Trail Pheromone Disruption of Red Imported Fire Ant

The fire ant, Solenopsis invicta (Hymenoptera: Formicidae), is considered one of the most aggressive and invasive species in the world. Toxic bait systems are used widely for control, but they also affect non-target ant species and cannot be used in sensitive ecosystems such as organic farms and national parks. The fire ant uses recruitment pheromones to organize the retrieval of large food resources back to the colony, with Z,E-α-farnesene responsible for the orientation of workers along trails. We prepared Z,E-α-farnesene, (91% purity) from extracted E,E-α-farnesene and demonstrated disruption of worker trail orientation after presentation of an oversupply of this compound from filter paper point sources (30 μg). Trails were established between queen-right colony cells and food sources in plastic tubs. Trail-following behavior was recorded by overhead webcam, and ants were digitized before and after presentation of the treatment, using two software approaches. The linear regression statistic, r ² was calculated. Ants initially showed high linear trail integrity (r ² = 0.75). Within seconds of presentation of the Z,E-α-farnesene treatment, the trailing ants showed little or no further evidence of trail following behavior in the vicinity of the pheromone source. These results show that trailing fire ants become disorientated in the presence of large amounts of Z,E-α-farnesene. Disrupting fire ant recruitment to resources may have a negative effect on colony size or other effects yet to be determined. This phenomenon was demonstrated recently for the Argentine ant, where trails were disrupted for two weeks by using their formulated trail pheromone, Z-9-hexadecenal. Further research is needed to establish the long term effects and control potential for trail disruption in S. invicta.     

Plant Volatiles Influence Electrophysiological and Behavioral Responses of <Emphasis Type="Italic">Lygus hesperus</Emphasis>

Previous laboratory studies have shown that the mirid Lygus hesperus is attracted to volatiles emitted from alfalfa; feeding damage increases the amounts of several of these volatiles, and visual cues can enhance attraction further. The present study tested single plant volatiles in electrophysiological and behavioral trials with L. hesperus. Electroantennogram (EAG) analyses indicated that antennae responded to most plant volatiles included in the test, and that when gender differences were observed, males usually were more responsive than females. Antennal responses to the alcohols ((E)-3-hexenol, (Z)-3-hexenol, 1-hexanol), the acetate (E)-2-hexenyl acetate, and the aldehyde (E)-2-hexenal were among the strongest. Moderate responses were observed for (E)-β-ocimene, (E,E)-α-farnesene, (±)-linalool, and methyl salicylate. A dose dependent response was not observed for several terpenes (β-myrcene, β-caryophyllene, (+)-limonene, or both (R)-(+)- and (S)-(−)-α-pinenes). EAG responses, however, were not always consistent with behavioral assays. In Y-tube bioassays, males did not exhibit a positive behavioral response to any of the compounds tested. Instead, males were repelled by (E)-2-hexenyl acetate, (±)-linalool, (E,E)-α-farnesene, and methyl salicylate. In contrast, female L. hesperus moved upwind towards (R)-(+)-α-pinene, (E)-β-ocimene, and (E,E)-α-farnesene, and showed a negative response towards (Z)-3-hexen-1-ol, (S)-(−)-α-pinene, and methyl salicylate. This study emphasizes the use of multiple approaches to better understand host plant finding in the generalist herbivore L. hesperus.     

The Tea Weevil, <Emphasis Type="Italic">Myllocerinus aurolineatus</Emphasis>, is Attracted to Volatiles Induced by Conspecifics

