One-Loop Radiative Corrections to Chargino Pair Production

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EMSA,凝胶阻滞实验技术文献,以及很好protocol,troubleshooting

EMSA,凝胶阻滞实验技术文献,以及很好protocol,troubleshooting

Aromatic Hydrocarbon Receptor(AhR)⅐AhR NuclearTranslocator-and p53-mediated Induction of theMurine Multidrug Resistance mdr1Gene by3-Methylcholanthrene and Benzo(a)pyrene in Hepatoma Cells*Received for publication,September18,2000,and in revised form,November10,2000Published,JBC Papers in Press,November28,2000,DOI10.1074/jbc.M008495200Marie-Claude Mathieu,Isabelle Lapierre,Karine Brault‡,and Martine Raymond§From the Institut de Recherches Cliniques de Montre´al,Montre´al,Que´bec H2W1R7,CanadaThe mouse multidrug resistance gene family consists of three genes(mdr1,mdr2,and mdr3)encoding P-gly-coprotein.We show that the expression of mdr1is in-creased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7with the polycyclic aromatic hydrocarbon3-methylcholanthrene(3-MC). This increase is not observed in the aromatic hydrocar-bon receptor(AhR)-defective TAOc1BP r c1and the AhR nuclear translocator(Arnt)-defective BP r c1variants, demonstrating that the induction of mdr1by3-MC re-quires AhR⅐Arnt.We show that the mdr1promoter (؊1165to؉84)is able to activate the expression of a reporter gene in response to3-MC in Hepa-1c1c7but not in BP r c1cells.Deletion analysis indicated that the re-gion from؊245to؊141contains cis-acting sequences mediating the induction,including a potential p53bind-ing sequence.3-MC treatment of the cells increased the levels of p53and induced p53binding to the mdr1pro-moter in an AhR⅐Arnt-dependent manner.Mutations in the p53binding site abrogated induction of mdr1by 3-MC,indicating that p53binding to the mdr1promoter is essential for the induction.Benzo(a)pyrene,a polycy-clic aromatic hydrocarbon and AhR ligand,which,like 3-MC,is oxidized by metabolizing enzymes regulated by AhR⅐Arnt,also activated p53and induced mdr1tran-scription.2,3,7,8-Tetrachlorodibenzo-p-dioxin,an AhR ligand resistant to metabolic breakdown,had no effect. These results indicate that the transcriptional induc-tion of mdr1by3-MC and benzo(a)pyrene is directly mediated by p53but that the metabolic activation of these compounds into reactive species is necessary to trigger p53activation.The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1independently of AhR⅐Arnt further supports the proposition that mdr1is transcriptionally up-regulated by p53in response to DNA damage.Multidrug resistance(MDR)1is characterized by cross-resis-tance of the cells to a large number of structurally and func-tionally unrelated cytotoxic agents used in chemotherapy.In cultured cells,MDR is frequently caused by the overexpression of P-glycoprotein(Pgp),an integral membrane protein belong-ing to the ATP-binding cassette superfamily of transporters and which functions as an energy-dependent efflux pump of cytotoxic drugs(1,2).Pgp is encoded by a small family of genes with two members in humans(MDR1and MDR2/MDR3)and three in rodents(mdr1/mdr1b,mdr2,and mdr3/mdr1a)(1,2). Only one human gene(MDR1)and two rodent genes(mdr1/ mdr1b and mdr3/mdr1a)can confer MDR upon overexpression in drug-sensitive cells(1,2).The different mdr genes and Pgp isoforms are expressed in a tissue-specific manner(1,2).In the mouse,mdr1is expressed mostly in the adrenal cortex,kidney,and pregnant uterus, mdr2in the liver at the canalicular face,and mdr3in the intestine and to a lesser extent in the heart,liver,lung,and capillaries of the brain(3).Pgps are localized on the apical membrane of epithelial cells lining luminal spaces,suggesting that they function in normal tissues as transporters of toxic substances and/or specific endogenous cellular products(4). Knockout mice experiments have demonstrated a role for the mdr3gene in the maintenance of the blood-brain barrier and drug elimination and for the mdr2gene in the transport of phospholipids in the bile(5,6).No physiological function has been attributed to the mouse mdr1gene so far,since knockout mdr1(Ϫ/Ϫ)mice display no obvious physiological abnormali-ties(7).However,different experimental evidence indicates that Pgp encoded by mdr1can serve in the transport of steroids(8).A number of factors have been found to modulate the level of mdr gene expression in the liver.For example,high levels of MDR1RNA have been found in human hepatocarcinomas,and overexpression of the mdr1isoforms has also been observed in rodent liver during cholestasis,during regeneration following partial hepatectomy,during chemically induced hepatocarcino-genesis,and following administration of various natural and synthetic xenobiotics(1,2).In particular,it has been shown that expression of the rat mdr1b gene is increased in liver cells in response to treatment with various polycyclic aromatic hy-*This work was supported by a research grant from the Cancer Research Society Inc.(to M.R).The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked“advertisement”in accordance with18 U.S.C.Section1734solely to indicate this fact.‡Supported by a studentship from the Medical Research Council ofCanada.Present address:Dept.of Biological Sciences,Bio-Mega Re-search Division,Boehringer Ingelheim(Canada)Ltd.,Laval,Que´bec H7S2G5,Canada.§Supported by a scholarship from Le Fonds de la recherche en sante´du Que´bec.To whom correspondence should be addressed:Institut de recherches cliniques de Montre´al,110Pine Ave.W.,Montre´al,Que´bec H2W1R7,Canada.Tel.:514-987-5770;Fax:514-987-5764;E-mail: raymonm@ircm.qc.ca.1The abbreviations used are:MDR,multidrug resistance;Pgp,P-glycoprotein;3-MC,3-methylcholanthrene;B(a)P,benzo(a)pyrene; TCDD,2,3,7,8-tetrachlorodibenzo-p-dioxin;DN,daunorubicin;CAT, chloramphenicol acetyl transferase;AhR,aromatic hydrocarbon recep-tor;Arnt,AhR nuclear translocator;EMSA,electrophoretic mobility shift assay;DME,drug metabolizing enzymes;PAH polycyclic aromatic hydrocarbon;XRE,xenobiotic response element;bp,base pair(s);kb, kilobase pair(s).T HE J OURNAL OF B IOLOGICAL C HEMISTRY Vol.276,No.7,Issue of February16,pp.4819–4827,2001©2001by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.This paper is available on line at 4819 at ZHEJIANG UNIVERSITY, on November 21, Downloaded fromdrocarbon(PAH)compounds,including3-methylcholanthrene (3-MC),and that this increased expression occurs at the tran-scriptional level(9–11).However,the precise molecular mech-anisms involved in mdr1b regulation in response to3-MC are still unknown.PAHs are carcinogenic compounds arising from the incom-plete combustion of organic matter and are widespread in the environment,including tobacco smoke and tar.PAHs such as 3-MC and benzo(a)pyrene(B(a)P)as well as halogenated aro-matic hydrocarbons such as2,3,7,8-tetrachlorodibenzo-p-di-oxin(TCDD)are specific inducers of genes coding for drug-metabolizing enzymes(DME),including cyp1a1and cyp1a2, that code for cytochromes P450involved in metabolic oxidation (12).PAHs and TCDD bind in the cytoplasm to the aromatic hydrocarbon receptor(AhR),a member of the bHLH-PAS(basic helix-loop-helix Per-Arnt-Sim)family of transcription factors (12,13).The ligand-bound AhR translocates to the nucleus, where it binds as a heterodimer with the AhR nuclear trans-locator(Arnt;another bHLH-PAS protein)to specific cis-acting regulatory DNA sequences located in the promoter of its tar-gets(known as AH-,dioxin-,or xenobiotic-responsive elements (or AHRE,DRE,or XRE,respectively))to enhance their tran-scription(12,13).Given that mdr1b expression is increased in liver cells in response to treatment with various PAHs,it was postulated that mdr1b may be under the control of the AhR(9). However,studies failing to show mdr1induction in the liver of mice treated with TCDD,one of the most potent agonists of the AhR,suggested that mdr1expression was not regulated by AhR(14).The involvement of AhR in the regulation of mdr1 has so far remained controversial.The mouse hepatoma cell lines Hepa-1c1c7(wild type), TAOc1BP r c1(AhR-defective),and BP r c1(Arnt-defective)con-stitute a powerful experimental system to investigate the tran-scriptional regulation of different AhR⅐Arnt targets in response to xenobiotics(12).The two mutant cell lines were derived as B(a)P-resistant variants of Hepa-1c1c7and were identified based on their inability to induce aryl hydrocarbon hydroxylase activity in response to TCDD treatment(15).TAOc1BP r c1cells have a decreased level of AhR(ϳ10%of wild-type cells)and therefore decreased induction of the cyp1a1promoter and lower aryl hydrocarbon hydroxylase activity in response to TCDD and other AhR ligands(15–18).BP r c1cells have a nor-mal cytosolic AhR,which fails to accumulate in the nucleus because of a defective Arnt(15).They have virtually no basal or inducible levels of cyp1a1expression and aryl hydrocarbon hydroxylase activity(15–17).In the present report,we have used this panel of cell lines to investigate the transcriptional regulation of the murine mdr1 gene by3-MC and other xenobiotic compounds.Our results demonstrate that mdr1is transcriptionally induced by3-MC and B(a)P and that this induction is mediated by p53but also requires AhR⅐Arnt.A model for the AhR⅐Arnt-and p53-medi-ated transactivation of mdr1in response to genotoxic stress is proposed.EXPERIMENTAL PROCEDURESCell Culture—Wild-type Hepa-1c1c7and Hepa1–6,AhR-defective TAOc1BP r c1,and Arnt-defective BP r c1cells were obtained from the American Type Culture Collection(ATCC;Manassas,VA)and main-tained in culture under the conditions recommended by the ATCC. Chinese hamster ovary LR73cell lines stably transfected with plasmid constructs carrying full-length cDNAs for the mouse mdr1,mdr2,or mdr3genes(LR73mdr1,LR73mdr2,and LR73mdr3,respectively;a gift from Dr.Philippe Gros,McGill University,Montre´al,Canada)were grown as described elsewhere(19,20).For inductions,cells atϳ50% confluence were exposed to different concentrations of xenobiotics for various periods of time(the exact conditions for each experiment are indicated in the figure legends).3-MC,B(a)P,and daunorubicin were obtained from Sigma,and TCDD was obtained from the Centre d’expertise en analyze environnementale du Que´bec(Laval,Canada).Stock solutions of3-MC(5m M)and B(a)P(25m M)were prepared in Me2SO,and the stock solutions of daunorubicin(1mg/ml)were pre-pared in water.TCDD was obtained in n-nonane at a concentration of 50␮g/ml and was stored at room temperature.Stock solutions of3-MC, B(a)P,and daunorubicin were stored atϪ80°C.RNA Preparation—Total RNA was prepared from3-MC-treated and untreated hepatocytes as well as from the LR73mdr1,LR73mdr2,and LR73mdr3cell lines by homogenizing the cells in a solution containing guanidium hydrochloride(6M)followed by sequential ethanol precipi-tation,as described previously(21).RNase Protection Assay—The plasmid constructed to detect the mdr1 RNA consisted of a165-bp Bam HI fragment isolated from the mdr1 cDNA(positions1926–2090relative to the ATG initiation codon(22)), blunt-ended with T4DNA polymerase,and cloned into plasmid pGEM-7Z(Promega,Madison,WI)at the Sma I site,giving plasmid pmdr1-G7.This plasmid was linearized with Eco RI and used as a template to synthesize an antisense mdr1probe using SP6RNA polym-erase(Amersham Pharmacia Biotech).The pKX10–3Z plasmid consist-ing of an Xba I–Kpn I mouse␤-actin cDNA fragment(positions724–969 in the␤-actin cDNA)cloned into pGEM-3Z at the Xba I and Kpn I sites (kindly provided by Dr.Rashmi Kothary,Institut du cancer de Mon-tre´al,Montre´al,Canada)was used to generate a control actin probe. pKX10–3Z was linearized with Xba I and used to synthesize an anti-sense actin RNA probe with T7RNA polymerase.The riboprobes were synthesized in the presence of[␣-32P]UTP,and the RNase protection assay was performed according to standard protocols(23).Nuclear Run-on Transcription Assay—The run-on experiment was performed essentially as described by Fisher et al.(24).Nuclei wereisolated from Hepa-1c1c7cells treated with Me2SO or with3-MC(5␮M) for48h and were used to label nascent RNAs with[␣-32P]UTP.Plas-mids pVT101-U/mdr1,carrying the full-length mouse mdr1cDNA(25); pmP1450–3Ј,carrying a1.2-kb Pst I cDNA fragment overlapping part of the mouse cyp1a1cDNA(26)(obtained from the ATCC);and pKX10–3Z were linearized with Stu I,Bam HI,and Xba I,respectively.The linear-ized plasmids were denatured,immobilized in duplicate onto a nylon membrane,and hybridized with the[␣-32P]UTP-labeled RNAs for48h at65°C.The membranes were washed and exposed for7days with two intensifying screens.Slot Blot Analyses—Slot blotting was performed as previously de-scribed(21).RNA samples(10␮g)were denatured in7ϫSSC-7.5% formaldehyde for15min at65°C and applied to a nylon membrane (Zeta-Probe).Detection of specific RNAs was performed by hybridiza-tion at65°C in0.5M NaPO4,pH7.2,1m M EDTA,7%SDS,1%bovine serum albumin,and100␮g/ml salmon sperm DNA with32P-labeled DNA probes.The mdr1probe was a4.2-kb Sph I–Eco RI fragment over-lapping the full-length mouse mdr1cDNA,isolated from plasmid pGEM7/mdr1(a gift from Dr.Philippe Gros,McGill University,Mon-tre´al);the cyp1a1probe was a 1.2-kb Pst I fragment isolated from plasmid pmP1450–3Ј;and the actin probe was a245-bp Xba I–Kpn I fragment isolated from pKX10–3Z.The membranes were washed twiceat65°C with a solution containing40m M NaPO4,pH7.2,5%SDS,1 m M EDTA,0.5%bovine serum albumin and twice with a solutioncontaining40m M NaPO4,pH7.2,5%SDS,and1m M EDTA before autoradiography.Chloramphenicol Acetyl Transferase(CAT)Expression Plasmids—Plasmid pMcat5.9consists of a482-bp DNA fragment containing the dioxin-responsive elements of the cyp1a1gene cloned upstream of the mouse mammary tumor virus promoter and the CAT gene(24)(kindly provided by Dr.Allan Okey,University of Toronto).Plasmids pmdr1, p-452,p-245,p-141,and p-93(previously referred to as pSacICAT, pExo6CAT,pExo2CAT,pExo1CAT,and pAluCAT,respectively)have been described elsewhere(27).The mdr1promoter sequence in these constructs ends at positionϩ84with respect to the transcription start site(27).To produce the p53mutant constructs,pM1and pM2,plasmid pSBM13was used.This plasmid consists of a1.2-kb Sac I–Hin dIII mdr1 promoter fragment(positionsϪ1165toϩ84)cloned into M13mp18. Single-stranded DNA was prepared from pSBM13and used as a tem-plate to perform site-directed mutagenesis of the p53binding site,using the mutant oligonucleotides M15Ј-TACCTGAA T AC A TAAAGACA and M25Ј-CGTAAAGA T AA A TCTATGTA(the base changes are shown in boldface type).The resulting M1and M2mdr1promoter fragments were then excised from pSBM13with Sac I and Hin dIII,blunt-ended with T4DNA polymerase,and cloned into plasmid pCAT at the Hin dIII site also blunt-ended with T4DNA polymerase,yielding plasmids pM1 and pM2.The presence of the mutations in the resulting constructs was confirmed by DNA sequencing.Transient Transfections and CAT Assays—Cells were plated at aInduction of the Mouse mdr1Gene by PAHs4820at ZHEJIANG UNIVERSITY, on November 21, Downloaded fromdensity of 8ϫ105/60-mm plate and transfected on the following day with 10␮g of plasmid DNA,using a standard calcium phosphate pre-cipitation method (28).After incubation with the DNA precipitate for 16h,the cells were washed twice with phosphate-buffered saline and supplied with fresh medium containing the different xenobiotics.After 48h,the cells were collected.Cell extracts were prepared,and protein concentrations were determined by the Bradford method (29).CAT activities were assayed by standard protocols as described previously,using 2␮g of proteins (27).Preparation of Nuclear Extracts—Nuclear extracts were prepared according to Schreiber et al .(30),with some modifications.Cells were harvested in cold phosphate-buffered saline,0.6m M EDTA and col-lected by centrifugation.The cell pellets were resuspended in 400␮l of ice-cold buffer A (10m M Tris,pH 8.0,10m M KCl,0.1m M EDTA,0.1m M EGTA,1m M dithiothreitol)containing 0.5m M phenylmethylsulfonyl fluoride,10␮g/ml aprotinin,1␮g/ml pepstatin,and 5␮g/ml leupeptin and swelled on ice for 15min.Subsequently,25␮l of 10%Nonidet P-40were added,and the tubes were vortexed vigorously.The nuclear pellets were collected by centrifugation and resuspended in 100␮l of cold buffer C (20m M Tris,pH 8.0,400m M NaCl,1m M EDTA,1m M EGTA,1m M dithiothreitol)in the presence of protease inhibitors.The suspen-sions were shaken vigorously at 4°C for 1h and centrifuged for 15min at 4°C,and the supernatants were frozen in aliquots at Ϫ80°C.Proteinconcentrations were determined by the Bradford method (29).ElectrophoreticMobility Shift Assay—Oligonucleotides overlapping the potential p53binding site in the mdr1promoter (5Ј-GAACACGTA-AAGACAAGTCTAT)and the p53consensus sequence in the p21waf1/cip1promoter (5Ј-GAACATGTCCCAACATGTTGAG)(31)were end-labeled with ␥-32P using T4polynucleotide kinase and annealed to their respec-tive in a M M 2.5m M dithiothreitol,4%Ficoll,1␮g of poly(dI-dC),and 20,000cpm of radiolabeled probe.The binding reactions were carried out at room temperature for 15min.Where needed,1␮g of the monoclonal anti-p53antibody pAb421(32)(Calbiochem)or of the polyclonal anti-Jun or anti-Skn-1antibodies (Santa Cruz Biotechnology,Inc.,Santa Cruz,CA)was added,and the incubation was continued for an additional 15min.The complexes were separated on 5%nondenaturing polyacrylamide gels in 1ϫTBE (90m M Tris,65m M boric acid,2.5m M EDTA,pH 8.0)at 200V.The gels were exposed to XAR films (Eastman Kodak Co.)for 16h with two intensifying screens at Ϫ80°C.Western Blotting—Total proteins from 3-MC-or Me 2SO-treated Hepa-1c1c7and BP r c1cells were extracted in ice-cold buffer (10m M Tris-HCl,pH 8.0,150m M NaCl,1m M EDTA,1%Nonidet P-40,and 1%sodium deoxycholate)containing 10␮g/ml leupeptin,10␮g/ml aproti-nin,1␮M sodium orthovanadate,and 1m M phenylmethylsulfonyl flu-oride.Total proteins (75␮g/sample)or nuclear extracts (30␮g/sample)were separated by SDS-polyacrylamide gel electrophoresis on a 10%acrylamide gel,transferred to a nitrocellulose membrane,and analyzed with the monoclonal anti-p53antibody pAb421(32)(Calbiochem)at a concentration of 5␮g/ml.Immune complexes were revealed by incuba-tion with a goat anti-mouse IgG antibody coupled to alkaline phospha-tase (Bio-Rad)and developed with 5-bromo-4-chloro-3-indolylphosphate p -toluidine salt and nitro blue tetrazolium chloride substrates as rec-ommended by the manufacturer (Life Technologies,Inc.).RESULTSTranscriptional Induction of the Mouse mdr1Gene by 3-MC in Hepatoma Cells—We have used an RNase protection assay to study the expression of mdr1in the hepatoma cell line Hepa-1c1c7upon exposure to 3-MC (Fig.1).An mdr1-specific riboprobe was prepared by cloning into pGEM7-Zf a mouse mdr1cDNA fragment overlapping the linker region of the protein,this domain displaying the lowest sequence homology among the three mouse mdr cDNAs (21).When tested with RNA prepared from LR73stable transfectants expressing each of the three mouse mdr cDNAs,the mdr1riboprobe was found to recognize the mdr1RNA but not the mdr2or mdr3RNA,thus confirming its specificity (Fig.1,top right ).The mdr1probe was then used with RNA from Hepa-1c1c7cells treated or not with 3-MC (Fig.1,top left ).This experiment showed that the amount of mdr1RNA detected is very low in untreated cells but is strongly increased in 3-MC-treated cells,demonstrating that expression of the mouse mdr1gene is induced by 3-MCtreatment.The use of an actin probe confirmed that equal quantities of RNA were used in the assay (Fig.1,bottom ).A similar experiment performed with mdr2-and mdr3-specific riboprobes showed that the expression of these genes is not induced under such conditions,demonstrating that the induc-tion of mdr1expression by 3-MC is isoform-specific (data not shown).A nuclear run-on experiment was performed to determine whether mdr1induction by 3-MC occurs at the transcriptional level (Fig.2).In addition to the mouse mdr1cDNA,cDNAs for the mouse cyp1a1gene (known to be transcriptionally regu-lated by 3-MC (12))and for the actin gene were also included as positive and negative controls,respectively.The data in Fig.2show that 3-MC induces an increase in the rate of mdr1mRNA synthesis,indicating that 3-MC acts at the transcriptional level to induce mdr1gene expression in Hepa-1c1c7cells.AhR ⅐Arnt-dependent Induction of mdr1Expression by 3-MC—To determine whether the increase in mdr1expression in response to 3-MC exposure is AhR ⅐Arnt-mediated,we ana-lyzed the mdr1RNA levels upon 3-MC treatment in two wild-type hepatoma cell lines Hepa-1c1c7and Hepa 1–6and in two variant cell lines derived from Hepa-1c1c7,TAOc1BP r c1(AhR-defective)and BP r c1(Arnt-defective)(15)(Fig.3).As controls,we also analyzed the level of cyp1a1and actin expression under the same conditions (Fig.3,middle and right ,respectively).This experiment showed that mdr1is expressed at low levels in the four cell lines in the absence of 3-MC induction (Fig.3,left panel ).Upon 3-MC treatment,the expression of mdr1is in-duced in the two wild-type hepatoma cell lines (by ϳ5-fold),this induction being completely abrogated in the AhR-defective or in the Arnt-defective variants (Fig.3,left panel ).The actin control probe confirmed that equal amounts of RNA had been applied to the membrane (Fig.3,right panel ).These data clearly demonstrate that the induction of mdr1in response to 3-MC requires an intact AhR ⅐Arnt complex,like cyp1a1(Fig.3,middle )(12).The Mouse mdr1Promoter Confers 3-MC-regulated Expres-sion in an AhR ⅐Arnt-dependent Manner—To determine if reg-ulatory sequences responsible for mdr1induction by 3-MC are present in the promoter region of the gene,plasmid pmdr1,consisting of a 1.2-kb Sac I–Hin dIII DNA fragment overlapping the mdr1promoter region (positions Ϫ1165to ϩ84with respect to the transcription start site (27))fused to the CAT reporter gene,was analyzed in transient transfection experiments.Plasmid pMcat5.9,which consists of a 482-bp fragment derived from the cyp1a1promoter fused to the mouse mammary tumorF IG .1.Increased mdr1expression in Hepa-1c1c7upon 3-MC treatment.The expression of mdr1was analyzed by RNase protection assay.Total RNAs (45␮g)from Hepa-1c1c7cells treated with 5␮M 3-MC (ϩMC )or with Me 2SO (ϪMC )for 56h and from the control cell lines LR73/mdr1,LR73/mdr2,and LR73/mdr3were analyzed with an mdr1riboprobe,which protects a 169-nt fragment within the mdr1transcript,or with a ␤-actin riboprobe,which protects a 245-nt actin transcript fragment.Autoradiography was for 15h with two intensify-ing screens (mdr1)or for 5h without intensifying screens (actin ).Induction of the Mouse mdr1Gene by PAHs4821at ZHEJIANG UNIVERSITY, on November 21, 2012 Downloaded fromvirus promoter and to the CAT gene (24),as well as the empty pCAT vector were also included as positive and negative con-trols,respectively.The three plasmids were transiently trans-fected into Hepa-1c1c7and BP r c1cells.The cells were treated with 3-MC or with Me 2SO for 48h,and the cellular extracts were prepared and assayed for CAT activity.This experiment showed that the mdr1promoter is transcriptionally active in Hepa-1c1c7cells and BP r c1cells,since it can drive the expres-sion of the CAT gene in both cell lines,albeit at low levels (Fig.4).This result is consistent with the basal level of expression of mdr1detected by slot blot analysis in these cells (Fig.3).3-MC treatment of the Hepa-1c1c7cells transfected with pmdr1re-sulted in a 10-fold induction in CAT activity as compared with untreated cells,reaching levels of CAT activity similar to those detected in the Hepa-1c1c7pMcat5.9transfectants upon 3-MC treatment.However,this induction was completely abrogated in BP r c1cells (Fig.4),consistent with the lack of mdr1induc-tion at the RNA level observed in the slot blot assay (Fig.3).Similar results were obtained upon transfection in TAOc1BP r c1cells (data not shown).These results,showing that the mdr1promoter is able to activate the expression of the reporter gene in response to 3-MC in Hepa-1c1c7but not in BP r c1and TAOc1BP r c1cells,demonstrate that (i)the mdr1promoter is able to confer 3-MC-mediated transcriptional acti-vation;(ii)this activation requires a functional AhR ⅐Arnt com-plex;and (iii)the sequences mediating this induction are lo-cated between positions Ϫ1165and ϩ84in the mdr1promoter.Two Putative XREs Located in the mdr1Promoter Are Dis-pensable for the Induction of mdr1by 3-MC—The AhR ⅐Arnt transcriptional complex binds to a specific DNA sequence,5Ј-(A/T)NGCGTG,known as an XRE to activate transcription (12).XREs render heterologous promoters responsive to xeno-biotics and function in a position-and orientation-independent manner (33,34).Examination of the mdr1promoter sequence indicated the presence of two potential XREs in an inverted orientation in the distal portion of the promoter at positionsϪ1129and Ϫ620(5Ј-CACGCAT and 5Ј-CACGCAA,respective-ly).To identify the cis -acting sequences responsible for the induction of mdr1by 3-MC and to investigate the possible involvement of these putative XREs,we analyzed the tran-scriptional activity of a series of mdr1promoter 5Ј-deletion CAT constructs after transient transfection into Hepa-1c1c7and treatment of the resulting transfectants with 3-MC (Fig.5A ).3-MC treatment of Hepa-1c1c7cells transfected with plas-mids p-452or p-245resulted in a level of CAT induction similar to that observed in cells transfected with plasmid pmdr1car-rying the full-length promoter,indicating that sequences lo-cated within positions Ϫ1165to Ϫ245are dispensable for the induction of mdr1by 3-MC,including the two putative XREs as well as a potential AP-1binding site (5Ј-TGACTCA;positions Ϫ265to Ϫ255(35))(Fig.5,B and C ).However,further deletion of a 104-bp region down to position Ϫ141(p Ϫ141)was found to greatly diminish the induction of CAT activity by 3-MC (Fig.5,B and C ),demonstrating that sequences important for the induction are located between positions Ϫ245and Ϫ141.CAT activity in the absence of 3-MC was reduced in the p Ϫ141transfectants when compared with the p Ϫ245transfectants,indicating that sequences between positions Ϫ245and Ϫ141are also involved in the basal transcriptional activity of the mdr1promoter in hepatoma cells.Finally,we found that alowF IG .2.Nuclear run-on experiment.Nuclei were isolated from Hepa-1c1c7cells treated with 5␮M 3-MC (ϩMC )or with Me 2SO (ϪMC )for 48h.Nascent RNAs were radiolabeled with [␣-32P]UTP and used to probe duplicate nylon membranes on which denatured cDNAs for mdr1,cyp1a1,and actin had been immobilized.The membranes were washed and exposed for 7days with two intensifyingscreens.F IG .3.AhR ⅐Arnt-dependent induction of mdr1expression by 3-MC.Total RNAs (10␮g)from wild-type Hepa-1c1c7and Hepa 1–6,AhR-defective TAOc1BP r c1,and Arnt-defective BP r c1cells treated (ϩMC )or not treated (ϪMC )with 3-MC at 5␮M for 56h were applied onto a nylon membrane.The membrane was hybridized sequentially with an mdr1(left ),a cyp1a1(middle ),and a ␤-actin (right )probe.Autoradiography was for 18h (mdr1and cyp1a1)or for 2h (actin)with two intensifyingscreens.F IG .4.AhR ⅐Arnt-dependent induction of the mdr1promoter by 3-MC.Plasmids pCAT (no promoter),pmdr1(mdr1promoter from position Ϫ1165to ϩ84),and pMcat5.9(pMcat;482-bp fragment from the cyp1a1promoter fused to the mouse mammary tumor virus pro-moter)were transiently transfected into Hepa-1c1c7and BP r c1cells by the calcium phosphate method.The cells were then treated with 3-MC (5␮M )or Me 2SO for 48h.Total cellular extracts were prepared,and equal quantities of proteins (2␮g)were assayed for CAT activity.A ,autoradiogram of a representative CAT assay,showing the activity of plasmids pCAT,pmdr1and pMcat in Hepa-1c1c7and BP r c1cells treated (ϩ)or not treated (Ϫ)with 3-MC (MC ).The position of the [14C]chloramphenicol (CM )and of its acetylated products (AcCM )is indicated on the left .B ,quantitative analysis of CAT activities.The percentage of conversion of [14C]chloramphenicol to its acetylated de-rivatives was quantitated by liquid scintillation counting.Open bars ,ϪMC ;filled bars ,ϩMC .The results presented are the averages of three independent transfections performed in duplicate.S.D.values are rep-resented by the bars .Induction of the Mouse mdr1Gene by PAHs4822 at ZHEJIANG UNIVERSITY, on November 21, 2012 Downloaded from。

