317 results on '"Hayashi T"'
Search Results
2. Three histidine residues of amyloid-[beta] peptide control the redox activity of copper and iron
- Author
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Nakamura, M., Shishido, N., Nunomura, Akihiro, Smith, Mark A., Perry, George, Hayashi, Y., Nakayama, K., and Hayashi, T.
- Subjects
Glycoproteins -- Chemical properties ,Histidine -- Chemical properties ,Oxidases -- Research ,Vitamin C -- Chemical properties ,Biological sciences ,Chemistry - Abstract
The ESR spectroscopic studies to measure the ascorbate oxidase activity and the intensity of ascorbate radicals generated by free Cu(II) is presented. The results showed that the redox activity of transition metals present in senile plaques is controlled by His6, His13, and His14 residues of amyloid-[beta]-42.
- Published
- 2007
3. Comparative characterization study of microporous carbons by HRTEM image analysis and gas adsorption
- Author
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Lozano-Castello, D., Cazorla-Amoros, D., Linares-Solano, A., Oshida, K., Miyazaki, T., Y.J. Kim, Hayashi, T., and Endo, M.
- Subjects
High resolution spectroscopy -- Research ,Charge transfer -- Research ,Porosity -- Research ,Gases -- Absorption and adsorption ,Gases -- Research ,Chemicals, plastics and rubber industries - Abstract
The verification of the transfer function of high-resolution transmission electron microscopy (HRTEM) was obtained by comparing the power spectrum of the HRTEM image with the logically calculated effective transfer function. The combination of HRTEM and image analysis is found to be useful for structural analysis of narrow micropores and closed porosity.
- Published
- 2005
4. Polymers for High Technology
- Author
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MURRAE J. BOWDEN, S. RICHARD TURNER, James H. O'Donnell, G. Arthur Salmon, J. K. Thomas, G. Beck, Adolphe Chapiro, Seiichi Tagawa, J. E. Guillet, Larry F. Thompson, Toshiaki Tamamura, Akinobu Tanaka, H. Shiraishi, N. Hayashi, T. Ueno, O. Suga, F. Murai, S. Nonogaki, Nigel R. Farrar, Geraint Owen, R. and MURRAE J. BOWDEN, S. RICHARD TURNER, James H. O'Donnell, G. Arthur Salmon, J. K. Thomas, G. Beck, Adolphe Chapiro, Seiichi Tagawa, J. E. Guillet, Larry F. Thompson, Toshiaki Tamamura, Akinobu Tanaka, H. Shiraishi, N. Hayashi, T. Ueno, O. Suga, F. Murai, S. Nonogaki, Nigel R. Farrar, Geraint Owen, R.
- Published
- 1987
5. Polymer Adsorption and Dispersion Stability
- Author
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E. D. GODDARD, BRIAN VINCENT, B. VINCENT, R. HOGG, A. TAKAHASHI, M. KAWAGUCHI, K. HAYASHI, T. KATO, M. A. COHEN STUART, J. M. H. M. SCHEUTJENS, G. J. FLEER, GERALD G. FULLER, JEN-JIANG LEE, M. S. AHMED, M. S. EL-AASSER, J. W. VANDERHOFF, J. E. GLASS, S. SHAH, D-L. LU, S. D. SENEKER, J. E. GLASS, H. and E. D. GODDARD, BRIAN VINCENT, B. VINCENT, R. HOGG, A. TAKAHASHI, M. KAWAGUCHI, K. HAYASHI, T. KATO, M. A. COHEN STUART, J. M. H. M. SCHEUTJENS, G. J. FLEER, GERALD G. FULLER, JEN-JIANG LEE, M. S. AHMED, M. S. EL-AASSER, J. W. VANDERHOFF, J. E. GLASS, S. SHAH, D-L. LU, S. D. SENEKER, J. E. GLASS, H.
- Published
- 1984
6. Protein Classes Predicted by Molecular Surface Chemical Features: Machine Learning-Assisted Classification of Cytosol and Secreted Proteins.
- Author
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Hu G, Moon J, and Hayashi T
- Abstract
Chemical structures of protein surfaces govern intermolecular interaction, and protein functions include specific molecular recognition, transport, self-assembly, etc. Therefore, the relationship between the chemical structure and protein functions provides insights into the understanding of the mechanism underlying protein functions and developments of new biomaterials. In this study, we analyze protein surface features, including surface amino acid populations and secondary structure ratios, instead of entire sequences as input for the classifier, intending to provide deeper insights into the determination of protein classes (cytosol or secreted). We employed a random forest-based classifier for the prediction of protein locations. Our training and testing data sets consisting of secreted and cytosol proteins were constructed using filtered information from UniProt and 3D structures from AlphaFold. The classifier achieved a testing accuracy of 93.9% with a feature importance ranking and quantitative boundary values for the top three features. We discuss the significance of these features quantitatively and the hidden rules to determine the protein classes (cytosol or secreted).
- Published
- 2024
- Full Text
- View/download PDF
7. Structure-Activity Relationship Studies in Benzothiadiazoles as Novel Vaccine Adjuvants.
- Author
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Belsuzarri MM, Sako Y, Brown TD, Chan M, Cozza R, Jin J, Sato-Kaneko F, Yao S, Pu M, Messer K, Hayashi T, Cottam HB, Corr M, Carson DA, and Shukla NM
- Subjects
- Structure-Activity Relationship, Humans, Animals, Mice, Dendritic Cells drug effects, Dendritic Cells immunology, Dendritic Cells metabolism, THP-1 Cells, Vaccines immunology, Vaccines chemistry, Mice, Inbred C57BL, Female, Extracellular Vesicles chemistry, Extracellular Vesicles immunology, Extracellular Vesicles metabolism, Adjuvants, Immunologic pharmacology, Adjuvants, Immunologic chemistry, Adjuvants, Immunologic chemical synthesis, Thiadiazoles chemistry, Thiadiazoles pharmacology, Thiadiazoles chemical synthesis
- Abstract
Extracellular vesicles (EVs) can transfer antigens and immunomodulatory molecules, and such EVs released by antigen-presenting cells equipped with immunostimulatory functions have been utilized for vaccine formulations. A prior high-throughput screening campaign led to the identification of compound 634 ( 1 ), which enhanced EV release and increased intracellular Ca
2+ influx. Here, we performed systematic structure-activity relationship (SAR) studies to investigate the scaffold for its potency as a vaccine adjuvant. Synthesized compounds were analyzed in vitro for CD63 reporter activity (a marker for EV biogenesis) in human THP-1 cells, induction of Ca2+ influx, IL-12 production, and cell viability in murine bone-marrow-derived dendritic cells. The SAR studies indicated that the ester functional group was requisite, and the sulfur atom of the benzothiadiazole ring replaced with a higher selenium atom ( 9f ) or a bioisosteric ethenyl group ( 9h ) retained potency. Proof-of-concept vaccination studies validated the potency of the selected compounds as novel vaccine adjuvants.- Published
- 2024
- Full Text
- View/download PDF
8. Why Is Surface-Enhanced Raman Scattering Insensitive to Liquid Water?
- Author
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Kamimura R, Maeda S, Hayashi T, Motobayashi K, and Ikeda K
- Abstract
Surface-enhanced Raman scattering (SERS) is widely recognized as a remarkably powerful analytical technique that enables trace-level detection of organic molecules on a metal surface in aqueous systems with negligible spectral interference of water. This insensitivity of SERS to liquid water is violated in a restrictive manner under specific electrochemical conditions. However, the origin of such different SERS sensitivities to liquid water remains unclear. Here, we show that hydrogen-bond networks of water play a pivotal role in losing SERS enhancement for liquid water, and SERS detection of water requires local defects in the hydrogen-bond networks, which are formed around hydration shells of solute ions or on a polarized electrode surface. This work gives a new perspective on in situ SERS investigations in aqueous systems, including electrochemical and biological reactions.
- Published
- 2024
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9. Correction to "Interlocking of Single-Walled Carbon Nanotubes with Metal-Tethered Tetragonal Nanobrackets to Enrich a Few Hundredths of a Nanometer Range in Their Diameters".
- Author
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Cheng G, Hayashi T, Miyake Y, Sato T, Tabata H, Katayama M, and Komatsu N
- Published
- 2024
- Full Text
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10. Elucidating Key Characteristics of PFAS Binding to Human Peroxisome Proliferator-Activated Receptor Alpha: An Explainable Machine Learning Approach.
- Author
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Maeda K, Hirano M, Hayashi T, Iida M, Kurata H, and Ishibashi H
- Subjects
- Humans, Liver metabolism, PPAR alpha metabolism, Fluorocarbons
- Abstract
Per- and polyfluoroalkyl substances (PFAS) are widely employed anthropogenic fluorinated chemicals known to disrupt hepatic lipid metabolism by binding to human peroxisome proliferator-activated receptor alpha (PPARα). Therefore, screening for PFAS that bind to PPARα is of critical importance. Machine learning approaches are promising techniques for rapid screening of PFAS. However, traditional machine learning approaches lack interpretability, posing challenges in investigating the relationship between molecular descriptors and PPARα binding. In this study, we aimed to develop a novel, explainable machine learning approach to rapidly screen for PFAS that bind to PPARα. We calculated the PPARα-PFAS binding score and 206 molecular descriptors for PFAS. Through systematic and objective selection of important molecular descriptors, we developed a machine learning model with good predictive performance using only three descriptors. The molecular size ( b_single ) and electrostatic properties ( BCUT_PEOE_3 and PEOE_VSA_PPOS ) are important for PPARα-PFAS binding. Alternative PFAS are considered safer than their legacy predecessors. However, we found that alternative PFAS with many carbon atoms and ether groups exhibited a higher affinity for PPARα. Therefore, confirming the toxicity of these alternative PFAS compounds with such characteristics through biological experiments is important.
