华广虻(Tabanusamaenus Walker)溶纤活性蛋白的纯化及生物活性分析

经过 ７５％ 饱和度硫酸铵沉淀、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 凝胶过滤层析、 Ｌｙｓ Ｓｅｐｈａｒｏｓｅ ４ Ｂ 亲和层析和电泳制备洗脱,从华广虻（ Ｔａｂａｎｕｓ ａｍ ａｅｎｕｓ Ｗ ａｌｋｅｒ）腹部组织匀浆液中分离纯化出分子量约为 ６７ｋ Ｄ 的溶纤活性蛋白 Ｔ Ａ Ｆ Ｐ经纤维蛋白平板测定表明, Ｔ Ａ Ｆ Ｐ 只具有纤溶酶作用,不具有激活纤溶酶原的作用;但 Ｔ Ａ Ｆ Ｐ 能分解纤溶酶原激活剂的生色底物—— Ｃｈｒｏｍ ｏｚｙｍ Ｕ Ｋ 及 Ｓ ２２８８还能水解胰蛋白酶专一底物 Ｂｚ Ｐｈｅ Ｖａｌ Ａｒｇ Ｎ Ａ 及 Ｃ Ｂ Ｚ Ｇｌｙ Ｐｒｏ Ａｒｇ Ｎ Ａ,表明 Ｔ Ａ Ｆ Ｐ具有类胰蛋白酶活性,专一水解精氨酸形成的酰胺键（或肽键） Ｔ Ａ Ｆ Ｐ无胰凝乳蛋白酶活性      

棕尾别麻蝇幼虫肠道溶纤活性蛋白酶的分离纯化和性质

从以富含纤维蛋白的血凝块为食物的棕尾别麻蝇幼虫肠道浸提液中分离纯化出3种具有溶纤活性的蛋白酶,分别命名为BPGFP1,BPGFP2和BPGFP3。其中,BPGFP1由两个分子量分别为32000和30000的亚基组成。BPGFP2和BPGFP3均为单体,分子量分别为40000和28000。这三种蛋白酶具有相似的底物特异性和抑制剂特性。三种蛋白酶均能降解溶纤活性蛋白酶的特异底物纤维蛋白,Chromzym,P,Chromzym UK和S－2288。三种酶还能够强烈降解类胰蛋白酶专一底物Bz-Phe-Val Arg NA,cBz Gly-Pro-Arg NA,Bz-Pro－Phe-Arg NA和Bz-Val-Gly-Arg NA.PMSF,STI,LBTI和SBBI能够对三种蛋白酶活怀有极强的抑制作用。三种溶纤活性蛋白酶均在pH9.0-10.0范围内表现出较高活性。     

华广虻溶纤活性蛋白(TAFP)的性质和对大鼠血液流变学的影响

华广虻溶纤活性蛋白 (TAFP)经血纤蛋白平板法和试管凝块法测定表明 ,TAFP只具有纤溶酶作用 ,不具有激活纤溶酶原的作用 .TAFP的最适 p H为 7.5,且在 p H为 6.0时最稳定 .蛋白水解酶抑制剂对 TAFP的抑制作用显示 :STI>antipain>SBBI>antitrypsin>TLCK>leupeptin>bacteracin>PMSF>TPCK,金属蛋白酶抑制剂 1 ,1 0 - phenanthroline对 TAFP没有抑制作用 .TAFP能显著的延长大鼠出血时间、抑制血小板聚集性 ;显著降低血浆中血纤蛋白原含量、全血粘度、血浆粘度、红细胞压积 ;减慢血沉速度     

SDS-PAGE纤维蛋白自显影法检测不同分子量的纤溶酶原激活剂

体内存在两种纤溶酶原激活剂(plasminogen activator, PA):血液中生理性的组织型纤溶酶原激活剂(t PA)及尿中的尿激酶型纤溶酶原激活剂(u PA)。它们通过将纤溶酶原转变成有活性的纤溶酶而启动纤溶过程使血栓溶解。目前模拟体内纤溶过程以纤维蛋白为底物的PA物质活性测定方法有纤维蛋白溶圈法和发光分析法,但这两种方法不能     

三七皂苷Rg1对tPA和PAI-1活性的调节作用

探讨三七皂苷Rg1对组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制物(PAI-1)活性的调节作用。运用发色底物方法测定三七皂苷Rg1在体外和静脉注射对家兔血浆纤溶酶原激活物(tPA)和血浆或血小板释放的纤溶酶原激活物抑制物(PAI-1)水平的影响。结果表明,三七皂苷Rg1在体外呈浓度依赖性明显抑制血浆PAI-1活性,同时提高血浆tPA活性;30和60 mg/kg的三七皂苷Rg1静脉注射显著抑制血浆PAI-1活性,提高血浆tPA活性,同时降低凝血酶激活的血小板所释放的PAI-1水平。本实验提示三七皂苷Rg1能抑制PAI-1活性,同时升高tPA活性可能是其抗血栓作用的分子机制之一。     

杭州虻纤溶酶的纯化及其生物活性分析

经过７５％硫酸铵沉淀、ＳｅｐｈａｄｅｘＧ－７５凝胶过滤层析、ＤＥＡＥ Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析、Ｂ ｅｎｚａｍｉｄｅ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,从杭州虻（Ｔａｂａｎｕｓ ｈｏｎｇｃｈｏｗｅｎｓｉｓ）腹部匀浆液中分离纯化出分子量约为３６．５ｋＤ的纤溶酶ＴＨＦＥ。经纤维蛋白平板测定表明,ＴＨＦＥ既具有纤溶酶作用,又具有激活纤溶酶原的作用。ＴＨＦＥ能分解纤溶酶原激活剂的生色底既具有纤     

