衍生自人ⅡA型磷脂酶A_2碳末端的多肽C-26杀菌作用研究

目的:考察衍生自人ⅡA型磷脂酶A(2ⅡA型PLA2)碳(C)末端的多肽C-26对不同细菌的体外杀菌作用。方法:根据人ⅡA型PLA2C末端26个氨基酸残基的顺序,合成多肽C-26。采用琼脂铺板计数法,将不同浓度的多肽C-26分别与6种细菌(金黄色葡萄球菌、炭疽杆菌、枯草杆菌、大肠杆菌、变形杆菌和绿脓杆菌)在37℃孵育2h,然后铺板并置于37℃恒温箱培养18～24h,记录每一琼脂板上的菌落形成单位(cfu),计算出多肽C-26作用后对细菌的杀菌率。结果:多肽C-26对金黄色葡萄球菌、枯草杆菌、炭疽杆菌等革兰阳性(G+)细菌的杀菌作用较强,其杀灭99%细菌的浓度在250～1000mg·L-1之间;对大肠杆菌、变形杆菌、绿脓杆菌等革兰阴性(G-)细菌的杀菌作用较弱,其浓度在500mg·L-1时对三者的杀菌率为41%～52%。结论:多肽C-26有杀菌作用,推测其具有与ⅡA型PLA2及其他抗菌多肽相似的杀菌机制。     

人Ⅰ型磷脂酶A_2的N末端衍生多肽PLA_2N_(1-15)的杀菌活性研究

目的:观察人Ⅰ型磷脂酶A2(PLA2)的N末端衍生多肽PLA2N1-15对不同细菌的体外杀菌活性。方法:以人Ⅰ型PLA2N末端的15个氨基酸残基为模板,合成多肽PLA2N1-15。采用琼脂铺板计数法,记录并计算不同质量浓度(1 000、250、62.5、15.625μg/ml)的PLA2N1-15对革兰阳性(G+)菌(金黄色葡萄球菌、粪肠球菌、枯草芽孢杆菌)和革兰阴性(G-)菌(大肠埃希菌、铜绿假单胞菌、变形杆菌)的杀菌率。结果:15.625、62.5、250、1 000μg/ml的PLA2N1-15对G+菌的杀菌率分别为10%¹9%、36.55%19%、36.55%⁸9.73%、83%89.73%、83%¹00%、96%100%、96%¹00%,对G-菌的杀菌率分别为8.86%100%,对G-菌的杀菌率分别为8.86%²2.63%、21.61%22.63%、21.61%⁴0.55%、40%40.55%、40%⁸3%、87%83%、87%⁹3%。结论:高浓度PLA2N1-15对G+和G-均具有较强的杀菌活性,特别是对G+。     

胸腺五肽及其衍生肽的杀菌活性研究

目的:研究胸腺五肽及其衍生肽的杀菌活性。方法:采用琼脂铺板计数法测定胸腺五肽[精氨酸(R)-赖氨酸(K)-天冬氨酸(D)-缬氨酸(V)-酪氨酸(Y),RKDVY]及其衍生肽1[RKN(天冬酰胺,N)VY]、衍生肽2(RKKVY)对革兰阴性(G-)菌(变形杆菌、大肠埃希菌)和革兰阳性(G+)菌(金黄色葡萄球菌、粪肠球菌)的杀菌活性,其中五肽剂量为15.625~1 000μg/ml,细菌数为102CFU。结果:3种五肽对G-菌均有杀菌活性,且RKKVY、RKNVY杀菌活性强于RKDVY(P<0.01),RKKVY与RKNVY间差异无统计学意义(P>0.05);对G+菌亦有杀菌活性,其杀菌活性由强到弱依次为RKKVY>RKNVY>RKDVY(P<0.01)。结论:胸腺五肽及其衍生肽对G-菌和G+菌均有杀菌活性,具有量效关系。     

