The effect of mercury and cadmium on the fatty acid and sterol composition of the marine diatom Asterionella glacialis

Cells of the marine diatom Asterionella glacialis treated with the organomercurial p-chloromercuribenzoate (PCMB) and cadmium, at growth retarding concentrations, exhibit decreased total fatty acid, polyunsaturated fatty acid and sterol contents. The level of individual fatty acids and sterols was also affected by metal treatment with significant decreases in the major polyunsaturated fatty acids 20:5Δ5,8,11,14,17, 16:1Δ9 and 16:3Δ3,6,9 in PCMB-treated, and 20:5Δ5,8,11,14,17 in cadmium-treated cells; increased cholest-5-en-3β-ol, particularly in PCMB-treated cells; and a decrease in the ratio of 24-ethylcholest-5-en-3β-ol to 24-ethylcholesta-5,24(28)Z-dien-3β-ol which was most notable in cadmium-treated cells. These results can be explained in terms of the formation of mercury and cadmium complexes with thiol-containing enzymes involved in lipid biosynthesis and metabolism, and thus provide further support for the hypothesis that transition metal toxicity is mediated by metal inactivation of physiologically essential, thiol-containing enzymes and co-factors.     

Caenorhabditis elegans Delta12-desaturase FAT-2 is a bifunctional desaturase able to desaturate a diverse range of fatty acid substrates at the Delta12 and Delta15 positions

Caenorhabditis elegans FAT-2 has been characterized as fatty acid Δ12-desaturase able to desaturate C16 and C18 fatty acids. However, in this report we show that when expressed in yeast cells this enzyme can also catalyze Δ15 desaturation. This results in the production of both linoleic acid (ω6 C18:2Δ9,12) and linolenic acid (ω3 C18:3Δ9,12,15) from oleic acid (C18:1Δ9) substrate, and hexadecadienoic acid (ω4 C16:2Δ9,12) and hexadecatrienoic acid (ω1 C16:3Δ9,12,15) from palmitoleic acid (C16:1Δ9) substrate. In addition, this enzyme can also produce C14:2Δ9,12, C15:2Δ9,12, C17:2Δ9,12, and C18:4Δ6,9,12,15 when C14:1Δ9, C15:1Δ9, C17:1Δ9, and C18:3Δ6,9,12 substrates are available in yeast cells. Mass spectrometry analysis of 2,4-dimethyloxazoline modification of fatty acid methyl esters confirms the positions of all newly formed double bonds. These results indicate that when expressed in yeast the C. elegans Δ12-desaturase CeFAT-2 shows a characteristic of a bifunctional Δ12/Δ15-desaturase and has a great deal of elasticity with respect to fatty acid chain length in being able to accept fatty acids ranging from C14 to C18. Interestingly, despite possessing a bifunctional Δ12/Δ15 desaturation activity, phylogenetic analysis suggests that C. elegans Δ12-desaturase CeFAT-2 might have arisen independently from other reported dual Δ12/Δ15-desaturases from fungi and protozoa.     

Effect of fatty acids on growth of Japanese encephalitis virus cultivated in BHK-21 cells and phospholipid metabolism of the infected cells.

Growth of Japanese encephalitis virus (JEV) in BHK-21 cells was stimulated in the presence of 20 to 40 mug of the sodium salt of oleic acid (cis-9-octadecenoic acid, 9-18:1) per ml supplemented in Waymouth medium. The stimulatory effect of the salt was highest when 9-18:1 was added after adsorption of the virus. Study of the effect of other fatty acids on growth of JEV showed the following results: the longer the chain length of the saturated fatty acid salt, the higher the stimulatory effect on viral growth. In contrast, polyunsaturated fatty acids had an inhibitory effect on viral growth. The effect of isomeric cis-octadecenoic acids on viral growth was variable, depending upon the position of the double bond. The cis-6-octadecenoic acid had the highest inhibitory effect on growth of JEV compared to other isomeric octadecenoic acids. The sodium salt of (1-14C) cis-9-octadecenoic acid (9-18:1, 20 mug/ml) was rapidly incorporated into control and JEV-infected cells. Specific radioactivity in phosphatidylcholine dropped 12 to 24 h after virus inoculation, whereas synthesis of phosphatidylethanolamine increased 12 to 24 h after virus inoculation in infected cells compared to uninfected cells. Results from these studies suggest that phospholipid metabolism of infected cells is markedly changed, which can be associated with altered fatty acid metabolism when using labeled 9-18:1 fatty acid as a marker.     

