on Degranulation in RBL-, H- Satoru Horigome, Izumi Yoshida, Chie Tamaki, Akihiro Yamaguchi, Nobuyuki Kibune, Takeshige Kamibe and Masatoshi Watai

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1 19 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. //, No. ++, /-/ /.* (,**2) 535 RBL-, H- Simple Screening Method for Inhibitory E# ects of Food Ingredients on Degranulation in RBL-, H- Cells Satoru Horigome, Izumi Yoshida, Chie Tamaki, Akihiro Yamaguchi, Nobuyuki Kibune, Takeshige Kamibe and Masatoshi Watai Japan Food Research Laboratories, 1..+, Asagi, Saito, Ibaraki-shi, Osaka /01 **2/ RBL-, H-rat mast cells were stimulated to secrete intracellular granules through multivalent binding of antigen to receptor-bound IgE and preferentially used for anti-allergic evaluation. b-hexosaminidase stored in the granules was used as a marker of mast cell degranulation. We developed a simple screening method to evaluate inhibitory e# ects of mast cell degranulation by e$ cient measurements of the enzyme activity in / a continuous 30-well format. The cells were inoculated into a 30 -well plate (,./ +* /well) and cultured overnight. Anti-dinitrophenyl-IgE, food extracts and dinitrophenyl-human serum albumin were successively added and incubated for each optimized interval of time. The supernatant was transferred to another well, and the cells were lysed with a passive lysis bu# er instead of conventional sonication. b-hexo- saminidase activity in the supernatant and the cells was measured using a color formation substrate with a microplate reader. The percentage of degranulation was calculated from the ratio of the enzyme activity (supernatant/cells). The proposed method can be easily completed by a multi-channel pipetting operation and adapted for high-throughput screening (,+ samples/plate, n, ) as compared to the conventional,.-well based method (- samples/plate, n, ). Reproducibility was also improved from RSD +,.- to /.. 3.* (within-run, n. ). The results agreed well with those of the previous method. (Received May,2,,**2 ; Accepted Jul.,,,,**2) Keywords : allergy, functional food, b-hexosaminidase, pollinosis, screening : b-hexosaminidase +, FceR IgE IgE -. 0 /01 **2/ 1..+ Corresponding author horigomes@jfrl.or.jp IgE IL-. IL-/ Th, IL-. IgE 12 MC3 RBL-, H- 3 +,

2 536 // ++,** / HEPES, *+. BSA, ph 02. MT buffer -1 / +** mmoll ph../ Glycine buffer, /* L -1,/ mol/l Glycine, ph +*.. Lysis buffer Passive Lysis / /* L Glycine buffer bu# er, Promega / p-nitrophenyl- 0/* nm.*/ nm,-acetamido-,-deoxy-(r)-d-glucopyranoside --. mmoll +** RBL-, H- +* FBS, Invitrogen Dulbecco s Modified Eagle s Medium DMEM, Sigma wortman- nin + mm MT buffer / GraphPad Prism /*+., : Dispergierantrieb T+* basic IKA Mann-Whitney : SpectraMax M, e - +* g /* ml - + *. + g /* 30 / ml +0 +* *** rpm, +* min + ml /** gml + DNP-IgE /* ngml,, + g /*,* ml DNP-HSA *+. gml +0 DNP-HSA + ml - - MT buffer +** -1 - /** gml. RBL-, H- bohexosamini-..2 / +* cellswell /* ngml DNP-IgE Sigma +** L, +** L MT buffer MT buffer wortmannin /* L -1 +* Watanabe +0 CaCl, + mmol/l MgCl, /. 0 mmol/l glucose,,* mmol/l buffer +** L +* +** ngml DNP-labeled human serum albumin Sigma /* L -1 - MT buffer + DNP-IgE MT buffer modified Tyrode +* bu# er +-1 mmol/l NaCl,,1. mmol/l KCl, +2. mmol/l Lysis,, Watanabe +0,. dase

21 : 537 + n. : : + : DNP-HSA *. + gml, DNP-IgE, DNP-HSA -*, : DNP-IgE /* ngml, DNP-IgE, DNP-HSA -* - : DNP-IgE /* ngml, DNP-HSA *+. gml, DNP-HSA -*. : DNP-IgE /* ngml, DNP-HSA *. + gml, DNP-IgE, ++ +0 30 Lysis buffer IgE +3,+ +, +1 +2 PI-- +.

