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Featured researches published by Takeshi Uemura.


Journal of Biological Chemistry | 2007

Polyamine Uptake by DUR3 and SAM3 in Saccharomyces cerevisiae

Takeshi Uemura; Keiko Kashiwagi; Kazuei Igarashi

It has been reported that GAP1 and AGP2 catalyze the uptake of polyamines together with amino acids in Saccharomyces cerevisiae. We have looked for polyamine-preferential uptake proteins in S. cerevisiae. DUR3 catalyzed the uptake of polyamines together with urea, and SAM3 was found to catalyze the uptake of polyamines together with S-adenosylmethionine, glutamic acid, and lysine. Polyamine uptake was greatly decreased in both DUR3- and SAM3-deficient cells. The Km values for putrescine and spermidine of DUR3 were 479 and 21.2 μm, respectively, and those of SAM3 were 433 and 20.7 μm, respectively. Polyamine stimulation of cell growth of a polyamine requiring mutant, which is deficient in ornithine decarboxylase, was not influenced by the disruption of GAP1 and AGP2, but it was diminished by the disruption of DUR3 and SAM3. Furthermore, the polyamine stimulation of cell growth of a polyamine-requiring mutant was completely inhibited by the disruption of both DUR3 and SAM3. The results indicate that DUR3 and SAM3 are major polyamine uptake proteins in yeast. We previously reported that polyamine transport protein kinase 2 regulates polyamine transport. It was found that DUR3 (but not SAM3) was activated by phosphorylation of Thr250, Ser251, and Thr684 by polyamine transport protein kinase 2.


Journal of Bacteriology | 2008

Identification of a Spermidine Excretion Protein Complex (MdtJI) in Escherichia coli

Kyohei Higashi; Hiroyuki Ishigure; Risa Demizu; Takeshi Uemura; Kunihiko Nishino; Akihito Yamaguchi; Keiko Kashiwagi; Kazuei Igarashi

A spermidine excretion protein in Escherichia coli was looked for among 33 putative drug exporters thus far identified. Cell toxicity and inhibition of growth due to overaccumulation of spermidine were examined in an E. coli strain deficient in spermidine acetyltransferase, an enzyme that metabolizes spermidine. Toxicity and inhibition of cell growth by spermidine were recovered in cells transformed with pUCmdtJI or pMWmdtJI, encoding MdtJ and MdtI, which belong to the small multidrug resistance family of drug exporters. Both mdtJ and mdtI are necessary for recovery from the toxicity of overaccumulated spermidine. It was also found that the level of mdtJI mRNA was increased by spermidine. The spermidine content in cells cultured in the presence of 2 mM spermidine was decreased, and excretion of spermidine from cells was enhanced by MdtJI, indicating that the MdtJI complex can catalyze excretion of spermidine from cells. It was found that Tyr4, Trp5, Glu15, Tyr45, Tyr61, and Glu82 in MdtJ and Glu5, Glu19, Asp60, Trp68, and Trp81 in MdtI are involved in the excretion activity of MdtJI.


Journal of Biological Chemistry | 2006

Identification of the Cadaverine Recognition Site on the Cadaverine-Lysine Antiporter CadB

Waraporn Soksawatmaekhin; Takeshi Uemura; Natsuko Fukiwake; Keiko Kashiwagi; Kazuei Igarashi

Amino acid residues involved in cadaverine uptake and cadaverine-lysine antiporter activity were identified by site-directed mutagenesis of the CadB protein. It was found that Tyr73, Tyr89, Tyr90, Glu204, Tyr235, Asp303, and Tyr423 were strongly involved in both uptake and excretion and that Tyr55, Glu76, Tyr246, Tyr310, Cys370, and Glu377 were moderately involved in both activities. Mutations of Trp43, Tyr57, Tyr107, Tyr366, and Tyr368 mainly affected uptake activity, and Trp41, Tyr174, Asp185, and Glu408 had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the Km value. Mutation of Arg299 mainly affected excretion, suggesting that Arg299 is involved in the recognition of the carboxyl group of lysine. These results indicate that amino acid residues involved in both uptake and excretion, or solely in excretion, are located in the cytoplasmic loops and the cytoplasmic side of transmembrane segments, whereas residues involved in uptake were located in the periplasmic loops and the transmembrane segments. The SH group of Cys370 seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys125, Cys389, or Cys394 together with Cys370. The relative topology of 12 transmembrane segments was determined by inserting cysteine residues at various sites and measuring the degree of inhibition of transport through crosslinking with Cys370. The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII.


