Amplification of acetylcholine-binding catenanes from dynamic combinatorial libraries

Directed chemical synthesis can produce a vast range of molecular structures, but the intended product must be known at the outset. In contrast, evolution in nature can lead to efficient receptors and catalysts whose structures defy prediction. To access such unpredictable structures, we prepared dynamic combinatorial libraries in which reversibly binding building blocks assemble around a receptor target. We selected for an acetylcholine receptor by adding the neurotransmitter to solutions of dipeptide hydrazones [proline-phenylalanine or proline-(cyclohexyl)alanine], which reversibly combine through hydrazone linkages. At thermodynamic equilibrium, the dominant receptor structure was an elaborate [2]-catenane consisting of two interlocked macrocyclic trimers. This complex receptor with a 100 nM affinity for acetylcholine could be isolated on a preparative scale in 67% yield.     

Combinatorial ShcA docking interactions support diversity in tissue morphogenesis

Changes in protein-protein interactions may allow polypeptides to perform unexpected regulatory functions. Mammalian ShcA docking proteins have amino-terminal phosphotyrosine (pTyr) binding (PTB) and carboxyl-terminal Src homology 2 (SH2) domains, which recognize specific pTyr sites on activated receptors, and a central region with two phosphorylated tyrosine-X-asparagine (pYXN) motifs (where X represents any amino acid) that each bind the growth factor receptor-bound protein 2 (Grb2) adaptor. Phylogenetic analysis indicates that ShcA may signal through both pYXN-dependent and -independent pathways. We show that, in mice, cardiomyocyte-expressed ShcA directs mid-gestational heart development by a PTB-dependent mechanism that does not require the pYXN motifs. In contrast, the pYXN motifs are required with PTB and SH2 domains in the same ShcA molecule for the formation of muscle spindles, skeletal muscle sensory organs that regulate motor behavior. Thus, combinatorial differences in ShcA docking interactions may yield multiple signaling mechanisms to support diversity in tissue morphogenesis.     

Ligand-binding properties of three odorant-binding proteins of the diamondback moth Plutella xylostella

Strategies for insect population control are currently targeting chemical communication at the molecular level. The diamondback moth Plutella xylostella represents one of the most serious pests in agriculture, however detailed information on the proteins mediating olfaction in this species is still poor. This species is endowed with a repertoire of a large number of olfactory receptors and odorant binding proteins(OBPs). As a contribution to map the specificities of these chemical sensors in the moth and eventually unrave l the complexity of chemodetection, we have measured the affinities of three selected OBPs to a series of potential odorants. Three proteins are highly divergent in their amino acid sequences and show markedly different expression profiles. In fact, PxylOBP3 is exclusively expressed in the antennae of both sexes, PxylOBP9 is male specific and present only in antennae and reproductive organs, while PxylOBP19, an unusual OBP with nine cysteines, is ubiquitously present in all the organs examined. Such expression pattern suggests that the last two proteins may be involved in non-chemosensory functions. Despite such differences, the three OBPs exhibit similar binding spectra, together with high selectivity. Among the 26 natural compounds tested, only two proved to be good ligands, retinol and coniferyl aldehyde. This second compound is particularly interesting being part of the chemical pathway leading to regeneration of lignin, one of the defense strategies of the plant against insect attack, and might find applications as a repellent for P. xylostella and other pests.     

草坪草高羊茅高温诱导抑制差减杂交文库的构建及其表达

为了深入研究草坪草对抗高温逆境的分子遗传机制,构建高羊茅在高温胁迫下相关的基因文库。研究以冷季型草坪草高羊茅为研究对象,在长期生理试验的基础上,通过构建高羊茅在高温胁迫下的SSH文库,挑选部分阳性克隆进行测序,并对所获得的差异基因序列进行分析,研究了在38℃/30℃(昼/夜)培养箱中进行高温胁迫6 h的高羊茅叶片和对照组高羊茅叶片的RNA差异。结果表明:试验得到的差减文库中大量组成型表达的基因已经被有效去除,使某些特有的差异基因得到了富集;两个文库的基因片段的长度分布在200~900 bp,平均片段长度约500 bp左右;在构建的高羊茅耐热cDNA文库中随机挑选了123个大小约500 bp左右的EST测序,共得到100个有效序列,其中38个是未知序列,其余62个为已报道序列,但是序列功能全部未知,这些EST中有25个与叶绿体染色体有关,其中9个与已提交的羊茅属植物叶绿体内的基因同源。本试验最终得到了合格的差减文库并对部分基因进行了测序,可为高羊茅耐热基因的研究提供依据,同时也为提高草坪草水肥调控措施提供了理论基础。     

