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1 PfNPC 5% O 10% AB 4, RPMI 5% CO % 30 sec 10% Giemsa 15 mins 100x oil-immersion lens parasitemia parasitemia 10 15% Parasitemia multiple invasion 4 Panel A B C parasitemia parasitemia 2% *Parasitemia % =(5 ) /(5 ) 14

2 Parasitemia parasitemia 10 15% parasitemia 5% 2.5% 10% 20% ring trophozoite schizont merozoite 48 hrs hrs Panel D PfNPC 10 ml parasitemia 12% ring trophozoit schizont R:T:S 3:7:2 25 cm 2 15 ml rpm 5 mins pipette PBS 4µl silane slides 15

3 70 30 mins retrival buffer 0.01 M citrate, ph % NP mins wash buffer 0.05 M Tris-HCl ph 7.2, 0.1% Tween 20 PfNPC pre-immune serum 0.05 M Tris-HCl ph 7.2, 0.1% Tween 20, 1% BSA, 15 mm NaN hr wash buffer slides 5 mins Biotinylated link anti-mouse and anti-rabbit Ig ( PBS, carrier protein 15mM NaN 3 10 mins wash buffer Streptavidin-HRP ( PBS, carrier protein 15mM NaN 3 10 mins wash buffer ( 0.75 mg/ml 3-amino-9-ethylcarbazole (ACE) in 2.5% N-dimethylformamide (DMF), 50 mm acetate buffer, ph 5.0, containing H 2 O 2, stabilizer, enhancers and 15 mm NaN 3 ) 10 mins wash buffer Vectashield (Vector Labs, Burlingame, CA) 100x oil-immersion lens 16

4 7, sorbitol 29 Percoll 30 late trophozoite schizont early trophozoite ring 5% sorbitol 37 2,000 rpm 5 mins 5 5% sorbitol mins 10 ml RPMI 17

5 65% percoll 37 5 ml 15 ml 5 ml 1x RPMI 65% percoll 2,000 rpm 10 mins 1x RPMI 1x RPMI Cell suspension 2,000 rpm, 15 mins 1x RPMI Late trophozoite, Schozont 65% percoll percoll Ring, Early trophozoite, RBC 31 5% sorbitol 95% schizont 65% percoll ring percoll 4 6 hr parasitemia 18

6 PfNPC 4% parasitemia 25 cm 2 15 ml rpm 5 mins pipette PBS 4 µl acetone 15 mins Pap Pen 20µl perforating reagent (0.05% saponin in PBS) 20 mins blocking reagent (0.2% fishskin gelatin % saponin in PBS) 30 mins PfNPC 0.04% fishskin gelatin % saponin PBS hr wash buffer 0.01% saponin in PBS 10 mins FITC-conjugated IgG 0.04% fishskin gelatin % saponin PBS hr wash buffer 0.01% saponin in PBS 10 mins well 10 µl Vectashield Vector Labs, Burlingame, CA 19

7 1 hr 100x oil-immersion lens PfNPC 酶 (protease) protease inhibitor 酶 25 ml parasitemia 15% 75 cm 2 50 ml rpm 5 mins 5 RBCM lysis buffer (0.1% saponin + cocktail protease inhibitor in PBS) 4000 rpm 5 mins 酶 PBS pellets pellets 2x SDS loading dye 10% SDS 95 20

8 5 mins denature SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ph SDS- (polyacrylamide gel) loading tips Mini-gels (80 mm x 70 mm x 0.75 mm) (Hoefer) 150V SDS running buffer 25 mm Tris, 190 mm glycine, 1% SDS dye front 1 cm Coomassie blue R-250 PVDF (western blotting ) Transfer buffer (48 mm Tris-base,39 mm glycine, 0.037% SDS, 20% methanol) PVDF (Immobilon-P, Millipore, USA) Transfer buffer 3M (Semi-Dry, Bio-Rad, USA) 250 ma 1 hr PVDF 1 min 21

9 Ponseus-S (Sigma, USA) ddh 2 O PVDF 30 mins PVDF blocking buffer (2% skim milk % Tween 20 in PBS) PfNPC pre-immune serum 1% skim milk, 0.05% Tween 20 PBS 1 hr wash buffer (0.05% Tween 20 in PBS) 10 mins Horseradish Peroxidase HRP conjugated IgG 1% skim milk, 0.05% Tween 20 PBS 1 hr wash buffer (0.05% Tween 20 in PBS) 10 mins (BioMax film, Kodak, USA) 3 mins ONE-D scan (One-Dimensional Gel Analysis) PfNPC 22

