40 resultados para Ficheiro GPX
em Chinese Academy of Sciences Institutional Repositories Grid Portal
Resumo:
谷胱甘肽过氧化物酶(GPX)是生物体内抗氧化应激酶系的重要成员,是一种含硒酶,它通过消除过氧化氢 (H_2O_2)和有机氢过氧化物(ROOH),保护机体免受活性的损伤或减少活笥氧对机伤程度。GPX的缺乏与多种疾病有关。天然GPX来源极其有限,因此研制具 GPX活力的人源抗体酶并且对其性质的研究对在临床上应用GPX治疗与其缺乏相关的疾病有重要意义。我们在本小组已从噬菌体展示的人单链抗体库中筛选得到了对S-2,4-二硝基苯基取代的谷胱甘肽二丁酯(Hp3)特异的单链抗体3B10并对之高效表达的基础上,进行了细菌培养 、包含体提取、蛋白体的变性与复性,纯化。并经过蛋白电泳和ELISA分析,证明所得的纯化后的蛋白为所需的半抗原特异的人单链抗体。并用化学修饰法把GPX的催化基团硒代半胱氨酸(Sec)组装到已复性、纯化的单链抗体3B10中,获得了具有GPX活力为75.4U/μmol的人源含硒抗体酶Sec-3B10。通过酶学性质的测定可知抗体酶在pH值为8.0、温度为37 ℃时生物活性最高。通过动力学性质的研究证明含硒抗体酶GPX的催化机制与天然GPX一样,符合Ping-Pong机制。光谱性质的研究可推知硒化位点位于蛋白的可变区CDR3区,在经过酶切,通过质谱测定后,比较硒化与未硒化的氨基酸片段的质量差可证实硒化位点位于蛋白的可变区CDR3。为鉴定经化学修饰法硒化的位置提供了一种手段。
Resumo:
谷胱甘肽过氧化物酶 (Glutathione Peroxidase, GPX),是生物体内抗氧化应激酶系的重要成员,它可以清除脂质氢过氧化物(ROOH)及过氧化氢 (H_2O_2),保护机体免受活性氧的损伤或减少活性氧对机体的损伤程度。为了解决鼠源抗体酶GPX应用于人体时会诱发人抗鼠反应 (Human Anti-Mouse Antibody)这一问题,我们实验室从人单链抗体库中筛选了特异的抗体,经化学修饰后得到了具有GPX活性的抗体酶。本文在此工作的基础上,对己筛选出的人单链抗体基因进行了可溶性表达,并用化学修饰的方法进行硒化,得到了GPX活力为80U/μmol的抗体酶。根据优化的表达条件,直接得到了具有天然结构的抗体蛋白,省去了原来的包含体表达中的变性、复性步骤。且所得的抗体为融合蛋白,在蛋白的末端接有6个连续的组氨酸,因此用Ni-NTA亲和层析柱对其进行了一步纯化,即得到了纯抗体蛋白。从而大大的简化了抗体酶的制备过程。本论文还对抗体酶的生物活性进行了研究。采用H_2O_2/Fe~(2+) 自由基发生系统,分别以线粒体的膨胀度、乙二醛生成量和自由基含量为不同的损伤指标进行测量,研究了抗体酶对自由基损伤的牛心线粒体的保护作用。实验结果表明,硒化后的抗体酶具有较强的保护作用。
Resumo:
对噬菌体展示人单链抗体库进行筛选 ,得到与半抗原 S-二硝基苯取代的谷胱甘肽二丁酯特异结合的单链抗体 3B1 0 .用计算机模拟分析了单链抗体的空间结构 ,发现抗原结合的 CDR3区位于抗体的表面 ,推测其可能进一步参加硒化反应 .利用突变引物 ,在大肠杆菌中表达了可溶性抗体蛋白 ,并用化学方法将催化必需基团硒代半胱氨酸 ( Sec)组装到 3B1 0抗原结合部位 ,获得了具有谷胱甘肽过氧化物酶活力的人源抗体酶 .动力学研究结果表明 ,抗体酶和天然酶一样 ,符合乒乓反应机制
Resumo:
