一种钙/钙调蛋白依赖性的蛇毒磷脂酶A_2

分别研究了钙离子和三价稀土离子对白眉蝮蛇 (Agkistrodon blomhoffii Ussurensis)蛇毒磷脂酶 A2(PLA2 )活性的影响以及钙调蛋白对它的激活作用 .实验结果表明 ,PLA2 的活性对钙离子表现出依赖性 ,钙调蛋白能够激活该蛇毒 PLA2 ,钙调蛋白的拮抗剂三氟甲基吩噻嗪 (Trifluoperazine)能够完全抑制它对 PLA2的激活作用 .三价稀土离子 La3+、Eu3+、Dy3+、Yb3+对该 PLA2 的活性表现出抑制作用 ,其中离子半径较大的La3+和 Eu3+对酶活的抑制程度要小于半径较小的 Dy3+和 Yb3+.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A(2) and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase AZ and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained, In addition to the dominant protein [M+H](+) ions, multimeric and multiply charged ions were also observed in the mass spectra, The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected, Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes, Mass accuracies of 0.1 and 0.3 % were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-palyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants, Copyright (C) 1999 John Wiley & Sons, Ltd.

MALDI/TOF MS法测定蛇毒纤溶酶及磷脂酶A_2的分子量和纯度

目的： 分析测定长白山白眉蝮蛇蛇毒纤溶酶和磷脂酶A2 的分子量和纯度。方法与结果： 应用MALDI/TOF MS法测定纤溶酶的分子量为23333±90，磷脂酶A2的分子量为14000±20，相对偏差在0138%以内。结论： 应用此方法未检测到杂蛋白质谱峰的存在， 酶的纯度较好， 测得结果要比电泳法准确。MALDI/TOF MS提供了一种测定蛋白质药物纯度快速准确的新方法。

Ca~(2+)和Tb~(3+)对白眉蝮蛇蛇毒磷脂酶A_2荧光光谱的影响

研究了金属离子Ca2+和Tb3+对长白山白眉蝮蛇蛇毒磷脂酶A2(phospholipase A2)荧光光谱的作用，发现Ca2+浓度的增加能够增强磷脂酶A2的荧光发射强度．而且Ca2+ 浓度的增大能够明显加快磷脂酶A2与其相应反应底物DPPC的反应速率．稀土离子Tb3+在低浓度条件下对磷脂酶A2起荧光淬灭作用， 而在浓度较高时能够提高磷脂酶A2荧光发射强度.

同步荧光法研究白眉蝮蛇蛇毒酶发色团的微环境

利用同步荧光技术对长白山白眉蝮蛇蛇毒中纯化得到的4种酶：磷脂酶A2(phospholipase A2,PLA2)、精氨酸酯酶(arginine esterase,AEase)、纤溶酶(fibrinolytic enzyme,FEase)和L-氨基酸氧化酶(L-amino acid oxidase,L-a a oxidase)进行了研究． 结果表明：当Δλ＝20nm时， 酶的荧光主要由酪氨酸(Tyr)残基所贡献； 当Δλ≥75nm时，酶的荧光主要由色氨酸(Try)所贡献．而且，长白山白眉蝮蛇蛇毒4种酶的Tyr和Trp残基所处的微环境并不相同.

La_(2-x)(Sr,Th)_xCuO_(4±λ)系催化剂的表征及对CO+NO反应催化性能的研究

合成了x值不同的La2 -xSrxCuO4±λ( 0 .0≤x≤ 0 .1 )和La2 -xThxCuO4±λ( 0 .0≤x≤ 0 .4)两系列K2 NiF4型复合氧化物催化剂 ,用XRD和IR研究了催化剂的晶体结构 .利用化学分析和XPS等方法测定了该系列含Cu氧化物中Cu离子的平均价态、非化学计量氧 (λ)和催化剂的表面和体相组成 .通过控制B位Cu离子的价态和氧化物的非化学计量氧在一定范围内有规律的变化 ,考察对NO +CO反应的催化性能 ;利用MS TPD法研究了该系列氧化物对NO和CO +NO等小分子的吸附和活化性能 ,在此基础上探讨了含Cu的A2 BO4型复合氧化物对NO +CO反应中的催化作用本质 .发现在低温反应条件下 ,NO分子的活化是控制步骤 ,催化剂的活性与低价离子及其含量有关 .在较高反应温度下 ,NO的吸附为控制步骤 ,催化活性与氧空位有关

