Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

Molecular cloning of a Pinguiochrysis pyriformis oleate-specific microsomal Δ12-fatty acid desaturase and functional analysis in yeasts and thraustochytrids

We isolated a putative desaturase gene from a marine alga, Pinguiochrysis pyriformis MBIC 10872, which is capable of accumulating eicosapentaenoic acid (C20:5(Δ5,8,11,14,17)). The gene possessed an open reading frame of 1,314 bp encoding a putative 437 amino acid residues showing high sequence identity (37-48%) with fungal and nematode Δ12-fatty acid desaturases. Yeast cells transformed with the gene converted endogenous oleic acid (C18:1(Δ9)) to linoleic acid (C18:2(Δ9,12)). However, no double bonds were introduced into other endogenous fatty acids or exogenously added fatty acids. Flag-tagged enzyme was recovered in the micosome fraction when expressed in yeast cells. To express the gene in thraustochytrids, a construct driven by the thraustochytrid-derived ubiquitin promoter was used. Interestingly, exogenously added oleic acid was converted to linoleic acid in the gene transformants but not mock transformants of Aurantiochytrium limacinum mh0186. These results clearly indicate that the gene encodes a microsomal Δ12-fatty acid desaturase and was expressed functionally in not only yeasts but also thraustochytrids. This is the first report describing the heterozygous expression of a fatty acid desaturase in thraustochytrids, and could facilitate a genetic approach towards fatty acid synthesis in thraustochytrids which are expected to be an alternative source of polyunsaturated fatty acids.     

少根根霉△6-脂肪酸脱氢酶基因在毕赤酵母中的表达

△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△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△6-脂肪酸脱氢酶基因.把少根根霉△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△6-脂肪酸脱氢酶基因的功能.     

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。     

Differential regulation of calmodulin-dependent and -independent cyclic AMP phosphodiesterases from oviduct by fatty acids.

Regulation of calmodulin-independent and -dependent cAMP phosphodiesterases from quail oviduct by various fatty acids was studied. The calmodulin-independent form was slightly activated by low concentrations (20 microM) of oleic, linoleic and arachidonic acid, higher concentrations were inhibitory. The basal activity of the calmodulin-dependent form was activated by linoleic acid and to a lesser extent by arachidonic acid at low concentrations and inhibited by higher concentrations of the two fatty acids. In contrast, arachidonic acid was a potent reversible inhibitor of calmodulin in the activation of this enzyme (IC50: 20 microM) whereas linoleic acid was inactive from 10 to 150 microM. The present results strongly suggest that the differential regulation of cAMP phosphodiesterases by these fatty acids could profoundly influence the level of cAMP in the oviduct and thus its subsequent effects.     

Inhibition of calcineurin by polyunsaturated lipids

From earlier studies on calcineurin, the presence of multiple double bonds in putative inhibitors was hypothesized as critical features for effective inhibition. Polyunsaturated fatty acids were tested as inhibitors of calcineurin and found to inhibit the phosphatase activity of calcineurin although effective inhibition was observed only in the absence of calmodulin. Calmodulin and fatty acids seemed to compete for the enzyme with the activation curve of calmodulin shifted approximately 100-fold in the presence of 50 microM eicosa-11Z,14Z-dienoic acid (20:2, n-6) or 50 microM eicosa-8Z,11Z,14Z-trienoic acid (20:3, n-6). Leukotriene B4 and derivatives also were screened as inhibitors. The most effective inhibition was caused by the 6-trans,12-epi-leukotriene B4 with an IC50 of 16.4 microM for the inhibition of calcineurin with pNPP as the substrate. Lipoxins A4 and B4 likewise caused inhibition in the presence of calmodulin with an IC50 of 42.7 microM for lipoxin B4. There was no protection by calmodulin, as found with the inhibition by the fatty acids. These data support the hypothesis that effective inhibition is bolstered by the presence of conjugated double bonds in the inhibitor. Consideration of cis- and trans-orientation of the double bonds suggests that presentation of the delocalized electron density is also a factor in effective inhibition of calcineurin.     

