微生物代谢产物在心血管疾病中的应用

心血管疾病严重威胁着人类健康,研发防治心血管疾病的有效药物一直是广大研究者研究的焦点。微生物的多种代谢产物在预防和治疗心血管疾病方面得到广泛应用,从微生物代谢产物中寻找预防和治疗心血管疾病的药物是比较传统而且简单的方法,文章就微生物代谢产物在心血管疾病方面的应用进行了阐述。     

构建高质量微生物天然产物库研究策略

微生物次级代谢产物历来是天然药物的重要来源,过去曾被称为"生物沙漠"的海洋,由于从中分离到大量新的微生物、基因及生物活性化合物,被重新认识逆转而成为一种"生物多样化的热带雨林".构建一个高质量微生物库及其天然产物库是保证药物和其他筛选成功的前提和关键.但如何高效建立高质量微生物天然产物库仍面临很多瓶颈问题.我们拟从:(1)扩大可培养微生物的多样性及去重复化;(2)扩大基因资源多样性及去重复化;(3)扩大微生物次级代谢产物多样性及去重复化;(4)寻找崭新次级代谢产物的新技术新方法,特别是针对多靶位药物的高通量互动筛选方面提出应对的研究策略.利用上述化学微生物学策略分离生物活性化合物不仅在生物技术和制药学应用中显现重要性,也增加了我们对微生物的多样性、生态系统功能和应用生物学的理解.     

特殊生境微生物及其活性代谢产物研究进展*

微生物是一种重要的生物资源,特别是近来年,从特殊生境微生物中寻找具有重要生物活性代谢产物成为一个热点研究领域。主要就特殊生境微生物代谢产物的特殊性、特殊生境适应机制及其活性代谢产物等方面的研究进展和前景进行了简要的阐述,为特殊生境微生物的研究与开发提供参考。     

特殊生境微生物及其活性代谢产物研究进展

微生物是一种重要的生物资源,特别是近来年,从特殊生境微生物中寻找具有重要生物活性代谢产物成为一个热点研究领域。主要就特殊生境微生物代谢产物的特殊性、特殊生境适应机制及其活性代谢产物等方面的研究进展和前景进行了简要的阐述,为特殊生境微生物的研究与开发提供参考。     

微生物——几种溶栓药物的重要来源

综述了几种来源于微生物的重要溶栓剂的性质及研究状况。微生物是溶栓剂的重要来源 ,从微生物中寻找溶栓药物是一种理想有效的途径。     

宏基因组技术在开拓天然产物新资源中的应用*

微生物代谢产物具有巨大的化学多样性,是多种抗生素和其它药物的重要来源。由于现有培养手段的局限性,可培养的微生物不到微生物总数的1%,使绝大部分微生物资源的开发利用受到制约。近年来,直接提取环境样品中混合微生物总基因组DNA,利用可培养的宿主细菌构建宏基因组文库,通过筛选目的克隆,寻找活性代谢产物,取得瞩目进展。对这一新领域的研究进展结合我们的研究概况进行了简要综述。     

宏基因组技术在开拓天然产物新资源中的应用

微生物代谢产物具有巨大的化学多样性,是多种抗生素和其它药物的重要来源。由于现有培养手段的局限性,可培养的微生物不到微生物总数的1％,使绝大部分微生物资源的开发利用受到制约。近年来．直接提取环境样品中混合微生物总基因组DNA,利用可培养的宿主细菌构建宏基因组文库,通过筛选目的克隆,寻找活性代谢产物,取得瞩目进展。对这一新领域的研究进展结合我们的研究概况进行了简要综述。     