COMPARISON OF THE BEHAVIOR OF CYCLIC AND LINEAR POLYSTYRENE IN DILUTE-SOLUTION

Using a low angle laser light scattering photometer, the second virial coefficients (A2) of both cyclic and linear polystyrene were determined in cyclohexane, toluene and methyl ethyl ketone (MEK) solutions. From the data obtained in cyclohexane solution the theta-temperature of cyclic polystyrene was determined to be 30-degrees-C. In toluene solution A2 of cyclic polystyrene is smaller than that of linear polystyrene with the same molecular weight, but in MEK the values are similar.

Analysis of the expression and antioxidative property of a peroxiredoxin 6 from Scophthalmus maximus

Peroxiredoxins (Prxs) are a group of antioxidant proteins that protect cells from oxidative damage caused by various peroxides. To date, six different isoforms of peroxiredoxin (Prx1 to Prx6) have been identified, of which, Prx6 belongs to the 1-Cys Prx subfamily. Although Prx6 of several fish species have been reported at sequence level, there are very few documented studies on the potential function of fish Prx6. In this report, we describe the identification and analysis of a Prx6 homologue, SmPrx6, from turbot Scophthalmus maximus. The full length cDNA of SmPrx6 contains a 5'- untranslated region (UTR) of 60 bp, an open reading frame of 666 bp, and a 3'-UTR of 244 bp. The deduced amino acid sequence of SmPrx6 shares 81-87% overall identities with known fish Prx6. In silico analysis identified in SmPrx6 a conserved Prx6 catalytic motif, PVCTTE, and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. Expression of SmPrx6 was detected in most fish organs, with the highest expression levels found in blood and heart and the lowest level in spleen. Experimental challenges with bacterial pathogens and poly(I:C) upregulated SmPrx6 expression in liver and spleen in a manner that is dependent on the challenging agent and the tissue type. Treatment of cultured primary hepatocytes with H2O2 enhanced SmPrx6 expression in a dose-dependent manner. Recombinant SmPrx6 expressed in and purified from Escherichia coli exhibited thiol-dependent antioxidant activity and could protect cultured hepatocytes from H2O2-induced oxidative damage. Taken together, these results indicate that SmPrx6 is a Prx6 homologue with antioxidative property and is likely to be involved in both cellular maintenance and protective response during host immune defense against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

Pharmacological and immunocytochemical investigation of the role of catecholamines on larval metamorphosis by beta-adrenergic-like receptor in the bivalve Meretrix meretrix

Catecholamines regulate several physiological processes in mollusks. Many pharmacological experiments have been conducted to determine the effects of adrenergic agonist and antagonist of catecholamine receptors on Meretrix meretrix metamorphosis. Results showed that adrenaline (AD) and noradrenaline (NA) had substantial effects (p < 0.05) on larval metamorphosis at concentrations ranging from 10 mu M to 100 mu M. 10 mu M beta-adrenergic receptor (AR) agonist isoproterenol showed the same inducement effect as that of NA and AD on metamorphosis, whereas the alpha-AR agonist phenylephrine had no significant effect at concentrations between 0.1 mu M and 100 mu M concentrations (p > 0.05). Furthermore, I mu M beta-AR antagonist propanolol, but not alpha-AR antagonist prazosin, depressed the larval metamorphosis induced by NA or AD. By immunocytochemistry, two cell bodies of beta-adrenergic-like receptor, C/A1, C/A2, were observed in the cerebral/apical ganglion of competent larvae. In addition, there were other immunoreactive dots near C/A1 and C/A2. The results of pharmacology and immunocytochemistry suggests that beta-adrenergic-like receptor located in the larval CNS, might play a considerable role in the larval metamorphosis of M meretrix by AD or NA. (c) 2006 Elsevier B.V. All rights reserved.

