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Yoshio Umezawa

Publications and source records attributed to Yoshio Umezawa.

At least 19 recordsLinked to original sources
PubMed2006

Cell-based indicator to visualize picomolar dynamics of nitric oxide release from living cells.

We report a novel cell-based indicator that is able to visualize picomolar dynamics of nitric oxide release from living cells. Cells from a pig kidney-derived cell line (PK15) endogenously express soluble guanylate cyclase (sGC), which is a receptor protein for the selective recognition of NO. Binding of NO by sGC causes the amplified generation of guanosine 3',5'-cyclic monophosphate (cGMP). To make the PK15 cells into NO indicators, the cells are transfected with a plasmid vector encoding a fluorescent indicator for cGMP and fluorescence resonance energy transfer is recorded at 480 +/- 15 and 535 +/- 12.5 nm upon excitation of the cells at 440 +/- 10 nm. The cell-based indicator exhibits exceptional sensitivity (detection limit of 20 pM), selectivity, reversibility, and reproducibility. The outstanding sensitivity of the present indicator has led us to uncover an oscillatory release of picomolar concentrations of NO from hippocampal neurons. We present evidence that Ca2+ oscillations in hippocampal neurons underlie the oscillatory NO release from the neurons during neurotransmission. We also have succeeded in visualizing the extent of diffusing NO from single vascular endothelial cells. The present cell-based indicator provides a powerful tool to uncover picomolar dynamics of NO that regulates a wide range of cell functions in biological systems.

Animals↗
PubMed2006

Genetically encoded optical probe for detecting release of proteins from mitochondria toward cytosol in living cells and mammals.

We developed a genetically encoded bioluminescence indicator for monitoring the release of proteins from the mitochondria in living cells. The principle of this method is based on reconstitution of split Renilla reniformis luciferase (Rluc) fragments by protein splicing with an Ssp DnaE intein. A target mitochondrial protein connected with an N-terminal fragment of Rluc and an N-terminal fragment of DnaE is expressed in mammalian cells. If the target protein is released from the mitochondria toward the cytosol upon stimulation with a specific chemical, the N-terminal Rluc meets the C-terminal Rluc connected with C-terminal DnaE in the cytosol, and thereby, the full-length Rluc is reconstituted by protein splicing. The extent of release of the target fusion protein is evaluated by measuring activities of the reconstituted Rluc. To test the feasibility of this method, here we monitored the release of Smac/DIABLO protein from mitochondria during apoptosis in living cells and mice. The present method allowed high-throughput screening of an apoptosis-inducing reagent, staurosporine, and imaging of the Smac/DIABLO release in cells and in living mice. This rapid analysis can be used for screening and assaying chemicals that would increase or inhibit the release of mitochondrial proteins in living cells and animals.

Animals↗
PubMed2006

A fluorescent indicator to visualize ligand-induced receptor/coactivator interactions for screening of peroxisome proliferator-activated receptor gamma ligands in living cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of nuclear receptors (NRs) superfamily and plays an important role for modulation of insulin sensitivity in type 2 diabetes. Ligand-dependent protein-protein interactions between NRs and NR coactivators are critical in regulation of transcription. To visualize the ligand-induced coactivator recruitment to PPARgamma in live cells, we developed a genetically encoded fluorescent indicator in which PPARgamma ligand binding domain (PPARgamma LBD) was connected to a steroid receptor coactivator peptide that contains LXXLL motif (L=leucine and X=any amino acid) through a flexible linker. This fusion protein was inserted between cyan and yellow fluorescent proteins (CFP and YFP, donor and acceptor fluorophore, respectively). Monitoring real-time ligand-induced conformational change in the PPARgamma LBD to interact with the coactivator allowed screening of natural and synthetic ligands (drugs against type 2 diabetes) in single living cells using intramolecular fluorescence resonance energy-transfer (FRET) microscopy. The high sensitivity of the present indicator made it possible to distinguish between strong and weak affinity ligands for PPARgamma in a dose-dependent fashion, immediately after adding a ligand to live cells. The indicator can discriminate agonist from antagonist compounds efficiently within a few minutes. The present system may be promising in the development of PPARgamma-targeted drugs against type 2 diabetes and inflammation.

