尖吻蝮蛇毒中一种具有抗补体活性金属蛋白酶的分离纯化及部分性质鉴定

通过离子交换和凝胶过滤层析从湖南产尖吻蝮蛇毒中分离纯化出1个抗补体活性蛋白AACI-I.还原与非还原SDS-PAGE测得其表观分子质量分别为26 kD和22 kD,等电聚焦电泳显示其等电点为pH 8.9.它能抑制补体经典途径和替代途径的溶血,且该作用具有量效和时效性.AACI-I可依次水解纤维蛋白原的Aα、Bβ、γ链,此活性能被EDTA、EGTA和1,10-phenanthroline完全抑制,不受PMSF、SBTI作用影响.同时,该蛋白无精氨酸酯酶活性,提示AACI-I是金属蛋白酶.另外,AACI-I具有azocasein水解活性和水肿活性,当0.01μg/g AACI-I注射小鼠足趾,其诱导肿胀率约为(14.06±6.78)%,而小鼠皮下注射2.6μg/g AACI-Ⅰ后没有发现其有出血毒活性.     

烙铁头蛇毒素纤维蛋白原溶酶研究:Ⅱ.对纤维蛋白原及凝血酶的作用

烙铁头（T.mucrosquamatus）蛇毒纤维蛋白原溶酶TMVFg能水解三肽底物Bz-Phe-Val-Arg-PNA，但对凝血酶的良好底物Cbz-Gly-Pro-Arg-PNA却活性甚低。TMVFg显著延长血浆凝血酶时间、血浆复钙时间及纤维蛋白原溶液凝血酶时间。同时，TMVFg体外也能延长全血凝固时间，表明具有抗凝作用。纤维蛋白原-纤维蛋白转换实验表明：TMVFg水解纤维蛋白原产生的纤维蛋白原断片（FDP）除具有抗凝血酶，抑制纤维蛋白聚合活性外，还能促进纤维蛋白的聚合。进一步用FPLC分离TMVFg水解人纤维蛋白原混合液，得两个FDP断片功能峰，FDP组分Ⅰ和FDP组分Ⅱ。其中FDP组分Ⅰ能抑制纤维蛋白凝块形成；FDP组分Ⅱ能促进纤维蛋白凝块形成，抑制TMVA（烙铁头蛇毒血小板活化素，它可不通过ADP、花生四烯酸途径而诱导血小板聚集），但对ADP诱导的家兔血小板聚集无影响。TMVFg对凝血酶水解三肽底物Cbz-Gly-Pro-Arg-PNA及凝固纤维蛋白原的活性也有一定抑制作用。实验证明，TMVFg抗凝的主要作用机理是其水解纤维蛋白原产生的断片对纤维蛋白原凝固的抑制作用、FDP断片抗凝血酶作用及TMVFg本身对凝血酶活性的抑制所引起的，但在二者之间，前者是主要的。从研究结果发现：TMVFg水解纤维蛋白原所产生的断片有一类能加速凝血酶凝固纤维蛋白原的过程，这就发现了FDP断片的新功能。它证明了FDP断片作为血液凝固、纤溶正反馈调节因子的功能。这一类FDP断片还能抑制TMVA诱导的血小板聚集，因此，烙铁头蛇毒纤维蛋白原溶酶TMVFg将成为研究血液凝固调节系统及血小板聚集第三条途径的强有力试剂。     

菜花烙铁头蛇毒中一种具有激肽释放酶与纤维蛋白原溶酶活性的Jerdonase

通过DEAESephadexA 5 0阴离子交换、超细SephadexG 10 0分子筛和反相高效液相C4 色谱层析 ,从菜花烙铁头蛇毒冻干粉中纯化出一种具有激肽释放酶活性和α纤维蛋白原溶酶活性的丝氨酸蛋白酶 ,命名为Jerdonase。在 12 .5 %胶浓度的SDS还原电泳条件下 ,该酶分子量大约为 5 5kD ,在非还原电泳条件下 ,分子量大约为 5 3kD。此酶是一种糖蛋白 ,含有约 35 .8%的中性糖。它的N末端氨基酸序列为IIGGDEENINEHPFLVALYDA ,其序列和蛇毒中其他丝氨酸蛋白酶具有非常高的序列相似性。Jerdonase能够催化BAEE、S 2 2 38和S 2 30 2的水解 ,其水解活性可被PMSF抑制 ,但是EDTA对此没有影响。Jerdonase能优先水解人纤维蛋白原的Aα链 ,同时伴随有微弱的Bβ链水解活性。另外 ,此酶能够水解牛低分子量的激肽原 ,释放舒缓激肽。总之 ,所有的结果表明Jerdonase是一个具有多功能活性的蛇毒丝氨酸蛋白酶     

