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Biomedical subjects

U Scheer

Publications and source records attributed to U Scheer.

At least 55 records · Page 3Linked to original sources
PubMed1988

Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells.

After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtK2) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.

Animals↗
PubMed1988

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus.

Certain autoimmune sera contain antibodies against a nucleolar ribonucleoprotein particle associated with 7-2-RNA (R. Reddy et al. (1983) J. Biol. Chem. 258, 1383; C. Hashimoto and J. A. Steitz (1983) J. Biol. Chem. 258, 1379). In this study, we showed by immunofluorescence microscopy that antibodies reactive with 7-2-ribonucleoprotein immunolocalized in the granular regions of actinomycin D and 5,6-dichloro-1-beta-D-ribofurano-sylbenzimidazole (DRB)--segregated nucleoli from Vero cells. By electron microscopic immunocytochemistry, antigen-antibody complexes were located in the granular component of transcriptionally active nucleoli from rat liver hepatocytes and HeLa cells. Anti-7-2-RNP antibodies from two autoimmune sera immunoprecipitated a major protein of Mr 40,000 from [35S] methionine--labeled HeLa cell extract. The immunolocalization data suggest that 7-2-ribonucleoprotein may be involved in stages of ribosome biogenesis which take place in the granular component of the nucleolus, i.e., assembly, maturation, and/or transport of preribosomes.

Animals↗
PubMed1988

Inhibition of nuclear accumulation of karyophilic proteins in living cells by microinjection of the lectin wheat germ agglutinin.

The lectin wheat germ agglutinin (WGA), which has been reported to inhibit nuclear protein uptake in vitro by isolated nuclei (Finlay et al. (1987) J. Cell Biol. 104, 189), also blocks, on microinjection into living cells, the migration of proteins into the cell nucleus. Radioactively labeled nuclear proteins were injected into the cytoplasm of Xenopus oocytes and their reentry into the nucleus was analyzed in the presence or absence of WGA by two-dimensional gel electrophoresis. In another set of experiments, fluorescently labeled nucleoplasmin was injected, alone or together with WGA, into the cytoplasm of rat hepatoma cells, and its nucleocytoplasmic distribution was studied by quantitative laser fluorescence microscopy. The results indicate that WGA inhibits the uptake of karyophilic proteins in general, independent of their sizes. Since the nucleocytoplasmic flux of a dextran with Mr 10,000 was not affected it can be excluded that WGA acts by a general blockade or constriction of the functional pore channel. At reduced WGA concentrations, the rate but not the final extent of nuclear protein accumulation was decreased. These findings support the concept that the O-glycosidically bound carbohydrates of certain nuclear pore complex proteins are exposed to the pore interior and that these regions are probably involved in nucleocytoplasmic translocation processes.

Animals↗
PubMed1988

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↗
PubMed1988

Localization of DNA within Ehrlich tumour cell nucleoli by immunoelectron microscopy.

The distribution of DNA in Ehrlich tumour cell nucleoli was investigated by means of an immunocytochemical approach involving a monoclonal antibody directed against double- and single-stranded DNA. Immunolabelling was performed either before or after the embedding process. The postembedding labelling method allows better ultrastructural preservation than the preembedding labelling method. In particular, the various nucleolar components are well preserved and identifiable. In the nucleolus, labelling is particularly concentrated over the perinucleolar chromatin and over its intranucleolar invaginations, which penetrate the nucleolar body and often terminate at the fibrillar centres. In addition, aggregates of gold particles are found in the fibrillar centres, preferentially towards the peripheral regions. By contrast, the dense fibrillar component is completely devoid of labelling. The results seem to indicate that DNA containing the rDNA genes is located in the fibrillar centres, with a preference for the peripheral regions. This finding suggests that transcription of the rDNA genes should occur within the confines of the fibrillar centre, probably close to the boundary region of the surrounding dense fibrillar component. The results are discussed in the light of present knowledge of the functional organization of the nucleolus.

Animals↗
PubMed1988

Immunoelectron microscopic study of nucleolar DNA during mitosis in Ehrlich tumor cells.

