Biosynthesis of lipids in chloroplasts isolated from jack pine needles

Suspensions of isolated pine needle chloroplasts were shown to incorporate galactose from UDP galactose-[¹⁴C] into galactolipids. The incorporation of the label among galactolipids was always considerably higher in the monogalactosyl diglycerides than in the digalactosyl diglycerides. The galactosyl incorporation into both galactolipid fractions was optimal at pH 8.0 and was inhibited by sulphydryl reagents (p-chloromercuribenzoate, N-ethyl maleimide and CdCl₂). The chloroplast preparations were also able to biosynthesize various phospholipids and galactolipids from palmitoyl-[1-¹⁴C]-CoA; the major portion of the label appeared in phosphatidyl choline. The incorporation of palmitic-[1-¹⁴C] acid into various lipids was very poor compared to that of palmitoyl-[1-¹⁴C]-CoA. However, addition of ATP and CoA markedly stimulated lipid biosynthesis from palmitic-[1-¹⁴C] acid, suggesting the presence of activating enzymes. These chloroplast suspensions did not show any de novo fatty acid synthesis.     

Inhibition of hydrocarbon biosynthesis in the housefly by 2-Octadecynoate

The elongation of [9,10-³H]oleoyl-CoA with malonyl-CoA to form 20, 22, and 24 carbon monounsaturated fatty acids was demonstrated in housefly microsomes by radio-GLC. These elongation reactions, which have been postulated to be involved in hydrocarbon biosynthesis, have not been previously demonstrated in insects. 2-Octadecynoate (18:1 Δ²⁼) inhibited the in vivo incorporation of [1-¹⁴C]acetate into both fatty acids and hydrocarbons in a dose-dependent manner. At doses of 10 μg per female housefly of the alkynoic acid, the incorporation of [1-¹⁴C]acetate into hydrocarbon was inhibited 93%, the incorporation of [9,10-³H]oleate into hydrocarbon was inhibited 64%, and the incorporation of [1-¹⁴C]acetate into total internal lipid was inhibited 65%. Partially purified FAS was inhibited 50% and 95% at 15 μM and 40 μM, respectively, of the alkynoic acid. These results show that 2-octadecynoate inhibits hydrocarbon biosynthesis in the housefly by inhibiting FAS, and the in vivo data suggest that the elongation of 18:1 to longer chain fatty acids is also inhibited.     

Fatty Acid Incorporation in Normal and Degenerating Rat Sciatic Nerve In Vivo

Abstract: Labeled palmitic acid ([16-¹⁴C]palmitate) (0).5 μCi) was injected into rat sciatic nerves in vivo to characterize thc incorporation of this fatty acid into complex peripheral nerve lipids after time lapses of 1 min to 2 weeks. For the first 30 min after intraneural injection, the label was concentrated in nerve diglycerides. Thereafter, the relative diglyccride label declined rapidly, and phospholipid radioactivity rose steadily. After 120 min, phospholipids contained over 70% of the total lipid radioactivity. Among the phospholipids, phosphatidylcholine had the largest percentage of total phospholipid label, and acylation of lysophosphatidylcholine accounted for approximately 75% of this label. With time, there was conversion of [16-¹⁴C]palmitate to other long-chain fatty acids by elongation and desaturation. Phosphatidic acid was labeled also, suggesting the operation of the de novo biosynthetic mechanism. However, the specific radioactivity of 1,2-diacylglycerol was much higher than that of phosphatidic acid, suggesting phosphorylation of diglycerides by diglyceride kinase. After nerve section and survival of 2 h to 50 days, the injection of [16-¹⁴C]palmitate into the degenerating distal segment revealed an overall decline of phospholipid labeling and a commensurate increase of triglyceride radioactivity. Phosphatidylcholine in degenerating nerve contained a larger percentage of the fatty acid label than that in normal nerve. Almost all of the labeling was due to acylation of lysophosphatidylcholine, implying a much smaller contribution of the de novo pathway. Phosphatidylethanolamine and phosphatidylserine showed a relative loss of radioactivity. The changes were apparent at 1 day, but not at 2 h, suggesting loss of homeostatic control, presumably by interruption of axonal flow. An incidental observation was the stimulation of phosphatidylcholine biosynthesis by acylation of lysophosphatidylcholine in the contralateral unoperated sciatic nerve.     