The Subleading Isgur-Wise Form Factor $chi_3(vcdot v')$ to Order $alpha_s$ in QCD Sum Rules

The Subleading Isgur-Wise Form Factor $chi_3(vcdot v')$ to Order $alpha_s$ in QCD Sum Rules

a rXiv:h ep-ph/9212266v116Dec1992SLAC–PUB–6017WIS–92/99/Dec–PH December 1992T/E The Subleading Isgur-Wise Form Factor χ3(v ·v ′)to Order αs in QCD Sum Rules Matthias Neubert Stanford Linear Accelerator Center Stanford University,Stanford,California 94309Zoltan Ligeti and Yosef Nir Weizmann Institute of Science Physics Department,Rehovot 76100,Israel We calculate the contributions arising at order αs in the QCD sum rule for the spin-symmetry violating universal function χ3(v ·v ′),which appears at order 1/m Q in the heavy quark expansion of meson form factors.In particular,we derive the two-loop perturbative contribution to the sum rule.Over the kinematic range accessible in B →D (∗)ℓνdecays,we find that χ3(v ·v ′)does not exceed the level of ∼1%,indicating that power corrections induced by the chromo-magnetic operator in the heavy quark expansion are small.(submitted to Physical Review D)I.INTRODUCTIONIn the heavy quark effective theory(HQET),the hadronic matrix elements describing the semileptonic decays M(v)→M′(v′)ℓν,where M and M′are pseudoscalar or vector mesons containing a heavy quark,can be systematically expanded in inverse powers of the heavy quark masses[1–5].The coefficients in this expansion are m Q-independent,universal functions of the kinematic variable y=v·v′.These so-called Isgur-Wise form factors characterize the properties of the cloud of light quarks and gluons surrounding the heavy quarks,which act as static color sources.At leading order,a single functionξ(y)suffices to parameterize all matrix elements[6].This is expressed in the compact trace formula[5,7] M′(v′)|J(0)|M(v) =−ξ(y)tr{(2)m M P+ −γ5;pseudoscalar meson/ǫ;vector mesonis a spin wave function that describes correctly the transformation properties(under boosts and heavy quark spin rotations)of the meson states in the effective theory.P+=1g s2m Q O mag,O mag=M′(v′)ΓP+iσαβM(v) .(4)The mass parameter¯Λsets the canonical scale for power corrections in HQET.In the m Q→∞limit,it measures thefinite mass difference between a heavy meson and the heavy quark that it contains[11].By factoring out this parameter,χαβ(v,v′)becomes dimensionless.The most general decomposition of this form factor involves two real,scalar functionsχ2(y)andχ3(y)defined by[10]χαβ(v,v′)=(v′αγβ−v′βγα)χ2(y)−2iσαβχ3(y).(5)Irrespective of the structure of the current J ,the form factor χ3(y )appears always in the following combination with ξ(y ):ξ(y )+2Z ¯Λ d M m Q ′ χ3(y ),(6)where d P =3for a pseudoscalar and d V =−1for a vector meson.It thus effectively renormalizes the leading Isgur-Wise function,preserving its normalization at y =1since χ3(1)=0according to Luke’s theorem [10].Eq.(6)shows that knowledge of χ3(y )is needed if one wants to relate processes which are connected by the spin symmetry,such as B →D ℓνand B →D ∗ℓν.Being hadronic form factors,the universal functions in HQET can only be investigated using nonperturbative methods.QCD sum rules have become very popular for this purpose.They have been reformulated in the context of the effective theory and have been applied to the study of meson decay constants and the Isgur-Wise functions both in leading and next-to-leading order in the 1/m Q expansion [12–21].In particular,it has been shown that very simple predictions for the spin-symmetry violating form factors are obtained when terms of order αs are neglected,namely [17]χ2(y )=0,χ3(y )∝ ¯q g s σαβG αβq [1−ξ(y )].(7)In this approach χ3(y )is proportional to the mixed quark-gluon condensate,and it was estimated that χ3(y )∼1%for large recoil (y ∼1.5).In a recent work we have refined the prediction for χ2(y )by including contributions of order αs in the sum rule analysis [20].We found that these are as important as the contribution of the mixed condensate in (7).It is,therefore,worthwhile to include such effects also in the analysis of χ3(y ).This is the purpose of this article.II.DERIV ATION OF THE SUM RULEThe QCD sum rule analysis of the functions χ2(y )and χ3(y )is very similar.We shall,therefore,only briefly sketch the general procedure and refer for details to Refs.[17,20].Our starting point is the correlatord x d x ′d ze i (k ′·x ′−k ·x ) 0|T[¯q ΓM ′P ′+ΓP +iσαβP +ΓM+Ξ3(ω,ω′,y )tr 2σαβ2(1+/v ′),and we omit the velocity labels in h and h ′for simplicity.The heavy-light currents interpolate pseudoscalar or vector mesons,depending on the choice ΓM =−γ5or ΓM =γµ−v µ,respectively.The external momenta k and k ′in (8)are the “residual”off-shell momenta of the heavy quarks.Due to the phase redefinition of the effective heavy quark fields in HQET,they are related to the total momenta P and P ′by k =P −m Q v and k ′=P ′−m Q ′v ′[3].The coefficient functions Ξi are analytic in ω=2v ·k and ω′=2v ′·k ′,with discontinuities for positive values of these variables.They can be saturated by intermediate states which couple to the heavy-light currents.In particular,there is a double-pole contribution from the ground-state mesons M and M ′.To leading order in the 1/m Q expansion the pole position is at ω=ω′=2¯Λ.In the case of Ξ2,the residue of the pole is proportional to the universal function χ2(y ).For Ξ3the situation is more complicated,however,since insertions of the chromo-magnetic operator not only renormalize the leading Isgur-Wise function,but also the coupling of the heavy mesons to the interpolating heavy-light currents (i.e.,the meson decay constants)and the physical meson masses,which define the position of the pole.1The correct expression for the pole contribution to Ξ3is [17]Ξpole 3(ω,ω′,y )=F 2(ω−2¯Λ+iǫ) .(9)Here F is the analog of the meson decay constant in the effective theory (F ∼f M√m QδΛ2+... , 0|j (0)|M (v ) =iF2G 2tr 2σαβΓP +σαβM (v ) ,where the ellipses represent spin-symmetry conserving or higher order power corrections,and j =¯q Γh (v ).In terms of the vector–pseudoscalar mass splitting,the parameter δΛ2isgiven by m 2V −m 2P =−8¯ΛδΛ2.For not too small,negative values of ωand ω′,the coefficient function Ξ3can be approx-imated as a perturbative series in αs ,supplemented by the leading power corrections in 1/ωand 1/ω′,which are proportional to vacuum expectation values of local quark-gluon opera-tors,the so-called condensates [22].This is how nonperturbative corrections are incorporated in this approach.The idea of QCD sum rules is to match this theoretical representation of Ξ3to the phenomenological pole contribution given in (9).To this end,one first writes the theoretical expression in terms of a double dispersion integral,Ξth 3(ω,ω′,y )= d νd ν′ρth 3(ν,ν′,y )1Thereare no such additional terms for Ξ2because of the peculiar trace structure associated with this coefficient function.possible subtraction terms.Because of theflavor symmetry it is natural to set the Borel parameters associated withωandω′equal:τ=τ′=2T.One then introduces new variables ω±=12T ξ(y) F2e−2¯Λ/T=ω0dω+e−ω+/T ρth3(ω+,y)≡K(T,ω0,y).(12)The effective spectral density ρth3arises after integration of the double spectral density over ω−.Note that for each contribution to it the dependence onω+is known on dimensionalgrounds.It thus suffices to calculate directly the Borel transform of the individual con-tributions toΞth3,corresponding to the limitω0→∞in(12).Theω0-dependence can be recovered at the end of the calculation.When terms of orderαs are neglected,contributions to the sum rule forΞ3can only be proportional to condensates involving the gluonfield,since there is no way to contract the gluon contained in O mag.The leading power correction of this type is represented by the diagram shown in Fig.1(d).It is proportional to the mixed quark-gluon condensate and,as shown in Ref.[17],leads to(7).Here we are interested in the additional contributions arising at orderαs.They are shown in Fig.1(a)-(c).Besides a two-loop perturbative contribution, one encounters further nonperturbative corrections proportional to the quark and the gluon condensate.Let usfirst present the result for the nonperturbative power corrections.WefindK cond(T,ω0,y)=αs ¯q q TT + αs GG y+1− ¯q g sσαβGαβq√y2−1),δn(x)=1(4π)D×1dλλ1−D∞λd u1∞1/λd u2(u1u2−1)D/2−2where C F=(N2c−1)/2N c,and D is the dimension of space-time.For D=4,the integrand diverges asλ→0.To regulate the integral,we assume D<2and use a triple integration by parts inλto obtain an expression which can be analytically continued to the vicinity of D=4.Next we set D=4+2ǫ,expand inǫ,write the result as an integral overω+,and introduce back the continuum threshold.This givesK pert(T,ω0,y)=−αsy+1 2ω0dω+ω3+e−ω+/T(16)× 12−23∂µ+3αs9π¯Λ,(17)which shows that divergences arise at orderαs.At this order,the renormalization of the sum rule is thus accomplished by a renormalization of the“bare”parameter G2in(12).In the9π¯Λ 1µ2 +O(g3s).(18)Hence a counterterm proportional to¯Λξ(y)has to be added to the bracket on the left-hand side of the sum rule(12).To evaluate its effect on the right-hand side,we note that in D dimensions[17]¯Λξ(y)F2e−2¯Λ/T=3y+1 2ω0dω+ω3+e−ω+/T(19)× 1+ǫ γE−ln4π+2lnω+−ln y+12T ξ(y) F2e−2¯Λ/T=αsy+1 2ω0dω+ω3+e−ω+/T 2lnµ6+ y r(y)−1+ln y+1According to Luke’stheorem,theuniversalfunction χ3(y )vanishes at zero recoil [10].Evaluating (20)for y =1,we thus obtain a sum rule for G 2(µ)and δΛ2.It reads G 2(µ)−¯ΛδΛ224π3ω00d ω+ω3+e −ω+/T ln µ12 +K cond (T,ω0,1),(21)where we have used that r (1)=1.Precisely this sum rule has been derived previously,starting from a two-current correlator,in Ref.[16].This provides a nontrivial check of our ing the fact that ξ(y )=[2/(y +1)]2+O (g s )according to (19),we find that the µ-dependent terms cancel out when we eliminate G 2(µ)and δΛ2from the sum rule for χ3(y ).Before we present our final result,there is one more effect which has to be taken into account,namely a spin-symmetry violating correction to the continuum threshold ω0.Since the chromo-magnetic interaction changes the masses of the ground-state mesons [cf.(10)],it also changes the masses of higher resonance states.Expanding the physical threshold asωphys =ω0 1+d M8π3 22 δ3 ω032π2ω30e −ω0/T 26π2−r (y )−ξ(y ) δ0 ω096π 248T 1−ξ(y ).It explicitly exhibits the fact that χ3(1)=0.III.NUMERICAL ANALYSISLet us now turn to the evaluation of the sum rule (23).For the QCD parameters we take the standard values¯q q =−(0.23GeV)3,αs GG =0.04GeV4,¯q g sσαβGαβq =m20 ¯q q ,m20=0.8GeV2.(24) Furthermore,we useδω2=−0.1GeV from above,andαs/π=0.1corresponding to the scale µ=2¯Λ≃1GeV,which is appropriate for evaluating radiative corrections in the effective theory[15].The sensitivity of our results to changes in these parameters will be discussed below.The dependence of the left-hand side of(23)on¯Λand F can be eliminated by using a QCD sum rule for these parameters,too.It reads[16]¯ΛF2e−2¯Λ/T=9T4T − ¯q g sσαβGαβq4π2 2T − ¯q q +(2y+1)4T2.(26) Combining(23),(25)and(26),we obtainχ3(y)as a function ofω0and T.These parameters can be determined from the analysis of a QCD sum rule for the correlator of two heavy-light currents in the effective theory[16,18].Onefinds good stability forω0=2.0±0.3GeV,and the consistency of the theoretical calculation requires that the Borel parameter be in the range0.6<T<1.0GeV.It supports the self-consistency of the approach that,as shown in Fig.2,wefind stability of the sum rule(23)in the same region of parameter space.Note that it is in fact theδω2-term that stabilizes the sum rule.Without it there were no plateau.Over the kinematic range accessible in semileptonic B→D(∗)ℓνdecays,we show in Fig.3(a)the range of predictions forχ3(y)obtained for1.7<ω0<2.3GeV and0.7<T< 1.2GeV.From this we estimate a relative uncertainty of∼±25%,which is mainly due to the uncertainty in the continuum threshold.It is apparent that the form factor is small,not exceeding the level of1%.2Finally,we show in Fig.3(b)the contributions of the individual terms in the sum rule (23).Due to the large negative contribution proportional to the quark condensate,the terms of orderαs,which we have calculated in this paper,cancel each other to a large extent.As a consequence,ourfinal result forχ3(y)is not very different from that obtained neglecting these terms[17].This is,however,an accident.For instance,the order-αs corrections would enhance the sum rule prediction by a factor of two if the ¯q q -term had the opposite sign. From thisfigure one can also deduce how changes in the values of the vacuum condensates would affect the numerical results.As long as one stays within the standard limits,the sensitivity to such changes is in fact rather small.For instance,working with the larger value ¯q q =−(0.26GeV)3,or varying m20between0.6and1.0GeV2,changesχ3(y)by no more than±0.15%.In conclusion,we have presented the complete order-αs QCD sum rule analysis of the subleading Isgur-Wise functionχ3(y),including in particular the two-loop perturbative con-tribution.Wefind that over the kinematic region accessible in semileptonic B decays this form factor is small,typically of the order of1%.When combined with our previous analysis [20],which predicted similarly small values for the universal functionχ2(y),these results strongly indicate that power corrections in the heavy quark expansion which are induced by the chromo-magnetic interaction between the gluonfield and the heavy quark spin are small.ACKNOWLEDGMENTSIt is a pleasure to thank Michael Peskin for helpful discussions.M.N.gratefully acknowl-edgesfinancial support from the BASF Aktiengesellschaft and from the German National Scholarship Foundation.Y.N.is an incumbent of the Ruth E.Recu Career Development chair,and is supported in part by the Israel Commission for Basic Research and by the Minerva Foundation.This work was also supported by the Department of Energy,contract DE-AC03-76SF00515.REFERENCES[1]E.Eichten and B.Hill,Phys.Lett.B234,511(1990);243,427(1990).[2]B.Grinstein,Nucl.Phys.B339,253(1990).[3]H.Georgi,Phys.Lett.B240,447(1990).[4]T.Mannel,W.Roberts and Z.Ryzak,Nucl.Phys.B368,204(1992).[5]A.F.Falk,H.Georgi,B.Grinstein,and M.B.Wise,Nucl.Phys.B343,1(1990).[6]N.Isgur and M.B.Wise,Phys.Lett.B232,113(1989);237,527(1990).[7]J.D.Bjorken,Proceedings of the18th SLAC Summer Institute on Particle Physics,pp.167,Stanford,California,July1990,edited by J.F.Hawthorne(SLAC,Stanford,1991).[8]M.B.Voloshin and M.A.Shifman,Yad.Fiz.45,463(1987)[Sov.J.Nucl.Phys.45,292(1987)];47,801(1988)[47,511(1988)].[9]A.F.Falk,B.Grinstein,and M.E.Luke,Nucl.Phys.B357,185(1991).[10]M.E.Luke,Phys.Lett.B252,447(1990).[11]A.F.Falk,M.Neubert,and M.E.Luke,SLAC preprint SLAC–PUB–5771(1992),toappear in Nucl.Phys.B.[12]M.Neubert,V.Rieckert,B.Stech,and Q.P.Xu,in Heavy Flavours,edited by A.J.Buras and M.Lindner,Advanced Series on Directions in High Energy Physics(World Scientific,Singapore,1992).[13]A.V.Radyushkin,Phys.Lett.B271,218(1991).[14]D.J.Broadhurst and A.G.Grozin,Phys.Lett.B274,421(1992).[15]M.Neubert,Phys.Rev.D45,2451(1992).[16]M.Neubert,Phys.Rev.D46,1076(1992).[17]M.Neubert,Phys.Rev.D46,3914(1992).[18]E.Bagan,P.Ball,V.M.Braun,and H.G.Dosch,Phys.Lett.B278,457(1992);E.Bagan,P.Ball,and P.Gosdzinsky,Heidelberg preprint HD–THEP–92–40(1992).[19]B.Blok and M.Shifman,Santa Barbara preprint NSF–ITP–92–100(1992).[20]M.Neubert,Z.Ligeti,and Y.Nir,SLAC preprint SLAC–PUB–5915(1992).[21]M.Neubert,SLAC preprint SLAC–PUB–5992(1992).[22]M.A.Shifman,A.I.Vainshtein,and V.I.Zakharov,Nucl.Phys.B147,385(1979);B147,448(1979).FIGURESFIG.1.Diagrams contributing to the sum rule for the universal form factorχ3(v·v′):two-loop perturbative contribution(a),and nonperturbative contributions proportional to the quark con-densate(b),the gluon condensate(c),and the mixed condensate(d).Heavy quark propagators are drawn as double lines.The square represents the chromo-magnetic operator.FIG.2.Analysis of the stability region for the sum rule(23):The form factorχ3(y)is shown for y=1.5as a function of the Borel parameter.From top to bottom,the solid curves refer toω0=1.7,2.0,and2.3GeV.The dashes lines are obtained by neglecting the contribution proportional toδω2.FIG.3.(a)Prediction for the form factorχ3(v·v′)in the stability region1.7<ω0<2.3 GeV and0.7<T<1.2GeV.(b)Individual contributions toχ3(v·v′)for T=0.8GeV and ω0=2.0GeV:total(solid),mixed condensate(dashed-dotted),gluon condensate(wide dots), quark condensate(dashes).The perturbative contribution and theδω2-term are indistinguishable in thisfigure and are both represented by the narrow dots.11。

