Inhibitory effects and mechanisms of high molecular-weight phlorotannins from Sargassum thunbergii on ADP-induced platelet aggregation

We evaluated the effects of high molecular-weight phlorotannins from Sargassum thunbergii (STP) on ADP-induced platelet aggregation and arachidonic acid (AA) metabolism in New Zealand white rabbits and Wistar rats. The inhibition of STP on platelet aggregation was investigated using a turbidimetric method, and the levels of the terminal products of AA metabolism were measured using the corresponding kits for maleic dialdehyde (MDA), thromboxane B-2 (TXB2) and 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) by colorimetry and radioimmunoassay, as appropriate. We found that STP could inhibit ADP-induced platelet aggregation, and the inhibitory ratio was 91.50% at the STP concentration of 4.0 mg/mL. Furthermore, STP markedly affected AA metabolism by decreasing the synthesis of MDA (P < 0.01) and increasing the synthesis of 6-keto-PGF(1 alpha), thus changing the plasma TXB2/6-keto-PGF(1 alpha) balance when the platelets were activated (P < 0.01). Therefore, STP altered AA metabolism and these findings

园艺产品采后品质的调控机制

该项目属农业科学技术学科，主要内容涉及到农产品贮藏、园艺采后生物学、植物学等领域。园艺产品品质是影响和制约其贮运保鲜的关键因素，该项目在园艺产品采后品质研究方面取得了重要进展：本项目首次从多酚氧化酶、花色素苷酶、褐变底物和能量亏损等多方面系统性提出了荔枝果实采后褐变机理；确定了香蕉、芒果、草莓等果实采后新的乙烯受体形成特点和规律以及贮藏环境和外源乙烯的响应特性；从乙烯结合位点、乙烯作用和扩展蛋白、苯丙氨酸解氨酶的基因表达等方面阐明了香蕉果实冷害发生的可能原因，丰富了果实冷害发生的作用机制；系统研究荔枝、龙眼、芒果、桃、甜樱桃、柑桔、冬枣、李、西兰花、康乃馨、月季切花等园艺产品采后的生理代谢特点，揭示了在不同贮藏条件下园艺产品品质的变化规律和调控机制；在技术和方法上，首次提出了果实组织ATP、ADP和AMP含量的快速HPLC测定技术；建立新的乙烯结合位点合成速率的测定技术；提出了以电导率为指标，建立了果实品质的定量评价方法，能更好评价/预测园艺产品的品质。该成果揭示了园艺产品采后品质调控的生理代谢特征和规律性变化，对影响品质的关键性问题（如衰老、褐变、冷害等）进行了深入研究，获得了最新成果，在研究方法上也取得了突破。研究成果在国际核心期刊上发表SCI收录论文72篇，获得国际科学基金Jubilee Award，得到国内外同行专家的关注并被广泛引用（被SCI论文引用323篇次）；在荔枝、龙眼等果实采后研究领域处于国际领先地位。

水稻幼苗的低温伤害及其和能量代谢的关系

本实验研究了水稻幼苗低温伤害的机理，并比较了两个抗寒性不同的水稻品种在2—4ºC低温处理一至四天期间和在30℃恢复期间，膜透性、呼吸作用和腺苷酸含量及能荷变化，丙酮酸激酶活性质膜ATPase水解活性和线粒体ATPase磷酸化活性的变化和蛋白质合成速率的变化。结果表明，水稻幼苗相对电导率随低温处理天数的增加而迅速地增加，在抗寒的早二六14中增加较慢，在抗寒性差的二九丰中增加较快。受冷四天的幼苗在3 0ºC下恢复一天后，早二六14的相对电导率能大幅度地下降，而二九丰仍然保持高水平。呼吸活性在低温处理l一3天起伏不定，但到第四天又剧烈地下降，若这时在30℃恢复一天后，呼吸活性又迅速增加。早二六14和二九丰的呼吸活性变化趋势十分相似。低温使幼苗中ATP含量和总腺苷酸含量显著地下降。处理一天后，早二六14和二九丰的ATP含量分别下降75%和78%，总腺苷酸含量都下降68%左右，ADP和AMP含量变化趋势与ATP相似，但下降幅度不如ATP明显，能荷分别从0.80和0.81降低至0.70和0.69 , ATP ∕ ADP比率分别从3∙ 4 7和4.45下降至1.60和1.46。之后继续低温处理，ATP、ADP和AMP含量和能荷及ATP/ADP比率基本上维持稳定。当受冷四天的幼苗在30ºC恢复一天后，ATP含量和总腺苷酸含量在这两个品种都显著地恢复上升，但上升的幅度在早二六1 4中大于二九丰。能荷也能恢复至正常的水平，AT P/ ADP在早二六14中从1.90上升至2.4 4，而在二九丰中没有增加。低温处理后，质膜ATPase活性明显上升，其变化幅度二九丰大于早二六14 ,尤其在2.5天的低温处理后，在二九丰中增加100%，而在早二六14中只增加12%。此外，通过分析质膜人ATPase对不同温度的反应情况，进一步证明了品种间的差异性。利用完整的线粒体在底物ADP的存在下直接侧定ATP产生速度证明了线粒体ATPase磷酸化活性在低温处理四天后显著下降。同时，丙酮酸激酶活性也下降。以3H一亮氨酸标记的实验结果表明，水稻幼苗经低温处理后，蛋白质合成速度显著下降，但在早二六1 4中下降幅度较小，在二九丰中下降幅度较大，并且在低温期间，早二六14的蛋白质合成速度总是明显大于二九丰。当受冷4天的幼苗在30ºC恢复一天后，蛋白质合成速度迅速上升，并超过未经低温处理的对照水平，在早二六14中恢复上升幅度大于二九丰。以上这些结果表明，低温损伤了水稻幼苗细胞膜的结构，使膜透性增加，引起非正常的呼吸，改变了与膜结合的质膜ATPase水解活性和线粒体ATPase磷酸化活性，同时也改变了糖酵解中的丙酮酸激酶活性，造成AT水平、能荷或ATP/ADP比值的下降。由于能量代谢的失调，蛋白质合成过程受到严重障碍，超过一定限定，幼苗不能正常生长而死亡。品种间抗寒性的差异也在上述变化中反映出来。