随着现代工农业的发展,环境污染日益加剧。农用土壤作为我们赖以生存的基础,大量含砷(Arsenic, As)和铜(Copper, Cu)的化肥和农药的应用造成As、Cu及其他重金属的复合污染日趋加重。蜈蚣草作为一种天然的砷超富集植物,在清除土壤砷污染的应用中备受人们关注。然而,目前的研究多集中在蜈蚣草对单一砷污染的吸收、转运和超富集等方面,几乎没有对土壤复合污染尤其是As、Cu复合污染的的响应和生理机制的研究。本文针对As、Cu复合污染的现实,研究了不同浓度砷、铜复合污染胁迫对蜈蚣草生长和发育的影响,探讨了蜈蚣草对砷、铜复合污染抗性和富集的生理生化机制,以期为蜈蚣草在植物修复复合污染的应用提供理论基础。 主要研究结果如下: 1、以砷超富集植物蜈蚣草成熟孢子为外植体材料,建立了一套成熟的蜈蚣草孢子植株再生体系,包括配子体分化和愈伤组织分化两条途径。为蜈蚣草的生理生化机制以及将来的遗传转化奠定了基础。同时证明了蜈蚣草愈伤组织与其孢子体及配子体一样具有对砷的抗性和超富集特性以及铜抗性。 2、以组培的蜈蚣草孢子体为材料,通过比较高浓度砷、低浓度砷以及高浓度铜、低浓度铜的相互组合对孢子体毒性的差异,表明适度浓度砷可以增强蜈蚣草对铜的抗性,但高、低浓度铜都不能增强砷的抗性。同时测定了不同浓度砷、铜处理对蜈蚣草体内砷和铜的吸收分配情况,探讨了蜈蚣草对砷铜复合污染土壤的植物修复的潜在可能性 。 3、以蜈蚣草组培的配子体为材料,研究了蜈蚣草对不同浓度铜砷复合处理的生理生化响应。结果表明砷的加入可以缓解铜对蜈蚣草配子体的植物毒性,而铜的加入并没有缓解砷的植物毒性。而且随着砷浓度的提高可以显著降低铜在配子体中的积累,显著提高了配子体细胞生存能力,降低了配子体细胞膜透性,且可以改变铜砷在配子体中亚细胞定位,暗示砷对铜的积累具有拮抗作用。同时观察到适度的铜也可以降低砷在孢子体根中的积累,对砷在叶柄以及羽叶中的积累有一定的降低作用,但并不显著。 4、以蜈蚣草愈伤组织为材料,研究了蜈蚣草愈伤组织对砷和铜复合污染胁迫的生理生化响应,结果表明适度的砷可以显著提高蜈蚣草愈伤组织中抗氧化酶系统,进而提高抵御铜胁迫引起的ROS胁迫的能力而显著缓解铜对蜈蚣草愈伤组织的毒性。低浓度砷加入显著诱导POD、CAT活性的升高,提高了蜈蚣草愈伤组织抵抗ROS胁迫能力,尤其是POD与生长呈显著的正相关;SOD活性在0-1.0 mM Na3AsO4条件下并没有显著增加,暗示在相对较低的砷胁迫和铜胁迫条件下POD对蜈蚣草愈伤组织解毒起到重要作用。同时在高砷或者高铜胁迫条件下GR活性和GPx活性与之的相关系数可以达到显著水平,这说明高砷或者高铜胁迫条件下GR和GPx在蜈蚣草解毒中起到重要作用。 5、首次证明NO可能参与砷缓解铜对蜈蚣草的植物毒性的过程。发现加入0.2 μM NO加入并没有显著改善蜈蚣草砷的植物毒性,清除蜈蚣草愈伤组织内的NO后,显著增强了砷胁迫条件下对蜈蚣草愈伤组织生长的抑制作用。同时加入NO后却显著改善了蜈蚣草铜胁迫条件下愈伤组织的生长。测定了As、Cu处理后蜈蚣草愈伤组织内源NO含量的变化以及CAT抗ROS能力的变化,结果表明蜈蚣草愈伤组织NO合成显著受As诱导,但不受铜的诱导,同时内源NO浓度的升高,伴随着抵抗ROS胁迫的抗氧化的CAT活性升高。暗示砷可能是通过诱导内源NO浓度升高提高蜈蚣草愈伤组织抵抗ROS胁迫能力来缓解对铜的植物毒性。
Resumo:
在果实采后贮藏过程中,病原真菌的侵染会引起果实腐烂,造成巨大的经济损失。利用生物和非生物因子诱导果实抗病性,已经成为采后病害防治领域的一个研究热点。本文主要利用RT-PCR和RACE技术克隆果实抗病相关基因,通过分子杂交和蛋白羰基化免疫检测技术,研究了外源SA和酵母拮抗菌诱导果实抗病性机理,结果表明: 1. 通过优化RNA提取方法,能从含有多糖的冬枣、葡萄、甜樱桃、桃、番茄等果实中提取到质量较好的RNA,用于RT-PCR和Northern杂交。 2. 采用RT-PCR和RACE方法,从甜樱桃果实克隆了两个抗氧化相关基因CAT2(Genbank:EF165590)和GPX(Genbank:EF165591)和两个PR基因GLU-1(Genbank:EF177487)和GLU-3(Genbank:EF177488)。