Publications by authors named "Yui Yamashita"

37 Publications

Global analysis of boron-induced ribosome stalling reveals its effects on translation termination and unique regulation by AUG-stops in Arabidopsis shoots.

Plant J 2021 Jun 26;106(5):1455-1467. Epub 2021 Apr 26.

Graduate School of Agricultural and Life Science, University of Tokyo, Tokyo, 113-8657, Japan.

We previously reported that ribosome stalling at AUG-stop sequences in the 5'-untranslated region plays a critical role in regulating the expression of Arabidopsis thaliana NIP5;1, which encodes a boron uptake transporter, in response to boron conditions in media. This ribosome stalling is triggered specifically by boric acid, but the mechanisms are unknown. Although upstream open reading frames (uORFs) are known in many cases to regulate translation through peptides encoded by the uORF, AUG-stop stalling does not involve any peptide synthesis. The unique feature of AUG-stops - that termination follows immediately after initiation - suggests a possible effect of boron on the translational process itself. However, the generality of AUG-stop-mediated translational regulation and the effect of boron on translation at the genome scale are not clear. Here, we conducted a ribosome profiling analysis to reveal the genome-wide regulation of translation in response to boron conditions in A. thaliana shoots. We identified hundreds of translationally regulated genes that function in various biological processes. Under high-boron conditions, transcripts with reduced translation efficiency were rich in uORFs, highlighting the importance of uORF-mediated translational regulation. We found 673 uORFs that had more frequent ribosome association. Moreover, transcripts that were translationally downregulated under high-boron conditions were rich in minimum uORFs (AUG-stops), suggesting that AUG-stops play a global role in the boron response. Metagene analysis revealed that boron increased the ribosome occupancy of stop codons, indicating that this element is involved in global translational termination processes.
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http://dx.doi.org/10.1111/tpj.15248DOI Listing
June 2021

Optineurin defects cause TDP43-pathology with autophagic vacuolar formation.

Neurobiol Dis 2021 01 6;148:105215. Epub 2020 Dec 6.

Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Electronic address:

We previously showed that optineurin (OPTN) mutations lead to the development of amyotrophic lateral sclerosis. The association between OPTN mutations and the pathogenesis of amyotrophic lateral sclerosis remains unclear. To investigate the mechanism underlying its pathogenesis, we generated Optn knockout mice. We evaluated histopathological observations of these mice and compared with those of OPTN- amyotrophic lateral sclerosis cases to investigate the mechanism underlying the pathogenesis of amyotrophic lateral sclerosis caused by OPTN mutations. The Optn (-/-) mice presented neuronal autophagic vacuoles immunopositive for charged multivesicular body protein 2b, one of the hallmarks of granulovacuolar degenerations, in the cytoplasm of spinal cord motor neurons at the age of 8 months and the OPTN- amyotrophic lateral sclerosis case with homozygous Q398X mutation. In addition, Optn (-/-) mice showed TAR-DNA binding protein 43/sequestosome1/p62 -positive cytoplasmic inclusions and the clearance of nuclear TAR-DNA binding protein 43. The axonal degeneration of the sciatic nerves was observed in Optn (-/-) mice. However, we could not observe significant differences in survival time, body weight, and motor functions, at 24 months. Our findings suggest that homozygous OPTN deletion or mutations might result in autophagic dysfunction and TAR-DNA binding protein 43 mislocalization, thereby leading to neurodegeneration of motor neurons. These findings indicate that the Optn (-/-) mice recapitulate both common and specific pathogenesis of amyotrophic lateral sclerosis associated with autophagic abnormalities. Optn (-/-) mice could serve as a mouse model for the development of therapeutic strategies.
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http://dx.doi.org/10.1016/j.nbd.2020.105215DOI Listing
January 2021

Comprehensive genome-wide identification of angiosperm upstream ORFs with peptide sequences conserved in various taxonomic ranges using a novel pipeline, ESUCA.

BMC Genomics 2020 Mar 30;21(1):260. Epub 2020 Mar 30.

Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan.

Background: Upstream open reading frames (uORFs) in the 5'-untranslated regions (5'-UTRs) of certain eukaryotic mRNAs encode evolutionarily conserved functional peptides, such as cis-acting regulatory peptides that control translation of downstream main ORFs (mORFs). For genome-wide searches for uORFs with conserved peptide sequences (CPuORFs), comparative genomic studies have been conducted, in which uORF sequences were compared between selected species. To increase chances of identifying CPuORFs, we previously developed an approach in which uORF sequences were compared using BLAST between Arabidopsis and any other plant species with available transcript sequence databases. If this approach is applied to multiple plant species belonging to phylogenetically distant clades, it is expected to further comprehensively identify CPuORFs conserved in various plant lineages, including those conserved among relatively small taxonomic groups.

Results: To efficiently compare uORF sequences among many species and efficiently identify CPuORFs conserved in various taxonomic lineages, we developed a novel pipeline, ESUCA. We applied ESUCA to the genomes of five angiosperm species, which belong to phylogenetically distant clades, and selected CPuORFs conserved among at least three different orders. Through these analyses, we identified 89 novel CPuORF families. As expected, ESUCA analysis of each of the five angiosperm genomes identified many CPuORFs that were not identified from ESUCA analyses of the other four species. However, unexpectedly, these CPuORFs include those conserved across wide taxonomic ranges, indicating that the approach used here is useful not only for comprehensive identification of narrowly conserved CPuORFs but also for that of widely conserved CPuORFs. Examination of the effects of 11 selected CPuORFs on mORF translation revealed that CPuORFs conserved only in relatively narrow taxonomic ranges can have sequence-dependent regulatory effects, suggesting that most of the identified CPuORFs are conserved because of functional constraints of their encoded peptides.

