Chain Unsaturated Fatty Acids Research Articles

Fatty acid uptake in the Saccharomyces cerevisiae mutant mon2

The yeast MON2 gene product is a scaffolding protein involved in vesicular budding on the trans‐Golgi membrane. We have found that the mon2Δ mutant shows increased growth sensitivity to undecylenic acid (11:1Δ10), a medium chain unsaturated fatty acid (UFA), compared to wild type cells under the same growth conditions. In order to better understand this effect, we examined wild type and mutant mon2Δ responses in unsupplemented, 18:2Δ9,12, and 11:1Δ10 fed conditions. We have found that when Mon2p is absent, there are altered molecular responses to 11:1Δ10 supplementation, including: small but significant changes in the fatty acid profile; differential localization of BODIPY 500/510 C1, C12, a fluorescent fatty acid analog; decreased expression of OLE1, the gene for the delta‐9‐desaturase; and, altered rates of fatty acid uptake. These changes result in decreased synthesis of endogenous UFAs, which may be responsible for the 11:1Δ10 toxicity. These findings suggest a role for Mon2p in the uptake and trafficking of exogenous medium chain fatty acids, possibly through vesicle transport. Supported by awards from the National Science Foundation‐Research Experiences for Undergraduates and a Research Scholar Award from Howard Hughes Medical Institute.

Antigonococcal activity of Ximenia caffra Sond. (Olacaceae) and identification of the active principle

The “large sour plum” tree Ximenia caffra Sond. (Olacaceae) is the only African representative of the genus Ximenia L., with a distribution covering East Africa, from Tanzania in the north to South Africa, as well as parts of Botswana and Namibia. It has many uses in traditional medicine, including treatment of wounds and infections as well as diarrhea, which have been verified through various pharmacological studies. Furthermore, the plant is also known for its nutritional and cosmetic value. Its edible fruits are known to be high in protein, potassium and vitamin C, while the seed oil, also referred to as “Ximenia oil” is rich in long chain unsaturated fatty acids and finds use as a domestic biofuel, with characteristics amenable to cosmetic development as topical applicants. Extracts of the plants are also widely used for treating sexually transmitted infections (STIs), which has been verified through ethnobotanical surveys and pharmacological screens. Based on these observations, X. caffra var. caffra was examined for its phytochemical composition and activity against the gonorrheal pathogen Neisseria gonorrhoeae. Initially, the leaf ethanolic extract was seen to be active (43.9% inhibition). The DCM partition of this extract (with 78.8% inhibition) was subjected to bioassay guided fractionation leading to the identification of the bisnorsesquiterpene vomifoliol which exhibited significant activity (63.1%) against N. gonorrhoeae.

Chemical Analysis ofBrassica juncea(L.) Czern var. Ensabi

The oil, protein and glucosinolate contents in the seeds of Brasica juncea var. Ensabi were determined as 34.6%, 32.1% and 34.8 μmol/g respectively. Fatty acid analysis of the Ensabi oil revealed the presence of significant amount of unsaturated fatty acids, namely oleic acid (18.2%) and linoleic acid (16.9%) while lower quantity of saturated fatty acids: stearic acid (1.4%) and palmitic acid (2.8%). It also contained a very high amount of erucic acid (42.0%) whereas other polyunsaturated fatty acids, such as linolenic acid (5.5%), eicosenoic acid (8.8%) showed moderate presence. This composition was similar to the one reported for rapeseed (high erucic) oil and could not meet the specifications for human nutrition due to the very high content of long chain unsaturated fatty acid, erucic acid against its absence as recommended by FAO and WHO. Therefore, Ensabi oil can not be used as a vegetable oil for human nutrition.

