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1、Metabolism of 5-fluorouracil(5-FU)5-FUCarac,Efudex5-Fluorouridine5-FUMP5-FdUMPUridine phosphorylase5-FluorodeoxyuridineThymidine phosphorylaseOrotatephosphoribosyltransferaseThymidine kinase5-FUTP5-FdUTPRNADNA5-nucleotidase5-nucleotidaseUridine kinaseATPADPATPADPPRPP第1页/共66页Western Blotting1.Resolve
2、 protein samples on SDS-PAGE6.Incubate the blot with chemiluminescent HRP substrate and expose to film 2.Electrophoretically transfer fractionated proteins from gel onto PVDF membrane3.Block the membrane with neutral protein (BSA or milk casein)4.Incubate the membrane with primary antibody specific
3、to target protein5.Incubate the membrane with HRP-labeled secondary antibody specific to primary antibody第2页/共66页第3页/共66页第4页/共66页CholesterolSterol ToolkitPregnenolonProgesteronAldosteronCortisolCorticosteroneAndrostendioneTestosteronEstriol17-Estradiol1,25-DihydroxycholecalciferolCholic acid第5页/共66页
4、Covalent modification of proteinsPoly(ADP ribosyl)Polymerase(PARP)Poly(ADP ribosyl)polymerase(PARP)NADNicotinamidePoly(ADP ribosyl)polymerase(PARP)Poly(ADP ribosyl)chain elongationand branchingNicotinamideNADNADDNA polymerase DNA polymerase DNA polymerase 第6页/共66页OligosacharyltransferaseN-glycosylat
5、ion pathway in ERMann-P-DolicholtransferasesFlippaseDolicholPDolicholPPDolicholPPDolicholPPDolicholPPGlcNAc-PtransferaseGlcNActransferaseDolicholPDolicholPPDolicholPPGDP-Mannosyl transferasesGlc-P-DolicholtransferasesDolicholPDolichol-pyrophofphataseCytosolER40S60S第7页/共66页UTPCTPTMPPyrimidine Nucleot
6、ide InterconversionsUMPATPADPH2OUDPNucleosidemonophosphatekinaseATPADPNucleosidediphosphatekinaseATPADPCTPsynthaseRibonucleotidereductasedeoxyUDPNucleosidediphosphatephosphohydrolasedeoxyUMPThymidylatesynthaseN5,N10-MethyleneTHFDHFTTPNucleosidemonophosphatekinaseNucleosidediphosphatekinase第8页/共66页My
7、o-Inositol-3-phosphateIns(3)P1Biosynthesis and metabolism of phosphoinositidesGlucose 6-pgosphateIns(3)P1synthaseIns(3)P1phosphataseMyo-InositolInsPtdInssynthasePtdInsMyo-inositolkinasePtdIns-4-PPtdIns4-kinasePtdIns5-kinasePtdIns-4,5-P2PI3KPtdIns-3,4,5-P3PLCPLCPI3KPtdIns-3,4-P2Ins-1,3,4-P3Ins-1,3,4,
8、5-P4Ins-1,4,5-P3PLCPTENPTENIns-1,4-P3PTENPTEN第9页/共66页Fatty acidsynthase-ketoacylACP-synthaseFatty acids synthesisAcetyl CoAcarboxylaseATPADP+PiAcetyl CoAMalonyl CoACitrateOxaloacetateATPADP+PiCitrate lyaseAcetyltransacylaseMalonyltransacylasePantetheine-ketoacylACP-reductasePantetheinePantetheine-hy
9、droxyacylACP-dehydratasePantetheineEnoyl-ACPreductasePantetheineNADPHNADPNADPHNADP第10页/共66页Cholesterol de novo synthesis IIsoprenyl formationAcetoacetyl-CoAThiolaseHMG-CoAsynthaseMevalonate3-Hydroxy-3-methylglutaryl-CoAMevalonatekinasePhosphomevalonatekinaseMevalonate-5-pyrophosphodecarboxylase5-Pho
10、sphomevalonate5-Pyrophosphomevalonate3-Isopentenylpyrophosphate3,3-DimethylalylpyrophosphateIsomeraseHMG-CoAreductase2 NADPH2 NADPATPADPADPATPADPATP第11页/共66页CholesterolBiosynthesis of bile acids7-HydroxycholesterolSterol 7 hydroxylaseCYP7A17-Hydroxy-4-cholesten-3-one3-Hydroxystroid dehydrogenaseHSD3
