Biogenesis of nonacosan-15-one in brassica oleracea

The incorporation of [4-¹⁴C]4-oxostearic acid, [4-¹⁴C]stearic acid, and [1-¹⁴C]eicosanoic acid, respectively, into the nonacosan-15-one of Brassica oleracea has been investigated. While the oxo-acid was found to be a poor precursor of nonacosan-15-one, the two n-fatty acids were efficiently incorporated as intact units into n-nonacosane and the related 15-ketone. In accordance with expectation, the label in the 4-position of stearic acid appeared in the carbonyl carbon of the ketone. These results suggest that the C₂₉ hydrocarbon is first formed and subsequently oxidized to nonacosan-15-one.     

Oxidation of [1-C]linoleic acid in isolated microsomes from pea leaves

The oxidation of [1-¹⁴C]linoleate in isolated microsomes from pea leaves was found to be stimulated by NADPH addition. The formation of one of the main metabolites, 12-hydroxy-9(Z)-dodecenoic acid is particularly NADPH-dependent. The predominant products in the absence of NADPH were hydroperoxides and in the presence of NADPH, 12-hydroxy-9(Z)-dodecenoic acid. Exogenous [1-¹⁴C]-13-hydroperoxy-9(Z), 11(E)-octadecadieoic acid and [1-¹⁴C]-12-oxo-9(Z)-dodecenoic acidwere the efficient precursors of 12-hydroxy9(Z)-dodecenoic acid. It was concluded that 12-hydroxy-9(Z)-dodecenoic acid is formed by NADPH-dependent enzymatic reduction of 12oxo-9(Z)-dodecenoic acid. The observed inhibition of linoleate oxidation in isolated microsomes by CO and metryapone suggests the involvement of cytochrome P-450 in the reaction. The relative contribution of lipoxygenase and monooxygenase activity to linoleate oxidation in microsomes is discussed.     

The sequence of initiation of RNA, DNA and protein synthesis in the wheat grains during germination

The syntheses of main macromolecular substances, in a whole wheat grain allowed to germinate, are triggered in the following order: RNA, protein, DNA. The RNA synthesis, as judged by [2-¹⁴C]uridine incorporation, is initiated almost immediately after the seeds are exposed to the optimal germination conditions, whereas [1-¹⁴C]leucine and [2-¹⁴C]thymidine incorporation begins to occur only 3 and 4 hr later, respectively. The initiation of protein synthesis is accompanied by an apparent cessation of uridine incorporation.     

On the mechanism of biosynthesis of leukotrienes and related compounds

[10D-³H; 3-¹⁴C]- and [10L-³H; 3-¹⁴C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B₄ and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the ³H/¹⁴C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8cis,11cis,14cis-eicosatetraenoic acid into leukotriene A₄ whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.     

Metabolism of some possible ecdysone precursors in Calliphora stygia

A study has been made, using Calliphora stygia at the time of puparium formation, of the incorporation of a number of labelled sterols into β-ecdysone. [1-³H]-Cholesterol and [4-¹⁴C]-cholesterol are incorporated to a similar extent (0·01-0·02%). [1-³H]-7-Dehydrocholesterol is better incorporated (0·025%) than cholesterol while [1-³H]-cholesterol sulphate, (22R)-22-hydroxy-[22-³H]-cholesterol, and 25-hydroxy-[26-¹⁴C]-cholesterol are not incorporated to a significant extent.     

The effect of exogenous gibberellic acid on gibberellin biosynthesis by Gibberella fujikuroi

The addition of gibberellic acid and some other gibberellins to cultures of Gibberella fujikuroi suppresses the incorporation of [2-¹⁴C]MVA and ¹⁴C-labelled ent-kaurene into the gibberellin metabolises.     

Determination of δC values of valine in protein hydrolysate by gas chromatography–combustion isotope ratio mass spectrometry

A gas chromatographic–combustion isotope ratio mass spectrometric (GC–C-IRMS) method for the determination of [1-¹³C]valine enrichments in protein hydrolysates is described. Using a quick derivatization method, δ¹³C values of the N-methoxycarbonyl methyl ester of valine can be determined from baseline separated GC peaks. Evaluation studies with respect to precision, accuracy, linearity, reduction capacity of the CuO combustion furnace and isotope dilution as a result of derivatization, showed that our GC–C-IRMS system allows robust measurement of enrichments of [1-¹³C]valine in the range 0 to 1.5 MPE (S.D.±0.01 MPE, n=3). Therefore this method is suited to determine fractional synthetic rates (FSRs) of proteins as low as one-tenth of the FSR of human albumin, in studies using a primed, continuous (6 h) infusion with [1-¹³C]valine plasma enrichments of approximately 15 MPE and an hourly sampling schedule.     