Selective HPLC method development for soy phosphatidylcholine fatty acids and its mass spectrometry

A novel, efficient and simple approach for soy phosphatidylcholine analysis according to its fatty acid composition was studied with reverse-phase high-performance liquid chromatography. The reverse-phase high-performance liquid chromatography analysis was performed isocratically using UV detector and simple mobile phase solvents consisting of isopropyl alcohol, methanol, and deionized water in the proportion of 70:8:22 v/v. The uniqueness of the proposed method was the separation of individual fatty acids of soy phosphatidylcholine. The high-performance liquid chromatography method for soy phosphatidylcholine was validated for linearity with correlation coefficient of above 0.99 for all the peaks separated according to their fatty acid composition. The intra-day and the inter-day precision studies provided the relative standard deviation of less than 2%. The limit of detection and limit of quantitation values were also calculated for all the resolved peaks of soy phosphatidylcholine. Also system performance parameters such as number of theoretical plates, capacity factor, tailing factor, separation factor, and peak resolution were studied systematically and found well within the acceptable range. The proposed high-performance liquid chromatography method was successfully applied to soy phosphatidylcholine extracted and purified from deoiled soy lecithin without any interference of impurities or solvent peaks. Individually, the collected peaks of sample soy phosphatidylcholine were subjected for mass spectroscopy. The mass spectra showed all the peaks having different saturated or unsaturated fatty acid chains attached to glyerophosphocholine moiety of soy phosphatidylcholine. The method developed is economic and well suited for estimation of soy phosphatidylcholine with its fatty acid composition.

Rice (Oryza sativa) lipase: Molecular cloning, functional expression and substrate specificity

Lipases are important biocatalysts showing many interesting properties with industrial applications. Previously, different isoforms of lipases, Lipase-I and Lipase-II from rice (Oryza sativa) have been purified and characterized. Lipase-II identified as the major lipase in rice bran is designated as rice bran lipase (RBL). In this study, we report the cloning and expression of the RBL in Escherichia coli and Pichia pastoris. An exploration of expression in four different E. coli expression systems analyzed: BL21(DE3)pLysS, RIL(DE3)pLysS, Rosetta(DE3)pLysS and Origami(DE3)pLysS indicated that E. coli was not a suitable host. Expression with supplement of rare codons in Rosetta (DE3)pLysS and RIL(DE3)pLysS resulted in highest expression as insoluble inclusion bodies. The hurdles of expression in E. coli were overcome by expression as a secretory protein in P. pastoris X-33. The expression of lipase in shake flasks was optimized to achieve the maximum recombinant lipase activity of 152.6U/mL. The purified recombinant lipase had a specific activity of 998U/mg toward triacetin. The pH and temperature optimum of native and recombinant enzymes were pH 7.4 and 25±2°C, respectively. Both the lipases showed higher activity toward short chain triacylglycerol and unsaturated fatty acid enriched oils. Computational modeling and molecular docking studies reveal that the catalytic efficiency of the lipase correlates with the distance of the nucleophilic Ser175-OH and the scissile ester bond. The shorter the distance, the greater is the turnover of the substrate.

Production of nutritionally desirable fatty acids in seed oil of Indian mustard (Brassica juncea L.) by metabolic engineering

Development of a designer oilseed crop with improved yield attributes and enhanced nutritional quality for the benefits of mankind and animal husbandry is now achievable with the combination of genetic engineering and plant breeding. In spite of their immense importance, the fatty acid profiles of most oilseed crops are imbalanced that necessitate the use of metabolic engineering strategies to overcome the various shortfalls in order to improve the nutritional quality of these edible oils. Indian mustard (Brassica juncea L.), being one of the important oilseed crops in Indian subcontinent naturally contains ~50 % nutritionally undesirable very long chain unsaturated fatty acids (VLCUFAs), e.g. erucic acid (C22:1). For the purpose of nutritional improvement of B. juncea seed oil, several metabolic engineering strategies have been employed to divert the carbon flux from the production of VLCUFAs to other important fatty acids. Stearic acid, being a saturated but nutritionally neutral fatty acid, is naturally inadequate in most of the conventional oil seeds. Due to its neutral effect on consumer’s health and as an important industrial ingredient, increased in planta production of stearic acid in the seed oil not only helps in reduction of production cost but also lessens the trans fatty acid production during commercial hydrogenation process. In this review metabolic engineering strategies to minimize the VLCUFAs along with increased production of stearic acid in the seed oil of B. juncea are discussed, so that further breeding attempts can be made to improve the nutritionally desirable fatty acid profile in the suitable cultivars of this important oilseed crop.

