Molecular Cloning of cDNA That Encodes Chymotrypsin Inhibitor ECI from Erythrina variegata Seeds and Its Expression in Escherichia coli

Synthetic oligonucleotides representing all possible sequences of the N-terminal and internal amino acid sequences of the chymotrypsin inhibitor ECI from Erythrina variegata seeds were used to generate a probe specific for ECI-related sequences by the polymerase chain reaction on the E. variegata genomic DNA. A lambda phage cDNA library constructed from poly(A⁺) RNA from maturing seeds was screened with the ECI gene thus obtained as a probe and characterized by DNA sequencing. The cloned ECI cDNA comprised 737 nucleotides and one open reading frame that encoded a polypeptide chain of 203 amino acids including a signal peptide composed of 24 amino acids. An expression plasmid was designed for export of the recombinant inhibitor into the periplasm. For this purpose, the cDNA fragment encoding matured ECI was ligated into the NcoI and BamHI sites following the pel B signal sequence in the expression vector pET-22b and expressed in Escherichia coli BL21 (DE3). However, this attempt failed as the recombinant inhibitor caused the formation of inclusion bodies in E. coli cells as a heterologous preprotein (SR-ECI), with the pel B upstream leader. SR-ECI was made soluble and renatured by refolding and reoxidation, and subsequently processed with pronase to give rise to recombinant ECI (R-ECI) that had an extra methionine residue attached to the N-terminal amino acid of ECI. Purified R-ECI inhibited chymotrypsin almost as strongly as authentic ECI.     

丝氨酸蛋白酶抑制剂的研究及应用

丝氨酸蛋白酶抑制剂（serpin）是一类结构、序列同源的蛋白酶抑制剂,它是体内许多蛋白水解级联反应的调节因子,其遗传性结构或分泌异常将导致许多疾病．因此对于其结构及作用机理的研究将为临床应用提供依据．     

Kazal型蛋白酶抑制剂结构与功能研究进展

蛋白酶抑制剂广泛存在于生物体内,在许多生命活动过程中发挥必不可少的作用,特别是对蛋白酶活性进行精确调控。其中Kazal型蛋白酶抑制剂是最重要的、研究最为广泛的酶抑制剂之一,该类抑制剂一般由一个或几个结构域组成,每一个结构域具有保守的序列和分子构象,同时发现该类抑制剂与蛋白酶作用的结合部位高度易变,它们大多数暴露于与溶剂接触的环上,其中P1部位是抑制作用的关键部位,抑制剂的专一性由P1部位氨基酸残基的性质决定,其它残基取代结合部位残基对抑制剂-酶的结合常数有显著的影响。Laskowski算法可直接从Kazal型丝氨酸蛋白酶抑制剂的序列推测其与6种丝氨酸蛋白酶之间的抑制常数(Ki)。目前在生物体内发现大量的Kazal型蛋白酶抑制剂,并证实其有重要的生物学功能。     

Binding of the Recombinant Proteinase Inhibitor Eglin c,of the Soybean Bowman-Birk Proteinase Inhibitor and of its Chymotrypsin and Trypsin Inhibiting Fragments to Leu-Proteinase,the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves: Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respetively) to Leuproteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of K_a (at 21°C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from ～6.9, in the free Leu-proteinase, to ～5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c - K_a = 2.2 × 10¹¹ M^-1, δG°= - 64kJ/mol, δH° = + 5.9kJ/mol, and δS° = + 240J/molK; Leu-proteinase:BBI - K_a = 3.2 × 10¹⁰ M^-1, δG° = - 59kJ/mol, δH°= + 8.8kJ/mol, and δS° = + 230J/molK; and Leu-proteinase:F-C - K_a = 1.1 × 10⁶ M^-1, δG°= - 34kJ/mol, δH° = + 18J/mol, and δS° = + 180J/molK (values of K_a, δG° and δS° were obtained at 21.0°C; values of δH° were temperature-independent over the range explored, i.e. between 10.0°C and 40.0°C). F-T does not inhibit Leu-proteinase up to an inhibitor concentration of 1.0 × 10^-3 M, suggesting that the upper limit of K_a is 1 × 10² M^-1. Considering the known molecular models, the observed binding behaviour of eglin c, BBI, F-C and F-T to Leu-proteinase has been related to the inferred stereochemistry of the enzyme/inhibitor contact region     

Three new constituents from the roots of Erythrina variegata and their antibacterial activity against methicillin-resistant Staphylococcus aureus

Two new isoflavonoids, eryvarins V and W (1 and 2, resp.), and a new chromen-4-one derivative, eryvarin X (3), along with three known isoflavonoids, 4-6, were isolated from the roots of Erythrina variegata. Their structures were established by spectroscopic analyses. Compound 1 is a rare naturally occurring isoflavanone which possesses a OH group at C(3). Among the new compounds 1-3, 2 exhibited a potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains.     

