Determination of lipopolysaccharide by a bioluminescence technique.

The determination of the lipid A content of bacterial lipopolysaccharide by using a dim mutant of the luminous bacterium Beneckea harveyi is described. The luminous bacteria emitted light upon the addition of an acid hydrolysate of lipopolysaccharide which contained myristic acid, thus making it possible to detect as little as 1 ng of lipopolysaccharide. By converting the 3-OH-myristic acid to myristic acid, it was possible to further increase the detection sensitivity and to establish a basis for a specific and highly sensitive bioassay for the detection of lipopolysaccharide.     

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.     

In rat hepatocytes, myristic acid occurs through lipogenesis, palmitic acid shortening and lauric acid elongation

The origin of myristic acid in mammalian cells and the regulation of its endogenous cellular low concentration are not known. Another intriguing question is the potential metabolic properties of endogenous myristic acid as compared with exogenous myristic acid. In the present paper, we hypothesised and demonstrated that, in liver cells, in addition to the usual fatty acid synthase (FAS) pathway that produces predominantly palmitic acid and minor amounts of myristic acid, part of endogenous cellular myristic acid also comes from a shortening of palmitic acid, likely by peroxisomal β-oxidation and from lauric acid by elongation. From a nutritional point of view, C16:0 is universally found in natural fats and its shortening to myristic acid could contribute to a non-negligible source of this fatty acid (FA) in the organism. Then, we measured the distribution of endogenously synthesised myristic acid in lipid species and compared it with that of exogenous myristic acid. Our results do not support the hypothesis of different metabolic fates of endogenous and exogenous myristic acid and suggest that whatever the origin of myristic acid, its cellular concentration and lipid distribution are highly regulated.     

A new,fast, and very sensitive bioluminescence assay for phospholipases A and C

A new, simple, and very sensitive assay for phospholipase A and C is described. The assay is based on the bioluminescence developed by the mutant of the bacterium Beneckea harveyi as a response to myristic acid released from dimyristoyl phosphatidylcholine by either phospholipase A or by a phospholipase C-lipase coupled system. It is possible to assay these enzymes at a rate corresponding to a release of as little as 1 to 2 pmol of myristic acid per minute.     

In vitro deacylation of lipopolysaccharide of Salmonella minnesota by Acanthamoeba castellanii enzymes

Enzymatic deacylation of the lipopolysaccharide isolated from a Salmonella Rd mutant by a cell-free preparation from Acanthamoeba castellanii has been studied. The degradation was found to be dependent on the presence of a surface-active component (Triton X-100) in the reaction mixture. The lipid A part of the lipopolysaccharide was the primary target of the enzymes, which cleaved with high efficiency the ester-bound long-chain nonhydroxylated and 3-hydroxylated acyl residues, i.e. lauric, myristic, palmitic and 3-hydroxymyristic acid. The cell-free preparation also exhibited amidase activity cleaving about 50% of the amide-bound 3-hydroxymyristic acid residues. In addition the extract proved to possess phosphatase activity liberating ester-bound and glycosidically bound phosphate groups of lipid A. On the other hand, the glucosaminyl-beta 1,6-glucosamine disaccharide was not degraded and remained bound to the oligosaccharide part (heptose/3-deoxyoctulosonic acid) of the lipopolysaccharide.     

The evaluation of the role of beta-hydroxy fatty acids on chronic inflammation and insulin resistance

Beta-hydroxy fatty acids are a major component of lipid A moiety of lipopolysaccharide. We aimed to investigate the role of free beta-hydroxy fatty acids on inflammation, as well as to evaluate their effects on cytokine release from human blood cells, and whether they exist in plasma of patients with chronic inflammatory diseases with/without insulin resistance. Peripheral venous blood was incubated with beta-hydroxy lauric and beta-hydroxy myristic acids (each 100 ng, 1 microg, 10 microg/mL) up to 24 hours. Cytokines were measured from culture media and plasma. Free fatty acids and biochemical parameters were also measured from patients' plasma. Only beta-hydroxy lauric acid significantly stimulated interleukin-6 production at 10 microg/mL compared to control (533.9 +/- 218.1 versus 438.3 +/- 219.6 pg/mL, P < .05). However, free beta-hydroxy lauric and myristic acids were not found in patients' plasma. Therefore, free beta-hydroxy lauric and myristic acids do not seem to have a role on sterile inflammation in chronic inflammatory diseases associated with insulin resistance.     

