Methyl Β-cyclodextrin Research Articles

Regulation of α1 Na/K-ATPase Expression by Cholesterol

We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol trafficking to the plasma membrane by compound U18666A had the same effect on α1 Na/K-ATPase. Similarly, the expression of α1, but not α2 and α3, Na/K-ATPase was significantly reduced in the target organs of Niemann-Pick type C mice where the intracellular cholesterol trafficking is blocked. Mechanistically, decreases in the plasma membrane cholesterol activated Src kinase and stimulated the endocytosis and degradation of α1 Na/K-ATPase through Src- and ubiquitination-dependent pathways. Thus, the new findings, taken together with what we have already reported, revealed a previously unrecognized feed-forward mechanism by which cells can utilize the Src-dependent interplay among Na/K-ATPase, caveolin-1, and cholesterol to effectively alter the structure and function of the plasma membrane.

Regulation of RhoA‐Dependent Sustained Contraction by Caveolin‐1 in Gastric Smooth Muscle

The main membrane proteins of caveolae (caveolin-1, −2 and −3) oligomerize within lipid rich domains of smooth muscle plasma membrane and participate in the regulation of G protein-coupled receptor signaling. AIM To identify the role of caveolin-1 in muscarinic m3-receptor mediated stimulation of Rho kinase and ZIP kinase activity in gastric smooth muscle. METHODS Muscle cells were isolated from rabbit stomach, and from wild type and caveolin-1−/− mice. Carbachol (CCh)-induced stimulation of Rho kinase and ZIP kinase activity was determined by immunokinase assay. Muscle contraction was measured by scanning micrometry. RESULTS CCh (0.1 μM) caused stimulation of both Rho kinase and ZIP kinase activity in gastric muscle cells. Y27632 (1 μM), a selective blocker of Rho kinase activity inhibited stimulation of both Rho kinase and ZIP kinase activity suggesting that Rho kinase is an upstream kinase of ZIP kinase. Disruption of caveolae with methyl β-cyclodextrin or suppression of caveolin-1 with caveolin-1 siRNA attenuated the increase in Rho kinase and ZIP kinase activity in response to CCh. Similarly, Rho kinase activation and sustained contraction in response to CCh were significantly attenuated in muscle cells isolated from caveolin-1−/− mice compared to wild type mice. CONCLUSION In gastric smooth muscle caveolin-1 positively regulates Rho kinase/ZIP kinase-dependent sustained muscle contraction.

Lipid raft-dependent activation of dual oxidase 1/H2O2/NF-κB pathway in bronchial epithelial cells

The present study addressed whether dual oxidase 1 (Duox1), a predominant isoform of NADPH oxidase in bronchial epithelial cells, is also activated through assembling of Duox1 and its partners such as p47(phox) due to lipid raft (LR) clustering. By gradient ultracentrifugation to isolate LR fractions in bronchial epithelial cells, it was found that Duox1 or p47(phox) was translocated into LR fractions when stimulated by tumor necrosis factor-α (TNF-α). Confocal microscopic analysis revealed that LRs were aggregated or clustered in the membrane, which were colocalized with Duox1 or p47(phox). Ceramide, a hydrolysis product of sphingomyelin, was also found colocalized with Duox1 or p47(phox) upon stimulation. In the presence of the commonly used LR disruptor, methyl-β-cyclodextrin (MCD), or the acid sphingomyelinase (ASMase) inhibitor, desipramine (DES), TNF-α-stimulated aggregation, translocation, and colocalization of LR components and Duox1 or its partners was abolished. Functionally, TNF-α-stimulated H(2)O(2) production was also blocked by MCD and DES (194.6 ± 15.4% vs. 90.6 ± 15.9% and 148.8 ± 20.4%), and the activation of the pivotal proinflammatory transcription factor, NF-κB, by TNF-α was reversed by MCD and DES as well as by small interfering RNAs of Duox1 or ASMase. Our results for the first time demonstrate that Duox1-mediated redox signaling in bronchial epithelial cells is associated with LR clustering dependent on the production of ceramide through ASMase.

