酿酒酵母氧化应激反应的研究进展

所有好氧生长的生物都必须经受由于氧分子的不完全还原形成活性氧类物质而产生的氧化应激反应。酵母细胞受氧化应激后会很快合成自我修复性的物质,以抵御这些不良环境。本文主要概述了酿酒酵母的氧化应激反应,并对该过程中的几种重要的转录调节因子进行阐述。     

人类超氧化物歧化酶在酿酒酵母系统中的高效表达

超氧化物歧化酶(SOD)是一种在生物界广泛存在的抗氧化酶,在抗衰老、抗肿瘤、抗免疫疾病和电磁辐射上都起着重要的作用．对一从人类胎肝cDNA文库中筛到的SOD基因片段进行重组、克隆,并得到带有启动子PADHZ—GAPDH和终止子TADHI的高效稳定的酿酒酵母表达载体pHC11-hSOD．转化酿酒酵母DCO4后获得工程菌DCO4／pHC11-hSOD．表达产物经破壁后SDS-PAGE电泳检测为20ku的条带;酶活性测定结果表明发酵液有40万U／L的hSOD活性．此外,还研究了工程菌的发酵条件和hSOD产物的纯化方法．纯化产物在SDS-PAGE上和Superdex分子筛检测显示,所得产物的纯度已达95％以上．     

基于GO的酵母膜系统蛋白相互作用规律研究

引进描述蛋白质相互作用倾向性参数(PIRB),基于GO(Gene Ontology)和相关的数据库中蛋白质功能注释,对酿酒酵母部分膜蛋白相互作用规律进行了研究,构建了基于PIRB的蛋白质相互作用网络图.结果表明各类膜蛋白质之间的相互作用具有明显的偏向性.对这种偏向性的生物学含义作了简要探讨.     

酿酒酵母的絮凝研究

酵母絮凝是由絮凝基因编码的絮凝蛋白与邻近细胞的胞壁甘露糖结合形成的多细胞聚集现象.利用31篇文献综述了酵母絮凝的遗传因子和遗传工程研究进展,并介绍了无机离子、营养成分、pH、温度、酒精浓度和菌龄等因素对细胞絮凝的影响.     

磁场对酿酒酵母微观结构及膜脂流动性的影响

以磁场为外加物理场,采用恒定磁场及循环磁处理方式对酿酒酵母进行处理,研究酿酒酵母微观结构及细胞膜流动性的变化.通过透射电子显微镜观察不同磁感应强度恒定磁场处理后酿酒酵母的微观结构.研究发现恒定磁场可以促进酵母细胞的生长,群体中以具有中央大液泡为特征的成熟细胞所占比率增大,同时线粒体呈现适应性膨大、增生.利用原子力显微镜观察循环磁处理下酵母细胞的三维结构,发现细胞壁表面出现皱缩、穿孔,胞质外流.用荧光偏振法对两种处理方式下细胞的膜脂流动性进行测定,发现不同强度恒定磁场处理后,膜脂各向异性下降,流动性增强,0.05T磁感应强度下各向异性值下降3.57%,而循环磁处理后各向异性值比对照提高了8.46%,膜脂流动减慢,证实细胞膜为受到磁场作用的亚细胞结构.     

蛋白质电子跳跃转移与溶剂化电子结构

本文综述了生物体内电荷跳跃转移的一些新特征。重点阐述了蛋白质电荷转移过程中一种可能的中间体--溶剂化电子--的结构性质特征以及在蛋白质长程电荷转移中的重要作用。蛋白质中存在着诸多基团、分子片或电正性区域,比如:质子化的碱性支链基团、芳香环、极性肽链、螺旋及结构水簇等,它们可以俘获电子、然后释放,扮演着电子跳跃转移中继站的作用。这种基于溶剂化电子的电子跳跃转移模式构成了蛋白质内电子长程转移的新途径。     