HPLC-ESI-ITMS在药品质量控制中确证胰岛素和胰岛素B链C端氨基酸序列的应用研究

在药品质量控制中应用HPLC-ESI-IT (Ion Trap) MS分别确证胰岛素和胰岛素B链的C端氨基酸序列。取胰岛素标准品溶液或者胰岛素B链溶液适量，加入羧肽酶P及羧肽酶Y溶液适量降解胰岛素或者胰岛素B链C端，在预定的时间点取出酶降解反应液适量，加入等体积1%甲酸停止反应，混旋均匀后制成供试液，供HPLC-ESI-IT MS分析测定。采用Zorbax Prosphere C₁₈柱(2.1 mm×150 mm，300A，5 μm)分离，以流动相进行梯度洗脱［A相：水-乙腈-三氟乙酸(98∶2∶0.02)，B相：乙腈-水-三氟乙酸(98∶2∶0.02)］。柱后修饰“TFAfix”溶液为丙酸-异丙醇(2∶8)的混合溶液。ESI-ITMS测定供试液中梯度多肽系列(彼此相差1个C端氨基酸残基)的分子质量，计算出分子质量最相近的相邻两肽段的系列分子质量差值，即可得到待测样品C端系列氨基酸残基质量的实验值。胰岛素的B链C端的第1个氨基酸残基丙氨酸残基可以被准确地确证；同样还原修饰的胰岛素B链C端的第1个氨基酸残基丙氨酸残基也能被准确地确证。本研究在nmol样品水平准确地确证胰岛素和胰岛素B链各一个C端氨基酸，符合重组DNA制品中试产品的质量控制要求。本实验方法不必将胰岛素经还原修饰将A链从B链裂解，直接确证胰岛素的C端氨基酸残基，避免还原修饰和纯化的烦琐操作，方法简便。     

天然环肽auyuittuqamide A的全合成研究

目的用Fmoc固相直链合成和液相环合的方法合成天然环肽auyuittuqamide A。方法以2–氯三苯甲基氯(CTC)树脂为固相载体,1,3–二异丙基碳二亚胺(DIC)和1–羟基苯并三氮唑(HOBT)为缩合剂,9–芴基甲氧基羰基(Fmoc)保护的氨基酸,按照序列依次缩合,以三氟乙醇(TFE)作为切割试剂,获得全保护直链肽。以六氟磷酸苯并三唑–1–基–氧基三吡咯烷基磷(PyBOP)和1–羟基苯并三氮唑(HOBT)为环合试剂,全保护直链肽在二氯甲烷(DCM)溶液中环合,以三氟乙酸(TFA)为脱保护试剂,获得天然环肽auyuittuqamide A。用高效液相制备色谱进行纯化,采用HR-Q-TOF-MS,500MHz ¹HNMR进行表征分析。结果获得纯度大于95%的天然环肽auyuittuqamide A,总收率5.48%。结论此法合成步骤简单,产率较高,首次建立天然环肽auyuittuqamide A的全合成方法,为auyuittuqamide A的进一步研究奠定基础。     

重组人ⅡA型磷脂酶A2对金黄色葡萄球菌超微结构的影响

摘 要 目的：观察重组人ⅡA型磷脂酶A2（PLA2）对金黄色葡萄球菌超微结构的影响。方法: 用琼脂平板计数法测定重组人ⅡA型PLA₂对金黄色葡萄球菌的杀菌活性,ⅡA型PLA₂与金黄色葡萄球菌共同孵育在RPMI-1640培养液中,37℃水浴,分别孵育30,60,120 min后,应用透射电镜观察细胞超微结构的变化。结果:重组人ⅡA型PLA₂杀灭99%金黄色葡萄球菌的最低浓度（MBC）为80 ng·ml^-1。ⅡA型PLA₂作用30～60 min后,金黄色葡萄球菌的菌体略显肿胀,细胞壁与细胞膜因局部分离而出现空隙,细胞膜、细胞壁在横隔结构处裂开。当ⅡA型PLA₂作用120 min后,可见细菌的细胞壁破坏严重,胞质外流。结论:ⅡA型PLA₂对金黄色葡萄球菌有较强的抗菌作用,并可能通过破坏细菌的细胞壁或细胞膜结构来抑制细菌的生长。     

变异链球菌UA140产生的细菌素(mutacin和mutacin)

细菌素是由细菌核糖体合成的肽类抗生素。根据翻译后的修饰作用情况 ,将革兰氏阳性菌细菌素分为2类 : 类 ,修饰的细菌素 ,即 lanbiotic类 ; 类 ,未修饰的细菌素 ,即非 lanbiotic类。lanbiotic类为含羊毛硫氨酸的小肽抗生素 ,通过翻译后的修饰作用形成脱水氨基酸残基和硫脂桥。非 lanbiotic类细菌素可以分为 2类 :单肽细菌素和双肽细菌素。非 lanbiotic类细菌素的生物合成涉及一个前肽的合成 ,该前肽由一个在加工位点具有 2个甘氨酸的先导肽和成熟肽部分组成 ,在成熟过程中 ,通过 1个专门的蛋白酶在 2个甘氨酸后的区域切下先导肽 ,释放出成熟…     

