[The most important stages of the mechanism of action in vivo of carcinogen diethylnitrosoamine and its effect on the synthesis of RNA, proteins, DNA, and deoxyribonucleotides].

The mechanisms of nitric oxide (NO) generation from exogenous and endogenous sources, induced by the addition of the carcinogen diethylnitrosoamine (DENA) to rat organism have been studied. Within 15 h after the addition of DENA, the carcinogen itselt acts as an exogenous NO donor. The products of protein degradation (the process induced by DENA) act as endogenous donors of NO. It was shown that the generation of nitric oxide from diethylnitrosoamine leads to deep hemic and tissue hypoxia and induces the inactivation of oxygen-dependent enzymes, including ribonucleotide reductase, and the inhibition of ATP synthesis. Under these conditions, the protein synthesis and as a consequence the synthesis of deoxyribonucleotides and DNA are strongly suppressed; i.e., diethylnitrosoamine produces the effect similar to the action of the antibiotic cycloheximide, an inhibitor of translation. The administration of cycloheximide to the animal organism also led to the appearance of a considerable amount of nitric oxide in the blood. It is assumed that nitric oxide initiates (on the administration of the carcinogen) or at least enhances (on the administration of cycloheximide) the blockage of the synthesis of the protein, deoxyribonucleotides, and DNA. In response to the disturbance of protein synthesis, the complex of enzymes is activated that accomplish the utilization of the degradation products of proteins, including the inducible form of NO synthase.

Multifactorial nature of high frequency of mitochondrial DNA mutations in somatic mammalian cells.

The high frequency of mitochondrial DNA (mtDNA) mutations in somatic mammalian cells, which is more than two orders of magnitude higher than the mutation frequency of nuclear DNA (nDNA), significantly correlates with development of a variety of mitochondrial diseases (neurodegenerative diseases, cardiomyopathies, type II diabetes mellitus, cancer, etc.). A direct cause-consequence relationship has been established between mtDNA mutations and aging phenotypes in mammals. However, the unclear nature of the high frequency of mtDNA mutations requires a comprehensive consideration of factors that contribute to this phenomenon: oxidative stress, features of structural organization and repair of the mitochondrial genome, ribonucleotide reductase activity, replication errors, mutations of nuclear genes encoding mitochondrial proteins.

[The use of EPR spectroscopy to control the changes in organism radioresistance. Experimental results].

The responses of deoxyribonucleotide (dNTP), DNA, and protein synthesis systems in blood-forming organs of animals (dogs, mice) as well as changes in Fe(3+)-transferrin (Fe(3+)-TF) and Cu(2+)-ceruloplasmin (Cu(2+)-CP) pools in blood to gamma-irradiation and the administration of radioprotectors have been studied. It has been shown that changes in Fe(3+)-TF and Cu(2+)-CP pools in blood are indices of changes of body radioresistance and are reliably controlled by the EPR technique. An increase in the Fe(3+)-TF pool promotes the activation of synthesis of dNTP, DNA, and Fe(3+)-containing proteins, which are essential for repair efficiency during early post-irradiation time as well as for the development of compensatory and restorative reactions of cellular systems; i.e., they are responsible for body resistance to DNA-damaging factors. It is important that the intensity of responses depends on the initial state of the organism. Thus, dogs with initial individual characteristics of blood typical for "suppressed" or "activated" states had abnormally high responses to irradiation by low doses of 0.25 and 0.5 Gy. This fact is important for the estimation of consequences of prolonged low-dose irradiation for human population. It has been shown that radioprotectors, efficient in survival test activate the synthesis of dNTP, DNA, and proteins in organs. The intensity of dNTP synthesis and the time when dNTP pools get maximum values determine the efficiency of protectors and the time of irradiation after their administration.

[The inhibitor of free radical processes decrease of protein biosynthesis in gun short wound tissues and weaken development of the general adaptation syndrome].

