Sn-2 Position Of Phosphatidylcholine Research Articles

The enzyme encoded by Myrmecia incisa, a green microalga, phospholipase A2 gene preferentially hydrolyzes arachidonic acid at the sn-2 position of phosphatidylcholine

The enzyme phospholipase A2 (PLA2) plays a crucial role in acyl remodeling of phospholipids via the Lands’ cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A2 (MiPLA2) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA2 contained 3 introns. Mature MiPLA2 (mMiPLA2) had a conserved Ca2+-binding loop and a catalytic site motif that has been recognized in plant secretory PLA2 (sPLA2) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA2 was clustered within GroupXIA of plant sPLA2 proteins. To ascertain the function of MiPLA2, the cDNA coding for mMiPLA2 was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA2 in Escherichia coli. Recombinant mMiPLA2 was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA2 indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA2. Although recombinant mMiPLA2 displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA2 illustrated that MiPLA2 was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA2 transcription and free ArA levels were established. Consequently, the role of mMiPLA2 in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.

Engineering Yarrowia lipolytica for sustainable ricinoleic acid production: A pathway to free fatty acid synthesis

Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.

Conjugation with Phospholipids as a Modification Increasing Anticancer Activity of Phenolic Acids in Metastatic Melanoma-In Vitro and In Silico Studies.

Phenolic acids possess many beneficial biological activities, including antioxidant and anti-inflammatory properties. Unfortunately, their low bioavailability restricts their potential medical uses, as it limits the concentration of phenolic acids achievable in the organism. The conjugation with phospholipids constitutes one of the most effective strategies to enhance compounds bioavailability in biological systems. In the present study, the conjugates of anisic (ANISA) and veratric acid (VA) with phosphatidylcholine (PC) were investigated. Since both ANISA and VA are inhibitors of tyrosinase, a melanocyte enzyme, the expression of which increases during tumorigenesis, anticancer potential of the conjugates was tested in several metastatic melanoma cell lines. The conjugates proved to be antiproliferative, apoptosis-inducing and cell-cycle-affecting agents, selective for cancerous cells and not affecting normal fibroblasts. The conjugates substituted by ANISA and VA, respectively, at both the sn-1 and sn-2 positions of PC, appeared the most promising, since they were effective against the vast majority of metastatic melanoma cell lines. Additionally, the conjugation of phenolic acids to PC increased their antioxidant activity. Molecular modeling was employed for the first time to estimate the features of the investigated conjugates relevant to their anticancer properties and membrane permeation. Again, the conjugates substituted by phenolic acid at both the sn-1 and sn-2 positions of PC seemed to be presumably most bioavailable.

The enrichment of eggs with docosahexaenoic acid and eicosapentaenoic acid through supplementation of the laying hen diet

The enrichment and transformation of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) enriched phospholipids for eggs deserve attention. The aim of the present study was to elucidate the comparative effects of DHA and EPA enriched phospholipids and triacylglycerols on egg fortification by determining the fatty acid composition of egg yolk after intervention with fish oil (15 g/kg) and krill oil (15 and 30 g/kg) for three consecutive weeks. The results indicated that laying hens could incorporate over 300 mg DHA and EPA into one egg. Greater retention efficiency of DHA and EPA in eggs was observed in fish oil supplementation compared with krill oil at equivalent dietary levels. DHA and EPA were prone to locate at the sn-2 position of phosphatidylcholine. Consequently, fish oil possessed high DHA content and conversion rate, and krill oil could raise the proportion of DHA-containing phospholipids in eggs.

Lipase Treatment of Dietary Krill Oil, but Not Fish Oil, Enables Enrichment of Brain Eicosapentaenoic Acid and Docosahexaenoic Acid.