3 21 : n. : : + : DNP-HSA *. + gml, DNP-IgE, DNP-HSA -*, : DNP-IgE /* ngml, DNP-IgE, DNP-HSA -* - : DNP-IgE /* ngml, DNP-HSA *+. gml, DNP-HSA -*. : DNP-IgE /* ngml, DNP-HSA *. + gml, DNP-IgE, Lysis buffer IgE +3,+ +, PI / *. +20 Lysis buffer +*0*. **33. p ** */. Lysis buffer +-. *-. p **/., wortmannin b-hexosaminidase Lysis buffer n.,,..,.. p **/. Lysis buffer wortmannin, *.*+ nm+** nm, IC +/. 1 nm +* nm.+ nm /*

4 538 // ++,** wortmannin, RSD RSD + -,4,,4, , * 34* ,34, +40 /4. +**41.4,.4,. -240,4/ 040, +*+4, -40-4/ RSD *+4,,42,42 + *.14/ /43 +,4-, * /4, n. +*/4+ 14* 041 wortmannin DNP-HSA,/ nm..42,4+.42 *. 2*41,40-4, / /41 3.4/ **42 /4- /4, 234, -42.4, /* , 2* / 2*43.40 / * *42,4*,4/ 3/ * * n. n. +,.- /.. 3* ,-,/ +0 RBL-, H- IgE - FceR,0 -,., */. +. 1*, 1* 2*, 2* 3* 1 3* +- 2* 2,,. /* , +* /

23 : 539. n. / n. +, -. / 0 1 2 3 +* ++ +, RBL-, H- 30 + +,+ n, Lysis buffer +* p **-. /* +, + Kaneko, Y.

5 23 : 539. n. / n. +, -. / * ++ +, RBL-, H ,+ n, Lysis buffer +* p **-. /* +, + Kaneko, Y., Motohashi, Y., Nakamura, H., Endo, T. and Eboshida, A., Increasing prevalence of Japanese cedar *. 20,/ p *.**-

