Lipid Raft Composition Research Articles

Vitamin E Prevents Lipid Raft Modifications Induced by an Anti-cancer Lysophospholipid and Abolishes a Yap1-mediated Stress Response in Yeast

We have previously established that the anti-cancer lysophospholipid edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, Et-18-OCH(3)) induces cell death in yeast by selective modification of lipid raft composition at the plasma membrane. In this study we determined that alpha-tocopherol protects cells from the edelfosine cytotoxic effect, preventing the internalization of sterols and the plasma membrane proton pump ATPase, Pma1p. Two non-mutually exclusive hypotheses were considered to explain the protective effect of alpha-tocopherol: (i) its classical antioxidant activity is necessary to break progression of lipid peroxidation, despite the fact Saccharomyces cerevisiae does not possess polyunsaturated fatty acids and (ii) due to its complementary cone shape, insertion of alpha-tocopherol could correct membrane curvature stress imposed by edelfosine (inverted cone shape). We then developed tools to distinguish between these two hypotheses and dissect the structural requirements that confer alpha-tocopherol its protective effect. Our results indicated its lipophilic nature and the H donating hydroxyl group from the chromanol ring are both required to counteract the cytotoxic effect of edelfosine, suggesting edelfosine induces oxidation of membrane components. To further support this finding and learn more about the early cellular response to edelfosine we investigated the role that known oxidative stress signaling pathways play in modulating sensitivity to the lipid drug. Our results indicate the transcription factors Yap1 and Skn7 as well as the major peroxiredoxin, Tsa1, mediate a response to edelfosine. Interestingly, the pathway differed from the one triggered by hydrogen peroxide and its activation (measured as Yap1 translocation to the nucleus) was abolished by co-treatment of the cells with alpha-tocopherol.

The lipid-modulating effects of a CD4-specific recombinant antibody correlate with ZAP-70 segregation outside membrane rafts

We previously reported that the anti-tumoral effects of the recombinant IgG 1 antibody 13B8.2, which is directed against the CDR3-like loop on the D1 domain of CD4, are linked to accumulation/retention of CD4 inside membrane rafts, recruitment of signaling molecules of the TCR/CD3 pathway and raft exclusion of the ZAP-70 kinase and its downstream targets Vav-1, PLCγ1 and SLP-76. We thus wanted to assess whether this compartmentalization could be related to a possible effect of 13B8.2 on the lipid composition of rafts. Here we show that 13B8.2 treatment of Jurkat T cells did not affect neutral lipids and particularly cholesterol content in GM1-positive membrane rafts, but decreased phosphatidylserine synthesis. C18:0 saturated fatty acid level in GM1-positive membrane rafts and ceramide release were concomitantly increased following treatment with 13B8.2. Antibody-induced ceramide release in membrane rafts occurred through enhanced acid sphingomyelinase activity and was blocked by the acid sphingomyelinase inhibitor imipramine, but was not affected by inhibitors of de novo ceramide synthesis, myriocin and fumonisin B1. Similarly to 13B8.2, addition of bacterial sphingomyelinase increased ceramide release and segregated ZAP-70 outside GM1-positive membrane rafts from Jurkat T cells. Besides CD4/ZAP-70 modulation in membrane rafts, the 13B8.2-induced activation of the acid sphingomyelinase/ceramide pathway is an important event for structuring raft platforms and transducing CD4-related intracellular signals, which can further fine-tune antibody-triggered tumoral effects.

Ursodeoxycholic acid and chemoprevention of colorectal cancer

Colorectal cancer is respectively the third and second most common cancer among men and women in France. Interest in chemoprevention for colorectal cancer has increased over the last two decades. Beside non-steroidal anti-inflammatory drugs, ursodeoxycholic acid (UDCA) may have chemopreventive action in colorectal cancer with a likely better tolerance. In high-risk populations such as patients with inflammatory bowel disease or prior colorectal adenoma or carcinoma, retrospective and prospective studies have suggested a beneficial effect of UDCA. In azoxymethane model, UDCA inhibits tumor development by countering the tumor-promoting effects of secondary bile acids, such as deoxycholic acid (DCA). The opposing effects of UDCA and DCA on lipid raft composition may be central to their effects on colonic tumorigenesis. Differential effects of DCA and UDCA on growth factor and inflammatory signals involved in colorectal carcinogenesis, such as epidermal growth factor receptors (EGFR) signaling and Cox-2 expression, likely mediate their opposing effects on colonic tumor promotion and tumor inhibition, respectively.