- Published
- 2024
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11. Identification of a Self-Assembling Small-Molecule Cancer Vaccine Adjuvant with an Improved Toxicity Profile.
- Author
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Zhuo SH, Noda N, Hioki K, Jin S, Hayashi T, Hiraga K, Momose H, Li WH, Zhao L, Mizukami T, Ishii KJ, Li YM, and Uesugi M
- Subjects
- Animals, Mice, Adjuvants, Vaccine, Adjuvants, Immunologic pharmacology, Adjuvants, Immunologic chemistry, T-Lymphocytes, Adjuvants, Pharmaceutic, Vaccines, Subunit, Peptides, Dendritic Cells, Cancer Vaccines therapeutic use, Neoplasms
- Abstract
Protein or peptide cancer vaccines usually include immune potentiators, so-called adjuvants. However, it remains challenging to identify structurally simple, chemically accessible synthetic molecules that are effective and safe as vaccine adjuvant. Here, we present cholicamideβ ( 6 ), a self-assembling small-molecule vaccine adjuvant with an improved toxicity profile and proven efficacy in vivo . We demonstrate that cholicamideβ ( 6 ), which is less cytotoxic than its parent compound, forms virus-like particles to potently activate dendritic cells with the concomitant secretion of cytokines. When combined with a peptide antigen, cholicamideβ ( 6 ) potentiated the antigen presentation on dendritic cells to induce antigen-specific T cells. As a therapeutic cancer vaccine adjuvant in mice, a mixture of cholicamideβ ( 6 ) and a peptide antigen protected mice from the challenges of malignant cancer cells without overt toxicity. Cholicamideβ ( 6 ) may offer a translational opportunity as an unprecedented class of small-molecule cancer vaccine adjuvants.
- Published
- 2023
- Full Text
- View/download PDF
12. Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase.
- Author
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Zhao J, Zhuo Y, Diaz DE, Shanmugam M, Telfer AJ, Lindley PJ, Kracher D, Hayashi T, Seibt LS, Hardy FJ, Manners O, Hedison TM, Hollywood KA, Spiess R, Cain KM, Diaz-Moreno S, Scrutton NS, Tovborg M, Walton PH, Heyes DJ, and Green AP
- Subjects
- Mixed Function Oxygenases, Oxidative Stress, Catalysis, Copper, Histidine
- Abstract
Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a Cu
II -(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled ( S = 1) CuII -tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.- Published
- 2023
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13. Nonadditivities of the Particle Sizes Hidden in Model Pair Potentials and Their Effects on Physical Adsorptions.
- Author
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Amano KI, Furukawa S, Kubo Y, Nakamura Y, Ishii R, Tanase A, Maebayashi M, Hayashi T, Nishi N, and Sakka T
- Abstract
It is important to understand the mechanism of colloidal particle assembly near a substrate for development of drug delivery systems, micro-/nanorobots, batteries, heterogeneous catalysts, paints, and cosmetics. Understanding the mechanism is also important for crystallization of the colloidal particles and proteins. In this study, we calculated the physical adsorption of colloidal particles on a flat wall mainly using the integral equation theory, wherein small and large colloidal particles were employed. In the calculation system, like-charged electric double-layer potentials were used as pair potentials. In some cases, it was found that the small particles are more easily adsorbed. This result is unusual from the viewpoint of the Asakura-Oosawa theory, and we call it a "reversal phenomenon". Theoretical analysis revealed that the reversal phenomenon originates from the nonadditivities of the particle sizes. Using the knowledge obtained from this study, we invented a method to analyze the size nonadditivity hidden in model pair potentials. The method will be useful for confirmation of various simulation results regarding the adsorption and development of force fields for colloidal particles, proteins, and solutes.
- Published
- 2023
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14. Vanillin Production Pathways in Alkaline Nitrobenzene Oxidation of Guaiacylglycerol-β-guaiacyl Ether.
- Author
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Hayashi T, Hosoya T, and Miyafuji H
- Subjects
- Nitrobenzenes, Lignin chemistry, Guaifenesin metabolism
- Abstract
Alkaline nitrobenzene oxidation (AN oxidation) is a significant method for chemical analysis of lignin. Despite its importance in lignin chemistry, the detailed chemical reactions involved in AN oxidation are not yet fully understood. Surprisingly, there is almost no experimentally supported information available regarding the reaction pathways in the AN oxidation of guaiacyl glycerol-β-guaiacyl ether ( GG ), a common model compound in lignin chemistry. This study reports the results of our investigation into the formation pathway of vanillin (4-hydroxy-3-methoxybenzaldehyde) in the AN oxidation of GG . Our series of experiments proposed a vanillin formation pathway involving an enol ether with a C
2 side chain, 2-methoxy-4-[2-(2-methoxyphenoxy)-ethenyl]-phenol C2EE , as an intermediate, in which C2EE is produced by the non-oxidative degradation of GG by alkali. Another enol ether with a C3 side-chain, Z -4-[3-hydroxy-2-(2-methoxyphenoxy)-1-propen-1-yl]-2-methoxyphenol ( C3EE ), and the condensation products formed under alkaline conditions were found to be insignificant as vanillin sources. On the other hand, the comparison of the vanillin yields from GG and isolated C2EE (80.7 and 86.5 mol %, respectively) in their AN oxidation to the C2EE yield from GG in the absence of nitrobenzene (69.9 mol %) also suggested that the vanillin formation from GG involved unknown pathways in which C2EE is not an intermediate.- Published
- 2023
- Full Text
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15. Designed Production of Atomic-Scale Nanowindows in Single-Walled Carbon Nanotubes.
- Author
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Nagata Y, Kukobat R, Furuse A, Otsuka H, Hayashi T, and Kaneko K
- Abstract
The controlled production of nanowindows in graphene layers is desirable for the development of ultrathin membranes. Herein, we propose a single-atom catalytic oxidation method for introducing nanowindows into the graphene layers of single-walled carbon nanotubes (SWCNTs). Using liquid-phase adsorption, copper(II) 2,3,9,10,16,17,23,24-octakis(octyloxy)-29 H ,31 H -phthalocyanine (CuPc) was adsorbed on SWCNT bundles at a surface coverage of 0.9. Subsequently, narrow nanowindows with a number density of 0.13 nm
-2 were produced by oxidation above 550 K, which is higher than the decomposition temperature of bulk CuPc. In particular, oxidation of the CuPc-adsorbed SWCNTs at 623 K increased the surface area from 280 to 1690 m2 g-1 owing to the efficient production of nanowindows. The nanowindow size was estimated to be similar to the molecular size of N2 based on the pronounced low-pressure adsorption hysteresis in the N2 adsorption isotherm. In addition, the enthalpy change for the nanowindow-formation equilibrium decreased by 4 kJ mol-1 when CuPc was present, further evidencing the catalytic effect of the Cu atoms supplied by the adsorbed CuPc molecules.- Published
- 2023
- Full Text
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16. Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx.
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Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, and Hayashi T
- Subjects
- Animals, Mice, Immunization, Small Molecule Libraries, Immunogenicity, Vaccine drug effects, Calcium metabolism, Extracellular Vesicles drug effects, Extracellular Vesicles metabolism, Immunologic Factors chemistry
- Abstract
Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca
2+ elevation enhances EV release, we screened 80 hit compounds and identified compound 634 as a Ca2+ influx inducer. 634 enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from 634 -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca2+ elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of 634 . The analogues that retained the ability to induce Ca2+ influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca2+ influx. The levels of Ca2+ induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, 634 , enhances the release of EVs with immunostimulatory potency via induction of Ca2+ influx. This agent is a novel tool for EV-based immune studies and vaccine development.- Published
- 2023
- Full Text
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17. Understanding Spatial Distributions of Dye Molecules Coupled to the Surface Lattice Resonance Mode through Electrochemical Reaction Control.
- Author
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Hayashi T, Minamimoto H, and Murakoshi K
- Abstract
The strong coupling, which is the light-matter interaction, leads to changes in the energy landscape of the chemical dynamics, resulting in the modulation of the reaction pathways. In this study, we achieved strong coupling between dye molecules dispersed in the polymer films and the surface lattice resonance mode, which is excited on plasmonic lattice arrays. In addition, we successfully tuned the coupling strength by introducing the electrochemical potential control method. Reversible decreases and increases in the coupling strength were observed as a result of the reversible electrochemical redox reactions of dye molecules. It is important that the spatial distribution of the molecules coupled to the lattice resonance mode was clarified by using various polymer film thicknesses. Our present electrochemical method for controlling strong coupling states represents a promising method for tuning the light-absorption properties of systems.
