Incorporation of thymidine-3H into the activated sea urchin egg with special reference to subthreshold stimulation with an activating reagent

It was ascertained that thymidine-³H added exogenously is incorporated periodically into the nucleus of the fertilized and the artificially activated eggs of Pseudocentrotus, Temnopleurus and Anthocidaris. Eggs stimulated insufficiently with butyric acid did not show any enhancement of incorporation of thymidine-³H. However, repetition of an insufficient stimulation induced incorporation of thymidine to some degree, although no visible cortical changes occurred.     

The stimulation of amino acid incorporation into the protein of unfertilized sea urchin eggs by gamma-irradiation

Control and γ-irradiated sea urchin (Arbacia punctulata) eggs were incubated with H³-leucine. No incorporation of the amino acid into the proteins of most unirradiated eggs was detectable by autoradiography. All irradiated eggs incorporated the amino acid and the amount of autoradiographic labelling varied greatly from cell to cell.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain

The incorporation of [³H]phenylalanine, [³H]tyrosine, and [³H]tryptophan into protein and amino acyl–tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl–tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl–tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl–tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both K_m and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than K_m.     

Alterations in Glial Cell Metabolism During Recovery from Chronic Osmotic Stress

NMR spectroscopy of F98 glioma cell extracts showed that chronic hypertonic conditions largely increased the intracellular content of small, osmotically active molecules. Moreover, hypertonic stress decreased the incorporation of ¹³C-labeled amino acids into the cellular proteins albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. Reincubation with isotonic medium restored almost completely the control values for the cytosolic metabolites but not for amino acid incorporation into the protein. An increased amount of ¹³C label was found in the phospholipids, which indicates stimulation of membrane synthesis processes due to the recovery-induced cell swelling. On the other hand, chronic hypotonic conditions largely decreased the steady state concentration and synthesis of small, cytosolic molecules, whereas the effect on the incorporation of ¹³C-labeled amino acids into the cellular proteins was variable. Reincubation with isotonic medium partially restored the depressed cytosolic metabolite content and also the incorporation of labeled amino acids into cellular protein, but induced an inhibition of phospholipid synthesis. The results verify that readaptation of glial cell metabolism during recovery from chronic osmotic stress is impaired or at least seriously retarded.     

Properties of RNA fractions from nuclei of brain cells which stimulate incorporation of amino acids by brain ribosomes

Abstract— The properties of RNA fractions from nuclei of brain cells which were capable of stimulating amino acid incorporation into proteins of an homologous ribosomal system were investigated. RNA was routinely prepared from crude nuclear preparations of rat brain by a method which involved treatment with sodium dodecyl sulphate and phenol at 65°. The capacity of this preparation to stimulate incorporation of radioactivity from a mixture of 15 l -[¹⁴C]amino acids was greatly enhanced by preliminary incubation of the ribosomal system from brain for 5–20 min. The response was markedly dependent upon the concentrations of ribosomes and of the pH 5 fraction. The optimal level of Mg²⁺ for basal incorporation of amino acids into protein was 8 mm ; however, incorporation in the presence of nuclear RNA was greater at higher concentrations of Mg²⁺. The response to nuclear RNA was also enhanced as the K⁺ concentration was increased from 25 to 100 mm . The stimulatory effect of nuclear RNA on incorporation of l -[¹²C]eucine was either unaltered or depressed by addition of a mixture of 19 l -[¹²C]amino acids each at concentrations, of 10^?8, 10^?2, or 10^?1 mm . Under appropriate conditions of incubation, basal rates of incorporation and rates of incorporation stimulated by nuclear RNA were linear for 30 min. The response was proportional to the concentration of nuclear RNA between 34 and 136 μg. RNA prepared from ribosomes of rat brain essentially failed to stimulate incorporation of amino acids over this range of concentrations. Fractionation of nuclear RNA by centrifugation in sucrose density gradients revealed that 75 per cent of the stimulatory activity was in the fraction which sedimented below 12 S and contained about 25 per cent of the total RNA. Most of the remaining activity was in the 18 S region. Less than 5 per cent of the RNA in the lightest fraction (< 12 S) exhibited amino acid-acceptor activity, The stimulatory action of nuclear RNA on incorporation of amino acids was readily destroyed by mild treatment with pancreatic ribonuclease, whereas amino acid-acceptor activity was relatively resistant to this treatment. The results suggest that the brain may contain low molecular weight RNA with properties of messenger RNA.     

