AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

The distribution of nuclear and cytoplasmic proteins in the blastoderm of the loach, Misgurnus fossilis L

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.     

Changes in pancreatic acinar cell nuclear number and DNA content during aging in the rat

Pancreatic acinar cells from rats 5 to 658 days (94 weeks) of age were isolated by enzymatic dissociation and stained with the DNA specific fluorochrome Hoechst 33258. The nuclear DNA content and the incidence of binucleation were estimated in these cells. Total pancreatic weight, RNA, protein and DNA, and the incorporation of 3H-thymidine into pancreatic acinar cell DNA were also estimated in similar animals as measures of pancreatic growth. From 5 to 17 days after birth, 95% of the cells were mononucleate diploid and 5% were binucleate diploid; but during the period of rapid pancreatic growth over the following 39 days, acinar cells became increasingly binucleate. By 56 days after birth, 64% of cells were binucleate with a diploid DNA content per nucleus; and the incidence of binucleation then remained constant. At 28 days of age, 4% of mononucleate cells were tetraploid, increasing to 6% at 658 days of age. At this time 3% of binucleate cells contained dual tetraploid nuclei. There is thus a rapid development towards diploid binucleate acinar cells in the growing, postnatal pancreas; and in the adult pancreas a small proportion of these cells develop tetraploid nuclei.     

contribution to the study of nuclear total proteins and DNA during the mitotic cycle in fibroblasts cultivated in vitro and in Ehrlich ascites cells

Summary The dry weight and total protein content of nuclei has been measured by interferometry in living or fixed cells cultivated in vitro (freshly prepared chick, mouse or rat embryo fibroblasts) and in fixed Ehrlich ascites tumor cells of the mouse growing in vivo. The DNA content was estimated by cytophotometry after Feulgen reaction in the same nuclei. The dry weight of nucleoli in fibroblasts and the dry weight and DNA content of chromosomes in dividing fibroblasts and Ehrlich tumor cells have also been measured.During the interphase in fibroblasts, the dry weight of the living nucleus and the nuclear total protein content as measured in fixed cells doubles during the preparation for mitosis, as the DNA content does. In chick and mammal fibroblasts and within the limits of accuracy of our measurements, the synthesis curves for nuclear proteins and DNA do not seem to be necessarily identical.In our fibroblasts, the nucleolar total dry weight per nucleus doubles during the interphase (nucleolar preparation for mitosis); it increases in proportion to the nuclear total protein content, even in polyploid nuclei.During the mitosis, the chromosomes contain all the DNA of the nucleus but some nuclear proteins (non chromosomal proteins) seem to move into the cytoplasm during the mitosis and return into the nucleus at the post-telophase.According to our observations, Ehrlich ascites mouse tumor cells are near-tetraploid as far as the number of chromosomes, nuclear total protein content and DNA content are concerned. During the preparation for mitosis, these amounts double but no necessary close time relation seems to link these premitotic syntheses. Prom this point of view, our results show no clear-cut differences between these tumor cells and the fibroblasts. Except the polyploidy, the behaviour of nuclear proteins and DNA during mitosis in the tumor cells is the same as that observed in our fibroblasts.The effects of various antimitotic agents on rat fibroblasts cultivated in vitro have also been studied with our cytochemical methods. Our measurements of nuclear protein, DNA and nucleolar material content have been made in cells in which mitosis was prevented by alkylating agents, beryllium sulphate, RNase or neutral DNase. The effects of colchicine on these cellular parameters have also been studied.     

Cytofluorometric study of the glycogen content in DNA-synthesizing and -nonsynthesizing hepatocytes in the regenerating liver

Using a combined cytochemical method that allows to determine glycogen, DNA and 3H-thymidine label in the same cell, glycogen amounts were measured both in 3H-TdR-marked and non-marked hepatocytes of the regenerating 3H-thymidine. During mitosis, the glycogen amount is reduced if compared with that in cells being in presynthetic phase. It is proposed that the decrease in glycogen content in the regenerating liver may partly depend on energetic expenses of cells that started DNA synthesis and mitotic division. The phenotypic expression of genes responsible for glycogen synthesis and splitting in di-, tetra- and octoploid hepatocytes of the regenerating liver liver was proportional to the corresponding values.     

