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收稿日期:2009-05-10作者简介:贾薇(1981-),男(汉族),辽宁铁岭人,博士研究生,E -m a i l :i d j i a w e i @126.c o m ;宫平(1964-),男(汉族),新疆乌鲁木齐人,教授,博士生导师,主要从事抗肿瘤、抗病毒和心脑血管药物的设计与合成研究,T e l :(024)23986426,E -m a i l :g o n g p i n g g p @126.c o m 。
文章编号:1005-0108(2010)01-0064-06法呢醇X 受体激动剂研究进展贾薇,李伟,宫平(沈阳药科大学制药工程学院,辽宁沈阳110016)摘 要:法呢醇X 受体(F X R )通过调节胆汁酸的合成、分泌和吸收来调节胆汁酸的代谢和稳态。
法呢醇X 受体激动剂有望成为治疗胆汁酸代谢相关疾病的新型药物。
该文对近年来报道的法呢醇X 受体激动剂进行简要综述。
关键词:法呢醇X 受体;激动剂;鹅脱氧胆酸;6-E C D C A ;W A Y -362450中图分类号:R 914 文献标志码:AP r o g r e s s o nt h e d e v e l o p m e n t o f f a r n e s o i dXr e c e p t o r a g o n i s t sJ I AWe i ,L I We i ,G O N GP i n g(S c h o o l o f P h a r m a c e u t i c a l E n g i n e e r i n g ,S h e n y a n g P h a r m a c e u t i c a l U n i v e r s i t y ,S h e n y a n g 110016,C h i n a )A b s t r a c t :F a r n e s o i d Xr e c e p t o r (F X R ),a k e yc o n t r o l l e r o f b i l e a c i d h o m e o s t a s i s a n dm e t a b o l i s m ,r e g u l a t e s b i l e a c i d s y n t h e s i s ,s e c r e t i o n ,a n d a b s o r p t i o n .F X Ra g o n i s t s a r e e x p e c t e d t o b e n e wk i n d o f t h e r a p e u t i c d r u g s f o r d i s e a s e s r e l a t e d t o b i l e a c i d m e t a b o l i s m .T h e p r o g r e s s o n t h e r e s e a r c h o f F X Ra g o n i s t s i n r e c e n t y e a r s w a s s u m m a r i z e d .K e y w o r d s :f a r n e s o i d Xr e c e p t o r ;a g o n i s t ;C D C A ;6-E C D C A ;W A Y -362450 法呢醇X 受体(F X R )是核受体超家族成员,在肝脏、肾脏、肾上腺和小肠组织中高表达,同时还存在于脂肪组织、心脏、脾脏和血管平滑肌中。
补阳还五汤乙醇、氯仿提取物挥发性成分及调控鼠骨髓间充质干细胞活性研究王春燕;曾和平;周亚东;陈薇【摘要】采用GC-MS对中药复方补阳还五汤的乙醇及氯仿提取物进行分析,结果发现2种溶剂提取物中挥发性成分大致相同;通过对补阳还五汤2种溶剂提取物进行骨髓间充质干细胞增殖活性的测定,发现2种溶剂提取物均促进鼠骨髓间充质干细胞的增殖(P＜0.05).【期刊名称】《华南师范大学学报（自然科学版）》【年(卷),期】2007(000)003【总页数】5页(P88-92)【关键词】补阳还五汤;气相-质谱联用(GC-MS);骨髓间充质干细胞(MSCs)【作者】王春燕;曾和平;周亚东;陈薇【作者单位】华南师范大学化学与环境学院,广东广州,510631;华南师范大学化学与环境学院,广东广州,510631;华南师范大学化学与环境学院,广东广州,510631;华南师范大学化学与环境学院,广东广州,510631【正文语种】中文【中图分类】O62补阳还五汤是中医治疗缺血性脑卒中的传统名方，它的主要组方是黄芪60 g，当归8 g，赤芍8 g，广地龙8 g，红花5 g，桃仁8 g.临床疗效确切，功效在补气活血、通经活络,常用于缺血性中风或出血性中风后遗症、周围性面瘫属气虚血瘀者的治疗[1].国内研究证实它有保护脑组织、减轻神经缺失症状.刘柏炎等人通过实验研究了中药补阳还五汤对大鼠局灶性脑缺血后内源性神经干细胞(NSC)反应的影响，从而得出局灶性脑缺血后，NSC激活、增殖，补阳还五汤能促进NSC的增殖的结论，其作用机制可能与其能调控微血管舒缩功能，改善大脑血供有关[2].此外，补阳还五汤还具有营养和促进神经干细胞生长的作用.孙晋浩等人利用Wistar雄性大鼠进行实验，观察不同培养时间内细胞的生长状况、细胞活性及神经元烯醇化酶和胶质纤维酸性蛋白的表达.结果显示，药物血清组神经干细胞的生长明显好于对照组，OD值高于对照组；免疫组化染色显示，药物血清组中NSE及GFAP 免疫阳性细胞均多于对照组，即药物血清组的神经干细胞更多地分化成神经元和神经胶质细胞[3].从而说明补阳还五汤药物血清可以促进神经干细胞的生长存活和向神经元及胶质细胞的分化.蔡光先等从新生Wistar大鼠海马分离培养神经干细胞(NSCs)，并低糖低氧体外模拟脑缺血损伤,运用MTT法、克隆计数观察损伤后NSCs的增殖情况，研究发现：低糖低氧损伤后，部分NSCs死亡，含药血清加入后克隆增大，克隆数增多；MMT实验测得吸光度值显示补阳还五汤可促进低糖低氧损伤NSCs的增殖[4].通常认为骨髓间质干细胞(MSCs)的主要功能是参与构成造血干细胞生存和分化的微环境，近年来发现，它还具有向多种细胞系分化的潜能[5-7]，为治疗神经损伤、帕金森病、早老性痴呆等神经退行性疾病带来了希望[8].但是骨髓中的MSC含量极少[9]，扩增速度慢，无法大量满足临床需求.因此，从传统中药寻找能够促进MSCs增殖的活性物质就成了一项有意义的研究.张国福等研究了补阳还五汤对骨髓间质干细胞移植治疗大鼠脊髓损伤的影响，实验用SD大鼠，采用改良Allen法制备大鼠T10脊髓损伤模型，研究发现：静脉注射移植的MSCs能够迁移到脊髓损伤组织，并促进神经功能的恢复.补阳还五汤能促进移植的MSCs迁移，同时有利于脊髓功能的恢复[10].虽然，基于补阳还五汤对干细胞作用的研究已经很多，但至今未报道补阳还五汤有机溶剂提取物对骨髓间质干细胞增殖作用的研究.GC-MS已广泛应用于单味药材挥发油的成分分析,大多数含挥发油的药材其化学成分已基本搞清,为中药复方挥发油的研究积累了丰富的数据，采用GC-MS对复方制剂挥发油研究,确定其化学成分可进一步提高对复方挥发油药效物质基础的认识.本研究用2种不同的溶剂(乙醇和氯仿)提取补阳还五汤，并对2种溶剂提取物进行GC-MS分析，发现2种溶剂提取物中挥发性成分大致相同.实验还证明2种溶剂提取物均具有促进骨髓间充质干细胞增殖的作用.1 实验部分1.1 实验仪器及材料复方补阳还五汤(购自广州采芝林药店)、无水乙醇(分析纯，广州试剂厂)、氯仿(分析纯，广州试剂厂)、索氏提取器、Trace GC Ultra Trace -DSQ气-质联用仪(FINNIGAN)，细胞培养及免疫组化试剂：低糖DMEM(L-DMEM)、 Percoll 和胎牛血清(FBS)为GIBCOL公司产品.抗体免抗CD34、CD44、CD45、生物素化二抗(羊抗兔)、SABC、DAB染色试剂盒均购自武汉博士德公司.MTT为SIGMA公司产品.清洁级Sprague-Dawley大鼠10只，220～250 g，雄性，月龄1个月，由广州中医药大学实验室动物中心提供(粤检证字2003A010号).1.2 实验方法1.2.1 无水乙醇提取补阳还五汤取复方中药补阳还五汤100 g，加入到索氏提取器，并加入300 mL无水乙醇，抽提3 h，溶剂挥发干，得补阳还五汤的乙醇提取物3.0 g.取10 mg用于测定干细胞活性，取少许用于GC-MS测定.1.2.2 氯仿提取补阳还五汤取复方中药补阳还五汤100 g，加入到索氏提取器，并加入300 mL氯仿，抽提3 h，溶剂挥发干，得补阳还五汤的氯仿提取物2.6 g.取10 mg用于测定干细胞活性，取少许用于GC-MS测定.1.2.3 气质联用及数据测定色谱条件：DB-5石英毛细管柱(30 m×φ0.25 mm),载气为氦气，柱前压50 kp，流速：1 mL/min，分流比为10∶1，进样口温度Tj：295 ℃.柱温Tc：60 ℃(5 min)，以8 ℃/min升至295 ℃(30 min).质谱条件：EI 源，离子源温度250 ℃，轰击电子能量70 Ev，电子倍增器电压1.7 kv，质量扫描范围28-500 amu.分别对补阳还五汤无水乙醇和氯仿提取物进行GC-MS研究.1.2.4 鼠骨髓间充质干细胞生物活性测定鼠骨髓间充质干细胞生物活性测定由广州中医药大学完成，活性测定采用MTT 法.实验对象为取自大鼠的骨髓间充质干细胞,样品用DMSO作溶剂，配成浓度4.95 g/L.实验分为空白对照组(只加培养基和细胞)、溶剂组(加DMSO溶剂和细胞)、2个样品的4.95 g/L组(加样品的DMSO溶液及细胞)及不加细胞组(加样品溶液不加细胞).结果显示，补阳还五汤的无水乙醇及氯仿提取物具有促进骨髓间充质干细胞增殖的作用.所得数据用均数±标准差(X±S)表示，采用SPSS软件进行方差分析.2 结果与讨论2.1 GC-MS测定结果图1 乙醇提取物挥发油总离子图2.1.1 GC-MS测定补阳还五汤乙醇提取物结果对补阳还五汤乙醇提取物进行GC-MS检测，得到乙醇提取物中挥发油的总离子图，如图1.解析质谱并参阅有关质谱图，共分离鉴定了20种成分，具体成分名称、含量及其保留时间见表1.其主要成分是9-烯十八酸乙酯(24.15%)，其次是5，7，8-三甲基二氢香豆素(15.06%)，6，9-十八二烯酸(12.21%).图2 氯仿提取物挥发油总离子图2.1.2 GC-MS测定补阳还五汤氯仿提取物结果对补阳还五汤氯仿提取物进行GC-MS检测，得到氯仿提取物中挥发油的总离子图，如图2.解析质谱并参阅有关质谱图，共分离鉴定了23种成分，具体成分名称、含量及其保留时间见表2.其主要成分是9-烯十八酸甲酯(20.93%)，其次是9-烯十八酸乙酯(17.74%)，6，9-十八二烯酸(12.34%).表1 补阳还五汤乙醇提取物挥发油成分序号保留时间/min化合物名称分子式相对含量/%17．95苯甲酸C7H6O21．74214．032，6-二叔丁基-4-甲基苯酚C15H24O1．32316．471-(2，4-二甲基苯基)-1-丙酮C11H14O1．29416．77丁烯基酞内酯C12H12O21．36517．665，7，8-三甲基-3，4-二氢香豆素C12H14O215．06618．58á-甲基苯丙醛C10H12O0．8720．2棕榈酸甲酯C17H34O21．58820．7棕榈酸C16H32O23．82921．1棕榈酸乙酯C18H36O24．671022．469-烯十八酸甲酯C19H36O29．451122．97亚油酸C18H32O212．211223．26油酸乙酯C20H38O224．151323．58十八酸乙酯C20H40O21．151424．81正十九烷C19H402．781526．199，17-十八二烯醛C18H32O3．131626．851-三十七烷醇C37H76O2．921729．2二十七烷C27H563．261832．2正二十九烷C29H604．071936．64二十八烷C28H582．982037．21胆固醇C27H46O2．26表2 补阳还五汤氯仿提取物挥发油成分序号保留时间/min化合物名称分子式相对含量/%16．95壬醛C9H18O1．2927．93苯甲酸C7H6O21．5339．852-癸烯酸C10H18O1．08416．464’-乙基苯丙酮C11H14O1．29516．77丁烯基酞内酯C12H12O21．48617．665，7，8-三甲基-3，4-二氢香豆素C12H14O27．66718．591-丙烯基卞醇C10H12O1．23819．013，7，11，15-四甲基-2-十六碳-1-醇C20H40O0．88919．62乙二醇-2-[9-十八烯醇]-醚C20H40O20．781020．22十六酸甲酯C17H34O22．611120．7十六酸C16H32O23．631221．1十六酸乙酯C18H36O21．971322．459-十八碳烯酸甲酯C19H36O220．931422．97亚油酸C18H32O212．341523．269-十八烯酸乙酯C20H38O217．741624．8正十九烷C19H402．191726．229，17-十八二烯醛C18H32O1．331826．75油酸C18H34O21．621929．2正二十七烷C27H568．62030．5711-(1-乙基丙基)-二十一烷C26H541．492131．652-十六醇C16H34O0．712236．64正二十四烷C24H503．142337．19胆固醇C27H46O2．8通过比较上述2种溶剂提取物的挥发性成分发现，2种溶剂提取物中挥发性成分的主要组分是相同的，它们是9-烯十八酸乙酯、6,9-十八二烯酸、5,7,8-三甲基二氢香豆素(15.06%)、9-烯十八酸甲酯等.2.2 补阳还五汤乙醇与氯仿提取物调控鼠骨髓间充质干细胞(MSCs)的增殖作用表3 rMSCs活性数据 (A值)空白乙醇氯仿0．3592±0．0357660．808±0．0852640．672±0．067177MTT法测定的MSCs的活性见表3.活性数值用吸光度(A)表示.A值结果显示相对于对照组(0.359 2±0.035 766)，2个药品组A增大；统计分析含乙醇和氯仿提取物(质量浓度4.950 g/L)2样品组的活性数据，得出：补阳还五汤乙醇和氯仿提取物均对MSCs的增殖起促进作用(p<0.05)；并且乙醇提取物的活性比氯仿提取物的活性强.3 结论中药绝大多数含低比重的挥发性成分，药材固有的气味(四气五味)是其中所含的成分决定的，这些成分是治病的主要药效物质.GC-MS测定补阳还五汤乙醇和氯仿提取物，发现挥发性成分大致相同，且2种溶剂提取物都具有促进骨髓间充质干细胞增殖的作用.据此可以知道，促进rMSCs增殖作用的物质之一是补阳还五汤中的挥发性成分.参考文献：[1] 侯玉鲁，秦元珠.补阳还五汤临床研究概况[J].中国中医药信息杂志，2003(增刊)：68-69.[2] 刘柏炎，蔡光先，林琳，等.补阳还五汤对大鼠局灶性脑缺血后神经干细胞影响的初步研究[J].中国临床康复，2004，8(22)：4532-4533.[3] 孙晋浩，杨琳，高英茂，等.补阳还五汤对神经干细胞生长分化的影响[J].山东大学学报：医学版,2002，40(5)：406-408.[4] DONALD O,JAN K,STEFANO C,et al.Bone marrow cells regenerate infarcted myocardium[J].Nature,2001，20(410)：701-705.[5] 蔡光先，刘柏炎.补阳还五汤对体外低糖低氧损伤海马神经干细胞增殖的影响[J].湖南中医学院学报，2005,25(2):1-3.[6] GIULIANA F,GABRIELLA C A,MARCELLO C,et al.Muscle regeneration by bone marrow-derived myogeinc progenitors[J].Science,1998,279(8):1528-1530.[7] ELIXABETH P.Bone marrow may provide musclepower[J].Science,1998,279(5):72-77.[8] DARWIN J,PROCKO P.Marrow stromal cells as stem cells for nonhematopoietic tissues.[J].Science,1997,276(1):71-74.[9] PITTENGER F,MACKAY A,BECK S,et al.Multilineage potenital of adult human mesenchymal stem cells[J].Science,1999,284(2):143-147.[10] 张国福，王和鸣.补阳还五汤对骨髓间质干细胞移植治疗大鼠脊髓损伤的影响[J].中国骨伤，2006,19(8):452-454.。
