Kyushu University Academic Staff Educational and Research Activities Database
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Yoshinori Fujimura Last modified date:2022.06.14

Associate Professor / Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture
Department of Bioscience and Biotechnology
Faculty of Agriculture

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Academic Degree
PhD, Agriculture
Country of degree conferring institution (Overseas)
Field of Specialization
Food Preventive Medicine, Food Chemical Biology, Nutrimetabolomics
ORCID(Open Researcher and Contributor ID)
Total Priod of education and research career in the foreign country
Research Interests
  • R&D of high-precision metabolic profiling system by multiple mass spectrometric analysis
    keyword : Metabolic profiling, Metabolomics, Mass spectrometry, Redox-related diseases, Biomarker, Pathological diagnosis
  • Chemical biology based food preventive medicine research
    keyword : Chemical biology, Target molecule, Preventive medicine, Nutrimetabolomics, Green tea catechin
Academic Activities
1. Fujimura Y, Tachibana H, Genomics, Proteomics and Metabolomics in Nutraceuticals and Functional Foods, 2nd Edition, Wiley-Blackwell, 2015.09.
2. Fujimura, Y. and Tachibana, H., Handbook of Green Tea and Health Research, Nova Science Publishers Inc., 2009.05.
1. Fujimura, Y. and Tachibana, H., Molecular basis for anti-cancer activity of EGCG in vivo: Molecular-targeting prevention of cancer by green tea catechin, 2009.05.
1. Metabolic profiling for evaluating the dipeptidyl peptidase-IV inhibitory potency of diverse green tea cultivars and determining bioactivity-related ingredients and combinations, Fustin, a flavanonol, synergically potentiates the anti-cancer effect of the green tea catechin EGCG with activation of the eNOS/cGMP axis, J. Agric. Food Chem., doi: 10.1021/acs.jafc.2c01693, 70, 21, 6455-6466, (Cover selected), 2022.06.
2. Shimada Y, Sato Y, Kumazoe M, Kitamura R, Fujimura Y, Tachibana H., Myricetin improves cognitive function in SAMP8 mice and upregulates brain derived neurotrophic factor and nerve growth factor, Biochem. Biophys. Res. Commun., doi: 10.1016/j.bbrc.2022.05.039, 616, 33-40, 2022.05.
3. Kumazoe M, Fujimura Y (Equal contribution), Yoshitomi R, Shimada Y, Shimada Y, Tachibana H., Fustin, a flavanonol, synergically potentiates the anti-cancer effect of the green tea catechin EGCG with activation of the eNOS/cGMP axis, J. Agric. Food Chem., doi: 10.1021/acs.jafc.1c07567, 70, 11, 3458-3466, (Cover selected), 2022.02.
4. Marugame Y, Takeshita N, Yamada S, Yoshitomi R, Kumazoe M, Fujimura Y, Tachibana H., Sesame lignans upregulate glutathione S-transferase expression and downregulate microRNA-669c-3p, Bioscience of Microbiota, Food and Health, doi: 10.12938/bmfh.2021-067, 41, 2, 66-72, 2022.01.
5. Kumazoe M, Takatatsu K, Yoshitomi R, Fujimura Y, Tachibana H., Methylated (−)-epigallocatechin 3-O-gallate potentiates the effect of split vaccine accompanied with upregulation of Toll-like receptor 5, Sci. Rep., doi: 10.1038/s41598-021-02346-4, 11, 1, 23101-1042, 2021.11.
6. Motofumi Kumazoe, Yasutake Tanaka, Ren Yoshitomi, Yuki Marugame, Kwan-Woo Lee, Hiroaki Onda, Yoshinori Fujimura, Madoka Yonekura, Yasuyo Shimamoto, Hirofumi Tachibana, Glucosyl-hesperidin enhances the cyclic guanosine monophosphate-inducing effect of a green tea polyphenol EGCG., Journal of Natural Medicines, 10.1007/s11418-021-01538-6, 75, 4, 1037-1042, 2021.09, Animal and clinical studies have revealed that (-)-epigallocatechin-3-O-gallate (EGCG), one of the major bioactive polyphenols in green tea, showed several pharmacological effects including anti-obesity effect and anti-inflammatory effect. We previously reported that the second messenger cyclic guanosine monophosphate (cGMP) mediates its anti-inflammatory and anti-cancer properties. Here we demonstrated that glucosyl-hesperidin, enhances the cGMP-inducing effects of green tea extract in vivo. Moreover, glucosyl-hesperidin intake potentiated the green tea-elicited upregulation of the anti-inflammatory factor, toll-interacting protein..
7. Ren Yoshitomi, Mao Yamamoto, Motofumi Kumazoe, Yoshinori Fujimura, Madoka Yonekura, Yasuyo Shimamoto, Akari Nakasone, Satoshi Kondo, Hiroki Hattori, Akane Haseda, Jun Nishihira, Hirofumi Tachibana, The combined effect of green tea and α-glucosyl hesperidin in preventing obesity: a randomized placebo-controlled clinical trial., Scientific Reports, 10.1038/s41598-021-98612-6, 11, 1, 19067-19067, 2021.09, Green tea, a widely consumed beverage in Asia, contains green tea catechins effective against obesity, especially epigallocatechin-3-O-gallate (EGCG), but must be consumed in an impractically huge amount daily to elicit its biological effect. Meanwhile, citrus polyphenols have various physiological effects that could enhance EGCG functionality. Here we investigated the antiobesity effect of a combination of EGCG and α-glucosyl hesperidin, a citrus polyphenol, at doses that have not been previously reported to exert antiobesity effects by themselves in any clinical trial. In a randomized, placebo-controlled, double-blinded, and parallel-group-designed clinical trial, 60 healthy Japanese males and females aged 30-75 years consumed green tea combined with α-glucosyl hesperidin (GT-gH), which contained 178 mg α-glucosyl hesperidin and 146 mg EGCG, for 12 weeks. Physical, hematological, blood biochemical, and urine examinations showed that GT-gH is safe to use. At week 12, GT-gH prevented weight gain and reduced body mass index (BMI) compared with the placebo. Especially in those aged < 50 years, triglyceride and body fat percentage decreased at week 6, visceral fat level and body fat percentage decreased at week 12; body weight, BMI, and blood LDL/HDL ratio also decreased. In conclusion, taking GT-gH prevents weight gain, and the antiobesity effect of GT-gH was more pronounced in people aged < 50 years..
8. Kumazoe M, Tanaka Y, Yoshitomi R, Marugame Y, Lee KW, Onda H, Fujimura Y, Yonekura M, Shimamoto Y, Tachibana H., Glucosyl-hesperidin enhances the cyclic guanosine monophosphate-inducing effect of a green tea polyphenol EGCG., J Nat Med, doi: 10.1007/s11418-021-01538-6, 75, 1037-1042, 2021.06.
9. Yoshinori Fujimura, Konatsu Fujino, Takanori Yoshimoto, Ayaka Nezu, Yuki Marugame, Jaehoon Bae, Motofumi Kumazoe, Hirofumi Tachibana, Eriodictyol-Amplified 67-kDa Laminin Receptor Signaling Potentiates the Antiallergic Effect of O-Methylated Catechin., Journal of Natural Products, 10.1021/acs.jnatprod.1c00337, 84, 6, 1823-1830, (Cover selected), 2021.06, (-)-Epigallocatechin-3-O-(3-O-methyl) gallate (1, EGCG3″Me), an antiallergic O-methylated catechin, is present in high quantities in the green tea cultivar "Benifuuki" (Camellia sinensis L.). Previous studies have shown that EGCG3″Me inhibited basophil degranulation mediated through the cell-surface 67-kDa laminin receptor (67LR), but the mechanisms are not fully elucidated. This study aimed to investigate the mechanisms underlying the inhibitory effect of EGCG3″Me on IgE/antigen (Ag)-mediated degranulation and the combined effect of EGCG3″Me with eriodictyol (2), a bioactive flavanone. EGCG3″Me inhibited β-hexosaminidase release from the rat basophilic/mast cell line RBL-2H3 stimulated by IgE/Ag and induced acid sphingomyelinase (ASM) activity. This induction was inhibited by anti-67LR antibody treatment. The ASM-specific inhibitor desipramine inhibited EGCG3″Me-induced suppression of degranulation. The soluble guanylate cyclase (sGC) inhibitor NS2028 weakened the potency of EGCG3″Me, and the sGC activator BAY41-2272 suppressed degranulation. The ability of EGCG3″Me to induce ASM activity and inhibit degranulation was amplified by eriodictyol. Furthermore, oral administration of the lemon-peel-derived eriodyctiol-7-O-glucoside (3) potentiated the suppressive effect of EGCG3″Me-rich "Benifuuki" green tea on the IgE/Ag-induced passive cutaneous anaphylaxis (PCA) reaction in BALB/c mice. These results suggest that EGCG3″Me inhibits IgE/Ag-mediated degranulation by inducing the 67LR/sGC/ASM signaling pathway, and eriodictyol amplifies this signaling..
10. Kumazoe M, Kadomatsu M, Bae J, Otsuka Y, Fujimura Y, Tachibana H, Src mediates epigallocatechin-3-O-gallate-elicited acid sphingomyelinase activation, Molecules, 25, 5481, 2020.11.
11. Kumazoe M, Fujimura Y, Tachibana H, 67-kDa Laminin Receptor mediates the beneficial effects of Green tea polyphenol EGCG, Curr. Pharmacol. Rep, 6, 280-285, 2020.07.
12. Motofumi Kumazoe, Shun Hiroi, Yousuke Tanimoto, Jyunichi Miyakawa, Maasa Yamanouchi, Yumi Suemasu, Ren Yoshitomi, Motoki Murata, Yoshinori Fujimura, Takashi Takahashi, Hiroshi Tanaka, Hirofumi Tachibana, Cancer cell selective probe by mimicking EGCG., Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2020.03.021, 525, 4, 974-981, 2020.05, Targeting proteins that are overexpressed in cancer cells is the major strategy of molecular imaging and drug delivery systems. The 67-kDa laminin receptor (67LR), also known as oncofetal antigen, is overexpressed in several types of cancer, including melanoma, multiple myeloma, cervical cancer and bile duct carcinoma. 67LR is involved in tumour growth, tumour metastasis and drug resistance. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) directly binds to cell-surface 67LR and induces apoptosis through the protein kinase B (Akt)/endothelial nitric oxide synthase/nitric oxide/cyclic GMP (cGMP) axis. Here we report the optimum hydroxyl group for the utilization of EGCG as a novel fluorescent EGCG-mimic imaging probe based on 67LR agonist characters, including Akt activation and inhibitory effect on viable cell number in cancer cells. 67LR specific targeting is unambiguously confirmed with the use of a non-labelled EGCG competitive assay and 67LR knockdown. Importantly, this probe strongly binds to multiple myeloma cells compared with its binding to normal cells..
13. Yoshitomi R, Nakayama K, Yamashita S, Kumazoe M, Lin TA, Mei CY, Marugame Y, Fujimura Y, Maeda-Yamamoto M, Kuriyama S, *Tachibana H, Plasma Homocysteine Concentration is Associated with the Expression Level of Folate Receptor 3, Sci Rep, 10.1038/s41598-020-67288-9, 10, 1, 2020.06.
