盐生杜氏藻和衣藻双结构域NAD+-GPD基因的生物信息学分析

盐生杜氏藻（Dunaliella salina）是极端耐盐的单细胞真核绿藻,依赖于NAD的3－磷酸甘油脱氢酶（NAD+-GPD）是盐生杜氏藻调渗物质——甘油合成的关键酶.盐生杜氏藻NAD+-GPD基因是第一个被发现含双结构域（SerB和GPD）的GPD基因.而双结构域可能是盐生杜氏藻具有极强耐盐性和快速合成甘油的关键.通过与莱茵衣藻基因组数据库比对,发现莱茵衣藻（Chlamydomonas reinhardtii）中也存在具有SerB和GPD结构域的NAD+-GPD基因序列.在对两个物种NAD+-GPD基因的基因结构、mRNA组成成分、编码蛋白及其理化性质和编码蛋白结构的分析中,发现二者具有较高的相似性.针对目前仅在盐藻和衣藻中发现含SerB和GPD结构域的NAD+-GPD基因这一现象,分别对SerB和GPD结构域同源基因的系统进化进行了分析.     

盐生杜氏藻的完整叶绿体分离

盐生杜氏藻(Dunaliella salina)是世界上最耐盐的一种单细胞真核绿藻,可在含0．1～5．0mol／L NaCl的培养液中正常生长．盐生杜氏藻细胞内的主要调渗物质是甘油,在甘油代谢途径中,3-磷酸甘油脱氢酶是一个关键酶．近年来的研究表明杜氏藻(Dunaliella tertiolecta)细胞质和叶绿体中都存在3-磷酸甘油脱氢酶的同工酶,且叶绿体上的同工酶与渗透调节作用直接相关,为了解D．satina中3-磷酸甘油脱氢酶同工酶的细胞定位和分布,我们通过冰浴超声波破碎和蔗糖密度梯度离心,获得盐生杜氏藻的完整叶绿体,用相差显微镜镜检、酶的活性分析等方法对其完整性作了初步分析。     

氮、磷、硫对盐生杜氏藻色素积累的影响

研究了盐生杜氏藻(Dunaliella salina)在不同浓度氮、磷、硫等营养条件下对细胞积累β-胡萝卜素和叶绿素的影响．发现盐生杜氏藻细胞经过10d生长,在营养缺乏条件下其β-胡萝卜素和叶绿素的比率达到最大,积累β-胡萝卜素的最适条件是无氮、无磷和无硫,但是不利于盐生杜氏藻的生长．讨论了此结果的机理和意义,为利用盐生杜氏藻大规模生产β-胡萝卜素提供了理论依据．     

根据β—胡萝卜素合成机制改进盐生杜氏藻(Dunaliella salina)培养方法的研究

根据β-胡萝卜素合成机制,在盐生杜氏藻(Dunaliella Salina)的培养过程中适当加入柠檬酸,Mg~(2+)和间断通CO_2可以提高盐生杜氏藻体内β—胡萝卜素的含量,其含量可达10.2％.     

盐生杜氏藻同步化及细胞周期的研究

盐生杜氏藻是一种海洋生活的单细胞藻类，细胞中具有细胞核，叶绿体等细胞器，细胞呈椭圆形，直径在15～20μm，人工配制的海水中培养。由于具有叶绿体，能进行光合作用，因此可以用光暗交替处理，诱导细胞同步法。Lor等人[1]报导用光暗交替诱导小眼虫得到较好的同步化结果，他指出对衣滴虫、小球藻。异变形藻等都可以用光暗交替法诱导同步化。盐生杜氏藻使用此法诱导同步化，并测定同步化后细胞周期，这方面还未见详细报道。本文就此作初步探讨。1材料与方法1.1盐生杜氏藻：来自山东海洋大学赠送，定名为Dunaliellabolt。a，采用人工配制…     

杜氏藻DNA酶活性比较研究

杜氏藻(Dunaliella,常称盐藻)是一属耐高盐度的单细胞绿藻,生长迅速,不具纤维素细胞壁,是基因工程潜在的DNA(基因)受体,而其DNA酶活性高低是衡量能否作为受体细胞的重要指标,越低越好。本研究中,DNA酶指能降解DNA的多种酶的总称。 采用三聚乙醛-二苯胺法分别测定了杜氏藻属6个种的DNA酶活性。从测定结果(Tab.1)看出,杜氏藻细胞的DNA酶活性偏低,并且不同种之间存在明显差异。其中D.minuta,     

温度对杜氏藻生长和脂肪酸组成的影响

研究了不同温度对盐生杜氏藻(Dunaliella.salina)生长及脂肪酸组成的影响.结果表明:在20～32℃之间,提高培养温度和增加光照强度促进了盐生杜氏藻的生长;盐生杜氏藻的脂肪酸组分以C16和C18脂肪酸为主,随着培养温度的升高和光照强度的增加,藻株内饱和脂肪酸所占百分比例增加,多不饱和脂肪酸所占百分比例减小.     

