Publications by authors named "Masaaki Ikeda"

41 Publications

Involvement of Oct4-type transcription factor Pou5f3 in posterior spinal cord formation in zebrafish embryos.

Dev Growth Differ 2021 Aug 18;63(6):306-322. Epub 2021 Aug 18.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama City, Japan.

In vertebrate embryogenesis, elongation of the posterior body is driven by de novo production of the axial and paraxial mesoderm as well as the neural tube at the posterior end. This process is presumed to depend on the stem cell-like population in the tail bud region, but the details of the gene regulatory network involved are unknown. Previous studies suggested the involvement of pou5f3, an Oct4-type POU gene in zebrafish, in axial elongation. In the present study, we first found that pou5f3 is expressed mainly in the dorsal region of the tail bud immediately after gastrulation, and that this expression is restricted to the posterior-most region of the elongating neural tube during somitogenesis. This pou5f3 expression was complementary to the broad expression of sox3 in the neural tube, and formed a sharp boundary with specific expression of tbxta (orthologue of mammalian T/Brachyury) in the tail bud, implicating pou5f3 in the specification of tail bud-derived cells toward neural differentiation in the spinal cord. When pou5f3 was functionally impaired after gastrulation by induction of a dominant-interfering pou5f3 mutant gene (en-pou5f3), trunk and tail elongation were markedly disturbed at distinct positions along the axis depending on the stage. This finding showed involvement of pou5f3 in de novo generation of the body from the tail bud. Conditional functional abrogation also showed that pou5f3 downregulates mesoderm-forming genes but promotes neural development by activating neurogenesis genes around the tail bud. These results suggest that pou5f3 is involved in formation of the posterior spinal cord.
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http://dx.doi.org/10.1111/dgd.12742DOI Listing
August 2021

Involvement of an Oct4-related PouV gene, pou5f3/pou2, in neurogenesis in the early neural plate of zebrafish embryos.

Dev Biol 2020 01 11;457(1):30-42. Epub 2019 Sep 11.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan. Electronic address:

In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4-related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3-expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2, and also soxB1, but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1, a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3, her5, and her9, further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19 cells showed that pou5f3 was repressed by neurog1, but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3-mediated gene regulatory network in neural development in vertebrate embryos.
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http://dx.doi.org/10.1016/j.ydbio.2019.09.002DOI Listing
January 2020

miR-222 regulates proliferation of primary mouse hepatocytes in vitro.

Biochem Biophys Res Commun 2019 04 27;511(3):644-649. Epub 2019 Feb 27.

Department of Biochemistry, Nagasaki University School of Medicine, Nagasaki, Japan. Electronic address:

It is well known that hepatocytes regenerate after liver injury, although it is difficult to reproduce this phenomenon in vitro. The goal of this research was to determine the factors that stimulate proliferation of primary mouse hepatocytes (PMHs) in vitro. We first tested knockdown (KD) of tumor protein 53 (p53) alone as well as partial hepatectomy (PH, performed 72 h prior to PMHs preparation) alone. However, neither intervention stimulated hepatocyte proliferation during the 72-h observation period in vitro. We then tested the combination of p53 KD with PH and found that these interventions together stimulated cell proliferation in vitro. Under these latter conditions we analyzed gene expression of these cells by mRNA sequencing (RNA-seq) and microRNA sequencing (miRNA-seq). TargetScan analysis, which determines the relationship between microRNAs and gene expression, found a relationship between downregulated mmu-mir-222 (miR-222) and upregulated genes such as mitogen-activated protein kinase kinase kinase 2 (Map3k2). To confirm this relationship, we performed miR-222 KD and overexpression (OE) and observed the expected changes in target gene expression. Furthermore, the finding that miR-222 KD or OE stimulates or suppresses, respectively, hepatocyte proliferation is well explained by the association between miR-222 and its target genes, which stimulate growth. Our results suggest that miR-222 is one of the key factors regulating PMH proliferation in vitro.
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http://dx.doi.org/10.1016/j.bbrc.2019.02.093DOI Listing
April 2019

Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth.

Sci Adv 2019 01 23;5(1):eaau9060. Epub 2019 Jan 23.

Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan.

Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.
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http://dx.doi.org/10.1126/sciadv.aau9060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357737PMC
January 2019

Evaluation of Teneligliptin Effects on Transcriptional Activity of PPARγ in Cell-Based Assays.

J Nippon Med Sch 2018 ;85(2):95-101

Department of Diabetes and Endocrinology, Saitama Medical University.

Background: The antidiabetic drug teneligliptin is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor with a thiazolidine-specific structure. This study aimed to investigate whether teneligliptin can activate PPARγ directly and/or indirectly in cell-based assays.

Methods: Promoter assays using the reporter construct driven under the control of the SV40 promoter and the PPAR response element (PPRE) were performed. Luciferase activity was measured after a 3-day incubation of vector-transduced cells with various concentrations of teneligliptin.

Results: Treatment of the cells with 50 μM teneligliptin significantly transactivated a reporter gene. The presence of the PPARγ antagonist, GW9662, did not affect the activation of PPRE-reporter expression by teneligliptin.

Conclusion: We found that teneligliptin could increase PPARγ activity in cell-based assays irrespective of the PPARγ ligand-binding domain.
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http://dx.doi.org/10.1272/jnms.2018_85-15DOI Listing
August 2018

In vitro analysis of the transcriptional regulatory mechanism of zebrafish pou5f3.

Exp Cell Res 2018 03 31;364(1):28-41. Epub 2018 Jan 31.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan; Saitama University Brain Science Institute, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan. Electronic address:

Zebrafish pou5f3 (previously named pou2), a close homologue of mouse Oct4, encodes a PouV-family transcription factor. pou5f3 has been implicated in diverse aspects of developmental regulation during embryogenesis. In the present study, we addressed the molecular function of Pou5f3 as a transcriptional regulator and the mechanism by which pou5f3 expression is transcriptionally regulated. We examined the influence of effector genes on the expression of the luciferase gene under the control of the upstream 2.1-kb regulatory DNA of pou5f3 (Luc-2.2) in HEK293T and P19 cells. We first confirmed that Pou5f3 functions as a transcriptional activator both in cultured cells and embryos, which confirmed autoregulation of pou5f3 in embryos. It was further shown that Luc-2.2 was activated synergistically by pou5f3 and sox3, which is similar to the co-operative activity of Oct4 and Sox2 in mice, although synergy between pou5f3 and sox2 was less obvious in this zebrafish system. The effects of pou5f3 deletion constructs on the regulation of Luc-2.2 expression revealed different roles for the three subregions of the N-terminal region in Pou5f3 in terms of its regulatory functions and co-operativity with Sox3. Electrophoretic mobility shift assays confirmed that Pou5f3 and Sox3 proteins specifically bind to adjacent sites in the 2.1-kb DNA and that there is an interaction between the two proteins. The synergy with sox3 was unique to pou5f3-the other POU factor genes examined did not show such synergy in Luc-2.2 regulation. Finally, functional interaction was observed between pou5f3 and sox3 in embryos in terms of the regulation of dorsoventral patterning and convergent extension movement. These findings together demonstrate co-operative functions of pou5f3 and sox3, which are frequently coexpressed in early embryos, in the regulation of early development.
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http://dx.doi.org/10.1016/j.yexcr.2018.01.023DOI Listing
March 2018

Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca signaling in human retinal pigment epithelial cells.

Sci Rep 2017 03 9;7:44175. Epub 2017 Mar 9.

Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama city, Toyama 930-8555, Japan.

Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks.
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http://dx.doi.org/10.1038/srep44175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343479PMC
March 2017

Structural and mechanistic insights into nuclear transport and delivery of the critical pluripotency factor Oct4 to DNA.

J Biomol Struct Dyn 2018 02 17;36(3):767-778. Epub 2017 Feb 17.

a Graduate School of Integrated Basic Sciences , Nihon University , 3-25-40 Sakurajousui, Setagaya-ku, Tokyo 156-8550 , Japan.

