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Whole mount electron microscopy of the nucleolus in salivary gland cells of Drosophila melanogaster.

The nucleolus of Drosophila melanogaster salivary gland cells, examined by whole mount electron microscopy, consists of a fibrillar core region and a peripheral region containing both fibres and granules. These regions appear to correspond to the fibrillar and granular components, respectively, seen in thin sections. Most of the nucleoli were attached to the chromocenter region of the polytene chromosomes, containing the nucleolar organizer. Bundles of relatively straight chromatin fibres, 13 nm in diameter, extended from the chromocenter into the core region of the nucleolus, however it was not possible to trace the path of these chromatin fibres through the nucleolus since they were obscured within the mass of nucleolar fibres. The nucleolar fibres in both the core and peripheral regions were irregular and knobby, with a diameter of about 15 nm. In the core region, the fibres appeared to be of considerable length and were characteristically clustered together to form small interconnected masses. The fibres in the peripheral region were relatively short and some appeared to blend with amorphous, poorly-defined pools of material. Electron dense granules 15-20 nm in diameter were also associated with this amorphous substance. It is hypothesized that the formation and subsequent packaging of the 28s rRNA may be represented by a morphological transition of the peripheral fibres, via an amorphous pool-like intermediate stage, into the nucleolar granules. The results of this study indicate that whole mount electron microscopy may be a useful alternative to thin sectioning in high resolution studies of the nucleolus.

Animals↗

Nucleolar and argyrophilic nucleolar organizer region counts in urothelial carcinomas with special emphasis on grade II tumors.

Prognostic assessment of bladder carcinomas of intermediate differentiation is difficult. This study therefore investigated the prognostic values of nucleolar status and silver staining of argyrophilic nucleolar organizer regions (AgNORs) in grade II bladder carcinomas. In biopsies from 34 grade II transitional cell carcinomas of the urinary bladder the number of nuclei with nucleoli, the location of nucleoli within the nucleus and the number of AgNORs were determined in 1000 or 200 nuclei per section respectively. Ten biopsies showing normal urothelium, 18 cases with mild to severe atypia, 27 grade I, 34 grade II and 12 grade III transitional cell carcinomas were also studied. Significantly differing nucleolar and AgNOR values were found comparing normal urothelium/grade I carcinomas with severe urothelial atypia/grade III carcinomas. Grade II carcinomas, however, were inhomogeneous. One subgroup had nucleolar and AgNOR values resembling grade I carcinomas while the second group had values similar to those of grade III carcinomas. This subdivision of grade II carcinomas correlates with results reported for DNA-cytometry. The results suggest a subdivision of patients with grade II transitional cell carcinomas into a low risk and high risk group.

Carcinoma, Transitional Cell↗

The nucleolus: a paradigm for cell proliferation and aging.

The nucleolus is the cellular site of ribosome biosynthesis. At this site, active ribosomal DNA (rDNA) genes are rapidly transcribed by RNA polymerase I (pol I) molecules. Recent advances in our understanding of the pol I transcription system have indicated that regulation of ribosomal RNA (rRNA) synthesis is a critical factor in cell growth. Importantly, the same signaling networks that control cell growth and proliferation and are deregulated in cancer appear to control pol I transcription. Therefore, the study of the biochemical basis for growth regulation of pol I transcription can provide basic information about the nuclear signaling network. Hopefully, this information may facilitate the search for drugs that can inhibit the growth of tumor cells by blocking pol I activation. In addition to its function in ribosome biogenesis, recent studies have revealed the prominent role of the nucleolus in cell senescence. These findings have stimulated a new wave of research on the functional relationship between the nucleolus and aging. The aim of this review is to provide an overview of some current topics in the area of nucleolus biology, and it has been written for a general readership.

Cell Division↗

Identification and definition of nucleolus-related fibrillar bodies in micronucleated cells.

Small nucleolus-related bodies which occur in the nucleoplasm of "micronuclei" lacking nucleolar organizers have been studied by immunofluorescence microscopy. These bodies stained specifically with three different antibodies directed against proteins that are normally associated with the dense fibrillar component of functional nucleoli, but not with antibodies specific for certain proteins of the granular component or the fibrillar centers. Our data show that, in the absence of rRNA genes, the various constituent proteins characteristic of the dense fibrillar component spontaneously assemble into spherical entities but that the subsequent fusion of these bodies into larger structures is prevented in these micronuclei. The similarity between these nucleolus-related bodies of micronuclei and the prenucleolar bodies characteristic of early stages of nucleologenesis during mitotic telophase is discussed.

