2H- and 31P-NMR studies of bilayers composed of 1-acyllysophosphatidylcholine and fatty acids

1-Palmitoyllysophosphatidylcholine has been mixed in equimolar amounts with specifically deuterated palmitic acid and the structural properties of the lipid/water phase have been studied by ²H- and ³¹P-nuclear magnetic resonance. The order profile of the free palmitic acid is very similar to that of the parent compound $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ at temperatures above the gel-to-liquid crystal phase transition. The bending of the $\text{sn-2}$ chain which is typical for diacyl lipids is not observed for the free palmitic acid. The mixture of lysolipid and palmitic acid exhibits well-defined quadrupole splittings even at temperatures below the gel-to-liquid crystal phase transition. Hence it is possible for the first time to establish an order profile in the gel-state of the lipid bilayer phase. Between carbon atoms 5 to 12 the palmitic acid chain is found to assume the extended all-trans conformation with a very small contribution from gauche defects. Towards the methyl terminal a distinct increase in the gauche probability can be noted. The motion of the phosphocholine headgroup was also studied by ²H- and ³¹P-NMR using selectively deuterated 1-palmitoyllysophosphatidylcholine. The headgroup has a considerably larger motional freedom in the mixture of lysolipid and palmitic acid than in $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$. In addition, the average headgroup conformations are also different in the two systems.     

The Uptake of 63Ni by the Diatom Phaeodactylum tricornutum

Kinetic studies of the uptake of ⁶³Ni by cultures of the diatom Phaeodactylum tricornutum Bohlin revealed that the uptake capacity of the cells depended strongly on their metabolic state. Phosphate-starved cells gave saturation-type uptake curves and had low nickel-binding capacity. This was increased markedly by the addition of phosphate to the cultures. When nickel ions were added before or together with the phosphate, the increase in nickel-binding capacity of phosphate-starved cells failed to appear more or less completely. Uptake of phosphate and formation of polyphosphate by the cells were not affected by the addition of nickel to the growth medium. The phosphate dependent synthesis of the principle involved in nickel binding was induced 15–20 h after the addition of phosphate to starved cells. Whether phosphate was directly or indirectly involved in this process could not be established.     

Spectrophotometric assay of long-chain unsaturated and hydroxy fatty acids in concentrated sulfuric acid

A spectrophotometric method has been developed for the determination of long-chain unsaturated and hydroxy fatty acids in concentrated sulfuric acid. The assay is based on the absorbance produced in the 290 to 300-nm range from their reaction with sulfuric acid at 100°C. α,β-Unsaturated aliphatic acids give absorption bands at 235–240 nm and thus can be easily differentiated from unsaturated fatty acids having the double bond(s) at positions not conjugated with the carboxyl group. A certain minimum chain length is required for full development of the absorption band at 300 nm. Position and intensity of the so-formed absorption band is independent on the position and number of the double bonds or hydroxyl groups. Carboxyl groups are not essential, as unsaturated hydrocarbons and higher alcohols likewise react with sulfuric acid to produce the absorbing species at 300 nm, providing a minimum chain length of 5 carbon atoms is present. The nature of the absorbing species at 300 nm is discussed.     

Hydroxy long-chain fatty acids in fungi

Hydroxy long-chain fatty acids occur widely in animals and plants and have important physiological activities in these eukaryotes. There are indications that these compounds are also common and important in fungi. The occurrence of hydroxy-polyunsaturated fatty acids (hydroxy-PUFAs) is of biotechnological importance, because these compounds are potentially high-value lipid products with medical applications. This review pays particular attention to the production of hydroxy-PUFAs by yeasts and other fungi. Hydroxy-PUFAs derived from lipoxygenase activity appear to be present in most fungi, while hydroxy-PUFAs from cyclooxygenase activity (i.e. prostaglandins) have mainly been implicated in the Oomycota and in yeasts from the genus Dipodascopsis. The occurrence of other hydroxy long-chain fatty acids in fungi is also discussed briefly; these include hydroxy fatty acids that are generally associated with cytochrome P-450 monooxygenase activity (i.e. terminal and sub-terminal hydroxy acids and diols derived from the corresponding epoxides) as well as 2-hydroxy-fatty acids and 3-hydroxy-fatty acids.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein, 9300, South Africa     

A collagenase assay using [3H-methyl]collagen

A new assay procedure for collagenase is presented. Highly radioactive substrate is prepared by methylation of native collagen. The ³H-labelled protein is readily attacked by bacterial as well as by mammalian collagenase and resistant to other proteinases. The sensitivity of this assay is higher than that of the enzymic methods hitherto available.     

