江浙蝮蛇毒碱性磷脂酶A2正交晶体分子置换法结构测定

江浙蝮蛇(Agkistrodon halys pallas)毒碱性磷脂酶A2具有很强的溶血作用.P2₁2₁2₁晶型的晶体学不对称单位中含有2个分子.用自身旋转函数研究了此2分子的相对空间关系,用交叉旋转函数和平移函数测定了2分子在晶胞中的取向与位置.在此基础上进行了初步的三维结构模型构建与结构修正.碱性磷脂酶A2正交晶体不对称单位中2个分子的排布呈现非晶体学二重对称关系.     

B链C端去七肽(B24～B30)胰岛素的分子置换法

一个几近失活的胰岛素类似物———B链C端去七肽 (B2 4～B30 )胰岛素(DHPI)已被结晶 .该晶体属于空间群P2 ₁2 ₁2 ₁,晶胞的 1个独立区内包含有 2个DHPI分子 .应用分子置换法确定了DHPI分子在晶胞中的取向和位置 ,并在 0 .3nm分辨率水平上构建了结构模型 .研究结果表明 ,一个独立区内的 2个DHPI单体分子由一个非晶体学 2次轴联系着 ,该局部 2次轴近似平行于晶体学c轴 .这一特性直接影响了自身旋转函数对该局部对称轴取向的确定     

云南48种木兰科野生植物资源遗传多样性研究

为了解云南省木兰科（Magnoliaceae）野生植物资源的遗传多样性,利用ISSR分子标记技术对48种木兰科野生植物资源进行研究。结果表明,10对引物共扩增出151条带,均为多态性条带,多态性条带百分率为100%。总的观测等位基因数（N_a）为2.000 0,有效等位基因数（N_e）为1.564 5,Nei’s基因多样性指数（H）0.337 9,Shannon’s信息指数（I）为0.510 1。总的基因多样性指数（H_t）为0.368 0,属间基因多样性指数（D_st）为0.251 9,占68.4%,基因分化系数（G_st）为0.684 0,基因流（N_m）为0.231 0。UPGMA聚类分析将48种木兰科植物划分为7个类群,各类群并非按照属聚在一起,而是不同属植物相间分布,长喙厚朴（Magnolia rostrata）、素黄含笑（Michelia flaviflora）和球花含笑（M.sphaerantha）可能为云南省木兰科植物中的原始种。48种木兰科野生植物总体具有较高的遗传多样性,但属间遗传变异较高,基因流较小,存在遗传漂变的风险,聚类结果与刘玉壶的分类系统存在分歧,这从分子水平为木兰科植物间的起源、进化与分类提供了重要依据。     

胰岛素单斜晶体(B型)结构的分子置换法研究

在含有ZnCl₂的柠檬酸缓冲体系中,保持苯酚浓度在0.76％～1.25％之间,获得了胰岛素单斜晶体(B型),空间群为P2₁,晶胞参数为:a=4.924 nm,b=6.094nm,c=4.818nm,β=95.8°,每个独立区包含有由6个胰岛素分子构成的1个六聚体。以四锌牛胰岛素六聚体作模型,用X-PLOR软件中的旋转函数程序和本实验室的分子密堆积程序,获得了胰岛素单斜晶体(B型)结构的初始相位。借助生物大分子刚体精化技术对模型进行了初步精化,用能量极小化的立体化学制约的最小二乘精化技术并辅以差值Fourier图人工分析对模型进行了调整和精化。最终R因子为22.4％,键长和键角与标准键长和键角的偏差分别为0.0022nm和4.7°。     

