Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism

A TOF MS/MS method to directly determine the amino acid sequence in a cyclic lipopeptide without its hydrolysis is described. The fragments of the peptide and the hydrocarbon chains were identified through comparing the MS of two analogues of the lipopeptide; the connecting relationship of amino acid residues in the lipopeptide was determined based on the difference of mass to charge ratio between peaks in the MS spectra and the amino acid analysis; and finally, according to the mechanism of double hydrogen transfer(DHT) the C-terminal of peptide and hydroxy aliphatic acid in the lipopeptide was directly determined without the hydrolysis. The determined sequence of amino acid residues in the cyclic lipopeptide is also supported by the rest peaks in the MS spectra grounded on simple fragmenting mechanism. This method can be used to determine the amino acid sequence in any aliphatic acid loop-inlaying cyclic lipopeptides.     

Determination of the structure of the fatty acid chain in a cyclic lipopeptide using GC–MS

A GC–EIMS method to determine the structure of the fatty acid chains in cyclic lipopeptides is described. The structure of the fatty acid chains can be determined by the characteristic peaks of the MS spectrogram according to the fact that the alpha cleavage predominates the MS of a fatty acid with amino and hydroxy groups, while the McLafferty rearrangement predominates the MS of one without amino or hydroxy group. The characteristics of the strongest peaks of 103 and 102 in MS spectrograms due to alpha cleavage represent the β-hydroxy-fatty acid and the β-amino fatty acid, respectively; the strongest peak of 117 due to alpha cleavage and the relatively weak peak of 88 due to McLafferty rearrangement indicate the β-hydroxy-fatty acid with a branched methyl group at its alpha position. The strongest peak of 74 due to McLafferty suggests the fatty acid without hydroxy or amino group. The ratio of relative intensity (I₄₃/I₅₇) characterizes the branches of alkyl chains. The greater I₄₃/I₅₇ corresponds to an iso alkyl, and the smaller I₄₃/I₅₇ corresponds to an anteiso alkyl. This method can be used to determine the full structure of the fatty acid chains in lipopeptides.     

Coarse-grained molecular dynamics simulation of interactions between cyclic lipopeptide Bacillomycin D and cell membranes

According to experimental studies, Bacillomycin D has strong antimicrobial activities, but the antimicrobial mechanism is still unknown. In this paper, the interaction mechanisms between this cyclic lipopeptide and three different charged cell membranes are studied via Coarse-Grained Molecular Dynamics (CG MD) simulations. A specific CG model for the cyclic lipopeptide Bacillomycin D was developed. The insertion of cyclic lipopeptide Bacillomycin D into DOPC, DOPC/DPPA and DOPC/DOTAP cell membranes was investigated. The position distribution and stability of Bacillomycin D in the three different cell membranes were analysed and compared based on density profile calculations. Additionally, we focused on the Radial Distribution Function (RDF) curves between amino acid residues with negative charges or strong hydrophobic properties and the head groups of two different cell membranes. Based on changes in the curvature of the three membranes, the cyclic lipopeptide Bacillomycin D can cause localised surface protrusions in DOPC/DOTAP membranes, inward depressions in the surface of DOPC/DPPA membranes and inhibition deformation in the surface of DOPC membranes. This study will help to further understand the antibacterial mechanism of the cyclic lipopeptide Bacillomycin D and provide a basis for the development of new antibiotics.     

Gramicidin induces the formation of non-bilayer structures in phosphatidylcholine dispersions in a fatty acid chain length dependent way

The hydrophobic peptide gramicidin is shown by ³¹P-NMR, freeze-fracture electron microscopy and small-angle X-ray diffraction, to induce a hexogonal H_II-phase lipid organization when incorporated in liquid crystalline saturated and unsaturated synthetic and natural phosphatidylcholines if the length of the fatty acids exceeds a 16 carbon atoms chain. The amount of hexagonally organized lipid increases with increasing fatty acid chain length. With phosphatidylcholines possessing shorter fatty acid chains, as well as with the longer phosphatidylcholines in the gel state, a lamellar organization results. Of the various possible models to explain the induction of the hexagonal H_II phase by gramicidin, one in which gramicidin dimers span adjacent cylinders of the hexagonal H_II phase seems most plausible. In phosphatidylcholines with intermediary chain lengths gramicidin induces intermediary structures, such as lipidic particles and possibly cubic phases. These experiments suggest that the balance between the length of the hydrophobic domain of a peptide and the membrane thickness is of critical importance for the structure of the membrane. In relation to this observation, the possible involvement of non-bilayer lipid structures in insertion and anchoring of membrane proteins is discussed.     

