Inactive nuclei of chick erythrocyte resume RNA synthesis and increase in volume and dry mass in heterokaryons made by virus-induced fusion of human tumor cells (HeLa) with chick erythrocytes.Nuclear growth is due primarily to migration of human macromolecules into the chick nucleus. Human nucleoplasmic antigens were detected in the nucleoplasm and human nucleolar antigens were detected in the nucleoli of reactivated chick erythrocyte nuclei. After some time, chick-specific nucleolar antigens appear in the nucleoli of both the reactivated chick nuclei and the HeLa cellnuclei. The increase and the condensed chromatin of the inactive erythrocyte nucleus is dispersed (3,5). This process is paralleled by an accelerated RNA synthesis (2), but nuclear growth occurs also if chick RNA synthesis is blocked by UV-irradiation of the erythrocytes before cell fusion (3, 5). Chickspecific antigen synthesis has not been detected until after the formation of the nucleoli. Nucleoli, which are visible by the light microscope, appear 2-4 days after cell fusion (6). Since the erythrocyte nuclei undergo a 5-to 8-fold increase in dry weight during the first 48 hr, these results suggest that the erythrocyte nuclei grow by taking up proteins from the cytoplasm, the majority of which must be of human origin. In order to obtain direct evidence for this conclusion and with the purpose of elucidating factors involved in the reactivation process, we have examined the distribution of human nuclear antigens in chick erythrocyte x HeLa heterokaryons with the aid of antinuclear antibodies from patients with autoimmune diseases.
MATERIAL AND METHODS CellsWe obtained chick erythrocytes from 15-day chick embryos by cutting the allantoic vessels and allowing blood to accumulate in the allantoic fluid. The erythrocytes were collected by centrifugation and washed twice in phosphate-buffered saline (pH 7.4).HeLa cells were grown on glass slides in Eagles Minimum Essential Medium containing 10% calf serum. Heterokaryons were made by addition of 20-50 haemagglutinating units of UV-inactivated Sendai virus and 30 X 106 chick erythrocytes in 10 ml of Earle's balanced salt solution to the HeLa cell monolayer. For further details of the procedure see ref. 7. The heterokaryons were maintained in Eagles MEM containing 10% calf serum. At various time intervals, slides were removed, rinsed in saline, and fixed in ethanol: acetone (1: 1).
Immunological methodsAntibody binding was demonstrated with the indirect immune fluorescence technique (7). Slides were transferred from the fixative to distilled water (two changes, 10 min each) and then exposed to antisera that were diluted 5-20 times with 10 mM sodium phosphate buffer (pH 7.0)-8% NaCl for 45 min, at 37°C. After rinsing (twice, 10 min each time) in buffer (pH 7.0) at 22°C, antibody binding was visualized by staining with fluorescein-conjugated rabbit anti-human immunoglobulin or sheep anti-rabbit immunoglobulin (Statens Bakteriologiska Laboratorium, Solna, Sweden), diluted 1:10 in buffer for 20 min ...
Administration of vitamin E into the coronary arteries before removal of the aortic cross-clamp can reduce myocardial cell injury and protect the myocardium from ischemia-reperfusion injury.
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