Selective effect of hormones on nucleic acid metabolism during germination of pear embryos

1. The effect of hormones on (32)P incorporation into various RNA fractions in germinating pear embryos was studied by fractionation on methylated albumin-kieselguhr columns. Abscisic acid inhibited labelling of soluble RNA, DNA-RNA hybrid and light-ribosomal RNA fractions with (32)P and this effect was reversed by both kinetin and gibberellic acid. 2. Kinetin reversed the inhibition by abscisic acid of (32)P incorporation into total ribosomal RNA and appeared to promote labelling of heavy-ribosomal RNA. Gibberellic acid was more active than kinetin in reversing the inhibition by abscisic acid of labelling of the DNA-RNA hybrid fraction with (32)P, but in contrast with kinetin appeared to increase further the inhibition by abscisic acid of labelling of total ribosomal RNA. 3. The percentage of radioactivity in various RNA fractions showed marked variation in response to hormones. 4. The pattern of labelling of RNA in pear embryos during reversal of inhibition by abscisic acid with a combination of kinetin and gibberellic acid was similar to that after cold-treatment of dormant pear embryos. This is suggestive of hormonal interplay in dormancy release by cold-treatment in pear embryos.     

Labelling kinetics of RNA containing poly(a) in liver subcellular fractions

Kinetics of incorporation of (3H) uridine into cytoplasmic RNA fractions of rat liver is investigated. The fractions include free and membrane bound polysomes, rough membranes sedimenting with mitochondria and free cytoplasmic RNA particles. (1) Poly(A) containing RNA, isolated by oligo-dT cellulose, amounts to 0.4% of the total RNA in the homogenate, 0.5% in bound polysomes, 3.4% in free polysomes and 16% in free cytoplasmic RNA particles. (2) The rate of (3H) uridine incorporation into RNA lacking poly(A) proceeds uniformly in all subcellular fractions except for free cytoplasmic RNA particles, which accumulate negligible amounts of radioactivity. (3) The initial labelling of RNA containing poly(A) is most active in free cytoplasmic RNA particles supporting their identity as mRNA en route to polysomes. The initial specific radioactivities decrease in the following order: homogenate, bound polysomes, rough membranes sedimenting with mitochondria, free polysomes. The data suggest that mRNA is supplied to free and membrane-bound polysomes via different routes. The kinetic analysis indicates that free cytoplasmic RNA particles may be a precursor of mRNA of free polysomes rather than that of bound polysomes. (4) The kinetic differences of free and membrane bound polysomes are also demonstrated by comparing the radioactivity of RNA containing poly(A) to the total radioactivity at various incorporation times. In bound polysomes this decreases from 31% at 1 h to 10% at 25 h, whereas in free polysomes the corresponding ratio increases from 10 to 13%. RNA containing poly(A) of free cytoplasmic RNA particles represents 64% of the total radioactivity throughout the experiment.     

Incorporation of [2-14C orotic] acid into chromatin RNA of tissues of animals of various ages

The structure and biological activity (the level of the labelled precursor incorporation into RNA) of active and repressed chromatin of the liver and small intestine mucosa were studied in adult (6-8 months) and old (24-26 months) rats. The content of repressed chromatin fraction in both tissues is found to increase with age. In the liver of old rats the level of [14C[ orotic acid incorporation into RNA of chromatin fractions decreases, radioactivity of the acid-soluble fraction being unchanged. In the small intestine mucosa a high leve of [14C] orotic acid incorporation into chromatin RNA with ageing is due to an increase in permeability of the mucosa cells.     

Uptake of amino acids and nucleic acid precursors by regenerating rat liver

1. At 0.5-1.0h after partial hepatectomy the intracellular acid-soluble fraction of rat liver took up twice as much radioactivity from [(3)H]orotic acid, [(3)H]uridine and [(3)H]thymidine as did similar fractions from sham-operated animals. This increase in penetration was not prevented by adrenalectomy or actinomycin, both of which decreased precursor uptake into nuclear RNA at this time. The increase in entry was still shown by thymidine at 22h after operation, at the height of the S period. Adenine penetration was not increased 1h after partial hepatectomy. 2. Plasma concentrations of ornithine and possibly methionine, tyrosine and lysine were raised 1.5h after partial hepatectomy. [(3)H]Lysine entry into regenerating liver at this time was increased by 60%; [(3)H]valine uptake was unaffected. Intracellular amounts of tyrosine, phenylalanine and ornithine in the liver were also increased. 3. The relation of these events to the start of liver regeneration is discussed.     

