論文 - 植村 武史
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Correlation of polyamines, acrolein-conjugated lysine and polyamine metabolic enzyme levels with age in human liver. 査読あり
Uemura Takeshi, Akasaka Yoshihisa, Ikegaya Hiroshi
Heliyon 6 ( 9 ) e05031 - e05031 2020年09月
担当区分：筆頭著者, 責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
The polyamines spermidine, spermine and putrescine are essential for normal cellular functions. The contents of polyamines in tissue decreased in aged mice compared to young mice. In this study, the polyamine contents and their metabolic byproduct acrolein-conjugated lysine (N (ε)-(3-formyl-3,4-dehydropiperidino)-lysine, FDP-Lys) in human liver tissue were measured and analyzed the correlation with age of the subjects. The putrescine and FDP-Lys levels were significantly increased with age. On the other hand, spermine level was decreased with age. Spermidine did not significantly correlate with age. The relative amount of spermine oxidase (SMOX) significantly correlated with the age of subjects whereas ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase (AMD1) significantly reduced by the age. Our results suggested that an increase in oxidation and reduction in polyamine synthesis may cause the change of polyamine profile in the elderly.
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A search for acrolein scavengers among food components 査読あり
Uemura T, Uchida M, Nakamura M, Shimekake M, Sakamoto A, Terui Y, Higashi K, Ishii I, Kashiwagi K, Igarashi K.
Amino Acids 2023年02月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
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Nα-(5-Fluoro-2,4-dinitrophenyl)-L-leucinamide-derivatized LC/MS/MS Analysis of Amino Acid Enantiomers in HepG2 Cells 査読あり
Takano Y, Takahashi M, Kobayashi M, Uemura T, Furuchi T.
Chromatography 42 ( 3 ) 143 - 149 2021年
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Structural change and degradation of cytoskeleton due to the acrolein conjugation with vimentin and actin during brain infarction. 査読あり
Uemura Takeshi, Suzuki Takehiro, Ko Kenta, Nakamura Mizuho, Dohmae Naoshi, Sakamoto Akihiko, Terui Yusuke, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
Cytoskeleton (Hoboken) 77 ( 10 ) 414 - 421 2020年10月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
We have found recently that dendritic spine extension is inhibited through acrolein conjugation with α- and β-tubulin proteins during brain infarction. In this current study, we looked for other acrolein-conjugated proteins in the 100,000g precipitate fraction, to clarify how cytoskeleton structure is modified by acrolein. Acrolein-conjugated proteins were sought from acrolein-treated mouse FM3A and Neuro2a cells and from tissues isolated from mouse brain infarction. It was found that vimentin was conjugated with acrolein, and the conjugated amino acid residue was Cys328, which is the only Cys residue in vimentin. It was also found that Cys207, 257, 285, and Lys118 in actin, another cytoskeleton protein, were conjugated with acrolein. The structure and localization of vimentin and actin filaments were changed greatly in infarct brain in photochemically induced thrombosis model mice and in acrolein-treated Neuro2a cells. In addition, degradation of cytoskeleton proteins was accelerated in the order vimentin > tubulin > actin in mouse brain infarction. These findings indicate that a dysfunction of the cytoskeleton by acrolein is strongly involved in the tissue damage during brain infarction, together with the apoptosis caused by glyceraldehyde-3-phosphate dehydrogenase and protein degradation by matrix metalloproteinase-9.
DOI： 10.1002/cm.21638
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Urinary Amino Acid-Conjugated Acrolein and Taurine as New Biomarkers for Detection of Dementia 査読あり
Yoshida M, Uemura T, Mizoi M, Waragai M, Sakamoto A, Terui Y, Kashiwagi K, Igarashi K.
J Alzheimers Dis. 2023年01月
担当区分：第二著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
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Polyamine analysis of acidophiles, alkaliphiles, halophiles and thermophiles belonging to the bacterial phyla Actinobacteria, Aquificae, Bacterioidetes, Cyanobacteria, Desulfobacterota, Firmicutes, Tenericutes, and Thermotogae – Polyamine catalogue of bacterial and archaeal extremophiles – (XII) 査読あり
Koei Hamana, Hidenori Hayashi, Takemitsu Furuchi, Takeshi Uemura, Masaru Niitsu, Takashi Itoh, Mitsuo Sakamoto, and Moriya Okuma
Journal of Japanese Society for Extremophiles 2022年
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Polyamines produced by an extreme thermophile are essential for cell growth at high temperature 査読あり
Akihiko Sakamoto, Masatada Tamakoshi, Toshiyuki Moriya, Tairo Oshima, Koichi Takao, Yoshiaki Sugita, Takemitsu Furuchi, Masaru Niitsu, Takeshi Uemura, Kazuei Igarashi, Keiko Kashiwagi, Yusuke Terui.
Journal of Biochemistry 172 ( 2 ) 109 - 115 2022年
記述言語：英語 掲載種別：研究論文（学術雑誌）
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The Polyamine Regulator AMD1 Upregulates Spermine Levels to Drive Epidermal Differentiation. 招待あり 査読あり 国際共著
Rahim Anisa B, Lim Hui Kheng, Tan Christina Yan Ru, Jia Li, Leo Vonny Ivon, Uemura Takeshi, Hardman-Smart Jonathan, Common John E A, Lim Thiam Chye, Bellanger Sophie, Paus Ralf, Igarashi Kazuei, Yang Henry, Vardy Leah A
J Invest Dermatol 141 ( 9 ) 2178 - 2188.e6 2021年09月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Maintaining tissue homeostasis depends on a balance between cell proliferation, differentiation, and apoptosis. Within the epidermis, the levels of the polyamines putrescine, spermidine, and spermine are altered in many different skin conditions, yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, Adenosylmethionine decarboxylase 1 (AMD1), as a crucial regulator of keratinocyte (KC) differentiation. AMD1 protein is upregulated on differentiation and is highly expressed in the suprabasal layers of the human epidermis. During KC differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown or inhibition of AMD1 results in reduced spermine levels and inhibition of KC differentiation. Supplementing AMD1-knockdown KCs with exogenous spermidine or spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signaling proteins that drive KC differentiation, including KLF4 and ZNF750. These findings show that human KCs use controlled changes in polyamine levels to modulate gene expression to drive cellular behavior changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or can be used in the treatment of hyperproliferative skin disorders.
