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SynthaseFNRPSG}˫;iÛÏ²@ÓͷŤϝ~ʧǫʣűǟϢ]:NRPS y9A-¼Ôà}º¾Üģ9T-¼Ôà~Þ@¼9C-¼Ôày~ɘǫͬě}ÍÊ¶¼Ʋǫbʧʣm:r9Ɨ͓9ATP ò˝~ÒÁȍÛ¡@±9tRNA ò˝~ÒÁȍ̪ùǧˆ}ÍÊ¶¼ƲǫʧʣűǟΧǬix]:g~^}ÍÊ¶¼ƦǜʧΕȶ~ʧǫʣ}ˠϠʢb\b9ÍÊ¶¼ǯŁp~~ɹŴ~Ĥa~űǟyʧǫʣi:ΨʻƖbʧȋmÍÊ¶¼ƦǜʧΕȶ9È¡ÁÑ¬àFPMG9N ι~È¾Ü¥Û¬àϘ̀ˡ}˜]x NVKDR z NVKDGPT ~͡Ђϖm͍ɄϿ~ǟ˔b˼x]:g~ˠϠʢaͪ˹mz9ÛÏ²@ÓŤϝ~ʧǫʣzǣ_}dd9r NRPS ~ɻǫ A-¼Ôà~ÒÁȍ~͑ȱͱņ̣ǀνy\gza9{~^|űǟyg~ C-ι~ˠϠʢb̀aaƎλbȪrrgza9p~ʧǫʣűǟ~ĳτȣr:
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kxǐΥþ̬ȕƺɚo|aur:¤Ú®´@͉}9A-¼ÔàȪv NRPS 1F649 ÒÁȍG9
T-¼ÔàȪv NRPS 2F81 ÒÁȍG9C-¼ÔàȪv NRPS 3F441 ÒÁȍGb˝Ȃkrb9gy NRPS ~[-¼ÔàyŋʢģirÒÁȍFϘ̀ˡG}ˣk9ÍÊ¶¼bŽłȁz|C-¼ÔàyɘǫiÍÊ¶¼ģǫΕʧʣmЀρ]:r9ATP ƲǫÕ¶@ÈȪv ORF ˝Ȃkrb9ATP ƲǫÕ¶@ÈȪvǧˆbÍÊ¶¼ũȶ}Ϣ]rЀ|]:pgy9{tbÍÊ¶¼ƲǫƤʣ}Ťϝmaþ̬ȕ͝ĵzˇř_ǧˆϢ]xɶǿ}Ʒ̹kr~yɳıkr]:1

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ˆFNRPSG}uxʧǫʣi:NRPS 9´àÄ¤ʢÒÁȍðļ~ÒÁȍũȶzkxϫϢ
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F25C35 Ȏũ, Fig. 1Gz streptothricin ~ !-Û­àÛ©ÍÊ¶¼ǟ˔F1〜7 Ȏũ Fig. 2Gb9ʋųͬěÔ ¾¯Óϖm NRPS }uxǫʣigzƺɚkrFNature Chem. Biol., 4, 766-772, 2008;
Nature Chem. Biol., 8, 791-797, 2012G:ζǦēy9gʋŪ NRPS ~ͬěÔ ¾¯ÓzǼƗ~˼ƺ}v]xɳım:Fig. 1

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streptothricin (ST)ǫʣǧˆ~ͬěÔ

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P01
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KɋɼÈô Bɟɵųʪ B˪ȓǱƮ G ĝʲɋĈã˼țʸïĈʴB GĎHº·HLϋ̣M ²ÈÚÏà9ÑÔĨ|{~ʁΕ}˝ȂmÈÚÏÁ¼Ͽy\9®»Þ§àϠŋʢǜȍģŋʢ̵Ȯmgzaű͖ʢʣΖzkx̉ϋix]:ʆ΁̇y̈́}ˬ̺}ˠdťx`9i}g²ÈÚÏàťϖϹˬ̺~ʣ̑}ͩ]Ξģmgzb˼x]:pgy9ζƸſyˬ̺~ͧİ}ͩ^ˬ̺²ÈÚÏàťϖϹ~Ƨọ̈̄Ξģ}v]xƷɵk9ű͖ʢ~Ǫ]ˬ̺
kb{~˺Ļy\a̏gz}kr:rp~Ȁ}9Ŀ²ÈÚÏàϿb̺΍zǴ΍y{~^|˝ȂͱyΖΆkx]a}v]x̏r:LΩΪ=ƲĩM ̲Ʋŏˍkrͧİ 092949697 ͎ϋ~ȕˬ̺k̈ɚk9HPLC Ϣ]x̛ϹǤ|ur:̛ϹΖʭ~Ʋĩ9ɁϤ²ÈÚÏà Daidzin z Genistin źp~ÑÞ¾Ü̷͡ˡy\gzbΖa
ur:r9ˏ²ÈÚÏàťϖϹͧİ 4 ͎ϋy˒Ħk9p~ǌǁɫkx]r:g~Ʋĩa9ű͖ʢ~Ǫ]ȕˬ̺kͧİ 4 ͎ϋy\gzbΖaur:Ȭ}ͧİ 4 ͎ϋ~ªàÊÜ}v]x9̲Ʋŏˍǌ~k̺΍zǴ΍}Ζe9̿Ϡ~ΩΪÿɚź̛ϹǤur:HPLC ̛ϹΖʭ~Ʋĩ9̺΍zǴ΍}`]x Genistein ƣ²ÈÚÏà ƕĂ}ΖΆkx]gzbΖaur:ĂΩ9Daidzein ƣ²ÈÚÏàǴ΍}ˠd9Glycitein ƣ²ÈÚÏàǴ΍}ˠd˝Ȃkx]gzbΖaur:

P02
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~ǓĩƷ̹kr:
LΩΪ=ƲĩM
¶ÈÚÆà TFThTGƪǑͧʄΪz̻ĭƣ̭ȕƼ͹ƏFTEMGΪϢ]x°ªÒàź°ªÒÁ@Ü~ A"1-42 ʻúƤʣˈĽǓĩ̏r:p~Ʋĩ;ThT ƪǑͧǑΪz TEM Ϊ~ϴΩy°ªÒÁ@Ü°ªÒà}ͱ;Ǫ] A"ʻúƤʣˈĽǓĩϖmgzbń͑ir:pgy;16 ɐЁ~Ϛʢ
SAMP8 } 0.05%°ªÒÁ@ÜǵǫȦϵ 4 áƴţȯϗʴɂiorǌ;ELISA ΪϢ]x͗̇~ A"Ϲ˙̛kr:p~Ʋĩ;°ªÒÁ@ÜʴɂƠy͗̇~ A"ϹƼ̋}ǁɫbń͑ir:gɇexͼȼ A"Ϲ~ǁɫ} A"ͷȋʧǧˆâ°¤Ý´@±~ŋʢģbūϝkx]~y|]az
ǣ_;ÛÜ´Ó PCR Ϊz®´àÉÞ·¹à¥ΪϢ]x A"1-42 ȋʧƧЅ}Ťϝm
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3

P03
Chlorogenic Acid and its Metabolites as Antimicrobial Agents!Faisal Kabir, Shigeru Katayama, Haruka Sugiyama, Soichiro Nakamura(Dept. of Bioscience and Biotechnology, Shinshu University)
LObjectivesM Chlorogenic acid (CGA) is a natural chemical ester composing of caffeic acid and(-)-quinic acid and it may be metabolized to active compounds, such as benzoic acid, ferulic acid,hippuric acid, etc, within the living body. Since it is well-known some of them elicited not only
bacteriostatic ability but also bactericidal effects, the antimicrobial activity and mechanism ofaction of CGA and its metabolites against a gram-negative bacterium Escherichia coli were
investigated.
LMethods and ResultsM E. coli IFO 3301 was used in this study. The susceptibility tests were
performed using agar disc diffusion method and minimum inhibitory concentration (MIC) was
measured by two-fold serial microdilution method. Chlorogenic acid and its metabolites exhibited
specific antimicrobial activity and corresponding MIC values. Ferulic acid, isoferulic acid, benzoic
acid and hydroxy benzoic acid were showed a remarkableantimicrobial effect against E. coli under thermal stress at
50ºC. E. coli cells (106/ml) completely disappeared in 2 mlof PBS coexisting with 30 mM ferulic acid and isoferulic
acid incubated at 50ºC for 3 hours, followed by benzoic acidand hydroxy benzoic acid incubated at 50ºC for 6 hours.
These results indicate that CGA and its metabolites
possess potent antimicrobial activities against E. coli.

P04ħçýȯŵðyğɇZ_¾~ʙʀɃgƎȗŠĳKɒɵĨžØ÷øǛǥƼÓ˛ɋ×ț˜ʖLϋ̣M
Acanthasteroside B3 ğ͊ȋ¾Å»ºa˱ϭirģǫΕy,ĦЁÑ®}ˣkxŴĠĶˀǓĩȮmgzaʏƧΞʢȵŖȭ϶ϐ~Û@¼zkxů˥ib, p~ˬϹƅƀ}ǲ͌bͩ^. pgy
acanthasterodside B3 ~^|ʧϬŋʢt, avǟ˔˱ɝģkr®¹Þ¼̷͡ˡ~ǫʣƸſbʔxcr. ǱĴʛϡʢ~ǔ
acanthasteroside B3ɹϋȘkʵƫirϿĕˡ 1 ~ǫʣϋ̣zm.
LΩΪ=ƲĩM
Ergosterol aǫʣi¬ÛÜ@¹Ü 2 ɚͧΕȶzkx, ˖Ǻʛȍũ̀͐kÜ¨@Ü 3zkr. Ȭ}Ð¢¬¼~ĺŚɾƵ`¥Û¨à~ʛȍũ̀͐ɾƵƷ̹k, ĺŚ}BF3-Et2O , ʛȍũ̀͐} OsO4 Ϣ]gz}kr. BF3-Et2O Ϣ]x, Ð¢¬¼~ĺŚǤ]Áà}̀]rǌ, ¥Û¨¬ÜģǤ^gzy­¥Û¨¬¼ 4 zkr. Ǳǌ OsO4 }­@Üģ`šƾ=˰ΤǎǤ], Ͽĕˡ 1 ǫʣmϜ̛y\.
3steps4steps

2

3

4

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Ͽĕˡ 1

P05
An antifungal compound, methyl 3-O-methylgallate from Tunisian halophyte, Tamarix aphylla
SAsma Ben Hmidene1, Zine Mighri2 and Mitsuru Hirota1 (1 Faculty of Agriculture,
Shinshu University, 2Faculty of science, University of Monastir, Tunisia)
Objectives:
Synthesized antifungal agents, such as o-phenylphenol (OPP) and ethyl p-hydroxybenzoate are used as foodand/or cosmetic preservatives. The use of these chemicals are nowadays raising concerns related to foodcontamination and toxicity towards human. Thus, the search for less toxic antifungal agents is still required. In
this study, we tried to search new antifungal compounds from some medicinal halophytes grown in Tunisia.
Methods and results:
Among some Tunisian halophytes, the methanol extract of Tamarix aphylla twigs showed remarked
antifungal activity against Fusarium oxysporium and Aspergillus awamori. Bioassay guided isolation led to thepurification of active compound, methyl 3-O-methylgallate (M3MG). Several related compounds, including
M3MG, were synthesized and subjected to antifungal assay. Their MIC values were determined based on
percent radial growth. Between the synthesized compounds, M3MG showed theH3COstrongest activity. Its effect (MIC 12 mM) was even stronger than OPP (MIC 18
OmM) and ethyl p-hydroxybenzoate (MIC19 mM) in the case of Fusarium. The HO
structure activity relationship study revealed that methylation of meta-hydroxyl
OCH3group in methyl gallate is a factor of antifungal efficiency enhancement.
HO

P06
Effects of gambir (Uncaria Gambir Roxb.) extract in a mouse model of allergic asthma!Pramita Sari Anungputri1,2BNami Nakagawa1BMasahiro Nishio1, Hayato Umekawa1 (1Grad. School of
Bioresources, Mie Univ., 2Fac. of Agriculture, Sriwijaya Univ., Indonesia)
LIntroductionM
Allergic asthma is a respiratory inflammatory disease, characterized by reversible obstruction andhyper-responsiveness in airway. Th2 cytokines, such as IL4, stimulate production ofallergen-specific IgE and may play important role in the pathophysiology of asthma. Gambier,Indonesian traditional food, has been reported to contain high amount of catechin. In this study,
we examined the effect of gambir extract on mouse model of asthma induced by ovalbumin (OVA).
LMethods and ResultsM
The mice (ddY, male, 7 week) were sensitized using OVA and alum in subcutaneous injection for 3
times every 7th day. Then the mice were immunized by gambir extract using nebulizer for 5 days,
followed by exposure to OVA for 5 days. Pathological study of lung tissues was performed by
HE-stain. The leukocyte count and the IL4 production were measured in both blood andbronchoalveolar lavage fluid (BALF). HE-stain showed that gambir extract-treated micesignificantly reduced the airway smooth muscle thickness. Moreover, gambir extract had atendency to reduce the levels of leukocyte and IL-4 both in blood and BALF. These observations
suggest that gambir extract reduced airway inflammation of the lung tissue in a mouse model ofallergic asthma.
Keywords: gambir extract, catechin, asthma

5

P07
ɰȰʸ˱́gƀUzƠgŵðf_Q[v§ ABCG2 ÍɴƮgÓàěɁōgÛĿgûȣ
Kɨ˗oVn BƕɝÓƇ Bɵɼ˩ƅ Bɟʧǆ̏ƌƟƮ BȤȩɚĮ Bɟȩɋ G ɟʴɋã˼țʸB ɟʴɋ×ã˼țʸB ȢåUzÀFB ̆Š "#HLϋ̣M Ş˓=ʖ˓=ɪ̎=͗|{~ˇʃ}ˠdͧǃkx] ABC ϓˑˡ ABCG2 ;ABC ϓˑˡ ABCB1zƇ}ǜbȁ͟ɚmgz}uxbǿΫ}ϐȁˤʢΌϝm<v;g~ ABC ϓˑˡzǜbȁz~ˎǋȄϢϬĳmgz;b~ģň϶Ϊʣǒ}̀d^_yƔxɖϤy\<
1 v~ǜbȁΒʟ~ ABC ϓˑˡ}ux͑ȱigzb˼x]~y;ζƸſy;
ABCB1 ~ũȶy\̧ˁΕϗϩ~ǜbȁz ABCG2 z~ˎǋȄϢĳʭmzƇ};ǜbȁz~ˎǋȄϢ}`e ABCG2 þ̬ȕɹ}˝ȂmĂĘũˠƣ~ĈƐĳʭkr<
LΩΪ=ƲĩM
 Flp-In System (Invitrogen Ȼ)ϫϢkx 12 Ͱʹʄˡ~˵ЉϺĀ}˝Ȃm FRT ͡Ђ} ABCB1` ABCG2;ABCG2 ~ĂĘũˠƣÃÛà»pq̀͐krǿΫ (Tamura et al., J. Exp.
Ther. Oncol., 6, 1-11, 2006.)pq 5 # 103 cells/well ~ν̰y 96 well plate }͛Ʉk;24 ȫţǌ}ǜbȁ̩Ħkx 72 ȫţͣϦkr<g~\z;MTT ȣϐͣϦƦ}̩Ħk;3 ȫţǌ} 20% SDS̩ĦkxǿΫϡĳkr. gĂͯ CO2 à¢ØÌ@´@͉}ʬ˾krǌ;630 nm ÛÈÝà®̑͜zkx;570 nm }`eŻǑ̰˙̛kxǿΫ~ʧ˝ϯͿĥkr<g~Ʋĩ;ABCB1 ~ũȶzkx˼x]ǜbȁ~̇y´¢ªàƦǜbȁb ABCG2 }ux͟ɚiħ͖ʢbƺ
ɚi;ABCG2 þ̬ȕɹ}˝ȂmĂĘũˠƣ~̇y͎ζʕ~ 3 ʕ} 1 ʕbΤϖm ABCG2 Q141K
ͧǃiorǿΫy;ύʧƣ~ ABCG2 ͧǃiorǿΫ´¢ªàƦǜbȁ}r]kxˤ
ʢȮmgzʋr}ƺɚkr<g~Ʋĩa;´¢ªàƦǜbȁbŖȼ}̳ϝmɻǫ};þ̬ȕʑ˹yŖȼ~þ̬ȕƣ͚ìkxa̳ϝ`p~Ϲư̛mcy\gzbǣ_
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P08
ț˧À~fęmwv»ÀgȜȞd§  dgȷŮƤ˼ûȣKƦɒˮȉ Bɵɼ˩ƅ BƕɝÓƇ B̛ːƔ Bǅ̡Õ BÝÏ̘ʮ ḄƜɉ Bɟȩɋ G ɟʴɋ×ã˼țʸB ˛ɋ×ț˜ʖB ɟʴɋțʸĠʕāʠŠĳǟB ɟʴɋã˼țʸH
Lϋ̣M
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x~Ĩṇ̌|ɵτɓΖ}Ǥx]|]<ζƸſy;ʧϐî˂}ȐϢyc^}mr
};ϐΕ̾˦ų̛mĄȕ~Ăvy\ ABC ϓˑˡ ABCB1 z~ˎǋȄϢͿĥkr<|`;ʧϐzkx;ͺw;ͺĔ;̃͘ɲ;̼̖}r]kxǓ͖ͧŭmgzb˼x]¬à (ʓô)ʾˮk;¬à}`eťϖϹbˠ]ģǫΕ˱ϭ=ʩʪk;Å» ABCB1 z~ˎǋȄϢ
Calcein assay ` ATPase assay }xͿĥkr<LΩΪ=ƲĩM
¬à (807 g)~Ô´Á@Ü̈ɚΕʘȍ¶ÜBÔ´Á@ÜBË¢ªàϢ]xΖ͡kB@Êà ÚÓ¤ÞÑ»¥ÚÈ@}xÛ¥½à epimagnolin (5.05 g)` fargesin (3.27 g)˱ϭ=ʩʪkr<g~Û¥½à sesamin zϿȨ~ǟ˔ϖkx]r~y;ζƸſy sesamin ťxͿĥǤu
r<Calcein assay y;ABCB1 î̛ͧǃmǿΫȣƽģǫΕz Calcein-AM ťͣϦČ̇yͣϦkBǿΫ͉}̃ʯkr calcein ƪǑƼ͹ƏĢyşȈkr<ĂΩ;ATPase assay yBABCB1 ťǿΫθįΖȣƽģǫΕ` ATP zǵǫk;ABCB1 bũȶ͑ȱmȀ}Ζĳm ATP ~Ϲ
37Q~ɾƵĢy˙̛kr<g~ƲĩBepimagnolin b˝Ȃkrɻǫ} calcein }ϗϩmƪǑƊ̰bǼƊd|;Ǽˠd~ ATP bΖĳir<krbuxBepimagnolin  ABCB1 ~ũȶy\;¬à˟~ϐΕzΘϢmɻǫ}ΘϢϐΕ~ϐΕ̾˦bΞģmħ͖ʢ}̉õΓ^ͻϤb\gzbȮǶir<6

P09
ĕǸ¬¯¬ destruxin E UƒİǎỳȔ[vƃȼgƙɭêfė[vŠĳKɀȓʡȈ Bɵɼ˩ƅ Bƕʁđ  BȤɵŏȎ BĭɵǤȈ BɻÏ̊Ɗ Bɟȩɋ G ɟʴɋ×ã˼țʸB ɽ˔ɋ×˧B ɵ˂ƦʫȞ˧B ɟʴɋã˼țʸH
Lϋ̣M
 Į?;ǳ̊΀ƿʢȞɿƖ M.anisopliae a˱ϭirŚɿºÊ¬ÍÊ¶¼ƦǜʧΕȶ destruxinE b
ED50 = 4.8 nM z]^̘͕̰}`]x͝ǯǿΫ}Ƥ˦øɽrkxǯŻɊϧʡmgzƺɚk(Nakagawa et al., Bone, 33, 443-455, 2003.);ǉˎǫʣΪϢ]r destruxin E ~˂ǫʣʠĹ}ʸƛex˯ʣkx] (Yoshida et al., Org. Lett., 12, 3792-3795, 2010.)<ζƸſy;͝ǯǿΫ~Ƥ˦øɽr
m destruxin E ~ǟ˔~Ϭĳ`ʋųǯŻɊϧʡȁ~ĺͧ}ǔer destruxin E ~ǟ˔~Ǽ̤ģϋ̣
zkx;͝ǯǿΫ~Ƥ˦}r]m destruxin E Ͽĕˡ~ĈƐͿĥkr<LΩΪ=ƲĩM
Slc:ddY Ñ®~Бǯ`ˬ˩ǯaǽɂkrǯʝǿΫ 100 ng/ml GST-RANKL ` 1 µg/ml
M-CSF;1 ng/ml TGF-!1 ~˝ȂĢ;100-mm diameter type I collagen-coated culture dish ɹy 72 ȫţͣϦkx˱ł͝ǯǿΫ̓r<g~˱ł͝ǯǿΫ 3 # 104 cells/well ~ν̰y half area 96 well plate }͛Ʉk;
100 ng/ml GST-RANKL ` 1 µg/ml M-CSF;1 ng/ml TGF-!1 ˝ȂĢy 24 ȫţͣϦkxˠł͝ǯǿΫ
̓r<͝ǯǿΫ~Ƥ˦}r]m destruxin E Ͽĕˡ~ĈƐ;ˠł͝ǯǿΫ 100 ng/ml GST-RANKL` 1 µg/ml M-CSF;Ͽĕˡ˝ȂĢyͣϦk;24 ȫţǌ}͝ǯǿΫ~Ñ@ @ǧˆy\Ɇʮȍ̝ǜʢȍʢÎ®È´@±~ũȶϢ]x͝ǯǿΫʹʄkrǌ;ǑňƼ͹ƏĢy͝ǯǿΫ~Ƥ˦şȈkxͿĥkr<p~Ʋĩ;ͿĥkrϿĕˡ~̇};destruxin E ̘͕̰y͝ǯǿΫ}Ƥ˦ø
ɽrm~˝Ȃk|aur<r;N-Ô¶ÜÚ¾àȎũ N-Ô¶ÜÃÛà\] N-Ô¶Ü²Þ¬à}˾řkx N-Ô¶ÜÚ¾àȎũ~˖ǺʞǪdkrɻǫ` "-Ú¾à¥Û¬à}˾řkrɻǫ}ŋʢ~ɯȲb͑r<g~Ʋĩa;destruxin E ~˖Ǻ~|n;Ś
ɿºÊ¬ÍÊ¶¼~ϰˡǟ˔͝ǯǿΫ~Ƥ˦øɽ~Ϙ̀}ɖϤ|ώŊ˲ux]zǣ_<P10Ȣåšƨ¿¨˽gȗʹfė[vŠĳ"ĭɵǏʼ Bƞɒźʝ , ȺɵÞǍ 2Bŉ̟˞Ƚ 1G1 Ȣåšɋ×ǽʬÜ˿B2(Ď)ɵĘæ˗ɱH
1

