Prostaglandin F2α produced by rabbit renal slices is not a metabolite of prostaglandin E2

Slices of rabbit renal medulla and rabbit renal papilla were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15-³H]-arachidonic acid. In both tissues, comparison of the isotope ratios of the radioactive products with the isotope ratio of the added arachidonic indicated that: (a) there was no discernable isotope effect in the biosynthesis of prostaglandin E₂; (b) prostaglandin F₂α was formed by reduction of prostaglandin H₂ and not by reduction of prostaglandin E₂; and (c) most of the radioactive product arose from arachidonic acid that had been incorporated into the tissue and not from the direct action of cyclooxygenase on arachidonic acid in the medium.     

Metabolism of 14C-arachidonate in rat isolated lung

Metabolism of ¹⁴C-arachidonate was investigated in rat isolated lungs perfused via the pulmonary circulation with Krebs solution. Only 10% of the radioactivity derived from an infusion of ¹⁴C-arachidonate through the pulmonary circulation of rat isolated lungs appeared in the effluent by 10 minutes. At 10 min, the major component of effluent radioactivity and 20–40% of that retained in lung was unchanged arachidonate. Between 10 and 20 min of perfusion, a further small amount of radioactivity was lost in lung effluent and at 20 min the retained radioactivity showed a decrease in the proportion present as free arachidonate. Between 20 and 60 min, there was no further loss of radioactivity in effluent and no further change in the distribution in lung. Addition of albumin to the Krebs solution perfusate during the infusion of ¹⁴C-arachidonate increased effluent radioactivity to 80%, but albumin added after 10 min only caused the efflux of a small amount of radioactivity (10%). Treatment of labelled lung at 20 min with the calcium ionophore A23187 released biologically active metabolites of arachidonate but very little radioactivity. Metabolism of arachidonate, either during the infusion or after retention in lung, in rat lung was closer to that in human lung than to that in guinea-pig lung.     

Incorporation of 2-14C-acetate into isolated nuclei and cytosolic lipids of rat thymus cells

It is generally recognized nowadays that active lipid metabolism takes place in the nucleus of a mammalian cell. Experimental data testify to the biosynthesis of polyphosphoinositides and phosphatidylcholine and reveal corresponding enzymes within nuclei of mammalian cells. These findings suggest that lipidmediated signaling pathways in nuclei operate independently of lipid-mediated regulatory mechanisms functioning in membranes and cytosol. To explore the pathways of intranuclear lipid biosynthesis, we studied incorporation of 2-¹⁴C-acetate into lipids of cytosol and isolated nuclei of rat thymus cells after separate and combined incubation with the labeled precursor. The most efficient incorporation of 2-¹⁴C-acetate into lipids (cholesterol, free fatty acids, and phospholipids) was observed in a reaction mixture containing cytosol. When the reaction mixture contained only nuclei, incorporation of the radioactive precursor into lipids also took place, but specific radioactivity of the lipids was essentially lower than in the cytosol. In both cases, 2-¹⁴C-acetate incorporated into phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylcholine, the most abundant membrane phospholipid, demonstrated the lowest radioactivity, which was significantly lower than that of phosphatidylethanolamine. Incorporation of newly synthesized free fatty acids in nuclear phospholipids was inhibited, if nuclei were incubated with cytosol. As a result, radioactive free fatty acids were accumulated in nuclei, while in cytosol they were efficiently incorporated into phospholipids. The levels of phospholipids and cholesterol remained constant regardless of incubation protocol, while the overall yield of free fatty acids decreased after combined incubation of nuclear and cytosolic fractions or after incubation of cytosol without nuclei. Putative mechanisms underlying the appearance of radioactive lipids in isolated nuclei of thymus cells are discussed.     

Incorporation of [14C]cinnamate into hydrolase-resistant components of the primary cell wall of spinach

[¹⁴C]Cinnamate was taken up very rapidly by cultured spinach cells and completely incorporated into low-MW conjugates within 20 min. The ¹⁴C-labelled products were similar whether the [¹⁴C]cinnamate was supplied continuously over a period of hours via a peristaltic pump or instantaneously. Radioactivity was slowly recruited from the low-MW pool into aromatic components of the cell-wall fraction. Saponification of the radioactive wall fraction yielded, in addition to radioactive ferulate and p-coumarate, large amounts of ethyl acetate-soluble radioactive material with the properties of oxidatively coupled phenols. The coupled material was associated with the most highly ‘Driselase’-resistant fractions of the cell wall. In contrast, ‘Driselase’ released most of the wall's ferulate and p-coumarate on disaccharide fragments. It is suggested that the oxidatively coupled phenols are formed from simpler phenols by peroxidase and that they cross-link the polysaccharides to which they are attached, making these polysaccharides relatively ‘Driselase’-resistant.     