螺环哌啶类似物作为DOR激动剂的特征结构研究

阿片受体激动剂与特定阿片受体亚型结合,常用来治疗与外伤、癌症或心脏病相关的严重疼痛,是十分有吸引力的药用物质．阿片受体有3种经典亚型(δ, κ, μ),均有与其对应的激动剂．δ阿片受体(DOR)激动剂因其还有明显的抗焦虑、抗抑郁和器官保护作用,是非常有前景的药物．本文研究了一批共102个N-取代螺环哌啶类似物作为δ阿片受体激动剂的分子,采用比较分子力场(CoMFA)和比较分子相似性指数(CoMSIA)两种分析方法对所有分子进行了三维定量构效关系(3D-QSAR)研究,其中基于疏水场和氢键供体场参数建立的CoMSIA模型最佳,其模型结果为：Q²=0.501,R²_ncv=0.787,R²_pre=0.780,证明模型自我吻合良好,同时有较强的内部及外部预测能力．而模型的等势线图分析表明,在R₁处引入疏水性的取代基及在R₂处引入亲水性的取代基或氢键供体基团对提高激动剂活性有利．这些结论有助于更好地理解N-取代螺环哌啶类似物作为DOR激动剂的机理,为新型的δ阿片受体激动剂的设计和优化提供一定的指导．     

苏云金芽孢杆菌辅助蛋白的研究进展

杀虫晶体蛋白是苏云金芽孢杆菌所产生的主要杀虫成分 ,在其超量表达与晶体形成过程中有时需要辅助蛋白的参与。在这些辅助蛋白中有的能够防止正在翻译过程中的杀虫晶体蛋白被菌体内的蛋白酶降解 ,起着分子伴侣的作用 ;有的在晶体形成过程中起着脚手架的作用。本文对辅助蛋白 P₂ 0、P1₉以及ORF₁、ORF_{2等在杀虫晶体蛋白表达及晶体形成过程中的作用作了综述。}     

牦牛MT-Ⅰ/-Ⅱ cDNA分子克隆及其蛋白质结构分析

利用基因特异引物Y_MTSP₁和Y_MTSP₂,通过RT-PCR从牦牛肝脏组织RNA中克隆出了牦牛MT-Ⅰ(GenbankAccessionNo:AY513744)和MT-Ⅱ(GenbankAccessionNo:AY513745)基因编码区全长。将牦牛MT-Ⅰ和MT-ⅡcDNA序列在CBI上进行同源性搜索发现,牦牛MT-Ⅰ/-Ⅱ编码区序列在不同哺乳动物中相当保守。牦牛MT-Ⅰ和MT-Ⅱ编码的MT-Ⅰ和MT-Ⅱ蛋白分别由61个氨基酸组成,其具有保守的短肽结构如:C-X-C,C-C-X-C-C,C-X-X-C等,其决定MT蛋白分子的整个三维结构,在分子进化上十分保守。同时对牦牛MT的疏水性和跨膜区分析表明,牦牛MT蛋白可能不存在跨膜区,也不存在信号肽,是1种非分泌蛋白。并通过同源比较模建,预测和构建了牦牛MT-Ⅰ和MT-Ⅱ蛋白的分子空间结构,表明牦牛MT-Ⅰ和MT-Ⅱ由α-和β-两个结构域组成,在α-结构域含有5个Cys短肽结构,β-结构域有4个Cys短肽结构,且2个结构域由保守的三肽序列KKS相连。     

高活性F1-ATP酶单分子旋转初步观察

从基因突变的F₁-ATP酶（基因突变质粒,α-C193S, γ-S107C,β亚基带有10个组氨酸标记（His-Tag）,转入到菌株大肠杆菌JM103）的菌株中筛选出一高表达菌株.该菌株表达的F₁-ATP酶经纯化后其水解活性明显高于文献值. 从单分子水平上进行观察,发现在水解ATP过程中,γ亚基上连接的荧光标记蛋白微丝,其旋转速度要比文献中同样条件下快约一倍.     

