Pseudogaps in the 2D half-filled Hubbard model

a r X i v :c o n d -m a t /9910226v 1 [c o n d -m a t .s t r -e l ] 15 O c t 1999Pseudogaps in the 2D half-ﬁlled Hubbard modelC.Huscroft,M.Jarrell,Th.Maier,S.Moukouri,A.N.TahvildarzadehDepartment of Physics,University of Cincinnati,Cincinnati,OH 45221-0011(February 1,2008)We study the pseudogaps in the spectra of the half-ﬁlled 2D Hubbard model using both ﬁnite-size and dynamical cluster approximation (DCA)quantum Monte Carlo calculations.A charge pseudogap,accompanied by non-Fermi liquid behavior in the self energy,is shown to persist in the thermodynamic limit.The DCA (ﬁnite-size)method systematically underes-timates (overestimates)the width of the pseudogap.A spin pseudogap is not seen at half-ﬁlling.Introduction For over a decade it has been recognized that the normal state properties of high-T c superconduc-tors are unusual and appear to have non-Fermi liquid characteristics.[1]One of the most remarkable features of the normal state is a suppression of the density of states at the Fermi energy in a temperature regime above T c in underdoped samples.Angular resolved photoemis-sion experiments [2,3]show that this pseudogap in the spectral function has a d-wave anisotropy,the same sym-metry as the superconducting order parameter in these materials.This,along with theories that short-ranged spin ﬂuctuations mediate pairing in the high-T c cuprates [4,5],emphasizes the importance of understanding the normal state,insulating phase.It is thought by many that the two-dimensional Hub-bard model,or closely related models,should capture the essential physics of the high-T c cuprates.[4]Yet,despite years of eﬀort,neither the precursor pseudogap nor d-wave superconducting order have been conclusively seen in the Hubbard model.Intuitively,one may expect that the Hubbard model should show pseudogap behavior.At half-ﬁlling,the ground state of the 2D Hubbard model is an antifer-romagnetic insulator [6,7]and the spectrum is there-fore gapped.However,the Mermin-Wagner theorem pre-cludes any transition at ﬁnite T ,so as the temperature is lowered one may anticipate that a pseudogap will de-velop.[8]This question has been previously addressed in the 2D Hubbard insulator by ﬁnite-size lattice Quantum Monte Carlo (QMC)[9,10]and approximate many-body techniques [11–13].The results have been contradictory and inconclusive as to the existence of a pseudogap at low temperatures,due to limitations of these ing the recently developed Dynamical Cluster Ap-proximation (DCA)[14,15]we ﬁnd that at suﬃciently low temperatures a pseudogap opens in the single par-ticle spectral weight A (k ,ω)of the 2D Hubbard modelwith a simultaneous destruction of the Fermi liquid state due to critical ﬂuctuations above the T =0transition temperature.This occurs in the weak-to-intermediate coupling regime U <W ,where U is the on-site Coulomb energy and W the non-interacting band width.Using ﬁnite-sized techniques,it is diﬃcult to determine if a gap persists in the thermodynamic limit.At half ﬁll-ing,ﬁnite-size QMC calculations display a gap in their spectra as soon as the correlation length exceeds the lat-tice size,so they tend to overestimate the pseudogap as it would appear in the thermodynamic limit.Finite-size scaling is complicated by the lack of an exact scaling ansatz for the gap and the cost of performing simula-tions of large systems.Calculations employing Dynami-cal Mean Field Approximation (DMFA)[16]in the para-magnetic phase do not display this behavior since they take place in the thermodynamic limit rather than on a ﬁnite-size lattice.However,the DMFA lacks the non-local spin ﬂuctuations often believed to be responsible for the pseudogap.The Dynamical Cluster Approxima-tion (DCA)is a fully causal approach which systemati-cally incorporates non-local corrections to the DMFA by mapping the problem onto an embedded impurity clus-ter of size N c .N c determines the order of the approx-imation and provides a systematic expansion parameter 1/N c .While the DCA becomes exact in the limit of large N c it reduces to the DMFA for N c =1.Thus,the DCA diﬀers from the usual ﬁnite size lattice calculations in that it is a reasonable approximation to the lattice prob-lem even for a “cluster”of a single site.Like the DMFA,the DCA solution remains in the thermodynamic limit,but the dynamical correlation length is restricted to the size of the embedded cluster.Thus the DCA tends to underestimate the pseudogap.Method The DCA is based on the assumption that the lattice self energy is weakly momentum dependent.This is equivalent to assuming that the dynamical inter-site correlations have a short spatial range b <∼L/2where L is the linear dimension of the cluster.Then,according to Nyquist’s sampling theorem [17],to reproduce these correlations in the self energy,we only need to sample the reciprocal space at intervals of ∆k ≈2π/L .Therefore,we could approximate G (K +˜k)by G (K )within the cell of size (π/L )D (see,Fig.1)centered on the cluster mo-mentum K (wherever feasible,we suppress the frequency labels)and use this Green function to calculate the self energy.Knowledge of these Green functions on a ﬁner scale in momentum is unnecessary,and may be discarded to reduce the complexity of the problem.Thus the clus-1ter self energy can be constructed from the coarse-grained average of the single-particle Green function within the cell centered on the cluster momenta:¯G(K )≡Ncsa−10−50510ω−3−2−101Σ(π,0,ω)β=1.3β=2.6β=3.0β=4.0β=5.000.10.20.30.4A (π,0,ω)0123ω0.20.4χ’’(ω)/χ(T )ωIm Σ(k,ω)Re Σ(k,ω)FIG. 3.The spectral density A (k ,ω),and the real ReΣ(k ,ω)and imaginary ImΣ(k ,ω)parts of the self-energy for the 2D Hubbard model via the DCA with a paramagnetic host at k =(π,0)for a 64-site cluster (N c =64)at various temperatures.The on-site Coulomb repulsion U =5.2,the band width W =8,and the ﬁlling n =1.As the tem-perature is lowered,the system ﬁrst builds a Fermi-liquid-like peak in A (k ,ω).By β=2.6,a pseudogap begins to develop in A (k ,ω)and simultaneously,ReΣ(k ,ω)develops a positive slope at ω=0,a signal of a non-Fermi liquid.The pseudogap deepens as the temperature is further lowered.The imaginary part of the dynamic spin susceptibility,divided by the static spin susceptibility is shown in the inset.No spin gap is seen.Results We study the 2D Hubbard Hamiltonian:H =−t i,j ,σ(c †iσc jσ+c †jσc iσ)+Ui(n i ↑−12)−µi,σn iσ.(4)where c †iσ(c iσ)creates (destroys)an electron at site i with spin σ,U is the on-site Coulomb potential,and n iσ=c †iσc iσis the number operator.We set the overlap integral t =1and measure all energies in terms of t .We work at µ=0where the system is half-ﬁlled ( n =1).We choose U =5.2,which is well below the value U >∼W believed to be necessary to open a Mott-Hubbard gap.We also calculate the angle integrated dynamical spin susceptibility shown in the inset.It does not have a pseudogap,as expected for the half-ﬁlled model since the spin-wave spectrum is gapless.Since a (spin)charge gap is generally deﬁned as one which appears in the (spin)charge dynamics or thermodynamics,we conclude that the pseudogap is only in the charge response and is due to short-ranged antiferromagnetic spin correlations.Fig.3shows the spectral density A (k ,ω),and the real ReΣ(k ,ω)and imaginary ImΣ(k ,ω)parts of the self-energy for the 2D Hubbard model via the DCA with a paramagnetic host at the Fermi surface X pointk =(π,0)for a 64-site cluster (N c =64)at various temperatures.We obtain the spectral function A (k ,ω)via the Maximum Entropy Method (MEM).[21]As the temperature is lowered,the system ﬁrst builds a Fermi-liquid-like peak in A (k ,ω).By β=2.6,a pseudogap begins to develop in A (k ,ω).The pseudogap builds as the temperature is further lowered.Fig.4shows the spectral function A (k ,ω)at the half-ﬁlled Fermi surface point k =(π/2,π/2).The qualitative features are similar to Fig.3,but the pseudogap opens at a lower temperature and the distance between the peaks is less than that seen at the X point.This behavior is reminiscent of the anisotropy of the pseudogap observed experimentally in the insulating [3]and in the supercon-ducting [5]cuprates,but is not large enough to be compa-rable with that seen experimentally.We speculate that the anisotropy seen here may be due to a diﬀerence in the number of states near the Fermi energy at these two points in the zone.−10−5510ω−3−2−101Σ(π/2,π/2,ω)β=1.3β=2.6β=3.0β=4.0β=5.000.10.20.30.4A (π/2,π/2,ω)Im Σ(k,ω)Re Σ(k,ω)FIG. 4.The spectral density A (k ,ω),and the real ReΣ(k ,ω)and imaginary ImΣ(k ,ω)parts of the self-energy for the 2D Hubbard model via the DCA at k =(π/2,π/2)and the same parameters as Fig.3.Again the system ﬁrst builds a Fermi-liquid-like peak in A (k ,ω)and then develops a pseudogap in A (k ,ω)with a simultaneous non-Fermi liq-uid behavior in ReΣ(k ,ω).Here,though,the pseudogap ﬁrst appears at a lower temperature than at k =(π,0).The DCA self-energy spectra Figs.3&4support the spectral evidence.At the X point,the slope of the real part ReΣ(k ,ω)becomes positive below β=2.6,the tem-perature at which we observed the opening of a pseudo-gap.This signals the appearance of two new solutions in the quasiparticle equation Re(ω−ǫk −Σ(k ,ω))=0in addition to the strongly damped solution at ω=0which is also present in the noninteracting system.These two new quasiparticle solutions for the same k -vector indi-cate precursor eﬀects of the onset of antiferromagnetic ordering which entails a doubling of the unit cell.They3are referred as shadow states and are caused by antiferro-magnetic spin ﬂuctuations in the paramagnetic state.At these temperatures,the imaginary part ImΣ(k ,ω)dis-plays a local minimum at ω=0indicating the break-down of the Fermi liquid behavior.We note that a diﬀer-ent conclusion was previously reached in a FLEX study [11],which found that ImΣ(k ,ω)has a local minimum at ω=0which was not accompanied by an opening of a pseudogap.Since the pseudogap is due to short-range spin correlations,we conclude that FLEX underestimates these correlations.024ω0.10.20.30.40.50.6A (π,0,ω)N c =64N c =36N c =16N c =10246ωN=64N=16246N c1/20.10.20.3T *∆/5.2(a)(b)FIG.5.The spectral density A (k ,ω)at k =(π,0)for the 2D Hubbard model via (a)the DCA and (b)ﬁnite-size Quan-tum Monte Carlo (QMC)at an inverse temperature times the bandwidth βW =40on various size clusters.The tempera-ture T ∗at which the pseudogap ﬁrst becomes apparent in the DCA spectra,as well as the full width ∆measured from peak to peak is plotted in the inset.The ﬁnite-size QMC over-estimates ∆and T ∗,whereas the DCA QMC systematically underestimates them.It is instructive to compare the DCA results with those obtained by ﬁnite-size QMC calculations.Fig.5shows the spectral density A (π,0,ω)obtained by analytically continuing both ﬁnite-size and DCA QMC data.In spite of the diﬀerence in the two methods,the information they provide is complimentary.In the ﬁnite-size results,(b),we see a similar opening of a pseudogap.However,as the length of the antiferromagnetic (AF)correlations reach the longest length on the ﬁnite-size lattice,the system develops a full gap.Thus,the ﬁnite-size QMC overesti-mates the size of the gap.In the DCA results,(a),the pseudogap emerges as soon as N c >1.The temperature T ∗at which the pseudogap ﬁrst becomes apparent in the spectra,as well as the full width ∆measured from peak to peak is plotted in the inset.Both T ∗and ∆increase with N c .Since the DCA calculation remains in the ther-modynamic limit,a full gap due to antiferromagnetic cor-relations alone cannot open until their correlation length diverges.However,since these correlations are restrictedto the size of the cluster,the DCA systematically under-estimates the size of the gap.Thus,if a pseudogap exists in the DCA for ﬁnite N c ,it should persist in the limit as N c →∞.In summary,we have employed the recently developed DCA to study the long-open question of whether the half-ﬁlled Hubbard model has a pseudogap due to AF spin ﬂuctuations.We ﬁnd conclusive evidence of a pseudogap in the charge dynamics and have shown unambiguously that the T =0phase transition of the half-ﬁlled model is preceded by an opening of a pseudogap in A (k F ,ω)accompanied by pronounced non-Fermi liquid behavior in Σ(k F ,ω).Acknowledgments We would like to acknowledge use-ful conversations with P.van Dongen,B.Gyorﬀy,M.Hettler,H.R.Krishnamurthy R.R.P.Singh and J.Za-anen.This work was supported by the National Sci-ence Foundation grants DMR-9704021,DMR-9357199,and the Ohio Supercomputing Center.。

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Bacillus spp.