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Polyamine-Rich Diet Elevates Blood Spermine Levels and Inhibits Pro-Inflammatory Status: An Interventional Study. 査読あり
Soda Kuniyasu, Uemura Takeshi, Sanayama Hidenori, Igarashi Kazuei, Fukui Taro
Med Sci (Basel) 9 ( 2 ) 2021年03月
記述言語：英語 掲載種別：研究論文（学術雑誌）
The Japanese diet and the Mediterranean diet are rich in polyamines (spermidine and spermine). Increased polyamine intake elevated blood spermine levels, inhibited aging-associated pro-inflammatory status (increases in lymphocyte function-associated antigen-1 (LFA-1) on immune cells), suppressed aberrant gene methylation and extended the lifespan of mice. To test the effects of increased polyamine intake by humans, 30 healthy male volunteers were asked to eat polyamine-rich and ready-to-eat traditional Japanese food (natto) for 12 months. Natto with high polyamine content was used. Another 27 male volunteers were asked not to change their dietary pattern as a control group. The volunteers' age of intervention and control groups ranged from 40 to 69 years (median 48.9 ± 7.9). Two subjects in the control group subsequently dropped out of the study. The estimated increases in spermidine and spermine intakes were 96.63 ± 47.70 and 22.00 ± 9.56 µmol per day in the intervention group, while no changes were observed in the control group. The mean blood spermine level in the intervention group gradually rose to 1.12 ± 0.29 times the pre-intervention level after 12 months, and were significantly higher (p = 0.019) than those in the control group. Blood spermidine did not increase in either group. LFA-1 on monocytes decreased gradually in the intervention group, and there was an inverse association between changes in spermine concentrations relative to spermidine and changes in LFA-1 levels. Contingency table analysis revealed that the odds ratio to decrease LFA-1 by increased polyamine intake was 3.927 (95% CI 1.116-13.715) (p = 0.032) when the effect of acute inflammation was excluded. The results in the study were similar to those of our animal experiments. Since methylation changes of the entire genome are associated with aging-associated pathologies and our previous studies showed that spermine-induced LFA-1 suppression was associated with the inhibition of aberrant gene methylation, the results suggest that dietary polyamine contributes to human health and longevity.
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A Raman algorithm to estimate human age from protein structural variations in autopsy skin samples: a protein biological clock. 査読あり
Miyamori Daisuke, Uemura Takeshi, Zhu Wenliang, Fujikawa Kei, Nakaya Takaaki, Teramukai Satoshi, Pezzotti Giuseppe, Ikegaya Hiroshi
Sci Rep 11 ( 1 ) 5949 - 5949 2021年03月
記述言語：英語 掲載種別：研究論文（学術雑誌）
The recent increase of the number of unidentified cadavers has become a serious problem throughout the world. As a simple and objective method for age estimation, we attempted to utilize Raman spectrometry for forensic identification. Raman spectroscopy is an optical-based vibrational spectroscopic technique that provides detailed information regarding a sample's molecular composition and structures. Building upon our previous proof-of-concept study, we measured the Raman spectra of abdominal skin samples from 132 autopsy cases and the protein-folding intensity ratio, R(PF), defined as the ratio between the Raman signals from a random coil an α-helix. There was a strong negative correlation between age and R(PF) with a Pearson correlation coefficient of r = 0.878. Four models, based on linear (R(PF)), squared (R(PF)(2)), sex, and R(PF) by sex interaction terms, were examined. The results of cross validation suggested that the second model including linear and squared terms was the best model with the lowest root mean squared error (11.3 years of age) and the highest coefficient of determination (0.743). Our results indicate that the there was a high correlation between the age and R(PF) and the Raman biological clock of protein folding can be used as a simple and objective forensic age estimation method for unidentified cadavers.
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Translational Regulation of Clock Genes BMAL1 and REV-ERBα by Polyamines. 査読あり
Sakamoto Akihiko, Terui Yusuke, Uemura Takeshi, Igarashi Kazuei, Kashiwagi Keiko
Int J Mol Sci 22 ( 3 ) 2021年01月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Polyamines stimulate the synthesis of specific proteins at the level of translation, and the genes encoding these proteins are termed as the "polyamine modulon". The circadian clock generates daily rhythms in mammalian physiology and behavior. We investigated the role of polyamines in the circadian rhythm using control and polyamine-reduced NIH3T3 cells. The intracellular polyamines exhibited a rhythm with a period of about 24 h. In the polyamine-reduced NIH3T3 cells, the circadian period of circadian clock genes was lengthened and the synthesis of BMAL1 and REV-ERBα was significantly reduced at the translation level. Thus, the mechanism of polyamine stimulation of these protein syntheses was analyzed using NIH3T3 cells transiently transfected with genes encoding enhanced green fluorescent protein (EGFP) fusion mRNA with normal or mutated 5'-untranslated region (5'-UTR) of Bmal1 or Rev-erbα mRNA. It was found that polyamines stimulated BMAL1 and REV-ERBα synthesis through the enhancement of ribosomal shunting during the ribosome shunting within the 5'-UTR of mRNAs. Accordingly, the genes encoding Bmal1 and Rev-erbα were identified as the members of "polyamine modulon", and these two proteins are significantly involved in the circadian rhythm control.
DOI： 10.3390/ijms22031307
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The Polyamine Putrescine Promotes Human Epidermal Melanogenesis. 査読あり
Sridharan Aishwarya, Shi Meng, Leo Vonny Ivon, Subramaniam Nagavidya, Lim Thiam Chye, Uemura Takeshi, Igarashi Kazuei, Tien Guan Steven Thng, Tan Nguan Soon, Vardy Leah A
J Invest Dermatol 140 ( 10 ) 2032 - 2040.e1 2020年10月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Hyperpigmentary conditions can arise when melanogenesis in the epidermis is misregulated. Understanding the pathways underlying melanogenesis is essential for the development of effective treatments. Here, we report that a group of metabolites called polyamines are important in the control of melanogenesis in human skin. Polyamines are cationic molecules present in all cells and are essential for cellular function. We report that polyamine regulator ODC1 is upregulated in melanocytes from melasma lesional skin. We report that the polyamine putrescine can promote pigmentation in human skin explants and primary normal human epidermal melanocytes through induction of tyrosinase which is rate-limiting for the synthesis of melanin. Putrescine supplementation on normal human epidermal melanocytes results in the activation of polyamine catabolism, which results in increased intracellular H(2)O(2.) Polyamine catabolism is also increased in human skin explants that have been treated with putrescine. We further report that inhibition of polyamine catabolism prevents putrescine-induced promotion of tyrosinase levels and pigmentation in normal human epidermal melanocytes, showing that polyamine catabolism is responsible for the putrescine induction of melanogenesis. Our data showing that putrescine promotes pigmentation has important consequences for hyperpigmented and hypopigmented conditions. Further understanding of how polyamines control epidermal pigmentation could open the door for the development of new therapeutics.
