86 results on '"Saito, K."'
Search Results
2. Effects of nutrition on the cell survival and gene expression of transplanted fat tissues in mice
- Author
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Matsumoto, F, primary, Bujo, H, additional, Kuramochi, D, additional, Saito, K, additional, Shibasaki, M, additional, Takahashi, K, additional, Yoshimoto, S, additional, Ichinose, M, additional, and Saito, Y, additional
- Published
- 2002
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3. Such Hydrophobic Peptides as Dansylated Mastoparan Can Elevate the Fertilization Membrane of Sea Urchin Eggs
- Author
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Saito, K., primary, Imoto, M., additional, Nagano, N., additional, Toriyama, M., additional, and Nakajima, T., additional
- Published
- 1995
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4. Conversion of D-Arabinose to D-Erythroascorbic Acid and Oxalic Acid in Sclerotinia sclerotiorum
- Author
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Loewus, F.A., primary, Saito, K., additional, Suto, R.K., additional, and Maring, E., additional
- Published
- 1995
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5. Evidence for Identity of β-Pyrazolealanine Synthase with Cysteine Synthase in Watermelon: Formation of β-Pyrazolealanine by Cloned Cysteine Synthase in Vitro and in Vivo
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Noji, M., primary, Murakoshi, I., additional, and Saito, K., additional
- Published
- 1993
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6. Molecular Characterization of the Gene Operon of Heat Shock Proteins HSP60 and HSP10 in Methicillin-Resistant Staphylococcus aureus
- Author
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Ohta, T., primary, Honda, K., additional, Kuroda, M., additional, Saito, K., additional, and Hayashi, H., additional
- Published
- 1993
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7. Heat Shock Promoter of Thermophilic Chaperonin Operon
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Ohta, T., primary, Honda, K., additional, Saito, K., additional, Hayashi, H., additional, Tano, H., additional, Hamamoto, T., additional, and Kagawa, Y., additional
- Published
- 1993
- Full Text
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8. Evidence for Identity of β-Pyrazolealanine Synthase with Cysteine Synthase in Watermelon: Formation of β-Pyrazolealanine by Cloned Cysteine Synthase in Vitroand in Vivo
- Author
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Noji, M., Murakoshi, I., and Saito, K.
- Abstract
The responsibility of cysteine synthase (EC 4.2.99.8) from watermelon (Citrullus vulgaris) for the formation of β-(pyrazole-1-yl)-L-alanine, a non-protein amino acid specifically accumulated in Curcubitaceae plants, was confirmed in vitroand in vivoby the cloned cDNA on expression vectors, pCCS11 and pCEN1. The cDNA sequence derived from pCCS11, an expression vector driven by the lacZpromoter, was placed under the transcriptional control of strong T7 promoter of pET3d to yield an over-expression vector, pCEN1, in Escherichia coli. The concentration of the exogenous cysteine synthase protein was increased up to approximately 10% of the total soluble protein of E. colicells by the expression of cDNA on pCEN1. β-(Pyrazole-1-yl)-L-alanine was formed in vitrofrom O-acetyl-L-serine and pyrazole by the action of cysteine synthase expressed in E. colicarrying pCCS11 or pCEN1. To confirm the responsibility of cysteine synthase for the formation of β-(pyrazole-1-yl)-L-alanine in vivo, the feeding experiments of pyrazole and serine or O-acetyl-L-serine were carried out using the transformed E. coliculture. β-(Pyrazole-1-yl)-L-alanine was produced in vivoby feeding the substrates to the culture of E. colicarrying pCEN1. These results provide the confirming evidence that the cloned cysteine synthase of watermelon catalyzes the formation of β-(pyrazole-1-yI)-L-alanine, indicating that β-pyrazolealanine synthase is identical with cysteine synthase in Cucurbitaceae plants.
- Published
- 1993
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9. The crystal and molecular structure of cytosine-glycyl-glycine-copper(II) complex, a biologically important ternary coordination complex
- Author
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Saito, K., primary, Terashima, R., additional, Sakaki, T., additional, and Tomita, K., additional
- Published
- 1974
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10. Roles of TRPM4 in immune responses in keratinocytes and identification of a novel TRPM4-activating agent.
- Author
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Otsuka Saito K, Fujita F, Toriyama M, Utami RA, Guo Z, Murakami M, Kato H, Suzuki Y, Okada F, Tominaga M, and Ishii KJ
- Subjects
- Humans, HEK293 Cells, Keratinocytes metabolism, Cytokines metabolism, Tumor Necrosis Factor-alpha metabolism, Immunity, Dermatitis, TRPM Cation Channels metabolism
- Abstract
The skin is a protective interface between the internal organs and environment and functions not only as a physical barrier but also as an immune organ. However, the immune system in the skin is not fully understood. A member of the thermo-sensitive transient receptor potential (TRP) channel family, TRPM4, which acts as a regulatory receptor in immune cells, was recently reported to be expressed in human skin and keratinocytes. However, the function of TRPM4 in immune responses in keratinocytes has not been investigated. In this study, we found that treatment with BTP2, a known TRPM4 agonist, reduced cytokine production induced by tumor necrosis factor (TNF) α in normal human epidermal keratinocytes and in immortalized human epidermal keratinocytes (HaCaT cells). This cytokine-reducing effect was not observed in TRPM4-deficient HaCaT cells, indicating that TRPM4 contributed to the control of cytokine production in keratinocytes. Furthermore, we identified aluminum potassium sulfate, as a new TRPM4 activating agent. Aluminum potassium sulfate reduced Ca
2+ influx by store-operated Ca2+ entry in human TRPM4-expressing HEK293T cells. We further confirmed that aluminum potassium sulfate evoked TRPM4-mediated currents, showing direct evidence for TRPM4 activation. Moreover, treatment with aluminum potassium sulfate reduced cytokine expression induced by TNFα in HaCaT cells. Taken together, our data suggested that TRPM4 may serve as a new target for the treatment of skin inflammatory reactions by suppressing the cytokine production in keratinocytes, and aluminum potassium sulfate is a useful ingredient to prevent undesirable skin inflammation through TRPM4 activation., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)- Published
- 2023
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11. MTR4 adaptor PICT1 functions in two distinct steps during pre-rRNA processing.
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Miyao S, Saito K, Oshima R, Kawahara K, and Nagahama M
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- Humans, Exosome Multienzyme Ribonuclease Complex genetics, Nuclear Proteins, Oligonucleotides, RNA Processing, Post-Transcriptional, RNA, Ribosomal, 5.8S, RNA, Small Interfering, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, RNA Precursors genetics, RNA Helicases genetics, Tumor Suppressor Proteins genetics
- Abstract
Ribosome biogenesis proceeds with the successive cleavage and trimming of the large 47S rRNA precursor, where the RNA exosome plays major roles in concert with the Ski2-like RNA helicase, MTR4. The recent finding of a consensus amino acid sequence, the arch-interacting motif (AIM), for binding to the arch domain in MTR4 suggests that recruitment of the RNA processing machinery to the maturing pre-rRNA at appropriate places and timings is mediated by several adaptor proteins possessing the AIM sequence. In yeast Saccharomyces cerevisiae, Nop53 plays such a role in the maturation of the 3'-end of 5.8S rRNA. Here, we investigated the functions of PICT1 (also known as GLTSCR2 or NOP53), a mammalian ortholog of Nop53, during ribosome biogenesis in human cells. PICT1 interacted with MTR4 and exosome in an AIM-dependent manner. Overexpression of PICT1 mutants defecting AIM sequence and siRNA-mediated depletion of PICT1 showed that PICT1 is involved in two distinct pre-rRNA processing steps during the generation of 60S ribosomes; first step is the early cleavage of 32S intermediate RNA, while the second step is the late maturation of 12S precursor into 5.8S rRNA. The recruitment of MTR4 and RNA exosome via the AIM sequence was required only during the late processing step. Although, the depletion of MTR4 and PICT1 induced stabilization of the tumor suppressor p53 protein in cancer cell lines, the depletion of the exosome catalytic subunits, RRP6 and DIS3, did not exert such an effect. These results suggest that recruitment of the RNA processing machinery to the 3'-end of pre-5.8S rRNA may be involved in the induction of the nucleolar stress response, but the pre-rRNA processing capabilities themselves were not involved in this process., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Masami Nagahama reports financial support was provided by Japan Society for the Promotion of Science., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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12. Deficiency of kynurenine 3-monooxygenase exacerbates impairment of prepulse inhibition induced by phencyclidine.
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Kubota H, Kunisawa K, Niijima M, Hirakawa M, Mori Y, Hasegawa M, Fujigaki S, Fujigaki H, Yamamoto Y, Saito K, Nabeshima T, and Mouri A
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- Animals, Kynurenic Acid metabolism, Mice, Mice, Inbred C57BL, Phencyclidine, Prepulse Inhibition, Quinolinic Acid metabolism, RNA, Messenger, Kynurenine metabolism, Kynurenine 3-Monooxygenase genetics, Kynurenine 3-Monooxygenase metabolism
- Abstract
Phencyclidine (PCP) causes mental symptoms that closely resemble schizophrenia through the inhibition of the glutamatergic system. The kynurenine (KYN) pathway (KP) generates metabolites that modulate glutamatergic systems such as kynurenic acid (KA), quinolinic acid (QA), and xanthurenic acid (XA). Kynurenine 3-monooxygenase (KMO) metabolizes KYN to 3-hydroxykynurenine (3-HK), an upstream metabolite of QA and XA. Clinical studies have reported lower KMO mRNA and higher KA levels in the postmortem brains of patients with schizophrenia and exacerbation of symptoms in schizophrenia by PCP. However, the association between KMO deficiency and PCP remains elusive. Here, we demonstrated that a non-effective dose of PCP induced impairment of prepulse inhibition (PPI) in KMO KO mice. KA levels were increased in the prefrontal cortex (PFC) and hippocampus (HIP) of KMO KO mice, but 3-HK levels were decreased. In wild-type C57BL/6 N mice, the PPI impairment induced by PCP is exacerbated by KA, while attenuated by 3-HK, QA and XA. Taken together, KMO KO mice were vulnerable to the PPI impairment induced by PCP through an increase in KA and a decrease in 3-HK, suggesting that an increase in the ratio of KA to 3-HK (QA and XA) may play an important role in the pathophysiology of schizophrenia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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13. A novel carotenoid biosynthetic route via oxidosqualene.
