Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVl

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (alpha), 20 kDa (beta(1)) and 22 kDa (beta(2)), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that beta(1) and beta(2) subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between 1 and 2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the U. and beta, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against UNA inhibited the aggregation response to stejnulxin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbalpha or alpha(2)beta(1) as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

蛋白质序列和结构的保守性与其功能的关系

在酪氨酸磷酸酶超家族分子的序列中,仅有3个与其催化功能密切相关的残基是高度保守的,但它们功能结构域的核心拓扑结构却明显类似,其中存在着#beta##alpha##beta#和#beta##alpha##beta##alpha#2个保守的结构单元;此外,它们活性位点的拓扑结构也极其相似。

Evolution of different oligorneric glycyl-tRNA synthetases

There are two oligomeric types of glycyl-tRNA synthetases (GlyRSs) in genome, the alpha(2)beta(2) tetramer and alpha(2) dimer. Here, we showed that the anticodon-binding domains (ABDs) of dimeric and tetrameric GlyRSs are non-homologous, although their catalytic central domains (CCDs) are homologous. The dimeric GlyRS_ABD is fused to the C-terminal of CCD in alpha-subunit, but the tetrameric GlyRS_ABD is to the C-terminal in beta-subunit during evolution. Generally, one species only contains one oligomeric type of GlyRS, but the both oligomeric GlyRSs with the multiple homologous domains can be observed in Magnetospirillum magnetotacticum genome, nevertheless, these homologous domains are probably from different genomes. (C) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A de novo originated gene depresses budding yeast mating pathway and is repressed by the protein encoded by its antisense strand

Recent transcription profiling studies have revealed an unexpectedly large proportion of antisense transcripts in eukaryotic genomes. These antisense genes seem to regulate gene expression by interacting with sense genes. Previous studies have focused on the non-coding antisense genes, but the possible regulatory role of the antisense protein is poorly understood. In this study, we found that a protein encoded by the antisense gene ADF1 acts as a transcription suppressor, regulating the expression of sense gene MDF1 in Saccharomyces cerevisiae. Based on the evolutionary, genetic, cytological and biochemical evidence, we show that the protein-coding sense gene MDF1 most likely originated de novo from a previously non-coding sequence and can significantly suppress the mating efficiency of baker's yeast in rich medium by binding MAT alpha 2 and thus promote vegetative growth. These results shed new light on several important issues, including a new sense-antisense interaction mechanism, the de novo origination of a functional gene, and the regulation of yeast mating pathway.

The three dimensional structure and biological domains of the p53 C-terminus.

It was expected that there are a coil (289 similar to 325) and two a helix (alpha(1)368 similar to 373, alpha(2)381 similar to 388) structures in p53 protein C-terminal region based on its mRNA secondary structure template and Chou-Fasman's protein secondary structure principle of prediction. The result was conformed by the other four methods of protein secondary structure prediction that are based on the multiple sequence alignment (accuracy = 73.20%). Combine with the 31 amino acids crystal structure of the oligomerization, the three dimensional conformation of p53 C-terminal 108 residues was built using the SGI INDIGO(2) computer. This structure further expounds the relationship among those biological function domains of p53 C- terminus at three-dimensional level.

Extract of Ginkgo biloba leaves reverses yohimbine-induced spatial working memory deficit in rats

Extract of Ginkgo biloba is used to alleviate age-related decline in cognitive function, which may be associated with the loss of catecholamines in the prefrontal cortex. The purpose of this study was to verify whether alpha-2 adrenergic activity is involved in the facilitative effects of extract of Ginkgo biloba on prefrontal cognitive function. Male Wistar rats were trained to reach criterion in the delayed alternation task (0, 25, and 50-s delay intervals). A pilot study found that 3 or 4 mg/kg of yohimbine (intraperitoneal) reduced the choice accuracy of the delayed alternation task in a dose and delay-dependent manner, without influencing motor ability or perseverative behaviour. Acute oral pre-treatment with doses of 50, 100, or 200 mg/kg (but not 25 mg/kg) of extract of Ginkgo biloba prevented the reduction in choice accuracy induced by 4 mg/kg yohimbine. These data suggest that the prefrontal cognition-enhancing effects of extract of Ginkgo biloba are related to its actions on alpha-2-adrenoceptors.

鲫鱼蛋白激酶PKR-like的Zα结构域与聚肌胞苷酸的结合

根据鲫鱼类PKR蛋白激酶基因(CaPKR-like)的全长cDNA序列,克隆CaPKR-likeZα结构域cDNA(Zα1、Zα2和Zα1Zα2),原核表达成功获得3种融合蛋白PZα1、PZα2和PZα1Zα2。凝胶阻滞实验结果显示:PZα1、PZα2、PZα1和PZα2混合表达蛋白不能与聚肌胞苷酸(PolyI∶C)结合,而表达完整的Zα结构域的PZα1Zα2与PolyI∶C有明显的结合现象。另外,3种表达多肽PZα1、PZα2和PZα1Zα2在体外分别都能聚合形成二聚体。与PZα1相比,PZα2和PZα

