CATECHOLAMINE METABOLISM IN THE DOG: COMPARISON OF INTRAVENOUSLY AND INTRAVENTRICULARLY ADMINISTERED [14C]DOPAMINE AND [3H]NOREPINEPHRINE

—The urinary excretion of labelled metabolites was measured in dogs which had been injected intravenously or intraventricularly with [³H]norepinephrine or [¹⁴C]dopamine. [³H]Norepinephrine injected by either route produced more labelled 3-methoxy-4-hydroxy-phenylglycol than 3-methoxy-4-hydroxymandelic acid, as did [¹⁴C]dopamine after intravenous administration. In contrast, following the intraventricular injection of [¹⁴C]dopamine, more [¹⁴C]3-methoxy-4-hydroxymandelic acid was formed than [¹⁴C]3-methoxy-4-hydroxyphenylglycol. These observations suggest that the metabolism of exogenously-administered and endogenously-formed norepinephrine may proceed through different routes and that the predominant metabolite of norepinephrine in canine brain may be 3-methoxy-4-hydroxymandelic acid rather than 3-methoxy-4-hydroxyphenylglycol.     

Cinnamate and shikimate incorporation into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins: Are there alternative pathways?

The incorporation of shikimate-[¹⁴C] and cinnamate-[¹⁴C] into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins was studied in isolated petals of Petunia hybrida. According to the dilution values, the incorporation of shikimate-[¹⁴C] was about 3–6 times better than that of cinnamate-[¹⁴C]. However a comparison of the incorporation of the 2 precursors on a relative basis showed no significant differences in the relative proportions of the specific activities of the 3′,4′-dihydroxysubstituted cyanidin-3-monoglucoside and the 3′,4′,5′-trihydroxysubstituted delphinidin-3-monoglucoside. This result and the [¹⁴C]-incorporation behaviour of the 3′-methoxy-4′-hydroxysubstituted peonidin-3-monoglucoside do not support the hypothesis that there are alternative pathways of flavonoid biosynthesis.     

Conversion of Indole-3-acetaldehyde Oxime to 3-Indolylmethylglucosinolate in Sinapis alba

dl -Tryptophan(methylene)-¹⁴C and indole-3-acetaldehyde oxime(methylene)-¹⁴C were supplied to cut shoots of 7-day-old plants of Sinapis alba L. Although both compounds were effective as precursors of 3-indolylmethylglucosinolate, the incorporation of the aldoxime radioactivity was more effective than the incorporation of the amino acid radioactivity. This, together with other information, suggests that indole-3-acetaldehyde oxime is an intermediate in the biosynthesis of 3-indolylmethylglucosinolate from tryptophan.     

1-Nitro-2-phenylethane,a possible intermediate in the biosynthesis of benzylglucosinolate

Results have been obtained consistent with the hypothesis that aci tautomers of nitro compounds are precursors of glucosinolates. When dl-[3-¹⁴C]phenylalanine and [¹⁴C]1-nitro-2-phenylethane were fed to shoots of Tropaeolum majus L., the incorporation of tracer from each compound into benzylglucosinolate was found to be similar. Conversion of ¹⁴C from 1-nitro-2-phenylethane into the aglycone moiety of benzyl-glucosinolate was specific. The natural occurrence of 1-nitro-2-phenylethane in T. majus and its formation in this plant from [1-¹⁴C]phenylacetaldoxime were demonstrated by gas chromatography and by means of a trapping experiment.     

GLYCOL SULPHATE ESTER FORMATION FROM [14C]NORADRENALINE IN BRAIN AND THE INFLUENCE OF A COMT INHIBITOR

A method is described allowing the identification and separation of the sulphate esters of the glycol metabolites of [¹⁴C]noradrenaline injected into the lateral ventricle of the rat. The esters of both the 3,4-dihydroxy derivative and the 3-methoxy-4-hydroxy derivative are formed in substantial amounts. It is suggested that the quantity of the former may have been underestimated in the past. Contemporaneous administration of pyrogallol, a catechol-O-methyl transferase inhibitor, with [¹⁴C] noradrenaline leads to a considerable fall in 3-methoxy-4-hydroxyphenylglycol sulphate without a rise in 3-4-dihydroxyphenylglycol sulphate although free 3-4-dihydroxyphenylglycol rises significantly. It is proposed that the latter may be an index of intraneuronal metabolism of noradrenaline and 3-methoxy-4-hydroxyphenylglycol that of released amine.     

