Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Society for Nutraceuticals & Functional Foods (ISNFF)  

Clove (Syzygium aromaticum L) is a precious spice that has been extensively used by many countries over the centuries to add flavor and for medicinal purposes. Because of its abundance of phytochemical compounds, clove has been shown to have positive benefits on human health. Hence, we investigated the anti-inflammatory activity of clove water extract (CWE) in LPS-stimulated RAW264.7 cells and mouse peritoneal. The results showed that CWE significantly inhibited the production of IL-6 and TNF-α in a dose-dependent manner, as well as the production of nitric oxide (NO), without any cytotoxic effects at less than 20 mg/mL, through down-regulating IL-6, TNF-α, and iNOS gene expression. Moreover, CWE impeded the MAPKs and inhibited the translocation of NF-kB from the cytosol to the nucleus. These results suggest that CWE possesses anti-inflammatory properties by inhibiting the MAPKs and NF-kB pathways.

DOI： 10.31665/jfb.2024.18373

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

DOI： 10.1016/j.focha.2023.100253

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Food Science and Technology  

DOI： 10.3136/fstr.fstr-d-23-00053

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

DOI： 10.33555/jffn.v5i1.119

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Language：English  

DOI： 10.1177/1934578X231183603

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

DOI： 10.1016/j.phytol.2022.10.002

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

DOI： 10.1016/j.fitote.2022.105298

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Cuminum cyminum L. (cumin) seeds are widely used as a spice. Although we previously reported that the aqueous extract of cumin seeds suppresses the degranulation of rat basophilic RBL-2H3 cells, it has not been clarified whether the extract alleviates actual allergy symptoms in vivo. Therefore, in this study, we investigated the effect of oral administration of cumin seed aqueous extract (CAE) in ovalbumin (OVA)-induced allergic rhinitis. BALB/c mice were randomly divided into the following three groups: control group (five mice), OVA group (five mice), and OVA + CAE group (five mice). Allergic rhinitis was induced by sensitization (intraperitoneal, 25 μg OVA and 1.98 mg aluminum hydroxide gel) followed by challenge (intranasal, 400 μg OVA). The oral administration of CAE (25 mg/kg) reduced the sneezing frequency of OVA-induced allergic rhinitis model mice. In addition to reducing the serum immunoglobulin E and IL-4 levels, the oral administration of CAE reduced the production of T-helper type-2 (Th2) cytokines (IL-4, IL-5, IL-10, and IL-13) in the splenocytes of the model mice. Furthermore, a significant increase in the ratio of Th1 to Th2 cells was observed in the CAE-administered group. Our findings suggest that the ingestion of CAE improves T cell balance, the dominant state of Th2, and alleviates allergic rhinitis symptoms.

DOI： 10.3390/foods11203224

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Coriander (Coriandrum sativum L.) is classified in the Apiaceae family and used as an herb. Coriander leaf has been reported to possess various health functions. Here, we report the anti-allergic effect of aqueous coriander leaf extract (ACLE). ACLE with 1.0 mg/mL or higher concentration significantly inhibited degranulation of RBL-2H3 cells in a concentration-dependent manner with no cytotoxicity. ACLE suppressed the increase in the intracellular Ca2+ concentration in response to antigen-specific stimulation. Immunoblot analysis demonstrated that ACLE significantly downregulates phosphorylation of phosphatidylinositol 3-kinase and tends to downregulate phosphorylation of Syk kinase in the signaling pathways activated by antigen-mediated stimulation. Oral administration of ACLE did not alter the sneezing frequency of pollinosis model mice stimulated with cedar pollen, but significantly reduced the serum IgE level. Our data show anti-allergic effects of coriander leaf in both cultured cells and pollinosis mice. These results suggest that coriander leaf has the potential to be a functional foodstuff with anti-allergy effects.

DOI： 10.3390/nutraceuticals2030013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Cuminum cyminum L. (cumin) is an annual plant of the Umbelliferae family native to Egypt. We previously showed that the aqueous extract of cumin seeds suppresses degranulation by downregulating the activation of antigen-induced intracellular signaling molecules in rat basophilic leukemia RBL-2H3 cells. However, the active substances in the extract have not yet been identified. Accordingly, herein, we aimed to ascertain the water-soluble substances present in cumin seeds that inhibit degranulation, which led to the identification of umbelliferose, a characteristic trisaccharide present in plants of the Umbelliferae family. Our study is the first to reveal the degranulation-suppressing activity of umbelliferose, and quantification studies suggest that cumin seed powder contains 1.6% umbelliferose. Raffinose, an isomer of umbelliferose, was also found to significantly suppress antigen-induced degranulation, but less so than umbelliferose. Both umbelliferose and raffinose contain sucrose subunits in their structures, with galactose moieties bound at different sites. These differences in structure suggest that the binding of galactose to the sucrose subunit at the α1-2 bond contributes to its strong degranulation-inhibiting properties.

DOI： 10.3390/molecules27134101

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Hasanuddin University, Faculty of Law  

Obesity is one of the major health problems which could trigger the development of many other non-communicable diseases. Tamarind (Tamarindus indica) is a multi-purpose plant that provides many health benefits, including anti-obesity potentials. In Indonesia, it is often used as a traditional food component in many dishes. A recent in vivo study proved that tamarind might reduce the bodyweight of obese rats. To further investigate the anti-obesity effect of tamarind, an in vitro study using 3T3-L1 pre-adipocytes was conducted. During differentiation, 3T3-L1 cells synthesize and accumulate lipid in the form of triglycerides in the cytoplasm. Tamarind water and ethanol extracts were incorporated into the medium during the differentiation stage of the cells. Oil red O staining assay was used to measure the total lipid accumulated. Results showed that both tamarind water and ethanol extracts exhibited non-cytotoxicity effects on 3T3-L1 cells. In addition, tamarind water extract at 10 mg/mL decreased the total lipid accumulated significantly to 80.5% compared with that of control, while tamarind ethanol extract showed less lipid accumulation inhibitory effect at any doses tested. This study revealed that tamarind water extract has the potential to reduce lipid accumulation, thus showing an anti-obesity effect.

DOI： 10.20956/canrea.v5i1.630

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

We found that p-synephrine inhibits the production of proinflammatory cytokines in LPS-stimulated RAW264.7 cells. This inhibitory effect could be attributed to the downregulation of p38 MAPK and NF-κB cascades mediated by β-adrenergic receptors.

DOI： 10.1039/d2fo00299j

PubMed

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

DOI： 10.1016/j.legalmed.2021.101988

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Media Peternakan  

DOI： 10.5398/tasj.2021.44.4.511

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

DOI： 10.33555/jffn.v3i1.68

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

Inflammation is related to various life-threatening diseases including cancer, neurodegenerative diseases, and metabolic syndrome. Because macrophages are prominent inflammatory cells, regulation of macrophage activation is a key issue to control the onset of inflammation-associated diseases. In this study, we aimed to evaluate the potential anti-inflammatory activity of Citrus unshiu leaf extract (CLE) and to elucidate the mechanism underlying its anti-inflammatory effect. We found the inhibitory activity of CLE on the secretion of proinflammatory cytokines and a chemokine from mouse macrophage-like RAW 264.7 cells and mouse peritoneal macrophages. The inhibitory activity of CLE was attributed to downregulated JNK, p38 MAPK, and NF-κB signaling pathways, leading to suppressed gene expression of inflammation-associated proteins. Oral administration of CLE significantly decreased the serum level of proinflammatory cytokines IL-6 and TNFα and increased that of anti-inflammatory cytokine IL-10 in lipopolysaccharide-induced systemic inflammation mice. In addition, oral administration of CLE decreased secretion and gene expression of several proinflammatory proteins in the liver and spleen of the model mice. Overall results revealed that C. unshiu leaf is effective to attenuate inflammatory responses in vitro and in vivo.

DOI： 10.3390/plants10081708

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

DOI： 10.3136/nskkk.68.356

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

DOI： 10.17509/ijost.v6i2.34544

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Research and Community Service Swiss German University  

Allergy rhinitis (AR), as reported by the World Allergy Organization (WAO), is one of the highest prevalence allergies affecting 10-30% of all adults and up to 40% of children. In Indonesia, current evidence showed that the prevalence of AR is increasing. Averrhoa bilimbi Linn. fruit (AF), or locally known as belimbing wuluh, has been scientifically proven to treat many diseases due to the abundant of polyphenol content which was shown to have the potential to treat allergies. Therefore, this study was aimed to investigate the anti-allergy potential of AF in vitro. The anti-allergy effect of Averrhoa bilimbi Linn. fruit water extract (AFWE) was examined using RBL-2H3 cells. At first, the cytotoxicity effect of AFWE was determined by WST-8 assay. The release of β-hexosaminidase by RBL-2H3 cells was also measured to evaluate degranulation suppression activity of AFWE. Lastly, calcium assay was employed to investigate the intracellular calcium concentration ([Ca­2+]i). Results demonstrated that AFWE does not show any cytotoxicity at any given concentration. In addition, AFWE at 1.25 mg/mL showed sufficient inhibitory effect towards degranulation by RBL-2H3 cells. Moreover, the degranulation-suppressing activity of AFWE was resulted from the inhibition of calcium-dependent signaling pathways. Unfortunately, the properties of active substances from AFWE have not been investigated. To conclude, this study indicated that AFWE has potential as an alternative treatment for allergic diseases.

DOI： 10.33555/jffn.v2i2.62

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

DOI： 10.1186/s41702-020-0061-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.bmcl.2020.127191

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.molimm.2019.12.020

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/acs.jmedchem.9b00994

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

Ficifolidione, a natural insecticidal compound isolated from the essential oils of Myetaceae species, is a spiro phloroglucinol with an isobutyl group at the C-4 position. We found that ficifolidione showed cytotoxicity against cancer cells via apoptosis. Replacement of the isobutyl group by n-propyl group did not influence the potency, but the effect of the replacement of this group by a shorter or longer alkyl group on the biological activity remains unknown. In this study, ficifolidione derivatives with alkyl groups such as methyl, n-pentyl, and n-heptyl group-instead of the isobutyl group at the C-4 position-were synthesized to evaluate their cytotoxicity against the human promyelocytic leukaemia cell line HL60 and their insecticidal activity against mosquito larvae. The biological activities of their corresponding 4-epimers were also evaluated. As a result, the conversion of the isobutyl group to another alkyl group did not significantly influence the cytotoxicity or insecticidal activity. In HL60 cells treated with the n-heptyl-ficifolidione derivative, the activation of caspase 3/7 and the early stages of apoptosis were detected by using immunofluorescence and flow cytometric techniques, respectively, suggesting that the cytotoxicity should be induced by apoptosis even though the alkyl group was changed.

