Inhibition of ceramide de novo synthesis as a postischemic strategy to reduce myocardial reperfusion injury.

The injury caused by myocardial reperfusion after ischemia can be contained by interventions aimed at reducing the inflammation and the oxidative stress that underlie exacerbation of tissue damage. Sphingolipids are a class of structural and signaling lipid molecules; among them, the inflammation mediator ceramide accumulates in the myocardium upon ischemia/reperfusion. Here, we show that, after transient coronary occlusion in mice, an increased de novo ceramide synthesis takes place at reperfusion in the ischemic area surrounding necrosis (area at risk). This correlates with the enhanced expression of the first and rate-limiting enzyme of the de novo pathway, serine palmitoyltransferase (SPT). The intraventricular administration at reperfusion of myriocin, an inhibitor of SPT, significantly protected the area at risk from damage, reducing the infarcted area by 40.9 % relative to controls not treated with the drug. In the area at risk, myriocin downregulated ceramide, reduced the content in other mediators of inflammation and reactive oxygen species, and activated the Nrf2-HO1 cytoprotective response. We conclude that an enhanced ceramide synthesis takes part in ischemia/reperfusion injury and that myriocin treatment can be proposed as a strategy for myocardial pharmacological postconditioning.

Comparative sex pherome biosynthesis in Thaumetopoea pityocampa and T. processionea: a rationale for the phenotypic variation in the sex pherome within the genus Thaumetopoea.

The female sex pheromones of the Mediterranean processionary moths (Thaumetopoea sp.) are conjugated dienes or enynes of 16 carbon atoms with the unsaturations located at C11 and C13. To investigate the biochemical basis of this phenotypic variation, the biosynthetic pathway of T. processionea sex pheromone, a diene acetate, has been elucidated and compared to that reported for the enyne-producing species T. pityocampa. Mass labeling experiments showed that T. processionea sex pheromone is biosynthesized from palmitic acid, by subsequent (Z)-11 and (Z)-13 desaturations and final reduction and acetylation. The Pheromone Biosynthesis Activating Neuropeptide (PBAN) activates this biosynthetic pathway downstream of the dienoate intermediate. When either 11-hexadecynoic acid or (Z)-13-hexadecen-11-ynoic acid were administered to T. processionea, this species was able to produce the enyne sex pheromone of T. pityocampa upon PBAN stimulation. In contrast, T. pityocampa does not produce either 11-hexadecynyl acetate or (Z,Z)-11,13-hexadecadienyl acetate, despite having the corresponding precursors in the pheromone gland. However, both acetates are detected after administration of the corresponding alcohols. These overall results suggest that the absence of delta(11) acetylenase and the existence of an enynoate specific reductase in the diene and enyne-producing Thaumetopeae, respectively, account for the different sex pheromones produced by the two groups.

Thiafatty acids as tracers to investigate biosynthetic pathways of lepidopteran sex pheromones.

In order to investigate the potential utility of thiafatty acids as tracers for biosynthetic studies of moth sex pheromones, a series of thiatetradecanoic acids, namely 8-, 9-, 10-, 11-, 12- and 13-thiatetradecanoic, were prepared and their metabolism was investigated in pheromone glands of Spodoptera littoralis. Analysis by gas chromatography coupled to mass spectrometry of extracts from pheromone glands treated with the above acids showed that only 8-thiatetradecanoic acid and 13-thiatetradecanoic acid were metabolized by desaturation and were incorporated into the sex pheromone biosynthetic pathway. 13-Thiatetradecanoic acid was converted into (E)- and (Z)-13-thiatetradec-11-enoic acids, (Z,E)-13-thiatetradeca-9,11-dienoic acid, 11-thiadodecanoic acid, (E)- and (Z)-11-thiadodec-9-enoic acids and 15-thiahexadecanoic acid. 8-Thiatetradecanoic acid gave rise to two monoenoic thiafatty acids and two dienoic thiafatty acids, which were assigned to (Z)- and (E)-8-thiatetradec-11-enoic acids, (Z,E)-8-thiatetradeca-9,11-dienoic acid and (E,E)-8-thiatetradeca-10,12-dienoic acid. The other thiafatty acids tested, 9-, 10-, 11- and 12-thiatetradecanoic acids, were not metabolized by desaturation, although the corresponding products of beta-oxidation and chain elongation were detected. The occurrence of sulfoxides was not detected in this case, in disagreement with results on the metabolism of some thiaacids previously reported by other authors in yeast, Saccharomyces cerevisiae.

