PAI－1糖基化位点突变体的构建及其在哺乳动物细胞中表达

Ⅰ型纤溶酶原激活物抑制剂（ＰＡＩ－１）是一种Ｍｒ为５０×１０３的单链糖蛋白，有３７９个氨基酸残基，３个Ｎ型糖基化位点。构建ＰＡＩ－１糖基化突变体，以便研究糖链的功能。用寡核苷酸定位突变技术将３个糖基化位点２０９，２６５，３２９位进行突变，把３个糖基化位点都发生了突变的ＰＡＩ－１ｃＤＮＡ组装到真核表达载体ｐＳＶ２中，得到真核表达质粒ｐＺＨ－ｐ１－Ｍ３Ｅ；在二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞（ＣＨＯｄｈｆｒ－）中进行短暂表达，用发色底物法和夹心ＥＬＩＳＡ方法检测培养液中ＰＡＩ－１的活性和含量。结果：糖基化位点突变的ＰＡＩ－１能在ＣＨＯ细胞中表达，但表达水平及活性较低。非糖基化ＰＡＩ－１的活性和抗原分别为４．３４ＩＵ／ｍｌ和３．１５μｇ／Ｌ结论：用寡核苷酸定位突变方法获得了ＰＡＩ－１糖基化突变体，并且在ＣＨＯ细胞中得到表达。     

Antibodies against dengue virus E protein peptide bind to human plasminogen and inhibit plasmin activity

Both mice and rabbits immunized with dengue virus E protein peptide spanning amino acids 100–119 (D4E) produced antibodies that reacted not only with the D4E peptide itself but also with human plasminogen, as shown by ELISA and Western blot. Sera from dengue virus-hyperimmunized mice and dengue patients also contained antibodies against D4E and plasminogen. Furthermore, such sera all contained plasmin inhibitory activity. Using affinity-purified anti-D4E antibodies and free D4E peptide for competitive inhibition, we demonstrated that the inhibition of plasmin activity was due to anti-D4E antibodies rather than other substances in the sera. Taken together, these results suggest dengue virus E protein amino acids 100–119 are a cross-reactive immunogenic region, and antibodies against this region may interfere with human fibrinolysis.     

组织型纤溶酶原激活剂及其抑制物的变化与冠心病的关系

目的：观察冠心病患者纤溶活性的变化及其在冠心病发病中的作用，探讨其临床意义。方法：用酶联免疫双抗夹心(ELISA)法测定58例冠心病患者血浆中组织型纤溶酶原激活剂(t—PA)及纤溶酶原激活抑制物-1(PAI-1)抗原含量，反映纤溶-抗纤溶活性的变化，并对冠心病患者组与对照组纤溶指标进行不同性别间的比较。结果：急性心肌梗死、不稳定心绞痛患者PM-1的含量、PAI-1／t—PA比值明显高于对照组，且急性心肌梗死患者PM-1的含量较不稳定心绞痛患者显著为高。但t—PA在急性心肌梗死、不稳定心绞痛患者均无显著降低。患者组及对照组不同性别间纤溶及抗纤溶水平均未见有显著差异。结论：血栓性疾病与纤溶系统的异常有密切关系。纤溶系统活性的变化及纤溶-抗纤溶的平衡失调在缺血性心脏病的发生、发展中起着重要作用。     

Risk factors for myocardial infarction during coronary artery bypass graft surgery

Patients with a particular thrombotic profile may be at greaterrisk of myocardial infarction during coronary artery bypassgraft surgery. The thrombotic profile of 50 patients admitted to hospital withstable angina pectoris was determined prior to haemodynamicinvestigation. ECG results and determination of cardiac enzymesshowed that 12 patients had suffered a perioperative myocardialinfarction. These patients had a higher mean atheroscleroticscore (42.1 ± 10.5 vs 32.9 ± 13, P<0.02), alonger aortic cross clamp time (59 ± 15.2 vs 45.7 ±16.3 min, P < 0.05), lower serum levels of protein C (101.2±26vs 124.7+ 31.4%, P<0.05) and tissue plasminogen activator(322 ± 580 vs 2307±2830 IU ml^–1, P<0.01). There were no differences between the two groups in Jenkin'scoronary score, the number and type of grafts, ejection fraction,left ventricular end-diastolic pressure, lipid profile or levelsof markers of platelet release. In addition to a more severe distal coronary atheroma and alonger aortic cross-clamp time, patients with impaired endothelialfibrinolytic activity appeared to be at greater risk of myocardialinfarction during coronary artery bypass graft surgery.     

