λgtlSfi-Not作载体构建兔输卵管上皮细胞表达型cDNA文库

λｇｔｌＳｆｉ┐Ｎｏｔ作载体构建兔输卵管上皮细胞表达型ｃＤＮＡ文库刘传聚沈虹顾正吕吉宁左嘉客（中国科学院上海细胞生物学研究所,上海２０００３１）兔输卵管上皮细胞合成并分泌的某些蛋白组份具克服小鼠早胚发育阻断的功能〔１,２〕．利用“功能缺失”分析从...     

造血干/祖细胞大规模测序克隆的一个ABC转运体蛋白基因

ＡＢＣ转运体家族是已知的最大基因家族之一〔１〕,在进化上具高度的保守性,其成员含有ＡＴＰ结合小匣（ＡＴＰｂｉｎｄｉｎｇｃａｓｓｅｔｅ,ＡＢＣ）。最近,我们在对具造血功能的ＣＤ３４＋造血干／祖细胞（ＨＳＰＣ）的基因表达谱的研究中,通过大规模的ｃＤＮＡ测...     

乙型肝炎病毒X蛋白的产生和作用机理

乙型肝炎病毒（ＨＢＶ）基因组的４个开放读框（ＯＲＦ）,分别编码包括Ｘ蛋白在内的７种病毒蛋白;其中Ｘ蛋白具有广泛的反式激活作用,并且可能和与ＨＢＶ感染相关的肝细胞癌的发生有直接关系〔１〕。ＨＢＶＸ蛋白已证明不与双链ＤＮＡ相结合,其反式激活作用的发挥有赖...     

昆虫中的mariner类转座子

转座子 (ｔｒａｎｓｐｏｓｏｎ) ,是一种不依靠同源重组而能在基因组内移动的遗传学元件（ＤＮＡ序列）。它通过在基因组内的插入和割离 ,引起靶位点处ＤＮＡ的缺失、重复或倒位重排 ,导致插入处基因突变或相邻基因表达活性的改变 ,是基因组不稳定性的主要内在因素之一 ,也是构成生物体生长、发育的遗传调控因子之一〔１〕。转座子最早是由ＢａｒｂａｒａＭｃｃｌｉｎｔｏｃｋ在玉米中发现的 ,之后相继在细菌、病毒、昆虫、酵母、线虫、啮齿类等哺乳动物及人类基因组中发现〔２〕。转座子的基本结构包括两端重复序列 ,中间是编码转座酶的可…     

高速PCR用DNA聚合酶

ＰＣＲ（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）技术自创始至今还不到１５年的时间 ,但它已以惊人的速度渗透到了工业、农业、医学、药学等各个领域〔１ ,２〕。ＰＣＲ技术的发明无疑给生物工程带来了历史性的转折 ,促进了生物工程 ,特别是基因工程研究的飞速发展〔３〕。近期 ,随着人类基因组计划等的实施 ,并伴随着生物芯片技术的推广 ,快速、高效的ＰＣＲ已成为ＰＣＲ技术发展的必然趋势。ＴａＫａＲａ推出的Ｚ ＴａｑＤＮＡ聚合酶（Ｚ是指Ａ、Ｂ……排列的最后一个字母 ,指最快的意思 ,为商品名）是为快速ＰＣＲ而研制成功…     

抑癌基因nm23—H1在逆转录病毒载体的表达

癌症转移是癌症病人死亡的主要原因之一。抑制肿瘤转移的基因ｎｍ２３－Ｈ１与ｎｍ２３－Ｈ２分别编码二磷酸核苷激酶（ＮＤＰＫ）的Ａ与Ｂ亚基〔１〕,ｎｍ２３－Ｈ１基因能有效地抑制肿瘤转移〔２〕。ｎｍ２３－Ｈ１的表达水平与黑色素瘤〔２,３〕、乳腺癌〔４〕、卵巢...     