Simultaneous photometry of the DNA and silver-grain count in liver cells labelled with 3H-thymidine or 3H-leucine

A modified method was proposed for reflected light simultaneous measurement of DNA content and of the silver grain number in the nucleus or cytoplasm of the same cell. Specimens-smears of isolated liver cells incorporated 3H-thymidine and 3H-leucine were prepared on coverslips and after processing were mounted on the slide glasses with smeared side facing downwards to avoid the influence of grains on DNA content measurements. To decrease the background, label measurements were carried out in polarized light. It was shown that the intensity of 3H-leucine incorporation in hepatocytes increases proportionally with cell ploidy degree.     

Effect of the alkylating agent, dipin, on induced hepatocyte proliferation. II. An increase in the DNA synthesis period]

The alkylating drug dipin was injected to mice 2 hours before a partial hepatectomy. The duration of DNA synthesis of proliferating hepatocytes was determined by means of cytophotometry of DNA mass in the nuclei, labeled with 3H-thymidine 30 hours after the operation. The DNA mass was doubled simultaneously in diploid and tetraploid nuclei within 18-26 hours. The DNA in labeled nuclei of the control mice was doubled in 8 hours. The kinetics of 3H-thymidine incorporation at different stages of S-period was similar in alkylated and normal cells.     

Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris. I. Manubria

During the development of an antheridium DNA content in manubria gradually increases to 8C-16C level. 3H-thymidine incorporation into the nuclei of the manubria lasts till the stage of quantitative predominance of the 16 celled antheridial filaments. The nucleus of the manubria is characterized by the low content of the condensed chromatin and the presence of nucleoli with the nucleolonema-like structure the number of which increases from 6-8 to 32-38 along with the increase of DNA content in a nucleus. In the cytoplasm of the manubria there are numerous secretive vesicles filled with fine-granular substance discharged outside plasmalemma, active Golgi apparatus, well-developed rough ER, numerous polysomes, mitochondria with the condensed structure and plastids with granar and inter-granar thylakoids as well as plastoglobules which increase in number and size along with the development of the antheridium. During spermiogenesis the cells are vacuolated, the number of the secretive vesicles decreases whereas the electron density of their content increases, smooth ER appears while rough ER is reduced. The manubria actively incorporate 3H-uridine, 3H-tryptophane and 3H-leucine. The increase of the incorporation activity is gradual in the period of increasing polyploidy of the manubria and rapid during the initiation of the spermatozoid differentiation. It has been suggested that the manubria should play an important role in the process of spermatogenesis and the induction of spermatozoid differentiation.     

Studies of nucleic acid metabolism in cultured chick embryo cells infected with Mycoplasma gallisepticum

M. gallisepticum infection of cultured chick embryo cells led to a sharp reduction the rate of 3H-thymidine and 3H-uridine incorporation into DNA and RNA cells, and almost completely suppressed the transposition of uridine label from the nucleus into the cytoplasm, this pointing to the inhibition of escape of RNA synthesized de novo into the infected cells cytoplasm. As suggested, weak labeling of the cytoplasm after prolonged (about several hours) incubation of cultured cells with labeled urine could indicate infection of cell cultures with the mycoplasmae.     

Reactivation of chick erythrocyte nuclei in young and senescent WI38 cells

The chromatin of the dormant chick nucleus is dispersed in the heterokaryons made by Sendai virus fusion of phase II WI38 cells with chick erythrocyte nuclei. The erythrocyte nucleus resumes RNA synthesis and enters into DNA synthesis with the host nucleus. In the heterokaryons of phase III WI38 cells and chick erythrocytes, the nuclear chromatin is not dispersed and RNA synthesis occurs at a reduced rate. The differences in the physiological state of the young and senescent cells measured by [³H]uridine incorporation into nuclear RNA is reflected in the extent of reactivation of the chick erythrocyte nuclei in the cytoplasm of these cells. The reactivation of the chick nucleus in enucleated fibroblasts parallels the nucleated cells. The results of these studies are interpreted as evidence that there is a specific loss of nuclear function in the senescent cells.     

H-thymidine incorporation into nuclear DNA of leaf cells

The incorporation of ³H-thymidine into nuclear DNA of leaf cells of Nanthium pennsylvanicum was studied as a function of concentration and specific activity of the radioisotope. From the assessment of the average number of grains per nucleus and the percent of labeled nuclei, it was concluded that the incorporation was a linear function of concentration of the exogenous radioisotopic solution and a logarithmic function of the incubation time. Ten microcuries per milliliter on the average yielded 20% of labeled nuclei with 18 grains per nucleus. Seven-fold increase in concentration only doubled the amount of ³H-thymidine incorporated. The lamina regions near the vein incorporated a significantly greater amount of the radioisotope than the lamina region at some distance from the vein. The specific activities of 2, 3.35, 6.7 and 15.3 c/mmole had no effect upon the amount of ³H-thymidine incorporated, if the amount of microcuries of the incubation solution was the same in each activity. Considering the total number of molecules, the estimated rates of incorporation indicated that at the activity of 2 c/mmole, the system operated with about 7 times higher rates as compared with the activity of 15.3 c/mmole.     