14. Motoki Murata, Yuki Shimizu, Yuki Marugame, Ayaka Nezu, Konatsu Fujino, Shuhei Yamada, Motofumi Kumazoe, Yoshinori Fujimura, Hirofumi Tachibana, EGCG down-regulates MuRF1 expression through 67-kDa laminin receptor and the receptor signaling is amplified by eriodictyol., Journal of Natural Medicines, 10.1007/s11418-020-01417-6, in press, 2020.05, (-)-epigallocatechin-3-O-gallate (EGCG) is a bioactive polyphenol in green tea. Previous studies have demonstrated the beneficial effects of EGCG on muscle mass and muscle atrophy. In the current study, we investigated the mechanisms underlying effect of EGCG on muscle atrophy. It was demonstrated that EGCG suppressed muscle-specific ubiquitin ligase, muscle RING Finger 1 (MuRF1) expression through 67-kDa laminin receptor (67LR). Previous studies have shown that eriodictyol potentiates the anti-tumor activities of EGCG by amplifying 67LR signaling. Therefore, we investigated the effects of EGCG and eriodictyol on the MuRF1 expression in C2C12 myotubes. The combined treatment of EGCG and eriodictyol significantly suppressed MuRF1 expression in dexamethasone-treated C2C12 myotubes. Tail suspension was maintained for 10 consecutive days using C57BL6/J mice, and during this time EGCG and eriodictyol were orally administered. In the gastrocnemius muscle, the muscle mass loss was inhibited by the combination of EGCG and eriodictyol. Therefore, EGCG may prevent muscle atrophy by inducing 67LR signaling and eriodictyol amplifies this pathway..
15. Bae J, Kumazoe M, Murata K, Fujimura Y, Tachibana H., Procyanidin C1 Inhibits Melanoma Cell Growth by Activating 67-kDa Laminin Receptor Signaling, Mol. Nutr. Food Res., 10.1002/mnfr.201900986, 64, 7, e1900986, (Cover selected), 2020.04, Scope: Procyanidin C1 (PC1) is an epicatechin trimer found mainly in grapes that is reported to provide several health benefits. However, little is known about the molecular mechanisms underlying these benefits. The aim of this study is to demonstrate the molecular mechanisms by which PC1 operates. Methods and results: A 67-kDa laminin receptor (67LR) is identified as a cell surface receptor of PC1, with a Kd value of 2.8 µm. PC1 induces an inhibitory effect on growth, accompanied by dephosphorylation of the C-kinase potentiated protein phosphatase-1 inhibitor protein of 17 kDa (CPI17) and myosin regulatory light chain (MRLC) proteins, followed by actin cytoskeleton remodeling in melanoma cells. These actions are mediated by protein kinase A (PKA) and protein phosphatase 2A (PP2A) activation once PC1 is bound to 67LR. Conclusion: It is demonstrated that PC1 elicits melanoma cell growth inhibition by activating the 67LR/PKA/PP2A/CPI17/MRLC pathway..
16. Bae J, Kumazoe M, Takeuchi C, Hidaka S, Fujimura Y, Tachibana H., Epigallocatechin-3-O-gallate induces acid sphingomyelinase activation through activation of phospholipase C, Biochem. Biophys. Res. Commun., 10.1016/j.bbrc.2019.09.102, 520, 1, 186-191, 2019.11, Epigallocatechin-3-O-gallate (EGCG)-induced cyclic guanosine monophosphate (cGMP) plays a crucial role in EGCG-induced cell death in various types of cancer cells. However, little is known regarding the early molecular events after cGMP induction. In this study, we showed that cGMP induction is sufficient to induce the phosphorylation of protein kinase C delta (PKCδ) at Ser664, the crucial kinase for EGCG-induced activation of acid sphingomyelinase (ASM). Using a chemical inhibitor library, we revealed that the inhibitors of the negative regulators of diacylglycerol strongly increase the effect of EGCG. We also showed that EGCG treatment increased phospholipase C (PLC) activity, and the same results were obtained with cGMP inducer treatment. EGCG-induced ASM activation was completely suppressed by pharmacological inhibition of PLC. Collectively, EGCG-induced cGMP activated the cGMP/PLC/PKCδ/ASM signaling axis in multiple myeloma cells..
17. Cuprizone-treated mice, a possible model of schizophrenia, highlighting the simultaneous abnormalities of GABA, serine and glycine in hippocampus..
18. Improvement of Sensitivity and Reproducibility for Imaging of Endogenous Metabolites by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry..
19. Bae J, Kumazoe M, Fujimura Y, Tachibana H, Diallyl disulfide potentiates anti-obesity effect of green tea in high-fat/high-sucrose diet-induced obesity, J Nutr Biochem, 10.1016/j.jnutbio.2018.10.014, 64, 152-161, 2019.02.
20. Masafumi Wasai, Yoshinori Fujimura, Haruna Nonaka, Ryo Kitamura, Motoki Murata, Hirofumi Tachibana, Postprandial glycaemia-lowering effect of a green tea cultivar Sunrouge and cultivar-specific metabolic profiling for determining bioactivity-related ingredients., Scientific Reports, 10.1038/s41598-018-34316-8, 8, 1, 16041-16041, 2018.10, Although the major green tea catechins can inhibit the activity of carbohydrate-hydrolyzing enzymes, there is a paucity of information describing the potential of other green tea ingredients and numerous green tea cultivars. Herein, we reveled that a green tea cultivar Sunrouge significantly suppressed the postprandial blood glucose level in mice. Unlike the most representative Japanese green tea cultivar, Yabukita, the suppression by Sunrouge was observed clearly during the initial period after oral dosing of starch. Sunrouge also strongly inhibited the carbohydrate-hydrolyzing enzymes α-glucosidase and α-amylase when compared with that of Yabukita and many other cultivars. Liquid chromatography-mass spectrometry (LC-MS)-based metabolic profiling (MP) of 42 Japanese green tea cultivars was performed. Multivariate statistical analysis enabled visualization of the differences among cultivars with respect to their ability to inhibit carbohydrate-hydrolyzing activities. Analysis of metabolites, contributing to the discrimination and prediction of the bioactivity of cultivars, showed that O-methylated catechins, epicatechin-3-O-(3-O-methyl) gallate (ECG3"Me) and epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me), were newly identified α-glucosidase inhibitors. Such ability was also observed in epigallocatechin-3-O-gallate (EGCG), epicatechin-3-O-gallate (ECG), delphinidin-3-O-glucoside and myricetin-3-O-glucoside. The amounts of these compounds in Sunrouge were higher than that in many other cultivars. These results suggest that Sunrouge has high potential for suppressing the elevation of the postprandial blood glucose level, and an MP approach may become a valuable strategy for evaluating the anti-hyperglycemic activity of green tea and for screening its active ingredients..
21. Visualizing Energy Charge in Breast Carcinoma Tissues by MALDI Mass-spectrometry Imaging Profiles of Low-molecular-weight Metabolites..
22. Miho Irie, Eisuke Hayakawa, Yoshinori Fujimura, Youhei Honda, Daiki Setoyama, Hiroyuki Wariishi, Fuminori Hyodo, Daisuke Miura, Analysis of spatiotemporal metabolomic dynamics for sensitively monitoring biological alterations in cisplatin-induced acute kidney injury, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2018.01.012, 496, 1, 140-146, 2018.01, Clinical application of the major anticancer drug, cisplatin, is limited by severe side effects, especially acute kidney injury (AKI) caused by nephrotoxicity. The detailed metabolic mechanism is still largely unknown. Here, we used an integrated technique combining mass spectrometry imaging (MSI) and liquid chromatography–mass spectrometry (LC–MS) to visualize the diverse spatiotemporal metabolic dynamics in the mouse kidney after cisplatin dosing. Biological responses to cisplatin was more sensitively detected within 24 h as a metabolic alteration, which is much earlier than possible with the conventional clinical chemistry method of blood urea nitrogen (BUN) measurement. Region-specific changes (e.g., medulla and cortex) in metabolites related to DNA damage and energy generation were observed over the 72-h exposure period. Therefore, this metabolomics approach may become a novel strategy for elucidating early renal responses to cisplatin, prior to the detection of kidney damage evaluated by conventional method..
23. Yoshinori Fujimura, Daisuke Miura, Hirofumi Tachibana, A Phytochemical-Sensing Strategy Based on Mass Spectrometry Imaging and Metabolic Profiling for Understanding the Functionality of the Medicinal Herb Green Tea, MOLECULES, 10.3390/molecules22101621, 22, 10, 22, E1621, 2017.10, Low-molecular-weight phytochemicals have health benefits and reduce the risk of diseases, but the mechanisms underlying their activities have remained elusive because of the lack of a methodology that can easily visualize the exact behavior of such small molecules. Recently, we developed an in situ label-free imaging technique, called mass spectrometry imaging, for visualizing spatially-resolved biotransformations based on simultaneous mapping of the major bioactive green tea polyphenol and its phase II metabolites. In addition, we established a mass spectrometry-based metabolic profiling technique capable of evaluating the bioactivities of diverse green tea extracts, which contain multiple phytochemicals, by focusing on their compositional balances. This methodology allowed us to simultaneously evaluate the relative contributions of the multiple compounds present in a multicomponent system to its bioactivity. This review highlights small molecule-sensing techniques for visualizing the complex behaviors of herbal components and linking such information to an enhanced understanding of the functionalities of multicomponent medicinal herbs..
24. Yoshinori Fujimura, Chihiro Kawano, Ayaka Maeda-Murayama, Asako Nakamura, Akiko Koike-Miki, Daichi Yukihira, Eisuke Hayakawa, Takanori Ishii, Hirofumi Tachibana, Hiroyuki Wariishi, Daisuke Miura, A Chemometrics-driven Strategy for the Bioactivity Evaluation of Complex Multicomponent Systems and the Effective Selection of Bioactivity-predictive Chemical Combinations, SCIENTIFIC REPORTS, 10.1038/s41598-017-02499-1, 7, 7, 2257, 2017.05, Although understanding their chemical composition is vital for accurately predicting the bioactivity of multicomponent drugs, nutraceuticals, and foods, no analytical approach exists to easily predict the bioactivity of multicomponent systems from complex behaviors of multiple coexisting factors. We herein represent a metabolic profiling (MP) strategy for evaluating bioactivity in systems containing various small molecules. Composition profiles of diverse bioactive herbal samples from 21 green tea extract (GTE) panels were obtained by a high-throughput, non-targeted analytical procedure. This employed the matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) technique, using 1,5-diaminonaphthalene (1,5-DAN) as the optical matrix for detecting GTE-derived components. Multivariate statistical analyses revealed differences among the GTEs in their antioxidant activity, oxygen radical absorbance capacity (ORAC). A reliable bioactivity-prediction model was constructed to predict the ORAC of diverse GTEs from their compositional balance. This chemometric procedure allowed the evaluation of GTE bioactivity by multicomponent rather than single-component information. The bioactivity could be easily evaluated by calculating the summed abundance of a few selected components that contributed most to constructing the prediction model. 1,5-DAN-MALDI- MS-MP, using diverse bioactive sample panels, represents a promising strategy for screening bioactivity-predictive multicomponent factors and selecting effective bioactivity-predictive chemical combinations for crude multicomponent systems..