DOI： 10.3390/molecules24224081

PubMed

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-019-52317-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa {UK} Limited  

DOI： 10.1080/09168451.2019.1618697

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.jff.2019.103422

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

DOI： 10.1007/s10616-019-00323-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/acs.jafc.9b03171

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

DOI： 10.1007/s10616-019-00309-2

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

DOI： 10.1007/s10616-019-00296-4

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

DOI： 10.1027/0269-8803/a000234

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.eujim.2018.11.003

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Netherlands  

Japanese black vinegar (JBV) is a traditional vinegar manufactured with steamed unpolished rice. After screening, beneficial effects of JBV on IgE-mediated allergic responses were found. In this study, acetic acid-free JBV was used to evaluate its antiallergic effects. JBV suppressed degranulation of rat basophilic leukemia RBL-2H3 cells in a dose-dependent manner without cytotoxicity. The inhibitory effect of JBV on the degranulation seemed to be caused by the bioactive ingredients other than proteins, because the activity was not affected by heat treatment or protease digestion. JBV inhibited the elevation in the intracellular Ca2+ concentration induced by antigen. Immunoblot analysis revealed that JBV suppresses degranulation of RBL-2H3 cells by downregulated phosphorylation of PI3K, Akt, and PLCγ1. In addition, oral administration of JBV significantly suppressed passive cutaneous anaphylaxis reaction in mice and an allergic symptom in Cry j1-induced pollinosis model mice. Thus, JBV has a potential as a health-promoting food with the antiallergy effect.

DOI： 10.1007/s10616-018-0208-6

Scopus

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Netherlands  

Lysozyme from hen egg has been reported to possess an anti-inflammatory effect. However, little is known about its detailed mechanism. The mechanism of anti-inflammatory effect of lysozyme was examined in this study. When mouse macrophage-like cell line RAW264.7 cells and mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and then treated with lysozyme, the production of tumor necrosis factor-α and interleukin-6 was significantly suppressed. The effect was induced by suppressing the gene expression levels of both cytokines. Phagocytosis activity of peritoneal macrophages was not altered by the treatment with lysozyme, suggesting that lysozyme shows the anti-inflammatory effect without inhibiting the phagocytotic response of macrophages. In addition, lysozyme inhibited phosphorylation of c-jun N-terminal kinase (JNK) and was taken up by macrophages within 1 h after treatment of the cells with lysozyme. Overall results suggest that lysozyme is taken up intracellularly and suppresses LPS-induced inflammatory responses by inhibiting JNK phosphorylation.

DOI： 10.1007/s10616-017-0184-2

Scopus

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Immunoglobulin E (IgE) is involved in the onset of allergic reaction, and the suppression of IgE production leads to alleviation of allergic symptoms. We found that mango peel ethanol extract (MPE) significantly suppresses IgE production by human myeloma cell line U266 cells, suggesting that MPE has an anti-allergic effect by inhibiting the production of IgE. Although mangiferin is contained in mango, which suppresses IgE production by U266 cells, it was not contained in MPE. We investigated the suppressive effect of MPE in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis model mice. The elevation of serum IgE level was significantly suppressed by oral administration of MPE. Intake of MPE also suppressed the expression level of IL-4 in the DNFB-challenged ears, suggesting that MPE suppresses the IL-4-mediated maturation into IgE-producing cells. Our findings indicate that MPE has a potential to alleviate the increase in serum IgE level that is feature of type I allergy.

DOI： 10.1080/09168451.2017.1412250

Scopus

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

BACKGROUNDCoriandrum sativum L. seed is generally used as a spice and crude drug. Although many functions of the various components in C. sativum L. seed have been reported, the immunostimulatory effect of water-soluble components in C. sativum L. seed has not been studied. In the present study, we focused on the immunostimulatory effect of C. sativum L. seed aqueous extract (CAE) on macrophages as a novel health function of C. sativum L. seed components.
RESULTSCAE significantly enhanced the production of tumor necrosis factor (TNF)- and interleukin (IL)-6 in both RAW264.7 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. CAE also stimulated nitric oxide (NO) production and the phagocytosis activity in RAW264.7 cells. We suggest that the activity of CAE is a result of the upregulation of mitogen-activated protein kinase and nuclear factor-B cascades via TLR4. In addition, IL-6 production by peritoneal macrophages collected from CAE-administered mice was significantly enhanced, suggesting that CAE could stimulate macrophage activity in vivo.
CONCLUSIONThe findings of the present study suggest that CAE contains a novel water-soluble component with an immunostimulatory effect on macrophages. CAE would contribute to activating host defense against pathogens by stimulating the innate immunity. (c) 2017 Society of Chemical Industry

DOI： 10.1002/jsfa.8341

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

A series of 1,3-(adamantan-1(2)-yl)imidazolidine-2,4,5-triones and 1,1'-(alkane-1,n-diyl)bis[3-(adamantan-1-yl)imidazolidine-2,4,5-triones] was synthesized via cyclization of 1,3-bis[adamantan-1(2)-ylureas] and 1,1'-(alkyl-1,n-diyl)bis[3-(adamantan-1-yl)ureas] with oxalyl chloride under mild conditions with high yields. All synthesized compounds were tested in vitro as inhibitors of soluble human epoxide hydrolase. A number of compounds have high inhibitory activity (IC50 1.6-650 nM), which makes them promising inhibitors of soluble epoxide hydrolase.

DOI： 10.1007/s10593-017-2174-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The new lignano-9,9'-lactones (alpha,beta-dibenzyl-y-butyrolactone lignans), which showed the higher cytotoxicity than arctigenin, were synthesized. The well-known cytotoxic arctigenin showed activity against HL-60 cells (EC50 =12 IA/I), however, it was inactive against HeLa cells (EC50 > 100 mu M). The synthesized (3,4-dichloro, 2'-butoxy)-derivative 55 and (3,4-dichloro, 4'-butyl)-derivative 66 bearing the lignano9,9'-lactone structures showed the EC50 values of 10 mu M and 9.4 mu M against HL-60 cells, respectively. Against HeLa cells, the EC50 value of the derivative 66 was 27 mu M. By comparing the activities with the corresponding 9,9'-epoxy structure (tetrahydrofuran compounds), the importance of the lactone structure of 55 and 66 for the higher activities was shown. The substituents on the aromatic ring of the lignano-9,9'-lactones affected the cytotoxicity level, observing more than 10-fold difference. (C) 2017 Published by Elsevier Ltd.

DOI： 10.1016/j.bmcl.2017.06.070

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Porang is a local plant of Indonesia, which has a high content of glucomannan. In this study, porang glucomannan (PG) was esterified with octenyl succinic anhydride (OSA) to enhance emulsion properties to be widely used in food industry. OSA-modified PG (OSA-PG) enhanced the phagocytosis activity of macrophage-like J774.1 cells and mouse peritoneal macrophages. In addition, OSA-PG increased the production of IL-6 and TNF-alpha by enhancing their gene expression. Immunoblot analysis displayed that OSA-PG tended to activate both nuclear factor-kappa B and mitogen-activated protein kinase cascades. Treatment of OSA-PG with polymyxin B revealed that cytokine production induced by OSA-PG was not caused by endotoxin contamination. Our findings also indicated that OSA-PG activates macrophages through not only Toll-like receptor (TLR) 4, but another receptor. Overall findings suggested that OSA-PG has a potential as an immunomodulatory food factor by stimulating macrophages.

DOI： 10.3390/molecules22071187

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

An array of foodstuffs was screened for the suppressive effect on production of IgE, a key molecule in the type I allergic reaction, using U266 cells. An ethanol extract from passion fruit seeds (PFS) has emerged as a food material with the IgE production-suppressive effect as the result of screening. Real-time RT-PCR analysis revealed that the IgE production-suppressive effect was caused by downregulated IgE gene expression. In addition, PFS selectively suppressed IgE production and did not affect the production of IgG, IgA, or IgM by mouse splenocytes. Resveratrol and piceatannol were identified as bioactive ingredients contained in passion fruit seeds. The effect of oral administration of PFS, resveratrol, and piceatannol was evaluated using a mouse model of allergic contact dermatitis. Both stilbenoids significantly suppressed IgE production in orally administered mice. Overall results indicated that passion fruit seeds are a promising candidate with the antiallergy effect for development of functional foods. (C) 2017 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2017.02.030

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

Dried bonito is a preserved food used in Japan, which contains abundant flavor ingredients and functional substances. We focused on the immunostimulatory effect of dried bonito extract (DBE) on mouse macrophage-like J774.1 cells, RAW264.7 cells, and mouse primary peritoneal macrophages. DBE significantly stimulated the production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by both J774.1 cells and peritoneal macrophages by enhancing the cytokine gene expression levels. In addition, DBE stimulated nitric oxide production by enhancing the expression of inducible nitric oxide synthase in RAW264.7 cells. DBE also increased the phagocytosis activity of J774.1 cells. Immunoblot analysis revealed that DBE has an immunostimulatory effect on macrophages through activation of mitogenactivated protein kinase and nuclear factor-jB cascades. TNF-alpha production enhanced by DBE was partially inhibited by treatment with TLR4 inhibitor TAK-242, whereas IL-6 production enhanced by DBE was almost inhibited. These results suggested that DBE is thought to strongly stimulate the TLR4 signaling pathway for macrophage activation, and its activation is also involved in other signaling. Finally, the phagocytosis activity of peritoneal macrophages from DBE-administered BALB/ c mice increased significantly, suggesting that DBE has the potential to stimulate macrophage activity in vivo. In conclusion, these data indicated that DBE contributes to activating host defense against pathogens by activating innate immunity.

DOI： 10.1007/s10616-016-0053-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Nobiletin is a major citrus flavonoid possessing several biological activities. Nobiletin has been reported to display anti-inflammatory activity and to reduce monocyte chemoattractant protein (MCP)-1 secretion by 3T3-L1 adipocytes. However, its mode of action remains unclear. In the present study, we investigated the suppressive effect of nobiletin on MCP-1 expression in 3T3-L1 cells and its underlying molecular mechanism. Nobiletin notably down regulated the MCP-1 mRNA level during adipocyte differentiation. Nobiletin also reduced MCP-1 secretion by preadipocytes (undifferentiated cells), differentiated adipocytes, and hypertrophied adipocytes. Immunoblot analysis showed that nobiletin inhibits the phosphorylation level of extracellular signal-regulated kinase (ERK) in both preadipocytes and differentiated adipocytes. Moreover, nobiletin suppressed MCP-1 secretion by adipocytes induced by tumor necrosis factor (TNF)-alpha. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2016.09.025

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

Snake fruit (Salacca edulis Reinw.) is a tropical fruit produced in Indonesia. Snake fruit peel is normally discarded as waste. In the present study, it was revealed that snake fruit peel has high bioactivities on stimulation of the immune system. Snake fruit peel extract (SFPE) was prepared by extracting snake fruit peel powder in water for 15 h at 4 A degrees C. SFPE enhanced phagocytotic activity of murine macrophage-like J774.1 cells. Production of cytokine such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 was also stimulated by SFPE. The gene expression levels for these cytokines were elevated. Immunoblot analysis revealed that SFPE enhanced not only nuclear factor (NF)-kappa B but also mitogen-activated protein kinases signalling cascades such as JNK and p38 in macrophage. Overall findings suggested that SFPE has a potential beneficial effect to promote our body health through the stimulation of macrophage.

