Brazilin inhibits activities of protein kinase C and insulin receptor serine kinase in rat liver

Hypoglycemic action of brazilin was found to be based on the improvement of peripheral glucose utility, and this action might be correlated with the insulin action pathway. In the present study we investigated the effect of brazilin on the insulin receptor autophosphorylation, protein kinase C (PKC), protein phosphatase and insulin receptor serine kinase in order to confirm whether the hypoglycemic mechanism is concerned with insulin action pathway. Brazilin was found to inhibit PKC and insulin receptor serine kinase, which are involved in the regulation of insulin signal pathway. But any significant effect was not shown on insulin receptor tyrosine kinase activity, autophosphorylation and phosphatase activity. These findings suggest that brazilin might enhance insulin receptor function by decreasing serine phosphorylation, which might mediate hypoglycemic effect of brazilin     

黄连素通过调节磷脂酰肌醇-3-激酶/丝氨酸/苏氨酸激酶/葡萄糖转运蛋白4信号途径改善胰岛素抵抗的机制研究

目的研究黄连素对胰岛素抵抗细胞3T3-L1细胞磷脂酰肌醇-3-激酶/丝氨酸/苏氨酸激酶(AKT)和葡萄糖转运蛋白4(GLUT4)mRNA和蛋白表达的影响,探讨黄连素改善胰岛素抵抗的分子机制。方法将3T3-L1脂肪细胞随机分为正常对照组、胰岛素抵抗、黄连素3组,应用地塞米松诱导细胞胰岛素抵抗,黄连素组同时加入黄连素。以葡萄糖氧化酶法测定3组细胞上清液中葡萄糖消耗量,观察黄连素对脂肪细胞葡萄糖摄取的影响;应用免疫印迹技术(Western blotting)技术测定脂肪细胞AKT和GLUT4的蛋白水平变化;应用实时荧光定量PCR技术检测脂肪细胞AKT和GLUT4的mRNA表达变化。结果与正常对照组相比,胰岛素抵抗组的AKT和GLUT4 mRNA和蛋白表达明显下降(P<0.05),经黄连素干预后,AKT和GLUT4 mRNA和蛋白表达水平有一定升高,但仍显著低于正常对照组(P<0.05)。结论黄连素通过上调AKT基因和蛋白的表达进而增加GLUT4的水平,改善胰岛素抵抗状态。     

Therapeutic applications of serine protease inhibitors

Serine proteases (SPs) are known to regulate important biological processes, which makes them attractive targets in therapy. In recent years, much effort has been directed to revealing physiological roles played by some of these enzymes, as well as to identify and characterise various members of the class as potential drug targets. These efforts materialised in the design of new drugs for thrombotic diseases, emphysema, asthma, herpesvirus protease, hepatitis C NS3 protease etc. and in emerging therapies for the treatment of cancer, based on the inhibition of plasminogen-type urokinase. However, some major issues, such as bioavailability, potency and selectivity of the first generation(s) of such inhibitors, must be improved for achieving better therapeutic profiles. This article presents an overview of the major important targets within the SP class, emphasising for each the recent advances in the design of effective SP inhibitors (SPIs) with potential therapeutic applications in a variety of medical fields.     

丝氨酸蛋白酶抑制剂的研究进展

目的对丝氨酸蛋白酶抑制剂的结构、功能等以及在黄粉虫中参与多种丝氨酸蛋白酶(serine protease,SP)级联的活性调控机制的研究进展进行综述。方法参考国外29篇文献,简述其结构、功能、与其相关的疾病及其研究的模式生物进行总结。结果丝氨酸蛋白酶抑制剂(serpin)是蛋白酶抑制因子中最大也是最重要的超家族,具有一定的特殊结构,serpin使用自杀底物原理与靶酶形成稳定的共价复合物,导致靶酶失活。该类型蛋白超家族成员遗传性结构或异常分泌将导致许多疾病的发生。在黄粉虫中丝氨酸蛋白酶抑制剂参与多种丝氨酸蛋白酶(serine protease,SP)级联的活性调控。结论目前对其生物学特性以及结构与功能关系的研究较多,对于临床应用及其模式生物的研究有待于更深入的研究。     