25. Junya Nakamura, Tomomi Morikawa-Ichinose, Yoshinori Fujimura, Eisuke Hayakawa, Katsutoshi Takahashi, Takanori Ishii, Daisuke Miura, Hiroyuki Wariishi, Hiroyuki Wariishi, Hiroyuki Wariishi, Hiroyuki Wariishi, Spatially resolved metabolic distribution for unraveling the physiological change and responses in tomato fruit using matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI), Analytical and Bioanalytical Chemistry, 10.1007/s00216-016-0118-4, 409, 6, 1697-1706, 2017.02, © 2016, The Author(s). Information on spatiotemporal metabolic behavior is indispensable for a precise understanding of physiological changes and responses, including those of ripening processes and wounding stress, in fruit, but such information is still limited. Here, we visualized the spatial distribution of metabolites within tissue sections of tomato (Solanum lycopersicum L.) fruit using a matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI) technique combined with a matrix sublimation/recrystallization method. This technique elucidated the unique distribution patterns of more than 30 metabolite-derived ions, including primary and secondary metabolites, simultaneously. To investigate spatiotemporal metabolic alterations during physiological changes at the whole-tissue level, MALDI–MSI was performed using the different ripening phenotypes of mature green and mature red tomato fruits. Although apparent alterations in the localization and intensity of many detected metabolites were not observed between the two tomatoes, the amounts of glutamate and adenosine monophosphate, umami compounds, increased in both mesocarp and locule regions during the ripening process. In contrast, malate, a sour compound, decreased in both regions. MALDI–MSI was also applied to evaluate more local metabolic responses to wounding stress. Accumulations of a glycoalkaloid, tomatine, and a low level of its glycosylated metabolite, esculeoside A, were found in the wound region where cell death had been induced. Their inverse levels were observed in non-wounded regions. Furthermore, the amounts of both compounds differed in the developmental stages. Thus, our MALDI–MSI technique increased the understanding of the physiological changes and responses of tomato fruit through the determination of spatiotemporally resolved metabolic alterations. [Figure not available: see fulltext.].
26. Yuhui Huang, Mami Sumida, Motofumi Kumazoe, Kaori Sugihara, Yumi Suemasu, Shuhei Yamada, Shuya Yamashita, Jyunichi Miyakawa, Takashi Takahashi, Hiroshi Tanaka, Yoshinori Fujimura, Hirofumi Tachibana, Oligomer formation of a tea polyphenol, EGCG, on its sensing molecule 67 kDa laminin receptor, CHEMICAL COMMUNICATIONS, 10.1039/c6cc09504f, 53, 12, 1941-1944, 2017.02, Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) has been attributed to the activation of its cell surface sensing receptor 67 kDa laminin receptor (67LR). However, the action of EGCG to activate 67LR remains unknown. Here we show that EGCG undergoes oligomer formation on its surface receptor 67LR..
27. Eisuke Hayakawa, Yoshinori Fujimura, Daisuke Miura, MSIdV: a versatile tool to visualize biological indices from mass spectrometry imaging data, BIOINFORMATICS, 10.1093/bioinformatics/btw548, 32, 24, 3852-3854, 2016.12, Mass spectrometry imaging (MSI) visualizes the simultaneous lateral distribution of multiple compounds on sample surface. However, it is still difficult to visualize biological indices such as energy charge index from multiple compounds because of the lack of publicly available tools. Here we present MSIdV, a visualization tool for biological indices calculated from mass spectrometry imaging data, which can effectively scan a series of mass spectra and process, calculate and visualize user-defined index measures accurately with a number of signal processing features.
Availability and Implementation: MSIdV is implemented in Python 2.7 and is freely available on the web at
Supplementary information: Supplementary data are available at Bioinformatics online..
28. Tsukamoto S, Huang Y, Kumazoe M, Lesnick C, Yamada S, Ueda N, Suzuki T, Yamashita S, Kim YH, Fujimura Y, Miura D, Kay NE, Shanafelt TD, Tachibana H, Sphingosine Kinase-1 Protects Multiple Myeloma from Apoptosis Driven by Cancer-Specific Inhibition of RTKs, Mol Cancer Ther, 10.1158/1535-7163, 14, 2303-2312, 2015.10.
29. Yukihira D, Fujimura Y, Wariishi H, Miura D, Bacterial metabolism in immediate response to nutritional perturbation with temporal and network view of metabolites, Mol Biosyst, 10.1039/c5mb00182j, 11, 2473-2482, 2015.09.
30. Shuntaro Tsukamoto, Yuhui Huang, Motofumi Kumazoe, Connie Lesnick, Suhei Yamada, Naoki Ueda, Takashi Suzuki, Shuya Yamashita, Yoon Hee Kim, Yoshinori Fujimura, Daisuke Miura, Nail E. Kay, Tait D Shanafelt, Hirofumi Tachibana, Sphingosine kinase-1 protects B-cell neoplasm and myeloid leukemia from apoptosis driven by cancer specific inhibition of RTKs., Molecular Cancer Therapeutics, 10.1158/1535-7163.MCT-15-0185, 14, 10, 1162-1168, 2015.09.
31. Yoshinori Fujimura, Small molecule-sensing strategy and techniques for understanding the functionality of green tea, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1080/09168451.2014.996205, 79, 5, 687-699, 2015.05, Various low-molecular-weight phytochemicals in green tea (Camellia sinensis L.), especially (-)-epigallocatechin-3-O-gallate (EGCG), are known to be involved in health promotion and disease risk reduction. However, the underlying mechanism has remained elusive because of the absence of an analytical technique that can easily detect the precise behavior of such a small molecule. Recently, we have identified a cell-surface EGCG-sensing receptor and the related signaling molecules that control the physiological functions of EGCG. We also developed a novel in situ label-free imaging technique for visualizing spatially resolved biotransformations based on simultaneous mapping of EGCG and its phase II metabolites. Furthermore, we established a chemometric method capable of evaluating the functionality of multicomponent green tea extracts by focusing on their compositional balances. This review highlights our proposed small molecule-sensing techniques for detecting the complex behavior of green tea components and linking such information to an enhanced understanding of green tea functionality..
32. Tomoaki Inoue, Toyoshi Inoguchi, Noriyuki Sonoda, Hari Hendarto, Hiroaki Makimura, Shuji Sasaki, Hisashi Yokomizo, Yoshinori Fujimura, Daisuke Miura, Ryoichi Takayanagi, GLP-1 analog liraglutide protects against cardiac steatosis, oxidative stress and apoptosis in streptozotocin-induced diabetic rats, ATHEROSCLEROSIS, 10.1016/j.atherosclerosis.2015.03.026, 240, 1, 250-259, 2015.05, Objective: Accumulating evidence has implicated that GLP-1 may have a beneficial effect on cardiovascular but the mechanism is not fully understood. Here we show that GLP-1 analog, liraglutide, inhibits cardiac steatosis, oxidative stress and apoptosis in streptozotocin (STZ)-induced type 1 diabetic rats, via activation of AMPK-Sirt1 pathway.
Methods: Diabetic rats were treated with subcutaneous injections of liraglutide (0.3 mg/kg/12 h) for 4 weeks. Myocardial steatosis (detected by oil red O staining and myocardial triglyceride and diacylglycerol (DAG) contents assay), expression of protein kinase C (PKC), heart NAD(P) H oxidase activity, oxidative stress markers (8-hydroxy-2'-deoxyguanosine staining), apoptosis (TUNEL analysis) and genes that affect apoptosis and lipid metabolism were evaluated.
Results: Administration of liraglutide did not affect plasma glucose and insulin levels or body weights in STZ-induced diabetic rats, but normalized myocardial steatosis, expression of PKC, NAD(P) H oxidase activity, oxidative stress markers and apoptosis, all of which were significantly increased in diabetic hearts. Additionally, expression of genes mediating lipid uptake, synthesis and oxidation were increased in the diabetic hearts, and these increases were all reduced by liraglutide. In addition, liraglutide increased expression of Sirt1 and phosphorylated AMPK in the diabetic hearts.
Conclusions: Liraglutide may have a beneficial effect on cardiac steatosis, DAG-PKC-NAD(P) H pathway, oxidative stress and apoptosis via activation of AMPK-Sirt1 pathway, independently of a glucose-lowering effect. (C) 2015 Elsevier Ireland Ltd. All rights reserved..
33. Keiko Iwasa, Daiki Setoyama, Hiroaki Shimizu, Harumichi Seta, Yoshinori Fujimura, Daisuke Miura, Hiroyuki Wariishi, Chifumi Nagai, Koichi Nakahara, Identification of 3-Methylbutanoyl Glycosides in Green Coffea arabica Beans as Causative Determinants for the Quality of Coffee Flavors, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 10.1021/jf5054047, 63, 14, 3742-3751, 2015.04, The quality of coffee green beans is generally evaluated by the sensory cupping test, rather than by chemical compound-based criteria. In this study, we examined the relationship between metabolites and cupping scores for 36 varieties of beans, using a nontargeted LCMS-based metabolic profiling technique. The cupping score was precisely predicted with the metabolic information measured using LC-MS. Two markers that strongly correlated with high cupping scores were determined to be isomers of 3-methylbutanoyl disaccharides (3MDs; 0.01-0.035 g/kg of beans) by spectroscopic analyses after purification, and one of them was a novel structure. Further, both the 3MDs were determined to be precursors of 3-methylbutanoic acid that enhance the quality of coffee. The applicability of 3MDs as universal quality indicators was validated with another sample set. It was concluded that 3MDs are the causative metabolites determining beverage quality and can be utilized for green bean selection and as key compounds for improving the beverage quality..
34. Motofumi Kumazoe, Yoshinori Fujimura, Shiori Hidaka, Yoonhee Kim, Kanako Murayama, Mika Takai, Yuhui Huang, Shuya Yamashita, Motoki Murata, Daisuke Miura, Hiroyuki Wariishi, Mari Maeda-Yamamoto, Hirofumi Tachibana, Metabolic Profiling-based Data-mining for an Effective Chemical Combination to Induce Apoptosis of Cancer Cells, SCIENTIFIC REPORTS, 10.1038/srep09474, 5, 2015.03, Green tea extract (GTE) induces apoptosis of cancer cells without adversely affecting normal cells. Several clinical trials reported that GTE was well tolerated and had potential anti-cancer efficacy.
Epigallocatechin-3-O-gallate (EGCG) is the primary compound responsible for the anti-cancer effect of GTE; however, the effect of EGCG alone is limited. To identify GTE compounds capable of potentiating EGCG bioactivity, we performed metabolic profiling of 43 green tea cultivar panels by liquid chromatography-mass spectrometry (LC-MS). Here, we revealed the polyphenol eriodictyol significantly potentiated apoptosis induction by EGCG in vitro and in a mouse tumour model by amplifying EGCG-induced activation of the 67-kDa laminin receptor (67LR)/protein kinase B/endothelial nitric oxide synthase/protein kinase C delta/acid sphingomyelinase signalling pathway. Our results show that metabolic profiling is an effective chemical-mining approach for identifying botanical drugs with therapeutic potential against multiple myeloma. Metabolic profiling-based data mining could be an efficient strategy for screening additional bioactive compounds and identifying effective chemical combinations..