A Dictyostelium long chain fatty acyl coenzyme A-synthetase mediates fatty acid retrieval from endosomes

We have identified a subset of Dictyostelium endosomes that carry a long chain fatty acyl coenzyme A-synthetase (LC-FACS 1) on their cytosolic surface. Immunofluorescence studies and observations using GFP-fusion proteins collectively suggest that LC-FACS 1 associates with endosomes a few minutes after their formation, remains bound through the acidic phase of endocytic maturation and dissociates early in the phase where the endosomal content is neutralised prior to exocytosis. Mutants in the fcsA gene, encoding the LC-FACS 1 protein, were constructed by homologous recombination. These cells show a strong defect in the intracellular accumulation of fatty acids, either taken up together with the liquid medium or bound to the surface of particles. Because the mutant cells are otherwise fully competent for macropinocytosis and phagocytosis, we conclude that the LC-FACS 1 protein mediates the retrieval of fatty acids from the lumen of endosomes into the cytoplasm.     

Effect of the microbial lipopeptide on tumor cell lines: apoptosis induced by disturbing the fatty acid composition of cell membrane

Microbial lipopeptides play an important role in apoptosis induction of tumor cells. However, there is little knowledge about the relationship between apoptosis induction and membrane fatty acids. The present study focused on the effects of lipopeptides produced by Bacillus subtilis HSO121 on Bcap-37 cell lines. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl (MTT) colorimetric assay and surface tension measurements, showed that the critical micelle concentration (CMC) was a critical level for the inhibitory activity of lipopeptides on the growth of Bcap-37 cells. Under the CMC, the order of least to greatest cytotoxicity effect on cancer cell lines by lipopeptides is C₁₃-lipopeptide<C₁₄-lipopepitde<C₁₅-lipopeptide. Above CMC, all lipopeptides directly exert cytolytic activity. The flow cytometric analysis and Hoechst33258 staining experiments confirmed the apoptosis of Bcap-37 cell lines induced by lipopeptides in a dose-dependent manner. This apoptosis was associated with a significant decrease of the unsaturated degree of the cellular fatty acids of Bcap-37 cell lines due to the changes in the cellular fatty acids composition induced by the lipopeptide treatment. These results indicated that disturbance of the cellular fatty acid composition of breast cancer cell lines were related to in the cell apoptosis. Furthermore, significant difference in IC₅₀ values of tumor cells and normal cell showed that the lipopeptide exerted selective cytotoxicity on the cancer cells. Thus HSO121 lipopeptides may have potential applications as an anticancer leads.     

Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus

This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene (EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host–parasite adaptation.     

红树植物红海榄脂肪酸的GC-MS分析

采用索氏提取法对红树植物红海榄(Rhizophora stylosa)叶中的脂肪酸进行提取,甲酯化后利用气相色谱-质谱联用仪对脂肪酸成分进行分离和鉴定,鉴定出了12种脂肪酸,占脂肪酸总量的84.95%,其中主要包括十六酸(棕榈酸)(40.49%),9-十八碳烯酸(油酸)(17.24%),9,12-十八碳二烯酸(亚油酸)(11.24%)等。     

Glucose stimulates a decrease of the fatty acid saturation degree in Acinetobacter calcoaceticus

The fatty acid composition of Acinetobacter calcoaceticus 69-V was determined under various growth conditions. Saturated, unsaturated, and hydroxy fatty acids with chain lengths of 12–18 carbon atoms predominated in the fatty acid profile. With acetate or propanol as growth substrates, the ratio of saturated to unsaturated fatty acids varied with changes in the temperature. This was the only adaptive mechanism detected that compensated for the physical effects of temperature alterations on the cell membranes. The fatty acid composition of A. calcoaceticus grown at 40 °C had a saturation degree of approximately 50%; after growth at 20 °C it was approximately 35%. In the presence of a carbon and energy source, A. calcoaceticus was able to respond to temperature reductions under oxic conditions regardless of whether fatty acid biosynthesis was inhibited or not. This suggests an aerobic mechanism of fatty acid biosynthesis and the involvement of a fatty acid desaturase system. Addition of the non-growth substrate, glucose, helped the organism to adapt to lower temperature. The molecular mechanism of the aid is not really understood. The oxidation of glucose could provide the desaturase either with electrons directly via a pyrrolo-quinoline-quinone-linked glucose dehydrogenase or with NADH after fatty acid degradation has been initiated by ATP generated by the oxidation of glucose. Received: 19 June 1998 / Accepted: 28 December 1998     

