Seasonal dynamics of fatty acids in the lipids of water moss <Emphasis Type="Italic">Fontinalis antipyretica</Emphasis> from the Yenisei River

Identification of lipid fatty acids (FA) and studying of their seasonal dynamics in water moss Fontinalis antipyretica from the Yenisei River were carried out by means of gas chromatography-mass spectrometry. FA composition of water moss was notable for a relatively low level of saturated acids and predominance of polyunsaturated acids (PUFA) with double bonds (accounting for more than 30% of total FA) and polyunsaturated acids with double and triple bonds (acetylenic acids, accounting for more than 40% of total FA). Among PUFA, α- and γ-linolenic (18:3ω3 and 18:3ω6), arachidonic (20:4ω6), and eicosapentaenoic (20:5ω3) acids prevailed. Relative content of PUFA from ω3-family was the greatest in spring, and the level of PUFA from ω6-group was essentially the same throughout all the seasons. In the biomass of water moss, we identified seven acetylenic acids; among them octadeca-9,12-dien-6-ynoic (6a,9,12-18:3), octadeca-9,12,15-trien-6-ynoic (6a,9,12,15-18:4), and eicosa-11,14-dien-8-ynoic (8a,11,14-20:3) acids were predominant. For the first time, in the lipids of water moss we identified an acetylenic eicosa-11,14,17-trien-8-ynoic acid (8a,11,14,15-20:4). Relative content of acetylenic acids in the total FA was great throughout the entire period of investigation with the peak accumulation in summer. Owing to a steadily high level in the biomass of water moss and the lack of other producers of these acids in the ecosystem, acetylenic FA are highly specific biochemical markers useful for the investigation of trophic interactions between higher aquatic plants and zoobenthos.     

Distribution and chemotaxonomic significance of acetylenic fatty acids in mosses of the dicranales

Thirty-eight moss species from four families of the order Dicranales were analysed for the fatty acid composition of their acyl lipids. In the Ditrichaceae and the Dicranaceae numerous species were found to contain acetylenic fatty acids in their triglycerides, 9,12,15-Octadecatrien-6-ynoic acid was the major component, often accounting for more than 80 mol%, whereas 9,12-octadecadien-6-ynoic acid was found in small amounts of less than 5 mol%. In some genera, all the species examined contained acetylenic fatty acids, e.g.Dicranella andDicranum, whereas in the genusCampylopus all five species tested were free of acetylenic compounds. Two genera, Ditrichum andDicranoweisia, were found to have a non-homogeous distribution of acetylenic fatty acids. The chemotaxonomic significance of the fatty acid composition in relation to morphological characters is discussed.     

Santalbic acid in the plant kingdom

The occurrence and distribution of santalbic acid in the plant kingdom are reported and discussed. This conjugated acetylenic fatty acid, octadeca-11-trans-en-9-ynoic acid (in “Delta Notation” 18:2Δ9a,11t), occurs in members of the santalalean plant families, mostly in tropical and subtropical areas. The range is from only traces up to 95.0?% of total fatty acids in the seed oil. However, some caution is required during the analysis, so the presence of this acid may have been overlooked sometimes. This fatty acid may be toxic to humans, but may be of interest for technical purposes because of a reactive functional group.     

9-octadecen-6-ynoic acid from Riccia fluitans

A new acetylenic fatty acid, 9-octadecen-6-ynoic acid, and the known compounds 9,12-octadecadien-6-ynoic acid and 9,12,15-octadecatrien-6-ynoic acid were isolated from the thalli of Riccia fluitans. The structure of the new compound was established by spectroscopic methods.     