KOLMOGOROV模型的无害时滞

1．引言考虑具有时滞的两种群相互作用模型的稳定性。其中τ_ij∈〔0,∞）,i,j=1,2．f和g对所含变元连续。假设存在正常数平衡点（x~*,y~*）,x~*＞0,y`*＞0,使得f（x~*,y~*)=g（x~*,y~*）＝0．系统（1）的特别情形是（1）中当τ_ij=0时得到著名的Kolmogorov模型。定义⑴系统（1"'）具有一类型的时滞,如果个时滞无论大小如何都不改变系统（1"）的平衡位置（x~*,y`*）的渐近稳定性,则称此时滞为无害时滞。文献〔1〕-〔3〕得到系统（1'）类型的时滞为无害时滞的充分条件。文献〔2〕提出这样一个有趣的问题：系统（1）类型的时滞…     

固氮酶铁钼辅基理化特性的研究

在紫外可见光谱区内,固氮酶铁钼辅基〔（Ｍｏ_２Ｆｅ_（１２）Ｓ_（１２））４－〕均无特征吸收峰,不含高柠檬酸盐。含双钼的铁硫簇〔（Ｍｏ_２Ｆｅ_（６～１２）Ｓ_（６～１２））~（１～４）－〕的电荷数、颜色与该金属簇中的亚铁量成对应关系,并都有较高的生物重组活性．     

用荧光指示剂Fura－2测定正常和电刺激后蛙肌胞浆自由Ca~（2＋）浓度

蛙半腱肌肌束负载Ｆｕｒａ－２／ＡＭ后,可用荧光信号Ｆ３４０、３８０ｎｍ波长比值（Ｒ３４０／３８０）反映胞浆内游离Ｃａ２＋浓度（〔Ｃａ２＋〕）。利用这一技术,我们发现长时间电刺激后的骨骼肌〔Ｃａ２＋〕,高于未刺激肌,Ｒ３４０／３８０分别为１．４９±０．５４（ｎ＝１０）和１．０２±０．２６（ｎ＝１０）。加入Ｃａ２＋载体伊屋诺霉素（ｉｏｎｏｍｙｃｉｎ,１μｍｏｌ／Ｌ）后,正常肌与电刺激肌〔Ｃａ２＋〕,均上升,但刺激肌上升幅度低,持续时间短。说明电刺激至力竭后,细胞内有较多的Ｃａ２＋负载。增加细胞外Ｃａ２＋浓度至１５ｍｍｏｌ／Ｌ,〔Ｃａ２＋〕ｉ下降。而给予Ｎａ＋－Ｃａ２＋交换阻断剂奎尼丁（１０４ｍｏｌ／Ｌ）后,正常肌〔Ｃａ２＋〕ｉ上升,刺激肌〔Ｃａ２＋〕ｉ下降。结果提示：Ｎａ＋－Ｃａ２＋交换是正常骨胳肌外排Ｃａ２＋的途径之一;而长时间肌肉活动则可能使细胞膜Ｎａ＋－Ｃａ２＋交换方式改变,从而导致力竭肌〔Ｃａ２＋〕ｉ上升。     

槲皮素对大鼠肝酪蛋白激酶Ⅱ的抑制作用

槲皮素（Ｑｕｅｒｃｅｔｉｎ,Ｑｕｅ）为天然的黄酮类化合物,具有广泛的药理作用,酪蛋白激酶Ⅱ（ＣＫⅡ）在细胞的增殖和分化及血小板的活化过程中起重要作用．依次采用ＤＥＡＥ－纤维素和肝素－Ｓｅｐｈａｒｏｓｅ４Ｂ层析法部分纯化了大鼠肝ＣＫⅡ;发现槲皮素对ＣＫⅡ活性有强烈的抑制作用,其ＩＣ_（５０）为１２．２μｍｏｌ／Ｌ;Ｌｉｎｅｗｅａｖｅｒ－Ｂｕｒｋ双倒数作图表明：Ｑｕｅ对ＣＫⅡ的抑制作用与酪蛋白呈竞争性,而与ＡＴＰ呈非竞争性抑制．这可能是槲皮素抑制血小板聚集的又一机制,也可能与槲皮素抑制癌细胞ＤＮＡ合成有关．     

Inhibition of platelet aggregation by a fibrinogenase from Naja nigricollis venom is independent of fibrinogen degradation

