Performance information

• Biosynthesis of unnatural polyisoprenes by engineered prenyltransferases on rubber particles
  Seiji Takahashi; Miki Suenaga-Hiromori; Tomoki Ishii; Nadia Nur Shazana Binti Abu Talib Khan; Tomoyo Mikami; Tomohiro Takahashi; Chiho Minakawa; Fumihiro Yanbe; Toshiyuki Waki; Toru Nakayama; Riki Imaizumi; Taro Yanai; Kunishige Kataoka; Satoshi Yamashita; Kohei Takeshita; Hiroaki Matsuura; Naoki Sakai; Masaki Yamamoto; Haruhiko Yamaguchi; Yukino Miyagi-Inoue; Kazuhisa Fushihara; Yuzuru Tozawa
  Jul. 2024
  Abstract

  Natural rubber (NR) is a sustainable biopolymer consisting mainly of cis-1,4-polyisoprene. Modifying an NR biosynthetic enzyme is a promising strategy to bioproduce novel polymers. Here, we have elucidated the NR biosynthetic mechanism and successfully developed novel enzymes that synthesise NR-sized polyisoprenes with unnatural substrates. NR is synthesised by a cis-prenyltransferase (cPT) on rubber particles (RPs), NR-harbouring lipid monolayer membrane organelles. However, the key to NR biosynthesis is not specialised cPTs, but the proper arrangement of cPTs on RPs since cPTs from various non-NR-producing organisms, such as humans, synthesise NR when introduced into the RPs. A tomato cPT, which condenses only one isoprene unit, was engineered to synthesise novel NR-sized polyisoprenes with artificial substrates by modifying residues for product size determination. Furthermore, the introduction of a modified trans-prenyltransferase into RPs led to the synthesis of NR-sized trans-1,4-polyisoprenes. This RP system could be used as a versatile platform for enzymatic polyisoprenoid synthesis.

  
  Springer Science and Business Media LLC
  DOI：https://doi.org/10.21203/rs.3.rs-3615345/v1
  DOI ID：10.21203/rs.3.rs-3615345/v1
• Partner-switching components PmgA and Ssr1600 regulate high-light acclimation in Synechocystis sp. PCC 6803
  Riku Nakamura; Yuji Takahashi; Shogo Tachibana; Arisa Terada; Kakeru Suzuki; Kumika Kondo; Yuzuru Tozawa; Yukako Hihara
  Plant Physiology, Volume：196, Number：1, First page：621, Last page：633, Jun. 2024
  Abstract
  
  Photomixotrophic growth A (PmgA) is a pleiotropic regulator essential for growth under photomixotrophic and prolonged high-light (HL) conditions in the cyanobacterium Synechocystis sp. PCC 6803. The overall similarity with the antisigma factor of the bacterial partner-switching system indicates that PmgA exerts a regulatory function via phosphorylation of its target proteins. In this study, we performed an in vitro phosphorylation assay and protein–protein interaction analysis and found that PmgA interacts with 4 antisigma antagonist homologs, Ssr1600, Slr1856, Slr1859, and Slr1912, but specifically phosphorylates Ssr1600. Phenotypic analyses using the set of gene disruption and overexpression strains of pmgA and ssr1600 revealed that phosphorylation by PmgA is essential for the accumulation of Ssr1600 protein in vivo. The ssr1600-disrupted mutant showed similar phenotypes as those previously reported for the pmgA-disrupted mutant, namely, no obvious phenotype just after the shift to HL, but higher chlorophyll content, 5-aminolevulinic acid synthesis activity, and psaAB transcript levels than those in the wild type after 6 h. These findings indicate that the phosphorylated form of Ssr1600 works as the output of the partner-switching system to coordinately repress chlorophyll biosynthesis and accumulation of photosystem I during HL acclimation.
  Oxford University Press (OUP), Scientific journal
  DOI：https://doi.org/10.1093/plphys/kiae323
  DOI ID：10.1093/plphys/kiae323, ISSN：0032-0889, eISSN：1532-2548
• Cell-free translation system with artificial lipid-monolayer particles as a unique tool for characterizing lipid-monolayer binding proteins.　　　　　　　　　　　　　　　
  Fu Kuroiwa; Hiraku Suda; Maho Yabuki; Kimie Atsuzawa; Haruhiko Yamaguchi; Masatsugu Toyota; Yasuko Kaneko; Satoshi Yamashita; Seiji Takahashi; Yuzuru Tozawa
  Bioscience, biotechnology, and biochemistry, Mar. 2024, ［International magazine］
  Methods for functional analysis of proteins specifically localizing to lipid monolayers such as rubber particles and lipid droplets are limited. We have succeeded in establishing a system in which artificially prepared lipid monolayer particles are added to a cell-free translation system to confirm the properties of proteins that specifically bind to lipid monolayers in a translation-coupled manner.
  English, Scientific journal
  DOI：https://doi.org/10.1093/bbb/zbae026
  DOI ID：10.1093/bbb/zbae026, PubMed ID：38444196
• Reconstitution of prenyltransferase activity on nanodiscs by components of the rubber synthesis machinery of the Para rubber tree and guayule.　　　　　　　　　　　　　　　
  Fu Kuroiwa; Akira Nishino; Yasuko Mandal; Masataka Honzawa; Miki Suenaga-Hiromori; Kakeru Suzuki; Yukie Takani; Yukino Miyagi-Inoue; Haruhiko Yamaguchi; Satoshi Yamashita; Seiji Takahashi; Yuzuru Tozawa
  Scientific reports, Volume：12, Number：1, First page：3734, Last page：3734, Mar. 2022, ［International magazine］
  Natural rubber of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity. The proteins HRT1, HRT2, and HRBP have been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Co-expression of HRT1 and HRBP in the presence of nanodiscs yielded marked polyisoprene synthesis activity. By contrast, neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP manifested such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (PaCPT1-3) manifested prenyltransferase activity only when co-expressed with PaCBP, the homolog of HRBP. Our results thus indicate that two heterologous subunits form the core prenyltransferase of the rubber biosynthetic machinery. A recently developed structure modeling program predicted the structure of such heterodimer complexes including HRT1/HRBP and PaCPT2/PaCBP. HRT and PaCPT proteins were also found to possess affinity for a lipid membrane in the absence of HRBP or PaCBP, and structure modeling implicated an amphipathic α-helical domain of HRT1 and PaCPT2 in membrane binding of these proteins.
  English, Scientific journal
  DOI：https://doi.org/10.1038/s41598-022-07564-y
  DOI ID：10.1038/s41598-022-07564-y, PubMed ID：35260628, PubMed Central ID：PMC8904820
• Catalytic promiscuity of rice 2-oxoglutarate/Fe(II)-dependent dioxygenases supports xenobiotic metabolism　　　　　　　　　　　　　　　
  Natsuki Takamura; Akihiko Yamazaki; Nozomi Sakuma; Sakiko Hirose; Motonari Sakai; Yukie Takani; Satoshi Yamashita; Masahiro Oshima; Makoto Kuroki; Yuzuru Tozawa
  Plant Physiology, Volume：187, Number：2, First page：816, Last page：828, Jun. 2021, [Reviewed], [Corresponding], ［International magazine］
  
  The rice (Oryza sativa) 2-oxoglutarate (2OG)/Fe(II)-dependent dioxygenase HIS1 mediates the catalytic inactivation of five distinct β-triketone herbicides (bTHs). By assessing the effects of plant growth regulators on HIS1 enzyme function, we found that HIS1 mediates the hydroxylation of trinexapac-ethyl (TE) in the presence of Fe2+ and 2OG. TE blocks gibberellin biosynthesis, and we observed that its addition to culture medium induced growth retardation of rice seedlings in a concentration-dependent manner. Similar treatment with hydroxylated TE revealed that hydroxylation greatly attenuated the inhibitory effect of TE on plant growth. Forced expression of HIS1 in a rice his1 mutant also reduced its sensitivity to TE compared with that of the nontransformant. These results indicate that HIS1 metabolizes TE and thereby markedly reduces its ability to slow plant growth. Furthermore, analysis of five rice HIS1-like (HSL) proteins revealed that OsHSL2 and OsHSL4 also metabolize TE in vitro. HSLs from wheat (Triticum aestivum) and barley (Hordeum vulgare) also showed such activity. In contrast, OsHSL1, which shares the highest amino acid sequence identity with HIS1 and metabolizes the bTH tefuryltrione, did not manifest TE-metabolizing activity. Site-directed mutagenesis of OsHSL1 informed by structural models showed that substitution of three amino acids with the corresponding residues of HIS1 conferred TE-metabolizing activity similar to that of HIS1. Our results thus reveal a catalytic promiscuity of HIS1 and its related enzymes that support xenobiotic metabolism in plants.
  Oxford University Press (OUP), English, Scientific journal
  DOI：https://doi.org/10.1093/plphys/kiab293
  DOI ID：10.1093/plphys/kiab293, eISSN：1532-2548, PubMed ID：34608958, PubMed Central ID：PMC8491036
• Lateral voltage as a new input for artificial lipid bilayer systems
  Teng Ma; Madoka Sato; Maki Komiya; Kensaku Kanomata; Takaya Watanabe; Xingyao Feng; Ryusuke Miyata; Daisuke Tadaki; Fumihiko Hirose; Yuzuru Tozawa; Ayumi Hirano-Iwata
  Faraday Discussions, Volume：233, First page：244, Last page：256, 2021, [Reviewed], [Invited]
  In this work, we propose lateral voltage as a new input for use in artificial lipid bilayer systems in addition to the commonly used transmembrane voltage. To apply a lateral...
  Royal Society of Chemistry (RSC), English, Scientific journal
  DOI：https://doi.org/10.1039/d1fd00045d
  DOI ID：10.1039/d1fd00045d, ISSN：1359-6640, eISSN：1364-5498
• Establishment of a cell-free translation system from rice callus extracts
  Kakeru Suzuki; Haruka Inoue; Satoshi Matsuoka; Ryugo Tero; Ayumi Hirano-Iwata; Yuzuru Tozawa
  Bioscience, Biotechnology, and Biochemistry, Volume：84, Number：10, First page：2028, Last page：2036, Oct. 2020
  
  Eukaryotic in vitro translation systems require large numbers of protein and RNA components and thereby rely on the use of cell extracts. Here we established a new in vitro translation system based on rice callus extract (RCE). We confirmed that RCE maintains its initial activity even after five freeze-thaw cycles and that the optimum temperature for translation is around 20°C. We demonstrated that the RCE system allows the synthesis of hERG, a large membrane protein, in the presence of liposomes. We also showed that the introduction of a bicistronic mRNA based on 2A peptide to RCE allowed the production of two distinct proteins from a single mRNA. Our new method thus facilitates laboratory-scale production of cell extracts, making it a useful tool for the in vitro synthesis of proteins for biochemical studies.
  Oxford University Press (OUP), Scientific journal
  DOI：https://doi.org/10.1080/09168451.2020.1779024
  DOI ID：10.1080/09168451.2020.1779024, ISSN：0916-8451, eISSN：1347-6947
• Characterization of mitochondrial carrier proteins of malaria parasite Plasmodium falciparum based on in vitro translation and reconstitution.　　　　　　　　　　　　　　　
  Akira Nozawa; Daisuke Ito; Mohamed Ibrahim; Herbert J Santos; Takafumi Tsuboi; Yuzuru Tozawa
  Parasitology international, Volume：79, First page：102160, Last page：102160, Jun. 2020, [Reviewed], [Corresponding]
  Members of the mitochondrial carrier (MC) family of membrane transporters play important roles in cellular metabolism. We previously established an in vitro reconstitution system for membrane transporters based on wheat germ cell-free translation system. We have now applied this reconstitution system to the comparative analysis of MC proteins from the malaria parasite Plasmodium falciparum and Saccharomyces cerevisiae. We synthesized twelve putative P. falciparum MCs and determined the transport activities of four of these proteins including PF3D7_1037300 protein (ADP/ATP translocator), PF3D7_1004800 protein (ADP/ATP translocator), PF3D7_1202200 protein (phosphate carrier), and PF3D7_1241600 protein (S-adenosylmethionine transporter). In addition, we tested the effect of cardiolipin on the activity of MC proteins. The transport activities of the yeast MCs, ScAac2p, ScGgc1p, ScDic1p, ScPic1p, and ScSam5p, which localize to the mitochondrial inner membrane, were increased by cardiolipin supplementation, whereas that of ScAnt1p, which localizes to the peroxisome membrane, was not significantly affected. Together, this indicates that the functional properties of the reconstituted MCs reflect the lipid content of their native membranes. Except for PF3D7_1241600 protein, these P. falciparum proteins manifested cardiolipin-dependent transport activities. Immunofluorescence analysis showed that PF3D7_1241600 protein is not mainly localized to the mitochondria of P. falciparum cells. We thus revealed the functions of four MC proteins of the malaria parasite and the effects of cardiolipin on their activities.
  DOI：https://doi.org/10.1016/j.parint.2020.102160
  DOI ID：10.1016/j.parint.2020.102160, PubMed ID：32574727
• Advances in artificial cell membrane systems as a platform for reconstituting ion channels　　　　　　　　　　　　　　　
  Maki Komiya; Miki Kato; Daisuke Tadaki; Teng Ma; Hideaki Yamamoto; Ryugo Tero; Yuzuru Tozawa; Michio Niwano
  The Chemical Record, Volume：accepted, 2020, []
• Substrate specificity of plastid phosphate transporters in a non-photosynthetic diatom and its implication in evolution of red alga-derived complex plastids　　　　　　　　　　　　　　　
  Daniel Moog; Akira Nozawa; Yuzuru Tozawa; Ryoma Kamikawa
  Volume：10, Jan. 2020, []
• Novel lineage-specific transmembrane β-barrel proteins in the endoplasmic reticulum of Entamoeba histolytica.　　　　　　　　　　　　　　　
  Herbert J Santos; Kenichiro Imai; Takashi Makiuchi; Kentaro Tomii; Paul Horton; Akira Nozawa; Kenta Okada; Yuzuru Tozawa; Tomoyoshi Nozaki
  The FEBS journal, Volume：286, Number：17, First page：3416, Last page：3432, Sep. 2019, []
  β-barrel outer membrane proteins (BOMPs) are essential components of outer membranes of Gram-negative bacteria and endosymbiotic organelles, usually involved in the transport of proteins and substrates across the membrane. Based on the analysis of our in silico BOMP predictor data for the Entamoeba histolytica genome, we detected a new transmembrane β-barrel domain-containing protein, EHI_192610. Sequence analysis revealed that this protein is unique to Entamoeba species, and it exclusively clusters with a homolog, EHI_099780, which is similarly lineage specific. Both proteins possess an N-terminal signal peptide sequence as well as multiple repeats that contain dyad hydrophobic periodicities. Data from immunofluorescence assay of trophozoites expressing the respective candidates showed the absence of colocalization with mitosomal marker, and interestingly demonstrated partial colocalization with endoplasmic reticulum (ER) proteins instead. Integration to organellar membrane was supported by carbonate fractionation assay and immunoelectron microscopy. CD analysis of reconstituted proteoliposomes containing EHI_192610 showed a spectrum demonstrating a predominant β-sheet structure, suggesting that this protein is β-strand rich. Furthermore, the presence of repeat regions with predicted transmembrane β-strand pairs in both EHI_192610 and EHI_099780, is consistent with the hypothesis that BOMPs originated from the amplification of ββ-hairpin modules, suggesting that the two Entamoeba-specific proteins are novel β-barrels, intriguingly localized partially to the ER membrane.
  DOI：https://doi.org/10.1111/febs.14870
  DOI ID：10.1111/febs.14870, ISSN：1742-464X, PubMed ID：31045303
• A Teflon-based system for applying multidirectional voltages to lipid bilayers as a novel platform for membrane proteins
  Maki Komiya; Kensaku Kanomata; Ryo Yokota; Yusuke Tsuneta; Madoka Sato; Daichi Yamaura; Daisuke Tadaki; Teng Ma; Hideaki Yamamoto; Yuzuru Tozawa; Albert Marti; Jordi Madrenas; Shigeru Kubota; Fumihiko Hirose; Michio Niwano; Ayumi Hirano-Iwata
  Jul. 2019
  Artificial bilayer lipid membranes (BLMs), along with patch-clamped
  
