嗜麦芽寡养单胞菌菌株间羟基化烟碱类杀虫剂吡虫啉的机制差异

嗜麦芽寡养单胞菌Stenotrophomonas maltophilia CGMCC1.1788,R551-3和K279a菌株均具有羟基化烟碱类杀虫剂吡虫啉IMI的能力,其中CGMCC1．1788菌株的羟基化能力明显高于其他两个菌株．细胞色素P450酶抑制剂胡椒基丁醚抑制CGMCC1,1788菌株的IMI羟基化活性,但不抑制R551-3和K279a菌株的IMI羟基化活性;黄素单加氧酶抑制剂甲硫咪唑均抑制3个菌株的IMI羟基化活性．上述结果表明在S．maltophilaCGMCC1．1788中存在细胞色素P450和黄素单加氧酶催化的两种IMI羟基化机制．R5513和K279a蛋白组比较分析表明,K279a菌株中没有细胞色素P450酶,其催化的IMI羟基化只有黄素单加氧酶参与．由此可见,不同S．maltophilia菌株中具有不同的IMI羟基化机制．该研究对于了解IMI的羟基化机制有一定的理论意义．     

芳香化酶在人类乳腺癌、睾丸癌发生过程中的作用

芳香化酶由细胞色素P450芳香化酶和NADPH-细胞色素P450还原酶组成,能够催化雌激素的合成及雄激素A环的羟化。目前研究发现,与非转基因鼠相比,芳香化酶转基因小鼠乳腺组织及睾丸组织中芳香化酶的过表达能引起相应部位雌激素的升高,进而引起乳房增生及睾丸增生,从而导致人类乳腺癌和睾丸癌的发生。     

昆虫细胞色素P450的多样性和功能

细胞色素P450是一类广泛存在的催化酶，能够在昆虫体内参与外源性物质的代谢和内源性物质的合成与降解，帮助昆虫进行正常的生理活动。尽管昆虫细胞色素P450的多样性和功能一直是研究的热点，但目前仍缺乏对这个领域的系统总结和归纳。本文综述了昆虫细胞色素P450数量、表达和反应类型的多样性以及细胞色素P450在昆虫中的功能和进化等方面的研究进展，为更好地研究和利用昆虫细胞色素P450的多样性和功能提供有力的参考依据，同时也能促进我们更好地理解昆虫的代谢和生理活动。     

细胞色素P450与农药相互作用及其机理研究

综述了细胞色素P450的种类和功能多样性,介绍了细胞色素P450参与除草剂代谢及其作用机理．同时,简述了细胞色素P450 14DM与杀菌剂特异性作用的机制．报道了我们从抗除草剂的瑞士黑麦草中克隆CYP8181同源基因及其功能的研究,以及柑橘绿霉菌细胞色素P450 14DM基因的克隆表达．为深入研究杀菌剂作用细胞色素P450 14DM的机理从而设计以P450 14DM为特异性靶标的新型农药、以及进一步研究细胞色素P450酶系与除草剂代谢的关系打下了良好的基础．     

重组家蝇细胞色素P450 6A1的大肠杆菌对艾氏剂的生物降解作用

以大肠杆菌为宿主表达家蝇细胞色素P450 6A1(CYP6A1)酶,在CYP6A1基因后面加入人细胞色素P450还原酶CPR基因,验证了CPR酶对CYP6A1酶催化功能的影响,和CYP6A1酶、重组大肠杆菌对艾氏剂的代谢能力.结果表明:CYP6A1蛋白对艾氏剂有环氧化作用,在CYP6A1对艾氏剂催化中起关键作用.将5μmol/L艾氏剂加入重组大肠杆菌菌液8 h可检测到163.36nmol/L艾氏剂环氧化产物狄氏剂,说明CYP6A1-CPR宿主菌对艾氏剂具有代谢能力,可用于艾氏剂污染的土壤的生物降解.     

苯并(a)芘对蚯蚓细胞色素P450和抗氧化酶影响

研究了苯并(a)芘(BaP)低剂量污染胁迫下对蚯蚓(Eiseniarfetida)体内细胞色素P450酶系和抗氧化酶系的影响.通过滤纸接触染毒法,按指数递增设计暴露质量浓度为10-6到10-2mg·mL-1,染毒时间48h,检测指标分别为蚯蚓细胞色素P450含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性.结果表明,在供试质量浓度范围内,蚯蚓体内P450含量和SOD活性与苯并(a)芘污染胁迫存在明显的响应关系,POD活性在BaP的最大暴露质量浓度下(10-2mg·mL-1)被显著诱导,而CAT活性对BaP暴露无明显的指示作用.这表明,BaP的代谢过程诱发了蚯蚓体内P450含量的增加,同时又抑制了SOD酶活性,从而使生物体对活性氧自由基的防御能力下降而更易受到毒害,这可能是BaP对生物体毒性作用的机制之一.     

