Detection of constitutive homomeric associations of the integrins Mac-1 subunits by fluorescence resonance energy transfer in living cells

Integrins alpha(M)beta(2) plays important role on leukocytes, such as adhesion, migration, phagocytosis, and apoptosis. It was hypothesized that homomeric associations of integrin subunits provide a driving force for integrins activation, and simultaneously inducing the formation of integrins clusters. However, experimental reports on homomeric associations between integrin subunits are still controversial. Here, we proved the homomeric associations of the isolated Mac-1 subunits in living cells using three-channel fluorescence resonance energy transfer (FRET) microscopy and FRET spectra methods. We found that the extent of homomeric associations between beta(2) subunits is higher than alpha(M) subunits. Furthermore, FRET imaging indicated that the extent of homomeric associations of the Mac-1 subunits is higher along the plasma membrane than in the cytoplasm. Finally, we suggested that homomeric associations of the transmernbrane domains or/and cytoplasmic domains may provide the driving force for the formation of constitutive homomeric associations between alpha(M) or beta(2) subunits. (c) 2006 Elsevier Inc. All rights reserved.

高山松的二倍体杂种起源-来自三个基因组的证据

利用松科植物特殊的遗传体系（叶绿体基因组一父系遗传、线粒体基因组—母系遗传、核基因组一双亲遗传），我们对高山松及其两个亲本种进行了广泛的群体取样，通过线粒体基因nadl、叶绿体基因rbcL和trnL-F基因间区以及低拷贝核基因4CL的序列分析或PCR-RFLP分析，为高山松同倍体杂种起源假说提供了翔实的遗传学证据，同时在个体水平上探讨了高山松不同群体的遗传组成、群体遗传结构、基因交流方向、群体建立过程以及杂种基因组的进化。具体结果如下： 1．细胞质基因组分析 1)线粒体基因nudl分析 本研究对油松、高山松和云南松的19个群体、295个个体的线粒体基因nadl的一个内含子进行了序列分析或PCR-RFLP分析，共检测到3种线粒体DNA单倍型-A、B和C。油松所有的取样群体仅含单倍型A;除BX群体外，所有的云南松群体仅含单倍型B; 10个高山松群体中，5个群体固定单倍型A，4个群体固定单倍型B，1个群体(ZD)分布有A和B两种单倍型。2)叶绿体rbcL基因分析 对同一组群体的rbcL基因进行序列分析或PCR-RFLP分析，共检测到两个变异位点和三种叶绿体单倍型（TT、TC和GC）。TT和GC分别是油松和云南松种特异性叶绿体单倍型，而在高山松群体里则三种单倍型均有分布，而且TC单倍型广泛地分布在7个杂种群体中，该单倍型很可能来源于点突变或第三个已灭绝的亲本。rbcL基因检测到的高山松群体分化系数很高(Gst=0.533)。 3)叶绿体trn L-F区序列分析 叶绿体trnL-F分子标记检测到的不同单倍型的差异主要是由引物“e”下游120碱基处一个多聚T结构的长度变异所致（叶绿体SSR位点）。10个高山松群体中共检测到5种叶绿体单倍型，其中两种主要的单倍型(9T和11T)分别为油松和云南松的种特异性单倍型，其他单倍型均为非典型单倍型。群体遗传结构分析表明：杂种群体表现最高的遗传多样性，而且trnL-F分析得到的高山松群体的分化系数也很高( Gst=0.443)。 总之，对高山松、油松和云南松的同一组群体取样进行的细胞质基因组分析表明：高山松群体分布有油松和云南松种特异性的线粒体和叶绿体单倍型，该细胞质DNA单倍型的地理分布为假说“高山松为油松和云南松的的二倍体杂种”提供了翔实的遗传学证据。油松和云南松在不同的杂种群体中分别做父本和母本，即两亲本在杂交过程中发生了双向基因交流。群体遗传结构分析发现高山松群体表现最高的遗传多样性，而且群体间的分化系数很高。不同的杂种群体在遗传组成上的差异表明他们经历过不同的建立和进化历史。从线粒体和叶绿体单倍型的地理分布可以看出杂种群体的建立曾经历强烈的奠基者效应和回交。青藏高原的隆升对高山松的起源、杂种群体的适应辐射以及保持产生了重要的影响。川西南和滇西北作为青藏高原的东边边界，很可能是当初云南松和油松分布的重叠区及杂交地带，即高山松的起源地。 2．核基因4CL分析 对高山松、油松和云南松的19个群体、32个个体的低拷贝核基因4CL进行了克隆及序列分析，获得的78条序列可分为两种类型（类型A和类型B）。这两种类型明显的差别是类型A相对于类型B在内含子区有- 20bp的缺失。以华山松的3条序列为外类群，对得到的78条序列进行基因谱系分析，发现所有的序列分成明显的两支，分别对应于类型A和类型B，而且每一支均包含三个种的部分序列，表明4CL基因在这三个种分化之前就已发生重复。另一个明显的特点是某个种的一条序列与另一个种的序列比其与同种的其他序列关系更近，可能因基因交流（杂交和渐渗）、非共祖、致同进化和重组等进化事件所致。三种松树中共检测到4CL基因序列的两种类型和六个亚类型，高山松群体中没有发现杂种独特的类型或亚类型。高山松和云南松共享三种序列亚类型以及最多的序列多态性，表明这两个种之间曾存在广泛的基因交流。

