Number Of Nucleolar Organizer Regions Research Articles

The RNA polymerase I transcription factor UBF and rDNA are located at the same major sites in both interphase and mitotic pig embryonic kidney (PK) cells.

Indirect immunolabeling with anti-UBF antibodies, in situ hybridization with an rDNA probe, and confocal scanning laser microscopy were used to study nucleolar organizer regions (NORs) during the cell cycle in pig embryonic kidney (PK) cells. The chromosomal distribution of the polymerase I transcription factor UBF and rDNA was compared with the number of silver-stained NORs (Ag-NORs) present and nucleolar size. It was shown, both at interphase and mitosis, that the majority of UBF and rDNA signals were located at the same foci and that the amounts of UBF and rDNA at any given site were in a striking positive correlation. At mitosis, only the NORs were labeled; at interphase, the signals for both UBF and rDNA were arranged in necklace-like structures around the nucleoli. No chromosomal NORs without Ag-proteins or UBF were present, indicating that all NORs in PK cells are active at interphase. It was concluded that (1) UBF and rDNA co-localize throughout the cell cycle in PK cells; (2) their association with mitotic NORs is determined by the number of rDNA repeats, rather than by any differential ability of NORs to recruit the transcription factor; and (3) the amount of UBF can be correlated with the size and activity of the nucleoli at interphase.

Ag-NOR studies in a human lymphocyte culture: are variants localized to specific chromosomes?

The genomic activity and polymorphisms of nucleolus organizer regions (NORs) were studied in an individual with routinely used AgNO3 and Q-banding techniques. The results demonstrate that (a) the mean number of NORs per metaphase spread was 7.4, (b) chromosomes 14, 13, 15, and 21 showed variations of Ag-NOR in decreasing order, (c) chromosome 22 did not show a polymorphism in any cell examined, and (d) both chromosomes 14 showed silver staining in most cells. Unlike many earlier reports indicating that NOR variants were constitutional in each individual, our present case represented a mosaic pattern of polymorphism involving most of the D- and G-group chromosomes.

Hypervariability of ribosomal DNA at multiple chromosomal sites in lake trout (Salvelinus namaycush).

Variation in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) of lake trout (Salvelinus namaycush) was examined. Digestion of genomic DNA with restriction enzymes showed that almost every individual had a unique combination of length variants with most of this variation occurring within rather than between populations. Sequence analysis of a 2.3 kilobase (kb) EcoRI-DraI fragment spanning the 3' end of the 28S coding region and approximately 1.8 kb of the IGS revealed two blocks of repetitive DNA. Putative transcriptional termination sites were found approximately 220 bases (b) downstream from the end of the 28S coding region. Comparison of the 2.3-kb fragments with two longer (3.1 kb) fragments showed that the major difference in length resulted from variation in the number of short (89 b) repeats located 3' to the putative terminator. Repeat units within a single nucleolus organizer region (NOR) appeared relatively homogeneous and genetic analysis found variants to be stably inherited. A comparison of the number of spacer-length variants with the number of NORs found that the number of length variants per individual was always less than the number of NORs. Examination of spacer variants in five populations showed that populations with more NORs had more spacer variants, indicating that variants are present at different rDNA sites on nonhomologous chromosomes.

Prognostic evaluation of HPV-associated precancerous and microinvasive Carcinoma of the Oral Cavity: Combined use of Nucleolar Organiser Regions (AgNOR) and Proliferating Cell Nuclear Antigen (PCNA)

Nucleolar Organiser Regions (NORs) and Proliferating Cell Nuclear Antigen (PCNA) were investigated on routine paraffin embedded histologic sections of 30 oral biopsy specimens (six cases of leukoplakia with low-degree of dysplasia, nine cases of leukoplakia with moderate-degree of dysplasia, nine cases of leukoplakia with severe-degree of dysplasia, six cases of squamous microinvasive carcinomas), tested for HPV-DNA by in situ hybridisation (ISH). The absolute number of NORs per nucleus and the percentage of nuclear positivity for PCNA were found to be different in each group of pathology, with further diversity due to the presence or absence of HPV-DNA. In the major part of HPV-positive lesions, the AgNOR number and percentage of cells positive for PCNA were found to be generally lower than in corresponding negative forms. Conversely, a few cases of HPV+ lesions showed significantly higher values both of AgNOR and PCNA, if compared to the other cases of HPV+ and HPV- lesions. These data suggested that high values of AgNOR and PCNA, in moderate and high grade oral dysplasia, could represent an "alarm signal" of a worse prognosis, and then a possible indication for a strict clinical management and/or a stronger treatment of some HPV-associated preneoplastic lesions.