Currently available omega-3 fatty acid supplements do not enrich the docosahexaenoic acid (DHA) of the adult brain because they are absorbed as triacylglycerol, whereas the transporter at the blood brain barrier requires lysophosphatidylcholine (LPC)-DHA. The hypothesis that treatment of krill oil (KO), which contains DHA/eicosapentaenoic acid (EPA) at the SN2 position of phosphatidylcholine, with SN1-specific lipase will generate LPC-DHA/EPA and which can be absorbed intact and transported into the brain, is tested. KO and fish oil (FO) are treated with Mucor meihei lipase, incorporated into AIN 93G diet, and fed to 2-month-old mice for 30 days. Fatty acid composition is analyzed by gas chromatography/mass spectroscopy. Brain derived neurotrophic factor (BDNF) is measured by ELISA. Lipase-treated (LT) KO increases brain DHA and EPA, respectively, 5-and 70-fold better than untreated (UT) KO. FO, whether lipase-treated or not, has no effect on brain DHA/EPA. LTKO is also more efficient in enriching liver DHA/EPA, but less efficient than UTKO and FO in enriching adipose tissue and heart. Brain BDNF is significantly increased by LTKO, but only marginally by other preparations. Pretreatment of dietary KO with lipase enables it to efficiently increase brain DHA/EPA because of the generation of LPC-DHA/EPA.

Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation

Docosahexaenoic acid (DHA, 22:6) is an n−3 polyunsaturated fatty acid (n−3 PUFA) that influences immunological, metabolic, and neurological responses through complex mechanisms. One structural mechanism by which DHA exerts its biological effects is through its ability to modify the physical organization of plasma membrane signaling assemblies known as sphingomyelin/cholesterol (SM/chol)-enriched lipid rafts. Here we studied how DHA acyl chains esterified in the sn-2 position of phosphatidylcholine (PC) regulate the formation of raft and non-raft domains in mixtures with SM and chol on differing size scales. Coarse grained molecular dynamics simulations showed that 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) enhances segregation into domains more than the monounsaturated control, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Solid state 2H NMR and neutron scattering experiments provided direct experimental evidence that substituting PDPC for POPC increases the size of raft-like domains on the nanoscale. Confocal imaging of giant unilamellar vesicles with a non-raft fluorescent probe revealed that POPC had no influence on phase separation in the presence of SM/chol whereas PDPC drove strong domain segregation. Finally, monolayer compression studies suggest that PDPC increases lipid-lipid immiscibility in the presence of SM/chol compared to POPC. Collectively, the data across model systems provide compelling support for the emerging model that DHA acyl chains of PC lipids tune the size of lipid rafts, which has potential implications for signaling networks that rely on the compartmentalization of proteins within and outside of rafts.

Profiling and Imaging of Phospholipids in Brains of Abcd1-Deficient Mice.

ABCD1 is a gene responsible for X-linked adrenoleukodystrophy (X-ALD), and is critical for the transport of very long-chain fatty acids (VLCFA) into peroxisomes and subsequent β-oxidation. VLCFA-containing lipids accumulate in X-ALD patients, although the effect of ABCD1-deficiency on each lipid species in the central nervous system has not been fully characterized. In this study, each phospholipid and lysophospholipid species in Abcd1-deficient mice brains were profiled by liquid chromatography-mass spectrometry. Among the phospholipid and lysophospholipid species that are significantly more enriched in Abcd1-deficient mice brains, VLCFA were present in 75, 15, 5, 4, and 1 species of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine, respectively. Most VLCFA were incorporated at the sn-1 position of phosphatidylcholine and phosphatidylethanolamine. Among the phospholipid species that are significantly less enriched in Abcd1-deficient mice brains, odd-numbered saturated or mono-unsaturated fatty acyl moieties are contained in all phosphatidylcholine species. In addition, a number of phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine species contained highly unsaturated fatty acyl moieties. Intriguingly, 44:1 phosphatidylcholine with VLCFA was mainly distributed in the gray matter, such as the cortex, but not in the white matter in the cerebrum and cerebellum. These results show that ABCD1-deficiency causes metabolic alternation of long-chain fatty acids and VLCFA. Moreover, our results imply a molecular mechanism for the incorporation of saturated or monounsaturated VLCFA into the sn-1 position of phospholipids, and also indicate that the distribution of phospholipids with VLCFA may correlate with the development of X-ALD.

Synthesis and Biological Evaluation of Novel Phosphatidylcholine Analogues Containing Monoterpene Acids as Potent Antiproliferative Agents.