S1核酸酶作用与微环DNA分子的克隆策略

米曲霉来源的S1核酸酶具有降解单链DNA或RNA的作用。在适当的条件下, 该酶能将不同的环形DNA分子从超螺旋转变成开环和线形结构,对质粒Puc19的实验证明, S₁核酸酶的这种转变作用与加入的酶量呈正相关。在25μL总反应体积中,按100ng DNA加入5u至17u的S1核酸酶,能获得较高比例的线形DNA。由于微环DNA分子太小,单酶切位点的出现率较低,很难用常规方式进行克隆,以S₁核酸酶进行线形化是微环DNA克隆的途径。pC3是已知最小的真核生物线粒体DNA类质粒（537bp）,经S₁核酸酶线形化后,成功地克隆到pMD18-T载体上。     

陆地棉产量性状QTLs的分子标记及定位

用我国的高产栽培品种泗棉3号和美国栽培品种TM-1为材料,构建F₂和F_2∶3作图群体,应用301对SSR引物和1040个RAPD引物,对产量性状QTLs进行了分子标记筛选,结果共筛选出了37对SSR多态性引物和10个RAPD多态性引物的49个位点,鉴定出了控制产量性状变异的主效QTLs。定位于第9染色体的连锁群,分别具有控制铃重、衣分和籽指的主效QTLs,铃重的2个QTLs分别解释F_2∶3群体表型变异的18.2%和21.0%;在F₂群体检测到的1个衣分QTL解释表型变异的25%,另一个衣分QTL在F₂群体和F_2∶3群体都检测到,解释F₂群体衣分的24.9%的表型变异,解释F_2∶3群体衣分的5.9%的表型变异;在F_2∶3群体铃重的一个QTL的同一位置同时检测到一个籽指QTL,它解释15.6%的表型变异,是一因多效或是紧密连锁的两个QTLs,有待进一步研究。本研究标记的产量性状主效QTLs可用于棉花产量性状的标记辅助选择。     

Expression and Functional Characterization of the Human Ether-à-go-go-Related Gene (<Emphasis Type="Italic">HERG</Emphasis>) K<Superscript>+</Superscript> Channel Cardiac Splice Variant in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes

HERG C_Cardiac, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG A), was identified and found to be 100% homologous to HERG_USO. Real-time polymerase chain reaction data indicated that in the human heart HERG C_Cardiac mRNA was expressed eight times more than HERG A, whereas in human ventricular tissue it was expressed six times more than HERG A. A HERG C_Cardiac-green fluorescence protein (GFP) construct was heterologously expressed in Xenopus oocytes. Confocal micrographs revealed that HERG C_Cardiac was mainly expressed in the plasma membrane. HERG C_Cardiac channel expressed in oocytes produced slower inactivating outward currents and faster deactivating tail currents than those of HERG A channel. Equal amounts of HERG A and HERG C_Cardiac cRNA coinjected into oocytes formed intermediate HERG A + HERG C_Cardiac heteromultimers, which was reconfirmed by immunoprecipitation experiments with a HERG A N-terminal antibody. These heteromultimers had different inactivation, deactivation and activation kinetics from those of HERG A and HERG C_Cardiac channels. HERG A + HERG C_Cardiac heteromultimers significantly reduced the model action potential mean amplitude and increased the fast and slow inactivation τ values of the action potential repolarization phase, suggesting involvement of HERG A and HERG C_Cardiac heteromultimers in modulation of the refractory interval.     

Selective modulation of amyloid-β peptide degradation by flurbiprofen, fenofibrate, and related compounds regulates Aβ levels

Gamma‐secretase modulators (GSMs) include selected non‐steroidal anti‐inflammatory drugs such as flurbiprofen that selectively lowers the neurotoxic amyloid‐β peptide Aβ_1–42. GSMs are attractive targets for Alzheimer’s disease, in contrast to ‘inverse GSMs,’ such as fenofibrate, which selectively increase the level of Aβ_1–42. A methodology for screening of Aβ modulating drugs was developed utilizing an Aβ‐producing neuroblastoma cell line stably transfected with mutant human amyloid precursor protein, immunoprecipitation of Aβ peptides, and mass spectroscopic quantitation of Aβ_1–37/Aβ_1–38/Aβ_1–40/Aβ_1–42 using an Aβ internal standard. The unexpected conclusion of this work was that in this system, drug effects are independent of γ‐secretase. The methodology recapitulated reported results for modulation of Aβ by GSMs. However, control experiments in which exogenous Aβ_1–40/Aβ_1–42 was added (i) to drug‐treated wild‐type cells or (ii) to conditioned media from these wild‐type cells, gave comparable patterns of Aβ modulation. These results, suggesting that drugs modulate the ability of cell‐derived factors to degrade Aβ, was interrogated by adding protease inhibitors and performing molecular weight cut‐off fractionation. The results confirmed that modulation of Aβ_1–40/Aβ_1–42 was mediated by selective proteolysis. Treatment of N2a cells with flurbiprofen or fenofibric acid selectively enhanced Aβ_1–42 clearance by extracellular proteolysis; treatment with HCT‐1026 or fenofibrate (esters of flurbiprofen and fenobric acid) inhibited clearance of Aβ_1–40 and Aβ_1–42.     