The dynamics of total protein biosynthesis and procollagen biosynthesis in skeletal muscle of injury tissues with the antioxidant BHT (dibunol) treatment and with common healing were studied. The obtained date indicate that the AO treatment reduce the rate of biosynthesis both the total proteins and procollagen at the 3th day of healing. Dibunol also considerably reduce the protein biosynthesis in adrenals and brake of corticosteroids biogenesis as measured by ESR-signals intensity of reduced adrenodoxine. AO treatment also reduce the protein biosynthesis in thymus, spleen and bone marrow. The lowering of functional activity of endocrine and immune systems indicate that the AO significantly inhibit the systemic reactions of organism induced by acute wound affect. It was suggested that as "primary mediator" of stress-reaction may be considered lipoperoxide radicals and decay products of lipohydroperoide.

[The mechanisms of nitric oxide production from exogenous and endogenous NO-donating compounds and its effect on deoxyribonucleotide and DNA synthesis].

The mechanisms of exogenous nitric oxide (NO) production through in vivo biotransformation of nitro-, nitroso- and amino-containing substances were discussed. In addition, the mechanisms of production and cellular sources of endogenous NO, appearing in the blood and tissues after the exposure to various DNA-damaging factors, have been considered. Considerable quantities of endogenous NO were detected in the body in the first hours after translation inhibition by cycloheximide or animal exposure to superlethal radiation doses, i.e., after the exposure to factors inducing destructive processes. The time and dose dependences of exogenous and endogenous NO production have been established. NO produced after a single or repeated administration of NO-donating compounds as well as endogenous NO proved to inhibit deoxyribonucleotide (dNTP) and DNA synthesis in animal tissues. Nonspecific compensatory responses to disturbed protein homeostasis included cyclic production of endogenous NO. The maximum levels of nitrosyl complexes were registered when the rate of protein synthesis decreased. The role of polyamines in the induction of macromolecule biosynthesis is discussed and NO production from these arginine-rich compounds is proposed. NO is released at the stage of polyamine inactivation. The inactivation mechanism includes the hydroxylation of aminogroups by NO synthase, the formation of nitroso intermediates, and their denitrosation with NO release.

[Activation of deoxyribonucleotide synthesis by radioprotectants and antioxidants as a key stage in formation of body resistance to DNA-damaging factors].

The responses of the systems of synthesis of deoxyribonucleotides (dNTPs), DNA, and proteins in hematopoietic organs and liver of animals to gamma-radiation, administration of radioprotectants and antioxidants as well as the dependence of these responses on the doses of radiation and drugs were studied. Radioprotectants of acute (indralin) and durable effects (indomethaphen) as well as natural (alpha2-tocopherol) and synthetic anti-oxidants (ionol or 2,6-di-tert-butyl-4-methylphenol) efficient in survival test were used. Three stages could be recognized in the standard unspecific response of the studied systems to radiation: (1) immediate increase in ribonucleotide reductase activity in the tissues within the first 30 min as a part of the integrated SOS response to DNA damage, which activates dNTP synthesis; (2) inhibition of the synthesis of dNTPs, DNA, and and (3) restoring ribonucleotide reductase activity and integral increase in the production of dNTPs, DNA, and total protein, which is essential for the development of compensatory and restorative responses of the organism. The radioprotectants significantly increased ribonucleotide reductase activity, which increased intracellular concentrations of the four dNTP types in organs during radiation exposure and three following days. Within this period, ribonucleotide reductase activity was inhibited by 40-50% in animals not treated with radioprotectants as compared to control. Balanced high pools of dNTPs in the organs of radioprotectant-treated animals provided for high-performance repair of DNA damage. The radioprotectant-induced activation of dNTP synthesis during the development of compensatory and restorative responses provides for an earlier restoration of the cellular composition and functioning of the organs. Antioxidants stimulated the synthesis of dNTPs, DNA, and proteins in animal tissues in a strict dose interval. Their effect on the studied syntheses was dose-dependent: single or multiple long-term administration of high antioxidant doses inhibited synthesis of dNTPs, DNA, and proteins. Radioprotectants and antioxidants affected the pool of blood protein Fe3+-transferrin controlling the synthesis of iron-containing ribonucleotide reductase activity in hematopoietic organs, and hence, the iron-dependent stage in DNA synthesis--dNTP synthesis. Activation of protein synthesis in organs by the studied substances increased the pools of Fe3+-transferrin and Cu2+-ceruloplasmin in the blood, which activated dNTP and DNA synthesis. Activated synthesis of dNTP, DNA, and proteins in the organs and increased pools of studied plasma proteins underlay the formation of body resistance to DNA-damaging factors.

