Koichiro Hayashi, Michihiro Nakamura, Hirokazu Miki, Shuji Ozaki, Masahiro Abe, Toshio Matsumoto, Sakamoto Wataru, Yogo Toshinobu and Kazunori Ishimura : Magnetically Responsive Smart Nanoparticles for Cancer Treatment with a Combination of Magnetic Hyperthermia and Remote-Control Drug Release, Theranostics, Vol.4, No.8, 834-844, 2014.
(要約)
We report the synthesis of smart nanoparticles (NPs) that generate heat in response to an alternating current magnetic field (ACMF) and that sequentially release an anticancer drug (doxorubicin, DOX). We further study the in vivo therapeutic efficacy of the combination of magnetic hyperthermia (MHT) and chemotherapy using the smart NPs for the treatment of multiple myeloma. The smart NPs are composed of a polymer with a glass-transition temperature (T g) of 44°C, which contains clustered Fe3O4 NPs and DOX. The clustered Fe3O4 NPs produce heat when the ACMF is applied and rise above 44°C, which softens the polymer phase and leads to the release of DOX. The combination of MHT and chemotherapy using the smart NPs destroys cancer cells in the entire tumor and achieves a complete cure in one treatment without the recurrence of malignancy. Furthermore, the smart NPs have no significant toxicity.
(キーワード)
Animals / Body Weight / Drug Liberation / Female / Hyperthermia, Induced / Magnetic Phenomena / Mice / Nanoparticles / Neoplasms / Organ Size / Telemedicine
Michihiro Nakamura, Kazunori Miyamoto, Koichiro Hayashi, Aziz Awaad, Masahito Ochiai and Kazunori Ishimura : Time-Lapse Fluorescence Imaging and Quantitative Single Cell and Endosomal Analysis of Peritoneal Macrophages Using Fluorescent Organosilica Nanoparticles, Nanomedicine : Nanotechnology, Biology, and Medicine, Vol.9, No.2, 274-283, 2013.
(要約)
Fluorescent thiol-organosilica nanoparticles with 100 nm diameter (F-thiol-OS-100) were applied for time-lapse fluorescence imaging. The evaluation of F-thiol-OS-100 for quantitative analysis demonstrated great advantages as compared with quantum dots and organic fluorescent dye. Time-lapse fluorescence imaging of mouse peritoneal macrophages using F-thiol-OS-100 clearly demonstrated cellular uptake, and single cell analysis showed various patterns of uptake kinetics that could be quantitatively evaluated. We also performed quantitative analysis of endosomal uptake and movements in single cells. A correlation between morphologic findings and endosomal uptake and movement over time was also observed and analyzed quantitatively. The F-thiol-OS-100 showed high potential as a new fluorescence marker for time-lapse fluorescence imaging and quantitative single cell functional analysis for nanomedicine development. In this study the authors report on 100 nm thiol-organosilica nanoparticles as time-lapse flurescent markers. F-thiol-OS-100 proved to be superior to quantum dots and organic flurescent dyes, and enabled quantitative single cell functional analysis.
Kazuyoshi Kitaoka, Noriyuki Shimizu, Koji Ono, Sachiko Chikahisa, Madoka Nakagomi, Koichi Shudo, Kazunori Ishimura, Hiroyoshi Sei and Kazuo Yoshizaki : The retinoic acid receptor agonist Am80 increases hippocampal ADAM10 in aged SAMP8 mice, Neuropharmacology, Vol.72, 58-65, 2013.
(要約)
The retinoic acid (RA, a vitamin A metabolite) receptor (RAR) is a transcription factor. Vitamin A/RA administration improves the Alzheimer's disease (AD)- and age-related attenuation of memory/learning in mouse models. Recently, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as a key molecule in RA-mediated anti-AD mechanisms. We investigated the effect of chronic administration of the RAR agonist Am80 (tamibarotene) on ADAM10 expression in senescence-accelerated mice (SAMP8). Moreover, we estimated changes in the expression of the amyloid precursor protein (APP), amyloid beta (Aβ), and hairy/enhancer of split (Hes), which are mediated by ADAM10. Spatial working memory and the levels of a hippocampal proliferation marker (Ki67) were also assessed in these mice. ADAM10 mRNA and protein expression was significantly reduced in the hippocampus of 13-month-old SAMP8 mice; their expression improved significantly after Am80 administration. Further, after Am80 administration, the expression levels of Hes5 and Ki67 were restored and the deterioration of working memory was suppressed, whereas APP and Aβ levels remained unchanged. Our results suggest that Am80 administration effectively improves dementia by activating the hippocampal ADAM10-Notch-Hes5 proliferative pathway.
Koichiro Hayashi, Michihiro Nakamura and Kazunori Ishimura : Near-Infrared Fluorescent Silica-Coated Gold Nanoparticle Clusters for X-Ray Computed Tomography/Optical Dual Modal Imaging of the Lymphatic System, Advanced Healthcare Materials, Vol.2, No.5, 756-763, 2013.
(要約)
Lymph nodes (LNs) are often removed to prevent the spread of cancer because they are frequently the first site of metastases. However, the enucleation of LNs requires difficult operative techniques and lymphedema can result as a complication. Although lymphedema can be cured by anastomosis of a lymph vessel (LV) to a vein, the operative procedure is extremely difficult because LNs and LVs are too small and indistinct to be identified. Therefore, visualization of LNs and LVs is important. The combination of X-ray computed tomography (CT) and fluorescence imaging, CT/fluorescence dual modal imaging, enables the visualization of LNs and LVs before and during surgery. To accomplish this, near-infrared fluorescent silica-coated gold nanoparticle clusters (Au@SiO2) with a high X-ray absorption coefficient are synthesized. Both fluorescence imaging and CT show that the Au@SiO2 nanoparticles gradually accumulate in LNs through LVs. CT determines the location and size of the LNs and LVs without dissection, and fluorescence imaging facilitates their identification. The Au@SiO2 nanoparticles have neither hepatotoxicity nor nephrotoxicity. The results demonstrate that CT/fluorescence dual modal imaging using Au@SiO2 nanoparticles provides anatomical information, including the location and size of LNs and LVs for determining a surgery plan, and provides intraoperative visualization of LNs and LVs to facilitate the operation.
Koichiro Hayashi, Michihiro Nakamura, Hirokazu Miki, Shuji Ozaki, Masahiro Abe, Toshio Matsumoto and Kazunori Ishimura : Gold Nanoparticle Cluster/Plasmon-Enhanced Fluorescent Silica Core-Shell Nanoparticles for X-Ray Computed Tomography/Fluorescence Dual-Mode Imaging of Tumors, Chemical Communications, Vol.49, 5334-5336, 2013.
(要約)
Owing to the surface plasmon resonance-enhanced electromagnetic field, clustered gold nanoparticles-fluorescent silica core-shell nanoparticles became excited within the therapeutic window and fluoresced strongly in this window. The nanoparticles enabled tumor detection using fluorescence imaging and X-ray computed tomography.
Koichiro Hayashi, Michihiro Nakamura, Wataru Sakamoto, Toshinobu Yogo, Hirokazu Miki, Shuji Ozaki, Masahiro Abe, Toshio Matsumoto and Kazunori Ishimura : "Superparamagnetic Nanoparticle Clusters for Cancer Theranostics Combining Magnetic Resonance Imaging and Hyperthermia Treatment, Theranostics, Vol.3, No.6, 366-376, 2013.
(要約)
Superparamagnetic nanoparticles (SPIONs) could enable cancer theranostics if magnetic resonance imaging (MRI) and magnetic hyperthermia treatment (MHT) were combined. However, the particle size of SPIONs is smaller than the pores of fenestrated capillaries in normal tissues because superparamagnetism is expressed only at a particle size <10 nm. Therefore, SPIONs leak from the capillaries of normal tissues, resulting in low accumulation in tumors. Furthermore, MHT studies have been conducted in an impractical way: direct injection of magnetic materials into tumor and application of hazardous alternating current (AC) magnetic fields. To accomplish effective enhancement of MRI contrast agents in tumors and inhibition of tumor growth by MHT with intravenous injection and a safe AC magnetic field, we clustered SPIONs not only to prevent their leakage from fenestrated capillaries in normal tissues, but also for increasing their relaxivity and the specific absorption rate. We modified the clusters with folic acid (FA) and polyethylene glycol (PEG) to promote their accumulation in tumors. SPION clustering and cluster modification with FA and PEG were achieved simultaneously via the thiol-ene click reaction. Twenty-four hours after intravenous injection of FA- and PEG-modified SPION nanoclusters (FA-PEG-SPION NCs), they accumulated locally in cancer (not necrotic) tissues within the tumor and enhanced the MRI contrast. Furthermore, 24 h after intravenous injection of the NCs, the mice were placed in an AC magnetic field with H = 8 kA/m and f = 230 kHz (Hf = 1.8×10(9) A/m∙s) for 20 min. The tumors of the mice underwent local heating by application of an AC magnetic field. The temperature of the tumor was higher than the surrounding tissues by ≈6°C at 20 min after treatment. Thirty-five days after treatment, the tumor volume of treated mice was one-tenth that of the control mice. Furthermore, the treated mice were alive after 12 weeks; control mice died up to 8 weeks after treatment.
