Mechanisms of cardiolipin oxidation by cytochrome c: relevance to pro- and antiapoptotic functions of etoposide

Execution of apoptotic program in mitochondria is associated with accumulation of cardiolipin peroxidation products required for the release of proapoptotic factors into the cytosol. This suggests that lipid antioxidants capable of inhibiting cardiolipin peroxidation may act as antiapoptotic agents. Etoposide, a widely used antitumor drug and a topoisomerase II inhibitor, is a prototypical inducer of apoptosis and, at the same time, an effective lipid radical scavenger and lipid antioxidant. Here, we demonstrate that cardiolipin oxidation during apoptosis is realized not via a random cardiolipin peroxidation mechanism but rather proceeds as a result of peroxidase reaction in a tight cytochrome c/cardiolipin complex that restrains interactions of etoposide with radical intermediates generated in the course of the reaction. Using low-temperature and ambient-temperature electron paramagnetic resonance spectroscopy of H(2)O(2)-induced protein-derived (tyrosyl) radicals and etoposide phenoxyl radicals, respectively, we established that cardiolipin peroxidation and etoposide oxidation by cytochrome c/cardiolipin complex takes place predominantly on protein-derived radicals of cytochrome c. We further show that etoposide can inhibit cytochrome c-catalyzed oxidation of cardiolipin competing with it as a peroxidase substrate. Peroxidase reaction of cytochrome c/cardiolipin complexes causes cross-linking and oligomerization of cytochrome c. With nonoxidizable tetraoleoyl-cardiolipin, the cross-linking occurs via dityrosine formation, whereas bifunctional lipid oxidation products generated from tetralinoleoyl-cardiolipin participate in the production of high molecular weight protein aggregates. Protein aggregation is effectively inhibited by etoposide. The inhibition of cardiolipin peroxidation by etoposide, however, is realized at far higher concentrations than those at which it induces apoptotic cell death. Thus, oxidation of cardiolipin by the cytochrome c/cardiolipin peroxidase complex, which is essential for apoptosis, is not inhibited by proapoptotic concentrations of the drug.     

6－甲氧基正丁苯酞在大鼠肝微粒体中的代谢研究

报道了用ＧＣ／ＭＳ方法及衍生化技术研究６甲氧基正丁苯酞（ＭＢＰ）在大鼠肝微粒体中的代谢转化结果。６甲氧基正丁苯酞在苯巴比妥（ＰＢ）诱导的大鼠肝微粒体中主要转化为３羟基、γ羟基取代物，６羟基正丁苯酞与一个环氧化代谢产物     

氯胺酮和硝苯吡啶对培养神经元谷氨酸兴奋毒性的保护作用

以体外培养的大鼠胚胎皮层神经元为对象,以培养上清液中乳酸脱氢酶活性为指标,研究了谷氨酸兴奋毒性及药物的保护作用。结果表明,培养10d的皮层神经元置于含10或50μmol·L^-1谷氨酸和低糖(1g·L^-1)的DMEM培养液中后,随着作用时间的延长,LDH漏出逐渐增加。在谷氨酸处理前,于培养液中加入氯胺酮或硝苯吡啶,则LDH漏出量明显低于对照组。氯胺酮和硝苯吡啶并用,LDH漏出量比单独使用氯胺酮或硝苯吡啶下降更加明显。结果表明,谷氨酸对培养的神经元可产生严重损伤。氯胺酮和硝苯吡啶单用或并用均有明显的保护作用。     

针灸治疗中风偏瘫选穴规律研究

目的对针灸治疗中风偏瘫的临床研究文献进行统计分析,为临床选穴与合理组穴提供依据。方法收集近28年来针灸治疗中风偏瘫的临床文献,对选用的腧穴种类、归经及使用频次进行统计分析。结果针灸治疗弛缓性偏瘫为主的文献276篇,共使用195个穴位,总频次为2 957次,十四经穴174个,总频次2 849次,占96%;经外奇穴108次,占4%。出现频率占10%以上的经穴29个,阳经穴21个,阴经穴8个。针灸治疗痉挛性偏瘫为主的文献52篇,共使用106穴,总频次为530次,十四经穴97个,总频次511次,占96%;经外奇穴19次,占4%。出现频率占10%以上的经穴31个,阳经穴21个,阴经穴10个。治疗弛缓性偏瘫和痉挛性偏瘫,均以十四经穴为主体,选用阳经穴为主。结论针灸治疗中风弛缓性瘫选用手足阳明、足少阳经、督脉四条阳经,治疗痉挛性瘫以手足少阳经、手阳明经及足太阳经四条经脉为主,上肢配以心包经,下肢配以足太阴经和足阳明经。     

