Publications by authors named "Kazuki Takeda"

73 Publications

The VKORC1 ER-luminal loop mutation (Leu76Pro) leads to a significant resistance to warfarin in black rats (Rattus rattus).

Pestic Biochem Physiol 2021 Mar 9;173:104774. Epub 2021 Jan 9.

Laboratory of Toxicology, Department of Environmental Sciences, Faculty of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan. Electronic address:

Well-known 4-hydroxycoumarin derivatives, such as warfarin, act as inhibitors of the vitamin K epoxide reductase (VKOR) and are used as anticoagulants. Mutations of the VKOR enzyme can lead to resistance to those compounds. This has been a problem in using them as medicine or rodenticide. Most of these mutations lie in the vicinity of potential warfarin-binding sites within the ER-luminal loop structure (Lys30, Phe55) and the transmembrane helix (Tyr138). However, a VKOR mutation found in Tokyo in warfarin-resistant rats does not follow that pattern (Leu76Pro), and its effect on VKOR function and structure remains unclear. We conducted both in vitro kinetic analyses and in silico docking studies to characterize the VKOR mutant. On the one hand, resistant rats (R-rats) showed a 37.5-fold increased IC value to warfarin when compared to susceptible rats (S-rats); on the other hand, R-rats showed a 16.5-fold lower basal VKOR activity (V/K). Docking calculations exhibited that the mutated VKOR of R-rats has a decreased affinity for warfarin. Molecular dynamics simulations further revealed that VKOR-associated warfarin was more exposed to solvents in R-rats and key interactions between Lys30, Phe55, and warfarin were less favored. This study concludes that a single mutation of VKOR at position 76 leads to a significant resistance to warfarin by modifying the types and numbers of intermolecular interactions between the two.
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http://dx.doi.org/10.1016/j.pestbp.2021.104774DOI Listing
March 2021

Sensitivity of turtles to anticoagulant rodenticides: Risk assessment for green sea turtles (Chelonia mydas) in the Ogasawara Islands and comparison of warfarin sensitivity among turtle species.

Aquat Toxicol 2021 Apr 25;233:105792. Epub 2021 Feb 25.

Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-ku, Sapporo, 060-0818, Japan. Electronic address:

Although anticoagulant rodenticides (ARs) are effectively used for the control of invasive rodents, nontarget species are also frequently exposed to ARs and secondary poisonings occur widely. However, little data is available on the effects of ARs, especially on marine organisms. To evaluate the effects of ARs on marine wildlife, we chose green sea turtles (Chelonia mydas), which are one of the most common marine organisms around the Ogasawara islands, as our primary study species. The sensitivity of these turtles to ARs was assessed using both in vivo and in vitro approaches. We administered 4 mg/kg of warfarin sodium either orally or intravenously to juvenile green sea turtles. The turtles exhibited slow pharmacokinetics, and prolongation of prothrombin time (PT) was observed only with intravenous warfarin administration. We also conducted an in vitro investigation using liver microsomes from green sea turtles, and two other turtle species (softshell turtle and red-eared slider) and rats. The cytochrome P450 metabolic activity in the liver of green sea turtles was lower than in rats. Additionally, vitamin K epoxide reductase (VKOR), which is the target enzyme of ARs, was inhibited by warfarin in the turtles at lower concentration levels than in rats. These data indicate that turtles may be more sensitive to ARs than rats. We expect that these findings will be helpful for sea turtle conservation following accidental AR-broadcast incidents.
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http://dx.doi.org/10.1016/j.aquatox.2021.105792DOI Listing
April 2021

Split conformation of Chaetomium thermophilum Hsp104 disaggregase.

Structure 2021 Feb 25. Epub 2021 Feb 25.

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 6 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.
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http://dx.doi.org/10.1016/j.str.2021.02.002DOI Listing
February 2021

Intermittency and Critical Scaling in Annular Couette Flow.

Entropy (Basel) 2020 Sep 4;22(9). Epub 2020 Sep 4.

Department of Mechanical Engineering, Tokyo University of Science, Chiba 278-8510, Japan.

The onset of turbulence in subcritical shear flows is one of the most puzzling manifestations of critical phenomena in fluid dynamics. The present study focuses on the Couette flow inside an infinitely long annular geometry where the inner rod moves with constant velocity and entrains fluid, by means of direct numerical simulation. Although for a radius ratio close to unity the system is similar to plane Couette flow, a qualitatively novel regime is identified for small radius ratio, featuring no oblique bands. An analysis of finite-size effects is carried out based on an artificial increase of the perimeter. Statistics of the turbulent fraction and of the laminar gap distributions are shown both with and without such confinement effects. For the wider domains, they display a cross-over from exponential to algebraic scaling. The data suggest that the onset of the original regime is consistent with the dynamics of one-dimensional directed percolation at onset, yet with additional frustration due to azimuthal confinement effects.
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http://dx.doi.org/10.3390/e22090988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7597309PMC
September 2020

Comparison of two reducing agents dithiothreitol and tris(3-hydroxypropyl)phosphine for kinetic assay of vitamin K epoxide reductase.

Vet Anim Sci 2020 Jun 15;9:100095. Epub 2020 Feb 15.

