Silver-stained nucleolar organizer proteins in chondrosarcoma.

Silver-stained nucleolar proteins (AgNORs) were counted in primary chondrosarcomas of three histologic grades and in metastatic chondrosarcomatous lesions in the lung. The AgNOR numbers of neoplastic cells in primary tumors increased stepwise from grade 1 (4.42 +/- 1.11) through grade 2 (4.94 +/- 1.31) to grade 3 (6.97 +/- 1.10). There was a significant difference in AgNOR numbers between grade 3 and both grades 1 and 2 (p less than 0.001). Furthermore, the mean number of AgNORs in metastatic lesions (9.75 +/- 0.83) was significantly higher than that in primary sites (p less than 0.001). The number of AgNORs therefore reflects the grade of the chondrosarcoma. The results in the present study indicate that silver colloid staining is a useful technique for determining the histologic grade and evaluating the proliferative activity of chondrosarcomas.     

Evaluation of nucleolar organizer region-associated proteins in endometrial pathology.

In the current study the argyrophil staining technique for NOR proteins (Ag-NORs) has been performed on cases of different endometrial lesions, trying to find an aid in differentiating atypical hyperplasia from well differentiated carcinoma in biopsy specimens. We conclude that the Ag-NOR count, even though in endometrial carcinoma is significantly exceeding that of atypical hyperplastic endometrium, could be a misleading discriminator, because of a wide overlap of values in individual cases.     

一组新的核仁组织者区结合蛋白

With the scleroderma autoantinucleolar antiserum which was discribed in our previous paper, we identified a novel group of nucleolar organizer associating proteins, ANOP. By means of immunoblotting, it was found that ANOP mainly consists of three polypeptides with molecular weights of 65, 76, and 78 KD respectively. Immunoelectron microscopy demonstrated that ANOP is localized in the shells of dense fibril components surrounding fibrillar centers of nucleolus. Moreover, indirect immunofluorescence studies on various cells of vertebrate animals indicated that ANOP is a group of highly conserved nucleolar antigens. The fibrillar centers of human, mouse, chinese hamster, chicken, frog and crucian carp cells can all be brightly stained by ANOP specific antiserum. However, it was found that in amphibian, the erythrocytes and the early embryo cells prior to gastrulation are devoid of ANOP. During gastrulation, ANOP begins to appear in the nuclei, and then, the content of ANOP in nucleoli apparently increases gradually. These phenomena suggest that ANOP may take a role in rRNA gene activity.     

Argyrophilic nucleolar organizer regions

Silver staining techniques developed to demonstrate argyrophilic nucleolar organizer regions (Ag-NORs) have been widely applied in a variety of cell kinetic studies, using the mean number of AgNORs in tumour cells as a marker for malignancy of certain types of neoplasms. However, the AgNOR techniques currently available are not entirely satisfactory, as unspecific silver precipitates readily form in the sections. On the other hand, the contrast staining, may be so weak as to render identification of the AgNORs difficult. In the present study, some of the key factors influencing the outcome of AgNOR staining were evaluated in a more systematic way. A modified AgNOR staining procedure is now proposed, giving highly contrasting AgNORs with minimal unspecific silver precipitation, thus facilitating both manual and computerized counting. The new technique involves the use of microwave irradiation in order to shorten the processing time, the use of gelatin as a protective colloid, and a Farmer's solution to optimize the specificity of the technique.     

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.     