- Published
- 2023
- Full Text
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18. Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric β-Barrel Protein Scaffold.
- Author
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Kato S, Onoda A, Schwaneberg U, and Hayashi T
- Abstract
Evolutionary engineering of our previously reported Cp*Rh(III)-linked artificial metalloenzyme was performed based on a DNA recombination strategy to improve its catalytic activity toward C(sp
2 )-H bond functionalization. Improved scaffold design was achieved with α-helical cap domains of fatty acid binding protein (FABP) embedded within the β-barrel structure of nitrobindin (NB) as a chimeric protein scaffold for the artificial metalloenzyme. After optimization of the amino acid sequence by directed evolution methodology, an engineered variant, designated NBHLH1 (Y119A/G149P) with enhanced performance and enhanced stability was obtained. Additional rounds of metalloenzyme evolution provided a Cp*Rh(III)-linked NBHLH1 (Y119A/G149P) variant with a >35-fold increase in catalytic efficiency ( kcat / KM ) for cycloaddition of oxime and alkyne. Kinetic studies and MD simulations revealed that aromatic amino acid residues in the confined active-site form a hydrophobic core which binds to aromatic substrates adjacent to the Cp*Rh(III) complex. The metalloenzyme engineering process based on this DNA recombination strategy will serve as a powerful method for extensive optimization of the active-sites of artificial metalloenzymes.- Published
- 2023
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19. Investigation of Hydration States of Ionic Liquids by Fourier Transform Infrared Absorption Spectroscopy: Relevance to Stabilization of Protein Molecules.
- Author
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Rajapriya Inbaraj N, Song S, Chang R, Fujita K, and Hayashi T
- Subjects
- Spectroscopy, Fourier Transform Infrared, Fourier Analysis, Proteins chemistry, Water chemistry, Hydrogen, Ionic Liquids chemistry
- Abstract
Among many kinds of ionic liquids, some hydrated ionic liquids (Hy ILs) have shown an exceptional capability to stabilize protein molecules and maintain their structure and functions over a long period. However, the complex IL-water interaction among these protein-stabilizing Hy ILs has yet to be elucidated clearly. In this work, we investigate the origin of the compatibility of ionic liquid with proteins from the viewpoint of hydration structure. We systematically analyzed the hydrogen-bonding state of water molecules around ionic liquid using Fourier transform infrared absorption (FT-IR) spectroscopy. We found that the native hydrogen-bonding network of water remained relatively unperturbed in the protein-stabilizing ILs. We also observed that the protein-stabilizing ILs have a strong electric field interaction with the surrounding water molecules and this water-IL interaction did not disrupt the water-water hydrogen-bonding interaction. On the other hand, protein-denaturing ILs perturb the hydrogen-bonding network of the water molecules to a greater extent. Furthermore, the protein-denaturing ILs were found to have a weak electric field effect on the water molecules. We speculate that the direct hydrogen bonding of the ILs with water molecules and the strong electric field of the ions lasting several hydration shells while maintaining the relatively unperturbed hydrogen-bonding network of the water molecules play an essential role in protein stabilization.
- Published
- 2023
- Full Text
- View/download PDF
20. Effect of Pretreatment Conditions on the Precise Nanoporosity of Graphene Oxide.
- Author
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Bairi P, Furuse A, Fujisawa K, Hayashi T, and Kaneko K
- Abstract
Nanoscale pores in graphene oxide (GO) control various important functions. The nanoporosity of GO is sensitive to low-temperature heating. Therefore, it is important to carefully process GO and GO-based materials to achieve superior functions. Optimum pretreatment conditions, such as the pre-evacuation temperature and time, are important during gas adsorption in GO to obtain accurate pore structure information. This study demonstrated that the pre-evacuation temperature and time for gas adsorption in GO must be approximately 333-353 K and 4 h, respectively, to avoid the irreversible alteration of nanoporosity. In situ temperature-dependent Fourier-transform infrared spectra and thermogravimetric analysis-mass spectrometry suggested significant structural changes in GO above the pre-evacuation temperature (353 K) through the desorption of "physically adsorbed water" and decomposition of unstable surface functional groups. The nanoporosity of GO significantly changed above the aforementioned pre-evacuation temperature and time. Thus, standard pretreatment is indispensable for understanding the intrinsic interface properties of GO.
- Published
- 2022
- Full Text
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21. Chiral Diene Ligands in Asymmetric Catalysis.
- Author
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Huang Y and Hayashi T
- Subjects
- Catalysis, Ligands, Stereoisomerism, Coordination Complexes
- Abstract
Asymmetric catalysis has emerged as a general and powerful approach for constructing chiral compounds in an enantioselective manner. Hence, developing novel chiral ligands and catalysts that can effectively induce asymmetry in reactions is crucial in modern chemical synthesis. Among such chiral ligands and catalysts, chiral dienes and their metal complexes have received increased attention, and a great progress has been made over the past two decades. This review provides comprehensive and critical information on the essential aspects of chiral diene ligands and their importance in asymmetric catalysis. The literature covered ranges from August 2003 (when the first effective chiral diene ligand for asymmetric catalysis was reported) to October 2021. This review is divided into two parts. In the first part, the chiral diene ligands are categorized according to their structures, and their preparation methods are summarized. In the second part, their applications in asymmetric transformations are presented according to the reaction types.
- Published
- 2022
- Full Text
- View/download PDF
22. Prediction of Serum Adsorption onto Polymer Brush Films by Machine Learning.
- Author
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Palai D, Tahara H, Chikami S, Latag GV, Maeda S, Komura C, Kurioka H, and Hayashi T
- Subjects
- Adsorption, Machine Learning, Surface Properties, Blood Proteins, Polymers
- Abstract
Using machine learning based on a random forest (RF) regression algorithm, we attempted to predict the amount of adsorbed serum protein on polymer brush films from the films' physicochemical information and the monomers' chemical structures constituting the films using a RF model. After the training of the RF model using the data of polymer brush films synthesized from five different types of monomers, the model became capable of predicting the amount of adsorbed protein from the chemical structure, physicochemical properties of monomer molecules, and structural parameters (density and thickness of the films). The analysis of the trained RF quantitatively provided the importance of each structural parameter and physicochemical properties of monomers toward serum protein adsorption (SPA). The ranking for the significance of the parameters agrees with our general understanding and perception. Based on the results, we discuss the correlation between brush film's physical properties (such as thickness and density) and SPA and attempt to provide a guideline for the design of antibiofouling polymer brush films.
- Published
- 2022
- Full Text
- View/download PDF
23. Interlocking of Single-Walled Carbon Nanotubes with Metal-Tethered Tetragonal Nanobrackets to Enrich a Few Hundredths of a Nanometer Range in Their Diameters.
- Author
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Cheng G, Hayashi T, Miyake Y, Sato T, Tabata H, Katayama M, and Komatsu N
- Abstract
We have separated carbon nanotubes through host-guest complexation using host molecules named "nanotweezers" and "nanocalipers". In this work, a host molecule named tetragonal "M-nanobrackets", consisting of a pair of dipyrrin nanocalipers corresponding to two brackets "[" and "]" tethered by two metals (M), is designed, synthesized, and employed to separate single-walled carbon nanotubes (SWNTs). A facile three-step process including one-pot Suzuki coupling is developed to synthesize M-nanobrackets in a 37% total yield (M = Cu). Upon extraction of SWNTs with a square nanobracket and Cu(II), in situ formed tetragonal M-nanobrackets are found to interlock SWNTs to disperse them in 2-propanol. The interlocking is confirmed by absorption and Raman spectroscopy as well as transmission electron and atomic force microscopy. Especially, Raman spectroscopy is utilized to prove the interlocking of SWNTs; Cu-nanobrackets are found to show inherent resonance Raman signals and affect the SWNT signals, or a radial breathing vibration, due to the rigid rectangular structure of Cu-nanobrackets. The interlocking is facilely and thoroughly released through demetalation to recover the pristine SWNTs as well as the square nanobracket. Such chemically controlled locking and unlocking for SWNTs are one of the characteristics of our separation process. This enables a precise evaluation by Raman, photoluminescence, and absorption spectroscopy of the diameter selectivity to SWNTs, revealing the diameter enrichment of only three kinds of SWNTs, (7,6), (9,4), and (8,5), in the 0.02 nm diameter range from 0.90 to 0.92 nm among ∼20 kinds of SWNTs from 0.76 to 1.17 nm in their diameter range.
- Published
- 2022
- Full Text
- View/download PDF
24. Glyco-Nanoadjuvants: Impact of Linker Length for Conjugating a Synthetic Small-Molecule TLR7 Ligand to Glyco-Nanoparticles on Immunostimulatory Effects.