蛇毒解离素的结构特征及其与生物学活性的关系

目前已从蛇毒中分离出 80余种解离素多肽,根据其结构及来源可分为五大类,即短链、中链、长链解离素、金属蛋白酶P Ⅲ的类解离素区释放的解离素以及二聚体解离素。不同解离素的分子结构共同点包括RGD活性中心、半胱氨酸残基配对形成的二硫键、RGD三肽的C 端和N 端的氨基酸序列以及解离素多肽分子的C 末端。这些结构决定了解离素的抑制血小板聚集、抑制细胞粘附以及血管形成等生物学活性。     

新型特异性磷脂酶A2抑制剂对急性胰腺炎肺损伤的疗效研究

目的探讨一种新型磷脂酶A2（PLA2）抑制剂对重症急性胰腺炎（SAP）并发急性肺损伤（ALI）的保护作用。方法24只Wistar大鼠随机分为3组：假手术组（SO组）、SAP模型组（SAP组）、抑制剂预处理组（抑制剂组）。采用5％牛磺胆酸钠逆行胰胆管注射的方法制备SAP模型,抑制剂组在术前30min经股静脉注射特异性PLA2抑制剂（5mg／kg）。3组于手术后12h处死大鼠并收集标本,测定3组血清淀粉酶、肺干湿比、大鼠血清和肺组织sPLA2活性,聚合酶链反应（PCR）法检测肺组织Ⅱ型分泌型PLA2抑制剂（sPLA2-ⅡA）mRNA的表达,光镜下观察胰腺和肺脏的病理变化并计算病理评分。结果SAP组血清淀粉酶、肺干湿比、血清和肺组织sPLA2-ⅡA、肺组织sPLA2-ⅡA mRNA表达、胰腺和肺组织病理评分明显高于其他2组（P〈0．01）;抑制剂组上述指标均低于SAP组（P〈0．01）,但仍高于SO组（P〈0．01）。结论该新型特异性PLA2抑制剂通过降低血清和肺组织PLA2的活性,对大鼠SAP并发肺损伤有一定的保护作用。     

磷脂酶A2对大鼠Ⅱ型肺泡上皮细胞NF-κB表达的影响

目的：研究磷脂酶A2（PLA2）及含有PLA2的急性出血坏死性胰腺炎（AHNP）大鼠血清对原代大鼠Ⅱ型肺泡上皮细胞的损伤作用及对NF-κB表达的影响.方法：对大鼠Ⅱ型肺泡上皮细胞进行分离纯化和原代培养,分别加入PLA2及含有PLA2的AHNP大鼠血清,并设PLA2抑制剂对照组,各组均孵育4h,离心收集细胞,破碎细胞核提取核蛋白,ELISA法测定NF-κB活性;测定培养上清液中乳酸脱氢酶（LDH）活性,对培养细胞进行透射电镜检查.结果：PLA2及AHNP大鼠血清可使Ⅱ型肺泡上皮细胞培养上清液中LDH活性及Ⅱ型肺泡上皮细胞NF-κB活性显著增高,透射电镜检查显示Ⅱ型肺泡上皮细胞受损严重.盐酸阿的平可显著抑制PLA2对Ⅱ型肺泡上皮细胞的损伤作用,降低LDH活性,抑制NF-κB的表达.结论：PLA2对Ⅱ型肺泡上皮细胞的损伤作用是造成胰腺炎肺损伤的重要因素,还与其激活NF-κB的表达有关.     

Antimicrobial activity of myotoxic phospholipases A2 from crotalid snake venoms and synthetic peptide variants derived from their C-terminal region.

