Identification of conjugated linoleic acid elongation and beta-oxidation products by coupled silver-ion HPLC APPI-MS

Atmospheric pressure photoionisation (APPI) was used in combination with silver-ion (Ag(+))-HPLC for detection of (conjugated) fatty acid methyl esters (FAME) by tandem-mass spectrometry. APPI-MS of methyl esters of conjugated linoleic acid showed an increase in signal-to-noise ratio by a factor of 40 compared to atmospheric pressure chemical ionization in the positive mode. It was possible to identify double bond position, configuration and chain length of FAME based on chromatographic separation and mass detection. The developed LC-MS method is useful for the analysis of CLA elongation and beta-oxidation products, especially with trans,trans-configuration, which are difficult to analyze by conventional GC-MS techniques.     

Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Structural Determination of Fatty Acid Components in the Seed Oils of Five Species of Euphorbiaceae

The fatty acid components of the seed oils of five species of Euphorbiaceae collected in Xishuanbanna district, Yunnan province—Aleurites montana (Lour.) Wis., Mallotus paniculatus (Lam.) Muell-Arg., Ostodes paniculata Bl., Mcaranga denticulata (Bl.) Muell-Arg., and Trewia nudiflora L.- have been analyzed. Long-chain fatty acids were first converted to corresponding 2-substituted 4,4-dimethyloxazoline derivatives (DMOX) by condensing with 2-amino- 2-methylpropanol (AMP) and subsequently analyzed by GC-MS. The position of unsaturation (double and triple bonds) and substituents (e.g., hydroxyl) in the aliphatic chain can easily be located by interpreting the mass spectra recorded in this way. The qualitative as well as quantitative results can be provided for all constituents in a single GC-MS run. This me thod proves simple and efficient, with good reliability, and is well suitable for the structure determination without reference specimens.     

Novel modification of glycosphingolipids by long-chain cyclic acetals: isolation and characterization of plasmalocerebroside from human brain.

A glycosphingolipid component of human brain, having long-chain cyclic acetals, has been isolated and characterized. This compound incorporates a novel type of natural glycan modification, in which a long-chain aliphatic aldehyde is conjugated through a cyclic acetal (plasmal) linkage to the galactosyl moiety of cerebroside. In addition to components normally observed by gas chromatography-mass spectrometry (GC-MS) following methanolysis of cerebroside (fatty acid methyl esters, methyl alpha- and beta-galactosides, sphingosine), this compound produced 16:0, 18:0, and 18:1 fatty aldehydes, unequivocally identified as their enol methyl ether derivatives. Results of positive ion fast atom bombardment mass spectrometry (FAB-MS) of the native compound, and GC-MS of partially methylated hexitol acetates derived from the permethylated derivative, were consistent with structures of galactocerebroside having 3,4- and 4,6-linked cyclic plasmal substituents, as shown. [formula: see text]     

Positional Isomerization of trans-3-Hexadecenoic Acid Employing 2-Amino-2-methyl-propanol as a Derivatizing Agent for Ethylenic Bond Location by Gas-Chromatography/Mass Spectrometry

The effect of derivatization with 2-amino-2-methyl-propanol on trans-3-hexadecenoic acid was investigated as part of the identification of the trans-3-hexadecenoic acid in two Nova Scotian seaweeds. After the extraction of the total fatty acids and their methylation, the monoenoic trans fraction was isolated by thin-layer chromatography on silica gels impregnated with silver nitrate. This fraction was first analyzed by gas chromatography and showed the presence of the trans-3-hexadecenoic acid; other fatty acids were not present. The isolated fraction was derivatized with 2-amino-2-methyl-propanol prior to analysis by gas chromatography/mass spectrometry. The chromatogram obtained showed the presence of a positional isomer formed during the derivatization of the trans-3-hexadecenoic acid. The mass spectrum showed a prominent [M + H]⁺ and diagnostic ions for the identification of the unknown isomer, corresponding to the 4,4-dimethyloxazoline (DMOX) derivative of a presumed 2-hexadecenoic acid. Definitive confirmation of the ethylenic bond position was obtained by oxidative ozonolysis of the DMOX derivatives of the fatty acids under investigation. Infrared spectroscopy showed that the artifact formed during the DMOX derivatization of trans-3-hexadecenoic acid was the DMOX derivative of cis-2-hexadecenoic acid.     

