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At least 811 records · Page 45Linked to original sources
PubMed1981

Membrane expression of Fc-receptors in cultured leukemic cell lines. I. Induction of Fc-receptor in undifferentiated types of cells after passive modulation of lipid viscosity.

The role of lipid environment of plasma membrane for the expression of Fc receptor (gamma) (FcR gamma) were studied by physicochemical modification of the membranes of myeloid leukemia cell line cells, M1. Sterol binding polyene antibiotics such as amphotericine B or filipin inhibit the expression of FcR gamma during the differentiation of M1- cells by the CM obtained from the culture of mouse embryonic fibroblasts, suggesting the possible involvement of cholesterol molecules in the induction mechanism of FcR gamma. Low-temperature incubation at either 20 degrees C or 4 degrees C results in the increment of their membrane microviscosity, when tested by the fluorescence depolarization method, in company with the appearance of FcR gamma on approximately 60% of M1 cells when assayed by the EA-rosetting method. FcR gamma is also induced on approximately 40% of M1- cells by incubating cells with cholesterol-phosphatidyl-choline liposomes with the concomitant increase of their membrane microviscosity. Nevertheless, no phagocytosis of EA is observed in these cells treated with such physicochemical procedures. These findings imply that FcR gamma is embedded in the membrane of M1- cells and that physicochemical modification of the membrane lipid bilayers can induce the FcR gamma independent on their functional differentiation marked by the expression of phagocytic activity.

Animals↗
PubMed1994

Detection of cholesterol associated with calcium mineral using confocal fluorescence microscopy.

BACKGROUND: Cholesterol and calcium are prominant components within human atherosclerotic lesions. Both accumulate predominantly within the central core region of lesions. Because of similarities in some crystallographic faces of cholesterol monohydrate and calcium apatite, it has been previously proposed that deposition of one may nucleate the deposition of the other. EXPERIMENTAL DESIGN: In this study, we have used the technique of confocal fluorescence microscopy to assess the spatial orientation of cholesterol in association with calcium mineral. Localization of cholesterol within mineral was carried out by staining cholesterol with the fluorescent probe, filipin. RESULTS: With this technique, it was possible to localize cholesterol associated with the surface of hydroxyapatite seeds, cholesterol incorporated within calcium phosphate-cholesterol agglomerates produced in vitro, and cholesterol within apatite isolated from human atherosclerotic lesions. CONCLUSIONS: The presence of cholesterol within the center of calcified granules from atherosclerotic plaque suggests that cholesterol or associated lipids may act to nucleate the deposition of apatite. Confocal fluorescence microscopy should be a useful technique by which to study the relationship of cholesterol associated with calcium minerals that occur not only in atherosclerotic blood vessels, but also in gallstones, and calcified cardiac valves.

Apatites↗
PubMed1994

Structure-specific inhibition of lysosomal cholesterol transport in macrophages by various steroids.

Cultured mouse peritoneal macrophages effectively take up and metabolize liposomes containing phosphatidylserine and cholesterol, resulting in massive accumulation of cholesteryl esters and triacylglycerols in their cytoplasm (Nishikawa, K., Arai, H. and Inoue, K. (1990) J. Biol. Chem. 265, 5226-5231). With this system, various steroid derivatives were assessed as to their ability to inhibit the cholesteryl ester formation from endocytosed cholesterol in macrophages. Among the steroids tested, one group of steroids having an oxo group at the C17 or C20 position, such as androstenedione, dehydroisoandrosterone, progesterone and pregnenolone, completely inhibited cholesteryl ester formation at 10 microM. Another group of steroids having a hydroxy group at the C17 position, such as testosterone and androstenediol, had a lesser effect; complete inhibition of cholesteryl ester formation was achieved with 100 microM or more. The mechanism underlying the inhibition by the former class of steroids was further studied. These steroids did not block the uptake or lysosomal hydrolysis of liposomes, nor esterification of fatty acyl chains into triacylglycerols. Moreover, dehydroisoandrosterone and pregnenolone, both of which possess a hydroxy group at the C3 position, had essentially no effect on 25-hydroxycholesterol-stimulated esterification of endogenous cellular cholesterol. On the other hand, androstenedione and progesterone, which possess an oxo group at the C3 position, had a mild inhibitory effect on the esterification of endogenous cholesterol. Upon incubation with a steroid having an oxo group at the C17 or C20 position, free cholesterol taken up by macrophages was accumulated in phagolysosomes, as judged from cytochemical study with filipin-cholesterol staining. These results indicate that a series of structurally-related steroids characterized by the presence of an oxo group at the C17 or C20 position inhibit cholesteryl ester formation in macrophages through blocking the intracellular transport of endocytosed cholesterol from lysosomes to endoplasmic reticulum.