Features of the biosynthesis of immunoglobulin G peculiar to the growth process

It is established that with partial hepatectomy, Shvets leukosis and hepatoma RS-1 the biosynthesis intensity of rat blood serum proteins producing aggregates in the acid medium is considerably higher than that of other serum proteins, the incorporation of the radioactive precursor into immunoglobulin G peculiar to intensive normal and malignant growth being particularly intensive. During liver regeneration as well as in malignant growth specific radioactivity of immunoglobulin G peculiar to the growth processes is three and five times, respectively, as high as this value for blood serum soluble proteins and proteins of alpha-globulin fractions.     

Formation, size, and solubility in chloroform/methanol of products of protein synthesis in isolated mitochondria of rat liver and Zajdela hepatoma.

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incroporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HC1) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [3H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [3H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions.     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

Hormonal stimulation of 3H-orotic acid incorporation into RNA by serum-free cultured hepatocytes

Insulin and dexamethasone greatly stimulate the incorporation of 3H-orotic acid into RNA. Such a stimulation is associated to an increase in the uptake of the labelled precursor into the acid soluble fraction as well as in the the specific radioactivity of the nucleoside plus nucleotide pool suggesting that hormone supplementation does not affect RNA synthesis by cultured cells. The lack of effect of insulin and dexamethasone on the level of total RNA polymerase activity in nuclei isolated from cultured hepatocytes is in line with this assumption. The hormone stimulated uptake of orotic acid is dependent on protein synthesis since it is completely abolished by cycloheximide.     

EFFECT OF ACTINOMYCIN-D ON LABELLED MATERIAL IN THE RETINA AND OPTIC TECTUM OF GOLDFISH AFTER INTRAOCULAR INJECTION OF TRITIATED RNA PRECURSORS

—After injection of [³H]guanosine or [³H]uridine into the eye of goldfish, labelled acid-soluble radioactivity and RNA appeared in the contralateral optic tectum. When 0·1 μg actinomycin-D was injected into the eye 4 h before the precursor, the labelled RNA in the retina by 18 h after the injection was only 23 per cent of normal, but the acid-soluble radioactivity in the retina and the small amount of labelled acid-soluble material conveyed to the tectum were not significantly affected; by 15–20 days after the injection the acid-soluble radioactivity in the retina was reduced and the amount of labelled material conveyed to the tectum, including both RNA and acid-soluble fractions, was less than normal. When the actinomycin was injected at various times before or after the precursor and measurements were made 6 days later, it was found that the amount of labelled RNA conveyed to the tectum was maximally decreased if the inhibitor was given simultaneously with or up to 4 h before the precursor, whereas the amount of RNA was normal if the incorporation of the precursor had been allowed to proceed for 12 h before the inhibitor was given. This result would be consistent with the view that much of the RNA conveyed to the tectum had been synthesized in the retina within 12 h of the injection of the precursor, and had then presumably been axonally transported in the optic nerve to the tectum. However, since the acid-soluble material conveyed to the tectum was also reduced as a result of the actinomycin treatment, the results of these experiments with actinomycin do not unequivocally rule out the possibility that the RNA appearing in the tectum had been locally synthesized from the axonally transported acid-soluble material. In the retina, both the labelled RNA and acid-soluble fractions were reduced, to about 15 and 60 per cent of normal, respectively, without any relationship to the time between the injection of inhibitor and precursor. The discrepancy between the effects of the labelling of the retina and the labelling of material conveyed to the tectum could be correlated with the fact that the actinomycin caused severe damage to the retinal receptor cells, while leaving the ganglion cells relatively intact. The more pronounced effect of actinomycin on the receptor cells could in turn be correlated with the fact that these cells had a higher rate of RNA synthesis than the ganglion cells. This was demonstrated autoradiographically by the higher rate of incorporation of [³H]uridine into the receptor cells. Intracranial injection of actinomycin did not affect significantly the amount of labelled RNA conveyed to the tectum, which would argue against the local synthesis of this RNA. It is not certain, however, that the actinomycin penetrated deeply enough into the tectum to be effective.     

Studies on distribution and metabolism of valproate in rat brain,liver, and kidney

Time-course studies on the distribution and metabolism of valproate (VPA) in rat brain, liver, and kidney, after intraperitoneal injection of a mixture of [¹⁴C]VPA and [³H]VPA, showed that: (1) maximal amount of radioactivity in the various tissues was observed after 30 min from the time the drug was administered; (2) at 30 min the distribution of labeled VPA in brain, liver, and kidney was 17%, 64%, and 19% of the total radioactivity, respectively; (3) at 24 hr more than 97% of the total radioactivity was lost from the tissues and the¹⁴C/³H ratios increased significantly with time. Studies on the regional distribution of the drug showed that it is relatively homogeneously distributed. Studies on the subcellular distribution of the drug showed that it is associated mostly with the soluble and mitochondrial fractions, with little radioactivity in the myelin and synaptosomal fractions. Radiochromatography of VPA metabolites in perchloric acid extracts from brain, liver, and kidney revealed the presence of four metabolites. VPA was not incorporated into phospholipids of the neuronal membranes. Furthermore, it had no significant effects on Mg²⁺-ATPase and (Na⁺+K⁺)-ATPase in synaptosomes and microsomes obtained either from control or from rats injected with VPA. It was concluded that this antiepileptic drug does not appear to act through its incorporation into neuronal membrane or through its action on the Na⁺ pump.Contribution No. 0601 from the Department of Cell and Molecular Biology, the Medical College of Georgia, Augusta, Georgia 30912.     