Growth factor-dependent activation of the MAPK pathway in human pancreatic cancer: MEK/ERK and p38 MAP kinase interaction in uPA synthesis

Increased expression of the hepatocyte growth factor (HGF) receptor (c-met) and urokinase type plasminogen (uPA) correlated with the development and metastasis of cancers. To investigate the role of HGF/c-met signaling on metastasis in cancer cells stimulated with HGF, we examined the effects of a specific MEK1 inhibitor (PD98059) and a p38 MAP kinase inhibitor (SB203580) on HGF-induced uPA expression in pancreatic cancer cell lines, L3.6PL and IMIM-PC2. Pretreatment of PD98059 decreased HGF-mediated phosphorylation of extracellular receptor kinase (ERK), uPA secretion and expression of matrix metalloproteinases (MMP-2 and MMP-9) in a dose-dependent manner. In contrast, SB203580 pretreatment increased HGF-stimulated ERK phosphorylation, uPA secretion and expression of MMPs. SB203580 also reversed the inhibition of HGF-mediated ERK activation and uPA secretion in the PD98059-pretreated cells. These results suggest that ERK activation by HGF might play important roles in the metastasis of pancreatic cancer and the p38 MAPK pathway also involved in the HGF-mediated uPA secretion and metastasis by regulation of ERK pathway. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plasminogen activator system modulates invasivecapacity and proliferation in prostatic tumor cells

The malignant phenotype of prostatic tumor cells correlates with the expression of both uPA and itscell-membrane receptor (uPAR); however, there is little information concerning the role of cell-bound uPAin matrix degradation and invasion. Our results suggest that cell-associated uPA plays a key role in regulat-ingthe amount of plasmin present at the surface of prostatic carcinoma (PRCA) cells and show that differ-entialproduction of uPA corresponds with the capacity to bind and activate plasminogen. In addition, weprovide direct evidence that both uPA secretion and the presence of uPA-uPAR complexes characterize theinvasive phenotype of PRCA cells and suggest the existence of several pathways by which tumor cells acquireplasmin activity. LNCaP cells (which do not produce uPA but express uPAR) may activate plasmin throughexogenous uPA. In vivo, the source of uPA may be infiltrating macrophages and/or fibroblasts as observedin several other systems. PAI-1 accumulation in the conditioned medium (CM) limits plasmin action to thepericellular microenvironment. Our results indicate that MMP-9 and MMP-2 are also activated by plasmingenerated by cell-bound but not by soluble, extracellular uPA. Plasmin activation and triggering of the pro-teolyticcascade involved in Matrigel invasion is blocked by antibodies against uPA (especially by anti- A-chainof uPA which interacts with uPAR) and by PA inhibitors such as p-aminobenzamidine which mayregulate levels of cell-bound uPA. uPA may also regulate growth in PRCA cells. Indeed, antibodies againstuPA A-chain (and also p-aminobenzamidine treatment) interfere with the ATF domain and inhibit cell growthin uPA-producing PC3 and DU145 prostate cancer cell lines, whereas exogenous uPA (HMW-uPA with ATF)induces growth of LNCaP prostate tumor cell line. These data support the hypothesis that in prostatic can-cerpatients at risk of progression, uPA/plasmin blockade may be of therapeutic value by blocking both growthof the primary tumor and dissemination of metastatic cells. ©Kluwer Academic Publishers     

Antibodies to PAI-1 alter the invasive and migratory properties of human tumour cells in vitro

