91 results on '"Haruo Hagiwara"'
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2. Myosin Light Chain Kinase / Actin Interaction in Phorbol Dibutyrate–Stimulated Smooth Muscle Cells
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Sean E. Thatcher, Mike E. Fultz, Hideyuki Tanaka, Haruo Hagiwara, Hou-Li Zhang, Ying Zhang, Kohichi Hayakawa, Shinji Yoshiyama, Akio Nakamura, Hong Hui Wang, Takeshi Katayama, Masaru Watanabe, Yuan Lin, Gary L. Wright, and Kazuhiro Kohama
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Therapeutics. Pharmacology ,RM1-950 - Abstract
Abstract.: Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun. 2008;369:135–143). Myosin ATPase activity measurements indicate that the 26 – 41 peptide of MLCK significantly decreases ATPase activity as the concentration of this peptide increases. Sliding velocity of actin-filaments on myosin and stress responses in skinned smooth muscle tissue are also inhibited. Peptide-mediated uptake and the microinjection technique in cells indicate that the peptide was necessary for actin-filament stabilization. Fluorescence resonance energy transfer analysis indicated that in the presence of MLCK, α-actin but not β-actin remodeled during phorbol 12,13-dibutyrate (PDBu)-induced contractions. PDBu also induced podosomes in the cell. When MLCK expression was down-regulated by introduction of RNAi for MLCK by lentivirus vector into the cells, we failed to observe the podosome induction upon PDBu stimulation. Rescue experiments indicate that the non-kinase activity of MLCK plays an important role in maintaining actin stress fibers and in the PDBu-induced reorganization of actin-filaments in smooth muscle cells. Keywords:: actin-remodeling, podosome, phorbol 12,13-dibutyrate (PDBu), FRET analysis, myosin light chain kinase (MLCK)-deficient cell
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- 2011
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3. Molecular Mechanisms and Drug Development in Aquaporin Water Channel Diseases: Water Channel Aquaporin-2 of Kidney Collecting Duct Cells
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Kuniaki Takata, Yuki Tajika, Toshiyuki Matsuzaki, Takeo Aoki, Takeshi Suzuki, Ablimit Abduxukur, and Haruo Hagiwara
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Therapeutics. Pharmacology ,RM1-950 - Abstract
Aquaporin-2 (AQP2) is one of the membrane water channel proteins expressed in principal cells of the kidney collecting ducts. In the basal state, AQP2 resides in the storage vesicles localized in the subapical cytoplasm. Upon stimulation with vasopressin, AQP2 is translocated to the apical plasma membrane by the exocytic fusion of the storage vesicles with the apical membrane. This translocation enables the transepithelial reabsorption of water from the lumen to the interstitium via AQP2 at the apical membrane and AQP3/AQP4 at the basolateral membrane. AQP2-storage vesicles are distinct from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and lysosomes. The early endosomal marker EEA1 is colocalized with some of AQP2 vesicles. Further analyses in Madin-Darby canine kidney (MDCK) cells transfected with AQP2 revealed that subapical Rab11-positive/EEA1-negative smaller vesicles constitute part of the AQP2 storage vesicles for the translocation to the apical membrane. Termination of stimulation results in the retrieval of AQP2 to the larger EEA1-positive early endosomal compartment. AQP2 is then transferred to the subapical storage compartment in a PI3-kinase-dependent manner. GLUT4 is an isoform of glucose transporters whose localization is also regulated by vesicular trafficking induced by insulin stimulation. Comparison of the intracellular localization of AQP2 with GLUT4 suggests distinct regulation of AQP2 trafficking. Keywords:: water channel, aquaporin-2, kidney, collecting duct, endosome
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- 2004
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4. Marked cortisol production by intracrine ACTH in GIP-treated cultured adrenal cells in which the GIP receptor was exogenously introduced.
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Hiroko Fujii, Mimi Tamamori-Adachi, Kousuke Uchida, Takao Susa, Takashi Nakakura, Haruo Hagiwara, Masayoshi Iizuka, Hiroko Okinaga, Yuji Tanaka, and Tomoki Okazaki
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Medicine ,Science - Abstract
The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing's syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (-) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.
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- 2014
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5. Exosomes as a Promising Therapeutic Strategy for Peripheral Nerve Injury
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Xiaohong Tian, Haruo Hagiwara, Tianhao Yu, Yingxi Xu, Rabia Javed, and Muhammad Arslan Ahmad
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0301 basic medicine ,Nervous system ,Schwann cell ,Exosomes ,Exosome ,03 medical and health sciences ,0302 clinical medicine ,Tissue engineering ,Peripheral Nerve Injuries ,Neurotrophic factors ,Humans ,Medicine ,Pharmacology (medical) ,Pharmacology ,business.industry ,Regeneration (biology) ,General Medicine ,Axons ,Microvesicles ,Nerve Regeneration ,Psychiatry and Mental health ,030104 developmental biology ,medicine.anatomical_structure ,Neurology ,Peripheral nerve injury ,Schwann Cells ,Neurology (clinical) ,business ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Peripheral nerve injury has a high incidence and often leads to severe losses of sensory and motor functions in the afflicted limb. Autologous nerve grafts are widely accepted as the gold standard for peripheral nerve repair, but the presence of inherent drawbacks dramatically reduces their usability. Numerous tissue engineering nerve grafts are developed as alternatives of autologous nerve grafts, and a variety of cells and neurotrophic factors were introduced into these grafts for improvement. However, they are still difficult to obtain satisfactory clinical results. Peripheral nerve regeneration following injury remains a significant challenge for researchers and clinicians. Exosomes are extracellular membranous nanovesicles that are secreted by most cells. As the key players of intercellular communication, exosomes play a fundamental role in the physiological and pathological processes of the nervous system. Accumulating evidence has suggested that exosomes can exert neurotherapeutic effects via mediating axonal regrowth, Schwann cell activation, vascular regeneration, and inflammatory regulation. Exosomes are emerging as a promising approach for treating peripheral nerve injury. Furthermore, they also provide possibilities for enhancing the repair capacity of various nerve grafts. This review primarily highlights the regenerative effects of exosomes on peripheral nerve injury. The exosomes from distinct sources reported so far in literature are summarized to understand their roles in the process of nerve repair. Moreover, the challenges that must be addressed in their clinical transformation are outlined as well. This review also provides further insight into the potential application of exosomes for peripheral nerve repair. Keywords: Exosome, nerve regeneration, peripheral nerve injury, Schwann cell, axonal regrowth, inflammation, vascular regeneration.
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- 2021
6. The Logic of Betrayal for Ethical Corporate Governance
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Haruo Hagiwara Horaguchi
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General Medicine - Published
- 2022
7. The Logic of Betrayal for Ethical Corporate Governance
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Horaguchi, Haruo Hagiwara, primary
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- 2022
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8. Fibronectin-integrin signaling regulates PLVAP localization at endothelial fenestrae by microtubule stabilization
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Takeshi Suzuki, Yoko Nekooki-Machida, Kotaro Horiguchi, Kenjiro Arisawa, Toshio Miyashita, Hideyuki Tanaka, Haruo Hagiwara, and Takashi Nakakura
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Male ,0301 basic medicine ,Integrins ,Histology ,Integrin ,Microtubules ,Pathology and Forensic Medicine ,03 medical and health sciences ,symbols.namesake ,chemistry.chemical_compound ,0302 clinical medicine ,Microtubule ,Animals ,Endomembrane system ,Transcellular ,Colcemid ,biology ,Endothelial Cells ,Membrane Proteins ,Cell Biology ,Golgi apparatus ,Fibronectins ,Rats ,Cell biology ,Fibronectin ,Disease Models, Animal ,030104 developmental biology ,chemistry ,Cytoplasm ,symbols ,biology.protein ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Endothelial fenestrae are the transcellular pores existing on the capillary walls which are organized in clusters referred to as sieve plates. They are also divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). In this study, we examined the involvement of fibronectin signaling in the formation of fenestra and diaphragm in endothelial cells. Results showed that Itga5 and Itgb1 were expressed in PECAM1-positive endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, and integrin α5 was localized at the fenestrated capillaries of the rat pituitary and cultured PECAM1-positive endothelial cells isolated from AL (CECAL). Inhibition of both integrin α5β1 and FAK, a key molecule for integrin-microtubule signaling, respectively, by ATN-161 and FAK inhibitor 14, caused the delocalization of PLVAP at the sieve plates and depolymerization of microtubules in CECAL. Paclitaxel prevented the delocalization of PLVAP by the inhibition of integrin α5β1. Microtubule depolymerization induced by colcemid also caused the delocalization of PLVAP. Treatment of CECAL with ATN-161 and colcemid caused PLVAP localization at the Golgi apparatus. The localization of PLVAP at the sieve plates was inhibited by BFA treatment in a time-dependent manner and spread diffusely to the cytoplasm. These results indicate that a constant supply of PLVAP proteins by the endomembrane system via the Golgi apparatus is essential for the localization of PLVAP at sieve plates. In conclusion, the endomembrane transport pathway from the Golgi apparatus to sieve plates requires microtubule cytoskeletons, which are regulated by fibronectin-integrin α5β1 signaling.
