甲壳动物蜕皮的调控

甲壳动物蜕皮的调控 甲壳动物蜕皮的调控:综述与展望 摘要:蜕皮是一个高度复杂的过程，它需要精确的协调才能完成。我们描述了早期经典的内分泌学实验来阐明激素和腺体在这一过程中所起的作用。然后，我们有描述了了一些近期实验，提供了一些关于蜕皮的细胞和分子调控方面的信息。除了提供这样一篇科学文献综述，也涉及了一些对未来研究的展望。 关键词:20—羟基蜕皮酮;甲壳动物;节肢动物;蜕皮激素;蜕皮酮;蜕皮 1. 前言 节肢动物要实现生长，必须快速脱掉角质层，摆脱其束缚，吸收水分或 空气来生长、延伸新的、柔软的外骨骼，然后再迅速硬化外骨骼来保护自己 和实现运动。节肢动物门中的甲壳纲和昆虫纲是研究蜕皮的内分泌调控很有 价值的模式动物。在这篇综述中，我们主要来谈甲壳动物。 2. 经典的内分泌学实验 蜕皮过程是一个周期性的事件，其高潮就是快速蜕皮，脱掉外骨骼(图1)。 [1]甾类激素的激素原—蜕皮激素，是从成百上千公斤的蛾蛹中分离出来的。几年后，我们确定了蜕皮激素的结构，并且知道它来源于前胸腺。从随后的许多实验中发现，在大部分物种中，20—羟基蜕皮酮(20E)是蜕皮激素的生理活性形式。在甲壳动物蜕皮激素的类似研究中，我们观察到，甲壳动物最重要的蜕皮激素是 [2]20E。 [3]经典的形态学观察和涉及器官切除的内分泌实验显示，在普通滨蟹中,其蜕皮腺是胸部的Y器官(YO)。在蟹类中，YO是类似球状的腺体，位于内庭腹侧较前的位置。大甲壳亚目的YO较难定位，因为它是一层很薄的单层细胞。在J.Dennis O’Connor的，Chang(EC)开始用躄鱼属蟹和厚纹蟹的YO做器官 [4]培养试验。这些实验涉及了作为YO重要的分泌物—蜕皮激素的特性研究。结果显示，蜕皮激素是20E的激素原。实验动物是EC晚上亲自在加利福尼亚Palos Verdes的岩石下捉的。 这些YO实验涉及合适的器官培养试验的确定。确定成分的培养基，比如M199比简单盐溶液更能延长YO的分泌活动。然而，鉴定分泌产物需要收集和浓缩成百上千毫升的培养基，而简单盐溶液中的分泌产物含杂质少，因此用于质谱分析的激素在生理盐溶液中培养更易纯化。 从小龙虾和三疣梭子蟹中也获得了相似的结果。更多的数据显示，在一些物种中，YO也分泌其它的蜕皮激素，包括3—脱氢蜕皮酮和25—脱氢蜕皮酮。未来研究的一个富有创造性的方面是不同甲壳动物的YO分泌的蜕皮激素的类型及其比例，不同的蜕皮激素，功能也可能不同，而这些方面现在还不太清楚。 loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: date case, agree to write off loans. Loan review signature: date of record form 16: qilu self credit for credit card cancellation application form name of borrower ID number and credit limit expiration date year month day to year month day year contract number self-service card-I do not use credit, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: 图1.小龙虾的蜕皮 整个过程共30分钟，蜕皮是这个过程的高潮，由甾类激素20- 羟基蜕皮酮调节。(A)小龙虾吸收了许多旧外骨骼的矿物质，并在胸部和腹部的连接处 出现裂口。(B)动物的前部通过向后面拖来从外骨骼中缩回(C)虾的后部往前拔(D) 虾摆脱了旧的外骨骼，开始吸收水分来延伸新的、柔软的外骨骼(E)蜕过皮的虾位于 旧外骨骼的上方，它需要几天的时间使几丁质、蛋白质和碳酸钙沉积到外骨骼中。蜕皮 周期将持续几天或几个月，根据动物的大小和年龄来决定。 在蜕皮周期中，血淋巴中的蜕皮激素波动很大(龙虾蜕皮后期<10pg/ul到前 [5]期>350pg/ul)，并且这些变化调节那些发生在蜕皮周期的生理、生化过程。在蜕皮周期中YO分泌或合成蜕皮激素的速率是不断变化着的，部分地解释了血淋巴中蜕皮激素滴度的波动。在蜕皮前期亚期之前，血淋巴中有高浓度的的蜕皮激 [6]素，而且发现移植的YO也分泌大量的蜕皮激素。血淋巴中低浓度的蜕皮激素与它较低的分泌速率相关联。这就出现了一个问题:什么控制着YO的分泌速率呢, 在一个世纪以前，移去蟹的两个眼柄造成一个蜕皮周期时间明显缩短。后来这在小龙虾和许多其他物种中被证实。通过这些观察，我们假定有一个内分泌因 [7]子存在于眼柄中，它正常地抑制蜕皮，即蜕皮抑制激素(MIH)。详细的镜检发现了几种十足目甲壳纲动物眼柄中的神经血器官，被称为窦腺，主要储存神经内分泌物质。而构成窦腺的膨大末端的一群神经内分泌细胞被称为X器官 [8](XO)。