Acceleration of the vertical migration of corneal epithelial cells in white rats under chronic immobilization stress

Autoradiography with 3H-thymidine was employed to study DNA synthesis and the rate of the migration of labeled nuclei from the basal layer to the surface layers of albino rat corneal epithelium after repeated immobilization stress. The studies were performed at once, and 24 and 72 hours after 3H-thymidine injection. Post-stressor activation of DNA synthesis and acceleration of the vertical migration of the cells were recorded.     

3H-thymidine and 3H-uridine incorporation into the heart cells of the freshwater crayfish Astacus astacus and the swimming crab Portunus holsatus

DNA and RNA syntheses in the heart cells of two decapod species were investigated with the aid of electron microscopic autoradiography. Isotopes were injected in the cavity of adult animals 4 hours before fixation. 3H-thymidine labeling was found in several satellite cell nuclei and in some particular epicardial cell nuclei. None of myonuclei was labeled. 3H-uridine incorporated in all the nuclei of muscle fibers. Satellite cells were labeled with 3H-uridine very slightly, if at all. Such a peculiarity of biosynthetic processes in the decapod heart satellite cell suggests their myoblastic nature similar to that of satellite cells of somatic muscles. The active 3H-thymidine uptake by the heart satellite cells of adult animals may be accounted for by the permanent growth of the decapods through their whole life span.     

Double-strand breaks of DNA of C57BL and mdx mouse cardiomyocytes after dynamic stress

One of the approaches to analysis of survival of cardiomyocytes during oxidative stress can be the use of animals with genetic defects—mdx mice. In mdx mice, disturbance of dystrophine synthesis is known to be accompanied by development of oxydative stress in contractile cells that in turn produces cell death. Earlier we established that dynamic stress leads to the formation of low molecular DNA fragments in the mdx mouse myocardium. It is beyond any doubt that the DNA fragmentation develops via formation of double-strand DNA breaks (DB). To record the dynamics of the appearance and disappearance of DB in the mdx mouse cardiomyocytes after dynamic stress, we used an antibody to the phosphorylated form of the γ-H2Ax histone. In the absence of stress, DB in myocardial cell nuclei are revealed both in C57Bl and in mdx mice. The percentage of cardiomyocyte nuclei with DB in C57Bl and in mdx mice was 0.05 ± 0.07% and 6.7 ± 0.2%, respectively (Table 1). In the C57Bl mice 1 h after dynamic stress the fraction of labeled cardiomyocyte nuclei rose to 1.0 ± 0.02%, while in the mdx mice—to 41.7 ± 11.4% (Table 1). At 24 h after the dynamic stress 5.7 ± 0.2% cardiomyocyte nuclei remained labeled in the mdx mouse myocardium (Table 1), whereas in C57Bl mice no labeled cardiomyocyte nuclei were revealed. One hour after the dynamic stress, 0.3 ± 0.2% of cardiomyocyte nuclei of the C57Bl mice incorporated ³H-thymidine. In the mdx mice, 2.9 ± 0.5% of cardiomyocyte nuclei incorporated ³H-thymidine. At 24 h after the stress and ³H-thymidine administration the percentage of cardiomyocyte nuclei in the mdx mice fell to 0.4 ± 0.2%. In the C57Bl mice primarily labeled nuclei were not revealed. The ³H-thymidine incorporation is not associated with entrance of cardiomyocytes into the mitotic cycle; we consider it as a manifestation of reparative DNA synthesis. We conclude is that the disappearance of DB in DNA from the mdx mouse myocardium 24 h after the dynamic stress is associated both with DNA reparation and the loss of cardiomyocytes.     