Comparison of the Effect of Calpain Inhibitors on Two Extralysosomal Proteinases: The Multicatalytic Proteinase Complex and m-Calpain

Abstract: The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of β-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC₅₀ values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.     

A Computer Modeling Study of the Interaction Between Tissue Factor Pathway Inhibitor and Blood Coagulation Factor Xa

Activation of blood coagulation factor X to factor Xa (FXa) is inhibited by tissue factor pathway inhibitor (TFPI). The second Kunitz-type inhibitory domain (K2) of TFPI binds a catalytic domain of FXa, whereas the first domain (K1) does not. We analyzed computer models of complexes of FXa with K1 or K2, which were made using a crystal structure of FXa. Favorable hydrophobic interaction was observed in the complex of FXa with K2. Furthermore, we constructed a tertiary structure of FXa using CHIMERA to assess the accuracy of a homology modeling method. The isolated model structure of FXa agreed well with the crystal structure, but analyses of complexes of this structure with K1 or K2 revealed that the models of complexes could not provide clear evidence of greater binding ability to K2 because of the positional difference of a few side chains interacting with the inhibitor.     

Serine Proteinase Inhibitors from Eggs and Larvae of Tick Boophilus microplus: Purification and Biochemical Characterization

The present study describes the purification, characterization, and comparison of serine proteinase inhibitors during the development of egg and larva phases of the tick Boophilus microplus. Samples were collected of eggs between the first day of hatching and the beginning of eclosion (defined as E1, E2, and E3) and of larvae between the first day of eclosion and the infectant phase (defined as L1, L2, and L3). Crude extracts of the samples (2.5% w/v in Tris-HCl buffer) were analyzed by SDS-PAGE, and showed three major protein bands of 42, 62, and 85 kDa, differing in intensity, from E1 to L3 samples. The total protein of the larva extracts was 34% less than that of the egg extracts, while no differences in active protein were detected. The apparent dissociation constant K _i determined for trypsin was 10-fold lower from E1 to L3 samples. Serine proteinase inhibitors from tick eggs and larvae (BmTIs) were purified on trypsin-Sepharose column and analyzed by SDS-PAGE. The results showed a slight difference in protein pattern, with a protein band of 20 kDa in the E1 and E2 samples which did not appear in the other samples. The K _i for neutrophil elastase was 10-fold lower in L3 than E1. BmTI reverse-phase chromatography showed two and one major peaks in egg and larva samples, respectively. The N-terminal amino acid sequence of the L3 main peak from a C8 column showed a mix of BmTIs with the major sequence AVDFDKGCVPTADPGPCKG. Changes indicated by molecular weight and inhibition activity suggest different roles for BmTIs during the development process.     

Eryvarins F and G,two 3-phenoxychromones from the roots of Erythrina variegata

Two 3-phenoxychromones, eryvarins F and G, were isolated from the roots of Erythrina variegata. Their structures were established to be 3-(2,4-dihydroxyphenoxy)-7-hydroxy-6,8-di(3,3-dimethylallyl)chromen-4-one and 3-(2,4-dihydroxyphenoxy)-8-(3,3-dimethylallyl)-2,2-dimethylpyrano[5,6:6,7]chromen-4-one on the basis of spectroscopic and chemical evidence. Eryvarins F and G are unusual 3-phenoxychromone derivatives with two isoprenoid groups.     

Heterogeneity of Proteinase Inhibitors in the Water-Soluble Organic Matrix from the Oyster Nacre

We extracted proteinase inhibitors from the nacre of the oyster Pinctada margaritifera with water. Mixing the nacre powder with water for 20 h led to a water-soluble fraction [0.24% (wt/wt) of nacre]. After dialysis of the water-soluble matrix through 6- to 8-kDa and 0.5-kDa membranes, the proteinase inhibitors were divided into low and high molecular weight fractions that contained inhibitors of papain, bovine cathepsin B, and human cathepsin L. We studied the heterogeneity of the inhibitors after separating the low molecular weight fraction according to charge and hydrophobicity. After multistep purification, mass spectrometry analysis revealed that a potent inhibitory fraction contained several molecules. This observation demonstrates the difficulties encountered in attempting to isolate individual metabolites from the complex mixture of molecules present in nacre matrix. Interestingly, the low molecular weight fraction contained specific inhibitors that could discern between cathepsin B and cathepsin L. The nacre organic inhibitors were active against several cysteine proteinases, yet they were more specific in relation to serine proteinases, because only proteinase K was inhibited. These results demonstrate, for the first time, the presence of active proteinase inhibitors in the mollusc shell, and it is possible that these inhibitors may play a role in either protection of proteins involved in shell formation or in defense against parasites, or both.     