• Use:
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strains well characterized – Study of gene regulation of the enzymes of
interest
Enhancing
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Commodity chemicals
• Amilases & Proteases • Bacillus:
– Good catalytic properties – High cellular density from low cost carbon and
nitrogen sources – Realise directly to the culture medium (low cost
• enzymes for human food industry (beverage,dairy products, bakery foods)
• enzymes for animal feeds
50-60% of this market are enzymes from Bacillus American, European, Japanese, Chinese companies (Novozymes, Danisco-Genencor, DSM)

法国海岸松树皮提取物碧萝芷对长链游离脂肪酸诱导的巨噬细胞perilipin2基因表达的影响

法国海岸松树皮提取物碧萝芷对长链游离脂肪酸诱导的巨噬细胞perilipin2基因表达的影响范斌;杜强;谷剑秋;张锦【摘要】Abstract Objective To investigate the effect of Pycnogenol on oleic acid-induced perilipin2 expression in macrophages. Methods Realtime PCR and Western blot were performed to detect perilipin2 expression. Transient transfection and luciferase assay were employed to measure perilipin2 promoter activity. Results Oleic acid significantly induced perilipiti2 expression in a dose-and time-dependent manner in macrophages; oleic acid markedly enhanced perilipin2 promoter activity; Pycnogenol significantly suppressed oleic acid-induced perihpin2 expression and promoter activity. Conclusion For the first time,we demonstrated that Pycnogenol significantly suppressed oleic acid-induced perilipin2 expression and promoter activity.%目的研究碧萝芷(PYC)对油酸诱导的巨噬细胞perilipin2表达的影响及其相关分子机制.方法应用Real-time PCR 和Western blot测定油酸及PYC对perilipin2 mRNA和蛋白水平表达影响.应用荧光素酶活性分析方法检测油酸及PYC时perilipin2启动子活性的影响.结果油酸以剂量和浓度依赖方式上调perilipin2 mRNA和蛋白水平表达,并促进perilipin2启动子活性.PYC以剂量依赖方式抑制了油酸诱导的perilipin2表达及启动子活性.结论PYC抑制了巨噬细胞中油酸诱导的perilipin2的表达.PYC通过抑制perilipin2启动子活性,从而直接抑制perilipin2的表达.【期刊名称】《中国医科大学学报》【年(卷),期】2011(040)007【总页数】5页(P611-615)【关键词】长链游离脂肪酸;泡沫细胞;碧萝芷;PAT家族蛋白【作者】范斌;杜强;谷剑秋;张锦【作者单位】中国医科大学附属盛京医院神经内科,沈阳110004;中国医科大学附属盛京医院内分泌科,沈阳110004;中国医科大学附属第一医院内分泌科,沈阳110001;中国医科大学附属第一医院内分泌科,沈阳110001【正文语种】中文【中图分类】R363单核细胞通过内皮细胞的间隙移入内膜下并分化成巨噬细胞，巨噬细胞吞噬体内过剩的脂质后变成泡沫细胞，泡沫细胞聚集并形成斑块是动脉粥样硬化发生、发展的主要病理基础［1，2］。

金线莲多糖抗衰老作用及其机制

疗组为阳性对照．６周后对小鼠进行行为学测试，８周后检测小鼠的大脑皮层ｐ?ＮＦ?κＢｐ６５表达，以及大脑皮
层过氧化氢酶、超氧化物歧化酶及血清总抗氧化能力活性和丙二醛浓度，并检测小鼠腹腔巨噬细胞的吞噬情
况．结果表明：与衰老小鼠相比较，经金线莲多糖治疗后，小鼠的抗氧化酶活性显著提高，其大脑皮层ｐ?ＮＦ?κＢ
ｐ６５表达下调，且运动、空间探索和学习记忆能力明显改善；同时，金线莲多糖能增强衰老小鼠清除抗原的能力；金线莲多糖抗衰老作用与其抗氧化、抑制ＮＦ?κＢ信号通路、增强衰老小鼠免疫能力有关．
关键词：金线莲多糖；ＮＦ?κＢ信号通路；抗氧化；认知能力；抗衰老
与衰老模型组小鼠相比经不同剂量金线莲多糖和ve治疗后小鼠的运动时间运动速度和站立次数均明显增加犘001犘005说明金线莲多糖和ve能使衰老小鼠的运动空间探索学习和记忆能力有明显改善
第４１卷第１期
２０２０年１月
华侨大学学报（自然科学版）ＪｏｕｒｎａｌｏｆＨｕａｑｉａｏＵｎｉｖｅｒｓｉｔｙ（ＮａｔｕｒａｌＳｃｉｅｎｃｅ）
衰老是生物体随着年龄增长在形态结构和生理功能方面出现的一系列退行性变化，是正常且自然
收稿日期：２０１９?０７?２２
通信作者：刘青（１９７０?），女，副教授，博士，主要从事天然药物的提取及活性的研究．Ｅ?ｍａｉｌ：ｙｕｎｌｉｕ＠１２６．ｃｏｍ．基金项目：福建省自然科学基金资助项目（２０１３Ｊ０１３３７）

Evolution of ATP-binding cassette transporter genes

AraG RbsA MalK UgpC KpsT
ABC gene
Transported substrate
Arabinose Ri bose Maltose G3P Polysaccharides
believed to transport tryptophan and a White-Brown dimer guanine [12]. The sterile 6 (STE6) gene encodes an ABC protein that extrudes the yeast mating factor, a modified oligopeptide [13]. STE6 is similar in organization and in amino acid sequence to the P-glycoproteins (see below). Characterization o f ABC gene sequences from the expressed sequence tag (EST) database suggests that there are at least 25 human ABC genes (our unpublished data). The organization of ABC genes in eukaryotes also varies significantly. The most common combination consists of four domains in succession, T M - A T P - T M - A T E H o w ever, there are examples o f A T P - T M - A T P - T M (PDR5), T M - A T P (ALD, PMP70, TAP, MDLI, MDL2, ATM1, PXA) and ATP-ATP (OAB) genes (Fig. lb). The evolutionary history and functional significance of this diversity in gene organization remains unclear. As for the bacterial genes, the functional protein always appears to contain two ATP and two transmembrane domains, the only described exception being OAB, which binds to 2'-5' oligoadenylate as part of the ribonuclease L complex [14]. Other ABC genes that have functions other than transport will undoubtedly be described.

Heritable epigenetic mutation of a transposon-flanked Arabidopsis gene due to lack of the chromatin