Candida cloacae oxidation of long-chain fatty acids to dioic acids

Candida cloacae cells oxidize long-chain fatty acids to their corresponding dicarboxylic acids (dioic acids) at rates dependent on their chain length and degree of saturation. This is despite the well-known toxicity of the fatty acids. Among the saturated substrates, the oxidation is limited to lauric acid (C12). The addition of pristane (5% v/v), which acts as an inert carrier for the poorly water-soluble substrate, boosts the oxidation of lauric acid to a rate that is comparable to that of dodecane. When dissolved in pristane, myristic (C14) and palmitic (C16) acids are effective carbon sources for C. cloacae, but dioic acid production is very low. Media glucose concentration and pH also influence cell growth and productivity. After the glucose is depleted, oxidation is optimal at a low pH. A two-phase (pristane/water) reaction was tested in a 2-l stirred tank bioreactor in which growth and oxidation were separated. A 50% w/w conversion of lauric acid (10 g/l) to dodecanedioic acid was achieved. The bioreactor also alleviated poor mass transfer characteristics experienced in shake flasks.     

Peroxisomes attenuate cytotoxicity of very long-chain fatty acids

One of the major functions of peroxisomes in mammals is oxidation of very long-chain fatty acids (VLCFAs). Genetic defects in peroxisomal β-oxidation result in the accumulation of VLCFAs and lead to a variety of health problems, such as demyelination of nervous tissues. However, the mechanisms by which VLCFAs cause tissue degeneration have not been fully elucidated. Recently, we found that the addition of small amounts of isopropanol can enhance the solubility of saturated VLCFAs in an aqueous medium. In this study, we characterized the biological effect of extracellular VLCFAs in peroxisome-deficient Chinese hamster ovary (CHO) cells, neural crest-derived pheochromocytoma cells (PC12), and immortalized adult Fischer rat Schwann cells (IFRS1) using this solubilizing technique. C20:0 FA was the most toxic of the C16–C26 FAs tested in all cells. The basis of the toxicity of C20:0 FA was apoptosis and was observed at 5 μM and 30 μM in peroxisome-deficient and wild-type CHO cells, respectively. The sensitivity of wild-type CHO cells to cytotoxic C20:0 FA was enhanced in the presence of a peroxisomal β-oxidation inhibitor. Further, a positive correlation was evident between cell toxicity and the extent of intracellular accumulation of toxic FA. These results suggest that peroxisomes are pivotal in the detoxification of apoptotic VLCFAs by preventing their accumulation.     

Cardiovascular disease and long-chain omega-3 fatty acids

PURPOSE OF REVIEW: Of all known dietary factors, long-chain omega-3 fatty acids may be the most protective against death from coronary heart disease. New evidence has confirmed and refined the cardioprotective role of these fatty acids. RECENT FINDINGS: Omega-3 fatty acid supplementation reduces the risk of sudden cardiac death and death from any cause within 4 months in post-myocardial infarction patients. Evidence continues to accrue for benefits in the primary prevention of coronary heart disease and stroke, and an anti-arrhythmogenic mechanism is emerging as the most likely explanation. SUMMARY: Current evidence suggests that individuals with coronary artery disease may reduce their risk of sudden cardiac death by increasing their intake of long-chain omega-3 fatty acids by approximately 1 g per day.     

Metabolism of exogenous long-chain fatty acids by spinach leaves

When applied in liquid paraffin to the upper surface of expanding spinach leaves, [1-14C]palmitic acid was efficiently and exclusively incorporated into the sn-1 position of cellular glycerolipids, principally phosphatidylcholine and triacylglycerol. A slow transfer of fatty acids from phosphatidylcholine to chloroplast glycolipids subsequently occurred with the positional specificity of the label remaining intact. Labeled palmitate at the sn-1 position of monogalactosyldiacylglycerol was desaturated to hexadecatrienoate so that 1-[14C]hexadecatrienoyl-2-linolenoyl-3-galactosoylglycerol became the major labeled species of the lipid between 8 and 24 h. There was no evidence of deacylation/reacylation reactions modifying the acyl compositions of spinach leaf glycerolipids for at least 48 h after labeling with [1-14C]palmitic acid; even the partially prokaryotic glycerolipids remained firmly labeled at the sn-1 position. Exogenous [1-14C]stearic acid was also incorporated into the sn-1 position of MGD, presumably by the same mechanism, and was there desaturated to [14C]linolenate. Exogenous [1-14C]oleic acid was initially incorporated equally into both sn-1 and sn-2 positions of phosphatidylcholine, and was desaturated to linoleate at both positions before the label was rapidly transferred to monogalactosyldiacylglycerol. There, desaturation of linoleate to linolenate took place. Galactolipids remained equally labeled at both positions throughout the 6 days of the experiment, but label was concentrated in the 1-saturated-2-[14C]linolenoyl molecular species of phosphatidylcholine as those species with two [14C]linoleoyl residues were drawn off for monogalactolipid synthesis. Glycerolipids synthesised from exogenous [1-14C]acetate by spinach leaves were labeled equally at both the sn-1 and the sn-2 positions. These results are interpreted as providing strong support for the two-pathway scheme of glycerolipid synthesis in plants.     

