Coupling of cholesterol-rich lipid phases in asymmetric bilayers

We showed previously that cholesterol-rich liquid-ordered domains with lipid compositions typically found in the outer leaflet of plasma membranes induce liquid-ordered domains in adjacent regions of asymmetric lipid bilayers with apposed leaflets composed of typical inner leaflet lipid mixtures [Kiessling, V., Crane, J. M., and Tamm, L. K. (2006) Biophys. J. 91, 3313-26]. To further examine the nature of transbilayer couplings in asymmetric cholesterol-rich lipid bilayers, the effects on the lipid phase behavior in asymmetric bilayers of different lipid compositions were investigated. We established systems containing several combinations of natural extracted and synthetic lipids that exhibited coexisting liquid-ordered (lo) and liquid-disordered (ld) domains in a supported bilayer format. We find that lo phase domains are induced in all quaternary inner leaflet combinations composed of PCs, PEs, PSs, and cholesterol. Ternary mixtures of PCs/PEs/Chol, PCs/PSs/Chol also exhibit lo phases adjacent to outer leaflet lo phases. However, with the exception of brain PC extracts, binary PC/Chol mixtures are not induced to form lo phases by adjacent outer leaflet lo phases. Higher melting lipid ad-mixtures of PEs and PSs are needed for lo phase induction in the inner leaflet. It appears that the phase behavior of the inner leaflet mixtures is dominated by the intrinsic chain melting temperatures of the lipid components, rather than by their specific headgroup classes. In addition, similar studies with synthetic, completely saturated lipids and cholesterol show that lipid oxidation is not a factor in the observed phase behavior.     

Synthesis of 5,9-hexacosadienoic acid phospholipids. 11. Phospholipid studies of marine organisms

The synthesis of phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines (PS) containing two acyl chains of the naturally occurring sponge fatty acid (5Z,9Z)-5,9-hexacosadienoic acid as well as its hitherto unknown geometrical isomers is described. The PCs were prepared by deacylation of natural lecithins, followed by reacylation with fatty acid anhydrides. The synthesis of mixed-acid PCs is also reported: a diacyl product was converted to the lyso-PC by treatment with phospholipase A2 and subsequent acylation of the secondary hydroxyl group to give the desired mixed-acid PCs. The PEs and the PSs were prepared from the corresponding PCs by enzymatic transphosphatidylation catalyzed by phospholipase D. Structural assignments of the compounds were confirmed by spectroscopy (1H-NMR and MS). Ammonia chemical ionization mass spectrometry provided molecular ion and significant fragment peaks for PCs and PEs.     

Characterisation of a potential biomarker of phospholipidosis from amiodarone-treated rats

A novel and relatively simple analytical method for the separation, characterisation and semi-quantitation of phospholipids (PLs) from extracts of complex biological samples has been developed. This methodology allows PL extracts from cells and tissues to be analysed by liquid chromatography (LC) coupled to electrospray ionisation mass spectrometry (ESI-MS). Complex mixtures of PLs were separated on a high-performance liquid chromatography (HPLC) system using 0.5% ammonium hydroxide in methanol/water/hexane/formate mixture with UV detection at 205 nm. Identification and structural characterisation of molecular species were carried out utilising ESI-MS and MS/MS in the negative ion mode.The abnormal accumulation of PLs (phospholipidosis) was induced in male Sprague-Dawley rats by administration of the cationic amphiphilic drug (CAD), amiodarone. Analysis of the PL profile of liver and lung tissues, lymphocytes and serum from treated rats was carried out using this analytical procedure (LC-ESI/MS/MS). Differences in PL profiles between treated and untreated animals were highlighted by principal component analysis (PCA). This led to the selection of a potential metabolic marker of phospholipidosis (PLD) identified as a lyso-bis-phosphatidic acid (LBPA) derivative, also known as bis(monoglycero)phosphate (BMP). This PL was absent in control animals but was present in quantifiable amounts in all samples from amiodarone-treated rats.     