Oct4 is a master regulator of the induction and maintenance of cellular pluripotency, and has crucial roles in early stages of differentiation. It is the only factor that cannot be substituted by other members of the same protein family to induce pluripotency. However, although Oct4 nuclear transport and delivery to target DNA are critical events for reprogramming to pluripotency, little is known about the molecular mechanism. Oct4 is imported to the nucleus by the classical nuclear transport mechanism, which requires importin α as an adaptor to bind the nuclear localization signal (NLS). Although there are structures of complexes of the NLS of transcription factors (TFs) in complex with importin α, there are no structures available for complexes involving intact TFs. We have therefore modeled the structure of the complex of the whole Oct4 POU domain and importin α2 using protein-protein docking and molecular dynamics. The model explains how the Ebola virus VP24 protein has a negative effect on the nuclear import of STAT1 by importin α but not on Oct4, and how Nup 50 facilitates cargo release from importin α. The model demonstrates the structural differences between the Oct4 importin α bound and DNA bound crystal states. We propose that the 'expanded linker' between the two DNA-binding domains of Oct4 is an intrinsically disordered region and that its conformational changes have a key role in the recognition/binding to both DNA and importin α. Moreover, we propose that this structural change enables efficient delivery to DNA after release from importin α.
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http://dx.doi.org/10.1080/07391102.2017.1289124DOI Listing
February 2018

Release from optimal compressive force suppresses osteoclast differentiation.

Mol Med Rep 2016 Nov 5;14(5):4699-4705. Epub 2016 Oct 5.

Department of Orthodontics, Hokkaido University Graduate School of Dental Medicine, Sapporo, Hokkaido 060‑8586, Japan.

Bone remodeling is an important factor in orthodontic tooth movement. During orthodontic treatment, osteoclasts are subjected to various mechanical stimuli, and this promotes or inhibits osteoclast differentiation and fusion. It has been previously reported that the release from tensile force induces osteoclast differentiation. However, little is known about how release from compressive force affects osteoclasts. The present study investigated the effects of release from compressive force on osteoclasts. The number of tartrate‑resistant acid phosphatase (TRAP)‑positive multinucleated osteoclasts derived from RAW264.7 cells was counted, and gene expression associated with osteoclast differentiation and fusion in response to release from compressive force was evaluated by reverse transcription‑quantitative polymerase chain reaction. Osteoclast number was increased by optimal compressive force application. On release from this force, osteoclast differentiation and fusion were suppressed. mRNA expression of NFATc1 was inhibited for 6 h subsequent to release from compressive force. mRNA expression of the other osteoclast‑specific genes, TRAP, RANK, matrix metalloproteinase‑9, cathepsin‑K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell‑specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC‑STAMP) was significantly inhibited at 3 h following release from compressive force compared with control cells. These findings suggest that release from optimal compressive force suppresses osteoclast differentiation and fusion, which may be important for developing orthodontic treatments.
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http://dx.doi.org/10.3892/mmr.2016.5801DOI Listing
November 2016

Circadian adaptation to cell injury stresses: a crucial interplay of BMAL1 and HSF1.

J Physiol Sci 2016 Jul 24;66(4):303-6. Epub 2016 Feb 24.

Department of Physiology, Faculty of Medicine, Saitama Medical University, 38 Morohongo Moroyama-machi, Iruma-gun, Saitama, 350-0495, Japan.

The circadian clock system confers daily anticipatory physiological processes with the ability to be reset by environmental cues. This "circadian adaptation system" (CAS), driven by cell-autonomous molecular clocks, orchestrates various rhythmic physiological processes in the entire body. Hence, the dysfunction of these clocks exacerbates various diseases, which may partially be due to the impairment of protective pathways. If this is the case, how does the CAS respond to cell injury stresses that are critical in maintaining health and life by evoking protective pathways? To address this question, here we review and discuss recent evidence revealing life-protective (pro-survival) molecular networks between clock (e.g., BMAL1, CLOCK, and PER2) and adaptation (e.g., HSF1, Nrf2, NF-κB, and p53) pathways, which are evoked by various cell injury stresses (e.g., heat, reactive oxygen species, and UV). The CK2 protein kinase-integrated interplay of the BMAL1 (clock) and HSF1 (heat-shock response) pathways is one of the crucial events in CAS.
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http://dx.doi.org/10.1007/s12576-016-0436-5DOI Listing
July 2016

The impact of HIF1α on the Per2 circadian rhythm in renal cancer cell lines.

