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PubMed2001

Cholesterol accumulates in senile plaques of Alzheimer disease patients and in transgenic APP(SW) mice.

Mounting evidence suggests that cholesterol may contribute to the pathogenesis of Alzheimer disease (AD). We examined whether cholesterol might be present in senile plaques, a hallmark neuropathological feature of AD. We employed 2 different fluorometric-staining techniques (filipin staining and an enzymatic technique) for the determination of cholesterol in brains of postmortem confirmed AD patients and in nondemented, age-matched histopathologically normal controls. AD patient brains showed abnormal accumulation of cholesterol in congophilic/birefringent dense cores of senile plaques that was essentially absent in histopathologically normal controls. To determine whether increased senile plaque-associated cholesterol occurred generally in all plaques or was restricted to a specific subset, quantitative analysis was performed. Data indicate abnormal accumulation of cholesterol in cores of mature plaques but not in diffuse or immature plaques. Additionally, transgenic mice that overexpress the "Swedish" amyloid precursor protein (Tg APP(SW), line 2576) exhibited a similar pattern of abnormal cholesterol accumulation in mature, congophilic amyloid plaques at 24 months of age that was absent in their control littermates or in 8-month-old Tg APP(SW) mice (an age prior to amyloid deposition). Taken together, our results imply a link between cholesterol and AD pathogenesis and suggest that cholesterol plays an important role in the formation and/or progression of senile plaques.

Aged↗

PubMed2004

Morphologic characteristics of lamellar channel deposits in the human eye: a case report.

OBJECTIVE: To report the histologic characteristics of lamellar channel deposits after polymethyl methacrylate intrastromal corneal ring segments in a human eye. METHODS: A 34-year-old man previously diagnosed with keratoconus received photorefractive keratectomy in his left eye that exacerbated his corneal ectasia. To delay or possibly prevent corneal transplant, the patient elected to have intrastromal corneal segments implanted in the left eye. One year later the patient underwent corneal transplant. Before transplant, lamellar channel deposits were noted on clinical microscopic examination of the left eye. We performed histologic evaluation to assess changes to the patient's excised corneal tissue and to further characterize the observed lamellar channel deposits. RESULTS: Lamellar channel deposits in humans are histologically similar to our previous descriptions of deposits in rabbits. These deposits primarily consist of intracellular lipid accumulations that stain positively with oil red O and filipin but not periodic acid Schiff reaction. Immunohistochemistry of cells located in the deposit region stained positively with vimentin, suggesting that these cells were keratocytes. Stains for cytokeratins 3 and 12 and for CD68 were both negative, indicating that epithelial cells and macrophages were not present in the deposit region. CONCLUSION: Lamellar channel deposits are a common clinical finding after intrastromal corneal implants. This case provides further evidence to support our hypothesis that lamellar channel deposits in humans are a tissue response to corneal implants that consist of intracellular lipid accumulations. We use this case to propose a theoretical construct for the observation of stromal lipid accumulation in response to corneal injury.

Adult↗

PubMed2005

Disruption of lipid rafts causes apoptotic cell death in HaCaT keratinocytes.

Lipid rafts are cholesterol-enriched microdomains in plasma membranes. The functional activity of many membrane proteins, including death and growth factor receptors, depends on their insertion in lipid rafts. We have previously demonstrated the presence of lipid rafts in keratinocytes and shown that lipid rafts are involved in the control of keratinocyte proliferation and metabolic activity. In this work, we investigated the effect of lipid-raft disruption on HaCaT keratinocyte survival. Lipid rafts could be disrupted or rearranged with cholesterol-targeting detergents: methyl-beta-cyclodextrin and filipin III. Moreover, cholesterol oxidation by a specific oxidase or blocking of cholesterol synthesis by mevastatin had a similar effect on lipid rafts. All cholesterol-modifying substances caused cell death in a concentration-dependent manner. More detailed studies on the effects of cyclodextrin revealed apoptotic cell death at concentrations >or=0.5% (w/v). The molecular mechanism of apoptosis precipitated by raft disruption remains unknown but does not seem to be dependent of either membrane permeabilization or cell-cycle arrest imposed by cholesterol-modifying compounds.

