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Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization  [PDF]
Jorgelina Buschiazzo, Come Ialy-Radio, Jana Auer, Jean-Philippe Wolf, Catherine Serres, Brigitte Lefèvre, Ahmed Ziyyat
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062919
Abstract: Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.
Membrane Partitioning of Anionic, Ligand-Coated Nanoparticles Is Accompanied by Ligand Snorkeling, Local Disordering, and Cholesterol Depletion  [PDF]
Paraskevi Gkeka ,Panagiotis Angelikopoulos,Lev Sarkisov,Zoe Cournia
PLOS Computational Biology , 2014, DOI: doi/10.1371/journal.pcbi.1003917
Abstract: Intracellular uptake of nanoparticles (NPs) may induce phase transitions, restructuring, stretching, or even complete disruption of the cell membrane. Therefore, NP cytotoxicity assessment requires a thorough understanding of the mechanisms by which these engineered nanostructures interact with the cell membrane. In this study, extensive Coarse-Grained Molecular Dynamics (MD) simulations are performed to investigate the partitioning of an anionic, ligand-decorated NP in model membranes containing dipalmitoylphosphatidylcholine (DPPC) phospholipids and different concentrations of cholesterol. Spontaneous fusion and translocation of the anionic NP is not observed in any of the 10-μs unbiased MD simulations, indicating that longer timescales may be required for such phenomena to occur. This picture is supported by the free energy analysis, revealing a considerable free energy barrier for NP translocation across the lipid bilayer. 5-μs unbiased MD simulations with the NP inserted in the bilayer core reveal that the hydrophobic and hydrophilic ligands of the NP surface rearrange to form optimal contacts with the lipid bilayer, leading to the so-called snorkeling effect. Inside cholesterol-containing bilayers, the NP induces rearrangement of the structure of the lipid bilayer in its vicinity from the liquid-ordered to the liquid phase spanning a distance almost twice its core radius (8–10 nm). Based on the physical insights obtained in this study, we propose a mechanism of cellular anionic NPpartitioning, which requires structural rearrangements of both the NP and the bilayer, and conclude that the translocation of anionic NPs through cholesterol-rich membranes must be accompanied by formation of cholesterol-lean regions in the proximity of NPs.
Impact of oxLDL on Cholesterol-Rich Membrane Rafts  [PDF]
Irena Levitan,Tzu-Pin Shentu
Journal of Lipids , 2011, DOI: 10.1155/2011/730209
Abstract: Numerous studies have demonstrated that cholesterol-rich membrane rafts play critical roles in multiple cellular functions. However, the impact of the lipoproteins on the structure, integrity and cholesterol composition of these domains is not well understood. This paper focuses on oxidized low-density lipoproteins (oxLDLs) that are strongly implicated in the development of the cardiovascular disease and whose impact on membrane cholesterol and on membrane rafts has been highly controversial. More specifically, we discuss three major criteria for the impact of oxLDL on membrane rafts: distribution of different membrane raft markers, changes in membrane cholesterol composition, and changes in lipid packing of different membrane domains. We also propose a model to reconcile the controversy regarding the relationship between oxLDL, membrane cholesterol, and the integrity of cholesterol-rich membrane domains. 1. Introduction Oxidative modifications of LDL (oxLDL) are considered to be one of the major risk factors for the development of coronary artery disease (CAD) and plaque formation (reviewed in [1, 2]). Indeed, elevated levels of oxLDL are associated with an increased risk of CAD [3–5] and correlate with plasma hypercholesterolemia both in humans [6, 7] and in the animal models of atherosclerosis [8, 9]. It is also well-known that exposure to oxLDL induces an array of proinflammatory and proatherogenic effects but the mechanisms that underlie oxLDL-induced effects remain controversial. The prevailing hypothesis is that oxLDL results in loading cells with cholesterol inducing formation of cholesterol-laden macrophages (foam cells) and dysfunctional endothelial cells. However, growing number of studies have shown recently that the effects of oxLDL on membrane cholesterol homeostasis are complex and may actually involve cholesterol depletion and disruption of cholesterol-rich membrane domains (membrane rafts) rather than cholesterol loading. Membrane rafts were originally described as cholesterol- and sphingolipid-rich microdomains that provide platforms for protein-protein interactions in multiple signaling cascades [10–12]. A consensus definition for membrane rafts was suggested at the Keystone Symposium on Lipid Rafts and Cell Function (March 23–28, 2006 in Steamboat Springs, CO): “Membrane rafts are small (10–200?nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes" [13]. Most recently, Simons and Gerl [11] further defined membrane rafts as “dynamic, nonoscale,