REUTILIZATION OF LYMPHOCYTE DNA BY CELLS OF INTESTINAL CRYPTS AND REGENERATING LIVER

Lymphoid cells from mice injected 54 hours and 30 hours earlier with ³H-thymidine were washed and transfused into isogenic recipients at 29 to 30 hours after partial hepatectomy. The recipients were killed 28 to 30 hours later, and liver, intestine, Peyer''s patch, spleen, and the transfused cells were examined in autoradiographs exposed 6 months. Approximately 80 per cent of the labeled transfused cells were classed as lymphocytes. The labeled DNA contained in the transfused cells was partitioned to about 14 times as many recipient liver and intestinal cells, appearing in 72 to 78 per cent of hepatocyte nuclei, in 30 to 35 per cent of liver reticuloendothelial nuclei, and in 90 to 95 per cent of intestinal crypt nuclei. The label was not comparably widespread in the lymphoid organs, but was limited to a few intensely labeled lymphocytes and a somewhat larger number of very weakly labeled cells. When heat-killed cells rather than living cells were transfused, intensely labeled lymphocytes were absent from the lymphoid organs, but the labeling of cells in the recipients was otherwise identical. The results suggest that (a) reutilized DNA is derived from dead cells, (b) reutilized DNA is mainly degraded to nucleosides and nucleotides, the usual immediate de novo DNA precursors, before reincorporation into DNA, and (c) DNA reutilization may occur in the lymphoid organs, but on a less active scale than in intestine or regenerating liver.     

Relative DNA content of human euchromatin and heterochromatin after G, C and Giemsa 11 banding.

Human chromosomes and interphase nuclei labeled with 3H-thymidine and treated with the ASG and trypsin technique for G banding show no DNA loss. However, after G 11 and C banding significantly more DNA is removed from euchromatin than from constitutive heterochromatin.     

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.     

The pattern of lymphocytes in the thymus and spleen after labeling with 3H-thymidine and 3H-deoxycytidine.

Adult male untreated mice (NMRI) were investigated after radioactive labeling with 3H-thymidine and 3H-deoxycytidine to find out whether the lymphocytes in the cortex and medulla of the thymus as well as in the perifollicular and periarteriolar regions of the spleen show a labeling pattern which allows a classification into T- and B-lymphocytes. The percentages of radioactively labeled small lymphocytes and their mean grain counts were determined. The percentages of radioactively labeled small lymphocytes after 3H-TdR and 3H-CdR showed no significant differences in both splenic zones. The grain counts over the lymphocyte nuclei in the periarteriolar zone showed lower values after 3H-TdR than after 3H-CdR. The lymphocytes in the perifollicular zone were strongly labeled with 3H-TdR and weakly labeled with 3H-CdR. In the thymus medulla, lymphocytes were weakly labeled with 3H-thymidine and strongly labeled with 3H-CdR. In the cortex no significant differences were observed. 75 to 80% of the small lymphocytes in the peripheral blood were weakly and 20-25% strongly labeled after 3H-TdR. Therefore there are similarities in the radioactive labeling pattern of thymic medulla lymphocytes and that of small lymphocytes of the periarteriolar zone of the spleen by both DNA precursors. The small lymphocytes in the peripheral T-dependent tissue zones, for example in the spleen, as well as in the mixed lymphocyte population of the peripheral blood can be differentiated from the B-lymphocytes through the difference in the amount of incorporation of 3H-thymidine and 3H-deoxycytidine.     

Ultrastructure of the cells and DNA synthesis in skeletal muscle regeneration. A study of the regeneration of the frog sartorius muscle by an electron microscopic autoradiographic method

The ultrastructure of cells of the regenerating frog's sartorius muscle and their capacity to synthesize DNA was studied by means of 3H-thymidine (3HT) electron microscope autoradiography. On the 8-17th post injury (p.i.) days, 2 hours following 3HT administration, only mononuclear cells were seen labeled, the myotube nuclei incorporating no 3HT. Along with the endothelial cells, fibroblasts, phagocytes and cells identified conventionally as myoblasts, satellite cells examined from both necrotic and viable parts of injured myofibers were labeled. No myoblast sequestration from the injured myofibers occurred. By the 13-15th p.i. days, numerous myoblast-like cells are accumulated beneath the glycocalix layer covering the free ends of myotubes which are rich in ribosomes and display an active sarcomerogenesis. Some of these myoblast-like cells become labeled after 3HT pulse. The 13 day p.i. regenerates examined 72 hours following 3HT injection display labeling in numerous myotube nuclei. This is indicative of the myoblast fusion, which is believed to play a principal role in the regenerative somatic myogenesis. Within the myonuclei adjacent to the areas of the regeneration, membranous and/or fibrillar structures of an unknown origin were frequently observed.     