- Author
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Shinchi H, Komaki F, Yuki M, Ohara H, Hayakawa N, Wakao M, Cottam HB, Hayashi T, Carson DA, Moroishi T, and Suda Y
- Subjects
- Adjuvants, Immunologic pharmacology, Animals, Gold, Immunization, Ligands, Mice, Toll-Like Receptor 7, Metal Nanoparticles, Thioctic Acid
- Abstract
Immunotherapy has become a powerful clinical strategy for treating infectious diseases and cancer. Synthetic small-molecule toll-like receptor 7 (TLR7) ligands are attractive candidates as immunostimulatory agents for immunotherapy. TLR7 is mainly localized in intracellular endosomal compartments so that the formulation of their small-molecule ligands with macromolecules enhances endocytic uptake of TLR7 ligands and improves the pharmaceutical properties. Previously, we demonstrated that gold nanoparticles co-immobilized with a TLR7 ligand derivative, that is, a conjugate of synthetic small-molecule TLR7 ligand (1V209) and thioctic acid (TA) via 4,7,10-trioxa-1,13-tridecanediamine, and α-mannose (1V209-αMan-GNPs: glyco-nanoadjuvants) significantly enhances immunostimulatory effects. In the present study, we designed a second-generation glyco-nanoadjuvant that possesses a poly(ethylene glycol) (PEG) chain as a spacer between 1V209 and GNPs and investigated the impact of linker length in 1V209 derivatives on the immunostimulatory activities. We used different chain lengths of PEG ( n = 3, 5, 11, or 23) as spacers between 1V209 and thioctic acid to prepare four 1V209-αMan-GNPs. In the in vitro study using primary mouse bone-marrow-derived dendritic cells, 1V209-αMan-GNPs that immobilized with longer 1V209 derivatives, especially the 1V209 derivative possessing PEG23 (1V209-PEG23-TA), showed the highest potency toward induction both for interleukin-6 and type I interferon production than those derivatives with shorter PEG chains. Furthermore, 1V209-αMan-GNPs that immobilized with 1V209-PEG23-TA showed significantly higher adjuvant effects for inducing both humoral and cell-mediated immune responses against ovalbumin in the in vivo immunization study. These results indicate that the linker length for immobilizing small-molecule TLR7 ligand on the GNPs significantly affects the adjuvant activity of 1V209-αMan-GNPs and that 1V209-αMan-GNPs immobilized with 1V209-PEG-23-TA could be superior adjuvants for immunotherapies.
- Published
- 2022
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25. Identification of Scytalone Dehydratase Inhibitors Effective against Melanin Biosynthesis Dehydratase Inhibitor-Resistant Pyricularia oryzae .
- Author
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Motoyama T, Kondoh Y, Shimizu T, Hayashi T, Honda K, Uchida M, and Osada H
- Subjects
- Ascomycota, Hydro-Lyases metabolism, Melanins, Plant Diseases microbiology, Magnaporthe, Oryza metabolism
- Abstract
Melanin is a secondary metabolite required for the infection of the rice blast fungus Pyricularia oryzae . Melanin biosynthesis enzymes are targets for controlling rice blast disease, and three types of commercial melanin biosynthesis inhibitors (MBIs) including MBI-R, MBI-D, and MBI-P have been developed. However, the occurrence of MBI-D-resistant strains containing scytalone dehydratase (SDH1/RSY1) with V75M mutations has been recently reported. In this study, we aimed to identify inhibitors of SDH1-V75M. We screened the RIKEN Natural Products Depository chemical library using chemical array technology and evaluated the inhibition of SDH1-V75M by candidate compounds. NPD13731 strongly inhibited the activity of wild-type and mutant SDH1. The structure-activity relationship data were used to create a more potent inhibitor 16 , which controlled rice blast disease in rice plants infected with MBI-D-resistant P. oryzae . Compound 16 , which we named melabiostin, may be used to develop fungicides for controlling rice blast infections.
- Published
- 2022
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26. Development of an Outward Proton Pumping Rhodopsin with a New Record in Thermostability by Means of Amino Acid Mutations.
- Author
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Yasuda S, Akiyama T, Kojima K, Ueta T, Hayashi T, Ogasawara S, Nagatoishi S, Tsumoto K, Kunishima N, Sudo Y, Kinoshita M, and Murata T
- Subjects
- Amino Acids genetics, Mutation, Protons, Proton Pumps chemistry, Rhodopsin genetics
- Abstract
We have developed a methodology for identifying further thermostabilizing mutations for an intrinsically thermostable membrane protein. The methodology comprises the following steps: (1) identifying thermostabilizing single mutations (TSSMs) for residues in the transmembrane region using our physics-based method; (2) identifying TSSMs for residues in the extracellular and intracellular regions, which are in aqueous environment, using an empirical force field FoldX; and (3) combining the TSSMs identified in steps (1) and (2) to construct multiple mutations. The methodology is illustrated for thermophilic rhodopsin whose apparent midpoint temperature of thermal denaturation T
m is ∼91.8 °C. The TSSMs previously identified in step (1) were F90K, F90R, and Y91I with Δ Tm ∼5.6, ∼5.5, and ∼2.9 °C, respectively, and those in step (2) were V79K, T114D, A115P, and A116E with Δ Tm ∼2.7, ∼4.2, ∼2.6, and ∼2.3 °C, respectively (Δ Tm denotes the increase in Tm ). In this study, we construct triple and quadruple mutants, F90K+Y91I+T114D and F90K+Y91I+V79K+T114D. The values of Δ Tm for these multiple mutants are ∼11.4 and ∼13.5 °C, respectively. Tm of the quadruple mutant (∼105.3 °C) establishes a new record in a class of outward proton pumping rhodopsins. It is higher than Tm of Rubrobacter xylanophilus rhodopsin (∼100.8 °C) that was the most thermostable in the class before this study.- Published
- 2022
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27. Interphase Protein Layers Formed on Self-Assembled Monolayers in Crowded Biological Environments: Analysis by Surface Force and Quartz Crystal Microbalance Measurements.
- Author
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Mondarte EAQ, Zamarripa EMM, Chang R, Wang F, Song S, Tahara H, and Hayashi T
- Subjects
- Adsorption, Hydrophobic and Hydrophilic Interactions, Surface Properties, Quartz Crystal Microbalance Techniques, Serum Albumin, Bovine chemistry
- Abstract
We investigated a viscous protein layer formed on self-assembled monolayers (SAMs) in crowded biological environments. The results were obtained through force spectroscopic measurements using colloidal probes and substantiated by exhaustive analysis using a quartz crystal microbalance with an energy dissipation technique. A hydrophobic SAM of n -octanethiol (C8 SAM) in bovine serum albumin (BSA) solution is buried under an adlayer of denatured BSA molecules and an additional viscous interphase layer that is five times more viscous than the bulk solution. C8 SAMs in fetal bovine serum induced a formation of a thicker adsorbed protein layer but with no observable viscous interphase layer. These findings show that a fouling surface is essentially inaccessible to any approaching molecules and thus has a new biological and physical identity arising from its surrounding protein layers. In contrast, the SAMs composed of sulfobetaine-terminated alkanethiol proved to be sufficiently protein-resistant and bio-inert even under crowded conditions due to a protective barrier of its interfacial water, which has implications in the accurate targeting of artificial particles for drug delivery and similar applications by screening any non-specific interactions. Finally, our strategies provide a platform for the straightforward yet effectual in vitro characterization of diverse types of surfaces in the context of targeted interactions in crowded biological environments.
- Published
- 2022
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28. Controlling the Rigidity of Kinesin-Propelled Microtubules in an In Vitro Gliding Assay Using the Deep-Sea Osmolyte Trimethylamine N -Oxide.
- Author
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Kabir AMR, Munmun T, Hayashi T, Yasuda S, Kimura AP, Kinoshita M, Murata T, Sada K, and Kakugo A
- Abstract
The biomolecular motor protein kinesin and its associated filamentous protein microtubule have been finding important nanotechnological applications in the recent years. Rigidity of the microtubules, which are propelled by kinesin motors in an in vitro gliding assay, is an important metric that determines the success of utilization of microtubules and kinesins in various applications, such as transportation, sensing, sorting, molecular robotics, etc. Therefore, regulating the rigidity of kinesin-propelled microtubules has been critical. In this work, we report a simple strategy to regulate the rigidity of kinesin-propelled microtubules in an in vitro gliding assay. We demonstrate that rigidity of the microtubules, propelled by kinesins in an in vitro gliding assay, can be modulated simply by using the natural osmolyte trimethylamine N -oxide (TMAO). By varying the concentration of TMAO in the gliding assay, the rigidity of microtubules can be modulated over a wide range. Based on this strategy, we are able to reduce the persistence length of microtubules, a measure of microtubule rigidity, ∼8 fold by using TMAO at the concentration of 1.5 M. Furthermore, we found that the decreased rigidity of the kinesin-propelled microtubules can be restored upon elimination of TMAO from the in vitro gliding assay. Alteration in the rigidity of microtubules is accounted for by the non-uniformity of the force applied by kinesins along the microtubules in the presence of TMAO. This work offers a facile strategy to reversibly regulate the rigidity of kinesin-propelled microtubules in situ , which would widen the applications of the biomolecular motor kinesin and its associated protein microtubule in various fields., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)
- Published
- 2022
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29. Small Molecule Calcium Channel Activator Potentiates Adjuvant Activity.