The synthesis of novel phosphatidylcholines with geranic and citronellic acids in sn-1 and sn-2 positions is described. The structured phospholipids were obtained in high yields (59–87%) and evaluated in vitro for their cytotoxic activity against several cancer cell lines of different origin: MV4-11, A-549, MCF-7, LOVO, LOVO/DX, HepG2 and also towards non-cancer cell line BALB/3T3 (normal mice fibroblasts). The phosphatidylcholines modified with monoterpene acid showed a significantly higher antiproliferative activity than free monoterpene acids. The highest activity was observed for the terpene-phospholipids containing the isoprenoid acids in sn-1 position of phosphatidylcholine and palmitic acid in sn-2.

Lecithin:Retinol Acyltransferase: A Key Enzyme Involved in the Retinoid (visual) Cycle.

Lecithin:retinol acyltransferase (LRAT) catalyzes the acyl transfer from the sn-1 position of phosphatidylcholine (PC) to all-trans-retinol, creating fatty acid retinyl esters (palmitoyl, stearoyl, and some unsaturated derivatives). In the eye, these retinyl esters are substrates for the 65 kDa retinoid isomerase (RPE65). LRAT is well characterized biochemically, and recent structural data from closely related family members of the NlpC/P60 superfamily and a chimeric protein have established its catalytic mechanism. Mutations in the LRAT gene are responsible for approximately 1% of reported cases of Leber congenital amaurosis (LCA). Lack of functional LRAT, expressed in the retinal pigmented epithelium (RPE), results in loss of the visual chromophore and photoreceptor degeneration. LCA is a rare hereditary retinal dystrophy with an early onset associated with mutations in one of 21 known genes. Protocols have been devised to identify therapeutics that compensate for mutations in RPE65, also associated with LCA. The same protocols can be adapted to combat dystrophies associated with LRAT. Improvement in the visual function of clinical recipients of therapy with recombinant adeno-associated virus (rAAV) vectors incorporating the RPE65 gene provides a proof of concept for LRAT, which functions in the same cell type and metabolic pathway as RPE65. In parallel, a clinical trial that employs oral 9-cis-retinyl acetate to replace the missing chromophore in RPE65 and LRAT causative disease has proven to be effective and free of adverse effects. This article summarizes the biochemistry of LRAT and examines chromophore replacement as a treatment for LCA caused by LRATmutations.

Synthesis of structured phosphatidylcholine containing punicic acid by the lipase-catalyzed transesterification with pomegranate seed oil

Incorporation of conjugated linolenic acid (punicic acid; 18:3, 9c,11t,13c) into egg-yolk phosphatidylcholine (PC) in the lipase-catalyzed transesterification process was the aim of this work. Pomegranate seed oil (PSO) containing over 77% of punicic acid was used as an acyl donor and three commercially available immobilized lipases were examined as biocatalysts. The effects of enzyme load, reaction time and molar ratio of substrates (PC:PSO) on the incorporation of punicic acid into the modified PC were tested. In all experiments the best results were obtained using lipase B from Candida antarctica. In optimum conditions (20% of enzyme load; 1:3 PC:PSO molar ratio; 72h), the incorporation of punicic acid into the sn-1 position of PC was 56%. Additionally, the total content of polyunsaturated fatty acids in modified PC was almost 50%.

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Objectives Because of the great diversity of human lipid species, discerning meaningful patterns in lipidomic data has proven daunting. Considering the function of lipid classes in maintaining cell membrane permeability and viscosity provides a tool to bring order to the chaos of lipidomic studies. Methods Literature search. Results Compared to controls, a decreased phosphatidyl choline (PC) to phophatidyl ethanolamine (PE) ratio is seen in preeclampsia, cold adaptation, and essential fatty acid deficiency lipidomics. Because PC tends to aggregate in a relatively impermeable lamellar pattern and PE in a more permeable hexagonal pattern, the altered sphingolipid ratios in these diseases may be interpreted as homeostatic responses to maintain membrane permeability. Cold adaptation occurs by remodeling phospholipids to reduce membrane stiffness. Lipids alter membrane flexibility in predictable ways: long-chain saturated fatty acids and trans fatty acids stiffen cell membranes, monounsaturated and medium-chain saturated fatty acids maintain moderate membrane viscosity, and polyunsaturated fatty acids increase membrane flexibility. Pulse wave velocity demonstrates that women with preeclampsia have significantly increased vascular stiffness, correlating positively with severity of disease. Dietary trans fatty acids are preferentially integrated into the sn-1 position of PCs, which contain PUFA at the sn-2 position; a rise in PC PUFAs is noted with diets enriched in trans fatty acids. Using metabolomics biomarkers, to predict preeclampsia in early pregnancy, long-chain PUFAs were found to be significantly increased in women destined to develop preeclampsia later in pregnancy compared to pregnant women who did not, therefore increasing the unsaturation index, a pattern similar to that of cold adaptation, but not found in essential fatty acid deficiency. Increased unsaturation index is positively correlated with increased oxidative stress, a known pathological element in preeclampsia. Conclusions Considering the effects of lipid species on membrane viscosity and permeability offers a novel means to interpret lipidomic studies. Disclosures N.R. Hart: None.

