Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, α-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred α-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from α-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.     

Responses to oleic,linoleic and α-linolenic acids in high-carbohydrate,high-fat diet-induced metabolic syndrome in rats

We investigated the changes in adiposity, cardiovascular and liver structure and function, and tissue fatty acid compositions in response to oleic acid-rich macadamia oil, linoleic acid-rich safflower oil and α-linolenic acid-rich flaxseed oil (C18 unsaturated fatty acids) in rats fed either a diet high in simple sugars and mainly saturated fats or a diet high in polysaccharides (cornstarch) and low in fat. The fatty acids induced lipid redistribution away from the abdomen, more pronounced with increasing unsaturation; only oleic acid increased whole-body adiposity. Oleic acid decreased plasma total cholesterol without changing triglycerides and nonesterified fatty acids, whereas linoleic and α-linolenic acids decreased plasma triglycerides and nonesterified fatty acids but not cholesterol. α-Linolenic acid improved left ventricular structure and function, diastolic stiffness and systolic blood pressure. Neither oleic nor linoleic acid changed the left ventricular remodeling induced by high-carbohydrate, high-fat diet, but both induced dilation of the left ventricle and functional deterioration in low fat-diet-fed rats. α-Linolenic acid improved glucose tolerance, while oleic and linoleic acids increased basal plasma glucose concentrations. Oleic and α-linolenic acids, but not linoleic acid, normalized systolic blood pressure. Only oleic acid reduced plasma markers of liver damage. The C18 unsaturated fatty acids reduced trans fatty acids in the heart, liver and skeletal muscle with lowered stearoyl-CoA desaturase-1 activity index; linoleic and α-linolenic acids increased accumulation of their C22 elongated products. These results demonstrate different physiological and biochemical responses to primary C18 unsaturated fatty acids in a rat model of human metabolic syndrome.     

Analysis of the total oil and fatty acid composition of seeds of some Boraginaceae taxa from Turkey

Mature seed samples of twenty-four Boraginaceae taxa collected from their natural habitats in Turkey were analysed by GC for total oil content and fatty acid composition. The range of total fat in the taxa varied between 7.0 and 35.7%. The amounts of palmitic (16:0) and stearic (18:0) acids determined were 5.65–17.81 and 1.49–5.08%, respectively. Mono-unsaturated fatty acids were in the range 8.83–55.32% for oleic, 0.22–6.21% for eicosenoic, 0.04–8.94% for erucic, and 0.08–2.71% for nervonic acid. Poly-unsaturated fatty acids were between 1.41 and 68.44% for linoleic, 0.12 and 43.0% for α-linolenic, 0.04 and 24.03% for γ-linolenic, and 0.02 and 14.59% for stearidonic acid. Total saturated (9.3–23.7%), mono-unsaturated (10.59–73.28%), and poly-unsaturated fatty acids (13.91–68.78%) varied substantially. Total unsaturated fatty acids ranged from 70.12 to 90.29%. There were significant differences between fatty acid profiles at taxa (P < 0.05) at genera levels, based on mono-unsaturated and poly-unsaturated fatty acid concentrations (P < 0.05). Segregation at the generic level by principle-component analysis was accomplished based on nine major fatty acids. The fatty acid patterns, their relative proportions, and quantities of unusual fatty acids as additional biochemical markers seem to be useful in the taxonomy of Boraginaceae at generic and infrageneric levels. All taxa are, in general, rich in linoleic and α-linolenic acids as essential fatty acids for dietary reference intakes. Seed oils of Symphytum, Anchusa, and Trachystemon orientalis for γ-linolenic acid and Echium for both γ-linolenic and stearidonic acid may be evaluated as alternative wild sources.     

一株富含α-亚麻酸栅藻的异养培养条件优化

HSJ296是本实验室分离纯化的1株能够异养生长、富含α-亚麻酸的栅藻(Scenedesmus sp.)。研究比较了不同温度、氮源和葡萄糖浓度对其生长的影响, 结果显示, 其最适培养条件为30℃、4 g/L尿素和20—40 g/L葡萄糖。通过分析不同培养条件下HSJ296总脂中的脂肪酸组成, 发现主要含有十六碳脂肪酸(C16:0)、油酸(C18:1)、亚油酸(C18:2)和α-亚麻酸(α-C18:3), 并且α-亚麻酸的含量稳定在35%—45%。栅藻HSJ296发酵产品或可用作鱼类饲料添加剂以补充α-亚麻酸等营养。     