11、B77,12-Dihydroxy-4-cholesten-3-oneSterol-12-hydroxylaseCYP8B15-Cholestan-3,7,12-TriolOxosteroid-5-reductaseAKR1D1Sterol 27-hydroxylaseCYP27A13,7,12-Trihydroxy-5-cholestanoic acidCholic acidAKR1D1AKR1C45-Cholestan-3,7-diol12-Deoxycholic acid3-Hydroxy-steroid dehydrogenaseAKR1C4第12页/共66页Biosynthesis o
12、f RNARNAPolymerase第13页/共66页Biosynthesis of DNADNAPolymerase第14页/共66页-oxidationMitochondrial matrixIntermembranespacePalmitoyl carnitinePalmitoyl carnitinePalmitoyl CoAPalmitoyl CoATrans 2 enolopalmitoyl CoA3-Hydroxypalmitoyl CoA3-Ketopalmitoyl CoAMyristoyl CoAAcetyl-CoAPalmitoyl CoAPalmitatePalmitoy
13、l CoAligaseATPAMPCarnitineacyltransferase ICarnitineCarnitineacyltransferase IIAcyl CoAdehydrogenaseEnoyl CoAhydratase3-Hydroxyacyl CoAdehydrogenaseCitric AcidCycleFADFADH2NADNADHCytoplasmH2O第15页/共66页Myo-Inositol-3-phosphateIns(3)P1Phospho-myo-inositol Toolkit 2Myo-Inositol-3,4-diphosphateIns(3,4)P2
14、Myo-Inositol-3,6-diphosphateIns(3,6)P2Myo-Inositol-3,4,6-triphosphateIns(3,4,6)P3Myo-Inositol-1,3,4-triphosphateIns(1,3,4,)P3Myo-Inositol-3,4,5,6-tetraphosphateIns(3,4,5,6)P4Myo-Inositol-1,4,5-triphosphateIns(1,4,5)P3Myo-Inositol-1,3,4,6-tetraphosphateIns(1,3,4,6)P4Myo-Inositol-1,3,4,5-tetraphosphat
15、eIns(1,3,4,5)P4Myo-Inositol-1,4,5,6-tetraphosphateIns(1,4,5,6)P4Myo-Inositol-1,2,3,4,6-pentaphosphateIns(1,2,3,4,6)P5Myo-Inositol-1,3,4,5,6-pentaphosphateIns(1,3,4,5,6)P5Myo-Inositol-1,2,3,4,5-pentaphosphateIns(1,2,3,4,5)P5Myo-Inositol-1,2,4,5,6-pentaphosphateIns(1,2,4,5,6)P5Myo-Inositol-1,2,3,4,5,6
16、-hexaphosphateIns(1,2,3,4,5,6)P6 (Phytic acid)第16页/共66页Myo-Inositol-3-phosphateIns(3)P1Phospho-myo-inositol ToolkitMyo-Inositol-3,4-diphosphateIns(3,4)P2Myo-Inositol-3,6-diphosphateIns(3,6)P2Myo-Inositol-3,4,6-triphosphateIns(3,4,6)P3Myo-Inositol-1,3,4-triphosphateIns(1,3,4,)P3Myo-Inositol-3,4,5,6-t
17、etraphosphateIns(3,4,5,6)P4Myo-Inositol-1,4,5-triphosphateIns(1,4,5)P3Myo-Inositol-1,3,4,6-tetraphosphateIns(1,3,4,6)P4Myo-Inositol-1,3,4,5-tetraphosphateIns(1,3,4,5)P4Myo-Inositol-1,4,5,6-tetraphosphateIns(1,4,5,6)P4Myo-Inositol-1,2,3,4,6-pentaphosphateIns(1,2,3,4,6)P5Myo-Inositol-1,3,4,5,6-pentaph
18、osphateIns(1,3,4,5,6)P5Myo-Inositol-1,2,3,4,5-pentaphosphateIns(1,2,3,4,5)P5Myo-Inositol-1,2,4,5,6-pentaphosphateIns(1,2,4,5,6)P5Myo-Inositol-1,2,3,4,5,6-hexaphosphateIns(1,2,3,4,5,6)P6 (Phytic acid)第17页/共66页GlycogenGlycogenin第18页/共66页8-oxoguanineA common DNA lesions Toolkit IO6-methylguanineUracilT
19、hymine glycol1,N6-ethenoadenine3-methyladenineCyclobutane pyrimidine dimersPhotoadduct第19页/共66页RiboseGlucuronateArabinoseFructoseGlucoseGalactoseMannoseFucoseSialic AcidInositolD-AbequoseL-RhamnoseA carbohydrate toolkit第20页/共66页PGH2TXA2TXB2Bioactive lipids-ProstanoidsPGD2PGJ2PGE2PGA2PGB2PGF2PGF-MPGI
