Biomedical subjects

R L Ochs

Publications and source records attributed to R L Ochs.

At least 55 records · Page 3Linked to original sources

PubMed1994

Coiled bodies in the nucleolus of breast cancer cells.

Coiled bodies are a special type of small round nuclear body, composed of coiled fibers and granules, especially prominent in the nucleoplasm of highly active cells (Brasch and Ochs (1992) Exp. Cell Res. 202, 211-223). Although no specific function has been assigned to coiled bodies, they contain spliceosome snRNAs and proteins, as well as the nucleolar U3 RNA-associated protein fibrillarin. In the present study, we have used antibodies to the coiled body-specific protein p80-coilin, together with double-label immunofluorescence, confocal microscopy and immunoelectron microscopy, to examine the distribution of coiled bodies in a number of different breast cancer cell lines. By immunofluorescence, all cell lines had prominent coiled bodies in the nucleoplasm and several cell lines appeared to have coiled bodies within the nucleolus itself. Double-label immunofluorescence and confocal laser scanning microscopy confirmed the nucleolar localization of coiled bodies. Besides containing p80-coilin, nucleoplasmic and nucleolar coiled bodies contained fibrillarin and Sm proteins. By conventional and immunoelectron microscopy, nucleolar coiled bodies appeared as discrete structures within the nucleolus in a number of different morphotypes, distinct from the normal nucleolar domains of granular component, dense fibrillar component, and fibrillar centers. While the significance of finding coiled bodies in the nucleolus of certain breast cancer cell lines is at present unknown, this represents the first report of coiled bodies and Sm staining in the nucleolus of mammalian cells.

Autoantigens↗

PubMed1993

Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.

Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.

Amino Acid Sequence↗

PubMed1993

Electron microscopic localization of ribosomal DNA in rat liver nucleoli by nonisotopic in situ hybridization.

We have used postembedding nonisotopic in situ hybridization, with biotinylated rat ribosomal DNA (rDNA) as a probe and streptavidin coupled to 10-nm colloidal gold particles as the detection system, to localize rDNA sequences in rat liver nucleoli at the electron microscopic level. For comparison purposes, immunoelectron microscopy was performed for the detection of DNA. Our results indicate that ribosomal DNA sequences are enriched in the dense fibrillar component of the rat liver nucleolus. These data are discussed in relation to the putative site(s) for transcription of ribosomal genes.

Animals↗

PubMed1993

Autoantibodies to a novel cell cycle-regulated protein that accumulates in the nuclear matrix during S phase and is localized in the kinetochores and spindle midzone during mitosis.

We have employed human autoantibodies to characterize a novel cell cycle-regulated nuclear protein, provisionally designated p330d (doublet polypeptide of 330 kDa). The expression and intracellular distribution of this protein was followed throughout the cell cycle using immunofluorescence microscopy, laser confocal microscopy, immunoelectron microscopy and flow cytometry. p330d was expressed only in proliferating cells and began accumulating in the nucleus during early S phase. The protein reached maximum expression levels during G2/M. In situ extractions with detergent, salt and nucleases failed to abolish the nuclear staining of interphase cells, suggesting a tight binding of p330d to the nuclear matrix during interphase. p330d was concentrated in the kinetochores during prophase but was relocated to the spindle midzone at the onset of anaphase. By late telophase, it was localized predominantly in the intercellular bridge regions flanking the midbody and disappeared gradually as the daughter cells separated. Immunoblotting analysis showed that the autoimmune sera recognized a doublet of 330 kDa, and affinity-purified antibodies from this doublet reproduced the fluorescence staining pattern of the whole serum. We propose that p330d is a novel member of the class of 'chromosomal passenger' proteins, which are associated transiently with centromeres during early mitosis and are then redistributed to other sites of the mitotic apparatus after the metaphase/anaphase transition. Possible in vivo functions for p330d and related proteins might include roles in centromere/kinetochore maturation and assembly, chromosome segregation, central spindle stabilization and cytokinesis.

Autoantibodies↗

PubMed1992

Antinuclear autoantibodies in women with silicone breast implants.

