SEARCH · Search PubMed

Results for “Filipin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources

PubMed1993

Heterogeneity of cellular cholesteryl ester accumulation by human monocyte-derived macrophages.

We have studied cholesteryl ester accumulation in human monocyte-derived macrophages, which together with smooth muscle cells, represent the major cell types that accumulate cholesterol in atherosclerotic lesions. Monocyte-derived macrophages were incubated with either acetylated low density lipoprotein (AcLDL) or non-lipoprotein cholesterol and the question as to whether all of the cells, or specific cell subpopulations could accumulate cholesteryl ester was examined. We stained cholesteryl ester in monocyte-macrophages with the fluorescent probe filipin. Cholesteryl ester accumulated as lipid droplets that were widely dispersed in the cell cytoplasm. Interestingly, no more than 65% of monocyte-macrophages accumulated cholesteryl ester during the 1st day of incubation with non-lipoprotein cholesterol. By 2 days of incubation, greater than 90% of cells displayed cholesteryl ester deposition. The cholesteryl ester which accumulated during the 2nd day of incubation was derived from unesterified cholesterol that had accumulated during the 1st day of incubation. This finding was substantiated by the following: (1) chemical measurements showed that the total cholesterol content of monocyte-macrophages did not increase further after the 1st day of incubation, and (2) all monocyte-macrophages had accumulated fluorescent tagged cholesterol during the 1st day of incubation. In contrast to the results obtained with non-lipoprotein cholesterol, more than 90% of monocyte-macrophages incubated with AcLDL for 1 day accumulated cholesteryl ester in two experiments. However, less than 62% of monocyte-macrophages accumulated cholesteryl ester in two other experiments, thereby resembling results obtained with non-lipoprotein cholesterol. Again, the lack of cholesteryl ester accumulation with AcLDL was not due to a lack of uptake of AcLDL, as greater than 90% of monocyte-macrophages accumulated fluorescent tagged AcLDL. The observed heterogeneity in cholesterol esterification among human monocyte-macrophages suggests that functional subpopulations of these cells may exist with respect to cholesterol processing. However, heterogeneity in cholesteryl ester accumulation did not seem to correlate with expression of HLA-DR antigen, a marker of immunological activation of macrophages. Other sources of heterogeneity most likely result from inter-cellular variation at one or more levels of regulation of the cholesterol trafficking and esterification process.

Acetylation↗

PubMed1989

The beta-adrenoceptor is precoupled to Gs in chicken erythrocyte membranes.

In this study we seek to elucidate the mechanism of hormone-independent adenylate cyclase stimulation by Gpp(NH)p in chicken erythrocyte membranes, and the inhibition of this stimulation by propranolol. Membrane treatment with isoprenaline + GMP increased Gpp(NH)p stimulation to near maximal levels [obtainable with isoprenaline + Gpp(NH)p], but reduced stimulation by NaF. The stimulation by Gpp(NH)p was stereoselectively inhibited by propranolol, but not by equal concentrations of the local anaesthetic lignocaine. Propranolol's inhibitory action was abolished following membrane treatment with isoprenaline/GMP. In contrast to its inhibition of Gpp(NH)p stimulation, propranolol did not alter Gpp(NH)p-mediated 3H-GDP release from membranes. The polyene antibiotic filipin, which uncouples receptor (R) from Gs, also abolished Gpp(NH)p stimulation and this effect was partly overcome by membrane treatment. These results are consistent with a model in which free R exists in equilibrium with precoupled R.Gs complexes in the absence of hormone. These complexes are activated by Gpp(NH)p and dissociated by antagonists. The existence of such complexes is a prerequisite for Gpp(NH)p stimulatory action.

Adenylyl Cyclases↗

PubMed2004

Membrane cholesterol content modulates activation of BK channels in colonic epithelia.

Changes in the level of membrane cholesterol regulate a variety of signaling processes including those mediated by acylated signaling molecules that localize to lipid rafts. Recently several types of ion channels have been shown to have cholesterol-dependent activity and to localize to lipid rafts. In this study, we have investigated the role of cholesterol in the regulation of ion transport in colonic epithelial cells. We observed that methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering molecule, activated transepithelial short circuit current (Isc), but only from the basolateral side. Similar results were obtained with a cholesterol-binding agent, filipin, and with the sphingomyelin-degrading enzyme, sphingomyelinase. Experiments with DeltaF508CFTR mutant mice indicated that raft disruption affected CFTR-mediated anion secretion, while pharmacological studies showed that this effect was due to activation of basolateral large conductance Ca2+-activated K+ (BK) channels. Sucrose density gradient centrifugation studies demonstrated that BK channels were normally present in the high-density fraction containing the detergent-insoluble cytoskeleton, and that following treatment with MbetaCD, BK channels redistributed into detergent-soluble fractions. Our evidence therefore implicates novel high-density cholesterol-enriched plasma membrane microdomains in the modulation of BK channel activation and anion secretion in colonic epithelia.