20、26-keto-PGF1 第21页/共66页Bioactive lipids-Leukotrienes5-HETELTA4LTB4LTC4LTD4LTE420-OH-LTE4LXA4LTB4LTF4LXALXB第22页/共66页Bioactive lipidsArachidonateCeramideSphingosine-1-phosphatePAFPhosphatidic acidAnandamide 2-Arachidonoylglycerol 第23页/共66页 qGTP NO mediated attenuation of adrenergic Gq signaling in VSMC
21、 qGDP PLC qGTPCasGCcGMPpGCANPSERCABKCaPKGPKCMLCKmyosinmyosinmyosinPPCaM MLCKCaCaCaCaCaCaCaCaCaCaCaCaCaCaCaCaK+CaCaCaCaCaCaCaCaCaCaCaCaCaCaCaCaCaCaM Ca PA1,4,5-InsP第24页/共66页 SGTP RGS function and regulation SGDP ACPKAcAMP cAMPcAMPcAMPcAMPcAMPcAMPAC SGTPLigandLigandRGSRGSRGS14-3-3RGSP14-3-3PcAMPcAMPcA
22、MPTNF 第25页/共66页Modular Structure of Cytokine ReceptorsIFN R2515 aaIFN R1 530 aaIL-6R 449 aaIL-11R 400 aaIL-10R1 557 aaIL-10R2325 aaSTATbindingIL-13R 1 427 aaIL-4R 825 aaFNIIIFNIIICCCCWS-WS FNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIIFNIIISOCSgp130 896 aaSTATbindingTyk2Jak1INF R1 489 aaSTATbindingFN
23、IIIFNIIIJak1IL-22R1 574 aaSTATbindingFNIIIFNIIIJak1Jak1INF R2 332 aaFNIIIFNIIIJak2STATbindingTyk2Jak1Jak2Jak1IL-13R 2 380 aaFNIIIFNIIICCCCWS-WS FNIIIFNIIICCCCWS-WS FNIIIFNIIIFNIIISTATbindingSTATbindingIgIgIgIgOSM-R952 aaJak3FNIIIFNIIIFNIIIFNIIISTATbindingIgFNIIIFNIIICCCCWS-WS FNIIIFNIIICCCCWS-WS FNI
24、IIFNIIICCCCWS-WS LIF-R 1053 aaJak3FNIIIFNIIIFNIIISTATbindingIgFNIIIWS-WS FNIIIFNIIICCCCWS-WS STATbindingSTATbindingSHPFNIIICCCCSTATbindingSTATbindingTyk2FNIIIFNIIICCCCWS-WS IgSOCSSHPFNIIIFNIIICCCCWS-WS IgJak3 c 349 aaIRSFNIIIFNIIICCCCWS-WS IgJak3FNIIIFNIIIFNIIIFNIIIFNIIICCCCWS-WS IL-12 662 aaFNIIIWS
25、-WS SOCSIRSSTATbindingIgTyk2Fibronectintype III domainIg-likeIRS-bindingFibronectintype III domainwith cysteinesSOCS-bindingSTAT-bindingJAK/TYK-bindingFibronectintype III domainwith WSXWSSHP-bindingSHP第26页/共66页Adaptor proteins-selected(human)SAMdomainSrc-homology 3 domainSH3SH2Src-homology 2 domainG
26、RB2PTBdomainPTBCysteine-richregion 1Calponinhomology domainC-1CalponinSproutySprouty domain1217SH2SH3SH31276SH2SH3SLAP1304SH2SH3SH31330SH2SH3SH3CrkGADS(GRID)1377SH2SH3SH3SH3Nck11456SH2SproutyBLNKRho-GEFRho-GEF homologydomainVav-11533SH2SAMSLP-761584SH2PTBSHC-11632SH2PHAPS1845SH2SH3SH3PHC-1Rho-GEFCal
27、poninSAMPHPlekstrin homologydomain第27页/共66页Protein separation using SDS-PAGE(Laemmli system)StackinggelResolvinggel1.Apply protein/dye samples into polyacrylamide gel wells2.Run the electrophoresis until dye reaches the end of the gel3.Remove the gel from the apparatus and stain for proteins第28页/共66
28、页A Column Chromatography Toolkit第29页/共66页Embryonic stem cellsEgg donorSperm donorEgg fertilizationCulture fertilized cells forseveral cell divisionsDissociate cell clusterfor subculturing andfurther utilizationFreeze in N2 forlong-term storageUse for In VitrofertilizationPotential use fortherapeutic
29、 cloning第30页/共66页Tet rAmp rOripBR3224361bpOripBluescript IIAmp rLacIMCSLacZf1 OriOripUC18Amp rMCSLacZCommon Cloning VectorsOripCR2.13929bpAmp rKanLacZf1 OriM13(-)APCRAT7M13(+)OripGEM 11Zf3221bpAmp rLacZf1 OriT7 SfiI-SacI-EcoRI-SalI-Xho-BamHI-ApaI-XbaI-NotI-SphI-NsiI-HindIII SP6OripGEM 9Zf2912bpAmp r