[Anabolic effect of natural and synthetic antioxidants]. / Anabolicheskii éffekt prirodnykh i sinteticheskikh antioksidantov.

The order of responses of cell systems of organs and changes in the content of some proteins of mouse and dog blood in response to addition of natural (alpha-tocopherol) and synthetic (ionol) antioxidants was studied at the whole-body level using ERP spectroscopy, radioisotope analysis, and chemiluminescence technique. Responses were evaluated by the temporary and concentration-dependence changes in the activity of ribonucleotide reductase and the rate of protein and DNA synthesis in organs of mice, as well as by the changes in the pools of Fe3+ -transferrin and Cu2+ -ceruloplasmin in blood and the antiradical activity of blood plasma of dogs and mice. During the first 24 h of exposure to alpha-tocopherol, the activity ribonucleotide reductase in bone marrow rapidly increased, whereas the activity of this enzyme and the rate of DNA synthesis in the thymus and spleen were suppressed by 30-50% compared to the control. The changes in these parameters had a phase mode with maxima on days 2-3 and 6-8. The stimulatory effect of the antioxidant on the processes of synthesis was concentration-dependent. We found that the optimal stimulation of the synthesis of deoxyribonucleotides, DNA, and protein was achieved by single administration of alpha-tocopherol at a dose of 20 mg per dog with an average weight of 15 kg and 17 mg/kg in the case of mice. Single or repetitive administration of higher doses of alpha-tocopherol was either ineffective or even suppressed the synthesis of DNA and deoxyribonucleotides. Ionol administered at a dose of 60 mg/kg increased DNA and protein synthesis in mouse organs in 2-4 and 1.2-1.5 times, respectively, compared to the control. It was also shown that single and repetitive administration of alpha-tocopherol to dogs increased the pool of Fe3+ -transferrin and Cu2+ -ceruloplasmin in blood in 2-3 times and by 20-30%, respectively, compared to the control. It is suggested that changes in Fe3+ -transferrin pool in peripheral blood may be used for evaluation of the stimulatory effect of antioxidants on the synthesis of macromolecules in organs and for the determination of dependence of this effect on the concentration of antioxidants.

[Reaction of rats organ cells to inhibition of protein biosynthesis by sublethal doses of cycloheximide]. / Reaktsii kletok organov krys na ingibirovanie biosinteza belka tsiklogeksimidom v subletal'noi doze.

Time-dependent responses of cellular systems in rat organs and Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in blood to the blocking of translation by sublethal doses of cycloheximide (CHI) was studied by EPR spectroscopy and radioisotope techniques. It was shown that, within the early post-CHI-treatment time, the suppression of deoxyribonucleotide and DNA biosynthesis, the activation of catabolic enzymes, the inhibition of electron transfer in the mitochondrial electron transport chain, the activation and the following inactivation of cytochrome P-450, and an intensive production of nitrosyl complexes in rat blood and organs occur. In addition, the activation of the synthesis of steroid hormones in adrenal gland was revealed within 1-24 h after cycloheximide injection. In response to these metabolic disturbances, nonspecific compensatory recovery reactions developed, first of all, the "reprograming" of the translation process to produce new protein-synthesizing elements instead of cycloheximide-blocked ones. The activation of protein synthesis promotes the recovery of deoxyribonucleotide and DNA synthesis, the restoration of the redox state of mitochondrial and microsomal electron transport chains in organs as well as an increase of Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in rat blood. These metabolic processes result in the full recovery of the functional ability of organs.

[Time- and dose-dependent post-irradiation changes of Fe3+-transferrin and Cu2+-ceruloplasmin pools in blood, their influence on ribonucleotide reductase activity in animal tissues and the effects of radioprotectors]. / Vremennye i dozozavisimye postradiatsionnye izmeneniia v soderzhanii Fe3+-transferrina i Cu2+ tseruloplazmina v krovi zhivotnykh i ikh vliianie na ribonukleotidreduktaznuiu aktivnost' tkanei.

