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At least 163 records · Page 9Linked to original sources
PubMed1992

Distribution of intramembranous particles and filipin-sterol complexes in the spermatid and spermatozoon of Culex quinquefasciatus (Culicidae).

A freeze-fracture study was carried out on spermatid and spermatozoon of the mosquito Culex quinquefasciatus. In the spermatid plasma membrane few and randomly distributed intramembranous particles were observed. In the spermatozoon the density of intramembranous particles was higher on the P- than on the E-fracture face of the plasma membrane. Two populations of particles were observed. Large particles (about 15 nm in diameter) are regularly arranged in double rows as a zipper-line, longitudinally oriented in relation to the main cell axis. These strands of particles were observed in the posterior head region, mainly associated with the E-fracture face. Filipin was used to analyse the presence and distribution of cholesterol in thin sections and freeze-fracture replicas. Filipin-sterol complexes were not homogeneously distributed throughout the spermatozoon plasma membrane. They were more abundant on the P-fracture face of the membrane lining the nuclear region. The results obtained show that Culex spermatozoon differs from those of other species in that its plasma membrane exhibits only a membrane domain, the zipper-line, localized in the postacrosomal region.

Animals↗
PubMed1979

Filipin prevents and reverses insulin stimulation of rat adipocyte phosphodiesterase.

A membrane fraction prepared from isolated rat adipocytes contained an insulin-sensitive cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which catalyzed the hydrolysis of both adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP). The rate of hydrolysis of cGMP was about one-third that of cAMP. The hydrolysis of the two nucleotides appeared to be assoicated with one catalytic site: one nucleotide interfered with the hydrolysis of the other, in a manner predictable from the kinetic constants in that the Km of one nucleotide as a substrate was comparable to its Ki as an inhibitor of the hydrolysis of the other nucleotide. Incubation of the adipocytes with insulin increased the Vmax of phosphodiesterase without affecting the Km values for either substrate. After adipocytes had been treated with filipin, a membrane perturbant, at a concentration that did not cause cell lysis, the response of phosphodiesterase to insulin was obliterated. Further, the insulin-stimulated phosphodiesterase activity was reversed when hormone-treated cells were subsequently incubated with this agent. These results suggest that the response of membrane phosphodiesterase to insulin is impaired once adipocytes have been exposed to filipin, either preceding or following the incubation with insulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Distribution of filipin-cholesterol complexes at sites of exocytosis - a freeze-fracture study of degranulating mast cells.

The distribution of membrane cholesterol at sites of exocytosis was studied with the cytochemical probe, filipin, in freeze-fractured rat peritoneal mast cells. When degranulation was induced in mast cells by polymyxin B, the membrane of all secretory granules, as well as the plasma membrane, including bulged areas signaling incipient exocytosis, appeared heavily labeled with filipin-sterol complexes (recognizable as 25-nm protuberances). This is evidence that cholesterol depletion is not an absolute requirement for membrane fusion.

Animals↗
PubMed1988

Changes in the distribution of intramembranous particles and filipin-sterol complexes during epididymal maturation of golden hamster spermatozoa.

Golden hamster spermatozoa in various segments of the excurrent duct system were studied by freeze-fracture with and without filipin treatment. Two types of regular IMP (intramembranous particle) patterns temporarily appear on the plasma membrane covering the sperm head. One is a hexagonal arrangement seen in the acrosomal region, and the other is a linear arrangement near the posterior ring. Both patterns are seen in the spermatozoa from the corpus epididymidis. The FSC (filipin-sterol complex) density in the plasma membrane covering the acrosome increases from about 400 to 500 FSC/microns2 during epididymal passage. In this region, the majority of the membrane sterols appears to reside on the outer leaflet of the lipid bilayer. When the spermatozoa reach the cauda epididymidis, FSCs in the outer acrosomal membrane virtually disappear from the apical segment, while they increase in the middle segment (250 FSC/microns2). These observations are discussed in relation to epididymal maturation.

Animals↗
PubMed2004

Effect of saponin and filipin on antagonist binding to AT 1 receptors in intact cells.

In the present study, [ 3H ]-candesartan binding experiments were performed on intact Chinese Hamster Ovary cells transfected with the human AT1 receptor (CHO-AT1 cells). Cells were pre-treated with 0.01mg/ml saponin or filipin. Both pre-treatments resulted in an increased dissociation rate and decreased affinity of the insurmountable non-peptide antagonist [3H ]-candesartan. A similar decrease in affinity was observed for the peptide antagonist Sar1-Ile8 angiotensin II and for other non-peptide antagonists, irrespectively of their degree of insurmountability. A similar discrepancy in [ 3H ]-candesartan binding was earlier observed when comparing intact CHO-AT1 cells and membrane preparations thereof. This similarity is further highlighted by the observations that saponin or filipin no longer affect [ 3H ]-candesartan binding to CHO-AT1 cell membranes and that both agents permeabilise the CHO-AT1 cells. This suggests that the intracellular composition and/or organisation of living cells play an active role with regard to antagonist-AT1 receptor interactions.