diastole 心舒期diatomaceous earth 硅藻土diatrizoate 3,5-双（乙酰氨基）-2,4,6-三碘苯甲酸（盐）diauxie growth curve 双峰生长曲线diazo 重氮基diazo compound 重氮化合物diazoacridine 重氮吖啶diazobenzyloxymethyl paper 重氮苄氧甲基纸，dbm纸diazonorleucine 重氮基正亮氨酸diazophenylthio paper 重氮苯硫醚纸，dpt纸diazotization 重氮化diazouridine 重氮尿苷dibucaine 狄步卡因dicarboxyl cellulose 二羧基纤维素dicarboxylic acid 二羧酸dicarboxylic amino acid 二羧基氨基酸dicentric chromosome 双着丝染色体dichlorodimethylsilane 二氯二甲硅烷dichlorofluorescein 二氯荧光黄dichloromethane 二氯甲烷dichlorovos 敌敌畏dichogamy 雌雄（蕊）异熟dichroism 二色性dick test 狄克试验[链球菌红斑毒素的皮肤试验] dicotyledons 双子叶植物dicoumarin 双羟香豆素，败坏翘摇素dictyosome （分散）高尔基体dictyostelium 盘基网柄菌属，网柄菌属dicyclohexylcarbodiimide 二环己基碳二亚胺[常用缩合剂]didanosine [商]2',3’-双脱氧肌苷didehydrothymidine 双脱氢胸苷dideoxy sequencing method 双脱氧测序法dideoxyadenosine triphosphate 双脱氧腺苷三磷酸dideoxycytidine 双脱氧胞苷dideoxycytidine triphosphate 双脱氧胞苷三磷酸dideoxyguanosine 双脱氧鸟苷dideoxyguanosine triphosphate 双脱氧鸟苷三磷酸dideoxyinosine 双脱氧肌苷dideoxyribonucleoside 双脱氧核苷dideoxyribonucleoside triphosphate 双脱氧核苷三磷酸dideoxythymidine triphosphate 双脱氧胸苷三磷酸dielectric constant 介电常数dielectric effect 介电效应dielectrometric titration 介电（常数）滴定（法）dielectrometry 介电（常数）滴定（法）dielectrophoresis 介电（电）泳diene 双烯diethyl pyrocarbonate 焦碳酸二乙酯diethyl sulfate 硫酸二乙酯diethylstilbestrol 乙酚，二乙基己烯雌酚difference spectrum 差光谱differential （示）差的，鉴别的；微分；微分的differential analysis 示差分析differential centrifugation 差速离心differential detection 示差检测，鉴别检测differential expression 差异表达differential flotation centrifugation 差速浮式离心differential hybridization 示差杂交（法）differential medium 鉴别培养基differential operation 示差操作differential permeability 差别透性，选择透性differential precipitation 示差沉淀differential refractive index detector 示差折光率检测器differential scattering 差散射differential screening 示差筛选differential sedimentation 差速沉降differential sepctrophotometry 示差分光光度法differential species 区别种differential spectrum （示）差光谱differential staining 鉴别染色（法）differential staining technique 鉴别染色技术[有时特指染色体显带技术]differential type detector 微分型检测仪differentiating solvent 鉴别剂，区分溶剂differentiation 分化differentiation antigen 分化抗原differentiation center 分化中心differentiation phase 分化时diffraction 衍射diffraction grating 衍射光栅diffraction symmetry 衍射对称性diffuser 扩散器；洗料器；[发酵罐]进气装置diffusion 扩散diffusion chamber 扩散盒，扩散小室diffusion coefficient 扩散系数diffusion controlled reaction 扩散控制（的）反应diffusion controlled termination 扩散控制的终止diffusional limitation 扩散限制diffusional resistance 扩散阻力diformazan 二甲digalactosyl diglyceride 双半乳糖甘油二酯digester 消化器，消化罐digestion 消化，（酶切）消化digestive enzyme 消化酶digital control 数字控制digital imaging microscope 数字成像显微镜digital imaging microscopy 数字成像显微术digitalis cardiac glycoside 毛地黄（类）强心苷digitalizer 数字化仪[用于计算机科学]digitonin 毛地黄皂苷digitoxigenin 毛地黄毒苷配基；beta-(丁烯酸内酯)-14-羟甾醇digitoxin 毛地黄毒苷diglyceride 甘油二酯digoxigenin 洋地黄毒苷，地高辛配基digoxin 异羟基洋地黄毒苷原，地高辛dihaploid 双单倍体dihedral angle 二面角，双面角dihydrobiopterin 二氢生物蝶呤dihydrochalcone 双氢查耳酮，二氢查耳酮dihydrofolate 二氢叶酸dihydrofolate reductase 二氢叶酸还原酶dihydrolipoamide 二氢硫辛酰胺dihydrolipoamide dehydrogenase 二氢硫辛酰胺脱氢酶dihydrolipoic acid 二氢硫辛酸dihydroorotase 二氢乳清酸酶dihydroorotate 二氢乳清酸dihydropteridine 二氢蝶啶dihydropteridine reductase 二氢蝶啶还原酶dihydropyridine 二氢吡啶dihydrotestosterone 双氢睾酮dihydrouracil 二氢尿嘧啶dihydrouracil arm 二氢尿嘧啶臂dihydrouracil loop 二氢尿嘧啶环dihydrouridine 二氢尿苷dihydroxyacetone phosphate 二氢丙酮磷酸dihydroxycholecalciferol 二羟胆钙化（固）醇dihydroxyphenylanaline 二羟苯丙氨酸，多巴dihydroxyphenylethylamine 二羟苯基乙胺，羟酪胺，多巴胺diisopropylfluorophosphate 二异丙基氟磷酸dikaryon 双核体diltiazem 硫氮酮diluent 稀释剂，稀释液dilution cloning 稀释克隆法[如用于获得细胞克隆株] dimensional electrophoresis 双向电泳dimer 二聚体dimerization 二聚化dimerization cofactor 二聚化辅因子dimethoxytrityl 二甲氧三苯甲基[在dna合成中用作羟基保护剂] dimethyl sulfate 硫酸二甲酯dimethyl sulfoxide 二甲基亚砜dimethylallylpyrophosphate 二甲（基）烯丙基焦磷酸dimethylaminoazobenzene 二甲基氨基偶氮苯dimethylformamide 二甲基甲酰胺dimorphism 二态二氢，双态现象dinitro benzene 二硝基苯dinitrochlorobenzene 二硝基氯苯dinitrofluorobenzene 二硝基氟苯dinitrogen 双氮，分子氮dinitrogenase 固氮酶dinitrogenase reductase 固氮酶还原酶dinitrophenol 二硝基苯酚dinitrophenyl 二硝基苯基dinoflagellate 甲藻dinoxanthine 甲藻黄素dinucleotide frequency 二核苷酸频率dinucletide 二核苷酸diodeelectrode 二极管电极dioecism 雌雄异体，雌雄异株diosgenin 薯蓣皂苷配基，薯蓣皂苷元dioxide 二氧化物dioxygen 双氧dioxygenase 双加氧酶dipeptidase 二肽酶dipeptide 二肽diphenylamine blue 二苯胺蓝diphenyloxazole 二苯基唑diphosphate 二磷酸diphosphatidylglycerol 双磷脂酰甘油diphosphoglycerate 二磷酸甘油酸diphosphoglycerate shunt 二磷酸甘油酸支路diphosphoinositide 二磷酸肌醇磷脂，磷脂酰肌醇磷酸diphthamide 白喉酰胺diphthera toxin 白喉毒素dipicolinic acid 2,6-吡啶二羧酸diplobacillus 双杆菌diploblastic 双胚层的diplococcus 双球菌diplococcus pneumoniae 肺炎双球菌diploid 二倍体diploid cell line 二倍体细胞系diploidization 二倍化diploidy 二倍性diplonema 双线期diplotene stage 双线期dipolar aprotic solvent 偶极非质子溶剂dipolar protophilic solvent 偶极亲质子溶剂dipolar protophobic solvent 极疏质子溶剂dipole 偶极dipole molecule 偶极分子dipole moment 偶极矩dipyrromethane 联吡咯甲烷direct cross 正交direct duplication 同向重复direct insertion 同向插入direct repeat 同向重复（序列）direct selection 正选择[使用只有突变体或重组体能生长的条件进行选择]directed cloning 定向克隆directed mutagenesis 定向诱变directed perturbation 定向微扰directed sequencing 定向测序direction selectivity 方向选择性directional cloning 定向克隆disaccharide 二糖disassembly 解装配，分解disc electrophoresis 圆盘电泳disc gel electrophoresis 圆盘凝胶电泳disc membrane 圆盘膜discharge 放电；卸下discoidal cleavage 盘状卵裂discontinuous epitope 非连续表位discontinuous gradient 不连续梯度discontinuous replication 不连续复制discontinuous variation 不连续变异discontinuous zone electrophoresis 不连续区带电泳discrete 分立的，不连续的discriminant analysis 判别分析discrimination 辨别，判别disease association 疾病相关disease resistance 抗病性dish 平皿disinfectant 消毒剂disinfection 消毒disinfestation 灭虫disinhibition 去抑制disintegration 蜕变，衰变；去整合，解整合；分解，破碎disintegrator 粉碎器，粉碎机disjunction 分离disk centrifuge 圆盘（式）离心机[带有成叠的有孔圆盘，常用于溶液的澄清化处理]dislocation 脱位，转位，位错dismutase 岐化酶disome 二体，双体disordered state 无序状态disordered structure 无序结构dispase 分散酶，中性蛋白酶[用于分散组织培养中的动物细胞] dispenser 分液器dispermy 双精人卵dispersant 分散剂disperse medium 分散介质disperse phase 分散相disperse system 分散系统dispersion 分散；色散dispersion force 分散力；色散力dispersion spectrum 色散谱displaced loop 替代环displacement 顶替，替代，置换displacement analysis 顶替（分析）法displacement chromatography 顶替层析displacement electrophoresis 顶替电泳displacement loop 替代环，d环[形如英文字母d] displacement reaction 置换反应disposable glove 一次性手套disposable microcentrifuge tube 一次性（使用的）微量离心管disposable tip 一次性（使用的）吸头disproportionation 岐化（反应）disrotatory 对旋disruption 破裂，破坏dissecting microscope 解剖显微镜dissection 解剖，剖分disseminated intravascular coagulation 弥漫性血管内凝血dissimilation 异化（作用）dissociation 解离，离解dissociation constant 解离常数dissolvability 溶（解）度，（可）溶（解）性dissolvant 溶剂distamycin 偏端霉素distance receptor 距离感受器distant hybirdization 远缘杂交distant hybrid 远缘杂种distorted peak 畸峰diterpene 双萜，二萜dithioerythritol 二硫赤藓糖醇dithiothreitol 二硫苏糖醇divergence 分散[用于神经系统]；趋异divergent 趋异进化diverse ion effect 异离子效应diversity gene d基因[为d区编码的基因]diversity region 多变区，d区[免疫球蛋白等分子重链的一个高变区]divinylbenzene 二乙烯苯dizygotic twins 二卵双生，异卵双生dna adduct dna加合物dna amplification in vitro dna体外扩增dna amplification polymorphism dna扩增多态性dna bending dna转折，dna弯曲dna blotting dna印迹（法）dna catenation dna连环dna circle dna环[指环状dna]dna cleavage dna裂解，dna切割dna cloning dna克隆（化）dna jumping technique dna跳查技术dna ladder dna梯[如大小不同的标准参照物的电泳谱]dna nicking dna切口形成dna pitch dna螺距dna sizing dna大小筛分dna sizing gene dna大小决定基因[如见于噬菌体，可决定所包装的dna量]dna typing dna分型dnaase dna酶dnaase i footprinting dna酶足迹法docking 停靠docking protein 船坞蛋白，停靠蛋白[内质网上与信号识别颗粒相互作用从而使蛋白质继续翻译的蛋白]dodecahedron 十二面体dodecane 十二烷dodecapeptide motif 十二肽基序dodecyl 十二烷基dolichol 多萜醇，长醇domain 域，区域，结构域，功能域domain assmbly 结构域装配domain deletion 结构域删除domain substitute 结构域置换dominance 显性；优势（度）dominance variance 显性方差dominant 显性的，优势的dominant acting gene 显性开放基因dominant allele 显性等位基因dominant gene 显性基因dominant hemisphere 优势半球dominant interference 显性干涉dominant lethal 