Recent reports suggest that elevated levels of plasminogen activator inhibitor-1 (PAI-1) may contribute to tumour progression. The studies reported here were designed to help elucidate PAI-1's contribution to the invasive and migratory phenotype. Antibodies to PAI-1 dose-dependently, and significantly, inhibited the invasive and migratory potential of human HT1080 fibrosarcoma cells, as did an antibody to uPA and the plasmin inhibitor aprotinin. Invasion of the human melanoma cell line, BLM, was also attenuated by the anti-PAI-1 monoclonal antibody MAI-12. The non-invasive human melanoma cell line, IF6, which does not express uPA, provided further confirmation of PAI-1 and uPA's role as, upon transfection with uPA, this cell line attained an invasive phenotype, which was again attenuated by MAI- 12. Although antibodies to PAI-1 did not affect the adhesion of HT1080 cells to vitronectin, the antibody to uPA reduced their attachment. Addition of exogenous PAI-1, however, prevented HT1080 cell adhesion (IC₅₀ 180nM) and promoted cell detachment from vitronectin. Furthermore melanoma cells transfected with a uPA variant, which had an impaired interaction with PAI-1, were not invasive and had impaired binding to vitronectin. These data highlight the importance of a balanced proteolysis and suggest an additional role for PAI-1 distinct from its role in proteolysis. These data also suggest that uPA and PAI-1 may co-operate in the migratory process by respectively facilitating the attachment to, and subsequent detachment from, vitronectin in the extracellular matrix. These results support the clinical findings and indicate that modulation of PAI-1 activity may be of therapeutic benefit for the treatment of cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Restriction site polymorphism at the LPA (Lp(a) apoliprotein; apoliprotein(a)) locus

A restriction site polymorphism in the Lp(a) apolipoprotein gene (the LPA gene) is reported. The basis for the polymorphism is presence or absence of an MspI restriction site that appears to be 3' to the last kringle IV structure of the gene. The "1" gene (presence of the restriction site) has a frequency of 0.316 and the "2" gene (absence of the restriction site) has a frequency of 0.684. Both members of each of 67 monozygotic (MZ) twin pairs had the same genotype and there was Mendelian segregation of the DNA variants in 40 families with a total of 75 children. There was a lower proportion of people with genotype 1-1 in the top quartile than in the 3 bottom quartiles of the population distribution of Lp(a) lipoprotein levels but the difference did not reach statistical significance.     

氧化修饰引起脂蛋白（a）结构和生化特性变化

本文观察了体外丙二醛（ＭＤＡ），铜离子（Ｃｕ２＋氧化修饰的脂蛋白（ａ）［Ｌｐ（ａ）］结构和生物学性质的变化。氧化修饰Ｌｐ（ａ）过氧化程度增高，负电荷增加，易被巨噬细胞—清道夫受体识别和摄取。ＭＤＡ修饰Ｌｐ（ａ）出现新的ＭＤＡ－ＬＤＬ位点；同纤维蛋白溶酶原（Ｐｇ）竞争抑制试验显示氧化、修饰Ｌｐ（ａ）同Ｐｇ同源性增加。提示载脂蛋白（ａ）状态同动脉粥样硬化的病理过程有关。     

尿激酶型纤溶酶原激活物系统对骨巨细胞瘤细胞基质金属蛋白酶-2和组织基质金属蛋白酶抑制物-3表达的影响

目的:研究尿激酶型纤溶酶原激活物(uPA)及其受体(uPAR)信号转导对骨巨细胞瘤基质金属蛋白酶-2(MMP-2)和金属蛋白酶组织抑制物-3(TIMP-3)的调节。方法:用免疫组化检测骨巨细胞瘤组织中uPAR、MMP-2和TIMP-3的表达。用免疫共沉淀法检测uPA对瘤细胞信号转导通路的p44蛋白磷酸化水平。用蛋白印迹法检测用uPA和uPAR抗体处理后瘤细胞MMP-2和TIMP-3蛋白表达。结果:(1)uPAR主要表达在部分单核基质细胞和一些多核巨细胞的胞膜上;(2)MMP-2主要表达在瘤细胞的胞浆,在多核巨细胞,其表达有明显的极向性;(3)在骨巨细胞瘤组织TIMP-3表达量低于MMP-2,在多核巨细胞也显示极向性表达;(4)将uPA-ATF加入培养的骨巨细胞瘤细胞后,细胞信号通路上的p44蛋白磷酸化水平明显增高。用uPAR抗体处理后,细胞p44蛋白磷酸化水平明显降低。说明uPA-ATF参与细胞信号转导,而且受uPAR拮抗剂的影响;(5)uPA-ATF信号通路上调MMP-2和TIMP-3的表达,而uPAR抗体则下调MMP-2和TIMP-3的表达。结论:本实验首次直接证明uPA-ATF通过信号转导能调节MMP-2和TIMP-3的表达,而后者则在骨巨细胞瘤局部骨质吸收中起重要作用。