Activity of nuclear RNA polymerases in the liver of rats of different ages after phenobarbital administration

DNA-dependent RNA-polymerases of nuclei isolated from the liver of rats aged 3- and 24 months were studied in different periods after phenobarbital administration. It is shown that 4h after single intraperitoneal injection of the preparation (80 mg per 1 kg of the body mass) to young animals the activity of the RNA-polymerase I rises, it remains higher, the following 12-20 h and then returns to the initial level. The activity of RNA-polymerases II and III under these conditions 16 h later remains higher the following 8 h and 24 h later the activity of the first enzyme returns to the initial value and that of the second one becomes still more. In old rats (24 months) the activity of all three classes of RNA-polymerases is lower than in young ones and increases only 48 h later. Under long-term administration of phenobarbital (40 mg per 1 kg of the body mass daily, 4 days) the activity of RNA-polymerase I was the same as in the intact animals of the corresponding ages and the activity of RNA-polymerases II and III increased both in young and old rats. Single administration of the preparation increases the liver mass in the young animals 24 h later and in the old ones--48 h later. Long-term administration of the preparation enhances its mass both in young and old animals, but its relative increase is more expressed in rats at the age of 3 months.     

Heliomycin suppression of RNA synthesis in a cell-free system]

Heliomycin inhibited in vitro the RNA-polymerase reaction catalyzed by the preparation of DNA-dependent RNA-polymerase from E. coli. The blocking effect increased with a rise in the antibiotic concentration. The inhibitory effect of heliomycin decreased, when the amount of RNA-polymerase in the system increased. Yet, it did not depend on the content of DNA and the nature of the DNA preparation. Preincubation of RNA-polymerase with DNA resulting in formation of the enzyme-matrix complex did not prevent blocking RNA synthesis by heliomycin. Suppression of the RNA-polymerase reaction did not depend on the time of the antibiotic addition to the polymerizing system. Heliomycin had a significant activity not only with respect to the bacterial RNA-polymerase, but also in the system containing the enzyme isolated from the cells of Crithidia oncopelti.     

Directed changes in the fatty acid spectrum of nuclear lipids and the functional state of the cellular genome

The content of polyenic fatty acids in lipids of nuclear membranes and chromatin of albino rat liver cells is established to decrease considerably in the process of ageing. It is also shown that the RNA-polymerase activity lowers with the animal age. With an increase of the content of polyunsaturated fatty acids in lipids of nuclear structures of liver cells in old animals with the aid of diet and phospholipid liposomes the RNA-polymerase activity enhances up to the level of young rats aged three months and older. However, if the relative content of polyunsaturated fatty acids in lipids of old test rats decreases with the aid of the diet to the level of the control rats aged 26 months, then the activity of RNA-polymerases falls to the level of activity in control rats of the given age group and younger.     

Inhibition of RNA synthesis in animal cells by sodium selenite

The effect of selenium-containing compounds on RNA synthesis in rat liver cells was studied in vivo. All the selenium derivatives under study inhibited the rRNA synthesis in liver cells. The most potent inhibiting effect was exerted by sodium selenite. It was shown that in a cell-free system of RNA biosynthesis sodium selenite selectively inhibited the activity of RNA-polymerase I in isolated nuclei and purified enzyme preparations of normal and tumour cells. A feasible mechanism of inhibition of the RNA-polymerase I activity by selenium and a hypothesis on the anticarcinogenic effect of selenium are postulated.     