Structural confirmation of dihydrocinnamic acids from Adiscanthus fusciflorus by 13C NMR

In the wood of Adiscanthus fusciflorus six known alkaloids 4-methoxy-2-quinolone, 1-methyl-4-methoxy-2-quinolone, dictamine, skimmianine, γ-fagarine and N-methylflindersine and two new dihydrocinnamic acids 3-[2′,6′-dimethoxy-6″,6″-dimethylpyrano(2″,3″:4′, 3′)phenyl]-propionic acid and its methyl ester were identified. The structures of the dihydrocinnamic acid derivatives were confirmed by ¹³C NMR.     

Synthesis of carbon-11-labeled 5-HT6R antagonists as new candidate PET radioligands for imaging of Alzheimer’s disease

Carbon-11-labeled serotonin (5-hydroxytryptamine) 6 receptor (5-HT₆R) antagonists, 1-[(2-bromophenyl)sulfonyl]-5-[¹¹C]methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (O-[¹¹C]2a) and 1-[(2-bromophenyl)sulfonyl]-5-methoxy-3-[(4-[¹¹C]methyl-1-piperazinyl)methyl]-1H-indole (N-[¹¹C]2a), 5-[¹¹C]methoxy-3-((4-methylpiperazin-1-yl)methyl)-1-(phenylsulfonyl)-1H-indole (O-[¹¹C]2b) and 5-methoxy-3-((4-[¹¹C]methylpiperazin-1-yl)methyl)-1-(phenylsulfonyl)-1H-indole (N-[¹¹C]2b), 1-((4-isopropylphenyl)sulfonyl)-5-[¹¹C]methoxy-3-((4-methylpiperazin-1-yl)methyl)-1H-indole (O-[¹¹C]2c) and 1-((4-isopropylphenyl)sulfonyl)-5-methoxy-3-((4-[¹¹C]methylpiperazin-1-yl)methyl)-1H-indole (N-[¹¹C]2c), 1-((4-fluorophenyl)sulfonyl)-5-[¹¹C]methoxy-3-((4-methylpiperazin-1-yl)methyl)-1H-indole (O-[¹¹C]2d) and 1-((4-fluorophenyl)sulfonyl)-5-methoxy-3-((4-[¹¹C]methylpiperazin-1-yl)methyl)-1H-indole (N-[¹¹C]2d), were prepared from their O- or N-desmethylated precursors with [¹¹C]CH₃OTf through O- or N-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (MA) at EOB was 370–740?GBq/μmol with a total synthesis time of ～40-min from EOB.     

Interaction between adenosine generated endogenously in neocortical tissues,and homocysteine and its thiolactone

[¹⁴C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted ¹⁴C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [¹⁴C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [¹⁴C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [¹⁴C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide ¹⁴C-derivative when homocysteine was 0.1 mM or greater. The total [¹⁴C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.     

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.     

Intermediates in fatty acid oxidation

1. Aqueous extracts of acetone-dried liver and kidney mitochondria, supplemented with NAD⁺, CoA and phenazine methosulphate, efficiently convert fatty-acyl-CoA compounds into acetyl-CoA; the process was followed with an O₂ electrode. 2. Label from [1-¹⁴C]octanoyl-CoA appears in acetyl-CoA more rapidly than that from [8-¹⁴C]octanoyl-CoA. 3. Oxidation of [8-¹⁴C]octanoyl-CoA was terminated by addition of neutral ethanolic hydroxylamine and the resulting hydroxamates were separated chromatographically. Hydroxamate derivatives of 3-hydroxyoctanoyl-, hexanoyl-, butyryl- and acetyl-CoA were obtained. 4. These and other observations suggest that oxidation of octanoyl-CoA by extracts involves participation of free intermediates rather than uninterrupted complete degradation of individual molecules to acetyl-CoA by a multienzyme complex. 5. Intact liver mitochondria studied by the hydroxamate technique were also shown to form intermediates during oxidation of labelled octanoates. In addition to octanoylhydroxamate, [8-¹⁴C]octanoate gave rise to small amounts of hexanoyl-, butyryl- and 3-hydroxyoctanoyl-hydroxamate. In contrast with extracts, however, where the quantity of intermediates found was a significant fraction of the precursors, mitochondria oxidizing octanoate contained much larger quantities of octanoyl-CoA than of any other intermediate.     