Biosynthetic studies of lactucin derivatives in hairy root cultures of Lactuca floridana

Biosynthetic studies of the guaianolide-type sesquiterpene lactones 11βH,13-dihydrolactucin-8-O-acetate and 8-desoxylactucin were performed in Agrobacterium rhizogenes—transformed hairy root cultures of blue-flowered lettuce, Lactuca floridana. The ¹³C NMR spectra of the two guaianolides labelled by incorporation of [1-¹³C], [2-¹³C], [1,2-¹³C₂]acetate and [2-¹³C]mevalolactone showed patterns of enrichment consistent with a previously proposed biogenetic pathway for guaianolide-type sesquiterpene lactones via the acetate-mevalonate-germacradiene route.     

Oxygenation of Hexadecane in the Biosynthesis of Cyclic Glycolipids in Torulopsis Apicola

Biotransformation of [1-¹³C] labelled hexadecane, hexadecanol and hexadecanoic acid have been investigated using the yeast Torulopsis apicola. The yeast produces a microcrystalline mixture of two glycolipids, the lipophilic moiety of which consists of ω- or (ω-l)-hydroxylated hexadecanoic acid. Biosynthesis of these glycolipids takes place via hydroxylation of hexadecane, oxidation to hexadecanoic acid and ω or (ω-l)-hydroxylation of hexadecanoic acid. Feeding the cell cultures with a mixture of hexadecane and [1-¹³C] labelled hexadecane derivatives one observes ¹³C enrichment ratios which indicate that neither of the biohydroxylation or oxidation steps are rate limiting in the formation of the glycolipids, furthermore, two different monooxygenase systems appear to be involved in hydroxylation of hexadecane and hexadecanoic acid.     

Phycomyces blakesleeanus car B mutants: Their use in assays of phytoene desaturase

Strains of car B (phytoene-accumulating) mutants of Phycomyces blakesleeanus have been characterized with respect to their carotene contents, in vitro formation of isoprenoids from [2-¹⁴C] mevalonic acid and their ability to produce [¹⁴C]phytoene in situ for use in coupled assays of phytoene desaturase activity. All strains produced predominantly (15-Z)-phytoene both in vivo and in vitro. Other isoprenoids were produced by cell extracts including squalene, sterols, prenyl diphosphates and prenyl alcohols. The addition of 1% Tween 60 to crude cell extracts of the mutants partially restored wild type carotenogenic activity and also altered the proportions of other isoprenoids formed. However, in a cytosolic fraction of the car B mutant, the addition of 1% Tween 60 did not result in the production of any carotenoid from phytoene. This fraction was the most effective source of [¹⁴C] phytoene for use in coupled assays of phytoene desaturase activity.     

Inhibitory effect of inorganic phosphate on the axonemal ATPase of cilia from Tetrahymena pyriformis

An inhibitory effect of inorganic phosphate on the axonemal ATPase of cilia from Tetrahymena pyriformis was shown. P_i inhibited the terminal phosphate liberation from [γ-³²P]ATP by 30-S dynein and inhibited the conversion of [8-¹⁴C]ATP to ADP and AMP by digitonin-extracted cilia.     

Regulation of pathways of glucose metabolism in kidney: Specific linking of pentose phosphate pathway activity with kidney growth in experimental diabetes and unilateral nephrectomy

The pentose phosphate pathway operates at an elevated level in rat kidney following induction of diabetes and in the compensatory hypertrophy following unilateral nephrectomy in control and alloxan-diabetic rats, as shown by the yields of ¹⁴Co₂ from [1-¹⁴C]glucose, [6-¹⁴C]glucose and ³H₂O yields from [2-³H]glucose. The elevated flux through the pentose phosphate pathway is correlated with the increased RNA content and weight of the kidney. The direct utilization of NADPH for reductive synthetic reactions and the potential for indirect utilization via the sorbitol route and the linked transhydrogenase reactions of the glucuronate-xylulose pathway, for NADH and ATP generation, are also discussed.     

The use of exogenous δ-aminolaevulinic acid for the studies of the regulation of haem synthesis in rabbit reticulocytes

δ-Amino [4-¹⁴C]laevulinate added to reticulocytes incubated in vitro is incorporated into haem. Exogenous δ-aminolaevulinate restores the incorporation of ⁵⁹Fe into haem in reticulocytes which had been treated with isonicotinic acid hydrazide (INH) or penicillamine and were hence unable to synthesize δ-aminolaevulinate. On the other hand, the addition of δ-aminolaevulinate does not restore the incorporation of Fe into reticulocytes incubated with haemin. The inhibition of the incorporation of iron is neither restored by δ-aminolaevulinate in reticulocytes incubated with cycloheximide (which inhibits globin synthesis and thus elevates the free intracellular haem pool). These results suggest that in intact reticulocytes haemin does not inhibit δ-aminolaevulinate synthetase. This conclusion is further supported by the finding that the pattern of incorporation of [2-¹⁴C]glycine and δ-amino[4-¹⁴C]-laevulinate into haem differs in reticulocytes incubated with an inhibitor of δ-aminolaevulinate synthetase (INH) and in reticulocytes incubated with haemin and cycloheximide.     

