较弱,而难治性癫痫患儿脑组织中MCP-1的表达较强。结论难治性癫痫患儿脑组织srGAP2和MCP-1表达上升而γ-氨基丁酸表达下降,srGAP2、γ-氨基丁酸及MCP-1与难治性癫痫发病密切相关,可能作为潜在治疗靶标。展开更多
目的:对免疫相关GTP结合蛋白2(GTPase of immunity-associated protein 2,GIMAP2)进行亚细胞定位分析,为深入研究GIMAP2蛋白的功能奠定基础。方法:使用国家生物技术信息中心(National Center for Biotechnology Information,NCBI)数据...目的:对免疫相关GTP结合蛋白2(GTPase of immunity-associated protein 2,GIMAP2)进行亚细胞定位分析,为深入研究GIMAP2蛋白的功能奠定基础。方法:使用国家生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库查询获取GIMAP2的蛋白序列,再利用生物信息学在线分析工具对GIMAP2蛋白的跨膜结构、核定位信号(nuclear localization signal,NLS)、核输出信号(nuclear export signal,NES)及亚细胞定位进行分析预测。采用PCR技术扩增GIMAP2基因片段,并插入至pQCXIP-mCherry-N1表达载体,用氨苄青霉素抗性筛选阳性克隆。测序正确的重组质粒pQCXIP-GIMAP2-mCherry经过提取、纯化步骤后,与逆转录病毒包装质粒VSVG、Gag/Pol在脂质体介导下共同转入HEK293FT细胞中进行病毒包装。转染48 h后收集病毒上清,直接感染人乳腺癌细胞系MDA-MB-436。使用免疫荧光染色方法检测内、外源性GIMAP2在MDA-MB-436胞内表达定位情况。使用绿色荧光化学染料分别标记稳定表达GIMAP2-mCherry融合蛋白的MDA-MB-436活细胞中的线粒体、内质网、脂滴,在超分辨率显微镜N-SIM下观察其与红色荧光的GIMAP2蛋白的定位情况。结果:生物信息学分析数据显示,由337个氨基酸组成的GIMAP2蛋白在羧基端可能有2个跨膜螺旋结构,其中跨膜螺旋含预期氨基酸数为40~41个,紧随跨膜螺旋结构之后的蛋白结构朝细胞质侧;羧基端第279~281位氨基酸有NES但无NLS;可能定位在内质网。测序结果表明,成功构建表达载体pQCXIP-GIMAP2-mCherry。荧光染色结果证实,GIMAP2-mCherry融合蛋白成功在MDA-MB-436细胞内表达,并与内源性GIMAP2定位一致,分布于内质网和脂滴。结论:免疫相关GTP结合蛋白2定位于内质网和脂滴,可能与脂代谢相关。展开更多
基金Supported by National Institute of Health Grants R37 AA-011290and DK-072909
文摘Alcoholism and acquired immune deficiency syndrome are associated with severe muscle wasting.This impairment in nitrogen balance arises from increased protein degradation and a decreased rate of protein synthesis.The regulation of protein synthesis is a complex process involving alterations in the phosphorylation state and protein-protein interaction of various components of the translation machinery and mammalian target of rapamycin(mTOR) complexes.This review describes mechanisms that regulate protein synthesis in cultured C2C12 myocytes following exposure to either alcohol or human immunodeficiency virus antiretroviral drugs.Particular attention is given to the upstream regulators of mTOR complexes and the downstream targets which play an important role in translation.Gaining a better understanding of these molecular mechanisms could have important implications for preventing changes in lean body mass in patients with catabolic conditions or illnesses.
文摘目的研究srGAP2(Slit-Robo GTPase activating Protein 2)、γ-氨基丁酸以及单核细胞趋化蛋白-1(MCP-1)在难治性癫痫患儿脑组织中的表达情况及意义。方法选取11例患儿的颞叶脑组织和同期11例正常标本的颞叶脑组织,使用免疫组化和免疫印迹等方法检测srGAP2、γ-氨基丁酸以及MCP-1的表达情况。结果难治性癫痫患儿脑组织中,srGAP2的阳性表达率显著高于正常脑组织。难治性癫痫患儿脑组织和正常脑组织可以看到染成棕褐色的神经元,但难治性癫痫患儿脑组织γ-氨基丁酸能神经元表达量明显低于正常脑组织。正常脑组织中MCP-1的表达较弱,而难治性癫痫患儿脑组织中MCP-1的表达较强。结论难治性癫痫患儿脑组织srGAP2和MCP-1表达上升而γ-氨基丁酸表达下降,srGAP2、γ-氨基丁酸及MCP-1与难治性癫痫发病密切相关,可能作为潜在治疗靶标。
文摘目的:对免疫相关GTP结合蛋白2(GTPase of immunity-associated protein 2,GIMAP2)进行亚细胞定位分析,为深入研究GIMAP2蛋白的功能奠定基础。方法:使用国家生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库查询获取GIMAP2的蛋白序列,再利用生物信息学在线分析工具对GIMAP2蛋白的跨膜结构、核定位信号(nuclear localization signal,NLS)、核输出信号(nuclear export signal,NES)及亚细胞定位进行分析预测。采用PCR技术扩增GIMAP2基因片段,并插入至pQCXIP-mCherry-N1表达载体,用氨苄青霉素抗性筛选阳性克隆。测序正确的重组质粒pQCXIP-GIMAP2-mCherry经过提取、纯化步骤后,与逆转录病毒包装质粒VSVG、Gag/Pol在脂质体介导下共同转入HEK293FT细胞中进行病毒包装。转染48 h后收集病毒上清,直接感染人乳腺癌细胞系MDA-MB-436。使用免疫荧光染色方法检测内、外源性GIMAP2在MDA-MB-436胞内表达定位情况。使用绿色荧光化学染料分别标记稳定表达GIMAP2-mCherry融合蛋白的MDA-MB-436活细胞中的线粒体、内质网、脂滴,在超分辨率显微镜N-SIM下观察其与红色荧光的GIMAP2蛋白的定位情况。结果:生物信息学分析数据显示,由337个氨基酸组成的GIMAP2蛋白在羧基端可能有2个跨膜螺旋结构,其中跨膜螺旋含预期氨基酸数为40~41个,紧随跨膜螺旋结构之后的蛋白结构朝细胞质侧;羧基端第279~281位氨基酸有NES但无NLS;可能定位在内质网。测序结果表明,成功构建表达载体pQCXIP-GIMAP2-mCherry。荧光染色结果证实,GIMAP2-mCherry融合蛋白成功在MDA-MB-436细胞内表达,并与内源性GIMAP2定位一致,分布于内质网和脂滴。结论:免疫相关GTP结合蛋白2定位于内质网和脂滴,可能与脂代谢相关。
