化学诱变抗体模拟谷胱甘肽过氧化物酶

用诱变剂苯甲基磺酰氟活化兔抗人ＩｇＭ片段上特殊活性部位的丝氨酸残基,经硒化氢处理,则将丝氨酸转变成 硒代半胱氨酸。诱变后的抗体具有谷胱甘肽过氧化物酶活性,其活性为世界最好的ＧＳＨ－Ｐｘ模拟物ＰＺ５１的７０多倍抗体效价为１：８,与没诱变的抗体相似。     

化学修饰单克隆抗体模拟谷胱甘肽过氧化物酶

化学修饰具有底物谷胱甘肽（ＧＳＨ）结合部位的单克隆抗体（４Ａ４）使其结合部位上的丝氨酸（Ｓｅｒ）转变成谷胱甘肽过氧化物酶（ＧＰＸ）的催化基因硒代半胱氨酸（ＳｅＣｙｓ）因而产生高活力的含硒抗体酶（Ｓｅ－ａｂｚｙｍｅ）突变的４Ａ４（ｍ４Ａ４）的ＧＰＸ活力达到了天然酶活力的１９％并对ｍ４Ａ４的酶学性质和动力学性质进行了研究;硒代谷胱甘肽（ＧＳｅＨ）连到４Ａ４结合部位,其ＧＰＸ活力由３．８６Ｕ／μｍｏｌ提     

植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生,其中包括超氧化物歧化酶(SOD,EC1.15.L1)、抗坏血酸过氧化物酶(APX,EC1.11.1.11)、过氧化氢酶(CAT,E.C.1.11.1.6)和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9).它们在清除活性氧过程中起着不同的作用.GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少.最近几年研究表明,植物体内也存在类似于哺乳动物的GPXs家族,并对其功能研究已初见端倪.本文综述了有关GPXs的结构以及植物GPXs功能的研究进展.     

化学修饰单克隆抗体模拟谷胱甘肽过氧化物酶

化学修饰具有底物谷胱甘肽（ＧＳＨ）结合部位的单克隆抗体（４Ａ４）,使其结合部位上的丝氨酸（Ｓｅｒ）转变成谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团硒代半胱氨酸（Ｓｅ－Ｃｙｓ）,因而产生高活力的含硒抗体酶（Ｓｅ－ａｂｚｙｍｅ）．突变的４Ａ４（ｍ４Ａ４）的ＧＰＸ活力达到了天然酶活力的１９％,并对ｍ４Ａ４的酶学性质和动力学性质进行了研究;硒代谷胱甘肽（ＧＳｅＨ）连到４Ａ４结合部位,其ＧＰＸ活力由３．８６Ｕ／μｍｏｌ提高到５９８．９Ｕ／μｍｏｌ用黄嘌呤氧化酶／次黄嘌呤为中心的心肌线粒体自由基损伤模型证明Ｓｅ－ａｂｚｙｍｅ（ｍ４Ａ４）可减轻活性氧对线粒体的损伤。     

具有谷胱甘肽过氧化物酶活性的含硒单链抗体酶制备

 利用RT PCR从分泌有谷胱甘肽结合部位的单克隆抗体杂交瘤细胞株 2F3中 ,扩增出单抗重链可变区和轻链可变区基因 .经DNA测序后 ,用Linker(Gly4 Ser1) 3 构建成单链抗体 (scFv)表达载体pTMF scFv ,将重组质粒pTMF scFv转化到大肠杆菌BL2 1(DE3) ,实现了单链抗体的高效表达 .表达的单链抗体占菌体总蛋白 2 5%～ 30 % .该重组蛋白以包涵体形式存在 ,分子量为 30kD .经过金属螯合亲和层析纯化、复性和凝胶过滤纯化 ,得到电泳均一的单链抗体 .再经化学诱变 ,得到含硒单链抗体酶 ,其谷胱甘肽过氧化物酶活性为 330 0U μmol.采用荧光滴定法测定了单链抗体对谷胱甘肽的结合常数     

植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生, 其中包括超氧化物歧化酶(SOD, EC1.15.1.1)、抗坏血酸过氧化物酶(APX, EC1.11.1.11)、过氧化氢酶(CAT, E.C.1.11.1.6 )和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9)。它们在清除活性氧过程中起着不同的作用。GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少。最近几年研究表明, 植物体内也存在类似于哺乳动物的GPXs家族, 并对其功能研究已初见端倪。本文综述了有关GPXs的结构以及植物GPXs功能的研究进展。     

化学诱变抗体模拟谷胱甘肽过氧化物酶

用诱变剂苯甲基磺酰氟（ＰＭＳＦ）活化兔抗人ＩｇＭ（Ｆｃμ）片段上特殊活性部位的丝氨酸（Ｓｅｒ）残基,经硒化氢（Ｈ_２Ｓｅ）处理,则将丝氨酸转变成硒代半胱氨酸（ＳｅＣｙｓ）。诱变后的抗体具有谷胱甘肽过氧化物酶（ＧＳＨ－Ｐｘ）活性,其活性为世界最好的ＧＳＨ－Ｐｘ模拟物ＰＺ５１的７０多倍。抗体效价为１：８,与没诱变的抗体相似。     