PLoS One 2014 21;9(10):e109693. Epub 2014 Oct 21.

Department of Physiology, Saitama Medical University, Saitama, Japan; Molecular Clock Project, Project Research Division, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan.

In mammals, the circadian rhythm central generator consists of interactions among clock genes, including Per1/2/3, Cry1/2, Bmal1, and Clock. Circadian rhythm disruption may lead to increased risk of cancer in humans, and deregulation of clock genes has been implicated in many types of cancers. Among these genes, Per2 is reported to have tumor suppressor properties, but little is known about the correlation between Per2 and HIF, which is the main target of renal cell carcinoma (RCC) therapy. In this study, the rhythmic expression of the Per2 gene was not detectable in renal cancer cell lines, with the exception of Caki-2 cells. In Caki-2 cells, HIF1α increased the amplitude of Per2 oscillation by directly binding to the HIF-binding site located on the Per2 promoter. These results indicate that HIF1α may enhance the amplitude of the Per2 circadian rhythm.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109693PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204850PMC
June 2015

Bmal1 is an essential regulator for circadian cytosolic Ca²⁺ rhythms in suprachiasmatic nucleus neurons.

J Neurosci 2014 Sep;34(36):12029-38

Department of Physiology, Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan.

The hypothalamic suprachiasmatic nucleus (SCN) plays a pivotal role in the mammalian circadian clock system. Bmal1 is a clock gene that drives transcriptional-translational feedback loops (TTFLs) for itself and other genes, and is expressed in nearly all SCN neurons. Despite strong evidence that Bmal1-null mutant mice display arrhythmic behavior under constant darkness, the function of Bmal1 in neuronal activity is unknown. Recently, periodic changes in the levels of intracellular signaling messengers, such as cytosolic Ca(2+) and cAMP, were suggested to regulate TTFLs. However, the opposite aspect of how clock gene TTFLs regulate cytosolic signaling remains unclear. To investigate intracellular Ca(2+) dynamics under Bmal1 perturbations, we cotransfected some SCN neurons with yellow cameleon together with wild-type or dominant-negative Bmal1 using a gene-gun applied for mouse organotypic cultures. Immunofluorescence staining for a tag protein linked to BMAL1 showed nuclear expression of wild-type BMAL1 and its degradation within 1 week after transfection in SCN neurons. However, dominant-negative BMAL1 did not translocate into the nucleus and the cytosolic signals persisted beyond 1 week. Consistently, circadian Ca(2+) rhythms in SCN neurons were inhibited for longer periods by dominant-negative Bmal1 overexpression. Furthermore, SCN neurons transfected with a Bmal1 shRNA lengthened, whereas those overexpressing wild-type Bmal1 shortened, the periods of Ca(2+) rhythms, with a significant reduction in their amplitude. BMAL1 expression was intact in the majority of neighboring neurons in organotypic cultures. Therefore, we conclude that proper intrinsic Bmal1 expression, but not passive signaling via cell-to-cell interactions, is the determinant of circadian Ca(2+) rhythms in SCN neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.5158-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608459PMC
September 2014

Identification of two nickel ion-induced genes, NCI16 and PcGST1, in Paramecium caudatum.

Eukaryot Cell 2014 Sep 7;13(9):1181-90. Epub 2014 Jul 7.

Department of Diabetes and Endocrinology, Saitama Medical University, Saitama, Japan.