P02-314 - Membrane lipid rafts are required for D2 dopamine receptor signaling

IntroductionLipid rafts are specialized membrane microdomains enriched in cholesterol and sphingolipids and are important in the organization of receptor-protein complexes and the regulation of signaling.Objective/aimsGiven the emerging significance of lipids with respect to receptor structure and activation, we investigated the role of lipid rafts and membrane cholesterol on D2 dopamine receptor (DAR) signaling. As the D2 DAR is the molecular target for all antipsychotic drugs, more information about its signaling may help refine therapeutics for schizophrenia.MethodsD2 DAR constructs were expressed in HEK293T cells. Sucrose density fractionation resolved lipid rafts from other membrane components. Methyl-β-cyclodextrin (MCD) was used to deplete membrane cholesterol and to disrupt lipid rafts.ResultsDetergent solubilization followed by sucrose gradient centrifugation resolved lipid rafts from heavier membrane fractions. The D2 DAR was equally distributed amongst both the lipid raft and heavier membrane fractions. Pretreatment with MCD, however, eliminated both lipid raft markers and the D2 DAR from lipid raft fractions, although the receptor was still found in heavier membrane fractions. We also found that MCD treatment abolished D2 DAR-mediated inhibition of cAMP accumulation. In contrast D1 DAR-stimulated cAMP accumulation was unaffected by MCD treatment.ConclusionsOur current results show that the D2 DAR is distributed in multiple membrane microdomains, including cholesterol-rich lipid rafts. We found that extraction of cholesterol disrupted lipid rafts and also an eliminated D2 DAR-mediated signaling. Thus, we hypothesize that lipid rafts are critical for D2 DAR signaling to occur.

αB-Crystallin Is Found in Detergent-resistant Membrane Microdomains and Is Secreted via Exosomes from Human Retinal Pigment Epithelial Cells

αB-crystallin (αB) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of αB has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that αB is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of αB in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize αB in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100-200 nm, were characterized by the presence of flotillin-1, α-enolase, and Hsp70, the same proteins that associate with detergent-resistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of αB is predominantly apical. Using OptiPrep gradients we demonstrate that αB resides in the DRM fraction. The secretion of αB is inhibited by the cholesterol-depleting drug, methyl β-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.

Transactivation of the Epidermal Growth Factor Receptor by Heat Shock Protein 90 via Toll-like Receptor 4 Contributes to the Migration of Glioblastoma Cells

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.

Eadie-Hofstee analysis of red blood cell deformability

In recent years, linear data transformation has become an accepted method to simplify the analysis of red blood cell (RBC) deformability curves obtained by ektacytometry. In this study, we introduce the Eadie-Hofstee transformation as an alternative linearization method for the analysis of RBC deformability. RBCs were treated with hydrogen peroxide (H(2)O(2)), tert-butyl-hydroperoxide (t-BuOOH), or methyl β-cyclodextrin (MβCD) and analyzed via ektacytometry (LORCA). RBC hemopathological clinical isolates (hereditary spherocytosis and α-thalassemia) were also analyzed by LORCA. Following ektacytometry, Eadie-Hofstee linearization was performed to obtain the maximum deformability (EI(max)) and shear stress at half maximal deformation (K(EI)) parameters. Significant changes in deformability parameters were observed with all agents tested. For H(2)O(2) and t-BuOOH, the K(EI) values increased significantly accompanied by marginal changes in EI(max), while treatment with MβCD resulted in a dose dependant decrease in EI(max). Contrasting deformability profiles were also observed in the two hematological disorders tested. In this study we have demonstrated the ability of Eadie-Hofstee linearization to detect and resolve changes in RBC deformability induced in vitro as well as deformability changes associated with in vivo hematological disorders. This technique shows promise in basic research, blood bank and clinical hematology settings.