Animals↗
PubMed1994

Acquisition of iron bound to low molecular weight chelates by human monocyte-derived macrophages.

The iron content of tissue macrophages increases under conditions of elevated extracellular iron. Studies of macrophage iron acquisition have generally focused on iron uptake from transferrin via receptor-mediated endocytosis. However, in vivo macrophages are also exposed to extracellular low m.w. iron chelates, particularly under conditions of iron overload. Therefore, we examined the mechanism of iron acquisition from low m.w. chelates by human monocyte-derived macrophages. Iron acquisition was influenced by the nature of the iron chelate: Fe-ascorbate > Fe-citrate > Fe-nitrilotriacetate (NTA) = Fe-ADP > Fe-glycyl-L-histidyl-L-lysine > Fe-diethylenetriamine pentaacetic acid >> Fe-EDTA = Fe-deferoxamine. With the exception of Fe-EDTA and Fe-deferoxamine, iron acquisition was greater than that with diferric transferrin. As assessed by using iron acquisition from NTA as a model, the process is temperature dependent, but pH independent (pH 5 to 8), and is influenced by the medium in which the cells are suspended. Acquisition is not affected by NaF, 2-deoxy-D-glucose, NaCN, or monocyte-derived macrophage exposure to trypsin, pronase, phenylarsine oxide, dihydrocytochalasin B, filipin, nystatin, or digitonin. The rate of iron acquisition from NTA is induced by iron pre-exposure as well as aluminum. In contrast, NTA chelates of other transition metals (Cd, Cu, Ga, Mn, and Zn) inhibited iron uptake by 20 to 80%. Unlike results obtained with Ga-NTA, Ga(NO3)3 increased iron uptake from NTA. Data obtained with neutrophils, and undifferentiated U937 and HL-60 cells were similar, which suggests that myeloid cells share this pathway for iron acquisition. Iron acquisition via this mechanism may allow macrophages and other leukocytes to clear local states of iron overload in vivo.

Adenosine Triphosphate↗
PubMed1978

Differential control of synergistic effect with polyene macrolide antibiotics upon Chinese hamster cells in vitro.

An amphotericin B-resistant cell (AMBR-1), which was isolated from aneuploid Chinese hamster cells (V79), was found to show much higher resistance than the parent V79 cells to other polyene antibiotics, such as pentamycin and filipin. To obtain the 50 to 60% inhibition of the control protein synthesis activity by a synergistic combination of fusidic acid and amphotericin B, 50 microgram fusidic acid per ml were combined with 10 microgram amphotericin B in V79 cells, whereas in AMBR cells 50 microgram fusidic acid per ml were combined with 100 microgram polyene antibiotic per ml. Bleomycin (10 microgram/ml), which alone did not affect cellular DNA synthesis, inhibited DNA synthesis of V79 cells by more than 90% of the control activity when combined with only 1 microgram pentamycin per ml, whereas a similar extent of inhibition in AMBR cells was observed by combination with more than 5 microgram pentamycin per ml.

Amphotericin B↗
PubMed1993

Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, blocks lysosomal cholesterol trafficking in macrophages.

Certain steroids having an oxo group at the C-17 or C-20 position such as pregnenolone and dehydroisoandrosterone inhibit the cholesterol transport from lysosomes to other cellular sites. Taking advantage of the fact that the inhibition is reversed upon removal of the steroids, we studied the factors that control the cholesterol transport from lysosomes to other cellular sites in macrophages. Macrophages that accumulated unesterified cholesterol in their lysosomes were prepared by incubating cells with liposomes containing cholesterol and phosphatidylserine in the presence of a steroid inhibitor. These cells were chased by means of steroid washout, and then the effects of various pharmacological agents on the subsequent metabolism of cholesterol were examined. When the cells were chased in the absence of the agents, some of the cholesterol was converted to cholesteryl esters in the cells, and others were desorbed into the medium as unesterified forms, suggesting recovery of lysosomal cholesterol trafficking. Among the agents tested, bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, completely blocked both cholesterol esterification and cholesterol desorption at 10 nM. Moreover, agents that neutralize the lysosomal proton gradient, such as ammonium chloride and chloroquine, also reduced both of the processes. Fluorescent microscopic examination of bafilomycin A1-treated cells revealed extensive filipin-cholesterol staining of perinuclear lysosomes. From these data, we conclude that acidic pH is required for the efflux of cholesterol from lysosomes to other cellular sites.

