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PubMed1984

Increase in membrane cholesterol: a possible trigger for degradation of HMG CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells.

The crystalloid endoplasmic reticulum (ER) houses large amounts of HMG CoA reductase, the rate-controlling enzyme in cholesterol synthesis. The crystalloid ER appears in UT-1 cells, a line of Chinese hamster ovary cells that has been chronically starved of cholesterol as a result of growth in the presence of compactin, an inhibitor of reductase. When cholesterol was provided to UT-1 cells in the form of low density lipoprotein (LDL), the reductase and crystalloid ER were destroyed. This destruction was preceded by an increase in the cholesterol content of crystalloid ER membranes, as judged by a 4- to 8-fold increase in their ability to form complexes with filipin, a cholesterol-binding compound that can be visualized in freeze-fracture electron micrographs. Filipin binding to other membranes was unchanged. Thus insertion of cholesterol into the crystalloid ER membrane may trigger the degradation of reductase and the membrane itself.

Animals↗
PubMed1988

Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2.

The highly selective fluorescent Ca2+ indicator 'quin 2' has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, 'quin 2/AM', which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean +/- S.E.M.) indicated a normal cytoplasmic free-calcium concentration, [Ca2+]i, of 193 nM +/- 0.2 (n = 10) for ejaculated ram sperm, 175 nM +/- 3.9 (n = 10) for cauda epididymal boar sperm and 105 nM +/- 10 (n = 10) for the caput sperm. After cold shock ejaculated ram and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a [Ca2+]i similar to control sperm. Sodium azide, propranolol and caffeine did not affect the [Ca2+]i of ram and boar sperm, however theophylline, dibutyryl c-AMP and La3+ significantly reduced it. The inhibitors rotenone and antimycin A, and the uncouplers 2,4-DNP and CCCP caused a transient elevation of [Ca2+]i, most likely resulting from release of mitochondrial calcium. The increased [Ca2+]i following addition of the ionophore A23187, was highly pH dependent in ram spermatozoa and it was critical to increase the pH of the medium above 7.5; the increase in [Ca2+]i was apparently not dependent on the oxidative metabolism of the sperm as addition of the uncouplers 2,4-DNP and CCCP had no effect on [Ca2+ )i. Addition of filipin to ram and boar sperm resulted in a large increase in [Ca2+]i but addition of filipin to ionophore-treated sperm caused [Ca2+]i to fall well below control levels.

Aminoquinolines↗
PubMed1989

Isolation and characterization of a highly purified flagellar membrane fraction from trypanosomatids.

The flagellar membrane of trypanosomatids has certain ultrastructural and antigenic characteristics that make its biochemical analysis very interesting. We have obtained a highly purified flagellar membrane fraction from epimastigotes of Trypanosoma cruzi and promastigotes of Herpetomonas samuelpessoai. The fractions consisted of regular spherical vesicles. Membrane fractions fixed in a glutaraldehyde solution containing filipin and freeze-fractured showed few intramembranous particles, and many protuberances indicative of filipin-sterol complexes. The protein composition of all the subflagellar fractions was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The flagellar membrane fraction showed more than twenty bands. We wish particularly to point out the presence of six bands corresponding to proteins of 160, 135, 41, 31, 26 and 18 kDa which were not present in the axoneme-paraxial structure fraction. The blot of flagellar membrane proteins successively incubated with concanavalin A and horseradish peroxidase showed intense binding of the lectin to proteins of 117 and 87 kDa, and less strong binding to several other minor bands. Our results showed that although the membrane of the flagellum of trypanosomatids had few proteins, it seemed rich in glycosylated elements.

Animals↗
PubMed1988

Morphological and functional reconsideration of the cytoplasmic bridges which connect male germ cells in snails.

Cytoplasmic bridges (CB) between male germ cells of three fresh-water snails have been examined by electron microscopy, using ultrathin sections and freeze-fracture replicas prepared by ordinary methods and after use of filipin for indicating the presence of membrane cholesterol. The bridge plasma membrane, which was formerly considered to be smooth, was found in these snails to be corrugated. The corrugations were periodically parallel and oriented parallel to the axis of the bridge. The mature bridges showed very low densities of intramembranous particles. No filipin-sterol complexes formed on either the P face or the E face of the bridge plasma membrane, in contrast to plasma membranes elsewhere. The numbers of corrugations in each CB varied with the species. The membrane corrugations overlie bundles of electron dense fibers measuring approximately 30 nm in diameter and 60 nm in center-to-center distance, fitting into convexities of the plasma membrane. The present observations lead us to the necessity of reconsidering the morphological and functional aspects of cytoplasmic bridges in vertebrate as well as in invertebrate germ cells.

