Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei

DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to α-amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 μg/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of α-amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates.     

THE ACTION OF α-AMANITIN ON RNA SYNTHESIS IN CHINESE HAMSTER OVARY CELLS : Ultrastructural and Biochemical Studies

α-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by α-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver α-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the α-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4–5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by α-amanitin in CHO cells.     

In Vitro Sensitivity of Torulopsis glabrata to Amphotericin B, 5-Fluorocytosine, and Clotrimazole (Bay 5097)

Thirty-five strains of Torulopsis glabrata were tested by a tube dilution method for their susceptibility to amphotericin B, 5-fluorocytosine, and clotrimazole (Bay 5097). Amphotericin B was the most active in vitro, inhibiting all strains at a concentration of 1 μg/ml and killing all strains at 2 μg/ml. 5-Fluorocytosine inhibited over 80% of strains at 0.24 μg/ml, but three strains required ≥7.8 μg/ml for killing. A concentration of 2 μg of clotrimazole per ml inhibited less than 50% of strains, and 8 μg/ml killed only 10% of strains. Most strains of T. glabrata were killed by therapeutically achievable concentrations of amphotericin B and 5-fluorocytosine, but not clotrimazole.     

Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by β-ecdysone

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

Effect of corticosterone, hydrocortisone and prostaglandin fatty acid precursors on the spontaneous motility of the uterus isolated from ovariectomized rats

The effect of corticosterone (CC, 0.4 and 1 μg/ml) and of hydrocortisone (HC, 20 and 40 μg/ml) on the spontaneous motility and on prostaglandin (PG) generation in the uterus from ovariectomized rats, was studied. Both concentrations of CC depressed significantly the frequency of contractions but the isometric developed tension was affected only by the higher dose. HC concentrations whereas the contractile frequency was only depressed by the higher one. The CC-inhibited motility was accompanied by a reduction in the amount of PGs released from the uterus into the bath solution. In addition, the influence of arachidonic acid (AA), linoleic acid (LA) and gamma-linolenic acid (γ-LA) - 1 or 2 μg/ml - on the depression evoked by CC, were also explored. The fatty acids had no effect on the spontaneous uterine motility except in the case of γ-LA at 1 μg/ml. γ-LA completely blocked the CC-evoked reduction of bothe tension and frequency; AA (1μg/ml) elicited a reversion only on frequency whereas LA had no effect at all. This reversion by a fatty acid PG-precursor might indicate that CC is able to diminish substrate availability for PG synthesis in the rat uterus.     

Embryoless Wheat Grain: A Natural System for the Study of Gibberellin-induced Enzyme Formation

Yorkstar wheat, grown in New York State, has a high percentage (10-11) of grains without embryos. The embryoless grains have viable aleurone layers and show no sign of injury. These grains are able to support α-amylase synthesis only in the presence of gibberellin A₃ (GA₃). In the absence of GA₃ some protein synthesis occurs in embryoless grains during the early hours of soaking, indicating that such activity occurs prior to and independent of GA₃ induction of α-amylase. The level of β-amylase on a dry weight basis is the same in embryoless and normal grains and decreases with time of soaking. In the presence of GA₃, β-amylase decreases at a slower rate. Isoenzymes of α-amylase from GA₃-treated embryoless and normal grains show quantitative as well as qualitative differences. Cycloheximide (60 μg/ml) completely inhibits the synthesis of α-amylase by embryoless grains. Of the RNA synthesis inhibitors, actinomycin D (60 μg/ml) was ineffective while 6-methylpurine (60 μg/ml) gave 65% inhibition without decreasing the number of isoenzymes.     

Stimulation by prostaglandin F2α of acidic glycosaminoglycan production in cultured fibroblasts

The effect of PGF2α on the synthesis of hexosamine-containing substances (acidic glycosaminoglycans and glycoproteins) was studied in cultured fibroblasts derived from a rat carrageenin granuloma. Treatment with PGF2α ranging from 0.01 μg/ml to 20 μg/ml resulted in a significant increase of the production of these macromolecules by the cells. The stimulatory effect was found significant even at the low concentration of 10 ng/ml, and could be seen as early as 3h after exposure to PGF2α. The hexosamine-containing substances increased by PGF2α revealed that 80% of the increase was due to acidic glycosaminoglycans and the rest was due to glycoproteins.     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E₂ and 6-keto-prostaglandin F_1α. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 μg/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 μg/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolyses are independently regulated.     