Animals↗
PubMed1995

[Niemann-Pick disease type C].

Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral lipid storage disorder, but the basic defect has not yet been clarified. Diagnostic biochemical makers are intracellular accumulation of free cholesterol or the decreased esterification of exogenous cholesterol. The differences in the severity of defective esterification are related to the onset of the disease. Genetically, an abnormal gene is located on the human chromosome 18. Biochemically, many drugs, i.e. imipraine, progesterone and bafilomycin A1 are known to interfere with cholesterol esterification. Clinically, vertical supranuclear gaze palsy and cataplexy are specific symptoms. The filipin stain of the foamy cell in bone marrow is available for rapid diagnosis. Many therapies, i.e. dimethyl sulfoxide, low-cholesterol diet and transplantations, have been challenged but improvement of neurological symptoms have not been reported.

Animals↗
PubMed1997

Bile acid-induced morphological changes in hepatoma cells with elevated sodium-dependent bile acid uptake capacity.

McNtcp.24 cells are rat hepatoma cells that were made competent to take up conjugated bile acids actively from the culture medium. Treatment of McNtcp.24 cells with certain species of bile acids caused significant changes in cell structure. Incubation of McNtcp.24 cells in medium containing 100 microM taurocholic acid induced a profound alteration of cellular morphology. Very larger vesicles, visible by phase contrast microscopy, were the most prominent feature of bile acid-treated McNtcp.24 cells. Staining of cells with Oil red O and filipin indicated that the vesicles did not contain neutral lipids or free cholesterol. The vesicles remained in the cells after efflux of radiolabeled taurocholic acid from bile acid loaded cells, indicating that these structures are not intracellular stores of bile acids. Electron microscopic analysis of bile acid-treated McNtcp.24 cells confirmed that the vesicles were localized within the cells. Taurine-conjugated bile acid species were generally potent inducers of the morphological changes, although tauroursodeoxycholic acid did not have a significant effect. Unconjugated bile acid species were ineffective or only mildly effective. Bile acid treatment also caused profound alteration of mitochondrial structure. Surprisingly, there was no significant effect on the ability of treated cells to oxidize fatty acids. The bile acid-treated cells remained viable and upon withdrawal of bile acids from the culture medium, the cells returned to normal morphology by 24 h. The morphological changes observed after treatment of McNtcp.24 with bile acids are reminiscent of the morphological changes observed in hepatocytes following induction of cholestasis.

Animals↗
PubMed1998

Caveolin is present in intestinal cells: role in cholesterol trafficking?

It was postulated that specialized microdomains of the plasma membrane, consistent with caveolae, might play a role in cholesterol trafficking in intestinal cells. The existence, therefore, of caveolin and the role of detergent-resistant microdomains of the plasma membrane in cholesterol trafficking were investigated in human small intestine and CaCo-2 cells. Caveolin mRNA was detected by RT-PCR in small intestinal brushings and biopsies and in CaCo-2 cells. Northern hybridization of caveolin mRNA detected 3 kb and 0.8 kb transcripts in CaCo-2 cells. From brushings of distal duodenum and in CaCo-2 cells, Western analysis for detection of caveolin protein demonstrated a 21 kDa-sized protein and a 600 kDa homooligomer. In CaCo-2 cells, caveolin was demonstrated by immunofluorescence in apical membranes as well as within cells. Using sucrose-density gradients, caveolin was localized to detergent-resistant microdomains of the plasma membrane. As determined by cholesterol oxidase-accessible cholesterol, 3-5% of plasma membrane cholesterol in CaCo-2 cells was estimated to be in these detergent-resistant microdomains. After the absorption of cholesterol from bile-salt micelles, more plasma membrane cholesterol moved to these specialized microdomains within the plasma membrane and was esterified. In CaCo-2 cells, filipin, N-ethyl maleimide, and cholesterol depletion, treatments that disrupt caveolar function, interfered with the transport of plasma membrane cholesterol to the endoplasmic reticulum, whereas okadaic acid, sphingomyelinase, and cholesterol oxidase did not. Changes in cholesterol flux at the apical membrane of the cell did not alter mRNA levels or mass of caveolin. The results suggest that caveolin is present in intestinal and CaCo-2 cells and is associated with detergent-resistant microdomains of cellular membranes. With the influx of micellar cholesterol from the lumen, plasma membrane cholesterol moves or "clusters" to these microdomains and is transported to the endoplasmic reticulum for esterification and eventual transport. Caveolin/caveolae may play a role in cholesterol trafficking in intestinal cells.