Anabolism versus catabolism of [5-3H]uridine and its relationship to ribonucleic acid labelling in mouse liver after partial hepatectomy

The balance between anabolism and catabolism of [5-(3)H]uridine was studied in the mouse after partial hepatectomy. Labelling of RNA and UDP-glucose was determined and evaluated in relation to changes in the specific radioactivity of UTP. The amounts of labelled catabolic products of uridine were increased several-fold in liver and blood after partial hepatectomy. The specific radioactivity of RNA decreased to about 60% of the control value at 6h and was in the same range as that of control liver at 24h after operation. Decreased labelling of RNA and UDP-glucose was attributable to decreased specific radioactivity of UTP. No changes in the size of the UTP pool or in the balance between uridine anabolism and catabolism were found that could explain the decreased specific radioactivity of UTP. Rather, the alterations in the labelling of this metabolite induced by the partial hepatectomy may be related to decreased phosphorylating capacity in the liver cells and/or dilution of the labelled precursor in an expanded uridine pool. The enhanced amounts of uridine catabolic products in liver and blood were probably a consequence of accumulation and altered incorporation of the metabolites from the blood into the liver cells. Despite the increased amounts of labelled catabolic products and the decreased labelling of RNA, the results reported here actually suggest decreased uridine catabolism and slightly increased RNA synthesis in mouse liver after partial hepatectomy. The results stress the importance of proper controls in determination of nucleic acid synthesis and in metabolic studies by use of labelled precursors.     

Metabolism of uridine and determination of liver ribonucleic acid synthesis in developing and adult mice

The metabolism of [5-3H]uridine and the incorporation of the precursor into liver RNA was studied in developing (13-day-old) and adult (45-day-old) mice. Different time-courses of labelling and increased amounts of labelled catabolic products of uridine were found in liver and blood of developing mice compared with adult animals. This is suggested to be a consequence of enlarged metabolite pools resulting from a lower total amount of uracil-degrading enzymes in the developing mice. The labelling of the uracil nucleotides was decreased in the developing liver. However, in spite of a lower specific radioactivity of UTP, the RNA-specific radioactivity of developing liver was increased compared with adult liver. Also the labelling of liver RNA with [6-14C]orotic acid was found to be increased in developing mice, thus indicating a higher rate of RNA synthesis in these animals. A more pronounced difference in liver RNA labelling between the developing and the adult mice obtained with the use of [14C]orotic acid than with [3H]uridine may suggest that the de novo pathway, relative to the salvage pathways, is more important in developing than in adult liver.     

Dégradation d'acides aminés à l'interface eau-sédiment réconstitué d'une lagune méditerranéenne (Golfe du Lion)

Degradation of Amino Acids at a Simulated Water-sediment Interface of a Mediterranean Lagoon Environment, Golfe du Lion The degradation of two ¹⁴C and ³H labelled amino acids at the simulated water-sediment interface of a Mediterranean lagoon was studied. The four day experiments included the respiratory activity measurement and the study of incorporation processes of the radioactivity in specific organic fractions of the ¹⁴C arginine. After active mineralization occurred during the first day, the radioactivity in the acid-soluble fraction decreased and polycondensed products were progressively incorporated. The radioactivity of the ³H lysine was included mainly in the acid soluble fraction. The nature of the substrate, and the reducing conditions in the environment affect the radioactivity distribution in the organic fractions. The identification of amino acids in the acid-soluble and base soluble fractions shows that the labelled arginine and lysine and other amino acids in the acid soluble and base soluble fractions shows that the labelled arginine and lysine released from the initial compounds are important quantitatively.     