  membranes, are frequently used for functional analyses of membrane proteins. In
  
  both methods, the electric properties of membranes are characterized by only
  
  one parameter, namely, transmembrane potential. Here the construction of a
  
  novel BLM system was reported, in which membrane voltages can be controlled in
  
  a lateral direction in addition to conventional transmembrane direction. A
  
  microaperture was fabricated in a Teflon film and Ti electrodes were evaporated
  
  around the aperture. BLMs were reproducibly formed in the aperture without
  
  being affected by the presence of the electrodes. The application of a lateral
  
  voltage induced no significant changes in the electric properties of the BLMs,
  
  such as baseline current, transmembrane resistance, and transmembrane
  
  capacitance. In contrast, lateral voltages clearly affected the activities of
  
  biological ion channels, suggesting that the lateral voltage might be a useful
  
  parameter for analyzing channel activities. The present Teflon-based system in
  
  which multidirectional voltages can be applied to BLMs represent a promising
  
  platform for the analysis of underlying functional properties of membrane
  
  proteins.
  arXiv ID：arXiv:1907.05643
• A rice gene that confers broad-spectrum resistance to β-triketone herbicides.　　　　　　　　　　　　　　　
  Maeda H; Murata K; Sakuma N; Takei S; Yamazaki A; Karim MR; Kawata M; Hirose S; Kawagishi-Kobayashi M; Taniguchi Y; Suzuki S; Sekino K; Ohshima M; Kato H; Yoshida H; Tozawa Y
  Science (New York, N.Y.), Volume：365, Number：6451, First page：393, Last page：396, Jul. 2019, []
  The genetic variation of rice cultivars provides a resource for further varietal improvement through breeding. Some rice varieties are sensitive to benzobicyclon (BBC), a β-triketone herbicide that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Here we identify a rice gene, HIS1 (HPPD INHIBITOR SENSITIVE 1), that confers resistance to BBC and other β-triketone herbicides. We show that HIS1 encodes an Fe(II)/2-oxoglutarate-dependent oxygenase that detoxifies β-triketone herbicides by catalyzing their hydroxylation. Genealogy analysis revealed that BBC-sensitive rice variants inherited a dysfunctional his1 allele from an indica rice variety. Forced expression of HIS1 in Arabidopsis conferred resistance not only to BBC but also to four additional β-triketone herbicides. HIS1 may prove useful for breeding herbicide-resistant crops.
  DOI：https://doi.org/10.1126/science.aax0379
  DOI ID：10.1126/science.aax0379, ISSN：0036-8075, PubMed ID：31346065
• N-myristoylation and S-acylation are common modifications of Ca2+-regulated Arabidopsis kinases and are required for activation of the SLAC1 anion channel　　　　　　　　　　　　　　　
  Shunya Saito; Shin Hamamoto; Koko Moriya; Aiko Matsuura; Yoko Sato; Jun Muto; Hiroto Noguchi; Seiji Yamauchi; Yuzuru Tozawa; Minoru Ueda; Kenji Hashimoto; Philipp Köster; Qiuyan Dong; Katrin Held; Jörg Kudla; Toshihiko Utsumi; Nobuyuki Uozumi
  New Phytologist, Volume：218, Number：4, First page：1504, Last page：1521, Jun. 2018, []
  N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca2+-regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5–CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5–CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca2+ signaling processes, and demonstrate their requirement for guard cell anion channel regulation.
  Blackwell Publishing Ltd
  DOI：https://doi.org/10.1111/nph.15053
  DOI ID：10.1111/nph.15053, ISSN：1469-8137, PubMed ID：29498046, SCOPUS ID：85042622802
• The checkpoint kinase TOR (target of rapamycin) regulates expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) and modulates chloroplast ribosomal RNA synthesis in a unicellular red alga　　　　　　　　　　　　　　　
  Sousuke Imamura; Yuhta Nomura; Tokiaki Takemura; Imran Pancha; Keiko Taki; Kazuki Toguchi; Yuzuru Tozawa; Kan Tanaka
  Plant Journal, Volume：94, Number：2, First page：327, Last page：339, Apr. 2018, []
  Chloroplasts are plant organelles that carry out oxygenic photosynthesis. Chloroplast biogenesis depends upon chloroplast ribosomes and their translational activity. However, regulation of chloroplast ribosome biogenesis remains an important unanswered question. In this study, we found that inhibition of target of rapamycin (TOR), a general eukaryotic checkpoint kinase, results in a decline in chloroplast ribosomal RNA (rRNA) transcription in the unicellular red alga, Cyanidioschyzon merolae. Upon TOR inhibition, transcriptomics and other analyses revealed increased expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) gene (CmRSH4b), which encodes a homolog of the guanosine 3′-diphosphate 5′-diphosphate (ppGpp) synthetases that modulate rRNA synthesis in bacteria. Using an Escherichia coli mutant lacking ppGpp, CmRSH4b was demonstrated to have ppGpp synthetase activity. Expression analysis of a green fluorescent protein-fused protein indicated that CmRSH4b localizes to the chloroplast, and overexpression of the CmRSH4b gene resulted in a decrease of chloroplast rRNA synthesis concomitant with growth inhibition and reduction of chloroplast size. Biochemical analyses using C. merolae cell lysates or purified recombinant proteins revealed that ppGpp inhibits bacteria-type RNA polymerase-dependent chloroplast rRNA synthesis as well as a chloroplast guanylate kinase. These results suggest that CmRSH4b-dependent ppGpp synthesis in chloroplasts is an important regulator of chloroplast rRNA transcription. Nuclear and mitochondrial rRNA transcription were both reduced by TOR inhibition, suggesting that the biogeneses of the three independent ribosome systems are interconnected by TOR in plant cells.
  Blackwell Publishing Ltd
  DOI：https://doi.org/10.1111/tpj.13859
  DOI ID：10.1111/tpj.13859, ISSN：1365-313X, PubMed ID：29441718, SCOPUS ID：85044217434
• Quantification of Drug Side Effects on Cell-Free Synthesized Ion-Channels Reconstituted in Bilayer Lipid Membranes　　　　　　　　　　　　　　　
  Kato Miki; Hirano Ayumi; Inoue Haruka; Yamaura Daichi; Yokota Reo; Komiya Maki; Tadaki Daisuke; Yamamoto Hidaka; Tozawa Yuzuru; Niwano Michio
  Abstract of annual meeting of the Surface Science of Japan, Volume：2018, Number：0, First page：181, Last page：181, 2018
  We aim to develop a drug screening system based on a reconstitution system in which ion channels are embedded in an artificial lipid bilayer membrane (BLM). We have been developing stable BLMs based on silicon (Si) microfabrication. We previously reported on efficient incorporation of ion channels in BLMs. Here we applied this system to a cell-free synthesized hERG channel and tried to quantify drug side effects on this channel.
  The Japan Society of Vacuum and Surface Science
  DOI：https://doi.org/10.14886/sssj2008.2018.0_181
  DOI ID：10.14886/sssj2008.2018.0_181, CiNii Articles ID：130007519109
• Mechanically stable solvent-free lipid bilayers in nano- and micro-tapered apertures for reconstitution of cell-free synthesized hERG channels　　　　　　　　　　　　　　　
  Daisuke Tadaki; Daichi Yamaura; Shun Araki; Miyu Yoshida; Kohei Arata; Takeshi Ohori; Ken-ichi Ishibashi; Miki Kato; Teng Ma; Ryusuke Miyata; Yuzuru Tozawa; Hideaki Yamamoto; Michio Niwano; Ayumi Hirano-Iwata
  SCIENTIFIC REPORTS, Volume：7, Number：1, First page：17736, Dec. 2017, []
  The self-assembled bilayer lipid membrane (BLM) is the basic component of the cell membrane. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for the functional analysis of ion channels and screening the effects of drugs that act on them. However, because BLMs are unstable, this limits the experimental throughput of BLM reconstitution systems. Here we report on the formation of mechanically stable solvent-free BLMs in microfabricated apertures with defined nano- and micro-tapered edge structures. The role of such nano- and micro-tapered structures on the stability of the BLMs was also investigated. Finally, this BLM system was combined with a cell-free synthesized human ether-alpha-go-go-related gene channel, a cardiac potassium channel whose relation to arrhythmic side effects following drug treatment is well recognized. Such stable BLMs as these, when combined with a cell-free system, represent a potential platform for screening the effects of drugs that act on various ion-channel genotypes.
  NATURE PUBLISHING GROUP
  DOI：https://doi.org/10.1038/s41598-017-17905-x
  DOI ID：10.1038/s41598-017-17905-x, ISSN：2045-2322, PubMed ID：29255199, Web of Science ID：WOS:000418323900048
• Molecular mutagenesis of ppGpp: turning a RelA activator into an inhibitor　　　　　　　　　　　　　　　
  Jelena Beljantseva; Pavel Kudrin; Steffi Jimmy; Marcel Ehn; Radek Pohl; Vallo Varik; Yuzuru Tozawa; Victoria Shingler; Tanel Tenson; Dominik Rejman; Vasili Hauryliuk
  SCIENTIFIC REPORTS, Volume：7, First page：41839, Feb. 2017, []
  The alarmone nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p) ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p) ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work.
  NATURE PUBLISHING GROUP
  DOI：https://doi.org/10.1038/srep41839
  DOI ID：10.1038/srep41839, ISSN：2045-2322, PubMed ID：28157202, Web of Science ID：WOS:000393552300001
• Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors　　　　　　　　　　　　　　　
  Liis Andresen; Vallo Varik; Yuzuru Tozawa; Steffi Jimmy; Stina Lindberg; Tanel Tenson; Vasili Hauryliuk
  SCIENTIFIC REPORTS, Volume：6, First page：35824, Oct. 2016, []
  The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecular tools for disarming and pacifying bacterial pathogens. By taking advantage of the valine amino acid auxotrophy of the Bacillus subtilis stringent response-deficient strain, we have set up a High Throughput Screening assay for the identification of stringent response inhibitors. By screening 17,500 compounds, we have identified a novel class of antibacterials based on the 4-(6-(phenoxy) alkyl)-3,5-dimethyl-1H-pyrazole core. Detailed characterization of the hit compounds as well as two previously identified promising stringent response inhibitors-a ppGpp-mimic nucleotide Relacin and cationic peptide 1018 - showed that neither of the compounds is sufficiently specific, thus motivating future application of our screening assay to larger and more diverse molecular libraries.
  NATURE PUBLISHING GROUP
  DOI：https://doi.org/10.1038/srep35824
  DOI ID：10.1038/srep35824, ISSN：2045-2322, PubMed ID：27775002, Web of Science ID：WOS:000385923600001
• Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis　　　　　　　　　　　　　　　
  Satoshi Yamashita; Haruhiko Yamaguchi; Toshiyuki Waki; Yuichi Aoki; Makie Mizuno; Fumihiro Yanbe; Tomoki Ishii; Ayuta Funaki; Yuzuru Tozawa; Yukino Miyagi-Inoue; Kazuhisa Fushihara; Toru Nakayama; Seiji Takahashi
  ELIFE, Volume：5, Oct. 2016, []
  Natural rubber (NR) is stored in latex as rubber particles (RPs), rubber molecules surrounded by a lipid monolayer. Rubber transferase (RTase), the enzyme responsible for NR biosynthesis, is believed to be a member of the cis-prenyltransferase (cPT) family. However, none of the recombinant cPTs have shown RTase activity independently. We show that HRT1, a cPT from Heveabrasiliensis, exhibits distinct RTase activity in vitro only when it is introduced on detergent-washed HeveaRPs (WRPs) by a cell-free translation-coupled system. Using this system, a heterologous cPT from Lactucasativa also exhibited RTase activity, indicating proper introduction of cPT on RP is the key to reconstitute active RTase. RP proteomics and interaction network analyses revealed the formation of the protein complex consisting of HRT1, rubber elongation factor (REF) and HRT1-REF BRIDGING PROTEIN. The RTase activity enhancement observed for the complex assembled on WRPs indicates the HRT1-containing complex functions as the NR biosynthetic machinery.
  ELIFE SCIENCES PUBLICATIONS LTD
  DOI：https://doi.org/10.7554/eLife.19022
  DOI ID：10.7554/eLife.19022, ISSN：2050-084X, PubMed ID：27790974, Web of Science ID：WOS:000388660900001
• Atomic force microscope observation of voltage-dependence K+ channel KAT1 reconstructed to supported lipid bilayer　　　　　　　　　　　　　　　
  Suzuki Yuya; Nozawa Akira; Tozawa Yuzuru; Tero Ryugo
  Abstract of annual meeting of the Surface Science of Japan, Volume：36, First page：149, Last page：149, 2016
  支持脂質二重膜(SLB)は細胞膜モデル系として膜タンパク質の挙動や構造を直接観察するために有用である。植物由来の電位依存性K⁺チャネルKAT1の構造と特性を明らかにすることを目的とし、大豆由来の粗精製リン脂質であるasolectinのSLBを作製しKAT1を再構成した。AFM観察によりKAT1に由来する突起物を観察した。SLB作製条件からKAT1の分子配向について議論する。
  The Surface Science Society of Japan
  DOI：https://doi.org/10.14886/sssj2008.36.0_149
  DOI ID：10.14886/sssj2008.36.0_149, CiNii Articles ID：130005175554
• Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3 '-Phosphoadenosine 5 '-Phosphosulfate and ATP　　　　　　　　　　　　　　　
  Fumika Mi-ichi; Akira Nozawa; Hiroki Yoshida; Yuzuru Tozawa; Tomoyoshi Nozaki
  EUKARYOTIC CELL, Volume：14, Number：11, First page：1144, Last page：1150, Nov. 2015, []
  Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i. e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3'-phosphoadenosine 5'-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids.
  AMER SOC MICROBIOLOGY
  DOI：https://doi.org/10.1128/EC.00130-15
  DOI ID：10.1128/EC.00130-15, ISSN：1535-9778, eISSN：1535-9786, PubMed ID：26385892, Web of Science ID：WOS:000363814000011
• An enzymatic method to estimate the content of L-hydroxyproline　　　　　　　　　　　　　　　
  Seiya Watanabe; Yoshinobu Hiraoka; Shiori Endo; Yoshiaki Tanimoto; Yuzuru Tozawa; Yasuo Watanabe
  JOURNAL OF BIOTECHNOLOGY, Volume：199, First page：9, Last page：16, Apr. 2015, []
  Post-translational hydroxylation of the L-proline residue mainly occurs in collagen; therefore, the L-hydroxyprolines (L-Hyp) synthesized, including trans-4-hydroxy-L-proline (T4LHyp) and trans-3-hydroxy-L-proline (T3LHyp), are important markers for directly measuring the content of collagen in several biological samples. The most frequently used method to estimate the content of L-Hyp is high-performance liquid chromatography (HPLC), which is inconvenient. In the present study, we attempted to estimate the content of L-Hyp using coupling systems with metabolic enzymes of the T4LHyp (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and T3LHyp pathways (T3LHyp dehydratase (T3LHypD) and Delta(1)-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. We constructed a functional expression system of recombinant HypDH with a heterooligomeric structure in Escherichia coli cells. Enzymological characterization revealed that the beta-subunit acted as a catalytic subunit, and also that assembly with other subunit(s) improved the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of T4LHyp and T3LHyp were successfully estimated within the ranges of 0.004-1 mM and 0.05-1 mM, respectively, and were consistent with those determined by HPLC. This enzymatic method was used to measure the content of T4LHyp in the acid-hydrolysate of collagen, and blood plasma. (C) 2015 Elsevier B.V. All rights reserved.
  ELSEVIER SCIENCE BV
  DOI：https://doi.org/10.1016/j.jbiotec.2015.01.026
  DOI ID：10.1016/j.jbiotec.2015.01.026, ISSN：0168-1656, eISSN：1873-4863, PubMed ID：25678137, Web of Science ID：WOS:000351660600002
• A Novel Mitosomal beta-Barrel Outer Membrane Protein in Entamoeba　　　　　　　　　　　　　　　
  Herbert J. Santos; Kenichiro Imai; Takashi Makiuchi; Kentaro Tomii; Paul Horton; Akira Nozawa; Mohamed Ibrahim; Yuzuru Tozawa; Tomoyoshi Nozaki
  SCIENTIFIC REPORTS, Volume：5, First page：8545, Feb. 2015, []
  Entamoeba possesses a highly divergent mitochondrion-related organelle known as the mitosome. Here, we report the discovery of a novel protein in Entamoeba, which we name Mitosomal beta-barrel Outer Membrane Protein of 30 kDa (MBOMP30). Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a beta-barrel protein. Circular dichroism analysis showed MBOMP30 has a predominant beta-sheet structure. Localization to Entamoeba histolytica mitosomes was observed through Percoll-gradient fractionation and immunofluorescence assay. Mitosomal membrane integration was demonstrated by carbonate fractionation, proteinase K digestion, and immunoelectron microscopy. Interestingly, the deletion of the putative beta-signal, a sequence believed to guide beta-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a,240 kDa complex. MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes.
  NATURE PUBLISHING GROUP
  DOI：https://doi.org/10.1038/srep08545
  DOI ID：10.1038/srep08545, ISSN：2045-2322, PubMed ID：25711150, Web of Science ID：WOS:000349894400005
• Incorporation of adenine nucleotide transporter, Ant1p, into proteoliposomes facilitates ATP translocation and activation of encapsulated luciferase　　　　　　　　　　　　　　　
  Akira Nozawa; Yuzuru Tozawa
  JOURNAL OF BIOSCIENCE AND BIOENGINEERING, Volume：118, Number：2, First page：130, Last page：133, Aug. 2014, []
  We prepared functional luciferase and membrane-integrated form of adenine nucleotide transporter (Ant1p) with a wheat germ cell-free system. The reconstituted Ant1p showed transport activity of ATP/AMP exchange across the membrane. Here we demonstrate that activity of the luciferase entrapped in the Ant1p-proteoliposomes is controllable by the external supply of ATP. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.
  SOC BIOSCIENCE BIOENGINEERING JAPAN
  DOI：https://doi.org/10.1016/j.jbiosc.2014.02.001
  DOI ID：10.1016/j.jbiosc.2014.02.001, ISSN：1389-1723, eISSN：1347-4421, PubMed ID：24656877, Web of Science ID：WOS:000341559000003
• Biochemical analyses of ppGpp effect on adenylosuccinate synthetases, key enzymes in purine biosynthesis in rice　　　　　　　　　　　　　　　
  Yuhta Nomura; Akira Nozawa; Yuzuru Tozawa
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：78, Number：6, First page：1022, Last page：1025, Jun. 2014, []
  The ppGpp-signaling system functions in plant chloroplasts. In bacteria, a negative effect of ppGpp on adenylosuccinate synthetase (AdSS) has been suggested. Our biochemical analysis also revealed rice AdSS homologs are apparently sensitive to ppGpp. However, further investigation clarified that this phenomenon is cancelled by the high substrate affinity to the enzymes, leading to a limited effect of ppGpp on adenylosuccinate synthesis.
  TAYLOR & FRANCIS LTD
  DOI：https://doi.org/10.1080/09168451.2014.910103
  DOI ID：10.1080/09168451.2014.910103, ISSN：0916-8451, eISSN：1347-6947, PubMed ID：25036129, Web of Science ID：WOS:000340133800016
• Diversity in Guanosine 3 ',5 '-Bisdiphosphate (ppGpp) Sensitivity among Guanylate Kinases of Bacteria and Plants　　　　　　　　　　　　　　　
  Yuhta Nomura; Atsushi Izumi; Yoshinori Fukunaga; Kensuke Kusumi; Koh Iba; Seiya Watanabe; Yoichi Nakahira; Andreas P. M. Weber; Akira Nozawa; Yuzuru Tozawa
  JOURNAL OF BIOLOGICAL CHEMISTRY, Volume：289, Number：22, First page：15631, Last page：15641, May 2014, []
  The guanosine 3,5-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a K-i of 2.8 m relative to the substrate GMP, whereas the K-m of this enzyme for GMP was 73 m. The IC50 of ppGpp for GKpm was approximate to 10 m. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers' yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp.
  AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
  DOI：https://doi.org/10.1074/jbc.M113.534768
  DOI ID：10.1074/jbc.M113.534768, ISSN：0021-9258, eISSN：1083-351X, PubMed ID：24722991, Web of Science ID：WOS:000337465400045
• Modifications of wheat germ cell-free system for functional proteomics of plant membrane proteins　　　　　　　　　　　　　　　
  Akira Nozawa; Yuzuru Tozawa
  Methods in Molecular Biology, Volume：1072, First page：259, Last page：272, 2014, []
  Functional proteomics of plant membrane proteins is an important approach to understand the comprehensive architecture of each metabolic pathway in plants. One bottleneck in the characterization of membrane proteins is the difficulty in producing sufficient quantities of functional protein for analysis. Here, we des-cribe three methods for membrane protein production utilizing a wheat germ cell-free protein expression system. Owing to the open nature of cell-free synthesis reaction, protein synthesis can be modified with components necessary to produce functional protein. In this way we have developed modifications to a wheat germ cell-free system for the production of functional membrane proteins. Supplementation of liposomes or detergents allows the synthesis of functional integral membrane proteins. Furthermore, supplementation of myristic acid enables synthesis of N-myristylated peripheral membrane proteins. These modified cell-free synthesis methods facilitate the preparation and subsequent functional analyses of a wide variety of membrane proteins. © 2014 Springer Science+Business Media, LLC.
  Humana Press Inc.
  DOI：https://doi.org/10.1007/978-1-62703-631-3_19
  DOI ID：10.1007/978-1-62703-631-3_19, ISSN：1064-3745, PubMed ID：24136528, SCOPUS ID：84934434965
• Identification and characterization of trans-3-hydroxy-L-proline dehydratase and Delta(1)-pyrroline-2-carboxylate reductase involved in trans-3-hydroxy-L-proline metabolism of bacteria　　　　　　　　　　　　　　　
  Seiya Watanabe; Yoshiaki Tanimoto; Seiji Yamauchi; Yuzuru Tozawa; Shigeki Sawayama; Yasuo Watanabe
  FEBS OPEN BIO, Volume：4, First page：240, Last page：250, 2014, []
  trans-4-Hydroxy-L-proline (T4LHyp) and trans-3-hydroxy-L-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to alpha-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685-6695; J Biol Chem (2012) 287, 32674-32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P) H-dependent Delta(1)-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and v-proline and D-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp -> -Pyr2C -> L-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/mu-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, D-proline and D-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Delta(1)-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and L-proline metabolism. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
  ELSEVIER SCIENCE LONDON
  DOI：https://doi.org/10.1016/j.fob.2014.02.010
  DOI ID：10.1016/j.fob.2014.02.010, ISSN：2211-5463, PubMed ID：24649405, Web of Science ID：WOS:000346278200030
• Ornithine cyclodeaminase/mu-crystallin homolog from the hyperthermophilic archaeon Thermococcus litoralis functions as a novel Delta(1)-pyrroline-2-carboxylate reductase involved in putative trans-3-hydroxy-L-proline metabolism　　　　　　　　　　　　　　　
  Seiya Watanabe; Yuzuru Tozawa; Yasuo Watanabe
  FEBS OPEN BIO, Volume：4, First page：617, Last page：626, 2014, []
  L-Ornithine cyclodeaminase (OCD) is involved in L-proline biosynthesis and catalyzes the unique deaminating cyclization of L-ornithine to L-proline via a Delta(1)-pyrroline-2-carboxyrate (Pyr2C) intermediate. Although this pathway functions in only a few bacteria, many archaea possess OCD-like genes (proteins), among which only AF1665 protein (gene) from Archaeoglobus fulgidus has been characterized as an NAD(+)-dependent L-alanine dehydrogenase (AfAlaDH). However, the physiological role of OCD-like proteins from archaea has been unclear. Recently, we revealed that Pyr2C reductase, involved in trans-3-hydroxy-L-proline (T3LHyp) metabolism of bacteria, belongs to the OCD protein superfamily and catalyzes only the reduction of Pyr2C to L-proline (no OCD activity) [FEBS Open Bio (2014) 4, 240-250]. In this study, based on bioinformatics analysis, we assumed that the OCD-like gene from Thermococcus litoralis DSM 5473 is related to T3LHyp and/or proline metabolism (TlLhpI). Interestingly, TlLhpI showed three different enzymatic activities: AlaDH; N-methyl-L-alanine dehydrogenase; Pyr2C reductase. Kinetic analysis suggested strongly that Pyr2C is the preferred substrate. In spite of their similar activity, TlLhpI had a poor phylogenetic relationship to the bacterial and mammalian reductases for Pyr2C and formed a close but distinct subfamily to AfAlaDH, indicating convergent evolution. Introduction of several specific amino acid residues for OCD and/or AfAlaDH by site-directed mutagenesis had marked effects on both AlaDH and Pyr2C reductase activities. The OCC_00387 gene, clustered with the TlLhpI gene on the genome, encoded T3LHyp dehydratase, homologous to the bacterial and mammalian enzymes. To our knowledge, this is the first report of T3LHyp metabolism from archaea. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
  ELSEVIER SCIENCE LONDON
  DOI：https://doi.org/10.1016/j.fob.2014.07.005
  DOI ID：10.1016/j.fob.2014.07.005, ISSN：2211-5463, PubMed ID：25161870, Web of Science ID：WOS:000346278200073
• Theophylline-dependent riboswitch as a novel genetic tool for strict regulation of protein expression in cyanobacterium synechococcus elongatus PCC 7942　　　　　　　　　　　　　　　
  Yoichi Nakahira; Atsushi Ogawa; Hiroyuki Asano; Tokitaka Oyama; Yuzuru Tozawa
  Plant and Cell Physiology, Volume：54, Number：10, First page：1724, Last page：1735, Oct. 2013, []
  The cyanobacterium Synechococcus elongatus PCC 7942 is a major model species for studies of photosynthesis. It is are also a potential cell factory for the production of renewable biofuels and valuable chemicals. We employed engineered riboswitches to control translational initiation of target genes in this cyanobacterium. A firefly luciferase reporter assay revealed that three theophylline riboswitches performed as expected in the cyanobacterium. Riboswitch-E* exhibited very low leaky expression of luciferase and superior and dose-dependent on/off regulation of protein expression by theophylline. The maximum magnitude of the induction vs. basal level was ∼190-fold. Furthermore, the induction level was responsive to a wide range of theophylline concentrations in the medium, from 0 to 2 mM, facilitating the fine-tuning of luciferase expression. We adapted this riboswitch to another gene regulation system, in which expression of the circadian clock kaiC gene product is controlled by the theophylline concentration in the culture medium. The results demonstrated that the adequately adjusted expression level of KaiC restored complete circadian rhythm in the kaiC-deficient arrhythmic mutant. This theophylline-dependent riboswitch system has potential for various applications as a useful genetic tool in cyanobacteria. © 2013 The Author.
  DOI：https://doi.org/10.1093/pcp/pct115
  DOI ID：10.1093/pcp/pct115, ISSN：0032-0781, PubMed ID：23969558, SCOPUS ID：84885929738
• Overproduction of Hyperthermostable beta-1,4-Endoglucanase from the Archaeon Pyrococcus horikoshii by Tobacco Chloroplast Engineering　　　　　　　　　　　　　　　
