Modification Of Phospholipids Research Articles

Hypochlorous Acid-Derived Modification of Phospholipids: Characterization of Aminophospholipids as Regulatory Molecules for Lipid Peroxidation

Hypochlorous acid (HOCl), an inflammatory oxidant derived from neutrophil myeloperoxidase, can chlorinate cytosolic proteins and nuclear DNA bases of target cells by passing through the cell membrane. However, little is known about the consequences of HOCl-derived modification of cell membrane components, including phospholipids. In this study, we characterize the reaction of HOCl with phospholipid molecules and found that aminophospholipids are the key molecules that chemically regulate lipid peroxidation. Upon incubation with HOCl, the peroxidation of egg yolk phosphatidylcholine was significantly enhanced in the presence of phosphatidylethanolamine (PE). In contrast, the peroxidation was significantly inhibited in the presence of phosphatidylserine (PS). On the basis of mass spectrometric and electron paramagnetic resonance characterization, the initiator of the peroxidation was identified as the nitrogen-centered radical originating from PE-derived chloramines, especially N,N-dichlorinated PE, a major product in the HOCl-modified PE. Although PS was also chlorinated upon reaction with HOCl, the formed chloramine rapidly decomposed to phosphatidylglycolaldehyde, a novel class of lipid aldehyde. Formation of phosphatidylglycolaldehyde was also confirmed in the porcine brain PS and erythrocyte cell membrane ghost exposed to HOCl. These results provide a novel mechanism for the HOCl-induced oxidative damage and its endogenous protection in the cell membrane at the site of inflammation.

Sodium and Epithelial Sodium Channels Participate in the Regulation of the Capacitation-associated Hyperpolarization in Mouse Sperm

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

Analysis of lecithin–cholesterol mixtures using Raman spectroscopy

FT-Raman spectroscopy has been used to investigate interactions between lecithin and cholesterol. Raman spectra of lecithin show multiple peaks which can be classified into three regions: hydrophobic chain, interfacial, and headgroup regions. Binary lipid mixtures (1:1, w/w, lecithin:cholesterol) were prepared by physical mixing, granulation, coprecipitation, hydration and heating (65 °C), and heating (120 °C). Regardless of the preparation method, no changes in the spectra were observed in the hydrophobic region. A shift in the wavenumber of the choline methyl asymmetric stretching mode was observed when the samples were prepared by coprecipitation, hydration and heating (65 °C), and heating (120 °C). This may indicate a modification of phospholipids in the headgroup region in these samples. The difference in degrees of frequency shift (physical mixing ≈ granulation < coprecipitation ≈ hydration and heating (65 °C) < heating (120 °C)) suggests that different levels of hydrogen bonding may have occurred in mixtures prepared with these methods. Multivariate analysis utilizing partial least squares regression based on selected wavenumber ranges was applied for the quantitative analysis of the amount of lecithin in lipid mixtures. Calibration models from physical mixing and heating (120 °C) exhibited lower R 2 and root mean square error of cross validation (RMSECV) values compared to the other models suggesting lower sample homogeneity for these preparation methods. Low values of the mean absolute residues and mean Mahalanobis distances imply that the calibration model generated from physical mixing samples may be appropriate for quantitative analysis of lecithin in lipid mixtures prepared by any of the other techniques.

Phospholipid Furan Fatty Acids and Ubiquinone-8: Lipid Biomarkers That May Protect Dehalococcoides Strains from Free Radicals

Dehalococcoides species have a highly restricted lifestyle and are only known to derive energy from reductive dehalogenation reactions. The lipid fraction of two Dehalococcoides isolates, strains BAV1 and FL2, and a tetrachloroethene-to-ethene-dechlorinating Dehalococcoides-containing consortium were analyzed for neutral lipids and phospholipid fatty acids. Unusual phospholipid modifications, including the replacement of unsaturated fatty acids with furan fatty acids, were detected in both Dehalococcoides isolates and the mixed culture. The following three furan fatty acids are reported as present in bacterial phospholipids for the first time: 9-(5-pentyl-2-furyl)-nonanoate (Fu18:2omega6), 9-(5-butyl-2-furyl)-nonanoate (Fu17:2omega5), and 8-(5-pentyl-2-furyl)-octanoate (Fu17:2omega6). The neutral lipids of the Dehalococcoides cultures contained unusually large amounts of benzoquinones (i.e., ubiquinones [UQ]), which is unusual for anaerobes. In particular, the UQ-8 content of Dehalococcoides was 5- to 20-fold greater than that generated in aerobically grown Escherichia coli cultures relative to the phospholipid fatty acid content. Naphthoquinone isoprenologues (MK), which are often found in anaerobically grown bacteria and archaea, were also detected. Dehalococcoides shows a difference in isoprenologue pattern between UQ-8 and MK-5 that is atypical of other bacteria capable of producing both quinone types. The difference in UQ-8 and MK-5 isoprenologue patterns strongly suggests a special function for UQ in Dehalococcoides, and Dehalococcoides may utilize structural modifications in its lipid armamentarium to protect against free radicals that are generated in the process of reductive dechlorination.

