機能性食品成分の標的受容体分子制御に基づく抗アレルギーシグナルの解明

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi:10.1016/j.bbrc.2023.05.112

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Many patients with allergies have anxiety about taking anti-allergic medicines due to their side effects and increased medical expenses. Thus, developing functional foods/agricultural products for allergy prevention is strongly desired. In this study, we revealed that a Citrus flavanone, hesperetin, amplified IgE/antigen-mediated degranulation-inhibitory potency of anti-allergic catechin, (-)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3 "Me), in the rat basophilic/mast cell line RBL-2H3. Hesperetin also significantly elevated the activation of acid sphingomyelinase (ASM), essential for eliciting anti-allergic effect of EGCG3 "Me through the cell surficial protein, 67-kDa laminin receptor (67LR). Furthermore, oral administration of the highly absorbent hesperidin, alpha-glucosyl hesperidin, also enhanced the inhibitory potency of EGCG3 "Me-rich 'Benifuuki' green tea (Camellia sinensis L.) on passive cutaneous anaphylaxis (PCA) reaction evoked by IgE/antigen in BALB/c mice. These observations indicate that hesperetin amplifies the ability of EGCG3 "Me to inhibit the IgE/antigen-mediated degranulation through activating ASM signaling.

DOI： 10.1007/s11418-022-01668-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biomedical Reports  

As pulmonary fibrosis (PF), a severe interstitial pulmonary disease, has such a poor prognosis, the development of prevention and treatment methods is imperative. (-)-Epigallocatechin-3-O-gallate (EGCG), one of the major catechins in green tea, exerts an antifibrotic effect, although its mechanism remains unclear. Recently, it has been reported that microRNAs (miRNAs or miRs) transported by extracellular vesicles (EVs) from vascular endothelial cells (VECs) are involved in PF. In the present study, the effects of EGCG on the expression of miRNAs in EVs derived from human umbilical vein endothelial cells (HUVECs) were assessed and miRNAs with antifibrotic activity were identified. miRNA microarray analysis revealed that EGCG modulated the expression levels of 31 miRNAs (a total of 27 miRNAs were upregulated, and 4 miRNAs were downregulated.) in EVs from HUVECs. Furthermore, TargetScan analysis indicated that miR-6757-3p in particular, which exhibited the highest degree of change, may target transforming growth factor-β (TGF-β) receptor 1 (TGFBR1). To evaluate the effects of miR-6757-3p on TGFBR1 expression, human fetal lung fibroblasts (HFL-1) were transfected with an miR-6757-3p mimic. The results demonstrated that the miR-6757-3p mimic downregulated the expression of TGFBR1 as well the expression levels of fibrosis-related genes including fibronectin and α-smooth muscle actin in TGF-β-treated HFL-1 cells. In summary, EGCG upregulated the expression levels of miR-6757-3p, which may target TGFBR1 and downregulate fibrosis-related genes, in EVs derived from VECs.

DOI： 10.3892/br.2023.1601

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Scientific Reports  

Lung fibrosis, including idiopathic pulmonary fibrosis, is an intractable disease accompanied by an irreversible dysfunction in the respiratory system. Its pathogenesis involves the transforming growth factorβ (TGFβ)-induced overproduction of the extracellular matrix from fibroblasts; however, limited countermeasures have been established. In this study, we identified osa-miR172d-5p, a plant-derived microRNA (miR), as a potent anti-fibrotic miR. In silico analysis followed by an in vitro assay based on human lung fibroblasts demonstrated that osa-miR172d-5p suppressed the gene expression of TGF-β activated kinase 1 (MAP3K7) binding protein 1 (Tab1). It also suppressed the TGFβ-induced fibrotic gene expression in human lung fibroblasts. To assess the anti-fibrotic effect of osa-miR172d-5p, we established bleomycin-induced lung fibrosis models to demonstrate that osa-miR172d-5p ameliorated lung fibrosis. Moreover, it suppressed Tab1 expression in the lung tissues of bleomycin-treated mice. In conclusion, osa-miR172d-5p could be a potent candidate for the treatment of lung fibrosis, including idiopathic pulmonary fibrosis.

DOI： 10.1038/s41598-023-29188-6

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： Doi: 10.1038/s41598-023-29188-6

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.3892/br.2023.1601

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BMFH Press  

<p>Quercetin, a flavonol present in many vegetables and fruits, has been identified as a chemoprevention agent in several cancer models. However, the molecular mechanism of quercetin’s anticancer activity is not entirely understood. MicroRNAs (miRNAs), small noncoding RNAs, have been reported to play key roles in various biological processes by regulating their target genes. We hypothesized that quercetin can exert an anticancer effect through the regulation of miRNAs. To test this hypothesis, we investigated the effects of quercetin on the expression of tumor-suppressive miRNAs in cervical cancer. Quercetin up-regulated the in vivo and in vitro expression of tumor-suppressive miRNAs miR-26b, miR-126, and miR-320a. Quercetin suppressed the level of β-catenin, encoded by catenin beta 1 (CTNNB1), by up-regulating miR-320a in HeLa cells. Moreover, quercetin increased the expression of mir-26b, mir-126, and mir-320a precursors in HeLa cells. The results from this study show that quercetin has the potential to prevent cervical cancer by regulating the expression of tumor-suppressive miRNAs.</p>

DOI： 10.12938/bmfh.2022-056

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DOI： doi: 10.3390/cimb44120426

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1007/s11418-022-01668-5

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Food and Chemical Toxicology  

Anticancer agents can cause various side effects, including tissue damages/inflammatory reactions. Drug-responsive biomarkers are essential for evaluating drug toxicity in disease processes. S100 calcium-binding proteins A8/A9 (S100A8/A9) are highly expressed in neutrophils and monocytes/macrophages accumulated at inflammatory sites and are known to be related to tissue damage/inflammation; however, their response to drug toxicity has not been reported. Herein, we investigated the effects of anticancer agents (doxorubicin, cisplatin, and docetaxel) on S100A8/A9 gene expression profiles in four representative tissues (heart, kidney, liver, and lung) in normal C57BL/6J mice. Both S100A8/A9 expression was transiently or time-dependently elevated in four tissues within 48 h after dosing of the three anticancer agents under toxicity-inducing conditions. S100A8/A9 patterns differed among agents and tissues. This result suggests that S100A8/A9 is useful for evaluating anticancer agent-induced tissue damage. Metabolomic analysis revealed that some metabolites showed temporal patterns similar to that of S100A8/A9 expression. The amounts of fumarate (doxorubicin-treated heart), tyrosine (cisplatin-treated kidney), acetylcarnosine (doxorubicin-treated liver), and 2-phosphoglycerate (docetaxel-treated lung) showed similar patterns to that of S100A8/A9 expression. Although these metabolites showed different behaviors between tissues and serum, they may be useful marker candidates for evaluating anticancer agent-induced tissue damage at an earlier stage after dosing.