Efek Antidiabetes dan Identifikasi Senyawa Dominan Fraksi Kloroform Herba Ciplukan (Physalis angulata L.)

Antidiabetic activity of ciplukan (Physalis angulata L.) herb has been widely observed, as an extracts and infusions, proven to reduce blood sugar levels in rats or mice induced by alloxan. The research objective was to evaluate the antidiabetic effect and identify the content of the chloroform fraction ciplukan herb eluated with methanol-ammonia. An- tidiabetic effects testing conducted using 30 male white mice (Mus musculus) ddY strain were divided into 6 groups, each consisting of 5 mice. K-1 is a negative control induced by alloxan, K-2, K-3 and K-4 induced by alloxan and treated with the test substance dose of 50 mg/kg bw/day, 100 mg/ kg bw/day, 200 mg/kg bw/day, K-5 is a positive control, induced by alloxan and treated with metformin dose of 65 mg/kg bw, K-6 as normal controls . Each mouse blood samples taken on day 7 and day 14, to test their blood sugar levels. Data were analyzed using one-way anova and multiple differences test. Identification of the content of the chloroform fraction eluated with methanol-ammonia, carried out by gas chroma- tography mass spectrometry (GC-MS). The results showed that all three doses showed a decrease in blood sugar levels significantly at day 7 , but the decline has not reach normal levels. Blood sugar levels drop significantly and the decline has reached normal occurred on day 14. Dominant content of the chloroform fraction eluated with methanol-ammonia is a class of unsaturated fatty acid chain length is Hexanoic acid, C16H1202, hexadecanoic acid methyl ester, C17H3402, 9-octadecenoic acid methyl ester, C19H3602, Oleic acid butyl ester, C22H4202, 9-octadecenoic acid, C19H3602, Octadecanoic acid, C18H3602, 1,2-Benzendicarboxylic acid, C8H604, and Aplysterylacetate, C31H5202. Another result is that Nordextromethorphan alkaloid compound, Cl 7H23NO. The conclusion is chloro- form fraction of ciplukan herb has antidiabetic effects and has a class of compounds con- tent of unsaturated fatty acids, Aplysterylacetate and alkaloid Nordextromethorphan.

Accelerated hydrothermal degradation of fibres of Phormium tenax (New Zealand flax)

Solid-phase microextraction (SPME)–gas chromatography (GC)–mass spectrometry (MS) has been applied to the analysis of acetic acid and furfural that are emitted from the fibres of Phormium tenax (Xanthorrhoeaceae) [New Zealand flax] during degradation. Accelerated hydrothermal ageing of fibres of the Ruawai cultivar of P. tenax for 55 days at 70 °C resulted in the production of acetic acid at a level greater or equal to 1.65 mg g–1 fibre. This corresponds to only 8.5% of the acetyl groups present in the fibres. These groups are an important source of acetic acid, which is capable of damaging the fibres. The rate of production of acetic acid suggests that a heritage object made from the fibres of P. tenax, that was stored in damp ambient conditions, would undergo significant deterioration after a relatively short period. Other volatile products released during ageing included furfural, a series of short to medium chain aliphatic aldehydes that were derived from the oxidation of long chain unsaturated fatty acids on the fibres and a small group of products that were derived from carotenoids. The levels of furfural were determined to be approximately 0.75% of the potential production but at these levels, furfural could contribute to unpleasant odours from unventilated items. Fibres from six cultivars of P. tenax were found to release acetic acid and furfural all at the same rate. The levels of acetic acid that can be generated from these fibres are sufficiently high that low moisture levels and continuous change of air is required to minimize degradation of cultural objects that are made from these fibres and displayed in museum halls.

