Molecular Characterization and Functional Analysis of OsPHY1

第50卷 第5期Vol.50, No.5, 463–4772021年9月GEOCHIMICASep., 2021收稿日期(Received): 2020-02-12; 改回日期(Revised): 2020-03-25; 接受日期(Accepted): 2020-04-01 基金项目: 国家自然科学基金(41872131)作者简介: 牛琮凯(1992–), 男, 博士研究生, 油气地球化学专业。

E-mail:******************** 通讯作者(Corresponding author):HOUDu-jie,E-mail:************.cn;Tel:+86-10-82322278Geochimica▌ Vol. 50▌ No. 5▌ pp. 463–477▌Sep., 2021原油含硫化合物分离及分析方法新进展牛琮凯1,2, 侯读杰1,2*(1. 中国地质大学(北京) 能源学院, 北京 100083; 2. 中国地质大学(北京) 海相储层演化与油气富集机理教育重点实验室, 北京 100083)摘 要: 原油中含硫化合物组成与结构信息广泛应用于油气勘探、石油运输和炼化以及环境保护。

目前, 挥发性和半挥发性含硫化合物的研究相对成熟, 但是对大分子含硫化合物的结构认识相对薄弱。

本次研究系统调研了国内外含硫化合物的分离和分析方法, 总结了各方法的原理、实验流程、优缺点、应用和意义。

含硫化合物分离方法主要包括化学转化方法和柱层析分离方法, 可以有效分离噻吩类、硫醚类、亚砜类、硫醇类以及含硫多环芳烃类化合物。

高分辨质谱和全二维气相色谱对高分子含硫化合物表征提供了技术支持。

总体而言, 对高分子含硫化合物的分子结构、来源、成岩演化过程及其生物标志物等信息的研究相对欠缺, 建议加强对低成熟高硫原油的研究, 探索高分子含硫化合物的赋存形式和地球化学意义, 为石油勘探开发进一步提供指导信息。