Remodelling of membrane phospholipids during transition to diapause and cold-acclimation in the larvae of Chymomyza costata (Drosophilidae)

The composition of molecular species of phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) was analysed in fat body and muscle tissues of Chymomyza costata larvae of different physiological states that markedly differed in their level of freeze-tolerance. Actively moving and feeding 3rd instar larvae had low (zero) capacity of freeze-tolerance and similar phospholipid (PL) compositions irrespective of their developmental destiny (non-diapause vs. diapause). Extensive remodelling of PL composition was found in these larvae in response to: (a) chilling of non-diapause larvae at 5 degrees C for 1 month; (b) developmental transition to diapause; and (c) chilling of diapause larvae. Transition to diapause and chilling led to an increase in freeze-tolerance. The increase in molar proportion of molecular species containing palmitic/linoleic (16:0/18:2) fatty acyls (FAs) esterified to sn-1/sn-2 positions of glycerol was the most prominent change, which was tightly statistically correlated with increasing freeze-tolerance. The increase of PLs with combination of 16:0/18:2 FAs was registered consistently in PEs and PCs in fat body and muscle tissues in response to chilling and to diapause onset. This increase was countered by a decreases of various molecular species, depending on tissue and lipid class. Most decreasing species shared one common theme: they had a saturated FA (palmityl, margaryl, stearyl) esterified at sn-1 position and a monounsaturate (palmitoleyl, oleyl) esterified at sn-2 position of glycerol. Possible adaptive meaning of PL molecular species remodelling is discussed.     

Effects of high-sugar,high-cholesterol,and high-fat diet on phospholipid profile of mouse tissues with a focus on the mechanism of plasmalogen synthesis

High-sugar diet (HSD), high-cholesterol diet (HCD), and high-fat diet (HFD) all modulate the levels of lipids. However, there is a lack of comparative data on the effects of different diets on phospholipids (PLs). Given their important role in physiology and disease, there has been an increasing focus on altered PLs in liver and brain disorders. This study aims to determine the effects of HSD, HCD, and HFD for 14-week feeding on the PL profile of the mouse liver and hippocampus. Quantitative analysis of 116 and 113 PL molecular species in liver and hippocampus tissues revealed that the HSD, HCD, and HFD significantly affected the PLs in liver and hippocampus, especially decreased the levels of plasmenylethanolamine (pPE) and phosphatidylethanolamine (PE). Overall, the impact of HFD on liver PLs was more significant, consistent with the morphological changes in the liver. Compared to HSD and HCD, HFD induced a significant decrease in PC (P-16:0/18:1) and an increase in LPE (18:0) and LPE (18:1) in liver. In the liver of mice fed with different diets, the expression of the key enzymes Gnpat, Agps in the pPE biosynthesis pathway and peroxisome-associated membrane proteins pex14p were decreased. In addition, all diets significantly reduced the expression of Gnpat, pex7p, and pex16p in hippocampus tissue. In conclusion, HSD, HCD, and HFD enhanced lipid accumulation in the liver, led to liver injury, significantly affected the liver and hippocampus PLs, and decreased the expression of genes related to plasmalogen synthesis in mouse liver and hippocampus, which caused severe plasmalogen reduction.     

Lipidomic analysis of phospholipids from human mammary epithelial and breast cancer cell lines

Alterations of phospholipid (PL) profiles have been associated to disease and specific lipids may be involved in the onset and evolution of cancer; yet, analysis of PL profiles using mass spectrometry (MS) in breast cancer cells is a novel approach. Previously, we reported a lipidomic analysis of PLs from mouse mammary epithelial and breast cancer cells using off‐line thin layer chromatography (TLC)‐MS, where several changes in PL profile were found to be associated with the degree of malignancy of cells. In the present study, lipidomic analysis has been extended to human mammary epithelial cells and breast cancer cell lines (MCF10A, T47‐D, and MDA‐MB‐231), using TLC‐MS, validated by hydrophilic interaction liquid chromatography‐MS. Differences in phosphatidylethanolamine (PE) content relative to total amount of PLs was highest in non‐malignant cells while phosphatidic acid was present with highest relative abundance in metastatic cells. In addition, the following differences in PL molecular species associated to cancer phenotype, metastatic potential, and cell morphology were found: higher levels of alkylacyl PCs and phosphatidylinositol (PI; 22:5/18:0) were detected in migratory cells, epithelial cells had less unsaturated fatty acyl chains and shorter aliphatic tails in PE and sphingomyelin classes, while PI (18:0/18:1) was lowest in non‐malignant cells compared to cancer cells. To date, information about PL changes in cancer progression is scarce, therefore results presented in this work will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for cancer therapy. J. Cell. Physiol. 228: 457–468, 2013. © 2012 Wiley Periodicals, Inc.     

