烙铁头蛇毒L-氨基酸氧化酶的分离、纯化及其生物学活性

通过DEAF Sephadex A-50阴离子交换柱，Sephadex G75分子筛，Resourse Q阴离子交换柱三步层析从湖南产的烙铁头蛇毒中分离、纯化得到一个L-氨基酸氧化酶( T1vf LAO)，它由两个非共价的亚基组成，每个亚基的分子量为55 kD。与台湾产的烙铁头蛇毒L-氨基酸氧化酶分子量(70 kD)不同。T1v+ LAO的N末端氨基酸序列是ADNKNPLEECFRETNYEEFLEIAR，与报 道的蜂科的L-氨基酸氧化酶的相似性比眼镜蛇科的要高。T1vf LAO能抑制大肠杆菌、金黄色葡萄球菌和痢疾杆菌的生长，杀死肿瘤细胞以及诱导血小板聚集。这些活性能被过氧化氢酶所抑制，说明T1vf LAO生理学功能主要是通过酶反应产生的过氧化氢( H202)介导的。

Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom

A novel disintegrin, jerdonin, was purified from the Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. Its coding cDNA was also isolated from the venom gland. The jerdonin coding cDNA is part of

A nerve growth factor from the venom of Chinese cobra (Naja naja atra) and its effects on male reproductive system in rats

A nerve growth factor (NGF) was isolated from the venom of Chinese cobra (Naja naja ntr a) by ion exchange chromatography, gel filtration and fast protein liquid chromatography (FPLC). The N-terminal sequence of 22 amino acid residues was identical with other NGFs previously purified from the venom of the same genus. The NGF monomer molecular weight was estimated to be 13 500 by reducing SDS-PAGE and the isoelectric point was determined to be 7.2 by isoelectric focusing electrophoresis. NGF improved the epididymal sperm motility of male rats and increased the pregnancy rate and fetus number of mated female rats. The serum levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) of male rats administrated NGF + gossypol was lower than that of male rats administrated gossypol. Histological sections of testes and epididymides showed that NGF reduced the destructive effects of gossypol on rat testes. (C) 1999 Elsevier Science Inc. All rights reserved.

A highly active anticomplement factor from the venom of Naja kaouthia

A highly active anticomplement factor (cobra venom factor) from the venom of Naja kaouthia in South Yunnan, China was isolated by sequential column chromatography (SP-Sephadex-C25, Q Sepharose HP and Sephadex G-150). It displays strong anticomplement acti

Purification and characterization of jerdofibrase, a serine protease from the venom of Trimeresurus jerdonii snake

A fibrin(ogen)olytic serine protease from Trimeresurus jerdonii venom was identified and purified to SDS-polyacrylamide gel electrophoresis homogeneity. It is a single chain polypeptide with a molecular weight of 32 kDa under reduced condition and 28 kDa

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5' nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes. (C) 2003 Elsevier Ltd. All rights reserved.

A new protein structure of P-II class snake venom metalloproteinases: it comprises metalloproteinase and disintegrin domains

A new metalloproteinase-disintegrin, named Jerdonitin, was purified from Trimeresurus jerdonii venom with a molecular weight of 36 kDa on SDS-PAGE. It dose-dependently inhibited ADP-induced human platelet aggregation with IC50 of 120 nM. cDNA cloning and sequencing revealed that Jerdonitin belonged to the class II of snake venom metalloproteinases (SVMPs) (P-II class). Different from other P-II class SVMPs, metalloproteinase and disintegrin domains of its natural protein were not separated, confirmed by internal peptide sequencing. Compared to other P-II class SVMPs, Jerdonitin has two additional cysteines (Cys219 and Cys238) located in the spacer domain and disintegrin domain, respectively. They probably form a disulfide bond and therefore the metalloproteinase and disintegrin domains cannot be separated by posttranslationally processing. In summary, comparison of the amino acid sequences of Jerdonitin with those of other P-II class SVMPs by sequence alignment and phylogenetic analysis, in conjunction with natural protein structure data, suggested that it was a new type of P-II class SVMPs. (C) 2003 Elsevier Inc. All rights reserved.

Jerdonase, a novel serine protease with kinin-releasing and fibrinogenolytic activity from Trimeresurus jerdonii venom

A novel kinin-releasing and fibrin (ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenymethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the Aalpha-chain of human fibrinogen with lower activity towards Bbeta-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.

Purification, characterization and biological activity of an L-amino acid oxidase from Trimeresurus mucrosquamatus venom

An L-amino acid oxidase (TM-LAO) from the venom of Hunan Trimeresurus mucrosquamatus was purified to homogenicity by three steps including DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration and Resourse Q ion-exchange chromatography. TM-LAO is composed of two identical subunits with a molecular weight of 55 kD by SDS-polyacrylamide gel electrophoresis. The molecular weight was different with that of LAO purified from the same species distributed in Taiwan that was 70 kD. The 24 N-terminal ammo acid sequence of TM-LAO is ADNKNPLEECFRETNYEEFLEIAR, which shares high similarity with other Viperid snake venom LAOs and has moderate similarity with Elapid snake venom LAOs. Further studies found that TM-LAO inhibited the growth of E. colt, S. aurues and B. dysenteriae. TM-LAO also showed cytotoxicity and platelet aggregation activity. All the biological activities were eliminated by catalase, a H2O2 scavenger. It shows that these biological effects are possibly due to the formation of H2O2 produced by TM-LAO.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity = 89%) to TSV-PA, a specific plasminogen activator from venom of T stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys(239) in TSV-PA to Gln(239) in jerdonobin-II might play an important role on their plasminogen activating activity difference. (C) 2005 Elsevier Ltd. All rights reserved.

