The relationships between ribosomal genes and fibrillar centers in thyroid cells cultivated in vitro

Despite the fact that the fibrillar centers of the nucleolus and the chromosomal nucleolar organisers (NORs) are similarly stained with the NOR-silver technique, there remain some questions about the identification of fibrillar centers as NORs. The distinct delineation of the fibrillar centers in porcine thyroid cells allowed us to determine whether there was a numerical equivalence or correlation between fibrillar centers and NORs. Hybridization in situ and silver staining performed on pig chromosomes showed that pairs 8 and 10 contained rDNA sites. Silver staining of thyroid cells in electron microscopy showed that the fibrillar centers and their surrounding layer of dense fibrils were the sites of silver deposit. Chromatin fibers were demonstrated within the fibrillar centers through the aid of the osmiumammine reaction and with the oxidized diaminobenzidine technique. It was observed that in cultured thyroid cells the fibrillar centers could be identified in the light microscope as argyrophilic spherules, and easily counted. The number of fibrillar centers was variable according to culture conditions. In cells cultured for 5 hr, the mean number of fibrillar centers was 1.7. After 5 days of culture, the number of fibrillar centers increased, reaching a mean value of 5.93. When thyroid cells were stimulated with thyrotropin, the number of fibrillar centers again increased to a mean value of 7.54. These results demonstrate that the relationship between fibrillar centers and NORs is not a simple proportionality: the number of fibrillar centers increases with increased cellular activity. These data imply that in active cells each NOR may pass through several fibrillar centers.     

The morphological relationship in electron microscopy between NOR-silver proteins and intranucleolar chromatin

The relative distribution of NOR proteins and chromatin fibers in the nucleoli was visualized in human cell line. The chromatin was revealed by a Feulgen-like procedure using osmium-ammine as DNA tracer. This selective staining was combined with NOR-silver staining. We provide morphological evidence for constant overlapping of the silver deposit sites with dispersed intranucleolar chromatin fibers. Silver stained proteins were sometimes observed in contact with the chromatin fibers, suggesting that at least some of the Ag-NOR proteins might be closely connected with the dispersed nucleolar DNA.     

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.     

Cytochemical variations in the nucleolus during spermiogenesis in man and monkey

Summary The fine structure, nature and fate of the components of the nucleolus were studied in young (steps 1, 2), intermediate (steps 3, 4, 5) and mature spermatids (steps 6, 7, 8) of man and monkey, by use of several cytochemical techniques (alcoholic PTA; sodium tungstate; EDTA; HAPTA; nuclease-gold complexes; NOR silver staining). As controls, comparative ultrastructural and cytochemical observations of the nucleolus in spermatids and Sertoli cells were made in the same sections of seminiferous tubules. In the young spermatids of the two species studied, the nucleolar masses exhibited identical features. Segregation of the nucleolar components took place in the nuclei of step 1 spermatids. No typical fibrillar center was observed. In spermatids at steps 1 and 2, the nucleolar masses appeared to be made up of two fibrillar components of equal density, one spherule-shaped, the other forming cords, both surrounded by clusters of 15–20 nm-diameter granules. Alcoholic PTA and sodium tungstate yielded a selective positive contrast of the two fibrillar components whereas EDTA and RNase-gold reacted with the peripheral granular material. Treatment with RNase-gold and DNase-gold complexes resulted in preferential labeling at the periphery of the fibrillar components. After NOR silver staining, numerous small silver grains were localized over the fibrillar cords, suggesting the persistence of specific acidic non-histone proteins. On the contrary, the spherule was never stained. In intermediate spermatids, when the nucleolar components were dissociated, scattered clusters of granules stained by EDTA and HAPTA remained in the entire nucleoplasm. Nucleolar disintegration was accompanied by dispersion of argyrophilic material. In mature spermatids granular material revealed by PTA and silver staining methods was found in the nuclear pockets bounded by the redundant nuclear envelope.     

Evidence from studies on segregated nucleoli that nucleolar silver staining proteins C23 and B23 are in the fibrillar component

Several procedures for the silver staining of nucleoli have been evaluated at the electron microscopic level to determine optimal conditions for ultrastructural preservation and staining specificity. The present study shows that a brief fixation with 1% buffered formaldehyde followed by methanol: acetic acid (3 : 1) fixation yielded optimal preservation and silver staining of nucleoli. Using this procedure for electron microscopic studies of interphase nucleoli, it was found that the punctate silver grains observed by light microscopy were composed of fine silver granules, of approx. 100 Å diameter, organized in discrete clusters. In similar studies on adriamycin-induced segregated nucleoli, it was observed that the silver staining reaction was mainly limited to the fibrillar portion of the nucleolus. Accordingly, nucleolar proteins C23 and B23, found earlier to be the major silver binding proteins of the nucleolus, are mainly concentrated in the fibrillar nucleolar component.     