Stereospecificity of the (Z)-9 desaturase that converts (E)-11-tetradecenoic acid into (Z,E)-9,11-tetradecadienoic acid in the biosynthesis of Spodoptera littoralis sex pheromone.

Moth pheromone glands contain desaturases that catalyze the formation of conjugated dienoic fatty acids. In this article we present the first stereochemical study on one of these enzymes, namely the Delta(9) desaturase of (E)-11-tetradecenoic acid, using the moth Spodoptera littoralis as a biological model and enantiopure deuterated probes derived from tridecanoic acid. Gas chromatography coupled to mass spectrometry analysis of methanolyzed lipidic extracts from glands incubated with each individual probe showed that in the transformation of (E)-11-tetradecenoic acid into (Z,E)-9,11-tetradecadienoic acid both pro-(R) hydrogen atoms at C9 and C10 are removed from the substrate.

Control of the biosynthetic pathway of Sesamia nonagrioides sex pheromone by the pheromone biosynthesis activating neuropeptide.

(Z)-11-Hexadecenyl acetate, the main pheromone component of Sesamia nonagrioides sex pheromone, is biosynthesized from palmitic acid by Delta(11)-desaturation followed by reduction and acetylation. Production of (Z)-11-hexadecenyl acetate is regulated by the Pheromone Biosynthesis Activating Neuropeptide (PBAN). Transformation of (Z)-11-hexadecen-1-ol into the corresponding acetate is a target step for PBAN in the regulation of this biosynthetic sequence, thus being the first example of a PBAN-activated acetylation. The production of the minor component (Z)-11-hexadecenal is also stimulated by PBAN. The usefulness of pentafluorobenzyloxime-derivatives for the analysis of aldehyde pheromone constituents by gas chromatography coupled to mass spectrometry is also reported.

Possible fatty acyl pheromone precursors in Spodoptera littoralis. Search for 11- and 12-hydroxytetradecanoic acids in the pheromone gland.

Lipid extracts of Spodoptera littoralis pheromone glands submitted to acid methanolysis using: (i) sulfuric acid/methanol/benzene (0.1:4:2, by vol) at 90 degrees C for 1 h; (ii) 12 N HCI/methanol (1:2, vol/vol) at 90 degrees C for 1 h, or (iii) 14% BF3-MeOH at 90 degrees C for 1 h did not reveal the presence of either 11- or 12-hydroxytetradecanoic acid in the extracts, as concluded from the gas chromatography-mass spectrometry analyses. Under the above methanolysis conditions, a synthetic sample of methyl (14, 14, 14-2H3) 12-hydroxytetradecanoate remained unaltered. These results may indicate that formation of (E)-11-tetradecenoic acid from tetradecanoic acid does not occur in the pheromone gland by dehydration of an intermediate hydroxyacid. Acid methanolysis of a lipidic extract using BF3-MeOH led to the formation of a mixture of methoxy fatty acid methyl esters, identified by gas chromatography-mass spectrometry. These methoxy derivatives should arise from BF3-catalyzed addition of methanol to the double bond of the natural monounsaturated fatty acyl derivatives present in the gland. Thus, under the same conditions, a synthetic sample of methyl (Z)-11-tetradecenoate was partially transformed into methyl 11-methoxytetradecanoate and methyl 12-methoxytetradecanoate. This reaction might be a useful alternative procedure to obtain methoxy derivatives of olefins, which are very helpful for the structural characterization of the parent alkenes.

Synthesis of gem-dideuterated tetradecanoic acids and their use in investigating the enzymatic transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid.