DOI： 10.1007/s10616-015-9925-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The effects of small dried sardine on IgE-mediated allergic responses are herein reported. Small dried sardine extract digested with an acidic protease from Aspergillus niger suppressed degranulation of rat basophilic leukemia RBL-2H3 cells in a dose-dependent manner without cytotoxicity. Size-exclusion chromatography analysis and a dialysis experiment indicated that the molecular weight of bioactive components contained in enzyme-treated dried sardine extract (EDS extract) ranges from 4800 to 14 000. Immunoblot analysis revealed that EDS extract does not affect the signaling pathway via the spleen tyrosine kinase Syk. Although degranulation of RBL-2H3 cells induced by either antigen or a calcium ionophore A23187 was suppressed by EDS extract, the elevation in intracellular Ca2+ concentration was not affected. Immunofluorescence staining revealed that EDS extract significantly suppresses microtubule formation in RBL-2H3 cells during degranulation induced by antigen. In addition, oral administration of EDS extract significantly suppressed passive cutaneous anaphylaxis reaction in mice and an allergic symptom in Cry j1-and Cry j2-induced pollinosis model mice. In conclusion, small dried sardine treated with a protease has potential as a health-promoting foodstuff with an anti-allergic effect.

DOI： 10.1039/c6ra14967g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

To estimate the effect of methyl group of dihydroguaiaretic acid, which shows many kinds of biological activities, on biological activity, both enantiomers of 9'-dehydroxyimperanene (5, 6) and 7,8-dihydro-9'-dehydroxyimperanene (7, 8) lacking one of the methyl groups of dihydroguaiaretic acid were synthesized. (S)-7,8-Dihydro-9'-dehydroxyimperanene (7) showed 4-6-fold higher cytotoxic activity than all stereoisomers of dihydroguaiaretic acid (2-4). The IC50 values of (S)-7,8-dihydro-9'-dehydroxyimperanene (7) against HL-60 and HeLa cells were 6.1 mu M and 5.6 mu M, respectively. Though only one of three stereoisomers of dihydroguaiaretic acid showed antibacterial activity against a gram negative bacterium, both enantiomers of 5-8 showed antibacterial activity against a gram negative bacterium. This is a Letter on biological activity of 9-norlignan, in which one of methyl groups of lignan is absent. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2016.05.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor- by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-B cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.

DOI： 10.1080/09168451.2016.1146068

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A novel in vivo selection method for active deoxyribonucleoside kinase proteins is described here. A pool of randomly mutated genes for deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) was prepared and inserted into an expression vector. Enzymatically active mutants were selected by repeated cycles, including (i) introduction into Escherichia coli, (ii) treatment of the E. coli pool with a mutagenic deoxyribonucleoside (2-hydroxy-dA), and (iii) selection of antibiotic-resistant colonies resulting from mutations by phosphorylated 2-hydroxy-dA and the subsequent isolation of the plasmid DNAs. The ratio of the resistant colonies increased by two orders of magnitude from the first cycle to the fifth cycle, and then reached a plateau. Fifteen Dm-dNK mutants selected after the seventh and eighth evolution cycles were actually active in vivo. Moreover, one of the mutant proteins was as active as the wild type protein in vitro. These results indicate that this novel in vivo evolution method was useful and that similar strategies would be applicable to other deoxyribonucleoside kinases. In addition, the distribution of mutated amino acids suggests important residues/regions in the Dm-dNK protein. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2016.04.046

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

DOI： 10.1080/09168451.2015.1123603

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Nobiletin and beta-lactoglobulin were previously demonstrated to suppress mast cell de granulation in vitro and in vivo. In this study, the multiple anti-allergic effects of both substances by simultaneous treatment were examined in order to determine if they could further mitigate allergic symptoms. Concurrent treatment with nobiletin and beta-lactoglobulin effectively suppressed de-granulation of RBL-2H3 cells by inhibiting increases in intracellular Ca2+ concentration. Immunoblot analysis showed that phosphorylation of phosphoinositide 3-kinase, Akt, and Syk in RBL-2H3 cells was all inhibited by the concurrent presence of nobiletin and beta-lactoglobulin. Moreover, concurrent administration of nobiletin and beta-lactoglobulin reduced passive cutaneous anaphylaxis (PCA) reaction in mice compared with nobiletin or beta-lactoglobulin administered alone. In addition, the concurrent administration of freeze-dried Citrus unshiu peel powder and yogurt whey containing these bioactive substances also reduced PCA reaction and allergic symptoms of a murine model of Japanese cedar pollinosis. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2016.01.045

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase (sEH) were investigated. First, a series of alkyl or aryl groups were substituted on the carbon alpha to the phosphonate function in amide compounds to see whether substituted phosphonates can act as a secondary pharmacophore. A tert-butyl group (16) on the alpha carbon was found to yield most potent inhibition on the target enzyme. A 4-50-fold drop in inhibition was induced by other substituents such as aryls, substituted aryls, cycloalkyls, and alkyls. Then, the modification of the O-substituents on the phosphonate function revealed that diethyl groups (16 and 23) were preferable for inhibition to other longer alkyls or substituted alkyls. In amide compounds with the optimized diethylphosphonate moiety and an alkyl substitution such as adamantane (16), tetrahydronaphthalene (31), or adamantanemethane (36), highly potent inhibitions were gained. In addition, the resulting potent amide-phosphonate compounds had reasonable water solubility, suggesting that substituted phosphonates in amide inhibitors are effective for both inhibition potency on the human sEH and water solubility as a secondary pharmacophore. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2015.10.016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

Methoxychlor, an organochlorine insecticide developed to replace DDT (dichlorodiphenyltrichloroethane), has been reported to induce mast cell degranulation and to enhance IgE-mediated allergic responses. However, the mechanisms underlying these effects are not clear. To clarify potential mechanisms, the effects of methoxychlor on degranulation of mast cells were examined. Degranulation responses were evaluated using RBL-2H3 cells and mouse bone marrow-derived mast cells with either the antigen-induced or calcium ionophore-induced stimulation. Phosphorylation of enzymes related to signaling events associated with mast cell degranulation was analyzed by immunoblotting. Effects on vascular permeability in the passive cutaneous anaphylaxis reaction were evaluated following oral administration of methoxychlor to BALB/c mice. The results indicated that methoxychlor caused increased mast cell degranulation in the presence of antigen, whereas it had no effect on calcium ionophore-induced degranulation of RBL-2H3 cells. Immunoblot analyses demonstrated that the phosphorylation level of phosphoinositide 3-kinase (which plays a central role in mast cell signaling) was increased by methoxychlor during antigen-induced degranulation. In addition, methoxychlor activated the signaling pathway via the high-affinity IgE receptor by inducing phosphorylation of Syk and PLC gamma 1/2, which transfer the signal for degranulation downstream. Lastly, oral administration of methoxychlor exhibited a tendency to promote vascular permeability in passive cutaneous anaphylaxis model mice. Taken together, the results here suggested that methoxychlor enhanced degranulation through Fc epsilon RI-mediated signaling and promoted allergenic symptoms involved in mast cell degranulation.

DOI： 10.3109/1547691X.2014.962122

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Ishizuchi dark tea is a microorganism-fermented tea produced in Ehime, Japan. We herein describe the anti-allergic effect of Ishizuchi dark tea. The water extract from Ishizuchi dark tea suppressed degranulation of RBL-2H3 cells in a dose-dependent manner with no apparent cytotoxicity. Fractionation and beta-hexosaminidase release assay of the tea components revealed theabrownins as the major bioactive substance in Ishizuchi dark tea. Phosphorylation of Syk, a tyrosine kinase, tended to be suppressed by treating RBL-2H3 cells with theabrownins. In addition, Ishizuchi dark tea suppressed an allergic symptom in the Cry j1-induced pollinosis model mice. Taken together, these data indicated that Ishizuchi dark tea would be a beneficial functional food with an anti-allergic effect. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2015.02.045

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Jellyfish has recently attracted much attention for various applications, including functional foods due to its abundance of collagen. Jellyfish collagen, extracted from Nemopilema nomurai, was found to stimulate murine macrophage-like J774.1 cells. However, few reports have determined the immunostimulatory effects of jellyfish collagen on the innate immune response. We herein demonstrate the effect of jellyfish collagen on mouse bone marrow-derived dendritic cells (BMDCs). Jellyfish collagen stimulated TNF-alpha, IL-6, IL-1 beta and IL-12 production by BMDCs as the result of the elevation of gene expression level of these cytokines. In addition, jellyfish collagen-treated BMDCs have more wrinkles and longer pseudopodia on the cell surface compared with the control cells. Jellyfish collagen also stimulated cell-surface MHC-II expression level. Furthermore, jellyfish collagen downregulated phagocytosis capacity of BMDCs. Thus, our findings suggest that JC has the potential to activate DCs and thereby contribute to health promotion. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2015.02.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

(-)-Dihydroguaiaretic acid (DGA) and its derivatives having 3-hydroxyphenyl (3-OH-DGA) and variously substituted phenyl groups instead of 3-hydroxy-4-methoxyphenyl groups were synthesized to measure their larvicidal activity against the mosquito Culex pipiens Linnaeus, 1758 (Diptera: Culicidae). Compared with DGA and 3-OH-DGA (LC50 (M), 3.52 x 10(-5) and 4.57 x 10(-5), respectively), (8R,8'R)-lignan-3-ol (3) and its 3-Me (10), 2-OH (12), 3-OH (13), and 2-OMe (15) derivatives showed low potency (ca. 6-8 x 10(-5) M). The 4-Me derivative (11) showed the lowest potency (12.1 x 10(-5) M), and the 2-F derivative (4) showed the highest (2.01 x 10(-5) M). All of the synthesized compounds induced an acute toxic symptom against mosquito larvae, with potency varying with the type and position of the substituents. The 4-F derivative (6), which killed larvae almost completely within 45 min, suppressed the O-2 consumption of the mitochondrial fraction, demonstrating that this compound inhibited mitochondrial O-2 consumption contributing to a respiratory inhibitory activity.