Cubic phases in phosphatidylcholine-cholesterol mixtures: cholesterol as membrane "fusogen"

X-ray diffraction reveals that mixtures of some unsaturated phosphatidylcholines (PCs) with cholesterol (Chol) readily form inverted bicontinuous cubic phases that are stable under physiological conditions. This effect was studied in most detail for dioleoyl PC/Chol mixtures with molar ratios of 1:1 and 3:7. Facile formation of Im3m and Pn3m phases with lattice constants of 30-50 nm and 25-30 nm, respectively, took place in phosphate-buffered saline, in sucrose solution, and in water near the temperature of the Lalpha-HII transition of the mixtures, as well as during cooling of the HII phase. Once formed, the cubic phases displayed an ability to supercool and replace the initial Lalpha phase over a broad range of physiological temperatures. Conversion into stable cubic phases was also observed for mixtures of Chol with dilinoleoyl PC but not for mixtures with palmitoyl-linoleoyl PC or palmitoyl-oleoyl PC, for which only transient cubic traces were recorded at elevated temperatures. A saturated, branched-chain PC, diphytanoyl PC, also displayed a cubic phase in mixture with Chol. Unlike the PEs, the membrane PCs are intrinsically nonfusogenic lipids: in excess water they only form lamellar phases and not any of the inverted phases on their own. Thus, the finding that Chol induces cubic phases in mixtures with unsaturated PCs may have important implications for its role in fusion. In ternary mixtures, saturated PCs and sphingomyelin are known to separate into liquid-ordered domains along with Chol. Our results thus suggest that unsaturated PCs, which are excluded from these domains, could form fusogenic domains with Chol. Such a dual role of Chol may explain the seemingly paradoxical ability of cell membranes to simultaneously form rigid, low-curvature raft-like patches while still being able to undergo facile membrane fusion.     

Lipid metabolism during aging of high-alpha-linolenate-phenotype potato tubers

Previous studies demonstrated that high levels of alpha-linolenate in cell membranes of potato tubers (achieved by overexpressing fatty acid desaturases) enhances lipid peroxidation, oxidative stress, and tuber metabolic rate, effectively accelerating the physiological age of tubers. This study details the changes in lipid molecular species of microsomal and mitochondrial membranes from wild-type (WT) and high-alpha-linolenate tubers during aging. The microsomal and mitochondrial polar lipids of high-alpha-linolenate tubers were dominated by 18:3/18:3 and 16:0/18:3 molecular species. Relative to WT tubers, high-alpha-linolenate tubers had a substantially higher 16:0/18:n to 18:n/18:n molecular species ratio in mitochondria and microsomes, potentially reflecting a compensatory response to maintain membrane biophysical properties in the face of increased unsaturation. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for 53 and 37% of polar lipids, respectively, in mitochondria from younger WT and high-alpha-linolenate tubers. The relative proportions of these phospholipids (PL) did not change during aging of WT tubers. In contrast, PE increased to dominate the PL pool of mitochondria during aging of high-alpha-linolenate tubers. While aging effected an increase in mitochondrial 18:3-bearing PCs and PEs in WT tubers, the concentration of 18:3-bearing PCs fell with a concomitant increase in 18:3-bearing PEs during aging of high-alpha-linolenate tubers. These age- and high-alpha-linolenate-induced changes had no effect on the respiration rate and functional integrity of isolated mitochondria. Differential increases in the respiration rates of WT and high-alpha-linolenate tubers during aging were therefore a consequence of unsaturation-dependent alterations in the microenvironments of cells. Microsomal 18:3-bearing PCs, PEs, digalactosyldiacylglycerols (DGDG), and monogalactosyldiacylglycerols all increased in WT tubers during aging. In contrast, a selective loss of 18:3-bearing PCs and DGDGs from microsomes of high-alpha-linolenate tubers likely reflects a greater susceptibility of membranes to peroxidative catabolism during aging. Aging resulted in an increase in sterol/PL ratio in microsomes from WT tubers, due primarily to a decline in PL. In high-alpha-linolenate tubers, the increase in sterol/PL ratio during aging was due to increases in Delta 5-avenasterol and stigmasterol, indicating membrane rigidification and likely contributing to increased membrane permeability. Age-induced changes in 18:3-bearing lipids in membranes of transformed tubers are discussed relative to the development of oxidative stress and accelerated aging.     