30、LacZf1 OriSP6 NsiI-HindIII-XbaI-EcoRI-SalI-SacI T7第31页/共66页Metabolism of capecitabine5-FU5-Fluorouridine5-FUMP5-FdUMPUridine phosphorylase5-FluorodeoxyuridineThymidine phosphorylaseOrotatephosphoribosyltransferaseThymidine kinase5-FUTP5-FdUTPRNADNA5-nucleotidase5-nucleotidaseUridine kinaseATPADPATPA
31、DPPRPPCapecitabineXeloda(prodrug)Carboxyl-esterase5-Fluoro-5-deoxycytidineCytidinedeaminaseThymidine phosphorylase5-Fluoro-5-deoxyuridinePharmacologicallyactive compound第32页/共66页PertussistoxinNADADPCholeratoxin ACATPcAMPAC SGTPLigandPharmacological modulation of cAMPLigandLigandLigand iGDP SGDP iGTP
32、 iGTPNADADPForskolinRolipramAMPPDE SGTP第33页/共66页Mechanism ofPenicillin(inhibition of crosslinkingpeptidoglycan)Tetra-peptidePenta-glycineGlycopeptidetranspeptidasePenicillin第34页/共66页40SInhibition of translation byRapamycin(Sirolimus)Growth Factor ReceptorsIP3KPDKPKBmTOR40S30SeIF4GeIF4EPABPeIF4A4E-BP
33、1eIF4EPP4E-BP1PPPPPS6KPPPPPRibosomalproteinseEF2eEF2PP40S30SeEF2kinase CaMeIF2RapamycinFKBP-12第35页/共66页TMPMechanism of 5-fluorouracil(5-FU)deoxyUMPThymidylatesynthaseTTP5-FUCarac,EfudexDNA synthesis5-Fluorouridine5-FUMP5-FdUMPUridine phosphorylaseUridine kinase5-FluorodeoxyuridineThymidine phosphory
34、laseOrotatephosphoribosyltransferaseThymidine kinaseN5,N10-methylene-THFDHF第36页/共66页The Life cycle of Plasmodium spp.Infected mosquito bitereleases sporozoitesinto human bloodBloodvesselEndotheliumHepatocytesKupffer cellsMerozoiteSporozoiteTrophozoiteSchizontMacrogametocyteMicrogametocyte1234Mosquit
35、o bitetakes up macrogametocytesand microgametocytesOocystGametogenesisMosquito gut第37页/共66页The Life cycle of HeartwormDirofilaria immitisMicrofilariaeLarvae14 daysMicrofilariaeLarvae14 daysLarvaeAdult worm6monthsLarvaeAdult worm8monthsMicrofilariaeMicrofilariae第38页/共66页Virus envelopes III第39页/共66页HI
36、V envelope第40页/共66页Bacteria&Bacteriophage ToolkitBacilliCoccispirillaBacteriophage Bam35Bacteriophage T4Bacteriophage T2第41页/共66页Adenovirus第42页/共66页Citric acid cycleCitrateSuccinyl-CoAsynthetaseAconitaseCitrate synthaseSuccinatedehydrogenaseGTPGDPOxaloacetateAconitaseIsocitratedehydrogenaseIsocitrat
37、edehydrogenase-KetoglutaratedehydrogenaseSuccinyl-CoA-KetoglutarateMalatedehydrogenaseFumarasecis-AconitateIsocitrateOxalosuccinateSuccinateFumarateMalateFADFADH2NADNADHCO2NADNADHCO2NADNADHH2OH2OH2OGlyoxylate cycleGlyoxylateGlyoxylate cycleH2OFattyacids第43页/共66页1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16
38、17 18 19 20 21 22 X YHuman chromosomes第44页/共66页第45页/共66页第46页/共66页第47页/共66页第48页/共66页Needle tip toolkit第49页/共66页A DNA Toolkit 3第50页/共66页RNA ToolkitMet-tRNA第51页/共66页A DNA Toolkit 4第52页/共66页第53页/共66页AstrocyteNeuron第54页/共66页Neutrophil Eosinophil BasophilMesenchymal cellSmooth muscle cellsEpithelial cellsMacrophageDendritic cellT-cellErythrocyteB-cell第55页/共66页第56页/共66页第57页/共66页Antibody Toolkit(IgG)第58页/共66页 helixVimentinActin and myosinCoiled-coilMicrotubule6 nm10 nm25 nm第59页/共66页第60页/共66页第61页/共66页第62页/共66页Nucleosomes 2第63页/共66页第64页/共66页ACACDDR1DDR1DDR176123454356217CD45PTP第65页/共66页感谢您的观看！第66页/共66页