Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.     

Proteins C23 and B23 are the major nucleolar silver staining proteins.

To examine the silver staining proteins of Novikoff hepatoma nucleoli, the nucleolar proteins were separated on two-dimensional polyacrylamide gels with an isoelectric focusing first dimension and an acid-urea gel second dimension. The nucleoli were sequentially extracted with (1) 0.6 M potassium acetate, pH 5.5 and (2) 2 M potassium acetate — 5 M urea — 10 mM Tris, pH 7.5. The silver staining method used for the detection of silver binding proteins in gels was similar to that used to stain the nucleolar granules on microscope slides. Two major silver staining proteins were found which were identified as (molecular weight × 10^?3/pI) proteins C23 (100/5.3) and B23 (37/5.1). These two proteins are the major acidic proteins in Novikoff hepatoma nucleoli.     

Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level

Summary The argyrophilic proteins of the nucleolar organizer region (Ag-NOR proteins) were specifically localized at the optical level with a modified one-step silver technique performed at 20° C.This method was applied to various materials including cells in smears, chromosomes, semi-thin sections of plastic-embedded cells and sections of paraffin-embedded human pathological tissues.In order to improve the visualization of the silver deposits we tested various modes of imaging, including bright-field, Nomarski contrast, reflected light and combined Nomarski contrast with reflected light. The use of Nomarski contrast is useful to define precisely the phases of mitosis. The use of reflected light, which is based on the ability of silver to reflect incident light specifically, gives images with an improved resolution compared to bright-field.     

A further study on nucleolar silver stained granules in some mononuclear-lymphoid bone marrow cells

Nucleolar silver stained granules (SSGs) representing nucleolus organizer regions (NORs) were investigated in human as well as rabbit bone marrows after visualization with standardized silver reaction for non-histone nucleolar argyrophilic proteins. The results indicated that few mononuclear lymphoid blast-like cells in investigated bone marrows are characterized by large irregularly shaped nucleoli which contain a larger number of SSGs than myeloblasts or proerythroblasts as well as immature or stimulated lymphocytes. Since according to previous studies the number of nucleolar SSGs decreased in the course of the erythroid, granulocytic and lymphocytic differentiation and maturation, a possibility exists that the described mononuclear lymphoid blast-like cells are even less differentiated and immature than committed stem cells for mentioned cell lines.     

Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining

A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.     

A quantitative study of Ag-positive nucleolar segments revealed by silver staining in the interphase nuclei of trophoblast cells from the rat placental connective zone

A cytomorphological study was made of silver stained nucleoli in interphasic nuclei of trophoblast cells from the rat placenta connective zone, in addition to calculation of Ag-positive spherules in the nucleoli. The prevalent number of Ag-positive nucleolar spherules in the nuclei was 6, corresponding to the number of nucleolar organizers (NOR's) in the diploid chromosome complement of the rat. The mean number of Ag-positive spherules in the nucleoli progressively increase in the course of polyploidization from 2c to 32c; variability of the spherule number also increasing. The mean area of nucleoli is found to increase in proportion to the ploidy degree. A high correlation is found between the number of Ag-positive spherules and the area of nucleoli in the nucleus (r = 0.78). This appropriateness is exhibited at all the ploidy levels. The number of Ag-spherules and the area of nucleoli are found to depend slightly on the number of nucleoli. The possibility to use the number of Ag-positive spherules as a criterion of the activity of the NOR in interphasic nuclei is discussed.     

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.     

Sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells

A method for sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells is described. Feulgen staining is done first and nuclear DNA estimated by absorption cytophotometry. Following this, the slides are stained with AgNO3. The method has been used to study the process of nucleolar fusion in garlic (Allium sativum L.) meristem root tip cells. It was found that during interphase nucleoli rarely fused, thus most fusion must have occurred before the G1 phase of the cell cycle.     

A combined staining method for argyrophilic nucleolar organizer regions and for glial fibrillary acidic protein in astrocytes of human brain

Summary Different protocols are described for the combined staining method by which argyrophilic nucleolar organizer region sites can be evaluated in human astrocytes that are immunoreactive for glial fibrillary acidic protein. Among the four protocols studied, the following method was superior to others in terms of unambiguous visualization of the regions in glial fibrillary acidic protein-positive astrocytes; the first step was immunostaining for the protein with a blue colour reaction of alkaline phosphatase, followed by sequential colloidal silver staining for the regions. By this double staining method, we have demonstrated that the reactive astrocytes found in white matter around the metastatic lesion of carcinoma and the infarction, contain more argyrophilic nucleolar organizer regions in terms of the count as well as the area than glial fibrillary acidic protein-positive astrocytes present in the white matter of the normal brain. In conclusion, the double staining may provide valuable information on the cellular activity of astroglia when performed on routine formalin-fixed paraffin sections of the human brain.     