Postsynthesis,Characterization,and Catalytic Properties in Alkene Epoxidation of HydrothermallyStable Mesoporous Ti-SBA-15Peng Wu and Takashi Tatsumi*Division of Materials Science&Chemical Engineering,Graduate School of Engineering, Yokohama National University,79-5Tokiwadai,Hodogaya-ku,Yokohama240-8501,JapanTakayuki Komatsu and Tatsuaki YashimaDepartment of Chemistry,Tokyo Institute of Technology,2-12-1Ookayama,Meguro-ku,Tokyo152-8551,JapanReceived September25,2001.Revised Manuscript Received January11,2002Mesoporous Ti-SBA-15has been postsynthesized by the titanation of pure silica SBA-15 in glycerol with the assistance of quaternary organic ammonium hydroxides,and has been extensively characterized by various techniques to investigate the mesostructural and catalytic properties,and the chemical nature of the incorporated Ti species,in comparison with those of Ti-MCM-41.To incorporate tetrahedral Ti species into the silica walls of SAB-15,the titanation needs to be carried out at elevated temperature and using an optimum amount of organic ammonium hydroxide.Ti-SBA-15samples with a Si/Ti ratio ranging from 376to16are successfully prepared with the retention of hexagonal mesostructure by elaborately conducting the titanation.The nature of the Ti species depends greatly on the Ti loading.At Si/Ti ratios higher than50,the Ti species are mainly tetrahedrally coordinated in isolated states,while poorly dispersed Ti species of octahedral coordination are formed at higher Ti incorporation levels;however,no anatase phase is formed in all cases.The IR band at948cm-1due to Si-O-Ti bonds can be used to characterize the tetrahedral Ti species in mesoporous materials,but the techniques of dehydration or trimethylsilylation should be adopted to get rid of the influence of silanol groups.In an actual catalytic reaction, Ti-SBA-15exhibits not only extremely high thermal stability but also outstanding endurance against Ti leaching not observed for Ti-MCM-41.Ti-SBA-15is thus presumed to be a promising liquid-phase oxidation catalyst.IntroductionMesoporous materials,having uniform mesopores and an extremely high specific surface area,have received extensive attention from researchers in the past decade because of their potential applications as catalysts, adsorbents for large organic molecules,and guest-host chemical supporters.1-4From the viewpoint of catalysis, many efforts have been made to introduce metal ions into the most representative mesoporous silica,MCM-41,by direct synthesis or postsynthesis methods.5-10Compared to the crystalline microporous materials,the mesoporous catalysts suffer two major disadvantages, that is,low intrinsic catalytic activity due to the amorphous nature of the pore walls and poor hydro-thermal and mechanical stability due to the high hydrophilicity derived from abundant surface silanol groups.11To overcome the former weakness,efforts have been made to introduce zeolite-like crystalline orders into the mesoporous materials.For example,with the aid of tetrapropylammonium ions,Al-containing MCM-41is reported to partially crystallize to form ZSM-5precur-sors within the mesopore walls,and then exhibits an enhanced catalytic activity for cumene cracking.12Par-tially dissolving zeolite in carefully controlled basic media and then assembling this zeolite source into mesostructures or pillaring lamellar zeolite with sur-factant to construct a micro-mesoporous hybrid struc-ture is an alternative way.13-15However,it seems that*To whom correspondence should be addressed.Phone:+81-45-339-3943.Fax:+81-45-339-3941.E-mail:ttatsumi@ynu.ac.jp.(1)Kresge C.T.;Leonowicz,M.E.;Roth,W.J.;Vartuli,J.C.;Beck, J.S.Nature1992,359,710.(2)Beck,J.S.;Vartuli,J.C.;Roth,W.J.;Leonowicz,M.E.;KresgeC.T.;Schmitt,K.D.;Chu,C.T-W.;Olson,D.H.;Sheppard,E.W.; McCullen,S.B.;Higgins,J.B.;Schlenker,J.L.J.Am.Chem.Soc. 1992,114,10834.(3)Sayari,A.Chem.Mater.1996,8,1840.(4)Corma,A.Chem.Rev.1997,97,2373.(5)Maschmeyer,T.;Rey,F.;Sanker,G.;Thomas,J.M.Nature 1995,378,159.(6)Corma,A.;Forne´s,V.;Navarro,M.T.;Pe´rez-Pariente,J.J. Catal.1994,148,569.(7)Blasco,T.;Corma,A.;Navarro,M.T.;Pe´rez-Pariente,J.J. Catal.1995,156,65.(8)Koyano,K.A.;Tatsumi,T.Microporous Mater.1997,10,259.(9)Mokoya,R.;Jones,mun.1997,2185.(10)Ryoo,R.;Jun,S.J.;Kim,J.M.;Kim,mun. 1997,2225.(11)Tatsumi,T.;Koyano,K.A.;Tanaka,Y.;Nakata,S.Chem.Lett. 1997,469.(12)Kloetstra,K.R.;van Bekkum,H.;Jansen,J. C.Chem. Commun.1997,2281.1657Chem.Mater.2002,14,1657-166410.1021/cm010910v CCC:$22.00©2002American Chemical SocietyPublished on Web03/12/2002many technical difficulties and limitations still remain in the above approaches.To increase the stability of mesoporous materials, postmodification and a search for more stable structures have been carried out.Trimethylsilylation effectively removes the surface silanols to greatly enhance the hydrophobicity.This enhances the hydrothermal stabil-ity and the catalytic activity especially for Ti-MCM-41-catalyzed liquid-phase oxidation involving water.16,17It is easy to consider the improvement of the stability through controlling the synthesis conditions to achieve an extensive condensation of the silica walls or by discovering new materials that are intrinsically stable. In this meaning,SBA-15,synthesized with triblock copolymer,has thicker walls and uniform mesopores ranging from5to30nm,and then reasonably shows a higher hydrothermal stability than conventional MCM-41.18,19Mesoporous pure silica MSU-G thereafter syn-thesized with novel gemini surfactants is reported to be more hydrothermally stable even than SBA-15 because of a high degree of SiO4unit cross-linking.20 However,to the best of our knowledge,the isomorphous substitution of heteroatoms into MSU-G has not been reported in the open literature so far.On the other hand,several papers concerning the incorporation of Al,Ti,or V into SBA-15have been reported.21-23In the case of Ti-SBA-15,although it can be synthesized directly under microwave hydrothermal conditions,24the postsynthesis method is still over-whelming mainly because SBA-15needs strongly acidic synthesis conditions.To apply Ti-SBA-15to a versatile catalyst in the selective oxidation with peroxide oxidants to produce fine chemicals and pharmaceutical sub-stances,Ti should be incorporated essentially as a tetrahedral species but not as a catalytically inactive anatase phase.Unfortunately,the catalytic data are still not available for the Ti-SBA-15samples either directly synthesized or prepared simply by the incipient-wetness impregnation.Nevertheless,they do not seem to serve as effective epoxidation catalysts because they contain a large amount of anatase especially at high levels of Ti incorporation as evidenced by UV-vis spectra.22,24 Thus,there is still high potential to prepare Ti-SBA-15 with highly qualitative Ti species applicable to liquid-phase oxidation.In the present study,we have postsynthesized hy-drothermally stable Ti-SBA-15of various Ti contents with the assistance of quaternary organic ammonium hydroxide.Various techniques have been used to char-acterize extensively both the pore properties and the Ti states of Ti-SBA-15.The stability against hydrothermal treatment and Ti leaching has been compared between Ti-SBA-15and Ti-MCM-41in the actual catalytic ep-oxidation of alkenes.Experimental SectionSample Preparation.Mesoporous silica SBA-15was hy-drothermally synthesized according to the procedures reported previously.18Triblock copolymer,poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)(PEG-PPG-PEG;average molecular weight5800,Aldrich),was dissolved in deionized water.Tetraethyl orthosilicate(TEOS;Wako)and hydrochloric acid(36wt%,Wako)were then added to the copolymer solution to obtain a homogeneous solution with a molar composition of SiO2/0.017PEG-PPG-PEG/6.55HCl/169 H2O.The resultant solution was heated at308K with stirring for20h and further aged statically at373K for24h.The white solid product(SBA-15)was filtered,washed with deion-ized water,dried,and then calcined at773K for10h.Ti-incorporated SBA-15,designated Ti-SBA,was prepared by the postsynthesis method using tetrabuthyl orthotitanate (95wt%,Wako)as a Ti source.In a typical preparation,a certain amount of TBOT was hydrolyzed in20mL of glycerol (99wt%,Wako)containing0-5mL of quaternary ammonium hydroxide to obtain a homogeneous solution.To the above solution was added1g of SBA-15without any pretreatment, and the mixture was heated statically to induce titanation at 303-393K for20-220h.The amount of TBOT added was varied to give Si/Ti atomic ratios of5.7-200.The quaternary ammonium hydroxides used were tetrapropylammonium hy-droxide(TPAOH;25wt%,TCI),tetraethylammonium hy-droxide(TEAOH;35wt%,Aldrich),and tetrabutylammonium hydroxide(TBAOH;45wt%,TCI).After the titanation,Ti-SBA was filtered and washed with deionized water,and the organic species were burned off at773K for4h.For control experiments,Ti-MCM-41samples,denoted Ti-MCM,with Si/Ti ratios of37-320were hydrothermally synthesized.7The trimethylsilylation of mesoporous materials was carried out using hexamethyldisilazane(HMDS).25After evacuation at473K for2h,1g of powder was added to20mL of anhydrous toluene and1g of HMDS in a flask equipped with a cooling condenser,and the mixture was refluxed for2h to induce the silylation of surface silanols.The silylated product, Ti-SBA-sil or Ti-MCM-sil,was then filtered,washed with acetone,and dried at373K for5h.Characterization Methods.Powder X-ray diffraction (XRD)patterns were recorded on a Rigaku Rint2000diffrac-tometer using Cu K R radiation,and UV-vis spectra were collected on a Shimadzu UV-2400PC spectrophotometer.The amount of Ti was quantified by inductively coupled plasma emission spectrometry(ICP;Rigaku JY38S).N2adsorption measurements at77K were carried out on a Coulter SA3100 volumetric adsorption analyzer after the samples were out-gassed for2h at473K.IR spectra were recorded on a Shimadzu FTIR-8100spectrophotometer using a KBr pellet technique.26Sample diluted in KBr(3wt%)was pressed into a wafer(31.9mg cm-2thickness).The wafer was evacuated at373-773K in a quartz cell sealed with KBr windows.The spectra were collected in absorbance mode after the cell was cooled to room temperature.Catalytic Reaction.The epoxidation of cyclohexene was carried out under vigorous stirring in a50mL glass flask which was heated with an oil bath.In a typical run,10mmol(13)Liu,Y.;Zhang,W.;Pinnavaia,T.J.Am.Chem.Soc.2000,122, 8791.