[Amyloid-like oligomers of presynaptic protein BASP1].

Brain protein BASP1 forms oligomers that resemble amyloid protein oligomers in some respects, including interaction with conformation-specific antibodies against amyloid oligomers. Aggregation-prone N-terminal myristoylated peptide myr-BASP1 (1-13) forms fibrillar aggregates of amyloid-like structure under physiological conditions in vitro, which is also the evidence of BASP1 similarity to amyloid proteins. Protein cross-linking by glutaraldehyde in situ demonstrated that BASP1 exists on the presynaptic membrane as lipid raft-associated oligomers. In addition, BASP1 is non-toxic to PC12 cells, when applied either as a monomer or oligomer. We conclude that BASP1 oligomer is a non-pathological physiologically relevant functional form of this protein.

[Characteristics of the spontaneously transformed human endothelial cell line ECV304. II. Functional responses of the ECV304 cells].

Functional responses of the spontaneously transformed human endothelial cell line ECV304 were studied in order to asses its applicability as an endothelial cell model for studying angiogenesis and signal transduction. The dependence of proliferation activity of this line on the presence of growth factor was shown. The absent serum in culture medium resulted in blocking of cells in G1-phase of a cell cycle which is not typical for tumor cell lines. Low doses of beta particles emitted during [3H]thymidine decay resulted in blocking the proliferation of these cells in G2M-phase in a dose-dependent manner. Incubation of the cells with another source of beta particles, 3H2O, under condition of equal specific activities of tritium resulted in preferable accumulation of the cells in S-phase. The different efficiency of beta particles of tritium as a part of 3H2O molecule or thymidine demonstrates that various mechanisms are responsible for various check points. The check point of G1/S is absent and that complies with the presence of deletion of chromosome 9 in locus p21. The level of NO produced by constitutive form of NO-synthase in ECV304 cells was relatively low and not modified by inducible NO-synthase inhibitors. The data obtained suggest that ECV304 line cells retained the properties of the initial spontaneously transformed cell line obtained from human umbilical vein (HUVEC) as well as they can be used as a model system for further studies of the properties of vascular endothelial.

[The blocking of the proliferation of the human endothelial cells in culture by beta-irradiation from internal and external sources of the radiation. S-block is induced by the 3H2O beta-particles].

Recently we shown that low doses (0.12-0.46 Gy) of (methyl-3H)-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. The temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the induction of the G2-block estimated by flow cytometry analysis of DNA content and in the induction of the chromosome aberrations (bridges and fragments in anaphase). The aim of this study was the comparative investigation of efficiency of beta-rays emitted 3H from 3H-thymidine and 3H2O by several of the cellular parameters. Here we shown that at the equal conditions of the incubation of the cells in medium with 3H2O induced the accumulation cells in S-phase without decreasing of the mitotic activity and without increasing of the chromosome aberrations level. Unlike from 3H2O the incubation of the cells with 3H-thymidine induced the accumulation cells in G2-phase with decrease of the mitotic activity and with increase of the chromosome aberrations level. Concurrent treatment cells with 3H-thymidine and thymidine abrogate these cellular effects of the 3H-thymidine. Inhibitor ATM-kinase caffeine abrogate as G2-block as S-phase block. These results suggest that the low-dose beta-radiation activates S-phase and G2-phase checkpoints requiring ATM-mediated signal transduction pathway. The factors, which impact on the efficiency of the internal and of the external sources of the irradiation, depend on theirs disposition in relation to radiosensitive target--DNA was discussed.

[Internal and external sources of the radiation induce the blocking of the proliferation of the human endothelial cells in culture. G2-block is induced by beta-particle 3H-thymidine and gamma-rays 137Cs].

We found that low doses (0.12-0.46Gy) of (methyl-) 3H-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. Temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the G2-block estimated by flow cytometry analysis of DNA content. Furthermore, the induced the high level of the chromosome aberrations (bridges and fragments in anaphases). 1Gy of gamma-ray 137Cs and 0.005 Gy of beta-rays induced the same per cent of the aberrant anaphases. Apparently, that the damages of the cellular hereditary structures are responsible for the blocking of the cellular proliferation in G2-phase. We suggest, that the disposition 3H-thymidine into radiosensitive target (DNA) defines the high cytotoxic of the beta-rays.