1

Lϋ̣M
 ßªÆ (Wasabia japonica Matsumura) ÉÚ½ĨßªÆ˛}˛kx`9ǴƩǇdaǩʓϵzkxsey|d9ɯɏȁǜƖȁzkxϢ]xcr:ßªÆ~Ƹſg~ǴƩ}Ťm~bˠ
d9ϣ~Ƹſ}ŤkxǜȍģȄϢ9ǜƖȄϢ9ǜĔɲȄϢ9ǜʹκȄϢ|{~ΧǬb\b9p~
ŋʢʣΖ}`]xμs΃τ|̫bˠ]:pgyζƸſy9ßªÆϣ~ŋʢʣΖ}̆ϋk9pg}
ťʣΖ~ǟ˔ĳʭz̛̿Ǥ]9p~ʧϬŋʢͿĥmgzyßªÆϣ~ϫϢĥ˻Ǫgzϋ̣zkr:
LΩΪ=ƲĩM ßªÆϣ~Ô´Á@Ü̈ɚΕË¢ªàzʛ9`ʘȍ¶ÜyɞȬΖ͡krǌ9ĿɄ¤ÞÑ»¥ÚÈ@}Ζįk9]dva~ʣΖ˱ϭkr:p MS9NMR ̵~ĿɄΖʭűŨϢ]xǟ˔ĳʭk99 Ʉ~È¾ÜÊÞÄÁ¼92 Ʉ~ÈÚÏÁ¼92 Ʉ~¹ÜÍÁ¼9` 1 Ʉ~ Þ¹Á¼̛̿kr:˱ϭkrģǫΕ~^t 6 Ʉ~È¾ÜÊÞÄÁ¼` 2 Ʉ~ÈÚÏÁ¼Ϣ]x FRAP assay }ǜȍģȣƽǤurzg9mx~È¾ÜÊÞÄÁ¼}`]
x Trolox ðɹ~ǜȍģ͖b͑9r9Trolox ~ 2 ͢ðɹ~ŋʢȮmģǫΕ~˝Ȃń͑kr:
r9̛̿kr Þ¹Á¼ all-trans-lutein y9Ɗ]ǜȍģ͖ȢϻĶˀȄϢȪvgzỷϋix]ģǫΕy\ur:ðɹ~^}ßªÆϣ}ű͖ʢϖmΒʟ~ģǫΕ~˝Ȃbń͑i9ϖǓȤǂzkxϫϢycħ͖ʢbƺɚir:7

P11ʍʽŧƨǺʸ Pterogyne nitens ˱́ nitensidine A d§ ABCB1 dgȷŮƤ˼ûȣ
Uɵɼ˩ƅ 1BƦɒˮȉ 1BƕɝÓƇ 1BȤȩɚĮ 2BLuis Octavio Regasini3BVanderlan da Silva Bolzani3B
Gert Fricker4Bɟȩɋ 1BThomas Efferth5
G1 ɟʴɋ×ãțB2 ̆Š CLSTB3 À¦¾ɋêĈB4 ¤~¼®¼ɋ˧B5 ³~Àɋ˧HLϋ̣M
Į?;͋ΛƿʧʁΕ Pterogyne nitens }ť¥¾­àÜ Þ¼ nitensidine A }ǯ˅Еɲȭ϶ϐǐΥzkx~ħ͖ʢƺɚkr[1]<ζƸſy;nitensidine A ȷƽ̾Ε}̳ϝmȀ~ˡ͉̾˦Ϭĳmr};ϐΕ~ˡ͉̾˦}ˬc|ĈƐźm ABC ϓˑˡ ABCB1 z nitensidine A z~ˎǋȄϢĳʭmgz}kr[2]<LΩΪ=ƲĩM
Å»żʢÛàÄİžʢͥƳ΀=T ÛàÄžϠǿΫŎ CCRF-CEM ǿΫ` CCRF-CEM ǿΫdoxorubicin ɟϬkx ABCB1 ĭɺ}ͧǃior CEM/ADR5000 ǿΫϢ];MTT assay ` Calcein
asay;ATPase assay Ǥ^gz}ux;ABCB1 z nitensidine A z~ˎǋȄϢĳʭkr<MTT assayy9CCRF-CEM ǿΫ}ͱkx CEM/ADR5000 ǿΫb nitensidine A }r]kxˤʢȮmgzbƺɚi;ABCB1 ~̦ƣ̣|ũȶy\ verapamil ̿ȫ}̳ϝmgz}ux;g~ˤʢbĳɧigzbƺɚir<Calcein assay y;CEM/ADR5000 ǿΫ nitensidine A z Calcein-AM ťͣϦČ̇yͣϦkBǿΫ͉}̃ʯkr calcein ƪǑƼ͹ƏĢyşȈkr<g~Ʋĩ; verapamil Ϣ]xȣƽkrɻǫz̿Ϡ};nitensidine A ǿΫ} calcein ~͟ɚϧʡmgzbƺɚir<
ATPase assay yBCEM/ADR5000 ǿΫa̏ʪkr ABCB1 ťǿΫθįΖ nitensidine A `
ATP zǵǫk;ABCB1 bũȶ͑ȱmȀ}Ζĳm ATP ~Ϲ 37Q~ɾƵĢy˙̛kr<g~ƲĩB ATP ~ΖĳϹ nitensidine A ~͕̰ò˝̣}˒Ħk;Hanes-Woolf ÊÞ·»a Vmax = 125 µM;
Km = 6.4 µM bȌɚir<r;ζƸſy;Autodock4 Ϣ]x ABCB1 }`e nitensidine A Ʋǫ΍ñzp~ƲǫÀÜ£@~Ϝ˙Ǥur<p~Ʋĩ; verapamil bƲǫmzϜˋi΍ñ}
nitensidine A bƲǫmħ͖ʢbȮǶi;p~ÀÜ£@˻ verapamil Ϣ]xȌɚkrɻǫzƗȨkx]r<g~Ʋĩa;nitensidine A  ABCB1 ~ũȶy\zǣ_;ȷƽ̾Ε}̳ϝm
Ȁ} ABCB1 b nitensidine A ~ŻɊ}ĈƐźmgzbȮǶir<
H1ITajima et al., Cytotechnology, in press. H2ITajima et al., Phytomedicine, in press.

P12ǽɟʇŃgʇȲƨțSsjČȖfɅ[v»À˱́¬¾|À| Àõʹg̀ȔŵðKȐƧ˱ô Bɼɀ˳Ʈ B˪ɵť̗ ȺɵǒÓ G Ȣåš̉ɋĈB ɽķ˧ïɋĈH
Lϋ̣M
 Į?gy};ĞʄÉ¼žƖ~ͅˆȋʧϧʡ`ͅˆŋʢˈĽʣΖÐÛÈÁ@ÜϿa˳ȇk;Ûà©ÐÛÈÁ@ÜϿ}p~ŋʢƺɚkx]<ζƸſy;ĞʄÉ¼žƖ~ͅˆȋʧ`ͅˆŋʢ}ˣmÛà©ÐÛÈÁ@Ü̇~ÊÞà»¬¾­àįΖ (ɖǫ̰Ν) ~ϧʡǓĩͿĥkr<LΩΪ=ƲĩM ĞʄÉ¼žƖ~ͅˆ staphylococcal enterotoxin A (SEA) ~ȋʧ` SEA z~ƲǫʐЈʢ}ˣmÛà©ϗϩÊÞà»¬¾­à~˅̈ɚįΖ`˱Ϲˡ〜6 Ϲˡðɹ~įΖ~ȄϢ®
´ à É Þ · »  Ϣ ] x ̏  r < æ SEA ȋ ʧ } ˣ m  Ĉ Ɛ : Ŀ į Ζ ( ɍ ͕ ̰ : 0.1~3.0 mM) 
Staphylococcus aureus C-29 (ɍ͕̰: 103~104 CFU/mL) zǵǫk;37 °C y 24 ȫţͣϦǌ;SEA Ϲ˙̛kr:çSEA z~ƲǫʐЈʢ: SEA (5 µg/mL) zĿįΖ (ɍ͕̰: 0.5 3.0 mM) ǵǫk;
37 °C y 24 ȫţͬěiorǌ;SEA Ϲ˙̛kr<p~Ʋĩ;3〜6 ϹˡįΖ}`]x;ͅˆȋʧϧʡkx]ħ͖ʢbȮǶir<r;SEA }ˣmǜ-SEA ǜˡ~Ʋǫ;2 Ϲˡðɹ~įΖyϢϹò˝̣}ˈĽi;6 Ϲˡðɹ~įΖyǼˈĽǓĩbǪaur<g~Ʋĩ;ÊÞà»¬¾­à~ɖǫ̰bɹb}krb];SEA ȋʧϧʡǓĩ` SEA Ζȕz~ƲǫʐЈʢbǪħ͖ʢbȮǶir<ǃȂ;ĿįΖ}`eͅˆŋʢˈĽ͖` SEA þ̬ȕͧǃ~ĈƐ}v]xƷ̹̇y\<8

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G ȢåšɋDǽʬÜ˿ Ƨ̅ɋDț˜ĕĺDəÐǽʸ GĎHxTYțČHLϋ̣M
 Retinol dehydrogenase 8FRDH8G9ωθ}˝ȂmŗǑΕȶÞ¼Ê¬à~ǻǫʣƧЅ~Ϯ˚˺Ļy\ all-trans-retinalFatRALGa all-trans-retinolFatROLG~ͬěʅͤmǧˆy\:RDH8 
atRAL a atROL ~ͬě}`]xɖϤy\9RDH8 ŋʢ~̘Ģ atRAL ˫Ƚ~̀đωθȵŖ~ϤĄz|gzbΧǬix]:p~r9ȢŅĶˀ} atRAL a atROL ~šƾͬěʅͤm
 RDH8 ŋʢģmgzbͻϤszǣ_9RDH8 ŋʢģmʣΖ}ȢŅĶˀbů˥ix]:ζƸſy9̴Ƹſȳ}xǟ́kr RDH8 ŋʢͿĥΪϢ]9ȢŅĶˀȄϢbΧǬix]à»¬¾àźŧ΀Ϝδ}Ǔị̃szǣ_x]̅ ¹¢à}v]x RDH8 ŋʢ~ĈƐͿĥǤ^gzy9ȢŅĶˀȄϢϖmʣΖ˳ȇźp~ȄϢűɥĳτmgzϋ̣zkr:
LΩΪ=ƲĩM Ñ®˨ȩϗϩʻúİǿΫ NIH3T3 }`]xȄʪkr RDH8 ͧǃǿΫϢ]x9à»¬¾à
Delphinidin-3-glucoside 9 Cyanidin-3-glucoside 9 Pelargonidin-3-glucoside ~ 3 Ʉ Ͽ 9 ̅ ¹ ¢ à Epigallocatechin gallate9Epicatechin gallate ~ 2 ɄϿ}`e RDH8 ŋʢ~ĈƐͿĥǤur: p~Ʋĩ9à»¬¾àź̅ ¹¢à~̩Ħ} atROL ʧʣϹb˒Ħkr:g9à»¬¾àź̅ ¹¢à~̩Ħ}ux RDH8 ŋʢb˕ʔkrgzbǣ_r:ǟ˔ŋʢ̣Ϳĥ}9à»¬¾ày B Ś~Å¼Þ¢¬ũFOH ũG~ʟbˠ] { atROL ʧʣϹbˠd|Ƣǔbƺr:Ǳǌ9˟~ÐÛÈÁ@ÜyͿĥǤ]9ģňǟ˔z RDH8 ŋʢ}ϝ_ĈƐ~ŤЄʢ}v]xi}Ʒɵʔx]dϜ̛y\:P14
,:(336:/065,05 ÍɴƮyʄʑZ_    ŎƿɳĔɃgûȣKŊɵǤʼBƸ̟ ȹBɵ̞ ƂGǿɋʖã˼ț˜H
Lϋ̣Mȝ˯;¾ÎàÂ· FMentha arvensisGƤȶ̪řʁΕˡϢ]rÈ»ÝÔº@¬Ùà~ħ͖ʢ̏ϋ̣yƸſǤux]<Ů};ȝ˯~Ƹſȳy;Agrobacterium Ϊ};Å»~ metallothionein þ̬ȕFMTL1G̀͐kr M<arvensis Ƥȶ̪řˡbWŎMa-B Ŏ;Ma-CŎȄɚix]<g~Ƥȶ̪řˡ~ρƖʁΕˡϢ];p~͂ˤʢ~̏ǸǤurƲĩ}v]
x;ĭƂ~ˏĲ}`]xΧǬkr<ǱĴ;ȝ˯;Ma-B Ŏź Ma-C Ŏ~ɞģʁΕˡϢ]x;͂ˤʢź͂̃ʯʢ~̏ǸǤur<r;͂ˤʢűǟ~ĳτ~˶ɢzkx;n;M<arvensis
ʐŎa;ČΫ~̈ɚȣr<
LΩΪ=ƲĩMM<arvensis Ƥȶ̪řˡ;Ma-B Ŏ\] Ma-C Ŏ} 0C1,000 mg/L ~͂̩Ħk;
26Q;16 ȫţτůyͣϦǤur<WɐţͣϦk;ʧā~şȈǤur<p~ǌ;ʁΕˡŏˍ=ĸ
ģF500C700QGǌ;͕ĘȍFϏ 6 MG}ϡĳkr<i};0.1 M Ęȍy 200C400 ͢}ŬȾǌ;ƿȕŻǑΪ};͕̰͂~˙̛Ǥur<p~Ʋĩ;Ma-B Ŏ, Ma-C Ŏz};ʐŎ}ͱkʧāϸǖy\ur<ʁΕˡ~Ă̛ɖϹ\r~͂~ťϖϹ;ǾͣɾƵ};ø|˻z|ur<ĂΩ;ÊÞ»ÊÚ®»̏ʪkrǌ;Ä@¨@ÜΉɹΪ}x;ȍʢÈ®È´@±ŋʢź¾Ø@»ÚÜ
Ý·¼ʹʄȘͽzkx;M<arvensis ʐŎaČΫ~̈ɚȣr<9

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B2, B4 ~ǫʣ}v]xΧǬm:n9phloroglucinol z methyl 3,4,5-trihydroxybenzoate ɚͧΕȶzkxŽ̭ȕȁǫʣkr:˜]x9Ž̭ȕȁzΝ̯ǫʣkrŽłȁ̵ϹϢ]rÜ®ȍ}ɘǫͬěǤ]9B1 yÜ®ȍ} Yb(OTf)3 Ϣ]xɊϯ 66%y9B2 yÜ®ȍ} AgBF4 Ϣ]xɊϯ 76%y9B4 yÜ®ȍ} Yb(OTf)3 Ϣ]xɊϯ 78%y 2 Ϲˡǫʣk9pq˂x~Τǎũ˰ΤǎmgzyÊÞºÜÈ¾­à B1 (3), B2 (4), B4 (5) ~ǫʣŒϳkr:ðɹ~^}kx̓rģǫΕzÐ­¹É¨à»Þ@Üzkx epigallocatechin gallate (EGCG) Ϣ]x9Å»ʿϰʽ¡à~ǿΫ}ˣkx¡àǿΫ˒ʂϧʡȣƽǤur:p~Ʋĩ9ǫʣkr 3 ɄϿ~ģǫΕ EGCG z
̟̰̿~ǜɅЏŋʢϖkx]rb; ¹¢àÇ ¹¢à~Û©Ñ@Ƽ̋|ŋʢȮi|aur:

P16
~·ɠǙʸgóʺǯœŨŵðd˰ŵȗʹgʃɪ
KˣŶǤȗBȅʾǓʻBˁƧ˞BɟɀǐÓ̗Gǿɋ×ʖH
Lϋ̣M®£īΗÝÜ£@;͎ζǭο~ 5 ʕ} 1 ʕbΤȪkx]zi;Jǭο΀KzǄx`;ʆ΁ϗϩ~î˂|īΗɲƬǁǓĩȪvϐȁ~ĺͧbů˥ix]:g^kxĮ?;ªÐ ¾ à  È  Á @ Ü ʢ ģ ǫ Ε | { ~ ˠ d ~ ʧ Ϭ ŋ ʢ Ε ȶ  ť  y ]  ¢ ¤  Õ F Helianthus
tuberosusG}̆ϋkr:ζƸſy¢¤ṎɚΕ~īΗɲƬǁǓĩ}v]x̏;p~ϖǓʣΖ̛̈́mgzϋ̣zkx]:LΩΪ=ƲĩM¢¤ÕΗι} 10 ͢Ϲ~ 70%Ô´Á@ÜĦ_;ȳġyŀЋF3min;12,000 rpmGk˅̈ɚΕ̏ʪkr:Cry j 1 ϡČ}ŗȄǤur Balb/c Ñ®Fȥ;5 ɐЁG};˅̈ɚΕǵǫkrȦϵ 40 ͎ţȯϗʴɂior~t;uac=dkĴʟ;Ƴʦ̇~Å®´Òà` IgE͕̰˙̛kr:p~Ʋĩ;pqy¨à»Þ@ÜƠzͱņkxƼ̋}̘Ģkr:r;˅̈ɚΕ HPLC Ζʭ}ƅk;ΖɂkįΖ A〜E ̓r:Balb/c Ñ®~̎ŜÄÜͭǿΫ}̩Ħǌ;Cry j
1 ȑƮĢy 3 ͎ţͣϦk;ͣϦɹʦ̇~ IL-4 ͕̰˙̛kr:p~Ʋĩ;Ç@¤ B } IL-4 ȋʧϧʡ
Ǔĩb\gzbȮǶir:i};ɉɿǿΫ~ǜƿ̞Ȯ͖}źmĈƐ}v]xƷ̹kr:m|
t;THP-1 ϗϩɉɿǿΫ}įΖ A〜E ̩Ħk;Cry j 1 ȑƮĢy 3 ͎ţͣϦkx; MHC classè~ǟʣΖȕy\ HLA-DR ~ͧǃϹÈÞ@ª»Ô»Û@}˙̛kr:p~Ʋĩ;įΖ B }`]x;Ƽ̋| HLA-DR ͧǃϧʡǓĩbń͑ir:pgy;įΖ B  EI-MS ` NMR ĳʭ}ƅkǟ˔ĳʭǤurƲĩ;g~ʣΖ; ÈȍϘ̀ˡ1-(3’,4’-dihydroxycinnamoyl)cyclopentane-2,3-diol       ʙ 1. ϖǓʣΖ~ǟ˔Ȱ
Fʙ 1Gz̛̿ir:

10

P17
Procyanidin B2,B3-gallate ̐gƎȗdƀǉ̢ČȖKȋɵȅȈ a, ìʁ Ž a, ŬɵÓˤ b, ǫ˗łƮ b, ȩŶƈÓ̗ b, òŧȠÓ c, ʵʴļǦ d,ʁÏ ʝ b, ȅʾǓʻ a (ǿǑɋĈɋĈ×ʖĈŠĳï ĠʕȖǽ̍āʠĈȨƁ a, ǿǑɋĈʖĈʴã˼ț˜ïĈï b ĎƽúǃÀF¼ c, ķɹ˧ïɋĈ ȵ˧ïĈő d )Lϋ̣M Procyanidin ϿȯˁĹ}`]xÉ¼̅ϣ}ťϖiÐÛÈÁ@Ü~ĂƠy\;ǜȍģŋ
ʢͼ̼}ˠd~ʧϬŋʢb˼x]<ze gallate Ϙ̀ˡ};DNA ÐÛÔÚ@±ˈĽŋʢǜɅЏŋʢbΧǬix`;ɖϤ|ʧϬŋʢΕȶzkx̉ϋɑx]<ζƸſ}`]x;ɘǫȁ} Yb(OTf)3 y˼Qè̘eǷŚcɾ̎gěƿgǘƎyƊQBgallate ˴ʄɃcPv procyanidin
B2-3-O-gallate, 3’’-O-gallate ; 3,3’’-di-O-gallate ź  procyanidin B3-3-O-gallate, 3’’-O-gallate ;3,3’’-di-O-gallate ~ǫʣmgzϋ̣zkr<rǫʣkr gallate Ϙ̀ˡ;Å»ʿϰʽŦǿΫϢ]rǜɅЏŋʢȣƽ}ƅmgzϋ̣zkr<
LΩΪ=ƲĩM
 (+)-Catechin ź($)-epicatechinɚͧΕȶzkxpqŽ̭ȕȁ=Ž+łȁǫʣk;p̵ϹϢ]rYb(OTf)3 fsvǘƎʢãyƊQ]w^wgɟĖɃyƎȗZ_Cʅtw_ɟĖɃfɅZ Pd/C y˼Q_ȌȲɲìʢãyƊ
+QBˠɬêƎʸgƎȗyɐȗZ_C ǫʣkr gallate Ϙ̀ˡ}v]xÅ»ʿϰʽŦǿΫϢ]rǜɅЏŋʢȣƽǤurɡ;ϖõ|ŋʢbƺbŮ}ŋʢb˼ EGCG ź prodelphinidin B3 zͱņkxɀ]ŋʢy\gzbͪτkr 1)<
OH

OBn

BnO

OBn

BnO

O

OBn

O

OBn

BnO

O

OR1

OBn

Yb(OTf)3

OR2

OBn OEE

OBn
BnO

OBn

OH

OR1

H2, Pd/C

OH
HO

OBn

53% (R1 = G, R2 = H)
22% (R1 = H, R2 = G)

PCB2-3-O-gallate : quant. (R1 = G, R2 = H)

43% (R1 = G, R2 = G)

PCB2-3,3''-di-O-gallate : quant. (R1 = G, R2 = G)

BnO

BnO

O

OBn

O

OR1

OBn OEE

O

OBn

BnO

OBn

OR2

Yb(OTf)3

PCB2-3''-O-gallate : quant. (R1 = H, R2 = G)

OH

求核剤

HO

OBn

OR1

OBn
BnO

OBn

O

OBn

求電子剤

OH

OH

OBn

OBn

OH

O

OR2

OR2

OBn

求核剤

求電子剤

OBn

O

O

HO

OBn

OR1

OBn

OR2

OBn

H2, Pd/C

O

OH

OR1

OH
HO

OH

O

OH

OR2

OH

68% (R1 = G, R2 = H)
28% (R1 = H, R2 = G)

PCB3-3-O-gallate : quant. (R1 = G, R2 = H)

68% (R1 = G, R2 = G)

PCB3-3,3''-di-O-gallate : quant. (R1 = G, R2 = G)

PCB3-3''-O-gallate : quant. (R1 = H, R2 = G)

1) Suda, M., Katoh, M., Toda, K., Matsumoto, K., Kawaguchi, K., Kawahara, S., Hattori, Y., Hujii, H.,
Makabe, H. Bioorg. Med. Chem. Lett. 2013, 23, 4935-4939.