Digital image analysis of silver-stained nucleolar organizer region--associated proteins in endometrial cytologic samples

OBJECTIVE: Digital image analysis was applied to determine the number, area and size of silver-stained nucleolar organizer regions (AgNORs) in cytologic samples from curettage in normal, hyperplastic and malignant endometrium. STUDY DESIGN: Thirty-two archival cytologic smears from curettage (previously stained by the Papanicolaou method) with the histologic diagnosis (4 inactive endometrium, 5 secretion, 5 proliferation, 5 simple hyperplasia, 5 complex hyperplasia, 3 atypical hyperplasia, 5 adenocarcinoma, grade 1) were analyzed with the AgNOR technique. Count, area and size of AgNORs were analyzed in 50 cells per sample using a magnification of 1,000x. Quantitative analysis was performed on an SFORM digital imaging system. Data were analyzed with the SPSS/PC+ program. Mann-Whitney and chi 2 tests were performed. RESULTS: The average value of AgNOR count increased from normal to hyperplastic endometrium and well-differentiated adenocarcinoma. Differences were significant except between atypical hyperplasia and adenocarcinoma. Four, five and more AgNORs in 40% or more of the nuclei were found in complex and atypical hyperplasia and adenocarcinoma. Proliferation, and simple and atypical hyperplasia had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic and well-differentiated adenocarcinoma. Differences were statistically significant. AgNOR size in well-differentiated adenocarcinoma was significantly different from that in normal endometrium and different grades of hyperplasia. CONCLUSION: Digital image analysis of AgNOR count, area and size enabled a distinction to be made between normal, hyperplastic and malignant endometrium.     

Polymorphisms of Ag-stained nucleolar organizer regions in man

Summary Polymorphisms of the NORs as tested by Ag-staining of metaphase G-banded chromosomes were investigated in cultured blood lymphocytes of karyotypically normal individuals from the Moscow population.The study of cell-to-cell variability in the number of Ag-stained NORs carried out on 14 monozygotic twin pairs showed the phenomenon to have some features of real intercellular variation.In 40 unrelated individuals the individual acrocentric chromosomes were compared by the number of Ag-stained NORs, their degree of staining, and their participation in acrocentric association. Chromosome 21 was found to be significantly more active than four others by all the criteria, and chromosome 15 was less active compared with the others by the size of the Ag deposits and the frequency of participation in NOR associations. The frequency distribution of homozygotes and heterozygotes for Ag-stained NORs in the same group of 40 individuals was in accordance with the Hardy-Weinberg law.     

Human nucleolar organizer chromosomes: satellite associations

The D and G group chromosomes from cultured human lymphocytes exhibit single and multiple satellite associations when stained with silver. Unlike earlier methods this simple and highly repeatable procedure shows physical attachments between satellited regions of various acrocentric autosomes. After studying 1,000 satellite associations from 118 normal individuals, it was found that both single and multiple associations occur with frequencies that correlate with random expectancies.     

Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D.

Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10(-2) micrograms/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10(-2) micrograms/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10(-1) micrograms/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.     

Importance of interphase nucleolar organizer regions in tumor pathology

The importance of the distribution of silverstained nucleolar organizer regions (Ag-NORs) in interphase nuclei for diagnostic and prognostic purposes in tumor pathology has been reviewed. The available data demonstrated that interphase Ag-NOR evaluation may be of help in distinguishing malignant from hyperplastic or normal cells. On the other hand, there is increasing evidence that a relationship exists between the quantity of interphase Ag-NORs and the prognosis of malignant tumors: the greater the number of interphase Ag-NORs, the worse is the prognosis. This can be explained by the observation that the interphase Ag-NOR quantity is strictly related to the cell proliferation rate. The procedures used for the measurement of the interphase Ag-NOR quantity are also critically discussed.     

Equivalence of nucleolar organizer activity among primate species

The number of ribosomal DNA (rDNA)-containing chromosomes and the amount of total rDNA vary widely among primate species. In spite of numerical variations, the mean number of ribosomal (rRNA) genes that participate in nucleolar organization is approximately the same.     

Silver staining of nucleolar organizer region associated proteins using polyethylene glycol as the protective colloidal developer

Summary A simple modification to the silver staining technique for the demonstration of nucleolar organizer region associated proteins is described. Polyethylene glycol 20 000 is used instead of gelatin as the colloidal developer. This modified technique remains a one stage procedure that is quick and easy to perform. It results in reduced precipitate and less non-specific staining with specimens in which it had been previously difficult to demonstrate these intranuclear silver staining structures.     

