Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells.
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Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function. (+info)
Proliferative activity in oral salivary gland tumors: the role of PCNA and AgNOR assessed by a double staining technique.
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The aim of this study was to evaluate the role of PCNA and AgNOR in the assessment of salivary gland tumor proliferation using a double staining technique. Ten cases of pleomorphic adenoma (PA) and seventeen cases of adenoid cystic carcinoma (ACC) were examined. Numeric and morphometric parameters of AgNOR were evaluated and compared in PCNA-positive and PCNA-negative nuclei. There were statistically significant differences in AgNOR numbers, perimeters and contour indices between PCNA-positive and -negative nuclei in the PA samples. The ACC samples demonstrated significant differences only in the AgNOR areas. Our results show that in salivary gland tumors there is not always a relationship between proliferative activity evaluated by AgNOR numeric and morphometric parameters and PCNA immunostaining. (+info)
Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells.
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One of the great mysteries of the nucleolus surrounds its disappearance during mitosis and subsequent reassembly at late mitosis. Here, the relative dynamics of nucleolar disassembly and reformation were dissected using quantitative 4D microscopy with fluorescent protein-tagged proteins in human stable cell lines. The data provide a novel insight into the fates of the three distinct nucleolar subcompartments and their associated protein machineries in a single dividing cell. Before the onset of nuclear envelope (NE) breakdown, nucleolar disassembly started with the loss of RNA polymerase I subunits from the fibrillar centers. Dissociation of proteins from the other subcompartments occurred with faster kinetics but commenced later, coincident with the process of NE breakdown. The reformation pathway also follows a reproducible and defined temporal sequence but the order of reassembly is shown not to be dictated by the order in which individual nucleolar components reaccumulate within the nucleus after mitosis. (+info)
Relationship of AgNOR counts and nuclear DNA content to survival in patients with parathyroid carcinoma.
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The aim of this study was to evaluate the usefulness of DNA flow cytometry to determine tumor nuclear DNA index (DI), and nucleolar organizer region protein counts visualized by the argyrophil (AgNOR) technique, in confirming diagnosis and predicting clinical outcome of patients with parathyroid carcinoma (PC). We reviewed paraffin-embedded tissue sections, from 15 patients (median age 63 years, range 30-68 years) with PC who died of the disease, which were randomly compared with tissue sections from 15 age- and sex-matched patients with parathyroid adenoma (PA). The proliferative activity in parathyroid tumours as detected by DI and AgNOR counts was evaluated in all specimens. Both DI (1.37 +/- 0.33 vs 1.0 +/- 0.1) and AgNOR (3.01 +/- 0.31 vs 1.54 +/- 0.35) counts were higher (P < 0.001) (Student's t-test) in patients with PC than in those with PA. Diploid (DI = 1), aneuploid (DI > 1) and hypoploid (DI < 1) neoplasms were found in 11 (PC = 4, PA = 7), 14 (PC = 11, PA = 3) and five (PC = 0, PA = 5) patients respectively. The average postoperative survival in patients with PC was 46.9 +/- 37.4 months (range 21-146 months). The survivals of patients with aneuploid (n = 11) and diploid (n = 4) PC were 74.0 +/- 58.1 and 34.1 +/- 18.4 months (P=0.21) respectively. There was a significant relationship between DI and AgNOR counts (R=0.69, P < 0.01), but no correlation was found between survival and both DI (Rho = 0.17, P = 0.55) and AgNOR counts (Rho = 0.26, P = 0.35). Moreover, there was no correlation (P = NS) between the main preoperative biochemical parameters and survival. In conclusion, DI and AgNOR are useful in confirming the diagnosis of PC, but they are of little value in predicting the clinical outcome of patients with PC. (+info)
AgNOR counts and determination of malignancy in stromal tumours of the stomach and small intestine.
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Twenty four primary stromal tumours of the stomach and small intestine were investigated by silver staining of interphase nucleolar organiser regions (AgNORs) in an attempt to obtain an objective criterion for prediction of malignant tumour behaviour. Malignant tumours tended to have higher AgNOR counts than their benign counterparts, but this increase was small and there was some overlap between the two groups. There was a correlation between the mean AgNOR count and the mitotic count. There was no correlation between tumour size and these two measurements. This study suggests that in these stromal tumours the AgNOR count is not a useful independent predictor of malignant behaviour. (+info)
Interferon plus ribavirin and interferon alone in preventing hepatocellular carcinoma: a prospective study on patients with HCV related cirrhosis.
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AIM: To determine the role of interferon (IFN) with or without ribavirin in preventing or delaying hepatocellular carcinoma (HCC) development in patients with hepatitis C virus (HCV) related cirrhosis. Data on the preventive effect of IFN plus ribavirin treatment are lacking. METHODS: A total of 101 patients (62 males and 39 females, mean age 55.1+/-1.4 years) with histologically proven HCV related liver cirrhosis plus compatible biochemistry and ultrasonography were enrolled in the study. Biochemistry and ultrasonography were performed every 6 mo. Ultrasound guided liver biopsy was performed on all detected focal lesions. Follow-up lasted for 5 years. Cellular proliferation, evaluated by measuring Ag-NOR proteins in hepatocytes nuclei, was expressed as AgNOR-Proliferative index (AgNOR-PI) (cut-off = 2.5). Forty-one patients (27 males, 14 females) were only followed up after the end of an yearly treatment with IFN-alpha2b (old treatment control group = OTCG). Sixty naive patients were stratified according to sex and AgNOR-PI and then randomized in two groups: 30 were treated with IFN-alpha2b + ribavirin (treatment group = TG), the remaining were not treated (control group = CG). Nonresponders (NR) or relapsers in the TG received further IFN/ribavirin treatments after a 6 mo of withdrawal. RESULTS: AgNOR-PI was significantly lowered by IFN (P<0.001). HCC incidence was higher in patients with AgNOR-PI>2.5 (26% vs 3%, P<0.01). Two NR in the OTCG, none in the TG and 9 patients in the CG developed HCC during follow-up. The Kaplan-Mayer survival curves showed statistically significant differences both between OTCG and CG (P<0.004) and between TG and CG (P<0.003). CONCLUSION: IFN/ribavirin treatment associated with re-treatment courses of NR seems to produce the best results in terms of HCC prevention. AgNOR-PI is a useful marker of possible HCC development. (+info)
Cytogenetic studies in three Pimelodella meeki populations (Pisces, Pimelodidae) from Tibagi River basin (Brazil).
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We analyzed cytogenetically specimens of Pimelodella meeki from Tibagi River at Limoeiro (LM) and from two tributaries, Couro do Boi (CB) and Gabriel da Cunha (GC) Rivers. All specimens presented 2n=46 chromosomes, which were the karyotypes composed by 15 pairs metacentric (M) + 6 pairs submetacentric (SM) + 2 pairs subtelocentric (ST). In specimens of GC, CB, and LM, the results of analyses of the nucleolus organizer regions (NORs), done by means of AgNO3 and CMA3 staining, showed that they are identical, located in terminal position on the short arm of a SM chromosome pair, and they were observed to be a size heteromorphism in some metaphase plates. FISH with 18S rDNA probe yielded evidence for these regions but not for the size variation, indicating that they are not due to a greater number of NOR cistrons in one of the homologue chromosomes. An interesting characteristic of these regions is that they could appear divided in blocks, as evidenced by all the techniques. This work makes clear the necessity for more deeply systematic studies, because of the confused taxonomic situation of the genus Pimelodella. (+info)
Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei.
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Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15-20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis. (+info)