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Saito T, Shukla NM, Sato-Kaneko F, Sako Y, Hosoya T, Yao S, Lao FS, Messer K, Pu M, Chan M, Chu PJ, Cottam HB, Hayashi T, Carson DA, and Corr M
- Subjects
- Animals, Humans, Mice, Cell Line, Cell Proliferation drug effects, Cytokines genetics, Cytokines metabolism, Gene Expression Regulation drug effects, Lipopolysaccharides pharmacology, Lymphocytes drug effects, Ovalbumin immunology, Receptors, Pattern Recognition genetics, Receptors, Pattern Recognition metabolism, Adjuvants, Immunologic pharmacology, Calcium Channel Agonists pharmacology
- Abstract
There remains an unmet need for reliable fully synthetic adjuvants that increase lasting protective immune responses from vaccines. We previously reported a high-throughput screening for small molecules that extended nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) activation after a Toll-like receptor 4 (TLR4) ligand, lipopolysaccharide (LPS), stimulation using a human myeloid reporter cell line. We identified compounds with a conserved aminothiazole scaffold including 2D216 [ N -(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide], which increased murine antigen-specific antibody responses when used as a co-adjuvant with LPS. Here, we examined the mechanism of action in human cells. Although 2D216 activated the major mitogen-activated protein kinases, it did not interact with common kinases and phosphatases and did not stimulate many of the pattern recognition receptors (PRRs). Instead, the mechanism of action was linked to intracellular Ca
2+ elevation via Ca2+ channel(s) at the plasma membrane and nuclear translocation of the nuclear factor of activated T-cells (NFAT) as supported by RNA-seq data, analysis by reporter cells, Ca2+ flux assays, and immunoblots. Interestingly, 2D216 had minimal, if any, activity on Jurkat T cells but induced cytokine production and surface expression of costimulatory molecules on cells with antigen-presenting functions. A small series of analogs of 2D216 were tested for the ability to enhance a TLR4 ligand-stimulated autologous mixed lymphocyte reaction (MLR). In the MLR, 2E151 , N -(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide, was more potent than 2D216 . These results indicate that a small molecule that is not a direct PRR agonist can act as a co-adjuvant to an approved adjuvant to enhance human immune responses via a complementary mechanism of action.- Published
- 2022
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30. Unsymmetric Dumbbell-Shaped Polyhedral Oligomeric Silsesquioxane (POSS) Compound as a Single-Component POSS Hybrid.
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Nagao M, Hayashi T, Imoto H, and Naka K
- Abstract
Dumbbell-shaped polyhedral oligomeric silsesquioxane (POSS) derivatives, in which two POSS units are linked through a bridge, have attracted attention in the last decade. Here, we prepared an unsymmetric dumbbell-shaped POSS derivative ( 3
Ph-iBu ) in which isobutyl- and phenyl-substituted POSS units are linked by a disiloxane unit and compared its thermal properties with those of the corresponding symmetric isobutyl- and phenyl-substituted dumbbell-shaped POSS derivatives ( 3iBu-iBu and 3Ph-Ph , respectively). The symmetric isobutyl- and phenyl-substituted dumbbell-shaped POSS derivatives, 3iBu-iBu and 3Ph-Ph , were almost completely phase-separated during a mixing process. This phase separation is due to the limited solubility of phenyl-substituted POSS compounds, which are only soluble in tetrahydrofuran (THF) and insoluble in hydrocarbons such as n -hexane and toluene, while the isobutyl-substituted POSS derivatives exhibit a wider spectrum of soluble solvents. The unsymmetric dumbbell-shaped POSS, 3Ph-iBu , showed hybrid properties of solubility in solvents and thermal behaviors. Differential scanning calorimetric (DSC) analysis showed that enthalpy of the phase transition of 3Ph-iBu was significantly lower than those of the mixture of 3iBu-iBu and 3Ph-Ph . No apparent melting behavior was observed above the phase transition. The thermal degradation of the weakest isobutyl substituents improves in the present single-component hybrid structure.- Published
- 2021
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31. Multifunctional Traceable Liposomes with Temperature-Triggered Drug Release and Neovasculature-Targeting Properties for Improved Cancer Chemotherapy.
- Author
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Yuba E, Takashima M, Hayashi T, Kokuryo D, Aoki I, Harada A, Aoshima S, Krishnan UM, and Kono K
- Subjects
- Animals, Antibiotics, Antineoplastic pharmacokinetics, Cell Line, Tumor transplantation, Disease Models, Animal, Doxorubicin administration & dosage, Doxorubicin analogs & derivatives, Doxorubicin pharmacokinetics, Drug Liberation, Female, Hot Temperature, Humans, Liposomes, Mice, Neoplasms blood supply, Neoplasms pathology, Neovascularization, Pathologic pathology, Peptides, Cyclic chemistry, Polyethylene Glycols administration & dosage, Polyethylene Glycols pharmacokinetics, Polymers chemistry, Antibiotics, Antineoplastic administration & dosage, Nanoparticle Drug Delivery System chemistry, Neoplasms drug therapy, Neovascularization, Pathologic drug therapy
- Abstract
Poor distribution of nanocarriers at the tumor site and insufficient drug penetration into the tissue are major challenges in the development of effective and safe cancer therapy. Here, we aim to enhance the therapeutic effect of liposomes by accumulating doxorubicin-loaded liposomes at high concentrations in and around the tumor, followed by heat-triggered drug release to facilitate low-molecular-weight drug penetration throughout the tumor. A cyclic RGD peptide (cRGD) was incorporated into liposomes decorated with a thermosensitive polymer that allowed precise tuning of drug release temperature (i.e., Polymer-lip) to develop a targeted thermosensitive liposome (cRGD-Polymer-lip). Compared with conventional thermosensitive liposomes, cRGD-Polymer-lip enhanced the binding of liposomes to endothelial cells, leading to their accumulation at the tumor site upon intravenous administration in tumor-bearing mice. Drug release triggered by local heating strongly inhibited tumor growth. Notably, tumor remission was achieved via multiple administrations of cRGD-Polymer-lip and heat treatments. Thus, combining the advantages of tumor neovascular targeting and heat-triggered drug release, these liposomes offer high potential for minimally invasive and effective cancer chemotherapy.
- Published
- 2021
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32. Asymmetric Synthesis of Chiral Bicyclo[2.2.1]hepta-2,5-diene Ligands through Rhodium-Catalyzed Asymmetric Arylative Bis-cyclization of a 1,6-Enyne.
- Author
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Sun C, Meng H, Chen C, Wei H, Ming J, and Hayashi T
- Abstract
A series of novel chiral diene ligands (1 R, 4 S ) -L
1 , which are based on the bicyclo[2.2.1]heptadiene skeleton and are substituted with methyl and an ester group at the bridgehead carbons, were synthesized through rhodium-catalyzed asymmetric arylative bis-cyclization of 1,6-enyne 1 as a key step. The rhodium catalyst with one of the (1 R, 4 S ) -L1 ligands was used for the asymmetric bis-cyclization of 1 giving bicyclic product (1 S, 4 R ) -2 of 99% ee, which is a synthetic precursor of (1 S, 4 R ) -L1 ligands.- Published
- 2021
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33. Functional Myoglobin Model Composed of a Strapped Porphyrin/Cyclodextrin Supramolecular Complex with an Overhanging COOH That Increases O 2 /CO Binding Selectivity in Aqueous Solution.
- Author
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Mao Q, Das PK, Le Gac S, Boitrel B, Dorcet V, Oohora K, Hayashi T, and Kitagishi H
- Subjects
- Hydrogen-Ion Concentration, Solutions, Temperature, Biomimetic Materials chemistry, Carbon Monoxide chemistry, Cyclodextrins chemistry, Myoglobin chemistry, Oxygen chemistry, Porphyrins chemistry, Water chemistry
- Abstract
A water-soluble strapped iron(III)tetraarylporphyrin ( Fe
III Por-1 ) bearing two propylpyridinium groups at the side chains and a carboxylic acid group at the overhanging position of the strap was synthesized to mimic the function of myoglobin with the distal polar functionality in aqueous solution. FeIII Por-1 forms a stable 1:1 inclusion complex with a per- O -methylated β-cyclodextrin dimer having a pyridine linker ( Py3OCD ), providing a hydrophobic environment and a proximal fifth ligand to stabilize the O2 -complex. The ferrous complex ( FeII PorCD-1 ) binds both O2 and CO in aqueous solution. The O2 and CO binding affinities ( P1/2 O2 and P1/2 CO ) and half-life time ( t1/2 ) of the O2 complex of FeII PorCD-1 are 6.3 and 0.021 Torr, and 7 h, respectively, at pH 7 and 25 °C. The control compound without the strap structure ( FeII PorCD-2 ) has similar oxygen binding characteristics ( P1/2 O2 = 8.0 Torr), but much higher CO binding affinity ( P1/2 CO = 3.8 × 10-4 Torr), and longer t1/2 (30 h). The O2 and CO kinetics indicate that the strapped structure in FeII PorCD-1 inhibits the entrance of these gaseous ligands into the iron(II) center, as evidenced by lower kon O2 and kon CO values. Interestingly, the CO complex of FeII PorCD-1 is significantly destabilized (relatively larger koff CO ), while the koff O2 value is much smaller than that of FeII PorCD-2 , resulting in significantly increased O2 /CO selectivity (reduced M value, where M = P1/2 O2 / P1/2 CO = 320) in FeII PorCD-1 compared to FeII PorCD-2 ( M = 21000).- Published
- 2021
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34. Amiodarone, Unlike Dronedarone, Activates Inflammasomes via Its Reactive Metabolites: Implications for Amiodarone Adverse Reactions.
