1

S01

Non Ribosomal Peptide

Synthase NRPS NRPS A-T- C-

ATP tRNA

PM N

NVKDR NVKDGPTNRPS A-

C-

PM NDpgA Type III PKS

NVKDR NVKDGPT NRPS

38NVKDR NVKDGPT C-

PM NVKDR NVKDGPTDHFGA- NRPS 1 649

T- NRPS 2 81 C- NRPS 3 441NRPS -

C- ATP ORFATP

2

S02

NRPS NRPS

NRPSPoly(!-L-lysine)

25 35 , Fig. 1 streptothricin !- 1～7 Fig. 2NRPS Nature Chem. Biol., 4, 766-772, 2008;

Nature Chem. Biol., 8, 791-797, 2012 NRPS

Fig. 2 streptothricin (ST)

Fig. 1 Poly(!-L-lysine)

3

P01

0 2 4 6 7 HPLCDaidzin Genistin

44 4

HPLCGenistein

Daidzein Glycitein

P02

" A" A" APP "

A"

APPA"

A" SAMP8

T ThT TEM

A"1-42 ThT TEMA" 16

SAMP8 0.05% 4 ELISA A"A"

A" A"PCR A"1-42

ADAM10 BACE1 "ADAM10

in vitro A"in vivo A"1-42

4

P03 Chlorogenic Acid and its Metabolites as Antimicrobial Agents

!Faisal Kabir, Shigeru Katayama, Haruka Sugiyama, Soichiro Nakamura (Dept. of Bioscience and Biotechnology, Shinshu University)

Objectives 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 of action of CGA and its metabolites against a gram-negative bacterium Escherichia coli were investigated.

Methods and Results 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 remarkable antimicrobial effect against E. coli under thermal stress at 50ºC. E. coli cells (106/ml) completely disappeared in 2 ml of PBS coexisting with 30 mM ferulic acid and isoferulic acid incubated at 50ºC for 3 hours, followed by benzoic acid and hydroxy benzoic acid incubated at 50ºC for 6 hours. These results indicate that CGA and its metabolites possess potent antimicrobial activities against E. coli. P04

Acanthasteroside B3 ,

, . acanthasterodside B3 ,

. 1 .

Ergosterol 2 , 3

. , BF3-Et2O , OsO4 . BF3-Et2O ,

, 4 . OsO4, 1 .

acanthasteroside B3

2 3 4

3steps

1

4steps

5

P05 An antifungal compound, methyl 3-O-methylgallate from Tunisian halophyte, Tamarix aphylla

Asma 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 food and/or cosmetic preservatives. The use of these chemicals are nowadays raising concerns related to food contamination 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 the purification 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 the strongest activity. Its effect (MIC 12 mM) was even stronger than OPP (MIC 18 mM) and ethyl p-hydroxybenzoate (MIC19 mM) in the case of Fusarium. The structure activity relationship study revealed that methylation of meta-hydroxyl group in methyl gallate is a factor of antifungal efficiency enhancement. P06

Effects of gambir (Uncaria Gambir Roxb.) extract in a mouse model of allergic asthma !Pramita Sari Anungputri1,2 Nami Nakagawa1 Masahiro Nishio1, Hayato Umekawa1 (1Grad. School of

Bioresources, Mie Univ., 2Fac. of Agriculture, Sriwijaya Univ., Indonesia)

Introduction Allergic asthma is a respiratory inflammatory disease, characterized by reversible obstruction and hyper-responsiveness in airway. Th2 cytokines, such as IL4, stimulate production of allergen-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).

Methods and Results 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 and bronchoalveolar lavage fluid (BALF). HE-stain showed that gambir extract-treated mice significantly reduced the airway smooth muscle thickness. Moreover, gambir extract had a tendency 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 of allergic asthma. Keywords: gambir extract, catechin, asthma

OCH3

O

H3CO

HO

HO

6

P07 ABCG2

ABC ABCG2 ABC ABCB1ABC

1 ABCABCB1 ABCG2

ABCG2

Flp-In System (Invitrogen ) 12 FRT ABCB1ABCG2 ABCG2 (Tamura et al., J. Exp.