Angiotensin II↗
PubMed1988

Lateral segregation of sterol and channel proteins in the mitochondrial outer membrane induced by phospholipase A2: evidence from negative-stain electron microscopy using filipin.

The channel protein in the mitochondrial outer membrane of Neurospora crassa aggregates laterally into crystalline arrays by the action of phospholipase A2. When mitochondrial outer membranes are reacted with filipin and examined by negative-stain electron microscopy, filipin-sterol complexes are found everywhere on the membranes except on the crystalline channel arrays. This suggests that the channel-rich membrane domains may have a relatively low content of accessible sterol. It is proposed that in vitro segregation of protein and lipid membrane components by phospholipase A2 may reflect a mechanism by which the endogenous enzyme organizes the native mitochondrial membrane into functional domains.

Filipin↗
PubMed1983

Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes.

A method is described for measuring rates of mitochondrial pyruvate carboxylation in hepatocytes treated with the polyene antibiotic, filipin, to render the plasma membrane permeable to substrates. With this approach it was possible to demonstrate that treatment of cells with glucagon or catecholamines results in a stimulation of mitochondrial CO2 fixation measured in situ comparable with that observed in the isolated mitochondria, in terms of time of onset of the response, hormone selectivity and sensitivity. In addition, angiotensin II and vasopressin were shown to enhance the activity of pyruvate carboxylase in both the intact mitochondria and filipin-treated cells, thus strengthening the postulate that this site is a major locus of hormone action in the control of gluconeogenesis. Addition of 3-mercaptopicolinic acid, to inhibit gluconeogenesis at the level of phosphoenolpyruvate carboxykinase, had no significant effect on the stimulation of pyruvate carboxylation by adrenaline, suggesting that the effect of the hormone at this site is independent of changes in activity of other enzymes further on in the pathway. The data presented preclude the possibility that acute effects of hormones on mitochondrial metabolism are solely artifacts of the preparation procedure.

Aminooxyacetic Acid↗
PubMed1979

Absence of filipin-sterol complexes from large coated pits on the surface of culture cells.

Monolayer cultures of normal or transformed fibroblasts and of liver cells fixed in a glutaraldehyde solution containing 300 microM filipin, a sterol-specific polyene antibiotic, were freeze-fractured to study the distribution of cholesterol within their plasma membranes. Filipin-sterol complexes, recognizable as 25- to 30-nm protuberances scattered in the fracture face of plasma membrane, were absent from invaginations corresponding to large, bristle-coated pits (and possibly also from small, flask-shaped invaginations). These results suggest that invaginating regions on the cell surface are specialized plasma membrane domains with a lower cholesterol content than the surrounding membrane. The localized change in membrane fluidity due to the low cholesterol concentration could play a role in endocytosis.

Animals↗
PubMed2002

Filipin prevents pathological prion protein accumulation by reducing endocytosis and inducing cellular PrP release.

Conversion of the normal membrane-bound prion protein (PrP-sen) to its pathological isoform (PrP-res) is a key event in the pathogenesis of transmissible spongiform encephalopathies. Although the subcellular sites of conversion are poorly characterized, several lines of evidence have suggested the involvement of membrane lipid rafts in the conversion process. Here we report that copper stimulates the endocytosis of PrP-sen via a caveolin-dependent pathway in both microglia and neuroblastoma cells. We show that the polyene antibiotic filipin both limits endocytosis of PrP-sen and dramatically reduces the amount of membrane-bound PrP-sen. This reduction results from a rapid and massive release of full matured PrP-sen into the culture medium. Finally, we demonstrate that filipin is a potent inhibitor of PrP-res formation into chronically infected neuroblastoma cells. Our results reinforce the role of rafts in PrP trafficking and raise the possibility that the release of PrP-sen from the plasma membrane decreases the amount of available substrate PrP-sen at the conversion sites.

Cell Line↗
PubMed1992

Fluorescence study of the macrolide pentaene antibiotic filipin in aqueous solution and in a model system of membranes.