The time- and dose-dependent changes of Fe(3+)-transferrin (Fe(3+)-TF) and Cu(2+)-ceruloplasmin (Cu(2+)-CP) pools, of superoxide dismutase activity and the inhibitory activity of alpha 2-macroglobulin in blood as well as changes in synthesis rates of deoxyribonucleotides (dNTP), DNA and proteins in organs (spleen, liver, bone marrow, thymus) of mice and dogs given total body irradiation have been studied using of ESR spectroscopy, radioisotope techniques and biochemical determination of enzymatic activity. The experimental data have allowed us to reveal the sequence of organism's response reactions against irradiation and their modifications by radioprotectors. Changes in blood Fe(3+)-TF pool is one of the most informative, highly radiosensitive and rapidly reactive marker against irradiation and drug administrations. This irontransport protein controls a rate-limiting iron-dependent stage for DNA synthesis--the synthesis of dNTP, catalyzed by iron-containing ribonucleotide reductase (Fe(3+)-RR). It has been shown that time-dependent post-irradiation changes of Fe(3+)-TP pool in blood are characterized by three distinct stages: 1) the prompt increase of pool (SOS-type response) playing the important role in protecting of cell's genetic apparatus from damage; 2) the decrease of its pool within 3-18 h after irradiation resulting in the loss of Fe(3+)-RR activity in tissues of blood-forming organs that make more stronger radiation-induced damage; 3) the following phase-dependent increase in Fe(3+)-TF pool at the 2-nd, 6th, 10-17th days after irradiation due to an increase in transferrin synthesis. This increase may be considered as compensatory reaction of blood-forming organs directed at restoring blood and organ's cells. The time-dependent courses of the reactions are independent from radiation doses indicating to the universal and nonspecific response of organism against irradiation. But, the intensity of this compensatory-adaptive response at 2-nd and 6th days grows with increasing radiation dose up to lethal that, and organism's response becomes abnormal and physiologically hypertrophic. The prolonged "stressful syndrome of biochemical tense state" should be attributed to negative effects for organism, since it may result in the failure of compensatory adaptive organism's reactions and animal killing. The radioprotectors ward off the appearance of this dangerous state. Dogs with initial individual characteristics of blood which were typical for "suppressed" or "activated" states had abnormal response against irradiation by low doses 0.25 or 0.5 Gy. In these cases the intensity of response reactions of organism was essentially increased and markedly deviated from linear dose dependence. The phase-dependent increase of Fe(3+)-TF pool in blood in post-irradiation time resulted to the increase of Fe(3+)-RR activity in blood-forming organs. The key event ensuring the development of compensatory adaptive reactions is the increase of capacity of protein-synthesizing apparatus, the activation of biosynthesis of dNTP and DNA against the treatment with damaging factors.

[Ecdysterone modulates antitumor activity of cytostatics and biosynthesis of macromolecules in tumor-bearing animals]. / Ekdisteron moduliruet protivoopukholevuiu aktivnost' tsitostatikov i ikh deistvie na biosintez makromolekul v organakh zhivotnykh-opukholenositelei.

The influence of therapeutic and half doses of cisplatin and adriamicin combination with the anabolic drug ecdisteron (20-hydroecdison) on development of subcutaneously and intraperitonially transplanted P388 and L1210 leukemia and metastasizing B16 melanoma was studied. Ecdisteron significantly stimulated the chemotherapeutic effect of low doses of the cytostatics: inhibition of tumor growth, mice survival rate, their lifespan, and the antimetastatic activity index were comparable or better than after therapy with high doses of the antitumor drugs. The influence of high and low doses of cisplatin and its low dose in combination with ecdisteron on the dynamics of protein and DNA biosynthesis in the liver, pancreas, thymus, spleen, and adrenals of tumor-bearing mice were also studied. Although the therapeutic effect of 4 mg/kg cisplatin by activated protein biosynthesis and DNA repair is comparable or better than that of its low dose (2 mg/kg) in combination with ecdisteron, in terms of chemotherapy the combination looks preferable since the therapeutic dose of cisplatin is toxic for the intact tissues.

