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At least 613 records · Page 34Linked to original sources
PubMed1990

Abnormal cholesterol metabolism in imipramine-treated fibroblast cultures. Similarities with Niemann-Pick type C disease.

Addition of low-density lipoprotein (LDL) to cholesterol-deprived human skin fibroblast cultures treated by imipramine at a 20 microM concentration induced a significant intracellular accumulation of unesterified cholesterol. Intracytoplasmic inclusions were already visible by histochemical filipin staining after 2 h of LDL uptake and were progressively mobilized towards the perinuclear region within 24 h. At this concentration of the drug, the rate of proteolytic 125I-LDL hydrolysis was similar in treated and untreated cells. Treated cells maintained in lipoprotein-deficient medium showed no abnormality, indicating the exogenous origin of the accumulated sterol. Further, the drug induced a drastic dose-dependent impairment of LDL-stimulated cholesterol esterification, not related to an inhibition of acyl CoA:cholesterol acyltransferase, and a significant delay in down-regulation of de novo cholesterol synthesis. However, imipramine did not affect 25-hydroxycholesterol-mediated regulation of the two latter processes. These results resemble those observed in Niemann-Pick type C disease and suggest an impaired mobilization of LDL-derived cholesterol in imipramine-treated cells.

Acetates↗
PubMed1986

Activation of phosphatidic acid metabolism of human erythrocyte membranes by perfringolysin O.

The effect of perfringolysin O on the lipid metabolism of human erythrocyte membranes was investigated. Erythrocytes were prelabeled with [3H]arachidonic acid and [32P]inorganic phosphate. In the presence of calcium ion(5.5 mM), the effect of perfringolysin O on lipid metabolism was very similar to that of an calcium-ionophore A23187. In the absence of calcium ion, the accumulation of phosphatidic acid and its following decreasing trend were observed during the reaction with the toxin. Such changes were not caused by filipin. These results suggest that perfringolysin O causes the activation of a diglyceride-phosphatidic acid cycle, which might be involved in the calcium transport.

Bacterial Toxins↗
PubMed1978

Ligand binding studies in the mouse olfactory bulb: identification and characterization of a L-[3H]carnosine binding site.

Binding sites for the dipeptide L-carnosine (beta-alanyl-L-histidine) have been detected in membranes prepared from mouse olfactory bulbs. The binding of L-[3H]-carnosine was saturable, reversible and stereospecific and had a Kd of about 770 nM. The stereospecific binding of L-carnosine represented about 30% of the total binding at pH 6.8, and decreased markedly with increasing pH. Binding was stimulated by calcium, unaffected by zinc, magnesium or manganese and inhibited by sodium and potassium. Carnosine binding was sensitive to trypsin and phospholipases A and C, but not to neuraminidase. Nystatin and filipin, which interact with membrane lipids, also interferred with binding. Some peptide analogues of carnosine were potent inhibitors of binding, but a variety of drugs serving as potent inhibitors in other binding systems had no effect on carnosine binding. Carnosine binding to mouse olfactory bulb membranes was 15-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than in cerebellum membranes and 3-fold higher than in membranes from spinal medulla and the olfactory tubercle-lateral olfactory tract area. Binding sites for 6 other radiolabeled receptor ligands were also detected in bulb membranes. Peripheral deafferentation of the olfactory bulbs by intranasal irrigation with ZnSO4 led to a loss greater than 90% of the L-[3H]carnosine binding in 4--5 days with much smaller losses in binding of the other 6 ligands over a 180-day observation period. This initial loss of carnosine binding after denervation was due to a loss of binding site stereo-specificity followed by a loss of binding sites. The characteristics of the carnosine binding site in olfactory bulb fulfil 6 of the 7 criteria considered relevant for a functional receptor.

Animals↗
PubMed1987

Endothelial cell plasma membrane obtained by chemically induced vesiculation.

