Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.     

Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared toward the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically expressed enzymes. Here, we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to fourfold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately fourfold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant fatty acid desaturase (FAD) not only provides a useful means for protein immunodetection using highly specific, commercially available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.     

The function of very long chain polyunsaturated fatty acids in the pineal gland

The mammalian pineal gland is a prominent secretory organ with a high metabolic activity. Melatonin (N-acetyl-5-methoxytryptamine), the main secretory product of the pineal gland, efficiently scavenges both the hydroxyl and peroxyl radicals counteracting lipid peroxidation in biological membranes. Approximately 25% of the total fatty acids present in the rat pineal lipids are represented by arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3). These very long chain polyunsaturated fatty acids play important roles in the pineal gland. In addition to the production of melatonin, the mammalian pineal gland is able of convert these polyunsaturated fatty acids into bioactive lipid mediators. Lipoxygenation is the principal lipoxygenase (LOX) activity observed in the rat pineal gland. Lipoxygenation in the pineal gland is exceptional because no other brain regions express significant LOX activities under normal physiological conditions. The rat pineal gland expresses both 12- and 15-lipoxygenase (LOX) activities, producing 12- and 15-hydroperoxyeicosatetraenoic acid (12- and 15-HpETE) from arachidonic acid and 14- and 17-hydroxydocosahexaenoic acid (14- and 17-HdoHE) from docosahexaenoic acid, respectively. The rat pineal also produces hepoxilins via LOX pathways. The hepoxilins are bioactive epoxy-hydroxy products of the arachidonic acid metabolism via the 12S-lipoxygenase (12S-LOX) pathway. The two key pineal biochemical functions, lipoxygenation and melatonin synthesis, may be synergistically regulated by the status of n-3 essential fatty acids.     

Fatty acid correlates of temperament in adolescent boys with attention deficit hyperactivity disorder

Atypical fatty acid metabolism has been reported in attention deficit hyperactivity disorder (ADHD), however, its relationship with temperament in this population is unclear. The current study investigated the association between blood levels of fatty acids implicated in brain structure and function (omega-3, omega-6, omega-9) and personality traits of stability (neuroticism, conscientiousness and agreeableness) and plasticity (extraversion and openness). Twenty right-handed adolescent boys with ADHD completed a self-report NEO-FFI personality questionnaire, and had fatty acid content assessed from red blood using gas chromatography. Pearson's correlations showed no significant associations between omega-3 levels and personality. After correction for multiple comparisons, Adrenic Acid (C22:4n6) was inversely associated with stability. Oleic acid (C18:1n9) was positively associated with plasticity. Results are in line with a role of fatty acids in brain function. They suggest that those fatty acids that are involved in myelination (Adrenic, Oleic) have the strongest associations with temperament in adolescents with ADHD.     

A synopsis of the process of lipid peroxidation since the discovery of the essential fatty acids

Eighty years ago, Burr and Burr, introduced for the first time the concept of essential fatty acids. Now is very well known that requirements for polyunsaturated fatty acids PUFAs can not be met by de novo metabolic processes within mammalian tissues. Animals are absolutely dependent on plants for providing the two major precursors of the n-6 and n-3 fatty acids, C18:2n-6; linoleic and C18:3n-3; α-linolenic acids. In animal tissues these precursors are transformed to fatty acids containing three to six double bonds. During the last four decades the interest in polyunsaturated fatty acids has augmented manifolds, and the number of published studies is rising each year. The current impetus for this interest has been mainly the observation that PUFAs and their metabolites have several physiological roles including: energy provision, membrane structure, cell signaling and regulation of gene expression. In addition the observation that PUFAs are targets of lipid peroxidation opens a new important area of investigation. Melatonin, the main secretory product of the pineal gland, efficiently scavenges both the hydroxyl and peroxyl radicals counteracting lipid peroxidation in biological membranes. In addition the two key pineal biochemical functions, lipoxygenation and melatonin synthesis may be synergistically regulated by the status of n-3 essential fatty acids. At the retina level, free radicals may preferentially react with the membrane polyunsaturated fatty acids leading to the release of lipoperoxide radicals. These lipoperoxides can induce oxidative stress linked to membrane lysis, damage to neuronal membranes may be related to alteration of visual function.     

