重组人尿激酶原氨基末端片段的制备及活性测定

尿激酶原的N-末端135个氨基酸片段包含了表皮生长因子结构域和环柄结构域，参与了尿激酶原和其受体的结合以及尿激酶原诱导的化学趋化活性．利用RT-PCR，从人脐带静脉内皮细胞中克隆ATF基因．将ATF基因插入pET-29a( )中构建表达质粒pET-29a( )／ATF，并转化至大肠杆菌BL21(DE3)中，经IPTG诱导表达及纯化后，从每升培养液中获得15mg rhATF，其纯度超过95％．活性测定结果表明rhATF能够阻断尿激酶原与尿激酶受体的结合，并能抑制尿激酶对纤溶酶原以及纤溶酶对尿激酶原的激活．     

Kunitz抑制剂家族成员抑肽酶对纤溶酶原活化的抑制作用

抑肽酶属Kunitz抑制剂家族成员，能够抑制激肽释放酶、纤溶酶及胰蛋白酶的蛋白水解活性．研究表明，抑肽酶能够抑制尿激酶型-纤溶酶原激活刹（u—PA）和组织型纤溶酶原激活剂（t—PA）对纤溶酶原的激活，但不影响u—PA和t—PA对小分子底物的酰胺水解活性．用u—PA研究了上述作用的机制，发现抑肽酶与u—PA的丝氨酸蛋白酶功能区特异性结合，而与纤溶酶原没有相互作用．抑肽酶与uPA的结合并不阻断u—PA的活性位点，因为u—PA对小分子底物的水解活性仍然保持．上述发现提示抑肽酶可能存在另一种抑制作用模式，该模式不同于以前报道的关于Kunitz抑制剂或纤溶酶原激活酶抑制剂的作用．由于人体内的Kunitz抑制剂与抑肽酶在结构上非常相似，根据研究结果，推测体内纤溶酶原的激活作用并非仅受丝氨酸蛋白酶抑制剂的控制。     

尿激酶体外抑制人体免疫缺损病毒的侵染能力

人体免疫缺损病毒的包膜蛋白gp120的V3环区包含一段在人类蛋白质中很少出现的高度保守序列，但这段序列与纤溶酶原被纤溶酶原激活剂酶切位点附近序列有同源性．由于V3环区在人体免疫缺损病毒侵染细胞过程中的重要性，评估了尿激酶对人体免疫缺损病毒侵染能力的影响．通过检测逆转录酶活力，P24抗原的表达和合胞体形成情况发现尿激酶可以抑制人体免疫缺损病毒对多种淋巴瘤和白血病细胞系，如MT4、CCM、H9和外周血单核细胞的侵染能力，并且这种抑制与尿激酶浓度呈剂量依赖关系．那些能够被尿激酶抑制的人体免疫缺损病毒株其V3环区序列必须与纤溶酶原激活区亭列同源，实验事常用病毒株包括BRU和RF以及某些野生病毒株．研究结果显示尿激酶在体外实验中可以抑制人体免疫缺损病毒的侵染能力．     

两种尿激酶原变体(Ala175→Ser,Tyr187→His,Lys300→His和Ala175→Ser,Tyr187→His)的构建及性质研究

利用定点突变方法 ,构建了尿激酶原变体基因muk1(Ala175→Ser,Tyr187→His ,Lys30 0→His)及muk2 (Ala175→Ser ,Tyr187→His) .两种尿激酶原变体在大肠杆菌BL2 1中表达得到包涵体 ,经体外变复性 ,SP Sepharose离子交换柱层析 ,Benzamidine Sepharose吸附除去双链尿激酶 ,得到的蛋白均为银染一条带 .用人工合成的发色底物S2 4 4 4测量muk1、muk2的动力学性质 ,muk1的内在酶活性比野生型 pro UK降低 8倍 ,muk2的内在酶活性比野生型 pro UK降低 2 .5倍 ;它们经纤溶酶 (plasmin)激活后的双链活性与 pro UK相比分别为muk1提高 50 % ,muk2和pro UK相当 .在实验基础上讨论了 pro UK高内源性催化活性的机制及其结构基础 .     

