丙型肝炎病毒核心蛋白下调沉默信息调节因子1导致肝星状细胞活化

目的研究丙型肝炎病毒(HCV)核心蛋白对肝星状细胞沉默信息调节因子1(SIRT1)表达及肝星状细胞活化的影响。方法 HepG2细胞或表达HCV核心蛋白的HepG2细胞与肝星状细胞(LX-2细胞)共培养。应用液体闪烁计数仪、实时荧光定量-PCR、Western印迹检测LX-2细胞SIRT1活性、mRNA及蛋白的表达。Western印迹检测LX-2细胞磷酸化AMP激活的蛋白激酶(p-AMPK)、脂联素受体2(AdipoR2)、转化生长因子-β1(TGF-β1)蛋白的表达。ELISA法检测共培养上清液中人Ⅳ胶原(ColⅣ)、Ⅲ型前胶原肽(PⅢNP)、透明质酸(HA)和人层黏连蛋白(LN)的水平。计量资料采用t检验。结果与LX-2细胞和HepG2细胞共培养组相比,LX-2细胞和表达HCV核心蛋白HepG2细胞共培养组SIRT1活性(0.4±0.1比1.0±0.2,t=6.573,P0.01)、mRNA(0.3±0.1比1.0±0.3,t=5.422,P0.01)和蛋白(0.4±0.1比0.8±0.2,t=4.382,P0.01)水平下降;p-AMPK(0.3±0.1比0.8±0.2,t=5.477,P0.01)和AdipoR2(0.4±0.1比0.8±0.2,t=4.382,P0.01)表达下降;TGF-β1(2.3±0.5比0.8±0.2,t=6.823,P0.01)表达增加;共培养上清液中ColⅣ、PⅢNP、HA和LN的水平增加。SIRT1激动剂白藜芦醇降低TGF-β1的表达。结论 HCV核心蛋白下调肝星状细胞SIRT1活性及表达,下调AdipoR2表达,上调TGF-βl表达,活化肝星状细胞。     

丙型肝炎病毒核心蛋白下调沉默信息调节因子1导致肝窦内皮细胞功能障碍

目的研究丙型肝炎病毒(HCV)核心蛋白对肝窦内皮细胞(LSEC)沉默信息调节因子1(SIRT1)表达及LSEC功能的影响。方法 HepG2细胞或表达HCV核心蛋白的HepG2细胞与LSEC共培养。应用液体闪烁计数仪、实时荧光定量-PCR(RT-PCR)、Western印迹检测LSEC SIRT1活性、mRNA和蛋白的表达,以及LSEC脂联素受体2(AdipoR2)、内皮型一氧化氮合酶(eNOS)、第Ⅷ相关抗原(Von Willebrand factor,vWf)、分化抗原簇31(CD31)、血管内皮细胞生长因子(VEGF)和CD14蛋白的表达。应用流式细胞仪检测细胞活性氧(ROS)水平,检测共培养上清液中丙二醛(MDA)、超氧化物歧化酶(SOD)、脂联素、一氧化氮(NO)和内皮素-1(ET-1)的水平。计量资料采用t检验。结果与LSEC和HepG2细胞共培养组相比,LSEC和表达HCV核心蛋白HepG2细胞共培养组SIRT1活性(0.3±0.1比1.0±0.3,t=5.422,P0.01)、mRNA(0.4±0.1比1.0±0.2,t=6.573,P0.01)和蛋白(0.3±0.08比1.0±0.3,t=5.613,P0.01)水平下降;脂联素[(3.41±0.61)比(5.82±0.87)μg/mL,t=5.556,P0.01]分泌减少且AdipoR2蛋白(0.3±0.1比0.8±0.2,t=5.477,P0.01)表达下降;eNOS蛋白(0.4±0.1比0.9±0.3,t=3.873,P0.01)表达下降;vWf蛋白(0.8±0.3比0.4±0.1,t=3.098,P0.01)、CD31蛋白(0.9±0.2比0.3±0.1,t=6.573,P0.01)、VEGF蛋白(0.9±0.3比0.5±0.1,t=3.873,P0.01)表达增加;CD14蛋白(0.4±0.1比0.9±0.3,t=3.873,P0.01)表达下降。共培养上清液中NO和SOD水平下降,ET-1和MDA水平升高。结论 HCV核心蛋白下调SIRT1活性及表达,下调脂联素及受体表达,引起LSEC收缩和肝窦毛细血管化,增加氧化应激反应,导致肝窦微循环障碍。     

