Membranes are not just rafts

A new definition for lipid rafts was coined at the Keystone Symposium of Lipid Rafts and Cell Function based on recent advances in the field. The revised definition lumps all membrane heterogeneities that are not rafts into a single class of "non-raft". In this commentary, we suggest that "non-raft" domains encompass a variety of membrane heterogeneities and are quite diverse in composition and origin. A good starting point for the study of this diversity would be phospholipids with unsaturated acyl chains, which display little affinity for cholesterol; these lipids are abundant in membranes such as the endoplasmic reticulum and that may form their own macro- or microdomains.     

Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with beta-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.     

Evaluation of the Neuroprotective Effects of Citicoline after Experimental Spinal Cord Injury: Improved Behavioral and Neuroanatomical Recovery

Spinal cord injury (SCI) caused by trauma mainly occurs in two mechanisms as primary and secondary injury. Secondary injury following the primary impact includes various pathophysiological and biochemical events. Methylprednisolone is the only pharmacological agent having clinically proven beneficial effects on SCI. Citicoline has been shown to have clinical and experimental beneficial effects on brain ischemia. This study aims to investigate the neuroprotective effect of citicoline in an experimental SCI model in rats. Sixty adult Wistar albino rats were randomized into five groups. SCI was performed by the weight-drop model. Group 1 underwent laminectomy alone. The Group 2 underwent laminectomy followed by SCI and received no medication. Group3, Group 4 and Group 5 underwent laminectomy followed by SCI and received medication. Group 3 and Group 5 received citicoline and Group 4 and Group 5 received methylprednisolone. The rats were divided into two subgroups for biochemical analysis (sacrificed at 24 h after surgery) and neurobehavioral and histopathological evaluation (sacrificed at 6 weeks after surgery). Malonildialdehyde levels, nitric oxide levels and trauma size ratios were lower and reduced glutathione levels were higher in Group 3, Group 4 and Group 5 as compared to Group 2. Posttraumatic neurological recovery after surgery was significantly better in Group 3, Group 4 and Group 5 compared to Group 2. In conclusion, this study demonstrates that citicoline is as effective as methylprednisolone. The efficacy of citicoline combined with methylprednisolone is not superior to either citicoline or methylprednisolone alone.     

Fe and Fe initiated peroxidation of sonicated and non-sonicated liposomes made of retinal lipids in different aqueous media

Retina is highly susceptible to oxidative damage due to its high content of polyunsaturated fatty acids (PUFAs), mainly docosahexaenoic acid (22:6 n3). Lipid peroxidation process is thought to be involved in many physiological and pathological events. Many model membranes can be used to learn more about issues that cannot be studied in biological membranes. Sonicated liposomes (SL) and non-sonicated liposomes (NSL) prepared with lipids isolated from bovine retina and characterized by dynamic light-scattering, were submitted to lipid peroxidation, under air atmosphere at 22 °C, with Fe²⁺ or Fe³⁺ as initiator, in different aqueous media. Conjugated dienes and trienes, determined by absorption at 234 and 270 nm respectively, and thiobarbituric acid-reactive substances were measured as a function of time. Peroxidation of SL or NSL initiated with 25 μM FeSO₄ in 20 mM Tris-HCl pH 7.4 resulted in an increase in TBARS production after a lag phase of 60 min. Incubation of both types of liposomes in water resulted in shortening of the lag phase at 30 min. When lipid peroxidation was performed in 0.15 M NaCl, lag phase completely disappeared. On the other hand, FeCl₃ (25 μM) induced a limited production of TBARS only just after 30 min of incubation. When Fe²⁺- or Fe³⁺-lipid peroxidation of both types of liposomes was carried out in water or 0.15 M NaCl, formation of conjugated dienes and conjugated trienes were higher than in reactions carried out in 20 mM Tris-HCl pH 7.4.Our results established that both liposome types were susceptible to Fe²⁺- and Fe³⁺-initiated lipid peroxidation. However, Fe²⁺ showed a clearly enhanced effect on peroxidation rate and steady state concentration of oxidation products.We verified that peroxidation of liposomes made of retinal lipids is affected not only by type of initiator but also by aqueous media. This model constitutes a useful system to study formation of lipid peroxidation intermediaries and products in an aqueous environment.     

