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Illumina-based transcriptomic analysis of the fast-growing leguminous tree Acacia crassicarpa: functional gene annotation and identification of novel SSR-markers

Shougo Ishio, Kazutaka Kusunoki, Michiko Nemoto, Tadayoshi Kanao, Takashi Tamura

Frontiers in Plant Science 15 2024年8月

掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers Media SA

Acacia crassicarpa is a fast-growing leguminous tree that is widely cultivated in tropical areas such as Indonesia, Malaysia, Australia, and southern China. This tree has versatile utility in timber, furniture, and pulp production. Illumina sequencing of A. crassicarpa was conducted, and the raw data of 124,410,892 reads were filtered and assembled de novo into 93,317 unigenes, with a total of 84,411,793 bases. Blast2GO annotation, Benchmark Universal Single-Copy Ortholog evaluation, and GO-term classification produced a catalogue of unigenes for studying primary metabolism, phytohormone signaling, and transcription factors. Massive transcriptomic analysis has identified microsatellites composed of simple sequence repeat (SSR) loci representing di-, tri-, and tetranucleotide repeat units in the predicted open reading frames. Polymorphism was induced by PCR amplification of microsatellite loci located in several genes encoding auxin response factors and other transcription factors, which successfully distinguished 16 local trees of A. crassicarpa tested, representing potentially exploitable molecular markers for efficient tree breeding for plantation and biomass exploitation.

DOI： 10.3389/fpls.2024.1339958

The Multiphasic Teeth of Chiton Articulatus, an Abrasion-Resistant and Self-Sharpening Tool for Hard Algae Collection 査読

Devis Montroni, Ezra Sarmiento, Ruoheng Zhao, Phani Saketh Dasika, John Michael Connolly, Richard Wuhrer, Yugang Zhang, Mikhail Zhernenkov, Taifeng Wang, Brenda Paola Ramirez-Santana, Leigh Sheppard, Omar Hernando Avila-Poveda, Atsushi Arakaki, Michiko Nemoto, Pablo Zavattieri, David Kisailus

Advanced Functional Materials 2024年

掲載種別：研究論文（学術雑誌）

Chiton articulatus is a species of mollusk living in the tropical Pacific intertidal rocky shores of Mexico. This species feeds on solid waste organic sources, including hard crustose algae that grow on rocky substrates, by grazing on them with its radula, a flexible chitinous membrane lined with mineralized major lateral teeth. In this study, the composition, morphology, and resulting mechanics of the mature teeth of this species, which have yet to be examined, are revealed. The results show the presence of multiphasic mature teeth, each consisting of aligned hard magnetite nanoparticles on the leading edge of the tooth underneath which are magnetite lamellae, followed by goethite, lepidocrocite, and eventually hydroxyapatite near the trailing edge. This multiregional structure demonstrates a gradation in hardness as well as different microstructural features integrated with tough interfaces. The combination of these microstructural and phase arrangements results in an abrasion-resistant tough structure with a self-sharpening ability. The results of this work will help contribute to developing new bioinspired designs while also helping to understand the evolution and feeding habits of these intriguing invertebrates.

DOI： 10.1002/adfm.202401658

Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation 査読

Koki Okada, Yu Morimoto, Yukine Shiraishi, Takashi Tamura, Shigeki Mayama, Takashi Kadono, Masao Adachi, Kentaro Ifuku, Michiko Nemoto

Marine Biotechnology 2023年12月

Fibrous anisotropy and mineral gradients Within the radula stylus of chiton: Controlled stiffness and damage tolerance in a flexible biological composite 査読

Jung-Eun Lee, John Connolloy, Wen Yang, Guillaume Freychet, Taifeng Wang, Steven A. Herrera, Satoshi Murata, Phani Saketh Dasika, Devis Montroni, Anna Pohl, Chenhui Zhu, Mikhail Zhernenkov, Richard Wuhrer, Leigh Sheppard, Michiko Nemoto, Atsushi Arakaki, Pablo Zavattieri, David Kisailus