AMINO ACID DISTRIBUTION AND INCORPORATION INTO PROTEINS IN ISOLATED, ELECTRICALLY-STIMULATED CEREBRAL TISSUES

—The uptake of radioactive amino acid by incubated cerebral cortex slices is found to be a first order process. Incorporation of the radioactive amino acid into tissue protein is from a precursor pool that has first equilibrated with the intracellular endogenous free amino acids. Ways of calculating the amino acid incorporation in molar quantities from the observed incorporation of radioactivity are discussed, and it is concluded that the specific radioactivity of the intracellular acid-soluble fraction is the best basis for such estimates. The in vitro incorporation of leucine into tissue protein is estimated to be approximately 1±2 mμnol/mg protein/h, and of valine 0±4 mμmol/mg protein/h. Addition of free amino acids to the media had little or no effect on the calculated rates of incorporation. On incubation for 1 h the total free valine in tissue and medium increased by 0±43 μmol/g and leucine increased by 0±55 μmol/g. Estimates of amino acid incorporation based on the specific radioactivity of the media amino acids can give misleading results if this considerable release of amino acids into the medium is not taken into account. Electrical stimulation of neocortical slices with a variety of types of pulses was either without effect or decreased incorporation into portein. The decrease could not be directly correlated with changes in tissue K⁺ nor with the utilization of ATP. Mild, local stimulation of the lateral olfactory tract of piriform cortex slices was without effect on tissue phosphocreatine, K⁺ or amino acid incorporation.     

Measurement of the incorporation rates of four amino acids into proteins for estimating bacterial production

In aquatic ecosystems, [³H]thymidine incorporation into bacterial DNA and [³H]leucine incorporation into proteins are usually used to estimate bacterial production. The incorporation rates of four amino acids (leucine, tyrosine, lysine, alanine) into proteins of bacteria were measured in parallel on natural freshwater samples from the basin of the river Meuse (Belgium). Comparison of the incorporation into proteins and into the total macromolecular fraction showed that these different amino acids were incorporated at more than 90% into proteins. From incorporation measurements at four subsaturated concentrations (range, 2–77 nm), the maximum incorporation rates were determined. Strong correlations (r > 0.91 for all the calculated correlations) were found between the maximum incorporation rates of the different tested amino acids over a range of two orders of magnitude of bacterial activity. Bacterial production estimates were calculated using theoretical and experimental conversion factors. The productions calculated from the incorporation rates of the four amino acids were in good concordance, especially when the experimental conversion factors were used (slope range, 0.91–1.11, and r > 0.91). This study suggests that the incorporation of various amino acids into proteins can be used to estimate bacterial production.     

Partial activation of sea-urchin eggs by bonellin

We here describe further studies on the action of bonellin on sea-urchin eggs. Bonellin brings about Some of the changes that are known to occur in the egg upon fertilization. In particular, it appears to cause the increased rate of incorporation of amino acids into proteins, the increase of the voltage noise, and the exocytosis of some of the cortical granules. A comparison with the effect of ammonia is discussed.     

Studies on the invitro formation of erythropoietin in sheep kidney medulla and the effect of cobalt thereon

Tissue slices of sheep kidney medulla catalyzed the incorporation of ¹⁴C-phenylalanine into protein when they were incubated in presence of twenty naturally occurring amino acids. Cobalt salt stimulated the incorporation significantly. Although slices from cortical or juxtaglomerular region of kidney catalyzed the incorporation of radioamino acid into proteins, yet no such stimulation of incorporation by cobalt salt was observed in those cases. The radioamino acid incorporated protein, produced under the influence of kidney medullary tissues in presence or absence of cobalt salt, was identified to be erythropoietin by its thermal stability, electrophoretic mobility on polyacrylamide gel and finally precipitation reaction with antierythropoietin.     

Relationship between leaf development and primary photosynthetic products in the C4 plant Portulaca oleracea L.

Summary The photosynthetic products of Portulaca oleracea differ greatly depending on leaf age and length of exposure to ¹⁴CO₂. Mature leaves of P. oleracea fix ¹⁴CO₂ primarily into organic and amino acids during a 10-s exposure period. Less than 2% of the ¹⁴CO₂ fixed appears in phosphorylated compounds. In contrast, incorporation into amino acids can account for over 60% of the total ¹⁴CO₂ fixed by young leaves in an equal time period, and incorporation into alanine alone can account for up to one half of this amount. Senescent leaves display a quantitative shift of primary products toward phosphorylated compounds with a concomitant reduction of the label residing in malate and asparate. About 8 times more phosphoglyceric acid is produced in senescent leaves than in mature leaves. The aspartate/ malate ratio is not constant and depends on the length of time the leaves are exposed to ¹⁴CO₂ and the age of the leaves under study. It appears as if the stage of leaf development is one of the most important factors determining the operation of a particular enzyme system in C₄ plants.     