Synthesis of Deoxyglucose-1-Phosphate, Deoxyglucose-1,6-Bisphosphate, and Other Metabolites of 2-Deoxy-D-[14C]Glucose in Rat Brain In Vivo: Influence of Time and Tissue Glucose Level

Abstract: Abstract: When the kinetics of interconversion of deoxy[¹⁴C]glucose ([¹⁴C]DG) and [¹⁴C]DG-6-phosphate ([¹⁴C]DG-6-P) in brain in vivo are estimated by direct chemical measurement of precursor and products in acid extracts of brain, the predicted rate of product formation exceeds the experimentally measured rate. This discrepancy is due, in part, to the fact that acid extraction regenerates [¹⁴C]DG from unidentified labeled metabolites in vitro. In the present study, we have attempted to identify the ¹⁴C-labeled compounds in ethanol extracts of brains of rats given [¹⁴C]DG. Six ¹⁴C-labeled metabolites, in addition to [¹⁴C]DG-6-P, were detected and separated. The major acid-labile derivatives, DG-1-phosphate (DG-1-P) and DG-1,6-bisphosphate (DG-1,6-P₂), comprised ?5 and ?10–15%, respectively, of the total ¹⁴C in the brain 45 min after a pulse or square-wave infusion of [¹⁴C]DG, and their levels were influenced by tissue glucose concentration. Both of these acid-labile compounds could be synthesized from DG-6-P by phosphoglucomutase in vitro. DG-6-P, DG-1-P, DG-1,6-P₂, and ethanol-insoluble compounds were rapidly labeled after a pulse of [¹⁴C]DG, whereas there was a 10–30-min lag before there was significant labeling of minor labeled derivatives. During the time when there was net loss of [¹⁴C]DG-6-P from the brain (i.e., between 60 and 180 min after the pulse), there was also further metabolism of [¹⁴C]DG-6-P into other ethanol-soluble and ethanol-insoluble ¹⁴C-labeled compounds. These results demonstrate that DG is more extensively metabolized in rat brain than commonly recognized and that hydrolysis of [¹⁴C]DG-1-P can explain the overestimation of the [¹⁴C]DG content and underestimation of the metabolite pools of acid extracts of brain. Further metabolism of DG does not interfere with the autoradiographic DG method.     

Studies of the Biochemical Background to Differences in Glucosinolate Content in Brassica napus L. II. Administration of Some Sulphur-35 and Carbon-14 Compounds and Localization of Metabolic Blocks

The aim of the investigation was to study the differences in the metabolism of substances that are utilized in the synthesis of glucosinolates between the Brassica napus cv. Bronowski, which is very low in glucosinolate content, and a cultivar (cv. Regina II) that contains approximately average amounts of these compounds. By experiments in which the plants were grown in nutrient solutions supplied with sulphate-³⁵S, it was shown that the rate of sulphate uptake was similar in the two cultivars. No accumulation of intermediate metabolites could be demonstrated by autoradiography in Bronowski. Sulphate-³⁵S, methionine-³⁵S, methionine-2-¹⁴C, 2-amino-6-(methylthio)caproic acid-2-¹⁴C, and S-(β-d -glucopyranosyl)-4-pentenethiohydroximic acid (desulpho-3-butenylglucosinolate) (glucose-U-¹⁴C or ³⁵S) were fed to shoots of the two cultivars. After incubation, the plant material was extracted with methanol. The extracts were separated into various fractions, and in some experiments glucosinolates or derivatives of their degradation products were isolated. When measuring the radioactivity of the various fractions or isolated products, the incorporation of radioactivity into glucosinolates was found to be poor in Bronowski from sulphate, methionine, and 2-amino-6-(methylthio)caproic acid. Desulpho-3-butenyl-glucosinolate was an efficient precursor of 3-butenylglucosinolate in Bronowski, but a poor precursor of 2-hydroxy-3-bute-nylglucosinolate, which suggests a metabolic block at the hydroxylation step in this cultivar. In Regina II desulpho-3-butenylglucosinolate was a good precursor of both 3-butenylglucosinolate and of 2-hydroxy-3-butenylglucosinolate, which demonstrates that these glucosinolates may be synthesized without prior formation of the corresponding co-methylthioalkyl glucosinolates and that the hydroxylation can take place after the formation of desulpho-3-butenylglucosinolate. The results indicate that the low glucosinolate content of Bronowski is caused by block(s) in the separate pathway leading to the biosynthesis of glucosinolates.     