[Modification of cellular radiosensitivity by NO-synthase inhibitors].

We recently reported that the treatment of V-79 and HeLa cells with nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly reduced the level of the radiation-induced unstable chromosome aberrations. The stereoisomer D-NAME had no effect. We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the NOS-activity. We tested this suggestion on the NO-resistant (ECV-304) and NO-sensitive (HeLa) cells, which were treated with L-NAME or aminoguanidine or D-NAME or cysteamine before gamma-irradiation. There are no significantly differences in radiosensitivity between these cells estimated after exposure by gamma-rays with different doses. However, the radioprotective effect of the NOS-inhibitors manifested only for HeLa. D-NAME had no radioprotective effect neither HeLa nor ECV-304. In contrast NOS-inhibitors, cysteamine treatment EVC-304 reduced the radiation-induced level chromosome aberrations almost twofold. The different mechanisms of the modification of cellular radiosensitivity are discussed.

[About the peculiar form of radiation induced genome instability].

The usually studied forms of the induced genome instability manifested as a steady change of mutability of proliferated cells, we compared with its peculiar form characterized by the inherited irreversible increase of the level of spontaneous damage of cells. These effects appear of the moment of reaching of the dose threshold according to the "all or nothing" principle, just after the relatively low-dose radiational action. These changes occur without association with the cell division and thereby have an exclusive significance in the pathogenesis of the radiational damage of low-renewing mammalian tissues. It is suggested that the considered changes could underlie the development of the delayed non-cancerogenic somatic postradiational sequelae and of the acceleration of natural aging.

[NO-dependent pathway of the modifying of the cellular sensitivity to gamma- and neutron irradiation]. / NO-zavisimyi put' modifikatsii kletochnoi chuvstvel'nosti k deistviiu gamma- i neitronnogo izlucheniia.

The modifying effect of L-NAME, the NO-synthase inhibitor and D-NAME, the inactive enantiomer was investigated in human carcinoma cells (HeLa) and Chinese hamster fibroblasts (V-79) exposed to different doses of gamma-rays and 0.85 MeV neutrons. We estimated the level of the chromosome aberrations manifested as the bridges and fragments in anaphases. Radioprotective effect of L-NAME showed the inverse dependence on the exposure dose and at low doses (1 Gy) it was higher in the V-79 cells, than in the HeLa cells. However, at high doses (3, 4, 6 Gy) the efficiency of L-NAME for these cellular lines was almost equal (DFR = 2). The modifying effect of L-NAME was almost equal for gamma-irradiation and neutrons, although the exposure of V-79 cells to neutrons induced more the asymmetric chromosome aberrations (RBE = 4). The D-NAME had no effect on the level of the radiation-induced chromosome aberrations, although D-NAME treatment of cells increased the chromatin condensation, as well as L-NAME. The counteractive condensation does not play the major role in the radioprotective effect of L-NAME. We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the inducible NO-synthase.

[Study of the radioprotective effect of the NO-synthase inhibitor L-NAME in Chinese hamster cell culture]. / Issledovanie radioprotektornogo éffekta ingibitora NO-sintazy L-NAME v kul'ture kletok kitaiskogo khomiachka.

A radioprotective effect of L-NAME was estimated by the yield of the aberrant anaphases after exposure of Chinese hamster cells to different doses of gamma-rays and beta-particles. It was shown that cell treatment with L-NAME before irradiation only decreased the frequency of radiation-induced chromosome aberrations. The equal yield of the aberrant anaphases was found in the cells treated with L-NAME and irradiated with 6 Gy gamma-rays later and in the cells non-treated with L-NAME and irradiated with 3 Gy gamma-rays. The treatment of cells with L-NAME just before and immediately after irradiation did not modify the radiation-induced frequency of the chromosome aberrations. The treatment of cells with L-NAME decreased the level of SH-groups (as estimated by UV-spectrophotometer) and increased the chromatin condensation (as estimated by flow cytometry). It was suggested that radioprotective effect of L-NAVE may be connected with its cooperation with DNA. Condensation of L-NAME on the surface of DNA resulted in increasing of probability of the chemical repair of DNA-radicals after irradiation. Thus, these results indicate the involvement of NO-dependent mechanism of the realization of the radiation-induced damage to the hereditary cell structure and optimal conditions for the realization as well as the conceivable mechanism of radioprotective effect one of the most inhibitors of NO-synthase--L-NAME.

