嗜热四膜虫的着丝粒蛋白检查

目前国际上的着丝粒蛋白研究工作几乎全是以酵母和高等生物为材料进行的,为了从起源与进化的角度考察着丝粒蛋白。我们以人喉癌培养细胞ＨｅｐＩＩ作为对照材料,以两种ＡＣＡ血清和ＣＥＮＰ－Ｂ单抗,多抗以及ＣＨＯ动粒蛋白单抗为探针,用间接免疫荧光和免疫印迹技术对嗜热四膜虫作检查,免疫荧光结果表明,ＨｅｐＩＩ细胞的着丝粒抗原间期核中呈点状分布;与ＨｅｐＩＩ细胞的不同,嗜热四膜虫的着丝粒抗原在间期核中的分布不规则     

一种新的核仁组成区胶银染色技术

核仁组成区(Nucleolar OrganizerRegions,NORs)是位于细胞核仁内的DNA 环,具有 rRNA 基因,与 rRNA 的转录活性有关、在蛋白质合成中起重要的作用。遗传学家曾利用银染技术显示染色体核仁组成区来研究各种遗传缺陷性疾病。1986年 Ploton 建立丁一科改良的一步法胶银染色技术显示核仁组成区相关蛋白(Nucleoar Organizer Region Associa-ted Protein,AgNORs),虽然现在对这些蛋白染色的性质还不清楚,但该技术提供了一种快速检测核仁结构和活性改变的有效手段,已受到病理学界的高度重视,     

十一种灵长类染色体核仁组织者(N0Rs)的比较研究

核仁组织者区域(NORs)存在185+285核糖体核糖核酸(rRNA)的密码基因(rDNA),可用银染色技术特异性地显示出来。Schwarzacher等(1978),用光学显微镜和电子显微镜研究了银染色体的原理,证明银可染物质是活性NORs周围或外面的酸性蛋白,而不是基因本身。 许多学者先后以银染色技术,测定了狨猴(Saguinusoedipus,Saguinus fuscicollis,Callithrix jacchus)、恒河猴(Macaca mulatta)、食蟹猴(M.fasciculars)、狒狒(Papio papio)等猴子的核仁组织者区域(Bedard等,1978;Goodpasture等,1975;Dutrillaux等1979)。Tantravahi等(1976)对人、黑猩猩猿(Pan troglodytes)、大猩猩(Gorlla gorilla)、猩猩(Pongo Pygmaeus)、银色长臂(Hylobates moloch)进行了NORs的比较研究。 本文报告了我国灵长类动物5个属11个种染色体的银染色的NORs(Ag—NORs)分布,并作了初步比较。     

小鼠肿瘤细胞染色体核仁组织者的细胞化学观察

本文报道用Ag-AS染色技术对几种小鼠肿瘤细胞(Ehrlich腹水瘤,肉瘤180,淋巴瘤1号)核仁组织者(NORs)的观察结果,发现肿瘤细胞的NORs即18 S+28 S rDNA或核糖体基因的位置和大小与正常细胞的不同。正常小鼠细胞有3—6条染色体带有银染色的核仁组织者(Ag-NORs),分布位置都紧靠在着丝点下方;而三种小鼠肿瘤细胞都有一个中等大小的近端着丝点染色体,其Ag-NOR的位置移至长臂的中部。小鼠淋巴瘤1号     

用免疫荧光技术研究细胞周期中核基质的分布变化

本文利用含有抗核基质自发抗体的硬皮人血清,以小鼠艾氏腹水癌细胞为材料,用间接免疫荧光染色的方法,追踪了对应核基质抗原在细胞周期中分布的变化。结果显示,在末期和间期之间存在一个核基质抗原从细胞质向细胞核内转移的过程。由于这一过程是通过核膜进行的,从而提示核基质结构可能有解聚和再聚合的行为。用酶化学结合间接免疫荧光染色的方法,初步研究了抗原的化学性质。染色形态的比较研究显示所用血清中可能含有不同于以前发现的、抗新的核基质抗原的自发抗体。     

不同染色方法揭示纤毛原生动物草履虫接合生殖过程的新细节

通过石炭酸品红、Hoechst 33342、蛋白银及免疫荧光标记等染色方法对草履虫接合生殖过程进行了重新观察,结果发现:1)新月核是第一次减数分裂前期小核的主要形态学特征,在核内有一未被石炭酸品红、Hoechst 33342着色区域,蛋白银染色则清楚显示该结构;2)4个单倍体减数分裂产物中的1个核进入口旁锥完成配前第三次核分裂,其余3核退化.蛋白银染色和抗α微管蛋白单克隆抗体进行免疫荧光标记显示,核进入口旁锥的时期在第二次减数分裂末期而非减数分裂结束后;3)配前第三次分裂末期,核间连丝的中间段有一被蛋白银识别的结构,但免疫荧光标记却无显示,只表现为纤维状结构与两侧核间连丝相连.观察结果为草履虫接合生殖过程中相关分子生物学机制研究奠定了必要的形态学基础.     