JOURNAL OF COMPOSITE MATERIALS 2022年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SAGE PUBLICATIONS LTD

Over hundreds of millions of years, organisms have evolved architected structures via precise control over hierarchically assembled components, including the integration of dissimilar materials. One such example is found in the radula system of chitons, intertidal mollusks that feed on algae growing on the rock. Their radula consists of multiple rows of ultrahard teeth, each integrated with a foldable belt-like substrate via a stiff, yet flexible stylus, which is essential for efficient rasping during the feeding process. Here, we investigate the nano and micro-scale components and architectures as well as regional mechanical properties of the stylus, and their subsequent role during the rasping of Cyptochiton stelleri. Three important factors were determined to contribute to the regio-specific stiffness of the stylus: the presence of mineral components, highly oriented chitinous fibers, and a chemically cross-linked protein matrix. All these factors are varied throughout the stylus. There is a high mineral content on the trailing edge close to the tooth and a cross-linked matrix on the leading edge, both with orientational specific oriented chitin fibers that provide force transduction to the tooth. Conversely, there is a significant lack of mineral or cross-linked matrix in the proximal end as well as a low degree of fiber orientation, resulting in a flexible region that can accommodate torsion and flexure during rasping. Understanding the graded composite structure of the stylus and applying this unique design to various engineering fields such as soft robotics, biotechnology, and the medical industry, can inspire the production of high-performance materials.

DOI： 10.1177/00219983221121867

Evaluation of the Geenius HIV 1/2 confirmatory assay for HIV-2 samples isolated in Japan 査読

Urara Shigemi, Yoshimi Yamamura, Masakazu Matsuda, Reiko Okazaki, Mai Kubota, Shiro Ibe, Michiko Nemoto, Masami Maejima-Kitagawa, Sayaka Sukegawa, Mayumi Imahashi, Tadashi Kikuchi, Wataru Sugiura, Yasumasa Iwatani, Atsuko Hachiya, Yoshiyuki Yokomaku

Journal of Clinical Virology 152 105189 - 105189 2022年7月

Nanoarchitected Tough Biological Composites from Assembled Chitinous Scaffolds 査読

Wei Huang, Devis Montroni, Taifeng Wang, Satoshi Murata, Atsushi Arakaki, Michiko Nemoto, David Kisailus

ACCOUNTS OF CHEMICAL RESEARCH 55 ( 10 ) 1360 - 1371 2022年5月

記述言語：英語出版者・発行元：AMER CHEMICAL SOC

Over hundreds of millions of years, organisms have derived specific sets of traits in response to common selection pressures that serve as guideposts for optimal biological designs. A prime example is the evolution of toughened structures in disparate lineages within plants, invertebrates, and vertebrates. Extremely tough structures can function much like armor, battering rams, or reinforcements that enhance the ability of organisms to win competitions, find mates, acquire food, escape predation, and withstand high winds or turbulent flow. From an engineering perspective, biological solutions are intriguing because they must work in a multifunctional context. An organism rarely can be optimally designed for only one function or one environmental condition. Some of these natural systems have developed well-orchestrated strategies, exemplified in the biological tissues of numerous animal and plant species, to synthesize and construct materials from a limited selection of available starting materials. The resulting structures display multiscale architectures with incredible fidelity and often exhibit properties that are similar, and frequently superior, to mechanical properties exhibited by many engineered materials. These biological systems have accomplished this feat through the demonstrated ability to tune size, morphology, crystallinity, phase, and orientation of minerals under benign processing conditions (i.e., near-neutral pH, room temperature, etc.) by establishing controlled synthesis and hierarchical 3D assembly of nano- to microscaled building blocks. These systems utilize organic-inorganic interactions and carefully controlled microenvironments that enable kinetic control during the synthesis of inorganic structures. This controlled synthesis and assembly requires orchestration of mineral transport and nucleation. The underlying organic framework, often consisting of polysaccharides and polypeptides, in these composites is critical in the spatial and temporal regulation of these processes. In fact, the organic framework is used not only to provide transport networks for mineral precursors to nucleation sites but also to precisely guide the formation and phase development of minerals and significantly improve the mechanical performance of otherwise brittle materials.Over the past 15 years, we have focused on a few of these extreme performing organisms, (Wang et al., Adv. Fund. Mater. 2013, 23, 2908; Weaver et al., Science 2012, 336, 1275; Huang et al., Nat. Mater. 2020, 19, 1236; Rivera et al., Nature 2020, 586, 543) investigating not only their ultrastructural features and mechanical properties but in some cases, how these assembled structures are mineralized. In specific instances, comparative analyses of multiscale structures have pinpointed which design principles have arisen convergently; when more than one evolutionary path arrives at the same solution, we have a good indication that it is the best solution. This is required for survival under extreme conditions. Indeed, we have found that there are specific architectural features that provide an advantage toward survival by enabling the ability to feed effectively or to survive against predatory attacks. In this Account, we describe 3 specific design features, nanorods, helicoids, and nanoparticles, as well as the interfaces in fiber-reinforced biological composites. We not only highlight their roles in the specific organisms but also describe how controlled syntheses and hierarchical assembly using organic (i.e., often chitinous) scaffolds lead to these integrated macroscale structures. Beyond this, we provide insight into multifunctionality: how nature leverages these existing structures to potentially add an additional dimension toward their utility and describe their translation to biomimetic materials used for engineering applications.