Correlation of nucleolar activity and nucleolar vacuolation in plant cells

In root meristematic cells nucleolar structure varies with the cell cycle. Apart from normal meristematic nucleoli one finds nucleoli with a big central vacuole surrounded by a loose cortex with individual fibrillar centres [22] clearly visible within it. There are also intermediate structures between both nucleolar types. In Pisum sativum nuclear tissue, the structure of the vacuolated nucleoli is similar and appears in periods of high metabolic activity during megasporogenesis. In both tissues, vacuolated nucleoli incorporate tritiated uridine more actively than 'normal' nucleoli. In this work the structure of spontaneous nucleolar vacuoles is compared with that induced by drugs such as cordycepin, and FUdR. The vacuolated nucleolus with its increased surface corresponds to a transient structure which not only shows higher metabolic activity but also supplies a storing and/or transporting mechanism for nucleolar products.     

Organization of argyrophilic nucleolar material throughout the division cycle of meristematic cells

Summary The silver impregnation of nucleolar material facilitated the study of the morphological changes which take place in the nucleolus throughout the division cycle in root tip cells ofAllium cepa. The nucleolus appears to undergo no morphological changes throughout the interphase. It undergoes disorganization during the prophase, while in the telophase it appears uniformly on the chromatin as condensing into prenucleolar bodies.The appearance of the prenucleolar bodies is unaffected by puromycin, cordycepin, or ethidium bromide. This suggests that the argyrophilic material does not undergo synthesis during the telophase, nor require RNA or protein synthesis to effect the aggregation into prenucleolar bodies. However, the organization of nucleoli from prenucleolar bodies is inhibited by both cordycepin and ethidium bromide, suggesting that RNA synthesis is involved in this proccess.In aneuploid nuclei induced by treatment with colchicine we observed the appearance of prenucleolar bodies during the telophase even in the absence of the nucleolar organizer, but in this case the formation of nucleoli fails to take place. The nucleolar organizers proved to be capable of acting only in the nucleus to which they belong, but not on other nuclei within the same cytoplasm belonging to multinucleate cells.It seems logical to assume that one of the roles of the nucleolar organizer is related with the above-mentioned RNA synthesis, which is required to the aggregation of prenucleolar bodies into nucleoli.The work reported in the paper was undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer.     

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.     

Non-ribosomal nucleolar proteins in HeLa cells

The study of nucleolar macromolecular components has previously emphasized ribosomal precursor and mature RNA, or ribosomal structural proteins. In this work, we have stressed the purification of nucleoli, and have studied their protein composition. The results indicate that there exists in highly purified nucleoli of HeLa cells, a class of high molecular weight polypeptides which have been identified by means of size, kinetics of turnover, and metabolic behavior to be non-ribosomal nucleolar-specific proteins. The possibility that these proteins play a part in the assembly of mature ribosomes is discussed.     

A technique for the simultaneous staining of both nucleolar organizer regions and kinetochores of human chromosomes with silver.

Pretreatment of human metaphase chromosomes with NaOH at a pH of 8.5, followed by staining with silver nitrate, differentially stains both the nucleolar organizer regions on the 10 acrocentric chromosomes as well as the kinetochore centers on all 46 chromosomes.     

An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes

Summary A reliable technique for staining human chromosomal nucleolar organizers (NOR's) with silver solutions is described. The NOR's can be selectively stained dark brown by silver solutions leaving the chromosome arms unstained and available for counterstaining with orcein or Giemsa dyes. Unequivocal identification of chromosome pairs bearing NOR's can be achieved using fluorescent banding techniques followed by silver staining. The silver staining procedure for NOR's was simplified and standardized through control of the chemical and physical conditions during silver impregnation and developing.     

A method for silver staining HMG chromosomal proteins in polyacrylamide electrophoretic gels

A method is presented for sensitive staining of the HMG14 and 17 proteins in polyacrylamide gels pre-stained with Coomassie Blue R250. The procedure involves binding negatively and positively charged polycyclic aromatic compounds to the proteins followed by staining with silver using the method of Wray et al. (1981).     

Selective staining of the same set of nucleolar phosphoproteins by silver and Giemsa

Gel electrophoresis of nucleolar isolates from Zajdela ascites hepatoma cells followed by various staining procedures revealed a common set of bands that stained selectively with silver and Giemsa. The gel bands, corresponding to molecular weights of 104, 78, 37, and 29 kilodaltons (kd), appeared to contain phosphoproteins that were at least partly associated with oligo-deoxyribonucleotides. Enzyme digestion studies showed that the Giemsastainability was due to the phosphorylated state of the proteins. The positive selective silver-staining reaction in gels could be most likely attributed to the high content of carboxyl groups present in these phosphoproteins. The significance of these findings in relation to cytological results produced by selective silver staining of nucleolus organizing regions (NORs) and by Giemsa N-banding is discussed.