细胞共养和BrdU对人和小鼠NORs活性的影响

不同种类细胞的共养可引起不同种细胞间的代谢协同现象,即在细胞相互接触的地方进行细胞间分子交换。它可降低姐妹染色单体互换频率、促进细胞DNA合成、细胞代谢缺陷互补和提高细胞对药物的敏感性。 核仁组织者(NORs)是细胞rDNA进行转录的地方,细胞银染NORs(Ag-NORs)数     

八种大鼠染色体银染核仁组织者的比较研究

本文对我国大鼠属(Rattus)8种鼠类的银染核仁组织者(NORs)进行了比较研究,结果表明:8种大鼠银染核仁组织者(Ag-NORs)的数目和分布均存在一定的差异,它们的NORs都呈现比较显著的多态性,并观察到了Ag-NORs的联合现象和异形现象。此外,将其银染核型差异数量化,通过模糊聚类分析,得到8种大鼠的聚类分析分支图,并结合它们的地理分布等对其分类地位进行了讨论。     

核仁组成区相关嗜银蛋白染色革新法

核仁组成区相关嗜银蛋白（ＡｇＮＯＲ）银染技术已广泛用以研究细胞生长活性,根据ＡｇＮＯＲ数目多少来判定肿瘤的良恶性和对肿瘤进行鉴别诊断。然而ＡｇＮＯＲ技术至今还存在背景非特异性银颗粒沉积问题而影响结果判定。本研究发现影响背景染色结果的主要因素是明胶的质量,采用Ｆａｒｍｅｒ’ｓ液可以清除背景染色,运用微波炉染色不仅可以缩短染色时间而且可以减少银用量。     

丁酸钠对人胃腺癌MGC-803细胞核仁纤维中心和银染蛋白的影响

用透射电镜观察到MGC-803细胞的核仁是网织型的,在网眼内分布有电子密度低的纤维中心。MGC-803细胞经丁酸钠作用后,其核仁的类型发生了改变,多呈环型的,核仁的中央有一个大的纤维中心;纤维中心和银染颗粒的大小和数目明显减低;用图像分析仪测得核仁银染蛋白所占面积与核总面积的比值也明显降低。结果提示:丁酸钠可能通过抑制rRNA合成和rDNA转录活性调控MGC-803细胞的增殖。     

Silver staining of the nucleoli of pig embryonic kidney cells during the hyperactivation and inhibition of the synthesis of nucleolar RNA by UV microirradiation

Silver staining of the nucleoli in pig embryo kidney cells (PK) was studied during the cell cycle and also upon mature nucleoli modifications induced by UV microirradiation. During anaphase only four silver-stained granules were revealed in each daughter set of chromosomes in the four nucleolus-organizing regions (NORs). In the following 1-2 hours, the number of granules in the NORs rapidly increased up to 25-30 per nucleus. During the next 20-25 hours of the cell cycle, the number of silver-stained granules was slowly doubling as the nucleoli grew in size. UV microirradiation of one nucleolus in the nucleus with two nucleoli induced a profound degradation of the injured nucleolus and a compensatory hypertrophy of the intact one. Such nucleolar modifications were accompanied by redistribution of the silver-stained granules between the injured and non-injured nucleoli and by alterations in the levels of nucleolar RNA synthesis in the NORs. These data support a hypothesis that silver-stained proteins may be involved in the regulation of the nucleolar activity.     

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.     

Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide

A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins in the RB were differentially extracted with acid, without any decrease in nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of Euplotes macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. An abundance of thiol groups was also demonstrated in the RBs and nucleoli by the fluorochrome 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin (coumarin maleimide). Histochemical studies, including blocking thiols with N-ethyl maleimide (NEM), indicated that thiols were not necessary for silver staining, and that proteins in the RBs and nucleoli reacting with coumarin maleimide were not acid extractable.     