Sun Mi Kim, Takashi Sakai, Huy Van Dang, HoangNam Tran, Koji Ono, Kazunori Ishimura and Kiyoshi Fukui : Nucling, a novel protein associated with NF-B, regulates endotoxin-induced apoptosis in vivo, The Journal of Biochemistry, Vol.153, No.1, 93-101, 2013.
(要約)
Nucling is a proapoptotic protein that regulates the apoptosome and nuclear factor-kappa B (NF-B) signalling pathways. Strong stimuli, such as Gram-negative bacterial lipopolysaccharide (LPS), induce the simultaneous secretion of cytokines following the activation of NF-B. Proinflammatory cytokines can induce liver damage through several mechanisms such as increases in oxidative stress and apoptotic reactions leading to tissue necrosis. Herein, we show that Nucling-knockout (KO) mice are resistant to LPS that consistently caused mortality in wild-type (WT) counterparts. Although serum levels of cytokines such as tumour necrosis factor (TNF)-, interleukin (IL)-1 and IL-6 did not differ significantly between WT and Nucling-KO mice after the LPS challenge, hepatocytes of Nucling-KO mice were refractory to LPS- or TNF--induced cell death. These results were consistent with the decreased expression of proapoptotic proteins including apoptosis-inducing factor and cleaved form of poly (ADP-ribose) polymerase and terminal deoxynucleotidyl transferase dUTP nick end-labelling positive cells in the liver of Nucling-KO mice after the administration of a lethal dose of LPS. Moreover, the upregulation of NF-B-regulated anti-apoptotic molecules including cellular inhibitor of apoptosis (cIAP) 1 and cIAP2 was observed in the liver of Nucling-KO mice after LPS treatment. These findings indicate that the Nucling deficiency leads to resistance to apoptosis in liver. We propose that Nucling is important for the induction of apoptosis in cells damaged by cytotoxic stressors through the NF-B signalling pathway.
Aziz Awaad, Michihiro Nakamura and Kazunori Ishimura : Imaging of size dependent uptake and identification of novel pathways in mouse Peyer's patches using fluorescent organosilica particles., Nanomedicine : Nanotechnology, Biology, and Medicine, Vol.8, 627-636, 2012.
Koichiro Hayashi, Michihiro Nakamura, Wataru Sakamoto, Toshinobu Yogo and Kazunori Ishimura : Synthesis and 3D Hierarchical Organization of 2D Structured Iron Oxide Based on Enzymatic Structure, Activity and Thermostability, Materials Research Bulletin, Vol.47, No.12, 3959-3964, 2012.
Michihiro Nakamura, Aziz Awaad, Koichiro Hayashi, Kazuhiko Ochiai and Kazunori Ishimura : Thiol-organosilica Particles Internally Functionalized with Propidium Iodide as a Multicolor Fluorescence and X-Ray Computed Tomography Probe and Application for Non-Invasive Functional Gastrointestinal Tract Imaging., Chemistry of Materials, Vol.24, No.19, 3772-3779, 2012.
Aziz Awaad, Michihiro Nakamura and Kazunori Ishimura : Histochemical and biochemical analysis of the size-dependent nanoimmunoresponse in mouse Peyer's patches using fluorescent organosilica particles., International Journal of Nanomedicine, Vol.7, 1423-1439, 2012.
(要約)
The size-dependent mucosal immunoresponse against nanomaterials (nanoimmunoresponse) is an important approach for mucosal vaccination. In the present work, the size-dependent nanoimmunoresponse of mouse Peyer's patches (PPs) and immunoglobulin A (IgA) level was investigated using fluorescent thiol-organosilica particles. Various sizes of fluorescent thiol-organosilica particles (100, 180, 365, 745, and 925 nm in diameter) were administered orally. PPs were analyzed histochemically, and IgA levels in PP homogenates, intestinal secretions around PPs, and bile were analyzed biochemically. When compared with the larger particles (745 and 925 nm), oral administration of smaller thiol-organosilica particles (100, 180, and 365 nm) increased the number of CD11b(+) macrophages and IgA(+) cells in the subepithelial domes of the PPs. Additionally, administration of larger particles induced the expression of alpha-L-fucose and mucosal IgA on the surface of M cells in the follicle-associated epithelia of PPs and increased the number of 33D1(+) dendritic cells in the subepithelial domes of the PPs. IgA contents in the bile and PP homogenates were high after the administration of the 100 nm particles, but IgA levels in the intestinal secretions were high after the administration of the 925 nm particles. Two size-dependent routes of IgA secretions into the intestinal lumen, the enterohepatic route for smaller particles and the mucosal route for larger particles were proposed. Thiol-organosilica particles demonstrated size-dependent nanoimmunoresponse after oral administration. The size of the particles may control the mucosal immunity in PPs and were useful in mucosal vaccination approaches.
Koichiro Hayashi, Michihiro Nakamura and Kazunori Ishimura : Silica-Porphyrin Hybrid Nanotubes for in vivo Cell Tracking by Near-Infrared Fluorescence Imaging, Chemical Communications, Vol.48, No.32, 3830-3832, 2012.
(要約)
Near-infrared fluorescent silica-porphyrin hybrid nanotubes (HNTs) were successfully synthesized by ϖ-ϖ stacking, electrostatic interaction and a sol-gel reaction. The HNTs-labeled macrophages were detected in vivo, and the minimum detectable number of cells was 200. Furthermore, the biodistribution of HNTs-labeled macrophages was tracked by fluorescence imaging.
Koichiro Hayashi, Michihiro Nakamura, Wataru Sakamoto, Toshinobu Yogo, Toshinari Kori and Kazunori Ishimura : Formation of TiO2 Nanostructures by Enzyme-Mediated Self-Assembly for the Destruction of Macrophages, Chemistry of Materials, Vol.23, No.14, 3341-3347, 2011.
Koichiro Hayashi, Michihiro Nakamura and Kazunori Ishimura : In Situ Synthesis and Photoresponsive Rupture of Organosilica Nanocapsules, Chemical Communications, Vol.47, No.5, 1518-1520, 2011.
(要約)
This communication describes in situ synthesis of thiocyanato-functionalized organosilica nanocapsules by a sol-gel reaction using 3-thiocyanatopropyltriethoxysilane as a precursor. Moreover, the nanocapsules are photoresponsive and burst under light irradiation, resulting in release of the encapsulated drug or dye.
(キーワード)
Anti-Inflammatory Agents, Non-Steroidal / Drug Carriers / Drug Delivery Systems / Ibuprofen / 光 (light) / Nanocapsules / ナノ粒子 (nanoparticles) / Organosilicon Compounds / Spectroscopy, Fourier Transform Infrared
Michihiro Nakamura, Shuji Ozaki, Masahiro Abe, Toshio Matsumoto and Kazunori Ishimura : One-pot synthesis and characterization of dual fluorescent thiol-organosilica nanoparticles as non-photoblinking quantum dots and their applications for biological imaging, Journal of Materials Chemistry, Vol.21, 4689-4695, 2011.
Michihiro Nakamura, Shuji Ozaki, Masahiro Abe, Hiroyuki Doi, Toshio Matsumoto and Kazunori Ishimura : Size-controlled synthesis, surface functionalization, and biological applications of thiol-organosilica particles., Colloids and Surfaces B:Biointerfaces, Vol.79, No.1, 19-26, 2010.