Syntheses of long-chain acids,part X acetylenic hydroxy-acids and 8,9,13-trihydroxydocosanoic acid

The dilithio-derivative of a terminal acetylenic secondary alcohol reacts with alkyl halide to form the long-chain acetylenic alcohol by C-alkylation. Similar condensation with the NN-dimethylamide of an ω-bromo-acid, followed by hydrolysis, gives the acetylenic hydroxy-acid in low yield. 13-Hydroxydocos-8-ynoic acid is obtained by this route and, more satisfactorily, by condensation of 7-bromo-NN-dimethylheptanamide with the lithium derivative of pentadec-1-yn-6-one ethylene glycol ketal, with subsequent hydrolysis and reduction. Catalytic partial hydrogenation of the acetylenic hydroxy-acid, and then cis-hydroxylation with potassium permanganate, gives two isomers of (±)-8,9 (erythro) 13-trihydroxydocosanoic acid. One of these corresponds to the (+)-8,9,13-trihydroxydocosanoic acid isolated by Stodola and his co-workers from the extracellular lipid produced by a yeast obtained from the frass of white spruce.     

Fatty acid and sterol compositions in mushrooms of ten species of polyporaceae

The simple lipids present in ten species of Polyporaceae (Piptororus betulinus, Coriolus pargamenus, C. versicolor, C. heteromorphus, Formitopsis cytisina, F. pinicola, Microporus flabelliformis, Gloephyllum saepiarium, Crytoderma citrinum and Grifola frondosa) were investigated. The fatty acids that these species had in common were C₁₆-saturated acids (except in P. betulinus) and C₁₈-unsaturated acids. Ergosterol and ergosta-7,22-dien-3β-ol were isolated from these mushrooms. Lupeol was obtained from G. saepiarium. Ergost-7-en-3β-ol, lanosterol and 24-methylene-24,25-dihydrolanosterol were tentatively identified.     

Synthesis and spectral characteristics of some unusual fatty esters of podophyllotoxin

Five fatty ester derivatives of podophyllotoxin have been prepared by reacting the corresponding fatty acids with the hydroxy group of podophyllotoxin in the presence of dimethylaminopyridine and N,N-dicyclohexylcarbodiimide. The fatty acids incorporated are: 9,12-epoxy-9,11-octadecadienoic acid, octadec-11E-en-9-ynoic acid, 11,12-E-epoxy-octadec-9-ynoic acid, octadeca-9Z,11E-dienoic acid and 9,10-dibromooctadecanoic acid. The average yield of esterification was >95% and the structures of the products were confirmed by a combination of nuclear magnetic resonance spectroscopic and mass spectrometric analyses.     

Fatty acids,part X. Raman spectra of all dimethylene interrupted methyl cis,cis-octadecadienoates and octadecadiynoates

The Raman spectra of all the dimethylene interrupted methyl cis, cis-octadecadienoates and octadecadiynoates have been studied. The Raman band positions and their relative intensities for the ν(CC), ν(CC), ν(CO), ν(CH) and δ(CH₂) modes are recorded. The height intensity of the bands arising from ν(CC) relative to ν(CO) provides a means of determining the number of cis-ethylenic bonds in a mono-ester. In the acetylenic series, the intensity of the bands arising from ν(CC) relative to ν(CO) failed to indicate with certainty the number of acetylenic bonds in the mono-esters studied, due to the weak intensity of the band due to ν(CO). However a better correlation between the relative intensities of the ν(CC) and δ(CH₂) bands is established instead. An attempt to correlate the areas under the bands due to ν(CC), (CC), (CO) and δ(CH₂) failed to produce any significant results. The Raman spectra of the methyl octadec-cis-10-en-5-ynoate and methyl octadeca-5, 10-diynoate are also recorded.     