Reversal of myofibroblast differentiation in valvular interstitial cells by long chain unsaturated fatty acids

Objectives: Most valve diseases are associated with the activation of valvular interstitial cells (VICs) from a quiescent fibroblastic to a myofibroblastic phenotype. Myofibroblasts are characterized by expression of α-smooth muscle actin (αSMA) and other components of the cytoskeleton. Cultivation of VICs on rigid cell culture surfaces also induces VIC activation resulting in contraction and spontaneous nodule formation. Here we test the hypothesis that activation of VICs in vitro can be reversed by exposure to long chain polyunsaturated fatty acids (PUFAs).

Reducing lipid peroxidation for improving colour stability of beef and lamb: on‐farm considerations

Meat colour and lipid stability are important sensory properties. Myoglobin oxidation is correlated with lipid oxidation, and one can exacerbate the occurrence of the other. Approaches to reduce both oxidative processes could focus on minimising oxidising agents in meat. In on-farm practices, dietary supplementation of antioxidants is a useful approach for improving lipid stability and then meat colour stability, and further improvement could be a combination of using hydrophobic and hydrophilic antioxidants. Genetic variations in the endogenous antioxidant systems could be identified for the development of a breeding strategy to bring a long-term benefit to the meat industry. To have a better understanding of oxidative stress and oxidation of long chain unsaturated fatty acids in live animals, the isoprostanes could be measured as an effective marker in vivo.

Preparation and properties of fully esterified erythritol

AbstractPure tetraesters of erythritol with C10, C12, C14, C16, C18 saturated, and C18:1 unsaturated (oleoyl) fatty acyl chains have been prepared for the first time and characterized using the acylating systems fatty acid/N,N′‐dicyclohexylcarbodiimide/4‐dimethylaminopyridine (DMAP), fatty acid anhydride/DMAP, fatty acyl chloride/pyridine, and fatty acyl chloride/boron trifluoride etherate. For the first three systems the yields were in the range of 80–90% while the fatty acyl chloride/pyridine system has the advantage of lower cost. The fatty acyl chloride/boron trifluoride etherate system gave lower (ca 70%) yields of the tetraesters. The tetraesters of erythritol may have applications analogues to those of triglycerides. In addition, new applications can be envisaged for these compounds, as a result of their differences in physical, chemical, and biochemical properties compared to triglycerides.Practical applications: The tetraesters of erythritol with saturated fatty acyl chains may have applications analogous to those of saturated triglycerides. However, tetraesters with unsaturated fatty acid chains may have greater prospects of having industrial uses after doing chemistry on the carbon–carbon double bonds.

Lipidomic Analysis of Apoptotic Hela Cells Induced by Paclitaxel

Apoptosis has been shown to be accompanied by changes in cellular phospholipids. In this study, lipidomic strategy was applied to investigate the early changes in Hela cell phospholipid metabolism in response to paclitaxel-induced apoptosis. A total of 240 phospholipid species were profiled and 202 of them were quantified using the NPLC-ESI/MS(n) procedure. Principal component analysis and partial least squares combined with variable influence on projection were applied to find the potential biomarkers for discrimination between the control and paclitaxel-treated cells. Lysophosphatidylethanolamine and lysophosphatidylcholine species with strong decreasing trends after 6 h of treatment were identified as possible biomarkers for discrimination, which could be backed up by the decreased expression of cPLA(2). In addition, some of phosphatidylethanolamine, phosphatidylinositol, and phosphatidylcholine species with unsaturated fatty acid chains and phosphatidic acid species with saturated fatty acid chains were also indentified as potential biomarkers, and their changes might be closely related to the alteration of cell membrane fluidity happened in apoptotic process. These results showed clearly that phospholipids and related phospholipases played important roles in paclitaxel-induced apoptosis in Hela cells. Our study also demonstrates that lipidomics provide a powerful tool for better understanding anticancer mechanisms of chemotherapeutic agents and for biomarker screening.

Regulation of mammalian physiology, development, and disease by the sphingosine 1-phosphate and lysophosphatidic acid receptors.