Cytochemical aspects of the nucleolar organizer in Allium cepa microspores

In Allium cepa mierospores, the nucleolar organizing region appears as an area of low density situated between two nucleolar masses. It consists of a series of zones with a density similar to that of the chromatin surrounded by areas of lower density. The dense zones are sometimes arranged in an orderly pattern of 2–4 rows. The organizing region consists of filaments, about 100 Å in diameter, which are seen to be concentrated in the dense areas, and more scattered in the rest of the region. — The alcoholic PTA staining technique reveals the presence of an appreciable quantity of arginine-Iysine rich histones in the organizing region, as well as a similarity between the dense areas of this region and the rest of the chromatin: a similarity which is also brought out by the thallium alcoholate technique, used for DNA staining. By means of uranyl-BDTA-lead RNP material can be shown to be predominantly located in the low-density areas of the organizing region in the form of fibres of about 80 Å in diameter, presumably representing the newly synthetized r-RNA (45 S RNA). A pattern is suggested for the organizing region, in which areas of functional chromatin (euchromatin) would appear alternating with areas of non-functional chromatin (heterochromatin).     

Dimorphic nucleolar organizer regions in the frog Rana blairi.

The nucleolar organizer-specific staining procedure, ammoniacal silver (Ag-AS), has been used to study the distribution and size of the nucleolar organizer regions (NORs) in chromosomes of the frog Rana blairi (Mecham, Littlejohn, Oldham, Brown and Brown). The somatic metaphase karyotype of this frog is similar to that of other frogs of the Rana pipiens species complex, numerically (2n=26) and morphologically. Secondary constrictions are detectable in untreated Giemsa-stained metaphase preparations as achromatic gaps in the long arms of a pair of submetacentric chromosomes (no. 10). These constrictions are the only regions which are deeply stained with the Ag-AS method and are thus identified as the nucleolar organizer regions (Ag-NORs). In each of the three individuals, the Ag-NORs as visualized on the homologues are of unequal length.     

Analysis of Y chromosome nucleolar organizer mutants in Drosophila melanogaster

Five Y chromosome nucleolar organizer (Y-NO) mutants were analyzed with respect to their rRNA gene numbers, phenotypes and additivity tests with other NO mutants. Four of these are indicative of a class of mutants in which most of the rRNA genes are transcribing functional rRNA. The other mutant has 80 genes, however, lethality and additivity tests suggests that many if not all of these rRNA genes are non-functional. The basis for the observed suppression of rRNA genes of the Y-NO region is discussed.     

Population polymorphism of silver-stained nucleolar organizer chromosome regions in man

Activity of nucleolar organizer regions (NORs) of acrocentric chromosomes determined by Ag-staining was comparatively studied in 40 individuals of Bulgarian and 40 individuals of Russian populations. Chromosome 21 was found to be significantly more often stained in both populations. The other NORs did not differ significantly in staining from the means. No differences were noted between individual NORs, in respect of the intensity of Ag-staining in both populations, except chromosome 15 which showed markedly decreased staining capacity in Russians. The data obtained are compared with those published in literature concerning four other populations.     

Polymorphism of nucleolar organizer regions of chromosomes in human embryos

NOR activity in metaphase chromosomes from extraembryonic and embryonic tissues of 9-12 week human fetuses was studied after standard silver staining. Significant interidividual variations in the average cummulative NOR activity was assessed by means of one-factor dispersion analysis. No significant intertissue fluctuation of NOR activity was found. Total number of NOR+ chromosomes demonstrated no correlation with the embryonic age. Steady growth of an average cummulative NOR activity respective of progressive embryonic age was proven by correlation analysis method. Unequal participation of NOR-bearing chromosomes of D- and G-groups during early embryonic development in human was shown.