jsv.umin.jpjsv.umin.jp/camp/15th/15th_slide2.pdfInsulin tolerance test CoreTg WT FDD Õ n i`lczo...
Transcript of jsv.umin.jpjsv.umin.jp/camp/15th/15th_slide2.pdfInsulin tolerance test CoreTg WT FDD Õ n i`lczo...
7 P 9) 9 BF BF+ BF-5 BF-6BF 6
Y/
i
BF 5
EB5
BF-5
BF-6
BF 5
BF+
7aD5
BF 6
< Y1(
:?6D0
R6%SWO)
I5D%5 6D ? L
<7I
i
??
B
FDD7
B
) + - . / 0 1
B 7
B
D P PWSUSWe% PN P L P#
7
K8
8
i FDD#5 YL aU SW LVPVM LWP%NUPL SW Y PL P %7 SD #
FDD wt
FLAG-Core
SPPKO cells YDD LGLGD
C
NHA
265219
DD
WT
SPPKO + SPP-HA
WT -
-FLAG-Core
-GFP
-Actin
IRES
GFPcoreFLAG
- FLAG-Core
- GFP
- Actin
- FLAG-Core(long exposure)
#1 #4 #2 #7WT SPPKO
Clone No
i z
WT or SPPKO cells
WT0 15 30 45 60 120
SPPKO
Core
GFP
Actin
0 15 30 45 60 (min)120Cycloheximide
ProteasomeLysosomeinhibitors
SPPKO cells
FLAG-Core
GFPCoreFLAG
FLAG-Core
GFPCoreFLAG
- 0.2 0.4 5 105 10 5 10EPO LAC ALLN MG132
GFP
Actin
Core
i
Core
GFP
Actin
p62
E64d PEP
BAF
SPP+ - + - + -
NT
i
FRS LT L P LU I ((.
9.5D
7 P
D5 0g
A SS RS P LU I ((+F b TS P LU I ((1
%:UL %7 P
% :D
%9.5D
%D5 0
%5N SW
D5 0FDD9.5D
% %%
%%
%%%
FDD D5 0 9.5D G?C
9.5D D5 0e pw z u
E9F
:DN P: 5
: 5 %7 P
+0%
?8L
% VVL P N P5W S%<5#
0%% % %FDD?C
FDDGE708?C
FDDGE70?CGE70%<5
: 5 %7 P%<5
GE70%<510%. %
% :D0%
%5N SW
% G LU N P5W S%: 5 #
0%
GE70w9+ i t
N P: <5
N P:
:D
<5
FDD
E9F
:DN P: <5
SEB59E 9+ US L P
EB5S N PPWSW
GE70
WRSMS S WGE70
i
7 PG l7 PG FDD % l
7 PG FDD %7 PG
+1%
. %
1/%
%5N SW
FDD
?8L ?8L0%
%N P
+0%%5N SW
7 PGFDD %7 PGJG
SPP+/+ SPP+/- SPP-/-
E13.5 13 21 11E18.5 22 22 2
Postnatal(~2wk) 19 40 0
FDDw
0
20
40
60
80
100
120
0 20 40 60 80
7 PG
JG
7 PG FDD %
FDD %DULVL
UN
P#
VSW#
FDD w
7 PG FDD %
C
FDD %
7 PGJG
FDD
< R/ NPUU
:
:
:D
:D
7 P
7 PE9F
i
VVL P N P
FDD
FDD
% 5N SW
% :D
?8L
0 %
+0 %
0 %
% 7 P5W S%: 5 #
RO-0492907LY-411575KDa
28 -
38 -
28 -
YO-01027
- Actin
- GFP
- Core(Anti-FLAG)
KDa
28 -
38 -
28 -
Semagacestat
- Actin
- GFP
- Core(Anti-FLAG)
Compound E Avagacestat
Compound 34 DAPT MK-0752
- Actin
- GFP
- Core(Anti-FLAG)
KDa
28 -
38 -
28 -
Presenilin1
Plasmamembrane
C
N
N C
SPP
ERmembrane
C
N
N
C
e% i FDD xx u wt
GT1 (J1)
GT1 (TN) GT2 (J6) GT2 (J8)
GT3 (S52) GT4 (ED43) GT5 (SA13) GT6 (HK6a) GT7 (QC69)
GT2 (JFH1)
- Actin
- GFP
- Actin
- GFP
KDa
28 -
38 -
28 -
KDa28 -
38 -
28 -
GT1 (H77)
1aTN2b
4a
KDa
28 -
38 -
28 -
YO-01027
- Actin
- GFP