尿激酶型纤溶酶原激活剂(uPA)系统在子宫内膜癌中的研究进展

尿激酶型纤溶酶原激活系统是纤溶系统的重要组成部分，它通过水解细胞外间质，参与组织改造和细胞迁移，在肿瘤细胞的侵袭和转移中发挥作用；讨论了uPA系统的结构、作用机理及与子宫内膜癌的关系，旨在为子宫内膜癌的诊断和治疗提供新方法。     

一种导向性溶栓剂的基因构建及其在大肠杆菌中的表达

以针对人活化血小板表面糖蛋白GMP140的单克隆抗体SZ51的单链抗体作为导向分子,以单链尿激酶32kd变体(scu-PA-32k)作为效应分子,构建了一种新型的导向性溶栓剂。通过聚合酶链式反应(PCR),分别从SZ51的Fab片段基因中扩增出V_K和V_H区;从尿激酶原基因中扩增出scu-PA-32k基因。通过合适的linker及酶切位点,将V_K, V_H及scu-PA-32k基因相连接并装入表达载体pET-5a中的Nde I位点,在大肠杆菌中经IPTG诱导表达了重组蛋白。Western-Blotting检测到在8mol/L尿素存在下,该重组蛋白与抗尿激酶的多克隆抗体之间仍有弱的结合反应。表达产物经变复性处理和部分分离纯化后,在纤维蛋白平板上表现有较好的纤溶活性,且这种活性是通过激活纤维蛋白溶酶原为纤溶酶而实现的。重组蛋白纤溶活性的比活达到17500IU/mg,较好地保留了尿激酶的纤溶活性,重组蛋白的产率约每100g湿菌为1.5mg。     

用三硝基苯磺酸(TNBS)比色法测尿激酶活性

尿激酶能激活纤维蛋白溶酶原,成纤维蛋白溶酶,后者能使含有大量精氨酸的鱼精蛋白充分水解,水解产物和2、4、6——三硝基苯磺酸反应生成在420nm 具光吸收的中间物。当尿激酶浓度低于每毫升40国际单位时,光吸收和尿激酶浓度成直线关系。本方法在灵敏度和重复性上都比较满意,也可用于纤维蛋白溶酶原的定量测定。     

赤子爱胜蚓纤溶酶活性的研究

研究了赤子爱胜蚓纤溶酶活性的提高条件及酶的最适作用条件。以纤维蛋白平板法测定赤子爱胜蚓纤溶酶活性。结果表明，纤溶酶在温度(57～59)℃，pH在8.0时，酶活性较稳定。赤子爱胜蚓纤溶酶干燥粉末浸泡在氯化钠等溶液中，可提高纤溶酶活性4-5倍。     

抗凝血蛋白药物的研究进展

除了血液中的抗凝因子外，自然界中还存在大量具有抗凝活性的生物大分子，如水蛭素、蚯蚓纤溶酶等．基础研究证实这些抗凝刑的作用一般是通过三方面得以实现：一是抑制凝血酶及凝血酶活化因子活性；二是水解血纤维蛋白或纤溶酶原；三是抑制血小板凝聚．由于这些活性物质具有高效的抗凝、溶栓作用，它们极有可能发展成治疗血栓性疾病的药物．     

中药红花与产纤溶酶菌株相互影响的研究

作者采用生长曲线法衡量不同浓度红花对产纤溶酶菌株C2-13的生长影响；纤维蛋白平板法测量纤溶酶活性；邻二氮菲-Fe^2 氧化法测量羟自由基清除率；乙酸酐直接测定法测量总胆固醇；HPLC法分析发酵产物．结果表明：产纤溶酶菌株C2-13能显著提高红花降血清总胆固醇的功效以及抗羟自由基氧化的能力；一定浓度红花的加入对C2-13的生长和纤溶酶活性有明显促进作用．HPLC分析还观察到红花经发酵炮制后，其成分发生了改变．说明中药红花与产纤溶酶菌株C2-13可相辅相成，互相促进．     

尿激酶原活性测定方法研究

以双链尿激酶国际标准品（IS）为对照，用显色底物法和凝块溶解法测定糖基化和非糖基化的尿激酶原的酶活性，结果表明与凝块法相比，显色底物法测定两种形式的尿激酶原的酶活力相近，测量误差小，重复性好，更适于产品的质量控制。     

重组人尿激酶原的工业化制备与性质研究

将含有 pET2 9a prouk重组质粒的人尿激酶原工程菌经 10L种子罐培养及 10 0L发酵 ,IPTG诱导表达 ,其表达量为占菌体总蛋白的 2 0 % ,表达产物经体外变复性 ,CM 纤维素离子交换层析 ,Superdex 75分子筛层析及Affi preppolymyxinsupport亲和层析去热源 ,每 10 0L发酵液得重组人尿激酶原纯品 6g ,纯度达 95 %以上 ,比活大于 1.2× 10 4 IU /mg ,双链含量低于0 .5 % ,内毒素及热源含量、宿主蛋白残留量、宿主DNA残留量等均达到临床使用标准 .其分子量 (质谱测定 )、氨基酸组成、N、C 端氨基酸序列分析等均与理论值相符 .进行了等电点及肽图等性质研究     