The moniker lysophospholipid incorporates two broad families of membrane-derived lipids: the glycerophospholipids and the sphingolipids1. Primary representatives of these two arms are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), the majority of whose activities are mediated via multiple G-protein-coupled receptors with a high degree of specificity for either LPA or S1P 2,3. Unlike S1P, which is a single molecular species (2S-amino-1-(dihydrogen phosphate)-4E-octadecene-1,3R-diol), LPA (1-O-acyl-2-hydroxy-sn-glycero-3-phosphate) is actually a diverse group of molecules consisting of either a saturated (e.g. 16:0; 18:0) or unsaturated (e.g. 16:1; 18:1; 18:2; 20:4) fatty acid chain esterified at the sn-1 or sn-2 position of a glycerol backbone 4,5. This can also be modified to alkyl or alkenyl at the sn-1 position 6. Other bioactive members of the LP family include the LPA analogues cyclic phosphatidic acid (cPA), sphigosylphosphotidylcholine (SPC), lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), and lysophosphatidylinositol (LPI) 1,5,7. Numerous emerging reports indicate roles in mammalian biology for other, less well-characterized LP members, as well 8. In the past decade, our understanding of LP biology has expanded exponentially, fueled by the identification of LP receptors, generation and analysis of LPA and S1P-receptor knockout mice, and small molecule compounds functioning as receptor-specific agonists or antagonists 3,9,10. Specific receptors for LPA and S1P were cloned and characterized beginning only about 15 years ago. The description of the first LPAR was reported in 1996, and the orphan receptor EDG-1, which was cloned as an immediate-early gene from endothelial cells, was identified as the first S1P receptor in 1998 11-13. Since then, four more S1P receptors and at least five more LPA receptors have been identified 3. The two families have been best characterized in specific organ systems; namely, LPARs in the nervous system and cancer, and S1PR in vascular biology and immunology. That is not to say that their contributions in other fields are minor, only that they are incompletely understood.

Phytochemical characterization of antimycobacterial crude extracts from medicinal plants traditionally used in Mozambique

A number of medicinal plants have long been used by traditional healers to treat tuberculosis and related diseases in Mozambique [1,2]. The present study was aimed to evaluate selected medicinal plants for their in vitro antimycobacterial activity, and reveal the main classes of compounds which might account for the observed activity. Four organic solvents (n-hexane, dichloromethane, ethylacetate, and 70% ethanol) were used for the sequential extraction. Decoction of each plant material was prepared according to traditional use. Different species of mycobacteria, namely, M. smegmatis, M. bovis BCG, M. avium, and M. tuberculosis were employed to screen extracts by broth microdilution method. The cytotoxicity against human macrophages from the monocytic THP-1 cells was also evaluated. Overall, n-hexane extracts of Maerua edulis Gilg & Gilg-Ben. and Securidaca longepedunculata Fresen, ethyl acetate extract of Tabernaemontana elegans Stapf and dichloromethane extract of Zanthoxylum capense (Thunb.) Harv. were found to possess considerable activity against M. bovis BCG and M. tuberculosis H37Ra with MIC=15.6–62.5µg/mL. Tabernaemontana elegans ethyl acetate extract displayed strong activity against M. tuberculosis H37Rv (MIC 15.6µg/mL) as well as potent cytotoxic effects in THP-1 cells (IC50 <4µg/mL). Based on 1H NMR spectroscopic analysis, major components in both Maerua edulis and Securidaca longepedunculata n-hexane extracts were linear chain unsaturated fatty acids. Zanthoxylum capense dichloromethane extract contained more complex constituents (mostly phenolic compounds), and the prominent compounds in ethyl acetate extract of Tabernaemontana elegans were identified as indole alkaloids.