[Effect of protein kinase C and NO-synthase inhibitors on the radiation-induced cytogenetic adaptive response in cultured Chinese hamster cells]. / Vliianie ingibitorov protein kinazy C i NO-sintazy na radiatsionno-indutsirovannyi tsitogeneticheskii adaptivnyi otvet v kletkakh kitaiskogo khomiachka v kul'ture.

The effects of the serin-threonin kinase inhibitor--staurosporin and inhibitor of NO-synthase--L-NAME on the radiation-induced adaptive response were studied in fibroblasts of Chinese hamster in culture. We have shown that the adaptive response as measured by the reduction of radiation-induced chromosome aberrations was completely blocked in the presence of staurosporin as well as L-NAME. These inhibitors themselves did not increase the chromosome aberration frequency when cultures were irradiated with 3 Gy alone in the presence of inhibitors. Furthermore, L-NAME noticeably decreased the gamma-rays-induced level of the chromosome aberrations. Our study confirms the participation of protein kinase C in the induction of the adaptive response and first indicates the participation of NO-synthase in this response.

[Cellular reactions induced by chronic exposure to 14C beta-rays]. / Kletochnye otvety, indutsirovannye khronicheskim oblucheniem beta-chastitsami, ispuskaemymi pri raspade 14C.

In V-79 cells the Effects of chronic exposure on induction of chromosome aberrations and abnormal metaphases as well as on efficiency of subsequent exposure to 2 Gy gamma-rays were investigated. It was found that chronic exposure increased the yield of chromosome aberrations as well as abnormal metaphases (spread-metaphases and apoptotic metaphases). In spite of the level of damages in cells, the chronic beta-exposure protected cells against the additional induction of chromosomal aberrations by subsequent exposure to higher acute dose of gamma-irradiation. Cytogenetic adaptive response was retained in the surviving progeny of irradiated cells which were cultured in free medium during 40, 70 days or one year after chronic exposure. At this time the level of residual chromosome aberrations, colony forming ability and distribution of the cells by the number of chromosomes were almost the same as in unirradiated cells. However, the high level of abnormal metaphases and half as much of cells in colony in the surviving progeny of irradiated cells in comparison with unirradiated cell, allow us to suggest that the chronic exposure induced the selection of adaptive forms in condition of the higher level of radiation.

[Changes in chromatin conformation induced by ionizing radiations with different LETs in resting and proliferating hepatocytes from rats of different ages]. / Izmenenie konformatsii khromatina, indutsirovannoe ioniziruiushchim izlucheniem s raznoi LPE v pokoiashchikhsia i proliferiruiushchikh gepatotsitakh krys raznogo vozrasta.

The method of flow cytometry was used to study conformation changes in rat hepatocyte chromatin exposed to various doses of X-rays and fission neutrons (mean energy of 0.85 MeV). Chromatin decondensation induces by both X-rays and neutrons was shown to be a function of dose. The saturation effect was observed for X-rays doses ranging from 4 to 6 Gy; for fission neutrons no saturation effect was noted within the dose range from 0.5 to 4 Gy. The initial chromatin level, that changed during the cell cycle and upon ageing, influence induction of conformation changes in the chromatin. The authors discuss the dependence of the processes of repair and fixation of damages to hereditary cell structure on the chromatin conformation at the time of exposure.

[The effect of inhibitors of DNA topoisomerase II on the radiosensitivity, repair and conformational changes in the chromatin in mammalian cells]. / Vliianie ingibitorov DNK-topoizomerazy II na radiochuvstvitel'nost', reparatsiiu i konformatsionnye izmeneniia khromatina v kletkakh mlekopitaiushchikh.

A comparative study was made of modification of the effects of UV-, X- and fission neutron-radiation by DNA-topoisomerase II and spermidine. The efficiency of the studied agents was the highest with UV-radiation of cells. Being insignificantly efficient in changing the radiosensitivity of cells, novobiocine and spermidine suppressed significantly the recovery of cells from radiation damages which were realized as chromosome aberrations upon X-ray dose fractionation. Adriamycin removed completely the effect of dose fractionation. Chromatin condensation was noted in cells after the treatment thereof with the agents under study. The authors discuss the possible mechanism of interaction between DNA-topoisomerase II and repair processes in mammalian cells.