糖脂代谢异常对丙型肝炎病毒感染肝细胞SIRT1-AMPK信号通路的影响

目的探讨不同浓度的葡萄糖、油酸对丙型肝炎病毒(HCV)感染肝细胞沉默信息调节因子1(SIRT1)-AMP激活蛋白激酶(AMPK)信号通路的影响。方法表达HCV核心蛋白的质粒转染HepG2细胞。表达HCV核心蛋白的HepG2细胞在不同终浓度的葡萄糖或油酸的无血清DMEM培养液中孵育24h。分别应用液体闪烁计数仪和蛋白质印迹检测SIRT1和AMPK活性及蛋白的表达。结果随着葡萄糖浓度(1、2、4.5g/L)不断升高,SIRT1活性(1.0±0.2、0.6±0.15、0.2±0.05)及SIRT1蛋白表达(0.9±0.2、0.4±0.1、0.1±0.05)下降,AMPK活性(1.0±0.2、0.5±0.08、0.22±0.05)及p-AMPK蛋白表达(0.9±0.2、0.4±0.1、0.2±0.05)下降;随着油酸浓度(0、300、500uM/L)不断升高,SIRT1活性(1.0±0.2、0.6±0.1、0.2±0.05)及SIRT1蛋白表达(0.9±0.2、0.5±0.1、0.2±0.05)下降,AMPK活性(1.0±0.2、0.5±0.1、0.1±0.05)及p-AMPK蛋白表达(0.9±0.2、0.5±0.1、0.2±0.05)下降。结论高糖、高油酸可下调HCV感染肝细胞SIRT1-AMPK信号通路的活性及表达。     

脂滴自噬在丙型肝炎病毒核心蛋白下调沉默信息调节因子1诱导小鼠肝脂肪变性中的作用

目的研究脂滴自噬在HCV核心蛋白下调沉默信息调节因子1(SIRT1)诱导小鼠肝脂肪变性中的作用。方法小鼠随机分为两组,每组10只。实验组小鼠尾静脉注射HCV core重组表达载体。对照组小鼠尾静脉注射磷酸盐缓冲溶液。1个月后处死小鼠。检测肝功能、脂联素、血清和肝内甘油三酰(TG)、肝脏组织病理学检查肝脂肪变性程度。蛋白质免疫法检测肝脏SIRT1蛋白、脂联素受体(AdipoR)蛋白以及脂滴自噬相关蛋白微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、脂肪分化相关蛋白(ADRP)、尾部作用蛋白(TIP-47)和P62蛋白的表达。计量资料采用t检验。结果与对照组相比,HCV组小鼠出现肝脂肪变性;肝脏TG含量明显增加[(80.9±20.1)比(45.8±10.5)μg/mg,t=4.964,P0.01];血清脂联素[(1.05±0.25)比(1.41±0.45)ng/mL,t=2.211,P0.05]水平下降;SIRT1蛋白水平(0.4±0.1比0.9±0.2,t=7.071,P0.01)和AdipoR2蛋白水平(0.4±0.1比0.8±0.2,t=5.656,P0.01)下降;LC3-Ⅱ蛋白(0.8±0.2比0.4±0.1,t=5.656,P0.01)、TIP-47蛋白(0.9±0.3比0.4±0.1,t=5.000,P0.01)和ADRP蛋白(0.8±0.3比0.4±0.1,t=4.000,P0.01)表达增加;而p62蛋白(0.7±0.2比0.8±0.3,t=0.877,P0.05)表达水平差异无统计学意义。结论 HCV核心蛋白下调SIRT1表达,下调脂联素及受体表达,引起不完全脂滴自噬导致肝脂肪变性。     

