银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A_1的cDNA克隆

分别取行天然雌核发育繁殖的银鲫和两性生殖的彩鲫的卵母细胞为材料 ,提取总RNA ,分离mRNA ,进而反转录合成cDNA并定向插入λgtllSfi Not克隆载体 ,经体外包装构建了银鲫与彩鲫卵母细胞的表达型cDNA文库。测试结果表明库容量分别达到 3 1× 1 0 6(银鲫 )和 1 6× 1 0 6(彩鲫 )。进一步人工合成CyclinA1 保守引物 ,采用PCR扩增文库的方法 ,克隆了银鲫 (1 61 6bp)与彩鲫 (1 62 6bp)的CyclinA1 全长cDNA。序列分析结果表明 :两种鱼编

A Proteomic Study on Cell Cycle Progression of Endothelium Exposed to Tumor Conditioned Medium and the Possible Role of Cyclin D-1/E

This study was designed to comprehensively analyze the differential expression of proteins from human umbilical vein endothelial cells (HUVECs) exposed to tumor conditioned medium (TCM) and to identify the key regulator in the cell cycle progression. The HUVECs were exposed to TCM from breast carcinoma cell line MDA-MB-231, then their cell cycle distribution was measured by flow cytometer (FCM). The role of protein in cell cycle progression was detected via two-dimensional polyacrylamide gel electrophoresis (2-DE) and western blotting. Following the stimulation of TCM, HUVECs showed a more cells in the S phase than did the negative control group (ECGF-free medium with 20% FBS), but the HUVECs' level was similar to the positive control group (medium with 25 mug/ml ECGF and 20% FBS). Increased expression of cyclin D-1/E and some changes in other related proteins occurred after incubation with TCM. From our results, we can conclude that breast carcinoma cell line MDA-MB-231 may secrete soluble pro-angiogenic factors that induce the HUVEC angiogenic switch, during which the expression of cell cycle regulator cyclin D-1/E increases and related proteins play an important role in this process.

Cyclin A2 is differentially expressed during oocyte maturation between gynogenetic silver crucian carp and gonochoristic color crucian carp

Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin A1 and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin A1 and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.

Biophysical studies on the full-length human cyclin A(2): Protein stability and folding/unfolding thermodynamics

Human cyclin A(2) participates in cell cycle regulation, DNA replication, and transcription. Its overexpression has been implicated in the development and progression of a variety of human cancers. However, cyclin A(2) or its truncated form is very unstable in the absence of binding partner, which makes it difficult to get a deep insight of structural basis of the interactions. Therefore, biophysical studies of the full-length human cyclin A, would provide important information regarding protein stability and folding/unfolding process.

Targeted RNA interference of cyclin A(2) mediated by functionalized single-walled carbon nanotubes induces proliferation arrest and apoptosis in chronic myelogenous leukemia K562 cells

Cyclin A(2) plays critical role in DNA replication, transcription, and cell cycle regulation. Its overexpression has been detected and related to many types of cancers including leukemia, suggesting that suppression of cyclin A(2) would be an attractive strategy to prevent tumor progression. Herein, we apply functionalized single wall carbon nanotubes (f-SWNTs) to carry small interfering RNA (siRNA) into K562 cells and determine whether inhibition of cyclin A(2) would be a potential therapeutic target for chronic myelogenous leukemia.

Knocking-Down Cyclin A(2) by siRNA Suppresses Apoptosis and Switches Differentiation Pathways in K562 Cells upon Administration with Doxorubicin

Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation.

Physical and spectral characterization of the human cyclin A gene and its interactions with anthracycline anticancer drugs

Over expression of cyclin A in human tumors has been linked to cancer by various experimental lines of evidence. However, physical and spectral characterization of the human cyclin A gene and its interactions with anticancer drugs have not been reported. Our gene sequence analysis, singular value decomposition method and melting studies in the presence of antitumor agents, daunomycin, doxorubicin and Hoechst 33258 showed that cyclin A gene had both AT-rich and GC-rich domains. For a ligand with unknown DNA binding specificity, this gene sequence can be used to differentiate its DNA binding preference.

Evaluation of different culture conditions for high-level soluble expression of human cyclin A(2) with pET vector in BL21 (DE3) and spectroscopic characterization of its inclusion body structure

In this paper, we evaluated various parameters of culture condition affecting high-level soluble expression of human cyclin A, in Escherichia coli BL21(DE3), and demonstrated that the highest protein yield was obtained using TB(no glycerol) + 0.5% glucose medium at 25 degrees C. By single immobilized metal ion affinity chromatography, we got highly purified human cyclin A(2) with a yield ranged from 20 to 30 mg/L. By amyloid-diagnostic dye ThT binding and Fourier transform infrared spectroscopy, we observed a significant decrease in alpha-helix content and an increase in beta-sheet structure in cyclin A(2) inclusion body in comparison to its native protein, and confirmed the resemblance of the internal organization of cyclin A(2) inclusion body and amyloid fibrils.