Heritable epigenetic mutation of a transposon-ﬂanked Arabidopsis gene due to lack of thechromatin-remodeling factor DDM1Hidetoshi Saze*and Tetsuji KakutaniDepartment of Integrated Genetics,National Institute of Genetics,Mishima,Shizuoka,JapanEpigenetically silent transposons and repeats constitute a substantial proportion of eukaryotic genomes,but their impact on cellular gene function remains largely unex-plored.In Arabidopsis ,transposons are silenced by DNA methylation,and this methylation is often abolished by mutations in a chromatin-remodeling gene DDM1(DECREASE IN DNA METHYLA TION 1).The ddm1muta-tion induces various types of developmental abnormalities through de-repression of transposons and repeats.Here,we report a novel mechanism for a ddm1-induced syn-drome,called bonsai (bns ).We identiﬁed the gene respon-sible for the bns phenotypes by genetic linkage analysis and subsequent transcriptional analysis.The bns pheno-types are due to silencing of a putative Anaphase-Promoting Complex (APC)13gene.The BNS gene silencing was associated with DNA hypermethylation,which is in contrast to the ddm1-induced hypomethylation in the other genomic regions.This paradoxical BNS hy-permethylation was reproducibly induced during self-pol-lination of the ddm1mutant,and it was mediated by a long interspersed nuclear element (LINE)retrotransposon ﬂanking the BNS gene.We discuss possible molecular mechanisms and the evolutionary implications of transpo-son-mediated epigenetic changes in the BNS locus.The EMBO Journal (2007)26,3641–3652.doi:10.1038/sj.emboj.7601788;Published online 12July 2007Subject Categories :chromatin &transcription;plant biology Keywords :anaphase promoting complex;BONSAI;epigenetic inheritance;heterochromatin;small RNAIntroductionMethylation of cytosine is a heritable epigenetic mark in-volved in several important biological processes,including genomic imprinting and transposon silencing (Jaenisch and Bird,2003;Rangwala and Richards,2004;Chan et al ,2005;Zilberman and Henikoff,2005).Transposons are methylated in diverse organisms,and loss of cytosine methylation leads to activation of transposons (Y oder et al ,1997;Walsh et al ,1998;Miura et al ,2001;Singer et al ,2001;Kato et al ,2003;Selker et al ,2003).Genome-wide mapping of DNA methyla-tion in the ﬂowering plant Arabidopsis demonstrated that a majority of cytosine methylation is concentrated in hetero-chromatic regions,where transposons and repetitive se-quences accumulate (Lippman et al ,2004;Zhang et al ,2006;Zilberman et al ,2007).Unexpectedly,however,recent high-resolution mapping studies revealed that B 20–30%of expressed genes have methylation within their transcribed regions,although the methylation level is generally lower than that in transposons (Zhang et al ,2006;Zilberman et al ,2007).Interestingly,the proportion of those methylated genes increases toward heterochromatic pericentromeric regions,possibly reﬂecting direct or indirect interaction(s)of epige-netic states between euchromatic genes and heterochromatic sequences (Zilberman et al ,2007).In the large genomes of plants and vertebrates,transposons and repeats are also scattered among and within genes.However,the impact of such local heterochromatin on activity of cellular genes remained largely unexplored.The impact of epigenetic changes on transposon activity can be directly examined using Arabidopsis mutants with defective genomic DNA methylation.In plants,cytosine methylation is found in both CG and non-CG contexts.In Arabidopsis ,methylation at CG sites is maintained by DNA methyltransferase MET1,an ortholog of Dnmt1in mammals,while methylation at non-CG sites depends on DNA methyl-transferase genes,CMT3and DRM2(Finnegan et al ,1996;Ronemus et al ,1996;Bartee et al ,2001;Lindroth et al ,2001;Cao et al ,2003;Kankel et al ,2003).Another gene involved in maintenance of methylation and silencing of heterochroma-tin loci is a chromatin-remodeling ATPase gene DDM1(DECREASE IN DNA METHYLA TION 1),which is involved in both CG and non-CG methylation (Vongs et al ,1993;Jeddeloh et al ,1998).In addition,chromatin and RNAi components involved in de novo DNA methylation have recently been identiﬁed using several reporter transgene systems (Aufsatz et al ,2002;Kanno et al ,2004,2005;Chan et al ,2005;Herr et al ,2005;Onodera et al ,2005;Pontier et al ,2005;Pontes et al ,2006).Notably,many of the putative endogenous targets of this pathway are located near transpo-son sequences,which might epigenetically regulate adjacent genes (Huettel et al ,2006).Several examples of developmental variants were recov-ered in both met1and ddm1mutant lines (Finnegan et al 1996;Kakutani et al ,1996,2004;Ronemus et al ,1996;Kankel et al ,2003;Saze et al ,2003).Genetic analysis of some of these ddm1-induced developmental variants revealed that each of the abnormalities is due to a heritable change in a locus other than DDM1(Kakutani et al ,1996).For example,a ddm1-induced dwarf phenotype named bal is produced by the overexpression of a cluster of disease resistance genes (Stokes et al ,2002).Another ddm1-induced developmental variation,characterized by a delay in ﬂowering onset,is dueReceived:28April 2007;accepted:13June 2007;published online:12July 2007*Corresponding author.Department of Integrated Genetics,National Institute of Genetics,Y ata 1111,Mishima,Shizuoka 411-8540,Japan.T el.:þ81559816805;Fax:þ81559816804;E-mail:hsaze@lab.nig.ac.jpThe EMBO Journal (2007)26,3641–3652|&2007European Molecular Biology Organization |All Rights Reserved 0261-4189/07to ectopic expression of the imprinted homeobox gene FW A (Kakutani,1997;Soppe et al ,2000;Kinoshita et al ,2004).Although these abnormalities behave as dominant traits,some of the ddm1-induced abnormalities behave as heritable recessive traits,suggesting that a different mechanism is responsible (Kakutani et al ,2004).Here,we report the identiﬁcation of the target gene of a ddm1-induced loss-of-function epigenetic abnormality called bns (Kakutani,1997;Kakutani et al ,2004).The loss of BONSAI gene function was due to gene silencing associated with DNA hypermethylation and small RNA accumulation.The de novo methylation of the BONSAI gene was induced reproducibly in independent ddm1mutant lines.This ectopic methylation depends on the presence of a long interspersed nuclear element (LINE)retrotransposon insertion within the 30non-coding region.The LINE insertion,which is found in the majority of natural accessions,generates a potential trigger for epigenetic variation with strong developmental effects.ResultsRepeated self-pollination of a ddm1mutant induced a combination of phenotypes named bnsRepeated self-pollination of the DNA hypomethylation mu-tant ddm1results in a variety of developmental abnormalities (Kakutani et al ,1996).Genetic analyses of some of the phenotypes have revealed that they are caused by gain-of-function alleles,which reﬂect overexpression of the respon-sible genes (Soppe et al ,2000;Stokes et al ,2002).However,not all of the developmental abnormalities are gain-of-func-tion alleles.An example is a ddm1-induced developmental syndrome that we named bns .The bns phenotypes were characterized by short,compact inﬂorescence,resulting in reduced plant height (Figure 1A and B).The bns variant showed disrupted phyllotaxis,re-duced apical dominance and production of clusters of bracts and ﬂowers at the apex of the inﬂorescence (Figure 1C and D).These phenotypes seem to reﬂect the inhibition of inter-node elongation and the termination of shoot growth at the apical meristems (Figure 1;Kakutani,1997;Kakutani et al ,2004).After backcrossing to the parental wild-type (WT)Columbia (Col),the bns phenotype was not detectable in the F 1population,suggesting that the abnormal phenotypes are not due to a gain-of-function mutation.In the self-pollinated progeny of an F 1plant,we recovered F 2plants showing the bns phenotype.The phenotypic plants included both ddm1/ddm1and DDM1/-genotypes.This observation suggests that the bns phenotypes are produced by a heritable change in a locus (or loci)other than DDM1.Identiﬁcation of the BNS geneT o further understand the basis of the heritable bns pheno-types,we examined their inheritance in the F 2progeny from a cross of a ddm1plant with bns phenotypes (Col)to a WT Landsberg erecta (L er )plant.The genotype was determined for 531F 2plants with clear bns phenotypes,which comprised about 10%of the F 2population.Characterization of Col/L er polymorphisms throughout the genome revealed that all of the phenotypic plants were homozygous for the Col haplo-type in one locus in the bottom arm of chromosome 1,suggesting that a loss-of-function allele in this locus is responsible for the bns trait.This locus was narrowed to an interval between genetic markers NGA111and BW54(ﬁve recombinants and two recombinants,respectively,out of the 1062chromosomes examined).We compared the transcript levels of 54predicted genes in this genetically deﬁned BNS region between WT and bns plants (backcrossed to DDM1/DDM1),using a reverse transcription (RT)–PCR assay.We found that one gene (AT1G73177)showed a severe reduction in its expression in bns DDM1compared to WT plants (Figure 2A).The AT1G73177transcript was also reduced in the self-pollinated ddm1plants with the bns phenotypes (data not shown).The identiﬁed gene consists of four exons and encodes a predicted 63-amino acid (aa)protein (Figure 2B),and this annotation is supported by the presence of full-length cDNA in nucleotide sequence databases (GenBank:A Y088589).A truncated non-LTR-type retrotran-sposon (LINE,long interspersed nuclear elements)sequence (AT1G73175)was found in the 30UTR in the WT Col genome (Figure 2B and see below).T wo ﬂanking genes,AT1G73170and AT1G73180,did not show a detectable reduction in their transcript level in bns plants (Figure 2A).T o test whether the bns phenotypes are due to the repres-sion of AT1G73177,this gene was knocked down by RNAi in WT Col plants by transformation with a transgenic construct producing double-stranded RNA (dsRNA)of the gene se-quence (Figure 2C).The transgenic lines showed the bns -like phenotypes (i.e.,reduced plant height and clustered ﬂowers)associated with a reduction in AT1G73177transcript abundance (Figure 2C and D).In addition,we analyzed the effect of a T -DNA insertion in the upstream non-coding region in the ﬁrst exon (SALK_027397).In the insertion mutant,a transcript was still detectable by RT–PCR,but the levelwasFigure 1The bns phenotypes.(A )WT Col plants (two on the left)and bns plants in a DDM1/DDM1background (two on the right;hereafter referred to as bns ).Both are six weeks old.(B )A close-up image of bns .(C )Inﬂorescences of bns .(D )A cluster of ﬂowers produced in bns .Epigenetic mutation of a transposon-ﬂanked gene H Saze and T Kakutaniless than that observed in WT plants(Figure2B and C).The plants homozygous for the T-DNA insertion showed similar phenotypes,although they were much milder(Figure2D), further conﬁrming that the reduction in AT1G73177transcript induces the bns phenotypes.From these results,together with the recessive nature of the bns mutation,we concluded that the loss or reduction in AT1G73177function is most likely to be responsible for the bns phenotypes.The BNS gene product has similarity to a subunit of the Anaphase-Promoting Complex/Cyclosome(APC/C) The predicted BNS protein has a high similarity to the mammalian Swm1/Apc13,a subunit of Anaphase-Promoting Complex/Cyclosome(APC/C)(Figure2E).The APC/C is a large ubiquitin–protein ligase complex that reg-ulates cell cycle progression in eukaryotic cells(Castro et al, 2005).Swm1/Apc13was originally identiﬁed for its role in spore wall assembly in Saccharomyces cerevisiae(Ufano et al, 1999),and was later found to be a core subunit of the APC/C (Y oon et al,2002;Hall et al,2003).The protein is evolution-arily conserved in a wide range of organisms(Schwickart et al,2004)(Figure2E).We detected BNS expression in all tissues examined in WT plants(Supplementary Figure1). bns is an epigenetic mutation associated with DNA hypermethylationDespite the marked reduction in BNS expression in the bns line,the nucleotide sequence of the BNS gene in the bnslineAT1G73170AT1G73177AT1G73180ACT2ACT2 (RT−)WT bns ddm1AT1G73170AT1G73177AT1G73180ACT2ACT2 (RT−)WT bns RNAi T-DNAMDS----EVQRDGRILDLIDDAWREDKLPYEDVAI-----PLNELPEP--EQDNG---GTMDS----EVQRDGRILDLIDDAWREDKLPYEDVAI-----PLSELPEP--EQDNG---GTMGGVEQEQLLSLGVLIDIVDEQWMRDTLPADDVPV-----PPAMAVKT--EEAEDPAPANMSGLELELGLSLGVLIDVVDEQWMRDTLPADDIPV-----PPAMAVKT--EDAEDPAPANMA------EVSLGMLIDIVDEEWMRDTLPDDDLPL-----PPVLAVKT--DDTEE---TNMA------ELSLGILIDIVDEEWMRDTLPDDDLPL-----PPTLVVRT--DDTED---SNMDSNYNYVHMNKPGVV-LFASDWLKDRLPVDDVEVRVEHLPPVTEDEMTIQHSSANLILM*:::..*.***:*::*.:..TESVKEQEMKWTDLALQYLHENVPPIGN74TESVKEQEMKWTDLALQGLHENVPPAGN74QESQPAQGDVWRDFTLENL---------72QESQPAQGDVWRDFALENL---------72QETQQADAETWRDLALDTQ---------63QETQQVNLDAWHDLAFGQE---------63KNKQLRHEPAWKDLELEDLVNAFAFIQ-86:..**:: Hs_APC13MmOsZmBNSGmSp_APC13Figure2Identiﬁcation of the BNS gene.(A)RT–PCR for the BNS gene(AT1G73177)and neighboring genes(AT1G73170and AT1G73180).T otal RNA isolated from wild-type Col(WT),bns(backcrossed to DDM1)and ddm1plants(before repeated self-pollination)was used.The BNS transcript was also reduced in ddm1lines after repeated self-pollination(not shown).Actin2(ACT2)was used as a control.(B)A schematic representation of the BNS locus.Boxes represent exons(coding sequences in black and UTRs in white for BNS and the neighboring genes,and in gray for the LINE sequence).Black arrows indicate the annotated transcription start sites and transcript orientation(). Horizontal white arrowheads represent the target site duplications of the LINE insertion.The position of the T-DNA insertion in theﬁrst exon of BNS in SALK_027397line is also indicated.The positions of primer pair,F2and R3,used for RT–PCR of AT1G73177,are also shown.(C)Knockdown of BNS transcripts in the RNAi lines and in the T-DNA insertion line.RT–PCR was performed with total RNA from wild-type Col (WT),bns,transgenic plants expressing dsRNA of BNS gene(RNAi)and SALK_027397line homozygous for the T-DNA insertion(T-DNA).