The role of vitamin E in the biosynthesis of the isoprenoid region of the ubiquinone molecule in the rat liver

A decrease in the CoA and acetyl-CoA amount in the rat liver tissue by 34.8 and 29.4%, respectively, as well as inhibition of the biosynthesis rate of mevalonic acid from [I-14C] acetyl-CoA in the postmitochondrial liver fraction by 17.9% as compared to the control are found against a background of E-hypovitaminosis. The last change is not associated with the inhibition of the 3-oxy-3-methylglutaryl-CoA-reductase activity and may not be one of reasons which cause the biosynthesis disturbances in the isoprenoid part of the ubiquinone molecule in this organ. alpha-Tocopherol activates the ubiquinone biosynthesis from [2-14C] sodium acetate in the liver of rats with E-hypovitaminosis under conditions of 30 min preincubation and is not efficient when added in combination with actinomycin D. Probably, such an effect of alpha-tocopherol is realized at the level of RNA synthesis and is associated with the biosynthesis activation of short-living RNA.     

The Effect of Actinomycin D and Cycloheximide on the Dedifferentiation of Cotyledon in Phaseolus radiatus L. and Its Nucleic Acid and Protein

Calli were induced from cotyledon segment of mung bean (Phaseolus radiatus L.) in Miller medium supplemented with NAA 4 mg/l, kinetin 10 mg/L. The callus formation was completely prevented by the addition of actinomycin D 15 μg/mL or cyclo- heximidc 0.5 μg/mL at 0 hour. The inhibitory effect of actinomycin D or cycloheximide was increased with the increment of concentration but decreased when the inhibitory agents were added a few hours later. If actinomycin D or cycloheximide was added at 24 hour culture it inhibits neither the induction of callus formation nor the proliferation. The content of RNA, DNA and protein were determined. RNA in each segment increased obviously in the early stage of callus formation, but DNA and protein increased slightly afterward. It is suggested that a large increase of RNA is the characteristic of dedifferentiation of cotyledon in P. radiatus. In addition, it has also been shown that an actinomycin D or cycloheximide-sensitive process in the early stage of dedifferentiation is crucial for the callus formation. Both RNA and protein synthesis are required for the initiation of dedifferentiation.     

A comparative study of the effect of aflatoxin B1 and actinomycin D on HeLa cells

1. The cytotoxic effects of aflatoxin B(1) on HeLa cells were examined and effects of short exposures of the cells to the toxin were found to be reversible. 2. Aflatoxin B(1) inhibited the synthesis of both ribosomal and heterodisperse RNA. It is proposed that the toxin's mechanism of action on ribosomal RNA synthesis is related to its inhibitory effect on the maturation of the 45s-ribosomal-RNA precursor. 3. Protein synthesis is inhibited to a greater extent by aflatoxin B(1) than by actinomycin D. In contrast with actinomycin D, aflatoxin B(1) was shown to disaggregate polyribosomes directly.     

Mechanism of the anabolic action of phytoecdisteroids in mammals

In experiments with white mice it has been established that phytoecdisteroides turkesteron, ecdisteron and 2-desoxy-alpha-ecdison in the dose of 5 mg on 1 kg of body mass stimulate the protein synthesis. Using the model of protein synthesis from mice liver it has been shown that the action of phytoecdisteroides is connected with the rise of poliribosome functional activity and rate increase of protein macromolecules formation. Preliminary administration of actinomycin D does not prevent the effect of protein synthesis stimulation. It has been concluded that the anabolic effect of phytoecdisteroides in mammals organism is connected not with induction of RNA synthesis but with the acceleration of translocation processes.     

