[Endogenous DNases as a tool for isolation of nuclear matrix: critical parameters of nucleolysis]. / Endogennye DNKazy kak instrument polucheniia preparatov iadernogo matriksa: kriticheskie parametry nukleoliza.

Degree of nucleolysis has critical significance for isolation of nuclear matrix (NM) specifically enriched in transcribed DNA sequences as demonstrated at the example of inactive (c-fos, c-myc, and Ck) and active (p53, albumin, and 28S rRNA) genes in resting hepatocytes. Optimal degree of nucleolysis features degradation of loop domains of chromatin with preserved relatively uniform molecular weight distribution of DNA. Deviation from these parameters leads to nonspecific fragmentation of chromatin in various gene loci and isolation of NM samples nonspecifically enriched or depleted of transcribed DNA sequences. Under optimal hydrolytic conditions, the transcribed chromatin is more resistant to endogenous DNase attack, which allows selective conservation of its association with the nuclear matrix.

[Bimodal physiological effect of picolinic acid derivatives is determined by the zwitterion structure characteristics]. / Bimodal'nyi fiziologicheskii éffekt riada proizvodnykh pikolinovoi kisloty obuslovlen osobennostiami stroeniia tsvitterionov.

In order to define the mechanism of action and causes for the manifestation of the bimodal physiological effect of certain picolinic acid derivatives we carried out quantum chemical calculations (QCC MO, 3-21G/6-31G* basis) of picolinic acid (1), 3,6-dichloropicolinic acid (2), acepox (3), and tetrapin (4) zwitterions. The exocyclic bond C(pyr)-Cexo is enhanced in zwitterions 3 and 4 with the bimodal physiological effect and loosened in zwitterions 1 and 2 without the effect. Zwitterions of the molecules 1-4 also differ in the pattern of the boundary molecular orbitals and electric charge distribution. On the basis of the obtained data, we propose that the difference in the physiological effect of compounds 1-4 is due to the different behavior of their zwitterions in aqueous solution.

[Structure-physiological activity correlation for picolinic acid derivatives based on quantum chemical data]. / Korreliatsiia struktura-fiziologicheskaia aktivnost' u proizvodnykh pikolinovoi kisloty po dannym kvantovokhimicheskikh raschetov.

Correlation between structure and properties for picolinic acid derivatives with and without bimodal effect was evaluated by quantum chemical calculations. Comparative data obtained by SCF MO method in the 3-21G/6-31G* basis indicate lower ionization potential in picolinic acid derivatives with bimodal effect as compared to those lacking this effect. This validates our previous assumption that physiological bimodal effect is defined by the electron structure of the molecules. Quantum chemical calculations allowed us to predict bimodal physiological activity in amino-substituted derivatives of picolinic acid.

[Markers of the metabolic changes arising as a result of ionizing radiation exposure]. / Markery metabolicheskikh izmenenii, voznikaiushchikh vsledstvie vozdeistviia ioniziruiushchei radiatsii.

Time-related changes have been studied in the content of extracellular DNA, Fe(3+)-transferrin (TF), and Cu(2+)-ceruloplasmin (CP) in the blood plasma and the activity of ribonucleotide reductase (RR) in the tumor cells and spleen of mice during the development of acute lympholeukosis P-388 and after ionizing irradiation. At the initial stages of leucosis P-388, the content of extracellular DNA increases, the TF and CP pools in the blood plasma enlarge, and the RR activity in the tumor cells and spleen of tumoral mice markedly increases. A dose-dependent increase in RR activity was also recorded in the spleen of 5-day-old rats within 15-30 min after irradiation. The causes of these changes and the possibility for these indices to be used in estimating leucosis risk are discussed. Radiation-induced increases in RR activity are discussed in relation to the SOS-response to DNA damage; an increased pool of deoxyribonucleotides is necessary for repair of DNA. The mean contents of extracellular DNA, TF and CP in the blood plasma were obtained from children of different ages degrees of radioactive contamination suffering the consequences of the accident at the Chernobyl' Nuclear Power Station (n = 155). Groups of children have been isolated with increased, sharply decreased, and close to normal levels of extracellular DNA, TF, and CP. The lowered TF pool was observed in children with thyroid glands damaged by incorporated radioactive iodine with the degree of suppression determined by the dose. For most children subject to general irradiation, the TF and CP pools in the blood were higher than in the control, suggesting an adaptive response to irradiation.

