The effects of electrical stimulation and ionic alterations on the metabolism of amino acids and proteins in excised superior cervical ganglia of the rat

Abstract— The effects of supramaximal electrical stimulation on the metabolism of amino acids and proteins in incubated superior cervical ganglia of the rat were studied by the use of a gas-liquid chromatographic (GLC) assay procedure. Stimulation at 5 Hz for 2 h caused an apparent increase in tissue levels of free amino acids, with alanine, serine, glycine, valine, threonine, isoleucine and aspartate (+ asparagine) most noticeably affected. The amino acid composition (partial) of the TCA-insoluble proteins of resting and stimulated ganglia was approximately the same after 60 min of incubation, but there was less TCA-insoluble protein in the stimulated ganglia. The addition of amino acids (at plasma concentrations) to the standard media had no apparent affect on the amino acid composition of this protein fraction. Stimulation for 0 , 5 h initially increased the efflux of alanine, valine, proline and ornithine into the incubation media but prolonged stimulation (for 4–0 h) decreased the efflux of alanine, serine, glycine and isoleucine and increased the efflux of lysine into the incubation media. The leakage of amino acids from the ganglia appeared to be a sodium-dependent process. The incorporation of ¹⁴C from [U-¹⁴C]glucose into glutamate (+ glutamine) and aspartate (+ asparagine) was greater in stimulated than in resting ganglia. However, the conversion of glutamate carbons from [U-¹⁴C]l -glutamate into aspartate was not affected by stimulation. Incorporation of ¹⁴C from [U-¹⁴C]glucose into glycine and serine was apparently not affected by stimulation during the 60 min of incubation. However, serine was the only amino acid which exhibited a higher specific radioactivity in stimulated ganglia than in resting ganglia incubated for 4 h in standard media. Lithium ions had the apparent specific effect of increasing the labelling with ¹⁴C from [U-¹⁴C]glucose into ornithine, and increasing the efflux and overall metabolism of serine in the ganglia. Incorporation of ¹⁴C from [U-¹⁴C]glucose into proteins was lower in the stimulated than in the resting ganglia if compensation was made for the higher radioactivity available in the total free amino acid pool of the stimulated ganglia. The rate of ¹⁴C incorporation from [U-¹⁴C]glutamate into the TCA-insoluble proteins of resting ganglia was greater when no other amino acids at concentrations approximating plasma levels were added to the bathing media; this rate was lower in stimulated than in resting ganglia.     

Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum

TMV inoculation is known to stimulate tyramine N-feruloyl-CoA transferase activity in Nicotiana tabacum cv Xanthi n.c. leaves during the hypersensitive reaction. When [2-¹⁴C]-tyramine is fed for 2 hr to TMV inoculated leaf discs or detached leaves, ca 1 % of the supplied radioactivity is integrated into cinnamoyl-, p-coumaroyl- and feruloyltyramine and up to 14 % is integrated into the cell wall residue. [2-¹⁴C]-tyramine can only be partially released from this residue by acid hydrolysis. After nitrobenzene oxidation, 97 % of the radioactivity found in the cell walls is made soluble but only 13 % is recovered in p-hydroxybenzaldehyde. Feruloyltyramine is very rapidly metabolised, ca 20 % of the administrated radioactivity is found after 2 hr feeding in unindentified methanoi soluble metabolites. Acid hydrolysis of the cell wall fraction, which hydrolyses the amide bond of feruloyltyramine, releases labelled tyramine, while radioactivity is still detected in the acid insoluble residue. Label from [¹⁴C]-feruloyltyramine is integrated into this residue more quickly than from free [2-¹⁴C]-tyramine.     