血清丝氨酸蛋白酶抑制剂、内脂素、趋化素在 2型糖尿病并发非酒精性脂肪性肝病中的表达及与胰岛素抵抗的关系

目的 探讨血清丝氨酸蛋白酶抑制剂(Vaspin)、内脂素(Visfatin)、趋化素(Chemerin)在2型糖尿病(T2DM)并发非酒精性脂肪性肝病(NAFLD)病人中的表达及其与胰岛素抵抗(IR)的关系.方法 选取2018年1月至2019年8月南阳市中心医院收治的T2DM,并发NAFLD病人76例作为观察组,未并发NAFLD病人62例作为对照组.比较两组血清Vaspin、Visfatin、Chemerin水平,受试者工作特征(ROC)曲线分析上述因子对T2DM并发NAFLD的诊断价值,并结合肝影像学诊断结果对比不同病情程度病人血清Vaspin、Visfatin、Chemerin水平、胰岛素参数[胰岛素抵抗指数(HOMA-IR)、胰岛素敏感指数(ISI)],分析各血清因子与胰岛素参数关联性及T2DM并发NAFLD病情程度的影响或关联作用因素.结果 观察组血清Vaspin[(82.03±12.46)ng/L比(70.81±11.22)ng/L]、Visfatin[(39.58±6.90)μg/mL比(33.27±5.73)μg/mL]、Chemerin[(53.37±8.61)ng/L比(45.39±8.02)ng/L]水平及HOMA-IR[(2.71±0.67)比(1.36±0.54)]高于对照组(P<0.05);Chemerin诊断T2DM并发NAFLD的AUC值最大,为0.758.随T2DM并发NAFLD病情加重血清Vaspin、Visfatin、Chemerin、HOMA-IR呈升高趋势,ISI呈降低趋势(P<0.05).血清Vaspin、Visfatin、Chemerin与HOMA-IR呈正相关,与ISI呈负相关(P<0.05).BMI、WHR、血清Vaspin、Visfatin、Chemerin、HOMA-IR、ISI是T2DM并发NAFLD病情程度的独立影响或关联作用因素(P<0.05).结论 T2DM并发NAFLD病人存在血清Vaspin、Visfatin、Chemerin异常表达情况,且表达水平与IR关系密切,可能通过诱导IR间接参与T2DM并发NAFLD的发病发展过程.     

Inhibition of serine proteases by steroids

Proteolysis of ¹⁴C-labeled globin, as well as the hydrolysis of the specific substrate benzoyl tyrosine ethyl ester, by purified bovine chymotrypsin was found to be inhibited by several steroid hormones. The inhibition of chymotrypsin by the steroids was of a competitive nature, with K_i values of 9.9 × 10^?5 M for triamcinolone (9-fluoro-11β, 16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione), 1.6 × 10^?4 M for cortisol (11β,17α,21-trihydroxypregn-4-ene-3,20-dione), 3.7 × 10^?4 M for testosterone (17β-hydroxy-4-androsten-3-one), 5.0 × 10^?4 M for dexamethasone (9-fluoro-11β,17,21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione), and 1.0 × 10^?4 M for epicortisol (11α,17,21-trihydroxy-4-pregnene-3,20-dione). The activity of purified bovine trypsin on its specific substrate, TAME (tosyl arginine methyl ester), also showed a similar pattern of inhibition by steroids. Both chymotrypsin and trypsin were found to bind ³H-labeled dexamethasone and cortisol. This binding was markedly inhibited by the general protease inhibitor, PMSF (phenylmethanesulfonyl fluoride), whereas the chymotrypsin-specific inhibitor, TPCK (l-[1-tosyl-amido-2-phenyl]ethylchloromethyl ketone), inhibited only the steroid binding to chymotrypsin but not to trypsin. These observations indicate that serine proteases recognize steroid hormones in a fashion similar to the recognition of their specific substrates and that the steroids inhibit activity of these enzymes at their binding sites.     