少根根霉Δ^6-脂肪酸脱氢酶基因在毕赤酵母中的表达

Δ^6-脂肪酸脱氢酶是一种膜整合蛋白,也是多不饱和脂肪酸合成途径中的限速酶。在前期工作中,通过RT-PCR和RACE技术,从少根根霉NK300037中克隆到一个潜在编码Δ^6-脂肪酸脱氢酶的序列,序列和功能分析结果表明该序列具有一个长度为1377bp、编码由458个氨基酸组成、大小为52kD的新的Δ^6-肪酸脱氢酶基因。把少根根霉Δ^6-脂肪酸脱氢酶基因（RAD6）亚克隆到表达载体pPIC3．5K,构建重组表达载体pPICRAD6,并转化到毕赤酵母菌株GS115进行表达。提取酵母细胞总脂肪酸和进行甲酯化,经气相色谱和气相色谱-质谱连用分析表明,目的基因的编码产物能将C16：1、C17：1、C18：1、亚油酸和α-亚麻酸在△6和7位间特异性脱氢而引入一个新的双键,生成更高不饱和的脂肪酸,该催化反应没有链长特异性,只有键位特异性。此外,按Kozak序列特点,改变目的基因转译起始密码子周边序列结构,并把改变后序列导入毕赤酵母GS115中进行功能表达分析,结果表明在毕赤酵母中这种改变同样能提高目的基因的表达水平。综合所有分析结果表明,巴斯德毕赤酵母更适合用来综合分析Δ^6-脂肪酸脱氢酶基因的功能。     

Isolation of 2-methyl branched unsaturated very long fatty acids from marine sponge Halichondria panicea and identification of them by GC-MS and NMR

In order to study the biomarker fatty acids of symbionts in the marine sponge Halichondria panicea, purification and structural identification of two new 2-methyl branched monoenoic very long fatty acids (2-Me-24:1 n-7 and 2-Me-26:1 n-9) were performed for the first time. These acids amounted to 7.1% of total sponge FAs, but our attempts to determine their structures by one-step GC-MS analysis were unsuccessful because of low yields of the correspondent N-acyl pyrrolidide derivatives. Silver-ion thin-layer chromatography isolated enriched fractions of monoenoic fatty acids extracted from the sponge. Further purification of unknown fatty acid methyl esters was carried out by reversed-phase high-performance liquid chromatography. Determination of the chain length, degree and position of unsaturations was achieved by gas chromatography-mass spectrometry on methyl esters and dimethyldisulfide adducts. Structures, position of methyl substitution, and double bonds cis isomery were confirmed by 1H nuclear magnetic resonance.     

The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates

Differentiation of Naegleria amebae into flagellates was used to examine the interaction between actin, actomyosin and microtubules in defining cell shape. Amebae, which lack microtubules except during mitosis, differentiate into flagellates with a fixed shape and a complex microtubule cytoskeleton in 120 min. Based on earlier models of ameboid motility it has been suggested that actomyosin is quiescent in flagellates. This hypothesis was tested by following changes in the cytoskeleton using three-dimensional reconstructions prepared by confocal microscopy of individual cells stained with antibodies against actin and tubulin as well as with phalloidin and DNase I. F-actin as defined by phalloidin staining was concentrated in expanding pseudopods. Most phalloidin staining was lost as cells rounded up before the onset of flagellum formation. Actin staining with a Naegleria-specific antibody that recognizes both F- and G-actin was confined to the cell cortex of both amebae and flagellates. DNase I demonstrated G-actin throughout all stages. Most of the actin in the cortex was not bound by phalloidin yet was resistant to detergent extraction suggesting that it was polymerized. The microtubule cytoskeleton of flagellates was intimately associated with this actin cortex. Treatment of flagellates with cytochalasin D produced a rapid loss of flagellate shape and the appearance of phalloidin staining while latrunculin A stabilized the flagellate shape. These results suggest that tension produced by an actomyosin network is required to maintain the flagellate shape. The rapid loss of the flagellate shape induced by drugs, which specifically block myosin light chain kinase, supports this hypothesis.     

