Kyowa Kako Co.

Machida, Japan

Kyowa Kako Co.

Machida, Japan
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PubMed | Ludwig Maximilians University of Munich, University of Florida, University of Tartu, Kyowa Kako Co. and University of Connecticut
Type: | Journal: Archaea (Vancouver, B.C.) | Year: 2017

Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in


Kimura T.,Hokkaido University | Joti Y.,Japan Synchrotron Radiation Research Institute | Shibuya A.,RIKEN | Song C.,RIKEN | And 9 more authors.
Nature Communications | Year: 2014

Emerging X-ray free-electron lasers with femtosecond pulse duration enable single-shot snapshot imaging almost free from sample damage by outrunning major radiation damage processes. In bioimaging, it is essential to keep the sample close to its natural state. Conventional high-resolution imaging, however, suffers from severe radiation damage that hinders live cell imaging. Here we present a method for capturing snapshots of live cells kept in a micro-liquid enclosure array by X-ray laser diffraction. We place living Microbacterium lacticum cells in an enclosure array and successively expose each enclosure to a single X-ray laser pulse from the SPring-8 Angstrom Compact Free-Electron Laser. The enclosure itself works as a guard slit and allows us to record a coherent diffraction pattern from a weakly-scattering submicrometre-sized cell with a clear fringe extending up to a 28-nm full-period resolution. The reconstructed image reveals living whole-cell structures without any staining, which helps advance understanding of intracellular phenomena.© 2014 Macmillan Publishers Limited. All rights reserved.


Yokoyama K.,Yamaguchi University | Jinnai K.,Yamaguchi University | Jinnai K.,Kyowa Kako Co. | Sakiyama Y.,Yamaguchi University | And 2 more authors.
Applied Soil Ecology | Year: 2012

This study was conducted to analyze the relative contribution of nitrifying microbial groups in tea field soils with relatively neutral pH where high ammonia availability was expected after heavy fertilization. The nitrification potential in tea field soils supplied with organic fertilizers (OF) or chemical fertilizers (CF) was determined by the chlorate-inhibition method. The response of the potential to the net ammonia concentration, which was employed as an index of ammonia availability, was tested by loading various levels of ammonium sulfate to soil slurries. The potential of tolerance to 10Pa acetylene was highlighted. The acetylene-tolerant nitrification potential occurred all year at a significant level. It was stimulated by ammonia availability of more than 10-5.5molNH3L-1, where the nitrification potential determined under an acetylene-free condition (total potential) was affected negatively. As the test results show, the contribution of the acetylene-tolerant potential ranged from 2 to 38% with OF soils and from 8 to 67% with CF soils. These indicated a significant contribution of heterotrophic microorganisms to the nitrification potential of these neutral tea soils. The addition of cycloheximide rather than streptomycin remarkably suppressed the acetylene-tolerant nitrification potential, suggesting the primary contribution of fungi. A new aspect of heterotrophic nitrification was identified. © 2012 Elsevier B.V.


Morimoto N.,Kwansei Gakuin University | Fukuda W.,Kwansei Gakuin University | Nakajima N.,Kwansei Gakuin University | Masuda T.,Kwansei Gakuin University | And 5 more authors.
Journal of Bacteriology | Year: 2010

Long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. Cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon Thermococcus kodakarensis. Spermidine [34] and N4-aminopropylspermine [3(3)43] were identified as major polyamines at 60°C, and the amounts of N 4-aminopropylspermine [3(3)43] increased as the growth temperature rose. To identify genes involved in polyamine biosynthesis, a gene disruption study was performed. The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N1-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher k cat/Km value for N1-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N1- aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85°C and 93°C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher k cat/Km value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N 1-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N1-aminopropylagmatine. Furthermore, spermine and N4-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Okada K.,Kwansei Gakuin University | Hidese R.,Kwansei Gakuin University | Fukuda W.,Ritsumeikan University | Niitsu M.,Josai University | And 8 more authors.
Journal of Bacteriology | Year: 2014

Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N4-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N4- bis(aminopropyl)spermidine, an isomer of N4-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N4-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N4-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap-time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N4-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N4-bis(aminopropyl)spermidine from spermidine via N4-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N4-aminopropylspermidine and N4- bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments. © 2014, American Society for Microbiology. All Rights Reserved.


Agari Y.,RIKEN | Sakamoto K.,RIKEN | Tamakoshi M.,RIKEN | Tamakoshi M.,Tokyo University of Pharmacy and Life Science | And 4 more authors.
Journal of Molecular Biology | Year: 2010

The clustered regularly interspaced short palindromic repeat (CRISPR) systems composed of DNA direct repeats designated as CRISPRs and several CRISPR-associated (cas) genes, which are present in many prokaryotic genomes, make up a host defense system against invading foreign replicons such as phages. In order to investigate the altered expression profiles of the systems after phage infection using a model organism, Thermus thermophilus HB8, which has 12 CRISPR loci, genome-wide transcription profiling of the strain infected with lytic phage ΦYS40 was performed by DNA microarray analysis. Significant alteration of overall mRNA expression gradually increased during infection (i.e., from the eclipse period to the period of host cell lysis). Interestingly, the expression of most cAMP receptor protein (CRP)-regulated genes, including two CRISPR-associated (cas) operons, was most markedly up-regulated, especially around the beginning of host cell lysis, although up-regulation of the crp gene was not observed. The expression of the CRP-regulated genes was less up-regulated in a crp-deficient strain than in the wild type. Thus, it is suggested that cAMP is a signaling molecule that transmits information on phage infection to CRP to up-regulate these genes. On the other hand, the expression of several cas genes and that of CRISPRs were up-regulated independent of CRP, suggesting the involvement of unidentified regulatory factor(s) induced by phage infection. On analysis of the expression profile of the entire genome, we could speculate that upon phage infection, the signal was transmitted to the cells, with host response systems including CRISPR defense systems being activated, while the overall efficiencies of transcription, translation, and metabolism in the cells decreased. These findings will facilitate understanding of the host response mechanism following phage infection. © 2009 Elsevier Inc. All rights reserved.