Metabolic kinetics of pulmonary surfactant in newborn infants using endogenous stable isotope techniques

We compared kinetic indices of pulmonary surfactant metabolism in premature infants (n = 41) with respect to i) tracer ([1-(13)C1]acetate, [U-(13)C6]glucose, and [1,2,3,4-(13)C4] palmitate), ii) phospholipid (PL) pool (total PLs or disaturated PLs), or iii) instrumentation [gas chromatography/mass spectrometry (GC/MS) or GC-combustion-isotope ratio mass spectometry (GC-C-IRMS)]. Tracer incorporation was measured in PLs extracted from serial tracheal aspirates after a 24 h tracer infusion. The fractional catabolic rate (FCR), representing the total fractional turnover from all sources of surfactant production, was independent of tracer. The fractional synthesis rate of surfactant PL from plasma palmitate was significantly higher than that from palmitate synthesized de novo from acetate, and these two sources of palmitate together accounted for only half of the total surfactant production in preterm infants. [U-(13)C6]glucose showed significant recycling of the (13)C label in intermediary metabolism, distinguishable by GC-MS but not by GC-C-IRMS, resulting in a slower apparent FCR when GC-C-IRMS was used. The extracted PL pool did not affect the surfactant metabolic indices. We suggest that FCR should be used as a primary measure of surfactant turnover kinetics and that tracers labeling both de novo synthesis (acetate and glucose) and preformed pathways (plasma palmitate) can be used to partition the fractional contribution of each pathway to total production.     

The transfer of VLDL-associated phospholipids to activated platelets depends upon cytosolic phospholipase A2 activity

We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 microM PL) to platelets (2 x 10(8)/ml) was measured after incubations of 1 h at 37 degrees C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 microM); esculetin, a 12-lipoxygenase inhibitor (20 microM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A(2) (PLA(2)) inhibitor (100 microM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca(2+) chelator (20 microM); bromoenol lactone (BEL), a Ca(2+)- independent phospholipase A(2) (iPLA(2)) inhibitor (100 nM); and 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 microM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A(2) (cPLA(2)) and iPLA(2), only cPLA(2) is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA(2)-dependent process. The inhibition of iPLA(2) by BEL had no effect on PL transfers.     

Modulation of Fetal Rat Brain and Liver Phospholipid Content by Intraamniotic Ethyl Docosahexaenoate Administration

Abstract: In an attempt to elucidate the role of docosahexaenoic acid (DHA; 22:6n-3) in the developing brain, a method was devised whereby rapid enrichment of fetal brain and liver lipid with DHA was achieved. Fetal rats at 17 days of gestation were injected intraamniotically with ethyl docosahexaenoate (EtDHA). Control fetuses were administered ethyl oleate (EtOle). Brain lipid DHA content increased by almost 21% (p = 0.02) 3 days after EtDHA administration as compared with EtOle-injected fetuses, whereas liver lipid DHA content increased by almost 60% (p = 0.0002). At this time brain phosphatidylinositol content doubled, whereas phosphatidylserine (PS) content increased by >50% (p = 0.03). Increases in liver PS (+25.8%; p = 0.015) and sphingomyelin (+43.6%; p = 0.01) content were observed. A redistribution of total brain phospholipid (PL) DHA was observed following Et-DHA administration, resulting in a 56.4% increase in PS-DHA abundance (p < 0.05) and an 8.8% decrease in phosphatidylethanolamine-DHA abundance (p = 0.05). These results suggest modulation of fetal brain and liver PL and provide a method for enrichment of DHA content in discrete PLs during intrauterine life.     

Membrane cholesterol uptake by recombinant lipoproteins

Recombinant lipoproteins, prepared with apo A-I isolated from human high density lipoprotein (HDL) and various phospholipids (PLs), were compared with respect to their ability to remove cholesterol (Chol) from labelled erythrocyte ghost membranes. It was found that uptake of Chol was essentially complete following an 8 h incubation at 37 degrees C. Quantitation of the amount of cholesterol taken up showed that recombinants prepared from bovine brain sphingomyelin (BBSM) or dipalmitoyl phosphatidylcholine (DPPC) acquired the highest proportion of Chol (80-140 mol/mol protein), whereas shorter chain phospholipids like dimyristoyl phosphatidylcholine (DMPC) acquired little or no membrane Chol. Chemical analysis of the incubation products indicated that this latter result was due to loss of PL, presumably to the membrane, with consequent disruption of the recombinant particle. Results with DPPC:A-I recombinants of differing PL/protein ratios and sizes showed that Chol uptake was fairly constant at 0.70 mol Chol/mol PL. It is concluded that discoidal, phospholipid-rich recombinant lipoproteins can effectively take up substantial amounts of Chol from physiological membranes, provided that the PLs utilized form micellar complexes which are capable of retaining their structural integrity during the incubation with the membranes.     