10 PfNPC PfNPC 25 ml schizont parasitemia 8% 75 cm 2 15 ml rpm 5 mins 1.5 ml 2% paraformaldehyde 2.5% glutaraldehyde 0.1 M PBS 10 ml 4 pellets petri-dish pellets 1 mm ml 0.2% glutaraldehyde 2 ml vial 4 1 hr glutaraldehyde 7% sucrose 0.1 M PBS wash 15 mins 50% 70% 86% 90% 95% 100% 20 mins 1:10 L.R. White 1.5 hr pure L.R. White 1 hr pure L.R. White pure L.R. White hr sample 23

11 0.3 x 0.3 x 0.3mm 3 ) blocks 2 Blocks sample thick section blocks Ultra S, Leica German 1.2 µm 10% % Toluidine blue mins dd H 2 O 3 blocks 90 nm (Ni-grid) grids 5% H 2 O 2 5 mins dd H 2 O 5 mins 0.05 M glycine PBS 30 mins PBS 5 mins 0.5% Tween % BSA PBS 24

12 90 mins PBS grids 5 mins 0.5% BSA blocking 30 mins PfNPC ( 0.5% BSA in PBS 1 40) 4 grids PBS 5 mins 0.5% Tween % BSA PBS 90 mins PBS 5 mins 0.5% BSA block 30 mins gold particle (10 nm ) conjugated IgG ( 0.5% BSA in PBS 1 30) (Aurion, Netherlands) 37 2 hr PBS 5 mins 0.5% Tween % BSA PBS 90 mins PBS grids 5 mins 2.5% glutaraldehyde 4% paraformaldehyde 0.1 M PBS 10 mins dd H 2 O 5 mins 1% OsO 4 OsO 4 dd H 2 O 5 mins 2% phosphotungstic acid lead citrate 33 25

13 PfNPC 10 ml parasitemia 15%, ring trophozoit schizont R:T:S 3:7:5 75 cm 2 O 10% AB RPMI 25 ml parasitemia 15% 50 ml rpm 5 mins 5 pellets S-PBS-EE 0.1% saponin in PBS with 25 mm EDTA, 5 mm EGTA and cocktail protease inhibitor 5 mins rpm 5 mins pellets 1.5 ml PBS-EE PBS with 25 mm EDTA, 5 mm EGTA and cocktail protease inhibitor pellets pellets 100 µl PBS-EE 20 sec 100 µl PBS 0.4 M Na 2 CO % Triton X hr 18 hrs 15,000 rpm 20 mins pellets 2x SDS loading dye denature 95 5 mins SDS-PAGE 26

14 PfNPC PfNPC 25 ml parasitemia 15% 75 cm 2 50 ml rpm 5 mins 5 pellets S-PBS-EE 5 mins rpm 5 mins pellets 1 ml PBS-EE pellets pelltes 200 µl lysis buffer 0.1% Ipegal in PBS-EE 20 sec 15 mins PfNPC PfNPC % BSA 37 blocking 2 hrs 12.5 µg 37 2 hrs PBST buffer wells Fluoresterol 37 2 hrs PBST buffer 27

15 wells PfNPC Fluoresterol Spectra Fluor Tecan, Austria 430 nm UV light Fluoresterol 535 nm PfNPC PfNPC 500 µg/ml 500 µl 1.5 ml 5 µl PfNPC 12 rpm 1 hr 40 µl protein A beads 12 rpm 2 hrs 13,500 rpm 20 mins beads pellets PBS buffer 12 rpm 15 mins 2x SDS loading buffer 95 5 mins 15,000 rpm 20 mins SDS-PAGE Coomassie Blue R-250 LC/MS/MS Thermo Finnigan, USA 1.5 ml 酶 buffer 37 2 hrs 28

16 polypeptide C18 polypeptide polypeptide polypeptide polypeptide He amide bonds polypeptide polypeptide polypeptide polypeptide polypeptide Correlation coefficient Xcorr 1 PfNPC fnpc PfNPC 1470 TMHMM2 29

17 fnpc (1) PfNPC NPC1 PTC1 Multin (version 5.4.1) ( (alignment) (2) NCBI Entrze ( sterol sensing domain SSD SSD PfNPC 30

材料與方法

材料與方法 10-12 250-350 Sprague-Dawley rat ( SD ) 12 ( AM 8 00-PM 8 00 ) (Control group) (HS) (Heat shock group) 15 0 4 6 16 24 (PAE) ( Pulmonary air embolism group) 20 40 (25 l/min) 40 17 20 (HS&PAE) 15 4 16 20