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
基本信息
- 批准号:10304061
- 负责人:
- 金额:$ 24.32万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (A).
- 财政年份:1998
- 资助国家:日本
- 起止时间:1998 至 2000
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
In most of chemical methods of analysis, selectivity of analytes against interfering substances is essentially governed by the respective binding constant, K_f, between the analyte, A, and its molecular recognition reagent, B, K_f=[AB]/[A][B] This is generally the basis for binding assay. A typical example is immunoassay. Recently, we have been studying analytical methods for bioactive substances, being based not only on binding to receptors but also the following biological process of signal transduction is, in part, taken into account. The idea has lead us to explore some new analytical methods that can evaluate physiologically more relevant selectivity of analytes (agonists or antagonists). Typical examples are ion channel, transporter proteins and kinase-type proteins embedded in lipid bilayer membranes. In contrast to the binding assay and related techniques, these sensors utilize the corresponding downstream signals, such as ion-channel currents or the extent of phosphorylation o … More f the receptor itself and its target proteins for yielding physiologically more relevant signals compared to those with the binding assay approach. This is achieved by both in vitro and in vivo analytical approaches using surface plasmon resonance (SPR) and fluorescent probe reagents involving part of these cellular signaling mechanisms. As to intracellular receptor proteins, Ca^<2+> signaling pathways for example based on a Ca^<2+>-dependent on/off switching of calmodulin, the agonist selectivity in terms of ion selectivity has been evaluated by SPR for the formation of a Ca^<2+> calmodulin target peptide ternary complex. By this method, not only Ca^<2+> ion, but also Sr^<2+>, Pb^<2+>, Cd^<2+> and many of lanthunum ions have been found to ride on "Ca^<2+> signaling" and behave as a strong agonist toward the Ca^<2+> signaling. Screening of agonists concerning their physiologically relevant selectivity is very important for biological studies as well as for pharmaceutical needs. The conventional binding assays can neither give sufficient information on the agonist activity nor discriminate between agonists and antagonists. Assay procedures based on cellular signal transduction mechanisms thus appear to become a general analytical method for bioactive analytes. Our further studies of this apporach include screening of antigen-specific IgEs based on intracellular Ca^<2+> singnaling, and also screening of anticancer drugs based on active transport involving multi-drug resistance proteins. Less
在大多数化学分析方法中,分析物对干扰物质的选择性基本上取决于分析物A与其分子识别试剂B之间的结合常数K_f,K_f=[AB]/[A][B]这通常是结合分析的基础。免疫分析就是一个典型的例子。最近,我们一直在研究生物活性物质的分析方法,不仅基于与受体的结合,还部分考虑了随后的生物信号转导过程。这个想法使我们探索了一些新的分析方法,可以评估分析物(激动剂或拮抗剂)在生理上更相关的选择性。典型的例子是嵌入在脂质双层膜中的离子通道、转运蛋白和激酶型蛋白。与结合分析和相关技术不同,这些传感器利用相应的下游信号,如离子通道电流或…的磷酸化程度与结合分析方法相比,更多的是受体本身及其目标蛋白,以产生更多的生理相关信号。这是通过使用表面等离子体共振(SPR)和涉及部分细胞信号机制的荧光探针试剂的体外和体内分析方法实现的。对于细胞内受体蛋白,例如基于钙调素依赖的钙离子开关开关的信号通路,已经用SPR评估了激动剂的选择性,以形成钙调素靶标多肽三元复合体。通过这种方法,不仅发现了Ca^<;2+>;离子,也发现了Sr^<;2+>;,Pb^<;2+>;,Cd^<;2+>;以及许多金属离子都依赖于“Ca^<;2+>;信号”,并且对Ca^<;2+>;信号具有很强的激动剂作用。筛选与生理相关的激动剂的选择性对于生物学研究和药物需求是非常重要的。传统的结合分析方法既不能提供关于激动剂活性的充分信息,也不能区分激动剂和拮抗剂。因此,基于细胞信号转导机制的分析方法似乎成为生物活性分析物的通用分析方法。我们的进一步研究包括基于细胞内Ca~(2+)~(2+)和GT~(2+)信号的抗原特异性免疫球蛋白的筛选,以及基于多药耐药蛋白主动转运的抗癌药物的筛选。较少
项目成果
期刊论文数量(29)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
M. Sato et al.: "A Fluorscent Indicator for Tyrosine Phosphorylation-Based Insulin Signaling Pathways"Anal. Chem.. 71. 3948-3954 (1999)
M. Sato 等人:“基于酪氨酸磷酸化的胰岛素信号通路的荧光指示剂”分析。
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T.Ozawa, et al.: "Split Luciferase as an Optical Probe for Detecting Protein-Protein Interactions in Mammalian Cells Based on Protein Splicing"Anal.Chem.. (in press).
T.Ozawa 等人:“基于蛋白质剪接将分裂荧光素酶作为光学探针用于检测哺乳动物细胞中蛋白质-蛋白质相互作用”Anal.Chem..(出版中)。
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S.Aketani, R.Teshima, J.Sawada and Y.Umezawa: "A Screening Method for Antigen-Specific IgE Using Mast Cells Based on Intracellular Calcium Signaling."Anal.Chem.. 72, No.11. 2653-2658 (2000)
S.Aketani、R.Teshima、J.Sawada 和 Y.Umezawa:“基于细胞内钙信号转导使用肥大细胞筛选抗原特异性 IgE 的方法。”Anal.Chem.. 72,第 11 期。
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H.Aoki, et al.: "Electrochemical Detection of a One-Based Mismatch in an Oligonucleotide Using Ion-Channel Sensors with Self-Assembled PNA Monolayers"Electroanalysis. 12. 1272-1276 (2000)
H.Aoki 等人:“使用具有自组装 PNA 单层的离子通道传感器对寡核苷酸中基于 1 的错配进行电化学检测”。
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- 影响因子:0
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A.Hirano, et al.: "Evaluation of Agonist Selectivity for the NMDA Receptor Ion Channel in Bilayer Lipid Membranes Based on Integrated Single-Channel Currents"Biosensors & Bioelectronics. 15. 173-181 (2000)
A.Hirano 等人:“基于集成单通道电流评估双层脂质膜中 NMDA 受体离子通道的激动剂选择性”生物传感器
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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
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
- 批准号:
15105003 - 财政年份:2003
- 资助金额:
$ 24.32万 - 项目类别:
Grant-in-Aid for Scientific Research (S)
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:
$ 24.32万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
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:
$ 24.32万 - 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:
$ 24.32万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
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:
$ 24.32万 - 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:
$ 24.32万 - 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:
$ 24.32万 - 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:
$ 24.32万 - 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:
$ 24.32万 - 项目类别:
Grant-in-Aid for General Scientific Research (B)