[Change in the level of sphingosine in rat liver nuclei and cells during oncogene superexpression induced by cycloheximide]. / Izmenenie urovnia sfingozina v iadrakh i kletkakh pecheni krys pri indutsirovannoi superékspressii onkogenov tsiklogeksimidom.

Changes in the sphingosine content in rat liver cells and nuclei have been studied with reference to the level of nuclear oncogene expression, induced by cycloheximide (0.1, 0.5 and 3.0 mg/kg). It has been found that only the sublethal (3 mg/kg) dose of cycloheximide which induces the superexpression of c-fos and c-myc oncogenes can promote sphingosine accumulation in the cell. At the moment of enhanced expression of nuclear oncogenes, the maximum content of free sphingosine exceeds the control level 1.5- and 3-fold in the cell and in the nuclei, respectively. The difference in the sphingosine accumulation patterns in the cell and in the nuclei testifies to the fact that sphingomyelin metabolism is more active in the nuclei than in the cell. Sphingosine accumulation in the nuclei is characterized by coordination of sphingomyelinase activity and changes in the sphingomyelin content. A comparative analysis of activities of enzymes of sphingomyelin (sphingomyelinase) and phosphatidyl inositol (phosphatidyl inositol kinase) cycles indicates that in the nuclei the activation of the sphingomyelin cycle forestalls the cycloheximide-induced activation of the phosphatidyl inositol cycle and the maximal accumulation of nuclear oncogene mRNAs. A model of activation of oncogene expression with participation of sphingosine inhibiting protein kinase C and activating casein kinase II, the key enzymes of the signal transduction system of cell proliferation and differentiation, is proposed.

[Structural changes of chromatin during proto-oncogene activation by cycloheximide: dose-effect]. / Strukturnye izmeneniia khromatina v protsesse aktivatsii protonkogenov tsklogeksimidom: doza-éffekt.

The level of expression of cellular proto-oncogens c-myc and c-fos in rat liver has been studied as a function of protein synthesis rate (cycloheximide dose). Activation of proto-oncogens has been established to be initiated by 50% inhibition of nuclear protein synthesis. This promotes a certain level in chromatin structural rearrangements which is manifested, in particular, in decreasing activity of chromatin cleavage by Ca2+, Mg(2+)-DNAase and increasing degree of chromatin condensation. A role of topoisomerase II in chromatin structural rearrangements during proto-oncogen activation is postulated.

[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.

[Effects of daunorubicin and its nitroxyl analog on the synthesis of nucleic acids]. / Vliianie daunorubitsina i ego nitroksil'nogo analoga na sintez nukleinovykh kislot.

The impact of daunorubicin, emoksil in sublethal doses and daunorubicin mixtures with a nitroxyl radical of 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (NR) on synthesis of DNA and RNA in some organs of rats was studied. Daunorubicin and emoksil induced marked (by 80 to 90%) inhibition of DNA synthesis in all the examined organs even within the first hours of administration. In the heart, DNA synthesis remained lower by the end of the experiment. In the spleen its partial recovery up to 40 to 60% of the control level was observed. In the liver the synthesis was stimulated in 24 hours. Its highest stimulation was recorded with the use of emoksil and daunorubicin in combination with NR. After administration of daunorubicin the maximum synthesis of DNA exceeded the control level 2.5 times. Addition of NR lowered 2-fold inhibition of DNA synthesis by daunorubicin within the first hours. It was of interest that the anthracyclines appeared to markedly stimulate RNA synthesis in the spleen, the fact not described in the literature. The stimulation of DNA synthesis in the liver was supposed to be one of the components of the compensatory mechanisms engaged by the cell in response to the drug's damaging effect.