The tea weevil, Myllocerinus aurolineatus (Voss) (Coleoptera: Curculionidae), is a leaf-feeding pest of Camellia sinensis (O.Ktze.) with aggregative behaviors that can seriously reduce tea yield and quality. Although herbivore-induced host plant volatiles have been shown to attract conspecific individuals of some beetle pests, especially members of the Chrysomelidae family, little is known about the volatiles emitted from tea plants infested by M. aurolineatus adults and their roles in mediating interactions between conspecifics. The results of behavioral bioassays revealed that volatile compounds emitted from tea plants infested by M. aurolineatus were attractive to conspecific weevils. Volatile analyses showed that infestations dramatically increased the emission of volatiles, (Z)-3-hexenal, (Z)-3-hexenol, (E)-β-ocimene, linalool, phenylethyl alcohol, benzyl nitrile, indole, (E, E)-α-farnesene, (E)-nerolidol, and 31 other compounds. Among the induced volatiles, 12 chemicals, including γ-terpinene, benzyl alcohol, (Z)-3-hexenyl acetate, myrcene, benzaldehyde, (Z)-3-hexenal, and (E, E)-α-farnesene, elicited antennal responses from both sexes of the herbivore, whereas (E)-β-ocimene elicited antennal responses only from males. Using a Y-tube olfactometer, we found that six of the 13 chemicals, γ-terpinene, benzyl alcohol, (Z)-3-hexenyl acetate, myrcene, benzaldehyde, and (Z)-3-hexenal, were attractive to both males and females; two chemicals, (E/Z)-β-ocimene and (E, E)-α-farnesene, were attractive only to males; and four chemicals, (E)-4,8-dimethyl-1,3,7-nonatriene, phenylethyl alcohol, linalool, and (Z)-3-hexenol, were attractive only to females. The findings provide new insights into the interactions between tea plants and their herbivores, and may help scientists develop new strategies for controlling the herbivore.     

(<Emphasis Type="Italic">E,E</Emphasis>)-α-Farnesene,an Alarm Pheromone of the Termite <Emphasis Type="Italic">Prorhinotermes canalifrons</Emphasis>

The behavioral and electroantennographic responses of Prorhinotermes canalifrons to its soldier frontal gland secretion, and two separated major components of the secretion, (E)-1-nitropentadec-1-ene and (E,E)-α-farnesene, were studied in laboratory experiments. Behavioral experiments showed that both the frontal gland secretion and (E,E)-α-farnesene triggered alarm reactions in P. canalifrons, whereas (E)-1-nitropentadec-1-ene did not affect the behavior of termite groups. The alarm reactions were characterized by rapid walking of activated termites and efforts to alert and activate other members of the group. Behavioral responses to alarm pheromone differed between homogeneous and mixed groups, suggesting complex interactions. Antennae of both soldiers and pseudergates were sensitive to the frontal gland secretion and to (E,E)-α-farnesene, but soldiers showed stronger responses. The dose responses to (E,E)-α-farnesene were identical for both soldiers and pseudergates, suggesting that both castes use similar receptors to perceive (E,E)-α-farnesene. Our data confirm (E,E)-α-farnesene as an alarm pheromone of P. canalifrons.     

Volatile Emissions from <Emphasis Type="Italic">Alnus glutionosa</Emphasis> Induced by Herbivory are Quantitatively Related to the Extent of Damage

Plant volatile organic compounds (VOCs) elicited in response to herbivory serve as cues for parasitic and predatory insects. Knowledge about quantitative relationships between the extent of herbivore-induced damage and the quantities of VOCs released is scarce. We studied the kinetics of VOC-emissions from foliage of the deciduous tree Alnus glutinosa induced by feeding activity of larvae of the geometrid moth Cabera pusaria. Quantitative relationships between the intensity of stress and strength of plant response were determined. Intensity of biotic stress was characterized by herbivore numbers (0–8 larvae) and by the amount of leaf area eaten. The strength of plant response was characterized by monitoring (i) changes in photosynthesis, (ii) leaf ultrastructure, and (iii) plant volatiles. Net assimilation rate displayed compensatory responses in herbivore-damaged leaves compared with control leaves. This compensatory response was associated with an overall increase in chloroplast size. Feeding-induced emissions of products of the lipoxygenase pathway (LOX products; (E)-2-hexenal, (Z)-3-hexenol, 1-hexanol, and (Z)-3-hexenyl acetate) peaked at day 1 after larval feeding started, followed by an increase of emissions of ubiquitous monoterpenes peaking on days 2 and 3. The emission of the monoterpene (E)-β-ocimene and of the nerolidol-derived homoterpene 4,8-dimethyl-nona-1,3,7-triene (DMNT) peaked on day 3. Furthermore, the emission kinetics of the sesquiterpene (E,E)-α-farnesene tended to be biphasic with peaks on days 2 and 4 after start of larval feeding. Emission rates of the induced LOX products, of (E)-β-ocimene and (E,E)-α-farnesene were positively correlated with the number of larvae feeding. In contrast, the emission of DMNT was independent of the number of feeders. These data show quantitative relationships between the strength of herbivory and the emissions of LOX products and most of the terpenoids elicited in response to feeding. Thus, herbivory-elicited LOX products and terpenoid emissions may convey both quantitative and qualitative signals to antagonists of the herbivores. In contrast, our data suggest that the feeding-induced homoterpene DMNT conveys the information “presence of herbivores” rather than information about the quantities of herbivores to predators and parasitoids.     