人细胞色素P450氧化还原酶的克隆表达和抗体的制备

细胞色素P450还原酶(CPR)是人细胞色素P450酶系的电子供给者,对后者活性的发挥起到重要作用.本研究将从人胚胎cDNA文库中得到的人CPR的编码基因,连接入pT7450表达载体中,在大肠杆菌BL21(DE3)中高效表达并纯化.每升发酵液可得到纯化蛋白49 mg,占总蛋白含量的10%,以细胞色素C为底物检测酶活性,比活为65 u/mg.利用纯化的CPR作为抗原,常规免疫纯系大耳家兔,获得高效价、高特异性的抗血清,抗体滴度为1∶200 000(ELISA法),纯化后抗体应用于不同组织中CPR含量的评价,证明重组CPR及其抗体可用于细胞色素P450的体外药物代谢及细胞色素P450与CPR作用机理的研究.     

深海微生物来源细胞色素P450酶与天然产物生物合成

细胞色素P450酶广泛分布于自然界各种生命体内,具有底物结构类型丰富和催化反应类型多样的两大特点,被誉为自然界中的"万能催化剂".P450酶可在温和条件下催化复杂有机化合物中惰性C-H键的区位或立体专一性高的氧化官能化,广泛参与天然产物的生物合成、异源物质的降解等生命活动,且在医药、化工、食品、农业等诸多领域具有广阔的...     

两个桂花品种花色色素相关基因的差异表达

结合桂花花青素、类胡萝卜素的含量与转录组测序检测到的花色代谢相关基因的差异表达,对品种为‘橙红丹桂’和‘早银桂’的花色变化过程进行了初步研究。结果发现:苯丙氨酸裂解酶、4-香豆酸—Co A连接酶、乙酰辅酶A合酶、查耳酮合酶、R2R3-MYB1、细胞色素P450、番茄红素ε环化酶基因的表达与花青素和类胡萝卜素含量变化相似,这两类色素代谢路径上的其他基因均在‘早银桂’中表达量较高。此外,这种现象很可能是多酚氧化酶(PPO)、细胞色素P450基因在转录因子R2R3-MYB调控下差异表达所致,并且还发现桂花类黄酮的代谢路径是从柚皮素开始转向二氢槲皮素进而在DFR、ANS催化下形成矢车菊色素。     

肉桂水煎液对肝微粒体细胞色素P450酶的影响

考察肉桂水煎液对肝微粒体细胞色素P450酶CYP1A2和CYP2E1活性的影响.采用双抗体夹心酶联免疫吸附试验(ELISA)对大鼠肝脏细胞色素P450酶CYP1A2和CYP2E1进行了初步研究.与空白对照组比较,肉桂水煎液对大鼠肝脏中的CYP1A2和CYP2E1含量有增加的趋势.肉桂水煎液对大鼠肝微粒体CYP1A2和CYP2E1活性有诱导作用,临床上当与经过CYP1A2和CYP2E1代谢的药物合用时应该注意药物间的相互作用.     

Odorant signal termination by olfactory UDP glucuronosyl transferase

The onset of olfactory transduction has been extensively studied, but considerably less is known about the molecular basis of olfactory signal termination. It has been suggested that the highly active cytochrome P450 monooxygenases of olfactory neuroepithelium are termination enzymes, a notion supported by the identification and molecular cloning of olfactory-specific cytochrome P450s (refs. 13-16). But as reactions catalysed by cytochrome P450 (refs 17, 18) often do not significantly alter volatility, lipophilicity or odour properties, cytochrome P450 may not be solely responsible for olfactory signal termination. In liver and other tissues, drug hydroxylation by cytochrome P450 is frequently followed by phase II biotransformation, for example by UDP glucuronosyl transferase (UGT), resulting in a major change of solubility and chemical properties. We report here the molecular cloning and expression of an olfactory-specific UGT. The olfactory enzyme, but not the one in liver microsomes, shows preference for odorants over standard UGT substrates. Furthermore, glucuronic acid conjugation abolishes the ability of odorants to stimulate olfactory adenylyl cyclase. This, together with the known broad spectrum of drug-detoxification enzymes, supports a role for olfactory UGT in terminating diverse odorant signals.     