大肠杆菌K1致病株外膜蛋白T体外功能

纤溶酶原激活及水解抗菌肽利于致病菌侵袭及体内存活。【目的】构建大肠杆菌K1致病株E44的ompT基因缺失突变株,证实E44外膜蛋白T(OmpT)体外激活纤溶酶原及水解抗菌肽鱼精蛋白的活性。【方法】采用基因同源重组技术敲除大肠杆菌K1株E44中的ompT基因,构建ompT缺失突变株;二步柱层析纯化E44外膜组分,S-2251发色底物法测定其纤溶酶原激活活性;考察野生株E44、ompT基因敲除株E44ompT及转化带有ompT完整阅读框的质粒pUCT的E44ompT/pUCT三者对0.1mg/mL阳离子抗菌肽鱼精蛋白的敏感程度。【结果】利用自杀性载体pCVD442和同源重组的原理构建E44的ompT基因敲除株E44ompT;纯化得到约37kDa的E44外膜组分,S-2251发色底物法证实其具有纤溶酶原激活活性,纤溶酶原激活与膜组分的加入量呈一定量效关系;与野生株E44相比,ompT敲除株E44ompT对0.1mg/mL鱼精蛋白敏感,转化入带有ompT完整序列的质粒pUCT有一定的回补作用,E44ompT部分恢复抗鱼精蛋白能力。【结论】外膜蛋白T在致病株E44中有表达,并具有激活纤溶酶原及水解鱼精蛋白的活性。     

蚯蚓纤溶酶原激活剂的分离纯化及其性质

为了从赤子爱胜蚓中获得主要表现为纤溶酶原激活活性的蛋白酶,采用盐析、离子交换层析、凝胶过滤层析和疏水吸附层析从蚯蚓组织匀浆中纯化出6种具有纤溶活性的酶组分(F1、F2、F3、F4、F5和F6).它们均为单一多肽链;表观分子量分别为28500、29500、26100、26300、14850和32800;经非还原型SDSPAGE和扫描仪扫描,纯度分别为100%、95.2%、96.5%、93.6%、98%和97.8%;等电点(pI)均不高于3;SDSPAGE后,用Schiff试剂染色显示F5为糖蛋白;纯化的6种酶于20℃～50℃保温1h,酶活力基本不变;F1和F2、F3和F4、F5和F6分别在pH4～10、4～11、7～12范围内稳定;水解BAEE试验及纤溶活性抑制试验表明,F6既是丝氨酸蛋白酶,又是含金属离子的蛋白酶,其它5种酶为胰蛋白酶样的丝氨酸蛋白酶;免疫双扩散试验结果初步表明,F1和F5以及它们和其它4种组分之间无共同的抗原决定簇;用纤维蛋白平板法及以ChromozymU和ChromozymPL为底物测定,除F1外,其余5种酶的纤溶酶原激活活性明显强于其直接纤溶活性.     

链霉菌产生的新型纤溶酶的纯化和性质的研究

从一株土壤链霉菌(Streptomyces sp.)Y405的发酵液中通过硫酸铵分级盐析、290树脂脱色、Sephadex G-75凝胶过滤、DEAESephadex A-25阴离子交换层析和LysineSepharose4B亲和层析,获得一种新型的具有纤溶活性的蛋白酶SW-1。每升发酵液中可获得4.2mg SW1样品,回收率为12.0%,每毫克蛋白活力达2952.3尿激酶单位,以发酵液作为起始,所获得样品纯度提高230.6倍,HPLC检测纯度约为83.5%。SW-1在SDS-聚丙烯酰胺凝胶电泳中是单肽链蛋白,分子量为30kD,等电点为8.5,测定其N-端17个氨基酸序列为Arg/Asn/Phe-Pro/Asp-Gly-Met-Thr-Met-Thr-Ala-Ile-Ala-Asn-Gln-Asn-Thr-Gln-Ile-Asn,N端第一和第二残基具有不均一性,根据氨基酸组成分析推算SW1由262个氨基酸组成。SW-1的纤溶活性可被10mmol/L PMSF、1mmol/L EDTA和1mol/L赖氨酸完全抑制,表明SW1其赖氨酸结合位点与活性有关。SW-1的纤溶活性在4～37℃和pH4.0～9.0具有较好的稳定性,最适pH为8.0。在纤维蛋白加热平板上,SW-1和SW-1与纤溶酶原的混合液显示相同的纤溶活性,表明SW-1对纤维蛋白具有直接的降解作用,而不具有激活纤溶酶原的活性,因而SW-1是一种纤溶酶而不是纤溶酶原激活剂。     

重组纤溶酶原激活剂抑制剂－2基因在HT1080亚克隆中的表达

重组纤溶酶原激活剂抑制剂－２基因在ＨＴ１０８０亚克隆中的表达曹祥荣（南京师范大学生物学系,２１００２４）关键词纤溶酶原激活剂,基因重组,纤溶酶原激活剂抑制剂活性表达目前认为纤溶酶系统是肿瘤细胞浸润和转移蛋白水解过程中重要的酶,纤溶酶能直接水解细胞外间...     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细