Methylation of newly synthesized ribonucleic acid by isolated rat liver nuclei. Characterization of the ribonucleic acid synthesized by nuclei from starved animals

1. Nuclei from rat liver incubated with S-adenosyl[methyl-(14)C]methionine incorporated radioactivity into RNA and into lipid and protein. 2. All of the labelled RNA was extracted from the nuclei with trichloroacetic acid at 90 degrees C. 3. The [(14)C]methyl-group incorporation into the hot-trichloroacetic acid extract was 30% inhibited by the addition of actinomycin D (100mug/mg of DNA) or by the omission of CTP, GTP and UTP. 4. Assuming that the main substrate for this triphosphate-dependent methylation was newly synthesized precursor rRNA containing one methyl group/30 uridylate residues, it was calculated that approx. 60% of the [(14)C]UMP incorporated under similar conditions represented precursor rRNA synthesis. 5. In agreement with this, low concentrations of actinomycin D (approx. 1mug/mg of DNA) sufficient to abolish the triphosphate-dependent incorporation of [(14)C]methyl group inhibited 68% of the [(14)C]UMP incorporation. 6. The incorporation of [(14)C]UMP by nuclei from starved animals decreased progressively with increasing periods of starvation, whereas the triphosphate-dependent [(14)C]methyl-group incorporation was not further decreased after 1 day of starvation. 7. This suggests that precursor rRNA synthesis decreased within 1 day whereas other species of RNA were affected only after longer periods of starvation.     

Estimation of regional circulation in rats: results obtained by using 15 and 50 micron diameter microspheres and rubidium]

Radioactive microspheres, 15 or 50 micron in diameter, were used to estimate the distrubtion of cardiac output and the degree of shunting of microspheres through the systemic and pulmonary circulations in anaesthetized rats. Extraction of 15 micron spheres by the pulmonary capillaries was nearly 100% and the amounts of microspheres per gram of lung tissue were not significantly different in the various lobes of lung. After injection into the left ventricle, the proportion of microspheres shunted to the lungs was almost identical using 15 or 50 micron spheres. Similar results were observed after injection into the internal of external carotid artery. The distribution of cardiac output showed a significant difference between 15 and 50 micron spheres, the proportion of 50 micron spheres found in the stomach being higher, which suggests the existence in this organ of arteriovenous shunts larger than 15 micron. The rubidium method yielded higher fractions of cardiac output in the liver (hepatic artery), lung and skin whereas the microspheres distribution to the heart, spleen and digestive tract exceeded that of rubidium. The origins of these differences are discussed.     

Effects of Growth Regulators on Ribonucleic Acid Metabolism of Barley Leaf Segments

Illumination or gibberellic acid treatment of etiolated barley leaf segments stimulates unrolling and results in an increased level of RNA. In contrast, segments treated with abscisic acid do not unroll and have a lower content of RNA. Gibberellic acid treatment enhanced the capacity of segments to incorporate radioactivity from ³²P-orthophosphate into all the RNA components detected by gel electrophoresis; abscisic acid greatly restricted the incorporation of precursors into all the RNA fractions. In conjunction with a changed capacity for RNA synthesis it was observed that abscisic acid-treated segments had a lowered soluble DNA-dependent RNA polymerase level in comparison to gibberellic acid-treated or illuminated segments. However, the influence of growth regulators on RNA polymerase content of the segments was associated with general effects on protein level rather than a specific effect on the synthesis of polymerase enzyme.     

Synthesis of beta-glucans in Prototheca zopfii. Evidence for the existence of a glycoprotein primer

Membrane preparations from the non-photosynthetic alga Prototheca zopfii incorporate glucose from UDP-[3H]glucose into the trichloroacetic-acid-insoluble fraction and the polysaccharides insoluble in hot alkali. Time course and pulse-chase experiments indicate that the acid-insoluble fraction was a precursor of the alkali-insoluble fraction. Isolation of 3H-labeled membrane or soluble fraction showed that only membrane fractions were able to transfer radioactivity into polysaccharides. Treatment of glucosylated membranes with trypsin or cellulase only partially affect their transfer ability, indicating that the precursor was internalized in vesicles. Analysis of the in vitro synthesized polysaccharides by enzymatic and acid hydrolysis showed that glucose and cellobiose were present as radioactive sugars. Permethylation of the polysaccharide indicates that 80% of the glucose was beta-1,4-bonded with 20% in beta-1,3-linkages. This polysaccharide was found to be identical with the cell-wall beta-glucan obtained in vivo [Rivas, L.A. & Pont Lezica, R. (1978) Planta (Berl.) 165, 348-353].     

Inhibition of RNA- and DNA-synthesis by citrinin and its effects on DNA precursor-metabolism in V79-E cells.

1. The RNA synthesis of V79-E cells was inhibited by the mycotoxin citrinin time- and concentration-dependently. 2. Among the different RNA species mainly the rRNA synthesis was found to be inhibited by 200 microM citrinin. 3. At different precursor concentrations DNA synthesis was inhibited by citrinin after 30 min at least whereas labelling of the acid soluble fractions was found to be 3-fold higher than in untreated cells. 4. Remarkable perturbation of the DNA precursor metabolism, including release of precursor into the medium, was found to occur during citrinin treatment.