Structural Basis for Aβ1-42 Toxicity Inhibition by Aβ C-Terminal Fragments: Discrete Molecular Dynamics Study

Amyloid β-protein (Aβ) is central to the pathology of Alzheimer's disease. Of the two predominant Aβ alloforms, Aβ_1-40 and Aβ_1-42, the latter forms more toxic oligomers. C-terminal fragments (CTFs) of Aβ were recently shown to inhibit Aβ_1-42 toxicity in vitro. Here, we studied Aβ_1-42 assembly in the presence of three effective CTF inhibitors and an ineffective fragment, Aβ_21-30. Using a discrete molecular dynamics approach that recently was shown to capture key differences between Aβ_1-40 and Aβ_1-42 oligomerization, we compared Aβ_1-42 oligomer formation in the absence and presence of CTFs or Aβ_21-30 and identified structural elements of Aβ_1-42 that correlated with Aβ_1-42 toxicity. CTFs co-assembled with Aβ_1-42 into large heterooligomers containing multiple Aβ_1-42 and inhibitor fragments. In contrast, Aβ_21-30 co-assembled with Aβ_1-42 into heterooligomers containing mostly a single Aβ_1-42 and multiple Aβ_21-30 fragments. The CTFs, but not Aβ_21-30, decreased the β-strand propensity of Aβ_1-42 in a concentration-dependent manner. CTFs and Aβ_21-30 had a high binding propensity to the hydrophobic regions of Aβ_1-42, but only CTFs were found to bind the Aβ_1-42 region A2-F4. Consequently, only CTFs but not Aβ_21-30 reduced the solvent accessibility of Aβ_1-42 in region D1-R5. The reduced solvent accessibility of Aβ_1-42 in the presence of CTFs was comparable to the solvent accessibility of Aβ_1-40 oligomers formed in the absence of Aβ fragments. These findings suggest that region D1-R5, which was more exposed to the solvent in Aβ_1-42 than in Aβ_1-40 oligomers, is involved in mediating Aβ_1-42 oligomer neurotoxicity.     

The Role of Phosphatidylinositol Contained in Lipoprotein Lipase Produced by Mucor javanicus IAM 6108

Respiratory inhibition by piericidin A was overcome by addition of vitamin K₃ to the inhibited respiratory chain in mammalian mitochondria but not in insect mitochondria.

Antagonistic effect of vitamin K₃ on the inhibition of piericidin A was apparently found in respiration, blood pressure and heart rate in rat in vivo. Furthermore, toxicity of piericidin A to mouse and rat decreased when piericidin A was administered as the mixture of vitamin K₃ in intraperitoneal route.

No antagonistic effect of vitamin K₃ was observed on the inhibition of piericidin A in TTC reaction of american cockroach nerve cord, femorals and digestive organs. Toxicity of piericidin A to some insects were not affected by vitamin K₃.     