DOI： 10.1021/jf504816a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Ficifolidione (1), a moderately active insecticidal compound from two species of Myrtaceae, and its derivatives were synthesized to evaluate their insecticidal activity. X-ray crystallographic analyses and specific rotation values of ficifolidione and its C-4 (2) demonstrated that the structure of ficifolidione differs from the reported absolute structure; that is, the C-4 configuration of ficifolidione should have an S configuration. The reported insecticidal activity of ficifolidione (1) and its C-4 epimer (2) against adult houseflies (Musca domestica), mosquito larvae (Culex pipiens), and cutworms (Spodoptera litura) was not observed. The cytotoxicities of ficifolidione and its derivatives (14) against four cell lines, Sf9, Colon26, HL60, and Vero, were also measured because ficifolidione has a phloroglucinol-derived moiety, a motif that is often present in the structure of cytotoxic chemicals. Compound 1 exhibited IC50 values of ca. 32, 9, 3, and 12 mu M for Sf9, Colon26, HL60, and Vero cells, respectively, indicating that ficifolidione possesses selective cytotoxicity against the four cell lines. In HL60 cells treated with 1, DNA fragmentation and the activation of procaspase 3 were observed, suggesting that the cytotoxicity is induced by apoptosis.

DOI： 10.1021/np5006746

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

We have already reported that lactate dehydrogenase (LDH) activates lymphocytes in vitro and in vivo. In this paper, we report the activating effects of LDH on the macrophage-like cell line J774.1. LDH was found to enhance production of IL-6 and TNF-alpha by J774.1 cells in a dose-dependent manner. Transcription levels of IL-6 and TNF-alpha in J774.1 cells were also enhanced by supplementation with LDH. From immunoblot analysis, it was revealed that LDH enhances the phosphorylation level of JNK in J774.1 cells. Moreover, the JNK inhibitor SP600125 decreased production of IL-6 and TNF-alpha induced by LDH. NF-kappa B translocation to the nucleus was also facilitated by LDH. These results was revealed that LDH enhances production of IL-6 and TNF-alpha by J774.1 cells via the increase of JNK phosphorylation and NF-kappa B translocation to the nucleus. Our data indicated that macrophages may be activated by LDH released from damaged tissues and cells in our body.

DOI： 10.1007/s10616-013-9646-3

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Various foodstuffs were screened for the ability to prevent mast cell degranulation using a rat basophilic leukaemia cell line (RBL-2H3 cells); whey emerged as a candidate to alleviate allergic symptoms. Further exploration of the suppressive effect of whey on mast cell degranulation led to identification of beta-lactoglobulin (BLG) as one of the active substances. The potential of BLG to modulate allergic responses of mast cells using the RBL-2H3 cells in vitro with the passive cutaneous anaphylaxis (PCA) reaction model mice in vivo is described. BLG was shown to suppress degranulation of RBL-2H3 cells. The molecular mechanism underlying the inhibitory effect of BLG on degranulation of RBL-2H3 cells included reduced phosphorylation of the tyrosine kinase Syk, phosphoinositide 3-kinase, and phospholipase C-gamma 1/2. Furthermore, oral administration of BLG tended to suppress the PCA reaction in the anti-dinitrophenol (DNP) IgE-sensitised, DNP-human serum albumin-challenged BALB/c mice, presenting a new biological functionality of BLG. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.idairyj.2014.05.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

All stereoisomers of methoxybutane and fluorobutane type of 1,7-seco-2,7'-cyclolignane were synthesized and cytotoxic activities of these compounds were compared with those of all stereoisomers of butane and butanol type compounds. Both enantiomers of butane type secocyclolignane showed higher cytotoxic activity (IC50 = 16-20 mu M) than methoxy type compounds, whereas none was observed for all the stereoisomers of butanol type secocyclolignane, however, (-)-Kadangustin J showed stereospecific cytotoxic activity (IC50 = 47-67 mu M). Since (R)-9'-fluoro derivative 23 was most potent (IC50 = 19 mu M) among the corresponding fluoro stereoisomers, (R)-9'-alkyl derivatives were synthesized, hydrophobic 9'-heptyl derivative 27 showing highest activity (IC50 = 3.7 mu M against HL-60, IC50 = 3.1 mu M against HeLa) in this experiment. Apoptosis induction caused by Caspase 3 and 9 for (R)-9'-heptyl derivative 27 was observed in the research on the mechanism. A degradation of DNA into small fragments was also shown by DNA ladder assay. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2014.07.033

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Chlorophyll c2 in a Sargassum horneri extract suppressed the degranulation of rat basophilic leukaemia RBL-2H3 cells. In this contribution, the mode of action of S. horneri extract was revealed. The extract inhibited the elevation of intracellular Ca2+ concentration induced by the antigen. Immunoblot analysis showed that S. horneri extract down-regulated the phosphorylation of both Syk and PI3K in the signalling pathways involved in degranulation triggered by antigen-antibody interaction. In addition, oral administration of S. horneri extract inhibited mice nasal rubbing behaviour sensitized with anti-DNP IgE. These findings suggest that S. horneri extract has anti-allergic activity via the suppression of degranulation of mast cells and basophils. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2014.06.002

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Bengkoang (Pachyrhizus erosus (L.) Urban) is an edible root tuber containing fairly large amounts of carbohydrates and crude fibers. Our previous studies showed that the bengkoang fiber extract (BFE) stimulates activation of macrophages, leading to induction of phagocytotic activity and cytokine production. In the present study we investigated the mechanism underlying activation of murine macrophages by BFE. BFE increased production of TNF-alpha, IL-6, and nitric oxide by J774.1 cells. In addition BFE also facilitated the gene expression levels of inducible nitric oxide synthase. We examined the effect of a TLR4 inhibitor on cytokine production to investigate the membrane receptor of macrophage activation by BFE. Treatment of J774.1 cells with the TLR4 inhibitor significantly inhibited production of IL-6 and TNF-alpha, suggesting that TLR4 is the target membrane receptor for BFE. The main signal molecules located downstream of TLR4 such as JNK, p38, ERK, and NF-kappa B were activated by BFE treatment. The immunostimulatory effect of BFE was cancelled by the pectinase treatment, suggesting that the active ingredient in BFE is pectin-like molecules. Overall results suggested that BFE activates J774.1 cells via the MAPK and NF-kappa B signaling pathways.

DOI： 10.1039/c3fo60360a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Cytotoxic activities of synthesized lignan derivatives were estimated by WST-8 reduction assay against HL-60 and HeLa cells to show the structure-activity relationship. The activities of some effective compounds were examined against Colon 26 and Vero cells. Dietary secoisolariciresinol (SECO, 1) and its metabolite, 9,9'-anhydrosecoisolariciresinol (2), did not show the cytotoxic activity. On the other hand, all stereoisomers of dihydroguaiaretic acid (DGA, 9,9'-dehydroxysecoisolariciresinol, 3-5) exhibited the activity (IC50: around 30 mu M). The IC50 value of (8R,8'R)-9-butyl DGA derivative 13 was around 6 mu M. This fact means that the hydrophobic group was advantageous for higher activity at 9- and 9'-positions. By the evaluation of the effect of 7and 7'-aryl group on the activity, we discovered the highest activity of (8R,8'R)-7-(3-hydroxy-4-methoxyphenyl)-7'-(2-ethoxyphenyl) DGA derivative 47 showing around 1 mu M of IC50 value, which is about 24-fold higher activity than that of natural (8R,8'R)-DGA. The derivative of dietary lignan showed the high cytotoxic activity.

DOI： 10.1021/jf5010572

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We previously reported that jellyfish collagen stimulates both the acquired and innate immune responses. In the acquired immune response, jellyfish collagen enhanced immunoglobulin production by lymphocytes in vitro and in vivo. Meanwhile, in the innate immune response jellyfish collagen promoted cytokine production and phagocytotic activity of macrophages. The facts that jellyfish collagen plays several potential roles in stimulating cytokine production by macrophages have further attracted us to uncover its mechanisms. We herein describe that the cytokine production-stimulating activity of jellyfish collagen was canceled by a Toll-like receptor 4 (TLR4) inhibitor. Moreover, jellyfish collagen stimulated phosphorylation of inhibitor of kappa B alpha (I kappa B alpha), promoted the translocation of nucleus factor-kappa B (NF-kappa B), and activated c-Jun N-terminal kinase (JNK). A JNK inhibitor also abrogated the cytokine production-stimulating activity of jellyfish collagen. These results suggest that jellyfish collagen may facilitate cytokine production by macrophages through activation of NF-kappa B and JNK via the TLR4 signaling pathways. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.molimm.2013.11.003

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We explored both structure-activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2013.12.027

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

DOI： 10.1016/j.jff.2013.12.005

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

Bengkoang (Pachyrhizus erosus (L.) Urban) is one of the most popular edible root vegetables in Indonesia. Bengkoang contains fairly large amounts of carbohydrates and crude fiber. The purpose of this research is to evaluate the immunomodulatory effect of the bengkoang fiber extract (BFE) in vitro and in vivo. BFE was prepared by heating the powder of bengkoang fiber suspended in distilled water at 121 degrees C for 20 min. BFE facilitated IgM production by the human hybridoma cell line HB4C5 cells. In addition, production of IgM, IgG, and IgA by mouse primary splenocytes was facilitated by BFE in a dose-dependent manner. BFE also significantly facilitated production of both interleukin-5 and interleukin-10 by splenocytes. Immunoglobulin production by lymphocytes from the spleen, Peyer's patch, and mesenteric lymph node were significantly activated by oral administration of BFE to mice for 14 days. The serum immunoglobulin levels of IgG, IgM, and IgA were also significantly enhanced. Furthermore, cytokine production by lymphocytes from the spleen, Peyer's patch, and mesenteric lymph node were also facilitated by oral administration of BFE. These results suggest that BFE has positive effects on the immune system in vitro and in vivo.