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- 2021
9. Effects of microRNA-338 Transfection into Sciatic Nerve on Rats with Experimental Autoimmune Neuritis
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Kai Gong, Yujun Wei, Zun-Cheng Zheng, Qiang-San Sun, Qiang Ao, Xiao-Jing Yuan, Haruo Hagiwara, and Tianrang Ao
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0301 basic medicine ,medicine.medical_specialty ,Neurology ,Neuritis ,Schwann cell ,Transfection ,Nerve conduction velocity ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Myelin ,0302 clinical medicine ,Internal medicine ,Animals ,Medicine ,Myelin Sheath ,biology ,business.industry ,Calcium-Binding Proteins ,Microfilament Proteins ,S100 Proteins ,General Medicine ,Neuritis, Autoimmune, Experimental ,Sciatic Nerve ,Nerve Regeneration ,Rats ,MicroRNAs ,RNAi Therapeutics ,030104 developmental biology ,medicine.anatomical_structure ,Endocrinology ,Rats, Inbred Lew ,biology.protein ,Female ,Schwann Cells ,Sciatic nerve ,Antibody ,business ,030217 neurology & neurosurgery - Abstract
Nerve demyelination or axonal lesions are characteristic of experimental autoimmune neuritis (EAN). Previous studies have demonstrated that microRNA-338 can regulate the differentiation and maturation of oligodendrocytes and Schwann cells and promote injured peripheral nerves in rats. In this study, we used microRNA-338 coded lentivirus vector (miR-338-LV) in a Lewis rat EAN model, in with the conjunction P0 peptide 180–199 which was injected into the footpads of animals to induce immunization. The clinical scores of miR-338-LV and intravenous immunoglobulin (IVIg) (positive drug) groups were significantly superior to those of untreated group at disease peak and disease plateau (p
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- 2020
10. Role of tubulin acetylation in cellular functions and diseases
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Haruo Hagiwara and Yoko Nekooki-Machida
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0301 basic medicine ,Centriole ,macromolecular substances ,Microtubules ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Cell Movement ,Tubulin ,Microtubule ,Neoplasms ,Cell polarity ,Animals ,Humans ,Molecular Biology ,biology ,Cell growth ,Chemistry ,Cilium ,Cryoelectron Microscopy ,Cytoplasmic Vesicles ,Cell Polarity ,Acetylation ,Neurodegenerative Diseases ,Cell migration ,General Medicine ,Ciliopathies ,Cell biology ,Kinetics ,030104 developmental biology ,030220 oncology & carcinogenesis ,biology.protein ,Protein Processing, Post-Translational - Abstract
Acetylation is a well-studied post-translational modification (PTM) of tubulin. Acetylated tubulin is present in the centrioles, primary cilia, and flagella, which contain long-lived stable microtubules. Tubulin acetylation plays an important role in cellular activities including cell polarity, cell migration, vesicle transport, and cell development. Cryo-electron microscopy reconstructions have revealed conformational changes in acetylated tubulin, revealing a reduction in intermonomer interactions among tubulins and an increase in microtubule elasticity. The kinetics of conformational changes in acetylated tubulin may elucidate microtubule functions in these cellular activities. Abnormal tubulin acetylation has been implicated in neurodegenerative disorders, ciliopathies, and cancers. Thus, it is important to elucidate the mechanisms underlying tubulin acetylation and its effects on cellular activity to understand these diseases and to design potential therapeutic strategies. This review discusses the cellular distribution and dynamics of acetylated tubulin and its role in regulating cellular activities.
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- 2020
11. Sociocultural Responses to COVID-19 and Their Consequences for International Business
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Horaguchi, Haruo Hagiwara, primary
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- 2021
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12. Fabrication and evaluation of an optimized acellular nerve allograft with multiple axial channels
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Xiaohong Wang, Jing He, Yizhan Ma, Lili Wen, Weizuo Wang, Ting Li, Qiang Ao, Jun Fan, Muhammad Arslan Ahmad, Tianhao Yu, Haruo Hagiwara, Yingxi Xu, and Xiaohong Tian
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Bioactive molecules ,0206 medical engineering ,Biomedical Engineering ,02 engineering and technology ,Biochemistry ,Biomaterials ,Extracellular matrix ,In vivo ,Mechanical strength ,Animals ,Prospective Studies ,Molecular Biology ,Decellularization ,Nerve allograft ,Chemistry ,General Medicine ,021001 nanoscience & nanotechnology ,Allografts ,020601 biomedical engineering ,Sciatic Nerve ,Nerve Regeneration ,Rats ,surgical procedures, operative ,Peripheral nerve injury ,Sciatic nerve ,Schwann Cells ,0210 nano-technology ,Biotechnology ,Biomedical engineering - Abstract
Acellular nerve allografts are promising alternatives to autologous nerve grafts, but still have many drawbacks which greatly limit their curative effects. Here, we developed an optimized acellular nerve allograft with multiple axial channels by a modified decellularization method. These allografts were confirmed to preserve more extracellular matrix components and factors, and remove cellular components effectively. Meanwhile, macrochannels and microchannels were introduced to optimize internal microstructure of allografts, which increases porosity and water absorption, without significant loss of mechanical strength. The in vitro experiments demonstrated that the multichannel allografts showed superior ability of facilitating proliferation and penetration of Schwann cells. Additionally, in the in vivo experiments, the multichannel allografts were used to bridge 10 mm rat sciatic nerve defects. They exhibited better capacity to guide regenerative nerve fibers through the defective segment and restore innervation of target organs, thus achieving better recovery of muscle and motor function, in comparison with conventional acellular allografts. These findings indicate that this multichannel acellular nerve allograft has great potential for clinical application and provides a new prospective for future investigations of nerve regeneration. STATEMENT OF SIGNIFICANCE: Acellular nerve allografts, with preservation of natural extracellular matrix, are officially approved to repair peripheral nerve injury in some countries. However, bioactive component loss and compact internal structure result in variable clinical effects of conventional acellular allografts. In the present study, we fabricated an optimized acellular nerve allograft with multiple axial channels, which could both enable decellularization to be easily accomplished and reduce the amount of detergents in the preparation process. Characterization of the multichannel acellular allografts was confirmed to have better preservation of ECM bioactive molecules and regenerative factors. Efficiency evaluation showed the multichannel allografts could facilitate Schwann cells to migrate inside them in vitro, and enhance regrowth and myelination of axons as well as recovery of muscle and motor function in vivo.
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- 2020
13. Expression and localization of forkhead box protein FOXJ1 in S100β-positive multiciliated cells of the rat pituitary
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Kotaro Horiguchi, Hideyuki Tanaka, Kenjiro Arisawa, Haruo Hagiwara, Takeshi Suzuki, Takehiro Tsukada, Yoko Nekooki-Machida, Anshin Asano-Hoshino, Yoshimi Nishijima, Takashi Nakakura, Ken Fujiwara, and Yoshiko Kiuchi
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Male ,0301 basic medicine ,medicine.medical_specialty ,Double negative ,Gene Expression ,S100 Calcium Binding Protein beta Subunit ,In situ hybridization ,Biology ,Stem cell marker ,Pathology and Forensic Medicine ,03 medical and health sciences ,SOX2 ,Pituitary Gland, Anterior ,Internal medicine ,Parenchyma ,medicine ,Animals ,Secretion ,Cilia ,Rats, Wistar ,Progenitor cell ,Molecular Biology ,In Situ Hybridization, Fluorescence ,SOXB1 Transcription Factors ,Stem Cells ,Cell Differentiation ,Forkhead Transcription Factors ,General Medicine ,Immunohistochemistry ,Rats ,Cell biology ,030104 developmental biology ,Endocrinology - Abstract
S100β-positive cells exist in the marginal cell layer (MCL) of the adenohypophysis and follicle structure in the parenchyma of anterior lobe (ALFS) in pituitary. They have multiple functions as phagocytes or cells that regulate hormone secretion. Majority of S100β-positive cells in the adenohypophysis express sex determining region Y-box 2 protein (SOX2), a stem cell marker; therefore, S100β/SOX2 double positive cells are also considered as one type of stem/progenitor cells. MCL and ALFS are consisting of morphologically two types of cells, i.e., multiciliated cells and non-ciliated cells. However, the relationship between the S100β-positive cells and multiciliated cells in the pituitary is largely unknown. In the present study, we first immunohistochemically verified the feature of multiciliated cells in MCL and ALFS. We then examined the expression patterns of FOXJ1, an essential expression factor for multiciliated cell-differentiation, and SOX2 in the S100β-positive multiciliated cells by in situ hybridization and immunohistochemistry. We identified anew the S100β/SOX2/FOXJ1 triple positive multiciliated cells, and revealed that they were dispersed throughout the MCL and ALFS. These results indicate that the MCL and ALFS are consisting of morphologically and functionally distinct two types of cells, i.e., S100β/SOX2 double positive non-ciliated cells and S100β/SOX2/FOXJ1 triple positive multiciliated cells.
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- 2016
14. Adrenalectomy facilitates ATAT1 expression and α-tubulin acetylation in ACTH-producing corticotrophs
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Yoshiko Kiuchi, Hideyuki Tanaka, Anshin Asano-Hoshino, Takashi Nakakura, Takahiro Nemoto, Haruo Hagiwara, Takeshi Suzuki, Yoshimi Nishijima, and Kenjiro Arisawa
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0301 basic medicine ,endocrine system ,medicine.medical_specialty ,Histology ,Arylamine N-Acetyltransferase ,Enteroendocrine cell ,Biology ,Cell morphology ,Pathology and Forensic Medicine ,03 medical and health sciences ,Adrenocorticotropic Hormone ,Tubulin ,Microtubule ,Internal medicine ,medicine ,Animals ,Secretion ,RNA, Messenger ,Rats, Wistar ,Corticotrophs ,Adrenal gland ,Acetylation ,Adrenalectomy ,Cell Biology ,Immunohistochemistry ,Isoenzymes ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,Corticotropic cell ,hormones, hormone substitutes, and hormone antagonists ,Intracellular - Abstract
Microtubules play an important role in the intracellular transport of secretory granules in endocrine cells and in mitosis and the maintenance of cell morphology and are composed of heterodimers of α- and β-tubulin. α-Tubulin N-acetyltransferase 1 (ATAT1), which acetylates the lysine residue at position 40 of α-tubulin, functions not only in stabilizing microtubule structures and forming the primary cilium assembly but also in vesicular trafficking in neurons. However, the localization of ATAT1 and the role of α-tubulin acetylation in endocrine cells in the pituitary are still poorly understood. Corticotrophs in the anterior lobe of the pituitary produce and secrete adrenocorticotropin (ACTH). Although removal of the adrenal gland, a target organ of ACTH, is reported to promote the synthesis and secretion of ACTH in corticotrophs and to induce structural alterations in their organelles, uncertainty remains as to whether the acetylation of α-tubulin is involved in such intracellular events of corticotrophs. We investigate the expression and localization of ATAT1 and the acetylation of α-tubulin in the pituitary of normal and adrenalectomized rats. We find that ATAT1 is localized to the Golgi apparatus of endocrine cells in the anterior lobe of normal pituitary and that the expression levels of ATAT1 and acetylation levels of α-tubulin increase following adrenalectomy. These results agree with the hypothesis that the acetylation of α-tubulin by ATAT1 regulates the intracellular transport of secretory granules in corticotrophs.