SYNTHETIC HYBRIDS OF NEW WORLD AND OLD WORLD AGROPYRONS II. AGROPYRON RIPARIUM X AGROPYRON REPENS

Five hybrids were obtained from 12 seeds formed in 26 emasculated florets of A. riparium pollinated by A. repens. The hybrid plants were morphologically intermediate between the parents for vegetative and spike characteristics, although they resembled A. repens more closely than A. riparium. The 28-chromosome A. riparium parent behaved cytologically as an allotetraploid and formed an average of 13.98 II and 0.04 I in 94 cells at metaphase I. An average of 20.27 II and 0.36 IV were observed at metaphase I in 55 cells of A. repens, which was designated as a segmental autoallohexaploid. The hybrids contained 35 chromosomes and averaged 6.75 I, 12.49 II, 1.05 III, 0.01 IV, and 0.01 V in 162 cells interpreted at metaphase I. Bivalent chromosome pairing in the hybrids was attributed to autosyndetic pairing of 2 A. repens genomes and allosyndetic pairing between 1 A. riparium genome and 1 A. repens genome. Multivalent chromosome associations were attributed to structural hybridity. A. repens and A. riparium apparently share a genome in common, and this genome is the one responsible for rhizomes in both species. A. riparium was given a genome formula of R₂R₂SS; whereas the A. repens genome formula was written as R₁R₁X₁X₁X₂X₂, and the hybrid genome formula was designated as R₁R₂X₁X₂S. The “S” genome of A. riparium was derived from A. spicatum, and the “R” genome is the genome shared by A. repens and A. riparium. The origin and distribution of the so-called “X” genomes of A. repens remain unknown. The hybrids produced from 3 to 10% stainable pollen; however, no seed was set on the hybrids during 2 years in the field.     

S100A8 and S100A9—oxidant scavengers in inflammation

S100A8 and S100A9 are generally considered proinflammatory. Hypohalous acids generated by activated phagocytes promote novel modifications in murine S100A8 but modifications to human S100A8 are undefined and there is no evidence that these proteins scavenge oxidants in human disease. Recombinant S100A8 was exquisitely sensitive to equimolar ratios of HOCl, which generated sulfinic and sulfonic acid intermediates and novel oxathiazolidine oxide/dioxide forms (mass additions, m/z +30 and +46) on the single Cys₄₂ residue. Met₇₈(O) and Trp₅₄(+16) were also present. HOBr generated sulfonic acid intermediates and oxidized Trp₅₄(+16). Evidence for oxidation of the single Cys₃ residue in recS100A9 HOCl was weak; Met₆₃, Met₈₁, Met₈₃, and Met₉₄ were converted to Met(O) in vitro. Oxidized S100A8 was prominent in lungs from patients with asthma and significantly elevated in sputum compared to controls, whereas S100A8 and S100A9 were not significantly increased. Oxidized monomeric S100A8 was the major component in asthmatic sputum, and modifications, including the oxathiazolidine adducts, were similar to those generated by HOCl in vitro. Oxidized Met₆₃, Met₈₁, and Met₉₄ were variously present in S100A9 from asthmatic sputum. Results have broad implications for conditions under which hypohalous acid oxidants are generated by activated phagocytes. Identification in human disease of the novel S100A8 Cys derivatives typical of those generated in vitro strongly supports the notion that S100A8 contributes to antioxidant defense during oxidative stress.     

Synthesis and Use of a Chromogenic Substrate Analog for Ap4A Catabolic Enzymes

ATP was coupled with 5-bromo-4-chloro-3-indolyl phosphate using a water-soluble carbodiimide to yield 5-bromo-4-chloro-3-indolyl tetraphospho-5′-adenosine (BClp₄A) which is an analog of diadenosine 5′,5′″-P¹,P⁴-tetraphosphate (Ap₄A). BClp₄A is a chromogenic substrate for three different types of Ap₄A catabolic enzyme in alkaline phosphatase-coupled reactions. Ap₄A phosphorylase I from Saccharomyces cerevisiae was used as a model enzyme to demonstrate that BClp₄A stains for enzymic activity in polyacrylamide gels under nondenaturing conditions. A yeast colony assay was developed to detect Ap₄A phosphorylase I activity in situ using BClp₄A as a chromogenic substrate. Ap₄A phosphorylase I was assayed in situ in yeast transformed with a multicopy plasmid containing APA1, the gene encoding Ap₄A phosphorylase I. BClp₄A should facilitate screening of genomic or cDNA libraries for genes encoding Ap₄A catabolic enzymes.     

