过度训练对大鼠心肌线粒体膜和基质游离钙的影响

采用模拟过度训练大鼠模型,观察了大鼠心肌线粒体的MDA、巯基（-SH）、还原型谷胱苷肽（GSH）、磷脂A2（PLA2）、游离钙（Fca2＋）等指标,结果表明：过度训练组大鼠心肌线粒体与对照组相比,-SH含量和GSH含量显著降低（P〈0．01）,PLA2活性显著增加（P〈0．01）;MDA、Fca2＋含量明显升高（P〈0．01）．本文根据以上结果探讨了运动时机体产生的活性氧的氧化过程与过度训练后心肌线粒体膜结构变化以及游离钙之间的关系,提出过度训练后PLA2活性增加可能是其发生机制的关键指标,通过对这一指标的进一步观察能更好地了解过度训练发生的分子机制。     

湘莲谷胱甘肽还原酶cDNA的克隆与分析

谷胱甘肽还原酶是植物体内一类重要的抗氧化酶.以湘莲叶片为材料,根据其它植物谷胱甘肽还原酶(GR)氨基酸保守区序列设计简并引物,通过RT-PCR扩增到1个408bp大小的cDNA片段(Genbank注册号为AY781786),5`和3`RACE获得5`和3`端序列,全长1580bp,包含一个1140bp的开放阅读框架,编码380个氨基酸,与其它植物谷胱甘肽还原酶氨基酸序列的同源性在77%～92%之间.     

Engineering human seleno-glutaredoxin containing consecutive rare codons as an artificial glutathione peroxidase

The active center of human glutaredoxin(hGrx1)shares a common thioredoxin fold and specific affinity for substrate glutathione (GSH)with natural glutathione peroxidase(GPx).hGrx1 was redesigned to introduce the catalytic selenocysteine residue to imi- tate the function of antioxidant selenoenzyme GPx in vivo.The human hGrx1 scaffold is a good candidate for potential medical application compared with other animal-originated protein scaffolds.Two consecutive rare codons(AGG-AGG)in the open reading frame of hGrx1 mRNA encoding Arg26-Arg27 residues can reduce seleno-hGrx1 expression level significantly in the Cys auxotrophic Escherichia coli strain BL21cysE51.Therefore,we optimized the rare codons,which resulted in a remarkable in- crease of the expression level in the Cys auxotrophic cells,which may be sufficient for future medical production.The engineered artificial selenoenzyme displays high GPx catalytic activity,rivaling that of some natural GPx proteins.Kinetic analysis of the engineered seleno-hGrx1 showed a typical ping-pong kinetic mechanism;its catalytic properties are similar to those of some nat- urally occurring GPx proteins.     

The interaction of GSSG modified magnetic nanoparticles with SPC-A1 cells in vitro

Magnetic nanoparticles(MNPs) are promising materials for various biomedical applications,including magnetic resonance imaging,stem cell tracking,gene/drug delivery,and cancer treatment.To increase the effectiveness of MNPs,high capture efficiency and controlled uptake of the particles by cells is required.In this paper we report the cytotoxicity and cellular uptake into SPC-A1 cells of oxidized glutathione(GSSG)-modified MNPs(GSSG@Fe3O4).Experimental findings indicated that GSSG@Fe3O4 were biocompatible,and could be efficiently taken up by SPC-A1 cells(up to 160 pg iron per cell).The internalized GSSG@Fe3O4 was retained in the cell cytoplasm for 6 generations.The uptake of GSSG@Fe3O4 into SPC-A1 cells was energy-,concentration-and time-dependent.Pinocytosis may be involved in the internalization process of GSSG@Fe3O4 into SPC-A1 cells,but this mechanism remains to be elucidated.The controlled and efficient localization of GSSG@Fe3O4 into the cytosol and long intracellular retention provides theoretical and experimental insight into the biomedical applications for these molecules.     

天然矿泉水与纯净水抗过氧化作用的比较

将6周龄纯种BALB/C小鼠随机分为4组,一组为皮下注射生理盐水的非致衰对照组,另三个组为注射D-半乳糖的致衰模型组,它们分别饮用天然矿泉水,纯净水和自来水.4个月后,测定红细胞超氧化物歧化酶(SOD)和全血谷胱甘肽过氧化物酶(GSH-Px)的活性.本实验表明,天然矿泉水具有一定的抗过氧化作用.天然矿泉水组与自来水致衰对照组比较,GSH-Px活力变化不显著,而SOD活力明显提高( 7.1%).而纯净水组与天然矿泉水组比较,SOD活力降低了10.2%,GSH-Px活力降低15.5%,两项指标均有显著差异.值得注意的是,纯净水组与自来水致衰对照组相比较,其抗过氧化作用也有所减弱,纯净水组的SOD活力下降3.9%,GSH-Px活力更是显著地下降了18.5%.     

