四膜虫细胞的核骨架及类中间纤维

采用非树脂包埋去包埋剂超薄切片结合选择性生抽提方法显示,原生动物四膜虫细胞大核具有发达的核骨架纤维网络,核周是一层完整的核纤层结构,在四膜虫细胞小核中,亦存在核骨架和核纤层。四膜虫细胞皮层中存在水下溶性纤维网架,其中含有类中间纤维蛋白组分,４９ＫＤ蛋白。     

PtK2细胞的核骨架—核纤层——中间纤维体系

本文用选择性系列抽提的方法结合整装细胞电镜技术和DGD包埋-去包埋超薄切片技术,在电镜下清晰地显示了PtK 2细胞的核骨架-核纤层-中间纤维体系的精细结构。处于分裂中期的细胞经抽提后可以看到,染色体残余与中间纤维仍然保持一定的联系。用免疫荧光技术对抽提后的PtK 2细胞进行分析结果表明:其中间纤维能同时与AE1和AE3反应;能与Lamin B反应的单抗可以特异地定位于其核周,而Lamin A(C)的单抗除了与其核纤层蛋白有很强的反应外还与中间纤维有交叉反应。此外,在分裂期细胞中可以看到Lamin A(C)可能与染色体能特异结合;与HeLa细胞不一样。PtK 2细胞的核骨架成份不能与280kD的核骨架蛋白单抗反应。双向电泳结果显示出PtK 2细胞的核骨架-核纤层-中间纤维体系的组成成份与HeLa细胞相比有较大的差异,而且这种差异主要反映在核骨架组份上,TdR的处理也能导致其组份发生变化。     

不同生理状态下膜状急纤虫大核DNA和线粒体DNA的多态性比较

为探索纤毛虫在营养及休眠条件下两套遗传系统的作用关系,对膜状急纤虫(Tachysomapellionella)营养细胞和休眠包囊大核DNA、线粒体DNA进行了RAPD比较。结果显示,在所选用的34条随机引物中,大核DNA共扩增出203条片段,其中以休眠包囊大核DNA为模板扩增出45条特有片段,以营养细胞大核DNA为模板扩增出36条特有片段,两者存在40%的差异。在所选用的32条随机引物中,线粒体DNA共扩增出216条片段,其中以休眠包囊线粒体DNA为模板扩增出35条特有片段,以营养细胞线粒体DNA为模板扩增出47条特有片段,两者有38%的差异。结果表明,膜状急纤虫休眠包囊与营养期的大核DNA结构存在显著的差异;两者的线粒体DNA结构也存在较大差异。这表明,膜状急纤虫在包囊形成过程中,大核及线粒体DNA结构可能都发生了一定的变化,并且这些变化可能与包囊形成过程中的形态结构和代谢活动等剧烈变化以及休眠状态下的生理生化变化密切相关。     

营养期和休眠期冠突伪尾柱虫的类中间纤维-核纤层-核骨架体系

近年来的研究表明 ,尽管不同类群的纤毛虫形成包囊过程中 ,细胞经历了不同程度的分化 ,形成不同类型的包囊 ,但是其休眠包囊生命活动中对细胞内物质的消化、能量利用和代谢等都具有共同的特征 (顾福康等 ,1995 ,1999;李恭楚等 ,1999;陈灵等 ,2 0 0 0 )。这一现象提示 ,纤毛虫在休眠条件下的生命活动可能具有一些相同的物质和结构基础。由于在高等真核细胞中普遍存在的中间纤维-核纤层 -核骨架体系对细胞生命活动起到多方面的重要作用 ,并且尽管目前仅对极少数原生动物观察到该结构体系 ,但它却预示了在这一原始的单细胞类群中以中间纤维为基…     