红树林植物海榄雌化学成分研究

本论文对红树林植物海榄雌的化学成分进行了研究，对其中分离得到的部分化合物进行了初步的生物活性筛选。 海榄雌采自海南东寨港，样品干燥后用氯仿甲醇（1：1）浸泡提取，合并提取物，先后用石油醚、乙酸乙酯和正丁醇萃取。得到石油醚相、乙酸乙酯相、正丁醇相和水相。 各部分采用常规的硅胶柱层析、制备薄层层析、凝胶Sephadex LH-20柱层析、反相硅胶柱层析、半制备HPLC以及重结晶等手段分离得到28个化合物。利用各种现代波谱技术(IR、UV、ESI-MS、FAB-MS、HR-FAB-MS、1D-NMR、2D-NMR等)，确定了其中20个化合物的结构，其中包括2个新化合物：化合物A1 2′-O-(5-phenyl-2E, 4E-pentadienoyl)mussaenosidic acid和化合物A2 2′-O-(p-methoxycinnamoyl)mussaenosidic acid，以及13个首次从海榄雌中报道的化合物。 对得到的20个化合物A1－A20进行了DPPH自由基清除活性筛选，化合物A4、A5、A6和A16表现出较好的活性，其IC50分别为9.61 μg/mL、8.55 μg/mL、11.72 μg/mL和7.73 μg/mL；化合物A13和A15表现出中等强度的DPPH自由基清除活性，其IC50分别为34.80 μg/mL和44.90 μg/mL；其他化合物只表现出微弱活性，其IC50均大于100 μg/mL；阳性对照BHT的IC50为18.00 μg/mL。 对分离得到的部分样品进行了抑菌活性测试，各样品在测试浓度下对测试菌均未表现出明显的抑菌活性。