Amiloride↗

PubMed2006

Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes.

Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.

Animals↗

PubMed2003

Degranulation and superoxide production depend on cholesterol in PLB-985 cells.

The effect of agents disrupting cholesterol-rich microdomains of the cell membrane was studied on the chemoattractant receptor (FPR and FRPL1) coupled effector responses of promyelocytic PLB-985 cells. Both methyl-beta-cyclodextrin (MbetaCD) and filipin III inhibited exocytosis of primary granules and O(2)(.-) production induced by stimulation of either chemotactic receptor. Alteration of calcium homeostasis of MbetaCD-treated cells does not account for the impairment of the effector responses. Disruption of microfilaments by cytochalasin B (CB) partially reverses the inhibitory effect of cholesterol depletion. Our results provide functional support for the involvement of cholesterol-rich membrane domains in the signaling of chemotactic receptors and call the attention to the possible role of microfilaments in the organization of lipid microdomains.

Amino Acid Sequence↗

PubMed2004

Chronic exposure to U18666A induces apoptosis in cultured murine cortical neurons.

Niemann-Pick disease type C (NPC) is a juvenile neurodegenerative disorder characterized by premature neuronal loss and altered cholesterol metabolism. Previous reports applying an 8-h exposure of U18666A, a cholesterol transport-inhibiting agent, demonstrated a dose-dependent reduction in beta-amyloid (Abeta) deposition and secretion in cortical neurons, with no significant cell injury. In the current study, we examined the chronic effect of 24-72h of U18666A treatment on primary cortical neurons and several cell lines. Our results showed caspase-3 activation and cellular injury in U18666A-treated cortical neurons but not in the cell lines, suggesting cell death by apoptosis only occurred in cortical neurons after chronic exposure to U18666A. We also demonstrated through filipin staining the accumulation of intracellular cholesterol in cortical neurons treated with U18666A, indicating the phenotypic mimic of NPC by U18666A. However, additions of 10 and 25microM pravastatin with 0.5microg/ml U18666A significantly attenuated toxicity. Taken together, these data showed for the first time that U18666A induces cell death by apoptosis and suggested an important in vitro model system to study NPC.

Androstenes↗

PubMed2004

Depletion of membrane cholesterol causes ligand-independent activation of Fas and apoptosis.

Fas is a member of the tumour necrosis factor receptor superfamily. Fas-mediated apoptosis is an essential mechanism protecting against skin cancer. Activation of Fas by specific ligand or agonistic antibodies leads to the formation of a membrane associated death-inducing signalling complex comprising aggregates of Fas, the Fas-associated death domain protein (FADD), and caspase-8. It has recently been suggested that activity of Fas is not only regulated by its cognate ligand but also by the association of this receptor with cholesterol-enriched lipid domains in the plasma membrane (lipid rafts). We report here that disruption of lipid rafts by cholesterol-depleting compounds (methyl-beta-cyclodextrin, filipin III, cholesterol oxidase, and mevastatin) leads to a spontaneous clustering of Fas in the non-raft compartment of the plasma membrane, formation of Fas-FADD complexes, activation of caspase-8, and apoptosis. We propose that in some cell types exclusion of Fas from lipid rafts leads to the spontaneous, ligand-independent activation of this death receptor, a mechanism that can potentially be utilized in anticancer therapy.

Apoptosis↗

PubMed2005

Transcriptional signaling from membrane raft-associated glucocorticoid receptor.

The contribution of plasma membrane-associated glucocorticoid receptor (GR) to transcriptional signaling is unclear. We observed GR in low-density detergent-resistant membrane (DRM) rafts derived from human hepatoma Hep3B cells in complexes with caveolin-1, HSP90, and STAT3. In transient transfection assays, GR-stimulated transcriptional signaling was reversibly inhibited by membrane-raft disrupters filipin III and progesterone. These data provide clear evidence for a functional contribution of DRM-associated GR to transcriptional signaling.

Base Sequence↗

PubMed2006

Mechanical strain increases gene transfer to skeletal muscle cells.