嗜盐微生物在生物医药领域的应用研究进展

近年来抗生素耐药性问题日趋严重,患癌人数也在逐年增加,亟需开发新型药物。嗜盐微生物作为一类特殊的极端环境微生物,具有代谢多样性丰富、营养需求较低和能适应恶劣条件等特点,是发现新型药物的希望。目前,国内外学者已从嗜盐微生物中分离出了多种代谢产物和酶,具有明显的抗菌和/或抗肿瘤等活性。文中综述了嗜盐微生物及其相关产物在抗菌、抗炎、抗肿瘤、抗氧化、生物医学材料以及药物载体等生物医学方面的作用,尤其对近年来在嗜盐微生物中发现的新型抗菌和抗肿瘤物质以及嗜盐微生物特有的代谢产物四氢嘧啶等进行了总结,并对其后续在生物医药领域的开发和产业化应用进行了展望。     

常见食源性致病菌代谢组学研究进展

食源性致病菌是一类以食品为传播媒介,可引起食物中毒的致病性微生物,是目前食源性疾病的主要诱因。食源性致病菌代谢组学通过研究致病菌代谢小分子物质产生的规律性和特征性,寻找新的检测靶标和建立基于代谢组学方法的食源性致病菌鉴别技术。随着目前代谢组学研究技术逐渐成熟和微生物挥发性代谢产物数据库的建立,食源性致病菌挥发性代谢产物分析已被建议作为临床和食品中致病菌鉴别的替代方法。本文综述了目前常见食源性致病菌代谢组学主要研究技术及其在临床和食品检测中的研究进展,以期为进一步建立食源性致病菌代谢产物数据库和研发基于代谢组学方法的食源性致病菌检测技术提供参考。     

抗雌激素药物的尿中代谢产物的检测和鉴定分析

目的:探讨分析抗雌激素药物的尿中代谢产物的检测和鉴定。方法:采用GC/MS技术检测和鉴定抗雌激素药物他莫昔芬的尿中代谢产物,建立常规检测抗雌激素药物他莫昔芬阳性尿样的方法,对抗雌激素药物他莫昔芬的尿中代谢产物进行结构鉴定。结果:抗雌激素药物他莫昔芬的尿中代谢产物主要为Met.C,分子量417,分子式C27H31NO3。结论:采用GC/MS技术检测和鉴定抗雌激素药物他莫昔芬的尿中代谢产物准确可靠,抗雌激素药物他莫昔芬的尿中代谢产物主要为Met.C。     

Regulation of plasmin activity by annexin II tetramer

Annexin II tetramer (AIIt) is a major Ca(2+)-binding protein of the endothelial cell surface which has been shown to stimulate the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. In the present report, we have examined the regulation of plasmin activity by AIIt. The incubation of plasmin with AIIt resulted in a 95% loss in plasmin activity. SDS-PAGE analysis established that AIIt stimulated the autoproteolytic digestion of plasmin heavy and light chains. The kinetics of AIIt-stimulated plasmin autoproteolysis were first-order, suggesting that binding of plasmin to AIIt resulted in the spontaneous autoproteolysis of the bound plasmin. AIIt did not affect the activity of other serine proteases such as t-PA or urokinase-type plasminogen activator. Furthermore, other annexins such as annexin I, II, V, or VI did not stimulate plasmin autoproteolysis. Increasing the concentration of AIIt on the surface of human 293 epithelial cells increased cell-mediated plasmin autoproteolysis. Thus, in addition to stimulating the formation of plasmin, AIIt also promotes plasmin inactivation. These results therefore suggest that AIIt may function to provide the cell surface with a transient pulse of plasmin activity.     

Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.     

Activation of human plasminogen by equimolar levels of streptokinase.