显性致死dominant mutation 显性突变dominant negative 显性阴性的，显性失活的dominant negative mutant 显性失活突变体dominant oncogenic 显性致癌的donnan dialysis 唐南透析donnan equilibrium 唐南平衡donnan potential 唐南膜电势donor 供体，给体donor splicing site 剪接供体dopamine 多巴胺dosage compensation 计量补偿（效应）dot blot 斑点印迹，斑点印迹膜dot blotting 点渍法，斑点印迹（法）dot hybridization 斑点杂交dotting 打点，打点杂交double antibody method 双抗体法[免疫测定方法之一种]double balloon catheter 双气囊导管double bar 重棒眼，双棒眼，超棒眼[黑腹果蝇唾液腺染色体的x 染色体上16a区段重复三次而出现的特殊表型]double beam mass spectrometer 双束质谱仪double beam spectrophotometer 双光束分光光度计double blind trial 双盲试验double bond migration 双键移位double coupling method 双偶联法，双偶合法double decomposition reaction 复分解double exchange 双交换double fertilization 双受精double focusing 双聚焦double focusing mass spectrometer 双聚焦质谱仪double helix 双螺旋double immunodiffusion 双向免疫扩散，免疫双扩散double innervation 双重神经支配double labeling 双重标记double minute chromosome 双微染色体[所携带基因得到扩增的成对额外小染色体]double recessive 双隐性double resonance 双共振doublet 双联体；双峰doubling time 倍增时间[培养物的生物质翻一番所需的时间]dower resin dower树脂[陶氏化学公司离子交换层析介质商品名] down promoter mutation 启动子减效突变downflow fixed bed 下流固定床doxorubicin 阿霉素drift （遗传）漂变drilling mud 钻探泥浆drinking center 饮水中枢drop method 点滴法droplet countercurrent chromatography 液滴反流层析，液滴逆流层析dropping mercury electrode 滴汞电极drosophila 果蝇属drug susceptibility 药物敏感性drug targeting 药物寻靶，药物导向duchenne muscular dystrophy duchenne型肌营养不良，假肥大型肌营养不良duocrinin 促十二指肠液素duplex 双链体；双螺旋；二显性组合duplicon 重复子duramycin 耐久霉素dwarf colony 侏儒型菌落dwarf plant 矮化植物[由遗传因素决定不能长高]；矮生植物[由认为措施或特殊环境决定不能长高]dyad 二分体，二联体dyad symmetry 二重对称dye exclusion test 染料排斥试验[用于检查细胞生活力]dynactin 动力蛋白激活蛋白dynamin 发动蛋白dynein 动力蛋白dynein arm 动力蛋白臂dynorphin 强啡肽dysbacteria 菌群失调dysbacteriosis 菌群失调dysentery bacillus 痢疾杆菌dysfunction 功能异常，机能障碍dysregulation 调节异常dystroglycan （肌）营养不良（蛋白）聚糖[与肌）营养不良蛋白相关的蛋白聚糖]dystrophin (肌）营养不良蛋白。
中科院上海研究所1998、2001生物化学与分子生物学,2002年生物化上海生化所 98 MOLECULAR GENETICS Paper一．名词解释拓扑异构酶组成型异染色质（constitutive heterochromation）阻遏蛋白RT-PCR 遗传重组（广义和狭义的）衰减子二．是非1．大肠杆菌染色体是一条由数百万个碱基对组成的环状DNA2．λ噬菌体能否整和进入宿主DNA或从宿主DNA上切除，主要取决于宿主细胞中λ噬菌体整和酶int的存在形式，同时还涉及到其他的一些蛋白因子。
3． Z型构象的DNA不存在于天然状态的细胞中4．生物体的结构与功能越复杂，其DNA的C值越大，但在结构与功能很相似的同一类生物中，在亲缘关系十分接近的物种之间，它们的DNA C值是相似的 5．在大肠杆菌，哺乳动物和人线粒体中，终止密码都是UAA，UAG两种。
6．反义RNA通过互补的碱基与特定的mRNA结合，从而抑制mRNA的翻译，因而反义RNA的调空机制显然只是翻译水平特异的7．转座过程通常是指DNA中的一段特殊序列（转座元）在转座酶以及其它蛋白因子的作用下，从DNA分子中的一个位置被搬移到另一位置或另一DNA分子中 8． DNA修复系统的作用是保证DNA序列不发生任何变化 9．持家基因（house keeping gene）是指那些在哺乳类各类不同细胞中都有表达的基因这组基因的数目约在一个万左右10．转座元两端通常都存在同向重复序列和倒转重复序列，研究表明这些序列对于转座元的转座作用非常重要11．荧光原位杂交（FISH）可以把同一个基因定位到特定的染色体位置上去12．在真核生物核内。
五种组蛋白（H1 H2 aH2b H3 和H4）在进化过程中，H4极为保守，H2A最不保守 13．还原病毒的线形双链DNA合成时，结合在病毒RNA3末端的用于第一链DNA合成的引物是在细胞中正常的TRNA分子 14． RNA聚合酶1负责转录TRNA和5SRNA，其启动子位于转录的DNA序列之内，称为下游启动子15．条件突变如温度敏感型突变不可被抑制基因所抑制16．多DNA酶1的优先敏感性不仅仅表现在活跃基因的转录区，而且也表现在活跃基因两侧的DNA区段上17．作为一个基因克隆的载体，主要是由药物抗性基因，供插入外源 DNA片段的克隆位点和外源DNA片段插入筛选标志这三大部分组成的 18．碱基序列排列方式可以严重影响DNA的解链温度19．大多数氨基酸由一个以上的密码子所编码，在A+T含量和G+C含量没有显著差异的基因组中，不同密码子的使用频率就近似相同了 20．在克隆载体PUC系列中，完整的LACZ基因提供了一个外源基因插入的筛选标记，兰色的转化菌落通常表明克隆是失败的三．1．下列--种碱基错配的解链温度最高 A T-T B G-G C A-C D G-A2．目前在转基因小鼠中常用的基因剔除技术（gene knockout）是根据--原理而设计的 A 反义核苷酸的抑制作用B转座成分的致突变作用C离体定向诱变D同源重组3．端粒酶与真核生物线形DNA末端的复制有关，它有一种--ARNA聚合酶（ RNA polymerase）B逆转录酶（reverse transcriptase）C核酸酶（nuclease）D 核糖核酸酶（ribonuclease rnase） 4．细胞合成的分泌蛋白质在N端都有一段信号肽，这些信号肽的长度是--个氨基酸残基A大于100 B15到30 C约50 D小于10 5．真核生物复制起始点的特征包括--A富含GC区 B富含AT区 CZ DNA D无明显特征6．细菌中，同源重组发生在一些热点周围，这和-- 在chi位点上的单链内切酶活性有关A RecAB RecBCRecCDRecD 7． HMG14和HMG17是属于--A一类分子量不大的碱性蛋白质B一类分子量很大的非组蛋白C一类分子量不大的非组蛋白D一类RNA聚合酶8． RNA修复体系可以使核酸突变频率下降--A10倍B1000倍C百万倍D不影响突变频率 9．真核生物DNA中大约有百分之2-7的胞嘧啶存在甲基化修饰，绝大多数甲基化发生--二核苷酸对上 A CC B CT C CG D CA 10．Holliday模型是用于解释--机制的A RNA逆转录酶作用B 转座过程C 噜噗切除D同源重组 11.化学诱变的结果与--因素有关A剂量 B细胞周期 C剂量时间率（Dose rate）D上述各因素12．外源基因在大肠杆菌中高效表达受很多因素影响，其中S-D序列的作用是： a提供一个mRNA转录终止子 b提供一个mRNA转录起始子 c提供一个核糖体结合位点 d提供了翻译的终点13．真核生物的组蛋白上的修饰作用，除了乙酰基化、甲基化和磷酸化外，还有一种特殊的修饰作用，即泛素化，这一个泛素（ubiquitin）与哪一种组蛋白连接？A．H3 B。
癌基因（oncogene）：通常表示原癌基因（proto oncogene）的突变体，这些基因编码的蛋白使细胞的生长失去控制，并转变成癌细胞，故称癌基因。
氨酰-tRNA合成酶（aminoacyl tRNA synthetase）：将氨基酸和对应的tRNA的3′端进行共价连接形成氨酰-tRNA的酶。
不同的氨基酸被不同的氨酰－tRNA合成酶所识别。
暗反应（light independent reaction）：光合作用中的另外一种反应，又称碳同化反应（carbon assimilation reaction)。
该反应利用光反应生成的ATP和NADPH中的能量，固定CO2生成糖类。
白介素-1β转换酶（interleukin-1β converting enzyme，ICE）：Caspase-1，Caspase家族成员之一，线虫Ced3在哺乳动物细胞中的同源蛋白，催化白介素-1β前体的剪切成熟过程。
半桥粒（hemidesmosome）：位于上皮细胞基底面的一种特化的黏着结构，将细胞黏附到基膜上。
胞间连丝（plasmodesma plasmodesma）：相邻植物细胞之间的联系通道，直接穿过两相邻细胞的细胞壁。
胞内体（endosome）：动物细胞内由膜包围的细胞器，其作用是转运由胞吞作用新摄取的物质到溶酶体被降解。
胞内体被认为是胞吞物质的主要分选站。
胞吐作用（exocytosis）：携带有内容物的膜泡与质膜融合，将内容物释放到胞外的过程。
胞吞作用（endocytosis）：通过质膜内陷形成膜泡，将细胞外或细胞质膜表面的物质包裹到膜泡内并转运到细胞内（胞饮和吞噬作用）。
胞外基质（extracellular matrix）：分布于细胞外空间、由细胞分泌的蛋白质和多糖所构成的网络结构，如胶原和蛋白聚糖等，在决定细胞形状和活性的过程中起着一种整合作用。
胞质动力蛋白（cytoplasmic dynein）：由多条肽链组成的巨型马达蛋白，利用ATP水解释放的能量将膜泡或膜性细胞器等沿微管朝负极转运。
文章编号：1007－8738（2014）01－0101－03炎症小体的活化及调控机制研究进展赵西宝，陈玮琳*（浙江大学医学院免疫学研究所，浙江杭州310058）收稿日期：2013－05－27;接受日期：2013－06－21基金项目：国家自然科学基金(31200682);浙江省自然科学基金(Y2110255);中央高校基本科研业务费专项资金资助(2013QNA7010)作者简介：赵西宝(1988－)，男，山东临沂人，硕士研究生Tel :0571-********;E-mail :xibaozhao@yeah．net*Corresponding author ，陈玮琳，E-mail :cwl@zju．edu．cn［摘要］炎症小体（inflammasome ）是细胞内模式识别受体（PＲＲ）参与组装形成的大分子蛋白复合体，是参与机体固有免疫的重要成分。
宿主细胞可通过PＲＲ识别病原体相关分子模式（PAMP ），组装成炎症小体，激活下游的信号通路，诱导炎症因子分泌，在抵抗病原菌入侵及维持机体免疫系统稳定中起重要作用。
本文主要综述了炎症小体的活化及调控机制研究进展。
［关键词］炎症小体；固有免疫；活化途径；调控机制［中图分类号］Ｒ392．11，Ｒ395，Ｒ364．5［文献标志码］A机体时刻都在病原微生物的包围之中，固有免疫系统是宿主抵御病原菌入侵的第一道防线，其中核苷酸结合寡聚结构域受体（nucleotide-binding oligomerization domain receptor ），简写为NOD 样受体（NOD-like receptor ，NLＲ）和HIN200蛋白家族组成的炎症小体（inflammasome ）起到重要作用。
炎症小体的研究越来越受到人们的重视，而且很多自身免疫病与炎症小体的非正常活化及调控有关，但是关于炎症小体活化调控的详细机制现在仍然知之甚少，所以相关研究有待进一步深入。
1炎症小体及其活化炎症小体是由多种蛋白组成的大分子复合物，通过招募pro-caspase-1并使2个相邻的pro-caspase-1发生自身水解，产生具有酶活性的caspase-1，介导IL-1β和IL-18等炎症因子由前体形式转变为活化形式，分泌到胞外发挥生物学功能。
神经系统中富含亮氨酸重复结构的蛋白【关键词】神经系统；富亮氨酸重复；蛋白彼此作用；功能【摘要】从细菌到哺乳动物，多种物种的神经系统内都存在有一3D结构为马蹄形的富亮氨酸重复结构（LRR）蛋白，其主要在信号转导，细胞黏连，神经系统发育等进程中起作用. 过去的几年内，在无脊椎动物及脊椎动物神经系统内发现了许多具LRRs的蛋白，它们参与了多种神经生理活动. 对这些蛋白的功能作进一步分析，有助于揭露该蛋白家族的分子作用机制，并对进一步熟悉神经系统的发育等重要生理活动有所裨益.【关键词】神经系统；富亮氨酸重复；蛋白彼此作用；功能随着对神经系统发育研究的进展,目前发现了多种参与神经系统发育的基因、蛋白，近几年内，在研究神经系统发育的进程中发此刻果蝇及一些脊椎动物神经系统内的多种蛋白均具有相同的结构域富亮氨酸重复结构域，目前已发现20多种具富亮氨酸重复结构域的蛋白存在于神经系统内.富亮氨酸重复结构（Leucinerich repeats，LRRs）是于1985年第一次发现存在于人血清中一种未知功能的糖蛋白，富亮氨酸α2糖蛋白（leucienrich α2glycoprotein）［1］,目前已在多种组织中的60多个功能相异及细胞定位不同的蛋白中发现了富亮氨酸重复结构.富亮氨酸重复结构不同于被普遍熟悉的亮氨酸拉链结构. 亮氨酸拉链存在于寡聚蛋白中，包括许多DNA结合蛋白，如cfos和cjun原癌基因产物. 亮氨酸拉链由重复的7个氨基酸残基组成，亮氨酸位于第7位氨基酸上. 这些亮氨酸位于其组成的α螺旋一侧，其侧链向外伸出，组成状如齿形排列的半拉链，与其异源的互补α螺旋接触后，可借助侧链疏水性交织对插，形成具有稳定卷曲螺旋结构的二聚体，即亮氨酸拉链［2］. 富亮氨酸重复结构与富亮氨酸拉链唯一相似的地方在于，二者的保守亮氨酸残基间都具有特定的距离，而且这些亮氨酸无法被其他疏水性氨基酸所替代. 但是二者间亮氨酸残基间的距离不同，只有在羧基端（C端）的LRRs中的特定亮氨酸之间距离有7个氨基酸. LRRs与亮氨酸拉链中的亮氨酸功能也完全不同，亮氨酸拉链中的亮氨酸残基参与了亮氨酸拉链的寡聚化形成，而LRR中亮氨酸只参与了结构组成，并非直接参与蛋白之间的作用.对含有LRRs蛋白的一级结构分析显示，LRRs 通常呈持续散布，有的蛋白只含有一个LRR，如血小板糖蛋白Ibβ，有的蛋白则含有多个LRR基序（motif），如Chaoptin则含有30个持续LRRs［3］. 不同蛋白中LRRs结构长度可变，通常含有20~29个氨基酸残基，其中最多见的LRR结构含有24个氨基酸残基，它们都含有一个长度为11个氨基酸残基的保守序列，排列为LxxLxLxxN/CxL(“x”可为任意氨基酸)，其中第1，4，6，11位氨基酸一般为亮氨酸(Leu)或其他脂肪族氨基酸，第9位氨基酸为天冬氨酸Asp或半光胺酸Cys［3］. 结构研究显示LRRs的排列越规则其3D结构也越规则. 当1，4，6，11位上的Leu被其他疏水性氨基酸如异亮氨酸，缬氨酸或苯丙氨酸所代替时，LRRs重复结构会变得不规则［4］.人们对LRRs 3D结构的熟悉来自对猪肝脏核糖核酸酶抑制因子（Ribonuclease Inhibitor, RI）X射线衍射分析［5］. RI为一种胞浆蛋白，其几乎完全由15个持续的LRRs结构组成，它可与包括催化位点在内的核糖核酸酶表面的大部份区域牢固结合，从而起到抑制核糖核酸酶对RNA的剪切作用. 对核糖核酸酶抑制因子晶体结构的研究揭露了LRR持续重复的3D结构组成，每一个独立的亮氨酸重复组成一个独立的βα单位，由一个短的β折叠和一个α螺旋组成，二者近乎平行排列. 另外，每一个持续重复的α螺旋围绕一个一路的轴，彼此也近乎平行地排列，组成一个弯曲的马蹄形结构的外层，β折叠围绕此轴平行排列为β片层结构组成马蹄结构的内层（图1）.正是LRR蛋白的这种马蹄状结构的特性，使得其容易和较小的球状蛋白相结合，并可增强它们之间的亲和力和彼此作用. 目前已知的LRR蛋白除具有重复结构的相似处之外，其另一个一路的特点就是富含亮氨酸重复序列蛋白质的LRR结构域为与其他蛋白彼此作用的结合区. LRR蛋白主要在信号转导，细胞黏连，发育，DNA修复、重组、转录，和RNA加工等方面起作用. 