Age-related changes of protein- and RNA-synthetic processes in experimental hyper- and hypothyroidism

The rate of liver and plasma protein synthesis and the activity of liver RNA polymerases 1 and 2 were investigated in rats of various age under experimental hyper- and hypothyroidism. The rate of plasma protein synthesis decreased with age more dramatically than that of liver proteins. Hyper- and hypothyroidism exerted opposite effects on protein synthesis in rats: stimulation and inhibition, respectively. The manifestation of these effects was age related. The thyroid status of animals also influenced the balance of protein synthesis. Thyroxin administration caused preferential incorporation of a label into blood plasma proteins. Changes of thyroid status of old animals insignificantly affected the absolute values of the label incorporation into proteins and the ratio of the label incorporation into local and secreted liver proteins. Age-related decrease of total hepatic nuclear RNA-polymerase activity was due to reduction of the template-bound functionally active forms of RNA-polymerases 1 and 2. Administration of thyroxin caused initial redistribution of the enzyme activity between template-bound and free fractions accompanied by the increase of template bound RNA-polymerases. Prolonged hormonal stimulus also caused an increase of free RNA-polymerases, which reflects the increased synthesis of these enzymes. Mecrazolyl administration reduced the activity of RNA-polymerase 1 and 2. All age groups were characterized by preferential reduction of the bound form. RNA-polymerase 2 activity decreased to a greater extent than that of RNA-polymerase 1. The data suggest age-determined reactions of the body to altered thyroid status.     

Additive effects of thyroid hormone,growth hormone and testosterone on deoxyribonucleic acid-dependent ribonucleic acid polymerase in rat-liver nuclei

1. The stimulations of DNA-dependent RNA polymerase in isolated rat-liver nuclei by thyroid hormone, human growth hormone and testosterone are compared. 2. Single or multiple administrations of growth-promoting doses of tri-iodo-l-thyronine, human growth hormone and testosterone stimulate the Mg²⁺-activated RNA-polymerase reaction in nuclei from thyroidectomized, hypophysectomized and castrated rats respectively. The magnitude of stimulation was proportional to the degree of enhancement of liver growth by each hormone. After a single injection, the latent period preceding the stimulation was 1, 2 and 10hr. for growth hormone, testosterone and tri-iodothyronine respectively. The time-course of stimulation of enzyme activity and the synthesis of rapidly labelled nuclear RNA in vivo were also different for each hormone. 3. Growth hormone administration failed to stimulate the Mn²⁺/ammonium sulphate-activated RNA-polymerase reaction. Thyroid hormone and testosterone, however, stimulated it but the effect was less pronounced and occurred several hours later than that observed for the Mg²⁺-activated RNA-polymerase reaction. 4. In combination experiments, hypophysectomized or the thyroidectomized rats were given growth hormone or tri-iodothyronine in a single or repeated doses at levels that produced the maximum stimulation of Mg²⁺-activated RNA-polymerase activity. Taking into account the different latent period for each hormone, a single administration of the second hormone caused an additional stimulation of the enzyme activity. Similar additive effects were observed in thyroidectomized–castrated rats after treatment with tri-iodothyronine and testosterone. The magnitude of the additional stimulation caused by the administration of the second hormone was compatible with the capacity of that hormone to promote liver growth in rats deprived of it. 5. It is concluded that, although these hormones have some similar effects, the regulation of nuclear RNA synthesis may be mediated via different routes for each hormone.     

Sequential changes in the macrostructure and RNA-synthesizing activity of nucleolar and extranucleolar chromatin from rat liver cells during induction of DNA synthesis

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.     

Enhancement of nuclear Ca2+-ATPase activity in regenerating rat liver: involvement of nuclear DNA increase

The alteration of calcium content, Ca²⁺-ATPase activity, DNA content and DNA fragmentation in the nuclei of regenerating rat liver was investigated. Liver was surgically removed about 70% of that of sham-operated rats. the reduced liver weight by partial hepatectomy was completely restored at 3 days after the surgery. Regenerating liver significantly increased Ca²⁺-ATPase activity and DNA content in the nuclei between 1 and 5 days after hepatectomy. The nuclear calcium content was clearly increased from 2 days after hepatectomy. The increase of Ca²⁺-ATPase activity in regenerating liver was clearly inhibited by the presence of trifluoperazine (10 M), staurosporine (2.5 M) and dibucaine (10 M), which are inhibitors of calmodulin and protein kinase, in the enzyme reaction mixture. However, the nuclear enzyme activity in normal rat liver was not significantly altered by these inhibitors. Meanwhile, the increase of nuclear DNA content in regenerating liver was completely blocked by the administration of trifluoperazine (2.5 mg/100 g body weight), suggesting an involvement of calmodulin. Now, the nuclear DNA fragmentation was significantly decreased in regenerating liver, suggesting that this decrease is partly contributed to the increase in nuclear DNA content. The present study clearly demonstrates that regenerating liver enhances nuclear Ca²⁺-ATPase activity and induces a corresponding elevation of nuclear calcium content. This Ca²⁺-signaling system may be involved in the regulation of nuclear DNA functions in regenerating rat liver.     