杉木生殖生物学特性的研究

本文就杉木大小孢子、雌雄配子体发育、受精作用、新细胞质形成以及细胞质遗传进行较为详细的研究，主要结果如下： 1.2月底小孢子母细胞进入减数分裂，其核相的变化与通常所描述的一致，细胞器集中排列在赤道板附近，其中质体有一重建的过程。当分裂完成时，在四个核之间产生胼胝质壁，细胞器随机分配在四分体中，每个四分孢子含有线粒体、质体、核糖体、高尔基体、脂体、RER和一些液泡。 2.花粉外壁的形成开始于四分体。颗粒状外壁外层和纤维状的原外壁几乎同时产生，具有三层结构的片层状外壁内层的形成晚于外壁外层。初期外壁外层的物质由小孢子和绒毡层共同提供，后期则主要由绒毡层形成的乌氏体所叠加;外壁内层则由小孢子本身分泌的物质形成，在液泡化之前厚度及数目达到最大，片层之间存在广泛的交叉和融合。3月底花粉成熟，花粉壁由颗粒状外层、片层结构的外壁内层和厚的纤维状内壁所组成，在喙处只有一层外壁内层和内壁。各种细胞器的活动在四分体及游离小孢子时期最为活跃。 3.绒毡层的变化与小孢子的发育密切相关，前者具有发生、发展和解体的过程。期间绒毡层分泌的圆球体在结构上有所不同，减数分裂时为中间电子密度深、外围浅的圆球体结构;四分体及游离小孢子时为中间电子密度浅、外围沉积有孢粉素的乌氏体;成熟花粉时为星芒状的乌氏体，它们共同形成花粉的我壁。绒毡层细胞中的细胞器也经历一个变化过程，造粉体的含量在四分体时达到高潮，在小孢子液泡期时基本消耗殆尽并解体;大量的粗糙内质网成零分布在质膜处，并与质膜走向平行。绒毡层解体时出现特殊的现象，具多个由多层膜组成的吞噬泡，并成堆分布，RER和线粒体为最后解体的细胞器。绒毡层外切向面的周绒毡层膜由两层膜组成，其上分布有大量的孢粉素和乌氏体。 4.萌发的花粉管4月底穿过珠心顶端，内含两个不育核和一精原细胞，不育核分布在具有淀粉、脂滴、线粒体和RER的原花粉细胞质中，位于精原细胞之前，这种关系保持到受精前。6月初精原细胞体积迅速增加到最大，相应的细胞质也发生变化，以线粒体为主的细胞质中出现少量质体，线粒体再资分化，细胞质中含有大量的可溶性多糖。 5.受精前，精原细胞分裂产生两个大小和形状相同的精细胞，彼此之间由胞间连丝的横壁联系在一起，并被原花粉细胞质所包被。精细胞中质体和淀粉粒的数量大幅度增加。精核们于细胞的中央，细胞质呈现区域分布的特点，据所含细胞器的差别，人核膜向外，细胞质分为五层，中间两层最为突出，即线粒体层和淀粉层，其间有大量核糖体的存在，线粒体层位于淀粉层的里面。这两层占据精细胞的大部分体积。 6.雌配子体游离核时期持续时间较长，近两个月，而从细胞化到卵细胞成熟则非常迅速，大约只需两周左右的时间。进一步发育，颈卵器中的液泡减少，细胞质变浓。初期少量的质体和淀粉粒被膨大的内质网片段和小泡的融合而与细胞质相隔，并最终退化。小内含物增加，亲锇颗粒出现。 7.成熟的卵细胞中具有大量各种形式的小内含物，细胞质被平行和环形的内质网所分隔。核膜外围有一圈疏松排列的亲锇颗粒，核仁变为多个基本为圆形的小核仁。没有质体的存在，大量脱分化的线粒体和核糖体集中分布在卵核的下部。 8.受精作用主要发生在6.9-6.16日期间，雄性细胞质始终伴随着精核向卵核移动，当两核接近时，朝向精核－面的卵核形成凹陷内，性细胞质覆盖在精核之上并最终包围两性核，而把雌性细胞质排除在外。因此，受精卵周围的胞质主要为由质体、线粒体和核糖体组成的新细胞质，且线粒体和质体的分布形式与精细胞的相同，即线粒体在里层，质体在外层。 9.融合后的合子核随即进行有丝分裂，形成两个原胚游离核，两个游离核同步分裂，并向基部移动，游离核始终分布在新细胞质中。八游离核时形成细胞壁，原胚属标准型。胚细胞中基本不含淀粉粒，具有大量的线粒体、高尔基体、RER和原质体。原胚之上的卵细胞质退化解体。杉木的质体和线粒体均为父本遗传。 10.幼胚的胚性细胞和胚柄细胞具有明显的差别，主要表现在质体的存在形式和高尔基体的数量上。胚性细胞中，原质体分布在核周围，大量的RER、高尔基体和线粒体平行于胚的走向;位于其上的胚柄细胞则含有淀粉粒和特别多的高尔基体。具简单多胚和裂生多胚，胚柄系统发达，7月底出现根原始细胞，8月中旬胚分化完成，具有两个子叶。

