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.     

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.     

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.     

Determination of membrane cholesterol partition coefficient using a lipid vesicle-cyclodextrin binary system: effect of phospholipid acyl chain unsaturation and headgroup composition

Lateral domain or raft formation in biological membranes is often discussed in terms of cholesterol-lipid interactions. Preferential interactions of cholesterol with lipids, varying in headgroup and acyl chain unsaturation, were studied by measuring the partition coefficient for cholesterol in unilamellar vesicles. A novel vesicle-cyclodextrin system was used, which precludes the possibility of cross-contamination between donor-acceptor vesicles or the need to modify one of the vesicle populations. Variation in phospholipid headgroup resulted in cholesterol partitioning in the order of sphingomyelin (SM) > phosphatidylserine > phosphatidylcholine (PC) > phosphatidylenthanolamine (PE), spanning a range of partition DeltaG of -1181 cal/mol to +683 cal/mol for SM and PE, respectively. Among the acyl chains examined, the order of cholesterol partitioning was 18:0(stearic acid),18:1n-9(oleic acid) PC > di18:1n-9PC > di18:1n-12(petroselenic acid) PC > di18:2n-6(linoleic acid) PC > 16:0(palmitic acid),22:6n-3(DHA) PC > di18:3n-3(alpha-linolenic acid) PC > di22:6n-3PC with a range in partition DeltaG of 913 cal/mol. Our results suggest that the large differences observed in cholesterol-lipid interactions contribute to the forces responsible for lateral domain formation in plasma membranes. These differences may also be responsible for the heterogeneous cholesterol distribution in cellular membranes, where cholesterol is highly enriched in plasma membranes and relatively depleted in intracellular membranes.     

Minor triterpenes from Orthopterygium huancuy

Two new triterpenes, 3-oxo-20-hydroxy-lupan-28-oic and 3β,6β-dihydroxy-olean-18-en-28-oic acids, along with oleanonic, morolic and sumaresinolic acids have been isolated from Orthopterygium huancuy. The structure of the first new terpene was determined by single crystal X-ray analysis.     

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants.     

An enzymatic method for the production of 6-oxohexanoic acid from 6-aminohexanoic acid by an enzyme oxidizing ω-amino compounds from Phialemonium sp. AIU 274

An enzymatic method for 6-oxohexanoic acid production was developed using 6-aminohexanoic acid and an ω-amino group-oxidizing enzyme (ω-AOX) from Phialemonium sp. AIU 274. 6-Oxohexanoic acid was produced from 6-aminohexanoic acid with 100% yield by incubation with 0.3 U of the ω-AOX and 20 U of catalase at 30 °C for 30 h in 0.1 M potassium phosphate buffer (pH 7.0).     

Production of 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) and Its Derivatives by Vibrio tyrosinaticus

Six strains of bacteria belonging to Vibrio and Pseudomonas were selected as good producers of L-DOPA from L-tyrosine out of various bacteria. The condition for the formation of L-DOPA by Vibrio tyrosinaticus ATCC 19378 was examined and the following results were obtained. (1) Intermittent addition of L-tyrosine in small portions gave higher titer of L-DOPA than single addition of L-tyrosine. (2) Higher amount of L-DOPA was produced in stationary phase of growth than in logarithmic phase. (3) Addition of antioxidant, chelating agent or reductant such as L-ascorbic acid, araboascorbic acid, hydrazine, citric acid and 5-ketofructose increased the amount of L-DOPA formed. (4) L-Tyrosine derivatives such as N-acetyl-L-tyrosine amide, N-acetyl-L-tyrosine, L-tyrosine amide, L-tyrosine methyl ester and L-tyrosine benzyl ester were converted to the corresponding L-DOPA derivatives.

In the selected condition about 4 mg/ml of L-DOPA was produced from 4.3 mg/ml of L-tyrosine.     

Prevention of UVB-Induced Production of the Inflammatory Mediator in Human Keratinocytes by Lactic Acid Derivatives Generated from Aromatic Amino Acids

The anti-inflammatory effects of lactic acid derivatives were investigated on ultraviolet B (UVB)-irradiated HaCaT human keratinocytes. A pretreatment with indole-3-lactic acid (ILA) and 4-hydroxyphenyllactic acid (HPLA) inhibited the UVB-induced production of interlekin-6 (IL-6). The inhibitory effect of L-HPLA was equivalent to that of a corresponding racemic mixture (DL-HPLA), suggesting that optical isomerism did not affect the anti-inflammatory activity of HPLA.     

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.     

Effect of Amino Acids on Lysosomal Proteolytic Activities in Rat Livers

(R)-2-(4′-Isobutylphenyl)propanoic acid (ibuprofen), (S)-3(4′-isobutylphenyl)butanoic acid and (S)-4-(4′-isobutylphenyl)pentanoic acid were obtained using microbial oxidation of (±)-l-isobutyl-4-(1′ -methyloctyl)benzene by Rhodococcus sp. BPM 1613.     

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 °C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH₂, CH, and terminal CH₃ groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.     