Role of cadmium in activating nuclear protein kinase C and the enzyme binding to nuclear protein.

Specific effects of cadmium on nuclear protein kinase C activity were found with 3T3/10T1/2 mouse fibroblast and rat liver nuclei. Treatment of the mouse fibroblasts in culture with 12-O-tetradecanoylphorbol-13-acetate resulted in the stimulation of nuclear protein kinase C activity in a "fixed" pool which is defined by its resistance to chelator extraction, whereas the chelator extractable enzyme activity, defined as the "labile" pool was unaffected. Cadmium was found to potentiate the effect of the phorbol ester, directed specifically to nuclei, since the particulate protein kinase C activity was not changed under similar treatment. In a reconstituted system consisting of rat liver nuclei and rat brain protein kinase C, cadmium stimulated the binding of the enzyme to a 105-kDa nuclear protein. The binding of a 105-kDa protein to protein kinase C is attributed strictly due to the cadmium effect, whereas a 50-kDa protein binding to protein kinase C was only enhanced by cadmium. We propose a mechanistic model, where cadmium substitutes zinc in the regulatory domain of protein kinase C rendering the putative protein-protein binding site exposed.     

Properties of the rat liver DNA-dependent RNA-polymerase stored at -25 degrees C in the presence of glycerol or polyethyleneglycol-400

The effect of slow freezing (1-2 degrees C/min) was studied as applied to the template activity of DNA in the RNA-polymerase reaction. It was found out that the activity of each RNA-polymerase form decreases after 24-hour storage by freezing at -25 degrees C in the presence of glycol or polyethylene glycol-400. The study of the enzyme in the glycerol concentration gradient showed sedimentation constant changes.     

Properties of DNA-dependent RNA-polymerase from spleens of mice infected with Rauscher leukemic virus]

The properties of RNA polymerases A, B and C isolated from the spleens of mice infected with Rauscher leukemic virus were studied. The solubilized RNA-polymerases A and B were purified 150--300-fold. The dynamic changes in the activities of all forms of RNA-polymerases at different stages of leukosis were studied. At the earliest steps of leukosis a 2-fold increase in the RNA-polymerase B activity followed by a 5-fold increase in the RNA-polymerase A activity was observed. At late stages of leukosis the activity of RNA-polymerase C also showed an increase. The properties of RNA-polymerases A and B from the spleens of virus-infected mice were compared to those of the controls. In leukemic tissues the specific activities of RNA-polymerases A and B were higher as compared to those of the enzymes isolated from the spleens of non-infected mice. However, no significant differences in the enzyme properties in normal and virus-infected animals were revealed. Dihydrorifampicine (200 mkg/ml) caused a 50% inhibition of RNA polymerase A in vitro but had no effect on the activities of RNA-polymerases B and C.     

3-Hydroxyisoquinolines as inhibitors of HCV NS5b RNA-dependent RNA polymerase

Isoquinoline-based non-nucleoside inhibitors of HCV NS5b RNA-dependent RNA-polymerase are described. The synthesis and structure–activity relationships are detailed, along with enzyme and cellular activity.     

