New Analytical Methods for Molecular Imaging in Single Live Cells and Interfacial Molecular Assemblies
单个活细胞和界面分子组装体分子成像的新分析方法
基本信息
- 批准号:15105003
- 负责人:
- 金额:$ 67.14万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (S)
- 财政年份:2003
- 资助国家:日本
- 起止时间:2003 至 2007
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
The purpose of the present research is to develop new analytical methods for molecular imaging.We have developed optical probes that the intracellular signaling can be monitored in vivo in living cells by genetically encoded intracellular fluorescent and bioluminescent probes or indicators, which include second messengers such as guanosine 3',5'-cyclic monophosphate(cGMP), inositol 1,4,5-trisphosphate(IP3), phosphatidylinositol 3,4,5-trisphosphate(PIP3), and nitric oxide(NO), protein phosphorylations, protein-protein interactions, and protein localizations. An amplifier-coupled fluorescent indicator for NO was developed to visualize physiological nanomolar dynamics of NO in living cells with a detection limit of 0.1 nM. Fluorescent indicators for PIP3 based on FRET allowed localized analysis of PIP3 concentrations. We analyzed the spatio-temporal regulation of the PIP3 production in single living cells. A intracellular fluorescent indicator for another lipid second messenger, diacylgly … More cerol was also reported.We developed genetically encoded fluorescent indicators for visualizing protein phosphorylations in living cells. As a result of the phosphorylation, FRET was induced between the two fluorescent units. Upon activation of the phosphatases, the phosphorylated substrate domain is dephosphorylated and the FRET signal is decreased.To monitor protein-protein interactions, a new method was developed by based on split-reporter reconstitution. One of the applications of the split-GFP reconstitution system is a probe for visualizing endogenous mRNA in single living cells. We developed genetically-encoded RNA probes for characterizing localization and dynamics of mtRNA in single living cells. We showed that ND6 mtRNA is localized within mitochondria and concentrated particularly on mtDNA. We demonstrated split-luciferase reconstitution system for a specific protein transported into the intracellular organelles. We developed a genetically encoded bioluminescence indicator for monitoring the release of proteins from the mitochondria in living cells. We also visualized movements of androgen receptor(AR), glucocorticoid receptor(GR) and STAT3 into the nucleus in living cells and mice. These methods allowed high-throughput screening of chemicals with multi-titer plates and imaging of the protein dynamics in living mice.Nucleobase molecular tips were prepared by chemical modification of underlying metal tips with thiol derivatives of adenine, guanine, cytosine, and uracil and the outmost single nucleobase adsorbate probes intermolecular electron tunneling to or from a sample nucleobase molecule. We found that the electron tunneling between a sample nucleobase and its complementary nucleobase molecular tip was much facilitated compared with its noncomplementary counterpart. Methods of analysis described above have been promised to become key analytical methods for studying chemistry and biology. Less
本研究的目的是开发新的分子成像分析方法,我们开发了可通过基因编码的细胞内荧光和生物发光探针或指示剂在活细胞中体内监测细胞内信号的光学探针,这些探针包括第二信使如鸟苷3 ',5'-环单磷酸(cGMP)、肌醇1,4,5-三磷酸(IP 3)、磷脂酰肌醇3,4,5-三磷酸(PIP 3)和一氧化氮(NO)、蛋白质磷酸化、蛋白质-蛋白质相互作用和蛋白质定位。开发了一种用于NO的荧光标记物偶联的荧光指示剂,以观察活细胞中NO的生理纳摩尔动力学,检测限为0.1 nM。基于FRET的PIP 3的荧光指示剂允许PIP 3浓度的局部分析。我们分析了单个活细胞中PIP 3产生的时空调控。另一种脂质第二信使二酰基甘氨酰的细胞内荧光指示剂 ...更多信息 我们开发了基因编码的荧光指示剂,用于观察活细胞中蛋白质磷酸化。作为磷酸化的结果,在两个荧光单位之间诱导FRET。磷酸酶激活后,磷酸化的底物结构域被去磷酸化,FRET信号减弱。分裂-GFP重建系统的应用之一是用于可视化单个活细胞中的内源性mRNA的探针。我们开发了基因编码的RNA探针,用于表征单个活细胞中mtDNA的定位和动态。我们发现ND 6 mtDNA定位于线粒体内,尤其集中在mtDNA上。我们证明了分裂荧光素酶重建系统的一个特定的蛋白质转运到细胞内的细胞器。我们开发了一种基因编码的生物发光指示剂,用于监测活细胞中线粒体蛋白质的释放。我们还观察了雄激素受体(AR)、糖皮质激素受体(GR)和STAT 3在活细胞和小鼠中进入细胞核的运动。这些方法允许高通量的化学品筛选与多滴定板和成像的蛋白质动力学在活mice.Nucleobase分子tips的制备通过化学修饰的基础金属提示与硫醇衍生物的腺嘌呤,鸟嘌呤,胞嘧啶,尿嘧啶和最外层的单个核碱基吸附探针分子间电子隧穿或从一个样本的核碱基分子。我们发现,与非互补碱基相比,样品碱基与其互补碱基分子尖端之间的电子隧穿更加容易。上述分析方法已有望成为研究化学和生物学的关键分析方法。少
项目成果
期刊论文数量(118)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Optical probes to identify the glucocorticoid receptor ligands in living cells
- DOI:10.1016/j.steroids.2007.08.006
- 发表时间:2007-12-01
- 期刊:
- 影响因子:2.7
- 作者:Awais, Muhammad;Sato, Moritoshi;Umezawa, Yoshio
- 通讯作者:Umezawa, Yoshio
A proinflammatory cytokine sensor cell for assaying inslammatory actvities of nanoparticles
用于测定纳米颗粒炎症活性的促炎细胞因子传感器细胞
- DOI:
- 发表时间:2007
- 期刊:
- 影响因子:0
- 作者:S. B. Kim;T. Ozawa;H. Tao;Y. Umezawa
- 通讯作者:Y. Umezawa
Complementary base-pair facilitated electron tunneling for electrically retrieving complementary nucleobases.