P18 ŗƎÀ¦ƿy˼Q_À¦ƿgȁĦ¨F}¬½~ˍgāʠdºÀ¼´F|~kgã˼KƆŶɏ̗ ;8@()(,<(89,3 ǥ̏Ŵ ̙ɼź˝ ȈŢȚ̊ ɟˣǓ˵  ˛ɋ×Dț˜ʖĈA ʖŠĠƃDǽȸŠA ˛ɋ×Dȵ˧ïĈ
L͠ƥzϋ̣M
ζƸſȳy9ÑÜ­Ùà͉y~  zρǿΫ´àÄ¤ȶǫʣƦˇǫorű͖ʢʧˡΖȕ   Â®Ü@Ê·»ĳʭɃΪ9Æ@¯º®ÊÝΪ~ĺͧ}ʣǒkx]:kak|bζɃΪϠ?|´àÄ¤ȶ=ǧˆű͖ĶΞ}Ϣ]r}9Æ@¯̴r~̞Ȯ´àÄ¤ȶΖȕʟ~˒ˬz9̞Ȯ´àÄ¤ȶΖȕʟ~ƕĂģbΰ:ζƸſy9 Ʋǫ´àÄ¤ȶy\ĂζǺ 31
F4'31GϢ]rÆ@¯º®ÊÝΪ~Ķϸϋ̣zkr:
LΩΪzƲĩMÕºÜÍÊ¶¼  Ʋǫ´àÄ¤ȶy\ 4'31 z~ϛǫ´àÄ¤ȶzkxͧǃio9p~Ʋǫ͡ЂϢ]xÆ@¯ɹ~̞ȮϹkrzg9ɔϩ~ǜÇ»@Ê´¥ǜˡıkrɻǫzͱ9Ƽ̋}Ǫ]̞ȮϹbń͑ir:r9ƕĂ|Č̢̇yρǿΫ´àÄ¤ȶǫʣͬěǤ^÷9.17
*1'64-0+ ŶɛϢ]xȄʪkrÑÜ­Ùà͉y9ρǿΫ´àÄ¤ȶǫʣƦ} 4'31 ~ͧǃǤ]9ÈÞ@ª»Ô»Û@}Æ@¯ɹ~ 4'31 ̞ȮϹ~ĳʭǤur:p~Ʋĩ9.17*1'64-0+}uxÑÜ­ÙàȄʪkrɻǫ~ 4'31 ̞ȮϹ9ɔϩ~®´@Ú@Ϣ]rɻǫ}ͱƕĂy9g~ŶɛbÚÉÚÛ@~ƕĂģ}ϖǓy\gzbȮir:ǃȂ9ĪƋǧˆy\Ñ®ϗϩ»Úà®¥Ü´Ò½@± ~´@§·»͡Ђ~˳ȇ}Ϣ]ĳʭʔx]:

11

P19
Cell-free Synthesis of Horseradish Peroxidase and Manganese Peroxidase
for Bead Display-based High-throughput Screening
KBo ZHU, Ryoko NINOMIYA, Takaaki KOJIMA, Yugo IWASAKI, Hideo NAKANO(Dept. Bioeng., Grad. Sch. Bioagr. Sci., Nagoya Univ.)
LPurposeM
A cell-free protein synthesis system can produce various types of proteins directly from DNA templates suchas PCR products, and therefore attracts great attention as an alternative protein synthesis system especially forhigh-throughput functional screening of proteins. Here, we report on successful expression of active horseradish
peroxidase (HRP) and Phanerochaete chrysosporium manganese peroxidase (MnP) in an Escherichia coli
cell-free protein synthesis system for bead display-based high-throughput screening.
LMethods and resultsM
The reaction conditions of cell-free protein synthesis of HRP and MnP, such as the concentrations of hemin,calcium ions, glutathione and disulfide bond isomerase were optimized to increase the solubility and activity ofthe synthesized enzyme. Then, the cell-free synthesized HRP and MnP purified using the hemagglutinin tag
showed similar and higher specific activity compared to the commercial wild-type enzymes, respectively,
suggesting that the cell-free system can be used as a preparative method for efficient synthesis of disulfide
bond-containing metalloenzymes such as HRP and MnP.
Moreover, the cell-free synthesis of HRP in hexadecane-based emulsion and its immobilization on
microbeads were successful. The activity of immobilized HRP was detected by a tyramide-based fluorogenic
assay using flow cytometer. A model bead library containing genes of wild-type HRP and inactive mutant was
screened, and an enrichment of the wild-type gene from the library has been achieved. Thus, the bead display
and fluorogenic assay are effective for high-throughput screening of HRP.

P20 g¼ºFÍɴƮºFfFYwvª{´»F ¼ºFgƋȲʆȖ
KǻɵɎBƸɵǭʼB̝ÃÚÉĐȅĥƮ˟ɀɮƚBÉĐ̘̗GƦǖɋĈHLϋ̣M ʁΕ~ǿΫΜ~ɁʣΖy\°ÜÞ@®Ζĳk9͔ϵģ9\]ƿϵģmr}9°ÜÚ@±~ϫϢbǣ_:Clostridium josui ~§ÁÓ}°ÜÚ@±Βǫˡy\°ÜÞ²@Ó~ʣΖ¨@¼mþ̬ȕ¤Ú®´@b˝Ȃm:ǱĴĮ?9g~þ̬ȕ¤Ú®´@}ť̷ȶΖĳǧˆÈÒÛ@9 ~°ÜÚ@±(Cel9A, Cel9B, Cel9C, Cel9D, Cel9E)}̆ϋk9̈́} Cel9A ź Cel9B~ǧˆ̈́ʢτa}mgzϋ̣zkr:
LΩΪ=ƲĩM
 C. josui ~°ÜÚ@±þ̬ȕ¤Ú®´@͉}¨@¼ix] cel9A, cel9B, cel9C, cel9D `
cel9E ~þ̬ȕ9pET-28a ÊÚ®Ò¼Ì¤´@Ϣ]x9ˬ̎Ɩ BL21 (DE3Gyͧǃio9ǧˆ̈́ʢ̏r:g~ǧˆ~ȡ̤ġ̰Ϗ 45-60Q9ȡ̤ pH  6.0-7.0 y\ur:r Cel9A, Cel9B,
Cel9C ź Cel9E  barley ã-D-glucan }9Cel9D  Konjac glucomannan }Ǫ]ŋʢbƺr:
Cel9A ź Cel9B ~̷~ΖĳϠȰ9ͦˊ¤ÞÑ»¥ÚÈϢ]x̏r:Cel9A ͷšƾι˶a
ʟ_x 3 Ͱϋ~ã1-4 ¥Û¨¬¼Ʋǫ}Ǫ]ĦʛΖĳŋʢȮk9r 2 Ͱϋ~ã1-4 ¥Û¨¬¼Ʋǫ}̘]ŋʢbƺr:Cel9B ͷšƾι˶˖aʟ_x 2 Ͱϋ~ã1-4 ¥Û¨¬¼Ʋǫ}Ǫ]ŋ
ʢbƺr:i}9Cel9A ź Cel9B apq CBM3c ź CBM4 ɧ]r´àÄ¤ȶǟ́k9ƾ~ǧˆzŋʢͱņkrƲĩ9Cel9A9Cel9B Ƈ}ǧˆŋʢb̘Ģkr:ðɹ~gza9
Cel9A ź Cel9B Ƈ}ÊÞ°·¬É|à¼¥Ü ½@±y\9ϴȼ}˝Ȃm CBM g~ǧˆ~ŋʢͧǃ}ͷɽ}ɖϤy\gzbȮǶir:     

12

P21
¼¾Fő¥~³ʹûƞŃ  gƋȲʷƎɃgʀƿŗƎ·¹F¼gŗƎʆȖKƯȩǛˆ BďɵǒÓ G Ʀǖɋ×əÐ~£®H
Lϋ̣M
 °ÜÞ@®ƦÃÑ®ϖϢ|ǻʧħ͖Ȥǂy\:´Á@Ü|{~ģǫΕ}̪řkxϫϢmr}ÃÑ®~ǧˆΖĳ~®¹·ÊͻϤzmb9p~Ǔϯͷɽ}ë]:g~Ǔϯǔɹior}9ij|ʿɟϬΪbψȇix]:°ÜÞ@®ƦÃÑ®ΖĳǿƖ
Clostridium josui ϗϩ~°ÜÞ@®Ʋǫ´àÄ¤ȶFCBM3G~ƲǫϹ˒Ħio^}ʿɟϬkrÃÑ®9Ǔϯ̣}ǧˆΖĳycgzbΧǬix]:ζƸſy9CBM3 ~Ʋǫ̈́ʢτa}mgz}ux9Ζĳkm]ÃÑ®~̈́̍~ϬĳϋȘkr:LΩΪ=ƲĩM
 Clostridium josui CipA CBM3GCBM3Hþ̬ȕ¤Þ@¾à¥k9ˬ̎Ɩ}xͧǃio9ʩʪmgz}9CBM3 ϡČ̏ʪkr:CBM3 ~°ÜÞ@®ƦÃÑ®ǟʣmˠ̷~ƲǫʢÑ¤ÞÝΪ}ư̛kr:CBM3 °ÜÞ@®~Ʋɮ;χ}Ʋǫm~zΧǬixcrb9
ͷƲɮʢ°ÜÞ@®y\ȍγɜ°ÜÞ@®9ħϡʢˠ̷y\¢¬Þ¥Ü àƲǫmgzbτaz|ur:r9CBM3 z°ÜÞ@®;χ~ƲǫͬěŻ̵̆ġĳʭ}xɶǿ}̏rƲĩ9
CBM3 Βȉ|°ÜÞ@®;χ~ɫ|dzWɄðɹ~ǟ˔͑ȱkxƲǫkx]gzbτaz|ur:g~Ʋĩ CBM3 b°ÜÞ@®ƦÃÑ®̇~ˠϠ|ǟ˔ƲǫmgzȮǶm:
CBM3 ~ƲǫϹij|ÃÑ®ΖĳǧˆbϫϢyc9ÃÑ®̇~;χʯ~ˬciͬĉkx]~zϜˋi:

P22˿ŃȖ~µdĸț[vȁĦ ȾƳǸŃUțƨ[v¼¾FʹûƋȲfė[vŠĳKɝÙţů ɵɟɋǡ Ċɀſ ưȌŦĵ ìʁøƭ G ˛ǴɋʖB ˛ɋ× ț˜ʖĈHLϋ̣M ¢¤Ó¬Ͽ9Ǡ̊Ͽ³Ó¬ɹĨ}˛mǳ̊y\:ϦƖʢ¢¤Ó¬zǈ¥Ü@ÊÑ à£zǈŨœt9pg}ƇʧƖΤϖkx]:ϦƖʢ¢¤Ó¬ɉϊ}ʺ
ǗkxǕ́ƤʣmȀ9Τϖkx]ƖϿǕ́Μχ}ʳɄk9p~ǌ9ʧākrƖϿʴɂm
z]^ʧŋϠȰvgzb˼x]:ϦƖʢ¢¤Ó¬ɉϊ}Ǖ́Ƥʣmsey|d9ȪtǰƖ}9ϊȃȶ~̘Ģɉϊ~ǊȜącŵgm:ϦƖʢ¢¤Ó¬bΤϖkx]ƖϿźǕ͉́~Ɩ9ϊȶ˴ˆǂzkxʧākx]gza9°ÜÞ@®ΖĳkxĊϦzkx]
gzbȮǶix]:ζƸſy¶­¤}ūʧmϦƖʢ¢¤Ó¬~Á¢¤Ó¬F    G~Τϖm°ÜÞ@®ΖĳƖ~˱ϭk9p~Ɩ~°ÜÞ@®Ζĳűǟĳτmgzϋ̣zm:LΩΪ=ƲĩM ʕǘȦākr   ~ǜ͉́Μȣϵǽɂk9°ÜÞ@®˱Ă~˴ˆǂzkrͣ˽yɑʯͣϦmgzy°ÜÞ@®ȤģycƖ®¤Û@¾à¥kr:˱ϭkrƖ~ !3~Ęũ͡Ђư̛k9!"ĳʭǤurzg9    z ~̿ĂʢȮmʋų~ ˛ȞɿƖbƺɚir:g~Ɩ°ÜÞ@®ťǼɫͣ˽yͣϦǌ9ͣϦЎČ̇~ŋʢ˙̛krzg9ͣϦ ͎ϋyǪ]°ÜÚ@±ŋʢbƷɚir:krbux9ΖϭirȞɿƖ°ÜÞ@®ΖĳƖyϖgzbΖaur:¥Ü¨@®~ź°ÜÞ@®~˴ˆǂzkrͣ˽y ˛ͣϦk9ǿΫļ}ΖͲir´àÄ¤ȶÊÞ¹@Óĳʭ}xͱņkr:p~Ʋĩ9°ÜÞ@®ͣ˽yˠʟ~´àÄ¤ȶb̈́ø̣}ͧǃkx]gzbτaz|ur:""!ź %4'154)%3',Ϣ]rĳʭ}v]xΘoxΧǬm:

13

P23
ɟƍÇǞ̆fsv¼ºFgʢã¶ µfaQb
Ŋŧȝ K̏Ǔɂ Bʁɵɚʓ B́Ƨɮʎ ƝǢǿʎ̗ G ǿǑɋ×ʖDĠʕǽB ²H
Lϋ̣M
 °ÜÞ@®ƦÃÑ®ʆ΁zƆǫk|]gza9Ã´Á@Ü~ƿϵzkx̉ϋix]:ͼȼ 90MPa ̟̰~̇ǪíɾƵĢy°ÜÚ@±ŋʢbǝʔmgzƺɚkr:gy}íϻò˝ʢb͑r Celluclast 1.5L Ϣ]xͬěɾƵ~Ʒ̹Ǥ]9ȋʧ̷Ϲ~˒ĦzĦíĢ}`e¥Ü¨@®~ĦʛΖĳϹb˒ĦmgzΧǬkr 1): ζƸſy9ĦíĢ}`e°ÜÚ@±ͬě}v]x9̷ˇʣ`¥Ü¨¬µ@±ŋʢɶǿ}Ʒ̹k9Ħí}ǓĩƷɵkr:
LΩΪ=ƲĩM
 °ÜÚ@± Celluclast 1.5L (Novozymes)9ͱņ}°ÜÚ@±ÑÁD4TE (̧ύà«Ó)9°
ÜÚ@±DÁ¯ E3S (×¤Ü») Ϣ]r:¶Ö·¤΄c˪}ũȶ (Avicel) 100mg90.25M ʘȍŝɴČ 5ml (pH4.5) ͐9°ÜÚ@± 1 mg ̩Ħk9ɽí (0.1MPa) r̇Ǫí (90MPa) Ģ950
}x 6 24 ȫţͬěǤur:ͬěǌ9ȺΔkĖʉkrǌɹʦĴɊk9RI ƷɚŨ΄ HPLC yΖʭk
r:r9p-¾»ÞÈ¾Ü¥Ü¨¬¼FpNPGG` D-¥Ü¨Á-1,5-Ú¤»àϢ]xb-¥Ü¨¬µ@±ŋʢ~ĈƐ̏Ǹkr:ɽí`ĦíĢ}`eͬě 24 ȫţǌ~°ÞÆ@®B¥Ü¨@®~ʧʣͱϯŽrƲĩ9
Celluclast1.5L9°ÜÚ@±ÑÁD4TE9°ÜÚ@±DÁ¯ E3S ~ɞyǪd|9ĦíĢ}`eȋʧ̷Ϲ~˒Ħ¥Ü¨@®~ʧʣ}ŵĄkx]gzbτaz|ur: ũȶ} pNPG Ϣ]x9"-¥Ü¨¬µ@±ŋʢ˙̛krƲĩ9ĦíĢ}`]xǧˆŋʢbǝʔmgzτa}kr:ͬěƦ̇}ˈĽȁy\ D-¥Ü¨Á-1,5-Ú¤»à̩Ħk9̿Ϡ~ͬěǤurzg9°ÞÆ@®~ʧʣbɽí=Ħíz}ͬě 6 ȫţðǨyˈĽigzń͑kr:ðɹ~Ʋĩa9Celluclast 1.5L ̇~"-¥Ü¨¬µ@±bíϻ}ŗɇʢȮmgzbȮǶir:1) ͎ζ͙ƭģňĲ 2013 ͓̰ˬĲǦēϤȚɑ, 2C12p11 (2013)P24ɤƍèȖɄʥgèɺdʨțʸŋgŒƹɬDňĖɬˀêgûȣKʶĘɏȈBɽǛʓBƢÏǣBÉĐȅĥƮB˟ɀɮƚBÉĐ̘̗GƦǖɋ×DțƶHLϋ̣M
 100Q}Ɨ]Ǫġ}ˢʹģy9ϖűΕ~Ζĳ˚̰b˚]zǣ_x`9p~^|ŚƉ̇}{~^|ǿƖbʧ˗kx]aŤʉbȪr:ĭƂ~Ƹſy91 ̫kdʟ̫ˣɷ}krġ̰~Ƨọ̈̄Ξģτa}ix]b9Ɯţ̣ΞģFʍi}ġ̰~ý]Gɶǿ}ĳτix]|]:pgyζƸſy9̐Ǫġ~ˢʹģĭ̟}`eġ̰z͹ʧΕƠ~Ƨọ̈̄=Ɯţ̣Ξģĳʭmgzϋ̣zkr:
LΩΪ=ƲĩM ˢʹģ9ę̵̠ɕ 13 mOĝ 7 m OǪi 3 m }ʯ9Ģ΍Ɗʡ̗Ųmgz}Ǥ:ˢʹ}Âà¼@¡yƱĺe9̭͒ˣ 25 cm ţŇyʵ˾kr̂~αǷkǰ9º@´Þ¡@Ϣ]x 1 ɐţ̟̰ġ̰˙̛Ǥur:p~ǌ9ġ̰˙̛Ǥur˽̫y˖χˢʹɧƂk9ˢʹ;χa̵ţŇ~ʍiyȣϵǽɂkr:ǽɂǌ9Ȏ~ˢʹʯɹfr:ʯɹfrˢʹ}ǻġ̰˙̛ː˾ʵ˾kr:g~ĂЄ~ˌȄƟΡkr:ǽɂkrˢʹϢ]x9ʧƖʟFͣϦΪź sFDA ΪGz˂ƖʟFEB ΪG˙̛kr:ġ̰˙̛~Ʋĩ9ʲΡk̓ǌ~ġ̰ƕĂy 65Q
̟̰y\urb 150 cm ɹ~ˊyɦ?}ġ̰ɹɭkr:̈́}ˢʹ~Ǽɹˊ(25~75 cm)yǼǪy 90QƗd}y˯kr:EB ΪyƧọ̈̄=Ɯţ̣|Ξģƺn9˂Ɩʟ 109 1010 cells/gyʚùkr:sFDA Ϊy9ʧƖʟ~Ɯţ̣ΖΆ}ý]ƺ|aurb9ͣϦΪy9Ģˊ(225 cm)~ʧƖʟɹˊ(25 cm)ˠ]ƢǔȮkr:r9sFDA ΪzͣϦΪ~ϴΩ}`]x9ˢʹģ͎ʟbƧĭm}ͩ]9ʧƖʟbǁɫmƢǔbƺr:ðɹ~Ʋĩa9ˢʹ~ġ̰bǪġ}úȪ
i˜ez9ʧƖʟbǁɫmgzbτa}|ur:

14

P25
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CMC %,7ÞÐ!3÷ß7Ē50℃ Üęî14 v7 CMC ÷ß ÷Ĕ
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4 v$16s rRNA cé¬öğqË7!541& Streptomyces Ô.&Bacillus Ô0' Chitinophaga Ô$Ô3£ ¥41% vÊ©3ÎÅÈý²/E][FuÈê7ã(3

P26
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©ÇY=LDX%Ęq&©jĒ$ä3o& Aspergillus Ô$3E][Fcé¬øġïbæè³uÈlg¬ ManR !dè³g¬ McmA $02ãÇ43!7¹9]=\ pH jë$y63è³g¬ PacC %Çk$ 3!.Ć1$ą & pH B@K\_@! ManR/McmA aÖæøÇ!%yĠ$qËĂăn A. nidulans &ďíý²E^Q<C$jë 7 ·%E][Fcé¬ø
3ĊÊv~' pacC órv$3ªÈ (pH 4.2)9]=\È (pH 8.2) iØ÷ß %41cé¬%ø7 qRT-PCR ă7ĒqËĊÊv &4%cé¬$.ªÈ½02.9]=\½$Ó/#è³x¨4pacC órv &"1%½$.
9]=\½ E^Q<Cďí¤%ĊÊvpacC órv %cé¬ø7 RNA-sequencing $02ćĕæ$qËnPacC aÖæ$ø3cé¬& ManR Çkcé¬!)*fà4& PacC  ManR %uÈ7Ç3!7±¡3pacC ór& manR %ø$h
7Đ#PacC ! ManR ě´$02Ç43cé¬$&üÆ%ÄåìċÒØcé¬z+42TLC þË1 pacC órv &E^Q<C«Ö3!Ć1!#`¼1PacC &E^Q<C%¶2 ,7pManR %uÈ7Ç3!143ą&ÊE_G%:NTBZ_Ñ¹{ÏæÄÃ¯%fw!64.%
3

15

P27
ǙöƋˈg||¼§½ɆȖfSWv¼ÀSsj~ ¼»Àg˦ċKƕʁ˭ɂBǫʁǗʩBȶȩĶưBɟȩɚƊĝʲɋ×ãțLϋ̣M
 °»ÜºÅ¼FAAG´àÄ¤ȶ|{~ʧˡΖȕz΄ĦˡƤʣmgzyƊ]ǿΫͅʢȮm®»Ý®ģǫΕ~zvy\b;Ϡ?|ʧˡ͉ͬě}`]xͻnʧʣi˫Ƚ̇ţˡy\<̈́};ɚİǧΦ~Ü¨@Üͧǧ}`]x AA ˠϹ}ʧʣigza;ɚİǧΦĤa~ AAͅʢĴͶ¬®¹ÓΤȪkx]gzbʚȈyc<Į?gy};ɚİǧΦ~ AA ˤʢűǟ}¥Ü´¶àFGSHGbɖϤ|ώŊȪvgzτa}kx`;šƾƣ GSH b̓ʳ AA »Ú·Êmgzy AA }ǿΫͅʢƬǁkx]zʚ̛kxcr 1)<ĂΩ;Ȇ͓;ɚİǧΦ}`]x
GSH ~ΖĳƧЅ}ǛɽʢúȪ~ʋr|˼ƺbΧǬi 2);AA ®»Ý®ˤʢűǟ}`]x GSH
˫Ƚ̇ţˡǣϲmͻϤbʧlxcr<pgy;ζƸſy GSH zp~˫ȽǛɽʢ~úȪ}Ťϝm
 DUG þ̬ȕƠ~ AA ˤʢűǟ}`eώŊ}v]xĳʭmgz}kr<LΩΪ=ƲĩM
 DUG þ̬ȕƠ;GSH a Cys-Gly ıkx Cys ȋʧm˫ȽƦ¨@¼mþ̬ȕƠy\gza;g~þ̬ȕƯ˞Ŏ dug1!;dug2!;dug3!~ AA ŗɇʢ}v]xȣƽǤur<p~Ʋĩ;dug2!Ŏb AA ŗɇʢȮkrgza;GSH a Cys-Gly ~ʧʣ}Ťϝkx] Dug2/3 b
AA ˤʢ}ɖϤ|ώŊȪvzǣ_<r;AA ®»Ý®ǿΫ͉~ GSH Ϲ̋kdǁɫiogza;AA ˤʢűǟ}`]x AA z̓ʳƲǫmgz}ͅʢĢfώŊȪvzǣ_
xcr 1)<kak; Cys-Gly  AA zƊǉ}Ʋǫmr 3);GSH ~|np~˫Ƚ̇ţˡ Cys-Gly̿Ϡ} AA ˤʢ}Ťϝmgzbʚ˙ir<1) AMB 97: 297 (2013);2) JBC 287: 4552 (2012);3) Alcohol Clin Exp Res 27: 1613 (2003).

