Lipopolysaccharides fromMastigocladus laminosus

A lipopolysaccharide (LPS) has been isolated by phenol-water extraction from the cells of the blue-green algaMastigocladus laminosus. It has been shown that the LPS contains polysaccharide and lipid components. The polysaccharide component includes a rhamnan fragment constructed of β-1,3- and, possibly, -1,2-bound L-rhamnose residues. The lipid component is constructed of glucosamine, glucose, and fatty acid residues, among which palmitic acid predominates.     

Lipid A components from Pseudomonas aeruginosa PAO1 (serotype O5) and mutant strains investigated by electrospray ionization ion-trap mass spectrometry

The lipid A components of the Pseudomonas aeruginosa strains PAO1 (wild-type) and derived mutants PAO1 algC::tet and PAO1 PDO100 were isolated after mild acetic acid hydrolysis of LPS. Their structural heterogeneities were characterized using electrospray ionization (ESI) ion-trap mass spectrometry (MS) with direct infusion in the negative ion mode without prior derivatization. The ESI-mass spectra revealed monophosphorylated molecules corresponding to known tetra-, penta- and hexaacylated structures of P. aeruginosa lipid A. The MS/MS fragmentation patterns allowed the location of fatty acyl chains on the disaccharide backbone of lipid A. In addition, a hexaacylated lipid A containing a hexadecanoyl chain was detected for the first time in strain P. aeruginosa PAO1. With multiple stages of fragmentation (MS(n)), the position of this hexadecanoyl chain O-linked to the decanoyl chain at the C-3(') position of the glucosamine backbone was determined. This sensitive method is suitable to reveal lipid A heterogeneity, i.e. the nature, number and distribution of acyl chains, without prior lipopolysaccharide purification. The lipid A from mutant strains were also characterized and significant differences were shown in the abundance of monophosphorylated lipid A components between the wild-type and the mutant strains.     

Characterization of complex,heterogeneous lipid A samples using HPLC–MS/MS technique I. Overall analysis with respect to acylation,phosphorylation and isobaric distribution

We established a new reversed phase‐high performance liquid chromatography method combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross‐reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2′ or the C3′ sites. The fragmentation of the various, C4′ monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri‐acylated, tetra‐acylated, penta‐acylated, hexa‐acylated and hepta‐acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa‐acylated ion at m/z 1716 with the E. coli‐type acyl distribution and the hepta‐acylated ion at m/z 1954 with the Salmonella‐type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative‐ion mode MS permitted efficient detection for non‐phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.     

Lipopolysaccharides fromMastigocladus laminosus

A lipopolysaccharide (LPS) has been isolated by phenol-water extraction from the cells of the blue-green algaMastigocladus laminosus. It has been shown that the LPS contains polysaccharide and lipid components. The polysaccharide component includes a rhamnan fragment constructed of -1,3- and, possibly, -1,2-bound L-rhamnose residues. The lipid component is constructed of glucosamine, glucose, and fatty acid residues, among which palmitic acid predominates.Pacific Ocean Institute of Bioorganic Chemistry, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 700–703, November–December, 1984.     

Flow-injection determination of phosphate with a cadmium ion-selective electrode

A flow-injection method is described, in which phosphate standards are introduced into a reagent stream containing Cd(2+) ,resulting in the formation of Cd(3)(PO(4))(2). The associated reduction in free metal concentration is sensed by a cadmium-selective electrode. With the exception of major interference from iodide and moderate interference from bromide and thiocyanate, the system exhibits excellent response to phosphate and selectivity over several common anions in solutions buffered at pH 8.4. A maximum sampling rate of 160/hr is possible for phosphate standards in the concentration range 10(-1)-10(-1)M with a 10(-4)M Cd(2+) reagent stream at a total flow-rate (carrier and reagent stream combined) of 8.4 ml/min.     

Synthesis and biological evaluation of a lipid A derivative that contains an aminogluconate moiety

A highly convergent strategy for the synthesis of several derivatives of the lipid A of Rhizobium sin-1 has been developed. The synthetic derivatives are 2-aminogluconate 3 and 2-aminogluconolactone 4, both of which lack C-3 acylation. These derivatives were obtained by the preparation of disaccharides in which the two amino groups and the C-3' hydroxy group could be modified individually with acyl or beta-hydroxy fatty acyl groups. Detailed NMR spectroscopy and MS analysis of 3 and 4 revealed that, even under neutral conditions, the two compounds equilibrate. The synthetic compounds lack the proinflammatory effects of Escherichia coli lipopolysaccharide (LPS), as indicated by an absence of tumor necrosis factor production. Although 3 and 4 were able to antagonize E. coli LPS, they were significantly less potent than the synthetic compound 2, which is acylated at C-3, and R. sin-1 LPS; these results indicate that the beta-hydroxy fatty acyl group at C-3 contributes to the antagonistic properties of R. sin-1 LPS. Based on a comparison of the biological responses of the synthetic lipid A derivatives with those of the R. sin-1 LPS and lipid A, the 3-deoxy-D-manno-octulosonic moieties appear to be important for the optimal antagonization of enteric LPS-induced cytokine production.     