DNA-dependent RNA-polymerase activity of isolated kinetoplasts of crithidia oncopelti]

DNA-dependent RNA-polymerase activity was found in the kinetoplasts of Crithidia oncopelti. Kinetic patterns of 14C-UTP incorporation into the acid-insoluble fraction of isolated kinetoplasts at 25 degrees, 30 degrees and 35 degrees C were estimated. The effects of different antibiotics and intercalating agents on RNA synthesis in kinetoplasts were studied. alpha-amanitin, a specific inhibitor of the nuclear enzyme, does not affect the RNA-polymerase activity of the kinetoplasts. Streptolidigin and rifampicin, inhibitors of bacterial RNA-polymerase, have little effect on RNA synthesis in the kinetoplasts even at high concentrations. Kinetoplasts preincubation in the phosphate buffer increases the permeability of their membranes for rifampicin. Intercalating agents, acriflavin and ethidium bromide, strongly inhibit the kinetoplast RNA synthesis. Similar specific effects of some antibiotics and intercalating agents on RNA synthesis in kinetoplasts and typical mitochondria may be indicative of similarity of functional properties of RNA-polymerases in those organelles.     

Murine catalase phenotypes

An examination of three inbred strains of mice differing with respect to liver and kidney catalase activity reveals two distinct genetic factors controlling the level of liver catalase activity. The first genetic factor controls the catalytic activity of the enzyme. Specific activity of purified enzyme from C57BL/6 and C57BL/Ha strains is 60% of that of the DBA/2 strain. The second factor controls the content of liver catalase. Liver catalase of C57BL/Ha is degraded in vivo at a rate one half that of liver catalase of DBA/2 and C57BL/6, resulting in the accumulation of twice as many catalase molecules in C57BL/Ha. The factor affecting turnover of catalase is apparently specific for catalase of liver since no differences exist in kidney catalase levels between C57BL/Ha and C57BL/6. Furthermore, this factor does not appear to alter the metabolism of total liver protein since no substantial difference in the turnover rate of liver protein is observed among the strains. It is particularly significant that the genetic factor affecting the amount of liver catalase does so by altering the rate of catalase degradation rather than the rate of synthesis, confirming the previously published report of Rechcigl and Heston (1967). Thus, these studies emphasize that the quantity of an enzyme in animal cells is a balance between the rate of synthesis and the rate of degradation of the enzyme.This paper was presented at a symposium entitled Genetic Control of Mammalian Metabolism held at The Jackson Laboratory, Bar Harbor, Maine, June 30–July 2, 1969. The symposium was supported in part by an allocation from NIH General Research Support Grant FR 05545 from the Division of Research Resources to The Jackson Laboratory.This investigation was supported by USPHS Research Grant GM 14931 from the Division of General Medical Sciences, and Grants PF-373 and P-427 from the American Cancer Society.     

Effect of vitamin E on transcription in isolated nuclei and rat liver chromatin in normal status and in E-hypovitaminosis]

The effect of alpha-tocopherol on the RNA-polymerase activity in isolated rat nuclei and chromatin from normal and E-deficient rats and the possible role of tocopherol-binding proteins in this process were studied. Some differences in the RNA-polymerase activities of the nuclei were found; however, in vitro added alpha-tocopherol had no effect on the level of the label incorporation into RNA. No effect of alpha-tocopherol on this process was observed after addition of cytosol either. Analysis of chromatins from normal and E-deficient rats revealed no differences in their RNA-polymerase activities. In vitro added alpha-tocopherol increased the RNA-polymerase activity of normal (but not of vitamin E-deficient) rats. Some differences in the RNA-polymerase activities were noted after addition to the incubation medium of the Triton X-100-solubilized nuclear fraction specifically binding alpha-tocopherol. This effect was enhanced in the presence of exogenous alpha-tocopherol. The susceptibility of chromatin from normal and E-deficient rats to DNAse I hydrolysis was also found to be different. It was concluded that vitamin E can influence the RNA-polymerase activity of the nuclei and chromatin as well as the chromatin structure and that alpha-tocopherol-binding proteins are necessary for the vitamin E effect on the RNA-polymerase activity to be manifested.