Conclusions: This study demonstrates that ESUCA is capable of efficiently identifying CPuORFs likely to be conserved because of the functional importance of their encoded peptides. Furthermore, our data show that the approach in which uORF sequences from multiple species are compared with those of many other species, using ESUCA, is highly effective in comprehensively identifying CPuORFs conserved in various taxonomic ranges.
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http://dx.doi.org/10.1186/s12864-020-6662-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106846PMC
March 2020

Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.

Nucleic Acids Res 2020 02;48(4):1985-1999

Division of Life Science, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which β-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the β-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.
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http://dx.doi.org/10.1093/nar/gkz1190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038982PMC
February 2020

Publisher Correction: Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Nature 2019 Jul;571(7764):E4

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Change history: In this Letter, the bottom blot in Fig. 2g (for 'IB: Myc') was missing. This has been corrected online.
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http://dx.doi.org/10.1038/s41586-019-1360-7DOI Listing
July 2019

Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Nature 2019 06 12;570(7762):538-542. Epub 2019 Jun 12.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails), whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis. Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.
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http://dx.doi.org/10.1038/s41586-019-1307-zDOI Listing
June 2019

The Pro-Oxidant Activity of Pheomelanin is Significantly Enhanced by UVA Irradiation: Benzothiazole Moieties Are More Reactive than Benzothiazine Moieties.

Int J Mol Sci 2018 Sep 23;19(10). Epub 2018 Sep 23.

Department of Chemistry, Fujita Health University School of Health Sciences, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan.

It is generally considered that eumelanin (EM) is photoprotective while pheomelanin (PM) is phototoxic. A recent study using a mouse model demonstrated that PM produces reactive oxygen species (ROS) that cause DNA damage and eventually lead to melanomagenesis. A biochemical study showed that PM possesses a pro-oxidant activity. PM consists of benzothiazine (BT) and benzothiazole (BZ) moieties, BT moieties being transformed to BZ moieties by heat or light. In this study, we compared the effects of ultraviolet A (UVA) irradiation using synthetic PMs with different BT to BZ ratios and using various coat color mouse hairs. We found that UVA irradiation of BZ-PM increased glutathione (GSH) depletion and generated more H₂O₂ than UVA irradiation of BT-PM. Non-irradiated controls did not exhibit strong pro-oxidant activities. Upon UVA irradiation, yellow mouse hairs oxidized GSH and produced H₂O₂ faster than black or albino mouse hairs. Next, to examine the mechanism of the pro-oxidant activity of BT-PM and BZ-PM, we examined the pro-oxidant activities of 7-(2-amino-2-carboxyethyl)-dihydro-1,4-benzothiazine-3-carboxylic acid (DHBTCA) and 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole (BZ-AA) as BT and BZ monomers, respectively. Their pro-oxidant activities were similar, but a large difference was seen in the effects of ROS scavengers, which suggests that the redox reactions may proceed via singlet oxygen in BZ-AA and via superoxide anions in DHBTCA. These results show that UVA enhances the pro-oxidant activity of PM, in particular BZ-PM.
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http://dx.doi.org/10.3390/ijms19102889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213070PMC
September 2018

Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death.

PLoS One 2018 22;13(3):e0194812. Epub 2018 Mar 22.

Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.

We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194812PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864047PMC
July 2018

Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Am J Physiol Cell Physiol 2018 06 7;314(6):C721-C731. Epub 2018 Mar 7.

Department of Nutritional Physiology, Institute of Medical Nutrition, Tokushima University Graduate School , Tokushima , Japan.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.
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http://dx.doi.org/10.1152/ajpcell.00184.2017DOI Listing
June 2018

Effects of growth hormone on thyroid function are mediated by type 2 iodothyronine deiodinase in humans.

Endocrine 2018 02 22;59(2):353-363. Epub 2017 Dec 22.

Department of Diabetes, Endocrinology and Nutrition, Kyoto University Graduate School of Medicine, 54 Kawaharacho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

Purpose: Growth hormone (GH) therapy in adults alters thyroid function, and acromegaly often involves thyroid disease. The present study aimed to elucidate roles and mechanisms of GH in regulating thyroid function.

Methods: We performed two retrospective observational studies, which focused on consecutive patients with severe adult GH deficiency who received recombinant human GH (rhGH) therapy (n = 20) and consecutive patients with acromegaly who underwent transsphenoidal surgery (TSS) (n = 25). In both studies, serum free triiodothyronine (fT3), free thyroxine (fT4), and fT3/fT4 ratio were examined before and after the interventions. We subsequently administered GH to four human cell lines (HepG2, TSA201, MCF7, and HTC/C3) in vitro, and examined changes in mRNA levels of iodothyronine deiodinases (D1, D2, and D3).

Results: Median serum fT3 level significantly increased after rhGH therapy from 2.38 to 2.78 pg/mL (p < 0.001), and fT4 decreased from 1.115 to 1.065 ng/dL (p = 0.081). TSS significantly decreased median serum fT3 from 3.03 to 2.53 pg/mL (p < 0.001), and increased fT4 from 1.230 to 1.370 ng/dL (p < 0.001). In vitro, GH significantly increased D2 expression at the mRNA level in HTC/C3 cells (p < 0.01), as well as D2 protein and its activity.

Conclusions: GH increased serum fT3 level and decreased serum fT4 level in humans. Our results suggest that its mechanism involves D2 upregulation. Considering this GH effect on thyroid hormone metabolism, data on thyroid function could be useful in the management of GH deficiency and acromegaly.
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http://dx.doi.org/10.1007/s12020-017-1495-yDOI Listing
February 2018

RASAL1 is a potent regulator of hepatic stellate cell activity and liver fibrosis.