- Author
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Kato R, Ijiri Y, and Hayashi T
- Subjects
- Amiodarone adverse effects, Cell Line, Dronedarone adverse effects, Hepatocytes drug effects, Hepatocytes immunology, Humans, Inflammasomes immunology, Macrophages drug effects, Macrophages immunology, THP-1 Cells, Amiodarone pharmacology, Anti-Arrhythmia Agents pharmacology, Dronedarone pharmacology, Inflammasomes agonists
- Abstract
Amiodarone is a benzofuran derivative used to treat arrhythmias, but its use is limited by adverse reactions. There is evidence that some of the severe adverse reactions such as liver injury and interstitial lung disease are immune-mediated; however, details of the mechanism have not been elucidated. We tested the ability of amiodarone to induce the release of danger-associated molecular patterns (DAMPs) that activate inflammasomes. Human hepatocarcinoma functional liver cell-4 (FLC-4) cells were used for drug bioactivation, and the detection of inflammasome activation was performed with the human macrophage cell line, THP-1 cells. Amiodarone is known to be oxidized to reactive quinone metabolites. The supernatant from the incubation of amiodarone with FLC-4 cells for 7 days increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells with amiodarone, the heat shock protein (HSP) 40 was significantly increased. Addition of a cytochrome P450 inhibitor to the FLC-4 cells prevented the release of HSP40 from the FLC-4 cells and activation of THP-1 inflammasomes by the FLC-4 supernatant. These results suggested that the reactive quinone metabolites of amiodarone can cause the release of DAMPs from hepatocytes which can activate inflammasomes. Dronedarone, a safer analog of amiodarone, did not activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by amiodarone, which in some patients, can cause immune-related adverse events. In addition, our data suggest that drugs that block the effects or the formation of IL-1β would provide better treatment of amiodarone-induced immune-related adverse reactions.
- Published
- 2021
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35. Cell-Specific Delivery Using an Engineered Protein Nanocage.
- Author
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Levasseur MD, Mantri S, Hayashi T, Reichenbach M, Hehn S, Waeckerle-Men Y, Johansen P, and Hilvert D
- Subjects
- Antibodies chemistry, Antibodies immunology, Cell Membrane Permeability, Drug Compounding, Drug Liberation, Humans, Immunoglobulins chemistry, Immunoglobulins immunology, Molecular Targeted Therapy, Protein Engineering, Surface Properties, Tissue Distribution, Biocompatible Materials chemistry, Multienzyme Complexes chemistry, Nanocapsules chemistry
- Abstract
Nanoparticle-based delivery systems have shown great promise for theranostics and bioimaging on the laboratory scale due to favorable pharmacokinetics and biodistribution. In this study, we examine the utility of a cage-forming variant of the protein lumazine synthase, which was previously designed and evolved to encapsulate biomacromolecular cargo. Linking antibody-binding domains to the exterior of the cage enabled binding of targeting immunoglobulins and cell-specific uptake of encapsulated cargo. Protein nanocages displaying antibody-binding domains appear to be less immunogenic than their unmodified counterparts, but they also recruit serum antibodies that can mask the efficacy of the targeting antibody. Our study highlights the strengths and limitations of a common targeting strategy for practical nanoparticle-based delivery applications.
- Published
- 2021
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36. Thermally Controlled Construction of Fe-N x Active Sites on the Edge of a Graphene Nanoribbon for an Electrocatalytic Oxygen Reduction Reaction.
- Author
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Matsumoto K, Onoda A, Kitano T, Sakata T, Yasuda H, Campidelli S, and Hayashi T
- Abstract
Pyrolytically prepared iron and nitrogen codoped carbon (Fe/N/C) catalysts are promising nonprecious metal electrocatalysts for the oxygen reduction reaction (ORR) in fuel cell applications. Fabrication of the Fe/N/C catalysts with Fe-N
x active sites having precise structures is now required. We developed a strategy for thermally controlled construction of the Fe-Nx structure in Fe/N/C catalysts by applying a bottom-up synthetic methodology based on a N-doped graphene nanoribbon (N-GNR). The preorganized aromatic rings within the precursors assist graphitization during generation of the N-GNR structure with iron-coordinating sites. The Fe/N/C catalyst prepared from the N-GNR precursor, iron ion, and the carbon support Vulcan XC-72R provides a high onset potential of 0.88 V (vs reversible hydrogen electrode (RHE)) and promotes efficient four-electron ORR. X-ray absorption fine structure (XAFS) and X-ray photoelectron spectroscopy (XPS) studies reveal that the N-GNR precursor induces the formation of iron-coordinating nitrogen species during pyrolysis. The details of the graphitization process of the precursor were further investigated by analyzing the precursors pyrolyzed at various temperatures using MgO particles as a sacrificial template, with the results indicating that the graphitized structure was obtained at 700 °C. The preorganized N-GNR precursors and its pyrolysis conditions for graphitization are found to be important factors for generation of the Fe-Nx active sites along with the N-GNR structure in high-performance Fe/N/C catalysts for the ORR.- Published
- 2021
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37. Incorporation of a Cp*Rh(III)-dithiophosphate Cofactor with Latent Activity into a Protein Scaffold Generates a Biohybrid Catalyst Promoting C(sp 2 )-H Bond Functionalization.
- Author
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Kato S, Onoda A, Grimm AR, Tachikawa K, Schwaneberg U, and Hayashi T
- Subjects
- Catalysis, Oximes chemistry, Silver chemistry, Biomimetic Materials chemistry, Carbon chemistry, Coordination Complexes chemistry, Hydrogen chemistry, Phosphates chemistry, Proteins chemistry, Rhodium chemistry
- Abstract
A Cp*Rh(III)-dithiophosphate cofactor with "latent" catalytic activity was developed to construct an artificial metalloenzyme representing a new type of biohybrid catalyst which is capable of promoting C(sp
2 )-H bond functionalization within the β-barrel structure of nitrobindin (NB). To covalently conjugate the Cp*Rh(III) cofactor into a specific position of the hydrophobic cavity of NB via a maleimide-Cys linkage, strong chelation of the dithiophosphate ligand is employed to protect the rhodium metal center against attack by nucleophilic amino acid residues in the protein. It is found that subsequent addition of the Ag+ ion induces dissociation of the dithiophosphate ligands, thereby activating the catalytic activity of the Cp*Rh(III) cofactor. The resulting "active" biohybrid catalyst promotes cycloaddition of acetophenone oxime with diphenylacetylene via C(sp2 )-H bond activation. This catalytic activity is enhanced 2.3-fold with the introduction of two glutamate residues (A100E/L125E) adjacent to the Cp*Rh(III) cofactor. The Cp*Rh(III) cofactor with switchable activity from a "latent" form to an "active" form provides a new strategy for generating biohybrid catalysts incorporating a variety of highly reactive transition metal complexes specifically within its protein scaffolds.- Published
- 2020
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38. Novel Tetrafunctional Probes Identify Target Receptors and Binding Sites of Small-Molecule Drugs from Living Systems.
- Author
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Miyajima R, Sakai K, Otani Y, Wadatsu T, Sakata Y, Nishikawa Y, Tanaka M, Yamashita Y, Hayashi M, Kondo K, and Hayashi T
- Subjects
- Animals, Binding Sites, Brain metabolism, CHO Cells, Central Nervous System Agents chemical synthesis, Central Nervous System Agents chemistry, Cricetulus, Cyclohexanones chemical synthesis, Cyclohexanones chemistry, Hydrazines chemistry, Mass Spectrometry, Mice, Molecular Probes chemical synthesis, Molecular Probes radiation effects, Proteomics methods, Receptors, AMPA chemistry, Receptors, AMPA metabolism, Receptors, Dopamine D2 chemistry, Receptors, Dopamine D2 metabolism, Receptors, GABA chemistry, Receptors, GABA metabolism, Tetrazoles chemical synthesis, Tetrazoles radiation effects, Ultraviolet Rays, Molecular Probes chemistry, Receptors, AMPA analysis, Receptors, Dopamine D2 analysis, Receptors, GABA analysis, Tetrazoles chemistry
- Abstract
Significant advancement of chemoproteomics has contributed to uncovering the mechanism of action (MoA) of small-molecule drugs by characterizing drug-protein interactions in living systems. However, cell-membrane proteins such as G protein-coupled receptors (GPCRs) and ion channels, due to their low abundance and unique biophysical properties associated with multiple transmembrane domains, can present challenges for proteome-wide mapping of drug-receptor interactions. Herein, we describe the development of novel tetrafunctional probes, consisting of (1) a ligand of interest, (2) 2-aryl-5-carboxytetrazole (ACT) as a photoreactive group, (3) a hydrazine-labile cleavable linker, and (4) biotin for enrichment. In live cell labeling studies, we demonstrated that the ACT-based probe showed superior reactivity and selectivity for labeling on-target GPCR by mass spectrometry analysis compared with control probes including diazirine-based probes. By leveraging ACT-based cleavable probes, we further identified a set of representative ionotropic receptors, targeted by CNS drugs, with remarkable selectivity and precise binding site information from mouse brain slices. We anticipate that the robust chemoproteomic platform using the ACT-based cleavable probe coupled with phenotypic screening should promote identification of pharmacologically relevant target receptors of drug candidates and ultimately development of first-in-class drugs with novel MoA.