Gene transfer techniques possess tremendous potential for treating diseases and for facilitating the study of basic physiological processes. However, further development of efficient and safe methods for gene transfer is needed. The purpose of this study was to test the hypothesis that mechanical strain increases the transfer of DNA to differentiated skeletal muscle cells. We tested this hypothesis by applying cyclic strain to cultured skeletal myotubes either prior to or immediately after the introduction of exogenous DNA complexed with lipids, with strains of varying magnitude (10%, 20% and 30%), number (1800, 3600 and 7200 strain cycles) and frequency (0.5, 1.0 and 1.5 Hz). Results demonstrated that DNA transfection was increased by exposing muscle cells to cyclic strain, and that strain magnitude, number and frequency each influenced DNA transfection. Optimal strain conditions (20% strain magnitude, 3600 cycles applied at 1 Hz) were utilized to examine the role of membrane transport systems in strain-induced increases in DNA transfection. Filipin III was used to inhibit caveolar transport and was found to inhibit strain-mediated increases in DNA transfection, whereas chlorpromazine, used to inhibit clathrin-coated vesicle transport, had no effect. These results indicate that mechanical strain may be an effective method for increasing DNA transfection in skeletal muscle through enhanced caveolar transport.

Animals↗

PubMed2006

Lipid raft disruption prevents apoptosis induced by 2-chloro-2'-deoxyadenosine (Cladribine) in leukemia cell lines.

To clarify the role of lipid rafts in 2-chloro-2'-deoxyadenosine (2CdA; Cladribine)-induced apoptosis, the effects of disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) and filipin on 2CdA-induced apoptosis were investigated in four human acute lymphoblastic leukemia (ALL) cell lines comprised of T cells (MOLT-4, Jurkat) and B cells (NALM, BALL-1). The disruption of lipid rafts significantly inhibited 2CdA-induced apoptosis, indicating the crucial role of lipid rafts in the induction of apoptosis in leukemia cells. These reagents significantly inhibited 2CdA-induced elevation of the intracellular calcium concentration ([Ca(2+)](i)) in MOLT-4 cells, and 2CdA-induced apoptosis was partly inhibited by the Ca(2+) chelators BAPTA-AM and EGTA, and the L-type Ca(2+) channel blocker nifedipine. On the other hand, they had no effects on the cellular uptake of 2CdA. These results indicated that lipid rafts partly contributed to 2CdA-induced apoptosis by regulating Ca(2+) influx via the plasma membrane.

Apoptosis↗

PubMed2006

Signal transduction events involved in human epithelial cell invasion by Campylobacter jejuni 81-176.

Analyses of invasive enteric bacteria (e.g. Shigella, Salmonella, Listeria, and Campylobacter) have shown that these pathogens initiate orchestrated signal transduction cascades in host cells leading to host cytoskeletal rearrangements that result in bacterial uptake. This current study was specifically aimed at examining the involvement of host membrane caveolae and certain protein kinases in epithelial cell invasion by C. jejuni strain 81-176, for which we have previously characterized the kinetics of entry and a unique microtubule-dependent mechanism of internalization. Utilizing in vitro cultured cell invasion assays with a gentamicin-kill step, disruption of membrane caveolae by pretreatment of INT407 cell monolayers with filipin III reduced C. jejuni 81-176 entry by >95%. Strain 81-176 uptake into INT407 cells was markedly inhibited by monolayer pretreatment with the protein kinase inhibitors genistein and staurosporine, or specific inhibitors of PI 3-kinase, wortmannin and LY294002. Western blot analysis using monoclonal anti-protein tyrosine phosphorylation antibody revealed distinctive changes during invasion in phosphorylation of at least nine proteins. Further inhibitor studies indicated that heterotrimeric G proteins, plus ERK and p38 MAP kinase activation are also involved in C. jejuni 81-176 invasion. These results suggest that C. jejuni 81-176 interact at host cell surface membrane caveolae with G protein-coupled receptors, which presumably trigger G-proteins and kinases to activate host proteins including PI 3-kinase and MAP kinases, that appear to be intimately involved in the events controlling 81-176 internalization.

Androstadienes↗

PubMed2004

The combinatorial extension method reveals a sphingolipid binding domain on pancreatic bile salt-dependent lipase: role in secretion.

Structure similarity searches using a combinatorial extension approach revealed that a protein fold structurally related to the sphingolipid binding domain (SBD) of HIV-1 gp120 (V3 loop) is present on pancreatic bile salt-dependent lipase (BSDL). A synthetic peptide derived from the predicted V3-like domain of BSDL interacted with reconstituted monolayers of sphingolipids such as GalCer and GlcCer. Using Chinese hamster ovary cells stably transfected with the cDNA encoding the rat BSDL (CHO-3B clone) or pancreatic SOJ-6 cells expressing the human BSDL as models, we showed that the enzyme cofractionates with caveolin-1. The secretion of BSDL by CHO-3B cells was inhibited by permeable drugs affecting rafts structure (D609, PDMP, and filipin). Data suggest that the functional interaction between the BSDL SBD and lipid rafts is physiologically relevant and could be essential for sensing the BSDL folding prior to secretion. A tentative model accounting for the phosphorylation-induced dissociation of BSDL from rafts is presented.