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Development of an ELISA for Measurement of Urinary 3-Hydroxypropyl Mercapturic Acid (3-HPMA), the Marker of Stroke. 査読あり
Sakamoto Akihiko, Uemura Takeshi, Terui Yusuke, Yoshida Madoka, Fukuda Kazumasa, Nakamura Takao, Kashiwagi Keiko, Igarashi Kazuei
Med Sci (Basel) 8 ( 3 ) 2020年08月
記述言語：英語 掲載種別：研究論文（学術雑誌）
We previously observed an inverse correlation between stroke and urinary 3-hydroxypropyl mercapturic acid (3-HPMA), an acrolein-glutathione metabolite, through its measurement by liquid chromatography with tandem mass spectrometry (LC-MS/MS). However, the cost of equipment for LC-MS/MS and its maintenance fee is very expensive and a cost-efficient method is required. In this study, we have developed a sensitive enzyme-linked immunosorbent assay (ELISA) system to measure 3-HPMA using a chicken antibody recognizing 3-HPMA-conjugated chicken albumin as antigen. Linearity to measure 3-HPMA was obtained from 0 to 10 μM, indicating that this ELISA system is useful for measurement of urine 3-HPMA. It was confirmed that 3-HPMA in urine of stroke patients decreased significantly compared with that of control subjects using the ELISA system. Using the ELISA kit, it became possible to evaluate the risk of brain stroke by not only plasma but also by urine. These results confirm that shortage of glutathione to detoxify acrolein is one of the major causes of stroke incidence. Our method contributes to maintenance of quality of life (QOL) of the elderly.
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Polyamines regulate gene expression by stimulating translation of histone acetyltransferase mRNAs. 査読あり
Sakamoto Akihiko, Terui Yusuke, Uemura Takeshi, Igarashi Kazuei, Kashiwagi Keiko
J Biol Chem 295 ( 26 ) 8736 - 8745 2020年06月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Polyamines regulate gene expression in Escherichia coli by translationally stimulating mRNAs encoding global transcription factors. In this study, we focused on histone acetylation, one of the mechanisms of epigenetic regulation of gene expression, to attempt to clarify the role of polyamines in the regulation of gene expression in eukaryotes. We found that activities of histone acetyltransferases in both the nucleus and cytoplasm decreased significantly in polyamine-reduced mouse mammary carcinoma FM3A cells. Although protein levels of histones H3 and H4 did not change in control and polyamine-reduced cells, acetylation of histones H3 and H4 was greatly decreased in the polyamine-reduced cells. Next, we used control and polyamine-reduced cells to identify histone acetyltransferases whose synthesis is stimulated by polyamines. We found that polyamines stimulate the translation of histone acetyltransferases GCN5 and HAT1. Accordingly, GCN5- and HAT1-catalyzed acetylation of specific lysine residues on histones H3 and H4 was stimulated by polyamines. Consistent with these findings, transcription of genes required for cell proliferation was enhanced by polyamines. These results indicate that polyamines regulate gene expression by enhancing the expression of the histone acetyltransferases GCN5 and HAT1 at the level of translation. Mechanistically, polyamines enhanced the interaction of microRNA-7648-5p (miR-7648-5p) with the 5'-UTR of GCN5 mRNA, resulting in stimulation of translation due to the destabilization of the double-stranded RNA (dsRNA) between the 5'-UTR and the ORF of GCN5 mRNA. Because HAT1 mRNA has a short 5'-UTR, polyamines may enhance initiation complex formation directly on this mRNA.
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Cytotoxic Mechanism of Excess Polyamines Functions through Translational Repression of Specific Proteins Encoded by Polyamine Modulon. 査読あり
Sakamoto Akihiko, Sahara Junpei, Kawai Gota, Yamamoto Kaneyoshi, Ishihama Akira, Uemura Takeshi, Igarashi Kazuei, Kashiwagi Keiko, Terui Yusuke
Int J Mol Sci 21 ( 7 ) 2020年03月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine-Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.
DOI： 10.3390/ijms21072406
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Assessing acrolein for determination of the severity of brain stroke, dementia, renal failure, and Sjögren's syndrome. 査読あり
Igarashi Kazuei, Uemura Takeshi, Kashiwagi Keiko
Amino Acids 52 ( 2 ) 119 - 127 2020年02月
記述言語：英語 掲載種別：研究論文（学術雑誌）
It was found recently that acrolein (CH(2)=CH-CHO), mainly produced from spermine, is more toxic than ROS (reactive oxygen species, O(2)(-·), H(2)O(2), and ·OH). In this review, we describe how the seriousness of brain infarction, dementia, renal failure, and Sjӧgren's syndrome is correlated with acrolein. In brain infarction and dementia, it was possible to identify incipient patients with high sensitivity and specificity by measuring protein-conjugated acrolein (PC-Acro) in plasma together with IL-6 and CRP in brain infarction and Aβ(40/42) in dementia. The level of PC-Acro in plasma and saliva correlated with the seriousness of renal failure and Sjӧgren's syndrome, respectively. Thus, development of acrolein scavenger medicines containing SH-group such as N-acetylcysteine derivatives is important to maintain QOL (quality of life) of the elderly.
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蛋白質抱合アクロレイン、IL-6並びにCRP測定に基づく脳梗塞リスク評価による脳梗塞発症者数の減少 査読あり
五十嵐一衛、植村武史、柏木敬子
未病と抗老化 29 30 - 34 2020年
担当区分：第二著者 記述言語：日本語 掲載種別：研究論文（学術雑誌）
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GSTΠ stimulates caveolin-1-regulated polyamine uptake via actin remodeling. 査読あり
Uemura Takeshi, Tsaprailis George, Gerner Eugene W
Oncotarget 10 ( 55 ) 5713 - 5723 2019年10月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Polyamines spermidine and spermine, and their diamine precursor putrescine, are essential for normal cellular functions in both pro- and eukaryotes. Cellular polyamine levels are regulated by biosynthesis, degradation and transport. Transport of dietary and luminal bacterial polyamines in gastrointestinal (GI) tissues plays a significant role in tissue polyamine homeostasis. We have reported that caveolin-1 play an inhibitory role in polyamine uptake in GI tissues. We investigated the mechanism of caveolin-1-regulated polyamine transport. We found that glutathione S-transferase Π(GSTΠ) was secreted from caveolin-1 knockdown cells and stimulated spermidine transport in human colon-derived HCT116 cells. GSTΠ secreted in the medium increased S-glutathionylated protein level in the plasma membrane fraction. Proteomic analysis revealed that actin was S-glutathionylated by GSTΠ. Immunofluorescence microscopy demonstrated that actin filaments around plasma membrane were S-glutathionylated in caveolin-1 knockdown cells. Inhibition of actin remodeling by jasplakinolide caused a decrease in polyamine uptake activity. These data support a model in which caveolin-1 negatively regulates polyamine uptake by inhibiting GSTΠ secretion, which stimulates actin remodeling and endocytosis.