6 540 // ++,** pollinosis : a meta-regression analysis. Int. Arch. Aller- Mentha piperita L. Biol. Pharm. Bull.,,/,,/0,/3 (,**,). gy.immunol., +-0, -0/ -1+ (,**/). +0 Watanabe, J., Shinmoto, H. and Tsushida, T., Coumarin, Okuda, M., Epidemiology of Japanese cedar pollinosis and flavone derivatives from estragon and thyme as throughout Japan. Ann. Allergy. Asthma. Immunol., 3+, inhibitors of chemical mediator release from RBL-, H-,22,30 (,**-). cells. Biosci. Biotech. Biochem., 03, + 0 (,**/) Yano, H., Nakanishi, S., Kimura, K., Hanai, N., Saitoh, Y., /*,3/ -*,,**- Fukui, Y., Nonomura, Y. and Matsuda, Y., Inhibition of. histamine secretion by wortmannin through the blockade pp +,1 +-3,**0 of phosphatidylinositol --kinase in RBL-, H- cells. J. / Oliver, J.M., Kepley, C.L., Ortega, E. and Wilson, B.S., Biol. Chem.,,02,,/2.0,/2/0 ( +33-). Immunologically mediated signaling in basophilis and +2 Yano, H., Agatsuma, T., Nakanishi, S., Saitoh, Y., Fukui, mast cells : finding therapeutic targets for allergic dis- Y., Nonomura, Y. and Matsuda, Y., Biochemical and eases in the human FceR+ signaling pathway. Immuno- Pharmacological studies with KT103, and LY,3.**, on pharmacology,.2,,03,2+ (,***). the role of phosphatidylinositol --kinase in FceR- 0 Lee, B., Bae, E.A., Trinh, H.T., Shin, Y.W., Phuong, T.T., mediated signal transduction. Biochem. J., -+,, +./ +/* Bae, K.H. and Kim, D.H., Inhibitory e# ect of Schizandrin ( +33/ ). on passive cutaneous anaphylaxis reaction and scratch- +3 Hirasawa, N., Sato, Y., Yomogida, S., Mue, S. and Ohuchi, ing behaviors in mice. Biol. Pharm. Bull., -*, ++/- ++/0 K., Role of phosphatidylinositol --kinase in degranula- (,**1). tion induced by IgE-dependent and -independent mechanisms 1 in rat basophilic RBL-, H - (ml) Cells. Cell Signal., 3, -*/ -+* ( +331).,* Cissel, D.S., Fraundorfer, P.F. and Beaven, M.A., Thapsi- /, /2. /3-,**/ gargin-induced secretion is dependent on activation of 2 Tachibana, H., Sunada, Y., Miyase, T., Sano, M., Maeda- a cholera toxin-sensitive and phosphatidylinositol--- Yamamoto, M. and Yamada, K., Identification of a kinase-regulated phospholipase D in a mast cell line. J. methylated tea catechin as an inhibitor of degranula- Pharmacol. Exp. Ther.,,2/, ++* ++2 ( +332). tion in human basophilic KU2+, cells. Biosci. Biotech.,+ Windmiller, D.A. and Backer, J.M., Distinct phospho- Biochem., 0.,./,./. (,***). inositide --kinases mediate mast cell degranulation in 3 Demo, S.D., Masuda, E., Rossi, A.B., Throndset, B.T., response to G-protein-coupled versus FceR receptors. Gerard, A.L., Chan, E.H., Armstrong, R. J., Fox, B.P., J. Biol. Chem.,,12, (,**-). Lorens, J.B., Payan, D.G., Scheller, R.H. and Fisher, J.M.,,, Suzuki, M., Nakamura, T., Iyoki, S., Fujiwara, A., Watanabe, Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-v binding assay. Y., Mohri, K., Isobe, K., Ono, K. and Yano, S., Elu- cidation of anti-allergic activities of curcumin-related Cytometry, -0, -.* -.2 ( +333). compounds with a special reference to their anti- +* Ito, N., Yokomizo, T., Sasaki, T., Kurosu, H., Penninger, oxidative activities. Biol. Pharm. Bull,,2, (,**/). J., Kanaho, Y., Katada, T., Hanaoka, K. and Shimizu, T.,,- Peng, C.H., Su, J.D., Chyau, C.C., Sung, T.Y., Ho, S.S., Requirement of phosphatidylinositol --kinase activa- Peng, C.C. and Peng, R.Y., Supercritical fluid extracts of tion and calcium influx for leucotriene B. -induced rosemary leaves exhibit potent anti-inflammation and enzyme release. J. Biol. Chem.,,11, *. (,**,). anti-tumor e# ects. Biosci. Biotech. Biochem., 1+,,,,- ++,,-, (,**1). RBL-, H-,. Swarup, V., Ghosh, J., Ghosh, S., Saxena, A and Basu, A., /-.+0.,,,**0 Antiviral and anti-inflammatory e# ects of rosmarinic acid in an experimental murine model of japanese encephalitis. +, Kitani, S., Teshima, R., Morita, Y., Ito, K., Matsuda, Y. and Nonomura, Y., Inhibition of IgE-mediated hista- Antimicrob. Agents. Chemother., /+, * (,**1). mine release by myosin light chain kinase inhibitors. Biochem. Biophys. Res. Commun, +2-,.2 /. ( +33, ).,/ Inoue, K., Takano, H., Shiga, A., Fujita, Y., Makino, H., Yanagisawa, R., Ichinose, T., Kato, Y., Yamada, T. and +- Yoshikawa, T., E# ects of volatile constituents of a rosemary ,**, extract on allergic airway inflammation related to +. Kanda, T., Akiyama, H., Yanagida, A., Tanabe, M., Goda, Y., Toyoda, M., Teshima, R. and Saito, Y., Inhibitory house dust mite allergen in mice. Int. J. Mol. Med., +0, -+/ -+3 (,**/). e# ects of apple polyphenol on induced histamine release,0 from RBL-, H- cells and rat mast cells. Biosci. Biotech. Biochem., 0,, +,2. +,23 ( +332). +* +,2 +--,**1 +/ Inoue, T., Sugimoto, Y., Masuda, H. and Kamei, C., Antiallergic e# ect of flavonoid glycosides obtaind from,* /,2,* 1,,

Inhibition of Degranulation of RBL-2H3 Cells by Casein Kinase II Substrate Peptide

Journal of Health Science, 49(3) 243 247 (2003) 243 Inhibition of Degranulation of RBL-2H3 Cells by Casein Kinase II Substrate Peptide Jun-ichi Onose, 1 Reiko Teshima,* Rhosuke Nakamura, and Jun-ichi Sawada