(14)Zhang,Z.;Han,Y.;Zhu,L.;Wang,R.;Yu,Y.;Qiu,S.;Zhao,D.;Xiao,F.S.Angew.Chem.,Int.Ed.2001,40,1258.(15)Cheng,J. C.;Degnan,T. F.;Beck,J.S.;Huang,Y.Y.; Kalyanaraman,M.;Kowalski,J.A.;Loehr,C.A.;Mazzone,D.N.Stud. Surf.Sci.Catal.1999,121,53.(16)Tatsumi,T.;Koyano,K.A.;Igarashi,mun.1998, 325.(17)Koyano,K.A.;Tatsumi,T.;Tanaka,Y.;Nakata,S.J.Phys. Chem.B1997,101,9436.(18)Zhao,D.;Feng,J.;Huo,Q.;Melosh,N.;Fredrickson,G.H.; Chmelka,B.F.;Stucky,G.D.Science1998,279,548.(19)Zhao,D.;Feng,J.;Huo,Q.;Melosh,N.;Chmelka,B.F.;Stucky,G.D.J.Am.Chem.Soc.1998,120,6024.(20)Kim,S.S.;Zhang,W.;Pinnavaia,T.J.Science1998,282,1302.(21)Cheng,M.;Wang,Z.;Sakurai,K.;Kumata, F.;Saito,T.; Komatsu,T.;Yashima,T.Chem.Lett.1999,131.(22)Luan,Z.;Maes,E.M.;van der Heide,P.A.W.;Zhao,D.;Czernuszewicz,R.S.;Kevan,L.Chem.Mater.1999,11,3680.(23)Luan,Z.;Bae,J.Y.;Kevan,L.Chem.Mater.2000,12,3202.(24)Newalkar,B.L.;Olanrewaju,J.;Komarneni,S.Chem.Mater. 2000,13,552.(25)Vansant,E.F.;Van Der Voort,P.;Vroancken,K.C.Stud.Surf. Sci.Catal.1995,93,83.(26)Wu,P.;Tatsumi,T.;Komatsu,T.;Yashima,T.J.Phys.Chem. B2001,105,2897.1658Chem.Mater.,Vol.14,No.4,2002Wu et al.of cyclohexene (99wt %,TCI),10mL of acetonitrile (99.5wt %,Wako)solvent,and 0.05g of catalyst were mixed in the flask and heated to 333K under agitation.The desirable amount of oxidant,TBHP (70wt %,Aldrich)or H 2O 2(30wt %,Wako),was then added to the mixture to start the reaction.After the catalyst powder was removed from the reaction mixture by centrifugal separation,the products were analyzed on a gas chromatograph (Shimadzu GC-14B)with an FID detector.Results and DiscussionPostsynthesis of Ti-SBA-15.Glycerol was chosen as the solvent for introducing Ti into pure silica SBA-15by the postsynthesis method,because it exhibits the advantage of serving as a stabilizing guest molecule due to its high viscosity,12,27and is considered to exert a less destructive effect than water solvent on the hydrother-mally unstable mesoporous structure.Table 1shows the influence of various titanation conditions on the Ti incorporation and the physicochem-ical properties of SAB-15.The effect of the treatment temperature was investigated at a treatment time of 70h,TPAOH/SBA-15liquid-to-solid ratio of 3mL g -1,and Si/Ti atomic ratio of 60(Table 1,nos.1-4).The amount of Ti incorporated into SBA-15was about half of that initially added,and did not change significantly in the range from 303to 393K.On the other hand,the amount of Ti incorporated at 373K increased with increasing treatment time to 70h and showed negligible change after 70h (Table 1,nos.3and 5-7),indicating the Ti incorporation took place slowly only to a low level within 20h.When the treatment temperature and the time were fixed at 373K and 70h,respectively,the relative amount of TPAOH aqueous solution to SBA-15solid was found to significantly influence the absolute amount of Ti incorporated;that is,a lower amount of TPAOH solution resulted in higher Ti incorporation (Table 1,nos.8-11).The use of TEAOH and TBAOH for the titanation resulted in no obvious difference in the Ti amount from that of TPAOH (Table 1,nos.3,12,and 13).Figure 1shows the XRD patterns of the parent SBA-15and three representative Ti-SBA-15samples.All the SBA-15and Ti-SBA-15samples exhibited very similar patterns where three well-resolved diffraction peaks dueto 100,110,and 200reflections were observed.Although a slightly lower intensity of the 100peak was observed for Ti-SBA-15prepared under serious conditions (Figure 1d),the well-retained 110and 200peaks strongly verified that the characteristic hexagonal features were maintained in Ti-SBA-15.N 2adsorption measurements were conducted to fur-ther investigate the structural change upon titanation because the XRD patterns may not be so sensitive as to show a slight textural change.Figure 2A shows N 2adsorption/desorption isotherms for SBA-15and various Ti-SBA-15samples.SBA-15showed a hysteresis loop which typically featured this kind of mesoporous mate-rial.The step of increase in the N 2adsorption amount due to multilayer absorption in the mesopores was observed in a higher pressure region than that of MCM-(27)Doughetry,J.;Iton,L.E.;White,J.W.Zeolites 1995,15,640.Table 1.Effect of Treatment Conditions on Ti Incorporation into SBA-15atitanation condit ionsSi/Ti rationo.sampl e temp (K)time (h)TPAOH/SBA-15(mL g -1)added incorporated BET (m 2g -1)micropore vol b(mL g -1)pore size (nm)1SBA-156830.152 5.82Ti-SBA1303703601016530.085 5.83Ti-SBA2373703601165610.020 5.84Ti-SBA3393703601105130.014 5.85Ti-SBA4373203602696130.031 5.86Ti-SBA53731203601205230.005 5.87Ti-SBA63732203601145050.001 5.88Ti-SBA737370022256540.056 5.89Ti-SBA8373701223410Ti-SBA937370322725540.015 5.811Ti-SBA10373705221264840.003 5.812Ti-SBA11373703c 60955840.016 5.813Ti-SBA12373703d601055420.0135.8a Other conditions:parent SBA-15,1g;glycerol,20mL;25wt %TPAOH,3mL;time,70h.b Calculated from t -plots.c 35wt %TEAOH.d45wt %TBAOH.Figure 1.X-ray diffraction patterns of pure silica SBA-15(a)and Ti-incorporated samples of Ti-SBA2(b),Ti-SBA4(c),and Ti-SBA6(d).For Ti-SBA samples,see Table1.Figure 2.N 2adsorption/desorption isotherms (A)and pore size distributions (B)for pure silica SBA-15(a)and Ti-incorporated samples of Ti-SBA2(b),Ti-SBA3(c),Ti-SBA4(d),and Ti-SBA6(e).For Ti-SBA samples,see Table 1.Alkene Epoxidation of Mesoporous Ti-SBA-15Chem.Mater.,Vol.14,No.4,2002165941,suggesting SBA-15has a larger pore size.24,28The sharpness of the hysteresis loop was maintained for Ti-SBA-15samples (Figure 2,curves b -d).Nevertheless,the sample prepared under severe conditions slightly lost the sharpness of the hysteresis loop (Figure 2,curve e),probably due to partial collapse of the mesostructure.The specific area (BET)and the BJH pore size distribution calculated from the desorption isotherms are summarized in Table 1and Figure 2B,respectively.The BET surface area of SBA-15is generally reported to vary in the range of 500-1000m 2g -1since the porous properties of this kind of mesoporous material are greatly dependent on the synthesis conditions.18,19,29The present SBA-15fell within the surface area range reported in the literature and was thus considered to be qualitatively good.The specific area was lowered by about 30m 2g -1after mild titanation,and further decreased gradually with increasing treatment temper-ature,treatment time,and relative amount of TPAOH solution (Table 1,nos.1-11).This can be reasonably explained in terms of a reduced order of the mesostruc-ture,because SBA-15,although consisting of thick silica walls,is still not hydrothermally stable enough to withstand the titanation treatment.Nevertheless,the average pore size was almost the same and the distri-bution was generally very narrow except for Ti-SBA-15prepared under severe conditions (Figure 2,curve e).Different from mesoporous MCM-41,SBA-15shows both mesoporous and microporous properties which greatly depend on the aging temperature.The presence of micropores has been confirmed either by the carbon replica method or by N 2adsorption measurement using R s -plot and t -plot methods.29-33The t -plot analysis was thus used to estimate the micropore volume in the mesoporous materials.The t -plots were obtained by using a reference isotherm of a nonporous silica as described previously.34As shown in Figure 3a,b,the extrapolation lines of the t -plots passed through the origin for MCM-41and Ti-MCM-41,indicating theylacked micropores.In contrast,SBA-15gave a straight line not passing through the origin (Figure 3c),which was indicative of distinct microporosity.The micropore volume of SBA-15obtained from the intercept of the y -axis accounted for about 15%of its total volume.Ti-SBA-15samples showed t -plots similar in shape to that of SBA-15,but the intercepts approached the origin (Figure 3d,e),indicating gradual disappearance of the micropores.The micropore volumes obtained from the t -plots are listed in Table bining these with the above BET analysis,we would propose that the decrease in the surface area during titanation is principally due to the loss of micropores.Since the micropores are probably generated by penetration of hydrophilic poly(ethylene oxide)chains of triblock copolymer into its thick silica walls,29-33a hydrothermal heating would shrink the silica walls to mend “holes”therein which are mi-cropores.Incorporation of Ti species into the micropores to remove this wall defect may also partially account for the reduced surface area.Both reasons would make Ti-SBA-15mesoporous but lack the bimodal porosity of SBA-15.However,very severe titanation at higher temperatures,for a longer time,and by using a larger amount of TAPOH would not only remove the mi-cropores but also destroy the mesopores as evidenced by a broader pore size distribution (Figure 2,curve e)and greatly reduced BET areas (Table 1,nos.4,7,11).UV -vis spectroscopy was used to characterize the effectiveness of the titanation conditions on the chemical nature and coordination states of the Ti species incor-porated.The bands at 200-230,330,and 260-290nm are usually taken as clear evidence for the isolated framework Ti,anatase Ti(IV)particles,and octahedrally coordinated Ti species with low dispersion,respec-tively.35The band position is thus indicative of the nature of the Ti species.Figure 4depicts the UV spectra of Ti-SBA-15samples prepared under various conditions.Ti-SBA-15obtained by titanation at 303K (Table 1,no.2)showed a main band at 260nm besides the 220nm band,and those prepared at higher temperatures (Table 1,nos.3and 4)exhibited essentially the 220nm band (Figure 4A),although the Ti contents were very similar to those described above.The reaction of the Ti species with the surface silanol groups is suggested to be requisite for Ti incorporation.A certain temperature is presumed to be necessary in crossing the activation energy barrier to cause this reaction to result in isolated Ti species.Titanation at an optimum temperature of 373K re-sulted in incorporation of Ti species almost tetrahedrally coordinated,increasing gradually with time (Figure 4B).The addition of TPAOH retarded the Ti introduction (Table 1,nos.8-11),but effectively controlled the nature of the Ti species as shown by the gradual blue shift of the band from 260to 220nm with increasing TPAOH/solid ratio (Figure 4C).The basic aqueous solution of TPAOH would promote the hydrolysis of TBOT,which then prevents the Ti species from ag-gregating to particles.Nevertheless,too much TPAOH solution added caused structural collapse.Other qua-ternary organic ammonium hydroxides,TEAOH and(28)Gregg,S.J.;Sing,K.S.Adsorption,Surface Area and Porosity ;Academic Press:London,1982.