PI3K Regulates Pleckstrin-2 in T-Cell Cytoskeletal Re-Organization.

Pleckstrin-2, a paralog of pleckstrin-1, is composed of two Pleckstrin Homology (PH) domains and a Disheveled-Egl 10-Pleckstrin (DEP) domain. Several studies have shown that PH domains mediate binding of their host proteins to inositol phosphates and phospholipids, and thus regulate protein function. PH domains are found in many molecules involved in cellular signaling, cytoskeletal organization, membrane trafficking, and phospholipid modification. Proteins containing the DEP domain also regulate a broad range of cellular functions and evidence is emerging that several signaling proteins may rely on their DEP domains for membrane association. We speculated that the function of pleckstrin-2 is dependent upon its ability to bind to specific polyphosphatidylinositols. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to the products of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bind to the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. To examine the effect of pleckstrin-2 in human T-cells, Jurkat T-cells were transfected with GFP-tagged plasmids that direct the expression of pleckstrin-2 variants. Using confocal video microscopy we demonstrated that upon activation of the T-cell antigen receptor or the integrin α4β1, pleckstrin-2 rapidly moves from the cytoplasm to the cellular membrane and enhances membrane ruffling. Quantitation of cell footprint size revealed a two-fold increase in cell spreading. Furthermore, the membrane association of pleckstrin-2 and its resultant cell spreading were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. Consistent with this observation, a pleckstrin-2 variant containing point mutations in both of its PH domains failed to associate with the cell membrane and had no effect on spreading under the same conditions, suggesting that pleckstrin-2 membrane association occurs through a pathway dependent on the phospholipid-binding pocket of its PH domains. Although still membrane-bound, a pleckstin-2 variant containing point mutations in the second β-turn and second α-helical coil of the DEP domain demonstrated a decreased ability of pleckstrin-2 to induce membrane ruffles and lamellipodia, without decreasing filopodia formation. The cell footprint size of the DEP domain mutants was also decreased compared to that of wild type pleckstrin-2. These results suggest that the pleckstrin-2 DEP domain may function to promote actin-rich membrane extensions and ruffling. The localization of receptors, signaling intermediates, and cytoskeletal components at the T-cell/APC interface is thought to be a major determinant of efficient T-cell activation. Our data indicate that in T-lymphocytes, pleckstrin-2 uses modular motifs to bind to membrane-associated phosphatidylinositols, such as those generated by PI3K, to organize the actin cytoskeleton and to promote lymphocyte spreading.

Lipid Peroxidation Induces Cholesterol Domain Formation in Model Membranes

Numerous reports have established that lipid peroxidation contributes to cell injury by altering the basic physical properties and structural organization of membrane components. Oxidative modification of polyunsaturated phospholipids has been shown, in particular, to alter the intermolecular packing, thermodynamic, and phase parameters of the membrane bilayer. In this study, the effects of oxidative stress on membrane phospholipid and sterol organization were measured using small angle x-ray diffraction approaches. Model membranes enriched in dilinoleoylphosphatidylcholine were prepared at various concentrations of cholesterol and subjected to lipid peroxidation at physiologic conditions. At cholesterol-to-phospholipid mole ratios (C/P) as low as 0.4, lipid peroxidation induced the formation of discrete, membrane-restricted cholesterol domains having a unit cell periodicity or d-space value of 34 A. The formation of cholesterol domains correlated directly with lipid hydroperoxide levels and was inhibited by treatment with vitamin E. In the absence of oxidative stress, similar cholesterol domains were observed only at C/P ratios of 1.0 or higher. In addition to changes in sterol organization, lipid peroxidation also caused reproducible changes in overall membrane structure, including a 10 A reduction in the width of the surrounding, sterol-poor membrane bilayer. These data provided direct evidence that lipid peroxidation alters the essential organization and structure of membrane lipids in a manner that may contribute to changes in membrane function during aging and oxidative stress-related disorders.