Significance of glycosphingolipid fatty acid chain length on membrane microdomain-mediated signal transduction

Lactosylceramide (LacCer), a neutral glycosphingolipid, is abundantly expressed on human neutrophils, and specifically recognizes several pathogenic microorganisms. LacCer forms membrane microdomains coupled with the Src family kinase Lyn on the plasma membrane, and ligand binding to LacCer activates Lyn, resulting in neutrophil functions. In contrast, neutrophilic differentiated HL-60 cells do not have Lyn-associated LacCer-enriched microdomains and lack LacCer-mediated functions. In neutrophil plasma membranes, the very long fatty acid C24:0 and C24:1 chains are the main components of LacCer, whereas plasma membrane of D-HL-60 cells mainly includes C16-LacCer species. Here, we suggest that LacCer species containing very long fatty acid chains are indispensable for the association of Lyn with LacCer-enriched microdomains and LacCer-mediated functions.     

Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases

Microbial biosynthesis of fatty acid-like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Free fatty acids can be produced by introducing an acyl–acyl carrier protein thioesterase gene into Escherichia coli. The presence of the acyl-ACP thioesterase will break the fatty acid elongation cycle and release free fatty acid. Depending on their sequence similarity and substrate specificity, class FatA thioesterase is active on unsaturated acyl-ACPs and class FatB prefers saturated acyl group. Different acyl-ACP thioesterases have different degrees of chain length specificity. Although some of these enzymes have been characterized from a number of sources, information on their ability to produce free fatty acid in microbial cells has not been extensively examined until recently. In this study, we examined the effect of the overexpression of acyl-ACP thioesterase genes from Diploknema butyracea, Gossypium hirsutum, Ricinus communis and Jatropha curcas on free fatty acid production. In particular, we are interested in studying the effect of different acyl-ACP thioesterase on the quantities and compositions of free fatty acid produced by an E. coli strain ML103 carrying these constructs. It is shown that the accumulation of free fatty acid depends on the acyl-ACP thioesterase used. The strain carrying the acyl-ACP thioesterase gene from D. butyracea produced approximately 0.2 g/L of free fatty acid while the strains carrying the acyl-ACP thioesterase genes from R. communis and J. curcas produced the most free fatty acid at a high level of more than 2.0 g/L at 48 h. These two strains accumulated three major straight chain free fatty acids, C14, C16:1 and C16 at levels about 40%, 35% and 20%, respectively.     

Surface plasmon resonance-enhanced fluorescence implementation of a single-step competition assay: demonstration of fatty acid measurement using an anti-fatty acid monoclonal antibody and a Cy5-labeled fatty acid

The development of a single-step, separation-free method for measurement of low concentrations of fatty acid using a surface plasmon resonance-enhanced fluorescence competition assay with a surface-bound antibody is described. The assay behavior was unexpectedly complex. A nonlinear coverage-dependent self-quenching of emission from surface-bound fluorescent label was deduced from the response kinetics and attributed to a surface plasmon-mediated energy transfer between adsorbed fluorophores, modified by the effects of plasmon interference. Principles of assay design to avoid complications from such effects are discussed. An anti-fatty acid mouse monoclonal antibody reacting to the alkyl chain was prepared and supported on a gold chip at a spacing appropriate for surface-plasmon field-enhanced fluorescence spectroscopy (SPEFS), by applying successively a self-assembled biotinylated monolayer, then streptavidin, then biotinylated protein A, and then the antibody, which was crosslinked to the protein A. Synthesis of a fluorescently (Cy5) tagged C-11 fatty acid is reported. SPEFS was used to follow the kinetics of the binding of the labeled fatty acid to the antibody, and to implement a competition assay with free fatty acid (undecanoic acid), sensitive at the 1 μM scale, a sensitivity limit caused by the low affinity of antibodies for free fatty acids, rather than the SPEFS technique itself. Free fatty acid concentration in human serum is in the range 0.1-1 mM, suggesting that this measurement approach could be applied in a clinical diagnostic context. Finally, a predictive, theoretical model of fatty acid binding was developed that accounted for the observed “overshoot” kinetics.     