中国明对虾病原检测新方法及酚氧化酶通路相关基因的研究

对虾病害在世界范围内肆虐，给水产养殖和沿海农村经济造成了重大损失。在水产养殖的实践中快速检测水产动物的病害并及时采取隔离等措施对于控制病害尤为重要，其中关键的环节就是快速检测出病害，并在对虾免疫机制上寻找对虾疾病防治的有效方法。研究表明当对虾等甲壳动物受到外界病原刺激时，极微量的微生物多糖就可以激活proPO系统。激活过程中涉及和产生一系列活性物质，如黑色素、酚氧化酶原激活因子（PPA）、模式识别蛋白（BGBP、PGBP、LGBP、LBP）及其膜上受体和A2巨球蛋白等，它们可通过多种方式参与防御反应，包括提供调理素，促进血细胞吞噬作用，形成结节或包囊以及介导凝集和凝固，产生杀菌物质并且黑色素化。黑色素常常在节肢动物的体表形成黑色斑点，形成的色素沉着对机体起到保护作用。所以，酚氧化酶原激活的级联反应是节肢动物免疫的关键因素。本论文研究开发了以环等温介导技术（LAMP）为基础的检测对虾白斑病毒（WSSV）和鳗弧菌（V. anguillarum）的快速检测方法。并从对虾对病害的免疫机制为切入点，从中国明对虾体内克隆了酚氧化酶原（PrpPO）和丝氨酸蛋白酶FcSP3这两个免疫系统中重要的基因，分析了它们的分子结构特征，组织分布及应答鳗弧菌病原刺激的表达变化模式。 建立的对虾常见病害对虾白班病毒（WSSV）和鳗弧菌（V. anguillarum）的LAMP检测方法，经过实验比对和Blast检索，发现本研究中使用的引物，比已经报导的LAMP方法或者PCR方法具有更宽的检测范围（更低的假阴性）。检测WSSV的LAMP方法使用病毒的VP28基因设计引物，而鳗弧菌的检测方法使用empA基因设计引物。在方法中，首次提出加入UNG酶和dUTP的措施来预防污染，在实际检测中非常有效。LAMP方法与PCR检测方法的灵敏性比较也进行了研究，二者灵敏性相当。 依据中国明对虾血液cDNA文库提供的部分片段信息，结合SMART-RACE技术，克隆了酚氧化酶原（PrpPO）基因，通过序列比对分析发现，PrpPO基因cDNA全长为3040 bp，其中开放阅读框2061 bp，编码686个氨基酸，其中推测的信号肽为12个氨基酸。推测的序列与斑节对虾（P. monodon）同源性为93%,与短钩对虾（P. semisulcatus.）同源性为92%。real time RT-PCR实验结果表明， ProPO在血细胞中的相对表达量最高，肝胰脏中表达量最低。弧菌刺激实验中注射弧菌，刺激了血细胞和淋巴器官中的ProPO mRNA显著增加，说明在血细胞和淋巴器官中存在快速反应的ProPO通路。而ProPO mRNA量在淋巴器官中在时间上早于血液中升至最高，说明该动物在在病原刚开始入侵的时候先有淋巴器官发挥主要的免疫作用，随着时间推移血细胞便变成主要的免疫器官。 根据中国明对虾肝胰脏cDNA文库提供EST信息，经过SMART-RACE克隆了一个丝氨酸蛋白酶FcSP3基因，通过序列比对分析发现，该丝氨酸蛋白酶基因cDNA全长为1622 bp，其中开放阅读框1431 bp，编码477个氨基酸，其中推测的信号肽为22个氨基酸。推测的序列与疟蚊的丝氨酸蛋白酶（A. gambiae）同源性为33%,与丽蝇蛹集金小蜂的酚氧化酶原激活因子（N. vitripennis）同源性为32%，与东北大黑鳃金龟的酚氧化酶原激活因子（H. diomphalia）同源性为34%。淋巴器官中PPAⅡ表达量约为血液中表达量的47560倍，肝胰脏中的FCSP3表达量为血细胞表达量的6226倍。鳗弧菌注射对虾后，淋巴器官中刺激组和对照组FcSP3的mRNA量在刺激后6小时显著降低，但是刺激组的表达量明显高于对照组。刺激组的血细胞与肝胰脏中FcSP3 mRNA的相对表达量增高。而病原刺激后的血液与肝胰脏中的FcSP3 mRNA的增长趋势也在时间上先与ProPO mRNA。这说明FcSP3对ProPO有正调控的作用，但这个调控有一个时间差，并且在不同组织中有不同的调控效率。

Experimental evaluation of heterobeltiosis and heterosis between two populations of Pacific abalone Haliotis discus hannai Ino

A2 x 2 factorial cross between two populations of Pacific abalone Haliotis discus hannai Ino, collected separately from Dalian (D) in China and Miyagi (M) in Japan, was conducted to compare performances in fertilization rate, hatching rate, metamorphosis rate and growth at days 20, 43, 160 and 330 between purebreds (DD and MM) and crossbreds (DM and MD) and investigate the magnitude of heterobeltiosis (better parent) and heterosis (mid-parent). Heterobeltiosis and heterosis for all the traits analyzed were evidently different between crossbreds DM and MD. Heterobeltiosis in the crossbred DM varied among traits, with values of 2.5% for the fertilization rate, 2.2% for the hatching rate, -1.9% for the metamorphosis rate and 7.4% for the growth at the (lay 330. The crossbred DM displayed positive heterotic values for fertilization rate (5.4%), hatching rate (7.4%), metamorphosis rate (7.6%) and growth (12.0%) at the day 330. However, both heterobeltiosis and heterosis for all the traits in the crossbred MD were negative except those for the growth at days 20 and 43. The results indicate the importance of selecting superior hybrid varieties if the exploitation of hybrid vigor is considered in the Pacific abalone breeding program.