Vitamin E deficiency in dogs does not alter preferential incorporation ofRRR-α-tocopherol compared withall rac-α-tocopherol into plasma

The plasma and lipoprotein transport ofRRR andall rac-α-tocopherols, labeled with different amounts of deuterium [2R,4′R,8′R-α-[5-C²H₃]tocopheryl acetate (d ₃ RRR-α-tocopheryl acetate] and 2RS, 4′RS, 8′RS-α-[5,7-(C²H₃)₂]tocopheryl acetate (d ₅ all rac-α-tocopheryl acetate), was studied in adult beagle dogs that had been fed a vitamin E-deficient (−E; two dogs) or supplemented (+E; two dogs) diet for two years. We set out to test the hypothesis that the activity of the hepatic tocopherol binding protein (which is thought to preferentially incorporateRRR-α-tocopherol into the plasma) is up-regulated by vitamin E deficiency. Labeled α-tocopherols increased and decreased similarly in plasma of both −E and +E dogs. Irrespective of diet,d ₃ RRR-α-tocopherol was preferentially secreted in plasma. Thus, vitamin E deficiency in dogs does not markedly increase the apparent function of the hepatic tocopherol binding protein. We also studied vitamin E transport in a German Shepherd dog with degenerative myelopathy (DM). Based on the coincident appearance ofd ₃ RRR-α-tocopherol in plasma and chylomicrons, we suggest that the abnormality in DM may be associated with abnormal vitamin E transport resulting from an impaired function of the hepatic tocopherol binding protein.     

Emission of Volatile Organic Compounds After Herbivory from <Emphasis Type="Italic">Trifolium pratense</Emphasis> (L.) Under Laboratory and Field Conditions

Plants emit a wide range of volatile organic compounds in response to damage by herbivores, and many of the compounds have been shown to attract the natural enemies of insect herbivores or serve for inter- and intra-plant communication. Most studies have focused on volatile emission in the laboratory while little is known about emission patterns in the field. We studied the emission of volatiles by Trifolium pratense (red clover) under both laboratory and field conditions. The emission of 24 compounds was quantified in the laboratory, of which eight showed increased emission rates after herbivory by Spodoptera littoralis caterpillars, including (E)-β-ocimene, the most abundant compound, (Z)-β-ocimene, linalool, (E)-β-caryophyllene, (E,E)-α-farnesene, 4,8-dimethyl-1,3,7-nonatriene (DMNT), 1-octen-3-ol, and methyl salicylate (MeSA). While most of these compounds have been reported as herbivore-induced volatiles from a wide range of plant taxa, 1-octen-3-ol seems to be a characteristic volatile of legumes. In the field, T. pratense plants with varying herbivore damage growing in established grassland communities emitted only 13 detectable compounds, and the correlation between herbivore damage and volatile release was more variable than in the laboratory. For example, the emission of (E)-β-ocimene, (Z)-β-ocimene, and DMNT actually declined with damage, while decanal exhibited increased emission with increasing herbivory. Elevated light and temperature increased the emission of many compounds, but the differences in light and temperature conditions between the laboratory and the field could not account for the differences in emission profiles. Our results indicate that the release of volatiles from T. pratense plants in the field is likely to be influenced by additional biotic and abiotic factors not measured in this study. The elucidation of these factors may be important in understanding the physiological and ecological functions of volatiles in plants.     