Incorporation of 3H-thymidine into glial cells of the parietal region and cells of the subependymal zone of two-week and adult mice under normal conditions and following brain injury]

Potencies of brain cells to DNA synthesis and proliferation were studied in two weeks old and adult mice in the norm and after the brain mechanical injury. No labeled large and middle neurons were found in the brain of intact and operated animals both under the pulse 3H-thymidine incorporation and saturation of mice with 3H-thymidine during 36 hrs. The same types of brains cells were labeled both in intact and operated two weeks old and adult mice: glial cells, cells of the subependymal zone, cells of the dentate gyrus inner margin, and sometimes, cells having characteristics of microneurons. The number of glial cells in the temporal cortex of intact mice diminished with the age. Under the brain trauma, the proliferative reaction of glia was expressed in a similiar way both in two weeks old and adult mice. The index of labeled cells in the subependymal zone is the same in these two age groups. With the age the cellular mass of subependymal zone decreases, rather than proliferative tendencies of supependymal zone. The brain traumatization resulted in the increase of labeled subependymal cell only under the direct injury of subependymal zone.     

Cytofluorometric determination of DNA and protein in the nuclei and nucleoli of Physarum polycephalum during the intermitotic period

Summary Cytofluorometric methods were used to study the syntheses of DNA and of some proteins during the second intermitotic period of isolated nuclei and nucleoli of Physarum polycephalum. Interferometric methods were used for the determination of their dry mass.The volume and dry mass of nuclei and nucleoli showed similar behaviour, the nuclei doubling their volume and dry mass, and the nucleoli tripling them.The DNA synthesis began immediately after mitosis; the content nearly doubled after the first 3 hours in the case of the nuclei, after the first 5 hours in the case of the nucleoli. In both cases a slow increase (10%) was registered until the next mitosis.Of all proteins investigated only protein-bound lysine of nuclei and nucleoli did not double in initial content, this is ascribed to the isolation method used. This fact was also reflected by the total protein analysis, as nuclei and nucleoli were very rich in lysine. Protein-bound arginine of the nuclei and nucleoli showed an evolution similar to DNA. The histones showed a synthesis independent of that of DNA. The comparison of total protein content and dry mass suggested that the quicker increase of the latter should be attributed to RNA.     

STUDIES ON THE GERMINATION OF SEEDS OF THE ROOT PARASITES,ALECTRA VOGELII AND STRIGA GESNERIOIDES. II. DNA SYNTHESIS AND DEVELOPMENT OF THE QUIESCENT CENTER IN THE RADICLE

DNA synthetic activity in the radicle meristem of embryos of germinating seeds of the obligate root parasites, Alectra vogelii and Striga gesnerioides was followed by autoradiography of ³H-thymidine incorporation. Incorporation of ³H-thymidine occurred in the nuclei of cells destined to form the vascular tissues, ground meristem and epidermis. An analysis of the distribution of labeled nuclei demonstrated the presence of a quiescent center of 2-4 cells in the radicle at the beginning of seed germination, becoming more prominent at later stages of germination. During continued growth of the radicle which resulted in a reduction in size of the meristem, cells of the original quiescent center were activated to undergo DNA synthesis.     