Native Glu-human plasminogen (Mr approximately 92,000 with NH2-terminal glutamic acid) is able to combine directly with streptokinase in an equivalent molar ratio, to yield a stoichiometric complex. The plasminogen moiety in the complex then undergoes streptokinase-induced conformational changes. As a result of such, an active center develops in the plasminogen moiety of the complex. This proteolytically active complex then activates plasminogen in the complex to plasmin and at least two peptide bonds are cleaved in the process. The data presented in this paper reveal that initially an internal peptide bond of plasminogen (in the complex) is cleaved to yield a two-chain, disulfide-linked plasmin molecule. The heavy chain (Mr approximately 67,000 with NH2-terminal glutamic acid) of this plasmin molecule has an identical NH2-terminal amico acid as the native plasminogen. The light chain (Mr approximately 25,000 with NH2-terminal valine) of plasmin is known to be derived from the COOH-terminal portion of the parent plasminogen molecule. A second peptide is then cleaved from the NH2-terminal end of the heavy chain of plasmin producing a proteolytically modified heavy chain (Mr =60.000 with NH2-terminal lysine). This cleavage of the NH2-terminal peptide from the heavy chain of plasmin is shown to be mediated by the dissociated free plasmin present in the activation mixture. Plasmin in the streptokinase-plasmin complex is unable to cleave this NH2-terminal peptide. This same NH2-terminal peptide can also be cleaved from native Glu-plasminogen or from the Glu-plasminogen-streptokinase complex by free plasmin and not by a complex of streptokinase-plasmin. From these studies we conclude (a) in the streptokinase-plasminogen complex, the NH2-terminal peptide need not be released prior to the cleavage of the essential Arg-Val peptide bond which leads to the formation of a two chain plasmin molecule and (b) that this peptide is cleaved from the native plasminogen or from the heavy chain of the initially formed plasmin in the streptokinase complex by free plasmin and not by the plasmin associated with streptokinase. In agreement with this, plasmin associated with streptokinase was unable to cleave the NH2-terminal peptide from the isolated native heavy chain possessing glutamic acid as the NH2-terminal amino acid; whereas free plasmin readily cleaved this peptide from the same isolated Glu-heavy chain.     

Analysis of plasmin binding and urokinase activation of plasminogen bound to the Heymann nephritis autoantigen, gp330.

Previously, we demonstrated that the Heymann nephritis autoantigen, gp330, can serve as a receptor site for plasminogen. This binding was not significantly inhibited by the lysine analogue epsilon-amino caproic acid (EACA), indicating that plasminogen binding was not just through lysine binding sites as suggested for other plasminogen binding sites. We now report that once plasminogen is bound to gp330, it can be converted to its active form of plasmin by urokinase. This conversion of plasminogen to plasmin proceeds at a faster rate when plasminogen is first prebound to gp330. Although there is a proportional increase in the Vmax of the urokinase-catalyzed reaction with increasing gp330 concentrations, no change in Km was observed. Once activated, plasmin remains bound to gp330 in an active state capable of cleaving the chromogenic tripeptide, S-2251. The binding of plasmin to gp330 did not significantly change its enzymatic activity; however, gp330 did have a stabilizing effect on plasmin activity at 37 degrees C. While bound to gp330, plasmin is protected from inactivation by its natural inhibitor alpha 2-antiplasmin. The binding of plasmin to gp330 as analyzed by ELISA was shown to be time dependent, reversible, saturable, and specific for gp330. Inhibition of binding of both plasminogen and plasmin to gp330 by benzamidine was similar, although EACA inhibited the binding of plasmin to gp330 slightly more than the binding of plasminogen to gp330. These results indicate that the binding of plasminogen to gp330 serves as an effective means of increasing the rate of plasmin production on the glomerular and tubular epithelial cell surface while protecting the active plasmin from natural inhibitors.     

Resistance of factor XIII to degradation or activation by plasmin

The effect of plasmin on the subunit polypeptides of factor XIII has been investigated. purified human plasma (a2b2) and platelet (a2) zymogens and the enzyme (a2) were incubated with plasmin at plasmin: factor XIII ratios of 0.03-0.5 casein units per mg protein. Under conditions in which plasmin readily digested fibrinogen and casein, it had no effect on either a2b2 or a2. There was no evidence for cleavage of peptide bonds in the zymogens, and all the potential catalytic activity was retained after prolonged incubation. Similarly a2*, either in the presence or absence of b subunit, was also unaffected by plasmin incubation. 90% of the activity was recovered after incubation of factor XIII with plasmin. b subunit was also not degraded. Additionally, no evidence was obtained that plasmin could activate factor Xiii. These results indicate that in purified systems there is no significant interaction between plasmin and factor XIII.     

