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961.
We have developed a modified particle bombardment method for plant transgenesis. An intein-tag and a 6×Cys-tag were successively fused to the N-terminus of a hyperactive Tn5 transposase. The modified transposase was immobilized on bare gold microscopic particles via covalent binding of a 6×Cys-tag sulfydryl groups to the gold surface. The tethered transposase can bind the transposon DNA in vitro to form the transposome in the absence of Mg2? ions. After bombardment of the gold particles carrying the transposomes into the plant cells, the transposomes will be released from the carrier due to the activated self-cleavage function of intein-tag. Our data showed this procedure integrated foreign DNA into the plant genome with an increased transformation frequency as compared to the conventional particle bombardment method. A single copy insertion can also be obtained by decreasing of the assembled transposon DNA amount in relation to plant cell biomass. 相似文献
962.
干旱是严重限制作物生长及产量的环境因子之一。经过长期的进化,植物形成了一套响应干旱胁迫的信号转导机制,包括对干旱胁迫信号的感知,第二信使的产生,信号转导和信号网络的形成。信号转导的结果是导致相关基因的表达和蛋白的合成,进而引起植物体渗透调节及抗氧化系统的改变,最终使植物适应干旱逆境或增强植物抗旱能力。干旱胁迫通常会促进ROS的积累及其他次级信号分子的产生。MAPK级联途径是真核生物信号转导最为保守的途径,在植物的生长发育及各种胁迫信号的传导中均起着较重要的作用。综述干旱胁迫信号及ROS→MAPK和ROS→Ca2+介导的信号途径,以及信号转导途径的调控机制。 相似文献
963.
964.
In cellular environments, coupled hydrolytic reactions are used to force efficient product formation in enzyme-catalyzed reactions. In the first step of protein synthesis, aminoacyl-tRNA synthetases react with amino acid and ATP to form an enzyme-bound adenylate that, in the next step, reacts with tRNA to form aminoacyl-tRNA. The reaction liberates pyrophosphate (PP(i)) which, in turn, can be hydrolyzed by pyrophosphatase to drive efficient aminoacylation. A potential polymorphic variant of human tryptophanyl-tRNA synthetase is shown here to sequester tryptophanyl adenylate. The bound adenylate does not react efficiently with the liberated PP(i) that normally competes with tRNA to resynthesize ATP and free amino acid. Structural analysis of this variant showed that residues needed for binding ATP phosphates and thus PP(i) were reoriented from their conformations in the structure of the more common sequence variant. Significantly, the reorientation does not affect reaction with tRNA, so that efficient aminoacylation is achieved. 相似文献
965.
Yan Ding Hua Yang Jing Bo Feng Ying Qiu Dong Sheng Li Yi Zeng 《In vitro cellular & developmental biology. Animal》2013,49(10):771-777
Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold great potential for their therapeutic use in various clinical diseases. Many publications have reported on human blood-derived alternatives to animal serum for culturing mesenchymal stem cells, such as human serum, allogenic umbilical cord blood serum, and human platelet derivatives. However, it is not clear whether human umbilical cord blood plasma (UCBP), as the surplusage of umbilical cord blood mesenchymal stem cell extraction, could be used. In this study, in order to make the best of umbilical cord blood, the human UCBP was dialyzed to replace fetal bovine serum (FBS) in the culture medium. hUC-MSCs were cultured in the new medium. Cell growth rate, specific biomarkers, and differentiation properties were detected to characterize the cell proliferation and MSC-specific properties. The hUC-MSCs cultured in such derived medium were verified with proliferation rate, cluster differentiation markers, cell cycle, as well as differentiation capabilities. Such dialyzed human UCBP is fully comparable with, if not superior to, FBS in deriving and culturing hUC-MSCs. 相似文献
966.
Ren X Zhao L Sivashanmugam A Miao Y Korando L Yang Z Reardon CA Getz GS Brouillette CG Jerome WG Wang J 《Biochemistry》2005,44(45):14907-14919
Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination. 相似文献
967.
968.
969.
Min Wu Qi Shen Yong Yang Sheng Zhang Wen Qu Jing Chen Hongying Sun Shuqing Chen 《Journal of industrial microbiology & biotechnology》2013,40(6):589-599
Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain. 相似文献
970.
We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control
in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to
measure the intracellular [Na+]i in these cells loaded with the Na+ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na+]i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations
up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA2 activity (10 μm AACOCF3) obviated EGF-induced increases in [Na+]i. In contrast, PGE2 (10 μg/ml) and cAMP (2 mm) increased [Na+]i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na+]i. Similarly, EGF failed to increase [Na+]i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na+]i followed by a decline towards its baseline value. EGF-induced increases in [Na+]i were unaltered by inhibition of K+ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA2 activity. Their stimulation increases PGE2 and cAMP levels resulting in PKA and NKCC activation.
Received: 18 December 2000/Revised: 24 May 2001 相似文献