Fibrinogenases, proteinases which release peptides from the carboxy-terminal end of fibrinogen, are classified as alpha-fibrinogenases or beta-fibrinogenases, based on their ability to preferentially attack the A alpha or B beta chain, respectively, of fibrinogen. alpha-Fibrinogenases have been shown to inhibit platelet aggregation whereas beta-fibrinogenases do not. We have studied the inhibition of platelet aggregation by proteinase F1, an alpha-fibrinogenase from Naja nigricollis venom. This proteinase inhibits whole blood aggregation in a dose-dependent manner, with an IC50 value of 145 micrograms. However, the proteinase fails to inhibit aggregation in washed platelet suspensions. Thus, proteinase F1 appears to require a plasma factor to cause inhibition. Since fibrinogen acts as an adhesive protein which links platelets during aggregation, and since proteinase F1 cleaves fibrinogen, we investigated the role of fibrinogen in the inhibition of platelet aggregation by proteinase F1. The degradation products of fibrinogen formed by the proteinase did not cause significant inhibition. Thus, the inhibition of platelet aggregation appears to be independent of the formation of fibrinogen degradation products. We also studied the effect of proteinase F1 on aggregation of platelets that were reconstituted with defibrinogenated plasma. The proteinase inhibited aggregation of platelets even in the absence of plasma fibrinogen. Proteinase F1 was about 4-fold more potent in inhibiting platelet aggregation in defibrinogenated blood. From these results, we conclude that the inhibition of platelet aggregation by proteinase F1 from N. nigricollis venom is independent of its action on fibrinogen.     

Secondary signals mediated by GPIIb/IIIa in thrombin-activated platelets

We have previously found that stimulation of aequorin-loaded platelets by thrombin produced a two-peaked increase in intracellular free calcium concentration ([Ca2+]i), and the development of the second peak of [Ca2+]i was closely related with the aggregation. In this report, we studied the interrelationship between the GPIIb/IIIa complex, aggregation, cytoskeletons and [Ca2+]i of platelets. The pretreatment of the platelets with dihydrocytochalasin B (4 microM), an actin polymerization inhibitor, did not inhibit aggregation and TXB2 production, but did inhibit both actin polymerization and the second peak of [Ca2+]i increase induced by thrombin, suggesting that actin polymerization and the second peak of [Ca2+]i are interrelated. GRGDSP (100 microM), a synthetic anti-adhesive peptide, has already been reported to inhibit platelet aggregation and the second peak of [Ca2+]i induced by thrombin. It also inhibited actin polymerization and TXB2 production, suggesting that aggregation was important for not only the generation of the second peak of [Ca2+]i but also for actin polymerization and TXB2 production. PGI2 (5 nM) did not abolish but only delayed aggregation, TXB2 production, actin polymerization and the second peak of [Ca2+]i increase. These findings suggest that the secondary signals are caused by aggregation (fibrinogen-binding to the GPIIb/IIIa) in thrombin-aggregated platelets, which results in the TXA2 production and the secondary peak of [Ca2+]i increase, and the latter was dependent on actin polymerization.     

Signaling pathways of the F11 receptor (F11R; a.k.a. JAM-1, JAM-A) in human platelets: F11R dimerization, phosphorylation and complex formation with the integrin GPIIIa

The F11 receptor (F11R) (a.k.a. Junctional Adhesion Molecule, JAM) was first identified in human platelets as a 32/35 kDa protein duplex that serves as receptor for a functional monoclonal antibody that activates platelets. We have sequenced and cloned the F11R and determined that it is a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules. The signaling pathways involved in F11R-induced platelet activation were examined in this investigation. The binding of M.Ab.F11 to the platelet F11R resulted in granule secretion and aggregation. These processes were found to be dependent on the crosslinking of F11R with the Fc gammaRII by M.Ab.F11. This crosslinking induced actin filament assembly with the conversion of discoidal platelets to activated shapes, leading to the formation of platelet aggregates. We demonstrate that platelet secretion and aggregation through the F11R involves actin filament assembly that is dependent on phosphoinositide-3 kinase activation, and inhibitable by wortmannin. Furthermore, such activation results in an increase in the level of free intracellular calcium, phosphorylation of the 32 and 35 kDa forms of the F11R, F11R dimerization coincident with a decrease in monomeric F11R, and association of the F11R with the integrin GPIIIa and with CD9. On the other hand, F11R-mediated events resulting from the binding of platelets to an immobilized surface of M.Ab.F11 lead to platelet adhesion and spreading through the development of filopodia and lammelipodia. These adhesive processes are induced directly by interaction of M.Ab.F11 with the platelet F11R and are not dependent on the Fc gammaRII. We also report here that the stimulation of the F11R in the presence of nonaggregating (subthreshold) concentrations of the physiological agonists thrombin and collagen, results in supersensitivity of platelets to natural agonists by a F11R-mediated process independent of the Fc gammaRII. The delineation of the two separate F11R-mediated pathways is anticipated to reveal significant information on the role of this cell adhesion molecule in platelet adhesion, aggregation and secretion, and F11R-dependent potentiation of agonist-induced platelet aggregation. The participation of F11R in the formation and growth of platelet aggregates and plaques in cardiovascular disorders, resulting in enhanced platelet adhesiveness and hyperaggregability, may serve in the generation of novel therapies in the treatment of inflammatory thrombosis, heart attack and stroke, and other cardiovascular disorders.     