(29)Miyazawa,K.;Inagaki,mun.2000,2121.(30)Kruk,M.;Jaroniec,M.;Ko,C.H.;Ryoo,R.Chem.Mater.2000,12,1961.(31)Jun,S.;Joo,S.H.;Ryoo,R.;Kruk,M.;Jaroniec,M.;Liu,Z.;Ohsuna,T.;Terasaki,O.J.Am.Chem.Soc.2000,122,10712.(32)Ryoo,R.;Hyun,K.C.;Kruk,M.;Antochshuk,V.;Jaroniec,M.J.Phys.Chem.B 2000,104,11465.(33)Imperor-Clere,M.;Davidson,P.;Davidson,A.J.Am.Chem.Soc.2000,122,11925.(34)Bhambhani,M.R.;Cutting,P.A.;Sing,K.S.W.;Turk,D.H.J.Colloid Interface Sci.1972,38,109.(35)Vayssilov,G.N.Catal.Rev.Sci.Eng.1997,39,209.Figure 3.t -plots of N 2adsorption for pure silica MCM-41(a),Ti-MCM-41(b),pure silica SBA-15(c),Ti-SBA2(d),and Ti-SBA3(e).For Ti-SBA samples,see Table 1.1660Chem.Mater.,Vol.14,No.4,2002Wu et al.TBAOH,showed basically the same effectiveness as TPAOH in forming highly dispersed Ti species (Figure 4D).Therefore,perusal of XRD,ICP,N 2adsorption,and UV -vis data leads to the conclusion that Ti-SBA-15containing the tetrahedral Ti species can be optimally postsynthesized by titanation in gycerol at an elevated temperature with organic ammonium hydroxide as a promoting agent for Ti dispersion.A series of Ti-SBA-15samples were thus prepared by varying the Ti content in the starting reaction mixture at the optimum conditions as described above.Table 2shows their physicochemical properties together with those of directly synthesized Ti-MCM-41samples.The amount of Ti incorporated into SBA-15silica decreased from about half to one-fourth of the amount initially added when the Si/Ti ratio of the starting mixture decreased,indicating a decreasing efficiency for Ti incorporation.The absorption band maximum was observed at 220nm in the UV -vis spectra for both Ti-SBA-15and Ti-MCM-41(Figure 5).The band gradually red-shifted to the longer wavelength region with increasing Ti content to result in a new shoulder band around 260nm especially when the Si/Ti ratio was lower than 50.This means octahedral Ti species formed partially within the mesoporous materials at high Ti levels irrespective of the Ti incorporation method (direct or postsynthesis).Nevertheless,no Ti-SBA-15sample showed the band around 330nm due to the anatase phase.The present postsynthesis method is presumed to be effective in achieving high Ti dispersion.IR Spectroscopy Study on Ti-SBA-15and Ti-MCM-41.IR spectroscopy is a useful technique for characterizing the framework Ti species in microporous crystalline titanosilicates such as TS-136and Ti-MOR.37Nevertheless,this method is not so useful for Ti meso-porous materials containing silanols groups or defect sites,7,26because those silanol groups or water molecules adsorbed therein also give a similar band.However,we have recently solved this problem by measuring IR spectra after evacuating the samples in situ at elevated temperatures.26This new technique was applied here to the characterization of Ti-SBA-15with IR spectros-copy.Figure 6shows the IR spectra of pure silica SBA-15and Ti-SBA-15recorded after evacuation at various temperatures.The sample was diluted in KBr pellets (15wt %)to obtain well-resolved and noncutoff bands in the region lower than 1000cm -1.SBA-15showed a band at 968cm -1before evacuation despite the absence of Ti (Figure 6B).However,the band gradually de-creased in intensity with increasing evacuation tem-perature,and was hardly observed after evacuation at 773K.This is a result of removal of adsorbed water and dehydroxylation of silanols as evidenced by the disap-pearance of the broad band at 3200-3600cm -1(Figure 6A).The same 968cm -1band was observed for Ti-SBA-15before evacuation,but the band split into two bands after evacuation at 573K and turned out to be a new band with a maximum adsorption at 948cm -1when evacuation was done at 773K (Figure 6D).These results suggest the 948cm -1band could be assigned to the tetrahedral Ti species in Ti-SBA-15.(36)Bellussi,G.;Rigguto,M.S.Stud.Surf.Sci.Catal.1994,85,177.(37)Wu,P.;Komatsu,T.;Yashima,T.J.Phys.Chem.1996,100,10316.Figure 4.UV -vis spectra for Ti-SBA-15samples prepared by titanation for 70h at different temperatures (A),at 373K for different periods of time (B),with different TPAOH/SBA-15ratios (C),and in various quaternary ammonium hydroxides (D).For detailed titanation conditions,see Table 1.Table 2.Postsynthesized Ti-SBA-15a andHydrothermally Synthesized Ti-MCM-41b with Various TiContents Si/Ti rationo.sample added incorporated BET (m 2g -1)pore size (nm)1Ti-SBA12003762Ti-SBA260116561 5.83Ti-SBA32280524 5.84Ti-SBA417575Ti-SBA51436542 5.86Ti-SBA610277Ti-SBA7720536 5.28Ti-SBA8 5.716515 5.29MCM-411158 2.710Ti-MCM12833201115 3.011Ti-MCM211013012Ti-MCM380761115 3.013Ti-MCM460581060 2.614Ti-MCM5353710412.6aPostsynthesis conditions:parent SBA-15,1g;glycerol,20mL;25wt %TPAOH,3mL;temperature,373K;time,70h.b Hydro-thermal synthesis conditions:see ref7.Figure 5.UV -vis spectra for postsynthesized Ti-SBA-15samples (A)and hydrothermally synthesized Ti-MCM-41samples (B)both with various Si/Ti ratios.For detailed preparation conditions,see Table 2.Alkene Epoxidation of Mesoporous Ti-SBA-15Chem.Mater.,Vol.14,No.4,20021661This assignment was further verified by the IR spectra of SBA-15and Ti-SBA-15after the trimethyl-silylation treatment.The silylation consumes the silanol groups effectively and greatly enhances the hydropho-bicity of mesoporous materials.17As shown in Figure 7,the silylation developed a band at 2975cm -1at-tributed to the C -H stretching vibration of methyl groups,25and greatly decreased the intensity of the terminal and hydrogen-bonded silanol bands at 3745and around 3500cm -1,respectively.Following the silylation,only a negligible band at 968cm -1was observed for pure silica SBA-15even before evacuation (Figure 7B).Silylated Ti-SBA-15,on the other hand,showed the tetrahedral Ti band at 948cm -1,whose intensity hardly changed with the evacuation temper-ature (Figure 7D).Combining the results of Figures 6and 7,we hypothesize that the presence of the tetra-hedral Ti species can be confirmed by IR spectroscopyonly either after the dehydroxylation at elevated tem-peratures or after the hydrophobic treatment by sily-lation.These new techniques would be effectively ap-plicable to the characterization of Ti-containing meso-porous materials.Figure 8shows the IR spectra of Ti-SBA-15and Ti-MCM-41samples with various Ti contents after elimi-nation of the influence of adsorbed water and silanols by evacuation at 773K.The 948cm -1band gradually increased in intensity with increasing Ti content.The intensity was proportional to the Ti content in the region lower than 0.35mmol g -1corresponding to a Si/Ti ratio of ca.50,and deviated from the linearity in the higher Ti content region (Figure 9).This is consistent with the findings obtained from UV -vis spectra that Ti species are incorporated into the silica framework of SBA-15to occupy initially the tetrahedral sites and then par-tially the octahedral sites with a gradual increase in Ti incorporation.A Comparison of Catalytic Properties and Sta-bility between Ti-SBA-15and Ti-MCM-41.Ti-MCM-41and Ti-MCM-48have been well-known to be prom-ising catalysts for the liquid-phase oxidation of bulky molecules.7,8,38Thus,postsynthesized Ti-SBA-15samples have been tested for the epoxidation of cyclohexene with tert -butyl hydroperoxide (TBHP).The products obtained were cyclohexene oxide,1,2-cyclohexanediols formed by a successive solvolysis of the oxide by water on acid sites such as surface silanol groups,and 2-cyclohexen-1-ol and 2-cyclohexen-1-one both due to allylic oxidation.The efficiency of TBHP calculated on the basis of the amount of tert -butyl alcohol was at least over 90mol %in all cases.As shown in Table 3,the main product was cyclohexene oxide,and its selectivity was comparable(38)Koyano,K.A.;Tatsumi,mun.1996,145.Figure 6.IR spectra for pure silica SBA-15(A,B)and Ti-SBA-15(Si/Ti )36)(C,D)without evacuation (a)and after evacuation at 373K (b),473K (c),573K (d),673K (e),and 773K (f)each for 1h.Figure 7.IR spectra for pure silica SBA-15-sil (A,B)and Ti-SBA-15-sil (Si/Ti )36)(C,D)without evacuation (a)and after evacuation at 373K (b),473K (c),573K (d),673K (e),and 773K (f)each for 1h.Both samples were obtained bytrimethylsilylation.Figure 8.IR spectra for Ti-SBA-15samples (A)with Si/Ti ratios of ∞(a),376(b),116(c),80(d),57(e),36(f),27(g),20(h),and 16(i)together with those of Ti-MCM-41samples (B)with Si/Ti ratios of ∞(a),320(b),76(c),58(d),and 37(e).All the spectra were recorded after evacuation at 773K for 2h.Figure 9.Dependence of the intensity of the 948cm -1band on the Ti content of Ti-SBA-15and Ti-MCM-41.1662Chem.Mater.,Vol.14,No.4,2002Wu et al.。