Human platelets have cholesteryl ester hydrolytic activity resulting in esterification of [1-14C]oleate to individual phospholipids of platelets

Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward cholesteryl [1-¹⁴C]oleate resulting in esterification of [1-¹⁴C]oleate to individual platelet phospholipids: choline-containing phospholipids (PC); ethanolamine-containing phospholipids (PE); phosphatidylserine (PS); phosphatidylinositol (PI); and sphingomyelin (SPH). Liberation of [1-¹⁴C]oleate and esterification of [1-¹⁴C]oleate to platelet phospholipids was enhanced by 100 nM iloprost (a stable analogue of prostacyclin that increases platelet cyclic adenosine monophosphate (c-AMP)), inhibited by 30 μM H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)) (a specific c-AMP dependent protein kinase (CADPK) inhibitor) and 500 μM 2′,5′ dideoxyadenosine (DDA) (an inhibitor of iloprost-induced rise in platelet c-AMP), but unaffected by 150 mM chloroquine diphosphate. These observations suggest that the CEH activity is mediated by a CADPK phosphorylation of an enzyme with the phosphorylated state representing the active form of the enzyme and that the CEH activity is extralysosomal.     

The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats

The concentration-dependent metabolism of 1-¹⁴C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [¹⁴C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-¹⁴C]20:5n-3 to [3-¹⁴C]22:6n-3 was more efficient than that of [1-¹⁴C]20:4n-6 to [3-¹⁴C]22:5n-6. At low substrate concentration (4 μM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 μM). The conversion of [1-¹⁴C]22:5n-3 to [1-¹⁴C]22:6n-3 was 1.7 times more efficient than that of [1-¹⁴C]22:4n-6 to [1-¹⁴C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-¹⁴C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-¹⁴C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-¹⁴C]20:4n-6 or [1-¹⁴C]22:4n-6 to [¹⁴C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-¹⁴C]20:5n-3 and [1-¹⁴C]22:5n-3 to [¹⁴C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.     

Elongation of acyl-CoAs by microsomes from etiolated leek seedlings

Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2-¹⁴C]malonyl-CoA was incorporated into fatty acids of C₁₆C₂₆. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C₁₂C₂₄) elongation showed that stearoyl-CoA, in the presence of [2-¹⁴C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C₂₀C₂₆. [1-¹⁴C]C₁₆CoA and [1-¹⁴C]C₁₈CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1-¹⁴C]C₁₈CoA was a better primer than [1-¹⁴C]C₁₆CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1-¹⁴C]C₁₈CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2-¹⁴C]malonyl-CoA in the presence of C₁₈CoA, on the one hand, and [1-¹⁴C]C₁₈CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.     

In Vivo Pathway of Oleate and Linoleate Desaturation in Developing Cotyledons of Cucumis sativus L. Seedlings

Exogenous [1-¹⁴C]oleic acid and [1-¹⁴C]linoleic acid were taken up and esterified to complex lipids by greening cucumber (Cucumis sativus L.) cotyledons. Both ¹⁴C-labeled fatty acids were initially esterified to phosphatidylcholine prior to eventual accumulation in triacylglycerols and galactolipids. Kinetic data suggest that esterification occurs prior to desaturation and that phosphatidylcholine is the initial site of both [¹⁴C]-oleate and [1-¹⁴C]linoleate esterification and of [1-¹⁴C]oleate desaturation to [1-¹⁴C]linoleate. [1-¹⁴C]Linoleic acid was esterified more rapidly than [¹⁴C]oleic acid and its desaturation product, [1-¹⁴C]α-linolenate, occurred mainly on monogalactosyl diacylglycerol, although some was also observed on the other major acyl lipids, including phosphatidylcholine.     

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.     

Biosynthetic preparation of cardenolides from [1-14C]acetic acid by stem discs of the milkweed Asclepias curassavica

[¹⁴C]Calotropin (11.2 μCi/mmol) and uscharidin (14.1 μCi/mmol) were biosynthesized by stem discs of Asclepias curassavica incubated in a medium containing [1-¹⁴C]acetic acid. Relative isotope enrichment sites determined by ¹³C NMR spectroscopy of [¹³C]calotropin prepared by the same method were at C-23 (0.71 %), C-2′ (0.28 %) and C-4′ (0.21 %).     