Department of Environmental Sciences, Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan.

Vitamin K epoxide reductase (VKOR) is a target enzyme for anticoagulants, such as warfarin, that are used as medicines or rodenticides. Assessing VKOR activity is required to ensure the proper usage of these drugs. Dithiothreitol (DTT) is a typical disulfide reductant that is used as a substrate for VKOR assays. However, DTT is considered problematic because of its side effects. Tris(3-hydroxypropyl)phosphine (THP) has been found to be a reliable alternative to DTT, as shown by kinetic analyses of the VKOR with them. THP showed significantly lower and values than those of DTT; however, there was no significant difference in their / and IC for warfarin.
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http://dx.doi.org/10.1016/j.vas.2020.100095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386777PMC
June 2020

Enantioselective 1,4-Addition Reaction of α,β-Unsaturated Carboxylic Acids with Cycloalkanones Using Cooperative Chiral Amine-Boronic Acid Catalysts.

Angew Chem Int Ed Engl 2020 Sep 29;59(39):17256-17260. Epub 2020 Jul 29.

Graduate School of Engineering, Nagoya University, B2-3(611), Furo-cho, Chikusa, Nagoya, 464-8603, Japan.

An enantioselective 1,4-addition of α,β-unsaturated carboxylic acids with cycloalkanones has been developed by using chiral amine-boronic acid cooperative catalysts. In the presence of a chiral amine and boronic acid, cycloalkanones and carboxylic acids are activated as chiral enamines and mixed anhydrides, respectively. The corresponding 1,4-adducts are obtained in high yield with high enantioselectivity. Furthermore, subsequent oxylactonization of the 1,4-adducts gives spirolactones with high diastereoselectivity.
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http://dx.doi.org/10.1002/anie.202007639DOI Listing
September 2020

One-Pot Tandem Michael Addition/Enantioselective Conia-Ene Cyclization Mediated by Chiral Iron(III)/Silver(I) Cooperative Catalysis.

Angew Chem Int Ed Engl 2020 Sep 10;59(38):16470-16474. Epub 2020 Jul 10.

Graduate School of Engineering, Nagoya University, B2-3(611), Furo-cho, Chikusa, Nagoya, 464-8603, Japan.

The first one-pot tandem Michael addition/enantioselective Conia-ene cyclization of N-protected prop-2-yn-1-amines with 2-methylene-3-oxoalkanoates promoted by chiral iron(III)/silver(I) cooperative catalysts has been developed. Alkyl 4-methylenepyrrolidine-3-acyl-3-carboxylates, which can be transformed into β-proline derivatives, are obtained in high yield with high enantioselectivity.
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http://dx.doi.org/10.1002/anie.202007180DOI Listing
September 2020

X-ray Crystal Structure of a Cyclic-PIP-DNA Complex in the Reverse-Binding Orientation.

J Am Chem Soc 2020 06 27;142(23):10544-10549. Epub 2020 May 27.

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

Elucidation of the details of the associating mode is one of the major concerns for the precise design of DNA-binding molecules that are used for gene regulation. Pyrrole-imidazole polyamide (PIP) is a well-established synthetic DNA-binding molecule that has sequence-specificity for duplex DNA. By the design of the sequence of pyrrole, imidazole, and other synthetic units, PIP is bound to the target DNA sequence selectively. Here, we report the X-ray crystal structure of newly synthesized chiral cyclic PIP (cPIP) complexed with DNA at 1.5 Å resolution and reveal that cPIP binds in the reverse orientation in the DNA minor groove. Analysis of the crystal structure revealed that the positions of the hydrogen bonds between the bases and the pyrrole-imidazole moieties of cPIP were similar for both forward- and reverse-binding orientations and that the distortion of the B-form DNA structure caused by cPIP binding was also similar for both orientations. We further found that new hydrogen bonds formed between the amino groups on the γ-turn units and DNA at both ends of the cPIP molecule. Additionally, by comparing the reverse PIP orientation with the forward orientation, we could clarify that the cause of the preference toward the reverse orientation in the -form cPIP as used in this study is the overall conformation of the cPIP-DNA complex, particularly the configuration of hydrogen bonds. These results thus provide an explanation for the different stereoselectivity of cPIP binding in the minor groove.
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http://dx.doi.org/10.1021/jacs.0c03972DOI Listing
June 2020

X-ray crystallographic studies on the hydrogen isotope effects of green fluorescent protein at sub-ångström resolutions.

Acta Crystallogr D Struct Biol 2019 Dec 19;75(Pt 12):1096-1106. Epub 2019 Nov 19.

Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

Hydrogen atoms are critical to the nature and properties of proteins, and thus deuteration has the potential to influence protein function. In fact, it has been reported that some deuterated proteins show different physical and chemical properties to their protiated counterparts. Consequently, it is important to investigate protonation states around the active site when using deuterated proteins. Here, hydrogen isotope effects on the S65T/F99S/M153T/V163A variant of green fluorescent protein (GFP), in which the deprotonated B form is dominant at pH 8.5, were investigated. The pH/pD dependence of the absorption and fluorescence spectra indicates that the protonation state of the chromophore is the same in protiated GFP in HO and protiated GFP in DO at pH/pD 8.5, while the pK of the chromophore became higher in DO. Indeed, X-ray crystallographic analyses at sub-ångström resolution revealed no apparent changes in the protonation state of the chromophore between the two samples. However, detailed comparisons of the hydrogen OMIT maps revealed that the protonation state of His148 in the vicinity of the chromophore differed between the two samples. This indicates that protonation states around the active site should be carefully adjusted to be the same as those of the protiated protein when neutron crystallographic analyses of proteins are performed.
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http://dx.doi.org/10.1107/S2059798319014608DOI Listing
December 2019

An enantioselective oxidative coupling reaction of 2-naphthol derivatives catalyzed by chiral diphosphine oxide-iron(ii) complexes.

Chem Commun (Camb) 2019 Nov 28;55(91):13677-13680. Epub 2019 Oct 28.

Graduate School of Engineering, Nagoya University, B2-3(611), Furo-cho, Chikusa, Nagoya 464-8603, Japan.

An enantioselective oxidative coupling of 2-naphthol derivatives is developed with the use of chiral Fe(ii)-diphosphine oxide complexes. Optically active 1,1-bi-2-naphthol derivatives can be synthesized in high yields when a 2 : 1 complex of (S)-xylyl-iPrO-BIPHEP-oxide and Fe(OTf) is used in the presence of t-butyl hydroperoxide as an oxidant. The non-linear effect, X-ray crystal structure and ESI-MS suggest that a 2 : 1 complex of (S)-xylyl-iPrO-BIPHEP-oxide and Fe(OTf) is a pre-catalyst for a Fe(iii)/Fe(iv) redox cycle.
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http://dx.doi.org/10.1039/c9cc07834gDOI Listing
November 2019

Genome-wide association study identifies 14 previously unreported susceptibility loci for adolescent idiopathic scoliosis in Japanese.

Nat Commun 2019 08 15;10(1):3685. Epub 2019 Aug 15.

Laboratory for Bone and Joint Diseases, Center for Integrative Medical Sciences, RIKEN, Tokyo, 108-8639, Japan.

Adolescent idiopathic scoliosis (AIS) is the most common pediatric spinal deformity. Several AIS susceptibility loci have been identified; however, they could explain only a small proportion of AIS heritability. To identify additional AIS susceptibility loci, we conduct a meta-analysis of the three genome-wide association studies consisting of 79,211 Japanese individuals. We identify 20 loci significantly associated with AIS, including 14 previously not reported loci. These loci explain 4.6% of the phenotypic variance of AIS. We find 21 cis-expression quantitative trait loci-associated genes in seven of the fourteen loci. By a female meta-analysis, we identify additional three significant loci. We also find significant genetic correlations of AIS with body mass index and uric acid. The cell-type specificity analyses show the significant heritability enrichment for AIS in multiple cell-type groups, suggesting the heterogeneity of etiology and pathogenesis of AIS. Our findings provide insights into etiology and pathogenesis of AIS.
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http://dx.doi.org/10.1038/s41467-019-11596-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695451PMC
August 2019

Activation of transient receptor potential vanilloid 4 channels dilates rat retinal arterioles through nitric oxide- and BK channel-dependent mechanisms in vivo.

Naunyn Schmiedebergs Arch Pharmacol 2020 01 8;393(1):35-41. Epub 2019 Aug 8.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

Transient receptor potential vanilloid 4 (TRPV4) channel, a cation channel expressed in nearly all cell types, plays an important role in the regulation of vascular tone. In the present study, we examined the effect of GSK1016790A, an activator of TRPV4 channels, on the diameter of retinal blood vessels in rats and the underlying mechanisms. Ocular fundus images were captured with an original high-resolution digital fundus camera in vivo and diameters of retinal blood vessels were measured. Intravenous infusion of GSK1016790A (0.2-2 μg kg min) increased retinal arteriolar diameter in a dose-dependent manner. The higher dose of GSK1016790A (2 μg kg min) slightly decreased blood pressure. These responses to GSK1016790A were significantly attenuated by intravenous injection of GSK2193874 (0.3 mg/kg), an antagonist of TRPV4 channels. Intravitreal injection of N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide (NO) synthase or iberiotoxin, an inhibitor of large-conductance Ca-activated K (BK) channel, significantly attenuated the GSK1016790A-induced increases in retinal arteriolar diameter. These results suggest that activation of TRPV4 channels dilates rat retinal arterioles through NO- and BK channel-dependent mechanisms in vivo.
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http://dx.doi.org/10.1007/s00210-019-01707-1DOI Listing
January 2020

Association of Susceptibility Genes for Adolescent Idiopathic Scoliosis and Intervertebral Disc Degeneration With Adult Spinal Deformity.

Spine (Phila Pa 1976) 2019 Dec;44(23):1623-1629

Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan.

Study Design: Genetic case-control study of single nucleotide polymorphisms (SNPs).

Objective: To examine the association of previously reported susceptibility genes for adolescent idiopathic scoliosis (AIS) and intervertebral disc (IVD) degeneration with adult spinal deformity (ASD).