Ther. Oncol., 6, 1-11, 2006.) 5 # 103 cells/well 96 well plate 2472 MTT 3 20% SDS

. CO2 630 nm570 nm ABCB1

ABCG2ABCG2 3 1 ABCG2 Q141K

ABCG2

P08

ABC ABCB1 (

) ABCB1Calcein assay ATPase assay

(807 g)

epimagnolin (5.05 g) fargesin (3.27 g)sesamin sesamin

Calcein assay ABCB1 Calcein-AMcalcein ATPase assay ABCB1

ATP ABCB1 ATP37 epimagnolin calcein

ATP epimagnolin ABCB1

7

P09 destruxin E

M.anisopliae destruxinEED50 = 4.8 nM (Nakagawa et al., Bone, 33, 443-455, 2003.) destruxin E

(Yoshida et al., Org. Lett., 12, 3792-3795, 2010.)destruxin E destruxin E

destruxin E

Slc:ddY 100 ng/ml GST-RANKL 1 µg/ml M-CSF 1 ng/ml TGF-!1 100-mm diameter type I collagen-coated culture dish 72

3 # 104 cells/well half area 96 well plate 100 ng/ml GST-RANKL 1 µg/ml M-CSF 1 ng/ml TGF-!1 24

destruxin E 100 ng/ml GST-RANKL1 µg/ml M-CSF 24

destruxin EN- N- N-

N- "-destruxin E

P10

" 1 1, 2 1 1 2( )

(Wasabia japonica Matsumura)

MS NMR9 2 2 1

6 2FRAP assay

Trolox Trolox 2all-trans-lutein

8

P11 Pterogyne nitens nitensidine A ABCB1

1 1 1 2 Luis Octavio Regasini3 Vanderlan da Silva Bolzani3

Gert Fricker4 1 Thomas Efferth5 1 2 CLST 3 4 5

Pterogyne nitens nitensidine A

[1] nitensidine AABC ABCB1 nitensidine A

[2]

T CCRF-CEM CCRF-CEMdoxorubicin ABCB1 CEM/ADR5000 MTT assay Calcein asay ATPase assay ABCB1 nitensidine A MTT assay

CCRF-CEM CEM/ADR5000 nitensidine AABCB1 verapamil

Calcein assay CEM/ADR5000 nitensidine A Calcein-AMcalcein verapamil

nitensidine A calceinATPase assay CEM/ADR5000 ABCB1 nitensidine AATP ABCB1 ATP 37

ATP nitensidine A Hanes-Woolf Vmax = 125 µMKm = 6.4 µM Autodock4 ABCB1 nitensidine A

verapamil nitensidine A verapamil

nitensidine A ABCB1ABCB1 nitensidine A

1 Tajima et al., Cytotechnology, in press. 2 Tajima et al., Phytomedicine, in press. P12

( )

staphylococcal enterotoxin A (SEA) SEA

～6SEA : ( : 0.1~3.0 mM)

Staphylococcus aureus C-29 ( : 103~104 CFU/mL) 37 °C 24 SEASEA : SEA (5 µg/mL) ( : 0.5 3.0 mM)

37 °C 24 SEA 3～6SEA -SEA 2

6SEA SEA

SEA

9

P13

Retinol dehydrogenase 8 RDH8all-trans-retinal atRAL all-trans-retinol atROL RDH8

atRAL atROL RDH8 atRALatRAL atROL

RDH8 RDH8

RDH8RDH8

NIH3T3 RDH8Delphinidin-3-glucoside Cyanidin-3-glucoside Pelargonidin-3-glucoside 3Epigallocatechin gallate Epicatechin gallate 2 RDH8

atROLRDH8

B OH atROLRDH8

P14

Mentha arvensis

Agrobacterium metallothionein MTL1 M arvensis Ma-B Ma-C

Ma-B Ma-CM arvensis

M arvensis Ma-B Ma-C 0 1,000 mg/L

26 16500 700 6 M 0.1 M 200 400

Ma-B , Ma-C

M arvensis

10

P15

B1, B2, B3, B4 C2

B3, C2 B1,

B2, B4 phloroglucinol methyl 3,4,5-trihydroxybenzoate

B1 Yb(OTf)3 66% B2 AgBF476% B4 Yb(OTf)3 78% 2

B1 (3), B2 (4), B4 (5) epigallocatechin gallate (EGCG)