Binding of sterols affects membrane functioning and sphingolipid composition in wheat roots

The present work was devoted to the exploration of the role of sterols in the functioning of membranes in root cells. Membrane characteristics and composition of the membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with exogenous cholesterol and antibiotic nystatin, which specifically binds with endogenous sterols, were analyzed. Cholesterol caused a fall of membrane potential, acidification of the incubation medium, decrease in potassium leakage of roots, and increase in the level of exogenous superoxide radical. Similarly to cholesterol, the application of nystatin also induced the depolarization of the plasma membrane, but in contrast with cholesterol it was accompanied by alkalinization of the incubation medium and decrease in the level of exogenous superoxide radical. Analysis of membrane lipids showed that following nystatin treatment the total sterol content in roots did not change, while the level of complex sphingolipids represented mainly by glycoceramides became higher. Using mass spectrometry with electrospray ionization ((+)ESI-MS) for the analysis of the glycoceramide composition, we showed that nystatin induced changes in the ratios of molecular species of glycoceramides. It was suggested that the modification of the sterol component of plasma membrane could influence membrane functioning by changing the sphingolipid composition of lipid rafts.

The nutritional and clinical significance of lipid rafts

Lipid rafts are potentially modifiable by diet, particularly (but not exclusively) by dietary fatty acids. This review examines the potential for dietary modification of raft structure and function in the immune system, brain and retinal tissue, the gut, and in cancer cells. In-vitro and ex-vivo studies suggest that dietary n-3 polyunsaturated fatty acids (PUFAs) may exert immunosuppressive and anticancer effects through changes in lipid raft organization. In addition, gangliosides and cholesterol may modulate lipid raft organization in a number of tissues, and recent work has highlighted sphingolipids in membrane microdomains as potential targets for inhibition of tumor growth. The roles of fatty acids and gangliosides, especially in relation to lipid rafts, in cognitive development, age-related cognitive decline, psychiatric disorders, and Alzheimer's disease are poorly understood and require further investigation. The roles of lipid rafts in cancer, in microbial pathogenesis, and in insulin resistance are starting to emerge, and indicate compelling evidence for the growing importance of membrane microdomains in health and disease. In-vitro and animal studies show that n-3 PUFAs, cholesterol, and gangliosides modulate the structure and composition of lipid rafts, potentially influencing a wide range of biological processes, including immune function, neuronal signaling, cancer cell growth, entry of pathogens through the gut barrier, and insulin resistance in metabolic disorders. The physiological, clinical, and nutritional relevance of these observations remains to be determined.

Haloperidol disrupts lipid rafts and impairs insulin signaling in SH-SY5Y cells

Haloperidol exerts its therapeutic effects basically by acting on dopamine receptors. We previously reported that haloperidol inhibits cholesterol biosynthesis in cultured cells. In the present work we investigated its effects on lipid-raft composition and functionality. In both neuroblastoma SH-SY5Y and promyelocytic HL-60 human cell lines, haloperidol inhibited cholesterol biosynthesis resulting in a decrease of the cell cholesterol content and the accumulation of different sterol intermediates (7-dehydrocholesterol, zymostenol and cholesta-8,14-dien-3β-ol) depending on the dose of the drug. As a consequence, the cholesterol content in lipid rafts was greatly reduced, and several pre-cholesterol sterols, particularly cholesta-8,14-dien-3β-ol, were incorporated into the cell membrane. This was accompanied by the disruption of lipid rafts, with redistribution of flotillin-1 and Fyn and the impairment of insulin-Akt signaling. Supplementing the medium with free cholesterol abrogated the effects of haloperidol on lipid-raft composition and functionality. LDL (low-density lipoprotein), a physiological vehicle of cholesterol in plasma, was much less effective in preventing the effects of haloperidol, which is attributed to the drug's inhibition of intracellular vesicular trafficking. These effects on cellular cholesterol homeostasis that ultimately result in the alteration of lipid-raft-dependent insulin signaling action may underlie some of the metabolic effects of this widely used antipsychotic.

