神经退化性疾病生物能量代谢和氧化应激研究进展

衰老是导致几种常见的神经系统退化性疾病的主要危险因素,包括帕金森氏病（Ｐａｒｋｉｎｓｏｎ’ｓ ｄｉｓｅａｓｅ ＰＤ）,肌萎缩性侧索硬化（Ａｍｙｏｔｒｏｐｈｉｃ ｌａｔｅｒａｌ ｓｃｌｅｒｏｓｉｓ,ＡＬＳ）,早老性痴呆（Ａｌｚｈｅｉｍｅｒ’ｓ ｄｉｓｅａｓｅ ＡＤ）和亨廷顿氏病（Ｈｕｎｔｉｎｇｔｏｎ’ｓ ｄｉｓｅａｓｅ ＨＤ）。最近研究表明,神经退化性疾病涉及到线粒体缺陷,氧化应激等因素。在脑和其它组织中,老化可导致线粒体功能的损伤和氧化损伤的增强。ＰＤ病人中,已发现线粒体复合酶体Ⅰ活性降低,氧化损伤增加和抗氧化系统活性的改变。在几例家族性ＡＬＳ病人中,也发现Ｃｕ、Ｚｎ超氧化物歧化酶（Ｃｕ,Ｚｎ ＳＯＤ）基因的突变,导致Ｃｕ、Ｚｎ超氧化物歧化酶活性减低;散发的ＡＬＳ病人氧化损伤增高。在ＨＤ病人中已发现能量代谢异常     

Cellular Mechanisms of Resistance to Chronic Oxidative Stress

Oxidative stress is implicated in several pathologies such as AIDS, Alzheimer’s disease, and Parkinson’s disease, as well as in normal aging. As a model system to study the response of cells to oxidative insults, glutamate toxicity on a mouse nerve cell line, HT-22, was examined. Glutamate exposure kills HT-22 via a nonreceptor-mediated oxidative pathway by blocking cystine uptake and causing depletion of intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species and, ultimately, apoptotic cell death. Several HT-22 subclones that are 10-fold resistant to exogenous glutamate were isolated and the mechanisms involved in resistance characterized. The expression levels of neither heat shock proteins nor apoptosis-related proteins are changed in the resistant cells. In contrast, the antioxidant enzyme catalase, but not glutathione peroxidase nor superoxide dismutase, is more highly expressed in the resistant than in the parental cells. In addition, the resistant cells have enhanced rates of GSH regeneration due to higher activities of the GSH metabolic enzymes γ-glutamylcysteine synthetase and GSH reductase, and GSH S-transferases activities are also elevated. As a consequence of these alterations, the glutamate resistant cells are also more resistant to organic hydroperoxides and anticancer drugs that affect these GSH enzymes. These results indicate that resistance to apoptotic oxidative stress may be acquired by coordinated changes in multiple antioxidant pathways.     

拟南芥神经酰胺酶基因对氧化胁迫的响应

以拟南芥哥伦比亚生态型（Col）和神经酰胺酶突变体为实验材料,通过对突变体的一系列生理生化指标的测定,来研究拟南芥神经酰胺酶基因（AtCER）对H2O2的响应。利用PCR和Northern blot获得了9个AtCER纯合单突变体。不同浓度H2O2处理野生型和突变体后,发现突变体对H2O2的反应比野生型更加敏感。H2O2处理后突变体叶片出现比野生型更严重的黄化现象和坏死斑点,总叶绿素含量也比野生型下降的更快,电导率测定也发现突变体比野生型的电导率增加得更多。抗氧化酶活性的分析结果发现H2O2处理后,突变体的抗氧化酶活性比野生型提高了1．5～3倍。上述研究结果说明AtCER参与了H2O2诱导的氧化胁迫反应。     

植物铁蛋白与氧化胁迫应激

植物铁蛋白是植物体重要的铁调节蛋白。许多研究表明植物铁蛋白与氧化胁迫抗性之间具有较强关联。植物铁蛋白不仅能抵御高铁产生的氧化毒性,在很多氧化胁迫及环境胁迫抗性中也发挥作用。对植物铁蛋白在氧化及逆境胁迫中的应激加以综述,为铁蛋白在生物工程领域的应用提供理论依据。     