- Author
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Otani Y, Maoka T, Kawai-Noma S, Saito K, and Umeno D
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Carotenoids chemistry, Escherichia coli genetics, Farnesyl-Diphosphate Farnesyltransferase genetics, Farnesyl-Diphosphate Farnesyltransferase metabolism, Microorganisms, Genetically-Modified, Oxidoreductases genetics, Oxidoreductases metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Squalene metabolism, Squalene Monooxygenase genetics, Squalene Monooxygenase metabolism, Biosynthetic Pathways physiology, Carotenoids metabolism, Escherichia coli metabolism, Squalene analogs & derivatives
- Abstract
Over 800 known carotenoids are synthesized from phytoene or 4,4'-diapophytoene (dehydrosqualene) characterized by three conjugated double bonds. In this paper, we report that carotenoid desaturase CrtN from Staphylococcus aureus and Methylomonas can accept oxidosqualene, which is the precursor for plant- or animal-type triterpenoids, yielding the yellow carotenoid pigments with 8, 9, or 10 conjugated double bonds. The resulting pathway is the second nonnatural route for carotenoid pigments and the first pathway for carotenoid pigments not biosynthesized via (diapo)phytoene., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Daisuke Umeno reports financial support was provided by Government of Japan Ministry of Education Culture Sports Science and Technology. Yusuke Otani reports financial support was provided by Government of Japan Ministry of Education Culture Sports Science and Technology., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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14. Effects of oxytocin on responses to nociceptive and non-nociceptive stimulation in the upper central nervous system.
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Saito H, Hidema S, Otsuka A, Suzuki J, Kumagai M, Kanaya A, Murakami T, Takei Y, Saito K, Sugino S, Toyama H, Saito R, Tominaga T, Nishimori K, and Yamauchi M
- Subjects
- Animals, Central Nervous System metabolism, Male, Mice, Mice, Inbred C57BL, Central Nervous System drug effects, Nociception drug effects, Oxytocin pharmacology
- Abstract
Oxytocin is known as a social bonding hormone, but it also functions as an anxiolytic or analgesic neurotransmitter. When oxytocin regulates pain or anxiousness centrally as a neurotransmitter, it is secreted by neurons and directly projected to targeted regions. Although the function of oxytocin at the spinal level is well studied, its effects at the supraspinal level are poorly understood. We aimed to investigate the effect of oxytocin at the supraspinal level in vivo using C57BL/6J (wild-type [WT]), oxytocin-deficient (Oxt
-/- ), oxytocin receptor-deficient (Oxtr-/- ), and oxytocin receptor-Venus (OxtrVenus/+ ) mice lines. Response thresholds in Oxtr-/- mice in Hargreaves and von-Frey tests were significantly lower than those in WT mice, whereas open field and light/dark tests showed no significant differences. Moreover, response thresholds in Oxt-/- mice were raised to those in WT mice after oxytocin administration. Following the Hargreaves test, we observed the co-localisation of c-fos with Venus or the oxytocin receptor in the periaqueductal gray (PAG), medial amygdala (MeA), and nucleus accumbens (NAc) regions in OxtrVenus/+ mice. Furthermore, in the PAG, MeA, and NAc regions, the co-localisation of oxytocin with c-fos and gamma-aminobutyric acid was much stronger in Oxtr-/- mice than in WT mice. However, following von-Frey test, the same findings were observed only in the MeA and NAc regions. Our results suggest that oxytocin exerts its analgesic effect on painful stimulation via the PAG region and a self-protective effect on unpleasant stimulation via the MeA and NAc regions., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hidehisa Saito reports was provided by Tohoku University Graduate School of Medicine Department of Anaesthesiology and Perioperative medicine, (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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15. Development of nanobody-based POLArIS orientation probes enabled multi-color/multi-target orientation imaging in living cells.
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Nakai N, Sato K, Tani T, Kawagishi M, Ka H, Saito K, and Terada S
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- Animals, Fluorescent Dyes chemical synthesis, Humans, LLC-PK1 Cells, Swine, Tumor Cells, Cultured, Color, Fluorescent Dyes chemistry, Optical Imaging
- Abstract
Fluorescence polarization microscopy (FPM) can visualize the dipole orientation of fluorescent molecules and has been used for analyzing architectural dynamics of biomolecules including cytoskeletal proteins. To monitor the orientation of target molecules by FPM, target molecules need to be labeled with fluorophores in a sterically constrained manner, so that the fluorophores do not freely rotate. Recently, a versatile probe for such labeling using fluorescent proteins, POLArIS (Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest), was reported. POLArIS is a fusion protein consisting of a non-immunoglobulin-based recombinant binder Affimer and a green fluorescent protein (GFP), where the Affimer and GFP are rigidly connected to each other. POLArIS probe for molecules of interest can be developed through phage display screening of Affimer. This screening is followed by the rigid connection of fluorescent proteins to the selected Affimers. The Affimer-based POLArIS, however, cannot be used with animal immune libraries for selecting specific binder clones. In addition, multi-color FPM by POLArIS was not available due to the lack of color variations of POLArIS. In this study, we have developed new versions of POLArIS with nanobodies, which are compatible with animal immune libraries, and expanded color variations of POLArIS with cyan/green/yellow/red fluorescent proteins, enabling multi-color orientation imaging for multiple targets. Using nanobody-based POLArIS orientation probes, we performed two-color FPM of F-actin and vimentin in living cells. Furthermore, we made nanobody-based POLArIS probes that have different dipole orientations for adjusting the orientation of fluorescence polarization with respect to the target molecules. These nanobody-based POLArIS with options of colors and dipole orientations will enhance the performance of this probe for broader applications of fluorescence polarization imaging in living cells, tissues, and whole organisms., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Sumio Terada, Keisuke Sato, Nori Nakai, Kenta Saito, and Masahiko Kawagishi have patent pending to Tokyo Medical and Dental University., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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16. CtBP2 confers protection against oxidative stress through interactions with NRF1 and NRF2.
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Kainoh K, Takano R, Sekiya M, Saito K, Sugasawa T, Ma Y, Murayama Y, Sugano Y, Osaki Y, Iwasaki H, Takeuchi Y, Yahagi N, Suzuki H, Miyamoto T, Nakagawa Y, Matsuzaka T, and Shimano H
- Subjects
- Amino Acid Sequence, Antioxidants metabolism, Gene Expression Regulation, Humans, NF-E2-Related Factor 1 chemistry, NF-E2-Related Factor 1 genetics, NF-E2-Related Factor 2 chemistry, NF-E2-Related Factor 2 genetics, Oxidative Stress, Protein Binding, Transcription, Genetic, Alcohol Oxidoreductases metabolism, Co-Repressor Proteins metabolism, NF-E2-Related Factor 1 metabolism, NF-E2-Related Factor 2 metabolism
- Abstract
While molecular oxygen is essential for aerobic organisms, its utilization is inseparably connected with generation of oxidative insults. To cope with the detrimental aspects, cells evolved antioxidative defense systems, and insufficient management of the oxidative insults underlies the pathogenesis of a wide range of diseases. A battery of genes for this antioxidative defense are regulated by the transcription factors nuclear factor-erythroid 2-like 1 and 2 (NRF1 and NRF2). While the regulatory steps for the activation of NRFs have been investigated with particular emphasis on nuclear translocation and proteosomal degradation, unknown redundancy may exist considering the indispensable nature of these defense systems. Here we unraveled that C-terminal binding protein 2 (CtBP2), a transcriptional cofactor with redox-sensing capability, is an obligate partner of NRFs. CtBP2 forms transcriptional complexes with NRF1 and NRF2 that is required to promote the expression of antioxidant genes in response to oxidative insults. Our findings illustrate a basis for understanding the transcriptional regulation of antioxidative defense systems that may be exploited therapeutically., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this short communication., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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17. Crumbs3 regulates the expression of glycosphingolipids on the plasma membrane to promote colon cancer cell migration.
- Author
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Iioka H, Saito K, and Kondo E
- Subjects
- Cell Line, Tumor, Colonic Neoplasms pathology, Humans, Membrane Glycoproteins deficiency, Cell Membrane metabolism, Cell Movement, Colonic Neoplasms metabolism, Glycosphingolipids biosynthesis, Membrane Glycoproteins metabolism
- Abstract
The cell polarity regulator Crumbs3 (Crb3) promotes colon cancer cell migration and metastasis. However, the underlying mechanism of cancer cell migration regulated by Crb3 has not been fully elucidated. Here, we demonstrated that Crb3 is associated with cell migration by regulating glycosphingolipid (GSL) expression in human colon cancer cells. Crb3-knockout (KO) cells showed a remarkable increase in ganglioside GM3 (GM3) on the cell surface. Reduced migration by Crb3-KO cells was restored by forced expression of both Crb3 and Neuraminidase3 (Neu3). Immunofluorescent staining revealed that most Crb3 is colocalized with the recycling endosome marker Rab11. These findings show that Crb3 may promote colon cancer cell migration by regulating the expression of GSLs on the cell surface., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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18. Circular orientation fluorescence emitter imaging (COFEI) of rotational motion of motor proteins.