Synthesis and Enzymatic Transformations of 5-Halo-6-Methoxy-5,6-Dihydro Derivatives of 5-[1-Methoxy-2-halo(or 2,2-dihalo)ethyl]-2′-deoxyuridines as Potential Herpes Simplex Virus Inhibitors

The 5-halo-6-methoxy-5,6-dihydro derivatives of 5-[1-methoxy-2-halo(or 2,2-dihalo)ethyl]-2′-deoxyuridines (3-12) were synthesized and investigated as potential anti-herpes agents. These 5,6-dihydro derivatives were designed to act as potential prodrugs to 5-[1-methoxy-2-halo(or 2,2-dihalo)ethyl]-2′-deoxyuridines (2a-e), with enhanced metabolic stability, and ready conversion to the parent molecules. These 5,6-disubstituted-5,6-dihydro analogs are stable to E. coli thymidine phosphorylase, and undergo regeneration of the 5,6-olefinic bond to provide parent moieties (2a-e), upon incubation with glutathione at 37°C. The compounds (3-12) themselves were found to be non-inhibitory against herpes simplex virus type-1 (HSV-1), likely due in part to their inability to undergo conversion to parent compounds in cell culture medium.     

Regulation of ketone body production from (14C)palmitate in rat liver mitochondria: effects of cyclic nucleotides and unlabeled fatty acids.

N⁶′, O²′-dibutyryl adenosine 3′, 5′-cyclic monophosphoric acid, but not other cyclic nucleotides stimulates [¹⁴C]ketone body production from [¹⁴C]palmitate in isolated rat liver mitochondria. Butyrate alone, as well as unlabeled acetate, octanoate and palmitate had similar effects. This redistribution of the oxidative products of [¹⁴C]palmitate can best be explained by exceeding the capacity of the Krebs cycle and/or changes in the acetyl coenzyme A/coenzyme A ratio. In contrast to [¹⁴C]palmitate, [¹⁴C]octanoate oxidation to [¹⁴C]O₂ and [¹⁴C]ketone bodies was inhibited by the addition of unlabeled fatty acids. This suggests that an additional mechanism by which unlabeled fatty acids may stimulate [¹⁴C]ketone body production is by enhancing the carnitine-dependent transport of [¹⁴C]palmitate into mitochondria.     

Identification of the metabolites of Indole-3-acetic acid in growing hypocotyls of Lupinus albus