蝙蝠侠:阿甘疯人院治疗中心、医疗设施、监狱谜题收集攻略

蝙蝠侠:阿甘疯人院治疗中心、医疗设施、监狱谜题收集攻略

蝙蝠侠:阿⽢疯⼈院-治疗中⼼、医疗设施、监狱谜题收集攻略I N T E N S I V E T R E AT M E N T应急治疗中⼼ 应急治疗中⼼-秘密地图 (S e c u r e Tr a n s i t) Yo u w o n't b e a b l e t o g r a b t h i s m a p u n t i l y o u'v e r e t u r n e d t o应急治疗中⼼i n s e a r c h o f K i l l e r C r o c's l a i r.O n c e y o u r e a c h t h e v e r yb o t t o m o f t h e f ac i l i t y-d o w n t w oe l e v a t o r s h af t s-l o o k f o r t h em o n s t e r's m e a t l o c k e r.O n t h e o p p o s i t e s i d e o f t h e r o o m i s as t a i r c a s e t h a t l e a d s t o a n o ffic e f u l l o f K i l l e r C r o c r e s e a r c h.T h e m a p i s t h e r e a s w e l l. (安全中转)(E r...这个房间的名字是什么意思啊。

)在你回到重症监护室寻找C r o c的藏⾝处之前你是不能够得到这张地图的。

⼀旦你到达了这个建筑的最底层——从两个电梯机井下——寻找那怪物的⾁柜。

在房间的对边有个楼梯通向装满了C r o c研究记录的办公室。

地图就在那。

%{p a g e-b r e a k|应急治疗中⼼-秘密地图|p a g e-b r e a k}% 应急治疗中⼼-谜题解答#1 (应急治疗中⼼L o b b y) "D o n't c u t y o u r s e l f o n t h i s S h a r p l y o b s e r v e d p o r t r a i t." I m p o s s i b l e t o m i s s,a s t h e g a m e h o l d s y o u r h a n d t h r o u g h t h i s fir s t r i d d l e.J u s t s c a n t h e p a i n t i n g o f Wa r d e n S h a r p,h a n g i n g o n t h e n e a r b y w a l l,t o p r o c e e d. (重症监护室⼤厅) “别让这幅肖像割伤你!” 不可能错过的⼀个,就好像游戏牵着你的⼿找到这第⼀个谜题。