Cholesterol Depletion Inactivates XMRV and Leads to Viral Envelope Protein Release from Virions: Evidence for Role of Cholesterol in XMRV Infection  [PDF]
Yuyang Tang, Alvin George, Thyneice Taylor, James E. K. Hildreth
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0048013
Abstract: Membrane cholesterol plays an important role in replication of HIV-1 and other retroviruses. Here, we report that the gammaretrovirus XMRV requires cholesterol and lipid rafts for infection and replication. We demonstrate that treatment of XMRV with a low concentration (10 mM) of 2-hydroxypropyl-β-cyclodextrin (2OHpβCD) partially depleted virion-associated cholesterol resulting in complete inactivation of the virus. This effect could not be reversed by adding cholesterol back to treated virions. Further analysis revealed that following cholesterol depletion, virus-associated Env protein was significantly reduced while the virions remained intact and retained core proteins. Increasing concentrations of 2OHpβCD (≥20 mM) resulted in loss of the majority of virion-associated cholesterol, causing disruption of membrane integrity and loss of internal Gag proteins and viral RNA. Depletion of cholesterol from XMRV-infected cells significantly reduced virus release, suggesting that cholesterol and intact lipid rafts are required for the budding process of XMRV. These results suggest that unlike glycoproteins of other retroviruses, the association of XMRV glycoprotein with virions is highly dependent on cholesterol and lipid rafts.
The role of cholesterol-sphingomyelin membrane nanodomains in the stability of intercellular membrane nanotubes
Lokar M, Kabaso D, Resnik N, Sep i K, Kralj-Igli V, Verani P, Zorec R, Igli A
International Journal of Nanomedicine , 2012, DOI: http://dx.doi.org/10.2147/IJN.S28723
Abstract: ole of cholesterol-sphingomyelin membrane nanodomains in the stability of intercellular membrane nanotubes Original Research (2809) Total Article Views Authors: Lokar M, Kabaso D, Resnik N, Sep i K, Kralj-Igli V, Verani P, Zorec R, Igli A Published Date April 2012 Volume 2012:7 Pages 1891 - 1902 DOI: http://dx.doi.org/10.2147/IJN.S28723 Received: 01 December 2011 Accepted: 05 January 2012 Published: 10 April 2012 Maru a Lokar1,*, Doron Kabaso1,2,*, Nata a Resnik3, Kristina Sepcic5, Veronika Kralj-Iglic4,6, Peter Veranic3, Robert Zorec2, Ale Iglic1,6 1Laboratory of Biophysics, Faculty of Electrical Engineering, 2Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, 3Institute of Cell Biology, Faculty of Medicine, 4Faculty of Health Sciences, 5Department of Biology, Biotechnical Faculty, 6Laboratory of Clinical Biophysics, Department of Orthopedic Surgery, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia *These authors equally share first authorship Abstract: Intercellular membrane nanotubes (ICNs) are highly curved tubular structures that connect neighboring cells. The stability of these structures depends on the inner cytoskeleton and the cell membrane composition. Yet, due to the difficulty in the extraction of ICNs, the cell membrane composition remains elusive. In the present study, a raft marker, ostreolysin, revealed the enrichment of cholesterol-sphingomyelin membrane nanodomains along ICNs in a T24 (malignant) urothelial cancer cell line. Cholesterol depletion, due to the addition of methyl-β-cyclodextrin, caused the dispersion of cholesterol-sphingomyelin membrane nanodomains and the retraction of ICNs. The depletion of cholesterol also led to cytoskeleton reorganization and to formation of actin stress fibers. Live cell imaging data revealed the possible functional coupling between the change from polygonal to spherical shape, cell separation, and the disconnection of ICNs. The ICN was modeled as an axisymmetric tubular structure, enabling us to investigate the effects of cholesterol content on the ICN curvature. The removal of cholesterol was predicted to reduce the positive spontaneous curvature of the remaining membrane components, increasing their curvature mismatch with the tube curvature. The mechanisms by which the increased curvature mismatch could contribute to the disconnection of ICNs are discussed.
BLOOD GLUCOSE & CHOLESTEROL CHANGES
Liaqat Ali
The Professional Medical Journal , 2001,
Abstract: Dietary intake of HCH by Oryctolagus Cuniculus rabbit for 4-weeks. (at 15 and 20 mg HCH/kgbody weight dose levels) resulted in hyperglycemia and hypercholesterolemia. The increase inblood glucose level was dose and time related, the increase was significant (P<0.01) with 15mg/kg body weight dose and highly significant (P<0.001) with 20 mg/kg dose treated animals in4 week of experiment. Increase in serum cholesterol was also dose and time related following almost thsimilar pattern. Marked changes in general physical condition; loss of appetite, diarrhoea, loss of hair andbehavioral changes, were observed in both groups of exposed animals. The hepatomegaly produced bydietary HCH was accompanied by marked histopathological changes. Whether these hematological changeswere purely the result of hepatotoxicity of drug or they were associated with the toxic effects of HCH onpancreas, requires further study.