Defects in very long chain fatty acid synthesis enhance alpha-synuclein toxicity in a yeast model of Parkinson's disease

We identified three S. cerevisiae lipid elongase null mutants (elo1Δ, elo2Δ, and elo3Δ) that enhance the toxicity of alpha-synuclein (α-syn). These elongases function in the endoplasmic reticulum (ER) to catalyze the elongation of medium chain fatty acids to very long chain fatty acids, which is a component of sphingolipids. Without α-syn expression, the various elo mutants showed no growth defects, no reactive oxygen species (ROS) accumulation, and a modest decrease in survival of aged cells compared to wild-type cells. With (WT, A53T or E46K) α-syn expression, the various elo mutants exhibited severe growth defects (although A30P had a negligible effect on growth), ROS accumulation, aberrant protein trafficking, and a dramatic decrease in survival of aged cells compared to wild-type cells. Inhibitors of ceramide synthesis, myriocin and FB1, were extremely toxic to wild-type yeast cells expressing (WT, A53T, or E46K) α-syn but much less toxic to cells expressing A30P. The elongase mutants and ceramide synthesis inhibitors enhance the toxicity of WT α-syn, A53T and E46K, which transit through the ER, but have a negligible effect on A30P, which does not transit through the ER. Disruption of ceramide-sphingolipid homeostasis in the ER dramatically enhances the toxicity of α-syn (WT, A53T, and E46K).     

Expression of yeast acyl-CoA-∆9 desaturase leads to accumulation of unusual monounsaturated fatty acids in soybean seeds

An acyl-CoA-Δ9 desaturase from Saccharomyces cerevisiae was expressed by subcellular-targeting in soybean (Glycine max) seeds with the goal of increasing palmitoleic acid (16:1Δ9), a high-valued fatty acid (FA), and simultaneously decreasing saturated FA in oil. The expression resulted in the conversion of palmitic acid (16:0) to 16:1Δ9 in soybean seeds. 16:1Δ9 and its elongation product cis-vaccenic acid (18:1Δ11) were increased to 17 % of the total fatty acids by plastid-targeted expression of the enzyme. Other lipid changes include the decrease of polyunsaturated FA and saturated FA, suggesting that a mechanism exists downstream in oil biosynthesis to compensate the FA alternation. This is the first time a cytosolic acyl-CoA-?9 desaturase is functionally expressed in plastid and stronger activity was achieved than its cytosolic expression. The present study provides a new strategy for converting 16:0 to 16:1Δ9 by engineering acyl-CoA-Δ9 desaturase in commercialized oilseeds.     

Growth inhibition of Monodus subterraneus by free fatty acids

Monodus subterraneus is a microalga, which is known for its high eicosapentaenoic acid (EPA; 20:5omega3) content. To produce EPA commercially, high volumetric productivities of microalgae are required. These high productivities can be reached in flat panel photobioreactors with small optical paths that have to be operated at high cell densities (>10 g/L). However, at these cell densities a reduction of productivity is observed. This growth inhibition is probably caused by growth inhibitors released by the microalgae, which have been suggested to be fatty acids. Our aim was to investigate if free fatty acids produced by M. subterraneus inhibited growth of this species. Therefore a bioassay was developed and saturated, unsaturated and poly-unsaturated fatty acids occurring in Monodus were tested on their growth inhibiting properties. Growth of M. subterraneus was completely inhibited at a saturated concentration (96 microM) of palmitoleic acid (16:1omega7). But, the saturated fatty acid palmitic acid (16:0) and the mono-saturated oleic acid (18:1omega9) were much stronger inhibitors. Growth was inhibited for 50% already at concentrations of 0.4 microM 16:0 and 3 microM 18:1omega9, respectively. These fatty acids probably cause the growth inhibition in high cell density cultures of M. subterraneus.     