Oncotarget 2017 Sep 4;8(39):64840-64852. Epub 2017 May 4.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Liver fibrosis, leading to cirrhosis and liver failure, can occur after chronic liver injury. The transition of hepatic stellate cells (HSCs) from quiescent cells into proliferative and fibrogenic cells is a central event in liver fibrosis. Here, we show that RAS protein activator like-1 (RASAL1), a RAS-GTPase-activating protein, which switches off RAS activity, is significantly decreased during HSC activation, and that HSC activation can be antagonized by forced expression of the RASAL1 protein. We demonstrate that RASAL1 suppresses HSC proliferation by regulating the Ras-MAPK pathway, and that RASAL1 suppresses HSC fibrogenic activity by regulating the PKA-LKB1-AMPK-SRF pathway by interacting with angiotensin II receptor, type 1. We also show that RASAL1-deficient mice are more susceptible to liver fibrosis. These data demonstrate that deregulated RASAL1 expression levels and the affected downstream intracellular signaling are central mediators of perpetuated HSC activation and fibrogenesis in the liver.
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http://dx.doi.org/10.18632/oncotarget.17609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630295PMC
September 2017

Circulating osteocrin stimulates bone growth by limiting C-type natriuretic peptide clearance.

J Clin Invest 2017 Nov 9;127(11):4136-4147. Epub 2017 Oct 9.

Department of Diabetes, Endocrinology and Nutrition, and.

Although peptides are safe and useful as therapeutics, they are often easily degraded or metabolized. Dampening the clearance system for peptide ligands is a promising strategy for increasing the efficacy of peptide therapies. Natriuretic peptide receptor B (NPR-B) and its naturally occurring ligand, C-type natriuretic peptide (CNP), are potent stimulators of endochondral bone growth, and activating the CNP/NPR-B system is expected to be a powerful strategy for treating impaired skeletal growth. CNP is cleared by natriuretic peptide clearance receptor (NPR-C); therefore, we investigated the effect of reducing the rate of CNP clearance on skeletal growth by limiting the interaction between CNP and NPR-C. Specifically, we generated transgenic mice with increased circulating levels of osteocrin (OSTN) protein, a natural NPR-C ligand without natriuretic activity, and observed a dose-dependent skeletal overgrowth phenotype in these animals. Skeletal overgrowth in OSTN-transgenic mice was diminished in either CNP- or NPR-C-depleted backgrounds, confirming that CNP and NPR-C are indispensable for the bone growth-stimulating effect of OSTN. Interestingly, double-transgenic mice of CNP and OSTN had even higher levels of circulating CNP and additional increases in bone length, as compared with mice with elevated CNP alone. Together, these results support OSTN administration as an adjuvant agent for CNP therapy and provide a potential therapeutic approach for diseases with impaired skeletal growth.
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http://dx.doi.org/10.1172/JCI94912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663354PMC
November 2017

Measurements of ionic concentrations along with endocochlear potential in wild-type and claudin 14 knockout mice.

Auris Nasus Larynx 2018 Jun 12;45(3):421-426. Epub 2017 Aug 12.

Department of Physiology, Faculty of Medicine, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan; Faculty of Nutrition, Koshien University, 10-1 Momijimachi, Takarazuka, Hyogo 665-0006, Japan.

Objective: To examine whether the changes in endolymphatic ion concentrations were involved in hair cells degeneration in claudin-14 knockout (KO) mice (Cldn14), we measured the endocochlear potential (EP) along with concentrations of K, Na, H, or Ca ([K], [Na], pH, [Ca]) in Cldn14, in which hair cells were selectively damaged, and compared with measurements in wild type mice (Wt).

Methods: We used the Cldn14 from 3 weeks of age, in which the auditory brain responses (ABR) was severely diminished. Using double-barreled ion-selective microelectrodes, we measured [K], [Na], pH, and [Ca] in both Wt and Cldn14 at 8-10 weeks of age.

Results: (1) In Wt, the EP was +92mV. [K], [Na], pH, and [Ca] were 169mM, ∼1.0mM, 7.50, and 395nM, respectively. In the Cldn14, the EP was +96mV. [K], [Na], pH, and [Ca] were 167mM, ∼1.0mM, 7.73, and 179nM, respectively. No significant differences in the above values were observed between Wt and Cldn14. (2) A significant linear correlation between EP and [Ca] (R=0.93) was observed for both Wt and Cldn14, but no correlation was observed between EP and K, Na, or H.

Conclusion: These findings suggest that (1) the changes in endolymphatic ion concentrations might not be involved in hair cells degeneration in Cldn14, (2) [Ca] might be regulated by EP in both Wt and Cldn14.
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http://dx.doi.org/10.1016/j.anl.2017.07.013DOI Listing
June 2018

Targeted Disruption of JCAD (Junctional Protein Associated With Coronary Artery Disease)/KIAA1462, a Coronary Artery Disease-Associated Gene Product, Inhibits Angiogenic Processes In Vitro and In Vivo.

Arterioscler Thromb Vasc Biol 2017 09 13;37(9):1667-1673. Epub 2017 Jul 13.

