The effect of gamma-irradiation on the poly(ADP-ribosylation) of nuclear proteins in rat thymocytes

Differences in the spectra of modified nuclear proteins of thymocytes of control and irradiated rats were investigated using antibodies specific for poly(ADP-ribose) and incorporation of a label from 14C-NAD in vitro. Two classes of modified proteins were identified differing in the rate of the polymer metabolism and the degree of poly(ADP-ribosylation). No postirradiation changes were detected in poly(ADP-ribosylation) of the nuclear sap proteins and chromatin. A pronounced increase in modification of proteins with the molecular mass of 72 and 83 kD and a sharp decrease in poly(ADP-ribosylation) of a protein group with the molecular mass of 47 to 65 kD were detected within the nuclear matrix by the second hour following irradiation. A study was made of the localization of modified proteins in polydeoxynucleotide fractions of different sizes (mononucleosomes and their oligomers).     

Early changes in the biosynthesis of proteins in the thymocytes of mice following exposure to lympholytic agents

The method of two-dimensional electrophoresis was used to study protein biosynthesis in cytoplasm, nuclei and nuclear matrix of mouse thymocytes 15-75 min after exposure to ionizing radiation and hydrocortisone. Irradiation was shown to inhibit some cytoplasm proteins and to induce synthesis of a few polypeptides within the total nuclear proteins and nuclear matrix. Hydrocortisone promotes synthesis of some new proteins in all the fractions leading to the formation of new polypeptides. At least one common peptide is found within the total nuclear proteins and nuclear matrix after the effect of both factors.     

Nature of breaks inducible in DNA at the early stages of interphase cell death

Dynamics of changes in 3'-OH- and 5'-OH-ends of DNA was determined by "nick"-translation and direct polynucleotide kinase reaction, respectively, in animal thymocytes after irradiation and administration of hydrocortisone. Breaks bearing both 3'-OH- and 5'-OH-ends were found in DNA after irradiation. In 40 min repair of single-strand breaks was almost completed, and enzymatic breaks were accumulated with 3'-OH-ends only. 60 min after the administration of hydrocortisone, the number of nuclear DNA breaks containing 3'-OH-ends, but not 5'-OH-ends, sharply increased. Upon DNA autolysis in isolated nuclei acid nuclease produced 5'-OH-ends, and Ca2+/Mg2+-dependent nuclease, 3'-OH-ends. No activity of Mg2+-dependent nuclease was registered either in the nuclei of control thymocytes or in the nuclei isolated from thymocytes of exposed rats.     

The characterization of the monoclonal antibody Th-10a, specific for a nuclear protein appearing in the S phase of the cell cycle in normal thymocytes and its unregulated expression in lymphoma cell lines

A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG_2a and had κ light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G₁, G₂ and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characteize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or***     

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.     

Immunomodulations of thymocytes and splenocytes in trained rats.

The aim of this study was to describe the effects of training (running) on thymus and spleen cells in the rat. Young Wistar control rats (n = 6), rats trained for 4 wk (n = 5), and rats trained for 4 wk followed by 1 wk of intensive training (3 h/day, n = 6) were studied. Various lymphocyte surface and nuclear markers were determined by immunocytochemistry. The results show that 4 wk of training 1) decreased the percentage of bromodeoxyuridine (BrdU+) thymocytes (cell in phase S of the cycle, immature thymocytes; P less than 0.05) and the viability of thymocytes stimulated with concanavalin A (Con A; P less than 0.05) and 2) increased the absolute number of CD8+ (suppressor/cytotoxic T cells; 29%) and the percentage of CD8+ splenocytes (P less than 0.01). An additional week of intensive training in the 4-wk trained rats induced 1) a decrease in the absolute number of thymocytes (25%, P less than 0.05), TCR+ thymocytes, splenocytes (28%, P less than 0.01), T, CD4+ (helper T cells; 34%), and CD8+ (31%) splenocytes (P less than 0.01) and 2) an increase in the viability of splenocytes after stimulation with Con A for 72 h (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Degree of chromatin fragmentation and frequency of nuclear pyknosis in Percoll-fractionated thymocytes of irradiated rats

