In vivo and in vitro inhibition of lipid-linked saccharide and glycoprotein synthesis in plants by amphomycin

The effect of the polypeptide antibiotic, amphomycin, on the in vitro and in vivo synthesis of polyprenyl-linked sugars and glycoproteins in plants was examined. This antibiotic blocked the transfer of mannose from GDP-[¹⁴C]mannose into mannosyl-phos-phoryl-dolichol by a particulate enzyme preparation from mung beans and also inhibited the transfer of GlcNAc from UDP-[³H]GlcNAc to GlcNAc-pyrophosphoryl-polyisoprenol. The in vitro incorporation of these sugars into trichloroacetic acid-insoluble material was also markedly inhibited by this antibiotic. Since most of the radioactivity incorporated into this insoluble material is rendered water-soluble by treatment with pronase, it seems likely that these sugars are incorporated into glycoproteins whose synthesis is sensitive to amphomycin. Amphomycin also inhibited the transfer of glucose from UDP-[¹⁴C]glucose to steryl glucosides, although this system was less sensitive to antibiotic than was synthesis of the polyprenyl-linked sugars. The antibiotic did not block the in vitro transfer of glucose from UDP-[¹⁴C]glucose to β-glucans. In carrot slice cultures, amphomycin also inhibited the incorporation of [¹⁴C]mannose into glycolipid and glycoprotein, but it did not prevent the incorporation of [¹⁴C]lysine into protein.     

Stimulation of Phospholipase A2 and Secretion of Catecholamines from Brain Synaptosomes by Potassium and A23187

Abstract: Potassium depolarization of rat brain synaptosomes (containing incorporated l-acyl-2-[¹⁴C]arachidonyl-phosphatidylcholine) stimulated endogenous phospholipase A₁ (EC 3.1.1.32) and A₂ (EC 3.1.1.4), as determined by the formation of [¹⁴C]lysophosphatidylcholine, [¹⁴C]arachidonate, and [¹⁴C]prostaglandins, and also stimulated the secretion of [³H]catecholamines. The phospholipase A₂ stimulation, dependent on calcium, was elicited in resting synaptosomes by A23187 and was demonstrated with incorporated 1-acyl-2-[^l4C]oleoyl-phosphatidylcholine but not with incorporated [^I4C]phosphatidylethanolamine or [^l4C]phosphatidylserine. Inhibitors of phospholipase A₂ [p-bromophenacylbromide (10 μM), trifluoperazine (3 μM), and quinacrine (3 μM) reduced the potassium-stimulated [³H]catecholamine release from synaptosomes to 78, 39. and 55%, respectively, of depolarized controls. The addition of lysophosphatidylcholine increased the release of [³H]norepinephrine to levels observed with potassium depolarization, whereas lysophosphatidylethanolamine, lysophosphatidylserine, and sodium dodecyl sulfate were much less effective. Potassium stimulation of synaptosomes increased the endogenous levels of free arachidonic acid and prostaglandins E₂ and F_2α. Indomethacin and aspirin decreased the amounts of prostaglandins formed, allowed the accumulation of free arachidonic acid, and diminished the potassium-stimulated release of [³H]dopamine. p-Bromophenacylbromide inhibited the formation of prostaglandin F_2α. Addition of prostaglandin E₂ inhibited, whereas prostaglandin F_2α enhanced the release of [³H]norepinephrine. These results suggest that calcium influx induced by synaptosomal depolarization activates endogenous phospholipase A₂, with subsequent formation of lysophosphatidylcholine and prostaglandins, both of which may modulate neurosecretion.     