From the Division of Cardiovascular Medicine, Department of Internal Medicine (T.H., T.M., K.M., T.O., M.O., T.I., K.-i.H.), Division of Cell Biology, Department of Physiology and Cell Biology (N.I., M.A., M.F.), Department of Oral and Maxillofacial Surgery (M.A.), Division of Evidence-Based Laboratory Medicine (R.T., Y.I.), Division of Integrated Medical Education, Department of Community Medicine and Social Healthcare Science (M.S.), and The Integrated Center for Mass Spectrometry (M.S.), Kobe University Graduate School of Medicine, Japan; Animal Resource Development Unit (Y.Y.) and Genetic Engineering Team (Y.Y., G.S.), RIKEN Center for Life Science Technologies, Kobe, Hyogo, Japan; and Division of Cell Structure, National Institute for Physiological Sciences, Okazaki, Aichi, Japan (M.F.).

Objective: Recent genome-wide association studies newly identified the human KIAA1462 gene as a new locus for coronary artery disease. However, the function of the gene product, named JCAD (junctional protein associated with coronary artery disease), is unknown. Because JCAD is expressed at cell-cell junctions in endothelial cells, we hypothesized and tested whether JCAD regulates angiogenic processes in vitro and in vivo.

Approach And Results: Cell culture experiments revealed impaired angiogenic ability (proliferation, migration, and cord formation) by the knockdown of JCAD with siRNA (<0.05 versus control siRNA). We have generated mice lacking JCAD (mKIAA1462) by gene-targeted deletion of JCAD to address in vivo angiogenic function. mKIAA1462 mice did not show morphological differences in development of retinal vasculature. Ex vivo aortic ring model demonstrated impaired neovascularization in aorta from mKIAA1462 mice than control wild-type mice (<0.05). Tumor growth was assessed by monitoring tumor volume after the subcutaneous injection of melanoma, LLC (Lewis lung carcinoma), and E0771 cells into the mice. mKIAA1462 mice exhibited significantly smaller tumor volume compared with wild-type mice (<0.001). Histological assessment of the tumor exhibited less smooth muscle actin-positive neovascularization determined by CD31-positive vascular structure in tumor of mKIAA1462 mice than wild-type mice, indicating that knockdown of JCAD inhibited the vascular maturation in pathological angiogenic process.

Conclusions: These in vitro and in vivo studies suggest that JCAD has a redundant functional role in physiological angiogenesis but serves a pivotal role in pathological angiogenic process after birth.
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http://dx.doi.org/10.1161/ATVBAHA.117.309721DOI Listing
September 2017

Identification of Arabidopsis thaliana upstream open reading frames encoding peptide sequences that cause ribosomal arrest.

Nucleic Acids Res 2017 Sep;45(15):8844-8858

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.

Specific sequences of certain nascent peptides cause programmed ribosomal arrest during mRNA translation to control gene expression. In eukaryotes, most known regulatory arrest peptides are encoded by upstream open reading frames (uORFs) present in the 5'-untranslated region of mRNAs. However, to date, a limited number of eukaryotic uORFs encoding arrest peptides have been reported. Here, we searched for arrest peptide-encoding uORFs among Arabidopsis thaliana uORFs with evolutionarily conserved peptide sequences. Analysis of in vitro translation products of 22 conserved uORFs identified three novel uORFs causing ribosomal arrest in a peptide sequence-dependent manner. Stop codon-scanning mutagenesis, in which the effect of changing the uORF stop codon position on the ribosomal arrest was examined, and toeprint analysis revealed that two of the three uORFs cause ribosomal arrest during translation elongation, whereas the other one causes ribosomal arrest during translation termination. Transient expression assays showed that the newly identified arrest-causing uORFs exerted a strong sequence-dependent repressive effect on the expression of the downstream reporter gene in A. thaliana protoplasts. These results suggest that the peptide sequences of the three uORFs identified in this study cause ribosomal arrest in the uORFs, thereby repressing the expression of proteins encoded by the main ORFs.
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http://dx.doi.org/10.1093/nar/gkx528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587730PMC
September 2017

Clinical Features of Nivolumab-Induced Thyroiditis: A Case Series Study.

Thyroid 2017 07 21;27(7):894-901. Epub 2017 Jun 21.

1 Department of Diabetes, Endocrinology and Nutrition, Kyoto University Graduate School of Medicine , Kyoto, Japan .

Background: The programmed cell death-1 (PD-1) pathway is a novel therapeutic target in immune checkpoint therapy for cancer. It consists of the PD-1 receptor and its two ligands, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). Nivolumab is an anti-PD-1 monoclonal antibody approved for malignant melanoma, advanced non-small cell lung cancer, and advanced renal cell carcinoma in Japan. Thyrotoxicosis and hypothyroidism have both been reported in international Phase 3 studies and national post-marketing surveillance of nivolumab in Japan.

Methods: This study analyzed five consecutive cases with thyroid dysfunction associated with nivolumab therapy. Second, it examined the mRNA and protein expressions of PD-L1 and PD-L2 by reverse transcription polymerase chain reaction and Western blotting.

Results: All patients were diagnosed with painless thyroiditis. Thyrotoxicosis developed within four weeks from the first administration of nivolumab and normalized within four weeks of onset in three of the five patients. Hypothyroidism after transient thyrotoxicosis developed in two patients, and preexisting hypothyroidism persisted in one patient. The other two patients were treated with glucocorticoids and discontinued nivolumab therapy for comorbid adverse events. One did not develop hypothyroidism, and the other developed mild, transient hypothyroidism. In addition, it was verified that normal thyroid tissue expresses PD-L1 and PD-L2 mRNA and those proteins.

