Molecular interactions of a soluble gibberellin receptor, GID1, with a rice DELLA protein, SLR1, and gibberellin

GIBBERELLIN INSENSITIVE DWARF1 (GID1) encodes a soluble gibberellin (GA) receptor that shares sequence similarity with a hormone-sensitive lipase (HSL). Previously, a yeast two-hybrid (Y2H) assay revealed that the GID1-GA complex directly interacts with SLENDER RICE1 (SLR1), a DELLA repressor protein in GA signaling. Here, we demonstrated, by pull-down and bimolecular fluorescence complementation (BiFC) experiments, that the GA-dependent GID1-SLR1 interaction also occurs in planta. GA(4) was found to have the highest affinity to GID1 in Y2H assays and is the most effective form of GA in planta. Domain analyses of SLR1 using Y2H, gel filtration, and BiFC methods revealed that the DELLA and TVHYNP domains of SLR1 are required for the GID1-SLR1 interaction. To identify the important regions of GID1 for GA and SLR1 interactions, we used many different mutant versions of GID1, such as the spontaneous mutant GID1s, N- and C-terminal truncated GID1s, and mutagenized GID1 proteins with conserved amino acids replaced with Ala. The amino acid residues important for SLR1 interaction completely overlapped the residues required for GA binding that were scattered throughout the GID1 molecule. When we plotted these residues on the GID1 structure predicted by analogy with HSL tertiary structure, many residues were located at regions corresponding to the substrate binding pocket and lid. Furthermore, the GA-GID1 interaction was stabilized by SLR1. Based on these observations, we proposed a molecular model for interaction between GA, GID1, and SLR1.     

恒定磁场出生前暴露对仔鼠生长发育的影响

目的:探讨不同强度恒定磁场出生前暴露对仔鼠生后生长发育的影响。方法:将30只孕鼠自怀孕第0天始分别置于不同强度的恒定磁场(0.04T;0.08T;0.12T)中饲养,10只孕鼠作为对照。孕鼠让其自然分娩。仔鼠生后第2、3、8、20、22天分别开始观察和记录耳廓张开、平面翻正、张眼、睾丸下降、阴唇张开的时间。结果:耳廓张开及睁眼的时间在0.12T组分别为4.42±0.48天及14.19±0.94天,较对照组耳廓张开时间4.01±0.47天和睁眼时间13.21±0.79天相比差异有统计学意义,与其他强度磁场组相比无显著性差异.平面翻正、阴唇张开及睾丸下降出现时间在各组间相比差异无统计学意义。结论:恒定磁场出生前暴露可能对仔鼠的生长发育起到一定的影响,这种影响可能与磁场强度具有相关性。随着磁场强度的增加各个器官生长发育的时间相对延长。     

观赏用的红肉苹果的组织培养

1植物名称红肉苹果[Malus pumila var.niedzwet- zkyana(Dieck)Schneid]。2材料类别春梢幼嫩茎段。3培养条件MS为基本培养基,(1)芽诱导培养基:MS 6-BA 0.2mg·L~(-1)(单位下同) NAA 0.2;(2)芽增殖培养基:MS 6-BA 0.5 NAA 0.5:(3)生根培养基:1/2MS IBA 0.1 NAA 0.1。以上培养基中均加入5.5 g·L~(-1)琼脂粉和30g·L~(-1)蔗糖,pH 5.8;     

松墨天牛对马尾松挥发物的触角电位和行为反应

利用触角电位和嗅觉测定技术,比较分析松墨天牛Monochamus alternatus Hope对马尾松(Pinus massoniana Lamb)健康和松材线虫危害的枝条挥发物的触角电位和行为反应特点。实验结果表明:未交配松墨天牛对健康枝条挥发物的EAG反应值大于被害枝条的,交配后天牛对被害枝条的EAG反应值明显大于健康枝条的。在“Y”型嗅觉仪中,未交配天牛对健康枝条挥发物表现为正趋性,对被害枝条挥发物表现为负趋性,而交配后天牛对被害枝条挥发物表现为正趋性,对健康枝条挥发物表现为负趋性。说明不同发育时期的松墨天牛成虫对不同生理状态的寄主具有不同的敏感性和选择性。     

Crystal structures of blasticidin S deaminase (BSD): implications for dynamic properties of catalytic zinc

The set of blasticidin S (BS) and blasticidin S deaminase (BSD) is a widely used selectable marker for gene transfer experiments. BSD is a member of the cytidine deaminase (CDA) family; it is a zinc-dependent enzyme with three cysteines and one water molecule as zinc ligands. The crystal structures of BSD were determined in six states (i.e. native, substrate-bound, product-bound, cacodylate-bound, substrate-bound E56Q mutant, and R90K mutant). In the structures, the zinc position and coordination structures vary. The substrate-bound structure shows a large positional and geometrical shift of zinc with a double-headed electron density of the substrate that seems to be assigned to the amino and hydroxyl groups of the substrate and product, respectively. In this intermediate-like structure, the steric hindrance of the hydroxyl group pushes the zinc into the triangular plane consisting of three cysteines with a positional shift of approximately 0.6 A, and the fifth ligand water approaches the opposite direction of the substrate with a shift of 0.4 A. Accordingly, the zinc coordination is changed from tetrahedral to trigonal bipyramidal, and its coordination distance is extended between zinc and its intermediate. The shift of zinc and the recruited water is also observed in the structure of the inactivated E56Q mutant. This novel observation is different in two-cysteine cytidine deaminase Escherichia coli CDA and might be essential for the reaction mechanism in BSD, since it is useful for the easy release of the product by charge compensation and for the structural change of the substrate.     