汽车总动员中英文字幕

汽车总动员中英文字幕

只为大家学英语《汽车总动员》Cars[Inhaling and exhaling deeply] [Male] OK... Here we go. Focus.好了,准备了,专心Speed. I am speed.速度,我是速度[Cars whizzing past]One winner, 41 losers.1个赢家,41个输家I eat losers for breakfast.我把输家当早餐吃[Car accelerating]Breakfast.早餐Wait, maybe Ishould have had breakfast. 也许我该吃早餐,可能会比较好A little breck-y could be good for me.No, no, no, stay focused. Speed.不不不不…保持专心,速度[Cars whizzing]I'm faster than fast.Quicker than quick. 疾如风,快如电I am lightning!我是“闪电”![Pounding on door][Male] Hey, Lightning! You ready?嘿,“闪电”,准备好了吗<i>[Sheryl Crow: Real Gone]</i>Oh, yeah. Lightning's ready没错,“闪电”早就准备好了[engine rewing][Engine revs][Crowd cheers]Ka-chow!咔嚓![Cars zooming]《汽车总动员》[Cars whooshing][Both scream][All cheer]Get your antenna balls here!来买天线球喔Go, Lightnin'!冲啊,“闪电”!- Whoo!- You got that right, slick. [whistles]- 哇喔!- 你说对了,滑头[Air wrench whirring]Uh! [screams]呀![Engine revs]- [Male] Welcome back to the Dinoco 400.- [Crowd cheers]欢迎回到戴诺可400I'm Bob Cutlass, herewith my good friend, Darrell Cartrip.我是鲍伯,跟我的好朋友达洛赛车进行到一半了,今天可能是赛车史上重要日子<i>We're midway through what maybe an historic day for racing.</i><i>Bob, my oil pressure'sthrough the roof.</i>鲍伯,现在我的油压暴升<i>If this gets more exciting,they'regonna have to tow me outta the booth!</i>比赛要是更刺激的话,恐怕我得被拖着出去了<i>Right, Darrell.</i>的确是的,达洛<i>[Bob] Three cars are tiedfor the season points lead,</i>三部领先的车,以相同的积分<i>heading into the final raceof the season.</i>进入本季最后一场比赛<i>And the winner of this race will winthe season title and the Piston Cup.</i>这场比赛的胜利者,将赢得季冠军头衔,跟活塞奖杯<i>Does The King, Strip Weathers,</i> 绰号“冠军”的史崔威勒<i>have one more victory in himbefore retirement?</i>是不是能在退休以前再夺得一次胜利呢<i>[Darrell] He's been Dinoco's golden boyfor years!</i>他是戴诺可赞助多年的金童<i>Can he win them one last Piston Cup?</i>能不能赢得最后一个活塞奖杯呢?<i>[Bob] And, as always, in thesecond place spot we find Chick Hicks.</i>还有就是一如往常的,第二名“路霸”在他的赛车生涯中,始终都位居第二<i>He's been chasingthat tailfin his entire career.</i><i>[Darrell] Chick thoughtthis was his year.</i>路霸以为今年是他的幸运年<i>His chance to finally emergefrom The King's shadow.</i>终于有机会脱离“冠军”史崔威勒的阴影<i>But the last thing he expected was...Lightning McQueen!</i>可是他万万没想到半路杀出个程咬金,“闪电”麦坤!<i>[Bob] You know, I don't thinkanybody expected this.</i>你知道吗,我看没有人想到这位菜鸟新秀,原本是个无名小子<i>The rookie sensationcame into the season unknown.</i> <i>But everyone knows him now.</i>现在大家都认识他了<i>[Darrell] Will he be the first rookieto win a Piston Cup and land Dinoco?</i>他会是第一个赢得活塞奖杯,跟戴诺可赞助的菜鸟吗?<i>[Bob] The legend, the runner-up,and the rookie!</i>赛车传奇,亚军,和菜鸟<i>Three cars, one champion!</i>三部车,一个冠军![Breaks screeching]哦,不,你休想!No you don't.- [Chuckling]- Hey!呵呵!- [Tires squealing]- [Crowd booing] What a ride!哦,闪电,快啊![Chuckling]Go get 'em, McQueen!Go get 'em!超过去,麦坤!超过去![Female] I love you, Lightning!我爱你!“闪电”!- Dinoco is all mine.- [Screaming]戴诺可是我的![Darrell] Trouble, turn three!有状况,第三弯道!- Get through that, McQueen.- [Bob] Huge crash behind the leaders!- 冲过来啊,麦坤- 不好,领先车的后面撞成了一团[Crowd gasps][Screaming][Giggling]- [Grunts]- [Gasps][Both screaming][Bob] Wait a second, Darrell.McQueen is in the wreckage.等一下,达洛,麦坤冲进车阵了[Darrell] There's no way the rookiecan make it through!那个菜鸟绝对不可能过得去!至少不是毫发无伤Not in one piece, that is. [Exhaling]Yeah!耶!Lightning! Oh!“闪电”!喔![Darrell] Look at that!McQueen made it through!你看看!麦坤过去了耶!天呐!“闪电”麦坤精彩突围[Bob] A spectacular moveby Lightning McQueen!Yeah! Ka-chow!耶!咔嚓!McQueen! McQueen! McQueen!McQueen! McQueen! McQueen!麦坤!麦坤!麦坤!麦坤!麦坤!麦坤!Yeah, McQueen! Ka-chow! [honking]耶,麦坤!咔嚓![Bob] While everyoneheads into the pits, 当其他赛车都进维修站的时候麦坤继续跑,取得领先McQueen stays out to take the lead! Don't take me out, coach.I can still race!别让我退出啊,教练我还可以跑![Air wrench whirring][Chuckling] What do you think?A thing of beauty.哈哈,你们觉得怎么样?很厉害吧- McQueen made it!- [Chick] What?- 麦坤他不休息!- 什么?He's not pitting!他不进站!You gotta get me out there!Let's go! Get me back out there!快,快让我出站快!快让我回去,快!McQueen's not going into the pits!麦坤居然不进站维修![Darrell] The rookie fired hiscrew chief. The third this season!那个菜鸟刚开除了他的总机械师,那是本季第三位了!- [Bob] Says he likes working alone.- Go, go!- 他自个儿说喜欢独立自主的- 跑!跑!跑!Looks like Chickgot caught up in the pits.路霸被维修给耽搁了Yeah, after a stop like that,he's got a lot of ground to make up.对,停了那么久,他可有一大段路要追呢Get ready, boys,we're coming to the restart!男士们,准备好,要开始喽![Crowd cheers]Come on, come on, come on!让开让开让开!We need tires now!Come on, let's go! 我们需要轮胎!快点!快点!- No, no, no, no! No tires, just gas!- [Male] What?- 不不不不!不要轮胎,汽油就好- 什么?You need tires, you idiot!你需要轮胎,你这白痴!看来麦坤今天只打算加了汽油就跑啊[Darrell] Looks like it'sgas-and-go's for McQueen today.没错,还是不换轮胎[Bob] Right. No tires again.通常我会说这就像是在炒短线可是对他挺有效的[Darrell] That's a short-termgain,long-term loss,but it's workin' for him. He obviouslyknows somethin' we don't know. 嘿嘿,显然他懂的比我们多啊[Cars whizzing][Bob] This is it, Darrell. One lap to goand Lightning McQueen has a huge lead. 决胜时刻到了,达洛,只剩一圈闪电麦坤遥遥领先哦,他赢定了,把庆功宴准备好吧!He's got it in the bag.Call in the dogs and put out the fire!新的赛车王就要产生喽!We're gonna crown us a new champion![Crowd cheers and whistles] [Screaming]方格旗,我来喽!- Checkered flag, here I come!- [Tire blows][Darrell] No! McQueen's blown a tire! 哦,糟了!麦坤爆了一个轮胎![Bob] And with only one turn to go!Can he make it?只剩最后一个弯道,他撑得住吗?- You fool!- [Grunts]傻瓜![Male] McQueen's blown a tire!He's blown a tire!麦坤爆胎了!麦坤爆胎了!Go, go, go!快快快!- [Grunting]- [Tire blows][Darrell] He's lost another tire!他又爆了一个轮胎!- King and Chick come up fast!- [Bob] They're entering turn three!- “冠军”跟“路霸”急起直追!- 他们进入第三弯道了Come on. [grunting]加油I don't believewhat I'm watching, Bob! 我真不敢相信我的眼睛!Lightning McQueenis 100 feet from his Piston Cup!“闪电”麦坤距离活塞奖杯只差一百英尺![Gasping][Growling][Bob] The King and Chickrounding turn four.“冠军”和“路霸”通过第四弯道[Darrell] Down the stretch they come!And it's, and it's...他们来到直线跑道了结果是……- It's too close to call! Too close!- I don't believe it!- 实在是太接近了,根本看不出来啊- 不敢相信!我真不敢相信!Lightning! 我们爱你,“闪电”!- The most spectacular, amazing...- I don't believe it!- 这真是我们赛车史上最精彩,最叹为观止,最不可思议的结局啊!- 不敢相信!我真不敢相信!我还是不敢相信!...unequivocally, unbelievable endingin the history of the world!- And we don't know who won!- Look at that!- 我们连谁赢了都不知道- 看看!<i>[Tape jittering]</i><i>[Slow motion drone]</i>- That's very close to call.- Can we play that again?- 太接近了,怎么看啊- 可以再放一次吗?请再放一次Hey, no cameras! Get outta here!嘿!不准照相!快走开!<i>We're here in Victory Lane,awaiting the results.</i><i>We're here in Victory Lane,awaiting the results.</i>我们在胜利之道等候赛车结果<i>McQueen, that was a risky move,not taking tires.</i>麦坤,不换胎是很冒险的举动哦<i>Tell me about it!</i>这还用说<i>Are you sorry you didn't havea crew chief out there?</i>你会不会后悔没有总机械师<i>Oh, Kori. There's a lot more to racingthan just winning.</i>呵呵,柯琳赛车不光是只为了赢,还有别的<i>I mean, taking the race by a full lap...Where's the entertainment in that?</i>我是说领先一整圈赢得冠军这样有什么看头啊<i>I wanted to give folks a little sizzle.</i>我要给赛车迷一点刺激<i>- Sizzle?- Am I sorry I don't have acrew chief?</i>- 剌激?- 我后不后悔没有总机械师?<i>No, I'm not.'Cause I'm a one-man show.</i>不,不后悔,因为我是个人秀<i>What? Oh, yeah, right.</i>什么?喔,是啊<i>That was a confidentLightning McQueen.</i>没错,那是非常有自信的“闪电”麦坤Live from Victory Lane,I'm Kori Turbowitz.记者柯琳·泰伯薇在胜利之道的现场报道- Get outta the shot.- Yo, Chuck.- 嘿,别挡镜头- 嘿,查理Chuck, what are you doing?You're blockin' the camera!你做什么啊?你挡到镜头了- Everyone wants to see the bolt.- What? - 大家都想看闪电- 什么?- Now, back away.- That's it! Come on, guys.- 退后一点- 嘿,够了,我们走吧- Whoa, team! Where are you going?- We quit, Mr. One-Man Show!- 唉呀,你们要去哪儿啊- 我们辞职,个人秀先生Oh, OK, leave. Fine.好啊,走啊How will I ever find anyone elsewho knows how to fill me up with gas?呵呵,我要上哪去找能帮我加油的组员呢[Crowd laughs]- Adios, Chuck!- And my name is not Chuck!- 再见,查理- 我的名字不叫查理!Oh, whatever.哦,随便了Hey, Lightning! Yo! McQueen!嘿,闪电!呦,麦坤!Seriously, that was somepretty darn nice racin' out there.说真的,刚才的冲刺可真精彩啊- By me!- Oh, yeah. 我是说我!- Zinger!- Welcome to the Chick era, baby!欢迎进入路霸的年代The Piston Cup...It's mine, dude. It's mine.活塞奖杯是我的,我的Hey, fellas, how do you thinkI'd look in Dinoco blue? Dinoco blue!嘿,各位,我开戴诺可蓝色车怎么样?戴诺可蓝!In your dreams, Thunder.别做梦了,雷鸣Yeah, right. Thunder?What's he talkin' about, "Thunder"?- 是呀,雷鸣?他在说什么?- 不知道啊You know, 'cause thunderalways comes after lightning.因为雷声总是在闪电后面Ka-ping! Ka-pow!呯!咔!- Who knew about the thunder thing?- I didn't.- 你们谁知道雷声这个玩笑- 我不知道- Give us the bolt!- That's right. Right in the lens.- Show me the bolt, baby!- Smile, McQueen!- Show me the bolt, McQueen!- That's it! [Electronic music][Crowd chattering]That was one close finish.You sure made Dinoco proud.好个平手的结局啊,你让戴诺可很骄傲哦Thank you, King.谢谢你,冠军Well, Tex, you've beengood to me all these years.老德啊,这些年来你对我很好It's the least I could do.这是我应该做的Whatever happens, you're a winnerto me, you old daddy rabbit.不论发生什么事你都是我心目中的赢家,老公Thanks, dear.Wouldn't be nothing without you.谢了,老婆没有你我什么也不是Kch-i-ka-chow!- I'm Mia.- I'm Tia.- 我是蜜亚- 我是蒂亚[Both] We're, like, your biggestfans!Ka-chow!我们最崇拜你了咔嚓!I love being me.哦,做我真好- [Police] OK, girls, that's it.- We love you, Lightning!- 好了,小姐们,够了- 我们爱你,“闪电”![Chuckles]Hey, buddy.You're one gutsy racer. 嘿,朋友,你真的很有胆识啊Oh, hey, Mr. The King.噢,冠军先生You got more talent in one lug nut你光是一个螺丝就比很多车子拥有更多赛车细胞than a lot of cars has gotin their whole body.- Really? Oh, that...- But you're stupid.- 真的?这个...- 但是你很笨- Excuse me?- This ain't a one-man deal, kid.- 你说什么?- 这不是独角戏啊,孩子You need to wise up and geta good crew chief and a good team.你要放聪明点给自己找一个一流的总机师和伙伴You ain't gonna winunless you got good folks behind you,你想赢,一定要有好人才[voice dwindling] and you let themdo their job, like they should.在背后支持,让他们做他们的工作- Like I tell the boys at the shop...- A good team.- 必须要每个人同心协力- 一流的伙伴- Yeah...- [electronic music]耶Ka-chow! Ka-pow![Crowd cheering][Screaming][Cheering]Oh, Lightning!哦!“闪电”!If you figure that outyou just gonna be OK.只要你想清楚了,前途无可限量Oh, yeah, that...That is spectacular advice.哦,对,真的是非常好的建议Thank you, Mr. The King.谢谢,冠军先生- [Fanfare]- [Bob] Ladies and gentlemen, 各位女士先生for the first timein Piston Cup history...活塞杯赛车史上头一遭[rewing] A rookiehas won the Piston Cup. 一位菜鸟赢得活塞奖杯Yes!耶![Bob]... we have a three-way tie.今天我们有三辆车平手- [Crowd cheers]- [Cameras flash] [Chuckling] Hey,that must be really embarrassing.嘿!麦坤,你一定很尴尬吧But I wouldn't worry about it.Because I didn't do it!可是我不会放在心上,因为尴尬的不是我[Bob] Piston Cup officialshave determined that a tiebreaker race因为今天的比赛有三位平手,所以裁判决定,一星期后between the three leaderswill be held in California in one week.将在加州举行三位领先者的决赛Well, thank you! Thanks toall of you out there! Thank you!谢谢!谢谢所有观众!谢谢![Whispering] Hey, first one toCalifornia gets Dinoco all to himself. 嘿,菜鸟,谁第一个到加州,就可以单独跟戴诺可谈No, not me!No, you rock, and you know that!不,不是我你们才是最棒的Oh, yeah! Whoo!喔耶!喔Yep! All right! Got it!谁第一个到加州,就可以单独跟戴诺可谈"First one to Californiagets Dinoco all to himself."Oh, we'll seewho gets there first, Chick.那就看看谁会先到,路霸Huh?嗯?- Hey, kid! Congrats on the tie.- I don't want to talk about it.- 嘿!小子,恭喜你争取到决赛- 我不想谈这件事Let's go, Mack. Saddle up.What'd you do with my trailer?好了,麦大叔,出发了我的拖车到哪去了?- I parked it at your sponsor's tent.- What?- 我把它停在你赞助者帐篷- 什么?Gotta make your personal appearance. 你必须亲自去露个脸No. No! No, no, no, no!不、不、不不不不…<i>Yes, yes, yes!Lightning McQueen here.</i>对对对,我是闪电麦坤<i>And I use Rust-eze MedicatedBumper Ointment, new rear end formula!</i>我用新配方“清锈”保险杆除锈药膏<i>Nothing soothes a rusty bumperlike Rust-eze.</i>“清锈”让生锈保险杆一擦见效<i>Wow! Look at that shine!</i>哇!马上亮晶晶!<i>Use Rust-eze and you toocan look like me!</i>使用“清锈”你也能像我一样帅<i>Ka-chow!</i>咔嚓![Both laughing]I met this car from Swampscott.我遇到来自沼泽的一辆车He was so rustyhe didn't even cast a shadow.他锈得很严重,连影子都没有- You could see his dirty undercarriage.- [Crowd laughs]你可以看见他肮脏的底盘[Groaning] I hate rusty cars.This is not good for my image.我讨厌生锈的车,这对我形象非常不好They did give you your big break.Besides, it's in your contract.你要感谢他们的赞助再说你签了合约Oh, will you stop, please?Just go get hooked up.哦,别说了,好吗?去牵拖车就好了- Winter is a grand old time.- Of this there are no ifs or buts.- 冬天是个好季节- 这一点没什么好争辩的[Male 1] But remember,all that salt and grime...可是要记住喔,那些盐跟灰尘Can rust your bolts and freeze your... 会让你的螺丝钉生锈,螺丝帽结冰Hey, look! There he is!嘿!你们看,他来了耶Our almost champ!冠军角逐者[Male 1] Get your rear end in here.快过来,小子Lightning McQueen,you are wicked fast! 闪电麦坤,你太快了- That race was a pisser!- You were booking!- 那场赛车真是精彩啊- 对,你好快,麦坤- Give me a little room.- You're my hero! - 拜托,让我过一下- 你是我的偶像!Yes, I know. [chuckles]- 麦坤先生- 是,我知道"Fred." Fred, thank you.- 弗瑞- 弗瑞,谢谢He knows my name.He knows my name!他知道我的名字!他知道我的名字!Looking good, Freddie!不错,弗瑞Thanks to you, Lightning,we had a banner year!多亏你了,让我们今年赚了不少We might clear enoughto buy you some headlights.很好,我们应该有足够的钱帮你买头灯喽You saying he doesn't have headlights? 你是说他没有头灯?That's what I'm telling ya.They're stickers!我就是这么说的,那些只是贴纸Well, you know,racecars don't need headlights,你们知道的赛车不需要什么头灯because the track is always lit.因为跑道啊,一向都是明亮的啊Yeah, well, so is my brother,but he still needs headlights.我弟是秃头,他还是需要头灯[Laughing][Forced laughter]- Ladies and gentlemen,- [both] Lightning McQueen!- 各位女士先生- 闪电麦坤[Crickets chirping]Free Bird!唱歌You know, the Rust-eze MedicatedBumper Ointment team各位知道吗?“清锈保险杆药膏”车队ran a great race today.今天表现的非常好And remember, with a little Rust-eze... 还有,别忘了,只要一点“清锈”[whispering] And an insaneamount of luck, 加上大量的运气...you too, can look like me.Ka-chow. 你也可以像我一样帅咔嚓- Hey, kid.- We love ya.- 嘿,小子- 我们爱你And we're looking forwardto another great year.而且我们期盼明年也是业绩长红Just like this year!跟今年一样!Not on your life.明年我就不干了- Don't drive like my brother!- Yeah, don't drive like my brother!- 别像我弟那样开车- 是吗?别像我哥那样开车<i>California, here we come!</i>亲爱的加州,我们来了Dinoco, here we come!戴诺可,我们来了!<i>[Rascal Flatts: Life is a Highway]</i>[Birds chirping]<i>[Phone ringing]</i>[Moaning] Oh... oh... uh,I needed this. Hello?好舒服喔,哪位?<i>[Male] Is this Lightning McQueen,the world's fastest racing machine?</i>请问是全世界最快的赛车,闪电麦坤吗?Is this Harv,the world's greatest agent? 你是最棒的经纪人哈威吗?<i>And it is such an honorto be your agent</i>当你的经纪人真荣幸啊<i>that it almost hurts me to taketen percent of your winnings.</i>我几乎不忍心拿你一成的奖金还有广告酬劳<i>Merchandising. And ancillary rights inperpetuity. Anyway, what a race, champ!</i>永久的经纪权了总之,好精彩的比赛<i>I didn't see it,but I heard you weregreat.</i>我没…我没看可是听说你很棒Thanks, Harv.谢了,哈威<i>Listen, they're giving you 20 ticketsfor the tiebreaker thing in Cali.</i>听着,你有二十张加州决赛的票我可以帮你拿给你朋友,你告诉我名字<i>I'll pass 'em on to yourfriends.Shoot me the names.</i>- 让哈威为你服务,好吗?宝贝- 是,朋友<i>- You let Harv rock it for you, baby.- Right. Friends.</i>Yes, there's...是,有,嗯……<i>OK, I get it, Mr. Popular.</i>好吧,我懂了,人气小子<i>So many you can't even narrow it down.Hey, when you get to town,</i>朋友太多,不知道给谁好嘿,你到城里来<i>you better make timefor your best friend!</i>最好抽空来看你最好的朋友<i>Break breadwith your mishpocheh here!</i>你一定要跟你的好友吃顿饭That'd be great!We should totally... 对,这样很好,我们是应该……<i>OK, I gotta jump, kid.Let me know how it goes. I'm out.</i>我得挂了,小子让我知道情况,再见<i>- [Dial tone hums]- [Sighing]</i> <i>- [Melodic beeping]- [Wheels squeaking]</i>[Lightning] What? A minivan?什么?小货车?<i>Come on, you're in the slow lane.</i> 拜托,麦大叔你开到慢车道了<i>This is Lightning McQueenyou're hauling here.</i>你载的是闪电麦坤耶Just stopping offfor a quick breather, kid. 我只要停一会就好了,小子<i>- Old Mack needs a rest.- Absolutely not.</i>绝对不可以<i>We're driving all night till weget to California. We agreed to it.</i>我们要整夜赶路,一直到加州去我们说好的All night? May I remind youfederal DOT regs state...- 整夜?让我提醒你国家交通部规定…-拜托,麦大叔<i>Come on, I need to get therebefore Chick and hang with Dinoco.</i>我要比路霸先到,跟戴诺可聊聊[Mack groans]All those sleeping trucks. 那些打盹的卡车<i>Hey, kid,I don't know if I can make it.</i>嘿,小子,我不知道我能不能做得到Oh, sure you can, Mack.你当然可以,麦大叔Look, it'll be easy.I'll stay up with you.很容易啊,我陪你熬夜<i>- All night?- All night long.</i> - 一整夜?- 一整夜[Snoring][Engines slowing][Snoring, gasping, blubbering] [Grunting][Snoring] Uh![Blubbering]- [Rap music]- [Engines rewing] [Sniffing] Ah-choo![Laughing]- Hey, yo, D.J.- What up?- 嘿呦,DJ- 干嘛?- We got ourselves a nodder.- [Chuckling]有一辆车在打瞌睡喔<i>[Kenny G: Songbird]</i>Pretty music. [snoring]音乐真好听Yo, Wingo! Lane change, man.嘿,文哥,换车道了,老兄- Right back at ya!- Yeah!回你那边啦- Oops! I missed.- You going on vacation? - 没有碰到- 你在渡假吗?[All laugh][Snoring][Snoring]- [Chuckling]- [Sniffling]- Oh, no, Snot Rod...- He's gonna blow! - 哦,糟了,大鼻!- 他要打喷嚏啦!Ah... Ah... Ah-choo!Gesundheit!保重身体One should never drive while drowsy. 绝对不要边打瞌睡边开车- [Tires squealing]- [Horns honking] - [Honking loudly]- [Screaming]- Uh! Ah!- [All honking]- 呃!啊![Breathing heavily] Mack!麦大叔!- [Grunting]- [Horns honking]Mack!麦大叔!Mack!麦大叔!Hey, Mack! Mack!嘿,麦大叔!麦大叔!Mack!麦大叔!Mack, wait for me! [engine rewing]麦大叔等等我![Tires squealing]Mack!麦大叔![Bell ringing][Horn bellowing]Mack! Mack!麦大叔!麦大叔!Mack! Mack...麦大叔!麦大叔......wait up! [coughing] Mack.等等!麦大叔!Mack! Mack!麦大叔!麦大叔!What? You're not Mack.你不是麦大叔Mack? I ain't no Mack!I'm a Peterbilt, for dang sake!麦大叔?我不是麦大叔我是大卡车,拜托[Peterbilt]Turn on your lights, you moron!开你的头灯,白痴Mack... The Interstate!麦大叔……州际公路[Tires squealing]- [Engine roaring]- Huh?Not in my town, you don't.在我辖区里休想超速[Siren wailing]Oh, no.哦,糗了- Oh, maybe he can help me!- [Sheriff backfiring]哦!也许他可以帮我He's shooting at me!Why is he shooting at me?他向我开枪?他为什么向我开枪?[Backfiring]I haven't gone this fast in years.我好多年没跑这么快了[Grunting] I'm gonnablow a gasket or somethin'.我的密封垫一定会裂开Serpentine! Serpentine, serpentine!蛇行!蛇行!蛇行!What in the blue blazes?搞什么呀?居然蛇行,飙车狂Crazy hot-rodder.[Siren continues wailing]<i>[Hank Williams:My Heart Would Know]</i>[Buzzing][史丹利我们的城镇奠基者 1909] [Water hose turning on][Snoring]I'm telling you, man,every third blinkis slower.我告诉你,老兄闪第三次都比较慢The sixties weren't good to you,were they?60年代太亏待你了,是吗?[Car backfires][Sirens blasting]What? That's not the Interstate!什么?那不是州际公路- [Sheriff backfires]- Ah! Oh!Ow, ow, ow, ow, ow!- [Screaming]- [Tires screeching] No, no, no, no, no, no!不不不Ow!I'm not the only one seeing this, right? 不是只有我看到,对吧?- Incoming!- Whoa, man.危险!- No!- Hey!嘿!你刮了我的漆Whoa, whoa, whoa, whoa!My tires!我的轮胎!- [Grunting]- [Gasping]- [Screaming]- [Gasping]- [Tires squealing]- [Cables vibrating] [Yelling][Shouting][Screaming]- [Cables whizzing]- [Straining]Fly away, Stanley. Be free!飞走吧,史丹利,自由自在[Gasping][Breathing heavily]Boy, you're in a heap of trouble.小子,你的麻烦可大了[Sighing]<i>We're live at the Los AngelesInternational Speedway</i>记者正在洛杉矶国际赛车场<i>as the first competitor,Lightning McQueen,</i>- 第一位决赛者闪电麦坤- 他来了<i>is arriving at the track.</i>- 刚刚到赛车场- 麦坤先生,旅途顺利吗?<i>Is it true he's gonna pose for Cargirl?</i>他真的要上《赛车女郎》杂志吗?闪电,你的策略是什么?<i>What's your strategy?</i>- [Crowd gasps]- [Cameras clicking]<i>What? Did I forgetto wipe my mud flaps?</i>怎么啦?我忘了擦挡泥板吗?[闪电麦坤失踪了!!]<i>McQueen's driver arrived in California,but McQueen was missing.</i> 麦柛的运送车到达加州但是麦坤不见了<i>- McQueen was reported missing....to race an unprecedented...</i>赛车“闪电麦坤”神秘失踪了麦坤应该参加一场前所未有的……<i>[male] Sponsor statedthey have no idea where he is.</i>赞助厂商说不知道他在什么地方I hope Lightning's OK. I'd hateto see anything bad happen to him.我只希望“闪电”没事我实在不希望他发生任何意外<i>I don't know what's harder to find,McQueen or a chief who'll work with him!</i>我不知道什么比较难找闪电麦坤还是肯跟他合作的总机械师<i>[German accent] Lightning McQueenmust be found at all costs!</i> 不计代价,一定要找到闪电麦坤!<i>They're all asking the same question:Where is McQueen?</i>他们都在问同一个问题:麦坤到底在哪里?[Groaning] Oh, boy.天啊Where am I?这是什么地方啊?- Mornin', sleepin' beauty!- Ah!早啊,睡美人!Boy, I was wonderin'when you was gonnawake up.我在想你要多久才会醒过来Take whatever you want!Just don't hurt me!你要什么就拿去,只要别伤害我A parking boot?Why do I have a parking boot on?轮胎锁?为什么给我上轮胎锁?- What's going on here? Please!- [Chuckling] You're funny.- 这是怎么回事?拜托- 哈哈,你真好笑I like you already. My name's Mater. 我喜欢你,我叫拖线Mater?拖线?Yeah, like "tuh-mater,"but without the "tuh".对,拖把的拖,不是脱衣服的脱- What's your name?- You don't know my name?- 你叫什么名字?- 你…你不知道我的名字?Uh...嗯…我知道你的名字你也叫拖线?No, I know your name.Is your name Mater too?What?什么?Look, I need to get to Californiaas fast as possible. Where am I?听我说,我必须在最短的时间内尽快赶到加州去这是什么地方啊?Where are you? Shoot!这是什么地方?嘿You're in Radiator Springs.这是化油器郡The cutest little townin Carburetor County.一个可爱的小镇,叫油车水镇Oh, great. Just great!好,好极了Well, if you think that's great,you should see the rest of the town.如果你认为这里很好你应该参观镇上其他的地方You know,I'd love to see the rest of the town!你知道吗?我很想参观其他的地方So if you could just open the gate,take this boot off,所以只要你打开门,解开轮胎锁you and me, we go cruisin',check out the local scene...你跟我就一起游览本地的风景好吗?- Dad-gum!- How'd that be, Tuhmater? - 耶,好- 怎么样,拖线?- Cool!- Mater!- 酷!- 拖线!What did I tell youabout talkin' to the accused?我说过,你可不可以跟被告说话?To not to.不可以Well, quit your yappin' and tow thisdelinquent road hazard to traffic court.那就废话少说把这个威胁道路的罪犯,拖去交通法庭Well, we'll talk later, Mater.等会聊了,小老弟[Chuckling] "Later, Mater."That's funny!小老弟,真好笑Ah![Sheriff] The Radiator SpringsTraffic Court will come to order!油车水镇交通法庭即将开庭Hey, you scratched my paint!I oughta take a blowtorch to you, man!你刮了我的漆真该放把火把你烧了You broke-a the road!You a very bad car! 我得重新叠罐头你弄坏道路,你是辆坏车- Fascist!- Commie!- 暴君!- 对啊,对啊Officer, talk to me, babe.How long is this gonna take?警长,请告诉我,这到底还要多久?I gotta get to California, pronto.我很忙,得立刻赶到加州去Where's your lawyer?你的律师呢?I don't know. Tahiti maybe.He's got a timeshare there.谁知道,在大溪地吧他在那里有一个度假屋When a defendant has no lawyer,the court will assign one to him. Hey!当被告没有律师的时候法庭会指派一个给他Anyone want to be his lawyer?谁要当他的律师啊?Shoot, I'll do it, Sheriff!对,我可以,警长All rise! The HonorableDoc Hudson presiding.请起立,法官大人韩大夫主审Show-off.爱现- May Doc have mercy on your soul.- [Door banging]希望韩大夫怜悯你这个可怜虫- [Gasping]- All right,好了,我要知道谁破坏我的镇警长I wanna know who's responsiblefor wreckin' my town, Sheriff.I want his hood on a platter!我要拿下他的车盖I'm gonna put him in jail till he rots!No, check that.我要他坐牢,直到他烂掉不,更正I'm gonna put him in jail tillthe jail rots on top of him,我要他坐牢,直到监牢烂掉,压在他车顶and then I'm gonna move himto a new jail and let that jail rot.然后再把他移到新的坚牢,让那个监牢也烂掉I'm...我……Throw him out of here, Sheriff.把他撵出去,警长I want him out of my courtroom.I want him out of our town! 我要他离开我的法庭我要他离开我的镇- Case dismissed.- Yes!- 本案终结- 好耶Boy, I'm purty goodat this lawyerin' stuff.我可是一流的律师耶Sorry I'm late, Your Honor!抱歉,我迟到了Holy Porsche! She's gotta befrom my attorney's office.保时捷美眉,她一定是我的律师派来的Hey, thanks for comin',but we're all set.谢谢你来,不过没事了- He's letting me go.- He's letting you go?- 他要放我走- 他要放你走?Yeah, your job's pretty easy today. 对,今天你的工作很轻松All you have to do now is stand thereand let me look at you.你只需要乖乖站在那儿让我好好的看看你Listen, I'm gonna cut to the chase.Me, you, dinner. Pi-cha-kow!好吧,我就不拐弯抹角了,我…你…晚餐,咔嚓- What the...? Ow! Oh!- Ka-chow!- 什么?噢!哦- 咔嚓!- Please!- I know. I get that reaction a lot.- 拜托!哦- 我知道,这种反应我见多了I create feelings in others thatthey themselves don't understand.我带给别人的感受连他们自己都无法理解- [Rewing]- Agh! Ow!Oh, I'm sorry. Did I scare you?对不起,吓着你了吗?A little bit, but I'll be all right. 嗯…有一点,但我不要紧OK. I'm gonna go talk to the judge.好吧,我要去跟法官谈谈Do what you gotta do, baby.But listen. Be careful.做你该做的,宝贝喔!但是小心一点。