A short peptide derived from the C-terminal region of Bothrops asper myotoxin II, a Lys49 phospholipase A(2) (PLA(2)), was previously found to reproduce the bactericidal activity of its parent molecule. In this study, a panel of eight PLA(2) myotoxins purified from crotalid snake venoms, including both Lys49 and Asp49-type isoforms, were all found to express bactericidal activity, indicating that this may be a common action of the group IIA PLA(2) protein family. A series of 10 synthetic peptide variants, based on the original C-terminal sequence 115-129 of myotoxin II and its triple Tyr-->Trp substituted peptide p115-W3, were characterized. In vitro assays for bactericidal, cytolytic and anti-endotoxic activities of these peptides suggest a general correlation between the number of tryptophan substitutions introduced and microbicidal potency, both against Gram-negative (Salmonella typhimurium) and Gram-positive (Staphylococcus aureus) bacteria. Peptide variants with high bactericidal activity also tended to be more cytolytic towards skeletal muscle C2C12 myoblasts, thus limiting their potential in vivo use. However, the peptide variant pEM-2 (KKWRWWLKALAKK) showed reduced toxicity towards muscle cells, while retaining high bactericidal potency. This peptide also showed the highest endotoxin-neutralizing activity in vitro, and was shown to functionally interact with lipopolysaccharide (LPS) using a chimeric bacteria model. The bactericidal and anti-endotoxic properties of pEM-2, combined with its relatively low toxicity towards eukaryotic cells, highlight it as a promising candidate for further evaluation of its antimicrobial potential in vivo.     

ISOLATION AND CHARACTERIZATION OF THE CYANOGEN BROMIDE FRAGMENTS OF LOBSTER ARGININE KINASE (HOMARUS VULGARIS)

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10%) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to different gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position; therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).     

Amino acid sequence of a phospholipase A2 from the venom of Trimeresurus gramineus (green habu snake)

Two phospholipases A2, named phospholipases A2-I and A2-II, were purified to homogeneity from the venom of Trimeresurus gramineus (green habu snake). The complete amino acid sequence of phospholipase A2-I was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I, clostripain, and chymotrypsin) and chemical (hydroxylamine) cleavages of the S-pyridylethylated derivative of the protein. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid snakes which belong to the category of Group II. A most striking feature of this protein is that tyrosine at the 28th position which is common in phospholipases A2 and is assumed to be a part of the Ca2(+)-binding loop is replaced by phenylalanine. Such replacement is the first finding in Group II phospholipases A2. Secondary structure compositions of phospholipase A2-I are similar to those of Crotalus atrox phospholipase A2. No appreciable Ca2(+)-induced difference spectrum was observed, due probably to the absence of the effective chromophoric groups in the neighborhood of the Ca2+ binding site although Ca2+ is bound with affinity similar to that for T. flavoviridis phospholipase A2.     

Chemical Pathways of Peptide Degradation. III. Effect of Primary Sequence on the Pathways of Deamidation of Asparaginyl Residues in Hexapeptides

Deamidation of Asn residues can occur either by direct hydrolysis of the Asn residue or via a cyclic imide intermediate. The effects of primary sequence on the pathways of deamidation of Asn residues were studied using Val-Tyr-X-Asn-Y-Ala hexapeptides with substitution on the C-terminal side (Y) and on the N-terminal side (X) of the Asn residue. In acidic media the peptides deamidate by direct hydrolysis of the Asn residue to yield only Asp peptides, whereas under neutral or alkaline conditions, the peptides deamidate by formation of the cyclic imide intermediates which hydrolyze to yield both isoAsp and Asp peptides. At neutral to alkaline pH's the rate of deamidation was significantly affected by the size of the amino acid on the C-terminal side of the Asn residue. The amino acid on the C-terminal side of the Asn residue has no effect on the rate of deamidation at acidic pH. Changes in the structure of the amino acid on the N-terminal side of the Asn residue had no significant effect on the rate of deamidation at all the pH's studied. For peptides that underwent deamidation slowly, a reaction involving the attack of the Asn side chain on the peptide carbonyl carbon resulting in peptide bond cleavage was also observed.     

Complete amino acid sequence of a phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

The acidic phospholipase A₂ has been isolated from Naja naja atra (Taiwan cobra) venom. The reduced and S-carboxymethylated enzyme was digested with trypsin, and eleven tryptic peptides which accounted for the whole molecule were isolated by a combination of chromatographic and gel filtration procedures, and their amino acid sequences were determined. The alignment of those eleven tryptic peptides was established by the analysis of nine major peptides obtained by Staphylococcus aureus protease digestion of the reduced and S-carboxymethylated enzyme. The phospholipase A₂ of Naja naja atra is a single polypeptide chain consisting of 120 amino acid residues including 14 intramolecularly linked half-cystines. It shows about 86% (103 out of 120) homology when compared with the Naja mossambica mossambica CM-II enzyme.     