The relationship between nuclear chromatin degradation to polydeoxyribonucleotides (PDN) and other features of interphase death were studied using thymocytes of normal and X-irradiated rats. Fractionation of the thymic cells in Percoll gradients was performed in order to separate dead from intact cells. The degree of radiation-induced chromatin fragmentation, as assessed by electrophoresis, was similar for PDN from all Percoll bands. Following irradiation 87-98 per cent of 'heavy' thymocytes were pyknotic and almost devoid of receptors to autologous erythrocytes thus comprising a dead cell population. A direct relationship between PDN content and nuclear pyknosis was noted throughout the gradient. The loss of autologous rosette-forming ability was directly related to other indices of interphase death. The possibility of PDN originating from pyknosis-prone cells and the capacity of radiosensitive thymocytes to form autologous rosettes are discussed.     

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.     

Plasminogen activator and protease inhibitor activities in isolated rat thymocytes

Summary Plasminogen Activator (PA) and its response to glucocorticoids and androgens was studied in viable rat thymocytes in suspension. PA was measured by its ability to convert plasminogen to plasmin, and the formed plasmin determined by cleavage of ¹⁴C-labeled globin. Using this functional assay, PA was found to be associated with the outer surface of thymic cells, and only negligible activity recovered from the incubation medium. Rat thymocytes also contain cytoplasmic and nuclear inhibitor(s) of the serine proteases plasmin, trypsin, chymotrypsin and thymic PA. Release of these inhibitors prevented determination of thymic PA activity in presence of lysed cells.The specific activity of PA in thymocytes isolated from adrenalectomized-castrated rats did not differ significantly from the specific activity associated with cells from intact animals. Furthermore, treatment of adrenalectomized-castrated rats with 0.1 mg of dexamethasone/ kg for 2 days induced thymic involution without affecting thymic PA activity. These observations suggest that PA activity of thymocytes is not involved in glucocorticoid-mediated thymic involution.     

Study of nuclear poly(ADP-ribose)polymerase and DNA-topoisomerase II of brain cells during postnatal development of rats

The nuclear poly(ADP-ribose)polymerase activity of neuronal and glial cells during postnatal development of rats was studied. It was shown that the poly(ADP-ribose)polymerase activity of nuclei and nuclear matrix of neuronal cells during postnatal development of rats is increased, whereas the polymerase activity of glial cell nuclei and nuclear matrix in newborn and adult rats is higher than in 14-day-old animals. The DNA-topoisomerase II activity of neuronal nuclear matrix during the postnatal development of rats does not change, whereas the topoisomerase activity of glial nuclear matrix decreases but is always higher than the DNA-topoisomerase II activity of neuronal cell matrix during the postnatal development of rats. It is suggested that ADP-ribosylation in the nuclear matrix of neuronal cells causes the inhibition of the DNA-topoisomerase II activity of nuclear matrix.     

Identification and characterization of glucocorticoid-regulated nuclease(s) in lymphoid cells undergoing apoptosis