35. Yoon Hee Kim, Yoshinori Fujimura, Masako Sasaki, Xue Yang, Daichi Yukihira, Daisuke Miura, Yumi Unno, Koretsugu Ogata, Hiroki Nakajima, Shuya Yamashita, Kanami Nakahara, Motoki Murata, I-Chian Lin, Hiroyuki Wariishi, Koji Yamada, Hirofumi Tachibana, In Situ Label-Free Visualization of Orally Dosed Strictinin within Mouse Kidney by MALDI-MS Imaging, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 10.1021/jf503143g, 62, 38, 9279-9285, 2014.09, Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualizing the distribution of a wide range of biomolecules within tissue sections. However, methodology for visualizing a bioactive ellagitannin has not yet been established. This paper presents a novel in situ label-free MALDI-MSI technique for visualizing the distribution of strictinin, a bioactive ellagitannin found in green tea, within mammalian kidney after oral dosing. Among nine representative matrix candidates, 1,5-diaminonaphthalene (1,5-DAN), harmane, and ferulic acid showed higher sensitivity to strictinin spotted onto a MALDI sample plate. Of these, 1,5-DAN enables visualization of a two-dimensional image of strictinin directly spotted on mouse kidney sections with the highest sensitivity. Furthermore, 1,5-DAN-based MALDI-MSI could detect the unique distribution of orally dosed strictinin within kidney sections. This in situ label-free imaging technique will contribute to the localization analysis of strictinin and its biological mechanisms..
36. Yoon Hee Kim, Yu Ninomiya, Shuya Yamashita, Motofumi Kumazoe, Yuhui Huang, Kanami Nakahara, Yeong Seon Won, Motoki Murata, Yoshinori Fujimura, Koji Yamada, Hirofumi Tachibana, Hirofumi Tachibana, Hirofumi Tachibana, IL-4 receptor α in non-lipid rafts is the target molecule of strictinin in inhibiting STAT6 activation, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2014.06.069, 450, 1, 824-830, 2014.07, Strictinin has been shown to suppress interleukin (IL)-4-induced signal transducer and activator of transcription (STAT)-6 phosphorylation, which is a critical event for IgE class switching. However, it is unclear how strictinin inhibits STAT6 activation. Strictinin inhibited STAT6 phosphorylation by suppressing IL-4 receptor α (IL-4Rα) activation. Strictinin was bound to the cell surface and only localized in non-lipid raft fraction of the cells where IL-4Rα was also located. In addition, strictinin directly bound to IL-4Rα and inhibited binding of IL-4 to IL-4Rα. These results suggest that IL-4Rα locating in non-lipid raft region is a target molecule for strictinin in inhibiting STAT6 activation. © 2014 Elsevier Inc. All rights reserved..
37. Miho Irie, Yoshinori Fujimura, Mayumi Yamato, Daisuke Miura, Hiroyuki Wariishi, Integrated MALDI-MS imaging and LC-MS techniques for visualizing spatiotemporal metabolomic dynamics in a rat stroke model, METABOLOMICS, 10.1007/s11306-013-0588-8, 10, 3, 473-483, 2014.06, Spatiotemporal information about biomolecules is indispensable for precise pathological analysis, but it remains largely unclear. Here we show a novel analytical platform combing mass spectrometry imaging (MSI) with its complementary technique, liquid chromatography-mass spectrometry (LC-MS), to elucidate more comprehensive metabolic behaviors, with spatiotemporal information, in tissues. Analysis of a rat transient middle cerebral artery occlusion (MCAO) brain tissue after ischemia-reperfusion was performed to characterize the detailed metabolomic response to pathological alterations. To compare the spatially resolved metabolic state between ischemic and contralateral hemispheres of the MCAO brain, coronally sliced tissues were subjected to MSI. We also measured the metabolites extracted from three different cerebral regions, including whole cortex (CTX), hippocampus (HI) and corpus striatum (CPu), by LC-MS. In the ischemic hemisphere, significant metabolic changes at the CTX and CPu were observed after reperfusion, while not at the HI. A region-specific metabolic behavior was observed in amino acid and nucleotide metabolism, as well as in the TCA cycle. Correlation between MSI and LC-MS data was relatively high in the CTX and CPu. Combination of both MS platforms visualized the diverse spatiotemporal metabolic dynamics during pathological progress. Thus, our proposed strategy will contribute to the understanding of the complex pathogenesis of ischemia-reperfusion..
38. MALDI Mass Spectrometry Imaging for Visualizing In Situ Metabolism of Endogenous Metabolites and Dietary Phytochemicals..
39. Yoshinori Fujimura, Naoki Ikenaga, Kenoki Ohuchida, Daiki Setoyama, Miho Irie, Daisuke Miura, Hiroyuki Wariishi, Masaharu Murata, Kazuhiro Mizumoto, Makoto Hashizume, Masao Tanaka, Mass Spectrometry-Based Metabolic Profiling of Gemcitabine-Sensitive and Gemcitabine-Resistant Pancreatic Cancer Cells, PANCREAS, 10.1097/MPA.0000000000000092, 43, 2, 311-318, 2014.03, Objectives
Gemcitabine resistance (GR) is one of the critical issues for therapy for pancreatic cancer, but the mechanism still remains unclear. Our aim was to increase the understanding of GR by metabolic profiling approach.
To establish GR cells, 2 human pancreatic cancer cell lines, SUIT-2 and CAPAN-1, were exposed to increasing concentration of gemcitabine. Both parental and chemoresistant cells obtained by this treatment were subjected to metabolic profiling based on liquid chromatography-mass spectrometry.
Multivariate statistical analyses, both principal component analysis and orthogonal partial least squares discriminant analysis, distinguished metabolic signature of responsiveness and resistance to gemcitabine in both SUIT-2 and CAPAN-1 cells. Among significantly different (P < 0.005) metabolite peaks between parental and GR cells, we identified metabolites related to several metabolic pathways such as amino acid, nucleotide, energy, cofactor, and vitamin pathways. Decreases in glutamine and proline levels as well as increases in aspartate, hydroxyproline, creatine, and creatinine levels were observed in chemoresistant cells from both cell lines.
These results suggest that metabolic profiling can isolate distinct features of pancreatic cancer in the metabolome of gemcitabine-sensitive and GR cells. These findings may contribute to the biomarker discovery and an enhanced understanding of GR in pancreatic cancer..
40. Tatsuhiko Nagao, Daichi Yukihira, Yoshinori Fujimura, Kazunori Saito, Katsutoshi Takahashi, Daisuke Miura, Hiroyuki Wariishi, Power of isotopic fine structure for unambiguous determination of metabolite elemental compositions: In silico evaluation and metabolomic application, ANALYTICA CHIMICA ACTA, 10.1016/j.aca.2014.01.032, 813, 70-76, 2014.02, In mass spectrometry (MS)-based metabolomics studies, reference-free identification of metabolites is still a challenging issue. Previously, we demonstrated that the elemental composition (EC) of metabolites could be unambiguously determined using isotopic fine structure, observed by ultrahigh resolution MS, which provided the relative isotopic abundance (RIA) of C-13, N-15, O-18, and S-34. Herein, we evaluated the efficacy of the RIA for determining ECs based on the MS peaks of 20,258 known metabolites. The metabolites were simulated with a <= 25% error in the isotopic peak area to investigate how the error size effect affected the rate of unambiguous determination of the ECs. The simulation indicated that, in combination with reported constraint rules, the RIA led to unambiguous determination of the ECs for more than 90% of the tested metabolites. It was noteworthy that, in positive ion mode, the process could distinguish alkali metal-adduct ions ([M + Na](+) and [M + K](+)). However, a significant degradation of the EC determination performance was observed when the method was applied to real metabolomic data (mouse liver extracts analyzed by infusion ESI), because of the influence of noise and bias on the RIA. To achieve ideal performance, as indicated in the simulation, we developed an additional method to compensate for bias on the measured ion intensities. The method improved the performance of the calculation, permitting determination of ECs for 72% of the observed peaks. The proposed method is considered a useful starting point for high-throughput identification of metabolites in metabolomic research. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (
41. Yukihira D, Miura D, Fujimura Y, Umemura Y, Yamaguchi S, Funatsu S, Yamazaki M, Ohta T, Inoue H, Shindo M, Wariishi H, A QSPR Study on Structural Properties of Metabolites for Preferred Ionization in MALDI-MS Analysis, J. Am. Soc. Mass Spectrom., 25, 1-5, 2014.01.
42. Battsetseg Batchuluun, Toyoshi Inoguchi, Noriyuki Sonoda, Shuji Sasaki, Tomoaki Inoue, Yoshinori Fujimura, Daisuke Miura, Ryoichi Takayanagi, Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells, ATHEROSCLEROSIS, 10.1016/j.atherosclerosis.2013.10.025, 232, 1, 156-164, 2014.01, Objective: Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress.
Methods: Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P) H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P) H oxidase pathway was assessed by translocation of GFP-fused PKC beta 2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P) H oxidase, in addition to endogenous PKC phosphorylation and NAD(P) H oxidase activity.
Results: High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKC beta 2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P) H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects.
Conclusions: Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P) H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications. (C) 2013 Elsevier Ireland Ltd. All rights reserved..
43. Daichi Yukihira, Daisuke Miura, Yoshinori Fujimura, Yoshikatsu Umemura, Shinichi Yamaguchi, Shinji Funatsu, Makoto Yamazaki, Tetsuya Ohta, Hiroaki Inoue, Mitsuru Shindo, Hiroyuki Wariishi, MALDI Efficiency of Metabolites Quantitatively Associated with their Structural Properties: A Quantitative Structure-Property Relationship (QSPR) Approach, JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 10.1007/s13361-013-0772-0, 25, 1, 1-5, 2014.01, Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) experiments require a suitable match of the matrix and target compounds to achieve a selective and sensitive analysis. However, it is still difficult to predict which metabolites are ionizable with a given matrix and which factors lead to an efficient ionization. In the present study, we extracted structural properties of metabolites that contribute to their ionization in MALDI-MS analyses exploiting our experimental data set. The MALDI-MS experiment was performed for 200 standard metabolites using 9-aminoacridine (9-AA) as the matrix. We then developed a prediction model for the ionization profiles (both the ionizability and ionization efficiency) of metabolites using a quantitative structure-property relationship (QSPR) approach. The classification model for the ionizability achieved a 91 % accuracy, and the regression model for the ionization efficiency reached a rank correlation coefficient of 0.77. An analysis of the descriptors contributing to such model construction suggested that the proton affinity is a major determinant of the ionization, whereas some substructures hinder efficient ionization. This study will lead to the development of more rational and predictable MALDI-MS analyses..
