膦酸酯单钠盐对丙酮酸脱氢酶活性的影响

以不同浓度(10-3~10-7g·mL-1)的O-甲基α-(2,4-二氯苯氧基乙酰氧基)甲基膦酸酯单钠盐(以下简称WT)分别在体外处理水稻幼芽和从水稻线粒体中提取的丙酮酸脱氢酶复合体,结果显示随着WT浓度的逐渐降低,其细胞相对存活率逐渐升高;随着WT浓度的逐渐增高,对丙酮酸脱氢酶的抑制活性也逐渐增强。WT对丙酮酸脱氢酶的抑制活性的I50为29·14μmol·L-1。由此说明化合物WT是丙酮酸脱氢酶的强抑制剂。     

大肠杆菌丙酮酸脱氢酶的克隆与表达优化

为了在大肠杆菌中表达大肠杆菌丙酮酸脱氢酶,用PCR法从大肠杆菌总DNA中克隆了pdc基因,将其插入载体pGEX-KG后转化大肠杆菌BL21(DE3),在IPTG的诱导下实现了表达.通过对表达条件的优化,在TB M9(1∶1)培养基中,33℃下使用终浓度0.5 g·L-1的乳糖诱导4 h,重组大肠杆菌丙酮酸脱氢酶的表达量可占全菌蛋白的45%左右,比未优化前提高了约20%,同时大大降低了成本.     

以植物丙酮酸脱氢酶为靶标设计除草剂的研究进展

在正努力探索的新农药靶标酶中,植物丙酮酸脱氢酶系是一个值得探索和研究的除草剂作用靶标。针对以丙酮酸脱氢酶为靶标设计除草剂的研究进展进行了综述,并对其发展趋势及前景进行了展望。     

靶向丙酮酸脱氢酶吡啶衍生物除草剂的设计与合成

丙酮酸脱氢酶系(pyruvate dehydrogenase,PDH)是一个有潜力的除草剂作用靶点。采用不同取代苯甲醛合成各种α-羟基芳基膦酸酯,然后与2,6-吡啶二甲酰氯反应得到五个不同取代基的2,6-吡啶二甲酰氧基烃基膦酸酯,并由IR与1H NMR波谱进行验证。进一步通过对比分析各个不同产物的产率得出苯甲醛上不同取代基对此反应的影响。     

重组苏云金芽孢杆菌丙酮酸脱氢酶克隆及表达条件的优化

根据Bc14579丙酮酸脱氢酶基因序列设计引物,从苏云金芽孢杆菌BMB171菌株总DNA中扩增得到相应的丙酮酸脱氢酶基因DNA片段.将DNA片段装载到大肠杆菌构建pET-E1表达系统,再通过优化重组菌的表达条件获得有生物活性的丙酮酸脱氢酶.结果表明, pET-E1表达系统构建获得成功;优化的表达条件如下:培养基为TB+M9(体积比1∶1)、起始菌体密度OD600为4～5.5、诱导剂IPTG浓度为0.04 mmol·L-1.     

人丙酮酸羧化酶异位表达提高中国仓鼠卵巢细胞补料分批培养后期活率

构建了人丙酮酸羧化酶(hPC)异位表达的重组CHO细胞,并检测了重组细胞生长情况的改变。利用分子生物学实验技术构建hPC-pcDNA 3.0表达载体,将其转染CHO-K1细胞;转染细胞经过G418抗性筛选后,通过荧光定量PCR检测目的基因表达,并挑选表达量最高的克隆进行补料分批培养。电泳及测序结果显示,构建的hPC-pcDNA3.0表达载体序列与预期一致;在mRNA水平鉴定目的基因显示,4#克隆的mRNA表达水平最高;选其进行补料分批培养,生长曲线显示在培养后期,其活细胞密度和细胞活率均高于对照。成功构建了细胞生长和活率改善的重组CHO细胞,获得了生长改善的细胞株,为后续的重组蛋白表达研究与细胞培养工艺优化奠定了基础。     

丙酮酸镁合成工艺的研究

研究了丙酮酸与碳酸镁反应合成丙酮酸镁的工艺过程.通过考察不同的原料配比、反应温度、反应时间以及终点控制,确定了最佳的合成条件:n(丙酮酸):n(碳酸镁)=2.1:1,在温度为65℃下反应60min,反应终点pH在4左右;在此条件下,丙酮酸镁的收率可达94.4%.     