Evidence for synthesis in vitro of neutral lipids by isolated oocytes of Perinereis cultrifera (Annelida,Polychaeta). A biochemical and autoradiographic study

Summary

Isolated oocytes of Perinereis cultrifera have been incubated in culture media with added [³H]glycerol, [¹⁴C]butyric acid or [¹⁴C]oleic acid. The principal neutral lipid synthesized was triacylglycerol, although incorporation of radioactivity into other lipid categories (sterol, fatty acid, wax ester) was also observed. A more significant percentage of triacylglycerol was labelled after incubation with [³H]glycerol and [¹⁴C]oleic acid than with [¹⁴C]butyric acid. With this precursor, monoacylglycerol appears to be the class of lipid compartment which initially show the most radioactivity. Electron microscopic autoradiography has revealed that labelling after incorporation of glycerol was mainly localized on the lipid droplets but not on the yolk granules. A second metabolic pathway is represented by phospholipid membrane synthesis.     

A new radiochemical assay for argininosuccinase with purified [14C]argininosuccinate

A sensitive and simple radiochemical assay is described to measure argininosuccinase activity in crude tissue homogenates and cultured cells. The method depends on the use of argininosuccinate labeled uniformly with ¹⁴C in the six carbons of the arginine moiety. On incubation in the presence of excess arginase, the [U-¹⁴C]arginine formed is measured as the sum of radioactivity in [U-¹⁴C]ornithine and [¹⁴C]urea. Separation from the substrate is accomplished on a small Domex 1-acetate column eluted with 25 mm acetic acid; ornithine and urea emerge in the first few milliliters while unutilized substrate remains on the column. [¹⁴C]Argininosuccinate was synthesized enzymatically from l-[U-¹⁴C]arginine and fumarate and isolated and purified as the barium salt. Development of a new purification step has brought the amino acid to a purity of 97% as judged by chromatographic and barium analysis. With the present specific radioactivity, as little as 5 to 10 nmol of product can be accurately measured under kinetically optimum conditions.     

Evidence of accumulation of ceramides containing [14C] nervonic acid in the rat lung following injection of [14C] erucic acid

A mixture of albumin-bound [¹⁴C]erucate and [³H]oleate was injected into rats fed a stock pellet diet containing 4% by weight of lipid. Chylomicrons containing the same labelled fatty acids were also injected into rats fed diets containing 15% by weight of rapeseed oil (48% of erucic acid), canbra oil (< 5% of erucic acid) or ground nut oil (no erucic acid). Lung lipids were analyzed at various times after injection.In all cases, except in the rapeseed oil diet group, ¹⁴C radioactivity of lung ‘monoacylglycerol’ was ten times higher than ³H radioactivity. More than 85% of this ¹⁴C radioactivity was found in nervonic acid (24:1). It was shown by TLC and GLC analysis that 85–90% of the ¹⁴C radioactivity of this fraction was in ceramides (N-acyl-4-sphingenine).Ceramides containing [¹⁴C] nervonic acid disappeared from the lung with time and their incorporation with time into sphingomyelin was also observed. The absence of accumulation of ³H and ¹⁴C (18:1) in ceramides showed that oleic acid was not incorporated into sphingomyelin in the same way as nervonic acid.In the rapeseed oil diet group, there was no accumulation of ¹⁴C radioactivity in ceramides and conversion of erucic acid into nervonic acid was less, and into oleic acid more, than in other diet groups indicating a possible enzyme adaptation.     

Inhibition of conjugation and cell division in Tetrahymena pyriformis by tunicamycin: A possible requirement of glycoprotein synthesis for induction of conjugation

Tunicamycin, a glucosamine-containing antibiotic inhibited the conjugation process of Tetrahymena pyriformis. Sexual pairing was prevented completely when 1.5 μg/ml of tunicamycin was added to a mixture of the two mating types. Tunicamycin caused preferential inhibition of glycoprotein synthesis in Tetrahymena pyriformis. At 1.5 μg/ml and 6 μg/ml tunicamycin inhibited by 40% and 60% respectively [³H]-glucosamine incorporation into material precipitated by ethanol, while it did not affect [¹⁴C]-leucine incorporation. Cell division was also inhibited when the drug was added either to the regular growth medium or to the starvation medium.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.     