Animals↗
PubMed1991

Type C Niemann-Pick disease: spectrum of phenotypic variation in disruption of intracellular LDL-derived cholesterol processing.

To investigate biochemical heterogeneity within Niemann-Pick type C disease (NPC), the two most characteristic abnormalities, namely (1) kinetics of LDL-stimulated cholesteryl ester formation and (2) intravesicular accumulation of LDL-derived unesterified cholesterol, evaluated by histochemical filipin staining, were studied in cultured skin fibroblasts from a population of 125 NPC patients. Profound alterations (esterification rates less than 10% of normal, very numerous and intensely fluorescent cholesterol-filipin granules) were demonstrated in 86% of the cases, depicting the 'classical' NPC phenotype. The remaining cell lines showed a graded less severe impairment and more transient delay in the induction of LDL-mediated cholesteryl esterification, along with an attenuated accumulation of unesterified cholesterol. In particular, cells from a small group (7%) of patients, which have been individualized as representative of a 'variant' phenotype, showed only slight alterations of esterification, restricted to the early phase of LDL uptake and undistinguishable from those in heterozygotes. In these cells, an abnormal cytochemical distribution of LDL-derived cholesterol, although moderate, was still evident provided rigorous experimental conditions were followed. A third, less clearly individualized group (7%), differing from the classical phenotype mostly by higher rates of cholesteryl ester formation, has been designated as an 'intermediary' phenotype to reflect a more difficult diagnosis of such patients. These findings have an important bearing with regard to diagnosis and genetic counselling, although the significance of such a phenotypic variation in terms of genetic heterogeneity has still to be demonstrated. A given biochemical phenotype was however a constant observation within a family (14 pairs of siblings tested so far). The unique feature of LDL-cholesterol processing alterations in NPC has been further established from comparative studies in Wolman disease and I-cell disease, showing normal or different intracellular distribution of unesterified LDL-derived cholesterol in the latter disorders. Correlation between biochemical and clinical NPC phenotypes was only partial, but a correlation between the severity of alterations in cholesterol processing and sphingomyelin catabolism could be established.

Adolescent↗
PubMed2004

Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells.

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8(+) T cells and induce cytolytic effector T cells in vitro.

Amiloride↗
PubMed1983

Regular structures on the microvillar surface membrane of ileal epithelial cells in suckling rat intestine.

Freeze-fracture deep-etch studies were done to examine regular patterns of surface membrane particles in the suckling rat ileal epithelium by using quick-freezing method. In addition to the presence on the luminal surfaces of well-developed endocytic complexes, latticed particles were consistently observed on almost the entire apical membrane between microvilli and frequently on the microvilli. These particles seemed to be partly integrated in the outer half layer of membranes. After rinsing with phosphate-buffered saline, patterns on the microvilli appeared to be parallel circle lines rather than latticed particles found in fresh preparations. Filipin treatment showed the presence of filipin-sterol complexes on most of the particle-covered membrane areas except small tubules and vesicles. Possibilities were suggested that these latticed particles were transferred to the apical surface along the outer half layer of membranes, and released or secreted into the intestinal lumen.

Animals↗
PubMed2003

Temporal and spatial changes of free cholesterol and neutral lipids in rat brain after transient middle cerebral artery occlusion.

In order to examine lipid metabolism in relation to neural process following brain ischemia, we investigated temporal and spatial changes of free cholesterol (FC) and neutral lipids (NLs) after 90 min of transient middle cerebral artery occlusion (MCAO). Filipin and Nile Red stainings were performed to detect mainly FC and NLs, respectively. Double stainings for Nile Red plus ED1, MAP2, or GFAP were performed in order to identify cell type of positive stainings. Filipin stanining decreased during 1-7 day and lost at 21 day after transient MCAO in the ischemic core, but did not change in the penumbra. Nile Red positive droplets reached the maximum at 7 day after transient MCAO and gradually decreased in the core, while the peak time delayed in the penumbra. MAP2 immunoreactivity lost at 7 day in the core, and increased in the penumbra during 7-56 day. Most Nile Red positive droplets were double positive for ED1 in the core, and were localized within GFAP positive cells in the penumbra. These results suggest that changes of FC and NLs are different temporally and spatially between the core and penumbra in relation to degenerative and regenerative neural processes following brain ischemia.