Apoptosis↗

PubMed1983

Measurement of binding of adenine nucleotides and phosphate to cytosolic proteins in permeabilized rat-liver cells.

1. A method is described for measuring the binding of metabolites to cytosolic proteins in situ in isolated rat-liver cells treated with filipin to render the plasma membrane permeable to compounds of low molecular weight. 2. There is no binding of ATP or inorganic phosphate to cytosolic proteins, either in the presence or in the absence of Mg2+. 3. Binding of ADP to cytosolic proteins occurs both in the absence and in the presence of Mg2+. The concentration of binding sites was 0.68 and 0.52 mumol/g dry weight of cells (n = 3-4) in the absence and presence of Mg2+, respectively. The corresponding Kd values were 320 microM and 235 microM.

Adenosine Diphosphate↗

PubMed1991

Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase).

1. Manganese(II) buffers were set up with inorganic triphosphate, trimethylenediaminetetraacetate and tetramethylenediaminetetraacetate to study the Mn dependence of beta 1,4-galactosyltransferase (lactose synthetase) in preparations of rat mammary gland. 2. In intact particulate preparations, treated with the calcium ionophore A23187, lactose synthesis was abolished by chelators and restored by bivalent transition metal ions in a manner characteristic of activation site I of the pure enzyme. Ni(II) also activated, as did Mg at high concentration. 3. Only Mn(II) could restore endogenous rates, giving an apparent Km of 0.1-0.2 microM, and eliciting about 70% full activity without addition of a site II activator. 4. In purified Golgi membrane vesicles, Mn gave an apparent Km of 0.4 microM. This increased sharply to about 10 microM on permeabilization with filipin, lysis with detergents, solubilization with Triton X-100, or in the pure enzyme. Preparations of chemically undamaged Golgi vesicles, known to include a proportion of the enzyme on exposed membranes, exhibited both low-Km and high-Km components. 5. The response of particulate galactosyltransferase to apparently physiological concentrations of free Mn(II) ion is interpreted as either due to a sensitizing factor within the Golgi lumen, or to the accumulation of Mn at elevated concentrations. Alternatively, the high Km of the soluble enzyme may reflect proteolytic damage.

Animals↗

PubMed1989

Characteristics of sorbitol uptake in rat glial primary cultures.

Uptake of [U-14C]sorbitol was studied in astrogliarich rat primary cultures. Initial rate of sorbitol uptake is proportional to sorbitol concentration between 20 microM and 400 mM. Sorbitol transport is not inhibited by glucose, fructose, and a variety of structurally related polyols, or by cytochalasin B, an inhibitor of glucose transport. Phloretin, phlorizin, filipin, and n-hexanol, all compounds that alter the properties of biological membranes, and the sulfhydryl reagent p-chloromercuribenzoate inhibit sorbitol uptake to various degrees. Variation in the concentrations of extracellular Na+ and K+ does not affect transfer of sorbitol across the cell membrane. It is concluded that sorbitol is taken up into glial cells by a diffusion process, not involving a carrier and probably not through the lipid bilayer, but through a proteinaceous channel-like structure.

Animals↗

PubMed1975

Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts.

A sensitive assay was developed for detection and quantitation of subtle permeability changes in the cytoplasmic membrane of human diploid fibroblasts. Release of the non-metabolizable amino acid [1-14C]alpha-aminoisobutyric acid (AIB; molecular weight (103) from the cytoplasm of prelabeled cells was used as an indicator of toxin-induced membrane damage. An optimal procedure for labeling these cells was designed after varying the conditions with regard to pH, temperature, concentration of AIB, composition of medium, and incubation time. Toxin-induced release of AIB was compared with release of a previously described nucleotide label, [3H]uridine. Melittin from bee venom and the polyene antibiotics filipin and amphotericin B in low concentrations induced a strikingly greater release of AIB than of nucleotide label. The sensitivity of this assay was furthermore demonstrated by treatment with the following bacterial cytolysins: phospholipase C and theta-toxin from Clostridium perfringens, alpha-, beta-, delta-, and gamma-toxins from Staphylococcus aureus, and streptolysin S from Streptococcus pyogenes. In spite of their different modes of action, all these membrane-active toxins at low concentrations induced a significant release of AIB label. For an equal release of nucleotide label, several times higher concentrations were required.