Journal of Biological Chemistry | 2003

Molecular Visualization of Immunoglobulin Switch Region RNA/DNA Complex by Atomic Force Microscope

Ryushin Mizuta; Kousuke Iwai; Masatsugu Shigeno; Midori Mizuta; Takeshi Uemura; Tatsuo Ushiki; Daisuke Kitamura

Immunoglobulin heavy-chain (IgH) class switch recombination (CSR) is initiated by DNA breakage in the switch (S) region featuring tandem repetitive nucleotide sequences. Various studies have demonstrated that S-region transcription and splicing proceed to genomic recombination and are indispensable for CSR in vivo, although the precise molecular mechanism is largely unknown. Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an atomic force microscope (AFM). The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate that may stall replication and increase the recombinogenicity of the S-region DNA.


Journal of Biological Chemistry | 2005

Excretion of Putrescine and Spermidine by the Protein Encoded by YKL174c (TPO5) in Saccharomyces cerevisiae

Ken Tachihara; Takeshi Uemura; Keiko Kashiwagi; Kazuei Igarashi

The properties of the protein encoded by YKL174c (TPO5) were studied. It was found that TPO5 excretes putrescine effectively and spermidine less effectively. γ-Aminobutyric acid slightly inhibited the excretion of putrescine, but basic amino acids did not affect excretion, suggesting that TPO5 preferentially recognizes polyamines. Accordingly, yeast cells transformed with the plasmid encoding YKL174c (TPO5) were resistant to toxicity caused by 120 mm putrescine or by 3 mm spermidine, and a mutant with a disrupted YKL174c (TPO5) gene was sensitive to toxicity by 90 mm putrescine. The growth of this mutant was faster than that of the wild-type strain. In parallel, there was an increase in putrescine and spermidine content of the YKL174c (TPO5) mutant compared with wild-type. It is noted that TPO5 functions as a suppressor of cell growth by excreting polyamines. The level of YKL174c (TPO5) mRNA was increased by the addition of polyamines to the medium. The degree of induction of the mRNA was spermine > spermidine > putrescine. The subcellular localization of TPO5 was determined by immunostaining of hemagglutinin-tagged TPO5, and it was found on Golgi or post-Golgi secretory vesicles. Excretion of putrescine and spermidine by TPO5 was reduced in cells that have mutations in the secretory or endocytic pathways, indicating that both processes are involved in the excretion of polyamines.


PLOS ONE | 2014

Identification of Functional Amino Acid Residues Involved in Polyamine and Agmatine Transport by Human Organic Cation Transporter 2

Kyohei Higashi; Masataka Imamura; Satoshi Fudo; Takeshi Uemura; Ryotaro Saiki; Tyuji Hoshino; Toshihiko Toida; Keiko Kashiwagi; Kazuei Igarashi

Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.


Biochemical and Biophysical Research Communications | 2016

Aggravation of brain infarction through an increase in acrolein production and a decrease in glutathione with aging

Takeshi Uemura; Kenta Watanabe; Misaki Ishibashi; Ryotaro Saiki; Kyoshiro Kuni; Kazuhiro Nishimura; Toshihiko Toida; Keiko Kashiwagi; Kazuei Igarashi

We previously reported that tissue damage during brain infarction was mainly caused by inactivation of proteins by acrolein. This time, it was tested why brain infarction increases in parallel with aging. A mouse model of photochemically induced thrombosis (PIT) was studied using 2, 6, and 12 month-old female C57BL/6 mice. The size of brain infarction in the mouse PIT model increased with aging. The volume of brain infarction in 12 month-old mice was approximately 2-fold larger than that in 2 month-old mice. The larger brain infarction in 12 month-old mice was due to an increase in acrolein based on an increase in the activity of spermine oxidase, together with a decrease in glutathione (GSH), a major acrolein-detoxifying compound in cells, based on the decrease in one of the subunits of glutathione biosynthesizing enzymes, γ-glutamylcysteine ligase modifier subunit, with aging. The results indicate that aggravation of brain infarction with aging was mainly due to the increase in acrolein production and the decrease in GSH in brain.


Toxicology | 2013

Acetaldehyde-induced cytotoxicity involves induction of spermine oxidase at the transcriptional level.