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Inhibition of dendritic spine extension through acrolein conjugation with α-, β-tubulin proteins. 査読あり
Uemura Takeshi, Suzuki Takehiro, Ko Kenta, Watanabe Kenta, Dohmae Naoshi, Sakamoto Akihiko, Terui Yusuke, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
Int J Biochem Cell Biol 113 58 - 66 2019年08月
記述言語：英語 掲載種別：研究論文（学術雑誌）
We have recently found that conjugation of acrolein with a 50 kDa protein(s) is strongly associated with tissue damage during brain infarction. In the current study, the identity and function of the 50 kDa protein(s) conjugated with acrolein during brain infarction were investigated. The 50 kDa protein(s) conjugated with acrolein were identified as α- and β-tubulins. Ten cysteine residues in α- and β-tubulins (Cys25, 295, 347 and 376 in α-tubulin and Cys12, 129, 211, 239, 303 and 354 in β-tubulin) were mainly conjugated with acrolein. Since two cysteine residues of α-tubulin (Cys347 and 376) and four cysteine residues of β-tubulin (Cys12, 129, 239 and 354) were located at the interaction site of α- and β-tubulins, association between α- and β-tubulins to form microtubules was strongly inhibited by conjugation with acrolein. Accordingly, dendritic spine extension consisting of microtubules was greatly inhibited in acrolein-treated Neuro2a cells. The results strongly suggest that acrolein contributes to the functional losses in brain signaling through its conjugation with α- and β-tubulins.
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Polyamine Regulator AMD1 Promotes Cell Migration in Epidermal Wound Healing. 査読あり
Lim Hui Kheng, Rahim Anisa B, Leo Vonny Ivon, Das Shatarupa, Lim Thiam Chye, Uemura Takeshi, Igarashi Kazuei, Common John, Vardy Leah A
J Invest Dermatol 138 ( 12 ) 2653 - 2665 2018年12月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Wound healing is a dynamic process involving gene-expression changes that drive re-epithelialization. Here, we describe an essential role for polyamine regulator AMD1 in driving cell migration at the wound edge. The polyamines, putrescine, spermidine, and spermine are small cationic molecules that play essential roles in many cellular processes. We demonstrate that AMD1 is rapidly upregulated following wounding in human skin biopsies. Knockdown of AMD1 with small hairpin RNAs causes a delay in cell migration that is rescued by the addition of spermine. We further show that spermine can promote cell migration in keratinocytes and in human ex vivo wounds, where it significantly increases epithelial tongue migration. Knockdown of AMD1 prevents the upregulation of urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor on wounding and results in a failure in actin cytoskeletal reorganization at the wound edge. We demonstrate that keratinocytes respond to wounding by modulating polyamine regulator AMD1 in order to regulate downstream gene expression and promote cell migration. This article highlights a previously unreported role for the regulation of polyamine levels and ratios in cellular behavior and fate.
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MINDY1 Is a Downstream Target of the Polyamines and Promotes Embryonic Stem Cell Self-Renewal. 査読あり
James Christina, Zhao Tian Yun, Rahim Anisa, Saxena Parul, Muthalif Nazreen Abdul, Uemura Takeshi, Tsuneyoshi Norihiro, Ong Sheena, Igarashi Kazuei, Lim Chin Yan, Dunn Norris Ray, Vardy Leah A
Stem Cells 36 ( 8 ) 1170 - 1178 2018年08月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Embryonic stem cells have the ability to self-renew or differentiate and these processes are under tight control. We previously reported that the polyamine regulator AMD1 is critical for embryonic stem cell self-renewal. The polyamines putrescine, spermidine, and spermine are essential organic cations that play a role in a wide array of cellular processes. Here, we explore the essential role of the polyamines in the promotion of self-renewal and identify a new stem cell regulator that acts downstream of the polyamines: MINDY1. MINDY1 protein levels are high in embryonic stem cells (ESCs) and are dependent on high polyamine levels. Overexpression of MINDY1 can promote ESC self-renewal in the absence of the usually essential cytokine Leukemia Inhibitory Factor (LIF). MINDY1 protein is prenylated and this modification is required for its ability to promote self-renewal. We go on to show that Mindy1 RNA is targeted for repression by mir-710 during Neural Precursor cell differentiation. Taken together, these data demonstrate that high polyamine levels are required for ESC self-renewal and that they function, in part, through promotion of high MINDY1 levels. Stem Cells 2018;36:1170-1178.
DOI： 10.1002/stem.2830
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Protective Effects of Brain Infarction by N-Acetylcysteine Derivatives. 査読あり
Uemura Takeshi, Watanabe Kenta, Ko Kenta, Higashi Kyohei, Kogure Noriyuki, Kitajima Mariko, Takayama Hiromitsu, Takao Koichi, Sugita Yoshiaki, Sakamoto Akihiko, Terui Yusuke, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
Stroke 49 ( 7 ) 1727 - 1733 2018年07月
記述言語：英語 掲載種別：研究論文（学術雑誌）
BACKGROUND AND PURPOSE: We recently found that acrolein (CH(2)=CH-CHO) is more strongly involved in brain infarction compared with reactive oxygen species. In this study, we looked for acrolein scavengers with less side effects. METHODS: Photochemically induced thrombosis model mice were prepared by injection of Rose Bengal. Effects of N-acetylcysteine (NAC) derivatives on brain infarction were evaluated using the public domain National Institutes of Health image program. RESULTS: NAC, NAC ethyl ester, and NAC benzyl ester (150 mg/kg) were administered intraperitoneally at the time of induction of ischemia, or these NAC derivatives (50 mg/kg) were administered 3× at 24-h intervals before induction of ischemia and 1 more administration at the time of induction of ischemia. The size of brain infarction decreased in the order NAC benzyl ester>NAC ethyl ester>NAC in both experimental conditions. Detoxification of acrolein occurred through conjugation of acrolein with glutathione, which was catalyzed by glutathione S-transferases, rather than direct conjugation between acrolein and NAC derivatives. The level of glutathione S-transferases at the locus of brain infarction was in the order of administration of NAC benzyl ester>NAC ethyl ester>NAC>no NAC derivatives, suggesting that NAC derivatives stabilize glutathione S-transferases. CONCLUSIONS: The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione S-transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.
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Acrolein toxicity at advanced age: present and future. 査読あり
Igarashi Kazuei, Uemura Takeshi, Kashiwagi Keiko
Amino Acids 50 ( 2 ) 217 - 228 2018年02月
記述言語：英語 掲載種別：研究論文（学術雑誌）
It is thought that tissue damage at advanced age is mainly caused by ROS (reactive oxygen species, O(2)(-), H(2)O(2), and ·OH). However, it was found that acrolein (CH(2)=CH-CHO) is more toxic than ROS, and is mainly produced from spermine (SPM), one of the polyamines, rather than from unsaturated fatty acids. Significant amounts of SPM are present normally as SPM-ribosome complexes, and contribute to protein synthesis. However, SPM was released from ribosomes due to the degradation of ribosomal RNA by ·OH or the binding of Ca(2+) to ribosomes, and acrolein was produced from free SPM by polyamine oxidases, particularly by SPM oxidase. Acrolein inactivated several proteins such as GAPDH (glycelaldehyde-3-phosphate dehydrogenase), and also stimulated MMP-9 (matrix metalloproteinase-9) activity. Acrolein-conjugated GAPDH translocated to nucleus, and caused apoptosis like nitrosylated GAPDH. Through acrolein conjugation with several proteins, acrolein causes tissue damage during brain stroke, dementia, renal failure, and primary Sjögren's syndrome. Thus, development of acrolein scavengers with less side effects is very important to maintain QOL (quality of life) of elderly people.