Cloning and Characterization of the cDNA Encoding the Masquerade-like Serine Proteinase Homologue Gene of the Silkworm, Bombyx mori

ABSTRACT From Bombyx mori larvae, RT-PCR and cDNA library screening isolated masquerade-like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined. The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio's 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disulfide bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis.     

Effects of Proteinase Inhibitor Ingestion on Survival,Learning Abilities and Digestive Proteinases of the Honeybee

The impact on beneficial insects of proteinase inhibitors expressed in pest-resistant transgenic crops needs to be assessed before the release of these plants into the environment. Three proteinase inhibitors, suitable for incorporation into oilseed rape, were tested on worker bees: the chicken egg white cystatin, oryzacystatin I (OCI) and Bowman-Birk soyabean inhibitor (BBI). Ingestion of low doses of the inhibitors did not cause short-term mortality, and a conditioned proboscis extension assay showed that olfactory learning performances were unchanged when the inhibitors were added to the reward. Long-term ingestion of BBI or OCI did not disrupt total digestive proteolytic activity, but ingestion of BBI induced a new proteinase form, suggesting the existence of a mechanism of control of proteinase synthesis in the honeybee.     

植物蛋白酶抑制剂在植物抗虫与抗病中的作用

综述了植物蛋白酶抑制剂抗虫与抗病作用的研究进展.蛋白酶抑制剂广泛存在于植物体内,与植物抗虫抗病密切相关.植物蛋白酶抑制剂能抑制昆虫肠道蛋白酶,使昆虫生长发育缓慢,甚至死亡.但取食蛋白酶抑制剂后,昆虫能迅速分泌对抑制剂不敏感的蛋白酶,而使蛋白酶抑制剂无效.食物蛋白的含量和质量也影响植物蛋白酶抑制剂的抗虫效果.病原菌的感染能诱导植物产生蛋白酶抑制剂,诱导产生的蛋白酶抑制剂能抑制病原菌的生长.     

The methyl group of N(alpha)(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-N(alpha)(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K(i) values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (K(i)=37pM), containing an N(alpha)(Me)Arg-Thr bond, was crystallized in complex with human alpha-thrombin. X-ray data were collected to 1.8A resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N(alpha)(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O(gamma) atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O(gamma) are separated from each other, impairing the nucleophilic attack of the Ser195 O(gamma) toward the N(alpha)(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.     

血栓弹力图指导食管癌患者临床输血的价值及其与常规凝血实验检测指标的相关性分析

目的:探讨血栓弹力图(TEG)指导食管癌患者临床输血的价值及其与常规凝血实验检测指标的相关性。方法:选取2017年1月-2019年3月在我院收治的食管癌手术治疗需输血的99例患者作为研究对象,将99例患者随机分为常规凝血功能检测组和TEG组,常规凝血功能检测组采用常规凝血实验检查结果指导输血,TEG组采用TEG检查结果指导输血,对比两组输血前后的常规凝血实验检测指标以及临床用血量,对比TEG组输血前后的TEG指标,分析TEG指标与常规凝血实验检测指标的相关性。结果:两组患者输血前凝血四项和血小板计数(PLT)差异无统计学意义(P>0.05),输血后两组活化凝血酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)、纤维蛋白原(FIB)均有不同程度的改善(P<0.05),TEG组PT、TT较常规凝血功能检测组低(P<0.05);输血后,TEG组患者凝血反应时间(R值)、血凝块形成时间(K值)较输血前降低,最大血凝块强度(MA值)、凝血综合指数(CI值)升高,凝血形成速率(Angle角)增大,差异有统计学意义(P<0.05);Pearson相关性分析结果显示,R值与APTT呈正相关(P<0.05),K值与PLT呈负相关,与FIB呈正相关(P<0.05),Angle角、MA值、CI值与FIB、PLT呈正相关(P<0.05);TEG组新鲜冰冻血浆、冷沉淀输注量少于常规凝血功能检测组,差异有统计学意义(P<0.05)。结论:TEG能更好地指导食管癌手术患者各种血液成分的合理输注,有效改善凝血异常情况,减少输血用量,TEG指标与常规凝血实验检测指标存在一定的相关性。     