Nuclear-cytoplasmic relationships in human cells in tissue culture. III. Auto-radiographic study of interrelation of nuclear and cytoplasmic ribonucleic acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H(3) for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H(3) in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H(3). It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.     

Quantitative changes in the total proteins of the cells of the hypothalamic supraoptic nucleus after extreme exposures: acute immobilization and deprivation of the paradoxical sleep phase

It has been shown that 1-hour acute immobilization of rats resulted in a decrease of total protein content (per 1 cell) in neurones (by 17% in cytoplasm and 22% in nucleus) of the supraoptic nucleus of the hypothalamus and in their glial satellite cells (by 10%), mainly at the expense of the nuclei of the latter. These changes remained at the same level in the neuronal cytoplasm and satellite cells within 6 hours after cessation of acute immobilization, being abolished in the neuronal nuclei. Deprivation of REM sleep for 24 hours affected these parameters to the same extent as acute immobilization. However, 6-hour rest was sufficient for normalisation in satellite cells and for decrease in protein deficiency in the neuronal nuclei; yet protein deficiency still sustained in the neuronal cytoplasm. REM sleep deprivation was also accompanied by a decrease in tritium incorporation into satellite cell proteins (by 18%); after 3-hour rest, incorporation of the label into satellite cells was restored to the initial level being increased by 20% in neuronal nuclei; another 3-hour rest resulted in normalisation of labeling in the neuronal nuclei and in 27% increase of labeling in the satellite cells.     

Changes in the amount and synthesis of DNA in the nuclei of large decidual cells in rats during their differentiation

Using cytophotometry of the Feulgen-stained nuclei, the quantity of DNA was measured in the nuclei of rat's large decidua cells (LDC) on tissue sections of the antimesometrial region within days 7-13 of gestation. The quantity of nuclear DNA was expressed in units of ploidy, the haploid DNA standard being the quantity of DNA in rat's spermatid nucleus. On different days of gestation, the nuclear DNA was seen to vary in cells located in different zones of decidua. The maximum DNA content was found in the LDC located on days 9-12 of gestation somewhat in the middle of the decidua thickness. On day 11, the quantity of nuclear DNA in these cells reached in average, 22c. The quantity of DNA in the nuclei of the least differentiated LDC located on the periphery of decidua never exceeded 4.9c, whereas that in the nuclei of the most differentiated LDC, located close to the embryo, varied from 2.9c to 9.3c. On days 10 and 11 of rat's false gestation, the maximum DNA contents in the nuclei were registered in the LDC located in the middle of the decidua thickness. 3H-thymidine incorporation into the nuclei of the most differentiated LDC located nearest to the embryo stopped starting from day 10 of gestation. Phenomena of lesser quantities of nuclear DNA in most differentiated LDC, compared to that in LDC in the previous steps of differentiation, are discussed.     

The modification of stored histones H3 and H4 during the oogenesis and early development of Xenopus laevis

In amphibian development large amounts of histone are accumulated at early stages and used in assembling nuclei at later stages, when cell proliferation is rapid. The acetate groups on stored H3 and H4 molecules turn over. In the oocyte nucleus H4 exists primarily in a diacetylated state, whereas it probably exists in a phosphorylated form in the cytoplasm. This represents, at steady state, what is normally a transitory stage in the transport of newly synthesized H4 into chromatin. Stored H3 of the oocyte has acetylated, and probably phosphorylated forms, in the same proportions as is normally seen in the chromatin of somatic cells. The various forms of H3 occur in similar proportions in the nucleus and cytoplasm of the oocyte. H3 does not therefore need to associate with DNA to be acetylated, even though the appearance of modified forms of H3 in cultured cells is seen only after the incorporation of H3 into chromatin.     

Nuclear-Cytoplasmic Relationships in Human Cells in Tissue Culture : III. Autoradiographic Study of Interrelation of Nuclear and Cytoplasmic Ribonucleic Acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H³ for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H³ in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H³. It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.     