Absorption and transport of deuterium-substituted 2R,4′R,8′R-α-tocopherol in human lipoproteins

Oral administration of a single dose of tri- or hexadeuterium substituted 2R,4′R,8′R-α-tocopheryl acetate (d₃- or d₆-α-T-Ac) to humans was used to follow the absorption and transport of vitamin E in plasma lipoproteins. Three hr after oral administration of d₃-α-T-Ac (15 mg) to 2 subjects, plasma levels of d₃-α-T were detectable; these increased up to 10 hr, reached a plateau at 24 hr, then decreased. Following administration of d₆-α-T-Ac (15–16 mg) to 2 subjects, the percentage of deuterated tocopherol relative to the total tocopherol in chylomicrons increased more rapidly than the corresponding percentage in whole plasma. Chylomicrons and plasma lipoproteins were isolated from 2 additional subjects following administration of d₃-α-T-Ac (140 or 60 mg). The percentage of deuterated tocopherol relative to the total tocopherol increased most rapidly in chylomicrons, then in very low density lipoproteins (VLDL), followed by essentially identical increases in low and high density lipoproteins (LDL and HDL, respectively) and lastly, in the red blood cells. This pattern of appearance of deuterated tocopherol is consistent with the concept that newly absorbed vitamin E is secreted by the intestine into chylomicrons; subsequently, chylomicron remnants are taken up by the liver from which the vitamin E is secreted in VLDL. The metabolism of VLDL in the circulation results in the simultaneous delivery of vitamin E into LDL and HDL.     

Isomers of alpha-tocopheryl acetate and their biological activity

α-Tocopheryl acetate, an ester of the naturally occurring form of vitamin E, has a biopotency of 1.66 IU/mg as determined by bioassays based on biological function. On a similar basis, 2l-α-tocopheryl acetate, the 2-epimer of α-tocopheryl acetate, has a biopotency of 0.35 IU/mg. Results of hemolysis bioassays indicate relative activities of 1.53 and 0.56 for α- and 2l-α-tocopheryl acetates, respectively, compared with 2dl- or all-rac-α-tocopheryl acetate. Responses to plasma and liver storage tests average 1.35 for α-tocopheryl acetate compared with all-rac-α-tocopheryl acetate. The configuration at C-2 is most important but the configurations at C-4’ and C-8’ may also be important in determining biopotency. There is no evidence of synergism between α-tocopheryl acetate and 2l-α-tocopheryl acetate. Since the two isomers comprising 2dl-α-tocopheryl acetate, the former International Standard, have different rates of absorption, tissue storage, metabolism and excretion, a better standard for vitamin E is needed. α-Tocopheryl acetate has both the chemical and biological properties required for a new International Standard for vitamin E. Redefinition of an International Unit in terms of 1 IU=0.60 mg of α-tocopheryl acetate would give a precise reference for future research and a reasonable correlation with data collected in the past. One of six papers to be published from the Symposium “Chemistry and Biochemistry of Tocopherols,” presented at the ISF-AOCS Congress, Chicago, September 1970.     

Evaluation of Lipophilic Antioxidant Efficacy in Vivo by the Biomarkers Hydroxyoctadecadienoic Acid and Isoprostane

The evaluation of antioxidant activity in vivo is difficult. In this study, the effects of dietary natural and synthetic antioxidants on the lipid peroxidation in mice were assessed using a biomarker, total hydroxyoctadecadienoic acid (tHODE). Biological samples such as plasma, erythrocytes, and tissues were first reduced and then saponified to convert various oxidation products of linoleates to tHODE. Subsequently, the absolute concentration of tHODE and its stereoisomer ratio, [9- and 13-(Z,E)-HODE)/[9- and 13-(E,E)-HODE], which is a measure of the hydrogen donor capacity of antioxidants, were determined by gas chromatography–mass spectrometry (GC–MS) analyses. These were then compared with total 8-iso-prostaglandin F_2α (t8-iso-PGF_2α) which was also assessed after reduction and saponification. Remarkable increases in tHODE and t8-iso-PGF_2α levels were observed in the plasma, erythrocytes, liver, and brain of mice that were fed an α-tocopherol (αT)-stripped (E-free) diet for 1 month when compared with those of mice that were fed a standard diet (αT = 0.002 wt%). When mice were fed for 1 month on an E-free diet supplemented with a lipophilic antioxidant (0.04 wt%), namely, αT, α-tocotrienol (αT3), γ-tocopherol (γT), or 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653), a potent synthetic antioxidant, the increases of tHODE and t8-iso-PGF_2α in the plasma, erythrocytes, liver, and brain were suppressed to the levels lower than those of mice fed a standard diet. The (Z,E/E,E) HODE ratio was decreased in the plasma and erythrocytes of mice fed the E-free diet when compared with that in mice fed the standard diet. This stereo-isomeric ratio was significantly recovered by the addition of αT and BO-653. These results show that the tHODE level and the (Z,E/E,E) HODE ratio are useful biomarkers for the assessment of antioxidant capacity in vivo and that the antioxidant capacity decreased in the order: BO-653 > αT3 ≧ αT, γT, as assessed by tHODE levels from blood, liver, and brain.     