Sphingolipids: the nexus between Gaucher disease and insulin resistance

Sphingolipids constitute a diverse array of lipids in which fatty acids are linked through amide bonds to a long-chain base, and, structurally, they form the building blocks of eukaryotic membranes. Ceramide is the simplest and serves as a precursor for the synthesis of the three main types of complex sphingolipids; sphingomyelins, glycosphingolipids and gangliosides. Sphingolipids are no longer considered mere structural spectators, but bioactive molecules with functions beyond providing a mechanically stable and chemically resistant barrier to a diverse array of cellular processes. Although sphingolipids form a somewhat minor component of the total cellular lipid pool, their accumulation in certain cells forms the basis of many diseases. Human diseases caused by alterations in the metabolism of sphingolipids are conventionally inborn errors of degradation, the most common being Gaucher disease, in which the catabolism of glucosylceramide is defective and accumulates. Insulin resistance has been reported in patients with Gaucher disease and this article presents evidence that this is due to perturbations in the metabolism of sphingolipids. Ceramide and the more complex sphingolipids, the gangliosides, are constituents of specialised membrane microdomains termed lipid rafts. Lipid rafts play a role in facilitating and regulating lipid and protein interactions in cells, and their unique lipid composition enables them to carry out this role. The lipid composition of rafts is altered in cell models of Gaucher disease which may be responsible for impaired lipid and protein sorting observed in this disorder, and consequently pathology. Lipid rafts are also necessary for correct insulin signalling, and a perturbed lipid raft composition may impair insulin signalling. Unravelling common nodes of interaction between insulin resistance and Gaucher disease may lead to a better understanding of the biochemical mechanisms behind pathology.

Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined.

Lipid Raft Composition Modulates Sphingomyelinase Activity and Ceramide-Induced Membrane Physical Alterations

Lipid rafts and ceramide (Cer)-platforms are membrane domains that play an important role in several biological processes. Cer-platforms are commonly formed in the plasma membrane by the action of sphingomyelinase (SMase) upon hydrolysis of sphingomyelin (SM) within lipid rafts. The interplay between SMase activity, initial membrane properties (i.e., phase behaviour and lipid lateral organization) and lipid composition, amount of product (Cer) generated, and how it modulates membrane properties was studied using fluorescence methodologies in model membranes. The activity of SMase was evaluated by following the hydrolysis of radioactive SM. It was observed that: i) enzyme activity and extent of hydrolysis is strongly dependent on membrane physical properties but not on substrate content, being higher in raft-like mixtures, i.e., those mixtures with ld-lo (liquid disordered-liquid ordered) phase separation, and ii) Cer-induced alterations are also dependent on membrane composition, namely on cholesterol (Chol) content. In the lowest-Chol range, Cer segregates together with SM into small, ∼8.5 nm, Cer/SM-gel domains. Upon increasing Chol, the ability of Cer to recruit SM and to form gel domains strongly decreases. In the high Chol range, a Chol-enriched/SM-depleted lo phase predominates. Together, these data suggest that in biological membranes Chol in particular, and raft domains in general, play an important role in the modulation of SMase activity and in the regulation of membrane physical properties by restraining Cer-induced alterations.

