Greater Proportion Of Radioactivity Research Articles

Interleukin-2 Causes an Increase in Saturated/Monounsaturated Phosphatidic Acid Derived from 1,2-Diacylglycerol and 1-O-Alkyl-2-acylglycerol

Phosphatidic acid generation through activation of diacylglycerol kinase alpha has been implicated in interleukin-2-dependent T-lymphocyte proliferation. To investigate this lipid signaling in more detail, we characterized the molecular structures of the diradylglycerols and phosphatidic acids in the murine CTLL-2 T-cell line under both basal and stimulated conditions. In resting cells, 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol subtypes represented 44 and 55% of total diradylglycerol, respectively, and both showed a highly saturated profile containing primarily 16:0 and 18:1 fatty acids. 1-O-Alk-1'-enyl-2-acylglycerol represented 1-2% of total diradylglycerol. Interleukin-2 stimulation did not alter the molecular species profiles, however, it did selectively reduce total 1-O-alkyl-2-acylglycerol by over 50% at 15 min while only causing a 10% drop in 1,2-diacylglycerol. When radiolabeled CTLL-2 cells were challenged with interleukin-2, no change in the cellular content of phosphatidylcholine nor phosphatidylethanolamine was observed thereby ruling out phospholipase C activity as the source of diradylglycerol. In addition, interleukin-2 failed to stimulate de novo synthesis of diradylglycerol. Structural analysis revealed approximately equal amounts of 1,2-diacyl phosphatidic acid and 1-O-alkyl-2-acyl phosphatidic acid under resting conditions, both containing only saturated and monounsaturated fatty acids. After acute (2 and 15 min) interleukin-2 stimulation the total phosphatidic acid mass increased, almost entirely through the formation of 1-O-alkyl-2-acyl species. In vitro assays revealed that both 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol were substrates for 1,2-diacylglycerol kinase alpha, the major isoform in CTLL-2 cells, and that the lipid kinase activity was almost totally inhibited by R59949. In conclusion, this investigation shows that, in CTLL-2 cells, 1,2-diacylglycerol kinase alpha specifically phosphorylates a pre-existing pool of 1-O-alkyl-2-acylglycerol to form the intracellular messenger 1-O-alkyl-2-acyl phosphatidic acid.

Distribution and metabolism of abamectin in larval stages of Spodoptera littoralis boisduval and Heliothis armigera hübner (Lepidoptera: Noctuidae)

AbstractComparisons were made between the amounts of abamectin and total radioactivity recovered from Spodoptera littoralis and Heliothis armigera larvae after topical application of [3H]abamectin. Penetration (as shown by wash‐off experiments) did not differ significantly between the Spodoptera instars. Significantly more abamectin was recovered from ventral nerve cord samples of larvae showing symptoms of poisoning than from larvae not showing these symptoms. Fifth‐instar S. littoralis larvae had a significantly lower proportion of radioactivity as abamectin in the ventral nerve cord than in sixth‐instar S. littoralis or fifth‐ and sixth‐instar H. armigera. The proportion of radioactivity present as abamectin (but not total radioactivity) was significantly increased when the fifth‐instar S. littoralis larvae were pre‐treated with piperonyl butoxide (PB) suggesting that the relative insensitivity of fifth‐instar S. littoralis larvae to abamectin is due at least in part to greater metabolism, particularly by microsomal oxidases. Fat body samples consistently had a greater proportion of radioactivity as abamectin than the nerve cord and the former may act as storage sites for abamectin.

Metabolism of apoprotein B in selectively bred baboons with low and high levels of low density lipoproteins.

Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) apoprotein (apo)-B turnover rates were measured simultaneously by injecting 131I-labeled VLDL and 125I-labeled LDL into fasting baboons (Papio sp.) selectively bred for high serum cholesterol levels and having either low or high LDL levels. The radioactivities in VLDL, intermediate density lipoprotein (IDL), LDL apoB, and urine were measured at intervals between 5 min and 6 days. Kinetic parameters for apoB were calculated in each baboon fed a chow diet or a high cholesterol, high fat diet (HCHF). VLDL apoB residence times were similar in the two groups of animals fed chow; they were increased by HCHF feeding in high LDL animals, but not in low LDL animals. Production rates of VLDL apoB were decreased by the HCHF diet in both high and low LDL animals. Most of the radioactivity from VLDL apoB was transferred to IDL. However, a greater proportion of radioactivity was removed directly from IDL apoB in low LDL animals than in high LDL animals, and only about one-third appeared in LDL. In high LDL animals, a greater proportion of this radioactivity was converted to LDL (61.4 +/- 7.2% in chow-fed animals and 49.2 +/- 10.9% in animals fed the HCHF diet; mean +/- SEM, n = 5). Production rates for LDL apoB were higher in high LDL animals than those in low LDL animals on both diets. The HCHF diet increased residence times of LDL apoB without changing production rates in both groups. VLDL apoB production was not sufficient to account for LDL apoB production in high LDL animals, a finding that suggested that a large amount of LDL apoB was derived from a source other than VLDL apoB in these animals.