- Published
- 2020
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39. Data-Driven Prediction of Protein Adsorption on Self-Assembled Monolayers toward Material Screening and Design.
- Author
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Kwaria RJ, Mondarte EAQ, Tahara H, Chang R, and Hayashi T
- Subjects
- Adsorption, Surface Properties, Proteins, Water
- Abstract
We attempt to predict the water contact angle (WCA) of self-assembled monolayers (SAMs) and protein adsorption on the SAMs from the chemical structures of molecules constituting the SAMs using machine learning with an artificial neural network (ANN) model. After training the ANN with data of 145 SAMs, the ANN became capable of predicting the WCA and protein adsorption accurately. The analysis of the trained ANN quantitatively revealed the importance of each structural parameter for the WCA and protein adsorption, providing essential and quantitative information for material design. We found that the degree of importance agrees well with our general perception on the physicochemical properties of SAMs. We also present the prediction of the WCA and protein adsorption of hypothetical SAMs and discuss the possibility of our approach for the material screening and design of SAMs with desired functions. On the basis of these results, we also discuss the limitation of this approach and prospects.
- Published
- 2020
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40. Methane Generation and Reductive Debromination of Benzylic Position by Reconstituted Myoglobin Containing Nickel Tetradehydrocorrin as a Model of Methyl-coenzyme M Reductase.
- Author
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Miyazaki Y, Oohora K, and Hayashi T
- Subjects
- Buffers, Halogenation, Hydrogen-Ion Concentration, Oxidation-Reduction, Benzene chemistry, Biomimetic Materials chemistry, Coordination Complexes chemistry, Corrinoids chemistry, Methane chemistry, Nickel chemistry, Oxidoreductases metabolism
- Abstract
Methyl-coenzyme M reductase (MCR), which contains the nickel hydrocorphinoid cofactor F430, is responsible for biological methane generation under anaerobic conditions via a reaction mechanism which has not been completely elucidated. In this work, myoglobin reconstituted with an artificial cofactor, nickel(I) tetradehydrocorrin (Ni
I (TDHC)), is used as a protein-based functional model for MCR. The reconstituted protein, rMb(NiI (TDHC)), is found to react with methyl donors such as methyl p -toluenesulfonate and trimethylsulfonium iodide with methane evolution observed in aqueous media containing dithionite. Moreover, rMb(NiI (TDHC)) is found to convert benzyl bromide derivatives to reductively debrominated products without homocoupling products. The reactivity increases in the order of primary > secondary > tertiary benzylic carbons, indicating steric effects on the reaction of the nickel center with the benzylic carbon in the initial step. In addition, Hammett plots using a series of para -substituted benzyl bromides exhibit enhancement of the reactivity with introduction of electron-withdrawing substituents, as shown by the positive slope against polar substituent constants. These results suggest a nucleophilic SN 2-type reaction of the Ni(I) species with the benzylic carbon to provide an organonickel species as an intermediate. The reaction in D2 O buffer at pD 7.0 causes a complete isotope shift of the product by +1 mass unit, supporting our proposal that protonation of the organonickel intermediate occurs during product formation. Although the turnover numbers are limited due to inactivation of the cofactor by side reactions, the present findings will contribute to elucidating the reaction mechanism of MCR-catalyzed methane generation from activated methyl sources and dehalogenation.- Published
- 2020
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41. Hydrated Silicate Layer Formation on Mica-Type Crystals.
- Author
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Sugiura M, Sueyoshi M, Seike R, Hayashi T, and Okada T
- Abstract
This study aims to investigate the growth of a cation-exchangeable hydrated layer on the surface of mica-type silicates based on a synthetic fluorophlogopite and a natural muscovite. Through the reaction of a synthetic fluorophlogopite using LiF, MgCl
2 , and a silica sol in water at 373 K for 48 h in the presence of urea, a hydrated phyllosilicate was formed on the fluoromica. As a result of examining the reaction in the alkali solution in the absence of Mg2+ , the uptake of the silica sol would be included as a chemical process to begin the crystallization on fluorophlogopite because the lithium and ammonium ions (generated by urea hydrolysis) are known to contribute to enhanced adsorption. We found that the urea hydrolysis increased the pH, which, in turn, assisted the formation of magnesium hydroxide after the isomorphic substitution of Li+ for Mg2+ . Bridging tetrahedral SiO4 with a magnesium-lithium double hydroxide afforded a 1 nm silicate layer. This facilitated the hectorite-like hydrated silicate layer to adhere closely to both the crystal edge and the cleaved face of the synthetic mica, which was found to coat the surface homogeneously. Only surface crystals were found to form through this process. The layered silicates included exchangeable hydrated cations for the cation-exchange reactions to expand the interlayer space by a cationic surfactant, dimethyldistearylammonium. The layered silicate also adsorbed methylene blue as a cationic dye in the aqueous phase. Apart from fluoromica, the natural muscovite also provided the surface to grow hydrated silicate layers, as a crystal turned dense blue when reacted with methylene blue.- Published
- 2020
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42. Correction to "How Does a Microbial Rodhopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained?"
- Author
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Hayashi T, Yasuda S, Suzuki K, Akiyama T, Kanehara K, Kojima K, Tanabe M, Kato R, Senda T, Sudo Y, Murata T, and Kinoshita M
- Published
- 2020
- Full Text
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43. Copper-Catalyzed Oxidative Trifluoromethylation of Terminal Alkenes Using AgCF 3 .
- Author
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Karuo Y, Hayashi T, Tarui A, Sato K, Kawai K, and Omote M
- Abstract
A mild and convenient reaction for oxidative trifluoromethylation of terminal alkenes was developed using in situ generated AgCF
3 in the presence of a copper catalyst. The reaction proceeded under an air atmosphere to afford trifluoromethylated allylic compounds in moderate to good yield. This reaction, with no need for highly hygroscopic or corrosive reagents, features not only a simple operation but also various functional group tolerances.- Published
- 2020
- Full Text
- View/download PDF
44. Methodology for Further Thermostabilization of an Intrinsically Thermostable Membrane Protein Using Amino Acid Mutations with Its Original Function Being Retained.
- Author
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Yasuda S, Akiyama T, Nemoto S, Hayashi T, Ueta T, Kojima K, Tsukamoto T, Nagatoishi S, Tsumoto K, Sudo Y, Kinoshita M, and Murata T
- Subjects
- Entropy, Mutation, Protein Conformation, Temperature, Amino Acids, Membrane Proteins genetics
- Abstract
We develop a new methodology best suited to the identification of thermostabilizing mutations for an intrinsically stable membrane protein. The recently discovered thermophilic rhodopsin, whose apparent midpoint temperature of thermal denaturation T
m is measured to be ∼91.8 °C, is chosen as a paradigmatic target. In the methodology, we first regard the residues whose side chains are missing in the crystal structure of the wild type (WT) as the "residues with disordered side chains," which make no significant contributions to the stability, unlike the other essential residues. We then undertake mutating each of the residues with disordered side chains to another residue except Ala and Pro, and the resultant mutant structure is constructed by modifying only the local structure around the mutated residue. This construction is based on the postulation that the structure formed by the other essential residues, which is nearly optimized in such a highly stable protein, should not be modified. The stability changes arising from the mutations are then evaluated using our physics-based free-energy function (FEF). We choose the mutations for which the FEF is much lower than for the WT and test them by experiments. We successfully find three mutants that are significantly more stable than the WT. A double mutant whose Tm reaches ∼100 °C is also discovered.- Published
- 2020
- Full Text
- View/download PDF
45. Preferential Recognition and Extraction to Pentoses over Hexoses by a D 6 h -Symmetrical Ethynylphenol Macrocycle with Six Inner Phenolic Hydroxy Groups.
- Author
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Hayashi T, Ohishi Y, Abe H, and Inouye M
- Abstract
A macrocycle consisting of six ethynylphenol units was developed as a host architecture for saccharides. The rigid framework of the macrocycle suppressed the intramolecular hydrogen-bonding between adjacent phenolic hydroxy groups and recognized saccharides by intermolecular hydrogen-bonding within the hole. The well-defined hydrogen-bonding sites enabled the size-selective guest recognition and showed preference to pentoses over hexoses.