Titles of related papers in other sections

Culture of Trypanosoma cruzi (Tulahuen strain) in the presence of ethidium bromide (1–20 μg/ml) resulted in dyskinetoplasty and inhibition of growth, to an extent depending on the dye concentration and the medium composition. The ethidium bromide-induced dyskinetoplasty caused a decrease of (a) the cytochrome content of epimastigotes (a,a₃ and b species); (b) the rate of respiration (endogenous or supported by D-glucose); and (c) the rate of production of ¹⁴CO₂ from [2-¹⁴C]acetate and [1-¹⁴C]glucose. [2-¹⁴C]Acetate oxidation to ¹⁴CO₂ was affected by dyskinetoplasty more than [1-¹⁴C]glucose oxidation, particularly at the exponential growth phase. With dyskinetoplastic epimastigotes, diminution of ¹⁴CO₂ production from [2-¹⁴C]acetate largely exceeded that of oxygen uptake, while with [1-¹⁴C]glucose, ¹⁴CO₂production and respiration were affected to about the same extent. Dyskinetoplasty also decreased the incorporation of [2-¹⁴C]acetate carbon into intermediates of the tricarboxylic acid cycle and related amino acids, and modified the distribution pattern of ¹⁴C in accordance with the decrease of respiration. Reduction of cytochrome content of epimastigotes by restriction of heme compounds during growth decreased ¹⁴CO₂ production from [2-¹⁴C]acetate, like the ethidium-induced dyskinetoplasty. The same occurred after inhibition of electron transfer by antimycin and cyanide, though to a much more significant extent, thus confirming the functional association of electron transport at the mitochondrial cytochrome system of T. cruzi and the enzymatic reactions of the tricarboxylic acid cycle.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Biosynthesis of Camptotheca acuminata alkaloids

Tracer feeding experiments with Camptotheca acuminata plants show that [1′-¹⁴C]L-tryptophan, [Ar-³H₄]L-tryptophan, [Ar-³H₄,1′-¹⁴C]tryptophan, [1′-¹⁴C]-tryptamine, [2-¹⁴C]DL-mevalonate, and [2-¹⁴C]geraniol-[2-¹⁴C]nerol are incorporated into camptothecin. Direct stem injection of the labeled precursors into C. acuminata plants resulted in a substantial increase in the activity of isolated Camptotheca alkaloids as compared to root feeding of the same tracer.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. III. Experiments with 14C-labeled substrates

An ecological substrate relationship between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten has been studied in experiments using ¹⁴C-labeled acetate and lactate as substrates. Fluoroacetate strongly inhibited the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate and β-fluorolactate gave an inhibition of similar magnitude of the breakdown of [U-¹⁴C]-l-lactate to ¹⁴CO₂ thus confirming earlier results on the specific action of these inhibitors. The turnover-rate constant of l-lactate was 2.37 hr^-1 and the average l-lactate pool size was 12.2 μg per gram of wet mud, giving a turnover rate of 28.9 μg of lactate/gram of mud per hr. The turnover-rate constant of acetate was 0.35 hr^-1 and the average pool size was 5.7 μg per gram of wet mud, giving a rate of disappearance of 1.99 μg of acetate/gram of mud per hr. Estimations of the acetate turnover rate based upon the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate or [1-¹⁴C]-acetate yielded figures of the same magnitude (range 0.45 to 1.74). These and other results suggest that only a portion of the lactate dissimilated is turned over through the acetate pool. The ratio of ¹⁴CO₂/¹⁴CH₄ produced from [U-¹⁴C]-acetate by mud was 1.32; indicating that 0.862 moles of CH₄ and 1.138 moles of CO₂ are formed per mole of acetate. From the rate of disappearance of acetate (0.027 μmoles/gram wet mud per hr) and the rate of methane production (0.034 μmoles/gram wet mud per hr), it may be concluded that acetate is an important precursor of methanogenesis in mud (approximately 70%). A substrate relationship between the two groups of bacteria is likely since ¹⁴CH₄ was formed from [U-¹⁴C]-l-lactate.     

