Effects of testosterone on nuclear ribonucleoprotein components of prostate epithelial cells

Summary— The changes of the nuclear components caused by castration and testosterone injection were studied in epithelial cells of the ventral prostate of the rat. Castration drastically diminishes the nuclear and nucleolar volume, as well as the fraction of the nuclear volume occupied by non-nucleolar ribonucleoprotein (RNP) fibrils. However, in castrated animals the frequency of perichromatin granules (PCG) is 79% higher than in controls. Testosterone injection causes a reduction of the number of PCG to 33% of the castrated level in 15 min, and increases the non-nucleolar RNP fibrils. Other parameters such as nuclear and nucleolar volume and the relative volume of the compact chromatin present only small changes in a period of 2 h following the hormone administration. High resolution quantitative autoradiography demonstrates that the transportation of previously synthesized RNA increases steeper than the RNA synthesis. All these effects are similar to those caused by ovariectomy and estradiol injection on the nuclear structures of endometrial epithelial cells. These similarities and other observations suggest that PCGs contain mRNA, of a few genes, stored in the nucleus by a restriction of its transportation to the cytoplasm.     

Isolation and partial characterization of perichromatin granules: A unique class of nuclear RNP particles

Perichromatin granules (PCG) have been isolated from cycloheximide-treated rat liver nuclei by a procedure that preserved their ultrastructural characteristics. Like the PCG particles in situ, the isolated granules were 300–400 Å in diameter; they had an approximate sedimentation coefficient of 40S. The Bernhard bleaching procedure showed that the isolated perichromatin granules are not chromatinous components. A low molecular weight 4.7S RNA approx. 100 nucleotides long was associated with the granules. Analysis of the proteins of the isolated perichromatin granules on SDS polyacrylamide gel electrophoresis showed one major polypeptide (mol. wt approx. 34 000) along with two other minor polypeptides (mol. wt 31 000 and 38 000). The major polypeptide found in the perichromatin granules had similar migration characteristics on SDS gels to a peptide found in both rat liver and HeLa cell heteronuclear ribonucleoprotein (hnRNP) particles.     

Correlation of the changes of the frequency of perichromatin granules with the RNA content of the interchromatin region of uterine cells in normal and ovariectomized rats. A high resolution in situ hybridization and stereological study.

The changes in the number of perichromatin granules (PCG) and the alterations in the RNA content of the interchromatin and perichromatin regions caused by ovariectomy and estradiol injection were studied in rat endometrial fibroblast and myometrial muscle cells. Twelve rats were divided in four groups. A group of rats was fixed without any treatment, the other three groups were ovariectomized and processed 21 days after the operation. One of them was studied without further treatment, and two groups were injected intraperitoneally with 20 micrograms of 17 beta-estradiol hemisuccinate and fixed 0.5 and 2 h after the injection. The frequency of PCG was evaluated in preparations stained with EDTA procedure preferential for RNP. The alterations of RNA content were estimated by post-embedding high resolution in situ hybridization using a total DNA probe labeled with biotinilated nucleotides revealed by streptavidin coupled with 10 nm gold grains. Most of the non-nucleolar labeling is associated to RNP containing fibrils. Perichromatin and interchromatin granules are labeled to a lesser extent. Castration brings about a reduction of the number of PCG and of the numerical density of labeling in endometrial fibroblasts. The injection of estradiol causes a rapid increase in both parameters. On the contrary, the frequency of PCG and intensity of labeling of epithelial endometrial cells and in muscle cells increase after ovariectomy and are reduced by estradiol administration. These results suggest that estradiol may affect differentially various types of target cells in the same organ, and also that PCG are not the only nuclear compartment of pre-mRNA or mRNA altered by the changes in estradiol, the RNP containing fibrils located in the perichromatin and in the interchromatin regions are also involved.     

Localization and characterization of newly synthesized nuclear RNA in isolated rat hepatocytes

The ultrastructural localization of extranucleolar RNA transcribed during short periods of labeling with [³H]UdR in isolated rat hepatocytes is studied using high resolution autoradiography combined with a preferential staining for ribonucleoproteins. The labeled RNA is characterized in parallel experiments by electrophoresis on exponential polyacrylamide gels under denaturing conditions. It is demonstrated, using ultrathin sections of Epon embedded cells, that after 2 or 5 min of labeling the radioactivity is predominantly associated with perichromatin fibrils localized frequently in proximity to condensed chromatin regions. Autoradiographs of ultrathin frozen sections confirm the perichromatin localization of the rapidly labeled RNA. The great majority of this label is represented by growing chains of pre-mRNA. After 2 or 4 h of non-radioactive chase following the radioactive incubation, the major part of silver grains is still associated with perichromatin fibrils but is found distributed rather homogeneously throughout the nucleoplasm. This would suggest a migration of a part of the labeled perichromatin fibrils towards the interchromatin regions. At this time the label is characterized as pre-mRNA of intermediate size. These findings are discussed in the context of other recent investigations of the localization of newly transcribed nuclear RNA.     