Animals↗

Protein kinase inhibitor H-7 blocks accumulation of unspliced mRNA of human T-cell leukemia virus type I (HTLV-I).

Rex, the post-transcriptional regulator of human T-cell leukemia virus type I (HTLV-I), is known to induce accumulation of the unspliced viral gag-pol mRNA. Rex is a phosphoprotein found in the cell nucleolus, whose function may be regulated by its localization and phosphorylation. We have examined the role of phosphorylation on Rex function by using a protein kinase inhibitor, H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine]. Treatment of an HTLV-I infected human T-cell line with H-7 blocked specifically accumulation of the unspliced gag-pol mRNA, resulting in the decreased Gag protein synthesis that corresponds with the decreased in vivo phosphorylation of Rex. In contrast, other viral and cellular products have not been influenced by the level of H-7 used. Therefore, the phosphorylation of Rex is required for the viral RNA partition of HTLV-I.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Effects of anti-fibrillarin antibodies on building of functional nucleoli at the end of mitosis.

During mitosis some nuclear complexes are relocalized at the chromosome periphery and are then reintegrated into the re-forming nuclei in late telophase. To address questions concerning translocation from the chromosome periphery to nuclei, the dynamics of one nucleolar perichromosomal protein which is involved in the ribosomal RNA processing machinery, fibrillarin, was followed. In the same cells, the onset of the RNA polymerase I (RNA pol I) activity and translocation of fibrillarin were simultaneously investigated. In PtK1 cells, RNA pol I transcription was first detected at anaphase B. At the same mitotic stage, fibrillarin formed foci of increasing size around the chromosomes, these foci then gathered into prenucleolar bodies (PNBs) and later PNBs were targeted into the newly formed nucleoli. Electron microscopy studies enabled the visualization of the PNBs forming the dense fibrillar component (DFC) of new nucleoli. Anti-fibrillarin antibodies microinjected at different periods of mitosis blocked fibrillarin translocation at different steps, i.e. the formation of large foci, foci gathering in PNBs or PNB targeting into nucleoli, and thereby modified the ultrastructural organization of the nucleoli as well as of the PNBs. In addition, antibody-bound fibrillarin seemed localized with blocks of condensed chromatin in early G1 nuclei. It has been found that blocking fibrillarin translocation reduced or inhibited RNA pol I transcription. It is postulated that when translocation of proteins belonging to the processing machinery is inhibited or diminished, a negative feed-back effect is induced on nucleolar reassembly and transcriptional activity.

Animals↗

In situ fluorescence visualization of bromouridine incorporated into newly transcribed nucleolar RNA.

Bromouridine-triphosphate is commonly used for in situ immunocytochemical labeling of newly synthesized RNA in living cells. While extranucleolar transcripts do not require special conditions for visualization, special treatment prior to fixation (e.g. incubation with alpha-amanitine) is necessary for immunofluorescence detection of bromouridine-labeled nucleolar RNA in previous studies. We show in the present investigation that bromouridine-triphosphate is efficiently used by both extranucleolar and nucleolar RNA polymerases in living cultured cells. The failure to detect incorporated bromouridine within nucleoli is entirely due to improper treatment of cells after bromouridine incorporation. When methanol/acetone fixation is used, fluorescence signals within nucleoli can be routinely found.

Animals↗

[The structure of antipodal complex in barley cultivars and hybrids in different growing conditions].

The comparative research of antipodal cell number and their karyometrical parameters in three two-rowed barley cultivars and their reciprocal F1 hybrids has been performed under different growing conditions. It was shown that the number of antipodes varied in different cultivars and depended on growing conditions. The essential level of antipodal complex functional activity is reached by definite combination of cell quantities and their dimensions. At greenhouse conditions the traits "number of antipodal cells" and "total volumes of nuclei and nucleoli" are inherited maternally.