麦芽酚对活性氧损伤人神经瘤细胞的保护作用

以人神经瘤细胞株 (SH SY5Y)为材料 ,使用过氧化氢 (H2 O2 )产生过量活性氧诱导SH SY5Y细胞株进入氧化应激状态 .研究麦芽酚对过量活性氧造成的SH SY5Y细胞株氧化损伤的保护作用 .分析活性氧对细胞膜蛋白和DNA的损伤 ,细胞线粒体功能变化 ,白介素 6 (IL 6 )的表达变化以及细胞核因子κB(NF κB)的激活 .结果显示 ,2mmol L麦芽酚保护细胞 2h后 ,对细胞膜蛋白和DNA的损伤均有明显的保护作用 ,减少了膜蛋白的氧化和细胞DNA片段化的形成 ,细胞线粒体功能损伤减小 ,细胞表达的IL 6减少 ,被激活的NF κB水平同时降低 .结果证明 ,麦芽酚可以有效保护活性氧对神经细胞的氧化损伤 ,维持细胞的正常生理功能     

血红素对人脐静脉内皮细胞氧化应激损伤的保护作用

采用过氧化氢作用于人脐静脉内皮细胞 ,成功地制作了氧化应激损伤的细胞模型 ,将培养的细胞分为 3组 :正常对照组 ,损伤组 (给予过氧化氢 )和血红素组 (同损伤组的制备 ,预先加入血红素 ) .观察各组细胞在光、电镜下形态学和蛋白质电泳的改变 ,用MTT法检测了各组细胞的生长状态 .结果表明 :在血红素组 ,细胞生长状态、MTT水平和蛋白质电泳结果与损伤组相比有明显好转且差异显著 ,证明血红素对人脐静脉内皮细胞氧化应激损伤有明显的保护作用 .     

Disturbance of Oxidative Phosphorylation in the Mitochondria of Late Post-Traumatic Epileptic Nidus

We measured the rate of oxygen consumption by the mitochondria from the brain tissues of rabbits within a remote period after light cranio-cerebral trauma. One and six months after traumatization, oxidative phosphorylation in rabbits of the experimental groups demonstrated no significant difference from that in the control group. Yet, after a 12-month-long interval, clear differences were observed within the cortical zone with post-traumatic epileptic nidus. The coefficient of energy production decreased, and the process of oxidative phosphorylation became uncoupled. When succinate was used as a substrate for oxidation, we observed significant decreases in the rate of oxygen consumption in ADP phosphorylation and in the coefficient of respiration control. A significant decrease in the rate of oxygen consumption in the resting state (V ₂), the absence of disturbances in the respiration control, and preservation of a sufficient reserve ATPase activity were characteristic features when glutamate was used as a substrate. It seems probable that such shifts in oxidative phosphorylation can result in creation of an excessive glutamate pool and provide excessive epileptogenic glutamatergic activation of the neurons.     

迷迭香酸对羟自由基所致小鼠肝线粒体损伤的保护作用

探索迷迭香酸对羟自由基致小鼠肝脏线粒体氧化损伤的保护作用。采用羟自由基(.OH),诱导小鼠肝线粒体损伤后,通过测定线粒体肿胀度、膜流动性、丙二醛(MDA)含量及琥珀酸脱氢酶(SDH)活性等指标以确定迷迭香酸对小鼠肝线粒体羟自由基损伤的保护作用。结果迷迭香酸剂量依赖地抑制线粒体肿胀,提高膜流动性,降低MDA的生成,增强SDH活性,差异显著。本实验证明迷迭香酸可以抑制.OH所致的线粒体损伤。     

活性氧对大豆下胚轴线粒体结构与功能的损伤

采用百草枯、H_2O_2和Fenton反应作为外源活性氧,探讨了活性氧对大豆下胚轴离体线粒体的部分功能与结构的伤害。结果表明,活性氧O_2~-,H_2O_2和·OH都能引起线粒体结构的迅速膨胀,呼吸控制比率和氧化磷酸化效率降低,以及细胞色素氧化酶活力下降。通过对线粒体膜脂氢过氧化物和脂质共轭二烯的吸收光谱分析,阐明了活性氧对线粒体的损伤是与膜脂过氧化作用密切相关的。     

豌豆叶片线粒体内甘氨酸氧化对苹果酸和异柠檬酸氧化的影响

豌豆幼苗叶片线粒体中,Gly,Mal和Isocit的氧化速率均受光促进。Gly的氧化抑制Mal和Isocit的氧化,而其本身不受影响。用INH抑制Gly氧化或提高NAD~+浓度均会降低其抑制程度。线粒体氧化Gly,Mal和Isocit的K_m(NAD~+)分别为66.67,119.1μmol/L和152.2μmol/L。Gly抑制Mal和Isocit氧化是由于Gly氧化在竞争NAD~+中占优势。     