转Rubisco 活化酶大型同工酶基因(rca)水稻的光合作用特性研究

Rubisco 是催化光合暗反应第一步反应的酶，是唯一能将CO2 转变成碳水化合物的酶，由它固定和最后转化成的碳水化合物提供了植物、动物和微生物的食物和能量。但是，Rubisco 催化该反应的效率十分低，使之成为光合作用的限速步骤。由于Rubisco 的合成和催化过程十分复杂，人们很难通过直接改造Rubisco 提高植物固定CO2 的能力。而Rubisco 活化酶能活化Rubisco，使植物在生理CO2 浓度下具有最大的CO2 同化速率，因此研究活化酶有重要意义。水稻活化酶有2 个同工酶，大型同工酶比小型同工酶C 端多37 个氨基酸，其中包括两个Cys 残基。这两个Cys 残基的存在使活化酶大型同工酶对ADP 的存在更加敏感，其活性在硫氧还蛋白的介导下能被基质中氧化还原状态的变化所调节。由于活化酶大型同工酶对调节Rubisco 的活性具有的这种特殊作用，在本研究中，将活化酶大型同工酶rca基因用正义和反义引入水稻基因组，获得了过量表达活化酶大型同工酶基因和反义抑制活化酶基因表达的转基因植株，对其光合作用进行了生理和生化分析。 本研究的主要结果如下： Rubisco 活化酶大型同工酶基因的克隆：从水稻镇恢249 中克隆了1525 bp 的活化酶大型同工酶cDNA 序列。经过测序，它与报道的粳稻品种活化酶大型同工酶cDNA 序列（rca）完全相同。 构建了4 个植物表达载体：3 个为过量表达rca的载体，分别是pCBUbirca，pCBSrca 和 pCBSUbirca ，其中rca分别在水稻中高效表达的玉米Ubiquitin 启动子、受光调控的Rubisco 小亚基基因启动子和由这两个启动子构成的双启动子控制下表达; 1 个在Ubiquitin 启动子控制的反义rca载体，即 pCBUbi-antirca。 获得了转化rca的水稻再生植株：用日本晴，台北309，武育梗7 号和籼稻品种培矮64S 水稻成熟种子诱导愈伤组织。用改良的农杆菌浸染法将rca基因转化这些愈伤组织，在潮霉素筛选压力下获得抗性愈伤组织，经过2 天的干燥处理后，转入到含山梨醇的高渗分化培养基上培养，能迅速获得大量的芽和转化体再生植株。 获得了转rca基因的水稻植株：抗性愈伤组织和再生水稻幼苗的叶片经GUS 染色呈蓝黑色。PCR 扩增转基因水稻基因组内的潮霉素基因和rca，大部分转基因水稻中含有841 bp 的潮霉素基因片段和1525 bp 长的rca cDNA 片段。251 粒T1 代转基因水稻种子中189粒呈现潮霉素抗性，抗性种子/非抗性种子的比率约为3：1，接近孟德尔分离规律。Southern杂交表明rca序列已整合到水稻基因组，一般含1－2个拷贝。Western 杂交显示Rubisco 活化酶含量在转pCBUbi －antirca 的水稻中和对照比，几乎看不出，被反义抑制;转pCBUbirca 的水稻与对照含量相差无几;转pCBSUbirca，pCBSrca 载体的水稻中活化酶的含量比对照有极显著的增加。 T1 代转rca水稻的光合作用发生显著变化：转pCBSrca 和pCBSUbirca 的水稻在饱和光强下的Rubisco 初始活性、羧化效率、光合速率都明显高于对照，但是表观量子效率、色素含量和Rubisco 总活性与对照相似。两者相比，前者比后者更高;转反义rca（pCBUbi-antirca）基因的水稻饱和光强下的光合速率、表观量子效率、羧化效率、Rubisco 初始活性明显降低，色素含量和Rubisco 总活性基本不变;转pCBUbirca 的水稻中，光合作用的各项参数与对照基本相似。 T1 代转rca水稻的叶绿素荧光明显改变：转pCBSrca 和pCBSUbirca 的水稻ΦPSII 的值明显高于对照，而且前者qP 的值明显高于对照。两者相比，前者的ΦPSII 和qP 的值比后者高;转反义rca的水稻ΦPSII，F′v/F′m，qP 值和对照比都明显降低，但qN 的值升高;转pCBUbirca 载体的水稻中，叶绿素荧光的各项参数与对照基本相似。 转rca基因的水稻生长发育的变化：转pCBUbirca 载体的水稻整个生长发育过程与对照相似;转化pCBSrca 和pCBSUbirca 载体的水稻和对照比，植株高大，生长发育速度加快，抽穗、开花和结籽的时间提前。两者本身相比，前者比后者明显;转反义rca（pCBUbi-antirca）基因的水稻生长发育延迟，植株矮小，种子败育。 由上可见，Rubisco 活化酶大型同工酶rca基因在Rubisco 小亚基基因启动子、Ubiquitin 基因启动子和Rubisco 小亚基基因启动子共同控制下正义转入水稻的转基因植物光合作用的参数最好，光合效率提高，植物表型最好，生长发育加快，提前开花结籽。这一研究可能为获得高光合效率和高产量的水稻奠定了基础。