(D)Phenotypes of a BNS RNAi line(left panel),and an inﬂorescence in a plant homozygous for the T-DNA insertion(right panel).(E)Multiple aa sequence alignment of BNS(Arabidopsis thaliana;AT1G73177)and APC13homologs in Homo sapiens(Hs,NP_056206),Mus musculus (Mm,NP_852059),Oryza sativa(Os,NP_001060376),Zea mays(Zm,A Y105005),Glycine max(Gm,CX701269)and Schizosaccharomyces pombe(Sp,NP_595754).The sequences were aligned using the ClustalW program that highlights the identical and conserved aa with asterisks and dots,respectively.Epigenetic mutation of a transposon-ﬂanked geneH Saze and T Kakutaniwas identical to that in the WT progenitor strain,Col(from À825toþ946;data not shown).These results suggested that the silencing of the BNS gene has an epigenetic basis.We therefore examined the level of DNA methylation in this region.T o detect DNA methylation,we used the following two methods:digestibility by methylation-sensitive restriction enzymes and bisulﬁte genomic sequencing.Bisulﬁte genomic sequencing revealed that the BNS gene in the WT Col genome was almost free of DNA methylation(WT Col in Figure3A). On the other hand,theﬂanking LINE sequence was heavily methylated,especially at CG sites(Figure3A and B).In the bns line,the BNS gene region was also heavily methylated (bottom diagram in Figure3A),which is in contrast to the situation in WT Col plants.The hypermethylation at the BNS locus was found at both CG and non-CG sites.The methylation status of the BNS region was conﬁrmed by digestion with methylation-sensitive restriction enzymes and subsequent PCR.WT Col samples did not show a PCR signal,reﬂecting the complete digestion of the genomic DNA (left panel in Figure3C).Samples derived from bns plants showed bands reﬂecting incomplete digestion due to methy-lation.These results are consistent with the results of the bisulﬁte sequencing.Repeated self-pollination of ddm1mutant reproducibly induced de novo DNA methylation in the BNS gene The hypermethylation in the BNS gene contrasts with the global DNA hypomethylation induced by the ddm1.We examined whether this paradoxical DNA hypermethylation in the BNS locus reﬂected one single purely stochastic event, or BNS hypermethylation could be reproducibly induced in a ddm1mutant background.In order to see the initial effect of the ddm1mutation, ddm1homozygotes were selected from progeny derived by self-pollination of a DDM1/ddm1heterozygote.This DDM1/ ddm1parent was generated by backcrossing a ddm1mutant six times to WT Col parent,in order to remove heritable effects from the original ddm1/ddm1mutant(Kakutani et al, 1996,1999).ddm1/ddm1plants segregated in the self-polli-nated progeny of the backcrossed DDM1/ddm1parent did not show signs of BNS gene hypermethylation(ddm1(1stG) in Figure3).In order to see the effect of repeated self-pollination of the ddm1mutant,seven ddm1homozygotes in the segregating family were independently self-pollinated seven times.The hypermethylation of the BNS gene was detected in all seven independent ddm1lines(ddm1(8thG) in Figure3).The ddm1mutation reproducibly induced BNS methylation,but this process was slow and required multiple generations.As a control,BNS methylation was also exam-ined in four DDM1/DDM1sibling lines segregated from the same DDM1/ddm1parent and self-pollinated seven times in parallel(DDM1(8thG)in Figure3).BNS methylation was not detected in any of the four DDM1control lines.The lack of methylation in the DDM1sibling families further conﬁrmed that the ddm1mutation was responsible for the de novo methylation of the BNS gene.Theﬂanking LINE sequence showed a reduction in DNA methylation in the self-pollinated ddm1plants(ddm1(8thG) in Figure3A and B).This result is consistent with the previous observations that DDM1activity is required for the maintenance of DNA methylation and silencing of endogen-ous transposons(Miura et al,2001;Singer et al,2001; Lippman et al,2004).The hypomethylation of the LINE was found only after repeated self-pollination,which is similar to the situation for the SINE-related sequence in the FW A promoter,which remains methylated in the initial gen-erations of ddm1inbreeding,but loses methylation stochas-tically in subsequent inbred generations(Soppe et al,2000). Interestingly,the LINE sequence was methylated to the WT level in the bns mutant line backcrossed into a DDM1/DDM1 background(Figure3A and B),suggesting de novo methyla-tion in the DDM1background.The hypermethylation and silencing of BNS gene is associated with small RNAsEpigenetic silencing of transposons and repeats are fre-quently associated with the production of small RNAs, which could be involved in RNA-directed DNA methylation (Zilberman et al,2003;Chan et al,2004;Matzke and Birchler, 2005).Because the BNS gene was methylated de novo in a ddm1background,we examined small RNAs corresponding to this region.As shown in Figure4,BNS gene silencing was associated with the accumulation of small RNA in the size of 24–25nt,the length of small RNA species often detected for heterochromatic sequences(Hamilton et al,2002;Xie et al, 2004;Henderson et al,2006;Pontes et al,2006).Small RNAs (24–25nt)were also detected in the ddm1mutant sample.A hybridization probe covering the30region of the BNS gene near the boundary with the LINE(BNS30probe)detected a weak but signiﬁcant signal in the WT Col sample(Figure4B and C),although the signal increased in the sample carrying a silent BNS allele.As is the case for many other silent transposons,small RNA was also detected for the LINE sequence(Figure4B)in the WT Col sample.Interestingly,the amount of the small RNA for this family of LINE increased in the ddm1and bns plants.The increase in the small RNA signal might mediate the de novo methylation of this LINE element,and that may explain the partial methylation of the LINE in the ddm1 mutant,and the de novo methylation of the element after introduction into a background with a WT DDM1allele (Figure3A and B).The LINE insertion was found at the BNS locus in majority of Arabidopsis natural accessionsThe LINE(AT1G73175)at the BNS locus belongs to a pre-viously uncharacterized subfamily of LINE sequences in Arabidopsis(Wright et al,1996;Noma et al,2000,2001). The presence of16-bp target site duplication(TSD)followed by a9-bp poly(A)sequence proximal to BNS indicates that the LINE sequence is inserted in a tail-to-tail orientation relative to the BNS gene(Figure3A).In WT Col,BNS mRNA extends into the LINE sequence over the TSD and poly(A)sequences(Supplementary Figure2).The Col gen-ome contains two other members of this LINE subfamily, which share more than97%nucleotide sequence identity (AT1G17390and AT5G36935;Figure5A).The copy on chro-mosome5(AT5G36935)is likely to be the full-length copy (Figure5A).This copy encodes three open reading frames, with a structure similar to A TLN-L class LINEs(Noma et al, 2001).The presence of these three copies in the Col genome was conﬁrmed by Southern blot analysis(Supplementary Figure3).Epigenetic mutation of a transposon-ﬂanked gene H Saze and T KakutaniCpGCpNpG 100 bpAsymmetricLINE0.750.50.25(1stG)(8thG)(8thG)(DDM1)(1stG)(8thG)(8thG)(DDM1)(1stG)(8thG)(8thG)(DDM1)ddm1(1stG)ddm1(8thG)DDM1(8thG)bns UndigestSau 3AI Bg /II ***Col*Figure 3DNA methylation pattern in the BNS locus.(A )Schematic representations of the BNS locus and cytosine methylation level analyzed by bisulﬁte sequencing.After treatment with bisulﬁte,DNA fragments were ampliﬁed using four pairs of primers separately (the positions indicated as short horizontal black bars),and cloned for sequencing (12clones for each ampliﬁed region).The percentage of methylated cytosine is indicated by vertical bars (black,CG;blue,CNG;red,asymmetric cytosine).Boxes below represent exons (coding sequences in black and UTRs in white for BNS ,and in gray for the LINE sequence).(B )Proportion of methylated cytosines in the BNS locus,which is based on the results shown in panel A.(C )Methylation of the BNS region detected by restriction digestion.Genomic DNA was digested by methylation-sensitive restriction enzymes Bgl II (50-AGATCT -30)or Sau 3AI (50-GATC-30)(),and was subsequently used as template for PCR ampliﬁcation.The positions of the restriction sites and primers used for the PCR are indicated in the bottom of panel A;primer pairs F2þR3and F3þR4were used after Bgl II and Sau 3AI digestion,respectively.Asterisks (*)indicate the samples used for bisulﬁte sequencing in panels A and B.Epigenetic mutation of a transposon-ﬂanked geneH Saze and T KakutaniIn order to evaluate the impact of the LINE insertion in natural populations,we examined the presence of the LINE insertion at the BNS locus in 96natural accessions of Arabidopsis thaliana .Among them,83accessions have the LINE insertion in the BNS 30UTR,while 13did not have the insertion (Figure 5B).This was conﬁrmed by Southern ana-lysis of the 96natural accessions (data not shown).Among the 13accessions without the LINE insertion,ﬁve contain sequences almost identical to Col apart from the LINE inser-tion (Figure 5C).In those accessions,the TSD sequence remained intact,suggesting that these are ancestral alleles before the LINE insertion.Presence of the LINE in the majority of natural accessions suggests that the LINE inser-tion per se does not have deleterious effects in natural populations.Dependence of the BNS hypermethylation on the ﬂanking LINE sequenceUsing Cvi,which does not have the LINE insertion at the BNS locus (Figure 5C),we tested whether the LINE sequence is necessary for the ddm1-induced de novo methylation at the BNS locus.WT Cvi was crossed to a ddm1heterozygote,which had already been backcrossed six times in the hetero-zygous state (Kakutani et al ,1996).A DDM1/ddm1hetero-zygote originating from this cross was self-pollinated,and from the progeny,we selected ddm1homozygotes with the BNS allele from genome of Col (BNS LINE /BNS LINE ;homozy-gous for the LINE insertion)or Cvi (BNS À/BNS À;without the LINE insertion)(Figure 6A).After three rounds of self-polli-nation,DNA methylation of the BNS locus in these ddm1plants was examined using methylation-sensitive restriction enzymes.All of seven independent ddm1lines homozygous for the BNS LINE allele (from Col)showed de novo DNA methylation of the BNS locus,whereas none of the ﬁve ddm1lines homozygous for the BNS Àallele (from Cvi)showed ectopic DNA methylation (Figure 6B).This result suggests that ddm1-induced de novo methylation at the BNS gene depends on the presence of the LINE insertion in the 30UTR.DiscussionMechanism for BNS gene hypermethylation triggered by the ddm1mutationHere,we report the identiﬁcation and characterization of a loss-of-function epigenetic developmental abnormality bns .The most striking feature of the bns trait is that the local hypermethylation of the BNS gene was induced in a back-ground of global DNA hypomethylation.The hypermethyla-tion of the BNS gene was not evident in newly segregated ddm1homozygous plants (1stG in Figure 3),but it was reproducibly induced in the self-pollinated progeny of ddm1mutants (8thG in Figure 3).These observations suggest that BNS hypermethylation may be due to an indirect effect of the globally hypomethylated ddm1background.Similar observa-tions have been previously reported for SUPERMAN (SUP )and AGAMOUS (AG )sequences;these sequences are stochas-tically hypermethylated in the absence of DDM1or MET1activity (Jacobsen and Meyerowitz,1997;Jacobsen et al ,2000).In both SUP and AG ,pyrimidine-rich sequences such as CT dinucleotide repeats are found in the hypermethylated target sequences,and the possible involvement of this sim-ple-sequence motif has been proposed (Jacobsen et al ,2000).However,a pyrimidine-rich sequence was not found in the BNS locus,suggesting that it is not the basis for BNS hyper-methylation (data not shown).Instead,our results suggest that BNS hypermethylation is mediated by the pre-existing LINE insertion in non-coding region of the BNS gene (Figure 6).The ectopic hypermethylation at BNS in ddm1background occurred in a spreading manner from the LINE into the BNS region (Figure 3A).Spread of heterochromatin into genic regions is also known in position-effect-variegation in Drosophila (Talbert and Henikoff,2006)and telomeric silen-cing in budding yeast (Grunstein,1997).Although the BNS locus resides in a euchromatic chromosomal arm,the dense DNA methylation on the LINE sequence at this locus suggests that the LINE sequence can function as local heterochroma-tin,which is maintained without affecting adjacent genes in the WT background.The DDM1gene is necessary for the maintenance of the heterochromatic characteristics of LINE and other transposons (Gendrel et al ,2002;Lippman et al ,2003,2004).DDM1might also be necessary to deﬁne a heterochromatin boundary (Figure 7A).In mammals,the chromatin insulator CTCF has a barrier function that blocks the extension of heterochromatin.The CTCF-dependent in-sulator activity was abolished by loss of an SNF2-like chro-modomain helicase/ATPase protein,leading to a decrease in euchromatic histone modiﬁcations and DNA hypermethyla-tion around the boundary sequences (Ishihara et al ,2006).Interestingly,the spreading of DNA methylation was not found in the other side (opposite from the BNS gene)of the LINE sequence.On that side,the expression of the gene AT1G73170,which has the transcription start site approxi-mately 150bp away from the TSD of the LINE,was not affected in bns and self-pollinated ddm1lines (Figure 2A and B and data not shown).These results suggest thattheAT1G73177(BNS )Colbns ddm1100 bp24-nt 21-nt 24-nt 21-nt 24-nt 21-nt24-nt 21-nt BNS middle probe EtBrLINE probe miR171BNS 3′probe (LNA)Col CviBNS3′probe (LNA)EtBrprobeprobeprobe (LNA)Figure 4Small RNA northern analysis of the BNS locus.(A )The positions of three hybridization probes used are indicated.(B )Small RNA was examined in WT Col,bns in DDM1background and self-pollinated ddm1plants with reduced BNS expression.The same membrane was used for hybridization with each of the three probes and for the control miR171probe.Ethidium bromide staining of the major RNA is shown as a control (EtBr).(C )WT Col and Cvi samples on a different membrane.Epigenetic mutation of a transposon-ﬂanked gene H Saze and T Kakutani。

