Robert Bittman

Docosahexaenoic Acid Enhances Segregation of Lipids between Raft and Nonraft Domains: 2H-NMR Study

Solid-state (2)H-NMR of [(2)H(31)]-N-palmitoylsphingomyelin ([(2)H(31)]16:0SM, PSM*), supplemented by differential scanning calorimetry, was used for the first time, to our knowledge, to investigate the molecular organization of the sphingolipid in 1:1:1 mol mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE, POPE) or 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE, PDPE) and cholesterol. When compared with (2)H-NMR data for analogous mixtures of [(2)H(31)]16:0-18:1PE (POPE*) or [(2)H(31)]16:0-22:6PE (PDPE*) with egg SM and cholesterol, molecular interactions of oleic acid (OA) versus docosahexaenoic acid (DHA) are distinguished, and details of membrane architecture emerge. SM-rich, characterized by higher-order, and PE-rich, characterized by lower-order, domains <20 nm in size are formed in the absence and presence of cholesterol in both OA- and DHA-containing membranes. Although acyl chain order within both domains increases on the addition of sterol to the two systems, the resultant differential in order between SM- and PE-rich domains is almost a factor of 3 greater with DHA than with OA. Our interpretation is that the aversion that cholesterol has for DHA--but not for OA--excludes the sterol from DHA-containing, PE-rich (nonraft) domains and excludes DHA from SM-rich/cholesterol-rich (raft) domains. We attribute, in part, the diverse health benefits associated with dietary consumption of DHA to an alteration in membrane domains.

A Single Subset of Dendritic Cells Controls the Cytokine Bias of Natural Killer T Cell Responses to Diverse Glycolipid Antigens

Summary Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α+ DEC-205+ dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α+ dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study

The Vibrio cholerae cytolysin (VCC) 63-kDa monomer has been shown to interact in aqueous suspension with cholesterol microcystals to produce a ring/pore-like heptameric oligomer approximately 8 nm in outer diameter. Transmission electron microscopy data were produced from cholesterol samples adsorbed to carbon support films, spread across the holes of holey carbon films, and negatively stained with ammonium molybdate. The VCC oligomers initially attach to the edge of the stacked cholesterol bilayers and with increasing time cover the two planar surfaces. VCC oligomers are also released into solution, with some tendency to cluster, possibly via the hydrophobic membrane-spanning domain. At the air/water interface, the VCC oligomers are likely to be selectively oriented with the hydrophobic domain facing the air. Despite some molecular disorder/plasticity within the oligomers, multivariate statistical analysis and rotational self-correlation using IMAGIC-5 strongly suggest the presence of sevenfold rotational symmetry. To correlate the electron microscopy data with on-going biochemical and permeability studies using liposomes of varying lipid composition, the direct interaction of VCC with several cholesterol derivatives and other steroids has been examined. 19-Hydroxycholesterol and 7 beta-hydroxycholesterol both induce VCC oligomerization. beta-Estradiol, which does not possess an aliphatic side chain, also efficiently induces VCC oligomer formation, as does cholesteryl acetate. Cholesteryl stearate and oleate and the C22 (2-trifluoroacetyl)naphthyloxy analogue of cholesterol fail to induce VCC oligomerization, but binding of the monomer to the surface of these steroids does occur. Stigmasterol has little tendency to induce oligomer formation, and oligomers are largely confined to the edge of the bilayers; ergosterol has even less oligomerization ability. Attempts to solubilize and stabilize the VCC oligomers from cholesterol suspensions have been pursued using the neutral surfactant octylglucoside. Although individual solubilized oligomers have been defined which exhibit a characteristic cytolysin channel conformation in the side-on orientation, a tendency remains for the oligomers to cluster via their hydrophobic domains.

Bax and Bcl-xL exert their regulation on different sites of the ceramide channel.

The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.

Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an omega-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K(m) of 35 microM for BODIPY-sphingosine 1-phosphate.

Cholesterol’s Aliphatic Side Chain Modulates Membrane Properties†

The influence of cholesterols alkyl side chain on membrane properties was studied using a series of synthetic cholesterol derivatives without a side chain or with a branched side chain consisting of 5 to 14 carbon atoms. Cholesterols side chain is crucial for all membrane properties investigated and therefore essential for the membrane properties of eukaryotic cells.