- Author
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Sugawa M, Masaike T, Mikami N, Yamaguchi S, Shibata K, Saito K, Fujii F, Toyoshima YY, Nishizaka T, and Yajima J
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- Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Bacillus enzymology, Bacterial Proteins metabolism, Fluorescence Polarization, Kinesins chemistry, Kinesins metabolism, Kinetics, Microscopy, Fluorescence, Molecular Motor Proteins metabolism, Protein Binding, Proton-Translocating ATPases metabolism, Rotation, Bacterial Proteins chemistry, Molecular Motor Proteins chemistry, Proton-Translocating ATPases chemistry, Single Molecule Imaging methods
- Abstract
Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F
1 -ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1 -ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
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19. The binding of TBK1 to STING requires exocytic membrane traffic from the ER.
- Author
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Ogawa E, Mukai K, Saito K, Arai H, and Taguchi T
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- Animals, Brefeldin A pharmacology, Cells, Cultured, Cytosol metabolism, Exocytosis, Fibroblasts metabolism, Gene Knockout Techniques, Golgi Apparatus metabolism, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Interferon Type I metabolism, Membrane Proteins deficiency, Membrane Proteins genetics, Mice, Protein Binding, Protein Transport, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Signal Transduction, Endoplasmic Reticulum metabolism, Membrane Proteins metabolism, Protein Serine-Threonine Kinases metabolism
- Abstract
Stimulator of interferon genes (STING) is essential for the type I interferon and pro-inflammatory responses against DNA pathogens. In response to the presence of cytosolic DNA, STING translocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK-binding kinase 1 (TBK1), a cytosolic kinase that is essential for the activation of STING-dependent downstream signalling. The organelles where TBK1 binds to STING remain unknown. Here we show that TBK1 binds to STING at the Golgi, not at the ER. Treatment with brefeldin A, an agent to block ER-to-Golgi traffic, or knockdown of Sar1, a small GTPase that regulates coat protein complex II (COP-II)-mediated ER-to-Golgi traffic, inhibited the binding of TBK1 to STING. Endogenous TBK1 was recruited to the Golgi when STING was transported to the Golgi, as shown by immunofluorescence microscopy. STING variants that constitutively induce the type I interferon response were found in patients with autoinflammatory diseases. Even these disease-causative STING variants could not bind to TBK1 when the STING variants were trapped in the ER. These results demonstrate that the Golgi is an organelle at which STING recruits and activates TBK1 for triggering the STING-dependent type I interferon response., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
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20. Oral administration of Euglena gracilis Z and its carbohydrate storage substance provides survival protection against influenza virus infection in mice.
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Nakashima A, Suzuki K, Asayama Y, Konno M, Saito K, Yamazaki N, and Takimoto H
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- Administration, Oral, Animals, Euglena gracilis chemistry, Female, Influenza A Virus, H1N1 Subtype drug effects, Influenza A Virus, H1N1 Subtype growth & development, Influenza A Virus, H1N1 Subtype pathogenicity, Interferon-gamma biosynthesis, Interferon-gamma immunology, Interleukin-10 biosynthesis, Interleukin-10 immunology, Interleukin-12 biosynthesis, Interleukin-12 immunology, Interleukin-1beta biosynthesis, Interleukin-1beta immunology, Interleukin-6 biosynthesis, Interleukin-6 immunology, Lung immunology, Lung virology, Mice, Mice, Inbred BALB C, Orthomyxoviridae Infections immunology, Orthomyxoviridae Infections mortality, Orthomyxoviridae Infections virology, Survival Analysis, Adjuvants, Immunologic administration & dosage, Dietary Supplements, Euglena gracilis immunology, Glucans administration & dosage, Lung drug effects, Orthomyxoviridae Infections diet therapy
- Abstract
Euglena gracilis Z is a micro-algae that is used as a food or nutritional supplement. Paramylon, the carbohydrate storage substance of Euglena gracilis Z has β-1, 3-glucan structure. Euglena gracilis Z and paramylon are reported to affect the immune system. In this study, we investigated the protective effects of Euglena gracilis Z and paramylon against influenza virus infection in mice. Euglena gracilis Z and paramylon were administered to mice as a 2% dietary mixture ad libitum. At 2 weeks after initiation of dietary administration, mice were infected intranasally with influenza virus A/PR/8/34 (H1N1). Survival rate was monitored 10 days after infection. In addition, we performed virus titer and cytokine profiles in the lung. High survival rates were observed for Euglena gracilis Z and paramylon-treated groups compared to the control group. Significantly lower virus titer in the lung was observed in the Euglena gracilis Z and paramylon-treated groups compared to the control group from day 1 after infection. Higher amount of IL-1β, IL-6, IL-12 (p70), IFN-γ, and IL-10 was observed in the paramylon groups compared to the control group. Our data therefore reveals a novel immunoregulatory role of the Euglena gracilis Z and paramylon which provides protection against influenza virus infection., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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21. Chemical and thermal sensitivity of medaka TRPA1 analyzed in heterologous expression system.
- Author
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Oda M, Saito K, Hatta S, Kubo Y, and Saitoh O
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- Animals, Caffeine toxicity, Cold Temperature, Cymenes, Female, Fish Proteins genetics, Hot Temperature, In Vitro Techniques, Isothiocyanates toxicity, Monoterpenes toxicity, Oocytes drug effects, Oocytes metabolism, Oryzias genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sensation genetics, Sensation physiology, TRPA1 Cation Channel genetics, TRPA1 Cation Channel physiology, Thermosensing genetics, Thermosensing physiology, Transient Receptor Potential Channels drug effects, Transient Receptor Potential Channels genetics, Xenopus, Zebrafish genetics, Zebrafish physiology, Zebrafish Proteins genetics, Zebrafish Proteins physiology, ortho-Aminobenzoates toxicity, Fish Proteins physiology, Oryzias physiology, Transient Receptor Potential Channels physiology
- Abstract
TRPA1 of insects and several tetrapod vertebrates except for those of rodents have been reported to be activated by noxious chemicals and also by high temperature with a relatively clear threshold. We previously analyzed the characteristics of two TRPA1 paralogs of zebrafish (zTRPA1a, b) and demonstrated that zTRPA1a is specialized for chemical sensing while zTRPA1b responds to thermal stimulations, that zTRPA1b responds to both cold and heat stimuli, and that heat stimulation gradually activates zTRPA1b without a clear threshold. In the medaka genome, a single TRPA1 (olTRPA1) gene is present. To examine if functional properties of olTRPA1 are similar to TRPA1 of land animals or either of zTRPA1a or zTRPA1b, we isolated a TRPA1 cDNA from medaka and performed functional analyses. OlTRPA1 showed a sensitivity to four noxious chemicals (allyl isothiocyanate, caffeine, carvacrol, methyl anthranilate). We observed that cold stimulation does not activate olTRPA1, but heat stimulation gradually activates olTRPA1 with an unclear threshold. Results suggested that a single TRPA1 functions as a chemical and thermal sensor in medaka, and that a gradual heat-activation without clear threshold might be a common feature for TRPA1 of fish living in water., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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22. Forced FOG1 expression in erythroleukemia cells: Induction of erythroid genes and repression of myelo-lymphoid transcription factor PU.1.
- Author
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Fujiwara T, Sasaki K, Saito K, Hatta S, Ichikawa S, Kobayashi M, Okitsu Y, Fukuhara N, Onishi Y, and Harigae H
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- Acetylation, Anion Exchange Protein 1, Erythrocyte metabolism, Basic Helix-Loop-Helix Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors metabolism, Blotting, Western, Cell Line, Chromatin genetics, Chromatin metabolism, GATA1 Transcription Factor genetics, GATA1 Transcription Factor metabolism, Histones metabolism, Humans, K562 Cells, Leukemia, Erythroblastic, Acute genetics, Leukemia, Erythroblastic, Acute metabolism, Leukemia, Erythroblastic, Acute pathology, Lysine metabolism, Nuclear Proteins metabolism, Promoter Regions, Genetic genetics, Protein Binding, Proto-Oncogene Proteins metabolism, RNA Interference, Reverse Transcriptase Polymerase Chain Reaction, T-Cell Acute Lymphocytic Leukemia Protein 1, Trans-Activators metabolism, Transcription Factors metabolism, Anion Exchange Protein 1, Erythrocyte genetics, Gene Expression Regulation, Leukemic, Nuclear Proteins genetics, Proto-Oncogene Proteins genetics, Trans-Activators genetics, Transcription Factors genetics
- Abstract
The transcription factor GATA-1-interacting protein Friend of GATA-1 (FOG1) is essential for proper transcriptional activation and repression of GATA-1 target genes; yet, the mechanisms by which FOG1 exerts its activating and repressing functions remain unknown. Forced FOG1 expression in human K562 erythroleukemia cells induced the expression of erythroid genes (SLC4A1, globins) but repressed that of GATA-2 and PU.1. A quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated increased GATA-1 chromatin occupancy at both FOG1-activated as well as FOG1-repressed gene loci. However, while TAL1 chromatin occupancy was significantly increased at FOG1-activated gene loci, it was significantly decreased at FOG1-repressed gene loci. When FOG1 was overexpressed in TAL1-knocked down K562 cells, FOG1-mediated activation of HBA, HBG, and SLC4A1 was significantly compromised by TAL1 knockdown, suggesting that FOG1 may require TAL1 to activate GATA-1 target genes. Promoter analysis and quantitative ChIP analysis demonstrated that FOG1-mediated transcriptional repression of PU.1 would be mediated through a GATA-binding element located at its promoter, accompanied by significantly decreased H3 acetylation at lysine 4 and 9 (K4 and K9) as well as H3K4 trimethylation. Our results provide important mechanistic insight into the role of FOG1 in the regulation of GATA-1-regulated genes and suggest that FOG1 has an important role in inducing cells to differentiate toward the erythroid lineage rather than the myelo-lymphoid one by repressing the expression of PU.1., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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23. Alteration of cell cycle progression by Sindbis virus infection.