The action of α-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of α-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10–20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by α-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

On the regulation of tyrosine transaminase, glutamatic dehydrogenase and aspartic transaminase in Tetrahymena

Aspartic transaminase, tyrosine transaminase, lactic dehydrogenase, and glutamic dehydrogenase were studied in Tetrahymena pyriformis in order to gain a better understanding of the control of the entrance and exit of metabolic intermediates to and from the major carbohydrate pathways. Glucose decreased the activity of aspartic transaminase, tyrosine transaminase and glutamic dehydrogenase but not lactic dehydrogenase. Actinomycin D (6 and 12 μg/ml) blocked the decrease in glutamic dehydrogenase and aspartic transaminase activity caused by glucose; 12 μg/ml partially prevented the decrease in tyrosine transaminase activity. Actinomycin D alone had little effect on enzyme activity. Uracil incorporation into RNA was doubled by 6 μg/ml actinomycin D, a concentration which did not alter the RNA content of the cells. At 12 μg/ml this drug caused a small decrease in RNA spec. act. Cycloheximide at 10 μg/ml, a concentration which inhibited protein synthesis by 70%, caused a three-fold increase in aspartic transaminase and a two-fold increase in glutamic dehydrogenase. In the presence of both cycloheximide and glucose, the drug effect predominated. Thus both actinomycin D and cycloheximide blocked the glucose-induced decrease in enzyme activity. These results suggest that the levels of aspartic transaminase, glutamic dehydrogenase, and probably tyrosine transaminase are regulated at least in part by a degradative control system.     

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.     

Prostaglandin synthesis by rheumatoid synovium and its stimulation by colchicine

The synthesis of prostaglandins by rheumatoid synovial tissue in organ culture was studied utilizing radioimmunoassay, with antisera to PGB₁, PGF_1α and PGF_2α. It was established that PGE₂ and PGF_2α were the major prostaglandins formed by analyses of culture media with the two antisera to PGF, before and after alkali treatment. Indomethacin at 5 μg/ml suppressed prostaglandin synthesis, usually to <1% of control cultures. Colchicine, 0.1 μg/ml resulted in marked stimulation of prostaglandin synthesis, in some cases over 10 fold. It is suggested, because of the colchicine effect, that the state of the microtubules may regulate the rate of prostaglandin biosynthesis. It is possible that prostaglandin E₂ produced by rheumatoid synovia may contribute to the pathogenesis of the inflammatory reaction and lead to destruction of juxta-articular bone in rheumatoid arthritis.     

Prostaglandin synthesis by rheumatoid synovium and its stimulation by colchicine

The synthesis of prostaglandins by rheumatoid synovial tissue in organ culture was studied utilizing radioimmunoassay, with antisera to PGB₁, PGF_1α and PGF_2α. It was established that PGE₂ and PGF_2α were the major prostaglandins formed by analyses of culture media with the two antisera to PGF, before and after alkali treatment. Indomethacin at 5 μg/ml suppressed prostaglandin synthesis, usually to <1% of control cultures. Colchicine, 0.1 μg/ml resulted in marked stimulation of prostaglandin synthesis, in some cases over 10 fold. It is suggested, because of the colchicine effect, that the state of the microtubules may regulate the rate of prostaglandin biosynthesis. It is possible that prostaglandin E₂ produced by rheumatoid synovia may contribute to the pathogenesis of the inflammatory reaction and lead to destruction of juxta-articular bone in rheumatoid arthritis.     