其中CAT2全长cDNA序列为1479 bp,编码492个氨基酸;GPX全长cDNA序列为513 bp,编码170个氨基酸;GLU-1全长cDNA序列为1050 bp,编码349个氨基酸;GLU-3部分cDNA序列为454 bp,编码141个氨基酸。 3. 酵母拮抗菌Pichia membranaefaciens处理不同成熟度的甜樱桃果实,能显著降低果实贮藏期间青霉病(Penicillium expansum)的发生,并且对低成熟度果实的病害防治效果更为明显。酵母拮抗菌的抑病机理与减轻了甜樱桃果实蛋白羰基化程度,诱导了果实抗氧化酶基因(CAT和GPX)和PR基因(GLU-1)的表达和提高了抗氧化酶(CAT和GPX)和β-1,3-葡聚糖酶的活性有关。 4. 四种酵母拮抗菌P. membranaefaciens, Cryptococcus laurentii, Candida guilliermondii和Rhodotorula glutinis处理桃果实,可显著降低贮藏期间的褐腐病(Monilinia fructicola)。这是由于酵母拮抗菌能抑制病原菌侵染造成的氧化胁迫和蛋白羰基化。此外,酵母拮抗菌处理还能显著诱导CAT、POD、几丁质酶、β-1,3-葡聚糖酶活性及相应基因的表达。 5. 水杨酸(SA,2 mM)处理采后不同成熟度的甜樱桃果实,能显著降低青霉病的危害。其抑病机理与SA处理能减轻P. expansum侵染引起的果实蛋白羰基化程度,显著提高CAT、GPX和β-1,3-葡聚糖酶基因的表达和相关的酶的活性有关。而2 mM的SA处理对P. expansum的生长没有直接抑制作用。 6. 水杨酸(SA,2 mM)与P. membranaefaciens(1×108 CFU/ml)配合处理能显著降低低温贮藏期间桃果实的褐腐病,并能提高几丁质酶、β-1,3-葡聚糖酶和POD的活性和相关基因的表达。另外,2 mM的SA对拮抗菌P. membranaefaciens的生长没有影响,但能够抑制病原菌M. fructicola的孢子萌发和菌丝扩展。
Resumo:
人类活动产生的氯氟烃化合物破坏了大气臭氧层,导致了到达地球表面的UV-B辐射大幅度增加。UV-B辐射增强可以影响到植物的生长、形态与发育等各个方面,因此有关增强UV-B辐射对植物的影响,及其与许多环境因子复合作用的研究都已经广泛开展。但是增强UV-B辐射与温度,特别是与低温的相互作用的研究报道很少。在北半球的晚秋至早春这段时期里,一些越冬生长的植物将面临着UV-B辐射增强和低温的双重胁迫,因此,迫切需要进行UV-B辐射和低温生长环境下植物的响应及其机制的研究。 以人工气候生长室中生长的冬小麦(Triticum aestivum)幼苗为试验材料,研究了低剂量(4.2 kJ m-2 d-1 UV-BBE,LUVB)和较高剂量(7.0 kJ m-2 d-1 UV-BBE,HUVB)UV-B辐射处理对20/16℃条件下幼苗抗寒力的交叉适应性及其抗氧化系统的反应;同时还研究了在两种生长温度(25/20℃和10/5℃)条件下,低剂量(4.2 kJ m-2 d-1 UV-BBE,LUVB)和超高剂量(10.3 kJ m-2 d-1 UV-BBE,SHUVB)UV-B辐射处理幼苗的生长速率、光合与荧光参数、叶黄素循环色素、抗氧化系统、以及抗寒性和酚类物质等生理反应,以期阐明不同温度条件下生长的冬小麦对UV-B辐射的生长、光合作用以及抗寒性响应与适应机制。主要结果如下: 1.在LUVB辐射处理下,在20/16℃和25/20℃条件下生长的冬小麦幼苗LT50值都显著降低,HUVB辐射处理对在20/16℃条件下生长的幼苗LT50值也可以显著降低,而SHUVB辐射对25/20℃条件下生长的幼苗LT50值没有显著影响。但是,LUVB和SHUVB辐射处理都导致了10/5℃条件下生长的幼苗LT50值的显著增加。