我们在观察切除眼柄的十足目动物中发现，蜕皮周期缩短，很可能是因loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: date case, agree to write off loans. Loan review signature: date of record form 16: qilu self credit for credit card cancellation application form name of borrower ID number and credit limit expiration date year month day to year month day year contract number self-service card-I do not use credit, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: 为循环蜕化类固醇的浓度迅速升高，这是由XO或窦腺的移去造成的。相反地，在切除眼柄的虾体内重新注入眼柄提取物，蜕皮激素的量降低，这个结果给MIH—YO蜕皮控制轴提供了额外的支持。Gersch et al为这个假设提供又一个证据。他证明:如果小龙虾在一开始就被注入了窦腺提取物，那接下来培养YO时，其产生的蜕皮激素的量就会减少。当虾的YO培养在条件培养基中，该培养基曾 [10]培养过移植的窦腺或加入过眼柄提取物时，YO分泌蜕皮酮会被抑制。EC的实验室证明:当在螯虾体内注入窦腺提取物会减少蜕皮酮的循环滴度，延长蜕皮期。 从普通滨蟹中提取的MIH是第一个被描述的MIH。EC的实验室就是从事H.americanus体内MIH的研究。当时，Keller正参观EC的实验室，他记住了普通滨蟹MIH的大部分序列，非常兴奋地看到这个序列的氨基酸与龙虾序列很相似。 MIH是一个新颖的神经肽家族成员之一。这个家族的代表到目前为止仅仅在节肢动物中发现。这个神经肽家族调控多种功能，如蜕皮、生殖和代谢。MIH [11]结合到YO细胞膜的受体上，可能通过环核苷酸第二信使或通过改变钙离子流量和蛋白激酶C(PKC)的活性来调节它的活性，PKC在类固醇生成中使酶磷酸化[12]。MIH活性调控方式将在下面介绍。 EC团队也研究了这个假设:即蜕皮酮可能反馈作用于XO、YO和其它靶组织来调节蜕皮。Chang 和O’Connor证明:各种组织羟化蜕皮酮成为20E的活动会被外源的20E所抑制。Cheng 和Chang证明通过注入外源20E，龙虾正常的蜕皮周期会被推迟。这些结果表明，要想完成正常的蜕皮周期过程，不仅需要提高蜕皮激素的浓度，还要在后来降低其量。蜕皮酮抑制机制尚不太清楚。El Haj et al.公布了一个发现:蜕皮酮受体位于眼柄的神经组织上。这些数据暗示，MIH的分泌可能存在一个正反馈或负反馈调控。最近，我们获得初步的数据:一个非甾族的蜕皮酮类似物(虫酰肼)能够抑制试管内蟹YO分泌蜕皮激素。这个结果是在浓度为50~100pg/ul的培养基中观察到的，不是在较低的浓度下观察到的。蜕皮酮抑制其自身的分泌可能解释了那个现象:即尽管眼柄组织没有再生，但是 [13]在去眼柄的龙虾体内，激素滴度仍呈周期性地升降。 3. 蜕皮的分子调控 3.1 附肢因素 有许多种信号影响YO的活动。外部因素，比如压力，极端温度，空间 [14]拥挤度，短光周期，都能抑制蜕皮。对于陆地蟹，失去5个或多个步行足被称为多足自切(MLA),能诱导蜕皮，因为肢芽(LBs)的生长局限于蜕皮前期。一旦开始蜕皮，肢芽开始延伸，并且变成多功能的附件。然而在蜕皮前期的早期，附肢自切(LBA)会延迟蜕皮2~3周，给出时间让附肢再度生长，这样虾蜕皮时 [15]会有完整的附肢。通过这些研究，Skinner提出:肢芽能产生刺激和抑制因子，loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: date case, agree to write off loans. Loan review signature: date of record form 16: qilu self credit for credit card cancellation application form name of borrower ID number and credit limit expiration date year month day to year month day year contract number self-service card-I do not use credit, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: 她分别把它们称之为肢自切的蜕皮间期因子(LAFan)和肢自切的蜕皮前期因子(LAFpro)。前者是第一个附肢自切引起的，它诱导产生蜕皮来应答MLA，LAFan的特性目前还不太清楚。后者是一个类似蜕皮抑制激素因子，由附肢的再生引起的，它通过降低血淋巴中蜕皮酮的浓度来抑制附肢的生长。