Lipocalin核糖体展示文库的构建及雌二醇特异性模拟抗体的筛选

[目的]构建Lipocalin核糖体展示文库,并筛选雌二醇特异性模拟抗体。[方法]以Lipocalin蛋白家族中的BBP(胆汁三烯结合蛋白)为模板,设计包含16个随机突变位点的引物,合成BBP基因文库;引入核糖体展示所必需的全部组件,采用重叠PCR技术将BBP库与核糖体展示组件进行拼接,构建核糖体展示Anticalin模拟抗体库;再将构建的Anticalin文库进行体外转录与翻译,产生模拟抗体-核糖体-mRNA三联复合体文库。以小分子抗原雌二醇为靶标,采用固相亲和方法筛选抗雌二醇抗体,通过改变Mg2+浓度将模拟抗体-核糖体-mRNA三联复合体解离,得到与模拟抗体对应的mRNA,经过RT-PCR得到筛选后的DNA文库并重新引入核糖体展示元件进行下一轮淘筛。[结果]通过5轮生物淘筛,模拟抗体得到富集,获得高特异性抗雌二醇抗体。其中有一株克隆的亲和力为54 mmol/L(Kon=4.975 6×104个/M·S;Koff=0.002 7个/S)。IC50与LOD值分别为50和0.071 ng/ml。[结论]获得的雌二醇模拟抗体可以作为抗体并用于检测动物体内雌二醇的残留。     

天然鼠源噬菌体抗体库的构建及抗羊抑制素基因工程单抗的筛选

 【目的】构建单链抗体（ScFv）噬菌体表面展示文库,从中筛选抗羊抑制素单抗并进行表达,建立制备羊抑制素单抗的新方法。【方法】用未经免疫的多种小鼠脾细胞作为基因来源,采用噬菌体抗体库技术,构建天然噬菌体表面抗体文库。用羊抑制素对其进行3轮吸附-洗脱-富集,筛选抗羊抑制素单抗,用ELISA检测其抗原结合活性。【结果】构建了天然鼠源噬菌体抗体库,为筛选和制备各种单链抗体提供了一个平台。用羊抑制素对其进行筛选,制备抗羊抑制素单抗,经ELISA检测,55/96克隆具有与羊抑制素结合活性,阳性率为57%。【结论】制备的羊抑制素可溶性ScFv及其表面展示噬菌体抗体有很好的抗原结合活性,为羊抑制素单抗的制备提供了一种新的方法。     

A family of AMPA-selective glutamate receptors

Four cloned cDNAs encoding 900-amino acid putative glutamate receptors with approximately 70 percent sequence identity were isolated from a rat brain cDNA library. In situ hybridization revealed differential expression patterns of the cognate mRNAs throughout the brain. Functional expression of the cDNAs in cultured mammalian cells generated receptors displaying alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective binding pharmacology (AMPA = quisqualate greater than glutamate greater than kainate) as well as cation channels gated by glutamate, AMPA, and kainate and blocked by 6,7-dinitroquinoxaline-2,3-dione (CNQX).     