DOI： 10.1016/j.fct.2022.113421

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Food factors, such as amino acids, vitamins, and polyphenols, have been reported to exert biological effects through a system that senses food factors. However, how food and its components affect food factor sensing (FFS) systems remains largely unknown. Japanese are known for their longevity, to which the unique Japanese food is believed to contribute their health. We hypothesized that the promotion of health by Japanese food was due to the enhancement of food functionality by improving FFS systems. To examine the effect of Japanese soup stocks (katsuo-dashi and kombu-dashi) on FFS-related gene expression in mice, we performed DNA microarray analysis. Katsuo-dashi upregulated the expression of 10 FFS-related genes in the quadriceps, induced and suppressed that of nine and two FFS-related genes in the small intestine, and increased two FFS-related genes in the perirenal fat, respectively. Kombu-dashi upregulated and downregulated the expression of 30 and one FFS-related genes in the quadriceps, induced and suppressed that of one and one FFS-related genes in the small intestine, and increased and reduced that of two and one FFS-related genes in the perirenal fat, respectively. These data suggest that Japanese soup stocks mediate FFS-related gene expressions and might regulate their sensitivity to food factors.

DOI： 10.1016/j.ijgfs.2022.100573

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Background: Polyphenols, including flavonoids, have been the focus of numerous studies that have revealed diverse health benefits. MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that function as posttranscriptional regulators of gene expression. miRNAs can be detected in the blood and these so-called circulating miRNAs are potential biomarkers of various diseases. This study aimed to explore circulating miRNAs in plasma as a means to predict the biological effects of functional food ingredients. Methods and results: We used miRNA microarray analysis to compare plasma miRNA levels in mice orally administered three flavonoids (daidzein, quercetin, and delphinidin). Several miRNAs were differentially expressed in plasma from mice in each treatment group compared with the vehicle-treated group. The plasma levels of miR-25-5p, miR-146b-5p, and miR-501-3p were increased in the flavonoid-treated and the plasma levels of miR-148b-3p, miR-669e-5p, and miR-3962 were decreased. Conclusions: Our findings suggested that flavonoids alter miRNA expression in plasma and identified promising plasma miRNAs for assessing the functionality of flavonoids.

DOI： 10.1007/s11033-022-07918-9

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Other Link： https://link.springer.com/article/10.1007/s11033-022-07918-9/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

INTRODUCTION: Considering that neurodegeneration is an irreversible process, an efficient, low-burden approach to prevent dementia is strongly needed. Here, we show that the daily intake of myricetin normalised cognitive dysfunction in senescence-accelerated mouse prone 8 (SAMP8) mice. METHODS: SAMP8 mice were fed a diet supplemented with myricetin and novel object recognition tests and Y-maze tests were performed. Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brains of SAMP8 mice were measured. The phosphorylation level of cAMP-response-element-binding protein (CREB) level in brains of SAMP8 mice were evaluated. Also, SH-SY5Y cells were treated with myricetin and cAMP levels were measured. RESULTS: In SAMP8 mice, neurotrophins, including BDNF and NGF, were downregulated relative to levels in their normal counterparts. In addition, myricetin intake upregulated the phosphorylation of CREB, the major transcription factor for BDNF and NGF. Also, myricetin induced cAMP upregulation, and CREB phosphorylation via a cAMP-dependent protein kinase-dependent manner in SH-SY5Y cells. CONCLUSION: Taken together, myricetin improves cognitive function in SAMP8 mice and upregulates BDNF and NGF.

DOI： 10.1016/j.bbrc.2022.05.039

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Molecules  

The body is equipped with a "food factor-sensing system" that senses food factors, such as polyphenols, sulfur-containing compounds, and vitamins, taken into the body, and plays an essential role in manifesting their physiological effects. For example, (-)-epigallocatechin-3-O-gallate (EGCG), the representative catechin in green tea (Camellia sinensi L.), exerts various effects, including anti-cancer, anti-inflammatory, and anti-allergic effects, when sensed by the cell surficial protein 67-kDa laminin receptor (67LR). Here, we focus on three representative effects of EGCG and provide their specific signaling mechanisms, the 67LR-mediated EGCG-sensing systems. Various components present in foods, such as eriodictyol, hesperetin, sulfide, vitamin A, and fatty acids, have been found to act on the food factor-sensing system and affect the functionality of other foods/food factors, such as green tea extract, EGCG, or its O-methylated derivative at different experimental levels, i.e., in vitro, animal models, and/or clinical trials. These phenomena are observed by increasing or decreasing the activity or expression of EGCG-sensing-related molecules. Such functional interaction between food factors is called "functional food pairing". In this review, we introduce examples of functional food pairings using EGCG.

DOI： 10.3390/molecules27165130

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Agricultural and Food Chemistry  

There are numerous cultivars of tea (Camellia sinensis L.), but the differences in their anti-hyperglycemic-related effects are largely unknown. The inhibition of the dipeptidyl peptidase (DPP)-IV enzyme plays an essential role in controlling hyperglycemia in diabetes by blocking the degradation of incretin hormones, which is necessary for insulin secretion. In this study, we examined the DPP-IV inhibitory activity of leaf extracts from diverse Japanese green tea cultivars. The inhibitory rates differed among tea extracts. Metabolic profiling (MP), using liquid chromatography-mass spectrometry, of all cultivars revealed compositional differences among cultivars according to their DPP-IV inhibitory capacity. Epigallocatechin-3-O-(3-O-methyl)gallate, kaempferol-3-O-rutinoside, myricetin-3-O-glucoside/galactoside, and theogallin were newly identified as DPP-IV inhibitors. The bioactivity of a tea extract was potentiated by adding these ingredients in combination. Our results show that MP is a useful approach for evaluating the DPP-IV inhibitory potency of green tea and for determining bioactivity-related ingredients and combinations.

