Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation.

Epidermal growth factor and platelet-derived growth factor can stimulate the production of the second messenger inositol trisphosphate in responsive cells, but the biochemical pathway for these signaling events has been uncertain because the reactions have not been reconstituted with purified molecules in vitro. A reconstitution is described that requires not only the growth factor, its receptor with tyrosine kinase activity, and the soluble phospholipase C-gamma 1, but also the small soluble actin-binding protein profilin. Profilin binds to the substrate phosphatidylinositol 4,5-bisphosphate and inhibits its hydrolysis by unphosphorylated phospholipase C-gamma 1. Phosphorylation of phospholipase C-gamma 1 by the epidermal growth factor receptor tyrosine kinase overcomes the inhibitory effect of profilin and results in an effective activation of phospholipase C-gamma 1.     

DNA gyrase and the supercoiling of DNA

Negative supercoiling of bacterial DNA by DNA gyrase influences all metabolic processes involving DNA and is essential for replication. Gyrase supercoils DNA by a mechanism called sign inversion, whereby a positive supercoil is directly inverted to a negative one by passing a DNA segment through a transient double-strand break. Reversal of this scheme relaxes DNA, and this mechanism also accounts for the ability of gyrase to catenate and uncatenate DNA rings. Each round of supercoiling is driven by a conformational change induced by adenosine triphosphate (ATP) binding: ATP hydrolysis permits fresh cycles. The inhibition of gyrase by two classes of antimicrobials reflects its composition from two reversibly associated subunits. The A subunit is particularly associated with the concerted breakage-and-rejoining of DNA and the B subunit mediates energy transduction. Gyrase is a prototype for a growing class of prokaryotic and eukaryotic topoisomerases that interconvert complex forms by way of transient double-strand breaks.     

Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis

The mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes were examined. When [3H]arachidonate labeling of phospholipids was used as an indicator of phospholipase C activation, transient increases in [3H]diacylglycerol were observed between 0.5 and 10 minutes after the onset of insulin treatment. With [3H]glycerol labeling as an indicator of de novo phospholipid synthesis, [3H]diacylglycerol was increased maximally at 1 minute and remained elevated for 20 minutes. [3H]Glycerol-labeled diacylglycerol was largely derived directly from phosphatidic acid. Insulin increased de novo phosphatidic acid synthesis within 5 to 10 seconds; within 1 minute, this synthesis was 60 times greater than that of controls. Thus, the initial increase in diacylglycerol is due to both increased hydrolysis of phospholipids and a burst of de novo phosphatidic acid synthesis. After 5 to 10 minutes, de novo phosphatidic acid synthesis continues as a major source of diacylglycerol. Both phospholipid effects of insulin seem important for generating diacylglycerol and other phospholipid-derived intracellular signaling substances.     

Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes.

Mammalian 3T3-L1 cells differentiate into adipocytes after continuous exposure to pharmacological doses of insulin or physiological doses of insulin-like growth factor I (IGF-1). Expression of transfected ras oncogenes led to differentiation of these cells into adipocytes in the absence of externally added insulin or IGF-I. Cells transfected with normal ras genes or the tyrosine kinase trk oncogene did not differentiate. Transfection with a dominant inhibitory ras mutant resulted in inhibition of differentiation. Exposure of untransfected 3T3-L1 cells to insulin stimulated formation of the active Ras.GTP complex. These observations indicate that Ras proteins participate in signal transduction pathways initiated by insulin and IGF-I in these cells.     

水稻转录因子DREB1的克隆与原核表达

DREB类转录因子特异地与DRE顺式作用元件结合,在非生物逆境胁迫(干旱、低温、高盐)中激活胁迫诱导基因的表达,使植物能够耐受水分胁迫的影响。以顺式元件rd29A3 cis构建诱饵质粒.通过酵母单杂交方法从水稻cDNA文库中筛选获得7个阳性质粒,序列分析显示阳性质粒中插入的cDNA序列一致,根据核苷酸序列推导出cDNA编码一219个氨基酸组成的多肽,该多肽有保守的EREBP/AP2结构域,N端为核定位信号(NLS),而C端则为转录激活区域,属于EREBP/AP2类转录因子,命名为RdreB1。重新合成的RdreB1 I基因降低了GC比含量,诱导处理产生了约45 kD的特异表达的融合蛋白。     

鸭肉肌内磷脂水解酶的提取及相关酶系的酶活测定

为了优化肌内磷脂水解酶的提取过程,建立各种鸭肉肌内磷脂水解酶活的测定方法。采用pH 7.5、8.0、8.5的0.1 mol.L-1的Tris-HCl缓冲液提取鸭肉肌内磷脂水解酶,用饱和度范围为0～90%的硫酸铵进行盐析,研究新鲜鸭肉及板鸭中酸性脂肪酶、中性脂肪酶、总磷脂酶、磷脂酶A2(PLA2)、磷脂酶C(PLC)和磷脂酶D(PLD)的活力。结果表明,肌内磷脂水解酶的最佳pH为8.0,最佳盐析条件为70%饱和度的硫酸铵溶液。透析过后的粗酶液中酸性脂肪酶活略大于中性脂肪酶,磷脂酶活次之。     