Proteomic analysis of major and minor allergens from isolated pollen cytoplasmic granules

Grass pollen is one of the most important vectors of aeroallergens. Under atmospheric conditions, pollen grains can release pollen cytoplasmic granules (PCGs). The allergens associated with these intrinsic subfractions induce, in laboratory animals as well as in asthmatic patients, allergic and inflammatory responses. The objectives of this study were to characterize the PCGs' intrinsic allergens and to compare them with those of pollen grains. The water-soluble proteins were extracted from pollen grains and their PCGs. IgE-binding proteins were analyzed and characterized through an allergomic strategy: 1- and 2-dimensional gel electrophoresis (1-DE and 2-DE), immunoblotting, using grass-pollen-sensitized patient sera, mass spectrometry (MS) analysis, and database searching. Several of the allergens listed in the IUIS nomenclature, Phl p 1, 4, 5, 6, and 12, were detected in pollen and PCG extracts, whereas Phl p 11 was found only in PCGs, and Phl p 2 as well as Phl p 13 were found only in pollen extract. Some other allergens not listed in the IUIS nomenclature were also characterized in both pollen and PCG extracts. Since the major grass pollen allergens were found in PCGs and because of their small size, these submicronic particles should be considered as very potent sensitizing and challenging respirable vectors of allergens.     

Fine Structure and Cytochemistry of the Nucleus and the Kinetoplast of Epimastigotes of Trypanosoma cruzi1

ABSTRACT. Ultrastructural cytochemical techniques were used to analyze the nucleus and the kinetoplast of epimastigotes of Trypanosoma cruzi. With the use of ethanolic phosphotungstic acid, which detects basic proteins, reaction product was seen in the chromatin and at the periphery of the kinetoplast. Thallium alcoholate, which interacts with DNA, stained strongly the whole kinetoplast and the chromatin. With the use of a silver impregnation method that detects acidic nucleolar proteins, silver granules were seen preferentially located in the central region of the nucleolus. With the EDTA method, which reveals the presence of ribonucleoproteins, staining was observed in the nuclear pores. Also 6–8 nm fibrils, 25 nm and 40 nm granules, which correspond to the perichromatin fibers, interchromatin granules and the perichromatin granules, respectively, were identified in the nucleus. The EDTA method also revealed the presence of 40 nm granules in the kinetoplast. These granules were seen mainly at the two extremities of the kinetoplast. Freeze-fracture images indicate that the nuclear membrane contains ca. 9 pores/μm² of nuclear surface area. The mean diameter of the pores was 80 nm. All these results suggest that epimastigotes of T. cruzi have a very active nucleus and a high rate of nucleocytoplasmic interchange.     

Fine structural organization of a non-reticulate plant cell nucleus

We studied the fine structural organization of the meristematic nucleus in roots of Lycopesicon esculentum (tomato) using ultracytochemical and immunocytochemical approaches. The nucleus has a non-reticulate (i.e. low DNA content) structure whose supramolecular organization differs in some respects from that in reticulate nuclei, principally in the organization of the chromocentres associated with the nuclear envelope, with which centromeric structures appear to be associated. The main difference at the nucleolar level is found in the fibrillar centres, which have a low amount of DNA labelling and in which inclusions of condensed chromatin are present only very rarely. The distribution of nucleolar DNA amongst the nucleolar compartments is similar to that in reticulate nucleoli as demonstrated using an anti-DNA monoclonal antibody. Tomato nuclei have nucleolus-associated bodies or karyosomes, like other plant species with a low DNA content and non-reticulate nuclear organization. The nuclear ribonucleoprotein structures in the inter- and perichromatin regions, namely inter- and perichromatin fibrils and granules, show similar ultrastructural and cytochemical characteristics in both types of nuclei.Abbreviations NAC nucleolus associated chromatin - CES centromeric structures - NOR nucleolar organizing region - NAB nucleolus associated body - IG interchromatin granules - RNP ribonucleoprotein - Mab monoclonal antibody by M.F. Trendelenburg     

Immunocytochemical identification of nuclear structures containing snRNPs in isolated rat liver cells