The polyene antibiotic filipin (a pentaene) has been studied using photophysical techniques. The polyene self-aggregates in water with a critical micellar concentration of 2 microM. Two approaches were used to evaluate the aggregate dimensions: (a) a lower limit of 10 nm for the aggregate radius was obtained from energy transfer experiments; (b) a formula for rationalizing the turbidity spectrum was derived, and from its application a spherical shape of radius about 50 nm was deduced. The low value for the fluorescence anisotropy of the aggregate (r = 0.02) is compatible with a very loose structure, i.e. the chromophore has very efficient depolarization dynamics that is not controlled by the aggregate size. The Stern-Volmer plot of aggregated filipin fluorescence quenching by iodide is non-linear, presenting a downward curvature. A model was used for the interpretation of these data, along with a study of the quenching in transient state; it was concluded that all the components of the decay are affected by the quencher, i.e. the aggregate has a very open structure with respect to the iodide ion. The partition constants of the polyene, Kp, between a model system of membranes (small unilamellar vesicles of dipalmitoylglycerophosphocholine) and the aqueous phase were determined from anisotropy measurements; the values obtained were Kp (gel phase) = (3.4 +/- 0.8) x 10(3) and Kp (liquid crystal phase) = (7.7 +/- 2.2) x 10(2). The observation that the polyene incorporation is efficient is at variance with the belief that the presence of sterols are essential for the interaction of polyene antibiotics with membranes [for review see Bolard, J. (1986) Biochim. Biophys. Acta 864, 257-304].

Energy Transfer↗
PubMedDate not supplied

Distribution of filipin-sterol complexes in sperm membranes from hypercholesterolaemic rabbits.

The distribution of membrane filipin-sterol complexes (FSC) was examined ultrastructurally in cauda epididymal sperm from normal and hypercholesterolaemic rabbits. Membrane FSC were quantitatively analysed on replicas of filipin-treated cells. We determined a significant difference in FSC concentration in the plasma membrane of the acrosome region (PMAR) of hypercholesterolaemic animals compared to normal rabbits. Hypercholesterolaemic animals had 0.56 +/- 0.05 FSC complex per micron 2 (enriched Cholesterol diet: Diet 2) in the marginal segment of PMAR; 0.62 +/- 0.05 FSC complex per micron 2 (enriched Cholesterol and fish oil diet: Diet 3) and only 0.28 +/- 0.01 FSC complex per micron 2 for normal animals (Control Diet 1). In the principal (anterior) segment we found 0.54 +/- 0.10 FSC complex per micron 2 (Diet 2), 0.56 +/- 0.03 FSC complex per micron 2 (Diet 3) and 0.30 +/- 0.04 FSC complex per micron 2 (Control Diet 1). We also counted 0.47 +/- 0.1 FSC complex per micron 2 in the equatorial segment of PMAR for Diet 2, 0.27 +/- 0.05 and 0.28 +/- 0.04 FSC complex per micron 2 in Diet 1 and Diet 3 respectively. Diet 4 (fish oil) did not differ from the control. An increase in the Cholesterol (Chol) level in biological membranes or a difference in the Chol membrane domains could cause a variation in the membrane rigidity that could modify the sperm membrane fusion capacity and functionality. The results presented in this paper are in agreement and could explain the decrease in the kinetic of the sperm acrosome reaction that we have observed in experimentally hypercholesterolaemic rabbits (Díaz-Fontdevila & Bustos-Obregón, 1992).

Acrosome↗
PubMed1987

Freeze-fracture localization of filipin-sterol complexes in plasma- and cyto-membranes of Pneumocystis carinii.

The polyene antibiotic, filipin, was used as the probe for demonstrating sterols in the freeze-fractured plasma- and cytomembranes of Pneumocystis carinii. The distribution of filipin-sterol complexes was homogeneous on the plasma membrane throughout all developmental stages from trophozoite to cyst; however, the density of the complexes gradually decreased with the progress of development. In the trophozoite, the density of the complexes was 485 +/- 42/micron2 on the P face and 341 +/- 27/micron2 on the E face. It was 249 +/- 50 on the P face and 132 +/- 48 on the E face in the precyst and 138 +/- 24 and 59 +/- 20, respectively, in the cyst. The membranes of nucleus, mitochondria, and small round bodies showed more or fewer complexes while no complexes were found in the membranes of one endoplasmic reticulum. In nuclear and mitochondrial membranes, some small scattered clusters of complexes were observed. Two types of vacuoles were distinguished: one having many complexes in its membrane and the other having none at all.

Animals↗
PubMed1978

Effects of the antidiuretic hormone, arginine vasotocin, theophylline, filipin and A23187 on cyclic AMP in isolated frog skin epithelium (Rana temporaria).

A method for measuring cAMP in frog skin epithelium was developed. The epithelia were isolated after collagenase-treatment. cAMP was extracted by boiling water and the extract was purified on dry Al2O3. The change with time of the cAMP level after addition of arginine vasotocin (AVT) was studied. The hormone caused a rapid increase in cAMP level with a maximum after 3-5 min, whereafter the cAMP level declined. Incubation with AVT made the epithelia refractory to a second dose of AVT, which indicates that the decline in cAMP level was caused by a feedback mechanism and not by inactivation of the hormone. cAMP appeared evenly distributed in all cell-layers of the epithelia both before and after stimulation with AVT. Theophylline caused a rapid increase in the cAMP level, which remained elevated for at least 45 min. Addition of the ionophore A23187 or of filipin had no effect on the cAMP level. However, in the presence of theophylline, A23187 enhanced the cAMP level, whereas filipin had no effect. Therefore the involvement of cAMP in the action of A23187 has to be considered.