Synthesis of 3-oxalinolenic acid and β-oxidation-resistant 3-oxa-oxylipins

3-Oxalinolenic acid (3-oxa-9(Z), 12(Z), 15(Z)-octadecatrienoic acid or (6(Z), 9(Z), 12(Z)-pentadecatrienyloxy)acetic acid) was synthesized from 5(Z), 8(Z), 11(Z), 14(Z), 17(Z)-eicosapentaenoic acid by a sequence involving the C15 aldehyde 3(Z), 6(Z), 9(Z), 12(Z)-pentadecatetraenal as a key intermediate. Conversion of the aldehyde by isomerization and two steps of reduction afforded 6(Z), 9(Z), 12(Z)-pentadecatrienol, which was coupled to bromoacetate to afford after purification by HPLC >99%-pure 3-oxalinolenic acid in 10–15% overall yield. 3-Oxalinolenic acid was efficiently oxygenated by soybean lipoxygenase-1 into 3-oxa-13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid, and this hydroperoxide could be further converted chemically into 3-oxa-13(S)-hydroxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid and 3-oxa-13-oxo-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The 3-oxa-hydroperoxide also served as the substrate for the plant enzymes allene oxide synthase, divinyl ether synthase, and hydroperoxide lyase to produce 3-oxa-12-oxo-10, 15(Z)-phytodienoic acid and other 3-oxa-oxylipins that were characterized by MS, 3-Oxalinolenic acid was not oxygenated by 9-lipoxygenase from tomato but was converted at a slow rate into 3-oxa-9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid by recombinant maize 9-lipoxygenase. Recombinant α-dioxygenase-1 from Arabidopsis thaliana catalyzed the conversion of 3-oxalinolenic acid into a 2-hydroperoxide, which underwent spontaneous degradation into a mixture of 6,9,12-pentadecatrienol and 6,9,12-pentadecatrienyl formate. A novel α-dioxygenase from the moss Physcomitrella patens was cloned and expressed and was found to display the same activity with 3-oxalinolenic acid as Arabidopsis thaliana α-dioxygenase-1. Lipoxygenase-generated 3-oxa-oxylipins are resistant toward β-oxidation and have the potential for displaying enhanced biological activity in situations where activity is limited by metabolic degradation.     

Simultaneous determination of RRR- and SRR-α-tocopherols and their quinones in rat plasma and tissues by using chiral high-performance liquid chromatography

We established a method to simultaneously determine RRR- and SRR-α-tocopherol (α-Toc) and their quinones in biological samples by chiral-phase high-performance liquid chromatography (HPLC). α-Toc had a shorter retention time than α-tocopherylquinones (α-TQ), and 2-ambo-α-Toc was completely separated into two peaks; the first peak was RRR-α-Toc and the second SRR-isomer by chiral HPLC connected Chiralcel OD-H column and Sumichiral OA4100 column. In contrast, of the two peaks of α-TQ, the first was the SRR-isomer. We also investigated differences in the distribution of RRR- and SRR-α-TQ in rat tissues after oral administration of 2-ambo-α-Toc by the above HPLC method. Rats deficient in vitamin E were divided into two groups, control and experimental, and tissues were collected at 3, 6, and 24 h after oral 2-ambo-α-Toc administration. The concentrations of RRR- and SRR-α-Toc and their quinones in plasma and each tissue were determined. The concentration of SRR-α-TQ in plasma and adrenal glands was not significantly different from RRR-α-TQ. However, the concentration of SRR-α-TQ in liver up to 6 h after oral administration was higher than that of RRR-α-TQ, and SRR- and RRR-α-TQ levels were similar at 24 h after oral administration. Therefore, we may assume that the formation of α-TQ in vivo was not different between RRR- and SRR-isomer and that it was not affected by the presence of α-Toc stereoisomers.     