WAters31843

WAters31843

Desolvation gas flow liters/hr 200 to 250 250 to 400 250 to 400 400 to 750
Higher desolvation temperatures give increased sensitivity. However, increasing the temperature above the range suggested reduces beam stability. Increasing the gas flow rate higher that the quoted values lead to unnecessary high nitrogen consumption. Avoid operating the desolvation heater for long periods of time without proper gas flow. To do so could damage the source.
©2004 Waters Corporation
MS Troubleshooting Strategy
Try to simplify -assess impact on lab efficiency -inspect the MS or /MS/MS -try to categorize troubleshoot the easiest to fix items first
Mass Spectrometry
Source: ESI APcI Nano-ESI Mass analyzers: magnetic sectors electric sectors time of flight quadrupole ion trap FT-ICR

甲壳动物中蜕皮激素新陈代谢

甲壳动物中蜕皮激素新陈代谢

Journal of Steroid Biochemistry &Molecular Biology 127 (2011) 196–203Contents lists available at ScienceDirectJournal of Steroid Biochemistry and MolecularBiologyj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j s b mbReviewEcdysteroid metabolism in crustaceans ଝDonald L.Mykles ∗Department of Biology,Colorado State University,Campus 1878,Fort Collins,CO 80523,USAa r t i c l ei n f oArticle history:Received 27July 2010Received in revised form 1September 2010Accepted 2September 2010Keywords:Crustacean MoltingEcdysteroid biosynthesis Ecdysteroid metabolism Ecdysteroid excretion Halloween genes Reviewa b s t r a c tThe molting gland,or Y-organ (YO),is the primary site for ecdysteroid synthesis in decapod crus-taceans.Ecdysteroid biosynthesis is divided into two stages:(1)conversion of cholesterol to 5␤-diketol and (2)conversion of 5␤-diketol to secreted products.Stage 1involves the conversion of cholesterol to 7-dehydrocholesterol (7DC)by 7,8-dehydrogenase,the “Black Box”reactions involving 3-oxo- 4intermediates,and the conversion of 4-diketol to 5␤-diketol by 5␤[H]-reductase.The stage 2reactions generate four major products,depending on species:ecdysone,3-dehydroecdysone (3DE),25-deoxyecdysone (25dE),and 3-dehydro-25-deoxyecdysone (3D25dE).Peripheral tissues convert these compounds to the active hormones 20-hydroxyecdysone (20E)and ponasterone A (25-deoxy-20-hydroxyecdysone or 25d20E).The hydroxylations at C25,C22,C2,and C20are catalyzed by cytochrome P-450mono-oxygenases,which are encoded by the Halloween genes Phantom ,Disembodied ,Shadow ,and Shade ,respectively,in insects.Orthologs of these genes are present in the Daphnia genome and a cDNA encoding Phantom has been cloned from prawn.Inactivation involves conversion of ecdys-teroids to polar metabolites and/or conjugates,which are eliminated in the urine and feces.The antennal gland is the major route for excretion of ecdysteroids synthesized by the YO.The hepatopancreas eliminates ingested ecdysteroids by forming apolar conjugates.The concentrations of ecdysteroids vary over the molt cycle and are determined by the combined effects biosynthesis,metabolism,and excretion.© 2010 Elsevier Ltd. All rights reserved.Contents 1.Introduction ..........................................................................................................................................1962.Ecdysteroid synthesis by the Y-organ ...............................................................................................................1973.Hemolymph ecdysteroids over the molt cycle ......................................................................................................1994.Ecdysteroid metabolism and excretion ..............................................................................................................2005.Conclusions and summary ...........................................................................................................................201Acknowledgements ..................................................................................................................................201References ...........................................................................................................................................2011.IntroductionEcdysteroids are polyhydroxylated C27steroids that control molting in arthropods.As the research on crustaceans has lagged behind that on insects,it comes as no surprise that what we have learned from insects has provided insights and guided the work on crustaceans.This review is restricted to ecdysteroid biosynthesis in the crustacean molting gland,or Y-organ (YO),and ecdysteroid metabolism and excretion by peripheral tissues.It emphasizesଝArticle submitted for the special issue on Marine organisms.∗Tel.:+19704917616;fax:+19704910649.E-mail address:don@ .research published over the last 20years.The reader is referred to [1–7]for comprehensive reviews of the earlier work.As most of the work has focused on decapod crustaceans,little is known about ecdysteroid biosynthetic pathways in other crustacean groups.Data from non-decapod crustaceans are included where appropriate.Due to space limitations,related topics are not covered.The reader is referred to reviews on the biochemistry of vertebrate-type steroids [8–10],endocrine disruptors [11–13],and the role of ecdysteroids on reproduction and larval development in crus-taceans [9,10,12,14].Moreover,recent reviews provide detailed treatment on the regulation of YO ecdysteroidogenesis by eyestalk neuropeptides [15–18].0960-0760/$–see front matter © 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.jsbmb.2010.09.001D.L.Mykles /Journal of Steroid Biochemistry &Molecular Biology 127 (2011) 196–2031972.Ecdysteroid synthesis by the Y-organCholesterol is the precursor for ecdysteroid synthesis in the YO.The YOs are located in the cephalothorax,anterior to the branchial chamber.They are epithelioid tissues with cells contain-ing numerous mitochondria and extensive endoplasmic reticulum,which is characteristic of steroidogenic tissues of vertebrates [1,2,19].Arthropods cannot synthesize cholesterol de novo ,and therefore acquire it from their diet [3,20,21].Cholesterol binds to high-density lipoproteins in the hemolymph and is taken up by the YO [20–26].The uptake of cholesterol is an important rate-limiting step in the synthesis of ecdysteroids and is regulated by eyestalk neuropeptides,such as molt-inhibiting hormone (MIH)[21–23,27,28].Ecdysteroid biosynthesis in the molting glands of insects and crustaceans is similar,but the crustacean YO synthesizes a greater diversity of ecdysteroids [1,3,4,29,30].The biosynthetic pathway in the crustacean YO is complex.For convenience,ecdysteroid biosyn-thesis can be divided into two stages:(1)conversion of cholesterol to 5␤-diketol (Fig.1)and (2)conversion of 5␤-diketol to secreted products (Fig.2).The first stage of ecdysteroid synthesis is common to all decapod species.The second stage has four pathways with branching points at 5␤-diketol and 5␤-ketodiol [1,31,32].These pathways are named for the final ecdysteroid product secreted by the YO:ecdysone,3-dehydroecdysone (3DE),25-deoxyecdysone (25dE),and 3-dehydro-25-deoxyecdysone (3D25dE).YOs usually secrete two products,indicating that only two of the potential four pathways are operational at any given time in one species.This appears to be due to the relative activities of enzymes at the branch points,which directs metabolites along particular pathways.Cholesterol7-Dehydrocholesterol (7DC)5β-Diketol (3D2,22,25,dE)Non-molting glossy (Nm-g)/Shroud (Sro)Spook (Spo)/Spookier (Spok)Spookiest (Spot)Neverland (Nvd)(7,8-Dehydrogenase)5β[H]-Reductase“B l a c k B o x ”Δ -Diketol4Fig.1.Stage 1of the ecdysteroid biosynthetic pathway in crustacean molting gland (Y-organ).In the first reaction,cholesterol is converted to 7-dehydrocholesterol (7DC)by 7,8-dehydrogenase,encoded by the Neverland (nvd )gene in insects.In a series of reactions involving enzymes encoded by non-molting glossy /shroud ,Spook /Spookier ,and Spookiest ,termed the “Black Box”,7DC is converted to 4-Diketol,which is converted to 5␤-diketol by 5␤[H]-reductase.Modified from [1,29,31].Much of what we know about the biosynthetic pathway in the YO comes from metabolic studies using radiolabelled precursors.The first step is the conversion of cholesterol to 7-dehydrocholesterol (7DC)by 7,8-dehydrogenase;the two H(␤)at carbons #7and #8are removed,resulting in the formation of a double bond between those carbons (Fig.1).Studies on a vari-ety of decapod crustacean species have shown that radiolabelled cholesterol injected into animals is converted to ecdysone,3DE,20-hydroxyecdysone (20E),25dE,3-dehydro-20-hydroxyecdysone (3D20E),and/or ponasterone A (PA;25-deoxy-20E or 25d20E)[21,26,33–35].A [3H]3␤-hydroxy- 4compound is poorly metab-olized by YO cells,indicating that the 3-oxo group is required for efficient 5␤-reduction [36].[3H]25-hydroxycholesterol (25C)is converted to 7-dehydro-25C by the YOs of green crab (Carci-nus maenas ),shrimp (Penaeus vannamei ),and crayfish (Orconectes limosus );the 7-dehydro-25C is converted to 3DE in C.maenas or to ecdysone and 3DE in P.vannamei and O.limosus [37].The YOs from Cancer antennarius can also synthesize 3DE from 25C [38].Although it is not a normal intermediate in the pathway,these results show that 25C can be a substrate for 7,8-dehydrogenase.The uptake of 25C is much faster than cholesterol,which makes it a useful tool for the study of the regulation of ecdysteroidogenesis [37,38].The 7DC undergoes a series of reactions involving 3-oxo- 4intermediates [36],the so-called “Black Box”,the details of which are becoming clearer [39].YO cells from C.maenas convert a 25-deoxy-3-oxo- 4intermediate to 3DE and 25dE;no ecdysone was detected [36].However,YO cells convert a 3-oxo- 4intermediate hydroxylated at C25to 3DE and ecdysone [36].This is consistent with studies showing that these ecdysteroids are synthesized from 25C as the precursor [32,37,38].The part of the pathway after the “Black Box”reactions is bet-ter understood,particularly the hydroxylations at C2,C22,and C25,which increase the polarity of the secreted products. 4-Diketol is first reduced at C5by 5␤[H]-reductase to form 5␤-diketol (Fig.2;3-dehydro-2,22,25-deoxyecdysone or 3D2,22,25dE).5␤-Diketol is converted to 5␤-ketodiol (2,22,25-deoxyecdysone or 2,22,25dE)by 3-dehydroecdysteroid-3␤-reductase (Fig.2)[31].In C.maenas ,[3H]5␤-diketol is ultimately converted to 3DE and 25dE by C.mae-nas YO cells [36].[3H]5␤-ketodiol is hydroxylated at C2,C22,or C25in C.maenas YOs to produce 22,25-deoxyecdysone (22,25dE),2,25-deoxyecdysone (2,25dE),or 2,22-dideoxyecdysone (2,22dE),respectively [40–42].2,22dE and 2,25dE are converted to ecdysone and 25dE,respectively,and secreted by the YO [40,41].22,25dE is not processed further and is not secreted [40,41].In the YOs of crayfish,[3H]5␤-diketol is converted to ecdysone and 3DE,whereas [3H]5␤-ketodiol is converted to ecdysone [31].The major secreted products of the YO are ecdysone,3DE,3D25dE,and 25dE,but the relative amounts vary among species.Four patterns are observed in decapod crustaceans (Table 1).TheTable 1Major ecdysteroid products secreted by the Y-organs of decapod crustaceans.The production of 25dE in U.pugilator is suggested by high hemolymph titers of ponas-terone A in premolt animals [30,49].SpeciesE a3DE a 25dE a 3D25dE aPachygrapsus crassipes •Cancer antennarius ••Orconectes limosus ••Procambarus clarkii ••Penaeus vannamei••Macrobrachium rosenbergii ••Carcinus maenas ••Callinectes sapidus ••Uca pugilator••Menippe mercenaria••aAbbreviations :E,ecdysone;3DE,3-dehydroecdysone;25dE,25-deoxyecdysone;3D25dE,3-dehydro-25-deoxyecdysone.198 D.L.Mykles /Journal of Steroid Biochemistry &Molecular Biology 127 (2011) 196–2035b- Diketol (3D2,22,25dE)5b -Ketodiol (2,22,25dE)5b- Ketotriol (2,22dE)20-Hydroxyecdysone (20E)b -reductase3-Dehydro-2,22-deoxyecdysone (3D2,22dE)3-Dehydro-2-deoxyecdysone (3D2dE)Disembodie 2,25-deoxyecdysone (2,25dE)Y-OrganPeripheral tissues3-Dehydroecdysone25-deoxyecdysone (25dE)Ponasterone A (PA)3-Dehydro-2,25-deoxyecdysone (3D2,25dE)3-Dehydro-25-deoxyecdysone (3D25dE)3-Dehydro-25-deoxyecdysone b -R(3D20E)Ponasterone A (PA)Fig.2.Stage 2of the ecdysteroid biosynthetic pathway in crustacean molting gland (Y-organ).Data indicate that there are two branching points,resulting in four pathways and four products secreted from the Y-organ.The products vary with species and molt stage,but no more than two major products are usually secreted at one time.3␤-Reductase and 20-hydroxylase activities in peripheral tissues convert the secreted products to the compounds circulating in the hemolymph.Therefore,the ecdysteroids in the hemolymph are determined by the compounds secreted by the YO and the activities of 3␤-reductase and 20-hydroxylase.All the compounds metabolized by peripheral tissues are detected in hemolymph samples;the major ecdysteroids in hemolymph are 20E,PA,3D20E,and 25d20E.In M.mercenaria ,a potential metabolite,3-dehydro-25-deoxy-20-hydroxyecdysone (3D25d20E),resulting from the 20-hydroxylation of 3D25dE has not been identified [32].The terminal hydroxylations at carbons #25,#22,#2,and #20are catalyzed by cytochrome P450mono-oxygenases encoded by Phantom (phm ),Disembodied (dib ),Shadow (sad ),and Shade (shd ),respectively.The specificities of these enzymes are such that the hydroxylations occur in the same order:C25→C22→C2→C20.Hence,hydroxylation at a specific carbon prevents hydroxylation by enzymes “upstream”of that step.For example,hydroxylation at C2prevents hydroxylations at C25and C22.Modified from [1,29,31,32].YOs in crayfish (O.limosus and Procambarus clarkii ),shrimp (P.vannamei and Macrobrachium rosenbergii ),and rock crab (C.anten-narius )secrete ecdysone and 3DE [25,31,43–46],the YOs in green crab (C.maenas )and blue crab (Callinectes sapidus )secrete ecdysone and 25dE [34,41](unpublished data on C.sapidus cited in [32]),theYOs in stone crab (Menippe mercenaria )secrete 3DE and 3D25dE [27,32],and the YOs in lined shore crab (Pachygrapsus crassipes )and fiddler crab (Uca pugilator )secrete ecdysone [47,48].However,the high levels of PA in the hemoymph of premolt U.pugilator indicate that the YO of this species also secretes 25dE in addition to ecdysoneD.L.Mykles/Journal of Steroid Biochemistry&Molecular Biology127 (2011) 196–203199[49].This suggests that the biosynthetic pathway has branching points at5␤-diketol and5␤-ketodiol,leading to fourfinal ecdys-teroid products(Fig.2).At thefirst branching point5␤-diketol can be converted to5␤-ketodiol,3-dehydro-2,22-deoxyecdysone (3D2,22dE),or3-dehydro-2,25-deoxyecdysone(3D2,25dE)(Fig.2). At the second branching point5␤-ketodiol can be converted to either5␤-ketotriol or2,25-deoxyecdysone(2,25dE),which leads to the production of ecdysone or25dE,respectively(Fig.2).In the pathway leading to ecdysone,5␤-ketodiol is hydroxylated sequen-tially at C25,C22,and C2[1].At thefirst branching point,5␤-diketol is hydroxylated at C25to3D2,22dE;subsequent serial hydroxyla-tions of3D2,22dE at C22and C2produce3DE[31].[3H]-3D2,22dE is converted to3DE and ecdysone by C.maenas YO cells[36].In M.mercenaria,5␤-diketol is apparently hydroxylated at C22to 3D2,25dE;subsequent hydroxylation at C2produces3D25dE[32]. At the second branching point,5␤-ketodiol is hydroxylated at C22 to2,25-deoxyecdysone(2,25dE);subsequent hydroxylation at C2 produces25dE.The branching points in the YO ecdsteroid synthetic pathway suggest that the P450mono-oxygenases,encoded by the Hal-loween genes Phantom(phm),Disembodied(dib),Shadow(sad), and Shade(shd)can bind multiple substrates.Phm apparently can hydroxylate5␤-diketol or5␤-ketodiol at C25;Dib apparently can hydroxylate5␤-diketol,3D2,22dE,5␤-ketodiol,or5␤-ketotriol at C22;Sad apparently can hydroxylate3D2dE,2,25dE,or2dE at C2; and Shd apparently can hydroxylate25dE,3DE,or ecdysone at C20 (Fig.2).However,the specificities of the Phm,Dib,Sad,and Shd enzymes are such that the C25→C22→C2→C20order of hydrox-ylations is maintained(Fig.2)[3,29].For example,compounds hydroxylated at C22(2,25dE and25dE)cannot be hydroxylated at C25in the YO from C.maenas[40].Hydroxylation at C2(22,25dE) prevents hydroxylations at C22and C25[1,40].Ecdysteroid biosynthetic reactions are catalyzed by reductases and P450mono-oxygenases(CYPs).The reduction of cholesterol to 7DC(Fig.1)is catalyzed by a7,8-dehydrogenase,which is encoded by the Neverland(nvd)gene in insects and nematode[50].The Nvd protein has two conserved domains involved in catalysis:a Rieske-like domain containing a2Fe-2S binding motif and a C-terminal domain containing a non-heme Fe(II)-binding motif[50]. Halloween genes constitute a family of P450mono-oxygenases, which have been well characterized in insects[29].They are char-acterized by havingfive conserved structural motifs(Helix-C or WxxxR sequence,Helix-I or GxE/DDT/S sequence,Helix-K or ExxR sequence,PERF motif or PxxFxPE/DRF sequence,and heme-binding or PFxxGxRxCxG/A sequence),as well as six substrate recogni-tion sites(SRS1,2/3,4,5,and6)[51].The“Black Box”reactions are catalyzed by non-molting glossy(nm-g)/shroud(sro)and sev-eral Halloween genes:spook(spo)and its paralog spookier(spok); and spookiest(spot)[51,52].Spo and Phm belong to the CYP2clade of P450enzymes and are associated with the endoplasmic reticu-lum(ER),whereas Dib,Sad,and Shd are associated with the inner membrane of the mitochondrion[29,53–55].Nm-g/sro encodes a short-chain NAD(P)H dehydrogenase/reductase,which appears to act upstream of Spo[52].Nm-g/sro has an N-terminal NADP/NAD binding domain and a central catalytic domain and is expressed primarily in prothoracic gland and ovary[52].Crustaceans appear to have the same enzymes for ecdysteroid biosynthesis as insects.An inhibitor of steroid5␣-reductase,L-645390,blocks the conversion of cholesterol to7DC in the YO of M.mercenaria[22].The5␤-reductase that converts 4-diketol to 5␤-diketol is a cytosolic enzyme in YO cells that requires NADPH for activity[36].Orthologs of nvd,nm-g/sro,spo,phm,dib,sad,and shd have been identified in the Daphnia pulex genome[52,54,56], and a cDNA encoding Phm has been cloned from Kuruma prawn, Marsupenaeus japonicus[57].Nvd and spo are located adjacent to each other in the D.pulex genome[54].The M.japonicus Phm and D.pulex Phm havefive conserved motifs present in insect Phm(WxxxR,GxE/DTT/S,ExxR,PxxFxPE/DRF,and PFxxGxRxCxG/A heme-binding motif),five of six substrate recognition sites(SRS 1,2/3,4,and5),and N-terminal ER-targeting and Pro/Gly-rich sequences[57].Mj-Phm is a target of eyestalk neuropeptides,as its expression in the YO is increased as much as7-fold during premolt and is decreased about2.5-fold by sinus gland extract and recom-binant MIH[57].None of the crustacean Halloween enzymes have been characterized biochemically.A member of the clade4of cytochrome P450enzymes has been cloned from O.limosus(CYP4C15)and C.maenas(CYP4C39) [58,59].Both genes encode proteins with hydrophobic N-terminal ER-targeting and Pro/Gly-rich sequences characteristic of microso-mal cytochrome P450enzymes[58,59].CYP4C15is expressed in the YO and the protein is associated with the ER[58,60].More-over,CYP4C15expression in the YO is increased during premolt and is decreased by MIH[58,60].These data suggest that they are involved in ecdysteroid biosynthesis,but their precise role remains to be determined.It has been hypothesized that the gene cat-alyzes7,8-dehydrogenation or25-hydroxylation[58,60].However, CYP4C15and CYP4C39have low sequence identity/similarity to nvd and phm,making it unlikely that these genes are involved in7,8-dehydrogenation and25-hydroxylation,respectively,in the YO. CYP4C39is expressed in the hepatopancreas,which suggests it may have a role in ecdysteroid metabolism by peripheral tissues[59].Ecdysteroids secreted by the YO are converted to20-hydroxyecdysone(20E)and related compounds by peripheral tissues,such as gonad,hindgut,abdominal ganglia,eyestalk ganglia,hepatopancreas,antennal gland,and epidermis(Fig.2) [38,61–66].YOs cannot convert3DE to ecdysone or ecdysone to20E[31,43,61].In peripheral tissues,ecdysone is hydroxy-lated at C20,probably by Shd,to produce20E[38,40,41,62–64]. A20-hydroxylase activity in the hepatopancreas of the blackback land crab(Gecarcinus lateralis)requires NADPH,is inhibited by metyrapone(an inhibitor of cytochrome P450enzymes),and is associated with the mitochondrial fraction[62].A3␤-reductase converts3DE to ecdysone,which is hydroxylated at C20,to produce 20E[32,38,45,61,63].In M.mercenaria,3␤-reductase in peripheral tissues converts3D25dE to25dE,which is then converted to PA [32].In C.maenas,25dE is converted to PA by a20-hydroxylase in the testis[34,40,41].PA can be hydroxylated at C25or C26 to produce20E or inokosterone,respectively[67].In crayfish(O. limosus),abdominal ganglia convert3DE to ecdysone,20E,and 3D20E[63].Antennal glands of crayfish express an aldoketore-ductase,but its precise function is unknown;it may catalyze 3␤-reduction or may have an osmoregulatory role by converting D-glucose to sorbitol[68].These data indicate that peripheral tissues have3␤-reductase,25-hydroxylase,and20-hydroxylase activities, which can convert products secreted by the YO to20E and related compounds.3.Hemolymph ecdysteroids over the molt cycleTotal ecdysteroid titers in the hemolymph vary over the molt cycle.In general,ecdysteroids are low during intermolt and postmolt;during premolt,concentrations rise and reach a peak shortly before molting(ecdysis)in a variety of crustacean species [2,5–7,46,62,69–89].A precipitous drop in hemolymph ecdys-teroids triggers the shedding of the exoskeleton[90].Eyestalk ablation,which removes the primary source of inhibitory neu-ropeptides(e.g.,MIH),activates the YO and increases hemolymph ecdysteroid levels[1,2,21,23,70,79,86,91,92].In addition to the peak in hemolymph titers at the end of the premolt stage,there are smaller peaks during postmolt and early premolt stages,which may have physiological functions[30,48,93–95].For species with terminal molts,the YOs degenerate and ecdysteroids remain at200 D.L.Mykles/Journal of Steroid Biochemistry&Molecular Biology127 (2011) 196–203low concentrations in the hemolymph,even after eyestalk ablation [2,96].The major ecdysteroids identified in the hemolymph of decapod crustaceans are ecdysone,20E,3D20E,and PA, but the specific compounds and their levels vary between species,developmental stage,molt stage,and season [32,44,48,49,61,64,66,67,70,78,81,82,87,93,97].In stone crabs (M.mercenaria)that have been eyestalk ablated to activate the YO, 20E,3D20E,and PA are present in about equal amounts;a fourth large peak eluting from a reverse-phase column was not identified [32].Infiddler crab(U.pugilator)induced to molt by eyestalk ablation,there are two transitory peaks of hemolymph ecdysteroid that differ in the relative titers of ecdysteroids.Peak I,which occurs at early premolt,has a higher ratio of ecdysone to20E than peak II,which occurs at late premolt[48].Eyestalk ablation alters the composition of ecdysteroids circulating in the hemolymph, but the effect differs between species.In blue crabs(C.sapidus) at late premolt,PA is the major ecdysteroid in intact animals, whereas20E is the major ecdysteroid in eyestalk-ablated animals [70].In U.pugilator,the PA:20E ratio in the hemolymph is higher in animals induced to molt by eyestalk ablation than in animals induced to molt by multiple leg autotomy[49].In Kuruma prawn (Penaeus japonicus),20E and PA are the major ecdysteroids during the postmolt and intermolt stages,while20E alone is the major ecdysteroid during early and late premolt stages[87].In C.maenas, ecdysone and20E are detected at low levels in the hemolymph of intermolt animals,whereas the PA level is higher than the20E level in the hemolymph of premolt animals[34,67,78].At late premolt(stage D3),the titers of PA in the hemolymph decline more rapidly than those of ecdysone and20E[78].This corresponds to a change in the relative amounts of ecdysone and25dE,a precursor to PA,secreted by the YO between intermolt and premolt animals; YOs from premolt animals secrete greater amounts of25dE than ecdysone than YOs from intermolt animals[40,41].This appears to depend on the activities of enzymes at the branching points (Fig.2).For example,in rock crab and crayfish YOs,an increase in3␤-reductase activity would shift production and secretion from3DE to ecdysone[31].In green andfiddler crabs,an increase in25-hydroxylase activity would shift secretion from25dE to ecdysone[31].Ecdysteroid composition can be affected by developmental stage.During the last three molts,blue crabs(C.sapidus)transi-tion through juvenile,prepubertal,and adult stages.PA is the major hemolymph ecdysteroid at all three stages.However,the titers of PA in juveniles are significantly higher than and the titers of20E significantly lower than the titers in prepubertal and adult stages [70].In juvenile C.sapidus,eyestalk ablation increases hemolymph ecdysteroid titers,but there is no peak in ecdysteroids prior to ecd-ysis and titers during postmolt remain elevated compared to intact animals[70].These data indicate that there are stage-specific dif-ferences in ecdysteroid metabolism,but the functional significance of these changes remains to be investigated.4.Ecdysteroid metabolism and excretion20E and PA are the major active ecdysteroids circulating in the hemolymph of decapod crustaceans.Inactivation involves conver-sion to more polar metabolites and/or formation of conjugates, which are excreted in the urine or eliminated in the feces.Thus,the two major organs responsible for removing ecdysteroids from the hemolymph are the antennal gland and hepatopancreas(midgut gland)in decapods.The diversity of inactivation pathways in crustaceans is reminiscent to those used by insects to detoxify ecdysteroids ingested from plants(see[98]for review).The pri-mary site for excretion of ecdysteroids is the antennal gland.In lobster(Homarus americanus),between81%and96%of excreted ecdysteroids is in the urine,depending on the molt stage;the remainder is in the feces[66,97].In crayfish(O.limosus),there is a preferential excretion of ecdysone over20E.About two-thirds of the[3H]ecdysone injected into the hemolymph appears in the water after1h[99].In contrast,most of the[3H]20E injected into the hemolymph remains in the hemolymph,with less than5%of the[3H]20E appearing in the water after1h[99].This suggests that selective excretion of unmetabolized ecdysone by the antennal gland keeps hemolymph ecdysone titers low.Conversion of ecdysteroids to more polar compounds involves two modifications of the side chain at the#26carbon:hydrox-ylation and oxidation.In insects,20E is inactivated by a cytochrome P45026-hydroxylase[100,101].In decapod crus-taceans,a26-hydroxylase activity in various tissues converts20E to20,26-dihydroxyecdysone(20,26E)and PA to inokosterone(25-deoxy-20,26-dihydroxyecdysone or25d20,26E)[63,65–67,97]. Oxidation at the C26forms ecdysonoic acids(EAs).For example, 20E and PA are metabolized to20-hydroxyecdysonoic acid(20EA) and25-deoxy-20-hydroxyecdysonoic acid(25d20EA),respec-tively,and are excreted in the urine[65–67,97,102].Ecdysteroids can also be converted to their3␣-hydroxy epimers by periph-eral tissues.In crayfish(P.clarkii),a3␣-reductase activity in the epidermis converts ecdysone and20E to3␣-hydroxyecdysone and3␣,20-hydroxyecdysone,respectively,which are selectively excreted[61].Various tissues can convert ecdysteroids and their metabolites to highly polar(HP)conjugates,which can be excreted in the urine[61,63,65,66,97].The levels of HP conjugates vary over the molt cycle and can at times be the predominant ecdysteroid in the hemolymph[61,77,82,87,88,93].In H.americanus,the rapid drop in ecdysteroids at the end of premolt is associated with an increase in HP metabolites in the hemolymph and excretion in the urine[93,97].Using[3H]ecdysone as a tracer,a greater proportion (about75%after72h)is converted into HP conjugates in postmolt (stage A)and intermolt(stage C)lobsters than is converted in early premolt(stages D0and D1)lobsters(about25%after72h)[66]. The conversion by late premolt(stage D2)lobsters increases after injection and reaches early premolt levels by96h as animals tran-sition through the late premolt ecdysteroid peak[66].In C.maenas, intermolt and premolt animals differ in the metabolism of[3H]PA injected into the hemolymph.Forty-eight hours after injection into intermolt animals,the major metabolites were conjugates and 20EA,whereas24h after injection into premolt animals,the major metabolites were conjugates only[67].Enzymatic hydrolysis with snail sulfatase or porcine liver esterase is used to analyze the com-position of conjugates[66,93].HP conjugates in the hemolymph contain various ecdysteroids and metabolites usually in proportion to their hemolymph concentrations[93,97].This indicates that the modifications are generally nonspecific.However,HP conjugates in the urine contain relatively high proportions of polar metabo-lites,such as20,26E,3␣-hydroxy compounds,and ecdysonoic acids [61,66,97].In addition,excretion rates of H.americanus are higher in intermolt and late premolt(D2)stages than early premolt(D0 and D1)stages[66].These data suggest that the conversion of ecdysteroids to inactive polar metabolites and their preferential excretion by the antennal gland can determine the ecdysteroid titers in the hemolymph,especially at the end of premolt when titers drop precipitously.The hepatopancreas is the primary site for the production of apolar conjugates(perhaps fatty acyl esters),which are then elim-inated in the feces[65,66,99].These conjugates can be formed from ecdysteroids in the hemolymph[63,65,66,84,97]or from ecdysteroids ingested with food[97].When lobsters were fed [3H]ecdysone,less than0.4%of the radioactivity appeared in the hemolymph and urine;essentially all of the[3H]ecdysone was in the form of apolar conjugates in the feces[97].The amounts of apo-。