Long-chain polyenoics and the essential dietary fatty acid requirement of the waxmoth, Galleria mellonella

The essential fatty acid requirement for normal pupal-adult ecdysis in Galleria mellonella was studied using non-axenic casein-based semisynthetic diets with or without various 99% pure fatty acids. The abilities of linoleic and linolenic acids to alleviate faulty adult emergence differed markedly, linolenic acid being 10-fold more potent than linoleic acid. One other ω6 polyunsaturated fatty acid, C20:2ω6, resembled its analogue, linoleic acid (18:2ω6), in efficacy at high dosage, but three others, C18:3ω6, C20: ω6 and C20:4ω6 (arachidonic acid), were without effect. Of five ω3 polyunsatures tested, C22:3ω3 and C20:3ω3 were as effective as linolenic acid (C18:3ω3), their shorter-chained analogue. Docosahexaenoic acid (C22:6ω3) was totally ineffective, but eicosapentaenoic acid (C20:5ω3), though supporting no perfect emergences, produced some active adults having wing malformations only, and was therefore considered partially active. It is suggested that a C18 polyunsaturate is physiologically required by G. mellonella and can be derived from various dietary longer-chained analogues by simple carbon chain shortening so long as there are no additional double bonds carboxylwards of an active di- or trienoic sequence. The partial activity of C20:5ω3 suggests there may additionally be a physiological requirement for this or a related long-chain polyunsaturate. The possibility of multiple essential fatty acid requirements in Lepidoptera in general is discussed.     

Inhibition of chymase activity by long chain fatty acids

The activity of chymase was markedly inhibited by fatty acids with carbon chain lengths of 14-22 at doses greater than 0.02 microM, irrespective of the number of double bonds. Cis acids with a carbon chain length of 18, such as stearic acid, oleic acid, linoleic acid, and linolenic acid were potent inhibitors, whereas the trans isomer of oleic acid, elaidic acid, showed less inhibitory activity. The extent of inhibition by oleyl alcohol was almost the same as that by oleic acid, suggesting that the acid moiety itself was not necessary for the inhibition; but a fatty acid with a terminal functional amide, oleamide, showed little inhibitory activity. The inhibition was noncompetitive and was reversible, and the Ki value of oleic acid was 2.7 microM. Stearic acid and oleic acid inhibited all chymotrypsin-type serine endopeptidases tested. The ID50 values of these fatty acids for atypical mast cell protease were higher than those for the other chymotrypsin-type serine endopeptidases tested. Other proteases, such as papain, trypsin, collagenase, and carboxypeptidase A, except cathespin D, were not affected by stearic or oleic acid.     

Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells.

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.     

Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.     

Unsaturated fatty acids inhibit ADP- arachidonate-induced platelet aggregation without affecting thromboxane synthesis

The inhibitory effects of three cis-unsaturated C18 fatty acids (oleic, linoleic, and linolenic acids, sodium salts) on ADP- and sodium-arachidonate-induced aggregation of washed rabbit platelets were investigated. When the platelets were suspended in protein-free medium containing dextran, it was found that these fatty acids at very low concentrations (2-45 microM) were potent inhibitors of platelet responsiveness and the inhibitory effect occurred within seconds. The inhibition of ADP-induced aggregation was not affected by abolishing the activity of platelet cyclooxygenase using aspirin. Human serum albumin relieved the inhibition caused by fatty acids for both ADP- and arachidonate-induced aggregation. The inhibitory effect of fatty acids does not seem to be due to decreased thromboxane formation (except possibly in the case of linolenate), and the relief of fatty acid inhibition by albumin does not potentiate thromboxane B2 formation from exogenous arachidonate. It is suggested that the inhibitory effect of polyunsaturated fatty acids on platelet aggregation is specific and not related to a general surfactant effect, since inhibition occurs far below the critical micelle concentration of fatty acid soaps.     