表明适当的UV-B辐射能增强较高温度(20/16℃或25/20℃)条件下冬小麦幼苗的抗寒力,即表现出对冷冻低温的交叉适应性,但低温(10/5℃)生长条件却削弱了UV-B辐射下冬小麦的抗寒能力。 2.在20/16℃条件下接受UV-B辐射预处理的幼苗在-6℃条件下冷冻胁迫6 h再缓慢恢复6 h后,与未进行UV-B辐射处理的对照相比,其叶片过氧化氢酶(CAT)、愈创木酚过氧化物酶(GPX)、谷胱甘肽还原酶(GR)活性,谷胱甘肽氧化还原比例(GSH/GSSG)都显著提高,而由硫代巴比妥酸反应物质(TBARS)代表的膜质过氧化程度显著低于对照。此外,UV-B辐射期间处理幼苗的H2O2含量较对照显著增加,而冷冻恢复以后却明显低于对照。表明UV-B辐射诱导的抗寒力的提高应该与冷冻恢复后植株体内抗氧化系统的上调表达有关,H2O2可能参与了UV-B辐射对低温的交叉适应的信号传导。 3.除25/20℃生长条件下的LUVB处理的小麦幼苗外,UV-B辐射显著降低幼苗的相对生长速率(RGR)、净光合速率(Pn)、光系统II最大量子产量(Fv/Fm)、光系统II实际量子产量((F΄m−Fs)/F΄m)以及光化学淬灭(qP),但是UV-B辐射并不影响叶片胞间CO2浓度(Ci),而且冬小麦幼苗生长和光合作用的抑制被增加的UV-B辐射剂量和降低的温度加强。UV-B辐射引起的光抑制由非气孔限制所导致,而且主要与PS II光化学效率降低有关。 4.UV-B辐射显著增加了两个温度条件(20/16℃或25/20℃)下生长的冬小麦幼苗叶黄素循环过程中紫黄素(V)的合成,但抑制了V向玉米黄质(Z)的转化,从而造成了对照与LUVB辐射处理幼苗之间的叶片中脱环氧化比例(DEPS)和NPQ无显著性差异,但SHUVB辐射处理幼苗叶片中DEPS和NPQ显著降低。因此,在本试验条件下,增强UV-B辐射处理的冬小麦可能并不通过热耗散形式形成光保护机制,光抑制形成的过剩激发能的耗散可能更多地通过代谢途径来实现。 5.UV-B辐射处理提高了在25/20℃条件下幼苗的超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和GR等活性,以及抗坏血酸氧化还原比例(AsA/DHA)和GSH/GSSG;但是在10/5℃下,UV-B辐射除了导致SOD和CAT活性升高之外,对APX活性和AsA/DHA并不产生明显影响,但GPX和GSH/GSSG则显著降低。说明UV-B辐射幼苗的抗氧化系统在较高生长温度下显著地增强,而在低温10/5℃下被严重地削弱或降低,即低温阻止了代谢途径的光保护机制的正常运转。 6.多酚物质在UV-B辐射或低温10/5℃条件下都能显著地累积,且在UV-B辐射和低温复合作用下增加尤其显著,表明多酚物质在两个温度生长条件下特别是低温条件下都参与了对UV-B辐射幼苗的保护。 7.在高温条件下仅仅SHUVB处理的幼苗TBARS含量显著增加,而低温10/5℃条件下两个UV-B辐射处理都非常显著地上升,说明与高温生长条件相比较,低温加重了UV-B辐射引起的氧化胁迫,低温10/5℃条件下幼苗多酚的增加以及抗氧化系统的部分增强都没有能阻止UV-B辐射对幼苗的伤害。
Resumo:
通过测定植物的丙二醛(MDA)含量、酸溶性SH含量以及两种抗氧化酶(超氧化物歧化酶SOD和愈创木酚过氧化物酶GPX)的酶活,研究了重金属镉(Cd)和铜(Cu)对美人蕉(Cannaindica Linn.)的氧化胁迫。结果表明,20μmol·L~(-1)和100μmol·L~(-1)的Cd~(2+)和Cu~(2+)均使其根部MDA含量显著增加,但除了100μmol·L~(-1)的Cd~(2+),叶部MDA含量无明显变化。与Cu~(2+)相比,Cd~(2+)能引起植物根部GSH的明显提高,并能诱导PCs的产生