Here, we describe the isolation of two nickel-induced genes in Paramecium caudatum, NCI16 and PcGST1, by subtractive hybridization. NCI16 encoded a predicted four-transmembrane domain protein (∼16 kDa) of unknown function, and PcGST1 encoded glutathione S-transferase (GST; ∼25 kDa) with GST and glutathione peroxidase (GPx) activities. Exposing cells to cobalt chloride also caused the moderate upregulation of NCI16 and PcGST1 mRNAs. Both nickel sulfate and cobalt chloride dose dependently induced NCI16 and PcGST1 mRNAs, but with different profiles. Nickel treatment caused a continuous increase in PcGST1 and NCI16 mRNA levels for up to 3 and 6 days, respectively, and a notable increase in H₂O₂ concentrations in P. caudatum. NCI16 expression was significantly enhanced by incubating cells with H₂O₂, implying that NCI16 induction in the presence of nickel ions is caused by reactive oxygen species (ROS). On the other hand, PcGST1 was highly induced by the antioxidant tert-butylhydroquinone (tBHQ) but not by H2O2, suggesting that different mechanisms mediate the induction of NCI16 and PcGST1. We introduced a luciferase reporter vector with an ∼0.42-kb putative PcGST1 promoter into cells and then exposed the transformants to nickel sulfate. This resulted in significant luciferase upregulation, indicating that the putative PcGST1 promoter contains a nickel-responsive element. Our nickel-inducible system also may be applicable to the efficient expression of proteins that are toxic to host cells or require temporal control.
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http://dx.doi.org/10.1128/EC.00112-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187627PMC
September 2014

A Novel Splicing Variant of Peroxisome Proliferator-Activated Receptor-γ (Pparγ1sv) Cooperatively Regulates Adipocyte Differentiation with Pparγ2.

PLoS One 2013 19;8(6):e65583. Epub 2013 Jun 19.

Department of Diabetes and Endocrinology, Saitama Medical University, Saitama, Japan.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate expression of a number of genes associated with the cellular differentiation and development. Here, we show the abundant and ubiquitous expression of a newly identified splicing variant of mouse Pparγ (Pparγ1sv) that encodes PPARγ1 protein, and its importance in adipogenesis. The novel splicing variant has a unique 5'-UTR sequence, relative to those of Pparγ1 and Pparγ2 mRNAs, indicating the presence of a novel transcriptional initiation site and promoter for Pparγ expression. Pparγ1sv was highly expressed in the white and brown adipose tissues at levels comparable to Pparγ2. Pparγ1sv was synergistically up-regulated with Pparγ2 during adipocyte differentiation of 3T3-L1 cells and mouse primary cultured preadipocytes. Inhibition of Pparγ1sv by specific siRNAs completely abolished the induced adipogenesis in 3T3-L1 cells. C/EBPβ and C/EBPδ activated both the Pparγ1sv and Pparγ2 promoters in 3T3-L1 preadipocytes. These findings suggest that Pparγ1sv and Pparγ2 synergistically regulate the early stage of the adipocyte differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065583PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686765PMC
October 2017

Droplet manipulation by an external electric field for crystalline film growth.

Langmuir 2013 Jul 16;29(30):9592-7. Epub 2013 Jul 16.

Center for Organic Photonics and Electronics Research, Kyushu University, 744 Motooka, Nishi, Fukuoka 819-0395, Japan.

Combining droplet manipulation by the application of an electric field with inkjet printing is proposed as a unique technique to control the surface wettability of substrates for solution-processed organic field-effect transistors (FETs). With the use of this technique, uniform thin films of 2,7-dioctyl[1]benzothieno[2,3,-b][1]benzothiopene (C8-BTBT) could be fabricated on the channels of FET substrates without self-assembled monolayer treatment. High-speed camera observation revealed that the crystals formed at the solid/liquid interface. The coverage of the crystals on the channels depended on the ac frequency of the external electric field applied during film formation, leading to a wide variation in the carrier transport of the films. The highest hole mobility of 0.03 cm(2) V(-1) s(-1) was obtained when the coverage was maximized with an ac frequency of 1 kHz.
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http://dx.doi.org/10.1021/la401729kDOI Listing
July 2013

In vitro circadian period is associated with circadian/sleep preference.

Sci Rep 2013 ;3:2074

Department of Psychophysiology, National Institute of Mental Health, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8553, Japan.

Evaluation of circadian phenotypes is crucial for understanding the pathophysiology of diseases associated with disturbed biological rhythms such as circadian rhythm sleep disorders (CRSDs). We measured clock gene expression in fibroblasts from individual subjects and observed circadian rhythms in the cells (in vitro rhythms). Period length of the in vitro rhythm (in vitro period) was compared with the intrinsic circadian period, τ, measured under a forced desynchrony protocol (in vivo period) and circadian/sleep parameters evaluated by questionnaires, sleep log, and actigraphy. Although no significant correlation was observed between the in vitro and in vivo periods, the in vitro period was correlated with chronotype, habitual sleep time, and preferred sleep time. Our data demonstrate that the in vitro period is significantly correlated with circadian/sleep preference. The findings suggest that fibroblasts from individual patients can be utilized for in vitro screening of therapeutic agents to provide personalized therapeutic regimens for CRSD patients.
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http://dx.doi.org/10.1038/srep02074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691610PMC
October 2013

Real-time analysis of the circadian oscillation of the Rev-Erb β promoter.