Autoantibodies to thyroxin and triiodothyronine.

Two clinically euthyroid patients with multinodular goiter were found to have high "free" thyroxin (Amerlex-M, Amersham and Coat-a-Count, DPC) and triiodothyronine (Amerlex-M) concentrations (FT4 and FT3, respectively). The presence of antibodies to T4 and T3 was confirmed by the finding that polyethylene glycol precipitated a far greater proportion of radioactivity when radiolabeled FT4 or FT3 analog (Amerlex-M) was incubated with serum from these patients than was true for normal subjects. With this method we could not demonstrate antibodies to thyroid hormones in 116 healthy volunteers. Of 101 hyperthyroid patients tested, one had antibodies to T4 but none had antibodies to T3. One patient had antibodies to T4, and one to T3, of 36 hypothyroid patients tested. All patients with thyroid hormone antibodies also demonstrated antithyroglobulin antibodies (measured immunoradiometrically). Evidently, the presence of thyroid hormone antibodies should be suspected when results of thyroid-function tests are discordant with the clinical state, and we suggest that measurement of thyrotropin by an assay with improved detection limits will aid in correctly determining thyroid status.

Intact biliary excretion of gastrically administered prostaglandin F2 alpha in rats: developmental differences.

Tritium-labeled prostaglandin F2 alpha was administered via orogastric tube to bile duct-cannulated suckling and weanling rats to determine if maturational differences were present in the biliary excretion of prostaglandin F2 alpha and metabolites. Animals were killed 2 h after radioactivity administration. Characterization of radioactivity present in bile revealed age-related differences in biliary prostaglandin F2 alpha excretion. Suckling rats had a greater proportion of radioactivity migrating in chromatographic regions of greater polarity than prostaglandin F2 alpha. Compared with the weanling, a significantly greater amount of radioactivity cochromatographed with intact, unmetabolized prostaglandin F2 alpha (33.08 +/- 1.99 vs. 21.38 +/- 1.46). These results indicate that orogastrically administered prostaglandin F2 alpha can be absorbed from the gastrointestinal tract, transported to the liver, and subsequently excreted into bile and detected in an unmetabolized form in suckling and weanling rats. The enterohepatic circulation of milk-derived prostaglandin present in bile may contribute to the overall content of intestinal prostaglandins.

Arachidonic acid metabolism in alveolar macrophages. A comparison of cells from healthy subjects, allergic asthmatics, and chronic bronchitis patients

Arachidonic acid (AA) metabolism was studied in preparations of purified human alveolar macrophages (AM) from healthy subjects (HS = 5), allergic asthmatics (ABA = 9) and chronic bronchitis patients (CB = 7). AM incubated for 6 to 24 h in the presence of labeled AA and for an additional 5 h without labelled AA, released cyclooxygenase and lipoxygenase products into the medium. The study of the metabolites showed that the most abundant sulfidopeptide-leukotriene, was LTD4 as analyzed by TLC and identified by reversed phase HPLC. The release of LTD4 was time-dependent but it was shown to be significantly higher (p ‹ 0,01) in AM from ABA or CB than in those from HS. TLC analysis of radioactivity distributed between the different lipid classes at 24 h revealed more labeling in AM phospholipids from ABA and CB than in those from HS, and was reflected in phosphatidylethanolamine and phosphatidylinositol species. After 5 h without labelled AA the distribution was marked, by different in triglycerides, with a greater proportion of radioactivity in the control cells than in the pathological macrophages. Thus, the pathological lung state is an important factor affecting the release of LTD4 and the distribution of AA into cellular phospholipids. The differences observed between HS and ABA or CB phospholipid distribution suggests the existence of 2 different sources of AA release, one for inflammatory macrophages and another for quiescent cells.