雌核发育银鲫和两性融合彩鲫卵母细胞体外诱导成熟过程中周期蛋白合成和MPF活性变化的比较研究

采用体外诱导卵母细胞成熟技术 ,比较研究了雌核发育银鲫和两性融合发育彩鲫卵母细胞成熟过程中周期蛋白合成和MPF活性变化情况。结果表明 :1 7α ,2 0 β 二氢孕酮(简称DP)浓度为 0 5μg/ml,温度为 2 4℃ ,光照时间 2 0小时为最适诱导条件。在相同条件下 ,两性生殖彩鲫卵母细胞体外诱导成熟速度明显快于银鲫。相应的细胞学研究表明 ,在体外诱导时 ,银鲫和彩鲫卵母细胞的发育成熟是正常的。银鲫卵母细胞生发泡破裂 (GVBD)时已有周期蛋白的合成 ,但生发泡破裂开始后则呈现合成速度下降直至没有

唾液酸转移酶Ⅰ（ST6GalⅠ）及甲胎蛋白（AFP）上肝癌特异糖链在肝细胞肝癌细胞系和肝病患者血液中的表达

肝癌（HCC）是一种重要的恶性肿瘤，具有高发病率和不良预后的特点，在中国有很多的肝癌患者。早期发现和精确区分肝癌与其他肝病，对于肝癌的临床诊断治疗有十分重要的作用。由于肝脏所处位置较深，以及检测仪器和手段的限制，肝癌的早期诊断相对困难，所以比较好的血清学标记物应用于早期区分肝癌和其他肝病显得尤为重要。目前应用较多的标记物是甲胎蛋白AFP。由于单独的AFP其敏感性和特异性并不高，更为理想的检测手段正在研究中。 Beta-galactoside alpha-2,6-sialyltransferase Ⅰ(ST6GalⅠ)是唾液酸转移酶家族中的一员，主要调控蛋白的唾液酸化。有研究发现ST6Gal在肝癌中表达升高，而且随着肝实体瘤的恶化而可能被释放到外周血中。所以血液中的ST6GalⅠ水平可能是一种有潜力的肝癌指标。另外，有研究显示，AFP的糖链结构在肝癌和其他肝病中是不同的，如果能用AFP上肝癌特异糖链作为检测目标，联合已有的检测体系，可能有助于早期精确诊断肝癌。 目的：我们选择ST6Gal Ⅰ和AFP上肝癌特异糖链TF（Thomsen-Friedenreich）抗原，H2血型抗原，Lewis Y血型抗原作为主要的研究内容，通过对其在肝癌细胞系和肝病血液中的表达研究，探讨其应用于肝癌早期诊断的可能。 方法：1）用合成的ST6Gal Ⅰ多肽免疫兔子，制备抗ST6Gal Ⅰ多克隆抗体；2）通过免疫细胞化学，ELISA和western blot等方法对制作的兔多克隆抗体进行效价分析；3）通过western blot方法对肝癌、肝硬化和正常人的血浆和血清中ST6Gal表达进行研究；4）通过免疫沉淀的方法对肝癌细胞系和肝癌血浆中AFP上肝癌特异糖链进行研究。 结果：1）制备的兔抗人ST多克隆抗血清，效价可达1:400000，能够通过 免疫细胞化学和western blot检测到肝癌细胞系中的ST6GalⅠ；2）所选的3例肝癌血浆和2例肝癌血清中都有ST6GalⅠ阳性，在所选的2例肝硬化血浆和2例肝硬化血清中，分别只有1例有ST6GalⅠ的阳性，在3例正常人血浆中有2例有ST6GalⅠ阳性；3）AFP上肝癌特异的糖链TF抗原、H2抗原、Lewis Y抗原可以通过免疫沉淀的方法在肝癌病人血浆和肝癌细胞系HepG2中检出。 结论：制备的兔抗人ST6GalⅠ多克隆抗体具有较高效价，并可以检测到自然状态和变性状态的目标蛋白，可以应用于ST6GalⅠ的研究；通过western blotting对肝病血液的检测，发现在肝癌、肝硬化和正常人血液中的ST6GalⅠ的表达有差异，为ST6GalⅠ作为一种肿瘤标记物进行血清学检测提供了可能的前提；免疫沉淀的结果，证实了在肝癌病人的AFP上有TF、H2、LewisY等糖链的存在，为AFP上肝癌特异糖链作一种区分肝癌和其他良性肝病的标记物提供了证据。