Animals↗
PubMedDate not supplied

Distribution of sterols and anionic lipids in human sperm plasma membrane: effects of in vitro capacitation.

Filipin, a membrane beta-hydroxysterol probe, and polymyxin B (PXB), a probe for anionic lipids, were used to study human sperm plasma membrane (PM) with particular reference to changes induced by capacitation in vitro. In washed but noncapacitated spermatozoa the density of filipin/sterol complexes (FSC) was uniformly high in the PM overlying the acrosome, without any differences between its anterior and equatorial regions. The postacrosomal region was poor in FSC. The exclusion of FSC from small areas of the PM covering the two acrosomal regions and reduction of their number in the postacrosomal region were the main changes induced by sperm capacitation. Unlike filipin, PXB produced a capacitation-independent reaction pattern characterized by a high reactivity of the PM covering the anterior portion of the acrosome, probably conferring to this region its potential fusigenicity temporarily antagonized by the elevated sterol content. Only an exceptional and slight PXB binding was seen in the nonfusigenic equatorial and postacrosomal regions.

Cell Membrane↗
PubMed2002

Bradykinin potentiation by ACE inhibitors: a matter of metabolism.

1. Studies in isolated cells overexpressing ACE and bradykinin type 2 (B(2)) receptors suggest that ACE inhibitors potentiate bradykinin by inhibiting B(2) receptor desensitization, via a mechanism involving protein kinase C (PKC) and phosphatases. Here we investigated, in intact porcine coronary arteries, endothelial ACE/B(2) receptor 'crosstalk' as well as bradykinin potentiation through neutral endopeptidase (NEP) inhibition. 2. NEP inhibition with phosphoramidon did not affect the bradykinin concentration-response curve (CRC), nor did combined NEP/ACE inhibition with omapatrilat exert a further leftward shift on top of the approximately 10 fold leftward shift of the bradykinin CRC observed with ACE inhibition alone. 3. In arteries that, following repeated exposure to 0.1 microM bradykinin, no longer responded to bradykinin ('desensitized' arteries), the ACE inhibitors quinaprilat and angiotensin-(1-7) both induced complete relaxation, without affecting the organ bath fluid levels of bradykinin. This phenomenon was unaffected by inhibition of PKC or phosphatases (with calphostin C and okadaic acid, respectively). 4. When using bradykinin analogues that were either completely or largely ACE-resistant ([Phe(8)psi(CH(2)-NH)Arg(9)]-bradykinin and [deltaPhe(5)]-bradykinin, respectively), the ACE inhibitor-induced shift of the bradykinin CRC was absent, and its ability to reverse desensitization was absent or significantly reduced, respectively. Caveolar disruption with filipin did not affect the quinaprilat-induced effects. Filipin did however reduce the bradykinin-induced relaxation by approximately 25-30%, thereby confirming that B(2) receptor-endothelial NO synthase (eNOS) interaction occurs in caveolae. 5. In conclusion, in porcine arteries, in contrast to transfected cells, bradykinin potentiation by ACE inhibitors is a metabolic process, that can only be explained on the basis of ACE-B(2) receptor co-localization on the endothelial cell membrane. NEP does not appear to affect the bradykinin levels in close proximity to B(2) receptors, and the ACE inhibitor-induced bradykinin potentiation precedes B(2) receptor coupling to eNOS in caveolae.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2001

Establishment and characterization of immortalized Schwann cells from murine model of Niemann-Pick disease type C (spm/spm).