两种纤毛虫营养细胞和休眠细胞蛋白组成的比较分析

应用生化抽提和SDS-PAGE方法显示,膜状急纤虫(Tachysoma pellionella)的营养细胞含38条蛋白谱带,休眠细胞含29条蛋白谱带,两者共有谱带为26条,特有谱带各为12条和3条,相似度为77.6%;休眠细胞的包囊壁含22条蛋白谱带,细胞脱包囊后残留的包囊壁含15条蛋白谱带,两者共有谱带为14条,特有谱带各为8条和1条,相似度为76%。包囊游仆虫(Euplotes encysticus)的营养细胞和休眠细胞均含23条蛋白谱带,两者共有谱带为19条,特有谱带各为4条,相似度为82.6%;休眠细胞的包囊壁和细胞脱包囊后残留的包囊壁均含20条蛋白谱带,两者共有谱带为19条,特有谱带均为1条,相似度为95%。结果表明,两种纤毛虫的营养细胞向休眠细胞转化过程中,细胞结构的主要蛋白质成分发生了明显变化,这些变化与细胞在不同生理状态下结构的分化及其生命活动特征相关。形成“毛基体吸收型包囊”的急纤虫与形成“毛基体非吸收型包囊”的游仆虫相比较,前者营养期和休眠期细胞蛋白组成有更明显的差异,这可能与其细胞结构更大程度的脱分化有关;根据纤毛虫休眠细胞的包囊壁和细胞脱包囊后残留的包囊壁两者蛋白组成的差异推测,前者包囊壁可能含有与休眠细胞生命活动相联系的“活性”成分。     

小鼠胚胎干细胞（ES—M13）核骨架—核纤层—中间纤维结构体系的研究

本文使用细胞的选择性抽提、ＤＧＤ包埋去包埋电镜制样、免疫荧光和免疫印迹技术研究了小鼠胚胎干细胞（ＥＳ－Ｍ１３）的核骨架－核纤层－中间纤维结构体系。在电镜下可以看到,ＥＳ细胞存在精细发达的核骨架结构,核骨架纤维同核纤层结构相连接,细胞质中有许多直径为１０ｎｍ的中间纤维单丝,细胞质中有许多直径为１０ｎｍ的中间纤维单丝,在免疫荧光分析中,使用角蛋白单克隆抗体有阳性反应,细胞质区域可以看到较强的荧光,没有     

北京鸭B淋巴细胞的中间纤维—核纤层—核内骨架体系

以北京鸭腔上囊为实验材料,应用细胞分级抽提方法与非树脂包埋－去包埋剂的电镜制样技术相结合,显示出Ｂ细胞中相互连结的中间纤维－核纤层－核内骨架体系的超结构及其分布,中间纤维交织成网络状,纤维直径在９－１１ｎｍ,其成份是分子量为６７ｋＤ,等电点约为６．２的波形蛋白,核纤民支呈片层状结构环绕在核区周围,其主要成份是分子量为６７ｋＤ,等电点偏酸性的Ｌａｍｉｎ Ｂ。核内骨架由粗细不一的纤维形成网络结构,其上     

PtK2细胞的核骨架—核纤层—中间纤维体系

本文用选择性系列抽提的方法结合整装细胞电技术和ＤＧＤ包埋－去包埋超薄切片技术,在电镜下清晰地显示了ＰｔＫ细胞的核骨架－核纤层－中间纤维绵精细结构。处于分裂中期的细胞经抽提后看到,染色体残３余与中间纤维仍然保持一定的联系。用免疫荧光技术对提提后的ＰｔＫ２细胞进行分析结果表明：其中间纤维能同时与ＡＥ１和ＡＥ３反应;能一ＬａｍｉｎＢ反应的单抗可以特异地定位于其核周,而ＬａｍｉｎＡ（Ｃ）的单抗除了与其核纤     

膜状急纤虫(原生动物,纤毛门)休眠期包囊与营养期细胞同工酶组成和活性比较

应用聚丙烯酰胺凝胶电泳酶化学技术显示,腹毛目纤毛虫膜状急纤虫（Tachysoma pellionella）休眠包囊和营养细胞中乳酸脱氢酶、α磷酸甘油脱氢酶、醇脱氢酶、细胞色素氧化酶、葡萄糖-6-磷酸脱氢酶、过氧化物酶和过氧化氢酶等7种同工酶的酶谱组成有明显差异,并且在休眠包囊中其同工酶成分少、活性低,部分同工酶酶谱表现出趋于简单的趋势。ATP酶、苹果酸脱氢酶和谷氨酸脱氢酶等3种同工酶在休眠包囊与营养细胞中有相同的酶谱,但在休眠期包囊酶的活性低于营养期细胞。     