Functional characterization of a desaturase from the tobacco hornworm moth (Manduca sexta) with bifunctional Z11- and 10,12-desaturase activity

The pheromone blend produced by the tobacco hornworm moth (Manduca sexta) (L.) female is unusually complex and contains two conjugated dienals and trienals together with two monounsaturated alkenals. Here, we describe the identification and construction of two genes encoding MsexKPSE and MsexAPTQ desaturases from a cDNA library prepared from the total RNA of the M. sexta pheromone gland. The MsexKPSE desaturase shares a high degree of similarity with Delta(9)-desaturases from different moth species. The functional expression of MsexAPTQ desaturase in Saccharomyces cerevisiae followed by a detailed GC-MS analysis of fatty acid methyl esters (FAME) and their derivatized products and gas-phase Fourier transform infrared (FTIR) spectroscopy of the extracted FAME confirms that this enzyme is a bifunctional Z-Delta(11)-desaturase. MsexAPTQ desaturase catalyses the production of Z11-hexadecenoate (Z11-16) and Z10E12- and E10E12-hexadecadienoates (Z10E12-16) via 1,4-desaturation of the Z11-16 substrate. The stereochemistry of 1,4-desaturation and formation of isomers is discussed.     

Viruses and flagellates sustain apparent richness and reduce biomass accumulation of bacterioplankton in coastal marine waters

To gain a better understanding of the interactions among bacteria, viruses and flagellates in coastal marine ecosystems, we investigated the effect of viral lysis and protistan bacterivory on bacterial abundance, production and diversity [determined by 16S rRNA gene polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE)] in three coastal marine sites with different nutrient supplies in Hong Kong. Six experiments were set up using filtration and dilution methods to develop virus, flagellate and virus+flagellate treatments for natural bacterial populations. All three predation treatments had significant repressing effects on bacterial abundance. Bacterial production was significantly repressed by flagellates and both predators (flagellates and viruses). Bacterial apparent species richness (indicated as the number of DGGE bands) was always significantly higher in the presence of viruses, flagellates and both predators than in the predator-free control. Cluster analysis of the DGGE patterns showed that the effects of viruses and flagellates on bacterial community structure were relatively stochastic while the co-effects of predators caused consistent trends (DGGE always showed the most similar patterns when compared with those of in situ environments) and substantially increased the apparent richness. Overall, we found strong evidence that viral lysis and protist bacterivory act additively to reduce bacterial production and to sustain diversity. This first systematic attempt to study the interactive effects of viruses and flagellates on the diversity and production of bacterial communities in coastal waters suggests that a tight control of bacterioplankton dominants results in relatively stable bacterioplankton communities.     

Rearrangement of 5S, 12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic acid during gas chromatography: formation of a cyclohexadiene derivative

The 5S, 12S-dihydroxy-6,8,10,14-(E,Z,E,Z,)-eicosatetraenoic acid, a product of double dioxygenation of arachidonic acid by lipoxygenases, undergoes severe decomposition during gas chromatography-mass spectrometric (GC-MS) analysis of the trimethylsilyl ether methyl ester derivative. The decomposition product was studied by GC-MS and identified as a cyclohexadiene derivative of the parent compound formed by ring closure at C6 and C11. Under identical GC conditions, two stereoisomers, i.e. 5S,12R-dihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic acid (leukotriene B4), and 6-trans-leukotriene B4 showed excellent chromatographic properties. These data indicated that the 5,12-dihydroxy derivative of arachidonic acid carrying the trans-cis-trans triene unit selectively undergoes cyclization during GC. These studies also provided an explanation to the controversial GC-MS data reported for this lipoxygenase product.     