Niemann-Pick disease type C (NPC) is characterized by an accumulation of unesterified cholesterol in the endosomal/lysosomal (E/L) system, resulting in progressive neurodegeneration and death during early childhood. To investigate the cellular pathomechanism of nervous system involvement in NPC, continuous neural cell lines are desirable. In this study, we obtained neuronal and Schwann cell cultures and established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of NPC model mouse (spm/spm). One of the cell lines, designated SPMS9, had distinct Schwann cell phenotypes and was maintained over 10 months without phenotypic alterations. The level of Npc1 mRNA was markedly decreased, and NPC1 protein was not detectable in SPMS9 cells. These cells contained intracytoplasmic granules positive for filipin cholesterol staining and immunoreactive for GM2 ganglioside. Electron-microscopically, intracytoplasmic polymorphous membranous inclusions and vacuoles were demonstrated in SPMS9 cells. The treatment with an inhibitor of ceramide-specific glucosyltransferase, N-butyldeoxynojirimysin (NB-DNJ) markedly reduced the intracytoplasmic granular immunofluorescence for GM2 ganglioside in SPMS9 cells, whereas the amount of filipin-positive granules remained unchanged. The SPMS9 cells retained vesicular fluorescence of cationic dye acriflavine 16-24 hours after loading, indicating the defect of transmembrane efflux pump activity of NPC1 in the E/L compartment in these cells. These immortalized Schwann cells can be useful in studies on the nervous system lesions in NPC.

Animals↗
PubMed2004

Targeted cytoplasmic irradiation induces bystander responses.

The observation of radiation-induced bystander responses, in which cells respond to their neighbors being irradiated, has important implications for understanding mechanisms of radiation action particularly after low-dose exposure. Much of this questions the current dogma of direct DNA damage driving response in irradiated systems. In this study, we have used a charged-particle microbeam to target individual helium ions ((3)He(2+)) to individual cells within a population of radioresistant glioma cells cultured alone or in coculture with primary human fibroblasts. We found that even when a single cell within the glioma population was precisely traversed through its cytoplasm with one (3)He(2+) ion, bystander responses were induced in the neighboring nonirradiated glioma or fibroblasts so that the yield of micronuclei was increased by 36% for the glioma population and 78% for the bystander fibroblast population. Importantly, the yield of bystander-induced micronuclei was independent of whether the cytoplasm or nucleus of a cell was targeted. The bystander responses were fully eliminated when the populations were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide or filipin, which scavenge nitric oxide (NO) and disrupt membrane rafts, respectively. By using the probe 4-amino-5-methylamino-2',7'-difluorofluorescein, it was found that the NO level in the glioma population was increased by 15% after 1 or 10 cytoplasmic traversals, and this NO production was inhibited by filipin. This finding shows that direct DNA damage is not required for switching on of important cell-signaling mechanisms after low-dose irradiation and that, under these conditions, the whole cell should be considered a sensor of radiation exposure.

Biological Factors↗
PubMed2001

Involvement of a triton-insoluble floating fraction in Dictyostelium cell-cell adhesion.

We have isolated and characterized a Triton-insoluble floating fraction (TIFF) from Dictyostelium. Ten major proteins were consistently detected in TIFF, and six species were identified by mass spectrometry as actin, porin, comitin, regulatory myosin light chain, a novel member of the CD36 family, and the phospholipid-anchored cell adhesion molecule gp80. TIFF was enriched with many acylated proteins. Also, the sterol/phospholipid ratio of TIFF was 10-fold higher than that of the bulk plasma membrane. Immunoelectron microscopy showed that TIFF has vesicular morphology and confirmed the association of gp80 and comitin with TIFF membranes. Several TIFF properties were similar to those of Dictyostelium contact regions, which were isolated as a cytoskeleton-associated membrane fraction. Mass spectrometry demonstrated that TIFF and contact regions shared the same major proteins. During development, gp80 colocalized with F-actin, porin, and comitin at cell-cell contacts. These proteins were also recruited to gp80 caps induced by antibody cross-linking. Filipin staining revealed high sterol levels in both gp80-enriched cell-cell contacts and gp80 caps. Moreover, sterol sequestration by filipin and digitonin inhibited gp80-mediated cell-cell adhesion. This study reveals that Dictyostelium TIFF has structural properties previously attributed to vertebrate TIFF and establishes a role for Dictyostelium TIFF in cell-cell adhesion during development.

Animals↗
PubMed1990

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.

Animals↗
PubMed1980

Specializations in filopodial membranes at points of attachment to the substrate.