Condensing and Fluidizing Effects of Gangliosides on Various Phospholipid Films

In model membrane mixtures that mimic lipid raft compositions, the more ordered domains are enriched in the ganglioside, GM1, which contains four neutral sugars and a negatively charged sialic acid. To understand the organization and partitioning of GM1 in cell membranes, the outer leaflet of the cell membrane was modeled using Langmuir monolayers of DPPC and varying concentrations of GM1. At low biologically relevant concentrations, GM1 condenses the DPPC monolayer while at higher concentrations, it fluidizes, with a switch-over point between the two behaviors at a ratio of 3:1 DPPC:GM1. Atomic force microscopy performed on deposited monolayers indicated that GM1 is located in nanoscale clusters within the condensed DPPC domains. The total surface area of these nanosize domains is larger than that attributable to GM1 molecules alone, suggesting the regions are due to GM1 and DPPC packing preferentially in condensed complexes due to variations in molecular geometry.To further study this effect, geometry of the phospholipid was varied. The zwitterionic lipid, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), with its smaller headgroup cross-sectional area compared to DPPC, was combined with various ratios of GM1. Additivity plots constructed for the mixtures to show deviations from ideal mixing indicated a 3:2 DMPE:GM1 ratio was most condensed compared to the individual components. Molecular geometry of the phospholipid headgroup plays a role in the condensation effect of GM1 on neighboring phospholipids. Additional experiments on certain monolayer mixtures of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) and GM1, components that are each fluid in pure monolayers, showed formation of condensed domains. This indicates the condensation effect of GM1 is strong enough to induce biologically relevant ordering phase transitions. Results will also be shown from experiments combining phospholipids containing negatively charged phosphatidylglycerol headgroups with GM1 to show the effect of electrostatic repulsion on the induced condensation.

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14alpha-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.

Altered mitochondria, energy metabolism, voltage-dependent anion channel, and lipid rafts converge to exhaust neurons in Alzheimer’s disease

Beta-amyloid (Abeta) deposition, in the form of plaques and amyloid angiopathy, and hyper-phosphorylated tau deposition forming neurofibrillary tangles, dystrophic neurites around beta-amyloid plaques and neuropil threads, are neuropathological hallmarks of Alzheimer's disease (AD) that accumulate in the brain with disease progression. These changes are accompanied by progressive loss of synapses and nerve cell death. Progressive cognitive impairment and dementia are the main neurological deficits. In addition, there is cumulative evidence demonstrating other metabolic disturbances that impair cell function and hamper neuron viability. The main components of the mitochondria are altered: complex IV of the respiratory chain is reduced; complex V which metabolizes ADP to form ATP is oxidatively damaged and functionally altered; and voltage-dependent anion channel VDAC, a major component of the outer mitochondrial membrane that regulates ion fluxes, is damaged as a result of oxidative stress. Mitochondria are a major source of reactive oxygen species that promote oxidative damage to DNA, RNA, proteins and lipids. Protein targets of oxidative damage are, among others, several enzymatic components of the glycolysis, lipid metabolism and cycle of the citric acid that fuel oxidative phosphorylation, mitochondrial respiration and energy production. The lipid composition of lipid rafts, key membrane specializations that facilitate the transfer of substrates, and protein-protein and lipid-protein interactions, is altered as a result of the abnormally low levels of n-3 long chain polyunsaturated fatty acids (mainly docosahexaenoic acid) that increase viscosity and augment energy consumption. Abnormal lipid raft composition may also modify the activity of key enzymes that modulate the cleavage of the amyloid precursor protein to form toxic Abeta. This is further complicated by the disruption of the complex VDAC with estrogen receptor alpha at the caveolae which participates, under physiological conditions, in the protection against beta-amyloid. Together, all these alterations converge in reduced energy production and increased energy demands in altered cells. Cell exhaustion is suggested as being a determining element to interpret impaired neuron function, reduced molecular turnover, and enhanced cell death.

Analysis of the Arabidopsis dry2/sqe1-5 mutant suggests a role for sterols in signaling

Sterols play multi-faceted roles in all eukaryotes. In plants, there are mounting evidences pointing to sterols, other than BRs, can act as signaling molecules. The Arabidopsis dry2/sqe1-5 mutant has multiple developmental defects caused by a point mutation in the SQE1 gene that generates a hypomorphic allele. SQE1 encodes a squalene epoxidase, which converts squalene into 2,3 oxidosqualene the precursor of plant sterols. Genetic, molecular and biochemical analyses suggest that dry2/sqe1-5 defective phenotypes cannot be simply explained by a depletion of bulk sterols but rather by altered ROS. It remains to be elucidated whether the altered ROS production of the mutant is caused by membrane composition, which in turn affect the lipid rafts composition and/or an altered signaling.