A method using low concentrations of formaldehyde and dithiothreitol was applied to obtain 'right-side out' luminal plasmalemma-derived vesicles from bovine aortic endothelial cells (EC) in culture, and from human umbilical vein and bovine or porcine aortas perfused ex vivo with the vesiculation solution. Vesicle formation and shedding were examined by phase-contrast microscopy and by transmission (TEM) and scanning electron microscopy (SEM). Vesicles showed the characteristic trilaminar pattern of the unit membrane and did not contain cellular organelles. As detected in freeze-fracture preparations, vesicle membrane displayed intramembrane particles and filipin-detectable cholesterol. Like EC plasmalemma, vesicle surface was heavily stained by Ruthenium Red and bound under a normal pattern cationized ferritin and ferritin hydrazide. As indicated by lectin agglutination assays and by ultrastructural cytochemistry, vesicles maintained on their ectodomains glycoconjugates bearing monosaccharides such as N-acetyl-neuraminic acid, beta-N-acetylglucosamine and beta-D-galactose, and expressed 5'-nucleotidase activity. The electrophoretic profiles of externally disposed 125I-labelled polypeptides of vesicles were found to be similar to those of intact EC. Chemically-induced vesiculation appears as a suitable method to obtain EC plasmalemma for studying its composition and functions in various vascular beds.

Animals↗
PubMed1986

Ruthenium red inhibits the mitochondrial Ca2+ uptake in intact bovine spermatozoa and increases the cytosolic Ca2+ concentration.

The uptake and cycling of Ca2+ by ejaculated bovine spermatozoa are almost completely abolished by ruthenium red, antimycin A or FCCP. The inhibitory effect of ruthenium red is also observed after washing of the dye-pretreated cells followed by addition of digitonin or filipin. In contrast, the inhibition is overcome by A23187 treatment. It is concluded that ruthenium red penetrates into intact spermatozoa, inhibits the mitochondrial Ca2+ uptake 'in situ', and causes the observed increase of the cytosolic free Ca2+ concentration.

Aminoquinolines↗
PubMed1987

Sub-compartmentation of the 'cytosolic' glucose 6-phosphate pool in cultured rat hepatocytes.

[14C]Glucose release either from endogenous 14C-prelabelled glycogen or from added 14C-labelled glucose 6-phosphate was measured in filipin-treated, permeabilized hepatocytes in 48 h culture. [14C]Glucose output from prelabelled glycogen was not altered by the addition of 5 mM glucose 6-phosphate to the incubation medium. Conversely, [14C]glucose release from 5 mM labelled glucose 6-phosphate was not influenced by different glycogen concentrations in the cells. Moreover, in the permeabilized cells the anion transport inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) inhibited only the liberation of [14C]glucose from labelled glucose 6-phosphate but not from glycogen. It is therefore concluded that there exist at least 2 separate, mutually non-accessible glucose 6-phosphate pools in cultured rat hepatocytes, one linked to glycogenolysis and the other to gluconeogenesis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Subendothelial accumulation of unesterified cholesterol. An early event in atherosclerotic lesion development.

This preliminary report describes the simplest and possibly earliest lipid-containing lesions induced in aortas of rabbits fed a cholesterol-containing diet. Subendothelial accumulation of unesterified cholesterol, detected with filipin dye, appears to precede morphologic changes in endothelium and accumulation of subendothelial lipid-containing "foam" cells. The origin of subendothelial unesterified cholesterol remains to be determined.

Animals↗
PubMed1987

Development of intracellular lipid deposits in the lipid-laden cells of atherosclerotic lesions. A cytochemical and ultrastructural study.

In atherosclerotic lesions of rabbits fed a cholesterol-rich diet, the lipid deposits of foam cells derived from monocytes, smooth muscle and endothelial cells were studied by physical, cytochemical and ultrastructural methods. Beginning with the third week of diet, the lipid material that could be visualized at the light microscope level by Oil red O and Nile red staining was progressively accumulated in the intimal cells of the atherosclerotic lesions. In the early stages of foam cell formation, the deposits occurred especially as intracytoplasmic non-membrane bound lipid inclusions (lipid droplets). In polarizing microscopy these appeared as a mixture of iso-, and anisotropic material. The latter were birefringent and showed an axial symmetry with a black cross image, suggesting that the lipids were in a liquid crystalline state. In chemically-fixed specimens, the content of lipid inclusions was preserved in various degrees. In freeze-fractured preparations they displayed a layered onion-like arrangement with smooth cleavage faces surrounding an amorphous core. Upon incubation with filipin, that specifically binds to 3 beta-hydroxysterols, the peripheral layers of the inclusions were labeled, revealing the existence of unesterified cholesterol. In the advanced stages of foam cell formation, lipids were additionally accumulated in the lysosomal compartment as polymorphic multilamellar structures concentrically arranged, with cleavage faces devoid of intralamellar particles. The presence of acid phosphatase showed that these features were modified lysosomes and were tentatively named lysosomal lipid bodies. In the latest stages examined cholesterol crystals developed within lysosomal lipid bodies usually enclosed in multilamellar structures. This lipid coat may represent the place of crystal formation and presumably acts as barrier for the turnover of the crystalline cholesterol, thus impeding plaque regression.

