Isolation and Characterization of a Δ5-Desaturase from Oblongichytrium sp.

We isolated a cDNA clone with homology to known desaturase genes from Oblongichytrium sp., recently classified as a new genus of thraustochytrids (Labyrinthulomycetes), and found that it encoded Δ5-desaturase by its heterologous expression in yeast. The enzyme had higher activity toward 20:4n-3 than 20:3n-6, indicating that this Δ5-desaturase can be used in the production of n-3 polyunsaturated fatty acids in transgenic organisms.     

Docosahexaenoate-containing phospholipids in sarcoplasmic reticulum and retinal photoreceptors. A proposal for a role in Ca2+-ATPase calcium transport

A critical review of the experimental literature concerning the metabolism of all-cis-4, 7, 10, 13, 16, 19-docosahexaenoate-containing phospholipids in muscle and retina suggests that it plays an essential role in maximizing the Ca²⁺/ATP stoichiometry of the Ca²⁺-ATPase of sarcoplasmic reticulum and retinal photoreceptor disks. Docosahexaenoate-phosphatidylcholine is proposed to participate in oligomerization of Ca²⁺-ATPase necessary for the establishment of a high Ca²⁺/ATP coupling ratio of the Ca²⁺ pump in these tissues. Possible tests of this hypothesis are presented.     

Dietary fat influences electric membrane properties of neurons in cell culture

1. SJL/J mice were maintained on semipurified diets which differed in the ratio of polyunsaturated/saturated fatty acid content (P/S). Exposure was from conception and was maintained for periods ranging from 6 to 34 weeks. 2. Neural cell cultures were prepared from dorsal root ganglia (DRG). After 6 and 20 days of culture, neuronal electric membrane properties were determined quantitatively by intracellular recording. 3. A number of significant differences were observed for the two dietary conditions. DRG from mice on the low-P/s diet had an increase in the rate of fall of both phases of repolarization which, in conjunction with the reduced action potential overshoot, led to a reduced action potential duration. This shift to shorter-duration action potentials was accompanied by a shift to more monophasic falling phases. The low-P/S neurons also exhibited a decreased afterhyperpolarization, decreased specific membrane resistance, and decreased membrane electrical time constant compared to high-P/S neurons. 4. It was concluded that the P/S ratio in the diet can have a significant effect on the electric properties of neurons. The high-P/S neurons tended to have action potentials with biphasic repolarizations and longer durations. In contrast, the low-P/S neurons tended to have action potentials with monophasic repolarizations and shorter durations. Moreover, the known ionic dependence of these two types of action potentials suggested that the low-P/S diet resulted in action potentials with a more exclusive Na dependence, while the high-P/S diet resulted in action potentials with both Na and Ca dependence.     

Mutation induction in Escherichia coli incubated in the reaction mixture of NADPH-dependent lipid peroxidation of rat-liver microsomes

Experiments were carried out to examine mutation induction in E. coli cells incubated in the reaction mixture of NADPH-dependent lipid peroxidation of microsomes isolated from rat liver. The results obtained were as follows: (1) Lipid peroxidation of microsomes occurred extensively on incubation with NADPH and Fe2+. In the E. coli WP2uvrA(pKM101) system, the mutation frequency to streptomycin resistance increased markedly when the cells were incubated in the reaction mixture of microsomal lipid peroxidation. The induced mutation frequencies were dependent on the extent of the lipid peroxidation. (2) It was also found that the mutations were induced at the same rate as in the case of (1) when the cells were added to the microsomal suspensions after the reactions due to the short-lived free radicals had terminated. (3) The cytotoxicity of the lipid peroxidation products was larger in the DNA repair-defective mutant, E. coli SR18 (uvrArecA) than the wild-type strain, SR749. From these results it is concluded that some DNA-damaging and mutagenic substances are indeed produced in the degradation process of peroxidized polyunsaturated fatty acids in liver microsomal lipids.     

The effects of bisulfite on growth and macromolecular synthesis in Escherichia coli

Bisulfite reversibly inhibits the growth of a variety of microorganisms and has been used as a preservative in foods and beverages for that reason. We have now measured macromolecule synthesis in Escherichia coli K12 after bisulfite treatment. RNA synthesis, the synthesis of total protein, and of an inducible enzyme, beta-galactosidase, stopped almost immediately upon addition of 2 mM (or higher concentrations) of bisulfite. These functions resumed after a lag whose duration depended on the concentration of bisulfite added. The synthesis of DNA was slowed upon bisulfite addition, but did not stop entirely. The inhibition of RNA synthesis by bisulfite took place in both stringent and relaxed strains of E. coli and was not relieved upon addition of chloramphenicol. Stringent control was therefore not involved in this effect. No effect on protein synthesis was observed in the cell-free system of E. coli (using poly(U) or MS2 RNA as messenger) at bisulfite concentrations up to 10 mM. Protein synthesis inhibition in vivo was apparently not due to a reaction of bisulfite with a component of this system. In additional experiments, RNA polymerase was not impaired by bisulfite, and the growth inhibition effect was shown to proceed in the presence of inhibitors of free radical chain reactions.     

