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New data concerning the functional organization of the mammalian cell nucleolus: detection of RNA and rRNA by in situ molecular immunocytochemistry.

We have investigated the fine spatial distribution of RNA and rRNA within the Ehrlich tumor cell nucleolus by in situ hybridization with a biotin-labeled probe and by two new strategies, the polyadenylate nucleotidyl transferase-immunogold technique and immuno-labeling with anti-RNA antibodies. Besides the presence, as expected, of RNA and rRNA in the granular component and the dense fibrillar component, we show, for the first time, significant label over all the fibrillar centers of the nucleoli. When RNA and DNA were detected simultaneously on the same sections, only the fibrillar centers were positive for both. These results throw light on the controversial subject of the precise location of transcribing rRNA genes within the nucleolus. The fibrillar centers, and not the dense fibrillar component, should thus be the site of rRNA synthesis.

Animals

Cytochemical and ultrastructural studies of changes in the nucleus and nucleolus during cell differentiation in the root cortex of Haemanthus katharinae.

Dimensions,dry mass content, binding of [3H]actinomycin D ([3H]AMD), and [3H]uridine incorporation in the nuclei and nucleoli as well as their ultrastructure during growth and differentiation of the root cortex cells of Haemanthus katharinae were compared. The dry mass of the nuclei did not change, the condensed chromatin content was slightly augmented, and the ability of the nuclei to bind [3H]AMD became somewhat decreased, whereas a considerable reduction was observed in the [3H]uridine incorporation. The highest capability for the binding of [3H]AMD and the highest intensity of RNA synthesis were demonstrated by nuclei in the meristematic zone and during the most intensive cell growth. The size of the nucleoli, their dry mass, the amount of granular component, and the intensity of [3H]uridine incorporation underwent a steady decrease as the cells grew and differentiated. The progressing differentiation of the cells was accompanied by an increasing participation of arginine-rich histones in restraining the capacity for [3H]AMD binding.

Cell Differentiation

Cytochemical distinction of various nucleolar components in insect cells.

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.

Animals

[Effect of vinblastine on the blast population of the thymus subcapsular zone in the mouse].

It is well admitted that blast cells of the thymic subcapsular zone play an important role in murine leukemogenesis. Previous ultrastructural investigations have revealed the morphological heterogeneity of this population which is formed by lymphoblasts, X cells and ring shaped nucleolus cells. These aspects could correspond to different positions in the cell cycle of an unique cell type. After administration of vinblastine, a drug blocking mitosis in metaphasis, the percentage of lymphoblasts decreases whereas the percentage of X cells increases. There is no modification of the number of ring-shaped nucleolus cells. This suggests that at least a part of lymphoblasts gives rise to x cells.

Animals

Effects of chimeric mutants of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex on nucleolar targeting signals.

Two chimeric mutant genes derived from rev of human immunodeficiency virus type 1 and rex of human T-cell leukemia virus type I were constructed to investigate the functions of the nucleolar-targeting signals (NOS) in Rev and Rex proteins. A chimeric Rex protein whose NOS region was substituted with the NOS of Rev was located predominantly in the cell nucleolus and functioned like the wild-type protein in the Rex assay system. However, a chimeric Rev with the NOS of Rex abolished Rev function despite its nucleolar localization. This nonfunctional nucleolar-targeting chimeric protein inhibited the function of both Rex and Rev. In the same experimental conditions, this mutant interfered with the localization of the functional Rex in the nucleolus.

Amino Acid Sequence

Cytologic correlates of chemical carcinogens in respiratory tracts of dogs.

During canine respiratory carcinogenesis studies with benzo[a]pyrene (BP) and N-methyl-N-nitrosourea (NMU), a stereotypic pattern of cytomorphology characteristic for each carcinogen was observed. In the early stage, BP induced changes primarily in the cytoplasm. These changes consisted of incresaed size, basophilia, and vacuolization. In contrast, NMU induced primarily nuclear enlargement and enhanced the prominence of the nucleolus. Subsequently, cells exposed to BP demonstrated pleomorphism of the cytoplasm and nucleus, whereas cells exposed to NMU formed bizarre spindle-shaped cells. Cells were generally more scattered from one another following BP treatment than after NMU treatment. When the mucosa was exposed to NMU after the previous use of BP, the cytologic findings promptly became those characteristic of NMU.

Animals

[Preliminary data on "fibrillary centers" of the nucleolus of various cells of the ovarian follicle of a lizard Lacerta muralis Laur].

The nucleolus of the thecal cells and little cells of the granulosa contains generally a single "fibrillar center" surrounded by the dense fibrous material of the nucleolus. The "fibrillar centers" are in contact with nucleolar associated heterochromatin. Cytochemical results show that "fibrillar centers" are chiefly composed of proteins which are revealed as ribonucleoproteins with the EDTA method, but the amount of RNA associated with these proteins seems to be small. It is possible that the fibrillar center also contains a little DNA.

Animals

Changes in the activity of RNA polymerase detected in situ and the intensity of 3H uridine incorporation into the nucleolus and the nucleus of interphase cells in antheridial filaments of Chara vulgaris L.