Acid Phosphatase↗
PubMed1987

Prelesional events in atherogenesis. Colocalization of apolipoprotein B, unesterified cholesterol and extracellular phospholipid liposomes in the aorta of hyperlipidemic rabbit.

The appearance and accumulation of apolipoprotein B and unesterified cholesterol in the lesion-prone areas of the aorta in rabbits with diet-induced hyperlipidemia were investigated by histo-, and cytochemical techniques. Apolipoprotein B was detected by an indirect immunoperoxidase procedure both in the light and electron microscopy. Unesterified cholesterol was revealed using filipin and tomatine as specific probes. In the prelesional stages of atherogenesis, before the appearance of any structurally detectable lesions, as demonstrated by bright-field and fluorescence microscopy, apolipoprotein B and free cholesterol accumulated progressively in the extracellular matrix of the subendothelial space. At ultrastructural level, extracellular phospholipid liposomes, unesterified cholesterol and apolipoprotein B concomitantly appeared and accumulated focally in the same areas. Apolipoprotein B was preferentially located on the outer surface of the free cholesterol-containing phospholipid lamellae of the extracellular liposomes. In the lesional stages leading to fatty streak formation, the extracellular liposomes, apolipoprotein B and unesterified cholesterol had also topographically a superimposed localization pattern. Intracellular apolipoprotein B and unesterified cholesterol were also colocalized in some intimal lipid-laden cells. In the prelesional stages of hyperlipidemia the prevalent localization of apolipoprotein B around individual unesterified cholesterol-rich extracellular phospholipid liposomes, progressively accumulating in the subendothelial space, suggests their possible origin from serum-derived lipoproteins.

Animals↗
PubMed1988

Lipoprotein uptake in primary cell cultures of rabbit atherosclerotic lesions. A fluorescence microscopic and flow cytometric study.

To characterize the lipoprotein metabolism of lipid-filled cells of atherosclerotic lesions, uptake of 3,3'-dioctadecylindocarbocyanine (DiI)-labelled low density lipoprotein (LDL), acetylated LDL (Ac-LDL) and beta-very low density lipoprotein (beta-VLDL) was studied by fluorescence microscopy and flow cytometry in primary cultures of enzymatically dispersed aortic cells from cholesterol-fed rabbits. Most of the foam cells were identified as macrophages on the basis of Fc-receptors and high activities of nonspecific esterase and acid lipase, although cholesteryl ester (CE) inclusions were found by filipin staining also in smooth muscle cells (SMCs). During the culture only SMCs proliferated and were confluent in about 1 week. After incubation with DiI-Ac-LDL most macrophage foam cells were brightly fluorescent, but also many SMCs accumulated fluorescence. In SMCs, an excess of LDL inhibited the uptake of DiI-beta-VLDL and DiI-LDL, indicating that these lipoproteins were taken up by the apoB,E receptor; the activity of this receptor was low 2 days after cell isolation but increased considerably during SMC proliferation. DiI-beta-VLDL was not taken up by the macrophage foam cells until after 7 days' culture, when their CE content had decreased, reflecting a feed-back regulation of these receptors as well. Our results indicate that, in primary cultures of enzyme-dispersed cells from rabbit atherosclerotic lesions, most of the foam cells have lipoprotein receptors resembling those described in macrophages and that also many SMCs accumulate Ac-LDL.

Animals↗
PubMed1989

Macrophage foam cells from human aortic fatty streaks take up beta-VLDL and acetylated LDL in primary culture.