Type I and type II GABAA-benzodiazepine receptors produced in transfected cells

GABAA (gamma-aminobutyric acid A)-benzodiazepine receptors expressed in mammalian cells and assembled from one of three different alpha subunit variants (alpha 1, alpha 2, or alpha 3) in combination with a beta 1 and a gamma 2 subunit display the pharmacological properties of either type I or type II receptor subtypes. These receptors contain high-affinity binding sites for benzodiazepines. However, CL 218 872, 2-oxoquazepam, and methyl beta-carboline-3-carboxylate (beta-CCM) show a temperature-modulated selectivity for alpha 1 subunit-containing receptors. There were no significant differences in the binding of clonazepam, diazepam, Ro 15-1788, or dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) to all three recombinant receptors. Receptors containing the alpha 3 subunit show greater GABA potentiation of benzodiazepine binding than receptors containing the alpha 1 or alpha 2 subunit, indicating that there are subtypes within the type II class. Thus, diversity in benzodiazepine pharmacology is generated by heterogeneity of the alpha subunit of the GABAA receptor.     

Novel pharmacology of substance K-binding sites: a third type of tachykinin receptor

The tachykinins are a family of peptides with the carboxyl terminal amino acid sequence Phe-X-Gly-Leu-Met-NH2. Three major mammalian tachykinins have been identified--substance K, neuromedin K, and substance P--but only two tachykinin receptors have been postulated. Three tachykinins were labeled with radioiodinated Bolton-Hunter reagent and their binding characteristics were determined in crude membrane suspensions from several tissues. In cerebral cortex labeled eledoisin exhibited high-affinity binding that was inhibited by tachykinins in a manner indicating a definitive SP-E receptor site. In gastrointestinal smooth muscle and bladder, high-affinity binding of labeled substance P was inhibited in a pattern indicating a definitive SP-P site. In intestinal smooth muscle and bladder, however, labeled substance K and labeled eledoisin were both bound in a pattern indicating a preference for substance K itself. The results suggest the existence of three distinct types of tachykinin receptors: SP-P, SP-E, and SP-K.     

适配体的研究进程与思考

适配体（aptamer）是指利用指数富集的配体系统进化（SELEX）技术，从人工合成的寡核苷酸文库中筛选获得的能够与靶分子特异结合的短单链DNA和RNA分子。筛选得到的适配体可以与DNA，RNA，蛋白质或其它靶分子结合，影响这些靶分子的性质，从而起到改变与靶分子相关的生物学功能的效果。而且研究发现，适配体在与靶分子相互作用时，不仅序列，它们所形成的三维结构也尤其重要。同时，适配体在生物医药研究方面显示出广阔的应用前景。介绍了适配体的发展历程，以及一些典型适配体的生物学功效。提出了RNA适配体三维结构在与靶分子进行相互作用时可能发挥重要作用，以期为RNA适配体的筛选提供理论思考。     

变处理对象通用库结构设计方法的探索

变处理对象的数据由静态数据和动态数据两部分构成。采用动静先分离后结合的设计方法，即将静态数据放在静态库中，将动态数据用键盘提供放入内存变量，校验后传送到临时文件中，最后将临时文件的内容作为处理对象的库结构。该方法与传统建库方法相比具有节约性、准确性、一致性、透明性、可移植性和可扩充性的特点。经实践效果较满意。     

适配体的研究进程与思考*

 适配体（aptamer）是指利用指数富集的配体系统进化（SELEX）技术，从人工合成的寡核苷酸文库中筛选获得的能够与靶分子特异结合的短单链DNA和RNA分子。筛选得到的适配体可以与DNA，RNA，蛋白质或其它靶分子结合，影响这些靶分子的性质，从而起到改变与靶分子相关的生物学功能的效果。而且研究发现，适配体在与靶分子相互作用时，不仅序列，它们所形成的三维结构也尤其重要。同时，适配体在生物医药研究方面显示出广阔的应用前景。介绍了适配体的发展历程，以及一些典型适配体的生物学功效。提出了RNA适配体三维结构在与靶分子进行相互作用时可能发挥重要作用，以期为RNA适配体的筛选提供理论思考。     

区域性“地方文献信息中心”构建探索——以驻马店市与黄淮学院共建图书馆为例

共建图书馆在构建地方文献保障体系和地方文献的收集、整理、整合等许多方面,真正实现了高校和地方人才优势、资源优势及技术优势的最优化组合,能够解决和弥补现行的图书馆事业管理体制的缺陷和障碍。     