[The induction of hepatocyte polyploidization in rats of various ages by ionizing irradiation with different LETs]. / Induktsiia poliploidizatsii gepatotsitov krys raznogo vozrasta ioniziruiushchim izlucheniem s razlichnoi LPE.

A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of nonproliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation.

[Polyploidization of rat hepatocytes due to cell fusion caused by radiations of different LET]. / Poliploidizatsiia gepatotsitov krys, obuslovlennaia sliianiem kletok pri deistvii izluchenii s raznoi LPE.

The method of flow cytometry was used to study polyploidization of hepatocytes following X-, gamma-, and neutron-irradiation. Ionizing radiation was shown to induce cell polyploidization by two different ways: (1) cells and nuclei fusion, and (2) restriction of mitosis after DNA replication. RBE of 14 MeV neutrons with respect to fusion was about 5.10(3). With neutron irradiation, the sensitivity of cells by fusion was not lower than that by chromosome mutations.

[Polyploidization of rat hepatocytes induced by x-ray radiation at different periods of the cell cycle]. / Poliploidizatsiia gepatotsitov krys, indutsirovannaia rentgenovskim izlucheniem v razlichnykh periodakh kletochnogo tsikla.

As determined by the yield of polyploid hepatocytes after X-irradiation of rats with a dose of 6 Gy the S-stage of the cell cycle was most radiosensitive; as to the yield of cells with chromosome aberrations the middle of the G1-stage was the most radiosensitive period of the cell cycle. The differences in the radiosensitivity of the cell cycle stages indicated that although primary lesions were similar molecular mechanisms leading to tre final effect were essentially different.

[Postradiation effect of a radioprotector depending on the degree of chromatin condensation in the cells]. / Postradiatsionnyi éffekt radioprotektora v zavisimosti ot stepeni kondensatsii khromatina v kletkakh.

It was shown on HeLa cells synchronized in mitosis and on rat liver hepatocytes in G0 stage that cysteamine administered after gamma-irradiation produced the radioprotective effect which was not observed in hepatocytes in G1 stage.

[Mechanism of the radioprotective effect of cysteamine]. / K voprosu o mekhanizme radiozashchitnogo deistviia tsisteamina.

The radioprotective effect of cysteamine combined with the modification of the chromatin state by sodium butyrate has been studied using V-79 and CHEL lines of Chinese hamster cells and HeLa cells. Sodium butyrate enhances the chromatin sensitivity to nucleases and removes the radioprotective effect of cysteamine as measured by the yield of cells with chromosome aberrations. As is indicated by changes in the intensity of fluorescence of the DNA-ethidium bromide complex, measured by laser flow cytometry, the protective agent decreases the binding of the dye with both irradiated and nonirradiated DNA whereas ionizing radiation and sodium butyrate increase thereof. It is concluded that the radioprotective effect of cysteamine depends in its ability to reduce the susceptibility of DNA to nucleases.

[Cell proliferation kinetics in the regenerating rat liver based on flow cytophotometric data]. / Kinetika kletochnoi proliferatsii v regeneriruiushchei krys po dannym protochnoi tsitofotometrii.

Redistribution of cells according to the ploidy levels permits studying kinetics of cell transitions from the state of mitotic rest to proliferation during the organ regeneration. In the first generation cycle about 95% of cells which accepted a proliferative stimulus leave the G0-phase and then, having completed DNA synthesis, some of them enter mitosis and some are blocked in the G2 phase. About 20% of cells which divide during the second mitosis are the "G2-population" formed after the first cycle.

[Genome mutation yield in the cells of intact and regenerating liver of normal rats and following gamma irradiation]. / Vykhod genomnykh mutatsii v kletkakh intaktnoi i regeneriruiushchei pecheni krys v norme i posle gamma-oblucheniia.

A comparative study was made of spontaneous and induced polyploidy in cells of resting and regenerating rat liver. Polyploidy was shown to play a major role in the ontogenesis and during regeneration after partial hepatectomy. An essential difference was revealed in the radiation response of cells of intact and regenerating liver with respect to the yield of polyploid cells. This distinction was referred to different effectiveness of the processes of repair and fixation of radiation damages in the actively proliferating and resting cells.