The activation of ribonucleotide reductase in animal organs as the cellular response against the treatment with DNA-damaging factors and the influence of radioprotectors on this effect.

Cellular requirements for deoxyribonucleotide (dNTP) pools during DNA synthesis are related to ensuring of the accuracy of DNA copying during replication and repair. This paper covers some problems on the reactions of dNTP synthesis system in organs of animals against the treatment with DNA-damaging agents. Ribonucleoside diphosphate reductase (NDPR) is the key enzyme for the synthesis of dNTP, since it catalyses the reductive conversion of ribonucleotides to deoxyribonucleotides. The results obtained show that the rapid and transient increase in NDPR activity in animal organs occurs as cellular response against the treatment with DNA-damaging agents (SOS-type activation). We have also found the intensive radioprotector-stimulated activation of deoxyribonucleotide synthesis as well as DNA and protein synthesis in mice organs within 3 days after the administration of two radioprotectors, indralin and indometaphen, that provide the high animal survival. Our studies suggest that these effects are the most important steps in the protective mechanism of the radioprotectors and are responsible for the high animal survival.

[The molecular mechanisms of the action of the radioprotector indometafen. The biosynthetic and bioenergetic aspects]. / Molekuliarnye mekhanizmy deistviia radioprotektora indometafena. Biosinteticheskie i bioénergeticheskie aspekty.

It was shown that indomethaphen (IM) is capable of stimulation of the synthesis of DNA, RNA, and protein precursors in mice. The IM-induced elevated level of the ribonucleotide reductase activity and, hence, deoxyribonucleotide pool in the spleen at the moment of irradiation and during the early postradiation period provides for complete DNA repair. As a result, the damaging effect of ionizing irradiation is weakened. At later stages (2-20 days) IM activates protein and DNA synthesis leading to the recovery of the ribonucleotide reductase activity in the spleen, on increased content of Fe3(+)-transferrin, cytochrome-c-oxidase, and ferrosulfuric components of the mitochondrial electron transport chain, and increased potential of the detoxication system due to the elevated content of cytochrome P-450. IM stimulates ATP synthesis. Thus, IM enhances compensatory-restorative reactions of the cell systems, more pronounced in the spleen than in the liver.

[The structural characteristics of the polyribosomes in the rat liver studied by means of a tritium label]. / Osobennosti struktury poliribosom pecheni krys, izuchennye metodom tritievoi metki.

Polyribosomes isolated from the rat liver in a medium with low ionic strength were irradiated by "hot" tritium atoms under conditions providing for the replacement of the hydrogen atoms located at the surface of polyribosomes by tritium. After fractionation of such polyribosomes, the radioactivity of the obtained fractions was measured and their proportions were calculated for the total surface accessible for the tritium atoms (in %), as well as their specific radioactivity. The material loosely associated with the polyribosomes and containing amino acyl-tRNA-synthetases is more radioactive than rRNA and r-proteins, especially concerning their specific radioactivity. This suggests that the material is organized as individual molecules located on the surface of ribosomes. The specific radioactivity of the RNA-component of this material (tRNA) is twice that of proteins, thus suggesting its surface localization in the composition of loosely associated material. Based on the pattern of labeling of rRNA and r-proteins of the native and preliminarily dissociated polyribosomes, we propose that the material, loosely associated with the polyribosomes, has affinity to both rRNA and r-proteins.

[The molecular aspects of the action of the radioprotector indralin]. / Molekuliarnye aspekty deistviia radioprotektora indralina.

The effect of indralin on the metabolic parameters in peripheral blood and organs of irradiated dogs and mice have been studied by EPR, NMR and radioisotope methods. It has been shown that indralin stimulated biosynthesis of DNA precursors as well as of DNA and proteins in the organs and stabilized the rate of ATP and glycogen synthesis. As a result indralin reduced considerably the changes produced by gamma-irradiation on the macromolecular biosynthesis during the early post-irradiation period. Indralin has induced marked favorable changes in the rate of macromolecular synthesis, normalized the ATP and glycogen content, induced ribonucleotide reductase activity and increased the Fe(3+)-transferrin content during development of compensatory-repair response in the irradiated animals. Indralin prevented hyperdevelopment of the repair response and its breakdown due to radiation-induced exhaustion of viability of many important cellular and body systems after irradiation with lethal doses.