互补碱基对促进电子隧道效应,用于电检索互补核碱基。
- DOI:
- 发表时间:2006
- 期刊:
- 影响因子:0
- 作者:T.Ohshiro;Y.Umezawa
- 通讯作者:Y.Umezawa
A Fullerene Molecular Tip Can Detect Localized and Rectified Electron Tuuneling within a Single Fuilerene-Porphyrin Pair
富勒烯分子尖端可以检测单个富勒烯-卟啉对内的局部和整流电子调谐
- DOI:
- 发表时间:2005
- 期刊:
- 影响因子:0
- 作者:T.Nishino.;T.Ito;Y.Umezawa
- 通讯作者:Y.Umezawa
High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase
- DOI:10.1073/pnas.0401722101
- 发表时间:2004-08-10
- 期刊:
- 影响因子:11.1
- 作者:Kim, SB;Ozawa, T;Umezawa, Y
- 通讯作者:Umezawa, Y
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UMEZAWA Yoshio其他文献
UMEZAWA Yoshio的其他文献
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{{ truncateString('UMEZAWA Yoshio', 18)}}的其他基金
Chemically modified STM tips for molecular imaging of DNA and cytochrome c
用于 DNA 和细胞色素 c 分子成像的化学修饰 STM 探针
- 批准号:
13440219 - 财政年份:2001
- 资助金额:
$ 67.14万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Intracellular fluorescent probes for chemical processes in single living cells
用于单个活细胞化学过程的细胞内荧光探针
- 批准号:
13554030 - 财政年份:2001
- 资助金额:
$ 67.14万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Scannning Tunneling Microscopy Based on Chemically Modified Tips
基于化学修饰尖端的扫描隧道显微镜
- 批准号:
10554044 - 财政年份:1998
- 资助金额:
$ 67.14万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Analytical Methods for Bioactive Substances Based on Cellular Signal Transduction Mechanisms
基于细胞信号转导机制的生物活性物质分析方法
- 批准号:
10304061 - 财政年份:1998
- 资助金额:
$ 67.14万 - 项目类别:
Grant-in-Aid for Scientific Research (A).
Design of Membrane Surfaces for Molecular Recognition and Signal Transduction, and Their Application for Chemical Sensing
用于分子识别和信号转导的膜表面设计及其在化学传感中的应用
- 批准号:
05403016 - 财政年份:1993
- 资助金额:
$ 67.14万 - 项目类别:
Grant-in-Aid for General Scientific Research (A)
Fundamental Study on Membrane Surface Chemistry for Ion-Selective Electrodes
离子选择电极膜表面化学基础研究
- 批准号:
03453038 - 财政年份:1991
- 资助金额:
$ 67.14万 - 项目类别:
Grant-in-Aid for General Scientific Research (B)
Development of Active Transport and Ion-Channel Sensors
主动传输和离子通道传感器的开发
- 批准号:
63430008 - 财政年份:1988
- 资助金额:
$ 67.14万 - 项目类别:
Grant-in-Aid for General Scientific Research (A)
Development of Novel Sensors Using Macrocyclic Polyamines as Sensory Elements
使用大环多胺作为传感元件的新型传感器的开发
- 批准号:
63840019 - 财政年份:1988
- 资助金额:
$ 67.14万 - 项目类别:
Grant-in-Aid for Developmental Scientific Research (B).
Fundamental Study on Ion Transport at Membrane Surface and Response Mechanism of Ion-Selective Electrodes.
膜表面离子输运及离子选择电极响应机制的基础研究。
- 批准号:
60470033 - 财政年份:1985
- 资助金额:
$ 67.14万 - 项目类别:
Grant-in-Aid for General Scientific Research (B)
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Research Grants