Caenorhabditis elegans Delta12-desaturase FAT-2 is a bifunctional desaturase able to desaturate a diverse range of fatty acid substrates at the Delta12 and Delta15 positions

Caenorhabditis elegans FAT-2 has been characterized as fatty acid Δ12-desaturase able to desaturate C16 and C18 fatty acids. However, in this report we show that when expressed in yeast cells this enzyme can also catalyze Δ15 desaturation. This results in the production of both linoleic acid (ω6 C18:2Δ9,12) and linolenic acid (ω3 C18:3Δ9,12,15) from oleic acid (C18:1Δ9) substrate, and hexadecadienoic acid (ω4 C16:2Δ9,12) and hexadecatrienoic acid (ω1 C16:3Δ9,12,15) from palmitoleic acid (C16:1Δ9) substrate. In addition, this enzyme can also produce C14:2Δ9,12, C15:2Δ9,12, C17:2Δ9,12, and C18:4Δ6,9,12,15 when C14:1Δ9, C15:1Δ9, C17:1Δ9, and C18:3Δ6,9,12 substrates are available in yeast cells. Mass spectrometry analysis of 2,4-dimethyloxazoline modification of fatty acid methyl esters confirms the positions of all newly formed double bonds. These results indicate that when expressed in yeast the C. elegans Δ12-desaturase CeFAT-2 shows a characteristic of a bifunctional Δ12/Δ15-desaturase and has a great deal of elasticity with respect to fatty acid chain length in being able to accept fatty acids ranging from C14 to C18. Interestingly, despite possessing a bifunctional Δ12/Δ15 desaturation activity, phylogenetic analysis suggests that C. elegans Δ12-desaturase CeFAT-2 might have arisen independently from other reported dual Δ12/Δ15-desaturases from fungi and protozoa.     

Production of arachidonic and eicosapentaenoic acids in plants using bryophyte fatty acid Delta6-desaturase, Delta6-elongase, and Delta5-desaturase genes

The liverwort Marchantia polymorpha L. synthesizes arachidonic (ARA) and eicosapentaenoic acids (EPA) from linoleic and alpha-linolenic acids respectively by a series of reactions catalyzed by Delta6-desaturase, Delta6-elongase, and Delta5-desaturase. Overexpression of the M. polymorpha genes encoding these enzymes in transgenic M. polymorpha plants resulted in 3- and 2-fold accumulation of ARA and EPA respectively, as compared to those in the wild type. When these three genes were introduced and co-expressed in tobacco plants, in which long-chain polyunsaturated fatty acids (LCPUFAs) are not native cellular components, ARA and EPA represented up to 15.5% and 4.9% respectively of the total fatty acid in the leaves. Similarly in soybean, C20-LCPUFAs represented up to 19.5% of the total fatty acids in the seeds. These results suggest that M. polymorpha can provide genes crucial to the production of C20-LCPUFAs in transgenic plants.     

The alternative pathway C20 Delta8-desaturase from the non-photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5-fusion desaturase

A cDNA encoding a C20 Delta8-desaturase was isolated from the free-living soil amoeba, Acanthamoeba castellanii and functionally characterised by heterologous expression. The open reading frame of the A. castellanii C20 Delta8-desaturase showed similarity to other microsomal front-end desaturases, but the N-terminal domain contained a variant form of the conserved heme-binding motif in which H-P-G-G is replaced by H-P-A-G. Co-expression of the A. castellani Delta8-desaturase with the Isochrysis galbana Delta9-elongase in transgenic Arabidopsis plants confirmed the activity observed in yeast and its role in the alternative pathway for C20 polyunsaturated fatty acid synthesis. Acyl-CoA profiles of these transgenic plants revealed an unexpected accumulation of C20 fatty acids in the acyl-CoA pool. This is the first report of an alternative pathway C20 Delta8-desaturase from a non-photosynthetic organism, and also the first report of a front-end desaturase lacking the canonical cytochrome b5 domain.     