DOI： 10.1021/acs.accounts.2c00110

Mesocrystalline Ordering and Phase Transformation of Iron Oxide Biominerals in the Ultrahard Teeth of Cryptochiton stelleri 査読

Taifeng Wang, Wei Huang, Cong Huy Pham, Satoshi Murata, Steven Herrera, Nathan D. Kirchhofer, Bassim Arkook, Dejan Stekovic, Mikhail E. Itkis, Nir Goldman, Luis Zepeda-Ruiz, Mikhail Zhernenkov, Michiko Nemoto, Atsushi Arakaki, David Kisailus

SMALL STRUCTURES 3 ( 4 ) 2022年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY

Biological organisms naturally synthesize complex, hierarchical, multifunctional materials through mineralization processes at ambient conditions and under physiological pH. One such example is the ultrahard and wear-resistant radular teeth found in mollusks, which are used to scape against the rock to feed on algae. Herein, the biologically controlled structural development of the hard, outer magnetite-containing shell of the chitin teeth is revealed. Specifically, the formation of a series of mesocrystalline iron oxide phases, templated by chitin-binding proteins, is identified. The initial domains, consisting of ferrihydrite mesocrystals with a spherulite-like morphology, undergo a solid-state phase transformation to form magnetite while maintaining mesocrystallinity, likely via a shear-induced solid-state reaction, without any noticeable architectural changes. Subsequent growth via Ostwald ripening leads to nearly single-crystalline rod-like elements. In addition, an interpenetrating organic matrix is identified that, at early stages of tooth development, potentially contains iron-binding proteins that guide the self-assembly of the mesocrystalline mineral and influence the preferred orientation of the later-formed magnetite nanorods, which ultimately determines the mechanical behavior of the mature chiton teeth.

DOI： 10.1002/sstr.202100202

Correction to: Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases. 国際誌

Michiko Nemoto, Sayako Iwaki, Hisao Moriya, Yuki Monden, Takashi Tamura, Kenji Inagaki, Shigeki Mayama, Kiori Obuse

Marine biotechnology (New York, N.Y.) 23 ( 1 ) 157 - 157 2021年2月

Multiple mutations in RNA polymerase β-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic sinefungin: modeling rif cluster region by density functional theory. 査読国際誌

Saori Ogawa, Hitomi Shimidzu, Koji Fukuda, Naoki Tsunekawa, Toshiyuki Hirano, Fumitoshi Sato, Kei Yura, Tomohisa Hasunuma, Kozo Ochi, Michio Yamamoto, Wataru Sakamoto, Kentaro Hashimoto, Hiroyuki Ogata, Tadayoshi Kanao, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

Bioscience, biotechnology, and biochemistry 2021年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

DOI： 10.1093/bbb/zbab011

Impact of long-term antiretroviral therapy on gut and oral microbiotas in HIV-1-infected patients. 査読国際誌

Mayumi Imahashi, Hirotaka Ode, Ayumi Kobayashi, Michiko Nemoto, Masakazu Matsuda, Chieko Hashiba, Akiko Hamano, Yoshihiro Nakata, Mikiko Mori, Kento Seko, Masashi Nakahata, Ayumi Kogure, Yasuhito Tanaka, Wataru Sugiura, Yoshiyuki Yokomaku, Yasumasa Iwatani

Scientific reports 11 ( 1 ) 960 - 960 2021年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

In HIV-1-infected patients, antiretroviral therapy (ART) is a key factor that may impact commensal microbiota and cause the emergence of side effects. However, it is not fully understood how long-term ART regimens have diverse impacts on the microbial compositions over time. Here, we performed 16S ribosomal RNA gene sequencing of the fecal and salivary microbiomes in patients under different long-term ART. We found that ART, especially conventional nucleotide/nucleoside reverse transcriptase inhibitor (NRTI)-based ART, has remarkable impacts on fecal microbial diversity: decreased α-diversity and increased ß-diversity over time. In contrast, dynamic diversity changes in the salivary microbiome were not observed. Comparative analysis of bacterial genus compositions showed a propensity for Prevotella-enriched and Bacteroides-poor gut microbiotas in patients with ART over time. In addition, we observed a gradual reduction in Bacteroides but drastic increases in Succinivibrio and/or Megasphaera under conventional ART. These results suggest that ART, especially NRTI-based ART, has more suppressive impacts on microbiota composition and diversity in the gut than in the mouth, which potentially causes intestinal dysbiosis in patients. Therefore, NRTI-sparing ART, especially integrase strand transfer inhibitor (INSTI)- and/or non-nucleotide reverse transcriptase inhibitor (NNRTI)-containing regimens, might alleviate the burden of intestinal dysbiosis in HIV-1-infected patients under long-term ART.