DOI： 10.1021/acs.jafc.2c01693

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1021/acs.jafc.2c01693

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/j.bbrc.2022.05.039

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

Epigallocatechin-3-O-gallate (EGCG), a catechin present in green tea, selectively elicits apoptosis in multiple myeloma cells by activating the endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) axis. However, the effects of EGCG alone are limited. Herein, we revealed that fustin, a flavanonol, enhances the EGCG-elicited activation of the cGMP/eNOS axis in multiple myeloma cells. Isobologram analysis demonstrated that EGCG/fustin synergistically elicited cell death in multiple myeloma cells. Importantly, this chemical combination significantly promoted cell death without affecting the normal cells. To assess the effects of EGCG and fustin in vivo, female BALB/c mice were inoculated with multiple myeloma MPC11 cells and then treated with each compound. The combination of EGCG/fustin suppressed tumor growth in vivo without affecting alanine aminotransferase/aspartate aminotransferase levels, the dose-limiting toxicity of EGCG. Consistent with in vitro findings, this combination increased eNOS phosphorylation at Ser1177 in the tumor. Collectively, fustin amplified EGCG-induced activation of the eNOS/cGMP axis.

DOI： 10.1021/acs.jafc.1c07567

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DOI： doi: 10.12938/bmfh.2021-067

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BMFH Press  

<p>Oxidative stress is associated with aging and pathologies such as cardiovascular diseases, Alzheimer’s disease, and cancer. Glutathione S-transferase (GST), a family of detoxification enzymes, plays a crucial role in countering oxidative stress. Therefore, there is a need for the development of physiologically functional foods and agricultural products, which enhance GST activity. Sesamin and episesamin are major lignans in refined sesame oil that exhibit beneficial properties including antioxidative stress effects. A previous study showed that sesamin upregulated GST activity. This study aimed to elucidate the mechanism underlying the GST activity enhancement elicited by sesame lignans. C57BL/6J mice were orally administered 20 mg/kg body weight sesame lignans (sesamin:episesamin=1:1) for 7 days. Oral administration of sesame lignans increased the GST activity in the mouse liver. Furthermore, the lignans upregulated GSTA1, GSTA4, and GSTM4 protein expression. Microarray analysis revealed that sesame lignans changed the expression of various microRNAs (miRNAs) (84 upregulated, 19 downregulated). We also found 16 miRNAs, including miR-669c-3p, that may negatively regulate GST expression among the 19 miRNAs with reduced expression caused by the sesame lignans. miR-669c is reportedly negatively correlated with GST. Additionally, we transfected NMuLi cells with an miR-669c-3p mimic and evaluated the effect of miR-669c-3p on GST mRNA and protein expressions. The results showed that the miR-669c-3p mimic suppressed the mRNA and protein levels of GSTA4 and GSTM4. In conclusion, sesame lignans increased GST protein expression and activity and downregulated miRNAs, including miR-669c-3p, which is a possible suppressor of GST.</p>

DOI： 10.12938/bmfh.2021-067

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1038/s41598-021-02346-4

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s41598-021-98612-6

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

(-)-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.

DOI： 10.1021/acs.jnatprod.1c00337

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DOI： doi: 10.1007/s11418-021-01538-6

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DOI： 10.1038/s41598-020-67288-9

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Epigallocatechin-3-O-gallate (EGCG) is one of the major bioactive compounds known to be present in green tea. We previously reported that EGCG shows selective toxicity through activation of the protein kinase B (Akt)/cyclic guanosine monophosphate (cGMP)/acid sphingomyelinase (ASM) axis via targeting its receptor 67-kDa laminin receptor (67LR), which is overexpressed in cancer. However, little is known about upstream mechanisms of EGCG-elicited ASM activation. In this study we show that the proto-oncogene tyrosine-protein kinase Src, also known as c-src, plays a crucial role in the anticancer effect of EGCG. We showed that EGCG elicits phosphorylation of Src at Tyr 416, a crucial phosphorylation site for its activity, and that the pharmacological inhibition of Src impedes the upstream events in EGCG-induced cell death signaling including upregulation of Akt activity, increase in cGMP levels, and activation of ASM. Moreover, focal adhesion kinase (FAK), which is involved in the phosphorylation of Src, is colocalized with 67LR. EGCG treatment enhanced interaction of FAK and 67LR. Consistent with these findings, pharmacological inhibition of FAK significantly neutralized EGCG-induced upregulation of Akt activity and activation of ASM. Taken together, FAK/Src play crucial roles in the upstream signaling of EGCG.

DOI： 10.3390/molecules25225481

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-020-67288-9

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.bbrc.2020.03.021

Language：English   Publishing type：Research paper (scientific journal)  

(-)-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.

DOI： 10.1007/s11418-020-01417-6

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1002/mnfr.201900986

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.bbrc.2019.09.102

Language：Others  

Cuprizone-treated mice, a possible model of schizophrenia, highlighting the simultaneous abnormalities of GABA, serine and glycine in hippocampus.

DOI： 10.1016/j.schres.2019.06.010

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Improvement of Sensitivity and Reproducibility for Imaging of Endogenous Metabolites by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry.

DOI： 10.1007/s13361-019-02221-7

Language：English   Publishing type：Research paper (scientific journal)  

Obesity is a major problem in developed countries and a burden on social health care systems. Several epidemiological studies showed the protective effects of green tea against obesity-related diseases. Cyclic guanosine monophosphate (cGMP) acts as a mediator for the physiological effects of (-)-epigallocatechin-3-O-gallate, the major constituent of green tea. Here, we showed that the level of phosphodiesterase 5, a negative regulator of cGMP, was up-regulated in adipose tissues of high-fat/high-sucrose (HF/HS) diet-fed mice and that this up-regulation was ameliorated by diallyl disulfide (DADS), the major organosulfur in garlic. A green tea extract (GT) and DADS in combination attenuated HF/HS diet-induced adipose increase and triglyceride accumulation in the liver. In these mechanisms, the combination regimen suppressed the HF/HS diet-induced up-regulation of fatty acid synthesis-related enzymes including sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase, and stearoyl-CoA desaturase-1. Moreover, this combination diet up-regulated thermogenesis-related genes including peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 alpha and uncoupling proteins in both white and brown adipose tissues. In conclusion, we identified DADS as an enhancer of the anti-obesity effect of GT accompanied by the suppression of SREBP-1 and activation of PPAR axis. The combination diet is a novel and easily applicable approach against obesity-related diseases.

DOI： 10.1016/j.jnutbio.2018.10.014

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s41598-018-34316-8

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Visualizing Energy Charge in Breast Carcinoma Tissues by MALDI Mass-spectrometry Imaging Profiles of Low-molecular-weight Metabolites.