一种联接DJS—130计算机和A_1型机箱控制器的CAMAC分支驱动器

本文介绍了一种并行CAMAC分支驱动器的设计方案。除具有一般CAMAC分支驱动器所应有的功能外,它的DMA交换可以与CAMAC程序型操作同时运行,从而大大提高了CAMAC系统的数传效率,并对CAMAC系统用于过程控制提供了良好条件。

车前科和苦苣苔科一些典型类群花对称性基因的进化发育生物学研究—兼论地黄属和崖白菜属的系统位置

在模式植物金鱼草中的花对称性分子发育与遗传学研究揭示出相关调控基因在花对称性形成过程中的功能和表达式样及其相互作用机制，但被子植物中花对称性的繁杂多样远非模式植物的表达模式所能概括。因此，我们选择车前科和苦苣苔科中与金鱼草较近缘的典型类群地黄属和非洲紫罗兰属作为研究对象，针对它们在花对称性形成方面区别于金鱼草的不同式样，开展这些类群中花对称性主控基因CYCLOIDEA(CYC)类基因的进化发育生物学研究。该研究旨在探讨CYC 类基因的功能和表达式样的变化在进化上的内在联系。 地黄花对称性基因的进化发育研究结果显示，地黄中CYC 类基因RgCYC 的表达模式与CYC 基因在金鱼草中和McCYC 基因在Mohavea 中的表达模式存在明显的差异。首先， RgCYC 基因在近轴雄蕊预期发生位置表皮细胞下的强烈表达与地黄近轴雄蕊的缺失密切相关。转录因子中的氨基酸替代所导致蛋白质功能的改变使RgCYC 基因对细胞周期基因cyclin D3b 抑制作用的增强可能是地黄中近轴雄蕊原基发生过程被彻底阻断的主要原因。由此看来，CYC 类基因的作用不仅导致近轴花器官生长缓慢或退化，而且可能与自然类群中花近轴器官丢失的现象有关。其次，同McCYC 基因在Mohavea 中的表达模式相似，RgCYC 基因的表达也从近轴雄蕊延伸到了两侧雄蕊，但是并没有强烈地抑制两侧雄蕊的发育，仅仅使得两侧雄蕊短于远轴雄蕊从而在地黄中形成二强雄蕊。这一现象可能是由于RgCYC 基因的表达与McCYC 基因的表达在时间和空间上的差异所造成的，并显示地黄中二强雄蕊的形成机制和金鱼草完全不同。第三，RgCYC 基因在近轴花冠裂片的表达没有象CYC 在金鱼草中一样明显促进它们的生长。此外，近轴花冠裂片明显的自身对称性显示在地黄中RgCYC 基因在两侧对称性形成方面可能单独对近轴花器官进行调控。地黄中RgCYC 基因的表达模式反映了广义唇形目中从五数花到四数花进化过程的一种新的进化机制。 两侧对称花向次生辐射对称花的反演进化机制在花对称性进化发育研究中倍受关注。我们在苦苣苔科中选择非洲紫罗兰栽培品种作为研究材料，通过 mTAIL-PCR 分别在两侧对称花和辐射对称花的栽培品种中分离出了包含完整的 ORF 的CYC 类基因：SiCYC1A 和SiCYC1B。这两个基因的完整序列在DNA 水平的相似性为88%，均包含了完整的TCP domain, R domain 和 5’ 端区段。令人意外的是SiCYC1A 和SiCYC1B 这两个基因的DNA 序列在两侧和辐射对称花品种中均完全一致。根据对导致辐射对称花产生机制的比较分析，我们认为在这两个栽培品种中的SiCYC1A 和 SiCYC1B 基因可能存在着某一共同的调节因子对其进行调控。其可能途径是该调节因子同时调控SiCYC1A 和 SiCYC1B 基因，这一共同的调节因子的改变导致了SiCYC1A 和 SiCYC1B 基因部分或完全失去功能，从而使两侧对称花转变为辐射对称花。 崖白菜属的花部器官发生研究显示其花萼和花冠裂片的发生顺序与毛地黄族和婆婆纳族相似，花冠裂片早期生长的迟滞和花冠裂片折叠式样介于毛地黄族和婆婆纳族之间。但是，近轴雄蕊的发育缺失完全不同于毛地黄族中的其它类群。对地黄属和崖白菜属以及它们近缘类群的ITS 或trnL-F 序列所构建的系统树的分析显示，地黄属和崖白菜属呈姊妹群。然而，分子系统学研究结果并不支持传统系统学和个体发育研究对这两个属科级系统位置的认识。毛地黄属与婆婆纳属和车前属构成一个单系分支，而地黄属与崖白菜属则形成另外一个独立的分支，并与泡桐属与透骨草科所形成的分支首先聚在一支。因此，毛地黄族可能并不是一个单系类群，地黄属和崖白菜属的科级系统位置可能需要重新考虑。