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用 100μg/L 的微囊藻毒素(MC-RR)处理细长聚球藻,研究了 MC-RR 在藻细胞中的积累及其对细长聚球藻的生长和抗氧化系统的影响.结果表明,MC-RR 能在细长聚球藻中迅速积累,而细长聚球藻具有较强的降解 MC-RR 的能力,MC-RR 对细长聚球藻的生长具有显著的抑制作用;MC-RR 处理后,活性氧(ROS)和膜脂过氧化产物丙二醛(MDA)含量明显升高,还原型谷胱甘肽(GSH)含量和谷胱甘肽 S-转移酶(GST)、谷胱甘肽过氧化物酶(GPX)活性则有一个先降后升的变化.以上结果说明,细长聚球藻
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采用0.1mg/L的微囊藻毒素-RR(MC-RR)处理烟草BY-2悬浮细胞,测定了细胞活力、细胞内活性氧(ROS)及抗氧化系统的变化.结果表明,处理144h后,BY-2的细胞活力与对照相比无显著差异,但是处理细胞的ROS含量随着MC-RR处理时间的延长而迅速上升,96h后,细胞的ROS含量与对照差异显著;毒素处理细胞的过氧化物酶(POD)和谷胱甘肽过氧化物酶(GPX)活性明显升高,分别在处理48h和72h后与对照差异显著;细胞中还原型谷胱甘肽(GSH)含量有一个先下降后升高的过程,96h后,MC-RR处理
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Objective To investigate the hispathological characteristics and antioxidant responses in liver of silver carp after intraperitoneal administration of microcystins (MCs) for further understanding hepatic intoxication and antioxidation mechanism in fish. Methods Phytoplanktivorous silver carp was injected intraperitoneally (i.p.) with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq./kg body weight, and liver histopathological changes and antioxidant responses were studied at 1, 3, 12, 24, and 48 h, respectively, after injection. Results The damage to liver structure and the activities of hepatic antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxide (GPX) were increased in a time-dependent manner. Conclusion In terms of clinical and histological signs of intoxication and LD50 (i.p.) dose of MC-LR, silver carp appears rather resistant to MCs exposure than other fishes. Also, the significantly increased SOD activity in the liver of silver carp suggests a higher degree of response to MCs exposure than CAT and GPX.