Effect of diets enriched in Delta6 desaturated fatty acids (18:3n-6 and 18:4n-3), on growth, fatty acid composition and highly unsaturated fatty acid synthesis in two populations of Arctic charr (Salvelinus alpinus L.)

This study aimed to test the hypothesis that diets containing relatively high amounts of the Delta6 desaturated fatty acids stearidonic acid (STA, 18:4n-3) and gamma-linolenic acid (GLA, 18:3n-6), may be beneficial in salmonid culture. The rationale being that STA and GLA would be better substrates for highly unsaturated fatty acid (HUFA) synthesis as their conversion does not require the activity of the reputed rate-limiting enzyme, fatty acid Delta6 desaturase. Duplicate groups of two Arctic charr (Salvelinus alpinus L.) populations with different feeding habits, that had been reported previously to show differences in HUFA biosynthetic capacity, were fed for 16 weeks on two fish meal based diets containing 47% protein and 21% lipid differing only in the added lipid component, which was either fish oil (FO) or echium oil (EO). Dietary EO had no detrimental effect on growth performance and feed efficiency, mortalities, or liver and flesh lipid contents in either population. The proportions of 18:2n-6, 18:3n-3, 18:3n-6, 18:4n-3, 20:3n-6 and 20:4n-3 in total lipid in both liver and flesh were increased by dietary EO in both populations. However, the percentages of 20:5n-3 and 22:6n-3 were reduced by EO in both liver and flesh in both strains, whereas 20:4n-6 was only significantly reduced in flesh. In fish fed FO, HUFA synthesis from both [1-(14)C]18:3n-3 and [1-(14)C]20:5n-3 was significantly higher in the planktonivorous Coulin charr compared to the demersal, piscivorous Rannoch charr morph. However, HUFA synthesis was increased by EO in Rannoch charr, but not in Coulin charr. In conclusion, dietary EO had differential effects in the two populations of charr, with HUFA synthesis only stimulated by EO in the piscivorous Rannoch morph, which showed lower activities in fish fed FO. However, the hypothesis was not proved as, irrespective of the activity of the HUFA synthesis pathway in either population, feeding EO resulted in decreased tissue levels of n-3HUFA and 20:4n-6. This has been observed previously in salmonids fed vegetable oils, and thus the increased levels of Delta6 desaturated fatty acids in EO did not effectively compensate for the lack of dietary HUFA.     

饲料中不同水平n-3 HUFA对军曹鱼生长及脂肪酸组成的影响

以鱼粉、豆粕和酪蛋白为蛋白源,通过调节饲料中的牛油和鱼油水平,使饲料中高度不饱和脂肪酸（n-3HU-FA）的含量分别达到0．60、0．96、1.24、1．56、1．77和2．11％,配制出6种等氮等能的实验饲料,并以此饲料投喂军曹鱼幼鱼（8．3±0、5g）8周。实验结果显示饲料中n-3HUFA水平对实验鱼成活率没有显著影响,但却显著影响了军曹鱼的特定生长率（SGR）。随着饲料中n-3HUFA水平的升高,SGR相应提高,但当饲料中n-3HUFA水平高于1．24％时,各处理之间的生长没有出现显著差异。随着饲料中n-3HUFA水平的升高,肌肉和肝脏中18：1n-9的含量逐渐下降,而22：6n-3的水平相应升高。根据实验结果,当以特定生长率为评价指标时,用折线模型估计出军曹鱼对饲料中n-3HUFA的需求量约为1．49％。     

Identification of a fatty acid Delta11-desaturase from the microalga Thalassiosira pseudonana

A set of genomic DNA sequences putatively encoding front-end desaturases were identified by in silico analysis of the draft genome of the marine microalga Thalassiosira pseudonana. Among these candidate genes, an open reading frame named TpdesN was found to be full-length, intronless, and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpDESN, exhibited typical features of desaturases involved in the production of polyunsaturated fatty acids (PUFAs) in algae, i.e. a cytochrome b5-like domain at the N-terminus and three conserved histidine-rich motifs in the desaturase domain. Expression of TpDESN in Saccharomyces cerevisiae revealed that this enzyme was not involved in PUFA synthesis, but specifically desaturated palmitic acid 16:0 to 16:1Delta11. To our knowledge, until this report, Delta11-desaturase activity had only been detected in insect cells.     