Aminobutyrates↗

PubMed2002

Role of cholesterol and the ganglioside GM(1) in entry and short-term survival of Brucella suis in murine macrophages.

Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. We have previously shown that the maturation inhibition of the Brucella-containing phagosome appears to be restricted at the phagosomal membrane, but the precise molecular mechanisms and factors involved in this inhibition have yet to be identified. Interestingly, recent studies have revealed that caveolae or lipid rafts are implicated in the entry of some microorganisms into host cells and mediate an endocytic pathway avoiding fusion with lysosomes. In this study, we investigated the role of cholesterol and the ganglioside GM(1), two components of lipid rafts, in entry and short-term survival of Brucella suis in murine macrophages, by using cholesterol-sequestering (filipin and beta-methyl cyclodextrin) and GM(1)-binding (cholera toxin B) molecules. Our results suggest that lipid rafts may provide a portal for entry of Brucella into murine macrophages under nonopsonic conditions, thus allowing phagosome-lysosome fusion inhibition, and provide further evidence to support the idea that the phagosome maturation inhibition is restricted at the phagosomal membrane.

Animals↗

PubMed2004

Mycoplasma fermentans binds to and invades HeLa cells: involvement of plasminogen and urokinase.

Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37 degrees C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with beta-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry.

Bacterial Adhesion↗

PubMed1961

Alterations in the permeability of Neurospora crassa due to polyene antibiotics.

Kinsky, Stephen C. (Washington University, St. Louis, Mo.). Alterations in the permeability of Neurospora crassa due to polyene antibiotics. J. Bacteriol. 82:889-897. 1961.-Thirty-three antibiotics and synthetic fungicides were examined for their effect on the growth and morphology of Neurospora crassa. Only the polyene antibiotics (nystatin, amphotericin B, and filipin) caused a decrease in the dry weight of mycelial mats, which was accompanied by the appearance of cytoplasmic constituents in the medium. The influence of polyene concentration, incubation time, mycelial age, various metabolic inhibitors, pH, and media composition on this phenomenon are described. The present evidence indicates that the polyenes exert their primarily fungicidal effect by an alteration of permeability, probably due to direct action on the membrane of sensitive organisms.

Amphotericin B↗

PubMed2004

Characterization of rotavirus cell entry.

While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-beta-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.

Animals↗

PubMed1994

Complementation studies in Niemann-Pick disease type C indicate the existence of a second group.

Niemann-Pick disease type C is a clinically heterogeneous storage disorder with an unknown primary metabolic defect. We have undertaken somatic cell hybridisation experiments using skin fibroblast strains from 12 patients representing a wide clinical spectrum. Preliminary experiments using filipin staining of free cholesterol as a marker for complementation indicated the existence of one major group (group alpha) and one minor group (group beta) represented by one mutant strain. Subsequent experiments in which sphingomyelinase activity was measured as a marker for complementation using five mutant strains showing activity consistently < 40% control levels confirmed the existence of the second group.

Cells, Cultured↗

PubMed1985

Phospholipid enrichment of Saccharomyces cerevisiae and its effect on polyene sensitivity.

Sensitivity to polyene antibiotics, e.g., nystatin, amphotericin B, and filipin, was determined in phosphatidylcholine (PC) or phosphatidylethanolamine (PE) or phosphatidylserine (PS) enriched Saccharomyces cerevisiae cells, using glutamic acid, phenylalanine, glycine, and lysine transport as an index of polyene antibiotic action. As compared with normal cells, phospholipid-enriched cells acquired resistance towards different polyenes. However, the sensitivity of glutamic acid transport towards nystatin remained unaffected in PC-, PE-, or PS-enriched cells. In contrast to nystatin, the other two polyenes were more effective in checking the influx of amino acids. Results demonstrated that the specific enrichment of PC, PE, or PS could selectively protect S. cerevisiae cells from polyene antibiotic action.