光谱法研究药物小分子与蛋白质大分子的相互作用的英文

Spectroscopic Study of the Interaction between Small Molecules and Large Proteins1. IntroductionThe study of drug-protein interactions is of great importance in drug discovery and development. Understanding how small molecules interact with proteins at the molecular level is crucial for the design of new and more effective drugs. Spectroscopic techniques have proven to be valuable tools in the investigation of these interactions, providing det本人led information about the binding affinity, mode of binding, and structural changes that occur upon binding.2. Spectroscopic Techniques2.1. Fluorescence SpectroscopyFluorescence spectroscopy is widely used in the study of drug-protein interactions due to its high sensitivity and selectivity. By monitoring the changes in the fluorescence emission of either the drug or the protein upon binding, valuable information about the binding affinity and the binding site can be obt本人ned. Additionally, fluorescence quenching studies can provide insights into the proximity and accessibility of specific amino acid residues in the protein's binding site.2.2. UV-Visible SpectroscopyUV-Visible spectroscopy is another powerful tool for the investigation of drug-protein interactions. This technique can be used to monitor changes in the absorption spectra of either the drug or the protein upon binding, providing information about the binding affinity and the stoichiometry of the interaction. Moreover, UV-Visible spectroscopy can be used to study the conformational changes that occur in the protein upon binding to the drug.2.3. Circular Dichroism SpectroscopyCircular dichroism spectroscopy is widely used to investigate the secondary structure of proteins and to monitor conformational changes upon ligand binding. By analyzing the changes in the CD spectra of the protein in the presence of the drug, valuable information about the structural changes induced by the binding can be obt本人ned.2.4. Nuclear Magnetic Resonance SpectroscopyNMR spectroscopy is a powerful technique for the investigation of drug-protein interactions at the atomic level. By analyzing the chemical shifts and the NOE signals of the protein in thepresence of the drug, det本人led information about the binding site and the mode of binding can be obt本人ned. Additionally, NMR can provide insights into the dynamics of the protein upon binding to the drug.3. Applications3.1. Drug DiscoverySpectroscopic studies of drug-protein interactions play a crucial role in drug discovery, providing valuable information about the binding affinity, selectivity, and mode of action of potential drug candidates. By understanding how small molecules interact with their target proteins, researchers can design more potent and specific drugs with fewer side effects.3.2. Protein EngineeringSpectroscopic techniques can also be used to study the effects of mutations and modifications on the binding affinity and specificity of proteins. By analyzing the binding of small molecules to wild-type and mutant proteins, valuable insights into the structure-function relationship of proteins can be obt本人ned.3.3. Biophysical StudiesSpectroscopic studies of drug-protein interactions are also valuable for the characterization of protein-ligandplexes, providing insights into the thermodynamics and kinetics of the binding process. Additionally, these studies can be used to investigate the effects of environmental factors, such as pH, temperature, and ionic strength, on the stability and binding affinity of theplexes.4. Challenges and Future DirectionsWhile spectroscopic techniques have greatly contributed to our understanding of drug-protein interactions, there are still challenges that need to be addressed. For instance, the study of membrane proteins and protein-protein interactions using spectroscopic techniques rem本人ns challenging due to theplexity and heterogeneity of these systems. Additionally, the development of new spectroscopic methods and the integration of spectroscopy with other biophysical andputational approaches will further advance our understanding of drug-protein interactions.In conclusion, spectroscopic studies of drug-protein interactions have greatly contributed to our understanding of how small molecules interact with proteins at the molecular level. Byproviding det本人led information about the binding affinity, mode of binding, and structural changes that occur upon binding, spectroscopic techniques have be valuable tools in drug discovery, protein engineering, and biophysical studies. As technology continues to advance, spectroscopy will play an increasingly important role in the study of drug-protein interactions, leading to the development of more effective and targeted therapeutics.。

赝势的介绍

l
Vl(r)|l l|.
Traditionally PPs are split into a local part, long-ranged and behaving like −Zv e2/r for r → ∞, and a short-ranged semilocal term: ˆ ps = V ˆloc + V ˆSL, V ˆloc ≡ Vloc(r), V ˆSL ≡ V
2 d −2π (rφ(r)) d
d ln φ(r) dr
rc
= 4π
rc 0
|φ(r)|2r2dr
valid for any regular solution of the Schr¨ odinger equation at energy . – non local: there is one potential per angular momentum: V ps(r) =
ps 2 fl|φps l (r )| l
(fl is the occupancy of state with angular momentum l).
Desirable characteristics of a Pseudopotential: • Transferability: can be estimated from atomic calculations on diﬀerent conﬁgurations. In many cases simple unscreening produces an unacceptable loss of transferability. May require the nonlinear core correction: Vlps(r) = Vl(r) − VH (nps(r)) − Vxc(nc(r) + nps(r)) where nc(r) is the core charge of the atom (Froyen, Louie, Cohen 1982) • Softness: atoms with strongly oscillating pseudo-wavefunctions (ﬁrst-row elements, elements with 3d and 4f valence electrons) will produce hard PPs requiring many PWs in calculations. Larger core radius means better softness but worse transferability. Various recipes to get optimal smoothness without compromising transferability: Troullier and Martins (1990), Rappe Rabe Kaxiras Joannopoulos (1990)

Tunneling Studies of Pseudogaps a Comment

a r X i v :co nd-mat/9802079v 1 [c o n d -m a t .s u p r -c o n ] 6 F e b 1998Tunneling Studies of Pseudogaps:a CommentR.S.Markiewicz and C.Kusko ∗Physics Department and Barnett Institute,Northeastern U.,Boston MA 02115The recent observation of the pseudogap in tunneling measurements on Bi 2Sr 2CaCu 2O 8+δ[1–3]should prove of great value in unravelling the mysteries of the ‘normal state’of the cuprates.However,several issues in these papers require clariﬁcation.Here,we discuss two impor-tant points.First,the gaps observed in the quasiparticle tunneling spectra are assumed to be superconducting gaps,and taken as evidence that the pseudogap is caused by super-conducting ﬂuctuations.However,a normal-state gap (due,e.g.,to charge or spin density waves)will also show up in the tunneling spectra [4].For illustrative purposes,we use the pinned [5]Balseiro-Falicov (BF)[6]model of competition between a charge density wave (CDW)and (s-wave)superconductivity (SC),which gives a good ac-count of the doping dependence of the pseudogap [7]and is a simple model for striped phases [8].For a pure CDW,the spectral function is of BCS form:A (k,ω)=2π[u 2k δ(ω−E k +)+v 2k δ(ω−E k −)],(1)with u 2k =1−v 2k =(1+ǫk −/˜E k )/2,E k ±=(ǫk +±˜E k )/2,ǫk ±=ǫk ±ǫk +Q and ˜Ek =[1]Ch.Renner,et al.,Phys.Rev.Lett.80,149(1998).[2]Y.DeWilde,et al.,Phys.Rev.Lett.80,153(1998).[3]N.Miyakawa,et al.,Phys.Rev.Lett.80,157(1998).[4]A.M.Gabovich,Sov.J.Low Temp.Phys.18,490(1992).[5]R.S.Markiewicz,Physica C 193,323(1992).[6]C.Balseiro and L.Falicov,Phys.Rev.B 20,4457(1979).[7]R.S.Markiewicz,Phys.Rev.Lett.73,1310(1994).[8]R.S.Markiewicz,Phys.Rev.B 56,9091(1997).[9]I.Aﬄeck and J.B.Marston,Phys.Rev.B 37,3774(1988).[10]A.Kampf and J.Schrieﬀer,Phys.Rev.B 42,7967(1990).[11]Summarized on p.1223of R.S.Markiewicz,J.Phys.Chem.Sol.58,1179(1997).1。