A sensitive staining method for NORs.

A sensitive staining method for nucleolar organizer regions (NORs) is described, using blue toning of AgNORs. NORs are loops of DNA which are transcribed into ribosomal RNA. NORs can be demonstrated by staining with silver nitrate, since NOR-associated proteins are argyrophilic, producing structures termed AgNORs. Normal blood lymphocytes were stained with both methods. The number and resolution of NORs increased 2-3 times by blue toning (30 mmol/l FeCl3, 11 mmol/l potassium hexacyanoferrate(III), and 33 mmol/l oxalic acid) compared with silver staining. A significant difference in the number of NORs was noticed between silver-stained and blue-toned cells (P < 0.001). The blue toning technique thus appears to be more sensitive in detecting NORs than the AgNOR method and may prove a useful alternative for applications in histopathology.

Number of nucleoli and coiled bodies and distribution of fibrillar centres in differentiating Purkinje neurons of chick and rat cerebellum.

We used differentiating chick and rat Purkinje cells to investigate in homologous neurons the influence of the number of nucleolar organizer regions (two in the chick and six in the rat) on the behaviour of the nucleolus and coiled bodies. We employed specific silver-staining methods on smear preparations and on semithin and ultrathin sections. In chick Purkinje cells the number of nucleolar silver-staining granules increased from 15.7 +/- 3 (mean +/- SD) at embryonic day 13 to 23.8 +/- 3 at post-hatching day 7. These nucleolar granules were unevenly distributed between the two nucleoli of binucleolated cells. Electron-microscopic cytochemistry showed that nucleolar granules are equivalent to the fibrillar centres with their associated shell of dense fibrillar component. A reduction in the number of nucleoli was found during the differentiation of both chick and rat Purkinje cells, although in mature cells the average number of nucleoli per cell was higher in the chick (1.60) than in the rat (1.07). The number of coiled bodies decreased from 1.33 in newborn rats to 0.47 at postnatal day 90 in the rat. Coiled bodies were not observed in homologous chick Purkinje cells. The dynamic behaviour of nucleoli and coiled bodies during neuronal differentiation and the relationship of these two nuclear organelles with the number of nucleolar organizer regions is discussed.

Use of Bromodeoxyuridine, Proliferating Cell Nuclear Antigen, and Nucleolar Organizer Regions in the Multiparametric Assessment of Proliferating Potency in Cervical Intraepithelial Neoplasia

The authors analysed of the proliferative activity of intraepithelial neoplasia of the cervix utilizing the labeling index of monoclonal antibody to Bromodeoxyuridine (BrdU) or proliferating cell nuclear antigen (PCNA), and the number of Nucleolar organizer regions (Ag-NOR). The BrdU labeling index (B.L.I.) for normal squamous epithelium, mild dysplasia, and moderate dysplasia were significantly lower than for severe dysplasia or carcinoma in situ. The PCNA labeling index (P.L.I.) for normal squamous epithelium, mild dysplasia, and moderate dysplasia were significantly lower than for severe dysplasia or carcinoma in situ. The number of Ag-NOR dots of normal squamous epithelium and mild dysplasia were lower than those of severe dysplasia or carcinoma in situ. There are significantly positive correlations of the B.L.I., P.L.I., and the number of Ag-NORs in cervical intraepithelial neoplasia.

