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.     

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.     

Expression and evolution of delta9 and delta11 desaturase genes in the moth Spodoptera littoralis

Desaturation of fatty acids is a key reaction in the biosynthesis of moth sex pheromones. The main component of Spodoptera littoralis sex pheromone blend is produced by the action of Δ¹¹ and Δ⁹ desaturases. In this article, we report on the cloning of four desaturase-like genes in this species: one from the fat body (Sls-FL1) and three (Sls-FL2, Sls-FL3 and Sls-FL4) from the pheromone gland. By means of a computational/phylogenetic method, as well as functional assays, the desaturase gene products have been characterized. The fat body gene expressed a Δ⁹ desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1:4.5) ratio, whereas the pheromone gland Sls-FL2 expressed a Δ⁹ desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1.5:1) ratio. Although both Δ⁹ desaturases produced (Z)-9-tetradecenoic acid from myristic acid, transformed yeast grown in the presence of a mixture of myristic and (E)-11-tetradecenoic acids produced (Z,E)-9,11-tetradecadienoic acid, but not (Z)-9-tetradecenoic acid. The Sls-FL3 gene expressed a protein that produced a mixture of (E)-11-tetradecenoic, (Z)-11-tetradecenoic, (Z)-11-hexadecenoic and (Z)-11-octadecenoic acids in a 5:4:60:31 ratio. Despite having all the characteristics of a desaturase gene, no function could be found for Sls-FL4.     

Δ-β-Sitosten-3-one from β-sitosterol by leaf homogenates

β-Sitosterol-4-¹⁴C is metabolized to Δ⁴-β-sitosten-3-one by Cheiranthus cheiri leaf homogenates. Greater than 60% conversion occurs within 2 hr. Under identical conditions, leaf homogenates of Strophanthus kombé fail to metabolize β-sitosterol, while Digitalis purpurea leaf homogenates yield only very small amounts of the metabolite.     

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.     

Pheromone biosynthetic pathways in the moths Heliothis subflexa and Heliothis virescens

Sex pheromones of many moth species have relatively simple structures consisting of a hydrocarbon chain with a functional group and one to several double bonds. These sex pheromones are derived from fatty acids through specific biosynthetic pathways. We investigated the incorporation of deuterium-labeled tetradecanoic, hexadecanoic, and octadecanoic acid precursors into pheromone components of Heliothis subflexa and Heliothis virescens. The two species utilize (Z)11-hexadecenal as the major pheromone component, which is produced by Delta11 desaturation of hexadecanoic acid. H. subflexa also produced (Z)11-hexadecanol and (Z)-11-hexadecenyl acetate via Delta11 desaturation. In H. subflexa, octadecanoic acid was used to biosynthesize the minor pheromone components (Z)9-hexadecenal, (Z)9-hexadecenol, and (Z)9-hexadecenyl acetate. These minor components are produced by Delta11 desaturation of octadecanoic acid followed by one round of chain-shortening. In contrast, H. virescens used hexadecanoic acid as a substrate to form (Z)11-hexadecenal and (Z)11-hexadecenol and hexadecenal. H. virescens also produced (Z)9-tetradecenal by Delta11 desaturation of the hexadecanoic acid followed by one round of chain-shortening and reduction. Tetradecanoic acid was not utilized as a precursor to form Z9-14:Ald in H. virescens. This labeling pattern indicates that the Delta11 desaturase is the only active desaturase present in the pheromone gland cells of both species.     

Biosynthesis of C-labeled branched polysaccharides by pestalotiopsis from C-labeled glucoses and the mechanism of formation

Biosynthesis of branched glucan by Pestalotiopsis from media containing D-(1-¹³C)glucose, D-(2-¹³C)glucose, D-(4-¹³C)glucose, D-(6-¹³C)glucose or a mixture of D-(1-¹³C)glucose and D-(2-¹³C)glucose was carried out to elucidate biosynthetic mechanism of branched polysaccharides. ¹³C NMR spectra of the labeled polysaccharides were determined and assigned. Analysis of ¹³C NMR spectra of glucitol acetates obtained from hydrolysates of the labeled branched polysaccharides indicated that transfer of labeling from C-1 to C-3 and C-6 carbons, from C-2 to C-1, C-3 and C-5 carbons, and from C-6 to C-1 carbon. From the results the percentages of routes via which the polysaccharide is biosynthesized are estimated. They show that the biosynthesis of the polysaccharide via the Embden-Meyerhof pathway and that from lipids and proteins are more active, and the pentose cycle is less active, than in the biosynthesis of cellulose and curdlan. As for the results, labeling at C-6 carbon in the branched polysaccharide cultured from D-(6-¹³C)glucose was low, compared to that of cellulose and curdlan.     

Hormonal control of female sex pheromone biosynthesis in the redbanded leafroller moth,Argyrotaenia velutinana