Studies on the nature of the catalytically essential ionizing group of plasmin with pK 8.4.

A fully carbamylated derivative of plasminogen having no free amino groups has been prepared and converted by urokinase to an active enzyme, called carbamyl plasmin A, with a single free NH2-terminal amino group (Val-561). Carbamyl plasmin A was shown to possess a catalytically essential ionizing group having pK 8.6. Carbamylation of the free NH2-terminal amino group of carbamyl plasmin A led to complete loss of catalytic activity. The results of solvent perturbation studies of normal plasmin (EC 3.4.21.7) indicate that the group with pK 8.4 is a neutral acid group. It is suggested that the catalytically essential ionizing group of plasmin having a pK of 8.4 is the alpha-ammonium group of the NH2-terminal Val-561 or the light chain of plasmin, forming an ion pair with a COO- group of an aspartate or glutamate residue.     

Effects of free fatty acids on fibrinolytic activity.

A novel method for the estimation of fibrinolytic activity is proposed. In this method, a fibrin clot suspension is used as a substrate (fibrin is known to be a physiological substrate of plasmin). The fibrin clot suspension was prepared by homogenization of human fibrin clots. With this method, we found that free fatty acids inhibited the plasmin activity, and long-chain, unsaturated free fatty acids had a particularly strong inhibitory action on plasmin. As regards the mechanism of the inhibitory action, free fatty acids may not inhibit complex formation between plasmin and fibirin, but may make it impossible for plasmin to act on fibrin due to deformation of the surface of the fibrin clot.     

Photoaffinity labeling of functionally different lysine-binding sites in human plasminogen and plasmin

Photoaffinity labeling of human plasmin using 4-azidobenzoylglycyl-L-lysine inhibits clot lysis activity, while the activity toward the active-site titrant, p-nitrophenyl-p'-guanidinobenzoate, or alpha-casein are maintained. Photoaffinity labeling of native Glu-plasminogen with the same reagent causes incorporation of approximately 1.5 mol label per mol plasminogen. This labeled plasminogen can be activated to plasmin by either urokinase or streptokinase. The resulting plasmin has full clot lysis activity and can be subsequently photoaffinity labeled with a loss of clot lysis activity. The rate of activation of labeled plasminogen by urokinase is increased relative to that of native plasminogen. epsilon-Aminocaproic acid blocks incorporation of photoaffinity label into both plasminogen and plasmin, indicating that the labeling is specific to the lysine-binding sites. The labels are located in the kringle 1+2+3 fragment in either photoaffinity-labeled plasminogen or plasmin. These results indicate that the specific lysine-binding site blocked in plasmin acts in concert with the active-site in binding and using fibrin as a substrate. This clot lysis regulating site is not available for labeling in plasminogen, but is exposed or changed upon activation to plasmin. The different lysine-binding sites labeled in plasminogen may regulate the conformation of the molecule as evidence by an enhanced rate of activation to plasmin.     

Synthesis and evaluation of tripeptidic plasmin inhibitors with nitrile as warhead

Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC₅₀ = 7.7–11 μM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of recombinant human single-chain urokinase-type plasminogen activator variants resistant to plasmin and thrombin

Single-chain urokinase-type plasminogen activator (scu-PA), a potential therapeutic reagent for thrombosis, is activated in plasma by plasmin. The activated enzyme is further digested by plasmin to generate low-molecular-weight urokinase (LMW-UK), which has no affinity for fibrin. To circumvent this dual effect of plasmin, we synthesized in Escherichia coli a variant of scu-PA, which is not converted to LMW-UK on treatment with plasmin. In another variant, the activation cleavage site was modified such that activation by plasmin was slowed down and that inactivation by thrombin was greatly diminished. The combination of these variants may be applicable as an effective thrombolytic reagent for clinical use.