Publications by authors named "Tomohiro Doura"

12 Publications

  • Page 1 of 1

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking.

Nat Commun 2021 02 5;12(1):831. Epub 2021 Feb 5.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, 464-8603, Japan.

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N-methyl-D-aspartate-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.
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http://dx.doi.org/10.1038/s41467-021-21082-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864911PMC
February 2021

Selective acetylcholinesterase inhibitors derived from muscle relaxant dantrolene.

Bioorg Med Chem Lett 2020 02 19;30(4):126888. Epub 2019 Dec 19.

School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392, Japan; Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

Dantrolene, the only therapeutic agent for malignant hyperthermia, is known to have not only a muscle relaxant effect, but also a neuroprotective effect and Alzheimer's disease improving effect. Recently, it has been reported that dantrolene has a weak inhibitory effect on acetylcholinesterase (AChE), which is a therapeutic drug target for Alzheimer's disease. Thus, we focused on developing of AChE inhibitors with benzylpiperidine/piperazine moieties that are based on the dantrolene skeleton. Several derivatives showed an inhibitory activity. Among them, ortho-nitro derivative 8c showed the most potent inhibitory activity with the IC value of 34.2 nM. Furthermore, Lineweaver-Burk plot analysis indicated that 8c is AChE-selective inhibitor, which shows only a weak inhibitory effect on butyrylcholinesterase (BuChE) and a non-competitive inhibition.
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http://dx.doi.org/10.1016/j.bmcl.2019.126888DOI Listing
February 2020

Hybrid cell reactor system from Escherichia coli protoplast cells and arrayed lipid bilayer chamber device.

Sci Rep 2018 08 6;8(1):11757. Epub 2018 Aug 6.

Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.

We developed a novel hybrid cell reactor system via functional fusion of single Escherichia coli protoplast cells, that are deficient in cell wall and expose plasma membrane, with arrayed lipid bilayer chambers on a device in order to incorporate the full set of cytosolic and membrane constituents into the artificial chambers. We investigated gene expression activity to represent the viability of the hybrid cell reactors: over 20% of hybrid cells showed gene expression activity from plasmid or mRNA. This suggests that the hybrid cell reactors retained fundamental activity of genetic information transduction. To expand the applicability of the hybrid cell reactors, we also developed the E. coli-in-E. coli cytoplasm system as an artificial parasitism system. Over 30% of encapsulated E. coli cells exhibited normal cell division, showing that hybrid cells can accommodate and cultivate living cells. This novel artificial cell reactor technology would enable unique approaches for synthetic cell researches such as reconstruction of living cell, artificial parasitism/symbiosis system, or physical simulation to test functionality of synthetic genome.
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http://dx.doi.org/10.1038/s41598-018-30231-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078950PMC
August 2018

Miniaturization of thiol-organosilica nanoparticles induced by an anionic surfactant.

J Colloid Interface Sci 2018 Sep 25;526:51-62. Epub 2018 Apr 25.

Department of Organ Anatomy and Nanomedicine, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. Electronic address:

Thiol-organosilica nanoparticles are a promising nanomaterial for biomedical applications. The enhanced permeability and retention (EPR) effect is useful for tumor targeting within the biomedical applications of nanomaterials, and nanomaterials with a size of less than 200 nm exhibit the maximum EPR effect. However, the synthesis of thiol-organosilica nanoparticles with a diameter of less than 200 nm is not efficient for the yield using the present conventional synthetic methods. Herein, we report the development of an efficient synthetic method of thiol-organosilica nanoparticles with a diameter of less than 200 nm using an anionic surfactant and discuss its mechanism. Compared with the conventional synthetic methods, a greater than 10-fold miniaturization of thiol-organosilica nanoparticles and an approximately 40-fold increase in the production efficiency of small thiol-organosilica nanoparticles were achieved using the sodium dodecyl sulfate (SDS)-addition synthetic method or sodium dodecylbenzenesulfonate (SDBS)-addition synthetic method. This is the first report about the miniaturization of organosilica nanoparticles induced by an anionic surfactant. The SDS-addition synthetic method or SDBS-addition synthetic method will accelerate the biomedical applications of thiol-organosilica nanoparticles.
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http://dx.doi.org/10.1016/j.jcis.2018.04.090DOI Listing
September 2018

Combretastatin A4-β-Galactosyl Conjugates for Ovarian Cancer Prodrug Monotherapy.

ACS Med Chem Lett 2017 Feb 20;8(2):211-214. Epub 2017 Jan 20.

Graduate School of Pharmaceutical Sciences, Chiba University , 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.

Chemotherapy for ovarian cancer often causes severe side effects. As candidates for combretastatin A4 (CA4) prodrug for ovarian cancer prodrug monotherapy (PMT), we designed and synthesized two β-galactose-conjugated CA4s (CA4-βGals), CA4-βGal-1 and CA4-βGal-2. CA4 was liberated from CA4-βGals by β-galactosidase, an enzyme more strongly expressed in ovarian cancer cells than normal cells. CA4-βGal-2, which has a self-immolative benzyl linker between CA4 and the β-galactose moiety, was more cytotoxic to ovarian cancer cell lines than CA4-βGal-1 without a linker. Therefore, CA4-βGal-2 can serve as a platform for the design and manufacture of prodrugs for ovarian cancer PMT.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5304305PMC
February 2017

Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution.