Thyroxine increases neonatal mouse submandibular gland mRNA-directed synthesis of epidermal growth factor

Thyroid hormones are known to modulate the concentrations of epidermal growth factor (EGF) in the mouse submandibular gland (SMG); this action is presumably mediated by the nuclear triiodothyronine receptor. To test the hypothesis that thyroid hormones act to increase SMG EGF concentrations by increasing the number of poly(A)+ -specific mRNA, poly(A)+ RNA was isolated from SMGs of neonatal mice which had been treated daily from birth through to 21 days of age with thyroxine (T4,0.4 microgram/g body weight). Poly(A)+ RNA also was extracted from SMGs of intact 21-day-old mice which had received vehicle alone. No significant differences in total nucleic acid, total RNA, or poly(A)+ RNA yields were noted between the two groups of animals. The isolated poly(A)+ RNAs from T4-treated and control mice were translated in an in vitro wheat germ system. Although no significant differences in efficiency of [35S]cysteine incorporation into trichloracetic acid precipitable material were noted between the two poly(A)+ RNA preparations, a significantly greater proportion of radioactivity was immunoprecipitable by anti-EGF antiserum in the translation medium derived from T4-treated mice (17.2 +/- 0.9%, mean +/- SEM) than in that of control mice (7.3 +/- 0.5%, P less than 0.001). Polyacrylamide gel electrophoresis of the immunoprecipitates (IMMP) revealed the presence of three radioactive bands with apparent relative masses (MrS) of 12,000, 9000, and 6000. The latter species comigrated with purified EGF, [125I]EGF, and an IMMP of a SMG extract. The translation product IMMPs following polyacrylamide gel electrophoresis were iodinated and digested with alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of methods of incorporation and source of14C on distribution of radioactivity in potato plants

Russet Burbank potato plants grown in the greenhouse were given14C as14CO2 and as uniformly labelled sucrose. Labelled sucrose was applied; (1) by injection into the main stem, (2) injection with a hypodermic needle into the debladed petiole and (3) uptake of labelled sucrose kept in a vial fastened to the main stem using thread as a wick. Plants harvested 6 hours later were analyzed for radioactivity in the foliage, roots, and tubers. The greatest proportion of radioactivity was found in the foliage. Tubers were analyzed for radioactivity in the basic (amino acids), acidic (organic acids and sugar phosphates), neutral (sugars) and alcohol insoluble solid (starch) fractions. Most of the radioactivity was found in the sugar component of the chemical fraction. Plants treated with147CO2 had a higher percentage of radioactivity in the acidic and starch fractions than plants treated with sucrose. Sucrose applied by petiole injection not only showed higher total incorporation but also exhibited more translocation of labelled material into the tubers than the other methods.

Distribution and fate of nicotine in the rat fetus.

Abstract[3H] (−)‐Nicotine was administered iv into pregnant Long‐Evans rats on day 19 of gestation or in three consecutive daily doses given on days 16 through 18 of gestation. The concentration of radioactivity and proportions of labeled compounds were determined at intervals from 5 min to 20 h after injection in maternal plasma and liver, decidua basalis, placenta, amniotic fluid, whole fetus, and fetal plasma, brain, lungs, heart, liver, adrenals, kidneys, gonads, and the stomach‐intestine‐spleen‐pancreas en bloc. Nicotine, cotinine, and other metabolites appeared in the fetus within 5 min of injection. The concentration of radioactivity in the fetal plasma exceeded that of the maternal plasma from 30 min through 20 h. Fetal liver had a greater proportion of radioactivity as nicotine than did maternal liver. Fetal plasma as well as most of the fetal tissues had a greater proportion of nicotine to metabolites than did the maternal plasma throughout the time period that was studied. By 60 min an equilibrium was established between maternal plasma and amniotic fluid with respect to concentration of radioactivity. This equilibrium was maintained through 20 h after single injections and also after the three daily injections. The proportions of nicotine to major metabolites were similar in maternal plasma and amniotic fluid at 4 h. The findings show that fetal rat tissues are exposed to levels of nicotine greater than those found in maternal plasma beginning within 30 min of the time nicotine enters the maternal circulation.

Metabolism of phospholipids and the characterization of fatty acids in bovine corpus luteum.