Purification and characterization of an extracellular lipase from a novel strain Penicillium sp. DS-39 (DSM 23773)

A newly isolated fungal strain, Penicillium sp. DS-39 (DSM 23773), was found to produce an inducible extracellular lipase when grown in a medium containing 1.0% (v/v) olive oil. Maximum lipase activity was obtained after 120 h of incubation at 28 °C. The lipase was purified 129-fold with a final specific activity of 308.73 IU/mg. The molecular weight of the homogeneous lipase was 43 kDa as determined by SDS-PAGE. It was optimally active at pH 5.5 and 45 °C. The lipase was most active on triolein and exhibited a broad substrate range with a preference for triacylglycerols containing long chain unsaturated fatty acids. It showed no regio-specificity for the ester bond in triolein. It was activated by Ca2+ and Mn2+, while significant inhibition was observed with Hg2+ and Zn2+. The lipase showed significant stability and activation in the presence of organic solvents with log P ≥ 2.0. These features render Penicillium sp. DS-39 lipase (PEL) a potential biocatalyst for applications such as biodiesel production, enzymatic restructuring, by interesterification of different oils and fats, and biodegradation of oil spills in the environment.

Antimycobacterial evaluation and preliminary phytochemical investigation of selected medicinal plants traditionally used in Mozambique

Several medicinal plants are traditionally used in Mozambique to treat tuberculosis and related symptoms. It was aimed to assess the in vitro antimycobacterial activity of crude extracts from fifteen medicinal plants and to reveal main classes of compounds which may account for the activity of extracts. The plant materials were sequentially extracted by n-hexane, dichloromethane, ethyl acetate, and 70% ethanol. Decoction of each plant material was also prepared according to traditional use. Broth microdilution method was employed to screen extracts against two mycobacterial species: Mycobacterium smegmatis ATCC 607 and Mycobacterium tuberculosis H37Rv. The extracts with minimum inhibitory concentration(s) (MIC) below 125 μg/mL were considered active and further tested against different mycobacterial species and strains, namely Mycobacterium tuberculosis H37Ra, Mycobacterium bovis BCG ATCC 35734, Mycobacterium smegmatis mc(2) 155, Mycobacterium avium DSM 44156 and DSM 44157. Cytotoxic effect was evaluated against human macrophages from the monocytic THP-1 cells. Main classes of compounds in these active extracts were proposed from their (1)H NMR spectroscopic characterizations. n-Hexane extracts of Maerua edulis and Securidaca longepedunculata, ethyl acetate extract of Tabernaemontana elegans and dichloromethane extract of Zanthoxylum capense were found to possess considerable activity against Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with MIC 15.6-62.5 μg/mL. Tabernaemontana elegans ethyl acetate extract displayed strong activity against Mycobacterium tuberculosis H37Rv (MIC 15.6 μg/mL). Except for Tabernaemontana elegans ethyl acetate extract which presented potent cytotoxic effects in THP-1 cells (IC(50)<4 μg/mL), the other three plant extracts showed moderate to none toxicity. Based on (1)H NMR spectroscopic analysis, major components in both Maerua edulis and Securidaca longepedunculata n-hexane extracts were linear chain unsaturated fatty acids. Zanthoxylum capense dichloromethane extract contained more complex constituents (mostly phenolic compounds). In the most potent extract, Tabernaemontana elegans ethyl acetate extract, the prominent compounds were identified as indole alkaloids. The pronounced antimycobacterial activity of the medicinal plants Maerua edulis, Securidaca longepedunculata, Zanthoxylum capense, and Tabernaemontana elegans suggested that they might provide compounds which could be potential anti-TB drug leads.

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

Analytical characterization of mannosylerythritol lipid biosurfactants produced by biosynthesis based on feedstock sources from the agrofood industry

Mannosylerythritol lipids (MELs) are currently one of the most promising biosurfactants because of their multifunctional applications and good biodegradability. Depending on the yeast strain and the feedstock used for the fermentation process, structural variations in the MELs obtained occur. Therefore, MELs produced by Pseudozyma aphidis DSMZ 70725 with a soybean oil feedstock were characterized by chromatography and mass spectrometry (MS). Column chromatography with silica provided fractionation of the different types of MEL. High-performance liquid chromatography combined with MS was employed for the analysis of the MEL fractions and crude mixtures. A characteristic MS pattern for the MELs was obtained and indications of the presence of new MEL homologues, showing the incorporation of longer and more unsaturated fatty acid chains than previously reported, were given. Gas chromatography-MS analysis confirmed the presence of such unsaturated fatty acid chains in the MELs, demonstrating the incorporation of fatty acids with lengths ranging from C(8) to C(14) and with up to two unsaturations per chain. The incorporation of C(16) and C(18) fatty acid chains requires further investigation. MS/MS data allowed the unambiguous identification of the fatty acids present in the MELs. The product ion spectra also revealed the presence of a new isomeric class of MELs, bearing an acetyl group on the erythritol moiety.