Adrenergic and cholinergic stimulation of arachidonate and phosphatidate metabolism in cultured astroglial cells

Primary cultures of neonatal rat brain polygonal astroglia, or cells of the C62B glioma line, were incubated with [1-¹⁴C]arachidonic acid (AA) in culture for 18 hr. In both culture systems, more than 80% of the added [1-¹⁴C]AA was taken up into cellular glycerolipids; less than 1% of the radioactivity in the cells was present in an unesterified form. When prelabeled C62B cells were stimulated with acetylcholine (ACh), there was a rapid accumulation of arachidonyl-phosphatidic acid (PA) accompanied by a liberation of [1-¹⁴C]AA. A variety of other neurotransmitters failed to activate this response in C62B glioma cells, primary astroglia generated PA and liberated [1-¹⁴C]AA in response to several neurotransmitters (i.e., ACh, norepinephrine, glutamate, and histamine) Treatment of astroglia with a combination of norephrine, ACh, and histamine resulted in a greater production of PA and free [1-¹⁴C]AA than did treatment with any one of these neurotransmitters alone. The results suggest that cultures of astroglia can respond to several different neurotransmitters with specific changes in AA and PA metabolism. Thus, a variety of neurotransmitters initiate cascades of lipid metabolism which may be of physiological significance in glial function.Special Issue dedicated to Prof. Eduardo De Robertis.     

Radioenzymatic determination of CMP-N-acetylsialic acid and free N-acetylsialic acid in biological material

Free N-acetylsialic acid (NeuNAc) and CMP-N-acetylsialic acids (CMP-NeuNAc) are extracted from freeze-clamped or liquid nitrogen-frozen biological material by sequential extraction with cold acetone and acetone/water. [¹⁴C]NeuNAc and [¹⁴C]CMP-NeuNAc (20,000 dpm each) are added to the frozen material to correct for small losses occurring during the subsequent steps. NeuNAc and CMP-NeuNAc are separated by anion-exchange chromatography. CMP-NeuNAc is hydrolyzed with formic acid and again chromatographed on an ion-exchange column. The NeuNAc-containing fractions (representing free NeuNAc and CMP-NeuNAc) are converted to [¹⁴C]CMP-NeuNAc in the presence of [¹⁴C]CTP and CMP-NeuNAc synthetase. [¹⁴C]CMP-NeuNAc is separated by paper chromatography and the radioactivity measured by liquid scintillation counting. The amount of NeuNAc is calculated from a calibration curve obtained with NeuNAc standards. The small amounts of [¹⁴C]NeuNAc and [¹⁴C]CMP-NeuNAc added initially do not interfere with the final assay. The method gives reliable values down to 50 pmol/assay, but the sensitivity can be easily increased by a factor of 10. Recoveries, with NeuNAc and CMP-NeuNAc added to biological extracts, were 98.3 and 98.5% for NeuNAc and CMP-NeuNAc, respectively. With this method values of 61.2 ± 12.8 and 24.4 ± 5.2 nmol/g wet wt were found in rat liver for free NeuNAc and CMP-NeuNAc, respectively. Values for free NeuNAc found in human blood plasma were 600 ± 476 and 373 ± 180 pmol/g plasma for healthy persons and patients with breast cancer, respectively. Free CMP-NeuNAc could not be found in plasma.     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-¹⁴C]linoleic acid or [5-¹⁴C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [¹⁴C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [¹⁴C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-¹⁴C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species.     