雅致枝霉△6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的表达

根据真菌△^6 -脂肪酸脱氢酶基因保守的组氨酸Ⅱ区和Ⅲ区附近保守序列设计兼并引物进行RT-PCR,得到雅致枝霉（Thamnidium elegans）As3．2806△^6 -脂肪酸脱氢酶基因459bp部分cDNA序列,然后通过快速扩增cDNA末端技术（RACE）向两端延伸得到1504bp的△^6 -脂肪酸脱氢酶基因全长cDNA序列。序列分析表明有一个1377bp、编码459个氨基酸的开放阅读框TED6。推测的氨基酸序列与已知其他真菌的△^6 -脂肪酸脱氧酶基因的氨基酸序列比对,具有3个组氨酸保守区、2个疏水区及N末端细胞色素b5融合区。将此编码区序列亚克隆到酿酒酵母缺陷型菌株INVSel的表达载体pYES2.0中,构建表达载体pYTED6,并在酿酒酵母INVSel中异源表达。通过气相色谱（GC）和气相色谱,质谱（GC-MS）分析表明,该序列在酿酒酵母中获得表达,产生γ-亚麻酸（GLA）的含量占酵母总脂肪酸的7．5％。证明此序列编码的蛋白能将外加的亚油酸转化为γ-亚麻酸,是一个新的有功能的△^6 -脂肪酸脱氢酶基因（GenBank．AY941161）。     

Activation of carbonyl reductase from pig lung by fatty acids.

The NADPH-linked reductase activity of pig lung carbonyl reductase was activated two- to fivefold by fatty acids with a carbon chain length greater than nine at pH 7.0. cis-Unsaturated fatty acids of C:18 and C:20 were potent activators, showing Ka values of 2-14 microM which were lower than the values of 21-125 microM for saturated fatty acids (C:9 to C:16). Of the fatty acids arachidonic acid (C20:4) gave the highest activation. No significant stimulatory effect was observed with acyl CoAs, fatty alcohols, phospholipids, and nonionic detergents. Anionic detergents (sodium dodecyl sulfate and sarkosyl) stimulated the enzyme activity more than ninefold, but the Ka values for them were much higher than those for the cis-unsaturated fatty acids. Although no change in molecular weight or in subunit composition was observed in the enzyme activated by C20:4, the activation led to a decrease in thermal stability of the enzyme. The binding of C20:4 to the enzyme was instantaneous and reversible, shifted the pH optimum of the activity from 5.8 to 6.5, and changed the inhibitor sensitivity. In addition, C20:4 acted as an allosteric effector abolishing the negative interaction of the enzyme with carbonyl substrates which was seen without the fatty acid, but the activation increased both Vmax and [S]0.5 values for the substrates. Kinetic analysis with respect to NADPH concentration, in which no cooperativity was detected with or without C20:4, indicated that C20:4 was a nonessential activator of mixed type showing a binding constant of 10 microM. These results suggest that cis-unsaturated fatty acids may be potential modulators of pulmonary carbonyl reductase.     

Polyunsaturated fatty acid biosynthesis from [1-14C]20:3 n-6 acid in rat cultured Sertoli cells. Linoleic acid effect

Primary culture is a suitable system to study lipid metabolism and polyunsaturated fatty acid biosynthesis. Sertoli cell-enriched preparations were used to determine the fatty acid composition after 5 and 7 days in culture (serum free) as well as the uptake and metabolism of [1-14C]eicosa-8,11,14-trienoic acid. The addition of unlabeled linoleic acid (0.2 and 2.0 microg/ml) was also evaluated. Fatty acid methyl esters derived from cellular lipids were analyzed by gas liquid chromatography and radiochromatography. After 5 days in culture, cells had significantly less 18:2, 20:4, 22:5 and 24:5 and more 18:3, 20:3, 22:4 and 24:4 n-6 fatty acids than non-cultured cells. On day 7, an additional increment in 22:4 n-6 and a decrease in linoleic, gamma-linoleic and 24:4 n-6 fatty acids were observed. The presence of linoleic acid (low dose) produced a significant decrease in saturated and monounsaturated acids and an increase in 18:2, 20:4 and 22:5 n-6 fatty acids. At a high concentration almost all fatty acids belonging to 18:2 n-6 increased significantly. The drop in 20:4 n-6/20:3 n-6 ratio was considered as an indirect evidence of a Delta 5 desaturase activity depression. This assumption was corroborated by studying the transformation of [1-14C]eicosa-8,11,14-trienoic acid into 20:4, 22:4, 22:5, 24:4 and 24:5 n-6 fatty acids. We conclude that Sertoli cells after 7 days in culture evidenced changes in the fatty acid profile similar to those described under fat deprivation. The addition of linoleic acid reverted this pattern and indicated that the Delta 5 desaturase activity is a limiting step in the polyunsaturated fatty acid biosynthesis.     