Huh7Various genotypes of HCV core
KC%()( / <7I
- Core (Anti-FLAG)
- Core (Anti-FLAG)
- Core (Anti-FLAG)
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
0.05 0.5 5 50
(log10)
(µM)
LY-411575
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
0.05 0.5 5 50
(log10)
(µM)
YO-01027
SPP阻害剤では耐性ウイルスは出現しにくい
Huh7.5.1 cells
passage
LY-411575YO-01027
orTPV
… X 5 ~20 times
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
0.0005 0.005 0.05 0.5
(log10)
(µM)
Telaprevir
Viru
s tit
er (F
FU/m
l) **
*
HCV
LY-411575YO-01027
orTPV
P-5No treatment
Evaluation ofdrug resistance
P-20No treatment
P-20No treatment
FDDw
0.0
0.4
0.8
1.2
1.6
Rela
tive
expr
essi
on(C
ore
/bet
a ac
tin)
N.D
4d 8d 12d 16dWT CoreTg LY-411575
Core mRNA in Liver
WT CoreTg 4 d 8 d 12 dLY-411575
-Actin
-Core
LY-411575 (10mg/kg) for 15 days
CoreTg
LY-411575 (10mg/kg) for 15 days
CoreTg
0
20
40
60
80
100
120
0 20 40 60 80 (min)
Plas
ma
gluc
ose
(%)
InsulinInsulin tolerance test
CoreTg
WT
FDD Gg w x
Oil Red O staining
Vehicle LY-411575
CoreTg+LY-411575
γ-secretase inhibitors with SPP inhibitionLY-411575 RO-0492907YO-01027
FDD v y w xwt h
LY-450139
DAPT
Avagacestat
MK-0752
Compound E
Compound 34
ILU +m UP#
DRP)1 mG Y#
5UL -1m P #
5 Y )15 Y .-
KC%()( /
< R/ FDD?C< R/JGI +:)1 J5 -1c
% 5N SW
% :D
% 7 P5W S%: 5 #
?8L
0 %
+0 %
0 %
� ���� KC%()( /
RFDD. %
1/ %
% 5N SW+0 %
FDD?C< R/JG I + :)1 J5 -1c?8L
: -0
GA/
GA.
ILU +
FDD
D P PWSUSW)
DRP -0
FDD
> SPP – EANNFAMLGLGDVV> PS1 – PEERGVKLGLGDFI
258
*****
FDD e% i DF)#
Ua -0
D P PWSUSW)
�
% :D
% 7 P5W S%: 5 #
% 5N SW
: -0JGJG : -07 VY WO 9
?8L
0 %
+0 %
0 %
KC%()( /7 VY WO 9 FDD : -0
2FDD
Acknowledgement
Yoshiharu Matsuura
SayakaAizawa
JunkiHirano
TatsuyaSuzuki
Yukari Sugiyama
ShinjiKusakabe
MakotoTokunaga
YukaMiyata
The University of TokyoKazuhiko Koike
Yamanashi UniversityKohji Moriishi
Kobe UniversityIkuo Shoji
NIBIOMasahiro Oka, Yoichi Miyamoto
Hamamatsu UniversityTetsuro Suzuki
NIIDRyosuke Suzuki, Hideki Aizaki, Takaji Wakita, Tatsuo Miyamura
NCGHMSachiyo Yoshio, Tatsuya Kanto
WEHISeong Lin Khaw, Guillaume LesseneJames Vince, David CS Huang
ImmunoparasitologyMiwa Sasai, Masahiro Yamamoto
iFReC, Osaka UniversityBiochemistryShigekazu NagataHost defense
Takashi Satoh, Shizuo Akira
RIMD, Osaka University, Molecular Virology
Experimantal Genome ResearchMasahito Ikawa