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Acrolein: An Effective Biomarker for Tissue Damage Produced from Polyamines. 査読あり
Igarashi Kazuei, Uemura Takeshi, Kashiwagi Keiko
Methods Mol Biol 1694 459 - 468 2018年
記述言語：英語 掲載種別：研究論文（学術雑誌）
It is thought that the major factor responsible for cell damage is reactive oxygen species (ROS), but our recent studies have shown that acrolein (CH(2)=CH-CHO) produced from spermine and spermidine is more toxic than ROS. Thus, (1) the mechanism of acrolein production during brain stroke, (2) one of the mechanisms of acrolein toxicity, and (3) the role of glutathione in acrolein detoxification are described in this chapter.
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加齢による脳梗塞悪化メカニズムと新規脳梗塞予防薬の探索 招待あり 査読あり
植村武史、渡辺健太、柏木敬子、五十嵐一衛
未病と抗老化 27 39 - 43 2018年
担当区分：筆頭著者 記述言語：日本語 掲載種別：研究論文（学術雑誌）
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Spermine oxidase promotes bile canalicular lumen formation through acrolein production. 査読あり
Uemura Takeshi, Takasaka Tomokazu, Igarashi Kazuei, Ikegaya Hiroshi
Sci Rep 7 ( 1 ) 14841 - 14841 2017年11月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Spermine oxidase (SMOX) catalyzes oxidation of spermine to generate spermidine, hydrogen peroxide (H(2)O(2)) and 3-aminopropanal, which is spontaneously converted to acrolein. SMOX is induced by a variety of stimuli including bacterial infection, polyamine analogues and acetaldehyde exposure. However, the physiological functions of SMOX are not yet fully understood. We investigated the physiological role of SMOX in liver cells using human hepatocellular carcinoma cell line HepG2. SMOX localized to the bile canalicular lumen, as determined by F-actin staining. Knockdown of SMOX reduced the formation of bile canalicular lumen. We also found that phospho-Akt (phosphorylated protein kinase B) was localized to canalicular lumen. Treatment with Akt inhibitor significantly reduced the formation of bile canalicular lumen. Acrolein scavenger also inhibited the formation of bile canalicular lumen. PTEN, phosphatase and tensin homolog and an inhibitor of Akt, was alkylated in a SMOX-dependent manner. Our results suggest that SMOX plays a central role in the formation of bile canalicular lumen in liver cells by activating Akt pathway through acrolein production.
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Determination of 3-hydroxypropylmercapturic acid in urine by three column-switching high-performance liquid chromatography with electrochemical detection using a diamond electrode. 査読あり
Higashi Kyohei, Shibasaki Mana, Kuni Kyoshiro, Uemura Takeshi, Waragai Masaaki, Uemura Kenichi, Igarashi Kazuei, Toida Toshihiko
J Chromatogr A 1517 79 - 85 2017年09月
記述言語：英語 掲載種別：研究論文（学術雑誌）
A three column-switching high-performance liquid chromatography (HPLC) using an electrochemical detector (ECD) equipped with a diamond electrode was established to determine 3-hydroxypropylmercapturic acid (3-HPMA) in urine. An extracted urine sample was consecutively fractionated using a strong anion-exchange column (first column) and a C8 column (second column) via a switching valve before application on an Octa Decyl Silyl (ODS) column (third column), followed by ECD analysis. The% recovery of 3-HPMA standard throughout the three-column process and limit of detection (LOD) were 94±1% and 0.1pmol, respectively. A solid phase extraction step is required for the sensitive analysis of 3-HPMA in urine by column-switching HPLC-ECD despite a decreased% recovery (55%) of urine sample spiked with 100pmol of 3-HPMA. To test the utility of our column-switching HPLC-ECD method, 3-HPMA levels of 27 urine samples were determined, and the correlation between HPLC-ECD and LC-Electrospray ionization (ESI)-MS/MS method was examined. As a result, the median values of μmol 3-HPMA/g Creatinine (Cre) in urine obtained by column-switching HPLC-ECD and LC-MS/MS were 2.19±2.12μmol/g Cre and 2.13±3.38μmol/g Cre, respectively, and the calibration curve (y=1.5171x-1.007) exhibited good linearity within a defined range (r(2)=0.907). These results indicate that the combination of column-switching HPLC and ECD is a powerful tool for the specific, reliable detection of 3-HPMA in urine.
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Activation of MMP-9 activity by acrolein in saliva from patients with primary Sjögren's syndrome and its mechanism. 査読あり
Uemura Takeshi, Suzuki Takehiro, Saiki Ryotaro, Dohmae Naoshi, Ito Satoshi, Takahashi Hoyu, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
Int J Biochem Cell Biol 88 84 - 91 2017年07月
記述言語：英語 掲載種別：研究論文（学術雑誌）
We have recently reported that the altered recognition patterns of immunoglobulins due to acrolein conjugation are at least partially responsible for autoimmune diseases in patients with primary Sjögren's syndrome (pSS). In the current study, it was found that the specific activity (activity/ng protein) of metalloproteinase-9 (MMP-9) in saliva was elevated about 2.4-fold in pSS patients. Accordingly, it was examined whether MMP-9 is activated by acrolein. It was found that the MMP-9 with 92kDa molecular weight was activated by acrolein. Under the conditions studied, Cys99, located in the propeptide, was conjugated with acrolein together with Cys230, 244, 302, 314, 329, 347, 361, 373, 388 and 516, which are located in fibronectin repeats and glycosyl domains, but not on the active site of MMP-9. In addition, 82 and 68kDa constructs of MMP-9s, lacking the NH(2)-terminal domain that contains Cys99, were not activated by acrolein. The results suggest that acrolein conjugation at Cys99 caused the active site of MMP-9 to be exposed. Activation of MMP-9 by acrolein was inhibited by cysteine, and slightly by lysine, because these amino acids inhibited acrolein conjugation with MMP-9. Conversely, MMP-9 activity in the presence of 50μM acrolein was enhanced by 100μM histidine. This was due to the inhibition of acrolein conjugation with His405 and 411 located at the Zn(2+) binding site of MMP-9. These results suggest that activation of 92kDa MMP-9 by acrolein is involved in tissue damage in pSS patients and is regulated by cysteine and histidine, and slightly by lysine. Activated 82 and 68kDa MMP-9s were not detected in saliva of pSS patients by Western blotting.