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin.Following supplementation with saturated fatty acids of longer than 15 carbons (100 μM) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth.Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35–41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72–82% after addition of unsaturated fatty acids.A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids.     

Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma

Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.     

Modulation of native T-type calcium channels by omega-3 fatty acids

Low voltage-activated, rapidly inactivating T-type Ca(2+) channels are found in a variety of cells where they regulate electrical activity and Ca(2+) entry. In whole-cell patch clamp recordings from bovine adrenal zona fasciculata cells, cis-polyunsaturated omega-3 fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid, and alpha-linolenic acid inhibited T-type Ca(2+) current (I(T-Ca)) with IC(50)s of 2.4, 6.1, and 14.4microM, respectively. Inhibition of I(T-Ca) by DHA was partially use-dependent. In the absence of stimulation, DHA (5microM) inhibited I(T-Ca) by 59.7+/-8.1% (n=5). When voltage steps to -10mV were applied at 12s intervals, block increased to 80.5+/-7.2%. Inhibition of I(T-Ca) by DHA was accompanied by a shift of -11.7mV in the voltage dependence of steady-state inactivation, and a smaller -3.3mV shift in the voltage dependence of activation. omega-3 fatty acids also selectively altered the gating kinetics of T-type Ca(2+) channels. DHA accelerated T channel recovery from inactivation by approximately 3-fold, but did not affect the kinetics of T channel activation or deactivation. Arachidonic acid, an omega-6 polyunsaturated fatty acid, also inhibited T-type Ca(2+) current at micromolar concentrations, while the trans polyunsaturated fatty acid linolelaidic acid was ineffective. These results identify cis polyunsaturated fatty acids as relatively potent, new T-type Ca(2+) channel antagonists. omega-3 fatty acids are essential dietary components that have been shown to possess remarkable neuroprotective and cardioprotective properties that are likely mediated through suppression of electrical activity and associated Ca(2+) entry. Inhibition of T-type Ca(2+) channels in neurons and cardiac myocytes could contribute significantly to their protective actions.     

Modulatory effect of omega-3 polyunsaturated fatty acids on osteoblast function and bone metabolism

Recent investigations indicate that the type and amount of polyunsaturated fatty acids (PUFA) influence bone formation in animal models and osteoblastic cell functions in culture. In growing rats, supplementing the diet with omega-3 PUFA results in greater bone formation rates and moderates ex vivo prostaglandin E(2) production in bone organ cultures. A protective effect of omega-3 PUFA on minimizing bone mineral loss in ovariectomized rats has also been reported. The actions of omega-3 fatty acids on bone formation appear to be linked to altering osteoblast functions. Herein we describe experiments with MC3T3-E1 osteoblast-like cells that support findings in vivo where omega-3 PUFA modulated COX-2 protein expression, reduced prostaglandin E(2) production, and increased alkaline phosphatase activity. Other studies indicate that the dietary source of PUFA may affect protein expression of Cbfa1 and nodule formation in fetal rat calvarial cells.     

Seasonal dynamics of fatty acid composition in female northern pike (Esox lucius L.)