Effects of the antipsychotic drug haloperidol on the somastostatinergic system in SH‐SY5Y neuroblastoma cells

Antipsychotics are established drugs in schizophrenia treatment which, however, are not free of side effects. Lipid rafts are critical for normal brain function. Several G protein-coupled receptors, such as somatostatin (SRIF) receptors, have been shown to localize to lipid rafts. The aim of this study was to investigate whether haloperidol treatment affects the composition and functionality of lipid rafts in SH-SY5Y neuroblastoma cells. Haloperidol inhibited cholesterol biosynthesis, leading to a marked reduction in cell cholesterol content and to an accumulation of sterol intermediates, particularly cholesta-8,14-dien-3beta-ol. These changes were accompanied by a loss of flotillin-1 and Fyn from the lipid rafts. We next studied the functionality of the SRIF receptor. Treatment with haloperidol reduced the inhibitory effect of SRIF on adenylyl cyclase (AC) activity. On the other side, haloperidol decreased basal AC activity but increased forskolin-stimulated AC activity. Addition of free cholesterol to the culture medium abrogated the effects of haloperidol on lipid raft composition and SRIF signaling whereas the AC response to forskolin remained elevated. The results show that haloperidol, by affecting cholesterol homeostasis, ultimately alters SRIF signaling and AC activity, which might have physiological consequences.

Lipid Raft Composition Modulates Sphingomyelinase Activity and Ceramide-Induced Membrane Physical Alterations

Lipid rafts and ceramide (Cer)-platforms are membrane domains that play an important role in several biological processes. Cer-platforms are commonly formed in the plasma membrane by the action of sphingomyelinase (SMase) upon hydrolysis of sphingomyelin (SM) within lipid rafts. The interplay among SMase activity, initial membrane properties (i.e., phase behavior and lipid lateral organization) and lipid composition, and the amount of product (Cer) generated, and how it modulates membrane properties were studied using fluorescence methodologies in model membranes. The activity of SMase was evaluated by following the hydrolysis of radioactive SM. It was observed that 1), the enzyme activity and extent of hydrolysis are strongly dependent on membrane physical properties but not on substrate content, and are higher in raft-like mixtures, i.e., mixtures with liquid-disordered/liquid-ordered phase separation; and 2), Cer-induced alterations are also dependent on membrane composition, specifically the cholesterol (Chol) content. In the lowest-Chol range, Cer segregates together with SM into small (∼8.5 nm) Cer/SM-gel domains. With increasing Chol, the ability of Cer to recruit SM and form gel domains strongly decreases. In the high-Chol range, a Chol-enriched/SM-depleted liquid-ordered phase predominates. Together, these data suggest that in biological membranes, Chol in particular and raft domains in general play an important role in modulating SMase activity and regulating membrane physical properties by restraining Cer-induced alterations.

Condensing And Fluidizing Effects Of Structurally Related Gangliosides On Phospholipid Films

In model membrane mixtures that mimic lipid raft compositions, the more ordered domains are enriched in the ganglioside, GM1, which contains four neutral sugars and a negatively charged sialic acid. To understand the organization and partitioning of GM1 in cell membranes, the outer leaflet of the cell membrane was modeled using Langmuir monolayers of DPPC and varying concentrations of GM1. At low biologically relevant concentrations, GM1 condenses the DPPC monolayer while at higher concentrations, it acts to fluidize, with a switch-over point between the two behaviors at a ratio of 3:1 DPPC:GM1. To examine phase morphology and organization of the components, the monolayers were transferred onto solid substrates and imaged with atomic force microscopy. At concentrations below the switch-over point, GM1 is located in nanoscale clusters within the condensed DPPC domains. The total surface area of these nanosize domains is larger than that attributable to GM1 molecules alone, suggesting the regions are due to GM1 and DPPC packing preferentially in condensed geometric complexes.To pinpoint the structural region of GM1 giving rise to the condensation effect, parallel experiments were run with ceramide and PEGylated lipids. Our results indicate that the bulky, rigid sugar ganglioside headgroup is necessary for the significant phase behavior effects on the surrounding lipid molecules. Ganglioside headgroup geometry and charge were further explored with binary mixtures of asialo-, disialo- and trisialo-gangliosides (containing zero, two, and three sialic acids) with DPPC. In all cases, a similar condensing and fluidizing effect that varies with ganglioside concentration is seen, suggesting the negatively charged sialic acid residue is not critical for the close-packing phenomenon. Variations in mole ratio of critical packing between different ganglioside molecules can be explained by global effects of headgroup geometry, charge, and resultant molecular dipole moments.