44. Daiki Setoyama, Yoshinori Fujimura, Daisuke Miura, Metabolomics reveals that carnitine palmitoyltransferase-1 is a novel target for oxidative inactivation in human cells, Genes to Cells, 10.1111/gtc.12098, 18, 12, 1107-1119, 2013.12, Oxidative dysfunction in the metabolism has long been implicated in diverse biological disorders. Although a substantial number of metabolic enzymes are targeted for inactivation by oxidative stress, identifying those targets remains difficult due to a lack of comprehensive observations of the metabolism acting through the stress response. We herein developed a metabolomics strategy using integrative liquid chromatography-mass spectrometry (LC-MS) and observing rapid metabolomic changes in response to hydrogen peroxide (H2O2)-induced oxidative stress in HeLa cells. Among the many metabolite changes detected, the most characteristic metabolites uniquely indicated carnitine palmitoyltransferase-1 (CPT1), the critical enzyme for mitochondrial β-oxidation of long-chain fatty acids, to be a target for oxidative inactivation. We showed that the enzymatic activity of CPT1 significantly declined by H2O2 in several human cells. Interestingly, the inactivation was shown to be a direct effect of H2O2 in vitro, but substantially occurred when cells were cultured with some reagents that generate reactive oxygen species (ROS). Thus, our results suggest the generality of CPT1 inhibition under various stress conditions associated with ROS generation, providing an insight into a mechanism for oxidative dysfunction in mitochondrial metabolism. Our metabolome data additionally suggest that certain methyltransferase(s) may be targets of oxidative stress as well. © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd..
45. Yoon Hee Kim, Yoshinori Fujimura, Takatoki Hagihara, Masako Sasaki, Daichi Yukihira, Tatsuhiko Nagao, Daisuke Miura, Shinichi Yamaguchi, Kazunori Saito, Hiroshi Tanaka, Hiroyuki Wariishi, Koji Yamada, Hirofumi Tachibana, In situ label-free imaging for visualizing the biotransformation of a bioactive polyphenol, SCIENTIFIC REPORTS, 10.1038/srep02805, 3, 2013.09, Although understanding the high-resolution spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmacological effects, there has been no analytical technique that can easily detect the naive molecular localization in mammalian tissues. We herein present a novel in situ label-free imaging technique for visualizing bioactive small molecules, using a polyphenol. We established a 1,5-diaminonaphthalene (1,5-DAN)-based matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) technique for visualizing epigallocatechin-3-O-gallate (EGCG), the major bioactive green tea polyphenol, within mammalian tissue micro-regions after oral dosing. Furthermore, the combination of this label-free MALDI-MSI method and a standard-independent metabolite identification method, an isotopic fine structure analysis using ultrahigh-resolution mass spectrometer, allows for the visualization of spatially-resolved biotransformation based on simultaneous mapping of EGCG and its phase II metabolites. Although this approach has limitations of the detection sensitivity, it will overcome the drawbacks associated with conventional molecular imaging techniques, and could contribute to biological discovery..
46. Daiki Setoyama, Keiko Iwasa, Harumichi Seta, Hiroaki Shimizu, Yoshinori Fujimura, Daisuke Miura, Hiroyuki Wariishi, Chifumi Nagai, Koichi Nakahara, High-Throughput Metabolic Profiling of Diverse Green Coffea arabica Beans Identified Tryptophan as a Universal Discrimination Factor for Immature Beans, PLOS ONE, 10.1371/journal.pone.0070098, 8, 8, 2013.08, The maturity of green coffee beans is the most influential determinant of the quality and flavor of the resultant coffee beverage. However, the chemical compounds that can be used to discriminate the maturity of the beans remain uncharacterized. We herein analyzed four distinct stages of maturity (immature, semi-mature, mature and overripe) of nine different varieties of green Coffea arabica beans hand-harvested from a single experimental field in Hawaii. After developing a high-throughput experimental system for sample preparation and liquid chromatography-mass spectrometry (LC-MS) measurement, we applied metabolic profiling, integrated with chemometric techniques, to explore the relationship between the metabolome and maturity of the sample in a non-biased way. For the multivariate statistical analyses, a partial least square (PLS) regression model was successfully created, which allowed us to accurately predict the maturity of the beans based on the metabolomic information. As a result, tryptophan was identified to be the best contributor to the regression model; the relative MS intensity of tryptophan was higher in immature beans than in those after the semi-mature stages in all arabica varieties investigated, demonstrating a universal discrimination factor for diverse arabica beans. Therefore, typtophan, either alone or together with other metabolites, may be utilized for traders as an assessment standard when purchasing qualified trading green arabica bean products. Furthermore, our results suggest that the tryptophan metabolism may be tightly linked to the development of coffee cherries and/or beans..
47. Tomoaki Inoue, Kunihisa Kobayashi, Toyoshi Inoguchi, Noriyuki Sonoda, Yasutaka Maeda, Eiichi Hirata, Yoshinori Fujimura, Daisuke Miura, Ken-ichi Hirano, Ryoichi Takayanagi, Downregulation of adipose triglyceride lipase in the heart aggravates diabetic cardiomyopathy in db/db mice, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2013.07.063, 438, 1, 224-229, 2013.08, Adipose triglyceride lipase (ATGL) was recently identified as a rate-limiting triglyceride (TG) lipase and its activity is stimulated by comparative gene identification-58 (CGI-58). Mutations in the ATGL or CGI-58 genes are associated with neutral lipid storage diseases characterized by the accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, is characterized by TG accumulation in coronary atherosclerotic lesions and in the myocardium. Recent reports showed that myocardial TG accumulation is significantly higher in patients with diabetes and is associated with impaired left ventricular diastolic function. Therefore, we investigated the roles of ATGL and CGI-58 in the development of myocardial steatosis in the diabetic state. Histological examination with oil red O staining showed marked lipid deposition in the hearts of diabetic fatty db/db mice. Cardiac triglyceride and diglyceride contents were greater in db/db mice than in db/+ control mice. Next, we determined the expression of genes and proteins that affect lipid metabolism, and found that ATGL and CGI-58 expression levels were decreased in the hearts of db/db mice. We also found increased expression of genes regulating triglyceride synthesis (sterol regulatory element-binding protein 1c, monoacylglycerol acyltransferases, and diacylglycerol acyltransferases) in db/db mice. Regarding key modulators of apoptosis, PKC activity, and oxidative stress, we found that Bcl-2 levels were lower and that phosphorylated PKC and 8-hydroxy-2'-deoxyguanosine levels were higher in db/db hearts. These results suggest that reduced ATGL and CGI-58 expression and increased TG synthesis may exacerbate myocardial steatosis and oxidative stress, thereby promoting cardiac apoptosis in diabetic mice. © 2013 Elsevier Inc..
48. Kim YH, Yoshimoto M, Nakayama K, Tanino S, Fujimura Y, Yamada K, Tachibana H, Tannic acid, a higher galloylated pentagalloylglucose, suppresses antigen-specific IgE production by inhibiting ε germline transcription induced by STAT6 activation, FEBS Open Bio, 3, 341-345, 2013.08.
49. Daisuke Miura, Yoshinori Fujimura, Hiroyuki Wariishi, In situ metabolomic mass spectrometry imaging: Recent advances and difficulties, JOURNAL OF PROTEOMICS, 10.1016/j.jprot.2012.02.011, 75, 16, 5052-5060, 75, 5052-5060, 2012.08, MS imaging (MSI) is a remarkable new technology that enables us to determine the distribution of biological molecules present in tissue sections by direct ionization and detection. This technique is now widely used for in situ imaging of endogenous or exogenous molecules such as proteins, lipids, drugs and their metabolites, and it is a potential tool for pathological analysis and the investigation of disease mechanisms. MSI is also thought to be a technique that could be used for biomarker discovery with spatial information. The application of MSI to the study of endogenous metabolites has received considerable attention because metabolites are the result of the interactions of a system's genome with its environment and a total set of these metabolites more closely represents the phenotype of an organism under a given set of conditions. Recent studies have suggested the importance of in situ metabolite imaging in biological discovery and biomedical applications, but several issues regarding the technical application limits of MSI still remained to be resolved. In this review, we describe the capabilities of the latest MSI techniques for the imaging of endogenous metabolites in biological samples, and also discuss the technical problems and new challenges that need to be addressed for effective and widespread application of MSI in both preclinical and clinical settings.
This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User's Guide to a New Technique for Biological and Biomedical Research. (C) 2012 Elsevier B.V. All rights reserved..
50. Yoshinori Fujimura, Mami Sumida, Kaori Sugihara, Shuntaro Tsukamoto, Koji Yamada, Hirofumi Tachibana, Green Tea Polyphenol EGCG Sensing Motif on the 67-kDa Laminin Receptor, PLOS ONE, 10.1371/journal.pone.0037942, 7, 5, e37942, 2012.05, Background: We previously identified the 67-kDa laminin receptor (67LR) as the cell-surface receptor conferring the major green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) responsiveness to cancer cells. However, the underlying mechanism for interaction between EGCG and 67LR remains unclear. In this study, we investigated the possible role of EGCG-67LR interaction responsible for its bioactivities.
Methodology/Principal Findings: We synthesized various peptides deduced from the extracellular domain corresponding to the 102-295 region of human 67LR encoding a 295-amino acid. The neutralizing activity of these peptides toward EGCG cell-surface binding and inhibition of cancer cell growth were assayed. Both activities were inhibited by a peptide containing the 10-amino acid residues, IPCNNKGAHS, corresponding to residues 161-170. Furthermore, mass spectrometric analysis revealed the formation of a EGCG-LR161-170 peptide complex. A study of the amino acid deletion/replacement of the peptide LR161-170 indicated that the 10-amino acid length and two basic amino acids, K-166 and H-169, have a critical role in neutralizing EGCG's activities. Moreover, neutralizing activity against the anti-proliferation action of EGCG was observed in a recombinant protein of the extracellular domain of 67LR, and this effect was abrogated by a deletion of residues 161-170. These findings support that the 10 amino-acid sequence, IPCNNKGAHS, might be the functional domain responsible for the anti-cancer activity of EGCG.
Conclusions/Significance: Overall, our results highlight the nature of the EGCG-67LR interaction and provide novel structural insights into the understanding of 67LR-mediated functions of EGCG, and could aid in the development of potential anti-cancer compounds for chemopreventive or therapeutic uses that can mimic EGCG-67LR interactions..
51. Tomoaki Inoue, Kunihisa Kobayashi, Kunihisa Kobayashi, Toyoshi Inoguchi, Toyoshi Inoguchi, Noriyuki Sonoda, Noriyuki Sonoda, Masakazu Fujii, Yasutaka Maeda, Yoshinori Fujimura, Daisuke Miura, Ken Ichi Hirano, Ryoichi Takayanagi, Reduced expression of adipose triglyceride lipase enhances tumor necrosis factor α-induced intercellular adhesion molecule-1 expression in human aortic endothelial cells via protein kinase C-dependent activation of nuclear factor-κB, Journal of Biological Chemistry, 10.1074/jbc.M111.285650, 286, 37, 32045-32053, 2011.09, We examined the effects of adipose triglyceride lipase (ATGL) on the initiation of atherosclerosis. ATGL was recently identified as a rate-limiting triglyceride (TG) lipase. Mutations in the human ATGL gene are associated with neutral lipid storage disease with myopathy, a rare genetic disease characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, shows massive TG accumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial triglyceride content is significantly higher in patients with prediabetes or diabetes and that ATGL expression is decreased in the obese insulin-resistant state. Therefore, we investigated the effect of decreased ATGL activity on the development of atherosclerosis using human aortic endothelial cells. We found that ATGL knockdown enhanced monocyte adhesion via increased expression of TNFα-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NFκB) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of IκBα were increased in ATGL knockdown human aortic endothelial cells. In addition, intracellular diacylglycerol levels and free fatty acid uptake via CD36 were significantly increased in these cells. Inhibition of the PKC pathway using calphostin C and GF109203X suppressed TNFα-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNFα-induced ICAM-1 expression via activation of NFκB and PKC. These results suggest that reduced ATGL expression may influence the atherogenic process in neutral lipid storage diseases and in the insulin-resistant state. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc..
52. Yoshinori Fujimura, Kana Kurihara, Megumi Ida, Reia Kosaka, Daisuke Miura, Hiroyuki Wariishi, Mari Maeda-Yamamoto, Atsushi Nesumi, Takeshi Saito, Tomomasa Kanda, Koji Yamada, Hirofumi Tachibana, Metabolomics-Driven Nutraceutical Evaluation of Diverse Green Tea Cultivars, PLOS ONE, 10.1371/journal.pone.0023426, 6, 8, e23426, 2011.08, Background: Green tea has various health promotion effects. Although there are numerous tea cultivars, little is known about the differences in their nutraceutical properties. Metabolic profiling techniques can provide information on the relationship between the metabolome and factors such as phenotype or quality. Here, we performed metabolomic analyses to explore the relationship between the metabolome and health-promoting attributes (bioactivity) of diverse Japanese green tea cultivars.
Methodology/Principal Findings: We investigated the ability of leaf extracts from 43 Japanese green tea cultivars to inhibit thrombin-induced phosphorylation of myosin regulatory light chain (MRLC) in human umbilical vein endothelial cells (HUVECs). This thrombin-induced phosphorylation is a potential hallmark of vascular endothelial dysfunction. Among the tested cultivars, Cha Chuukanbohon Nou-6 (Nou-6) and Sunrouge (SR) strongly inhibited MRLC phosphorylation. To evaluate the bioactivity of green tea cultivars using a metabolomics approach, the metabolite profiles of all tea extracts were determined by high-performance liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses, principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA), revealed differences among green tea cultivars with respect to their ability to inhibit MRLC phosphorylation. In the SR cultivar, polyphenols were associated with its unique metabolic profile and its bioactivity. In addition, using partial least-squares (PLS) regression analysis, we succeeded in constructing a reliable bioactivity-prediction model to predict the inhibitory effect of tea cultivars based on their metabolome. This model was based on certain identified metabolites that were associated with bioactivity. When added to an extract from the non-bioactive cultivar Yabukita, several metabolites enriched in SR were able to transform the extract into a bioactive extract.
Conclusions/Significance: Our findings suggest that metabolic profiling is a useful approach for nutraceutical evaluation of the health promotion effects of diverse tea cultivars. This may propose a novel strategy for functional food design..
53. Miura, Daisuke, Fujimura, Yoshinori, Yamato, Mayumi, Hyodo, Fuminori, Utsumi, Hideo, Tachibana, Hirofumi, Wariishi, Hiroyuki, Ultrahighly Sensitive in Situ Metabolomic Imaging for Visualizing Spatiotemporal Metabolic Behaviors, ANALYTICAL CHEMISTRY, 10.1021/ac101998z, 82, 23, 9789-9796, 2010.12, A sensitive and simultaneous analytical technique for visualizing multiple endogenous molecules is now strongly required in biological science Here, we show the applicability of a matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) system for getting chemically diverse metabolite profiles on a single mammalian cell This ultrahighly sensitive MALDI MS technique enabled a spatially resolved detection of a broad range of metabolites including nucleotides, cofactors, phosphorylated sugars, amino acids, lipids, and carboxylic acids in normal mouse brain tissue with their unique distributions Furthermore, a combination of MS imaging and metabolic pathway analysis of a rat transient middle cerebral artery occlusion model visualized a spatiotem poral behavior of metabolites in the central metabolic pathway regulated by an ischemia reperfusion These findings highlight potential applications of an in situ metabolomic imaging technique to visualize spatiotem poral dynamics of the tissue metabolome, which will facilitate biological discovery in both preclinical and clinical settings.
54. Eui Hong Byun, Yoshinori Fujimura, Koji Yamada, Hirofumi Tachibana, TLR4 Signaling Inhibitory Pathway Induced by Green Tea Polyphenol Epigallocatechin-3-Gallate through 67-kDa Laminin Receptor, JOURNAL OF IMMUNOLOGY, 10.4049/jimmunol.0903742, 185, 1, 33-45, 2010.07, Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to downregulate inflammatory responses in macrophages; however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor that mediates the anticancer action of EGCG at physiologically relevant concentrations (0.1-1 mu M). In this study, we show the molecular basis for the downregulation of TLR4 signal transduction by EGCG at 1 mu M in macrophages. Anti-67LR Ab treatment or RNA interference-mediated silencing of 67LR resulted in abrogation of the inhibitory action of EGCG on LPS-induced activation of downstream signaling pathways and target gene expressions. Additionally, we found that EGCG reduced the TLR4 expression through 67LR. Interestingly, EGCG induced a rapid upregulation of Toll-interacting protein (Tollip), a negative regulator of TLR signaling, and this EGCG action was prevented by 67LR silencing or anti-67LR Ab treatment. RNA interference-mediated silencing of Tollip impaired the TLR4 signaling inhibitory activity of EGCG. Taken together, these findings demonstrate that 67LR plays a critical role in mediating anti-inflammatory action of a physiologically relevant EGCG, and Tollip expression could be modulated through 67LR. These results provide a new insight into the understanding of negative regulatory mechanisms for the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases. The Journal of Immunology, 2010, 185: 33-45..
55. Hiroaki Kanouchi, Mayumi Shibuya, Shuntaro Tsukamoto, Yoshinori Fujimura, Hirofumi Tachibana, Koji Yamada, Tatsuzo Oka, Comparisons of uptake and cell surface binding among pyridoxal, pyridoxine, and pyridoxamine in RAW264.7 cells, NUTRITION, 10.1016/j.nut.2009.08.005, 26, 6, 648-652, 2010.06, Objective: Vitamin B6 (B6) suppresses the expression of cyclooxygenase-2 stimulated by lipopolysaccharide in mouse macrophage RAW264.7 cells. The greatest effect is recognized for pyridoxal (PL) compared with pyridoxamine (PM), pyridoxine (PN), and pyridoxal 5'-phosphate (PLP). However, it has not been elucidated why PL has the strongest effect. We compared the uptakes and cell surface interactions among PL, PM, PN, and PLP in RAW264.7 cells.
Methods: Cyclo-oxygenase-2 mRNA expression was evaluated by real-time polymerase chain reaction. Intracellular B6 concentrations were measured by high-performance liquid chromatography. Interactions of B6s with the cell surface were analyzed using a surface plasmon resonance biosensor. B6 uptake speeds were measured using [(3)H]-PN.
Results: The intracellular PLP levels did not change significantly when cells were cultured in medium containing PL, PM, PN, or PLP. Only PL interacted with the cell surface. Although PM and PN were associated with the cell surface, their binding was only recognized during sample loading. After the change to phosphate buffered saline after sample loading, the binding resonances of PM and PN returned to baseline, whereas that of PL did not. Uptake of [(3)H]-PN was inhibited by non-labeled PN, PL, or PLP, but not PM, at 1 mu M. The inhibition rate of PL was higher than those of PN and PLP.
Conclusion: The inhibition of cyclo-oxygenase-2 mRNA expression by PL may be related to the cell surface interaction of PL, rather than the intracellular PLP level. The uptake mechanism for PN and PL may differ from that for PM. (C) 2010 Elsevier Inc. All rights reserved..
56. Yuki Matsumoto, Takumi Ishida, Tomoki Takeda, Takayuki Koga, Misaki Fujii, Yuji Ishii, Yoshinori Fujimura, Daisuke Miura, Hiroyuki Wariishi, Hideyuki Yamada, Maternal exposure to dioxin reduces hypothalamic but not pituitary metabolome in fetal rats: a possible mechanism for a fetus-specific reduction in steroidogenesis, JOURNAL OF TOXICOLOGICAL SCIENCES, 10.2131/jts.35.365, 35, 3, 365-373, 2010.06, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) reduces the synthesis of pituitary gonadotropins in a fetal age-specific manner. The pituitary synthesis of gonadotropins is regulated by the hypothalamus and, thus, needs the differentiation and development of the hypothalamus requiring a number of factors including energy supply and neurotransmitters. To investigate the mechanism whereby TCDD reduces fetal gonadotropins, we carried out a comparative study on the metabolomes of the hypothalamus and pituitary using fetal and mature Wistar rats. Male fetuses at gestational day (GD)20 were removed from dams treated orally with TCDD (1 mu g/kg) at GD15, and the metabolome profiles were analyzed by gas chromatography-mass spectrometry (GC-MS). The principal component analysis of GC-MS data revealed that TCDD caused a change in the profile of fetal metabolome more markedly in the hypothalamus than in the pituitary. In sharp contrast, TCDD did not cause any marked alteration in hypothalamic as well as pituitary metabolomes in male rats born of untreated dams and treated with TCDD at postnatal day 49. It was also demonstrated that a number of fetal hypothalamic components, including glutamine and gamma-aminobutyric acid, are reduced by TCDD. These results demonstrate a possibility that TCDD may reduce the metabolic activity of the hypothalamus in a fetus-specific fashion, resulting in the reduced synthesis of gonadotropins..
57. Miura, D., Fujimura, Y., Tachibana, H., and Wariishi, H. , Functional evaluation of anticancer drugs in human leukemia cells based on metabolic profiling technique, Animal Cell Technology, 2010.05.
58. Daisuke Miura, Yoshinori Fujimura, Hirofumi Tachibana, Hiroyuki Wariishi, Highly Sensitive Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry for High-Throughput Metabolic Profiling, ANALYTICAL CHEMISTRY, 10.1021/ac901083a, 82, 2, 498-504, 2010.01, In the present study, a high-throughput and nontargeted metabolomic technique using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was developed for the rapid analysis of cellular metabolites. Either the detection limit or die linearity between concentrations of several standards and peak intensities was examined, indicating a detection limit lower than 10 fmol/well with a high linearity at low concentrations. To verify the validity of this method, metabolites from human acute lymphoblastic leukemia Jurkat cells were analyzed. Only 2 500 cells suspended in PBS were directly dropped onto a stainless MALDI sample plate, followed by mixing with., matrix on the sample plate. Up to 150 metabolite peaks were detected from a single analysis within 90 s. For multivariate analysis of Jurkat cells against drug-treatment, three anticancer drugs were utilized. Principal component analysis of metabolites showed clear independent clusters for cells treated with these anticancer drugs. Furthermore, several metabolites involved in nucleotide synthesis were found to contribute to the separation of each cluster. These data suggest that the high-throughput MALDI-MS-based metabolomic technique proposed in the present study can be utilized for drug screening and validation of drug efficacy and safety..