Reversible inhibition of bacterial bioluminescence by long-chain fatty acids

Long-chain unsaturated fatty acids, as well as certain saturated fatty acids such as lauric acid, are inhibitors of the in vivo luminescence of wild-type strains of four species of luminous bacteria (Beneckea harveyi, Photobacterium phosphoerum, P. fischeri, andP. leiognathi) as well as the myristic acid-stimulated luminescence in the aldehyde dim mutant M17 ofB. harveyi. Based on studies with the system in vivo, the principal site of action of all the fatty acids appears to be the reductase activity that converts myristic acid to myristyl aldehyde. This was confirmed by in vitro studies: Reductase activity in crude cell-free extracts is strongly inhibited by oleic acid.     

Production of long-chain hydroxy fatty acids by microbial conversion

Hydroxy fatty acids (HFAs) are very important chemicals for versatile applications in biodegradable polymer materials and cosmetic and pharmaceutical industries. They are difficult to be synthesized via chemical routes due to the inertness of the fatty acyl chain. In contrast, these fatty acids make up a major class of natural products widespread among bacteria, yeasts, and fungi. A number of microorganisms capable of producing HFAs from fatty acids or vegetable oils have been reported. Therefore, HFAs could be produced by biotechnological strategies, especially by microbial conversion processes. Microorganisms could oxidize fatty acids either at the terminal carbon or inside the acyl chain to produce various HFAs, including α-HFAs, β-HFAs, mid-position HFAs, ω-HFAs, di-HFAs, and tri-HFAs. The enzymes and their encoded genes responsible for the hydroxylation of the carbon chain have been identified and characterized during the past few years. The involved microbes and catalytic mechanisms for the production of different types of HFAs are systematically demonstrated in this review. It provides a better view of HFA biosynthesis and lays the foundation for further industrial production.     

Binding of long-chain fatty acids to bovine serum albumin

We have studied the binding of long-chain free fatty acids (FFA) to crystalline bovine serum albumin (BSA) that had been extracted with charcoal to remove endogenous fatty acids. The data were analyzed in terms of a model consisting of six high-energy binding sites and a large number of weak binding sites. The high-energy sites were resolved into two distinct classes, each containing three sites. At 37 degrees C and pH 7.4, k'(1) (the apparent association constant of a class of binding sites) was about 10(6) m(-1) for binding to the three primary sites, and k'(2) was about 10(5) m(-1) for binding to the three secondary sites. The number of weak (tertiary) sites was estimated to be 63 with a k'(3) of 10(3) m(-1). In general, palmitate and palmitoleate were bound more tightly than oleate, linoleate, stearate, or myristate, and much more tightly than laurate. The association of palmitate with human and rabbit albumin also was analyzed in terms of this model. Palmitate was bound less firmly by human or rabbit albumin than by BSA. Palmitate binding to BSA was dependent upon the pH and temperature of the incubation medium. Long-chain hydrocarbons that did not contain a free carboxyl group (methyl palmitate, cetyl alcohol, and hexadecane) were bound to a limited extent and weakly. The presence of positively charged protein sites and native protein tertiary structure were required for maximal binding of palmitate to BSA. Of nine other proteins tested, only -lactoglobulin exhibited a significant capacity to bind palmitate.     

Enzymes for transgenic biosynthesis of long-chain polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are important for the normal development and function of all organisms, and are essential in maintaining human health. Impaired PUFA metabolism is thought to be associated with pathogenesis of many chronic diseases. Dietary supplementation of PUFAs, such as gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which bypass the defective or dysfunctional steps of the biosynthetic pathway has been found to significantly alleviate the symptoms of the disease. These findings have drawn a great deal of interest from general public and food manufacturers. As the demand of these beneficial PUFAs has drastically increased in recent years, there are also increasing efforts in finding the alternate sources of PUFAs that are more economical and sustainable. One option is to modify the oil-seed crops to produce PUFAs through genetic engineering technique. This review examines the isolation, identification and expression of genes encoding the enzymes required for the biosynthesis of the above mentioned PUFAs in plants.