Fatty acid utilisation and metabolism in caecal enterocytes of rainbow trout (Oncorhynchus mykiss) fed dietary fish or copepod oil

A combined fatty acid metabolism assay was employed to determine fatty acid uptake and relative utilisation in enterocytes isolated from the pyloric caeca of rainbow trout. In addition, the effect of a diet high in long-chain monoenoic fatty alcohols present as wax esters in oil derived from Calanus finmarchicus, compared to a standard fish oil diet, on caecal enterocyte fatty acid metabolism was investigated. The diets were fed for 8 weeks before caecal enterocytes from each dietary group were isolated and incubated with [1-14C]fatty acids: 16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:1n-9, 20:4n-6, 20:5n-3, and 22:6n-3. Uptake was measured over 2 h with relative utilisation of different [1-14C]fatty acids calculated as a percentage of uptake. Differences in uptake were observed, with 18:1n-9 and 18:2n-6 showing the highest rates. Esterification into cellular lipids was highest with 16:0 and C18 fatty acids, accounting for over one-third of total uptake, through predominant incorporation in triacylglycerol (TAG). The overall utilisation of fatty acids in phospholipid synthesis was low, but highest with 16:0, the most prevalent fatty acid recovered in intracellular phosphatidylcholine (PC) and phosphatidylinositol (PI), although exported PC exhibited higher proportions of C20/C22 polyunsaturated fatty acids (PUFA). Other than 16:0, incorporation into PC and PI was highest with C20/C22 PUFA and 20:4n-6 respectively. Recovery of labelled 18:1n-9 in exported TAG was 3-fold greater than any other fatty acid which could be due to multiple esterification on the glycerol 'backbone' and/or increased export. Approximately 20-40% of fatty acids taken up were beta-oxidised, and was highest with 20:4n-6. Oxidation of 20:5n-3 and 22:6n-3 was also surprisingly high, although 22:6n-3 oxidation was mainly attributed to retroconversion to 20:5n-3. Metabolic modification of fatty acids by elongation-desaturation was generally low at <10% of [1-14C]fatty acid uptake. Dietary copepod oil had generally little effect on fatty acid metabolism in enterocytes, although it stimulated the elongation and desaturation of 16:0 and elongation of 18:1n-9, with radioactivity recovered in longer n-9 monoenes. The monoenoic fatty acid, 20:1n-9, abundant in copepod oil as the homologous alcohol, was poorly utilised with 80% of uptake remaining unesterified in the enterocyte. However, the fatty acid composition of pyloric caeca was not influenced by dietary copepod oil.     

The effect of two isomeric octadecenoic acids on the lipid metabolism and growth of Novikoff hepatoma cells.

The origin and metabolism of octadecenoic acid (18 : 1) was examined in intact Novikoff rat hepatoma cells by using labeled precursors and two isomeric octadecenoic acids which differed in their abilities to stimulate cell growth in a serum-free medium. The isomers (ci-6-18 : 1 and cis-9-18 : 1) were measured in the cellular lipid by ozonolysis and reduction of the ozonides. The results indicate that the 18 : 1 fatty acid accumulated in the cell lipid by uptake of the preformed acid from the medium. The cis-9-18 : 1 to 16 : 1 and 20 : 1 fatty acids by chain shortening and chain elongation. Both isomers inhibited de novo fatty acid synthesis from acetate by cells suspended in a serum-free medium. The isomers did not exert coordinate control of both fatty acid and cholesterol biosynthesis in the Novikoff cells.     