- Author
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Yi R, Saito K, Isegawa N, and Shirasawa H
- Subjects
- HeLa Cells, Humans, Sindbis Virus physiology, Virus Replication, Cell Cycle, Sindbis Virus pathogenicity
- Abstract
We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G1 phase preferred to proliferate during S/G2 phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G1 phase than in cells infected during S/G2 phase., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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24. The JAK2 inhibitor AZD1480 inhibits hepatitis A virus replication in Huh7 cells.
- Author
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Jiang X, Kanda T, Nakamoto S, Saito K, Nakamura M, Wu S, Haga Y, Sasaki R, Sakamoto N, Shirasawa H, Okamoto H, and Yokosuka O
- Subjects
- Animals, COS Cells, Cell Line, Chlorocebus aethiops, Hepatitis A metabolism, Hepatitis A virology, Hepatitis A virus genetics, Hepatitis A virus physiology, Humans, RNA, Viral genetics, STAT3 Transcription Factor metabolism, Antiviral Agents pharmacology, Hepatitis A drug therapy, Hepatitis A virus drug effects, Janus Kinase 2 antagonists & inhibitors, Pyrazoles pharmacology, Pyrimidines pharmacology, Virus Replication drug effects
- Abstract
The JAK2 inhibitor AZD1480 has been reported to inhibit La protein expression. We previously demonstrated that the inhibition of La expression could inhibit hepatitis A virus (HAV) internal ribosomal entry-site (IRES)-mediated translation and HAV replication in vitro. In this study, we analyzed the effects of AZD1480 on HAV IRES-mediated translation and replication. HAV IRES-mediated translation in COS7-HAV-IRES cells was inhibited by 0.1-1 μM AZD1480, a dosage that did not affect cell viability. Results showed a significant reduction in intracellular HAV HA11-1299 genotype IIIA RNA levels in Huh7 cells treated with AZD1480. Furthermore, AZD1480 inhibited the expression of phosphorylated-(Tyr-705)-signal transducer and activator of transcription 3 (STAT3) and La in Huh7 cells. Therefore, we propose that AZD1480 can inhibit HAV IRES activity and HAV replication through the inhibition of the La protein., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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25. A novel evaluation method of survival motor neuron protein as a biomarker of spinal muscular atrophy by imaging flow cytometry.
- Author
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Arakawa M, Arakawa R, Tatsumi S, Aoki R, Saito K, and Nomoto A
- Subjects
- Base Sequence, Cells, Cultured, DNA Primers, Enzyme-Linked Immunosorbent Assay, Humans, Reverse Transcriptase Polymerase Chain Reaction, Subcellular Fractions metabolism, Biomarkers metabolism, Flow Cytometry methods, Muscular Atrophy, Spinal metabolism, Survival of Motor Neuron 1 Protein metabolism
- Abstract
Spinal muscular atrophy (SMA) is caused by mutations within the survival motor neuron 1 (SMN1) gene. These mutations result in the reduction of survival motor neuron (SMN) protein expression and SMN complex in spinal motor neurons and other tissues. SMN protein has been used as a therapeutic biomarker in recent SMA clinical studies using enzyme-linked immunosorbent assay (ELISA). Here, we investigated whether imaging flow cytometry can be a viable source of quantitative information on the SMN protein. Using a FlowSight imaging flow cytometer (Merck-Millipore, Germany), we demonstrated that imaging flow cytometry could successfully identify different expression patterns and subcellular localization of SMN protein in healthy human fibroblasts and SMA patient-derived fibroblasts. In addition, we could also evaluate the therapeutic effects of SMN protein expression by valproic acid treatment of SMA patient-derived cells in vitro. Therefore, we suggest that imaging flow cytometry technology has the potential for identifying SMN protein expression level and pattern as an evaluation tool of clinical studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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26. Developmental expression profiles of axon guidance signaling and the immune system in the marmoset cortex: potential molecular mechanisms of pruning of dendritic spines during primate synapse formation in late infancy and prepuberty (I).
- Author
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Sasaki T, Oga T, Nakagaki K, Sakai K, Sumida K, Hoshino K, Miyawaki I, Saito K, Suto F, and Ichinohe N
- Subjects
- Animals, Callithrix growth & development, Callithrix immunology, Cerebral Cortex growth & development, Cerebral Cortex immunology, DNA, Complementary genetics, Female, Male, Oligonucleotide Array Sequence Analysis, Axons, Callithrix genetics, Cerebral Cortex metabolism, Dendritic Spines, Gene Expression Profiling, Sexual Maturation, Signal Transduction, Synapses
- Abstract
The synapse number and the related dendritic spine number in the cerebral cortex of primates shows a rapid increase after birth. Depending on the brain region and species, the number of synapses reaches a peak before adulthood, and pruning takes place after this peak (overshoot-type synaptic formation). Human mental disorders, such as autism and schizophrenia, are hypothesized to be a result of either too weak or excessive pruning after the peak is reached. Thus, it is important to study the molecular mechanisms underlying overshoot-type synaptic formation, particularly the pruning phase. To examine the molecular mechanisms, we used common marmosets (Callithrix jacchus). Microarray analysis of the marmoset cortex was performed in the ventrolateral prefrontal, inferior temporal, and primary visual cortices, where changes in the number of dendritic spines have been observed. The spine number of all the brain regions above showed a peak at 3 months (3 M) after birth and gradually decreased (e.g., at 6 M and in adults). In this study, we focused on genes that showed differential expression between ages of 3 M and 6 M and on the differences whose fold change (FC) was greater than 1.2. The selected genes were subjected to canonical pathway analysis, and in this study, we describe axon guidance signaling, which had high plausibility. The results showed a large number of genes belonging to subsystems within the axon guidance signaling pathway, macrophages/immune system, glutamate system, and others. We divided the data and discussion of these results into 2 papers, and this is the first paper, which deals with the axon guidance signaling and macrophage/immune system. Other systems will be described in the next paper. Many components of subsystems within the axon guidance signaling underwent changes in gene expression from 3 M to 6 M so that the synapse/dendritic spine number would decrease at 6 M. Thus, axon guidance signaling probably contributes to the decrease in synapse/dendritic spine number at 6 M, the phenomenon that fits the overshoot-type synaptic formation in primates. Microglial activity (evaluated by quantifying AIF1 expression) and gene expression of molecules that modulate microglia, decreased at 6 M, just like the synapse/dendritic spine number. Thus, although microglial activity is believed to be related to phagocytosis of synapses/dendritic spines, microglial activity alone cannot explain how pruning was accelerated in the pruning phase. On the other hand, expression of molecules that tag synapses/dendritic spines as a target of phagocytosis by microglia (e.g., complement components) increased at 6 M, suggesting that these tagging proteins may be involved in the acceleration of pruning during the pruning phase., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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27. Developmental genetic profiles of glutamate receptor system, neuromodulator system, protector of normal tissue and mitochondria, and reelin in marmoset cortex: potential molecular mechanisms of pruning phase of spines in primate synaptic formation process during the end of infancy and prepuberty (II).
- Author
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Sasaki T, Oga T, Nakagaki K, Sakai K, Sumida K, Hoshino K, Miyawaki I, Saito K, Suto F, and Ichinohe N
- Subjects
- Animals, Animals, Newborn, Callithrix, Cerebral Cortex growth & development, Oligonucleotide Array Sequence Analysis, Reelin Protein, Sexual Maturation, Cell Adhesion Molecules, Neuronal metabolism, Cerebral Cortex metabolism, Extracellular Matrix Proteins metabolism, Gene Expression Profiling, Gene Expression Regulation, Developmental, Mitochondria metabolism, Nerve Tissue Proteins metabolism, Neurotransmitter Agents physiology, Receptors, Glutamate genetics, Serine Endopeptidases metabolism, Synapses
- Abstract
This is the second report of a series paper, which reports molecular mechanisms underlying the occurrence of pruning spine phase after rapid spinogenesis phase in neonates and young infant in the primate brain. We performed microarray analysis between the peak of spine numbers [postnatal 3 months (M)] and spine pruning (postnatal 6M) in prefrontal, inferior temporal, and primary visual cortices of the common marmoset (Callithrix jacchus). The pruning phase is not clearly defined in rodents but is in primates including the marmoset. The differentially expressed genes between 3M and 6M in all three cortical areas were selected by two-way analysis of variance. The list of selected genes was analyzed by canonical pathway analysis using "Ingenuity Pathway Analysis of complex omics data" (IPA; Ingenuity Systems, Qiagen, Hilden, Germany). In this report, we discuss these lists of genes for the glutamate receptor system, G-protein-coupled neuromodulator system, protector of normal tissue and mitochondria, and reelin. (1) Glutamate is a common neurotransmitter. Its receptors AMPA1, GRIK1, and their scaffold protein DLG4 decreased as spine numbers decreased. Instead, GRIN3 (NMDA receptor) increased, suggesting that strong NMDA excitatory currents may be required for a single neuron to receive sufficient net synaptic activity in order to compensate for the decrease in synapse. (2) Most of the G protein-coupled receptor genes (e.g., ADRA1D, HTR2A, HTR4, and DRD1) in the selected list were upregulated at 6M. The downstream gene ROCK2 in these receptor systems plays a role of decreasing synapses, and ROCK2 decreased at 6M. (3) Synaptic phagosytosis by microglia with complement and other cytokines could cause damage to normal tissue and mitochondria. SOD1, XIAP, CD46, and CD55, which play protective roles in normal tissue and mitochondria, showed higher expression at 6M than at 3M, suggesting that normal brain tissue is more protected at 6M. (4) Reelin has an important role in cortical layer formation. In addition, RELN and three different pathways of reelin were expressed at 6M, suggesting that new synapse formation decreased at that age. Moreover, if new synapses were formed, their positions were free and probably dependent on activity., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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28. Sexual dimorphisms in zonal gene expression in mouse liver.
