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Evaluation of the optimum induction gap for the GEM-MIGAS Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 600, Issue 3, 11 March 2009, Pages . Mir, . Rhodes, . Schooneveld, . Concei�0�4�0�0o, . Maia, . Veloso, . dos Santos Show preview | PDF (201 K) | Related articles | Related reference work articles 2Structure–function studies of the G-domain from human gem, a novel small G-protein FEBS Letters, Volume 580, Issue 25, 30 October 2006, Pages 5959-5964Yarden Opatowsky, Yehezkel Sasson, Isabella Shaked, Yvona Ward, Orna Chomsky-Hecht, Yael Litvak, Zvi Selinger, Kathleen Kelly, Joel A. Hirsch Show preview | PDF (787 K) | Supplementary content | Related articles | Related reference work articles 3Tanzanite as conflict gem: Certifying a secure commodity chain in Tanzania Original Research ArticleGeoforum, Volume 41, Issue 1, January 2010, Pages 56-65Richard A. Schroeder Show preview | PDF (234 K) | Related articles | Related reference work articles 4GEM simulation methods development Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 478, Issues 1-2, 1 February 2002, Pages 452-459V. Tikhonov, R. Veenhof Show preview | PDF (381 K) | Related articles | Related reference work articles 5BoNus: Development and use of a radial TPC using cylindrical GEMs Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 592, Issue 3, 21 July 2008, Pages 273-286H. Fenker, N. Baillie, P. Bradshaw, S. Bueltmann, V. Burkert, M. Christy, G. Dodge, D. Dutta, R. Ent, J. Evans, R. Fersch, K. Giovanetti, K. Griffioen, M. Ispiryan, C. Jayalath, N. Kalantarians, C. Keppel, S. Kuhn, G. Niculescu, I. Niculescu, et al. Show preview | PDF (1819 K) | Related articles | Related reference work articles 6Progress on large area GEMs Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 628, Issue 1, 1 February 2011, Pages 182-186Marco Villa, Serge Duarte Pinto, Matteo Alfonsi, Ian Brock, Gabriele Croci, Eric David, Rui de Oliveira, Leszek Ropelewski, Hans Taureg, Miranda van Stenis Show preview | PDF (1022 K) | Related articles | Related reference work articles 7Properties of the GEM, double GEM and GEM+MGC combination Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 443, Issues 2-3, 1 April 2000, Pages 375-385T. L. van Vuure, F. D. van den Berg, C. W. E. van Eijk, R. W. Hollander Show preview | PDF (395 K) | Related articles | Related reference work articles 8Development of thick-foil and fine-pitch GEMs with a laser etching technique Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 608, Issue 3, 21 September 2009, Pages 390-396T. Tamagawa, A. Hayato, F. Asami, K. Abe, S. Iwamoto, S. Nakamura, A. Harayama, T. Iwahashi, S. Konami, H. Hamagaki, . Yamaguchi, H. Tawara, K. Makishima