烙铁头蛇毒纤维蛋白原溶酶研究——Ⅱ.对纤维蛋白原及凝血酶的作用

烙铁头(T.mucrosquamatus)蛇毒纤维蛋白原溶酶TMVFg能水解三肽底物Bz-Phe-Val-Arg-PNA,但对凝血酶的良好底物Cbz-Gly-Pro-Arg-PNA却活性甚低。TMVFS显著延长血浆凝血酶时间。血浆复钙时间及纤维蛋白原溶液凝血酶时间。同时,TMVFg体外也能延长全血凝固时间,表明具有抗凝作用。纤维蛋白原-纤维蛋白转换实验表明:TMVFg水解纤维蛋白原产生的纤维蛋白原断片(FDP)除具有抗凝血酶,抑制纤维蛋白聚合活性外,还能促进纤维蛋白的聚合。 进一步用FPLC分离TMVFg水解人纤维蛋白原混合液,得两个FDP断片功能峰,FDP组分Ⅰ和FDP组分Ⅱ。其中FDP组分Ⅰ能抑制纤维蛋白凝块形成;FDP组分Ⅱ能促进纤维蛋白凝块形成,抑制TMVA(烙铁头蛇毒血小板活化素,它可不通过ADP、花生四烯酸途径而诱导血小板聚集),但对ADP诱导的家兔血小板聚集无影响。TMVFg对凝血酶水解三肽底物Cbz-Gly-Pro-Arg-PNA及凝固纤维蛋白原的活性也有一定抑制作用。 实验证明,TMVFg抗凝的主要作用机理是其水解纤维蛋白原产生的断片对纤维蛋白原凝固的抑制作用、FDP断片抗凝血酶作用及TMVFg本身对凝血酶活性的抑制所引起的,但在二者之间,前者是主要的。 从研究结果发现:TMVFg水解纤维蛋白原所产生的断片有一类能加速凝血酶凝固纤维蛋白原的过程,这就发现了FDP断片的     

中草药徐长卿(Cynanchum paniculatum)治疗毒蛇咬伤的分子机制研究

徐长卿(Cynanchum paniculatum)作为一种传统中草药,被广泛应用于毒蛇咬伤治疗,然而其疗效的科学实验证据缺乏,治疗的分子机制也不清楚.本文采用有机溶剂萃取、柱层析及高效液相色谱法(HPLC)相结合,从徐长卿中筛选出和分离得到一个功效组分,经质谱和核磁共振鉴定为丹皮酚原苷.动物活体实验与组织病理切片观察表明,该组分显著地抑制尖吻蝮(Deinagkistrodon acutus)蛇毒引起的出血、水肿及肌肉组织坏死等活性.体外酶活检测显示,丹皮酚原苷可以抑制尖吻蝮蛇毒水解纤维蛋白原活性和PLA2活性.进一步采用分子模拟软件技术,揭示出丹皮酚原苷是通过与蛇毒中主要成分金属蛋白酶和PLA2发生相互作用,从而抑制尖吻蝮蛇毒发挥致伤作用.本研究首次揭示了中草药徐长卿治疗蛇伤的分子机制,为中医治疗毒蛇咬伤使用徐长卿找到了科学实验证据,也为蛇伤药物的研发提供了新的思路.     

肺腺癌细胞中STAT3表达对细胞生长及药物敏感性的影响

目的:探讨STAT3表达变化对细胞生长及化疗药物敏感性的影响.方法:采用AG490处理细胞、SOCS3基因转染A549细胞后.Western blot检测STAT3蛋白酪氨酸磷酸化水平变化;MTT法检测细胞增殖情况;不同浓度泰素处理细胞后观察细胞对药物的敏感性.结果:AG490处理细胞、SOCS3基因转染细胞后,Western blot证实其能显著抑制STAT3蛋白酪氨酸磷酸化水平(P<0.01);MTT法结果示细胞增殖明显受到抑制;细胞对泰素敏感性显著增高.结论:STAT3能促进细胞增殖,AG490、SOCS3能显著抑制A549细胞中STAT3蛋白的活性,从而抑制A549细胞生长并增加其对化疗药物的敏感性.     