- Author
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Saito K, Negishi M, and James Squires E
- Subjects
- Animals, Female, Male, Mice, Mice, Inbred C3H, Quality Control, Transcription, Genetic, Gene Expression Profiling, Liver metabolism, Sex Characteristics
- Abstract
Many of the metabolic functions of the liver are localized either in the pericentral region (zone 3) or in the periportal region (zone 1). However, a systematic analysis of the heterogeneity and sexual dimorphism of gene expression in the liver is lacking. Our objective was to obtain sections of intact tissue from zone 1 and zone 3 from both male and female mouse liver, and to measure the patterns of gene expression in these sections. Zone 1 and zone 3 areas were isolated by laser capture microdissection of liver sections, total RNA was isolated and microarray analysis was conducted using Agilent Whole Mouse Genome oligo arrays. To investigate functional characteristics as well as upstream regulators of specific gene lists, we used Ingenuity Pathway Analysis. We identified more than 925 genes in zone 1 and more than 450 genes in zone 3 of both male and female mice. Sexual dimorphism in metabolic functions was present in zone 1 but not zone 3. In zone 1, canonical pathways related to gluconeogenesis were male predominant, while canonical pathways related to hepatic progenitor cells were female predominant. In addition, we also analyzed the upstream regulators of zone-specific genes. SREBF1 was male-specific in zone 1, while TRIM24 was female-specific in zone 3. These results demonstrate the heterogeneity and sexually dimorphic differences in gene expression in the liver., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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29. A hypoxia-inducible factor (HIF)-3α splicing variant, HIF-3α4 impairs angiogenesis in hypervascular malignant meningiomas with epigenetically silenced HIF-3α4.
- Author
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Ando H, Natsume A, Iwami K, Ohka F, Kuchimaru T, Kizaka-Kondoh S, Ito K, Saito K, Sugita S, Hoshino T, and Wakabayashi T
- Subjects
- Alternative Splicing, Animals, Apoptosis Regulatory Proteins, Basic Helix-Loop-Helix Transcription Factors metabolism, Cell Line, Tumor, Cell Proliferation, DNA Methylation, Epigenesis, Genetic, Gene Expression Regulation, Neoplastic, Gene Silencing, Glucose metabolism, Humans, Meningeal Neoplasms metabolism, Meningeal Neoplasms pathology, Meningioma metabolism, Meningioma pathology, Mice, Mice, Nude, Neoplasm Invasiveness, Promoter Regions, Genetic, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Repressor Proteins, Transplantation, Heterologous, Basic Helix-Loop-Helix Transcription Factors genetics, Meningeal Neoplasms blood supply, Meningeal Neoplasms genetics, Meningioma blood supply, Meningioma genetics, Neovascularization, Pathologic genetics
- Abstract
Hypoxia inducible factor is a dominant regulator of adaptive cellular responses to hypoxia and controls the expression of a large number of genes regulating angiogenesis as well as metabolism, cell survival, apoptosis, and other cellular functions in an oxygen level-dependent manner. When a neoplasm is able to induce angiogenesis, tumor progression occurs more rapidly because of the nutrients provided by the neovasculature. Meningioma is one of the most hypervascular brain tumors, making anti-angiogenic therapy an attractive novel therapy for these tumors. HIF-3α has been conventionally regarded as a dominant-negative regulator of HIF-1α, and although alternative HIF-3α splicing variants are extensively reported, their specific functions have not yet been determined. In this study, we found that the transcription of HIF-3α4 was silenced by the promoter DNA methylation in meningiomas, and inducible HIF-3α4 impaired angiogenesis, proliferation, and metabolism/oxidation in hypervascular meningiomas. Thus, HIF-3α4 could be a potential molecular target in meningiomas., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
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30. XLGαolf regulates expression of p27Kip1 in a CSN5 and CDK2 dependent manner.
- Author
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Akita K, Takahashi Y, Takata N, Hashimoto M, Kataoka M, Tomigahara Y, and Saito K
- Subjects
- COP9 Signalosome Complex, Cyclin-Dependent Kinase 2 antagonists & inhibitors, Cyclin-Dependent Kinase 2 genetics, Cyclin-Dependent Kinase Inhibitor p27 genetics, Down-Regulation, GTP-Binding Protein alpha Subunits genetics, Gene Silencing, HEK293 Cells, Humans, Intracellular Signaling Peptides and Proteins genetics, Peptide Hydrolases genetics, RNA, Small Interfering genetics, Two-Hybrid System Techniques, Cyclin-Dependent Kinase 2 metabolism, Cyclin-Dependent Kinase Inhibitor p27 biosynthesis, GTP-Binding Protein alpha Subunits metabolism, Intracellular Signaling Peptides and Proteins metabolism, Peptide Hydrolases metabolism
- Abstract
XLGα(olf) is an extra large transcriptional variant of the heterotrimeric G protein, Gα(olf), which we previously reported to be localized in the Golgi apparatus and interacted with Rab3A and Rab8A through its N-terminal region. However, many physiological functions of XLGα(olf) remain to be elucidated. In this study, performance of yeast two-hybrid screening with XLGα(olf) allowed isolation of COP9 signalosome subunit 5 (CSN5), known to regulate the p27(Kip1) protein level through a proteasome dependent pathway. Co-immunoprecipitation experiments followed by Western blotting also showed association of CSN5 with XLGα(olf) linked to down-regulation of p27(Kip1). Gene silencing of endogenous CSN5 by siRNA attenuated the XLGα(olf)-mediated down-regulation, which was also demonstrated to require CDK2. Both knock down of CDK2 and the treatment with a CDK2 inhibitor reversed the reduction of p27(Kip1) due to XLGα(olf). Our findings provide important clues to understanding physiological functions of XLGα(olf)., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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31. Mild caloric restriction up-regulates the expression of prohibitin: a proteome study.
- Author
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Takahashi S, Masuda J, Shimagami H, Ohta Y, Kanda T, Saito K, and Kato H
- Subjects
- Animals, Body Weight, Liver anatomy & histology, Liver metabolism, Male, Organ Size, Prohibitins, Proteome genetics, Proteomics, RNA, Messenger metabolism, Rats, Rats, Wistar, Repressor Proteins genetics, Caloric Restriction, Longevity, Proteome biosynthesis, Repressor Proteins biosynthesis
- Abstract
Caloric restriction (CR) is well known to expand lifespan in a variety of species and to retard many age-related diseases. The effects of relatively mild CR on the proteome profile in relation to lifespan have not yet been reported, despite the more extensive studies of the stricter CR conditions. Thus, the present study was conducted to elucidate the protein profiles in rat livers after mild CR for a relatively short time. Young growing rats were fed CR diets (10% and 30% CR) for 1month. We performed the differential proteomic analysis of the rat livers using two-dimensional electrophoresis combined with MALDI-TOF mass spectrometry. The most remarkable protein among the differentially expressed proteins was found to be prohibitin, the abundance of which was increased by 30% CR. Prohibitin is a ubiquitously expressed protein shown to suppress cell proliferation and to be related to longevity. The increase in prohibitin was observed both in 10% and 30% CR by Western blot analysis. Furthermore, induction of AMP-activated kinase (AMPK) protein, related to the actions of prohibitin in promoting longevity, was observed. The increased prohibitin level in response to subtle CR suggests that this increase may be one of the early events leading to the expansion of lifespan in response to CR., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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32. The LIM protein Ajuba is required for ciliogenesis and left-right axis determination in medaka.
- Author
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Nagai Y, Asaoka Y, Namae M, Saito K, Momose H, Mitani H, Furutani-Seiki M, Katada T, and Nishina H
- Subjects
- Animals, Cilia genetics, Cilia metabolism, Cilia physiology, Cloning, Molecular, Gene Knockdown Techniques, Heart embryology, Homeodomain Proteins genetics, Liver abnormalities, Microtubules genetics, Microtubules metabolism, Oryzias genetics, Oryzias metabolism, Spleen abnormalities, Homeodomain Proteins metabolism, Microtubules physiology, Oryzias embryology
- Abstract
Cilia are microtubule-based organelles that are present on the surfaces of almost all vertebrate cells. Most cilia function as sensory or molecular transport structures. Malfunctions of cilia have been implicated in several diseases of human development. The assembly of cilia is initiated by the centriole (or basal body), and several centrosomal proteins are involved in this process. The mammalian LIM protein Ajuba is a well-studied centrosomal protein that regulates cell division but its role in ciliogenesis is unknown. In this study, we isolated the medaka homolog of Ajuba and showed that Ajuba localizes to basal bodies of cilia in growth-arrested cells. Knockdown of Ajuba resulted in randomized left-right organ asymmetries and altered expression of early genes responsible for left-right body axis determination. At the cellular level, we found that Ajuba function was essential for ciliogenesis in the cells lining Kupffer's vesicle; it is these cells that induce the asymmetric fluid flow required for left-right axis determination. Taken together, our findings identify a novel role for Ajuba in the regulation of vertebrate ciliogenesis and left-right axis determination., ((c) 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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33. Interaction between LPS-induced NO production and IDO activity in mouse peritoneal cells in the presence of activated Valpha14 NKT cells.