Determination of S-phenylmercapturic acid by GC-MS and ELISA: a comparison of the two methods

Abstract

S-phenylmercapturic acid (PMA) is a specific urinary biomarker of benzene at exposure levels lower than 1?ppm. However, measuring PMA in urine is an expensive task by either GC or HPLC due to the necessity of extensive sample pretreatment. In the present study, a commercial chemiluminescence enzyme-linked immunosorbent assay (ELISA) test for PMA and GC-MS were used for screening urine samples of 60 workers employed in petrochemical settings. The ELISA results were evaluated by comparison with the GC-MS. Overall, the ELISA test proved sensitive (limit of detection?=?0.1?µg?l^?1), rapid, robust and reliable, affording results in good agreement with the GC-MS (54% of measurements) and no false-negatives. On the other hand, 46% of the ELISA assays were assigned as false-positives (arbitrarily established when ELISA >5?µg?l^?1, GC-MS <5?µg?l^?1) and a correlation coefficient of 0.687 was calculated between the two methods. It appears that urinary PMA routine biomonitoring on large numbers of samples is carried out in a cost-effective and rapid approach by preliminary screening with the ELISA assay followed by GC-MS confirmation of concentrations exceeding the biological exposure index for PMA.     

蜀葵种油的脂肪酸组成分析

The seed oil from Althaea rosea (L.) Caven was extracted with Hexane.After saponification and formation,the oil was determined by Capillary GC-MS.16 compounds have been determined and its unsaturation is 72.59%,Among them the content of linoleic acid is 69.24%.     

Fatty acid sensor for low-cost lifetime-assisted ratiometric sensing using a fluorescent fatty acid binding protein

Elevated free fatty acid (FA) levels lead to insulin resistance, hypertension, and microangiopathy, all of which are associated with type 2 diabetes. On the other hand, deficiencies of FA are indicative of certain neurodegenerative diseases, including autism. Thus, free FA levels are a diagnostic indicator for a variety of disorders. Here we describe the use of a commercially available FA binding protein labeled with acrylodan (ADIFAB), which we modified with a ruthenium metal-ligand complex with the intention of creating a low-cost FA sensor. The dual-labeled FA binding protein was used in lifetime-assisted ratiometric sensing (LARS) of oleic acid. For both steady-state and time-resolved luminescence decay experiments, the protein is responsive to oleic acid in the range of 0.02-4.7 microM. The emission at 432 nm, which is associated with the acrylodan occupying the FA binding site, decreases in intensity and red shifts to 505 nm on the addition of oleic acid. The intensities of the 505-nm peak due to the acrylodan displaced from the binding site by FA and of the 610-nm emission peak of ruthenium remained nearly unchanged. Fitting of the fluorescence decay data using the method of least squares revealed three emitting components with lifetimes of approximately 0.60, 4.00, and 370 ns. Fractional intensities of the emitting species indicate that changes in modulation between 2 and 10 MHz on binding of the protein with oleic acid are due mainly to the 4.00-ns component. The 0.60- and 370-ns components are assigned to acrylodan (505 nm) and ruthenium, respectively. Note that because ruthenium has a lifetime that is two orders of magnitude longer than that of acrylodan, the FA measurements were carried out at excitation frequencies lower than what can be done with acrylodan alone. Thus, low-cost instrumentation can be designed for a practical FA sensor without sacrificing the quality of measurements.     