Signaling Pathways of the F11 Receptor (F11R; a.k.a. JAM-1, JAM-A) in Human Platelets: F11R Dimerization,Phosphorylation and Complex Formation with the Integrin GPIIIa

The F11 receptor (F11R) (a.k.a. Junctional Adhesion Molecule, JAM) was first identified in human platelets as a 32/35 kDa protein duplex that serves as receptor for a functional monoclonal antibody that activates platelets. We have sequenced and cloned the F11R and determined that it is a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules. The signaling pathways involved in F11R-induced platelet activation were examined in this investigation. The binding of M.Ab.F11 to the platelet F11R resulted in granule secretion and aggregation. These processes were found to be dependent on the crosslinking of F11R with the FcγRII by M.Ab.F11. This crosslinking induced actin filament assembly with the conversion of discoidal platelets to activated shapes, leading to the formation of platelet aggregates. We demonstrate that platelet secretion and aggregation through the F11R involves actin filament assembly that is dependent on phosphoinositide-3 kinase activation, and inhibitable by wortmannin. Furthermore, such activation results in an increase in the level of free intracellular calcium, phosphorylation of the 32 and 35 kDa forms of the F11R, F11R dimerization coincident with a decrease in monomeric F11R, and association of the F11R with the integrin GPIIIa and with CD9. On the other hand, F11R-mediated events resulting from the binding of platelets to an immobilized surface of M.Ab.F11 lead to platelet adhesion and spreading through the development of filopodia and lammelipodia. These adhesive processes are induced directly by interaction of M.Ab.F11 with the platelet F11R and are not dependent on the FcγRII. We also report here that the stimulation of the F11R in the presence of nonaggregating (subthreshold) concentrations of the physiological agonists thrombin and collagen, results in supersensitivity of platelets to natural agonists by a F11R-mediated process independent of the FcγRII. The delineation of the two separate F11R-mediated pathways is anticipated to reveal significant information on the role of this cell adhesion molecule in platelet adhesion, aggregation and secretion, and F11R-dependent potentiation of agonist-induced platelet aggregation. The participation of F11R in the formation and growth of platelet aggregates and plaques in cardiovascular disorders, resulting in enhanced platelet adhesiveness and hyperaggregability, may serve in the generation of novel therapies in the treatment of inflammatory thrombosis, heart attack and stroke, and other cardiovascular disorders.     

Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis

Spice active principles are reported to have anti-diabetic, anti-hypercholesterolemic, antilithogenic, anti-inflammatory, anti-microbial and anti-cancer properties. In our previous report we have shown that spices and their active principles inhibit 5-lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles viz., eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulphide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggregation induced by different agonists, namely ADP (50 μM), collagen (500 μg/ml), arachidonic acid (AA) (1.0 mM) and calcium ionophore A-23187 (20 μM). Spice active principles showed preferential inhibition of arachidonic acid-induced platelet aggregation compared to other agonists. Among the spice active principles tested, eugenol and capsaicin are found to be most potent inhibitors of AA-induced platelet aggregation with IC50 values of 0.5 and 14.6 μM, respectively. The order of potency of spice principles in inhibiting AA-induced platelet aggregation is eugenol>capsaicin>curcumin>cinnamaldehyde>piperine>allyl sulphide>quercetin. Eugenol is found to be 29-fold more potent than aspirin in inhibiting AA-induced human platelet aggregation. Eugenol and capsaicin inhibited thromboxane B2 (TXB2) formation in platelets in a dose-dependent manner challenged with AA apparently by the inhibition of the cyclooxygenase (COX-1). Eugenol-mediated inhibition of platelet aggregation is further confirmed by dose-dependent decrease in malondialdehyde (MDA) in platelets. Further, eugenol and capsaicin inhibited platelet aggregation induced by agonists—collagen, ADP and calcium ionophore but to a lesser degree compared to AA. These results clearly suggest that spice principles have beneficial effects in modulating human platelet aggregation.     