Abstract A34: Cholesterol depletion in cell membrane induces apoptosis of oral cavity cancer cells

Abstract Cell membrane cholesterol is not distributed evenly, but it mainly exists in a specific microdomain of cell membrane called lipid raft. The lipid rafts consist of cholesterol and glycosphingolipid, and are associated with the regulation of transmembrane receptors. Cholesterol depletion has been reported to bring structural disruption of the raft and functional change of associated receptors which leads to the induction of apoptosis in certain types of cells. In this study, the cell membrane cholesterol of oral cavity cancer cells such as SCC-15, SCC-25, and SCC-QLL1 cell line was depleted by treating with methyl β-cyclodextrin (MβCD), and the effect of cholesterol depletion on the induction of apoptosis was evaluated. Treating oral cavity cancer cells for 1 hour with 1% MβCD inhibited cell proliferation. The cholesterol analysis and the filipin fluorescence staining demonstrated that MβCD selectively depleted cholesterol in oral cavity cancer cells. Furthermore, flow cytometry, poly (ADP-ribose) polymerase (PARP) cleavage assay, ELISA cell death assay showed that this inhibition of cell proliferation was caused by apoptosis. To evaluate the relationship between Mitogen-activated protein kinases (MAPK) and this apoptosis, Western blot analysis of the oral cavity cancer cells treated with MβCD was performed using the antibodies of Phospho-Extracellular signal-regulated kinase (p-ERK), phospho-p38 MAPK, and phosho-Jun-N-terminal kinases (p-JNK). The activity of ERK, p38 MAPK, and JNK in each oral cavity cancer cells was changed after treating with MβCD. In conclusion, cholesterol depletion of cell membrane may be considered as one of treatment modalities for oral cavity cancer, but further studies on its mechanism will be needed. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A34.

Emodin suppresses lipopolysaccharide-induced pro-inflammatory responses and NF-κB activation by disrupting lipid rafts in CD14-negative endothelial cells.

Emodin [1,3,8-trihydroxy-6-methylanthraquinone] has been reported to exhibit vascular anti-inflammatory properties. However, the corresponding mechanisms are not well understood. The present study was designed to explore the molecular target(s) of emodin in modifying lipopolysaccharide (LPS)-associated signal transduction pathways in endothelial cells. Cultured primary human umbilical vein endothelial cells (HUVECs; passages 3-5) were pre-incubated with emodin (1-50 µg·mL(-1) ). LPS-induced expression of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6] and chemokines (IL-8; CCL2/MCP-1) were determined by reverse transcription-PCR and elisa. Nuclear factor-κB (NF-κB) activation, inhibitor of κB (IκB)α degradation and Toll-like receptor-4 (TLR-4) were detected by immunocytochemistry and Western blotting. Cholesterol depletion [by methyl β-cyclodextrin (MBCD), a specific cholesterol binding agent] and cholesterol replenishment were further used to investigate the roles of lipid rafts in activation of HUVECs. Emodin inhibited, concentration-dependently, the expression of LPS-induced pro-inflammatory cytokines (IL-1β, IL-6) and chemokines (IL-8, CCL2) and, in parallel, inhibited NF-κB activation and IκBα degradation in HUVECs. However, emodin did not inhibit the NF-κB activation and IκBα degradation induced by IL-1β. The cholesterol binding agent, MBCD, inhibited LPS-induced NF-κB activation in passaged HUVECs [which also lack the LPS receptor, membrane CD14 (mCD14)], showing that lipid rafts played a key role in LPS signalling in mCD14-negative HUVECs. Moreover, emodin disrupted the formation of lipid rafts in cell membranes by depleting cholesterol. Lipid rafts were crucial in facilitating inflammatory responses of mCD14-negative HUVECs to LPS. Emodin disrupted lipid rafts through depleting cholesterol and, consequently, inhibited inflammatory responses in endothelial cells.