它们散布普遍，从细菌到哺乳动物中都有存在，而且在多种组织以至细胞器中也有发现. 过去的几年内，在无脊椎动物及脊椎动物神经系统内发现了多种具LRRs的蛋白(表1). 而咱们最感兴趣的正是这些存在于神经系统内的LRR蛋白.表1神经系统中含有富亮氨酸重复结构的蛋白（略）咱们对上述这些蛋白进行了初步的结构及功能域分析，和细胞定位分析，结果显示这些蛋白中绝大部份位于细胞膜上，只有Slit 蛋白分泌到胞外，LANP蛋白定位到核内（图2），在整个蛋白序列中，LRR 结构域均位于蛋白的N端且占整个蛋白结构的绝大部份. 它们通过LRR结构与相应的配体或受体蛋白结合、彼此作用，从而在胚胎发育，神经发育，细胞极化，基因表达调控，信号转导等方面发挥作用.将上述蛋白与典型的LRR蛋白核酶/血管生成素抑制因子一同进行系统进化分析，结果显示，这些富含LRR结构域的蛋白主要分为三个系统发生群. 不同种属来源的LRR结构域散布于这三个系统发生群中，揭露这些蛋白有可能由一个或几个一路的先人进化而来(图3).上述发现的LRR蛋白有多种位于果蝇（Drosophila）的神经系统内，它们大多参与细胞细胞彼此作用，多作为细胞黏连分子，在神经发育进程中扮演重要角色. 咱们在这里对上述蛋白进行一下简单的介绍.Toll果蝇Toll基因编码一个跨膜蛋白,由803个氨基酸组成的19个富含亮氨酸重复序列的胞外区、跨膜区和269个氨基酸组成的胞内区组成. 其中,17个潜在的糖基化位点和17个半胱氨酸残基均散布于胞外(图2). 自合子期起，Toll蛋白表达贯穿于果蝇胚胎发育的整个进程，其主要散布于胚胎的腹侧，在胚胎发育的后期，主要使肌肉组织形成的进程中，其表达量明显升高. 当RP3或其他运动神经元生长锥伸经肌细胞时，生长锥表面会表达大量Toll蛋白，其作用为支配突触末梢的肌细胞，当神经肌肉接头形成后，Toll表达量随之下降. 在Toll缺失突变体中，RP3生长锥有时会错误支配非目的靶肌细胞. 另外，在非Toll表达期人为表达Toll蛋白，虽然能促使生长锥抵达正确的目的细胞，可是会抑制神经肌肉接头的形成. 因此以为，Toll局部作用并制约特定运动神经元生长锥支配其目的细胞，其时空表达调控对其在胚胎发育进程中的作用十分重要［6］.Slit在果蝇、线虫、大鼠和人类等均发现Slit基因的存在，它是由发育期神经管的腹侧中线胶质细胞分泌的一组Mr为170000~190000的分泌型糖蛋白，散布于胶质细胞表面并在所有中枢神经系统轴突表面低水平表达，若是缺乏会致使纵行传导路和交叉(连合)神经元轴突在中线的异样聚集. 其结构从氨基结尾到羧基结尾有一段N端短的信号肽序列，4个富亮氨酸重复(LRRs)序列,7～9个EGF重复序列,一个层粘素(laminin) G序列和一个C结尾富含的胱氨酸的序列，相对于EGF或G序列，LRR结构域几乎组成了Slit蛋白的绝大部份结构. Slit可结合于轴突及生长锥表面的3种Roundabout 受体（Robo，Robo2，Robo3）（图2），研究表明，Slit/ Robo参与多种轴突导向进程，其主要功能在于对轴突的排斥性导向作用，并能增进轴突的分枝和延伸及引导神经细胞的迁移［7］.体外研究表明，LRR结构域是Slit作为排斥信号的必需结构. 编码LRR结构域的单个氨基酸发生点突变即可降低Slit的排斥作用，转基因显示主如果该结构域影响轴突的导向. 实验证明，Slit和Robo 的结合及排斥作用需要LRR结构域的存在，LRR缺失后，Slit无法与Robo相结合［8］.Connectin果蝇Connectin蛋白,属于具有LRR重复的细胞细胞黏附分子家族,它含有一个信号肽，10个LRR结构域，依托糖基磷脂酰肌醇（GPI）锚定在细胞外膜上（图2），可作为吸引或排斥特定神经细胞的分子,参与运动神经元生长锥导向及突触形成. 在中枢神经系统，Connectin最初表达于果蝇腹侧的8块肌肉组织和支配这些肌肉的运动神经元表面，和沿运动神经元轴突伸展路径上的胶质细胞表面. 在突触形成进程中，Connectin蛋白定位于神经肌肉接头形成时的突触上，突触形成后，则检测不到Connectin的表达. 另外，Connectin还具有促使同型细胞黏连的作用，在肌肉发育的初期表达于成肌细胞，并促使这些成肌细胞成束化. 在运动神经元的轴突生长锥延伸通过外周神经系统时，Connectin蛋白还表达于两种胶质细胞PG1和PG3内. Connectin主要通过同型细胞黏附，协调细胞间彼此作用，从而在靶位点识别中起到重要作用［9］.Capricious在果蝇胚胎发育后期，运动神经元轴突生长锥抵达靶目标区域时，会一度搜寻多个可能的靶位点的肌细胞表面，但只会和其中的一个正确靶细胞成立稳定的突触联系，果蝇体内Capricious（caps）基因即调控此进程. caps编码一含有14个LRRs结构域的跨膜蛋白，其胞外区几乎完全由14个LRRs及其N端、C端侧翼结构组成（图2）. CAPS蛋白定位于发育期的运动神经元表面，在神经肌肉接头形成进程中，caps表达于很少数量的突触后靶目标肌肉中，和表达于支配这些肌肉的运动神经元中. 体内研究显示，当位于其第一个外显子内蹬LRRs编码区缺失突变后，运动神经元对靶目标选择的特异性就会发生改变，显示caps具有调控特异性突触形成的作用，其可能通过LRRs 结构域调控突触靶位点的识别［10］.Tartan果蝇Tartan蛋白为一跨膜蛋白，胞外区由10个LRR结构域及其N端，C端侧翼结构组成，占整个蛋白结构的绝大部份，近接为跨膜区和短的胞内区. Tartan的表达与果蝇胚胎幼虫分节发育，神经发生相关，它参与了神经母细胞，感觉母细胞和外周神经的形成进程，其表达几乎贯穿了胚胎发育的整个进程. tartan等位基因缺失突变体会致使隐性致死，造成周围感觉器官内细胞位置和数量的缺点，影响外周神经的投射，并造成中枢神经系统内神经连合的组织错误. 其缺失突变还可影响到肌肉组织的排列［11］.Chaoptin果蝇Chaoptin蛋白是目前发现的具有LRR结构域最多的蛋白，其几乎完全由41个串联的LRR结构域组成（图2）. 其作为一种特异性细胞黏附分子，为光感神经元特异性的黏附分子，在成体果蝇复眼中，Chaoptin表达于光感神经元胞体、轴突，起光导作用的微绒毛，和视神经纤维的细胞膜上，它还表达于单眼及果蝇幼虫的光感组织中，通过糖基磷脂酰肌醇（GPI）锚定在细胞膜上. 其功能是作为细胞黏附分子，参与视神经的发育进程，尤其是对视杆微绒毛的排列组成发挥作用［12］.Kekkonkekkon基因家族的两个产物Kek1和Kek2均为跨膜蛋白，二者的胞内区不同较大，只有19%同源性，而胞外区相同，均含有6个LRR结构和一个C2型免疫球蛋白结构域（图2），这两种结构域均能介导蛋白蛋白间彼此作用. 二者在胚胎发育进程CNS多种神经元内都有表达. 作为细胞黏合和信号分子，它们可能参与了胚胎CNS 神经元的分化，通过其细胞细胞间彼此作用功能，使得神经元在分化进程中能够识别相邻细胞及分子，从而引导轴突生长及导向［13］.近20年来，随着对LRR家族蛋白研究的深切，在高等的脊椎动物，包括哺乳动物体内也发现了多种LRR家族新成员.Trk神经营养素（NTs）高亲和性受体Trk为原癌基因trk编码的跨膜蛋白，其包括有与配体结合的胞外区，跨膜区和胞内酪氨酸蛋白激酶三部份. NTs的信号主如果通过Trk家族酪氨酸蛋白激酶受体抵达神经元的. NTs各个因子特异地识别Trk家族特定的酪氨酸蛋白激酶受体：NGF特异地识别TrkA，BDNF、NT4特异地识别TrkB，NT3识别TrkC. 所有Trk家族受体胞外区包括3个串联的LRR结构域及其N端和C端富含半胱氨酸的侧翼结构，紧接2个Ig样结构. 而第二个LRR motif正是与神经营养因子结合的位点（图2）. 实验表明，单独的一个由24个氨基酸残基组成的LRR结构多肽可高效结合NGF，阻碍NGF结合于TrkA胞外的相应区域. TrkB受体上的相应的第二个LRR结构域也具有特异性结合BDNF，NT4的作用［14］.LANPLeucinerich acidic nuclear protein （LANP）是酸性富亮氨酸核蛋白，包括247个氨基酸，含两种不同的结构域，其中N端为5个串联的LRR结构域，C端结构域为第105~247位氨基酸残基，为一段富含酸性氨基酸天冬氨酸和谷氨酸的高度重复序列并包括一段核定位信号（图2）. LANP普遍散布于大鼠的中枢神经系统内，尤以小脑散布最多，免疫组化研究表明，该蛋白主要位于小脑蒲肯野细胞核内. 其表达量在诞生后发育前期一度升高，在大鼠诞生后第7天，该基因mRNA在小脑外粒层及蒲肯野细胞内适度表达，而在内粒层细胞内弱表达. 在诞生后第2周，该基因在上述细胞内，尤其是蒲肯野细胞内表达量升高，诞生3wk后回落到成体表达水平. 从该蛋白的上述生物学特性推测，LANP有可能在小脑神经元分化进程中起信号转导作用［15］.NgRNgR为轴突生长抑制性蛋白Nogo的受体，其含有473个氨基酸，氨基端有一易位信号序列，其后为8个富含亮氨酸的重复区（LRR）和1个LRRC结尾区（LRRCT）（图2）. 作为一个糖基醇磷脂结合蛋白，NgR并非跨越细胞膜，其信号的转导必然要激活其他跨膜受体. 实验表明：CNS髓磷脂中另外2种轴突生长抑制性蛋白MAG 与OMgp均通过NgR及与其相连的受体复合物发挥作用. Nogo66与MAG结合于NgR的不同位点上，而Nogo66与OMgp在NgR上的结合位点有重叠，故二者存在竞争. NgR似乎是CNS髓磷脂中各类轴突生长抑制性蛋白发挥作用的集中点［16］. OMgpOMgp(少突胶质细胞/髓鞘糖蛋白)是Mr为110000的糖蛋白,由440个氨基酸组成,通过GPI锚定在髓鞘膜外层. OMgp由四个结构域组成,即氨基端一个较短的富含半胱氨酸的基序,7个富含亮氨酸的串联重复序列,一个丝/苏氨酸富含区和一个疏水的羧基端片段［17］（图2）. OMgp表达在CNS髓鞘、培育的少突胶质细胞表面和外周神经的节旁区部份神经元上. 它具有诱使生长锥溃变和抑制神经突起再生的作用，这一作用是通过与nogo66等神经再生抑制因子竞争结合同一受体NgR而实现的. 有实验表明，在OMgp与NgR的黏附结合进程中，OMgp的亮氨酸富集重复结构域是必需的，只有含该结构域的OMgp蛋白片段才能黏附表达有NgR的CHO细胞，并抑制神经突起的生长［18］. 还有实验证明，去除LRR的OMgp失去了对COS7细胞的生长抑制功能［19］. 因此推测OMgp LRR结构域有可能在CNS损伤后神经生长抑制进程中起重要作用.LINGO其基因染色体定位为,被命名为“LINGO1”(LRR and Ig domain containing Nogo receptor interacting protein)即“含亮氨酸重复序列和免疫球蛋白结构域的Nogo受体作用蛋白”. LINGO为脑专有蛋白,高表达于脑内,与NgR1共散布;在脊髓中低水平表达,但不存在于机体其他组织. LINGO有四个异构体,LINGO1是主要活性分子,含12个富含亮氨酸的重复序列(12 leucine rich repeats, LRR),一个免疫球蛋白结构域,一个跨膜结构域和一个较短的胞质区（图2）. 胞质区上第591位氨基酸残基磷酸化是其发挥活性的结构基础. 在生物体内,LINGO1，NgR1和p75以复合体形式存在于神经元细胞膜,被神经再生抑制因子活化后,一路完成对RhoA的激活,以实现对轴突生长的抑制作用［20］.Alivin 1Alivin 1在小鼠和人体神经细胞内都有表达，Alivin 1蛋白结构类似于Kek和Trk家族蛋白，也为一跨膜蛋白，胞外区含有7个LRR结构域，1个IgC2样环状结构域（图2），它在神经元被激活后表达，可增进神经元存活. Alivin 1基因mRNA的表达受电压门控Ca2+通道引发的Ca2+内流所调控，Ca2+浓度升高可引发该基因上调表达，其表达与去极化依赖的细胞存活和NMDA依赖的细胞存活相关，当河豚毒素阻断自发点位后，其在神经元内的表达也受到抑制. 因此该基因的表达是与神经活化状态相关的. 另外，在濒死细胞内该蛋白表达量明显低于正常水平，而高表达该基因可增进凋亡细胞的存活［21］.NGL1NGL1为NetrinG1蛋白配体，而NetrinG1是轴突导向分子Netrin家族的一个成员. NGL1为一含640个氨基酸残基的跨膜蛋白，其胞外区占整个蛋白结构的绝大部份，含有9个LRR结构域，外加双侧的LRR N端和C端结构，后接一个Ig结构域，其胞内区仅有92个氨基酸残基（图2）. NGL1通过LRR结构域与NetrinG1特异性彼此作用. NetrinG1高度表达于丘脑神经元轴突中，而NGL1在其投射的中间和最终靶目标区纹状体和大脑皮层中表达量最高. 在体实验表明，NGL1可结合在发育期的丘脑轴突表面受体上，增进胚胎丘脑轴突的生长，而游离的NGL1胞外区可显著抑制发育期丘脑轴突在鸡胚前脑内的生长［22］.LIG1LIG1为一膜糖蛋白（含1091个氨基酸残基），其胞外区（794个氨基酸残基）包括一个信号肽，15个LRR结构域，3个Ig样结构域，跨膜区由23个氨基酸残基组成，胞内区有274个氨基酸残基（图2）. LIG1主要表达于小鼠脑中，尤其是小脑和嗅球内胶质细胞，另外在小鼠胚胎瘤P19细胞分化为神经元样细胞进程中，其表达水平明显升高. 按照LIG1结构和表达特征分析，LIG1极可能作为胶质细胞表面的细胞特异性黏附分子或受体，在神经系统发育，胶质细胞分化，和神经功能的维持等方面发挥功能［23］.最近几年来，又有多种含LRR结构的蛋白在神经系统内被发现，如Pal［24］，含有5个LRR结构域和1个Ig结构域，表达于视网膜光感细胞中；AMIGO［25］，含有6个LRR结构域及1个Ig 结构域，存在于大脑和小脑中，参与轴突束的发育进程；GAC1［26］含12个LRR结构域及1个Ig结构域，主要表达于神经胶质瘤中；NLRR1/2/3［27-28］，含有11个LRR结构域，在神经系统发育和再生进程中发挥作用；zfNLRR［29］含有12个LRR结构域及1个Ig 结构域，参与斑马鱼神经系统损伤修复进程.综上所述，自从20世纪80年代在α2糖蛋白内发现LRR结构域以来，所发现的含LRR蛋白已达数十种，它们组成了LRR蛋白家族，参与了多种生物进程，如有的作为细胞黏附分子，有的作为跨膜受体，还有一些是可溶性的结合蛋白或配体. 对于上述这些主要表达于神经系统内的LRR蛋白来讲，它们更多的在神经生理活动中扮演重要角色. 如NgR,OMpg之于轴突生长抑制，Alivin1之于神经元存活，和Slit之于轴突导向，等等. 对于为何神经系统内存在这么多的具LRR结构的蛋白？它们在神经系统内的作用是什么？它们是如何发挥生理作用的？这些问题都有待于咱们在此后去作出解答.相信随着现代分子生物学技术的发展，尤其是蛋白彼此作用技术的发展，咱们此后有可能在更深层次上探讨存在于神经系统内的这些LRR蛋白的功能，这将有助于揭露该蛋白家族的分子作用机制，并对进一步熟悉神经系统的发育等重要生理活动有所裨益.【参考文献】［1］Takahashi N, Takahashi Y, Putnam FW. Periodicity of leucine and tandem repetition of a 24amino acid segment in the primary structure of leucinerich 2glycoprotein of human serum ［J］. 