Clinical Significance of Argyrophilic Nucleolar Organizer Regions(AgNORs) in Squamous Cell Carcinoma of the Lung

Background: Nucleolar organizer regions(NORs) are chromosomal segments encoding for ribosomal RNA and associated with argyrophilic nonhistone protein. Ribosomal RNA genes ultimately direct ribosome and protein synthesis, and it has been suggested the numbers of NORs detected in the cell may reflect nuclear and cellular activity. This study was performed to evaluate the applicability of AgNORs to the diagnosis of squamous cell carcinoma of the lung. Method: The one step silver methods(AgNORs) was used to stain NORs in the routinely processed, formalin fixed, paraffin embedded sections of 36 cases of squamous cell carcinoma of the lung obtained by surgical resection of primary tumor. In each specimen, 100 tumor cells and 100 normal cells adjacent to the tumor chosen at random were examined under an oil immersion lens at a magnification of . The mean number of AgNORs per nucleus was calculated for each specimen. Results: The mean number of AgNORs per nucleus(mAgNORs) of normal bronchial epithelium and squamous cell carcinoma of the lung was and , respectively. The difference of mAgNOR between normal and tumor tissue was statistically significant(p

Absence of geographic chromosomal variation in the roan and sable antelope and the cytogenetics of a naturally occurring hybrid.

The determination of geographic chromosomal variation in rare or endangered species, or those of special management concern, is important, since geographically defined cytotypes can negatively influence breeding programs involving founders drawn from widely divergent localities. We cytogenetically analyzed specimens of the roan (Hippotragus equinus) and sable antelope (H. niger) collected from widely divergent localities throughout their respective ranges. Each species was characterized by a diploid number of 60 and an invariant karyotype. In contrast to the absence of intraspecific variation, however, the two species differ with respect to centromeric constitutive heterochromatin and numbers of nucleolar organizer regions. These cytogenetic landmarks were subsequently used to verify an anecdotal account of a naturally occurring roan x sable hybrid. The data show that, despite their markedly distinct phenotypes, the roan and sable antelope are nonetheless sufficiently similar genetically to produce viable offspring. Hybridization, although a rare event between these species, is probably partly promoted by behavioral differences which are not always sufficient to prevent mating between them.

Nucleolar organizer regions in bladder cancer: Application to urinary cytology

Objectives We investigated the relationship between the numbers of nucleolarorganizer regions (NORs) in nonmalignant reactive cells and those in transitional cell carcinomas using urinary exfoliated cell specimens. Another aim of this study was to determine whether higher numbers of NORs are correlated with tumors with higher pathologic grade. Methods Nucleolar organizer regions, which are important for regulating protein synthesis,were counted by means of a silver staining technique in urinary exfoliated cells from 34 patients with transitional cell carcinoma (TCC) and in 30 patients with other urologic diseases (controls). Results The number of argyrophilic proteins of the nucleolar organizer region (AgNORs)per cell (mean ± SD) was 2.9 ± 0.5 in the control group and 5.5 ± 1.9 in patients with TCC, which was significantly higher than that in the controls (p < 0.001). It was 4.0 ± 1.0 in patients with grade 1 TCC, 5.4 ± 2.0 in those with grade 2 TCC, and 6.5 ± 1.2 in those with grade 3 TCC. Although no statistically significant difference was observed between the groups, the patients with grade 2 TCCs with higher numbers of Ag-NORs showed a tendency to have more diffuse and/or invasive lesions. Conclusions This method is simple and quick, and can identify low-grade malignancy. Itcould be used as a tool for further grading of grade 2 TCCs and for determining prognosis.

Cytogenetic characteristics of cell lines from Ixodes scapularis (Acari: Ixodidae).