Schistosoma mansoni: utilization of exogenous metabolites by eggs in vitro

Schistosoma mansoni eggs were isolated from the livers of mice infected for 8 wk and were purified by a series of settling and sieving procedures. Aliquots of eggs were suspended in saline with added Eagle's minimal essential medium supplemented with NaHCO₃, glutamine, penicillin, and streptomycin to which a variety of radioisotope labelled metabolites were added. The uptake and utilization of tritiated thymidine demonstrated a high rate of DNA synthesis, particularly in the more immature eggs studied. RNA synthesis as shown with tritiated uridine was also significant. Large amounts of ¹⁴C-labelled isoleucine and arginine were incorporated into protein. Little glycolytic activity was demonstrated on prolonged incubation with ¹⁴C-labelled glucose. A high rate of catabolism of amino acids to CO₂ was observed, as was a very high rate of acetate metabolism. Degradation of radiolabelled glutamate after incubation of eggs with ¹⁴C acetate revealed labelling consistent with metabolism via the Krebs cycle. Thus, Schistosoma mansoni eggs utilize a wide variety of exogenous metabolites. They show active DNA and RNA metabolism, incorporation of amino acids into protein, and intermediary metabolism characterized by a low rate of glycolysis and an active Krebs cycle.     

A self-inhibitor of protein synthesis in the conidia of Glomerella cingulata

Summary A diffusible self-inhibitor of germination of conidia of Glomerella cingulata appears to act as a regulator of protein synthesis. Both uptake of labeled amino acids and their incorporation into protein are reduced by the inhibitor or by crowding. Compared to conidia incubated without self-inhibitor, conidia incubated with self-inhibitor incorporated no labeled amino acids into protein in the first hour and 80% less in 6h. Thoroughly washed conidia were more permeable to amino acids and incorporated 6 times more precursor into proteins than unwashed conidia. At high density in nutrient medium, conidia of G. cingulata preferentially form secondary conidia instead of germ tubes and a mycelium. This inhibition of germination of conidia and regulation of development is mimicked by exposing them to an auto-inhibitor extracted from used culture medium and conidial washings. Germination of conidia of G. cingulata involves two steps, an initial step of 5 h duration which continues unaffected by crowing (1.7×10⁸/ml) and a subsequent 2 h step which conidia do not take unless they are sufficiently diluted. It is this step for which protein synthesis may be required.Non-Standard Abbreviations CHM cyloheximide - NM Neurospora minimal medium - psi pound per square inch - RPH reconstituted algal protein hydrolysate - TCA trichloroacetic acid     

The effect of 2,4-dichlorophenoxyacetic acid upon the metabolism and composition of soybean hypocotyl mitochondria

Summary Dark-grown, 3-day-old soybean seedlings were sprayed with 1 mM 2,4-dichlorophenoxyacetic acid 24 hours before harvest. Mitochondria from 2,4-D-treated lower hypocotyls were found to be larger and showed greater incorporation in vivo, of amino acids into protein and phosphate into phospholipids and RNA, than mitochondria from untreated tissue. Mitochondria isolated from 2,4-D-treated hypocotyls showed an enhanced energy-dependent incorporation of amino acids into protein, although the incorporation of nucleoside triphosphates into the RNA of isolated mitochondria was not affected. No effect of 2,4-D, applied in vitro, was noted, and no enhancement of mitochondrial respiratory efficiency followed auxin treatment. A method of isolating mitochondria with a very low level of bacterial contamination is reported.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

AMINO ACID INCORPORATION INTO PROTEIN IN NEURONAL CELL BODY AND NEUROPIL FRACTIONS IN VITRO

Abstract—

• 1 The incorporation of radioactivity from [³H]lysine into acid-insoluble material in vitro in mixed cell suspensions and isolated neuronal and neuropil fractions has been followed.
• 2 In the mixed cell suspension, incorporation was linear in fresh preparations for up to 60 min. In cold stored preparations, incorporation began to fall off after 30 min. Incorporation, at 4-11 pmol/mg protein/h, was intermediate between that in the tissue slice and in a cell-free preparation. Addition of a mixture of non-labelled amino acids at 1 mM produced a 30-40 per cent inhibition of incorporation. Molar rates of incorporation of glutamate and tryptophan into the mixed cell fractions were respectively 73 and 1-4 times that of lysine.
• 3 Only 8 per cent of the incorporated radioactivity could be recovered in soluble as opposed to particulate material. After hydrolysis of the protein, followed by paper chromatography and autoradiography, radioactivity was detected only in the position corresponding to lysine.
• 4 Incorporation in the separated cell fractions was not markedly reduced by the centri-fugation procedure. Incorporation into the neuronal fraction was 2-2-6 times that into the neuropil fraction, depending on the amino acid used. Incorporation into both was reduced by some 40 per cent by addition of an amino acid mixture.
• 5 Comparison of in vivo and in vitro data suggest that the differences in rate of incorporation are characteristic of neurons and neuropil in situ.