Coexpressing Escherichia coli Cyclopropane Synthase with Sterculia foetida Lysophosphatidic Acid Acyltransferase Enhances Cyclopropane Fatty Acid Accumulation

Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation.

Toxicity of ricinoleic acid production in fission yeast Schizosaccharomyces pombe is suppressed by the overexpression of plg7, a phospholipase A2 of a platelet-activating factor (PAF) family homolog

In an effort to produce ricinoleic acid (RA), an important natural raw material with great values as a petrochemical replacement, in Schizosaccharomyces pombe, we introduced Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12) to S. pombe, putting it under the control of an inducible nmt1 promoter. However, RA was toxic to S. pombe and the cells expressing CpFAH12 grew poorly at the normal growth temperature 30°C. To address its toxic mechanism in S. pombe, we screened for a S. pombe cDNA library and identified plg7, which encodes a phospholipase A2, as a suppressor that restored the growth defect without affecting the RA production. A lacZ fusion experiment showed that the expression of plg7 was inducible by RA. Thin layer chromatographic analysis confirmed a reduction in RA moiety in phospholipids and a concomitant increase in free RA in the plg7 overexpressed strain. Since RA is synthesized at the sn-2 position of phosphatidylcholine by Fah12p, and phospholipase A2 hydrolyzes the sn-2 acyl bond of phospholipids, we speculate that plg7 is a stress-responsive gene, and removal of RA moieties from phospholipids, major components of lipid bilayer membrane, by Plg7p would be its suppression mechanism.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

Human Lysophosphatidylcholine Acyltransferases 1 and 2 Are Located in Lipid Droplets Where They Catalyze the Formation of Phosphatidylcholine

Phosphatidylcholine (PC) is synthesized by two different pathways, the Lands cycle and the Kennedy pathway. The recently identified key enzymes of the Lands cycle, lysophosphatidylcholine acyltransferase 1 and 2 (LPCAT1 and -2), were reported to localize to the endoplasmic reticulum and to function in lung surfactant production and in inflammation response. Here, we show in various mammalian cell lines that both enzymes additionally localize to lipid droplets (LDs), which consist of a core of neutral lipids surrounded by a monolayer of phospholipid, mainly PC. This dual localization is enabled by the monotopic topology of these enzymes demonstrated in this study. Furthermore, we show that LDs have the ability to locally synthesize PC and that this activity correlates with the LPCAT1 and -2 expression level. This suggests that LPCAT1 and -2 have, in addition to their known function in specialized cells, a ubiquitous role in LD-associated lipid metabolism.

As simple as ACB – new insights into the role of acyl‐CoA‐binding proteins in Arabidopsis

As simple as ACB – new insights into the role of acyl‐CoA‐binding proteins in Arabidopsis

CDNA cloning and characterization of human and mouse Ca 2+-independent phosphatidylethanolamine N-acyltransferases