Conclusions: In the present cases, nivolumab-induced thyrotoxicosis seemed to be associated with painless thyroiditis, while no patient with Graves' disease was observed. A transient and rapid course with subsequent hypothyroidism was observed in nivolumab-induced thyroiditis. In addition, it was verified that PD-L1 and PD-L2 are expressed in normal thyroid tissue. This suggests that nivolumab therapy reduces immune tolerance, even in normal thyroid tissue, and leads to the development of thyroiditis. Treating thyrotoxicosis with only supportive care and considering levothyroxine replacement therapy once subsequent hypothyroidism occurs is proposed. Further investigations are required to confirm whether glucocorticoid therapy and discontinuation of nivolumab therapy prevent subsequent hypothyroidism.
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http://dx.doi.org/10.1089/thy.2016.0562DOI Listing
July 2017

Neuronal cells derived from human induced pluripotent stem cells as a functional tool of melanocortin system.

Neuropeptides 2017 Oct 7;65:10-20. Epub 2017 Apr 7.

Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; Kyoto University Graduate School of Medicine Medical Innovation Center, Kyoto, Japan.

Background: The preparation of human neurons derived from human induced pluripotent stem (iPS) cells can serve as a potential tool for evaluating the physiological and pathophysiological properties of human neurons and for drug development.

Methods: In the present study, the functional activity in neuronal cells differentiated from human iPS cells was observed.

Results: The differentiated cells expressed mRNAs for classical neuronal markers (microtubule-associated protein 2, β-tubulin III, calbindin 1, synaptophysin and postsynaptic density protein 95) and for subunits of various excitatory and inhibitory transmitters (NR1, NR2A, NR2B, GABA α1). Moreover, the differentiated cells expressed neuropeptides and receptors which are predominantly present in the hypothalamus. The expression of mRNA for preopiomelanocortin, agouti-related protein (AgRP), melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) increased in culture with a peak on Day 30 which subsequently decreased at Day 45. Immunoreactivities for MC3R and MC4R were also observed in cells differentiated from human iPS cells. Application of a potent agonist for MC3R and MC4R, [Nle4, D-Phe7]-α-melanocyte-stimulating hormone, significantly increased intracellular cAMP levels, but this was suppressed by AgRP (83-132) and SHU9119.

Conclusions: These findings offer the possibility for drug developments using neurons differentiated from normal or disease-associated human iPS cells.
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http://dx.doi.org/10.1016/j.npep.2017.04.004DOI Listing
October 2017

Sucrose sensing through nascent peptide-meditated ribosome stalling at the stop codon of Arabidopsis bZIP11 uORF2.

FEBS Lett 2017 05 10;591(9):1266-1277. Epub 2017 Apr 10.

Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Germany.

Arabidopsis bZIP11 is a transcription factor that modulates amino acid metabolism under high-sucrose conditions. Expression of bZIP11 is downregulated in a sucrose-dependent manner during translation. Previous in vivo studies have identified the second upstream open reading frame (uORF2) as an essential regulatory element for the sucrose-dependent translational repression of bZIP11. However, it remains unclear how uORF2 represses bZIP11 expression under high-sucrose conditions. Through biochemical experiments using cell-free translation systems, we report on sucrose-mediated ribosome stalling at the stop codon of uORF2. The C-terminal 10 amino acids (29-SFSVxFLxxLYYV-41) of uORF2 are important for ribosome stalling. Our results demonstrate that uORF2 encodes a regulatory nascent peptide that functions to sense intracellular sucrose abundance. This is the first biochemical identification of the intracellular sucrose sensor.
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http://dx.doi.org/10.1002/1873-3468.12634DOI Listing
May 2017

Rapid exacerbation of lymphocytic infundibuloneurohypophysitis.

Medicine (Baltimore) 2017 Mar;96(9):e6034

Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, Japan Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan. Department of Neurosurgery, Graduate School of Medicine, Kyoto University, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, Japan.

Rationale: Lymphocytic hypophysitis is a relatively rare autoimmune disease defined by lymphocytic infiltration to the pituitary. Its rarity and wide spectrum of clinical manifestations make clarification of the pathology difficult. Here, we describe a case we examined from the primary diagnosis to final discharge, showing the serial progression of lymphocytic infundibuloneurohypophysitis (LINH) to panhypopituitarism with extrapituitary inflammatory invasion in a short period, and responding favorably to high-dose glucocorticoid treatment.

Patient Concerns: Polyuria, General fatigue and Nausea/Vomiting.

Diagnoses: Central diabetes insipidus (CDI), Lymphocytic infundibuloneurohypophysitis (LINH).

Interventions: Desmopressin acetate, High-dose glucocorticoid (GC) treatment.

Outcomes: He was prescribed desmopressin acetate and subsequently discharged. A month later, he revisited our hospital with general fatigue and nausea/vomiting. A screening test disclosed hypopituitarism with adrenal insufficiency. MRI revealed expanded contrast enhancement to the peripheral extrapituitary lesion. He received high-dose GC treatment and the affected lesion exhibited marked improvement on MRI, along with the recovery of the anterior pituitary function.

Lessons: This case demonstrates the potential for classical LINH to develop into panhypopituitarsim. We consider this is the first documentation of approaching the cause of atypical LINH with progressive clinical course from the pathological viewpoint.
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http://dx.doi.org/10.1097/MD.0000000000006034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5340433PMC
March 2017

CAPS1 RNA Editing Promotes Dense Core Vesicle Exocytosis.

Cell Rep 2016 11;17(8):2004-2014

Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address:

Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis in vivo, affecting physical activity.
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http://dx.doi.org/10.1016/j.celrep.2016.10.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199237PMC
November 2016

The Minimum Open Reading Frame, AUG-Stop, Induces Boron-Dependent Ribosome Stalling and mRNA Degradation.