人线粒体tRNALeu(UUR)基因氧化损伤与修复研究

人mtDNA比核DNA更易受到自由基的氧化损伤,这些损伤可以被线粒体内的DNA修复机制所修复,损伤与修复是决定突变是否产生的两个重要因素.为了确定氧化损伤与损伤后修复对mtDNA突变的具体影响,采用四氧嘧啶处理LO2细胞,这种试剂进入细胞后,经氧化还原反应生成的自由基与线粒体自身代谢产生的自由基类似,然后观察自由基对细胞mtDNA的氧化损伤与损伤后DNA修复的动力学变化.由于线粒体的正常功能为修复机制所必需,采用MTT细胞活力实验检测不同浓度四氧嘧啶处理下线粒体酶活力,发现9 mmol/L四氧嘧啶培养细胞1h后,线粒体琥珀酸脱氢酶功能在撤去药物后0,2,8和24 h时间点均无明显变化.提取各组细胞的mtDNA,用EndoⅢ和Fgp两种酶切除受氧化损伤的核苷酸,然后用碱性琼脂糖凝胶电泳分离大小不等的mtDNA,进行DNA印迹实验,地高辛-抗体-碱性磷酸酶系统显色,检测完整与断裂的mtDNA量,利用Poisson公式(s=-lnP0/P,P0为未断裂链光密度值,P为所有链光密度值总和)计算一个mtDNA分子的平均损伤频率,结果显示,9 mmol/L四氧嘧啶处理细胞1 h,链平均损伤频率由对照的0.11个/分子增加至5.60个/分子,明显增加了mtDNA上核苷酸的氧化损伤,除去药物后8 h,绝大部分损伤可被修复,损伤频率减至0.40个/分子,除去药物后24h核苷酸的氧化损伤恢复至正常水平.采用接头介导PCR(LM-PCR)检测MTTL1基因区域内单个核苷酸的损伤与修复动力学.这种方法可以检测各组mtDNA上MTTL1基因75 bp区域内单个核苷酸损伤的部位及频率.结果显示,人MTTL1基因存在20个易受氧化损伤的核苷酸热点,经与相应区域内文献报道的16个突变热点比较,有12个热点部位重合,而修复未显示热点部位或区域.结果提示,自由基对核苷酸的选择性氧化损伤是决定mtDNA点突变发生及发生部位的主要原因.     

PINCH in the Cellular Stress Response to Tau-Hyperphosphorylation

Particularly interesting new cysteine- histidine- rich protein (PINCH) is an adaptor protein that our data have shown is required for neurite extension under stressful conditions. Our previous studies also report that PINCH is recalled by neurons showing decreased levels of synaptodendritic signaling proteins such as MAP2 or synaptophysin in the brains of human immunodeficiency virus (HIV) patients. The current study addressed potential role(s) for PINCH in neurodegenerative diseases. Mass spectrometry predicted the interaction of PINCH with Tau and with members of the heat shock response. Our in vitro data confirmed that PINCH binds to hyperphosphorylated (hp) Tau and to E3 ubiquitin ligase, carboxy-terminus of heat shock-70 interacting protein. Silencing PINCH prior to induction of hp-Tau resulted in more efficient clearance of accumulating hp-Tau, suggesting that PINCH may play a role in stabilizing hp-Tau. Accumulation of hp-Tau is implicated in more than 20 neuropathological diseases including Alzheimer''s disease (AD), frontotemporal dementia (FTD), and human immunodeficiency virus encephalitis (HIVE). Analyses of brain tissues from HIVE, AD and FTD patients showed that PINCH is increased and binds to hp-Tau. These studies address a new mechanism by which AD and HIV may intersect and identify PINCH as a contributing factor to the accumulation of hyperphosphorylated Tau.     