We report the preparation of the deuterated tetradecanoic acids [2,2,3,3-(2)H(4)]-, [2,2,3,3,10,10-(2)H(6)]-, and [2,2,3,3,13,13-(2)H(6)]-tetradecanoic acids (1, 2, and 3, respectively) and their use to investigate the mechanism of the enzymatic transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid. Probes 2 and 3 were prepared from intermediate ketones 7 and 10, which were transformed into the labeled bromides 17 and 18 by reduction with NaBD(4), tosylation of the resulting alcohol, replacement of the tosyloxy group by deuteride with LiAlD(4), hydrolysis, and reaction with N-bromosuccinimide. The resulting bromides were converted into the alpha-acetylenic esters 21 and 22, respectively, and the additional deuterium labels were introduced by reduction of the conjugated triple bond with Mg in deuterated methanol. The same sequence of reactions starting with 11-bromoundecane afforded 27. Saponification of the labeled esters 23, 24, and 27 gave the deuterated acids 2, 3, and 1, respectively. The results of the biochemical experiments showed that C10-H removal, but not elimination of C13-H, was sensitive to deuterium substitution in the transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid, which is consistent with the hypothesis that this desaturase reaction involves a first slow, C10-H bond cleavage, with probable formation of an unstable allylic intermediate, followed by a second fast C13-H bond removal and concomitant rearrangement.

Synthesis of [14,14,14-2H3] 12-hydroxytetradecanoic acid and [13,14-2H2] 11-hydroxytetradecanoic acid useful as tracers to study a (11E)-desaturation reaction in Spodoptera littoralis.

The synthesis of deuterium labeled 11- and 12-hydroxytetradecanoic acids to study a (11E) desaturase in the moth Spodoptera littoralis is reported. [14,14,14-2H3] 12-hydroxytetradecanoic acid was synthesized in four steps from 11-iodo-1-undecene in 49% overall yield. Deuterium was introduced by reaction of an epoxy ester with (CD3)2CuLi. The preparation of [13,14-2H2] 11-hydroxytetradecanoic acid was carried out in six steps from 11-bromoundecanoic acid in 55% overall yield. In this case, label was introduced by deuteration of an homoallyl alcohol with D2, using the Wilkinson catalyst. Incubation of pheromone glands with either of both acids did not lead to the formation of the labeled (11E)-tetradecenoic acid.

Cryptoregiochemistry of the delta11-myristoyl-CoA desaturase involved in the biosynthesis of Spodoptera littoralis sex pheromone.

Many moth species biosynthesize their sex pheromones by the action of unique desaturases. These membrane-bound family of enzymes are especially interesting, since some of them produce (E)-unsaturated fatty acids either exclusively or along with the (Z)-isomer. In this article we present the first mechanistic study on one of these enzymes, namely, the Delta11-myristoyl-CoA desaturase of the moth Spodoptera littoralis. Intermolecular primary isotope effect determinations were performed in competition experiments. The unusual use of odd-number fatty acids, tridecanoic acid and deuterium-labeled tridecanoic acid, in these experiments showed the existence of a large isotope effect for the carbon-hydrogen bond cleavage at C11, but no isotope discrimination occurred in the removal of C12-H. The results of the competitive experiments are consistent with the hypothesis that this Delta11-desaturase involves a first slow, isotope-sensitive C11-H bond cleavage, with probable formation of an unstable intermediate, followed by a second fast C12-H bond removal. We suggest that a single enzyme may be responsible for the formation of both (Z)- and (E)-11-tetradecenoic acids by accommodating both gauche and anti conformers of the substrate, respectively. It is also possible that two mechanistically identical discrete enzymes are involved in each desaturation. In this case, the geometry of the resulting double bond would result from the different conformation adopted by the acyl substrate at each enzyme active site.

Inhibition of sex pheromone production in female lepidopteran moths by 2-halofatty acids.

Inhibition of sex pheromone production has been observed after topical treatment of pheromonal glands with DMSO solutions of 2-bromohexadecanoic acid, in three lepidopteran insects: Spodoptera littoralis, Thaumotopoea pityocampa, and Bombyx mori. It has been shown that this effect was brought about by action on the reductases and acetyltransferases of the final steps of the pheromones biosynthesis. Other halofatty acids, such as 2-fluoro- and 2-chlorohexadecanoic acids, were less active than the above bromoderivative whereas 2-bromotetradecanoic acid and 2-bromooctanoic acid exhibited activities quite comparable to the C-16 bromoacid. These results indicate that bromosubstitution is very important for this inhibitory action and chain length is of secondary importance.

Sex pheromone biosynthetic pathway in Spodoptera littoralis and its activation by a neurohormone.