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Oxidative stress response after prolonged exposure to a low dose of microcystins (MCs) was studied in liver, kidney and brain of domestic rabbits. Rabbits were treated with extracted MCs (mainly MC-LR and MC-RR) at a dose of 2 MC-LReq. mu g/kg body weight or saline solution every 24 h for 7 or 14 days. During the exposure of MCs, increase of lipid peroxidation (LPO) levels were detected in all the organs studied, while antioxidant enzymes responded differently among different organs. The enzyme activities Of Superoxide dismutase (SOD). catalase (CAT) and glutathione reductase (GR) in liver decreased in the MCs treated animals. In brain, there were obvious changes in glutathione peroxidase (GPx) and GR, while only CAT was obviously influenced in kidney. Therefore, daily exposure at a lower dosage of MCs, which mimicked a natural route of MCs. could also induce obvious oxidative stress in diverse organs of domestic rabbits. The oxidative stress induced by MCs in brain was as serious as in liver and kidney, suggesting that brain may also be a target of MCs in mammals. And it seems that animals may have more time to metabolize the toxins or to form an adaptive response to reduce the adverse effects when exposed to the low dose of MCs. (C) 2008 Elsevier B.V. All rights reserved.
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A novel multi-cell device made of organic glass was designed to study morphological and physiological characteristics of Microcystis population trapped in simulated sediment conditions. Changes of colonial morphology and antioxidant activities of the population were observed and measured over the range of 31-day incubation. During the incubation, the antioxidant enzyme activities fluctuated significantly in sediment environments. The activities of catalase (CAT), glutathione peroxidase (GPx) and malondialdehyde (NIDA) reached the highest on the 11(th) day, 6(th) day and 6(th) day. respectively, and then dropped down remarkably in the following days. The ratios of Fv/Fm and the maximal electron transfer rate (ETRm) declined during the initial days (1 similar to 11(th) day), but rebounded on the 16(th) day, which were consistent with the variations of total protein. In the end of incubation. gas vacuoles were hard]), observed and the gelatinous sheath was partly disappeared in the population of Microcystis. Nevertheless, the remaining populations. upon transferred to culture medium, were able to grow though experiencing a longer lag phase of nine days. The results indicated that the sediment environments were able to cause negative effects on M. aeruginosa cells. The cells, however, responded to against the possible damage afterwards. It is thus proposed the acute responses in the population during the early stage of sedimentation could be of importance in aiding the long-term survivor of Microcystis and recruitment in lake sediments. The present study also demonstrated the utility of the device in simulating the sediment environments for further investigation.
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This study was undertaken to investigate the role of the glutathione-involved detoxifying mechanism in defending the tobacco BY-2 suspension cells against microcystin-RR (MC-RR). Analysis showed that exposure of the cells to different concentrations of MC-RR (0.1, 1 and 10 mu g/mL) for 0-6 days resulted in a time and concentration-dependent decrease in cell viability and increase in reactive oxygen species (ROS) content. Reduced glutathione (GSH) and total glutathione (tGSH) content as well as glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) activities significantly increased after 3-4 days exposure in the highest two concentration treated groups, while decreased until reaching the control values except for GPX at day 6. Oxidized glutathione (GSSG) content markedly increased compared with control in high concentration MC-RR treated group after 6 days exposure. The GSH/GSSG ratio was much higher than control in 10 mu g/mL MC-RR treated group at day 4, but after 6 days exposure, the ratios in all treated groups were lower than that of the control group.
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Physiological and biochemical responses of four fishes with different trophic levels to toxic cyanobacterial blooms were studied in a large net cage in Meiliang Bay, a hypereutrophic region of Lake Taihu. We sampled four fishes: the phytoplanktivorous Hypophthalmichthys molitrix and Aristichthys nobilis, the omnivorous Carassius auratus, and the carnivorous Culter ilishaeformis. Alterations of the antioxidant (GSH) and the major antioxidant enzymes (CAT, SOD, GPx, GST) in livers were monitored monthly, and the ultrastructures of livers were compared between the bloom and post-bloom periods. During the cyanobacterial blooms, the phytoplanktivorous fishes displayed only slight ultrastructural changes in liver, while the carnivorous fish presented the most serious injury as swollen endomembrane system and morphologically altered nuclei in hepatocytes. Biochemically, the phytoplanktivorous fishes possessed higher basal GSH concentrations and better correlations between the major antioxidant enzymes in liver, which might be responsible for their powerful resistance to MCs. This article provided physiological and toxicological evidences for the possible succession of fish communities following occurrence of toxic cyanobacterial blooms and also for the applicability of using phytoplanktivorous fish to counteract toxic cyanobacterial blooms in natural waters. (C) 2007 Elsevier Ltd. All rights reserved.