3 EGCG

P16

5 1

Helianthus tuberosus

10 70% 3min 12,000 rpm

Cry j 1 Balb/c 540 IgE

HPLC A～E Balb/c Cry j 1 3 IL-4 B IL-4

THP-1 A～E Cry j 1 3 MHC classHLA-DR

BHLA-DRB EI-MS NMR

1-(3’,4’-dihydroxycinnamoyl)cyclopentane-2,3-diol 1. 1

11

P17 Procyanidin B2,B3-gallate

a, a, b, b, b, c, d, b, a ( a,

b c, d )

Procyanidingallate DNA

Yb(OTf)3 gallate procyanidin B2-3-O-gallate, 3’’-O-gallate 3,3’’-di-O-gallate procyanidin B3-3-O-gallate, 3’’-O-gallate3,3’’-di-O-gallate gallate

(+)-Catechin ($)-epicatechin

Yb(OTf)3

Pd/C

gallate

EGCG prodelphinidin B31)

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

Yb(OTf)3O

OBn

OBn

OBn

OEE

BnO

OR1

O

OBn

OBn

OBnBnO

OR2

H2, Pd/C

Yb(OTf)3O

OBn

OBn

OBn

OEE

BnO

OR1

O

OBn

OBn

OBnBnO

OR2

H2, Pd/C

求核剤

求核剤

O

OR1OBn

OBn

OBnBnO

BnO O

OBn

OBn

OBn

OR2

O

OR1OBn

OBn

OBnBnO

BnO O

OBn

OBn

OBn

OR2

O

OR1OH

OH

OHHO

HO O

OH

OH

OH

OR2

O

OR1OH

OH

OHHO

HO O

OH

OH

OH

OR2

+

求電子剤

53% (R1 = G, R2 = H)22% (R1 = H, R2 = G)43% (R1 = G, R2 = G)

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

+

求電子剤

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

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

12

P19 Cell-free Synthesis of Horseradish Peroxidase and Manganese Peroxidase

for Bead Display-based High-throughput Screening Bo ZHU, Ryoko NINOMIYA, Takaaki KOJIMA, Yugo IWASAKI, Hideo NAKANO

(Dept. Bioeng., Grad. Sch. Bioagr. Sci., Nagoya Univ.)

PurposeA cell-free protein synthesis system can produce various types of proteins directly from DNA templates such

as PCR products, and therefore attracts great attention as an alternative protein synthesis system especially for high-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.

Methods and results 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 of the 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

Clostridium josui

9 (Cel9A, Cel9B, Cel9C, Cel9D, Cel9E) Cel9A Cel9B

C. josui cel9A, cel9B, cel9C, cel9D

cel9E pET-28a BL21 (DE345-60 pH 6.0-7.0 Cel9A, Cel9B,

Cel9C Cel9E barley -D-glucan Cel9D Konjac glucomannanCel9A Cel9B Cel9A

3 1-4 2 1-4Cel9B 2 1-4

Cel9A Cel9B CBM3c CBM4Cel9A Cel9B

Cel9A Cel9B CBM

13

P21

Clostridium josui CBM3CBM3

Clostridium josui CipA CBM3 CBM3CBM3 CBM3

CBM3

CBM3CBM3

CBM3CBM3

P22

14

P23

90MPaCelluclast 1.5L

1)

Celluclast 1.5L (Novozymes) 4T ( )