DOI： 10.21873/anticanres.12723

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.bbrc.2018.01.012

Language：English  

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.

DOI： 10.3390/molecules22101621

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s41598-017-02499-1

Language：English   Publishing type：Research paper (scientific journal)  

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)
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.

DOI： 10.1007/s00216-016-0118-4

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1039/c6cc09504f

Language：English   Publishing type：Research paper (scientific journal)  

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 https://sourceforge.net/projects/msidv/.
Contact: eisuke.hayakawa@gmail.com
Supplementary information: Supplementary data are available at Bioinformatics online.

DOI： 10.1093/bioinformatics/btw548

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1158/1535-7163

Language：English   Publishing type：Research paper (scientific journal)  

Sphingosine kinase-1 protects B-cell neoplasm and myeloid leukemia from apoptosis driven by cancer specific inhibition of RTKs.
Activation of acid sphingomyelinase (ASM) leads to ceramide accumulation and induces apoptotic cell death in cancer cells. In the present study, we demonstrate that the activation of ASM by targeting cancer-overexpressed 67-kDa laminin receptors (67LR) induces lipid raft disruption and inhibits receptor tyrosine kinase (RTK) activation in multiple myeloma cells. Sphingosine kinase 1 (SphK1), a negative regulator of ceramide accumulation with antiapoptotic effects, was markedly elevated in multiple myeloma cells. The silencing of SphK1 potentiated the apoptotic effects of the green tea polyphenol epigallocatechin-3-O-gallate (EGCG), an activator of ASM through 67LR. Furthermore, the SphK1 inhibitor safingol synergistically sensitized EGCG-induced proapoptotic cell death and tumor suppression in multiple myeloma cells by promoting the prevention of RTK phosphorylation and activation of death-associated protein kinase 1 (DAPK1). We propose that targeting 67LR/ASM and SphK1 may represent a novel therapeutic strategy against multiple myeloma.

DOI： 10.1158/1535-7163.MCT-15-0185

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c5mb00182j

Language：English  

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.

DOI： 10.1080/09168451.2014.996205

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.atherosclerosis.2015.03.026

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1021/jf5054047

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/srep09474

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1021/jf503143g

Language：English   Publishing type：Research paper (scientific journal)  

IL-4 receptor alpha in non-lipid rafts is the target molecule of strictinin in inhibiting STAT6 activation
Strictinin has been shown to suppress interleukin (IL)-4-induced signal transducer and activator of transcription (STAT)-6 phosphoiylation, 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 a (IL-4R alpha) activation. Strictinin was bound to the cell surface and only localized in non-lipid raft fraction of the cells where IL-4R alpha was also located. In addition, strictinin directly bound to IL-4R alpha and inhibited binding of IL-4 to IL-4R alpha. These results suggest that IL-4R alpha locating in non-lipid raft region is a target molecule for strictinin in inhibiting STAT6 activation. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2014.06.069

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1007/s11306-013-0588-8

Language：Others  

MALDI Mass Spectrometry Imaging for Visualizing In Situ Metabolism of Endogenous Metabolites and Dietary Phytochemicals.

DOI： 10.3390/metabo4020319

Language：English   Publishing type：Research paper (scientific journal)  

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.
Methods
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.
Results
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.
Conclusions
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.

DOI： 10.1097/MPA.0000000000000092

Language：English   Publishing type：Research paper (scientific journal)  

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&#37; 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&#37; 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&#37; 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 (http://creativecommons.org/licenses/by-nc-nd/3.0/).

DOI： 10.1016/j.aca.2014.01.032

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

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 &#37; 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.

DOI： 10.1007/s13361-013-0772-0

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.atherosclerosis.2013.10.025

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1111/gtc.12098

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/srep02805

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1371/journal.pone.0070098

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.bbrc.2013.07.063

Language：English  

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.

DOI： 10.1016/j.jprot.2012.02.011

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1371/journal.pone.0037942

Language：English   Publishing type：Research paper (scientific journal)  

Reduced Expression of Adipose Triglyceride Lipase Enhances Tumor Necrosis Factor alpha-induced Intercellular Adhesion Molecule-1 Expression in Human Aortic Endothelial Cells via Protein Kinase C-dependent Activation of Nuclear Factor-kappa B
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 TNF alpha-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NF kappa B) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of I kappa B alpha 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 TNF alpha-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNF alpha-induced ICAM-1 expression via activation of NF kappa B 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.

DOI： 10.1074/jbc.M111.285650

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1371/journal.pone.0023426

Language：English   Publishing type：Research paper (scientific journal)  

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

DOI： 10.1021/ac101998z

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.4049/jimmunol.0903742

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.nut.2009.08.005

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.2131/jts.35.365

Language：English   Publishing type：Research paper (international conference proceedings)  

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1021/ac901083a

Language：English   Publishing type：Research paper (international conference proceedings)  

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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&#37; 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.

DOI： 10.1016/j.lfs.2009.01.004

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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.

DOI： 10.1016/j.abb.2008.03.002

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.bbrc.2007.09.095

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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.

DOI： 10.1007/s00394-007-0658-7

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1021/jf071176o

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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.

DOI： 10.1016/j.bbrc.2006.07.086

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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.

DOI： 10.1021/jf0607361

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Water-soluble component in dried chrysanthemum flower stimulates tumor necrosis factor-alpha production by mouse macrophage-like cell line RAW264.7
To clarify immunoregulatory activity of "Shiranui Himekiku" (Chrysanthemum indicam x Erigeron annus), we examined the effect on tumor necrosis factor alpha (TNF-alpha) 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-alpha 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 degrees C for 30 min enhanced the activity of water extract markedly, and freeze-thawing only moderately. The TNF-alpha production enhancing activity of the extract was observed within 3 h. These results suggest that the TNF-alpha production enhancing activity of CF extract can be recovered efficiently by hot water extraction and preserved stably by freezing.