- Author
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Ohtaki H, Ito H, Ando K, Ishikawa T, Hoshi M, Tanaka R, Osawa Y, Yokochi T, Moriwaki H, Saito K, and Seishima M
- Subjects
- Animals, Galactosylceramides immunology, Indoleamine-Pyrrole 2,3,-Dioxygenase genetics, Interferon-gamma immunology, Lipopolysaccharides immunology, Lymphocyte Activation, Mice, Mice, Inbred C57BL, Mice, Knockout, Nitric Oxide Synthase Type II metabolism, Peritoneum enzymology, Peritonitis immunology, Receptors, Antigen, T-Cell, alpha-beta immunology, Immune Tolerance, Indoleamine-Pyrrole 2,3,-Dioxygenase metabolism, Natural Killer T-Cells immunology, Nitric Oxide biosynthesis, Peritoneum immunology
- Abstract
In this study, we demonstrated that lipopolysaccharide (LPS) markedly increased nitric oxide (NO) production and indoleamine 2,3-dioxygenase (IDO) activity in mouse peritoneal cells in the presence of activated Valpha14 natural killer T cells. Moreover, LPS-induced NO production in peritoneal cells from IDO-knockout (KO) mice was more increased than that from wild-type mice. However, there was no significant difference in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein between the wild-type and IDO-KO mice. No significant difference was also observed in the ratio of CD3- and DX5-positive cells and F4/80- and TLR4-positive cells in peritoneal cells between the wild-type and IDO-KO mice. Since the IDO activity was enhanced by an NO inhibitor, NO may be post-translationally consumed by inhibiting the IDO activity. IDO is well known to play an important role in immunosuppression during inflammatory disease. Therefore, the inhibition of IDO by NO may exacerbate inflammation in the peritoneal cavity.
- Published
- 2009
- Full Text
- View/download PDF
34. Subcellular localization of a novel G protein XLGalpha(olf).
- Author
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Akita K, Takahashi Y, Kataoka M, Saito K, and Kaneko H
- Subjects
- Animals, COS Cells, Cell Nucleus enzymology, Chlorocebus aethiops, Endosomes enzymology, Golgi Apparatus enzymology, Humans, rab GTP-Binding Proteins metabolism, rab3A GTP-Binding Protein metabolism, GTP-Binding Protein alpha Subunits metabolism
- Abstract
XLGalpha(olf) was identified as a transcriptional variant of the heterotrimeric G protein, Galpha(olf). Previous work showed that XLGalpha(olf) couples with adenosine A2a receptor and dopamine D1 receptor in vitro. However, physiological functions of XLGalpha(olf) remain to be elucidated. In this study, we performed indirect immunofluorescence confocal analyses to examine the subcellular localization of XLGalpha(olf). With overexpression, surprisingly, many large endosomes resulted. We also observed that XLGalpha(olf) localizes at the Golgi apparatus. The N-terminal region of XLGalpha(olf) appears necessary for both endosome formation and the Golgi localization. The results indicate that XLGalpha(olf) and Galpha(olf) play distinctly separate roles. Moreover, XLGalpha(olf) colocalized with Rab3A and Rab8A, as well as partially with Rab11A, but not with other endocytotic endosomes. We could confirm the interaction between XLGalpha(olf) and Rab3A/Rab8A by co-immunoprecipitation experiments. Our study provides important clues toward understanding physiological functions of XLGalpha(olf).
- Published
- 2009
- Full Text
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35. A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver.
- Author
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Shikanai M, Asahina K, Iseki S, Teramoto K, Nishida T, Shimizu-Saito K, Ota M, Eto K, and Teraoka H
- Subjects
- Animals, Cell Differentiation, Female, Fetus cytology, Fetus physiology, Hepatocytes physiology, Liver cytology, Liver physiology, Mice, Organogenesis, Uterus, Hepatocytes transplantation, Liver embryology, Stem Cell Transplantation methods, Stem Cells cytology
- Abstract
Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or alpha-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.
- Published
- 2009
- Full Text
- View/download PDF
36. Dynamic regulation of bHLH-PAS-type transcription factor NXF gene expression and neurotrophin dependent induction of the transcriptional control activity.
- Author
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Ooe N, Kobayashi K, Motonaga K, Saito K, and Kaneko H
- Subjects
- Animals, Gene Expression, Mice, Mice, Inbred C57BL, Mitogen-Activated Protein Kinase Kinases metabolism, PC12 Cells, Phosphatidylinositol 3-Kinases metabolism, Promoter Regions, Genetic, Rats, Rats, Sprague-Dawley, Receptor, Nerve Growth Factor metabolism, Transcription, Genetic, Basic Helix-Loop-Helix Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors metabolism, Brain metabolism, Brain-Derived Neurotrophic Factor metabolism, Gene Expression Regulation, Protein Processing, Post-Translational
- Abstract
While neurotrophin is known to be involved in a variety of neuronal functions inducing several immediate early genes and activating several signaling molecules, the correspondence with downstream cascades remains to be defined in detail. Here we show that a bHLH-PAS transcription factor, NXF, is a new member genes under the control of neurotrophin. The PI3K-Akt system, an important cell-protection-signaling cascade under the control of the neurotrophin receptor, was also revealed to contribute to the mechanism of NXF mRNA induction. Activation of MAPK under the control of the neurotrophin receptor resulted in NXF protein phosphorylation as well as enhancement of NXF transcriptional activity. This newly identified NXF gene system may provide a new insight into neurotrophin biology, which reflects the target gene functions.
- Published
- 2009
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37. Valpha14 NKT cells activated by alpha-galactosylceramide augment lipopolysaccharide-induced nitric oxide production in mouse intra-hepatic lymphocytes.
- Author
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Ohtaki H, Ito H, Ando K, Ishikawa T, Saito K, Imawari M, Yokochi T, Moriwaki H, and Seishima M
- Subjects
- Animals, Cytokines metabolism, Interferon-gamma genetics, Lipopolysaccharides immunology, Mice, Mice, Knockout, Natural Killer T-Cells metabolism, Nitric Oxide Synthase Type II genetics, Toll-Like Receptor 4 immunology, Galactosylceramides immunology, Liver immunology, Lymphocyte Activation, Natural Killer T-Cells immunology, Nitric Oxide biosynthesis
- Abstract
Valpha14 natural killer T (Valpha14 NKT) cells activated by alpha-galactosylceramide (alpha-GalCer) secrete a large amount of Th1 and Th2 cytokines. IFN-gamma plays a crucial role in the inflammation response, and is also known as an activator of nitric oxide (NO) production. We previously reported that lipopolysaccharide (LPS)-induced NO production is augmented by alpha-GalCer in mouse peritoneal cells. Since the liver is susceptible to LPS stimulation via the portal vein, we examined the effect of alpha-GalCer on LPS-induced NO production in murine intra-hepatic lymphocytes (IHLs). Although IHLs augmented LPS-induced NO production by alpha-GalCer administration, such an augmentation was not observed in non-treated mice. Furthermore, alpha-GalCer did not augment LPS-induced NO production in IHLs from IFN-gamma knockout mice. In flow cytometry analysis of IHLs from alpha-GalCer-treated mice, the ratio and number of F4/80- and TLR4-positive cells rose as compared with non-treated mice. The liver injury may be induced by LPS and NO under the condition where Valpha14 NKT cells were activated.
- Published
- 2009
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38. Telmisartan inhibits methylglyoxal-mediated cell death in human vascular endothelium.
- Author
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Baden T, Yamawaki H, Saito K, Mukohda M, Okada M, and Hara Y
- Subjects
- Angiotensin II Type 1 Receptor Blockers, Caspase 3 metabolism, Cells, Cultured, Endothelium, Vascular cytology, Enzyme Activation drug effects, Humans, Pyruvaldehyde toxicity, Telmisartan, Apoptosis drug effects, Benzimidazoles pharmacology, Benzoates pharmacology, Endothelium, Vascular drug effects, Pyruvaldehyde antagonists & inhibitors
- Abstract
Methylglyoxal (MGO) is a metabolite of glucose. Since serum MGO level is increased in diabetic patients, MGO is implicated in diabetic complications related to vascular injury. We have recently demonstrated that glucose metabolite is a more powerful stimulant for endothelial cells (ECs) injury rather than glucose or advanced glycation-end products. Recent clinical trials suggest that angiotensin receptor blockers are effective to prevent diabetes-associated cardiovascular disorders beyond blood pressure lowering effect. To explore the mechanisms, we examined effects of telmisartan on MGO-induced ECs injury. Treatment of human umbilical vein ECs with MGO (560 microM) induced time-dependent (0-24 h) cell death. MGO-induced cell death was apoptosis since MGO increased cleaved caspase-3 expression. Telmisartan (0.1-10 microM) inhibited MGO-induced cell death and caspase-3 activation. These results indicate that telmisartan prevents MGO-induced apoptosis by inhibiting caspase-3 activation, which might explain at least in part the beneficial effects of telimisartan against diabetes-related cardiovascular diseases.
- Published
- 2008
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39. Proteomic identification of ZO-1/2 as a novel scaffold for Src/Csk regulatory circuit.
- Author
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Saito K, Enya K, Oneyama C, Hikita T, and Okada M
- Subjects
- Amino Acid Sequence, Animals, CSK Tyrosine-Protein Kinase, Cell Line, Chickens, Humans, Membrane Proteins chemistry, Mice, Molecular Sequence Data, Phosphoproteins chemistry, Protein Binding, Protein Interaction Mapping, Protein-Tyrosine Kinases chemistry, Rats, Zonula Occludens-1 Protein, Zonula Occludens-2 Protein, src Homology Domains, src-Family Kinases, Membrane Proteins metabolism, Phosphoproteins metabolism, Protein-Tyrosine Kinases metabolism, Proteomics methods
- Abstract
To elucidate the regulatory mechanism of cell transformation induced by c-Src tyrosine kinase, we performed a proteomic analysis of tyrosine phosphorylated proteins that interact with c-Src and/or its negative regulator Csk. The c-Src interacting proteins were affinity-purified from Src transformed cells using the Src SH2 domain as a ligand. LC-MS/MS analysis of the purified proteins identified general Src substrates, such as focal adhesion kinase and paxillin, and ZO-1/2 as a transformation-dependent Src target. The Csk binding proteins were analyzed by a tandem affinity purification method. In addition to the previously identified Csk binding proteins, including Cbp/PAG, paxillin, and caveolin-1, we found that ZO-1/2 could also serve as a major Csk binding protein. ZO-2 was phosphorylated concurrently with Src transformation and specifically bound to Csk in a Csk SH2 dependent manner. These results suggest novel roles for ZO proteins as Src/Csk scaffolds potentially involved in the regulation of Src transformation.
