Biomedical subjects

Yoshio Umezawa

Publications and source records attributed to Yoshio Umezawa.

11 recordsLinked to original sources

PubMed2003

Fluorescent indicators for Akt/protein kinase B and dynamics of Akt activity visualized in living cells.

Akt/protein kinase B (PKB) is a serine/threonine kinase that regulates a variety of cellular responses. To provide information on the spatial and temporal dynamics of Akt/PKB activity, we have developed genetically encoded fluorescent indicators for Akt/PKB. The indicators contain two green fluorescent protein mutants, an Akt/PKB substrate domain, flexible linker sequence, and phosphorylation recognition domain. A phosphorylation of the substrate domain in the indicators caused change in the emission ratio based on fluorescent resonance energy transfer between the two green fluorescent protein mutants. To let the fluorescent indicators behave as endothelial nitric-oxide synthase and Bad, which are endogenous Akt/PKB substrates, they were fused with the Golgi target domain and mitochondria target domain, respectively. The indicators thus colocalized with the endogenous substrates conferred their susceptibilities to phosphorylation by Akt/PKB. We showed that the Golgi-localized indicator responded to the stimulation with 17beta-estradiol (E2) and insulin in endothelial cells. In addition, E2 elicited the phosphorylation of the mitochondria-localized indicator in the endothelial cells, but no phosphorylation was observed by E2 or by insulin of the diffusible indicator that has no targeting domain. The difference in the results with the three indicators suggests that the activated Akt/PKB is localized to subcellular compartments, including the Golgi apparatus and/or mitochondria, rather than diffusing in the cytosol, thereby efficiently phosphorylating its substrate proteins. E2 triggered the phosphorylation of the mitochondria-localized indicator, whereas insulin did not induce this phosphorylation, which suggests that the localization of the activated Akt/PKB to the mitochondria is directed differently between insulin and E2 via distinct mechanisms.

Amino Acid Sequence↗

PubMed2003

A genetic approach to identifying mitochondrial proteins.

The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.

Amino Acid Sequence↗

PubMed2003

Seeing what was unseen: new analytical methods for molecular imaging.

For nondestructive analysis of chemical processes in living cells, we developed novel intracellular fluorescent indicators for second messengers, protein phosphorylation, and protein/protein interactions that work in single living cells. Key molecules and steps of cellular signaling pathways were visualized under a confocal laser microscope in target live cells using developed fluorescent indicators. A second new approach to molecular imaging is also described. When chemically modified tips were used for STM measurements, contrast enhancements at specific regions in the STM images occurred on the basis of hydrogen bond and metal-coordination interactions. This enabled us to detect not only the distribution of specific chemical species and functional groups but also the orientation of functional groups. The contrast enhancements reflect the increase in a tunneling current due to the overlap of electronic wave functions induced by the chemical interactions between tip and sample.

Animals↗

PubMed2003

Voltammetric detection of inorganic phosphate using ion-channel sensing with self-assembled monolayers of a hydrogen bond-forming receptor.

A voltammetric ion-channel sensing for phosphate based on gold electrodes modified with the self-assembled monolayers of a bis-thiourea receptor was developed to detect phosphate. The working principle of this voltammetric sensor conceptually mimics that of ligand gated ion-channel proteins, as to chemically stimulated changes in membrane permeability. The response to analytes is based on the change in electron transfer rate constant of the redox reaction of [Fe(CN)(6)](4-/3-) marker, before and after binding of phosphate to the receptor on the electrode surface; where the electrostatic repulsion between a phosphate-receptor complex and the marker induced the decrease in the rate constant. In a solution of pH 7.0, a high selectivity was observed for phosphate and the sensor was virtually insensitive at all to many of other anions, such as SO(4)(2-), AcO(-), NO(3)(-), and Cl(-). The sensor response was obtained with phosphate concentrations above 5.0 x 10(-4) M using cyclic voltammetry and differential pulse voltammetry.

Coated Materials, Biocompatible↗

PubMed2003

Trace analysis of an oligonucleotide with a specific sequence using PNA-based ion-channel sensors.

The gold electrodes modified with self-assembled monolayers of a 13-mer peptide nucleic acid (PNA) probe and 8-amino-1-octanethiol were used for the detection of a complementary oligonucleotide at a femtomolar level using the ion-channel sensor technique. No response to a one-base mismatched oligonucleotide was observed. The electrode surface was positively charged in a pH 7.0 buffer solution due to the protonation of an amine group of the thiol, where the electron transfer between the positively charged marker [Ru(NH3)6]3+ and the surface was hindered because of the charge-charge repulsion between them. Binding of the negatively-charged complementary oligonucleotide to the probe cancels the positive charge at the surface, and provides an excess negative charge at the surface, thereby facilitating the access of the marker to the electrode surface and its redox reaction. Using a 13-mer PNA probe for this sensing mode, we achieved the detection of the oligonucleotide at a femtomolar (approximately 10-15 M) level, improved by five orders of magnitude than the previously used 10-mer PNA probe.

DNA, Complementary↗

PubMed2003

A screening method for estrogens using an array-type DNA glass slide.

