Postirradiation changes in the chromatin structure of thymocytes in irradiated rats

In this work the antibodies were obtained against chromatin isolated from thymocytes of intact and irradiated rats (2 h after exposing to 10 Gy) and against polydeoxyribonucleotides (PDN) extracted from thymus nuclei 6 h following irradiation. All the antibodies under study reacted more readily with the chromatin obtained from the thymus of exposed rats than with the control chromatin. The complexes of DNA with the most firmly bound non-histone proteins, obtained from the three objects under study, reacted with the antibodies with equal efficiency. Thus, a higher reactivity of PDN and chromatin from thymocytes of exposed rats was associated with the decondensation of the latter leading to an increase in availability of a part of antigenic determinants. Using the immunoblotting method we failed to discover any qualitative differences in the protein composition of the chromatin from control and exposed rats.     

Surface changes in irradiated thymocytes studied by partition in 2-phase systems and electrophoresis

A study was made of thymocyte surface changes 1-6 hour after the irradiation in a dose of 4 Gy by means of two phase partition in dextran-polyethylenglycole systems and of electrophoretic mobility registration. A decrease in the two phase partition coefficient (by 20% per cent in one hour after irradiation) was registered. The electrophoretic mobility of irradiated cells did not change.     

Immunochemical analysis of changes in the structure of the nuclear matrix in thymocytes of irradiated rats

It was shown that reactivity of the nuclear matrix of thymocytes for antibodies against chromatin of the control and irradiated thymocytes and PDN did not change immediately and increased markedly 2 h following irradiation of rats with a dose of 10 Gy. The method of immunoblotting failed to reveal any qualitative differences in the protein content of the thymocyte nuclear matrix of the control and exposed rats.     

Synthesis of lipids in the thymocytes of irradiated rats

During the first two hours following whole-body gamma-irradiation of rats with a dose of 7.5 Gy, the content of free cholesterol, phosphatidylcholine and phosphatidylethanolamine in the thymus decreases. Incorporation of 2-14C-acetate into cholesterol and fatty acids of thymus phospholipids in vitro is inhibited. At a dose of 4 Gy, incorporation of 2-14C-acetate into cholesterol and total lipids of thymocytes is activated.     

Effect of cycloheximide on the morphological and biochemical changes in chromatin in non-irradiated and irradiated rat thymocytes

The level of chromatin degradation was studied and the method of electron-microscopy was used to estimate the changes in the ultrastructure of irradiated and nonirradiated thymocytes of rats treated with cycloheximide. The latter was found to decrease the degree of derangement of nuclear ultrastructure and the level of chromatin degradation in exposed animals and to increase the yield of these damages in thymocytes of nonirradiated animals. The electronmicroscopic determinations showed that the percentage of thymocytes with the impaired nucleus structure is twice as high as that of degraded chromatin. The causes of the quantitative disagreement between the morphological and biochemical indices of the interphase thymocyte death are discussed.     

Analysis of DNA degradation in the thymocytes of irradiated rats by flow cytometry

The method of flow cytofluorometry of cells treated with probes specifically bound to AT- or GC-pairs of DNA was used to study DNA degradation in thymocytes of irradiated and hydrocortisone-treated rats. Death of thymocytes was shown to be accompanied by the decrease in the DNA content. The main regularities in the formation and accumulation of cells, the DNA content of which being lower than that of diploid cells, were the same as those of the internucleosome DNA fragmentation.     

Endonucleolysis of chromatin in thymocytes of irradiated and non-irradiated rats

It was shown that in conditions optimal for Ca/Mg endonuclease, chromatin endonucleolysis in the nuclei and thymocytes occurs due to internucleosome fragmentation of DNA. Irradiation activates chromatin degradation in thymocytes washed by a buffer containing 0.25 M sucrose, 10 mM tris-HCl, pH 7.2, 3 mM MgCl2, and does not influence this process in thymocytes washed by 10 mM tris-HCl, pH 7.2, 3 mM MgCl2.     

Biosynthesis of chromatin proteins in the thymocytes of irradiated rats

Biosynthesis of chromatin proteins in thymus cells of irradiated rats has been studied. During the period immediately preceding DNA degradation, new proteins, which are not found in the chromatin of the control cells, are synthesized in the exposed thymocytes and enter the chromatin. Besides, the rate of biosynthesis of a large number of other chromatin proteins decreases. The data obtained indicate that interphase death of lymphoid cells is an active, genetically programmed process.     

Biosynthesis of nuclear sap proteins in the thymocytes of irradiated rats

A study was made of biosynthesis of nuclear sap proteins in rat thymus cells exposed to 8 Gy gamma-radiation. Four hours following irradiation, changes were observed in the nuclear sap protein spectrum which were not associated with proteolysis.     

The intercellular interactions in the interphase death of irradiated thymocytes. A study of the nature of the mediator of the interaction of irradiated thymocytes]

A study was made of the influence of cytosol and conditioned medium, obtained at different times of incubation of irradiated thymocytes, on native thymocytes, as well as of the influence of mixing the suspension and changing the medium and the enzyme inhibitors, involved in metabolism of arachidonic acid, on the radiation injury to thymocytes. Cytosol of thymocytes, incubated for 60 min after irradiation, was shown to exert an insignificant toxic effect on native thymocytes. The conditioned medium of irradiated thymocytes is virtually ineffective. The mixing of the suspension upon incubation and the changing of the medium reduced the damage to exposed thymocytes while inhibitors of phospholipase A2 and lipoxygenase prevented completely the radiation-induced pyknosis of nuclei, whereas a inhibitor of cyclooxygenase did not influence it. It is inferred from the data obtained that unstable lipoxygenase products may serve as mediators of intercellular interactions of exposed thymocytes.     

Patterns in the degradation of individual genes of thymocytes in irradiated rats

Degradation of genes of actin, albumin, histones, heat shock protein, and ribosomal RNA within DNA of irradiated animal thymocytes has been investigated. It has been shown that single strand enzymatic breaks occurred in thymocyte DNA 2 h following irradiation are localized in linker sites of nucleosomes. All the transcribed genes under study degrade to fragments that correspond by their length to DNA of nucleosomes and their oligomers. The albumin gene nontranscribed in thymocytes also degrades; however, no low molecular weight fragments are found. The degree of gene degradation is invariable in time.     

Study of changes in the surface of irradiated thymocytes using a method of separating cells in a 2-phase water-polymer system

The coefficient of cell distribution in a charged biphase water-polymeric system "polyethyleneglycol-dextran" decreased 60 min after irradiation of rat thymus lymphocytes with doses of 1 to 8 Gy. This dose-dependent effect was mainly related to radiosensitive thymocytes of a low fraction of density gradient; it disappeared with the uncharged system and was probably connected with the reduction in the negative electric charge of the irradiated cell surface.     

Thymidine excretion from thymocytes in irradiated and nonirradiated rats during endonucleolysis

The concentration of free thymidine (dT), excreted from thymocytes of irradiated and nonirradiated rats, was determined after incubation of cells in various digestive buffers. The release of dT from thymocytes depended upon the rate of DNA fragmentation in conditions of chromatin endonucleolysis. The increase in the thymidine content, in conditions of chromatin endonucleolysis in buffers containing no calcium ions, was only noted in thymocytes of exposed rats: this was the consequence of chromatin DNA damages already available in these cells.