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Time dependent transition of the levels of protein-conjugated acrolein (PC-Acro), IL-6 and CRP in plasma during stroke. 査読あり
Yoshida Madoka, Kato Naoki, Uemura Takeshi, Mizoi Mutsumi, Nakamura Mizuho, Saiki Ryotaro, Hatano Keisuke, Sato Kunitomo, Kakizaki Shota, Nakamura Aya, Ishii Takuya, Terao Tohru, Murayama Yuichi, Kashiwagi Keiko, Igarashi Kazuei
eNeurologicalSci 7 18 - 24 2017年06月
記述言語：英語 掲載種別：研究論文（学術雑誌）
OBJECTIVE: Measurement of plasma levels of protein-conjugated acrolein (PC-Acro) together with IL-6 and CRP can be used to identify silent brain infarction (SBI) with high sensitivity and specificity. The aim of this study was to determine how these biomarkers vary during stroke. METHODS: Levels of PC-Acro, IL-6 and CRP in plasma were measured on day 0, 2, 7 and 14 after the onset of ischemic or hemorrhagic stroke. RESULTS: After the onset of stroke, the level of PC-Acro in plasma was elevated corresponding to the size of stroke. It returned to near control levels by day 2, and remained similar through day 14. The degree of the decrease in PC-Acro on day 2 was greater when the size of brain infarction or hemorrhage was larger. An increase in IL-6 and CRP occurred after the increase in PC-Acro, and it was well correlated with the size of the injury following infarction or hemorrhage. The results suggest that acrolein becomes a trigger for the production of IL-6 and CRP, as previously observed in a mouse model of stroke and in cell culture systems. The increase in IL-6 and CRP was also correlated with poor outcome judging from mRS. CONCLUSION: The results indicate that the degree of the decrease in PC-Acro and the increase in IL-6 and CRP from day 0 to day 2 was correlated with the size of brain infarction, and the increase in IL-6 and CRP with poor outcome at discharge.
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Correlation between brain damage, associated biomarkers, and medication in psychiatric inpatients: A cross-sectional study. 査読あり
Yoshida Madoka, Kanzaki Tetsuto, Mizoi Mutsumi, Nakamura Mizuho, Uemura Takeshi, Mimori Seisuke, Uju Yoriyasu, Sekine Keisuke, Ishii Yukihiro, Yoshimi Taro, Yasui Reiko, Yasukawa Asuka, Sato Mamoru, Okamoto Seiko, Hisaoka Tetsuya, Miura Masafumi, Kusanishi Shun, Murakami Kanako, Nakano Chieko, Mizuta Yasuhiko, Mishima Shunichi, Hayakawa Tatsuro, Tsukada Kazumi, Kashiwagi Keiko, Igarashi Kazuei
Clin Chim Acta 464 50 - 56 2017年01月
記述言語：英語 掲載種別：研究論文（学術雑誌）
BACKGROUND: We clarified the correlation between brain damage, associated biomarkers and medication in psychiatric patients, because patients with schizophrenia have an increased risk of stroke. METHODS: The cross-sectional study was performed from January 2013 to December 2015. Study participants were 96 hospitalized patients (41 men and 55 women) in the Department of Psychiatry at Kohnodai Hospital, National Center for Global Health and Medicine, Ichikawa, Chiba, Japan. Patients were classified into schizophrenia (n=70) and mood disorders (n=26) by psychiatric diagnoses with DSM-IV-TR criteria. RESULTS: The incidence of brain damage [symptomatic and silent brain infarctions (SBIs) and white matter hyperintensity (WMH)] was correlated more with mood disorders than with schizophrenia. It has been previously shown that the concentrations of protein-conjugated acrolein (PC-Acro) and interleukin-6 (IL-6) increased in plasma of brain infarction patients together with C-reactive protein (CRP). The concentration of PC-Acro was significantly higher in patients with mood disorders than in those with schizophrenia. The concentration of IL-6 in both groups was nearly equal to that in the control group, but that of CRP in both groups, especially in mood disorders, was higher than that in the control group. Accordingly, the relative risk value for brain infarction was higher in patients with mood disorders than with schizophrenia. Medication with atypical antipsychotics reduced PC-Acro significantly in all psychiatric patients and reduced IL-6 in mood disorder patients. CONCLUSION: Measurement of 3 biomarkers (CRP, PC-Acro and IL-6) are probably useful for judgement of severity of brain damage and effectiveness of medication in psychiatric patients.
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Decrease in acrolein toxicity based on the decline of polyamine oxidases. 査読あり
Uemura Takeshi, Nakamura Mizuho, Sakamoto Akihiko, Suzuki Takehiro, Dohmae Naoshi, Terui Yusuke, Tomitori Hideyuki, Casero Robert A Jr, Kashiwagi Keiko, Igarashi Kazuei
Int J Biochem Cell Biol 79 151 - 157 2016年10月
記述言語：英語 掲載種別：研究論文（学術雑誌）
We have shown recently that acrolein is strongly involved in cell damage during brain infarction and chronic renal failure. To study the mechanism of acrolein detoxification, we tried to isolate Neuro2a cells with reduced sensitivity to acrolein toxicity (Neuro2a-ATD cells). In one cell line, Neuro2a-ATD1, the level of glutathione (GSH) was increased. We recently isolated a second cell line, Neuro2a-ATD2, and found that acrolein-producing enzymes [polyamine oxidases (PAO); i.e. acetylpolyamine oxidase (AcPAO), and spermine oxidase (SMO)] are reduced in this cell line due to changes at the level of transcription. In the Neuro2a-ATD2 cells, the IC(50) of acrolein increased from 4.2 to 6.8μM, and the levels of FosB and C/EBPβ - transcription factors involved in the transcription of AcPAO and SMO genes - were reduced. Transfection of siRNAs for FosB and C/EBPβ reduced the levels of AcPAO and SMO, respectively. In addition, the synthesis of FosB and AcPAO was also decreased by siRNA for C/EBPβ, because C/EBPβ is one of the transcription factors for the FosB gene. It was also found that transfection of siRNA for C/EBPβ decreased SMO promoter activity in Neuro2a cells but not in ATD2 cells confirming that a decrease in C/EBPβ is involved in the reduced SMO activity in Neuro2a-ATD2 cells. Furthermore, transfection of the cDNA for AcPAO or SMO into Neuro2a cells increased the toxicity of acrolein. These results suggest that acrolein is mainly produced from polyamines by PAO.
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Aggravation of brain infarction through an increase in acrolein production and a decrease in glutathione with aging. 査読あり
Uemura Takeshi, Watanabe Kenta, Ishibashi Misaki, Saiki Ryotaro, Kuni Kyoshiro, Nishimura Kazuhiro, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
Biochem Biophys Res Commun 473 ( 2 ) 630 - 635 2016年04月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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.
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Toxic acrolein production due to Ca(2+) influx by the NMDA receptor during stroke. 査読あり
Nakamura Mizuho, Uemura Takeshi, Saiki Ryotaro, Sakamoto Akihiko, Park Hyerim, Nishimura Kazuhiro, Terui Yusuke, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
Atherosclerosis 244 131 - 137 2016年01月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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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Increase in acrolein-conjugated immunoglobulins in saliva from patients with primary Sjögren's syndrome. 査読あり
Hirose Tadao, Saiki Ryotaro, Uemura Takeshi, Suzuki Takehiro, Dohmae Naoshi, Ito Satoshi, Takahashi Hoyu, Ishii Itsuko, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
Clin Chim Acta 450 184 - 189 2015年10月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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 Sjögren's 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 Sjögren's 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.