B7家族在肿瘤免疫治疗中的应用B7家族B7家族是具有协同刺激因子作用的属于免疫球蛋白超家族的一类跨膜蛋白。

首先于1982年Yokochi等发现［1］。

至今共发现的B7家族共有6个成员，分别是B7-1(CD80) 、B7-2(CD86)、B7-H1(PD-L1)、B7-H2(BPR7-1) 、B7-H3(BPR7-2)、B7-DC(PD-L2)，其中研究得比较早与比较多的是B7-1、B7-2［2、3、4］。

B7家族属免疫球蛋白超家族的跨膜蛋白，分布广泛，如B细胞、T细胞、单核细胞、树突状细胞、心肾、肺和肿瘤细胞等等。

其中B7-1的编码基因位于人类染色体3q13.3～q21区内, B7-2的编码基因则位于3q13.3～q23区内。

B7-1分子量44kd, 胞外功能区有216个氨基酸残基,跨膜区有27个氨基酸残基,胞内19个氨基酸残基。

B7-2由306个氨基酸组成,胞外区有220个氨基酸残基,跨膜区有23个氨基酸残基,胞内60个氨基酸残基。

CD28和CTLA-4(cytotoxic T lymphocyte antigen 4)是B7-1、B7-2的受体，也属于免疫球蛋白超家族,以单体或同源二聚体形式存在于T细胞表面。

PD-1(programmed death 1)则是B7-H1、B7-DC的受体，ICOS(inducible costimulator)是B7-H2受体，B7-H3的受体则还不清楚。

一般认为，B7(主要是抗原呈递细胞表面的B7)与细胞(主要是T细胞)的受体结合，作为第二信号，发挥作用。

如抗原呈递细胞(APC)表面的B7-1或B7-2与T细胞表面的CD28结合，促进T细胞增殖、分泌、上调抗凋亡基因；而与CTLA-4结合，则抑制T细胞的进一步活化［5］。