Double bond localization in minor homoallylic fatty acid methyl esters using acetonitrile chemical ionization tandem mass spectrometry

Double bond position in natural fatty acids is critical to biochemical properties, however, common instrument-based methods cannot locate double bonds in fatty acid methyl esters (FAME), the predominant analysis form of fatty acids. A recently described mass spectrometry (MS) method for locating double bonds in FAME is reported here for the analysis of minor (<1%) components of real FAME mixtures derived from three natural sources; golden algae (Schizochytrium sp.), primate brain white matter, and transgenic mouse liver. Acetonitrile chemical ionization tandem MS was used to determine double bond positions in 39 FAME, most at concentrations well below 1% of all fatty acid methyl esters. FAME identified in golden algae are 14:1n-6, 14:3n-3, 16:1n-7, 16:2n-6, 16:3n-6, 16:3n-3, 16:4n-3, 18:2n-7, 18:3n-7, 18:3n-8, 18:4n-3, 18:4n-5, 20:3n-7, 20:4n-3, 20:4n-5, 20:4n-7, 20:5n-3, and 22:4n-9. Additional FAME identified in primate brain white matter are 20:1n-7, 20:1n-9, 20:2n-7, 20:2n-9, 22:1n-7, 22:1n-9, 22:1n-13, 22:2n-6, 22:2n-7, 22:2n-9, 22:3n-6, 22:3n-7, 22:3n-9, 22:4n-6, 24:1n-7, 24:1n-9, and 24:4n-6. Additional FAME identified in mouse liver are 26:5n-6, 26:6n-3, 28:5n-6, and 28:6n-3. The primate brain 22:3n-7 and algae 18:4n-5 are novel fatty acids. These results demonstrate the usefulness of the technique for analysis of real samples. Tables are presented to aid in interpretation of acetonitrile CIMS/MS spectra.     

Synthesis of racemic 9-methyl-10-hexadecenoic acid

The marine bacterial fatty acid 9-methyl-10-hexadecenoic acid was conveniently prepared in 6 steps and in a 22% overall yield, starting from commercially available methyl 10-hydroxydecanoate. The naturally occurring fatty acid has the E double bond configuration as confirmed by gas chromatographic co-elution experiments.     

A desaturase-like protein from white spruce is a Delta(9) desaturase

Gymnospermae seed lipids are characterized by a high degree of desaturation, most having a Delta(9) double bond. By degenerate polymerase chain reaction (PCR) we have isolated a white spruce (Picea glauca) cDNA clone that encodes an amino acid sequence sharing a high degree of homology with other putative plant acyl-coenzyme A (CoA) Des9 desaturases. Both in vivo and in vitro expression studies in a Delta(9) desaturase-deficient yeast strain demonstrated the desaturation functionality of the white spruce clone, and gas chromatography-mass spectrometry (GC-MS) analyses confirmed the regioselectivity of the encoded enzyme. This is the first report of the functional characterization of a plant membrane-bound acyl-CoA-like protein Delta(9) desaturase by heterologous expression in yeast.     

Grazing Characteristics and Growth Efficiencies at Two Different Temperatures for Three Nanoflagellates Fed with Vibrio Bacteria at Three Different Concentrations

Small inocula of one of the flagellates Paraphysomonas imperforata, Pteridomonas danica, and Cafeteria roenbergensis were added to suspensions of the bacterium Vibrio natriegens at each of three concentrations between 107 and 108 cells ml-1 and incubated at each of the temperatures 10 degrees C and 25 degrees C. Samples were taken at intervals for counting the flagellates and bacteria to determine the timing of the maximum of flagellate numbers and the concentrations at that time. Measurements of the protein concentration of the suspensions during incubation were used to determine the gross growth efficiency (GGE) or yield of flagellate grazing in each experiment. The most effective grazer was Pteridomonas, followed by Paraphysomonas, with Cafeteria being least effective, as judged by the threshold bacterial concentrations at which flagellate multiplication ceased, which were about 2 x 105, 2 x 106, and 2 x 107, respectively, and by the finding that Pteridomonas consumed 99%, Paraphysomonas about 95%, and Cafeteria only 60-70% of the available bacteria in the experiments. Peak concentrations of flagellates were reached later at the lower temperature, but the numbers of flagellates produced and of bacteria eaten were of a similar order at the two temperatures and the GGE was only slightly higher at the lower temperature. The time taken to reach peak flagellate numbers changed little with a threefold increase in bacterial concentrations, but the GGE increased and the numbers of bacteria eaten to produce one flagellate decreased when the bacterial concentration was increased. The three flagellates show clear evidence of niche specialization in differences in thresholds of bacterial prey concentration.     