Summary Of Background Data: ASD is a spinal deformity that develops and progresses with age. Its etiology is unclear. Several ASD susceptibility genes were recently reported using a candidate gene approach; however, the sample sizes were small and associations with ASD development were not determined.

Methods: ASD was defined as structural scoliosis with a Cobb angle more than 15° on standing radiographs, taken of patients at age 40 to 75 years in this study. Subjects in whom scoliosis was diagnosed before age 20 were excluded. We recruited 356 Japanese ASD subjects and 3341 healthy controls for case-control association studies of previously reported SNPs. We genotyped four known AIS-associated SNPs (rs11190870 in LBX1, rs6570507 in GPR126, rs10738445 in BNC2, and rs6137473 in PAX1) and three IVD degeneration-associated SNPs (rs1245582 in CHST3, rs2073711 in CILP, and rs1676486 in COL11A1) by the Invader assay.

Results: Among the AIS-associated SNPs, rs11190870 and rs6137473 showed strong and nominal associations with ASD (P = 1.44 × 10, 1.00 × 10, respectively). Of the IVD degeneration-associated SNPs, rs1245582 and rs2073711 showed no association with ASD, while rs1676486 showed a nominal association (P = 1.10 × 10). In a subgroup analysis, rs11190870 was significantly associated with a Cobb angle more than 20° in the minor thoracic curve (P = 1.44 × 10) and with a left convex lumbar curve (P = 6.70 × 10), and nominally associated with an apical vertebra higher than L1 (P = 1.80 × 10).

Conclusion: rs11190870 in LBX1, a strong susceptibility SNP for AIS, may also be a susceptibility SNP for ASD. Thus, ASD and AIS may share a common genetic background.

Level Of Evidence: 4.
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http://dx.doi.org/10.1097/BRS.0000000000003179DOI Listing
December 2019

Characterization of perdeuterated high-potential iron-sulfur protein with high-resolution X-ray crystallography.

Proteins 2020 02 10;88(2):251-259. Epub 2019 Aug 10.

Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan.

Perdeuteration in neutron crystallography is an effective method for determining the positions of hydrogen atoms in proteins. However, there is shortage of evidence that the high-resolution details of perdeuterated proteins are consistent with those of the nondeuterated proteins. In this study, we determined the X-ray structure of perdeuterated high-potential iron-sulfur protein (HiPIP) at a high resolution of 0.85 å resolution. The comparison of the nondeuterated and perdeuterated structures of HiPIP revealed slight differences between the two structures. The spectroscopic and spectroelectrochemical studies also showed that perdeuterated HiPIP has approximately the same characteristics as nondeuterated HiPIP. These results further emphasize the suitability of using perdeuterated proteins in the high-resolution neutron crystallography.
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http://dx.doi.org/10.1002/prot.25793DOI Listing
February 2020

Subatomic resolution X-ray structures of green fluorescent protein.

IUCrJ 2019 May 3;6(Pt 3):387-400. Epub 2019 Apr 3.

Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

Green fluorescent protein (GFP) is a light-emitting protein that does not require a prosthetic group for its fluorescent activity. As such, GFP has become indispensable as a molecular tool in molecular biology. Nonetheless, there has been no subatomic elucidation of the GFP structure owing to the structural polymorphism around the chromophore. Here, subatomic resolution X-ray structures of GFP without the structural polymorphism are reported. The positions of H atoms, hydrogen-bonding network patterns and accurate geometric parameters were determined for the two protonated forms. Compared with previously determined crystal structures and theoretically optimized structures, the anionic chromophores of the structures represent the authentic resonance state of GFP. In addition, charge-density analysis based on atoms-in-molecules theory and noncovalent interaction analysis highlight weak but substantial interactions between the chromophore and the protein environment. Considered with the derived chemical indicators, the lone pair-π interactions between the chromophore and Thr62 should play a sufficient role in maintaining the electronic state of the chromophore. These results not only reveal the fine structural features that are critical to understanding the properties of GFP, but also highlight the limitations of current quantum-chemical calculations.
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http://dx.doi.org/10.1107/S205225251900246XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503917PMC
May 2019

Bi-allelic loss of function variants of causes a spectrum of malformation of spine and rib including congenital scoliosis and spondylocostal dysostosis.

J Med Genet 2019 09 22;56(9):622-628. Epub 2019 Apr 22.

Laboratory of Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences, Tokyo, Japan

Background: Congenital scoliosis (CS) is a common vertebral malformation. Spondylocostal dysostosis (SCD) is a rare skeletal dysplasia characterised by multiple vertebral malformations and rib anomalies. In a previous study, a compound heterozygosity for a null mutation and a risk haplotype composed by three single-nucleotide polymorphisms in have been reported as a disease-causing model of CS. Another study identified bi-allelic missense variants in a SCD patient. The purpose of our study is to identify variants in CS and SCD and examine their pathogenicity.

Methods: We recruited 200 patients with CS or SCD and investigated variants. We evaluated the pathogenicity of the variants by in silico prediction and in vitro experiments.