Glioprotective Effects of Lingonberry Extract Against Altered Cellular Viability,Acetylcholinesterase Activity,and Oxidative Stress in Lipopolysaccharide-Treated Astrocytes

Altered astrocytic function is a contributing factor to the development of neurological diseases and neurodegeneration. Berry fruits exert neuroprotective effects by modulating pathways involved in inflammation, neurotransmission, and oxidative stress. The aim of this study was to examine the effects of the lingonberry extract on cellular viability and oxidative stress in astrocytes exposed to lipopolysaccharide (LPS). In the reversal protocol, primary astrocytic cultures were first exposed to 1 µg/mL LPS for 3 h and subsequently treated with lingonberry extract (10, 30, 50, and 100 μg/mL) for 24 and 48 h. In the prevention protocol, exposure to the lingonberry extract was performed before treatment with LPS. In both reversal and prevention protocols, the lingonberry extracts, from 10 to 100 μg/mL, attenuated LPS-induced increase in reactive oxygen species (around 55 and 45%, respectively, P?<?0.01), nitrite levels (around 50 and 45%, respectively, P?<?0.05), and acetylcholinesterase activity (around 45 and 60%, respectively, P?<?0.05) in astrocytic cultures at 24 and 48 h. Also, in both reversal and prevention protocols, the lingonberry extract also prevented and reversed the LPS-induced decreased cellular viability (around 45 and 90%, respectively, P?<?0.05), thiol content (around 55 and 70%, respectively, P?<?0.05), and superoxide dismutase activity (around 50 and 145%, respectively, P?<?0.05), in astrocytes at both 24 and 48 h. Our findings suggested that the lingonberry extract exerted a glioprotective effect through an anti-oxidative mechanism against LPS-induced astrocytic damage.     

Growth Response of Clostridium perfringens to Oxidative Stress

The sensitivity of Clostridium perfringens strain 13 to oxygen and its toxic derivatives was investigated in a new, defined medium (MMP). Exponentially growing cells in MMP medium were very sensitive to exposure to air by vigorous shaking. When exposed to air, the cells survived only 1hour and then rapidly died. Addition of cysteine, ascorbic acid, or yeast extract to the medium significantly increased vegetative cell survival without inducing sporulation. The level of toxicity of peroxyl and hydroperoxyl radicals, generated by H₂O₂, t-butyl hydroperoxide or ethanol, was very similar with and without air exposure. By contrast, plumbagin or menadione, which generate superoxide radicals in the presence of oxygen, caused high levels of cell death only in aerobiosic culture. Growth-arrested cells were more resistant to H₂O₂and to redox-cycling agents than were exponentially growing cells, but the resistance required de novo synthesis of proteins. An adaptive response to oxidative stress was also suggested by the higher level of cell resistance to H₂O₂and to ethanol when cells were pretreated with sublethal doses of these oxidants.     

酵母SOD1基因缺失突变体应答刚果红胁迫的转录组学分析

SOD1基因编码的铜锌超氧化物歧化酶是酵母细胞中最重要的抗氧化酶. 前期研究发现,SOD1基因缺失(sod1Δ)导致酵母细胞对真菌细胞壁抑制剂刚果红(Congo red, CR)的敏感性增加,提示细胞抗氧化能力与细胞壁稳定性相关. 本研究采用酵母全基因组表达谱芯片,比较了CR胁迫条件下,野生型酵母细胞和sod1Δ酵母细胞的转录表达谱. 结果表明,与野生型酵母细胞相比,sod1Δ酵母细胞中260个基因发生了显著差异表达(140个基因表达上调、120个基因表达下调). 随机选取12个差异表达基因采用定量PCR验证,结果与芯片分析结果一致. 差异表达基因功能主要涉及细胞壁(几丁质合成)、细胞代谢、细胞防御(抗氧化和热冲击蛋白)、蛋白质合成以及大量功能未知基因. 进一步研究发现,CR处理后,细胞壁几丁质含量和细胞内氧化应激指标丙二醛(MDA)含量在sod1Δ酵母细胞中显著升高,而在野生型酵母细胞中无明显变化,与芯片筛选差异表达基因的生物学功能分析结果一致. 本研究提供了在全基因组水平上对SOD1基因与细胞壁应激反应之间关联的新认识.     