Absence of 12/15 Lipoxygenase Reduces Brain Oxidative Stress in Apolipoprotein E-Deficient Mice

The enzyme 12/15 lipoxygenase (12/15LO) has been implicated in the oxidative modification of lipoproteins and phospholipids in vivo. In addition, mice deficient in apolipoprotein E (ApoE-/-) are characterized by spontaneous hypercholesterolemia and a systemic increase in oxidative stress. Whereas the absence of 12/15LO reduces lipid peroxidation in the plasma and urine of ApoE-/- mice, the relative contribution of this enzyme to oxidative stress in the central nervous system remains unknown. Here, we provide the first in vivo evidence that 12/15LO modulates brain oxidative stress reactions using ApoE-/- mice crossbred with 12/15LO-deficient (12/15LO-/-) mice (12/15LO-/-/ApoE-/-). In chow-fed 12-month-old 12/15LO-/-/ApoE-/- mice, the amount of brain isoprostane iPF2alpha-VI, a marker of lipid peroxidation, and carbonyls, markers of protein oxidation, were significantly reduced when compared with 12/15LO-expressing controls (12/15LO+/+/ApoE-/-). These results were observed despite the fact that cholesterol, triglyceride, and lipoprotein levels were similar to those of ApoE-/- mice. These data indicate a functional role for 12/15LO in the modulation of oxidative reactions in the central nervous system, supporting the hypothesis that inhibition of this enzymatic pathway may be a novel therapeutic target in clinical settings involving increased brain oxidative stress.

The mechanism of lipopolysaccharide infiltration through HUVEC membrane surface in direct endotoxin injury

In this study, the HUVEC's cellular biomechanical properties of HUVEC (elastic modulus K 1, K 2 and viscoefficient μ) were determined with micropipette aspiration system and analyzed after being directly damaged with lipopolysaccharide (LPS). The phospholipid compositions of HUVEC membrane were analyzed with high-performance capillary electrophoresis and PLA 2 activity was determined to research the modification and metabolism of HUVEC membrane phospholipid. Infiltration of LPS on HUVEC membrane was studied by observation with confocal microscopy and fluorescent microscopy. Results showed that LPS direct injuring HUVEC can cause the changes of HUVEC biomechanical properties and membrane lipid contents; HUVEC directly damage by LPS could also activate HUVEC phospholipase A 2 (PLA 2), influencing membrane lipid metabolism; LPS could directly infiltrate and intercalate HUVEC membrane, causing and membrane contents variation. Based on these experimental results, the mechanism of lipopolysaccharide infiltration VEC membrane surface in direct LPS injury was studied and analyzed in view of the cellular biomechanical mechanism.

Endurance training limits the functional alterations of heart rat mitochondria submitted to in vitro anoxia-reoxygenation

Background Studies analysing the effect of endurance training on heart mitochondrial function submitted to in vitro anoxia-reoxygenation (A-R) are missing. The present study aimed to investigate the effect of moderate endurance treadmill training (14 weeks) against rat heart mitochondrial dysfunction induced by in vitro A-R. Methods Respiratory parameters (state 3, state 4, ADP/O and respiratory control ratio—RCR) and oxidative damage markers (carbonyl groups and malondialdehyde) were determined in isolated mitochondria before and after 1 min anoxia followed by 4 min reoxygenation. Levels of heat shock protein 60 kDa (HSP60) and 70 kDa (HSP70) were measured before A-R in mitochondria and whole muscle homogenate, respectively. Results A-R significantly impaired the rate of state 3 and state 4 respiration, as well as the RCR and ADP/O in the sedentary group. However, mitochondrial state 3 respiration was significantly higher in trained than in the sedentary group both before and after A-R. The impairments in RCR, ADP/O ratio and state 4 induced by A-R in sedentary group were significantly attenuated in endurance-trained group. The inhibition of state 4 induced by GDP was significantly higher in trained than in sedentary group. Oxidative modifications of mitochondrial proteins and phospholipids were found in sedentary group after A-R, although limited in trained group. Increased levels of mitochondrial HSP60 and tissue HSP70 accompanied the lower decrease in the respiratory function after A-R observed in trained group. Conclusion We therefore concluded that endurance training limited the impairments on rat heart mitochondria caused by the oxidant insult inflicted by in vitro A-R.