Modulation of protein kinase C activity by NaF in bone marrow derived macrophages

Stimulation of murine bone marrow derived macrophages with NaF, prelabeled with [1-¹⁴C]oleate and [³H]inositol, increased the production of inositol phosphates and the release of 1,2-[¹⁴C]diacylglycerol (DAG). Moreover, NaF also induced activation of protein kinase C. These results indicate that bone marrow derived macrophages exhibit a phosphatidyl-4,5-bisphosphate phospholipase C activity, sensitive to NaF, which might be modulated by G-proteins. Activation of protein kinase C could have been mediated by NaF-induced release of DAG.     

Ascorbic acid catabolism by gut microflora: studies in germ-free and conventional guinea pigs

The role of gut microflora in ascorbic acid catabolism was investigated in both conventional and germ-free guinea pigs. In vitro studies demonstrated extensive degradation of the vitamin by fresh feces, cecal, and colonic contents of conventional guinea pigs. Direct injection of [1-¹⁴C] ascorbic acid into the cecum of conventional guinea pigs in vivo yielded a 70% recovery of the label as respiratory ¹⁴CO₂ within 6 hr compared with only 5% recovery following injection into the virtually sterile peritoneum in a comparable group of conventional guinea pigs. Thus, ascorbic acid not absorbed prior to reaching the lower gastrointestinal tract stands to be extensively decarboxylated by microflora in the cecum. In a companion study of germ-free guinea pigs, 10% of an administered dose of [1-¹⁴C] ascorbic acid was expired as ¹⁴CO₂ within 36 hr post-injection following intraperitoneal injection compared with 16% recovery in a matched group of conventional animals injected at the same site. Results of this series of studies suggest that hepatic decarboxylation and gut microflora, in tandem, contribute to ascorbic acid decarboxylation in this species.     

Uptake to adenosine in a marine bacterium is not an active transport process

The uptake and intracellular interconversions of [8-¹⁴C]adenosine in a marine bacterium Vibrio harveyi were investigated under varying physiological conditions. The results indicated that in contrast with the current views, translocation of adenosine across the cytoplasmic membrane in Vibrio harveyi was not driven by respiration. The uptake of adenosine was dependent upon its intracellular utilization and was inhibited under conditions preventing its metabolic conversions.     

Haem-binding proteins of the rat liver cytosol.

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino[3H]laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [3H]haem-labelled mitochondria, [3H]haem-labelled microsomes or with [3H]haemin. These results are discussed with particular reference to ligandin.     

Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine

Abstract: This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S -adenosyl-homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4-¹⁴C]putrescine, the specific radioactivity (sra) of the newly formed [¹⁴C]spermidine remained unchanged. Conversely, after i.vt. l -[3,4-¹⁴C]methionine, the other polyamine precursor, significantly higher sra values for [¹⁴C]spermidine and [¹⁴C]spermine were recorded in the brains of the MSO-treated animals. [¹⁴C] S - adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1-¹⁴C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4-¹⁴C]-methionine. The results of these experiments show both significantly lower sra values for [¹⁴C]spermidine and [¹⁴C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules.     

Diverse function of aromatase and the N-terminal sequence deleted form

The diverse function of human placental aromatase including estradiol 6-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6,7-³H₂,4-¹⁴C]estradiol, 7-ethoxycoumarin, and [N-methyl-³H₃]cocaine. 6-Hydroxy[7-³H,4-¹⁴C]estradiol was isolated as the metabolite of estradiol and the ³H-water release method based on the 6-³H label was established. The initial rate kinetics of the 6-hydroxylation gave K_m of 4.3 μM, V_max of 4.02 nmol min⁻¹mg⁻¹, and turnover rate of 0.27 min⁻¹. Testosterone competed dose-dependently with the 6-hydroxylation and showed the K_i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K_m of 200 μM, V_max of 12.5 nmol min⁻¹mg⁻¹ and turnover rate of 1.06 min⁻¹. The N-demethylation of cocaine was analysed by the ³H-release method, giving K_m of 670 μM, V_max of 4.76 nmol min⁻¹mg⁻¹, and turnover rate of 0.49 min⁻¹. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min⁻¹mg⁻¹ (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min⁻¹.     

Feeding of xenobiotic ω-phenylalkanoic acids remarkably changes the chemistry and toxicity of the defensive secretion of Oxytelus sculpturatus Grav. (Coleoptera: Staphylinidae: Oxytelinae)

Feeding experiments with 12-phenyl[2,2-²H₂]dodecanoic acid and the correponding methylester resulted in the formation of 2-phenylethanol (probably produced via β-oxidation) and high amounts of 2-phenylethylesters of free fatty acids from the defensive secretion of Oxytelus sculpturatus rove beetles. The extraordinarily high amount of the metabolites occurring in the glands after administration of methyl-12-phenyl[2,2-²H₂]dodecanoate had a significant effect on the toxicity of the toluquinone-saturated defensive secretion mixture. Analogous experiments with 11-phenyl-[2-²H]undecanoic acid revealed a less efficient incorporation of this precursor leading to esters of 1-phenylethanol and 4-phenylbutan-2-ol and traces of 10-phenyl-1-decene probably formed via oxidative decarboxylation.