星形胶质细胞条件培养液对活性氧所致的神经元损伤的防护作用

本文以活性氧叔丁基脂氢过氧化物tbOOH应激PC12神经元细胞,造成PC12神经元损伤、死亡;以分离、纯化的SD大鼠大脑皮层星形胶质细胞制备的无血清条件培养液培养tbOOH损伤的神经元细胞。采用快速灵敏的MTT比色法测定神经细胞活力,用Olym—pus光学显微镜观察神经细胞的形态学变化。结果发现神经元细胞活力由0．179±0．037上升至0．563±0．025,细胞数目明显增多,细胞死亡残留碎片明显减少,表明星形胶质细胞条件培养液可能有防护tbOOH致的神经元损伤作用,具抗氧化能力。     

谷胱甘肽/谷胱甘肽过氧化物酶系统在微生物细胞抗氧胁迫系统中的作用

谷胱甘肽(GSH)/谷胱甘肽过氧化物酶(GPx)系统在不同微生物细胞抵抗氧胁迫中的生理功能不尽相同。该系统在真核模式微生物酿酒酵母中是必需存在的,在维持胞内氧化还原平衡和抵抗氧胁迫中发挥主要作用。然而,在原核微生物中,该系统只是条件性的,即部分胞内存在谷胱甘肽还原酶和GPx的原核微生物,如流感嗜血杆菌和乳酸乳球菌,可通过从胞外吸收GSH,形成条件性的依赖于GSH的GPx系统,参与抵抗氧胁迫。     

谷胱甘肽/谷胱甘肽过氧化物酶系统在微生物细胞抗氧胁迫系统中的作用

谷胱甘肽(GSH)/谷胱甘肽过氧化物酶(GPx)系统在不同微生物细胞抵抗氧胁迫中的生理功能不尽相同。该系统在真核模式微生物酿酒酵母中是必需存在的,在维持胞内氧化还原平衡和抵抗氧胁迫中发挥主要作用。然而,在原核微生物中,该系统只是条件性的,即部分胞内存在谷胱甘肽还原酶和GPx的原核微生物,如流感嗜血杆菌和乳酸乳球菌,可通过从胞外吸收GSH,形成条件性的依赖于GSH的GPx系统,参与抵抗氧胁迫。     

GPX4缺失介导的铁死亡与精子发生

铁死亡是一种不同于坏死和凋亡的程序性细胞死亡,其特征为铁依赖性的脂质过氧化物及活性氧蓄积。铁死亡可影响生精细胞分裂分化、血睾屏障及睾酮分泌,从而影响精子发生,导致男性不育。谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)作为将脂质过氧化物还原为醇的唯一关键还原酶,在铁死亡中发挥着关键作用,并与氧化应激、炎症、焦亡、自噬等病理生理过程密切相关。本文就GPX4缺失介导睾丸细胞铁死亡调控氧化应激、炎症、焦亡、自噬等影响精子发生的相关研究进展进行综述,以期为男性生精障碍的防治提供理论参考。     

泥蚶谷胱甘肽过氧化物酶基因的全长克隆与表达分析

为了探讨谷胱甘肽过氧化物酶（Glutathione peroxidase,GPx）基因在泥蚶应激反应中的作用,研究采用RACE技术克隆了泥蚶GPx基因（TgGPx）cDNA全长,其cDNA全长1195 bp,包含45 bp 5’-UTR,639 bp开放阅读框（ORF）和511 bp 3’-UTR。ORF编码212个氨基酸残基,预测蛋白分子量为24.3 kD,理论等电点为8.33,其中,第53个氨基酸U是由密码子202UGA204编码的硒代半胱氨酸（Se-Cys）。在3’-UTR上存在一段序列,形成一种独特的茎环结构,即SECIS元件。SECIS元件在密码子UGA翻译为Se-Cys的过程中起决定性作用。通过序列比对与系统进化分析,发现软体动物中也存在不同种类的GPx基因,TgGPx与GPx1和GPx2的亲缘关系较近。利用qRT-PCR技术对TgGPx在泥蚶的不同组织以及重金属刺激后的表达量进行分析,结果表明,TgGPx在泥蚶的5个组织中都有表达,但存在组织特异性,在外套膜中的表达量最高,在血细胞中的表达量最低。用重金属铅、铜、镉刺激后,TgGPx在肝胰脏中的表达量显著升高,表明TgGPx在维护机体正常功能方面及泥蚶抵御外界刺激的应激反应中发挥作用。     