Comparative Study of Cultured Burkitt Tumor Cells by Immunofluorescence, Autoradiography, and Electron Microscopy

Cultured Burkitt cells were examined by immunofluorescence, autoradiography, and electron microscopy in an effort to identify the stainable cells with those harboring herpes-type virus particles. Immediately after a 2-hr pulse of (3)H-thymidine, from 30 to 60% of the cells revealed heavy nuclear labeling. In most cases the grains were evenly dispersed, but in about 3 to 5% the grains showed a focal distribution and occasionally they extended into the cytoplasm. Such nuclear foci were rarely seen at 8 hr after the pulse. When the analysis was restricted to preselected immunofluorescent cells, up to 80% showed label at 8 hr and cytoplasmic grains were prominent. To reduce cellular deoxyribonucleic acid (DNA) synthesis, cells were X-irradiated with 3,000 to 6,000 R, and the isotope pulse was applied 1, 4, or 7 days later. Whereas the total number of labeled cells decreased in roughly twofold steps at the respective intervals (from 40 to 10%), the incorporation of (3)H-thymidine into fluorescent cells was not affected by X irradiation. In each series, about 70% of the fluorescent cells contained label when they were examined at 24 and 48 hr after the pulse, whereas at 8 and 72 hr fewer were positive. At the earlier intervals, unlabeled fluorescent cells most likely represented cells which had completed viral DNA synthesis prior to the pulse; at the later intervals, unlabeled fluorescent cells were probably cells which commenced viral replication after the pulse. These data support the conclusion that the immunofluorescent cells are the ones which harbor virus, and also confirm the expectation that the virus is a DNA virus from a member of the herpes group. This conclusion was firmly established by sectioning and electron microscopic examination of individual fluorescent cells, all of which contained numerous virus particles, whereas the nonstained cells prepared in a similar manner were free of them.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

Intracellular traffic of oligodeoxynucleotides in and out of the nucleus: effect of exportins and DNA structure

The delivery of oligodeoxynucleotides (ODNs) into cells is widely utilized for antisense, antigene, aptamer, and similar approaches to regulate gene and protein activities based upon the ODNs' sequence-specific recognition. Short pieces of DNA can also be generated in biological processes, for example, after degradation of viral or bacterial DNA. However, the mechanisms that regulate intracellular trafficking and localization of ODNs are not fully understood. Here we study the effects of major transporters of microRNA, exportin-1 (Exp1) and exportin-5 (Exp5), on the transport of single-stranded ODNs in and out of the nucleus. For this, we employed a fluorescent microscopy-based assay to quantitatively measure the redistribution of ODNs between the nucleus and cytoplasm of live cells. By measuring the fluorescent signal of the nuclei we observed that after delivery into cells via cationic liposomes ODNs rapidly accumulated inside nuclei. However, after removal of the ODN/liposome containing media, we found re-localization of ODNs from the nuclei to cytoplasm of the cells over the time course of several hours. Downregulation of the Exp5 gene by siRNA resulted in a slight increase of ODN uptake into the nucleus, but the kinetics of ODN efflux to the cytoplasm was not affected. Inhibition of Exp1 with leptomycin B somewhat slowed down the clearance of ODNs from the nucleus; however, within 6 hours most of the ODN were still being cleared form the nucleus. ODNs that could form intramolecular G-quadruplex structures behaved differently. They also accumulated in nuclei, although at a lesser extent than unstructured ODN, but they remained there for up to 20 hours after transfection, causing significant cell death. We conclude that Exp1 and Exp5 are not the major transporters of our ODNs out of the nucleus, and that the transport of ODNs is strongly affected by their secondary structure.     

DNA replication in mammalian cells after the action of physical, chemical or biological factors. VI. The recovery of DNA synthesis in a culture of irradiated cells

The kinetics of DNA synthesis restoration in cultured HeLa cells and in L-929 mouse fibroblasts irradiated by gamma-rays of 60Co with a dose of 10 Gy was studied. Early after irradiation the rate of DNA synthesis in HeLa cells measured with 3H-thymidine incorporation was seen to decrease. Two hours later the incorporation starts to increase to reach the control level 4 hours after irradiation and then becomes even higher than this level. The distribution of cells among phases of the cell cycle measured with flow cytometry undergoes changes. 4-6 hours after irradiation part of S-phase cells increased contributing presumably to the elevating of 3H-thymidine incorporation observed at this time. The restoration of the incorporation was suppressed by inhibitors of protein and RNA synthesis--cycloheximide and actinomycin D. It is suggested that the processes of restoration of DNA synthesis in irradiated cells can be of inducible nature. In irradiated HeLa and L-929 cells the restoration of DNA synthesis is resistant to novobiocin, an inhibitor of DNA replication.