UTILIZATION OF ACETATE AND PYRUVATE FOR THE SYNTHESIS OF'TOTAL','BOUND'AND'FREE'ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

—Slices of tissue of the electric organ of Torpedo marmorata were incubated in vitro in a salineurea-sucrose solution containing a labelled precursor of the acetyl moiety of ACh ([1-¹⁴C]glucose, [2-¹⁴C]pyruvate, or [1-¹⁴C]acetate) either alone or in the presence of another unlabelled precursor. The incorporation of ¹⁴C from [1-¹⁴C]acetate into ACh was considerably higher than from the other two substrates. The specific radioactivities (SRA) of the‘total',‘bound’and‘free’ACh were compared in experiments with [2-¹⁴C]pyruvate and [1-¹⁴C]acetate. With both precursors, the SRA of the‘bound’ACh were lower than those of‘total’ACh; consequently, the‘free’ACh pool was more labelled than the‘bound’pool. After short incubations with [2-¹⁴C]pyruvate the SRA of'bound’ACh were closer to the SRA of‘total’ACh than with [1-¹⁴C]acetate. A simple method is described for the labelling of ACh and its separation from other labelled compounds in experiments with the electric organ using [¹⁴C]acetate as the labelled precursor.     

Incorporation of intravenously injected (2-(14)C) acetate into tissue lipids of hamsters during the last hour of 3-, 6-, and 24-hour periods of hypothermia.

The in vivo incorporation of total lipid ¹⁴C from [2-¹⁴C]acetate is decreased in kidney, liver, and small intestine tissue from 3-, 6-, and 24-hr hypothermic hamsters compared to tissues from normothermic animals. The length of time in hypothermia affects hamster tissues differently; thus, ¹⁴C activity: decreases with time in kidney; increases with time in liver; and increases at 3 and 6 hr but decreases from 6 to 24 hr of hypothermia in small intestine.Tissues from hypothermic hamsters incorporated a greater percentage of [2-¹⁴C] acetate into free sterols and diglycerides and a smaller percentage into phospholipid than did corresponding tissues from normothermic hamsters.The percentage of total fatty acid ¹⁴C activity found as polyunsaturated fatty acid ¹⁴C activity increases in hypothermic kidney, liver, and small intestine with a decrease in the percentage of ¹⁴C activity measured in the saturated fatty acids. Esterification of fatty acid was inhibited in all tissues taken from hypothermic hamsters.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

Fatty Acid Transport Through the Blood-Brain Barrier

Across the cerebral capillaries, the anatomical locus of the blood-brain barrier, the unidirectional influxes of the saturated fatty acids, octanoic and myristic acids, and the unsaturated essential fatty acid, linoleic acid, were measured. Employing an in situ rat brain perfusion technique that allows control of perfusate composition and accurate measurement of perfusate-to-brain fatty acid transport, we found that both [¹⁴C]octanoic and [¹⁴C]myristic acids were transported through the blood-brain barrier in vivo, in large part, by a specific, probenecid-sensitive transport system. However, the transport of [¹⁴C]linoleic acid was not probenecid sensitive. With 0.5 μM fatty acid but no plasma proteins in the perfusate, the permeability-surface area constant was higher for myristic acid (4.8 × 10^--²× s^-1) than for octanoic and linoleic acids (1.5 and 1.2 × 10^--²× s^-1, respectively). Approximately 70, 30, and 25% of the [¹⁴C]myristic, [¹⁴C]octanoic, or [¹⁴C]linoleic acids, respectively, were extracted from the perfusate.     

Biosynthesis of lysine and other amino acids in the developing maize endosperm

Tracer studies with aspartic acid-[4-¹⁴C], alanine-[1-¹⁴C] acetate-[2-¹⁴C] and diaminopimelic acid-[1,(7)-¹⁴C] injected into the developing endosperm of maize revealed that the biosynthesis of lysine and other amino acids occurs in this organ. The data suggest that lysine is synthesized via the diaminopimelic acid pathway.     

Incorporation of Label from Acetate and Laurate into the Mannan of Leishmania donovani via the Glyoxylate Cycle

Leishmania donovani promastigotes in late-stationary phase incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into the mannose residues of mannan, thus confirming the presence of a functional glyoxylate bypass in these parasitic protozoa. Isolated, washed calls also incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into mannan during a 1-hr incubation in buffer. Glucose had no effect on label incorporation into mannan, but glutamate caused over a four-fold increase in incorporation from [2-¹⁴C]acetate and a 2.4-fold increase from [1-¹⁴C]laurate. Staurosporine, a protein kinase inhibitor that inhibits glutamate and alanine oxidation, did not inhibit label incorporation from [2-¹⁴C]acetate into mannan. Hyperosmolality caused about a 33% inhibition of label incorporation into mannan. These results show the glyoxylate cycle and/or the subsequent biosynthetic pathway from fructose-6-phosphate to mannan are subject to regulation.