Two types of somatostatin receptors differentiated by cyclic somatostatin analogs

Somatostatin receptors in rat brain, pituitary, and pancreas were labeled with two radioiodinated analogs of somatostatins 14 and 28. Two cyclic analogs of somatostatin, SMS201-995 and cyclo(Ala-Cys-Phe-D-Trp-Lys-Thr-Cys), showed biphasic displacement of binding to somatostatin receptors by these radioligands. In contrast, all other somatostatin analogs, including somatostatin-14, competed for the receptor sites with monophasic displacement of radioligand receptor binding. Thus two types of somatostatin receptors were identified. It was found that the pituitary and pancreas have predominantly one type of somatostatin receptor whereas the brain has both, and that different regions of the brain have various proportions of the two types. These findings suggest methods to characterize other types of somatostatin receptors subserving somatostatin's diverse physiological functions, including a potential role in cognitive function and extrapyramidal motor system control.     

Fluorescent guest molecules report ordered inner phase of host capsules in solution

Despite recent strides in the synthesis of elaborate nanometer-scale molecular hosts, the internal structure of these self-assembled cages remains ill characterized. We used fluorescent probe molecules, pyrene butyric acid (PBA), as guests in C-hexylpyrogallol[4]arene capsules to relay information about the chemical environment on the interior of the assemblies. Spectroscopic and single-crystal x-ray diffraction studies show that, in both solution and the solid state, the host can encapsulate two PBA guests and keep them well separated through specific interactions with the capsule walls.     

Identification of nuclear receptors for VIP on a human colonic adenocarcinoma cell line

Vasoactive intestinal peptide (VIP) is a neuropeptide with broad tissue distribution. Although its precise function is unknown, it is thought to exert its effect, at least in part, by interacting with cell surface receptors. Nuclear receptors for VIP have now been identified by specific binding of 125I-labeled VIP to nuclei of a human colonic adenocarcinoma cell line (HT29) and by cross-linking of 125I-labeled VIP to its receptor on intact nuclei. In contrast, 125I-labeled transferrin shows only background binding to nuclei but significant binding to intact cells. Purity of the isolated nuclei was further substantiated by electron microscopy. The apparent molecular sizes of the VIP--cross-linked nuclear and cell surface receptors are similar but not identical.     

Divergent immunoglobulin g subclass activity through selective Fc receptor binding

Subclasses of immunoglobulin G (IgG) display substantial differences in their ability to mediate effector responses, contributing to variable activity of antibodies against microbes and tumors. We demonstrate that the mechanism underlying this long-standing observation of subclass dominance in function is provided by the differential affinities of IgG subclasses for specific activating IgG Fc receptors compared with their affinities for the inhibitory IgG Fc receptor. The significant differences in the ratios of activating-to-inhibitory receptor binding predicted the in vivo activity. We suggest that these highly predictable functions assigned by Fc binding will be an important consideration in the design of therapeutic antibodies and vaccines.     

浅析物理空间及其功能的变化对高校图书馆发展的影响

图书馆的物理空间及其收藏功能是图书馆重要部分,而在不同时期,这二者的相对形态和联系发生了巨大变化。遵循事物的客观基本规律,用创新精神对所属资源进行重新整合对于管理者规划未来至关重要。所以,对于图书馆的发展来说,面临的重要问题则是图书馆的物理空间及其收藏功能的动态变化对其未来发展的重大影响。文中以广西大学图书馆为例,分析这两个方面相对形态及其变化,继而探讨由此而引起的高校图书馆的一系列问题。     

六甲基六元瓜环与紫精衍生物作用体系及其除草活性研究

以紫精衍生物为研究对象,利用核磁共振技术、紫外吸收光谱和循环伏安法等方法,发现六甲基六元瓜环包结了紫精衍生物的烷基部分,形成2∶1比例的哑铃型包结配合物.结果表明,随着紫精衍生物烷基链的增长,紫精衍生物对杂草的除草活性依次减弱,且六甲基六元瓜环可增强紫精衍生物的除草活性.