DOI： 10.3136/fstr.12.144

Language：Japanese  

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A lipid raft-associated 67 kDa laminin receptor mediates suppressive effect of epigallocatechin-3-O-gallate on Fc epsilon RI expression
(-)-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 (Fc epsilon RI). 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 Fc epsilon RI expression correlated with the amount of 67LR. Thus, these results suggest that the lipid raft-associated 67LR plays an important role in mediating the Fc epsilon RI-suppressive action of EGCG. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.08.146

Language：English   Publishing type：Research paper (international conference proceedings)  

Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/nsmb743

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Lipid raft-associated catechin suppresses the Fc epsilon RI expression by inhibiting phosphorylation of the extracellular signal-regulated kinase1/2
The major green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has a suppressive effect on the expression of the high-affinity IgE receptor FcepsilonRI, 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 FcepsilonRI-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 FcepsilonRI 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 FcepsilonRI expression, and ERK1/2 may be involved in this suppression signal. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/S0014-5793(03)01432-7

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DOI： 10.1016/S0006-291X(02)02139-3

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DOI： 10.1021/jf025680z

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DOI： 10.1021/jf001392w

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DOI： 10.1271/bbb.64.2298

Publisher：Food Chemistry Molecular Sciences  

Matcha, a traditional Japanese green tea, has health-promoting effects. However, little is known about its bioactive components, except for polyphenols, caffeine, and amino acids. Here, we revealed the presence of diverse miRNAs, a type of functional RNAs, as new components of Matcha, using next-generation sequencing. Quantitative reverse-transcription PCR analysis of 10 different Matcha showed that miRNA levels varied depending on the cultivar and harvest season. As extraction methods of miRNAs, we found that soaking at 95 °C significantly enhanced total RNA and miRNA yields. Furthermore, a positive correlation was observed between total RNA and miRNA yields extracted from 27 plant-based dried powders. Notably, Matcha exhibited the highest levels of four representative miRNAs: lja-miR166-3p, csn-miR396d-5p, gma-miR396e, and csn-miRn409. The miRNA yields in Matcha were correlated with the major Matcha components. These results highlight Matcha as a source of miRNAs and candidate bioactive components. These findings provide new insights into the functionality of Matcha.

DOI： 10.1016/j.fochms.2025.100265

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Delphinidin, a plant anthocyanidin, suppresses disuse muscle atrophy in mice. However, its effect on muscle fiber type shift is unclear. To examine whether delphinidin affects skeletal muscle fiber type, differentiated C2C12 cells were treated with delphinidin. Results revealed that delphinidin upregulated the mRNA expression of myosin heavy chain type I (MyHCI), troponin C1, troponin I1, and MyHCIIx and increased slow MyHC protein level in C2C12 myotubes. Delphinidin also enhanced succinic dehydrogenase (SDH) activities and suppressed lactate dehydrogenase (LDH) activity. Adenosine monophosphate–activated protein kinase (AMPK) inhibition attenuated delphinidin-induced MyHCI upregulation and MyHCIIb downregulation. We investigated the effect of delphinidin on the upstream factors involved in AMPK activation. Delphinidin increased liver kinase B1 (LKB1) phosphorylation and nuclear respiratory factor 1 (NRF1) and calcium/calmodulin-dependent protein kinase 2 (CaMKK2) protein levels. In conclusion, delphinidin induced muscle fiber type conversion from fast-twitch to slow-twitch muscles through the AMPK signaling pathway.

DOI： 10.1016/j.bbrep.2024.101884

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Deficit of oxygen and nutrients in the tumor microenvironment (TME) triggers abnormal angiogenesis that produces dysfunctional and leaky blood vessels, which fail to adequately perfuse tumor tissues. Resulting hypoxia, exacerbation of metabolic disturbances, and generation of an immunosuppressive TME undermine the efficacy of anticancer therapies. Use of carefully scheduled angiogenesis inhibitors has been suggested to overcome these problems and normalize the TME. Here, we propose an alternative agonist-based normalization approach using a derivative of the C-type natriuretic peptide (dCNP). Multiple gene expression signatures in tumor tissues were affected in mice treated with dCNP. In several mouse orthotopic and subcutaneous solid tumor models including colon and pancreatic adenocarcinomas, this well-tolerated agent stimulated formation of highly functional tumor blood vessels to reduce hypoxia. Administration of dCNP also inhibited stromagenesis and remodeling of the extracellular matrix and decreased tumor interstitial fluid pressure. In addition, treatment with dCNP reinvigorated the antitumor immune responses. Administration of dCNP decelerated growth of primary mouse tumors and suppressed their metastases. Moreover, inclusion of dCNP into the chemo-, radio-, or immune-therapeutic regimens increased their efficacy against solid tumors in immunocompetent mice. These results demonstrate the proof of principle for using vasculature normalizing agonists to improve therapies against solid tumors and characterize dCNP as the first in class among such agents.

DOI： 10.1126/scitranslmed.adn0904

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フスチンのメラノーマに対する阻害効果について検討した。フスチンはB16メラノーマ細胞の増殖を阻害した。フスチンはメラノーマ細胞のアクチン構造の立体構造変化を誘導し、アクチン構造の調節因子であるミオシン軽鎖2(MLC2)のリン酸化抑制がみられた。プロテインキナーゼA(cAMP依存性プロテインキナーゼ)阻害剤のH89は、MLC2の脱リン酸化に関与するリン酸化ミオシンホスファターゼ標的サブユニット1のフスチンによる下方制御を軽減させた。マウスモデルにて、フスチンは有害作用なしにB16メラノーマ細胞における腫瘍増殖を抑制した。

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bioscience, Biotechnology and Biochemistry  

Melanoma, a cancer arising from melanocytes, requires a novel treatment strategy because of the ineffectiveness of conventional therapies in certain patients. Fustin is a flavanonol found in young fustic (Cotinus coggygria). However, little is known about its antimelanoma effects. Our study demonstrates that fustin suppresses the growth of B16 melanoma cells. Phalloidin staining of cytoskeletal actin revealed that fustin induced a conformational change in the actin structure of melanoma cells, accompanied by suppressed phosphorylation of myosin regulatory light chain 2 (MLC2), a regulator of actin structure. Furthermore, the protein kinase A (cAMP-dependent protein kinase) inhibitor H89 completely attenuated fustin-induced downregulation of phosphorylated myosin phosphatase targeting subunit 1, which is involved in dephosphorylation of MLC2. In a mouse model, administration of fustin suppressed tumor growth in B16 melanoma cells without adverse effects. In conclusion, our findings suggest that fustin effectively suppresses melanoma cell growth both in vitro and in vivo.