Show preview | PDF (623 K) | Related articles | Related reference work articles 9Atmospheric gaseous elemental mercury (GEM) over a coastal/rural site downwind of East China: Temporal variation and long-range transport Original Research ArticleAtmospheric Environment, Volume 45, Issue 15, May 2011, Pages 2480-2487Zhijia Ci, Xiaoshan Zhang, Zhangwei Wang, Zhenchuan Niu Show preview | PDF (1306 K) | Supplementary content | Related articles | Related reference work articles Highlights�7�8 Elevated GEM levels were observed at a coastal/rural site downwind of East China. �7�8 GEM showed high levels in cold seasons and low levels in warm seasons. �7�8 GEM showed peak levels in daytime and low levels in late night and early morning. �7�8 Complicated patterns of GEM transport from different source regions were recorded. 10The PANDA GEM-based TPC prototype Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 628, Issue 1, 1 February 2011, Pages 204-208L. Fabbietti, H. Angerer, R. Arora, R. Beck, M. Berger, P. Bühler, M. Cargnelli, S. Doerheim, J. Hehner, N. Herrmann, C. H�0�2ppner, D. Kaiser, B. Ketzer, K. Mladen, V. Kleipa, I. Konorov, J. Kunkel, R. Lalik, M. Lang, Y. Leifels, et al. Show preview | PDF (697 K) | Related articles | Related reference work articles 11Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1 Original Research ArticleBiochemical Pharmacology, Volume 49, Issue 7, 30 March 1995, Pages 929-939Ruiwen Zhang, Robert B. Diasio, Zhihong Lu, Tiepu Liu, Zhiwei Jiang, Wayne M. Galbraith, Sudhir Agrawal Show preview | PDF (1639 K) | Related articles | Related reference work articles 12Chordal co-gem-free and (P5,gem)-free graphs have bounded clique-width Original Research ArticleDiscrete Applied Mathematics, Volume 145, Issue 2, 15 January 2005, Pages 232-241Andreas Brandst�0�1dt, Hoàng-Oanh Le, Raffaele Mosca Show preview | PDF (232 K) | Related articles | Related reference work articles 13Further studies of the GEM photomultiplier Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 442, Issues 1-3, 11 March 2000, Pages 68-73A. Buzulutskov, A. Breskin, R. Chechik, G. Garty, F. Sauli, L. Shekhtman Show preview | PDF (301 K) | Related articles | Related reference work articles 14Charge transfer of GEM structures in high magnetic fields Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 535, Issues 1-2, 11 December 2004, Pages 330-333S. Roth Show preview | PDF (296 K) | Related articles | Related reference work articles 15Short induction gap gas electron multiplier (GEM) for X-ray spectroscopy Original Research ArticleNuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Volume 573, Issues 1-2, 1 April 2007, Pages . Mir, R. Stephenson, . Rhodes, . Schooneveld, . Veloso, . Dos Santos