丙型肝炎病毒核心蛋白对低氧诱导因子-1α和血管内皮生长因子表达的影响

目的 探讨HCV核心蛋白对低氧诱导因子1 α(HIF-1 α)和血管内皮生长因子(VEGF)表达的影响. 方法 将HCV核心蛋白基因的真核表达载体flag2B-core和HIF-1 αsiRNA转染Huh7.5.1细胞;采用RT-PCR和Western blot法分别检测HIF-1 α、VEGF在mRNA和蛋白水平的表达变化,采用酶联免疫吸附试验检测细胞上清液中VEGF的含量.对各组间数据进行t检验.结果 Huh7.5.1细胞转染flag2B-core后,HIF-1 α和VEGF的mRNA和蛋白表达水平升高,细胞上清液中VEGF含量明显高于对照组[(654.5±43.7) pg/ml与(365.9±26.8)pg/ml,t=653.1％,P＜ 0.01)];Huh7.5.1细胞共转染flag2B-core和HIF-1 α siRNA后,HIF-1 α和VEGF的mRNA和蛋白表达水平降低,细胞上清液VEGF含量明显低于对照组[(389.2±29.6) pg/ml与(768.8±47.3) pg/ml,t=1330.22,P＜0.01).结论 HCV核心蛋白能够上调HIF-1 α和VEGF的表达;HCV可能通过核心蛋白来调节HIF-1 α和VEGF的表达.     

过氧化物酶体增殖活化受体-α激活对HepG2细胞脂肪变性及血红素加氧酶-1表达的影响

目的 研究过氧化物酶体增殖活化受体-α(PPAR-α)激活对油酸诱导的HepG2细胞脂肪变性及血红素加氧酶-1 (HO-1)表达的影响.方法 以油酸(OA)诱导人肝癌HepG2细胞脂肪变性为模型组,采用不同浓度的PPAR-α激动剂非诺贝特(FF)处理HepG2细胞24h,油红O染色观察细胞内脂滴数量,甘油-3-磷酸氧化酶法检测细胞内甘油三酯(TG)含量,采用实时荧光定量PCR检测各组HepG2细胞PPAR-α、HO-1 mRNA水平,采用免疫细胞化学法检测PPAR-α与HO-1蛋白表达.采用SPSS13.0软件,采用单因素方差分析及Pearaon直线相关进行相关分析.结果 (1)模型组HepG2细胞内TG含量为(379.98±23.19) mg/g,对照组为(185.03±12.68) mg/g,模型组HepG2细胞内TG含量明显增加,t=24.385,P＜0.01.PPAR-α的mRNA及蛋白相对表达水平模型组分别为0.42±0.38和0.47±0.14,对照组分别为1.00±0.00和1.85±0.12,模型组明显降低,t=0.583和1.382P值均＜0.01.HO-1的mRNA及蛋白相对表达水平模型组分别为0.36±0.66和0.26±0.10,对照组分别为1.00±0.00和1.22±0.12,模型组明显降低,t=0.637和t=0.967,P值均＜0.01;(2)FF浓度在5、10、50μmol/L时,HepG2细胞内TG值分别为(294.00±19.80) mg/g、(250.33±9.96)mg/g和(196.99±9.14) mg/g,t值分别为10.747、16.200和22.873, F=148.555;P值均＜0.01 ;PPAR-α的mRNA相对表达量分别为0.55±0.65,0.85±0.61,1.31±0.36,t值分别为0.137、0.430和0.893,F=177.637,P值均＜0.01;PPAR-α蛋白累积光密度值分别为0.82±0.11、1.31±0.16和1.75±0.13,t值分别为0.352,0.840,1.280,F=120.764,P值均＜0.01;HO-1的mRNA相对表达量分别为0.62±0.05、0.84±0.07和1.30±0.11,t值分别为0.257,0.480,0.937,F=74.768,P值均＜0.01;HO-1蛋白累积光密度值分别为0.44±0.08、0.81±0.08和1.20±0.10,t值分别为0.180,0.553,0.943,F=119.903,P值均＜0.01.结论 PPAR-α的激活可以抑制HepG2细胞脂肪变性,HO-1可能是其重要下游因子.     

一种用于脂肪肝脂毒性药理研究的体外模型

目的 建立和复制一种适合大鼠药物血清进行脂肪肝脂毒性药理研究的体外模型.方法 以大鼠血清替代胎牛血清培养HepG2细胞,添加长链游离脂肪酸(油酸1 mmol/L、棕榈酸0.5 mmol/L)刺激,观察上清液中肿瘤坏死因子(TNF)α含量、细胞内甘油三酯含量,细胞脂肪油红染色及电镜下观察超微结构变化;同时观察细胞TNF α蛋白及其基因表达,细胞组织蛋白酶B(ctsb)表达和分布的变化.结果 游离脂肪酸刺激24 h后,HepG2细胞内甘油三酯显著沉积,高达627.24 mg/g(t=23.6,P＜0.01);上清液中TNF α含量显著升高,增至52.04 pg/mg(t=2.6,P＜0.05);细胞ctsb、TNF α的蛋白表达及其mRNA表达均显著增强.结论 在大鼠血清培养环境下,游离脂肪酸可通过对ctsb作用显著诱导HepG2细胞脂肪变性和TNF α分泌,方法简易经济,可作为一种较理想的抗脂肪肝脂毒性药理研究模型.     