研究报告重组人GPx7的原核表达、纯化及其抗肿瘤活性检测

研究大肠杆菌表达重组人谷胱甘肽过氧化物酶7（glutathione peroxidase 7,GPx7）,并对纯化精制的重组人GPx7进行体内抗肿瘤活性检测。以质粒pCDNA3.1（-）-GPx7为模板,PCR 扩增获得N-端去除信号肽的 GPx7 基因片段,构建的重组质粒pET24A-GPx7在E.coli BL21（DE3）中诱导表达。采用Sulfopropyl强阳离子交换层析和Superdex 75分子排阻层析纯化精制获得原核表达的重组人GPx7蛋白。该蛋白的体外催化活性微弱,但S180荷瘤小鼠连续14d注射GPx7蛋白后,肿瘤生长受到抑制,抑制率为29.26%,与对照组相比具有显著差异。实验结果表明,采用阳离子交换和分子排阻层析两步法可纯化精制原核表达的重组人GPx,重组人GPx7蛋白在荷瘤小鼠模型中表现出体内抗肿瘤活性。     

砷诱导蚕豆气孔保卫细胞死亡的毒性效应

采用蚕豆(Vicia faba L.)叶面气孔保卫细胞,研究砷对细胞的毒性效应。结果表明,0.3—10 mg/L的NaAsO_2能降低保卫细胞活性,使部分细胞死亡,死亡率随砷浓度升高而增高。死细胞中呈现核固缩、核崩解等典型程序性死亡特征,且泛caspase抑制剂Z-Asp-CH_2-DCB能阻止NaAsO_2诱发的细胞死亡。过氧化氢清除剂过氧化氢酶与NaAsO_2共同作用时,细胞死亡率显著低于砷单独处理组,保卫细胞内Ca~(2+)水平降低,具程序性死亡特征的细胞数减少;Ca~(2+)特异性螯合剂EGTA亦能降低NaAsO_2诱发的细胞死亡。研究结果表明,NaAsO_2能诱发蚕豆保卫细胞程序性死亡,该过程由胁迫引发的ROS升高引起,ROS可能通过激活质膜Ca~(2+)通道,使胞外Ca~(2+)内流,造成胞内Ca~(2+)浓度升高,进而诱导细胞程序性死亡。     

hTNFRII-Fc融合基因的克隆及在CHO细胞中的表达

构建人肿瘤坏死因子受体II胞外区（TNFRII）和人免疫球蛋白（IgG1）Fc段的融合基因真核表达载体,并在中国仓鼠卵巢细胞（CHO）中表达融合蛋白hTNFRII-Fc。用全合成重叠延伸PCR方法克隆TNFRII胞外区的基因片段,再与（IgG1）Fc段拼接起来形成hTNFRII-Fc融合基因,经HindIII、EcoRI双酶切后插入至pEE14中构建pEE14-TNFRII-Fc真核表达载体,脂质体转染至CHO细胞中进行表达及鉴定。ELISA检测到转染hTNFRII-Fc基因的CHO细胞培养上清中含有hTNFRII-Fc蛋白,SDS-PAGE与Western-blot鉴定证实其为hTNFRII-Fc蛋白,为进一步研究该融合蛋白在CHO细胞稳定表达及应用于类风湿关节炎等自身免疫性疾病临床治疗奠定了一定的研究基础。     

长效山羊促卵泡素类似物真核表达载体的构建及其在CHO细胞内的稳定表达

通过PCR重叠延伸法将人绒毛膜促性腺激素（hCG）的羧基末端延长肽（CTP）基因连接至山羊促卵泡素（FSH）15亚基的羧基末端,克隆入双表达载体pVITRO,测序证实后将表达载体转染CHO细胞,用潮霉素B筛选稳定表达的CHO细胞株。结果表明获得了长效山羊FSH基因,并得到稳定表达的CHO细胞株,表达量为0．105mU／mL,为进一步研究CTP结构与功能的关系,以及长效激素的理化特性奠定了基础。     

人重组白细胞介素12在CHO细胞中的表达

将人白细胞介素12（interleukin 12, IL-12）两条链p35及p40全长cDNA分别亚克隆至真核表达载体pcDNA3中,构建了pcDNA3/p35a,pcDNA3/p40a,pcDNA3/p35b,pcDNA3/p40b四种真核细胞重组表达质粒,利用磷酸钙共沉淀法转染中国苍鼠卵巢(CHO)细胞. 通过对阳性克隆的筛选鉴定,获得了稳定表达人IL-12的CHO细胞株,活性最高的一株表达量为105 U/ml.此细胞株经半年的传代培养,能够稳定地分泌IL-12.结果显示：IL-12在CHO细胞中的稳定表达受到重组质粒结构、DNA整合、mRNA转录、蛋白质翻译等多因素的影响,IL-12两个亚基在CHO细胞中的共同表达是产生有生物活性IL-12的基础.     