A potential basis for the thrombotic risks associated with lipoprotein(a)

Lipoprotein(a) (Lp(a)) has been strongly linked with atherosclerosis and is an independent risk factor for myocardial infarction. Distinguishing Lp(a) from other low-density lipoprotein particles is its content of a unique apoprotein, apo(a). The recently described sequence of apo(a) indicates a remarkable homology with plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin. Lp(a) may contain 37 or more disulphide-looped kringle structures, which are 75-85% identical to the fourth kringle of plasminogen. Plasminogen receptors are widely distributed on blood cells and are present at extremely high density on endothelial cells. These receptors promote thrombolysis by accelerating plasminogen activation and protecting plasmin from inhibition. If, by molecular mimicry, Lp(a) competes with plasminogen for receptors, then thrombolysis would be inhibited and thrombosis promoted. Here we provide support for such a mechanism being responsible for the thrombotic risks associated with elevated Lp(a) by demonstrating that Lp(a) inhibits plasminogen binding to cells.     

一种具有抗肿瘤活性的新型尿激酶原Kringle结构域变体蛋白的研究

利用PCR技术获得人尿激酶原Kringle结构域基因,将其克隆至具有T7启动子的表达质粒pET29a中,并进而插入plasminogen Kringle 5一段16肽片段构建了其变体,重组质粒转化至大肠杆菌BL21中,经IPTG诱导表达,其产物以包涵体形式存在．通过体外复性,得到可溶性的prourokinase Kringle及其变体．所得蛋白质经过动物肿瘤模型测活,确定变体蛋白具有抑制肿瘤生长作用．     

Serpin-resistant mutants of human tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) converts the inactive zymogen, plasminogen, into the powerful protease, plasmin, which then degrades the fibrin meshwork of thrombi. To prevent systemic activation of plasminogen, plasma contains several inhibitors of t-PA, the most important of which is plasminogen activator inhibitor-1 (PAI-1), a member of the serpin superfamily. As the ability to produce serpin-resistant variants of t-PA could increase the potential of this enzyme as a thrombolytic agent, we have used the known three-dimensional structure of the complex between trypsin and bovine pancreatic trypsin inhibitor (BPTI) to model the interactions between the active site of human t-PA and PAI-1. On the basis of this model we then altered by site-directed mutagenesis those amino acids of t-PA predicted to make contact with PAI-1 but not with the substrate plasminogen. We report here that although the resulting mutants have enzymatic properties similar to those of wild-type t-PA, they display significant resistance to inhibition by PAI-1. For example, following incubation with an amount of the serpin that completely inhibits the wild-type enzyme, one variant retains 95% of its initial activity. This mutant is also resistant to inhibition by the complex mixture of serpins present in human plasma.     

大批量制备人血浆纤维蛋白溶酶原

血浆经大豆胰蛋白酶抑制物、苯甲基磺酰氟和苯甲脒预处理,保持低温操作,以及赖氨酸—Sepharose 4B 亲和载体合成手续的改进,可以从近千毫升血浆中以140mg/L 血浆的产率制得低纤维蛋白溶解酶活性的纤维蛋白溶酶原制剂。     

Activation of specific interneurons improves V1 feature selectivity and visual perception

Inhibitory interneurons are essential components of the neural circuits underlying various brain functions. In the neocortex, a large diversity of GABA (γ-aminobutyric acid) interneurons has been identified on the basis of their morphology, molecular markers, biophysical properties and innervation pattern. However, how the activity of each subtype of interneurons contributes to sensory processing remains unclear. Here we show that optogenetic activation of parvalbumin-positive (PV+) interneurons in the mouse primary visual cortex (V1) sharpens neuronal feature selectivity and improves perceptual discrimination. Using multichannel recording with silicon probes and channelrhodopsin-2 (ChR2)-mediated optical activation, we found that increased spiking of PV+ interneurons markedly sharpened orientation tuning and enhanced direction selectivity of nearby neurons. These effects were caused by the activation of inhibitory neurons rather than a decreased spiking of excitatory neurons, as archaerhodopsin-3 (Arch)-mediated optical silencing of calcium/calmodulin-dependent protein kinase IIα (CAMKIIα)-positive excitatory neurons caused no significant change in V1 stimulus selectivity. Moreover, the improved selectivity specifically required PV+ neuron activation, as activating somatostatin or vasointestinal peptide interneurons had no significant effect. Notably, PV+ neuron activation in awake mice caused a significant improvement in their orientation discrimination, mirroring the sharpened V1 orientation tuning. Together, these results provide the first demonstration that visual coding and perception can be improved by increased spiking of a specific subtype of cortical inhibitory interneurons.