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N R Ringertz

Publications and source records attributed to N R Ringertz.

At least 37 records · Page 2Linked to original sources
PubMed1986

Direct evidence for the non-random localization of mammalian chromosomes in the interphase nucleus.

Indirect immunofluorescence staining with human anti-centromere autoantibodies from a patient (LU 851) suffering from the CREST form of scleroderma was used to analyse chromosome topology in interphase nuclei of rat-kangaroo (PTO) and Indian muntjac (IM) cells. In some cells, centromeres were arranged in pairs suggesting association of homologous chromosomes. Clustering of centromeres at one pole of the nucleus (Rabl configuration) and other patterns suggesting higher order organization were also observed. In one fifth of the IM cells it was possible to identify the intranuclear location of each single chromosome on the basis of the morphology of the immunostained centromeres. In 30% of the IM cells in which centromeres could be identified, homologous chromosomes occupied adjacent territories within the interphase chromatin.

Animals↗
PubMed1986

Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese hamster ovary cells.

The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo[a]pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 microns), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possibly be used as an index of the degree of mitotic disturbances induced by mineral fibers.

Animals↗
PubMed1986

Intranuclear localization of snRNP antigens.

Anti-Sm antibodies recognize a group of small, nuclear RNA-protein complexes (snRNPs) containing U1, U2, U4, U5, and U6 snRNAs. Anti-RNP antibodies only react with U1 snRNA-containing complexes. The intranuclear distribution of snRNP particles was studied by double immunofluorescence staining of human fibroblasts. Mouse monoclonal anti-Sm antibodies and polyclonal patient sera reacting with different peptides in the snRNP complexes were used. The immunofluorescence patterns obtained with fluorescein isothiocyanate-conjugated anti-mouse Ig and tetramethylrhodamine isothiocyanate-conjugated anti-human Ig second antibodies were examined using computer analysis of digitized images. With this approach the similarity of different patterns could be visualized and estimated with mathematical methods. It was found that human anti-Sm serum as well as three different anti-RNP sera produced speckled patterns overlapping with the anti-Sm monoclonal pattern. Thus, Sm antigenic intranuclear domains also reacted with anti-RNP antibodies, suggesting a high degree of co-localization of the antigenic structures. A partial overlap was found between speckles detected by mouse anti-Sm antibodies and a human La-antiserum. No significant co-localization occurred between speckles detected by mouse anti-Sm antibodies and speckles detected by human antisera reacting with Scl-70 and centromeric antigens. As the U1 snRNP complex is believed to play a role in the splicing of RNA polymerase II transcripts, it appears that the speckles detected by Sm and RNP antibodies may be regions of hnRNA synthesis and mRNA processing. Although no function has been demonstrated for the U2, U4, U5, and U6 snRNPs, the co-localization with the U1 RNA complexes shown in this report indicate that they too participate in some aspect of mRNA processing. The results suggest that computer-assisted analysis of nuclear immunofluorescence patterns will be a useful tool in studies of the spatial and functional organization of the interphase nucleus.

Antibodies, Monoclonal↗
PubMed1985

Density-dependent arrest of DNA replication is accompanied by decreased levels of c-myc mRNA in myogenic but not in differentiation-defective myoblasts.

Myoblasts from primary rat cultures and established mouse (Cl10) and rat (L6, Ama 420) cell lines were examined for c-oncogene expression during exponential growth and under conditions which allowed myogenic differentiation. The abundance of c-Ki-ras transcripts in mRNA from confluent, quiescent cultures was reduced to 15-40% of that in mRNA from exponentially growing cells. This reduction was found both in primary myoblast cultures, myoblast lines that formed myotubes (L6 and Cl10) and in a differentiation defective subline (Ama 420). The level of c-myc transcripts was lowered when myogenic rat L6 myoblasts reached a high cell density, stopped DNA synthesis and formed myotubes. At the same cell density, growth arrested myoblasts of differentiation defective Ama 420 cells maintained a high level of c-myc expression. This shows that DNA replication and c-myc expression are independently regulated. All myoblast lines also showed expression of c-abl during exponential growth phase. Reduced expression was seen in differentiated L6 and Cl10 cultures. No expression was detected when mRNA from multiplying and differentiating myoblasts cultures were probed for c-myb, c-erbA, c-erbB, c-mos, c-fes, and c-src. The observations are consistent with a role for c-Ki-ras in myoblast proliferation and suggest that a reduction in c-myc expression may be a necessary prerequisite for terminal myogenic differentiation.

Animals↗
PubMed1985

DNA replication and H5 histone exchange during reactivation of chick erythrocyte nuclei in heterokaryons.