Gyrotaxis: Interaction between algae and flagellates

We describe the behaviour of motile microorganisms (e.g. flagellates) attracted by “gyrotaxis” to a sinking, non-motile particle (e.g. an algal cell). The model is based on the application of Stokes' solution for the flow field around the settling cell. The volume within which the flagellate is attracted to the sinking particle is determined from the trajectories of the flagellate. The model of gyrotaxis has several applications; these include the colonization of sinking marine snow particles with motile microoganisms and suspension feeding by protozoa.     

Antimicrobial activity of fatty acid methyl esters of some members of Chenopodiaceae

Fatty acid methyl ester (FAME) extracts of four halophytic plants, viz. Arthrocnemum indicum, Salicornia brachiata, Suaeda maritima and Suaeda monoica belonging to the family Chenopodiaceae, were prepared and their composition was analyzed by GC-MS. The FAME extracts were also screened for antibacterial and antifungal activities. The GC-MS analysis revealed the presence of more saturated fatty acids than unsaturated fatty acids. Among the fatty acids analyzed, the relative percentage of lauric acid was high in S. brachiata (61.85%). The FAME extract of S. brachiata showed the highest antibacterial and antifungal activities among the extracts tested. The other three extracts showed potent antibacterial and moderate anticandidal activities.     

The structure of extrusive organelles in alveolate and other heterotrophic flagellates

The structure of ejective organelles (extrusomes) in 12 species of heterotrophic flagellates belonging to 8 taxonomic groups is considered. Trichocysts of various structures have been found in colpodellids, Spumella and Metromonas. Kinetocysts of amoeboid flagellates, thaumatomonads, contain a cylindrical element. These kinetocysts are attached to bacteria after discharge. When being discharged, trichocysts of colpodellids produce filaments which have transversal striation. Discobolocysts have been found in excavate flagellate Reclinomonas, and ejectisomes--in Goniomonas. Toxicysts have been marked in carnivorous alveolate flagellate Colponema. The most of studied extrusomes lie inside the cytoplasm, while exstrusomes in chrysomonad Spumella are also found inside the nucleus. Trichocysts of colpodellids and Metromonas as well as ejectisomes of Goniomonas are located near the branches of Golgi apparatus. The comparison of the data obtained confirms the hypothesis about the correlation of taxonomic position of flagellates and the structure of their extrusomes that allows in some cases using these features as phylogenetic markers.     

Distribution of heterotrophic flagellates at the littoral of estuary of Chernaya River (Kandalaksha Bay, White Sea)

Species composition, distribution, and the character of structural changes in the heterotrophic flagellate community were studied along environmental gradients in the Chernaya River estuary. There were 99 species and forms of heterotrophic flagellates, subdivided into three groups: prevalently marine species and euryhaline species preferring biotopes either of higher or decreased salinity. The heterotrophic flagellate community of the estuary was continuously divided into two distinct variants: (1) cenosis of halophilic species, prevalently of sea forms and euryhaline species preferring biotopes of increased salinity; (2) cenosis of halophobic species with prevalence of euryhaline forms gravitating to fresh biotopes. The arbitrary and indistinct boundary between the variants of the community ran at a salinity of 9–10‰. The response of estuarine communities of heterotrophic flagellates and infusorians to variation of abiotic factors was similar and differed from response of communities of microphyto-, meiozoo-and macrozoobenthos; this implied similarity of the response mechanism to environmental factors in organisms of one level of organization.