The acyl-acyl carrier protein synthetase from Synechocystis sp. PCC 6803 mediates fatty acid import

The transfer of fatty acids across biological membranes is a largely uncharacterized process, although it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum. Here, we analyzed loss-of-function mutants of the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a model system to circumvent redundancy problems encountered in eukaryotic organisms. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided α-linolenic acid, whereas wild-type cells bleached upon this treatment. Bleaching of wild-type cells was accompanied by a continuous increase of α-linolenic acid in total lipids, whereas no such accumulation could be observed in SynAas-deficient cells (Δsynaas). When SynAas was disrupted in the tocopherol-deficient, α-linolenic acid-hypersensitive Synechocystis mutant Δslr1736, double mutant cells displayed the same resistance phenotype as Δsynaas. Moreover, heterologous expression of SynAas in yeast (Saccharomyces cerevisiae) mutants lacking the major yeast fatty acid import protein Fat1p (Δfat1) led to the restoration of wild-type sensitivity against exogenous α-linolenic acid of the otherwise resistant Δfat1 mutant, indicating that SynAas is functionally equivalent to Fat1p. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate α-[(14)C]linolenic acid retrieval from preloaded liposome membranes via the synthesis of [(14)C]linolenoyl-acyl carrier protein. Taken together, our data show that an acyl-activating enzyme like SynAas is necessary and sufficient to mediate the transfer of fatty acids across a biological membrane.     

培养条件对头孢霉脂肪酸链长和ω-3脱饱和的影响

为了获得高产量的长链ω-3多不饱和脂肪酸,用十八碳脂肪酸和十六碳脂肪酸的比值考察碳链延长,用α-亚麻酸和亚油酸的比值考察ω-3脱饱和;探讨了八种因子对脂肪酸链长和ω-3脱饱和的影响。有利于碳链延长的条件为：麦芽糖10g/L、(NH4)2SO4 3g/L、起始pH为4.0、500mL三角瓶装50mL培养基、接种20%（V/V）、20℃培养6d。有利于ω-3脂肪酸生成的条件为：蔗糖30g/L、NH4Cl 3g/L、培养基起始pH为4.0、500mL三角瓶装50mL培养基、接种20%（V/V）、10℃培养10d。     

培养条件对头孢霉脂肪酸链长和ω-3脱饱和的影响*

为了获得高产量的长链ω-3多不饱和脂肪酸,用十八碳脂肪酸和十六碳脂肪酸的比值考察碳链延长,用α-亚麻酸和亚油酸的比值考察ω-3脱饱和;探讨了八种因子对脂肪酸链长和ω-3脱饱和的影响。有利于碳链延长的条件为：麦芽糖10g/L、(NH4)2SO4 3g/L、起始pH为4.0、500mL三角瓶装50mL培养基、接种20%（V/V）、20℃培养6d。有利于ω-3脂肪酸生成的条件为：蔗糖30g/L、NH4Cl 3g/L、培养基起始pH为4.0、500mL三角瓶装50mL培养基、接种20%（V/V）、10℃培养10d。     

七种植物籽油中脂肪酸成分的比较分析(英文)

为寻求新的食用油资源,发展了一种快速可靠的气相色谱-质谱联用方法,用于植物籽油中脂肪酸成分的定性鉴定和含量测定。所建立的方法成功用于葡萄籽、南瓜籽和猕猴桃籽等七种植物籽油中的棕榈酸、十八烷酸、油酸、亚油酸和α-亚麻酸的定性定量分析。结果表明,刺葡萄籽油、普通葡萄籽油、国外葡萄籽油、南瓜籽油、枸杞籽油和西番莲籽油均具有相似的脂肪酸谱,尽管其中它们所含上述五种脂肪酸含量不同,由于均存在人体所必需的饱和与不饱和脂肪酸,故可以用作替代食用油。猕猴桃籽油因为其存在高含量的α-亚麻酸成分,可能是更好的食用油和营养油资源。本文首次对枸杞籽油、西番莲籽油和猕猴桃籽油脂肪酸成分进行绝对含量分析,为新的食用油资源的开发提供了重要的依据。     