到蜕皮前期的中期，虾开始要蜕皮，诸如附肢自切和蜕皮抑制激素的注入之类的干预不能延缓蜕皮前期，虾的蜕皮也不会推迟。 地蟹是一种很好的实验动物来研究蜕皮的内分泌调控。它们遍及加勒比海，在沙滩上的沙丘上挖洞并生活在这些洞穴中。因为它们在实验室中易于饲养，且蜕皮也易于控制，Dorothy Bliss率先在19世纪50年代使用地蟹作为实验动物。1962年，Skinner发表了她的关于地蟹的第一篇，报告了在地蟹蜕皮周期中， [16]其表皮结构的微观变化分析，地蟹自此成为Skinner最初的实验动物，并长达40年。地蟹后来又成为Mykles的实验动物，当他在1979年进入Skinner的实验室，即橡树岭国家实验室，研究蜕皮诱导的螯虾肌肉萎缩(1966年发表)。当Mykles在1985年搬到美国科罗拉多州大学后，他继续研究地蟹。当Mykles在1998-1999年居住在Bodega Marine实验室时，他开始和Chang合作研究地蟹蜕 [128]皮的内分泌调控，即研究LAFpro的特性。随后的工作直接转向YO神经肽信 [17]号通路的研究和肌肉萎缩的蜕化类固醇的研究。由此，地蟹在60多年来已经成为研究甲壳动物蜕皮生理学的一种很重要的实验动物。 3.2 眼柄神经肽对YO的调控 MIH和一种相关肽，即CHH，甲壳动物高血糖激素，抑制YO蜕皮酮的产生，它通过不同的信号通路，集中于环鸟苷酸(cGMP)。YO细胞膜上有CHH和MIH的特异受体。CHH受体是膜结合的鸟苷酰环化酶(GC),然而MIH受体的特性还不确定。受体与CHH的结合直接导致cGMP含量的升高。与之相比，MIH信号经常涉及环腺苷酸(cAMP)含量的短暂升高，紧接着是cGMP含量大 [18]量持久地增高。cGMP的延时增长暗示MIH活化一个可溶的GC，就像活化一个膜结合GC将导致cGMP的快速增长一样。cGMP 和cAMP都抑制离体YO中蜕皮酮的产生。我们近期的工作揭示，对NO敏感的GC参与了反应，即GC-1 2+竞争性抑制了YO蜕皮酮的产生。此外，YO表达了一种Ca/CaM依赖性的NO合酶(NOS)和GC-1的催化亚基。 2+很多数据支持MIH信号通路由两部分组成，即cAMP/ Ca依赖的“触发”阶段和一个NO/cGMP依赖的“总和”阶段，两阶段通过钙调蛋白(CaM)连接。 2cAMP的短暂升高引发Ca流入细胞内，激活CaM(图2)。NOS的持续激活是 2+2+由两个结果造成的，即Ca/CaM直接结合到NOS，通过CaN(Ca/CaM依赖性 [19]的磷酸酶)使NOS去磷酸化，从而活化NOS。NOS的磷酸化降低了它的活性，而通过CaN的去磷酸化作用增强NOS的活性。NOS在活化的YO中是磷酸化的，在缺少MIH时，YO中NOS由于蛋白激酶的作用始终处于失活状态。这个类似于信号发送机制，通过十肽菌素刺激液体分泌物流到果蝇的马氏管中。MIH以loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: date case, agree to write off loans. Loan review signature: date of record form 16: qilu self credit for credit card cancellation application form name of borrower ID number and credit limit expiration date year month day to year month day year contract number self-service card-I do not use credit, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: 脉冲的形式释放，每个脉冲的半衰期是5-10分钟，这造成在大部分处于蜕皮间期动物的血淋巴中MIH效价很低(<5fmol/ml)。在这个例子中，持续活化GC-1提供一个机制:即在脉冲间抑制YO，这样就解释了处于蜕皮间期的动物，其MIH的平均效价很低时，YO是怎样被抑制的。 3.3蜕皮周期中YO的变化 YO是一个动态器官，在蜕皮周期中有4个生理状态的过渡(图3)。在蜕皮后期和间期，YO处于基本状态，血淋巴中的蜕皮酮效价很低。MIH的减少引发YO由基本状态到活化状态的转变，血淋巴中越来越多的蜕皮酮启动了表皮中(比如上表皮与外骨骼的分离)蜕皮前期的过程，以及蜕皮前期的早期肢再生。在蜕皮前期的中期，YO由活化态向committed状态转变，YO提高了蜕皮酮的生物合成，血淋巴中其浓度也达到一个峰值(>300pg/ul),爪肌肉萎缩，胃石也完全形成。高浓度的蜕皮酮引起YO由committed状态到抑制状态的转变。在蜕皮期，当蜕皮酮浓度很低时，YO又回到基态。 YO在D期活化是由窦腺中MIH的减少所引发的。它使YO去抑制，可能0 通过MIH信号组分的失活引起的，最终导致YO快速生长并提高蜕皮酮的生物合成能力(图3)。