Phosphoprotein pp135 is an essential component of the nucleolus organizer region (NOR)

The association of phosphoproteins pp135 and pp105 with distinct substructures of the nucleolus was studied by cytochemical and immunological methods at the light microscopic and electron microscopic level. Both phosphoproteins exhibited a very high affinity for silver and Giemsa staining compared to other nucleolar proteins. Immunolocalization of pp135 and pp105 during mitosis by light microscopy revealed a tight association of pp135 with the silver staining nucleolus organizer region (NOR), whereas pp105 (cross-reacting with C23) appeared to be only partially associated with the NOR, exclusively at telophase. At the immunoelectron microscopic level the distribution of pp135 and pp105 was investigated in interphase nucleoli. Phosphoprotein pp135 was located in the fibrillar shell and pp105 in the fibrillar shell and the granular zone. The fibrillar centers were essentially free of both phosphoproteins..     

Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.     

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.     

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.     

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.     

AgNOR proteins from morphologically intact isolated nucleoli.

AgNOR staining has been proposed as a useful tool for the diagnosis and prognosis of cancer. The AgNOR proteins, however, have not yet been clearly identified and characterized, possibly due to the partial character of the results obtained when studying the proteins extracted from altered nucleoli isolated by "standard" methods. In the present study, we analysed, on western blots, the AgNOR staining profiles obtained with protein extracts from Ehrlich tumor cell nucleoli isolated by a recent procedure that preserves the nucleolar ultrastructure. In addition to the well-known C23 and B23 protein bands, we readily detected an extra band at approximately 125 Kda. By immunoblotting, we showed that this polypeptide may be related to the nucleolar phosphoprotein pp135 evidenced in rat-cell nucleoli. By immunoelectron microscopy, we detected this protein in the dense fibrillar component and fibrillar center of the nucleoli as well as the coiled bodies. The distribution coincides with the cytochemical AgNOR staining pattern obtained at the ultrastructural level.     

Distribution of nucleolar proteins B23 and nucleolin during mouse spermatogenesis

The intracellular distribution of nucleolar phosphoproteins B23 and nucleolin was studied during mouse spermatogenesis, a process that is characterized by a progressive reduction of nucleolar activity. Biochemical analyses of isolated germ cell fractions were performed in parallel with the in situ ultrastructural immunolocalization of these two proteins by means of specific antibodies and colloidal gold markers, and by silver staining. RNA blot experiments showed that mRNA for nucleolin progressively decreased during spermatogenesis whereas mRNA for B23 increased in amount during early spermatogenic stages. Immunoblotting confirmed that both proteins were present during early spermatogenesis up to the round spermatid stage and absent from mature sperm. Immunoelectron microscopy revealed that in spermatogonia, leptotene and pachtyene spermatocytes, and in Golgi phase spermatids, B23 and nucleolin were localized in the dense fibrillar component and granular component of the nucleolus but not in the fibrillar centers. In the dense fibrillar residue of the cap phase spermatids, labeling with anti-nucleolin but not with anti-B23 was observed. During nucleolar inactivation, neither of the two polypeptides was dispersed to the nucleoplasm. Silver salts stained the fibrillar centers and dense fibrillar component but not the granular component of the nucleolus. Our results suggest that there is no direct relationship between nucleolar activity and the occurrence of B23 and nucleolin or silver staining. Moreover, we confirm that silver staining and the presence of B23 or nucleolin are not directly related to each other.by M. Trendelenburg     

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.     

胃粘膜病变核仁组成区嗜银蛋白的银染计量和能谱分析

对77例胃粘膜活检标本进行银染,显示核仁组成区相关的嗜银蛋白(Ag-NOR),进行计数和统计分析,并对银染标本作电镜观察及能谱分析。结果表明,Ag-NOR 颗粒计数的多少,对胃肿瘤恶性程度的诊断有重要参考价值。电镜证实银染物质定位于核仁组成区,银染阳性区能谱分析显示 Ag 峰,说明银染反应具特异性.