Enzymatic methods for the determination of L-serine concentration and L-[14C]serine specific radioactivity in blood plasma

Methods are desribed for the use of l-serine dehydratase purified from Clostridium acidiurici for the determination of l-serine concentration and l[¹⁴C]serine specific radioactivity in sheep plasma. A spectrophotometric assay using this enzyme accurately measured the concentration of l-serine in standard solutions and in a commercially available mixture of amino acids and related compounds. This assay was shown to be suitable for measurement of plasma l-serine concentrations in excess of 30 μm. The reverse isotope dilution method was used for plasma l-[¹⁴C]serine specific radioactivity measurements. Carrier l-serine was added to plasma and separated from neutral and anionic compounds using ion-exchange chromatography. The l-serine was then converted to pyruvate with l-serine dehydratase and this was purified as the phenylhydrazone derivative. After recrystallization, drying and weighing, the derivative was assayed for radioactivity. The accuracy of this method was verified by adding l-[U-¹⁴C]serine to plasma and comparing the experimentally determined l-[¹⁴C]serine specific radioactivity with the calculated value. The method yielded a value which was 98.6 ± 0.8% (5) of this calculated value.     

Biosynthesis of triterpenoid sapogenols in soybean and alfalfa seedlings

By incubation of germinating soybeans with mevalonate-[2-¹⁴C] (MVA), radioactivity was incorporated into four sapogenols which were identified by TLC. Unequivocal evidence for the identity of three of the four sapogenols was provided by co-crystallization to constant specific radioactivity. The partition of incorporated radioactivity into lipid- and water-soluble fractions and the pattern of radioactivity of individual sapogenols varied with the mode of administering labeled substrates to soybean seedlings, such as incubation of germinating soybeans with MVA-[2-¹⁴C], immersion of roots into MVA-[2-¹⁴C] or foliar application of squalene-[¹⁴C]. When alfalfa seedlings were incubated with MVA-[2-¹⁴C], about two-thirds of the radioactivity incorporated into the sapogenols was associated with medicagenic acid.     

Cytokinin biosynthesis in crown-gall tissue ofVinca rosea

When care was taken to minimise the effects of phosphatase activity during extraction ofVinca rosea crown-gall tumour tissue, a large proportion of extractable cytolinin activity was present in the nucleotide fraction. Analysis using ion-exchange chromatography followed by enzymic or chemical degradation and subsequent identification of the biologically active material indicated that this activity was due to zeatin riboside 5′-monophosphate. This was also the major radiolabelled cytokinin formed when this tissue was supplied with [¹⁴C]adenine. The incorporation of radioactivity from [¹⁴C]adenosine into free cytokinins was also shown, but no incorporation of radioactivity was found when [³H]mevalonic acid lactone was supplied to this tissue under the same conditions. In parallel experiments using normal stem callus tissue ofV. rosea, no incorporation of [¹⁴C]adenine into free cytokinins was observed. The significance of these results is discussed in relation to a possible transfer-RNA-independent pathway of cytokinin biosynthesis, operating primarily at the mononucleotide level.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.     

Preparation, Characterization, and Microbial Degradation of Specifically Radiolabeled [14C]Lignocelluloses from Marine and Freshwater Macrophytes

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [¹⁴C]phenylalanine, [¹⁴C]tyrosine, and [¹⁴C]cinnamic acid as precursors. Specifically radiolabeled [¹⁴C-polysaccharide]lignocelluloses were prepared by using [¹⁴C]glucose as precursor. The rates of microbial degradation varied among [¹⁴C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [¹⁴C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [¹⁴C]phenylalanine and [¹⁴C]tyrosine were found associated with protein, although very little (3%) radioactivity from [¹⁴C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [¹⁴C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [¹⁴C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to ¹⁴CO₂; during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.     

The metabolism of [3−3H]oleanolic acid-3-O-mono-[14C]glucoside in isolated cells from Calendula officinalis leaves

The radioactive precursor, [3?³H]oleanolic acid-3-O-mono-[¹⁴C]glucoside was administrated to isolated cells obtained from the leaves of Calendula officinalis. The radioactivity of the precursor was incorporated into fractions containing free oleanolic acid, individual glucosides, glucuronide F and other glucuronides. The ratio of ³H: ¹⁴C radioactivity in these fractions indicated that glucosides were formed in a process involving direct glycosylation of the precursor, whereas the glucuronides were formed from oleanolic acid released by hydrolysis of the precursor. Dynamics curves showed that glucoside II formed by direct glycosylation of the precursor was intensively transformed to other derivatives.     