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- 2016
15. Intracellular localization of α-tubulin acetyltransferase ATAT1 in rat ciliated cells
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Takeshi Suzuki, Hideyuki Tanaka, Takahiro Nemoto, Yoshimi Nishijima, Kenjiro Arisawa, Yoshiko Kiuchi, Haruo Hagiwara, Anshin Asano-Hoshino, and Takashi Nakakura
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Male ,0301 basic medicine ,Blotting, Western ,Intracellular Space ,Biology ,Pathology and Forensic Medicine ,Cell membrane ,03 medical and health sciences ,symbols.namesake ,Acetyltransferases ,Antibody Specificity ,Microtubule ,medicine ,Animals ,Humans ,Basal body ,Cilia ,Rats, Wistar ,Molecular Biology ,Retina ,Cilium ,General Medicine ,respiratory system ,Golgi apparatus ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Organ Specificity ,Motile cilium ,symbols ,Oviduct ,Female - Abstract
Cilia are microtubule-based hair-like organelles on basal bodies located beneath the cell membrane in various tissues of multicellular animals, and are usually classified into motile cilia and primary cilia. Microtubules are assembled from the heterodimers of α- and β-tubulin. The lysine residue at position 40 (K40) of α-tubulin is an important site for acetylation, and this site is acetylated in the cilium. α-Tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to the K40 residue of α-tubulin; however, its intracellular distribution in mammalian tissues remains unclear. In this study, we analyzed ATAT1 localization in rat trachea, oviduct, kidney, retina, testis and the third ventricle of the brain by immunohistochemical techniques using a specific antibody against ATAT1. ATAT1 was distributed to the motile cilia of multiciliated cells of the trachea, third ventricle of the brain and oviduct, and in the primary cilia of the renal medullary collecting duct. ATAT1 also localized to the primary cilia, inner and outer segments of retinal photoreceptor cells, and at the Golgi apparatus of spermatocytes and spermatids of testis. These results indicated that α-tubulin acetylation by ATAT1 at distinct subcellular positions may influence the functional regulation of microtubules and cilia in a variety of ciliated cells.
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- 2015
16. Dynamic localization of α-tubulin acetyltransferase ATAT1 through the cell cycle in human fibroblastic KD cells
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Toshio Miyashita, Hideyuki Tanaka, Kenjiro Arisawa, Yoshiko Kiuchi, Yoko Nekooki-Machida, Haruo Hagiwara, Takashi Nakakura, and Yoshimi Nishijima
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0301 basic medicine ,Centriole ,Transcription, Genetic ,Microtubules ,Pathology and Forensic Medicine ,Cell Line ,03 medical and health sciences ,Microtubule ,Acetyltransferases ,Basal body ,Humans ,Telophase ,Molecular Biology ,Mitosis ,Chemistry ,Cell Cycle ,Acetylation ,General Medicine ,Cell cycle ,Fibroblasts ,Cell biology ,Midbody ,Protein Transport ,030104 developmental biology ,Microtubule Proteins ,Cytokinesis - Abstract
Acetylation of α-tubulin is a well-studied posttranscriptional modification, which is mostly catalyzed by α-tubulin N-acetyltransferase (ATAT1). ATAT1 possibly affects various cellular functions related with microtubules, such as intracellular transport, cell motility, cilia formation, and neuronal signaling. Here, we analyzed the subcellular localization of immunolabeled ATAT1 in human fibroblast KD cells through the cell cycle using confocal laser scanning microscopy. ATAT1 dramatically changed its localization through the cell cycle, depending on the mitotic phase. In interphase, immunolabeled ATAT1 was observed in centrioles, nuclei, and basal bodies if the cells projected primary cilia. ATAT1 was intensely detected as clusters in the nuclei in the G1–G2 phase. In telophase, ATAT1 colocalized with chromatids and spindle poles, and ultimately migrated to the daughter nucleus, newly synthesized centrioles, and midbody. The nucleolus is a core region of ribosomal RNA transcription, and the midbody is associated with severing and depolymerizing of microtubules in the stembody. The specific distributions of ATAT1 through the cell cycle suggest multiple functions of ATAT1, which could include acetylation of microtubules, RNA transcription activity, severing microtubules, and completion of cytokinesis.
- Published
- 2018
17. Loss of zinc finger MYND-type containing 10 (zmynd10) affects cilia integrity and axonemal localization of dynein arms, resulting in ciliary dysmotility, polycystic kidney and scoliosis in medaka (Oryzias latipes)
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Takahiko Yokoyama, Masato Kinoshita, Takashi Nakakura, Toshiyuki Nishimaki, Satoshi Ansai, Daisuke Kobayashi, Motoyuki Ogawa, Anshin Asano-Hoshino, and Haruo Hagiwara
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0301 basic medicine ,Male ,medicine.medical_specialty ,Axoneme ,Embryo, Nonmammalian ,Movement ,Oryzias ,Motility ,Biology ,Flagellum ,Morpholinos ,03 medical and health sciences ,0302 clinical medicine ,Intraflagellar transport ,Internal medicine ,medicine ,Animals ,Amino Acid Sequence ,Cilia ,RNA, Messenger ,Molecular Biology ,Primary ciliary dyskinesia ,Body Patterning ,Zinc finger ,Gene knockdown ,Polycystic Kidney Diseases ,Base Sequence ,Cilium ,Tumor Suppressor Proteins ,Dyneins ,Gene Expression Regulation, Developmental ,Epistasis, Genetic ,Zinc Fingers ,Cell Biology ,medicine.disease ,Spermatozoa ,Cell biology ,030104 developmental biology ,Endocrinology ,Phenotype ,Scoliosis ,Motile cilium ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Cilia and flagella are hair-like organelles that project from the cell surface and play important roles in motility and sensory perception. Motility defects in cilia and flagella lead to primary ciliary dyskinesia (PCD), a rare human disease. Recently zinc finger MYND-type containing 10 ( ZMYND10 ) was identified in humans as a PCD-associated gene. In this study, we use medaka fish as a model to characterize the precise functions of zmynd10. In medaka, zmynd10 is exclusively expressed in cells with motile cilia. Embryos with zmynd10 Morpholino knockdown exhibited a left-right (LR) defect associated with loss of motility in Kupffer's vesicle (KV) cilia. This immotility was caused by loss of the outer dynein arms, which is a characteristic ultrastructural phenotype in PCD. In addition, KV cilia in zmynd10 knockdown embryos had a swollen and wavy morphology. Together, these results suggest that zmynd10 is a multi-functional protein that has independent roles in axonemal localization of dynein arms and in formation and/or maintenance of cilia. The C-terminal region of zmynd10 has a MYND-type zinc finger domain (zf-MYND) that is important for its function. Our rescue experiment showed that the zmynd10–ΔC truncated protein, which lacks zf-MYND, was still partially functional, suggesting that zmynd10 has another functional domain besides zf-MYND. To analyze the later stages of development, we generated a zmynd10 knockout mutant using transcription activator-like effector nuclease (TALEN) technology. Adult mutants exhibited sperm dysmotility, scoliosis and progressive polycystic kidney.
- Published
- 2017
18. Myosin Light Chain Kinase / Actin Interaction in Phorbol Dibutyrate–Stimulated Smooth Muscle Cells
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Ying Zhang, Shinji Yoshiyama, Hideyuki Tanaka, Yuan Lin, Takeshi Katayama, Hou-Li Zhang, Kohichi Hayakawa, Masaru Watanabe, Gary L. Wright, Haruo Hagiwara, Mike E. Fultz, Akio Nakamura, Hong-Hui Wang, Kazuhiro Kohama, and Sean E. Thatcher
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Myosin light-chain kinase ,Guinea Pigs ,Myocytes, Smooth Muscle ,macromolecular substances ,In Vitro Techniques ,Myosins ,Biology ,Cell Line ,Myosin ,medicine ,Animals ,Protein Isoforms ,Myocyte ,Gene Silencing ,Enzyme Inhibitors ,Phosphorylation ,RNA, Small Interfering ,Kinase activity ,Myosin-Light-Chain Kinase ,Cytoskeleton ,Phorbol 12,13-Dibutyrate ,Actin ,Pharmacology ,Smooth muscle tissue ,lcsh:RM1-950 ,Microfilament Proteins ,Actin remodeling ,Muscle, Smooth ,Molecular biology ,Actins ,Peptide Fragments ,Rats ,Cell biology ,Kinetics ,lcsh:Therapeutics. Pharmacology ,medicine.anatomical_structure ,Molecular Medicine ,Cell Surface Extensions ,medicine.symptom ,Chickens ,Protein Processing, Post-Translational ,Muscle Contraction ,Muscle contraction - Abstract
Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun. 2008;369:135–143). Myosin ATPase activity measurements indicate that the 26 – 41 peptide of MLCK significantly decreases ATPase activity as the concentration of this peptide increases. Sliding velocity of actin-filaments on myosin and stress responses in skinned smooth muscle tissue are also inhibited. Peptide-mediated uptake and the microinjection technique in cells indicate that the peptide was necessary for actin-filament stabilization. Fluorescence resonance energy transfer analysis indicated that in the presence of MLCK, α-actin but not β-actin remodeled during phorbol 12,13-dibutyrate (PDBu)-induced contractions. PDBu also induced podosomes in the cell. When MLCK expression was down-regulated by introduction of RNAi for MLCK by lentivirus vector into the cells, we failed to observe the podosome induction upon PDBu stimulation. Rescue experiments indicate that the non-kinase activity of MLCK plays an important role in maintaining actin stress fibers and in the PDBu-induced reorganization of actin-filaments in smooth muscle cells. Keywords:: actin-remodeling, podosome, phorbol 12,13-dibutyrate (PDBu), FRET analysis, myosin light chain kinase (MLCK)-deficient cell
- Published
- 2011
19. Characterization of the medaka (Oryzias latipes) primary ciliary dyskinesia mutant, jaodori: Redundant and distinct roles of dynein axonemal intermediate chain 2 (dnai2) in motile cilia
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Keiichiro Kamura, Haruo Hagiwara, Daisuke Kobayashi, Hiroyuki Takeda, Norio Iijima, and Takahiko Yokoyama
- Subjects
Fish Proteins ,Tail ,Embryo, Nonmammalian ,Positional cloning ,Movement ,DNA Mutational Analysis ,Molecular Sequence Data ,Dynein ,Oryzias ,Motility ,Biology ,Flagellum ,Kidney ,Primary ciliary dyskinesia ,Intraflagellar transport ,medicine ,Animals ,Cilia ,RNA, Messenger ,Molecular Biology ,Body Patterning ,Genetics ,Base Sequence ,Kartagener Syndrome ,Cilium ,Gene Expression Regulation, Developmental ,Axonemal Dyneins ,Cell Biology ,medicine.disease ,Medaka ,Cell biology ,Cyst ,Phenotype ,Organ Specificity ,Mutation ,Motile cilium ,Rheology ,Biomarkers ,Developmental Biology - Abstract
Cilia and flagella are highly conserved organelles that have diverse motility and sensory functions. Motility defects in cilia and flagella result in primary ciliary dyskinesia (PCD). We isolated a novel medaka PCD mutant, jaodori (joi). Positional cloning showed that axonemal dynein intermediate chain 2 (dnai2) is responsible for joi. The joi mutation was caused by genomic insertion of the medaka transposon, Tol1. In the joi mutant, cilia in Kupffer's vesicle (KV), an organ functionally equivalent to the mouse node in terms of left–right (LR) specification, are generated but their motility is disrupted, resulting in a LR defect. Ultrastructural analysis revealed severe reduction in the outer dynein arms in KV cilia of joi mutants. We also found the other dnai2 gene in the medaka genome. These two dnai2 genes function either redundantly or distinctly in tissues possessing motile cilia.