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Mini-ReviewLeucine-Rich Repeat Proteins of SynapsesJaewon Ko and Eunjoon Kim*National Creative Research Initiative Center for Synaptogenesis and Department of Biological Sciences, Korea Advanced Institute of Science and Technology(KAIST),Yuseong-Ku,Kuseong-Dong, Daejeon,KoreaLeucine-rich repeats(LRRs)are20–29-aa motifs that mediate protein–protein interactions and are present in a variety of membrane and cytoplasmic proteins.Many LRR proteins with neuronal functions have been reported.Here,we summarize an emerging group of syn-aptic LRR proteins,which includes densin-180,Erbin, NGL,SALM,and LGI1.These proteins have been impli-cated in the formation,differentiation,maintenance,and plasticity of neuronal synapses.V C2007Wiley-Liss,Inc.Key words:LRR;cell adhesion molecule;densin-180; Erbin;NGL;SALM;LGI1Neuronal development involves many events, including the outgrowth and migration of neuronal processes and the formation and differentiation of neuro-nal synapses.Neuronal proteins containing leucine-rich repeats(LRRs),such as the Nogo-66receptor,Slit, AMIGO,LINGO,NGL,and NLRR,have been impli-cated in the regulation of neurite outgrowth and migra-tion(Wong et al.,2002;Filbin,2003;Chen et al., 2006),but relatively little is known about the role of LRR proteins in the regulation of neuronal synapses. Several recent studies have identified LRR proteins that regulate the structure and function of neuronal synapses. They include densin-180,Erbin,NGL,SALM,and LGI1.LRRSThe LRR is a20–29-aa motif that mediates pro-tein–protein interactions and contains a conserved11-aa sequence,LxxLxLxxN/CxL(where x is any amino acid; Kobe and Kajava,2001).Analysis of the human genome reveals that there are*330LRR-containing proteins, indicating that LRRs are common.In proteins,LRRs usually occur in tandem arrays of a few to more than a dozen(2–52;Matsushima et al.,2005).Early X-ray crys-tallographic studies of ribonuclease inhibitor,which has 15LRRs,revealed that each LRR contains a beta-strand and an alpha-helix connected by loops(Kobe and Dei-senhofer,1993).Multiple LRRs are arranged so that they form a nonglobular,horseshoe-shaped structure, wherein parallel beta-sheets line the inner circumference of the horseshoe(the concave side)and alpha-helices decorate the outer circumference(the convex side). Although the sequences and numbers of LRRs can dif-fer,diverse LRRs share this overall horseshoe shape (Kobe and Kajava,2001).Mutagenesis studies and struc-tural analyses of LRR-ligand complexes have revealed that the concave surface of LRRs,which contains paral-lel beta-strands and adjacent loops,is involved mainly in ligand binding(Kobe and Deisenhofer,1995;Papageor-giou et al.,1997;Kobe and Kajava,2001).DENSIN-180AND ERBINDensin-180was discovered as an LRR protein concentrated in the postsynaptic density(PSD;Apperson et al.,1996),a postsynaptic membrane specialization containing macromolecular complexes of membrane,sig-naling,and scaffolding proteins(Kennedy,2000). Densin-180contains,from the N-terminus,16LRRs,an LAPSD(LAP-specific domain),a mucin-like domain,a transmembrane domain,and a C-terminal PDZ domain (Fig.1).Alternative splicing in the C-terminal region pro-duces four variants of densin-180that are differentially expressed during development(Strack et al.,2000). Based on its similarity to adhesion molecules,including GPIb-alpha,a surface membrane protein in platelets that binds to von Willebrand factor,densin-180has been suggested to be a type I transmembrane protein media-ting synaptic adhesion.This is further supported by the fact that densin-180contains a predicted transmembrane domain and a site for glycosylation by sialic acid in its extracellular region.Studies in cultured hippocampal neu-rons,however,have shown that endogenous densin-180Contract grant sponsor:Creative Research Initiatives Program of the Korean Ministry of Science and Technology(to E.K.).*Correspondence to:Eunjoon Kim,National Creative Research Initia-tive Center for Synaptogenesis and Department of Biological Sciences, Korea Advanced Institute of Science and Technology(KAIST), Yuseong-Ku,Kuseong-Dong,Daejeon305-701,Korea.E-mail:kime@ kaist.ac.krReceived4December2006;Revised29January2007;Accepted1 February2007Published online30April2007in Wiley InterScience(www. ).DOI:10.1002/jnr.21306Journal of Neuroscience Research85:2824–2832(2007) '2007Wiley-Liss,Inc.is not accessible to extracellular biotin labeling (Izawa et al.,2002),suggesting that it may not be a membrane protein.Proteins interacting with densin-180include a -actinin,the a subunit of calcium/calmodulin-dependent kinase II (CaMKII a ),d -catenin,MAGUIN,and Shank (Fig.2).The C-terminal region of densin-180binds to CaMKII a and a -actinin,and a -actinin binds to CaMKII a ,so that the three proteins form a ternary complex (Strack et al.,2000;Walikonis et al.,2001;Robison et al.,2005).Autophosphorylated CaMKII a binds more strongly to densin-180,whereas densin-180phosphorylation by CaMKII a has little effect on their interaction,suggesting that densin-180is involved in the synaptic localization of activated CaMKII a (Walikonis et al.,2001);however,another study reported that auto-phosphorylation of CaMKII a does not affect its binding to densin-180(Strack et al.,2000).This discrepancy could be due to the use of different assay methods (Walikonis et al.,2001).In addition todensin-180,Fig.2.Schematic diagram of the postsynaptic organization by LRR proteins.The C-terminal cytoplasmic tails of membrane proteins are indicated by black lines.Specific protein–protein interactions are indicated by direct contacts of the proteins or rin-G is tethered to the plasma membrane through a GPI anchor.Densin-180may be either membrane or cytoplasmic protein.Proteins:CaMKII a ,the a subunit of calcium/calmodulin-dependent kinase II;ADAM22,a disintegrin and metalloprotease 22;PSD-95,postsynaptic density 95;GKAP,guanylate kinase-associated protein;Shank,SH3and ankyrin repeat-containing protein;MAGUIN,membrane-associated guanylate kinase-interacting protein;NR1,NMDA receptor subunit 1;NR2,NMDA receptor subunit2.Fig.1.Domain structures of synaptic LRR proteins.The five LRR proteins found at neuronal synapses are shown along with their do-main structures.LRR,leucine-rich repeat;LAPSD,LAP-specific do-main;TM,transmsmbrane segment;PDZ,PSD-95,Dlg,and ZO-1;LRRNT,N-terminal LRR;LRRCT,C-terminal LRR;Ig,immu-noglobulin;FNIII,fibronectin type III;EPTP,Epitempin.Proteins:Erbin,ErbB2interacting protein;NGL,netrin-G ligand;SALM,syn-aptic adhesion-like molecule;LGI1,leucine-rich,glioma-inactivated 1.Scale bar ¼100amino acids.LRR Proteins of Synapses 2825Journal of Neuroscience Research DOI 10.1002/jnrCaMKII a binds the NR2B subunit of N-methyl-D-aspartate(NMDA)receptors(Strack and Colbran,1998; Gardoni et al.,1999;Leonard et al.,1999;Bayer et al., 2001),although densin-180and NR2B do not compete for CaMKII a binding(Strack et al.,2000).Together, these results suggest that synaptic localization of CaMKII a requires binding to multiple synaptic proteins, including densin-180,a-actinin,and NR2B.The C-terminal PDZ domain of densin-180binds the C-terminus of d-catenin(Izawa et al.,2002). Densin-180forms a complex with d-catenin and N-cad-herin in the brain,suggesting that densin-180may regu-late N-cadherin-based synaptic adhesion and plasticity. The PDZ domain of densin-180also associates with MAGUIN(Ohtakara et al.,2002),a mammalian homo-log of Drosophila CNK and a multidomain adaptor that regulates the Ras-ERK/MAPK pathway(Kolch,2005). Because MAGUIN interacts with PSD-95(Yao et al., 1999),an abundant PDZ protein in the PSD(Funke et al.,2004;Kim and Sheng,2004),and forms self-mul-timers(Ohtakara et al.,2002),MAGUIN multimers may link PSD-95and densin-180,forming a ternary com-plex.The C-terminal region of densin-180interacts with Shank(Quitsch et al.,2005),an abundant PDZ protein in the PSD that regulates the maturation of den-dritic spines(Sala et al.,2001).The N-terminal LRR domain of densin-180mediates its plasma membrane association infibroblasts and targeting to the basolateral membrane in epithelial cells(Quitsch et al.,2005). Overexpression of densin-180in cultured neurons pro-motes dendritic