Non-forward double Pomeron exchange in QCD

a r X i v :h e p -p h /9810359v 1 14 O c t 1998NON-FOR W ARD DOUBLE POMERON EXCHANGE IN QCDH.M.NAVELET,R.B.PESCHANSKI Service de Physique Theorique,CEA-Saclay,F-91191Gif-sur-Yvette Cedex,France E-mail:pesch@spht.saclay.cea.frWe derive the analytic expression of the two one-loop dipole contributions to the elastic 4-gluon amplitude in QCD for arbitrary transverse momentum.The first one corresponds to the double QCD pomeron exchange,the other to an order α2correction to one-pomeron exchange.1AimsIt is well-known that the bare pomeron singularity in QCD 1is violating the Froissart bound.The computation in the QCD framework of unitarity correc-tions to the bare pomeron is thus required.The first order correction implies the computation of the two-pomeron contribution to the elastic gluon-gluon amplitude.The aim of our paper is to give the first complete derivation of the analytical expression of the one-loop dipole contribution to the elastic am-plitude in the QCD-dipole picture 2of BFKL dynamics.The solution for the forward amplitude has been already derived in ref.3,and the non-forward ex-pression conjectured on the basis of assuming conformal invariance.Here we give the full proof of the result and thus of the conformal invariant property.Note that the QCD dipole formulation is known 4to be equivalent at tree level to the derivation of the BFKL amplitude in terms of Feynman graphs.The idea of our derivation is to use this formalism as an effective theory defining the propagation and interaction vertices of two QCD Pomerons,which are colorless compound states of reggeized gluons in the BFKL representation 1.We first introduce the SL (2,C )-invariant formalism for the 4-gluon elastic amplitude A Q (k,k ′;Y )in the BFKL derivation.The solution of the BFKL equation is more easily expressed 5in terms of the Fourier transformed ampli-tude f Q (ρ,ρ′;Y )given by the relationA Q (k,k ′;Y )=12)−iρ′(k ′−Q16dh ¯E h Q (ρ′)E h Q (ρ)d (h )e ω(h )Y ,(2)1where the factorα2comes from the coupling to incident dipoles.In equation(2),the symbolic notation dh≡ ∞n=−∞ dνcorresponds to the integration over the SL(2,C)quantum numbers with h=iν+1−nπχ(h)≡¯αN c2+iν ,(3)whereΨ≡(logΓ)′.The SL(2,C)Eigenvectors are defined byE h Q(ρ)=2π22,b+ρ2,b+ρb2−ρ2¯b2−¯ρ24ν2+(n+1)21γ(1Γ(1−˜z).Note that an analytic expression of the Eigenvectors E h Q(ρ)in the mixed rep-resentation has been provided4in terms of a combination of products of two Bessel functions.For simplicity,we did not include the impact factors5,6.Note also that the leading contribution to the amplitude(2)is the n=0component which corresponds to the BFKL Pomeron.2FormulationThe formulation of the general one-loop amplitude in the QCD dipole model can be written:f(one−loop)(ρ0ρ1;ρ′0ρ′1|Y=y+y′)=1|ρaρb|2d2ρa′d2ρa′1d2ρb′d2ρb′1×n2(ρ0ρ1;ρa0ρa1,ρbρb1|y−¯y,¯y)¯n2 ρ′0ρ′1;ρ′a0ρ′a1,ρ′b0ρ′b1|y′−¯y′,¯y′×T(ρa0ρa1,ρa′ρa′1)¯T(ρbρb1,ρb′ρb′1),(7)whereρ0ρ1are the transverse coordinates of one of the initially colliding dipoles(resp.ρ′0ρ′1for the second one),ρa0ρa1andρbρb1,the two interacting dipolesemerging from the dipoleρ0ρ1after evolution in rapidity(resp.ρi→ρi′,for the second one).It is important to notice that one has to introduce the probability distributions n2(···|y−¯y,¯y)of producing two dipoles after a mixed rapidity evolution,namely with a rapidity y−¯y with one-Pomeron type of evolution and a rapidity¯y with two-Pomeron type of evolution and then one has to integrateover¯y.The interaction amplitudes T(ρa0ρa1,ρa′ρa′1)and T(ρbρb1,ρb′ρb′1)arethe elementary two-gluon exchange amplitudes between two colorless dipoles, namelyT(ρa0ρa1,ρa′ρa′1)= d2q”e i q”π dhdh a dh bω1−ω(h) dωeωya(h)a(h a)a(h b) d2ραd2ρβd2ργE h a(ρa0α,ρa1α)E h b(ρb0β,ρb1β)E h(ρ0γ,ρ1γ)¯R h,h a,h bα,β,γ,(10)withR h,h a,h bα,β,γ≡ d2r0d2r1d2r2where g3P(h,h a,h b)is the celebrated triple Pomeron coupling as obtained in the QCD dipole model,namely:g3P= d2r0d2r1d2r2r0r2 h a × r12¯r0¯r2 ˜h a ¯r12+Q w2w+v2 −1+h−h a+h bPlugging in the general formula(7)the results obtained in formulae(8)to(14)and integrating overδ-fuctions,one gets:f Q(ρ,ρ′;Y)=α4 αN C(ω(h a)+ω(h b)−ω)(ω1−ω(h))×eω′¯y′+ω′1(y′−¯y′)¯Q h−h′ 22(h−˜h)γ(h−h′) d2td2t′× t−1+h+h b−h a(1−t)−1+h−h b+h a t′h a−h b−h′(1−t′)−h a+h b−h′ ×{a.h.}.(17) The remaining integral is of a type which has already been met11.Following the method of paper10the result can be expressed as followsH Q(h,h′)=1+˜h′−˜h1−h−h′e iπsinπ(2−2h)×γ(h−h a+h b)γ(1−h′+h a−h b).(18) The integral over h′can now easily be performed.The remaining poles at h′=h and h′=1−h give twice the same contribution due to the over completeness relation of the E h Q generators5.Onefinally obtains:f Q(ρ,ρ′;Y)=α4 αN C b(h a)b(h b)b(h) 2 E h Q(ρ)¯E h Q(ρ′)× y0d¯y Y−y0d¯y′ dωdω1dω′dω′1eω¯y+ω1(y−¯y)eω′¯y′+ω′1(y′−¯y′)×b(h a)b(h b)b(h) 2eω(h)Yb(h a)b(h b)b(h) 2e(ω(h a)+ω(h b))Y×the forward amplitudes(Q=0)are obtained by replacing E h Q(ρ)→(ρ)1。

