植物细胞中间纤维角蛋白性质的分析

角蛋白是植物细胞中间纤维的主要。应用选择性抽提和生物化学技术,分离纯化了豌豆根尖细胞５８、５２ｋＤ、白菜叶５２ｋＤ和胡萝卜悬浮细胞６４ＫＤ角蛋白,测定了它们的氨基酸组成,结果表明上述角蛋白与动物细胞中间纤维角蛋白 氨基酸组成有较大的相似性。比较了动、植物细胞角蛋白肽谱、结果显示它们之间存在较大的差异,但是植物细胞嶂角蛋白肽谱比较一致,这提示它们属于同一蛋白家族,为植物中间纤维及其蛋白 存在提供了新     

植物细胞中含有类角蛋白多肽及角蛋白cDNA的同源序列

根据中间纤维对去圬剂和高盐溶液的抗性,用类似动物细胞中间纤维抽提的方法,对玉米和烟草的叶肉细胞进行选择性抽提处理,提取中间纤维的蛋白成份。蛋白经SDS聚丙烯酰胺凝胶电泳后,用动物细胞角蛋白的单克隆抗体进行免疫印迹反应,主要显示了分子量为52kD和64kD两种多肽成份。同时,提取玉米和烟草的DNA与α-~(32)P标记的兔K_3cDNA片段RB_3进行点杂交,均呈现较强的阳性反应,说明它们的基因组DNA中存在角蛋白基因的同源序列。     

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

甲藻(涡鞭毛虫,dinoflagellate)的细胞核是现存真核生物中最原始的。我们采用整装细胞制样和非树脂包埋去包埋剂超薄切片电镜技术,结合选择性生化抽提方法显示在寇氏隐甲藻(Cryptheccdinium cohnii)细胞中存在一个以水不溶性纤维蛋白成份为主的,贯穿于细胞核和细胞质的纤维网架系统,即核骨架-中间纤维结构体系,而Lamina结构不明显。免疫印迹法显示,甲藻细胞中存在类角蛋白组分,分子量为63kD和67kD,哺乳动物Lamin抗体与甲藻细胞全蛋白反应阴性。实验结果表明,在原始真核细胞中已经出现了类似于哺乳动物细胞的核骨架和中间纤维,并提示核骨架-中间纤维细胞骨架体系可能起源于真核细胞起源早期。本文对Lamina与中间纤维在进化上的关系及Lamina在真核细胞进化中的功能意义作了讨论。     

拟南芥细胞中存在中间纤维的研究

利用整装电镜制样与选择性抽提技术,在拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ （Ｌ．） Ｈｅｙｎｈ） 愈伤组织细胞质中观察到直径１０ ｎｍ 左右的纤维网络结构。免疫印迹分析表明纤维的主要成分是６ 种多肽,它们分别与动物角蛋白单克隆抗体ＡＥ１ 、ＡＥ３ 有免疫交叉反应。利用间接免疫荧光技术,与ＡＥ１ 和ＡＥ３ 反应的抗原呈弥散状定位于整个细胞质中,而且１０ ｎｍ 纤维可以在体外重新组装。以上结果表明,在拟南芥细胞质中存在类角蛋白的中间纤维。以动物中间纤维基因的保守序列为引物,采用ＲＴ＿ＰＣＲ技术,进一步从这一模式植物中克隆到一个ｃＤＮＡ片段,这可能为从分子水平上证明植物中间纤维的存在提供了一个线索     

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。     

植物角蛋白中间纤维在体外的装配特性

经选择性抽提与纯化的植物角蛋白在体外进行重组装 ,扫描隧道显微镜及负染电镜显示 ,酸性角蛋白和碱性角蛋白在体外能自组装成 10nm中间纤维 .在装配的初期阶段 ,可观察到角蛋白二聚体 ,在二聚体中部有非螺旋的连接肽链 ,它是二聚体进一步装配的基础 .对完整 10nm纤维的观察可发现 ,每根 10nm纤维是由多根直径为 3nm的纤维组成 ,这反映了中间纤维组装过程中所形成的原丝等中间结构状态 .在 10nm纤维及原丝的纵向都存在 2 3～ 2 5nm的重复周期 ,这一周期是所有中间纤维的典型特性 ,它说明了角蛋白分子在组装过程中发生了半分子交错 .     