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

A SOLUBLE PREPARATION FROM RAT BRAIN THAT INCORPORATES INTO ITS OWN PROTEINS [14C]ARGININE BY A RIBONUCLEASE-SENSITIVE SYSTEM AND [14C]TYROSINE BY A RIBONUCLEASE-INSENSITIVE SYSTEM

Abstract— A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G-25-40 was able, after addition of some factors, to incorporate [^I4C]arginine (apparent K_m= 5 μM) and [¹⁴C]tyrosine (apparent K_m= 20 μM) into its own proteins. The factors required for the incorporation of [¹⁴C]arginine were: ATP (optimal concentration = 0-25-2 μM) and Mg²⁺ (optimal concentration 5 mM). For the incorporation of [^I4C]tyrosine the required factors were: ATP (apparent K_m= 0-75 μM), Mg²⁺ (optimalconcentration 8-16 mM) and K⁺ (apparent K_m= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [¹⁴C]arginine and [¹⁴C]tyrosine, respectively, 7-4 and 7-0 in phosphate buffer and 7–9 and 7-3-8-1 in tris-HCl buffer. Pancreatic ribonuclease abolished the incorporation of [¹⁴C]arginine but had practically no effect in the incorporation of [¹⁴C]tyrosine. Furthermore, [¹⁴C]arginyl-tRNA was a more effective donor of arginyl groups than [¹⁴C]arginine, whereas [¹⁴C]tyrosyl-tRNA was considerably less effective than [¹⁴C]tyrosine. The incorporations of [¹⁴C]arginine and [¹⁴C]tyrosine into brain proteins were from 25- to 2000-fold higher than for any other amino acid tested (12 in total). In brain [¹⁴C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [¹⁴C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [¹⁴C]Arginine was bound so that its a-amino group remained free. Maximal incorporation of [¹⁴C]tyrosine in brain of 30-day-old rats was about one-third of that in the 5-day-old rat. The changes with postnatal age in the incorporation of [¹⁴C]arginine were not statistically significant.     

INCORPORATION OF [14C]GLYCINE INTO PROTEIN OF THE ADULT RAT PERIPHERAL NERVE: EFFECTS OF INHIBITORS

—(1) The rate of incorporation in vitro of [¹⁴C]glycine into adult rat peripheral nerve protein was studied and found to be linear up to a concentration of 4.5 μc/ml. It was also linear with time of incubation up to at least 6 hr. (2) Anaerobiosis, potassium cyanide, dinitrophenol and diphtheria toxin inhibited the incorporation of [¹⁴C]glycine into protein, in a manner comparable to other tissues.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

Acetylcholine Synthesis and CO2 Production from Variously Labeled Glucose in Rat Brain Slices and Synaptosomes

Abstract: The molecular basis of the close linkage between oxidative metabolism and acetylcholine (ACh) synthesis is still unclear. We studied this problem in slices and synaptosomes by measurement of ACh synthesis from [U-¹⁴C]glucose, and ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose, an index of glucose decarboxylation by the pyruvate dehydrogenase complex (PDH) and the enzymes of the Krebs cycle, respectively. We examined both under conditions that either inhibited (low O₂ or antimycin) or stimulated (2,4- dinitrophenol [DNP] or 35 mm -K⁺) ¹⁴CO₂ production from [2-¹⁴C]- or [3,4-¹⁴C]glucose. Incorporation of [U-¹⁴C]glucose into ACh was reduced under low O₂ and by antimycin or DNP (by 51-93%) and stimulated by 35 mm -K⁺ (by 30-60%). Under all of these conditions, ACh synthesis and the decarboxylation of [3,4-¹⁴C]- and [2-¹⁴C]glucose were linearly related (r= 0.741 and 0.579, respectively). The difference in the rate of ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose was used as a measure of the amount of glucose that was not oxidatively decarboxylated (efflux). We found that efflux was reduced (low 0₂ and antimycin), unchanged (DNP in slices), or increased (DNP in synaptosomes and K⁺ stimulation in slices) compared with control values under 100% O₂. ACh synthesis and efflux were more closely related (r= 0.860) than ACh synthesis and ¹⁴CO₂ production from variously labeled glucoses.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

The fate of [14C]glutamate and [14C]malate in birch roots is strongly modified under inoculation with Paxillus involutus