Seasonal changes in the fatty acid composition of neutral and polar lipids were measured in the ovary, liver, white muscle, and adipopancreatic tissue of northern pike. The role of environmental and physiological factors underlying these changes was evaluated. From late summer (August–September) to winter (January–March), the weight percentage of n-3 polyunsaturated fatty acids (especially 22:6n3) declined significantly in the neutral lipids of all somatic tissues examined. However, large quantities of n-3 polyunsaturated fatty acids accumulated in the recrude cing ovaries during fall and the weight percentage of n-3 polyunsaturated fatty acids in ovary polar lipids also increased significantly. Additionally, the n-3 polyunsaturated fatty acid content of somatic polar lipids increased significantly during fall due to increases in the total polar lipid content of the somatic tissues. This suggests that during fall n-3 polyunsaturated fatty acid are diverted away from somatic neutral lipids and thereby conserved for use in ovary construction and for incorporation into tissue polar lipids. The percentage of n-3 polyunsaturated fatty acid in ovary neutral lipids also declined during fall and early winter, perhaps as an adaptation to conserve these fatty acids for storage in oocyte polar lipids and later incorporation into cellular membranes of the developing embryo. Reductions in the n-3 polyunsaturated fatty acids content of somatic and ovarian neutral lipids during fall were compensated for specifically by increases in the percentage of monounsaturated fatty acids rather than saturated fatty acids. This suggests that the ratio of saturated to unsaturated fatty acids in pike neutral lipid, is regulated physiologically, and hence may influence the physiological functioning of these lipids. During fall and early winter the percentage of saturated fatty acids declined significantly in the polar lipids of all tissues examined. This change was consistent with the known effects of cold acclimation on the fatty acid composition of cellular membranes. As the ovaries were recrudescing from September to January, liver polar lipids exhibited significant decreases in the percentage of total polyunsaturated fatty acids and n-3 polyunsaturated fatty acids and increases in monounsaturated fatty acids, and acquired a fatty acid composition very similar to that of ovary polar lipids. Therefore, seasonal changes in the percentage of polyunsaturated and monounsaturated fatty acids in liver polar lipids probably reflect the liver's role in vitellogenesis rather than the effects of temperature on membrane fatty acid composition. At all times of year, the fatty acid compositions of white muscle and adipopancreatic tissue neutral lipids were very similar, which may indicate a close metabolic relationship between these lipid compartments.Abbreviations AP adipopancreatic - BHT butylated hydroxytoluene - CI confidence interval - EFA essential fatty acids - MUFA monounsaturated fatty acids - NL neutral lipids - PL polar lipids - PUFA polyunsaturated fatty acids - SFA saturated fatty acids     

Dietary manipulation implicates lipid signaling in the regulation of germ cell maintenance in C. elegans

Reproduction in C. elegans relies on continuously proliferating germ cells which, during germline development, exit mitosis, undergo meiosis and differentiate into gametes. Supplementing the diet of C. elegans with dihommogamma-linolenic acid (20:3n-6, DGLA), a long chain omega-6 polyunsaturated fatty acid, results in sterile worms that lack germ cells. The effect is remarkably specific for DGLA, as eicosapentaenoic acid (20:5n-3, EPA) and other long-chain polyunsaturated fatty acids with similar physical properties have little or no effect on fertility. Germ cells undergoing mitosis during larval stages are especially sensitive to DGLA, but exposure to DGLA during adulthood also reduces germ cells and brood size, in part by inducing inappropriate apoptosis of meiotic germ cells. Mutant strains with defects in fatty acid desaturation and elongation display altered susceptibility to DGLA, indicating that the sterility effect of the dietary lipid depends on the amount of DGLA present in membranes as well as on the capacity to convert DGLA to other fatty acids. We propose that DGLA produces a signal that interacts with one or more pathways regulating germ cell survival. Our DGLA findings are the first report of a role for a specific fatty acid affecting the development and maintenance of germ cells in C. elegans.     

Effect of long-chain fatty acids in the culture medium on fatty acid composition of WEHI-3 and J774A.1 cells

As a first step in determining the mechanism of action of specific fatty acids on immunological function of macrophages, a comparative study of the effect of long-chain polyunsaturated fatty acids (PUFA) in the medium was conducted in two macrophage cell lines, J774A.1 and WEHI-3. The baseline fatty-acid profiles of the two cell lines differed in the % distribution of saturated (SFA) and unsaturated fatty acids (UFA). J774A.1 cells had a higher % of SFA (primarily palmitic acid) than WEHI-3 cells. Conversely, WEHI-3 cells had a higher % of UFA (primarily oleic acid) than J774A.1 cells. Neither cell line had detectable amounts of alpha-linolenic acid (ALA) or eicosapentaenoic acid (EPA). The most abundant polyunsaturated fatty acid in both cells lines was arachidonic acid (AA). The efficiency of transport of fatty acids from the medium to the macrophages by two delivery vehicles (BSA complexes and ethanolic suspensions) was compared. Overall, fatty acids were transported satisfactorily by both delivery systems. Alpha-linolenic acid and doscosahexenoic acid (DHA) were transported more efficiently by the ethanolic suspension system. Linoleic acid (LA) was taken up more completely than ALA, and DHA was taken up more completely than EPA by both cell cultures and delivery systems. A dose-response effect was demonstrated for LA, ALA, EPA and DHA in both J774A.1 and WEHI-3 cells. Addition of polyunsaturated fatty acids (PUFA) to the cell cultures modified the total lipid fatty acid composition of the cells. The presence of ALA in the culture medium resulted in a significant decrease in AA in both cell lines. The omega-3/omega-6 fatty acid ratio (omega-3/omega-6), polyunsaturated/saturated fatty acid ratio (P/S), and unsaturation index (UI) increased directly with the amount of PUFA and omega-3 fatty acid provided in the medium. The results indicate that the macrophage cell lines have similar, but not identical, fatty acid profiles that may be the result of differences in fatty acid metabolism. These distinctions could in turn produce differences in immunological function. The ethanol fatty-acid delivery system, when compared with the fatty acid-BSA complex system, is preferable for measurement of dose-response effects, because the cellular fatty acid content increased in proportion to the amount of fatty acid provided in the medium. Similar dose-response results were observed in a previous in vivo study using flaxseed, rich in ALA, as a source of PUFA.     