[Circadian rhythm of tRNA aminoacylation in hepatocyte culture]. / Okolochasovoi ritm aminoatsilirovaniia tRNK v kul'ture gepatotsitov.

In the monolayer of rat hepatocytes in vitro, the circadian rhythms were revealed of 3H-leucine incorporation in proteins and in aminoacyl-tRNA(Leu) fraction. The oscillations were mainly synphasic, though coordination between aminoacylation and protein synthesis was not stable in the coarse of time. In cell free system with the excess of ATP and aa-tRNA, the rhythm of 3H-leucine incorporation was also clear. This means, that oscillatory kinetics of the protein synthesis rate is not caused by oscillation of ATP which has been revealed earlier in the hepatocyte monolayer.

[Expression of oncogenes in the rat liver under conditions of template biosynthesis uncoupling by sublethal doses of cycloheximide]. / Ekspressiia onkogenov v pecheni krys v usloviiakh razobshcheniia matrichnykh biosintezov subletal'nymi dozami tsiklogeksimida.

Injection of sublethal doses of cycloheximide (CHI) to rats allowed to reveal three stages in the dynamics of protein synthesis: 1) suppression stage (0-6 hrs), 2) regeneration stage (6-12 hrs), 3) stimulation stage (6-12 hrs). RNA-polymerases are activated when protein synthesis is inhibited. The stimulation stage precedes the activation of DNA replication. This model of DNA replication induced by CHI is specified by the expression of various cell oncogenes (c-fos, c-mys, p53, c-Ha-ras, c-sis, c-src). The investigated oncogenes may be divided into 4 groups according to the character of their expression. 1. Oncogenes (c-fos, c-myc) are switched on step-by-step 1 hour after CHI injection, the superexpression of the oncogenes being comparatively short. Maximum expression of c-fos and c-myc oncogenes is registered after 2-3 hours, respectively. 2./p53 oncogene expression increases within a few hours' after CHI injection and manifests itself at all three stages of protein synthesis till DNA replication. 3. c-Ha-ras oncogene is expressed at a high level in control and experimental animals. 4. Expression of c-sis and c-src oncogenes are absent both before and after CHI injection. Sublethal doses of CHI have the same effect on oncogene expression as the lethal ones.

[Effect of cycloheximide on lipid metabolism in cells, nuclei and subcellular fractions in the rat liver]. / Vliianie tsiklogeksimida na lipidnyi metabolizm v kletkakh, iadrakh i sub''iadernykh fraktsiiakh pecheni krys.

Well coordinated stages of inhibition, restoration and stimulation of protein, DNA and RNA synthesis were observed after administration of cycloheximide (3 mg/kg). The changes in lipid synthesis and composition in the nuclei and intranuclear structures were studied at different stages of cycloheximide action. The accumulation and stimulation of lipid synthesis in the nuclei during the inhibition and restoration of protein and DNA syntheses were followed by electron microscopy and labeled precursors methods. Dramatic changes were observed in the phospholipid composition of chromatin and nuclear matrix. The accumulation of minor phospholipid fractions in intranuclear structures was observed during DNA synthesis. The sphingomyelin concentration was predominant and commensurable with those of phosphatidylcholine and phosphatidylethanolamine.

[Characteristics of [3H]cycloheximide distribution, protein and DNA biosynthesis in rat organs after sublethal blocking of translation]. / Osobennosti raspredeleniia [3H]tsiklogeksimida, biosintez belka i DNK v organakh krysy dosle subletal'nogo bloka transliatsii.