The effect of bio-converted polyunsaturated fatty acids on the oxidation of TAG containing highly unsaturated fatty acids

Microbial modification of vegetable fatty acids can often lead to special changes in their structure and in biological function. A bacterial strain, Pseudomonas aeruginosa PR3, is known to carry out multiple hydroxylations on polyunsaturated fatty acids containing 1,4-cis, cis diene structural units, resulting in antibacterial activity. In this paper, in an effort to understand the overall mechanism involved in the varied biological functions of the complicated metabolites of bio-converted polyunsaturated fatty acids, we performed bioconversion of several polyunsaturated fatty acids using PR3, and determined their oxidative activities against fish oil. Bio-converted linoleic acid, eicosapentanoic acid, and docosahexanoic acid promoted effectively oxidation of fish oil. It is assumed that this oxidative effect could plausibly play an important role in the antimicrobial function of these bio-converted fatty acids.     

Regulation of growth performance and lipid metabolism by dietary n-3 highly unsaturated fatty acids in juvenile grass carp, Ctenopharyngodon idellus

To determine the effects of n-3 highly unsaturated fatty acids (HUFAs) in grass carp (Ctenopharyngodon idellus), a 75-day feeding experiment was conducted using five isonitrogenous and isoenergetic semi-purified diets containing 0% (control), 0.26%, 0.52%, 0.83% or 1.13% n-3 HUFAs. Weight gain, specific growth rate, feed efficiency and protein efficiency increased by increasing the dietary HUFAs content from 0% to 0.52%, and declined thereafter. Intraperitoneal fat content and the hepatopancreatic lipid levels were lowest in the 0.52% group. The tissue fatty acid level was well correlated with dietary HUFAs content. Hepatic lipoprotein lipase (LPL) activity was significantly higher in the 0.52% group, while that of malate dehydrogenase (MDH) was stable in the 0-0.52% groups, and was significantly lower in the 1.13% group. Superoxide dismutase (SOD) activity increased significantly with increasing dietary HUFAs content, consistent with the level of malondialdehyde (MDA). Hepatic mRNA expression of peroxisome proliferator-activated receptor-α (PPAR-α) was greatest in the 0.83% group and that of the LPL gene increased with increasing dietary HUFAs content up to 0.83%. These results indicate that adequate dietary HUFAs supplementation significantly promotes growth performance and lipid metabolism in freshwater fish grass carp. However, excess HUFAs fortification may exert adverse effects, which might be due to oxidative stress.     

Chemical synthesis of highly unsaturated fatty acids. IV. A new procedure for the preparation of substituted propargyl iodides

An improved method for the preparation of substituted propargyl iodides is reported. These compounds are easily accessible by the reaction of the corresponding methanesulfonates with sodium or magnesium iodide. The yields do not depend on the number of triple bonds. Even substituted propargyl iodides with five acetylenic bonds are obtained in very high yields.     

Cloning and characterization of unusual fatty acid desaturases from Anemone leveillei: identification of an acyl-coenzyme A C20 Delta5-desaturase responsible for the synthesis of sciadonic acid

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Delta(5,11,14); SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C(20) Delta(5cis)-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Delta(5)-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C(18) Delta(9)-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Delta(5,11,14,17)). Thus, AL10 acted only on C(20) polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b(5) domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Delta(5)-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Delta(5)-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Delta(5), 18:1Delta(5), and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Delta(5)-desaturases using acyl-CoA substrates.     