branching through the N-terminal LRRs,and this effect is reversed by coexpression of Shank(Quitsch et al.,2005),suggesting that Shank antagonizes the effects of densin-180on dendrites.Densin-180belongs to the LAP(LRR and PDZ) family(Bilder et al.,2000),which has three other mem-bers:Erbin(Borg et al.,2000;Huang et al.,2001), hScrib(Nakagawa and Huibregtse,2000),and Lano (Saito et al.,2001).LAP family proteins have16LRRs at their N-termini and zero to four PDZ domains at their C-termini:Erbin and hScrib have one and four PDZ domains,respectively,and Lano does not have PDZ domains but has a PDZ-binding C-terminus(Fig.1).Erbin,hScrib,and Lano mRNAs are expressed in a wide variety of tissues,including the brain(Borg et al., 2000;Nakagawa and Huibregtse,2000;Huang et al., 2001;Saito et al.,2001).In neurons,Erbin is concentrated in the PSD (Huang et al.,2001).A C-terminal region of Erbin excluding the PDZ domain associates with PSD-95 (Huang et al.,2001).In addition,the PDZ domain of Erbin mediates interactions with proteins,including d-catenin(Laura et al.,2002)and ErbB2/HER2(Huang et al.,2001;Fig.2),a receptor tyrosine kinase for neure-gulin that regulates neuronal development and synaptic plasticity(Huang et al.,2000;Holbro and Hynes,2004; Esper et al.,2006).Erbin suppresses the Ras-Raf-MEK pathway(Huang et al.,2003),and the N-terminal LRR domain of Erbin is both required and sufficient for this inhibition(Dai et al.,2006).Interestingly,the LRR do-main of Erbin interacts with Sur-8,an LRR-containing scaffold protein that associates with Ras and Raf and enhances ERK activation(Li et al.,2000),and this inter-action suppresses the association of Sur-8with active Ras and Raf and the activation of ERK(Dai et al., 2006).Thesefindings suggest that Erbin inhibits the Ras-Raf-MEK pathway through Sur-8binding and that the LRR domain of Erbin has a novel role in the regu-lation of intracellular signaling.This regulation is likely to have neuronal implications,because Sur-8mRNAs are detected in the brain(Sieburth et al.,1998).It should be noted that the majority of the proteins that interact with densin-180and Erbin bind to the C-termi-nal region.Identification of additional proteins that asso-ciate with the N-terminal LRR domain may help to clarify the functions of densin-180and Erbin.NGLThe NGL(netrin-G ligand)family of synaptic cell adhesion molecules has three known members;NGL-1, NGL-2(also known as LRRC4),and NGL-3(Lin et al., 2003;Zhang et al.,2005;Kim et al.,2006).Northern blot analyses with rat and human tissues indicate that NGLs are expressed mainly in the brain,although low-level expression is observed in liver(NGL-1)and heart (NGL-3;Lin et al.,2003;Zhang et al.,2005;Kim et al., 2006).NGL proteins have nine LRRs and an extracellu-lar immunoglobulin(Ig)domain,followed by a trans-membrane domain and a PDZ domain-binding motif at the C-terminus.The LRRs of NGL areflanked by cys-teine-rich LRR N-terminal(LRRNT)and LRR C-ter-minal(LRRCT)domains(Fig.1).NGL is modified by N-glycosylation.NGL associates with netrin-G through its extracellular region and with PSD-95through the PDZ-binding C-terminus(Lin et al.,2003;Kim et al., 2006;Fig.2).Classical netrins are diffusible axon-guidance mole-cules related to laminin that bind to receptors such as DCC and Unc5(Chisholm and Tessier-Lavigne,1999). Netrin-G(also called laminet)is a family of netrin-like adhesion proteins that has two known members,netrin-G1and netrin-G2(Nakashiba et al.,2000,2002;Yin et al.,2002).Northern blot analysis indicates that netrin-G1and netrin-G2mRNAs are expressed mainly in the brain(Nakashiba et al.,2000,2002;Yin et al.,2002). Netrin-G is distinct from classical netrins in several ways (Nakashiba et al.,2000).First,netrin-G1is linked to the plasma membrane through a glycosylphosphatidylinositol (GPI)anchor.Second,netrin-G1does not bind to the netrin receptors(DCC or Unc5).Netrin-G1and netrin-G2share a similar domain structure,with the laminin N-terminal domain(LamNT;also called domain VI)in the N-terminal half and three laminin-type EGF-like domain(LEGF)in the C-terminal half.Interestingly,the distributions of netrin-G1and netrin-G2mRNAs in the brain minimally overlap(Nakashiba et al.,2000,2002; Yin et al.,2002;Kim et al.,2006),suggesting that their2826Ko and KimJournal of Neuroscience Research DOI10.1002/jnrassociation with specific ligands may contribute to the formation of specific neural networks in the brain.Netrin-G1is expressed on the surface of thalamo-cortical axons(Nakashiba et al.,2002).The trajectory of thalamocortical axons is precisely controlled during their migration in the embryonic brain,suggesting that the migration is regulated by multiple chemorepellent and chemoattractant activities(Braisted et al.,1999).NGL-1 (netrin-G1ligand1)was identified as a specific receptor for netrin-G1in a screen for extracellular molecules with the ability to influence thalamocortical axon guid-ance(Lin et al.,2003).The LRR region in the extracel-lular region of NGL-1is sufficient for netrin-G1bind-ing.NGL-1-coated substrate promotes axonal outgrowth in cultured thalamic neurons.Conversely,soluble NGL-1,which competes with endogenous NGL-1,suppresses axon outgrowth in chick embryo thalamic neurons. These results suggest that the interaction between netrin-G1and NGL-1regulates the outgrowth and migration of thalamocortical axons.Unlike soluble NGL-1,however,soluble netrin-G1does not suppress the growth of thalamocortical axons,suggesting that netrin-G1is not the only NGL-1receptor.The expression of NGL is higher in postnatal brains than in embryonic brains(Kim et al.,2006),sug-gesting that NGL has additional roles in later stages of brain development.A recent study revealed that NGL can induce the formation of neuronal synapses(Kim et al.,2006).NGL-2expressed on nonneuronal cells or linked to beads induces morphological and functional presynapses in contacting axons of cocultured neurons. Also,overexpression of NGL-2in cultured neurons increases the number of excitatory synapses.Direct aggregation of NGL-2on the dendritic surface induces the clustering of postsynaptic proteins,including PSD-95 and NMDA receptors.Furthermore,knockdown of NGL-2reduces the number and function of excitatory synapses,but not inhibitory synapses,and soluble NGL-2reduces the number of excitatory synapses in a domi-nant negative manner.These results suggest that NGL-2 regulates the formation of excitatory synapses via a trans-synaptic interaction with netrin-G2and a cytoplasmic interaction with PSD-95.The netrin-G and NGL families have multiple members,indicating that there may be isoform-specific interactions.In addition to the previously identified interaction between netrin-G1and NGL-1(Lin et al., 2003),a recent study further revealed that netrin-G2 binds NGL-2but not NGL-1or NGL-3and that netrin-G1does not bind NGL-2or NGL-3(Kim et al., 2006).In addition,neither netrin-G1nor netrin-G2 associates with NGL-3(Kim et al.,2006).These results suggest that netrin-G and NGL associate in an isoform-specific manner and that NGL-3may have a novel ligand.The adhesion between presynaptic neurexins and postsynaptic neuroligins is a well-known example of het-erophilic and synaptogenic adhesion(Ichtchenko et al., 1995).Their transsynaptic adhesion induces pre-and postsynaptic differentiation in a bidirectional manner (Scheiffele et al.,2000;Graf