Role of melittin-like region within phospholipase A(2)-activating protein in biological function.

Phospholipase A(2)-activating protein (PLAA) has been implicated in the production of prostaglandins (e.g. PGE(2)) via activation of phospholipases in various stimulated cell types. Human PLAA, with 738 amino acid (aa) residues, contains a region of 38% homology (aa 503-538) with the 26-aa long melittin peptide, a major component of bee venom and a reported regulator of phospholipase A(2) and phospholipase D activity. To learn more about the role of PLAA in the production of eicosanoids and other inflammatory mediators, we synthesized a murine PLAA peptide (36-aa long) having homology to melittin, as well as to human and rat PLAA. The PLAA peptide and melittin increased the expression of genes encoding the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) and cyclooxygenase-2 (COX-2), which is involved in PGE(2) production. We determined that the C-terminal region of the PLAA peptide (aa 515-538) was essential, since truncation of the C-terminal end of the PLAA peptide significantly reduced expression of genes encoding TNFalpha and COX-2 in macrophages. We concluded that PLAA could be important in the regulation of the inflammatory response because of its stimulatory effects on eicosanoid and cytokine synthesis. Consequently, control of plaa gene expression could be a target for the development of new drugs to control the inflammatory response.     

Antimicrobial activity of arginine‐ and tryptophan‐rich hexapeptides: the effects of aromatic clusters,d‐amino acid substitution and cyclization

Abstract: Many antimicrobial peptides bear arginine (R)‐ and tryptophan (W)‐rich sequence motifs. Based on the sequence Ac‐RRWWRF‐NH₂, sets of linear and cyclic peptides were generated by changes in the amino acid sequence, l ‐d ‐amino acid exchange and naphthylalanine substituted for tryptophan. Linear RW‐peptides displayed moderate activity towards Gram‐positive Bacillus subtilis (15 < MIC < 31 μm ) and were inactive against Gram‐negative Escherichia coli at peptide concentrations <100 μm . Cyclization induced high antimicrobial activity. The effect of cyclization was most pronounced for peptides with three adjacent aromatic residues. Incorporation of d ‐amino acid residues had minor influence on the biological activity. The haemolytic activity of all RW‐peptides at 100 μm concentration was low (<7% lysis for linear R/W‐rich peptides and <28% for the cyclic analogues). Introduction of naphthylalanine enhanced the biological activities of both the linear and cyclic peptides. All peptides induced permeabilization of large unilamellar vesicles (LUVs) composed of lipids of the membrane of B. subtilis and erythrocytes, but surprisingly had no effect on LUVs composed of lipids of the E. coli inner membrane. The profiles of peptide activity against B. subtilis and red blood cells correlated with the permeabilizing effects on the corresponding model membranes and were related to hydrophobicity parameters as derived from reversed phase high‐performance liquid chromatography (HPLC). The results underlined the importance of amphipathicity as a driving force for cell lytic activity and suggest that conformational constraints and an appropriate position of aromatic residues allowing the formation of hydrophobic clusters are highly favourable for antimicrobial activity and selectivity.     

New antifungal antibiotics,bacillopeptins and fusaricidins

We isolated four strains of bacteria producing antifungal antibiotics from the rhizosphere of garlic with basal rot caused by the plant pathogenic fungal strain Fusarium oxysporum. Among them, Bacillus subtilis FR-2 was found to produce new antifungal antibiotics, named bacillopeptins A, B, and C. Their structures have been determined by 1D and 2D NMR and MS experiments, and amino acid analysis coupled with chiral HPLC, to be cyclic lipopeptides each containing a long-chain beta-amino acid. Another bacterial strain, Bacillus polymyxa KT-8, was shown to produce new antifungal antibiotics named fusaricidins A, B, C, and D which are more potent than bacillopeptins in their antimicrobial activity. The structures of the fusaricidins have been elucidated similarly as bacillopeptins to be cyclic hexadepsipeptides all containing 15-guanidino-3-hydroxypentadecanoic acid as a side chain. Fusaricidins strongly inhibit the growth of various kinds of fungi and moreover surprisingly show strong inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus or Micrococcus luteus.