C.parvum全基因组序列

DOI: 10.1126/science.1094786, 441 (2004);304Science et al.Mitchell S. Abrahamsen,Cryptosporidium parvum Complete Genome Sequence of the Apicomplexan, (this information is current as of October 7, 2009 ):The following resources related to this article are available online at/cgi/content/full/304/5669/441version of this article at:including high-resolution figures, can be found in the online Updated information and services,/cgi/content/full/1094786/DC1 can be found at:Supporting Online Material/cgi/content/full/304/5669/441#otherarticles , 9 of which can be accessed for free: cites 25 articles This article 239 article(s) on the ISI Web of Science. cited by This article has been /cgi/content/full/304/5669/441#otherarticles 53 articles hosted by HighWire Press; see: cited by This article has been/cgi/collection/genetics Genetics: subject collections This article appears in the following/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprints Information about obtaining registered trademark of AAAS.is a Science 2004 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m3.R.Jackendoff,Foundations of Language:Brain,Gram-mar,Evolution(Oxford Univ.Press,Oxford,2003).4.Although for Frege(1),reference was established rela-tive to objects in the world,here we follow Jackendoff’s suggestion(3)that this is done relative to objects and the state of affairs as mentally represented.5.S.Zola-Morgan,L.R.Squire,in The Development andNeural Bases of Higher Cognitive Functions(New York Academy of Sciences,New York,1990),pp.434–456.6.N.Chomsky,Reflections on Language(Pantheon,New York,1975).7.J.Katz,Semantic Theory(Harper&Row,New York,1972).8.D.Sperber,D.Wilson,Relevance(Harvard Univ.Press,Cambridge,MA,1986).9.K.I.Forster,in Sentence Processing,W.E.Cooper,C.T.Walker,Eds.(Erlbaum,Hillsdale,NJ,1989),pp.27–85.10.H.H.Clark,Using Language(Cambridge Univ.Press,Cambridge,1996).11.Often word meanings can only be fully determined byinvokingworld knowledg e.For instance,the meaningof “flat”in a“flat road”implies the absence of holes.However,in the expression“aflat tire,”it indicates the presence of a hole.The meaningof“finish”in the phrase “Billfinished the book”implies that Bill completed readingthe book.However,the phrase“the g oatfin-ished the book”can only be interpreted as the goat eatingor destroyingthe book.The examples illustrate that word meaningis often underdetermined and nec-essarily intertwined with general world knowledge.In such cases,it is hard to see how the integration of lexical meaning and general world knowledge could be strictly separated(3,31).12.W.Marslen-Wilson,C.M.Brown,L.K.Tyler,Lang.Cognit.Process.3,1(1988).13.ERPs for30subjects were averaged time-locked to theonset of the critical words,with40items per condition.Sentences were presented word by word on the centerof a computer screen,with a stimulus onset asynchronyof600ms.While subjects were readingthe sentences,their EEG was recorded and amplified with a high-cut-off frequency of70Hz,a time constant of8s,and asamplingfrequency of200Hz.14.Materials and methods are available as supportingmaterial on Science Online.15.M.Kutas,S.A.Hillyard,Science207,203(1980).16.C.Brown,P.Hagoort,J.Cognit.Neurosci.5,34(1993).17.C.M.Brown,P.Hagoort,in Architectures and Mech-anisms for Language Processing,M.W.Crocker,M.Pickering,C.Clifton Jr.,Eds.(Cambridge Univ.Press,Cambridge,1999),pp.213–237.18.F.Varela et al.,Nature Rev.Neurosci.2,229(2001).19.We obtained TFRs of the single-trial EEG data by con-volvingcomplex Morlet wavelets with the EEG data andcomputingthe squared norm for the result of theconvolution.We used wavelets with a7-cycle width,with frequencies ranging from1to70Hz,in1-Hz steps.Power values thus obtained were expressed as a per-centage change relative to the power in a baselineinterval,which was taken from150to0ms before theonset of the critical word.This was done in order tonormalize for individual differences in EEG power anddifferences in baseline power between different fre-quency bands.Two relevant time-frequency compo-nents were identified:(i)a theta component,rangingfrom4to7Hz and from300to800ms after wordonset,and(ii)a gamma component,ranging from35to45Hz and from400to600ms after word onset.20.C.Tallon-Baudry,O.Bertrand,Trends Cognit.Sci.3,151(1999).tner et al.,Nature397,434(1999).22.M.Bastiaansen,P.Hagoort,Cortex39(2003).23.O.Jensen,C.D.Tesche,Eur.J.Neurosci.15,1395(2002).24.Whole brain T2*-weighted echo planar imaging bloodoxygen level–dependent(EPI-BOLD)fMRI data wereacquired with a Siemens Sonata1.5-T magnetic reso-nance scanner with interleaved slice ordering,a volumerepetition time of2.48s,an echo time of40ms,a90°flip angle,31horizontal slices,a64ϫ64slice matrix,and isotropic voxel size of3.5ϫ3.5ϫ3.5mm.For thestructural magnetic resonance image,we used a high-resolution(isotropic voxels of1mm3)T1-weightedmagnetization-prepared rapid gradient-echo pulse se-quence.The fMRI data were preprocessed and analyzedby statistical parametric mappingwith SPM99software(http://www.fi/spm99).25.S.E.Petersen et al.,Nature331,585(1988).26.B.T.Gold,R.L.Buckner,Neuron35,803(2002).27.E.Halgren et al.,J.Psychophysiol.88,1(1994).28.E.Halgren et al.,Neuroimage17,1101(2002).29.M.K.Tanenhaus et al.,Science268,1632(1995).30.J.J.A.van Berkum et al.,J.Cognit.Neurosci.11,657(1999).31.P.A.M.Seuren,Discourse Semantics(Basil Blackwell,Oxford,1985).32.We thank P.Indefrey,P.Fries,P.A.M.Seuren,and M.van Turennout for helpful discussions.Supported bythe Netherlands Organization for Scientific Research,grant no.400-56-384(P.H.).Supporting Online Material/cgi/content/full/1095455/DC1Materials and MethodsFig.S1References and Notes8January2004;accepted9March2004Published online18March2004;10.1126/science.1095455Include this information when citingthis paper.Complete Genome Sequence ofthe Apicomplexan,Cryptosporidium parvumMitchell S.Abrahamsen,1,2*†Thomas J.Templeton,3†Shinichiro Enomoto,1Juan E.Abrahante,1Guan Zhu,4 Cheryl ncto,1Mingqi Deng,1Chang Liu,1‡Giovanni Widmer,5Saul Tzipori,5GregoryA.Buck,6Ping Xu,6 Alan T.Bankier,7Paul H.Dear,7Bernard A.Konfortov,7 Helen F.Spriggs,7Lakshminarayan Iyer,8Vivek Anantharaman,8L.Aravind,8Vivek Kapur2,9The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals,and causes an unrelenting infection in immuno-compromised individuals such as AIDS patients.We report the complete ge-nome sequence of C.parvum,type II isolate.Genome analysis identifies ex-tremely streamlined metabolic pathways and a reliance on the host for nu-trients.In contrast to Plasmodium and Toxoplasma,the parasite lacks an api-coplast and its genome,and possesses a degenerate mitochondrion that has lost its genome.Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected.Elu-cidation of the core metabolism,including enzymes with high similarities to bacterial and plant counterparts,opens new avenues for drug development.Cryptosporidium parvum is a globally impor-tant intracellular pathogen of humans and animals.The duration of infection and patho-genesis of cryptosporidiosis depends on host immune status,ranging from a severe but self-limiting diarrhea in immunocompetent individuals to a life-threatening,prolonged infection in immunocompromised patients.Asubstantial degree of morbidity and mortalityis associated with infections in AIDS pa-tients.Despite intensive efforts over the past20years,there is currently no effective ther-apy for treating or preventing C.parvuminfection in humans.Cryptosporidium belongs to the phylumApicomplexa,whose members share a com-mon apical secretory apparatus mediating lo-comotion and tissue or cellular invasion.Many apicomplexans are of medical or vet-erinary importance,including Plasmodium,Babesia,Toxoplasma,Neosprora,Sarcocys-tis,Cyclospora,and Eimeria.The life cycle ofC.parvum is similar to that of other cyst-forming apicomplexans(e.g.,Eimeria and Tox-oplasma),resulting in the formation of oocysts1Department of Veterinary and Biomedical Science,College of Veterinary Medicine,2Biomedical Genom-ics Center,University of Minnesota,St.Paul,MN55108,USA.3Department of Microbiology and Immu-nology,Weill Medical College and Program in Immu-nology,Weill Graduate School of Medical Sciences ofCornell University,New York,NY10021,USA.4De-partment of Veterinary Pathobiology,College of Vet-erinary Medicine,Texas A&M University,College Sta-tion,TX77843,USA.5Division of Infectious Diseases,Tufts University School of Veterinary Medicine,NorthGrafton,MA01536,USA.6Center for the Study ofBiological Complexity and Department of Microbiol-ogy and Immunology,Virginia Commonwealth Uni-versity,Richmond,VA23198,USA.7MRC Laboratoryof Molecular Biology,Hills Road,Cambridge CB22QH,UK.8National Center for Biotechnology Infor-mation,National Library of Medicine,National Insti-tutes of Health,Bethesda,MD20894,USA.9Depart-ment of Microbiology,University of Minnesota,Min-neapolis,MN55455,USA.*To whom correspondence should be addressed.E-mail:abe@†These authors contributed equally to this work.‡Present address:Bioinformatics Division,Genetic Re-search,GlaxoSmithKline Pharmaceuticals,5MooreDrive,Research Triangle Park,NC27009,USA.R E P O R T S SCIENCE VOL30416APRIL2004441o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mthat are shed in the feces of infected hosts.C.parvum oocysts are highly resistant to environ-mental stresses,including chlorine treatment of community water supplies;hence,the parasite is an important water-and food-borne pathogen (1).The obligate intracellular nature of the par-asite ’s life cycle and the inability to culture the parasite continuously in vitro greatly impair researchers ’ability to obtain purified samples of the different developmental stages.The par-asite cannot be genetically manipulated,and transformation methodologies are currently un-available.To begin to address these limitations,we have obtained the complete C.parvum ge-nome sequence and its predicted protein com-plement.(This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the project accession AAEE00000000.The version described in this paper is the first version,AAEE01000000.)The random shotgun approach was used to obtain the complete DNA sequence (2)of the Iowa “type II ”isolate of C.parvum .This isolate readily transmits disease among numerous mammals,including humans.The resulting ge-nome sequence has roughly 13ϫgenome cov-erage containing five gaps and 9.1Mb of totalDNA sequence within eight chromosomes.The C.parvum genome is thus quite compact rela-tive to the 23-Mb,14-chromosome genome of Plasmodium falciparum (3);this size difference is predominantly the result of shorter intergenic regions,fewer introns,and a smaller number of genes (Table 1).Comparison of the assembled sequence of chromosome VI to that of the recently published sequence of chromosome VI (4)revealed that our assembly contains an ad-ditional 160kb of sequence and a single gap versus two,with the common sequences dis-playing a 99.993%sequence identity (2).The relative paucity of introns greatly simplified gene predictions and facilitated an-notation (2)of predicted open reading frames (ORFs).These analyses provided an estimate of 3807protein-encoding genes for the C.parvum genome,far fewer than the estimated 5300genes predicted for the Plasmodium genome (3).This difference is primarily due to the absence of an apicoplast and mitochondrial genome,as well as the pres-ence of fewer genes encoding metabolic functions and variant surface proteins,such as the P.falciparum var and rifin molecules (Table 2).An analysis of the encoded pro-tein sequences with the program SEG (5)shows that these protein-encoding genes are not enriched in low-complexity se-quences (34%)to the extent observed in the proteins from Plasmodium (70%).Our sequence analysis indicates that Cryptosporidium ,unlike Plasmodium and Toxoplasma ,lacks both mitochondrion and apicoplast genomes.The overall complete-ness of the genome sequence,together with the fact that similar DNA extraction proce-dures used to isolate total genomic DNA from C.parvum efficiently yielded mito-chondrion and apicoplast genomes from Ei-meria sp.and Toxoplasma (6,7),indicates that the absence of organellar genomes was unlikely to have been the result of method-ological error.These conclusions are con-sistent with the absence of nuclear genes for the DNA replication and translation machinery characteristic of mitochondria and apicoplasts,and with the lack of mito-chondrial or apicoplast targeting signals for tRNA synthetases.A number of putative mitochondrial pro-teins were identified,including components of a mitochondrial protein import apparatus,chaperones,uncoupling proteins,and solute translocators (table S1).However,the ge-nome does not encode any Krebs cycle en-zymes,nor the components constituting the mitochondrial complexes I to IV;this finding indicates that the parasite does not rely on complete oxidation and respiratory chains for synthesizing adenosine triphosphate (ATP).Similar to Plasmodium ,no orthologs for the ␥,␦,or εsubunits or the c subunit of the F 0proton channel were detected (whereas all subunits were found for a V-type ATPase).Cryptosporidium ,like Eimeria (8)and Plas-modium ,possesses a pyridine nucleotide tran-shydrogenase integral membrane protein that may couple reduced nicotinamide adenine dinucleotide (NADH)and reduced nico-tinamide adenine dinucleotide phosphate (NADPH)redox to proton translocation across the inner mitochondrial membrane.Unlike Plasmodium ,the parasite has two copies of the pyridine nucleotide transhydrogenase gene.Also present is a likely mitochondrial membrane –associated,cyanide-resistant alter-native oxidase (AOX )that catalyzes the reduction of molecular oxygen by ubiquinol to produce H 2O,but not superoxide or H 2O 2.Several genes were identified as involved in biogenesis of iron-sulfur [Fe-S]complexes with potential mitochondrial targeting signals (e.g.,nifS,nifU,frataxin,and ferredoxin),supporting the presence of a limited electron flux in the mitochondrial remnant (table S2).Our sequence analysis confirms the absence of a plastid genome (7)and,additionally,the loss of plastid-associated metabolic pathways including the type II fatty acid synthases (FASs)and isoprenoid synthetic enzymes thatTable 1.General features of the C.parvum genome and comparison with other single-celled eukaryotes.Values are derived from respective genome project summaries (3,26–28).ND,not determined.FeatureC.parvum P.falciparum S.pombe S.cerevisiae E.cuniculiSize (Mbp)9.122.912.512.5 2.5(G ϩC)content (%)3019.43638.347No.of genes 38075268492957701997Mean gene length (bp)excluding introns 1795228314261424ND Gene density (bp per gene)23824338252820881256Percent coding75.352.657.570.590Genes with introns (%)553.9435ND Intergenic regions (G ϩC)content %23.913.632.435.145Mean length (bp)5661694952515129RNAsNo.of tRNA genes 454317429944No.of 5S rRNA genes 6330100–2003No.of 5.8S ,18S ,and 28S rRNA units 57200–400100–20022Table parison between predicted C.parvum and P.falciparum proteins.FeatureC.parvum P.falciparum *Common †Total predicted proteins380752681883Mitochondrial targeted/encoded 17(0.45%)246(4.7%)15Apicoplast targeted/encoded 0581(11.0%)0var/rif/stevor ‡0236(4.5%)0Annotated as protease §50(1.3%)31(0.59%)27Annotated as transporter ࿣69(1.8%)34(0.65%)34Assigned EC function ¶167(4.4%)389(7.4%)113Hypothetical proteins925(24.3%)3208(60.9%)126*Values indicated for P.falciparum are as reported (3)with the exception of those for proteins annotated as protease or transporter.†TBLASTN hits (e Ͻ–5)between C.parvum and P.falciparum .‡As reported in (3).§Pre-dicted proteins annotated as “protease or peptidase”for C.parvum (CryptoGenome database,)and P.falciparum (PlasmoDB database,).࿣Predicted proteins annotated as “trans-porter,permease of P-type ATPase”for C.parvum (CryptoGenome)and P.falciparum (PlasmoDB).¶Bidirectional BLAST hit (e Ͻ–15)to orthologs with assigned Enzyme Commission (EC)numbers.Does not include EC assignment numbers for protein kinases or protein phosphatases (due to inconsistent annotation across genomes),or DNA polymerases or RNA polymerases,as a result of issues related to subunit inclusion.(For consistency,46proteins were excluded from the reported P.falciparum values.)R E P O R T S16APRIL 2004VOL 304SCIENCE 442 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mare otherwise localized to the plastid in other apicomplexans.C.parvum fatty acid biosynthe-sis appears to be cytoplasmic,conducted by a large(8252amino acids)modular type I FAS (9)and possibly by another large enzyme that is related to the multidomain bacterial polyketide synthase(10).Comprehensive screening of the C.parvum genome sequence also did not detect orthologs of Plasmodium nuclear-encoded genes that contain apicoplast-targeting and transit sequences(11).C.parvum metabolism is greatly stream-lined relative to that of Plasmodium,and in certain ways it is reminiscent of that of another obligate eukaryotic parasite,the microsporidian Encephalitozoon.The degeneration of the mi-tochondrion and associated metabolic capabili-ties suggests that the parasite largely relies on glycolysis for energy production.The parasite is capable of uptake and catabolism of mono-sugars(e.g.,glucose and fructose)as well as synthesis,storage,and catabolism of polysac-charides such as trehalose and amylopectin. Like many anaerobic organisms,it economizes ATP through the use of pyrophosphate-dependent phosphofructokinases.The conver-sion of pyruvate to acetyl–coenzyme A(CoA) is catalyzed by an atypical pyruvate-NADPH oxidoreductase(Cp PNO)that contains an N-terminal pyruvate–ferredoxin oxidoreductase (PFO)domain fused with a C-terminal NADPH–cytochrome P450reductase domain (CPR).Such a PFO-CPR fusion has previously been observed only in the euglenozoan protist Euglena gracilis(12).Acetyl-CoA can be con-verted to malonyl-CoA,an important precursor for fatty acid and polyketide biosynthesis.Gly-colysis leads to several possible organic end products,including lactate,acetate,and ethanol. The production of acetate from acetyl-CoA may be economically beneficial to the parasite via coupling with ATP production.Ethanol is potentially produced via two in-dependent pathways:(i)from the combination of pyruvate decarboxylase and alcohol dehy-drogenase,or(ii)from acetyl-CoA by means of a bifunctional dehydrogenase(adhE)with ac-etaldehyde and alcohol dehydrogenase activi-ties;adhE first converts acetyl-CoA to acetal-dehyde and then reduces the latter to ethanol. AdhE predominantly occurs in bacteria but has recently been identified in several protozoans, including vertebrate gut parasites such as Enta-moeba and Giardia(13,14).Adjacent to the adhE gene resides a second gene encoding only the AdhE C-terminal Fe-dependent alcohol de-hydrogenase domain.This gene product may form a multisubunit complex with AdhE,or it may function as an alternative alcohol dehydro-genase that is specific to certain growth condi-tions.C.parvum has a glycerol3-phosphate dehydrogenase similar to those of plants,fungi, and the kinetoplastid Trypanosoma,but(unlike trypanosomes)the parasite lacks an ortholog of glycerol kinase and thus this pathway does not yield glycerol production.In addition to themodular fatty acid synthase(Cp FAS1)andpolyketide synthase homolog(Cp PKS1), C.parvum possesses several fatty acyl–CoA syn-thases and a fatty acyl elongase that may partici-pate in fatty acid metabolism.Further,enzymesfor the metabolism of complex lipids(e.g.,glyc-erolipid and inositol phosphate)were identified inthe genome.Fatty acids are apparently not anenergy source,because enzymes of the fatty acidoxidative pathway are absent,with the exceptionof a3-hydroxyacyl-CoA dehydrogenase.C.parvum purine metabolism is greatlysimplified,retaining only an adenosine ki-nase and enzymes catalyzing conversionsof adenosine5Ј-monophosphate(AMP)toinosine,xanthosine,and guanosine5Ј-monophosphates(IMP,XMP,and GMP).Among these enzymes,IMP dehydrogenase(IMPDH)is phylogenetically related toε-proteobacterial IMPDH and is strikinglydifferent from its counterparts in both thehost and other apicomplexans(15).In con-trast to other apicomplexans such as Toxo-plasma gondii and P.falciparum,no geneencoding hypoxanthine-xanthineguaninephosphoribosyltransferase(HXGPRT)is de-tected,in contrast to a previous report on theactivity of this enzyme in C.parvum sporo-zoites(16).The absence of HXGPRT sug-gests that the parasite may rely solely on asingle enzyme system including IMPDH toproduce GMP from AMP.In contrast to otherapicomplexans,the parasite appears to relyon adenosine for purine salvage,a modelsupported by the identification of an adeno-sine transporter.Unlike other apicomplexansand many parasitic protists that can synthe-size pyrimidines de novo,C.parvum relies onpyrimidine salvage and retains the ability forinterconversions among uridine and cytidine5Ј-monophosphates(UMP and CMP),theirdeoxy forms(dUMP and dCMP),and dAMP,as well as their corresponding di-and triphos-phonucleotides.The parasite has also largelyshed the ability to synthesize amino acids denovo,although it retains the ability to convertselect amino acids,and instead appears torely on amino acid uptake from the host bymeans of a set of at least11amino acidtransporters(table S2).Most of the Cryptosporidium core pro-cesses involved in DNA replication,repair,transcription,and translation conform to thebasic eukaryotic blueprint(2).The transcrip-tional apparatus resembles Plasmodium interms of basal transcription machinery.How-ever,a striking numerical difference is seenin the complements of two RNA bindingdomains,Sm and RRM,between P.falcipa-rum(17and71domains,respectively)and C.parvum(9and51domains).This reductionresults in part from the loss of conservedproteins belonging to the spliceosomal ma-chinery,including all genes encoding Smdomain proteins belonging to the U6spliceo-somal particle,which suggests that this par-ticle activity is degenerate or entirely lost.This reduction in spliceosomal machinery isconsistent with the reduced number of pre-dicted introns in Cryptosporidium(5%)rela-tive to Plasmodium(Ͼ50%).In addition,keycomponents of the small RNA–mediatedposttranscriptional gene silencing system aremissing,such as the RNA-dependent RNApolymerase,Argonaute,and Dicer orthologs;hence,RNA interference–related technolo-gies are unlikely to be of much value intargeted disruption of genes in C.parvum.Cryptosporidium invasion of columnarbrush border epithelial cells has been de-scribed as“intracellular,but extracytoplas-mic,”as the parasite resides on the surface ofthe intestinal epithelium but lies underneaththe host cell membrane.This niche may al-low the parasite to evade immune surveil-lance but take advantage of solute transportacross the host microvillus membrane or theextensively convoluted parasitophorous vac-uole.Indeed,Cryptosporidium has numerousgenes(table S2)encoding families of putativesugar transporters(up to9genes)and aminoacid transporters(11genes).This is in starkcontrast to Plasmodium,which has fewersugar transporters and only one putative ami-no acid transporter(GenBank identificationnumber23612372).As a first step toward identification ofmulti–drug-resistant pumps,the genome se-quence was analyzed for all occurrences ofgenes encoding multitransmembrane proteins.Notable are a set of four paralogous proteinsthat belong to the sbmA family(table S2)thatare involved in the transport of peptide antibi-otics in bacteria.A putative ortholog of thePlasmodium chloroquine resistance–linkedgene Pf CRT(17)was also identified,althoughthe parasite does not possess a food vacuole likethe one seen in Plasmodium.Unlike Plasmodium,C.parvum does notpossess extensive subtelomeric clusters of anti-genically variant proteins(exemplified by thelarge families of var and rif/stevor genes)thatare involved in immune evasion.In contrast,more than20genes were identified that encodemucin-like proteins(18,19)having hallmarksof extensive Thr or Ser stretches suggestive ofglycosylation and signal peptide sequences sug-gesting secretion(table S2).One notable exam-ple is an11,700–amino acid protein with anuninterrupted stretch of308Thr residues(cgd3_720).Although large families of secretedproteins analogous to the Plasmodium multi-gene families were not found,several smallermultigene clusters were observed that encodepredicted secreted proteins,with no detectablesimilarity to proteins from other organisms(Fig.1,A and B).Within this group,at leastfour distinct families appear to have emergedthrough gene expansions specific to the Cryp-R E P O R T S SCIENCE VOL30416APRIL2004443o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mtosporidium clade.These families —SKSR,MEDLE,WYLE,FGLN,and GGC —were named after well-conserved sequence motifs (table S2).Reverse transcription polymerase chain reaction (RT-PCR)expression analysis (20)of one cluster,a locus of seven adjacent CpLSP genes (Fig.1B),shows coexpression during the course of in vitro development (Fig.1C).An additional eight genes were identified that encode proteins having a periodic cysteine structure similar to the Cryptosporidium oocyst wall protein;these eight genes are similarly expressed during the onset of oocyst formation and likely participate in the formation of the coccidian rigid oocyst wall in both Cryptospo-ridium and Toxoplasma (21).Whereas the extracellular proteins described above are of apparent apicomplexan or lineage-specific in-vention,Cryptosporidium possesses many genesencodingsecretedproteinshavinglineage-specific multidomain architectures composed of animal-and bacterial-like extracellular adhe-sive domains (fig.S1).Lineage-specific expansions were ob-served for several proteases (table S2),in-cluding an aspartyl protease (six genes),a subtilisin-like protease,a cryptopain-like cys-teine protease (five genes),and a Plas-modium falcilysin-like (insulin degrading enzyme –like)protease (19genes).Nine of the Cryptosporidium falcilysin genes lack the Zn-chelating “HXXEH ”active site motif and are likely to be catalytically inactive copies that may have been reused for specific protein-protein interactions on the cell sur-face.In contrast to the Plasmodium falcilysin,the Cryptosporidium genes possess signal peptide sequences and are likely trafficked to a secretory pathway.The expansion of this family suggests either that the proteins have distinct cleavage specificities or that their diversity may be related to evasion of a host immune response.Completion of the C.parvum genome se-quence has highlighted the lack of conven-tional drug targets currently pursued for the control and treatment of other parasitic protists.On the basis of molecular and bio-chemical studies and drug screening of other apicomplexans,several putative Cryptospo-ridium metabolic pathways or enzymes have been erroneously proposed to be potential drug targets (22),including the apicoplast and its associated metabolic pathways,the shikimate pathway,the mannitol cycle,the electron transport chain,and HXGPRT.Nonetheless,complete genome sequence analysis identifies a number of classic and novel molecular candidates for drug explora-tion,including numerous plant-like and bacterial-like enzymes (tables S3and S4).Although the C.parvum genome lacks HXGPRT,a potent drug target in other api-complexans,it has only the single pathway dependent on IMPDH to convert AMP to GMP.The bacterial-type IMPDH may be a promising target because it differs substan-tially from that of eukaryotic enzymes (15).Because of the lack of de novo biosynthetic capacity for purines,pyrimidines,and amino acids,C.parvum relies solely on scavenge from the host via a series of transporters,which may be exploited for chemotherapy.C.parvum possesses a bacterial-type thymidine kinase,and the role of this enzyme in pyrim-idine metabolism and its drug target candida-cy should be pursued.The presence of an alternative oxidase,likely targeted to the remnant mitochondrion,gives promise to the study of salicylhydroxamic acid (SHAM),as-cofuranone,and their analogs as inhibitors of energy metabolism in the parasite (23).Cryptosporidium possesses at least 15“plant-like ”enzymes that are either absent in or highly divergent from those typically found in mammals (table S3).Within the glycolytic pathway,the plant-like PPi-PFK has been shown to be a potential target in other parasites including T.gondii ,and PEPCL and PGI ap-pear to be plant-type enzymes in C.parvum .Another example is a trehalose-6-phosphate synthase/phosphatase catalyzing trehalose bio-synthesis from glucose-6-phosphate and uridine diphosphate –glucose.Trehalose may serve as a sugar storage source or may function as an antidesiccant,antioxidant,or protein stability agent in oocysts,playing a role similar to that of mannitol in Eimeria oocysts (24).Orthologs of putative Eimeria mannitol synthesis enzymes were not found.However,two oxidoreductases (table S2)were identified in C.parvum ,one of which belongs to the same families as the plant mannose dehydrogenases (25)and the other to the plant cinnamyl alcohol dehydrogenases.In principle,these enzymes could synthesize protective polyol compounds,and the former enzyme could use host-derived mannose to syn-thesize mannitol.References and Notes1.D.G.Korich et al .,Appl.Environ.Microbiol.56,1423(1990).2.See supportingdata on Science Online.3.M.J.Gardner et al .,Nature 419,498(2002).4.A.T.Bankier et al .,Genome Res.13,1787(2003).5.J.C.Wootton,Comput.Chem.18,269(1994).Fig.1.(A )Schematic showing the chromosomal locations of clusters of potentially secreted proteins.Numbers of adjacent genes are indicated in paren-theses.Arrows indicate direc-tion of clusters containinguni-directional genes (encoded on the same strand);squares indi-cate clusters containingg enes encoded on both strands.Non-paralogous genes are indicated by solid gray squares or direc-tional triangles;SKSR (green triangles),FGLN (red trian-gles),and MEDLE (blue trian-gles)indicate three C.parvum –specific families of paralogous genes predominantly located at telomeres.Insl (yellow tri-angles)indicates an insulinase/falcilysin-like paralogous gene family.Cp LSP (white square)indicates the location of a clus-ter of adjacent large secreted proteins (table S2)that are cotranscriptionally regulated.Identified anchored telomeric repeat sequences are indicated by circles.(B )Schematic show-inga select locus containinga cluster of coexpressed large secreted proteins (Cp LSP).Genes and intergenic regions (regions between identified genes)are drawn to scale at the nucleotide level.The length of the intergenic re-gions is indicated above or be-low the locus.(C )Relative ex-pression levels of CpLSP (red lines)and,as a control,C.parvum Hedgehog-type HINT domain gene (blue line)duringin vitro development,as determined by semiquantitative RT-PCR usingg ene-specific primers correspondingto the seven adjacent g enes within the CpLSP locus as shown in (B).Expression levels from three independent time-course experiments are represented as the ratio of the expression of each gene to that of C.parvum 18S rRNA present in each of the infected samples (20).R E P O R T S16APRIL 2004VOL 304SCIENCE 444 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m。