Structural characterizations of lipids A by MS/MS of doubly charged ions on a hybrid linear ion trap/orbitrap mass spectrometer

Here, a new 'one pot' and fast approach is described, based on electrospray ionization (ESI) of negative ions by using a hybrid linear ion trap/orbitrap mass spectrometer (LTQ/orbitrap) for MS and MS/MS analysis. By this method the distribution of the primary and secondary acyl residues of the intact lipid A is inferred by analysis of the ESI spectra measured in positive and negative mode. The analysis of these data allows an unequivocal assignment of the fatty acid distribution. This methodology was successfully tested on two different lipid A with known structures, deriving from the Agrobacterium tumefaciens and Escherichia coli lipopolysaccharides (LPS).     

Modular synthesis of bis(monoacylglycero)phosphate for convenient access to analogues bearing hydrocarbon and perdeuterated acyl chains of varying length

Bis(monoacylglycero)phosphate is an important phospholipid that controls the structure of late endosomes as well as biological activities that occur there. The study of this lipid is complicated by the fact that acyl migrations are known to generate different regioisomers. Herein, we describe a modular synthesis of 3,3′-BMP derivatives that allows for the incorporation of a range of different acyl chains at a late stage. This approach was exploited to produce eight BMP analogues bearing normal saturated and perdeuterated acyl chains of varying length.     

Extraction of trivalent lanthanum,cerium and yttrium thiocynate complexes by tributyl phosphate

The distribution of La(III), Ce(III) and Y(III) from potassium thiocyanate solutions by tributyl phosphate is described. The dependence of extraction on pH, thiocyanate concentration, metal and extractant concentration, diluent type and temperature, was thoroughly investigated. Solvation numbers and thermodynamic data were calculated and discussed. A method has been suggested for the separation of Th(IV) from such elements.     

Metal ion complexes in the structural analysis of phospholipids by electrospray ionization tandem mass spectrometry

The effects of metal cationization on collisionally activated dissociation (CAD) of phospholipids were investigated by electrospray ionization with quadrupole ion trap tandem mass spectrometry. The metal ions include Li(+), Na(+), K(+), Sr(2+), Ba(2+), and the first transition series. CAD of the transition metal ion-bound lipid complexes gave significant yields of product ions that identify the positions of the two fatty acyl substituents on the glycerophospholipid backbone. The cobalt(II) ion, which has a single naturally occurring isotope, was expected to be a better cationization reagent as it produces simpler mass spectra than other transition metal ions. CAD of the cobalt(II) ion complexes of glycerophosphoethanolamines, glycerophosphoglycerols and glycerophosphoserines yielded product ions that revealed information regarding both the lipid classes and the regiospecific positions of the two fatty acyl substituents.     

Determination of lipids in infant formula powder by direct extraction methylation of lipids and fatty acid methyl esters (FAME) analysis by gas chromatography.

Fatty acid methyl esters (FAMEs) of pure triglyceride standards, oils, and fat from dry matrixes were formed by transesterification using sodium methoxide in methanol-hexane. FAMEs were produced by direct addition of sodium methoxide-hexane to samples and heating to simultaneously extract and transesterify acyl lipids. FAMEs were quantitated by capillary gas chromatography (GC) over a fatty acid concentration range of 0 to 1.7 mg/mL (r > or = 0.9997). Total fat was calculated as the sum of individual fatty acids expressed as triglyceride equivalents, in accordance with nutrition labeling guidelines. Saturated, polyunsaturated, and monounsaturated fats were calculated as sums of individual free fatty acids. Absolute recoveries determined from individual fatty acids in test samples ranged from 69.7 to 106%. Recoveries (relative to the C13:0 internal standard) for individual fatty acids in test samples ranged from 95 to 106%. Reproducibility was constant at each fatty acid level in the reaction mixture (n = 5, coefficient of variation [CV] < 2%). Absolute recovery determined from the sum of total fatty acids in standard reference material (SRM) 1846 (powdered infant formula) was 96.4%. Analysis of SRM 1846 gave results that agreed closely with the certified fat and fatty acid values. Analysis of commercial infant formula gave results that were comparable to those obtained with AOAC Method 996.01. The direct extraction methylation procedure is rapid, and the transesterification of acyl lipids to form FAMEs is complete within 15 min. Classical saponification and refluxing are not required. This method provides FAMEs free of interferences and easily quantitated by GC or confirmed by GC/mass spectrometry (MS). Unambiguous MS identification of individual FAMEs derived from pure standards, SRM 1846, and powdered infant formula product was obtained.     

