論文 - 植村 武史
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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.