A mouse cell line (LM), which grows predominantly as spindle-shaped cells with numerous filopodia, was employed in this study. These filopodial projections appear to be important as sites of attachment to the substratum in LM cells. Morphologically the filopodia are slender projections from the cell body which usually attach to the substrate at their distal ends (filopodial footpads). Freeze-fracture of monolayer cultures in situ preserves the spatial relationship of filopodial processes to that of the cell body. Examination of these freeze-fracture preparations reveals a striking difference in the density of intramembrane particles (IMP) in the filopodial-footpad plasmalemma compared with the plasmalemma of the cell body (number of IMP in footpad > cell body). Additionally, there is a marked difference in the number of filipin-sterol complexes on the cell body, compared with the filopodial footpad, implying a difference in the cholesterol content in these regions (filipin-sterol complexes in footpad < cell body). These data suggest a structural and functional specialization of the filopodial-footpad plasma membrane which may be related to cell adhesion.

Animals↗
PubMed1987

Effect of verapamil and sulfhydryl reagents on calcium transport in bovine spermatozoa.

Calcium uptake by intact bovine epididymal spermatozoa is not affected by low concentrations (up to 0.75 mM) of the calcium transport blocker verapamil. Under these conditions, calcium transport into sperm mitochondria is highly inhibited. At higher verapamil concentrations (1.0, 1.5 mM), calcium transport into intact sperm is also inhibited, and this inhibition cannot be relieved by disrupting the plasma membrane with filipin. Calcium uptake into intact sperm is highly inhibited by mersalyl and this inhibitory effect can be completely relieved when the plasma membrane is disrupted by filipin. This effect of mersalyl is not dependent on the presence of phosphate in the incubation medium. Phosphate itself, up to 2 mM, enhances calcium uptake into the cells; this effect decreases at higher concentrations and is depressed 57% at 10 mM phosphate. This inhibitory effect of high phosphate concentration can be blocked by mersalyl. It is suggested that the calcium carrier itself and not a phosphate carrier of the plasma membrane is inhibited by mersalyl. It is possible that there is a symporter for calcium and phosphate in the plasma membrane of bovine spermatozoa.

Animals↗
PubMed1994

25-Hydroxycholesterol induces reorganization of lysosomes in normal but not Niemann-Pick disease type C astrocytes.

25-hydroxycholesterol (25-OHC), an oxysterol that potently regulates cellular cholesterol metabolism, induced formation of novel fibrillar structures in normal mouse astrocytes as observed by fluorescence microscopy with the cholesterol probe, filipin. These fibrils were identified as lysosomes by their immunoreactivity for the lysosome associated membrane glycoprotein (LAMP). In contrast, astrocytes derived from the Niemann-Pick disease type C (NPC) mutant mouse were resistant to this oxysterol-induced lysosomal reorganization. NPC astrocytes have abnormal intracellular cholesterol storage as observed by brightly positive filipin staining of their lysosomes. These results show that lysosomal cholesterol storage in NPC astrocytes is associated with a block in oxysterol-mediated fibrillar reorganization of lysosomes.

Animals↗
PubMed2003

Histologic evaluation of corneal stroma in rabbits after intrastromal corneal ring implantation.

PURPOSE: Previous refractive corneal implants have produced histologic changes characteristic of nonspecific lipid keratopathy. Intacs intrastromal corneal ring segments are polymethylmethacrylate (PMMA) corneal inserts approved for the correction of low myopia by the U.S. Food and Drug Administration. The purpose of this study was to describe histologic changes associated with these corneal implants in rabbit eyes. METHODS: Two 150-degree PMMA arc segments, 0.30 mm thick, were surgically implanted through a single radial incision at two-thirds stromal depth in the mid-peripheral cornea of five New Zealand white rabbits. We collected corneas 6 months after implantation that were prepared for light and electron microscopy. We analyzed tissue with oil red O, filipin, and periodic acid-Schiff (PAS) staining. Additional studies with energy-dispersive x-ray analysis and autofluorescent microscopy were conducted. RESULTS: In all samples, we observed new collagen formation with lamellar organization adjacent to the implant and increased keratocyte density. Intracellular accumulations of osmophilic and saturated lipid material corresponded to stromal opacification visible by slit-lamp microscopy. These same regions were autofluorescent and stained positively with oil red O, and filipin but negatively with PAS. Granular lipid inclusions characteristic of lipofuscinosis were absent on transmission electron microscopy. Energy-dispersive x-ray analysis demonstrated elevated levels of calcium compared with adjacent tissue. CONCLUSION: Tissue response to these corneal inserts includes keratocyte activation, intracellular lipid accumulation, and new collagen formation. Our histologic findings suggest that these deposit formations are not lipofuscin accumulations.

Animals↗