- Published
- 2010
20. Immunohistochemical and electron microscopic observations of stromal cells in the human oviduct mucosa
- Author
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Takeo Aoki, Haruo Hagiwara, Takeshi Suzuki, Kuniaki Takata, and Nobuo Ohwada
- Subjects
Pathology ,medicine.medical_specialty ,Lamina propria ,Mucous Membrane ,animal structures ,Stromal cell ,Cilium ,General Medicine ,Biology ,Actins ,Pathology and Forensic Medicine ,medicine.anatomical_structure ,Pregnancy ,medicine ,Ultrastructure ,Animals ,Humans ,Oviduct ,Immunohistochemistry ,Female ,Decidual cells ,Stromal Cells ,Molecular Biology ,Myofibroblast ,Fallopian Tubes - Abstract
Stromal cells in the lamina propria of the human oviduct mucosa are unique cells that can differentiate into decidual cells during ectopic pregnancy in the oviduct. The nature of stromal cells is still unknown. In the present study, we investigated human oviductal stromal cells with transmission electron microscopy and immunohistochemistry and revealed that they had ultrastructural features similar to myofibroblasts and expressed alpha-smooth muscle actin, a marker used to identify myofibroblasts. Primary cilia were also one of the characteristic profiles of the stromal cells. These findings showed that the connective tissue-stromal cells in the human oviduct mucosa are myofibroblasts. They are considered to play an important role in the transport of oocytes by bringing about contraction of the mucosal folds.
- Published
- 2008
21. Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins
- Author
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Sumito Koshida, Ritsu Kamiya, Hanswalter Zentgraf, Manfred Fliegauf, Chikako Hara, Tatsuya Tsukahara, Atsushi Miyawaki, Richard Reinhardt, Daisuke Kobayashi, Hiroyuki Takeda, Eileen T. O'Toole, Heymut Omran, Hideaki Mizuno, Hiroyuki Kawano, David R. G. Mitchell, Gerard Leblond, Niki T. Loges, Toshiki Yagi, H. Seithe, Qi Zhang, Haruo Hagiwara, Yoshinori Watanabe, and Heike Olbrich
- Subjects
Fish Proteins ,Male ,Axoneme ,Movement ,Molecular Sequence Data ,Dynein ,Oryzias ,Genes, Recessive ,Flagellum ,Biology ,Article ,Mice ,Otolithic Membrane ,Intraflagellar transport ,Dynein ATPase ,Testis ,medicine ,Animals ,Humans ,HSP70 Heat-Shock Proteins ,Disease ,Cilia ,Cloning, Molecular ,Primary ciliary dyskinesia ,Multidisciplinary ,Sequence Homology, Amino Acid ,Kartagener Syndrome ,Cilium ,Chlamydomonas ,Dyneins ,Proteins ,Epithelial Cells ,Forkhead Transcription Factors ,Inner dynein arm ,medicine.disease ,Cell biology ,Mutation ,Sperm Motility ,Growth and Development ,Protein Binding - Abstract
Cilia and flagella are highly conserved organelles that have diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia and flagella often result in primary ciliary dyskinesia. However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a new gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in primary ciliary dyskinesia patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.
- Published
- 2008
22. Immunolocalization of Water Channel Aquaporins in the Vomeronasal Organ of the Rat: Expression of AQP4 in Neuronal Sensory Cells
- Author
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Toshiyuki Matsuzaki, Takeshi Suzuki, Takeo Aoki, Shigeru Takami, Abduxukur Ablimit, Haruo Hagiwara, and Kuniaki Takata
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Vomeronasal organ ,Physiology ,Aquaporin ,Nerve fiber ,Sensory system ,Biology ,Aquaporins ,Behavioral Neuroscience ,Olfactory Mucosa ,Physiology (medical) ,medicine ,Animals ,Neurons, Afferent ,Rats, Wistar ,Axon ,Microscopy, Immunoelectron ,Aquaporin 4 ,Apical membrane ,Immunohistochemistry ,Sensory Systems ,Epithelium ,Rats ,Cell biology ,medicine.anatomical_structure ,Pheromone ,sense organs ,Vomeronasal Organ - Abstract
The vomeronasal organ comprises a pair of narrow tubes in the mammalian nasal septum, serving as a chemosensory system for pheromones. We examined the expression and localization of water channel aquaporins (AQPs) in the rat vomeronasal organ. AQP1 was localized in blood vessels, being particularly abundant in cavernous tissues of the nonsensory mucosa. AQP5 was found in the apical membrane of the gland acinar cells in the vomeronasal organ. AQP3 was detected in the basal cells of the nonsensory epithelium, whereas it was absent in the sensory epithelium. AQP4 was found in both the sensory and the nonsensory epithelia. Interestingly, AQP4 was highly concentrated in the sensory cells of the sensory epithelium. Immunoelectron microscopic examination clearly showed that AQP4 was localized at the plasma membrane in the cell body and lateral membrane of the dendrite, except for the microvillous apical membrane. Nerve fiber bundles emanating from neuronal sensory cells were positive for AQP4, whereby the plasma membrane of each axon was positive for AQP4. These observations clearly show that neuronal sensory cells in the vomeronasal organ are unique in that they express abundant AQP4 at their plasma membrane. This is in marked contrast to the olfactory and central nervous systems, where AQPs are not detectable in neurons, and instead, AQP4 is abundant in the supporting cells and astrocytes surrounding them. The present findings suggest a unique water-handling feature in neuronal sensory cells in the vomeronasal organ.
- Published
- 2008
23. Transformational and Transactional Leadership on Affective Commitment: The Mediating Role of Trust
- Author
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Hong, Gahye, primary, Shin, Mannsoo, additional, and Horaguchi, Haruo Hagiwara, additional
- Published
- 2017
- Full Text
- View/download PDF
24. International Internship: Business Education for Leadership and Trust
- Author
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Horaguchi, Haruo Hagiwara, primary, Kodama, Yasushi, additional, and Yukimoto, Seiki, additional
- Published
- 2017
- Full Text
- View/download PDF
25. Recent Advances in Fluorescent Labeling Techniques for Fluorescence Microscopy
- Author
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Haruo Hagiwara, Takeo Aoki, Kuniaki Takata, Toshiyuki Matsuzaki, and Takeshi Suzuki
- Subjects
fluorescent dyes ,Histology ,Physiology ,fluorescent proteins ,fluorescent labeling ,immunofluorescent staining ,Nanotechnology ,Cell Biology ,Biology ,Biochemistry ,Fluorescence ,fluorescence microscopy ,Pathology and Forensic Medicine ,Green fluorescent protein ,Fluorescent labelling ,Technical Advancement ,Microscopy ,Fluorescence microscope ,Fluorescent staining ,Biophysics - Abstract
Tremendous progress in recent computer-controlled systems for fluorescence and laser-confocal microscopy has provided us with powerful tools to visualize and analyze molecular events in the cells. Various fluorescent staining and labeling techniques have also been developed to be used with these powerful instruments. Fluorescent proteins such as green fluorescent protein (GFP) allow us to directly label particular proteins of interest in living cells. This technique has been extended over a large area of cell biology, and a variety of fluorescent protein-derived techniques have been developed to visualize the functions and conditions of the molecules within living cells. In this review, we summarize the techniques for fluorescent staining and labeling for recent fluorescence microscopy.
- Published
- 2007
26. Apical Localization of Sodium-Dependent Glucose Transporter SGLT1 is Maintained by Cholesterol and Microtubules
- Author
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Yukiko Tajika-Takahashi, Takeshi Suzuki, Toshiyuki Matsuzaki, Takeo Aoki, Haruo Hagiwara, and Kuniaki Takata
- Subjects
Confluency ,Histology ,Tight junction ,Colcemid ,Physiology ,fungi ,Glucose transporter ,cholesterol ,Regular Article ,Cell Biology ,Apical membrane ,Cycloheximide ,Biology ,Biochemistry ,Pathology and Forensic Medicine ,Cell biology ,cell polarity ,chemistry.chemical_compound ,chemistry ,Microtubule ,Cell polarity ,apical membrane ,SGLT ,microtubule - Abstract
A GFP-labeled sodium-dependent glucose transporter SGLT1 (SGLT-GFP) was transfected into MDCK cells. SGLT-GFP was localized at the apical membrane in confluent cells. When cellular cholesterol was depleted by methyl-beta-cyclodextrin (MbetaCD) treatment, the localization of SGLT-GFP gradually switched from apical to whole plasma membrane. Time-lapse microscopy revealed that the effect of MbetaCD appeared within 30 min, and that the transition of SGLT-GFP to the whole plasma membrane was completed within 2 hr after the administration. Immunofluorescence microscopy revealed that the tight junction framework remained steady during this process. The effect of MbetaCD on SGLT-GFP localization was counter-balanced by the addition of cholesterol into the culture medium. Disruption of microtubules by colcemid also perturbed SGLT-GFP localization. SGLT-GFP localized to the whole plasma membrane by colcemid treatment, and apical localization was restored within 1 hr after -removal of colcemid. Inhibition of protein synthesis by cycloheximide had no effect on the transition of SGLT-GFP induced by the MbetaCD or colcemid. These results indicated that the apical localization of SGLT-GFP is maintained by cellular cholesterol and microtubules, possibly with an apical recycling machinery.