唾液酸转移酶Ⅰ（ST6GalⅠ）及甲胎蛋白（AFP）上肝癌特异糖链在肝细胞肝癌细胞系和肝病患者血液中的表达

肝癌（HCC）是一种重要的恶性肿瘤，具有高发病率和不良预后的特点，在中国有很多的肝癌患者。早期发现和精确区分肝癌与其他肝病，对于肝癌的临床诊断治疗有十分重要的作用。由于肝脏所处位置较深，以及检测仪器和手段的限制，肝癌的早期诊断相对困难，所以比较好的血清学标记物应用于早期区分肝癌和其他肝病显得尤为重要。目前应用较多的标记物是甲胎蛋白AFP。由于单独的AFP其敏感性和特异性并不高，更为理想的检测手段正在研究中。 Beta-galactoside alpha-2,6-sialyltransferase Ⅰ(ST6GalⅠ)是唾液酸转移酶家族中的一员，主要调控蛋白的唾液酸化。有研究发现ST6Gal在肝癌中表达升高，而且随着肝实体瘤的恶化而可能被释放到外周血中。所以血液中的ST6GalⅠ水平可能是一种有潜力的肝癌指标。另外，有研究显示，AFP的糖链结构在肝癌和其他肝病中是不同的，如果能用AFP上肝癌特异糖链作为检测目标，联合已有的检测体系，可能有助于早期精确诊断肝癌。 目的：我们选择ST6Gal Ⅰ和AFP上肝癌特异糖链TF（Thomsen-Friedenreich）抗原，H2血型抗原，Lewis Y血型抗原作为主要的研究内容，通过对其在肝癌细胞系和肝病血液中的表达研究，探讨其应用于肝癌早期诊断的可能。 方法：1）用合成的ST6Gal Ⅰ多肽免疫兔子，制备抗ST6Gal Ⅰ多克隆抗体；2）通过免疫细胞化学，ELISA和western blot等方法对制作的兔多克隆抗体进行效价分析；3）通过western blot方法对肝癌、肝硬化和正常人的血浆和血清中ST6Gal表达进行研究；4）通过免疫沉淀的方法对肝癌细胞系和肝癌血浆中AFP上肝癌特异糖链进行研究。 结果：1）制备的兔抗人ST多克隆抗血清，效价可达1:400000，能够通过免疫细胞化学和western blot检测到肝癌细胞系中的ST6GalⅠ；2）所选的3例肝癌血浆和2例肝癌血清中都有ST6GalⅠ阳性，在所选的2例肝硬化血浆和2例肝硬化血清中，分别只有1例有ST6GalⅠ的阳性，在3例正常人血浆中有2例有ST6GalⅠ阳性；3）AFP上肝癌特异的糖链TF抗原、H2抗原、Lewis Y抗原可以通过免疫沉淀的方法在肝癌病人血浆和肝癌细胞系HepG2中检出。 结论：制备的兔抗人ST6GalⅠ多克隆抗体具有较高效价，并可以检测到自然状态和变性状态的目标蛋白，可以应用于ST6GalⅠ的研究；通过western blotting对肝病血液的检测，发现在肝癌、肝硬化和正常人血液中的ST6GalⅠ的表达有差异，为ST6GalⅠ作为一种肿瘤标记物进行血清学检测提供了可能的前提；免疫沉淀的结果，证实了在肝癌病人的AFP上有TF、H2、LewisY等糖链的存在，为AFP上肝癌特异糖链作一种区分肝癌和其他良性肝病的标记物提供了证据。

In situ UV-vis and CD thin-layer spectroelectrochemistry study of the electrochemical behavior of L-noradrenaline in alkaline solution

Electrochemical redox behavior of noradrenaline in alkaline solution on a glassy carbon electrode has been investigated by in situ UV-vis and CD spectroelectrochemistry by using a long optical path thin-layer cell. The experimental data were processed by using a double logarithmic method of analysis together with nonlinear regression which confirmed that the first step in both the oxidation of noradrenaline and reduction of noradrenochrome is a two-electron irreversible process governed by an EE mechanism. The kinetic parameters of the electrode reactions, i.e., charge transfer coefficient and the number of electrons transferred, alpha(1)n(1) = 0.11 and alpha(2)n(2) = 0.23, formal potentials modified with kinetics, E-1(0') = 0.65 (+/- 0.01) V and E-2(0') = 0.72V and standard rate cnstants, k(1)(0) = 7.0(+/-0.5)x10(-5) cm s(-1), for the first and second steps in the oxidation process of noradrenaline, and similarly, alpha(1)n(1) = 0.33, alpha(2)n(2) = 0.58, E-1(0') = 0.37(+/-0.01) V, E-0' = -0.25 (+/-0.01) V and k(1)(0) approximate to k(2)(0) = 1.06 (+/-0.05)x10(-4) cm s(-1) for the first and second steps in the reduction process of noradrenochrome were also determined.

SYNTHESIS AND STRUCTURAL CHARACTERIZATION OF THE DAWSON-TYPE TUNGSTOPHOSPHATE HETEROPOLY COMPLEXES SUBSTITUTED BY NIOBIUM

Eight new complexes alpha(2)-M(7-m)H(m)[P2W17NbO62]. H2O and alpha-1, 2, 3-M(g-m)H(m) [P2W15Nb3O62]. XH(2)O(M=K, TMA, TEA, TBA) were synthesized and characterized by IR and UV spectroscopy, polarography, XPS and XRD methods. P-31 and W-183 NMR studies show that the niobium atoms in the anions are on the polar sites. The crystal of alpha-1, 2, 3-K7H2 [P2W15Nb3O62]. 30H(2)O is hexagonal, its cell parameters: a=1.9836(4), b=1.9836(9), c=1.5498(6)nm, alpha=beta=90 degrees, gamma=120 degrees.

G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.

An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.

Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.

Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.