DOI： 10.1007/s10616-013-9539-5

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

DOI： 10.1016/j.bmcl.2013.06.067

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

ortho-Phenylphenol has been employed in post-harvest treatment of citrus fruits. Although o-phenylphenol has been reported to cause carcinomas in the urinary tract in rats, toxicity to the immune organs is still unknown. Herein, we report that administration of o-phenylphenol induces thymic atrophy and loss of thymocytes in female BALB/c mice. The influence seems to result from inhibition of the thymocyte development, because increased and decreased populations of the CD4(-) CD8(-) double-negative and CD4(+) CD8(+) double-positive thymocytes were observed in the o-phenylphenol-administered mice, respectively. ortho-Phenylphenol is metabolized to phenylhydroquinone by cytochrome P450 monooxygenases. Phenylhydroquinone made cell cycle of thymocytes to be arrested through reduced expression of the genes associated with G(2)/M phase and through phosphorylation of p53 at Ser15. Phosphorylation of p53 at Ser15 was upregulated by activation of not only ATR but also Erk1/2 and p38, leading to increase of apoptosis. Gene expression of cytochrome P450 1A1 (CYP1A1) was promoted in thymocytes from the o-phenylphenol-administered mice. Overall, our results suggest that o-phenylphenol induces CYP1A1 expression and is metabolized into phenylhydroquinone by the expressed GYP 1A1 in thymocytes. The produced phenylhydroquinone in turn induces inhibition of thymocyte development through cell cycle arrest and apoptosis in the p53-dependent pathway.

DOI： 10.2131/jts.38.325

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

Bengkoang (Pachyrhizus erosus (L) Urban) is an edible root vegetable containing fairly large amounts of carbohydrates and crude fibers. The purpose of this study is to evaluate the effects of the bengkoang fiber extract (BFE) on macrophages in vitro and in vivo. BEF was prepared by heat-extraction from the bengkoang fiber in distilled water at 121. °C for 20. min. BFE stimulated the phagocytotic activity of J774.1 cells. In addition, BFE significantly facilitated production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 by J774.1 cells and mouse peritoneal macrophages. BFE also facilitated the gene expression levels of inducible nitric oxide synthase and cyclooxygenase-2. The phagocytotic activity of mouse peritoneal macrophages was significantly enhanced by oral administration of BFE to BALB/c mice for 7. days. Ex vivo analysis revealed that production and gene expression of TNF-α and IL-6 in peritoneal macrophages from the BFE-administered mice were significantly enhanced. These results demonstrated that BFE activates macrophages in vitro and in vivo. © 2012 Elsevier Ltd.

DOI： 10.1016/j.jff.2012.12.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The inhibitory effect of an aqueous extract from spinach on degranulation of RBL-2H3 cells is herein reported. The extract significantly suppressed antigen-induced degranulation in a dose-dependent manner without affecting cell viability. Active substances in the extract were heat-stable and trypsin-resistant with molecular weights ranging from 500 Da to 14 kDa. The extract inhibited elevation of the intracellular Ca2+ concentration caused by stimulation by antigen, while not suppressing degranulation induced by a calcium ionophore A23187. Immunoblot analysis revealed that the inhibitory effect results from down-regulation of phosphorylation of both Syk kinase and phosphatidylinositol 3-kinase in the signalling pathways involved in degranulation caused by the antigen-antibody interaction. Taken together, these findings suggest that aqueous spinach extract has an anti-allergic activity that controls degranulation. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.foodchem.2012.08.079

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

In multifunctional type I restriction enzymes, active methyl-transferases (MTases) are constituted of methylation (HsdM) and specificity (HsdS) subunits. In this study, the crystal structure of a putative HsdM subunit from Vibrio vulnificus YJ016 (vvHsdM) was elucidated at a resolution of 1.80 angstrom. A cofactor-binding site for S-adenosyl-L-methionine (SAM, a methyl-group donor) is formed within the C-terminal domain of an alpha/beta-fold, in which a number of residues are conserved, including the GxGG and (N/D) PP(F/Y) motifs, which are likely to interact with several functional moieties of the SAM methyl-group donor. Comparison with the N6 DNA MTase of Thermus aquaticus and other HsdM structures suggests that two aromatic rings (Phe199 and Phe312) in the motifs that are conserved among the HsdMs may sandwich both sides of the adenine ring of the recognition sequence so that a conserved Asn residue (Asn309) can interact with the N6 atom of the target adenine base (a methyl-group acceptor) and locate the target adenine base close to the transferred SAM methyl group.

DOI： 10.1107/S0907444912038826

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Lactate dehydrogenase (LDH) has been demonstrated to have an immunoglobulin production-stimulating effect on an IgM-producing human hybridoma cell line and on human peripheral blood lymphocytes in vitro. Here, we examined the immunostimulatory activity of LDH on mouse immune system in vitro and in vivo. LDH stimulated IgA, IgG, and IgM production by lymphocytes from spleen, mesenteric lymph node, and Peyer's patch in vitro. BALB/c mice were administered with LDH at 1.0 or 5.0 mg/kg/day for 2 weeks to examine the immunostimulatory activity of LDH in vivo. The IgA level in mouse serum was increased by administration at 5.0 mg/kg/day. Moreover, IgA and IgG production by Peyer's patch lymphocytes from mice administered at 1.0 mg/kg/day of LDH was accelerated. IgA, IL-4, IL-5, IL-10, IFN-gamma, and TNF-alpha production by spleen lymphocytes were also facilitated by administration of LDH. It was revealed that oral administration of LDH stimulates immunoglobulin and cytokine production by lymphocytes in vivo. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2012.07.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Effects of auraptene on immune cells in vitro and in vivo were examined. IgM production by human hybridoma HB4C5 cells was enhanced by auraptene. Auraptene also stimulated IgA and IgG production by mouse primary splenocytes, and IgA and IgM production by lymphocytes from mesenteric lymph nodes in vitro. To examine the effect of auraptene on immune system in vivo, auraptene was orally administered to mice for 14 days. Immunoglobulin production by splenocytes and lymphocytes from mesenteric lymph nodes were significantly activated by intake of auraptene. Moreover, IL-4, IFN-gamma, and TNF-alpha production by splenocytes was accelerated under concanavalin A-activated condition. B cell population in spleen was increased by auraptene intake. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2012.06.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Substituted ureas with a carboxylic acid ester as a secondary pharmacophore are potent soluble epoxide hydrolase (sEH) inhibitors. Although the ester substituent imparts better physical properties, such compounds are quickly metabolized to the corresponding less potent acids. Toward producing biologically active ester compounds, a series of esters were prepared and evaluated for potency on the human enzyme, stability in human liver microsomes, and physical properties. Modifications around the ester function enhanced in vitro metabolic stability of the ester inhibitors up to 32-fold without a decrease in inhibition potency. Further, several compounds had improved physical properties. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2012.07.074

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Oysters are one of foodstuffs containing a relatively high amount of cadmium. Here we report on establishment of an immunochromatographic assay (ICA) method of cadmium levels in oysters. Cadmium was extracted with 0.1 mol L-1 HCl from oysters and cleaned up from other metals by the use of an anion-exchange column. The behavior of five metals Mn, Fe, Cu, Zn, and Cd was monitored at each step of extraction and clean-up procedure for the ICA method in an inductively coupled plasma-mass spectrometry (ICP-MS) analysis. The results revealed that a simple extraction method with the HCl solution was efficient enough to extract almost all of cadmium from oysters. Clean-up with an anion-exchange column presented almost no loss of cadmium adsorbed on the column and an efficient removal of metals other than cadmium. When a spiked recovery test was performed in the ICA method, the recovery ranged from 98% to 112% with relative standard deviations between 5.9% and 9.2%. The measured values of cadmium in various oyster samples in the ICA method were favorably correlated with those in ICP-MS analysis (r(2)=0.97). Overall results indicate that the ICA method established in the present study is an adequate and reliable detection method for cadmium levels in oysters. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.talanta.2012.04.028

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Beta-cryptoxanthin (beta-Crp), a mono-hydroxylated beta-carotene, is a major dietary provitamin A xanthophyll. The immunostimulatory effect of beta-Crp was examined using human hybridoma HB4C5 cells and mouse primary lymphocytes in vitro and using mice in vivo. IgM production by the HB4C5 cells and both IgA and IgG productions by mouse primary lymphocytes from mesenteric lymph node and spleen were accelerated in vitro by the treatment of beta-Crp in dose-dependent manners. Afterwards 6-week-old female BALB/c mice were administered with a low or high dose of beta-Crp for 14 days, mesenteric lymph node and spleen were excised and the immunoglobulin production levels were evaluated using lymphocytes from mesenteric lymph node and spleen in vivo. Results exhibited that all of IgA, IgG, and IgM productions by lymphocytes from mesenteric lymph node were increased. Especially, the IgG production increased with statistically significant differences in both of the low and high dose groups against the control group. The immunostimulatory effect on splenocytes was also observed although not as clear as on lymphocytes from mesenteric lymph node. Overall results suggest that beta-Crp may stimulate the humoral immunity in mammals. Hence, beta-Crp might have a potentially significant impact on human health and on prevention of diseases. (c) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2012.04.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background: Nobiletin is a citrus flavonoid which possesses the flavone structure with six methoxy groups. Although nobiletin has been reported to display anti-inflammatory, anti-tumor, and anti-diabetes activities, its effect on adipocyte differentiation remained unclear. In the present study, we investigated the effect of nobiletin on the differentiation of 3T3-L1 preadipocytes into adipocytes.
Methods: 3T3-L1 preadipocytes were treated with nobiletin under various differentiation conditions. The effect of nobiletin on adipocyte differentiation was evaluated by oil red 0 staining, real-time RT-PCR, and Western blotting.
Results: Nobiletin significantly suppressed the differentiation of 3T3-L1 preadipocytes into adipocytes, upon induction with insulin together with a cAMP elevator such as 3-isobutyl-1-methylxanthine (IBMX), by downregulating the expression of the gene encoding peroxisome proliferator-activated receptor (PPAR) gamma 2. In addition, nobiletin decreased the phosphotylation of CAMP-response element-binding protein (CREB) and strongly enhanced the phophorylation of signal transducer and activator of transcription (STAT) 5.
General significance: Nobiletin has a suppressive effect on the differentiation of preadipocytes into adipocytes when cells were induced with a general differentiation cocktail such as insulin, IBMX, and dexamethasone. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2011.11.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

As we previously reported, jellyfish collagen stimulates immunoglobulin and cytokine productions by lymphocytes in vitro and in viva Herein, the effects of collagens from jellyfish and bovine Achilles tendon on the innate immune response, especially on the activity of macrophages were evaluated. Macrophage functions were examined by the phagocytotic activity, cytokine production activity, and gene expression. The phagocytosis of the mouse J774.1 cells was enhanced by both collagens. Collagens from jellyfish and bovine Achilles tendon also stimulated the productions of tumor necrosis factor-alpha and interleukin-6 by J774.1 cells and mouse primary peritoneal macrophages as the result of elevated gene expression levels of these cytokines. Oral administration of collagens elevated cytokine production by peritoneal macrophages in mice. On the contrary, collagens suppressed gene expression of PPAR gamma 1 in macrophages. It is supposed from this result that collagens may stimulate the cytokine production by suppressing the PPAR gamma 1 expression. Collagens from jellyfish and bovine Achilles tendon enhanced not only the acquired immune response but also the innate immune response through the activation of macrophages. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2012.02.011

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

Arrowroot (Maranta arundinacea. L) is an underutilized local crop potentially to be developed as carbohydrate source and functional food in Indonesia. The objectives of this research are to evaluate the immunostimulatory effects of arrowroot extracts in vitro by using animal cell culture techniques, and in vivo by using BALB/c mice. The arrowroot tuber extracts were prepared by heat-treatment at 121 degrees C for 20 min in distilled water. The IgM production stimulatory activity of arrowroot tuber extracts against human hybridoma HB4C5 cells and mouse splenocytes was assessed. The result indicated that the arrowroot tuber extract stimulated IgM production by HB4C5 cells and immunoglobulin (IgG, IgA and IgM) production by splenocytes in vitro. In addition, the arrowroot tuber extracts strongly enhanced interferon c production by splenocytes. In vivo study indicated that the diet containing arrowroot extracts increased the serum IgG, IgA and IgM levels in mice. These results revealed that the arrowroot tuber extracts have immunostimulatory effects in vivo as well as in vitro.