影响酿酒酵母电击转化率的条件

以酿酒酵母为材料,用穿梭质粒ｐＹＥＳ２进行电击转化的条件实验．实验表明电击中电压大小、细胞的生长状态、处理条件和电击后细胞的培养时间以及质粒浓度等对电击转化率均有影响．取对数生长期酵母细胞,用二硫苏糖醇（ＤＴＴ）处理以疏松细胞壁,加０．７μｇ／ｍＬ质粒ＤＮＡ,５ｋＶ电压电击,转化后细胞在完全培养基中培养６０ｍｉｎ后,涂布选择培养基,转化率可达４．８×１０５个转化子／μｇ质粒ＤＮＡ,细胞的存活率为３９．２％,比完整细胞转化的效率高,存活率偏低     

超声波对酿酒酵母诱变作用的初探

通过利用现有超声波仪器对酿酒酵母进行诱变作用试验,初步探讨了超声波对酿酒酵母致突变的因果效应.试验结果表明,在相同的时间内,超声波频率越高对试验菌株的致死率越高;同一频率,超声波辐射时间增加也可使试验菌株的致死率增高,同时经过诱变处理后的菌株其生理生化特性也有变化.     

酿酒酵母超微结构观察方法的研究

本文用常规电镜技术对不同细胞周期的酿酒酵母ＳａｃｃｈａｒｏｍｙｃｅｓＣｅｒｅｖｉｓｉａｅ菌体细胞和原生质体进行超微结构的对比观察，结果发现，用减数分裂时期的原生质体进行超微结构的观察，效果最为理想。包括细胞核在内的许多细胞器都可观察到。纺锤极体，微管，染色质，染色体，核仁等核内结构也都十分清晰。     

酵母双杂交系统的改进和发展

酵母双杂交系统是研究蛋白质间相互作用的一种非常有效的分子生物学方法，具有快速和高效的特点，但也存在一些局限性．人们针对它的缺点相应地进行了一些完善和改进，并衍生出单杂交系统、三杂交系统等一系列相关的技术，从而拓宽了该系统的应用领域．     

Regulation of cellular response to oncogenic and oxidative stress by Seladin-1

Expression of multiple oncogenes and inactivation of tumour suppressors is required to transform primary mammalian cells into cancer cells. Activated Ha-RasV12 (Ras) is usually associated with cancer, but it also produces paradoxical premature senescence in primary cells by inducing reactive oxygen species followed by accumulation of tumour suppressors p53 and p16(INK4a) (ref. 4). Here we identify, using a direct genetic screen, Seladin-1 (also known as Dhcr24) as a key mediator of Ras-induced senescence. Following oncogenic and oxidative stress, Seladin-1 binds p53 amino terminus and displaces E3 ubiquitin ligase Mdm2 from p53, thus resulting in p53 accumulation. Additionally, Seladin-1 associates with Mdm2 independently of p53, potentially affecting other Mdm2 targets. Ablation of Seladin-1 causes the bypass of Ras-induced senescence in rodent and human fibroblasts, and allows Ras to transform these cells. Wild-type Seladin-1, but not mutants that disrupt its association with either p53 or Mdm2, suppresses the transformed phenotype. The same mutants are also inactive in directing p53-dependent oxidative stress response. These results show an unanticipated role for Seladin-1, previously implicated in Alzheimer's disease and cholesterol metabolism, in integrating cellular response to oncogenic and oxidative stress.     

Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics

Integrins are important adhesion receptors in all Metazoa that transmit conformational change bidirectionally across the membrane. Integrin alpha and beta subunits form a head and two long legs in the ectodomain and span the membrane. Here, we define with crystal structures the atomic basis for allosteric regulation of the conformation and affinity for ligand of the integrin ectodomain, and how fibrinogen-mimetic therapeutics bind to platelet integrin alpha(IIb)beta3. Allostery in the beta3 I domain alters three metal binding sites, associated loops and alpha1- and alpha7-helices. Piston-like displacement of the alpha7-helix causes a 62 degrees reorientation between the beta3 I and hybrid domains. Transmission through the rigidly connected plexin/semaphorin/integrin (PSI) domain in the upper beta3 leg causes a 70 A separation between the knees of the alpha and beta legs. Allostery in the head thus disrupts interaction between the legs in a previously described low-affinity bent integrin conformation, and leg extension positions the high-affinity head far above the cell surface.     