     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

Experimental alcoholism in rats: protein synthesis in subcellular fractions from cerebellum, cerebral cortex and liver after long term treatment

Abstract— The incorporation in vivo of [³H]leucine into protein from subcellular fractions was determined in rats chronically ingesting 15 per cent ethanol for 8 months. Mitochondrial, microsomal and cell sap fractions from cerebellum, cortex cerebri and liver were investigated. The results showed a minor over-all depression of protein synthesis in cerebellum and cortex cerebri and a slight stimulation of the incorporation of leucine into protein from liver subcellular fractions. If the animals were abstinent 24 h before injection of the isotope, the incorporation of labelled amino acids into protein was markedly increased in cerebellum and cerebral cortex but not in liver.     

Autoradiographic study of protein and RNA formation during early development of Drosophila eggs

Permeabilized eggs of Drosophila melanogaster were incubated in tritiated uridine, valine, and phenylalanine. The uptake and incorporation into TCA-insoluble material were measured by scintillation counting. There was very little incorporation of uridine before the blastoderm stage. At the blastoderm stage, the egg took up 2.4 pmoles/hr of uridine and incorporated 0.13 pmoles into RNA (assuming no dilution of specific activity of the precursor). The uptake of amino acids varied with the age of the embryo; virgin eggs synthesized about as much protein as fertilized eggs. Autoradiography of eggs incubated in uridine showed a lack of RNA synthesis in nuclei until the start of the blastoderm formation. The small amount of uridine incorporation before this stage was due to mitochondria. Incorporation of amino acids was uniform in the cytoplasm until the blastoderm; there was no incorporation by yolk granules. Regional difference in labeling appeared during gastrulation. The pole cells did not form RNA during the blastoderm stage, formation started during gastrulation. Protein labeling of the pole cells, on the contrary, was very strong in the blastoderm and early gastrula. These results indicate that the expression of zygotic genome before the blastoderm stage is unlikely.     

Atypical incorporation of single amino acids into myelin proteins.

—Purified myelin incorporated l -[¹⁴C]leucine and l -[¹⁴C]lysine into myelin proteins in an enzymatic process similar to that of renal brush border membranes. The system was not inhibited by cycloheximide or puromycin or by pretreatment with ribonuclease; the reaction was inhibited by cetophenicol. ATP was an effector, shifting the optimal pH from 7.2 to 8.3. In the presence of ATP, myelin was less dense in a sucrose gradient. Ammonia was released from the membrane during the incorporation of amino acids. Myelin preloaded with cold leucine did not incorporate [¹⁴C]leucine but did incorporate [¹⁴C]lysine; there was no cross inhibition between the two amino acids. The incorporation was into or onto proteins of the Wolfgram proteolipid fraction of myelin. The incorporation was of the high affinity type with a K_m of 10^?7m and was restricted to the natural amino acids.     

THE INFLUENCE OF INTRAVENTRICULARLY INJECTED AMINO ACID EXCITANTS ON THE LABELLING OF ENDOGENOUS BRAIN AMINO ACIDS FROM [U-14C]ACETATE IN NEMBUTALIZED MICE

Mice were anaesthetized with nembutal and the effects of intraventricularly injected excitant amino acids on [U-¹⁴C]acetate metabolism were investigated. The natural excitant amino acids, l -glutamate and l -aspartate, reduced the incorporation of ¹⁴C from [U-¹⁴C]acetate into glutamine, GAB A and possibly alanine. The synthetic excitant amino acid, N-methyl-d -aspartate caused a reduction in the incorporation of ¹⁴C from intraventricularly injected [U-¹⁴C]acetate into all of the brain amino acids labelled by [U-¹⁴C]acetate within 5 min. It is suggested that these effects may be due to changes in pool sizes of tricarboxylic cycle intermediates, to inhibition of acetyl-CoA formation, or both. Differences in the metabolic effects of the synthetic and natural excitants are interpreted in terms of the uptake of the natural amino acids into glutamine-forming pool(s) of glutamate metabolism.