挤压协同酶法制备高粱蛋白ACE抑制肽及其稳定性

【目的】 利用挤压协同酶法制备高粱蛋白ACE抑制肽,为提高高粱蛋白资源的利用效率提供参考。【方法】 以高粱粉为原料,先经挤压处理,再经淀粉酶酶解,最后通过碱性蛋白酶酶解,获得高粱蛋白ACE抑制肽。研究物料水分、挤压温度和淀粉酶活力对高粱蛋白酶解液的水解度和ACE抑制活性的影响,并探讨高粱蛋白ACE抑制肽的稳定性。【结果】 随着物料水分含量和挤压温度的增加,挤压过程中单位机械能耗（SME）逐渐降低。在挤压环境下,高粱中淀粉和蛋白质的相互结合变得松散,淀粉-蛋白质包埋体系被破坏;同时高粱中球形蛋白质体被打破,提高所获得高粱蛋白的酶敏感性,在碱性蛋白酶的作用下生成更多具有抑制活性的短肽。挤压过程中物料水分含量和挤压温度以及α-淀粉酶活力对高粱蛋白酶解液的水解度和ACE抑制率有显著影响。随着物料水分的增加,蛋白质分子的聚合程度下降,使得高粱蛋白酶解液的水解度和ACE抑制率随之增加,当物料水分增加至19%后,挤压过程对蛋白质周围的淀粉分子的破坏作用降低,水解度和ACE抑制率的上升趋势趋于平缓;当挤压温度从120℃增加至180℃时,高粱内部的蛋白质-淀粉包埋体系破坏加剧,同时蛋白质的空间结构在高温作用下的变性程度加大,高粱蛋白酶解液的水解度由7.42%增加至11.06%,同时高粱蛋白ACE抑制肽的抑制率也由46.57%增加至53.41%;挤压后高粱粉经α-淀粉酶处理,进一步去除包裹在蛋白质周围的淀粉,发现随着α-淀粉酶活力的增加,高粱内部的蛋白质-淀粉包埋体系破坏程度加剧,为制备高粱蛋白ACE抑制肽提供更多原料,导致高粱蛋白酶解液的水解度和ACE抑制率随之增加,当α-淀粉酶活力增加至2.0 U·g ^-1时,淀粉酶与淀粉结合达到饱和状态,水解度和ACE抑制率趋于稳定。高粱蛋白ACE抑制肽经温度和酸碱处理后,ACE抑制活性在68.1%—71.31%,保持了良好的抑制活性;高粱蛋白ACE抑制肽在体外经胃肠道酶系消化酶解后,ACE抑制活性均高于73%,依然保持了较强的ACE抑制活性,说明挤压协同酶法制备的高粱蛋白ACE抑制肽具有长期保存有效性,同时能够在胃肠道消化后保持生物活性。 【结论】 采用挤压协同酶法可以显著提高高粱蛋白酶解液的水解度和ACE抑制肽的活性,同时制备的高粱蛋白ACE抑制肽具有良好的稳定性,为拓宽高粱的利用和制备功能性食品配料提供了一条新途径。     

T cell antigen receptor-mediated activation of phospholipase C requires tyrosine phosphorylation

Triggering of the antigen-specific T cell receptor-CD3 complex (TCR-CD3) stimulates a rapid phospholipase C-mediated hydrolysis of inositol phospholipids, resulting in the production of second messengers and in T cell activation and proliferation. The role of tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited TPK activity and tyrosine phosphorylation of the TCR zeta subunit, but not phospholipase C activity, genistein prevented TCR-CD3-mediated phospholipase C activation, interleukin-2 receptor expression, and T cell proliferation. These findings indicate that tyrosine phosphorylation is an early and critical event that most likely precedes, and is a prerequisite for, inositol phospholipid breakdown during receptor-mediated T cell activation.     

一个葡萄抗逆相关转录因子VvPF1基因的in silico克隆及生物信息学分析

AP2/EREBP是植物所特有的一类转录因子,广泛参与植物生长、发育以及多种生理生化反应的信号传导.本文利用in silico克隆方法获得葡萄VvPF1 基因,并利用生物信息学方法对该基因编码产物从氨基酸组成、理化性质、进化关系、二级及三级结构、功能等方面进行预测和分析.结果表明:VvPF1 是一个含有明显AP2结构域,具有潜在核定位能力的ERF类转录因子.此外,VvPF1与CaPF1, JERF1等抗逆相关的转录因子具有较高的同源性,因此推测,VvPF1很可能参与葡萄对生物和非生物胁迫的信号传导.     

Positive regulation of Itk PH domain function by soluble IP4

Pleckstrin homology (PH) domain-mediated protein recruitment to cellular membranes is of paramount importance for signal transduction. The recruitment of many PH domains is controlled through production and turnover of their membrane ligand, phosphatidylinositol 3,4,5-trisphosphate (PIP3). We show that phosphorylation of the second messenger inositol 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) establishes another mode of PH domain regulation through a soluble ligand. At physiological concentrations, IP4 promoted PH domain binding to PIP3. In primary mouse CD4+CD8+ thymocytes, this was required for full activation of the protein tyrosine kinase Itk after T cell receptor engagement. Our data suggest that IP4 establishes a feedback loop of phospholipase C-gamma1 activation through Itk that is essential for T cell development.     

植物响应水分胁迫的分子机制研究进展

水分胁迫是影响植物生长发育及农作物产量和品质的主要限制因子.论述植物响应水分胁迫的分子机制,其信号转导整个过程包括细胞感知水分胁迫信号、胁迫信号的传递、第二信使激活一些相关的转录因子、转录因子与顺式元件相瓦作用诱导基因的表达.在水分胁迫起始信号和靶基因表达之间至少存在4条独立的信号转导途径,包括两条ABA依赖型和两条非...