Superoxide Dismutase (Sod-1) Null Mutants of Neurospora Crassa: Oxidative Stress Sensitivity, Spontaneous Mutation Rate and Response to Mutagens

Enzymatic superoxide-dismutase activity is believed to be important in defense against the toxic effects of superoxide. Although superoxide dismutases are among the best studied proteins, numerous questions remain concerning the specific biological roles of the various superoxide-dismutase types. In part, this is because the proposed damaging effects of superoxide are manifold, ranging from inactivation of certain metabolic enzymes to DNA damage. Studies with superoxide-deficient mutants have proven valuable, but surprisingly few such studies have been reported. We have constructed and characterized Neurospora crassa mutants that are null for sod-1, the gene that encodes copper-zinc superoxide dismutase. Mutant strains are sensitive to paraquat and elevated oxygen concentrations, and they exhibit an increased spontaneous mutation rate. They appear to have near wild-type sensitivities to near- and far-UV, heat shock and γ-irradiation. Unlike the equivalent Saccharomyces cerevisiae mutant and the sodA sodB double mutant of Escherichia coli, they do not exhibit aerobic auxotrophy. These results are discussed in the context of an attempt to identify consensus phenotypes among superoxide dismutase-deficient mutants. N. crassa sod-1 null mutant strains were also employed in genetic and subcellular fractionation studies. Results support the hypothesis that a single gene (sod-1), located between Fsr-12 and leu-3 on linkage group I, is responsible for most or all CuZn superoxide dismutase activity in this organism.     

Myocardial Creatine Levels Do Not Influence Response to Acute Oxidative Stress in Isolated Perfused Heart

Background

Multiple studies suggest creatine mediates anti-oxidant activity in addition to its established role in cellular energy metabolism. The functional significance for the heart has yet to be established, but antioxidant activity could contribute to the cardioprotective effect of creatine in ischaemia/reperfusion injury.

Objectives

To determine whether intracellular creatine levels influence responses to acute reactive oxygen species (ROS) exposure in the intact beating heart. We hypothesised that mice with elevated creatine due to over-expression of the creatine transporter (CrT-OE) would be relatively protected, while mice with creatine-deficiency (GAMT KO) would fare worse.

Methods and Results

CrT-OE mice were pre-selected for creatine levels 20–100% above wild-type using in vivo ¹H^–MRS. Hearts were perfused in isovolumic Langendorff mode and cardiac function monitored throughout. After 20 min equilibration, hearts were perfused with either H₂O₂ 0.5 µM (30 min), or the anti-neoplastic drug doxorubicin 15 µM (100 min). Protein carbonylation, creatine kinase isoenzyme activities and phospho-PKCδ expression were quantified in perfused hearts as markers of oxidative damage and apoptotic signalling. Wild-type hearts responded to ROS challenge with a profound decline in contractile function that was ameliorated by co-administration of catalase or dexrazoxane as positive controls. In contrast, the functional deterioration in CrT-OE and GAMT KO hearts was indistinguishable from wild-type controls, as was the extent of oxidative damage and apoptosis. Exogenous creatine supplementation also failed to protect hearts from doxorubicin-induced dysfunction.

Conclusions

Intracellular creatine levels do not influence the response to acute ROS challenge in the intact beating heart, arguing against creatine exerting (patho-)physiologically relevant anti-oxidant activity.     

氧化应激下植物线粒体自噬分析

线粒体自噬,是指通过选择性的识别并清除损伤、衰老及功能紊乱的线粒体,对维持细胞内线粒体质量和数量的平衡产生了重要作用。与动物和酵母中线粒体自噬的研究进展相比,植物线粒体自噬的途径及具体调控机制尚不明确。基于GFP标签,本文探究了氧化胁迫下植物线粒体自噬发生情况。研究发现甲基紫精诱导线粒体在液泡中积累,并呈现两种状态:1) GFP小体包含的线粒体; 2)不含GFP的线粒体。本研究发展的GFP标签策略可为植物线粒体自噬关键调控因子的筛选提供借鉴。     

