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R L Ochs

Publications and source records attributed to R L Ochs.

At least 73 records · Page 4Linked to original sources
PubMed1987

Studies on satellite nucleoli in rat hepatocytes and Novikoff hepatoma cells.

The present study was undertaken to provide information on the presence and frequency of satellite nucleoli in cells with increased nucleolar biosynthetic activity. The number of hepatocytes containing satellite nucleoli was analyzed in rat liver, regenerating liver 18 h after partial hepatectomy and in Novikoff hepatoma ascites cells. In comparison with hepatocytes of normal liver, the number of both stimulated hepatocytes and malignant hepatoma cells containing satellite nucleoli was significantly reduced. The results also indicated that whereas most satellite nucleoli contain protein C23, a smaller percentage contain protein B23.

Animals↗
PubMed1987

Ultrastructural and biochemical comparisons of nuclear matrices prepared by high salt or LIS extraction.

We have directly compared two independently published methods for isolating operationally defined nuclear matrices by studying EM ultrastructure, protein composition and distribution of replicating DNA. Nuclear matrices prepared by extraction with 2 M NaCl consisted of fibrous pore complex lamina, residual fibrillar and granular components of nucleoli and interchromatin granules, and an extensive anastomosing internal fibrous network. These matrices were enriched in high molecular weight nonhistone proteins but were virtually devoid of histones. Consistent with previously published data, newly-replicated DNA was resistant to this high salt extraction. Nuclear matrices prepared by extraction of nuclei with 25 mM lithium 3,5-diiodosalicylate, LIS, also contained fibrous pore complex lamina, but lacked morphologically distinct residual nucleoli and were markedly depleted in internal structure. The reduced amounts and complexity of proteins associated with the LIS matrix were consistent with the ultrastructural data. Moreover, much less newly-replicated DNA was recovered in LIS matrices. The data show that LIS dissociates nuclear ultrastructure and extracts both protein and DNA in proportion to the concentration used, regardless of whether nuclei or high salt nuclear matrices are used as starting material. While the data suggest that LIS may not necessarily be an optimal reagent for preparing nuclear matrices containing internal structural elements from all tissue sources, it may be useful for selectively solubilizing and analyzing components of the nuclear matrix.

Animals↗
PubMed1986

Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: further evidence for an in situ nuclear matrix.

Polyclonal antibodies have been produced which react with a nuclear protein having a molecular weight of 107kD and a pI of 8.7-8.8 (designated p107). This protein is shown to be a component of the residual ribonucleoprotein (RNP) network of the nuclear matrix. P107 localized exclusively to the nuclear interior but not within nucleolar or chromatin domains. We have taken advantage of this unique probe to examine whether the RNP network of the isolated nuclear matrix has a physical counterpart in situ. We show that RNA, p107, divalent cations and the 28 kD Sm antigen of U-snRNPs are components of in situ macromolecular assemblies. While the morphology and intranuclear distribution of these assemblies are insensitive to the removal of chromatin, they are markedly altered by degradation of RNA. Digestion in situ of RNA in the presence of EDTA followed by extraction with high ionic strength buffers solubilized the components of these assemblies. Electron microscopic and immunobiochemical data are presented which support the concept that the residual RNP network of the nuclear matrix is an isolate of a pre-existing structure, and that perturbations in this internal network can be created by RNA degradation, depletion of essential metal ions and proteolysis.

Animals↗
PubMed1986

Intermediate filament proteins in human sperm heads.

Monoclonal antibodies made against human sperm cells have been characterized with regard to binding patterns and molecular coordinates of the recognized antigens. Antibodies T5 and T6 gave uniform binding to the acrosomal cap in an intact cell, and decreased to equatorial segment binding in an 'acrosome-reacted' cell. Monoclonal antibody T15 gave the reverse: equatorial segment binding in intact cells and uniform acrosomal cap binding in reacted cells. From staining patterns on cultured cell lines, determination of molecular coordinates, immunoblots, and partial peptide analysis, we have determined that T15 is directed against the cytoskeletal protein, vimentin, while T5 and T6 recognize a keratin-like protein which may be unique to sperm cells. This is the first immunological and biochemical study to analyse both types of intermediate filament proteins in human sperm cells.

Antibodies, Monoclonal↗
PubMed1985

Purification and partial characterization of a nucleolar scleroderma antigen (Mr = 34,000; pI, 8.5) rich in NG,NG-dimethylarginine.