DOI： 10.1038/s41598-020-80247-8

Radular stylus of Cryptochiton stelleri: A multifunctional lightweight and flexible fiber-reinforced composite. 査読国際誌

Anna Pohl, Steven A Herrera, David Restrepo, Ryo Negishi, Jae-Young Jung, Chris Salinas, Richard Wuhrer, Tomoko Yoshino, Joanna McKittrick, Atsushi Arakaki, Michiko Nemoto, Pablo Zavattieri, David Kisailus

Journal of the mechanical behavior of biomedical materials 111 103991 - 103991 2020年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Chitons are herbivorous invertebrates that use rows of ultrahard magnetite-based teeth connected to a flexible belt (radula) to rasp away algal deposits growing on and within rocky outcrops along coastlines around the world. Each tooth is attached to the radula by an organic structure (stylus) that provides mechanical support during feeding. However, the underlying structures within the stylus, and their subsequent function within the chiton have yet to be investigated. Here, we investigate the macrostructural architecture, the regional material and elemental distribution and subsequent nano-mechanical properties of the stylus from the Northern Pacific dwelling Cryptochiton stelleri. Using a combination of μ-CT imaging, optical and electron microscopy, as well as elemental analysis, we reveal that the stylus is a highly contoured tube, mainly composed of alpha-chitin fibers, with a complex density distribution. Nanoindentation reveals regiospecific and graded mechanical properties that can be correlated with both the elemental composition and material distribution. Finite element modeling shows that the unique macroscale architecture, material distribution and elemental gradients have been optimized to preserve the structural stability of this flexible, yet robust functionally-graded fiber-reinforced composite tube, providing effective function during rasping. Understanding these complex fiber-based structures offers promising blueprints for lightweight, multifunctional and integrated materials.

DOI： 10.1016/j.jmbbm.2020.103991

Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases. 査読国際誌

Michiko Nemoto, Sayako Iwaki, Hisao Moriya, Yuki Monden, Takashi Tamura, Kenji Inagaki, Shigeki Mayama, Kiori Obuse

Marine biotechnology (New York, N.Y.) 2020年6月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.

DOI： 10.1007/s10126-020-09976-1

Antiviral effect of sinefungin on in vitro growth of feline herpesvirus type 1. 査読

Yudai Kuroda, Haruka Yamagata, Michiko Nemoto, Kenji Inagaki, Takashi Tamura, Ken Maeda

The Journal of antibiotics 72 ( 12 ) 981 - 985 2019年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.

DOI： 10.1038/s41429-019-0234-4

Redox-tuning of oxidizing disulfide oxidoreductase generates a potent disulfide isomerase. 査読国際誌

Shinya Sutoh, Yuko Uemura, Yuko Yamaguchi, Asako Kiyotou, Rena Sugihara, Makiko Nagayasu, Mihoko Kurokawa, Koreaki Ito, Naoki Tsunekawa, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

Biochimica et biophysica acta. Proteins and proteomics 1867 ( 3 ) 194 - 201 2019年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was -172 mV, representing a less-oxidizing catalysis than the wild type DsbA (-122 mV), but one that is closer to yeast protein disulfide isomerase (-175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of -172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.

DOI： 10.1016/j.bbapap.2018.12.005

Integrated transcriptomic and proteomic analyses of a molecular mechanism of radular teeth biomineralization in Cryptochiton stelleri. 査読国際誌

Michiko Nemoto, Dongni Ren, Steven Herrera, Songqin Pan, Takashi Tamura, Kenji Inagaki, David Kisailus

