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At least 361 records · Page 20Linked to original sources
PubMed2005

[Presentation of Niemann-Pick type C disease with psychiatric disturbance in an adult].

INTRODUCTION: Niemann-Pick Type C disease (NPC) is an autosomal recessive neurovisceral lysosomal lipid storage disorder. CASE REPORT: A 31-year-old right-handed woman had suffered from schizophrenia for 13 years. At 25 years of age, she developed a gait disorder with a static and kinetic cerebellar syndrome, dysarthria, vertical supranuclear gaze palsy and cognitive impairment. Brain MRI was normal. Abdominal ultrasonography was performed because of hypercholesterolemia and elevated transaminases and revealed hepatosplenomegaly, which in conjunction with other signs and symptoms, suggested the diagnosis of NPC. The diagnosis was confirmed by demonstration of lysosomal storage of unesterified cholesterol (filipin staining) and of a reduced rate of LDL-induced cholesterol esterification. Implication of the NPC1 gene was assessed by genetic complementation analysis. DISCUSSION: The phenotypic presentation of NPC is remarkably variable. The rarer adult-onset form has a slowly progressive course. Psychotic manifestations are often prominent and may precede neurologic symptoms. Exposure to neuroleptics delays the diagnosis of NPC. CONCLUSION: Psychotic manifestations associated with cerebellar syndrome, vertical supranuclear gaze palsy, and splenomegaly are very suggestive of NPC disease which can be reliably diagnosed on cultured skin fibroblasts by filipin staining.

Adult↗
PubMed1998

Contribution of membrane cholesterol to outer hair cell lateral wall stiffness.

The outer hair cell can be divided into three domains: the apex, the base, and the lateral wall. With the use of filipin, a polyene fluorescent antibiotic that binds to cholesterol, we found under fluorescence microscopy that the lateral wall membranes were less intensely stained than the apical and basal membranes. This difference in filipin fluorescence between the lateral walls and the ends diminished when cells were incubated in water-soluble cholesterol before staining, suggesting that exogenous cholesterol enters the lateral wall. Under confocal microscopy, we studied the incorporation pattern of a fluorescent cholesterol analogue, NBD-cholesterol. NBD-cholesterol did not stain the apical membranes whereas it intensely labeled the lateral wall. The micropipette aspiration technique was used to assess the effect of cholesterol on lateral wall stiffness. The lateral wall stiffness parameter of cells treated with water-soluble cholesterol (n = 23) was significantly higher than that of controls (n = 27): 0.76+/-0.24 (mean +/- SD) versus 0.46+/-0.10 (Student's t-test, p < 0.001). In conclusion, cholesterol has different distributions among outer hair cell membranes, and when water-soluble cholesterol is incorporated into the cells, the outer hair cell lateral wall stiffness parameter increases.

Affinity Labels↗
PubMed2002

Cholesterol storage defect in RSH/Smith-Lemli-Opitz syndrome fibroblasts.

The RSH/Smith-Lemli-Opitz syndrome (SLOS) is a multiple malformation/mental retardation syndrome caused by an inborn error of cholesterol synthesis. Mutations in the 3beta-hydroxysteroid Delta(7)-reductase gene result in impaired enzymatic reduction of 7-dehydrocholesterol (7-DHC) to cholesterol. Cells obtain cholesterol by either de novo synthesis or from exogenous sources by the binding and uptake of low density lipoprotein (LDL) particles. Because de novo synthesis of cholesterol is impaired in SLOS, current investigational therapy for SLOS consists of dietary cholesterol supplementation. However, the potential effects of elevated intracellular levels of 7-DHC on intracellular LDL metabolism have not been described. We now report that in addition to the primary defect in de novo cholesterol synthesis, SLOS fibroblasts have a secondary defect of LDL cholesterol metabolism. Staining of fibroblasts with filipin, a fluorescent polyene antibiotic which binds unesterified sterols, shows that SLOS fibroblasts accumulate unesterified sterols. Further studies show that this increased filipin staining was due to an abnormal accumulation of LDL derived cholesterol rather than due to storage of endogenously synthesized 7-dehydrocholesterol (7-DHC). We have also found that SLOS fibroblasts failed to degrade LDL at a normal rate, and examination of SLOS fibroblasts by electron microscopy demonstrated the formation of lysosomal inclusions similar to that seen in Niemann-Pick type C (NPC) cells. We propose that 7-DHC may directly or indirectly inhibit the function of the NPC protein through its sterol-sensing domain (SSD), and that 7-DHC may perturb the function of other SSD containing proteins in SLOS.

Cell Line↗
PubMedDate not supplied

Vasopressin stimulates formation of coated pits in rat kidney collecting ducts.