Polar lipid ester-linked fatty acid composition of Lake Vechten seston: an ecological application of lipid analysis

Abstract In order to relate the benthic lipid composition to possible sources in the water column, the sestonic communities of a monomictic lake were profiled using their saponifiable polar lipid fatty acids, which were identified by capillary gas chromatography-mass spectrometry (GC-MS). The epilimnion, dominated by the dinoflagellate alga Ceratium hirundella , was characterized by C_20:5 and C_22:6 polyunsaturated fatty acids. The photic anoxic metalimnion supported a radically different community, dominated by photosynthetic sulfur-oxidizing bacteria ( Chromatium and Chloronema spp.) and a Synechococcus -like cyanobacterium, and was characterized by high concentrations of C₁₆ and C₁₈ monounsaturated fatty acids. The fatty acid compositions of the hypolimnetic seston and the sediment were qualitatively similar to that of the metalimnion. Methyl-branched acids, commonly found in eubacteria, increased with depth in the water column. The concentrations of several unusual fatty acids found in Desulfovibrio spp. Desulfobacter spp. and Desulfotomaculum spp. were inversely related to sulfate concentration in the metalimnion. After the water column mixed in the winter, steep gradients of respiratory terminal electron acceptors developed in the surface sediment and were reflected in the polar lipid fatty acid compositions. The results show that fatty acids derived from the membranes of epilimnetic phytoplankton were efficiently metabolized in the oxic portion of the water column. The fatty acids synthesized by prokaryotic microorganisms at and below the oxycline dominated the sediment. The polar lipid fatty acid composition of the sediment showed seasonal changes which can be associated with concentrations of terminal electron acceptors of microbial respiration, and thus with physiologically distinct bacterial groups.     

Mutants of Arabidopsis with alterations in seed lipid fatty acid composition

Summary A diverse collection of mutants of Arabidopsis with altered seed lipid compositions was isolated by determining the fatty acid composition of samples of seed from 3,000 mutagenized lines. A series of mutations was identified that caused deficiencies in the elongation of 181 to 201, desaturation of 181 to 182, and desaturation of 182 to 183. In each of these cases the wild type exhibited incomplete dominance over the mutant allele. These results, along with results from earlier studies, point to a major influence of gene dosage in determining the fatty acid composition of seed lipids. A mutation was also isolated that resulted in increased accumulation of 183. On the basis of the effects on fatty acid composition, the nature of the biochemical lesion in three of the mutants could be tentatively attributed to deficiencies in activities of specific enzymes. The other mutant classes had relatively less pronounced changes in fatty acid composition. These mutants may represent alterations in genes that regulate lipid metabolism or seed development. The availability of the mutants should provide new opportunities to investigate the mechanisms that control seed lipid fatty acid composition.Abbreviations FAMES (fatty acid methyl esters) - PC (phosphatidylcholine) - 181 (oleic acid) - 182 (linoleic acid) - 183 (linolenic acid) - 201 (eicosenoic acid) Supported in part by grants from the USDA (No. 89-37262-4388), USDA/NSF/DOE Plant Science Center Program, the U.S. Department of Energy (No. AC02-76ER01338), Karlshamns Research Foundation, and the WSU Research and Arts Committee     

皱皮木瓜果实中有机酸成分的GC-MS分析

木瓜为常用中药,其原植物为木瓜属（Chaenomeles Lindl．）植物皱皮木瓜[Chaenomeles speciosa（Sweet）Nakai],又称贴梗海棠、贴梗木瓜等,中国南方各地均有栽培。其花可供观赏;果实入药,能驱风强壮、舒筋、镇痛消肿,民间常用于浸泡药酒;种仁富含油酸和亚油酸。对皱皮木瓜有机酸的脂肪性成分已有研究报道,但尚无其水溶性有机酸成分的研究报道。皱皮木瓜的药用功效主要为镇痛解痉,但至今未明确其主效成分,有机酸虽具有一定的镇痛功效,但有机酸的种类不同,功效也不相同。鉴于长期以来皱皮木瓜还是作为汤药服用,所以就其水溶性有机酸组成及含量开展研究,特别是与其有机酸中的脂肪性成分进行比较研究尤为必要,对于揭示皱皮木瓜主要功效成分具有重要的意义。     

莱菔子脂肪酸成分的GC-MS分析

采用索氏提取法提取莱菔(Raphanussativus)种子中的脂肪酸成分,进行甲酯化处理后用气相色谱-质谱联用技术分离和鉴定脂肪酸成分的组成和含量。结果表明,从莱菔子中共分离鉴定出12种脂肪酸成分,占总量的99.32%,主要包括芥酸(32.47%)、油酸(29.07%)、亚油酸(9.45%)、亚麻酸(8.41%)、棕榈酸(4.31%)和硬脂酸(2.08%)等脂肪酸,其中饱和脂肪酸占9.25%,不饱和脂肪酸高达90.07%。