Oshima T.,Kyowa kako Co.
Methods in molecular biology (Clifton, N.J.) | Year: 2011

Unusual long polyamines such as caldopentamine and caldohexamine, and branched polyamines such as tetrakis(3-aminopropyl)ammonium and N (4)-aminopropylspermidine were often found in cells of extreme thermophiles and hyperthermophiles belonging to both Bacteria and Archaea domains. Some of these unusual polyamines are essential for life at extreme temperatures. In some cases, the unusual polyamines also exist in cells of nonthermophilic organisms and play important physiological roles under normal conditions. Methods for chromatographic analysis, isolation, and chemical syntheses of unusual polyamines as well as experimental methods for measuring their physiological roles are discussed. Especially, many newly improved methods for chemical syntheses are presented in this article.


Hori H.,Ehime University | Terui Y.,Chiba Institute of Science | Nakamoto C.,Ehime University | Iwashita C.,Ehime University | And 3 more authors.
Journal of Biochemistry | Year: 2016

Thermus thermophilus is an extreme-thermophilic eubacterium, which grows at a wide range of temperatures (50-83°C). This thermophile produces various polyamines including long and branched polyamines. In tRNAs from T. thermophilus, three distinct modifications, 2'-O-methylguanosine at position 18 (Gm18), 5-methyl-2-thiouridine at position 54 and N1-methyladenosine at position 58, are assembled at the elbow region to stabilize the L-shaped tRNA structure. However, the structures of unmodified tRNA precursors are disrupted at high temperatures. We hypothesize that polyamine(s) might have a positive effect on the modification process of unmodified tRNA transcript. We investigated the effects of eight polyamines on Gm18 formation in the yeast tRNAPhe transcript by tRNA (Gm18) methyltransferase (TrmH). Higher concentrations of linear polyamines inhibited TrmH activity at 55°C, while optimum concentration increased TrmH activity at 45-75°C. Exceptionally, caldohexamine, a long polyamine, did not show any positive effect on the TrmH activity at 55°C. However, temperature-dependent experiments revealed that 1 mM caldohexamine increased TrmH activity at 60-80°C. Furthermore, 0.25 mM tetrakis(3-aminopropy)ammonium, a branched polyamine, increased TrmH activity at a broad range of temperatures (40-85°C). Thus, caldohexamine and tetrakis(3-aminopropy)ammonium were found to enhance the TrmH activity at high temperatures. © 2015 The Authors.


Oshima T.,Kyowa kako Co.
Plant Physiology and Biochemistry | Year: 2010

Thermus thermophilus, an extreme thermophile belonging to Domain Bacteria, produces unusual polyamines in addition to standard polyamines. To understand mechanisms of changes of polyamine compositions of the thermophile upon change of growth conditions such as environmental temperature, metabolic pathways of polyamine biosyntheses of T. thermophilus have been studied and a new polyamine metabolic pathway was proposed. However, many enigmas remain to be solved in future studies. In this paper, biosyntheses of two non-standard polyamines, thermospermine and sym-homospermidine which are also produced and play important roles in plant cells, of the extreme thermophile are discussed in relation to the biosynthetic reactions in plants. © 2010 Elsevier Masson SAS.


PubMed | Ehime University, Kyowa Kako Co. and Chiba Institute of Science
Type: Journal Article | Journal: Journal of biochemistry | Year: 2016

Thermus thermophilus is an extreme-thermophilic eubacterium, which grows at a wide range of temperatures (50-83C). This thermophile produces various polyamines including long and branched polyamines. In tRNAs from T. thermophilus, three distinct modifications, 2-O-methylguanosine at position 18 (Gm18), 5-methyl-2-thiouridine at position 54 and N(1)-methyladenosine at position 58, are assembled at the elbow region to stabilize the L-shaped tRNA structure. However, the structures of unmodified tRNA precursors are disrupted at high temperatures. We hypothesize that polyamine(s) might have a positive effect on the modification process of unmodified tRNA transcript. We investigated the effects of eight polyamines on Gm18 formation in the yeast tRNA(Phe) transcript by tRNA (Gm18) methyltransferase (TrmH). Higher concentrations of linear polyamines inhibited TrmH activity at 55C, while optimum concentration increased TrmH activity at 45-75C. Exceptionally, caldohexamine, a long polyamine, did not show any positive effect on the TrmH activity at 55C. However, temperature-dependent experiments revealed that 1 mM caldohexamine increased TrmH activity at 60-80C. Furthermore, 0.25 mM tetrakis(3-aminopropy)ammonium, a branched polyamine, increased TrmH activity at a broad range of temperatures (40-85C). Thus, caldohexamine and tetrakis(3-aminopropy)ammonium were found to enhance the TrmH activity at high temperatures.

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