  Yoichi Nakahira; Kazuhiko Ishikawa; Kunisuke Tanaka; Yuzuru Tozawa; Takashi Shiina
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：77, Number：10, First page：2140, Last page：2143, Oct. 2013, []
  One of the most cost-effective methods of producing industrial enzymes is by the use of transgenic plants. We demonstrated successful high-level expression of a hyperthermostable archaeal beta-1,4-endoglucanase in mature tobacco leaves by transformation of chloroplasts by homologous recombination. The active recombinant enzyme was readily recovered not only from fresh but also from dried leaves.
  TAYLOR & FRANCIS LTD
  DOI：https://doi.org/10.1271/bbb.130413
  DOI ID：10.1271/bbb.130413, ISSN：0916-8451, eISSN：1347-6947, PubMed ID：24096651, Web of Science ID：WOS:000326486500028
• Characterization of the Plastidic Phosphate Translocators in the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum　　　　　　　　　　　　　　　
  Shin Kore-eda; Akira Nozawa; Yusuke Okada; Kazuki Takashi; Muhammad Abul Kalam Azad; Jun-ichi Ohnishi; Yoshitaka Nishiyama; Yuzuru Tozawa
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：77, Number：7, First page：1511, Last page：1516, Jul. 2013, []
  In plant Mesembryanthemum crystallinum, which has the inducible crassulacean acid metabolism (CAM), isoforms of plastidic phosphate translocators (pPTs) are categorized into three subfamilies: the triose phosphate/phosphate translocator (McTPT1), the phosphoenolpyruvate/phosphate translocator (McPPT1), and the glucose 6-phosphate/phosphate translocator (McGPT1 and McGPT2). In order to elucidate the physiological roles of these pPTs in M. crystallinum, we determined the substrate specificity of each pPT isoform. The substrate specificities of McTPT1, McPPT1, and McGPT1 showed overall similarities to those of orthologs that have been characterized. In contrast, for glucose 6-phosphate, McGPT2 showed higher selectivity than McGPT1 and other GPT orthologs. Because the expression of McGTP2 is specific to CAM while that of McGTP1 is constitutively expressed in both the C-3- and the CAM-state in M. crystallinum, we propose that McGPT2 functions as a CAM system-specific GPT in this plant.
  TAYLOR & FRANCIS LTD
  DOI：https://doi.org/10.1271/bbb.130174
  DOI ID：10.1271/bbb.130174, ISSN：0916-8451, eISSN：1347-6947, PubMed ID：23832369, Web of Science ID：WOS:000323533800026
• Cell-free expression - making a mark　　　　　　　　　　　　　　　
  Frank Bernhard; Yuzuru Tozawa
  CURRENT OPINION IN STRUCTURAL BIOLOGY, Volume：23, Number：3, First page：374, Last page：380, Jun. 2013, []
  Cell-free protein production opens new perspectives for the direct manipulation of expression compartments in combination with reduced complexity of physiological requirements. The technology is therefore in particular suitable for the general synthesis of difficult proteins including toxins and membrane proteins as well as for the analysis of their functional folding in artificial environments. A further key application of cell-free expression is the fast and economic labeling of proteins for structural and functional applications. Two extract sources, wheat embryos and Escherichia coli cells, are currently employed for the preparative scale cell-free production of proteins. Recent achievements in structural characterization include cell-free synthesized membrane proteins and even larger protein assemblies may become feasible.
  CURRENT BIOLOGY LTD
  DOI：https://doi.org/10.1016/j.sbi.2013.03.012
  DOI ID：10.1016/j.sbi.2013.03.012, ISSN：0959-440X, PubMed ID：23628286, Web of Science ID：WOS:000321408300008
• Effect of counter ions on the transport current across membranes containing KAT1 potassium channel　　　　　　　　　　　　　　　
  Shintaro Kubota; Osamu Shirai; Takao Hibi; Yuzuru Tozawa; Kenji Kano
  Analytical Sciences, Volume：29, Number：1, First page：161, Last page：164, 2013, []
  Ion transports from one aqueous phase (W1) to another (W2) across bilayer lipid membranes (BLM) containing KAT1 potassium channel were investigated by recording current fluctuations. KAT1 channel is a voltage-gated K+channel from Arabidopsis thaliana and has been suggested to have a key role in stomatal opening by osmoregulation in guard cells. Although KAT1 channel is a K+-specific transporter, the species of the counter anions significantly affected the level of the single channel current, suggesting that KAT1 also transported these anions. When various potassium salts were used as electrolytes, the single channel conductance decreased with an increase of the ionic radius of the coexisting anions except for F-. This indicates the existence of selective permeation for anions, but the anion-selectivity is less noticeable than the cation-selectivity.© The Japan Society for Analytical Chemistry.
  DOI：https://doi.org/10.2116/analsci.29.161
  DOI ID：10.2116/analsci.29.161, ISSN：0910-6340, PubMed ID：23303104, SCOPUS ID：84873869267
• Identification and Characterization of D-Hydroxyproline Dehydrogenase and Delta(1)-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel L-Hydroxyproline Metabolism of Bacteria METABOLIC CONVERGENT EVOLUTION　　　　　　　　　　　　　　　
  Seiya Watanabe; Daichi Morimoto; Fumiyasu Fukumori; Hiroto Shinomiya; Hisashi Nishiwaki; Miyuki Kawano-Kawada; Yuuki Sasai; Yuzuru Tozawa; Yasuo Watanabe
  JOURNAL OF BIOLOGICAL CHEMISTRY, Volume：287, Number：39, First page：32674, Last page：32688, Sep. 2012, []
  L-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert L-hydroxyproline to alpha-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, D-hydroxyproline dehydrogenase and Delta(1)-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. D-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (D-hydroxyproline) and some artificial electron acceptor( s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (alpha(4)beta(4)gamma(4)), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in alpha beta gamma of the heterotrimeric unit. These results suggested that the L-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on L-hydroxyproline (as well as D-hydroxyproline) but not L-and D-proline, indicating that this pathway is related only to L-hydroxyproline degradation, which is not linked to proline metabolism.
  AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
  DOI：https://doi.org/10.1074/jbc.M112.374272
  DOI ID：10.1074/jbc.M112.374272, ISSN：0021-9258, PubMed ID：22833679, Web of Science ID：WOS:000309168300035
• Exploration of a Possible Partnership among Orphan Two-Component System Proteins in Cyanobacterium Synechococcus elongatus PCC 7942　　　　　　　　　　　　　　　
  Hiroaki Kato; Satoru Watanabe; Kaori Nimura-Matsune; Taku Chibazakura; Yuzuru Tozawa; Hirofumi Yoshikawa
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：76, Number：8, First page：1484, Last page：1491, Aug. 2012, []
  To understand the induction of the adaptive response under various stress conditions, it is important to determine the partnership between histidine kinase and response regulators in the bacterial two-component system (TCS). The genes encoding TCS partners are usually comprised of an operon in the genome, but many of them are orphans in the cyanobacterial genome. There is little information on their partnerships in Synechococcus elongatus PCC 7942. Our comprehensive analysis of protein-protein interactions among all 37 full-length proteins and the truncated domains of 24 orphans revealed a number of specific interactions. They involved evolutionarily well-conserved orphan proteins among cyanobacterial species such as Synpcc7942_0453/Ycf29, NblS/RpaB, NblS/SrrA, SasA/RpaA, and SasA/Synpcc7942_2466. Our investigation of the transphosphorylation of interaction partners indicates that orphan TCSs comprise a complex signaling network.
  TAYLOR & FRANCIS LTD
  DOI：https://doi.org/10.1271/bbb.120172
  DOI ID：10.1271/bbb.120172, ISSN：0916-8451, eISSN：1347-6947, PubMed ID：22878191, Web of Science ID：WOS:000308835000010
• Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp(0) mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome　　　　　　　　　　　　　　　
  Kazumi Tagami; Hideaki Nanamiya; Yuka Kazo; Marie Maehashi; Shota Suzuki; Eri Namba; Masahiro Hoshiya; Ryo Hanai; Yuzuru Tozawa; Takuya Morimoto; Naotake Ogasawara; Yasushi Kageyama; Katsutoshi Ara; Katsuya Ozaki; Masaki Yoshida; Haruko Kuroiwa; Tsuneyoshi Kuroiwa; Yoshiaki Ohashi; Fujio Kawamura
  MICROBIOLOGYOPEN, Volume：1, Number：2, First page：115, Last page：134, Jun. 2012, []
  To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the Delta relA Delta yjbM Delta ywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG-induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.
  WILEY-BLACKWELL
  DOI：https://doi.org/10.1002/mbo3.16
  DOI ID：10.1002/mbo3.16, ISSN：2045-8827, PubMed ID：22950019, Web of Science ID：WOS:000209077500004
• Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase　　　　　　　　　　　　　　　
  Yusuke Yagi; Yoko Ishizaki; Yoichi Nakahira; Yuzuru Tozawa; Takashi Shiina
  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Volume：109, Number：19, First page：7541, Last page：7546, May 2012, []
  Plastid transcription is mediated by two distinct types of RNA polymerases (RNAPs), bacterial-type RNAP (PEP) and phage-type RNAP (NEP). Recent genomic and proteomic studies revealed that higher plants have lost most prokaryotic transcription regulators and have acquired eukaryotic-type proteins during plant evolution. However, in vivo dynamics of chloroplast RNA polymerases and eukaryotic-type plastid nucleoid proteins have not been directly characterized experimentally. Here, we examine the association of the a-subunit of PEP and eukaryotic-type protein, plastid transcriptionally active chromosome 3 (pTAC3) with transcribed regions in vivo by using chloroplast chromatin immunoprecipitation (cpChIP) assays. PEP a-subunit preferentially associates with PEP promoters of photosynthesis and rRNA genes, but not with NEP promoter regions, suggesting selective and accurate recognition of PEP promoters by PEP. The cpChIP assays further demonstrate that the peak of PEP association occurs at the promoterproximal region and declines gradually along the transcribed region. pTAC3 is a putative DNA-binding protein that is localized to chloroplast nucleoids and is essential for PEP-dependent transcription. Density gradient and immunoprecipitation analyses of PEP revealed that pTAC3 is associated with the PEP complex. Interestingly, pTAC3 associates with the PEP complex not only during transcription initiation, but also during elongation and termination. These results suggest that pTAC3 is an essential component of the chloroplast PEP complex. In addition, we demonstrate that light-dependent chloroplast transcription is mediated by light-induced association of the PEP-pTAC3 complex with promoters. This study illustrates unique dynamics of PEP and its associated protein pTAC3 during light-dependent transcription in chloroplasts.
  NATL ACAD SCIENCES
  DOI：https://doi.org/10.1073/pnas.1119403109
  DOI ID：10.1073/pnas.1119403109, ISSN：0027-8424, PubMed ID：22529394, Web of Science ID：WOS:000304090600082
• ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro　　　　　　　　　　　　　　　
  Yuhta Nomura; Taito Takabayashi; Hiroshi Kuroda; Yasushi Yukawa; Kwanchanok Sattasuk; Mitsuru Akita; Akira Nozawa; Yuzuru Tozawa
  PLANT MOLECULAR BIOLOGY, Volume：78, Number：1-2, First page：185, Last page：196, Jan. 2012, []
  Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(beta,gamma-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.
  SPRINGER
  DOI：https://doi.org/10.1007/s11103-011-9858-x
  DOI ID：10.1007/s11103-011-9858-x, ISSN：0167-4412, PubMed ID：22108865, Web of Science ID：WOS:000297815400014
• In Vitro Protein Import of a Putative Amino Acid Transporter from Arabidopsis thaliana into Chloroplasts and Its Suborganellar Localization　　　　　　　　　　　　　　　
  Kwanchanok Sattasuk; Akira Nozawa; Yuzuru Tozawa; Yoshimi Kakinuma; Mitsuru Akita
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：75, Number：11, First page：2200, Last page：2206, Nov. 2011, []
  We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin etal., Proc. Natl. Acad. Sci. USA, 97,559-564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.
  TAYLOR & FRANCIS LTD
  DOI：https://doi.org/10.1271/bbb.110489
  DOI ID：10.1271/bbb.110489, ISSN：0916-8451, eISSN：1347-6947, PubMed ID：22056429, Web of Science ID：WOS:000297610500020
• Cell-free synthesis, reconstitution, and characterization of a mitochondrial dicarboxylate-tricarboxylate carrier of Plasmodium falciparum　　　　　　　　　　　　　　　
  Akira Nozawa; Ryoji Fujimoto; Hiroki Matsuoka; Takafumi Tsuboi; Yuzuru Tozawa
  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume：414, Number：3, First page：612, Last page：617, Oct. 2011, []
  The malaria parasite, Plasmodium falciparum, was recently shown to operate a branched pathway of tricarboxylic acid (TCA) metabolism. To identify and characterize membrane transporters required for such TCA metabolism in the parasite, we isolated a cDNA for a dicarboxylate-tricarboxylate carrier homolog (PfDTC), synthesized the encoded protein with the use of a cell-free translation system, and determined the substrate specificity of its transport activity with a proteoliposome reconstitution system. PfDTC was found to mediate efficient oxoglutarate-malate, oxoglutarate-oxaloacetate, or oxoglutarate-oxoglutarate exchange across the liposome membrane. Our results suggest that PfDTC may mediate the oxoglutarate-malate exchange across the inner mitochondrial membrane required for the branched pathway of TCA metabolism in the malaria parasite. (C) 2011 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE
  DOI：https://doi.org/10.1016/j.bbrc.2011.09.130
  DOI ID：10.1016/j.bbrc.2011.09.130, ISSN：0006-291X, PubMed ID：21986531, Web of Science ID：WOS:000298519500029
• Signalling by the global regulatory molecule ppGpp in bacteria and chloroplasts of land plants　　　　　　　　　　　　　　　
  Y. Tozawa; Y. Nomura
  Plant Biology, Volume：13, Number：5, First page：699, Last page：709, Sep. 2011, []
  The hyperphosphorylated guanine ribonucleotide ppGpp mediates the stringent response in bacteria. Biochemical and genetic studies of this response in Escherichia coli have shown that the biosynthesis of ppGpp is catalysed by two homologous enzymes, RelA and SpoT. RelA is activated in response to amino acid starvation, and SpoT responds to abiotic physical stress beside nutritional stress. All free-living bacteria, including Gram-positive firmicutes, contain RelA-SpoT homologues (RSH). Further, novel ppGpp biosynthetic enzymes, designated small alarmone synthetases (SASs), were recently identified in a subset of bacteria, including the Gram-positive organism Bacillus subtilis, and were shown to consist only of a ppGpp synthetase domain. Studies suggest that these SAS proteins contribute to ppGpp signalling in response to stressful conditions in a manner distinct from that of RelA-SpoT enzymes. SAS proteins currently appear to always occur in addition to RSH enzymes in various combinations but never alone. RSHs have also been identified in chloroplasts, organelles of photosynthetic eukaryotes that originated from endosymbiotic photosynthetic bacteria. These chloroplast RSHs are exclusively encoded in nuclear DNA and targeted into chloroplasts. The findings suggest that ppGpp may regulate chloroplast functions similar to those regulated in bacteria, including transcription and translation. In addition, a novel ppGpp synthetase that is regulated by Ca 2+ as a result of the presence of two EF-hand motifs at its COOH terminus was recently identified in chloroplasts of land plants. This finding indicates the existence of a direct connection between eukaryotic Ca 2+ signalling and prokaryotic ppGpp signalling in chloroplasts. The new observations with regard to ppGpp signalling in land plants suggest that such signalling contributes to the regulation of a wider range of cellular functions than previously anticipated. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.
  DOI：https://doi.org/10.1111/j.1438-8677.2011.00484.x
  DOI ID：10.1111/j.1438-8677.2011.00484.x, ISSN：1435-8603, PubMed ID：21815973, SCOPUS ID：79961029191
• Differences of two polychaete species reflected in enzyme activities　　　　　　　　　　　　　　　
  Katsutoshi Ito; Mana Nozaki; Takashi Ohta; Chiemi Miura; Yuzuru Tozawa; Takeshi Miura
  MARINE BIOLOGY, Volume：158, Number：6, First page：1211, Last page：1221, Jun. 2011, []
  Abstract Polychaetes constitute most of the benthic macroinvertebrates in estuarine and coastal environments. We investigated the utilization of organic matter in two polychaete species, Capitella sp. I and Perinereis nuntia brevicirris, living in different coastal habitats. The protease activity of Capitella sp. I (89.7 mu g mg(-1)) was about 10 times that of P. nuntia brevicirris (8.0 mu g mg(-1)). High cellulase (endo-beta-1,4-glucanase) activity was detected in P. nuntia brevicirris (3.2 mu g mg(-1)), whereas scarcely any was detected in Capitella sp. I. We isolated cDNA clones of protease mRNA from Capitella sp. I and of cellulase mRNA from P. nuntia brevicirris. The high protease activity of Capitella sp. I enabled it to survive in the sediment under a fish farm, where it degrades organic matter. In contrast, the high cellulase activity of the estuarydwelling P. nuntia brevicirris allowed it to degrade organic matter originating from terrestrial areas.
  SPRINGER HEIDELBERG
  DOI：https://doi.org/10.1007/s00227-011-1641-7
  DOI ID：10.1007/s00227-011-1641-7, ISSN：0025-3162, eISSN：1432-1793, Web of Science ID：WOS:000290806500003
• Co-translational function of Cosmc, core 1 synthase specific molecular chaperone, revealed by a cell-free translation system　　　　　　　　　　　　　　　
  Yoshiki Narimatsu; Tomomi Kubota; Sanae Furukawa; Mie Shimojima; Hiroko Iwasaki; Yuzuru Tozawa; Kouichi Tachibana; Hisashi Narimatsu
  FEBS LETTERS, Volume：585, Number：9, First page：1276, Last page：1280, May 2011, []
  The core 1 structure of the mucin type O-glycan is synthesized by core 1 beta 1,3-galactosyltransferase (C1GalT). Core 1 synthase specific molecular chaperone (Cosmc), a molecular chaperone specific for C1GalT, is essential for the expression of functional C1GalT in mammalian cells. In this study, we have established a procedure for detecting the chaperone activity of Cosmc by using a wheat germ cell-free translation system. Active C1GalT was expressed following simultaneous translation with Cosmc or translation in the presence of recombinant Cosmc protein. Moreover, we show that recombinant Cosmc must be present during the translation of C1GalT, as it is ineffective when added after translation. These results indicate that Cosmc mediates the co-translational activation of C1GalT and that it may prevent the unfavorable aggregation of C1GalT.
  Structured summary of protein interactions:
  C1GalT and Cosmc bind by enzymatic study (View Interaction 1, 2, 3, 4) (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  ELSEVIER SCIENCE BV
  DOI：https://doi.org/10.1016/j.febslet.2011.04.010
  DOI ID：10.1016/j.febslet.2011.04.010, ISSN：0014-5793, CiNii Articles ID：80021796477, PubMed ID：21496458, Web of Science ID：WOS:000290248300004
• Anti-viral effects of interferon administration on sevenband grouper, Epinephelus septemfasciatus　　　　　　　　　　　　　　　
  Takashi Ohta; Yusuke Ueda; Katsutoshi Ito; Chiemi Miura; Hirofumi Yamashita; Takeshi Miura; Yuzuru Tozawa
  FISH & SHELLFISH IMMUNOLOGY, Volume：30, Number：4-5, First page：1064, Last page：1071, Apr. 2011
  Interferon (IFN) plays crucial roles in innate immune responses against viral infections. In the present study, we report cloning and characterization of the IFN gene from the sevenband grouper (Epinephelus septemfasciatus), and the anti-viral effects of its recombinant IFN protein in vivo. The isolated cDNA from sevenband grouper IFN encoded a protein consisting of 178 amino acids, and its first 22 amino acids represented a putative signal peptide. We named the identified sevenband grouper IFN gene as SgIFNa1 based on the result from phylogenetic analysis that categorized the deduced protein sequence into fish IFNa family. The expression of SgIFNa1 mRNA in the head kidney cells was induced by synthetic Poly(1:C), which is known as an inducer of IFN. It has also been confirmed that injection of recombinant SgIFNa1 protein (rSgIFNa1) upregulates expression of the Mx gene, which is known as an IFN-responsive gene, in head kidney cells. Moreover, we observed that preliminarily injection of rSgIFNa1 provided significant protection against a lethal challenge of nervous necrosis virus (NNV), which is a serious disease of sevenband grouper. These results demonstrate that SgIFNa1 has anti-viral activity and the administration of rSgIFNa1 to sevenband grouper is effective in preventing severe symptom development after NNV infection. (C) 2011 Elsevier Ltd. All rights reserved.
  ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
  DOI：https://doi.org/10.1016/j.fsi.2011.02.003
  DOI ID：10.1016/j.fsi.2011.02.003, ISSN：1050-4648, PubMed ID：21316457, Web of Science ID：WOS:000289969000009
• Tolerance of Spermatogonia to Oxidative Stress Is Due to High Levels of Zn and Cu/Zn Superoxide Dismutase　　　　　　　　　　　　　　　
  Fritzie T. Celino; Sonoko Yamaguchi; Chiemi Miura; Takashi Ohta; Yuzuru Tozawa; Toshiharu Iwai; Takeshi Miura
  PLOS ONE, Volume：6, Number：2, First page：e16938, Feb. 2011, []
  Background: Spermatogonia are highly tolerant to reactive oxygen species (ROS) attack while advanced-stage germ cells such as spermatozoa are much more susceptible, but the precise reason for this variation in ROS tolerance remains unknown.
  Methodology/Principal Findings: Using the Japanese eel testicular culture system that enables a complete spermatogenesis in vitro, we report that advanced-stage germ cells undergo intense apoptosis and exhibit strong signal for 8-hydroxy-29-deoxyguanosine, an oxidative DNA damage marker, upon exposure to hypoxanthine-generated ROS while spermatogonia remain unaltered. Activity assay of antioxidant enzyme, superoxide dismutase (SOD) and Western blot analysis using an anti-Copper/Zinc (Cu/Zn) SOD antibody showed a high SOD activity and Cu/Zn SOD protein concentration during early spermatogenesis. Immunohistochemistry showed a strong expression for Cu/Zn SOD in spermatogonia but weak expression in advanced-stage germ cells. Zn deficiency reduced activity of the recombinant eel Cu/Zn SOD protein. Cu/Zn SOD siRNA decreased Cu/Zn SOD expression in spermatogonia and led to increased oxidative damage.
  Conclusions/Significance: These data indicate that the presence of high levels of Cu/Zn SOD and Zn render spermatogonia resistant to ROS, and consequently protected from oxidative stress. These findings provide the biochemical basis for the high tolerance of spermatogonia to oxidative stress.
  PUBLIC LIBRARY SCIENCE
  DOI：https://doi.org/10.1371/journal.pone.0016938
  DOI ID：10.1371/journal.pone.0016938, ISSN：1932-6203, Web of Science ID：WOS:000287482800022
• Ribosome rescue by Escherichia coli ArfA (YhdL) in the absence of trans-translation system　　　　　　　　　　　　　　　
  Yuhei Chadani; Katsuhiko Ono; Shin-ichiro Ozawa; Yuichiro Takahashi; Kazuyuki Takai; Hideaki Nanamiya; Yuzuru Tozawa; Kazuhiro Kutsukake; Tatsuhiko Abo
  MOLECULAR MICROBIOLOGY, Volume：78, Number：4, First page：796, Last page：808, Nov. 2010, []
  P>Although SsrA(tmRNA)-mediated trans-translation is thought to maintain the translation capacity of bacterial cells by rescuing ribosomes stalled on messenger RNA lacking an in-frame stop codon, single disruption of ssrA does not crucially hamper growth of Escherichia coli. Here, we identified YhdL (renamed ArfA for alternative ribosome-rescue factor) as a factor essential for the viability of E. coli in the absence of SsrA. The ssrA-arfA synthetic lethality was alleviated by SsrADD, an SsrA variant that adds a proteolysis-refractory tag through trans-translation, indicating that ArfA-deficient cells require continued translation, rather than subsequent proteolysis of the truncated polypeptide. In accordance with this notion, depletion of SsrA in the Delta arfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codon-independent mimic of aminoacyl-tRNA, rescued the bacterial growth under such conditions. That ArfA takes over the role of SsrA was suggested by the observation that its overexpression enabled detection of the polypeptide encoded by a model non-stop mRNA, which was otherwise SsrA-tagged and degraded. In vitro, purified ArfA acted on a ribosome-nascent chain complex to resolve the peptidyl-tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3' end of a non-stop mRNA without involving trans-translation.
  WILEY-BLACKWELL
  DOI：https://doi.org/10.1111/j.1365-2958.2010.07375.x
  DOI ID：10.1111/j.1365-2958.2010.07375.x, ISSN：0950-382X, CiNii Articles ID：80021439724, Web of Science ID：WOS:000284072200003
• Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents　　　　　　　　　　　　　　　
  Takahisa Genji; Akira Nozawa; Yuzuru Tozawa
  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume：400, Number：4, First page：638, Last page：642, Oct. 2010, []
  Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield. (c) 2010 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE
  DOI：https://doi.org/10.1016/j.bbrc.2010.08.119
  DOI ID：10.1016/j.bbrc.2010.08.119, ISSN：0006-291X, CiNii Articles ID：80021313088, PubMed ID：20807510, Web of Science ID：WOS:000282850500032
• Control of translational initiation in the wheat-embryo cell-free protein expression system for producing homogenous products　　　　　　　　　　　　　　　
  Takashi Ohta; Hiroki Matsuoka; Yuhta Nomura; Yuzuru Tozawa
  PROTEIN EXPRESSION AND PURIFICATION, Volume：73, Number：1, First page：15, Last page：22, Sep. 2010, []