协同刺激与肿瘤免疫逃避当机体发生肿瘤时,肿瘤细胞可以凭借多种机制逃避机体免疫系统的监控而生长、分裂、增殖。

现代研究表明,荷瘤机体不是不能对肿瘤抗原进行识别、加工和递呈等等免疫反应,只是功能状态常常十分低下,不能产生有效的杀灭肿瘤作用。

Molecular Characterization of Propionyllysines in Non-histone Proteins*□SZhongyi Cheng‡§¶,Yi Tang§ʈ,Yue Chen‡,Sungchan Kim**,Huadong Liu‡‡,Shawn S.C.Li‡‡,Wei Guʈ§§,and Yingming Zhao‡¶¶¶Lysine propionylation and butyrylation are protein modi-fications that were recently identified in histones.The molecular components involved in the two protein modi-fication pathways are unknown,hindering further func-tional studies.Here we report identification of the first three in vivo non-histone protein substrates of lysine pro-pionylation in eukaryotic cells:p53,p300,and CREB-bind-ing protein.We used mass spectrometry to map lysine propionylation sites within these three proteins.We also identified the first two in vivo eukaryotic lysine propionyl-transferases,p300and CREB-binding protein,and the first eukaryotic depropionylase,Sirt1.p300was able to perform autopropionylation on lysine residues in cells. Our results suggest that lysine propionylation,like lysine acetylation,is a dynamic and regulatory post-translational modification.Based on these observations,it appears that some enzymes are common to the lysine propiony-lation and lysine acetylation regulatory pathways.Our studies therefore identified first several important players in lysine propionylation pathway.Molecular&Cellular Proteomics8:45–52,2009.Acetylation of the␧-amino group of lysine residues,or lysine acetylation(Lys Ac),is one of several abundant post-translational modifications of the lysine side chain,and it has important roles in cellular physiology.Lysine acetylation was first identified in the1960s in histones(1).Discovery of en-zymes responsible for adding and removing acetyl groups (histone acetyltransferases(HATs)1and histone deacetylases (HDACs))as well as non-histone substrate proteins(e.g.p53) in the mid-1990s marked a turning point in the field of lysine acetylation biology(2–4).Extensive studies over the past decade have established that lysine acetylation has diverse cellular functions and plays an important role in multiple dis-eases(5–10).The high abundance of lysine acetylation in mammalian cells,as demonstrated in a proteomics screen(11),led us to hypothesize that the␧-amino group of lysine residues under-goes the structurally similar modifications of propionylation and butyrylation(Lys Prop and Lys Buty,respectively)(12).We confirmed our hypothesis in human histones and verified the discovery by1)comparing the tandem mass spectra of the modified histone peptides with spectra obtained from syn-thetic peptides and2)identifying the first two in vitro propio-nyl-and butyryltransferases,p300and CBP(12).We also demonstrated that these two enzymes,which are also acetyl-transferases,can carry out in vitro autopropionylation and autobutyrylation on lysine residues.In unpublished work2we have also found lysine propionylation and butyrylation of his-tones from Saccharomyces cerevisiae.Lysine propionylation was subsequently reported in the Salmonella enterica propi-onyl-CoA synthetase enzyme PrpE(13).Elegant enzymologi-cal studies by Smith and Denu(14)suggest that some HDACs have measurable activity toward peptides containing propio-nyllysine and butyryllysine residues.Given the unique meta-bolic roles of acetyl-CoA,propionyl-CoA,and butyryl-CoA, which are the co-substrates for the modification reactions,as well as subtle structural differences among the modifications, we propose that lysine propionylation and lysine butyrylation have different biological functions than lysine acetylation. However,in vivo regulatory enzymes and non-histone sub-strates in eukaryotic cells remain to be characterized,hinder-ing biological studies of the two modification pathways.p53is a short-lived protein whose activity is maintained at low levels in normal cells.Tight regulation of p53is essential for its effect on tumorigenesis as well as maintaining normal cell growth.The cellular functions of p53are rapidly activated in response to stress.Although the mechanisms of p53acti-vation are not fully understood,they are generally thought to entail post-translational modifications of p53,such as ubiq-uitination,phosphorylation,and acetylation(15–17).In fact,From the‡Department of Biochemistry,University of Texas South-western Medical Center,Dallas,Texas75390,࿣Institute for CancerGenetics,College of Physicians and Surgeons,Columbia University,New York,New York10032,**Department of Biochemistry,College ofMedicine,Hallym University,Chuncheon,Kangwon-Do,200-702,Ko-rea,and the‡‡Department of Biochemistry and the Siebens-DrakeResearch Institute,Schulich School of Medicine and Dentistry,Uni-versity of Western Ontario,London,Ontario N6A5CI,CanadaReceived,May21,2008,and in revised form,August21,2008Published,MCP Papers in Press,August26,2008,DOI10.1074/mcp.M800224-MCP2001The abbreviations used are:HAT,histone acetyltransferases;CBP,CREB-binding protein;HDAC,histone deacetylase;Lys Ac,ly-sine acetylation;Lys Buty,lysine butyrylation;Lys Prop,lysine propiony-lation;MS,spectrometry;MS/MS,tandem mass spectrometry;HPLC,high pressure liquid chromatography;HA,hemagglutinin,PCAF,p300/CBP associate factor;MOF,male-absent on the first;HBO1,histone acetyltransferase bound to ORC1.2Z.Cheng and Y.Zhao,unpublished data.Research©2009by The American Society for Biochemistry and Molecular Biology,Inc.Molecular&Cellular Proteomics8.145 This paper is available on line at p53was the first non-histone protein found to be acetylated on lysine residues(3).Lysine acetylation regulates the pro-tein’s stability(by competing with ubiquitination for modifica-tion of specific sites),its interactions with binding partners (e.g.Mdm2and Mdmx),and its DNA-binding activity(18). Lysine acetylation status modulates p53-regulated effects in both cell cycle arrest and apoptosis.We recently demon-strated that p53can also be lysine propionylated and butyry-lated in vitro,catalyzed by p300and CBP.Nevertheless,it remains unclear whether p53is also propionylated and bu-tyrylated by the same enzymes in human cells.Research into any biological pathway at the molecular level is dependent on the identification and isolation of the key players,which remain unknown for lysine propionylation path-way.The present work was undertaken to identify in vivo non-histone substrates of lysine propionylation in eukaryotic cells and the enzymes responsible for adding and removing the modification.MATERIALS AND METHODSMaterials:Plasmids,Antibodies,and Other Reagents—Plasmids used in this study were described previously(12,22).p300HAT-dead mutant,p300DY,was generated by mutation of aspartic acid in1399 to tyrosine;CBP HAT-dead mutant,CBP-LD,was generated by mu-tation of both leucine in1345and aspartic acid in1346to alanine; Sirt1,enzymatic defective mutant was generated by replacing histi-dine in363by tyrosine.Generation of anti-propionyllysine antibody was described in Supplemental Fig.S1;anti-acetyllysine antibody was from(ImmunoChem Pharmaceuticals Inc.(Burnaby,British Co-lumbia,Canada);anti-HA antibody was from Roche Diagnostics; anti-Sirt1antibody was purchased from Upstate(Upstate,Charlottes-ville,VA);anti-FLAG M2antibody,FLAG-M2beads,and HA-agarose beads were purchased from Sigma-Aldrich.Modified porcine trypsin was purchased from Promega Inc.(Madison,WI),HPLC-grade ace-tonitrile,water,and ethanol were purchased from EMD Chemicals Inc. (Gibbstown,NJ),and acetic acid was purchased from Sigma.Cell Culture,Cell Transfection,and Treatment—H1299or293T cells were maintained in Dulbecco’s modified Eagle’s medium in the presence of10%fetal bovine serum at37°C in a humidified atmosphere of5%CO2.Cell transfection was carried out with Lipofectamine2000as described by the manufacturer(Invitrogen). For H1299cells,cells were treated with5m M sodium butyrate;for 293T cells,cells were treated with HDAC inhibitor mixture(2␮M trichostatin A,30m M nicotinamide,and50m M sodium butyrate)for six hours to inhibit the activity of endogenous HDACs before har-vesting the cells.Detection of Lysine Propionylation of p53,p300,and CBP in Cells—Twenty-four hours after transfection with various plasmids,as indicated,transfected cells were treated with described HDAC inhib-itors for6h,harvested,and lysed in FLAG-lysis buffer(50m M Tris-HCl,pH7.9,137m M NaCl,10m M NaF,1m M EDTA,1%Triton X-100, 0.2%Sarkosyl,10%glycerol,and fresh protease inhibitor mixture (Sigma))plus10m M sodium butyrate.Propionylation of p53in the total cell extracts or in material immunoprecipitated with M2-agarose beads was detected by Western blotting analysis using the anti-propionyllysine antibody described above.Propionylation of p300or CBP in material immunoprecipitated with M2or HA-agarose beads was detected by Western blot analysis using the anti-propionyllysine antibody.Purification of p53,p300,and CBP Proteins—Transfected cells were lysed by NETN buffer(20m M Tris-HCl pH8.0,100m M NaCl,1 m M EDTA,0.5%Nonidet P-40)containing described HDAC inhibitors and protease inhibitor mixture.Cell lysates were clarified by centrif-ugation at100,000ϫg for20min at4°C and incubated with either FLAG-M2beads or HA beads(Sigma-Aldrich)at4°C for1h.Anti-body beads were washed five times with NETN buffer.The bound proteins were eluted with SDS-PAGE sample buffer.In-gel and In-solution Digestion with Trypsin—The proteins of in-terest purified above were separated by SDS-PAGE and visualized by staining with colloidal Coomassie Blue.The protein bands were ex-cised,in-gel digested,and extracted as described previously(19). The extracted peptides were pooled,dried in a SpeedVac,and de-salted using a␮C18Ziptip(Millipore,Billerica,MA)prior to HPLC/ MS/MS analysis.HPLC/MS/MS Analysis and Database Searching—The dried pep-tide sample was dissolved in3␮l of HPLC buffer A(acetonitrile:water: acetic acidϭ2:97.9:0.1,v/v/v)and loaded onto a home-made cap-illary column(10cm lengthϫ75␮m ID)packed with Jupiter C12resin (4␮m particle size,90Åpore diameter;Phenomenex,St.Torrance, CA)connected to an Agilent1100nanoflow HPLC system(Agilent, Palo Alto,CA).Peptides were eluted with a gradient of10%to90% HPLC buffer B(acetonitrile:water:acetic acidϭ90:9.9:0.1,v/v/v). Peptides were ionized and introduced into an LTQ mass spectrome-ter using a nano-spray source.The mass spectrometric data were acquired in data-dependant mode.Tandem mass spectra were ana-lyzed using PTMap software,an algorithm we recently developed to identify all possible post-translational modifications in a substrate protein(20).The specified parameters for protein sequence database searching included Met oxidation as a variable modification,trypsin as the enzyme,three allowed missing cleavages,and mass errors of 4Da for precursor ions and0.6Da for fragment ions.All identified peptides bearing modifications were manually inspected according to a procedure we described previously(21).RESULTSp300and CBP Are Propionyltransferases for p53in Vivo—Our previous experiments(12)demonstrated that p300and CBP can catalyze in vitro lysine propionylation of p53.Given that p300and CBP can catalyze both acetylation and propio-nylation reactions in vitro and that they can acetylate p53in vivo,we reasoned that the enzymes are likely able to perform propionylation of p53in vivo.To test this hypothesis,we used co-transfection experiments in H1299cells followed by West-ern blotting analysis using a pan-propionyllysine antibody.As expected,lysine propionylation of p53was dramatically in-creased when cells were co-transfected with either p300or CBP(Fig.1A),but not mutated,HAT-dead forms of these enzymes(B).These data in combination with our earlier in vitro results showing p53lysine propionylation by p300and CBP(12)suggest that these two enzymes can catalyze p53 propionylation in vivo.We tested the ability of other acetyltransferases to propi-onylate p53in vivo.For example,Tip60is an acetyltransferase that can acetylate p53at lysine-120.This modification site is critical for p53-dependent apopotosis,but not p53-mediated growth arrest(22).In contrast to p300and CBP,little lysine propionylation activity was detected for Tip60either in vitro (12)or in vivo(Fig.1B).Co-transfection experiments usingPropionyllysines in Non-histone Proteins 46Molecular&Cellular Proteomics8.1four HATs(MOF,PCAF,Tip60,and HBO1)suggest that these proteins are not major enzymes responsible for propionylating lysine residues in p53(Fig.1B).Sirt1Can Catalyze Lysine Depropionylation in p53—Sirt1is a known lysine deacetylase for p53(23).To test if Sirt1have enzymatic activity toward lysine propionylation in p53,we detected lysine propionylation levels in293T cell by Western blotting analysis using a pan-propionyllysine antibody follow-ing co-transfection of p53with wild type Sirt1or its HDAC-defected mutant.As expected,treatment of cells with HDAC inhibitors induced lysine propionylation(Fig.1C).Lysine pro-pionylation level was decreased when co-transected with wild type Sirt1but not its HDAC-defected mutant.Our results suggest that Sirt1is likely a lysine depropionylation enzyme for p53.Mapping Lysine Propionylation Sites in p53by Mass Spec-trometry—Although we previously identified the lysine resi-dues within p53that