小鼠胚胎干细胞(ES-M_(13))核骨架-核纤层-中间纤维结构体系的研究

本文使用细胞的选择性抽提、DGD包埋去包埋电镜制样、免疫荧光和免疫印迹技术研究了小鼠胚胎干细胞(ES-Ml_(13))的核骨架-核纤层-中间纤维(NM-L-IF)结构体系。在电镜下可以看到,ES细胞存在精细发达的核骨架结构,核骨架纤维同核纤层结构相连接,细胞质中有许多直径为10nm的中间纤维单丝。在免疫荧光分析中,使用角蛋白单克隆抗体有阳性反应,细胞质区域可以看到较强的荧光,没有极性分布现象,也没有观察到纤维状的荧光染色。ES细胞对波形蛋白和结蛋白抗体呈阴性反应,同对照组一样,只能看到非特异性的很微弱的荧光染色。在免疫印迹分析中,使用角蛋白单克隆抗体AF6检测到三条角蛋白多肽,分子量分别为65KD,62KD和52KD。     

Proteomic Approach to Reveal the Proteins Associated with Encystment of the Ciliate Euplotes encysticus

In order to identify and reveal the proteins related to encystment of the ciliate Euplotes encysticus, we analyzed variation in the abundance of the proteins isolated from the resting cyst comparing with proteins in the vegetative cell. 2-D electrophoresis, MALDI-TOF MS techniques and Bioinformatics were used for proteome separation, quantification and identification. The comparative proteomics studies revealed 26 proteins with changes on the expression in the resting cysts, including 12 specific proteins and 14 differential proteins. 12 specific proteins and 10 out of the 14 differential proteins were selected and identified by MALDI-TOF MS. The identified specific proteins with known functions included type II cytoskeletal 1, keratin, Nop16 domain containing protein, protein arginine n-methyltransferase, epsilon-trimethyllysine hydroxylase and calpain-like protein. The identified differential proteins with known functions included Lysozyme C, keratinocyte growth factor, lysozyme homolog AT-2, formate acetyltransferase, alpha S1 casein and cold-shock protein. We discussed the functions of these proteins as well as their contribution in the process of encystment. These identified proteins covered a wide range of molecular functions, including gene regulation, RNA regulation, proteins degradation and oxidation resistance, stress response, material transport and cytoskeleton organization. Therefore, differential expression of these proteins was essential for cell morphological and physiological changes during encystment. This suggested that the peculiar proteins and differential proteins might play important roles in the process of the vegetative cells transforming into the resting cysts. These observations may be novel findings that bring new insights into the detailed mechanisms of dormancy.     

BHK_(21)细胞的中间纤维-lamina-核骨架体系

以系列选择性抽提技术与显示细胞骨架的整装电镜技术为基础,应用免疫胶体金标记与蛋白质成份的双向电泳分析技术,研究了BHK_(21)细胞的中间纤维-lamina与核骨架(核基质)结构体系及其主要的蛋白成份。BHK_(21)细胞的中间纤维-lamina与核骨架是在结构上相互联系,贯穿于核与质的网络体系。中间纤维单丝直径为10nm,能很好地被抗波形蛋白抗体-金颗粒所标记,生化分析同样说明BHK_(21)细胞中间纤维的主要成份是波形蛋白(vimentin),其分子量为55KD,等电点为5.6。中间纤维网在胞质内呈极性分布,与lamina密切联结。BHK_(21)细胞的lamina能被抗lamin A与C的单克隆抗体-金颗粒标记。双向电泳分析证明,lamina含有三种蛋白成份,即lamin A,B,C,其分子最分别为68KD,70KD与62KD,lamin A,C等电点均为6.9—7.2,而lamin B偏酸,其等电点为5.8。BHK_(21)细胞核骨架纤维网也可以被清晰的显示,其蛋白成份较为复杂,在双向电泳谱上经常出现多个清晰的斑点,很可能含有肌动蛋白(actin)。298KD核基质蛋白的单克隆抗体-金颗粒能准确的标记核骨架纤维。     

寇氏隐甲藻（Crypthecodinium cohnii）细胞核骨架与中间纤维...

