Determination and mass spectrometric investigation of a new mixed halogenated persistent component in fish and seal.

An unknown component that caused an intense signal in sample extracts of fish tissue was enriched and investigated using a variety of mass spectrometric techniques coupled to gas chromatographic separation. With the help of electron capture negative ion mass spectrometry (ECNI-MS) and electron impact mass spectrometry (EI-MS) it was established that the component carries 2Br and 3Cl atoms and forms a molecular ion at m/z 396. A concentrated solution of this mixed halogenated compound (MHC-1) was investigated by gas chromatography interfaced to electron impact high-resolution mass spectrometry (GC/EI-HRMS). Using full scan and SIM techniques, the molecular formula of MHC-1 was established to be C10H13Br2Cl3. This points toward MHC-1 having a monoterpene backbone. No chemical with this molecular formula has been synthesized, but two components with this composition have been earlier isolated from marine algae.

Rapid HPLC screening of jasmonate-induced increases in tobacco alkaloids, phenolics, and diterpene glycosides in Nicotiana attenuata.

A rapid, HPLC-based screening procedure for the main classes of secondary metabolites in Nicotiana attenuata leaves (alkaloids, phenolics, and diterpene glycosides) is reported. In a single step, leaves are extracted in aqueous acidified (0.5% acetic acid) methanol, and the extracted compounds are separated by reversed-phase HPLC with an acidic water/acetonitrile gradient in <30 min. The utility of the method in quantifying changes in the secondary metabolites after methyl jasmonate treatment of the plants, a treatment known to elicit resistance to herbivores in nature, is illustrated. Methyl jasmonate treatment elicited dramatic increases in some secondary metabolites (caffeoylputrescine, nicotine, and diterpene glycosides increased 12.5-, 1.4-, and 1.9-fold, respectively) but left others, such as rutin, unchanged. Such broad-based analytical screens will help characterize environmental and genetic changes in secondary metabolite profiles.

Detection and removal of an artefact fatty acid from the binding site of recombinant Bombyx mori pheromone-binding protein.

Recombinant Bombyx mori pheromone-binding protein (PBP), purified from an Escherichia coli expression system, has been found to contain (11Z)-octadecenoic acid (cis-vaccenic acid) as an artefact ligand. An efficient delipidation procedure is described to overcome what would appear to be a general problem with recombinant lepidopteran PBPs.

The biosynthesis of benzoic acid glucosinolate esters in Arabidopsis thaliana.

The siliques and seeds of Arabidopsis thaliana accumulate a series of glucosinolates containing an alkyl side chain of varying length with a terminal benzoate ester function. The biosynthesis of these unusual nitrogen- and sulfur-containing natural products was investigated by feeding isotopically-labeled precursors to detached flowering stems. Glucosinolates were extracted, purified and analyzed by tandem mass spectrometry. Phenylalanine and benzoic acid were incorporated into the benzoate ester function, and methionine and acetate were incorporated into the aliphatic portion of the side chain in a position-specific manner. The labeling patterns observed were consistent with the chain extension of methionine by a three-step elongation cycle which begins with the condensation of acetyl-CoA with a 2-oxo acid derived from methionine and ends with an oxidative decarboxylation forming a new 2-oxo acid with an additional methylene group. Incorporation of desulfo-4-methylthiobutyl glucosinolate into 4-benzoyloxybutyl olucosinolate suggested chain-extended methionine derivatives are first converted to their corresponding methylthioalkyl glucosinolates with further side chain modification occurring later. Transformation of the methylthiol function to a hydroxyl group is followed by esterification with benzoic acid. The siliques appear to possess the complete machinery for carrying out all of the reactions in the biosyntheis of these complex glucosinolates.

Iridoid biosynthesis in staphylinid rove beetles (Coleoptera: Staphylinidae, Philonthinae).

The biosynthesis of chrysomelidial and plagiodial was studied in the rove beetle subtribe Philonthina (Staphylinidae). Glandular homogenates were found to convert synthetic (2E,6E)-[trideuteromethyl-5,5-(2)H(5)]octa-2,6-diene-1,8-diol (10) into nor-chrysomelidial (14) and nor-plagiodial (13). The overall transformation requires; i) oxidation of the substrate at C(1) and C(8), ii) cyclization of the resulting dialdehyde to nor-plagiodial followed by iii) isomerization to give nor-chrysomelidial. The oxidase requires molecular oxygen as a cofactor and operates with removal of the pro-R hydrogen from C(1) and C(8) of synthetic (1R,8R,2E,6E)-[1,8-(2)H(2)]-2,6-dimethyl-octa-2,6-diene-1,8-diol (15), producing a dialdehyde along with H(2)O(2). Unlike enzymes from iridoid-producing leaf beetle larvae, the Philonthus enzyme is able to oxidize saturated substrates such as citronellol. Crude protein extracts prepared from Philonthus glands by ammonium sulfate precipitation, were found to produce hydrogen peroxide at a rate of 0.085+/-0.003 ng H(2)O(2) (ng protein)(-1) hr(-1) with nerol as an oxidase substrate. The cyclase operates with opposite stereochemistry to the enzyme(s) from Phaedon cochleariae and other herbivorous leaf beetles, specifically removing the C(5)-H(R) hydrogen atom from (4R,5S,2E,6E)-[4,5-(2)H(2)]-2-methyl-octa-2,6-diene-1,8-diol (17). These findings have enabled us to construct a detailed account of iridoid biosynthesis in rove beetles, which resembles the biosynthetic route in leaf beetle larvae, but exhibits distinct stereochemical differences.