眼柄切除的动物，YO中的NOS是磷酸化的，表明在缺少MIH时，NOS是失活的，下游的目标大部分是不知道的，但是活化可能包括转录、翻译和翻译后调控。YO活化对数百个蛋白质表达产生重大影响。在转录水平，phantom(phm0，蜕皮酮生物合成途径中的一个关键酶，在蜕皮前期上调表达，它编码一个细胞色素P-450单加氧酶，能羟化蜕皮酮的前体(第25个C原子)。在虾中，YO中phm的表达在蜕皮前期增长了7倍，而通过注入窦腺提取物和MIH，则其表达降低了2.5倍。 YO的活化也涉及了翻译调控。在躄鱼的YO中，翻译抑制剂放线菌酮抑制了蛋白质的合成和蜕皮酮的合成。与之相比，转录抑制剂放线菌素D抑制了RNA的合成，而对蜕皮酮的合成没有任何影响。MIH信号发送的潜在目标是mTOR，它的功能是作为营养传感器促进动物细胞的生长。在昆虫中，mTOR刺激前胸腺中蜕皮酮的合成。营养物质和胰岛素样的肽段活化mTOR，使蛋白质合成装置中的组分磷酸化，比如p70-S6激酶和elF4E结合蛋白，提高mRNA的转录，胰岛素能刺激家蚕前胸腺中蜕皮酮的合成。在黑腹果蝇中，mTOR途径控制前胸腺的 [20]大小和蜕化类固醇基因表达能力。mTOR活动可能需要甲壳动物YO中所有蜕化类固醇基因表达，正如雷帕霉素，一种mTOR抑制剂，它是地蟹和滨蟹离体YO中蜕化类固醇的潜在抑制剂。 处于committed状态的YO，它的一个显著特点是对MIH和CHH变得不敏感。在蜕皮前期，滨蟹和小龙虾对MIH的敏感度降低，在蜕皮前期的中期最后和蜕皮前期的后期，它们对MIH的敏感度最低。在蜕皮前期，MIH和CHH结合高亲和力受体的能力下降，敏感性的降低是由下游信号发送组分的下调造成的。增强了活性的磷酸二酯酶(PDE)通过保持细胞内的环核苷酸处于低水平来loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: date case, agree to write off loans. Loan review signature: date of record form 16: qilu self credit for credit card cancellation application form name of borrower ID number and credit limit expiration date year month day to year month day year contract number self-service card-I do not use credit, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: 降低对MIH的应答。然而，PDE活性增长量小于MIH敏感性减小量，表明MIH信号通路中的其他组分，比如NOS、GC-1的表达都下调。在地蟹和滨蟹的YO中，NOS和GC-1 mRNA在眼柄切除后水平上升，表明这些基因的表达对MIH和其他神经肽的急速下降还是很敏感的。 从活化态到committed态转变的调控还不太清楚，它可能是由MIH的升高所触发的。然而可能也涉及有自分泌或旁分泌因素。转化生长因子β超家族成员是可能的因素，它们都属于TGFβ/Activin/Nodal和BMP/GDF/MIS家族。TGFβ激活Smad转录因子，该因子可以通过转绿激活或抑制来调节基因表达。唯一的TGFβ/Activin/Nodal成员在十足目动物中具有类似肌肉生长抑制素因子的特点。cDNAs已经从地蟹和其他十足目动物中克隆出来，并且可能在肌肉蛋白的转运 [30]中起作用。据报道在GenBank数据库中，龙虾和虾的编码TGFβ因子的序列与编码Mstn序列明显不同。我们假定一个因子像TGFβ因子一样作为一个自分泌因子在蜕皮前期的早期合成，并且通过下调MIH信号通路中的成分和上调mTOR信号通路中组分和蜕化类固醇合成酶，比如phm来改变YO中基因表达。这使YO转变到committed状态。动物现在开始蜕皮，此时(诸如LBA或注入 [5]MIH之类的)干预都不能推迟蜕皮。 在蜕皮前期的后期，YO的抑制包括蜕皮酮的负反馈抑制。在蜕皮前期的最后，即蜕皮期的那几天内，血淋巴中蜕皮酮含量迅速下降，蜕皮酮含量的下降决定蜕皮期的时间，当人工地提高蜕皮酮的含量(在蜕皮前期的后期)会推迟蜕皮期，它是由蜕皮酮排泄量升高和YO中蜕皮酮产生下降所造成的。当将20E注入小龙虾体内时，它会抑制YO蜕皮酮的产生。RH-5849,一种非甾族蜕皮酮拮抗剂，能模仿昆虫体内20E的作用，抑制小龙虾离体YO中蜕皮酮的分泌。这两种处理均能使蜕皮酮在1h内大量下降，表明通过G蛋白偶联受体或膜结合受体这样一 [3]个非基因组应答所调节。在注入20E后，这种抑制至少持续24h,表明蜕皮酮可能也影响基因表达。 loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: date case, agree to write off loans. Loan review signature: date of record form 16: qilu self credit for credit card cancellation application form name of borrower ID number and credit limit expiration date year month day to year month day year contract number self-service card-I do not use credit, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: 图2 十足目甲壳动物蜕皮腺中MIH调控蜕皮酮生成的信号通路 触发阶段从MIH结 2+合G蛋白偶联受体和活化腺苷环化酶开始;cAMP通过cAMP的蛋白激酶A磷酸化Ca通 2+道提高细胞内Ca的量。对MIH的敏感性由PDE1的活性所决定，而其活性在蜕皮周期中变化很大。总和阶段由NO和cGMP调节。CaM通过直接活化NOS和间接通过CaN连接两个阶段。通过CaN将NOS去磷酸化可以潜在地延长对MIH的应答。CaM也能活化PDE1来抑制触发阶段(PDE1也能水解cGMP来抑制总和阶段)。cGMP依赖的PKG抑制蜕皮酮的合成。PKA的慢性活化可能直接抑制蜕皮酮的合成。缓慢提高细胞内cAMP能直接抑制蜕皮酮的合成，可能通过抑制蛋白合成实现的。 loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: date case, agree to write off loans. Loan review signature: date of record form 16: qilu self credit for credit card cancellation application form name of borrower ID number and credit limit expiration date year month day to year month day year contract number self-service card-I do not use credit, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: 图3 陆地蟹蜕皮的激素调控 图表显示蜕皮阶段、YO阶段、YO对MIH的敏感性、肢再生、YO蜕皮酮合成能力和血淋巴蜕皮酮滴度之间的关系。在蜕皮后期(A,B,C),蜕1-3皮间期(C),蜕皮前期的早期(D)和蜕皮前期的中期(D)，血淋巴蜕皮酮滴度与YO401,2 合成能力相关;在蜕皮前期的后期(D),高浓度的蜕皮酮抑制YO蜕皮酮的合成，蜕皮酮3,4 滴度下降。YO在蜕皮周期有四个生理状态的转变:基态(B)，活化态(A)，committed(C)和抑制态(R)。B到A的转变由MIH的减少所触发，YO肥大，但是依然对MIH敏感，正如通过注入MIH或肢芽自切(LBA)能使蜕皮前期暂停。在A到C的转变过程中，动物开始蜕皮，正如YO对MIH不太敏感，蜕皮前期不能被LBA所暂停;这个转变可能通过增加MIH或不确定的热因素所触发。在C到R的转变过程中,YO蜕皮酮的合成能力依然很高，但是高浓度蜕皮激素滴度抑制蜕皮酮的分泌。蜕皮(E)标志着R到B的转变，在这个过程中，YO萎缩，并且变得对MIH敏感。 4. 展望 很多年来，甲壳动物内分泌学家一直被一个很简单的模式所引导，即蜕皮仅仅由血淋巴中MIH循环水平所控制。如果是这样的话，在蜕皮间期和后期，MIH水平应该很高，而在蜕皮前期水平较低。然而，在蜕皮周期中对血淋巴中的MIH进行测量，并不是这种情况。在蜕皮间期，MIH水平并不是一直很高，在蜕皮 [20]前期的早期到后期，MIH水平比蜕皮间期的水平还高。这表明蜕皮的激素调控比较复杂。任何一种动物在蜕皮周期中都必须遵循YO的动态变化。MIH从XO或窦腺复合体中的释放是断断续续的而不是连续的，致使只有少量MIH存在于血淋巴中。虽然YO一次性释放大量MIH对蜕皮酮的产生具有持续作用，loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: date case, agree to write off loans. Loan review signature: date of record form 16: qilu self credit for credit card cancellation application form name of borrower ID number and credit limit expiration date year month day to year month day year contract number self-service card-I do not use credit, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: 但是我们认为MIH的定期释放高效地保持了YO在蜕皮间期不那么活跃。