Principles of Plasma Discharges and Materials Processing9

CHAPTER8MOLECULAR COLLISIONS8.1INTRODUCTIONBasic concepts of gas-phase collisions were introduced in Chapter3,where we described only those processes needed to model the simplest noble gas discharges: electron–atom ionization,excitation,and elastic scattering;and ion–atom elastic scattering and resonant charge transfer.In this chapter we introduce other collisional processes that are central to the description of chemically reactive discharges.These include the dissociation of molecules,the generation and destruction of negative ions,and gas-phase chemical reactions.Whereas the cross sections have been measured reasonably well for the noble gases,with measurements in reasonable agreement with theory,this is not the case for collisions in molecular gases.Hundreds of potentially signiﬁcant collisional reactions must be examined in simple diatomic gas discharges such as oxygen.For feedstocks such as CF4/O2,SiH4/O2,etc.,the complexity can be overwhelming.Furthermore,even when the signiﬁcant processes have been identiﬁed,most of the cross sections have been neither measured nor calculated. Hence,one must often rely on estimates based on semiempirical or semiclassical methods,or on measurements made on molecules analogous to those of interest. As might be expected,data are most readily available for simple diatomic and polyatomic gases.Principles of Plasma Discharges and Materials Processing,by M.A.Lieberman and A.J.Lichtenberg. ISBN0-471-72001-1Copyright#2005John Wiley&Sons,Inc.235236MOLECULAR COLLISIONS8.2MOLECULAR STRUCTUREThe energy levels for the electronic states of a single atom were described in Chapter3.The energy levels of molecules are more complicated for two reasons. First,molecules have additional vibrational and rotational degrees of freedom due to the motions of their nuclei,with corresponding quantized energies E v and E J. Second,the energy E e of each electronic state depends on the instantaneous con-ﬁguration of the nuclei.For a diatomic molecule,E e depends on a single coordinate R,the spacing between the two nuclei.Since the nuclear motions are slow compared to the electronic motions,the electronic state can be determined for anyﬁxed spacing.We can therefore represent each quantized electronic level for a frozen set of nuclear positions as a graph of E e versus R,as shown in Figure8.1.For a mole-cule to be stable,the ground(minimum energy)electronic state must have a minimum at some value R1corresponding to the mean intermolecular separation (curve1).In this case,energy must be supplied in order to separate the atoms (R!1).An excited electronic state can either have a minimum( R2for curve2) or not(curve3).Note that R2and R1do not generally coincide.As for atoms, excited states may be short lived(unstable to electric dipole radiation)or may be metastable.Various electronic levels may tend to the same energy in the unbound (R!1)limit. Array FIGURE8.1.Potential energy curves for the electronic states of a diatomic molecule.For diatomic molecules,the electronic states are speciﬁedﬁrst by the component (in units of hÀ)L of the total orbital angular momentum along the internuclear axis, with the symbols S,P,D,and F corresponding to L¼0,+1,+2,and+3,in analogy with atomic nomenclature.All but the S states are doubly degenerate in L.For S states,þandÀsuperscripts are often used to denote whether the wave function is symmetric or antisymmetric with respect to reﬂection at any plane through the internuclear axis.The total electron spin angular momentum S (in units of hÀ)is also speciﬁed,with the multiplicity2Sþ1written as a preﬁxed superscript,as for atomic states.Finally,for homonuclear molecules(H2,N2,O2, etc.)the subscripts g or u are written to denote whether the wave function is sym-metric or antisymmetric with respect to interchange of the nuclei.In this notation, the ground states of H2and N2are both singlets,1Sþg,and that of O2is a triplet,3SÀg .For polyatomic molecules,the electronic energy levels depend on more thanone nuclear coordinate,so Figure8.1must be generalized.Furthermore,since there is generally no axis of symmetry,the states cannot be characterized by the quantum number L,and other naming conventions are used.Such states are often speciﬁed empirically through characterization of measured optical emission spectra.Typical spacings of low-lying electronic energy levels range from a few to tens of volts,as for atoms.Vibrational and Rotational MotionsUnfreezing the nuclear vibrational and rotational motions leads to additional quan-tized structure on smaller energy scales,as illustrated in Figure8.2.The simplest (harmonic oscillator)model for the vibration of diatomic molecules leads to equally spaced quantized,nondegenerate energy levelse E v¼hÀv vib vþ1 2(8:2:1)where v¼0,1,2,...is the vibrational quantum number and v vib is the linearized vibration frequency.Fitting a quadratic functione E v¼12k vib(RÀ R)2(8:2:2)near the minimum of a stable energy level curve such as those shown in Figure8.1, we can estimatev vib%k vibm Rmol1=2(8:2:3)where k vib is the“spring constant”and m Rmol is the reduced mass of the AB molecule.The spacing hÀv vib between vibrational energy levels for a low-lying8.2MOLECULAR STRUCTURE237stable electronic state is typically a few tenths of a volt.Hence for molecules in equi-librium at room temperature (0.026V),only the v ¼0level is signiﬁcantly popula-ted.However,collisional processes can excite strongly nonequilibrium vibrational energy levels.We indicate by the short horizontal line segments in Figure 8.1a few of the vibrational energy levels for the stable electronic states.The length of each segment gives the range of classically allowed vibrational motions.Note that even the ground state (v ¼0)has a ﬁnite width D R 1as shown,because from(8.2.1),the v ¼0state has a nonzero vibrational energy 1h Àv vib .The actual separ-ation D R about Rfor the ground state has a Gaussian distribution,and tends toward a distribution peaked at the classical turning points for the vibrational motion as v !1.The vibrational motion becomes anharmonic and the level spa-cings tend to zero as the unbound vibrational energy is approached (E v !D E 1).FIGURE 8.2.Vibrational and rotational levels of two electronic states A and B of a molecule;the three double arrows indicate examples of transitions in the pure rotation spectrum,the rotation–vibration spectrum,and the electronic spectrum (after Herzberg,1971).238MOLECULAR COLLISIONSFor E v.D E1,the vibrational states form a continuum,corresponding to unbound classical motion of the nuclei(breakup of the molecule).For a polyatomic molecule there are many degrees of freedom for vibrational motion,leading to a very compli-cated structure for the vibrational levels.The simplest(dumbbell)model for the rotation of diatomic molecules leads to the nonuniform quantized energy levelse E J¼hÀ22I molJ(Jþ1)(8:2:4)where I mol¼m Rmol R2is the moment of inertia and J¼0,1,2,...is the rotational quantum number.The levels are degenerate,with2Jþ1states for the J th level. The spacing between rotational levels increases with J(see Figure8.2).The spacing between the lowest(J¼0to J¼1)levels typically corresponds to an energy of0.001–0.01V;hence,many low-lying levels are populated in thermal equilibrium at room temperature.Optical EmissionAn excited molecular state can decay to a lower energy state by emission of a photon or by breakup of the molecule.As shown in Figure8.2,the radiation can be emitted by a transition between electronic levels,between vibrational levels of the same electronic state,or between rotational levels of the same electronic and vibrational state;the radiation typically lies within the optical,infrared,or microwave frequency range,respectively.Electric dipole radiation is the strongest mechanism for photon emission,having typical transition times of t rad 10À9s,as obtained in (3.4.13).The selection rules for electric dipole radiation areDL¼0,+1(8:2:5a)D S¼0(8:2:5b) In addition,for transitions between S states the only allowed transitions areSþÀ!Sþand SÀÀ!SÀ(8:2:6) and for homonuclear molecules,the only allowed transitions aregÀ!u and uÀ!g(8:2:7) Hence homonuclear diatomic molecules do not have a pure vibrational or rotational spectrum.Radiative transitions between electronic levels having many different vibrational and rotational initial andﬁnal states give rise to a structure of emission and absorption bands within which a set of closely spaced frequencies appear.These give rise to characteristic molecular emission and absorption bands when observed8.2MOLECULAR STRUCTURE239using low-resolution optical spectrometers.As for atoms,metastable molecular states having no electric dipole transitions to lower levels also exist.These have life-times much exceeding10À6s;they can give rise to weak optical band structures due to magnetic dipole or electric quadrupole radiation.Electric dipole radiation between vibrational levels of the same electronic state is permitted for molecules having permanent dipole moments.In the harmonic oscillator approximation,the selection rule is D v¼+1;weaker transitions D v¼+2,+3,...are permitted for anharmonic vibrational motion.The preceding description of molecular structure applies to molecules having arbi-trary electronic charge.This includes neutral molecules AB,positive molecular ions ABþ,AB2þ,etc.and negative molecular ions ABÀ.The potential energy curves for the various electronic states,regardless of molecular charge,are commonly plotted on the same diagram.Figures8.3and8.4give these for some important electronic statesof HÀ2,H2,and Hþ2,and of OÀ2,O2,and Oþ2,respectively.Examples of both attractive(having a potential energy minimum)and repulsive(having no minimum)states can be seen.The vibrational levels are labeled with the quantum number v for the attrac-tive levels.The ground states of both Hþ2and Oþ2are attractive;hence these molecular ions are stable against autodissociation(ABþ!AþBþor AþþB).Similarly,the ground states of H2and O2are attractive and lie below those of Hþ2and Oþ2;hence they are stable against autodissociation and autoionization(AB!ABþþe).For some molecules,for example,diatomic argon,the ABþion is stable but the AB neutral is not stable.For all molecules,the AB ground state lies below the ABþground state and is stable against autoionization.Excited states can be attractive or repulsive.A few of the attractive states may be metastable;some examples are the 3P u state of H2and the1D g,1Sþgand3D u states of O2.Negative IonsRecall from Section7.2that many neutral atoms have a positive electron afﬁnity E aff;that is,the reactionAþeÀ!AÀis exothermic with energy E aff(in volts).If E aff is negative,then AÀis unstable to autodetachment,AÀ!Aþe.A similar phenomenon is found for negative molecular ions.A stable ABÀion exists if its ground(lowest energy)state has a potential minimum that lies below the ground state of AB.This is generally true only for strongly electronegative gases having large electron afﬁnities,such as O2 (E aff%1:463V for O atoms)and the halogens(E aff.3V for the atoms).For example,Figure8.4shows that the2P g ground state of OÀ2is stable,with E aff% 0:43V for O2.For weakly electronegative or for electropositive gases,the minimum of the ground state of ABÀgenerally lies above the ground state of AB,and ABÀis unstable to autodetachment.An example is hydrogen,which is weakly electronegative(E aff%0:754V for H atoms).Figure8.3shows that the2Sþu ground state of HÀ2is unstable,although the HÀion itself is stable.In an elec-tropositive gas such as N2(E aff.0),both NÀ2and NÀare unstable. 240MOLECULAR COLLISIONS8.3ELECTRON COLLISIONS WITH MOLECULESThe interaction time for the collision of a typical (1–10V)electron with a molecule is short,t c 2a 0=v e 10À16–10À15s,compared to the typical time for a molecule to vibrate,t vib 10À14–10À13s.Hence for electron collisional excitation of a mole-cule to an excited electronic state,the new vibrational (and rotational)state canbeFIGURE 8.3.Potential energy curves for H À2,H 2,and H þ2.(From Jeffery I.Steinfeld,Molecules and Radiation:An Introduction to Modern Molecular Spectroscopy ,2d ed.#MIT Press,1985.)8.3ELECTRON COLLISIONS WITH MOLECULES 241FIGURE 8.4.Potential energy curves for O À2,O 2,and O þ2.(From Jeffery I.Steinfeld,Molecules and Radiation:An Introduction to Modern Molecular Spectroscopy ,2d ed.#MIT Press,1985.)242MOLECULAR COLLISIONS8.3ELECTRON COLLISIONS WITH MOLECULES243 determined by freezing the nuclear motions during the collision.This is known as the Franck–Condon principle and is illustrated in Figure8.1by the vertical line a,showing the collisional excitation atﬁxed R to a high quantum number bound vibrational state and by the vertical line b,showing excitation atﬁxed R to a vibra-tionally unbound state,in which breakup of the molecule is energetically permitted. Since the typical transition time for electric dipole radiation(t rad 10À9–10À8s)is long compared to the dissociation( vibrational)time t diss,excitation to an excited state will generally lead to dissociation when it is energetically permitted.Finally, we note that the time between collisions t c)t rad in typical low-pressure processing discharges.Summarizing the ordering of timescales for electron–molecule collisions,we havet at t c(t vib t diss(t rad(t cDissociationElectron impact dissociation,eþABÀ!AþBþeof feedstock gases plays a central role in the chemistry of low-pressure reactive discharges.The variety of possible dissociation processes is illustrated in Figure8.5.In collisions a or a0,the v¼0ground state of AB is excited to a repulsive state of AB.The required threshold energy E thr is E a for collision a and E a0for Array FIGURE8.5.Illustrating the variety of dissociation processes for electron collisions with molecules.collision a0,and it leads to an energy after dissociation lying between E aÀE diss and E a0ÀE diss that is shared among the dissociation products(here,A and B). Typically,E aÀE diss few volts;consequently,hot neutral fragments are typically generated by dissociation processes.If these hot fragments hit the substrate surface, they can profoundly affect the process chemistry.In collision b,the ground state AB is excited to an attractive state of AB at an energy E b that exceeds the binding energy E diss of the AB molecule,resulting in dissociation of AB with frag-ment energy E bÀE diss.In collision b0,the excitation energy E b0¼E diss,and the fragments have low energies;hence this process creates fragments having energies ranging from essentially thermal energies up to E bÀE diss few volts.In collision c,the AB atom is excited to the bound excited state ABÃ(labeled5),which sub-sequently radiates to the unbound AB state(labeled3),which then dissociates.The threshold energy required is large,and the fragments are hot.Collision c can also lead to dissociation of an excited state by a radiationless transfer from state5to state4near the point where the two states cross:ABÃðboundÞÀ!ABÃðunboundÞÀ!AþBÃThe fragments can be both hot and in excited states.We discuss such radiationless electronic transitions in the next section.This phenomenon is known as predisso-ciation.Finally,a collision(not labeled in theﬁgure)to state4can lead to dis-sociation of ABÃ,again resulting in hot excited fragments.The process of electron impact excitation of a molecule is similar to that of an atom,and,consequently,the cross sections have a similar form.A simple classical estimate of the dissociation cross section for a level having excitation energy U1can be found by requiring that an incident electron having energy W transfer an energy W L lying between U1and U2to a valence electron.Here,U2is the energy of the next higher level.Then integrating the differential cross section d s[given in(3.4.20)and repeated here],d s¼pe24021Wd W LW2L(3:4:20)over W L,we obtains diss¼0W,U1pe24pe021W1U1À1WU1,W,U2pe24021W1U1À1U2W.U28>>>>>><>>>>>>:(8:3:1)244MOLECULAR COLLISIONSLetting U2ÀU1(U1and introducing voltage units W¼e E,U1¼e E1and U2¼e E2,we haves diss¼0E,E1s0EÀE11E1,E,E2s0E2ÀE1EE.E28>>>><>>>>:(8:3:2)wheres0¼pe4pe0E12(8:3:3)We see that the dissociation cross section rises linearly from the threshold energy E thr%E1to a maximum value s0(E2ÀE1)=E thr at E2and then falls off as1=E. Actually,E1and E2can depend on the nuclear separation R.In this case,(8.3.2) should be averaged over the range of R s corresponding to the ground-state vibrational energy,leading to a broadened dependence of the average cross section on energy E.The maximum cross section is typically of order10À15cm2. Typical rate constants for a single dissociation process with E thr&T e have an Arrhenius formK diss/K diss0expÀE thr T e(8:3:4)where K diss0 10À7cm3=s.However,in some cases E thr.T e.For excitation to an attractive state,an appropriate average over the fraction of the ground-state vibration that leads to dissociation must be taken.Dissociative IonizationIn addition to normal ionization,eþABÀ!ABþþ2eelectron–molecule collisions can lead to dissociative ionizationeþABÀ!AþBþþ2eThese processes,common for polyatomic molecules,are illustrated in Figure8.6.In collision a having threshold energy E iz,the molecular ion ABþis formed.Collisionsb andc occur at higher threshold energies E diz and result in dissociative ionization,8.3ELECTRON COLLISIONS WITH MOLECULES245leading to the formation of fast,positively charged ions and neutrals.These cross sections have a similar form to the Thompson ionization cross section for atoms.Dissociative RecombinationThe electron collision,e þAB þÀ!A þB Ãillustrated as d and d 0in Figure 8.6,destroys an electron–ion pair and leads to the production of fast excited neutral fragments.Since the electron is captured,it is not available to carry away a part of the reaction energy.Consequently,the collision cross section has a resonant character,falling to very low values for E ,E d and E .E d 0.However,a large number of excited states A Ãand B Ãhaving increasing principal quantum numbers n and energies can be among the reaction products.Consequently,the rate constants can be large,of order 10À7–10À6cm 3=s.Dissocia-tive recombination to the ground states of A and B cannot occur because the potential energy curve for AB þis always greater than the potential energycurveFIGURE 8.6.Illustration of dissociative ionization and dissociative recombination for electron collisions with molecules.246MOLECULAR COLLISIONSfor the repulsive state of AB.Two-body recombination for atomic ions or for mol-ecular ions that do not subsequently dissociate can only occur with emission of a photon:eþAþÀ!Aþh n:As shown in Section9.2,the rate constants are typically three toﬁve orders of magnitude lower than for dissociative recombination.Example of HydrogenThe example of H2illustrates some of the inelastic electron collision phenomena we have discussed.In order of increasing electron impact energy,at a threshold energy of 8:8V,there is excitation to the repulsive3Sþu state followed by dissociation into two fast H fragments carrying 2:2V/atom.At11.5V,the1Sþu bound state is excited,with subsequent electric dipole radiation in the ultraviolet region to the1Sþg ground state.At11.8V,there is excitation to the3Sþg bound state,followedby electric dipole radiation to the3Sþu repulsive state,followed by dissociation with 2:2V/atom.At12.6V,the1P u bound state is excited,with UV emission tothe ground state.At15.4V,the2Sþg ground state of Hþ2is excited,leading to the pro-duction of Hþ2ions.At28V,excitation of the repulsive2Sþu state of Hþ2leads to thedissociative ionization of H2,with 5V each for the H and Hþfragments.Dissociative Electron AttachmentThe processes,eþABÀ!AþBÀproduce negative ion fragments as well as neutrals.They are important in discharges containing atoms having positive electron afﬁnities,not only because of the pro-duction of negative ions,but because the threshold energy for production of negative ion fragments is usually lower than for pure dissociation processes.A variety of pro-cesses are possible,as shown in Figure8.7.Since the impacting electron is captured and is not available to carry excess collision energy away,dissociative attachment is a resonant process that is important only within a narrow energy range.The maximum cross sections are generally much smaller than the hard-sphere cross section of the molecule.Attachment generally proceeds by collisional excitation from the ground AB state to a repulsive ABÀstate,which subsequently either auto-detaches or dissociates.The attachment cross section is determined by the balance between these processes.For most molecules,the dissociation energy E diss of AB is greater than the electron afﬁnity E affB of B,leading to the potential energy curves shown in Figure8.7a.In this case,the cross section is large only for impact energies lying between a minimum value E thr,for collision a,and a maximum value E0thr for8.3ELECTRON COLLISIONS WITH MOLECULES247FIGURE 8.7.Illustration of a variety of electron attachment processes for electron collisions with molecules:(a )capture into a repulsive state;(b )capture into an attractive state;(c )capture of slow electrons into a repulsive state;(d )polar dissociation.248MOLECULAR COLLISIONScollision a 0.The fragments are hot,having energies lying between minimum and maximum values E min ¼E thr þE affB ÀE diss and E max ¼E 0thr þE af fB ÀE diss .Since the AB Àstate lies above the AB state for R ,R x ,autodetachment can occur as the mol-ecules begin to separate:AB À!AB þe.Hence the cross section for production of negative ions can be much smaller than that for excitation of the AB Àrepulsive state.As a crude estimate,for the same energy,the autodetachment rate is ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃM R =m p 100times the dissociation rate of the repulsive AB Àmolecule,where M R is the reduced mass.Hence only one out of 100excitations lead to dissociative attachment.Excitation to the AB Àbound state can also lead to dissociative attachment,as shown in Figure 8.7b .Here the cross section is signiﬁcant only for E thr ,E ,E 0thr ,but the fragments can have low energies,with a minimum energy of zero and a maximum energy of E 0thr þE affB ÀE diss .Collision b,e þAB À!AB ÀÃdoes not lead to production of AB Àions because energy and momentum are not gen-erally conserved when two bodies collide elastically to form one body (see Problem3.12).Hence the excited AB ÀÃion separates,AB ÀÃÀ!e þABunless vibrational radiation or collision with a third body carries off the excess energy.These processes are both slow in low-pressure discharges (see Section 9.2).At high pressures (say,atmospheric),three-body attachment to form AB Àcan be very important.For a few molecules,such as some halogens,the electron afﬁnity of the atom exceeds the dissociation energy of the neutral molecule,leading to the potential energy curves shown in Figure 8.7c .In this case the range of electron impact ener-gies E for excitation of the AB Àrepulsive state includes E ¼0.Consequently,there is no threshold energy,and very slow electrons can produce dissociative attachment,resulting in hot neutral and negative ion fragments.The range of R s over which auto-detachment can occur is small;hence the maximum cross sections for dissociative attachment can be as high as 10À16cm 2.A simple classical estimate of electron capture can be made using the differential scattering cross section for energy loss (3.4.20),in a manner similar to that done for dissociation.For electron capture to an energy level E 1that is unstable to autode-tachment,and with the additional constraint for capture that the incident electron energy lie within E 1and E 2¼E 1þD E ,where D E is a small energy difference characteristic of the dissociative attachment timescale,we obtain,in place of (8.3.2),s att¼0E ,E 1s 0E ÀE 1E 1E 1,E ,E 20E .E 28>><>>:(8:3:5)8.3ELECTRON COLLISIONS WITH MOLECULES 249wheres 0%p m M R 1=2e 4pe 0E 1 2(8:3:6)The factor of (m =M R )1=2roughly gives the fraction of excited states that do not auto-detach.We see that the dissociative attachment cross section rises linearly at E 1to a maximum value s 0D E =E 1and then falls abruptly to zero.As for dissociation,E 1can depend strongly on the nuclear separation R ,and (8.3.5)must be averaged over the range of E 1s corresponding to the ground state vibrational motion;e.g.,from E thr to E 0thr in Figure 8.7a .Because generally D E (E 0thr ÀE thr ,we can write (8.3.5)in the forms att %p m M R 1=2e 4pe 0 2(D E )22E 1d (E ÀE 1)(8:3:7)where d is the Dirac delta ing (8.3.7),the average over the vibrational motion can be performed,leading to a cross section that is strongly peaked lying between E thr and E 0thr .We leave the details of the calculation to a problem.Polar DissociationThe process,e þAB À!A þþB Àþeproduces negative ions without electron capture.As shown in Figure 8.7d ,the process proceeds by excitation of a polar state A þand B Àof AB Ãthat has a separ-ated atom limit of A þand B À.Hence at large R ,this state lies above the A þB ground state by the difference between the ionization potential of A and the electron afﬁnity of B.The polar state is weakly bound at large R by the Coulomb attraction force,but is repulsive at small R .The maximum cross section and the dependence of the cross section on electron impact energy are similar to that of pure dissociation.The threshold energy E thr for polar dissociation is generally large.The measured cross section for negative ion production by electron impact in O 2is shown in Figure 8.8.The sharp peak at 6.5V is due to dissociative attachment.The variation of the cross section with energy is typical of a resonant capture process.The maximum cross section of 10À18cm 2is quite low because autode-tachment from the repulsive O À2state is strong,inhibiting dissociative attachment.The second gradual maximum near 35V is due to polar dissociation;the variation of the cross section with energy is typical of a nonresonant process.250MOLECULAR COLLISIONS。