Purif ication and Characterization of Jerdonitin ,a Non2hemorrhagic Metalloproteinase fromTrimeresurus jerdonii Venom

以前从菜花烙铁头蛇毒中分离纯化到J erdonitin 。与其他Ⅱ型蛇毒金属蛋白酶相比, J erdonitin 由金属蛋白酶和去整合素两个结构域组成。但没有检测到其出血和纤维蛋白原降解活性, 推测可能高压液相色谱的有机溶液影响了其酶活性。采用不含高压液相色谱柱层析的新分离手段分离得到J erdonitin 。J erdonitin 在还原和非还原SDS2PAGE 电泳中分别呈现一条表观分子量为38 和36 kDa 的条带。像其他典型的蛇毒金属蛋白酶一样, J erdonitin 优先降解人纤维蛋白原的alpha 链, 并且该活性能被EDTA 完全抑制, 而PMSF 对其没有影响。J er2 donitin 不诱导小白鼠皮下出血。

Sox2基因3'非翻译区保守元件对基因表达的调控作用

转录因子Sox2是脊椎动物早期发育中最早表达的神经系统特异性基因之一,同时在干细胞的维持中也起着关键作用.通过生物信息学分析,作者发现在脊椎动物Sox2 mRNA 3'非翻译区中存在4段非常保守的富含AU的区域.将这些片段按照不同的组合克隆到GFP和荧光素酶两种报告基因载体中,在非洲爪蟾胚胎和培养细胞中检测了这些片段对报告基因表达的影响.结果显示,Sox2的3'UTR可影响报告基因的表达水平,特别是其中的保守片段2可显著提高报告基因的表达水平,表明Sox2 3'非翻译区有可能参与Sox2表达的转录后调控.

Physical Blocking Neural Tube Closure Affects Radial Intercalation and Neural Crest Midline-directed Migration in Xenopus Dorsal Explants

神经管闭合缺陷(NTDs)是一种严重的先天畸形疾病,在新生儿中有千分之一的发病率.神经管融合前后,多种组织参与形态发生运动.神经管一经融合,神经嵴细胞就会向背侧中线方向产生单极突出并向此方向迁移形成神经管的顶部.与此同时,神经管从腹侧开始发生辐射状切入以实现单层化.在此,我们在非洲爪蟾的移植体中机械阻断神经管的闭合以检测其细胞运动及随后的图式形成.结果显示神经管闭合缺陷的移植体不能形成单层化的神经管,并且神经嵴细胞滞留在侧面区域不能向背侧中线迁移,而对神经前体标记基因的检测显示神经管的背腹图式形成并未受到影响.以上结果表明神经管的融合对于辐射状切入和神经嵴细胞向背侧中线方向的迁移过程是必需的,而对于神经管的沿背腹轴方向的图式形成是非必需的.

Developmental Expression of an Amphioxus (Branchiostoma belcheri) Gene Encoding a GATA Transcription Factor

GATA基因在脊椎动物和非脊椎动物的发育中行使重要的功能,该家族的成员在进化上也足非常保守的.脊椎动物的GATA基因分为两个亚群:GATA1/2/3和GATA4/5/6.通过生物信息分析,在文吕鱼的基因缓中找到了3个GATA基因:一个GATA1/2/3业家族基因,两个GATA4/5/6亚家族基因:还找到一个类GATA基因.还克隆了白氏文昌鱼(Branchiostoma belcheri)GATA123的一段序列,并研究了它在早期胚胎发育中的表达图式.结果表明GATA123在原肠胚的中内胚层表达,而在神经胚晚期和幼体早期,GATA123在脑泡和消化道中部区域表达.这种表达模式与头部发育的重要基因Otx相类似.结果提示在文吕鱼脑泡的发育过程中GATA123和Otx很可能共同发挥着重要的作用.

Expansion of the Actin Gene Family in Amphioxus

肌动蛋白是一种分布广泛而且在进化上十分保守的蛋白,是构成细胞骨架的关键组分.肌动蛋白通常被分成肌肉型和胞质型两种类型,各自行使着不同的功能.在此,作者对弗罗里达文昌鱼基因组中的肌动蛋白基因家族进行了系统分析,发现文昌鱼中该基因家族成员多达30多个,其中很多都是连锁分布的.基因结构趋于多样,分别包含2～7个外显子.进化分析的结果显示,文昌鱼的肌动蛋白基因家族可能通过串联重复而发生了扩增.作者还克隆了厦门文昌鱼两个不同的肌肉犁肌动蛋白基因,并比较了它们在文昌鱼早期胚胎中的表达图式.结果显示,这两个基因在表达上有着细微的差别,提示文昌鱼肌动蛋白基因家族成员在功能上的分化.上述结果将有助于阐明肌动蛋白基因家族及其功能在脊索动物中的演化.