  Wheat-embryo cell-free protein expression system allows efficient production of a wide variety of proteins. Homogeneity of the end products is an important characteristic of an advanced cell-free system that will be used in a field of protein science such as structural biology. A translation enhancer such as the omega sequence derived from tobacco mosaic virus, that allows cap-independent translation of the mRNA in the cell-free system, is required for low-cost preparation of template mRNAs in the cell-free translation system. However, the use of translational enhancers often leads to unexpected byproducts. Several AUU codons in the omega sequence can potentially function as translation initiators. We confirmed that the in-frame AUU in the omega sequence functions as a non-canonical start codon and results in the extension of the N-terminus of the target protein in some cases. Investigation of the selectivity of non-canonical initiation codon under the control of omega sequence in the wheat-embryo cell-free system revealed that seven non-AUG codons, CUG, AUA, AUU, GUG, ACG, AUC, and UUG, are recognized as translation initiators. We found that the introduction of an in-frame stop codon just upstream of the target open reading frame is an efficient way to avoid unexpected byproducts. This minor but effective modification facilitates production of homogeneous proteins within the wheat-embryo cell-free protein expression system at the preparative scale. (C) 2010 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE
  DOI：https://doi.org/10.1016/j.pep.2010.03.011
  DOI ID：10.1016/j.pep.2010.03.011, ISSN：1046-5928, CiNii Articles ID：10030014301, PubMed ID：20304073, Web of Science ID：WOS:000279312700003
• The consensus motif for N-myristoylation of plant proteins in a wheat germ cell-free translation system　　　　　　　　　　　　　　　
  Seiji Yamauchi; Naoki Fusada; Hidenori Hayashi; Toshihiko Utsumi; Nobuyuki Uozumi; Yaeta Endo; Yuzuru Tozawa
  FEBS JOURNAL, Volume：277, Number：17, First page：3596, Last page：3607, Sep. 2010, []
  Protein N-myristoylation plays key roles in various cellular functions in eukaryotic organisms. To clarify the relationship between the efficiency of protein N-myristoylation and the amino acid sequence of the substrate in plants, we have applied a wheat germ cell-free translation system with high protein productivity to examine the N-myristoylation of various wild-type and mutant forms of Arabidopsis thaliana proteins. Evaluation of the relationship between removal of the initiating Met and subsequent N-myristoylation revealed that constructs containing Pro at position 3 do not undergo N-myristoylation, primarily because of an inhibitory effect of this amino acid on elimination of the initiating Met by methionyl aminopeptidase. Our analysis of the consensus sequence for N-myristoylation in plants focused on the variability of amino acids at positions 3, 6 and 7 of the motif. We found that not only Ser at position 6 but also Lys at position 7 affects the selectivity for the amino acid at position 3. The results of our analyses allowed us to identify several A. thaliana proteins as substrates for N-myristoylation that had previously been predicted not to be candidates for such modification with a prediction program. We have thus shown that a wheat germ cell-free system is a useful tool for plant N-myristoylome analysis. This in vitro approach will facilitate comprehensive determination of N-myristoylated proteins in plants.
  WILEY-BLACKWELL
  DOI：https://doi.org/10.1111/j.1742-4658.2010.07768.x
  DOI ID：10.1111/j.1742-4658.2010.07768.x, ISSN：1742-464X, CiNii Articles ID：80021279820, PubMed ID：20716180, Web of Science ID：WOS:000280996300014
• Cell-free protein N-myristoylation system utilizing wheat-germ extracts facilitates update of consensus motif for the modification of plant proteins　　　　　　　　　　　　　　　
  Yamauchi, S; Fusada, N; Hayashi, H; Utsumi, T; Uozumi, N; Tozawa, Y
  FEBS. J., Jul. 2010, []
• Expression of bacterial tyrosine ammonia-lyase creates a novel p-coumaric acid pathway in the biosynthesis of phenylpropanoids in Arabidopsis　　　　　　　　　　　　　　　
  Yasutaka Nishiyama; Choong-Soo Yun; Fumio Matsuda; Tadamasa Sasaki; Kazuki Saito; Yuzuru Tozawa
  PLANTA, Volume：232, Number：1, First page：209, Last page：218, Jun. 2010, []
  Some flavonoids are considered as beneficial compounds because they exhibit anticancer or antioxidant activity. In higher plants, flavonoids are secondary metabolites that are derived from phenylpropanoid biosynthetic pathway. A large number of phenylpropanoids are generated from p-coumaric acid, which is a derivative of the primary metabolite, phenylalanine. The first two steps in the phenylpropanoid biosynthetic pathway are catalyzed by phenylalanine ammonia-lyase and cinnamate 4-hydroxylase, and the coupling of these two enzymes forms a rate-limiting step in the pathway. For the generation of p-coumaric acid, the conversion from phenylalanine to p-coumaric acid that is catalyzed by two enzymes can be theoretically performed by a single enzyme, tyrosine ammonia-lyase (TAL) that catalyzes the conversion of tyrosine to p-coumaric acid in certain bacteria. To modify the p-coumaric acid pathway in plants, we isolated a gene encoding TAL from a photosynthetic bacterium, Rhodobacter sphaeroides, and introduced the gene (RsTAL) in Arabidopsis thaliana. Analysis of metabolites revealed that the ectopic over-expression of RsTAL leads to higher accumulation of anthocyanins in transgenic 5-day-old seedlings. On the other hand, 21-day-old seedlings of plants expressing RsTAL showed accumulation of higher amount of quercetin glycosides, sinapoyl and p-coumaroyl derivatives than control. These results indicate that ectopic expression of the RsTAL gene in Arabidopsis enhanced the metabolic flux into the phenylpropanoid pathway and resulted in increased accumulation of flavonoids and phenylpropanoids.
  SPRINGER
  DOI：https://doi.org/10.1007/s00425-010-1166-1
  DOI ID：10.1007/s00425-010-1166-1, ISSN：0032-0935, CiNii Articles ID：80021069995, Web of Science ID：WOS:000277792700018
• Transcription Activity of Individual rrn Operons in Bacillus subtilis Mutants Deficient in (p)ppGpp Synthetase Genes, relA, yjbM, and ywaC　　　　　　　　　　　　　　　
  Yousuke Natori; Kazumi Tagami; Kana Murakami; Sawako Yoshida; Osamu Tanigawa; Yoonsuh Moh; Kenta Masuda; Tetsuya Wada; Shota Suzuki; Hideaki Nanamiya; Yuzuru Tozawa; Fujio Kawamura
  JOURNAL OF BACTERIOLOGY, Volume：191, Number：14, First page：4555, Last page：4561, Jul. 2009, []
  In Bacillus subtilis a null mutation of the relA gene, whose gene product is involved in the synthesis and/or hydrolysis of (p) ppGpp, causes a growth defect that can be suppressed by mutation(s) of yjbM and/or ywaC coding for small (p) ppGpp synthetases. All 35 suppressor mutations newly isolated were classified into two groups, either yjbM or ywaC, by mapping and sequencing their mutations, suggesting that there are no (p) ppGpp synthetases other than RelA, YjbM, and YwaC in B. subtilis. In order to understand better the relation between RelA and rRNA synthesis, we studied in the relA mutant the transcriptional regulation of seven rRNA operons (rrnO, -A, -J, -I, -E, -D, or -B) individually after integration of a promoter- and terminatorless cat gene. We identified the transcriptional start sites of each rrn operon (a G) and found that transcription of all rrn operons from their P1 promoters was drastically reduced in the relA mutant while this was almost completely restored in the relA yjbM ywaC triple mutant. Taken together with previous results showing that the intracellular GTP concentration was reduced in the relA mutant while it was restored in the triple mutant, it seems likely that continuous (p)ppGpp synthesis by YjbM and/or YwaC at a basal level causes a decrease in the amounts of intracellular GTP.
  AMER SOC MICROBIOLOGY
  DOI：https://doi.org/10.1128/JB.00263-09
  DOI ID：10.1128/JB.00263-09, ISSN：0021-9193, CiNii Articles ID：80020423770, PubMed ID：19447912, Web of Science ID：WOS:000267372900008
• Novel Bacterial N-Acetyltransferase Gene for Herbicide Detoxification in Land Plants and Selection Maker in Plant Transformation　　　　　　　　　　　　　　　
  Choong-Soo Yun; Hisakazu Hasegawa; Hideaki Nanamiya; Teruhiko Terakawa; Yuzuru Tozawa
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：73, Number：5, First page：1000, Last page：1006, May 2009, []
  Phosphinothricin (PPT) is the active ingredient in bialaphos, which specifically inhibits glutamine synthetase in land plants. We isolated a novel PPT-resistant gene from a soil bacterium, Nocardia sp., and characterized it. The encoded protein, consisting of 177 amino acids, showed significant similarity to bacterial N-acetyltransferases, and we originally designated the gene MAT (methionine sulfone N-acetyltransferase). The recombinant MAT protein exhibited functions as a methionine sulfone and PPT N-acetyltransferase in vitro. The PPT N-acetyltransferase activity reached the maximum at pH 8-8.5, indicating that the protein might optimally function in chloroplasts. We therefore constructed a MAT gene, encoding the enzyme with a chloroplast-localizing signal in its amino-terminus. Plant transformation with the construct resulted in the generation of PPT-resistant rice and Arabidopsis. Furthermore, the transformed Arabidopsis was selectable in a synthetic medium containing PPT. The MAT gene thus facilitated establishment of herbicide-resistant plants, and as a new selectable gene marker.
  TAYLOR & FRANCIS LTD
  DOI：https://doi.org/10.1271/bbb.80777
  DOI ID：10.1271/bbb.80777, ISSN：0916-8451, eISSN：1347-6947, CiNii Articles ID：10027540472, Web of Science ID：WOS:000266620100005
• The contribution of endogenous cellulase to the cellulose digestion in the gut of earthworm (Pheretima hilgendorfi: Megascolecidae)　　　　　　　　　　　　　　　
  Mana Nozaki; Chiemi Miura; Yuzuru Tozawa; Takeshi Miura
  SOIL BIOLOGY & BIOCHEMISTRY, Volume：41, Number：4, First page：762, Last page：769, Apr. 2009, []
  Cellulase activity has been detected in the digestive tract of earthworms. However, it has not been well clarified whether the origin of those cellulases are the earthworm themselves or the symbionts. In our study, zymogram analysis suggests that one cellulase (endo-beta-1,4-glucanase, EC3.2.1.4) mainly works to digest cellulose in Pheretima hilgendorfi. To identify the cellulase in P. hilgendorfi, we carried out cDNA cloning of the cellulase gene from the digestive tract. A novel cellulase gene was identified from the gut of earthworm. The cDNA encoding cellulase of P. hilgendorfi (phhEG) is 1606 bp with an open reading frame encoding a protein of 449 amino acid residues. The deduced amino acid sequence of P. hilgendorfi cellulase showed higher homology to invertebrate cellulases than bacterium cellulases belonging to the glycosyl hydrolase family (GHF) 9. The phhEG gene was detected in intestinal epithelium cell of mid-foregut using Northern blot and in situ hybridization. Similarly, specific cellulase activity against carboxymethyl cellulose (CMC) was significantly higher in midforegut tissue. Recombinant phhEG produced by wheat germ cell-free protein synthesis system had a cellulase activity which degrade CIVIC In zymogram analysis, the molecular size of cellulase was detected as a single band of 51 kDa from the whole gut contents extracts of P. hilgendorfi, and was very similar to the predicted molecular size of the mature phhEG protein. These results strongly suggested that the earthworm has the capacity to produce the endogenous and functional cellulase around the midforegut, and use this cellulase for their cellulose digestion with the support of intestinal caecum. (C) 2009 Elsevier Ltd. All rights reserved.
  PERGAMON-ELSEVIER SCIENCE LTD
  DOI：https://doi.org/10.1016/j.soilbio.2009.01.016
  DOI ID：10.1016/j.soilbio.2009.01.016, ISSN：0038-0717, CiNii Articles ID：80020272776, Web of Science ID：WOS:000265325300013
• (3) The Role of the Earthworm, Pheretima (Metaphire) hilgendorfi, in Terrestrial Ecosystem Nutrient Cycling.　　　　　　　　　　　　　　　
  Nozaki Mana; Miura Chiemi; Tozawa Yuzuru; Miura Takeshi
  Interdisciplinary Studies on Environmental Chemistry— Biological Responses to Chemical Pollutants, First page：275, Last page：279, Dec. 2008, []
• Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan　　　　　　　　　　　　　　　
  Tetsuya Yamada; Fumio Matsuda; Koji Kasai; Shuichi Fukuoka; Keisuke Kitamura; Yuzuru Tozawa; Hisashi Miyagawa; Kyo Wakasa
  PLANT CELL, Volume：20, Number：5, First page：1316, Last page：1329, May 2008, []
  Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenyl-propanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size.
  AMER SOC PLANT BIOLOGISTS, English
  DOI：https://doi.org/10.1105/tpc.107.057455
  DOI ID：10.1105/tpc.107.057455, ISSN：1040-4651, eISSN：1532-298X, Web of Science ID：WOS:000257320600012
• Expression of parsley flavone synthase I establishes the flavone Biosynthetic pathway in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Choong-Soo Yun; Tomio Yamamoto; Akira Nozawa; Yuzuru Tozawa
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：72, Number：4, First page：968, Last page：973, Apr. 2008, []
  Arabidopsis thaliana lacks the flavone biosynthetic pathway, probably because of a lack or low activity of a flavone synthase. To establish this biosynthetic pathway in Arabidopsis, we subjected this model plant to transformation with the parsley gene for flavone synthase type I (FNS-I). Transgenic seedlings expressing FNS-I were cultured in liquid medium with or without naringenin, and plant extracts were then analyzed by high-performance liquid chromatography. In contrast to wild-type seedlings, the transgenic seedlings accumulated substantial amounts of apigenin, which is produced from naringenin by FNS-I, and the apigenin level correlated with the abundance of FNS-I mRNA in three different transgenic lines. These results indicate that the FNS-I transgene produces a functional enzyme that catalyzes the conversion of naringenin to apigenin in Arabidopsis. These FNS-I transgenic lines should prove useful in investigating the in vivo functions of enzymes that mediate the synthesis of the wide variety of flavones found in other plants.
  TAYLOR & FRANCIS LTD, English
  DOI：https://doi.org/10.1271/bbb.70692
  DOI ID：10.1271/bbb.70692, ISSN：0916-8451, eISSN：1347-6947, CiNii Articles ID：10027526457, Web of Science ID：WOS:000255501100007
• The bacterial stringent response, conserved in chloroplasts, controls plant fertilization　　　　　　　　　　　　　　　
  Shinji Masuda; Kazuki Mizusawa; Takakuni Narisawa; Yuzuru Tozawa; Hiroyuki Ohta; Ken-ichiro Takamiya
  PLANT AND CELL PHYSIOLOGY, Volume：49, Number：2, First page：135, Last page：141, Feb. 2008, [Reviewed]
  The chloroplast, an essential organelle for plants, performs a wide variety of metabolic processes for host cells, which include photosynthesis as well as amino acid and fatty acid biosynthesis. The organelle conserves many bacterial systems in its functions, implicating its origin from symbiosis of a photosynthetic bacterium. In bacterial cells, the stringent response acts as a global regulatory system for gene expression mediated by a small nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), that is necessary for cell adaptation to diverse environmental stimuli such as amino acid starvation. Recent studies indicated that proteins similar to the bacterial ppGpp synthase/hydrolyase are conserved in plants, although their precise roles are not known. Here we show that the stringent response in chloroplasts is crucial for normal plant fertilization. Specifically, one of the Arabidopsis ppGpp synthase homologs, CRSH (Ca(2+)-activated RelA/SpoT homolog), exhibits calcium-dependent ppGpp synthesis activity in vitro, and is localized in chloroplasts in vivo. A knockdown mutation of CRSH in Arabidopsis results in a significant reduction in silique size and seed production, indicating that plant reproduction is under the control of chloroplast function through a ppGpp-mediated stringent response.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcm177
  DOI ID：10.1093/pcp/pcm177, ISSN：0032-0781, CiNii Articles ID：10027328528, PubMed ID：18178586, Web of Science ID：WOS:000253021400001
• Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis　　　　　　　　　　　　　　　
  Hideaki Nanamiya; Koji Kasai; Akira Nozawa; Choong-Soo Yun; Takakuni Narisawa; Kana Murakami; Yousuke Natori; Fujio Kawamura; Yuzuru Tozawa
  MOLECULAR MICROBIOLOGY, Volume：67, Number：2, First page：291, Last page：304, Jan. 2008, [Reviewed], [Last, Corresponding]
  Bacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram-positive bacteria such as Bacillus subtilis have been thought to possess only a single RelA-SpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins (similar to 25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA-SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA-SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA-SpoT homologues.
  BLACKWELL PUBLISHING, English, Scientific journal
  DOI：https://doi.org/10.1111/j.1365-2958.2007.06018.x
  DOI ID：10.1111/j.1365-2958.2007.06018.x, ISSN：0950-382X, Web of Science ID：WOS:000251765900008
• Possible targets of "magic spots" in plant signalling　　　　　　　　　　　　　　　
  Shinji Masuda; Yuzuru Tozawa; Hiroyuki Ohta
  Plant Signaling and Behavior, Volume：3, Number：11, First page：1021, Last page：1023, 2008, [Reviewed]
  The prokaryotic signalling molecules (p)ppGpp, also called "magic spots", regulate a wide variety of physiological activities in bacteria, including transcription, translation, and replication as well as some enzymatic activities such as those of some GTP-binding proteins, which are necessary for bacterial cells to adapt their physiology to different environmental stimuli. This response is called the stringent response. Recently, (p)ppGpp molecules and (p)ppGpp synthetase homologues, designated RSHs, have been identified in plants. At least some of the RSHs are targeted to chloroplasts. A knockdown mutation in one of the RSHs results in unusual flower development in Arabidopsis, suggesting that the plastid stringent response has important roles in the physiology of higher plants. Possible (p)ppGpp target proteins are investigated. ©2008 Landes Bioscience.
  Landes Bioscience, English, Scientific journal
  DOI：https://doi.org/10.4161/psb.6766
  DOI ID：10.4161/psb.6766, ISSN：1559-2324, PubMed ID：19704441, SCOPUS ID：56049119193
• Calcium-activated (p)ppGpp synthetase in chloroplasts of land plants　　　　　　　　　　　　　　　
  Yuzuru Tozawa; Akira Nozawa; Takuya Kanno; Takakuni Narisawa; Shinji Masuda; Koji Kasai; Hideaki Nanamiya
  JOURNAL OF BIOLOGICAL CHEMISTRY, Volume：282, Number：49, First page：35536, Last page：35545, Dec. 2007, [Reviewed], [Lead, Corresponding]
  The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5 '-triphosphate ((p) ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p) ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of ( p) ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH2 terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca2+ and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca2+-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca2+ and (p)ppGpp signaling in regulation of the genetic system of these organelles.
  AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, English, Scientific journal
  DOI：https://doi.org/10.1074/jbc.M703820200
  DOI ID：10.1074/jbc.M703820200, ISSN：0021-9258, PubMed ID：17938177, Web of Science ID：WOS:000251458100013
• A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters　　　　　　　　　　　　　　　
  Akira Nozawa; Hideaki Nanamiya; Takuji Miyata; Nicole Linka; Yaeta Endo; Andreas P. M. Weber; Yuzuru Tozawa
  PLANT AND CELL PHYSIOLOGY, Volume：48, Number：12, First page：1815, Last page：1820, Dec. 2007, [Reviewed], [Corresponding]
  We describe here a novel proteoliposome reconstitution system for functional analysis of plant membrane transporters that is based on a modified wheat germ cell-free translation system. We established optimized conditions for the reconstitution system with Arabidopsis thaliana phosphoenolpyruvate/phosphate translocator 1 (AtPPT1) as a model transporter. A high activity of AtPPT1 was achieved by synthesis of the protein in the presence of both a detergent such as Brij35 and liposomes. We also determined the substrate specificities of three putative rice PPT homologs with this system. The cell-free proteoliposome reconstitution system provides a valuable tool for functional analysis of transporter proteins.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcm150
  DOI ID：10.1093/pcp/pcm150, ISSN：0032-0781, CiNii Articles ID：10027006502, PubMed ID：17981875, Web of Science ID：WOS:000252507200016
• The plastid sigma factor SIG1 maintains photosystem I activity via regulated expression of the psaA operon in rice chloroplasts　　　　　　　　　　　　　　　
  Yuzuru Tozawa; Masayoshi Teraishi; Tadamasa Sasaki; Kintake Sonoike; Yoshitaka Nishiyama; Mitsuhiro Itaya; Akio Miyao; Hirohiko Hirochika
  PLANT JOURNAL, Volume：52, Number：1, First page：124, Last page：132, Oct. 2007, [Lead, Corresponding]
  Sigma factors encoded by the nucleus of plants confer promoter specificity on the bacterial-type RNA polymerase in chloroplasts. We previously showed that transcripts of OsSIG1, which encodes one such sigma factor in rice, accumulate relatively late during leaf development. We have now isolated and characterized two allelic mutants of OsSIG1, in which OsSIG1 is disrupted by insertion of the retrotransposon Tos17, in order to characterize the functions of OsSIG1. The OsSIG1(-/-) plants were found to be fertile but they manifested an approximately one-third reduction in the chlorophyll content of mature leaves. Quantitative RT-PCR and northern blot analyses of chloroplast gene expression revealed that the abundance of transcripts derived from the psaA operon was markedly reduced in OsSIG1(-/-) plants compared with that in wild-type homozygotes. This effect was accompanied by a reduction in the abundance of the core protein complex (PsaA-PsaB) of photosystem I. Analysis of chlorophyll fluorescence also revealed a substantial reduction in the rate of electron transfer from photosystem II to photosystem I in the OsSIG1 mutants. Our results thus indicate that OsSIG1 plays an important role in the maintenance of photosynthetic activity in mature chloroplasts of rice by regulating expression of chloroplast genes for components of photosystem I.
  BLACKWELL PUBLISHING, English, Scientific journal
  DOI：https://doi.org/10.1111/j.1365-313X.2007.03216.x
  DOI ID：10.1111/j.1365-313X.2007.03216.x, ISSN：0960-7412, Web of Science ID：WOS:000249828700011
• Oxidation of elongation factor G inhibits the synthesis of the D1 protein of photosystem II　　　　　　　　　　　　　　　
  Kouji Kojima; Masaru Oshita; Yohei Nanjo; Koji Kasai; Yuzuru Tozawa; Hidenori Hayashi; Yoshitaka Nishiyama
  MOLECULAR MICROBIOLOGY, Volume：65, Number：4, First page：936, Last page：947, Aug. 2007, [Reviewed]
  Oxidative stress inhibits the repair of photodamaged photosystem II (PSII). This inhibition is due initially to the suppression, by reactive oxygen species (ROS), of the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, at the level of translational elongation. To investigate in vitro the mechanisms whereby ROS inhibit translational elongation, we developed a translation system in vitro from the cyanobacterium Synechocystis sp. PCC 6803. The synthesis of the D1 protein in vitro was inhibited by exogenous H2O2. However, the addition of reduced forms of elongation factor G (EF-G), which is known to be particularly sensitive to oxidation, was able to reverse the inhibition of translation. By contrast, the oxidized forms of EF-G failed to restore translational activity. Furthermore, the overexpression of EF-G of Synechocystis in another cyanobacterium Synechococcus sp. PCC 7942 increased the tolerance of cells to H2O2 in terms of protein synthesis. These observations suggest that EF-G might be the primary target, within the translational machinery, of inhibition by ROS.
  WILEY-BLACKWELL, English, Scientific journal
  DOI：https://doi.org/10.1111/j.1365-2958.2007.05836.x
  DOI ID：10.1111/j.1365-2958.2007.05836.x, ISSN：0950-382X, eISSN：1365-2958, CiNii Articles ID：80018361516, PubMed ID：17617168, Web of Science ID：WOS:000249126500007
• Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system　　　　　　　　　　　　　　　
  Takuya Kanno; Michiko Kitano; Rika Kato; Akira Omori; Yaeta Endo; Yuzuru Tozawa
  PROTEIN EXPRESSION AND PURIFICATION, Volume：52, Number：1, First page：59, Last page：65, Mar. 2007, [Reviewed], [Last, Corresponding]
  Recent improvements in wheat-embryo cell-free translation resulted in a highly productive system for protein preparation. To clarify N-terminal processing of the cell-free system in a preparative-scale (> mg protein product per ml), 20 mutant variants of maltose-binding protein (MalE), each having a different penultimate residue in the sequence Met-Xaa-Ile-Glu-, and 20 glutathione S-transferase (GST) variants, having Met-Xaa-Pro-Ile-sequence, were designed and synthesized. The MalE and GST proteins were purified by amylose-resin and glutathione columns, respectively, followed by analysis of their N-terminal sequences. These investigations revealed that sequence specificity and efficiency of the N-terminal Met (N-Met) elimination in the cell-free system are similar to those reported from investigations in cellular systems or in the wheat-embryo cell-free protein expression system in analytical scale (similar to 10 mu g protein product per ml). Cleavage of the N-Met is basically determined by the penultimate amino acid in the polypeptide sequence. In the case of MalE, the cleavage was efficient when the penultimate residue was Ala, Cys, Gly, Pro, Ser or Thr. But, in the case of GST with Pro as the antepenultimate residue, the efficiency was significantly reduced when the penultimate residue was Gly or Thr. We also confirmed that substitution of the antepenultimate residue in MalE to Pro drastically reduced the efficiency of N-Met cleavage when the penultimate residue was Ala, Gly, Pro, Ser or Thr, indicating inhibitory effects of antepenultimate residue Pro on N-Met elimination. These results clarified sequence-specific functions of the endogenous N-terminal processing machinery in the scaled-up wheat-embryo cell-free translation system. (c) 2006 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE, English, Scientific journal
  DOI：https://doi.org/10.1016/j.pep.2006.09.007
  DOI ID：10.1016/j.pep.2006.09.007, ISSN：1046-5928, CiNii Articles ID：80018650020, PubMed ID：17123829, Web of Science ID：WOS:000244772900008
• Functional similarities of a thermostable protein-disulfide oxidoreductase identified in the archaeon Pyrococcus horikoshii to bacterial DsbA enzymes　　　　　　　　　　　　　　　
  Toshihiro Kuroita; Takuya Kanno; Atsushi Kawai; Bunsei Kawakami; Masanori Oka; Yaeta Endo; Yuzuru Tozawa
  EXTREMOPHILES, Volume：11, Number：1, First page：85, Last page：94, Jan. 2007, [Reviewed], [Last, Corresponding]