Analogously as in the 32-cell stage [20], in the 4-, and 8-cell generations of antheridial filaments of Chara vulgaris, the activity of DNA dependent RNA polymerase detected in situ as well as the 3H uridine incorporation increase in the middle S phase and in the middle G2 phase, while they decrease considerably at the end of S phase and in late G2 phase. The diphasic changes occur both in the nucleolar and extranucleolar (nucleoplasmic) activity of RNA polymerase as well as in the 3H uridine incorporation. However, the maximum nucleolar activity, in both S and G2 phases, precedes the peak of nucleoplasmic activity. During the increased nucleolar activity RNA polymerase (as calculated per nucleus) shows a higher level as compared with nucleoplasmic RNA polymerase, whereas the intensity of 3H uridine incorporation into nucleous and nucleoplasm is similar. It may be supposed that the incubation environment containing Mg2+ used in vitro is more stimulating for the nucleolar RNA polymerase than for the nucleoplasmic RNA polymerase. The mean transcriptional activity of the nucleus and the activity of RNA polymerase in the 8-cell generation is about 20% lower than in the 4-cell generation, in proportion to the decrease in cell sizes.

Cell Cycle

Phosphorylation of the Rex protein of human T-cell leukemia virus type I.

Rex protein, the posttranscriptional regulator of human T-cell leukemia virus type I (HTLV-I), is required for the control of viral structural protein expression and virus replication. Rex is a phosphoprotein found predominantly in the cell nucleolus, whose function is thought to be regulated by its nucleolar localization and phosphorylation. Therefore, we investigated the in vivo phosphorylation of Rex protein in more detail. Phosphorylation of Rex occurred in all HTLV-I-infected cell lines examined in vivo, primarily at serine residues and to a very small extent at threonine residues. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) led to significant but transient enhancement of the incorporation of [32P]orthophosphate into Rex protein. N-terminal truncation of Rex protein abolished TPA-dependent phosphorylation. Chymotryptic digestion of phosphorylated Rex yielded two phosphopeptides. In vivo phosphorylation sites were identified as serine residues 70 and 177 and threonine residue 174. Serine 70 was a TPA-dependent phosphorylation site within a regulatory domain. We have already shown that the protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) specifically blocked accumulation of viral unspliced gag-pol mRNA. Therefore, the phosphorylation at serine 70 may be involved in the regulation of Rex function in response to extracellular stimuli.

Amino Acid Sequence

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

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

Morphologic study of virus-like particles in a case of acute leukemia.

Virus-like particles, grouped in clusters not bound by a membraine, were seen in electron micrographs of fresh leucoblasts of an acute leukemic patient. They consisted of large (100-nm diameter), round particles apparently composed of subunits. Tubular structures (30-50-nm diameter) were also seen in leucoblasts of the same patient. The two types of structures were never seen simultaneously in the same cell. The authors review the literature pertaining to the morphology of virus-like particles associated with malignant hematologic disease.

Cell Nucleolus

Metastatic cystosarcoma phyllodes: a light and electron microscopic study.

Two cases of metastatic cystosarcoma phyllodes, one in lung and the other in axillary lymph nodes, were studied with the light and the electron microscopes. The malignant element of these tumors appears to be a poorly differentiated mesenchymal cell. No evidence supporting the presence of epithelial cells was found. The tumor metastatic to the axilla exhibited intracellular virus-like particles similar to those described in adenocarcinoma of breast and other sarcomas.

Cell Nucleolus

So-called "minute pulmonary chemodectoma": a tumor not related to paragangliomas.

Twenty-six cases of so-called "minute pulmonary chemodectoma" are presented. The patient population showed a marked female preponderance, and there appeared to be an association of the lesion with pulmonary injury from a variety of causes including cardiac failure, chronic bronchitis and emphysema, and thromboemboli. Half the cases had multiple tumors. Microscopically, the tumors consisted of nests of cells in the interstitial tissue near small veins. Argentaffin and argyrophil stains failed to demonstrate cytoplasmic granules in any case. By electron microscopy, the nests were composed of large cells with broadly interdigitating processes connected by many well-formed desmosomes. The cytoplasm was filled with numerous 60-A filaments. The Golgi apparatus was prominent, while other organelles were sparse. No secretory granules were identified. It is concluded that the fine structure and lack of silver-positive granules are inconsistent with the morphology of previously reported paragangliomas, but that there is a resemblance at the light and electron microscopic level to meningeal arachnoed cells and the cells of meningiomas.

Adolescent

Small cell undifferentiated carcinoma of salivary gland origin: an ultrastructural study.

A case of small cell carcinoma of salivary gland was studied by light and electron microscopy. Light microscopy showed sheets of small uniform cells with scanty cytoplasm and pyknotic nuclei. Electron microscopy showed two distinct cell types; an electron-lucent epithelial-appearing cell, and a denser cell with processes containing bundles of filaments and other features suggesting myoepithelial differentiation. Neurosecretory granules were absent. These findings support a salivary duct origin, and are evidence against a neurendocrine derivation for this tumor.

Adult