Stream mosses as chemically-defended refugia for freshwater macroinvertebrates

Marine and terrestrial studies show that small, sedentary herbivores that utilize plants as both food and habitat can gain enemy-free space by living on hosts that are chemically defended from larger, generalist consumers. Although large herbivores are increasingly recognized as important consumers of macrophytes in freshwater communities, the potential indirect effects of herbivory on plant-associated macroinvertebrates have rarely been studied. Here, we show that the large, generalist consumers in a riverine system, Canada geese, Branta canadensis , and crayfish, Procambarus spiculifer , both selectively consumed riverweed, Podostemum ceratophyllum , over an aquatic moss, Fontinalis novae-angliae, even though moss comprised 89% of the total plant biomass on riverine rocky shoals. Moss supported twice as many plant-associated macroinvertebrates as riverweed, suggesting that it might provide a spatial refuge from consumption by these larger consumers. Bioassay-guided fractionation of moss extracts led to the isolation of a C₁₈ acetylenic acid, octadeca-9,12-dien-6-ynoic acid, that deterred crayfish feeding. In contrast to results with Canada geese and crayfish, both the amphipod Crangonyx gracilis and the isopod Asellus aquaticus consumed significant amounts of moss but rejected riverweed in laboratory feeding assays. Moreover, neither amphipod nor isopod feeding was deterred by the crude organic extract of Fontinalis , suggesting that these mesograzers tolerate or circumvent the chemical defenses that deterred larger consumers. Thus, herbivory by large, generalist herbivores may drive freshwater plant community structure towards chemically defended plants and favor the ecological specialization of smaller, less mobile herbivores on unpalatable hosts that represent enemy-free space.     

Production of 6-Phenylacetylene Picolinic Acid from Diphenylacetylene by a Toluene-Degrading Acinetobacter Strain

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene. Acinetobacter sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.     

Physiology of Sporeforming Bacteria Associated with Insects II. Lipids of Vegetative Cells

Lipid composition was studied in two strains each of mid-log phase cells of Bacillus thuringiensis, B. larvae, B. popilliae, B. alvei, and B. lentimorbus. Total lipids varied from 2.5 to 3.5% of the cell dry weight of B. thuringiensis to 4.3 to 5.0% of B. popilliae. Phospholipids in the organisms examined ranged from 55 to 79% of total lipids; neutral lipids averaged from 13 to 45%. Common phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and lysophosphatidylethanolamine. 1,2-Diglycerides, methyl esters, free fatty acids, and hydrocarbons were found in all the organisms studied. Branched-chain fatty acids constituted more than 50% of the total fatty acids in B. thuringiensis, B. larvae, B. popilliae, and B. alvei, whereas, in B. lentimorbus, normal-chain acids constituted more than 50%. Anteiso-C₁₅ (12-methyltetradeconoate) was the most abundant acid (30 to 50%) in B. alvei, B. larvae, B. popilliae, and B. lentimorbus. In contrast, B. thuringiensis contained more iso-C₁₃ (7%), iso-C₁₅ (17%), normal-C₁₆ (24%), and iso-C₁₇ (18%) than anteiso-C₁₅ (6%). The distribution of individual fatty acids was similar in the phospholipids and neutral lipids of each organism. However, the total amount of iso, anteiso, and normal isomers differed.     

Acetylenic fatty acids from Porcelia macrocarpa (Annonaceae) against trypomastigotes of Trypanosoma cruzi: Effect of octadec-9-ynoic acid in plasma membrane electric potential

Porcelia macrocarpa (Warm.) R. E. Fries (Annonaceae) is an endemic plant in Brazil where its tasty pulp has been eaten fresh. The hexane extract from its flowers was subjected to chromatographic procedures to afford four acetylene derivatives identified as octadec-9-ynoic (stearolic acid – 1), (11E)-octadec-11-en-9-ynoic (santalbic acid – 2), 8-hydroxyoctadec-9,11-diynoic (3) and 8-hydroxyoctadec-17-en-9,11-diynoic (isanolic acid – 4) acids by NMR and HRESIMS. Among tested compounds against trypomastigote forms of T. cruzi, octadec-9-ynoic acid (1) displayed higher potential with IC₅₀ = 27.6 µM and a selectivity index (SI) higher than 7. Compounds 2 and 3 showed IC₅₀ of approximately 60 µM while compound 4 was inactive. The lethal action of the compound 1 was investigated using spectrofluorometric techniques to detect ROS content, plasma membrane permeability and plasma membrane potential by flow cytometry. Compound 1 showed no alteration in the production of ROS of treated trypomastigotes and no alteration of the plasma membrane permeability was observed as detected by the fluorescent probe SYTOX-green after 120 min of incubation. However, by using the potential-sensitive fluorescent probe DiSBAC₂(3), compound 1 caused depolarization of the plasma membrane potential when compared to untreated parasites. Our results demonstrated the anti-T. cruzi effects of compounds 1–3 isolated from flowers of P. macrocarpa and indicated that the lethal effect of compound 1 in T. cruzi could be associated to the plasma membrane disturbance of the parasite.     