Interplay between the Maillard Reaction and Lipid Peroxidation in Biochemical Systems

The Maillard reaction and lipid peroxidation are two of the most important chemical reactions that take place in biochemical systems. Both include a whole network of different reactions in which an extraordinarily complex mixture of compounds is produced in very different amounts, with both positive and negative consequences. In addition, both reactions are intimately interrelated, and the products of each reaction influence the other. Furthermore, there are common intermediates and products in both pathways; these products are usually known as advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). Moreover, other AGE/ALEs are analogous and participate similarly in both amino acid degradation and amino phospholipid/protein polymerization by identical mechanisms. All these data suggest that the Maillard reaction and lipid peroxidation are so closely interrelated that both reactions should be considered simultaneously to understand the reaction mechanisms, kinetics, and products in the complex mixtures of carbohydrates, lipids, and proteins occurring in biochemical systems. In these systems, lipids and carbohydrates are competing in the chemical modification of amino phospholipids and proteins. Therefore, although there are significant differences between the Maillard reaction and lipid peroxidation, many aspects of both reactions can be better understood if they are included in only one general carbonyl pathway that can be initiated by both lipids and carbohydrates.

Membrane assisted coupled enzyme system for phospholipid modification

Phospholipase D obtained from a culture of Streptomyces sp. strain PMF, was immobilised in a hollow fiber membrane by ultrafiltration. The reactor was used to contact an organic solution containing phosphatidylcholine (PC) and a water phase; the rate of formation of the hydrolysis product (phosphatidic acid, PA) depending on different parameters (pH, temperature, PC concentration and enzyme units) was measured. Subsequently, the water phase was replaced with a 2–3 M solution of different alcohols ( l-serine, glycerol and ethanolamine) and the rate of transphosphatidylation was studied and finally optimized using glycerol as model alcohol. The ratio of formation of phosphatidylglycerol (PG) (transphosphatidylation reaction) over PA (hydrolysis reaction) was minimized working at pH 4.5 where hydrolysis rate is lower. The operational stability of the system was excellent during a period of several months. Purification of the reaction mixture from PA was effected by selectively hydrolysing it with phosphatase (Pase). Co-immobilization of PLD and Pase resulted in effective PA removal. Although the space–time yield of the transphosphatidylation reaction is lower than the one observed in a biphasic system, product purification is unnecessary, product isolation easier and enzyme stability higher.

The Bacillus subtilis desaturase: a model to understand phospholipid modification and temperature sensing

Most fatty acid desaturases are members of a large superfamily of integral membrane, O2-dependent, iron-containing enzymes that insert double bonds into previously synthesized fatty acyl chains. The cold shock-induced, membrane-bound desaturase from Bacillus subtilis (Delta5-Des) uses existing phospholipids as substrates to introduce a cis-double bond at the fifth position of the fatty acyl chain. While essentially no three-dimensional structural information is available for these difficult-to-purify enzymes, experimental analysis of the topology of Delta5-Des has provided a model that might be extended to most acyl-lipid desaturases. In addition, studies of the cold-induced expression of Delta5-Des led to the identification of a two-component system composed of a membrane-associated kinase, DesK, and a transcriptional regulator, DesR, which stringently controls the transcription of the des gene, coding for the desaturase. A model for sensing and transduction of low-temperature signals has emerged from our results, which we discuss in the context of transcriptional regulation of membrane lipid fluidity homeostasis.

Enzyme‐Catalyzed modification of oilseed materials to produce eco‐friendly products

AbstractNovel products produced from seed oil materials (TAG, phospholipids, and minor components such as tocopherols, sterols, stanols, and fatty acyl esters of the latter two) by enzyme‐mediated purification or chemical modification are reviewed. The primary focus is on “value‐added products” of current and potential use (particularly in the food, cosmetics, and pharmaceutical industries) that require the selectivity of enzymes and mild operating conditions, the latter being beneficial for polyunsaturated and oxygenated acyl groups. The paper briefly reviews the biochemistry of enzymes in lipid modification (lipases, phospholipases, and lipoxygenases) and discusses and assesses the current and future applications, current state of the art, and areas for future research for the following enzyme‐mediated processes: isolation of polyunsaturated and oxygenated FFA; formation of structured TAG as nutraceuticals; formation of MAG, saccharide‐FA esters, and other polyhydric alcohol ester as emulsifiers and surfactants; isolation and/or modification of tocopherols and sterols as antioxidants; formation of hydroperoxides as chemical intermediates; and modification of phospholipids for use in liposomes.