Amino Acid Sequence↗

PubMed2006

Disruption of lipid rafts enhances activity of botulinum neurotoxin serotype A.

Botulinum neurotoxin serotype A (BoNT/A), one of seven serotypes of botulinum neurotoxin, is taken up by neurons of the peripheral nervous system. Within the neurons it catalyzes cleavage of the synaptosomal-associated protein having a mass of 25kDa, SNAP-25, thereby blocking neurotransmission. BoNT/A has been shown to interact with SV2, as well as gangliosides that are often found in lipid rafts. Lipid rafts are microdomains that can be found on the outer leaflet of the plasma membrane and are enriched in cholesterol and glycosphingolipids. To determine whether lipid rafts are needed for BoNT/A activity, those associated with the plasma membranes of murine N2a neuroblastoma cells were disrupted using either methyl-beta-cyclodextrin or filipin. Disruption of cholesterol-containing lipid rafts by either reagent did not prevent the action of BoNT/A on N2a cells, in fact activity was enhanced. While our results indicate that disruption of lipid rafts enhances BoNT/A activity, disruption of clathrin-dependent endocytosis appeared to be inhibitory.

Animals↗

PubMed2005

Evidence that membrane transduction of oligoarginine does not require vesicle formation.

The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 16 degrees C as compared to the 37 degrees C control, and was only partially inhibited at 4 degrees C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITC-dextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine.

Amiloride↗

PubMed2005

The complement inhibitor, CRIT, undergoes clathrin-dependent endocytosis.

Complement C2 receptor inhibitor trispanning (CRIT) is a receptor for the second component of complement and is found in various tissues and hemopoietic cells. On binding to CRIT, C2 cannot be activated to potentially form a variant-C3 convertase as it is rendered non-cleavable by C1s. CRIT thus limits the amount of C3 convertase formed on the cell surface. In this study we have shown, using flow cytometry and immunofluorescence microscopy, that human CRIT undergoes endocytosis from the plasma membrane. The endocytosis, possibly ligand mediated, occurs via clathrin-coated pits as it can be inhibited by prior incubation of cells in hypertonic medium or with chlorpromazine, at 37 degrees C. However, inhibition of endocytosis was not possible after treatment with nystatin, or filipin, inhibitors of caveolae/raft-dependent endocytosis. In the presence of C2 alone, CRIT associates with the adapter protein, beta-arrestin-2, and whether in association with C2 or not, then appears in the perinuclear region, but does not appear to be translocated into the nucleus. Apart from the C3aR and C5aR that internalize the anaphylatoxic peptides, this is the first report of the internalization via the clathrin pathway of a receptor for a complement serum protein.

Arrestins↗

PubMed1990

Cholesterol localization in ultrathin frozen sections in Schnyder's corneal crystalline dystrophy.

We examined a 57-year-old woman who had bilateral corneal crystalline deposits associated with xanthelasma. The patient's son had bilateral stromal haze. Plasma cholesterol and apolipoprotein A-I and B levels were normal. Histopathologic examination disclosed lipid deposits, particularly in the superficial stroma and Bowman's layer. These deposits stained with oil red O and filipin, a fluorescent probe that specifically detects unesterified cholesterol. Cryoultramicrotomy disclosed more specific ultrastructural localization of unesterified cholesterol with an array of crystals resembling multiple plates in extracellular corneal tissue.

Cataract↗

PubMed2000

cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera.

To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling.

Animals↗

PubMed2003

Real-time analysis of the effects of cholesterol on lipid raft behavior using atomic force microscopy.

Cholesterol plays a crucial role in cell membranes, and has been implicated in the assembly and maintenance of sphingolipid-rich rafts. We have examined the cholesterol-dependence of model rafts (sphingomyelin-rich domains) in supported lipid monolayers and bilayers using atomic force microscopy. Sphingomyelin-rich domains were observed in lipid monolayers in the absence and presence of cholesterol, except at high cholesterol concentrations, when separate domains were suppressed. The effect of manipulating cholesterol levels on the behavior of these sphingomyelin-rich domains in bilayers was observed in real time. Depletion of cholesterol resulted in dissolution of the model lipid rafts, whereas cholesterol addition resulted in an increased size of the sphingomyelin-rich domains and eventually the formation of a single raftlike lipid phase. Cholesterol colocalization with sphingomyelin-rich domains was confirmed using the sterol binding agent filipin.

Cholesterol↗