P28
ǙöƋˈg||¼§ɆȖĠƃfSWv +/ 7 Ssj 3+7 g˦ċKǫƧ˝ìBɂɵȅ˱ʩBɵɼƛʋBȶȩĶưBɟȩɚƊGĝʲɋ×ãțïã˼ț˜H
Lϋ̣M°»ÜºÅ¼FAAG;´àÄ¤ȶ DNA |{zʧˡ͉y΄ĦˡƤʣmgzyͅʢȮmŭͧʢģňΕȶy\<AA ´Á@Ü˫Ƚĭ̟yͻnȋʧi a;Ü¨@Üͧǧ}`eɖϤ|˫Ƚ̇ţˡy\<g~gza;ɚİǧΦ AA }ˣmĤa~ˤʢűǟŃ̓kx]
~zǣ_<ζƸſy;´Á@Ü˫Ƚ}`]xɖϤ|rcȪvgzb˼x]Ü¨@Ü˰ʛˆǧˆFADHGz AA ˰ʛˆǧˆFALDG}̆ϋk;AA ˤʢűǟ}`e´Á@Ü˫ȽƦ~ώŊ}v]xƷ̹kr<
LΩΪ=ƲĩM ɚİǧΦ 5 v~ ADH ²«Óz 5 v~ ALD ²«ÓȪv<n ADH ~ȒɖƯ˞Ŏ~´Á@Ü` AA }ˣmŗɇʢşȈkrzg;adh2!adh3!adh4!adh5! ŎbύʧŎƊ]´Á@Ü` AA ˤʢ͖Ȯkr<v;Adh1p bϴ®»Ý®ˤʢ}`]xɖϤ|ώŊȪvgzbʚ˙ir<ĂΩ;ALD þ̬ȕƯ˞Ŏ~´Á@Ü` AA ˤʢ͖şȈkrzg;
ald6! ŎbǼƊ]ŗɇʢȮkr<r;ald6! Ŏ~ AA ɯƂ͖şȈkrzg;ald6! ŎύʧŎͣ˽̇~ AA ǁɫio͖ϻb̋kd̘Ģkx]r<i};¥Ü¨@®`¥Û°Þ@Üʧāȫ}`e ald6! Ŏ~ AA ŗɇʢşȈkrzg;¥Ü¨@®ʧā}ͱxÍà»@®Û
àȍƧЅFPPPGy˫Ƚi|]¥Û°Þ@Üʧāy;ald6! Ŏ AA }ˣkxƊ]ŗɇʢȮkr<ux;AA ˤʢűǟ}`]x Ald6p  AA ~ɯƂ~|n;PPP zƇ} NADPH ǻʧƦzkxɖϤ|ώŊĩrkx]gzbʚ˙i<

16

P29
¶¾¾FªƋˈ     gËǈÍɴƮʠŪőkgã˼yˠƱZ_
ȁ_eɖȲũgɕƥd  |~~µgʠŪʆȖgûȣKǆƧŐƬBȶȩĶưɟȩɚƊGĝʲɋãțïDã˼ț˜H
Lϋ̣M
 P. methanolica ;Ɗϻ|ͧǃϻȪvÔ´Á@ÜϘ̀ʢ MOD1 ÊÞÕ@´ϖmgza;p~͖ϻϫϢkrøɄþ̬ȕͧǃƦbĺͧi;ǙdϫϢix]<kak;ζǧΦͧǃě̶b
ø| 2 Ʉ~Ü¨@Ü¢¬µ@±FAODGþ̬ȕ MOD1 z MOD2 Ȫt;p~þ̬ȕȋΕa| AOD ²«ÓȪv<v;ϴ AOD þ̬ȕ~ͧǃ̈́ʢɶǿ}ĳʭk;p~͖ϻϖǓŋϢmgzy;ϖĎ|ʋųøɄþ̬ȕͧǃƦ~ěϢbů˥yc<ĂΩ;Ô¶Þ»Þ@ÈǧΦ~Ô´Á@ÜʧāǪƖˡɊϹbƺǰ|]gza;ʧā˕ʔav AOD þ̬ȕͧǃˈĽk|]ʋr|˴ˆǂ~ʾͨbżπy\<g~^|͠ƥa;ζƸſy P. methanolica AOD ²«
Ó~ͧǃ̈́ʢ~ɶǿĳʭk;i}ϴ AOD þ̬ȕͧǃ}Ǽ̤|ͣϦ˴ˆǂ~ʾͨϋ̣zkr<
LΩΪ=ƲĩM
 P. methanolica ~ʧā̈́ʢ;i} AOD ²«Ó~ͧǃˈĽk|]˴ˆǂ~ʾͨϋ̣};Ϡ?|˴ˆǂ} 1%Ô´Á@Ü̩Ħkrͣ˽Ȅʣk;p~ʧāz AOD ²«Ó~ͧǃ̈́ʢşȈkr<p~Ʋĩ;ÑàÁ@®;ÚÆÁ@®;°ÞÆ@®˴ˆǂzkrɻǫ;Ô´Á@Ü̩Ħkrͣ˽y;Ô´Á@Ü~zͱϏ 3 ͢}ƖˡɊϯbɹɭkr<rg~ AOD 
²«Ó~ͧǃŋʢʹʄΪ}xşȈkrzg;Mod1p ` Mod2p ~ͧǃbƺr<r;
AOD ²«Ó~Ϙ̀ȫţͱņkrzg;Mod1p ʧāɠů}Ϙ̀i;̀x Mod2p ~Ϙ̀bƺr<g~gza;ÑàÁ@®˴ˆǂzkxϫϢmgzyǪǓϯ|ϖϢ´àÄ¤ȶʧȋbů˥i;Ô´Á@Ü͕̰Ϙ̀ȫţʡǍmgzy 2 v~ AOD ÊÞÕ@´ŋϢkrʋųøɄþ̬ȕͧǃƦ~ěϢů˥yczʚ˙i<P30
Simple, Rapid and Simultaneous Determination of Sugar and Ethanol Contents in Fermentation Medium!Vioni Derosya, Naoto Isono, Akira Yamaoka, Ken-ichiro Suehara, Takaharu Kameoka, Atsushi
Hashimoto（Graduate School of Bioresources, Mie University）
LPurposeM
Low utilization of sago starch (Metroxylon sagu) as food source gives a possibility to use it forbioethanol production. Considering waste reduction, both starch and fiber of sago should be usedin ethanol fermentation with glucose and xylose as the hydrolysis products. To accurately control
the fermentation process, we tried to develop a simple, rapid and simultaneous determination of
the sugar and ethanol contents in the medium based on the infrared spectroscopic information.
LExperimental Methods & ResultsM
The fermentation media containing different concentrations of glucose, xylose and ethanol inyeast nitrogen based (YNB) media were prepared for the infrared spectroscopic measurements.
The spectra were collected using a Fourier transform infrared (FT-IR) spectrometer equipped withan attenuated total reflectance (ATR) accessory in the range from 4000 to 400 cm-1.
The strongest peaks characterizing glucose, xylose and ethanol in YNB media were observed at
1036, 1050 and 1046 cm-1, respectively. All calibration curves for each metabolite at thesewavenumbers presented excellent linearities. Moreover, the intercepts of the calibration curves,which may theoretically show the absorbance of the medium without metabolite, were almost
same as the experimental values. The metabolite contents in the mixture media were
simultaneously calculated under the assumption that the spectral additivities could be satisfied.
Consequently, the good agreements were observed between the actual and calculated values. This
study could contribute the simple, rapid and simultaneous monitoring of the metabolites duringbioethanol fermentation.

17

P31
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 svPLA2 ~Ģϱ}ǜϪͥÛ³¶@Ó˱ǺǜˡЄƲkrͧǃÊÚ®Ò¼ǟ́k9pET ¬®¹ÓϢ]xͧǃior:ͣϦġ̰9Ϙ̀ȫţ9ĿɄ¬ÖÍÞà~ϫϢ̵}ŤkxͧǃɾƵƷ̹krƲĩ9Skp ¬ÖÍÞàzƇͧǃmgzyϛǫ´àÄ¤ȶ~ͣϦɹʦ̇~ΖͲbr:Ȭ}ͧǃkrϛǫ´àÄ¤ͣϦɹʦaʩʪkrzg9100 mL ~ÈÚ®¨ͣϦa 0.1 mg ~ɊϹyʩʪ´àÄ¤b̓9ǜˡŋʢń͑kr:i}9ʩʪ PLA2-scFv z PLA2 ~ͧʄũȶϢ]gzyǜƿy\Û³¶@Ó~ ELISA Ʒɚbħ͖y\ur:P32°ª{¼~£F¼ƎȗČȖy˰[v°°»¦F  g   ʆËȖgŷǳKƑĚȧɚAȤɵśAɵɟǓǛAɟˣǓ˵AĚƣ˵ů ˛ɋ×Dț˜ʖDț˜ĪǚLϋ̣MÎ®È¶­ÜÁ¬»@ÜȠȶ˫ȽĶˀȄϢ|{b͑ű͖ʢÛàȠȶy\:ζƸſȳy9ƾϩ  ǫʣŋʢȪr|]ΨʻƖϗϩÎ®ÎÛÄ@± ´àÄ¤ǘṇ̌}ĶΞmgzy9̿ŋʢ΄ϝmȧ}ʣǒkr:kak9ĶΞƣ  }  ~ǫʣͬěy9ǧ
ˆ~ Á¬»@Ü}ˣmñ˾̈́øʢbŒ˂y|]r̧ˁƣ~  }Ħ_x9p~ñ˾øʢˡy\  ǫʣixk^z]^ό˭b\ur:pgyζƸſy9  ~Ǫɝ̰ǫʣϋ̣
zkx9 ǫʣƣ  ĶΞmgzyi|ñ˾̈́øʢ~ǔɹȣr:
LΩΪ=ƲĩMǃϖmĶΞƣ  ~^t9$ # $  $$  Ξøˡ  ϕʸ̣}ʧʣk9p~   ͱϏ  y\:$ ~Á¬»@ÜƲǫÐ¦·»ǟʣm   a   ~Ȏũa|Ü@Êǟ˔}̆ϋk9pq~Ȏũ˟~  ÒÁȍ}˾řkrΞø  þ̬ȕȄʪkr:ȄʪkrΞøþ̬ȕˬ̎Ɩyͧǃio9̓r˅  Ϣ]x  ǫʣͬěǤ]9ʧʣm  ~øʢˡˇʣΖʭkr:p ~ Ʋ ĩ 9   `      ~ Ξ ø b ʾ ˮ ʢ ǔ ɹ } ϖ Ǔ
y \  9̈́ }  " Ξ ø ˡ y    ͱ b Ϗ   ̟ ̰ } ǔ ɹ k r :ǃ Ȃ ;  ñ z  ñ Ξ ø ˡ ~ ̇ a  ̈́ ø ʢ ~ ϕ  r Ʉ Ͽ n v  ʾ ˮ k 9p    ŉ e ǫ  o r ͍ ɖ Ξøˡ~Ȅʪzñ˾ʾˮʢ~ͿĥǤux]:

18

P33
Uniform Emulsion for Biochemical Applications Using Inexpensive Flow-Focusing Device!Marsel MURZABAEV1, Takaaki KOJIMA1, Isao KOBAYASHI2, Hideo NAKANO1
1( Dept. Bioeng., Grad. Sch. Bioagr. Sci., Nagoya Univ., 2Food Engineering Div., National Food ResearchInst.)[Purpose]
Carrying out biochemical reactions such as PCR or cell-free protein synthesis in water-in-oil
emulsion allows high-throughput screening of large libraries of biomolecules, where each droplet
functions as an independent reactor. But droplets generated by bulk emulsification greatly vary insize, which leads to uneven distribution of reagents and causes difficulties in statistical analyses.
Microfluidic devices can generate monodisperse emulsion but are expensive and require special
equipment. We are working on development of inexpensive device which can generatemonodisperse emulsion from biological samples.
[Methods and results]
We could make a relatively simple flow-focusing device which can produce monodisperse
emulsion. It consists of two syringes driven by syringe pumps, standard fittings and tubes and a
specially fabricated nozzle. This nozzle is made of a larger glass capillary for oil phase, smaller
steel needle for water phase and a thin insulin needle for emulsification connected to the exit
capillary. Using this device we could produce emulsion droplets that have an average diameter of
76,4 "m with coefficient of variation of 3,7%, which can be considered as monodisperse. Averagevolume was 0,000235 "l or 0,235 nl, which means 100 "l sample can be partitioned into around
425 500 compartments. We used PCR buffer with magnetic beads as a water phase, so this device
can be used for emulsion PCR.

P34
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ȐƧȠ BɟƓdsƮ BɟƓƻ B˥ʴʯ˵   BŅĘīŐ   G ĝʲɋ×ãțB ĝʲɋãțB ńģɋ˧B ĝʲɋ×̓ʖB
˛ūæɋ×ț˜ʖBGĎH˝ƻDŠĳ˗ʴB ɥƣɋ×ÎƷ˧ȸƎHLϋ̣M Ɓ͏ϗϩ´àÄ¤ȶÚ¤»ÈÛàF28 kDa;ðĢ LP28G` 18 kDa ÈÚ¥Ôà»FðĢ LP18G
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bǿΫ͉}ɂǰgzbͻʗy\aƷɵmgzϋ̣zm<LΩΪ=ƲĩM
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r<p~Ʋĩ;SDS  LP ~ŗʹˈĽ͖}ˣkxĈƐv~~;native-PAGE }ʩʪy;ªàÊÜbζϩvŗʹˈĽ͖ΤȪmgzbaur<ǃȂ;native-PAGE } N ƣ̷ǺɧƂ
LP18 ~ʩʪ`ŗʹˈĽȣƽȣx]<g~Ʋĩa;LP18 bŗʹˈĽȄϢȮmr}
N ƣ̷ǺıkxǿΫ}ʈ͐mgzbͻʗy\~a{^abτa}|s^<

19

P35

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Û©̷ DFAIFº@È@ßàGbʧȋi:DFAI ǹ̷~ 50R̟̰~śλϖk9̘ÞÛ@śλϵ|{zkx~ϫϢbů˥i:ǱĴʫʌƹ͉FǚʿȖGaǽɂkṟɼȣϵa¿ÛàȤģmǿƖ H10-2 ŎΖϭkr:ζƖŎͣϦɹʦ̇}¿Ûàa DFAI Û©̷ʧʣmǧˆʧȋmgzbͪτkr:ζƖŎ}v]xΖϿ=̛̿}ŤmƷ̹Ǥ^zz}9ζǧˆʩʪkǧˆṇ̌|ɣ̈́ʢĳτmgzϋ̣zkxƷ̹Ǥur:LΩΪ=ƲĩM
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98.6R~ÎÕÞ­@Ȯkr:kak9ΖȕƦ̸ĳʭ~Ʋĩ Arthrobacter ~Ů˼~Ʉzø|9ɄÝÌÜy͆ϰkrΖȕƦ̸ñ˾Ȯkr:g~Ʋĩa9ζƖŎ Arthrobacter sp. H10-2 z̛̿ir:ζƖŎbͣϦɹʦ}ʧȋm DFAI Û©̷ǫʣǧˆ[EC4.2.2.17]àǏř¤ÞÑ»9˃ʛ¤ÞÑ»|{}ux̭Ųċ̣̾}ƕĂ}ʩʪkr:ʩʪ~ĭ̟yͱŋʢϏ 35 ͢}ɹɭk9ɊϯϏ 17Ryʩʪǧˆ̓gzbycr:ζǧˆ~ͬěȡ̤ pH  5.59ͬěȡ̤ġ̰ 40Qy\ur:ˤ͒ʢ}v]xƷ̹mz 75Q930 Ζyî̛z]^Ʋĩb̓r:ζǧˆ~ΖȕϹ
SDS-PAGE yʚ̛mz 39kDa z]^ĥb̓r:

P36ŝĢȖƞŃgțƨ[väǼǼȲ&"d¼ºFČȖfė[vŠĳKÉĐȅĥƮB˟ȱ̤ɂ̗A˟ɀɮˢBÉĐ̘̗GƦǖɋ×țƶHLϋ̣M ƶŲʢǿƖ~̇}ͣϦ̇} YAS ʧȋmƖb]:
     ΃ϡʢ°ÜÞ@®ũȶzkxͣϦkrȀ9˒ʂ̇} $! ʧȋm:ζƸſy9   ~ʧȋm $! ~°ÜÚ@±ŋʢ}źmǓĩΧǬm:
LΩΪ=ƲĩM   "   Ŏ΃ϡʢ°ÜÞ@®È½°ÜũȶzkxͣϦkƖˡĴɊk9g
}°»à̩Ħkx $! ̈ɚkr:̈ɚkr $! È½°Ü}Ż̆io9gǧˆͬě~ũȶzkr:$! Ż̆iorÈ½°Üũȶzkrɻǫ9   ~̧ˁ°ÜÞ²@Ó9   ~°ÜÚ@±y\ ).9).9&, ~ŋʢ9$! Ż̆kx]|]È½°Üũȶzkrȫ}ͱx9pq  ͢9  ͢9 ͢9  ͢z˒Ħkr:r ).9).9&,
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20

P37
·¼țʸdZbg¶fSWvÀ¦ƿñľêƋȲgțêĈɬŠĳ   Kǥò̌ɂAȑǬ˛ɧĤAĬɵˡôAƍɵˬĥAɷ˂˭ƮAɑȩÞǍAȈŢȚ̊G˛ɋ×ȵ˧ïĈHLϋ̣M
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̣ĳʭǤur:LΩΪ=ƲĩM þ̬ȕƷȇ}9Ôµ }´àÄ¤ȶĪƋģǧˆbYɄϿ˝Ȃmgzƺɚkr:pq
}v]x˂͡Ђư̛kr:ǧˆ~ĂȬǟ˔a9Å»~²«Ó}ˣěmÜ²Þ¥Fˎ̿
þ̬ȕGǧˆ 5 Ʉư̛kr:r{~²«Ó}ľ̴k|]92 Ʉ~Ü²Þ¥ƺɚkr:p}v]x9ˬ̎Ɩkdǳ̊ǿΫͧǃƦ}ux9ˇř_˷ͥȶ̓gzbycrr9ʧģṇ̌|ĳʭǤur:i}XɄ~ˇř_´àÄ¤}v]xÕÁ¤Þ@½ÜǜˡȄʪkr:ύʧƣÔµ ˇʃʲΟzÕÁ¤Þ@½ÜǜˡϢ]x9Ŀǧˆ~ˇʃΖΆτa}kr:ǃȂ
TALEN Ϊkd9TILLING ΪzǈɃΪ}ux9þ̬ȕƯȲΞøˡ~ȄʪǤux`9ζǧˆŋʢb̘Ģ=Ư˞krÔµ ~;ǃƣĳʭkx]:P38À¦ƿñľêƋȲgțɃʌȳǻʹʰgûȣKɋľȀɂ̗AÄʴʋɹʩAɑȩÞǍAȈŢȚ̊G˛ɋ×ȵ˧ïĈHL͠ƥ=ϋ̣M´àÄ¤ȶĪƋģǧˆ»Úà®¥Ü´Ò½@±FðĢ TGaseG9´àÄ¤ȶ̇~¥Ü´ÒàȎũzÛ­àȎũţ Ü¬Óò˝̣}ĪƋģmǧˆÈÒÛ@y\:TGase Њ͏Ͽy 8 Ʉ~
²«Ób˝Ȃkx`9Ϡ?|ˇʃÿ́ø̣}ͧǃkx9ƳČƑǉͳΊƤʣ|{~ʧϬ̣ώŊĩrkx]b9g~²«Ó~ɶǿ|ˇʃΖΆ}v]x9μsτa}|ux]|]̫bˠ]:ζƸſyÑ®~˂ʒʲΟϢ]x TGase ~ƓȂ΍ñωϨ̣}Ʒɚk9ũ˄̣|º@´̓gzϋ̣zm:˂ʒʲΟ~ŋʢħȢģφčʹʄy²«Ómx˘_gzǃɿǲ͌y\r9n9²«Óhz~ mRNA ÝÌÜy~˝ȂÄ´@à}v]x
RT-PCR, Real- Time PCR, in situ hybridization Ϣ]xĳʭǤur:ɠ}ɁϤ|²«Óy\ TGase1 , TGase2 }v]x~ͿĥǤur:LΩΪ=ƲĩM
RT-PCR }uxÑ®~ 21 ɄϿ~ˇʃ}`e mRNA ÝÌÜ~ΖΆτa}kr:r9
Real-Time PCR }ux TGase1 bϋ9ͳΊ9ʉ˓9В˓9r9TGase2 bϋ9ʉ˓9ʖ˓9Ş˓}`]xǪ]ͧǃϹȮmgzτa}kr:In situ Hybridization Ϣ]xÑ®ʋʧȩ~˂ˇʃʲΟ}`e TGase1, TGase2 ~ mRNA ÝÌÜy~ƓȂΖΆħȢģmgz}ʣǒkr:Ʋĩ9
TGase1 В˓9ƍʽ9ͳΊ9Ş˓}`]xͧǃbƺ9TGase2 g~΍ñ}Ħ_xʰ̕9û9ʖ˓}`]xͧǃb͑r:Ǳǌ9˟~²«Ó}`]x9̛Ϲ̣=ωϨ̣|ĳʭɞȬʔx]d:21