Interaction of the hepatitis B spliced protein

Interaction of the Hepatitis B Spliced Protein with Cathepsin B Promotes Hepatoma Cell Migration and InvasionWan-Nan Chen,a,b Jin-Yan Chen,a Bo-Yan Jiao,b Wan-Song Lin,b Yun-Li Wu,b Ling-Ling Liu,b and Xu Lin a,bKey Laboratory of Ministry of Education for Gastrointestinal Cancer,Research Center for Molecular Medicine,Fujian Medical University,Fuzhou,Fujian,People’s Republic of China,a and Key Laboratory of Tumor Microbiology,Department of Medical Microbiology,Fujian Medical University,Fuzhou,Fujian,People’s Republic of China bHepatitis B spliced protein(HBSP)is involved in the pathogenicity and/or persistence of hepatitis B virus(HBV).Chronic HBV infection is one of the most important risk factors for the development of hepatocellular carcinoma(HCC).However,whether or not HBSP contributes to the progression of HBV-associated HCC remains unknown.This study reports that overexpression of HBSP in human hepatoma cells increased cell invasion and motility.Conversely,small interfering RNA(siRNA)-mediated knockdown of HBSP expression inhibited migration and invasion.By glutathione S-transferase(GST)pulldown,coimmunopre-cipitation,and a mammalian two-hybrid assay,HBSP was found to directly interact with cathepsin B(CTSB).Similar to HBSP knockdown,knocking down CTSB also reduced cell migration and invasion.Furthermore,the HBSP-overexpressing hepatoma cells were shown to have increased expression and activity of matrix metalloproteinase-9(MMP-9)and urokinase-type plasmin-ogen activator(uPA),and overexpression of HBSP signiﬁcantly enhanced tumor-induced vascularization of endothelial cells.In contrast,knockdown of either HBSP or CTSB by siRNA resulted in inhibition of the two proteolytic enzymes and of the in vitro angiogenesis.Expression of HBSP in the hepatoma cells appeared to activate the mitogen-activated protein kinase(MAPK)and Akt signaling pathway,as evidenced by increases in phosphorylation of p38,Jun N-terminal protein kinase(JNK),extracellular signal-regulated kinase(ERK),and Akt.Taken together,theseﬁndings imply that interaction of HBSP with CTSB may promote hepatoma cell motility and invasion and highlight new molecular mechanisms for HBSP-induced HCC progression that involve the secretion and activation of proteolytic enzymes,increased tumor-induced angiogenesis,and activation of the MAPK/Akt signaling,thereby leading to the aggressiveness of hepatoma cells.C hronic hepatitis B virus(HBV)infection has been proven tobe one of the most important risk factors for the development of hepatocellular carcinoma(HCC)(3,9).However,the patho-genesis of cancer in HBV infection is still not fully understood, and it appears that multiple factors and cellular signaling path-ways are involved in hepatocarcinogenesis(28).Integration of HBV genome into host DNA can lead to alterations in cellular gene function or generate chromosomal instability(1,10,43). Expression of some oncogenic HBV proteins such as HBx and truncated Pre-S2/S has been shown to have a direct effect on malignant transformation of the liver(42)and to promote metas-tasis of the malignant cells,and there is thus a very high mortality rate for HCC patients(18).Another HBV protein,encoded by a singly spliced2.2-kb HBV DNA and referred to as the hepatitis B spliced protein(HBSP),is found to be expressed in liver biopsy tissues from four out ofﬁve chronic patients but not from two hepatitis C virus-infected patient samples and one normal liver sample(39).Our prior study also showed that a2.2-kb splice variant was present in all tumor and peritumor samples from12 HCC patients studied(20).Exogenous expression of HBSP in transfected Huh-7cells can induce cell apoptosis(40).However, the cytopathic effect of the HBSP was unclear,and its role in pro-gression,invasion,and metastasis of HBV-related HCC has not yet been elucidated.We previously showed that cathepsin B(CTSB)was one of the major intracellular interacting partners of HBSP,and it was iden-tiﬁed using a yeast two-hybrid screening assay(8).This led us to investigate whether the HBSP could interact with CTSB in the context of hepatoma cells to affect cell migration,invasion,and metastasis,all of which can be modulated by CTSB acting directly and indirectly on extracellular matrix(ECM)component remod-eling and degradation(26,47).CTSB is a lysosomal cysteine pro-tease that plays an important role in physiological protein turn-over and processing(19,41).In nonmalignant cells,CTSB is mainly stored in the lysosome,whereas in malignant cells,CTSB redistributes into exocytic vesicles at the cell periphery,leading to its secretion and association with binding partners on the tumor cell surface(22,33).CTSB can cleave and activate a wide variety of substrates in proteolytic pathways that increase neoplastic pro-gression.It has been shown that activation of urokinase-type plas-minogen activator(uPA)and matrix metalloproteinases(MMPs) by CTSB not only enhances ECM degradation and cancer cell motility and invasion(14)but also induces in vitro angiogenesis (2).In addition to interacting with other proteases,CTSB may regulate the activity of kinase signaling networks,cell surface re-ceptors,and signaling molecules such as chemokines,cytokines and growth factors(25).However,such interactions are usually bidirectional because kinases can also regulate many proteases through phosphorylation while proteases can control the actions of a multitude of kinases(21).High expression levels of CTSB have been linked to aggressive-ness and poor prognosis in several cancers,such as colorectal and ovarian carcinomas(15,29,34,37,38).Compared to the amount Received10August2012Accepted24September2012Published ahead of print3October2012Address correspondence to Xu Lin,linxu@.W.-N.C.and J.-Y.C.contributed equally to this work.Copyright©2012,American Society for Microbiology.All Rights Reserved.doi:10.1128/JVI.02095-12December2012Volume86Number24Journal of Virology p.13533–13533of information on the role of CTSB in colon and ovarian cancers, very little information about the precise function of CTSB in HCC and its interactions with other signaling molecules and pathways regulating the progression of HCC is available.In this study,we attempted to investigate the interaction of HBSP with CTSB both in vitro and in vivo,examine the function of this interaction by manipulating their expression in various hepatoma cell lines, and explore potential molecular mechanisms underlying the changes in hepatoma cell migratory behaviors and metastatic potential.We found that HBSP interacting with CTSB en-hances the migration and invasion of hepatoma cells and pro-motes in vitro angiogenesis via activation of MMP-9and uPA and the pathways involving mitogen-activated protein kinase (MAPK)and Akt signaling.MATERIALS AND METHODSVector construction.A2.2-kb spliced,defective HBV DNA was previ-ously isolated from a patient with chronic HBV infection(8).The CTSB and cathepsin D(CTSD)genes were reverse transcribed from the total RNA isolated from Huh-7cells and cloned into pCMVTNT at the XhoI and SalI sites of the vector(Promega,Madison,WI).The paired forward and reverse primers were5=-CCGCTCGAGGCCACCTGGCAGCTCTG GGCCTCCCT-3=and5=-ACGCGTCGACTTAGATCTTTTCCCAGT-3= (for CTSB)and5=-CCGCTCGAGCAGCCCTCCAGCCTTCTGC-3=and 5=-ACGCGTCGACTTAGAGGCGGGCAGCCTCG-3=(for CTSD).pACT-CTSB,used for the mammalian two-hybrid assay,was generated by cloning the CTSB gene into the SalI and NotI sites of pACT(Promega, Madison,WI).p3ϫFLAG-CMV-10-HBSP,used for establishing a sta-ble HBSP-expressing cell line,was constructed by the insertion of an HBSP open reading frame(ORF)into p3ϫFLAG-CMV-10(Sigma,St. Louis,MO).Cell lines and transfection.The human hepatoma cell lines Huh-7, HepG2,and Hep3B and human dermal microvascular endothelial cell line HMEC-1(obtained from the American Type Culture Collection,Ma-nassas,VA)were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM;Invitrogen,Carlsbad,CA)supplemented with10%fetal bovine serum(FBS)and maintained in a humidiﬁed atmosphere containing5% CO2at37°C.Transfection was performed using Lipofectamine2000 transfection reagent(Invitrogen)according to the manufacturer’s in-structions.Generation of stable HBSP-expressing hepatoma cell lines.To es-tablish stably transfected cell lines expressing HBSP N-terminally fused to 3ϫFLAG,the Huh-7,HepG2,and Hep3B hepatoma cells were transfected with the respective vectors and selected in the presence of800␮g/ml Geneticin for4weeks.The Geneticin-resistant clones were individually expanded into cell lines and screened for the extent of HBSP protein expression by Western blotting.Western blot analysis.Cell lysates were prepared using radioimmu-noprecipitation assay(RIPA)protein lysis buffer(Pierce Company,Rock-ford,IL),and the protein extracts were quantiﬁed and then subjected to electrophoresis on a4to15%SDS-PAGE gel.The proteins were trans-ferred to polyvinylidene diﬂuoride(PVDF)membranes and blocked in Tris-buffered saline(TBS)containing5%bovine serum albumin(BSA). After the membranes were incubated sequentially with primary and sec-ondary antibodies,the immunoreactive protein bands were visualized using CDP-Star reagents(Roche,Mannheim,Germany)and analyzed densitometrically by a ChemiImager(Alpha Innotech Corporation,San Leandro,CA).The speciﬁc antibodies used in this study included anti-FLAG(1:2,000;Sigma),anti-GAL4(1:1,000;Clontech,Palo Alto,CA), anti-CTSB,anti-MMP9(1:200;Santa Cruz Biotechnology,Santa Cruz, CA),anti-uPA,anti-p38,anti-pp38,anti-Jun N-terminal protein kinase (anti-JNK),anti-pJNK,anti-extracellular signal-regulated kinase(anti-ERK),anti-pERK,anti-Akt,and anti-pAkt(1:1,000;Cell Signaling Tech-nology,Beverly,MA).In vitro cell invasion assay.The invasive capabilities of cells weredetermined using24-well BD BioCoat Matrigel invasion chambers(BDBiosciences,San Jose,CA).Cells were serum starved overnight,and analiquot of5ϫ104cells in500␮l DMEM was added to the upper chamberof transwell while750␮l DMEM containing10%FBS and10␮g/mlﬁbronectin(BD Sciences,San Diego,CA)were added to the lower cham-ber.After36h of incubation,Matrigel and cells remaining in the upperchamber were manually removed by cotton swabs,and the inserts wereﬁxed in4%paraformaldehyde and stained with0.5%crystal violet.Cellsin at least six randomly selected microscopicﬁelds were counted andphotographed.All experiments were performed in duplicate and repeatedﬁve times.Wound healing migration assay.Cells were grown to90%conﬂuenceon6-well plates,and a scratch through the cell monolayer was introducedby using a pipette tip.Baseline(time zero)images were captured and cellmigration was assessed at48h by counting the number of cells that hadmigrated across the scratch,and this number was normalized to thescratch area.Data were expressed as percentages of the control value.Cellmigration data were obtained fromﬁve independent wound healing ex-periments.GST pulldown assay.Construction,expression,and puriﬁcation ofglutathione S-transferase(GST)-fused HBSP or HBx were described pre-viously(8,12).An in vitro T7-coupled reticulocyte lysate system(Pro-mega)was used to generate35S-labeled CTSB protein by the addition of2␮g of pCMVTNT-CTSB with50␮Ci of[35S]methionine(Amersham Pharmacia Biotech,Arlington,IL).35S-labeled CTSB was added to theGST-HBSP recombinant-protein-immobilized beads and incubatedovernight at4°C.Beads were washed three times with1%Triton X-100inphosphate-buffered saline(PBS),resuspended in SDS sample buffer,andsubjected to12%SDS-PAGE.35S-CTSB was detected by autoradiogra-phy.Both GST-fused HBx protein and35S-labeled CTSD generated bypCMVTNT-CTSD were used as irrelevant negative controls.Co-IP assay.For the in vivo coimmunoprecipitation(Co-IP)experi-ments,transiently transfected Huh-7cells were lysed and the soluble pro-teins were precleared with100␮l of a50%slurry of protein A agarose(Invitrogen).The clear lysates were then mixed with2␮g of goat poly-clonal anti-CTSB IgG(Santa Cruz Biotechnology)and100␮l of a50%slurry of protein A agarose.The immunoprecipitated complexes wereanalyzed by10%SDS-PAGE and immunoblotting using anti-GAL4DNA-binding domain(GAL4BD)monoclonal antibody(Clontech)andanti-CTSB antibody(Santa Cruz Biotechnology).Mammalian two-hybrid assay.A mammalian two-hybrid assay wasperformed using the CheckMate mammalian two-hybrid system(Pro-mega)according to the manufacturer’s protocol.Brieﬂy,pACT-CTSBand each of the pBIND-HBSP,pBIND-HBSP1–47,and pBIND-HBSP48–111vectors were cotransfected with the pG5luc reporter plas-mid into Huh-7cells by using Lipofectamine2000(Invitrogen).Pairedempty plasmids pBIND and pACT were used as negative controls.Cells were harvested48h posttransfection,and renilla-normalizedﬁreﬂy luciferase activities were measured using the Dual-Luciferasereporter assay system(Promega).RNA interference assay.The small interfering RNA(siRNA)speciﬁ-cally targeting HBSP(HBSP-siRNA)or the human CTSB(CTSB-siRNA)was designed at the Whitehead Institute webserver(http://sirna.wi.mit.edu/)and chemically synthesized by Shanghai GenePharma Co.