Scientific reports 9 ( 1 ) 856 - 856 2019年1月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Many species of chiton are known to deposit magnetite (Fe3O4) within the cusps of their heavily mineralized and ultrahard radular teeth. Recently, much attention has been paid to the ultrastructural design and superior mechanical properties of these radular teeth, providing a promising model for the development of novel abrasion resistant materials. Here, we constructed de novo assembled transcripts from the radular tissue of C. stelleri that were used for transcriptome and proteome analysis. Transcriptomic analysis revealed that the top 20 most highly expressed transcripts in the non-mineralized teeth region include the transcripts encoding ferritin, while those in the mineralized teeth region contain a high proportion of mitochondrial respiratory chain proteins. Proteomic analysis identified 22 proteins that were specifically expressed in the mineralized cusp. These specific proteins include a novel protein that we term radular teeth matrix protein1 (RTMP1), globins, peroxidasins, antioxidant enzymes and a ferroxidase protein. This study reports the first de novo transcriptome assembly from C. stelleri, providing a broad overview of radular teeth mineralization. This new transcriptomic resource and the proteomic profiles of mineralized cusp are valuable for further investigation of the molecular mechanisms of radular teeth mineralization in chitons.

DOI： 10.1038/s41598-018-37839-2

Novel Compound Heterozygous TYK2 Mutations in Primary Immunodeficiency with T-Cell Lymphopenia 査読

Hattori Hiroyoshi, Nemoto Michiko, Maeda Naoko, Akita Nobuhiro, Moritani Suzuko, Muramatsu Hideki, Kawasaki Tomonori, Maejima Masami, Ode Hirotaka, Hachiya Atsuko, Sugiura Wataru, Yokomaku Yoshiyuki, Iwatani Yasumasa, Horibe Keizo

PEDIATRIC BLOOD & CANCER 65 S45 2018年11月

Compound heterozygous TYK2 mutations underlie primary immunodeficiency with T-cell lymphopenia. 査読国際誌

Michiko Nemoto, Hiroyoshi Hattori, Naoko Maeda, Nobuhiro Akita, Hideki Muramatsu, Suzuko Moritani, Tomonori Kawasaki, Masami Maejima, Hirotaka Ode, Atsuko Hachiya, Wataru Sugiura, Yoshiyuki Yokomaku, Keizo Horibe, Yasumasa Iwatani

Scientific reports 8 ( 1 ) 6956 - 6956 2018年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Complete tyrosine kinase 2 (TYK2) deficiency has been previously described in patients with primary immunodeficiency diseases. The patients were infected with various pathogens, including mycobacteria and/or viruses, and one of the patients developed hyper-IgE syndrome. A detailed immunological investigation of these patients revealed impaired responses to type I IFN, IL-10, IL-12 and IL-23, which are associated with increased susceptibility to mycobacterial and/or viral infections. Herein, we report a recessive partial TYK2 deficiency in two siblings who presented with T-cell lymphopenia characterized by low naïve CD4+ T-cell counts and who developed Epstein-Barr virus (EBV)-associated B-cell lymphoma. Targeted exome-sequencing of the siblings' genomes demonstrated that both patients carried novel compound heterozygous mutations (c.209_212delGCTT/c.691C > T, p.Cys70Serfs*21/p.Arg231Trp) in the TYK2. The TYK2 protein levels were reduced by 35% in the T cells of the patient. Unlike the response under complete TYK2 deficiency, the patient's T cells responded normally to type I IFN, IL-6, IL-10 and IL-12, whereas the cells displayed an impaired response to IL-23. Furthermore, the level of STAT1 was low in the cells of the patient. These studies reveal a new clinical entity of a primary immunodeficiency with T-cell lymphopenia that is associated with compound heterozygous TYK2 mutations in the patients.

DOI： 10.1038/s41598-018-25260-8

An Internal Sequence Region Catalytically Essential for the Human Selenophosphate Synthetase 2 査読

Muneaki Takahata, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

FREE RADICAL BIOLOGY AND MEDICINE 112 65 - 65 2017年11月

Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590. 査読国際誌

Masaya Hayashi, Akane Okada, Kumiko Yamamoto, Tomomi Okugochi, Chika Kusaka, Daizou Kudou, Michiko Nemoto, Junko Inagaki, Yuu Hirose, Toshihide Okajima, Takashi Tamura, Kenji Soda, Kenji Inagaki

Journal of biochemistry 161 ( 4 ) 389 - 398 2017年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

DOI： 10.1093/jb/mvw083

A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. 査読国際誌

Saho Kamada, Takahiro Okugochi, Kaori Asano, Ryuta Tobe, Hisaaki Mihara, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

Bioscience, biotechnology, and biochemistry 80 ( 10 ) 1970 - 2 2016年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：TAYLOR & FRANCIS LTD

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-(14)C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min(-1), respectively.

DOI： 10.1080/09168451.2016.1200458

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