PEG引发预防大豆种子吸胀冷害的研究

黑河3号大豆种子对低温吸胀非常敏感，PEG引发处理明显提高抗吸胀冷害能力。引发处理的效果与引发后种子含水量的提高有关。引发3天种子含水量达到34%左右。 引发处理过程中种子呼吸强度提高，呼吸商大于1。引发3天大豆种子胚根细胞ATP酶活性定位于质膜、细胞壁及细胞间隙。 引发种子和对照种子低温吸胀后，在常规增减条件下生理生化过程有显著的不同，这些差异与二者不同的种子活力水平有关。引发种子浸种过程中细胞物质损失少，膜选择性通透能力强。对照种子有大量物质外渗，特别是K+。引发种子常规培养条件下呼吸强度上升很快，RQ较低。对照种子呼吸强度很低。引发种子线粒体以L－Mal、α－Kg和Succ为底物时，具有较高的ADP／O和RC，并且氧化磷酸化活性出现早。对照种子线粒体氧化磷酸化功能不健全。引发种子胚根细胞ATP酶活性定位于质膜、核仁、胞间连丝及液泡膜。对照种子ATP酶活性在各亚细胞结构大为降低。 本文讨论了吸胀冷害的普遍性以及吸胀冷害对农业生产的影响，并推论低温吸胀主要导致敏感种子吸胀初期正在进行结构转化的膜系统不可逆损伤从而种子活力下降。PEG引发处理具有防止低温吸胀时膜系统伤损和修复种子成熟、加工、干燥贮藏阶段损伤的作用。PEG引发还预先活化了种子代谢系统。从而使种子萌发迅速、整齐，显著地提高敏感种子搞吸胀冷害能力，对于不存在吸胀冷害问题的种子，PEG引发也有提高种子活力的效果。

吸血节肢动物唾液腺来源的抗止血物质

下载PDF阅读器在采食脊椎动物血液能力的进化过程中,吸血节肢动物的唾液腺形成了丰富的抗止血因子,如血小板聚集抑制因子,他们通过不同机制抑制ADP、凝血酶和胶原等诱导的血小板聚集.抗凝因子能扰乱内源性和外源性止血通路.血管扩张因子包括储藏、运输一氧化氮的nitrophorins,模拟内源性血管扩张的多肽和催化或水解内源性血管收缩因子的酶.吸血节肢动物的唾液腺蛋白可以通过直接作用或协同作用起到抗止血的效果.复杂多样的唾液腺生物活性分子解释了吸血节肢动物成功获得血餐的分子机制,也提供了新的抗止血药物分子.