Distinct MHC Class II Molecules Are Associated on the Dendritic Cell Surface in Cholesterol-dependent Membrane Microdomains

Very small amounts of MHC class II-peptide complexes expressed on the surface of antigen-presenting cells (APCs) are capable of stimulating antigen-specific CD4 T cells. There is intense interest to elucidate the molecular mechanisms by which these small amounts of MHC-II can cluster, cross-link T cell receptors, and promote T cell proliferation. We now demonstrate that a significant fraction of the total pool of MHC-II molecules on the surface of dendritic cells is physically associated in macromolecular aggregates. These MHC-II/MHC-II interactions have been probed by co-immunoprecipitation analysis of the MHC-II I-A molecule with the related I-E molecule. These molecular associations are maintained in gentle detergents but are disrupted in harsh detergents such as Triton X-100. MHC-II I-A/I-E interactions are disrupted when plasma membrane cholesterol is extracted using methyl β-cyclodextrin, suggesting that lipid raft microdomains are important mediators of these MHC-II interactions. Although it has been proposed that tetraspanin proteins regulate molecular clustering, aggregation, and co-immunoprecipitation in APCs, genetic deletion of the tetraspanin family members CD9 or CD81 had no effect on MHC-II I-A/I-E binding. These data demonstrate that the presence of distinct forms of MHC-II with plasma membrane lipid rafts is required for MHC-II aggregation in APCs and provides a molecular mechanism allowing dendritic cells expressing small amounts of MHC-II-peptide complexes to cross-link and stimulate CD4 T cells.

Involvement of cholesterol-enriched microdomains in class A scavenger receptor-mediated responses in human macrophages

ObjectiveLipid rafts are cholesterol-enriched microdomains on cell membranes. We hypothesized that these microdomains could involve modified low-density lipoprotein (LDL) uptake. Methods and resultsCo-localizations of cholesterol-enriched microdomains and CD204 during the uptake of acetyl LDL (AcLDL) and oxidized LDL were observed using Alexa488-labeled polyethylene glycol cholesteryl ester, which is a sensitive probe used to analyze the dynamics of cholesterol-rich lipid microdomains in living cells. The lipid raft disruptors, methyl-β cyclodextrin and filipin, inhibited the uptake of AcLDL. CD204 siRNA treatments significantly reduced AcLDL uptake by 80%. We also demonstrated the presence of CD204 in the detergent-resistant membrane fraction (DRM) by immunoblotting analysis. The ratio of CD204/flotillin-1 in DRM was increased 11.5-fold by modified LDL administration. The PI3 kinase inhibitor LY294002, but not the Src kinase inhibitor PP1 or the Gαi/o inhibitor pertussis toxin, inhibited modified LDL uptake. The production of interleukin (IL)-8, but not CCL2, CXCL2, CXCL3, IL-6 or tumor necrosis factor-α was increased by AcLDL administration. The AcLDL-induced IL-8 production was inhibited by LY294002 and filipin. ConclusionsThese data firstly demonstrated that PI3 kinase-associated cholesterol-enriched microdomains are involved in CD204-mediated modified LDL uptake in human macrophages. Cholesterol-enriched microdomains may play a critical role in inflammatory processes.

Leishmania-Induced Inactivation of the Macrophage Transcription Factor AP-1 Is Mediated by the Parasite Metalloprotease GP63

Leishmania parasites have evolved sophisticated mechanisms to subvert macrophage immune responses by altering the host cell signal transduction machinery, including inhibition of JAK/STAT signalling and other transcription factors such as AP-1, CREB and NF-κB. AP-1 regulates pro-inflammatory cytokines, chemokines and nitric oxide production. Herein we show that upon Leishmania infection, AP-1 activity within host cells is abolished and correlates with lower expression of 5 of the 7 AP-1 subunits. Of interest, c-Jun, the central component of AP-1, is cleaved by Leishmania. Furthermore, the cleavage of c-Jun is dependent on the expression and activity of the major Leishmania surface protease GP63. Immunoprecipitation of c-Jun from nuclear extracts showed that GP63 interacts, and cleaves c-Jun at the perinuclear area shortly after infection. Phagocytosis inhibition by cytochalasin D did not block c-Jun down-regulation, suggesting that internalization of the parasite might not be necessary to deliver GP63 molecules inside the host cell. This observation was corroborated by the maintenance of c-Jun cleavage upon incubation with L. mexicana culture supernatant, suggesting that secreted, soluble GP63 could use a phagocytosis-independent mechanism to enter the host cell. In support of this, disruption of macrophage lipid raft microdomains by Methyl β-Cyclodextrin (MβCD) partially inhibits the degradation of full length c-Jun. Together our results indicate a novel role of the surface protease GP63 in the Leishmania-mediated subversion of host AP-1 activity.