Phospholipid regulation of a cyclic AMP-specific phosphodiesterase (PDE4) from U937 cells

The regulation of phosphodiesterase-4 (PDE4) by various phospholipids was explored using PDE4s partially purified from U937 cells. Preincubation (5 min, 4°C) of the large molecular weight PDE4 denoted “Peak 2 PDE4” with mixed phosphatidic acids (PAs) produced a 2-fold increase in its V_max without changing its K_m ( 2 μM) for cyclic AMP. This “activation” was not limited to PAs with specific fatty acid substituents: Synthetic PAs containing saturated and/or unsaturated fatty acids 16-20 carbons long produced similar effects. Lysophosphatidic acids (LPAs) and phosphatidylserines (PSs) also induced PDE4 activation, whereas phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and diacylglycerol did not. Antibodies to a peptide region near the PDE4 catalytic site specifically inhibited PA-induced activation. The data demonstrate that anionic phospholipids can act as non-essential activators of a leukocyte PDE4, and suggest biochemical crosstalk between phospholipid-dependent and cyclic AMP-dependent signalling pathways in human leukocytes.     

Cloning of the pectate lyase genes from Erwinia carotovora and their expression in Escherichia coli

A hybrid cosmid coding for pectate lyase (PL) activity was identified from an Erwinia carotovora genomic library by an immunological screening method. A 7-kb DNA fragment was identified which codes for three proteins identical in size to proteins with PL activity purified from E. carotovora culture supernatants. The three proteins had apparent Mrs of 41, 44 and 44 X 10(3) as estimated by SDS-PAGE. None of the PLs were exported from Escherichia coli strain HB101 but all were found in the periplasmic space. Plant tissue was macerated by the PLs made in E. coli.     

Effects of alkylphenols on glycerophospholipids and cholesterol in liver and brain from female Atlantic cod (Gadus morhua)

Offshore oil production releases large amounts of lipophilic compounds in produced water into the ocean. In 2004, 143 million m(3) produced water, containing approximately 13 tons of long-chain (>C(4)) alkylphenols (AP), was discharged from installations in the Norwegian sector of the North Sea. Long-chain APs are known to cause endocrine disruption in a number of species. However, relatively little is known about their long-term effects in the marine environment. In the present study, Atlantic cod (Gadus morhua) were exposed (0.02 to 80 mg AP/kg) to a mixture (1:1:1:1) of APs (4-tert-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol and 4-n-heptylphenol) or 17 beta-estradiol (5 mg E2/kg) for 5 weeks and the effect on the fatty acid profile and cholesterol content in the membrane lipids from the liver and the brain was studied. We also determined the interaction between different para-substituted APs and glycerophospholipids (native phospholipids extracted from cod liver and brain) and model phosphatidylcholine (PC 16:0/22:6 n-3) in monolayers with the Langmuir-Blodget technique. The study demonstrated that APs and E2 alter the fatty acid profile in the polar lipids (PL) from the liver to contain more saturated fatty acids (SFA) and less n-3 polyunsaturated fatty acids (n-3 PUFA) compared with control. In the brain of the exposed groups a similar effect was demonstrated, although with higher saturation of the fatty acids found in the neutral lipids (mainly cholesterol ester), but not in the polar lipids. The AP and E2 exposure also gave a decline in the cholesterol levels in the brain. The in vitro studies showed that APs increased the mean molecular areas of the PLs in the monolayers at concentrations down to 5 microM, most likely due to intercalation of the APs between PL molecules. The increase in molecular area increased with the length of the alkyl side chain.     

Analysis of pectate lyases produced by soft rot bacteria associated with spoilage of vegetables.