Substitution of dietary oleic acid for myristic acid increases the tissue storage of α-linolenic acid and the concentration of docosahexaenoic acid in the brain, red blood cells and plasma in the rat

Various strategies have been developed to increase the cellular level of (n-3) polyunsaturated fatty acids in animals and humans. In the present study, we investigated the effect of dietary myristic acid, which represents 9% to 12% of fatty acids in milk fat, on the storage of α-linolenic acid and its conversion to highly unsaturated (n-3) fatty acid derivatives. Five isocaloric diets were designed, containing equal amounts of α-linolenic acid (1.3% of dietary fatty acids, i.e. 0.3% of dietary energy) and linoleic acid (7.0% of fatty acids, i.e. 1.5% of energy). Myristic acid was supplied from traces to high levels (0%, 5%, 10%, 20% and 30% of fatty acids, i.e. 0% to 6.6% of energy). To keep the intake of total fat and other saturated fatty acids constant, substitution was made with decreasing levels of oleic acid (76.1% to 35.5% of fatty acids, i.e. 16.7% to 7.8% of energy) that is considered to be neutral in lipid metabolism. After 8 weeks, results on physiological parameters showed that total cholesterol and low-density lipoprotein-cholesterol did not differ in the diets containing 0%, 5% and 10% myristic acid, but were significantly higher in the diet containing 30% myristic acid. In all the tissues, a significant increasing effect of the substitution of oleic acid for myristic acid was shown on the level of both α-linolenic and linoleic acids. Compared with the rats fed the diet containing no myristic acid, docosahexaenoic acid significantly increased in the brain and red blood cells of the rats fed the diet with 30% myristic acid and in the plasma of the rats fed the diet with 20% myristic acid. Arachidonic acid also increased in the brain of the rats fed the diet with 30% myristic acid. By measuring Δ6-desaturase activity, we found a significant increase in the liver of the rats fed the diet containing 10% of myristic acid but no effect at higher levels of myristic acid. These results suggest that an increase in dietary myristic acid may contribute in increasing significantly the tissue storage of α-linolenic acid and the overall bioavailability of (n-3) polyunsaturated fatty acids in the brain, red blood cells and plasma, and that mechanisms other than the single Δ6-desaturase activity are involved in this effect.     

缅甸蟒脂肪酸分析

用气相色谱法测定了缅甸蟒油20种脂肪酸,其中不饱和脂肪酸含量达67.5%,多不饱和脂肪酸含量达10.3%.含量较高的脂肪酸有油酸、棕榈酸、亚油酸、棕榈油酸,特有脂肪酸DHA、α-亚麻酸,并且明显不同于其他蟒和蛇的脂肪酸含量.缅甸蟒油具有重要的药用和保健品开发利用价值.     

Barramundi (Lates calcarifer) desaturase with Δ6/Δ8 dual activities

Barramundi is a commercially farmed fish in Australia. To examine the potential for barramundi to metabolise dietary α-linolenic acid (ALA, 18:3 n-3), the existence of barramundi desaturase enzymes was examined. A putative fatty acid Δ6 desaturase was cloned from barramundi liver and expressed in yeast. Functional expression revealed Δ6 desaturase activity with both the 18 carbon (C(18)) and C(24) n-3 fatty acids, ALA and 24:5 n-3 as well as the C(18) n-6 fatty, linoleic acid (LA, 18:2 n-6). Metabolism of ALA was favoured over LA. The enzyme also had Δ8 desaturase activity which raises the potential for synthesis in barramundi of omega-3 (n-3) long chain polyunsaturated fatty acids from ALA via a pathway that bypasses the initial Δ6 desaturase step. Our findings not only provide molecular evidence for the fatty acid desaturation pathway in the barramundi but also highlight the importance of taking extracellular fatty acid levels into account when assessing enzyme activity expressed in Saccharomyces cerevisiae.     

Lipid composition of azolla caroliniana biomass and its seasonal variation

The chemical composition of the lipid fraction of the aquatic fern, Azolla caroliniana, grown in outdoor mass culture in Florence, was investigated. Palmitic acid was the main fatty acid, particularly in summer when it reached 51.6% of the total fatty acids. In autumn a decrease in palmitic acid content and an increase in α-linolenic acid content occurred. The percentage of unsaturated fatty acids changed from 36.1% in summer to 54% in the biomass harvested in November.     