J Atheroscler Thromb 2013 7;20(3):267-76. Epub 2012 Dec 7.

Department of Neurology, Meitan General Hospital, Beijing, China.

Aim: Rev-Erb β gene plays crucial roles in circadian rhythm, lipid and glucose metabolism, and several diseases. The molecular mechanisms of the transcriptional regulation of Rev-Erb β that generate and determine the phase of the circadian oscillation remain unclear.

Methods: We analyzed the Rev-Erb β promoter by luciferase reporter assays, real-time bioluminescence monitoring assays and electrophoretic mobility shift assays.

Results: Luciferase reporter assays indicated that only the 5' region and exon 1 have obvious promoter activity. Real-time bioluminescence monitoring assays revealed that E1, E2, E3, D boxes are important for maintenance of the amplitude of Rev-Erb β oscillation. Based on EMSA results, REV-ERBβ binds ROREs in the Bmal1 promoter region and inhibits Bmal1 promoter activity.

Conclusion: We provide direct evidence that three E-boxes and one D-box located in the first intron are crucial for the phase of circadian oscillation in Rev-Erb β expression and that the sequences upstream from its transcription start site function as a promoter with no circadian regulation. We also found that the E1 box affects the Rev-Erb β oscillation phase. Our results offer new insight into the role of Rev-Erb β in the circadian rhythm system.
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http://dx.doi.org/10.5551/jat.14381DOI Listing
September 2013

A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands.

PLoS One 2012 15;7(5):e37093. Epub 2012 May 15.

Health Research Institute, National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka, Japan.

In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co-culture system is a useful tool to analyze humoral communication between different tissues or cell populations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037093PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3352896PMC
January 2013

Organic transistors with high thermal stability for medical applications.

Nat Commun 2012 Mar 6;3:723. Epub 2012 Mar 6.

Department of Applied Physics, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

The excellent mechanical flexibility of organic electronic devices is expected to open up a range of new application opportunities in electronics, such as flexible displays, robotic sensors, and biological and medical electronic applications. However, one of the major remaining issues for organic devices is their instability, especially their thermal instability, because low melting temperatures and large thermal expansion coefficients of organic materials cause thermal degradation. Here we demonstrate the fabrication of flexible thin-film transistors with excellent thermal stability and their viability for biomedical sterilization processes. The organic thin-film transistors comprise a high-mobility organic semiconductor, dinaphtho[2,3-b:2',3'-f]thieno[3,2-b]thiophene, and thin gate dielectrics comprising a 2-nm-thick self-assembled monolayer and a 4-nm-thick aluminium oxide layer. The transistors exhibit a mobility of 1.2 cm(2) V(-1)s(-1) within a 2 V operation and are stable even after exposure to conditions typically used for medical sterilization.
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http://dx.doi.org/10.1038/ncomms1721DOI Listing
March 2012

Contact resistance and megahertz operation of aggressively scaled organic transistors.

Small 2012 Jan 17;8(1):73-9. Epub 2011 Nov 17.

Max Planck Institute for Solid State Research, Heisenbergstr. 1, 70569 Stuttgart, Germany.

Bottom-gate, top-contact organic thin-film transistors (TFTs) with excellent static characteristics (on/off ratio: 10(7) ; intrinsic mobility: 3 cm(2) (V s)(-1) ) and fast unipolar ring oscillators (signal delay as short as 230 ns per stage) are fabricated. The significant contribution of the transfer length to the relation between channel length, contact length, contact resistance, effective mobility, and cutoff frequency of the TFTs is theoretically and experimentally analyzed.
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http://dx.doi.org/10.1002/smll.201101677DOI Listing
January 2012

Contact doping and ultrathin gate dielectrics for nanoscale organic thin-film transistors.