Selective Up-regulation of Human Selenoproteins in Response to Oxidative Stress

Selenocysteine is inserted into selenoproteins via the translational recoding of a UGA codon, normally used as a stop signal. This process depends on the nature of the selenocysteine insertion sequence element located in the 3′ UTR of selenoprotein mRNAs, selenium bioavailability, and, possibly, exogenous stimuli. To further understand the function and regulation of selenoproteins in antioxidant defense and redox homeostasis, we investigated how oxidative stress influences selenoprotein expression as a function of different selenium concentrations. We found that selenium supplementation of the culture media, which resulted in a hierarchical up-regulation of selenoproteins, protected HEK293 cells from reactive oxygen species formation. Furthermore, in response to oxidative stress, we identified a selective up-regulation of several selenoproteins involved in antioxidant defense (Gpx1, Gpx4, TR1, SelS, SelK, and Sps2). Interestingly, the response was more efficient when selenium was limiting. Although a modest change in mRNA levels was noted, we identified a novel translational control mechanism stimulated by oxidative stress that is characterized by up-regulation of UGA-selenocysteine recoding efficiency and relocalization of SBP2, selenocysteine-specific elongation factor, and L30 recoding factors from the cytoplasm to the nucleus.     

Exogenous Calcium Improves Viability of Biocontrol Yeasts Under Heat Stress by Reducing ROS Accumulation and Oxidative Damage of Cellular Protein

In this article, we investigated the effect of exogenous calcium on improving viability of Debaryomyces hansenii and Pichia membranaefaciens under heat stress, and evaluated the role of calcium in reducing oxidant damage of proteins in the yeast cells. The results indicated that high concentration of exogenous calcium in culture medium was beneficial for enhancing the tolerance of the biocontrol yeasts to heat stress. The possible mechanism of calcium improving the viability of yeasts was attributed to enhancement of antioxidant enzyme activities, decrease in ROS accumulation and reduction of oxidative damage of intracellular protein in yeast cells under heat stress. D. hansenii is more sensitive to calcium as compared to P. membranaefaciens. Our results suggest that application of exogenous calcium combined with biocontrol yeasts is a practical approach for the control of postharvest disease in fruit.     

人线粒体tRNALeu（UUR）基因氧化损伤与修复研究

人mtDNA比核DNA更易受到自由基的氧化损伤,这些损伤可以被线粒体内的DNA修复机制所修复,损伤与修复是决定突变是否产生的两个重要因素.为了确定氧化损伤与损伤后修复对mtDNA突变的具体影响,采用四氧嘧啶处理LO₂细胞,这种试剂进入细胞后,经氧化还原反应生成的自由基与线粒体自身代谢产生的自由基类似,然后观察自由基对细胞mtDNA的氧化损伤与损伤后DNA修复的动力学变化.由于线粒体的正常功能为修复机制所必需,采用MTT细胞活力实验检测不同浓度四氧嘧啶处理下线粒体酶活力,发现9 mmol/L四氧嘧啶培养细胞1h后,线粒体琥珀酸脱氢酶功能在撤去药物后0,2,8和24 h时间点均无明显变化.提取各组细胞的mtDNA,用EndoⅢ和Fgp两种酶切除受氧化损伤的核苷酸,然后用碱性琼脂糖凝胶电泳分离大小不等的mtDNA,进行DNA印迹实验,地高辛-抗体-碱性磷酸酶系统显色,检测完整与断裂的mtDNA量,利用Poisson公式（s=－lnP₀/P,P₀为未断裂链光密度值,P为所有链光密度值总和）计算一个mtDNA分子的平均损伤频率,结果显示,9 mmol/L四氧嘧啶处理细胞1 h,链平均损伤频率由对照的0.11个/分子增加至5.60个/分子,明显增加了mtDNA上核苷酸的氧化损伤,除去药物后8 h,绝大部分损伤可被修复,损伤频率减至0.40个/分子,除去药物后24 h核苷酸的氧化损伤恢复至正常水平.采用接头介导PCR（LM-PCR）检测MTTL1基因区域内单个核苷酸的损伤与修复动力学.这种方法可以检测各组mtDNA上MTTL1基因75 bp区域内单个核苷酸损伤的部位及频率.结果显示,人MTTL1基因存在20个易受氧化损伤的核苷酸热点,经与相应区域内文献报道的16个突变热点比较,有12个热点部位重合,而修复未显示热点部位或区域.结果提示,自由基对核苷酸的选择性氧化损伤是决定mtDNA点突变发生及发生部位的主要原因.