An innate bactericidal oleic acid effective against skin infection of methicillin-resistant Staphylococcus aureus: a therapy concordant with evolutionary medicine

Free fatty acids (FFAs) are known to have bacteriocidal activity and are important components of the innate immune system. Many FFAs are naturally present in human and animal skin, breast milk, and in the bloodstream. Here, the therapeutic potential of FFAs against methicillin-resistant Staphylococcus aureus (MRSA) is demonstrated in cultures and in mice. Among a series of FFAs, only oleic acid (OA) (C18:1, cis-9) can effectively eliminate Staphylococcus aureus (S. aureus) through cell wall disruption. Lauric acid (LA, C12:0) and palmitic acid (PA, C16:0) do not have this ability. OA can inhibit growth of a number of Gram-positive bacteria, including hospital and community-associated MRSA at a dose that did not show any toxicity to human sebocytes. The bacteriocidal activities of FFAs were also demonstrated in vivo through injection of OA into mouse skin lesions previously infected with a strain of MRSA. In conclusion, our results suggest a promising therapeutic approach against MRSA through boosting the bacteriocidal activities of native FFAs, which may have been co-evolved during the interactions between microbes and their hosts.     

Two Fatty Acid Elongases Possessing C18-Δ6/C18-Δ9/C20-Δ5 or C16-Δ9 Elongase Activity in Thraustochytrium sp. ATCC 26185

Thraustochytrids, unicellular eukaryotic marine protists, accumulate polyunsaturated fatty acids. Here, we report the molecular cloning and functional characterization of two fatty acid elongase genes (designated tselo1 and tselo2), which could be involved in the desaturase/elongase (standard) pathway in Thraustochytrium sp. ATCC 26185. TsELO1, the product of tselo1 and classified into a Δ6 elongase group by phylogenetic analysis, showed strong C18-Δ6 elongase activity and relatively weak C18-Δ9 and C20-Δ5 activities when expressed in the budding yeast Saccharomyces cerevisiae. TsELO2, classified into a Δ9 elongase subgroup, showed only C16-Δ9 activity. When expressed in Aurantiochytrium limacinum mh0186 using a thraustochytrid-derived promoter and a terminator, TsELO1 exhibited almost the same specificity as expressed in the yeast but TsELO2 showed weak C18-Δ9 activity, in addition to its main C16-Δ9 activity. These results suggest that TsELO1 functions not only as a C18-Δ6 and a C20-Δ5 elongase in the main route but also as a C18-Δ9 elongase in the alternative route of standard pathway, while TsELO2 functions mainly as a C16-Δ9 elongase generating vaccenic acid (C18:1n?7) in thraustochytrids. This is the first report describing a fatty acid elongase harboring C16-Δ9 activity in thraustochytrids.     

Fatty acid profiling of Chlamydomonas reinhardtii under nitrogen deprivation

The Chlamydomonas reinhardtii starch-less mutant, BAF-J5, was found to store lipids up to 65% of dry cell weight when grown photoheterotrophically and subjected to nitrogen starvation. Fourier transform infrared spectroscopy was used as a high-throughput method for semi-quantitative measurements of protein, carbohydrate and lipid content. The fatty acids of wild-type and starch mutants were identified and quantified by gas chromatography mass spectrometry. C. reinhardtii starch mutants, BAF-J5 and I7, produce significantly elevated levels of 16:0, 18:1(Δ9), 18:2(Δ9,12) and 18:3(Δ9,12,15) fatty acids. Long-chain saturated, mono- and polyunsaturated fatty acids were found under nitrogen starvation. Oleosin-like and caleosin-like genes were identified in the C. reinhardtii genome. However, proteomic analysis of isolated lipid bodies only identified a key lipid droplet associated protein. This study shows it is possible to manipulate algal biosynthetic pathways to produce high levels of lipid that may be suitable for conversion to liquid fuels.     