  We have isolated and characterized a gene for a putative protein-disulfide oxidoreductase (phdsb) in the archaeon Pyrococcus horikoshii. The open reading frame of phdyb encodes a protein of 170 amino acids with an NH2-terminal extension similar to the bacterial signal peptides. The putative mature region of PhDsb includes a sequence motif, Cys-Pro-His-Cys (CPHC), that is conserved in members of the bacterial DsbA family, but otherwise the archaeal and bacterial sequences do not show substantial similarity. A recombinant protein corresponding to the predicted mature form of PhDsb behaved as a monomer and manifested oxidoreductase activities in vitro similar to those of DsbA of Escherichia coli. The catalytic activity of PhDsb was thermostable and was shown by mutation analysis to depend on the NH2-terminal cysteine residue of the CPHC motif. Thus, in spite of their low overall sequence similarities, DsbA-like proteins of archaea and bacteria appear to be highly similar in terms of function.
  SPRINGER TOKYO, English, Scientific journal
  DOI：https://doi.org/10.1007/s00792-006-0015-4
  DOI ID：10.1007/s00792-006-0015-4, ISSN：1431-0651, CiNii Articles ID：80018656207, PubMed ID：16896527, Web of Science ID：WOS:000244249100011
• Two novel nuclear genes, OsSIG5 and OsSIG6, encoding potential plastid sigma factors of RNA polymerase in rice: Tissue-specific and light-responsive gene expression　　　　　　　　　　　　　　　
  Yoshiki Kubota; Akio Miyao; Hirohiko Hirochika; Yuzuru Tozawa; Hiroyuki Yasuda; Yuichi Tsunoyama; Yasuo Niwa; Sousuke Imamura; Makoto Shirai; Munehiko Asayama
  PLANT AND CELL PHYSIOLOGY, Volume：48, Number：1, First page：186, Last page：192, Jan. 2007, [Reviewed]
  Two novel nuclear genes, OsSIG5 and OsSIG6, encoding potential plastid sigma factors of RNA polymerase (RNAP) were identified in Oryza sativa. The deduced amino acid sequences contain conserved regions, regions 1.2-4.2, and a novel region A/B at the N-terminus. Tissue-specific and light-responsive transcripts of OsSIG5 and OsSIG6 were observed. The N-terminal region of OsSig5 conferred import of green fluorescent protein into the chloroplast. Specific transcripts of rice psbA were synthesized in vitro by reconstituted OsSig5-RNAP holoenzymes. These results indicated that OsSig5 is a plastid sigma factor. This is the first report of the Sig5-type sigma factor in crops.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcl050
  DOI ID：10.1093/pcp/pcl050, ISSN：0032-0781, eISSN：1471-9053, CiNii Articles ID：10018520749, PubMed ID：17148693, Web of Science ID：WOS:000243993900019
• Roles of 11 beta-hydroxysteroid dehydrogenase in fish spermatogenesis　　　　　　　　　　　　　　　
  Yuichi Ozaki; Masato Higuchi; Chiemi Miura; Sonoko Yamaguchi; Yuzuru Tozawa; Takeshi Miura
  ENDOCRINOLOGY, Volume：147, Number：11, First page：5139, Last page：5146, Nov. 2006, [Reviewed]
  In fish spermatogenesis, the main action of progestins is generally regarded as the induction of sperm maturation. Our previous in vitro study demonstrated that a progestin, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP), induced the initiation of meiosis in spermatogenesis in the Japanese eel (Anguilla japonica). In the present study, to elucidate the molecular mechanisms underlying the action of DHP, we attempted to clone cDNAs encoding genes whose expression was induced by DHP in eel testis, using cDNA subtraction. One of the cDNAs we isolated encodes eel 11 beta-hydroxysteroid dehydrogenase short form (e11 beta-HSDsf), and Northern blot and RT-PCR analysis showed that transcripts of e11 beta-HSDsf in testis were induced by DHP. The recombinant e11 beta-HSDsf had 11 beta-dehydrogenase activity, metabolizing cortisol to cortisone, and 11 beta-hydroxytestosterone to 11-ketotestosterone (11-KT). In vitro experiments revealed that eel immature testis had 11 beta-dehydrogenase activity, and DHP treatment enhanced the activity. To understand the role of 11 beta-HSD in spermatogenesis, we examined the direct effects of cortisol on eel spermatogenesis using an organ culture system. Cortisol induced DNA replication in spermatogonia and enhanced the spermatogonial proliferation induced by 11-KT. However, excess cortisol inhibited proliferation. In addition, 11-KT production was induced in testicular fragments incubated with cortisol. These results suggest that optimal levels of cortisol induced spermatogonial mitosis by increasing 11-KT production. Furthermore, two possible roles of DHP on spermatogenesis, via the up-regulation of 11 beta-HSD expression, are suggested: positive feedback control of 11-KT production and the modulation of cortisol levels to protect testes from excess circulating cortisol.
  ENDOCRINE SOC, English, Scientific journal
  DOI：https://doi.org/10.1210/en.2006-0391
  DOI ID：10.1210/en.2006-0391, ISSN：0013-7227, Web of Science ID：WOS:000241324900017
• Covalent circularization of exogenous RNA during incubation with a wheat embryo cell extract　　　　　　　　　　　　　　　
  Shin-ichi Makino; Tatsuya Sawasaki; Yuzuru Tozawa; Yaeta Endo; Kazuyuki Takai
  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume：347, Number：4, First page：1080, Last page：1087, Sep. 2006, [Reviewed]
  Cell extracts from wheat embryos have been widely used for mRNA-directed protein production. Here, we found that a significant fraction of exogenous linear RNAs are circularized in a wheat embryo extract. The circularization was seen only in uncapped RNAs. The amount of the circular species reached around 1% of the initial RNA and increased along with an increase in the initial concentration more than proportionally. The circular RNAs were stable but unable to be translated in the extract. The circularization was competitively inhibited in the presence of a known substrate of a wheat embryo RNA ligase. Thus, we cloned the RNA ligase cDNAs. Three isoform sequences were homologous to the other plant RNA ligases. An addition of a cell-free synthesized wheat RNA ligase abolished the inhibition, which indicates a participation of its activity in the circularization. A possible role in RNA metabolism, RNA silencing in particular, is discussed. (c) 2006 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE, English, Scientific journal
  DOI：https://doi.org/10.1016/j.bbrc.2006.07.011
  DOI ID：10.1016/j.bbrc.2006.07.011, ISSN：0006-291X, CiNii Articles ID：80019022189, PubMed ID：16870150, Web of Science ID：WOS:000239681100033
• High-level tryptophan accumulation in seeds of transgenic rice and its limited effects on agronomic traits and seed metabolite profile　　　　　　　　　　　　　　　
  Kyo Wakasa; Hisakazu Hasegawa; Hiroshi Nemoto; Fumio Matsuda; Haruna Miyazawa; Yuzuru Tozawa; Keiko Morino; Akira Komatsu; Tetsuya Yamada; Teruhiko Terakawa; Hisashi Miyagawa
  JOURNAL OF EXPERIMENTAL BOTANY, Volume：57, Number：12, First page：3069, Last page：3078, Sep. 2006, [Reviewed]
  Metabolic manipulation of plants to improve their nutritional quality is an important goal of plant biotechnology. Expression in rice (Oryza sativa L.) of a transgene (OASA1D) encoding a feedback-insensitive alpha subunit of rice anthranilate synthase results in the accumulation of tryptophan (Trp) in calli and leaves. It is shown here that the amount of free Trp in the seeds of such plants is increased by about two orders of magnitude compared with that in the seeds of wild-type plants. The total Trp content in the seeds of the transgenic plants was also increased. Two homozygous lines, HW1 and HW5, of OASA1D transgenic rice were generated for characterization of agronomic traits and aromatic metabolite profiling of seeds. The marked overproduction of Trp was stable in these lines under field conditions, although spikelet fertility and yield, as well as seed germination ability, were reduced compared with the wild type. These differences in agronomic traits were small, however, in HW5. In spite of the high Trp content in the seeds of the HW lines, metabolic profiling revealed no substantial changes in the amounts of other phenolic compounds. The amount of indole acetic acid was increased about 2-fold in the seeds of the transgenic lines. The establishment and characterization of these OASA1D transgenic lines have thus demonstrated the feasibility of increasing the Trp content in the seeds of rice (or of other crops) as a means of improving its nutritional value for human consumption or animal feed.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/jxb/erl068
  DOI ID：10.1093/jxb/erl068, ISSN：0022-0957, CiNii Articles ID：80018891538, PubMed ID：16908506, Web of Science ID：WOS:000240696800014
• Tolerance for random recombination of domains in prokaryotic and eukaryotic translation systems: Limited interdomain misfolding in a eukaryotic translation system　　　　　　　　　　　　　　　
  N Hirano; T Sawasaki; Y Tozawa; Y Endo; K Takai
  PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, Volume：64, Number：2, First page：343, Last page：354, Aug. 2006, [Reviewed]
  It has been proposed that eukaryotic translation systems have a greater capacity for cotranslational. folding of domains than prokaryotic translation systems, which reduces interdomain misfolding in multidomain proteins and, therefore, leads to tolerance for random recombination of domains. However, there has been a controversy as to whether prokaryotic and eukaryotic translation systems differ in the capacity for cotranslational. domain folding. Here, to examine whether these systems differ in the tolerance for the random domain recombination, we systematically combined six proteins, out of which four are soluble and two are insoluble when produced in an Escherichia coli and a wheat germ cell-free protein synthesis systems, to construct a fusion protein library. Forty out of 60 two-domain proteins and 114 out of 120 three-domain proteins were more soluble when produced in the wheat system than in the E. coli system. Statistical analyses of the solubilities and the activities indicated that, in the wheat system but not in the E. coli system, the two soluble domains comprised mainly of beta-sheets tend to avoid interdomain misfolding and to fold properly even at the neighbor of the misfolded domains. These results demonstrate that a eukaryotic system permits the concomitance of a wider variety of domains within a single polypeptide chain than a prokaryotic system, which is probably due to the difference in the capacity for cotranslational folding. This difference is likely to be related to the postulated difference in the tolerance for random recombination of domains.
  WILEY-LISS, English, Scientific journal
  DOI：https://doi.org/10.1002/prot.21008
  DOI ID：10.1002/prot.21008, ISSN：0887-3585, Web of Science ID：WOS:000238624300005
• Identification of three shikimate kinase genes in rice: characterization of their differential expression during panicle development and of the enzymatic activities of the encoded proteins　　　　　　　　　　　　　　　
  K Kasai; T Kanno; M Akita; Y Ikejiri-Kanno; K Wakasa; Y Tozawa
  PLANTA, Volume：222, Number：3, First page：438, Last page：447, Oct. 2005, [Reviewed], [Last, Corresponding]
  The shikimate pathway is common to the biosynthesis of the three aromatic amino acids and that of various secondary metabolites in land plants. Shikimate kinase (SK; EC 2.7.1.71) catalyzes the phosphorylation of shikimate to yield shikimate 3-phosphate. In an attempt to elucidate the functional roles of enzymes that participate in the shikimate pathway in rice ( Oryza sativa), we have now identified and characterized cDNAs corresponding to three SK genes-OsSK1, OsSK2, and OsSK3-in this monocotyledenous plant. These SK cDNAs encode proteins with different NH2-terminal regions and with putative mature regions that share sequence similarity with other plant and microbial SK proteins. An in vitro assay of protein import into intact chloroplasts isolated from pea ( Pisum sativum) seedlings revealed that the full-length forms of the three rice SK proteins are translocated into chloroplasts and processed, consistent with the assumption that the different NH2-terminal sequences function as chloroplast transit peptides. The processed forms of all three rice proteins synthesized in vitro manifested SK catalytic activity. Northern blot analysis revealed that the expression of OsSK1 and OsSK2 was induced in rice calli by treatment with the elicitor N-acetylchitoheptaose, and that expression of OsSK1 and OsSK3 was up-regulated specifically during the heading stage of panicle development. These results suggest that differential expression of the three rice SK genes and the accompanying changes in the production of shikimate 3-phosphate may contribute to the defense response and to panicle development in rice.
  SPRINGER, English, Scientific journal
  DOI：https://doi.org/10.1007/s00425-005-1559-8
  DOI ID：10.1007/s00425-005-1559-8, ISSN：0032-0935, eISSN：1432-2048, CiNii Articles ID：80017625599, PubMed ID：15891897, Web of Science ID：WOS:000232611600005
• Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway　　　　　　　　　　　　　　　
  T Kanno; A Komatsu; K Kasai; JG Dubouzet; M Sakurai; Y Ikejiri-Kanno; K Wakasa; Y Tozawa
  PLANT PHYSIOLOGY, Volume：138, Number：4, First page：2260, Last page：2268, Aug. 2005, [Reviewed], [Last, Corresponding]
  Rice (Oryza sativa) anthranilate synthase alpha-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.105.062885
  DOI ID：10.1104/pp.105.062885, ISSN：0032-0889, CiNii Articles ID：80017515978, PubMed ID：16040654, Web of Science ID：WOS:000231206600040
• Use of a feedback-insensitive at subunit of anthranilate synthase as a selectable marker for transformation of rice and potato　　　　　　　　　　　　　　　
  T Yamada; Y Tozawa; H Hasegawa; T Terakawa; Y Ohkawa; K Wakasa
  MOLECULAR BREEDING, Volume：14, Number：4, First page：363, Last page：373, Nov. 2004, [Reviewed]
  A selection system based on a mutant rice gene for a feedback-insensitive alpha subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.
  KLUWER ACADEMIC PUBL, English, Scientific journal
  ISSN：1380-3743, Web of Science ID：WOS:000226417400002
• Characterization of a rice nuclear-encoded plastid RNA polymerase gene OsRpoTp　　　　　　　　　　　　　　　
  K Kusumi; A Yara; N Mitsui; Y Tozawa; K Iba
  PLANT AND CELL PHYSIOLOGY, Volume：45, Number：9, First page：1194, Last page：1201, Sep. 2004, [Reviewed]
  We isolated and characterized two rice genes, OsRpoTp and OsRpoTm, that encode putative phage-type RNA polymerases. Predicted amino acid sequences showed high homology of these genes to known RpoT genes. A transient expression assay using green fluorescent protein indicated that the encoded proteins were localized to plastids and mitochondria, respectively. We demonstrated by reverse transcription-PCR experiments and immunoblot analysis that OsRpoTp expression occurred at an early stage of leaf development, prior to the transcript accumulation of the genes that were transcribed by the nuclear-encoded plastid RNA polymerase (NEP). Expression analyses of the chloroplast-deficient rice mutant, virescent-1, showed a discrepancy between OsRpoTp protein accumulation and the level of transcripts of NEP-transcribed genes. Our results suggest that NEP activation is regulated by a process after transcription, and is affected by the developmental state of chloroplast biogenesis.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pch133
  DOI ID：10.1093/pcp/pch133, ISSN：0032-0781, J-Global ID：200902270080615115, CiNii Articles ID：10013734472, PubMed ID：15509842, Web of Science ID：WOS:000224706900010
• The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early stage of chloroplast differentiation　　　　　　　　　　　　　　　
  H Sugimoto; K Kusumi; Y Tozawa; J Yazaki; N Kishimoto; S Kikuchi; K Iba
  PLANT AND CELL PHYSIOLOGY, Volume：45, Number：8, First page：985, Last page：996, Aug. 2004, [Reviewed]
  The rice virescent-2 mutant (v(2)) is temperature conditional and develops chlorotic, chloroplast-deficient leaves at the restrictive temperature. In the v(2) mutant, plastid-encoded proteins involved in photosynthesis and plastid transcriptional regulation were not detectable at any time during chloroplast differentiation. However, the plastid transcripts for these two classes of proteins behaved differently in the mutant, with those for the plastid transcription/translation apparatus accumulating to wild-type levels and those for photosynthetic apparatus being suppressed. Polysome analysis showed that translation of the plastid transcripts encoding the plastid transcription/translation apparatus was blocked at an early stage of chloroplast differentiation. Accumulation of transcripts of nuclear-encoded photosynthetic genes, such as cab and rbcS, was strongly suppressed in the mutant at later stages of chloroplast differentiation, whereas transcripts of genes for the plastid transcription apparatus, such as OsRpoTp and OsSIG2A, accumulated to abnormally high levels at these stages. These results suggest that activation of the plastid translation machinery at an early stage of chloroplast differentiation is important for triggering the transmission of information about plastid developmental state to the nucleus, which in turn is required for the induction of nuclear-encoded chloroplast proteins at later stages of chloroplast differentiation.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pch111
  DOI ID：10.1093/pcp/pch111, ISSN：0032-0781, J-Global ID：200902225111246436, CiNii Articles ID：10013525420, PubMed ID：15356324, Web of Science ID：WOS:000223730800005
• Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments　　　　　　　　　　　　　　　
  N Tanaka; M Fujita; H Handa; S Murayama; M Uemura; Y Kawamura; T Mitsui; S Mikami; Y Tozawa; T Yoshinaga; S Komatsu
  MOLECULAR GENETICS AND GENOMICS, Volume：271, Number：5, First page：566, Last page：576, Jun. 2004, [Reviewed]
  Despite recent progress in sequencing the complete genome of rice (Oryza saliva), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antiftingal defense and signal systems in the membranes. Edman degradation revealed that 60-98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.
  SPRINGER HEIDELBERG, English, Scientific journal
  DOI：https://doi.org/10.1007/s00438-004-1002-z
  DOI ID：10.1007/s00438-004-1002-z, ISSN：1617-4615, CiNii Articles ID：10016454375, PubMed ID：15069638, Web of Science ID：WOS:000222752700006
• Differential expression of three plastidial signia factors, OsSIG1, OsSIG2A, and OsSIG2B, during leaf development in rice　　　　　　　　　　　　　　　
  K Kasai; M Kawagishi-Kobayashi; M Teraishi; Y Ito; K Ochi; K Wakasa; Y Tozawa
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：68, Number：4, First page：973, Last page：977, Apr. 2004, [Reviewed], [Last, Corresponding]
  We isolated and characterized two rice nuclear genes, OsSIG2A and OsSIG2B, encoding the putative a-factor of the plastid RNA polymerase. Deduced protein sequences predicted a plastid-localizing signal in the N-terminus and subsequent polypeptides similar to known SIG2 proteins. Gene expression analysis revealed that the OsSIG2A transcript is more abundant than the OsSIG2B transcript in all tissues tested and that both rice SIG2s are expressed from earlier stages of leaf development than that in the case of OsSIG1. These results indicate differential expression of SIG genes in leaf morphogenesis, suggesting the existence of tissue-and stage-specific functions of SIG proteins for transcriptional regulation of chloroplast genes in plant development.
  TAYLOR & FRANCIS LTD, English, Scientific journal
  DOI：https://doi.org/10.1271/bbb.68.973
  DOI ID：10.1271/bbb.68.973, ISSN：0916-8451, eISSN：1347-6947, CiNii Articles ID：10013144286, PubMed ID：15118338, Web of Science ID：WOS:000221175600038
• Guanosine tetra- and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline　　　　　　　　　　　　　　　
  K Kasai; T Kanno; Y Endo; K Wakasa; Y Tozawa
  NUCLEIC ACIDS RESEARCH, Volume：32, Number：19, First page：5732, Last page：5741, 2004, [Reviewed], [Last, Corresponding]
  Chloroplasts possess bacterial-type systems for transcription and translation. On the basis of the identification of a Chlamydomonas reinhardtii gene encoding a RelA-SpoT homolog (RSH) that catalyzes the synthesis of guanosine tetra- or pentaphosphate [(p)ppGpp], we have previously suggested the operation of stringent control in the chloroplast genetic system. Although RSH genes have also been identified in several higher plants, the activities of the encoded enzymes and their mode of action in chloroplasts have remained uncharacterized. We have now characterized the intrinsic (p)ppGpp synthase activity of chloroplast extracts prepared from pea (Pisum sativum). Fractionation by ultracentrifugation suggested that the (p)ppGpp synthase activity of a translationally active chloroplast stromal extract was associated with 70S ribosomes. Furthermore, this enzymatic activity was inhibited by tetracycline, as was the peptide elongation activity of the extract. Structural comparisons between rRNA molecules of Escherichia coli and pea chloroplasts revealed the conservation of putative tetracycline-binding sites. These observations demonstrate the presence of a ribosome-associated (p)ppGpp synthase activity in the chloroplasts of a higher plant, further implicating (p)ppGpp in a genetic system of chloroplasts similar to that operative in bacteria.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/nar/gkh916
  DOI ID：10.1093/nar/gkh916, ISSN：0305-1048, eISSN：1362-4962, J-Global ID：200902204999264987, CiNii Articles ID：80017051820, PubMed ID：15507686, Web of Science ID：WOS:000225259100019
• In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the alpha subunits OASA1 and OASA2　　　　　　　　　　　　　　　
  T Kanno; K Kasai; Y Ikejiri-Kanno; K Wakasa; Y Tozawa
  PLANT MOLECULAR BIOLOGY, Volume：54, Number：1, First page：11, Last page：23, Jan. 2004, [Reviewed], [Last, Corresponding]
  Anthranilate synthase ( AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, alpha and beta, and converts chorismate to anthranilate. Two or more AS alpha-subunit genes have been identified and characterized in several land plants. Although alpha subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS alpha-subunit genes (OASA1 and OASA2) in rice (Oryza sativa). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS beta subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two beta subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a beta subunit to achieve maximal enzymatic activity even with NH4+ as the amino donor. The V-max and K-i for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.
  KLUWER ACADEMIC PUBL, English, Scientific journal
  DOI：https://doi.org/10.1023/B:PLAN.0000028729.79034.07
  DOI ID：10.1023/B:PLAN.0000028729.79034.07, ISSN：0167-4412, J-Global ID：200902250576251139, CiNii Articles ID：80016758958, PubMed ID：15159631, Web of Science ID：WOS:000221591000002
• Ribosome engineering and secondary metabolite production　　　　　　　　　　　　　　　
  K Ochi; S Okamoto; Y Tozawa; T Inaoka; T Hosaka; J Xu; K Kurosawa
  ADVANCES IN APPLIED MICROBIOLOGY, VOL 56, Volume：56, First page：155, Last page：+, 2004
  ELSEVIER ACADEMIC PRESS INC, English
  DOI：https://doi.org/10.1016/S0065-2164(04)56005-7
  DOI ID：10.1016/S0065-2164(04)56005-7, ISSN：0065-2164, Web of Science ID：WOS:000226025600005
• Expression profiling of translation-associated genes in sporulating Bacillus subtilis and consequence of sporulation by gene inactivation　　　　　　　　　　　　　　　
  Y Ohashi; T Inaoka; K Kasai; Y Ito; S Okamoto; H Satsu; Y Tozawa; F Kawamura; K Ochi
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：67, Number：10, First page：2245, Last page：2253, Oct. 2003, [Reviewed]
  A DNA microarray technique was used to demonstrate global changes in the transcription pattern of translation-associated genes that encode fifty-four ribosomal proteins including a putative ribosomal gene, and eleven translation factors in sporulating B. subtilis. We found that the mRNA levels of nine genes involved in the translation system, which include the genes for three ribosomal proteins (rpmA, rpmGB, and ctc) and two translation factors (efp, and frr), were maintained at a high level at the onset of sporulation. The ypfD gene, which encodes the ribosomal protein S1 homologue, was also found to be expressed significantly during the early sporulation stage. In order to demonstrate the significance of these genes for sporulation, mutants were constructed using the pMutinT3 disruption vector. We detected an impaired sporulation in the mutants of rpmA (gene for the ribosomal protein L27), efp (elongation factor P), frr (ribosome recycling factor), and ypfD. The effect was especially pronounced in the efp mutant, sporulation of which was entirely abolished without affecting growth. The reduced expression of rpmGB (ribosomal protein L33) resulted in an impaired sporulation only at a high temperature (47degreesC). Only the rplI mutant, which encodes the ribosomal protein L9, could not be obtained, implying that its function is essential for viability. Thus, we successfully demonstrated the significance of several translation-associated genes in sporulation by using the results of the gene expression profiling.
  TAYLOR & FRANCIS LTD, English, Scientific journal
  DOI：https://doi.org/10.1271/bbb.67.2245
  DOI ID：10.1271/bbb.67.2245, ISSN：0916-8451, eISSN：1347-6947, CiNii Articles ID：110002698767, PubMed ID：14586115, Web of Science ID：WOS:000186486900025
• OsRALyase1, a putative F-box protein identified in rice, Oryza sativa, with enzyme activity identical to that of wheat RALyase　　　　　　　　　　　　　　　
  Y Ito; A Ozawa; T Sawasaki; Y Endo; K Ochi; Y Tozawa
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Volume：66, Number：12, First page：2727, Last page：2731, Dec. 2002, [Reviewed], [Corresponding]
  A rice gene, OsRALyase1, encoding a product similar to wheat ribosomal RNA apurinic site specific lyase (RALyase), was isolated and expressed in vitro. An open reading frame of the gene predicted a protein of 476 amino acid residues with 75% identity to RALyase and contained an F-box-like motif in its amino terminal region. The rice gene product expressed in a wheat-germ protein expression system had the same characteristics as its wheat counterpart, cleaving a specific depurinated site of the 28S rRNA sarcin-ricin domain.
  TAYLOR & FRANCIS LTD, English, Scientific journal
  DOI：https://doi.org/10.1271/bbb.66.2727
  DOI ID：10.1271/bbb.66.2727, ISSN：0916-8451, eISSN：1347-6947, PubMed ID：12596877, Web of Science ID：WOS:000180151700035
• A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro　　　　　　　　　　　　　　　
  K Kasai; S Usami; T Yamada; Y Endo; K Ochi; Y Tozawa
  NUCLEIC ACIDS RESEARCH, Volume：30, Number：22, First page：4985, Last page：4992, Nov. 2002, [Reviewed], [Last, Corresponding]