(要約)
Thiol-organosilica particles of a narrow size distribution, made from 3-mercaptopropyltrimethoxysilane (MPMS), were prepared by means of a one-pot synthesis. We examined three synthetic conditions at high temperature (100 degrees C), including the Stöber synthesis and two entirely aqueous syntheses. Under all conditions, the sizes of MPMS particles were well controlled, and the average of the coefficient of variation for the size distribution was less than 20%. The incubation times required for formation of MPMS particles were shorter at high temperature than at low temperature. MPMS particles internally functionalized with fluorescent dye were also prepared by means of the same one-pot synthesis. On flow cytometry analysis these MPMS particles showed distinct peaks of scattering due to well-controlled sizes of particles as well as due to fluorescence signals. Real-time observation of interaction between fluorescent MPMPS particles and cultured cells could be observed under fluorescent microscopy with bright light. The surface of the as-prepared MPMS particles contained exposed mercaptopropyl residues, and the ability to adsorb proteins was at least 6 times higher than that of gold nanopaticles. In addition, fluorescein-labeled proteins adsorbed to the surface of the particles were quantitatively detected at the pg/ml level by flow cytometry. MPMS particles surface functionalized with anti-CD20 antibody using adsorption could bind with lymphoma cells expressing CD20 specifically. In this paper, we demonstrated the possibility of size-controlled thiol-organosilica particles for wild range of biological applications.
Hiroshi Nakagawa, Akio Hiura, Masato Mitome and Kazunori Ishimura : Nerve fibers that were not stained with the non-specific acetylcholinesterase (NsAchE) method, and TRPV1- and IB4-positive nerve fibers in the rat cornea.(Cited in "Journal of Chemical Neuroanatomy, 01/2014"), The Journal of Medical Investigation : JMI, Vol.56, No.3,4, 157-165, 2009.
(要約)
Previously, we noticed the presence of nerve fiber-like structures in a whole mount preparation of the rat cornea that had not been stained with the non-specific acetylcholinesterase (NsAchE) method. These nerve-like fibers were projected into the central area of the cornea, forming a mesh-like pattern. The aim of this study is to examine the properties of these mesh-like fibers using the following two methods: their sensitivity to capsaicin and the detection of isolectin B4 (IB4)- and capsaicin receptor TRPV1 (transient receptor potential vanilloid 1)-reactivities. The mean disappeared area of non-stained fibers after NsAchE treatment was 26% of the total areas in the neonatally capsaicin-treated cornea. Bunches composed of fine IB4-positive nerve fibers were seen in a whole mount preparation. There were connections between the bunches, producing a mesh-like pattern similar to that of the fibers that were not stained with NsAchE. Fine TRPV1-immunoreactive (ir) nerve fibers were also shown to form bunches, with connections between each bunch observed in whole mount preparations. Thus, TRPV1-ir nerve fibers seem to densely innervate the rat corneal subepithelial stroma and are distinct from the NsAchE-positive nerve fibers. The TRPV1-ir fine nerve fibers overlapped with the IB4-positive nerve fibers, suggesting that the mesh-like fibers that were not stained with NsAchE are fine nociceptive sensory nerve fibers because of their sensitivity to capsaicin and similar distribution pattern to IB4- and TRPV1-positive nerve fibers.
Michihiro Nakamura and Kazunori Ishimura : Size-controlled, One-pot Synthesis, Characterization, and Biological Applications of Epoxy-Organosilica Particles Possessing Positive Zeta Potential as Prepared. Langmuir, Langmuir, Vol.24, No.21, 12228-12234, 2008.
(要約)
Epoxy-organosilica particles made from 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (EpoMS) as a single silica source were synthesized by means of a one-pot method. We evaluated three sets of synthesis conditions, including traditional Stober conditions and two variations. Although the traditional conditions did not afford EpoMS particles, the variations did. The size distributions of the particles were evaluated by means of transmission electron microscopy. The mean diameters and size distributions of the particles depended on the EpoMS concentration, and the best coefficient of variation for the size distribution was 5.9%. The surface of the particles had unique properties, such as a positive zeta potential. The particles bound strongly to proteins as well as to DNA. The particles made from EpoMS, allowing particles internally functionalized with fluorescent dye to be prepared by means of a one-pot synthesis. EpoMS particles doped and tuned with fluorescent dye showed strong fluorescence signals and distinct peaks on flow cytometry, and the fluorescent particles could be used to label cells. The labeled cells showed clear fluorescence under a fluorescence microscope, and electron microscopy showed many particles in the cytoplasm. This is the first report describing the synthesis of epoxy-organosilica particles with a positive zeta potential and describing differences in the characteristics of particle formations due to changes in synthesis conditions. We also discuss the advantages of EpoMS particles, as well as the potential biological applications of these particles.
Michihiro Nakamura and Kazunori Ishimura : One-pot synthesis and characterization of three kinds of thiol-organosilica nanoparticles, Langmuir, Vol.24, No.9, 5099-5108, 2008.
(要約)
Using a one-pot synthesis, thiol-organosilica nanoparticles (NPs) made from (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)triethoxysilane, and (3-mercaptopropyl)methyldimethoxysilane have been successfully prepared. We compared the synthesis processes of thiol-organosilica NPs made of these three kinds of organosilicates, as well as particles made from tetraethoxysilicate (TEOS), at concentrations varying between 6.25 and 200 mM. We examined three types of synthetic conditions: the Stöber method, in which particles are prepared in 65% ethanol, and two entirely aqueous solvent syntheses, containing either 2% or 27% ammonium hydroxide. The synthetic mixtures were examined using transmission electron microscopy (TEM) to evaluate the as-prepared NPs. The formation trends and rates for these organosilica NPs vary with differing organosilicate precursors, concentrations, and synthetic conditions. The Stöber method is not suitable for formation of thiol-organosilica NPs as compared with the case of TEOS, but the conditions without ethanol and with 27% ammonium hydroxide are suitable for the formation of thiol-organosilica NPs. The size distributions of the formed NPs were evaluated using TEM and dynamic light scattering. The mean diameters of NPs increase with increasing concentrations of silicate, but the size distributions of NPs prepared under various conditions also differ between silicate sources. Thiol-organosilica NPs internally functionalized with fluorescent dye were also prepared using a one-pot synthesis and were characterized using fluorescence microscopy. The thiol-organosilica NPs retain fluorescent dye maleimide very well. In addition, rhodamine B-doped thiol-organosilica NPs show higher fluorescence than thiol-organosilica NPs prepared with rhodamine red maleimide. The surface of thiol-organosilica NPs contains exposed thiol residues, allowing the covalent attachment of fluorescent dye maleimide and protein maleimide. This is the first report describing the synthesis of thiol-organosilica NPs made of three kinds of thiol-organosilicates, differences in nanoparticle formation due to the kinds and concentrations of thiol-organosilicate and due to synthetic conditions, and the advantages of thiol-organosilica NPs due to the existence of both interior and exterior thiol residues.
Michihiro Nakamura and Kazunori Ishimura : Synthesis and characterization of organosilica nanoparticles prepared from 3-mercaptopropyltrimethoxysilane as the single silica source., The Journal of Physical Chemistry C, Vol.111, No.51, 18892-18898, 2007.
Michihiro Nakamura, Masayuki Shono and Kazunori Ishimura : Synthesis, Characterization, and Biological Applications of Multifluorescent Silica Nanoparticles, Analytical Chemistry, Vol.79, No.17, 6507-6514, 2007.
(要約)
Multifluorescent silica nanoparticles were synthesized by the Stöber method using conjugates of (3-aminopropyl)triethoxysilane and fluorescent dye-N-hydroxysuccinimide esters. The nanoparticles containing the fluorescent dyes were well dispersed and showed high, stable, and tunable fluorescence intensities. In addition, we prepared multifluorescent silica nanoparticles containing two kinds of fluorescent dyes and used the nanoparticles in biological applications. Flow cytometry analysis showed high and tuned fluorescence and multiple fluorescences from single nanoparticles with diameters of approximately 400 nm. Fluorescence microscopy analysis also showed high and tuned fluorescence, as well as multiple fluorescences from single nanoparticles and from cells labeled with multifluorescent silica nanoparticles. The intracellular distribution of nanoparticles was evaluated by confocal microscopy and electron microscopy. We discuss the advantages and demonstrate the usefulness of our nanoparticles in relation to commercially available fluorescent nanoparticles including quantum dots.
Toshiko Suzuki-Yamamoto, Yukihiko Sugimoto, Atsushi Ichikawa and Kazunori Ishimura : Co-localization of prostaglandin F synthase, cyclooxygenase-1 and prostaglandin F receptor in mouse Leydig cells., Histochemistry and Cell Biology, Vol.128, No.4, 317-322, 2007.