美国海滨桤木和薄叶桤木水分生理特性的比较

采取盆栽、人工控水的方式, 研究并比较了美国本土海滨桤木(Alnus maritima)和薄叶桤木(A. incana)的气孔导度(G_s)、叶片水势(ψ_leaf)以及渗透调节能力对土壤水分条件的响应, 以探讨引起两种桤木生态分布差异巨大的生理生态原因。结果表明: 1)正常水分条件下, 海滨桤木的G_s低于薄叶桤木, 其与大气温度、相对湿度和水蒸气亏缺等气象因子的相关性低于薄叶桤木; 干旱胁迫下, 海滨桤木的G_s对其自身ψ_leaf下降信号的敏感度低于薄叶桤木; 复水后, 其G_s恢复更为缓慢。2)正常水分条件下, 海滨桤木的ψ_leaf高于薄叶桤木, 且引起气孔关闭的ψ_leaf临界值较高; 干旱胁迫下, 海滨桤木的ψ_leaf下降幅度高于薄叶桤木。3)正常水分条件下, 海滨桤木和薄叶桤木的渗透调节能力无显著差异; 干旱胁迫下, 尽管两种桤木均表现出饱和状态渗透势(ψ_s^sat)下降、膨压与水势关系的最大变化率降低、初始失膨点渗透势(ψ_s^tlp)增加、细胞渗透调节能力范围(ψ_s^sat-ψ_s^tlp, Dψ_s)减小的趋势, 但与薄叶桤木相比, 海滨桤木的ψ_s^tlp较高, Dψ_s较小。从以上生理生态指标可以看出, 较高的叶片水势、较低的气孔调节能力、干旱下较低的渗透调节能力是造成海滨桤木分布范围狭小的重要原因。     

Structural modification of Escherichia coli lipid A by myristoyltransferase gene from Klebsiella pneumoniae

Lipid A in lipopolysaccharide (LPS) of Escherichia coli mutant strains was modified by the introduction of myristoyltransferase gene cloned from Klebsiella pneumoniae. When the gene was introduced into the mutant having lipid A containing only 3‐hydroxymyristic acids, it produced lipid A with two additional myristic acids (C_14:0). When the same gene was introduced into the mutant with pentaacylated lipid A containing one lauric acid (C_12:0), C_12:0 was replaced by C_14:0. IL‐6‐inducing activity of LPS with modified lipid A structure suggested that C_12:0 in lipid A could be replaced by C_14:0 without changing the immunostimulating activity.     

The regulatory role of adenosine-activated T-lymphocyte subset on the immune response in humans: I. Mitogenic response and production of mediators

Human T lymphocytes, rerosetted with sheep erythrocytes in the presence of adenosine, yield two subpopulations: a major one (E_R), still capable of forming E rosettes; and a minor nonrosetting (E_S) one. The two subpopulations differed in their proliferative responses to various mitogens. E_R cells responded well to galactose oxidase (GO), soybean agglutinin (SBA), and phytohemagglutinin (PHA) but responded poorly to concanavalin A (Con A). The response of E_S cells was poor to GO and SBA, intermediate to PHA, and significantly high to Con A. The different response of E_R and E_S subsets to Con A was not greatly affected by adherent cells, but an enhancing effect on the proliferation of E_S cells to Con A was observed when prostaglandin synthesis was inhibited by indomethacin. Addition of E_S cells to E_R cells in a ratio of 1:5 resulted in an enhanced synergistic effect of Con A-induced proliferation. A soluble mitogenic factor released from Con A-activated T cells appeared involved in this enhanced proliferation. This factor (E_SF) was produced only by the minor T-cell subpopulation which is sensitive to adenosine (E_S). The induction of E_SF was not dependent on the addition of adherent cells and required 72 hr of incubation for its production. E_SF was mitogenic to nonactivated and Con A-activated PBL as well as to T, E_R, and E_S subpopulations. Following incubation of E_R cells with E_SF, a suppressor factor (E_RSF) was produced which abolished the mitogenic activity of E_SF. Differences between these factors and a known mediator like Interleukin-2 (IL-2) and suppressor factors are discussed.