- Published
- 2020
- Full Text
- View/download PDF
46. How Does a Microbial Rhodopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained?
- Author
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Hayashi T, Yasuda S, Suzuki K, Akiyama T, Kanehara K, Kojima K, Tanabe M, Kato R, Senda T, Sudo Y, Murata T, and Kinoshita M
- Subjects
- Crystallography, X-Ray, Models, Molecular, Protein Folding, Solvents chemistry, Actinobacteria chemistry, Halobacterium salinarum chemistry, Proton Pumps chemistry, Rhodopsins, Microbial chemistry, Thermodynamics
- Abstract
We often encounter a case where two proteins, whose amino-acid sequences are similar, are quite different with regard to the thermostability. As a striking example, we consider the two seven-transmembrane proteins: recently discovered Rubrobacter xylanophilus rhodopsin (RxR) and long-known bacteriorhodopsin from Halobacterium salinarum (HsBR). They commonly function as a light-driven proton pump across the membrane. Though their sequence similarity and identity are ∼71 and ∼45%, respectively, RxR is much more thermostable than HsBR. In this study, we solve the three-dimensional structure of RxR using X-ray crystallography and find that the backbone structures of RxR and HsBR are surprisingly similar to each other: The root-mean-square deviation for the two structures calculated using the backbone C
α atoms of the seven helices is only 0.86 Å, which makes the large stability difference more puzzling. We calculate the thermostability measure and its energetic and entropic components for RxR and HsBR using our recently developed statistical-mechanical theory. The same type of calculation is independently performed for the portions playing essential roles in the proton-pumping function, helices 3 and 7, and their structural properties are related to the probable roles of water molecules in the proton-transporting mechanism. We succeed in elucidating how RxR realizes its exceptionally high stability with the original function being retained. This study provides an important first step toward the establishment of a method correlating microscopic, geometric characteristics of a protein with its thermodynamic properties and enhancing the thermostability through amino-acid mutations without vitiating the original function.- Published
- 2020
- Full Text
- View/download PDF
47. Thermoresponsive Micellar Assembly Constructed from a Hexameric Hemoprotein Modified with Poly( N -isopropylacrylamide) toward an Artificial Light-Harvesting System.
- Author
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Hirayama S, Oohora K, Uchihashi T, and Hayashi T
- Abstract
Artificial protein assemblies inspired by nature have significant potential in development of emergent functional materials. In order to construct an artificial protein assembly, we employed a mutant of a thermostable hemoprotein, hexameric tyrosine-coordinated heme protein (HTHP), as a building block. The HTHP mutant which has cysteine residues introduced on the bottom surface of its columnar structure was reacted with maleimide-tethering thermoresponsive poly( N -isopropylacrylamide), PNIPAAm, to generate the protein assembly upon heating. The site-specific modification of the cysteine residues with PNIPAAm on the protein surface was confirmed by SDS-PAGE and analytical size exclusion chromatography (SEC). The PNIPAAm-modified HTHP (PNIPAAm-HTHP) is found to provide a 43 nm spherical structure at 60 °C, and the structural changes observed between the assembled and the disassembled forms were duplicated at least five times. High-speed atomic force microscopic measurements of the micellar assembly supported by cross-linkage with glutaraldehyde indicate that the protein matrices are located on the surface of the sphere and cover the inner PNIPAAm core. Furthermore, substitution of heme with a photosensitizer, Zn protoporphyrin IX (ZnPP), in the micellar assembly provides an artificial light-harvesting system. Photochemical measurements of the ZnPP-substituted micellar assembly demonstrate that energy migration among the arrayed ZnPP molecules occurs within the range of several tens of picoseconds. Our present work represents the first example of an artificial light-harvesting system based on an assembled hemoprotein oligomer structure to replicate natural light-harvesting systems.
- Published
- 2020
- Full Text
- View/download PDF
48. Gold Nanoparticles Coimmobilized with Small Molecule Toll-Like Receptor 7 Ligand and α-Mannose as Adjuvants.
- Author
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Shinchi H, Yamaguchi T, Moroishi T, Yuki M, Wakao M, Cottam HB, Hayashi T, Carson DA, and Suda Y
- Subjects
- Adenine chemistry, Adenine pharmacology, Adjuvants, Immunologic chemistry, Animals, Bone Marrow Cells drug effects, Bone Marrow Cells immunology, Bone Marrow Cells metabolism, Dendritic Cells drug effects, Dendritic Cells immunology, Dendritic Cells metabolism, Humans, Immunization, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear immunology, Leukocytes, Mononuclear metabolism, Ligands, Metal Nanoparticles chemistry, Mice, Mice, Inbred C57BL, Ovalbumin immunology, Small Molecule Libraries chemistry, Splenomegaly immunology, Splenomegaly pathology, Toll-Like Receptor 7 immunology, Adenine analogs & derivatives, Adjuvants, Immunologic pharmacology, Gold chemistry, Mannose chemistry, Metal Nanoparticles administration & dosage, Small Molecule Libraries pharmacology, Splenomegaly prevention & control, Toll-Like Receptor 7 agonists
- Abstract
Adjuvants enhance the immune response during vaccination. Among FDA-approved adjuvants, aluminum salts are most commonly used in vaccines. Although aluminum salts enhance humoral immunity, they show a limited effect for cell-mediated immune responses. Thus, further development of adjuvants that induce T-cell-mediated immune response is needed. Toll-like receptors (TLRs) recognizing specific pathogen-associated molecular patterns activate innate immunity, which is crucial to shape adaptive immunity. Using TLR ligands as novel adjuvants in vaccines has therefore attracted substantial attention. Among them a small molecule TLR7 ligand, imiquimod, has been approved for clinical use, but its use is restricted to local administration due to unwanted adverse side effects when used systematically. Since TLR7 is mainly located in the endosomal compartment of immune cells, efficient transport of the ligand into the cells is important for improving the potency of the TLR7 ligand. In this study we examined gold nanoparticles (GNPs) immobilized with α-mannose as carriers for a TLR7 ligand to target immune cells. The small molecule synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine (1V209), and α-mannose were coimmobilized via linker molecules consisting of thioctic acid on the GNP surface (1V209-αMan-GNPs). The in vitro cytokine production activity of 1V209-αMan-GNPs was higher than that of the unconjugated 1V209 derivative in mouse bone marrow-derived dendritic cells and in human peripheral blood mononuclear cells. In the in vivo immunization study, 1V209-αMan-GNPs induced significantly higher titers of IgG2c antibody specific to ovalbumin as an antigen than did unconjugated 1V209, and splenomegaly and weight loss were not observed. These results indicate that 1V209-αMan-GNPs could be useful as safe and effective adjuvants for development of vaccines against infectious diseases and cancer.
- Published
- 2019
- Full Text
- View/download PDF
49. Structure-Activity Relationship Studies To Identify Affinity Probes in Bis-aryl Sulfonamides That Prolong Immune Stimuli.
- Author
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Chan M, Lao FS, Chu PJ, Shpigelman J, Yao S, Nan J, Sato-Kaneko F, Li V, Hayashi T, Corr M, Carson DA, Cottam HB, and Shukla NM
- Subjects
- Cell Line, Humans, Kinetics, NF-kappa B metabolism, Structure-Activity Relationship, Benzene chemistry, Immunologic Factors chemistry, Immunologic Factors pharmacology, Sulfonamides chemistry, Sulfonamides pharmacology
- Abstract
Agents that safely induce, enhance, or sustain multiple innate immune signaling pathways could be developed as potent vaccine adjuvants or coadjuvants. Using high-throughput screens with cell-based nuclear factor κB (NF-κB) and interferon stimulating response element (ISRE) reporter assays, we identified a bis-aryl sulfonamide bearing compound 1 that demonstrated sustained NF-κB and ISRE activation after a primary stimulus with lipopolysaccharide or interferon-α, respectively. Here, we present systematic structure-activity relationship (SAR) studies on the two phenyl rings and amide nitrogen of the sulfonamide group of compound 1 focused toward identification of affinity probes. The murine vaccination studies showed that compounds 1 and 33 when used as coadjuvants with monophosphoryl lipid A (MPLA) showed significant enhancement in antigen ovalbumin-specific immunoglobulin responses compared to MPLA alone. SAR studies pointed to the sites on the scaffold that can tolerate the introduction of aryl azide, biotin, and fluorescent rhodamine substituents to obtain several affinity and photoaffinity probes which will be utilized in concert for future target identification and mechanism of action studies.
- Published
- 2019
- Full Text
- View/download PDF
50. Site-Specific Modification of Proteins through N-Terminal Azide Labeling and a Chelation-Assisted CuAAC Reaction.
- Author
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Inoue N, Onoda A, and Hayashi T
- Subjects
- Catalysis, Cycloaddition Reaction, Humans, Models, Molecular, Protein Conformation, Staining and Labeling, Alkynes chemistry, Azides chemistry, Chelating Agents chemistry, Copper chemistry, Proteins chemistry
- Abstract
Site-specific modification of peptides and proteins is an important method for introducing an artificial function to the protein surface. Recently, we found that new bioconjugation reagents, 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives, allow specific N-terminal modification and enhance the reaction rate of the subsequent bioconjugation in a chelation-assisted CuAAC reaction. The N-terminal specific azide-labeling of bioactive peptides and proteins occurs under mild reaction conditions with 6AMPC derivatives (angiotensin I: 90%, ribonuclease A: 90%). Kinetic analysis of the CuAAC reaction with azide-labeled proteins reveals that the ligation is promoted in the presence of a copper-chelating pyridine moiety. Importantly, the introduction of an electron-donating methoxy group to the pyridine moiety further accelerates the CuAAC ligation. We demonstrate that this method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol.
- Published
- 2019
- Full Text
- View/download PDF
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