Three new cell lines, IDE8 and IDE12 from embryos of northern specimens of Ixodes scapularis Say and ISE18 from southern specimens of I. scapularis, were compared cytogenetically via conventional karyotyping, C- and G-banding, and nucleolar organizing regions (NORs). The karyotypes were very similar. The standard karyotype in the three cell lines consisted of 28 chromosomes with 26 autosomes and XX (female) or XY (male) sex chromosomes. The X chromosome was the largest, and the Y chromosome the smallest chromosome of the karyotype. Constitutive heterochromatin (C-bands) was almost entirely restricted to the centromeric region. An additional interstitial C-band in chromosome 7 was an important notable characteristic of the three cell lines. In sets showing a similar degree of condensation, individual chromosomes of the three lines had identical G-banding patterns. In addition, there was no difference among the cells in number and position of NORs. There were approximately 100 G-bands per haploid set in chromosomes from cells in metaphase, with three to 18 G-bands in each chromosome arm. After staining with silver nitrate, interstitial NORs were identified in chromosomes 7, 10, and the X chromosome. Male cells had five and female cells had six NORs. These findings support the notion that I. scapularis and I. dammini Spielman et al. are conspecific.

Peripheral benzodiazepine stimulates secretion of growth hormone and mitochondrial proliferation in pituitary tumour GH3cells

High affinity receptors for peripheral benzodiazepines (PBD) are present within the pituitary gland. Whether these receptors influence cell growth, mitochondrial morphology and secretion of growth hormone was examined in pituitary tumour GH3 cells. Cells were incubated in medium with either 10% calf serum, or serum free medium, or serum free medium plus 10 nM of selective PBD ligands (PK11195 or Ro5-4864) or a central benzodiazepine ligand (clonazepam). Peripheral or central ligands had no effect on cell growth. Quantitative electron micrography, however, revealed that cells treated with PBDs had a 2.5-fold increase in the total mitochondrial area within cells, a 1.6-fold increase in the number of mitochondria, and a 2.0 to 2.7-fold increase on the number of dividing mitochondria compared to serum free controls. The activity of DNA polymerase gamma, which replicates mitochondrial DNA, was increased approximately 3 times the initial value after 6 h exposure to PBDs. The number of nucleolar organizer regions (NORs), which relate to the ultimate synthesis of cellular protein, showed a 1.4-fold increase in PBDs treated cells. Secretion of growth hormone was stimulated (240% of serum free control) by exposure to PBDs for 12 h. The central benzodiazepine ligand, clonazepam, had little effect on mitochondrial morphology or secretion of growth hormone. These findings suggest that PBD receptors may be involved in mitochondrial proliferation and may affect the secretion of pituitary hormones.

Promoting effects of ethinyl estradiol on development of renal proliferative lesions induced by N-nitrosobis (2-oxopropyl)amine in female Syrian golden hamsters.

The modulating effects of female sex hormones, ethinylestradiol and levonorgestrel, on the development of renal proliferative lesions after initiation with N-nitrosobis(2-oxopropyl)amine (BOP) were investigated. Three groups of female Syrian golden hamsters, each comprising 30 animals, were given four weekly s.c. injections of BOP (10 mg/kg body weight) and then fed diet containing 1 ppm ethinylestradiol (group 1: BOP/EE), diet containing 10 ppm levonorgestrel (group 2) or basal diet (group 3) for the next 27 weeks. As hormone controls, two groups of 20 female hamsters each received diet containing 1 ppm ethynylestradiol (group 4) and 10 ppm levonorgestrel (group 5) from week 3 for 27 weeks without the prior initiation treatment. The severity of diffuse anisokarya, characterized by varied nuclear size and the incidence of dysplasias of the proximal tubular epithelia induced by BOP, were significantly increased in the BOP/EE group, indicating the promoting effects of the hormone. In the renal dysplastic lesions (small-cluster, cystic, clear-cell and acidophilic cell types), adenomas and nephroblastomas, increases in the numbers of argyrophilic proteins associated with nucleolar organizer regions (NOR) in the nucleus, suggesting a high proliferative activity, were seen in dysplasia of acidophilic cell types and adenomas. In addition, the number of bizarre NOR per nucleus was significantly higher in adenomas than in dysplasias and highest in nephroblastomas. This morphological change in NOR should therefore be a useful parameter for the diagnosis of malignancy of renal tumors.