Recruitment pheromone in the harvester ant genus Pogonomyrmex.

Workers of the harvester ant genus Pogonomyrmex employ recruitment trail pheromones discharged from the poison gland. In P. barbatus, P. maricopa, P. occidentalis and P. rugosus we identified three pyrazines [2,5-dimethylpyrazine, trimethylpyrazine and 3-ethyl-2,5-dimethylpyrazine (EDMP)] as major compounds of the volatile part of the poison-gland secretions. Laboratory and field tests revealed EDMP to be the main recruitment pheromone.

Accumulation of soluble and wall-bound indolic metabolites in Arabidopsis thaliana leaves infected with virulent or avirulent Pseudomonas syringae pathovar tomato strains.

The chemical structures and accumulation kinetics of several major soluble as well as wall-bound, alkali-hydrolyzable compounds induced upon infection of Arabidopsis thaliana leaves with Pseudomonas syringae pathovar tomato were established. All identified accumulating products were structurally related to tryptophan. Most prominent among the soluble substances were tryptophan, beta-d-glucopyranosyl indole-3-carboxylic acid, 6-hydroxyindole-3-carboxylic acid 6-O-beta-d-glucopyranoside, and the indolic phytoalexin camalexin. The single major accumulating wall component detectable under these conditions was indole-3-carboxylic acid. All of these compounds increased more rapidly, and camalexin as well as indole-3-carboxylic acid reached much higher levels, in the incompatible than in the compatible P. syringae/A. thaliana interaction. The only three prominent phenylpropanoid derivatives present in the soluble extract behaved differently. Two kaempferol glycosides remained largely unaffected, and sinapoyl malate decreased strongly upon bacterial infection with a time course inversely correlated with that of the accumulating tryptophan-related products. The accumulation patterns of both soluble and wall-bound compounds, as well as the disease resistance phenotypes, were essentially the same for infected wild-type and tt4 (no kaempferol glycosides) or fah1 (no sinapoyl malate) mutant plants. Largely different product combinations accumulated in wounded or senescing A. thaliana leaves. It seems unlikely that any one of the infection-induced compounds identified so far has a decisive role in the resistance response to P. syringae.

Rapid and sensitive analysis of azadirachtin and related triterpenoids from Neem (Azadirachta indica) by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Based on reversed-phase high-performance liquid chromatography (RP-HPLC) and atmospheric pressure chemical ionization (APCI) mass spectrometry, a HPLC-MS method was developed to permit the rapid qualitative and quantitative analysis of azadirachtin and related tetranortriterpenoids from seeds and tissue cultures of Neem (Azadirachta indica). APCI+ standard scanning mass spectra of the major Neem triterpenoids were recorded and utilized to select suitable ions for selected ion monitoring (SIM). Transitions for selective reaction monitoring (SRM) were based on MS-MS experiments. Using SIM, major Neem triterpenoids were detected in callus culture material and seed kernels of A. indica. The limit of detection for azadirachtin in extract samples (approximately 1 ng ml(-1) or 10 pg in SIM mode) was determined to be (with respect to injected absolute amounts) approximately 1000-times lower than values quoted in the literature for existing HPLC methods (approximately 200 ng ml(-1) or 10 ng). In addition to high sensitivity, the HPLC-MS method is able to tolerate minimal sample preparation and purification, dramatically reducing total analysis time.

The methionine chain elongation pathway in the biosynthesis of glucosinolates in Eruca sativa (Brassicaceae).

Glucosinolates are nitrogen- and sulfur-containing plant natural products that have become increasingly important in human affairs as flavor precursors, cancer-prevention agents, and crop protectants. While many glucosinolates are biosynthesized from common amino acids, the major glucosinolates in economically important species of the Brassicaceae, such as Brassica napus (oilseed rape), are thought to be formed from chain-elongated derivatives of methionine or phenylalanine. We investigated the chain elongation pathway for methionine that is involved in glucosinolate biosynthesis in Eruca sativa. Isotopically labeled methionine and acetate were administered to cut leaves and the major product, 4-methylthiobutylglucosinolate (isolated as its desulfated derivative), was analyzed by MS and NMR. Administration of ¿U-(13)Cmethionine showed that the entire carbon skeleton of this amino acid, with the exception of the COOH carbon, is incorporated as a unit into 4MTB. Administration of ¿(13)C- and ¿(14)Cacetate gave a labeling pattern consistent with the operation of a three-step chain elongation cycle which begins with the condensation of acetyl-CoA with a 2-oxo acid derived from methionine and ends with an oxidative decarboxylation forming a new 2-oxo acid with one additional methylene group. Administration of ¿(15)Nmethionine provided evidence for the transfer of an amino group to the chain-elongated 2-oxo acid, forming an extended amino acid which serves as a substrate for the remaining steps of glucosinolate biosynthesis. The retention of a high level of (15)N in the products suggests that the amino transfer reactions and the chain elongation cycle are localized in the same subcellular compartment.