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.     

Oxalate synthesis from [14C1]glycollate and [14C1]glycoxylate in the hepatectomized rat

Hepatectomy significantly altered the metabolism of [1-¹⁴C]glyoxylate and [1-¹⁴C]glycollate in the rat. The production of ¹⁴CO₂ was reduced by 47% and 77%–86%, respectively, indicating the involvement of the liver in the oxidation of both substrates. Unidentified intermediates, assumed to be primary glycine, serine and ethanolamine, were also reduced by over 50%, was would be expected from the removal of the aminotransferase enzymes through the hepatectomy. The biosynthesis of [¹⁴C]oxalate from [1-¹⁴C]glycollate was reduced by more than 80% in the hepatectomized rat. This suggests that this oxidation is primarily catalyzed by the liver enzymes, glycolic acid oxidase and glycolic acid dehydrogenase, in the intact rat. The limited formation of [¹⁴C]oxalate from [¹⁴₁]glycollate observed in the hepatectomized rat is probably catalyzed by lactate dehydrogenase or extrahepatic glycolic acid oxidase. Hepatectomy did not significantly alter the rate of formation of [¹⁴C]oxalate from [¹⁴₁]glyoxylate. However, since saturating concentrations of glyoxylate could not be used because of the toxicity of this substrate, the involvement of glycollic acid oxidase in this oxidation reaction in the intact rat can not be ruled out. In the hepatectomized rat, lactate dehydrogenase appears to be the enzyme making the major contribution, although other as yet not identified enzymes may be contributing. The increased deposition of oxalate in the tissues, oxalosis, may result from the shift in oxalate synthesis from the liver to the extrahepatic tissues.     

Incorporation of [14C]-2 deoxy-D-glucose into the lipids of normal cells as compared to virus-transformed cells

[¹⁴C]-2 deoxy-D-glucose is incorporated into the glycolipids of both normal and transformed cells. The chromatographic pattems of [¹⁴C]-2 deoxy-D-glucose labeled lipids differ markedly in oncornavirus and herpes simplex virus-transformed cells as compared to normal and virus-infected but not transformed cells. Deoxyglucoselabeled lipids with intermediate chromatographic mobility were enriched in normal and virus-infected but not transformed cells. Studies with a murine sarcoma virus-infected cell line which is temperature-sensitive for transformation indicated that the altered chromatographic pattern of [¹⁴C]-2 deoxy-D-glucose labeled lipids was related to the expression of the transformed phenotype.     

Metabolism of [6-14C]orotate by shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus

Incorporation of radioactivity from [6-¹⁴C]orotate into the pyrimidine constituents of shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus was examined with special reference to the unusual pyrimidine constituents. With each species, although 80% of the orotate supplied was catabolized to β-alanine, all the pyrimidine derivatives became radioactively labelled. With Pisum, the major part of the radioactivity incorporated into pyrimidines was located in UMP and the uracil derivatives, including the uracilyl amino acids willardiine and isowillardiine. With Phaseolus, UMP and the uracil derivatives were again the major radioactive products; incorporation of radioactivity into 5-ribosyluracil (pseudouridine), which accumulates in Phaseolus tissues, was comparable to the incorporation into orotidine and twice that found in cytidine. Lathyrus incorporated a substantially larger part of the presented [6-¹⁴C] orotate into pyrimidine derivatives than did the other two species. CMP was the most highly radioactive product, followed next by lathyrine and UMP. Surprisingly, 20% of the total radioactivity incorporated into pyrimidines by Lathyrus was located in the pyrimidine amino acid lathyrine. This confirms previous evidence that lathyrine is essentially a product of the orotate pathway. The overall recovery of radioactivity in all three species was 93–95%. The data emphasize the necessity of including the less common pyrimidine constituents, as well as the common ones, in quantitative studies of pyrimidine metabolism in plants.     