Leptin reverts pro-apoptotic and antiproliferative effects of α-linolenic acids in BCR-ABL positive leukemic cells: involvement of PI3K pathway

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.     

Brain Phospholipids as Dietary Source of (n-3) Polyunsaturated Fatty Acids for Nervous Tissue in the Rat

Abstract: In a previous work, we calculated the dietary α-linolenic requirements (from vegetable oil triglycerides) for obtaining and maintaining a physiological level of (n-3) fatty acids in developing animal membranes as determined by the cervonic acid content [22:6(n-3), docosahexaenoic acid]. The aim of the present study was to measure the phospholipid requirement, as these compounds directly provide the very long polyunsaturated fatty acids found in membranes. Two weeks before mating, eight groups of female rats (previously fed peanut oil deficient in α-linolenic acid) were fed different semisynthetic diets containing 6% African peanut oil supplemented with different quantities of phospholipids obtained from bovine brain lipid extract, so as to add (n-3) polyunsaturated fatty acids to the diet. An additional group was fed peanut oil with rapeseed oil, and served as control. Pups were fed the same diet as their respective mothers, and were killed at weaning. Forebrain, sciatic nerve, retina, nerve endings, myelin, and liver were analyzed. We conclude that during the combined maternal and perinatal period, the (n-3) fatty acid requirement for adequate deposition of (n-3) polyunsaturated fatty acids in the nervous tissue (and in liver) of pups is lower if animals are fed (n-3) very long chain polyunsaturated fatty acids found in brain phospholipids [this study, ˜60 mg of (n-3) fatty acids/100 g of diet, i.e., ˜130 mg/1,000 kcal] rather than α-linolenic acid from vegetable oil triglycerides [200 mg of (n-3) fatty acids/100 g of diet, i.e., ˜440 mg/1,000 kcal].     

Identification and characterization of Δ12 and Δ12/Δ15 bifunctional fatty acid desaturases in the oleaginous yeast Lipomyces starkeyi

Fatty acid desaturases play vital roles in the synthesis of unsaturated fatty acids. In this study, Δ12 and Δ12/Δ15 fatty acid desaturases of the oleaginous yeast Lipomyces starkeyi, termed LsFad2 and LsFad3, respectively, were identified and characterized. Saccharomyces cerevisiae expressing LsFAD2 converted oleic acid (C18:1) to linoleic acid (C18:2), while a strain of LsFAD3-expressing S. cerevisiae converted oleic acid to linoleic acid, and linoleic acid to α-linolenic acid (C18:3), indicating that LsFad2 and LsFad3 were Δ12 and bifunctional Δ12/Δ15 fatty acid desaturases, respectively. The overexpression of LsFAD2 in L. starkeyi caused an accumulation of linoleic acid and a reduction in oleic acid levels. In contrast, overexpression of LsFAD3 induced the production of α-linolenic acid. Deletion of LsFAD2 and LsFAD3 induced the accumulation of oleic acid and linoleic acid, respectively. Our findings are significant for the commercial production of polyunsaturated fatty acids, such as ω-3 polyunsaturated fatty acids, in L. starkeyi.

     

alpha-linolenic acid biosynthesis in Cyanidium caldarium.

Although α-linolenic acid is nearly absent from Cyanidium caldarium cultured at 53 °C, it is the most abundant unsaturated fatty acid in 20 °C-grown cells. A sudden growth temperature shift of 55 to 25 °C does not stimulate the immediate biosynthesis of α-linolenic acid. However, after an induction period of 48 h, synthesis of α-linolenic acid from acetate can be detected, and the fatty acid accumulates in phosphatidyl choline and sulfolipid. The newly synthesized α-linolenic acid appears to be formed primarily by de novo synthesis and to a much lesser extent from the elongation of a previously formed hexadecatrienoic acid precursor. On the other hand, when a cell-free algal preparation was presented with a hexadecatrienoic acid precursor in the presence of [¹⁴C] malonyl-CoA, the α-linolenic acid formed demonstrated a synthesis by elongation of the precursor. While the cell appears enzymatically capable of α-linolenic acid biosynthesis by both the de novo and elongation processes, de novo synthesis of α-linolenic acid appears to be the more significant mode of synthesis.     