Clinical syndromes resembling autoimmune diseases have been reported in women who have had breast augmentation procedures. To see whether there is a humoral immune response in these diseases that is similar to the immune response in their idiopathic counterparts, we assessed the immunological specificity of antinuclear antibodies (ANAs) and certain epidemiological features in 24 patients, all of whom (with 1 exception) had received silicone gel breast implants. ANA specificities were identified by indirect immunofluorescence, immunodiffusion, western blot analysis, and immunoprecipitation of radiolabelled intracellular proteins. Of 11 patients who had symptoms and signs that met criteria for defined autoimmune diseases, 7 had scleroderma or subsets of this disorder and the others had systemic lupus erythematosus, rheumatoid arthritis, or overlapping autoimmune diseases. High ANA titres were present in 10 of these 11 patients and the ANA specificities were similar to those found in the idiopathic forms of the corresponding autoimmune diseases. Trauma, with resultant rupture of implants, accelerated onset of symptoms. 13 other patients had autoimmune disorders of a less clearly defined nature and low titres of ANAs whose specificities could not be identified. ANAs are associated with the development of autoimmune complications in women with silicone breast implants. Further studies are needed to see whether this relation is one of cause and effect and whether ANAs might be early serological markers preceding development of autoimmune symptoms.

Academies and Institutes↗

PubMed1992

Nuclear bodies (NBs): a newly "rediscovered" organelle.

Nuclear bodies (NBs) were first described in detail some 30 years ago, by conventional electron microscopy, as prominent interchromatin structures found primarily in the nuclei of malignant or hyperstimulated animal cells. Subsequent studies have shown that NBs are ubiquitous organelles, but they are numerically and morphologically quite varied. With the recent discovery of human autoantibodies against several key nuclear antigens present in some NBs, these structures are once again the subject of much attention. At least one class of NBs, coiled bodies, has been shown to be nucleolus-derived and to contain not only nucleolus-associated antigens, but also many of the snRNP components involved in pre-mRNA splicing. These data suggest that coiled bodies, and perhaps other NBs as well, are multifunctional and may be involved in the processing or transport of both pre-mRNA and pre-rRNA. Further evidence is provided showing that NBs constitute distinct nuclear domains whose functional significance is just now emerging.

Animals↗

PubMed1992

Centromere autoantigens are associated with the nucleolus.

Because of their importance as target antigens in scleroderma and since all other major autoantigens in scleroderma can be localized to the interphase nucleolus, we were interested in a further investigation of the potential relationship between interphase centromeres and the nucleolus. Using human anticentromere autoantibodies (ACA) from patients with the CREST form of scleroderma as probes in indirect immunofluorescence microscopy, we observed nonrandom interphase "clumping" of centromeres in a distribution suggestive of nucleoli. By double-label immunofluorescence comparing the localization of centromeres to nucleolar proteins Ki-67, fibrillarin, or protein B23 (nucleophosmin), interphase centromeres appeared to be localized around and within nucleoli. A number of different ACA sera were tested on HEp-2, HeLa, PtK2, Indian muntjac, 3T3, and NRK cells, all with identical results indicating colocalization between centromeres and nucleoli. Immunoelectron microscopy revealed that interphase centromeres were distributed free in the nucleoplasm, in contact with the nuclear envelope, in contact with and on the periphery of nucleoli, and totally embedded within the confines of the nucleolus itself. Interestingly, actinomycin D treatment dissociated centromeres from localization within the segregated nucleolus. To determine if interphase centromeres were integral components of nucleoli, nucleoli were isolated according to classical methods. By double-label immunofluorescence, immunoelectron microscopy, and Western blotting, it was demonstrated that centromere autoantigens copurified with isolated nucleoli. These studies offer proof that some interphase centromeres can be associated with, and may even be considered part of, the interphase nucleolus. Furthermore, all of the major autoantigens in scleroderma can now be localized to the nucleolus.

Autoantigens↗

PubMed1992

Nucleolar antigens and autoantibodies in hepatocellular carcinoma and other malignancies.

Patients with hepatocellular carcinoma (HCC), gastrointestinal, lung, and ovarian cancers were shown to have autoantibodies to nuclear and nucleolar antigens as detected by immunofluorescence on cell substrates. The frequency of antinuclear antibodies (ANAs) was significantly higher (P less than 0.001) in patients with HCC (57/184 = 31%) than in patients with chronic hepatitis or liver cirrhosis (25/187 = 13%). Although a range of fluorescence patterns was observed, a higher percentage of nucleolar fluorescence was detected in HCC, and three of these nucleolar antigens were identified. They were NOR-90, nucleolus organizer region doublet polypeptides of 93 and 89 kDa involved in RNA polymerase I transcription; fibrillarin, a 34 kDa protein of the nucleolar U3 ribonucleoprotein particle which is engaged in preribosomal RNA processing; and nucleophosmin/protein B23, a 37 kDa polypeptide which is associated with ribosome maturation and cellular proliferation. All these antigens are nucleolar components that are engaged in some aspect of ribosome biosynthesis. Since autoantibodies to these nucleolar antigens have also been found in systemic autoimmune diseases, they do not represent autoimmune reactions unique to cancer but might reflect reaction pathways related to immune responses that are antigen-driven. The ANA response in HCC appears to be dynamic reactions to this antigen-drive since some patients with chronic liver disease showed seroconversion to ANA positivity, marked increase in titer and/or change in antibody specificity preceding or coincident with clinical detection of HCC. These changes in ANA showed a close temporal relationship with transformation from long-established chronic liver disease to HCC.