水松生殖生物学的研究

本文运用组织化学技术、透射电镜技术，就水松雌雄配子体发育，精卵细胞形成和结构，受精作用以及细胞质遗传等问题进行了较为详细的研究。主要结果如下： 1 绒毡层发育与雄配子体发育，尤其与花粉外壁的形成关系密切。绒毡层组织在小孢子母细胞和减数分裂时期达到发育高峰。四分体阶段和游离小孢子发育前期，绒毡层细胞中内质网、脂体、线粒体非常活跃，参与孢粉素和乌氏体的形成和转移。乌氏体有球形、星芒状两种类型，它们主要参与花粉外壁内层和外壁外层的建成。小孢子发育后期绒毡层细胞开始明显解体。 2 减数分裂阶段，质体、线粒俺等细胞器和造粉体随细胞核和染色体的变化，出现有规律的迁移现象。减数分裂前期，细胞核移到细胞的一侧;各种细胞器和造粉体迁移到细胞的另一侧，并随染色体移向细胞中部而转移到细胞质周缘。减数分裂中期l和后期，各种细胞器和造粉体汇聚在赤道板丙侧分布。说明细胞质中微管系统在起调节作用。 3 花粉壁形成开始于四分体时期。片层状结构的外壁内层随纤维状原外壁的出现雨形成。外壁外层和花粉内壁在小孢子发育中期几乎同时形成。小孢子细胞和绒毡层组织共同参与了外壁内层和外壁外层孢粉素物质的合成、转运。水松成熟花粉由孢粉素组成的外壁外层、片层状结构的外壁内层及有分层状结构的内壁构成。 4 传粉到受精间隔要四个月左右的时间。六月上旬，花粉管和精原细胞抵达颈卵器上部。精原细胞富含淀粉粒、质体、线粒体和异形泡并分区分布。受精前，精原细胞分裂形成两个大小和形状相同豹精细胞。精细胞含有质体、线粒体和异型泡等细胞器。 5 雌配子体游离核持续时间长，从颈卵器原始细胞形成到卵细胞发育成熟所需时间较短。复合颈卵器结构，颈细胞和套层细胞形态结构特殊。中央细胞不经分裂，直接行使卵细胞的功能。 6 成熟卵细胞中除弋量各种形式的内含物外，细胞质被庞大的内质网包围、分割，形成网膜系统。质体、淀粉等细胞器被包含成大内含物。在卵核周围细胞质中没有发现质体和发育完好的线粒体存在。 7 受精作用发生在六月十日左右。雌雄核融合时，朝向精核一面的卵核形成凹陷，精核陷入其中与卵核进一步融合。精卵融合可发生在颈卵器的上部、中部、下部甚至底部。有旋转受精、两个精核同时与一个卵核受精及多卵细胞现象等。 8 新细胞质主要为雄性细胞质成分。合子转移到颈卵器基部分裂，形成原胚游离核。合子、新细胞质和游离核周围淀粉鞘显著。八游离核时期形成细胞壁，新细胞质和淀粉粒转移到原胚细胞中。 9 水松的质体和线粒体为父系遗传。