DOI： 10.1093/bbb/zbae072

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Cirrhosis is becoming one of the most common diseases worldwide. Abnormal upregulation of transforming growth factor β (TGF-β) signaling plays a pivotal role in the excess activation of hepatic stellate cells. However, an efficient countermeasure against abnormal hepatic stellate cell activation is yet to be established because TGF-β signaling is involved in several biological processes. Herein, we demonstrated the antifibrotic effect of miR-12135, a microRNA with unknown function upregulated by isoflavone. Comprehensive transcriptome assay demonstrated that miR-12135 suppressed Integrin Subunit Alpha 11 (ITGA11) and that ITGA11 expression is correlated with alpha smooth muscle actin expression in patients with cirrhosis. miR-12135 suppressed the expression level of ITGA11 and liver fibrosis. Importantly, ITGA11 is overexpressed in activated hepatic stellate cells, whereas ITGA11 knockout mice are viable and fertile. In conclusions, the miR-12135/ITGA11 axis can be an ideal therapeutic target to suppress fibrosis by precisely targeting abnormally upregulated TGF-β signaling in hepatic stellate cells.

DOI： 10.1016/j.isci.2023.108730

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Natural Medicines  

Recently, the number of patients diagnosed with dementia has increased. The World Health Organization (WHO) estimates that 50 million patients suffer from dementia. Although several therapeutic strategies have been proposed, currently, there is no curative approach for treating dementia. Neurodegeneration is an irreversible process. As this disease gradually progresses over 15-20 years, a low-cost and sustainable method for preventing these diseases is desired. Cacao nib is consumed in many countries, and a recent clinical study indicated that cocoa intake upregulates brain-derived neurotrophic factor (BDNF), which plays a significant role in memory formation and neuronal cell survival. In the present study, neural cells were treated with cacao nib extract or the 17 characteristic components of cacao nib. Treatment with Cacao nib extract upregulates BDNF mRNA expression. In addition, cacao nib extract elicits the phosphorylation of cAMP-response-element-binding protein (CREB), which regulates the transcription of BDNF. Among the 17 species screened, isovaleraldehyde (IVA), also known as an aroma component of cacao nibs extract, improved BDNF mRNA expression without SH-SY5Y cell toxicity. IVA also promoted CREB phosphorylation through a cAMP-dependent protein kinase (PKA)-dependent mechanism. In conclusion, IVA could be responsible for the BDNF upregulation effect of cacao nib, and IVA upregulated BDNF expression via the PKA-CREB axis.

DOI： 10.1007/s11418-023-01763-1

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Language：English   Publisher：シュプリンガー・ジャパン(株)  

神経細胞をカカオニブエキスまたはその17成分で処理し、その作用について検討した。カカオニブエキスによって脳由来神経栄養因子(BDNF) mRNA発現が上方制御された。カカオニブエキスはBDNFの転写を調節するcAMP応答配列結合蛋白質(CREB)のリン酸化を促進した。17化合物のうち、カカオニブエキスの香り成分のイソバレルアルデヒド(IVA)が、SH-SY5Y細胞に対して毒性なしにBDNF mRNA発現を改善した。IVAはcAMP依存性蛋白質キナーゼ(PKA)依存性機序を介してCREBのリン酸化を促進した。カカオニブのIVAはPKA-CREB軸を介してBDNFを上方制御すると考えられた。

Web of Science

Web of Science

Web of Science

Language：English   Publisher：シュプリンガー・ジャパン(株)  

柑橘類フラバノンのヘスペレチンが、ラット好塩基球/肥満細胞株RBL-2H3の抗アレルギー性カテキンの(-)-エピガロカテキン-3-O-(3-O-メチル)ガラート(EGCG3''Me)によるIgE/抗原媒介性脱顆粒の阻害活性を増幅することを見出した。ヘスペレチンは、細胞表面蛋白質の67-kDaラミニン受容体(67LR)を介して、EGCG3''Meの抗アレルギー効果発現に必須の酸性スフィンゴミエリナーゼ(ASM)の活性化を有意に促進した。強く吸収されるα-グルコシルヘスペリジンを経口投与すると、BALB/cマウスにおけるIgE/抗原の受動的皮膚アナフィラキシー(PCA)反応に対するEGCG3''Meの豊富な「べにふうき緑茶」の阻害活性を促進した。ヘスペレチンはASMシグナル伝達の活性化を介して、IgE/抗原媒介性脱顆粒を阻害するEGCG3''Meの活性を増幅することが示された。

Language：English   Publisher：BMFH出版会  

ヒト子宮頸癌細胞中のmiRNAと代表的フラボノイドのケルセチンとの関係を二つの試験で調べた。試験1ではヌードマウスにHeLa細胞を皮下注入して腫瘍が形成された後、マウスを対照群、10mg/kg b.w.ケルセチン群、50mg/kg b.w.ケルセチン群に割り付けた。11日後に子宮頸癌からRNAを抽出した。試験2ではマウスを対照群と50mg/kg b.w.ケルセチン群に割り付け48時間後に血漿からRNAを抽出した。試験1では、ケルセチン投与群の初期の子宮頸癌のmiR-26b、miR-126、miR-320aの発現が亢進された。試験2では、ケルセチンの単回投与は血漿中のmiR-26b、miR-126a、miR-320発現に影響を与えなかった。これらの結果から、ケルセチンは子宮頸癌内の腫瘍抑制miRNA分子の発現を上昇させることが示唆された。HeLa細胞を用いたin vitro試験の結果、ケルセチンは癌細胞に直接作用してmiRNAの亢進を促進することが示された。In vitro試験の結果、ケルセチンはmiR-320aの亢進を介してβ-カテニン濃度を低下させることが判明した。ケルセチンはpre-miR-26bとpre-miR-320aより早い段階でpre-miR-126の発現を増加させることが判明した。

Language：English   Publisher：Current Issues in Molecular Biology  

Epigallocatechin 3-O-gallate (EGCG) is a predominant component in green tea with various health benefits. The 67 kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that is overexpressed in various types of cancer; 67LR was identified a cell surface EGCG target that plays a pivotal role in tumor growth, metastasis, and resistance to chemotherapy. However, the plasma concentration of EGCG is limited, and its molecular mechanisms remain unelucidated in colon cancer. In this study, we found that the phosphodiesterase 5 (PDE5) inhibitor, vardenafil (VDN), potentiates EGCG-induced apoptotic cell death in colon cancer cells. The combination of EGCG and VDN induced apoptosis via activation of the endothelial nitric oxide synthase/cyclic guanosine monophosphate/protein kinase Cδ signaling pathway. In conclusion, the PDE5 inhibitor, VDN, may reduce the intracellular PDE5 enzyme activity that potentiates EGCG-induced apoptotic cell death in Caco-2 cells. These results suggest that PDE5 inhibitors can be used to elevate cGMP levels to induce 67LR-mediated, cancer-specific cell death. Therefore, EGCG may be employed as a therapeutic candidate for colon cancer.