Antibodies, Neoplasm↗

PubMed1991

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix.

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.

Animals↗

PubMed1991

Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies.

Studies with human autoimmune sera identified auto-antibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the coiled body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the coiled body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function.

Animals↗

PubMed1990

Immunocytochemistry of the cell nucleus.

This electron microscopic review addresses in situ immunocytochemistry of the mammalian cell nucleus with special reference to the use of autoantibodies, which are the major source of antinuclear antibodies. The localization of many key nuclear antigens is documented and immunocytochemical data are related to the major functional processes of transcription and processing of RNA and to replication of DNA.

Animals↗

PubMed1990

Studies on satellite nucleoli in human blastic cells of acute leukaemias.

Blastic cells of acute lymphoid, acute myeloid and acute myelomonocytic leukaemias were studied by means of indirect immunofluorencence to provide more information on the presence of satellite nucleoli in blood cells. According to results, satellite nucleoli were found in a small but constant number of blastic cells disregarding their type and type of acute leukaemia. Satellite nucleoli exhibited a positive immunoreaction for fibrillarin and protein B23 which are characteristic for main nucleolar components. These findings suggest that satellite nucleoli contain fibrillar centers as well as dense fibrillar and granular components or at least proteins characteristic for these nucleolar components. Similarly as in normal and pathological cells of completely different origin, in blastic cells of acute leukaemias the number of satellite nucleoli per cells ranged between 1 and 2.

Cell Nucleolus↗

PubMed1989

Fibrillar center distribution in nucleoli of PHA-stimulated human lymphocytes.

We have utilized acidic toluidine blue staining for RNA and immunofluorescence staining for RNA polymerase 1 to visualize the distribution of fibrillar centers (FCs) in nucleoli of PHA-stimulated human lymphocytes. At 0 h, there is a single large fibrillar center in each nucleolus which splits into smaller and more numerous FCs until the number of FCs reaches five, the number of nucleolus organizers in normal haploid human cells. With time, each FC then "unwinds" to form linear arrays of smaller FCs until the maximum number of FCs approaches the ribosomal gene copy number of 200 at 48 h in culture. It is hypothesized that in the most active state, each nucleolar FC visualized by RNA polymerase 1 staining actually represents a single transcription unit and the distance between adjacent FCs is occupied by the nontranscribed spacer region. We conclude that the number of fibrillar centers per nucleolus can be used as a direct quantitative measure of nucleolar transcriptional activity.

Cell Cycle↗

PubMed1989

Nucleologenesis: use of non-isotopic in situ hybridization and immunocytochemistry to compare the localization of rDNA and nucleolar proteins during mitosis.

Using in situ hybridization and immunocytochemistry during interphase and mitosis, we have compared the distribution of ribosomal DNA (rDNA) to that of the nucleolar proteins fibrillarin and RNA polymerase I. During interphase, nucleolar proteins were localized at sites throughout the nucleolus while the bulk of rDNA was localized in a single restricted nucleolar area. During metaphase and anaphase, all six NORs were detected by in situ hybridization, Ag-staining, or by the immunolocalization of RNA polymerase I. During telophase, rDNA and RNA polymerase I were found in a distinct subset of the prenucleolar bodies (PNBs) which obviously must contain the nucleolar organizers. Other numerous PNBs are smaller in size and do not contain detectable amounts of rDNA or RNA polymerase I. Therefore, reconstruction of the nucleolus originates in telophase-specific domains which contain both rDNA and RNA polymerase I.

Animals↗

PubMed1988

Intranucleolar localization of human proliferating cell nucleolar antigen p120.