Kinetic Studies on the Inhibition of GABA-T by γ-Vinyl GABA and Taurine

γ-Aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of γ-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: γ-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by γ-vinyl GABA and taurine at concentrations of 51.0 and 78.5?mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (K_i) values for γ-vinyl GABA and taurine were found to be 26±3?mM and 68±7?mM respectively. The transamination process of α-ketoglutarate was not affected by the presence of γ-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when α-ketoglutarate was the substrate. The inhibition dissociation constant (K_ii) for this system was found to be 96±10?mM. The Michaelis-Menten constant (K_m) in the absence of inhibition, was found to be 0.79±0.11?mM, and 0.47±0.10?mM for GABA and α-ketoglutarate respectively.     

西蒙得木茎段组织培养中腋芽生长和增殖的激素调节

6-苄基嘌呤(6-BA);赤霉素(GA_3)和吲哚乙酸(IAA)对西蒙得木茎段的腋芽生长和增殖都可起促进作用,三种激素间有复杂的相互关系。6-BA的作用尤为明显和重要,适宜浓度为1—2mg/L。GA_3浓度为1mg/L时对腋芽生长和增殖有促进进作用,但浓度再增加,长出的腋芽数减少,且芽条生长不良。6-BA与GA_3组合使用,能明显促进腋芽生长,最好组合为6-BA_1—2mg/L GA_3 0.5mg/L:若6-BA浓度升高时,出芽量虽增多,但芽条生长不良。IAA1mg/L或2mg/L时添加不同浓度的6-BA,长出的腋芽数均比单独使用IAA要多;在加低浓度的6-BA(1—2mg/L)时抽出的腋芽数比单独使用6-BA(1—2mg)的少,但芽条生长健壮;在加入较高温度的6-BA(3—5mg/L)时抽出的腋芽数比单独使用6-BA(3—5mg/L)时多。     

Serum 11-deoxycorticosterone levels in adrenal-regeneration hypertension under conditions of quiescence and stress.

A time course study to measure adrenal cortical function was undertaken for the period prior to the development of hypertension until the onset of hypertension in the adrenal-regeneration hypertension (ARH) model. Quiescent rat kills were used so that all adrenal cortical parameters investigated would reflect basal or resting levels for controls. Thus a more accurate determination of the differences between control and experimental animals could be made. A radioimmunoassay procedure for deoxycorticosterone was developed to measure this steroid in individual rat serum samples. Elevated serum deoxycorticosterone levels were observed in rats with regenerating adrenals when they were killed under quiescent conditions. This agreed with our recently reported in vitro finding of restoration of cholesterol side chain cleavage activity while 11beta-hydroxylase activity remained imparied 25 days after adrenal enucleation. When rats were killed after ether stress, deoxycorticosterone levels were elevated in both control rats and in rats with regenerating adrenals but the difference was not significant. In contrast, after ether stress serum corticosterone levels were lower in rats with regenerating adrenals than in controls. These studies, in conjunction with our previous in vitro findings, point to the importance of deoxycorticosterone in the pathogenesis of adrenal regeneration hypertension and help to explain the anomalous corticosteroid secretion rate data found in this experimental hypertension model.     

Nutritional intervention with omega-3 fatty acids enhances tumor response to anti-neoplastic agents

Nutritional intervention with specific fatty acids depresses tumor growth and enhances tumor responsiveness to chemotherapy. Supplementation of tumors with long chained omega-3 polyunsaturated fatty acids results in enrichment of tumor phospholipid fractions with omega-3 fatty acids resulting in an altered membrane composition and function. Tumors enriched with long chained omega-3 polyunsaturated fatty acids possess membranes with increased fluidity, an elevated unsaturation index, enhanced transport capabilities that results in accumulation of selective anti-cancer agents, increased activity of selected drug activating enzymes, and alteration of signaling pathways important for cancer progression. These nutritionally induced changes in tumor fatty acid composition result in increased sensitivity to chemotherapy, especially in tumor lines that are resistant to chemotherapy and cause specific enhancement of cytotoxicity to tumor cells and protection of normal cells. Pre-disposing tumors to increased chemo-sensitivity through nutritional intervention with specific fatty acids has the potential to improve patient response to chemotherapy with fewer untoward side effects if these pre-clinical findings carry over into a clinical setting.     

The nervous system has an absolute molecular species requirement for proper function

A considerable body of biological evidence has accumulated that suggests that docosahexaenoic acid (22:6n3) is an essential component in the nervous system. Moreover, it appears from these studies that long chain polyunsaturates of the n-6 family such as arachidonate (20:4n6) and docosapentaenoate (22:5n6) cannot substitute for 22:6n3. This evidence is briefly reviewed and two hypotheses centering upon either biochemical or biophysical aspects of polyunsaturate function are presented and discussed. It is concluded that a bioactive metabolite of 22:6n3 is not responsible for its function in brain and that the best hypothesis asserts that a membrane function of a 22:6n3-containing species of phospholipid, such as phosphatidylserine, is critical for optimal neural function. Moreover, data are presented indicating that the biophysical properties of various highly unsaturated species of phospholipid are distinguishable. It is further contended that these species are not randomly distributed in membranes and thus the differences in physical properties may be amplified. It is concluded that a conceptual framework is needed in which the distinct membrane roles of phospholipid species may be understood as a function of the positions and numbers of double bonds. Only then may the critical role of the highly unsaturated n-3 polyunsaturates in the brain and retina be understood.     

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.