Cooperative Substrate Oxidation by Mitochondria from Castor Bean Hypocotyls

Cooperative oxidation of succinate and exogenous NADH was followed in the mitochondria from five- to six-day-old castor bean (Ricinus communisL.) seedlings. Although succinate was oxidized at a much higher rate than NADH, the former inconsiderably (less than 15%) inhibited the oxidation of the latter substrate in state 4, while, in state 3 (in the presence of ATP), the two substrates did not compete and were jointly oxidized. When two substrates were oxidized by the mitochondria with the alternative CN-resistant oxidase (AO) inhibited with salicylhydroxamic acid, the rate of NADH oxidation in state 4 dropped by over 40% as compared to the initial rate. Meanwhile, the rate of succinate oxidation was not considerably affected by AO inhibition. We believe that one of the AO functions in the mitochondria is to provide for noncompeting oxidation of two (or more) substrates by employing two (or several) dehydrogenases of the respiratory chain.     

依达拉奉通过Nrf2信号分子调节氧化应激减轻脑缺血再灌注诱导的神经损伤

本研究旨在探讨依达拉奉(edaravone,ED)在脑缺血再灌注损伤中发挥神经元保护作用与Nrf2信号分子间的关系。体内实验利用脑内脑中动脉闭塞(middle cerebral artery occlusion model,MCAO)建立SD大鼠脑缺血再灌注损伤模型,体外实验采用过氧化氢(H2O2)损伤PC12细胞建立氧化应激模型。通过TTC染色、HE染色、Nissl染色来检测大脑的病理状态。测定活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活性,来反映氧化应激水平。此外,通过Hoechst 33342染色和线粒体膜电位(mitochondrial membrane potential,MTP)测定,探究细胞水平的损伤。采用免疫组织化学和蛋白质印记测定Nrf2的表达。构建Nrf2敲除的PC12细胞系,证实Nrf2信号分子抑制氧化应激损伤的作用。结果提示,经依达拉奉给药后,在动物体内水平,TTC染色证实,脑缺血再灌注损伤(cerebral ischemia reperfusion injury,CIRI)大鼠的脑组织梗死体积减小(P<0.001),ROS和MDA水平下降(P<0.01),SOD活性上升(P<0.01);在细胞水平,凋亡细胞减少(P<0.05),MTP上升(P<0.01),ROS和MDA水平下降,SOD活性上升(P<0.01);在分子水平,免疫组化和Western印迹结果均提示,Nrf2蛋白质含量较正常组增加。H2O2诱导Nrf2基因敲除的PC12细胞损伤加重,且依达拉奉的治疗效果明显削弱。综上所述,Nrf2在依达拉奉减轻脑缺血再灌注诱导的氧化应激损伤中发挥关键作用。     

Protection Against Ischemic Brain Injury by Inhibition of Mitochondrial Oxidative Stress

Mitochondria are both targets and sources of oxidative stress. This dual relationship is particularly evident in experimental paradigms modeling ischemic brain injury. One mitochondrial metabolic enzyme that is particularly sensitive to oxidative inactivation is pyruvate dehydrogenase. This reaction is extremely important in the adult CNS that relies very heavily on carbohydrate metabolism, as it represents the sole bridge between anaerobic and aerobic metabolism. Oxidative injury to this enzyme and to other metabolic enzymes proximal to the electron transport chain may be responsible for the oxidized shift in cellular redox state that is observed during approximately the first hour of cerebral reperfusion. In addition to impairing cerebral energy metabolism, oxidative stress is a potent activator of apoptosis. The mechanisms responsible for this activation are poorly understood but likely involve the expression of p53 and possibly direct effects of reactive oxygen species on mitochondrial membrane proteins and lipids. Mitochondria also normally generate reactive oxygen species and contribute significantly to the elevated net production of these destructive agents during reperfusion. Approaches to inhibiting pathologic mitochondrial generation of reactive oxygen species include mild uncoupling, pharmacologic inhibition of the membrane permeability transition, and simply lowering the concentration of inspired oxygen. Antideath mitochondrial proteins of the Bcl-2 family also confer cellular resistance to oxidative stress, paradoxically through stimulation of mitochondrial free radical generation and secondary upregulation of antioxidant gene expression.     

克山病心肌线粒体氧化损伤的实验研究

用克山病区粮喂养豚鼠证明其红细胞、肝等组织及其线粒体、上清中的硒及谷胱甘肽过氧化物酶活性明显降低,心肌线粒体脂质氢过氧化物、荧光色脂等增加、同时膜脂组成异常,内膜界面脂的心磷脂(CL)减少,细胞色素C氧化酶(CCO)活性降低;园二色性(CD)异常,近208nm及222nm区的峰值降低,病区粮加喂青菜的动物,随其线粒体硒及CL水平的增加。CCO活性和CD谱均明显恢复;通过外源性磷脂与纯化CCO的重组复性实验,只有CL可使病区粮组酶活性完全达到正常对照水平。表明心肌线粒体膜结合酶CCO活性的降低是CL含量降低引起酶的二级结构(构象)改变的结果。