Amino Acids↗

PubMed2003

Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures.

The preferential association of cholesterol and sphingolipids within plasma membranes forms organized compartments termed lipid rafts. Addition of caveolin proteins to this lipid milieu induces the formation of specialized invaginated plasma membrane structures called caveolae. Both lipid rafts and caveolae are purported to function in vesicular transport and cell signaling. We and others have shown that disassembly of rafts and caveolae through depletion of plasma membrane cholesterol mitigates mechanotransduction processes in endothelial cells. Because osteoblasts are subjected to fluid-mechanical forces, we hypothesize that cholesterol-rich plasma membrane microdomains also serve the mechanotransduction process in this cell type. Cultured human fetal osteoblasts were subjected to either sustained hydrostatic pressure or laminar shear stress using a pressure column or parallel-plate apparatus, respectively. We found that sustained hydrostatic pressure induced protein tyrosine phosphorylation, activation of extracellular signal-regulated kinase (ERK)1/2, and enhanced expression of c-fos in both time- and magnitude-dependent manners. Similar responses were observed in cells subjected to laminar shear stress. Both sustained hydrostatic pressure- and shear stress-induced signaling were significantly reduced in osteoblasts pre-exposed to either filipin or methyl-beta-cyclodextrin. These mechanotransduction responses were restored on reconstitution of lipid rafts and caveolae, which suggests that cholesterol-rich plasma membrane microdomains participate in the mechanotransduction process in osteoblasts. In addition, mechanical force-induced phosphoproteins were localized within caveolin-containing membranes. These data support the concept that lipid rafts and caveolae serve a general function as cell surface mechanotransduction sites within the plasma membrane.

Anti-Bacterial Agents↗

PubMed1991

Type C Niemann-Pick disease: use of hydrophobic amines to study defective cholesterol transport.

Niemann-Pick Type C (NPC) disease is a cholesterol lipidosis resulting from defective postlysosomal cholesterol transport. In normal cells this segment of cholesterol trafficking is inhibited by treatment with either U18666A or imipramine. Other compounds are also capable of blocking postlysosomal cholesterol transport: stearylamine, RV-538, and sphinganine inhibit low-density lipoprotein-induced esterification of cholesterol and cause unesterified cholesterol to accumulate in perinuclear vesicles. These vesicles can be stained with filipin to give a staining pattern indistinguishable from that seen in NPC fibroblasts. Because all of these compounds are hydrophobic amines, we conclude that most, if not all, hydrophobic amines block the postlysosomal transport of cholesterol. These results also raise the possibility that an endogenous amine, e.g., sphinganine, may inhibit cholesterol transport in NPC.

Amines↗

PubMed1996

Aging affects the conformation of cholesterol in the human eye lens.

The distribution of 3-beta-OH-cholesterol along the optical axis of 20 human eye lenses of different ages was measured using confocal Raman microspectroscopy in combination with the 3-beta-hydroxysteroid-specific probe filipin. This non-destructive technique allow a spatially very detailed study of the distribution of 3-beta-OH-cholesterol in individual lenses. It revealed age-related changes of the 3-beta-OH-cholesterol distribution along the optical axis. The 3-beta-OH-cholesterol was found to be distributed asymmetrically along the optical axis and this distribution was found to change with age. The relative 3-beta-OH-cholesterol content in the anterior part of the lens decreases significantly with increasing age compared with its equal posterior counterpart. Additionally all the lenses have a very low 3-beta-OH-cholesterol content in the central part of their nuclear region, the area where the primary lens fibres are located.

Adolescent↗

PubMed2003

Lipid raft compartmentalization of urokinase receptor signaling in human neutrophils.