Animals↗
PubMedDate not supplied

Cholesterol content of focal opacities and multilamellar bodies in the human lens: filipin cytochemistry and freeze fracture.

Adult human lenses with focal opacities were processed for normal as well as freeze fracture electron microscopy. Cholesterol was demonstrated using filipin cytochemistry. Filipin cytochemistry in combination with freeze fracture revealed that the amount of cholesterol in the normal fibre membranes was fully comparable with the amount of cholesterol in the deviating membranes of the focal opacities and the multilamellar bodies. As regards the cholesterol/phospholipid ratio, the deviating membranes have the same state of maturation as the surrounding normal lens fibre membranes, which implies that their synthesis is as highly coordinated as the synthesis of the normal lens fibre membranes.

Cataract↗
PubMed1985

Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct.

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.

Animals↗
PubMed1987

Filipin and digitonin studies of cell membrane changes during junction breakdown in the dystrophic rat retinal pigment epithelium.

We have previously found that a breakdown of tight junctions in retinal pigment epithelial cells of Royal College of Surgeons' rats is associated with a redistribution of intramembrane particles and Na-K-ATPase activity. Changes in the lipid and sterol composition of membranes can alter their fluidity, permeability and enzyme activity, and may contribute to changes in cell barrier function in the dystrophic epithelium. We have now used filipin and digitonin, which bind to membrane sterols and produce membrane deformations recognizable by freeze-fracture and thin-section electron microscopy, to study the distribution of cholesterol and related 3-B-hydroxysterols in the dystrophic epithelium. The results of these studies show that in the normal pigment epithelium and prior to tight junction breakdown in the dystrophic epithelium, filipin- and digitonin-sterol complexes are rare in the membranes between tight junctions and adhering junctions, and in areas of attachment between the plasma membrane and basal lamina. Complexes are more numerous in the basal infoldings, and most densely packed in the lateral and apical microvillous membranes. During junction breakdown, complexes increase substantially in apical, basal, junctional, and nuclear membranes. Later, after the junctions disappear, complexes decrease. These results indicate that alterations in the expression of membrane sterols accompany the changes in structure and function of tight junctions in the dystrophic retinal pigment epithelium.

Animals↗
PubMed2004

[Diagnosis of adult type of Niemann-Pick disease (type C) in two brothers by filipin staining of bone marrow smears].

Niemann-Pick disease, type C (NPC) is a neurometabolic genetic disorder that is distinguished from other types of Niemann-Pick disease by its later onset, more insidious progression, variable visceromegaly, and abnormalities of intracellular cholesterol metabolism. We report cases in 18-year-old and 20-year-old brothers who presented with disinhibition and involuntary movement of their hands. Both brothers presented various signs such as dementia, vertical supranuclear ophthalmoplegia (VSO), dysarthria, axial and limb dystonia, hyperreflexia, pathologic reflex, cerebellar ataxia, as reported. They also presented startle response. Brain MRI showed diffuse cerebral atrophy and abdominal CT reveals hepato-splenomegaly in both patients. These cases were suspected to be NPC based on dementia, VSO, cerebellar ataxia, hepato-splenomegaly and foam cells in the bone marrow. Generally, the diagnosis of NPC is based on deficient cholesterol esterification and excessive lysosomal filipin staining in cultured skin fibroblasts. However, culture of fibroblasts obtained from a biopsied skin samples is slow. We have rapidly made the diagnosis of NPC in our patients by filipin staining of foam cells from bone marrow. This diagnostic process using a bone marrow smear is more convenient and rapid than previous methods using cultured skin fibroblasts.

Adolescent↗
PubMed1992

Filipin labeling in Chlamydomonas gametes exposes site-specific lipid specializations.

A thin section study of mating Chlamydomonas cell wall-less CW 15 mating type plus (mt+) and mating type minus (mt-) gametes utilized filipin. The results show extensive labeling of mt+ and mt- plasma membranes. No labeling was seen on the mating structure membranes of activated mt+ or mt- gametes. These results indicate that differences exist between the plasma membrane and the mating structure membrane of gametes. If filipin is specific for the 3-beta-OH sterol, ergosterol and/or other Chlamydomonas sterols, then these results imply that the fusing mating structure membranes may be altered or reduced in sterol content. Such lipid specializations may increase local membrane fluidity and thereby facilitate the site-specific cell fusion associated with mating Chlamydomonas gametes.

Animals↗