硬化性肌膜炎患者血清ACA的发现及对小鼠细胞着丝点的研究

首次在硬化性肌膜炎患者血清中发现有抗着丝点抗体(ACA),可使 Hep-2间期细胞核及 M 期细胞染色体着丝点显示分散的荧光斑点.用 ACA 血清对小鼠几种器官的冰冻切片或涂片进行免疫荧光研究,可见无论幼龄或老龄小鼠的肝、十二指肠、脑皮层细胞及老龄小鼠睾丸生精细胞均有明亮的荧光斑点.绒毛顶端细胞呈均匀性荧光.     

HL－60细胞分化对动粒蛋白及其基因表达的影响

以人早幼粒白血病细胞ＨＬ－６０为实验材料，用终浓度１μｍｏｌ·Ｌ－１甲酸（ＲＡ）和二甲亚砜（ＤＭＳＯ）分别连续处理细胞６ｄ后，可使ＨＬ－６０细胞按粒系途径定向成熟分化，即表现出核分叶，具有ＮＢＴ还原能力的正常粒细胞的形态和功能特征。利用ＡＣＡ间接免疫荧光方法，观察到在终未分化的细胞核中荧光斑点强度要明显弱于对照细胞。用ＲＮＡＮｏｒｔｈｅｒｎ和斑点杂交方法研究发现，在ＨＬ－６０细胞诱导分化过程中，Ｉｄ，ＣｙｃｌｉｎＢ１基因表达很快受到明显抑制，在终末分化的细胞中已基本被关闭；ＣＥＮＰ－Ｂ基因表达水平也明显降低，但表达变化过程与Ｉｄ和ＣｙｃｌｉｎＢ１基因有所差异。     

Progress and perspective of the kinetochore-associated motor proteins

The kinetochore is structurally composed of four layers,We know that three microtubule-based motor protcins such as CENP-E,dynein,and MCAK are located at the outmost region of the kinctochore,Experimentation of these motot functions betters our understanding of mitotic regulation,and chromaosme movements in particular,With real-time studies of chromosome movements in live cells,we hope to illustrate the molecular mechanisms under lying mitotic regulation.     

Protein kinase TTK interacts and co-localizes with CENP-E to the kinetochore of human cells

Spindle checkpoint is an important biochemical signaling cascade during mitosis which monitors the fidelity of chromosome segregation, and is mediated by protein kinases Mps1 and Bub1/BubR1. Our recent studies show that kinesin-related motor protein CENP-E interacts with BubR1 and participates in spindle checkpoint signaling. To elucidate the molecular mechanisms underlying spindle checkpoint signaling, we carried out proteomic dissection of human cell kinetochore and revealed protein kinase TTK, human homologue of yeast Mps1. Our studies show that TTK is localized to the kinetochore of human cells, and interacts with CENP-E, suggesting that TTK may play an important role in chromosome segregation during mitosis.     

Functions of spindle check-point and its relationship to chromosome instability

It is generally believed that the equal distribution of genetic materials to two daughter cells during mitosis is the key to cell health and development. During the dynamic process, spindle checkpoint plays a very important role in chromosome movements and final sister chromatid separation. The equal and precise segregation of chromosomes contributes to the genomic stability while aberrant separations result in chromosome instability that causes pathogenesis of certain diseases such as Down’s syndrome and cancers. Kinetochore and its regulatory proteins consist of the spindle checkpoint and determine the spatial and temporal orders of chromosome segregation.     

taxol对V79—8系细胞微核化机理的探讨

用多种方法探讨了 v_(79-8)系细胞在 taxol 作用下微核化的时期和机理.早熟染色体凝集(PCC)法发现此种 G_1 及 G_2 期缺陷的细胞株,在一定的生长条件下有相当数量的 G_1 和 G_2 细胞存在.taxol 作用后,首先是有丝分裂指数(I_m)的增加,然后随 I_m 的下降细胞微核化增加.秋水仙酰胺阻断的 M 期细胞,在释放的同时加入 taxol,细胞微核化速率增加迅速.微管的间接免疫荧光法显示,在 taxol 作用下,微管集合成不规则的束.微核化细胞可以继续合成 DNA.每个微核中含有可被抗着丝点抗体(ACA)血清荧光染色的着丝点.     

ACA的间接免疫荧光法显示多种动、植物细胞着丝点

用硬化性肌膜炎病人ACA血清的间接免疫荧光法显示了多种动、植物细胞的着丝点,其中包括Hep-2,文昌鱼、水螅、螽蜥、蚯蚓、草履虫、洋葱、大蒜,吊兰、蚕豆和葫芦藓等。表明在进化不同阶段的动、植物细胞均有同源的、高度保守性的着丝点蛋白。     

Inhibition of protein deacetylation by trichostatin A impairs microtubule-kinetochore attachment

Inhibition of protein deacetylation arrests cells in mitosis, but the mechanism is unknown. To understand why inhibiting protein deacetylation causes cell cycle arrest, we treated HeLa cells beyond G1/S transition with trichostatin A (TSA), a potent protein deacetylase inhibitor, and found that the cells arrested at prometaphase with ectopic spindles and unaligned chromosomes. The hyper-acetylated cells encountered a serious microtubule (MT)-kinetochore attachment problem, although the kinetochores are intact at ultrastructural level. By immunofluorescence staining of kinetochore proteins, we found that the pericentromeric H3K9Me3-HP1 pathway was disrupted and that the CENP-A-dependent outer plate protein dynamics of kinetochores was greatly diminished by the drug treatment. The treatment also caused the loss of chromosome passenger complex (CPC), the proposed error checking system, from centromere and impaired the microtubule dynamics of the cells. Overall, we propose that deacetylation inhibition impairs MT-kinetochore attachment through disrupting the centromere function and altering the kinetochore composition and MT dynamics. Received 30 April 2008; received after revision 28 July 2008; accepted 14 August 2008     

Effects of sense and antisense centromere/kinetochore complex protein-B （CENP-B） in cell cycle regulation

This paper investigates the effects of sense and antisense centromere/kinetochore complex protein-B （CENP-B） in cell cycle regulation. Full-length cenpb cDNA was subcloned into pBI-EGFP eukaryotic expression vector in both sense and antisense orientation. HeLa-Tet-Off cells were transfected with sense or antisense cenpb vectors. Sense transfection of HeLa-Tet-Off cells resulted in the formation of a large centromere/kinetochore complex, and apoptosis of cells following several times of cell division. A stable antisense cenpb transfected cell line, named HACPB, was ob- tained. The centromere/kinetochore complex of HACPB cells became smaller than control HeLa-Tet-Off cells and scattered, and the expression of CENP-B was down-regulated. In addition, delayed cell cycle progression, inhibited malignant phenotype, restrained ability of tumor formation in nude mice, and delayed entry from G2fM phase into next G1 phase were observed in HACPB cells. Furthermore, the expression of cyclin-dependent kinases （CDKs）, cyclins, and CDK inhibitors （CKIs） were modulated during different phases of the cell cycle. CENP-B is an essential protein for the maintenance of the structure and function of centromere/kinetochore complex, and plays important roles in cell cycle regulation.