Pheromone biosynthesis in female redbanded leafroller moths (RBLR) is under control of a neuropeptide produced in the brain. A bioassay consisting of isolated abdomens was developed to test the mode of action of the pheromone biosynthesis activating neuropetide (PBAN). Pheromone titer and incorporation of radiolabeled acetate into pheromone could be monitored with this bioassay. Synthetic PBAN with sequences identical to PBAN isolated from Heliothis zea and Bombyx mori were active in inducing synthesis of pheromone in RBLR. Removal of the ventral nerve cord in isolated abdomens did not inhibit the action of PBAN. Small amounts of PBAN-like activity was found in hemolymph collected from normal females but not from decapitated females. Severing the VNC in vivo in normal females did not lower the pheromone titer. These data indicate that PBAN is released into the hemolymph and then travels to its site of action. A two-fold increase in both pheromone titer and radiolabeled acetate incorporation upon incubation with PBAN was shown with isolated pheromone glands. However, the differences between control and PBAN-induced values were smaller than those obtained with the isolated abdomen culture bioassay where a seven-fold increase was observed. A decrease in pheromone titer was seen upon the in vivo removal of the corpus bursae from normal females. Removal of the corpus bursae in the isolated abdomen cultures also abolished the activity of PBAN. However, cutting the cervix bursae and leaving the corpus bursae in the abdomen culture increased both titer and radiolabeled acetate incorporation into pheromone without the presence of PBAN. An aqueous extract made from the corpus bursae of 5-day-old females was also active by itself in inducing pheromone biosynthesis in the isolated abdomen cultures. Experiments performed using newly emerged females confirmed that the corpus bursae extracts will induce pheromone biosynthesis. These results indicate that both PBAN and the corpus bursae are involved in controlling pheromone biosynthesis in RBLR.     

Regulation of pheromone biosynthesis in the "Z strain" of the European corn borer, Ostrinia nubilalis

The regulation of pheromone biosynthesis by the neuropeptide PBAN in the Z strain of the European corn borer, Ostrinia nubilalis, was investigated using labeled intermediates. Injection of radiolabeled acetate showed PBAN did not influence the de novo synthesis of saturated fatty acids in the gland. When deuterium-labeled myristic acid was topically applied to the gland, females injected with PBAN produced more labeled pheromone than did control females, indicating that PBAN controls one of the later steps of pheromone biosynthesis. Although more myristic acid was Delta11-desaturated in the gland in the presence of PBAN, this was counterbalanced by less Delta11-desaturation of palmitic acid, indicating that desaturase activity did not change overall. This change in flux of myristic acid through to pheromone was shown to be caused by increased reduction of fatty acid pheromone precursors occurring in the presence of PBAN.     

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.     

Assessment of temporal changes in haem and protein levels in Ixodid ticks following blood engorgement, and their use as age-grading parameters

Nymphs of Rhipicephalus appendiculatus and Hyalomma anatolicum anatolicum were fed on rabbits and maintained in the laboratory to allow moulting and further development. At specified time intervals from 0 to 500 days after engorgement, samples of ticks were ground individually in distilled water within the wells of an agglutination plate. A 0.1 ml aliquot was removed from each and levels of haemin and protein assessed from optical density values at specific wavelengths in a spectrophotometer.

Both protein and haemin levels showed an initial rapid decrease after engorgement; values did not fall to zero in either case but showed marked fluctuation throughout the study period. These fluctuations combined with the high standard errors of the results, made assessments of the physiological age of individual ticks impossible.

Such fluctuating values, however, suggested the possibility that haem biosynthesis might be taking place, and this was tested by injecting the radioactively-labelled haem precursor [4-¹⁴C]δ-amino laevulinic acid into engorged nymphs, immediately following their detachment. Both tick species revealed an incorporation of this compound into their haematin content, although neither incorporated the non-haem precursor [1-¹⁴C]2-amino isobutyric acid. These results indicate an ability of ticks to synthesize haem in vivo, although the underlying reasons for such a mechanism remain unknown.     

Investigations into the biosynthetic pathways for classical and ring B-unsaturated oestrogens in equine placental preparations and allantochorionic tissues

In on-going studies of ‘classical’ and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-²H₅]-testosterone and [16,16,17-²H₃]-5,7-androstadiene-3β,17β-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [²H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography–mass spectrometry (GC–MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell™ cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [²H₅]-Testosterone was converted into [²H₄]-oestradiol-17β, [²H₄]-oestrone and [²H₃]-6-dehydro-oestradiol-17 in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse.

On the basis of GC–MS characteristics (M⁺ m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M⁺). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [²H₄]-17β-dihydroequilin was also shown, and a biosynthetic pathway is proposed.

In static incubations of placental microsomal fractions, the 17β-dihydro forms of both equilin and equilenin were shown to be major metabolites of [²H₃]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17β-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17β-isomer of dihydroequilenin. Confirmation of the identity of 17β-dihydroequilin and 17β-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/z 414 (17β-dihydroequilin) and 412 (17β-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3β,17β-diol are proposed.     

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.     

Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

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.     

RNA interference of PBAN receptor suppresses expression of two fatty acid desaturases in female Plutella xylostella

Pheromone biosynthesis-activating neuropeptide (PBAN) stimulates sex pheromone biosynthesis by activating PBAN receptor (PBANr), which triggers a specific signal transduction in the pheromone gland cells. We have shown that RNA interference (RNAi) of PBANr of Plutella xylostella significantly suppressed pheromone biosynthesis and subsequent mating behavior. In order to assess molecular events occurring downstream of PBAN signaling, we cloned partial sequences of Δ9 and Δ11 fatty acid desaturases of P. xylostella. Phylogenetic analysis indicated that these two desaturase genes were highly clustered with other desaturases associated with sex pheromone biosynthesis in other insects. RT-PCR analysis showed that Δ9 desaturase was dominantly expressed in adult females, whereas Δ11 desaturase was expressed in all P. xylostella developmental stages. When PBANr expression was suppressed by PBANr-RNAi, the treated females also showed significant suppression of expression of both desaturases. These results suggest that expressions of the two desaturases are controlled by PBAN and that the two desaturases may be involved as downstream components in sex pheromone biosynthesis of P. xylostella.     

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.     

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.