Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17

BACKGROUND Streptococcus agalactiae capsular type III strains are a leading cause of invasive neonatal infections. Many pathogens have developed mechanisms to escape from host defense response using the host membrane microdomain machinery. Lipid rafts play an important role in a variety of cellular functions and the benefit provided by interaction with lipid rafts can vary from one pathogen to another. OBJECTIVES This study aims to evaluate the involvement of membrane microdomains during infection of human endothelial cell by S. agalactiae. METHODS The effects of cholesterol depletion and PI3K/AKT signaling pathway activation during S. agalactiae-human umbilical vein endothelial cells (HUVEC) interaction were analysed by pre-treatment with methyl-β-cyclodextrin (MβCD) or LY294002 inhibitors, immunofluorescence and immunoblot analysis. The involvement of lipid rafts was analysed by colocalisation of bacteria with flotillin-1 and caveolin-1 using fluorescence confocal microscopy. FINDINGS In this work, we demonstrated the importance of the integrity of lipid rafts microdomains and activation of PI3K/Akt pathway during invasion of S. agalactiae strain to HUVEC cells. Our results suggest the involvement of flotillin-1 and caveolin-1 during the invasion of S. agalactiae strain in HUVEC cells. CONCLUSIONS The collection of our results suggests that lipid microdomain affects the interaction of S. agalactiae type III belonging to the hypervirulent ST-17 with HUVEC cells through PI3K/Akt signaling pathway.

Simvastatin treatment reduces the cholesterol content of membrane/lipid rafts, implicating the N -methyl-D-aspartate receptor in anxiety: a literature review

ABSTRACT Membrane/lipid rafts (MLRs) are plasmalemmal microdomains that are essential for neuronal signaling and synaptic development/stabilization. Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (statins) can disable the N-methyl-D-aspartate (NMDA) receptor through disruption of MLRs and, in turn, decrease NMDA-mediated anxiety. This hypothesis will contribute to understanding the critical roles of simvastatin in treating anxiety via the NMDA receptor.

Choice of cyclodextrin for cellular cholesterol depletion for vascular endothelial cell lipid raft studies: Cell membrane alterations, cytoskeletal reorganization and cytotoxicity.

The use of cyclodextrins as tools to establish the role of cholesterol rafts in cellular functions has become a widely accepted procedure. However, the adverse effects of cyclodextrins as the cholesterol-depleting agents on cellular structure and functions are not reported in detail. Therefore, in the current study, we investigated the membrane-perturbing actions and cytotoxicity of the two widely used cellular cholesterol-depleting cyclodextrins methyl-b-cyclodextrin (MbCD) and hydroxypropyl-b-cyclodextrin (HPCD) in our well-established bovine pulmonary artery endothelial cell (BPAEC) in vitro model system. BPAECs treated with different concentrations of MβCD and HPCD (2% and 5%, wt/vol.) for 15-180 min showed significant loss of membrane cholesterol, cytotoxicity, cell morphology alterations, actin cytoskeletal reorganization, alterations in cellular proteins and membrane fatty acid composition, and decrease in trans-endothelial electrical resistance (TER). MbCD induced a marked loss of cellular proteins, as compared to that caused by HPCD under identical conditions. More noticeably, MbCD caused a drastic loss of membrane lipid fatty acids in BPAECs, as compared to HPCD which failed to cause such alteration. Removal of cholesterol by cyclodextrin (especially MβCD) treatment apparently caused loss of fluidity of the cell membrane and leakage of vital cellular molecules including proteins and fatty acids, and thus caused cytotoxicity and loss of cell morphology in BPAECs. Replenishment of cells with cholesterol following its depletion by MbCD treatment significantly attenuated the depletion of cellular cholesterol, cytotoxicity and morphological alterations in BPAECs, indicating the importance of membrane cholesterol in vascular EC integrity. Also, the current study offered a safer method of cholesterol removal from membranes and lipid rafts by HPCD, suggesting its use in studies to investigate the role of lipid raft-associated cholesterol in cellular functions.

GD3 Accumulation in Cell Surface Lipid Rafts Prior to Mitochondrial Targeting Contributes to Amyloid-beta-induced Apoptosis

Neuronal apoptosis induced by amyloid beta-peptide (A beta) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying A beta-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-alpha-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in A beta-induced apoptosis is still unclear. Here, we investsigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during A beta-induced apoptosis using human brain-derived TE671 cells. Extracellular A beta induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular A beta, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-beta-cyclodextrin significantly prevented both GD3 accumulation in cell surface and A beta-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in A beta-induced apoptosis.