- Published
- 2008
- Full Text
- View/download PDF
40. Differentially expressed genes among motor and prefrontal areas of macaque neocortex.
- Author
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Sato A, Nishimura Y, Oishi T, Higo N, Murata Y, Onoe H, Saito K, Tsuboi F, Takahashi M, Isa T, and Kojima T
- Subjects
- Animals, Brain metabolism, Brain Mapping, Cluster Analysis, Gene Expression Profiling, Macaca, Oligonucleotide Array Sequence Analysis, Gene Expression Regulation, Motor Cortex metabolism, Neocortex metabolism, Prefrontal Cortex metabolism
- Abstract
In higher primates, the motor-related areas of the neocortex are highly differentiated into several subareas based on both functional and cytoarchitectural aspects. To assess the molecular basis of this areal specialization, we investigated the gene expression profiles of the primary motor area (M1), premotor area (dorsal and ventral, PMd and PMv, respectively), and prefrontal area (A46) in rhesus monkeys using DNA microarray. We discovered that 476 genes were differentially expressed among these 4 areas. More than half of these genes were most abundantly expressed in M1, and most genes were complementarily expressed between M1 and A46. The expression profiles of PMd and PMv were quite similar to each other and different from those of M1 and A46. The data will provide a fundamental basis for the further analysis of the structure-function relationship of the primate brain.
- Published
- 2007
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41. Small GTPase Rho signaling is involved in beta1 integrin-mediated up-regulation of intercellular adhesion molecule 1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation.
- Author
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Hirai F, Nakayamada S, Okada Y, Saito K, Kurose H, Mogami A, and Tanaka Y
- Subjects
- ADP Ribose Transferases genetics, ADP Ribose Transferases metabolism, Acid Phosphatase metabolism, Amides pharmacology, Botulinum Toxins genetics, Botulinum Toxins metabolism, Cell Line, Tumor, Cells, Cultured, Coculture Techniques, Collagen Type I pharmacology, Enzyme Activation drug effects, Enzyme Inhibitors pharmacology, Flow Cytometry, Humans, Isoenzymes metabolism, Monocytes cytology, Monocytes drug effects, Monocytes metabolism, Osteoblasts cytology, Osteoblasts drug effects, Pyridines pharmacology, Signal Transduction drug effects, Tartrate-Resistant Acid Phosphatase, Transfection, Up-Regulation drug effects, Vascular Cell Adhesion Molecule-1 metabolism, rho GTP-Binding Proteins antagonists & inhibitors, Integrin beta1 physiology, Intercellular Adhesion Molecule-1 metabolism, Osteoblasts metabolism, RANK Ligand metabolism, Signal Transduction physiology, rho GTP-Binding Proteins metabolism
- Abstract
We assessed the characteristics of human osteoblasts, focusing on small GTPase Rho signaling. Beta1 Integrin were highly expressed on osteoblasts. Engagement of beta1 integrins by type I collagen augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on osteoblasts. Rho was activated by beta1 stimulation in osteoblasts. Beta1 Integrin-induced up-regulation of ICAM-1 and RANKL was inhibited by transfection with adenoviruses encoding C3 transferase or pretreated with Y-27632, specific Rho and Rho-kinase inhibitors. Engagement of beta1 integrin on osteoblasts induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) in a coculture system of osteoblasts and peripheral monocytes, but this action was completely abrogated by transfection of C3 transferase. Our results indicate the direct involvement of Rho-mediated signaling in beta1 integrin-induced up-regulation of ICAM-1 and RANKL and RANKL-dependent osteoclast maturation. Thus, Rho-mediated signaling in osteoblasts seems to introduce major biases to bone resorption.
- Published
- 2007
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42. Key adhesin gene in community-acquired methicillin-resistant Staphylococcus aureus.
- Author
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Otsuka T, Saito K, Dohmae S, Takano T, Higuchi W, Takizawa Y, Okubo T, Iwakura N, and Yamamoto T
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Bacterial Toxins genetics, Bacterial Toxins metabolism, Carrier Proteins genetics, Carrier Proteins metabolism, Exotoxins genetics, Exotoxins metabolism, Leukocidins, Polymerase Chain Reaction, Staphylococcal Infections microbiology, Staphylococcus aureus genetics, Adhesins, Bacterial genetics, Community-Acquired Infections microbiology, Methicillin Resistance genetics, Staphylococcus aureus metabolism
- Abstract
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) possessing the Panton-Valentine leukocidin (PVL) gene (luk(PV)) is associated with skin and soft tissue infections, osteomyelitis, and necrotizing pneumonia. There are geographically two types of CA-MRSA: one (sequence type ST30) that is worldwide (pandemic) and the other (sequence types, e.g., ST1, ST8 or ST80) that is continent-specific. The pandemic type, but not continent-specific type, possessed the bone sialoprotein-adhesin gene (bbp), which was associated with osteomyelitis. No recent hospital-acquired MRSA had the bbp gene, while past PVL-positive nosocomial outbreak-derived strains did possess it. The collagen-adhesin gene (cna) was associated with pandemic CA-MRSA, though with positive cases even in continent-specific CA-MRSA and PVL-negative Japanese region-specific CA-MRSA. Thus, the pandemic type is characterized by the combination of luk(PV) and bbp (and cna) genes. A specific real-time PCR assay for the bbp gene was developed, and dual assay for bbp and luk(PV) in one test tube became possible.
- Published
- 2006
- Full Text
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43. Phosphatidylserine is involved in gene expression from Sindbis virus subgenomic promoter.
- Author
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Saito K, Nishijima M, and Kuge O
- Subjects
- Animals, CHO Cells, Cells, Cultured, Cricetinae, Female, Genome, Viral physiology, Mutation, Ovary cytology, Phosphatidylserines deficiency, RNA, Viral genetics, RNA, Viral metabolism, RNA-Dependent RNA Polymerase genetics, RNA-Dependent RNA Polymerase metabolism, Sindbis Virus metabolism, Transfection, Virus Replication genetics, Virus Replication physiology, Gene Expression Regulation, Viral physiology, Genome, Viral genetics, Phosphatidylserines physiology, Promoter Regions, Genetic, Sindbis Virus genetics
- Abstract
Sindbis virus replication is mediated by an RNA replicase translated from viral RNA genome. The replicase synthesizes progeny genomic RNA and shorter RNA (subgenomic RNA) carrying viral structural genes in association with cytoplasmic membranes. Here we examined the involvement of a membrane lipid, phosphatidylserine (PS), in Sindbis virus gene expression using Chinese hamster ovary cell mutants. When the mutant cells were transfected with viral replicon RNA, in which the structural genes downstream of the subgenomic promoter were replaced by a reporter gene, reporter expression was inhibited under PS-deficient conditions. In contrast, reporter expression from an SV40 promoter-driven construct was normal under similar conditions. Furthermore, expression of a viral replicase protein from the genomic RNA and accumulation of the subgenomic RNA were not inhibited by PS deficiency. These findings indicate that reduced cellular PS level impairs a posttranscriptional event of Sindbis virus subgenomic promoter-driven gene expression.
- Published
- 2006
- Full Text
- View/download PDF
44. Hepatocyte growth factor is constitutively produced by donor-derived bone marrow cells and promotes regeneration of pancreatic beta-cells.
- Author
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Izumida Y, Aoki T, Yasuda D, Koizumi T, Suganuma C, Saito K, Murai N, Shimizu Y, Hayashi K, Odaira M, Kusano T, Kushima M, and Kudano M
- Subjects
- Animals, Cell Proliferation, Diabetes Mellitus, Experimental metabolism, Diabetes Mellitus, Experimental therapy, Hyperglycemia metabolism, Hyperglycemia therapy, Insulin metabolism, Liver metabolism, Male, Pancreas metabolism, Pancreatic Ducts cytology, Pancreatic Ducts physiology, Proto-Oncogene Proteins c-met metabolism, Rats, Regeneration, Signal Transduction, Spleen metabolism, Bone Marrow Cells metabolism, Bone Marrow Transplantation, Hepatocyte Growth Factor metabolism, Insulin-Secreting Cells physiology
- Abstract
Recent studies have demonstrated that the transplantation of bone marrow cells following diabetes induced by streptozotocin can support the recovery of pancreatic b-cell mass and a partial reversal of hyperglycemia. To address this issue, we examined whether the c-Met/hepatocyte growth factor (HGF) signaling pathway was involved in the recovery of b-cell injury after bone marrow transplantation (BMT). In this model, donor-derived bone marrow cells were positive for HGF immunoreactivity in the recipient spleen, liver, lung, and pancreas as well as in the host hepatocytes. Indeed, plasma HGF levels were maintained at a high value.The frequency of c-Met expression and its proliferative activity and differentiative response in the pancreatic ductal cells in the BMT group were greater than those in the PBS-treated group, resulting in an elevated number of endogenous insulin-producing cells. The induction of the c-Met/HGF signaling pathway following BMT promotes pancreatic regeneration in diabetic rats., (© 2005 Elsevier Inc. All rights reserved.)
- Published
- 2005
- Full Text
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45. Photoreceptor cells from mouse ES cells by co-culture with chick embryonic retina.
