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To characterize thyroid hormone action in the ovary, the direct effect of triiodothyronine (T3) was investigated in vitro using a culture system of porcine theca cells (Tcs) and granulosa cells (Gcs) in mono- and co-culture (GT), the latter resembling follicles in vivo. The cells were cultured in the absence or presence of human chorionic gonadotrophin (hCG) with or without T3 (10(-7), 10(-9) or 10(-11) M). Follicular cells were obtained from follicles of different size (small, medium and large), and steroid secretion into the culture medium was detected by radioimmunoassay. T3 alone did not influence steroid secretion by Tcs and Gcs isolated from follicles that were small and medium in size. In preovulatory follicles, an increase in basal androgen secretion and a simultaneous decrease in oestradiol secretion were observed with Tcs and Gcs in both mono- and co-culture. T3 together with hCG decreased hCG-stimulated androgen secretion in Tcs isolated from medium-sized follicles and had a simultaneous stimulatory effect on hCG-stimulated oestradiol secretion by Gcs. In cultures of follicular cells obtained from large follicles, T3 decreased hCG-stimulated secretion of both androgen and oestrogen by Tcs and simultaneously stimulated oestradiol secretion in GT co-cultures. Thus, the interaction of T3 with gonadotrophin hormone modulated follicular steroidogenesis, depending on follicle size and cell type used in culture. The observed T3-induced increase in basal androgen secretion by Tcs could account for the atresia of follicles, since it is accompanied by a decrease in oestradiol secretion in GT co-culture. In its co-activity with hCG, an adequate level of T3 prevents excessive androgen production by Tcs, probably influencing aromatization processes in the follicle. An increase in hCG-stimulated oestradiol secretion in GT co-cultures is then observed. Further investigations are required to clarify whether this is linked with an effect on the aromatization processes occurring in the follicle.

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The characteristics of the postirradiation degradation of chromatin in thymocytes in vivo were compared with the features of chromatin fragmentation in isolated thymocyte nuclei in vitro by endogenous chromatin-bound nucleases. Nuclease which degrades chromatin produces in vivo fragments of nucleosomal size; the double-strand breaks appear as the result of the accumulation of single-strand breaks with 3'-OH ends; the nuclease is inhibited by Zn2+ and DTNB and its activity is depressed by cycloheximide pretreatment. In experiments on in vitro degradation of chromatin in isolated thymocyte nuclei similar properties were observed for the Ca, Mg-dependent, but not for acid nuclease. The results bring further evidence of the involvement of an enzyme of the Ca, Mg-dependent nuclease-type in chromatin degradation in irradiated thymocytes.

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The administration of actinomycin D in toxic doses (4 mg/kg i.p.) to mice induces a progressive degradation of thymus chromatin. The course of this damage is slower than the radiation-induced chromatin degradation; it starts at 6-8h and culminates at 24 h after actinomycin D injection, when already most of the treated mice are dying. The inhibition of the [2-14C] orotic acid incorporation into rapidly labelled RNA by actinomycin D is substantially less expressed in the thymus than in the liver. The course of chromatin degradation correlates with the onset of the inhibition of protein synthesis in the thymuses of actinomycin D-treated mice. This demonstrates that, starting from the 6th h after actinomycin D administration, irreversible changes appear in the thymocytes that lead to chromatin degradation and cellular death. Contrary to the action of cycloheximide, the treatment with actinomycin D aggravates the chromatin degradation induced by irradiation. A comparative electrophoretic analysis of the DNA fragments (in native state and denatured) isolated from thymus chromatin of mice treated with actinomycin D (and/or irradiated) demonstrated that the chromatin DNA is degraded only in the internucleosomal segments yielding DNA fragments of nucleosomal size. Similarly to the effect of other agents leading to chromatin damage in vivo, the actinomycin D-induced degradation does not affect the DNA in the nucleosomal segments of chromatin.

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Irradiation of mice with doses of 2 and 4 Gy induced extensive chromatin degradation in the thymocytes within 6 hours accompanied by an increase in polydeoxynucleotide (PDN) content (36 and 42 times, respectively). Fifteen hours after irradiation the PDN level was considerably lower, however, still being 4.7 and 14 times the control values after doses of 2 and 4 Gy. The PDN content in control LS/BL lymphosarcoma cells was similar as that in the thymocytes of non-irradiated mice. Unlike in the thymocytes, irradiation of lymphosarcoma cells did induce no statistically significant increase in the PDN level 6 and 15 hours after the irradiation, respectively. It has been reported previously (Matyásová et al. 1973) that chromatin of LS/BL cells degraded similarly as that in the irradiated thymocytes. The results of the present experiments thus provide additional evidence for changes of LS/BL cell properties due to long term cultivation. These cells, however, are still able to react by chromatin fragmentation to nitrogen mustard treatment.

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The action of 1,10-phenanthroline-copper complex under aerobic conditions in the presence of 2-mercaptoethanol on chromatin DNA in murine thymocyte nuclei was studied. At limited oxygen supply primarily multiple single-strand breaks of DNA in the spacer segments are observed while the core DNA segments in chromatin remain intact. After the single-strand breaks accumulate in both DNA strands (under conditions of improved oxygen supply), double-strand cleavage of DNA to fragments of nucleosomal size becomes apparent. This regular character of DNA degradation in chromatin is apparently due to the preferential binding of the 1,10-phenanthroline-copper complex to DNA of the spacer segments and to the localized generation of damaging radicals.