The kinetics of accumulation and release of [3H]cycloheximide (CHI) as well as protein and DNA biosyntheses in some organs of the rats injected with sublethal doses of CHI were studied. It was shown that in the majority of organs under study (especially in the liver, kidneys and adrenals) the inhibition is completed within 12 hours after CHI injection followed by the resumption of protein and DNA syntheses. In the thymus and pancreas the levels of these biosyntheses remain below control values up to the 72nd hour. A positive correlation was observed between the decrease of CHI (or its metabolites) concentration and the beginning of protein and DNA syntheses in different organs. However, there was a reverse correlation between the high values of squares below the kinetic curves of CHI release from the liver, kidneys and adrenals and the intensive resumption of protein and DNA biosyntheses in these organs. It was thus assumed that in these particular organs CHI is subjected to intensive biotransformations. The contribution of the endocrine system to the induction of intensive compensatory protein and DNA syntheses in the liver were estimated from the viewpoint of the nature of reconstructive processes occurring in the appropriate organs.

[Phase-specific protein synthesis in human fibroblasts after synchronization by a double thymidine block]. / Fazospetsificheskii sintez belkov v fibroblastakh cheloveka posle sinkhronizatsii dvoinym timidinovym blokom.

Kinetics of acid-insoluble non-histone protein synthesis during S and G2 cell cycle phases of diploid human fibroblasts and heteroploid transformed cells was investigated. Two distinct groups of protein with different kinetic pattern depending on the cell culture type were revealed. Export of one group of protein and turnover of the other group of acid-insoluble non-histone protein is arrested in heteroploid transformed cells.

[Correlation of the activity of the key enzymes of glycolysis and respiration in hepatomas with different growth rates]. / Sootnoshenie aktivnosti kliuchevykh fermentov glikoliza i dykhaniia v gepatomakh s razlichnoi skorost'iu rosta.

It is shown that an increase in the activity of glycolysis resulting from the rise of the hepatoma growth rate is accompanied by a decrease in the activity of the pentose-phosphate pathway and respiratory chain. It is supposed that variations in the activity both of different carbohydrate catabolism ways and the respiration with a rise of the hepatoma growth rate reflect changes in the relative content of cells at different phases of the cell cycle.

[Glucose metabolism and template synthesis in the mitotic cycle of human diploid fibroblasts]. / Metabolizm gliukozy i matrichnye sintezy v mitoticheskom tsikle diploidnykh fibroblastov cheloveka.

The correlation between the rates of protein and nucleic acid synthesis and the activity of the key enzymes of glycolysis (hexokinase, phosphofructokinase) and pentose phosphate cycle (glucose-6-phosphate dehydrogenase) in the mitotic cycle of human diploid fibroblasts synchronized by double thymidine block was studied. It was found that the removal of the thymidine block is followed by short-term (presumably, non-specific) simultaneous stimulation of matrix syntheses, as well as by glycolytic and pentose phosphate cycle enzyme syntheses. By the beginning of the S-phase, all the processes appear to be inhibited, followed by gradual activation of glycolysis and pentose phosphate cycle reactions. The implementation of the cell cycle is concomitant with stepwise transitions of protein and hexokinase synthesis rates and ATP content to one of the following levels--basal, intermediate or maximal. Changes in the activity of glucose-6-phosphate dehydrogenase in the course of the cell cycle appear as oscillations, those in phosphofructokinase as alternative states. At stage M, the oscillatory processes are temporarily quenched, whereas the ATP content occupies an intermediate level. In contrast with diploid fibroblasts, in transformed T9 cells the enzyme activity is much higher, and the fluctuations in activity throughout the cell cycle are less noticeable. Presumably, in transformed cells the enzyme activity is at the maximum level and is not prone to effector regulation.

[Sequence of activation of template biosynthesis in normal and transformed human cells after synchronization by a double thymidine block]. / Posledovatel'nost' aktivatsii matrichnykh biosintezov v normal'nykh i transformirovannykh kletkakh cheloveka posle sinkhronizatsii dvoinym timidinovym blokom.