P39ƍʢãȖěƿ¯¬y˼Q_ȊȻfSWvÀ¦ƿñľêƋȲgČȖŞǙKūȩśɂ̗AƧƣ̅ƖAƧƔʩǍAɑȩÞǍAȈŢȚ̊G˛ɋ×ȵ˧ïĈHLϋ̣M ´àÄ¤ȶĪƋģǧˆy\»Úà®¥Ü´Ò½@±FðĢ TGaseG9Ǫ̵̾Ε}`eʧσű͖}ˣkxˠϠ|ώŊĩrk9´àÄ¤ȶ̇~̛̈́~¥Ü´ÒàȎũz´àÄ¤ȶ̇~Û­àȎũ~ţ}²ÍÊ¶¼ƲǫƤʣmǧˆy\:TGase ̛̈́~¥Ü´ÒàȎũ͑ȱkxͬě̇ţˡƤʣmr9¥Ü´ÒàȎũz~ͬěϠȰbɖϤz|:Å»y 8 ɄϿ~²«Ób˝Ȃmb9ͬěmũȶ~¥Ü´ÒàȎũ~ɋó~ǟ˔ø|9̈́øʢbʧl:Į?Ŀ TGase}ˣm912 Ȏũa|Ǫͬěʢũȶ͡Ђ(¥Ü´ÒàȎũ˖~ũȶ)ÚàµÓÍÊ¶¼ÚÉÚÛa®¤Û@¾à¥kxɂ̓kr: ζƸſyg~ǪͬěʢũȶÍÊ¶¼Ϣ]x9ʖ˓ť z{~ˇʃ}˝Ȃm TG2 z9ʖ˓}ƓȂmbʧϬṇ̌õŷb΃τ|ʋų²«Óy\ TG7 ~ϴȼ}v]x9Û­àȎũ˖~ũȶ˳ȇϋ̣zkr:
LΩΪ=ƲĩM
 TG29TG7 pq}ˣmǪͬěʢũȶÍÊ¶¼FÆ¶àͽȱGÑ®ʖ˓̈ɚČzͬěior:͉Ȃʢ TGase }uxͽȱũȶÍÊ¶¼zĪƋirÛ­àȎũ˖~ũȶ´àÄ¤ȶ~ͬěȋΕ}v]x͍Ȭƾ̭Ųċ̾Ǥ]9Æ­àĭȍģǧˆyƷɚkr:p~Ʋĩ9τa}ϴȼyø|ĪƋȋΕb̓9pq~ǧˆ}̈́ø̣|ũȶbƷɚix]gzƺɚkr:ĂΩy9ͽȱũȶzĪƋirÛ­à˖~ũȶ̓r}9Æ­à§Ü}ʩʪǤurzg9pq}ˣkẍ́ø̣|ĪƋͬěȋΕb̓r:ǃȂ9ȶϹΖʭΪ}uxp~ũȶ~̛̿ȣx]
: Kuramoto and Yamasaki et al. Arch. Biochem. Biophys, (2013)P40
«ºª}¹y˼Q_ DNA ʷȞªFgíƵêKɽƧŏ̆Ʈ BŊɒśư B|Á¸¼ŏ̆Ʈ BǠˋȇ BɵĘƅ BâɀƐǝ GƦǖɋ×țƶD ț˜ĠʕB ȌŜțʸH
Lϋ̣MʎłʧΕ~ DNA ΒʪǿΫɋů~ S ů}ŵg9ΒʪÈ@¤ΒʪĺȔ̫ (ori) aϴΩǔ}ʔǤm:̑ˬ|ʹʄˡ DNA ;\̟̰zurϺĀhz} S ů~̛̈́~´Òà¥yΒʪmΒʪ¼ÔàƤʣkx`9§ÁÓ~î̛ʢΤux]zǣ_x]:kak9Аͧʧ~ĭ̟yƤʣ=ńϰizǣ_JΒʪ´Òà¥¼ÔàKb]v{~^|ʡǍ~z}Ƥ
ʣi~a}v]x~ɶǿΖaux]|]:g~ǃɷτa}mr}9±ÉÚÈ·¬Ø}̉ϋkr:±ÉÚÈ·¬Ø9ɇʩϪa~ͧʧĭ̟b̻τ|ˡ̗kxşȈycɹ9ͧʧ˚̰b˚dɇʩǌϏ 72 ȫţyɁϤ˓ŨbŒʣm:r9ʰ̾̕Εy\r9ɁϤ˓Ũ=ˇʃ~ͧʧ=ǟ˔bÅ»zdȨx`9Ƹſ~ÕºÜʧΕzkxǙdϢ]x]:ǱĴ9DNA Βʪ}Ťk
xº@´~ɫ|]±ÉÚÈ·¬Ø}v]x9nˡǿΫϗϩ~ͣϦǿΫϢ]x DNA ΒʪÈ@¤~ħȢģǤ]9Å»ͣϦǿΫzͱņmgz9i}9±ÉÚÈ·¬ØɠůА}v]xΒʪͽȱΪ̵Ʒ̹mgzϋ̣zkr:
LΩΪ=ƲĩM
ͣϦǿΫzkx AB.9 ǿΫ(±ÉÚÈ·¬Ø͸ÅÝϗϩǿΫŎ)Ϣ]9nǿΫ~˒ʂ˚̰~˙
̛9ÈÞ@ª»Ô»Û@}ǿΫɋů~ĳʭȣr:DNA Βʪ΍ñ~ħȢģ}9Ζȕ¨@Òà¥Ϊz]^9DNA ΖȕĂΖȕ˱ñyƞɿ}ƕĂ|ʇyƼ͹ƏϢ Ã@¡Ú®}̌΄eΩΪϢ]r:ǿΫÂÞ§àģ¿¤Ý¬¼yΒʪͽȱk9DNA ÈÃ@ɹ}Βʪ΍ñƪǑƷ
ɚ=Ƽ͹ƏÔ@­à¥kx9Βʪ΍ñ~̑i˙̛mgzy9DNA ΒʪÈ@¤~ʔǤ˚̰ĳʭȣr:ĂΩ9±ÉÚÈ·¬ØɠůА}v]xΒʪͽȱΪƷ̹k9Аͧʧȫ}`e DNAΒʪ΍ñ DNA ÈÃ@ΪyħȢģmgz}ʣǒkr:

22

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Ȅþʨțʸ     ˱́  À¡½|Ffė[vŠĳKƕĮĖÓȤ Bưɵʭʮ G ǿɋ×̆ŻĈőã˼țʸH
Lϋ̣M
DNA ˞ɨ~^tłȍĘũb˰ϭkr̷ÛàȍǯŁ~΍Ζ AP ΍ñ(apurinic/apyrimidinic site)zǈ9g~ AP ΍ñ͑ȱkxϾʳmÎ®Î­®¹ÜƲǫʲ˹m DNA ɌΏǧˆ AP à¼¿¤Ý@±z]^:Ǳy}9ƿłʧΕaǪ̵ʎłʧΕyϠ?|ʧΕ~ AP à¼¿¤Ý@±}v]xƸſbixcr:kak9ʍķ͹ʧΕ~ AP à¼¿¤Ý@±}Ťkx]sƸſix]|]:ζƸſyʍķz]^̐Ǫí9̘ġĢy̽]x]zȗʍķ͹ʧΕ Shewanella
violacea ϗϩ AP à¼¿¤Ý@±(SviExo)~̈́ʢ̏r:LΩΪ=ƲĩM
 SviExo ͧǃmÊÚ®Ò¼ǟ́k9ˬ̎ƖyˬϹͧǃio9His ´¥È¾¹@¤ÞÑ»¥ÚÈ@9ϥàǏř¤ÞÑ»¥ÚÈ@yʩʪkr:ǫʣ DNA Ϣ]x SviExo ~ǧˆŋʢ̏rzg9AP à¼¿¤Ý@±z 3’T5’¢²¿¤Ý@±~ŋʢȪux]gzbΖau
r:3’#5’¢²¿¤Ý@±ŋʢˬ̎Ɩ Exoé~pƊaur:SviExo ~ȡ̤ͬěɾƵ
34Q9250 mM NaCl9pH 9 y\ur:ǧˆŋʢ934QǪġ˖yżƮ}̘Ģk9̘ġ}|}ɔuxŝa}̘ĢmgzbΖaur:g~gz9a|~̘ġĀyŋʢΤȪkx]~}ͬkx9ȡ̤ġ̰ðɹyΞʢbŵgm]gzbʚ˙ir:SviExo ~͒î̛ʢ̏
rzg9˟~ʧΕɄ~ AP à¼¿¤Ý@±zͱņk9͒î̛ʢbͷɽ}̘]gzbΖaur:
ǃȂ9SviExo ΞøˡȄʪk9͒î̛ʢb̘]ƿĄ}v]xɶǿ|Ƹſʔx]zgy\:P42­~±¼TtŢaT`_ȁĦ¼ªºF|~ªFµd]ĝSsj̦gʠųkgė˷KʿǟĈBūľ˙ʋBƕȩȇůBɋǵ˳ÓG˛ɋ×ț˜ʖHLϋ̣M Î´Ü, ʣ̊~ͧǑŨsey|d, ϪДͧǑmgzbǇda˼x]. Д~ͧǑ}v]x, Д~ˡb̻ͫτy\r}, Д~̇yƤʣirʣ̊ƣͧǑŨ~Ǒb̻ex]
~szǣ_x]r. Î´Ü~ͧǑ9ͧǑũȶÜ¬ÈÛàzͧǑǧˆÜ¬ÈÚ@±, ATP,Mg2+}uxącŵgi. Ë¦Ï´Ü Luciola lateralis }9Ğϼʄ~ͧǑʄȮmÜ¬È
Ú@±bƺvaux`, Ϟ̊`ʣ̊~ͧǑŨ~ͧǑ}Ȑx]gzbΖaux]. kak, ~şȈ}, ϪzДϼʄ~ͧǑkx]gzba, Ů˼Ü¬ÈÚ@±z
Ν~Ü¬ÈÚ@±~ŤϝbȮǶir. ζƸſy, Ë¦Ï´ÜϢ]x, ϪzД~ͧǑ}ŤĄȕτa}mgzϋ̣zm.
LΩΪ=ƲĩM Ë¦Ï´Üa, gy}˼x]rÜ¬ÈÚ@±(LlLuc1)zø|Ü¬ÈÚ@±þ̬ȕ LlLuc2 ̛̿kr. Ȭ}, ÛÜ´Ó PCR Ϣ]x 2 v~Ü¬ÈÚ@±þ̬ȕ~Ŀʣ̑˺Ļy~ͧǃÄ´@àĳʭkr. i}, in vitro z invivo yͧǑ®Í¤»Üͱņkr. g~Ʋĩa, Ϫy LlLuc2 bͧǃkx]gzbτa}|ur. r, Дy LlLuc1 z LlLuc2 ~ϴþ̬ȕbͧǃkx`, ʣ̊ƣͧǑŨa~Ğϼʄ~Ǒ
LlLuc1 }ϗϩk, ˡ˂ˡ~zkrϼʄ~Ǒ LlLuc2 }ϗϩmgzbτaz|ur.

23

P43
°¼¼ª»ÀgțƎȗŒ̕fė[vŠĳKĀƄǓȘBĭɵǦțBɋǵ˳ÓG˛ɋ×ț˜ʖH
Lϋ̣M ζƸſ;Î´Ü~ͧǑũȶy\Î´ÜÜ¬ÈÛà~ʧǫʣ˱ñτa}mgzϋ̣zm<Î´Ü~ͧǑǧˆFÜ¬ÈÚ@±G~þ̬ȕmy}ĳτix`;ÃÔ@­à¥ŶɛzkxʧσĨňΖύyǙdϫϢix]<p~ĂΩy;Î´ÜÜ¬ÈÛà~ʧǫʣƧЅμsτay|]<;p~ʧǫʣǧˆτa}mgzyʋk]Ô@­à¥Ŷɛ}v|bzǣ_;ʧǫʣƧЅ~ĳτǤux]<LΩΪ=ƲĩM Î´ÜÜ¬ÈÛà~ʧǫʣ˱ñτa}mr};Ϝˋiʧǫʣũȶ~î̛̿ñˡͽȱˡË¦Ï´Üʣ̊}̉ȹk;24 ȫţǌ}̉ȹǆˡ~Î´ÜÜ¬ÈÛà LC/ESI-TOF-MS Ϣ
]xΖʭkr<p~Ʋĩ;Î´ÜÜ¬ÈÛàb 2 Ζȕ~ L-¬®¹àz 1 Ζȕ~Ìà³¢Áà\]Â¼Þ¢Áàaʧǫʣigzbτaz|ur<rÎ´Üˡ͉};Â¼Þ¢Áà~a}ÜÉ¶àFÂ¼Þ¢Áà~̷͡ˡGb˝Ȃmgzbτaz|ur<g~gz;ͅʢ~Ɗ]Â¼Þ¢Áàϙϭ~ɿ˦y|d;̷͡ˡzkxÎ´ÜbΤϖkx]gzȮǶm<

P44¶Àțȗ|F| Methanosarcina acetivorans ˱́ȁĜ¬½ ¼½FgĠʕûȣKǥȩɎƚAÒʴŌ˲AȃśAĭɀɯA̧ŢĮG˛ɋ×ț˜ʖHLϋ̣M@¢}ǟ˔̣}̈́ø|²ÊÝÁ¼ģǫΕbʟˠdƺɚi:@¢~θȠ
ȶ~˴ģʛˆǺ²ÊÝÁ¼y\b9kkšƾʛȍģ9Ζȕ͉Śģ9͍Ϲģz]urɌʀɇex]:r9ΒʟɄ~@¢aǈŻǺ¢Áàp~Ͽĕˡ9̷¢ÖÛȠȶb˱ϭi
9ˠϠ|ϠȰy²ÊÝÁ¼Ǻb΍ΖέЈkx]gzbΧǬix]:kak9p~ʧǫʣƧЅ~ z{΃Ζτy\:ǃȂ9Į?ˬ̎ƖɗɁzkr@¢ƣθȠȶʧȋƦϢ
]x9μ̛̿~θȠȶʧǫʣǧˆ˳ȇkx]:ζͧ;y9p~ĭ̟yͧƺkrʋŪÊÝ¾ÜÝ
µ¤´@±}v]xΧǬm:LΩΪzƲĩMM. acetivorans ~§ÁÓ}¨@¼i Þ¹Á¼ʧǫʣǧˆ~ÎÕÞ¥þ̬ȕ}̉ϋkr:̿þ̬ȕ@¢ƣθȠȶʧȋˬ̎ƖŎ}̀͐k9Ƞȶ̈ɚΕ LC-MSn Ζʭ}ƅk
r:p~Ʋĩ9²ÊÝÁ¼Ǻzkx9͍ɖƲǫbV ɡšƾir§Ú¾Ü§Ú¾Üũϖm@¢ƣθȠȶ~ʧȋbȮǶir:pgy9ˇř_ǧˆɂ̓kẍ́ʢͿĥǤur:̿ǧˆ§Ú¾Ü§Ú¾ÜũvģǫΕ}̈́ø̣y\9ʧʣΕ~ GC-MS ĳʭ~Ʋĩa9šƾ΍ñ $ι˶~͍ɖƲǫ 1 ɡ~y\gzbȮir:gy}̛̿ir@¢~̷¢ÖÛȠȶ
9$-ι˶˖~ 1 v|]kΒʟ~͍ɖƲǫbέЈkr¼Û¨@Üy\:r9M. acetivorans }`]xθȠȶ~²ÊÝÁ¼Ǻ~Œ˂έЈʅͤmǧˆ9§Ú¾Ü§Ú¾ÜÝµ¤´@±Į?bŮ}̛̿kx]:ðɹ~gz9ǱĴƺɚiršƾǧˆbθȠȶ̷¢ÖÛȠȶ~ʧǫʣ}Ťϝmħ͖ʢșȪkx`9g~̫}ŤmΖʭƲĩΘoxΧǬkr]:

24

P45
ƳǸŃ  fSWvÀ¬ÀʹûƋȲÍɴƮŋgɳǂČȖêÖƮ 4?! gʦ˴ʄǷŚécgƞˎƿŁơ˹ƽgûȣKćśɂ B˕ɵɚ˳ Bâɀ˳ĥ BưȌŦĵ Bǥ̏ɮʮ Bìʁøƭ  ˛ǴɋĈ×DʖB ˛ɋ×Dț˜ʖLϋ̣M Aspergillus ˛~ºàÊàΖĳǧˆƠ~ʧȋ9̪ȸŋʢģĄȕ AmyR }uxʡǍi:
A. nidulans }`]x AmyR ʧϬ̣Ϙ̀Εȶy\²ÑÜ»@® (IM) b˝Ȃmz9ǿΫȶał͉ùǤk9ÒÚ@±þ̬ȕ~ÊÞÕ@´ϺĀ}ƲǫmgzyÒÚ@±þ̬ȕ~ͧǃϘ̀kx]gzbΧǬix]:kak|b9ͷϘ̀ɾƵĢ}`]x AmyR bǿΫȶy{~^}ǿΫȶ}ƓȂģkx]a}v]xɓΖĳʭix]|]:ζƸſy9GFP ϛǫkr AmyR 
A. nidulans yͧǃio9Ƈɰ̫Ý@«@Ƽ͹ƏϢ]x9ͷϘ̀ɾƵĢ}`e GFP-AmyR ~ƓȂɶǿ}ĳʭmgzϋ̣zkr:
LΩΪ=ƲĩM GFP-AmyR ͧǃm A. nidulans SGZ5 ¥Ü¨@®˴ˆǂzmͣ˽yͣϦǌ9Ƈɰ̫Ý@«@Ƽ͹Ə(ZEISS9LSM700 ƣ) ~´ÓÚÊ®ű͖Ϣ]x9GFP-AmyR ~ƓȂϠȰƧọ̈̄}ĳʭ
krzg9ǿΫȶ}ƕȶ}ƓȂm~y|d9ʼúɿ~ǟ˔ˡ}΄̆k9av9ƖˡΩǔ}Ēu
xʊ̾m^}ƓȂkx]r:i}9Rhodamine phalloidin Ϣ]xǿΫǯŁ´àÄ¤ȶʹʄkrzg9GFP-AmyR ~ƓȂ΍ñzĂ˿kx]r:ðɹ~Ʋĩ9AmyR bǿΫȶy¤¶àÈÚÔà}΄̆k9ƓȂkx]zǣ_r:i}9¤¶àÈÚÔà»~˶}Ʋǫk9ɖǫˈĽm¤¶àɖǫˈĽȁ~ª» Ú¬ D (CD) ̩Ħkrzg9GFP-AmyR ~ł͉ùǤbşȈir:r9¤¶àÈÚÔà»ʲ˹k9˱Ϲˡ¤¶àzƲǫkxgŇϭk9¤¶à˰ɖǫio¤¶àɖǫˈĽȁ~Ò ÞÚ¼ B (MB) ̩Ħkrzg9̿Ϡ} GFP-AmyR~ł͉ùǤbşȈir:g~gza AmyR z¤¶àÈÚÔà»bˎǋȄϢk9ǿΫȶyƓȂkx]gzbȮǶir:

P46
ɔƮŃ Coprinopsis cinerea g¬¾FµûȣfsvƮǀɃŎȗĠƃgŠĳKȷɼʋá BƧɵɘëLBưȌŦĵ  Bìʁøƭ  G ˛ǴɋʖB ˛Ǵɋ×ʖHLϋ̣M ˲ȕƖ9Ǒɱȹġ̰|{ŚƉΞģ}ělxƖȞˡaȕȷˡ~Ƥ˦ΞģǤ^:g~r9˲ȕƖǿΫΖģ`Ƥ˦Ƥʣűǟĳτm}\r9ƔxƎλʍ]´@§·»z|zǣ_r:kak|b9ȕȷˡƤʣűǟ}v]xŒ˂}τaz|ux]|]:˲ȕƖ Coprinopsis
cinerea 9§ÁÓÊÞ­¤»bŒϳkx`9˵ůţyȕȷˡbƤʣigza˲ȕƖϿ~ÕºÜʧΕzkxϢ]x]:ζƸſy9ÊÞ¹@ÓĳʭΪϫϢmgzy9C. cinerea ~ȕȷˡ}̈́ø̣}ͧǃm´àÄ¤ȶ˳ȇmgzϋ̣zkx]:LΩΪ=ƲĩM
 C. cinerea  Yeast Malt Glucose ͣ˽}ʳɄǌ 3 ͎ţ (30 °C) ïɡĢ}xͣϦkr:ŮΧ}ɔ] 4 ͎ϋa 12 ȫţ~τïª¤ÜyͣϦmgzyȕȷˡƤʣϘ̀kr:ƤʣkrȕȷˡzƖȞˡapq´àÄ¤ȶ̈ɚk9͍Ȭƾ̭Ųċ̾Ǥurzg9pq 300 ̐_®Ð·»bƷɚir:p~^tƖȞˡ}̈́ø̣}ͧǃm~b 309ȕȷˡ}̈́ø̣}ͧǃm~b 40 Ʒɚir:§Üɹ}Ʒɚir®Ð·»ʲɚk»ÛÊ¬àɟϬkr: MALDI-TOF/TOF-MS }xĳʭmgzy̓rȶϹ®Í¤»Üa9MASCOT ª@¶Ǥ]ƖȞˡzȕȷˡyͧǃm´àÄ¤ȶ̛̿kr:ǃȂ9100 ̐_®Ð·»}v]x̛̿bycx]:g~̇}ȕȷˡ}̈́
ø̣}ͧǃmgzbΧǬix]¡Ý¤¶àťx]r:r9ȕȷˡ}̈́ø̣}ͧǃkrÝ¤¶à hypothetical protein ¨@¼mþ̬ȕ}v]xÛÜ´Ó PCR Ǥ]9mRNA ÝÌÜyȕȷˡ}̈́ø̣}ͧǃkx]gzń͑kr:ǃȂ9i}p~þ̬ȕ~ǪͧǃŎÁ·¤µàŎƤȶ̪ř}Ȅʪk9ȕȷˡ̈́ø̣}ͧǃkx]r´àÄ¤ȶbȕȷˡƤʣ}ϝ_ĈƐĳʭkx]:

25

P47
äǼ«ĲŃÀ¾À  ƨțĎg˻ŃfÛĿ[vĕĺÖƮgû˝KǁɵǩƻBɼɀ˳ƮȺɵǒÓGȢåš̉ɋĈɋĈ×HLϋ̣M
 Į?;gy} 2 Ʉ~ĞʄÉ¼žƖŎ~¨Þ¾@bƝˁʳʅkrȀ};ĂΩ~É¼žƖà¹Þ»¢¬à A (SEA) ȋʧŎ~¨Þ¾@bϡƖk;˟Ω~ͷȋʧƖŎ} SEA þ̬ȕb̬͛mz]^ʋr|˼ƺτa}kx]<pgyζƸſy;¨Þ¾@ţy~ SEA þ̬ȕ̬͛~΂̰Ɩ~˒ʂŚƉ}ˬcdΞģmzǣ_;SEA ȋʧŎ~ϡƖ}ĈƐźmŚƉĄȕɾƵ}v]xƷ̹Ǥur<LΩΪ=ƲĩM SEA ȋʧŎzkx Staphylococcus aureus No.29 (EMr;SEA+;ϡƖŎ);ͷȋʧŎzkx S. aureus
No.77 (KMr9SEA-) ƅȣƖŎzkx;¨Þ¾@ţy~ SEA ȋʧŎ~ϡƖ˕ʔmɾƵƷ̹kr<p~Ʋĩ;No.29 Ŏ` No.77 Ŏ~ͣϦ 6 ȫţ (ˣʟ˒ʂů) ~ͣϦČ pH 6.0 ~Ő̧ͣ˽}̮Άk;37 °C y¨Þ¾@ƤʣiorzgǼˠd~¨Þ¾@bϡƖkr<rͣ˽ʣΖʧāɿƌbϸd;NaCl ͕̰bǪ] (7.5%) ɻǫ}ϡƖ¨Þ¾@bˠdƤʣir<g~Ʋĩ;¨Þ¾@bʳʅmȀ~Ő̧ͣ˽ɹy~ʧā˚̰bϡƖ}ĈƐkx]ħ͖ʢbȮǶir<r;MgCl2(100 mM) ~̩Ħ}uxϡƖ¨Þ¾@~ƤʣbϧʡiƢǔb͑r<i};No.29 Ŏz
No.77 Ŏǵǫmz;ĿƖŎ~˱͆ͣϦȫzͱņkx;ÃÈÜÓƤʣ͖~ɹɭb͑r<ÃÈÜÓƖ~ǉˡ;χ~΄̆}ͩ];̛̈́~þ̬ȕ~ͧǃbǪgza;ǃȂ;ÃÈÜÓƤʣȫ~ SEA þ̬ȕͧǃϹ}v]xƷ̹kx]<P48
cis-ǘĕy˰[vɁĕȖƒĄg̉Ƀȭɍɬƃɜ
Sɛɵ ̃BɟƣʊʮBȟȩǛʮG˛ɋ×ț˜ʖHLϋ̣M cis-ɘŚϖmˠŚʢǯŁ9͉Ąʢ­£´Û®ϠΕȶFEDLFG K+¶ÖÀÜˈĽŋʢȪv¨ÝÛ¼|{~^|®¹Þ¼¹ÜÍà}ǙdƺɇeũζǯŁy\:ζƸſy9Ζȕ͉ Diels-AlderFIMDAGͬě} cis-ɘŚǟ˔ϰˡʾˮ̣}ǟ́k9ˠŚʢģǫΕ~Ǔϯ̣|ǫʣϋȘkr:
LΩΪ=ƲĩM
Ȗͮ~ 2-Ô¶ÜÉ¹½@Üa 5 ˺Ļy̏ʪkrÁ@Ü®¹Ü 1 ~ IMDA ͬěƷ̹kr:p~Ʋĩ9Á@Ü®¹Ü~Ś~ăʟ}n9ΰ cis-ɘŚkrȊŚʢ¦»à 2 ̓gzbɚϩr:Ǧēȫ}9IMDA ͬě~ɾƵƷ̹z EDLF ~Ξř}v]xŸЇm:
O
O
O

n

O
R

1 (n = 0, 1)