Angew Chem Int Ed Engl 2016 08 12;55(33):9620-4. Epub 2016 Jul 12.

Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

The LacZ gene, which encodes Escherichia coli β-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.
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http://dx.doi.org/10.1002/anie.201603328DOI Listing
August 2016

PAMAM Dendron Lipid Assemblies That Undergo Structural Transition in Response to Weakly Acidic pH and Their Cytoplasmic Delivery Capability.

Langmuir 2015 May 1;31(18):5105-14. Epub 2015 May 1.

Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.

Dendron lipids designed to consist of amine-terminated polyamidoamine G1 dendron and two octadecyl chains were used for the preparation of pH-responsive molecular assemblies having phase structures that are changed through their dynamic molecular shape. The dendron lipid contains two primary amines and two tertiary amines in the dendron moiety, changing its charged state in the pH region between pH 10 and pH 4. The assemblies were shown to take a vesicle structure at neutral and alkaline pHs, but their structure changed to a micelle-like structure below pH 6.4. Because this pH region corresponds to one in which tertiary amines of the dendron lipid became protonated, protonation of tertiary amines in addition to primary amines in the dendron moiety might affect its dynamic molecular shape, resulting in a sharp pH response of the assemblies. The assemblies tended to form aggregates when taking on a vesicle form with a gel phase, but incorporation of a poly(ethylene glycol)-lipid provided dendron lipid vesicles with both sharp pH response and high colloidal stability. The poly(ethylene glycol)-incorporated dendron lipid vesicles tightly retained ovalbumin molecules in their internal aqueous space but released them almost completely at pH 6.0. In addition, the vesicles were shown to achieve efficient ovalbumin delivery into cytosol of DC2.4 cells (mouse dendritic cell line) after internalization through endocytosis.
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http://dx.doi.org/10.1021/acs.langmuir.5b00183DOI Listing
May 2015

Phenylboronic acid-based (19)F MRI probe for the detection and imaging of hydrogen peroxide utilizing its large chemical-shift change.

Anal Sci 2015 ;31(4):331-5

INAMORI Frontier Research Center, Kyushu University.

Herein, we report on a new (19)F MRI probe for the detection and imaging of H2O2. Our designed 2-fluorophenylboronic acid-based (19)F probe promptly reacted with H2O2 to produce 2-fluorophenol via boronic acid oxidation. The accompanying (19)F chemical-shift change reached 31 ppm under our experimental conditions. Such a large chemical-shift change allowed for the imaging of H2O2 by (19)F chemical-shift-selective MRI.
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http://dx.doi.org/10.2116/analsci.31.331DOI Listing
January 2016

An adhesive (19)F MRI chemical probe allows signal off-to-on-type molecular sensing in a biological environment.

Chem Commun (Camb) 2013 Dec;49(97):11421-3

INAMORI Frontier Research Center, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, Japan.

We report a new strategy for designing a signal off-to-on-type (19)F MRI chemical probe that operates in biological environments. The present strategy is based on the control of adherence of a (19)F MRI chemical probe to certain blood proteins, accompanied by a change in transverse relaxation time of (19)F nuclei.
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http://dx.doi.org/10.1039/c3cc46471gDOI Listing
December 2013

A platform for designing hyperpolarized magnetic resonance chemical probes.

Nat Commun 2013 ;4:2411

INAMORI Frontier Research Center, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819 0395, Japan.

Hyperpolarization is a highly promising technique for improving the sensitivity of magnetic resonance chemical probes. Here we report [(15)N, D(9)]trimethylphenylammonium as a platform for designing a variety of hyperpolarized magnetic resonance chemical probes. The platform structure shows a remarkably long (15)N spin-lattice relaxation value (816 s, 14.1 T) for retaining its hyperpolarized spin state. The extended lifetime enables the detection of the hyperpolarized (15)N signal of the platform for several tens of minutes and thus overcomes the intrinsic short analysis time of hyperpolarized probes. Versatility of the platform is demonstrated by applying it to three types of hyperpolarized chemical probes: one each for sensing calcium ions, reactive oxygen species (hydrogen peroxide) and enzyme activity (carboxyl esterase). All of the designed probes achieve high sensitivity with rapid reactions and chemical shift changes, which are sufficient to allow sensitive and real-time monitoring of target molecules by (15)N magnetic resonance.
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http://dx.doi.org/10.1038/ncomms3411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778512PMC
April 2014

Design of a 13C magnetic resonance probe using a deuterated methoxy group as a long-lived hyperpolarization unit.

Angew Chem Int Ed Engl 2012 Oct 7;51(40):10114-7. Epub 2012 Sep 7.

INAMORI Frontier Research Center, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, Japan.

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http://dx.doi.org/10.1002/anie.201202885DOI Listing
October 2012

Atom arrangement strategy for designing a turn-on 1H magnetic resonance probe: a dual activatable probe for multimodal detection of hypochlorite.

Chem Commun (Camb) 2012 Feb 17;48(10):1565-7. Epub 2011 May 17.

INAMORI Frontier Research Center, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.

The first dual activatable hypochlorite ((-)OCl)-sensing probe was developed, based on a new proof-of-concept design involving signal-activatable (1)H chemical probes using the triple-resonance NMR technique. The probe enabled fluorescence-(1)H MR dual turn-on detection of (-)OCl in solution and in crude tissue extracts.
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http://dx.doi.org/10.1039/c1cc12044aDOI Listing
February 2012