The sequence of matrix biosyntheses of DNA, RNA and various proteins in normal and transformed human fibroblasts in the first mitotic cycle after synchronization of cells by double thymidine block was studied. Two important regularities of synthesis of acid-soluble histone-like and acid-insoluble proteins in normal and transformed cells were established. In normal fibroblasts, the synthesis of both acid-soluble and acid-insoluble proteins is minimal before DNA replication and maximal in the G2-phase; that in transformed cells is maximal after removal of the thymidine block and decreased in the G2-phase. In normal fibroblasts, the synthesis of acid-insoluble proteins is maximal before, while that of acid-soluble ones--after the maximum of DNA synthesis. In transformed cells the situation is opposite. RNA synthesis in normal and transformed cells is stimulated at the end of the G2-phase. In normal cells, protein synthesis is coupled with the activation of RNA synthesis, whereas in transformed fibroblasts protein synthesis occurs, in all probability, in the next mitotic cycle. These differences are especially well-pronounced in the expression of some LMG proteins. It is concluded that in transformed cells the regulatory control over the coupling of matrix biosyntheses is impaired.

[Activation of nuclear polypeptide synthesis in rat liver cells after inhibition of template protein synthesis. The role of nuclear polypeptide synthesis in the changes of the chromatin structure]. / Aktivatsiia iadernogo polipeptidnogo sinteza v kletkakh pecheni krys pri tormozhenii matrichnogo sinteza belkov. Vklad iadernogo polipeptidnogo sinteza v izmenenie struktury khromatina.

The correlation between the rates of nuclear polypeptide synthesis (NPS) and matrix protein synthesis in rat liver cells was investigated. It was shown that NPS is activated under conditions of protein synthesis inhibition in the cytoplasm. Model experiments revealed that the NPS and ADP ribosylation systems compete for chromatin structure: ADP ribosylation induces condensation, while NPS--decondensation of chromatin.

[Sequential changes in the macrostructure and RNA-synthesizing activity of nucleolar and extranucleolar chromatin from rat liver cells during induction of DNA synthesis]. / Posledovatel'nye izmeneniia makrostrukturnogo sostoianiia i RNK-sinteziruiushchei aktivnosti iadryshkovogo i ékstraiadryshkovogo khromatina kletok pecheni krys v protsesse induktsii sinteza DNK.

The dynamics of structural changes and RNA-polymerase activity in rat liver cell chromatin caused by drastic changes in the rates of protein synthesis was investigated. Inhibition of protein synthesis after a single injection of animals with cycloheximide (0.3 mg/100 g of body weight) increased the total condensibility of chromatin. Under these conditions, the stepwise activation of RNA-polymerases I and II correlated with decondensation of chromatin. By the 6-12th hour following cycloheximide injection, a chromatin fraction enriched with RNA-polymerase I and a RNA-polymerase II-rich fraction could be isolated from liver cell nuclei.

[Relation of the biosynthesis of intracellular and export proteins in rat liver cells during induction of proliferation by cycloheximide]. / Sootnoshenie biosinteza vnutrikletochnykh i éksportnykh belkov v kletkakh pecheni krys v protsesse induktsii proliferatsii tsikloeksimidom.

After a single injection of a sublethal dose of cycloheximide (CHI) the biosynthesis of extracellular proteins in rat hepatocytes was rapidly suppressed, the reconstitution being very slow. On the contrast, the biosynthesis of intracellular proteins (e.g., histones, and other acid-soluble liver proteins) was more resistant to CHI. The activation of biosynthesis of acid-soluble and acid-insoluble proteins was found to occur stepwise. It was assumed that the activation of synthesis and accumulation of intracellular proteins after CHI release accompanied by a decreased synthesis of extracellular proteins is one of possible causes of stimulation of DNA synthesis in the hepatocytes following a single injection of CHI.