Reactivities of highly unsaturated fatty acids for the hydrogenation on nickel catalyst

The reactivities of the C₂₀ highly unsaturated fatty acids (20:5, 20:4, 20:3 etc.…) during heterogeneous hydrogenation on nickel catalyst have been studied using a computer program. Three parameters are required to determine the hydrogenation rate constant of the different fatty acid classes; the C₂₀ fatty acid composition of the starting oil, the C₂₀ fatty acid composition of a partially hydrogenated oil (PHO) and the time of reaction. It is shown that, in order to minimize the experimental errors, the PHO must be selected in such a way that the induction period is over and that this oil still contains appreciable amounts of 20:5 and 20:4.Very little difference was found for the reactivities of the 20:5, 20:4 and 20:3 acids. The major difference among unsaturated fatty acids was found to be between the 20:2 and 20:1 isomers for a hydrogenation effected according to common commercial practices.The computer program is a general one also useful for the prediction of the fatty acid composition of slightly hydrogenated oils, but is not suitable for oils hydrogenated to very low iodine values, or for those containing high proportions of trans fatty acids.     

Screening for strains of the genusMortierella,showing elevated production of highly unsaturated fatty acids

The content of oligousaturated fatty acids was investigated in 13 strains of filamentous fungi of the genusMortierella. M. isabellina producing up to 500 mg/L of γ-linolenic acid andM. verticillata containing up to 300 mg/L of arachidonic acid were found to be perspective producers of these acids. InM. parvispora dihomo-γ-linolenic acid (100 mg/L) could be detected.     

A chemical and autoradiographic study of cellulose synthesis during the encystment of Acanthamoeba castellanii

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of ¹⁴C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-³H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway.Fractionation data of cells grown on [³H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the ³H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.     

The incorporation of unsaturated fatty acids of the n-9, n-6, and n-3 families into individual phospholipids by isolated hepatocytes of thermally-acclimated rainbow trout, Salmo gairdneri

Rates of incorporation of 1-14C-oleic (18:1n9), -linoleic (18:2n6), and -linolenic (18:3n3) acids into individual phosphatides were determined in isolated hepatocytes from cold (5 degrees C)- and warm (20 degrees C)-acclimated rainbow trout, Salmo gairdneri. Fatty acid incorporation into phosphatidylcholine (PC) exceeded that into all other phospholipids, but at assay and acclimation temperatures of 5 degrees C, incorporation into phosphatidylethanolamine (PE) was generally intermediate between that of PC and the remaining phosphatides. Specific radioactivities (ratios of percentage isotope incorporation-to-mole percentage of phosphatide) were consistently less than one for both PC and PE, and greater than one for phosphatidic acid (PA), lysophosphatidylcholine (LPC), phosphatidylserine (PS), and cardiolipin (CL). For PS, specific radioactivities were greater in cold- than warm-acclimated trout, and greater at 5 degrees C than 20 degrees C. Rates of oleate incorporation were generally higher, and rates of incorporation of 18:2 and 18:3 lower in cold- than warm-acclimated trout. Most phospholipids demonstrated a clear preference for the incorporation of 18:2 when assayed at 20 degrees C; however, at 5 degrees C the incorporation of 18:2 was reduced and 18:3 was generally the preferred substrate. A reduction in assay temperature from 20 degrees C to 5 degrees C also shifted the incorporation of 18:2 away from PC into PS and PA. These data were interpreted to indicate 1) a cold-induced activation of PS metabolism, possibly resulting in elevated levels of PE; 2) lower rates of general acyl group turnover in animals acclimated to 5 degrees C than 20 degrees C; 3) a specificity to the acclimation response that favors the incorporation at cold temperatures of polyunsaturated fatty acids, but not the parent acids from which they are derived; and 4) the participation of a deacylation-reacylation cycle in the metabolism of phospholipids, particularly at cold temperatures.     