石斛碱生物合成途径及萜类化合物CYP450酶的研究进展

为了对石斛碱生物合成途径进一步解析,对石斛碱生物合成途径研究进展、5 种药用萜类生物 合成的 CYP450 酶功能、CYP450 酶研究手段三个方面进行文献综述。 通过综述石斛碱生物合成研究进展, 提出 CYP450 酶在石斛碱合成途径中羟基化中间体的作用;总结对药用萜类青蒿酸、丹参酮、鼠尾草酸、人参 皂苷、甘草甜素等合成途径中 CYP450 酶的功能研究与鉴定,发现 CYP450 酶在萜类化合物中的主要功能为 羟基化、酮基化等氧化功能;总结 CYP450 酶的研究手段:一方面基因的筛选可以通过数据库检索或植物组 织、多因子诱导表达差异筛选,另一方面基因的表达和功能鉴定通过基因工程的多种手段,转入原核或真核 宿主细胞中表达出酶蛋白,然后加入底物鉴定其催化活性。 最后,展望下一步石斛碱生物合成 CYP450 酶的 挖掘,认为可以通过已有的转录组中筛选出差异 CYP450 酶基因并进行蛋白表达,然后通过上游酶催化或从 植物中分离纯化的方式获得底物,从酶蛋白与底物的催化挖掘与石斛碱合成相关 CYP450 酶类。     

金庸武侠剧接受心理论

金庸武侠剧之所以广受欢迎，从某种意义上说，主要在于它和大众读者（观众）、精英读者（观众）的接受心理实现了双重沟通。一方面，它通过宣泻、代入、补偿等心理效应，实现了表层的娱乐功能；一方面，它以内在的人文关怀，体现了深层的精神蕴涵。     

Crystal structure of human cytochrome P450 2C9 with bound warfarin

Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug-drug interactions.     

Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation.

Enzyme-based chemical transformations typically proceed with high selectivity under mild conditions, and are becoming increasingly important in the pharmaceutical and chemical industries. Cytochrome P450 monooxygenases (P450s) constitute a large family of enzymes of particular interest in this regard. Their biological functions, such as detoxification of xenobiotics and steroidogenesis, are based on the ability to catalyse the insertion of oxygen into a wide variety of compounds. Such a catalytic transformation might find technological applications in areas ranging from gene therapy and environmental remediation to the selective synthesis of pharmaceuticals and chemicals. But relatively low turnover rates (particularly towards non-natural substrates), low stability and the need for electron-donating cofactors prohibit the practical use of P450s as isolated enzymes. Here we report the directed evolution of the P450 from Pseudomonas putida to create mutants that hydroxylate naphthalene in the absence of cofactors through the 'peroxide shunt' pathway with more than 20-fold higher activity than the native enzyme. We are able to screen efficiently for improved mutants by coexpressing them with horseradish peroxidase, which converts the products of the P450 reaction into fluorescent compounds amenable to digital imaging screening. This system should allow us to select and develop mono- and di-oxygenases into practically useful biocatalysts for the hydroxylation of a wide range of aromatic compounds.     

Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino-acid residue

As a family of structurally-related enzymes, cytochrome P450 (P450) monooxygenases exhibit paradoxical characteristics: although collectively the enzymes display a broad range of substrate specificities, individually they are characterized by a high degree of substrate and product selectivity. Mouse P45015 alpha and P450coh, for example, which are expressed in female liver and male kidney cells, catalyse 15 alpha-hydroxylation of delta 4 3-ketone steroids, such as testosterone and 7-hydroxylation of coumarin, respectively. In spite of their divergent catalytic activities, however, these enzymes differ by only 11 amino acids within their 494 residues. To determine the structural basis of the different substrate specificities of P45015 alpha and P450coh we therefore altered each of these 11 residues by site-directed mutagenesis, expressing the mutant cytochromes in COS-1 cells. We report that the activities of both cytochromes depend critically on the identities of the amino acids at positions 117, 209 and 365 and, moreover, that a single mutation in which Phe 209 is substituted by Leu is sufficient to convert the specificity of P450coh from coumarin to steroid hydroxylation.     

高等教育大众化背景下素质教育的几点思考

中国的高等教育处在一个新旧体制交替变革的时期.一方面,许多原有的观念、体制、教学方法和模式都不能适应高校自身发展的要求和社会对高等教育培育出来的人才的期望;另一方面,无论是理论界还是实际的管理者都还在高校教育改革的路上摸索前进着,新的,合理的,顺应社会发展形势的理念、体制和模式还没有真正被建立起来.     

论明代女词人的怀亲词

词发展到明代,随着社会思潮的变化,一方面大量的女性参与到词的创作中来,一方面女性词人大力开拓词体情感领域,出现了大量的怀念亲人的词作。本文揭橥于此,初步探讨了明代女性怀亲词的形成原因与基本特色。