A new screening assay was described for the determination of endocrine disrupting chemicals (EDCs), such as synthetic estrogens, with an array-type DNA glass slide having characteristics of 1) a high sample throughput, 2) a compact size allowing a small sample volume, and 3) a sensitive determination based on the estrogen-dependent binding of the human estrogen receptor a (hERalpha) with its estrogen responsive element (ERE; Vit. A2 gene promoter). We devised a glass slide on which a thin agarose gel was mounted. Avidin was then covalently immobilized on each well of the glass slide after the gel was activated by a NaIO4 solution. Also, the biotinylated ERE as a DNA probe was immobilized on the gel layer through avidin-biotin binding. After the estrogen-dependent binding of a yellow fluorescent protein-fused hERalpha (YFP-hERalpha) to ERE on the gel layer, the fluorescence intensity of YFP-hERalpha quantitatively extracted into the gel was directly determined with a fluorescence microplate reader. Pre-incubation of YFP-hERalpha with estrogen at 37 degrees C for 30 min enhanced the estrogen-dependent hERalpha-ERE binding. The determined hormonal activities of estrogens on the interaction of YFP-hERalpha with ERE were as follows in their decreasing order: diethylstilbestrol (DES) > 17beta-estradiol (E2) <==> ethynylestradiol (EE2) > 4-hydroxy tamoxifen (OHT) > clomiphene (Clo). The present method provides a sensitive estrogen-dependent dose-response curve down to approximately 10(-13) M in the case of DES. This method will become a competitive alternative to the conventional in vitro assays, such as a DNA-binding assay using radioisotopes.

Avidin↗

PubMed2002

Carbon nanotube scanning tunneling microscopy tips for chemically selective imaging.

Carboxyl-terminated single-walled carbon nanotubes (SWNTs) were successfully immobilized from solution phases onto the apexes of gold tips for scanning tunneling microscopy (STM). Gold STM tips were first modified with self-assembled monolayers of 4-mercaptobenzoic acid, and its carboxyl groups were used to anchor carboxylated SWNTs through Zn2+ ion-bridged coordination. These SWNT tips gave high-resolution STM images of a diether monolayer formed on the graphite surface. In addition and more importantly, the ether oxygens of the sample molecules were selectively observed as bright spots with the SWNT tips with significantly high reproducibility, which is due to the facilitation of electron tunneling through hydrogen bond interactions between the ether oxygens and carboxyl groups at the end of the SWNT tips.

Journal Article↗

PubMed2002

Assay and screening methods for bioactive substances based on cellular signaling pathways.

Assay and screening methods for bioactive substances based on cellular signaling pathways are presented. Examples include: (1) intracellular protein phosphorylation and protein-protein interaction, (1-i) a new assay method for evaluating chemical selectivity of agonists for insulin signaling pathways based on agonist-induced phosphorylation of a target peptide, (1-ii) an SPR-based screening method for agonist selectivity for insulin signaling pathways based on the binding of phosphotyrosine to its specific binding protein, (1-iii) a fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways, and (1-iv) split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing; (2) a screening method for antigen-specific IgE using mast cells based on intracellular calcium signaling; (3) a screening method for substrates of multidrug resistance-associated protein (MRP); and (4) fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2002

Fluorescent indicators for imaging protein phosphorylation in single living cells.

To visualize signal transduction based on protein phosphorylation in living cells, we have developed genetically encoded fluorescent indicators, named phocuses. Two different color mutants of green fluorescent protein (GFP) were joined by a tandem fusion domain composed of a substrate domain for the protein kinase of interest, a flexible linker sequence, and a phosphorylation recognition domain that binds with the phosphorylated substrate domain. Intramolecular interaction of the substrate domain and the adjacent phosphorylation recognition domain within a phocus was dependent upon phosphorylation of the substrate domain by protein kinase, which influenced the efficiency of fluorescence resonance energy transfer (FRET) between the GFPs within a phocus. In the present study, we employed phocuses composed of insulin signaling proteins to visualize protein phosphorylation by the insulin receptor. This method may provide a general approach for studying the dynamics of protein phosphorylation-based signal transduction in living cells.

Animals↗

PubMed2002

Ion-channel sensors for electrochemical detection of DNA based on self-assembled PNA monolayers.

The gold electrodes modified with self-assembled monolayers composed of a 10-mer peptide nucleic acid (PNA) probe and 8-amino-1-octanethiol were used for the detection of the complementary oligonucleotide with a detection limit of 5.1 x 10(-10) M in a pH 7.0 phosphate buffer solution. In contrast, no response to a non-complementary oligonucleotide was observed. The electrode surface was positively charged in the phosphate buffer solution due to the protonated amine group of the thiol, where the electron transfer reaction between the electroactive marker [Ru(NH3)6]3+ and the electrode was hindered because of the electrostatic repulsion between them. Binding of the complementary oligonucleotide to the PNA probe monolayer cancels the positive charge at the electrode surface, and provides an excess negative charge at the surface, thereby facilitating the access of [Ru(NH3)6]3+ to the electrode surface and its redox reaction.

DNA↗

PubMed2002

Methods of analysis for chemicals that promote/disrupt cellular signaling.

Methods of analysis were presented for chemicals that promote or disrupt cellular signaling pathways. The developed analytical methods are based not only on receptor binding, but also on the following known molecular-level processes involved in signal transduction along signaling pathways, reconstituted in vitro or taken in part in living cells. The methods were discussed in relation to receptor binding assay and/or bioassay. Examples include: (1) Insulin signaling pathways; (1-i) Chemical selectivity of agonists for insulin signaling pathways based on agonist-induced phosphorylation of a target peptide; (1-ii) An SPR-based screening method for agonist selectivity for insulin signaling pathways based on the binding of phosphotyrosine to its specific binding protein; (1-iii) A fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways; (2) An optical method for evaluating ion selectivity for calcium signaling pathways in the cell; (3) Assay and screening of chemicals that disrupt cellular signaling pathways, potential endocrine disruptors in particular; (4) Protein conformational changes, and (5) A screening method for antigen-specific IgE using mast cells, based on intracellular calcium signaling.

Animals↗