Biokinetics of dietaryRRR-α-tocopherol in the male guinea pig at three dietary levels of vitamin C and two levels of vitamin E. Evidence that vitamin C does not “spare” vitamin Ein vivo

The net rates of uptake of “new” and loss of “old”2R,4′ R,8′ R-α-tocopherol (RRR-α-TOH, which is natural vitamin E) have been measured in the blood and in nine tissues of male guinea pigs over an eight week period by feeding diets containing deuterium-labelled α-tocopheryl acetate (d ₆-RRR-α-TOAc). There was an initial two week “lead-in” period during which 24 animals [the “high” vitamin E (HE) group] received diets containing 36 mg of unlabelled (d ₀)RRR-α-TOAc and 250 mg of ascorbic acid per kg diet, while another 24 animals [the “low” vitamin E (LE) group] received diets containing 5 mgd ₀-RRR-α-TOAc and 250 mg ascorbic acid per kg diet. The HE group was then tivided into three equal subgroups, which were fed diets containing 36 mgd ₆-RRR-α-TOAc and 5000 mg [the “high” vitamin C (HEHC) subgroup], 250 mg [the “normal” vitamin C (HENC) subgroup] and 50 mg [the “low” vitamin C (HELC) subgroup] ascorbic acid per kg diet. One animal from each group was sacrificed each week and the blood and tissues were analyzed ford ₀- andd ₆-RRR-α-TOH by gas chromatography-mass spectrometry. The LE group was similarly divided into three equal subgroups with animals receiving diets containing 5 mgd ₆-RRR-α-TOAc and 5,000 mg (LEHC), 250 mg (LENC) and 50 mg (LELC) ascorbic acid per kg diet with a similar protocol being followed for sacrifice and analyses. In the HE group the totald ₀-+d ₆-)RRR-α-TOH concentrations in blood and tissues remained essentially constant over the eight week experiment, whereas in the LE group the totalRRR-α-TOH concentrations declined noticeably (except in the brain, an organ with a particularly slow turnover of vitamin E). There were no significant differences in the concentrations of “old”d ₀-RRR-α-TOH nor in the concentrations of “new”d ₆-RRR-α-TOH found in any tissue at a particular time between the HEHC, HENC and HELC subgroups, nor between the LEHC, LENC and LELC subgroups. We conclude that the long-postulated “spring” action of vitamin C on vitamin E, which is well documentedin vitro, is of negligible importancein vivo in guinea pigs that are not oxidatively stressed in comparison with the normal metabolic processes which consume vitamin E (e.g., by oxidizing it irreversibly) or elminate it from the body. This is true both for guinea pigs with an adequate, well-maintained vitamin E status and for guinea pigs which are receiving insufficient vitamin E to maintain their body stores. The biokinetics of vitamin E uptake and loss in the HE guinea pigs are compared with analogous data for rats reported previously (Lipids 22, 163–172, 1987). For most guinea pig tissues the uptake of vitamin E under “steadystate” conditions was faster than for the comparable rat tissues. However, the brain was an exception with the turnover of vitamin E occurring at only one-third of the rate for the rat. NRCC Publication No. 30775.     

Catalytic properties of allene oxide synthase from flaxseed (Linum usitatissimum L.)

We investigated the catalytic and kinetic properties of allene oxide synthase (AOS; E.C. 3.2.1.92) from flaxseed (Linum usitatissimum L.). Both Michaelis constant and maximal initial velocity for the conversion of 9(S)-and 13(S)-hydroper-oxides of linoleic and linolenic acid were determined by a photometric assay, 13(S)-Hydroperoxy-9(Z), 11(E)-octadecadienoic acid [13(S)-HPOD] as the most effective substrate was converted at 116.9±5.8 nkat/mg protein by the flax enzyme extract. The enzyme was also incubated with a series of variable conjugated hydroperoxy dienyladipates. Substrates with a shape similar to the natural hydroperoxides showed the best reactivity. Monoenoic substrates as oleic acid hydroperoxides were not converted by the enzyme. In contrast, 12-hydroperoxy-9(Z), 13(E)-octadecadienoic acid was a strong competitive inhibitor for AOS catalyzed degradation of 13(S)-HPOD. The inhibitor constant was determined to be 0.09 μM. Based on these results, we concluded that allene oxide synthase requires conjugated diene hydroperoxides for successful catalysis. Studying the enantiomeric preference of the enzyme, we found that AOS was also able to metabolize (R)-configurated fatty acid hydroperoxides. Conversion of these substrates into labile allene oxides was confirmed by steric analysis of the stable α-ketol hydrolysis products.     