IMDAreaction

OH
n

R

*

HO
O
O
2

H
OH

H
OH OH
O
HO
possible structure of
Inagami-Tamura's EDLF

H

H

OH

OAc
OAc
OAc

O
AcO

OAc
correolide

26

O
CO2Me

P49
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§˱́gȆŒʈĩǾɥČȖʸƿfė[vŠĳKŧɵ̤ɂAƧɵ̆ǄAǥƼÓ˛ɋ×ț˜ʖLϋ̣M Ɨ͓~ǪЁģ}ͩ]Ü¸ÂÑ@ƣ͑˼ɲ~Ŗȼʟˬcd˒Ħkx]b9Ǵζ̣ȭ϶Ϊń
ϰix]|]:NGF (ʏƧʣ̑Ąȕ) ʏƧǿΫ~Ζģ=ʧ˝}ͻʗ~ĄȕzkxʏƧΞʢȵŖȭ϶ϐzkxů˥irb9̳ϝΪ}Ĭ˭b\:̴Ƹſȳ}`]xʏƧÕºÜǿΫy\ PC12 ǿΫϢ]r NGF ϠΕȶ~˳ȇǤurƲĩ9ğ͊ȋ¾Å»º Acanthaster planci  acanthasteroside B3 b˱ϭi9Ñ®}ˣmŴĠĶˀǓĩͪτkr:ζƸſy9¾Å»ºa acanthasteroside B3 ðļ} NGF ϠΕȶ˳ȇmgz9` acanthasteroside B3 ~ȄϢűǟτa}mgzϋ̣zkr:  acanthasteroside B3
LΩΪ=ƲĩM
 ¾Å»ºȴɖϹ 9.6 kg Ô´Á@Ü̈ɚk9ODS ÚÓ}ux 4 įΖ}Ζϭkr:g~^tacanthasteroside B3 ťįΖ (Fr. 2)  silica gel ÚÓ}uxΖϭk9ʏƧ͈ŵʇ̑ŋʢȮ
m 2 v~įΖ Fr. 2-29Fr. 2-6 ̓r:g~^t Fr. 2-6  acanthasteroside B3 (70 mg) ɝʜ}̓r:r9Fr. 2-2 ʏƧ͈ŵʇ̑ŋʢȘͽ}Ζϭʔ9ŋʢȮmΕȶ̓rbɊϹb͹Ϲy\urr9ϹɹfǤ]ǟ˔ư̛Ǥ^Ϝ̛y\: ĂΩ9ʩʪkr acanthasteroside B3 Ϣ]xȄϢűɥ~ĳʭǤur:Acanthasteroside B3 
PC12 ǿΫ}ˣm NGF ~͈ŵʇ̑ŋʢ˒Ɗm:ͣ˽̇y 3 ȫţðɹà¢ØÌ@»kr NGF
 PC12 ǿΫ}̳ϝmzŋʢ~̘Ģbƺrb9acanthasteroside B3 Ƈ˝iozŋʢ\̟̰ΤȪirgza9acanthasteroside B3  NGF ~î̛ģ}ūϝkx]gzȮǶkr:Ǳǌ9i}ɶǿ}ȄϢűǟĳʭmϜ̛y\:P50":8,7:6:/80*05 țƎȗÍɴƮʗüĎfSWvțƎȗɟĖɃgʃɪĘƧȧɸȕ BE˗ƧţȈ BȪȩʩˇ BȁîÓɗ BǥǫŲ Bɛɵƻț B̠ˣĭǕ G ʶÏšɋB B ƨȸŠB ˔̇ɋH
IˠɬJstreptothricinFSTG9p~ǟ˔} streptolidine lactam9carbamoyl
gulosamine9!-lysine ϖmǜʧΕȶy\9ΨʻƖ}uxʧȋi:ST 9Ɗ]ǜƖŋʢȮmzz}9ʎłʧΕ}ˣkxͅʢȮmgzaϽɬϫϢ}ȡux]|]:streptolidine lactam ǟ˔9
ST ~ʧϬŋʢ}ɖϤy\gzb˼x]gza9g~ģňǟ˔ʧǫʣǘň}uxĶΞyc9ʋr|ÿϐ΁Û@¼ģǫΕ~ˉ
ʪ}v|bzǣ_:ǼƗĮ?9ST ʧǫʣþ̬Ơɂ̓k9
orf18 þ̬ȕ~͝ĵŎa streptolidine lactam z carbamoyl gulosamine ~a|ʧǫʣ̇ţˡstreptothrisamine ̛̿krFNature Chem. Biol., 8, 791-797, 2012G:pgyζƸſy9p~˟~þ̬ȕ͝ĵŎa ST ʧǫʣ̇ţˡ~̛̿Ǥur:IˋˍDŗðJ¨®Ò¼Ì¤´@}¤Þ@¾à¥ix] ST ʧǫʣþ̬ȕƠ~ĿɄþ̬ȕ%-Red recombinase ϫϢkx in-frame }þ̬ȕ͝ĵǤur:͝ĵþ̬ȕťþ̬ȕƠFϏ 34 kbGʹʄˡˇǰƣÌ¤´@}¤Þ@¾à¥k9øɄΨʻƖStreptomyces avermitilis SUKA17 Ŏ}̀͐kr:̓rƤȶ̪řˡ~ͣϦČ LCMS yΖʭkrzg
 9 carbamoyl transferase  ¨ @ ¼ m  orf17 þ ̬ ȕ ͝ ĵ Ŏ } ` ] x 9 decarbamoyl-N-acetyl
streptothrisamine zϜˋiģǫΕbƷɚir:i}9orf89orf119orf13 þ̬ȕ~͝ĵŎ}`]x9streptolidine lactam ~ʧǫʣ̇ţˡzȗģǫΕƷɚkr~y9gƸſʣĩ}v]x
ΧǬm:

27

P51
ɰȰʸ˱́gƀUzƠɆȖĠƃfSWv§ ABCG2 g˘˨Űǒǹgė˷gûȣKƕɝÓƇ Bɨ˗oVn Ʀɒˮʎ Bɵɼ˩ƅ Bɟʧǆ̏ƌƟƮ BȤȩɚĮ B
ɟȩɋ G ɟʴɋ×ã˼țʸB ɟʴɋĈã˼țʸB ȢåUzÀFB ̆Š "#HLϋ̣MŞ˓=ʖ˓=ɪ̎=͗|{~ˇʃ}ˠdͧǃkx] ABC ϓˑˡ ABCG2 ;ǜbȁ͟ɚmgz}uxbǿΫ}ϐȁˤʢΌϝmθ´àÄ¤ȶy\;Ζȕţ­®ÜÈ¼ƲǫƤʣkxÎÕ͍Ϲˡzkxű͖mzƇ};N Ʋǫƣ~̷ǺɌʀɇe<ζƸſy;Ζȕţ­®ÜÈ¼Ʋǫz N Ʋǫƣ̷Ǻ΄Ħbŵg|] ABCG2 Ϣ]x;ABCG2 }ǜbȁˤʢűǟ}`e ABCG2 ~ηϑǌɌʀ~ŤϝƷ̹kr<
LΩΪ=ƲĩM
Flp-In System (Invitrogen Ȼ)ϫϢkx 12 Ͱʹʄˡ~˵ЉϺĀ}˝Ȃm FRT ͡Ђ}ύʧƣ~
ABCG2(WT)` N Ʋǫƣ̷Ǻ΄Ħbŵg|] ABCG2(N596Q);Ζȕţ­®ÜÈ¼Ʋǫbŵg
|] ABCG2(C603G);Ζȕţ­®ÜÈ¼Ʋǫz N Ʋǫƣ̷Ǻ΄Ħ~]nŵg|]
ABCG2(N596Q/C603G)pq̀͐kr<g~ǿΫ~ǜbȁ}r]mŗɇʢÌ¤´@~̀͐krǿΫzͱņkrƲĩ;]n~ ABCG2 ͧǃmǿΫÌ¤´@~̀͐krǿΫzͱkxƼ̋| SN-38 ` Mitoxantrone ˤʢȮkr<ĂΩ; ABCG2(N596Q)\]
ABCG2(C603G);ABCG2(N596Q/C603G)ͧǃmǿΫ;ABCG2(WT)ͧǃmǿΫzͱkx
Ǫ] SN-38 ŗɇʢȮkr<ABCG2 ͧǃmǿΫţy ABCG2 mRNA Ϲ}Ƿ͑|aurb;ABCG2(N596Q)` ABCG2(C603G);ABCG2(N596Q/C603G)~ǿΫ͉˝ȂϹ]n
ABCG2(WT)~Ϗ 2 Ζ~ 1 y\ur<g~Ʋĩ;Ζȕţ­®ÜÈ¼Ʋǫz N Ʋǫƣ̷Ǻb
ABCG2 ~ǿΫ͉˝ȂϹų̛mɖϤ|Ąȕy\gzȮkx]<r;g~Ʋĩa;Ζȕţ­®ÜÈ¼Ʋǫz N Ʋǫƣ̷Ǻ~]nƯ]x ABCG2 bǿΫ}ϐȁˤʢΌϝycgzȮir<P52
KˣʳƂ BƏɉƪ«º£¾~ǋ˸ɃȴĂƠgȵǙBɟˣ˵ƪ  B0*/(,36:/685 G Ȣɋ×ʖB Ȣɋ»FÀŠB ̆êĈŠĳǟDěĒŠĳǟB "#!"#B (> 3(5*2"6*0,:?H¬Þ¿½¯½~ÉÚ¬Á®¹Þ¼FBRGɇϟˡFBRI1;BRL1;` BRL3G BR zΒǫˡƤʣk;Ƈɇϟˡy\¢½@±FSERK1-4GzǿΫθ~͉ļyˎǋȄϢmgzy¬¥½Ü̬˯Ϙ̀k;ǿΫʇ̑ǑƤ˦Ƥʣ|{~ BR ě̶ącŵgm:ĂȆ͓ΧǬir BRI1-ÉÚ¬ÁÚ¼FBLGΒǫˡ~Ʋɮǟ˔ƺz;BL Ɂzkx®¹Þ¼Ś~"χ`˖Ǻ˶΍y BRI1zˎǋȄϢkx]:ĂΩ;®¹Þ¼Ś~&χɹ;ǟ˔ŋʢˎŤƸſ}`]x BR ŋʢ}Ɗdūϝmgzbaux] A Ś 2&,3&-diol ťxǙdƜ]x`;BRI1 z~ˎǋȄϢε{ƺ|]:g~gz;BL "χy BRI1 }Ʋǫkvv;&χ;̈́} A Ś 2&,3&-diol ıkx SERKs zˎǋȄϢmgzy;BRI1 z SERKs ~pq~ǿΫļ¼Ôàʳ̆ioώŊ˲^Ζȕy\gzƊdȮǶkr:pgyēȼ;BL ~ 2&,3&-diol °»¾¼}Ξřkr BL-2,3-acetonideǫʣkxʧΕŋʢ̏z;BR ʧΕŋʢ ɯȲkx`;˫} BR }ˣmЌǜŋʢϖkx]r:v;BL-2,3-acetonide  BRI1 }Ʋǫmb;SERKs }Ʋǫyc|]Û¡à¼
FBRI1 à´©¾®»Gy\ħ͖ʢbȮǶir:ȄϢűɥ~Ǟ|Ʒ̹Ȣύ}92 ñ` 3
ñʛȍũ~{t~ĶΞb BRI1 à´©¾®»ŋʢ}ϖǓy\~a˳ȇmr~ģǫΕˉɚkr:ǫʣ¨®»Ţ;BL y|d;BL z ̵̿~ŋʢϖmÎÕÉÚ¬ÁÚ¼FHBLG
Φłzk;2-O-alkyl-HBL z 3-O-alkyl-HBL pqʟɄǫʣk;ʧΕŋʢͱņkrƲĩ;2-O-alkyl-HBL ~b BL ЌǜŋʢȮkr:g~Ʋĩz}ǃȂ;Ǟ|ǟ˔ĶΞǤux]:

28

P53
 ČȖêʸƿ.3()80+05 g !! ¼ɴɐŒ̕fɅ[vƤ˼KǔƧɚʩ BȡƧ˯ð B˟ɀţÓ ɛŧĻ  ̣Ɯɉ ɋȟȲƮ 
G ɟʴɋãțB ĚǇɋʖ GĎH¾©¼£ÀFH
Lϋ̣MÊÞ¹àÎ®È´@± 2C(PP2C)9PPM ÈÒÛ@}˛m Ser/Thr Î®È´@±
y\9®»Ý®ě̶ǿΫɋů~ʡǍ}Ťϝmgzb˼x]:Į?gy}9PP2Cb͝ǯǿΫΖģĄȕ(RANKL)}uxŋʢģi¬¥½Ü̬˯ƧЅϧʡk9͝ǯǿΫΖģ΋}ʡǍmgzΧǬkxcr:r9PP2C ŋʢģΕȶzkxƺɚkr glabridin b9RANKL }uxŋʢģm MAPK ¬¥½Ü̬˯ƧЅϧʡk9NFATc1 ~ͧǃϧʡmgzy͝ǯǿΫΖģˈĽmgzτa}kr:kak|b9glabridin ~ȄϢ̫}v]xμsτay|]:pg
y RANKL/RANK ¬¥½Ü̬˯ƧЅ}`e glabridin ~ȄϢűɥ}v]x9ɶǿ}Ʒ̹kr~yΧǬm:
LΩΪ=ƲĩMÑ®Ñ¤ÞÈ@­=˱žϗϩ RAW264 ǿΫ}`e RANKL/RANK ¬¥½Ü̬˯ƧЅ~ glabridin ~ĈƐ9®´à=ÉÞ·»Ϊ}Ʒ̹kr:n9RANKL }uxŋʢģm NF-åB ¬¥½Ü̬˯ƧЅ~ glabridin ~ĈƐ̏rƲĩ9glabridin  IåB ~Ûàȍģz IKKâ/ã~Ûàȍģz}ϧʡmgzbΖaur:IKK  NF-åB ~Ʋǫ´àÄ¤ȶzkx˼ IåB ~ÛàȍģzΖĳącŵgk9NF-åB ŋʢģmgzb˼x]:g~gza9glabridin  NF-åB ~ŋʢģϧʡmħ͖ʢbȮǶir:i}9MAPK ¬¥½Ü̬˯ƧЅz NF-åB ¬¥½Ü̬˯ƧЅ~Ƈ̗~Ąȕy\9PP2Cã~ũȶzkx˼ TAK1 ~Ûà
ȍģ}ˣm glabridin ~ĈƐ̏rzg9glabridin  TAK1 ~Ûàȍģϧʡkr:ðɹ~Ʋĩa9glabridin  PP2Cãŋʢģmgz}ux9TAK1 ~Ûàȍģϧʡk9RANKL/RANK¬¥½Ü̬˯ƧЅˈĽmħ͖ʢbȮǶir:P54
ņ˚ǋ˸Ƀ #" ! hÀgņ˚yǋ˸[v 1)
KƧ﨑ˏǀ Aȗȩȅ̊ A˒řˊƮ AƦƢż Aɷ˂ɐʮ  ȢåšɋD×DǽÜA ɽɋD×DʖțïLϋ̣M ϼ̅}ť ¹¢àϿƚλ̚m:ƚλ 25 ɄϿ˝Ȃmƚλɇϟˡ}ŗ˼igzb˼x]:pgy9ζƸſyͣϦǿΫϢ]x ¹¢à~ƚλ͑ȱmƚλɇϟˡ~̛̿ȣr:
LΩΪ=ƲĩM Å»ƚλɇϟˡ hTAS2Rs z¢ÔÚ G ´àÄ¤ȶ G#16gust44  HEK293T ǿΫ}Ƈͧǃio9 Ü¬ÓŗɇʢƪǑʄˆɂǰor 96 well ÊÝ@»}]r:FLEXstationTMè}x9 ¹¢à̳ϝǌ~ǿΫ͉ Ü¬Óà͕̰[Ca2+]i ~˒ĦϹ˙̛kͿĥkr:p~Ʋĩ9Ů˼~ ¹¢à~ƚλɇϟˡ TAS2R 392)~˟} TAS2R14 ͧǃǿΫy ¹¢à̳ϝǌ9[Ca2+]i ~˒Ħbƺrr9g~Wv~ƚλɇϟˡb ¹¢à~ƚλɇϟˡy\gzbaur:TAS2R39 ~Ωb̘͕̰~ ¹¢
à}ŋʢģirr9 ¹¢à~ƚλɁ} TAS2R39 yɇϟizǣ_:TAS2R39y ECg bĂͰƊ]©¾®»y\ur~}ˣk9TAS2R14 y EGCg buz̘͕̰yŋʢģiogzbτa}|ur:
1) T. Yamazaki, et al., Biosci. Biotechnol. Biochem. 77 (9) (2013), in press
2) M. Narukawa, et al., Biochem. Biophys. Res. Commun., 405, 620-625 (2011)

29

P55
|~gȳǻʚ˿¼g˴ʄdȗʹʹȣ
Kɋŧśƫ BĭɵĮʩ Bɧ̔șȈ G ˛ɋ×ǶˉïĈB ˛ǴɋʖH
Lϋ̣M
 ­ªFHydrangea macrophylla~ːʀīFГG~īʄà»¬¾à}~y\;̿Ă~à»¬¾àFºÜÈ¾­à 3-¥Ü¨¬¼Gť|b;ČΫ~ pH;ČΫ͉~ɤʄˆ~ˇʣ`ÜÒ¾Óà}uxʫ;ȟ;ʱzЄ˜̣}Ξģm<ĂΩ;­ªƊ]ȍ
ʢ̱ɼˤʢȪvgzy˼;ǼƗĮ?~¥Ü@Ê;ɠxǿΫθƣ`ČΫƣ~ÜÒ¾
Óϓˑˡτa}kr<ʧΕň=ĐƭňƸſ~|n;īʄΞø~ȏˇÜÒ¾Óˤʢűǟ;i};ʆ΁Ćϵ~ʄˆěϢϋȘmɹy;­ª~ˇʃͣϦ Ü®~Ϙ̀ɖϤy\b;gyƔxǲ͌zi; z{ΧǬb|]<ζƸſ;­ªaˇʃͣϦ Ü®Ϙ̀k;g~ị̛̂|úȪzà»¬¾àʧȋɾƵ~ńϰ}­ª~īʄ}ŤΖȕ
űǟ~ĳτϋ̣zkxǤur<LΩΪ=ƲĩM ­ªF΁Ʉυķʫ`υķʱG~ϣʣ̫̑ͣϦmgz}ρƖʁΕˡ̓r<g~ρƖʁΕˡa̨ĺkrϣɂ;ʁΕʣ̑̏ʤΕȶ9ǉƤͣ˽~˴ˆǂ`̄ˆǂ;Ǒɱȹ~ɾƵƷ̹`g|^gz}; Ü®Ϙ̀ȣr<0.3R~§Úà¡Óť MS ͣ˽} 5 mg/L ~ 2,4­¤ÞÞÈÁ¢¬ʘȍ̩Ħkr~t;24Q;16 ȫţτɾƵ(4500 lx)y 3 áƴţͣϦmz Ü®
~Ĝʨ|˒ʂzƊ]̆ʄb˕ʔigzba;;χ}ʱȟʄ~ʄˆǿΫȪv Ü®~Ƥʣ`˒ʂ}ʣǒkr< Ü®Čˡ̄ˆy̲Ʋǌ̈ɚk;HPLC }ʣΖΖʭǤur<ɁʄˆГ~ʄˆzø|;¬¾­à 3-¥Ü¨¬¼`¬¾­à 3-ªàÉÆ¬¼y\gzbτaz|ur<ɤʄˆ~ˇʣȿŔ~ý]b͑r<ͣ˽}ÜÒ¾Ó̩Ħkrb; Ü®~ʫʄģ͑|aur<īΣzμΖģǿΫ( Ü®)yø|ʧǫʣʡǍbǤx]gzbτ
az|ur<ǃȂ;ČˡͣϦɾƵƷ̹̇y\<P56»ÀƩSsj©¾»ÀƩʔɺhǺʸgŎɈŎȗf˝ąeÛĿyįl[Kʶɵ̥˱ BȓǳǲƬLB30,81(50 ȮɼșīLG ˛ɋ×Dț˜ʖB ɽķĈŔɋDĽÑDț˜H
Lϋ̣M ¬Þ¿½¯½~ČΫθÊÞ»àÇÞÎ®È´@± (H+-PPase);ǿΫȶÇÞÛàȍ(PPi)~Ħʛ
ΖĳÀÜ£@zƇώkxÊÞ»àČΫ͉}͖̾ϓˑmű͖Ȫv:PPi 9łȍ´àÄ¤ȶ9̷ȶ|{~ǫʣ~Ȁ}ΎȋΕzkxʧʣm:PPi ~ĭɺ|̃ʯ9g~ɖϤ|Ζȕ~ǫʣͬěģňΙǥЇ̣}ϧʡˈĽm~y9ǿΫȶ̇~ PPi ̤ʲ|͕̰}Τvgz˫Ƚ̏ʶɹɖϤy\: H+-PPase ~ű͖Ư˞Ŏ fugu5 9̄ˆǂzkx NO3-~ť MGRL ʛǢͣ˽yʧāioz9ϣʒzϣΚ~ţ~ǿΫbǊȜk9ζϣbüɘmʧāøɽbşȈir:ζƸſ9ζϣ~üɘ;ǃƣącŵgmƿĄΕȶĤa9r H+-PPase ~Ư˞}uxŵgÊÞ»à~ϓˑű͖Ư˞z PPi ~̃ʯ~{t}ŵĄm~aτa}mgzϋ̣zkr:
LΩΪ=ƲĩM ǧΦϗϩ~ħϡʢ PPase y\ IPP1 ̀͐kr fugu5 Ϣ]x;ʧāͱņǤur:p~Ʋĩ;
IPP1/fugu5 ;ǃƣȮin9WT z̿Ϡ}ʧākr:ħϡʢ PPase }ux PPi ɧƂkrgzy;
ǃƣbϧʡirgza9g~;ǃƣ PPi ~ĭɺ|̃ʯbƿĄyŵggzbȮǶir:r9
ͣ˽} NH4+̩Ħm9\]ͣ˽}À·»·cͣ˽zϣ~ʳʅδțmgzy;ǃƣ~ɚǃϧʡycgzbͪτkr:NH4+~̽c}v]xͪτkx]|]b9;ǃƣȮkx]ű͖Ư˞Ŏʥɽ}ʧākx]ű͖Ư˞ŎÛàȍ(Pi)ťϹbˠ]gza9ϣ~ʸ˶~͟ʛˇʃaĭɺ}ɂǰr Pi b;ǃƣ~ɚǃ~ĂĄz|ux]gzbȮǶir:m|t9ĭɺ~ Pi PPi ~ΖĳģňΙǥЇ̣}ϧʡm~y9PPi ~̃ʯ}ƨbzǣ_x]:

30

P57
Ǻʸgț̆ČȖ¯¬g¬¾ÀƋȲfė[vŠĳKìʁȗȈ Bńʁ̋ʪ G ˛ɋ×ț˜ʖH
Lϋ̣M ʁΕ~Ϡ?|ˇʃmx9Ʃ̒ΖЃˇʃFSAMG}˝Ȃmɫʟ~ŕǿΫ}ϗϩm:¬Þ¿½¯½˱ϭir clavata ΞøŎ9SAM bʹˬģmz]^;ǃƣȮm:g~Ξø~ƿĄþ̬ȕ~Ăvy\ CLV3 ÍÊ¶¼ÎÜÕà~ʿƛˡ¨@¼kx]:CLV3 ~ģṇ̌ζˡ C ι˶΄Ɨ~Τ˝ϺĀFCLE Õ¶@ÈG}ϗϩm 13 Ȏũ~ɌʀÍÊ¶¼y\9497 Ȏũϋ~ Pro bʛȍģi97 Ȏũϋ}i}̷ǺɌʀɇex]: gy~ CLV3 ~ηϑǌɌʀ}ŤmƸſa9 ÛÈÚß@~̈ɚČ̇}9CLV3 ʿƛˡ´àÄ¤ȶ̇~ CLE Õ¶@È~Ǽɠ~ Arg ~ N ι˶˖ʲ˹mǧˆŋʢb\gzbΧǬix]: pgyζƸſy9 ÛÈÚß@a̏ʪkrÐÊÚ®»̈ɚČʩʪɚͧƿϵzkx9CLV3
ʿƛˡʲ˹ŋʢϖmǧˆ˱ϭ=̛̿mgzϋ̣zm:LΩΪ=ƲĩM
ÛÈÚß@~ħʆ΍a̏ʪkrÐÊÚ®»Č9ˇř_´àÄ¤ȶzkx̏ʪkr CLV3 ʿƛˡzǵǫk9ͬěǌ~˅ÍÊ¶¼įΖ~ MALDI-TOF MS ˙̛krƲĩ9Ϝˋ̗ Arg Ȏũ~ Nι˶˖y~ʲ˹bń͑ir: pgyȬ}9g~ÐÊÚ®»ČϥàǏř¤ÞÑ»¥ÚÈ@}ƅkrƲĩ9Wv~ϭr
įΖ}ǧˆŋʢbń͑ir:̿Ϡ}ćàǏř¤ÞÑ»¥ÚÈ@}uxÐÊÚ®»ČΖįkrƲĩ9Wv~įΖ}ŋʢbń͑ir:g~ŋʢįΖ SDS-PAGE }ƅkrzg980
kDa ΄Ɨ}Βʟ~Ãà¼bşȈir:ζĲyg~ΖįΪˇǫoxʩʪkrƲĩ}v]xǫoxͧ;mϜ̛y\:P58
Ǻʸgț̆ČȖ¯¬g˘˨Űǒǹfė[vŠĳKʕȓ˾Bɵ̧˰ĨŏBńʁ̋ʪG˛ɋ×ț˜ʖH
Lϋ̣M ʁΕ~˽ɹ΍~ˠd~ˇʃ9Ʃ̒ΖЃˇʃ (SAM) }˝ȂmŕǿΫ}ϗϩm:CLV3 þ̬ȕ9ŕǿΫʟ~̏ʶ΋}ʡǍmʧϬŋʢÍÊ¶¼~ʿƛˡ¨@¼kx`9ʧϬŋʢȪr|]ʿƛˡb9ÊÞÛàȎũ~ʛȍģ9̷Ɍʀɇerǌ9ÊÞ¹@±}ʲ˹ɇe9ʧϬŋʢȪvʣəƣÍÊ¶¼}|zǣ_x]:kak9g~ĭ̟}Ťϝmǧˆ̵}Ťkxs
΃τ|̫bˠ]:pgy9ζƸſyn9CLV3 ~ÊÞÛàȎũ~ʛȍģ~űǟ}v]xτa}mgzy9ɩϩ̣}ʣəƣ~ÍÊ¶¼ϟö}̓ɃΪńϰmgzϋ̣zm:LΩΪ=ƲĩM ¬Þ¿½¯½§ÁÓɹ}9Å»´Ã¨~ÊÞÛà-4-ʛȍģǧˆ (P4H) z~ˎ̿ʢa9
P4H ÎÕÞ¥þ̬ȕb 11 ǆ¨@¼ix]gzbΧǬix]:ʣəƣ CLV3 ÍÊ¶¼ʧȋm͖ϻb\gzbͪux]¬Þ¿½¯½ Ü®a total RNA ̏ʪk RT-PCR ǤurƲĩ9 Ü®}`]x 11 ǆ~^t 8 ǆ~ P4H ÎÕÞ¥bͧǃkx]gzbΖaur:pg
y9g~Zǆ~þ̬ȕˬ̎ƖϢͧǃÌ¤´@}¤Þ@¾à¥k9ˇř_´àÄ¤ȶ̓r:̿Ϡ}̏ʪkr CLV3 ʿƛˡ´àÄ¤ȶũȶzkx in vitro ǧˆͬěǤurƲĩ9CLV3 ʿƛˡ~ CLEÕ¶@È}˝ȂmÊÞÛàȎũʛȍģmgz~yc P4H Βʟͧƺkr:

31

P59
NMOgʎ̎ɃêU¼ ¢ºgŎɈf˷RvÛĿfaQbgŠĳ
KȓǳǲƬB˕ˣƖɘBƦɌÅȫåȅ̆ƮȮɼșīG˛ɋ×ț˜ʖH
Lϋ̣M
 Į?9ÕºÜʁΕ¬Þ¿½¯½~ČΫθ}ƓȂmÊÞ»àÐàÊ AtVHP1 ~ GFP ϛǫ´àÄ¤ȶb9̇ĚČΫθ}Ħ_xÃÜÉǟ˔zǈű͖μ˼~ČΫθªÉ¼Ôà}Ɗd̃ʯmgzƺɚkr:ζƸſy9̈́} VHP1-GFP bˠdͧǃmΖЃ=ʇ̑ŋʢ~Ǫ]ǿΫyÃÜÉǟ
˔bˠ]gz}̉ϋk9ÃÜÉǟ˔~ƤʣɾƵzp~ʧϬ̣õŷvczgzϋ̣zm:LΩΪ=ƲĩM
 VHP1-GFP ͧǃϹ~ø|Úàţ}`]xÃÜÉǟ˔~ʟͱņkrzg9ͧǃϹ~Ǫ]ÚàyƼ̋}ÃÜÉǟ˔b˒Ħkx]r:kak9͉Ȃʢ VHP1~ϖρÃÜÉǟ˔~ʟ}urdĈƐkx]|]gza9ÃÜÉǟ˔ GFP Ζȕ~ʟ}ò˝kx˒Ħkx]gzτa}kr:pgy9GFP ~ɀ]͍ϹˡģȄϢ(Kd = 110 µM)}̉ϋk9˱ϹˡģΞøzkx˼ Ara206Lys Ξø GFP }̀͐krzg9ͧǃϹ}ŤnÃÜÉǟ˔burdƺ|d|ur:v9ÃÜÉǟ˔ƗʳkrČΫθ̿ȓb GFP ~ŹΙǤƣ~͍Ϲˡģ}Ɗǉ}ϒ̆krƲĩ~ȋΕy\gzbτaz|ur:ζƸſ9GFP ~͍ϹˡģρȢycÝÌÜ~ϻy|d9Ǫ]ͧǃϹ=̤ʲ|ϰˡǟ˔=źθţʳ̆bŵgɻɡz]^ɾƵκrkrɻǫ9Ü¡ÀÚ~Ƥ˦}yĈƐϝ_gzϋ}ƺ_Ƥyτń}Ȯkx]:ǃȂɚĴux]ßà¤Ú§ GFP ź YFP |{Ͽĕˡ9Ϡ?|Ƹſ}ˣkx˿σ̣|ĈƐ
ϝ_ħ͖ʢb\r9Ara206Lys ~̀͐}˱ϹˡģƊdő:

P60
ĢŹʹêyɣȦ[v 9:64(.,5 gʱ˃Ȗfė[vŠĳKȟ̟ʩɚ BɀǳÓʘ BʑƄÓƅ Bńʁ̋ʪ BƢȆ˺ƺ G ˛ɋ×ț˜ʖB ķɋ×ʖHLϋ̣M ǃ˝mǪ̵ʁΕy9 z{~ʁΕ}`]xŲǗ~ƤʣbşȈi:Ɨ͓~¬Þ¿½¯½Ϣ]rƸſa9ŲǗΖģʥ}ʡǍmΖͲƣÍÊ¶¼ÎÜÕàzkx stomagen b̛̿ir:gy9§ÁÓbĳ͇irˠd~ʁΕɄ}`]x stomagen Ü²Þ¥þ̬ȕbƺvaux]
b9ʣəƣ stomagen ~ǟ˔ű͖}Ťkx9¬Þ¿½¯½ðļ~ʁΕyμsΧǬb|]:ζ
Ƹſy;Â¤ª9µ¯Ϣ]x stomagen ~Έ΢ʢ}v]x̏gzϋ̣zm:LΩΪ=ƲĩM
 n9ÍÊ¶¼ǫʣűϢ]xÂ¤ª`µ¯ stomagen Ü²Þ¥ǫʣkr:ǫʣ
stomagen ̩ĦkrČˡͣ˽yʧāiorÂ¤ª~;ͳşȈkrƲĩ9ŲǗν̰bɹɭk9ŲǗ¤Ú®´@bƤʣir:ĂΩµ¯y9ǫʣÍÊ¶¼ɟϬ}ŲǗν̰~ɹɭşȈmgzɚϩ|aur: r9ǜ stomagen ǜˡϢ]rφč̔Ǩȷƽ}9͹Ϲ stomagen ~ƷɚΩΪńϰk9g
Ϣ]x̧ˁ stomagen ~Ʒɚȣr:¬Þ¿½¯½ύʧŎ~ÐÊÚ®»̈ɚǤ]9φč̔ǨȷƽǤurƲĩ9̧ˁ stomagen Ʒɚmgzbycr:i}9̿Ϡ~ΩΪyÂ¤ª9µ¯~ÐÊÚ®»̈ɚČ}v]xφč̔ǨȷƽǤurƲĩ9ϴȼa stomagen Ü²Þ¥~ΖȕϹz Ă˿m¬¥½ÜƷɚmgz}ʣǒkr:g~Ʋĩ9Â¤ªµ¯y9¬Þ¿½¯½̿Ϡ9stomagen Ü²Þ¥ÊÞ°¬à¥ɇer C ι˶ 45 Ȏũ~ÍÊ¶¼zkx
ű͖kx]gzbƊdȮǶir:

32

P61
ºÀh  ʠŪɩéyùZb½¾F¼İǎỳȔ[vKǥƾPrʩBƧ˗ɐħB˟ə˳ʩAɋʞÅʩAɼɵǩˢAɥå̄GĝʲɋĈBã˼țʸïĈʴH
Lϋ̣M
̴ƸſȳyͧƺirƁ͏ %-Ú¤»¥ÞÉÛàϗϩ~ʋųƳʦ¨Ý®¹Þ@Ü̘ǁģÍÊ¶¼y\Ú¤»®´¶àFIIAEKG9Å»̎ÕºÜǿΫy\ Caco-2 ǿΫ}`]x¨Ý®¹Þ@ÜŻɊϧʡmgzτa}kr<kak;p~ɶǿ|Ζȕűǟ΃τ|̫bˠ]<pgyζƸſy;Ζģ Caco-2 ǿΫϢ]r in vitro ȣƽ}`]xÚ¤»®´¶à~ͤımʋk]¨Ý®¹Þ@ÜŻɊ̏ʶƦĳτmgzϋ̣zkr<
LΩΪ=ƲĩM
 Ζģ Caco-2 ǿΫ};¨Ý®¹Þ@ÜÒ°Üz 2mM Ú¤»®´¶àťͣ˽ 24 ȫţ̩Ħk;¨Ý®¹Þ@Ü̎ǿΫ~ũ̜θFˡ͉G˖}ϓˑm ATP-binding cassette transporter A1
FABCA1G;¨Ý®¹Þ@Ü 27 ñʛȍģǧˆFCYP27A1Gp~˟~¨Ý®¹Þ@Ü˫ȽŤЄþ̬
ȕ~ mRNA ÝÌÜ~ĈƐƷ̹kr<|`;ABCA1 }v]x´àÄ¤ȶÝÌÜ~ĈƐƷ̹
kr<r;Ζģ Caco-2 ǿΫ}Å» ABCA1 þ̬ȕÊÞÕ@´@ЄƲior luciferase ÊÚ®Ò¼
Fύʢƣ-928 +107Gź;p~ΞøˡĂĭʢ}̀͐k;¨Ý®¹Þ@ÜÒ°Üz 2mM Ú¤»®
´¶àťͣ˽F12 ȫţ̩ĦG~ ABCA1 þ̬ȕ̪ȸŋʢ}ˣmĈƐƷ̹kr<p~Ʋĩ;Ú¤»®´¶à ABCA1 þ̬ȕ̪ȸŋʢ̘Ģio;mRNA `´àÄ¤ȶÝÌÜϖõ}̘Ģior<r;ABCA1 þ̬ȕÊÞÕ@´@~-126 +107 ϺĀF̪ȸĄȕ LXR/RXR ~ƲǫϺĀ-77〜-55
ťGȪvΞøˡϢ]rȣƽy ABCA1 þ̬ȕ̪ȸŋʢb̘Ģkr<Ú¤»®´¶à
CYP27A1 ~ mRNA ÝÌÜϖõ}ǁɫior<ðɹ;Ú¤»®´¶à ABCA1 þ̬ȕ~̪ȸ}ȄϢk, mRNA ź´àÄ¤ȶÝÌÜ̘Ģiogzτa}kr<r;Ú¤»®´¶à} ABCA1 ~ͧǃ̘Ģ}, CYP27A1 ź̪ȸĄȕ LXR/RXR bŤϝmħ͖ʢbȮǶir<

P62ɞÀfsv ! ǋ˸ɃČȖêh " q 55,>05 Uʛù[vK˔ɀŽʼBƦɼǏʼBɼɵǩˢBɥå̄GĝʲɋĈBã˼țʸïĈʴH
Lϋ̣M
 Ǫ]ƳЍ̘ν̰ÛÐ´àÄ¤ȶ(LDL)ÝÌÜ̾ξǡģɲ̵~Û®¤˒ˬiogzb˼x]<LDL ÝÌÜɁ}Ş˓yͧǃkx] LDL ɇϟˡ(LDLR)}̏ʶi<Ɨ͓;LDLR ´àÄ¤ȶ~Ζĳ˕ʔmĄȕzkx Proprotein convertase subtilisin/kexin type 9 (PCSK9)b̛̿i;ƳЍ LDL ÝÌÜ}ĈƐϝ_ʋr|Ąȕzkx̉ϋɑx]<Į?;̅}ťɁϤ|ÐÛÈÁ@Üy\ Epigallocatechin gallate (EGCG)bÅ»ͣϦŞ˓ǿΫ HepG2 }`]x;
LDLR mRNA ÝÌÜ˒ĦiogzΧǬkr(Br. J. Nutr. 107, 769-773 (2012))<pgy;ζƸſy EGCG } LDLR mRNA ÝÌÜɹɭȄϢ~űǟĳτź PCSK9  Annexin A2FPCSK9
Ζĳ˕ʔĄȕG}ˣm EGCG ~ĈƐ}v]x;HepG2 ǿΫϢ]xĳʭmgzϋ̣zkr.
LΩΪ=ƲĩM
HepG2 ǿΫ} JNK;ERK;p38 ~ĿƧЅ~ˈĽȁź EGCG ̩Ħk;24 ȫţǌ~ LDLR mRNAÝÌÜ˙̛krƲĩ;EGCG ~ȄϢb ERK ƧЅ~ˈĽȁ̩Ħ}ɯȲkr<˜]x;EGCG ź Annexin A2 ǜˡ̩Ħk;24 ȫţǌ~ LDLR mRNA ÝÌÜ˙̛krƲĩ;EGCG ~ȄϢbǜˡ̩Ħ}ɯȲkr<r;PCSK9 ~´àÄ¤ȶź mRNA ÝÌÜ~ĈƐƷ̹k;EGCG ͣ˽̇ PCSK9 ´àÄ¤ȶ̋ǁiorb;mRNA }ĈƐk|aur<3;12;18 ȫţǌ~ PCSK9mRNA źͣ˽̇ PCSK9 ´àÄ¤ȶ~ EGCG ̩ĦĈƐƷ̹krƲĩ;mRNA ϖõ}Ξģk|aurb;ͣ˽̇´àÄ¤ȶ EGCG ~ 3 ȫţ̩Ħy̋ǁkx]r<ðɹ~Ʋĩ;EGCG PCSK9 ~Ƽ̋|ǁɫϘ̀mzz}9Annexin A2TERK ƧЅıkx LDLR mRNA ÝÌÜ˒ĦioʋųÔ ¾¯Óıkx9LDL ɇϟˡŋʢģmgzͧƺkr:

33

P63
ƴ˓ȮŇƞˎ #  ʹêfSWvʹʤǥˎFµfęmwv"! gʠŪûȣSsjʠŪ̀ȔgÛĿKɡȩ̌AşɨɐǛAƍŧǮɦAǁɵǓǍA˕șʭG˛ɋ×Dț˜ʖH! 2,142,1.-2-(4'3%/&.%4)9ðĢ !'3 "6&&8 ®@Ä@ÈÒÛ@}˛k9ɔϩ9Ƞȶ͍ɖθ}`]xÛàȠȶǵǫioǧˆszǣ_x]r:kakǼƗ9!'3 þ̬ȕÁ·¤»kxÛàȠȶǵǫbòˁzkxŵggz9`9ÛàȠȶʧˡ͉yǵǫioʎ~Ąȕy\ "  ~˝Ȃbτaz|9!'3 ˷ͥȶÈÒÛ@®¤ÚàÉÚA±ŋʢzø|Ν~ű͖vzǣ_x]:gy !'3  Ü­ÛÇà~ǫʣÐ»@¬®~Ťϝ|{bΧǬix]:r9Į?gy}ǿΫļ} !'3 bΖͲi9¤²²@Óz]^θɪΫ}˝Ȃmgzτa}kr:i} !'3 Á·¤»Ñ®Α΍Ƞβ~̃ʯ9Ƴ̇~Ƞȶ͕̰~ɹɭ9à®Ûà̝ǜʢ~ʧŋɎŘ΀ɲɿȮmgzb˼x]:ζƸſy9!'3 ͧǃzȠȶ̃ʯ~Ťơτa}mr}9Ñ®~ȠβʿƛǿΫŎy\ "  ǿΫϢ]xĳʭǤur:"  ǿΫ~ȠβǿΫΖģ}`e !'3 ͧǃϹ /  ÝÌÜz´àÄ¤ȶÝÌÜyͱņkr: !
3  ÛÈÝà®þ̬ȕzkx̛Ϲ̣ÛÜ´Ó  ΪǤurzg9!'3 ~ /  ˝ȂϹΖģǌyϏ ǁɫkr:ĂΩ9!'3 ̈́ø̣ǜˡϢ]r®´àÉÞ·»y9ǿΫ͉}˝Ȃm
 !'3 ´àÄ¤ȶ~ϹΖģǌyϏ ǁɫk9ȠβǿΫΖģʿǌyǿΫļ}ΖͲi !'3 ´àÄ¤ȶ~Ϲ}Ƿ͑|aur:r9 "  ǿΫΖģϘ̀ʿ} !'3 Á·¤µàmzΖģϘ̀ǌ9»Û¥Û°Û¼̃ʯϹbǁɫkr:g~gza !'3  "  ǿΫΖģ}Ťϝkx]zǣ_:P64¨´À  ŕˑ˴ʄȖƍ°·~ÀŘǯfɅ[vȥ©F³ÀSsj°½ÀgýȯŵðKȒȃɋǍ BƧ˗Ƈʼ ɟȩɚƊ  Ôŉ̊ʝ ıéɐˢ Ɨ˖ ˫ BȶȩĶư   G ĝɋ×ãțï ĝɋ×̓ƎʖĈ ɫɨƧɋ ¶ĎƽúǃHLϋ̣MÆ´Òà BF
6 B 6GÔ¶¾à˫Ƚ}ŤΥǧˆy\<gy B6 ƯήǪÎÕ¬®¹àFHcyGƳɲącŵgk;Ǫ Hcy ƳɲϣȍƊģ}Ķˀycgzτa}k;B6 Ưήȫ}`eϣȍ~ɖϤʢȘ̡kr<ζƸſy;B6 ˠdťʆ΁y\ʱÇ@ÑàzϣȍˠdťÎÝà
²~ B6 ƯήϘ̀ʢǪ Hcy ƳɲĶˀ}ˣmϖǓʢ}v]xƷ̹kr<
LΩΪ=ƲĩM
ȷƽæ>4 ɐЁ~ Wistar ƦϚÚ·» 6 Ơ}ΖeFnP7G;Ȧϵ 1 kg ̇} 9 g ~ L-Met ť¨à»Þ@ÜȦϵFC ʆGz B6 ƯήȦϵFD ʆG;`Ȧϵ̇~ʱÇ@ÑàΗι~ťϖϹb 0.49%;0.98%;
1.97%;3.94%z|^}̩Ħkr B6 ƯήȦϵϝ_r<D Ơ}ˣmÍÈ@ºà¥Ǥ];35 ͎ţȦāk;ĳίkr<ĳίǌ;ƳЍ Hcy ͕̰|{˙̛kr<ȷƽç>Ú·» 6 Ơ}ΖeFn
P7G;C ʆz D ʆ;`Ȧϵ̇~ťϖϹb 1.98%;3.97%;7.94%z|^}ÎÝà²Ηι̩Ħkr B6 ƯήȦϵzťϖϹb 0.061% z|^} B6 ̩Ħkr B6 ̩ĦȦϵϝ_;ȷƽæz̿Ϡ}ȷƽǤur<p~Ʋĩ;ȷƽæy;ƳЍ Hcy ͕̰ C Ơzͱņkx D Ơyɹɭk;D ƠzͱņkxʱÇ@Ñà 0.49R̩ĦyɓΖĶˀir<ȷƽçy;ƳЍ Hcy ͕̰ C Ơzͱņkx D Ơy
ɹɭk;D ƠzͱņkxÎÝà² 1.98R̩ĦyɓΖĶˀir<ðɹ~Ʋĩa;B6 Ưή}
Met ˫ȽøɽɫϹ~ʱÇ@ÑàrÎÝà²̩Ħ}Ķˀigzτa}kr<