Hydroxy-alkenals from the peroxidation of n-3 and n-6 fatty acids and urinary metabolites

4-Hydroxy-2E-hexenal (4-HHE) and 4-hydroxy-2E-nonenal (4-HNE) have been characterized as prominent by-products of n-3 and n-6 hydroperoxy derivatives of n-3 and n-6 fatty acids, respectively. We also have characterized the homolog 4-hydroxy-2E,6Z-dodecadienal (4-HDDE) as a specific by-product of the 12-lipoxygenase product of arachidonic acid 12-hydroperoxy-eicosatetraenoate (12-HpETE). The three hydroxy-alkenals have been found in human plasma with 4-HHE being the most prominent followed by 4-HNE. They were found increased in tissues submitted to oxidative stress, according to the fatty acid characteristic of those tissues, e.g., 4-HNE and 4-HDDE in blood platelets and 4-HHE in the retina. We have shown they covalently bind to the primary amine moiety of ethanolamine phospholipids (PE), especially the plasmalogen subclass, with the highest hydrophobic alkenal (4-HDDE) being the most reactive. Their carboxylic acid metabolites, 4-hydroxy-2E-hexenoic acid (4-HHA), 4-hydroxy-2E-nonenoic acid (4-HNA) and 4-hydroxy-2E,6Z-dodecadienoic acid (4-HDDA), respectively, were found in human urine and measured in higher amounts in situations in which oxidative stress has been reported such as aging and diabetes. As reported above with their hydroxy-alkenals precursors, 4-HHA proved to be the most prominent followed by 4-HNA. Altogether, the three hydroxy-alkenals, either in their free form or bound to membrane PE, may be considered as specific markers of lipid peroxidation able to discriminate between n-3 and n-6 fatty acids. This is corroborated by the measurement of their urinary carboxylic acid metabolites.     

Food quality for Eudiaptomus gracilis: the importance of particular highly unsaturated fatty acids

1. Development times and survival of nauplii and copepodites of the freshwater calanoid Eudiaptomus gracilis were measured on comparably sized Cryptomonas sp. and two strains of Chlamydomonas reinhardii under food saturated conditions (1 mg C L−1) to investigate the nutritional quality of these algae.
2.  Cryptomonas sp. supported complete ontogenesis of nauplii to adults, whereas both strains of C. reinhardii were inadequate for the development of nauplii and copepodites and resulted in high mortality. The nutritional deficiency of both strains of C. reinhardii was compensated for by Cryptomonas sp. when the latter constituted ≥50% of dietary carbon. Pulses of Cryptomonas sp. had a similar compensatory effect.
3. In comparison to Cryptomonas sp., both strains of C. reinhardii were deficient in the three highly unsaturated fatty acids (HUFAs) stearidonic, eicosapentaenoic and docosahexaenoic acid. We manipulated the fatty acid content of C. reinhardii by externally adding these three HUFAs to the alga so that the fatty acid profile resembled that of Cryptomonas sp. This supplementation did not improve food quality, however, indicating that the nutritional deficiency of both strains of C. reinhardii for the ontogenesis of E. gracilis is not due to a lack of these three HUFAs.     

Effect of essential amino acids on the phosphorylation of a 40S ribosomal protein and protein synthesis in Acanthamoeba castellanii

Reversible and multiple phosphorylation of a 40S ribosomal protein is observed in a variety of eukaryotic cells. In the primitive eukaryote Acanthamoeba, one or three phosphorylated S3 derivatives are observed during growth phase in nondefined nutrient medium (ND cells) or in chemically defined nutrient medium (D cells), respectively. In both cases, stationary phase cells exhibit nonphosphorylated S3; however, transfer of these cells into the respective fresh nutrient media results in a transient accumulation of four phosphorylated S3 derivatives. Transfer of D cells into nutrient medium, deficient in all or any single essential amino acids, leads to reversible inhibition of S3 phosphorylation and growth arrest. The low level of phosphorylated S3 is not simply the consequence of growth arrest, since in cells where growth is arrested differently, the level of phosphorylated S3 can be high. In response to amino acid deficiency, a number of other changes can be observed. These include a 2-3-fold decrease of total protein synthesis, 13 changes in the cellular protein pattern, and specific alterations in the ribosome absorbance profiles and in the distribution of poly-A+ RNA within subribosomal and ribosomal fractions. While the rate of total protein synthesis seems to be associated with the level of phosphorylated S3, the level of the synthesis of at least 10 of the particular proteins can be dissociated from the level of S3 phosphorylation.