Salt stress-induced programmed cell death in tobacco protoplasts is mediated by reactive oxygen species and mitochondrial permeability transition pore status

The status of mitochondrial permeability transition pore (PTP) and levels of reactive oxygen species (ROS) play key roles in regulating apoptosis in animal cells. To investigate if the PTP and cellular oxidation-reduction state are also involved in salt stress-induced programmed cell death (PCD) in tobacco (Nicotiana tabacum, cultivar BY-2) protoplasts, flow cytometry was used to simultaneously monitor ROS levels, PTP status and PCD. Increased ROS and decreased mitochondrial membrane potential (delta psi(m)) were observed before the appearance of PCD. Pre-treatment with an inhibitor of the PTP opening, cyclosporin A (CsA), effectively retarded the onset of PCD, the delta psi(m) decrease and the ROS content increase. Addition of ascorbic acid (AsA) during the salt stress significantly decreased the percentage of protoplasts undergoing PCD and ROS levels but increased delta psi(m). Hydrogen peroxide effectively induced the appearance of PCD and caused an increase in ROS and a decrease in delta psi(m). Pre-treatment of protoplasts with CsA weakened the effects of H2O2. All these results suggest that the open state of PTP and ROS are necessary elements for salt stress-induced PCD in tobacco protoplasts. The open states of PTP and ROS could promote each other suggesting that ROS could lead to a self-amplifying process. This positive feedback loop may act as an all-or-nothing switch, which is in good accordance with the hypothesis that PTP is an important coordinator and executioner of PCD in both animals and plants.     

Glutathione peroxidase-1 protects from CD95-induced apoptosis

Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis.     

Taurine alleviates ochratoxin A-induced pyroptosis in PK-15 cells by inhibiting oxidative stress

Ochratoxin A (OTA) is one of the most harmful mycotoxins, which can cause multiple toxicological effects, especially nephrotoxicity in animals and humans. Taurine is an essential amino acid with various biological functions such as anti-inflammatory and anti-oxidation. However, the protective effect of taurine on OTA-induced nephrotoxicity and pyroptosis had not been reported. Our results showed that OTA exposure induced cytotoxicity and oxidative stress in PK-15 cells, including reactive oxygen species (ROS) accumulation, increased mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), and decreased mRNA levels of catalase (CAT), glutathione peroxidase 1 (GPx1), and glutathione peroxidase 4 (GPx4). In addition, OTA treatment induced pyroptosis by increasing the expressions of pyroptosis-related proteins NLRP3, GSDMD, Caspase-1 P20, ASC, Pro-caspase-1, and IL-1β. Meanwhile, taurine could alleviate OTA-induced pyroptosis and cytotoxicity, as well as reduce ROS level, COX-2, and iNOS mRNA levels, and increase the mRNA levels of the antioxidant enzyme in PK-15 cells. Taken together, taurine alleviated OTA-induced pyroptosis in PK-15 cells by inhibiting ROS generation and altering the activity of antioxidant enzymes, thereby attenuating its nephrotoxicity.     

Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.     

Wld(S) reduces paraquat-induced cytotoxicity via SIRT1 in non-neuronal cells by attenuating the depletion of NAD

Wld(S) is a fusion protein with NAD synthesis activity, and has been reported to protect axonal and synaptic compartments of neurons from various mechanical, genetic and chemical insults. However, whether Wld(S) can protect non-neuronal cells against toxic chemicals is largely unknown. Here we found that Wld(S) significantly reduced the cytotoxicity of bipyridylium herbicides paraquat and diquat in mouse embryonic fibroblasts, but had no effect on the cytotoxicity induced by chromium (VI), hydrogen peroxide, etoposide, tunicamycin or brefeldin A. Wld(S) also slowed down the death of mice induced by intraperitoneal injection of paraquat. Further studies demonstrated that Wld(S) markedly attenuated mitochondrial injury including disruption of mitochondrial membrane potential, structural damage and decline of ATP induced by paraquat. Disruption of the NAD synthesis activity of Wld(S) by an H112A or F116S point mutation resulted in loss of its protective function against paraquat-induced cell death. Furthermore, Wld(S) delayed the decrease of intracellular NAD levels induced by paraquat. Similarly, treatment with NAD or its precursor nicotinamide mononucleotide attenuated paraquat-induced cytotoxicity and decline of ATP and NAD levels. In addition, we showed that SIRT1 was required for both exogenous NAD and Wld(S)-mediated cellular protection against paraquat. These findings suggest that NAD and SIRT1 mediate the protective function of Wld(S) against the cytotoxicity induced by paraquat, which provides new clues for the mechanisms underlying the protective function of Wld(S) in both neuronal and non-neuronal cells, and implies that attenuation of NAD depletion may be effective to alleviate paraquat poisoning.     