Takeshi Uemura; Yuka Tanaka; Kyohei Higashi; Daisuke Miyamori; Tomokazu Takasaka; Tatsuo Nagano; Toshihiko Toida; Kanji Yoshimoto; Kazuei Igarashi; Hiroshi Ikegaya

Ethanol consumption causes serious liver injury including cirrhosis and hepatocellular carcinoma. Ethanol is metabolized mainly in the liver to acetic acid through acetaldehyde. We investigated the effect of ethanol and acetaldehyde on polyamine metabolism since polyamines are essential factors for normal cellular functions. We found that acetaldehyde induced spermine oxidase (SMO) at the transcriptional level in HepG2 cells. The levels and activities of ornithine decarboxylase (ODC) and spermidine/spermine acetyltransferase (SSAT) were not affected by acetaldehyde. Spermidine content was increased and spermine content was decreased by acetaldehyde treatment. Knockdown of SMO expression using siRNA reduced acetaldehyde toxicity. Acetaldehyde exposure increased free acrolein levels. An increase of acrolein by acetaldehyde was SMO dependent. Our results indicate that cytotoxicity of acetaldehyde involves, at least in part, oxidation of spermine to spermidine by SMO, which is induced by acetaldehyde.


Clinica Chimica Acta | 2015

Increase in acrolein-conjugated immunoglobulins in saliva from patients with primary Sjogren's syndrome.

Tadao Hirose; Ryotaro Saiki; Takeshi Uemura; Takehiro Suzuki; Naoshi Dohmae; Satoshi Ito; Hoyu Takahashi; Itsuko Ishii; Toshihiko Toida; Keiko Kashiwagi; Kazuei Igarashi

BACKGROUND We previously reported that the level of protein-conjugated acrolein (PC-Acro), a marker of cell or tissue damage, was increased in saliva from patients with primary Sjögrens syndrome (pSS), and that the level of PC-Acro was well correlated with the severity of pSS. METHODS Acrolein-conjugated immunoglobulins were measured in saliva from pSS patients. RESULTS The activities of autoantibodies recognizing Sjögrens syndrome SSA (Ro) and SSB (La) proteins in saliva from pSS patients were approximately 3- to 5-fold higher than those from control subjects. We also found that autoantibody activities recognizing SSA (Ro) and SSB (La) proteins increased after acrolein treatment of saliva from control subjects. When an antibody against human serum albumin was treated with acrolein, the ability to recognize albumin was reduced but the ability to recognize other proteins was increased. Twenty-four and eleven kinds of acrolein-conjugated amino acids were found at the variable and constant regions of peptides, respectively, obtained from the immunoglobulins in saliva from pSS patients. CONCLUSION The altered recognition patterns of immunoglobulins due to acrolein conjugation are at least partially involved in autoimmune diseases.


Atherosclerosis | 2016

Toxic acrolein production due to Ca2+ influx by the NMDA receptor during stroke

Mizuho Nakamura; Takeshi Uemura; Ryotaro Saiki; Akihiko Sakamoto; Hyerim Park; Kazuhiro Nishimura; Yusuke Terui; Toshihiko Toida; Keiko Kashiwagi; Kazuei Igarashi

BACKGROUND AND PURPOSE N-Methyl-d-aspartate (NMDA) receptors have a high permeability to Ca(2+), contributing to neuronal cell death after stroke. We recently found that acrolein produced from polyamines is a major toxic compound during stroke. Thus, it was determined whether over-accumulation of Ca(2+) increases the production of acrolein from polyamines in a photochemically-induced thrombosis mouse model of stroke and in cell culture systems. METHODS A unilateral infarction was induced in mouse brain by photoinduction after injection of Rose Bengal. The volume of the infarction was analyzed using the public domain National Institutes of Health image program. Protein-conjugated acrolein levels at the locus of infarction and in cells were measured by Western blotting. Levels of polyamines were measured by high-performance liquid chromatography. RESULTS When the size of brain infarction was decreased by N(1), N(4), N(8)-tribenzylspermidine, a channel blocker of the NMDA receptors, levels of Ca(2+) and protein-conjugated acrolein (PC-Acro) were reduced, while levels of polyamines were increased at the locus of infarction. When cell growth of mouse mammary carcinoma FM3A cells and neuroblastoma Neuro2a cells was inhibited by Ca(2+), the level of polyamines decreased, while that of PC-Acro increased. It was also shown that Ca(2+) toxicity was decreased in an acrolein toxicity decreasing FM3A mutant cells recently isolated. In addition, 20-40 μM Ca(2+) caused the release of polyamines from ribosomes. The results indicate that acrolein is produced from polyamines released from ribosomes through Ca(2+) increase. CONCLUSION The results indicate that toxicity of Ca(2+) during brain infarction is correlated with the increase of acrolein.

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Keiko Kashiwagi

Chiba Institute of Science

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Yusuke Terui

Chiba Institute of Science

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Akihiko Sakamoto

Chiba Institute of Science

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