Animals↗
PubMed2006

Ginsenoside Re, a main phytosterol of Panax ginseng, activates cardiac potassium channels via a nongenomic pathway of sex hormones.

Ginseng root is one of the most popular herbs throughout the world and is believed to be a panacea and to promote longevity. It has been used as a medicine to protect against cardiac ischemia, a major cause of death in the West. We have previously demonstrated that ginsenoside Re, a main phytosterol of Panax ginseng, inhibits Ca(2+) accumulation in mitochondria during cardiac ischemia/reperfusion, which is attributable to nitric oxide (NO)-induced Ca(2+) channel inhibition and K(+) channel activation in cardiac myocytes. In this study, we provide compelling evidence that ginsenoside Re activates endothelial NO synthase (eNOS) to release NO, resulting in activation of the slowly activating delayed rectifier K(+) current. The eNOS activation occurs via a nongenomic pathway of each of androgen receptor, estrogen receptor-alpha, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase, Akt, and eNOS are sequentially activated. However, ginsenoside Re does not stimulate proliferation of androgen-responsive LNCaP cells and estrogen-responsive MCF-7 cells, implying that ginsenoside Re does not activate a genomic pathway of sex hormone receptors. Fluorescence resonance energy transfer experiments with a probe, SCCoR (single cell coactivator recruitment), indicate that the lack of genomic action is attributable to failure of coactivator recruitment. Thus, ginsenoside Re acts as a specific agonist for the nongenomic pathway of sex steroid receptors, and NO released from activated eNOS underlies cardiac K(+) channel activation and protection against ischemia-reperfusion injury.

Animals↗
PubMed2006

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe.

Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.

Animals↗
PubMed2006

Intein-mediated reporter gene assay for detecting protein-protein interactions in living mammalian cells.

For nondestructive analysis of chemical processes in living mammalian cells, here we developed a new reporter gene assay for detecting cytosolic protein-protein interactions based on protein splicing of transcription factors with DnaE inteins. The protein splicing induces connection of a DNA-binding protein (modified LexA; mLexA) with a transcription activation domain of a herpes simplex virus protein (VP16AD). We thereby circumvented the limitation of earlier methods for monitoring protein-protein interactions, including the two-hybrid systems, protein complementation systems (PCS), and protein reconstitution systems, and rather combined their advantages. To test the applicability of this method, we monitored epidermal growth factor (EGF)-induced interactions on cell membranes of a known partner, an oncogenic product Ras and its target Raf-1. Ras was connected with N-terminal DnaE and mLexA, while Raf-1 was connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggered folding of the DnaEs, thereby inducing protein splicing to form mLexA-VP16AD fusion protein, and transcription of a reporter gene, firefly luciferase. The extent of Ras-Raf-1 interaction was quantified by measuring the luciferase activity. The interaction was not able to be monitored by two-hybrid systems nor by PCS of split firefly luciferases; however, by using the protein splicing elements and the reporter gene, we obtained the bioluminescence signals sufficient for evaluation of the interactions close to cell membranes.

Animals↗
PubMed2006

Imaging diacylglycerol dynamics at organelle membranes.

Fluorescence imaging is a powerful technique to visualize spatiotemporal dynamics of biomolecules in living cells. We describe fluorescent indicators for a lipid second messenger, diacylglycerol (DAG), which allow the localized analysis of DAG dynamics at subcellular membranes. We have thus pinpointed that DAG concentrations increase and/or decrease at not only the plasma membrane but also organelle membranes such as endomembranes and mitochondrial outer membranes.

Animals↗
PubMed2006

Ion-channel sensors based on ETH 1001 ionophore embedded in charged-alkanethiol self-assembled monolayers on gold electrode surfaces.

An ion-channel sensor was demonstrated by immobilizing ETH 1001, an ionophore for ion-selective electrodes, on a gold electrode surface. The approach for preparing the sensor was to incorporate the ionophore into a mixed self-assembled monolayer of 10-mercaptodecanesulfonate and 11-hydroxy-1-undecanethiol formed on the surface. The voltammetric responses for the thus prepared sensor to the primary cation Ca(2+) were observed by using [Fe(CN)(6)](3-/4-) as an electroactive marker. The ionophore was stably immobilized on the electrode surface with the hydrophobic interaction between its alkyl chains and those of the alkanethiol. The introduction of a proper charge density to the electrode surface improved the sensor sensitivity with retaining the selective response to Ca(2+) against Mg(2+) with concentrations above 10(-4) M.