Intimal cells from human aortic fatty streak lesions were isolated with collagenase-elastase digestion and the cellular uptake of lipoproteins fluorescently labeled with 3,3'-dioctadecylindocarbocyanine (DiI) was studied in primary culture. The majority of the cells in primary culture contained lipid droplets and the foam cells consisted of both macrophages and smooth muscle cells (SMC), identified with electron microscopy and the macrophages also using the monoclonal anti-Leu-M3 antibody. The lipid inclusions contained cholesteryl ester, as visualized with filipin staining. Arterial macrophages took up DiI-labeled acetylated low density lipoprotein (DiI-acetyl-LDL) in the same way as did monocyte-macrophages isolated from blood. DiI-labeled beta-very low density lipoprotein (DiI-beta-VLDL) isolated from cholesterol-fed rabbits, was taken up by both macrophages and SMCs. In macrophages DiI-beta-VLDL was internalized also in the presence of excess unlabeled low density lipoprotein (LDL), whereas in SMCs the uptake was partially prevented. DiI-LDL uptake was only seen in SMCs free of lipid inclusions and especially during cell growth. The present results show that, in human aortic fatty streaks, (a) both macrophages and SMCs accumulate cholesteryl ester, (b) macrophage foam cells possess active scavenger receptors capable of mediating the uptake of acetyl-LDL, and (c) macrophages are also capable of accumulating cholesteryl ester by receptor-mediated uptake of beta-VLDL.

Adult↗
PubMed1989

Cytochemical localization of beta-lipoproteins and their components in successive stages of hyperlipidemic atherogenesis of rabbit aorta.

We have investigated cytochemically the correlative distribution of lipoprotein (LP) components in successive lesional stages of plaque formation in hyperlipoproteinemic atherogenesis in the rabbit. Apoprotein B (apo B) was detected by an immunoperoxidase procedure, unesterified cholesterol (UC) by filipin and tomatine and phospholipid lamellae of the extracellular liposomes (EL) as they appear in standard EM. The changes were evaluated in relation to the state of endothelial cells and their transport pathways, and the reaction of the cellular and extracellular components of the intima. Each lesional stage has a relatively characteristic pattern distribution of the LP components. In fatty streaks with no endothelial denudation, apo B reaction product occurs mostly in non-particulate form associated with UC-rich EL; this suggests that transcytosed LP upon partial degradation and interaction with the extracellular components, reassemble as polymorphic EL-UC-apo B complexes. Serofibrinous insudates, although commonly devoid of EL and apo B, may contain UC presumably transported by a carrier other than LP. In advanced fibrolipidic lesions with open endothelial junctions and deendothelialized areas, a bulky intramural insudation of plasma results in the presence of large amounts of apparently little modified LP. This may represent what several investigators have isolated as 'aortic LP', which may be insudated rather than transported plasma LP.

Animals↗
PubMed1995

Analysis of anticholesterol antibodies using hydrophobic membranes.

An analytical immunoblotting procedure and a serological enzyme-linked immunosorbent assay (ELISA) for the characterization of antibodies to cholesterol are described. Hydrophobic membranes consisting of polyvinylidene fluoride (PVDF) are used to immobilize cholesterol for immunodetection by anti-sterol antibodies. To determine whether antibodies to cholesterol were induced after immunization with liposomal cholesterol, we separated total lipid extracts of very-low density lipoproteins by thin layer chromatography (TLC) on silica gel plates and transferred the separated lipid classes to PVDF membranes using isopropanol to facilitate passive diffusion. Lipid transfer was confirmed by exposure of membranes to iodine vapors or by staining of cholesterol with filipin complex. Serum from immunized mice reacted with cholesterol, whereas pre-immune serum or serum from mice injected with control liposomes did not bind. To determine the amount of anti-cholesterol activity in serum, we coated microtiter plates consisting of PVDF membrane wells with cholesterol. The PVDF membrane-based ELISA was found to be more reproducible and four-fold more sensitive than the conventional ELISA on polystyrene plates. These techniques may be useful in the analysis of anti-sterol antibodies and antibodies to other hydrophobic antigens.

Antibodies↗
PubMed1995

Amiodarone induced lipidosis similar to Niemann-Pick C disease. Biochemical and morphological study.