Plant Cell 2016 11 19;28(11):2830-2849. Epub 2016 Oct 19.

Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan

Upstream open reading frames (uORFs) are often translated ahead of the main ORF of a gene and regulate gene expression, sometimes in a condition-dependent manner, but such a role for the minimum uORF (hereafter referred to as AUG-stop) in living organisms is currently unclear. Here, we show that AUG-stop plays an important role in the boron (B)-dependent regulation of NIP5;1, encoding a boric acid channel required for normal growth under low B conditions in Arabidopsis thaliana High B enhanced ribosome stalling at AUG-stop, which was accompanied by the suppression of translation and mRNA degradation. This mRNA degradation was promoted by an upstream conserved sequence present near the 5'-edge of the stalled ribosome. Once ribosomes translate a uORF, reinitiation of translation must take place in order for the downstream ORF to be translated. Our results suggest that reinitiation of translation at the downstream NIP5;1 ORF is enhanced under low B conditions. A genome-wide analysis identified two additional B-responsive genes, SKU5 and the transcription factor gene ABS/NGAL1, which were regulated by B-dependent ribosome stalling through AUG-stop. This regulation was reproduced in both plant and animal transient expression and cell-free translation systems. These findings suggest that B-dependent AUG-stop-mediated regulation is common in eukaryotes.
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http://dx.doi.org/10.1105/tpc.16.00481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155345PMC
November 2016

C-type natriuretic peptide restores impaired skeletal growth in a murine model of glucocorticoid-induced growth retardation.

Bone 2016 11 2;92:157-167. Epub 2016 Sep 2.

Department of Diabetes, Endocrinology and Nutrition, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawahara-cho, Sakyo-ku, 606-8507 Kyoto, Japan. Electronic address:

Glucocorticoids are widely used for treating autoimmune conditions or inflammatory disorders. Long-term use of glucocorticoids causes impaired skeletal growth, a serious side effect when they are used in children. We have previously demonstrated that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. In this study, we investigated the effect of CNP on impaired bone growth caused by glucocorticoids by using a transgenic mouse model with an increased circulating CNP level. Daily administration of a high dose of dexamethasone (DEX) to 4-week-old male wild-type mice for 4weeks significantly shortened their naso-anal length, which was restored completely in DEX-treated CNP transgenic mice. Impaired growth of the long bones and vertebrae by DEX was restored to a large extent in the CNP transgenic background, with recovery in the narrowed growth plate by increased cell volume, whereas the decreased proliferation and increased apoptosis of the growth plate chondrocytes were unaffected. Trabecular bone volume was not changed by DEX treatment, but decreased significantly in a CNP transgenic background. In young male rats, the administration of high doses of DEX greatly decreased N-terminal proCNP concentrations, a marker of CNP production. In organ culture experiments using fetal wild-type murine tibias, longitudinal growth of tibial explants was inhibited by DEX but reversed by CNP. These findings now warrant further study of the therapeutic potency of CNP in glucocorticoid-induced bone growth impairment.
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http://dx.doi.org/10.1016/j.bone.2016.08.026DOI Listing
November 2016

Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice.

Peptides 2016 07 25;81:38-50. Epub 2016 Mar 25.

Kyoto University Graduate School of Medicine Medical Innovation Center, 53, Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice. Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.
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http://dx.doi.org/10.1016/j.peptides.2016.03.014DOI Listing
July 2016

Cognitive Function Related to the Sirh11/Zcchc16 Gene Acquired from an LTR Retrotransposon in Eutherians.

PLoS Genet 2015 Sep 24;11(9):e1005521. Epub 2015 Sep 24.

School of Health Sciences, Tokai University, Isehara, Kanagawa, Japan.

Gene targeting of mouse Sushi-ichi-related retrotransposon homologue 11/Zinc finger CCHC domain-containing 16 (Sirh11/Zcchc16) causes abnormal behaviors related to cognition, including attention, impulsivity and working memory. Sirh11/Zcchc16 encodes a CCHC type of zinc-finger protein that exhibits high homology to an LTR retrotransposon Gag protein. Upon microdialysis analysis of the prefrontal cortex region, the recovery rate of noradrenaline (NA) was reduced compared with dopamine (DA) after perfusion of high potassium-containing artificial cerebrospinal fluid in knockout (KO) mice. These data indicate that Sirh11/Zcchc16 is involved in cognitive function in the brain, possibly via the noradrenergic system, in the contemporary mouse developmental systems. Interestingly, it is highly conserved in three out of the four major groups of the eutherians, euarchontoglires, laurasiatheria and afrotheria, but is heavily mutated in xenarthran species such as the sloth and armadillo, suggesting that it has contributed to brain evolution in the three major eutherian lineages, including humans and mice. Sirh11/Zcchc16 is the first SIRH gene to be involved in brain function, instead of just the placenta, as seen in the case of Peg10, Peg11/Rtl1 and Sirh7/Ldoc1.
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http://dx.doi.org/10.1371/journal.pgen.1005521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4581854PMC
September 2015

The Transcriptional Modulator Interferon-Related Developmental Regulator 1 in Osteoblasts Suppresses Bone Formation and Promotes Bone Resorption.

J Bone Miner Res 2016 Mar 22;31(3):573-84. Epub 2015 Oct 22.

Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School, Kanazawa, Ishikawa, Japan.