COMPARATIVE ANALYSIS OF KARYOTYPES AND NUCLEOLAR ORGANIZER REGIONS IN DIFFERENT POPULATIONS OF MYTILUS TROSSULUS, MYTILUS EDULIS AND MYTILUS GALLOPROVINCIALIS

The cylotaxonomical characteristics of Mytilus tros-sulus, M. edulis and M. galloprovincialis were studied using karyometric analysis and silver staining. The karyotype of M. trossulus, reported here for the first time, consists of six metacentric and eight submetacentric-subtelocentric chromosome pairs. The occurrence of five metacentric chromosome pairs in M. galloprovincialis differentiates this taxon from M. trossulus and M. edulis which both have six meta-centrics. The number of submetacentric to subtelo-centric chromosome pairs was variable between populations and between taxa. We suggest that this variability has arisen from differential chromosome condensation rather than from structural change. Intercomparison of chromosome relative length showed strong similarity among the three taxa. Chromosomal nucleolar organizer regions (NORs) were identically located on two submetacentric-subtelocentric chromosome pairs in all three taxa. However, M. trossulus showed a characteristic difference in the occurrence of NORs on one metacentric pair, which was absent in M. galloprovincialis and M. edulis. Variability in the number of NORs per cell was observed in all populations studied. We conclude that the three taxa of Mytilus studied here cannot be differentiated by asingle karyological character, but that a combination of karyological characters is virtually diagnostic

Proliferating cell nuclear antigen and nucleolar organizer regions in CNS tumors: correlation with histological type and tumor grade. A comparative study of 82 cases on paraffin sections.

We investigated the expression of proliferating cell nuclear antigen (PCNA) and the number of nucleolar organizer regions (NORs) in 82 cases of CNS tumors. PCNA is a nuclear protein maximally elevated in the S phase of the cell cycle and recognized immunohistochemically in paraffin sections by the monoclonal antibody PC-10. On the other hand, NORs are loops of DNA that carry the rRNA genes and can be demonstrated in paraffin sections using an argyrophilic method (AgNORs). The present study shows a significant correlation of PCNA index and of AgNOR number with the histological grade (PCNA: I versus II, p < 0.01; II versus III, p < 0.01; and III versus IV, p < 0.05; AgNORs: I versus II, p < 0.001; II versus III, p < 0.05; and III versus IV, p < 0.001). Higher values of PCNA index (0.01 < p < 0.05) were found in recurrent tumors. Metastatic carcinomas were characterized by high PCNA indices and AgNOR numbers, similar to grade IV tumors, whereas in CNS lymphomas the malignancy grade was reflected in PCNA indices and AgNOR numbers. A wide range of PCNA and AgNOR values has been observed within each histological type and grade, probably reflecting variations in the biological behavior, but little overlap in PCNA values was present between grades II and III. The latter finding might be of importance in distinguishing between low- and high-grade CNS tumors. The linear regression coefficient between PCNA index and AgNOR number was excellent (0.91). We suggest that PCNA and AgNORs may be successfully applied in routine material to assess the growth potential of CNS tumors. Their prognostic value, however, must be validated with clinical studies.

Leuprolide acetate depot decreases the number of nucleolar organizer regions in uterine leiomyomata

Women with symptomatic uterine myomas were randomized to receive LA depot or placebo for 12 weeks before myomectomy. Silver staining nucleolar organizer regions (AgNORs) per nuclei were assessed in the tissue obtained at the time of myomectomy. Myoma tissue from women treated with LA depot before myomectomy had significantly fewer AgNORs per nuclei than myoma tissue obtained from women treated with placebo. Leuprolide acetate depot may reduce the number of proliferating cells in myomas.

Morphological changes of the nucleolar organizer regions induced by 7,12-dimethylbenz[a]anthracene in the hamster cheek pouch.

The staining of nucleolar organizer regions (NORs), which equate to rDNA transcription, was applied to chemically induced-lesions of the hamster cheek pouch. The cheek pouches of 16 male golden Syrian hamsters were treated three times a week with 0.5% 7,12-dimethylbenz[a]anthracene (DMBA) in mineral oil for 16 weeks. The percentage of gathered-type NORs with high activity nucleoli increased in the pouch epithelium during DMBA treatment and reached the highest values in malignant tumors. The percentage of dispersed-type NORs also increased in the malignant lesions. However, the absolute number of NORs was not affected by DMBA treatment. These results suggest that DMBA induces modification of NOR activity at the early stages of carcinogenesis and shows the potential of this model for studying NOR alterations in neoplasia.