Fusion of terminally differentiated chick erythrocytes (CE) with replicating quail myoblasts or established L6J1 rat myoblasts results in reactivation of DNA synthesis in the dormant CE nuclei and in suppression of DNA synthesis in the myoblast nuclei. The nuclei of primary quail myoblasts are more effectively inhibited than the nuclei of established rat myoblasts. Inhibition of DNA replication occurs not only by preventing G1 nuclei from entering S-phase but also by blocking nuclei in S-phase and by delaying nuclei in G2 from undergoing mitosis and starting a new DNA replication cycle. No inhibition of DNA synthesis could be observed when mouse erythrocytes, i.e., erythrocytes lacking nuclei, were fused with rat myoblasts to generate mouse-globin-containing L6J1 cybrids.--Reactivation of CE nuclei is associated with a loss of the tissue-specific H5 histone variant. Complete elimination of H5 histone, however, does not seem to be a necessary prerequisite for the initiation or completion of DNA replication in CE nuclei since H5 antigens are found on reactivated G1, S, and G2 nuclei.

Animals↗
PubMed1985

Expression of cellular oncogenes in teratoma-derived cell lines.

The expression of ten proto-oncogenes was studied in cell lines derived from transplantable mouse teratomas. The cell lines represent different forms of early embryonic cell specialization. The analysis included two embryonal carcinoma (EC) lines (PCC3 and F9), and four differentiated cell lines derived from teratocarcinoma, namely trophoblastoma (3-TDM), parietal endoderm (PYS-2), visceral endoderm (PSA5-E) and skeletal myoblasts (Cl10). The expression of c-oncogenes was studied by analysing poly(A)+RNA for complementary sequences by dot blot and Northern blot hybridization. The results were related to the rate of cell multiplication and the state of differentiation by examining [3H]thymidine incorporation, growth curves and tissue-specific differentiation markers. Expression of c-myc and c-Ki-ras was found in all cell lines. In dot blot assays, poly(A)+RNA from all cell lines also hybridized with v-abl and v-sis probes. A marked decrease in c-myc expression was found in teratoma-derived myoblasts differentiating into myotubes. A similar reduction was found when 'nullipotent' F9 cells were induced by retinoic acid (RA) to form primitive endoderm. However, reduction of the growth rates of the parietal and visceral endodermal cell lines were not accompanied by decreased expression of c-myc or c-Ki-ras. Hybridization signals obtained with a v-sis probe was low in all teratoma-derived cell lines tested, except for the myogenic cell line Cl10. Both in exponentially growing and differentiated cultures of this line, two size classes of transcripts hybridized strongly to the v-sis probe. However, these transcripts, 7 and 3 kb, most likely represent endogenous retroviral transcripts and not c-sis transcripts. Expression of c-myb, c-mos, c-fes, c-src and c-erb A and c-erb B could not be detected in any of the cell lines studied.

Animals↗
PubMed1984

Changes in nuclear antigens during reactivation of chick erythrocyte nuclei in heterokaryons.

Reactivation of dormant chick erythrocyte (CE) nuclei was studied by fusing chick red cells with rat myoblasts, HeLa cells, and chick fibroblasts. Heterokaryons representing different stages of nuclear reactivation were fixed and examined for nuclear antigens using polyclonal patient autoantisera reacting with mammalian (human, mouse, and rat) nuclear envelope, nucleoplasm, and chromatin (DNA) antigens. Reactivation of CE nuclei was associated with marked changes in nuclear antigenicity. In rat myoblast and HeLa heterokaryons the CE nuclei acquired mammalian nucleoplasmic and nuclear envelope antigens in the corresponding nuclear subcompartments. Drastic changes in nuclear antigenicity were noted also in heterokaryons stained with DNA antisera. The compact chromatin of nuclei in mature chick erythrocytes showed little binding of DNA antibodies. Isolated nuclei on the other hand gave a strong immunofluorescence. CE nuclei in heterokaryons were strongly positive during the early stages of nuclear reactivation but then exhibited decreased reactivity. An unexpected finding was a marked reduction in the capacity of mammalian nuclei in heterokaryons to bind DNA-antibodies. This observation is discussed in relation to the previous finding that in CE-heterokaryons these nuclei often show reduced transcription and replication. The present results indicate that in heterokaryons both types of nuclei exchange macromolecules of regulatory importance via the common cytoplasm.

Animals↗
PubMed1984

Globin synthesis in heterokaryons formed between chick erythrocytes and human K562 cells or rat L6 myoblasts.

Chick globin synthesis was studied in heterokaryons formed between chick erythrocytes and human K562 erythroleukemia cells or rat L6J1 myoblasts. It was found that chick globin synthesis was activated after fusion of definitive (17 to 19-day) chick erythrocytes obtained from 17 to 19-day embryos with K562 cells. Chick globins appeared to be of the adult alpha A, alpha D and beta types, whereas no embryonic globin synthesis could be detected. The pattern and time-course of globin synthesis was investigated after fusion of 4 to 5-day embryonic erythrocytes with rat L6J1 myoblasts. The level of globin synthesis was high at early time points but then decreased. Globin synthesis, however, was still detectable at 9 days in these heterokaryons. Chick alpha A, alpha D and epsilon-globin chain synthesis was observed both at early and late time points after fusion.

Animals↗
PubMed1983

Migration of rat RNA polymerase I into chick erythrocyte nuclei undergoing reactivation in chick-rat heterokaryons.