Biosynthesis of Sesterterpenes,Head‐to‐Tail Triterpenes,and Sesquarterpenes in Bacillus clausii: Identification of Multifunctional Enzymes and Analysis of Isoprenoid Metabolites

We performed functional analysis of recombinant enzymes and analysis of isoprenoid metabolites in Bacillus clausii to gain insights into the biosynthesis of rare terpenoid groups of sesterterpenes, head‐to‐tail triterpenes, and sesquarterpenes. We have identified an (all‐E)‐isoprenyl diphosphate synthase (E‐IDS) homologue as a trifunctional geranylfarnesyl diphosphate (GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate (HepPP) synthase. In addition, we have redefined the function of a tetraprenyl‐β‐curcumene synthase homologue as that of a trifunctional sesterterpene/triterpene/sesquarterpene synthase. This study has revealed that GFPP, HexPP, and HepPP, intermediates of two isoprenoid pathways (acyclic terpenes and menaquinones), are biosynthesized by one trifunctional E‐IDS. In addition, GFPP/HexPP and HepPP are the primary substrates for the biosynthesis of acyclic terpenes and menaquinone‐7, respectively.     

From Terpenoids to Aliphatic Acids: Further Evidence for Late-Instar Switch in Osmeterial Defense as a Characteristic Trait of Swallowtail Butterflies in the Tribe Papilionini

We compared the chemical compositions of the osmeterial secretions of fourth and fifth (last) instars of eight swallowtail species of the tribe Papilionini. Four species (Papilio demoleus, P. polytes, P. paris, and P. macilentus) are Asian Rutaceae-feeding swallowtails. The other four (Chilasa epicydes, C. agestor, P. troilus, and P. glaucus) represent more distant clades within the Papilionini and species with larval hosts in other plant families. We conducted a quantitative analysis for six species, but only qualitative analysis for P. glaucus and C. agestor. In all eight species, regardless of larval host plant, secretions of the fourth instar principally consisted of mono- and sesquiterpene hydrocarbons, whereas those of the fifth instar comprised aliphatic acids and their esters. Consistent with earlier findings, our results suggest that this “heterogeneous” pattern of osmeterial chemistry, not seen in other tribes, may characterize the Papilionini as a whole. Unlike those of most Papilio species, the fourth and fifth instars of Chilasa species resemble each other in body coloration. Thus, the heterogeneous osmeterial pattern is not necessarily associated with color change in papilionid larvae. The major terpenoids identified in fourth instar larval secretions from the six species were α-pinene, sabinene, β-myrcene, limonene, β-phellandrene, (Z)-β-ocimene, (E)-β-ocimene, p-mentha-1,4(8)-diene, β-elemene, β-caryophyllene, (E)-β-farnesene, (3Z,6E)-α-farnesene, (Z)-α-bisabolene, germacrene-A, (E)-α-bisabolene, and germacrene-B. The profiles for individual species differed both qualitatively and quantitatively from one another, and certain species also secreted methyl 3-hydroxy-n-butyrate and oxygenated sesquiterpenes in relatively large proportions. Secretions from fifth instars were composed of varying proportions of isobutyric, 2-methylbutyric, and acetic acids, and methyl and ethyl (minor) esters of both isobutyric and 2-methylbutyric acids. The heterogeneity of osmeterial chemistry in the tribe Papilionini may represent fine-tuning of chemical defense in response to shifting predation pressures as the larvae age and grow.     

Volatiles from a Mite-Infested Spruce Clone and Their Effects on Pine Weevil Behavior

Induced responses by Norway spruce (Picea abies) seedlings to feeding damage by two mite species were studied by analyzing the volatiles emitted during infestation. Four specimens of a Norway spruce (Picea abies L.) clone were infested with mites of Nalepella sp., another four with Oligonychus ununguis, and four were kept mite-free as controls. After a year of infestation, spruce volatiles were collected, analyzed, and identified using SPME-GC-MS. In addition, enantiomers of chiral limonene and linalool were separated by two-dimensional GC. Methyl salicylate (MeSA), (-)-linalool, (E)-β-farnesene, and (E,E)-α-farnesene were the main volatiles induced by both species of mites, albeit in different proportions. The ability of the main compounds emitted by the mite-infested spruces to attract or repel the pine weevil, Hylobius abietis (L.), was tested. (E)-β-farnesene was found to be attractive in the absence of spruce odor, whereas methyl salicylate had a deterrent effect in combination with attractive spruce odor. The other tested compounds had no significant effects on the behavior of the weevils.     