1. Phosphatidylcholine was the predominant phospholipid in bovine corpora lutea; it accounted for about 50% of the total phospholipid phosphorus. Phosphatidylethanolamine (13%) and ethanolamine plasmalogen (8-9%) were the next two major components. 2. After incubation of the tissue with [(32)P]orthophosphate the total radioactivity and specific radioactivity of phosphatidylinositol were higher than those of any other lipid. 3. Luteinizing hormone failed to increase significantly the incorporation of [(32)P]orthophosphate into total phospholipids from luteal tissue slices, but did stimulate progesterone synthesis and lactate production. 4. The proportion of oleate (18:1) in the neutral lipids and phospholipids was higher than that of any other fatty acid. 5. The proportion of unsaturated fatty acid in the tissue lipids exceeded 60%, and almost half of this was polyunsaturated. Arachidonate (20:4), docosatetraenoate (22:4) and docosapentaenoate (22:5) were the principal polyunsaturated fatty acids. 6. After incubation of luteal tissue with [1-(14)C]acetate, the greatest proportion of radioactivity in the fatty acids isolated from the total lipid fraction was in palmitate (16:0) and docosatetraenoate (22:4). Polyunsaturated fatty acids accounted for almost 50% of the (14)C radioactivity incorporated and this pattern was observed in phospholipids, triglycerides and free fatty acids.

Absorption of 14C triolein in the bile fistula rat.

SummaryGlyceride triolein-1-14C was fed to bile fistula and sham operated rats which were killed after 4 hours. In the bile fistula animals there was a diminished absorption and decreased lipolysis of the triolein. Analysis of small gut mucosal lipid showed a greater proportion of radioactivity in the FFA and lower glycerides of the bile fistula animals than of the controls. This supports the concept that bile facilitates mucosal esterification of absolrbed long chain fat.

Steroid metabolism in the rabbit. Biliary and urinary excretion of metabolites of [4-14C]progesterone

1. [4-(14)C]Progesterone was administered intravenously to anaesthetized male and female New Zealand White rabbits as a single injection or as a 45-60min. infusion. 2. After a single dose about 60% of the radioactivity was recovered in 6hr., and twice as much radioactivity was present in bile as in urine. After infusion total recovery of radioactivity was only about 40% in 6hr., but the relative proportions of metabolites in bile and urine were about the same as after a single dose. 3. Bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid. 4. In bile the major proportion of metabolites appeared in the glucuronide fraction; in urine beta-glucuronidase hydrolysis yielded the greatest amounts of ether-extractable radioactivity, but the greatest proportion of radioactivity could not be extracted by ether from an alkaline solution of the hydrolysed urine. 5. There was no apparent difference in the quantity or distribution of metabolites excreted by male and female animals.

Preparation of radiochemically pure tritium nuclear-labelled morphine.

MORPHINE has been labelled with carbon-14 in the N-methyl position by chemical synthesis1,2 and randomly by biosynthesis3,4. Because of the metabolic lability of the N-methyl group and the need for considerable quantities of a product of high specific activity, studies on the preparation of tritium-nuclear labelled morphine were initiated. The Wilzbach procedure5 of exposure to tritium gas was utilized for the introduction of the tracer element into the morphine molecule. The preparation of biologically stable tritium-labelled morphine by this technique had been described previously6, and the radiochemical purity of the product was reported. We have found, however, that the exposure to tritium gas of morphine base or morphine sulphate in finely divided form, and subsequent purification to remove the labile tritium and radio-decomposition products, uniformly gave a seemingly pure crystalline morphine which showed two distinct areas of radioactivity when paper chromatograms were developed using correctly buffered paper and the proper solvent mixtures. One area contained a greater proportion of radioactivity (approximately 65–70 per cent of total) but negligible mass of material (not detectable by spray reagents or by visualization with ultra-violet light, less than 1 per cent). Similarly when normorphine base or its hydrochloride was tritiated in the same manner and purified, the morphine synthesized from it by the method of Andersen and Woods1 showed the same trace radio contaminant. When paper chromatograms were prepared from the above tritium-labelled ‘morphine’ using non-buffered paper and several solvent mixtures, that is, n-butanol, formic acid, and water (12 : 1 : 7 v/v); 2.3 per cent p-toluene sulphonic acid in n-butanol and water (100: 14-v/v), etc., all the radioactivity was located in one area. This area was the alkaloidal spot as localized by spraying with iodoplatinate alkaloidal reagent or by visualization of the shadow with ultra-violet light. The R F values were identical with parallel non-labelled morphine.