et al.,2004).The netrin-G-NGL complex shares some but not all features of the neurexin-neuroligin complex.As with neuroligin,NGL associates with PSD-95through its C-terminus(Irie et al.,1997;Kim et al.,2006),suggesting that PSD-95is an important postsynaptic scaffolding protein that coor-dinates synaptic adhesion and synaptogenesis.The ecto-domain of NGL mediates heterophilic adhesion(Ichtch-enko et al.,1995;Lin et al.,2003)and induces presyn-aptic differentiation(Scheiffele et al.,2000;Kim et al., 2006).A small difference between them is that NGL binds mainly to thefirst two PDZ domains of PSD-95 whereas neuroligin binds to the third PDZ domain(Irie et al.,1997;Kim et al.,2006),suggesting that NGL and neuroligin do not compete for PSD-95binding and that they may even function synergistically.Neuroligin-1and neuroligin-2differentially distrib-ute to excitatory and inhibitory synapses,respectively (Song et al.,1999;Graf et al.,2004;Varoqueaux et al., 2004;Chih et al.,2005;Levinson et al.,2005).PSD-95 enhances synaptic localization of neuroligin-1and trans-locates neuroligin-2from inhibitory to excitatory synap-ses,promoting excitatory synapse formation at the expense of inhibitory synapses and thus regulating the balance of excitation and inhibition in a single neuron (Graf et al.,2004;Prange et al.,2004;Chih et al., 2005).Because NGL-2is recruited to synapses by PSD-95binding and regulates excitatory but not inhibitory synapse formation(Kim et al.,2006),NGL may act in concert with neuroligins and PSD-95to regulate the excitatory/inhibitory balance.The netrin-G-NGL interaction differs from the neurexin-neuroligin interaction in that netrin-G2ex-pressed in nonneuronal cells lacks the ability to induce postsynaptic differentiation in contacting dendrites, whereas NGL-2is able to induce presynaptic differentia-tion in contacting axons(Kim et al.,2006).This unidir-ectional synaptogenic activity contrasts with the bidirec-tional synaptogenesis mediated by neurexins and neuroli-gins(Scheiffele et al.,2000;Graf et al.,2004).This difference is consistent with the notion that netrin-G might not be the only ligand of NGL(Lin et al.,2003).Netrin-G and NGL are implicated in brain dys-functions.Single nucleotide polymorphism analyses indi-cate that netrin-G1and netrin-G2are highly associated with schizophrenia,and netrin-G1expression is reduced in schizophrenic brain(Aoki-Suzuki et al.,2005).Fur-thermore,truncation of the netrin-G1gene by a bal-anced chromosomal translocation leads to Rett syndrome (Borg et al.,2005),although whether alterations in netrin-G1are the main cause of Rett syndrome remains to be determined(Archer et al.,2006).Several issues remain to be addressed regarding syn-aptogenic function of netrin-G and NGL.First,there are a large number of splice variants of netrin-G1and netrin-G2,and they are regulated spatiotemporally (Nakashiba et al.,2000,2002;Yin et al.,2002;Meera-bux et al.,2005),similar to neurexin(Ullrich et al.,LRR Proteins of Synapses2827Journal of Neuroscience Research DOI10.1002/jnr1995).Therefore,alternative splicing of netrin-G may affect NGL binding.The function of the Ig domain of NGL is currently unknown,and,although the extracel-lular regions are relatively conserved between the differ-ent NGLs,their cytoplasmic regions are markedly differ-ent(Lin et al.,2003;Zhang et al.,2005;Kim et al., 2006),suggesting that the variants have different func-tions.Unlike netrin-G mRNAs,which have largely nonoverlapping distributions in the rat brain(Nakashiba et al.,2000,2002;Yin et al.,2002;Kim et al.,2006), NGL mRNAs have overlapping distributions(i.e.,py-ramidal neurons in the CA1region of hippocampus express mRNAs for all three NGLs;Kim et al.,2006).It is possible that different NGL isoforms segregate into different dendritic regions of a neuron to mediate syn-apse formation with axons of different origins.Alterna-tively,a single synapse may contain multiple NGLs that could act synergistically.A recent study reported that NGL-1binds whirlin (Delprat et al.,2005),a PDZ protein implicated in deaf-ness(Belyantseva et al.,2003;Mburu et al.,2003).This interaction between NGL-1and whirlin is mediated by the C-terminus of NGL-1and thefirst two PDZ domains of whirlin.Whirlin is encoded by the Whrn gene,and mutation of this gene causes autosomal reces-sive deafness in mice and humans(Holme et al.,2002; Mburu et al.,2003).Whirler mice,which have a muta-tion in the Whrn gene,have shorter stereocilia,which are stiff microvilli located at the apex of cochlear inner hair cells,and they exhibit degeneration of hair cells (Holme et al.,2002).It has been suggested that NGL-1 links adjacent stereocilia through homophilic adhesion, leading to the formation of a bundle of stereocilia(Del-prat et al.,2005).In support of this idea,NGL-1mole-cules homophilically interact with each other in GST pull-down assays in the presence of Ca2+concentrations (5–250l M)similar to that found in endolymph,the extracellularfluid surrounding the hair cells(Delprat et al.,2005).These results suggest a novel function of NGL in hair cell regulation.However,the homophilic adhesion in NGL-1remains to be further characterized; it is unclear whether the interaction occurs in a cis or trans conformation.Finally,NGL-2/LRRC4has been implicated in the suppression of glioma,a common primary malignant tumor in the central nervous system(Zhang et al.,2005; Wu et al.,2006).Northern blot analysis indicates that NGL-2/LRRC4expression is reduced in glioblastoma cell lines and gliomas.Exogenous overexpression of NGL-2/LRRC4suppresses the proliferation of glioblas-toma cells in a manner requiring the protein’s LRR region.These results suggest that NGL-2functions as a tumor suppressor.SALMThe SALM(synaptic adhesion-like molecule)fam-ily is a novel group of synaptic LRR proteins that includesfive known members(Ko et al.,2006;Wang et al.,2006).Shortly after the initial reports,these pro-teins were independently described as members of the Lrfn(leucine-rich repeat andfibronectin III domain-containing)family(Morimura et al.,2006).Northern blot analysis indicates that the mRNAs for all of the SALM isoforms are expressed mainly in the brain, although lower levels of SALM3/Lrfn4and SALM4/ Lrfn3mRNAs are found in other tissues(Ko et al., 2006;Morimura et al.,2006).SALMs contain six LRRs, an Ig domain,and afibronectin III domain in the extracellular region,followed by a transmembrane do-main and a C-terminal PDZ-binding motif(Fig.1).The LRRs of SALMs areflanked by LRRNT and LRRCT domains.SALM is similar to NGL in its overall domain structure and its ability to bind PSD-95(Ko et al.,2006; Wang et al.,2006;Fig.2).Unlike NGL,SALM has a fibronectin III domain as an additional adhesion domain. Also,SALM4and SALM5lack the PDZ-binding motif, and the ligands for SALMs are not known.SALMs are modified by N-glycosylation(Ko et al.,2006;Morimura et al.,2006).When overexpressed in cultured neurons,SALM1 promotes neurite outgrowth in early-stage cultured hip-pocampal neurons(4–6days in vitro)but not in late-stage neurons(14–16days in vitro;Wang et al.,2006), suggesting that SALM1regulates an important aspect of early neuronal development.Interestingly,overexpres-sion of SALM1induces the surface clustering of NMDA receptors on dendrites by a mechanism that requires PDZ interaction(Wang et al.,2006),suggesting that dendritic coclustering