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.

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.

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.

蛇毒Ⅱ类磷脂酶A2 功能分化的分析研究

研究蛇毒Ⅱ类磷脂酶A2 (PLA2 ) 中D49 PLA2 和K49 PLA2 的功能分化及其功能分化决定位点的鉴定。方法: 运 用序列比较分析, 进化树构建和DIVERGE v1104 软件计算研究D49 PLA2 和K49 PLA2 的功能分化情况及其分化位点。结果: 序列比较分析, 进化树构建和DIVERGE v1104 软件计算结果表明蛇毒Ⅱ类PLA2 中D49 PLA2 和K49 PLA2 的确发生了功能分 化, 对于K49 PLA2 来说, 1S , 7K, 11Q , E12 , R34 , T56 , N88 , L92 , E108 , K116 , K128 可能为功能分化决定位点。对于 D49 PLA2 , L2 , G33 , G35 , F46 和Y118 可能为功能分化决定位点。结论: 我们首次通过序列比较分析, 进化树构建和DI2 VERGE v1104 软件计算鉴定出蛇毒Ⅱ类PLA2 中D49 PLA2 和K49 PLA2 可能的功能分化位点, 为今后通过基因重组和定点突 变方法研究蛇毒Ⅱ类PLA2 结构功能关系提供了线索。

眼镜王蛇泛素融合蛋白和核糖体蛋白L30的分子克隆与分析及大蹼铃蟾膜联蛋白A2样蛋白的纯化与活性研究

本论文分为两个部分。第一部分利用分子生物学手段对广西产眼镜王蛇（OPhiophagushannah）泛素融合蛋白基因和核糖体蛋白L30基因进行了克隆及分析；第二部分利用生物化学手段对大蹼铃蟾（Bombinamaxima）膜联蛋白A2样蛋白进行了初步研究。首先，从广西产眼镜王蛇毒腺中抽提总RNA，经mRNA纯化后构建眼镜王蛇毒腺。DNA文库，得到大概1.8*105个独立的克隆。从所构建的cDNA文库中，我们随机筛选200个克隆测序，得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBahk登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp，后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体；后者编码1巧个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明，泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白，C末端含有一个"锌指"结构域。与16个物种比较的结果表明，眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高，具有高度的保守性。我们克隆到的眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因为泛素家族和核糖体家族提供新的序列信息，有助于深入研究泛素蛋白和核糖体蛋白的结构、功能以及与其他物种的类似分子之间的相互关系。其次，通过阴离子交换，凝胶过滤和阳离子交换层析我们从大蹼铃蟾皮肤匀浆物中纯化了一个表观分子量为33000Da的单链蛋白，N-末端序列比较分析显示它与脊椎动物膜联蛋白AZ亚群有较高的同源性，与来自非洲爪蟾、红色原鸡和人膜联蛋白AZ序列的annexin核心的第一个重复区结构域序列一致性分别为78.9％、89％和68％，因此命名为大蹼铃蟾膜联蛋白AZ样蛋白（BAllLP）。BAllLP具有钙依赖性的抑制专一性血小板膜受体GPVI激动剂一Stejnulxin诱导人血小板聚集的生物学功能。

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.