Computational analysis and physico-chemical characterization of an inclusion compound between praziquantel and methyl-β-cyclodextrin for use as an alternative in the treatment of schistosomiasis

Schistosomiasis is still an endemic disease in many regions, with 250 million people infected with Schistosoma and about 500,000 deaths per year. Praziquantel (PZQ) is the drug of choice for schistosomiasis treatment, however it is classified as Class II in the Biopharmaceutics Classification System, as its low solubility hinders its performance in biological systems. The use of cyclodextrins is a useful tool to increase the solubility and bioavailability of drugs. The aim of this work was to prepare an inclusion compound of PZQ and methyl-β-cyclodextrin (MeCD), perform its physico-chemical characterization, and explore its in vitro cytotoxicity. SEM showed a change of the morphological characteristics of PZQ:MeCD crystals, and IR data supported this finding, with changes after interaction with MeCD including effects on the C–H of the aromatic ring, observed at 758 cm−1. Differential scanning calorimetry measurements revealed that complexation occurred in a 1:1 molar ratio, as evidenced by the lack of a PZQ transition temperature after inclusion into the MeCD cavity. In solution, the PZQ UV spectrum profile in the presence of MeCD was comparable to the PZQ spectrum in a hydrophobic solvent. Phase solubility diagrams showed that there was a 5.5-fold increase in PZQ solubility, and were indicative of a type AL isotherm, that was used to determine an association constant (Ka) of 140.8 M−1. No cytotoxicity of the PZQ:MeCD inclusion compound was observed in tests using 3T3 cells. The results suggest that the association of PZQ with MeCD could be a good alternative for the treatment of schistosomiasis.

The fusion of synaptic vesicle membranes studied by lipid mixing: the R18 fluorescence assay validity

The various experimental approaches and octadecyl rhodamine B chloride (R18) assay's capability to meet the criteria for examining the Ca2+dependent synaptic vesicles (SVs) fusion with target membranes have been investigated. The existence of at least two simultaneous processes one of which attributed to real Ca2+-dependent membrane fusion, while another is considered to be non-specific probe transfer has been shown. The differences in response to temperature changes were found for R18 fluorescence dequenching upon stimulation of membrane fusion or nonspecific probe transfer. The temperature dependences of the probe dequenching rate were the same for heterotypic and homotypic membrane systems and increased with the temperature growth. The combination of R18 fluorescence studies with the data obtained by dynamic light scattering (DLS) offers a unique opportunity for the determination of SVs aggregation and the membrane fusion. The cholesterol content of the synaptosomal plasma membrane was modulated by methyl-β-cyclodextrin (MCD). The MCD molecule has proven to bind directly the membrane cholesterol and interact with lipophilic probe R18 that affects its fluorescence. The obvious distinctions in probe dequenching due to the membrane mixing or the MCD effect were observed. The cholesterol depletion from the synaptosomal plasma membranes was found to inhibit the process of Ca2+-induced membrane fusion with SVs. Thus, the manipulations with conditions of R18 probe dequenching at the model conditions, specific for the Ca2+-triggered fusion steps of regulated exocytosis, allowed us to determine the relative contribution of probe transfer and genuine membrane fusion to the overall fluorescence signal.