of SALM1and NMDA receptors requires a PDZ scaffold.In addition,SALM1forms a complex with NMDA receptors in brain and associates with the NR1but not the NR2subunit of NMDA receptors in heterologous cells(Wang et al.,2006).Previous studies have identified several NMDA re-ceptor-associated adhesion molecules.EphB receptor ty-rosine kinases directly interact with NR1,and ephrin stimulation of EphB receptors induces coclustering of EphB and NMDA receptors,tyrosine phosphorylation of NMDA receptors,and an increase in the number of syn-apses(Dalva et al.,2000;Takasu et al.,2002).Overex-pression of neuroligin-1induces dendritic NMDA re-ceptor clustering through mechanisms that partially require PSD-95binding(Chih et al.,2005),and neu-rexin presented to dendrites induces clustering of neuro-ligin and NMDA receptors(Graf et al.,2004).The neu-ral cell adhesion molecule(NCAM)forms a complex with NMDA receptors in brain,and antibody-induced dendritic clustering of NCAM induces coclustering of NMDA receptors(Sytnyk et al.,2006).In addition, NCAM-associated polysialic acid negatively regulates NR2B-containing NMDA receptors(Hammond et al., 2006).Direct aggregation of NGL-2on dendrites indu-ces secondary clustering of NMDA receptors(Kim et al., 2006).Therefore,SALM1,along with EphB receptors, neuroligin,NCAM,and NGL,may participate in den-dritic or synaptic clustering of NMDA receptors.The SALM1-NMDA receptor interaction may also contrib-2828Ko and KimJournal of Neuroscience Research DOI10.1002/jnrute to reciprocal modulation of synaptic adhesion and NMDA receptor activity(Wang et al.,2006).SALM2has also been characterized(Ko et al., 2006).SALM2expressed in nonneuronal cells does not induce presynaptic differentiation in contacting axons, suggesting that,unlike NGL,SALM2does not have syn-aptogenic activity;however,overexpression of SALM2 in cultured neurons increases the number of excitatory synapses.SALM2knockdown by RNA interference reduces the number and functions of excitatory but not inhibitory synapses,as determined by pre-and postsy-naptic markers and miniature postsynaptic currents. Direct aggregation of SALM2on the surface membrane of dendrites induces secondary clustering of excitatory postsynaptic proteins,including PSD-95,a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA) receptors,and to a lesser extent NMDA receptors,sug-gesting that SALM2clustering is sufficient to drive post-synaptic differentiation.These results suggest that SALM2regulates the differentiation or maturation of excitatory synapses,whereas NGL contributes to excita-tory synapse formation.There are several differences between SALM2and SALM1.SALM2associates with both AMPA and NMDA receptors but is more strongly associated with AMPA receptors,whereas SALM1preferentially associ-ates with NMDA receptors(Ko et al.,2006;Wang et al.,2006).They also differ in their temporal expres-sion patterns:SALM2expression gradually increases dur-ing postnatal synaptic development,and SALM1expres-sion reaches a plateau at early stages and is maintained throughout development(Ko et al.,2006;Wang et al., 2006).Therefore,SALM2may promote the maturation of excitatory synapses at later stages,whereas SALM1 exerts its actions in wider developmental stages.The effect of SALM2on synaptic maturation is reminiscent of Dasm1,an Ig family adhesion-like molecule that interacts with S-SCAM and Shank PDZ proteins and regulates synaptic maturation through a selective effect on AMPA receptors(Shi et al.,2004a,b).Several aspects of SALM function remain to be studied.Ligands of SALMs,if any,have to be identi-fied.The ligands could be SALMs,as suggested previ-ously(Wang et al.,2006).Although SALM2does not mediate homophilic adhesion(Ko et al.,2006),other SALM isoforms may participate in homophilic or heterophilic adhesion.In support of this possibility, SALM1and SALM2are found in both axons and den-drites(Ko et al.,2006;Wang et al.,2006).Another issue that remains to be addressed is whether SALMs have lateral or cis-type interactions on the same plasma membrane surface,forming homo-or heteromultimers. Finally,different SALMs may have different functions. Notably,the cytoplasmic regions of SALMs are essen-tially unrelated except for their extreme C-termini, although their ectodomains are more conserved.In addition,SALM4and SALM5do not contain C-termi-nal PDZ-binding motifs,suggesting that they might have distinct functions.LGI1LGI1(leucine-rich,glioma inactivated gene1)is an LRR protein implicated in epilepsy(Chernova et al., 1998;Kalachikov et al.,2002;Morante-Redolat et al., 2002).LGI1contains four LRRs in the N-terminal half and seven epitempin(EPTP)repeats in the C-terminal half(Fig.1).Mutations in LGI1cause a rare form of epilepsy known as autosomal dominant partial epilepsy with auditory features(ADPEAF;Kalachikov et al.,2002;Mor-ante-Redolat et al.,2002).LGI1is modified by N-gly-cosylation and secreted from cells in a manner requiring the EPTP repeats(Senechal et al.,2005;Sirerol-Piquer et al.,2006).LGI1mutants found in ADPEAF patients are not secreted(Senechal et al.,2005;Sirerol-Piquer et al.,2006),suggesting that the limited secretion of LGI1may cause epileptic conditions.LGI1has been implicated in tumor suppression (Chernova et al.,1998),although a recent study does not support this possibility(Piepoli et al.,2006).An im-portant clue to the function of LGI1has come from the tight association of LGI1with Kv1.1(Schulte et al., 2006),a presynaptic voltage-dependent potassium chan-nel subunit.Kv1.1,which is a noninactivating channel, is transformed into a rapidly inactivating channel when coassembled with the Kv1.4or Kv b1subunit.In the Kv1.1protein complex,LGI1inhibits Kv b1-medi-ated Kv1.1inactivation,and LGI1mutants found in ADPEAF patients do not have this activity,suggesting that the rapid presynaptic Kv1.1inactivation caused by defective LGI1may induce epileptic activities.How does LGI1inhibit the effect of Kv b1on Kv1.1?A possi-bility is that LGI1mutants,which cannot be secreted, may associate with and trap Kv1.1in the early secretory pathway.This may lead to the surface expression of Kv1.1complexes that lack LGI1and thus have rapid inactivation,as recently suggested(Sirerol-Piquer et al., 2006).Another important clue to the function of LGI1 was thefinding that LGI1associates with ADAM22 (Fukata et al.,2006),a transmembrane protein associated with epilepsy in mice(Sagane et al.,2005;Fig.2).This interaction is mediated by EPTP repeats in LGI1and the disintegrin domain in ADAM22.An LGI1mutant found in ADPEAF patients fails to bind ADAM22.The PDZ-binding motif at the C-terminus of ADAM22also associates with the second half of PSD-95,which con-tains the third PDZ domain(Fukata et al.,2006;Fig.2). Furthermore,LGI1,ADAM22,and PSD-95form a tri-partite complex in heterologous cells.These results sug-gest that LGI1is an extracellular ligand for ADAM22 and that PSD-95clusters LGI1and ADAM22at excita-tory synapses.Interestingly,addition of soluble LGI1to hippo-campal slices increases surface AMPA receptor expres-sion,the AMPA/NMDA receptor ratio,and the fre-quency and amplitude of miniature excitatory postsynap-tic currents(Fukata et al.,2006).These effects on synaptic transmission are blocked by preincubating LG11 with soluble ADAM22,suggesting that LGI1actsLRR Proteins of Synapses2829Journal of Neuroscience Research DOI10.1002/jnr。