Are variations in cuticular hydrocarbons of queens and workers a reliable signal of fertility in the ant Harpegnathos saltator?

One of the key features of insect societies is the division of labor in reproduction between one or a few fertile individuals and many sterile nestmates that function as helpers. The behavioral and physiological mechanisms regulating reproduction in ant societies are still not very well understood, especially in species in which all colony members are reproductively totipotent. In the ponerine ant Harpegnathos saltator, queen-worker dimorphism is very limited, and a few mated workers reproduce ("gamergates") once the founding queen becomes senescent. Worker oviposition is regulated by highly directed aggressive interactions among nestmates, who can recognize different levels of ovarian activity. We show that variations in cuticular hydrocarbons (CHC) correlate with oogenesis, both for queens and workers. 13,23-Dimethylheptatriacontane is present in egg-layers, but not in infertile workers and queens. Proportions of other CHCs vary as well, resulting in clear separation of the ants in a multivariate analysis. Egg-layers are characterized by an elongation of the chain length of CHCs. We used solid-phase microextraction to measure CHCs in live ants that were experimentally induced to start producing eggs. Over a period of 118 days, CHC profiles of infertile workers changed completely to that of reproductives. The effect of age can be excluded in this modification. This striking correlation of ovarian activity with CHC variation and its correspondence with the observed recognition behavior exhibited by the workers toward egg-laying nestmates suggests that CHCs serve as a fertility signal in the ant H. saltator, a reliable basis for regulating reproduction.

Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-beta-ocimene synthase.

The Arabidopsis genome project has recently reported sequences with similarity to members of the terpene synthase (TPS) gene family of higher plants. Surprisingly, several Arabidopsis terpene synthase-like sequences (AtTPS) share the most identity with TPS genes that participate in secondary metabolism in terpenoid-accumulating plant species. Expression of a putative Arabidopsis terpene synthase gene, designated AtTPS03, was demonstrated by amplification of a 392-bp cDNA fragment using primers designed to conserved regions of plant terpene synthases. Using the AtTPS03 fragment as a hybridization probe, a second AtTPS cDNA, designated AtTPS10, was isolated from a jasmonate-induced cDNA library. The partial AtTPS10 cDNA clone contained an open reading frame of 1665 bp encoding a protein of 555 amino acids. Functional expression of AtTPS10 in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate (C(10)) into the acyclic monoterpenes beta-myrcene and (E)-beta-ocimene and small amounts of cyclic monoterpenes. Based on sequence relatedness, AtTPS10 was classified as a member of the TPSb subfamily of angiosperm monoterpene synthases. Sequence comparison of AtTPS10 with previously cloned monoterpene synthases suggests independent events of functional specialization of terpene synthases during the evolution of terpenoid secondary metabolism in gymnosperms and angiosperms. Functional characterization of the AtTPS10 gene was prompted by the availability of Arabidopsis genome sequences. Although Arabidoposis has not been reported to form terpenoid secondary metabolites, the unexpected expression of TPS genes belonging to the TPSb subfamily in this species strongly suggests that terpenoid secondary metabolism is active in the model system Arabidopsis.

3-hydroxy-3-phenylpropanoic acid is an intermediate in the biosynthesis of benzoic acid and salicylic acid but benzaldehyde is not.

Stable-isotope-labelled (2H6, 18O) 3-hydroxy-3-phenylpropanoic acid, a putative intermediate in the biosynthesis of benzoic acid (BA) and salicylic acid (SA) from cinnamic acid, has been synthesized and administered to cucumber (Cucumis sativus L.) and Nicotiana attenuata (Torrey). Analysis of the products by gas chromatography-mass spectrometry revealed incorporation of labelling into BA and SA, but not into benzaldehyde. In a separate experiment, 3-hydroxy-3-phenylpropanoic acid was found to be a metabolite of phenylalanine, itself the primary metabolic precursor of BA and SA. These data suggest that cinnamic acid chain shortening is probably achieved by beta-oxidation, and that the proposed "non-oxidative" pathway of side-chain degradation does not function in the biosynthesis of BA and SA, in cucumber and N. attenuata.

Volatile secretions of old world army antAenictus rotundatus and chemotaxonomic implications of army ant dufour gland chemistry.

The Dufour glands ofAenictus rotundatus contain a complex mixture of terpenoids with geranylgeraniol comprising over 50% of the secretion. Some novel compounds have been tentatively identified as higher homologs of 1,3,3-trimethyl-2,7-dioxabicyclo[2,2,1]heptane based on GC-MS data. The Dufour gland secretion ofA. rotundatus is more similar in composition to the secretions of members of the subfamily Ecitoninae than to its closer relatives from the tribe Dorylini, a result that further complicates studies on the phylogeny of army ants. The mandibular glands ofA. rotundatus contain a mixture of 4-methyl-3-heptanone and limonene in trace amounts, and the well-developed postpygidial glands contain methyl anthranilate only.