Lipid Areas Obtained from the Simultaneous Analysis of Neutron and X-ray Scattering

Despite their importance to biophysical research, published lipid areas have been relatively scarce and for the most part, inconsistent. Noteworthy are the discrepancies between lipid areas as determined by standalone X-ray and neutron scattering experiments - arguably two of the most commonly used techniques in structural biology. Although they each have their advantages and disadvantages, when used in combination their advantages can be maximized. In particular, the large scattering contrast in the case of neutrons best resolves the overall bilayers thickness that is directly related to lipid lateral area. On the other hand, high resolution X-ray experiments yield detailed intra molecular structural information [Kucerka et al., Biophys. J. 95, 2356 (2008)].We have utilized our recently developed method to characterize lipid areas of various phospholipids with varying numbers of carbons and double bonds. In the case of lipids with unsaturated fatty acid chains our results suggest that lipid areas change with increasing hydrocarbon chain length, but not linearly - lateral lipid area is the result of the fine balance between the hydrocarbon chain length and double bond position. Furthermore, we discovered that the most dramatic change in lipid area occurs after the introduction of the first double bond to the lipid's acyl chains.Besides their importance in biology, lipid areas play a central role in molecular dynamics (MD) simulations, where their inconsistencies have been highlighted by the disparate results arising from MD simulations using different force fields. Since MD force fields are considered to be “well tuned” if they are able to reproduce experimental data, more reliable experimental information is necessary for their future development.

Fatty acid composition modulates sensitivity of Legionella pneumophila to warnericin RK, an antimicrobial peptide

Warnericin RK is an antimicrobial peptide, produced by a Staphyloccocus warneri strain, described to be specifically active against Legionella, the pathogenic bacteria responsible for Legionnaires’ disease. Warnericin RK is an amphiphilic alpha-helical peptide, which possesses a detergent-like mode of action. Two others peptides, δ-hemolysin I and II, produced by the same S. warneri strain, are highly similar to S. aureus δ-hemolysin and also display anti-Legionella activity. It has been recently reported that S. aureus δ-hemolysin activity on vesicles is likewise related to phospholipid acyl-chain structure, such as chain length and saturation. As staphylococcal δ-hemolysins were highly similar, we thus hypothesized that fatty acid composition of Legionella's membrane might influence the sensitivity of the bacteria to warnericin RK. Relationship between sensitivity to the peptide and fatty acid composition was then followed in various conditions. Cells in stationary phase, which were already described as less resistant than cells in exponential phase, displayed higher amounts of branched-chain fatty acids (BCFA) and short chain fatty acids. An adapted strain, able to grow at a concentration 33 fold higher than minimal inhibitory concentration of the wild type (i.e. 1μM), was isolated after repeated transfers of L. pneumophila in the presence of increased concentrations of warnericin RK. The amount of BCFA was significantly higher in the adapted strain than in the wild type strain. Also, a transcriptomic analysis of the wild type and adapted strains showed that two genes involved in fatty acid biosynthesis were repressed in the adapted strain. These genes encode enzymes involved in desaturation and elongation of fatty acids respectively. Their repression was in agreement with the decrease of unsaturated fatty acids and fatty acid chain length in the adapted strain. Conclusively, our results indicate that the increase of BCFA and the decrease of fatty acid chain length in membrane were correlated with the increase in resistance to warnericin RK. Therefore, fatty acid profile seems to play a critical role in the sensitivity of L. pneumophila to warnericin RK.