In order to investigate the DNA localization within Ehrlich tumor cell nucleoli during mitosis, two recent immunocytochemical methods using either an anti-DNA or an anti-bromodeoxyuridine (BrdU) monoclonal antibody have been applied. In both cases, the immunogold labeling has been performed on ultrathin sections of cells embedded either in Lowicryl K4M or in Epon, respectively. Identical results are observed with both immunocytochemical approaches. In the interphase nucleolus, besides the labeling of the perinucleolar chromatin shell and of its intranucleolar invaginations which penetrate into the nucleolar body and often terminate at the fibrillar centers, a few gold particles are also preferentially found towards the peripheral region of the fibrillar centers. In contrast, the dense fibrillar component and the granular component are never labeled. During mitosis, the fibrillar centers persist at the chromosomal nucleolus organizing regions (NOR's) and can be selectively stained by the silver method. However, these metaphase fibrillar centers are no longer decorated by the DNA- or BrdU antibodies. These results indicate that until the end of prophase, rRNA genes are present inside the fibrillar center material, disappear during metaphase and reappear in reconstituting nucleoli during telophase. Thus, fibrillar centers appear to represent structures sui generis, which are populated by rRNA genes only when the nucleolus is functionally active. In segregated nucleoli after actinomycin D treatment, the DNA labeling is exclusively restricted to the perinucleolar chromatin blocks. These findings also suggest that the DNA content of the fibrillar center material varies according to the rRNA transcription level of the cells. The results are discussed in the light of the present knowledge of the functional organization of the nucleolus.

Animals↗
PubMed1987

Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells.

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.

Animals↗
PubMed1987

Autoantibody to RNA polymerase I in scleroderma sera.

Autoantibodies to components of the nucleolus are a unique serological feature of patients with scleroderma. There are autoantibodies of several specificities; one type produces a speckled pattern of nucleolar staining in immunofluorescence. In actinomycin D and 5,6-dichloro-beta-D-ribofuranosylbenzimidazole-treated Vero cells, staining was restricted to the fibrillar and not the granular regions. By double immunofluorescence, specific rabbit anti-RNA polymerase I antibodies stained the same fibrillar structures in drug-segregated nucleoli as scleroderma sera. Scleroderma sera immunoprecipitated 13 polypeptides from [35S]methionine-labeled HeLa cell extract with molecular weights ranging from 210,000 to 14,000. Similar polypeptides were precipitated by rabbit anti-RNA polymerase I antibodies, and their common identities were confirmed in immunoabsorption experiments. Microinjection of purified IgG from a patient with speckled nucleolar staining effectively inhibited ribosomal RNA transcription. Autoantibodies to RNA polymerase I were restricted to certain patients with scleroderma and were not found in other autoimmune diseases.

Antibody Specificity↗
PubMed1987

High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody.

A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.

Animals↗
PubMed1986

Immunolocalization and partial characterization of a nucleolar autoantigen (PM-Scl) associated with polymyositis/scleroderma overlap syndromes.

Precipitating anti-PM-Scl antibodies are present in sera from patients with polymyositis, scleroderma, and polymyositis/scleroderma overlap syndromes. By indirect immunofluorescence microscopy, anti-PM-Scl antibodies stained the nucleolus in cells of different tissues and species, suggesting that the antigen is highly conserved. By electron microscopy, anti-PM-Scl antibodies reacted primarily with the granular component of the nucleolus. Drugs that inhibit rRNA synthesis had a marked effect on the expression of PM-Scl antigen. In actinomycin D-treated cells, immunofluorescence staining by anti-PM-Scl was significantly reduced with residual staining restricted to the granular regions of nucleoli. Treatment with 5,6-dichloro-beta-D-ribofuranosylbenzimidazole (DRB) also selectively reduced nucleolar staining. On a molecular level, anti-PM-Scl antibodies precipitated 11 polypeptides with molecular weights (Mr) ranging from 110,000 to 20,000. The Mr 80,000 and 20,000 polypeptides were phosphorylated. Evidence suggests that the PM-Scl antigen complex may be related to a preribosomal particle.