The structure of the lipid A component of Sphaerotilus natans

The lipopolysaccharide of Sphaerotilus natans afforded a ladder-like pattern of bands in sodium deoxycholate-polyacrylamide gel electrophoresis, indicating the presence of a S-form lipopolysaccharide. The chemical analysis showed neutral sugars (rhamnose, glucose, l-glycero-d-manno-heptose), 3-deoxy-octulosonic acid (Kdo), amino compounds (glucosamine, glucosamine phosphate, ethanolamine and ethanolamine phosphate), and phosphorus. The lipid A fraction contained saturated and unsaturated capric, lauric, and myristic acids, and 3-hydroxy capric acid (3-OH-10:0). Its chemical structure was consisting of a glucosamine disaccharide, glycosidically substituted by a phosphomonoester, and substituted at C-4 by a pyrophosphodiester esterified with ethanolamine. The amino groups of both glucosamines are acylated by 3-hydroxy capric acids and these in turn are substituted by saturated and unsaturated capric, lauric, and myristic acids. Hydroxyl groups of the backbone disaccharide at C-3 and C-3 were also esterified by 3-hydroxy capric acid, those at C-4 and C-6 were unsubstituted. The latter provides the attachment site for Kdo.Abbreviations Kdo 3-deoxy-d-manno-octulosonic acid - 3-OH-10:0 3-hydroxy capric acid - DOC-PAGE deoxycholate-polyacrylamide gel electrophoresis - GC-MS gas chromatography/mass spectrometry - LD-MS laser desorption mass spectrometry - LPS lipopolysaccharide - PS polysaccharide     

Myristic acid specifically stabilizes diacylglycerol kinase δ protein in C2C12 skeletal muscle cells

Decreased levels of the δ isozyme of diacylglycerol kinase (DGK) in skeletal muscle attenuate glucose uptake and, consequently, are critical for the pathogenesis of type 2 diabetes. We recently found that free myristic acid (14:0), but not free palmitic acid (16:0), increased the DGKδ protein levels and enhanced glucose uptake in C2C12 myotube cells. However, it has been unclear how myristic acid regulates the level of DGKδ2 protein. In the present study, we characterized the myristic acid-dependent increase of DGKδ protein. A cycloheximide chase assay demonstrated that myristic acid, but not palmitic acid, markedly stabilized DGKδ protein. Moreover, other DGK isozymes, DGKη and ζ, as well as glucose uptake-related proteins, such as protein kinase C (PKC) α, PKCζ, Akt and glycogen synthase kinase 3β, failed to be stabilized by myristic acid. Furthermore, DGKδ was not stabilized in cultured hepatocellular carcinoma cells, pancreas carcinoma cells or neuroblastoma cells, and only a moderate stabilizing effect was observed in embryonic kidney cells. A proteasome inhibitor and a lysosome inhibitor, MG132 and chloroquine, respectively, partly inhibited DGKδ degradation, suggesting that myristic acid prevents, at least in part, the degradation of DGKδ by the ubiquitin-proteasome system and the autophagy-lysosome pathway. Overall, these results strongly suggest that myristic acid attenuates DGKδ protein degradation in skeletal muscle cells and that this attenuation is fatty acid-, protein- and cell line-specific. These new findings provide novel insights into the molecular mechanisms of the pathogenesis of type 2 diabetes mellitus.     

Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues

To establish a non-radioactive, cell-free detection system for protein N-myristoylation, metabolic labeling in a cell-free protein synthesis system using bioorthogonal myristic acid analogues was performed. After Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) with a biotin tag, the tagged proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted on a polyvinylidene fluoride (PVDF) membrane, and then protein N-myristoylation was detected by enhanced chemiluminescence (ECL) using horseradish peroxidase (HRP)-conjugated streptavidin. The results showed that metabolic labeling in an insect cell-free protein synthesis system using an azide analogue of myristic acid followed by CuAAC with alkynyl biotin was the most effective strategy for cell-free detection of protein N-myristoylation. To determine whether the newly developed detection method can be applied for the detection of novel N-myristoylated proteins from complementary DNA (cDNA) resources, four candidate cDNA clones were selected from a human cDNA resource and their susceptibility to protein N-myristoylation was evaluated using the newly developed strategy. As a result, the products of three cDNA clones were found to be novel N-myristoylated protein, and myristoylation-dependent specific intracellular localization was observed for two novel N-myristoylated proteins. Thus, the metabolic labeling in an insect cell-free protein synthesis system using bioorthogonal azide analogue of myristic acid was an effective strategy to identify novel N-myristoylated proteins from cDNA resources.     

Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog.

The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.     