Journal Article↗
PubMed2006

Nitrogenous synergists induced potentiometric response to metal ions with polymeric liquid membranes containing thenoyltrifluoroacetone as an ionophore.

The effects of nitrogenous synergists on the potentiometric responses to divalent transition metal ions were investigated concerning polymeric liquid membranes containing thenoyltrifluoroacetone (Htta) as an ionophore. The tested synergists were pyridine (py) and 4,4'-dioctyl-2,2'-bipyridyl (C8bpy). The potentiometric responses to metal ions, such as Cd2+, Co2+, Ni2+ and Zn2+, were induced by adding the synergists into the liquid membrane systems. The coexistence of Htta and a synergist was necessary for generating the membrane potential. The tta- anion adsorbed at the liquid membrane/solution interface and the complex formation between the synergist and a given metal ion appeared to participate in preferential uptake of metal ions.

Cadmium↗
PubMed2005

Complementary base-pair-facilitated electron tunneling for electrically pinpointing complementary nucleobases.

Molecular tips in scanning tunneling microscopy can directly detect intermolecular electron tunneling between sample and tip molecules and reveal the tunneling facilitation through chemical interactions that provide overlap of respective electronic wave functions, that is, hydrogen-bond, metal-coordination-bond, and charge-transfer interactions. 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. The complementary nucleobase tip was thereby capable of electrically pinpointing each nucleobase. Chemically selective imaging using molecular tips may be coined "intermolecular tunneling microscopy" as its principle goes and is of general significance for novel molecular imaging of chemical identities at the membrane and solid surfaces.

Base Pairing↗
PubMed2005

Quantitative determination of protein nuclear transport induced by phosphorylation or by proteolysis.

Nucleocytoplasmic transport of proteins in eukaryotic cells is a fundamental process for gene expression. The transport is regulated by posttranslational modifications of the proteins, such as ligand-binding, phosphorylation, and proteolysis. For monitoring the nuclear transport of proteins induced by a ligand binding, we have recently developed a genetically encoded bioluminescent indicator based on reconstitution of split fragments of Renilla reniformis (RLuc) by protein splicing with DnaE inteins. We herein describe that the technique is used for detecting phosphorylation- or proteolysis-induced nuclear transports of a target protein. Two model proteins, signal transducer and activator of transcription 3 (STAT3) and sterol-regulatory element binding protein-2 (SREBP-2), were exemplified as phosphorylation- and proteolysis-induced nuclear transport, respectively. Each STAT3 or SREBP-2 is connected with C-terminal halves of RLuc and DnaE. If the protein translocates into the nucleus, the C-terminal fragment of RLuc meets the N-terminal fragment of RLuc, and full-length RLuc is reconstituted by protein splicing in the nucleus. The indicator with SREBP-2 enabled us to quantify the intracellular concentrations of cholesterol. The indicator with STAT3 quantified the extent of the nuclear transport induced by representative cytokines. This simple assay based on protein nuclear transports allows the selection of suitable drugs among candidates and has significant potential for risk assessments, such as carcinogenic chemical screening in vitro and in vivo.

Active Transport, Cell Nucleus↗
PubMed2005

Genetically encoded stress indicator for noninvasively imaging endogenous corticosterone in living mice.

Physical and emotional stress is one of the major controllers of physiological reactions and homeostasis in living animals. A stress hormone, corticosterone, is secreted from the adrenal cortex into the blood vessels when animals sense the stress. The quantitative evaluation of corticosterone in living animals has been limited because of the unavailability of suitable methods in vivo. For a noninvasive molecular imaging of the stress, we developed a method for detecting physiological increases in the endogenous corticosterone caused by exo- and endogenous stress in living animals. We constructed a pair of genetically encoded indicators composed of cDNAs of glucocorticoid receptor (GR), split Renilla luciferase (RLuc), and a Synechocystis sp. DnaE intein. The GR fused with C-terminal halves of RLuc and DnaE is localized in the cytosol, whereas a fusion protein of N-terminal halves of RLuc and DnaE is localized in the nucleus. If corticosterone induces GR translocation into the nucleus, the C-terminal RLuc meets the N-terminal one in the nucleus, and full-length RLuc is reconstituted by protein splicing with DnaE. Cell-based methods provided a quantitative bioluminescence assay of the extent of GR translocation into the nucleus. We further demonstrated that the indicator enabled noninvasive imaging against two different types of imposed stress: a forced swimming and metabolic perturbation caused by 2-deoxy-D-glucose. This stress indicator should be valuable for screening pharmacological compounds and for tools to study the mechanism of physiological stress.

