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.

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。

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

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.

Expression, purification and spectral characterization of p21(Waf1/Cip1)

p21(Waf1/Cip1), best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, can interact with various target proteins, and this ability relies on its structural plasticity. Therefore, studies on the structural properties of p(21) are very important to understand its structure-function relationship. However, detailed studies on its secondary structure and biophysical properties have been comparatively sparse. A human p(21) gene was cloned into the temperature expression vector pBV220 and transformed into Escherichia coli strain JM109.