异烟肼诱导HepG2细胞损伤及其Fas/Fas配体的表达

目的 通过建立异烟肼致HepG2细胞坏死或凋亡模型,观察HepG2细胞Fas/Fas配体(FasL)的表达.方法 以HepG2细胞为模型,分别用含1、2、4、6、8 mg/mL异烟肼的细胞培养液,空白对照组加入新鲜培养液,培养24 h后观察各组细胞形态,膜联蛋白(Annexin)V和碘化丙啶染色,流式细胞仪检测HepG2细胞的坏死和凋亡情况以及其Fas/FasL的表达.数据采用单因素方差分析,各不同浓度药物组与空白对照组的比较采用Dunnett t检验.结果 随异烟肼浓度的增加(4、6、8 mg/mL),HepG2细胞出现逐渐增多的坏死和凋亡,总死亡率分别为(32.1±7.5)％、(34.9±8.1)％和(38.2±9.4)％,与正常对照组的(7.2±1.5)％相比,差异有统计学意义(t=4.62、5.14、5.75,均P＜0.01);Fas的表达也随之增加,异烟肼2、4、6和8 mg/mL浓度组Fas表达率分别为(8.7±2.2)％、(11.5±2.8)％、(12.3±3.0)％和(10.6±2.9)％,与正常对照组的(3.1±0.8)％比较,差异有统计学意义(t=2.97,P＜0.05;t=4.46,P＜0.01;t=4.88,P＜0.01;t=3.98,P＜0.05).异烟肼4、6、8 mg/mL浓度组FasL表达率分别为(16.2±3.5)％、(21.7±4.8)％、(18.7±4.9)％,与正常对照组的(7.4±1.4)％相比,差异有统计学意义(t=3.11,P＜0.01;t=5.06,P＜0.01;t=3.99,P＜0.05).异烟肼浓度为8 mg/mL时,HepG2细胞的死亡增加,主要以坏死为主,凋亡发生率未再增加.结论 异烟肼可以诱导HepG2细胞变性、坏死和凋亡,这种凋亡的发生可能与异烟肼诱导肝细胞表达Fas/FasL增多有关.     

丙型肝炎病毒抑制DNA损伤修复相关基因生长阻滞和DNA损伤诱生蛋白45α的表达

目的 探讨HCV感染对DNA损伤修复相关基因生长阻滞和DNA损伤诱生蛋白45 α(GADD45 α)表达的影响. 方法 建立全基因HCV JFH1感染的Huh7,5.1细胞模型.用相对荧光定量PCR和Western blot分别检测HCV JFH1感染和未感染的Huh7.5.1细胞中GADD45 α mRNA和蛋白质的表达水平.组间数据比较用单因素方差分析.结果 HCV JFH1感染的Huh7.5.1细胞内有HCV RNA高水平复制及HCV NS5A蛋白质和核心蛋白的表达.与未感染Huh7.5.1细胞相比,HCV JFH1感染72h的细胞内GADD45αmRNA和蛋白质相对表达量均明显降低,分别为0.57±0.09比1.00±0.11和0.28±0.03比1.00±0.07,差异均有统计学意义(F值分别为75.407和560.04,P值均＜0.01). 结论 HCV下调GADD45 α的转录和蛋白质表达,影响DNA损伤修复,这可能是HCV感染致肝癌发生的机制之一.     