Polyunsaturated fatty acids in zooplankton: variation due to taxonomy and trophic position

1. Food quality has major effects on the transfer of energy and matter in food webs, and essential long‐chained polyunsaturated fatty acids (PUFAs) can affect the quality of phytoplankton as food. In a study of oligotrophic lakes in north‐western Sweden, we investigated the fatty acid composition of four planktonic cladocerans and two calanoid copepods, representing herbivorous and carnivorous species. We also collected seston samples. 2. The proportions of long‐chain PUFAs in the organisms increased with their increasing trophic position. Thus, both their quality as food for other organisms, as well as their requirement for fatty acids (FAs), differed among taxa and depended on their trophic position. 3. We found taxon‐specific differences in the FA composition of zooplankton that were not related to sestonic FA composition. This implies that the variation in zooplankton FA composition is constrained by phylogenetic origin, life history characteristics, or both. 4. The cladoceran taxa contained 12–23% eicosapentaenoic acid (EPA) but only 0.9–2.1% docosahexaenoic acid (DHA) of the total FA content. In contrast, the calanoid copepods contained 7–11% EPA and 14–21% DHA. Thus, our results show that differences in the PUFA content among zooplankton species could have repercussions for both food web structure and function.     

Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω-3) and Arachidonic Acid (20:4ω-6)

Elongated, highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.     

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.     

Glycerolipid analysis during desiccation and recovery of the resurrection plant Xerophyta humilis (Bak) Dur and Schinz

Xerophyta humilis is a poikilochlorophyllous monocot resurrection plant used as a model to study vegetative desiccation tolerance. Dehydration imposes tension and ultimate loss of integrity of membranes in desiccation sensitive species. We investigated the predominant molecular species of glycerolipids present in root and leaf tissues, using multiple reaction monitoring mass spectrometry, and then analysed changes therein during dehydration and subsequent rehydration of whole plants. The presence of fatty acids with long carbon chains and with odd numbers of carbons were detected and confirmed by gas chromatography. Dehydration of both leaves and roots resulted in an increase in species containing polyunsaturated fatty acids and a decrease in disaturated species. Upon rehydration, lipid saturation was reversed, with this being initiated immediately upon watering in roots but only 12–24 hr later in leaves. Relative levels of species with short‐chained odd‐numbered saturated fatty acids decreased during dehydration and increased during rehydration, whereas the reverse trend was observed for long‐chained fatty acids. X. humilis has a unique lipid composition, this report being one of the few to demonstrate the presence of odd‐numbered fatty acids in plant phosphoglycerolipids.     

Expression of E-FABP in PC12 cells increases neurite extension during differentiation: involvement of n-3 and n-6 fatty acids

Epidermal fatty acid-binding protein (E-FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E-FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E-FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n-3 docosahexaenoic acid (DHA), C20:5n-3 eicosapentaenoic acid (EPA), and C20:4n-6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E-FABP expression that is blocked by mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor-differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12-AS) which exhibit low E-FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12-S). Replenishing NGFDPC12-AS cells with biotinylated recombinant E-FABP (biotin-E-FABP) protein restores normal neurite outgrowth. Cellular localization of biotin-E-FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E-FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12-S than in NGFDPC12-AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E-FABP.     

Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms

Although some interest in growing microalgae heterotrophically for the production of pigments was generated in the 1960s, only minimal commercial research was focused on this type of production technology until the 1980s. Recent developments indicating the nutritional and pharmaceutical importance of long chain omega-3 polyunsaturated fatty acids in the human diet have stimulated interest in microalgae as a source of these vital compounds, for they are the primary producers of these fatty acids in marine food webs. Food and pharmaceutical quality production can be enhanced both by the degree of process control and by the sterility achieved through a fermentation process, when compared to outdoor solar pond production. The data presented illustrate that microalgal-based heterotrophic production systems can exhibit omega-3 fatty acid productivities 2–3 orders of magnitude greater than those of outdoor pond systems. Additionally, long chain omega-3 fatty acid productivities reported for the microalgal fermentation systems are 1–2 orders of magnitude greater than productivities reported for fungal or bacterial systems.     

A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1

A phosphopantetheinyl transferase (PPTase) gene (pfaE), cloned from the docosahexaenoic acid (DHA)-producing bacterium Moritella marina strain MP-1, has an open reading frame of 861 bp encoding a 287-amino acid protein. When the pfaE gene was expressed with pfaA-D, which are four out of five essential genes for biosynthesis of eicosapentaenoic acid (EPA) derived from Shewanella pneumatophori SCRC-2738 in Escherichia coli, the recombinant produced 12% EPA of total fatty acids. This suggests that pfaE encodes a PPTase required for producing n-3 polyunsaturated fatty acids, which is probably involved in the synthesis of DHA in M. marina strain MP-1.     

Cryptophyceae and rhodophyceae; chemotaxonomy, phylogeny, and application

The biochemical compositions of seven strains of marine cryptomonad and a rhodophyte were determined in logarithmic phase batch (1.4 L flask) and semi-continuous (10 L carboy) culture. Lipid ranged from 13% to 28%, protein ranged from 53% to 68%, and carbohydrate ranged from 9% to 24% of the organic weight. The major lipid classes in the species examined were polar lipids (78-88% of total lipid). The major sterol in the Cryptophyceae and the Rhodophyceae was 24-methylcholesta-5,22E-dien-3beta-ol (62-99% of total sterols); which is also the major sterol in some diatoms and haptophytes. Smaller proportions of cholest-5-en-3beta-ol (1-17.7%) were also found in the Cryptophyceae. Most cryptomonads contained high proportions of the n-3 polyunsaturated fatty acids (PUFA), 18:3n-3 (20.7-29.9% of the total fatty acids), 18:4n-3 (12.5-30.2%), 20:5n-3 (7.6-13.2%) and 22:6n-3 (6.4-10.8%). However, the blue-green cryptomonad Chroomonas placoidea was characterized by a low proportion of 22:6n-3 (0.2% of total fatty acids), and a significant proportion of 22:5n-6 (4.5%), and the presence of 24-ethylcholesta-5,22E-dien-3beta-ol (35.5% of total sterols). The fatty acid composition of the rhodophyte Rhodosorus sp. was similar to those of the Cryptophyceae except for lower proportions of 18:4n-3 and lack of C21 and C22 PUFA. It is postulated that the primary endosymbiosis of a photosynthetic n-3 C18 PUFA-producing prokaryote and a eukaryotic host capable of chain elongation and desaturation of exogenous PUFA, resulted in the Rhodophyceae capable of producing n-3 C20 PUFA. The secondary endosymbiosis of a photosynthetic n-3 C20 PUFA-producing eukaryote (such as a Rhodosorus sp. like-rhodophyte) and a eukaryotic host capable of further chain elongation and desaturation, resulted in the Cryptophyceae being capable of producing n-3 C20 and C22 PUFA de novo. Selected isolates were examined further in feeding trials with juvenile Pacific oysters (Crassostrea gigas). Rhodomonas salina CS-24(containing elevated 22:6n-3) produced high growth rates in oysters; equivalent to the microalga commonly used in aquaculture, Isochrysis sp. (T.ISO).