Follicle stimulating hormone and prolactin release induced by prostaglandins in rat

Ten to 60 minutes following a single i.v. injection of PGE₂ (500 μg/rat) into male rats of 30 to 35 days of age FSH concentration in the serum was raised significantly. The rise in FSH was maintained from 10 to 60 minutes after treatment, then at 90 minutes FSH had declined and was not significantly different from that of the control before treatment. Prostaglandin E₁, E₂ or F_2α (670μg/rat) significantly increased the serum prolactin level 10 to 60 minutes after a single i.v. injection in spayed rats primed with estrogen and progesterone. And, rats primed with estrogen and progesterone. And, increases in prolactin in the serum were observed with as little as 2μg of PGE₁ or E₂, and 20μg of PGF_2α. Twenty μg of PGE₂, and 200μg of PGE₁ or F_2α gave the maximum stimulation. These results indicate that release of pituitary hormones is affected by prostaglandins.Prostaglandins (PGs) are widely distributed in mammalian tissues, and they have been reported to have an almost equally wide variety of endocrine and metabolic effects. It was recently postulated that PGs may be involved in the process of ovulation because ovulation was blocked by inhibitors of PG synthesis (1–5).     

Protective effect of curcumin on γ-radiation induced DNA damage and lipid peroxidation in cultured human lymphocytes

The present work is aimed at evaluating the radioprotective effect of curcumin, a naturally occurring phenolic compound on γ-radiation induced toxicity. The cellular changes were estimated by using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analysed by using cytokinesis blocked micronucleus assay and dicentric aberration (DC). The γ-radiation at different doses (1, 2 and 4 Gy) were found to significantly increase micronuclei (MN), DC frequencies and TBARS level whereas the levels of GSH and antioxidant enzymes were significantly decreased. The maximum damage to lymphocytes was observed at 4 Gy irradiation. Curcumin pretreatment (1, 5 and 10 μg/ml) significantly decreased the frequency of MN and DC. The levels of TBARS decreased and activities of SOD, CAT and GPx significantly increased along with GSH levels. At 1 Gy irradiation all the concentrations of curcumin (1, 5 and 10 μg/ml) significantly protected the lymphocytes from radiation damage. At 2 Gy irradiation, 5 and 10 μg/ml of curcumin showed significant radioprotection. Since the highest damage was observed at 4 Gy irradiation both 1 and 5 μg/ml of curcumin pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10 μg/ml of curcumin significantly protected the cultured lymphocytes from radiation damage. Thus, pretreatment with curcumin gives protection to lymphocytes against γ-radiation induced cellular damage.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

DNA fingerprinting pattern and susceptibility to antifungal drugs in Cryptococcus neoformans variety grubii isolates from Barcelona city and rural environmental samples

Cryptococcus neoformans var. grubii (serotype A) was isolated from 12 soil samples mixed with pigeon droppings (16.9%) from 71 soil samples in Barcelona and rural areas of Catalonia. C. neoformans was not isolated from indoor dust and Eucalyptus debris. PCR fingerprinting was performed in 22 representative isolates and all of them corresponded to the VNI pattern. Susceptibility testing for the 22 isolates of C. neoformans var. grubii showed that all of them were susceptible to amphotericin B. Three isolates presented MICs (Minimal Inhibitory Concentrations) ≥ 1 μg/ml to Itraconazole, five MICs ≥ 1 μg/ml to ketoconazole and four were fluconazole resistant, (MICs ≥ 64 μg/ml), while three of them were shown to have MICs ≥ 1 μg/ml to voriconazole. In spite that all isolates presented the same DNA fingerprinting pattern, the susceptibility to antifungals is very variable. The possibility of acquiring cryptococcosis infection with primarily resistant environment strains is feasible.     

Synthesis of ribonucleic acid in baby-hamster kidney cells in the presence of actinomycin D

1. RNA molecules with sedimentation values in sucrose gradients of 12–20s are synthesized in baby-hamster kidney cells even after prolonged incubation in medium containing 1μg. of actinomycin D/ml. 2. The rate of formation of this RNA is dependent on the age of the cultures and is greatest during the exponential phase of growth. 3. Growth of cells on nutritionally poor medium causes degradation and inhibits the synthesis of these RNA fractions. 4. Replacement of the nutritionally poor medium with a rich medium stimulates the synthesis of actinomycin-resistant RNA. This stimulation is blocked by cycloheximide. 5. The base composition of this RNA is characterized by low cytidine and high guanosine values.