唇形科益母草等7种植物果实化学成分分析及形态特征

益母草、白花益母草、紫苏、野紫苏、小鱼仙草、石荠苎和细风轮菜系唇形科常见的药用植物。常生于路旁、沟边及空旷草地 ,民间也有广泛栽培。分布于广东、广西、湖南、湖北、云南、贵州、浙江、安徽、福建等省区。益母草、白花益母草民间全草入药 ,有活血调经、去痰生津的功效 ,果实称茺蔚子。用于月经不调 ,产后子宫出血及复位不全等症 [14 ]。野紫苏和紫苏的果实有镇咳、平喘和祛痰的功效 ,用于治疗咳嗽气喘、肠热便秘等症 [3 ] 。小鱼仙草用于治疗感冒头痛 ,胃炎 ,肾积水等症。石荠苎全草入药 ,用于治疗胃炎 ,慢性气管炎和肺积水等症[1] …     

Rapid and efficient gas chromatographic method for measuring the kinetics of lipase-catalyzed transesterification of phosphatidylcholine

Both n-3 polyunsaturated fatty acids (n-3 PUFA) and phospholipids have a lot of special functions on human body. n-3 PUFA includes α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Among them, ALA is the precursor of EPA and DHA. The synthesis of ALA-containing phospholipids can solve the problems of low content of EPA and DHA in phospholipids and show an increased double effect on human body. At a water activity of 0.33, a rapid and efficient method was used for kinetics of the transesterification of phosphatidylcholine (PC) with α-linolenic acid ethyl ester (ALAEE). The rate equation was obtained using palmitic acid ethyl ester (PAEE) as index for reaction rate and the reaction proved to proceed via a Ping-Pong mechanism without inhibition by both the substrates.     

Oxidative Stability of Polyunsaturated Fatty Acids in an Aqueous Solution

The relative oxidative stability of six kinds of typical polyunsaturated fatty acids (PUFAs) was investigated in an aqueous solution (pH=7.4 at 37°C) with Fe²⁺-ascorbic acid as a catalyst. The highest stability was shown by docosahexaenoic acid (22: 6n-3, DHA), followed by eicosapentaenoic (20: 5n-3), arachidonic (20: 4n-6), α-linolenic (18: 3n-3), γ-linolenic (18: 3n-6), and linoleic (18: 2n-6, LA) acids, indicating that the stability increased with increasing degree of unsaturation. The significant difference found between α-linolenic and γ-linolenic acids also suggests the higher oxidative stability of n-3 PUFAs than of n-6 PUFAs in an aqueous solution. Moreover, when a mixture of DHA and LA was oxidized in an aqueous solution, the stability increased with increasing molar ratio of DHA to LA in the mixture. This characteristic oxidative stability of PUFAs in the aqueous phase is quite different from that in the neat phase, and can be explained by correlating with the conformation of PUFAs in the aqueous medium.     

In Silico Structural Studies and Molecular Docking Analysis of Delta6-desaturase in HUFA Biosynthetic Pathway

Fish are an important source of highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid EPA (20:5 n-3) and docosahexaenoic acid DHA (22:6 n-3) and play a significant role in human nutrition. The fatty acyl delta6-desaturase (Δ6 desaturase) is a rate-limiting enzyme in the biosynthetic pathway of highly unsaturated fatty acids (HUFA) that converts polyunsaturated fatty acids (PUFA) such as linoleic (18:2n-6) and α-linolenic (18:3n-3) acids into HUFA. In this study, fatty acyl Δ6 desaturase was identified from pangasius (Pangasianodon hypophthalmus) and further analyzed for sequenced-based characterization and 3D structural conformation. Sequenced-based analysis revealed some important secondary information such as physicochemical property. e.g., isoelectric point, extinction coefficient, aliphatic index, and grand average hydropathy, among others, and also post-translational modification sites were identified. An evolutionary-conserved stretch of amino acid residue and a functionally significant conserved structural ancestor, N-terminal cytochrome b5 and membrane FADS-like superfamily, were identified. Protein association analysis showed a high confidence score with acyl-CoA synthetase, elovl5, elovl2, and phospholipase A2. Herein, we report, for the first time, a 3D native structure of Δ6 desaturase protein by homology modeling approach; molecular docking analysis was performed with linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, which are the two key substrates in the HUFA biosynthetic pathway. This work provides insight into the structural and functional characterization of Δ6 desaturase, which is involved in HUFA biosynthesis as a rate-limiting enzyme.