《粒子物理学教学讲义》6.4-节重夸克和重夸克偶素

6
确定了J/Ψ粒子子的夸克组分——正反粲夸克对,我们就可以解
释它的窄宽度了:
质量最轻的含粲D介子子质量为1870MeV,两个D介子子的阈能
为3740MeV,所以J/Ψ不能衰变到D介子子末态,它的强衰变末
态只能是由u,d,s夸克组成的轻强子子。
J/Ψ的强子子衰变的费曼图可以画作,
u d
c
d
c
c
d
c
d
它和J/Ψ粒子子类似, 也只能通过把b 和 b 湮没掉的Zweig禁 戒过程做强衰变,衰变概率被大大大大地压低,表现出突出的“重 质量,窄宽度”性质. 同样ϒ (2S), ϒ(3S)也具有同样的性质。ϒ (4S)以及更高高质量的 态则可以衰变到两个B介子子。因而而具有很大大的宽度。
17
对前3个ϒ共振态, ϒ→ 2B 的衰变道没有打开,他们所有的强衰 变道都是OZI禁戒过程而受到压低, 衰变宽度很窄。 但对下面更高质量的ϒ高激发态, 均可以衰变到两个B介子,因 而具有很大的几十个MeV的宽度
16
ϒ粒子子所有的强衰变道都是OZI 禁戒过程而而受到压低,
分支支比比最大大的衰变道也是二二级电磁衰变的 e+e− 道,µ +µ − 道 和 τ +τ − 道.
含底夸克b的最轻的粒子子是 B+(bu) 和 B0 (bd) 粒子子,其质量为
m = (5279)MeV,因此ϒ粒子子不可能衰变为一一对正反B介子子。
14
如果粲夸克存在,那么可以将u,d,s的SU(3) 味道对称性扩 充到u,d,s,c的SU(4) 对称性,则应该存在含有若干(反)粲夸 克的重子子和介子子多重态,
1976年发现了含粲介子子态( D0 = cu , D+ = cd ), 1977年又又发现了含粲奇异介子子 Ds+ = cs ,

单晶结构解析技巧

单晶结构解析技巧1. 通常,H原子的处理方法作者要给出:(1)一般通过理论加H,其温度因子为固定值,可通过INS等文件查看(2) 水分子上H原子可通过Fourier syntheses得到(3)检查理论加上的H原子是否正确,主要看H原子的方向。

若不正确则删去再通过Fourier syntheses合成得到(4) 检查H原子的键长、键角、温度因子等参数是否正常。

通过检查分子间或分子内的H键是否合理最易看出H键的合理性(5) 技巧:有时通过Fourier syntheses得到的H原子是正确的,可一计算其温度因子等参就变得不正常,则可以固定其参数后再精修(如在INS中的该H原子前用afix 1,其后加afix 0)(6) 各位来说说方法与心得?2.胡老师,下面的问题怎么解决啊?谢谢您。

220_ALERT_2_B Large Non-Solvent C Ueq(max)/Ueq(min) ... 3.70 Ratio222_ALERT_3_B Large Non-Solvent H Ueq(max)/Ueq(min) ... 4.97 Ratio342_ALERT_3_B Low Bond Precision on C-C bonds (x 1000) Ang (49)B 级提示当然得重视了。

建议你先把H撤消,精修到C的热椭球不太变形和键长趋正常。

如做不到就要看空间群?衍射点变量比太小?以至追查到原始数据的录取参数和处理等。

这些粗略意见仅供参考,如何?3.在XP中画图时,只有一部分,想长出另外的对称部分。

我是envi完了,然后sgen长出来的,可是和symm显示的对称信息不一样。

比如:我根据envi的结果用sgen O1 4555得到的是O1A而不是O1D,这跟文献中标注的不一样啊,怎么统一呢?很困扰,忘达人指教。

xp里是按顺序编号的,第一个sgen出的的统一为A,依次标号。

你如果想一开始就统一D的话,重新name一下4.高氯酸根怎么精修呀?我用的SHETXL6.1版的,最好告诉我怎么用其中的XSHELL来做,我觉得他好用!Method 1DFIXDfix 1.42 0.02 Cl1 O1 Cl1 O2 Cl1 O3 Cl1 O4Dfix 1.42 0.02 O1 O2 O1 O3 O1 O4 O2 O3O2 O4O3 O4Method 2SADISadi 0.01 Cl1 O1 Cl1 O2 Cl1 O3 Cl1 O4Sadi 0.01 O1 O2 O1 O3 O1 O4 O2 O3 O2 O4 O3 O45. 晶体的无序是怎么造成的呀,是晶体培养的问题吗?如果无序太多,在解单晶的时候怎么办?我指的是很多的点,没有结构,他们的峰值都大于了0.5大于0.5没什么的,解完后都在1以下就可以了。

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The Z gauge boson vertex has the following form factors:
with
χ˜+i
Z
= i GZijχχ
χ˜−j
GZijχχ = (1 + γ5) FZ+0γµ + FZ+1k1µ + FZ+2k2µ + (1 − γ5) FZ−0γµ + FZ−1k1µ + FZ−2k2µ (3)
we change the gaugino masses to
mν˜e = 150
M = 2M
GeV, and M = 2M ′ = 200 GeV.
In Fig. 1 we consider a constant, one–loop corrected, chargino mass mχ˜+ 1 = 170 GeV, a sneutrino mass given by mν˜e = 150 GeV, and gaugino masses M = 2M ′ = 300 GeV. The parameter tan β is varied along the curve with extreme values
and k1µ (k2µ) is the 4–momenta of the chargino χ˜−j (χ˜+i ). Analogous expressions are valid for the photon form factors. In GZχχ we include triangular 1PI graphs,
ν˜e
e−
χ˜−j
= iC−1(1 + γ5)Fν˜−e ,
where C is the charge conjugation matrix. The form factor Fν˜−e receive contributions from chargino mixing and chargino wave function renormalization. In order to be non–vanishing the charginos have to be a mixing between higgsino and gaugino. In this case, corrections proportional to logarithms of squark masses are enhanced by large Yukawa couplings.
Figure 1: Tree–level and one–loop corrected production cross section of a pair of charginos as a function of the LSP mass. We take mχ˜+ 1 = 170 GeV, mν˜e = 150 GeV, and M = 2M ′ = 300 GeV.
hep-ph/9910445 UCCHEP/4-99 FSU-HEP-991020
ONE–LOOP RADIATIVE CORRECTIONS TO CHARGINO PAIR PRODUCTION
arXiv:hep-ph/9910445v1 22 Oct 1999
MARCO AURELIO D´IAZ
They are produced in the s–channel via Z and γ gauge bosons, and in the t–channel via electron–sneutrinos:
e+ Z, γ
e−
χ˜ቤተ መጻሕፍቲ ባይዱi
e+
+
χ˜−j
e−
χ˜+i ν˜e
χ˜−j
The measurement of the total production cross section, the chargino mass, and the neutralino mass (a decay product), give enough information that can be used to find the fundamental parameters of the theory 1. This analysis also has been extended to CP violating scenarios and to polarized beams 2, and to the neutralino sector 3.
chargino mixing and self energies, and gauge bosons mixing and self energies. The
corrections turn out to be enhanced by large Yukawa couplings ht and hb, and proportional to logarithms of the squark masses. The corrections for this center of
In the following four figures we plot the tree–level (dashes) and one–loop corrected (solid) total production cross section σ(e+e− → χ˜+1 χ˜−1 ) as a function of the neutralino mass (LSP). We keep constant the squark mass parameters MQ = MU = MD = 1 TeV as well as the t√rilinear couplings AU = AD = 1 TeV, and we work with a center of mass energy s = 500 GeV.
Figure 2: Tree–level and one–loop corrected production cross section of a pair of charginos as a
function of the
In Fig. 2
LSP mass. We take mχ˜+ 1 = 170 GeV,
U∗MCV−1 = diag(mχ˜± 1 , mχ˜± 2 ) ,
(2)
and every chargino interaction depends on the matrix elements Uij and Vij . Electron–Positron colliders are a specially clean environment for chargino searches.
2
0.5 < tan β < 100 and, considering that the chargino mass is constant, this fixes the value of |µ|. Two branches appear according to the sign of µ. In parenthesis, and indicated by arrows it is shown the extreme values of tan β and µ. It is obvious from the figure that the largest deviations occur for extreme values of tan β.
is well known:

MC =
√M 2mW sin β
2mW cos β µ
(1)
where M is the gaugino mass associated to the SU (2) group, µ is the supersymmetric higgsino mass, and tan β = v2/v1 is the ratio of the two Higgs vacuum expectation values (vev). This mass matrix is diagonalized by two rotation matrices U and V such that
e+ Z, γ
χ˜+i
e+
+
χ˜+i ν˜e
e−
χ˜−j
e−
χ˜−j
with the renormalized vertices parametrized by form factors as defined in 4. Each sneutrino vertex has only one form factor:
In the Minimal Supersymmetric Standard Model (MSSM) the supersymmetric
fermionic partners of the W gauge bosons and the charged Higgs H± mix to form
a set of two Dirac fermions called charginos χ˜±i , i = 1, 2. The chargino mass matrix
Facultad de F´ısica, Universidad Cat´olica de Chile Av. Vicun˜a Mackena 4860, Santiago, Chile and
Department of Physics, Florida State University Tallahassee, Florida 32306, USA
We report on the effect that one–loop radiative corrections to the chargino pair production cross section has on the determination of the fundamental parameters of the theory. We work in the context of electron–positron colliders with √s = 500 GeV. We conclude that the inclusion of these corrections is crucial in precision measurements, specially at large and small values of tan β.
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