用pLexA的5′和3′端载体臂引物测序，证实插入子HERPUD1读码框正确、无碱基突变。人胎脑文库扩增及质粒DNA的提取Human Fetail Brain MATCHMAKER cDNA文库构建于pB42AD质粒载体?17修回日期：2005； QIAGEN Plasmid Mega Kit 购自 QIAGEN公司。关键词神经病学神经元蜡样脂褐质沉积症蛋白质相互作用酵母双杂交HerpBax inhibitor?α.1诱饵质粒pLexA?HERPUD1的构建PCR扩增HERPUD1编码区，正反向引物分别为5′，有自激活性?磷酸多萜醇低聚糖和其他物质、指纹状，低熔点琼脂糖回收目的带，收集于锥形瓶中;?Trp培养基Bax Inhibitor?1与Herp的相互作用中国生物工程杂志China Biotechnology，探讨编码该蛋白的基因HERPUD1的调节机制，为研究NCLs发病的分子机理提供依据, 材料胎脑MATCHMAKER LexA cDNA文库，将Herp与筛选到的相互作用蛋白再一对一回复进行酵母双杂交实验。细胞中溶酶体水解酶出现生化异常，去除假阳性?Ura/。每皿加LB培养液10ml，刮下菌落,铺于SD /?1收稿日期，则菌落成蓝色，否则为白色。质粒+EGY48（p8op，线粒体ATP酶的亚基C在多型NCLs中显著沉积?Trp/，包括金属和蛋白质等〔2〕。根据溶酶体蛋白酶基因缺陷和结构蛋白的功能失调〔3〕，这组神经变性疾病被认为是一种新的溶酶体脂褐质沉积疾病。1,EGY48（p8op?lacZ），HERPUD1基因序列由美国纽约州立发育不全研究所Nanbert Zhong博士惠赠。结果得到其中1个阳性克隆的插入子序列与TEGT基因序列一致、进行性智力障碍，X?Ura。对阳性克隆插入子的DNA序列测序，在GenBank中作匹配及生物信息学分析，编码蛋白为Bax inhibitor?1。得出结论, 25(11)?3′, 5′?GGTGAATTCGTTTGCGATGGCTGGGGGGCCTTC?3′.6阳性克隆质粒的分离抽提阳性酵母克隆质粒，电转化至大肠杆菌kc8中。目前，已经鉴定了6个致病基因（CLN1~3?Leu培养基，X?α?gal等购自Clontech公司;Amp+/Trp?培养基上筛选分离得到靶质粒。重建Herp和相互作用蛋白的相互关系消除假阳性按相互作用重建阳性和阴性对照的4组合：（1）pLexA?HERPUD1+pB42AD?Y；（2）pB42AD?Y自激活性检测：pLexA + pB42AD?Y ；（3）相互作用阳性对照pLexA?Pos+ pB42AD?T；（4）相互作用阴性对照pLexA?Lam+pB42AD。初步验证其相互作用，其中再次变蓝色者进行测序。相互作用蛋白基因Y的DNA测序和生物信息学分析以pB42AD的5′端和3′端载体臂引物测序，对重建相互作用仍为阳性的pB42AD?Y测序，测序结果在互联网上与NCBI GenBank，EMBL等生物信息数据库做BLAST分析，应用GCG软件分析可能的阅读框。2结果诱饵质粒的验证pLexA?HERPUD1转化大肠杆菌DH5α培养后提取质粒，对质粒进行PCR扩增，产生约925bp的条带（图1），经EcoRI酶切，也产生约925bp的条带（图2）。图1pLexA?HERPUD1 PCR产物电泳图Fig. 1Electrophoretic map of PCR product of pLexA?HERPUD 11: Product of PCR; M: 200bp DNA step ladder DNA marker图2pLexA?HERPUD1经EcoRI酶切产物电泳图Fig. 2Electrophoretic map of product of pLexA?HERPUD 1 by EcoRI digestionM：Lambda DNA/EcoRI+HindIII marker；1: pLexA?HERPUD1；2: pLexA；3: Product of pLexA?HERPUD1 digested by EcoRIpLexA?HERPUD1测序结果证实诱饵质粒的插入子无突变、阅读框正确。测序结果见图3。阳性克隆的DNA测序及生物信息学分析对反复检测Leu和LacZ 2个报告基因呈阳性表达的1个相互作用蛋白克隆DNA测序，测序获得680bp序列（图4），在GenBank做BLAST分析，发现与TEGT基因的序列一致，GCG软件分析编码区读框正确，TEGT基因编码蛋白为Bax inhibitor?1，生物信息学分析Bax inhibitor?1是一种基因增强转录因子，与BCL2和BCL?X相互作用，调节细胞凋亡。图3pLexA?HERPUD1融合质粒部分序列 Fig. 3Partial sequence of pLexA?HERPUD1 fusion plasmid图4从人胎脑cDNA文库筛选得到的一个阳性克隆质粒的插入子DNA部分测序结果Fig. 4Partial sequencing result of one positive clone screened from the human fetal cDNA library3讨论NCLs是一组致死性疾病，目前尚无有效的治疗方法。了解CLN基因及其编码蛋白的功能，对阐明病理、遗传及发病的分子机制具有重要意义。目前已知NCLs至少由8种不同的基因（CLN1～8）发生突变引起。青少年型（JNCL，NCL3）的致病基因为CLN3。CLN3编码蛋白Battenin位于溶酶体膜，为溶酶体必需的膜糖蛋白，参与哺乳动物细胞溶酶体pH的调节并且具有营养神经作用〔11〕。Herp是Battenin的相互作用蛋白质，Sai等〔12〕发现Herp与早老素（presenilin）相互作用，增加淀粉β蛋白（amyloid beta?protein，Abeta）的退化，说明Herp与细胞生长发育相关，因此研究Herp的功能对阐明NCLs的发病机理有重要意义。体内许多生化反应及代谢过程都存在蛋白质与蛋白质之间的相互作用，蛋白质之间的互作在生命过程中起着极其重要的作用，本研究应用酵母双杂交体系筛选得到一个与Herp相互作用的蛋白——Bax inhibitor?1，经查阅相互作用蛋白数据库IntAct和DIP，发现它是一种新的Herp互作蛋白。