南极鱼两种寄生海蛭的研究

从南极海纽喀姆湾(南纬66°17′,东经110°32′)的南极鱼属Notothenia鱼体上得到两个海蛭标本。经鉴定为多皱海蛭Pontobdella rugosa Moore,1938和椎蛭属Notobdella的一新种。前者的环带后区环的分割明显并因许多大、小结节变得崎岖不平。在一完全体节(ⅫⅠ-ⅩⅫⅠ)里环的通常大小关系是8_2(?)a_1>b_5=b_(60)扭椎蛭新种Notobdella streptocheles sp.nov.呈S形弯曲井从稍膨大的中部向两端渐渐变细。在前吸盘上没有眼。尾吸盘与

Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps

Silver crucian carp (Carassius auratus gibelio) is a unique triploid bisexual species that can reproduce by gynogenesis. As all other gynogenetic animals, it keeps its chromosome integrity by inhibiting the first meiosis division (no extrusion of the first pole body). To understand the molecular events governing this reproduction mode, suppression subtractive hybridization was used to identify the genes differentially expressed in fully-grown oocytes of the gynogenetic and gonochoristic crucian carp (gyno-carp and gono-carp). From two specific subtractive cDNA libraries, the clones screened out by dot blots and virtual Northern blots were chosen to clone, full-length cDNA by RACE. Four differentially expressed genes were obtained. Two are novel genes and are expressed specifically in the oocytes. The gyno-carp stores much more mRNA of cyclin A2, a new member of the fish A-type cyclin gene, in its fully-grown oocyte than in the gono-carp. The last gene is histone H2A. The histone H2As of these two closely related crucian carps are quite different in the C-terminus. Preliminary characterization of the four genes has been analyzed by nucleotide and deduced amino acid sequence and Northern analysis. (C) 2001 Elsevier Science B.V. All rights reserved.