59. Ohta, S., Fujimura, Y., Yamada, K., and Tachibana, H., Involvement of 67 kDa laminin receptor on cellular uptake of green tea polyphenol, epigallocatechin-3-O-gallate, in Caco2 cells, Animal Cell Technology, 15, 211-215, 2009.11.
60. Hyodo, F., Miura, D., Fujimura, Y., Yasukawa, K., Sakai, K., Ichikawa, K., Wariishi, H., and Utsumi, H., Visualization of nitroxyl probes for molecular redox imaging by Overhauser MRI and mass spectrometry imaging, Free Radic. Biol. Med., 47, 144, 2009.11.
61. Miura, D., Yamato, M., Fujimura, Y., Hyodo, F., Tachibana, H., Utsumi, H., and Wariishi, H., In situ metabolomics imaging of a rat brain section of transient middle cerebral artery occlusion model, Free Radic. Biol. Med., 47, 146-147, 2009.11.
62. Fujimura, Y., Miura, D., Hyodo, F., Yasukawa, K., Tachibana, H., Utsumi, H., and Wariishi, H, Evaluation of cisplatin-induced nephrotoxicity by Overhauser MRI and mass spectrometry imaging, Free Radic. Biol. Med., 47, 141-142, 2009.11.
63. Ju-Hye Lee, Hirofumi Tachibana, Yoshiko Morinaga, Yoshinori Fujimura, Koji Yamada, Modulation of proliferation and differentiation of C2C12 skeletal muscle cells by fatty acids, LIFE SCIENCES, 10.1016/j.lfs.2009.01.004, 84, 13-14, 415-420, 2009.03, Aims: This study was performed to elucidate whether mitogen-activated protein kinases (MAPKs) are involved in the modulation of the proliferation and differentiation of skeletal muscle cells by fatty acids.
Main methods: C2C12 myoblasts were cultured in differentiation medium containing 2% horse serum for 3 days, and treated with each fatty acid. Phosphorylation levels of MAPKs were examined by immunoblot analysis.
Key findings: The mono- unsaturated fatty acids (MUFAs), oleic acid (OA) and n-6 polyunsaturated fatty acids (n-6 PUFAs), linoleic acid (LA), gamma-linoleic acid (GLA), and arachidonic acid (AA) increased the proliferation of C2C12 cells. On the other hand, n-3 polyunsaturated fatty acids (n-3 PUFAs) and saturated fatty acids (SFs) did not affect the proliferation of C2C12 cells. In addition, the treatment of cis-9, trans-11 conjugated linoleic acid (c9,t11 CIA) showed an increased cell proliferation. However, trans-10, cis-12 conjugated linoleic acid (t10,c12 CIA) significantly inhibited cell proliferation. Treatment of C2C12 cells with LA, OA, and c9,t11 CIA increased phosphorylation levels of ERK1/2 and JNK during proliferation. During cell differentiation, OA, LA, and c9,t11 CIA stimulated differentiation of C2C12 cells, whereas t10,c12 CIA inhibited differentiation. We also found that OA, LA and c9, t11 CLA increased phosphorylation level of ERK1/2, but not JNK during differentiation.
Significance: These results suggest that fatty acids are able to modulate the proliferation and differentiation of skeletal muscle and MAPKs may be involved in the modulation of the proliferation and differentiation of skeletal muscle cells by fatty acids. (C) 2009 Elsevier Inc. All rights reserved..
64. Yano, S., Fujimura, Y., Umeda, D., Miyase, T., Yamada, K., and Tachibana, H., Relationsip between the biological activities of methylated derivatives of EGCG and their cell surface binding activities, J. Clin Biochem. Nutr., 43, 473-476, 2008.11.
65. Fujimura, Y., Umeda, D., Maeda-Yamamoto, M., Yamada, K., and Tachibana, H., The 67 kDa laminin receptor mediates anti-allergic effects of (-)-epigallocatechin-3-O-(3-O-methyl) gallate, J. Clin Biochem. Nutr., 43, 477-480, 2008.10.
66. Yoshinori Fujimura, Daisuke Umeda, Koji Yamada, Hirofumi Tachibana, The impact of the 67 kDa laminin receptor on both cell-surface binding and anti-allergic action of tea catechins, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 10.1016/, 476, 2, 133-138, 2008.08, Here, we investigated the structure-activity relationship of major green tea catechins and their corresponding epimers on cell-surface binding and inhibitory effect on histamine release. Galloylated catechins; (-)-epigallocatechin-3-O-gallate (EGCG), (-)-gallocatechin-3-O-gallate (GCG), (-)-epicatechin-3-O-gallate (ECG), and (-)-catechin-3-O-gallate (CG) showed the cell-surface binding to the human basophilic KU812 cells by surface plasmon resonance analysis, but their non-galloylated forms did not. Binding activities of pyrogallol-type catechins (EGCG and GCG) were higher than those of catechol-type catechins (ECG and CG). These patterns were also observed in their inhibitory effects on histamine release. Previously, we have reported that biological activities of EGCG are mediated through the binding to the cell-surface 67 kDa laminin receptor (67LR). Downregulation of 67LR expression caused a reduction of both activities of galloylated catechins. These results suggest that both the galloyl moiety and the B-ring hydroxylation pattern contribute to the exertion of biological activities of tea catechins and their 67LR-dependencies. (C) 2008 Elsevier Inc. All rights reserved..
67. Yoshinori Fujimura, Daisuke Umeda, Satomi Yano, Mari Maeda-Yamamoto, Koji Yamada, Hirofumi Tachibana, The 67 kDa laminin receptor as a primary determinant of anti-allergic effects of O-methylated EGCG, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2007.09.095, 364, 1, 79-85, 2007.12, Previously we have reported that the O-methylated derivative of (-)-epigallocatechin-3-O-gallate (EGCG), (-)-epigallocatechin-3-O(3-O-methyl) gallate (EGCG3"Me), possesses anti-allergic activities such as inhibition of histamine release and suppression of the high-affinity IgE receptor (Fc epsilon RI) expression. However, the underlying mechanism is still unclear. Recently we have identified the 67 kDa laminin receptor (67LR) as a cell-surface receptor that can mediate biological activities of EGCG. Here we show that the suppression of myosin II regulatory light chain (MRLC) phosphorylation through the cell-surface binding to the 67 LR contributes to the inhibitory effect of EGCG3"Me on the histamine release from the human basophilic KU812 cells. The 67LR also mediated the EGCG3"Me-induced suppression of Fc epsilon RI expression by reducing ERK1/2 phosphorylation. These results suggest that anti-allergic effects of EGCG3"Me may be triggered by the inhibition of MRLC or ERK1/2 phosphorylation mediated through the cell-surface 67LR. (C) 2007 Elsevier Inc. All rights reserved..
68. Satomi Yano, Daisuke Umeda, Tatsunori Yamashita, Yu Ninomiya, Mami Sumida, Yoshinori Fujimura, Koji Yamada, Hirofumi Tachibana, Dietary flavones suppresses IgE and th2 cytokines in OVA-immunized BALB/c mice, EUROPEAN JOURNAL OF NUTRITION, 10.1007/s00394-007-0658-7, 46, 5, 257-263, 2007.08, Background The flavonoids are a diverse family of chemicals commonly found in fruits and vegetables. Previously, we have shown that the two flavones, chrysin and apigenin could suppress the expression of the high affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells. We also demonstrated that dietary apigenin decreased IgE level in C57BL/6N mice sera. Aim of the study To evaluate the anti-allergic effect of the two flavones in vivo, we evaluated the effect of the two flavones, chrysin and apigenin, on the immune system in BALB/c mice sensitized with ovalbumin (OVA). Methods Mice were fed experimental diets containing either of the flavones for 3 weeks and immunized with OVA. After the experimental feeding period, measurement of Igs concentration in the mice sera was performed using a sandwich ELISA. Cytokines expression in mice sera was assessed using a cytokine array. Furthermore, cytokines mRNA levels in spleen lymphocytes from mice sensitized with OVA were measured by RT-PCR. Results The total IgE level in mice fed one of the two flavones were suppressed, whereas levels of IgG, IgM, and IgA were not affected. The production of interleukin (IL)-4, which is known as one of Th2 cytokines and regulates the production of IgE, was down-regulated by the chrysin or the apigenin diet. Moreover, OVA-induced mRNA expression of Th2 cytokines in spleen lymphocytes from mice sensitized with OVA, such as IL-4 and IL-13 were down-regulated by the chrysin or the apigenin diet. Conclusions The results suggest that the diet containing one of the two flavones might suppress the up-regulation of serum IgE induced by OVA-immunization through the suppression of Th2-type immune response..
69. Satomi Yano, Yoshinori Fujimura, Daisuke Umeda, Toshio Miyase, Koji Yamada, Hirofumi Tachibana, Relationship between the biological activities of methylated derivatives of (-)-epigallocatechin-3-O-gallate (EGCG) and their cell surface binding activities, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 10.1021/jf071176o, 55, 17, 7144-7148, 2007.08, It was previously reported that (-)-epigallocatechin-3-O-gallate (EGCG) suppresses the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic cells and that this suppressive effect is associated with EGCG binding to the cell surface. This study examined the effects of five methylated derivatives of EGCG, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (EGCG 3 '' Me), (-)-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4 '' Me), (-)-4'-O-methyl-epigallocatechin-3-O-gallate (EGCG 4'Me), (-)-epigallocatechin-3-O-(3,4-O-methyl)gallate (EGCG 3 '' 4 '' diMe), and (-)-4'-O-methyl-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4'4 '' diMe) on Fc epsilon RI expression and ERK1/2 phosphorylation, and each of their cell surface binding activities was measured. Of these five methylated derivatives, three that are methylated at the 3 ''- and/or 4 ''-position, EGCG 3 '' Me, EGCG 4 '' Me, and EGCG 3 '' 4 '' diMe, suppressed Fc epsilon RI expression and ERK1/2 phosphorylation, although the suppressive effects were lower than that of EGCG. EGCG 4'Me and EGCG 4'4 '' diMe, both of which are methylated at the 4'-position, did not demonstrate a suppressive effect. Furthermore, it was found that EGCG 3 '' Me, EGCG 4 '' Me, EGCG 3 '' 4 '' diMe, and EGCG 4'Me, which are methylated at the 3 ''- and/or 4 ''-positions or the 4'-position, could bind to the cell surface even though their binding activities were lower than that of EGCG. Only EGCG 4'4 '' diMe, which is methylated at both the 4'- and 4 ''-positions, could not bind. These results suggest that the trihydroxyl structure of the B ring is essential for EGCG to exert the suppressive effects and that the hydroxyl groups on both the 4'-position in the B ring and the 4 ''-position in the gallate are crucial for the cell surface binding activity of EGCG..