合成类丝氨酸蛋白酶抑制剂的研究进展

合成类丝氨酸蛋白酶抑制剂是人工合成的非肽类小分子化合物，具有广谱抑制性，无抗原性，相对分子质量小，可以自由进入血管及胰腺组织，能直接作用于胰腺细胞上，抑制胰酶（trypsin）、血纤维蛋白溶酶（plasmin）、血浆激肽释放酶（plasmakellilrein）、凝血酶（throbin）及补体酯酶C1和C1r。目前已获准上市的药物有加贝酯、奈莫司他、卡莫司他和司匹司他，临床主要用于急、慢性胰腺炎和弥散性血管内凝血的治疗。     

Targeting serine proteases in asthma

Leukocytes and lung structural cells contribute to the pathophysiology of asthma through the production of numerous mediators including serine proteases. Such proteases include mast cell tryptase and chymase; neutrophil elastase, cathepsin G and myeloblastin (proteinase 3); bronchial epithelial cell-derived transmembrane protease, serine 11D (human airway trypsin-like protease); cytotoxic T lymphocyte- and natural killer cell-derived granzyme B; and, eosinophil serine protease 1 (testisin). Considerable effort to develop potent and selective inhibitors, mostly non-peptidic, especially targeting tryptase and chymase have been made in the last few years. This review presents promising inhibitors, currently in the research and development pipeline. Some endogenous inhibitors and other compounds purified from non-human species are also discussed.     

丝氨酸对宁南霉素生物合成的影响

研究宁南霉素发酵过程中如何通过加入前体来提高宁南霉素的产量，进行了添加前体种类选择、前体加入量和加入时间对比实验。结果表明，丝氨酸是较佳的合成前体。添加间接前体物A和B，对宁南霉素生物合成也有明显促进作用。在发酵24、36和42h分别添加丝氨酸、前体物A和B，宁南霉素发酵效价为对照的143％、130%和11141％。     

Inhibition of serine proteases by anti-inflammatory triterpenoids

The lupane triterpenoid lupeol, the ursane triterpenoid alpha-amyrin and esters of these compounds are present in the bark of roots of Alstonia boonei (Apocynaceae) and have anti-inflammatory properties. alpha-Amyrin is a competitive inhibitor of bovine trypsin and chymotrypsin (Ki values 29 microM and 18 microM, respectively). Lupeol linoleate, lupeol palmitate and alpha-amyrin linoleate are non-competitive inhibitors of trypsin (Ki values 7 microM, 10 microM and 16 microM, respectively). alpha-Amyrin linoleate is also a non-competitive inhibitor of chymotrypsin (Ki value 28 microM). Lupeol is a competitive inhibitor of both trypsin and chymotrypsin (Ki values 22 and 8 microM, respectively). alpha-Amyrin palmitate is a potent non-competitive inhibitor of chymotrypsin (Ki 6 microM). Lupeol, alpha-amyrin and the palmitic and linoleic acid esters of these compounds are ineffective or very weak as inhibitors of porcine pancreatic elastase and of Lucilia cuprina and Helicoverpa punctigera leucine aminopeptidases. These hydrophobic triterpenoids represent further examples of anti-inflammatory triterpenoids that are PKA inhibitors as well as being selective protease inhibitors.     

Lanthionine macrocyclization by in situ activation of serine

The present report details a straightforward, solid-phase approach to cyclolanthionine peptides. After stepwise assembly of the linear sequence and transformation of a single exposed serine to bromoalanine using P(Ph)₃/CBr₄, the detritilation of a cysteine side-chain sets the stage for a base-promoted macrocyclization. The entire procedure can be carried out in a solid-phase vessel using conventional 9-fluorenylmethyloxycarbonyl/tert-butyl-based chemistry and is amenable to automated format. The utility of this novel procedure is demonstrated by the synthesis of two previously reported lanthionine-containing cyclic peptides.     