Results: We identified five 16p11.2 deletions, one splice-site variant and five missense variants in 10 patients. In vitro functional assays for missense variants identified in the previous and present studies demonstrated that most of the variants caused abnormal localisation of TBX6 proteins. We confirmed mislocalisation of TBX6 proteins in presomitic mesoderm cells induced from SCD patient-derived iPS cells. In induced cells, we found decreased mRNA expressions of and its downstream genes were involved in somite formation. All CS patients with missense variants had the risk haplotype in the opposite allele, while a SCD patient with bi-allelic missense variants did not have the haplotype.

Conclusions: Our study suggests that bi-allelic loss of function variants of cause a spectrum of phenotypes including CS and SCD, depending on the severity of the loss of function.
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http://dx.doi.org/10.1136/jmedgenet-2018-105920DOI Listing
September 2019

A multiethnic meta-analysis defined the association of rs12946942 with severe adolescent idiopathic scoliosis.

J Hum Genet 2019 May 21;64(5):493-498. Epub 2019 Feb 21.

Laboratory of Bone and Joint Diseases, Center for Integrative Medical Sciences, RIKEN, Tokyo, Japan.

Adolescent idiopathic scoliosis (AIS) is the most common type of scoliosis. Controlling its curve progression is the most important clinical task. Although recent genome-wide association studies (GWASs) identified several susceptibility loci associated with the development of AIS, the etiology of curve progression has been still unknown. Our previous GWAS has identified that rs12946942 showed significant association with severe AIS. To confirm the association, we conducted an international meta-analysis using four cohorts with different ethnicity. We analyzed 2272 severe AIS cases and 13,859 controls in total, and found the replication of significant association of rs12946942 (combined P = 7.23×10; odds ratio = 1.36, 95% confidence interval = 1.25-1.49). In silico analyses suggested that SOX9 is the most likely susceptibility gene for AIS curve progression in the locus.
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http://dx.doi.org/10.1038/s10038-019-0575-7DOI Listing
May 2019

TBX6-associated congenital scoliosis (TACS) as a clinically distinguishable subtype of congenital scoliosis: further evidence supporting the compound inheritance and TBX6 gene dosage model.

Genet Med 2019 07 14;21(7):1548-1558. Epub 2019 Jan 14.

Department of Orthopedic Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China.

Purpose: To characterize clinically measurable endophenotypes, implicating the TBX6 compound inheritance model.

Methods: Patients with congenital scoliosis (CS) from China(N = 345, cohort 1), Japan (N = 142, cohort 2), and the United States (N = 10, cohort 3) were studied. Clinically measurable endophenotypes were compared according to the TBX6 genotypes. A mouse model for Tbx6 compound inheritance (N = 52) was investigated by micro computed tomography (micro-CT). A clinical diagnostic algorithm (TACScore) was developed to assist in clinical recognition of TBX6-associated CS (TACS).

Results: In cohort 1, TACS patients (N = 33) were significantly younger at onset than the remaining CS patients (P = 0.02), presented with one or more hemivertebrae/butterfly vertebrae (P = 4.9 × 10), and exhibited vertebral malformations involving the lower part of the spine (T8-S5, P = 4.4 × 10); observations were confirmed in two replication cohorts. Simple rib anomalies were prevalent in TACS patients (P = 3.1 × 10), while intraspinal anomalies were uncommon (P = 7.0 × 10). A clinically usable TACScore was developed with an area under the curve (AUC) of 0.9 (P = 1.6 × 10). A Tbx6 mouse model supported that a gene dosage effect underlies the TACS phenotype.

Conclusion: TACS is a clinically distinguishable entity with consistent clinically measurable endophenotypes. The type and distribution of vertebral column abnormalities in TBX6/Tbx6 compound inheritance implicate subtle perturbations in gene dosage as a cause of spine developmental birth defects responsible for about 10% of CS.
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http://dx.doi.org/10.1038/s41436-018-0377-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6659397PMC
July 2019

Identification of novel LFNG mutations in spondylocostal dysostosis.

J Hum Genet 2019 Mar 10;64(3):261-264. Epub 2018 Dec 10.

Laboratory of Bone and Joint Diseases, Center for Integrative Medical Sciences RIKEN, Tokyo, Japan.

Spondylocostal dysostosis (SCDO) is a heterogeneous group of skeletal disorders characterized by multiple segmentation defects involving vertebrae and ribs. Seven disease genes have been reported as causal genes for SCDO: DLL3, MESP2, TBX6, HES7, RIPPLY2, DMRT2, and LFNG. Here we report a Japanese SCDO case with multiple severe vertebral anomalies from cervical to sacral spine. The patient was a compound heterozygote for c.372delG (p.K124Nfs*) and c.601G>A (p.D201N) variants of LFNG, which encodes a glycosyltransferase (O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase). The missense variant was in the DxD motif, an active-site motif of the glycosyltransferase, and its loss of the enzyme function was confirmed by an in vitro enzyme assay. This is the second report of LFNG mutations in SCDO.
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http://dx.doi.org/10.1038/s10038-018-0548-2DOI Listing
March 2019

Crystal structure of heme A synthase from .

Proc Natl Acad Sci U S A 2018 11 5;115(47):11953-11957. Epub 2018 Nov 5.