- Published
- 2006
27. Differential regulation of AQP2 trafficking in endosomes by microtubules and actin filaments
- Author
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Toshiyuki Matsuzaki, Michio Kuwahara, Yuki Tajika, Abdushukur Ablimit, Sei Sasaki, Takeo Aoki, Kuniaki Takata, Haruo Hagiwara, and Takeshi Suzuki
- Subjects
Time Factors ,Histology ,Endosome ,Vesicular Transport Proteins ,Arp2/3 complex ,Endosomes ,Biology ,urologic and male genital diseases ,Microtubules ,chemistry.chemical_compound ,Microtubule ,Humans ,Cytoskeleton ,Molecular Biology ,Actin ,Cytochalasin D ,Colcemid ,urogenital system ,Membrane Proteins ,Epithelial Cells ,Cell Biology ,Immunohistochemistry ,Cell biology ,Actin Cytoskeleton ,Protein Transport ,Medical Laboratory Technology ,Nocodazole ,Gene Expression Regulation ,chemistry ,rab GTP-Binding Proteins ,biology.protein - Abstract
Vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct cells is critical to regulate the urine concentration. To better understand the mechanism of subcellular trafficking of AQP2, we examined MDCK cells expressing AQP2 as a model. We first performed double-immunolabeling of AQP2 with endosomal marker proteins, and showed that AQP2 is stored at a Rab11-positive subapical compartment. After the translocation to the plasma membrane, AQP2 was endocytosed to EEA1-positive early endosomes, and then transferred back to the original Rab11-positive compartment. When Rab11 was depleted by RNA interference, retention of AQP2 at the subapical storage compartment was impaired. We next examined the role of cytoskeleton in the AQP2 trafficking and localization. By the treatment with microtubule-disrupting agent such as nocodazole or colcemid, the distribution of AQP2 storage compartment was altered. The disruption of actin filaments with cytochalasin D or latrunculin B induced the accumulation of AQP2 in EEA1-positive early endosomes. Altogether, our data suggest that Rab11 and microtubules maintain the proper distribution of the subapical AQP2 storage compartment, and actin filaments regulate the trafficking of AQP2 from early endosomes to the storage compartment.
- Published
- 2005
28. Expression and immunolocalization of water-channel aquaporins in the rat and mouse mammary gland
- Author
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Haruo Hagiwara, Abdushukur Ablimit, Takeo Aoki, Natsuko Machida, Takeshi Suzuki, Yuki Tajika, Kuniaki Takata, and Toshiyuki Matsuzaki
- Subjects
medicine.medical_specialty ,Histology ,Alveolar Epithelium ,Immunoblotting ,Mammary gland ,Gene Expression ,Biology ,Aquaporins ,Mice ,Mammary Glands, Animal ,Antibody Specificity ,Pregnancy ,Internal medicine ,medicine ,Animals ,Lactation ,Interlobular duct ,Rats, Wistar ,Molecular Biology ,Epithelial polarity ,Aquaporin 3 ,Aquaporin 2 ,Venule ,Aquaporin 1 ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Biology ,Immunohistochemistry ,Molecular biology ,Rats ,Mice, Inbred C57BL ,Medical Laboratory Technology ,medicine.anatomical_structure ,Endocrinology ,Microscopy, Fluorescence ,Female ,Duct (anatomy) - Abstract
We examined the expression and immunolocalization of water-channel aquaporins in the mammary gland by reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry. RT-PCR and immunoblotting revealed the expression of aquaporin-1 (AQP1) and AQP3 in the lactating rat mammary gland. AQP3 was detected in the alveolar epithelium and duct system whereas AQP1 was found in the capillaries and venules. AQP3 was present in the basolateral membrane of secretory epithelial cells and intralobular and interlobular duct epithelial cells. The main duct near the orifice in the nipple, which is comprised of a stratified epithelium, bore AQP3 in its basal and intermediate layers. AQP1 was located in both the apical and basolateral membranes of capillary and venule endothelia. AQP3 was not detected in virgin females. AQP3 was found in some differentiating mammary epithelial cells in the pregnant rat. AQP1 was present in capillaries and venules in the differentiating mammary gland of the pregnant rat and in the mammary fat pad of virgin females. We found a similar distribution of AQP1 and AQP3 in the mouse. AQP1 and AQP3 seem to play roles in the synthesis and/or secretion of milk.
- Published
- 2005
29. Aquaporin Water Channels in the Kidney
- Author
-
Yuki Tajika, Takeo Aoki, Haruo Hagiwara, Abdushukur Ablimit, Kuniaki Takata, Toshiyuki Matsuzaki, and Takeshi Suzuki
- Subjects
medicine.medical_specialty ,Kidney ,Histology ,urogenital system ,Physiology ,Reabsorption ,Aquaporin ,Cell Biology ,Biology ,Nephrogenic diabetes insipidus ,medicine.disease ,Biochemistry ,Pathology and Forensic Medicine ,Cell biology ,Endocrinology ,Aquaglyceroporins ,medicine.anatomical_structure ,Aquaporin 2 ,Internal medicine ,Diabetes insipidus ,medicine ,Epithelial polarity - Abstract
Aquaporins (AQPs) are water channel proteins of cellular membranes serving in the permeation of water across the membrane. AQP families are found virtually in all types of life ranging from bacteria to plant and animal cells. In mammals, at least 13 isoforms of AQPs have been identified. They are classified into three subtypes: classical aquaporins, aquaglyceroporins, and superaquaporins. These AQPs are differentially expressed in a wide variety of cells and tissues in the body, and play important roles in water metabolism. In the kidney, at least 6 isoforms of AQPs, namely AQP1, AQP2, AQP3, AQP4, AQP6, and AQP7, are reported to be expressed. Water transfer occurs mainly in the proximal tubules and collecting ducts in the kidney. In the proximal tubules, AQP1 and AQP7 are expressed, among which AQP1 plays a major role in water reabsorption. In the collecting ducts, AQP2, AQP3, AQP4, and AQP6 are expressed. AQP3 and AQP4 are localized at the basolateral membrane. AQP2 is stored in the cytoplasmic vesicles and is translocated to the apical plasma membrane in response to antidiuretic hormone. Mutations of AQP2 lead to either loss of channel function or mistrafficking and result in nephrogenic diabetes insipidus, the inability to concentrate urine.
- Published
- 2005
30. Anti-calreticulin Antibody Binds to a Membrane Protein in Caveolae
- Author
-
Ryuji Nomura, Takao Senda, Takeo Aoki, Toyoshi Fujimoto, and Haruo Hagiwara
- Subjects
Vesicle-associated membrane protein 8 ,Histology ,biology ,Physiology ,Immunocytochemistry ,chemistry.chemical_element ,Cell Biology ,Calcium ,Biochemistry ,Pathology and Forensic Medicine ,Cell biology ,medicine.anatomical_structure ,Membrane protein ,chemistry ,Caveolae ,medicine ,biology.protein ,Antibody ,Fibroblast ,Calreticulin - Published
- 2005
31. Water Channel Aquaporin 1 (AQP1) Is Present in the Perineurium and Perichondrium
- Author
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Abdushukur Ablimit, Toshiyuki Matsuzaki, Yuki Tajika, Takeo Aoki, Haruo Hagiwara, Kuniaki Takata, and Takeshi Suzuki
- Subjects
Pathology ,medicine.medical_specialty ,Histology ,Physiology ,Chemistry ,Cartilage ,Nerve fiber ,Cell Biology ,Anatomy ,Perineurial Cell ,Biochemistry ,Pathology and Forensic Medicine ,medicine.anatomical_structure ,Aquaporin 1 ,medicine ,Perichondrium ,Sugar transporter ,Sciatic nerve ,Perineurium - Abstract
Aquaporin 1 (AQP1) is an isoform of membrane water channels. We examined the distribution of AQP1 in peripheral nerves. Immunofluorescence microscopy with specific antibodies to AQP1 was performed in the tissue sections of the rat tongue, esophagus, trachea, and sciatic nerve. AQP1 was present in the perineurium of nerve fiber bundles in the sciatic nerve and nerve fiber bundles in the tongue, esophagus, and trachea. Laser confocal microscopy of the double-labeled specimens for AQP1 and sugar transporter GLUT1 revealed that AQP1 was localized in the outer layer(s) of the perineurial cell layers, whereas GLUT1 was present throughout the entire perineurial cell layers. AQP1 and GLUT1 were also present in blood vessels inside the nerve fiber bundle. They were rarely found in the same blood vessels, namely, AQP1 was hardly detectable in GLUT1-positive blood vessels and GLUT1 was hardly detectable in AQP1-positive vessels. AQP1 in these sites of the blood-nerve barrier may participate in the water transfer across the barrier and contribute to the maintenance of the milieu of the nerve. In addition, we showed the presence of AQP1 in the perichondrium of the tracheal cartilage suggesting a possible role in water transfer between the cartilage and surrounding connective tissues.
- Published
- 2005
32. Aquaporin-2 Is Retrieved to the Apical Storage Compartment via Early Endosomes and Phosphatidylinositol 3-Kinase-Dependent Pathway
- Author
-
Yuki Tajika, Kuniaki Takata, Michio Kuwahara, Toshiyuki Matsuzaki, Takeshi Suzuki, Sei Sasaki, Takeo Aoki, and Haruo Hagiwara
- Subjects
Monosaccharide Transport Proteins ,Endosome ,Molecular Sequence Data ,Muscle Proteins ,Endosomes ,Biology ,Aquaporins ,Kidney ,Transfection ,urologic and male genital diseases ,Antibodies ,Cell Line ,EEA1 ,Phosphatidylinositol 3-Kinases ,symbols.namesake ,Endocrinology ,Lysosome ,Receptors, Transferrin ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Aquaporin 2 ,Glucose Transporter Type 4 ,urogenital system ,Vesicle ,Cell Polarity ,Golgi apparatus ,Apical membrane ,Cell Compartmentation ,Cell biology ,Protein Transport ,medicine.anatomical_structure ,Cytoplasm ,symbols - Abstract
Aquaporin-2 (AQP2) is one of the water-channel proteins expressed in principal cells of kidney collecting ducts, where it is stored in the intracellular compartment. Previous studies have demonstrated that AQP2 vesicles constitute a distinct intracellular compartment partially overlapping with early endosomes. In this report, we performed in vitro experiments using the renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, stably expressing AQP2 (MDCK-hAQP2). In nonpolarized cells, AQP2 vesicles were scattered in the cytoplasm and did not colocalize with Golgi 58K or TGN38. Small portions of AQP2 vesicles were positive for the lysosome marker cathepsin D. An early endosome antigen (EEA1) localized around AQP2 vesicles in close proximity, suggesting involvement of the endosomal system in the trafficking of AQP2. AQP2 vesicles are distinct from other recycling molecules, such as glucose transporter 4 (GLUT4) and endocytosed transferrin. In polarized MDCK-hAQP2 cells, AQP2 vesicles were localized in the subapical recycling compartment and distinct from the Golgi apparatus, trans-Golgi network, lysosome, and early endosome in the nonstimulated state. When the cells were treated with forskolin, translocation of AQP2 to the apical membrane was observed. Washout of forskolin induced retrieval of AQP2 into the cytoplasm, and AQP2 was transiently colocalized with EEA1-positive endosomes. Then, AQP2 moved from EEA1-positive endosomes to the subapical AQP2-storage compartment, which is sensitive to wortmannin and LY294002. These results suggest that AQP2 resides in a recycling compartment at the apical side in polarized MDCK-hAQP2 cells, and its retrieval uses the apical endosomal system and the phosphatidylinositol 3-kinase-dependent pathway.