Somatic polyploidy in neurons of the gastropod molluscs. II. Dynamics of DNA synthesis in the process of postnatal growth of CNS neurons in Succineid snail

A study was made of the age dynamics of polyploidization and dynamics of DNA synthesis in neuron cell nuclei during the postnatal growth of the gastropod pulmonate snail Succinea lauta. According to cytophotometrical results, the degree of polyploidization in neuron nuclei increases from young to adult individuals, varying from 2c to 16,384c. In the visceral complex, the maximum and medium ploidy values of the neuron nuclei are higher by almost 4-8 times than those in cerebral and pedal ganglia. The medium level of ploidy in adult snails increases by 5.7 times in the visceral complex of ganglia and by 4.1-4.2 times in the pedal and cerebral ganglia. According to 3H-thymidine autoradiography, DNA synthesis in neuron nuclei occurs during the whole life of the snail. In young individuals the neurons have the highest activity of DNA synthesis--the index of labeled nuclei of neurons making in total 50.2%. In older age, a steady decrease in the index of labeled nuclei is observed--in total to 35.8% and 7.0% in small and large adult snails, respectively. The state of summer hibernation completely stops DNA syntheses in neurons, but emergency from hibernation is accompanied by restoration of DNA syntheses.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G₂, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.     

The Role of Cytokinin in the Regulation of Growth, DNA Synthesis and Cell Proliferation in Cultured Soybean Tissues (Glycine max var. Biloxi)

Changes in protein content and cell proliferative activity were followed after a cytokinin-requiring strain of cultured Glycine max tissue was transferred to freshly prepared media which either contained or lacked cytokinin. Cell numbers doubled within the first two days after transfer, both in the presence and absence of cytokinin. However, after the second day no further increase in cell number was observed in the absence of cytokinin, while cell numbers continued to increase logarithmically in the presence of cytokinin. The size of the cell population attained after the first six days of growth was a function of the cytokinin concentration of the culture medium. However, the amount of ³H-thymidine incorporated into nuclear DNA bore no relation to the rate of cell proliferation. Tissues cultured on medium lacking cytokinin incorporated the greatest amount of ³H-thymidine per microgram of DNA, while the actively dividing tissues incorporated somewhat less. Using autoradiography and isopycnic CsCl gradient centrifugation, it was shown that the radioactivity derived from ³H-thymidine was associated with nuclear DNA in the cytokinin-deprived cells. Biochemical measurements demonstrated that cells cultured for six days without cytokinin had approximately twice the DNA content of the actively proliferating cells cultured on cytokinin-containing medium. Furthermore, in autoradiographs labeled cells were found to average nearly three times as many silver grains per nucleus in tissues cultured without cytokinin as the cytokinin-grown tissues. This suggests that the ³H-thymidine incorporation in the non-proliferating soybean cells results from nuclear DNA synthesis and that some of the cells became polypoid in the absence of cytokinin. These findings would be consistent with the idea that cytokinin acts as a specific trigger for cytokinesis.     

Mitotic activity of the endothelium of different-diameter blood microvessels in the mesentery of Wistar rats

The technique of intravascular autoradiography of 3H-thymidine labeled nuclei of microvessels endothelia was developed. The density difference of labeled cells in microvascular bed of mesentery was estimated. The maximal density of labelled nuclei was found in pre- and postcapillary vessels, and minimal--in capillaries and large arteries and veins of mesenteric bed. The number of labeled nuclei per vessel was found to be relatively constant. The density of labeled cells in 4-months old rats is less than that in 3 weeks-old ones.     

Association of newly replicated DNA with the nuclear matrix of Physarum polycephalum.