The formation of N-acylphosphatidylethanolamine by N-acylation of phosphatidylethanolamine (PE) is the initial step in the biosynthetic pathway of bioactive N-acylethanolamines, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. We recently cloned a rat enzyme capable of catalyzing this reaction, and referred to the enzyme as Ca 2+-independent N-acyltransferase (iNAT). Here we report cDNA cloning and characterization of human and mouse iNATs. We cloned iNAT-homologous cDNAs from human and mouse testes, and overexpressed them in COS-7 cells. The purified recombinant proteins abstracted an acyl group from both sn-1 and sn-2 positions of phosphatidylcholine, and catalyzed N-acylation of PE as well as phospholipase A 1/A 2-like hydrolysis. The iNAT activity was mainly detected in soluble rather than particulate fractions, and was only slightly increased by Ca 2+. These results demonstrated that the human and mouse homologues function as iNAT. As for the organ distribution of iNAT, human testis and pancreas and mouse testis exhibited by far the highest expression level, suggesting its physiological importance in the specific organs. Moreover, mutagenesis studies showed crucial roles of His-154 and Cys-241 of rat iNAT in the catalysis and a possible role of the N-terminal domain in membrane association or protein–protein interaction.

Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain

Anandamide is an endogenous signaling lipid that binds to and activates cannabinoid receptors in the brain and peripheral tissues. The endogenous precursors of anandamide, N-arachidonoyl phosphatidylethanolamines (NArPEs), are a family of complex glycerophospholipids that derive from the exchange reaction of an arachidonoyl group between the sn-1 position of phosphatidylcholine and the primary amine of phosphatidylethanolamine catalyzed by N-acyl transferase activity. A precise characterization of the molecular composition of NArPE species generating anandamide has not yet been reported. In the present study, using liquid chromatography coupled to electrospray ionization ion-trap mass spectrometry, we identified the major endogenous NArPE species, which mainly contained sn-1 alkenyl groups (C16:0, C18:0, C18:1) and monounsaturated (C18:1) or polyunsaturated (C20:4, C22:4, C22:6) acyl groups at the sn-2 position of the glycerol backbone. Using rat brain particulate fractions, we observed a calcium-dependent increase in both NArPEs and anandamide formation after incubation at 37 degrees C for 30 min. Furthermore, a targeted lipidomic analysis showed that Ca(2+) specifically stimulated the formation of PUFA-containing NArPE species. These results reveal a previously unrecognized preference of brain N-acyl transferase activity for polyunsaturated NArPE and provide new insights on the physiological regulation of anandamide biosynthesis.

Sequestration of Retinyl Esters Is Essential for Retinoid Signaling in the Zebrafish Embryo

For vertebrate development, vitamin A (all-trans retinol) is required in quantitative different amounts and spatiotemporal distribution for the production of retinoic acid, a nuclear hormone receptor ligand, and 11-cis retinal, the chromophore of visual pigments. We show here for zebrafish that embryonic retinoid homeostasis essentially depends on the activity of a leci-thin:retinol acyltransferase (Lratb). During embryogenesis, lratb is expressed in mostly non-overlapping domains opposite to retinal dehydrogenase 2 (raldh2), the key enzyme for retinoic acid synthesis. Blocking retinyl ester formation by a targeted knock down of Lratb results in significantly increased retinoic acid levels, which lead to severe embryonic patterning defects. Thus, we provide evidence that a balanced competition between Lratb and Raldh2 for yolk vitamin A defines embryonic compartments either for retinyl ester or retinoic acid synthesis. This homeostatic mechanism dynamically adjusts embryonic retinoic acid levels for gene regulation, concomitantly sequestering excess yolk vitamin A in the form of retinyl esters for the establishment of larval vision later during development.

Multi-component analysis of marine lipids in fish gonads with emphasis on phospholipids using high resolution NMR spectroscopy

High resolution NMR has been applied for assessment of lipid classes and acyl stereospecific positions of fatty acids in marine phospholipids and triacylglycerols. 1D and 2D NMR techniques in combination with recording of a number of reference standards have been used to interpret the 1H and 13C NMR spectra of fish gonads. 13C NMR spectra gave information regarding the polyunsaturated fatty acids (PUFAs) in phosphatidylcholine (PC) and phoshatidylethanolamine (PE). The carbonyl resonances showed that n − 3 PUFAs primarily were esterified in the sn-2 position of PC and PE. The glycerol resonances showed that the PC/PE ratio was higher in roe than in milt and that roe comprised more triacylglycerols than milt. Thin layer chromatography showed that milt contained 2.4 times more cholesterol than roe, which was also found by integrating the 1H NMR spectra. Concentration (mol%) of n − 3 fatty acids were calculated from the 1H NMR data and showed 44.8 and 36.3% in roe and milt, respectively.