Cell Nucleolus↗

[Selectivity and localized nature of exposure in laser microirradiation of cells].

Some modern applications of laser microirradiation to studies of relationship between structure and function are reported. The main advantage of the method is the ability to act on various subcellular parts and organelles, both locally and selectively. However, the degrees of laser microirradiation locality and selectivity are not always sufficiently high. These properties of laser microirradiation are analysed in addition to factors of deteriorating and improving the methods. It is assumed that the most important influence in cellular response to microirradiation is exerted by laser on different cellular membrane systems, particularly on the surface membrane and mitochondria.

Animals↗

Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei.

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.

Cell Nucleolus↗

Morphometric study by image analysis of Ag-stained nucleoli in thyroids bearing proliferating lesions.

The Ag-NOR staining technique and image analysis were used to evaluate morphological parameters (area, perimeter and axis ratio) in nucleoli from normal thyroids and from thyroids bearing proliferating lesions (carcinomas, adenomas and hyperplasias). Regions with normal appearance located close to adenomatous and carcinomatous regions, in the thyroid of every patient, were also analyzed for comparison with the respective pathological regions and with normal thyroids. Statistical analysis of data for the nucleolar area and perimeter allowed the separation of adenomas and carcinomas from hyperplasias and normal tissue but not the two components in each of these two groups. However, if we look at the numbers, a sequence of increasing nucleolar mean areas in the order: normal, hyperplasia, adenoma and carcinoma may be observed, indicating the sequence of increasing rRNA requirements in these different kinds of cells. The axis ratio that denotes the nucleolar shape (round or oblong) did not show significant differences among tissues, suggesting that shape is not important in the characterization of these pathologies. Differences in nucleolar areas and perimeter between normal and affected regions from each patient were statistically significant for adenomas and carcinomas. When these normal regions were compared with the normal thyroids, significant differences were not obtained in the three evaluated parameters. The observations and their importance for histopathological diagnosis are discussed.

Adenocarcinoma↗

Distribution of argyrophilic proteins in the nuclei of hepatocytes.

The ultrastructural study on the distribution of argyrophilic proteins of hepatocytes was carried out. Because it has been suggested that the numbers of silver grains were connected with transcriptional activity of nucleoli, we used in our studies as a model the regenerating liver. Among extranucleolar structures affinity to silver interchromatin granules and weakly perichromatin granules was demonstrated. Deposits of silver grains were always located within dense fibrillar components and fibrillar centers. Our study does not enable to indicate a direct relationship between numbers of silver grains deposits and transcriptional activity of nucleoli.

Animals↗

Image analysis of argyrophil nucleolar organizer regions (AgNORs) in oestrogen-induced rat anterior pituitary hyperplasia: comparison of automatized (LUCIA M) and non-automatized evaluation.

Argyrophil nucleolar organizer regions (AgNORs) were morphometrically evaluated in rat anterior pituitaries of control and oestrogenized rats using an automatized (LUCIA M--Laboratory Universal Computer Image Analysis Micro) and a non-automatized approaches to investigate not only the sensitivity and effectiveness of the automatized procedure but also the morphology of AgNORs in oestrogen-induced rat anterior pituitary hyperplasia. In the experimental oestrogen-induced rat anterior pituitary hyperplasia, the image analysis system LUCIA M was compared to a non-automatized morphometric procedure and proved to be very efficient and yielded analogical results. The AgNOR morphology in the oestrogenized pituitaries was characterized by an increase of the total AgNOR area and clustering of AgNORs in the large nucleoli.

Animals↗

[the effect of colonystimulating macrophage factor on activity of the nuclear organisers in central macrophages of the cultures of the bone marrow erythroblastic islands].

Erythroblastic islands of the bone marrow are morpho-functional units of erythropoiesis. The functional state of erythroblastic islands' cells of the bone marrow was for the first time defined by estimation of activity of the nuclear organisers of the central macrophages in the erythroblastic islands cultivated for 24 hrs in presence of various doses of the colony-stimulating macrophage factor. The findings indicate that increased doses of the colonystimulating macrophage factor was accompanied by a respective enhancement of the activity of nucleolar organisers in central macrophages of erythroblastic islands.

Animals↗

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.

Apoptosis↗