中华硬蜱血小板集聚抑制剂的分离纯化与活性研究

目的研究蜱类抗血小板集聚的生物活性成分, 了解其与其宿主相互作用的分子机制。方法用葡聚糖 Sephadex G-50 凝胶过滤及高效液相从半饱吸血的中华硬蜱(Ixodes sinensis) 唾液腺中分离纯化具有血小板集聚抑制活性 的蛋白质, 用飞行质谱测定该抑制剂的分子量, 并用兔富血小板血浆研究其血小板集聚抑制活性。结果通过液相分离, 从中华硬蜱唾液腺中得到了一种血小板集聚抑制剂, 用飞行质谱测定该抑制剂的相对分子质量(M r ) 为8 065。该抑制 剂对二磷酸腺苷(ADP) 诱导的血小板集聚表现强烈的抑制活性, 在浓度为10 μg/mL 时, 可以抑制90% 以上的血小板集 聚。结论首次分离获得中华硬蜱唾液腺血小板集聚抑制剂, 该抑制剂对硬蜱顺利获取宿主血餐至关重要。

Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions

In mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel two domain TFF protein from frog Bombina maxima skin secretions (Bm-TFF2) has been purified and cloned. It activated human platelets in a dose-dependent manner and activation of integrin a(11b)beta(3) was involved. Aspirin and apyrase did not largely reduce platelet response to Bm-TFF2 (a 30% inhibition), indicating that the aggregation is not substantially dependent on ADP and thromboxane A2 autocrine feedback. Elimination of external Ca2+ with EGTA did not influence the platelet aggregation induced by Bm-TFF2, meanwhile a strong calcium signal (cytoplasmic Ca2+ release) was detected, suggesting that activation of phospholipase C (PLC) is involved. Subsequent immunoblotting revealed that, unlike in platelets activated by stejnulxin (a glycoprotein VI agonist), PLC gamma 2 was not phosphorylated in platelets activated by Bm-TFF2. FITC-labeled Bm-TFF2 bound to platelet membranes. Bm-TFF2 is the first TFF protein reported to possess human platelet activation activity. (c) 2005 Elsevier Inc. All rights reserved.

Antihemostatic molecules from saliva of blood-feeding arthropods

The ability to feed on vertebrate blood has evolved many times in various arthropod clades. Consequently, saliva of blood-feeding arthropods has proven to be a rich source of antihemostatic molecules. A variety of platelet aggregation inhibitors antagonize platelet responses to wound-generated signals, including ADP, thrombin, and collagen. Anticoagulants disrupt elements of both the intrinsic and extrinsic pathways. Vasodilators include nitrophorins (nitric oxide storage and transport heme proteins), a variety of peptides that mimic endogenous vasodilatory neuropeptides, and proteins that catabolize or sequester endogenous vasoconstrictors. Multiple salivary proteins may be directed against each component of hemostasis, resulting in both redundancy and in some cases cooperative interactions between antihemostatic proteins. The complexity and redundancy of saliva ensures an efficient blood meal for the arthropod, but it also provides a diverse array of novel antihemostatic molecules for the pharmacologist.

Characterization and cloning of a novel phospholipase A(2) from the venom of Trimeresurus jerdonii snake

A phospholipase A(2) (PLA(2)) called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 mumol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 mug/ml enzyme was incubated for 90 min in the presence of PC and Ca2+. No detectable hemolysis was noticed when PC was not added. Ca2+ was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca2+ was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC50 of 1.0 muM. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D49 PLA(2)s. Also, the residues concerned to Ca2+ binding were conserved. This is the first report of cDNA sequence of T jerdonii venom PLA(2). (C) 2002 Elsevier Science Ltd. All rights reserved.

Biochemical and biological properties of Trimeresurus jerdonii venom and characterization of a platelet aggregation-inhibiting acidic phospholipase A(2)

Several biochemical and biological activities such as phospholipase A(2), arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA(2)) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA(2) had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA(2) was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.

A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor,;encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization. (C) 2004 Elsevier Inc. All rights reserved.

GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) lb is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLCgamma2. Mucetininduced phosphorylation of the Fcgamma chain of platelet was greatly promoted by inhibition of alpha(llb)beta(3) by the peptidomimetic EMD 132338, suggesting that phosphatases downstream Of alpha(llb)beta(3) activation are involved in dephosphorylation of Fcgamma. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates alpha(llb)beta(3). Inhibition Of alpha(llb)beta(3) strongly reduced the aggregation response to mucetin, indicating that activation Of alpha(llb)beta(3) and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxaneA(2) are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.