Iron-catalyzed reaction products of α-tocopherol with methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate

α-Tocopherol was reacted with methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate in the presence of an iron-chelate, Fe(III)-acetylacetonate, at 37°C in benzene. The reaction was carried out either aerobically or anaerobically. The main products of α-tocopherol under air were isolated and identified as two stereoisomers of 4a,5-epoxy-8a-hydroperoxy-α-tocopherone, four stereoisomers of methyl 9-(8a-dioxy-α-tocopherone)-12,13-epoxy-10(E)-octadecenoate, four stereoisomers of methyl 11-(8a-dioxy-α-tocopherone)-12,13-epoxy-9(Z)-octadecenoate, two stereoisomers of methyl 13(S)-(8a-dioxy-α-tocopherone)-9(Z),11(E)-octadecadinoate, and α-tocopherol dimer. Besides the 8a-(lipid-peroxy)-α-tocopherones, two stereoisomers of methyl 11-(α-tocopheroxy)-12(S),13(S)-epoxy-9(E)-octadecenoate, two stereoisomers of methyl 9-(α-tocopheroxy)-12(S),13(S)-epoxy-10(E)-octadecenoate, and two isomers of methyl (α-tocopheroxy)-octadecadienoate were obtained under nitrogen atmosphere. The results indicate that the peroxyl radicals from lipid hydroperoxides prefer to react with the 8a-carbon radical of α-tocopherol and the carbon-centered radicals react with the phenoxyl radical of α-tocopherol.     

A new glucoceramide from the watermelon begonia, <Emphasis Type="Italic">Pellionia repens</Emphasis>

A new glucoceramide named pellioniareside (1) was isolated from the aqueous ethanolic extract of whole plants of Pellionia repens, together with lupeol (2), uracil (3), (22E,20S,24R)-5α,8α-epidioxyergosta-6,22-dien-3-β-ol (4), and daucosterol (5). The structure and relative configurations of pellioniareside were identified as (2S,3S,4R,6E,8E)-1-O-β-d-glucopyranosyl-2-[(2R)-2-hydroxytetracosanoylamino]-1,3,4-octadecanetriol-6,8-diene by analysis of spectral data and by chemical evidence.     

Vitamin E supplementation increases circulating vitamin E metabolites tenfold in end-stage renal disease patients

Vitamin E supplementation could elevate circulating vitamin E metabolites while modulating oxidative and inflammatory status in end-stage renal failure patients undergoing hemodialysis. Plasma concentrations of carboxyethyl-hydroxychromanols (α-and γ-CEHC), ascorbic acid, α-and γ-tocopherols, E₂-isoprostanes, and inflammatory biomarkers [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), ferritin, and C-reactive protein (CRP)] were measured in blood samples obtained from patients (n=11) before and after dialysis on two occasions prior to, and at 1 and 2 mon of daily vitamin E supplementation (400 IU RRR-α-tocopherol). Supplementation nearly doubled plasma α-tocopherol concentrations (from 18±0.5 to 31±1.7 μM, P<0.0001), whereas γ-tocopherol concentrations decreased (from 2.8±0.3 to 1.7±0.2 μM, P=0.001). Serum α-CEHC increased 10-fold from 68±3 to 771±175 nM (P<0.0001), and γ-CEHC increased from 837±164 to 1136±230 nM (P=0.008). Vitamin E supplementation also increased postdialysis hematocrits from 38±1% to 41±1% (P<0.001). Dietary antioxidant intakes (vitamins E and C) were low in most subjects; plasma ascorbic acid levels (88±27 μM) decreased significantly with dialysis (33±11 μM, P=0.01). Plasma Il-6, CRP, TNF-α, and free F₂-isoprostane concentrations were elevated throughout the study. There is a complex relationship between chronic inflammation and oxidative stress that is not mitigated by short-term vitamin E supplementation. Importantly, serum vitamin E metabolite concentrations that increased 10-fold within 30 d of supplementation did not increase further, suggesting routes other than urine for removal of metabolites.