DOI： 10.1007/s10616-011-9403-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Coplanar polychlorinated biphenyls (Co-PCBs) consisting of non-ortho and mono-ortho-chlorinated PCBs are dioxin-like compounds and cause wide contamination in the environment. To monitor Co-PCB residues, it was attempted to establish an enzyme-linked immunosorbent assay (ELISA) with monoclonal and recombinant antibodies selective to Co-PCBs. When 3,3',5,5'-tetrachlorobiphenoxybutyric acid (PCBH)-keyhole limpet hemocyanin conjugate was immunized into mice, two monoclonal antibodies, Mab-0217 and Mab-4444, were obtained. 3,3'5,5'-Tetrachlorobiphenyl (PCB80) was determined with an IC50 value of 2.6 and 0.46 ng mL(-1) in ELISA based on Mab-0217 and Mab-4444, respectively. Mab-4444 cross-reacted with Co-PCB congeners, except for PCB77 and PCB81. Mab-0217 reacted with PCB80 and cross-reacted with PCB111. A single-chain variable fragment (scFv) antibody derived from Mab-4444 was produced in recombinant Escherichia coli cells. The scFv antibody showed nearly the same sensitivity toward PCBH as the parent monoclonal antibody in ELISA. These results clearly suggested that Mab-4444 and its scFv antibodies were suitable for monitoring the representative congeners of Co-PCBs.

DOI： 10.1021/jf203585b

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Immunostimulating effects of water extract from bulbus arteriosus in tuna were examined and found to enhance IgM production by human hybridoma HB4C5 cells. When tuna bulbus arteriosus extract was heated, the IgM production-stimulating activity was decreased or lost. To identify the active substance, the extract was partially purified by anion-exchange chromatography, and proteins contained in positive fractions were analyzed by LC-MS/MS. The positive fractions contained lactate dehydrogenase, triosephosphate isomerase, enolase, and haemoglobin. Each of these four proteins was verified to enhance IgM production by HB4C5 cells with the commercially available purified proteins, suggesting that all of these four proteins are active substances in the extract. The immunostimulatory effect of these proteins was also examined on mouse primary spleen lymphocytes in vitro. Lactate dehydrogenase enhanced IgA, IgG, and IgM production and triosephosphate isomerase enhanced IgA and IgG production. In particular, lactate dehydrogenase had the strongest Ig production-stimulating effect on HB4C5 cells and on mouse primary lymphocytes. Thus, fish viscera may serve as an important raw material for the enhancement of the acquired immune system, once processed. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2011.12.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Flaxseed lignan, secoisolariciresinol has been reported to possess health benefits. We previously synthesized each stereoisomer of secoisolariciresinol and found that (-)-secoisolariciresinol reduces lipid accumulation and induces adiponectin production in 3T3-L1 adipocytes. Here we show the effects of (-)-secoisolariciresinol on high-fat diet-induced obesity in C57BL/6 male mice. Oral administration of (-)-secoisolariciresinol for 28 consecutive days significantly suppressed the gain of body weight. Increased serum adiponectin level and decreased gene expression of fatty acid synthase and sterol regulatory element-binding protein-1c in liver, which are related to fatty acid synthesis, were observed in the mice orally administered with (-)-secoisolariciresinol. In addition, subcutaneous injection of (-)-secoisolariciresinol also significantly suppressed the gain of body weight. Serum leptin levels were significantly increased by treating with (-)-secoisolariciresinol or (-)-enterolactone. Subcutaneous injection of (-)-secoisolariciresinol, (-)-enterolactone, or (-)-enterodiol promoted gene expression of acyl-CoA oxidase, carnitine palmitoyl transferase-1, and peroxisome proliferator-activated receptor alpha, which are related to beta-oxidation. Overall results suggest that (-)-secoisolariciresinol exerts a suppressive effect on the gain of body weight of mice fed a high-fat diet by inducing gene expression of adiponectin, resulting in the altered expression of various genes related to the synthesis and beta-oxidation of fatty acids.

DOI： 10.1039/c1fo10166h

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Matairesinol is one of the lignan compounds found in a variety of plant foodstuffs. We investigated the immunomodulatory effects of (-)-matairesinol in vivo and ex vivo by using mice. Although we found no significant differences in the IgG, IgA and IgM levels in the serum, the IgE level was strongly suppressed by the uptake of (-)-matairesinol in both intact and ovalbumin-immunized mice. The immunoglobulin produced by lymphocytes from the spleen was not activated by the intake of (-)-matairesinol. However, lymphocytes in such gut-associated lymphatic tissues as Peyer's patches and mesenteric lymph nodes were activated by the administration of (-)-matairesinol.

DOI： 10.1271/bbb.100781

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The immunostimulation effects of yellowtail heart extracts were examined. Screening various parts of the yellowtail viscera, we found that extracts from the yellowtail heart enhanced IgM production by human hybridoma HB4C5 cells. Yellowtail heart extracts heated at 121 degrees C for 20 min and dialyzed showed the highest IgM production-stimulating activity toward HB4C5 cells. Also, immunoglobulin production by mouse spleen lymphocytes was stimulated by yellowtail heart extracts in vitro, and lymphocytes derived from mice administered the extract for 20d were activated in vivo. Yellowtail heart extracts were partially purified by anion-exchange chromatography, and fractions containing a 33 kDa-protein exhibited immunostimulating activity. LC-MS/MS analysis revealed that the 33 kDa-protein was most similar to tropomyosin-4 from various fishes. Purified tropomyosin from porcine muscle enhanced IgM production by HB4C5 cells. This means that tropomyosin-4 is one of the immunostimulating substances in the yellowtail heart.

DOI： 10.1271/bbb.100713

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Structure-activity relationships of cycloalkylamide compounds as inhibitors of human sEH were investigated. When the left side of amide function was modified by a variety of cycloalkanes, at least a C6 like cyclohexane was necessary to yield reasonable inhibition potency on the target enzyme. In compounds with a smaller cycloalkane or with a polar group on the left side of amide function, no inhibition was observed. On the other hand, increased hydrophobicity dramatically improved inhibition potency. Especially, a tetrahydronaphthalene (20) effectively increased the potency. When a series of alkyl or aryl derivatives of cycloalkylamide were investigated to continuously optimize the right side of the amide pharmacophore, a benzyl moiety functionalized with a polar group produced highly potent inhibition. A nonsubstituted benzyl, alkyl, aryl, or biaryl structure present on the right side of the cycloalkylamide function induced a big decrease in inhibition potency. Also, the resulting potent cycloalkylamide (32) showed reasonable physical properties.

DOI： 10.1021/jm101431v

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The water-soluble fraction of kale (Brassica oleracea L. var. acephala DC.) had immunoglobulin (Ig) production stimulating activity in human hybridoma HB4C5 cells and human peripheral blood lymphocytes. The biochemical and physical properties of the main active substance in kale were found to be a heat-stable protein with a molecular weight higher than 50 kDa. The Ig production-stimulating factors were assumed to act on the translational and/or secreting processes of Igs. This Ig production-stimulating effect was also observed in lymphocytes from the mesenteric lymph node and Peyer's patches of mice that had been administered with the kale extract for 14 d. The partially purified kale extract was analyzed by LC-ESI-MS/MS, the result indicating ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) as an active substance. Rubisco from spinach indeed exhibited Ig production-stimulating activity in HB4C5 cells. These findings provide another beneficial aspect of kale as a health-promoting foodstuff.

DOI： 10.1271/bbb.100490

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

HlyU is a transcription factor of the ArsR/SmtB family and activates the expression of the pathogenic Vibrio vulnificus RTX toxin. In contrast to the other metal-responding ArsR/SmtB proteins, HlyU does not sense metal ions. To provide its structural information, we elucidated the crystal structure of HlyU from V. vulnificus CMCP6 (HlyU_Vv). The monomeric HlyU_Vv architecture of five alpha-helices and two beta-strands, some of which constitute a typical DNA-binding winged helix-turn-helix (wHTH) motif, is very similar to that of other transcription regulators. Nonetheless, the homo-dimeric HlyU_Vv structure shows several different, three-dimensional features in the spatial position and the detailed dimeric interaction, which were not observed in the modeling study based on the same protein family and sequence similarity.
Structured summary:
MINT-7710072, MINT-7710086: HlyU_Vv ( uniprotkb: Q8DES3) and HlyU_Vv (uniprotkb: Q8DES3) bind (MI: 0407) by X-ray crystallography (MI: 0114) (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2010.02.052

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Soluble epoxide hydrolase (sEH) is a key enzyme involved in the metabolism of epoxy fatty acid mediators such as epoxyeicosatrienoic acids with emerging roles in the regulations of hypertension and inflammation. Inhibitors of human sEH (hsEH) are effective drug candidates for the treatment of cardiovascular diseases. Preparation of hsEH for enzyme inhibition studies has been carried out by using baculovirus expression system. We herein explored the feasibility of expression of hsEH in Escherichia coli cells for the study of high-throughput screening assays of enzyme inhibitors, because the bacterial expression system is easier to handle and more cost-effective than the baculovirus expression system. The functional target enzyme was successfully produced in prokaryotic expression system by an auto-induction method and exhibited comparable enzyme activity to that yielded in baculovirus expression system. The bacterial-hsEH showed similar sensitivity to the baculovirus-hsEH against six reported inhibitors. Overalls indicate that bacterial expression of hsEH employed in the present study is useful for preparing enzymatically active hsEH, leading to effective performance of high-throughput screening assay of hsEH inhibitors and to rapid identification of novel drug candidates for the treatment of cardiovascular diseases. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pep.2009.09.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Modification (HsdM) and specificity (HsdS) subunits are constituents of an active methyltransferase (MTase) of multifunctional type I restriction enzymes. To provide a molecular background on HsdM, a putative hsdM gene from Vibrio vulnificus YJ016 (HsdM_Vv) was cloned and the expressed protein was purified and crystallized from 22%(w/v) polyethylene glycol 8000, 0.02 M imidazole pH 7.5 and 5 mM beta-mercaptoethanol. Diffraction data were collected to 1.86 angstrom resolution using synchrotron radiation. The crystal belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 78.9, c = 165.8 angstrom. With one molecule in the asymmetric unit, the crystal volume per unit protein weight was 2.12 angstrom(3) Da(-1), with a solvent content of 42%.