采用AFLP分子标记和分子生物学方法对资源植物的评价

中国资源植物丰富，蕴藏着优异的基因资源，开发和利用这些优异资源是植物学研究的重要课题。本文面向国家重大需求选择两种资源植物一羊草(Leymuschinensis (Trin.)Tzvel)和向日葵(llelia thus annuus L．)，采用分子标记技术和分子生物学方法对其进行评价和研究，以期为资源利用提供依据。由于两种植物本身的差别和采用的研究方法各异，故分别论述。 羊草，隶属禾本科赖草属，是欧亚大陆草原区东部重要建群种之一。羊草是牧草之王，是我国比较有优势的战略性生物资源，对我国北方畜牧业的发展以及生态环境的保育均具有重要意义。近年来，由于缺乏科学管理、过度放牧等不利影响，加之羊草本身固有的“三低”问题（即抽穗率低、结实率低、发芽率低）已对羊草生物多样性维持构成了严重的威胁，限制了我国人工草地建设和天然草地的改良及沙化治理的步伐。因此，如何通过形态调查结合生物技术手段评价羊草遗传多样性为建立核心种质及改良羊草、快速评价和创造新的种质、如何加快育种进程便成为当前亟待解决的问题。本文围绕这些问题开展了系统的研究并取得如下结果： 1． 对羊草的形态调查和AFLP分析，表明羊草是一种形态变异较大但是遗传变 异较小的物种。两种生态型的表现显著差异，其中灰绿生态型羊草比黄绿生 态型差异大。羊草遗传多样性与包括长期的栽培驯化、地理分布有很大的相 关性，地理来源相同的几乎全部聚到了一组。 2． 通过主成分分析和通径分析，简化了羊草31个性状分析的复杂性，了解到 羊草无性繁殖受好的营养生长促进。 3． AFLP分子标记技术在分析羊草遗传多样性方面有显著优势，尤其是对于羊 草这样多态性不高的物种是一种非常有效的分析工具。在分析AFLP数据时 采用聚类分析和主坐标分析相结合的方法，既兼顾了亲缘关系较近的种质之 间的关系调查也兼顾了亲缘关系较远的种质之间的关系调查。 4．羊草AFLP反应，不同引物所获得的总带数和多态性带数差别明显。羊草基因 组对3’端有选择性碱基TN的所有EcoRJ选择性引物扩增效果很差，前人 的有关赖草属的遗传研究也支持这一结果。 向日葵(n=17)，属于菊科( Compositae)向日葵属(Helia thus)，向日葵的研究重要领域是向日葵杂种生产，而细胞质雄性不育系的使用是杂种优势育种的核心。全世界90%以上的向日葵杂交种生产仍然在使用同一个细胞质类型PETI，玉米遗传单一给生产带来的毁灭性打击仍然令研究者和生产者记忆犹新，因此寻找更多的细胞质类型仍然是研究者的重要任务。本研究围绕一个新的不育源(G20023)的发现及鉴定，通过使用不育的G20023的保持系、恢复系、恢复的Fi代、回交一代之间比较以及与属于PETI细胞质类型的不育系的相应材料进行比较，找出与这一新的细胞质类型不育表型有关的可能差异序列，来探讨其不育机制，得到如下结果： 1、 通过田间杂交试验，证明G20023的保持系有很多（已证实有24份）， 目前找到的恢复系只有一个，H.maximiliani。G20023不育源作为一 种新的细胞质类型可以成为将来杂交育种的候选资源。同时，我们找 到一些表型证据，除了无花粉之外，G20023与PETI表型的典型不同 之处还在于前者的花药上下均为分离状态，而后者花药的基部联合， 顶部分离。显然，不同的细胞质类型在解剖结构上可能表现不尽相同。 2、 与线粒体基因组特异基因的核酸序列比较，结果表明，G20023线粒体 基因组上没有orfH522序列，与PETI表现出差异;此外，在基因atp6 位点也与PETI不同，而且在该位点也与同属向日葵ANTI不相同。同 时由于orf873并没有出现在ANTI中而出现在G20023中，因此我们可 以认为G20023这一个新的不育系是与ANTI和PETI不同的细胞质类 型。 3、 在参考常规线粒体DNA提取方法的基础上，我们做了很多改进，建立 了自己的向日葵线粒体DNA提取方法。该方法更快更简单，提取的线 粒体DNA完全可以用于酶切和杂交。 4、 G20023不育源由于其稳定的不育性状，可以作为培育无花粉彩色向日 葵杂交种的亲本材料，我们通过此不育源选育适当花色的无花粉观赏 向日葵生产杂交种。