(要約)
In order to promote better understanding of the physiological roles of prostaglandin F(2alpha) in the mouse testis, we investigated the protein expression and the cellular localization of the enzymes cyclooxygenase and prostaglandin F synthase that are essential for the production of prostaglandin F(2alpha), and the binding site, which is the prostaglandin F(2alpha )receptor (FP). Western blot exhibited the expression of FP protein in wild type mouse testis, and that of prostaglandin F synthase and cyclooxygenase-1 proteins in the both of wild type mouse and FP-deficient mouse testes. The expression of prostaglandin F synthase and cyclooxygenase-1 were detected intensely in Leydig cell-rich fraction, and that of FP was detected equally in Leydig cell-rich fraction and the other fraction. Immunohistochemistry for cyclooxygenase-1 and prostaglandin F synthase demonstrated their co-localization in mouse Leydig cells. Histochemistry for FP demonstrated the localization in Leydig cells and in spermatids of seminiferous tubules. Double histochemical staining confirmed the co-localization of cyclooxygenase-1, prostaglandin F synthase and FP in the Leydig cells. These findings indicate that prostaglandin F(2alpha) may have an effect on the functions of Leyding cells in an autocrine fashion. It implies that prostaglandin F synthase and FP are involved in the control of testosterone release from Leydig cells and in spermatogenesis via the local pathway and the hypothalamo-hypophysial-testis pathway, and affect the testicular function.
Emi Kiyokage, Kazunori Toida, Toshiko Suzuki-Yamamoto and Kazunori Ishimura : Localization of 5α-Reductase in the Rat Main Olfactory Bulb, The Journal of Comparative Neurology, Vol.493, No.3, 381-395, 2005.
(要約)
The enzyme steroid 5alpha-reductase catalyzes the production of dihydroprogesterone and dihydrotestosterone, which were recently recognized as neurosteroids in the brain with variably potential neuroactivity. The present study reports for the first time detailed localization of 5alpha-reductase type 1 in the rat main olfactory bulb. The occurrence of 5alpha-reductase in the olfactory bulb was detected by reverse transcription-polymerase chain reaction and Western blotting analyses. In addition, the enzyme activity was also detected by thin layer chromatography. Immunocytochemistry showed that 5alpha-reductase immunoreactive cells of variable intensity were present in all layers of the olfactory bulb. Multiple immunolabeling revealed that 5alpha-reductase was mainly localized in glial cells, namely, in S-100beta- and glial fibrillary acidic protein-immunoreactive astrocytes, 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-immunoreactive oligodendrocytes, and in S-100beta- and neuropeptide-Y-immunoreactive olfactory ensheathing cells, whereas the bulbar neurons exhibited little immunoreactivity. Quantitative analysis revealed that the number of 5alpha-reductase-immunoreactive cells was greatest in the olfactory nerve layer. The most intense 5alpha-reductase-immunoreactivity was found in the olfactory ensheathing cells, and next in the CNPase-immunoreactive cells. The 5alpha-reductase in the olfactory bulb was expressed constantly throughout different ages and sexes and in neutered and hypophysectomized rats. Thus, 5alpha-reductase may contribute via 5alpha-reduced metabolites to the formation and maintenance of olfactory inputs and outputs, which were closely associated with the olfactory ensheathing cells and the oligodendrocytes, respectively.
Nociceptin (NOC), an endogenous ligand of the opioid receptor-like 1 receptor, is thought to be involved in learning and memory processes. Since acetylcholine (ACh) is involved in hippocampal function, and the hippocampus plays a critical role on the learning and memory function, hippocampal ACh release in NOC-receptor knockout mice was examined using an in vivo microdialysis method. The release of hippocampal ACh was largely increased in the knockout mice. Furthermore, in the knockout mice, an enhanced hippocampal theta rhythm, which is known to be linked to hippocampal memory function, was also observed. Immunohistochemically, in septum, co-existence of NOC receptor with cholinergic, but not with GABAergic neurons, was verified. The findings demonstrate that the NOC receptor is involved in hippocampal cholinergic function.
Nami Yamamoto, Sakie Tamura, Junko Matsushita and Kazunori Ishimura : Fracture Properties and Microstructure of Chicken Breasts Frozen by Electromagnetic Freezing, Journal of Home Economics of Japan, Vol.56, No.3, 141-151, 2005.
Hiromichi Yokoi, Yoshihiro Tsuruo, Shiro Kominami, Takeshi Yamazaki, 石村 和敬 : Distributions of Steroid 5α-Reductase and 17α-Hydroxylase/C17,20-lyase (P450c17) Immunoreactivities in Rat Gastric Mucosa, Acta Histochemica et Cytochemica, Vol.38, No.1, 61-67, 2005年.
(要約)
We examined the immunohistochemical localization of steroid 5α-reductase type 1 and 17α-hydroxylase/C17, 20-lyase (P450c17) in rat gastric mucosa. Immunoreactivity of 5α-reductase was localized in the chief cells and the mucous neck cells. P450c17 immunoreactivity was localized in the parietal cells. Subcellular localizations of both 5α-reductase and P450c17 were on the membrane of endoplasmic reticulum. P450c17 is a key enzyme of androgen biosynthesis, and 5α-reductase has a high affinity for testosterone and catalyzes the conversion of testosterone to its 5α-reduced metabolite. Thus, it is suggested that there is an androgenic metabolic pathway in rat gastric mucosa.
Kumiko Tominaga, Junko Matsuda, Makiko Kido, Etsuo Naito, Ichiro Yokota, Kazunori Toida, Kazunori Ishimura, Kunihiko Suzuki and Yasuhiro Kuroda : Genetic Background Markedly Influences Vulnerability of the Hippocampal Neuronal Organization in the ''Twitcher'' Mouse Model of Globoid Cell Leukodystrophy, Journal of Neuroscience Research, Vol.77, No.4, 507-516, 2004.
(要約)
The twitcher mouse is well known as a naturally occurring authentic mouse model of human globoid cell leukodystrophy (GLD; Krabbe disease) due to genetic deficiency of lysosomal galactosylceramidase. The twitcher mice used most commonly are on the C57BL/6J background. We generated twitcher mice that were on the mixed background of C57BL/6J and 129SvEv, the standard strain for production of targeted mutations. Twitcher mice on the mixed background were smaller and had a shorter lifespan than were those on the C57BL/6J background. Many twitcher mice on the mixed background developed generalized seizures around 30 days that were never seen in twitcher mice on the C57BL/6J background. Neuropathologically, although the degree of the typical demyelination with infiltration of macrophages was similar in the central and peripheral nervous systems, in both strains, marked neuronal cell death was observed only in twitcher mice on the mixed background. In the hippocampus, the neuronal cell death occurred prominently in the CA3 region in contrast to the relatively well-preserved CA1 and CA2 areas. This neuropathology has never been seen in twitcher mice on the C57BL/6J background. Biochemically, the brain of twitcher mice on the mixed background showed much greater accumulation of lactosylceramide. Genetic background must be carefully taken into consideration when phenotype of mutant mice is evaluated, particularly because most targeted mutants are initially on a mixed genetic background and gradually moved to a pure background. These findings also suggest an intriguing possibility of important function of some sphingolipids in the hippocampal neuronal organization and maintenance.
Takashi Sakai, Li Liu, Xichuan Teng, Rika Mukai-Sakai, Hidenori Shimada, Ryuji Kaji, Tasuku Mitani, Mitsuru Matsumoto, Kazunori Toida, Kazunori Ishimura, Yuji Shishido, Tak W. Mak and Kiyoshi Fukui : Nucling Recruits Apaf-1/Pro-caspase-9 Complex for the Induction of Stress-Induced Apoptosis, The Journal of Biological Chemistry, Vol.279, No.39, 41131-41140, 2004.
(要約)
Nucling is a novel protein isolated from murine embryonal carcinoma cells with an up-regulated expression during cardiac muscle differentiation. We show here that Nucling was up-regulated by proapoptotic stimuli and important for the induction of apoptosis after cytotoxic stress. We further demonstrated that overexpressed Nucling was able to induce apoptosis. In Nucling-deficient cells, the expression levels of Apaf-1 and cytochrome c, which are the major components of an apoptosis-promoting complex named apoptosome, were both down-regulated under cellular stress. A deficiency of Nucling also conferred resistance to apoptotic stress on the cell. After UV irradiation, Nucling was shown to reside in an Apaf-1/pro-caspase-9 complex, suggesting that Nucling might be a key molecule for the formation and maintenance of this complex. Nucling induced translocation of Apaf-1 to the nucleus, thereby distributing the Nucling/Apaf-1/pro-caspase-9 complex to the nuclear fraction. These findings suggest that Nucling recruits and transports the apoptosome complex during stress-induced apoptosis.