The human proliferation-associated nucleolar antigen p120 was localized to substructures within HeLa cell nucleoli by immunofluorescence and immunoelectron microscopy of cells whose nucleoli were segregated by drug treatment or extracted with nucleases. By indirect immunofluorescence, protein p120 was localized diffusely throughout all interphase nucleoli. However, high resolution immunoelectron microscopy demonstrated that protein p120 staining delineated a network of 20-30-nm diameter beaded fibrils distributed throughout the nucleolus. This distribution was unique compared to that of the nucleolar proteins p145, RNA polymerase I, or B23 which were examined simultaneously. Drug-induced segregation of nucleoli by actinomycin D or dichlorobenzimidazole riboside, followed by immunoelectron microscopy, indicated that protein p120 was concentrated at the periphery of the granular region in segregated nucleoli. In situ nuclease digestion of cells with DNase I and/or RNase A did not release p120 from the nucleolus. Instead, p120 immunoreactivity was retained within phase-dense residual nucleoli. These results provide evidence that protein p120 is associated with, and in fact delineates, a network of fibrils which is retained in the nucleolar residue fraction of proliferating cells.

Antigens, Neoplasm↗

PubMed1987

Effect of differentiation on the expression of a nucleolar antigen with a molecular weight of 145,000 in HL-60 cells.

Mr 145,000 nucleolar protein antigen (p145) is associated with growing cells (R. L. Ochs et al., J. Cell Biol., 101: 211a, 1985) and has been found in a broad range of human cancers (J. W. Freeman et al., Cancer Res., 46: 3593-3598, 1986). In this study the presence of nucleolar antigen p145 was examined in the human promyelocytic tumor cell line HL-60 which was induced to differentiate by retinoic acid. Differentiation was monitored by morphological changes, [3H]thymidine accumulation, the ability of cells to reduce nitroblue tetrazolium, and cell number. The monoclonal antibody to nucleolar antigen p145 produced bright immunofluorescence in all cycling interphase HL-60 cells; during mitosis only diffuse staining was detected. Nucleolar antigen p145 in HL-60 cells was undetectable after 132 h of treatment with retinoic acid. The absence of nucleolar antigen p145 was associated with an 81% decline in thymidine accumulation and apparent inactivation of ribosomal and nonribosomal DNA transcription as observed by electron microscopy. The loss in expression of the antigen also correlated with increased nitroblue tetrazolium-positive cells, appearance of morphologically distinct myeloid cells, and termination of cell proliferation. These data indicate that the expression of nucleolar antigen p145 occurred in cycling HL-60 cells but not in terminally differentiated noncycling HL-60 cells.

Antibodies, Monoclonal↗

PubMed1987

Monoclonal autoantibody from a (New Zealand black x New Zealand white)F1 mouse and some human scleroderma sera target an Mr 34,000 nucleolar protein of the U3 RNP particle.

A monoclonal IgG2a antinucleolar autoantibody (72B9) was obtained by fusion of spleen cells from a (New Zealand black x New Zealand white)F1 mouse with myeloma cells (P3x63Ag8.653). Antibody 72B9 recognized a highly conserved nucleolar antigen present in both animal and plant cells. The staining pattern produced by antibody 72B9 in different cell substrates was identical with those obtained by scleroderma antibodies reactive with a basic (pI 8.5) nucleolar protein of Mr 34,000, which is associated with the U3 RNP particle. Western blotting further confirmed its reactivity with this scleroderma-related U3 RNP protein.

Animals↗

PubMed1987

Modulation of fibronectin, laminin, and cellular adhesion in the transformation and differentiation of murine AKR fibroblasts.

The functional relationship between membrane/cell surface expression of fibronectin and laminin and transformation/differentiation was examined in an AKR mouse fibroblastic cell model. This model consisted of the untransformed AKR-2B cells, their chemically transformed counterpart (AKR-MCA cells) and the chemically differentiated form of the AKR-MCA cells. The transformed AKR-MCA cells were found to express more surface laminin and less fibronectin than the untransformed AKR-2B cells. The transformed AKR-MCA cells were slower to attach and spread on both plastic and type IV collagen-coated dishes in comparison to the AKR-2B cells. However, a higher percentage of the AKR-MCA cells ultimately attached and spread on the type IV collagen-coated dishes. The induction of differentiation in the AKR-MCA cells by N,N-dimethylformamide (DMF) restored fibronectin to the surface of the AKR-MCA cells but reduced laminin expression only slightly. The DMF-treated AKR-MCA cells resembled the AKR-2B cells in that they rapidly attached and spread on plastic dishes and dishes coated with type IV collagen. They also resembled the AKR-MCA cells in that a high proportion ultimately attached and spread on the collagen-coated dishes.

Animals↗