3S ( ) (Avicel) 100mg 0.25M 5ml (pH4.5) 1 mg (0.1MPa) (90MPa) 506 24 RI HPLC

p- pNPG D- -1,5- b-

24Celluclast1.5L 4T 3S

pNPG "-

D- -1,5-6

Celluclast 1.5L "- 1) 2013 , 2C12p11 (2013) P24

1001

13 m 7 m 3 m25 cm

1

sFDA EB 65150 cm (25~75 cm)

90 EB 109 1010 cells/gsFDA (225 cm)

(25 cm) sFDA

15

P25

CO2

CMC 50℃ CMCCongoRed

PASC CMC16s rRNA Streptomyces

Bacillus Chitinophaga

P26

AspergillusManR McmA

pH PacC pHManR/McmA

A. nidulans 7pacC (pH 4.2) (pH 8.2)

qRT-PCRpacC

pacC RNA-sequencing PacC ManR

PacC ManR pacC manRPacC ManR

TLC pacCPacC ManR

16

P27

AA

AA AAAA

GSH GSH AAAA 1)

GSH 2) AA GSHGSH

DUG AA

DUG GSH Cys-Gly Cysdug1! dug2! dug3! AA

dug2! AA GSH Cys-Gly Dug2/3AA AA GSH

AA AA1) Cys-Gly AA 3) GSH Cys-Gly

AA 1) AMB 97: 297 (2013) 2) JBC 287: 4552 (2012) 3) Alcohol Clin Exp Res 27: 1613 (2003). P28

AA DNA

AAAA

ADH AA ALD AA

5 ADH 5 ALD ADH

AA adh2!adh3!adh4!adh5!AA Adh1p

ALD AAald6! ald6! AA ald6!

AAald6! AA

PPP ald6! AAAA Ald6p AA PPP NADPH

17

P29

P. methanolica MOD1

2 AOD MOD1 MOD2AOD AOD

AODP. methanolica AOD

AOD

P. methanolica AOD1% AOD

3 AODMod1p Mod2p

AOD Mod1p Mod2p

2 AOD

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） Purpose Low utilization of sago starch (Metroxylon sagu) as food source gives a possibility to use it for

bioethanol production. Considering waste reduction, both starch and fiber of sago should be used in 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.

Experimental Methods & Results The fermentation media containing different concentrations of glucose, xylose and ethanol in

yeast nitrogen based (YNB) media were prepared for the infrared spectroscopic measurements. The spectra were collected using a Fourier transform infrared (FT-IR) spectrometer equipped with an 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 these wavenumbers 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 during bioethanol fermentation.

18

P31 A2

pelB Streptomyces violaceoruberA2 svPLA2

svPLA2ELISA

svPLA2 pET

Skp100 mL 0.1 mg

PLA2-scFv PLA2ELISA

P32

19

P33 Uniform Emulsion for Biochemical Applications Using Inexpensive Flow-Focusing Device !Marsel MURZABAEV1, Takaaki KOJIMA1, Isao KOBAYASHI2, Hideo NAKANO1

(1Dept. Bioeng., Grad. Sch. Bioagr. Sci., Nagoya Univ., 2Food Engineering Div., National Food Research Inst.)

[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 in size, 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 generate monodisperse 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. Average volume 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

28 kDa LP28 18 kDa LP18

LP1877 N LP18

LP18 NLP18

LP18

LP18 N N LP18

MA104N

SDS-PAGE native-PAGESDS LP native-PAGE

native-PAGE NLP18 LP18N

20

P35

DFAI DFAI 50

H10-2 DFAI

H10-2 16S DNADNA Arthrobacter ramosus

98.6 ArthrobacterArthrobacter sp. H10-2

DFAI [EC4.2.2.17]35

17 pH 5.5 4075 30

SDS-PAGE 39kDa P36

YAS

21

P37

Oryzias latipes

8

5 2

TALEN TILLING

P38

TGaseTGase 8

TGase

mRNART-PCR, Real- Time PCR, in situ hybridization

TGase1 , TGase2

RT-PCR 21 mRNAReal-Time PCR TGase1 TGase2

In situ HybridizationTGase1, TGase2 mRNA

TGase1 TGase2

22

P39

TGase

TGase8

TGase12 ( )