Prostaglandin A2 and 35S uptake in connective tissue

Rats deficient in essential fatty acids (EFA) incorporated lesser amounts of radioactive sulfate into lung, kidney, spleen, heart, costal cartlidge, long bone and skull bone than did normal control animals. Administration of prostaglandin A₂ stimulated ³⁵S uptake by lung, kidney and aorta while ³⁵S levels in costal cartilage, tibial cap and long bone were strikingly reduced. Comments are presented suggesting that this metabolic mechanism may explain, in part, cartilage and bone resorption in areas of inflammation, such as arthritis, both rheumatoid arthritis and osteoarthritis.     

The lack of movement of [14C]-phenylalanine and [14C]-cinnamate after administration to leaves of Polygonum and wheat

U-¹⁴C-phenylalanine and 3-¹⁴C-cinnamic acid were fed to detached Polygonum leaves through the cut petioles and to the bases of detached wheat leaves. After feeding, the leaves were divided into basal, middle and terminal segments; for each treatment of each plant more than 80% of the total radioactivity incorporated remained in the basal segment. The distribution of radioactivity between ethanol-soluble and insoluble fractions in each segment was examined. The basal segments contained more insoluble radioactivity than the terminal ones; the differences were far more marked for both plants when cinnamate rather than phenylalanine was administered. In view of the gross differences in distribution of radioactivity between the basal and terminal segments of each leaf, it is concluded that basal infusion of precursors is not the most suitable technique for studies of phenolic biosynthesis.     

Biosynthesis of radiolabelled alkaloids from 14C-tyrosine in Erythrina crista-galli

Alkaloid biosynthesis has been demonstrated in the young expanding tissues of Erythrina crista-galli shoots, the stem part of which elaborated, from [2-^14C]-tyrosine, alkaloid of specific radioactivity 2μuCi mmol^?1, 10-fold greater than previously reported.     

Ovarian sequestration of [14C]-inulin by an insect: An index of vitellogenesis

[¹⁴C]-Inulin injected into the blood of female milkweed bugs (Oncopeltus fasciatus) undergoing vitellogenesis is sequestered in the eggs. Determination of ovarian radiocarbon uptake gives a reproducible index of the progression of vitellogenesis that permits quantitative measurements prior to oviposition or under conditions where vitellogenesis is incomplete. This assay allows intra- and interspecific comparisons of the rates of juvenile hormone biosynthesis by corpora allata (CA) when these endocrine glands are transplanted into milkweed bug females, whose CA have been chemically destroyed with precocene. The method has the potential of distinguishing between nervous and hormonal regulation of the CA.     

Desaturation of oleate-[14C] in leaves of barley

Etiolated barley leaves when exposed to light desaturate oleate-[¹⁴C] to linoleate. The production of substantial amounts of radioactive linolenate was found only in very young, tightly rolled leaves. In oleate-[¹⁴C] pulse experiments, radioactive linolenate first appeared in phosphatidylcholine (PC) and only after a lag period did it begin to accumulate in monogalactosyldiacylglycerol (MGDG). The results indicate that in young, immature barley leaves linolenate is synthesized from oleate on the parent lipid, PC, and is then transferred to MGDG.     

Plasmodium lophurae: Quantitative in vitro incorporation of 14C-1-acetate into lipids

Blood from ducks parasitized with Plasmodium lophurae and normal duck blood were incubated with sodium ¹⁴C-1-acetate. After release of the parasites from infected red blood cells (RBC) and concurrent treatment of normal blood, lipids were extracted from cellular material and plasma and lipid classes separated by thin-layer chromatography. Specific activity (dpm/mg lipid) of lipid classes was measured quantitatively by liquid scintillation radioassay and gravimetric analysis. The data indicated that the parasite within the RBC incorporated ¹⁴C-labeled lipid precursors.Experiments employing sodium ¹⁴C-1-acetate in two concentrations, 50 μCi ¹⁴C in 0.91 μmole sodium acetate/50 ml blood and 500 μCi ¹⁴C in 9.1 μmole sodium acetate/50 ml blood (1.82 × 10^?5M and 1.82 × 10^?4M), showed higher ¹⁴C incorporation into parasitized blood than normal blood preparations at the higher substrate concentration at 5 hr of incubation. At $\text{1.82 × 10}{}^{\mathrm{?5}}\text{M}{}^{14}$C-1-acetate, the highest specific activity in P. lophurae was associated with lipid alcohols. Monoglycerides and diglycerides were significantly labeled. At the higher acetate concentration (1.82 × 10^?4M), monoglyceride and diglyceride lipid classes had the highest specific activity in preparations of partially purified P. lophurae.Lipids of plasma from parasitized blood incubated for 5 hr with both concentrations of labeled acetate exhibited the highest specific activity in the free fatty acid class and sterols.At 24 hr of incubation, the lipids of partially purified P. lophurae had increased specific activity in free fatty acids, diglycerides, monoglycerides, phospholipids, and triglycerides.In plasma from parasitized blood incubated 24 hr with ¹⁴C-1-acetate, the highest specific activity and greatest percent of ¹⁴C incorporation was found in free fatty acids.     