Chae等〔13〕研究发现Bax inhibitor?1调节与内质网应激相关的凋亡途径，在内质网诱导的凋亡中通过抑制Bax活化和线粒体移位介导保护、稳定线粒体膜电压，抑制Caspase活化。Bax inhibitor?1是一个进化保守的内质网蛋白，是一种凋亡调节因子，与BCL2（B细胞淋巴瘤/白血病?2）和BCL?X （B细胞淋巴瘤/白血病?X）相互作用，BCL2和BCL?X这2种基因是凋亡相关基因，Herp能与Bax inhibitor?1相互作用，提示Herp与凋亡调节相关。NCLs是一种神经元退行性病变，Herp、Bax inhibitor?1、BCL2、BCL?X在NCLs的发病过程中通过相互协调或拮抗促进神经细胞凋亡或死亡，但其中具体机制，如基因表达调控网络、细胞信号传导通路的改变、细胞离子的具体变化等机制仍需进一步探索。参考文献〔1〕 Wisniewski K E, Kida E, Connell F, et al. Neuronal ceroid lipofuscinoses: research update. Neurol Sci， 2000, 21(Suppl): 49~56〔2〕 Prasal V V, Pullarkat R K. Brain lysosomal hydrolases in neuronal ceroid lipofuscinoses. Mol Chem Neuropathol, 1996,29(2?3):169~179〔3〕 Mole S, Zhong N, Sarpong A, et al. New mutations in the neuronal ceroid lipofuscinosis genes. Eur J Pediatr Neurol, 2001,5 (Suppl):7~10〔4〕 Goebel H H. The neuronal ceroid lipofuscinosis. J Child Neurol,1995,10:424~437〔5〕 Mole S E. The genetic spectrum of human neuronal ceroid lipofuscinoses. Brain Pathol, 2004, 14(1):70~76〔6〕 Sohar I, Sleat D E, Jadot M, et al. Biochemical characterization of a lysosomal protease deficient in classical late infantile neuronal ceriod lipofuscinoses(LINCL) and development of an enzyme?based assay for diagnosis and exclusion of LINCL in human specimens and animal models. J Neurochem, 1997,73：700~711〔7〕 Ranta S, Savukoski M, Santavuori P, et al. Studies of homogenous populations∶CLN5 and CLN8. Adv Genet, 2001,45：123~140〔8〕 Zhong N, Ju W, Moroziewicz D N, et al. Molecular pathogenic studies of Batten disease: identification and charaterization of Battenin?interactive proteins (Herp). J Mol Diag, 1999,1(1):G23〔9〕 Zhong N, Ju W, Moroziewicz D, et al. Prenatal diagnostic testing for infantile and late\|infantile neuronal ceroid lipofusinoses(NCL) using allele specific primer extension(ASPE). Journal of Peking University(Health Sciences), 2005,37(1):20~25〔10〕 Zhong N, Ju W, Moroziewicz D, et al. Finding of Battenin?interactive protein (BIP) may lead to understanding the pathogenic mechanism for neuronal ceroid lipofuscinoses. Genet Med, 2001, 3(3):140〔11〕 Puranam K L ,Guo W X, Qian W H,et al. CLN3 defines a novel antiapototic pathway operative in neurodegeneration and mediated by ceramide. Mol Genet Metab, 1999, 66:294~308〔12〕 Sai X, Kokame K, Shiraishi H, et al. The ubiquitin?like domain of Herp is involved in Herp degradation, but not necessary for its enhancement of amyloid beta?protein generation. FEBS Lett, 2003,553(1?2):151~156〔13〕 Chae H J, Kim H R, Xu C, et al. BI?1 regulates an apoptosis pathway linked to endoplasmic reticulum stress. Mol Cell, 2004,15(3):355~366Screening and Bioinformatic Analysis of the Proteins Interacting With HerpLI Bin?yuan1HE Shu?ya1WANG Gui?liang1MA Yun1XIAO Wei?chun1LI Jie1SUN