DOI： 10.1107/S1744309109043115

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Among four types of bacterial restriction enzymes that cleave a foreign DNA depending on its methylation status, type I enzymes composed of three subunits are interesting because of their unique DNA cleavage and translocation mechanisms performed by the restriction subunit (HsdR). The elucidated N-terminal fragment structure of a putative HsdR subunit from Vibrio vulnificus YJ016 reveals three globular domains. The nucleolytic core within an N-terminal nuclease domain (NTD) is composed of one basic and three acidic residues, which include a metal-binding site. An ATP hydrolase (ATPase) site at the interface of two RecA-like domains (RDs) is located close to the probable DNA-binding site for translocation, which is far from the NTD nucleolytic core. Comparison of relative domain arrangements with other functionally related ATP and/or DNA complex structures suggests a possible translocation and restriction mechanism of the HsdR subunit. Furthermore, careful analysis of its sequence and structure implies that a linker helix connecting two RDs and an extended region within the nuclease domain may play a central role in switching the DNA translocation into the restriction activity.

DOI： 10.1093/nar/gkp603

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Alkylphenol polyethoxylates and alkylphenols are widely distributed contaminants in the environment. Two anti-alkylphenol polyethoxylate monoclonal antibodies MOF3-139 and AP-14 were established to measure these chemicals by enzyme immunoassays in previous studies. Interestingly, these two monoclonal antibodies showed different specificity; AP-14 cross-reacts with nonylphenoxyacetic acid and nonylphenol, whereas MOF3-139 does not. To understand the molecular basis of the difference in specificity, single-chain Fv (scFv) antibodies derived from the monoclonal antibodies were each produced in Escherichia coli cells and characterized in competitive enzyme-linked immunosorbent assay. The scFv antibodies exhibited comparable reactivity profiles to the derived parent monoclonal antibodies. It was found that the VH domain of AP-14 play an important role in the cross-reaction when specificity tests were performed using variable domain-swapped scFv antibodies. An experiment using complementarity-determining region (CDR)-grafted scFv antibodies revealed that CDRI and CDR2 of AP-14 are involved in the cross-reaction to nonylphenoxyacetic acid and nonylphenol, respectively. Site-directed mutagenesis was introduced in both regions and the assay revealed that 33rd Thr and 35th His in VH domain of AP-14 were highly involved in the cross-reaction with nonylphenoxyacetic acid and that 33rd Thr, 57th Asp, and 59th Glu were involved in the cross-reaction with nonylphenol. The findings herein would contribute to the antibody engineering for specificity modification and to the generation of an alkylphenol-specific recombinant antibody by antibody engineering. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.molimm.2009.05.182

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We investigated N-adamantyl-N'-phenyl urea derivatives as simple sEH inhibitors. Salicylate ester derivatives have high inhibitory activities against human sEH, while the free benzoic acids are less active. The methyl salicylate derivative is a potent sEH inhibitor, which also has high metabolic and chemical stabilities; suggesting that such inhibitors are potential lead molecule for bioactive compounds acting in vivo. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2009.01.069

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

This is a report on the development of immunoaffinity chromatography using a column of silica gel with an immobilized single-chain variable fragment (scFv) antibody specific to bisphenol A (BPA) for cleanup of BPA-contaminated water samples. The BBA-2187 scFv antibody specific to BPA was purified from the periplasmic fractions of the recombinant Escherichia coli. After a sample of BPA-contaminated river water was applied to the immunoaffinity column, the background signal intensity observed in high-performance liquid chromatography (HPLC) analysis of the eluates was markedly lower than that observed in HPLC analysis of the eluates from an Oasis HLB cartridge treated with the same sample. The immunoaffinity column efficiently concentrated BPA from actual river water samples with different matrices. Our results demonstrate that the immunoaffinity column with immobilized BBA-2187 scFv antibody is efficient for the cleanup of BPA-contaminated water samples from different sources.

DOI： 10.1021/jf802781t

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Type I restriction enzymes are multimeric proteins that consist of three subunits. The HsdS and HsdM subunits form a complex protein that shows methyltransferase activity, while the HsdR subunit functions as an endonuclease as well as as a translocase. Of these three subunits, no structural information on the HsdR subunit is yet available. The putative HsdR gene from Vibrio vulnificus YJ016 (HsdR_Vv) was cloned and expressed and the expressed protein HsdR_Vv was purified. HsdR_Vv was crystallized from 8%(w/v) polyethylene glycol 3350, 0.15 M ammonium chloride, 0.1 M HEPES pH 7.5 and 2 mM beta-mercaptoethanol. Diffraction data were collected to 2.60 angstrom resolution using synchrotron radiation. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.01, b = 89.04, c = 113.66 angstrom. With one HsdR_Vv molecule in the asymmetric unit, the Matthews coefficient was 2.14 angstrom(3) Da(-1) and the solvent content was 42%.

DOI： 10.1107/S1744309108027516

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

Nitroreductases (NTR) are enzymes that reduce hazardous nitroaromatic compounds and are of special interest due to their potential use in bio-remediation and their activation of prodrugs in directed anticancer therapies. We elucidated the crystal structures of ydjA from Escherichia coli (Ec_ydjA), one of the smallest NTRs, in its flavin mononucleotide (FMN)-bound and cofactor-free forms. The alpha + beta mixed monomeric Ec_ydjA forms a homodimeric structure through the interactions of the long central helices and the extended regions at both termini. Two FMN molecules are bound at the dimeric interface. The absence of the 30 internal amino acids in Ec_ydjA, which forms two helices and restricts the cofactor and substrate binding in other NTR family members, creates a wider and more flexible active site. Unlike the bent FMN ring structures present in most NTR complexes currently known, the flavin system in the Ec_ydjA structure maintains a flat ring conformation, which is sandwiched between a Trp and a His residue from each monomer. The analysis of our Ec_ydjA structure explains its specificity for larger substrates and provides structural information for the rational design of novel prodrugs with the ability to reduce nitrogen-containing hazardous molecules. (c) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2008.01.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

Esterases are one of the most common enzymes and are involved in diverse cellular functions. ybfF protein from Escherichia coli (Ec_ybfF) belongs to the esterase family for the large substrates, palmitoyl coenzyme A and malonyl coenzyme A, which are important cellular intermediates for energy conversion and biomolecular synthesis. To obtain molecular information on ybfF esterase, which is found in a wide range of microorganisms, we elucidated the crystal structures of Ec_ybfF in complexes with small molecules at resolutions of 1.1 and 1.68 angstrom, respectively. The structure of Ec-ybfF is composed of a globular alpha/beta hydrolase domain with a three-helical bundle cap, which is linked by a kinked helix to the alpha/beta hydrolase domain. It contains a catalytic tetrad of Ser-His-Asp-Ser with the first Ser acting as a nucleophile. The unique spatial arrangement and orientation of the helical cap with respect to the alpha/beta hydrolase domain form a substrate-binding crevice for large substrates. The helical cap is also directly involved in catalysis by providing a substrate anchor, viz., the conserved residues of Arg123 and Tyr208. The high-resolution structure of Ec_ybfF shows that the inserted helical bundle structure and its spatial orientation with respect to the alpha/beta hydrolase domain are critical for creating a large inner space and constituting a specific active site, thereby providing the broad substrate spectrum toward large biomolecules. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2007.12.062

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir, and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i. e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC50) and binding kinetics (k(i)). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1 H NMR, X-ray crystallography, and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom b to the ketone contributes an increase (similar to 5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2007.10.081

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Nitroreductases that reduce hazardous nitroaromatic compounds are of interest because of their central role in nitroaromatic toxicity, their potential use in bioremediation and their utility in activating prodrugs in directed anticancer therapies. To provide the molecular background to the enzymatic mechanism of the ydjA nitroreductase, which is one of the smallest nitroreductases, the ydjA gene from Escherichia coli K12 was cloned and expressed and the expressed protein Ec_ydjA was purified. Ec_ydjA was crystallized from 20%(w/v) polyethylene glycol 1000, 0.2 M lithium sulfate and 0.1 M phosphate-citrate pH 4.2. Diffraction data were collected to 2.00 angstrom resolution using synchrotron radiation. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 87.55, b = 129.28, c = 36.88 angstrom, alpha = 90, beta = 103.8, gamma = 90 degrees. With two Ec_ydjA molecules in the asymmetric unit, the Matthews coefficient was 2.43 angstrom(3) Da(-1) and the solvent content was 48.33%.

DOI： 10.1107/S1744309107057636

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Soluble epoxide hydrolase (sEH) is a therapeutic target for treating hypertension and inflammation. 1,3-Disubstituted ureas functionalized with an ether group are potent sEH inhibitors. However, their relatively low metabolic stability leads to poor pharmacokinetic properties. To improve their bioavailability, we investigated the effect of incorporating various polar groups on the ether function on the inhibition potencies, physical properties, in vitro metabolic stability, and pharmacokinetic properties. The structure- activity relationship studies showed that a hydrophobic linker between the urea group and the ether function is necessary to keep their potency. In addition, urea-ether inhibitors having a polar group such as diethylene glycol or morpholine significantly improved their physical properties and metabolic stability without any loss of inhibitory potency. Furthermore, improved pharmacokinetic properties in murine and canine models were obtained with the resulting inhibitors. These findings will facilitate the usage of sEH inhibitors in animal models of hypertension and inflammation.