- Published
- 2004
33. Molecular Mechanisms and Drug Development in Aquaporin Water Channel Diseases: Water Channel Aquaporin-2 of Kidney Collecting Duct Cells
- Author
-
Yuki Tajika, Takeshi Suzuki, Haruo Hagiwara, Toshiyuki Matsuzaki, Ablimit Abduxukur, Takeo Aoki, and Kuniaki Takata
- Subjects
Pharmacology ,Aquaporin 2 ,Endosome ,urogenital system ,Vesicle ,Endoplasmic reticulum ,lcsh:RM1-950 ,Biology ,Apical membrane ,Golgi apparatus ,Aquaporins ,urologic and male genital diseases ,Cell biology ,symbols.namesake ,lcsh:Therapeutics. Pharmacology ,Aquaporin 3 ,symbols ,Molecular Medicine ,Animals ,Humans ,Technology, Pharmaceutical ,Kidney Diseases ,Kidney Tubules, Collecting ,Epithelial polarity - Abstract
Aquaporin-2 (AQP2) is one of the membrane water channel proteins expressed in principal cells of the kidney collecting ducts. In the basal state, AQP2 resides in the storage vesicles localized in the subapical cytoplasm. Upon stimulation with vasopressin, AQP2 is translocated to the apical plasma membrane by the exocytic fusion of the storage vesicles with the apical membrane. This translocation enables the transepithelial reabsorption of water from the lumen to the interstitium via AQP2 at the apical membrane and AQP3/AQP4 at the basolateral membrane. AQP2-storage vesicles are distinct from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and lysosomes. The early endosomal marker EEA1 is colocalized with some of AQP2 vesicles. Further analyses in Madin-Darby canine kidney (MDCK) cells transfected with AQP2 revealed that subapical Rab11-positive/EEA1-negative smaller vesicles constitute part of the AQP2 storage vesicles for the translocation to the apical membrane. Termination of stimulation results in the retrieval of AQP2 to the larger EEA1-positive early endosomal compartment. AQP2 is then transferred to the subapical storage compartment in a PI3-kinase-dependent manner. GLUT4 is an isoform of glucose transporters whose localization is also regulated by vesicular trafficking induced by insulin stimulation. Comparison of the intracellular localization of AQP2 with GLUT4 suggests distinct regulation of AQP2 trafficking. Keywords:: water channel, aquaporin-2, kidney, collecting duct, endosome
- Published
- 2004
34. Cryosectioning of Cultured Cells on Permeable Support
- Author
-
Takeshi Suzuki, Takeo Aoki, Toshiyuki Matsuzaki, Yuki Tajika, Haruo Hagiwara, and Kuniaki Takata
- Subjects
Immunolabeling ,Histology ,Physiology ,Homogeneous ,Cell polarity ,Cultured cell ,Cell Biology ,Single section ,Biology ,Biochemistry ,Pathology and Forensic Medicine ,Cell biology - Abstract
Immunolabeling of cultured cell lines of epithelial origin is often performed to study cell polarity. To explore the localization of cellular molecules in detail, we devised an efficient and convenient method to prepare semithin and ultrathin cryosections of cells cultured on permeable support. Multiple specimens were piled up to obtain many vertical images of cells in a single section. Freezing them in cooled n-hexane resulted in clear and homogeneous frozen specimens, from which semithin and ultrathin cryosections of epitherial cells on permeable support were easily obtained. With this method, it is possible to prepare many cryosections of good quality for histochemical examination of vertical images of cells.
- Published
- 2003
35. Introduction and Expression of Glucose Transporters in Pancreatic Acinar Cells by In Vivo Electroporation
- Author
-
Minako Yokoo-Sugawara, Takeo Aoki, Takeshi Suzuki, Haruo Hagiwara, Hiroyuki Kuwano, Kuniaki Takata, Yasuo Shinoda, and Toshiyuki Matsuzaki
- Subjects
Histology ,biology ,Physiology ,Electroporation ,Glucose transporter ,Cell Biology ,Apical membrane ,Biochemistry ,Molecular biology ,Pathology and Forensic Medicine ,Cell biology ,Membrane protein ,Zymogen ,biology.protein ,GLUT1 ,GLUT3 ,Epithelial polarity - Abstract
cDNAs encoding facilitated-diffusion glucose transporters GULT1, GULT3, GULT4, and GULT5 were introduced into the rat pancreas by in-vivo electroporation method, and their expression and localization in pancreatic acinar cells were examined immunohistochemically. GLUT1 was localized at the basolateral membrane, whereas GLUT3 and GLUTS were at the apical membrane. Restriction of GLUT3 and GLUTS to the apical membrane makes a marked contrast to their localization to the entire plasma membrane when expressed in hepatocytes in situ. Such differential localization may be due to differential apical targeting mechanism: direct targeting in acinar cells and indirect transcytotic delivery in hepatocytes in situ. GLUT4 was present in the membrane of zymogen secretory granules in the cytoplasm in acinar cells. This observation suggests that GLUT4 is segregated to the regulated secretory pathway. Expression of glucose transporters in situ is a useful method in analyzing the targeting mechanism of membrane proteins in cells of tissues in situ.
- Published
- 2003
36. Immunolocalization of the water channel, aquaporin-5 (AQP5), in the rat digestive system
- Author
-
Haruo Hagiwara, Toshiyuki Matsuzaki, Yuki Tajika, Kuniaki Takata, Takeshi Suzuki, and Takeo Aoki
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Histology ,Duodenum ,Biology ,Aquaporins ,Occludin ,Exocytosis ,Tongue ,stomatognathic system ,medicine ,Animals ,Tissue Distribution ,Pyloric region ,Rats, Wistar ,Salivary gland ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Membrane ,Myoepithelial cell ,Membrane Proteins ,Water ,Biological Transport ,Apical membrane ,Immunohistochemistry ,Submandibular gland ,Aquaporin 5 ,Protein Structure, Tertiary ,Rats ,Cell biology ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Gastric Mucosa ,Adenomere ,Digestive System - Abstract
Aquaporin-5 (AQP5), an isoform of membrane water channel aquaporins, is expressed in the salivary and lacrimal glands. We surveyed the expression and immunohistochemical localization of AQP5 in the rat digestive system. RT-PCR analysis revealed that AQP5 is expressed in the submandibular gland, tongue, gastric corpus, pyloric region, duodenum, and liver. Immunofluorescence microscopy using AQP5-specific antibodies showed that AQP5 protein is present in the minor salivary glands of the tongue, the pyloric glands, and duodenal glands. To distinguish apical and basolateral domains of the plasma membrane of epithelial cells, double-immunofluorescence staining for AQP5 and tight junction protein occludin was performed. In the minor salivary gland, AQP5 was present in both the serous and mixed secretory end portions. AQP5 was found in the apical membrane of the secretory cells including intercellular secretory canaliculi demarcated with occludin. At higher magnifications, omega-shaped indentations of AQP5 labeling were seen along the apical membrane, suggesting a dynamic process for the apical membrane in exocytosis. Only weak labeling for AQP5 was detected in the basolateral domain. In the stomach, AQP5 was detected in the apical membrane of the pyloric gland secretory cells. In the duodenum, AQP5 was restricted to duodenal glands, where it was localized to the apical membrane. AQP5 was not detected in the intestinal glands or cells in the villi. These observations show that AQP5 is localized mainly in the apical membrane, including intercellular secretory canaliculi of secretory cells in the minor salivary glands, pyloric glands, and duodenal glands. AQP5 appears to play an important role in water transfer in these glands.
- Published
- 2003
37. A Simple Electroporation Method for the Introduction of Plasmids into Cells Cultured on Coverslips for Histochemical Examination
- Author
-
Yuki Tajika, Takeo Aoki, Takeshi Suzuki, Yukiko Takahashi, Haruo Hagiwara, Toshiyuki Matsuzaki, and Kuniaki Takata
- Subjects
Histology ,biology ,Physiology ,Electroporation ,Cell Biology ,Transfection ,biology.organism_classification ,Biochemistry ,Molecular biology ,Pathology and Forensic Medicine ,Green fluorescent protein ,Cell biology ,HeLa ,Plasmid ,Plasmid dna - Abstract
Expression of genes followed by the examination of the localization of their products by histochemical methods is one of important and powerful approaches to elucidate the role of genes of interest. We show here a simple and versatile electroporation method for the introduction of plasmids into cells cultured on coverslips for histochemical examination. Electroporation apparatus was assembled using a 35-mm plastic dish and aluminum wire. The apparatus was placed over the coverslip and plasmid DNA was applied onto it. Green fluorescent protein (GFP) was used as a reporter of transfection. Under the appropriate settings of voltage, duration, and pulse numbers, successful transfection was easily achieved in 3T3-L1 fibroblasts, HeLa cells, Cos7 cells, and MDCK cells. The electroporation method described here is simple, easy, rapid, and economical, and may be best suited for the screening of many genes for their histochemical examination.