Chun?li1MIN Ling?feng1YU Jia1Nanbert Zhong2(1 Department of Biochemistry and Molecular Biology, Nanhua UniversityHengyang421001, China)(2 Department of Human Genetics, New York State Institute for Basic ResearchNew YorkNY10314, USA )AbstractThe aim is to screen the interactive proteins of Herp by yeast two hybrid system. The eukaryotic expression vector,pLexA HERPUD1, was constructed, MATCHMAKER LexA was used to screen the human fetal cDNA library, and the inserts of positive clones were regarded as the candidate interactive proteins of Herp. The yeast two hybrid was repeated to screen the proteins and remove the false positive clones. One positive inserted fragment is accordant with the sequence of TEGT which codes Bax inhibitor?1. Herp interacts with Bax inhibitor?1 with the characteristic of regulating apoptosis, hinting that Herp is involved in regulating wordsNeurologyNeuronal ceroid lipofuscinosesProtein interactionYeast two hybrid systemHerpBax inhibitor?1。37℃培养12h，根据克隆的生长数计算文库滴度, 25(11)李斌元 等：Bax Inhibitor?1与Herp的相互作用中国生物工程杂志 China Biotechnology 20051。1材料与方法1，用T4 DNA连接酶将HERPUD1插入pLexA?HTU+X?LacZ）融合菌株的建立小量分步转化法将已经构建的Bait质粒pLexA?HERPUD1全部转化EGY48（p8op?LacZ）储备待用。，MATCHMAKER LexA酵母双杂交系统,酵母SD培养基，SD/, SD/，5：2005?05，电子信箱：神经元蜡样脂褐质沉积症 (neuronal ceroid lipofuscinoses，其它化学试剂均为国内购买。测序公司为上海生工生物工程技术服务有限公司。方法1：Herp与Bax inhibitor?1相互作用，Bax inhibitor?1具有调节凋亡特性，提示Herp可能参与凋亡调节；kc8大肠杆菌，Bait质粒（pLexA?HERPUD1）与pB42AD共转EGY48（p8op?LacZ），铺于SD;? Ura/,7〕。Battenin存在于溶酶体和线粒体中，其功能尚不清楚。Zhong等〔8~10〕研究发现Herp能与Battenin相互作用，文库独立克隆数（cfu）是×106。根据滴度和独立克隆数计算所需文库原始菌液体积和铺皿个数。液体培养液（Amp+）稀释文库原始菌液，铺于150mm的LB皿（Amp+），共铺皿80个, SD/，以基因HERPUD1的编码序列（coding sequence）为诱饵（bait）?07?22*国家自然科学基金资助项目（30370795）** 通讯作者、运动失调和行为变化为主要特征〔1〕,SD/, NCLs)是一组人类进行性神经元变性疾病;?Leu/Raf/Gal，培养4～6d，8），所致疾病分别为NCL1~3，5，6，8〔4,5〕;?Ura/His, SD/，在M9/.2，其主要功能也不清楚，醋酸锂等购自Sigma公司;?His/;?Ura/His?Trp/、曲线状，多为常染色体隐性遗传。临床上以快速的视力恶化、癫痫发作;?His?Trp 。用QIANGEN Plasmid Mega Kit 抽提质粒，置-20℃保存待用。诱饵质粒的毒性和自激活性检测采用小规模共转化法、直线状和混合状样沉积，导致以神经细胞和视网膜细胞为主的细胞凋亡。NCLs至少可分为10种疾病亚型。共同病理特征主要为溶酶体内蜡样脂褐质以颗粒状;? Ura/.5文库的转化与筛选制备酵母菌（已经转化pLexA?HERPUD1表达质粒）感受态，醋酸锂法转入文库质粒;?His?Trp，培养 4～6d。刮取菌液铺于SD/，6、2，另外，在沉积的脂褐质中还可以发现磷脂、中性脂质。CLN3编码的Battenin蛋白被认为是跨膜蛋白〔6，从人胎脑cDNA文库筛选与Herp有相互作用的蛋白质，以研究Herp的功能，Herp是定位于内质网的高半胱氨酸应答的类似泛素的区域构件蛋白1，编码该蛋白的基因为HERPUD1?gal皿，30℃倒置培养4~6d。如Bait有毒性则无菌落生长?TTCGAATTCATGGAGTCCGAGACCGAACC。本研究采用MATCHMAKER LexA 双杂交系统（MATCHMAKER LexA Two?Hybrid System）.2；半乳糖(Gal)，棉子糖(Raf)，HERPUD1插入T载体，T? HERPUD1与pLexA分别用EcoRI酶切?gal滤膜分析克隆变为蓝色者初步认为是阳性克隆（设为pB42AD?Y）:16～20李斌元1何淑雅1**王桂良1马云1肖卫纯1李洁1孙春丽1闵凌峰1虞佳1Nanbert Zhong2（1 南华大学生物化学与分子生物学研究室衡阳4210012 纽约州立基础研究所人类遗传研究室纽约NY10314）摘要应用酵母双杂交技术筛选Herp的相互作用蛋白。构建编码Herp的基因HERPUD1真核表达载体HERPUD1?plexA，应用MATCHMAKER LexA酵母双杂交系统筛选人胎脑cDNA文库，获得的阳性克隆的插入子为Herp的候选相互作用蛋白质