Small 2011 May 15;7(9):1186-91. Epub 2011 Apr 15.

Max Planck Institute for Solid State Research, Heisenbergstr. 1, 70569 Stuttgart, Germany.

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http://dx.doi.org/10.1002/smll.201002254DOI Listing
May 2011

Organic electronics on banknotes.

Adv Mater 2011 Feb 6;23(5):654-8. Epub 2010 Dec 6.

Max Planck Institute for Solid State Research, Heisenbergstrasse 1, Stuttgart, Germany.

Organic transistors and circuits are fabricated directly on the surface of banknotes. The transistors operate with voltages of 3 V and have a field-effect mobility of about 0.2 cm2 V−1s−1. For an array of 100 transistors a yield of 92% is obtained.
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http://dx.doi.org/10.1002/adma.201003374DOI Listing
February 2011

Alkylated dinaphtho[2,3-b:2',3'-f]thieno[3,2-b]thiophenes (C(n)-DNTTs): organic semiconductors for high-performance thin-film transistors.

Adv Mater 2011 Mar 20;23(10):1222-5. Epub 2010 Aug 20.

Department of Applied Chemistry, Graduate School of Engineering, Hiroshima University, Higashi-Hiroshima, Japan.

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http://dx.doi.org/10.1002/adma.201001283DOI Listing
March 2011

Dual-color luciferase mouse directly demonstrates coupled expression of two clock genes.

Biochemistry 2010 Sep;49(37):8053-61

National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.

We have established a dual-color transgenic mouse that simultaneously reports the expression of two clock genes, Bmal1 and Per2, in a single tissue. The expression of the two genes is monitored with green- and red-emitting beetle luciferases with a single luminescent substrate. Antiphasic oscillations of Bmal1 and Per2, consistent with their endogenous mRNA profiles, were clearly monitored in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, and in the peripheral tissues, demonstrating that the system allows the long-term, quantitative, and simultaneous monitoring of the expression of the two genes. We also showed that although the expression patterns of Bmal1 and Per2 in each organ are strictly antiphasic, the recorded circadian phases and periods of both genes varied between organs. The phase shifts in the expression of both genes in the SCN, induced by a change of medium, also occurred in a similar manner. Therefore, this dual-color luciferase mouse allows noninvasive and continuous monitoring of the coupled expression of two clock genes. This system provides a simple technique with which to unravel the complex interactions of two genes in the body.
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http://dx.doi.org/10.1021/bi100545hDOI Listing
September 2010

A promoter in the novel exon of hPPARgamma directs the circadian expression of PPARgamma.

J Atheroscler Thromb 2010 Feb 21;17(1):73-83. Epub 2010 Jan 21.

Department of Diabetes and Endocrinology, Saitama Medical University, Saitama 350-0495, Japan.

Aim: PPARgamma (peroxisome proliferator-activated receptor gamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes associated with lipid metabolism. Herein, we show that expression levels of the novel PPARgamma transcript exhibit circadian oscillation. To study the mechanisms controlling PPARgamma expression, a novel PPARgamma gene promoter was cloned and characterized.

Methods: We analyzed the novel PPARgamma promoter by luciferase reporter assays and gel shift analysis.

Results: Surprisingly, it was not an intron but rather the novel first exon of PPARgamma that was found to have functional minimal promoter activity. Luciferase reporter assays and gel shift assays revealed that the novel first exon is essential for novel PPARgamma promoter activation and that DBP (albumin gene D-site binding protein) and E4BP4 (E4 promoter A binding protein 4) bind directly to D-sites in the novel first exon.

Conclusion: Our results demonstrate that the PAR-bZIP (bZIP, basic leucine zipper) family and E4BP4 are the main regulatory factors involved in oscillation of novel PPARgamma expression. This regulatory mechanism clearly differs from that of the circadian expression of PPARalpha.
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http://dx.doi.org/10.5551/jat.2410DOI Listing
February 2010

One-pot synthesis of benzo[b]thiophenes and benzo[b]selenophenes from o-halo-substituted ethynylbenzenes: convenient approach to mono-, bis-, and tris-chalcogenophene-annulated benzenes.