DOI： 10.1021/jm070705c

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Fatty acid amide hydrolase (FAAH) is a pharmaceutical target whose inhibition may lead to valuable therapeutics. Sensitive substrates for high-throughput assays are crucial for the rapid-screening FAAH inhibitors. Here we describe the development of novel and highly sensitive fluorescent assays for FAAH based on substituted aminopyridines. Examining the relationship between the structure and the fluorescence of substituted aminopyridines suggested that a methoxy group in the para position relative to the amino group in aminopyridines greatly increased the fluorescence (i.e., quantum yields approach unity). These novel fluorescent reporters had a high Stokes' shift of 94 nm, and their fluorescence in buffer systems increased with pH values from neutral to basic. Fluorescent substrates with these reporters displayed a very low fluorescent background and high aqueous solubility. Most importantly, fluorescent assays for FAAH based on these substrates were at least 25 times more sensitive than assays using related compounds with published colorimetric or fluorescent reporters. This property results in shorter assay times and decreased protein concentrations in the assays. Such sensitive assays will facilitate distinguishing the relative potency of powerful inhibitors of FAAH. When these fluorescent substrates were applied to human liver microsomes, results suggested that there was at least one amide hydrolase in addition to FAAH that could hydrolyze long-chain fatty acid amides. These results show that these fluorescent substrates are very valuable tools in FAAH activity assays including screening inhibitors by high-throughput assays instead of using the costly and labor-intensive radioactive ligands. Potential applications of novel fluorescent reporters are discussed. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2006.10.041

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in blood pressure regulation and vascular inflammation. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid (AUDA, 1) is a very active inhibitor of sEH both in vitro and in vivo. However, its relatively high melting point and limited solubility in either water or oil-based solvents leads to difficulties in formulating the compound and often results in poor in vivo availability. We investigated the effect of derivatization of the acid functional group of inhibitor I on the inhibition potencies, physical properties, and pharmacokinetic properties. For human sEH, similar inhibition potency was obtained when the acid of compound I was modified to esters (2-15). The resulting compounds exhibited improved physical properties (23-66 degrees C lower melting point and 5-fold better solubility in oil). Pharmacokinetic studies showed that the esters possess improved oral bioavailability in mice. On the other hand, amide derivatives of AUDA I did not show significant improvement in inhibition potencies or physical properties (higher melting points and lower solubility). The esterification of 1 results in compounds that are easier to formulate in animal food and in triglycerides for gavage and other routes of administration, making it easier to study the biological effects of sEH inhibition in vivo. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2006.09.057

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Esterases play a crucial role in industrial chemical synthesis, maintaining normal physiological metabolism and detoxifying exogenous ester-containing toxicants. To meet the rapidly increasing industrial need for all kinds of esterases, especially enantioselective esterases used to generate highly pure chiral compounds, general substrates are necessary for rapid screening, monitoring, purification, and characterization. In this study, general fluorescent substrates including phenolic derivatives and alpha-cyanoesters were evaluated for sensitivity in detecting esterases in buffer systems. Results with two different esterases and different incubation times suggested that the alpha-cyanoesters examined were significantly more sensitive at detecting esterases than the corresponding tested phenolic derivatives. More importantly, a-cyanoesters, containing a secondary alcohol, possess at least one chiral center; thus, they are tools to screen for enantioselective hydrolysis. Results indicated that the enantioselectivity of esterases toward general alpha-cyanoesters strongly depended on the esterase and the substrate, but the majority of esterases examined preferred S-isomers to their corresponding alpha-enantiomers. Most appealing was the very high enantioselectivity displayed in cytosolic esterases of the house fly. The potential utility of such esterases is discussed. In addition, the use of alpha-cyanoesters as chiral fluorescent substrates was demonstrated for monitoring in enantioselective esterases.

DOI： 10.1021/jf0526083

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Carboxylesterases hydrolyze a large array of endogenous and exogenous ester-containing compounds, including pyrethroid insecticides. Herein, we report the specific activities and kinetic parameters of human carboxylesterase (hCE)-1 and hCE-2 using authentic pyrethroids and pyrethroid-like, fluorescent surrogates. Both hCE-1 and hCE-2 hydrolyzed type I and II pyrethroids with strong stereoselectivity. For example, the trans-isomers of permethrin and cypermethrin were hydrolyzed Much faster than corresponding cis-counterparts by both enzymes. Kinetic values of hCE-1 and hCE-2 were determined using cypermethrin and 11 stereoisomers of the pyrethroid-like. fluorescent surrogates. K-m values for the authentic pyrethroids and fluorescent Surrogates were in general lower than those for other ester-containing substrates of hCEs. The pyrethroid-like, fluorescent Surrogates were hydrolyzed at rates similar to the authentic pyrethroids by both enzymes, suggesting the potential of these Compounds as tools for high throughput screening of esterases that hydrolyze pyrethroids. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2005.11.005

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Mammalian hepatic carboxylesterases (CEs) play important roles in the detoxification of ester-containing pyrethroids, which are widely used for the control of agricultural pests and disease vectors such as mosquitoes. Pyrethroids and pyrethroid-like fluorescent substrates exhibit a consistent pattern of stereoselective hydrolysis by a recombinant murine hepatic CE. We sought to understand whether this pattern is maintained in other hepatic CEs and to unravel the origin of the stereoselectivity. We found that all hepatic CEs tested displayed a consistent pattern of stereoselective hydrolysis: the chiral center(s) in the acid moiety more strongly influenced stereoselective hydrolysis than the chiral center in the alcohol moiety. For cypermethrin analogues with a cyclopropane ring in the acid moiety, trans-isomers were generally hydrolyzed faster than the corresponding cis-isomers. For fenvalerate analogues without a cyclopropane ring in the acid moiety, 2R-isomers were better substrates than 2S-isomers. These general hydrolytic patterns were examined by modeling the pyrethroid-like analogues within the active site of the crystal structure of human carboxylesterase 1 (hCE1). Stereoselective steric clashes were found to occur between the acid moieties and either the catalytic Ser loop (residues 219-225) or the oxyanion hole (residues 140 - 144). These clashes appeared to explain the stereopreference between trans- and cis-isomers of cypermethrin analogues, and the 2R- and 2S-isomers of fenvalerate analogues by hCE1. The implications these findings have on the design and use of effective pesticides are discussed.

DOI： 10.1021/tx050072+

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A monoclonal antibody (mab) selective for the thiomethyl-s-triazine herbicide simetryn was obtained and characterized in enzyme-linked immunosorbent assay (ELISA). An IC50 value for simetryn was 8.5 ng/mL, and the detection range extended from 1.1 to 70 ng/mL in ELISA. The cIDNAs encoding variable heavy chain (VH) and variable light chain (VL) of the mab were cloned to produce various recombinant antibodies. Single-chain variable fragment (scFv) antibodies derived from the mab were characterized in ELISA and showed similar reactivities and specificities to the parent mab. A urea denaturation test revealed that the scFv antibodies bound to simetryn were more stable than those in the absence of antigen. A sandwich ELISA based on VH and VL fragments of the mab was successfully developed and showed similar sensitivity to those based on the mab and scFv antibodies in ELISA. In the recovery experiments using spiked environmental samples, the results obtained in ELISA based on the mab were favorably correlated with those by HPLC.

DOI： 10.1021/jf050246t

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in the regulation of blood pressure and inflammation. 1,3-Disubstituted ureas with a polar group located on the fifth atom from the carbonyl group of urea function are active inhibitors of sEH both in vitro and in vivo. However, their limited solubility in water and relatively high melting point lead to difficulties in formulating the compounds and poor in vivo efficacy. To improve these physical properties, the effect of structural modification of the urea pharmacophore on the inhibition potencies, water solubilities, octanol/water partition coefficients (log P), and melting points of a series of compounds was evaluated. For murine sEH, no loss of inhibition potency was observed when the urea pharmacophore was modified to an amide function, while for human sEH 2.5-fold decreased inhibition was obtained in the amide compounds. In addition, a NH group on the right side of carbonyl group of the amide pharmacophore substituted with an adamantyl group (such as compound 14) and a methylene carbon present between the adamantyl and amide groups were essential to produce potent inhibition of sEH. The resulting amide inhibitors have 10-30-fold better solubility and lower melting point than the corresponding urea compounds. These findings will facilitate synthesis of sEH inhibitors that are easier to formulate and more bioavailable.

DOI： 10.1021/jm0500929

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Previously, immunological detection of a small hapten was only possible in competitive format, which needed a competitor antigen either labeled by a reporter or attached to a carrier protein. Recently, we proposed the open sandwich (OS) immunoassay, a simple immunoassay that can noncompetitively determine monovalent antigen concentration by measuring the antigen-dependent change in a heavy-chain variable region (V-H)/light-chain variable region (V-L) interaction of an antibody. However, there was a limitation in the assay that the antibody used should have a suitable property such that the V-H/V-L interaction would become fairly strong along with the addition of antigen. Here, we devised a phage-based "split-Fv system" to rapidly evaluate and select antibody variable region (Fv) fragments that are suitable to OS immunoassay. When three antibodies raised against endocrine disruptor bisphenol A were tested with this system, all were more or less suitable to OS-ELISA. Among them, the best Fv selected was used to construct fusion proteins of V-H tethered to an alkaline phosphatase and a tagged V-L that can be site-specifically biotinylated to perform direct OS-ELISA. The results showed that the OS-ELISA detects bisphenol A with higher sensitivity than the corresponding competitive assay, also implying that many antibodies to small haptens have suitable properties for OS-ELISA.

DOI： 10.1021/ac034280n

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

DOI： 10.1584/jpestics.28.301

CiNii Books

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Four anti-bisphenol A monoclonal antibodies (mabs) were obtained and each characterized by an enzyme-linked immunosorbent assay (ELISA). Among these mabs, BBA-2187 was the most reactive towards bisphenol A. The quantitation limit of the ELISA assay for bisphenol A was 0.13 ng/ml, which is more sensitive than the other immunoassays reported. Then, the cDNA clones encoding variable heavy and variable light chains of these four mabs were isolated, and used for construction of four single-chain Fv (scFv) antibody genes, which were expressed in Escherichia coli cells. The reactivity of four scFv antibodies towards bisphenol A in ELISA was comparable to those of the parent mabs. The most sensitive assay was achieved with BBA-2187scFv. Its cross-reactivity to the related compounds was similar to that of the parent mab. Based on the reactivity of heterologous combinations of VH and VL fragments, it was found that the unique structure of the framework region 2 in the VL of BBA-2187 appeared to be important for specific assembly together with the VH.

DOI： 10.1271/bbb.67.1358

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Pesticide Science Society of Japan  

DOI： 10.1584/jpestics.27.157

Scopus

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

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DOI： 10.1063/1.4982287

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：TAYLOR & FRANCIS INC  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：TAYLOR & FRANCIS INC  

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Language：Japanese   Publisher：Pesticide Science Society of Japan  

CiNii Books

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CiNii Books

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CiNii Books

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