Effects of propranolol and pindolol on platelet aggregation and serotonin release

The aggregation of human platelets by adrenaline and adenosine di-phosphate (ADP) and its inhibition by β-blockers was studied by measuring the light transmission of plateletrich plasma (PRP) and suspensions of washed platelets exposed to these agents. Inhibition of aggregation of PRP and washed platelets was dose related in the two β-blockers tested: propranolol and pindolol. The potent β-blockers pindolol was less inhibitory than propranolol when adrenaline and ADP were used to induce platelet aggregation. The aggregation of platelets by adrenaline has two phases. With low doses of the blockers only the second phase was inhibited whereas higher doses blocked both phases. Preincubation of human platelets (PRP and washed platelets) with both blockers per se resulted in release of ¹⁴C-labelled serotonin. Propranolol released more serotonin than pindolol. There was no concomitant release of lactic dehydrogenase. It is concluded that the effects of propranolol and pindolol on platelets do not correlate with the β-blocking activity of these agents. Rather, the more lypophilic agent, propranolol, is more active both in inhibition of aggregation and in releasing platelet serotonin. It is suggested that these actions of the drugs are related to their non-specific membrane effects.     

槲皮素硫酸酯对猪血小板胞浆游离钙浓度和蛋白激酶C的影响

槲皮素（ｑｕｅｒｃｅｔｉｎ,Ｑｕｅ）,是一种天然的黄酮类化合物,具有多种生物活性［１］,但是Ｑｕｅ水溶性差,口服时胃肠难以吸收［２］．因此,为进一步开发和利用Ｑｕｅ,人工合成水溶性Ｑｕｅ——槲桷皮素硫酸酯（ｓｏｄｉｕｍｑｕｅｒｃｅｔｉｎｓｕｌｆａｔｅ...     

A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation.

In the accompanying paper (Nemoto, Y., Namba, T., Teru-uchi, T., Ushikubi, F., Morii, N., and Narumiya, S. (1992) J. Biol. Chem. 267, 20916-20920), we have identified rhoA protein as the sole substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human blood platelets. Here we examined the role of rhoA protein in platelet functions. C3 exoenzyme added to washed platelets dose- and time-dependently ADP-ribosylated rhoA protein in situ in the cells. Concomitant with this modification, inhibition of thrombin-induced platelet aggregation was observed. This inhibition was not reversed by washing the treated platelets, but was not found when C3 exoenzyme was pretreated with mouse monoclonal anti-C3 exoenzyme antibody. C3 exoenzyme treatment did not affect thrombin-induced inositol 1,4,5-trisphosphate production. Secretion of preloaded [14C]serotonin was delayed by the enzyme treatment, but the extent of the secretion was not influenced. In addition, the enzyme treatment did not change the expression of the glycoprotein IIb-IIIa complex on the platelet surface. The enzyme treatment also suppressed platelet aggregation induced by phorbol myristate acetate. These results suggest that rhoA protein plays a role mainly in the aggregation process downstream from receptor-phospholipase C coupling. This, together with the previous finding that rhoA protein modulates stress fiber formation in cultured fibroblasts (Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K., and Hall, A. (1990) J. Cell Biol. 111, 1001-1007), suggests that rhoA protein regulates the assembly of actin filaments and the avidity of the platelet integrin (glycoprotein IIb-IIIa) in the aggregation process.     

Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

Aggregation of platelets by muscle actin. A multivalent interaction model of platelet aggregation by ADP

Filamentous muscle actin (F-actin) aggregated blood platelets while G-actin was ineffective. This aggregation could be blocked by ATP suggesting a possible role of actin-bound ADP in this process. Actin-bound ADP caused platelet aggregation at concentrations significantly lower than equivalent concentrations of free ADP. Thus, actin potentiates the aggregating action of ADP. An actin antibody or DNase I inhibited this aggregation showing the requirement of actin in this process. Like other physiological agents, Ca⁺⁺ was necessary for platelet aggregation by actin. Platelets fixed in formaldehyde were not aggregated by actin showing the need for viable platelets. Since F-actin contains 1 mole of bound ADP/mole protein, it is postulated that actin potentiates ADP-induced aggregation by providing multiple interaction sites for platelets.