Urokinase plasminogen activator (uPA) receptors (uPAR) can be engaged for activation signaling either by aggregation or by binding exogenous uPA. These signaling mechanisms require uPAR to associate with two distinct adhesion proteins, L-selectin and complement receptor 3 (CR3), respectively. uPAR contains a glycosylphosphatidylinositol anchor, suggesting that it is concentrated within glycosphingolipid-enriched microdomains, or "lipid rafts". This study was undertaken to determine the extent to which uPAR-mediated signaling is compartmentalized to lipid rafts. Human neutrophil uPAR was cross-linked or stimulated with uPA after pretreatment with the lipid raft-disrupting agents, methyl-beta-cyclodextrin or filipin III. Both agents suppressed increases in intracellular Ca(2+) concentrations ([Ca(2+)](i)) triggered by cross-linking, but did not affect [Ca(2+) ](i) in response to uPA. Neutrophil membranes were separated into lipid raft and non-raft fractions, revealing the presence of uPAR and L-selectin, but the virtual absence of CR3 alpha chain in lipid rafts, either constitutively or in response to uPAR aggregation. Fluorescence resonance energy transfer experiments confirmed close proximity of a lipid raft marker to both uPAR and L-selectin, but not CR3. We conclude that uPAR can engage distinct signaling pathways involving different partner proteins that are functionally and physically segregated from one another in both lipid raft and non-raft domains of the plasma membrane.

Animals↗

PubMed2004

Basal laminar deposit formation in APO B100 transgenic mice: complex interactions between dietary fat, blue light, and vitamin E.

PURPOSE: Dietary fat intake has been proposed as a mechanism of sub-RPE deposit formation. It has been demonstrated recently that sub-RPE deposits develop in 16- to 18-month-old C57BL/6 mice fed a high-fat diet and exposed to blue-green light. Hyperlipidemia also develops in these mice after they consume a high-fat diet. Because hyperlipidemia also develops in young C57BL/6 mice that overexpress APO B100, the major apolipoprotein in LDL cholesterol, this research was conducted to determine whether high-fat diet and plasma hyperlipidemia correlate with formation of basal laminar deposits (BLD) in young transgenic mice. METHODS: APO B100 and wild-type C57BL/6 2-month-old mice were fed a high-fat diet for 4.5 months. After the first month, the right eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light (20 mJ of argon 488 nm) over 2 weeks. Three months later, transmission electron microscopy (TEM) of the retina was performed to evaluate whether sub-RPE deposits correlate with plasma cholesterol and triglyceride levels. Several eyes were stained with filipin to detect cholesterol and osmium-thiocarbohydrazide-osmium (OTO) to detect neutral lipids in Bruch's membrane (BrM). A third group of APO B100 2-month-old mice were pretreated with vitamin E subcutaneously twice a week throughout the experiment and underwent the same light-exposure protocol. RESULTS: Mice fed a high-fat diet had a more elevated plasma triglyceride and cholesterol level than those that consumed a regular diet. Young APO B100 mice fed a high-fat diet had blood lipid levels higher than those in young wild-type mice that consumed high-fat diets, and these two groups had higher lipid levels than animals with regular diets, as shown previously in wild-type C57BL/6 (old and young). Eyes of APO B100 mice treated with blue-green light showed a high frequency of "moderate BLD", whereas the nonexposed eyes did not. In contrast, no BLD formed in either eye of the wild-type young mice fed a high-fat diet. In individual affected mice, only a weak correlation was observed between deposit severity and plasma lipid concentration. None of the eyes in mice with sustained hyperlipidemia with or without BLD demonstrated obvious widespread neutral lipid or cholesterol deposition in BLD or BrM. However, vitamin E-treated mice showed minimal formation of BLD. CONCLUSIONS: Although a high-fat diet is a necessary precondition for this model of BLD, the findings demonstrated a convincing direct correlation between plasma lipidemia and deposit severity. The results suggest that age, as shown in previous studies, and high-fat predispose to formation of BLD by altering hepatic and/or RPE lipid metabolism in ways more complicated than plasma hyperlipidemia alone.

Animals↗