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward. During training and incorporation for 20-30 min, both [³H]leucine and [1-¹⁴C]leucine underwent a significant increase in catabolism, resulting in a decreased radioactivity in the free amino acids. [2-²H]Methionine underwent a rapid loss of isotope, so that 90% of the radioactivity was in the form of tritiated water at the end of training, and this phenomenon masked any possible effect of training. The brain uptake of [³⁵S]methionine increased during the training, resulting in an increased radioactivity in the proteins. Uptake of [³H]lysine increased slightly during training only after 1 h incorporation and not after 20 or 30 min, as judged from a time course of radioactivity in the free amino acids. Incorporation into nuclear proteins increased selectively during 20 min, and into nuclear and cytosol proteins after 60 min incorporations. It is concluded that changes in the observed rate of incorporation of a precursor into brain subcellular proteins under the influence of behaviour might be the result of changes in precursor catabolism or uptake, or both, and that each amino acid behaves in a different way. Even the same amino acid gives different results depending on the isotope and its position in the amino acid.     

ENHANCED CEREBRAL PROTEIN SYNTHESIS IN DEVELOPING HYPOTHYROID RATS: EVIDENCE FOR DELAYED MATURATION

(1) Neonatal hypothyroidism resulted in a 40% increase in the incorporation of [¹⁴C]leucine into protein by cerebral cortical slices from 25-day-old rats. The uptake of the [¹⁴C]-labelled amino acid into the acid-soluble free amino acid pool was similar in hypothyroid and control groups which excluded the possibility that transport differences contributed to the observed differences in incorporation. (2) The conversion of [¹⁴C]leucine in the free amino acid pool to other metabolites was substantially greater in the hypothyroid state compared to euthyroid controls. (3) The correction of the incorporation data for radioactivity associated with [¹⁴C]leucine in the precursor pool, provided an estimate of cerebral protein synthetic rate which was markedly higher in thyroid hormone-deficient-rats compared to litter mate controls. (4) The administration of L-thyroxine to hypothyroid animals for two successive days essentially returned the accelerated metabolism of the precursor pool leucine to normal but failed to ameliorate the increased incorporation into protein. (5) Incubations conducted in the presence of high exogenous leucine levels, to eliminate possible differences in intracellular free amino acid pool size, provided additional evidence for an increased rate of cerebral protein synthesis in 25-day-old hypothyroid rats compared to controls. (6) The results are compatible with a retardation in the normal developmental decline in the rate of cerebral protein synthesis associated with hypothyroidism.     

Coronatine biosynthesis: Incorporation of l-[U-14C]isoleucine and l-[U-14C] threonine into the 1-amido-1-carboxy-2-ethylcyclopropyl moiety

Addition of either l-[U-¹⁴C]threonine or l-[U-¹⁴C]isoleucine to 2.7-day-old shaking liquid cultures of Pseudomonas syringae pv. atropurpurea resulted in incorporation of radioactivity into coronatine, but not into N- coronafacoylvaline, another phytotoxin excreted by P.s. atropurpurea. In contrast, addition ofl-[U-¹⁴C]valine did not lead to incorporation of radioactivity into coronatine, but instead into coronafacoylvaline. Acid hydrolysis of the purified [¹⁴C] coronatine obtained after incorporation of either [¹⁴C]isoleucine or [¹⁴C]threonine demonstrated that > 94% of the radioactivity was present in the 1-amido-1-carboxy-2-ethylcyclopropyl moiety of coronatine, and < 6 % was in the coronafacoyl moiety. These findings are used to propose a biosynthetic pathway for coronatine.