TG2

TG7

TG2 TG7

TGase

Kuramoto and Yamasaki et al. Arch. Biochem. Biophys, (2013) P40

DNA

DNA S (ori) DNA S

72DNADNA

AB.9 ( )DNA

DNADNA

DNADNA

DNA

23

P41

DNA AP (apurinic/apyrimidinic site)AP DNA AP

APAP

Shewanella violacea AP (SviExo)

SviExo His

DNA SviExoAP 3’ 5’

3’#5’ Exo SviExo34 250 mM NaCl pH 9 34

SviExoAP

SviExo P42

, , . , ,

. , ATP, Mg2+ . Luciola lateralis

, . , , ,

. , , .

,

(LlLuc1)LlLuc2 . , PCR

2. , in vitro in

vivo . , LlLuc2

. , LlLuc1 LlLuc2,

LlLuc1 , LlLuc2 .

24

P43

24 LC/ESI-TOF-MS2 L- 1

P44

Methanosarcina acetivorans

M. acetivorans

LC-MSn

GC-MS $-1

$- 1 M. acetivorans

25

P45

Aspergillus AmyR

A. nidulans AmyR (IM)

AmyR GFP AmyR

A. nidulans GFP-AmyR

GFP-AmyR A. nidulans SGZ5

(ZEISS LSM700 ) GFP-AmyR

Rhodamine phalloidin GFP-AmyR AmyR

D (CD) GFP-AmyR

B (MB) GFP-AmyR AmyR

P46

Coprinopsis cinerea

Coprinopsis cinerea

C. cinerea

C. cinerea Yeast Malt Glucose 3 (30 °C) 4

12 300

30 40 MALDI-TOF/TOF-MS

MASCOT100

hypothetical protein PCR mRNA

26

P47

2A (SEA) SEA

SEASEA

SEA Staphylococcus aureus No.29 (EMr SEA+ ) S. aureus No.77 (KMr SEA-) SEA

No.29 No.77 6 ( ) pH 6.037 °C

NaCl (7.5%) MgCl2

(100 mM) No.29No.77

SEA P48

cis-

cis- EDLF K+

Diels-Alder IMDA cis-

2- 5 1 IMDA

cis- 2IMDA EDLF

n RO

O

O

RO

O

OnIMDAreaction

OH

OH

HO

OH

OHHHO

possible structure ofInagami-Tamura's EDLF

*

1 (n = 0, 1)2

OO

OH

OAcOAc

H

H

OAcOAcAcO

OCO2MeH

correolide

27

P49

NGF ( )

PC12 NGFAcanthaster planci acanthasteroside B3

acanthasteroside B3 NGFacanthasteroside B3

9.6 kg ODS 4

acanthasteroside B3 (Fr. 2) silica gel2 Fr. 2-2 Fr. 2-6 Fr. 2-6 acanthasteroside B3 (70 mg)

Fr. 2-2

acanthasteroside B3 Acanthasteroside B3PC12 NGF 3 NGF

PC12 acanthasteroside B3acanthasteroside B3 NGF

P50

streptothricin ST streptolidine lactam carbamoyl

gulosamine !-lysineST

streptolidine lactamST

STorf18 streptolidine lactam carbamoyl gulosaminestreptothrisamine Nature Chem. Biol., 8, 791-797, 2012

STST %-Red recombinase in-frame

34 kbStreptomyces avermitilis SUKA17 LCMS

carbamoyl transferase orf17 decarbamoyl-N-acetyl streptothrisamine orf8 orf11 orf13

streptolidine lactam

acanthasteroside B3

28

P51 ABCG2

ABC ABCG2

NN ABCG2 ABCG2

ABCG2

Flp-In System (Invitrogen ) 12 FRTABCG2(WT) N ABCG2(N596Q)