Animals↗
PubMed1986

Immunolocalization of nucleolar proteins after D-galactosamine-induced inhibition of transcription in rat hepatocytes. Maintenance of association of RNA polymerase I with inactivated nucleolar chromatin.

The fate of defined nucleolar constituents during D-galactosamine-induced inhibition of transcription and the accompanying extensive structural changes such as nucleolar segregation, fragmentation and disappearance of the granular components was studied by light and electron microscopic immunolocalization, using antibodies to different nucleolar components. In contrast to other inhibitors such as actinomycin D, we show that preribosomal components as monitored by a ribosomal protein leave the nucleolus, while a large proportion of RNA polymerase I remains associated with the nucleolar chromatin, i.e. probably the pre-rRNA genes, during inactivation of transcription. These small structures containing the RNA polymerase I are characterized by low electron density and resemble the 'fibrillar centers' of normal nucleoli. The results are discussed in relation to current concepts of the functional topology of the nucleolus.

Animals↗
PubMed1985

Ribocharin: a nuclear Mr 40,000 protein specific to precursor particles of the large ribosomal subunit.

Using a monoclonal antibody (No-194) we have identified, in Xenopus laevis and other amphibia, an acidic protein of Mr 40,000 (ribocharin) which is specifically associated with the granular component of the nucleolus and nucleoplasmic 65S particles. These particles contain the nuclear 28S rRNA and apparently represent the precursor to the large ribosomal subunit in nucleocytoplasmic transit. By immunoelectron microscopy ribocharin has been localized in the granular component of the nucleolus and in interchromatin granules. During mitosis ribocharin-containing particles are associated with surfaces of chromosomes and are recollected in the reconstituting nucleoli in late telophase. We suggest that ribocharin is a specific component of precursor particles of the large ribosomal subunit, which dissociates from the 65S particle before passage through the nuclear envelope, and is reutilized in ribosome biogenesis.

Animals↗
PubMed1985

Localization of ribosomal protein S1 in the granular component of the interphase nucleolus and its distribution during mitosis.

Using antibodies to various nucleolar and ribosomal proteins, we define, by immunolocalization in situ, the distribution of nucleolar proteins in the different morphological nucleolar subcompartments. In the present study we describe the nucleolar localization of a specific ribosomal protein (S1) by immunofluorescence and immunoelectron microscopy using a monoclonal antibody (RS1-105). In immunoblotting experiments, this antibody reacts specifically with the largest and most acidic protein of the small ribosomal subunit (S1) and shows wide interspecies cross-reactivity from amphibia to man. Beside its localization in cytoplasmic ribosomes, this protein is found to be specifically localized in the granular component of the nucleolus and in distinct granular aggregates scattered over the nucleoplasm. This indicates that ribosomal protein S1, in contrast to reports on other ribosomal proteins, is not bound to nascent pre-rRNA transcripts but attaches to preribosomes at later stages of rRNA processing and maturation. This protein is not detected in the residual nucleolar structures of cells inactive in rRNA synthesis such as amphibian and avian erythrocytes. During mitosis, the nucleolar material containing ribosomal protein S1 undergoes a remarkable transition and shows a distribution distinct from that of several other nucleolar proteins. In prophase, the nucleolus disintegrates and protein S1 appears in numerous small granules scattered throughout the prophase nucleus. During metaphase and anaphase, a considerable amount of this protein is found in association with the surfaces of all chromosomes and finely dispersed in the cell plasm. In telophase, protein S1-containing material reaccumulates in granular particles in the nucleoplasm of the newly formed nuclei and, finally, in the re-forming nucleoli. These observations indicate that the nucleolus-derived particles containing ribosomal protein S1 are different from cytoplasmic ribosomes and, in the living cell, are selectively recollected after mitosis into the newly formed nuclei and translocated into a specific nucleolar subcompartment, i.e., the granular component. The nucleolar location of ribosomal protein S1 and its rearrangement during mitosis is discussed in relation to the distribution of other nucleolar proteins.

Animals↗