乙型肝炎病毒X蛋白对HepG2细胞羧末端结合蛋白反应蛋白表达的影响

目的 研究乙型肝炎病毒X蛋白(HBx)对博莱霉素诱导HepG2细胞DNA双链断裂(DSB)损伤修复关键因子羧末端结合蛋白反应蛋白(CtIp)的影响.方法 建立稳定表达HBx的HepG2肝癌细胞株(HepG2-HBx)及空质粒对照细胞株(HepG2-vec).用博莱霉素诱导细胞DSB损伤后,用流式细胞仪检测细胞周期和细胞凋亡情况,用实时定量PCR及Western blot检测CtIP的mRNA与蛋白表达水平,并用激光共聚焦显微镜观察CtIp蛋白在细胞内的定位情况.多组数据采用单因素方差分析和SNK-q检验;两组数据间比较用t检验.结果 经检测转染HBx基因的HepG2细胞(HepG2-HBx)能稳定表达HBx.博莱霉素处理后,HepG2-HBx细胞与HepG2-vec细胞的凋亡比例分别为16.90%±0.89%和15.30%±0.86%,差异无统计学意义(q=2.074,P＞0.05),但死亡细胞比例分别为8.71%±0.74%和4.90%±0.46%,差异有统计学意义(q=7.126,P＜0.01) ;同时两种细胞株都出现了细胞周期G2/M期阻滞,差异有统计学意义(F=11.401,P＜0.05).HBx使CtIP蛋白表达水平和mRNA表达水平均下调,HepG2-HBx细胞与HepG2-vec细胞CtIp蛋白的相对表达量分别为0.66±0.04、0.73±0.05,差异有统计学意义(t=2.314,P＜0.05); CtIP mRNA相对表达量分别为1.00±0.06、1.23±0.08,差异有统计学意义(t=2.732,P＜0.05).同时观察到CtIP蛋白主要在细胞胞核内表达.结论 HBx能干扰肿瘤抑制蛋白CtIp的表达,可能影响细胞DSB损伤的修复.     

Tau protein and beta protein

The main pathological features of Alzheimer's disease are Alzheimer neurofibrillary tangles and senile plaques. Recent biochemical research revealed that tangles are composed of tau protein and ubiquitin and amyloid in senile plaques is composed of beta protein which is a fragment of the membrane receptor protein. Although fraction, other data suggest that whole molecules become abnormal and aggregate into PHF. On the other hand, beta protein is a small cleavage product of the precursor protein. It is not concluded yet whether abnormal precursor proteins exist or not. Recent research in this field is reviewed.     

乙型肝炎病毒X蛋白结合蛋白

随着人和各种生物的基因和基因组测序的完成,生物学和医学正处在一深刻变革的时代.基因组学(genomics)是指对人和其他生物类型基因组结构与功能的分析.基因组学可以分为结构基因组学(structural genomics)和功能基因组学(functional genomics),功能基因组学是指应用整体的研究技术阐明这些基因和蛋白的生物学功能.各种生物系统是一个复杂的系统,基因组中基因的序列是一个庞大的数据库,因此,需要发展一些强大的分析技术,代替传统的分析技术,对这些基因和蛋白质的功能进行研究.基因是 DNA 中的一些具有功能的单位,在遗传信息的流向中,首先转录生成中间产物 RNA,然后再翻译成具有生物学功能的蛋白质.蛋白质是执行生命活动的基本成分.作为基因的一段 DNA,一般包括调节基因序列和编码基因序列.功能基因组学的主要任务就是阐明这些基因及其编码产物的结构与功能、表达与调控.第一,人类不同个体之间的基因序列之间的区别;第二,人和人之间决定疾病状态和疾病的易感性基因;第三,引起疾病的各种病原体,还有包括大肠杆菌、酵母、果蝇、线虫、人等合成的每一种蛋白的功能;第四,这些不同的蛋白协同完成生命的活动的机制;第五,在特定的细胞类型和特定的时间内,并不是所有的基因都具有表达活性,决定选择性的表达和活动机制;第六,在多细胞生物中,不同的基因表达是形成不同的细胞和组织的机制.中国人民解放军第302医院传染病研究所基因治疗研究中心,在成军博士、教授、主任医师、博士生导师的领导下,应用功能基因组学的研究技术,研究乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)感染之后,肝细胞基因组表达调节的改变及机制.这是肝炎病毒感染肝细胞之后,引起病毒性肝炎、肝硬化、肝细胞癌的发病机制.基因组计划完成之后,提供能了大量的人和其他生物类型的基因序列,即将完成了结构基因组学的任务,但是,功能基因组学的任务还有许多的工作要做.利用抑制性消减杂交(SSH)、基因芯片技术,酵母单杂交技术、酵母双杂交技术、噬菌体展示技术等对于调节肝炎病毒基因的复制和表达,肝炎病毒蛋白结合蛋白,肝炎病毒蛋白表达对于肝细胞基因表达谱的影响,从而为揭示肝炎病毒感染肝细胞的致病机制的研究,开辟新的研究方向.     