ABCG2(C603G) NABCG2(N596Q/C603G)

ABCG2SN-38 Mitoxantrone ABCG2(N596Q)

ABCG2(C603G) ABCG2(N596Q/C603G) ABCG2(WT)SN-38 ABCG2 ABCG2 mRNAABCG2(N596Q) ABCG2(C603G) ABCG2(N596Q/C603G)

ABCG2(WT) 2 1 NABCG2

N ABCG2

P52

BR BRI1 BRL1 BRL3 BR

SERK1-4BR BRI1-

BL BL " BRI1& BR

A 2&,3&-diol BRI1BL " BRI1 & A 2&,3&-diol SERKs

BRI1 SERKsBL 2&,3&-diol BL-2,3-acetonide

BR BRBL-2,3-acetonide BRI1 SERKs

BRI1 2 3BRI1BL BL HBL

2-O-alkyl-HBL 3-O-alkyl-HBL2-O-alkyl-HBL BL

29

P53

2C(PP2C) PPM Ser/Thr

PP2C(RANKL)

PP2C glabridin RANKLMAPK NFATc1

glabridinRANKL/RANK glabridin

RAW264 RANKL/RANK

glabridin RANKLNF- B glabridin glabridin I B

IKK / IKK NF- BI B NF- B

glabridin NF- B MAPKNF- B PP2C TAK1

glabridin glabridin TAK1glabridin PP2C TAK1 RANKL/RANK

P54

1)

﨑

25

hTAS2Rs G G#16gust44 HEK293T96 well FLEXstationTM

[Ca2+]iTAS2R 392) TAS2R14 [Ca2+]i

TAS2R39TAS2R39 TAS2R39

ECg TAS2R14 EGCg

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)

30

P55

Hydrangea macrophylla3- pH

0.3 MS 5 mg/L 2,4-24 16 (4500 lx) 3

HPLC3- 3-

( )

P56

(H+-PPase) (PPi)PPi

PPiPPi

H+-PPase fugu5 NO3- MGRL

H+-PPase PPi

PPase IPP1 fugu5

IPP1/fugu5 WT PPase PPiPPi

NH4+

NH4+

(Pi)Pi Pi

PPi PPi

31

P57

SAMclavata SAM

CLV3 CLV3 CCLE 13 4 7 Pro

7 CLV3 CLV3

CLE Arg N CLV3

CLV3

MALDI-TOF MS Arg N

SDS-PAGE 80 kDa

P58

(SAM) CLV3

CLV3

-4- (P4H)

P4H 11 CLV3total RNA RT-PCR

11 8 P4H

CLV3 in vitro CLV3 CLEP4H

32

P59

AtVHP1 GFP

VHP1-GFP

VHP1-GFPVHP1

GFPGFP (Kd = 110 µM)

Ara206Lys GFPGFP

GFP

GFP YFP

Ara206Lys P60

stomagenstomagen

stomagenstomagen

stomagen

stomagen

stomagen stomagen

stomagenstomagen

stomagen

stomagen C 45

33

P61

%-IIAEK Caco-2

Caco-2 in vitro

Caco-2 2mM 24

ATP-binding cassette transporter A1ABCA1 27 CYP27A1

mRNA ABCA1Caco-2 ABCA1 luciferase

-928 +107 2mM12 ABCA1

ABCA1 mRNAABCA1 -126 +107 LXR/RXR -77～-55

ABCA1CYP27A1 mRNA ABCA1

, mRNAABCA1 , CYP27A1 LXR/RXR

P62

(LDL)LDL LDL (LDLR) LDLR

Proprotein convertase subtilisin/kexin type 9 (PCSK9)LDL

Epigallocatechin gallate (EGCG) HepG2LDLR mRNA (Br. J. Nutr. 107, 769-773 (2012))

EGCG LDLR mRNA PCSK9 Annexin A2 PCSK9EGCG HepG2 .