Regulation of mammalian desaturases by myristic acid: N-terminal myristoylation and other modulations

Myristic acid, the 14-carbon saturated fatty acid (C14:0), usually accounts for small amounts (0.5%-1% weight of total fatty acids) in animal tissues. Since it is a relatively rare molecule in the cells, the specific properties and functional roles of myristic acid have not been fully studied and described. Like other dietary saturated fatty acids (palmitic acid, lauric acid), this fatty acid is usually associated with negative consequences for human health. Indeed, in industrialized countries, its excessive consumption correlates with an increase in plasma cholesterol and mortality due to cardiovascular diseases. Nevertheless, one feature of myristoyl-CoA is its ability to be covalently linked to the N-terminal glycine residue of eukaryotic and viral proteins. This reaction is called N-terminal myristoylation. Through the myristoylation of hundreds of substrate proteins, myristic acid can activate many physiological pathways. This review deals with these potentially activated pathways. It focuses on the following emerging findings on the biological ability of myristic acid to regulate the activity of mammalian desaturases: (i) recent findings have described it as a regulator of the Δ4-desaturation of dihydroceramide to ceramide; (ii) studies have demonstrated that it is an activator of the Δ6-desaturation of polyunsaturated fatty acids; and (iii) myristic acid itself is a substrate of some fatty acid desaturases. This article discusses several topics, such as the myristoylation of the dihydroceramide Δ4-desaturase, the myristoylation of the NADH-cytochrome b5 reductase which is part of the whole desaturase complex, and other putative mechanisms.     

Acylation of Rat Brain Myelin Proteolipid Protein with Different Fatty Acids

The acylation of rat brain proteolipid protein (PLP) with tritiated palmitic, oleic, and myristic acids was studied in vivo and in vitro and compared with the acylation of lipids. Twenty-four hours after intracranial injection of [3H]myristic acid, only 16% of the PLP-bound label appeared as myristic acid, with 66% as palmitic, 9% as stearic, and 6% as oleic acid, whereas greater than 63% of the label in total or myelin phospholipid was in the form of myristic acid. In contrast, after labelling with [3H]palmitic or oleic acids, 75% and 86%, respectively, of the radioactivity in PLP remained in the original form. When brain tissue slices were incubated for short periods of time, the incorporation of palmitic and oleic acids into PLP exceeded that of myristic acid by a factor of 8. In both systems and with all precursors studied, the label associated with PLP was shown to be in ester linkage. The results suggest a preferential acylation of PLP with palmitic and oleic acids as compared with myristic acid. This is consistent with the fatty acid composition of the isolated PLP.     

Dietary myristic acid at physiologically relevant levels increases the tissue content of C20:5 n-3 and C20:3 n-6 in the rat

This study was designed to investigate the effect of myristic acid on the biosynthesis and metabolism of highly unsaturated fatty acids, when it is supplied in a narrow physiological range in the diet of the rat (0.2-1.2% of total dietary energy). Three experimental diets were designed, containing 22% of total dietary energy as lipids and increasing doses of myristic acid (0.71, 3.00 and 5.57% of total fatty acids). Saturated fat did not exceed 31% of total fat and the C18:3 n-3 amount in each diet was strictly equal (1.6% of total fatty acids). After 7 weeks, the diets had no effect on plasma cholesterol level but greatly modified the liver, plasma and adipose tissue saturated, monounsaturated and polyunsaturated fatty acid profiles. Firstly, daily intakes of myristic acid resulted in a dose-dependent tissue accumulation of myristic acid itself. Palmitic acid was significantly increased in the tissues of the rats fed the higher dose of myristic acid. A dose-response accumulation of tissue C16:1 n-7 as a function of dietary C14:0 was also shown. Secondly, a main finding was that, among n-3 and n-6 polyunsaturated fatty acids, a dose-response accumulation of liver and plasma C20:5 n-3 and C20:3 n-6 (two precursors of eicosanoids) as a function of dietary C14:0 was shown. This result suggests that dietary myristic acid may participate in the regulation of highly unsaturated fatty acid biosynthesis and metabolism.     

A lipopolysaccharide fromAspergillus flavus conidia

A lipopolysaccharide was isolated by extraction ofAspergillus flavus conidia with 45 % phenol at 68–70 °C. Quantitative analysis revealed 7 % nucleic acids, 5.5 % proteins, 46 % polysaccharides and 49 % lipids, of which 12 % were covalently bound. Glucose, mannose, galactose and fucose were detected as monosaccharide components of the polysaccharide moiety by gas chromatography; palmitic acid, stearic acid, oleic acid, linoleic acid and myristic acid were mainly present in the lipidic fraction. This material differs from the bacterial lipopolysaccharides, both in composition of the polysaccharide moiety and representation of fatty acids in the lipidic fraction.     

Studies on the attachment of myristic and palmitic acid to cell proteins in human squamous carcinoma cell lines: evidence for two pathways.