  A gene encoding a putative guanosine 3',5'-bispyrophosphate (ppGpp) synthase-degradase, designated Cr-RSH, was identified in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii. The encoded Cr-RSH protein possesses a putative chloroplast-targeting signal at its NH2-terminus, and translocation of Cr-RSH into chloroplasts isolated from C.reinhardtii was demonstrated in vitro. The predicted mature region of Cr-RSH exhibits marked similarity to eubacterial members of the RelA-SpoT family of proteins. Expression of an NH2-terminal portion of Cr-RSH containing the putative ppGpp synthase domain in a relA, spoT double mutant of Escherichia coli complemented the growth deficits of the mutant cells. Chromatographic analysis of P-32-labeled cellular mononucleotides also revealed that expression of Cr-RSH in the mutant bacterial cells resulted in the synthesis of ppGpp. SpoT, which catalyzes (p)ppGpp degradation, is dispensable in E.coli only if cells also lack RelA, which possesses (p)ppGpp synthase activity. The complementation analysis thus indicated that Cr-RSH possesses both ppGpp synthase and degradase activities. These results represent the first demonstration of ppGpp synthase-degradase activities in a eukaryotic organism, and they suggest that eubacterial stringent control mediated by ppGpp has been conserved during evolution of the chloroplast from a photosynthetic bacterial symbiont.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/nar/gkf628
  DOI ID：10.1093/nar/gkf628, ISSN：0305-1048, CiNii Articles ID：80015682695, PubMed ID：12434003, Web of Science ID：WOS:000179253800021
• A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2)　　　　　　　　　　　　　　　
  J Xu; Y Tozawa; C Lai; H Hayashi; K Ochi
  MOLECULAR GENETICS AND GENOMICS, Volume：268, Number：2, First page：179, Last page：189, Oct. 2002, [Reviewed]
  In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK (relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant (rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp.
  SPRINGER-VERLAG, English, Scientific journal
  DOI：https://doi.org/10.1007/s00438-002-0730-1
  DOI ID：10.1007/s00438-002-0730-1, ISSN：1617-4615, CiNii Articles ID：10010276045, PubMed ID：12395192, Web of Science ID：WOS:000179526100005
• Genetic and physiological characterization of rpoB mutations that activate antibiotic production in Streptomyces lividans　　　　　　　　　　　　　　　
  Caixia Lai; Jun Xu; Yuzuru Tozawa; Yoshiko Okamoto-Hosoya; Xingsheng Yao; Kozo Ochi
  Microbiology, Volume：148, Number：11, First page：3365, Last page：3373, 2002, [Reviewed]
  Antibiotic production in Streptomyces lividans can be activated by introducing certain mutations (rif) into the rpoB gene that confer resistance to rifampicin. Working with the most typical (rif-17) mutant strain, KO-417, the rif-17 mutation was characterized. The rif-17 mutation was shown to be responsible for activating antibiotic production and for reducing the growth rate of strain KO-417, as demonstrated by gene-replacement experiments. Gene-expression analysis revealed that introduction of rif into S. lividans elevates expression of the pathway-specific regulatory gene actII-ORF4 to nearly the same level seen in Streptomyces coelicolor. The rif effect on antibiotic production was still evident in the genetic background of relC, indicating that the rif mutation can provoke its effect without depending on ppGpp. Accompanying the restoration of antibiotic production, rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a nutritionally rich medium, suggesting that the mutant RNA polymerases may behave like 'stringent' RNA polymerases. These results indicate that the rif mutation can alter the gene-expression pattern independent of ppGpp. The impaired growth of strain KO-417 (rif-17) was largely restored by introducing the second rif mutation (rif-18) just adjacent to the rif-17 position. Proteome analysis using two-dimensional PAGE revealed that the rif mutant strain KO-418 (rif-17 rif-18) displayed a temporal burst of expression especially of two enzymes, glutamine synthetase (type II) and oxidoreductase, during the late growth phase.
  Society for General Microbiology, English, Scientific journal
  DOI：https://doi.org/10.1099/00221287-148-11-3365
  DOI ID：10.1099/00221287-148-11-3365, ISSN：1350-0872, PubMed ID：12427928, SCOPUS ID：0036856382
• Characterization of rice anthranilate synthase alpha-subunit genes OASA1 and OASA2. Tryptophan accumulation in transgenic rice expressing mutant of OASA1　　　　　　　　　　　　　　　
  Y Tozawa; H Hasegawa; T Terakawa; K Wakasa
  PLANT PHYSIOLOGY, Volume：126, Number：4, First page：1493, Last page：1506, Aug. 2001, [Reviewed], [Lead]
  Anthranilate synthase (AS) is a key enzyme in the synthesis of tryptophan (Trp), indole-3-acetic acid, and indole alkaloids. Two genes, OASA1 and OASA2, encoding AS a-subunits were isolated from a monocotyledonous plant, rice (Oryza sativa cv Nipponbare), and were characterized. A phylogenetic tree of AS alpha -subunits from various species revealed a close evolutionary relationship among OASA1 and Arabidopsis ASA2, Ruta graveolens AS alpha2, and tobacco ASA2, whereas OASA2, Arabidopsis ASA1, and R. graveolens AS alpha1 were more distantly related. OASA1 is expressed in all tissues tested, but the amount of its mRNA was greater in panicles than in leaves and roots. The abundance of OASA2 transcripts is similar among tissues and greater than that of OASAI transcripts; furthermore, OASA2 expression was induced by a chitin heptamer, a potent elicitor, suggesting that OASA2 participates in secondary metabolism. Expression of wild-type OASA1 or OASA2 transgenes did not affect the Trp content of rice calli or plants. However, transformed calli and plants expressing a mutated OASAI gene, OASA1(D323N), that encodes a protein in which aspartate-323 is replaced with asparagine manifested up to 180- and 35-fold increases, respectively, in Trp accumulation. These transgenic calli and plants were resistant to 300 mum 5-methyl-Trp, and AS activity of the calli showed a markedly reduced sensitivity to Trp. These results show that OASA1 is important in the regulation of free Trp concentration, and that mutation of OASAI to render the encoded protein insensitive to feedback inhibition results in accumulation of Trp at high levels. The OASA1(D323N) transgene may prove useful for the generation of crops with an increased Trp content.
  AMER SOC PLANT PHYSIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.126.4.1493
  DOI ID：10.1104/pp.126.4.1493, ISSN：0032-0889, CiNii Articles ID：80012556707, PubMed ID：11500548, SCOPUS ID：0034865082, Web of Science ID：WOS:000170413800017
• Efficient transformation of suspension-cultured rice cells mediated by Agrobacterium tumefaciens
  S. Urushibara; Y. Tozawa; M. Kawagishi-Kobayashi; K. Wakasa
  Breeding Science, Volume：51, Number：1, First page：33, Last page：38, 2001, [Reviewed]
  The efficiency of Agrobacterium tumefaciens-mediated gene transfer in rice was improved by air-drying of the suspension-cultured cells. The suspension-cultured rice calli showed less efficient transformation mediated by Agrobacterium than usual calli cultured on the solid medium. However, simple air-drying of the calli suspension-cultured for 10 to 15 min increased the transformation frequency 10-fold or more. The suspension cultures were established from the calli derived from mature rice seeds and subcultured every week. The frequency of transformation varied with the period of suspension culture and the size of the callus. The calli suspension-cultured for 4 weeks showed a higher transformation frequency than the control (not suspension-cultured), and the highest frequency was as high as 20%. However, the frequency was dramatically lowered by the extension of suspension-culture period up to 7 weeks or more. The transformation occurred frequently in the callus 1 to 2 mm in diameter. Plant regeneration was observed in transformed calli suspension-cultured for 1 to 12 weeks, but plants abnormal in morphology and seed fertility were regenerated from the calli suspension-cultured for 7 weeks. In conclusion, rice calli suspension-cultured for less than 4 weeks were useful as targets of transformation mediated by Agrobacterium.
  English, Scientific journal
  DOI：https://doi.org/10.1270/jsbbs.51.33
  DOI ID：10.1270/jsbbs.51.33, ISSN：1344-7610, SCOPUS ID：0035084386
• The calcium-independent protein kinase C participates in an early process of CD3/CD28-mediated induction of thymocyte apoptosis　　　　　　　　　　　　　　　
  A Asada; Y Zhao; H Komano; T Kuwata; M Mukai; K Fujita; Y Tozawa; R Iseki; H Tian; K Sato; Y Motegi; R Suzuki; M Yokoyama; M Iwata
  IMMUNOLOGY, Volume：101, Number：3, First page：309, Last page：315, Nov. 2000, [Reviewed]
  Thymocyte negative selection eliminates self-reactive clones and involves both a T-cell receptor (TCR)/CD3-mediated signal and a costimulatory signal, which can be delivered via CD28. Anti-CD3/anti-CD28-triggered apoptosis in isolated CD4(+)CD8(+) thymocytes in vitro provides a basic model for negative selection. Effects of isoform-selective and non-isoform-selective inhibitors of protein kinase C (PKC) on this apoptotic process suggest that activation of Ca2+-independent PKC isoforms during the first 2-3 hr of culture is essential for inducing apoptosis, and that Ca2+-dependent PKC isoforms may be influential, but not essential, for apoptosis. To assess the CD3/CD28-mediated activation of PKC in the apoptotic process, we prepared CD4(+)CD8(+) thymocytes (without contamination with cells that had received negative or positive selection signals in vivo) by establishing TCR-transgenic mice with RAG-2-deficient and non-selecting major histocompatibility complex (MHC) backgrounds, in addition to a CD4(+)CD8(+) thymocyte-enriched population from normal mice. Translocation of Ca2+-independent PKC from the cytosolic fraction to the particulate fraction of CD4(+)CD8(+) thymocytes was induced by CD3/CD28-mediated stimulation, but not by CD3- or CD28-mediated stimulation alone, and peaked 2 hr after the start of culture. The kinase activity of the translocated Ca2+-independent PKC was dependent on cofactors in vitro, indicating that novel (n)PKC, but not atypical (a)PKC or a proteolytic PKC fragment, was responsible for the activity. Immunoblotting analysis indicated that the nPKC-theta isoform was the major contributor among nPKC isoforms, and that the classical (c)PKC-alpha isoform was the major contributor among cPKC isoforms. These results suggest that activation of nPKC (especially the theta isoform) in CD4(+)CD8(+) thymocytes is involved in a pathway for negative selection.
  BLACKWELL SCIENCE LTD, English, Scientific journal
  ISSN：0019-2805, Web of Science ID：WOS:000165663100003
• Nuclear encoding of a plastid sigma factor in rice and its tissue- and light-dependent expression　　　　　　　　　　　　　　　
  Y Tozawa; K Tanaka; H Takahashi; K Wakasa
  NUCLEIC ACIDS RESEARCH, Volume：26, Number：2, First page：415, Last page：419, Jan. 1998, [Reviewed], [Lead]
  A full-length cDNA encoding a putative sigma factor for a plastid RNA polymerase was isolated from the higher plant Oryza sativa, The nucleotide sequence of the corresponding nuclear gene, named Os-sigA (O.sativa sigma A), predicts a polypeptide of 519 amino acids that contains a putative plastid-targeting sequence in its N-terminal region, The predicted mature protein shows extensive sequence homology to bacterial sigma factors, encompassing the conserved regions 1.2, 2.1, 2.2, 2.3, 2.4, 3, 4.1 and 4.2 implicated in binding to -10 promoter elements, promoter melting and interaction with the core RNA polymerase enzyme, RNA blot analysis revealed that the abundance of Os-sigA transcripts was markedly greater in green shoots than in roots or in dark-grown etiolated shoots of rice seedlings, Furthermore, exposure of dark-grown etiolated seedlings to light resulted in a rapid increase in the amount of Os-sigA mRNA in the shoot, These observations suggest that regulation of expression of the nuclear gene for this putative plastid RNA polymerase sigma factor by light contributes to light-dependent transcriptional regulation of plastid genes.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/nar/26.2.415
  DOI ID：10.1093/nar/26.2.415, ISSN：0305-1048, CiNii Articles ID：80010098998, PubMed ID：9421493, SCOPUS ID：0032518161, Web of Science ID：WOS:000071779000004
• Characterization of three cDNA species encoding plastid RNA polymerase sigma factors in Arabidopsis thaliana: Evidence for the sigma factor heterogeneity in higher plant plastids　　　　　　　　　　　　　　　
  K Tanaka; Y Tozawa; N Mochizuki; K Shinozaki; A Nagatani; K Wakasa; H Takahashi
  FEBS LETTERS, Volume：413, Number：2, First page：309, Last page：313, Aug. 1997, [Reviewed]
  By database search analysis, we identified three Arabidopsis EST (Expression Sequence Tag) entries having similarity to eubacterial RNA polymerase sigma factors, cDNA clones corresponding to these partial sequences were isolated, and the complete nucleotide sequences mere determined, All three sequences encode proteins highly homologous to cyanobacterial and plastid sigma factors, and the gene products have N-terminal extensions which are assumed to function as plastid-targeting transit peptides, Thus we have concluded that the gene products are RNA polymerase sigma factors of plastids, and named sigA, sigB and sigC, respectively, Expression of these genes was analyzed by RNA gel-blot analysis and shown to be induced by illumination after a short-term dark adaptation, This strongly suggests that light regulation of the nuclear encoded sigma factor genes is involved in light-dependent activation of plastid promoters, (C) 1997 Federation of European Biochemical Societies.
  ELSEVIER SCIENCE BV, English, Scientific journal
  DOI：https://doi.org/10.1016/S0014-5793(97)00906-X
  DOI ID：10.1016/S0014-5793(97)00906-X, ISSN：0014-5793, CiNii Articles ID：80009857878, PubMed ID：9280303, SCOPUS ID：0030843866, Web of Science ID：WOS:A1997XT08500025
• Molecular cloning of the gene encoding putative chloroplast RNA polymerase sigma subunit from rice.
  Tozawa Y; Tanaka K; Wakasa K
  Plant Physiology, Volume：114, Number：3, First page：1276, 1997, [Reviewed], [Lead]
  English, Scientific journal
  ORCID：16605403
• Differential induction of helper and killer T cells from isolated CD4(+)CD8(+) thymocytes in suspension culture　　　　　　　　　　　　　　　
  M Iwata; T Kuwata; M Mukai; Y Tozawa; M Yokoyama
  EUROPEAN JOURNAL OF IMMUNOLOGY, Volume：26, Number：9, First page：2081, Last page：2086, Sep. 1996, [Reviewed]
  Thymocytes of T cell receptor transgenic mice with nonselecting and RAG-2(-/-) backgrounds were developmentally arrested at the CD4(+)CD8(+) stage before positive selection. These thymocytes underwent lineage commitment upon transient stimulation with a combination of ionomycin, a calcium ionophore, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in suspension culture. The effective drug doses were limited within narrow ranges and much lower than those which induce proliferation of mature T cells. The doses corresponded to those which inhibit glucocorticoid-induced apoptosis in these thymocytes. CD4 lineage commitment required longer duration, higher intensity of the stimulation, or both, than CD8 lineage commitment. Functional helper T cells (Th1 and Th2) were induced from the CD4 lineage-committed cells upon secondary stimulation with a combination of ionomycin and PMA followed by lymphokine treatment. Cytotoxic T cells were induced from the CD8 lineage-committed cells upon incubation with concanavalin A and irradiated splenic dendritic cells, but not with the combination of ionomycin and PMA. These results indicate that positive selection is mimicked by the pharmacological stimulation in the absence of other cell types, but that final maturation of CD8 T cells may require a different signal.
  VCH PUBLISHERS INC, English, Scientific journal
  DOI：https://doi.org/10.1002/eji.1830260918
  DOI ID：10.1002/eji.1830260918, ISSN：0014-2980, CiNii Articles ID：10005503306, PubMed ID：8814250, Web of Science ID：WOS:A1996VG26900017
• In vitro differentiation and commitment of CD4+CD8+ thymocytes to the CD4 lineage without TCR engagement　　　　　　　　　　　　　　　
  Y Ohoka; T Kuwata; Y Tozawa; Y Zhao; M Mukai; Y Motegi; R Suzuki; M Yokoyama; M Iwata
  INTERNATIONAL IMMUNOLOGY, Volume：8, Number：3, First page：297, Last page：306, Mar. 1996, [Reviewed]
  Thymocyte positive selection is based on protection of immature CD4/CD8 double-positive (DP) thymocytes from apoptosis and their differentiation into CD4 or CD8 single-positive (SP) cells, Intracellular signals essential for positive selection appear to be induced through the TCR and some of the accessory molecules including LFA-1, CD4 and CD8 upon interaction with thymic stromal cells, The signals, however, still remain to be identified, Since physiological levels of glucocorticoids potentially induce or enhance thymocyte apoptosis even in vivo, the signals are likely to inhibit the apoptotic effect of glucocorticoids. We have previously shown that proper cross-linking of TCR-CD3 with LFA-1, CD4 or CD8 inhibited glucocorticoid-induced thymocyte apoptosis in vitro, and that a proper combination of the calcium ionophore, ionomycin and the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), mimicked the inhibitory effect, Here we determined whether this combination of ionomycin and PMA induces differentiation of isolated DP thymocytes from normal and Ton transgenic mice, We found that pretreatment of DP thymocytes with ionomycin and PMA followed by 1 day culture of the cells without the reagents resulted in the differentiation of the cells into CD4 SP and CD4(+)CD8(lo) T cells that have mostly committed to the CD4 lineage, The changes in expression of other differentiation markers were also in good accordance with those associated with positive selection, except the final maturation, The results indicate that moderate and transient increases in intracellular Ca2+ level and PKC activity induce differentiation and commitment of DP thymocytes to the CD4 lineage, and suggested that the biochemical pathway leading to positive selection is based on a similar mechanism.
  OXFORD UNIV PRESS UNITED KINGDOM, English, Scientific journal
  DOI：https://doi.org/10.1093/intimm/8.3.297
  DOI ID：10.1093/intimm/8.3.297, ISSN：0953-8178, PubMed ID：8671615, Web of Science ID：WOS:A1996TZ60800002
• DISTINCT ROLES OF THE RECEPTOR TYROSINE KINASES TIE-1 AND TIE-2 IN BLOOD-VESSEL FORMATION　　　　　　　　　　　　　　　
  TN SATO; Y TOZAWA; U DEUTSCH; K WOLBURGBUCHHOLZ; Y FUJIWARA; M GENDRONMAGUIRE; T GRIDLEY; H WOLBURG; W RISAU; Y QIN
  NATURE, Volume：376, Number：6535, First page：70, Last page：74, Jul. 1995, [Reviewed]
  TIE-1 and Tie-2 define a new class of receptor tyrosine kinases that are specifically expressed in developing vascular endothelial cells. To study the functions of Tie-1 and Tie-2 during vascular endothelial cell growth and differentiation in vivo, targeted mutations of the genes in mice were introduced by homologous recombination. Embryos deficient in Tie-1 failed to establish structural integrity of vascular endothelial cells, resulting in oedema and subsequently localized haemorrhage. However, analyses of embryos deficient in Tie-2 showed that it is important in angiogenesis, particularly for vascular network formation in endothelial cells. This result contrasts with previous reports on Tie-2 function in vasculogenesis and/or endothelial cen survival. Our in vivo analyses indicate that the structurally related receptor tyrosine kinases Tie-1 and Tie-2 have important hut distinct roles in the formation of blood vessels.
  MACMILLAN MAGAZINES LTD, English, Scientific journal
  DOI：https://doi.org/10.1038/376070a0
  DOI ID：10.1038/376070a0, ISSN：0028-0836, CiNii Articles ID：80008391175, PubMed ID：7596437, Web of Science ID：WOS:A1995RH11100064
• CALCINEURIN ACTIVATION PROTECTS T-CELLS FROM GLUCOCORTICOID-INDUCED APOPTOSIS　　　　　　　　　　　　　　　
  Y ZHAO; Y TOZAWA; R ISEKI; M MUKAI; M IWATA
  JOURNAL OF IMMUNOLOGY, Volume：154, Number：12, First page：6346, Last page：6354, Jun. 1995, [Reviewed]
  In T cell hybridomas, TCR/CD3 complex-mediated stimulation induces apoptosis but inhibits that induced by glucocorticoids. A combination of ionomycin (IM), a calcium ionophore, and PMA, a protein kinase C activator, mimics the effects of the TCR/CD3-mediated stimulation. Glucocorticoid-induced apoptosis is, however, markedly inhibited by IM alone, and less markedly by PMA alone. The immunosuppressant FK506 canceled the inhibition by IM but not that by PMA. As calcineurin (CN) is one of the target molecules of FK506, we examined whether CN activation might have an anti-apoptotic effect. BOG8, a T cell hybridoma, was stably transfected with a mutant CN catalytic subunit with Ca2+/calmodulin-independent, constitutive but FK506-sensitive phosphatase activity. The transfectant clones were fairly resistant to glucocorticoid-induced death. Their resistance, however, was hardly affected by FK506 when added simultaneously with glucocorticoid, but was lost after a prolonged preincubation with FK506. In the parent BOG8 cells, FK506 failed to cancel the inhibitory effect of IM on glucocorticoid-induced death when the addition of FK506 was delayed for 1 h or more. These results suggest that CN activation is required for the resistance only as an early event. The transfectant clones produced IL-2 but failed to undergo apoptosis upon stimulation with PMA alone, whereas apoptosis was induced by a combination of IM and PMA. These results suggest that activation-induced cell death may require a higher level of CN activity than IL-2 production or may require another Ca2+-dependent pathway.
  AMER ASSOC IMMUNOLOGISTS, English, Scientific journal
  ISSN：0022-1767, Web of Science ID：WOS:A1995RC61000015
• INVOLVEMENT OF PROTEIN KINASE-C-EPSILON IN GLUCOCORTICOID-INDUCED APOPTOSIS IN THYMOCYTES　　　　　　　　　　　　　　　
  M IWATA; R ISEKI; K SATO; Y TOZAWA; Y OHOKA
  INTERNATIONAL IMMUNOLOGY, Volume：6, Number：3, First page：431, Last page：438, Mar. 1994, [Reviewed]
  Apoptosis is induced in immature thymocytes by physiological peak levels of glucocorticoid hormones, especially in murine and rat cells. Endogenous glucocorticoids may have some role in thymic selection. Glucocorticoid-induced thymocyte apoptosis appears to be dependent on protein kinase C (PKC), since it is inhibited by PKC inhibitors. PKC is a family of closely related enzymes, consisting of Ca2+-dependent (PKC-alpha, -betaI, -betaII, and -gamma) and Ca2+-independent (PKC-delta, -epsilon, -eta (L), -theta, -zeta, and -lambda) isozymes. In the present study, we analyzed the role of PKC in glucocorticoid-induced apoptosis in murine thymocytes and found that glucocorticoid selectively induces an increase in Ca2+-independent PKC activity in the particulate fraction of immature thymocytes but not in that of mature T cells. The increase as well as the apoptosis was inhibited by actinomycin D, cycloheximide, or the glucocorticoid receptor antagonist, RU 38486. Immunoblotting studies revealed the selective translocation of PKC-epsilon from the cytosolic fraction to the particulate fraction upon glucocorticoid treatment. These results suggest that glucocorticoid-induced apoptosis in immature thymocytes involves glucocorticoid receptor-mediated and selective activation of PKC-epsilon through de novo synthesis of macromolecules.
  OXFORD UNIV PRESS UNITED KINGDOM, English, Scientific journal
  DOI：https://doi.org/10.1093/intimm/6.3.431
  DOI ID：10.1093/intimm/6.3.431, ISSN：0953-8178, CiNii Articles ID：80007508525, PubMed ID：8186194, Web of Science ID：WOS:A1994NC47900011
• Isolation and Characterization of the gro ES and groEL genes of Bacillus subtilis Marburg　　　　　　　　　　　　　　　
  Yuzuru Tozawa; Hirofumi Yoshikawa; Fujio Kawamura; Hideo Takahashi; Mitsuhiro Itaya
  Bioscience, Biotechnology and Biochemistry, Volume：56, Number：12, First page：1995, Last page：2002, 1992, [Reviewed], [Lead]
  The complete set of gro ES and groEL gene homologues from Bacillus subtilis Marburg 168 was identified, cloned, and characterized. The nucleotide sequence indicated the presence of two open reading frames corresponding to the gro ES and groEL genes. The presumptive gro ES and GroEL proteins were calculated to be Polypeptides of 10,175 and 57,175 Da, respectively, and showed extensive sequence similarities with the known gro ES and GroEL proteins of Escherichia coli and Mycobacterium tuberculosis. A heat-inducible transcript initiated upstream of the groES coding region was identified by primer-extension analysis of in vivo transcripts, indicating that the two genes consist of an Operon. At least six heat-shock inducible proteins were identified in the cell extract of heat treated B. subtilis. Two proteins of 10 and 60 kDa overproduced in B. subtilis cells carrying a multi-copy groES and groEL plasmid were demonstrated to correspond to two out of the six heat-shock inducible proteins. The groES and groEL genes of B. subtilis were physically mapped on the 60° region of a 360° map and genetically mapped at the position of 40% linkage with the purB locus using PBS1 transduction of the groEL genes tagged with a chloramphenicol resistance (chlr) marker. © 1992, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.
  English, Scientific journal
  DOI：https://doi.org/10.1271/bbb.56.1995
  DOI ID：10.1271/bbb.56.1995, ISSN：1347-6947, CiNii Articles ID：110002691720, PubMed ID：1369494, SCOPUS ID：0027015407
• CHARACTERIZATION OF DIMETHYLSULFOXIDE REDUCTASE, A NOVEL MOLYBDO-ENZYME FROM PHOTOSYNTHETIC BACTERIA, Rhodobacter sphaeroides f.s. denitrificans　　　　　　　　　　　　　　　
  Sunao Yamazaki; Naoyuki Kobayashi; Akira Okubo; Toshio Sato; Yuzuru Tozawa; Etsuro Yoshimura; Hideyuki Kajiwara; Hisashi Hirano; Shozo Toda
  ANALYTICAL SCIENCES, Volume：7, First page：821, Last page：824, 1991, [Reviewed]
  Dimethylsulfoxide reductase (DMSO-R), a novel molybdoenzyme isolated from photosynthetic bacteria, Rhodobacter sphaeroides f.s. denitrificans, have been studied in relation to structure and activity. On limited proteolysis with trypsin and V8 protease, the DMSO-R activity increased more than twofold. Higher order structure was maintained by disulfide bonds even after nicking by proteases and was changed to be more susceptible to substrate.
  JAPAN SOC ANALYTICAL CHEMISTRY, English, Scientific journal
  ISSN：0910-6340, eISSN：1348-2246, Web of Science ID：WOS:000209357900224