Alterations of cell wall structure and metabolism accompany reduced susceptibility to vancomycin in an isogenic series of clinical isolates of Staphylococcus aureus

A series of isogenic methicillin-resistant Staphylococcus aureus isolates recovered from a bacteremic patient were shown to acquire gradually increasing levels of resistance to vancomycin during chemotherapy with the drug (K. Sieradzki, T. Leski, L. Borio, J. Dick, and A. Tomasz, J. Clin. Microbiol. 41:1687-1693, 2003). We compared properties of the earliest (parental) vancomycin-susceptible isolate, JH1 (MIC, 1 microg/ml), to two late (progeny) isolates, JH9 and JH14 (vancomycin MIC, 8 microg/ml). The resistant isolates produced abnormally thick cell walls and poorly separated cells when grown in antibiotic-free medium. Chemical analysis of the resistant isolates showed decreased cross-linkage of the peptidoglycan and drastically reduced levels of PBP4 as determined by the fluorographic assay. Resistant isolates showed reduced rates of cell wall turnover and autolysis. In vitro hydrolysis of resistant cell walls by autolytic extracts prepared from either susceptible or resistant strains was also slow, and this abnormality could be traced to a quantitative (or qualitative) change in the wall teichoic acid component of resistant isolates. Some change in the structure and/or metabolism of teichoic acids appears to be an important component of the mechanism of decreased susceptibility to vancomycin in S. aureus.     

Adaptation of anaerobically grown Thauera aromatica,Geobacter sulfurreducens and Desulfococcus multivorans to organic solvents on the level of membrane fatty acid composition

The effect of different solvents and pollutants on the cellular fatty acid composition of three bacterial strains: Thauera aromatica, Geobacter sulfurreducens and Desulfococcus multivorans, representatives of diverse predominant anaerobic metabolisms was investigated. As the prevailing adaptive mechanism in cells of T. aromatica and G. sulfurreducens whose cellular fatty acids patterns were dominated by palmitic acid (C16:0) and palmitoleic acid (C16:1cis), the cells reacted by an increase in the degree of saturation of their membrane fatty acids when grown in the presence of sublethal concentrations of the chemicals. Next to palmitic acid C16:0, the fatty acid pattern of D. multivorans was dominated by anteiso-branched fatty acids which are characteristic for several sulfate-reducing bacteria. The cells responded to the solvents with an increase in the ratio of straight-chain saturated (C14:0, C16:0, C18:0) to anteiso-branched fatty acids (C15:0anteiso, C17:0anteiso, C17:1anteisoΔ9cis). The results show that anaerobic bacteria react with similar mechanisms like aerobic bacteria in order to adapt their membrane to toxic organic solvents. The observed adaptive modifications on the level of membrane fatty acid composition can only be carried out with de novo synthesis of the fatty acids which is strictly related to cell growth. As the growth rates of anaerobic bacteria are generally much lower than in the so far investigated aerobic bacteria, this adaptive response needs more time in anaerobic bacteria. This might be one explanation for the previously observed higher sensitivity of anaerobic bacteria when compared with aerobic ones.     

Effect of feeding and lack of food on the growth, gross biochemical and fatty acid composition of juvenile catfish

The effect of feeding and starvation on growth, gross body composition and fatty acid composition of body muscle and liver were investigated in juvenile African catfish ( Clarias gariepinus ). The relative composition of fatty acids was influenced by starvation. In both liver and muscle there were marked relative decreases of 14: 0, 16: 1n9 and 18: 1n9 fatty acids and relative increases of 20: 5n3 and 22: 6n3 when these were compared to the controls. Preference of utilization of fatty acids from liver and body muscle during starvation was highest for 14: 0>16: 1n9>18: 1n9 and conservation highest for 22: 6n3>20: 5n3. The changes of these fatty acids were smaller in muscle than in the liver.     