Direct and indirect effects of single walled carbon nanotubes on RAW 264.7 macrophages: role of iron

Single-walled carbon nanotubes (SWCNT), nano-cylinders with an extremely small diameter (1-2 nm) and high aspect ratio, have unique physico-chemical, electronic and mechanical properties and may exhibit unusual interactions with cells and tissues, thus necessitating studies of their toxicity and health effects. Manufactured SWCNT usually contain significant amounts of iron that may act as a catalyst of oxidative stress. Because macrophages are the primary responders to different particles that initiate and propagate inflammatory reactions and oxidative stress, we utilized two types of SWCNT: (1) iron-rich (non-purified) SWCNT (26 wt.% of iron) and (2) iron-stripped (purified) SWCNT (0.23 wt.% of iron) to study their interactions with RAW 264.7 macrophages. Ultrasonication resulted in predominantly well-dispersed and separated SWCNT strands as evidenced by scanning electron microscopy. Neither purified nor non-purified SWCNT were able to generate intracellular production of superoxide radicals or nitric oxide in RAW 264.7 macrophages as documented by flow-cytometry and fluorescence microscopy. SWCNT with different iron content displayed different redox activity in a cell-free model system as revealed by EPR-detectable formation of ascorbate radicals resulting from ascorbate oxidation. In the presence of zymosan-stimulated RAW 264.7 macrophages, non-purified iron-rich SWCNT were more effective in generating hydroxyl radicals (documented by EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide, DMPO) than purified SWCNT. Similarly, EPR spin-trapping experiments in the presence of zymosan-stimulated RAW 264.7 macrophages showed that non-purified SWCNT more effectively converted superoxide radicals generated by xanthine oxidase/xanthine into hydroxyl radicals as compared to purified SWCNT. Iron-rich SWCNT caused significant loss of intracellular low molecular weight thiols (GSH) and accumulation of lipid hydroperoxides in both zymosan-and PMA-stimulated RAW 264.7 macrophages. Catalase was able to partially protect macrophages against SWCNT induced elevation of biomarkers of oxidative stress (enhancement of lipid peroxidation and GSH depletion). Thus, the presence of iron in SWCNT may be important in determining redox-dependent responses of macrophages.     

高三尖杉酯碱在大鼠及兔肝微粒体的代谢研究

采用动物肝微粒体体外代谢法对高三尖杉酯碱进行了代谢转化的研究。应用梯度洗脱—反相HPLC结合二极管陈列检测器对体外代谢体系进行了分析。判定在体外代谢体系中,高三尖杉酯碱主要产生一个代谢产物。用HPLC法制备出一定量代谢物纯品,经光谱分析及与化学制备的对照品相比较,推定其代谢物结构为:2′-羟基-2′(α-乙酸)-6′-甲基-6′-羟基-庚酰三尖杉碱。     

选择性κ阿片激动剂K-Ⅱ与U-50488的药理作用比较

K-Ⅱ系k阿片激动剂U-50488的同类物。通过部分离体和整体实验比较了K-Ⅱ与U-50488的药理作用。实验发现,K-Ⅱ抑制电刺激兔输精管收缩的IC₅₀值为0.42 nmol/L,U-50488为26.5 nmol/L;K-Ⅱ抑制小鼠运动功能(横筛法)的ED₅₀值为1.7 mg/g,U-50488为15.3 mg/kg;K-Ⅱ的小鼠LD₅₀值为152.5 mg/kg,U-50488为118.4 mg/g;K-Ⅱ明显降低小鼠自发活动的作用比U-50488强5倍。结果表明,K-Ⅱ是一个药理作用较U-50488强的k受体激动剂。     