Deuterium-labeled fatty acids have been used to elucidate the sex pheromone biosynthetic pathway in Spodoptera littoralis. Label from palmitic acid was incorporated during the scotophase into all the pheromone acetates and their corresponding fatty acyl intermediates. (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone blend, is synthesized from palmitic acid via tetradecanoic acid, which, by the action of a specific (E)-11 desaturase and subsequently a (Z)-9 desaturase, is converted into (Z,E)-9,11-tetradecadienoate. By further reduction and acetylation, this compound leads to the dienne acetate. Deuterated precursors applied to the pheromone gland during the photophase were also incorporated into the pheromone. The percentage of labeled (Z,E)-9,11-tetradecadienyl acetate relative to natural compound was significantly higher during the light period. Label incorporation from different intermediates into the pheromone was stimulated by injection of brain-subesophageal ganglion extract during the photophase. The influence of the pheromone biosynthesis-activating neuropeptide on the biosynthetic pathway is discussed.

Sex pheromone biosynthesis in the processionary moth Thaumetopoea pityocampa by delta-13 desaturation.

In vivo treatments of female sex pheromone glands of the processionary moth, Thaumetopoea pityocampa, with mass-labeled fatty acids showed that (Z)-13-hexadecen-11-ynyl acetate, the main sex pheromone component, is biosynthesized from palmitic acid by the combined action of delta-11 and delta-13 desaturases. The involvement of this unusual delta-13 has been proven by application of [16,16,16-2H3] [1,2-13C2]-hexadecanoic acid to the glands with a resultant incorporation of all labeled atoms into the pheromone and each one of the corresponding intermediates. These results seem to exclude alternative biosynthetic pathways, such as chain shortening and elongation combined with delta-11 desaturation. The delta-11 desaturase responsible for the formation of the triple bond in both the 11-hexadecynoyl and (Z)-13-hexadecen-11-ynoyl intermediates is also an unusual enzyme not previously reported in lepidopteran sex pheromone biosynthesis.

Direct inhibition of (Z)-9 desaturation of (E)-11-tetradecenoic acid by methylenehexadecenoic acids in the biosynthesis of Spodoptera littoralis sex pheromone.

Analysis by gas chromatography coupled to mass spectrometry demonstrated that 11,12-methylenehexadec-11-enoic acid and 12,13-methylenehexadec-12-enoic acid are beta-oxidized in Spodoptera littoralis sex pheromone gland to 9, 10-methylenetetradec-10-enoic acid and 10,11-methylenetetradec-10-enoic acid, respectively. This result supported our previous hypothesis that inhibition of the (Z)-9 desaturation of (E)-11-tetradecenoic acid by those C-16 fatty acids is actually caused by their corresponding beta-oxidation products. However, although (2,2,3,3-2H4)-11,12-methylenehexadec-11-enoic acid was not chain-shortened to the C-14 derivative, its activity as inhibitor of the (Z)-9 desaturation reaction was similar to that exhibited by 11,12-methylenehexadec-11-enoic acid. Therefore, the C-16 cyclopropene fatty acids may inhibit the (Z)-9 desaturation enzyme by themselves, probably through interaction of the cyclopropene ring with a binding site of the enzyme with the substrate double bond.

Identification and biosynthesis of (E,E)-10,12-tetradecadienyl acetate in Spodoptera littoralis female sex pheromone gland.

A minor component of the sex pheromone gland of the Egyptian cotton leafworm, Spodoptera littoralis, has been identified as (E,E)-10,12-tetradecadienyl acetate. Structural elucidation has been carried out by isobutane-chemical ionization mass spectrometry of the fatty acyl biosynthetic precursor-derived methyl ester. To assign the stereochemistry of the double bonds, the four isomers of both 10,12-tetradecadienyl acetates and methyl 10,12-tetradecadienoates have been synthesized and their gas chromatography retention times and mass spectra have been compared to those of the corresponding natural compounds. Mass-labeling experiments showed that the (E,E)-10,12-tetradecadienoyl moiety is biosynthetically derived from (Z)-11-tetradecenoic acid in the insect pheromone gland.

Dihydroceramide delta(4) desaturase initiates substrate oxidation at C-4.