Animals↗
PubMed2005

A genetically encoded indicator for assaying bioactive chemicals that induce nuclear transport of glucocorticoid receptor.

Glucocorticoids, the adrenal steroid hormones secreted during stress, are essential to homeostasis and metabolism in the human body. An impaired glucocorticoid signaling due to dysfunction of the glucocorticoid receptor (GR) by synthetic chemicals can cause diseases and disruptions of the homeostasis and metabolism. Here we demonstrate the development of a method for screening endocrine-disrupting chemicals and potent risk factors of human diseases based on the nuclear trafficking of the GR. We constructed a new assay using a pair of genetic indicators with the full length of the GR, split Renilla luciferase (RLuc), and split DnaE (a protein splicing element). The GR-containing fusion protein with C-terminal halves of DnaE and RLuc is localized in cytosol due to the cytosolic character of the GR, whereas the fusion protein with N-terminal halves of DnaE and RLuc stays in the nucleus due to the cofused nucleus localization signal. On being stimulated with a ligand, the GR is translocated into the cellular nucleus. Thus, a protein splicing occurs in the nucleus by an interaction between the splicing junctions of each DnaE fragment. The enzymatic activities from the reconstituted RLuc allow the ligand-dependent luminescence intensities. The feasibility of the method was evaluated by quantifying the hormonal activities of 20 different kinds of steroids and synthetic chemicals using the NIH 3T3 cells carrying the pair of indicators. The hormonal activities of tested ligands are discussed based on the chemical structure-activity relationship. We found that androgens, testosterone, and 19-nortestosterone weakly induce the nuclear transport of the GR. The current assay allows high-throughput screening of risk chemicals and drug candidates influential to a signal transduction pathway of the GR.

Active Transport, Cell Nucleus↗
PubMed2005

Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells.

Nitric oxide (NO) is a small uncharged free radical that is involved in diverse physiological and pathophysiological mechanisms. NO is generated by three isoforms of NO synthase, endothelial, neuronal, and inducible ones. When generated in vascular endothelial cells, NO plays a key role in vascular tone regulation, in particular. Here, we describe an amplifier-coupled fluorescent indicator for NO to visualize physiological nanomolar dynamics of NO in living cells (detection limit of 0.1 nM). This genetically encoded high-sensitive indicator revealed that approximately 1 nM of NO, which is enough to relax blood vessels, is generated in vascular endothelial cells even in the absence of shear stress. The nanomolar range of basal endothelial NO thus revealed appears to be fundamental to vascular homeostasis.

Animals↗
PubMed2005

Locating inositol 1,4,5-trisphosphate in the nucleus and neuronal dendrites with genetically encoded fluorescent indicators.

Inositol 1,4,5-trisphosphate (InsP3) is a key second messenger in many cell types and also in distinct subcellular regions of single living cells; however, little is examined about the subcellular dynamics of InsP3 in a variety of cell types. We have developed fluorescent indicators to locate InsP3 dynamics in single living cells based on an intramolecular fluorescence resonance energy transfer. Our indicator has visualized InsP3 dynamics in the cytoplasm of cultured cells and even in single thin dendrites of hippocampal neurons, which has been unseen previously. We have further localized the present indicator in the nucleus and pinpointed nuclear InsP3 dynamics. The observation with our nuclear InsP3 indicator has solved a question on nuclear propagation of InsP3 from the cytoplasm and has drawn a conclusion that the nuclear InsP3 dynamics synchronously occurs with cytosolic InsP3 dynamics evoked by agonist stimulations. The present approach contributes to the understanding of when, where, and how InsP3 is generated and removed in a variety of living cells.

Animals↗