Perspectives on the origin of microfilaments, microtubules, the relevant chaperonin system and cytoskeletal motors - a commentary on the spirochaete origin of flagella

The origin of cytoskeleton and the origin of relevant intracellular transportation system are big problems for understanding the emergence of eukaryotic cells. The present article summarized relevant information of evidences and molecular traces on the origin of actin, tubulin, the chaperonin system for folding them, myosins, kinesins, axonemal dyneins and cytoplasmic dyneins. On this basis the authors proposed a series of works, which should be done in the future, and indicated the ways for reaching the targets. These targets are mainly: 1) the reconstruction of evolutionary path from MreB protein of archaeal ancestor of eukaryotic cells to typical actin; 2) the finding of the MreB or MreB-related proteins in crenarchaea and using them to examine J. A. Lake's hypothesis on the origin of eukaryote from "eocytes" (crenarchaea); 3) the examinations of the existence and distribution of cytoskeleton made of MreB-related protein within coccoid archaea, especially in amoeboid archaeon Thermoplasm acidophilum; 4) using Thermoplasma as a model of archaeal ancestor of eukaryotic cells; 5) the searching for the homolog of ancestral dynein in present-day living archaea. During the writing of this article, Margulis' famous spirochaete hypothesis on the origin of flagella and cilia was unexpectedly involved and analyzed from aspects of tubulins, dyneins and spirochaetes. Actually, spirochaete cannot be reasonably assumed as the ectosymbiotic ancestor of eukaryotic flagella and cilia, since their swing depends upon large amount of bacterial flagella beneath the flexible outer wall, but not depends upon their intracellular tubules and the assumed dyneins. In this case, if they had "evolved" into cilia and lost their bacterial flagella, they would immediately become immobile! In fact, tubulin and dynein-like proteins have not been found in any spirochaete.

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from greater than or equal to 1000 mum diameter follicles

Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions

In mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel two domain TFF protein from frog Bombina maxima skin secretions (Bm-TFF2) has been purified and cloned. It activated human platelets in a dose-dependent manner and activation of integrin a(11b)beta(3) was involved. Aspirin and apyrase did not largely reduce platelet response to Bm-TFF2 (a 30% inhibition), indicating that the aggregation is not substantially dependent on ADP and thromboxane A2 autocrine feedback. Elimination of external Ca2+ with EGTA did not influence the platelet aggregation induced by Bm-TFF2, meanwhile a strong calcium signal (cytoplasmic Ca2+ release) was detected, suggesting that activation of phospholipase C (PLC) is involved. Subsequent immunoblotting revealed that, unlike in platelets activated by stejnulxin (a glycoprotein VI agonist), PLC gamma 2 was not phosphorylated in platelets activated by Bm-TFF2. FITC-labeled Bm-TFF2 bound to platelet membranes. Bm-TFF2 is the first TFF protein reported to possess human platelet activation activity. (c) 2005 Elsevier Inc. All rights reserved.

Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (greater than or equal to 1000 mum in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%)f 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P<0.05). The lowest (P<0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca2+ permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASICla-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca2+ elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.

与烟草细胞质雄性不育相关的线粒体基因atp9的mRNA研究

下载PDF阅读器已知导入未编辑atp9 mRNA的烟草表现细胞质雄性不育(CMS),因此认为线粒体基因atp9是引起高等植物CMS的主要基因.为了解atp9在CMS中的作用机制,从3对烟草不育系及其同型保持系中提取atp9,利用实验与理论结合来分析其mRNA在编辑前后以及在不育系及其同型保持系中的一维、三维信息差别.结果表明,atp9 mRNA一维信息方面的差异,更重要的是二级结构的差异和稳定性,可能是影响ATP合成而导致CMS的根本原因.

Molecular modeling on human CCR5 receptors and complex with CD4 antigens and HIV-1 envelope glycoprotein gp120

AIM: To investigate the interaction between human CCR5 receptors (CCR5) and HIV-1 envelope glycoprotein gp120 (HIV-1 gp120) and HIV-1 receptor CD4 antigens (CD4). METHODS: The structurally con served regions (SCR) of human CCR5 was built by the SYBYL/Biopolymer module using the corresponding transmembrane (TM) domain of bacteriorhodopsin (bR) as the template. The coordinates for amino-ter minal residue sequence, and carboxyl-terminal residue sequence, extracellular and cytoplasmic loops were generated using LOOP SEARCH algorithm. Subsequently the structural model was merged into the complex with HIV-1 gp120 and CD4. RESULTS: Human CCR5 interacted with both an HIV-1 gp120 and CD4. The N-terminal residues (especially Met1 and Gln4) of human CCR5, contacted with CD4 residues, mainly 7Nith one span (56 - 59) of CD4 in electrostatic interaction and hydrogen-bonds. The binding sites of human CCR5 were buried in a hydrophobic center surrounded by a highly basic periphery. On the other hand, direct interatomic contacts were made between ? CCR5 residues and 6 gp120 amino-acid residues, which included van der Waals contacts, hydrophobic interaction, and hydrogen bonds. CONCLUSION: The interaction model should be helpful for rational design of novel anti-HIV drugs.

Toll-like receptor 4 signaling pathway can be triggered by grass carp reovirus and Aeromonas hydrophila infection in rare minnow Gobiocypris rarus

Toll-like receptor 4 (TLR4) is critical for LPS recognition and cellular responses. It also recognizes some viral envelope proteins. Detection mostly results in the inflammation rather than specific antiviral responses. However, it's unclear in fish. In this report, a TLR4 gene (named as GrTLR4b) was cloned and characterized from rare minnow Gobiocypris rarus. The full length of GrTLR4b cDNA consists of 2766 nucleotides and encodes a polypeptide of 818 amino acids with an estimated molecular mass of 94,518 Da and a predicted isoelectric point of 8.41. The predicted amino acid sequence comprises a signal peptide, six leucine-rich repeat (LRR) motifs, one leucine-rich repeat C-terminal (LRRCT) motif, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic region of 167 amino acids containing one Toll - interleukin 1 - receptor (TIR) motif. It's closely similar to the zebrafish (Danio rerio) TLR4b amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed GrTLR4b mRNA was constitutive expression in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus or Aeromonas hydrophila, GrTLR4b expressions were up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). These data implied that TLR4 signaling pathway could be activated by both viral and bacterial infection in rare minnow. (C) 2009 Elsevier Ltd. All rights reserved.

Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O-2) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.

Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (Q. Western blotting analysis revealed that three mAbs (1145, IE10, and 11-17) recognized specifically to a single protein of 47 kDa (N), the mAb 3G4 reacted with, two SVCV0504 proteins of 69 kDa (G) and 47 kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69 kDa (G), 50 kDa (P), and 47 kDa (N). By indirect ELISA, two mAbs (1H5 and 11-17) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID50 (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28 h after inoculation with the virus (0.01 PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus. (c) 2008 Elsevier B.V. All rights reserved.