- Published
- 2003
38. Aquaporins: a water channel family
- Author
-
Nomingerel Tserentsoodol, Toshiyuki Matsuzaki, Yuki Tajika, Haruo Hagiwara, Takeshi Suzuki, Kuniaki Takata, and Takeo Aoki
- Subjects
Gene isoform ,Chemistry ,Major intrinsic proteins ,Water ,Aquaporin ,General Medicine ,Aquaporins ,Rats ,Cell biology ,Aquaporin 4 ,Membrane ,Water channel ,Cytoplasm ,Animals ,Humans ,Protein Isoforms ,Anatomy ,Integral membrane protein - Abstract
Water channel proteins, aquaporins, are integral membrane proteins serving in the permeation of water and some other small molecules. Eleven isoforms of aquaporins have been identified from various tissues to date. They are expressed in tissue- and cell-specific manners, and are closely related to the specific functions of tissues and cells. Aquaporins are usually localized to the plasma membrane. Some isoforms are present in cytoplasmic compartments, and their translocation to the plasma membrane is crucial in the regulation of water transfer. This review focuses on the localization of aquaporins in mammalian tissues and discusses the physiological importance of water channels.
- Published
- 2002
39. Marked cortisol production by intracrine ACTH in GIP-treated cultured adrenal cells in which the GIP receptor was exogenously introduced
- Author
-
Kousuke Uchida, Haruo Hagiwara, Hiroko Okinaga, Masayoshi Iizuka, Takao Susa, Mimi Tamamori-Adachi, Yuji Tanaka, Hiroko Fujii, Takashi Nakakura, and Tomoki Okazaki
- Subjects
medicine.medical_specialty ,endocrine system ,Pro-Opiomelanocortin ,Hydrocortisone ,lcsh:Medicine ,Adrenocorticotropic hormone ,Gastric Inhibitory Polypeptide ,Biology ,Cell Line ,Receptors, Gastrointestinal Hormone ,Endocrinology ,Proopiomelanocortin ,Adrenocorticotropic Hormone ,Internal medicine ,Adrenal Glands ,medicine ,Medicine and Health Sciences ,Humans ,ACTH receptor ,RNA, Messenger ,RNA, Small Interfering ,Receptor ,lcsh:Science ,Cyclic GMP ,Molecular Biology ,Multidisciplinary ,Adrenal gland ,Cholesterol side-chain cleavage enzyme ,lcsh:R ,Colforsin ,Steroid 17-alpha-Hydroxylase ,Biology and Life Sciences ,Cell Biology ,Molecular biology ,Up-Regulation ,medicine.anatomical_structure ,Glucocorticoid secretion ,Cell culture ,biology.protein ,lcsh:Q ,RNA Interference ,Steroid 21-Hydroxylase ,hormones, hormone substitutes, and hormone antagonists ,Research Article - Abstract
The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing’s syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (–) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.
- Published
- 2014
40. The elongation of primary cilia via the acetylation of α-tubulin by the treatment with lithium chloride in human fibroblast KD cells
- Author
-
Hideyuki Tanaka, Takashi Nakakura, Haruo Hagiwara, Anshin Asano-Hoshino, Kazuhiro Kawai, Kenjiro Arisawa, Takeshi Suzuki, Yoshiko Kiuchi, and Yoshihisa Sekino
- Subjects
Time Factors ,Cell ,Blotting, Western ,macromolecular substances ,Biology ,Pathology and Forensic Medicine ,Cell Line ,chemistry.chemical_compound ,Glycogen Synthase Kinase 3 ,Microscopy, Electron, Transmission ,Acetyltransferases ,Tubulin ,Organelle ,medicine ,Humans ,Cilia ,Phosphorylation ,Fibroblast ,Molecular Biology ,GSK3B ,Glycogen Synthase Kinase 3 beta ,Dose-Response Relationship, Drug ,Reverse Transcriptase Polymerase Chain Reaction ,Cilium ,Acetylation ,General Medicine ,Fibroblasts ,Cell biology ,medicine.anatomical_structure ,chemistry ,Microscopy, Fluorescence ,Cell culture ,Lithium chloride ,RNA Interference ,Lithium Chloride ,Adenylyl Cyclases - Abstract
Primary cilium, an organelle found on nearly every cell in the human body, typically serves as the mechanical sensor of the cell. Lithium ion is known to promote the elongation of primary cilia in a variety of cell types, but it is unknown whether lithium is involved in the acetylation of α-tubulin which is essential for the assembly of primary cilia. In order to reveal the relationship between the elongation of primary cilia with lithium and the acetylation of α-tubulin, we first observed the formation and structure of primary cilia in KD cells, a cell line deriving fibroblasts in human labium. Subsequently, by immunohistochemical and western blot analysis we elucidated that the length of primary cilia and acetylation of α-tubulin are regulated by lithium chloride (LiCl) in the medium in a time- and concentration-dependent manner. We next performed the RT-PCR, RNAi-based experiments and biochemical study using an inhibitor of glycogen synthase kinase-3βGSK-3β). We found that LiCl mobilizes the α-tubulin N-acetyltransferase 1 (αTAT1) in the signaling pathway mediating GSK-3β and adenylate cyclase III. In conclusion, our results suggested that LiCl treatments activate αTAT1 by the inhibition of GSK-3β and promote the α-tubulin acetylation, and then elongate the primary cilia.
- Published
- 2014
41. [Untitled]
- Author
-
Kuniaki Takata, Kenji Mogi, Haruo Hagiwara, and Akio Kano
- Subjects
Cell type ,Centriole ,Cilium ,Myoepithelial cell ,Striated duct ,Cell Biology ,Anatomy ,Columnar Cell ,Biology ,Submandibular gland ,medicine.anatomical_structure ,medicine ,Apical cytoplasm - Abstract
Using an antibody specific to striated rootlets, we investigated the immuolocalization of striated rootlets in cells constituting human submandibular glands. Striated rootlets were positively stained in all cell types constituting acini, intercalated ducts, striated ducts, and interlobular ducts, but their shapes were different. The mean lengths of striated rootlets were 1.46 ± 0.49, 3.15 ± 1.35 and 3.99 ± 1.02 μm in acinar secretory cells, myoepithelial cells, and columnar cells of the striated duct, respectively. The rootlets were the longest in columnar cells of the striated duct, in which paired centrioles were located in the apical cytoplasm away from nuclei. These findings suggest that striated rootlets play important roles in the positioning of centrioles in the cell. 2–8% of striated rootlets in myoepithelial cells were associated with solitary cilia, but they were not associated with solitary cilia in acinar cells and columnar cells of the striated duct. These observations suggest that striated rootlets may be associated with centrioles under normal physiological conditions, without formation of solitary cilia.
- Published
- 2001
42. Immunocytochemistry of the striated rootlets associated with solitary cilia in human oviductal secretory cells
- Author
-
Takeo Aoki, Akio Kano, Haruo Hagiwara, and Nobuo Ohwada
- Subjects
Histology ,medicine.drug_class ,Blotting, Western ,Immunocytochemistry ,Apical cell ,Biology ,Monoclonal antibody ,medicine ,Animals ,Humans ,Cilia ,Fluorescent Antibody Technique, Indirect ,Microscopy, Immunoelectron ,Molecular Biology ,Fallopian Tubes ,Cilium ,Antibodies, Monoclonal ,Membrane Proteins ,Cell Biology ,Anatomy ,Immunohistochemistry ,Epithelium ,Rats ,Cell biology ,Medical Laboratory Technology ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Microscopy, Electron, Scanning ,Oviduct ,Female ,Clone (B-cell biology) ,Developmental biology - Abstract
The human oviduct epithelium is a simple columnar structure that consists primarily of ciliated and secretory cells. Solitary cilia usually extend from the apical cell surface of secretory cells. By injecting crude preparations of striated rootlets into rats, we successfully obtained six monoclonal antibodies (R38, R67, R95, R149, R155, R213) that commonly labeled ciliary rootlets. Using these antibodies, proteins of 205-215 kDa were identified by immunoblotting. Using a clone, R67, we investigated the morphology of the striated rootlets associated with solitary cilia by immunocytochemistry. It was found that the shapes of the rootlets were not simple but varied considerably. The rootlets had branched, radiated, arched, and looped shapes. This is the first report of the rootlets having a variety of shapes. The 205- to 215-kDa antigens identified by the six different antibodies were mostly localized to dark bands of striations, suggesting that they are constitutive components of dark striations of the rootlet.
- Published
- 2000
43. A 190-kDa Antigen Is Present Around Lipid Droplets of Rat Pulmonary Lipofibroblasts
- Author
-
Takeo Aoki, Seu-Mei Wang, Toyoshi Fujimoto, and Haruo Hagiwara
- Subjects
Histology ,Colcemid ,Physiology ,Vimentin ,macromolecular substances ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,Epitope ,Pathology and Forensic Medicine ,Cell biology ,chemistry.chemical_compound ,chemistry ,Cytoplasm ,Lipid droplet ,biology.protein ,Cytoskeleton ,Cytochalasin B ,Actin - Abstract
By using a monoclonal antibody (mAb A2) raised against the cytoskeleton of goldfish xanthophores, we found that a 190−kDa protein is localized along the rim of lipid droplets in rat pulmonary lipofibroblasts (lung septal cells). By immunofluorescence microscopy of frozen sections, we observed mAb A2 bound to the rim of lipid droplets in the lipofibroblast in vivo. The circular labeling around lipid droplets as well as filamentous labeling in the cytoplasm was observed in lipofibroblasts in culture. In cells cultured for several days, the number and size of lipid droplets decreased and the circular labeling became indistinct, whereas the filamentous labeling persisted. In Western blotting, mAb A2 recognized 190−kDa and 58−kDa bands in the cultured lipofibroblast; the latter was found to be vimentin. Treatment of lipofibroblasts for 4hr with Colcemid extinguished the labeling around lipid droplets, and only the coarse filaments that matched the anti−vimentin labeling remained. Interestingly, after more than 12hr of Colcemid treatment, the circular labeling reappeared around lipid droplets. The disappearance of the circular labeling also occurred when lipofibroblasts were treated with Taxol or cytochalasin B for more than 4hr. From Western blotting, this mAb A2 appears to recognize a common epitope shared by vimentin and the 190−kDa antigen. The result indicates that the 190−kDa protein recognized by mAb A2 is localized around lipid droplets of lipofibroblasts and that this localization may be related to the cytoskeleton.