Characterization of the programmed cell death induced by metabolic products of Alternaria alternata in tobacco BY-2 cells

Alternaria alternata has received considerable attention in current literature and most of the studies are focused on its pathogenic effects on plant chloroplasts, but little is known about the characteristics of programmed cell death (PCD) induced by metabolic products (MP) of A. alternata, the effects of the MP on mitochondrial respiration and its relation to PCD. The purpose of this study was to explore the mechanism of MP-induced PCD in non-green tobacco BY-2 cells and to explore the role of mitochondrial inhibitory processes in the PCD of tobacco BY-2 cells. MP treatment led to significant cell death that was proven to be PCD by the concurrent cytoplasm shrinkage, chromatin condensation and DNA laddering observed in the cells. Moreover, MP treatment resulted in the overproduction of reactive oxygen species (ROS), rapid ATP depletion and a respiratory decline in the tobacco BY-2 cells. It was concluded that the direct inhibition of the mitochondrial electron transport chain (ETC), alternative pathway (AOX) capacity and catalase (CAT) activity by the MP might be the main contributors to the MP-induced ROS burst observed in tobacco BY-2 cells. The addition of adenosine together with the MP significantly inhibited ATP depletion without preventing PCD; however, when the cells were treated with the MP plus CAT, ROS overproduction was blocked and PCD did not occur. The data presented here demonstrate that the ROS burst played an important role in MP-induced PCD in the tobacco BY-2 cells.     

Protective effect of ebselen on cytotoxicity induced by cholestane-3 beta, 5 alpha, 6 beta-triol in ECV-304 cells

The protective effect of ebselen, with documented glutathione peroxidase-like activity and antioxidative and anti-inflammatory properties, on the cytotoxicity induced by oxysterol was investigated in ECV-304 cells with cholestane-3beta, 5alpha, 6beta-triol (3-triol), one of the most toxic oxysterols. 3-triol exhibited significant cytotoxicity to ECV-304 cells in dose- and time-dependent manners. Pre-incubations with ebselen at different concentrations for 4 h effectively inhibited the decreases of the cell viability and the intracellular thiols level induced by 3-triol; suppressed the 3-triol-caused increases of the GPx and NOS activities, the LDH leakage and MDA formation. The inhibition of ebselen to the generation of intracellular ROS induced by 3-triol was monitored by luminol-, lucigenin-derived chemiluminescence and DCFH-DA-derived fluorescence assays. Our results suggest that ebselen inhibited 3-triol-induced enhancement of intracellular ROS level and the cytotoxicity of 3-triol is contributed to, for the most part, an enhanced formation of intracellular O2.-; nevertheless, the mitochondria were not the main source of intercellular O2.- contributed to the cytotoxicity of 3-triol. Ebselen lost its high protection against 3-triol-induced injuries in the presence of GSH probably due to the formation of the ebselen-GSH adduct. In conclusion, our investigations provide new utility for ebselen as a prospective antiatherosclerotic in both clinical and non-clinical situations.     

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.     

Cell death in wheat roots induced by the powdery mildew fungus Blumeria graminis f. sp. tritici

Inoculation of wheat (Triticum aestivum L. cv. Huamai 8) leaves with wheat powderly mildew fungus (Blumeria graminis f. sp. tritici) induced cell death in wheat adventitious roots, where no fungal structures were observed. The cytological and molecular characterization of this cell death was shown as following: cell nuclei were TUNEL positive labeled; genomic DNA was fragmented and showed DNA laddering; chromatin condensed and formed peripheral conglomeration in nuclei; and perinuclear spaces partly dilated. These results suggested that, without pathogen spread, the infection could induce systemic PCD in adventitious roots. Comparison with a leaf-cutting experiment (LC)enabled us to speculate that lack of assimilates was not the only reason for the systemic PCD in wheat roots in powdery mildew experiment and that such systemic PCD might be mediated by long-distance signals. In addition, reactive oxygen species (ROS) and Ca²⁺ were related to the systemic PCD.     

Changes in the antioxidant systems as part of the signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobacco Bright-Yellow 2 cells

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for the activation of the hypersensitive reaction, a defense response induced in the noncompatible plant-pathogen interaction. However, its involvement in activating programmed cell death (PCD) in plant cells has been questioned. In this paper, the involvement of the cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition to the culture medium of NO and/or ROS generators. The individual increase in NO or ROS had different effects on the studied parameters than the simultaneous increase in the two reactive species. NO generation did not cause an increase in phenylalanine ammonia-lyase (PAL) activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death when it was generated at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed. The involvement of ASC and GSH as part of the transduction pathway leading to PCD is discussed.