We have studied the role of the nuclear matrix in DNA replication in a naturally synchronized eucaryote, Physarum polycephalum. When P. polycephalum. When P. polycephalum macroplasmodia were pulse labeled with 3H-thymidine, the DNA remaining tightly associated with the matrix was highly enriched in newly synthesized DNA. This enrichment was found both in nuclei that had just initiated DNA replication as well as in nuclei isolated later during S phase. Pulse chase experiments showed that the association of newly replicated DNA with the matrix is transient, since most of the newly replicated DNA could be chased from the matrix by incubating pulse labeled macroplasmodia in media containing unlabeled thymidine. Studies measuring the size distribution of the matrix DNA supported the hypothesis that replication forks are attached to the nuclear matrix. Reconstitution controls indicated that these results were unlikely to be due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. These results with P. polycephalum in combination with previous studies in non-synchronized rodent cells, suggest that the association of newly replicated DNA with the nuclear matrix may be a general feature of eucaryotic DNA replication.     

OBSERVATIONS ON SYNAPTONEMAL COMPLEXES IN PREMEIOTIC INTERPHASE OF WHEAT

Synaptonemal complexes (SCs) were found in stage 3, premeiotic (S phase) pollen mother cell (PMC) nuclei of wheat which were labeled with ³H-thymidine. Three nucleoli are present in PMC nuclei at the beginning of stage 3, premeiotic interphase (S3). During S3, nucleoli move toward the nuclear envelope and fuse to form one nucleolus near the end of the stage. PMC nuclei labeled with ³H-thymidine were serially sectioned to show that more than one nucleolus was present and that SCs were also present in these DNA synthetic nuclei. Entire S3 PMC nuclei were serially sectioned to show the presence of SCs and all three nucleoli. Entire leptotene nuclei were also serially sectioned and segments of SCs were found. It is concluded that the association of homologous chromosomes in S3 of wheat is an early step in SC formation which proceeds through leptotene and is completed in zygotene and pachytene. Thus there is evidence that the continuum of chromosome pairing in wheat starts much earlier than was once thought.     

Incorporation of tritiated thymidine into mitochondrial DNA of the liver and kidney cells of chickens and mice in tissue culture

Summary ³H-thymidine incorporation into mitochondrial DNA of the liver and the kidney cells of chick embryos and newborn mice in tissue culture was shown by means of electron microscope radioautography with accurate localization. In these cells, about 20% of all the mitochondria were labeled at their matrices between the cristae within 4 hours in contact with the radioisotope, which were removed by DN'ase.From the results, it is clear that the mitochondria of avian and mammalian cells in tissue culture synthesize DNA.     

Cumulative indices of DNA synthesizing myocytes in different compartments of the working myocardium and conductive system of the rat’s heart muscle following extensive left ventricle infarction

Ten successive³H-thymidine injections at 12h intervals (which is a little shorter than the adult heart myocyte S phase) were performed for labeling of the majority of cardiac myocytes synthesizing DNA at any moment of such a 5 days experiment. In the hearts of control unoperated rats ten-fold repeated³H-thymidine administration results in labeling of 2–3% myocyte nuclei, in both atria, ca. 1% of the specialized muscle cell nuclei in the atrioventricular conductive system, only occasional muscle cells being labeled in the working ventricular myocardium. When ten successive³H-thymidine injections were made between the 5th and 10th days following extended left ventricle infarction, the percentage of labeled myocytes in left and right atria reaches, respectively, 51.4±4.4% and 34.7±3.6%. In the left ventricle labeled muscle nuclei are accumulated predominantly (9.3±2.1%) within the thin subepicardial layer of the surviving myofibers, while myofibers located in other perinecrotic areas contained only 1.3±0.5% labeled muscle nuclei. The number of these nuclei in the atrioventricular system remains at the level observed in control hearts (up to 2%), approaching closely the zero level in the working myocardium of both the ventricles and interventricular septum, located at the considerable distance from the infarcted region. When similar experiments with ten-fold repeated³H-thymidine injections were performed between 15th and 20th post-infarction days the number of labeled myocyte nuclei was found to be reduced 4–6 times in atria, being changed rather a little in the perinecrotic ventricular myocardium and in the specialized myocardium of the atrioventricular system. Some possible reasons of the observed differences in the proliferative behaviour of cardiac myocytes in terms of their topology and/or specialization are discussed