Isoelectric focusing (IEF) profiles of pectate lyases (PLs) produced by five different groups of soft rot bacteria were analyzed by using the combined techniques of thin-layer polyacrylamide gel IEF and agarose-pectate overlay activity staining. Four strains of soft rot Erwinia spp. produced three or more PL isozymes. All of eight Pseudomonas viridiflava strains examined produced one single PL with a pI of 9.7. All 10 of Pseudomonas fluorescens strains produced two PLs; the major one had a pI of 10.0 and the minor one had a pI of 6.7. A single PL with a pI of greater than or equal to 10.0 was detected in one strain each of Xanthomonas campestris and Cytophaga johnsonae. PLs of six representative strains were purified from culture supernatants by ammonium sulfate precipitation and anion-exchange chromatography. All purified PL samples macerated potato slices, but to different degrees. The Mrs of alkaline PLs produced by P. viridiflava, P. fluorescens, X. campestris, and C. johnsonae were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000, 41,000, 41,500, and 35,000, respectively. IEF profiles of PLs were distinct among the bacterial species. Profiles of non-Erwinia spoilage bacteria were considerably simpler than those of Erwinia spp. The PL with an alkaline pI appeared to be the principal or the sole enzymatic factor involved in tissue maceration caused by most strains of soft rot bacteria.     

Analysis of pectate lyases produced by soft rot bacteria associated with spoilage of vegetables

Isoelectric focusing (IEF) profiles of pectate lyases (PLs) produced by five different groups of soft rot bacteria were analyzed by using the combined techniques of thin-layer polyacrylamide gel IEF and agarose-pectate overlay activity staining. Four strains of soft rot Erwinia spp. produced three or more PL isozymes. All of eight Pseudomonas viridiflava strains examined produced one single PL with a pI of 9.7. All 10 of Pseudomonas fluorescens strains produced two PLs; the major one had a pI of 10.0 and the minor one had a pI of 6.7. A single PL with a pI of greater than or equal to 10.0 was detected in one strain each of Xanthomonas campestris and Cytophaga johnsonae. PLs of six representative strains were purified from culture supernatants by ammonium sulfate precipitation and anion-exchange chromatography. All purified PL samples macerated potato slices, but to different degrees. The Mrs of alkaline PLs produced by P. viridiflava, P. fluorescens, X. campestris, and C. johnsonae were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000, 41,000, 41,500, and 35,000, respectively. IEF profiles of PLs were distinct among the bacterial species. Profiles of non-Erwinia spoilage bacteria were considerably simpler than those of Erwinia spp. The PL with an alkaline pI appeared to be the principal or the sole enzymatic factor involved in tissue maceration caused by most strains of soft rot bacteria.     

Ethanolamine plasmalogens prevent the oxidation of cholesterol by reducing the oxidizability of cholesterol in phospholipid bilayers

The aims of the present study are to establish an appropriate system for assessing the oxidizability of cholesterol (CH) in phospholipid (PL) bilayers, and to explore the effect of ethanolamine plasmalogens on the oxidizability of CH with the system, through comparing with those of choline plasmalogens, phosphatidylethanolamine, and antioxidant alpha-tocopherol (Toc). Investigation of the effects of oxidants, vesicle lamellar forms, saturation level, and constituent ratio of PLs in vesicles on CH oxidation revealed the suitability of a system comprising unilamellar vesicles and the water-soluble radical initiator 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH). As CH oxidation in the system was found to follow the rate law for autoxidation without significant interference from oxidizable PLs, the oxidizability of CH in PL bilayers could be experimentally determined from the equation: k (p)/(2k (t))(1/2)=R (p)/[LH]R(i)(1/2) by measuring the rate of CH oxidation. It was found with this system that bovine brain ethanolamine plasmalogen (BBEP), bovine heart choline plasmalogen, and egg yolk phosphatidylethanolamine lower the oxidizability of CH in bilayers. Comparison of the dose-dependent effects of each PL demonstrated the greatest ability of BBEP to reduce the oxidizability. A time course study of CH oxidation suggested a novel mechanism of BBEP for lowering the oxidizability of CH besides the action of scavenging radicals.     

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A_1/2 (PLA_1/2) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA_1/2 and acyltransferase activities.     

Dimeric galectin-1 binds with high affinity to alpha2,3-sialylated and non-sialylated terminal N-acetyllactosamine units on surface-bound extended glycans

Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an approximately 29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent K(d) approximately 2-4 microM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Galbeta1-4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (-3Galbeta1-4GlcNAcbeta1-)(n)) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to alpha3-sialylated and alpha2-fucosylated terminal LN, but not to alpha6-sialylated and alpha3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo-beta-galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both approximately 3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.