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Raman spectroscopy of human skin: looking for a quantitative algorithm to reliably estimate human age. 査読あり
Pezzotti Giuseppe, Boffelli Marco, Miyamori Daisuke, Uemura Takeshi, Marunaka Yoshinori, Zhu Wenliang, Ikegaya Hiroshi
J Biomed Opt 20 ( 6 ) 065008 - 065008 2015年06月
記述言語：英語 掲載種別：研究論文（学術雑誌）
The possibility of examining soft tissues by Raman spectroscopy is challenged in an attempt to probe human age for the changes in biochemical composition of skin that accompany aging. We present a proof-of-concept report for explicating the biophysical links between vibrational characteristics and the specific compositional and chemical changes associated with aging. The actual existence of such links is then phenomenologically proved. In an attempt to foster the basics for a quantitative use of Raman spectroscopy in assessing aging from human skin samples, a precise spectral deconvolution is performed as a function of donors' ages on five cadaveric samples, which emphasizes the physical significance and the morphological modifications of the Raman bands. The outputs suggest the presence of spectral markers for age identification from skin samples. Some of them appeared as authentic "biological clocks" for the apparent exactness with which they are related to age. Our spectroscopic approach yields clear compositional information of protein folding and crystallization of lipid structures, which can lead to a precise identification of age from infants to adults. Once statistically validated, these parameters might be used to link vibrational aspects at the molecular scale for practical forensic purposes.
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Experimental studies of remarkable monoamine releases and neural resistance to the transient ischemia and reperfusion. 査読あり
Yoshimoto Kanji, Namera Akira, Arima Yousuke, Nagao Takahiro, Saji Hiroh, Takasaka Tomokazu, Uemura Takeshi, Watanabe Yoshihisa, Ueda Shuichi, Nagao Masataka
Pathophysiology 21 ( 4 ) 309 - 316 2014年11月
記述言語：英語 掲載種別：研究論文（学術雑誌）
INTRODUCTION: The literature described that neural damage caused by ischemia definitely occurs in brain areas. However, few studies have shown real-time changes of extracellular monoamine levels at the time of transient ischemia. METHODS: We examined changes in the responses of dopamine (DA) and serotonin (5-HT) release in the nucleus accumbens (ACC) of rats treated with four-vessel occlusion (4VO) in experiment 1. In the second experiment, we investigated the selective neural vulnerabilities among the ACC, lateral hypothalamus (LH), and frontal cortex (FC) of rats treated with 4VO and four days of reperfusion. RESULTS: The extracellular levels of DA and 5-HT were remarkably increased 200- and 20-fold upon the 10-min clipping of both common carotid arteries in transient cerebral ischemia, respectively. Each increased monoamine release returned to the baseline levels immediately. The release of DA in the ACC and FC was significantly decreased in the rats treated with the coagulation of bilateral vertebral arteries (2VO), compared with that of sham-operated rats. K(+)-induced DA release in the ACC and FC of 4VO-treated rats was increased without alteration of DA content. DISCUSSION: Surviving dopaminergic neurons in the ACC and FC showed neural hyperfunction associated with the monoamine release, serotonergic neurons in particular these areas exhibiting functional resistance to the transient ischemic change. CONCLUSION: It is suggested that the remarkable extracellular release of DA and 5-HT was not the cause of the ischemic delayed neural degeneration in each brain area, and that the functions of neurotransmitter release involved remarkable resistance to the transient ischemia.
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Identification of functional amino acid residues involved in polyamine and agmatine transport by human organic cation transporter 2. 査読あり
Higashi Kyohei, Imamura Masataka, Fudo Satoshi, Uemura Takeshi, Saiki Ryotaro, Hoshino Tyuji, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
PLoS One 9 ( 7 ) e102234 - e102234 2014年
記述言語：英語 掲載種別：研究論文（学術雑誌）
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.
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Acetaldehyde-induced cytotoxicity involves induction of spermine oxidase at the transcriptional level. 査読あり
Uemura Takeshi, Tanaka Yuka, Higashi Kyohei, Miyamori Daisuke, Takasaka Tomokazu, Nagano Tatsuo, Toida Toshihiko, Yoshimoto Kanji, Igarashi Kazuei, Ikegaya Hiroshi
Toxicology 310 1 - 7 2013年08月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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.
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Polyamine transport systems in mammalian cells and tissues. 査読あり
Uemura Takeshi, Gerner Eugene W
Methods Mol Biol 720 339 - 348 2011年
記述言語：英語 掲載種別：研究論文（学術雑誌）
Polyamine transport plays an important role in the homeostatic regulation of the polyamine levels. In animals, dietary polyamines are absorbed efficiently in the intestinal tract. In the colon, luminal bacterial derived polyamines are important contributors to cellular polyamine contents. Polyamine transport involves unique uptake and export mechanisms. The amino acid transporter SLC3A2 acts as a polyamine exporter in colon cancer-derived cells. Polyamine uptake is mediated by caveolin-1 dependent -endocytosis. The K-RAS oncogene signals increased polyamine uptake and decreased polyamine export. Here, we describe the methods of polyamine transport analysis in the colon and the small intestine using -membrane vesicles, culture cells, and mouse models.
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Identification and functions of amino acid residues in PotB and PotC involved in spermidine uptake activity. 査読あり
Higashi Kyohei, Sakamaki Yoshiharu, Herai Emiko, Demizu Risa, Uemura Takeshi, Saroj Sunil D, Zenda Risa, Terui Yusuke, Nishimura Kazuhiro, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
J Biol Chem 285 ( 50 ) 39061 - 39069 2010年12月
記述言語：英語 掲載種別：研究論文（学術雑誌）
Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.
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Polyamine transport is mediated by both endocytic and solute carrier transport mechanisms in the gastrointestinal tract. 査読あり
Uemura Takeshi, Stringer David E, Blohm-Mangone Karen A, Gerner Eugene W
Am J Physiol Gastrointest Liver Physiol 299 ( 2 ) G517 - G522 2010年08月
記述言語：英語 掲載種別：研究論文（学術雑誌）
The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.
