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.

杂交水稻威优64及其亲本叶绿体D1蛋白基因psbA的分子克隆与结构比较研究

水稻是我国及东南亚广大地区的主要粮食作物之一。已发现由叶绿体基因组编码的某些多肽与光合效率之间存在着密切的联系。但是，根据我们所掌握的资料，直至目前为止，还没有见到从分子水平上阐明高光效植物与低光效植物之间相互关系的研究报导。 本工作主要从编码D1蛋白的psbA基因着手研究。D1蛋白是光系统Ⅱ反应中心的组成之一,它是均三氮苯类(triazine)除草剂的结合受体。 实验采用无水法从杂交水稻威优64及其亲本V20A和测64的幼苗叶片中提联并纯化各自的ctDNA，然后用限制性内切酶BamHI、EcoRI、Hind III、PstⅠ分别进行切割消化。Southern吸印杂交结果表明，在水稻ctDNA2.2Kb的EcoRI限制片段上，编码着psbA基因的全序列。据此，我们用2.0-2.5 Kb范围的ctDNA的EcoR I酶切片段和pBR322质粒载体进行重组，并转化到E. coli HB101菌株中，构成水稻叶绿体DNA专一性基因文库。用分子杂交方法分别从三种水稻品种的专一性基因文库中调到了各自的psbA基因，它们的重组体质粒分别定名为pWsbA, pVsbA和ZsbA并构建了这三个基因的核酸内切酶图谱。结果发现，在本实验体检测的若干种核酸内切酶切割位点分布上，这三个基因并无差异，但同已发表的双子叶植物（豆，烟草等）比较，则有明显的不同。

光系统II反应中心D1/D2/Cyt b559复合物的光谱特性及其光破坏作用的研究

采用柱层析法从菠菜叶绿体中分离纯化得到高等植物光系统Ⅱ(PSⅡ)反应中心色素蛋白复合体Dl/D2/Cyt b559，并对其性质，特别是光破坏作用的分子机理进行了研究。主要结果如下： 1、PSⅡ反应中心复合物所含的色素比大约为Chla／2 Pheo a=6.0。其四阶导数光谱在红区有两个峰，表明该反应中心至少存在两种结合状态的Chla。 2、Dl/D2/Cyt b559复合物的荧光相对产率及发射光谱的谱带位置与样品的浓度直接相关。只有当样品的浓度达到足够稀的程度(Chla和Pheo a总浓度小于1μg／ml)，才能得到较真实的荧光光谱，其峰位在681nm处。 3、Dl/D2/Cyt b559复合物的CD光谱在红区（Qy带）有一对反向谱带，正蜂为680nm,负峰为660nm，而在β-胡萝卜素的吸收区没有明显的CD信号。当该反应中心复合物受光破坏后，CD信号明显下降，而且当正峰完全消失后，负峰仍然存在，说明负峰不仅包含P680 的信号，也包含其它色素分子的信号，很可能有部分来源于Pheo a。 4、Dl/D2/Cyt b559复合物在488nm处激发的共振拉曼光谱显示四个主要谱带，其峰位分别在1532(ν1)、1165(ν2)、1010(ν3)和970cm-1（ν4）处，表明PSⅡ反应中心结合的B-胡萝卜素分子是全反式构型。Dl/D2/Cyt b559复合物的色素抽提液的拉曼光谱也显示四个主要的拉曼峰，其中ν4谱带的强度急剧下降，说明PSⅡ反应中心内部结合的β-胡萝卜素分子与抽提液中自由的β-胡萝卜素分子的构象不同，而与光合细菌反应中心内部的类胡萝卜素分子的构象相似，其共轭多烯链的平面也处于扭曲状态。 5、光照使PSⅡ反应中心的原初电子供体P680受到破坏，在光照后的暗放置过程中P680分子继续受到破坏，表明在光照过程中很可能有一个相对稳定的反应中间体产生，以至于光照后暗放置过程中Dl/D2/Cyt b559复合物的光谱特性继续发生变化。也就是说，PSⅡ反应中心Dl/D2/Cyt b559复合物的光破坏不是一步反应，而是一个多步反应或多条途径。 6、光照使Dl/D2/Cyt b559复合物中的组氨酸(His)残基受到很大程度的破坏，甲硫氨酸(Met）残基的含量也略有下降，而其它氨基酸的含量基本保持不变。His残基的破坏很可能与光照后暗放置过程中Dl/D2/Cyt b559复合物的光谱特性变化相关。我们认为His残基的光照破坏很可能是Dl/D2/Cyt b559复合物受光照破坏的另一分子机理。 7、人工电子受体癸基质体醌(DPQ)可以与Dl/D2/Cyt b559复合物进行重组。Dl/D2/Cyt b559复合物的荧光衰减分析表明，在DPQ重组之后，两个长寿命荧光组分（24ns和73ns）的寿命减小，而且占整个荧光的分数也下降，表明这两个长寿命荧光衰减组分均来源于电荷重组过程。同时，β-胡萝卜素分子在DPQ重组之后更易于被光照破坏，这个过程可能与β-胡萝卜素分子的生理功能相关。 8、在没有外加人工电子受体的情况下，光照使DDl/D2/Cyt b559 复合物的多肽组成发生一定变化。SDS-PAGE图谱中出现一个约40KDa的新谱带，同时Dl与D2多肽的表观分子量增加，谱带染色强度下降。 9、本文根据以上实验结果，着重对Dl/D2/Cyt b559复合物光破坏的分子机理进行了分析和讨论，并在D1蛋白裂解的两种可能途经中又增加了一个新的可能导致Dl蛋白裂解的途径，即：His残基的光照破坏可以作为Dl/D2/Cyt b559复合物光破坏及Dl蛋白裂解的又一分子机理，这为深入研究PSⅡ反应中心的光破坏提供了新的线索，也为今后研究活体内光抑制现象的分子机制打下了良好的基础

Sexual Behavior Modulates Contextual Fear Memory Through Dopamine D1/D5 Receptors

Traumatic events always lead to aversive emotional memory, i.e., fear memory. In contrast, positive events in daily life such as sex experiences seem to reduce aversive memory after aversive events. Thus, we hypothesized that post-traumatic pleasurable ex

Dose-dependent effects of the dopamine D1 receptor agonists A77636 or SKF81297 on spatial working memory in aged monkeys

With advancing age, monkeys develop deficits in spatial working memory resembling those induced by lesions of the prefrontal cortex (PFC). Aged monkeys also exhibit marked loss of dopamine from the PFC, a transmitter known to be important for proper PFC cognitive function. Previous results suggest that D1 agonist treatment can improve spatial working memory abilities in aged monkeys. However, this research was limited by the use of drugs with either partial agonist actions or significant D2 receptor actions. In our study, the selective dopamine D1 receptor full agonists A77636 and SKF81297 were examined in aged monkeys for effects on the working memory functions of the PFC. Both compounds produced a significant, dose-related effect on delayed response performance without evidence of side effects: low doses improved performance although higher doses impaired or had no effect on performance. Both the improvement and impairment in performance were reversed by pretreatment with the D1 receptor antagonist, SCH23390. These findings are consistent with previous results demonstrating that there is a narrow range of D1 receptor stimulation for optimal PFC cognitive function, and suggest that very low doses of D1 receptor agonists may have cognitive-enhancing actions in the elderly.

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.