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Histones precipitate from a solution of 0.14 mol/l NaCl with increasing concentrations of the polyanions polypentosesulphate; dextran sulphate; inorganic polyphosphate; heparin; or copolymer ethylene-maleic acid forming complexes from which histones cannot be extracted by 0.25 mol/l HCl. Affinities of the histone classes for polypentosesulphate appeared in the order from greatest to least: H4 approximately H3 greater than H2A greater than H2B greater than H1. At increased concentrations of most polyanions studied, the complexes of histones with polyanions remained partially soluble. Complexes of histones with all polyanions used were completely soluble in 2% SDS electrophoresis buffer, in 0.14 mol/l NaCl buffered at pH 12, and in 2 mol/l NaCl buffered at pH 7.2. Solubilisation of the complex polypentosesulphate-histone in 2 mol/l NaCl proved to be due to its dissociation.

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Some characteristics of the postirradiation degradation of chromatin in the thymuses of mice were studied. The results proved that the main wave of chromatin degradation becomes evident between 2 and 4 h postirradiation, when considerable amounts of degradation products leach from nuclei during their isolation and are solubilized by lysis of nuclei. Similarly the degradation is manifested in the increase of salt-soluble chromatin fraction as well as of the fractions released from chromatin by various solutions (EDTA, heparin, deoxycholate, alkaline buffer). Later on, within 24 h after irradiation, only little changes in the relative amounts of the degradation products take place. Evidently only a certain thymocyte population is involved. Electrophoretic analyses of DNA fragments from various fractions in native and denatured state demonstrated that chromatin was degraded into nucleosomes and their oligomers by an endonuclease activity. The DNA bears, however, no signs of intranucleosomal regular single-strand fragmentation. This fact makes improbable the participation in this process of DNase I, DNase II and Ca,Mg-dependent endonuclease. No appreciable amount of smaller DNA fragments (products of further degradation of nucleosomes) were found even at 24 h postirradiation interval. Thus the nucleosomes and their oligomers must be considered as the only "long-lived" chromatin fragments in damaged lymphoid cells.

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The authors studied the capability of chromatin for structural reorganization in a medium of ionic strength, close to physiological one, under the effect of polyions of polycationic nature--summary histone of chromatin. Electrophoretic analysis demonstrated that like polyanions, summary histone forces out histone HI and partially histones H2B and H2A from chromatin. However, in contrast to polyanions, summary histone makes chromatin resistant to DNase-II, apparently potentiating the aggregation capability of chromatin despite the removal of histone HI.

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The aim of the present study was to reassume the chromatin changes occurring in lymphoid tissues of mice treated with alkylating agents of the nitrogen-mustard type in relation to recent evidence on the nucleosomal organization of chromatin and to our new data on the regular character of chromatin degradation in lymphoid tissues of irradiated mice. DNA was isolated from nuclei at various intervals (1-18 h) after treatment of mice and subjected to gel electrophoresis in polyacrylamide gels. Thymus chromatin from treated mice has been shown to degrade in a regular fashion and to yield discrete DNA fragments, resembling those that originate in lymphoid tissues of irradiated mice or in thymus nuclei digested with micrococcal nuclease in vitro. With increasing interval after treatment higher amounts of smaller DNA fragments appear. Chromatin in spleen cells responds to treatment in a similar way, whilst no degradation in vivo takes place in liver chromatin. Chromatin of LS/BL lymphosarcoma cells in mice treated with alkylating agents or with irradiation suffers from a similar regular degradation. The results stress the significance of the action of liberated or activated endogenous nuclease(s) in the development of chromatin damage in lymphoid cells after treatment with alkylating agents.

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The degradation of DNA in modified deoxyribonucleoprotein samples by DNase II in the presence of a polyanion (polypentose sulphate) was studied. The behaviour of DNP samples partially depleted of histone and treated with acetic anhydride is characterized by an increased accessibility of their DNA to DNase II, by a decreased polyanion amount needed for the maximum enzymatic degradation of their DNA, and, finally, by a more profound inhibitory effect of higher polyanion amounts on the DNase II-catalyzed degradation. On the other hand, the behaviour of DNP samples treated with formaldehyde is characterized by a decreased accessibility of their DNA to DNase II, by a limited increase in the enzymatic degradation of their DNA in the presence of the polyanion, and finally by a less profound inhibitory effect of higher polyanion amounts on the DNase II-catalyzed degradation.

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Increasing amounts of DNA and proteins are released from the suspensions of chromatin from thymuses and spleens of irradiated mice (6 hours after 600R whole-body) by the action of alkaline solutions (pH 8-10) at physiological ionic strengths. The suspension of chromatin from normal tissues releases in this pH range only a small amount of proteins and negligible amount of DNA. The behaviour of liver and kidney chromatin to alkaline solutions shows no difference between normal and irradiated tissues. The time of onset and dose relation of the increased sensitivity of thymus and spleen chromatin from irradiated mice to alkaline solutions show a similar course as earlier described signs of postirradiation damage to chromatin of these tissues.

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