Biosynthesis of lipoic acid in the rat: Incorporation of 35S- and 14C-labeled precursors

After injection of various ³⁵S- and ¹⁴C-containing compounds, the incorporation of the label into the lipoic acid present in the liver of growing rats has been determined. The best precursor for sulfur atoms, after 24 h, is cysteine; methionine and cystamine are scarcely incorporated and thiosulfate not at all. Good precursors of the carbon moiety are acetate and octanoate, whereas the incorporation of butyrate and cysteine is very low. It is concluded that lipoic acid is biosynthesized in the rat liver, and that sulfur atoms probably originate from cysteine.     

Effect of Vanadium Pentoxide on the Incorporation of (2-14C)-Acetate into Fungal Lipids

Mycelia of Glomerella cingulata grown in the presence of vanadium pentoxide (V₂O₅), incorporated many times less (2-¹⁴C)-acetate into their lipids than mycelia of the same organism grown in the absence of V₂O₅. All neutral and polar lipid fractions investigated were affected. These data suggested that V₂O₅ depressed lipid biosynthesis in G. cingulata.     

Amo 1618 effects on incorporation of 14C-MVA and 14C-acetate into sterols in Nicotiana and Digitalis seedlings and cell-free preparations from Nicotiana

Incorporation of radioactivity from acetate-[¹⁴C] and MVA-[¹⁴C] into sterols and sterol precursors in tobacco was inhibited by Amo 1618; differing patterns of accumulation were obtained with the two precursors, suggesting more than one point of inhibition. This was borne out with cell-free preparations with which it was demonstrated that both HMG-CoA reductase and squalene-2,3-epoxide cyclase were inhibited, the latter more strongly than the former. GLC analysis of gross sterol and hydrocarbon fractions confirmed previous indications that incorporation of radioactivity into individual sterols was inhibited by Amo 1618. Finally, incorporation of MVA-[¹⁴C] into sterols and sterol precursors of Digitalis was significantly altered by the retardant, thus expanding the generality of the relationship between sterol (particularly 4-desmethylsterol) biosynthesis inhibition and retardant effect.     

14CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

Uptake of [3H]-arachidonic acid by human endometrium. Differences between normal and menorrhagic tissue

Human proliferative and secretory endometrium from normal women and from menorrhagic patients was maintained in culture for up to a 24 h in the presence of [³H]-arachidonic acid (³H-AA). This prostaglandin (PG) precursor was incorporated into endometrial neutral lipids and phospholipids in a time-dependent manner. Uptake of ³H-AA into phospholipids was significantly higher in normal secretory endometrium than in normal proliferative endometrium. However, this increased uptake of ³H-AA into phospholipids between the 2 phases of the cycle did not occur in menorrhagic endometrium. In contrast, uptake of ³H-AA into neutral lipids (especially triglyceride) was approximately 2-fold higher in menorrhagic endometrium compared to normal endometrium at both stages of the cycle, particularly during the proliferative phase. Abnormalities apparently exist in menorrhagic endometrium in the uptake processes which control arachidonic acid (AA) turnover. These abnormalities may be responsible, in part for abnormal PG production by menorrhagic endometrium.     

Double-label autoradiography on polyacrylamide gels with 3H and 14C

A method of autoradiography is deseribed which allows discrimination between ³H- and ¹⁴C-labeled materials on polyacrylamide gels. The method relies on a very simple procedure for switching from fluorography to autoradiography which results in a decrease in ¹⁴C detection sensitivity of about 10-fold but a concomitant decrease in ³H sensitivity of over 800-fold. With the proper ratio of ³H to ¹⁴C radioactivities, there is little or no “spillover” of one isotope during the detection of the other. No special equipment is required.