Chemical‐physical properties of lipoproteins in anorexia nervosa

Anorexia nervosa (AN), a psychosomatic disorder, has serious negative effects on multiple organs and systems of the human body. Anorexia nervosa usually runs a chronic course and is associated with significant morbidity and mortality. In order to elucidate the role played by lipids in AN, in the present study we compared the plasma lipid profile and the chemical-physical properties of lipoproteins obtained from subjects affected by AN. The study was performed on lipoproteins of AN subjects and of age-matched healthy subjects used as controls. We tested the susceptibility to oxidative stress in vitro, the fatty acid content, the fluidity using 2-dimethylamino-(6-lauroyl)-naphthalene (Laurdan) and 1,6-difenil-1,3,5-esatriene (DPH) probes. Present results indicate that AN patients present a deep alteration of the composition and of chemical-physical properties in circulating lipoproteins, even in the absence of significant modifications to clinical metabolic parameters. A significantly decreased body mass index (BMI) was found in AN patients in comparison with controls. Anorexia nervosa patients showed a significant modification of phospholipids to protein ratio and a significantly increased percentage of unsaturated fatty acids compared with control subjects as well as a decreased fluidity, a significantly increased percentage of liquid-crystalline phase in VLDL, and a significantly reduced susceptibility to oxidative stress, more pronounced in LDL. These results confirm the hypothesis that anorexia is accompanied by changes of lipid metabolism in the central nervous system (CNS).

Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac.

After an acute phase of inflammation or injury, restoration of the endothelial barrier is important to regain vascular integrity and to prevent edema formation. However, little is known about mediators that control restoration of endothelial barrier function. We show here that oxidized phospholipids that accumulate at sites of inflammation and tissue damage are potent regulators of endothelial barrier function. Oxygenated epoxyisoprostane-containing phospholipids, but not fragmented oxidized phospholipids, exhibited barrier-protective effects mediated by small GTPases Cdc42 and Rac and their cytoskeletal, focal adhesion, and adherens junction effector proteins. Oxidized phospholipid-induced cytoskeletal rearrangements resulted in a unique peripheral actin rim formation, which was mimicked by coexpression of constitutively active Cdc42 and Rac, and abolished by coexpression of dominant-negative Rac and Cdc42. Thus, oxidative modification of phospholipids during inflammation leads to the formation of novel regulators that may be critically involved in restoration of vascular barrier function.

Lipase‐catalyzed acidolysis and phospholipase D‐catalyzed transphosphatidylation of phosphocholine

AbstractTwo approaches on enzymatic phospholipid modification were studied: (1) transphosphatidylation of the 1,2‐dilauroyl‐sn‐glycero‐3‐phosphocholine (DLPC) and ethanolamine in biphasic and anhydrous organic solvent systems by phospholipase D (PLD) and (2) incorporation of oleic acid into the sn1‐position of DLPC in organic solvents with different immobilized lipases at controlled water activity. First, DLPC was chemically synthesized from glycerophosphocholine and lauric acid. Next, PLD‐catalyzed head group exchange of DLPC with ethanolamine was studied using an enzyme from Streptomyces antibioticus expressed recombinantly in E. coli. A comparison of the free PLD with the biocatalyst activated by a salt‐activation technique using KCl showed that the salt‐activated enzyme (PLD‐KCl) was 10–12 folds more active based on the amount of protein used. Thus, DLPC was quantitatively converted to 1,2‐dilauroyl‐sn‐glycero‐3‐phosphoethanolamine in an anhydrous solvent system within 12 h at 60 °C. For the acidolysis of DLPC with oleic acid, among the four lipases studied (CAL‐B, Lipozyme TL IM, Lipozyme RM IM and lipase D immobilized on Accurel EP‐100), Lipozyme TL IM showed the highest activity and incorporation of oleic acid. A quantitative incorporation was achieved at 40 °C using a 8‐fold molar excess of oleic acid in n‐hexane at a water activity of 0.11.

Involvement of thempooperon in resistance to class IIa bacteriocins inListeria monocytogenes

High resistance to class IIa bacteriocins in Listeria monocytogenes has been clearly linked to lack of expression of the mptACD operon, encoding the EIIt Man mannose PTS permease. Also, intermediate resistance has been associated with membrane phospholipid modifications in the spontaneous mutants L. monocytogenes B73-V1 and B73-V2. We constructed a new mutant of L. monocytogenes that was interrupted in mpoA, and which also exhibited an intermediate resistance phenotype. The mpoABCD operon putatively encodes a PTS permease of the mannose family similar to that encoded by the mpt operon. In silico analysis indicated that mpo transcription might be dependent on sigma54. Our study demonstrated that the three intermediate resistant mutants have a slight decrease in mptACD expression, showing that the level of sensitivity is correlated to the level of mpt expression. We show a cross-regulation between mpo and mpt. In particular, the mpo mutant has a defect in mpt expression that possibly could explain its intermediate resistance phenotype.