Transcriptionally inactive chick erythrocyte nuclei were reactivated by Sendai virus-induced fusion of erythrocytes with rat L6J1 myoblasts. We used antibodies to trace the appearance of a specific protein engaged in transcription of a defined class of genes, those coding for rRNA, during reactivation. Using immunofluorescence microscopy, we found increasing amounts of rat RNA polymerase I to appear, during a certain period of time after fusion, in the reforming nucleoli of the chick nuclei. Amounts of rat RNA polymerase I sufficient to be detected by immunofluorescence microscopy had accumulated in the newly developed chick nucleoli 72-190 h after fusion was initiated. This time interval coincides with the time when chick rRNA synthesis can first be detected. The results raise the possibility that during these stages of the reactivation process chick rRNA genes are transcribed by heterologous RNA polymerase I molecules of rat origin.

Animals↗
PubMed1982

Transcription of chick genes by mammalian RNA polymerase II in chick erythrocyte-mammalian cell heterokaryons.

The introduction of chick erythrocyte nuclei into mammalian cell cytoplasms results in their reactivation as evidenced by the de novo transcription of chick genes and the synthesis of both globin and constitutive proteins. In the present study, chick erythrocytes have been fused to L6 rat myoblasts and to alpha-amanitin-resistant variants of L6 to determine whether the chick or the mammalian RNA polymerase II was responsible for transcription of chick genes. Heterokaryons formed by fusing chick erythrocytes with alpha-amanitin-resistant L6 myoblasts synthesize both chick globin and chick constitutive proteins in the continued presence of 5 micrograms/ml alpha amanitin ten days postfusion. Both the synthesis of globin and other chick polypeptides occurs at levels comparable to those observed for untreated heterokaryons. Synthesis occurs under conditions in which insignificant chick RNA polymerase II activity can be detected in wild-type heterokaryons by autoradiography. These results demonstrate that RNA polymerase II is one of the mammalian proteins that is selectively taken up by the chick nucleus during reactivation in the presence of alpha amanitin. Furthermore, the mammalian RNA polymerase II alone can account for the transcription of both differentiation specific and constitutive genes in the chick nucleus.

Amanitins↗
PubMed1982

Pattern of chick gene activation in chick erythrocyte heterokaryons.

The reactivation of chicken erythrocyte nuclei in chick-mammalian heterokaryons resulted in the activation of chick globin gene expression. However, the level of chick globin synthesis was dependent on the mammalian parental cell type. The level of globin synthesis was high in chick erythrocyte-rat L6 myoblast heterokaryons but was 10-fold lower in chick erythrocyte-mouse A9 cell heterokaryons. Heterokaryons between chick erythrocytes and a hybrid cell line between L6 and A9 expressed chick globin at a level similar to that of A9 heterokaryons. Erythrocyte nuclei reactivated in murine NA neuroblastoma, 3T3, BHK and NRK cells, or in chicken fibroblasts expressed less than 5% chick globin compared with the chick erythrocyte-L6 myoblast heterokaryons. The amount of globin expressed in heterokaryons correlated with globin mRNA levels. Hemin increased beta globin synthesis two- to threefold in chick erythrocyte-NA neuroblastoma heterokaryons; however, total globin synthesis was still less than 10% that of L6 heterokaryons. Distinct from the variability in globin expression, chick erythrocyte heterokaryons synthesized chick constitutive polypeptides in similar amounts independent of the mammalian parental cell type. Approximately 40 constitutive chick polypeptides were detected in heterokaryons after immunopurification and two-dimensional gel electrophoresis. The pattern of synthesis of these polypeptides was similar in heterokaryons formed by fusing chicken erythrocytes with rat L6 myoblasts, hamster BHK cells, or mouse neuroblastoma cells. Three polypeptides synthesized by non-erythroid chicken cells but less so by embryonic erythrocytes were conspicuous in heterokaryons. Two abundant erythrocyte polypeptides were insignificant in non-erythroid chicken cells and in heterokaryons.

Animals↗
PubMed1981

Reactivation of chicken erythrocyte nuclei in heterokaryons results in expression of adult chicken globin genes.

Activation of chicken globin gene transcription has been demonstrated in chicken erythrocyte--rat L6 myoblast heterokaryons. The globin mRNA is polyadenylylated and is translated into adult chicken alpha A-, alpha D-, and beta-globin polypeptides. No fetal globin mRNA or globin polypeptides were detected. Heterokaryons between chicken erythrocytes and mouse neuroblastoma cells or hamster BHK cells also synthesized adult chicken globins.

Animals↗
PubMed1980

Production of chick interferon by reactivating chick erythrocytes.

When chick erythrocytes were fused with mouse L929 or A9 cells and the heterokaryons induced for interferon production on consecutive days, considerable amounts of mouse interferon were produced every day. Small amounts of chick interferon were produced 2 to 3 days after fusion, coincident with the appearance of a chick enzyme, appearance of nucleoli and increase in chick cell nuclear diameter.

Animals↗