The products of indole-3-acetic acid (IAA) metabolism by incubating hypocotyl sections and decapitated seedlings of Lupinus albus were investigated. Single treatments using [1-¹⁴C]-IAA, [2-¹⁴C]-IAA or [5-³H]-IAA and double treatments using [1-¹⁴C]-IAA+[5-³H]-IAA were carried out. Extracts from treated plant material were analyzed by paper chromatography (PC), Thin layer chromatography (TLC), and high performance liquid chromatography (HPLC). When hypocotyl sections were incubated in [2-¹⁴C]-IAA, several IAA decarboxylation products including indole-3-aldehyde (IA1), indole-3-methanol (IM), 3-hydroxymethyloxindole (HMOx), methyleneoxindole (MOx) and 3,3-bisindolylmethane (BIM) were detected in the 95% ethanol extract; a latter extraction with 1M NaOH rendered IAA, IM and BIM, suggesting that conjugated auxins were formed in addition to conjugated IM. In sections incubated with [1-¹⁴C]-IAA, the 1M NaOH extraction also produced IAA so confirming the formation of conjugated auxins. The same decarboxylation products and two conjugated auxins, indole-3-acetylaspartic acid (IAAsp) and 1-O-(indole-3-acetyl)--D-glucose (IAGlu), were detected in the acetonitrile extracts from decapitated seedlings treated with [5-³H]-IAA. After a double isotope treatment ([1-¹⁴C]-IAA+[5-³H]-IAA) of decapitated seedlings, the ratio ¹⁴C/³H measured in the HPLC fractions of the acetonitrile extracts confirmed the presence of decarboxylation products as well as conjugated auxins.     

The Conversion of 3-Indolylmethylglucosinolate to 3-Indolylacetonitrile by Myrosinase, and its Relevance to the Clubroot Disease of the Cruciferae

[Methylene-¹⁴C]-3-indolylmethylglucosinolate (¹⁴C-IMG) was convertedin vitro to [methylene-¹⁴Cl-3-indolylacetonitrile (¹⁴C-IAN)by myrosinase over a pH range of 4.0-6.0 and this conversionwas enhanced by ferrous ions. Other products of the reactionincluded 3-indolylmethanol, 3, 3'-diindolylmethane and ascorbigenA. Trace amounts of ¹⁴C-IAN were produced non-enzymically from¹⁴C-IMG in the presence of ferrous ion over a similar _pH range.Furthermore, swede tissues (Brassica napus cv. Danestone) infectedwith Plasmodiophora brassicae Woron. could convert ¹⁴C-IMG to¹⁴C-IAN. These results were consistent with the hypothesis thatthe overgrowth symptoms of the clubroot disease are caused bythe conversion of IMG to the auxin precursor IAN.     

IN VITRO EXPERIMENTS ON THE METABOLISM, ACCUMULATION AND RELEASE OF 5-HT IN THE NERVOUS SYSTEM OF THE SNAIL HELIX POMATIA

Abstract— The accumulation, metabolism and stimulated-induced release of 5-HT in the nervous system of the snail was studied. When nervous tissue was incubated at 24°C in a medium containing [¹⁴C]5-HT or [³H]tryptophan, tissue: medium ratios of about 25:1 and 4:1 respectively were obtained after 45 min incubation. The process responsible for [¹⁴C]5-HT accumulation showed properties of an active transport system: it was temperature sensitive and was greatly inhibited by dinitrophenol and ouabain. Furthermore, the accumulation process was inhibited by imipramine and desipramine. Of a number of analogues of indole, N-acetyl-5-HT and 5-hydroxytryptophan were the most potent in the inhibition of the accumulation of [¹⁴C]5-HT. The presence of a large molar excess of amino acids had little effect. A small amount (less than 14 per cent) of the accumulated [¹⁴C]5-HT was metabolized to form 5-hydroxyindole acetic acid, even after long periods (2 h) of incubation. The accumulated [³H]tryptophan was metabolized to form 5-hydroxytryptophan and 5-HT; the content of formed [³H]5-HT increased with incubation time whilst the [³H]5-hydroxytryptophan remained more or less constant. The presence of p-chlorophenylalanine in the incubation medium did not interfere with the accumulation of [³H]tryptophan, though it inhibited the formation of [³H]5-hydroxytryptophan and to a greater extent [³H]5-HT. A rapid efflux of the accumulated [¹⁴C]5-HT from snail nervous tissue was observed on electrical stimulation. Slower release resulted when the Ca²⁺ ion content of the incubation medium was replaced by Mg²⁺ ions. There is also a slight efflux of radioactive substances following electrical stimulation in tissues previously incubated in [³H]tryptophan. Most of this radioactivity was attributed to the formed [³H]5-HT. The data support the idea that 5-HT is a transmitter-substance in the snail Helix pomatia, and that re-uptake of the substance is a method of inactivating the released amine.