The intermolecular primary deuterium isotope effects on the individual C-H bond cleavage steps involved in dihydroceramide Delta(4) desaturation have been determined for the first time by incubating rat liver microsomes with 1:1 mixtures of nonlabeled substrate and the appropriate regiospecifically dideuterated analogue. Analysis of the enzymatic products via gas chromatography coupled to mass spectrometry showed that the introduction of the (E) double bond between C-4 and C-5 occurs in two discrete steps: cleavage of the C4-H bond was found to be very sensitive to isotopic substitution (k(H)/k(D) = 8.0 +/- 0.8), while a negligible isotope effect (k(H)/k(D) = 1.02 +/- 0.07) was observed for the C5-H bond-breaking step. According to a mechanistic model that we have previously proposed, these results suggest that initial oxidation for this desaturation reaction occurs at C-4. This finding correlates nicely with the observation that 4-hydroxylated products are produced from a similar substrate by a closely related oxidative enzyme in yeast.

Synthesis of dideuterated and enantiomers of monodeuterated tridecanoic acids at C-9 and C-10 positions.

We report a route for the preparation of mono and dideuterated tridecanoic acids: (R)-[9-(2)H(1)]-, (S)-[9-(2)H(1)]-, (R)-[10-(2)H(1)]-, (S)-[10-(2)H(1)]-, [9,9-(2)H(2)]-, and [10, 10-(2)H(2)]-tridecanoic acids required as probes for biochemical studies on desaturases. The key intermediates in the synthesis of all these probes are ketones 9, which give rise to the corresponding alcohols 10 and 13 by reduction with LiAlD(4) and LiAlH(4), respectively. Derivatization of nondeuterated racemic alcohols 13 with (S)-(+)-9-anthranylmethoxyacetic acid ((S)-(+)-9-AMA) and chromatographic resolution of both diastereoisomers allowed us to determine the absolute configuration of the stereogenic centers by (1)H NMR using an adaptation of the model proposed by Riguera and co-workers which was validated with alcohols of known absolute configuration. Both enantiomeric alcohols (R)- and (S)-13 were recovered by reduction of each diastereomeric ester with LiAlH(4). Mesylation of alcohols 10 and 13 followed by nucleophilic substitution by LiAlD(4) generated the saturated methoxymethyl derivatives 12 and 16, respectively. Final deprotection and Jones oxidation of the resulting alcohols afforded the above deuterated tridecanoic acids.

Action of PBAN and related peptides on pheromone biosynthesis in isolated pheromone glands of the redbanded leafroller moth, Argyrotaenia velutinana.

Isolated pheromone glands from the redbanded leafroller moth, Argyrotaenia velutinana, were utilized to demonstrate the action of pheromone biosynthesis activating neuropeptide (PBAN) and bursa pheromonotropic peptide plus several other related peptides on pheromone biosynthesis. All peptides belonging to the PBAN family and the bursa peptide stimulated pheromone biosynthesis as measured by pheromone titer and incorporation of radiolabeled acetate. These peptides required the presence of extracellular Ca2+ for expression of full activity and several inorganic Ca2+ channel blockers inhibited the stimulation of pheromone biosynthesis. The Ca2+ ionophore A23187 alone stimulated pheromone biosynthesis as did a cAMP analogue. Stimulation by the cAMP analogue in the absence of extracellular Ca2+ was observed. Maximum pheromone titers were observed in 16 hr gland incubations; however, 2-6 hr incubations were required if pheromone biosynthesis was measured by incorporation of radiolabeled acetate. Radiolabeled glucose incorporation was not increased in the presence of PBAN. These results are discussed in the context of how the pheromone biosynthetic pathway is stimulated by these peptides.

Analogs of sex pheromone of processionary moth,Thaumetopoea pityocampa: Synthesis and biological activity.

The synthesis and biological activity of some analogs of (Z)-13-hexadecen-11-ynyl acetate1, the major component of the sex pheromone of the processionary mothThaumetopoea pityocampa is described. The analogs have been formally derived by structural modification of the enyne and acetate functions of the parent compound1. In field tests, trifluoroacetate ester16 and the analog,11, with fluorine substitution at the olefin site, decreased the pheromone action, whereas epoxy derivative,10, from epoxidation of the olefin moiety in1, and propionate ester15 gave synergistic activity. The formate14 had a variable effect according to the composition of the lure. Formal reduction of the enyne to give the acetylene2 was found to retain activity. Alcohols12 and13, resulting from hydrolysis of the enyne1 and acetylene2, respectively, inhibited the action of their parent compounds.