Department of Chemistry, Graduate School of Science, Kyoto University, 606-8502 Kyoto, Japan;

Heme A is an essential cofactor for respiratory terminal oxidases and vital for respiration in aerobic organisms. The final step of heme A biosynthesis is formylation of the C-8 methyl group of heme molecule by heme A synthase (HAS). HAS is a heme-containing integral membrane protein, and its structure and reaction mechanisms have remained unknown. Thus, little is known about HAS despite of its importance. Here we report the crystal structure of HAS from at 2.2-Å resolution. The N- and C-terminal halves of HAS consist of four-helix bundles and they align in a pseudo twofold symmetry manner. Each bundle contains a pair of histidine residues and forms a heme-binding domain. The C-half domain binds a cofactor-heme molecule, while the N-half domain is vacant. Many water molecules are found in the transmembrane region and around the substrate-binding site, and some of them interact with the main chain of transmembrane helix. Comparison of these two domain structures enables us to construct a substrate-heme binding state structure. This structure implies that a completely conserved glutamate, Glu57 in , is the catalytic residue for the formylation reaction. These results provide valuable suggestions of the substrate-heme binding mechanism. Our results present significant insight into the heme A biosynthesis.
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http://dx.doi.org/10.1073/pnas.1813346115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255202PMC
November 2018

Genome-wide meta-analysis and replication studies in multiple ethnicities identify novel adolescent idiopathic scoliosis susceptibility loci.

Hum Mol Genet 2018 11;27(22):3986-3998

Sarah M. & Charles E. Seay Center for Musculoskeletal Research, Texas Scottish Rite Hospital for Children, Dallas, TX, USA.

Adolescent idiopathic scoliosis (AIS) is the most common musculoskeletal disorder of childhood development. The genetic architecture of AIS is complex, and the great majority of risk factors are undiscovered. To identify new AIS susceptibility loci, we conducted the first genome-wide meta-analysis of AIS genome-wide association studies, including 7956 cases and 88 459 controls from 3 ancestral groups. Three novel loci that surpassed genome-wide significance were uncovered in intragenic regions of the CDH13 (P-value_rs4513093 = 1.7E-15), ABO (P-value_ rs687621 = 7.3E-10) and SOX6 (P-value_rs1455114 = 2.98E-08) genes. Restricting the analysis to females improved the associations at multiple loci, most notably with variants within CDH13 despite the reduction in sample size. Genome-wide gene-functional enrichment analysis identified significant perturbation of pathways involving cartilage and connective tissue development. Expression of both SOX6 and CDH13 was detected in cartilage chondrocytes and chromatin immunoprecipitation sequencing experiments in that tissue revealed multiple HeK27ac-positive peaks overlapping associated loci. Our results further define the genetic architecture of AIS and highlight the importance of vertebral cartilage development in its pathogenesis.
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http://dx.doi.org/10.1093/hmg/ddy306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488972PMC
November 2018

Screening of known disease genes in congenital scoliosis.

Mol Genet Genomic Med 2018 11 9;6(6):966-974. Epub 2018 Sep 9.

Laboratory of Bone and Joint Diseases, Center for Integrative Medical Sciences, RIKEN, Tokyo, Japan.

Background: Congenital scoliosis (CS) is defined as a lateral curvature of the spine due to the vertebral malformations and has an incidence of 0.5-1/1,000 births. We previously examined TBX6 in Japanese CS patients and revealed that approximately 10% of CS was caused by TBX6 mutations. However, the genetic cause of remaining CS is unknown.

Methods: We recruited 78 CS patients without TBX6 mutations and major comorbidities, and investigated the genes previously reported to be associated with CS and congenital vertebral malformations by whole-exome sequencing.

Results: We identified the compound heterozygous missense variants in LFNG in one patient. No likely disease-causing variants were identified in other patients, however. LFNG encodes a GlcNAc-transferase. The LFNG variants showed loss of their enzyme function.

Conclusions: A LFNG mutation is reported in a case of spondylocostal dysostosis (SCD), a skeletal dysplasia with severe malformations of vertebra and rib. The CS patient with LFNG mutations had multiple vertebral malformations including hemivertebrae, butterfly vertebrae, and block vertebrae, and rib malformations. LFNG mutations may cause a spectrum of phenotypes including CS and SCD. The current list of known disease genes could explain only a small fraction of genetic cause of CS.
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http://dx.doi.org/10.1002/mgg3.466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305645PMC
November 2018

X-ray structure analysis of bacteriorhodopsin at 1.3 Å resolution.

Sci Rep 2018 09 3;8(1):13123. Epub 2018 Sep 3.

Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.

Bacteriorhodopsin (bR) of Halobacterium salinarum is a membrane protein that acts as a light-driven proton pump. bR and its homologues have recently been utilized in optogenetics and other applications. Although the structures of those have been reported so far, the resolutions are not sufficient for elucidation of the intrinsic structural features critical to the color tuning and ion pumping properties. Here we report the accurate crystallographic analysis of bR in the ground state. The influence of X-rays was suppressed by collecting the data under a low irradiation dose at 15 K. Consequently, individual atoms could be separately observed in the electron density map at better than 1.3 Å resolution. Residues from Thr5 to Ala233 were continuously constructed in the model. The twist of the retinal polyene was determined to be different from those in the previous models. Two conformations were observed for the proton release region. We discuss the meaning of these fine structural features.
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http://dx.doi.org/10.1038/s41598-018-31370-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120890PMC
September 2018

Comparison of xenobiotic metabolism in phase I oxidation and phase II conjugation between rats and bird species.