were propionylated by p300and CBP in vitro(12),the in vivo modification sites remain unknown.To identify these sites,we co-transfected p53into H1299cells with either p300or CBP,then isolated p53by immunopre-cipitation(Fig.2A).The isolated proteins were subjected to SDS-PAGE,in-gel digestion,and HPLC/MS/MS analysis to map sites of lysine acetylation and propionylation.As ex-pected,our analysis identified11lysine acetylation sites in p53(Supplemental Fig.S2).We also identified one lysine propionylation site and three lysine butyrylation sites(Fig.2,B and C).The propionyllysine sites were identified in p53from cells co-transfected with p300/CBP,but not from cells with-out p300/CBP co-transfection(Fig.2B),further suggesting that p53is an in vivo propionyltransferase substrate for p300/CBP.As expected,we found that several lysine residues(Lys372, Lys373,and Lys382)at the C terminus can be butyrylated, supporting the notion that multiple protein modifications of the C terminus including ubiquitination,acetylation,and methylation are involved in functional regulation of p53.Inter-estingly,we found that Lys292is propinylated in cells,sug-gesting that modification of Lys292by propinylation may me-diate specific functions in vivo.Moreover,tumor-associated mutations of Lys292have been observed in several different types of human tumors.Thus,it is important to understand the precise functional consequence of this modification in p53-mediated tumor suppression,which warrants future investigations.p300and CBP Are Lysine Propionylated in Vivo—We pre-viously found that p300and CBP can perform auto-lysine propionylation in vitro(12).To determine whether these pro-teins are propionylated in vivo,we transfected293T cells with p300or CBP,immnoprecipitated the proteins,and analyzedF IG.1.In Vivo propionylation of p53determined by p300/CBP and Sirt1.A,p300/CBP catalyzes p53propionylation in vivo.H1299cells were transfected with the plasmid DNA-expressing FLAG-p53with or without FLAG-p300or CBP-HA.The immunoprecipitated proteins by M2 beads were analyzed by Western blotting using anti-propionyllysine pan antibody,anti-p53,and anti-p300antibodies.CBP in the total cell extracts was detected by anti-HA antibody.B,p300/CBP are specific propionyltransferases in p53propionylation.The total cell extracts from H1299cells transfected with the plasmid DNA-expressing p53and/or various HATs were analyzed by Western blot using anti-propionyllysine and anti-p53antibodies.C,Sirt1de-propionylates p53in vivo.293T cells were transfected with plasmid DNA-expressing FLAG-p53with or without Sirt1-V5-His.Indicated transfection cells were treated with HDAC inhibitor mixture for six hours before harvesting cells for immuno-precipitation.Immunoprecipitates were analyzed by Western blotting using anti-propionyllysine and anti-FLAG antibodies.Ectopic expression of Sirt1was detected by Sirt1antibody.Propionyllysines in Non-histone ProteinsMolecular&Cellular Proteomics8.147Propionyllysines in Non-histone Proteins48Molecular&Cellular Proteomics8.1for propionyllysine by Western blotting.Lysine propionylation of p300was detected in these cells (Fig.3A ).In a subsequent attempt to increase lysine propionylation levels,we treated the cells with HDAC inhibitors for six hours before they were harvested.This treatment increased the amount of lysine propionylation detected (Fig.3A ),suggesting that deacety-lases contribute to regulate the propionyllysine status.Like-wise,in vivo propionylation of CBP was also detected under this experimental condition (Fig.3B ).To test if Sirt1,a class III HDAC,can remove propionyl group from p300,we co-trans-fected p300into 293T cells along with Sirt1or its HDAC-dead version of Sirt1.Lysine propionylation was significantly de-creased when p300was co-transfected with wild type Sirt1,but not the mutated form (Fig.3C ),implying that Sirt1can catalyze in vivo lysine depropionylation of p300.Next,we carried out mass spectrometric analysis of endog-enous p300and CBP immunoprecipitated directly from H1299cells.To boost lysine propionylation levels,the cells were treated with HDAC inhibitors for six hours before the cells were harvested.HPLC/mass spectrometric analysis in combination with protein sequence database searching identified 27lysine acetylation sites (Supplemental Fig.S3),4propionylation sites,and 4lysine butyrylation sites in p300(Fig.4,A and C ).Likewise,we identified 31acetylation sites (Supplemental Fig.S4),and 2lysine butyrylation sites (Fig.4B )in CBP.We were not able to identify propionylation sites in CBP with mass spectrometry possibly because of low stoichiometry of the modification.Nevertheless,together with Western blotting analysis,these results demonstrate that (i)p300is in vivo lysine propionylation substrate;(ii)F IG .2.Mapping lysine propionylation sites in p53by mass spectrometry.A ,purification of p53,p300,and CBP in vivo .H1299cells were transfected with plasmids DNA encoding FLAG-p53(lane 1),FLAG-p53with FLAG-p300(lane 2),or FLAG-p53with FLAG-p300and SIRT1-V5-His (lane 3),or FLAG-p53with HA-CBP (lane 4),FLAG-p300(lane 5),and HA-CBP (lane 6),respectively.Twenty-four hours after transfection,cells were lysed and subjected to immunoprecipitation as described in “Materials and Methods”.The purified target proteins were resolved in SDS-PAGE for mapping modification sites of interest.B ,in vivo lysine propionylated and butyrylated peptides and sites identified in p53when it was co-transfected with (ϩ)or without (Ϫ)an enzyme.C ,MS/MS spectrum of “K Prop GEPHHELPPGST K Ac R”that identified Lys 292as in vivo lysine propionylation site in p53.F IG .3.p300and CBP are lysine propionylated in vivo .A ,the p300propionylation was induced in vivo by HDAC inhibitor mixture.293T cells were transfected to express FLAG-p300.The indicated transfection cells were treated with HDAC inhibitor mixture for six hours before harvesting for immunoprecipitation.The immunoprecipitated proteins were analyzed by Western blotting using anti-propionyllysine,anti-acetyllysin,and anti-FLAG antibodies,respectively.B ,the CBP propionylation levels were enhanced in vivo under HDAC inhibitor mixture treatment.293T cells were transfected to express HA-CBP.The indicated transfection cells were treated with HDAC inhibitor mixture for six hours before cell harvesting for immunoprecipitation.The immuno-isolated proteins were analyzed by Western blot using anti-propionyllysine,anti-acetyllysine,and anti-FLAG antibodies,respectively.C ,Sirt1depropionylates p300in vivo .293T cells were transfected with plasmid DNA expressing FLAG-p300with or without Sirt1-V5-His.The indicated transfection cells were treated with HDAC inhibitor mixture for six hours before the cells were harvested for immunoprecipitation.The isolated proteins were subjected to Western blotting analysis using anti-propionyllysine and anti-FLAG antibodies.Ectopic expression of Sirt1was detected by Sirt1antibody.Propionyllysines in Non-histone ProteinsMolecular &Cellular Proteomics 8.149Propionyllysines in Non-histone ProteinsF IG.4.Mapping lysine propionylation sites in p300by mass spectrometry.In Vivo lysine propionylated and butyrylated peptides and sites identified in p300(A)and in CBP(B).C,MS/MS spectrum of“NNKAcKPropTSKAcNKAcSSLSR”that identified Lys1555as in vivo lysine propionylation site in p300.50Molecular&Cellular Proteomics8.1p300and CBP are in vivo lysine butyrylation substrates;and (iii)Sirt1can catalyze p300in vivo lysine depropionylation reaction.DISCUSSIONLysine propionylation is originally identified in histones(12), but whether it,like lysine acetylation,is present in non-histone proteins remains unknown,as do the identities of the en-zymes responsible for regulating the modification.Here,we show that(1)lysine propionylation is present in three non-histone proteins(p53,p300,and CBP);(2)p300and CBP can catalyze lysine propionylation of p53;(3)p300and CBP have auto-lysine propionylation activities;and(4)Sirt1is a depro-pionylase that removes lysine propionylation from p53and p300.These results indicate that lysine acetylation and lysine propionylation pathways are likely to share many substrates and regulatory enzymes in common.Although three regula-tory enzymes for lysine acetylation(Sirt1,p300,and CBP)also affect lysine propionylation,Tip60and the other three HATs tested seem to have much higher selectivity and are likely to restrict their enzymatic activity to lysine acetylation or have much lower activity to lysine propionylation,at least for the substrate p53.Our analysis indicates that stoichiometry for lysine acetyla-tion is much higher than that for lysine propionylation and lysine butyrylation.For example,the mass spectrometric sig-nal for propionylated lysine(Lys292)is25times less that that of acetylated lysine(data not shown).Nevertheless,the stoi-chiometric ratios for the three modifications are not sequen-tially decreased with the lengths of the short acyl chains.This is evidenced by the fact that two lysine butyrylation sites (Lys1595and Lys1597)were identified in CBP and three lysine butyrylation sites(Lys372,Lys373,and Lys382)in p53,but not their propionyllysine counterparts.This result suggests that there might be the existence of enzymes that prefer lysine butyrylation to lysine propionylation.Lysine propionylation status can be enhanced moderately by HDAC inhibitors among three substrate proteins,raising the possibilities of other depropionylation enzymes in cells the activities of which cannot be blocked by HDAC inhibitors.It should also be pointed that we are likely to fail to identify lower stoichiometric propionyllysine and butyryllysine sites among the three sub-strate proteins as only0.5–1pmol of p300/CBP andϳ3pmol of p53were used in our experiment.Our studies raise a few interesting questions.How are the relative levels of acetylation and propionylation at a particular lysine residue regulated by the same enzyme?In the case of the transferase reaction,Lys Ac and Lys Prop status may be regulated by the intrinsic activity of the enzyme toward the substrate compounds(acetyl-CoA and propionyl-CoA)and also by the relative concentrations of the two acyl-CoA mol-ecules.Propionyl-CoA is generated by the catabolism of short-chain lipids and certain amino acids.Therefore,diet may affect the concentrations of the acyl-CoAs,which in turn modulate the lysine modification status.It is also possible that some transferases prefer one acyl-CoA to the other as a substrate.If this is so,cells will contain Lys Ac-or Lys Prop-specific transferases.The question of how specificity is achieved also arises for the depropionylases.Because no modification-specific cofactors are involved in the demodifi-cation reaction,this reaction is mainly dependent on intrinsic enzymatic activity.Finally,what are functional differences between Lys Ac and Lys Prop in a given substrate protein?Al-though acetyllysine and propionyllysine are structurally simi-lar,they may cause differences in protein-protein interactions. For example,acetylated and propionylated proteins may as-sociate differently with bromo-domain proteins.Because Lys Prop has been identified in several proteins known to be substrates for lysine acetylation(histones,p53, p300,and CBP),we anticipate that a large number of other Lys Ac substrates may also be lysine propionylated and lysine butyrylated.Similarly,additional regulatory enzymes for Lys Prop and Lys Buty may be found among enzymes known to be acetyltransferases or deacetylases.*This work was supported by,in whole or in part,by NCI,National Institutes of Health Grant CA126832(to Y.Z.)and NCI,National Institutes of Health grant(to W.G.).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 accord-ance with18U.S.C.Section1734solely to indicate this fact.□S The on-line version of this article(available at http://www. )contains supplemental Figures S1,S2,S3,and S4.§Both authors contributed equally to this work.¶Present address:Ben May Department for Cancer Research,The University of Chicago,Chicago,IL60637.§§To whom correspondence may be addressed.E-mail:wg8@ .¶¶To whom correspondence may be addressed.E-mail: Yingming.Zhao@.REFERENCES1.Allfrey,V.G.,Faulkner,R.,and Mirsky,A.E.(1964)Acetylation and meth-ylation of histones and their possible role in the regulation of RNA synthesis.Proc.Natl.Acad.Sci.U.S.A.51,786–7942.Brownell,J.E.,Zhou,J.,Ranalli,T.,Kobayashi,R.,Edmondson,D.G.,Roth,S.Y.,and Allis,C.D.(1996)Tetrahymena histone acetyltransferase A:a homolog to yeast Gcn5p linking histone acetylation to gene activa-tion.Cell84,843–8513.Gu,W.,Shi,X.L.,and Roeder,R.G.(1997)Synergistic activation oftranscription by CBP and p53.Nature387,819–8234.Taunton,J.,Hassig,C.A.,and Schreiber,S.L.(1996)A mammalian histonedeacetylase related to the yeast transcriptional regulator Rpd3p.Science 272,408–4115.Kouzarides,T.(2000)Acetylation:a regulatory modification to rival phos-phorylation?EMBO J.19,1176–11796.Haigis,M.C.,and Guarente,L.P.(2006)Mammalian sirtuins–emergingroles in physiology,aging,and calorie restriction.Genes Dev.20, 2913–29217.McKinsey,T. 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神经内分泌前列腺癌的研究进展[摘要]神经内分泌前列腺癌(neuroendocrine prostate cancer, NEPC)是一种恶性程度极高的前列腺癌亚型,目前临床对其早期诊断与治疗的认知仍存在不足。