34

P65
ȁĦG¬¾H½ Àǋ˸ɃŗƎÀ¦ƿgʃɪdûȣKŬǫȧ˳BƉȩ̘ĞB̜ȓidnBɟȩȧǜBþ̖ŧǰ̗B̑˟ʻǪBɟȩʉGĝɋã˼țʸïĈHLϋ̣M(ÊÞ)Ý¾àɇϟˡH(P)RRIɭíǧˆÝ¾à`p~΃ŋʢʿƛˡÊÞÝ¾àÛ¡à
¼zmĂĴθŠ̗ƣɇϟˡy\:(P)RR Ɂ}ǿΫ͉΍}˝Ȃkx`9C ι˶~ǿΫȶϺĀ}ɪΫˡƓȂ¬¥½ÜȪvgzbΧǬix]:kak9(P)RR ~ C ι˶ϺĀ(θŠ̗ϺĀ`ǿΫȶϺĀ)Ư˞ioxˬ΍ΖbǿΫ͉}˝Ȃkrgza9N ι˶~ǿΫļϺĀıkẍ̛́~ΖȕzƲǫmgzyǿΫ͉}ƓȂkx]ħ͖ʢbǣ_r:pgyζƸſy9(P)RR ~ǿΫ͉ƓȂűǟ}Ťmʋk]˼ƺ̓gzϋ̣zkx9ʚ̛ɹ~(P)RR Ʋǫ´àÄ¤ȶ˳ȇkr:
LΩΪMFLAG ´¥yͽȱkrˇř_ƣÅ»(P)RRHðĢ FLAG-h(P)RRIî̛ͧǃmÅ»˨ȩʖ˓ϗϩǿΫ(293T)ɉϰkr:FLAG-h(P)RR ~ǿΫ͉ƓȂ9ǿΫǜ FLAG ´¥ǜˡzǿΫɪŨ
œÑ@ @}ˣmǜˡϢ]x͍ɖʹʄkrǌ9ƪǑƼ͹ƏzƇɰ̫Ƽ͹ƏyşȈkr:(P)RR Ʋǫ´àÄ¤ȶ̓r}9FLAG-h(P)RR ͧǃǿΫaǿΫ̈ɚČ̏ʪk9ǜ FLAG ǜˡbƲǫkrÆ@¯Ϣ]rφč̔Ǩ}ux FLAG-h(P)RR `p}Ʋǫm´àÄ¤ȶĴɊkr:
̓r´àÄ¤ȶΒǫˡ SDS-PAGE yΖϭǌ9ƙʹʄ}ux´àÄ¤ȶÃà¼ħȢģkr:ϋ̣´àÄ¤ȶȶϹΖʭ}̛̿kr:Ʋǫ~ʢȶÊÜµà·°}ĳʭkr:
LƲĩ=ǣȈMFLAG-h(P)RR ~ƓȂˬ΍ΖbɪΫˡÑ@ @zĂ˿kr:FLAG-h(P)RR ͧǃǿΫϢ]rφč̔Ǩ~Ʋĩ9À¡¹É¨à»Þ@Ü}ρ]9ʚ̛ΖȕϹϏ 70 k ~Ãà¼(p70)bƷ
ɚir:(P)RR z p70 ~Ʋǫ Ü¬Ó͕̰ò˝̣}˒Ħkr:g~Ʋĩa9(P)RR ~ǿΫ͉ƓȂűǟzkx9ǿΫȶϺĀ}˝ȂmɪΫˡƓȂ¬¥½Ü}Ħ_x9ǿΫļϺĀy~ Ü¬Ó͕̰ò˝̣| p70 z~Ʋǫz]^ʋk]űǟb˝Ȃmħ͖ʢƺɚkr:

P66¶¾¾FªƋˈ      gƩȲãɿfSWv ?* 7 g˦ċKåɵÓȅ BŊ˗Ǥɂ BȶȩĶư  ɟȩɚƊ  G ĝʲɋ×ãțB ĝʲɋãțHLϋ̣M Ô¶Þ»Þ@ÈǧΦ~Ô´Á@Ü˫Ƚ;ÍÜ¢¬²@ÓFPsGƓȂƣÜ¨@Ü¢¬µ@±FAOXG}ȍˆò˝̣|Ô´Á@Üȍģ}ĺȔi<ĂΩ;Ô´Á@Ü˫Ƚ~ι˶}\rÒ»¨à¼ÛFMtGǈŻǺ~¬»¤ÞÓ c ¢¬µ@±ˠϹ~ȍˆũȶzkxϤŽmgza;Ô´Á@Ü˫Ƚďō}Ǥ^r} Mt-Ps ţ~ȍˆɯ͵ÃÚà®̏ʶm¬®¹Ób˝Ȃmzǣ_<ζƸſy;ǈŻǺǟʣmĄȕ¬»¤ÞÓ cFCyc1pG}̆ϋk;Pichia pastoris}`e PpCyc1p ~ AOX1 ͧǃ~Ťϝ}v]xĳʭǤur<LΩΪ=ƲĩM AOX1 ÊÞÕ@´~ͧǃƄ̾şȈkrzg;ǖŲɾƵĢyǪ]ͧǃÝÌÜȮkrb;ƶŲɾƵĢyp~ͧǃŒ˂}ϧʡix]r<v;P. pastoris ȍˆɿ˦͑ȱk;p}ǫ
ox AOX1 ~ͧǃʡǍkx]gzbȮir<̿Ϡ} PpCYC1 ÊÞÕ@´~ͧǃƄ̾şȈkrzg;̘ȍˆɾƵĢy;¥Ü¨@®;Ô´Á@Üz}ǖŲɾƵ}ͱp~̪ȸŋʢͫΖðĢ}y̘Ģkx]r<g~gz PpCYC1 ~ͧǃ;ɚİǧΦ CYC1 z̿Ϡ}ȍˆò˝̣}̪ȸbʡǍix]gzȮkx]<i};þ̬ȕ͝ĵŎ Ppcyc1$ ŎȄʪk;p~;ǃƦĳʭkr<
Ppcyc1$ Ŏ}`]xÔ´Á@Ü˫ȽǧˆƠ~ͧǃϘ̀şȈkrzg; ´Ú@±ŋʢ`ÎÜÓÜºÅ¼˰ʛˆǧˆŋʢύʧŎz̟̰̿~Ϙ̀bşȈirb;AOX ŋʢύʧŎ}ͱx̋kd̘˻Ȯkr<r;AOX1 ÊÞÕ@´ŋʢύʧŎzͱņkx;Ppcyc1$ Ŏ̘˻Ȯkr<ðɹ~gza;AOX ~ͧǃϘ̀}`]x PpCyc1p ɖϤ|ώŊȪvgzbʚ˙ir<

35

P67

    ª¼F ¨»ÀƩ|¼ºF|~~µgĠʕd˦ċKʶĉđ̊BȶȩĶưBɟȩɚƊGĝʲɋãțïã˼ț˜H
Lϋ̣M Ô¶Þ»Þ@ÈǧΦÔ´Á@Ü˫Ƚ~Ȁ;ÍÜ¢¬²@Ó͉yÔ´Á@Üȍģk;˫Ƚ̇ţˡ~ÎÜÓÜºÅ¼¢¬ÜÞ@® 5-ÛàȍFXul5PG}ǉ̛mgzy;Ô´Á@Üϗϩ˴ˆǿΫǟʣʣΖ}̀]x]<g~ Xul5P ~ƅƀ~r;˫Ƚ̇ţˡÍà»@®ÛàȍƧЅ̀dÈÜ¤»@®-1,6-Æ®ÛàȍÜ¼Ú@±FFBAGbɖϤ|ώŊ˲ux]gzbʚ˙i<g
y FBA ĳ̷ƦǧˆzkxɒΖ|Ƹſb|ixcrb;Ô´Á@Ü˫Ƚ}`eɖϤʢű͖}Ťmɶǿ|ΧǬ z{|]<pgyζƸſy P. pastoris ~Ô´Á@Ü˫Ƚ}Ťϝm FBA~ĳʭǤur<LΩΪ=ƲĩM
 P. pastoris }`]x FBA ~ǧˆŋʢ˙̛krzg;Ô´Á@Ü}`]xƊ]Ϙ̀bƺr<pgy§ÁÓº@´Ì@®̏rzg;Class-II FBA zǪ]ˎ̿ʢȮm 2 v~þ̬ȕ PpFBA1` PpFBA2 ƺ]skr<r;pq~ͧǃě̶şȈkrzg PpFBA1 ¥Ü¨@®}`]x;PpFBA2 Ô´Á@Ü}`]xǪ]ͧǃÝÌÜȮkr<r;Ô´Á@ÜϘ̀ɾƵĢ}`]x FBA ~ǿΫ͉ƓȂşȈkrzg9PpFba2p bÍÜ¢¬²@ÓùǤ¬¥½Üϖm~~;p~ƓȂǿΫȶy\ur<g~gza;P. pastoris  FBA  2 Ʉux`;ϴȼ
Ƈ}ǿΫȶƓȂƣǧˆy\b9PpFba1p ĳ̷Ʀ}Ťϝmǟʣǧˆ;PpFba2p Ô´Á@Ü}Ϙ̀iÔ´Á@Ü˫Ƚ}Ťϝmʋų FBA y\gzbȮǶir<

P68½||Fh¶¾¾FªƞŃg¶£F¼țÑgŤÖƮcPvKʏʣˣ˅ʩ BƦÏ̌ƪ Bɒ˝ț BɵɊȂˢ BȶȩĶư BɟȩɚƊ G ĝʲɋ×ãțïã˼ț˜B åƧ̆ɋ̆țê åƧɋƶũǺʸïŠHLϋ̣M Ô´Á@ÜϔĂ~˴ˆǂzkxϫϢycÔ¶Þ»Þ@ÈǿƖ;ɁϤ|ʁΕƇʧǿƖzkx˼x]<Ɨ͓;ʁΕ~ϣ΍;ˊ}˝ȂmɁϤ´àÄ¤ȶb̛̿i;p~zvzkx
Methylobacterium extorquens ϗϩ XoxF b̛̿ir 1)<ȝrt~¥Ü@Êy XoxF ~ű͖ĳʭʔ̇;XoxF1 bÝ@®FREEGò˝ƣÔ´Á@ÜºÅ¼Þ§½@±FMDHGy\gzɠxƺ]sk;p~ű͖̣ĳʭǤuxcr<ζƸſy;M. extorquens AM1 Ŏ~Ô´Á@Üʧā}`e REE ~̈́øʢz XoxF1 ~ű͖ͧǃ}`e REE ~ώŊ}v]xĳʭǤur<LΩΪ=ƲĩM Ô´Á@Üʧā}`eƘ˛ò˝ʢşȈkrzg;Ca ~|n;REE y\ La;Ce;Pr;Nd ̩Ħkrͣ˽y AM1 ŎɓΖ|Ô´Á@ÜʧāȮkr<AM1 Ŏ Ca ò˝ƣ MDH þ̬ȕz
kx mxaF 9La ò˝ƣ MDH þ̬ȕzkx xoxF1 Ȫvgza 2);pq~þ̬ȕ͝ĵŎ !mxaFŎ;!xoxF1 ŎϢ]xÔ´Á@ÜʧāşȈkr<p~Ʋĩ;!mxaF Ŏ Ca yÔ´Á@ÜʧāȮin;!xoxF1 Ŏ REE y z{ʧāyc|]~|n;Ca yʧā̀đbşȈir<i}ύʧŎ~ÎÜÓÜºÅ¼ʧāşȈkrzg;Ƙ˛}ʧā~ˎýƺ|aur<ðɹ;AM1 Ŏ~Ô´Á@Üʧā}`]x;REE  MDH ~ŋʢͧǃ}ˬcdĈƐϝ_x`;Ô
¶Þ»Þ@ÈǿƖ~ʁΕϣɹy~ű͖ͧǃ}ˬcdŤϝkx]gzbʚ˙ir<1)Delmotte et al., 2009. PNAS 106: 16428, 2) Nakagawa T et al., 2012. PLoS ONE 7: e50480.

36

P69
ǙöƋˈg||¼§ɆȖĠƃfSWv|´ÀgĠʕd˦ċKĮ˄ɵ̘óBȶȩĶưBɟȩɚƊGĝʲɋãțïã˼ț˜H
Lϋ̣M ɚİǧΦ´Á@Üͧǧ~ĭ̟y˫Ƚ̇ţˡzkx°»ÜºÅ¼FAAGȋʧmb;g~AA }ȯ~˫ȽbˈĽi<g~r;ɚİǧΦ´Á@Üͧǧ~Ȁ;AA ~ͅʢĴͶmͻϤb\<Aranda ɚİǧΦ~ AA Ϙ̀ʢþ̬ȕƠΧǬkx`;g}¶ÒàǫʣƦFTHI4;THI6Gz¶ÒàϓˑƦFTHI7THI72Gbťx]r 1)<g~gz;ɚİǧΦb AA®»Ý®Ģy¶ÒàϤŽkx]ħ͖ʢȮkx`;¶Òàb AA ˤʢűǟ}`eƻĄȕ~zvy\gzbʚ˙i~~;p~ɶǿτay|]<pgy;ζƸſyɚİǧΦ~ AA ˤʢűǟ}`e¶Òà~ɖϤʢτa}m<LΩΪ=ƲĩM                   ɚİǧΦ;ʧāͣ˽~¶Òà~̩ĦϹ}ͱЀkx AA ˤʢ͖Ń̓krgza;¶Òàb AA ˤʢ}`]xɖϤ|ώŊȪvgzbτaz|ur<pgy;thi4! Ŏ;thi6! Ŏ;thi7! Ŏ;thi72! Ŏ~ AA ŗɇʢşȈkrzg;thi7! Ŏ~b AA ŗɇʢȮk;ɚİǧΦǿΫļa¶Òàŋͧ}ɂǰgzy AA ˤʢ͖Ń̓kx]gzbȮir<r;¶Òà~ AA z~ͬěʢ in vitro yşȈkrzg;¶Òà̓ʳ AA z~ͬěʢȮi|aur<ux;¶Òà AA ® Ìà­Ö@zkx~ű͖|d;ǿΫ͉yΥǧˆzkx AA ˤʢ}ūϝkx]gzbʚ˙ir<¶Òà»Úà®¦»Ú@±~Υǧˆy\;»Úà®¦»Ú@±bű͖mÍà»@®ÛàȍƧЅ NADPH ƅƀƦzkx AA ˤʢűǟyɖϤ|ώŊȪv 2)<g~gz;AA ®»Ý®Ģy¶ÒàbɖϤ|ΥĄȕy\;NADPH ˠϹ}ƅƀmÍà»@®ÛàȍƧЅ~ŋʢͧǃ}`]xɖϤ|ώŊȪvgzȮkr<1) Aranda A, et al. (2004) Appl Environ Microbiol, 70: 1913. 2) Matsufuji Y, et al. (2008) Yeast, 25: 825.

P70ʹ̒ƋˈgȁĦŒƹǌ˜ßɥÖƮ  !gʃɪdûȣKǊÏƲ̇ƌ Bɋɨ˔ɶ Bǥȩȅű BȩɀÞǧ Bɀǳƅƭ Bŀǵƅʻ 
G ˛ɋ×ȵ˧B ˛ɋ×ț˜ʖB ɟáɋ×̆ŻHLϋ̣M ΖЃǧΦ9ɈσƸſ}`]xϖϢ|ÕºÜʧΕy\9gy}Ϡ?|ƧȫɈσđ̑þ̬ȕbͧƺix]:ƧȫɈσΖЃk|]ɿƌĢy~ǿΫɑ˸~ʧ˝ϯz̛ŷi:ζƸſ9Ǫͧǃ}ΖЃǧΦ~ƧȫɈσđ̑mʋr|þ̬ȕ~˳ȇzp~ű͖~ĳʭϋ̣zkx]:LΩΪ=ƲĩM
 ΖЃǧΦ~§ÁÒ·¤ÚÉÚÛ@Ϣ]x9Ϗ 3000 Ŏ~®¤Û@¾à¥Ǥur:p~Ʋĩ97
ǆ ~ Ƨ ȫ Ɉ σ đ ̑ þ ̬ ȕ  ͧ ƺ k r : g ~ ̇ a  ű ͖ b μ ˼ y \ u r SPBC16A3.08c (ð Ģ 9
16A3.08c )}̉ϋkĳʭǤur:16A3.08c ~þ̬ȕȋΕ in vitro y¥¾àȒɖǺǟ˔ (G4) }ƲǫmɚİǧΦ~ Ylr150w ´àÄ¤ȶzÒÁȍ͡Ђɹ~ϿȨʢȮk916A3.08c ȯʒ G4 z invitro yƲǫkr:i}916A3.08c ɚİǧΦ Ylr150w Ư˞Ŏ~ ÈàŗɇʢˎΥkr:g
~Ʋĩa 16A3.08c  Ylr150w ~ű͖̣ÎÕÞ¥szȮǶirr9 oga1!" (ortholog of
G4-associated protein) zσςkr: oga1!~ĭɺͧǃ}9ƧȫɈσđ̑ącŵgm˟}Ǫġ=̘ġŗɇʢ9ʳǫ͖~̘Ģ|{Ȯmgzbτaz|ur:r9 oga1!~ÎÕÞ¥y\
Ylr150w 9Ɉσ~ʡǍ}ŤzȮǶir TOR ƧЅ}Ťϝkx]zϜ˙ix]: ĭƂ}ΧǬir9TOR ƧЅ~ǟʣĄȕy\ tor1!Ư˞krŎ~;ǃƣ Oga1 ǪͧǃŎ~;ǃƣzϿȨkx]r:p~r9 oga1!b tor1!~Ģϱy΋}ʡǍix]z]^ħ͖ʢbȮǶir:

37

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Œƹǌ˜ßɥÖƮ *3 h   ½fãɿZbɳǂÖƮ :- fsuČȖêYwvKɼƣȏƫ Bɋɨ˔ɶ BʌʁɘëƮ Bɀǳƅƭ Bŀǵƅʻ 
G ˛ɋ×ȵ˧ïĈB ˛ɋ×ț˜ʖB ɟáɋ̆ŻHLϋ̣M
 Ecl1 Ǫͧǃ}ΖЃǧΦ~ƧȫɈσđ̑mþ̬ȕzkx̴Ƹſȳyʋų}ͧƺir:
Ecl1  80 ÒÁȍ~ɪi|´àÄ¤ȶy\9Ů˼~ÎÕÞ¥þ̬ȕb˝Ȃon9Ϝˋycű͖¼Ôàń͑ycx]|]r9p~ű͖μs}΃τ|̫bˠ]:Ecl1 ~Ǫͧǃ H2O2 ®»Ý
®}ˣkxˤʢȮmgza9Ecl1 ƧȫɈσ~đ̑}Ħ_9H2O2 ®»Ý®~ě̶}ʍdŤϝkx]gzbˋ̛ix]:Į?g~ Ecl1 z H2O2 ®»Ý®z~ŤЄ}̆ϋk9Ɉσzø|urş̫a Ecl1 ~ĳʭȣr:
LΩΪ=ƲĩM
 Ecl1 ~Ǫͧǃ H2O2 ®»Ý®}ˣkxˤʢȮmb9ͬˣ} Ecl1 ~Ư˞ H2O2 ®»Ý®}ˣkx ŗ ɇ ʢ  Ȯ k r : H2O2 ® » Ý ® }   ecl1+ ~ ͧ ǃ Ϲ ~ Ξ ģ  ̏  r Ʋ ĩ 9 H2O2 ̩ Ħ ǌ }ecl1+-mRNA Ϲb˒Ħkx`9H2O2 ®»Ý®b ecl1+-mRNA ~̪ȸϘ̀mgzbͪτkr:i
}9g~̪ȸϘ̀bΖЃǧΦ~ H2O2 ®»Ý®ě̶}`]xɖϤ|ώŊ˲^ Sty1 MAPK }ò˝mgzbτaz|urr9p~Ģϱyű͖m̪ȸĄȕ Atf1 z ecl1+~ H2O2 ®»Ý®}̪ȸϘ̀z~Ťơ}v]xǞ|ĳʭǤur: atf1+~Ư˞Ŏy H2O2 ®»Ý®} ecl1+~ͧǃϹ~˒Ħbƺ|aurgza9ecl1+~̪ȸϘ̀ Atf1 }ò˝mgzbτaz|9i} ChIP assay }ux Atf1 b ecl1+~ɹϱ}̓
ʳƲǫmgzbń͑ir:˟Ω9Ecl1 ~Ǫͧǃ}ux ´Ú@±¨@¼m ctt1+þ̬ȕ~ͧǃɹɭbŵgb9g~ͧǃɹɭ9H2O2 ˤʢ~ɹɭ atf1+Ư˞Ŏ}`]x͑r:Ħ_x9
Sty1NAtf1 ƧЅŋʢģmz Ecl1 ͷò˝̣} ctt1+~ͧǃbɹɭkr:g~Ʋĩa9Atf1 Ecl1 }ò˝mƧЅ|}ò˝k|]ƧЅy ctt1+~ͧǃϘ̀Ǥ^ħ͖ʢbȮǶir:P72ʹ̒Ƌˈh ,B'5 gŭčcȖʹêyĩX[KȤɵ˙ÌƮ Bɋɨ˔ɶ BʌʁɘëƮ Bɀǳƅƭ Bŀǵƅʻ 
G ˛ɋ×ȵ˧ïĈB ˛ɋ×ț˜ʖB ɟáɋ̆ŻHLϋ̣M
ΖЃǧΦ;ĊϦά΅|ɾƵĢyϖȞΖЃǤ];ĊϦbǊŌmzΖЃ̙țm<p~Ȁ9ǿΫ̿ȓbʳǫk;ǁʟΖЃƧxΫȕƤʣm<g~ĭ̟ʢΖģzǄ];̄ˆǂ˴ˆǂ~ǊŌĢyϘ̀igzb˼x]<̴Ƹſȳy;ǪͧǃmzΖЃǧΦ~ƧȫɈσđ̑mʋų|ˎ̿þ̬ȕƠ ecl1+, ecl2+, ecl3+(Extender of Chronological Lifespan) ƺɚk;g Ecl1 ÈÒÛ@zσςkr<kakp~
̽c}Ťkx΃τ|̫bˠ]<gy} Ecl1 ÈÒÛ@Ǫͧǃmz;ʳǫΫȕƤʣϯbɹɭmgzƺɚkx]<ζƸſy Ecl1 ÈÒÛ@zʢΖģz~Ťơʢτa}mgz
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