Role of serine esterases in mast cell activation

1. A variety of chymotryptic substrates and inhibitors prevented the release of histamine and prostaglandin D₂ from rat peritoneal mast cells stimulated with anti-IgE but not the calcium ionophore A23187 or a variety of polyamines.
2. The activity of the compounds was strikingly increased in cells reversibly permeabilized with ATP, indicating the importance of their effective incorporation into the cytosol.
3. The compounds produced a comparable inhibition of immunological, but not pharmacological, histamine release from human mast cells and basophils.
4. Treatment of rat mast cells with anti-IgE led to a marked increase in the total chymotryptic activity expressed by the cells.
5. Immunological, but not pharmacological, stimulation of permeabilized rat mast cells loaded with a fluorescent chymotryptic substrate led to a pronounced and rapid increase in fluorescence, indicating activation of the enzyme and hydrolysis of the substrate. These changes were attenuated by chymotryptic inhibitors.
6. In total, these data provide compelling evidence for the direct involvement of a serine protease in IgE-mediated histamine release from mast cells.

     

Inner blood-retinal barrier mediates l-isomer-predominant transport of serine

D-serine, a coagonist for N-methyl-D-aspartate-type glutamate receptors, which mediate visual signal transmission, is thought to be generated from L-serine via serine racemase in the retina. However, the source of L-serine and D-serine in the retina are yet to be determined. The purpose of the present study was to investigate the characteristics of the blood-to-retina transport of serine at the inner blood-retinal barrier (BRB). In vivo study revealed the blood-to-retina transport of [(3) H]L-serine with an influx clearance of 49.9 μL/(min·g retina), which is greater than that of [(3) H]D-serine. This was consistent with the L-isomer-predominant uptake of serine by conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), an in vitro inner BRB model. [(3) H]L-Serine and [(3) H]D-serine uptake by TR-iBRB2 cells took place in an Na(+)-dependent and a concentration-dependent manner with Michaelis constant values of 97.5 μM and 9.63 mM, respectively. The uptake process of [(3) H]L-serine and [(3) H]D-serine was significantly inhibited by system ASC (alanine-serine-cysteine) substrates. Polymerase chain reaction analysis and immunocytochemistry revealed the expression of ASC transporters ASCT1 and ASCT2 in TR-iBRB2 cells. These results suggest that the system ASC at the inner BRB is a potent pathway for supplying serine in the form of the L-isomer from the circulating blood to the retina.     

Rat liver kininase,a serine peptidase

A serine peptidase (RLK₁) was partially purified from rat liver homogenates. Its molecular weight was 80,000, and its optimum pH was 7.5. Bz-Tyr-O-Et was hydrolyzed by the enzyme, which was inhibited by Ip₂PF, PMSF and by Tos-Phe-CH₂Cl. The bonds cleaved by the enzyme were Phe⁵-Ser⁶ and Phe⁸-Arg⁹, when bradykinin was used as substrate.     

Azaphenylalanine-based serine protease inhibitors induce caspase-mediated apoptosis

Molecules regulating cell death constitute prominent therapeutic targets. The pro-apoptotic role of serine protease inhibitors prompted us to search for novel modulators of this process. We have tested some recently synthesized antithrombotic compounds for their potential to induce apoptotic cell death. Cell based analyses revealed that inhibitors built on the azaphenylalanine scaffold are, for B-cell lymphoma cells, severely cytotoxic, while other compounds tested were moderate or non-cytotoxic. These inhibitors induced the time and concentration dependent biochemical and morphological characteristics of apoptosis, such as DEVDase activation, loss of mitochondrial membrane potential, nuclear degradation and genomic DNA fragmentation. Most of the inhibitors proved to be selective for thrombin, with inhibition constants (K_i) in the nanomolar range. However, they could also inhibit at least one additional serine protease (trypsin, chymotrypsin and/or coagulation factor X) with K_i values in the nanomolar or low micromolar range. These serine protease inhibitors constitute novel apoptosis inducing compounds in B-cell lymphoma cells.     

Human insulin genome sequence map,biochemical structure of insulin for recombinant DNA insulin

Insulin is a essential molecule for type I diabetes that is marketed by very few companies. It is the first molecule, which was made by recombinant technology; but the commercialization process is very difficult. Knowledge about biochemical structure of insulin and human insulin genome sequence map is pivotal to large scale manufacturing of recombinant DNA Insulin. This paper reviews human insulin genome sequence map, the amino acid sequence of porcine insulin, crystal structure of porcine insulin, insulin monomer, aggregation surfaces of insulin, conformational variation in the insulin monomer, insulin X-ray structures for recombinant DNA technology in the synthesis of human insulin in Escherichia coli.