An unsaturated fatty acid mutant of Aspergillus niger with partially defective delta 9-desaturase

The wild-type Aspergillus niger (V35) does not require fatty acids for growth. Four unsaturated fatty acid auxotrophs designated as UFA1, UFA2, UFA3, and UFA4 have been produced from this organism by treating the conidia of the wild-type strain with a mutagen, N-methyl-N'-nitro-N-nitrosoguanidine, followed by isolation on media containing monounsaturated fatty acids and the nonionic detergent, Brij 58. Optimal growth of the mutants comparable with that of the wild type was achieved with medium supplemented with C16 or C18 unsaturated fatty acids containing at least one cis double bond at the delta 9 position. Some other fatty acids (18:1 delta 11 cis and 16:1 delta 9 trans) support growth to some extent. The mutants do not grow at all in the presence of saturated fatty acids. Fatty acid analyses of the mutant, UFA2, grown in the presence of different fatty acid supplements reveal that it may be defective in a desaturase system. Experiments with unlabeled and [1-14C]palmitoyl-CoA have shown that the microsomes of the mutant (UFA2) contain a partially defective delta 9-desaturase system.     

Lipids of the antarctic sea ice diatom Nitzschia cylindrus

The sterol and neutral, glyco- and phospholipid fatty acid profiles of the sea ice diatom Nitzschia cylindrus, isolated from McMurdo Sound, Antarctica, are reported. Two sterols were detected, trans-22-dehydrocholesterol (66% of total sterols) and cholesterol (34%); no sterols containing alkyl groups at the C₂₄ position were present. The major fatty acids in N. cylindrus, 16:1Δ9c, 14:0, 16:0, 20:5Δ5,8,11,14,17 and 20:4Δ5,8,11,14, were typical of previous reports of diatom fatty acids. A number of long-chain monounsaturated fatty acids were also detected, with higher relative proportions present in the phospholipid fraction. GC-MS analysis of the dimethyldisulphide adducts of these monounsaturated components showed that 24: 1Δ13c, 24:1Δ15c, 26:1Δ15c and 26:1Δ17c were the major components. The distribution of these fatty acids suggests that chain elongation of monounsaturated fatty acids was occurring in N. cylindrus. The proposed chain lengthening occurring for N. cylindrus represents, to our knowledge, the first report of possible chain lengthening of monounsaturated fatty acids in microscopic algae. These features, the presence of long-chain monounsaturated fatty acids and the sterol profile, may allow the input of this alga into benthic marine sediments or food webs to be monitored.     

培养条件对海洋假单胞菌脂肪酸的影响

研究了不同温度条件下一株海绵附生假单胞菌(Pseudomonassp.)在不同碳源培养基中的生长情况及脂肪酸变化.结果表明,该海洋菌生长最适温度为30℃,在以淀粉作为外加碳源的培养基中生长最好;实验菌含13种脂肪酸,主要是c16:1(n7)、c15:0、c16:0、c17:0、c18:1(n6)、c18:1(n9)、9,10cp c17:0和其同分异构体.在30℃温度条件下,不饱和脂肪酸的相对含量急剧减少.在有外加碳源(葡萄糖和淀粉)的培养基中生长的细菌,奇数脂肪酸和环丙基脂肪酸含量远比未外加碳源的低.聚类分析结果表明,两种环境因子中,温度比碳源的影响更明显.     

Interaction of acridine orange with teichoic acids of Staphylococcus aureus]

Teichoic acids of the cell wall of Staphylococcus aureus 209-P belong to the class of ribitol teichoic acids and can bind actively the molecules or acridine orange, changing its optical properties. The intravital fluorochromy of the cells with acridine orange may be caused by the sorption of its molecules by the teichoic acids of the cell wall.