Protein Profiling of Plasma Membranes and Lipid Rafts Defines Aberrant Signaling Pathways in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) remains incurable with current therapeutic approaches. Proteins expressed aberrantly at the MCL plasma membrane are candidate molecules for the development of targeted therapy. In the current study, we characterized globally the expression of proteins in the MCL plasma membrane and in purified lipid raft fractions in comparison with normal B cells using proteomic and bioinformatic analyses. Plasma membrane fractions were prepared from MCL patients in leukemic phase of disease and were then separated on 1D SDS-PAGE gels; sequential gel slices were digested with trypsin and the extracted peptides identified by LC/MS-MS. This approach overcomes the limitations of 2D gels for membrane proteins and also provides information on protein localization and post-translational modifications. Bioinformatics identified 111 intrinsic transmembrane proteins, from which we profiled selected proteins across primary MCL cases, MCL derived cell lines and normal B cells by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. Several transmembrane proteins, including CD27/CD70 and CD31 (PECAM) were abnormally expressed when compared to normal B cells. CD70 was significantly up-regulated (> 10 fold) in MCL patients along with its cognate receptor, CD27 which was also up-strongly up-regulated (4–9 fold), suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and PKC-βII were also detected in plasma membranes, suggesting constitutive activation of these proteins in MCL. Since activated PKC-βII has been reported to be recruited to lipid rafts during BCR signaling and to control IêB kinase lipid raft recruitment and activation, we undertook shotgun proteomics and protein profiling of MCL lipid rafts, purified using sucrose gradient centrifugation. This analysis revealed an abnormal composition of lipid rafts in MCL. Raftlin a myristoylated lipid raft B-cell specific protein, required for the integrity of lipid rafts and BCR signal transduction, was markedly down-regulated in MCL, as was the lymphoid transmembrane adaptor protein, Cbp/PAG (PAG1), which controls lymphocyte activation. These data were confirmed by RT-PCR which showed significant down-regulation of both genes in MCL. In comparison, other constitutive lipid raft proteins, such as Csk, Blk, Fyn and Lyn kinases and flotillin 1 were expressed within lipid rafts at levels similar to normal B lymphocytes. However, PKC-bII was not localized within lipid rafts indicating aberrant signaling via this molecule in MCL. Conversely, 5-lipoxygenase (5-LO/ALOX5) a key enzyme in leukotriene biosynthesis, which is normally expressed in either the nucleus or cytoplasm, was unexpectedly associated with lipid rafts isolated from MCL cells and was up-regulated ~7-fold in MCL as compared to normal B cells. Comparable results were obtained in chronic lymphocytic leukemia (CLL). Aberrant expression of 5-lipoxygenase has been associated with increased proliferation and suppression of apoptosis in other malignancies. To assess the possible functional activity of this pathway in malignant B-cells, the effects of inhibitors of 5-LO activity (AA861) and FLAP (MK886) its activating enzyme, were assessed on MCL cell lines and primary CLL cells. Both inhibitors induced high levels of apoptosis in a time-dependent and concentration-dependent manner in MCL cell lines and CLL cells, indicating an important role for this enzyme and the leukotriene biosynthetic pathway in MCL and other B-cell malignancies. Thus, using shotgun proteomics and protein expression profiling we have identified a subset of transmembrane proteins with aberrant expression and aberrant subcellular localization in MCL plasma membranes that may contribute to the pathology of the disease and are potential therapeutic targets in treating MCL.