The impact of inoculation with Paxillus involutus on the utilization of organic carbon compounds by birch roots was studied by feeding [¹⁴C]Glu or [¹⁴C]malate to the partners of the symbiosis, separately or in association, and by monitoring the subsequent distribution of ¹⁴C. Inoculation increased [¹⁴C]Glu and [¹⁴C]malate absorption capacities by up to eight and 17 times, respectively. Six- and 15-d-old mycorrhizal roots showed about four-fold higher [¹⁴C]Glu and [¹⁴C]malate absorption capacities compared with 60-d-old mycorrhizal roots, suggesting that the early stages of mycorrhiza formation induced higher requirements for C skeletons. Moreover, the results demonstrated that inoculation strongly modified the fate of [¹⁴C]Glu and [¹⁴C]malate. It was demonstrated that exogenously supplied Glu and malate might serve as C skeletons for amino acid synthesis in mycorrhizal birch roots and in the free-living fungus. Gln was the major ¹⁴C-sink in mycorrhizal roots and in the free-living P. involutus. In contrast, citrulline and insoluble compounds were the major ¹⁴C sinks in non-mycorrhizal roots, whatever the ¹⁴C source. It was concluded that mycorrhiza formation leads to a profound alteration of the metabolic fate of exogenously supplied C compounds. The ecological significance of amino acid and organic acid utilization by mycorrhizal plants is further discussed.     

Effect of α-Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+-Dependent and Na+-Independent Components

Abstract: We studied the effect of α-latrotoxin (αLTX) on [¹⁴C]acetylcholine ([¹⁴C]ACh) release, intracellular Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10^?10-10^?8M) caused an elevation of the [Ca²⁺]_i as detected by Fura 2 fluorescence and evoked [¹⁴C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na⁺ in the external medium and another that did not. Displacement of Na⁺ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca²⁺]_i and [¹⁴C]ACh release by αLTX. The Na⁺-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca²⁺]_i and [¹⁴C]ACh release remained in the absence of Na⁺. Both the Na⁺-dependent and the Na⁺-independent components of the αLTX-evoked [¹⁴C]ACh release partly required the presence of either Mg²⁺ or Ca²⁺. The nonneurotransmitter [¹⁴C]choline was released along with [¹⁴C]ACh, but this release did not depend on the presence of either Na⁺ or Ca²⁺, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na⁺-dependent divalent cation entry and (b) Na⁺-independent divalent cation entry, and (2) nonspecific Na⁺- and divalent cation-independent leakage.     

α-Ketoisocaproate Alters the Production of Both Lactate and Aspartate from [U-13C]Glutamate in Astrocytes: A 13C NMR Study

Abstract: The present study determined the metabolic fate of [U-¹³C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mMα-ketoisocaproate (α-KIC). When astrocytes were incubated with 0.2 mM [U-¹³C]glutamate, 64.1% of the ¹³C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3-¹³C₃]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [¹³C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [¹³C]glutamate into aspartate in astrocytes incubated in the presence of α-KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [¹³C]glutamate into the 1,2,3-¹³C₃-isotopomer of lactate in cells incubated in the presence of α-KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of α-KIC. Altogether more of the [¹³C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of α-KIC than in control cells. Overall, the results demonstrate that the presence of α-KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate-aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

MECHANISMS FOR THE EFFLUX OF [14C]DOPA AND [14C]DOPAMINE FROM THE CSF OF RHESUS MONKEYS

—Clearance of [¹⁴C]DOPA and [¹⁴C]dopamine from CSF was investigated in anaesthetized rhesus monkeys (M. Mulatta) subjected to ventriculocisternal perfusion. The efflux coefficients, k_VE, at tracer concentrations (3–5 m ) in the perfusate were 0.0487 ml/min and 0.0325 ml/min for [¹⁴C]DOPA and [¹⁴C]dopamine, respectively. Carrier DOPA (10 mm ) in the perfusate decreased the efflux of [¹⁴C]DOPAsignificantly, but carrier dopamine had no appreciable effect on the clearance of [¹⁴C]dopamine. These findings suggest that DOPA is cleared from CSF in part by a saturable mechanism which may be located in the choroid plexus, whereas dopamine leaves the ventricular system by passive diffusion. Radioactivity in the caudate nucleus immediately adjacent to the perfused ventricle averaged 15.5 % and 12.6% of the radioactivity in the perfusates with [¹⁴C]DOPA or [¹⁴C]dopamine, respectively. These distribution percentages were similar to those found for various extracellular indicators after ventriculocisternal perfusion and may indicate that the efflux of intraventricularly-administered exogenous DOPA and dopamine occurs in part through extracellular channels.     