Nucleolar and dispersed nucleolar organiser regions (NORs) in differentiating neoplastic from atypical non-neoplastic lesions of the pancreas.

Our objective was to compare the accuracy of differentiation of pancreatic carcinoma from the benign ductular aggregation of chronic pancreatitis using two methods, nuclear morphometric analysis and modified argyrophilic nucleolar organiser region (AgNOR) staining for nucleoli and dispersed NORs (AgNU staining). We evaluated 35 metastatic or invasive lesions of pancreatic carcinoma with low-grade atypia, 16 lesions of ductular aggregation and 12 lesions of proper epithelium. Four parameters, consisting of the size of nucleus: mean area, mean minor axis, mean major axis and mean perimeter, and two parameters of AgNu staining: mean number of AgNu-stained dots per nucleus (equal to mean number of nucleoli and dispersed NORs) and mean area of the largest AgNu-stained dot (equal to mean area of the largest nucleolus) of pancreatic carcinomas were significantly larger than those of ductular aggregations and of proper epithelia. In the discriminant analysis of four parameters of nuclear size, the discrimination of carcinomas was correct in 82.9% and that of ductular aggregations in 81.3%. Discriminant analysis by two parameters of AgNu staining showed 2 correct discrimination of lesions in 88.6% and 100%. Thus, AgNu staining was more accurate than nuclear morphometric analysis in discriminating a pancreatic carcinoma from ductular aggregation.

Prognostic assessment of superficial squamous cell carcinoma of the esophagus using karyometric analysis and nucleolar organizer regions.

To investigate prognostic values for squamous cell carcinoma, we measured nuclear area (NA), nuclear shape factor (NSF), DNA content and mean number of nucleolar organizer regions (NOR), using formalin-fixed, paraffin-embedded tissue sections from 39 patients with superficial squamous cell carcinoma of the esophagus. NA and DNA content were significantly higher in patients with lymph node metastasis than in those without metastasis. NA and the NOR number were also significantly higher in patients with recurrence than in those surviving for more than 3 years without recurrence. To determine the optimal combination of these parameters for prognostic assessment, we used a stepwise discriminant analysis to obtain two linear discriminant functions. One (f = 0.331 x NA + 6.64 x NSF + 9.85) gave an accuracy of 87.2% in predicting lymph node metastasis, and the other (f = -0.071 x NA - 2.34 x NOR + 13.0) yielded an overall accuracy of 88.9% in classifying patients into two groups with a better and worse prognosis. These results suggest that karyometric analysis and determination of the NOR number are useful for the prediction of disease outcome in patients with superficial squamous cell carcinoma of the esophagus.

Numerical variation of nucleolar organizer regions after silver staining in domestic and wild Suidae (Mammalia).

Selective silver staining was used to investigate the cellular distribution of numbers of nucleolar organizer regions (NORs) in domestic pigs (Sus scrofa) of eight different breeds, the European wild boar (S. scrofa scrofa), Indonesian wild boar (S. scrofa vittatus), Javan warty pig (S. verrucosus), Sulawesi warty pig (S. celebensis), and pigmy hog (S. salvanius). In the domestic pig as well as in the wild (sub)species of Sus, actively transcribing ribosomal RNA genes were found to be present in the secondary constrictions of chromosome pairs 10 and 8. Chromosomes 10 were consistently Ag-positive. Chromosomes 8 less frequently showed Ag-NORs, resulting in different mean numbers of Ag-NORs per individual animal. Mean Ag-NOR numbers per breed or (sub)species were generally higher in the wild representatives of Sus than in the domestic breeds. The highest mean numbers of Ag-NORs were observed in the Meishan breed and in S. celebensis and S. salvanius. The Meishan breed appears to be conservative in Ag-NOR staining pattern, being more comparable to the Asian wild Suidae than to the European breeds.