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Critical developmental periods, such as fertilization, involve metabolic activation, membrane fusion events such as sperm-egg or plasma membrane-cortical granule merger, and production and hydrolysis of phospholipids. However, there has been no large-scale quantification of phospholipid changes during fertilization. Using an enzymatic assay, traditional FA analysis by TLC and gas chromatography, along with a new method of phospholipid measurement involving HPLC separation and evaporative light-scattering detection, we report lipid levels in eggs, sperm, and during fertilization in Xenopus laevis. Sperm were found to contain different amounts of phospholipids as compared with eggs. During fertilization, total phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, and phosphatidylserine decreased, and ceramide increased, whereas there was no change in phosphatidylcholine, cardiolipin, or phosphatidylethanolamine. FA analysis of phospholipids found numerous changes during fertilization. Because there is an increase in sn-1,2-diacylglycerol at fertilization, the FAs associated with this increase and the source of the increase in this neutral lipid were examined. Finally, activation of phospholipase C, phospholipase D, phospholipase A2, autotoxin, and sphingomyelinase at fertilization is discussed.

Interaction of N-myristoylethanolamine with cholesterol investigated in a Langmuir film at the air–water interface

The dramatic increase in the content of N-acylethanolamines (NAEs) having different acyl chains in various tissues when subjected to stress has resulted in significant interest in investigations on these molecules. Previous studies suggested that N-myristoylethanolamine (NMEA) and cholesterol interact to form a 1:1 (mol/mol) complex. In studies reported here, pressure-area isotherms of Langmuir films at the air–water interface have shown that at low fractions of cholesterol, the average area per molecule is lower than that predicted for ideal mixing, whereas at high cholesterol content the observed molecular area is higher, with a cross-over point at the equimolar composition. A plausible model that can explain these observations is the following: addition of small amounts of cholesterol to NMEA results in a reorientation of the NMEA molecules from the tilted disposition in the crystalline state to the vertical and stabilization of the intermolecular interactions, leading to the formation of a compact monolayer film, whereas at the other end of the composition diagram, addition of small amounts of NMEA to cholesterol leads to a tilting of the cholesterol molecules resulting in an increase in the average area per molecule. In Brewster angle microscopy experiments, a stable and bright homogeneous condensed phase was observed at a relatively low applied pressure of 2 mN.m − 1 for the NMEA:Chol. (1:1, mol/mol) mixture, whereas all other samples required significantly higher pressures (> 10 mN.m − 1 ) to form a homogeneous condensed phase. These observations are consistent with the formation of a 1:1 stoichiometric complex between NMEA and cholesterol and suggest that increase in the content of NAEs under stress may modulate the composition and dynamics of lipid rafts in biological membranes, resulting in alterations in signaling events involving them, which may be relevant to the putative cytoprotective and stress-combating ability of NAEs.

Long-Chain Polyunsaturated Fatty Acids Influence the Immune System of Infants1,2,

Several events occur during the first months of life that allow the immune system to become competent and functional. The aim of this article is to review the rationale and evidence of an influence of (n-3) long-chain PUFA (LCPUFA) on the immune system of infants. The (n-3) LCPUFA exert their immunomodulatory activities at different levels. The (n-3) LCPUFA metabolites induce eicosanoid production, alter gene expression, and modify lipid raft composition, altering T-cell signaling; all contribute to immunological functional changes. However, the roles of these mechanisms and the types of T or other immunological cells involved remain unclear at present. Moreover, the effect of (n-3) LCPUFA on the immune system of infants may vary according to dose, time of exposure, and profile of the immune system (T-helper, Th1/Th2). Most of the interventional studies in infancy have been performed for the prevention of allergy. They all confirmed influence on T-cell function and cytokine profiles, but clinically beneficial effects are more conflicting. Supplementation of the maternal diet in pregnancy or early childhood with (n-3) LCPUFA is potentially a noninvasive intervention strategy to prevent the development of allergy, infection, and possibly other immune-mediated diseases. However, any long-term in vivo effects on (n-3) LCPUFA early in life for immunomodulatory defense in infants and later on immune status and health remain to be assessed.