Kayoko Uezu, Hiroyoshi Sei, Atsuko Sano, Kazunori Toida, Toshiko Suzuki-Yamamoto, Takeshi Houtani, Tetsuo Sugimoto, Hiroshi Takeshima, Kazunori Ishimura and Yusuke Morita : Lack of nociceptin receptor alters body temperature during resting period in mice, NeuroReport, Vol.15, No.5, 751-755, 2004.
(要約)
The role of nociceptin (NOC) receptor on body core temperature (Tcore) control was examined using NOC receptor knockout mice. In homozygote NOC receptor-knockout, wild-type, and control C57BL/6J and 129/SV mice, Tcore was continuously recorded under 12:12 h light:dark (LD) and conditions of constant darkness (DD). The Tcore values during the resting period were higher in the NOC receptor-knockout mice than in both wild-type and control mice under both LD and DD conditions. Spontaneous activity during the resting period and plasma cortisol levels were not different between the NOC receptor-knockout and control mice. The findings herein indicate that the NOC receptor is involved in the control of Tcore during the resting period and is independent of light, physical activity and/or cortisol regulation.
Moe Kyaw, Masanori Yoshizumi, Koichiro Tsuchiya, Shoji Kagami, Yuki Izawa, Yoshiko Fujita, Nermin Ali, Yasuhisa Kanematsu, Kazunori Toida, Kazunori Ishimura and Toshiaki Tamaki : Src and Cas are essentially but differentially involved in angiotensin II-stimulated migration of vascular smooth muscle cell via extracellur signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase activation, Molecular Pharmacology, Vol.65, No.4, 832-841, 2004.
(要約)
Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang II-stimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (DeltaSD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2'-amino-3'-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src- and DeltaSD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.
(キーワード)
Angiotensin II / Animals / Cell Adhesion / Cell Movement / Cells, Cultured / Cellular Apoptosis Susceptibility Protein / Enzyme Activation / Genes, src / JNK Mitogen-Activated Protein Kinases / Male / Mitogen-Activated Protein Kinase 3 / Mitogen-Activated Protein Kinases / Muscle, Smooth, Vascular / リン酸化 (phosphorylation) / Rats / Rats, Sprague-Dawley / Tyrosine / Vinculin / src-Family Kinases
Hisashi Ueta, Hideko Nagasawa, Yuriko Oyabu-Manabe, Kazunori Toida, Kazunori Ishimura and Hitoshi Hori : Localization of enolase in synaptic plasma membrane as an alpha gamma heterodimer in rat brain., Neuroscience Research, Vol.48, No.4, 379-386, 2004.
Toshiko Suzuki-Yamamoto, Kazunori Toida, Kikuko Watanabe and Kazunori Ishimura : Immunocytochemical localization of prostaglandin F synthase II in the rat spinal cord., Brain Research, Vol.969, No.1-2, 27-35, 2003.
(要約)
Prostaglandin F synthase has at least two isozymes, i.e. prostaglandin F synthase I and II. Recently, we demonstrated immunocytochemically that prostaglandin F synthase I was localized in neuronal dendrites and somata, and in endothelial cells of blood vessels in the whole area of rat spinal cord. In the present study, we immunocytochemically localized prostaglandin F synthase II in ependymal cells and tanycytes surrounding the central canal and in endothelial cells of blood vessels, but not in any neuronal elements at all segmental levels of the rat spinal cord. Immunoelectron microscopy and confocal laser scanning microscopy confirmed these findings and further revealed that strong immunoreactivity was found in the basal processes of the tanycytes. Our present and recent studies using antibodies against the two isozymes of prostaglandin F synthase clearly indicated that they were localized differentially in ependymal (prostaglandin F synthase II) and neuronal elements (prostaglandin F synthase I), but were co-localized in blood vessels in the rat spinal cord. The distinct localization of the two isozymes suggests that prostaglandin F(2) has different transcellular biological actions via different cell groups.
Akihiko Tsuji, Kensuke Sakurai, Emi Kiyokage, Takahito Yamazaki, Shizuyo Koide, Kazunori Toida, Kazunori Ishimura and Yoshiko Matsuda : Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix. Potential role of PACE4 in the activation of proproteins in the extracellular matrix, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol.1645, No.1, 95-104, 2003.
Michihiro Nakamura, Kouhei Tsumoto, Izumi Kumagai and Kazunori Ishimura : A morphologic study of filamentous phage infection of Escherichia coli using biotinylated phages., FEBS Letters, Vol.536, No.1, 167-172, 2003.
(キーワード)
ファージ (phage) / 感染 (infection) / biotin / periplasm / membrane / electron microscopy / FD ADSORPTION PROTEIN / PERIPLASMIC PROTEINS / BACTERIOPHAGE-FD / COAT PROTEIN / TOLA / RELEASE / DOMAIN / SITES / EXCC / GENE
Michihiro Nakamura, Kouhei Tsumoto, Kazunori Ishimura and Izumi Kumagai : The effect of an agglutogen on virus infection: biotinylated filamentous phages and avidin as a model., FEBS Letters, Vol.520, No.1-3, 77-80, 2002.
(要約)
To address the effect of an agglutogen on virus infection, we studied the avidin-associated inhibition of infection by biotinylated M13 phages (BIO-phages). Microscopic observation of mixtures of BIO-phages and avidin-fluorescein conjugates revealed many aggregates. Even at low phage concentrations, avidin induced inhibition of infection significantly. Anti-M13 phage antibody also made aggregates and inhibited the infection but in a different manner from avidin. The inhibition by avidin was at > or = 2 microg/ml, time dependent and marked until 10 min after the mixing of the BIO-phages and Escherichia coli. On the other hand, antibody inhibited the infection at > or = 0.1 microg/ml dose dependently, and the inhibition was time dependent and marked until 45 min after the mixing at moderate and low phage concentrations. These results indicate that avidin against BIO-phages and antibodies are agglutogens, and the inhibition of the BIO-phages by avidin is closely related to the tetramerization of avidin. Agglutogens may be novel alternative antiviral drugs.
(キーワード)
Avidin / Bacteriophage M13 / Biotin / Dose-Response Relationship, Drug / 大腸菌 (Escherichia coli) / Time Factors / Viruses
Michihiro Nakamura, Kouhei Tsumoto, Kazunori Ishimura and Izumi Kumagai : Detection of biotinylated proteins in polyacrylamide gels using an avidin-fluorescein conjugate., Analytical Biochemistry: Methods in the Biological Sciences, Vol.304, No.2, 231-235, 2002.
(要約)
Biotinylated proteins are widely used as a molecular tool in biotechnological applications. In this paper, we demonstrated that biotinylated proteins after electrophoresis were detected directly in gels using an avidin-fluorescein conjugate with a fluorescence image analyzer. Upon analysis of the purified and chemically biotinylated protein, the sensitivity of this method was almost equal to that of silver staining. Chemically biotinylated proteins of Escherichia coli cell surfaces could also be specifically detected with our method. Furthermore, recombinant proteins fused with the biotin acceptor domain and biotinylated enzymatically in vivo were also detected in a lysate of E. coli specifically. The sensitivity and specificity of our method are high, and the procedure is simple. Therefore, our method would benefit detection of biotinylated proteins via gel electrophoresis and also various fields of study using avidin-biotin technology.
Michihiro Nakamura, Kouhei Tsumoto, Kazunori Ishimura and Izumi Kumagai : Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay: application of selected VH to histochemistry., Journal of Immunological Methods, Vol.261, No.1-2, 65-72, 2002.
(要約)
Comprehensive preparations of antibodies against various kinds of proteins in cells would be useful in proteome research and antibody-based research. Here we report the panning of a human antibody heavy chain variable domain (VH) phage library against a cytosolic fraction of rat liver to obtain antibodies specific for certain cytoplasmic proteins. Rat liver specimens were homogenized and subjected to differential centrifugation. A 125000 x g supernatant (rat liver cytosol, RLC) was immobilized onto a nitrocellulose membrane and subjected to phage VH library panning. For efficient assessment of binding phages, we established a system that was a combination of monoclonal phage ELISA and quantitative dot blotting of phages. The VH genes of the binding phages were selected and expressed as VH--bacterial alkaline phosphatase (PhoA) conjugates (VH/RLC--PhoAs) in Escherichia coli. One of the VH/RLC--PhoAs stained one major band on Western blotting of RLC and also stained the cytoplasm of hepatocytes histochemically. This is the first report of phage library panning against the cytosolic fraction of cells to obtain human VH fragments, and the application of those human VH fragments to histochemical study.