The extraction and identification of lipid rafts / 医学研究生学报

Objective: To establish a method using non-ionic detergent for extracting lipid rafts. Methods: Because lipid rafts can resist solubilization by non-ionic detergents under 4℃, we use non-ionic detergent (Triton X-100) to treat with epicyte fractions, the non-raft membrane would be solubilized. Then we utilize sucrose gradient centrifugation, preparations enriched in lipid rafts could be obtained.caveolin-1 was used as markers of lipid-raft structures. Results:A white light-scattering band under light illumination located at the interface between 15%-20% sucrose was detectable, and a brown stripe which comparative molecular quantity is 24 000 was identified by Western-Blot analysis. Conclusion: The method using non-ionic detergent is simple and useful for extracting lipid rafts, extracting lipid rafts would be prerequisite in studying the function of lipid rafts.

Interleukin-2? receptor in membrane lipid rafts / 肠外与肠内营养

Objective: To determine lipid rafts on the membrane of T cells is the functional microdomains for IL-2? receptor. Methods: Flow cytometric analysis was performed for expression of CD25(IL-2R?) on the surface of T cells. Lipid rafts were isolated by discontinuous sucrose density gradient ultracentrifugation. The localization of IL-2R? in fractions isolated from sucrose density gradients was determined by immunoblotting and detected by chemiluminescence. Results: cells were stimulated with IL-2 and expression of CD25(IL-2R?) on the surface of T cells was 37.08%. Immunoblot analysis of fractions from sucrose gradients revealed that a large proportion of IL-2R? was localized in lipid rafts. Conclusions: lipid rafts is the functional microdomains for IL-2? receptors.

EICOSAPENTAENOIC ACID ALTERS INTERLEUKIN-2 RECEPTOR DISTRIBUTION IN DETERGENT-INSOLUBLE MEMBRANE DOMAIN / 营养学报

Objective: To determine whether EPA exert effectively immunosuppressive function by altering distribution of IL-2R in membrane subdomains. Method: The human Jurkat E6-1 T cells were cultured in EPA-supplemented medium and the cells treated with stearic acid served as control. The effect of EPA on CD25 (IL-2? receptor) expression on the surface of T cells was investigated by flow cytometry. The lipid rafts were isolated by discontinuous sucrose density gradient ultracentrifugation. The localization of IL-2R?, IL-2R?, and IL-2R?c in fractions isolated and the effect of EPA treatment were determined by immunoblot and chemiluminescence. Results: EPA suppressed CD25 expression on the surface of T cells. IL-2R?, IL-2R?, and IL-2R?c were associated with lipid rafts of T cells, and these subunits were partly displaced from lipid rafts of EPA-treated T cells. Conclusion: Lipid rafts are functional subdomains for IL-2R signaling. EPA enrichment modifies distribution of IL-2R?, IL-2R?, and IL-2R?c in lipid rafts and EPA plays immunosuppressive roles probably by partly removing IL-2R from lipid rafts.

A study of localization of NMDA receptor protein labelling with colloid-gold on membrane by Scanning Electron Microscopy / 解放军医学杂志

Objective To investigate the distribution rule of NMDA receptor protein localizated on neuron membrane surface. Methods After the primary culture model of forebrain cortex neuron of rat had been set up,the NR1 subunit protein molecule of NMDA receptor has been combined with primary antibody IgG,and marked by Staphylococcus protein A-colloid gold particles (SPA-G). Then the neuron was observed with Field-emission SEM,and the content of AU element was calcalated by software. Results The cluster of NMDA receptor combined with SPA-G particulates was clearly observed as some globular being distributed mainly under some niches of lipid rafts,mainly localized at originating section of dendritic protuberance of neuron membrane surface. Conclusion NMDA receptor molecule protein was distributed on the membrane surface lipid rafts of originating section of dendritic protuberance of neuron. It was in accord with the distribution rule of postsynaptic membrane on neuron. The Field-emission SEM could distinguish single NMDA receptor melocule cluster after being marked with SPA-G molecule in neuron.