The fatty acid profile changes in marine invertebrate larval cells during cryopreservation

The development of cryopreservation methods for embryonic cells and larvae of sea animals offers a great potential for marine biotechnology. Larval cells of bivalves and sea urchins were frozen to −196 °C using traditional cryoprotectants (Me₂SO and trehalose) and the cryoprotective mixture developed by us. In addition to Me₂SO and trehalose, this mixture contained an exogenous lipid extract from mussel tissues and antioxidants. A positive effect of antioxidants (α-tocopherol acetate, ascorbic acid or echinochrome, the quinoid pigment of sea urchins) on cell viability became significant only in the presence of exogenous lipids. Antioxidants added to cryoprotective mixtures did not reveal visible cryoprotective activity when used separately. To better understand the mechanism of the protective effect of exogenous lipids on cell membranes of sea animals, a comparative analysis of the fatty acid (FA) composition of total lipids in larval cells before and after freezing was carried out using a gas–liquid chromatography. The results indicate that freezing–thawing has direct effects on the FA composition of major lipid classes in marine invertebrate cells, and these effects can vary depending on the provenance of the cells. We have found that (I) both cell viability and the FA profile of cell lipids after cryopreservation depend on the cryoprotectants used; (II) an amount of saturated, monoenic and polyenic FAs changes significantly after cryopreservation. We assume that the addition of the exogenous lipid extract in form of liposomes could promote a renewal of disturbance areas and prevent from membrane damages during freezing–thawing.     

The glycolipid transfer protein interacts with the vesicle-associated membrane protein-associated protein VAP-A

The glycolipid transfer protein (GLTP) is a cytoplasmic protein with an ability to bind glycolipids and catalyze their in vitro transfer. In this study, we have found a FFAT-like motif in GLTP. The FFAT (two phenylalanines in an acidic tract) motif in lipid-binding proteins has previously been shown to interact with the VAPs (vesicle-associated membrane protein-associated proteins) in the endoplasmic reticulum. Here we used glutathione S-transferase pull-down experiments to confirm that GLTP and VAP-A interact. By displacing different amino acids in the motif we clearly show that the interaction is dependent on the FFAT-like motif in GLTP. The potential role of GLTP in the endoplasmic reticulum association is discussed.     

FRET analysis of domain formation and properties in complex membrane systems

The application of Förster Resonance Energy Transfer (FRET) to the detection and characterization of phase separation in lipid bilayers (both in model systems and in cell membranes) is reviewed. Models describing the rate and efficiency of FRET for both uniform probe distribution and phase separation, and recently reported methods for detection of membrane heterogeneity and determination of phase boundaries, probe partition coefficients and domain size, are presented and critically discussed. Selected recent applications of FRET to one-phase lipid systems, gel/fluid phase separation, liquid ordered/liquid disordered phase separation (lipid rafts), complex systems containing ceramide and cell membranes are presented to illustrate the wealth of information that can be inferred from carefully designed FRET studies of membrane domains.     