The water permeability of collecting ducts is greatly increased by the antidiuretic hormone, vasopressin. This permeability increase is associated with the appearance of intramembrane particle (IMP) clusters on the luminal plasma membranes of principal cells of the collecting duct epithelium. IMP aggregates have also been related to an increase in water permeability of two other vasopressin-sensitive epithelia, the amphibian urinary bladder and the amphibian epidermis, and it has been proposed that these specialized membrane domains might represent specific water-permeable membrane patches, induced by the hormone in their respective epithelia. Using a cytochemical probe for membrane cholesterol, filipin, we show here that the membrane patches in rat kidney are selectively devoid of filipin-sterol complexes and that when identified in thin sections, they have a cytoplasmic bristle coat: both of these features are characteristics of coated pits which, in other systems, are involved in receptor-mediated endocytosis. We also show that vasopressin induces the appearance of coated pits on collecting duct luminal membranes in Brattleboro homozygous rats, which have hereditary diabetes insipidus.

Animals↗
PubMed2004

Heparan sulphate proteoglycans modulate fibroblast growth factor-2 binding through a lipid raft-mediated mechanism.

We investigated how lipid raft association of HSPG (heparan sulphate proteoglycans) modulates FGF-2 (fibroblast growth factor-2/basic fibroblast growth factor) interactions with vascular smooth-muscle cells. When lipid rafts were disrupted with sterol-binding agents, methyl-beta-cyclodextrin and filipin, FGF-2 binding to HSPG was reduced 2-5-fold, yet the amount and turnover of cell-surface HSPG were unaffected [corrected]. Approx. 50-65% of bound FGF-2 was in lipid raft-associated fractions based on insolubility in cold Triton X-100 and flotation in OptiPrep density gradients, and this level was increased with higher FGF-2 concentrations [corrected]. Less FGF-2 (50-90%) was associated in raft fractions when cholesterol was depleted or HSPG were degraded with heparinase III. To investigate how lipid raft-HSPG interactions altered binding, we compared the rates of FGF-2 dissociation with native, MbetaCD (methyl-beta-cyclodextrin)- and filipin-treated cells. We found that FGF-2 dissociation rates were increased when lipid rafts were disrupted. These results suggest that localization of HSPG within lipid rafts creates high local concentrations of binding sites such that dissociation of FGF-2 is hindered. The localization of FGF-2 and HSPG to lipid rafts also correlated with the activation of protein kinase Calpha. Thus raft association of HSPG might create growth factor traps resulting in increased binding and signal transduction to enhance cell sensitivity.

Animals↗
PubMed2003

Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin.

Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.

Animals↗
PubMed1999

Requirement of caveolae microdomains in extracellular signal-regulated kinase and focal adhesion kinase activation induced by endothelin-1 in primary astrocytes.

Endothelin-1 (ET-1) mitogenic activity in astrocytes is mediated by the activation of the extracellular signal-regulated kinase (ERK) pathway together with the Rho-dependent activation of the focal adhesion kinase (FAK) pathway. To clarify the mechanisms responsible for the coordinate activation of both pathways in the ET-1 signal propagation, the involvement of caveolae microdomains, suggested to play a role in signal transduction, was evaluated. In this study, it is reported that caveolae of primary astrocytes are enriched in endothelin receptor (ETB-R). Furthermore, signaling molecules such as the adaptor proteins Shc and Grb2, and the small G protein Rho, also reside within these microdomains. Selective disassembly of caveolae by filipin III impairs the ET-1-induced tyrosine phosphorylation of proteins including ERK and FAK. In agreement with these observations, astrocytes pretreated with filipin III also failed to form stress fibers and focal adhesions and did not undergo the associated morphological changes in response to ET-1. This study reveals that structural integrity of caveolae is necessary for the adhesion-dependent mitogenic signals induced by ET-1 in astrocytes, through compartmentation of ETB-R with the upstream signaling molecules of the ERK and FAK pathways.

Actins↗
PubMed2001

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts).

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCtheta) of perfringolysin O (theta-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BCtheta on a sucrose gradient. BCtheta was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCtheta binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCtheta binding to FLDF. The staining patterns of BCtheta and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCtheta binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCtheta binding does not cause any damage to cell membranes, indicating that BCtheta is a useful probe for the detection of membrane rafts in living cells.

Bacterial Toxins↗
PubMed1977

Animal cell mutants defective in sterol metabolism: a specific selection procedure and partial characterization of defects.