Inhibition of the ryanodine receptor calcium channel in the sarcoplasmic reticulum of skeletal muscle by an ADP/ATP translocase inhibitor, atractyloside.

The effects of an inhibitor of ADP/ATP translocase (AAT) mainly expressed in the mitochondria inner membrane, atractyloside (ATR), on the gating property of the Ca2+ channels in the sarcoplasmic reticulum (SR) vesicles from the rabbit skeletal muscle were investigated using ion flux measurement and single channel recording. At 10 microM of cytoplasmic Ca2+, ATR decreased the rate constant of choline+ influx through the Ca2+ channels up to about 60% and perfectly inhibited about half the population of single Ca2+ channels incorporated into planar bilayers. Furthermore, the inhibition of the Ca2+ channels by ATR was effective at lower Ca2+. These results support the previous results that AAT exists in the skeletal muscle SR and plays a key role in the Ca2+ mobilization of the skeletal muscle cell [Yamaguchi, N., and Kasai, M. (1998) Biochem. J. 335, 541-547], and the number of Ca2+ channels regulated by AAT is thought to depend on the cytoplasmic Ca2+ concentration.     

Induction of cell shape changes through activation of the interleukin-3 common beta chain receptor by the RON receptor-type tyrosine kinase.

The RON receptor-type tyrosine kinase, a member of the hepatocyte growth factor receptor family, is a receptor for macrophage-stimulating protein (MSP). Recently, we observed that MSP induces morphological changes in interleukin (IL)-3-dependent Ba/F3 cells ectopically expressing RON. We show here that stimulation of those cells with either MSP or IL-3 increases tyrosine phosphorylation of proteins of 130, 110, 90, 62, and 58 kDa and induces similar morphological changes, accompanied by unique nuclear shape and redistribution of F-actin. A tyrosine kinase inhibitor, genistein, blocked both the increase in tyrosine phosphorylation and morphological changes. Upon stimulation with either MSP or IL-3, prominent tyrosine-phosphorylated pp90 was similarly co-immunoprecipitated with the common beta chain of IL-3 receptor (betac). Unlike IL-3, stimulation with MSP increased tyrosine phosphorylation of betac without activation of JAK2, resulting in morphological changes with modest cell growth. Confocal immunofluorescence analyses showed colocalization of RON, betac, and tyrosine-phosphorylated proteins. In vitro kinase assays revealed that autophosphorylated RON phosphorylated betac. These results suggest that the signaling pathway for morphological changes through betac and its associated protein pp90 is distinct from the pathway for cell growth in the IL-3 signal transduction system.     

Cloning and sequence analysis of cDNA encoding active phosphoenolpyruvate carboxylase of the C4-pathway from maize.

A recombinant clone, pM52, containing cDNA for maize phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) was isolated from a maize leaf cDNA library constructed using an expression vector in Escherichia coli. The screening of the clone was conveniently performed through its ability to complement the phenotype (glutamate requirement) of PEPCase-negative mutant of E. coli. The enzyme encoded by this clone was identical with the major PEPCase in maize, a key enzyme in the C4-pathway, as judged from its allosteric properties and immunological reactivity. The cloned cDNA (3093 nucleotides in length) contained an open reading frame of 2805 nucleotides, the 3'-untranslated region of 222 nucleotides and the poly(dA) tract of 64 nucleotides. The deduced amino acid sequence (935 residues) of the enzyme showed higher homology with that of an enterobacterium, E. coli (43%) than that of a cyanobacterium (blue-green alga), Anacystis nidulans (33%).     

EF—Tumt和EF—Tsmt在不同发育阶段小鼠各组织中的表达分析

线粒体蛋白质翻译延长因子Ｔｕ和Ｔｓ（ｍｉｔｏｃｈｏｎｄｒｉａｌｅｌｏｎｇａｔｉｏｎｆａｃｔｏｒＴｕａｎｄＴｓ,ＥＦＴｕｍｔａｎｄＥＦＴｓｍｔ）是由核基因编码的两个蛋白质,它们的功能和调控对细胞的生长发育有重要意义。采用ＥＦＴｕｍｔ和ＥＦＴｓｍｔ重组蛋白分别制备了抗ＥＦＴｕｍｔ和抗ＥＦＴｓｍｔ特异抗体并以此检测了它们在小鼠不同发育时期心肌、骨骼肌、肝、脑、脾等组织中的表达。蛋白质印迹结果表明ＥＦＴｕｍｔ和ＥＦＴｓｍｔ在各组织中的表达水平不同、有明显的组织差异性,并都受发育的调节。ＥＦＴｕｍｔ在同一发育时期各组织中的表达及随发育的变化趋势与ＥＦＴｓｍｔ基本一致。结果提示ＥＦＴｕｍｔ和ＥＦＴｓｍｔ的表达水平与组织细胞能量代谢水平密切相关,它们不仅在体内以复合体形式发挥作用,其基因表达可能受同一机制的调控。     

含谷胱甘肽硫转移酶基因工程菌表达条件研究

将重组谷胱甘肽硫转移酶 (GST)大肠杆菌表达株BL2 1(DE3 )PGEX 作为研究对象 ,进行了生长及表达条件的优化研究 ,分析比较了不同培养条件对菌体生长的影响以及诱导剂的添加量对产物表达量的影响。