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Polyamine modulon in yeast-Stimulation of COX4 synthesis by spermidine at the level of translation. 査読あり
Uemura Takeshi, Higashi Kyohei, Takigawa Miki, Toida Toshihiko, Kashiwagi Keiko, Igarashi Kazuei
Int J Biochem Cell Biol 41 ( 12 ) 2538 - 2545 2009年12月
記述言語：英語 掲載種別：研究論文（学術雑誌）
We proposed that a group of genes whose expression is enhanced by polyamines at the level of translation in Escherichia coli and mammalian cells be referred to as a "polyamine modulon". In Saccharomyces cerevisiae, proteins whose synthesis is enhanced by polyamines at the level of translation were searched for using a polyamine-requiring mutant of S. cerevisiae deficient in ornithine decarboxylase (YPH499 Deltaspe1). Addition of spermidine to the medium recovered the spermidine content and enhanced cell growth of the YPH499 Deltaspe1 mutant by 3-5-fold. Under these conditions, synthesis of COX4, one of the subunits of cytochrome C oxidase (complex IV), was enhanced by polyamines about 2.5-fold at the level of translation. Accordingly, the COX4 gene is the first member of a polyamine modulon in yeast. Polyamines enhanced COX4 synthesis through stimulation of the ribosome shunting of the stem-loop structures (hairpin structures) during the scanning of the 5'-untranslated region (5'-UTR) of COX4 mRNA by 40S ribosomal subunit-Met-tRNA(i) complex.
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Identification and characterization of a diamine exporter in colon epithelial cells. 査読あり
Uemura Takeshi, Yerushalmi Hagit F, Tsaprailis George, Stringer David E, Pastorian Kirk E, Hawel Leo 3rd, Byus Craig V, Gerner Eugene W
J Biol Chem 283 ( 39 ) 26428 - 26435 2008年09月
記述言語：英語 掲載種別：研究論文（学術雑誌）
SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N(1)-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.
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Identification of a spermidine excretion protein complex (MdtJI) in Escherichia coli. 査読あり
Higashi Kyohei, Ishigure Hiroyuki, Demizu Risa, Uemura Takeshi, Nishino Kunihiko, Yamaguchi Akihito, Kashiwagi Keiko, Igarashi Kazuei
J Bacteriol 190 ( 3 ) 872 - 878 2008年02月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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.
DOI： 10.1128/JB.01505-07
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Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae. 査読あり
Uemura Takeshi, Kashiwagi Keiko, Igarashi Kazuei
J Biol Chem 282 ( 10 ) 7733 - 7741 2007年03月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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 K(m) values for putrescine and spermidine of DUR3 were 479 and 21.2 mum, respectively, and those of SAM3 were 433 and 20.7 mum, 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 Thr(250), Ser(251), and Thr(684) by polyamine transport protein kinase 2.
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Identification of the cadaverine recognition site on the cadaverine-lysine antiporter CadB. 査読あり
Soksawatmaekhin Waraporn, Uemura Takeshi, Fukiwake Natsuko, Kashiwagi Keiko, Igarashi Kazuei
J Biol Chem 281 ( 39 ) 29213 - 29220 2006年09月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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 Tyr(73), Tyr(89), Tyr(90), Glu(204), Tyr(235), Asp(303), and Tyr(423) were strongly involved in both uptake and excretion and that Tyr(55), Glu(76), Tyr(246), Tyr(310), Cys(370), and Glu(377) were moderately involved in both activities. Mutations of Trp(43), Tyr(57), Tyr(107), Tyr(366), and Tyr(368) mainly affected uptake activity, and Trp(41), Tyr(174), Asp(185), and Glu(408) had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the K(m) value. Mutation of Arg(299) mainly affected excretion, suggesting that Arg(299) 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 Cys(370) seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys(125), Cys(389), or Cys(394) together with Cys(370). 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 Cys(370). The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII.
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Excretion of putrescine and spermidine by the protein encoded by YKL174c (TPO5) in Saccharomyces cerevisiae. 査読あり
Tachihara Ken, Uemura Takeshi, Kashiwagi Keiko, Igarashi Kazuei
J Biol Chem 280 ( 13 ) 12637 - 12642 2005年04月
記述言語：英語 掲載種別：研究論文（学術雑誌）
The properties of the protein encoded by YKL174c (TPO5) were studied. It was found that TPO5 excretes putrescine effectively and spermidine less effectively. Gamma-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.
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Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae. 査読あり
Uemura Takeshi, Kashiwagi Keiko, Igarashi Kazuei
Biochem Biophys Res Commun 328 ( 4 ) 1028 - 1033 2005年03月
記述言語：英語 掲載種別：研究論文（学術雑誌）
It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.
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Characteristics of the polyamine transporter TPO1 and regulation of its activity and cellular localization by phosphorylation. 査読あり
Uemura Takeshi, Tachihara Ken, Tomitori Hideyuki, Kashiwagi Keiko, Igarashi Kazuei
J Biol Chem 280 ( 10 ) 9646 - 9652 2005年03月
記述言語：英語 掲載種別：研究論文（学術雑誌）
The subcellular localization of the polyamine transporter TPO1 of Saccharomyces cerevisiae was determined by sucrose gradient centrifugation and indirect immunofluorescence microscopy. When expressed from a multi-copy vector, TPO1 was located mainly on the plasma membrane, but with some localization on the vacuolar membrane. Polyamine transport by TPO1 was dependent on pH. Uptake of spermidine and spermine occurred at alkaline pH (pH 8.0), whereas inhibition of spermidine uptake, but not spermine uptake, was observed at acidic pH (pH 5.0). This suggests that TPO1 catalyzes polyamine excretion at acidic pH, similar to the PotE transporter in Escherichia coli. Paraquat, a polyamine analogue, was excreted by TPO1 at a rate comparable with the excretion of spermidine (deduced from the inhibition of spermidine uptake) at pH 5.0. However, excretion of preloaded radiolabeled spermidine and spermine was not observed in intact cells, suggesting that preloaded spermidine (or spermine) exists mainly as spermidine (or spermine)-ribosome complex in cells. The transport activity of TPO1 was enhanced through phosphorylation at Ser19 by protein kinase C and at Thr52 by casein kinase 1. Sorting of TPO1 from the endoplasmic reticulum to the plasma membrane was enhanced through phosphorylation at Ser342 by cAMP-dependent protein kinases 1 and 2.
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Uptake of GABA and putrescine by UGA4 on the vacuolar membrane in Saccharomyces cerevisiae. 査読あり
Uemura Takeshi, Tomonari Yuki, Kashiwagi Keiko, Igarashi Kazuei
Biochem Biophys Res Commun 315 ( 4 ) 1082 - 1087 2004年03月
記述言語：英語 掲載種別：研究論文（学術雑誌）
The product of the UGA4 gene in Saccharomyces cerevisiae, which catalyzes the transport of 4-aminobutyric acid (GABA), also catalyzed the transport of putrescine. The Km values for GABA and putrescine were 0.11 and 0.69 mM, respectively. The UGA4 protein was located on the vacuolar membrane as determined by the effects of bafilomycin A1 and by indirect immunofluorescence microscopy. Uptake of both GABA and putrescine was inhibited by spermidine and spermine, although these polyamines are not substrates of UGA4. The UGA4 mRNA was induced by exposure to GABA, but not putrescine over 12h. The growth of an ornithine decarboxylase-deficient strain was enhanced by putrescine, and both putrescine and spermidine contents increased, when the cells were expressing UGA4. The results suggest that a substantial conversion of putrescine to spermidine occurs in the cytoplasm even though UGA4 transporter exists on vacuolar membranes.