Retention of human skin fibroblast fatty acid modifications during maintenance culture

Summary The fatty acid composition of cultured human skin fibroblasts was modified by adding either oleic or linoleic acid to the growth medium. After the cultures became confluent, they were washed and transferred to different maintenance media in order to determine the stability of the various fatty acyl modifications. Some changes in fatty acid composition occurred under all conditions. When the maintenance medium was supplemented with fatty acid, the cellular neutral lipid and phospholipid fatty acyl composition were altered markedly within 16 to 24 hr. If no supplemental fatty acid was available during the maintenance period, however, the modified fatty acyl compositions were sufficiently retained so that appreciable differences between the cells enriched with oleate and linoleate persisted for at least 48 to 72 hr. This considerable degree of stability occurred when either 10% delipidized fetal bovine serum or 10% fetal bovine serum containing its inherent lipids were present in the maintenance medium. Although the triglyceride content of the fatty acid-modified cells was quite labile, neither the cholesterol nor phospholipid content changed appreciably during culture in any of the maintenance media. Since the fatty acid compositional differences persisted during several days of maintenance under certain conditions, these modified cultures appear to be a useful experimental system for assessing the effect of lipid structure on fairly long-term cellular functions. This work was supported by Arteriosclerosis Specialized Center of Research Grant HL14230 from the National Heart, Lung and Blood Institute, National Institutes of Health.     

Vitamin D level and its relation to muscle and fat mass in adult male Arabs

Low levels of vitamin D have been linked with increased adiposity and diminished muscle strength. Whether it is also related to fat deposition in muscle tissues is not studied well. This study explored the associations between circulating 25-hydroxyvitamin D (25(OH)D) and fat deposition in muscle tissues of adult Arab males. A total 465 adult Saudi males were included in this cross-sectional study. Anthropometrics, body composition and muscle strength were assessed. Serum 25(OH)D was determined and quantified enzymatically. They were grouped according to vitamin D status: deficient (25(OH)D < 50 nmol/l) N = 325 (69.9%) and sufficient (25(OH)D > 50 nmol/l)140 (30.1%). Mean level of lean/height², lean-arm-legs and lean-arms-legs/height² were significantly higher in 25(OH)D deficient participants (p-values 0.03; 0.05 and 0.01 respectively). Thigh strength was significantly higher in 25(OH)D sufficient participants than their deficient counterparts (p = 0.02). In all participants, a significant correlation between 25(OH)D was observed with age and thigh-strength (p-values < 0.05), while a significant inverse correlation between 25(OH)D and lean/height², lean-arms-legs, lean-arms-legs/height², fat (%) region, fat arms, fat legs, fat trunk, lean legs were noted. In conclusion, low circulating 25(OH)D is associated with enhanced fat infiltration in muscle tissues of adult Arab males.     

Enzymatic and Non-Enzymatic Esterification of long Polyunsaturated Fatty Acids and Lysophosphatidylcholine in Isooctane

Phosphatidylcholine containing large amounts of long polyunsaturated fatty acid, eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), was synthesized in isooctane. Immobilized phospholipase A₂ was used as a catalyst. A parallel non-enzymatic esterification reaction was investigated in separate experiments.

The concentrations of lyso-phosphatidylcholine, polyunsaturated fatty acids, water and the enzyme were varied over wide ranges as were the temperature and the reaction time. The type of enzyme, carrier and immobilization procedure were held constant.

The yield of phosphatidylcholine was relatively high (about 21%) when the concentration of polyunsaturated fatty acids was high (300 mg/g of reaction mixture) and the water content was low (below 30% of the dry immobilized enzyme). The highest yield of phosphatidylcholine was found at 80 hours and 75°C. However, at this temperature an extensive non-enzymatic reaction between polyunsaturated fatty acids and lyso-phosphatidylcholine occurred. At 80°C the polyunsaturated fatty acids were partly oxidized. Therefore, a temperature of 45°C to 65°C is probably the optimum temperature for the reaction.     

Enzymatic Esterification of Long Polyunsaturated Fatty Acids and Lyso-Phosphatidylcholine in Isooctane and Ethanol

Phosphatidylcholine (PC) was synthesized from lyso-PC and long poly-unsaturated (n-3) fatty acids (PUFA) using immobilized phospholipase A₂. The esterification was performed using the fatty acids as the main solvent and isooctane or ethanol (99.5%) at low concentrations (7–45%) as additional solvents. The temperature was kept constant at 45d`C and the water concentrations were carefully controlled.

The best yield of PC (22%) was found in the isooctane system at a low water content (22% of the dry immobilized enzyme). In the ethanol system, the yield of PC was only half. The best yields were attained when the concentrations of both isooctane and ethanol were below 7% and the reaction time was very long (9 days). Improved contact between the enzyme and the substrates would probably increase the reaction rate.     

A fish oil-rich diet reduces vascular oxidative stress in apoE–/– mice

Abstract

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE^–/– mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O₂^.–) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22^phox expression and O₂^.–production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.