Critical role of lipid rafts in virus entry and activation of phosphoinositide 3′ kinase/Akt signaling during early stages of Japanese encephalitis virus infection in neural stem/progenitor cells

Japanese encephalitis virus (JEV), the leading cause of acute encephalitis in South-East Asia is a neurotropic virus infecting various CNS cell types. Most Flaviviruses including JEV get internalised into cells by receptor-mediated endocytosis, which involve clathrin and membrane cholesterol. The cholesterol-enriched membrane microdomains referred to as lipid rafts act as portals for virus entry in a number of enveloped viruses, including Flavivirus. However, the precise role played by membrane lipid rafts in JEV internalisation into neural stem cells is still unknown. We have established neural stem/progenitor cells and C17.2 cell line as models of productive JEV infection. Increase in membrane fluidity and clustering of viral envelope proteins in lipid rafts was observed in early time points of infection. Localisation of non-structural proteins to rafts at later infection stages was also observed. Co-localisation of JEV glycoprotein with Cholera toxin B confirmed that JEV internalisation occurs in a lipid-raft dependent manner. Though JEV entry is raft dependent, however, there is requirement of functional clathrin during endocytosis inside the cells. Besides virus entry, the lipid rafts act as signalling platforms for Src tyrosine kinases and result in activation of phosphoinositìde 3'-kinase/Akt signalling during early JEV infection. Disruption of lipid raft formation by cholesterol depletion using Methyl β-cyclodextrin, reduced JEV RNA levels and production of infectious virus particles as well as impaired phosphoinositìde 3'-kinase/Akt signalling during initial infection. Overall, our results implicate the importance of host membrane lipid rafts in JEV entry and life cycle, besides maintaining survival of neural stem/progenitor cells during early infection.

Cyclodextrin complexed insulin encapsulated hydrogel microparticles: An oral delivery system for insulin

An oral insulin delivery system based on methyl-β-cyclodextrin (MCD) complexed insulin encapsulated polymethacrylic acid (PMAA) hydrogel microparticles was evaluated in this investigation. Poly(methacrylic acid)-chitosan-polyethylene glycol (PCP) microparticles were prepared by ionic gelation method. The insulin–MCD (IC) complex prepared was characterized by fluorescence spectroscopic and isothermal titration micro-calorimeteric (ITC) methods. MCD complexed insulin was encapsulated onto PCP microparticles by diffusion filling method. Loading and release properties of the complexed insulin from microparticles were evaluated under in vitro conditions. The effect of MCD complexation on the permeability of insulin was studied using Caco 2 cell monolayers and excised intestinal tissue with an Ussing chamber set-up. In vivo experiments were carried on streptozotocin induced diabetic rats to evaluate the efficacy of MCD complexed insulin encapsulated PCP microparticles to deliver insulin by the oral route. IC complex formation was established by fluorescence and ITC investigations. Insulin loading and release properties from the hydrogel matrix was rather unaffected by the MCD complexation. However MCD complexation was effective in enhancing insulin transport across Caco 2 cell monolayers, when applied in combination with the PMAA hydrogel system. Both insulin and MCD complexed insulin encapsulated PCP microparticles were effective in reducing blood glucose level in diabetic animal models. Cyclodextrin complexed insulin encapsulated hydrogel microparticles appear to be an interesting candidate for oral delivery of insulin.

Endocytosis of chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments.

BackgroundThe replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells.Methodology/Principal FindingsEntry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl β-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island.ConclusionsTogether our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.