[A model of positive rRNA gene expression regulation based on complementary interaction of transcripts of the prealbumin gene introns with its promotors]. / Model' pozitivnoi reguliatsii ékspressii gena rRNK na osnove komplementarnogo vzaimodeistviia s ego promotorami transkriptov intronov gena preal'bumina.

The present paper is concerned with computation of the homology between the promoter and spacer sequences of the rRNA gene and the prealbumin introns. Both the sense and antisense strands of the promoters were compared with the introns. The rRNA promoters were found to contain a considerable number of prealbumin-homologous intron sites on the coding strand only, whereas the spacer sequence had the intron-homologous sites on both strands. The fragments homologous to the ribosome gene promoter regions are mainly concentrated in the first prealbumin intron. The oncogene, glycoprotein and the interleukin intron fragments revealed no sites homologous to the ribosome gene promoter. A model has been proposed for positive regulation of the rRNA gene expression via complementary interaction between the transcripts of the prealbumin gene introns with sense and antisense strands of the rRNA promoters. It is believed that either positive or negative regulation of the ribosome biogenesis can be achieved in this way.

[Radiosensitivity of coding and noncoding DNA sequences of various organisms]. / Porazhaemost' radiatsiei kodiruiushchikh i nekodiruiushchikh posledovatel'nostei DNK razlichnykh organizmov.

Relationship between pyrimidine distribution patterns and radiosensitivity (Z) of DNA molecules of different species was derived by computer analysis of recurrence frequency of pyrimidine clusters. Blocking factors (beta) and Z for coding and non-coding DNA sequences of species from different taxonomic classes have been calculated within a new model. The radiosensitivity of coding DNA sequences practically does not vary whereas Z values were increased during evolution from simplest to higher organisms. The beta and Z values calculated for several groups of individual genes were shown to vary considerably.

[Effects of cycloheximide on protein, RNA and DNA synthesis in cultures of CHO cells and human diploid fibroblasts]. / Vliianie tsiklogeksimida na sintez belka, RNK i DNK v kul'turakh kletok CHO i diploidnykh fibroblastov cheloveka.

In CHO cell line and primary human diploid fibroblasts culture an incorporation of protein, RNA and DNA biosyntheses precursors was investigated under different conditions of inhibition of translation by cycloheximide (CHM). Both CHO and human fibroblasts transitory treatment by CHM in the serumfree medium resulted in inhibition of protein and DNA syntheses during S-period while RNA synthesis increased up to 130% (CHM concentration from 0.003 to 2 Mg/ml), as well as in Go--an incorporation of 3H-U increased to 200% (CHM concentration-100 Mg/ml). Long-term treatment (48 hours) in the serum-free medium resulted in decreased uptake of 3H-T and 3H-L during first 6 hours of experiment, while incorporation of 3H-U increased to 160%. By 16-th hour of treatment characters of protein, RNA and DNA syntheses came back to control levels.

[Reorganization of chromatin superstructure during marked changes in the rate of protein synthesis]. / Reorganizatsiia superstruktur khromatina v usloviiakh rezkikh kolebanii skorosti sinteza belkov.

The changes in the structure and RNA-polymerase activity of rat liver cell chromatin after a single injection of cycloheximide (3 mg/kg of body mass) were studied. The cycloheximide-induced fluctuations in protein synthesis rates are concomitant with episodes of drastic changes in the chromatin structure. The reorganization of the general protein structure is associated with an increase or a decrease of the RNA-polymerase II activity. The data obtained suggest that the activation-inactivation of RNA-polymerase II in cell nuclei is due to reorganization of chromatin infrastructures--from higher levels of the electron-dense chromatin package to the unfolded nucleosomes of the transcriptionally active protein.