宽能隙半导体氮化镓的合成、相变与性质的研究

GaN是最有前途的宽能隙半导体材料之一。它具有优异的光电子特性及稳定的物理性能，因此引起了各国科研工作者的广泛兴趣，各国争相研制GaN基蓝紫光和紫外波段的光电器件，并取得了令人瞩目的结果。关于GaN的研究每年有大量的文献报道，GaN基发光二极管已经产业化。通过文献调研发现，在GaN的研究中存在一些重要的基础工作有待完成。如，GaN陶瓷体的制备，结构相变研究，材料的室温合成方法等。当前 GaN器件的研究的关键问题之一是获得高质量的GaN薄膜，而影响膜质量的关键因素是选择合适的与GaN晶格匹配和热膨胀匹配的衬底材料。GaN晶体是公认的最佳材料，但是GaN晶体制备困难，迄今为止，尚未得到足够尺寸的大单晶材料。GaN陶瓷体的制备有可能替代单晶体作为理想的衬底材料。由于GaN热稳定性差，烧结时易挥发，到目前为止，尚无合成GaN陶瓷体的报道。闪锌矿结构立方GaN具有比纤锌矿结构的GaN更容易进行P型掺杂、发光效率高的特点，因而成为该材料研究的热点问题之一。虽然己有一些立方相GaN粉末合成和膜制备的报道，但是六方纤锌矿GaN向立方闪锌矿的相变研究尚属空白。研究六方相到立方相的相变规律，有助于相变的认识及立方GaN的合成。降低GaN的合成温度对该材料的制备有重要的意义。低温合成GaN的工作已有一些报道，但室温下的合成研究尚待探索。本文通过建立高氨压合成新技术，首次实现了GaN陶瓷体的合成。利用机械合金化产生的机械力化学效应，实现了h－GaN向c－GaN的相变。利用机械力化学反应方法完成了非晶纳米GaN粉末的室温反应合成。此外，还对GaN的衬底材料LiGaO_2晶体的气相合成进行了研究。GaN粉末是在常压下，通过Ga_2O_3和NH_3反应制备的。在实验中发现，反应温度直接影响产物的颜色、结晶状态和纯度。实验中得到的制备GaN粉末的最佳条件是温度为950 ℃左右、反应时间为4小时。以GaN粉末为原料，通过建立高氨压合成新方法，在高压佩刃成功的实现GaN陶瓷体的烧结。通过在组装中添加适量的LiNH_2使之在高温高压下分解，从而形成高氨压的烧结环境。所得到的GaN的烧结体的结晶状态明显提高，在空气中的 稳定性也明显增强。随着温度的变化，所制得的GaN烧结体分别呈现为棕褐色（<900 ℃）、墟拍色（900-1100 ℃)及淡黄色（>1100 ℃）。通过对不同条件下制备的烧结体进行密度测试及破碎表面的SEM分析发现，4GPa、1000 ℃为GaN陶瓷体的最佳制备条件。通过对GaN烧结体的光谱性质进行研究发现GaN陶瓷体的荧光发射峰及红外光谱的吸收峰位置均发生了位移。实验采用机械合金化装置，在机械力化学效应的诱导下实现了GaN由纤锌矿的六方结构向闪锌矿立方结构的相转变。六方结构的GaN的XRD的图谱的明显特征是三个最高衍射强度所对应的峰的位置依次为36.7°, 32.3°和34. 5°（2θ）。经过机械球磨后的XRD的图谱在34.5°、57.8°和69.1°（2θ）位置的衍射峰为三个最强峰，而且在40.4°(2θ位置出现了一个较弱的衍射峰（该衍射峰为立方氮化嫁的特征峰）。以上结果表明，在机械球磨的作用下，六方GaN发生了向立方GaN结构的相转变。通过Raman散射光谱对比测试，六方GaN的光谱图中535, 557和567cm~(-1)的散射峰分别对应与A_1(TO), E_1(TO)和E_2声子模的振动频率，其中Ez为六方相的特有的声子模。经过球磨后的粉末的尺aman散射光谱图中551、716cm~(-1)分别对应于立方相的To和LO声子模的振动频率。光谱中六方结构的特征峰El的消失及立方相特征峰LO的出现进一步证实了在机械合金化的作用下，GaN发生了由六方相向立方相的转变。根据机械球磨可发生机械力化学反应的原理，实验中通过选择合适的嫁源和氮源，在机械合金化装置中实现了纳米非晶态GaN的室温合成。以化学计量比为1；1：1的Ga, NaN_3和NH_4Cl为原料，通过机械球磨进行合成反应。分析产物的XRD图谱发现，所有结晶相的衍射峰与NaCl的标准卡片吻合，在32-38°（2θ）区间出现一非晶相的宽峰（与GaN主峰位置对应）。证实了经过机械球磨发生了合成反应（Ga+NaN_3+NH_4Clx→GaN+NaCl+N_2+NH_3+H_2），得到了纳米非晶态GaN。通过对所得的粉末进行TEM表征，其产物颗粒为5nm左右。根据Li_2o和Ga_2o易挥发的特点，采用LiCO_3、Ga和Ca_2O_3为原料通过气相法制备了LiGaO_2晶体。

分子激发态寿命及其应用研究

寿命测量现在使用的有位相偏移法和脉冲法。本工作是利用脉冲法进行的。本论文做了三方面的工作：1.分子激发态寿命在分析化学中的应用。荧光寿命是荧光物质的特征参数，可利用来进行定性，定量分析工作。从荧光强度与荧光寿命的基本公式I＝A_ie~(-t/τi)出发，可以得到c_1/c_2=(a_1τ_1ε_2φ_2)/(α_2τ_2ε_1φ_1)其中C_1，C_2是同一体系中两种不同物质的浓度，τ是荧光寿命，ε为激发波长下的消光系数，φ为量子产率，A是幅度常数。ε与φ可预先测量或查表得到。τ，A可根据荧光衰减曲线与标准衰减函数相拟合，解卷积后同时得到。因此可用上式进行定量分析。本论文利用上式分析了卟吩类化合物，其中包括四苯基卟吩；四－邻氯苯基卟吩；锌－四苯基卟吩络合物在不同浓度下的组合，分析误差绝大多数在10％以内。从（1）式出发得到另一个基本公式A＝KCT其中T为时间相关单光子计数时间，K是一常数。根据（3）式进行了多环芳香碳氢化合物分析。稀土发光材料的荧光寿命研究。本工作者首先使用不同的激发光源，不同分光装置和不同的讯号探没器及讯号处理方式，建立了测量范围较宽的荧光寿命测量装置。测量了Eu~(3+), Eu~(2+), Ce~(3+), Dy~(3+), Nd~(3+), Tb~(3+)在不同基质中的荧光寿命。其数值从10~(-8)秒到10~(-3)秒，探测波长包括可见和近红外，并进行了要必的讨论。直接测定单线态氧在溶液中寿命的研究。本工作装置了一套近红外弱信号探测系统测定了O_2(~1△_g)在液相中的磷光寿命。