70. Ikeda, Y., Murakami, A., Fujimura, Y., Tachibana, H., Yamada, K., Masuda, D., Hirano, K., Yamashita, S., and Ohigashi, H., Aggregated ursolic acid, a natural triterpenoid, binds to CD36 for inducing interleukin-1 release from murine peritoneal macrophages, J. Clin Biochem. Nutr., 41, 99, 2007.05.
71. Fujimura, Y., Umeda, D., Maeda-Yamamoto, M., Yamada, K., and Tachibana, H., The 67 kDa laminin receptor mediates anti-allergic effects of (-)-epigallocatechin-3-O-(3-O-methyl) gallate, J. Clin Biochem. Nutr., 41, S145, 2007.05.
72. Tachibana, H., Daisuke, U., Fujimura, Y., and Yamada, K., Green tea polyphenol EGCG signaling through 67 kDa laminin receptor, J. Clin Biochem. Nutr., 41, S27, 2007.05.
73. Ikeda, Y., Murakami, A., Fujimura, Y., Tachibana, H., Yamada, K., Hirano, K., and Ohigashi, H., Aggregated ursolic acid, a natural triterpenoid, induces interleukin-1 release in murine peritoneal macrophages: Role of CD36, J. Immunol., 178, 4854-4864, 2007.05.
74. Tachibana, H., Fujimura, Y., Ogawa, N., Sumida, M., Tsuruda, S., and Yamada, K., Identification of the binding site of the green tea polyphenol EGCG receptor, J. Clin Biochem. Nutr., 41, S145, 2007.05.
75. Yoshinori Fujimura, Daisuke Umeda, Yuko Kiyohara, Yousuke Sunada, Koji Yamada, Hirofumi Tachibana, The involvement of the 67 kDa laminin receptor-mediated modulation of cytoskeleton in the degranulation inhibition induced by epigallocatechin-3-O-gallate, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2006.07.086, 348, 2, 524-531, 2006.09, Recently, we have reported that (-)-epigallocatechin-3-O-gallate (EGCG) acts as an inhibitor of degranulation. However, the inhibitory mechanism for degranulation is still poorly understood. Here we show that suppression of exocytosis-related myosin II regulatory light chain phosphorylation and alteration of actin remodeling are involved in the inhibitory effect of EGCG on the calcium ionophore-induced degranulation from human basophilic KU812 cells. Surface plasmon resonance assay also revealed that EGCG binds to the cell surface, and the disruption of lipid rafts resulted in reduction of EGCG's ability. We have previously identified the raft-associated 67 kDa laminin receptor (67LR) as an EGCG receptor on the cell surface. Treatment of the cells with anti-67LR antibody or RNA interference-mediated downregulation of 67LR expression abolished the effects of EGCG. These findings suggest that EGCG-induced inhibition of the degranulation includes the primary binding of EGCG to the cell surface 67LR and subsequent modulation of cytoskeleton. (c) 2006 Elsevier Inc. All rights reserved..
76. S Yano, D Umeda, N Maeda, Y Fujimura, K Yamada, H Tachibana, Dietary apigenin suppresses IgE and inflammatory cytokines production in C57BL/6N mice, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 10.1021/jf0607361, 54, 14, 5203-5207, 2006.07, Flavonoids ubiquitously exist in plants, vegetables, fruits, and teas. We evaluated the effect of dietary apigenin, one of the well-known flavonoids, on the immune system in C57BL/6N mice. Mice were fed experimental diets containing apigenin for 2 weeks. After the experimental period, there was no significant difference in body and organ weights between the control and the apigenin group. The total immunoglobulin (Ig) E levels in mice fed apigenin were significantly suppressed, whereas levels of IgG, IgM, and IgA were not affected. We also examined the effect of the apigenin diet on cytokine expression in mice sera using a cytokine array. The production of regulated upon activation normal T cell expressed and secreted (RANTES) and soluble tumor necrosis factor receptor I (sTNFRI) in mice sera was down-regulated by the apigenin diet. These results suggest that a diet containing apigenin can reduce serum IgE and inflammatory cytokines such as RANTES and sTNFRI in mice..
77. Satomi Kobayashi, Naoto Ogawa, Yoshinori Fujimura, Hirofumi Tachibana, Hirofumi Tachibana, Koji Yamada, Water-soluble component in dried chrysanthemum flower stimulates tumor necrosis factor-α production by mouse macrophage-like cell line RAW264.7, Food Science and Technology Research, 10.3136/fstr.12.144, 12, 2, 144-147, 2006.05, To clarify immunoregulatory activity of "Shiranui Himekiku" (Chrysanthemum indicam × Erigeron annus), we examined the effect on tumor necrosis factor α (TNF-α) production by mouse macrophage-like cell line RAW264.7. The dried chrysanthemum flower (CF) petals were extracted with water, and the cells were cultured in the presence of the extract. CF extract significantly enhanced TNF-α production of the cells by the dose-dependent manner. Diluted CF solution did not significantly affect the cell number and viability; however, non-diluted solution suppressed the cell proliferation and decreased the cell viability. Heating CF extract at 100°C for 30 min enhanced the activity of water extract markedly, and freeze-thawing only moderately. The TNF-α production enhancing activity of the extract was observed within 3h. These results suggest that the TNF-α production enhancing activity of CF extract can be recovered efficiently by hot water extraction and preserved stably by freezing..
78. Yoshinori Fujimura, Koji Yamada, Hirofumi Tachibana, A lipid raft-associated 67 kDa laminin receptor mediates suppressive effect of epigallocatechin-3-O-gallate on FcεRI expression, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2005.08.146, 336, 2, 674-681, 2005.10, (-)-Epigallocatechin-3-O-gallate (EGCG), a major green tea polyphenol, has previously exhibited a suppressive effect on the expression of the high-affinity IgE receptor (FcεRI). This effect has been shown to be elicited by interaction with the plasma membrane microdomain lipid rafts. Recently, we have identified the 67 kDa laminin receptor (67LR) as a cell surface EGCG receptor that mediates an anti-cancer action. Here we show that the 67LR is highly associated with lipid rafts on human basophilic KU812 cells. Experiments using 67LR-enhanced and -reduced cells revealed that the EGCG's ability to downregulate FcεRI expression correlated with the amount of 67LR. Thus, these results suggest that the lipid raft-associated 67LR plays an important role in mediating the FcεRI-suppressive action of EGCG. © 2005 Elsevier Inc. All rights reserved..
79. Fujimura, Y., Tachibana, H., and Yamada, K., Negative regulation of the basophil activation by natural ligands for Peroxisome proliferator-activated receptors, Animal Cell Technology, 13, 369-374, 2004.05.
80. Tachibana, H., Fujimura, Y., and Yamada, K., Epigallocatechin gallate associated with cell surface lipid rafts downregulates high affinity IgE receptor through the inhibition of extracellular signal-regulated kinase1/2 phosphorylation, Biofactors, 21, 1-4, 383-385, 2004.05.
81. Fujimura, Y., Tachibana, H., and Yamada, K., A difference between Epigallocatechin-3-gallate and Epicatechin-3-gallate on anti-allergic effect is dependent on their distinct distribution to lipid rafts, Biofactors, 21, 1-4, 133-135, 2004.05.
82. Hirofumi Tachibana, Kiyoshi Koga, Yoshinori Fujimura, Koji Yamada, A receptor for green tea polyphenol EGCG, Nature Structural and Molecular Biology, 10.1038/nsmb743, 11, 4, 380-381, 2004.04, The major polyphenol in green tea, (-)-epigallocatechin-3-gallate (EGCG), has been shown to prevent carcinogenesis. We have identified a receptor that mediates the anticancer activity of EGCG. Expression of the metastasis-associated 67-kDa laminin receptor confers EGCG responsiveness to cancer cells at physiologically relevant concentrations. Experiments using surface plasmon resonance demonstrate binding of EGCG to the 67-kDa laminin receptor with a nanomolar Kd value..
83. Yoshinori Fujimura, Hirofumi Tachibana, Koji Yamada, Lipid raft-associated catechin suppresses the FcεRI expression by inhibiting phosphorylation of the extracellular signal-regulated kinase1/2, FEBS Letters, 10.1016/S0014-5793(03)01432-7, 556, 1-3, 204-210, 2004.01, The major green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has a suppressive effect on the expression of the high-affinity IgE receptor FcεRI, which is key molecule in the IgE-mediated allergic reactions. Here we show that EGCG binds to the cell surface and highly associates with plasma membrane microdomains, lipid rafts, on the human basophilic KU812 cells. The disruption of these lipid rafts caused a reduction of the amount of raft-associated EGCG and the FcεRI-suppressive effect of EGCG. We also found that EGCG has an ability to inhibit the phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2) and that the ERK1/2 specific inhibitor also reduced FcεRI expression. Moreover, the inhibitory effect elicited by EGCG on ERK1/2 was prevented by disruption of rafts. Thus, these results suggest that the interaction between EGCG and the lipid rafts is important for EGCG's ability to downregulate FcεRI expression, and ERK1/2 may be involved in this suppression signal. © 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved..
84. Fujimura, Y., Tachibana, H., and Yamada, K., Downregulation of high affinity IgE receptor FcepsilonRI expression in the human basophilic KU812 cells by a tea catechin, Animal Cell Technology, 12, 365-370, 2002.05.
85. Fujimura, Y., Tachibana, H., Maeda-Yamamoto, M., Miyase, T., Sano, M., and Yamada, K., Antiallergic tea catechin, (-)-epigallocatechin-3-O-(3-O-methyl)-gallate, suppresses FcepsilonRI expression in human basophilic KU812 cells., J. Agric. Food Chem., 10.1021/jf025680z, 50, 20, 5729-5734, 2002.05.
86. Fujimura, Y., Tachibana, H., and Yamada, K., Peroxisome proliferator-activated receptor (PPAR) ligands negatively regulate the expression of the high-affinity IgE receptor FcepsilonRI in human basophilic KU812 cells, Biochem. Biophys. Res. Commun., 10.1016/S0006-291X(02)02139-3, 297, 2, 193-201, 2002.05.
87. Fujimura, Y., Tachibana, H., and Yamada, K., A tea catechin suppresses the expression of the high-affinity IgE receptor FcepsilonRI in human basophilic KU812 cells, J. Agric. Food Chem., 10.1021/jf001392w, 49, 5, 2527-2531, 2001.05.
88. Fujimura, Y., Tachibana, H., Eto, N., and Yamada, K., Antigen binding of an ovomucoid specific antibody is affected by a carbohydrate chain located on the light chain variable region, Biosci. Biotechnol. Biochem., 10.1271/bbb.64.2298, 64, 11, 2298-2305, 2000.05.
1. Fujimura Y, Miura D, Tachibana H, Metabolic Profiling Strategy for the Bioactivity Evaluation of Green Tea and the Selection of Bioactivity-related Chemical Combinations, The 3rd Symposium of Kyoto Biomolecular Mass Spectrometry Society, 2019.02.
Membership in Academic Society
  • International Conference on Food Factors for Health Promotion (ICoFF2007) Poster Award
    The 67 kDa laminin receptor mediates anti-allergic effects of (-)-epigallocatechin-3-O-(3-O-methyl) gallate