Cobalt carbonyl complexes as probes for alkyne-tagged lipids

Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a “tag” that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV_349nm. The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity “pull-down” strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection.     

The absolute configuration of the acetylenic compound falcarindiol

The absolute configuration of the acetylenic compound falcarindiol is established as (3R,8S) and falcarindiol is thus (+)-(3R,8S)-(Z)-heptadeca-1,9-dien-4,6-diyn-3,8-diol. (R)-Heptadecan-8-ol and (S)-heptadecan-8- ol are synthesized and (3R,8S)-heptadecan-3,8-diol is characterized.     

Epicuticular wax of Pinus radiata needles

Epicuticular wax isolated from the cotyledons and primary needles of 10-week-old Pinus radiata seedlings is similar in composition and contains 86% neutral compounds, viz. alkyl esters (25%, C₂₄–C₆₄), nonacosan-10-ol (52%), heptacosane-5,10-diol (2%), nonacosane-4,10-diol, nonacosane-5,10-diol, and nonacosane-10,13-diol (total 12%) and estolides, MW ca 800 (2%), MW ca 1100 (6%), and MW ca 1500 (1%). The acidic fraction (14%) contains n-acids (78%, C₁₂–C₃₂) and diterpene acids (22%, mainly abieta-8,11,13-trien-18-oic, with lesser amounts of pimara-8(14),15-dien-18-oic, isopimara-7,15-dien-18-oic and hydroxylated aromatic, diene and mono-ene acids). Wax isolated from primary needles of 1-yr-old seedlings had a similar neutral fraction composition, but the acidic fraction contained predominantly the diterpene acid mixture, with only trace amounts of n-acids. The wax from 1-yr-old secondary, needles from P. radiata forest trees aged 5 yr and 40 yr contained an acid fraction (12% 5 yr, 17% 40 yr trees) comprising the diterpene acid mixture, with trace amounts of n-acids together with ω-hydroxy acids (C₁₂, C₁₄ and C₁₆). The neutral fraction from both young and old trees had a similar composition containing alkyl esters (7%, C₂₄–C₆₆), estolides (90%, MW 566-ca 1500), nonacosan-10-ol (2%) and the heptacosane and nonacosane diols (1%). During growth and maturation of P. radiata, the nonacosan-10-ol content of the needle wax decreases while the proportion of estolides and diterpene acids increases, the latter probably being located around the stomatal pore.     

Differential inhibition of thromboxane B2 and leukotriene B4 biosynthesis by two naturally occurring acetylenic fatty acids

The seed oil of the plant Ixiolaena brevicompta is a rich source of crepenynic acid (octadec-cis-9-en-12-ynoic acid), which has been linked with extensive sheep mortalities in Western New South Wales and Queensland, Australia. A number of acetylenic fatty acids have been found to interfere with lipid and fatty acid metabolism and inhibit cyclooxygenase and lipoxygenase enzymes in a variety of tissues. We have investigated the effects of crepenynic acid and ximenynic acid (octadec-trans-11-en-9-ynoic acid) on leukotriene B4 and thromboxane B2 production in rat peritoneal leukocytes and compare them with non-acetylenic compounds linoleic and ricinoleic acids. In concentrations ranging from 10 to 100 microM linoleic acid and ricinoleic acid had only minimal effects on leukotriene B4 and thromboxane B2 production in ionophore-stimulated cells. Ximenynic acid gave dose-dependent inhibition of leukotriene B4, thromboxane B2 and 6-ketoprostaglandin F1 alpha production. Ximenynic acid appears to be a more effective inhibitor of leukotriene B4 than crepenynic acid with an IC50 of 60 microM compared to 85 microM. On the other hand, crepenynic acid is a much more effective inhibitor of the cyclooxygenase products, having an IC50 for thromboxane B2 of less than 10 microM. Both acetylenic fatty acids inhibited phospholipase activity in these cells by 40-50% at a concentration of 100 microM but had no inhibitory effect at 10 microM. These results indicate that crepenynic acid and ximenynic acid differentially inhibit the cyclooxygenase and lipoxygenase products of stimulated leukocytes, and that at high doses of these fatty acids the effect on these products may be partially due to inhibition of phospholipase A2.     