马来酸罗格列酮对人肺腺癌（A549）细胞中PGC-1α表达及增殖的影响

摘要：为探讨马来酸罗格列酮对肺腺癌（A549）细胞中PGC-1a表达和活性的影响,以及抑制细胞增殖的机制,我们首先构建了pGL3-PGC-1a promoter重组质粒,转染人肺腺癌A549细胞,用双荧光素酶报告基因系统检测马来酸罗格列酮对PGC-1a启动子转录活性的影响;实时定量PCR检测胞内PGC-1a mRNA的表达;用Mitotracker green染色, 流式细胞仪检测线粒体质量;细胞计数检测马来酸罗格列酮对A549细胞体外增殖的影响。结果显示,马来酸罗格列酮能够抑制pGL3-PGC-1apromoter重组质粒的转录活性,降低A549细胞PGC-1amRNA水平的表达及线粒体质量,具有明显的剂量依赖性（P<0.05）。其抑制PGC-1a表达和活性的IC50（约为80 umol/L）与马来酸罗格列酮抑制A549细胞增殖的IC50（约为80 umol/L）相符。结论 马来酸罗格列酮能够抑制A549细胞中PGC-1a的表达与活性,进而降低细胞中的线粒体质量。这有可能是罗格列酮抑制A549细胞增殖的原因之一。     

菜花烙铁头蛇毒金属蛋白酶Jerdonitin的分离纯化和理化性质

以前从菜花烙铁头蛇毒中分离纯化到Jerdonitin。与其他Ⅱ型蛇毒金属蛋白酶相比,Jerdonitin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Jerdonitin。Jerdonitin在还原和非还原SDS—PAGE电泳中分别呈现一条表观分子量为38和36kDa的条带。像其他典型的蛇毒金属蛋白酶一样,Jerdonitin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。Jerdonitin不诱导小白鼠皮下出血。     

肿瘤特异的sea基因真核表达质粒的构建及在肺癌细胞中的功能研究

构建Survivin启动子调控的葡萄球菌肠毒素A(SEA)的真核表达质粒,检测其在人肺腺癌A549细胞中的特异性表达以及表达产物的超抗原活性.以PCR法扩增sea基因,以含有Survivin启动子为调控序列的pGL-S-RED质粒为基础,构建pGL-S-SEA真核表达质粒.用脂质体转染A549细胞,以RT-PCR法检测sea基因表达水平.制备健康献血者PBMC,用pGL-S-SEA质粒转染A549细胞的上清液和细胞裂解液进行PBMC刺激试验,以MTT法检测其促细胞增殖效应.结果显示,成功构建Survivin启动子调控的真核表达质粒pGL-S-SEA.重组质粒在A549细胞中启动了sea基因的表达,其转录强度为内参(GADPH基因)的65.96%,在对照MRC-5细胞中无明显表达.该质粒转化A549细胞的裂解液具有促进人PBMC增殖活性、上清液无明显促PBMC增殖作用,裂解液组、上清液组和PHA阳性对照组的刺激指数分别为1.29、0.95和1.58.结论成功构建pGL-S-SEA质粒,其在A549细胞的表达产物具有诱导人PBMC增殖的超抗原活性,为下一步将其作为基因疫苗治疗肺癌奠定了基础.     

哇巴因抑制Wnt/β-catenin信号通路和对肿瘤细胞的细胞毒作用

哇巴因是一种强心苷类药物,临床上常用于治疗充血性心力衰竭与心律失常.近期的研究发现,哇巴因具有抗肿瘤活性,但其作用机制不甚明了.Wnt/β-catenin信号通路的异常激活与多种肿瘤的发生发展密切相关.本项研究发现,哇巴因是一种新颖的Wnt/β-catenin信号通路抑制剂,其作用靶点是低密度脂蛋白受体相关蛋白6(LRP6)和β-连环蛋白(β-catenin).在人大肠癌HCT116和非小细胞肺癌A549细胞中,纳摩尔浓度的哇巴因有效地降低磷酸化LRP6、总LRP6、活化β-catenin和总β-catenin的蛋白水平.另外,哇巴因还可有效地抑制Wnt/β-catenin信号通路靶基因CD44和纤维蛋白连接素(fibronection)的表达.进一步研究发现,哇巴因对HCT116和A549细胞具有明显的杀伤活性,并能够诱导其凋亡.该研究首次阐明了哇巴因抑制Wnt/β-catenin信号通路的分子机制.     