Small nuclear ribonucleoproteins (snRNPs) containing U1, U2, U4, U5, and U6 small nuclear RNAs were detected by ultrastructural immunocytochemistry in the nuclei of isolated rat hepatocytes using Fab fragments of anti-Sm and anti-RNP autoantibodies. Their localization was carried out in normal cells and in cells treated with two drugs, the adenosine analog DRB and CdCl2, which alter the number and distribution of nuclear RNP components. It was found that more precise determination of the distribution of these small RNAs could be obtained by using two complementary procedures in parallel rather than either one alone. They consisted of an indirect immunoperoxidase labeling carried out before embedment and an indirect immunogold labeling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that snRNPs are associated with all extranucleolar perichromatin fibrils and granules and interchromatin fibrils, which confirms that they occur in structures involved in the synthesis and processing of hnRNA. The snRNPs are not associated with nucleolar perichromatin granules induced by DRB, which confirms that there may be two kinds of perichromatin granules. The snRNPs are also associated with the still enigmatic interchromatin granules which apparently do not contain hnRNA but at least in DRB-treated cells, also contain ribosomal RNA.     

Correlation between isolated lampbrush chromosomes and nuclear structures of Pleurodeles waltl oocytes: An electron microscopic study

The organization of the lampbrush chromosomes of Pleurodeles waltl was studied by fixation and embedding of oocytes in toto and correlated with that observed in end-embedded preparations of isolated chromosomes. Particular attention was focused on marker loops, like the granular and globular loops, and atypical structures known as spheres (S) and M. In both types of preparations, the majority of the loops, the so-called normal loops, and the granular loops appeared to share a common basic organization, with ribonucleoprotein (RNP) fibrils appearing as strings of 30 nm particles, as described by earlier authors. Some new types of loop organization were observed: (i) P loops with 45 nm RNP particles; (ii) dense granular loops; (iii) loops with a cylindrical organization. RNP fibrils formed by 60 nm particles were found to occur in association with the globular loops. EDTA staining suggested the presence of large amounts of RNP in the sphere but very little in M. Three morphologically different types of RNP granules could be observed free in the nucleoplasm.     

The effects of zinc chloride on the RNP structures in HEp-2 cells: accumulation of perichromatin granules

The effects of zinc on the ribonucleoprotein (RNP) constituents of HEp-2 cells have been analyzed. Pulse-chase autoradiographic experiments show a preferential inhibition of nucleolar RNA synthesis and a block in the transport of nucleolar and extranucleolar RNA in zinc-treated cells. Concomitantly with the disturbance in RNA metabolism and in protein synthesis, nucleolar condensation, accumulation of perichromatin granules and fibrils, condensation of interchromatin fibrils, and appearance of dense granular bodies occur. Accumulation of perichromatin fibrils and condensation of interchromatin fibrils appear to be related to the block in the transport of heterogeneous nuclear RNA. Depletion of certain proteins required for the assembly of RNP particles could share in the abnormal behavior of RNA and lead to the accumulation of perichromatin granules and the appearance of dense granular bodies.     

Electron microscope study of ribonucleoprotein polyparticles and their relation to perichromatin granules

Nuclear ribonucleoprotein particles were studied in the central nervous system of the rat after fixation by perfusion with hypotonic-detergent formaldehyde. Beads-on-a-string structures (polyparticles or chains) formed of 14-nm granules related by a 7-nm thick filament were found. These polyparticles are positively contrasted by a preferential method for ribonucleoproteins and they are sensitive to RNase. Perichromatin granules are loosened by this fixation and their internal structure is clearly depicted. They are composed of a tangled mass of 3-nm thick filaments, but lack 14-nm granules. The internal filaments of the perichromatin granules display frequent continuities with polyparticles. These results show that polyparticles correspond to perichromatin fibrils visualized in nuclei by standard fixation procedures.     

Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions

The self-assembly of proteins and peptides into polymeric amyloid fibrils is a process that has important implications ranging from the understanding of protein misfolding disorders to the discovery of novel nanobiomaterials. In this study, we probe the stability of fibrils prepared at pH 2.0 and composed of the protein insulin by manipulating electrostatic interactions within the fibril architecture. We demonstrate that strong electrostatic repulsion is sufficient to disrupt the hydrogen-bonded, cross-β network that links insulin molecules and ultimately results in fibril dissociation. The extent of this dissociation correlates well with predictions for colloidal models considering the net global charge of the polypeptide chain, although the kinetics of the process is regulated by the charge state of a single amino acid. We found the fibrils to be maximally stable under their formation conditions. Partial disruption of the cross-β network under conditions where the fibrils remain intact leads to a reduction in their stability. Together, these results support the contention that a major determinant of amyloid stability stems from the interactions in the structured core, and show how the control of electrostatic interactions can be used to characterize the factors that modulate fibril stability.