The influence of branched-chain and ω-alicyclic fatty acids on the transition temperature of bacillus subtilis lipids

The influence of branched-chain and ω-alicyclic fatty acids on the transition temperature of Bacillus subtilis lipids was studied by measuring the fluorescence depolarisation of the probe 1,6-diphenyl-1,3,5-hexatriene incorporated into lipid bilayers. Only anteiso-C₁₅ and C₁₇ fatty acid-enriched lipids showed no transition in the observed temperature range. Compared to the transition of normal lipids iso-fatty acid-enriched lipids have a slightly higher transition temperature. The incorporation of ω-alicyclic fatty acids with increasing size of the alicycle leads to a decrease in the transition temperature. A possible role of ω-cyclohexane fatty acids in Bacillus acidocaldarius is proposed.     

The Influence of Growth Rate on 2H/1H Fractionation in Continuous Cultures of the Coccolithophorid Emiliania huxleyi and the Diatom Thalassiosira pseudonana

The hydrogen isotope (²H/¹H) ratio of lipids from phytoplankton is a powerful new tool for reconstructing hydroclimate variations in the geologic past from marine and lacustrine sediments. Water ²H/¹H changes are reflected in lipid ²H/¹H changes with R² > 0.99, and salinity variations have been shown to cause about a 1‰ change in lipid δ²H values per unit (ppt) change in salinity. Less understood are the effects of growth rate, nutrient limitation and light on ²H/¹H fractionation in phytoplankton. Here we present the first published study of growth rate effects on ²H/¹H fractionation in the lipids of coccolithophorids grown in continuous cultures. Emiliania huxleyi was cultivated in steady state at four growth rates and the δ²H value of individual alkenones (C_37:2, C_37:3, C_38:2, C_38:3), fatty acids (C_14:0, C_16:0, C_18:0), and 24-methyl cholest-5,22-dien-3β-ol (brassicasterol) were measured. ²H/¹H fractionation increased in all lipids as growth rate increased by 24‰ to 79‰ (div d^-1)^-1. We attribute this response to a proportional increase in the fraction of NADPH from Photosystem I (PS1) of photosynthesis relative to NADPH from the cytosolic oxidative pentose phosphate (OPP) pathway in the synthesis of lipids as growth rate increases. A 3-endmember model is presented in which lipid hydrogen comes from NADPH produced in PS1, NADPH produced by OPP, and intracellular water. With published values or best estimates of the fractionation factors for these sources (α_PS1 = 0.4, α_OPP = 0.75, and α_H2O = 0) and half of the hydrogen in a lipid derived from water the model indicates α_lipid = 0.79. This value is within the range measured for alkenones (α_alkenone = 0.77 to 0.81) and fatty acids (α_FA = 0.75 to 0.82) in the chemostat cultures, but is greater than the range for brassicasterol (α_{brassicasterol} = 0.68 to 0.72). The latter is attributed to a greater proportion of hydrogen from NADPH relative to water in isoprenoid lipids. The model successfully explains the increase in ²H/¹H fractionation in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol from marine centric diatom T. pseudonana chemostat cultures as growth rate increases. Insensitivity of α_FA in those same cultures may be attributable to a larger fraction of hydrogen in fatty acids sourced from intracellular water at the expense of NADPH as growth rate increases. The high sensitivity of α to growth rate in E. huxleyi lipids and a T. pseudonana sterol implies that any change in growth rate larger than ~0.15 div d^-1 can cause a change in δ²H_lipid that is larger than the analytical error of the measurement (~5‰), and needs to be considered when interpreting δ²H_lipid variations in sediments.