MEDIATION BY β-ADRENERGIC RECEPTORS OF EFFECT OF NOREPINEPHRINE ON PINEAL SYNTHESIS OF [14C]SEROTONIN AND [14C]MELATONIN

Rat pineal organs maintained in organ culture converted [¹⁴C]tryptophan to [¹⁴C]serotonin and [¹⁴C]melatonin. The synthesis of both indoles was stimulated by the presence of norepinephrine or dibutyryl adenosine 3′,5′-monophosphate. This effect of norepinephrine could be blocked by the α-adrenergic blocking drug, propranolol, but was not modified by the a-adrenergic blocking agent, phenoxybenzamine. Neither blocking agent modified the pineal response to dibutyryl adenosine 3′,5′-monophosphate. Unlike dibutyryl adenosine 3′,5′-monophosphate, the naturally occurring adenosine phosphates did not stimulate synthesis of [¹⁴C]melatonin in vitro.     

Cytokinin Function: Increase in [Methyl−14C] Metabolism to Phosphatidylcholine and a Decrease in Oxidation to Carbon Dioxide−14C

Axillary buds of Nicotiana tabacum L. cv. Maryland Catterton normally suppressed by apical dominance are released from dormancy with 6-benzylaminopurine. This work was done to determine the change in the [methyl^?14C] metabolism from methionine during bud stimulation with cytokinin. Dormant buds metabolize [methyl^?14C] -methionine to ¹⁴CO₂ more effectively than buds released from dormancy. This oxidation of the methyl group is inhibited with benzylaminopurine. On the other hand, the methylation of polar membrane components, including phosphatidylcholine, is enhanced by the cytokinin during the period preceding the increase in bud weight. The interpretation is presented that the enhanced synthesis of membrane components, as well as the preservation of the methyl groups, are mechanisms for the cytokinin release of bud dormancy with 6-benzylaminopurine.     

Incorporation of [C]Glucosamine and [C]Mannose into Glycolipids and Glycoproteins in Cotyledons of Pisum sativum L

Developing pea cotyledons incorporate radioactivity in vivo from [¹⁴C]glucosamine and [¹⁴C]mannose into glycolipids and glycoproteins. Several different lipid components are labeled including neutral, ionicnonacidic, and acidic lipids. The acidic lipids labeled in vivo appear similar to the polyisoprenoid lipid intermediates formed in vitro in pea cotyledons. Radioactivity from [¹⁴C]glucosamine and [¹⁴C]mannose is also incorporated into glycopeptides. Considerable redistribution of [¹⁴C]mannose into other glycosyl components found in endogenous glycoproteins is observed. An N-acetylglucosamine to asparagine glycopeptide linkage has been isolated from [¹⁴C]glucosamine-labeled glycoproteins.     

DECARBOXYLATION STUDIES OF GLUTAMATE, GLUTAMINE, AND ASPARTATE FROM BRAIN LABELLED WITH [1-14C] ACETATE, l-[U-14C]-ASPARTATE, AND l-[U-14C]GLUTAMATE

Studies in vivo and in vitro of the distribution of label in C-1 of glutamate and glutamine and C-4 of aspartate in the free amino acids of brain were carried out. [1-¹⁴C]-Acetate was used both in vivo and in vitro and l -[U-¹⁴C]aspartate and l -[U-¹⁴C]glutamate were used in vitro.

• 1 The results obtained with labelled acetate and aspartate suggest that CO₂ and a 3-carbon acid may exchange at different rates on a CO_a-fixing enzyme.
• 2 The apparent cycling times of both glutamate and glutamine show fast components measured in minutes and slow components measured in hours.
• 3 With [1-¹⁴C]acetate in vitro glutamine is more rapidly labelled in C-1 than is glutamate at early time points; the curves cross over at about 7 min.
• 4 The results support and extend the concept of metabolic compartmentation of amino acid metabolism in brain.