Ceramide-1-phosphate inhibits cigarette smoke-induced airway inflammation

Sphingolipids are involved in the pathogenesis of inflammatory diseases. The central molecule is ceramide, which can be converted into ceramide-1-phosphate (C1P). Although C1P can exert anti- and pro-inflammatory effects, its influence on cigarette smoke (CS)-induced lung inflammation is unknown. We aimed to clarify the role of C1P in the pathogenesis of CS-triggered pulmonary inflammation and emphysema in humans and mice. The effects of C1P were addressed on CS-induced lung inflammation in C57BL/6 mice, CS extract-triggered activation of human airway epithelial cells (AECs) and neutrophils from patients with chronic obstructive pulmonary disease. Differential cell counts in bronchoalveolar lavage fluid were determined by flow cytometry and pro-inflammatory cytokines were measured by ELISA. Expression and DNA binding of nuclear factor (NF)-κB and neutral sphingomyelinase (nSMase) were quantified by PCR, electrophoretic mobility shift and fluorometric assays. C1P reduced CS-induced acute and chronic lung inflammation and development of emphysema in mice, which was associated with a reduction in nSMase and NF-κB activity in the lungs. nSMase activity in human serum correlated negatively with forced expiratory volume in 1 s % predicted. In human AECs and neutrophils, C1P inhibited CS-induced activation of NF-κB and nSMase, and reduced pro-inflammatory cytokine release. Our results suggest that C1P is a potential target for anti-inflammatory treatment in CS-induced lung inflammation. Ceramide-1-phosphate, a potential target for anti-inflammatory treatment in cigarette smoke-induced lung inflammation http://ow.ly/E7tUS

Organization of fluorescent cholesterol analogs in lipid bilayers - lessons from cyclodextrin extraction.

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor.

Tracking sphingosine metabolism and transport in sphingolipidoses: NPC1 deficiency as a test case.

The late endosomal/lysosomal compartment (LE/LY) plays a key role in sphingolipid breakdown, with the last degradative step catalyzed by acid ceramidase. The released sphingosine can be converted to ceramide in the ER and transported by ceramide transfer protein (CERT) to the Golgi for conversion to sphingomyelin. The mechanism by which sphingosine exits LE/LY is unknown but Niemann–Pick C1 protein (NPC1) has been suggested to be involved. Here, we used sphingomyelin, ceramide and sphingosine labeled with [3H] in carbon‐3 of the sphingosine backbone and targeted them to LE/LY in low‐density lipoprotein (LDL) particles. These probes traced LE/LY sphingolipid degradation and recycling as suggested by (1) accumulation of [3H]‐sphingomyelin‐derived [3H]‐ceramide and depletion of [3H]‐sphingosine upon acid ceramidase depletion, and (2) accumulation of [3H]‐sphingosine‐derived [3H]‐ceramide and attenuation of [3H]‐sphingomyelin synthesis upon CERT depletion. NPC1 silencing did not result in the accumulation of [3H]‐sphingosine derived from [3H]‐sphingomyelin/LDL or [3H]‐ceramide/LDL. Additional evidence against NPC1 playing a significant role in LE/LY sphingosine export was obtained in experiments using the [3H]‐sphingolipids or a fluorescent sphingosine derivative in NPC1 knock‐out cells. Instead, NPC1‐deficient cells displayed an increased affinity for sphingosine independently of protein‐mediated lipid transport. This likely contributes to the increased sphingosine content of NPC1 cells.

PARTIALLY DEUTERATED PHOSPHOLIPIDS AS IR STRUCTURE PROBES OF CONFORMATIONAL ORDER IN BULK AND MONOLAYER PHASES

Abstract Acyl chain conformations of the positional isomers 1-palmitoyl- d 31 , 2-oleoylphosphatidylcholine (P- d 31 OPC) and 1-oleoyl-2-palmitoyl- d 31 -phosphatidylcholine (OP- d 31 PC) were investigated by FT-IR spectroscopy. The temperature dependence of the CH 2 and CD 2 stretching frequencies showed that P- d 31 OPC displayed a gel → liquid crystal phase transition between −6 and −3°C, with parameter changes similar to, but slightly reduced from, those found in disaturated PCs. In contrast, the oleoyl chain of OP- d 31 PC showed a broadened (nearly abolished) transition with substantial conformational disorder in the gel phase. The palmitoyl chain of this species also revealed a broadened transition, with, however, the retention of an ordered gel phase. Examination of the CH 2 wagging region, which reveals the occurrence of particular two- and three-bond conformational states in disordered phospholipid phases, shows that the sn -1 oleoyl chains of OP- d 31 PC chains have a somewhat different set of these conformations than the sn -2 oleoyl chains of P- d 31 OPC. A simple structural model based on the putative existence of a bend at the 2′ position of the sn -2 chain is proposed to explain these observations. Examination of OP- d 31 PC and P- d 31 OPC monolayers in situ at the air/water interface by IR reflection-absorption spectroscopy (IRRAS) showed the oleoyl chains of the latter to be somewhat more ordered at surface pressures > approx. 25 mN m −1 , and the palmitoyl- d 31 chains to possess a similar level of conformational order. The conformational order in these monolayers at high pressure, as judged by the CH 2 or CD 2 stretching frequencies, is similar to that in the bulk phase. Finally, IRRAS studies of tail-end deuterated (both chains) 1,2-dipalmitoylphosphatidylcholine- d 34 showed that the chains possess more conformational order adjacent to the interface than at their tails. Overall, these studies confirm the utility of partially deuterated derivatives for structural investigations of phospholipids both in monolayer and bulk phases.