The hydrating effect of lathyrogenic compounds on chick-embryo cartilage in vivo

1. Injection of 0.16mug. of actinomycin D into pupae of the beetle Tenebrio molitor L. results in the development of modified adults in which the head and thorax are essentially adult while the abdomen and wings remain pupal-like. It is suggested that the messenger RNA for the development of head and thorax is present in the animal from the first day of pupation. 2. Injection of 0.16mug. of actinomycin D brings about 51-67% inhibition of labelled uridine incorporation into RNA. 3. When thymus DNA is mixed with actinomycin D before injection into pupae the latter develop into normal adults. This protection does not occur when DNA and actinomycin D are injected separately. 4. The inhibition of incorporation of labelled uridine into RNA by actinomycin is diminished to some extent when DNA and actinomycin D are injected separately and abolished if they are injected together. 5. Inhibition of RNA synthesis by actinomycin D in vitro is fully reversible. DNA or deoxyguanosine can reverse the effect of actinomycin D. 6. Incorporation of labelled glycine into protein is not affected by actinomycin D injection during the first 6 days of pupation. On the seventh day it becomes diminished in control pupae but this effect is prevented by actinomycin D. It is suggested that the template for protein synthesis is stable during the first 6 days of metamorphosis and that on the seventh day there is a qualitative change in the protein synthesized on the template.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

Inhibitor interaction in the suppression of ribosomal RNA synthesis in Ehrlich ascites carcinoma cells

Kinetic analysis of inhibitory action of papaverine, 2,4-dinitrophenol (DNP) and actinomycin D on RNA synthesis in the intact Ehrlich ascites carcinoma cells has shown that the action of these agents is mediated by their effect on the same step of rRNA synthesis.     

Effect of actinomycin D on DNA replication and c-myc expression during rat liver regeneration

In an attempt to elucidate the mechanisms that control the hepatic DNA replication, effect of low doses of actinomycin D on DNA synthesis and c-myc expression during the early stage of liver regeneration was investigated. Small amounts of actinomycin D, in amounts that had no effect on the rate of RNA synthesis in normal rats, were multiple injected at 0, 2 and 4 hours after partial hepatectomy. DNA synthesis and c-myc expression in these rats were compared with those in untreated rats. Hepatic DNA synthesis in the treated rats was delayed about 4 to 6 hours in comparison with control rats. In contrast, time course of c-myc expression in inhibitor treated rats was very similar to that in control rats.     

Phospholipid Synthesis in Sindbis Virus-Infected Cells

We investigated the metabolic requirements for the decrease in phospholipid synthesis previously observed by Pfefferkorn and Hunter in primary cultures of chick embryo fibroblasts infected with Sindbis virus. The incorporation of (32)PO(4) into all classes of phospholipids was found to decline at the same rate and to the same extent; thus, incorporation of (14)C-choline into acid-precipitable form provided a convenient measure of phospholipid synthesis that was used in subsequent experiments. Experiments with temperature-sensitive mutants suggested that some viral ribonucleic acid (RNA) synthesis was essential for the inhibition of choline incorporation, but that functional viral structural proteins were not required. The reduction in phospholipid synthesis was probably a secondary effect of infection resulting from viral inhibition of the cellular RNA and protein synthesis. All three inhibitory effects required about the same amount of viral RNA synthesis; the inhibition of host RNA and protein synthesis began sooner than the decline in phospholipid synthesis; and both actinomycin D and cycloheximide inhibited (14)C-choline incorporation in uninfected cells. In contrast, incorporation of (14)C-choline into BHK-21 cells was not decreased by 10 hr of exposure to actinomycin D and declined only slowly after cycloheximide treatment. Growth of Sindbis virus in BHK cells did not cause the marked stimulation of phospholipid synthesis seen in picornavirus infections of other mammalian cells; however, inhibition was seen only late in infection.     

Gibberellin-induced Yeast Sporulation in Relation to RNA and Protein Metabolism

Gibberellic acid (GA) promoted sporulation in yeast when added to the sporulation medium. When added together with GA, metabolic inhibitors of RNA synthesis such as 8-azaguanine, 2-thiouracil, and actinomycin D suppressed selectively the promoting effect of GA on sporulation. The effect of 8-azaguanine and 2-thiouracil was alleviated by simultaneous addition of guanine and uracil, respectively. The promoting effect of GA on sporulation was also suppressed by inhibitors of protein synthesis such as ethionine, chloramphenicol, and puromycin. Methionine eliminated the inhibitory effect of ethionine on the GA action.     