(キーワード)
Amino Acid Sequence / Animals / Base Sequence / Blotting, Western / Cytosol / DNA (DNA) / Enzyme-Linked Immunosorbent Assay / Humans / Immunoblotting / Immunoglobulin Heavy Chains / Immunoglobulin Variable Region / Liver / Male / Peptide Library / Rats / Rats, Wistar
Michihiro Nakamura, Kouhei Tsumoto, Kazunori Ishimura and Izumi Kumagai : A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages., Biochemical and Biophysical Research Communications, Vol.289, No.1, 252-256, 2001.
(要約)
Direct visualization of filamentous phage infection in Escherichia coli (E. coli) was attempted using biotinylated phages (BIO-phages). The biotinylation of the phages did not influence their infectivity into E. coli. E. coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E. coli could be directly observed by using the avidin-biotin-peroxidase complex method with electron microscopy. This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin-avidin interaction. This simple and powerful method is applicable to the study of infection by various viruses.
Michihiro Nakamura, H. Watanabe, Y. Nishimiya, K. Tsumoto, Kazunori Ishimura and I. Kumagai : Panning of a phage VH library using nitrocellulose membranes: application to selection of a human VH library., The Journal of Biochemistry, Vol.129, No.2, 209-212, 2001.
(要約)
We have established a method for selecting binding phages from a phage immunoglobulin heavy chain variable region (VH) library by panning with nitrocellulose membranes (membrane panning). To evaluate the concentrating ability of membrane panning for binding phages, a phage VH library containing clones that bind to hen egg white lysozyme (HEL) was used for panning against HEL. The efficiency of our method was as high as that of panning with magnetic beads. In addition, we performed membrane panning against target proteins and isolated the binding phages. The human VH genes of these phages were cloned and expressed as VH-bacterial alkaline phosphatase (PhoA) conjugates (VH-PhoA) in Escherichia coli. The dose-dependent binding of VH-PhoA to target proteins was confirmed by dot blotting. When applied to disease-associated antibodies, these methods will likely benefit clinical research. In addition, these techniques may be applicable to systematic analysis in proteome studies.
(キーワード)
Alkaline Phosphatase / Animals / Egg White / Humans / Immunoblotting / Immunoglobulin Variable Region / Muramidase / Peptide Library
(文献検索サイトへのリンク)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11173521
Toshiko Suzuki-Yamamoto, Kazunori Toida, Yoshihiro Tsuruo, Kikuko Watanabe and Kazunori Ishimura : Immunocytochemical localization of lung-type prostaglandin F synthase in the rat spinal cord., Brain Research, Vol.877, No.2, 391-395, 2000.
(要約)
Prostaglandin F synthase, producing prostaglandin F(2 alpha) and 9 alpha,11 beta-prostaglandin F(2), has at least two isozymes, lung-type and liver-type ones. The present study including double immunolabelling with microtubule-associated protein 2 indicated that the lung-type isozyme was present in neuronal dendrites and somata of gray matter (relatively intense in lamina I and II in dorsal horn, and IX in ventral horn) and vascular endothelial cells in the rat spinal cord at all segmental levels.
Toshiko Suzuki-Yamamoto, Hiromichi Yokoi, Yoshihiro Tsuruo, Kikuko Watanabe and Kazunori Ishimura : Identification of prostaglandin F-producing cells in the liver., Histochemistry and Cell Biology, Vol.112, No.6, 451-456, 1999.
(要約)
Prostaglandin (PG) F synthase forms PGF(2alpha) and 9alpha, 11beta-PGF2 from PGH2 and PGD2, respectively. PGH2 is synthesized from arachidonic acid by cyclooxygenase (COX) and then metabolized to various PGs and thromboxane by specific enzymes. PGD2 is synthesized from PGH2 by PGD synthase. To identify PGF2-producing cells in the rat liver, the occurrence and localization of PGF synthase and COX were studied with immunochemical and immunohistochemical techniques using anti-liver-type PGF synthase and anti-COX antibodies. In Western blot analyses, positive bands of liver-type PGF synthase and constitutive COX-1 were observed at positions approximately 37 kDa and 70-72 kDa, respectively. However, inducible COX-2 was not detected. In the immunohistochemical study, PGF synthase was present in the cytoplasm of the sinusoidal endothelial cells. COX-1 was present on the membranes of the nucleus and endoplasmic reticulum of the endothelial cells and Kupffer cells. Double immunostaining for PGF synthase and COX-1 showed that both enzymes were present in the same endothelial cells. These results suggest that the main site of PGF2 synthesis in the liver is the sinusoidal endothelial cell.
Kyoji Morita, Yoshihiro Tsuruo, Kazunori Ishimura, Song Her, Rose Ann Bell and Dona L. Wong : Influence of serum-free culture conditions on steroid 5a-reductase mRMA expression in rat C6 glioma cells, Brain Research, Vol.830, No.1, 179-182, 1999.
Kyoji Morita, Kazunori Ishimura, Yoshihiro Tsuruo and Dona L. Wong : Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors, Brain Research, Vol.816, No.2, 309-316, 1999.
(要約)
Glucocorticoids have been shown to be neurotoxic and appear to play a role in neuronal cell loss during aging and following neuropathological insults. However, very little is known about the effects of these steroid hormones on glial cells. The effect of the synthetic glucocorticoid dexamethasone (DEX) on glial cell viability was therefore examined by measuring neutral red uptake into rat C6 glioma cells. Serum deprivation markedly reduced cell viability, and this effect was significantly enhanced by DEX. Electrophoretic analysis showed that the cell damage induced by either serum deprivation alone or in combination with DEX was not accompanied by the degradation of DNA into nucleosomic fragments. Electron microscopic studies confirmed that serum deprivation and glucocorticoid treatment caused necrotic cell death. Furthermore, the effect of DEX on cell viability could be mimicked by the glucocorticoid receptor agonist RU28362, and completely prevented by the glucocorticoid receptor antagonist RU38486. These results indicate that dexamethasone can enhance the necrotic death of glioma cells induced by serum deprivation, suggesting that glucocorticoids may be involved in the chronic alteration of brain function arising from neuropathological damage to glial cells.
Yoshihiro Tsuruo, Kazunori Ishimura and Kyoji Morita : Influence of serum-free culture conditions on subcellular localization of steroid 5alpha-reductase in rat C6 glioma cells, Brain Research, Vol.801, No.1-2, 130-136, 1998.
(要約)
Rat C6 glioma cells are considered to be well characterized, and therefore commonly used as a model system to investigate the function of glial cells. However, recent study has shown that an alteration in the expression of their phenotypic antigens is observed when the cells are maintained under the serum-free conditions, proposing the possibility that various properties of glioma cells can be altered by the growth conditions. To test this possibility, the effects of serum-free culture conditions on the expression of steroid 5alpha-reductase (5alpha-R) type 1 isozyme in glioma cells were examined using immunocytochemical technique. Immunoreactivity of 5alpha-R type 1 was confined to the perinuclear region of glioma cells cultured in serum-containing medium, and observed in the cytoplasmic space as well as the perinuclear region of the cells cultured in serum-free medium. In contrast, serum deprivation failed to affect the expression of phenotypic antigens, glial fibrillary acidic protein (GFAP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Further studies showed that the expression of cytoplasmic 5alpha-R immunoreactivity induced by serum deprivation was reversible, and might be attributed to removal of serum proteins rather than biologically active small molecules from culture medium. This alteration in the expression of 5alpha-R immunoreactivity is therefore considered to reflect the translocation of the enzyme from the perinuclear region to the cell cytoplasm rather than the induction of cytoplasmic enzyme, and suggest that the culture conditions cause an alteration in the subcellular localization of 5alpha-R type 1 isozyme without phenotypic change of the glioma cell.
Kyoji Morita, Shuichi Hamano, Kazuhiko Teraoka and Kazunori Ishimura : Possible involvement of intracellular Ca2+ in hyposmosis-evoked catecholamine release from adrenal chromaffin cells, Neurochemistry International, Vol.31, No.5, 731-737, 1997.
(要約)
The influence of hyposmotic conditions on catecholamine release was studied using cultured adrenal chromaffin cells. Incubation of the cells in hyposmotic solution led to the enhancement of catecholamine release in a manner dependent on the reduction of osmolarity. Hyposmosis-evoked catecholamine release was similarly observed in the presence or absence of extracellular Ca2+, and was not significantly affected by organic and inorganic Ca2+ entry blockers. These results indicated that the hyposmosis-evoked release might be associated with a rise in the intracellular Ca2+ concentration. Further studies showed that neither ryanodine nor thapsigargin caused any significant effect on hyposmosis-evoked catecholamine release, whereas pretreatment of chromaffin cells with carbonyl cyanide m-chlorophenyl hydrazone significantly enhanced the hyposmosis-evoked release. Catecholamine release evoked by exposure to hyposmotic medium is therefore thought to be mediated through intracellular Ca2+, which may be mainly sequestered by the mitochondrial pools. Neither caffeine- nor inositol 1,4,5-trisphosphate-sensitive Ca2+ pools seems likely to be involved in hyposmosis-evoked catecholamine release, although the Ca2+ pools that contribute to the elevation of intracellular Ca2+ observed under hyposmotic conditions are not yet completely identified.