处于 ，[67]蜕皮前期后期的YO对MIH不敏感，这至少部分是由PDEs大量降解环核苷 酸所造成。蜕皮酮抑制YO其自身的分泌，导致血淋巴中蜕皮酮的含量在蜕皮前 期的最后阶段大量下降。 未来的研究应直接转向了解生理状态转变下的信号通路机制，需要一个系统 的方法来全面了解在转录、翻译及翻译后水平YO的调控，以及它们是如何与环 [18]境因素相互作用的。高速发展的测序水平和光谱测定技术使获得完整的YO转 录组和蛋白质组数据库，以及在蜕皮周期的一些重要转变点定量测定mRNA水 平、蛋白质水平和翻译后修饰成为可能(图3)。一个完整的转录组有助于通过质 谱分析法来测定蛋白质和磷酸化位点。我们期望这种分析将有助于我们鉴定 MIH、mTOR、TGFβ信号通路中的基因，并鉴定在YO调控中起重要作用的其 他基因，它也可能鉴定编码MIH受体的candicate基因。 参考文献 [1] K.S. Kim, J.M. Jeon, H.-W. 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Tsutsui, Crustacean hyperglycemic hormone (CHH) loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. 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J. 430 (2010) 191–198. loan management (post) review: 1, approval criteria have been implemented; 2, the contract is correct; 3, in line with the contract, payment; 4, no other major cases against loan security;、Reviewed by: date of record form 9: qilu bank borrowing by single years of maximum amount a maximum no-loan borrowers: borrowers: ... Borrower name number, line of credit expiry date year month day to year month day maximum number of years in the loan contract are the top line opened, the borrowed and requested your bank opened credit lines above, is hereby requested. Borrower signature: date agreed to open credit lines. Customer Manager signature: date agreed to open credit lines. Loan review signature: date line cancellation-I did not use the loan amount, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: date case, agree to write off loans. Loan review signature: date of record form 16: qilu self credit for credit card cancellation application form name of borrower ID number and credit limit expiration date year month day to year month day year contract number self-service card-I do not use credit, your bank credit lines to be cancelled. -Credit loan principal repayment, your bank credit lines to be cancelled. Borrower signature: date-unused credit lines, agree to write off loans. -Credit loan principal repayment, agree to write off loans. Customer Manager signature: