Active gamma-carboxylated human factor IX expressed using recombinant DNA techniques

Factor IX (Christmas factor), a vitamin K-dependent plasma protein made in the liver, functions in the middle phase of the intrinsic pathway of blood coagulation. A functional deficiency of factor IX underlies haemophilia B, a chromosome X-linked recessive disease for which the major therapeutic approach is replacement treatment using factor IX concentrates. The cloning and characterization of the gene for human factor IX would mean that human factor IX could be produced in greater yield and purity through using recombinant DNA techniques. We have now used a human factor IX cDNA clone, inserted into a vaccinia virus-derived vector, to infect human hepatoma cells which normally produce no factor IX, and mouse fibroblasts. Fully active factor IX was produced by the hepatoma cells, whereas the fibroblasts produced a protein less active than natural factor IX, even in the presence of high levels of vitamin K. Human factor IX is extensively post-translationally modified, and thus represents probably the most complex protein produced in active form by recombinant DNA techniques to date. Our study also illustrates the potential of vaccinia virus-based vectors for expressing significant amounts of complex, clinically useful proteins in eukaryotic cells, in addition to its already demonstrated usefulness for producing live recombinant vaccines.     

Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2

Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational gamma-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.     

Identification of the gene for vitamin K epoxide reductase

Vitamin K epoxide reductase (VKOR) is the target of warfarin, the most widely prescribed anticoagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-used because of the difficulty of controlling dosage and the fear of inducing bleeding. Although identified in 1974 (ref. 2), the enzyme has yet to be purified or its gene identified. A positional cloning approach has become possible after the mapping of warfarin resistance to rat chromosome 1 (ref. 3) and of vitamin K-dependent protein deficiencies to the syntenic region of human chromosome 16 (ref. 4). Localization of VKOR to 190 genes within human chromosome 16p12-q21 narrowed the search to 13 genes encoding candidate transmembrane proteins, and we used short interfering RNA (siRNA) pools against individual genes to test their ability to inhibit VKOR activity in human cells. Here, we report the identification of the gene for VKOR based on specific inhibition of VKOR activity by a single siRNA pool. We confirmed that MGC11276 messenger RNA encodes VKOR through its expression in insect cells and sensitivity to warfarin. The expressed enzyme is 163 amino acids long, with at least one transmembrane domain. Identification of the VKOR gene extends our understanding of blood clotting, and should facilitate development of new anticoagulant drugs.     

见血青总生物碱凝血活性的研究

用超声波辅助提取见血青总生物碱(TALN),配置成10mg/mL见血青总生物碱药液(TASLN),测试药液凝血活性.实验以生理盐水(NS)为空白对照、云南白药做阳性对照,体内凝血实验以小白鼠为研究对象测定其出血时间和出血量;局部创面止血实验以兔子为实验对象,记录背部创伤止血时间;体外凝血实验选用抗凝兔血,分别测定凝血时间、凝血酶原时间以及复钙时间.结果显示:TALN的体内和体外凝血实验与空白对照组相比,明显缩短了老鼠出血时间(212.95s)和出血量(42.5格)、家兔的凝血时间(凝血板法时间为192.23s、试管法时间为245.48s)、凝血酶原时间(PT)(12.22s)及血浆复钙时间(RT)(180.78s);局部创面止血实验结中,TASLN组平均止血时间为81.50s,相对于NS组125.20s明显缩短且与云南白药组75.20s的结果差异不显著.说明:TALN具有良好的凝血活性.     

Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation

Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting. Here we present the 2.2 A crystal structures of human alpha-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as 'molecular sexuality'.     

苦瓜的营养成分研究

用自动定氮仪、氨基酸自动分析仪分析测定苦瓜 (Momordica charantia L .) 5个品种 (当地栽培苦瓜、长白苦瓜、长白 1号苦瓜、 89- 1苦瓜、 92 - 2苦瓜 )的干物质含量、维生维 C含量、可溶性糖含量、蛋白质和氨基酸、矿物质含量及其种子的蛋白质含量。结果表明 ,苦瓜果实含干物质 6 %～ 7% ,品种间的差异不显著 (P>0 .0 5 ) ;每 10 0 g嫩苦瓜中含维生素 C6 0～ 80 mg,可溶性糖 1.0 8～ 1.5 5 mg,品种间的差异都不显著 (P >0 .0 5 ) ;苦瓜果实的蛋白质含量为 1.3%～ 1.8%、总氨基酸含量为 1.3%～ 1.5 % ,种子蛋白质含量高达 39%～4 5 % ,各种成分相对含量有显著性差异 (P <0 .0 1) ,不同品种之间存在蛋白质多态性变化。苦瓜种子的营养价值很高。     

Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability

Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin. At the metaphase-anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K). The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine-a destabilizing residue in the N-end rule. Here we show that the SCC1 fragment is short-lived (t1/2 approximately 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of a long-lived derivative of the SCC1 fragment is lethal. In ubr1Delta cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss. The bulk of increased chromosome loss in ubr1Delta cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment. This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue. A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.     

血必净注射液对严重肢体创伤患者凝血功能的影响

目的:探讨应用血必净注射液对严重肢体创伤患者凝血系统的影响.方法:将36例严重肢体创伤患者随机分为2组,对照组18例给予常规综合治疗,治疗组18例在对照组治疗基础上早期加用血必净注射液,分别检测2组伤后治疗前、治疗后1、3、7d的血小板计数、凝血酶原时间、活化部分凝血活酶时间、凝血酶时间、纤维蛋白原.结果:治疗组于治疗3、7d后上述检测指标改善且与对照组比较有显著性差异.结论:应用血必净注射液对严重肢体创伤患者凝血系统有良性调节作用.     

Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1

A magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 lacZ2 transposon mutagenesis, and a 3073-bp fragment flanking mini-Tn5 lacZ2 in NM21 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved three putative ORFs; the mini-Tn5 lacZ2 was inserted into ORF1. Functional complementary test indicated that the 3073-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The majority of proteins, which had h...     

Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein

The three-dimensional structure of the amino-terminal 44K ATPase fragment of the 70K bovine heat-shock cognate protein has been solved to a resolution of 2.2 A. The ATPase fragment has two structural lobes with a deep cleft between them; ATP binds at the base of the cleft. Surprisingly, the nucleotide-binding 'core' of the ATPase fragment has a tertiary structure similar to that of hexokinase, although the remainder of the structures of the two proteins are completely dissimilar, suggesting that both the phosphotransferase mechanism and the substrate-induced conformational change intrinsic to the hexokinases may be used by the 70K heat shock-related proteins.     

云南白药保险子在重度创伤性失血急救中的应用

创伤性失血是急救领域的重要挑战,可导致凝血-抗凝系统紊乱,云南白药保险子是中医经典急救药物,兼有止血和抗凝的功效。以2018年1月至2021年12月期间在呼和浩特市第一医院急诊中心收治的骨科创伤患者为研究对象,通过前瞻性平行对照临床研究,对保险子的止血疗效进行评价,指标包括术中出血量、术后引流量、血红蛋白下降值、凝血酶原时间（PT）、激活部分凝血酶原时间、纤维蛋白原（FBG）、输血率,预后出血/凝血不良反应等。结果显示,保险子组（n=27）的术中出血量、术后引流量、血红蛋白下降值、输血率、PT以及预后出血/凝血不良反应均显著小于对照组（n=24）,保险子组FBG显著高于对照组;说明保险子能够维持机体凝血-抗凝系统的平衡,在显著降低出血量的同时,兼具抗凝的功效,可预防创伤性凝血病,减少预后不良反应。     

魔芋葡甘低聚糖醛酸丙酯硫酸酯钠盐生物活性研究

为了研究低聚KGM醛酸丙酯硫酸酯钠盐的抗凝和抗栓作用,应用玻片法研究了对小鼠凝血时间的影响;应用大鼠体外颈动脉－颈静脉血流动旁路法形成血小板血栓模型研究了其对血栓形成的影响;并进行了对兔凝血活酶时间（PT）和纤维白原（Fg）的影响的研究。结果表明剂量在0.9-3.6g/kg时,能明显延长小鼠的纤维蛋朱含量效果显著,其LD50为8.8lg/kg。说明该物质具有明显的高凝血和抗血栓作用,毒性极微,是一种有前途的防治心血管疾病的新型低分子类肝素物质。     

Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli

High-level expression of many eukaryotic genes has proved difficult to achieve even when a strong promoter and the ribosome binding sequence from highly expressed Escherichia coli genes have been placed in front of the coding sequences. To overcome this problem, many eukaryotic proteins have been efficiently produced as hybrids after fusion of their genes with a coding sequence of E. coli genes. However, such hybrid proteins are not suitable for functional studies or clinical use unless the authentic protein sequence can be released by specific cleavage. Here, we have inserted the sequence Ile-Glu-Gly-Arg between the 31 amino-terminal residues of lambda cII protein and Val 1 of human beta-globin, and produced this hybrid in high yield in E. coli. We then cleaved the hybrid specifically at the single arginine, using blood coagulation factor Xa and thus liberated the authentic beta-globin chain. As factor Xa is specific for the tetrapeptide Ile-Glu-Gly-Arg, which is rare in protein sequences, our expression/cleavage system is applicable to the efficient production of many eukaryotic proteins.     

Enhancing protein C interaction with thrombin results in a clot-activated anticoagulant.

Human protein C is a vitamin K-dependent plasma glycoprotein that circulates as an inactive zymogen. At the endothelial cell surface, thrombin in complex with the integral membrane protein thrombomodulin converts protein C to its active form by specific cleavage of an activation peptide. The activated form of protein C has potent anticoagulant activity as a feedback regulator of thrombin generation (reviewed in refs 4-6), and also has profibrinolytic, anti-ischaemic and anti-inflammatory properties. Protein C is effective in the treatment of model and human thrombotic diseases but, except when it has been used to treat genetic or acquired deficiencies and microvascular thrombosis, it is administered as the activated enzyme, which has a short biological half-life. We have altered two putative inhibitory acidic residues near the thrombin cleavage site, which results in a 30-fold increase in substrate utilization by alpha-thrombin. We combined these changes with a genetically altered glycoform to generate a zymogen protein C with a 60-fold increased cleavage rate by free alpha-thrombin, independent of its cofactor thrombomodulin. We show that this 'proform' of protein C, unlike the natural circulating zymogen, can be activated by thrombin generated in clotting human plasma, resulting in an inhibition of further clot formation. Our data therefore show that we have engineered a site-activated agent, which only has anticoagulant activity when significant amounts of thrombin are being generated.     

维生素E对小白鼠骨髓细胞染色体畸变效应的实验研究

灌胃有毒源污水或皮下注入苯诱发小白鼠骨髓细胞染色体畸变率分别为4.0%和5.0%,4h后灌胃维生素E,染色体畸变率分别下降为1.0%和1.5%,P相似文献   

Association of a Ras-related protein with cytochrome b of human neutrophils

Activation of the superoxide generating system in human neutrophils is thought to involve the interaction or assembly of cytochrome b with other cytosolic and membrane proteins. We have now co-isolated by conventional purification procedures a protein of relative molecular mass 22,000 with cytochrome b. This Ras-related protein is not a fragment of either of the subunits of cytochrome b, and its primary structure, as determined by the sequencing of its complementary DNA, is identical to that predicted from a recently cloned ras-related gene, rap1 (also termed Krev-1). Immunoaffinity purification on anti-cytochrome and anti-Ras immunoaffinity matrices indicates an association between cytochrome b and the Ras-related protein. The association of a Ras-related GTP-binding protein with cytochrome b of human neutrophils could indicate a role for such a protein in the transduction, regulation or structure of the superoxide generating system.     

维医异常体液型冠心病与ACE、eNOS、FVII及ICAM-1基因多态性关系的研究

研究维医不同异常体液型维吾尔族冠心病患者血管紧张素转换酶(ACE)基因、内皮型-氧化氦合酶(eNOS)基因、凝血因子Ⅶ(FVⅡ)基因、细胞黏附分子-1(ICAM-1)基因等基因的多态性变化.采用聚合酶链式反应-限制性片段多态性(PCR-RFLP)方法,分别检测不同异常体液型维吾尔族冠心病患者(共92例)及健康对照组(共30例)4种基因型及其等位基因频率.结果显示,冠心病组、不同异常体液型冠心病组与健康对照组相比,ACE基因、FVⅡ基因、ICAM-1基因在基因型及等位基因频率方面均无显著性差异(p>0.05);异常胆液质、异常黑胆质及异常血液质型冠心病组在eNOS基因型和等位基因频率分布两方面与健康对照组比较,均有显著性差异(P<0.05).但在异常黏液质型冠心病组无显著性差异(P>0.05).由此得出结论:ACE基因、FVⅡ基因、ICAM-1基因多态性与不同异常体液型维吾尔族冠心病患者的关系不明显,而eNOS基因多态性与异常胆液质、异常黑胆质、异常血液质型冠心病患者有相关性.     

维生素类制药废水处理工艺

将好氧共基质工艺、厌氧-好氧工艺、混凝以及Fenton试剂处理工艺用于维生素类制药废水的处理并加以比较．结果表明：在好氧共基质条件下,葡萄糖人工配水和制药废水的最佳COD浓度比为1：5;厌氧反应器的串联使用提高了反应器中的生物降解效果,有利于反应器的长期稳定运行;混凝工艺适合作为生化处理的预处理工艺,Fenton试剂则不适于处理此种制药废水．     

磷脂酰肌醇-3激酶结构与功能研究进展

磷脂酰肌醇-3激酶家族成员在生长因子等因素激活后可产生磷脂类物质,作为第二信使绑定和激活不同的靶细胞,形成复杂的信号级联反应,最终在细胞增殖、分化、趋化、存活、蛋白交通、糖代谢等活动中发挥核心作用.在详细介绍磷脂酰肌醇-3激酶家族成员分类、结构特征的基础上,对磷脂酰肌醇-3激酶依赖性的信号通路及其相关功能进行了介绍.     

Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex

The serine proteinase alpha-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1-6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin-TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.