Nucleolar behavior during meiosis in four species of phyllostomid bats (Chiroptera, Mammalia)

We analyzed the behavior of the nucleolus, nucleolar structures and nucleolus organizer regions (NORs) during meiotic division in four species of phyllostomid bats that have different numbers and locations of NORs. Nucleoli began disassembly at leptotene, and the subcomponents released from the nucleolus were dispersed in the nucleoplasm, associated with perichromosomal regions, or they remained associated with NORs throughout division. In Phyllostomus discolor, a delay in nucleolus disassembly was observed; it disassembled by the end of pachytene. The RNA complexes identified by acridine orange staining were observed dispersed in the nucleoplasm and associated with perichromosomal regions. FISH with rDNA probe revealed the number of NORs of the species: one NOR in Carollia perspicillata, one pair in Platyrrhinus lineatus and P. discolor, and three pairs in Artibeus lituratus. During pachytene, there was a temporary dissociation of the homologous NORs, which returned to pairing at diplotene. The variation in the number (from one to three pairs) and location of NORs (in sex or autosomal chromosomes, at terminal or interstitial regions) did not seem to interfere with the nucleolar behavior of the different species because no variation in nucleolar behavior that could be correlated with the variation in the number and chromosomal location of NORs was detected.     

Nucleolin is an Ag-NOR protein; this property is determined by its amino-terminal domain independently of its phosphorylation state.

The Ag-NOR proteins are defined as markers of "active" ribosomal genes. They correspond to a set of proteins specifically located in the nucleolar organizer regions (NORs), but have not yet been clearly identified. We adapted the specific detection method of the Ag-NOR proteins to Western blots in order to identify these proteins. Using a purified protein, Western blots, and immunological characterization, the present study brings the first direct evidence leading to the identity of one Ag-NOR protein. We found that nucleolin is specifically revealed by Ag-NOR staining. Using different nucleolin fragments generated by CNBr cleavage and by overexpression in Escherichia coli, we demonstrate that the amino-terminal domain of nucleolin and not the carboxy-part of the protein is involved in silver staining. Moreover, as the pattern of staining does not vary using casein kinase II- and cdc2-phosphorylated nucleolin or dephosphorylated nucleolin, we conclude that the reduction of the silver ions is not linked to the phosphorylation state of the molecule. We propose that the concentration of acidic amino acids in the amino-terminal domain of nucleolin is responsible for Ag-NOR staining. This hypothesis is also supported by the finding that poly L-glutamic acid peptides are silver stained. These results provide data that can be used to explain the specificity of Ag-NOR staining. Furthermore, we clearly establish that proteolysis of the amino-terminal Ag-NOR-sensitive part of nucleolin occurs in vitro, leading to the accumulation of the carboxy-terminal Ag-NOR-negative part of the protein. We argue that this cleavage occurs in vivo as already proposed, bearing in mind that nucleolin is present in the fibrillar and in the granular component of the nucleolus, whereas no Ag-NOR staining is observed in the latter nucleolar component.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

Ultrastructural localization of Ag-NOR stained proteins in the nucleolus during the cell cycle and in other nucleolar structures

EM investigation of Ag-AS-NOR staining after short glutaraldehyde prefixation followed by Carnoy fixation maintained good ultrastructural preservation and reactive selectivity. This enables exact localization of silver deposits both in the fibrillar centers of typical or segregated nucleoli during interphase, and in chromosome NORs during mitosis. These results argue in favour of the possibility that fibrillar centers are the interphasic counterpart of chromosome NORs. Special structures such as nucleolar blobs and remnants usually considered to be of nucleolar origin, were also stained. — These findings seem to indicate a relationship between the distribution of the silver-stained proteins, the arrangement of the nucleolar structures and the degree of nucleolar activity resulting from the experimental conditions. These results are of interest at the time when the concept of the nucleolar matrix is gradually emerging.     

Silver staining,immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23

Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.     

Nucleolar assembly of the rRNA processing machinery in living cells

To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein-tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time-lapse microscopy was used. In early telophase, fibrillarin is concentrated simultaneously in prenucleolar bodies (PNBs) and NORs, whereas PNB-containing Nop52 forms later. These distinct PNBs assemble at the chromosome surface. Analysis of PNB movement does not reveal the migration of PNBs towards the nucleolus, but rather a directional flow between PNBs and between PNBs and the nucleolus, ensuring progressive delivery of proteins into nucleoli. This delivery appeared organized in morphologically distinct structures visible by electron microscopy, suggesting transfer of large complexes. We propose that the temporal order of PNB assembly and disassembly controls nucleolar delivery of these proteins, and that accumulation of processing complexes in the nucleolus is driven by pre-rRNA concentration. Initial nucleolar formation around competent NORs appears to be followed by regroupment of the NORs into a single nucleolus 1 h later to complete the nucleolar assembly. This demonstrates the formation of one functional domain by cooperative interactions between different chromosome territories.