Involvement of the mpo operon in resistance to class IIa bacteriocins in Listeria monocytogenes

High resistance to class IIa bacteriocins in Listeria monocytogenes has been clearly linked to lack of expression of the mptACD operon, encoding the EII t Man mannose PTS permease. Also, intermediate resistance has been associated with membrane phospholipid modifications in the spontaneous mutants L. monocytogenes B73-V1 and B73-V2. We constructed a new mutant of L. monocytogenes that was interrupted in mpoA, and which also exhibited an intermediate resistance phenotype. The mpoABCD operon putatively encodes a PTS permease of the mannose family similar to that encoded by the mpt operon. In silico analysis indicated that mpo transcription might be dependent on σ 54. Our study demonstrated that the three intermediate resistant mutants have a slight decrease in mptACD expression, showing that the level of sensitivity is correlated to the level of mpt expression. We show a cross-regulation between mpo and mpt. In particular, the mpo mutant has a defect in mpt expression that possibly could explain its intermediate resistance phenotype.

Enzymatic synthesis of structured lipids.

Structured lipids (SLs) are defined as lipids that are modified chemically or enzymatically in order to change their structure. This review deals with structured triacylglycerols (STGs) and structured phospholipids (SPLs). The most typical STGs are MLM-type STGs, having medium chain fatty acids (FAs) at the 1- and 3-positions and a long chain fatty acid at the 2- position. MLM-type STGs are synthesized by: 1) 1,3-position-specific lipase-catalyzed acyl exchange of TG with FA or with FA ethylester (FAEt); 2) 1,3-position-specific lipase-catalyzed acylation of glycerol with FA, giving symmetric 1,3-diacyl-sn-glycerol, followed by chemical acylation at the sn-2 position, and; 3) 1,3-position-specific lipase-catalyzed deacylation of TG, giving 2-monoacylglycerol, followed by reacylation at the 1- and 3-positions with FA or with (FAEt). Enzymatic preparation of SPLs requires: 1) acyl group modification, and 2) head group modification of phospholipids. Acyl group modification is performed using lipases or phospholipase A2-mediated transesterification or ester synthesis to introduce arbitrary fatty acid to phospholipids. Head group modification is carried out by phospholipase D-catalyzed transphosphatidylation. A wide range of compounds can be introduced into the polar head of phospholipids, making it possible to prepare various SPLs.

Phosphatidylethanolamine modification by oxidative stress product 4,5(E)-epoxy-2(E)-heptenal.

Phosphatidylethanolamine and ethanolamine were incubated with 4,5(E)-epoxy-2(E)-heptenal to analyze the changes produced in amino phospholipids as a consequence of their reaction with this lipid oxidation product. The reactions, which were followed by GC/MS or HPLC/MS, produced in a first step the corresponding imines, which later evolved into two main products. These products, which were isolated and characterized by (1)H and (13)C NMR and mass spectrometry, resulted in the corresponding phosphatidylethanolpyrroles (PEPs) and phosphatidylethanol-2-(1-hydroxypropyl)pyrroles (PEHPs). PEPs were relatively stable, but PEHPs polymerized spontaneously. This polymerization, which was followed in 4,5(E)-epoxy-2(E)-heptenal/ethanolamine incubations with the detection of the corresponding dimers and trimers, was related to the brown color and fluorescence development produced. In addition, when hydroxyalkylpyrroles were incubated in the presence of single pyrroles, mixed polymers of the two pyrroles were obtained, and these polymers were produced in a higher extent than those derived exclusively from PEHP. These results confirm that lipid oxidation products are able to react with amino phospholipids analogously to protein amino groups, and therefore, both amino phospholipids and proteins may compete for lipid oxidation products. Although these results do not demonstrate the formation of pyrrolyzed phospholipids in vivo, they suggest that analogously to pyrrolyzed proteins, these modified phospholipids are likely to be produced during the phospholipid peroxidation process. In addition, these results also provide new bases for assessing the role of phospholipid modification in certain diseases, pathophysiological conditions, and aging.