高山被孢霉ATCC16266△^6—脂肪酸脱氢酶基因在酿酒酵母中的表达

△^6－脂肪酸脱氢酶基因是形成γ－亚麻酸的关键酶。从含有高山被孢霉△^6－脂肪酸脱氢酶基因的重组质粒pT-MACL6中,酶切出1.4kb的目的片段,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒pYMAD6,用醋酸昔方法转化到酿洒酵母的缺陷型菌株INCSc1中,在SC－Ura合成培养基中,选择得到酿酒酵母工程株YMAD6。在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体。通过GC－MS对酵母工程株进行脂肪酸色谱分析,结果表明,产生了31.6％的γ－亚麻酸,边是迄今为止,国内外△^6－脂肪酸脱氢酶基因在酿酒酵母中表达量最高的报道。     

Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.     

Arachidonyl trifluoromethyl ketone induces lipid body formation in leukocytes.

Leukocyte lipid bodies, abundant in cells associated with inflammation, can be induced to form in response to stimuli that include cis -unsaturated, but not saturated, fatty acids. Arachidonyl trifluoromethyl ketone (AACOCF(3)), a non-esterifiable arachidonate analog and an inhibitor of cytosolic phospholipase A(2)enzymes (PLA(2)), dose-dependently (0-20 microM) stimulated neutrophil lipid body formation, but this stimulation was not attributable to PLA(2)inhibition. Palmitoyl trifluoromethyl ketone, also a PLA(2)inhibitor, failed to stimulate lipid body formation, like palmitic acid itself, and did not inhibit stimulated lipid body formation. Moreover, aspirin, indomethacin and ibuprofen, which inhibit cis -unsaturated fatty acid-induced lipid body formation, inhibited AACOCF(3)-induced lipid body formation. Lipid body induction with AACOCF(3)reflected its structural basis as a cis -unsaturated fatty acid analog. These results indicate that cytosolic PLA(2)enzymes are not active in lipid body induction and cis -fatty acid stimulation of lipid body formation does not require esterification of cis -fatty acids into glycerolipids.     

A novel Delta12-fatty acid desaturase gene from methylotrophic yeast Pichia pastoris GS115

The methylotrophic yeast Pichia pastoris GS115, a widely used strain in production of various heterologous proteins, especially membrane-bound enzymes, can also produce linoleic and linolenic acids, which indicates the existence of membrane-bound Delta12 and Delta15-fatty acid desaturases. This paper describes the cloning and functional characterization of a novel Delta12-fatty acid desaturase gene from this methylotrophic yeast. The open reading frame of the gene (named Pp-FAD12) is 1263 bp in size and encodes a 420-amino-acid peptide. The deduced Pp-FAD12 protein shows high identity (50-67%) with Delta12-fatty acid desaturases from other fungi. It also shows a high identity (57%) with Delta15-fatty acid desaturase (named Sk-FAD15) from Saccharomyces kluyveri. Expression of Pp-FAD12 in polyunsaturated fatty acids non-producing yeast Saccharomyces cerevisiae demonstrated that its product converted oleic acid (18 : 1) to linoleic acid (18 : 2). This result suggests that Pp-FAD12 encodes a novel Delta12-fatty acid desaturase in P. pastoris GS115. This is the first report about the cloning and functional characterization of Delta12-fatty acid desaturase gene in methylotrophic yeast.