- Published
- 2000
44. A Possible Mechanism of Primary Ciliary Dyskinesia. A Case of a Segmental Defect in Ciliary Microtubules
- Author
-
Masatomo Mori, Kunio Dobashi, Sumiyasu Ishii, Mayumi Yonezu, Kunihiko Iizuka, Toshio Fukusato, Haruo Hagiwara, Kenju Shimomura, and Koichi Minato
- Subjects
Pathology ,medicine.medical_specialty ,Adolescent ,Microtubules ,Internal Medicine ,medicine ,Humans ,Basal body ,Cilia ,Sinusitis ,Bronchitis ,Radionuclide Imaging ,Primary ciliary dyskinesia ,business.industry ,Cilium ,Respiratory disease ,Technetium ,General Medicine ,Anatomy ,respiratory system ,medicine.disease ,Bronchiectasis ,Microscopy, Electron ,Dyskinesia ,Sputum ,Female ,medicine.symptom ,business ,Ciliary Motility Disorders - Abstract
We report here a 13-year-old woman with cough, sputum and fever. The patient had both chronic sinusitis and bronchitis. Chest X-ray and computed tomographic scan of the chest revealed mucous bronchial filling and bronchiectasia in bronchi of bilateral lower lobes, right middle lobe and left upper lobe. Aerosol inhalation scintigraphy with 99mTechnetium demonstrated delays of the discharged tracer. On the basis of these findings, primary ciliary dyskinesia was suggested. This was confirmed by the findings from nasal biopsy with transmission electron microscopy where all of the microtubules were segmentally defected near the basal body in the cilia. On the basis of these findings, we diagnosed the patient with primary ciliary dyskinesia which may be due, at least in part, to segmental defect of ciliary microtubules.
- Published
- 1999
45. Review Caveolae: from a morphological point of view
- Author
-
Hiroshi Kogo, Ryuji Nomura, Takeo Aoki, Toyoshi Fujimoto, and Haruo Hagiwara
- Subjects
Membrane ,Chemistry ,Caveolae ,medicine ,Compartment (development) ,medicine.symptom ,Instrumentation ,Function (biology) ,Confusion ,Cell biology - Abstract
Caveolae in the plasma membrane have been a focus of intensive research during the past several years. There has been confusion concerning caveolae and caveola-like membrane domains, but it is now generally thought that the latter is a region distinct from caveolae. However, due to similar buoyancy of caveolae and caveola-like membranes, whether caveolae in situ are enriched with a given molecule is often difficult to be concluded by biochemical techniques alone. Furthermore, relatively shallow caveolae may be detected by some techniques, but not by others. Thus whether a molecule is enriched in caveolae should be confirmed by methods based on different principles. Among many putative caveolar molecules, those related to Ca2+ influx and extrusion were shown to be concentrated in caveolae by both immunocytochemical and biochemical techniques. In conjunction with other characteristics, the result implies that caveolae may function as a mobile compartment for Ca2+ signalling.
- Published
- 1998
46. Development of striated rootlets during ciliogenesis in the human oviduct epithelium
- Author
-
Toyoshi Fujimoto, Nobuo Ohwada, Takeo Aoki, and Haruo Hagiwara
- Subjects
Adult ,Periodicity ,Histology ,Biology ,Pathology and Forensic Medicine ,law.invention ,law ,Ciliogenesis ,Morphogenesis ,Humans ,Basal body ,Cilia ,Fallopian Tubes ,Budding ,Cilium ,Epithelial Cells ,Cell Biology ,Anatomy ,Middle Aged ,Cell biology ,Microscopy, Electron ,Ultrastructure ,Oviduct ,Female ,Electron microscope ,Striation - Abstract
Striated rootlets in ciliated cells are conical banded structures composed of longitudinally aligned filaments. The formation of striated rootlets during ciliognesis in the human oviduct epithelium was studied by electron microscopy. Primitive rootlets appeared at the proximal side of basal bodies before or at the same time as ciliary budding. After the formation of several striations, the tip of the rootlets extended deeply toward the interior of the cell and became differentiated into two distinct parts, viz., the proximal conical part connected to the basal body and the distal fibrillar part. The periodicity of the striations in the fibrillar part was 68. 5+/-2.95 nm, about 5 nm longer than that of the conical part (63. 9+/-2.25 nm). The dark band in the striation was thicker in the fibrillar part than in the conical part. Since the fibrillar part was not observed in the mature cilium, this part was considered as being either degraded or changed into the conical part during ciliogenesis.
- Published
- 1997
47. Peculiar Distribution of Fodrin in Fat-Storing Cells
- Author
-
Toyoshi Fujimoto, Takeo Aoki, and Haruo Hagiwara
- Subjects
Male ,Cytoplasm ,Immunoelectron microscopy ,Vimentin ,Lipid droplet ,Cell cortex ,Adipocytes ,Animals ,Humans ,Rats, Wistar ,Microscopy, Immunoelectron ,Cells, Cultured ,Actin ,biology ,Microfilament Proteins ,Cell Biology ,Fibroblasts ,Immunohistochemistry ,Lipids ,Rats ,Cell biology ,Cytoskeletal Proteins ,stomatognathic diseases ,Tubulin ,Liver ,Microscopy, Fluorescence ,biology.protein ,Desmin ,Carrier Proteins - Abstract
Fat-storing cells (FSCs) show unique morphology containing many lipid droplets in the cytoplasm. In this study, we found that a membrane skeletal protein, fodrin, shows peculiar distribution in FSCs of rat liver. By immunofluorescence microscopy of FSCs in culture, intense labeling for fodrin was seen as coarse filaments in the cytoplasm. Especially in FSCs isolated from vitamin A-treated rats, the labeling was often seen as many small rings in the cytoplasm. In contrast, labeling for fodrin in human fibroblasts or rat adipocytes in culture was seen diffusely in the cell cortex. Distribution of actin, tubulin, vimentin, and desmin in FSCs was also examined, but none of them appeared correlated with fodrin. By immunoelectron microscopy using nanogold labeling with silver enhancement, positive labeling for fodrin was seen around some lipid droplets in FSCsin vivo.We assume that the peculiar distribution of fodrin may be related to the morphological characteristics of FSCs.
- Published
- 1997
48. Ultrastructural observation on 'transitional tubules' in human oviductal ciliogenic cells
- Author
-
Toyoshi Fujimoto, Takeo Aoki, and Haruo Hagiwara
- Subjects
Adult ,Histology ,Biology ,Epithelium ,Ciliogenesis ,medicine ,Humans ,Basal body ,Cilia ,Apical cytoplasm ,Molecular Biology ,Fallopian Tubes ,Ecology, Evolution, Behavior and Systematics ,Cilium ,Cell Biology ,Anatomy ,Middle Aged ,Cell biology ,Microscopy, Electron ,Tubule ,medicine.anatomical_structure ,Ultrastructure ,Oviduct ,Female ,Research Article ,Developmental Biology - Abstract
In the human oviduct epithelium during ciliogenesis, short tubular structures were found in the transitional zone between the basal body and cilium. The tubules, termed transitional tubules from their location, were 34-36 nm in diameter and 0.13 +/- 0.06 micron in length; the number around a basal body was variable, but usually 4-6. The cytoplasmic leaflets of the tubule membranes were coated by electron-dense material and appeared to be connected to alar sheets. The transitional tubules existed transiently during ciliogenesis. The exact role of transitional tubules is unknown, but considering their location, they may fix the basal body in the apical cytoplasm during ciliary elongation and/or may be related to formation of alar sheets.
- Published
- 1997
49. Variously-formed inclusions in the rough endoplasmic reticulum of plasma cells in the human oviduct mucosa
- Author
-
Haruo Hagiwara, Susumu Shibasaki, and Nobuo Ohwada
- Subjects
Lamina propria ,Pathology ,medicine.medical_specialty ,Endoplasmic reticulum ,Russell bodies ,nutritional and metabolic diseases ,Ovary ,General Medicine ,Plasma cell ,Biology ,Cisterna ,Inclusion bodies ,Pathology and Forensic Medicine ,medicine.anatomical_structure ,medicine ,Oviduct ,Anatomy ,Molecular Biology - Abstract
Human oviducts, obtained from 60 women having undergone total hysterectomy due to diseases of the uterus or ovary, were studied for cyclic changes by electron microscopy. In the studies, plasma cells were frequently observed in the lamina propria of the oviductal mucosa. Russell bodies were occasionally encountered in the plasma cells. In the oviduct mucosa obtained from a patient with cervical carcinoma, a plasma cell contained C-shaped or worm-like structures in an extensively-distended cisterna of the rER. These structures were morphologically quite similar to type A curvilinear bodies in lysosomes of certain lysosomal diseases. Additionally, intracisternal tubules were noted in slightly dilated cisternae of other rER. The origins and functional significance of curvilinear inclusions and intracisternal tubules in the plasma cell remain obscure.
- Published
- 1995
50. Electron Microscopic Observation of the Transitional Portion in the Human Eccrine Sweat Gland
- Author
-
Haruo HAGIWARA and Susumu SHIBASAKI
- Subjects
Adult ,Histology ,Chemistry ,Myoepithelial cell ,Columnar Cell ,Anatomy ,Eccrine Glands ,Middle Aged ,Epithelium ,Microscopy, Electron ,medicine.anatomical_structure ,Excretory system ,Cytoplasm ,Ultrastructure ,medicine ,Humans ,Female ,Eccrine sweat gland ,Mitosis ,Duct (anatomy) - Abstract
The transitional portion in human eccrine sweat glands was observed by transmission electron microscope (TEM). The transitional portion, consisting of columnar epithelial cells (columnar cells) and basal cells, formed a very short segment about 25 to 50 microns in length. The portion was abruptly connected to the secretory segment, and shifted to the excretory duct without marked ultrastructural changes. The columnar cells were morphologically characterized by small apical vesicles as reported in previous reports. Considering the frequency of the mitosis of the columnar cells, the transitional portion seemed to be one of the areas of cell proliferation in the gland. Basal cells were sparse in the region connecting the secretory segment, but dense near the duct. Basal cells in the secretory segment side of the transitional portion, containing thin microfilament-bundles in the infranuclear cytoplasm, were considered to be immature myoepithelial cells. On its ductal side, however, the basal cells showed morphological profiles similar to ductal peripheral cells. These findings suggest that the differentiation of myoepithelial cells occurs in the transitional portion of eccrine sweat glands.
- Published
- 1994
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