Polyunsaturated fatty acid-cholesterol interactions: Domain formation in membranes

Polyunsaturated fatty acids (PUFA) constitute an influential group of molecules that promote health by an as yet unknown mechanism. They are structurally distinguished from less unsaturated fatty acids by the presence of a repeating CH-CH₂-CH unit that produces an extremely flexible chain rapidly reorienting through conformational states. The most highly unsaturated case in point is docosahexaenoic acid (DHA) with 6 double bonds. This review will summarize how the high disorder of DHA affects the properties of the membrane phospholipids into which the PUFA incorporates, focusing upon the profound impact on the interaction with cholesterol. Results obtained with model membranes using an array of biophysical techniques will be presented. They demonstrate DHA and the sterol possesses a mutual aversion that drives the lateral segregation of DHA-containing phospholipids into highly disordered domains away from cholesterol. These domains are compositionally and organizationally the opposite of lipid rafts, the ordered domain enriched in predominantly saturated sphingolipids “glued” together by cholesterol that is believed to serve as the platform for signaling proteins. We hypothesize that DHA-rich domains also form in the plasma membrane and are responsible, in part, for the diverse range of health benefits associated with DHA.     

Demethylallosamidin, a chitinase inhibitor, suppresses airway inflammation and hyperresponsiveness

Acidic mammalian chitinase is upregulated in response to allergen exposure in the lung. We investigated the effects of chitinase inhibitors, allosamidin (Allo) and demethylallosamidin (Dma), on asthmatic responses. Mice were subjected to IL-13 instillation into the airways or to ovalbumin sensitization plus exposure with or without treatment of Allo or Dma. Airway hyperresponsiveness (AHR) and inflammation were evaluated. Allo and Dma attenuated airway eosinophilia and the upregulation of eotaxin after IL-13 instillation, while Dma, but not Allo, suppressed AHR in IL-13-induced asthma. Allo or Dma suppressed the elevated chitinase activity in BAL fluids after IL-13 to similar levels. The bronchoprotective PGE₂ levels in BAL fluids were elevated after IL-13 instillation. Allo, but not Dma, suppressed the overproduction of PGE₂ and the expression of COX-2 and PGE synthase-1 induced by IL-13. In ovalbumin-induced asthma, Dma suppressed AHR more strongly than Allo. These findings suggest that Dma attenuates asthmatic responses induced by IL-13 without affecting PGE₂ synthesis. Dma may have potential as therapeutic agents for asthma.     

The interaction of estrogen receptor α and caveolin-3 regulates connexin43 phosphorylation in metabolic inhibition-treated rat cardiomyocytes

Caveolin-3, the major caveolin isoform in cardiomyocytes, plays an important role in the rapid signaling pathways initiated by stimulation of the membrane-associated molecules. To examine the role of caveolin-3 in regulating estrogen receptor α in cardiomyocytes, we investigate whether the membrane estrogen receptor α associates with caveolin-3 and whether this association is linked to the 17β-estradiol-mediated signals. In control cardiomyocytes, following discontinuous sucrose gradient centrifugation, caveolin-3 was found predominantly in the lipid raft buoyant fractions, whereas it was distributed to both the buoyant and non-lipid raft heavy fractions following metabolic inhibition treatment. Confocal microscopy showed that estrogen receptor α co-localized with caveolin-3 on the plasma membrane of neonatal and adult rat cardiomyocytes. This membrane labeling of estrogen receptor α was not seen following treatment with the cholesterol-depleting agent methyl-β-cyclodextrin (5 mM), whereas metabolic inhibition had little effect on the membrane distribution of estrogen receptor α. Metabolic inhibition induced tyrosine phosphorylation of caveolin-3 and decreased its association with estrogen receptor α, both effects being mediated via a Src activation mechanism, since they were inhibited by the selective tyrosine kinase inhibitor PP2. Metabolic inhibition also induced tyrosine phosphorylation of connexin43 and increased its association with c-Src, both effects being prevented by 17β-estradiol (200 nM). The effect of 17β-estradiol on metabolic inhibition-induced tyrosine phosphorylation of connexin43 was inhibited by the specific estrogen receptor antagonist ICI182780. These data identify cardiac caveolin-3 as juxtamembrane scaffolding for estrogen receptor α docking at caveolae, which provide a unique compartment for conveying 17β-estradiol-elicited, rapid signaling to regulate connexin43 phosphorylation during ischemia.