基于尿动力学客观指标的神经原性膀胱概率预测模型构建

目的:建立概率预测的Logistic回归模型,分析影响神经原性膀胱尿动力学的主要危险因素和保护因素,并评价模型的灵敏度、特异度和准确性。方法:收集2004-03/2006-03在中山大学附属第一医院尿流动力学室行尿动力学检查的患者80例,对其尿动力学图的客观指标进行回顾性分析。①80例中尿流动力学图正常者29例为对照组,尿流动力学图显示神经原性膀胱者51例为病例组。②将两组资料采用统一的变量指标(包括性别、年龄以及尿流动力学仪器自动采集计算的34个数据)输入SPSS12.0版本数据库,进行主成分分析,将贡献率高的主成分进行单因素分析,取其中有统计学意义的主成分作多因素Logistic回归分析,建立Logistic回归方程,计算各因素的OR值,并计算模型的灵敏度、特异度和准确度。结果:①尿动力学34个客观指标进行主成分分析后得出8个主成分(C1～8),取贡献率高的5个主成分进行单因素分析,结果有2个主成分可以进入多因素Logistic回归分析。获得Logistic回归概率预测模型,此概率预测模型灵敏度为82.4%,特异度75.9%,准确度为80.0%。②主成分C1的OR值=4.606,C1中的首次尿意膀胱压力和逼尿肌压力、正常尿意膀胱压力和逼尿肌压力、强烈尿意膀胱压力和逼尿肌压力、尿急尿意膀胱压力和逼尿肌压力、充盈期最大逼尿肌压力的系数分别是0.823,0.834,0.781,0.913,0.924,0.932,0.883,0.916,0.857,高于C1中其他变量的系数,故可把主成分C1看作是一个“压力型”指标。③C3的OR值=0.183,C3中系数较高的变量是最大流率、平均流率、压力流率中最大流率和平均流率,分别是0.694,0.777,0.768,0.771,因此把C3看作为“流率”变量指标。结论:①成功构建了人神经原性膀胱尿流动力学图的概率预测模型,其中“压力”因子主成分是危险因素,“流率”因子主成分是保护因素。②概率预测模型的灵敏度、特异度和准确性显示其有较好的代表性。     

Selective early cardiolipin peroxidation after traumatic brain injury: an oxidative lipidomics analysis

OBJECTIVE: Enhanced lipid peroxidation is well established in traumatic brain injury. However, its molecular targets, identity of peroxidized phospholipid species, and their signaling role have not been deciphered. METHODS: Using controlled cortical impact as a model of traumatic brain injury, we employed a newly developed oxidative lipidomics approach to qualitatively and quantitatively characterize the lipid peroxidation response. RESULTS: Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry analysis of rat cortical mitochondrial/synaptosomal fractions demonstrated the presence of highly oxidizable molecular species containing C(22:6) fatty acid residues in all major classes of phospholipids. However, the pattern of phospholipid oxidation at 3 hours after injury displayed a nonrandom character independent of abundance of oxidizable species and included only one mitochondria-specific phospholipid, cardiolipin (CL). This selective CL peroxidation was followed at 24 hours by peroxidation of other phospholipids, most prominently phosphatidylserine, but also phosphatidylcholine and phosphatidylethanolamine. CL oxidation preceded appearance of biomarkers of apoptosis (caspase-3 activation, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positivity) and oxidative stress (loss of glutathione and ascorbate). INTERPRETATION: The temporal sequence combined with the recently demonstrated role of CL hydroperoxides (CL-OOH) in in vitro models of apoptosis suggest that CL-OOH may be both a key in vivo trigger of apoptotic cell death and a therapeutic target in experimental traumatic brain injury.     

Selective oxidation and externalization of membrane phosphatidylserine: Bcl-2-induced potentiation of the final common pathway for apoptosis.

The induction of apoptosis in PC12 cells by the enediyne neocarzinostatin (NCS) is paradoxically potentiated by overexpression of bcl-2. The enhanced activation of NCS seen in bcl-2-overexpressing cells cannot by itself be responsible for the potentiation of apoptosis, since Bcl-2 would be expected to block apoptosis at a point distal to NCS activation (e.g., in the apoptosis final common pathway). We now report that overexpression of bcl-2 in PC12 cells does not protect the cells from NCS-induced oxidation of membrane phosphatidylserine (PS), and results in potentiation of NCS-induced externalization of membrane PS, two events associated with the apoptosis final common pathway. The mechanism of potentiation of apoptosis by Bcl-2 is related to the enhanced reducing potential of bcl-2-overexpressing PC12 cells.