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Although interferon-related developmental regulator 1 (Ifrd1) has been identified as a transcriptional coactivator/repressor in various cells, little attention has been paid to its role in osteoblastogenesis and bone homeostasis thus far. Here, we show that Ifrd1 is a critical mediator of both the cell-autonomous regulation of osteoblastogenesis and osteoblast-dependent regulation of osteoclastogenesis. Osteoblast-specific deletion of murine Ifrd1 increased bone formation and decreased bone resorption, causing high bone mass. Ifrd1 deficiency enhanced osteoblast differentiation and maturation along with increased expression of Runx2 and osterix (Osx). Mechanistically, Ifrd1 deficiency increased the acetylation status of p65, a component of NF-κB, at residues K122 and K123 via the attenuation of the interaction between p65 and histone deacetylase (HDAC). This led to the nuclear export of p65 and a decrease in NF-κB-dependent Smad7 expression and the subsequent enhancement of Smad1/Smad5/Smad8-dependent transcription. Moreover, a high bone mass phenotype in the osteoblast-specific deletion of Ifrd1 was markedly rescued by the introduction of one Osx-floxed allele but not of Runx2-floxed allele. Coculture experiments revealed that Ifrd1-deficient osteoblasts have a higher osteoprotegerin (OPG) expression and a lower ability to support osteoclastogenesis. Ifrd1 deficiency attenuated the interaction between β-catenin and HDAC, subsequently increasing the acetylation of β-catenin at K49, leading to its nuclear accumulation and the activation of the β-catenin-dependent transcription of OPG. Collectively, the expression of Ifrd1 in osteoblasts repressed osteoblastogenesis and activated osteoclastogenesis through modulating the NF-κB/Smad/Osx and β-catenin/OPG pathways, respectively. These findings suggest that Ifrd1 has a pivotal role in bone homeostasis through its expression in osteoblasts in vivo and represents a therapeutic target for bone diseases.
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http://dx.doi.org/10.1002/jbmr.2720DOI Listing
March 2016

miR-199a Links MeCP2 with mTOR Signaling and Its Dysregulation Leads to Rett Syndrome Phenotypes.

Cell Rep 2015 Sep 3;12(11):1887-901. Epub 2015 Sep 3.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; Laboratory of Molecular Neuroscience, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Electronic address:

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Although emerging evidence suggests that MeCP2 deficiency is associated with dysregulation of mechanistic target of rapamycin (mTOR), which functions as a hub for various signaling pathways, the mechanism underlying this association and the molecular pathophysiology of RTT remain elusive. We show here that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs) as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that miR-199a positively controls mTOR signaling by targeting inhibitors for mTOR signaling. miR-199a and its targets have opposite effects on mTOR activity, ameliorating and inducing RTT neuronal phenotypes, respectively. Furthermore, genetic deletion of miR-199a-2 led to a reduction of mTOR activity in the brain and recapitulated numerous RTT phenotypes in mice. Together, these findings establish miR-199a as a critical downstream target of MeCP2 in RTT pathogenesis by linking MeCP2 with mTOR signaling.
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http://dx.doi.org/10.1016/j.celrep.2015.08.028DOI Listing
September 2015

Increased Bone Turnover and Possible Accelerated Fracture Healing in a Murine Model With an Increased Circulating C-Type Natriuretic Peptide.

Endocrinology 2015 Jul 10;156(7):2518-29. Epub 2015 Apr 10.

Departments of Diabetes, Endocrinology, and Nutrition (E.K., A.Y., T.F., Y.Y., Y.U.-S., N.K., M.M., N.I.) and Maxillofacial Surgery (Kazum. N.) and Medical Innovation Center (Kazuw. N.), Kyoto University Graduate School of Medicine, 606-8507 Kyoto, Japan; Health Care Service Center (H.A.), Kyoto Institute of Technology, Matsugasaki-Hashikami-cho, 606-8585 Kyoto, Japan; and Department of Nephrology (M.K.), Graduate School of Medical Science, Kumamoto University, 860-8556 Kumamoto City, Japan.

Recent studies have revealed that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. Nevertheless, the effect of CNP on bone turnover has not yet been well studied. To elucidate this issue, we investigated the bone phenotype of a mouse model with elevated plasma CNP concentrations (SAP-CNP-Tg mice) in the present study. Microcomputed tomography (CT) analysis revealed less bone in femurs, but not in lumber vertebrae, of young adult SAP-CNP-Tg mice than that of wild-type mice. Bone histomorphometry of the tibiae from 8-week-old SAP-CNP-Tg mice showed enhanced osteoblastic and osteoclastic activities, in accordance with elevated serum levels of osteocalcin and tartrate-resistant acid phosphatase-5b, respectively. Next we performed an open and stabilized femoral fracture using 8-week-old SAP-CNP-Tg mice and compared the healing process with age-matched wild-type mice. An immunohistochemical study revealed that CNP and its receptors, natriuretic peptide receptor-B and natriuretic peptide clearance receptor, are expressed in hard calluses of wild-type mice, suggesting a possible role of CNP/natriuretic peptide receptor-B signaling in fracture repair, especially in bone remodeling stage. On micro-CT analysis, a rapid decrease in callus volume was observed in SAP-CNP-Tg mice, followed by a generation of significantly higher new bone volume with a tendency of increased bone strength. In addition, a micro-CT analysis also showed that bone remodeling was accelerated in SAP-CNP-Tg mice, which was also evident from increased serum osteocalcin and tartrate-resistant acid phosphatase-5b levels in SAP-CNP-Tg mice at the remodeling stage of fracture repair. These results indicate that CNP activates bone turnover and remodeling in vivo and possibly accelerates fracture healing in our mouse model.
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http://dx.doi.org/10.1210/en.2014-1801DOI Listing
July 2015

Expression of ocular albinism 1 (OA1), 3, 4- dihydroxy- L-phenylalanine (DOPA) receptor, in both neuronal and non-neuronal organs.