By using a chemically defined medium, a general and highly specific procedure was devised to select for mutant cells with less abundant or structurally altered sterol in their surface membranes. Within a certain concentration range, the polyene antibiotic filipin was shown to kill only cells with normal (as opposed to decreased) membrane sterol levels. Sterol-requiring derivatives of LM cells were isolated by chemical mutagenesis, filipin treatment, and cloning followed by replica plating in soft agar. Mutants (S1 and S2) are described which, when compared to normal cells, show decreased synthesis of demosterol in vivo from acetate and mevalonate relative to cell number or to fatty acid synthesis. When exogenous sterol is supplied, mutants S1 and S2 grow normally in suspension culture. However, when deprived of sterol supplement, mutant S1 grows slower than wild type cells and mutant S2 lyses within one to two generations. Gas/liquid chromatography revealed that the mutants contained a normal spectrum of fatty acids including unsaturated fatty acyl groups but, unlike wildtype cells, they have less abundant (mutant S1) or no (mutant S2) desmosterol in either the presence or absence of exogenous cholesterol. In vitro experiments with mevalonate as the substrate suggest that the defect in both mutants is in a demethylation reaction subsequent to lanosterol synthesis. The selection method developed here may permit the isolation of mutants with defective membrane incorporation of sterols and other polyisoprenoids as well as defective synthesis of these compounds.

Acetates↗
PubMed1981

Effects of amphotericin B, nystatin, and other polyene antibiotics on chitin synthase.

The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations greater than or equal to 10 micrograms/ml, filipin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a Ki value of about 0.13 mM. the effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at approximately 50 micrograms/ml. Our findings suggest the existence of binding sites (sterols?) on the chitosome that are selective for certain polyenes and that play a role in the operation of chitin synthase. Because the minimal growth inhibitory concentrations of AmB or nystatin are lower than the concentrations that inhibit chitin synthase in vitro, the possibility of chitosomal chitin synthase being a primary target for the antifungal action of these polyenes seems unlikely.

Amphotericin B↗
PubMed1981

Membrane lipid heterogeneity associated with acetylcholine receptor particle aggregates in Xenopus embryonic muscle cells.

Filipin, digitonin, and saponin react with membrane cholesterol to produce unique membrane alterations (sterol-specific complexes) that are easily discernible in freeze-fracture replicas. We have treated both noninnervated and innervated Xenopus embryonic muscle cells in culture with these agents. Freeze-fracture of these treated muscle cells showed that most areas of the muscle plasma membrane contain sterol-specific complexes (19- to 40-nm protuberances and dimples with filipin, a scalloped appearance with digitonin, or an irregular, rough appearance with saponin). However, these complexes were virtually absent from membrane areas of junctional and nonjunctional aggregates of acetylcholine receptor particles. This result suggests that the membrane matrix of these aggregates is low in cholesterol and that this membrane lipid heterogeneity may be linked to the mechanisms involved in their formation and stabilization on muscle cells in culture.

Animals↗
PubMed1998

Separation of "glycosphingolipid signaling domain" from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling.

Two membrane subfractions, one enriched in GM3 ganglioside and the other containing caveolin, were separated from low density detergent-insoluble membrane fraction prepared by sucrose density gradient centrifugation of postnuclear fraction of mouse melanoma B16 cells. The GM3-enriched subfraction, separated by anti-GM3 monoclonal antibody DH2, contained sphingomyelin, cholesterol, c-Src, and Rho A but not caveolin. In contrast, the caveolin-containing subfraction, separated by anti-caveolin antibody, contained neither GM3, c-Src, nor Rho A but did contain glucosylceramide, Ras, a very small quantity of sphingomyelin, and a very large quantity of cholesterol. The GM3/c-Src-enriched membrane subfraction was characterized by (i) maintenance of GM3-dependent adhesion and (ii) susceptibility to being activated for signal transduction through GM3. 32P-Phosphorylation of c-Src (Mr 60,000) together with two other components (Mr 45,000 and 29,000) was enhanced in the fraction bound to dishes coated with asialo-GM2 (Gg3) or with anti-GM3 monoclonal antibody DH2, detected by incubation with [gamma-32P]ATP at 37 degreesC for 5 min. GM3-dependent adhesion of B16 cells to Gg3-coated dishes and associated signaling were not reduced or abolished in the presence of either filipin or nystatin, which are cholesterol-binding reagents known to abolish caveolae structure and function. B16 melanoma cells incubated with filipin (0.16-0.3 micrograms/ml) or with nystatin (25 micrograms/ml) for 30 min showed depletion of cholesterol in detergent-insoluble membrane fraction but were still capable of binding to Gg3-coated plate and capable of the associated signaling. Thus, the GM3-enriched subfraction, involved in cell adhesion and capable of sending signals through GM3, represents a membrane domain distinguishable from caveolin-containing subfraction or caveolae. This microdomain is hereby termed the "glycosphingolipid signaling domain" or "glycosignaling domain".

Animals↗
PubMed2000

Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway.

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.

ATP Binding Cassette Transporter 1↗
PubMed2002

Caveolar localization dictates physiologic signaling of beta 2-adrenoceptors in neonatal cardiac myocytes.