Effect of nonprotein thiols on protein synthesis in isolated rat hepatocytes

The ability of nonprotein thiols to modulate rates of protein synthesis was investigated in isolated rat hepatocytes. Addition of cysteine stimulates protein labelling by [¹⁴C] Leucine. Glutahione depletion, induced by in vivod administration of L-buthionine sulfoximine and diethylmaleate, did not alter the effect of cysteine, although it decreased the rate of protein synthesis by 32%. The effect of cysteine on protein synthesis does not seem to be related to a perturbatin of the redox state of the NAD⁺/NADH system or to changes in the rate of gluconeogenic pathway. The following observations indicate that cysteine may stimulate protein syntheis by increasing intracellular levels of aspartate: 1. Amino-oxyacetate, an inhibitor of pyridoxyal-dependent enzymes, inhibits protein labelling and decreases aspartate cellular content, whereas most amino acids accumulate or remain unchanged; 2. Cysteine, in the absence or in the presence of amino-ocycetate, stimulates protein labelling and induces aspartate accumulation, although mot amino acids diminish or remain unchanged.     

谷胱甘肽合成酶系基因协调表达体系的构建

通过 PCR获得 E.coli B谷胱甘肽合成酶系基因 ( gsh I,gsh II)片段 ,结合定点突变稀有起始密码子 ,设定 gsh I与 gsh II位置与距离 ,构建双顺反子重组表达载体 p Trc99A/gsh I- gsh II,建立GSHI、GSH- II蛋白表达体系。结果表明 :以 0 .0 8mmol/L IPTG于 2 8℃诱导工程菌 E.coli BL2 1( DE3) ( p Trc99A/gsh I- gsh II) ,GSH- I、GSH- II蛋白比为 4.5∶ 1 ( m∶ m)时谷胱甘肽合成能力最高 ,达到每克湿菌体 8.5 mg。通过构建单顺反子重组表达载体 p Trc99A/gsh I、p Trc99A/gsh II测定GSH- I与 GSH- II蛋白的最适配比 ,结果表明 :在 GSH- I、GSH- II蛋白总量恒定的情况下 ,要提高谷胱甘肽产率 ,两者比例以 3∶ 1～ 6∶ 1为宜     

固定化E.coli BL21(pTrc-gsh)细胞催化合成谷胱甘肽

分别以卡拉胶、明胶、海藻酸钠包埋E.coli BL21 (pTrc-gsh)细胞催化合成谷胱甘肽(GSH).从酶活收率及机械强度方面进行比较,选择卡拉胶为包埋载体,其最适pH为7.0,最适温度为40°C.相关物质对GSH的合成均有影响半胱氨酸、甘氨酸的最适浓度为20mmol/L,谷氨酸的最适浓度为60mmol/L;Mg2+/ATP为1～5(V/V)较合适;腺苷二磷酸(ADP)浓度为5mmol/L时对酶活的抑制为20%.优化条件下罐式反应器中GSH的产量为0.84g/L,操作稳定性较好;延迟加入甘氨酸时GSH产量可提高17.5%;与酵母生产ATP体系相耦联的共固定化体系在填充床中反应,GSH合成量达1.24g/L,收率比直接加入ATP提高24.2%.     

日本七鳃鳗含硒谷胱甘肽过氧化物酶4的克隆与生物学特性分析

谷胱甘肽过氧化物酶4（GPx4）是一类抗氧化酶,能够减少脂质氢过氧化物、氢过氧化物和有机氢过氧化物。从日本七鳃鳗中克隆得到了 G Px4（L jG Px4）编码区和基因组DNA序列,揭示了其基因组特征为含有6个外显子结构和5个内含子结构,并将日本七鳃鳗GPx4序列同其他脊椎动物进行了同源序列比对。使用实时定量PCR分析了 LjGPx4在日本七鳃鳗中的组织分布。脂多糖（LPS）体内刺激七鳃鳗后发现,LjGPx4 mRNA在白细胞中表达升高。日本七鳃鳗GPx4具有高度保守的基因序列和蛋白质结构,并参与抗菌免疫应答,为抗氧化剂酶在无颌脊椎动物的先天免疫反应中发挥至关重要的作用提供了实验依据。     

Characterization of the separate kinase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The bifunctional enzvme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase consists of two dis tinct domains which catalyze the synthesis and hydrolysis of fructose-2, 6-bisphosphate, respectively. In this work the properties of the separate 6-phosphofructo-2-kinase domain were investigated. Purification of the expressed separate do main or isolation of this domain from purified glutathione S-transferase (GST) fusion protein with thrombin cleavage led to the loss of its kinase activity. Thus the domain in the GST-tagged form was characterized. The two forms of the do main with different lengths (amino acids 1 ～ 249 and 1 ～ 286) were very similar in kinetic property and could catalyze the formation of fructose-2,6-bisphosphate with a kcat 4-fold lower than that of the full-length enzyme. In addition, the domain was much more sensitive to guanidine inactivation and heat denaturation, and less stable at pH values below 7 than the full-length enzyme. The results suggest that the separate kinase domain of the bifunctional enzyme is far less perfect in structure in the absence of the bisphosphatase domain, though it still possesses the 6-phosphofructo-2-kinase activity.