水溶性丙酮酸锌的合成工艺研究

采用丙酮酸与乙酸锌反应合成水溶性丙酮酸锌,确定了适宜的工艺条件。合成水溶性丙酮酸锌最佳的反应条件是:以乙酸乙酯和丙酸为溶剂,其体积比为1∶0.7,丙酮酸与乙酸锌摩尔配比为1∶0.55,在20~25℃温度下反应。得到的产品的结构经红外和核磁的确证,其结构式为(CH3COCOO)2Zn.1/2H2O,得到的产品在水中具有较好的溶解性。     

在杀螨剂评价中几种生测方法的比较研究

在室内用琼脂法、浸玻片法和浸渍法分别测定了氟氯氰菊酯、乐果和齐墩螨素对棉红蜘蛛的毒力.结果表明每种药剂以三种方法测得的对红蜘蛛的毒力均无显著的差异.对三种方法进行分析比较,琼脂法具明显优势,建议在新化合物杀螨活性筛选中采用此法.     

乳酸合成丙酮酸的研究

介绍了由乳酸制备丙酮酸的各种催化工艺方法 ,并对其产率、前景、优缺点等进行了简单的评述     

Computational Study on New Natural Compound Inhibitors of Pyruvate Dehydrogenase Kinases

Pyruvate dehydrogenase kinases (PDKs) are key enzymes in glucose metabolism, negatively regulating pyruvate dehyrogenase complex (PDC) activity through phosphorylation. Inhibiting PDKs could upregulate PDC activity and drive cells into more aerobic metabolism. Therefore, PDKs are potential targets for metabolism related diseases, such as cancers and diabetes. In this study, a series of computer-aided virtual screening techniques were utilized to discover potential inhibitors of PDKs. Structure-based screening using Libdock was carried out following by ADME (adsorption, distribution, metabolism, excretion) and toxicity prediction. Molecular docking was used to analyze the binding mechanism between these compounds and PDKs. Molecular dynamic simulation was utilized to confirm the stability of potential compound binding. From the computational results, two novel natural coumarins compounds (ZINC12296427 and ZINC12389251) from the ZINC database were found binding to PDKs with favorable interaction energy and predicted to be non-toxic. Our study provide valuable information of PDK-coumarins binding mechanisms in PDK inhibitor-based drug discovery.     

Phosphonate Inhibitors of Pyruvate Dehydrogenase Perturb Homeostasis of Amino Acids and Protein Succinylation in the Brain

Mitochondrial pyruvate dehydrogenase complex (PDHC) is essential for brain glucose and neurotransmitter metabolism, which is dysregulated in many pathologies. Using specific inhibitors of PDHC in vivo, we determine biochemical and physiological responses to PDHC dysfunction. Dose dependence of the responses to membrane-permeable dimethyl acetylphosphonate (AcPMe₂) is non-monotonous. Primary decreases in glutathione and its redox potential, methionine, and ethanolamine are alleviated with increasing PDHC inhibition, the alleviation accompanied by physiological changes. A comparison of 39 brain biochemical parameters after administration of four phosphinate and phosphonate analogs of pyruvate at a fixed dose of 0.1 mmol/kg reveals no primary, but secondary changes, such as activation of 2-oxoglutarate dehydrogenase complex (OGDHC) and decreased levels of glutamate, isoleucine and leucine. The accompanying decreases in freezing time are most pronounced after administration of methyl acetylphosphinate and dimethyl acetylphosphonate. The PDHC inhibitors do not significantly change the levels of PDHA1 expression and phosphorylation, sirtuin 3 and total protein acetylation, but increase total protein succinylation and glutarylation, affecting sirtuin 5 expression. Thus, decreased production of the tricarboxylic acid cycle substrate acetyl-CoA by inhibited PDHC is compensated by increased degradation of amino acids through the activated OGDHC, increasing total protein succinylation/glutarylation. Simultaneously, parasympathetic activity and anxiety indicators decrease.     

钻井液处理剂溶液活度测量方法对比

室内通过吸附等温曲线法与饱和湿度法测定了钻井液处理剂溶液的活度。结果表明：两种方法均具有较好的重复性,测量结果基本一致,吸附等温曲线法需要的测试周期较长,饱和湿度法的测试周期明显缩短。     

三种脂肪酶活力测定方法的比较及改进

比较了碱滴定法、对硝基苯酚法和铜皂法等三种常用的脂肪酶活力测定方法的测定时间、准确性和重复性.为对硝基苯酚法找到了有效的终止反应方法,改进的对硝基苯酚法具有检测快捷、结果稳定的特点.对铜皂法进行改进,用甲苯代替了苯,改进的铜皂法具有毒性小、结果稳定、重复性好的优点.     

薄膜压电性能的测量方法

压电薄膜在微传感器和微致动器方面的应用日益受到重视.薄膜压电性能的测量方法与体材相比有很大不同.本文介绍了几种测量薄膜压电特性的方法,包括:激光干涉仪法,扫描探针显微镜法,垂直加载法等,并比较了这些方法的优缺点.对已有的测量结果进行了简要分析.