A new scleroderma antigen of Mr = 34,000; pI, 8.5 has been identified. This 34-kDa protein is a nucleolar protein as determined by immunostaining procedures with affinity-purified antibodies. The 34-kDa protein was shown to localize to the fibrillar regions of the nucleolus by immunoelectron microscopy. Antibodies against the 34-kDa protein precipitate U3 RNA-containing particles. The 34-kDa protein has been isolated from Novikoff hepatoma cell nucleoli by ion exchange and reverse-phase column chromatography. The protein contains 4.1 mol % NG,NG-dimethylarginine (DMA) and 22.8 mol % glycine. It is the most highly arginine-methylated protein thus far detected in higher eukaryotes. This nucleolar 34-kDa protein resembles several nucleoplasmic proteins that are associated with heterogeneous nuclear RNA with respect to isoelectric point, Mr, presence of NG,NG-dimethylarginine, and its high glycine content. The amino-terminal sequence of the first 31 residues of the 34-kDa protein is: Met-Lys-Pro-Gly-Phe-Ser-Pro-DMA-Gly-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly- Phe-Gly-Asp-DMA-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-DMA. In the first 31 residues, there are 16 glycine, 6 DMA, and 3 phenylalanine residues. This is a novel demonstration of clusters of glycine and DMA in a protein.

Amino Acid Sequence↗
PubMed1985

Nucleologenesis: composition and fate of prenucleolar bodies.

A time course study was conducted on nucleologenesis after release from a mitotic block in the presence and absence of actinomycin D to determine the composition and fate of prenucleolar bodies (PNBs). Prenucleolar bodies, whether naturally occurring or induced by actinomycin D treatment, stain with silver and contain phosphoproteins B23 and C23, two of the major proteins of the interphase nucleolus as determined by double label immunofluorescence with specific antibodies. The nucleolus is formed by fusion of PNBs, which subsequently "reorganize" and form internal fibrillar and peripheral granular regions. Actinomycin D prevents fusion of PNBs, which are then randomly dispersed throughout the nucleus but they still contain proteins B23 and C23. These results demonstrate that the nucleolus is formed by fusion of prenucleolar structures whose biochemical composition resembles the mature nucleolus, since PNBs contain at least two of the major nucleolar proteins.

Animals↗
PubMed1985

Alterations in chromatin conformation are accompanied by reorganization of nonchromatin domains that contain U-snRNP protein p28 and nuclear protein p107.

The intranuclear distribution of nuclear matrix-associated protein p107 and the 28-kD Sm antigen of U-snRNPs have been studied using double-label immunofluorescence and immunoperoxidase electron microscopy. In interphase nuclei of HeLa cells, Novikoff hepatoma cells, and rat kangaroo kidney cells, p107 was confined to discrete interchromatin domains. The domains had an irregular contour, with an average diameter of 1-1.5 micron. Each domain appeared to be composed of interconnected granules. The Sm antigen colocalized and appeared concentrated in these domains but also showed some general nucleoplasmic distribution. During mitosis, the interchromatin domains disassembled such that the Sm portion redistributed to the perichromosomal and spindle regions and the p107 component redistributed throughout the mitotic cytoplasm. During anaphase, p107 assembled into discrete clusters throughout the mitotic cytoplasm. The Sm antigen was not a component of these clusters. Double-label immunofluorescence with anti-p107 and the anti-DNA tight-binding protein, AhNa1, showed that the extranuclear p107 domains assumed an interchromatin localization only after the chromosomes had decondensed. The correlation between chromosome decondensation and the occurrence of p107 within interchromatin domains was also observed during chicken erythrocyte nuclear reactivation. We propose that the discrete interchromatin domains that contain p107 and p28 may be important for processing and splicing of RNA and that their structural assembly within nuclei is sensitive to the presence of the transcriptionally active conformation of chromatin.

Animals↗
PubMed1985

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera.

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.

Animals↗
PubMed1985

The effect of harmine on the localization of the nucleolar proteins C23 and B23.

In this study we show that harmine treatment (10 mg/l for 2 or 24 h) of PtK2 cells had a marked effect on the localization of the nucleolar phosphoproteins C23 and B23. C23 was localized with silver staining in the fibrillar areas of completely segregated nucleoli. B23 was localized mainly on the periphery of the nucleoli with the aid of immunofluorescence.

Alkaloids↗
PubMed1984

Silver staining, immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23.

Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.

Animals↗
PubMed1984

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein.

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.

Animals↗
PubMed1983

Nuclear matrix, hnRNA, and snRNA in friend erythroleukemia nuclei depleted of chromatin by low ionic strength EDTA.