Apoptosis is a physiological process by which selected cells are deleted from a population in response to specific regulatory signals. A hallmark of apoptosis is the internucleosomal degradation of DNA prior to cell death. We are studying glucocorticoid-induced lymphocytolysis as a model system for apoptosis within the immune system. In rat thymocytes, the internucleosomal DNA cleavage which occurs following glucocorticoid treatment is both time- and dose-dependent, and is blocked by the glucocorticoid antagonist RU 486, indicating that this effect is mediated by the glucocorticoid receptor. Similar experiments using glucocorticoid-responsive (wt) and glucocorticoid-resistant (nt⁻) S49.1 lymphoma cell lines confirm that internucleosomal DNA degradation and cell death are glucocorticoid receptor-mediated events and thus reflect the direct effects of glucocorticoids on lymphocytes. In an effort to identify the nuclease(s) responsible for the DNA degradation, we have developed two assays to detect nucleases whose activity is altered by glucocorticoid treatment. The first assay involves electrophoresing extracts of nuclear protein from control and glucocorticoid-treated lymphoid cells into SDS-polyacrylamide gels containing [³²P]DNA within the gel matrix. This assay is used to estimate the molecular mass of the nuclease, based on the observed in situ nuclease activity. The second assay uses HeLa nuclei as a substrate to detect internucleosomal cleavage activity present in nuclear extracts of control and glucocorticoid-treated lymphoid cells. Using these assays we have identified a novel Ca²⁺, Mg²⁺-dependent nuclease with an apparent molecular weight of 18 kDa in both S49 wt cells and rat thymocytes treated with glucocorticoids. Furthermore, nuclear extracts of glucocorticoid-treated, but not control, rat thymocytes and S49 wt cells were capable of cleaving HeLa chromatin at internucleosomal sites. In an effort to determine the identity of the nuclease capable of internucleosomal cleavage of DNA, nuclear extracts from dex-treated rat thymocytes were fractionated by gel filtration chromatography under non-denaturing conditions, and the fractions were analyzed using the [³²P]DNA SDS-PAGE and HeLa nuclei assays. When analyzed under native conditions, the 18 kDa nuclease described previously appears to exist as a 25 kDa protein which may be part of a high molecular weight complex. Interestingly, only the 25 kDa form of the protein was associated with internucleosomal DNA cleavage activity where as the high molecular weight form of the enzyme was devoid of this activity.     

Radiation-induced formation of apoptotic bodies in rat thymus

The process of interphase death of thymocytes in whole-body X-irradiated rats were studied. Cell size distribution analysis indicates that cell fragments (= apoptotic bodies) appeared in the thymus and increased in number depending on dose (200-1000 R) and time (2-6 hr) after irradiation with corresponding decrease in normal-size thymocytes. Occurrence of nuclear fragmentation in association with the cellular fragmentation was proved with cytofluorometric determination of DNA content in individual cells. Scanning electron microscopic observations also revealed extensive fragmentation of cells in the irradiated rat thymus. The results show clearly that cells as well as nuclei fragment rapidly into smaller pieces of various sizes in the irradiated rat thymus as commonly observed with apoptosis.     

The microelectrophoresis of the DNA in individual intact and gamma-irradiated thymocytes

The in vitro gamma-irradiated mouse thymocytes were embedded in low melting agarose at 37 degrees C. After getting at 4 degrees C, the cells were lysed in neutral detergent solution containing proteinase K and ethidium bromide. Microscopic visualization of single lysed and stained cells showed the presence of the central "core" (nuclear matrix) surrounded with "halo" (relaxed nuclear DNA). During electrophoresis (2-5 V/sm, 5 min) this "halo" migrated towards the anode forming a "tail". The use of microdensitometric system provided measuring the size of the tail (L) and quantity of migrated DNA (S) for individual cells as well as obtaining the distribution of these parameters among the cells. The latter may be characteristic of heterogeneity of the cell population. It was shown that L and S increased linearly with the dose irradiation at least between 0.2 and and 5.0 Gy. In irradiated thymocyte (3 Gy) the DNA repair occurred within 10-20 min, but residual DNA damage could be observed even after 60 min of incubation. These damages may initiate the degradation of DNA in irradiated thymocytes that was observed after the repair of DNA.     

Inhibitor of apoptosis protein from Orgyia pseudotsugata nuclear polyhedrosis virus provides a costimulatory signal required for optimal proliferation of developing thymocytes

The inhibitors of apoptosis proteins (IAPs) constitute a family of endogenous inhibitors that control apoptosis in the cell by inhibiting caspase processing and activity. IAPs are also implicated in cell division, cell cycle regulation, and cancer. To address the role of IAPs in thymus development and homeostasis, we generated transgenic mice expressing IAP generated from the baculovirus Orgyia pseudotsugata nuclear polyhedrosis virus (OpIAP). Developing thymocytes expressing OpIAP show increased nuclear levels of NF-kappaB and reduced cytoplasmic levels of its inhibitor, IkappaBalpha. In mature thymocytes, OpIAP induces optimal activation and proliferation after TCR triggering in the absence of a costimulatory signal. OpIAP expression in immature thymocytes blocks TCR-induced apoptosis. Taken together, our data illustrate the pleiotropism of OpIAP in vivo.     