Separation of trace amounts of palladium (II) with crown ether from hydrochloric acid and potassium thiocyanate media

A new method for the separation of trace amounts of palladium from hydrochloric acid and potassium thiocyanate media has been established based on the formation of an ion-pair complex of palladium thiocyanate anion Pd(SCN)4(2-) and the cationic potassium complex of dicyclohexyl- 18-crown-6 (DC18C6) in chloroform. The effect of various factors (solvent, crown ether, potassium thiocyanate, hydrochloric acid, reagent concentration, shaking time, phase volume ratio, composition of the extracted species, foreign ions, etc.) on the extraction and back-extraction of palladium has been investigated. The method can be combined with subsequent FAAS determination of palladium. The procedure was applied to determine palladium traces in chloroplatinic acid and rhodium chloride.     

1 H and 13C NMR Assignments of 2.3-Diacetylamido-2,3-Dideoxy-D-Glucopyranose

Abstract

Lipid A exhibits thp most important biological attributes of lipopolysaccharide (LPS) of gram-negative bacteria including endotoxicity, adjuvanticity and antitu-mor activity.′ The lipid A backbone, in general, is found to consist of a pyranosidic β 1,6-linked D-glucosamine disaccharide [β-D-Glc_pN-(1→6)-α-D-Glc_pN] phospho-rylated at 1 and 4′ positions and bearing two amide bound and two ester linked hydroxy and/or acyloxy fatty acids.² However, the lipid A moiety of LPS from var-ious strains of the two gram-negative, photosynthetic bacteria, Rhodopseudomonas virtdia and Rhodopseudomonas palustrts, possesses 2,3-diamino-2,3-dideoxy-D-glucose as a constituent sugar. 3 This diamino sugar has been also reported to occur in LPS from several other bacterial specie^4.5 Recently we found that the lipid X of Brucella abortus contains p(1→6)-linked 2,3-diamino-2,3-dideoxy-D- glucopyranose disaccharide moiety with a phosphate group at the 4′ position and amide bound acyloxy and hydroxy fatty acids.⁶     

基于超高效液相色谱-四极杆-静电场轨道离子阱串联质谱的冷链贮藏过程中滩羊肉的脂质组学研究

该文建立了超高效液相色谱一四极杆一静电场轨道离子阱串联质谱(UHPLC-Q-Orbitrap MS/MS)结合脂质组学分析滩羊肉在冷链贮藏过程中脂质变化规律和脂质分子碎裂机理的方法.样品经异丙醇提取后,采用质谱全扫描模式和二级扫描模式对目标物质进行定性.共鉴定出48个变化显著性脂质,包括8个脂肪酰基肉碱、23个磷脂酰胆...     

Free pyrene probes in gel and fluid membranes: perspective through atomistic simulations

We consider the properties of free pyrene probes inside gel- and fluidlike phospholipid membranes and unravel their influence on membrane properties. For this purpose, we employ atomic-scale molecular dynamics simulations at several temperatures for varying pyrene concentrations. Molecular dynamics simulations show that free pyrene molecules prefer to be located in the hydrophobic acyl chain region close to the glycerol group of lipid molecules. Their orientation is shown to depend on the phase of the membrane. In the fluid phase, pyrenes favor orientations where they are standing upright in parallel to the membrane normal, while, in the gel phase, the orientation is affected by the tilt of lipid acyl chains. Pyrenes are found to locally perturb membrane structure, while the nature of perturbations in the gel and fluid phases is completely different. In the gel phase, pyrenes break the local packing of lipids and decrease the ordering of lipid acyl chains around them, while, in the fluid phase, pyrenes increase the ordering of nearby acyl chains, thus having an opposite effect. Interestingly, this proposes a similarity to effects induced by cholesterol on structural membrane properties above and below the gel-fluid transition temperature. Further studies express a view that the orientational ordering of pyrene is not a particularly good measure of the acyl chain ordering of lipids. While pyrene ordering provides the correct qualitative behavior of acyl chain ordering in the fluid phase, its capability to predict the correct temperature dependence is limited.     

Separation of trace amounts of palladium (II) with crown ether from hydrochloric acid and potassium thiocyanate media

A new method for the separation of trace amounts of palladium from hydrochloric acid and potassium thiocyanate media has been established based on the formation of an ion-pair complex of palladium thiocyanate anion Pd(SCN)₄ ^2– and the cationic potassium complex of dicyclohexyl-18-crown-6 (DC18C6) in chloroform. The effect of various factors (solvent, crown ether, potassium thiocyanate, hydrochloric acid, reagent concentration, shaking time, phase volume ratio, composition of the extracted species, foreign ions, etc.) on the extraction and back-extraction of palladium has been investigated. The method can be combined with subsequent FAAS determination of palladium. The procedure was applied to determine palladium traces in chloroplatinic acid and rhodium chloride.     