Structural basis for AMP binding to mammalian AMP-activated protein kinase

AMP-activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy and is therefore a target for type II diabetes treatment. It senses changes in the ratio of AMP/ATP by binding both species in a competitive manner. Thus, increases in the concentration of AMP activate AMPK resulting in the phosphorylation and differential regulation of a series of downstream targets that control anabolic and catabolic pathways. We report here the crystal structure of the regulatory fragment of mammalian AMPK in complexes with AMP and ATP. The phosphate groups of AMP/ATP lie in a groove on the surface of the gamma domain, which is lined with basic residues, many of which are associated with disease-causing mutations. Structural and solution studies reveal that two sites on the gamma domain bind either AMP or Mg.ATP, whereas a third site contains a tightly bound AMP that does not exchange. Our binding studies indicate that under physiological conditions AMPK mainly exists in its inactive form in complex with Mg.ATP, which is much more abundant than AMP. Our modelling studies suggest how changes in the concentration of AMP ([AMP]) enhance AMPK activity levels. The structure also suggests a mechanism for propagating AMP/ATP signalling whereby a phosphorylated residue from the alpha and/or beta subunits binds to the gamma subunit in the presence of AMP but not when ATP is bound.     

酿酒酵母CMK2在氧化胁迫应答中的作用

CMK2是酵母中和哺乳动物钙调蛋白激酶CaM kinasesⅡ同源的一种丝氨酸/苏氨酸蛋白激酶,具有重要调控作用.本文利用PCR方法扩增含内源启动子的CMK2基因序列,克隆到pRS316上并在C末端连接6个组氨酸标签,构建真核表达载体,转化不同酵母进行表达,并通过免疫印迹鉴定.发现氧化胁迫后细胞中CMK2发生磷酸化.生长实验结果显示cmk2缺失的细胞对氧化胁迫非常敏感,而Δcmk2中转入pRS316- cmk2-HIS6可以部分恢复Δcmk2细胞对双氧水的敏感性.表明CMK2参与细胞氧化胁迫应答途径,并发挥正调控作用.     

Structural basis for glycosphingolipid transfer specificity

Lipid transfer proteins are important in membrane vesicle biogenesis and trafficking, signal transduction and immunological presentation processes. The conserved and ubiquitous mammalian glycolipid transfer proteins (GLTPs) serve as potential regulators of cell processes mediated by glycosphingolipids, ranging from differentiation and proliferation to invasive adhesion, neurodegeneration and apoptosis. Here we report crystal structures of apo-GLTP (1.65 A resolution) and lactosylceramide-bound (1.95 A) GLTP, in which the bound glycosphingolipid is sandwiched, after adaptive recognition, within a previously unknown two-layer all-alpha-helical topology. Glycosphingolipid binding specificity is achieved through recognition and anchoring of the sugar-amide headgroup to the GLTP recognition centre by hydrogen bond networks and hydrophobic contacts, and encapsulation of both lipid chains, in a precisely oriented manner within a 'moulded-to-fit' hydrophobic tunnel. A cleft-like conformational gating mechanism, involving two interhelical loops and one alpha-helix of GLTP, could enable the glycolipid chains to enter and leave the tunnel in the membrane-associated state. Mutation and functional analyses of residues in the glycolipid recognition centre and within the hydrophobic tunnel support a framework for understanding how GLTPs acquire and release glycosphingolipids during lipid intermembrane transfer and presentation processes.     

Structural basis for template-independent RNA polymerization

The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates. Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.     

Sch9通过Cdc34调控酵母细胞的寿命和胁迫应答

为研究Sch9激酶的生理功能,本文首先通过同步细胞周期的方法发现Sch9的PDK1位点的磷酸化随细胞周期变化.接下来在酵母中过表达Cdc34显示Cdc34的组成性表达可以进一步延长s ch9△突变体的寿命并且增强其对氧化胁迫及热胁迫的抗性.我们的研究结果首次表明Cdc34作为Sch9的底物参与细胞胁迫应答和衰老的调控.     

Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis

Enzyme-catalysed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.