盐生杜氏藻(6-4)光裂合酶在大肠杆菌CPD缺陷株SY2中的功能鉴定

将盐生杜氏藻（6—4）光裂合酶基因Ds64PHR的编码序列构建到表达载体pET32a中,利用高效转化法将构建好的表达载体转入E．coli．CPD光裂合酶缺陷菌株SY2中,诱导表达（6-4）光裂合酶融合蛋白,对其功能进行验证．在含有Amp的LB平板上涂布相同数量的大肠杆菌,改变紫外照射强度、光照修复时间,统计最终的菌株存活率．经研究发现,在基因水平上,SY2中表达的盐生杜氏藻（6—4）光裂合酶具有修复紫外诱导损伤的功能,光照是修复功能实现的必需条件．     

盐生杜氏藻Dunaliella salina的生物学特性与培养研究

盐生杜兵藻富含β－胡萝卜素和蛋白质，适宜在高纬度、光照强的各种咸水环境中生长，对它的生物学特性和培养条件进行了研究，发现适宜于盐生杜氏藻生长的培养基主要成分为：２ｍｏｌ／ＬＮａＣｌ，５ｍｍｏｌ／ＬＫＮＯ３，６．５ｍｍｏｌ／ＬＮａＨＣＯ３，５ｍｍｏｌ／ＬＭｇＳｏｒ，０．３ｍｍｏｌ／ＬＫＨ２ＰＯ４。     

杜氏藻中β—胡萝卜素的提取分离

从盐生杜氏藻中提取分离得β-胡萝卜素，探讨了不同提取条件对收率的影响．     

武汉地区丙型肝炎病毒包膜蛋白E1的生物信息学研究

使用生物信息学的方法,分析武汉地区不同基因型、亚型的丙肝病毒的包膜E1蛋白,预测其二级结构、核苷酸变异性、糖基化位点、亲水性、跨膜区、信号肽、蛋白修饰位点、B细胞抗原.结果显示各HCV的包膜E1蛋白二级结构差距不大;序列始末段有数个氨基酸残基的差距,序列上存在高变异位点,基因型2a的型内一致性最高;序列大量糖基化,有多个糖基化基化位点;序列分布着亲水性区域,基因型1b的分值很高;基因型1b、6a、3b、3a多数亚型有1个跨膜区,基因型2a多数亚型有两个跨膜区;基因型1b、6a、3b、3a无信号肽,基因型2a都有信号肽;蛋白序列上存在多个不同修饰位点和B细胞抗原,各基因型、亚型之间有明显差距,具有较大异质性.该研究为揭示病毒感染机制和研制地区性疫苗提供一定的科学依据.     

一个烟草Mlo基因的电子克隆及其序列特性分析

电子克隆并探讨了一个烟草Mlo基因的结构和功能．以GenBank公布的水稻Mlo基因序列为信息探针对烟草的EST数据库进行同源搜索,并将获得的同源性高的EST序列进行序列拼接,最后得到了一个烟草Mlo基因的cDNA序列．采用生物信息学方法分析该基因编码蛋白的一级、二级结构,并对其功能进行预测．结果表明,此烟草Mlo基因...     

Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7?? resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.     

X-ray structure of a protein-conducting channel

A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 A. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two linked halves, transmembrane segments 1-5 and 6-10, clamped together by the gamma-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation.     

Crystal structure of bacterial multidrug efflux transporter AcrB

AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel.     

轴对称ELF磁场中的球细胞感应电场及跨膜电位

为揭示低频磁场作用下人体的电生理特性,研究轴对称ELF(extremelylowfrequency)磁场作用下球细胞的感应电场和跨膜电位,并结合相关实验讨论了磁场对[Ca2 ]i的影响,对不同磁场激励模式、不同频率下的跨膜电位进行了仿真分析.结果表明,ELF磁场可以改变球细胞的跨膜电位,并与频率相关.     

A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.     

Crystal structure of the plasma membrane proton pump

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement.     

Structural changes in the calcium pump accompanying the dissociation of calcium

In skeletal muscle, calcium ions are transported (pumped) against a concentration gradient from the cytoplasm into the sarcoplasmic reticulum, an intracellular organelle. This causes muscle cells to relax after cytosolic calcium increases during excitation. The Ca(2+) ATPase that carries out this pumping is a representative P-type ion-transporting ATPase. Here we describe the structure of this ion pump at 3.1 A resolution in a Ca(2+)-free (E2) state, and compare it with that determined previously for the Ca(2+)-bound (E1Ca(2+)) state. The structure of the enzyme stabilized by thapsigargin, a potent inhibitor, shows large conformation differences from that in E1Ca(2+). Three cytoplasmic domains gather to form a single headpiece, and six of the ten transmembrane helices exhibit large-scale rearrangements. These rearrangements ensure the release of calcium ions into the lumen of sarcoplasmic reticulum and, on the cytoplasmic side, create a pathway for entry of new calcium ions.     

ETMD减振系统及其在海洋平台振动控制中的应用

扩展调谐质量阻尼器(ETMD)减振系统是TMD减振系统的延伸,是利用装置内部设备进行装置振动被动控制的技术.理论分析表明,随着ETMD与剩余平台质量比和频率比的增加,振动控制的频率使用范围扩大,共振的频带缩小.有限元分析结果表明,ETMD减振系统在频率接近激励的激振频率时,有较大的振动位移,可以更多地吸收海洋平台的振动能量;质量比在3.75到5之间时具有比较好的减振效果;频率比在0.3到0.35、0.35到0.5两个区间具有比较好的减振效果.当频率比在0.5到2之间时,系统总体振动都比较小.仿真研究同时表明,在传统导管架平台上ET-MD减振系统也具有良好的减振效果.