Increased basolateral sorting of carcinoembryonic antigen in a polarized colon carcinoma cell line after cholesterol depletion-Implications for treatment of inflammatory bowel disease  [cached]
Robert Ehehalt, Markus Krautter, Martin Zorn, Richard Sparla, Joachim Füllekrug, Hasan Kulaksiz, Wolfgang Stremmel
World Journal of Gastroenterology , 2008,
Abstract: AIM: To investigate a possible increase of basolateral expression of carcinoembryonic antigen (CEA) by interfering with the apical transport machinery, we studied the effect of cholesterol depletion on CEA sorting and secretion.METHODS: Cholesterol depletion was performed in polarized Caco-2 cells using lovastatin and methyl-β-cyclodextrin.RESULTS: We show that CEA is predominantly expressed and secreted at the apical surface. Reduction of the cholesterol level of the cell by 40%-50% with lovastatin and methyl-β-cyclodextrin led to a significant change of the apical-to-basolateral transport ratio towards the basolateral membrane.CONCLUSION: As basolateral expression of CEA has been suggested to have anti-inflammatory properties, Cholesterol depletion of enterocytes might be a potential approach to influence the course of inflammatory bowel disease.
Cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in MARC-145 cells
Ying Sun,ShaoBo Xiao,Dang Wang,Rui Luo,Bin Li,HuanChun Chen,LiuRong Fang
Science China Life Sciences , 2011, DOI: 10.1007/s11427-011-4236-0
Abstract: Cholesterol represents one of the key constituents of small, dynamic, sterol- and sphingolipid-enriched domains on the plasma membrane. It has been reported that many viruses depend on plasma membrane cholesterol for efficient infection. In this study, the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus (PRRSV) infection of MARC-145 cells was investigated. Pretreatment of MARC-145 cells with methyl-β-cyclodextrin (MβCD), a drug used to deplete cholesterol from cellular membrane, significantly reduced PRRSV infection in a dose-dependent manner. This inhibition was partially reversed by supplementing exogenous cholesterol following MβCD treatment, suggesting that the inhibition of PRRSV infection was specifically mediated by removal of cellular cholesterol. Further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry, especially virus attachment and release. These results indicate that the presence of cholesterol in the cellular membrane is a key component of PRRSV infection.
Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells  [PDF]
Bárbara Hissa,Jacqueline G. Duarte,Ludmila F. Kelles,Fabio P. Santos,Helen L. del Puerto,Pedro H. Gazzinelli-Guimar?es,Ana M. de Paula,Ubirajara Agero,Oscar N. Mesquita,Cristina Guatimosim,Egler Chiari,Luciana O. Andrade
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001583
Abstract: Background Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. Methodology/Principal Finding In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. Conclusion/Significance Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.
Cholesterol Depletion in Adipocytes Causes Caveolae Collapse Concomitant with Proteosomal Degradation of Cavin-2 in a Switch-Like Fashion  [PDF]
Michael R. Breen, Marta Camps, Francisco Carvalho-Simoes, Antonio Zorzano, Paul F. Pilch
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034516
Abstract: Caveolae, little caves of cell surfaces, are enriched in cholesterol, a certain level of which is required for their structural integrity. Here we show in adipocytes that cavin-2, a peripheral membrane protein and one of 3 cavin isoforms present in caveolae from non-muscle tissue, is degraded upon cholesterol depletion in a rapid fashion resulting in collapse of caveolae. We exposed 3T3-L1 adipocytes to the cholesterol depleting agent methyl-β-cyclodextrin, which results in a sudden and extensive degradation of cavin-2 by the proteasome and a concomitant movement of cavin-1 from the plasma membrane to the cytosol along with loss of caveolae. The recovery of cavin-2 at the plasma membrane is cholesterol-dependent and is required for the return of cavin-1 from the cytosol to the cell surface and caveolae restoration. Expression of shRNA directed against cavin-2 also results in a cytosolic distribution of cavin-1 and loss of caveolae. Taken together, these data demonstrate that cavin-2 functions as a cholesterol responsive component of caveolae that is required for cavin-1 localization to the plasma membrane, and caveolae structural integrity.
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