- Author
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Sugie Y, Yoshikawa M, Ouji Y, Saito K, Moriya K, Ishizaka S, Matsuura T, Maruoka S, Nawa Y, and Hara Y
- Subjects
- Animals, Cell Differentiation physiology, Cells, Cultured, Chick Embryo, Mice, Coculture Techniques methods, Photoreceptor Cells cytology, Retina cytology, Retina embryology, Stem Cells cytology, Tissue Engineering methods
- Abstract
Degeneration of photoreceptors is a consistent and common endpoint in retinal diseases. Herein, we report the efficient induction of photoreceptor-like cells from mouse embryonic stem (ES) cells using chick embryonic retina tissue. Undifferentiated mouse ES cells were initially cultured in a preferential condition into a neural lineage, and ES cells were then co-cultured with chick embryonic day 6 (E6) retina tissues. After a 10-day co-culture, approximately 20% of the mouse ES derivatives became immuno-positive for rhodopsin. RT-PCR analysis demonstrated expression of the transcription factor crx and a distinct increase of rod photoreceptor-specific markers, IRBP and recoverin, after the start of the co-culture. These results indicate that co-culture of ES cells with chick embryonic retina tissue is a useful and efficient method for the induction of photoreceptor-like cells.
- Published
- 2005
- Full Text
- View/download PDF
46. Molecular dynamics of STAT3 on IL-6 signaling pathway in living cells.
- Author
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Watanabe K, Saito K, Kinjo M, Matsuda T, Tamura M, Kon S, Miyazaki T, and Uede T
- Subjects
- Cell Line, Tumor, Diffusion, Green Fluorescent Proteins analysis, Green Fluorescent Proteins chemistry, Humans, Kinetics, Microscopy, Confocal, STAT3 Transcription Factor, Spectrometry, Fluorescence, DNA-Binding Proteins analysis, Interleukin-6 pharmacology, Signal Transduction, Trans-Activators analysis
- Abstract
Signal transducer and activator of transcription 3 (STAT3) is a critical signal transducer of interleukin-6 (IL-6) signaling. To investigate the mobility and the dynamics of STAT3 complex on IL-6 signaling in living cells, we generated a chimeric gene consisting of STAT3 fused to enhanced green fluorescence protein, STAT3-GFP. STAT3-GFP was expressed in Hep3B cells and the dynamics of this protein were analyzed by fluorescence correlation spectroscopy. After IL-6 stimulation, STAT3 translocated from the cytoplasm to the nucleus, as shown previously. According to the analysis of STAT3 diffusion in stable transformants, the number of STAT3 molecules at the cytoplasmic membrane and in the cytoplasm decreased after IL-6 stimulation. In the nucleus, the diffusion speed of STAT3 complex strongly decreased after IL-6 stimulation. Furthermore, we found that STAT3 existed as a complex whose molecular weight was less than 400kDa before IL-6 addition. However, IL-6 stimulation induced the formation of STAT3 dimer as a megacomplex form whose molecular weight was more than 1MDa at the cytoplasm and a very slow diffusion complex in the nucleus.
- Published
- 2004
- Full Text
- View/download PDF
47. Direct detection of caspase-3 activation in single live cells by cross-correlation analysis.
- Author
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Saito K, Wada I, Tamura M, and Kinjo M
- Subjects
- Apoptosis, Caspase 3, Enzyme Activation, Green Fluorescent Proteins metabolism, HeLa Cells, Humans, Kinetics, Luminescent Proteins metabolism, Models, Statistical, Peptide Hydrolases chemistry, Peptide Hydrolases metabolism, Plasmids metabolism, Polymerase Chain Reaction, Recombinant Fusion Proteins chemistry, Time Factors, Red Fluorescent Protein, Caspases metabolism, Spectrometry, Fluorescence methods
- Abstract
Dual color fluorescence cross-correlation spectroscopy (FCCS) provides information about the coincidence of spectrally well-defined two fluorescent molecules in a small observation area at the single-molecule level. To evaluate the activity of caspase-3 in vivo directly, FCCS was applied to single live cells. We constructed chimeric proteins that consisted of tandemly fused enhanced green FP (EGFP) and monomeric red FP (mRFP). In control experiments, the protease reaction was monitored in solution, where a decrease in cross-correlation amplitude was observed due to specific cleavage of the amino acid sequence between EGFP and mRFP. Moreover, a decrease in cross-correlation amplitude could be detected in a live cell, where caspase-3 activation was induced by apoptosis. This is the first report of FP-based in vivo cross-correlation analysis. FP-based FCCS may become the most versatile method for analysis of protein-protein interactions in live cells.
- Published
- 2004
- Full Text
- View/download PDF
48. TAP/NXF1, the primary mRNA export receptor, specifically interacts with a neuronal RNA-binding protein HuD.
- Author
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Saito K, Fujiwara T, Katahira J, Inoue K, and Sakamoto H
- Subjects
- Animals, Binding Sites, ELAV Proteins, ELAV-Like Protein 4, HeLa Cells, Humans, Nerve Tissue Proteins chemistry, Nerve Tissue Proteins genetics, Neurites metabolism, Nucleocytoplasmic Transport Proteins chemistry, Nucleocytoplasmic Transport Proteins genetics, PC12 Cells, Protein Binding, Protein Structure, Tertiary, RNA, Messenger genetics, RNA-Binding Proteins chemistry, RNA-Binding Proteins genetics, Rats, Sequence Deletion genetics, Nerve Tissue Proteins metabolism, Neurons metabolism, Nucleocytoplasmic Transport Proteins metabolism, RNA Transport, RNA, Messenger metabolism, RNA-Binding Proteins metabolism
- Abstract
Hu proteins are RNA-binding proteins that are implicated in the control of stabilization, nuclear export, and/or translation of specific mRNAs with AU-rich elements (AREs) in the 3'-untranslated region. Three neuron-specific Hu proteins (HuD, HuB, and HuC), but not a ubiquitously expressed Hu protein HuR, have an activity to induce neurite outgrowth when they are overexpressed in a rat neuronal cell line PC12. Here we show that TAP/NXF1, the primary export receptor for the bulk mRNA, is a specific binding partner for HuD. In vitro binding experiments using recombinant proteins revealed that the interaction between TAP and HuD is direct and that HuD can form a ternary complex together with both TAP and RNA. Interestingly, HuR does not interact with TAP. These results suggest that HuD acts as a novel adaptor protein to recruit TAP for efficient export of ARE-containing mRNAs in neuronal cells.
- Published
- 2004
- Full Text
- View/download PDF
49. Genetic variations in humans associated with differences in the course of hepatitis C.
- Author
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Saito T, Ji G, Shinzawa H, Okumoto K, Hattori E, Adachi T, Takeda T, Sugahara K, Ito JI, Watanabe H, Saito K, Togashi H, Ishii K, Matsuura T, Inageda K, Muramatsu M, and Kawata S
- Subjects
- Adult, Aged, Aged, 80 and over, Comorbidity, Disease Progression, Female, Genetic Variation, Hepatitis C blood, Humans, Japan epidemiology, Male, Middle Aged, Polymorphism, Single Nucleotide genetics, Viremia blood, Genetic Predisposition to Disease epidemiology, Genetic Predisposition to Disease genetics, Genetic Testing methods, Hepatitis C epidemiology, Hepatitis C genetics, Phenotype, Viremia epidemiology, Viremia genetics
- Abstract
The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.
- Published
- 2004
- Full Text
- View/download PDF
50. Differentiation of bone marrow cells into cells that express liver-specific genes in vitro: implication of the Notch signals in differentiation.
- Author
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Okumoto K, Saito T, Hattori E, Ito JI, Adachi T, Takeda T, Sugahara K, Watanabe H, Saito K, Togashi H, and Kawata S
- Subjects
- Animals, Bone Marrow Cells cytology, Calcium-Binding Proteins, Cells, Cultured, Hepatocytes cytology, Immunomagnetic Separation, Intercellular Signaling Peptides and Proteins, Jagged-1 Protein, Male, Membrane Proteins genetics, Phenotype, Proteins metabolism, Rats, Rats, Sprague-Dawley, Receptor, Notch1, Serrate-Jagged Proteins, Signal Transduction physiology, Trans-Activators genetics, Trans-Activators metabolism, Bone Marrow Cells physiology, Cell Differentiation physiology, Hepatocytes physiology, Membrane Proteins metabolism, Receptors, Cell Surface, Transcription Factors
- Abstract
Bone marrow (BM) stem cells have been shown to differentiate into liver cells. It remains difficult to sort and culture BM stem cells, and the gene expression of liver-specific proteins in these cells has not been fully investigated. We used a negative selective magnetic cell separation system to obtain stem cell-enriched BM cells. The cells obtained were cultured with hepatocytes or with hepatocyte growth factor (HGF), and the differentiation of BM cells into cells expressing liver-specific genes, hepatocyte nuclear factor (HNF) 1alpha, cytokeratin (CK) 8, alpha-fetoprotein (AFP), and albumin was investigated by the reverse transcription-polymerase chain reaction. We also investigated the gene expressions of Notch receptor-1 (Notch-1) and its ligand Jagged-1 in BM cell differentiation. Sorted BM cells showed positive for Sca-1 (Ataxin-1) by immunofluorescence staining. Fluorescence activated cell sorter analysis showed that 32.6% of sorted BM cells had a high level of expression of the hematopoietic stem cell marker CD90 (Thy-1). When cultured with hepatocytes, these cells expressed the liver-specific genes HNF1alpha and CK8 on culture day 3, AFP and albumin on culture day 7. When cultured with HGF (20ng/ml), the cells expressed HNF1alpha on day 3 and CK8 on day 7. Gene expressions of Notch-1 and Jagged-1 were detected in cultured BM cells on day 3. These results suggest that the negative selective magnetic cell separation system is useful for the rapid preparation of stem cell-enriched BM cells, and that the Notch signaling pathway plays a role in BM cell differentiation into a hepatocyte lineage in vitro.
- Published
- 2003
- Full Text
- View/download PDF
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