There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that beta(1)- and beta(2)-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from beta(1)AR and beta(2)AR knockout mice, we found that stimulation of the beta(1)AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the beta(2)AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the beta(2)AR but not the beta(1)AR in neonatal cardiac myocytes. Evidence for beta(2)AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the beta(2)AR and caveolin 3, and co-migration of the beta(2)AR with a caveolin-3-enriched membrane fraction. The beta(2)AR-stimulated increase in the myocyte contraction rate is increased by approximately 2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of beta(2)AR and caveolin 3 and co-migration of beta(2)AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on beta(1)AR signaling. These observations suggest that beta(2)ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.

Animals↗
PubMed2000

Replacement of the transmembrane anchor in angiotensin I-converting enzyme (ACE) with a glycosylphosphatidylinositol tail affects activation of the B2 bradykinin receptor by ACE inhibitors.

To investigate further the relationship of angiotensin I-converting enzyme (ACE) inhibitors to activation of the B(2) bradykinin (BK) receptor, we transfected Chinese hamster ovary cells to stably express the human receptor and either wild-type ACE (WT-ACE), an ACE construct with most of the cytosolic portion deleted (Cyt-del-ACE), or ACE with a glycosylphosphatidylinositol (GPI) anchor replacing the transmembrane and cytosolic domains (GPI-ACE). BK or its ACE-resistant analogue were the agonists. All activities (arachidonic acid release and calcium mobilization) were blocked by the B(2) antagonist HOE 140. B(2) was desensitized by repeated administration of BK but resensitized to agonist by ACE inhibitors in the cells expressing both B(2) and either WT-ACE or Cyt-del-ACE. In GPI-ACE expressing cells, the B(2) receptor was still activated by the agonists, but ACE inhibitors did not resensitize. Pretreatment with filipin returned the sensitivity to inhibitors. In immunocytochemistry, GPI-ACE showed patchy, uneven distribution on the plasma membrane that was restored by filipin. Thus, ACE inhibitors were inactive as long as GPI-ACE was sequestered in cholesterol-rich membrane domains. WT-ACE and B(2) receptor in Chinese hamster ovary cells co-immunoprecipitated with antibody to receptor, suggesting an interaction on the cell membrane. ACE inhibitors augment BK effects on receptors indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2004

Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements.

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.

ADP-Ribosylation Factor 6↗
PubMed1996

Regionalization and redistribution of membrane phospholipids and cholesterol in mouse spermatozoa during in vitro capacitation.

Fracture-label, surface-replica, and routine freeze-fracture techniques were used in combination with phospholipase A2-colloidal gold (PLA2-CG) and filipin as probes to study changes in the distribution of phospholipids and cholesterol, respectively, in morphologically defined plasma membrane domains of mouse spermatozoa during in vitro capacitation. In noncapacitated spermatozoa, quantitative analysis revealed that the fractured plasma membrane overlying the equatorial segment carried the highest PLA2-CG labeling density. The next highest labeling densities were found in the anterior acrosome region and the post-acrosomal region. On the external surface of the plasma membrane revealed by surface replicas, a uniform distribution of PLA2-CG was confined mainly to the acrosomal region of the head. The plasma membrane of the sperm tail had a relatively low labeling density for PLA2-CG. In freeze-fracture replicas of filipin-treated spermatozoa, the labeling density of filipin/sterol complexes (FSCs) was high in the plasma membrane over the acrosomal region where the FSCs were uniformly distributed. The postacrosomal region was weakly labeled. After in vitro capacitation, the densities of PLA2-CG and FSCs were significantly reduced in the fractured plasma membrane of the sperm head and the middle piece of the tail. However, surface replicas revealed an increased PLA2-CG labeling on the external surface of the plasma membrane covering the postacrosomal region, the middle piece, and the principal piece. Another major change detected in capacitated spermatozoa was the presence of small aggregates and patches of elevated, membrane-associated particles on the surface-replicated plasma membrane in the upper portion of the postacrosomal domain. Here the PLA2-CG labeling density was found to be higher than in noncapacitated spermatozoa. These results provide new information with respect to the reorganization and redistribution of phospholipids in specific regions of the plasma membrane during capacitation and provide further support for the concept that removal or loss of antifusigenic sterol from the sperm plasma membrane constitutes an important step of the capacitation process.

Animals↗
PubMedDate not supplied

Heterogeneous distribution of membrane cholesterol at the attachment site of Cryptosporidium muris to host cells.

Distribution of membrane cholesterol at the attachment site of Cryptosporidium muris was investigated by freeze-fracture cytochemistry using a polyene antibiotic filipin. Since the host plasma membrane enveloped C. muris, the inner and outer membranes were continuous with the parasite plasma membrane at the annular ring and with host membrane at the dense band, respectively. Although many filipin-cholesterol complexes were observed on the plasma membrane of host cells and parasites, a line showing no complexes was evident at the above two membrane junctures. These observations indicate that parasitic infection of C. muris altered the organization of membrane cholesterol.

Animals↗