DOI： 10.3390/cimb44120426

Web of Science

Scopus

PubMed

Language：English  

DOI： 10.1016/j.jbc.2022.101962

Web of Science

PubMed

Language：Japanese  

Language：Japanese  

Language：Japanese   Publisher：(公社)日本栄養・食糧学会  

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Language：Japanese   Publisher：(公社)日本栄養・食糧学会  

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Language：English   Publisher：BMFH出版会  

マウスの肝臓中のグルタチオンS-トランスフェラーゼ(GST)とmiRNAの発現に及ぼすゴマリグナンの効果を解明した。セサミン/エピセサミン混合物(SE)を7日間マウスに経口投与した。SE摂取はマウスの体重または肝臓重量を変化させずに肝臓中のGST活性を亢進した。リアルタイムPCRでSE投与後のGSTA1、GSTA4、GSTM4のmRNA発現量は変化しなかったが、GSTA1、GSTA4、GSTM4のタンパク質発現量は増大した。これらの結果から、SEはGSTタンパク質の発現増大によってGST活性を上昇していることが示された。肝臓組織のmiRNAマイクロアレイ解析により、SEは肝臓中の84個のmiRNA発現を亢進し19個のmiRNA発現を抑制することが判明した。miRNA標的予測アルゴリズムにより、SE投与で発現が抑制した19個のmiRNAのうち16個がGSTを標的にしている可能性が示された。定量リアルタイムPCRにより、miR-669c-3pに及ぼすSEの効果が確認された。NMuLi細胞へのmiR-669c-3p mimicのトランスフェクションにより、miR-669c-3pはマウス肝臓中のGSTA4とGSTM4のmRNAとタンパク質の発現を抑制していることが示唆された。

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：(一財)バイオインダストリー協会  

J-GLOBAL

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Language：Japanese  

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植物における質量分析イメージング法の最適化

Language：Japanese  

質量分析イメージング法を用いたトマト果実の生理応答に対する代謝動態解析

Language：Japanese  

要素還元的手法の限界とメタボロミクスの挑戦:食品機能性評価への応用

Language：Japanese  

DOI： 10.20632/vso.90.1_26

Language：Japanese  

DOI： 10.5702/massspec.S15-28

Language：Japanese  

Language：English  

Tea (Camellia sinensis L.) is a popular beverage worldwide, and because of its possible health effects, it has received considerable attention as a medicinal herb. Unfermented green tea constituents show various biological and pharmacological activities. Recently, metabolomic approaches to study the functionality and quality of tea are becoming more common place. One such metabolomic approach, metabolic profiling, can provide information on the relationships between the metabolome and factors such as phenotype or quality. Mass spectrometry (MS) and proton nuclear magnetic resonance (1H-NMR) spectroscopy combined with multivariate statistical analyses are employed for tea metabolomic studies. The highlighted topics are divided into three sections: (1) tea chemical composition, (2) metabolic responses to tea consumption, and (3) biotransformation of dietary tea components. In this chapter, we describe the latest metabolomic techniques applicable to new avenues of tea research.

DOI： 10.1002/9781118930458.ch31

Language：Japanese  

1P-126 Development of a novel method based on MS/MS spectral network for database-assisted identification of metabolites

Language：Japanese  

MALDI mass spectrometry imagingによる凍結組織アレイ包埋乳がん組織の低分子代謝産物プロファイリング

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要素還元的手法を補完するメタボロミクス:緑茶の機能性評価への応用

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Sensomicsによるコーヒーの品質を決定する原料生豆中の成分指標の発見

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MALDIマトリックスの構造機能相関と戦略的合成展開

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イメージング質量顕微鏡によるファイトケミカルの高感度分布解析

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メトホルミンとリラグルチドはPKC‐NAD(P)Hオキシダーゼの経路を介して酸化ストレスを減少させる

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メトホルミンおよびリラグルチドはPKC‐NAD(P)Hオキシダーゼ系の抑制により高血糖誘導酸化ストレスを抑制する

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質量分析イメージングによる傷害ストレス条件下でのトマト果実代謝物分布の可視化

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凍結組織アレイおよび質量分析イメージングをもちいた乳癌における癌関連代謝産物の高効率マッピング実証

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緑茶カテキンEGC代謝の可視化に向けた質量分析イメージング法の開発

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質量分析イメージング法を用いた緑茶カテキンならびにその代謝産物の生体組織内分布の非標識可視化

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質量分析イメージングを用いた植物代謝物分布の可視化

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2P-187 Metabolite Network Structure of Metabolic System in Escherichia coli for Sensing the Nutritional Environment

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質量分析イメージングによる熟度に応じたトマト果実代謝物分布の比較

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メトホルミンとリラグルチドの添加効果:PKC‐NAD(P)Hオキシダーゼの経路を介して酸化ストレスを減少させる。

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3P-031 Metabolic Variance in Mouse Kidney upon Cisplatin-Induced Acute Kidney Injury

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低分子食品成分の組織内空間分布の非標識可視化戦略

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組織内微小領域における機能性食品因子の時空間分解可視化

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質量分析イメージングを用いたトマト果実における代謝物分布の可視化

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質量分析イメージング法による緑茶カテキンおよびその代謝物の生体組織内分布情報の非標識可視化

Language：English  

DOI： 10.1016/j.freeradbiomed.2012.10.325

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3Ga14 Analysis of Comprehensive and Spatiotemporal Metabolic Dynamics by Integrated MS Technique

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超高分解能質量分析を用いた組成式決定法の理論的検証

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緑茶カテキンEGCのin situ質量分析イメージング法の開発

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IgE産生阻害緑茶成分ストリクチニンのin situ質量分析イメージング法の開発

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3Ga15 Development of Metabolite/Protein Imaging Technique using MALDI-MS in a Single Tissue Section

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レドックス関連疾患の理解とニュートリメタボロミクス

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組織内微小領域における緑茶カテキンの二次元可視化に向けた質量分析イメージング法の開発

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緑茶カテキンのin situ質量分析イメージング

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超高分解能質量分析による組成式決定アルゴリズムの開発

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質量分析によるmulti‐omicsイメージング

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緑茶カテキンEGCGの抗腫瘍効果に対する酸素応答性の理解に向けた細胞内代謝物プロファイリング

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メタボロミクスを駆動力とする緑茶品種の新たな機能性評価

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酸化ストレス疾患のためのOMRI/MSI法の開発

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メタボリック・プロファイリングによる血管内皮障害抑制作用を有する茶葉成分の探索