针对这一现状,本文通过综述与神经内分泌性前列腺癌分化发生、机制及模型进展相关的最新研究成果，为同行诊治与研究神经内分泌性前列腺癌提供新思路。

[关键词]前列腺癌; 神经内分泌分化[中图分类号]R 737.25 [文献标识码] A [文章编号]Advance in the researches ofneuroendocrine prostate cancerHU Junchao,LI Hubo,JIA Mingfei,Kang Shaosan,(North ChinaUniversity of Science and Technology TangShan,063000,China)[ABSTRACT]Neuroendocrine prostate cancer (NEPC) is a highly malignant subtype of prostate cancer. At present, the early diagnosis and treatment of NEPC are still insufficient.In view of this situation, this article reviews the latest research results related to the differentiation, mechanism and model progress of neurosecretoryprostate cancer, so as to provide new ideas for peer diagnosis, treatment and research of neurosecretory prostate cancer.[KEY WORDS] P rostate cancer; Neuroendocrine differentiation.前列腺癌是全球男性最常见的恶性肿瘤之一, 近年我国前列腺癌发病率也持续上升。

与试验研究犬食道异物诊断与治疗曹百灵1陆优兰2（1，上海骐奇动物诊疗有限公司201499；2，上海三米宠物用品有限公司201107）摘要：犬食道异物是临床常见病，因为异物体积太大而不能通过食道或其尖端刺入食道黏膜里，异物通常在胸腔入口、心肌或膈区域，因为这些部位的食道外解剖位置限制了食道膨胀。

本文通过一只7个月的约克夏犬的临床病例诊治，总结并分析了犬食道异物的病因、临床症状及诊断方法，并介绍手术治疗本病的方法、诊治要点及对本病的治疗体会。

关键词：犬食道异物；诊断；治疗犬食道异物是指犬吞下难以排岀或消化的东西（如骨头、布料、玩具、石头、果核、木块、塑料瓶盖、针线等），因无法通过比较狭窄的食道而引起食道梗阻，临床表现为拒食、呕吐、咳喘、流口水、吞咽困难、消瘦、衰竭等急、慢性临床症状。

在临床当中食道异物可以引起完全梗阻和不完全梗阻，异物在食道内不完全梗阻使大多数动物表现为食欲不振、呕吐、少量饮水，消瘦、衰竭、局部血液循环不良、缺血和坏死。

发生完全梗阻时，食欲废绝、精神萎靡、咳喘，并伴有呼吸困难、消瘦、衰竭、局部血液循环不良等情况，病程发展迅速。

由于异物压迫，局部血液循环不良、缺血、食道水肿、缺氧、作者简介：曹百灵（1983.9-），女，甘肃省平凉市人，大学本科，兽医师，坏死，甚至引起食道穿孔破裂，最终休克、衰竭而死亡。

1基本信息约克夏犬，8个月，公犬，最近几天有咳嗽，干呕，呕吐有白沫，大便少，没食欲，精神不佳，免疫驱虫全。

2病因该犬由于长期饮食比较单一，平时以人吃的东西为主，包括吃一些骨头之类的东西，而就在5d前主人刚好饲喂过排骨，怀疑由于吃排骨时误吞造成食道梗阻。

3症状该犬来我院诊治时已经5d不吃东西，精神状况较差，刚开始主要从事兽医诊疗工作。

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