A novel and rapid procedure is described for the preparation of chromatin-depleted nuclei (CDN) from Friend erythroleukemia cells under conditions that avoid the use of high salt concentrations. By this procedure isolated nuclei that had previously been incubated with DNase I to partially digest DNA were washed twice in 2 mM EDTA to extract the chromatin. The resulting structures contained 1% of DNA, 65% of total RNA, 60-80% of hnRNA, 74% of snRNA, 29% of protein, and 2% of histones contained in isolated nuclei. Electron microscopy revealed intact, spherical structures similar in diameter to isolated nuclei and consisting of dense networks of fibrils 50-100 A thick surrounded by distinct nuclear laminae. Although no morphological evidence was found for residual nucleoli, C23, a nucleolus-specific phospho-protein, remained centrally localized in CDN, while Sm antigen specific for snRNPs was diffusely localized but absent from central regions. Addition of 2 mM MgCl2 to CDN resulted in the reformation of morphologically distinguishable residual nucleoli. These studies suggest that nucleolar morphology is, in part, dependent upon divalent cations and components unique to the nucleolar matrix and demonstrate that little randomization of nuclear and nucleolar matrix fibrils occurs during CDN isolation in the absence of divalent cations.

Animals↗
PubMed1982

A functional in vitro model for studies of intracellular motility in digitonin-permeabilized erythrophores.

Phase contrast cine results demonstrate that erythrophores maintain saltatory particle motion for hours after permeabilization with 0.001% digitonin in a cytoskeletal stabilizing solution at 23 degrees C. High voltage electron microscopy (HVEM) studies reveal that cytoskeletal elements are retained intact, except in immediate subplasmalemmal regions where the plasma membrane is punctured by digitonin. During digitonin treatments, cells are permeable to ions, small molecules, and antibodies. We find that motion is Ca2+ and ATP-sensitive, and optimal in PIPES buffer (pH 7.2 containing 1 mM Mg2+/ATP and EGTA-CA2+ (10(-7) M Ca2+) at 37 degrees C. Experiments testing the inhibitory effects of vanadate (0.4-10 microM), ouabain (100-600 microM), N-ethyl maleimide, and the cytochalasins B and D indicate that a dyneinlike ATPase may provide the motive force for driving saltatory pigment motion in erythropores.

Actins↗
PubMed1982

The role of microtubules in cell shape and pigment distribution in spreading erythrophores.

In order to investigate the role of microtubules in determining overall shape of the cell and distribution of pigment granules, a correlative whole cell electron microscopic and immunofluorescence light microscopic study was done of microtubule distribution during spreading of cultured erythrophores from scales of the squirrel fish, Holocentrus. After dissociation from the scale and immediately after attachment, erythrophores are round with long thin processes containing bundles of microtubules with associated pigment granules. In time these processes attach, and elongate, and cytoplasmic ground substance fills areas between microtubule bundles, preceding the appearance of microtubules in these areas of the cell. By 8 h cells are fully spread (star-shaped), prominent microtubule bundles have disappeared, and microtubules (along with pigment granules) are more evenly dispersed throughout the cell. After 24 h cells have increased in size, in number of microtubules, and in the length of preexisting tubules. The sequence of events in normal spreading is altered if cytochalasin D, cycloheximide, or nocodazole is included in the culture medium. Cytochalasin D, a microfilament-disrupting drug, prevents attachment and spreading, but allows elongation to occur. Cycloheximide, a protein synthesis inhibitor, allows for attachment and elongation, but no spreading. Nocodazole, a microtubule-disrupting drug, allows for attachment and spreading, but an irregular cell outline is the result. Even though spreading can occur without them, it is concluded that a normal number and distribution of microtubules are required for the development of normal cell shape and pigment distribution.

Animals↗
PubMedDate not supplied

Association between the nucleolus and the coiled body.

By means of light and electron microscopic immunocytochemistry, we have localized p80-coilin, a specific protein marker for coiled bodies, in mammalian cell lines as well as in primary rat neuron cultures. p80-coilin-stained nuclear bodies, which also contained fibrillarin, could be subsequently silver stained by a method specific for the visualization of nucleolar organizer regions. In cycling cells, most coiled bodies were not associated with nucleoli, whereas in rat neurons such as association was frequent. The treatment of cycling cells with actinomycin D or 5,6-dichloro-1-beta-D-ribo furanosyl-benzimidazole led to nucleolar segregation and/or disintegration, and to an association of p80-coilin staining structures with nucleoli. p80-coilin-positive structures contained fibrillarin in both untreated and treated cells. These results support the opinion that there might be a special association between coiled bodies and nucleoli, particularly in neuronal cells.

Animals↗