眼镜蛇毒纤维蛋白原溶解因子的纯化及理化性质的研究

经Heparin-SepharoseCL-6B亲和层析和SephadexG-150分子筛层析,从中国眼镜蛇毒(Najanajaatra)中纯化出一种可水解纤维蛋白原Aα链的蛋白酶——组分F.SDS-PAGE测得分子量为47100,为一含2%中性己糖的单肽链糖蛋白;它对纤维蛋白、酪蛋白和苯甲酰-L-精氨酸乙酯(BAEE)均无水解作用,也无激活纤溶酶原的作用;100μg皮下或皮内注射,未见出血反应。组分F对酸和热均不稳定。EDTA,EGTA,PMSF和肝素等可抑制其纤维蛋白原溶解活性,benzami-dine,aprotinin和大豆胰蛋白酶抑制因子(SBTI)则无抑制作用。     

Telomerase inhibition is a specific early event in salvicine-treated human lung adenocarcinoma A549 cells

The telomere and telomerase have been suggested as targets for anticancer drug discovery. However, the mechanisms by which conventional anticancer drugs affect these targets are currently unclear. The novel topoisomerase II inhibitor, salvicine, suppresses telomerase activity in leukemia HL-60 cells. To further determine whether this activity of salvicine is specific to the hematological tumor and distinct from those of other conventional anticancer agents, we studied its effects on telomere and telomerase in a solid lung carcinoma cell line, A549. Differences in telomerase inhibition and telomere erosion were observed between salvcine and other anticancer agents. All anticancer agents (except adriamycin) induced shortening of the telomere, which was identified independent of replication, but only salvicine inhibited telomerase activity in A549 cells under conditions of high concentration and short-term exposure. At the low concentration and long-term exposure mode, all the tested anticancer agents shortened the telomere and inhibited telomerase activity in the same cell line. Notably, salvicine inhibited telomerase activity more severely than the other agents examined. Moreover, the compound inhibited telomerase activity in A549 cells indirectly in a concentration- and time-dependent manner. Salvicine did not affect the expression of hTERT, hTP1, and hTR mRNA in A549 cells following 4 h of exposure. Okadaic acid protected telomerase from inhibition by salvicine. These results indicate specificity of salvicine and diversity of anticancer agents in the mechanism of interference with telomerase and the telomere system. Our data should be helpful for designing the study in the development of agents acting on telomere and/or telomerase.     

Purification and properties of a fibrinogenase from the venom of Naja nigricollis

A fibrinogenolytic proteinase from the venom of Naja nigricollis was purified by chromatography on Bio-Rex 70 and Phenyl-Sepharose. The purified enzyme, designated proteinase F1, was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis, and consisted of a single chain with a molecular weight of 58 000. Purified proteinase F1 had approximately 15-fold more proteinase activity than the crude venom, based on its ability to inactive α₂-macroglobulin. The enzyme acted on only the Aα-chain of fibrinogen and left the Bβ- and γ-chains intact. The pH optimum for this fibrinogenolytic activity was in the range of pH 8 to 10. In addition to its activity on fibrinogen, proteinase F1 was active on α₂-macroglobulin and fibronectin, but did not degrade casein, hemoglobin or bovine serum albumin. The enzyme was not inhibited by inhibitors of serine proteinases, cysteine proteinases or acid proteinases, but only by the metalloproteinase inhibitor, EDTA. The inhibition by EDTA could be prevented by Zn²⁺, but not by Ca²⁺ or Mg²⁺.     

Caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis through modulating GSK3β/β-catenin pathway

In this study, we investigate the anti‐cancer activity of caudatin in carcinomic human alveolar basal epithelial cell line A549 and anti‐angiogenic activity in human umbilical vein endothelial cells (HUVECs). We show that caudatin impairs the cell viability and induces G₀/G₁ phase arrest in A549 cells with a dose dependent manner. A549 cells, not HUVECs, dealing with caudatin exhibited typical characteristics of apoptosis, which were accompanied by activation of caspase‐3, caspase‐9 and Poly(ADP–Ribose) Polymerase (PARP). In addition, caudatin treatment resulted in a decrease of β‐catenin and increase of phosphorylation of β‐catenin, and inhibited phosphorylation levels of GSK3β (Ser 9) in A549 cells. Conditional medium of A549 cells‐induced or growth factors‐induced tube formation of HUVECs was markedly inhibited by caudatin treatment, which was associated with the inhibiting VEGF secretion from A549 cells by caudatin. Our findings suggest that caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis by targeting GSK3β/β‐catenin pathway and suppressing VEGF production. J. Cell. Biochem. 113: 3403–3410, 2012. © 2012 Wiley Periodicals, Inc.     

Purification and biochemical characterization of Eumiliin from Euphorbia milii var. hislopii latex

A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatographic steps using DEAE-Sephacel and gel-filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS-PAGE under reducing conditions and gave one main peak at 29,814 KDa in MALDI-TOF/TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the Aα-chain of fibrinogen and, more slowly, the Bβ-chain. Its fibrinogenolytic activity is inhibited by β-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 °C; activity was completely lost at ?80 °C. Intraplantar injection of Eumiliin (1-25 μg/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1-5 h after enzyme injection. Intraplantar injection of Eumiliin (1-25 μg/paw) also caused an oedematogenic response that was maximal after 1 h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection.     

Purification and characterization of a fibrinogenase from the venom of Western Diamondback rattlesnake (Crotalus atrox)

A fibrinogenolytic enzyme was isolated from the venom of Western Diamondback rattlesnake (Crotalus atrox) by a three-step procedure involving gel filtration and anion-exchange chromatography. The molecular weight was estimated as 22 900 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 4.65. The enzyme rapidly destroyed the ability of bovine fibrinogen to form a clot on incubation with thrombin. Incubation of fibrinogen with the fibrinogenolytic enzyme for 5 min resulted in the disappearance of the beta-chain of fibrinogen and the appearance of lower molecular weight fragments. Thus the enzyme can be classified as a beta-fibrinogenase. However, on prolonged incubation of the fibrinogen there was also a partial digestion of the alpha-chain. The fibrinogenase showed no activity towards fibrin or casein or arginine esters. The fibrinogenolytic activity was inhibited by phenylmethanesulphonyl fluoride (PMSF) but was unaffected by EDTA.     

Dexamethasone and cyclosporin A do not inhibit interleukin-15 expression in the human lung carcinoma cell line A549

A549 cells constitutively expressed IL-15 mRNA which could be upregulated by stimulation with TNF-alpha- or IL-1beta. Constitutive and induced levels of IL-15 mRNA were not decreased in the presence of 10- 6 M dexamethasone. Control experiments revealed that 10- 6 M dexamethasone inhibited the TNF-alpha- or IL-1beta-mediated increase of IL-8 mRNA in A549 cells, which showed that the glucocorticoid was functional. A549 cells did not secrete relevant amounts of IL-15 protein. The constitutive expression and the TNF-alpha- or IL-1beta-mediated upregulation of intracellular IL-15 protein was not inhibited by dexamethasone, in contrast, the release of IL-8 protein was inhibited. Also, cyclosporin A at 250 ng/ml did not inhibit the TNF-alpha-induced upregulation of IL-15 mRNA and intracellular IL-15 protein. The data suggest that the synthesis of IL-15 mRNA and protein is not influenced by immunosuppressive glucocorticoids or by cyclosporin A.     

Curcumin inhibits interferon-alpha induced NF-kappaB and COX-2 in human A549 non-small cell lung cancer cells

The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-alpha treatment. The IFN-alpha-treated A549 cells showed increase in protein expression levels of NF-kappaB and COX-2. IFN-alpha induced NF-kappaB binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-alpha-induced COX-2 expression in A549 cells. Within 10 min, IFN-alpha rapidly induced the binding activity of a gamma-(32)P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-alpha-induced activations of NF-kappaB and COX-2 were inhibited by the addition of curcumin in A549 cells.