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メタボリック・プロファイリングによる新規高アントシアニン系の活性成分の探索

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NMRと質量分析を用いた高精度メタボリック・プロファイリング法

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OMRI/MSIによるレドックスプローブのin vivo/in situ解析

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MALDI‐MSを用いたメタボロミクスイメージング

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緑茶カテキン投与マウス尿のメタボリック・プロファイリング

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新規高アントシアニン茶のメタボリック・プロファイリング

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メタボリック・プロファイリング法によるシスプラチン誘導性酸化ストレスに対する緑茶ポリフェノールの活性評価

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緑茶ポリフェノールEGCGのニュートリメタボロミクス

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DOI： 10.1271/kagakutoseibutsu.47.111

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Aggregated ursolic acid, a natural triterpenoid, induces IL-1 beta release from murine peritoneal macrophages: Role of CD36
IL-1 beta has been shown to play a pivotal role in the development of inflammatory disorders. We recently found that a natural triterpene, ursolic acid (UA), enhanced MIF release from nonstimulated macrophages. In this study, we examined the effects of UA on the production of several cytokines in resident murine peritoneal macrophages (pM phi). UA increased the protein release of IL-1 beta, IL-6, and MIF, but not of TNF-alpha, in dose- and time-dependent manners. This triterpene also strikingly induced the activation of p38 MAPK and ERK1/2 together with that of upstream kinases. The release of UA-induced IL-1 beta was significantly inhibited by the inhibitors of p38 MAPK, MEK1/2, ATP-binding cassette transporter, and caspase-1. Furthermore, UA induced intracellular ROS generation for IL-1 beta production, which was suppressed by an antioxidant. Pretreatment with an anti-CD36 Ab significantly suppressed IL-1 beta release, and surface plasmon resonance assay results showed that UA bound to CD36 on macrophages. In addition, the amount of IL-1 beta released from UA-treated pM phi of CD36-deficient mice was markedly lower than that from those of wild-type mice. Interestingly, UA was found to aggregate in culture medium, and the aggregates were suggested to be responsible for IL-1 beta production. In addition, i.p. administration of UA increased the levels of IL-1 beta secretion and MPO activity in colonic mucosa of ICR mice. Taken together, our results indicate that aggregated UA is recognized, in part, by CD36 on macrophages for generating ROS, thereby activating p38 MAPK, ERK1/2, and caspase-1, as well as releasing IL-1 beta protein via the ATP-binding cassette transporter.

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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 K-d value.

DOI： 10.1038/nsmb743

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We previously found that the O-methylated derivative of (-) epigallocatechin 3 O-gallate (EGCg), (-)-epigallocatechin 3 O-(3-O-methyl)-gallate (EGCG"3Me) has potent antiallergic activity The high-affinity IgE receptor, FcepsilonRI, is found at high levels on basophils and mast cells and plays a key role in a series of acute and chronic human allergic reactions To understand the mechanism of action for the antiallergic EGCG"3Me, the effect of EGCG"3Me on the cell surface expression of FcepsilonRI in human basophilic KU812 cells was examined Flow cytometric analysis showed that EGCG"3Me was able to decrease the cell surface expression of FcepsilonRI Moreover, immunoblot analysis revealed that total cellular expression of the FcepsilonRI alpha chain decreased upon treatment with EGCG"3Me FcepsilonRI is a tetrameric structure comprising one alpha chain, one beta chain, and two gamma chains The level of mRNA production of each subunit in KU812 cells was investigated EGCG"3Me reduced FcepsilonRI alpha and gamma mRNA levels The cross-linkage of FcepsilonRI causes the activation of basophils which leads to the secretion of inflammatory mediators including histamine EGCG"3Me treatment inhibited the FcepsilonRI cross-linking induced histamine release These results suggested that EGCG"3Me can negatively regulate basophil activation through the suppression of FcepsilonRI expression.

DOI： 10.1021/jf025680z

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Antiallergic tea catechin, (-)-epigallocatechin-3-O-(3-O-methyl)-gallate, suppresses Fc epsilon RI expression in human basophilic KU812 cells
We previously found that the O-methylated derivative of (-) epigallocatechin 3 O-gallate (EGCg), (-)-epigallocatechin 3 O-(3-O-methyl)-gallate (EGCG"3Me) has potent antiallergic activity The high-affinity IgE receptor, FcepsilonRI, is found at high levels on basophils and mast cells and plays a key role in a series of acute and chronic human allergic reactions To understand the mechanism of action for the antiallergic EGCG"3Me, the effect of EGCG"3Me on the cell surface expression of FcepsilonRI in human basophilic KU812 cells was examined Flow cytometric analysis showed that EGCG"3Me was able to decrease the cell surface expression of FcepsilonRI Moreover, immunoblot analysis revealed that total cellular expression of the FcepsilonRI alpha chain decreased upon treatment with EGCG"3Me FcepsilonRI is a tetrameric structure comprising one alpha chain, one beta chain, and two gamma chains The level of mRNA production of each subunit in KU812 cells was investigated EGCG"3Me reduced FcepsilonRI alpha and gamma mRNA levels The cross-linkage of FcepsilonRI causes the activation of basophils which leads to the secretion of inflammatory mediators including histamine EGCG"3Me treatment inhibited the FcepsilonRI cross-linking induced histamine release These results suggested that EGCG"3Me can negatively regulate basophil activation through the suppression of FcepsilonRI expression.

DOI： 10.1021/jf025680z

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We cloned the variable regions of heavy and light chain genes of an anti-ovomucoid monoclonal antibody (MAb-OM21) produced by the mouse hybridoma cell line OM21. DNA sequence analysis showed that the light chain of the MAb-OM21 has only one potential N-glycosylation consensus sequence in the complementarity determining region 2 of the light chain. To find whether carbohydrate chains are located on the light chain, we assayed for the size of the light chain, after treatment with N-glycosidase, by western blotting, and also detection of the carbohydrate chains on the light chain was done using the lectin blot assay. A N-linked carbohydrate chain has been shown to bind to the light chain. To clarify the role of this carbohydrate chain in the light chain, we produced carbohydrate variant antibodies by N-deglycosylation using glycosidase or by expressing the antibody from different host cells. The N-deglycosylated variant antibody has greater antigen binding, and the antibody produced from the different host cells showed a reduced antigen binding activity and acquired the ability to react to ovalbumin. These results suggest that antigen binding of the ovomucoid specific antibody MAb-OM21 can be affected by the carbohydrate chain on the light chain variable region.

DOI： 10.1271/bbb.64.2298