Effects of psychotropic drugs of the phenothiazine series on matrix activity of thymocyte DNA and glucocorticoid receptor interactions

The experiment on adrenalectomized rats has shown that the concentration rates of 3.5.10(-4)M aminazine and 7.0.10(-4)M tizercine reduce 3H-acetonide triamcinolone-receptor interaction, and the concentration rate of 7.0.10(-4) tizercine increases 3H-cortisol receptor interaction in liver cytosol. The concentration rates of 3.5.10(-7)M aminazine and 3.5.10(-5)M tizercine suppress 3H-uridine inclusion into the acid-resisting mRNA thymocyte fraction. Associated introduction of aminazine and tizercine with acetonide triamcinolone (10(-8)M) increases the inhibiting effect on 3H-uridine inclusion into mRNA thymocytes. This suggests that aminazine and tizercine suppress matrix activity of DNA thymocytes through their effect on glucocorticoid receptors of type II.     

Ovariectomy causes cell proliferation and matrix synthesis in the growth plate cartilage of the adult rat

The in vivo effects of ovariectomy in rats have been studied on cell proliferation and matrix synthesis in the growth plate cartilage by assessing immunohistochemically the levels of proliferating cell nuclear antigen and chondroitin sulphate proteoglycan(s). The serum levels of insulin-like growth factor-I and growth hormone were also measured by radioimmunoassay procedures. At 5 weeks after ovariectomy, the serum levels of the growth factor were significantly higher than those in sham-operated rats. In contrast, the level of growth hormone was lower. The nuclear staining of proliferating cell nuclear antigen was generally seen in the zone of proliferative chondrocytes from both groups of rats. Whereas almost all chondrocytes in the proliferative zone of ovariectomized rats expressed proliferating cell nuclear antigen immunoreactivity, fewer did so in that of the sham rats. Quantitative image analysis by ACAS 570 laser cytometry demonstrated that the n uclear antigen-positive sites in ovariectomized rats had significantly higher integrated values (staining intensity), areas and perimeters than those in sham rats. In addition, the number of chondroitin sulphate proteoglycan-immunoreactive cells in the proliferative chondrocytes was also higher in ovariectomized rats than in sham ones. These results suggest that ovariectomy significantly stimulates the cell proliferation and matrix synthesis in the growth plate cartilage, probably through the higher serum level of insulin-like growth factor-I.     

Activity of cell-detached 5'-nucleotidase in thymocytes of various densities

The rat thymocytes submitted to heating at 45 degrees C for 1 hr liberate plasma membrane fragments containing 5'-nucleotidase activity in the supernatant. The thymocytes were separated by ficoll density gradient centrifugation. High activity of 5'-nucleotidase per 10(6) cells was found in the supernatant of low density (1.069) subset of thymocytes. Thymocyte supernatant of rats treated with hydrocortisone demonstrated higher 5'-nucleotidase activity per 10(6) cells than in intact animals. This is due to an increase of the low density population of thymocytes in treated rats since the 5'-nucleotidase activity per 10(6) cells of the supernatant obtained from this density fraction is the same both in treated with hydrocortisone and intact rats. Hydrocortisone seems to induce a selection of the thymocytes with high 5'-nucleotidase activity.     

DNA-polymerase activity and DNA synthesis in the hepatocyte nuclear matrix

The comparative analysis of DNA-synthetase activity of hepatocytes, isolated nuclei and nuclear matrix from normal and regenerating rat liver was performed. The highest enrichment with newly-synthesized DNA was registered in the DNA fraction associated with the nuclear matrix both in vivo and in vitro. The functioning of DNA polymerases alpha and beta in the matrix was shown. Our results indicate that DNA polymerase beta is more firmly bound with the nuclear matrix in the cells of normal liver but this enzyme is eluted almost completely from the nuclei of regenerating liver cells. At the first moment after gamma-irradiation of rats the preferential initiation of unscheduled DNA synthesis in vivo has been observed on the nuclear skeletal structures. This may serve as an indication on the possibility that DNA repair process occurs on the nuclear matrix.