Evolution of protein structural classes and protein sequence families

In protein structure space, protein structures cluster into four elongated regions when mapped based solely on similarity among the 3D structures. These four regions correspond to the four major classes of present-day proteins defined by the contents of secondary structure types and their topological arrangement. Evolution of and restriction to these four classes suggest that, in most cases, the evolution of genes may have been constrained or selected to those genetic changes that results in structurally stable proteins occupying one of the four "allowed" regions of the protein structure space, "structural selection," an important component of natural selection in gene evolution. Our studies on tracing the "common structural ancestor" for each protein sequence family of known structure suggest that: (i) recently emerged proteins belong mostly to three classes; (ii) the proteins that emerged earlier evolved to gain a new class; and (iii) the proteins that emerged earliest evolved to become the present-day proteins in the four major classes, with the fourth-class proteins becoming the most dominant population. Furthermore, our studies also show that not all present-day proteins evolved from one single set of proteins in the last common ancestral organism, but new common ancestral proteins were "born" at different evolutionary times, not traceable to one or two ancestral proteins: "the multiple birth model" for the evolution of protein sequence families.     

Gene and protein sequences of adenovirus protein VII, a hybrid basic chromosomal protein.

The sequences of both the gene and the corresponding protein of adenovirus major core protein VII have been determined. The precise location of this gene is between 43.37 and 44.90 map coordinates on the viral genome. Protein VII is 173 residues long and has a molecular weight of 19,258. Detailed analysis of its sequence has revealed four basic domains separated by several predicted alpha helices. It is proposed that intrachain folding of protein VII is driven by hydrophobic interactions of the alpha helices, leaving the basic domains of the protein to interact with DNA phosphates. Protein monomers may further associate with each other in the formation of hexameric nucleosome-like particles. The displacement and replacement of protein VII during the viral infectious cycle in the host cell appears to mimic the biology of nucleoprotamine during the processes of spermatogenesis and fertilization. The presence of a protamine-like domain affirms a hybrid histone/protamine molecular structure for protein VII, although it may resemble the protamine in function.     

Expression of hepatitis C virus core protein as a fusion protein with maltose binding protein

Putative hepatitis C virus core sequence was amplified from a serum sample positive for anti-C-100-3 and expressed inEscherichia coli. Approximately 62 kDa fusion protein with maltose binding protein containing 20 kDa hepatitis C core protein was obtained. The antibody to this protein was detected in 53 of 54 (98%) sera from hepatitis C virus ribonucleic acid-positive patients including 40 sera positive for anti-C-100-3 and 13 sera negative for anti-C-100-3. The antibody was also detected in all of 12 patients with acute hepatitis C showing the earlier detectability of the antibody than anti-C-100-3. Thus, the protein expressed from the amplified hepatitis C core sequence by the polymerase chain reaction would be useful for the diagnosis of hepatitis C.     

Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases caused by the conversion of prion protein (PrP^C) into an infectious isoform (PrP^Sc). How this event leads to pathology is not fully understood. Here we demonstrate that protein synthesis in neurons is enhanced via PrP^C interaction with stress-inducible protein 1 (STI1). We also show that neuroprotection and neuritogenesis mediated by PrP^C–STI1 engagement are dependent upon the increased protein synthesis mediated by PI3K-mTOR signaling. Strikingly, the translational stimulation mediated by PrP^C–STI1 binding is corrupted in neuronal cell lines persistently infected with PrP^Sc, as well as in primary cultured hippocampal neurons acutely exposed to PrP^Sc. Consistent with this, high levels of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation were found in PrP^Sc-infected cells and in neurons acutely exposed to PrP^Sc. These data indicate that modulation of protein synthesis is critical for PrP^C–STI1 neurotrophic functions, and point to the impairment of this process during PrP^Sc infection as a possible contributor to neurodegeneration.     

Synaptic regulation of protein synthesis and the fragile X protein

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use "synaptoneurosomes," a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.     

糖尿病患者尿液蛋白质质谱分析

H4蛋白芯片结合表面增强激光解吸电离飞行时间质谱技术检测糖尿病患者和健康人尿液样本的蛋白质质谱,按糖尿病肾病的分期分组比较,结果得到一组特异性蛋白.筛选出预测准确率最高的4个差异蛋白质建立早期糖尿病肾病的人工神经网络模型并验证.