(Zhangjiang Hi-Tech Park,Shanghai,China).A nontargeting siRNA(NC-siRNA)was used as a negative control.Transfections were per-formed using the Lipofectamine2000transfection reagent(Invitrogen)according to the manufacturer’s instructions.Real-time PCR analysis.Total RNA was extracted using the TRIzolreagent(Invitrogen)and converted to cDNA by using the ExScript reversetranscription-PCR(RT-PCR)kit(TaKaRa,Japan).Quantitative real-time PCR was performed in the ABI StepOne real-time PCR system(Ap-plied Biosystems,Foster City,CA)in the presence of SYBR Premix Ex Taq(TaKaRa).The GAPDH(glyceraldehyde-3-phosphate dehydrogenase)Chen et al. Journal of Virologygene was used as a reference gene,and relative mRNA levels were deter-mined using the 2Ϫ⌬⌬CT method.The paired forward and reverse primers were 5=-ACCTCGAACTTTGACAGCGACA-3=and 5=-GATGCCATTCA CGTCGTCCTTA-3=(for MMP-9),5=-CTGTGAGATCACTGGCTTT G-3=and 5=-TTGGAGGGAACAGACGAG-3=(for uPA),and 5=-TGCAC CACCAACTGCTTAGC-3=and 5=-AGCTCAGGGATGACCTTGCC-3=(for GAPDH).Gelatin zymography.Activities of MMP-9in the culture medium of cells were analyzed using gelatin zymography.Cells were serum starved for 24h,after which the cell-conditioned medium was collected and equal amounts of proteins were then separated under nonreducing condition on 10%zymogram gels containing 0.1%gelatin (Invitrogen).After elec-trophoresis,the gel was incubated in the zymogram-renaturing buffer for 30min at room temperature,followed by an overnight incubation at 37°C in the zymogram-developing buffer.The gel was then stained with Coo-massie blue R-250(Sigma)for 30min and destained with the destaining solution for 1h.Clear bands appear on the Coomassie blue R-250-stained blue background in the areas of gelatinolytic activity.In vitro angiogenesis assay.An in vitro angiogenesis assay was imple-mented as described previously,with some modiﬁcations (24).Brieﬂy,conditioned medium was collected from the transfected cells grown in serum-free medium for 16h and used to culture HMEC-1cells,which were seeded in 96-well plates coated with 50␮l Matrigel and incubated for 16h.The formation of the microtubule networks was examined and pho-tographed.The angiogenic effect was measured by counting the lengths of capillary tubes in ﬁve different ﬁelds and expressed as a percentage of the control value.Statistical analysis.Statistical analysis was performed using SPSS software (SPSS 17.0;SPSS Inc.,Chicago,IL).Data are expressed as the means Ϯstandard deviations (SDs).Statistical signiﬁcance (P Ͻ0.05)of differences between groups was determined by Student’s t test.RESULTSHBSP enhances the invasion and migration of hepatoma cells.To explore the functional signiﬁcance of the HBSP protein in hep-atoma cells,we ﬁrst examined the effects of its expression in Huh-7cells on cell invasive and migratory ability.Three individ-ual clones from the Huh-7cells stably transfected with p3ϫFLAG-CMV-10-HBSP or an empty control (p3ϫFLAG-CMV-10)were randomly selected for the following studies.As shown in Fig.1a ,p3ϫFLAG-CMV-10-HBSP-transfected clones HBSP-1,HBSP-2,and HBSP-3expressed a strong band corresponding to the HBSP protein,whereas this band was not detectable in the empty-vec-tor-transfected clones FLAG-1,FLAG-2,and FLAG-3.The effect on invasive potential was examined using a modiﬁed Boyden chamber invasion assay.The number of cells that invadedthroughFIG 1HBSP enhances invasion and migration of hepatoma cells.(a)The three individual clones stably transfected with p3ϫFLAG-CMV-10-HBSP (HBSP-1,HBSP-2,and HBSP-3)or those stably transfected with empty control p3ϫFLAG-CMV-10(FLAG-1,FLAG-2,and FLAG-3)were randomly selected and examined for the presence of HBSP protein by Western blotting.(b)The Matrigel invasion assay was performed with the three HBSP-positive clones and the three negative controls.Thirty-six hours after incubation,the invaded cells were stained with crystal violet,photographed,and counted.(c)The number of invaded cells in each well was counted from six microscopic ﬁelds and expressed as a percentage of the control value.Values are means ϮSDs (n ϭ5).*,P Ͻ0.01(versus controls).(d)Relative motility as visualized by the ability of the cells to close a wound made by creating a scratch through a lawn of conﬂuent cells.(e)Cell migration was assessed at 48h by counting the number of cells that had migrated across the scratch,normalized to the scratch area and expressed as a percentage of FLAG-1controls.Values are means ϮSDs (n ϭ5).*,P Ͻ0.05(versus controls).HBSP Promotes Hepatoma Cell MotilityDecember 2012Volume 86Number 24 13535a layer of Matrigel was analyzed at 36h after the cells were plated on Matrigel-coated transwell inserts.Figure 1b andc show that compared with empty-vector-transfected controls,HBSP-ex-pressing cells signiﬁcantly increased their invasive potential.Like-wise,HBSP-expressing cells migrated through the wound scratch more rapidly than the control cells (Fig.1d and e ).Owing to the fact that all three clones demonstrated very similar results,HBSP-3cells were randomly chosen for all subsequent experi-ments.The FLAG-1cells were used as a negative control.To further conﬁrm that HBSP expression promotes invasion and migration of hepatoma cells,another two HCC cell lines (HepG2and Hep3B)were used to assess the effects of HBSP ex-pression on migratory behaviors.Similarly,when HBSP was ex-pressed in HepG2and Hep3B cells,cell invasion and migration were enhanced signiﬁcantly in the HBSP-expressing HepG2and Hep3B cells compared with those of vector control cells (data not shown).This result indicates that the effect of HBSP on cell mi-gration and invasion is not conﬁned to a single hepatoma cell model.HBSP interacts with CTSB both in vitro and in vivo .We havepreviously shown the evidence of interaction between HBSP and CTSB in a yeast two-hybrid system (8).To test the association between these two proteins in vitro ,we carried out a GST pull-down assay.As shown in Fig.2,GST and the fusion proteins GST-HBSP and GST-HBx were expressed in Escherichia coli and immobilized on glutathione Sepharose beads (Fig.2a ).35S-labeled CTSB,but not the irrelevant 35S-labeled CTSD protein,was re-tained when reacted with bead-bound GST-HBSP fusion protein,while 35S-labeled CTSB was not retained with bead-bound GST-HBx control protein (Fig.2b ).To conﬁrm and extend the result of the GST pulldown assay,we performed an in vivo Co-IP study with the Huh-7cells tran-siently transfected with a pBIND vector that expressed full-length HBSP,HBSP 1–47(lacking the 64-residue C-terminal domain [amino acids 48to 111]),or HBSP 48–111(lacking the 47-residue N-terminal domain [amino acids 1to 47]).As shown in Fig.2c ,full-length HBSP and HBSP 1–47,but not HBSP 48–111,were able to interact with endogenous CTSB,as evidenced by efﬁcient coim-munoprecipitation with the antibody to CTSB (upper panel,lanes 6and 7from the left),indicating that the N-terminal 47aminoFIG 2In vitro and in vivo interactions between HBSP and CTSB.(a)Coomassie blue-stained SDS-PAGE gel showing that GST,GST-HBSP,and GST-HBx recombinant proteins were expressed in E.coli and bound to glutathione Sepharose beads.(b)Representative autoradiogram of in vitro -translated 35S-CTSB captured by GST-HBSP fusion proteins from a GST pulldown assay.Several independent experiments yielded consistent results.The input lane was loaded with 1/10the amount of 35S-labeled proteins used in the binding reactions.GST-HBx fusion protein and 35S-CTSD were used as irrelevant negative controls,demonstrating that HBSP and CTSB bound speciﬁcally under the conditions employed.(c)Coimmunoprecipitation of HBSP and endogenous CTSB after Huh-7cells were transfected with pBIND-HBSP,pBIND-HBSP 1–47,or pBIND-HBSP 48–111and immunoprecipitates (IP)were immunoblotted (IB)for GAL4(upper panel)or CTSB (lower panel).(d)Western blot analysis conﬁrmed expression of full-length and truncated HBSP fused with GAL4DNA-binding domain (left panel)and CTSB fused with VP16activation domain (right panel)in Huh-7hepatoma cells.(e)In vivo association of HBSP with CTSB.A mammalian two-hybrid assay was used to assess the interaction between HBSP and CTSB in Huh-7cells cotransfected with same amount of pBIND-HBSP or pBIND containing truncated-HBSP variants,pACT-CTSB and pG5luc ,and measured for luciferase activity.Empty vectors pBIND and pACT together with pG5luc -cotransfected cells are the negative controls.Values are means ϮSDs (n ϭ5).*,P Ͻ0.01(versus controls).Chen et al.13536Journal of Virologyacid residues of HBSP were indispensable for its interaction with CTSB.In order to conﬁrm the coimmunoprecipitation data and to further validate the domains of HBSP necessary and sufﬁcient for interactions with CTSB,we carried out HBSP-CTSB interaction studies using the mammalian two-hybrid system.Immunoblot analysis revealed that each protein was efﬁciently expressed in the Huh-7hepatoma cells when fused to the GAL4BD or VP16acti-vation domain (AD)(Fig.2d ).Signiﬁcantly increased luciferase activity was observed in cells cotransfected with pBIND-HBSP or pBIND-HBSP 1–47and pACT-CTSB (Fig.2e ).In contrast,there was no discernible difference between the luciferase activities of the pBIND-HBSP 48–111group and negative control (Fig.2e ).These results indicate that the N-terminal 47amino acid residues of HBSP participate in the interaction with CTSB,which conﬁrms the results of the coimmunoprecipitation study.Interaction of HBSP and CTSB contributes to the invasion and migration of hepatoma cells.Given that expression of HBSP in Huh-7cells promotes motility and that we have demonstrated the existence of its interaction with CTSB,we examined the effects of knockdown of either HBSP or CTSB on cell migration and invasion.Small interfering RNAs (siRNAs)targeting HBSP or CTSB expression were separately introduced into HBSP-3cells.An siRNA containing a sequence that does not target any known mammalian gene was used as a control.The expression levels of HBSP and CTSB were analyzed by Western blotting,which showed marked reductions in HBSP and CTSB protein levels (Fig.3a and b )in HBSP-3cells.Wound healing and transwell invasion assays were again used to assess the effects of HBSP and CTSB expression on cell pared with control siRNA-trans-fected cells (negative control),HBSP-3cells with HBSP-siRNA or CTSB-siRNA showed signiﬁcantly impaired cell migration in the wound healing assay (Fig.3c and d )and cell invasion through the Matrigel (Fig.3e and f ).These results provide further evidence that HBSP and CTSB may function in a way to promote the mi-gration and invasion of the hepatoma cells.Interaction of HBSP and CTSB increases expression of MMP-9and uPA.Recent evidence has revealed that proteases,once regarded as little more than cellular garbage disposals or ECM degraders,play more and more important roles in regulating multiple processes during malignant progression,including an-giogenesis,invasion,and metastasis,etc.CTSB has been impli-cated in a proteolytic cascade through interactions with MMPs and the serine protease uPA (14).The fact that high-level expres-sion of proteases has been found to be correlated with invasive growth in many human malignancies suggests that this doesnotFIG 3Downregulation of HBSP or CTSB expression inhibits hepatoma cell invasion and migration.siRNA-mediated knockdown of HBSP (a)or CTSB (b)expression in HBSP-3cells.(c)Relative motility as determined by the ability of the HBSP-3cells,when HBSP or CTSB was knocked down,to close a wound made by creating a scratch through a lawn of conﬂuent cells.(d)Cell migration was assessed by counting the number of cells that had migrated across the scratch.The number of migrated cells was normalized to the scratch area and expressed as a percentage of the nontargeting siRNA control value.Values are means ϮSDs (n ϭ5).*,P Ͻ0.01(versus controls).(e)The Matrigel invasion assay was performed with the HBSP-siRNA,CTSB-siRNA,and NC-siRNA cells.Thirty-six hours after incubation,the invaded cells were stained with crystal violet,photographed,and counted.(f)The number of invaded cells in each well was counted from six microscopic ﬁelds and expressed as a percentage of the control.Values are means ϮSDs (n ϭ5).*,P Ͻ0.01(versus controls).HBSP Promotes Hepatoma Cell MotilityDecember 2012Volume 86Number 24 13537occur by chance.To understand the potential mechanisms under-lying HBSP-induced increased invasion and migration of hepa-toma cells,the relative expression levels of MMP9and uPA in the HBSP-3cells were quantiﬁed by quantitative reverse transcrip-tion-PCR (qRT-PCR).The mRNA levels of MMP9and uPA in the HBSP-3cells were 3.69-fold and 8.23-fold higher,respectively,than those of control cells (Fig.4a ).Conversely,when HBSP or CTSB was knocked down in the HBSP-3cells by siRNA,the ex-pression of MMP-9or uPA was signiﬁcantly reduced.Consistent with the changes in mRNA level,the level of MMP-9or uPA pro-tein also changed accordingly with the manipulations of HBSP and CTSB expression (Fig.4b and c ).Thus,both HBSP and CTSB may regulate expression of MMP-9and uPA at the mRNA and protein levels.Next,gelatin zymography was utilized to evaluate the enzy-matic activity of MMP-9(Fig.4d and e ).Both active forms of MMP-2(62kDa)and MMP-9(82kDa)were detectable in cul-ture-conditioned media from all the cell lines used,including the controls.Speciﬁcally,enzymatic activity of MMP-9in HBSP-3cells was enhanced compared to that in FLAG-1control cells.On the other hand,siRNA knockdown of HBSP or CTSB led to a moderate decrease in the MMP activity present in these cells.Interaction of HBSP and CTSB promotes in vitro angiogen-esis.Angiogenesis is an important aspect of tumor progression and metastasis.CTSB,MMP,and uPA are well known to be over-expressed and implicated in angiogenesis during tumor progres-sion (2,23).To assess the effects of HBSP-CTSB interactions on tumor-induced vessel formation,HMEC-1cells were cultured in the conditioned medium collected from transfected or control cells.Figure 5a and b show that the endothelial cells formed well-deﬁned capillary-like networks after 16h of growing under the conditioned medium from HBSP-3cells.In contrast,the condi-tioned media harvested from cells transfected with HBSP-siRNA or CTSB-siRNA decreased endothelial capillary formation.These results suggest that both HBSP and CTSB are likely to play prom-inent roles in tumor-induced angiogenesis in vitro .Interaction of HBSP and CTSB interferes with intracellular signaling events of MAPK and PI3K pathways.To identifypos-FIG 4Interaction of HBSP and CTSB increases expression of MMP9and uPA.(a)The relative mRNA levels (RQ)of MMP-9and uPA in FLAG-1,HBSP-3,HBSP-3–HBSP-siRNA,HBSP-3–CTSB–siRNA and HBSP-3–NC-siRNA cells were measured by quantitative RT-PCR.Values are means ϮSDs (n ϭ5).*,P Ͻ0.01(versus FLAG-1negative control);**,P Ͻ0.01(versus NC-siRNA control).(b and c)Western blot analysis of MMP9and uPA expression in the ﬁve cell lines.The protein expression levels in the HBSP-3,HBSP-3–HBSP-siRNA,and HBSP-3–CTSB-siRNA cells relative to that in the FLAG-1cells was calculated densitometrically after normalization to the level of ␤-actin.Values are means ϮSDs (n ϭ5).*,P Ͻ0.01(versus FLAG-1negative control);**,P Ͻ0.01(versus HBSP-3–NC-siRNA control).(d and e)Gelatin zymography to determine the activities of MMP-9in the cells was performed as described in Materials and Methods.Bands or signals detected by zymography were quantiﬁed using densitometric analysis.Values in panel e are means ϮSDs (n ϭ5).*,P Ͻ0.01(versus FLAG-1negative control);**,P Ͻ0.01(versus HBSP-3–NC-siRNA control).Chen et al.13538Journal of Virology。

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