玉蜀黍轴及苞叶对凝血作用的影响

采用体外血浆复钙时间法,以维生素k1作为促凝血和灯盏花素作为抗凝血作用阳性对照,对玉蜀黍轴及苞叶石油醚、乙酸乙酯和正丁醇提取物及玉蜀黍苞叶甲醇总浸膏对体外血浆复钙时间的影响进行测定。结果显示玉蜀黍轴正丁醇及苞叶乙酸乙酯提取物,均可显著缩短体外血浆复钙时间(P<0.001);玉蜀黍苞叶石油醚提取物可显著延长体外血浆复钙时间(P<0.001)。提示玉蜀黍轴正丁醇及苞叶乙酸乙酯提取物具有较好的促凝血活性,玉蜀黍苞叶石油醚提取物具有较好的抗凝作用。     

不同化疗方案对非小细胞肺癌患者凝血纤溶功能及血小板参数的影响及其临床意义

目的:探讨不同化疗方案对非小细胞肺癌患者凝血纤溶功能及血小板参数的影响及临床意义。方法:回顾性分析2013年2月至2016年2月于我院进行治疗的96非小细胞肺癌患者的临床资料,按照化疗方案的不同分为TP组(50例)及GP组(46例),TP组患者给予多西他赛联合卡铂方案(TP方案)进行化疗,GP组患者给予吉西他滨联合卡铂方案(GP方案)进行化疗,另选择45例健康体检者作为对照组。比较三组治疗前者血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、血浆凝血酶时间(TT)、血浆纤维蛋白原含量(FIB)以及D-二聚体(D-dimer)、血小板数(PLT)、血小板比积(PCT)、血小板平均体积(MPV)、血小板体积分布宽度(PDW)以及大体积血小板比率(P-LCR)及TP组和GP组治疗后以上指标的差异。结果:TP组和GP组患者化疗前PT、APTT、TT、FIB、D-dimer、PLT、PCT、MPV、PDW以及P-LCR水平均明显高于对照组(P0.05),而TP组与GP组以上指标比较无显著差异(P0.05)。治疗后,TP组和GP组患者PT、APTT、TT、FIB、D-dimer、PLT、PCT、MPV、PDW以及P-LCR水平均较治疗前有所下降,且GP组TT明显长于TP组(P0.05),PLT、PCT明显低于TP组(P0.05)。结论:凝血纤溶功能及血小板参数对于评价不同化疗方案治疗非小细胞肺癌患者预后具有积极的临床价值,不同化疗方案选择对于患者凝血系统均具有一定的影响。     

水稻半胱氨酸蛋白酶抑制剂基因的克隆及其mRNA在水稻中的组成型表达

从40年代发现豆科植物中存在蛋白酶蛋白抑制剂以来,在动物、植物和微生物体内已发现普遍存在着多种类型的蛋白酶抑制剂(PI)。人们往往是为了研究某种蛋白酶的作用机制或出于某种应用目的去分离和研究PI的,对PI的真正生理功能尚不十分清楚。一般认为除防止体内不必要的蛋白降解作用、调节蛋白代谢及调节各种蛋白酶的生理活性外,很多植物的PI还具有抑制某些病源微生物及某些昆虫体内蛋白酶的作用,从而对植物有防卫功能。Hilder等和Johnson等已分别将属于丝氨酸蛋白酶抑制剂的豇豆蛋白酶抑制剂及马铃薯PⅠⅠ和PⅠⅡ基因转入烟草,结果转基因烟草对烟芽夜蛾(He-     

Circadian Variations in Coagulation and Fibrinolytic Factors among Four Different Strains of Mice

This study examined circadian variation in coagulation and fibrinolytic parameters among Jcl:ICR, C3H/HeN, BALB/cA, and C57BL/6J strains of mice. Plasma plasminogen activator inhibitor 1 (PAI‐1) levels fluctuated in a circadian manner and peaked in accordance with the mRNA levels at the start of the active phase in all strains. Fibrinogen mRNA levels peaked at the start of rest periods in all strains, although plasma fibrinogen levels remained constant. Strain differences in plasma antithrombin (AT) activity and protein C (PC) levels were then identified. Plasma AT activity was circadian rhythmic only in Jcl:ICR, but not in other strains, although the mRNA levels remained constant in all strains. Levels of plasma PC and its mRNA fluctuated in a circadian manner only in Jcl:ICR mice, whereas those of plasma prothrombin, factor X, factor VII, prothrombin time (PT), and activated partial thrombin time (APTT) remained constant in all strains. These results suggest that genetic heterogeneity underlies phenotypic variations in the circadian rhythmicity of blood coagulation and fibrinolysis. The circadian onset of thrombotic events might be due in part to the rhythmic gene expression of coagulation and fibrinolytic factors. The present study provides fundamental information about mouse strains that will help to understand the circadian variation in blood coagulation and fibrinolysis.