HepG2 JNK ERK p38 EGCG 24 LDLR mRNA

EGCG ERK EGCGAnnexin A2 24 LDLR mRNA EGCG

PCSK9 mRNA EGCGPCSK9 mRNA 3 12 18 PCSK9

mRNA PCSK9 EGCG mRNAEGCG 3 EGCG

PCSK9 Annexin A2 ERK LDLR mRNALDL

34

P63

P64

B6 B6 B6 Hcy

Hcy B6B6

B6 Hcy

4 Wistar 6 n 7 1 kg 9 g L-MetC B6 D 0.49% 0.98%

1.97% 3.94% B6 D35 Hcy 6 n

7 C D 1.98% 3.97% 7.94%B6 0.061% B6 B6

Hcy C D D0.49 Hcy C D

D 1.98 B6Met

35

P65

( ) (P)RR

(P)RR C(P)RR C (

) N(P)RR

(P)RR FLAG (P)RR FLAG-h(P)RR

(293T) FLAG-h(P)RR FLAG(P)RR

FLAG-h(P)RR FLAGFLAG-h(P)RR

SDS-PAGE

FLAG-h(P)RR FLAG-h(P)RR70 k (p70)

(P)RR p70 (P)RR

p70 P66

PsAOX

Mt cMt-Ps

c Cyc1p Pichia pastorisPpCyc1p AOX1

AOX1

P. pastorisAOX1 PpCYC1

PpCYC1 CYC1Ppcyc1$

Ppcyc1$AOX

AOX1 Ppcyc1$AOX PpCyc1p

36

P67

5- Xul5PXul5P

-1,6- FBAFBA

P. pastoris FBA

P. pastoris FBA

Class-II FBA 2 PpFBA1PpFBA2 PpFBA1

PpFBA2FBA PpFba2p

P. pastoris FBA 2PpFba1p PpFba2p

FBA P68

Methylobacterium extorquens XoxF 1) XoxFXoxF1 REE MDH

M. extorquens AM1REE XoxF1 REE

Ca REE La Ce Pr

Nd AM1 AM1 Ca MDHmxaF La MDH xoxF1 2) !mxaF!xoxF1 !mxaF Ca

!xoxF1 REE Ca

AM1 REE MDH

1) Delmotte et al., 2009. PNAS 106: 16428, 2) Nakagawa T et al., 2012. PLoS ONE 7: e50480.

37

P69

AAAA AA

Aranda AATHI4 THI6 THI7 THI72 1) AA

AA

AA

AAAA thi4! thi6! thi7!

thi72! AA thi7! AAAA AA

in vitro AAAA AA

NADPH AA 2)

AA NADPH

1) Aranda A, et al. (2004) Appl Environ Microbiol, 70: 1913. 2) Matsufuji Y, et al. (2008) Yeast, 25: 825.

P70

!

3000 7SPBC16A3.08c (

16A3.08c ) 16A3.08c in vitro (G4) Ylr150w 16A3.08c G4 in

vitro 16A3.08c Ylr150w16A3.08c Ylr150w oga1!" (ortholog of

G4-associated protein) oga1!oga1!

Ylr150w TOR TOR tor1! Oga1

oga1! tor1!

38

P71

Ecl1Ecl1 80

Ecl1 H2O2Ecl1 H2O2

Ecl1 H2O2Ecl1

Ecl1 H2O2 Ecl1 H2O2

H2O2 ecl1+ H2O2ecl1+-mRNA H2O2 ecl1+-mRNA

H2O2 Sty1 MAPKAtf1 ecl1+ H2O2

atf1+ H2O2 ecl1+ ecl1+

Atf1 ChIP assay Atf1 ecl1+Ecl1 ctt1+H2O2 atf1+

Sty1 Atf1 Ecl1 ctt1+ Atf1Ecl1 ctt1+ P72

ecl1+, ecl2+, ecl3+

(Extender of Chronological Lifespan) Ecl1Ecl1

Ecl1

ecl1+,

ecl2+, ecl3+

Ecl1 Ecl1

Ecl1