• Model-Free Idealization: Adaptive Integrated Approach for Idealization of Ion Channel Currents (AI2)　　　　　　　　　　　　　　　
  Madoka Sato; Masanori Hariyama; Komiya Maki; Kae Suzuki; Yuzuru Tozawa; Hideaki Yamamoto; Ayumi Hirano-Iwata
  arXiv, 14 Feb. 2023
  English
  DOI：https://doi.org/10.48550/arXiv.2302.06792
  DOI ID：10.48550/arXiv.2302.06792
• Solubilization of natural rubber biosynthetic enzyme complexes from rubber particles of the Para rubber tree (Hevea brasiliensis) by the amphiphilic copolymer treatments　　　　　　　　　　　　　　　
  KHAN Nadia Nur Shazana Binti Abu Talib; 小島幸治; 山口晴彦; 三上智世; 廣森美樹; 和氣駿之; 宮城ゆき乃; 山下哲; 戸澤譲; 中山亨; 高橋征司
  日本植物生理学会年会(Web), Volume：64th, 2023
  J-Global ID：202302261107495062
• 高等植物のシス型プレニルトランスフェラーゼの活性制御におけるcPT-like proteinの役割　　　　　　　　　　　　　　　
  KHAN Nadia Nur Shazana Binti Abu Talib; 高橋朋宏; 廣森美樹; 山口真琴; 皆川知歩; 山家史大; 大場崇史; 山下哲; 戸澤譲; 山口晴彦; 宮城ゆき乃; 和氣駿之; 中山亨; 高橋征司
  Volume：32nd (CD-ROM), 2022
  J-Global ID：202202210485248037
• 脂肪滴およびゴム粒子の構造維持に関わる膜タンパク質の比較解析　　　　　　　　　　　　　　　
  中山隆司; 開琢海; 三輪幸祐; 廣森美樹; 和氣駿之; 山下哲; 戸澤譲; 山口晴彦; 宮城ゆき乃; 岩井雅子; 太田啓之; 中山亨; 高橋征司
  Volume：95th, 2022
  J-Global ID：202302287459103588
• Identification and functional analysis of interaction domains of rubber transferase protein complex from Hevea brasiliensis　　　　　　　　　　　　　　　
  NUR SHAZANA BINTI ABU TALIB KHAN Nadia; 廣森美樹; 和氣駿之; 山下哲; 戸澤譲; 山口晴彦; 宮城ゆき乃; 中山亨; 高橋征司
  日本農芸化学会大会講演要旨集(Web), Volume：2022, 2022
  ISSN：2186-7976, J-Global ID：202202225311742510
• Functional analysis for reconstituted rubber synthetic enzyme on nanodiscs　　　　　　　　　　　　　　　
  黒岩風; 西野輝; 廣森美樹; MANDAL Yasuko; 山口晴彦; 宮城ゆき乃; 山下哲; 高橋征司; 戸澤譲
  日本農芸化学会大会講演要旨集(Web), Volume：2021, 2021
  ISSN：2186-7976, J-Global ID：202102231726715483
• In vitro biosynthesis of isoprenoid polymers by trans-prenyltransferases identified from Manilkara zapota　　　　　　　　　　　　　　　
  三輪幸祐; 廣森美樹; 青木裕一; 和氣駿之; 小島幸治; 山下哲; 山口晴彦; 宮城ゆき乃; 戸澤譲; 中山亨; 高橋征司
  日本農芸化学会大会講演要旨集(Web), Volume：2021, 2021
  ISSN：2186-7976, J-Global ID：202102247197845981
• Establishment of a system for in vitro translation-coupled external protein introduction into lipid droplets from Chlamydomonas reinhardtii towards elucidation of lipid droplet transit sequence motifs.　　　　　　　　　　　　　　　
  開琢海; 廣森美樹; 和氣駿之; 山下哲; 戸澤譲; 山口晴彦; 宮城ゆき乃; 岩井雅子; 太田啓之; 中山亨; 高橋征司
  日本農芸化学会大会講演要旨集(Web), Volume：2021, 2021
  ISSN：2186-7976, J-Global ID：202102256839519018
• サポジラ(Manilkara zapota)由来trans型プレニルトランスフェラーゼの酵素機能解析　　　　　　　　　　　　　　　
  三輪幸祐; 廣森美樹; 青木裕一; 和氣駿之; 小島幸治; 山下哲; 山口晴彦; 宮城ゆき乃; 戸澤譲; 中山亨; 高橋征司
  Volume：31st (CD-ROM), 2021
  J-Global ID：202102215838782832
• パラゴムノキおよびグアユール由来タンパク質によるナノディスク上へのプレニルトランスフェラーゼ活性の再構成　　　　　　　　　　　　　　　
  黒岩風; 西野輝; 廣森美樹; モンドル慶子; 山口晴彦; 宮城ゆき乃; 山下哲; 高橋征司; 戸澤譲
  Volume：31st (CD-ROM), 2021
  J-Global ID：202102248299172157
• 無細胞翻訳系を用いた脂肪滴への外来酵素導入によるイソプレノイドポリマー生産　　　　　　　　　　　　　　　
  開琢海; 三輪幸祐; 廣森美樹; 和氣駿之; 山下哲; 戸澤譲; 山口晴彦; 宮城ゆき乃; 岩井雅子; 太田啓之; 中山亨; 高橋征司
  Volume：31st (CD-ROM), 2021
  J-Global ID：202102253026207253
• 完全インビトロ再構成系によるパラゴムノキ(Hevea brasiliensis)由来ポリイソプレン合成酵素の機能解析　　　　　　　　　　　　　　　
  黒岩風; 西野輝; 廣森美樹; 山口晴彦; 宮城ゆき乃; 山下哲; 高橋征司; 戸澤譲
  Volume：30th, 2020
  J-Global ID：202002246313093860
• Functional analysis for reconstituted rubber synthesis enzyme　　　　　　　　　　　　　　　
  黒岩風; 西野輝; 廣森美樹; MANDAL Yasuko; 山口晴彦; 宮城ゆき乃; 山下哲; 高橋征司; 戸澤譲
  日本農芸化学会大会講演要旨集(Web), Volume：2020, 2020
  ISSN：2186-7976, J-Global ID：202002249198737453
• Trial for in vitro reconstitution of rubber synthesis machinery　　　　　　　　　　　　　　　
  西野輝; 黒岩風; MANDAL Yasuko; 山口晴彦; 宮城ゆき乃; 山下哲; 高橋誠司; 戸澤譲
  日本農芸化学会大会講演要旨集(Web), Volume：2020, 2020
  ISSN：2186-7976, J-Global ID：202002284391363210
• 長鎖シス型プレニルトランスフェラーゼのパートナーであるNogo-B receptorファミリーのタンパク質構造について　　　　　　　　　　　　　　　
  矢内太朗; 今泉璃城; 高畑佳佑; 山口晴彦; 宮城ゆき乃; 竹下浩平; 戸澤譲; 高橋征司; 山下哲
  Volume：30th, 2020
  J-Global ID：202002235766899575
• Development of a hERG channel sensor for evaluation of drug side effects　　　　　　　　　　　　　　　
  渡辺恭也; 横田澪央; 加藤美生; 佐藤まどか; 但木大介; 小宮麻希; 山本英明; 井上遥香; 戸澤譲; 庭野道夫; 平野愛弓
  電子情報通信学会技術研究報告, Volume：119, Number：431(CPM2019 91-109), 2020
  ISSN：0913-5685, J-Global ID：202002258145057027
• Construction of an artificial cell membrane device and its application to a sensor for drug side effects　　　　　　　　　　　　　　　
  平野 愛弓; 但木 大介; 山浦 大地; 加藤 美生; 小宮 麻希; 井上 遥香; 戸澤 譲; 馬 騰; 山本 英明; 庭野 道夫
  Volume：119, Number：9, First page：23, Last page：26, 18 Apr. 2019
  Japanese
  ISSN：0913-5685, CiNii Articles ID：40021885351, CiNii Books ID：AN10012954
• パラゴムノキの天然ゴム生合成に関与するcis-prenyltransferaseとパートナータンパク質間の相互作用解析　　　　　　　　　　　　　　　
  今泉璃城; 山口晴彦; 宮城ゆき乃; 片岡邦重; 戸澤譲; 伏原和久; 高橋征司; 山下哲
  Volume：92nd, 2019
  J-Global ID：202002283188587712
• Quantitative assessment of drug side effect on hERG channels based on artificial bilayer lipid membranes and cell-free expression system　　　　　　　　　　　　　　　
  横田澪央; 加藤美生; 山浦大地; 常田悠介; 佐藤まどか; 但木大介; 小宮麻希; 山本英明; 井上遥; 戸澤譲; 庭野道夫; 平野愛弓
  電子情報通信学会技術研究報告, Volume：118, Number：461(CPM2018 101-120), 2019
  ISSN：0913-5685, J-Global ID：201902219959543041
• パラゴムノキの天然ゴム生合成マシナリを構成する因子の相互作用と機能相関　　　　　　　　　　　　　　　
  小島幸治; 山口真琴; 石井智樹; 廣森美樹; 和氣駿之; 山下哲; 戸澤譲; 山口春彦; 井之上ゆき乃; 伏原和久; 中山亨; 高橋征司
  日本植物生理学会年会(Web), Volume：60th, First page：435 (WEB ONLY), 2019
  Japanese
  J-Global ID：201902226936188372
• パラゴムノキの天然ゴム合成反応におけるゴム粒子の役割　　　　　　　　　　　　　　　
  小島幸治; 廣森美樹; 山家史大; 石井智樹; 和氣駿之; 山下哲; 戸澤譲; 山口晴彦; 井之上ゆき乃; 伏原和久; 中山亨; 高橋征司
  日本農芸化学会東北支部大会プログラム・講演要旨集, Volume：153rd, First page：51, 22 Sep. 2018
  Japanese
  J-Global ID：201802235741194636
• Stable formation of an artificial cell membrane sensor and its application to sensing drug side effects　　　　　　　　　　　　　　　
  平野 愛弓; 但木 大介; 山浦 大地; 荒田 航平; 大堀 健; 井上 遥香; 馬 騰; 山本 英明; 戸澤 譲; 庭野 道夫
  Volume：118, Number：9, First page：37, Last page：40, 19 Apr. 2018
  Japanese
  ISSN：0913-5685, CiNii Articles ID：40021538724, CiNii Books ID：AN10012954
• 非光合成性珪藻類葉緑体トリオースリン酸輸送体の基質特異性と紅藻類由来葉緑体の進化　　　　　　　　　　　　　　　
  神川 龍馬; Daniel Moog; 野澤 彰; 戸澤 譲
  Mar. 2018
• 微細加工チップ中に形成した脂質二分子膜への無細胞合成イオンチャネルの包埋と薬物副作用評価の定量　　　　　　　　　　　　　　　
  横田澪央; 加藤美生; 吉田美優; 山浦大地; 荒田航平; 但木大介; 山本英明; 戸澤譲; 庭野道夫; 平野愛弓; 平野愛弓
  Volume：67th, 2018
  J-Global ID：201802223449111951
• 微細加工チップ中に形成した脂質二分子膜への無細胞合成イオンチャネルの包埋と薬物副作用の定量　　　　　　　　　　　　　　　
  加藤美生; 吉田美優; 山浦大地; 荒田航平; 但木大介; 山本英明; 戸澤譲; 庭野道夫; 平野愛弓; 平野愛弓
  Volume：65th, 2018
  J-Global ID：201802240025100442
• 微細加工シリコンチップ中に再構成した脂質二分子膜における無細胞合成hERGチャネルの薬物副作用の定量　　　　　　　　　　　　　　　
  常田悠介; 加藤美生; 山浦大地; 荒田航平; 但木大介; 小宮麻希; 山本英明; 井上遥香; 戸澤譲; 庭野道夫; 平野愛弓; 平野愛弓
  Volume：79th, 2018
  J-Global ID：201802269986179413
• 脂質粒子におけるシス型プレニルトランスフェラーゼの発現と精製の試み　　　　　　　　　　　　　　　
  佐田京香; 山口晴彦; 宮城ゆき乃; 片岡邦重; 戸澤譲; 伏原和久; 高橋征司; 山下哲
  日本生化学会大会(Web), Volume：91st, First page：ROMBUNNO.1P‐109 (WEB ONLY), 2018
  Japanese
  J-Global ID：201902246837938521
• 天然ゴム生合成マシナリを構成するNgBRファミリータンパク質の機能解析　　　　　　　　　　　　　　　
  高畑佳佑; 今泉璃城; 山口晴彦; 宮城ゆき乃; 片岡邦重; 戸澤譲; 伏原和久; 高橋征司; 山下哲
  日本生化学会大会(Web), Volume：91st, First page：ROMBUNNO.1P‐108 (WEB ONLY), 2018
  Japanese
  J-Global ID：201902282333864882
• 大腸菌無細胞翻訳系を用いた天然ゴム合成酵素の再構成　　　　　　　　　　　　　　　
  小島幸治; 山下哲; 戸澤譲; 山口春彦; 井之上ゆき乃; 伏原和久; 中山亨; 高橋征司
  日本植物生理学会年会(Web), Volume：59th, First page：ROMBUNNO.P.408 (WEB ONLY), 2018
  Japanese
  J-Global ID：201802240902763361
• Evolutionary cross talk between the non-photosynthetic plastids and the cytosol of Nitzschia　　　　　　　　　　　　　　　
  Ryoma Kamikawa; Daniel Moog; Akira Nozawa; Yuzuru Tozawa
  The IVth Molecular Life of Diatoms, Jul. 2017, [Reviewed]
  English
• 支持脂質二重膜に再構成したイオンチャネルの膜内分子配向と分子分布　　　　　　　　　　　　　　　
  手老 龍吾; 福本 幸平; 鈴木 祐也; 吉田 美優; 平野 愛弓; 庭野 道夫; 野澤 彰; 戸澤 譲
  Volume：117, First page：37, Last page：40, 2017
• 電位依存性K⁺チャネル KAT1の支持脂質二重膜への再構成過程と分子配向 (ケミカルセンサ バイオ・マイクロシステム合同研究会 統合化バイオサーキットおよびエレクトロバイオロジー)　　　　　　　　　　　　　　　
  鈴木 祐也; 野澤 彰; 戸澤 譲; 手老 龍吾
  Volume：2016, Number：32, First page：5, Last page：10, 21 Dec. 2016
  Japanese
  CiNii Articles ID：40021052866, CiNii Books ID：AA1146975X
• パラゴムノキの天然ゴム合成酵素の分子解析
  山下哲; 山下哲; 山口晴彦; 和氣駿之; 山家史大; 青木裕一; 宮城ゆき乃; 伏原和久; 戸澤譲; 中山亨; 高橋征司
  イソプレノイド研究会例会講演要旨集, Volume：26th, First page：22, 20 Sep. 2016
  Japanese
  J-Global ID：201602228274455885
• 日本型イネから見出した4‐HPPD阻害型除草剤抵抗性遺伝子の有効性　　　　　　　　　　　　　　　
  山崎明彦; 関野景介; 山田祐司; 前田英郎; 村田和優; 加藤浩; 吉田均; 川岸万紀子; 廣瀬咲子; 谷口洋二郎; 川田元滋; 大島正弘; 戸澤譲
  育種学研究, Volume：18, First page：27, 21 Mar. 2016
  Japanese
  DOI：https://doi.org/10.1270/jsbbr.18.27
  DOI ID：10.1270/jsbbr.18.27, ISSN：1344-7629, J-Global ID：201602281603394823
• シロイヌナズナPAPS輸送体PAPST2の解析　　　　　　　　　　　　　　　
  名樂仁; 井上寛之; 佐々木孝行; 山本洋子; 戸澤譲; 澤崎達也; 野澤彰
  Volume：39th, 2016
  J-Global ID：201702264255851067
• 電位依存性K+チャネル KAT1の支持脂質二重膜への再構成過程と分子配向　　　　　　　　　　　　　　　
  鈴木 祐也; 野澤 彰; 戸澤 譲; 手老 龍吾
  Volume：CHS-16, First page：033, 2016
• 細菌によるトランス‐3‐ヒドロキシ‐L‐プロリン代謝経路の解明
  渡辺誠也; 谷本佳彰; 山内清司; 戸澤譲; 渡部保夫
  日本生化学会大会(Web), Volume：87th, First page：3P-180 (WEB ONLY), 2014
  Japanese
  J-Global ID：201502287720005863
• 膜輸送体タンパク質の完全インビトロ機能解析系　　　　　　　　　　　　　　　
  野澤 彰; 戸澤 譲
  Volume：72, First page：190, Last page：191, 2014
• 細菌によるコラーゲン由来ヒドロキシプロリン代謝に関する新知見
  渡辺誠也; 谷本佳彰; 西脇寿; 戸澤譲; 渡部保夫
  日本生化学会大会(Web), Volume：86th, First page：3T18P-04(1P-155) (WEB ONLY), 2013
  Japanese
  J-Global ID：201402256710473630
• 細菌由来の新規L‐ヒドロキシプロリン代謝経路の解明　　　　　　　　　　　　　　　
  渡辺誠也; 森本大地; 福森文康; 四宮博人; 西脇寿; 河田美幸; 笹井雄貴; 戸澤譲; 渡部保夫
  日本生化学会大会(Web), Volume：85th, First page：WEB ONLY 3T03-09, 2012
  Japanese
  J-Global ID：201402272214685428
• マダイI型インターフェロンの機能解析　　　　　　　　　　　　　　　
  太田史; 加藤千絵; 上田祐輔; 岩井俊治; 三浦智恵美; 三浦猛; 戸澤譲; 戸澤譲
  Volume：2011, 2011
  J-Global ID：201102208485910620
• マハタ組み換えインターフェロンの効果
  上田祐輔; 太田史; 伊藤克敏; 岩井俊治; 浦崎慎太郎; 山下浩史; 戸澤譲; 三浦智恵美; 三浦猛
  日本水産学会大会講演要旨集, Volume：2010, First page：52, 22 Sep. 2010
  Japanese
  J-Global ID：201002298368180124
• ppGpp biosynthesis systems and their physiological roles in plants　　　　　　　　　　　　　　　
  Tozawa Yuzuru; Kasai Koji
  Regulation of Plant Growth & Development, Volume：45, Number：2, First page：119, Last page：124, 2010
  For the last fifty years, unique signal molecule, ppGpp, has been received broad attention by a number of molecularbiologists. Recently, it has been revealed that the ppGpp biosynthesis system also exists in chloroplasts of photosynthetic eukaryotes including land plants. For elucidating physiological functions of the ppGpp in plants, currently some different approaches such as biochemical analysis of synthetic enzymes, genetic analysis of mutant plants, and in vitro reconstitution analysis of ppGpp-signaling in chloroplast extracts, have been utilized. This review overviews recent progress and perspectives in understanding of plant ppGpp-signaling systems.
  The Japanese Society for Chemical Regulation of Plants, Japanese
  DOI：https://doi.org/10.18978/jscrp.45.2_119
  DOI ID：10.18978/jscrp.45.2_119, ISSN：1346-5406, CiNii Articles ID：110008006480, CiNii Books ID：AA11550064
• Production of membrane proteins through the wheat-germ cell-free technology.　　　　　　　　　　　　　　　
  Volume：607, First page：213, Last page：218, 2010
  DOI：https://doi.org/10.1007/978-1-60327-331-2_17
  DOI ID：10.1007/978-1-60327-331-2_17
• Protein engineering accelerated by cell-free technology.　　　　　　　　　　　　　　　
  Volume：607, First page：85, Last page：99, 2010
  DOI：https://doi.org/10.1007/978-1-60327-331-2_9
  DOI ID：10.1007/978-1-60327-331-2_9
• ppGpp biosynthesis systems and their physiological roles in plants　　　　　　　　　　　　　　　
  Tozawa Yuzuru; Kasai Koji
  The Japanese Society for Chemical Regulation of PlantsRegulation of Plant Growth & Development, Volume：45, Number：2, First page：119, Last page：124, 2010
  For the last fifty years, unique signal molecule, ppGpp, has been received broad attention by a number of molecularbiologists. Recently, it has been revealed that the ppGpp biosynthesis system also exists in chloroplasts of photosynthetic eukaryotes including land plants. For elucidating physiological functions of the ppGpp in plants, currently some different approaches such as biochemical analysis of synthetic enzymes, genetic analysis of mutant plants, and in vitro reconstitution analysis of ppGpp-signaling in chloroplast extracts, have been utilized. This review overviews recent progress and perspectives in understanding of plant ppGpp-signaling systems.
  The Japanese Society for Chemical Regulation of Plants, Japanese
  DOI：https://doi.org/10.18978/jscrp.45.2_119
  DOI ID：10.18978/jscrp.45.2_119, ISSN：1346-5406, CiNii Articles ID：110008006480, CiNii Books ID：AA11550064
• Production of membrane proteins through the wheat-germ cell-free technology.　　　　　　　　　　　　　　　
  Humana Press Inc., Totowa, NJ.Methods in Molecular Biology, Volume：607, First page：213, Last page：218, 2010
  DOI：https://doi.org/10.1007/978-1-60327-331-2_17
  DOI ID：10.1007/978-1-60327-331-2_17
• Protein engineering accelerated by cell-free technology.　　　　　　　　　　　　　　　
  Humana Press Inc., Totowa, NJ.Methods in Molecular Biology, Volume：607, First page：85, Last page：99, 2010
  DOI：https://doi.org/10.1007/978-1-60327-331-2_9
  DOI ID：10.1007/978-1-60327-331-2_9
• ゴカイ類が有する有機物分解能の分子生物学的解析　　　　　　　　　　　　　　　
  伊藤克敏; 野崎真奈; 太田史; 戸澤譲; 三浦智恵美; 三浦猛
  Volume：2010, 2010
  J-Global ID：201002228400588590
• Functional analysis of a novel ppGpp synthetase, YwaC, and regulatory mechanism for the dimerization of ribosome, in Bacillus subtilis　　　　　　　　　　　　　　　
  Kazumi Tagami; Kenta Masuda; Marie Maehashi; Yoshitaka Hirohata; Masaki Yoshida; Haruko Kuroiwa; Tsuneyoshi Kuroiwa; Hideaki Nanamiya; Yuzuru Tozawa; Shenghao Liu; Yasushi Kageyama; Katsutoshi Ara; Katsuya Ozaki; Fujio Kawamura
  GENES & GENETIC SYSTEMS, Volume：84, Number：6, First page：455, Last page：455, Dec. 2009
  GENETICS SOC JAPAN, English, Summary international conference
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000277071300090
• [Membrane protein production using wheat germ cell-free system].　　　　　　　　　　　　　　　
  Nozawa A; Tozawa Y; Sawasaki T; Endo Y
  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, Volume：54, Number：12 Suppl, First page：1443, Last page：1447, Sep. 2009, [Reviewed]
  Japanese
  ISSN：0039-9450, CiNii Articles ID：40016800815, CiNii Books ID：AN00140437, PubMed ID：21089567
• コムギ胚芽無細胞系を用いた膜タンパク質生産　　　　　　　　　　　　　　　
  Volume：54, Number：12, First page：1443, Last page：1447, 2009
• Functional Analysis of Membrane Proteins by Cell-free Translation System　　　　　　　　　　　　　　　
  TOZAWA Yuzuru; NOZAWA Akira; GENJI Takahisa
  KAGAKU TO SEIBUTSU, Volume：47, Number：2, First page：98, Last page：103, 2009
  Japan Society for Bioscience, Biotechnology, and Agrochemistry, Japanese
  DOI：https://doi.org/10.1271/kagakutoseibutsu.47.98
  DOI ID：10.1271/kagakutoseibutsu.47.98, ISSN：0453-073X, CiNii Articles ID：10026434746, CiNii Books ID：AN00037573
• Functional Analysis of Membrane Proteins by Cell-free Translation System　　　　　　　　　　　　　　　
  Volume：47, Number：2, First page：98, Last page：103, 2009
• Functional analysis of a novel ppGpp synthetase, YwaC, in Bacillus subtilis　　　　　　　　　　　　　　　
  Kazumi Tagami; Tetsuya Wada; Masaki Yoshida; Haruko Kuroiwa; Tsuneyoshi Kuroiwa; Hideaki Nanamiya; Yuzuru Tozawa; Fujio Kawamura
  GENES & GENETIC SYSTEMS, Volume：83, Number：6, First page：513, Last page：513, Dec. 2008
  GENETICS SOC JAPAN, English, Summary international conference
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000265228400140
• Metabolic Engineering of The Phenylpropanoid Pathway in Arabidopsis thaliana Using Foreign Gene　　　　　　　　　　　　　　　
  Yun Choong-Soo; Matsuda Fumio; Yamamoto Tomio; Nozawa Akira; Saito Kazuki; Tozawa Yuzuru
  Plant and Cell Physiology Supplement, Volume：2008, Number：0, First page：142, Last page：142, 2008
  The first step of phenylpropanoid pathway in plants starts from phenylalanine. In plant cells, phenylalanine is converted to courmaric acid via cinnamic acid, then courmaric acid branches to various secondary metabolites. One more important branch point of this pathway is flavanone that is converted to flavone or isoflavone. In this study, we attempted to alter this pathway by using tyrosine ammonia-lyase (TAL), which directly converts tyrosine to coumaric acid instead of phenylalanine, and parsley flavone synthase (FNS). Expressions of the introduced genes were examined by RT-PCR or RNA gel-blot analysis, and the resulted transgenic plants were analyzed by LC-MS and HPLC to investigate profiles of their secondary metabolites. We observed accumulations of specific flavonoids that were dependent on the expression of transgene, TAL or FNS. We thus confirmed the in vivo functions of each trasgene that can alter the biosynthetic pathway of phenylpropanoids in <I>Arabidopsis</I>.
  日本植物生理学会
  CiNii Articles ID：130006992950
• Regulation of transcription of an rrn operon by relA, yjbM and ywaC in Bacillus subtilis　　　　　　　　　　　　　　　
  Murakami Kana; Natori Yousuke; Nanamiya Hideaki; Tozawa Yuzuru; Kawamura Fujio
  GENES & GENETIC SYSTEMS, Volume：82, Number：6, First page：548, Last page：548, Dec. 2007
  GENETICS SOC JAPAN, English, Summary international conference
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000254238200204
• Improvement of analysis system for plant membrane proteins by using cell-free protein synthesis system.　　　　　　　　　　　　　　　
  Akira Nozawa; Takuji Miyata; Yuzuru Tozawa
  PLANT AND CELL PHYSIOLOGY, Volume：48, First page：S88, Last page：S88, 2007
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000245922700347
• Functional analysis of arogenate dehydratase of rice by means of wheat-embryo cell-free protein expression system　　　　　　　　　　　　　　　
  Koji Kasai; Yuki Obayashi; Tetsuya Yamada; Takuya Kanno; Kyo Wakasa; Yuzuru Tozawa
  PLANT AND CELL PHYSIOLOGY, Volume：48, First page：S219, Last page：S219, 2007
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000245922701353
• Functional roles of plastid SIG1 for gene expression of photosystem I components　　　　　　　　　　　　　　　
  Tozawa Yuzuru; Teraishi Masayoshi; Sasaki Tadamasa; Sonoike Kintake
  Plant and Cell Physiology Supplement, Volume：2007, Number：0, First page：73, Last page：73, 2007, [Lead, Corresponding]
  Nuclear-encoding sigma factors confer promoter specificity on bacterial-type RNA polymerase in plant chloroplasts. To date, 6 SIG genes encoding sigma factor have been identified in rice and Arabidopsis. To clarify functions of one of the SIG genes, SIG1, we isolated and characterized two allelic mutants of rice SIG1, in which SIG1 is disrupted by insertion of a retrotransposon, Tos17. The mutants impaired in SIG1 were fertile, however, they showed one-third reduction of chlorophyll content in mature leaves. Semi-comprehensive analysis for chloroplast genes revealed that accumulation of psaA operon in mutant heterozygotes reduced to ~30% compared to that in wild type. The reduction of PSI complex resulted in 70% reduction of light-reaction activity. These results demonstrated that SIG1 takes significant roles in maintenance of photosynthetic activity of PSI in matured chloroplasts in leaves by regulating transcription of the genes encoding photosystem I apparatus.
  The Japanese Society of Plant Physiologists
  DOI ID：10.14841/jspp.2007.0.073.0, ISSN：0032-0781, CiNii Articles ID：130006990401, Web of Science ID：WOS:000245922700135
• Function analysis of relA gene and it's paralogous genes, yjbM and ywaC, in Bacillus subtilis.　　　　　　　　　　　　　　　
  Kana Murakami; Yousuke Natori; Makiko Sato; Hideaki Nanamiya; Fujio Kawamura; Yuzuru Tozawa
  GENES & GENETIC SYSTEMS, Volume：81, Number：6, First page：442, Last page：442, Dec. 2006
  GENETICS SOC JAPAN, English, Summary international conference
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000245138300140
• Chloroplast ppGpp synthesis system　　　　　　　　　　　　　　　
  PROTEIN, NUCLEIC ACID AND ENZYME, Volume：50, Number：14, First page：1860, 2005
• The Wheat Germ Cell-Free Expression System　　　　　　　　　　　　　　　
  Humana Press Inc.Methods in Molecular Biology, Volume：310, First page：131, Last page：144, 2005
• 葉緑体のppGpp合成系　　　　　　　　　　　　　　　
  Volume：50, Number：14, First page：1860, 2005
• Functional analysis of rice virescent-2 gene essential for chloroplast development　　　　　　　　　　　　　　　
  H Sugimoto; K Kusumi; A Yoshimura; S Kikuchi; Y Tozawa; K Iba
  PLANT AND CELL PHYSIOLOGY, Volume：45, First page：S208, Last page：S208, 2004
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000220592700820
• "Magic spot" synthase in chloroplasts　　　　　　　　　　　　　　　
  Y Tozawa
  SEIKAGAKU, Volume：75, Number：7, First page：600, Last page：604, Jul. 2003
  JAPANESE BIOCHEMICAL SOC, Japanese
  ISSN：0037-1017, Web of Science ID：WOS:000184465400005
• Cell-free Protein Synthesis by Wheat Germ Extracts　　　　　　　　　　　　　　　
  戸澤 譲; 澤崎 達也; 遠藤 弥重太
  Volume：45, Number：1, First page：3, Last page：8, 2003
  DOI：https://doi.org/10.5940/jcrsj.45.3
  DOI ID：10.5940/jcrsj.45.3, ISSN：0369-4585
• For the regulation and utilization of primary and secondary metabolisms in the tryptophan biosynthetic pathways　　　　　　　　　　　　　　　
  Wakasa Kyo; Tozawa Yuzuru
  Regulation of Plant Growth & Development, Volume：38, Number：2, First page：249, Last page：254, 2003
  The Japanese Society for Chemical Regulation of Plants, Japanese
  DOI：https://doi.org/10.18978/jscrp.38.2_249
  DOI ID：10.18978/jscrp.38.2_249, ISSN：1346-5406, CiNii Articles ID：110001845961, CiNii Books ID：AA11550064
• Cell-free Protein Synthesis by Wheat Germ Extracts　　　　　　　　　　　　　　　
  Yuzuru TOZAWA; Tatsuya SAWASAKI; Yaeta ENDO
  Nihon Kessho Gakkaishi, Volume：45, Number：1, First page：3, Last page：8, 2003
  With the sequencing of the genomes of various species, attention has turned to the structure, properties, and functional activities of proteins. However, rapid progress in the area of proteomics is premised on the availability of sufficient amounts of a large number of proteins. Here we described a novel cell-free system from wheat embryos for the high-throughput screening/ synthesis of gene products. Our system should open up many possibilities in the post-genome era.
  The Crystallographic Society of Japan, Japanese
  DOI：https://doi.org/10.5940/jcrsj.45.3
  DOI ID：10.5940/jcrsj.45.3, ISSN：0369-4585, CiNii Articles ID：10012821848, CiNii Books ID：AN00188364
• “Magic spot” synthase in chloroplasts　　　　　　　　　　　　　　　
  Volume：75, First page：600, Last page：604, 2003
• Expression analysis of chloroplast proteins in virescent mutants (v(1), v(3)) of rice　　　　　　　　　　　　　　　
  K Kusumi; T Kouno; H Sugimoto; Y Tozawa; A Yoshimura; S Kikuchi; K Iba
  PLANT AND CELL PHYSIOLOGY, Volume：44, First page：S57, Last page：S57, 2003
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000181914300223
• Analysis of rice transformants with the mutated anthranilate synthase gene driven by five different promoters for the development of selection marker　　　　　　　　　　　　　　　
  A Komatsu; A Yoshikawa; H Hasegawa; M Kawagishi-Kobayashi; Y Nishizawa; K Sugimoto; Y Tozawa; K Wakasa
  PLANT AND CELL PHYSIOLOGY, Volume：43, First page：S121, Last page：S121, 2002
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000174726400437
• The analysis of the function of the gene for the chloroplast RNApolymerase σ factor from Oryza sative　　　　　　　　　　　　　　　
  TSUNOYAMA Yuichi; TOZAWA Yuzuru; WAKASA Kyo
  Volume：39, First page：S95, Last page：S95, May 1998
  English
  ISSN：0032-0781, CiNii Articles ID：10003751857, CiNii Books ID：AA0077511X
• Roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation　　　　　　　　　　　　　　　
  Volume：14, First page：88, Last page：91, 1996
• Roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation　　　　　　　　　　　　　　　
  EXPERIMENTAL MEDICINE, Volume：14, First page：88, Last page：91, 1996
• INVOLVEMENT OF PROTEIN-KINASE C-EPSILON IN GLUCOCORTICOID-INDUCED APOPTOSIS IN MURINE THYMOCYTES　　　　　　　　　　　　　　　
  M IWATA; R ISEKI; Y TOZAWA; K SATO; Y ZHAO; Y OHOKA
  FASEB JOURNAL, Volume：8, Number：5, First page：A786, Last page：A786, Mar. 1994
  FEDERATION AMER SOC EXP BIOL, English, Summary international conference
  ISSN：0892-6638, Web of Science ID：WOS:A1994ND19701169
• INVOLVEMENT OF CALCIUM-INDEPENDENT LIPID-STIMULATED KINASE IN GLUCOCORTICOID-INDUCED APOPTOSIS IN THYMOCYTES　　　　　　　　　　　　　　　
  M IWATA; R ISEKI; Y TOZAWA; K SATO
  JOURNAL OF IMMUNOLOGY, Volume：150, Number：8, First page：A31, Last page：A31, Apr. 1993
  AMER ASSOC IMMUNOLOGISTS, English, Summary international conference
  ISSN：0022-1767, Web of Science ID：WOS:A1993KX95600170