Brain Res 2015 Mar 16;1602:62-74. Epub 2015 Jan 16.

Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. Electronic address:

Oa1 is the casual gene for ocular albinism-1 in humans. The gene product OA1, alternatively designated as GPR143, belongs to G-protein coupled receptors. It has been reported that OA1 is a specific receptor for 3, 4-dihydroxy- L-phenylalanine (DOPA) in retinal pigmental epithelium where DOPA facilitates the pigmentation via OA1 stimulation. We have recently shown that OA1 mediates DOPA-induced depressor response in rat nucleus tractus solitarii. However, the distribution and function of OA1 in other regions are largely unknown. We have generated oa1 knockout mice and examined OA1 expression in both neuronal and non-neuronal tissues by immunohistochemical analyses using anti-mouse OA1 monoclonal antibodies. In the telencephalon, OA1 was expressed in cerebral cortex and hippocampus. Predominant expression of OA1 was observed in the pyramidal neurons in these regions. OA1 was also expressed in habenular nucleus, hypothalamus, substantia nigra, and medulla oblongata. The expression of OA1 in the nucleus tractus solitarii of medulla oblongata may support the reduction of blood pressure by the microinjection of DOPA into this region. Outside of the nervous system, OA1 was expressed in heart, lung, liver, kidney and spleen. Abundant expression was observed in the renal tubules and the splenic capsules. These peripheral regions are innervated by numerous sympathetic nerve endings. In addition, substantia nigra contains a large population of dopaminergic neurons. Thus, the immunohistochemical analyses suggest that OA1 may modulate the monoaminergic functions in both peripheral and central nervous systems.
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http://dx.doi.org/10.1016/j.brainres.2015.01.020DOI Listing
March 2015

A U-system approach for predicting metabolic behaviors and responses based on an alleged metabolic reaction network.

BMC Syst Biol 2014 12;8 Suppl 5:S4. Epub 2014 Dec 12.

Background: Progress in systems biology offers sophisticated approaches toward a comprehensive understanding of biological systems. Yet, computational analyses are held back due to difficulties in determining suitable model parameter values from experimental data which naturally are subject to biological fluctuations. The data may also be corrupted by experimental uncertainties and sometimes do not contain all information regarding variables that cannot be measured for technical reasons.

Results: We show here a streamlined approach for the construction of a coarse model that allows us to set up dynamic models with minimal input information. The approach uses a hybrid between a pure mass action system and a generalized mass action (GMA) system in the framework of biochemical systems theory (BST) with rate constants of 1, normal kinetic orders of 1, and -0.5 and 0.5 for inhibitory and activating effects, named Unity (U)-system. The U-system model does not necessarily fit all data well but is often sufficient for predicting metabolic behavior of metabolites which cannot be simultaneously measured, identifying inconsistencies between experimental data and the assumed underlying pathway structure, as well as predicting system responses to a modification of gene or enzyme. The U-system approach was validated with small, generic systems and implemented to model a large-scale metabolic reaction network of a higher plant, Arabidopsis. The dynamic behaviors obtained by predictive simulations agreed with actually available metabolomic time-series data, identified probable errors in the experimental datasets, and estimated probable behavior of unmeasurable metabolites in a qualitative manner. The model could also predict metabolic responses of Arabidopsis with altered network structures due to genetic modification.

Conclusions: The U-system approach can effectively predict metabolic behaviors and responses based on structures of an alleged metabolic reaction network. Thus, it can be a useful first-line tool of data analysis, model diagnostics and aid the design of next-step experiments.
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http://dx.doi.org/10.1186/1752-0509-8-S5-S4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4305983PMC
September 2015

The N-terminal cleavable pre-sequence encoded in the first exon of cystathionine γ-synthase contains two different functional domains for chloroplast targeting and regulation of gene expression.

Plant Cell Physiol 2014 Oct 20;55(10):1779-92. Epub 2014 Aug 20.

Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan Graduate School of Life Science, Hokkaido University, Sapporo, 060-0810 Japan

Chloroplast transit peptide sequences (cTPs) located in the N-terminal region of nuclear-encoded chloroplast proteins are essential for their sorting, and are generally cleaved from the proteins after their import into the chloroplasts. The Arabidopsis thaliana cystathionine γ-synthase (CGS), the first committed enzyme of methionine biosynthesis, is a nuclear-encoded chloroplast protein. Arabidopsis CGS possesses an N-terminal extension region that is dispensable for enzymatic activity. This N-terminal extension contains the cTP and several functional domains including an MTO1 region, the cis-element for post-transcriptional feedback regulation of CGS1 that codes for CGS. A previous report suggested that the cTP cleavage site of CGS is located upstream of the MTO1 region. However, the region required for protein sorting has not been analyzed. In this study, we carried out functional analyses to elucidate the region required for chloroplast targeting by using a chimeric protein, Ex1:GFP, in which the CGS1 exon 1 coding region containing the N-terminal extension was tagged with green fluorescent protein. The sequence upstream of the MTO1 region was responsible for efficient chloroplast targeting and for avoidance of missorting to the mitochondria. Our data also showed that the major N-terminus of Ex1:GFP is Ala91, which is located immediately downstream of the MTO1 region, and the MTO1 region is not retained in the mature Ex1:GFP accumulated in the chloroplast. These findings suggest that the N-terminal cleavable pre-sequence harbors dual functions in protein sorting and in regulating gene expression. Our study highlights the unique properties of Arabidopsis CGS cTP among chloroplast-targeted proteins.
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http://dx.doi.org/10.1093/pcp/pcu110DOI Listing
October 2014
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