Comp Biochem Physiol C Toxicol Pharmacol 2018 Dec 31;214:28-35. Epub 2018 Aug 31.

Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N18 W9, Kita-ku, Sapporo 060-0818, Japan.

There have been many reports regarding toxic chemicals in birds. Chemicals are mainly metabolized in the liver through phase I oxidation by cytochrome P450 (CYP) and phase II conjugation by conjugated enzymes, such as UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), glutathione-S-transferase (GST), etc. Xenobiotic metabolism differs among bird species, but little detailed information is available. In the present study, the four-ring polycyclic aromatic hydrocarbon (PAH), pyrene, was used as a model xenobiotic to clarify the characteristics of xenobiotic metabolism in birds compared with laboratory animals by in vivo and in vitro studies. Plasma, bile, and excreta (urine and feces) were collected after oral administration of pyrene and analyzed to clarify xenobiotic metabolism ability in chickens and quails. Interestingly, pyrenediol-glucuronide sulfate (PYDOGS) and pyrenediol-diglucuronide (PYDOGG) were present in chickens and quails but not in rats. In addition, the area under the curve (AUC), maximum plasma concentration (C), and time to maximum plasma concentration (T) of pyrene-1-sulfate (PYOS) were higher than those of the parent molecule, pyrene, while the elimination half-life (t) and mean residence time (MRT) were faster than those of the parent pyrene. With regard to sulfation of 1-hydroxypyrene (PYOH), the maximum velocity (V) and Michaelis constant (K) of rat liver cytosol were greater than those of chicken and quail liver cytosol. Furthermore, V/K of UGT activity in rat liver microsomes was also greater than those of chicken and quail liver microsomes. Characterization of xenobiotic metabolism revealed species differences between birds and mammals, raising concerns about exposure to various xenobiotics in the environment.
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http://dx.doi.org/10.1016/j.cbpc.2018.08.007DOI Listing
December 2018

Co-translational folding of α-helical proteins: structural studies of intermediate-length variants of the λ repressor.

FEBS Open Bio 2018 Aug 27;8(8):1312-1321. Epub 2018 Jun 27.

Department of Chemistry Graduate School of Science Kyoto University Japan.

Nascent polypeptide chains fold cotranslationally, but the atomic-level details of this process remain unknown. Here, we report crystallographic, modeling, and spectroscopic studies of intermediate-length variants of the λ repressor N-terminal domain. Although the ranges of helical regions of the half-length variant were almost identical to those of the full-length protein, the relative orientations of these helices in the intermediate-length variants differed. Our results suggest that cotranslational folding of the λ repressor initially forms a helical structure with a transient conformation, as in the case of a molten globule state. This conformation subsequently matures during the course of protein synthesis.

Database: Structural data are available in the PDB under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZCA and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3WOA.
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http://dx.doi.org/10.1002/2211-5463.12480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070647PMC
August 2018

A multi-ethnic meta-analysis confirms the association of rs6570507 with adolescent idiopathic scoliosis.

Sci Rep 2018 08 1;8(1):11575. Epub 2018 Aug 1.

Laboratory of Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences, Tokyo, Japan.

Adolescent idiopathic scoliosis (AIS) is the most common type of spinal deformity and has a significant genetic background. Genome-wide association studies (GWASs) identified several susceptibility loci associated with AIS. Among them is a locus on chromosome 6q24.1 that we identified by a GWAS in a Japanese cohort. The locus is represented by rs6570507 located within GPR126. To ensure the association of rs6570507 with AIS, we conducted a meta-analysis using eight cohorts from East Asia, Northern Europe and USA. The analysis included a total of 6,873 cases and 38,916 controls and yielded significant association (combined P = 2.95 × 10; odds ratio = 1.22), providing convincing evidence of the worldwide association between rs6570507 and AIS susceptibility. In silico analyses strongly suggested that GPR126 is a susceptibility gene at this locus.
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http://dx.doi.org/10.1038/s41598-018-29011-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070519PMC
August 2018

Investigation of hepatic warfarin metabolism activity in rodenticide-resistant black rats (Rattus rattus) in Tokyo by in situ liver perfusion.

Pestic Biochem Physiol 2018 Jun 1;148:42-49. Epub 2018 Apr 1.

Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan. Electronic address:

Anti-blood coagulation rodenticides, such as warfarin, have been used all over the world. They inhibit vitamin K epoxide reductase (VKOR), which is necessary for producing several blood clotting factors. This inhibition by rodenticides results in lethal hemorrhage in rodents. However, heavy usage of these agents has led to the appearance of rodenticide-resistant rats. There are two major mechanisms underlying this resistance, i.e., mutation of the target enzyme of warfarin, VKOR, and enhanced metabolism of warfarin. However, there have been few studies regarding the hepatic metabolism of warfarin, which should be related to resistance. To investigate warfarin metabolism in resistant rats, in situ liver perfusion of warfarin was performed with resistant black rats (Rattus rattus) from Tokyo, Japan. Liver perfusion is an in situ methodology that can reveal hepatic function specifically with natural composition of the liver. The results indicated enhanced hepatic warfarin hydroxylation activity compared with sensitive black rats. On the other hand, in an in vitro microsomal warfarin metabolism assay to investigate kinetic parameters of cytochrome P450, which plays a major role in warfarin hydroxylation, the V of resistant rats was slightly but significantly higher compared to the results obtained in the in situ study. These results indicated that another factor like electron donators may also contribute to the enhanced metabolism in addition to high expression of cytochrome P450.
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http://dx.doi.org/10.1016/j.pestbp.2018.03.018DOI Listing
June 2018

An international meta-analysis confirms the association of BNC2 with adolescent idiopathic scoliosis.

Sci Rep 2018 03 16;8(1):4730. Epub 2018 Mar 16.

Laboratory of Bone and Joint Diseases, Center for Integrative Medical Sciences, RIKEN, Tokyo, Japan.

Adolescent idiopathic scoliosis (AIS) is a common spinal deformity with the prevalence of approximately 3%. We previously conducted a genome-wide association study (GWAS) using a Japanese cohort and identified a novel locus on chromosome 9p22.2. However, a replication study using multi-population cohorts has not been conducted. To confirm the association of 9p22.2 locus with AIS in multi-ethnic populations, we conducted international meta-analysis using eight cohorts. In total, we analyzed 8,756 cases and 27,822 controls. The analysis showed a convincing evidence of association between rs3904778 and AIS. Seven out of eight cohorts had significant P value, and remaining one cohort also had the same trend as the seven. The combined P was 3.28 × 10 (odds ratio = 1.19, 95% confidence interval = 1.14-1.24). In silico analyses suggested that BNC2 is the AIS susceptibility gene in this locus.
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http://dx.doi.org/10.1038/s41598-018-22552-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856832PMC
March 2018

A functional variant in MIR4300HG, the host gene of microRNA MIR4300 is associated with progression of adolescent idiopathic scoliosis.

Hum Mol Genet 2017 10;26(20):4086-4092

Laboratory of Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences, Tokyo 108-8639, Japan.

Adolescent idiopathic scoliosis (AIS) is a common spinal deformity affecting millions of children. Since treatment and prognosis of AIS depend on curve progression, identifying factors related to AIS curve progression is important in its management. Although several genetic loci for AIS occurrence are reported, no locus for curve progression has been identified. To identify genes associated with AIS progression, we conducted a genome-wide association study followed by a replication study using a total of 2,543 AIS subjects who were evaluated for the curve progression. We identified a significantly associated locus on chromosome 11q14.1 (P = 1.98 × 10-9, odds ratio = 1.56). In silico and in vitro analyses identified a functional variant, rs35333564 in MIR4300HG, the host gene of a microRNA, MIR4300. The genomic region containing rs35333564 had enhancer activity, which was decreased in its risk allele. Our data suggest that decrease of MIR4300 is related to AIS progression.
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http://dx.doi.org/10.1093/hmg/ddx291DOI Listing
October 2017

A Replication Study for the Association of rs11190870 With Curve Severity in Adolescent Idiopathic Scoliosis in Japanese.

Spine (Phila Pa 1976) 2018 05;43(10):688-692

Laboratory of Bone and Joint Diseases, RIKEN Center for Integrative Sciences, Tokyo, Japan.

Study Design: Case-only study.

Objective: The aim of this study was to confirm the association of rs11190870 with adolescent idiopathic scoliosis (AIS) severity in Japanese patients with AIS.

Summary Of Background Data: Although the association of rs11190870 with AIS susceptibility is replicated in multiple ethnics, the association of rs11190870 with curve severity is controversial. Since the previous studies are of small, we performed a replication study using far larger number of patients than previous studies.

Methods: A total of 1860 Japanese patients with AIS who had reached skeletal maturity or undergone surgical fusion were included in the study. We evaluated the association between rs11190870 and AIS progression for the entire group, and then for patients grouped according to a severe curve (a Cobb angle of ≥40°) or mild curve (a Cobb angle <30°). Because braces could affect the results of the present study, patients in the mild-curve group were divided according to whether or not they had worn a brace. We then evaluated associations between rs11190870 genotype and curve severity in these groups.

Results: The mean Cobb angles were 54.8° ± 12.1° in the severe-curve group and 24.4° ± 4.0° in the mild-curve group. The difference in rs11190870 risk-allele frequency between the severe- and mild-curve groups was evaluated. No significant differences were observed. We then examined the association of rs11190870 risk-allele frequency between patients in the mild- and severe-curve groups using the χ test for three models, and found a marginal association between rs11190870 and curve severity in the dominant model (P = 0.035, odds ratio = 1.51).

Conclusion: We found no association between rs11190870 and curve severity using the criteria of previous study. However, we found a marginal association between rs11190870 and curve severity. Large-scale replication studies that consider skeletal maturity and brace history, including replication studies in other ethnic groups, would be helpful for clarifying the association.

Level Of Evidence: 4.
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http://dx.doi.org/10.1097/BRS.0000000000002413DOI Listing
May 2018