铁线蕨中间纤维的研究及某些植物类角蛋白的比较分析

应用整装电镜制样技术,结合选择性抽提方法在铁线蕨（ＡｄｉａｎｔｕｍｐｈｉｌｉｐｐｅｎｓｅＬ．）叶细胞中观察到直径１０ｎｍ的纤维网络结构。免疫印迹结果显示：经选择性抽提得到的纤维蛋白与动物角蛋白抗体有免疫交叉反应,间接免疫荧光标记也得到类似结果,而且此类蛋白能在体外自组装为１０ｎｍ或更粗的纤维。说明蕨类植物细胞中存在类角蛋白中间纤维网络。免疫印迹结果表明,螺旋藻（ＳｐｉｒｕｌｉｎａｓｕｂｔｉｌｌｉｓｉｎａＫｕｔｚ．）细胞,地钱（ＭａｒｃｈａｎｔｉａｐｏｌｙｍｏｒｐｈａＬ．）叶状体,铁线蕨（Ａ．ｐｈｉｌｉｐｐｅｎｓｅＬ．）、银杏（ＧｉｎｋｇｏｂｉｌｏｂａＬ．）、白菜（ＢｒａｓｉｃａｐｅｋｉｎｅｎｓｉｓＲｕｐｒ．）的叶组织经选择性抽提后得到的蛋白均与动物角蛋白抗体有免疫交叉反应。其中,螺旋藻仅含有两种类酸性角蛋白,而其余４种植物材料均含有３种类酸性角蛋白及３种类碱性角蛋白。结合以往实验结果,我们认为类角蛋白在植物细胞中是普遍存在的。     

植物细胞类角蛋白的纯化、序列分析与类中间纤维cDNA的克隆

从胡萝卜悬浮培养细胞中纯化了64,55 ku两种类角蛋白,并对其部分氨基酸序列进行了测定,同源性比较结果表明64 ku蛋白与β-葡糖苷酶同源,55 ku蛋白未发现同源序列.以动物中间纤维蛋白的保守序列为引物,从胡萝卜悬浮培养细胞中克隆到一个cDNA片段,Southern,Northern杂交结果表明该片段为单拷贝,并在胡萝卜悬浮培养细胞及叶片中均表达,同源性比较揭示其与一些含有α-螺旋的基因序列有一定的同源性.以上结果为从分子水平上证明植物中间纤维的存在提供了一条线索.     

不同条件对植物中间纤维体外装配的影响(简报)

细胞骨架的研究是当今细胞生物学中最为活跃的领域之一,而中间纤维是三种主要骨架纤维中研究较少的一种。从60年代发现至今,人们对动物细胞中间纤维的研究已经比较深入,近来又发现它在基因表达等重要生命活动中起一定的作用。中间纤维有一个显著的特征,就是能够在体外进行自我装配,不需要核苷酸和结合蛋白参加,也不依赖于蛋白质的浓度。植物细胞中是否存在中间纤维一直是未解决的问题。从80年代起,有一些研究发现在高等植物细胞中存在能与动物细胞中间纤维抗体进行     

角蛋白与肿瘤

角蛋白属于I型和II型中间纤维,是上皮细胞中间纤维的主要组成蛋白。角蛋白对上皮细胞及组织的稳定性和完整性具有重要的功能,此外,许多角蛋白还参与细胞内的信号转导通路。角蛋白的基因突变会导致一系列的遗传性皮肤病,还能引起肝脏、口腔粘膜、食管、外阴、直肠粘膜的白海绵痣等疾病。最近大量研究发现,角蛋白在人类多种类型肿瘤中也存在特异性表达,角蛋白及其抗体在肿瘤的免疫化学诊断、核转移、精确的分型或分类等方面具有重要的作用,并且还有助于预测肿瘤治疗反应和预后情况。因此,研究角蛋白与肿瘤之间的相互联系,揭示它们的作用机制对肿瘤的诊断和治疗有着重要意义。该文回顾了近年来角蛋白的分子生物学研究概况及临床应用,对各种角蛋白与肿瘤发生、进展、诊断以及预后的关系进行综述,同时对存在的问题及困难作了探讨并对未来的研究进行了展望。     