Flagellar synthesis in Salmonella typhimurium: requirement for ribonucleic acid synthesis

The micro-complement-fixation assay has been demonstrated to be a sensitive assay for flagella which occur in nanogram amounts. By use of this assay, it was found that flagellar synthesis occurs during starvation of Salmonella typhimurium for tryptophan, an amino acid not present in flagellar protein. Under these conditions net ribonucleic acid (RNA) synthesis was reduced to approximately 10% of the control rate. Less than 1 mug of actinomycin D per ml further reduced RNA synthesis to less than 1% of the control rate in a culture sensitized by prior treatment for 5 min at 37 C with 5 x 10(-4)m ethylenediaminetetraacetate in 0.33 m tris(hydroxymethyl)aminomethane-chloride (pH 8.0). In the presence of actinomycin D, no synthesis of flagellar protein could be detected. Analysis of fractions of RNA separated by zone centrifugation indicated that actinomycin D reduces the production of template RNA as well as of ribosomal RNA. This suggests that in S. typhimurium the production of flagellar protein requires the concomitant synthesis of RNA. There is no evidence that a stable messenger RNA specific for flagellar synthesis is present.     

LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HEPATIC CELL NUCLEOLI IN MICE TREATED WITH ACTINOMYCIN D

Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology was studied when actinomycin D treatment preceded labeling with tritiated orotic acid. Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible if a dosage of 25 µg of actinomycin D was used, but nucleolar RNA was still synthesized. After a dosage of 400 µg of actinomycin D, nucleolar RNA synthesis was completely stopped If labeling with tritiated orotic acid preceded treatment with 400 µg of actinomycin D, labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component. At 30 min after actinomicyn D treatment all labeling was lost. Since labeling was associated with the granular component the progressive loss of label as a result of actinomycin D treatment indicated a release of nucleolar granules. The correlation between this release and the loss of 28S RNA from actinomycin D treated nucleoli as described in the literature is discussed.     

Synthesis of ribonucleic acid by isolated rat liver mitochondria

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.     

Contribution of a change in mRNA half-life to the accumulation of the tissue-specific S-100 protein during postnatal development of the mouse brain

The S-100 protein accumulates rapidly in the mouse brain between 15 and 21 days of postnatal development. The accumulation of this protein is brought about mainly by an increased rate of its synthesis. The present study focuses on attempts to determine if a change in the half-life of messenger RNA (mRNA) is involved in bringing about the increased rate of synthesis of the S-100 protein. Utilizing the inhibition of RNA synthesis by actinomycin D, we were able to show that the halflife of mRNA increases concurrently with an increase in the rate of synthesis of the S-100 protein. Utilization of actinomycin D does present hazards in interpretation of results on mRNA stability; experiments were performed to determine if the results obtained were due to side effects of the drug. As far as could be determined, the possible side effects of actinomycin D did not affect our results.     

Microexudation phenomenon in the testing of antibiotics]

Changes in formation of the surface protein-polysaccharide layer (microexudate) on the cell surface under the action of inhibitor antibiotics, such as puromycin, actinomycin D and mitomycin C, as well as protein substances with adhesive action, such as horse serum and triprotamine in low doses were characterized quantitatively on a model of HeLa cells ellipsometrically. Under the action of puromycin, actinomycin D and mitomycin C formation of the microexudate ceased, which was in full accordance with the data on ceasation of the intracellular synthesis of protein, RNA and DNA under the action of the above antibiotics respectively. Inhibition of the microexudate formation was reversible. Still, the time of the inhibitory effect of puromycin and actinomycin D was longer than that of mitomycin C. Under the action of horse serum and triprotamine production of the microexudate by the cells was increased and accelerated. Accounting with the relative simplicity of the ellipsometric method and possibility of rapid estimation of the results, the data substantiate the expediency of using the phenomenon of microexudation as a cytopharmacological test.