Amiodarone is effective in the treatment of supraventricular and ventricular cardiac arrhythmia, however a high incidence of toxic side effects has been observed in various organs and tissues during chronic treatment. Ultrastructural observation of affected tissues reveals myelinoid inclusion bodies. The exact pathogenetic mechanism of these changes is still unknown. In this study we investigated the biochemical effects of this drug on lysosomal hydrolases and the alterations induced in subcellular organelles of fibroblasts cultured for 24 h with different concentrations of amiodarone in the medium. Of the enzyme activities assayed, we only observed a significant reduction in sphingomyelinase. Ultrastructural observation of fibroblasts showed swollen lysosomes and a few onionoid inclusion bodies at lower concentrations of the drug; at higher concentrations the lysosomal system was severely impaired. Cytochemical staining of unesterified cholesterol with filipin showed accumulation of cholesterol. We conclude that chronic amiodarone treatment in experimental conditions induces inhibition in sphingomyelinase activity through interaction with membrane lipids and modification of bilayer structure. Higher concentrations of the drug impair cholesterol transport and induce lipid accumulation. These results may be useful for understanding the pathogenesis of induced lipidosis in patients in chronic treatment with amiodarone.

Amiodarone↗
PubMed1985

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture.

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Effects of DNA and synthetic oligodeoxyribonucleotides on the binding properties of a cGMP-binding protein from Dictyostelium discoideum.

Previously, we identified a cGMP-binding protein (cGBP) in Dictyostelium discoideum that can exist in two forms: a fast-dissociating (F-type) activity and a slow-dissociating (S-type) activity. Moreover, the F-type activity was converted effectively to S-type by the addition of nucleic acids, especially DNA (Parissenti, A.M. and Coukell, M. B. (1989) J. Cell Sci. 92, 291-301). In this study, we examined the effects of heterologous DNA and various synthetic homo-oligodeoxyribonucleotides on the cGMP-binding properties of partially purified F-type activity. Equilibrium and kinetic binding experiments revealed that DNA increased the affinity of the protein for cGMP without altering the number of binding sites. However, the presence of DNA decreased only slightly the apparent Kd of the protein for cGMP because the nucleic acid also reduced the rate of cGMP association. Addition of oligo(dGMP)8 or oligo(dCMP)8 to the protein increased both total cGMP binding and the conversion of F-type activity to S-type; in contrast, oligo(dAMP)8 or oligo(dTMP)8, at the same concentration, had no effect. Oligodeoxycytidylic acids with chain lengths less than about eight nucleotides were also ineffective or inhibitory. Analysis of cGMP binding to intact, filipin-permeabilized cells revealed a binding activity with association and dissociation rates comparable to isolated S-type activity. This observation suggests that in vivo the cGBP might exist in its S-form.

Carrier Proteins↗
PubMed1992

The role of exocytosis in the apocrine secretion of milk lipid globules in mouse mammary gland during lactogenesis.

Functional relations between exocytotic vesicle membranes, plasmalemma and milk fat globule membranes (MFGM) were studied during the final stages of mouse mammary gland differentiation, in the gland during full lactation and in the postpartum gland in which the synthesis of secretory products was partly inhibited by application of 2-Br-alpha-ergocryptine. Analysis of ultrathin sections, freeze-fracture replicas, scanning electron microscopy and application of a cytochemical marker filipin showed that the apocrine secretion of lipid globules was closely related to the exocytosis of milk proteins. During the last days of gestation the secretion of lipid globules resulted from many exocytotic events of the secretory vesicles that accumulated and fused around the cytoplasmic lipid droplets. Seldom the lipid droplet protruded partly into the gland lumen and a part of its surface became covered with the apical plasmalemma. Although apical plasmalemma became more important in the formation of MFGM in the postpartum period, we could still confirm a direct contribution of secretory vesicle membranes to the final detachment of the lipid globule. The application of 2-Br-alpha-ergocryptine hindered the apocrine secretion of the lipid globules and a situation similar to the situation in the prepartum gland was observed.

Animals↗
PubMed1994

Clinical features of Niemann-Pick disease type C. An example of the delayed onset, slowly progressive phenotype and an overview of recent literature.

Analysis of recent literature on Niemann-Pick disease type C (NPC) reveals a broad clinical spectrum with diverse neurological manifestations. Diagnosis and assessment are discussed. We present a case with symptomatology that is in concordance with a specific phenotype. The major clinical features are highlighted in a review of recently published cases. NPC could be underdiagnosed and we argue that in the work-up of progressive neurological disorders NPC should be seriously considered, at all ages, whenever there is any combination of visceromegaly, psychomotor deterioration, ataxia, vertical gaze disturbances or developmental delay. The Filipine staining of fibroblasts is a helpful asset in the diagnostic process, to be concluded with the detection of defective intracellular cholesterol esterification.

Adolescent↗