The assembly of keratins from higher plant cells

Summary In vitro assembly and morphological characteristics of purified 58 kDa, 52 kDa, 50 kDa, and 45 kDa polypeptides in the leaves and the cotyledons of the cabbage (Brassica pekinensis Rupt.) were investigated by electron microscopy and scanning tunneling microscopy. The three or four purified intermediate filament (IF) polypeptides can spontaneously assemble into intermediate filaments in vitro with a 23–24 nm axial repeat, which indicates that keratin IFs in higher plant cells have the same molecular arrangement as in animal cells. STM images suggest that the plant keratin filaments display a pronounced structural polymorphism, which can be composed of 3 nm, 4.5 nm, or 6 nm wide keratin protofilaments.Abbreviation IF intermediate filament - STM scanning tunneling microscopy - SDS sodium dodecyl sulfate - BCIP 5-bromo-4-chloro-3-indolyl phosphate-toluidine - NBC p-nitroblue tetrazolium chloride - PMSF phenylmethyl sulfonylfluoride - HOPG high oriented pyrolytic graphite     

The keratin intermediate filament—like system in maize protoplasts

The application of Penman‘s method of cell fractionation to plant protoplasts leads to our finding of keratin intermediate filament(IF)-like system in maize protoplasts,which was identified by using immunogold labelling with monoclonal antibody of cytokeratin from animal cells.Many gold particles were found to be bound on filaments,linked by 3 nm filaments.After further digestion and extraction with DNase I and ammonium sulphate.IF-like framework-lamina-nuclear matrix system was shown under electron microscope.That IF system exists in plant protoplasts just like in animal cells,and their main component is keratin-like protein.     

衣藻(Chlamydomonas sp)中间纤维及核纤层存在的证实(简报)

衣藻(Chlamydomonas sp)是属于绿藻门的最低等单细胞植物,为典型的真核生物。迄今以衣藻为材料所作的有关细胞骨架方面的研究多集中在微管蛋白(tubulin)。C.J.Miller等曾以衣藻(Chlamydomonas reinhardtii)全蛋白与几种中间纤维抗体进行免疫印迹实验有阳性反应,但是衣藻中是否存在中间纤维与核纤层是不清楚的问题。衣藻中间纤维与核纤层的形态研究更未见报道。目前认为中间纤维-核纤     

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembled in vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filaments in vitro. In higher magnification images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24—25 nm periodic structural repeat alone the axis of beth the 10 nm filaments and protofilaments. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

Assembly characteristics of plant keratin intermediate filamentsin vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembledin vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filamentsin vitro. In higher mcation images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24–25 nm periodic structural repeat alone the axis of both the 10 nm filaments and protofilarnents. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembledin vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filamentsin vitro. In higher mcation images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24–25 nm periodic structural repeat alone the axis of both the 10 nm filaments and protofilarnents. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments. Project supported by the National Natural Science Foundation of China (Grant No. 39370352) and the Doctor Foundation of Minishy of Education of China.     

The epidermal intermediate filament proteins of tunicates are distant keratins; a polymerisation-competent hetero coiled coil of the Styela D protein and Xenopus keratin 8

Two novel cytoplasmic intermediate filament (IF) proteins (C and D) from the tunicate (urochordate) Styela are characterised as putative keratin orthologs. The coexpression of C and D in all epidermal cells and the obligatory heteropolymeric IF assembly of the recombinant proteins argue for keratin orthologs, but the sequences do not directly reveal which protein behaves as a keratin I or II ortholog. This problem is solved by the finding that keratin 8, a type II keratin from man or Xenopus, forms chimeric IF when mixed with Styela D. Mutant proteins of Styela D and keratin 8 with a single cysteine in equivalent positions show that these chimeric IF are, like vertebrate keratin filaments, based on the hetero coiled coil. We propose that Styela D retains, in spite of its strong sequence drift, important molecular features of type I keratins. By inference Styela C reflects a type II ortholog. We discuss that type I to III IF proteins are expressed along the chordate branch of metazoa.     

In vitro assembly and structure of trichocyte keratin intermediate filaments: a novel role for stabilization by disulfide bonding

Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from "microfibrils." These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.     

The coiled-coil molecules of intermediate filaments consist of two parallel chains in exact axial register

Amino acid sequence studies of helical particles derived from proteolytic digests of mouse epidermal keratin intermediate filaments (IF) have shown that their coiled-coil molecules are heterodimers of Type I and Type II keratins, with a parallel arrangement of the two chains. From a reappraisal of published chemical cross-linking data, it is concluded that the coiled-coil molecules in all IF consist of pairs of parallel chains in precise axial register.