Lipid rafts and membrane cholesterol are involved in regulating D2 dopamine receptor signaling

Lipid rafts are specialized membrane microdomains enriched in cholesterol, sphingolipids and caveolin. They are important in the organization of GPCR‐protein complexes and the regulation of signaling. Given the emerging significance of lipids with respect to GPCR structure and activation, we have investigated the role of lipid rafts and membrane cholesterol on D2 dopamine receptor (DAR) localization and function. Sucrose density fractionation revealed that D2 DARs are enriched in lipid rafts from both striatum and transfected HEK293 cells. Depletion of membrane cholesterol with methyl‐β‐cyclodextrin (MCD) diminished cell‐surface D2 DAR expression in HEK293T cells by approximately 75% without altering total receptor number. MCD treatment also completely abolished DA signaling at the D2 DAR. Importantly, when D2 DAR transfections were titrated to achieve surface expression values comparable to MCD‐treatment, a DA‐dependent decrease in cAMP was still observed. Diminished signaling of Gi/o‐coupled muscarinic receptors was also observed following MCD‐treatments. Notably, MCD‐treatment did not inhibit Gs‐linked D1 DAR function or forskolin‐stimulated cAMP accumulation. These results suggest that lipid rafts and/or membrane cholesterol are critical for D2 DAR signaling.

Membrane Cholesterol Depletion by Methyl-β-Cyclodextrin Enhances the Expression of Cardiac Differentiation Markers

Cholesterol is a sterol lipid that plays pleiotropic roles in the plasma membrane; it is involved in maintaining membrane fluidity and permeability and the structure of lipid microdomains. Despite its importance, the consequences of membrane cholesterol depletion during cardiac differentiation have not been described. Therefore, we investigated the cellular and molecular mechanisms associated with cholesterol depletion in cultures of chick cardiac cells. We used methyl-β-cyclodextrin (MCD) to deplete membrane cholesterol and investigate its role in cardiac differentiation by following the expression of several markers including the transcriptional factor Nkx2.5, the myofibrillar protein tropomyosin, the cytoskeletal intermediate filament protein desmin, the caveolar protein caveolin-3, the cadherin/β-catenin adhesion complex, and the junctional protein connexin 43. Confocal microscopy showed that desmin-positive cells were located more externally in the aggregates in relation to the more internally located caveolin-3-positive cells. Desmin and caveolin-3 were co-localized in filamentous structures in the subsarcolemmal region of well-spread cells outside the aggregates. β-Catenin was concentrated in regions of cell-cell contact, and tropomyosin in sarcomeric structures. Western blot tests showed that immediately following cholesterol depletion, there was a slight decrease in the expression of caveolin-3 and desmin, and at the same time there was a sharp increase in the expression of cadherin, tropomyosin, Nkx2.5 and connexin 43. Further, we found an increase in the expression of cardiac β-myosin heavy chain 7, a marker of the cardiac hypertrophic phenotype. These observations suggest that membrane cholesterol plays a significant role in regulating cardiomyocyte differentiation.

Involvement of Ceramide in the Propagation of Japanese Encephalitis Virus

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus and one of the most important flaviviruses in the medical and veterinary fields. Although cholesterol has been shown to participate in both the entry and replication steps of JEV, the mechanisms of infection, including the cellular receptors of JEV, remain largely unknown. To clarify the infection mechanisms of JEV, we generated pseudotype (JEVpv) and recombinant (JEVrv) vesicular stomatitis viruses bearing the JEV envelope protein. Both JEVpv and JEVrv exhibited high infectivity for the target cells, and JEVrv was able to propagate and form foci as did authentic JEV. Anti-JEV envelope antibodies neutralized infection of the viruses. Treatment of cells with inhibitors for vacuolar ATPase and clathrin-mediated endocytosis reduced the infectivity of JEVpv, suggesting that JEVpv enters cells via pH- and clathrin-dependent endocytic pathways. Although treatment of the particles of JEVpv, JEVrv, and JEV with cholesterol drastically reduced the infectivity as previously reported, depletion of cholesterol from the particles by treatment with methyl beta-cyclodextrin enhanced infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and propagation of JEVrv, and these enhancements were inhibited by treatment with an SMase inhibitor or C(6)-ceramide. These results suggest that ceramide plays crucial roles in not only entry but also egress processes of JEV, and they should assist in the clarification of JEV propagation and the development of novel therapeutics against diseases caused by infection with flaviviruses.