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

Background: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. Objectives: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. Methods: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. Results: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD 106). Coronary MaVEC released significantly less EMP than MiVEC. Conclusion: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium. (C) 2003 Elsevier Science Ltd. All rights reserved.

污水生化处理过程仿真及在A~2/O工艺改进中的应用

基于国际水协会IWA发布的国际评价基准Benchmark,建立了与污水处理厂实际特性相近的标准仿真平台。对污水处理传统A2/O工艺做了合理改进,在不影响出水水质的情况下,取消内回流。应用该平台分别对晴天、雨天与暴雨三种天气的工艺过程模拟,验证了工艺改进的合理性。仿真结果表明,与传统A2/O工艺相比,出水指标差别不大,但污泥泵能耗降低约50%。

A/O工艺城市污水生化处理过程建模与优化控制研究

可持续发展的要求增强了人类有效利用资源和环境保护的意识，而城市污水处理过程是水环境保护的一项重要内容。本文针对我国城市污水处理系统能耗高、出水水质不稳定以及工业毒水经常入侵的现状，以A/O工艺城市污水生化处理过程为研究对象，研究通过建模、控制和优化手段提高污水处理系统运行性能和降低运行降耗的理论和方法。论文工作是在国际水协会发布的典型工艺与污水处理厂实际运行环境相结合的基础上完成，内容主要涉及活性污泥模型简化、基于简化模型的工艺改进与过程模拟、仿真平台的建立、关键控制回路设计、工业毒水诊断与应对系统开发以及优化控制方法与应用研究。具体内容包括： (1) 基于国际评价基准benchmark实现了污水生化处理过程的稳态模拟和动态模拟，对A2/O工艺的改进进行了仿真研究和分析，结果表明在保证出水水质稳定达标的前提下，改进工艺由于取消了内回流，大大节约了回流泵的能耗。 (2) 在国际水协会活性污泥模型ASM1模型的基础上，结合我国污水处理过程特点，并充分考虑我国污水处理厂现场测量信息严重不足的现状，对ASM1模型的组分和反应过程进行简化，建立了简化的活性污泥模型，研究了难以测量的模型组分浓度与易测的常规水质指标的转换方法，针对温度对反应速率的影响，研究了模型参数的校正方法。在此基础上，开发了A/O工艺污水生化处理过程模拟与仿真平台，并对辽宁某污水处理厂的实际运行过程进行了模拟，取得了较好的模拟结果。 (3) 建立了完整的城市污水处理过程控制系统设计框架，引入入水有机负荷和比耗氧速率分别表征微生物（活性污泥）的“食物”（有机物）数量和微生物活性，进行了系统动态特性分析，在此基础上完成了入水流量串级控制、污泥浓度前馈－反馈控制、溶解氧前馈－串级控制三个控制回路的设计。针对溶解氧控制过程采用了仿人智能控制方法，在实际应用中取得了较好的控制效果。 (4) 针对城市污水处理厂经常遇到的工业毒水侵入的问题，开发了基于专家经验的工业毒水诊断与应对系统，编写了较为完备的专家规则，系统能够在毒水侵入时及时发出警报，并采取相应的应对措施，最大程度的降低污水处理厂的损失，该系统在辽宁某污水处理厂取得了较好的应用效果。 (5) 在优化控制方面，针对污水处理过程能耗过高的问题，设计了A/O工艺城市污水处理过程的优化控制策略。建立了只考虑底物与微生物两种组分的简化活性污泥模型，以溶解氧浓度和污泥排放量为决策变量，以污水处理厂日运行费用为性能指标，以物料平衡、水质排放标准等限制为约束条件，建立了完整的优化控制模型，实现了污水处理过程的最优控制，采用基于最优步长参数动态搜索的改进型梯度法进行最佳运行工况寻优。通过对辽宁某城市污水处理厂的优化，获得了当前溶解氧浓度设定值和污泥排放量的最优值，在保证出水水质稳定达标的前提下，污水处理厂日运行费用显著降低，为污水处理厂的实际操作提供了指导。