Org Lett 2009 Jun;11(11):2473-5

Department of Applied Chemistry, Graduate School of Engineering, Hiroshima University, Higashi-Hiroshima 739-8527, Japan.

A convenient one-pot procedure for the synthesis of benzo[b]thiophenes and selenophenes from readily available o-halo-ethynylbenzene precursors is described. Regardless of the substituent on the acetylene terminus or the number of cyclization moieties on the precursors, various benzo[b]thiophenes and selenophenes, including not only the parent, alkyl-, and phenyl-substituted derivatives but also benzo[1,2-b:4,5-b']dithiophenes and diselenophenes and benzo[1,2-b:3,4-b':5,6-b'']trithiophenes and triselenophenes can be prepared in good to high yields.
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http://dx.doi.org/10.1021/ol900809wDOI Listing
June 2009

The molecular mechanism regulating the autonomous circadian expression of Topoisomerase I in NIH3T3 cells.

Biochem Biophys Res Commun 2009 Feb 10;380(1):22-7. Epub 2009 Jan 10.

Department of Physiology, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan.

To identify whether Topoisomerase I (TopoI) has autonomous circadian rhythms regulated by clock genes, we tested mouse TopoI (mTopoI) promoter oscillation in NIH3T3 cells using a real-time monitoring assay and TopoI mRNA oscillations using real-time RT-PCR. Analysis of the mTopoI promoter region with Matlnspector software revealed two putative E-box (E1 and E2) and one DBP/E4BP4-binding element (D-box). Luciferase assays indicated that mTopoI gene expression was directly regulated by clock genes. The real-time monitoring assay showed that E-box and D-box response elements participate in the regulation of the circadian expression of mTopoI. Furthermore, a gel-shift assay showed that E2 is a direct target of the BMAL1/CLOCK heterodimer and DBP binds to the putative D-site. These results indicate that TopoI is expressed in an autonomous circadian rhythm in NIH3T3 cells.
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http://dx.doi.org/10.1016/j.bbrc.2008.12.186DOI Listing
February 2009

Statins Activate Human PPARalpha Promoter and Increase PPARalpha mRNA Expression and Activation in HepG2 Cells.

PPAR Res 2008 24;2008:316306. Epub 2008 Dec 24.

Department of Biochemistry, Faculty of Medicine, Saitama Medical University, Saitama 350-0495, Japan.

Statins increase peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA expression, but the mechanism of this increased PPARalpha production remains elusive. To examine the regulation of PPARalpha production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin) on human PPARalpha promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1) Majority of statins enhanced PPARalpha promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPARalpha promoter. This enhancement may be mediated by statin-induced HNF-4alpha. (2) PPARalpha mRNA expression was increased by statin treatment. (3) The PPARalpha levels in nuclear fractions were increased by statin treatment. (4) Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPARalpha/RXRalpha expression vectors. In summary, these data demonstrate that PPARalpha production and activation are upregulated through the PPARalpha promoter activity by statin treatment.
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http://dx.doi.org/10.1155/2008/316306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610383PMC
July 2011

Establishment of human cell lines showing circadian rhythms of bioluminescence.

Neurosci Lett 2008 Nov 17;446(1):40-4. Epub 2008 Sep 17.

Department of Human Genetics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

We have established human retinal pigment epithelial cell lines stably expressing the luciferase gene, driven by the human Bmal1 promoter, to obtain human-derived cells that show circadian rhythms of bioluminescence after dexamethasone treatment. The average circadian period of bioluminescence for the obtained clones was 24.07+/-0.48 h. Lithium (10 mM) in the medium significantly lengthened the circadian period of bioluminescence, which is consistent with previous reports, while 2 mM or 5 mM lithium had no effect. This is the first report on the establishment of human-derived cell lines that proliferate infinitely and show circadian rhythms of bioluminescence, and also the first to investigate the effects of low-dose lithium on the circadian rhythms of human-derived cells in vitro. The established cells will be useful for various in vitro studies of human circadian rhythms and for the development of new therapies for human disorders related to circadian rhythm disturbances.
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http://dx.doi.org/10.1016/j.neulet.2008.08.091DOI Listing
November 2008
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