Nuclear matrix (NM) and intermediate filament (IF) scaffold in primitive eukaryote Crypthecodinium cohnii were shown using selective extraction together with embedment-free electron microscopy, whole mount cell preparation and immunoblot techniques. There exists a delicate NM-IF network spreading over cytoplasm and nucleus in dinoflagellate cells, however, nuclear lamina is undeveloped. The diameter of NM fiber is about 3-5 nm and IF is 10 nm. Chromosomes are connected with NM filament network. Immunoblot analysis showed that dinoflagellate contained keratin-like polypeptides (63 kD and 67 kD) while mammalian lamin antibodies did not crossreact with dinoflagellate total protein. Our experiment results demonstrated that a framework similar to NM-IF scaffold in mammalian cell appeared in primitive eukaryote. We propose that: (1) NM-IF scaffold is not restrict to vertebrate cell, and it may be originated from early stages of eukaryote evolution; (2) Keratin is probably very conservative; (3) Compared with IF, lamina might appear late in evolution, and some of primitive characteristics of dinoflagellate nucleus may be related to the lack of lamina.     

The intermediate filament-lamina-nuclear matrix system of BHK-21 cells

We have employed collodial gold immuno-labelling in whole-mount cell and 2-D gel electrophoresis to demonstrate the intermediate filament (IF)-lamina-nuclear matrix (NM) system in BHK-21 (Baby Hamster Kidney) cells. Grown on grids, cells were gently extracted with salt solutions as previously described by S. Penman to preserve intact IF-lamina-NM systems. The extracted samples were fixed, postfixed, dehydrated and dried through the CO2 critical point, then examined under high voltage electron microscope (HVEM). The results revealed that the IF-lamina-NM system is a interconnecting network throughout the cell from cytoplasma to nuclear. The IF unit is 10 nm in diameter. IFs radiate away from the nuclear region into the spreading cytoplasm and the polarity of their distributing is obvious. The IF system closely connected to lamina. Immuno-gold labelling and 2-D gel proved that vimentin, a 55 KD protein (pI 5,6), is the major component of IFs in BHK-21 cells. Lamina can be precisely and specifically labelled with anti-lamin A, C proteins and as well as 2-D gel electrophoresis indicated that there are lamin A, B, C proteins in BHK-21 cells, whose molecular weights are 68 KD, 70 KD, 62 KD respectively. Its components are more complicated, but a few dots of NM proteins can be clearly distinguished in 2-D gel map, in which actin, a 45 KD protein (pI 4.5), might be involved. The nuclear matrix network was also clearly presented under HVEM. Its filaments can be labelled with anti-NM 298 KD protein precisely.     

Regulated Expression of Human Filaggrin in Keratinocytes Results in Cytoskeletal Disruption, Loss of Cell–Cell Adhesion, and Cell Cycle Arrest

Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.     

Mannuronan C-5 epimerases and cellular differentiation of Azotobacter vinelandii

Differentiation in Azotobacter vinelandii involves the encystment of the vegetative cell under adverse environmental circumstances and the germination of the resting cell into the vegetative state when growth conditions are satisfactory again. Morphologically, the encystment process involves the development of a protective coat around the resting cell. This coat partly consists of multiple layers of alginate, which is a co-polymer of β- d -mannuronic acid (M) and α- l -guluronic acid (G). Alginate contributes to coat rigidity by virtue of a high content of GG blocks. Such block structures are generated through a family of mannuronan C-5 epimerases that convert M to G after polymerization. Results from immunodetection and light microscopy, using stains that distinguish between different cyst components and types, indicate a correlation between cyst coat organization and the amount and appearance of mannuronan C-5 epimerases in the extracellular medium and attached to the cells. Specific roles of individual members of the epimerase family are indicated. Calcium and magnesium ions appear to have different roles in the structural organization of the cyst coat. Also reported is a new gene sharing strong sequence homology with parts of the epimerase-encoded R-modules. This gene is located within the epimerase gene cluster of Azotobacter vinelandii .