The ability of human keratinocytes and squamous carcinoma cell lines to attach lipid covalently to cell proteins has been examined using both palmitic and myristic acids. SDS-polyacrylamide gel analyses of the proteins labelled with these lipids demonstrated that each labelled a different set of proteins. Covalently protein bound palmitic acid could be removed from the proteins by mild alkali hydrolysis but the bound myristic acid required prolonged acid hydrolysis to release it from the associated proteins. H.p.l.c. analyses of the released lipid confirmed that both lipids were attached to proteins directly and that the labelling was not due to the lipids being catabolised. Cycloheximide could prevent the attachment of myristic acid to cell proteins, but only reduced the levels of palmitic acid incorporation. Pulse chase experiments indicated that there was little turnover of the attached myristic acid whereas this was significant for covalently bound palmitic acid. These observations show for the first time that two different protein populations are labelled by different lipids in eukaryotic cells, and that there appear to be two separate pathways for the acylation of proteins in such cells.     

Single-step purification of myristoylated and nonmyristoylated recoverin and substrate dependence of myristoylation level

Recoverin is cotranslationally modified by the covalent linkage of a myristoyl group to its N terminus. It is a member of a family of Ca(2+)-myristoyl switch proteins. Recombinant myristoylated revoverin is currently produced by the cotransformation of bacteria with recoverin and an enzyme that allows N-myristoylation and by supplementing the culture medium with myristic acid. A large variation in the myristoylation level of recoverin and in the amount of myristic acid supplied to the culture medium can be found in the literature. Moreover, although it is known to strongly affect bacterial growth, the amount of ethanol used to solubilize myristic acid is only scarcely mentioned. To improve our understanding of the parameters responsible for recombinant recoverin myristoylation, the effects of myristic acid and ethanol on recoverin myristoylation and expression levels have been systematically studied. In addition, a single-step purification procedure to produce purified myristoylated and nonmyristoylated recombinant recoverin has also been devised. Finally, sodium myristate has been used as an efficient alternative substrate to achieve high myristoylation and expression levels of recoverin. Given that a large number of proteins are myristoylated, these procedures could be applied to several other proteins in addition to recoverin.     

Effect of hydroxylamine hydrochloride on lipopolysaccharide fatty acid composition in Shigella dysenteriae 1

Changes in the fatty acid composition of S. dysenteriae 1 lipid A after the treatment of lipopolysaccharide (LPS) with hydrosylamine hydrochloride (HH) and 4 degrees C, 37 degrees C and 56 degrees C were studied with the use of gas-liquid chromatographicmass-spectrometry. The treatment with HH led to a decrease in the toxicity of LPS, but produced no changes in the content of the main fatty acid components of lipid A (lauric, myristic, oxymyristic and palmitic acids). At the same time the total number of minor fatty acid derivatives decreased from 11 (in the original LPS) to 5 in LPS treated with HH at 56 degrees C.     

Exchange and aggregation in dispersions of dimyristoyl phosphatidylcholine vesicles containing myristic acid

Processes occurring in dispersions of dimyristoyl phosphatidylcholine containing myristic acid have been studied by light scattering of dilute dispersions (concn. ≤ 1 mg/ml) at temperatures above and below the phase transition temperatures of these dispersions. The transition temperatures increase with increasing mol fraction of myristic acid. Above these temperatures, vesicles with different mol fractions of myristic acid exchange lipid molecules. The exchange process leads to vesicles having phase transition temperatures and radii, which are both intermediate between the initial transitions and radii, respectively. In contrast with the observations above the phase transitions, it was found that when dimyristoyl phosphatidylcholine/myristic acid vesicles were cooled to a few degrees below the phase transition, larger particles were formed. These observations are consistent with a mechanism consisting of vesicle aggregation followed by fusion of the aggregated vesicles. The aggregation process is of second order in the vesicle concentration, and its rate increases with increasing mol fraction of myristic acid.     

Metabolism of fatty acids by bovine spermatozoa

The incorporation of (14)C-labelled myristic, palmitic, stearic, oleic and linoleic acids in vitro into the lipids of bovine spermatozoa was measured at intervals from 2min to 2h. All acids were rapidly incorporated into diglycerides, myristic acid being metabolized to the greatest extent. Whereas the low incorporation of acids into total phospholipids reflected the relative stability of the major phospholipid fractions in sperm, the minor phospholipids, particularly phosphatidylinositol, showed comparatively high metabolic activity. Although, in general, saturated acids were incorporated more actively than unsaturated substrates, stearic acid was poorly incorporated into all lipids except phosphatidylinositol. In regard to fatty acid composition of sperm lipids it was notable that diglycerides contained myristic acid as the major component, and this acid was also a prominent moiety of phosphatidylinositol. Docosahexaenoic acid was the principal fatty acid of the major phospholipid classes. These findings have been discussed in relation to the role of lipids in the metabolism of spermatozoa.