Michihiro Nakamura, N Ueda, Koji Kishimoto, T Yoshimoto, S Yamamoto and Kazunori Ishimura : Immunocytochemical localization of platelet-type arachidonate 12-lipoxygenase in mouse blood cells., The Journal of Histochemistry and Cytochemistry, Vol.43, No.3, 237-244, 1995.
53.
Yumiko Tomita, Chie Yuasa, Runzhou Ni, Kazunori Ishimura and Akira Ichihara : Long-term maintenance of functional hepatocytes in primary culture by additions of pyruvate and various hormones, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1243, No.3, 329-335, 1995.
(要約)
Mature adult rat hepatocytes were cultured as monolayers in serum-free Williams medium E containing 10(-7) M each of insulin (Ins), dexamethasone (Dex) and triiodothyronine (T3) and 30 mM pyruvate. The hepatocytes remained morphologically intact for at least 14 days, during which period they maintained normal liver functions such as the expressions of cytochrome P-450 mRNA and glucokinase and secretion of albumin. They also retained the ability to resume proliferation. Cells cultured with pyruvate had a much higher ATP level than those without pyruvate, suggesting that pyruvate can sustain functional hepatocytes for a long period in culture in the presence of Ins, Dex and T3, probably by producing enough energy for their maintenance.
Ramesh Gala Reddy, Natsuo Ueda, Toshiko Suzuki, Shozo Yamamoto, Kazunori Ishimura, Norifumi Kawada and Yasuhiro Mizoguchi : Characterization of arachidonate 12-lipoxygenase found in the liver of mongrel dog and its immunohistochemical localization in neutrophil., The Tokushima Journal of Experimental Medicine, Vol.42, 27-35, 1995.
55.
Runzhou Ni, Yumiko Tomita, Koichi Matsuda, Akira Ichihara, Kazunori Ishimura, Jun Ogasawara and Shigekazu Nagata : Fas-Mediated Apoptosis in Cultured Mouse Hepatocytes, Experimental Cell Research, Vol.215, 332-337, 1994.
56.
Yoshihiro Tsuruo, Kazunori Ishimura, Masato Tamura, Susumu Kagawa and Kyoji Morita : Biochemical and histochemical studies of the effects of cerebral metabolism-improving drugs on NADPH diaphorase in the mouse brain, The Japanese Journal of Pharmacology, Vol.65, No.3, 285-288, 1994.
(要約)
The effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in the mouse brain were studied, and we found that diaphorase activity in the post-mitochondrial fraction of brain homogenate was enhanced by idebenone in a concentration-dependent manner. Histochemical studies also indicated that diaphorase staining was intensified by idebenone at the same concentration. These results suggest that idebenone may stimulate the production of nitric oxide, probably through its direct action on nitric oxide synthase, thus producing its protective action on neurological disorders due to cerebral hypoxia or ischemia as a consequence of dilating the cerebral blood vessels.
Masato Tamura, Susumu Kagawa, Yoshihiro Tsuruo, Kazunori Ishimura and Kyoji Morita : Effects of flavonoid compounds on the activity of NADPH diaphorase prepared from the mouse brain, The Japanese Journal of Pharmacology, Vol.65, No.4, 371-373, 1994.
(要約)
The effects of flavonoids on NADPH diaphorase activity were studied in vitro, and we found that the enzyme activity was markedly inhibited by quercetin. This inhibitory action was shown to be accompanied by an increase in the apparent Km value of the enzyme for the cofactor NADPH, with a decrease in the Vmax, and an increase in the apparent Km for the substrate nitro blue tetrazolium, without any significant change in the Vmax. These results indicate that quercetin may directly inhibit NADPH diaphorase, thus suggesting the possibility that this compound may be able to inhibit the production of nitric oxide in the brain.
C. Yuasa, Y. Tomita, Masayuki Shono, Kazunori Ishimura and Akira Ichihara : Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes., American Journal of Physiology, Cell Physiology, Vol.156, No.3, 522-530, 1993.
(キーワード)
liver / hepatocytes
59.
Kazunori Ishimura, Toshiko Suzuki, K Fukui, Akira Yamamoto, Y Omoto, Natsuo Ueda and Shozo Yamamoto : Immunohistochemical localization of prostaglandin endoperoxide synthase in the bovine intestine., Histochemistry, Vol.99, No.6, 485-490, 1993.
(要約)
The localization of prostaglandin (PG) endoperoxide synthase in bovine intestine was examined immunocytochemically with polyclonal antibody raised against PG endoperoxide synthase purified from bovine seminal glands. The most intense positive staining reaction for the enzyme was present in mast cells. Mast cells were found to be widely distributed in the intestinal wall, and were particularly numerous in the lamina propria. Most of the mast cells in the lamina propria of the intestinal villi were elongated and oriented with their long axis parallel to the plane of the absorptive epithelium. In whole mount preparations of jejunal villi, mast cells were seen to form a two-dimensional network in the lamina propria. In addition to mast cells, smooth muscle cells of the inner circular muscle layer and muscularis mucosae, nerve cells and fibers, endothelial cells of arterioles, and serosal epithelial cells also showed faint to moderate staining for the enzyme. These results suggested that mast cells are the major source of PGs in the bovine intestinal wall. The characteristic arrangement of mast cells in the intestinal villi may be related to their functions in this portion of the bovine intestine.
Chie Yuasa, Yumiko Tomita, Masayuki Shono, Kazunori Ishimura and Akira Ichihara : Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes, Journal of Cellular Physiology, Vol.156, No.3, 522-530, 1993.
(要約)
Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, approximately 80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was approximately 70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G0 phase, but that when they formed monolayers, they progressed to the G1 phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions.
kawada Norifumi, Ueda Natsuo, Mizoguchi Y., Kobayashi K., Monna T., Morisawa S., Kazunori Ishimura, Toshiko Suzuki and Yamamoto Shozo : Increased 5-lipoxygenase activity in massive hepatic cell necrosis in the rat correlates with neutrophil infiltration, Hepatology, Vol.16, No.2, 462-468, 1992.
(要約)
Rats were treated with heat-killed Propionibacterium acnes and subsequent injection of a small amount of lipopolysaccharide after 7 days. After 24 hr most of the rats died of massive liver cell necrosis. Nonparenchymal liver cells were isolated from this liver injury model and incubated with arachidonic acid. Reverse-phase high-pressure liquid chromatography detected the 5-lipoxygenase metabolites (leukotriene B4 and 5-hydroxy-arachidonic acid), whereas these compounds were produced in negligible amounts when the rats were treated with P. acnes only. Immunohistochemical studies with 5-lipoxygenase antiserum revealed that the injured livers contained a large number of positively stained round cells with segmented nuclei, which were rarely found in the livers treated with P. acnes only. These positively stained cells were histologically identified as neutrophils. The results suggested that the increased 5-lipoxygenase activity in the injured rat liver is attributable to the infiltrating neutrophils rather than to nonparenchymal hepatic cells.
Koji Ono, Kobahashi Anna and Kazunori Ishimura : Immunohistochemical analysis of aromatase and estrogen receptor in rat hippocampus, The Journal of Physiological Sciences, Vol.61, 2011.
3.
Reddy .R. G., Ueda Natsuo, Toshiko Suzuki, Yamamoto Shozo, Kazunori Ishimura, Kawada Norifumi and Mizoguchi Y. : Characterization of arachidonate 12-lipoxygenase found in the liver of mongrel dog and its immunohistochemical localization in neutrophil., The Tokushima Journal of Experimental Medicine, Vol.42, No.1-2, 27-35, 1995.