     

SIMULTANEOUS INCORPORATION OF ORALLY ADMINISTERED [9, 10-3H2]OLEIC AND [1-14C]LINOLEIC ACIDS INTO LIPIDS OF THE ADULT RAT BRAIN

—The incorporation of an orally administered mixture of [9,10-³H₂joleic acid and [1-¹⁴C]linoleic acid into the brain and spinal cord lipids was maximal after 24 h compared with 4 h for extraneural tissue. In the latter, both acids were utilized equally well for triglyceride biosynthesis, but linoleate entered phosphatidylcholine more rapidly than oleate. Oleic acid was preferentially incorporated into newly synthesized cholesterol esters although 4 h after dosing most cholesterol esters present in serum were formed preferentially from linoleate presumably by the action of lecithin-cholesterol acyl transferase. In neural tissue, a considerable amount of [1-¹⁴C]linoleate was metabolized to higher polyunsaturated fatty acids, whereas in the case of oleate, 90 per cent of the tritium activity remained in monoenic acids at all time periods studied. Both acids were initially incorporated most rapidly into the lecithin fraction of brain and spinal cord, but after 7 days diacyl phosphatidylethanolamine had the highest specific activity. These data are consistent with the view that the uptake of labelled fatty acids by the brain takes place principally as free acids but that some uptake of esterified forms, probably largely as phosphatidylcholine, also occurs. The low linoleate content of the brain and probably also of cerebrospinal fluid cannot be explained on the basis of a selective restriction on the uptake of this lipid from plasma.     

Half-life of tRNA Containing N6(Δ2-isopentenyl)adenosine in Lactobacillus acidophilus ATCC 4963

tRNA containing N⁶-(Δ²-isopentenyl)adenosine may be precursors for the plant hormone cytokinin. To discriminate between tRNA containing and not containing cytokinin nucleotides, double labelling experiments were made by the use of [2¹⁴C]-mevalonic acid and [³H-methyl]-methionine. At a generation cycle of 2 h for Lactobacillus acidophilus ATCC 4963, the half-lives of tRNA labelled with [³H-methyl]-methionine and [2-¹⁴C]-mevalonic acid are similar, namely 3 h. Isopentenylation of tRNA could be measured to be maximally 1:10.     

Alterations in the Production of 14CO2 and [14C]Acetylcholine from [U-14C]Glucose in Brain Subregions Following Transient Forebrain Ischemia in the Rat

Abstract: The production of ¹⁴CO₂ and [¹⁴C]acetylcholine from [U-¹⁴C]glucose was determined in vitro using tissue prisms prepared from the dorsolateral striatum (a region developing extensive neuronal loss following ischemia) and the paramedian neocortex (an ischemia-resistant region) following 30 min of forebrain ischemia and recirculation up to 24 h. Measurements were determined under basal conditions (5 mMK⁺) and following K⁺ depolarization (31 mM K⁺). The production of ¹⁴CO₂ by the dorsolateral striatum was significantly reduced following 30 min of ischemia for measurements in either 5 or 31 mM K⁺ but recovered toward preischemic control values during the first hour of recirculation. Further recirculation resulted in ¹⁴CO₂ production again being reduced relative to control values but with larger differences (20–27% reductions) detectable under depolarized conditions at recirculation times up to 6 h. Samples from the paramedian neocortex showed no significant changes from control values at all time points examined. [¹⁴C]Acetylcholine synthesis, a marker of cholinergic terminals that is sensitive to changes in glucose metabolism in these structures, was again significantly reduced only in the dorsolateral striatum. However, even in this tissue, only small (nonstatistically significant) differences were seen during the first 6 h of recirculation, a finding suggesting that changes in glucose oxidation during this period were not uniform within all tissue components. The results of this study provide evidence that in a region susceptible to ischemic damage there were specific changes during early recirculation in the metabolic response to depolarization. This apparent inability to respond appropriately to an increased need for energy production could contribute to the further deterioration of cell function in vivo and ultimately to the death of some cells.