• Chemical Genomics: Reviews and protocols. Methods in Molecular Biology　　　　　　　　　　　　　　　
  2005
• Chemical Genomics: Reviews and protocols. Methods in Molecular Biology　　　　　　　　　　　　　　　
  Humana Press, 2005
• 日本結晶学会誌　　　　　　　　　　　　　　　
  2003

• Japanese Society for Plant Cell and Molecular Biology

• 無細胞タンパク質合成系の保健衛生および畜産分野への応用　　　　　　　　　　　　　　　
  2005 - 2005

• Molecular dissection of plant specialized metabolism machineries　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (S), 12 Apr. 2023 - 31 Mar. 2028
  Tohoku University
  Grant amount(Total)：203710000, Direct funding：156700000, Indirect funding：47010000
  Grant number：23H05470
• 未知ストリゴラクトン代謝経路におけるHIS1/HSLファミリーの機能的役割の解明　　　　　　　　　　　　　　　
  01 Apr. 2022 - 31 Mar. 2025
  Grant amount(Total)：17420000, Direct funding：13400000, Indirect funding：4020000
  Grant number：22H02270
• Understanding the core structures and dynamics of membrane protein complexes related in productions of valuable compounds from plants　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), 01 Apr. 2021 - 31 Mar. 2024
  Kanazawa University
  Grant amount(Total)：17810000, Direct funding：13700000, Indirect funding：4110000
  Grant number：21H02115
• 固液界面機能と脂質二重膜を用いた人工生体膜反応場の構築　　　　　　　　　　　　　　　
  01 Apr. 2020 - 31 Mar. 2024
  Grant amount(Total)：17550000, Direct funding：13500000, Indirect funding：4050000
  Grant number：20H02690
• 単一イオンチャネル分子／バイオ二次元物質ハイブリッド膜の機能解析と応用　　　　　　　　　　　　　　　
  01 Apr. 2019 - 31 Mar. 2023
  Grant amount(Total)：45500000, Direct funding：35000000, Indirect funding：10500000
  Grant number：19H00846
• Study on molecular regulatory mechanisms in thermo-sensitive male sterility of rice based on cell-free translation system　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 01 Apr. 2019 - 31 Mar. 2022
  Tozawa Yuzuru, Saitama University
  Grant amount(Total)：4290000, Direct funding：3300000, Indirect funding：990000
  The rapid global warming has a great impact on the production of major cereals such as rice, and the high-temperature conditions during the flowering season not only deteriorate the quality of seeds but also sometimes cause sterility. This problem of high-temperature sterility is one of the important issues to be solved in order to maintain food production in the future. The principal investigators predicted transcription factors that it might be involved in high-temperature male sterility in rice. To conduct a functional analysis of these transcription factors at the protein level, the candidates were synthesized and purified as a full-length protein by using a cell-free translation system based on the wheat germ extract. It has been confirmed that one transcription factor, OsMyb80, shows specific binding to the regulatory region of the gene CYP703, which is essential for pollen formation.
  Grant number：19K05822
• Development of screening system for high-affinity artificial ligand against memblane proteins　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Research (Exploratory), 30 Jun. 2017 - 31 Mar. 2019
  Nemoto Naoto; NAKAI JYUNICHI, Saitama University
  Grant amount(Total)：6500000, Direct funding：5000000, Indirect funding：1500000
  Over the past 20-year, bio-pharmaceuticals as molecular target drugs, especially antibody drugs have been given some effective ways to treat difficult disorder diseases (i.e., cancer). On the other hand, any antibodies against G protein- coupled receptors (GPCR) which are around 50% of drug targets, have not been developed because high titer of antibodies can not be obtained. Recently, peptide aptamers have been attracting much attention because of its thermal stability and low-cost to overcome antibody’s problems. In this study, we have developed a novel method to select some peptide aptamers against GPCR using cDNA display method that is one of powerful evolutionary molecular engineering tools.
  Grant number：17K19471
• Elucidation of molecular mechanism in ATP-depending translation activity in plant seed cells　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Exploratory Research, 01 Apr. 2016 - 31 Mar. 2019
  Tozawa Yuzuru; Suzuki Kakeru, Saitama University
  Grant amount(Total)：3640000, Direct funding：2800000, Indirect funding：840000
  In this study, we have established a new plant cell-free translation system based on rice callus extracts. We revealed that the rice cell-free system allows efficient protein expression in the absence of GTP. We confirmed generation of low level GTP from GMP, which is degradation product of mRNA by endogenous nuclease. On the other hand, By using polyuridylic acid (polyU), which contains only uridine but not guanine nucleotide, as a template, we demonstrated that translation elongation activity of the system is not affected by the lack of GTP in the reaction. This GTP-independence in the translation elongation activity is different from yeast cell-free system, and seems to be unique to plant seed cell system.
  Grant number：16K14665
• Analysis of effects of lipids on function and structure of membrane proteins　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 01 Apr. 2015 - 31 Mar. 2018
  Nozawa Akira; TOZAWA Yuzuru, Ehime University
  Grant amount(Total)：5070000, Direct funding：3900000, Indirect funding：1170000
  In this study, by using mitochondrial carriers and a lipid, cardiolipine, which is known to interact with the mitochondrial carrier, as a model membrane protein-lipid system, we clarified roles of lipids against the function and structure of the membrane protein. In this study, the effect of cardiolipin on transport activity of yeast and malaria parasite mitochondrial carriers was analyzed. As a result, it was clarified that addition of cardiolipin increases the transport activity of the mitochondrial carriers localized in mitochondrial membrane, but such a effect was not observed in the mitochondrial carriers present in other membrane systems.
  Grant number：15K07006
• Cooperative effects of natural-derived lipids and membrane proteins in lipid bilayer membranes　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), 01 Apr. 2015 - 31 Mar. 2018
  Tero Ryugo; FUKUMOTO Kohei; SUZUKI Yuya; NIIYAMA Yuya; OKAMOTO Yoshiaki, Toyohashi University of Technology
  Grant amount(Total)：17680000, Direct funding：13600000, Indirect funding：4080000
  This project aimed to elucidate the active state created by a plurality of membrane proteins and surrounding lipid molecules, and relating phenomena using a cell membrane model system. We reconstructed ion channel proteins that were obtained from cultured cells and cell-free protein synthesis system, in supported lipid bilayers (SLBs). Distribution and molecular structures of lipids and membrane proteins were observed by fluorescence microscopy and atomic force microscopy. We propose a model to explain the mechanism of reconstitution by clarifying the role of cell membrane derived components in SLB. We revealed that the role of intra-membrane microdomains in the reconstitution process. It is revealed that the molecular orientation of the channel protein expressed by cell-free synthesis varies depending on the reconstitution process.
  Grant number：15H03768
• Establishment of post-translational modification system in the cell free protein system　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Young Scientists (B), 01 Apr. 2013 - 31 Mar. 2017
  Izumi Atsushi; YUZURU TOZAWA
  Grant amount(Total)：4030000, Direct funding：3100000, Indirect funding：930000
  We introduced a simple method to synthesize and purify a membrane-associated protein in the wheat germ cell free system as a model production system. We used ferrochelatase from Saccharomyces cerevisiae, catalyzing the insertion of ferrous ion into the tetrapyrrole center of protoporphyrin IX, as a model protein. The luminol-horse radish peroxidase assay indicated that the active ferrochelatase was only synthesized in the presence of assolectin liposomes. The Accudenz density gradient centrifugation experiment indicated that ferrochelatase clearly localized at the liposome fraction and the complex of the synthesized protein with liposomes can be simply separated from soluble protein fraction by a single step centrifugation.
  Grant number：25850071
• 細胞内物質代謝系とオルガネラ膜輸送体の共進化　　　　　　　　　　　　　　　
  01 Apr. 2014 - 31 Mar. 2016
  Grant amount(Total)：7670000, Direct funding：5900000, Indirect funding：1770000
  Grant number：26117717
• Functional analysis of transporters regulating translocation of energy metabolites in rice organelle　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 01 Apr. 2012 - 31 Mar. 2015
  NOZAWA Akira; TOZAWA Yuzuru, Ehime University
  Grant amount(Total)：4810000, Direct funding：3700000, Indirect funding：1110000
  We improved a method for functional analysis of transporter proteins based on liposome-supplemented cell-free system. In the new system, detection range of transport activity against non-specific background activity was improved by up-regulated synthetic efficiency of membrane proteins in cell-free reaction. By using this method, we analyzed several transporter proteins localized in plant organelle. Substrate specificity of sugar-phosphate transporters in rice and iceplant plastids and sulfate metabolite transporter in Arabidopsis plastids.
  Grant number：24580094
• Characterization of plant stringent regulation by introducing artificial ppGpp-signal system　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 01 Apr. 2012 - 31 Mar. 2015
  TOZAWA Yuzuru; OGAWA Atsushi
  Grant amount(Total)：5460000, Direct funding：4200000, Indirect funding：1260000
  In this study, we have performed experiments for clarifying function of ppGpp in plant chloroplasts. By establishing in vitro translation system from isolated chloroplasts, we have demonstrated that chloroplast translation activity is inhibited by ppGpp. Further, we have shown that activity of guanylate kinase, a key enzyme for GTP biosynthesis in chloroplasts, is potently inhibited by ppGpp. We have thus proven that ppGpp regulates chloroplast translation and GTP biosynthesis in plants. Moreover, we have established cyanobacterial genetic system that enables induction of protein-expression by theophylline administration based on riboswithch system. The system has also been applied to transformation of tobacco chloroplast genome.
  Grant number：24570054
• 細胞内物質代謝系の統合と変遷に伴うミトコンドリア輸送体の獲得と共進化　　　　　　　　　　　　　　　
  01 Apr. 2012 - 31 Mar. 2014
  Grant amount(Total)：5720000, Direct funding：4400000, Indirect funding：1320000
  Grant number：24117516
• Study on stringent response in plant chloroplasts　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 2009 - 2011
  TOZAWA Yuzuru; YUKAWA Yasushi; NOMURA Yuhta, Ehime University
  Grant amount(Total)：4680000, Direct funding：3600000, Indirect funding：1080000
  Chloroplasts are derived from symbiosis of photosynthetic bacteria, and have ppGpp-mediated signal transduction system. In order to clarify the functional roles of ppGpp-signal in chloroplasts, we have investigated functions of ppGpp in the protein synthesis. Here we have established in vitro translation system based on extracts of isolated pea chloroplasts, and demonstrated that ppGpp directly regulates protein synthesis activity of chloroplasts in vitro.
  Grant number：21570047
• Calcium signal transduction network in plant innate immunity　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research on Innovative Areas (Research a proposed research project), 2008 - 2010
  NAKAHIRA Yoichi; YUKAWA Yasushi; SHIINA Takashi; YOSHIOKA Hirofumi; SUGIURA Masahiro; TOZAWA Yuzuru; KUCHITU Kazuyuki, Kyoto Prefectural University
  Grant amount(Total)：31980000, Direct funding：24600000, Indirect funding：7380000
  Mitochondria and chloroplasts are not only energy source for eukaryotic cells, but also play additional important roles to modulate cellular functions. For example, mammal mitochondria contribute to the initiation of apoptosis, diseases and aging. Plant chloroplast also serves several important functions in plant immunity, including biosynthesis of salicylic acid (SA) and the generation of reactive oxygen species (ROS), key signaling molecules in plant immune response. Interestingly, previous studies have shown that chloroplasts contain proteins involved in plant immune responses. Furthermore, several pathogen effectors target chloroplasts to suppress chloroplast-derived defense signals. Therefore, it is anticipated that chloroplasts play a role in plant innate immunity. However, how chloroplasts sense pathogen signals and control immune signaling network remains unclear. This study illustrates a previously unknown crosstalk between chloroplasts and innate immunity through chloroplast Ca^<2+> signaling in plants.
  Mitochondrial Ca^<2+> homeostasis plays a key role in apoptosis by switching Ca^<2+>-induced release of cytochrome C (caspase cofactor). However, very little is known about Ca^<2+> homeostasis in chloroplasts. Here, we report that pathogen associated molecular pattern (PAMP) signals induce a rapid increase in the stromal Ca^<2+> concentration. Pharmacological studies demonstrate that the cytoplasmic Ca^<2+> signal is transferred to the chloroplasts and induces the stromal Ca^<2+> transient. We further demonstrate that chloroplast-localized putative Ca^<2+> binding protein, CAS is involved in the generation of stromal Ca^<2+> signals. These studies suggest a novel mechanism relaying PAMP signals into chloroplasts at the beginning of the immune process.
  The PAMP-induced stromal Ca^<2+> signaling may control or activate the innate immune response. Thus, we examined the role of CAS in plant innate immunity. Remarkably, CAS deficient plants (cas-1) are compromised in broad range of innate immune responses including PAMP-induced basal defense and R-gene mediated hypersensitive cell death. It is unlikely that the compromised immunity of cas-1 plants is caused by limited cellular resources, since CAS deficient mutant lines exhibit normal phenotype and photosynthetically competent. Furthermore, a comprehensive analysis of plant hormone dynamics demonstrated that CAS is specifically required for PAMP-induced accumulation of SA, a key signal molecule in plant immune responses. These results suggest that chloroplast protein CAS acts as a positive regulator of plant innate immunity through SA-dependent signaling.
  CAS regulates SA biosynthesis most likely at transcriptional level. It is therefore anticipated that chloroplasts release retrograde signals that control defense gene expression during immune responses. PAMP induces expression of numerous defense related genes in nucleus. Using a genome wide transcriptome analysis we show that CAS is indispensable for the global induction of defense gene expression before SA biosynthesis starts, suggesting that CAS is involved in the chloroplast-to-nucleus retrograde signaling and mediates chloroplast control of plant innate immunity. Chloroplast-derived ^1O_2^- signaling has been implicated with the control of stress defense-related gene expression. Here we show that most genes downregulated in CAS deficient plants were enriched for ^1O_2^- responsive genes, providing evidence for a role of CAS in ^1O_2^- signaling to control nuclear-encoded defense genes.
  Collectively this study illustrates previously unknown mechanisms that coordinate chloroplast functions with nuclear-encoded defense gene expression. Upon infection, PAMP signals are relayed to chloroplasts through the cytoplasmic Ca^<2+>-induced stromal Ca^<2+> signaling pathway. This study further implicates a role of ^1O_2^- retrograde signaling as underlying mechanisms of chloroplast control of plant innate immunity. Chloroplast protein CAS plays a critical role in both processes. Identification of the chloroplast-dependent immune pathway may provide a novel strategy for the disease control of plants.
  Grant number：20200060
• 無細胞翻訳系を応用した植物膜輸送体の機能解析　　　　　　　　　　　　　　　
  2008 - 2009
  Grant amount(Total)：4400000, Direct funding：4400000
  Grant number：20053016
• Molecular mechanisms of the response of photosynthesis via redox regulation of translation　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 2007 - 2008
  NISHIYAMA Yoshitaka; TOZAWA Yuzuru; HISABORI Toru
  Grant amount(Total)：4550000, Direct funding：3500000, Indirect funding：1050000
  Grant number：19570043
• Study on functional roles of calcium-dependent stringent factor in plant chloroplasts　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 2007 - 2008
  TOZAWA Yuzuru; NANAMIYA Hideaki, Ehime University
  Grant amount(Total)：4420000, Direct funding：3400000, Indirect funding：1020000
  Grant number：19570042
• 無細胞蛋白質合成技術を応用した植物膜輸送タンパク質の機能解析　　　　　　　　　　　　　　　
  2006 - 2007
  Grant amount(Total)：5200000, Direct funding：5200000
  Grant number：18056016
• Molecular mechanisms of the response of photosynthesis to oxidative stress　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 2005 - 2006
  NISHIYAMA Yoshitaka; TOZAWA Yuzuru, Ehime University
  Grant amount(Total)：3500000, Direct funding：3500000
  Ph00otoinhibition of photosystem II (PSII) is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has dual effects on PSII ; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein. Moreover, analysis of polysomes has determined that a primary target for inhibition by ROS is the elongation step of translation. Investigations using a cyanobacterial translation system in vitro have revealed that elongation factor G might be the primary target, within the translational machinery, of inhibition by ROS. Here we present a new paradigm for the molecular action of ROS in photoinhibition.
  Grant number：17570040