(要約)
The cytosol fraction of non-parenchymal cells isolated from the liver of adult mongrel dogs converted arachidonic acid to 12S-hydroxy-5,8,10,14-eicosatetraenoic acid. The arachidonate 12-lipoxygenase enzyme reacted with linoleic and alpha- and gamma-linolenic acids as well as arachidonic acid. The enzyme was immunoprecipitable with an antibody against the leukocyte 12-lipoxygenase, but not with an antibody against the platelet 12-lipoxygenase. By immunohistochemical observation with anti-leukocyte 12-lipoxygenase antiserum, hepatocytes, Kupffer cells and sinusoidal endothelial cells were not immunostained, but a large number of neutrophils located in sinusoidal cavities were positively stained. The 12-lipoxygenase activity thus detected may be attributed predominantly to the neutrophils appearing in sinusoidal cavities rather than the non-parenchymal cells in the liver of mongrel dog upon infection. In agreement with this finding there was essentially no neutrophil accumulation in the liver of Beagle dog which had no bacterial and parasitic infection.
Kazunori Ishimura and Hisao Fujita : Light and electron microscopic immunohistochemistry of the localization of adrenal steroidogenic enzymes., Microscopy Research and Technique, Vol.36, No.6, 445-453, Mar. 1997.
(要約)
Recent immunohistochemical studies have revealed the precise localization of the enzymes involved in adrenal steroidogenesis. Light microscopical investigations showed that cytochromes P450 of cholesterol side-chain cleavage enzyme (P450scc) and of 11 beta-hydroxylase (P45011 beta), 3 beta-hydroxysteroid dehydrogenase/ delta 5-4 isomerase (3 beta HSD), and 21-hydroxylase (P450C21) are localized in all the adrenocortical cells, especially in those of the zona fasciculata-reticularis. 17 alpha-Hydroxylase/C17-C20 lyase (P45017 alpha,lyase) is present in the zona fasciculata-reticularis cells of human, bovine, pig, and guinea-pig adrenals, but absent in the adrenals of some rodents such as rat, hamster, and mouse. Aldosterone synthase (P450aldo) is contained only in the zona glomerulosa cells. In the rat adrenal, P45011 beta, which catalyzes the conversion of deoxycorticosterone to corticosterone, is localized in the zona fasciculata-reticularis cells. Electron microscopic investigations demonstrated that P450scc and P45011 beta are colocalized in the matrix side of inner mitochondrial membrane including cristae, while 3 beta HSD, P450C21, and P45017 alpha, lyase are present in the membranes of smooth endoplasmic reticulum (SER). These results clearly indicate that aldosterone, the most potent mineralocorticoid, is synthesized in the zona glomerulosa cells, and glucocorticoids, such as corticosterone and cortisol, are produced in the zona fasciculata-reticularis cells. The conversion of cholesterol to pregnenolone and the final steps of corticosteroid synthesis occur in the mitochondria, while the intermediate steps, leading to the synthesis of deoxycorticosterone or deoxycortisol from pregnenolone, take place in the SER membranes.
Michihiro Nakamura, Koichiro Hayashi, Hitoshi Kubo, Masafumi Harada, Keisuke Izumi and Kazunori Ishimura : Organosilica Nanoparticles for Multimodal imaging, 7th International Symposium on Nanomedicine (ISNM2013), Nov. 2013.
2.
Takuya Takashima, Michihiro Nakamura, Koichiro Hayashi, K Miyamoto, M Nakano, S Akiyama and Kazunori Ishimura : Nano-biodistribution study using organosilica nanoparticles containing near-infrared fluorescent dye., 6th International Symposium on Nanomedicine (ISNM2012), Shimane, Nov. 2012.
3.
Koji Ono and Kazunori Ishimura : Change in steroidogenic enzymes expression in the rat hippocampus after ovariectomy, 14th International Congress of Histochemistry and Cytochemistry, Kyoto, Aug. 2012.
4.
Michihiro Nakamura, Koichiro Hayashi and Kazunori Ishimura : Preparation of Novel Multifunctional Fluorescent Nanoprobes using Organosilica Particles Technology, 14th International Congress of Histochemistry and Cytochemistry, Kyoto, Aug. 2012.
5.
Aziz Awaad, Michihiro Nakamura and Kazunori Ishimura : Imaging of size dependent uptake and identification of novel pathway in mouse Peyer's patches using fluorescent organosilica particles, The 11th US-Japan Symposium on Drug Delivery Systems Conference, Maui, Dec. 2011.
6.
Takafumi Kanadani, Michihiro Nakamura, Koichiro Hayashi, Aziz Award, Motoko Higashiguchi, Hiroshi Nakaoka and Kazunori Ishimura : Quantitative Single Cell Analysis of Macrophages using Fluorescent Organosilica Nanoparticles Surface-modified with Polyethylene Glycol, The 11th US-Japan Symposium on Drug Delivery Systems Conference, Maui, Dec. 2011.
7.
Michihiro Nakamura, Koichiro Hayashi and Kazunori Ishimura : Preparation of Novel Multifunctionalized Nanoparticles for Multimodal DDI using Organosilica Particles Technology, The 11th US-Japan Symposium on Drug Delivery Systems Conference, Maui, Dec. 2011.
8.
Koichiro Hayashi, Michihiro Nakamura and Kazunori Ishimura : Silica/Porphyrin Hybrid Nanotubes for Macrophage Tracking by Fluorescence Imaging, International Symposium on EcoTopia Science 2011, Nagoya, Dec. 2011.
9.
Michihiro Nakamura, Koichiro Hayashi and Kazunori Ishimura : Preparation of Novel Multifunctionalized Hybrid Nanoparticles using Organosilica Particles Technology, 3rd International Congress on Ceramics, Osaka, Nov. 2010.
10.
Koichiro Hayashi, Michihiro Nakamura and Kazunori Ishimura : In-Situ Template-Free Synthesis of Organosilica Nanocapsules, 3rd International Congress on Ceramics, Osaka, Nov. 2010.
11.
Michihiro Nakamura, Koichiro Hayashi and Kazunori Ishimura : Preparation of Novel Multimodal Imaging Nanoparticles using Organosilica Particles Technology, 2010 World Molecular Imaging Congress, Kyoto, Sep. 2010.
12.
Takashi Sakai, HoangNam Tran, Sun Mi Kim, Li Liu, Xichuan Teng, Yuji Shishido, Mukai-Sakai Rika, Mitsuru Matsumoto, Kazunori Ishimura, Yoshio Hayashi, Ryuji Kaji and Kiyoshi Fukui : Nucling, a novel stress-sensitive protein, regulates NF-kappa B activation, The 4th International Congress on Stress Responses in Biology and Medicine, The 4th Annual Meeting of the Biomedical Society for Stress Response, Sapporo, Oct. 2009.
13.
Michihiro Nakamura, M. Shono and Kazunori Ishimura : Preparation and characterization of novel biofunctionalized fluorescent silica nanoparticles and their possibility for photodynamic therapy, 12th Congress of the European Society for Photobiology, Bath, UK, Sep. 2007.
14.
Michihiro Nakamura, Kazunori Ishimura, Hirokazu Miyoshi and H Satake : Synthesis And Characterization Of Silica Nanoparticles With High Fluorescence, Satellite for 15th Annual Meeting of Bioimaging Society, Kyoto, Oct. 2006.
15.
Michihiro Nakamura, K. Tsumoto, I Kumagai and Kazunori Ishimura : Agglutogen as a new concept of antiviral drug: Biotinylated filamentous phages and avidin as a model., Antibody Engineering Conference~from Milstein C. Human antibody~, Kagoshima, Jun. 2006.
16.
Michihiro Nakamura, Kazunori Ishimura, Hirokazu Miyoshi and H Satake : Synthesis And Characterization Of Silica Nanoparticles With High Fluorescence, Particles 2006 Medical/Biochemical Diagnostic, Pharmaceutical, and Drug Delivery Application, Orlando, May 2006.
17.
Toshiko Suzuki-Yamamoto, Yutaka Fujii, Masashi Miyano, Lan-Ying Chen, Tomohiro Takahashi, Hiromichi Yokoi, Yoshihiro Tsuruo, Kazunori Ishimura and Watanabe Kikuko : cDNA cloning and mutagenesis study of liver-type prostaglandin F synthase, and identification of the prostaglandin F producing cells in the liver., Advances in Experimental Medicine and Biology, Vol.507, 263-268, Boston, 2002.
Kikuko Watanabe, Toshiko Suzuki-Yamamoto, Mikio Nishizawa, Motonari Fukui, Kazunori Ishimura and Seiji Ito : Roles of prostaglandin F synthase., Adv. Prostaglandin Leukotriene Res., Vol.16, 73-76, Florence, 2001.
19.
S Yamamoto, H Suzuki, Koji Kishimoto, Michihiro Nakamura and Kazunori Ishimura : Arachidonate 12-lipoxygenase isozymes., International Conference on Lipoxygenases and Their Products: Biological Functions (special lecture), Vol.447, 37-44, St. Julians, May 1997.