Effects of covalent modification by 4‐hydroxy‐2‐nonenal on the noncovalent oligomerization of ubiquitin

When lipid membranes containing ω‐6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4‐hydroxy‐2‐nonenal (HNE)—a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non‐covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd.     

pH-Metric determination of acid values in oilseeds without titration

A new pH-metric method for determination of acid values in oilseeds without titration has been developed in the range 0.6-10 and more mg KOH/g. The method is based on a rapid (1-2 min) selective and complete extraction of free fatty acids from an oilseed test portion into a special reagent A, separation of the solution from the solid oilseed material by centrifugation or filtration, transfer of an aliquot of the solution into a pH-metric cell with reagent B for measurement of conditional pH(1)' of the formed mixture, addition of standard acid (HCl or H(2)SO(4)) and pH(2)' measurement. The reagents are non-toxic, and the method is rapid. Its metrological parameters for Soybean, Canola and Sunflower oilseeds are satisfactory for practical purposes.     

The Structure of a Novel Neutral Lipid A from the Lipopolysaccharide of Bradyrhizobium elkanii Containing Three Mannose Units in the Backbone

The chemical structure of the lipid A of the lipopolysaccharide (LPS) from Bradyrhizobium elkanii USDA 76 (a member of the group of slow‐growing rhizobia) has been established. It differed considerably from lipids A of other Gram‐negative bacteria, in that it completely lacks negatively charged groups (phosphate or uronic acid residues); the glucosamine (GlcpN) disaccharide backbone is replaced by one consisting of 2,3‐dideoxy‐2,3‐diamino‐D ‐glucopyranose (GlcpN3N) and it contains two long‐chain fatty acids, which is unusual among rhizobia. The GlcpN3N disaccharide was further substituted by three D ‐mannopyranose (D ‐Manp) residues, together forming a pentasaccharide. To establish the structural details of this molecule, 1D and 2D NMR spectroscopy, chemical composition analyses and high‐resolution mass spectrometry methods (electrospray ionisation Fourier‐transform ion cyclotron resonance mass spectrometry (ESI FT‐ICR MS) and tandem mass spectrometry (MS/MS)) were applied. By using 1D and 2D NMR spectroscopy experiments, it was confirmed that one D ‐Manp was linked to C‐1 of the reducing GlcpN3N and an α‐(1→6)‐linked D ‐Manp disaccharide was located at C‐4′ of the non‐reducing GlcpN3N (α‐linkage). Fatty acid analysis identified 12:0(3‐OH) and 14:0(3‐OH), which were amide‐linked to GlcpN3N. Other lipid A constituents were long (ω‐1)‐hydroxylated fatty acids with 26–33 carbon atoms, as well as their oxo forms (28:0(27‐oxo) and 30:0(29‐oxo)). The 28:0(27‐OH) was the most abundant acyl residue. As confirmed by high‐resolution mass spectrometry techniques, these long‐chain fatty acids created two acyloxyacyl residues with the 3‐hydroxy fatty acids. Thus, lipid A from B. elkanii comprised six acyl residues. It was also shown that one of the acyloxyacyl residues could be further acylated by 3‐hydroxybutyric acid (linked to the (ω‐1)‐hydroxy group).     

Thin-layer chromatography-pyrolysis-gas chromatography-mass spectrometry: a multidimensional approach to marine lipid class and molecular species analysis

A new multidimensional chromatographic method is described in which material separated into lipid-class bands on silica-coated quartz thin-layer chromatography (TLC) rods (Chromarods) is desorbed using a pyrolysis unit interface and introduced directly into a gas chromatograph-mass spectrometer for molecular species analysis. Steryl esters, wax esters, hydrocarbons, ketones, and fatty-acid methyl esters (FAMEs) are thermally desorbed without pretreatment. In order to desorb free sterols, monoacylglycerols (MAGs), aliphatic alcohols, and free fatty acids, the esters are converted to trimethylsilyl derivatives on the rod. Triacylglycerols and phospholipids are converted to FAMEs by thermochemolysis with tetramethylammonium hydroxide. The method's utility is demonstrated with lipids from seawater particulate matter by first confirming the identity of lipid bands with the appropriate standards. The wax ester-steryl ester TLC band contained no more than 8% steryl esters. Wax esters of up to C42 are detected. In six individual acyl lipid classes, C14-C22 fatty acids are detected with C16 acids predominant in all but wax esters. C16-C22 MAGs are identified in the complex acetone-mobile polar lipid band. The method successfully extends the scope of latroscan TLC-flame-ionization detection on Chromarods, which is a widely used technique for lipid-class analysis. Modification of the pyrolysis probe to handle intact TLC rods is a future objective.