Biological Transport↗
PubMed1988

Establishment of three bleomycin-resistant human carcinoma cell lines and their cross-resistance to other antitumor agents.

Three bleomycin-resistant (BLMr) human carcinoma cell lines (HeLa-BLMr, KB-BLMr, and Hepd-uvBLMr) were established in culture by progressively increasing the concentration of bleomycin (BLM). HeLa-BLMr and KB-BLMr were obtained after 5 and 2 months of incubation with BLM, whereas the establishment of Hepd-uvBLMr required 3 months of incubation with BLM after ultraviolet treatment. These cells have been successfully subcultured for more than 150 passages during more than 2 years in the presence of 1 microgram/ml of BLM. The degrees of BLM resistance were 20-fold, 11.6-fold, and 186-fold for HeLa-BLMr, KB-BLMr, and Hepd-uvBLMr, respectively, and the resistant phenotype of both HeLa-BLMr and Hepd-uvBLMr was stable when they were cultured for 30 passages in BLM-free medium, but unstable in KB-BLMr. Although each cell line exhibited cross-resistance to 3 to 5 other antitumor agents including peplomycin, combined use of BLM with a polyene antibiotic, a calcium channel blocker, or a poly(ADP-ribose) polymerase inhibitor overcame the BLM resistance in vitro to various degrees.

Aged↗
PubMed2000

Lysenin-sphingomyelin binding at the surface of oligodendrocyte lineage cells increases during differentiation in vitro.

We have investigated the relationship between the developmental expression of sphingomyelin, a major component of myelin, and oligodendrocyte lineage. Using lysenin as a cytochemical probe for membrane sphingomyelin, we have now determined the distribution pattern of sphingomyelin on the plasma membrane of rat cultured oligodendrocytes. Although lysenin does not bind to A2B5(+)/NG2(+) bipolar oligodendrocyte progenitors, lysenin recognizes sphingomyelin on the cell bodies of multipolar A2B5(+) cells, but not on their processes. O4(+) and O1(+) immature and MBP(+) mature oligodendrocytes are strongly labeled by lysenin from cell bodies to the tips of processes. The content of sphingomyelin in immature and mature oligodendrocytes is approximately 2-fold higher than that in oligodendrocyte progenitors. These findings show that sphingomyelin increases during differentiation of cells in the oligodendrocyte lineage. In multipolar oligodendrocyte progenitors exposed to Triton X-100 at 4 degrees C, lysenin labels cell processes in addition to cell bodies. In contrast, Triton X-100 extraction does not alter the distribution of lysenin binding on O4(+), O1(+) and MBP(+) cells, although the immunocytochemical intensities of the lysenin bindings increase. Our data suggest that the alteration in sphingomyelin content and distribution in the oligodendrocyte lineage cells could have important consequences for cell recognition and downstream signaling events through sphingomyelin-rich domains.

Animals↗
PubMed2000

The mitogenic activity of fibroblast growth factor-1 correlates with its internalization and limited proteolytic processing.

The fibroblast growth factor-1 (FGF-1) mitogenic signal transduction pathway is not well characterized, and evidence indicates that FGF-1 binding to and activation of cell-surface receptors is not solely sufficient for a full mitogenic response. Although initiation of the phosphorylation signaling cascades are likely important in FGF-1-induced mitogenic signaling, there appear to be additional signaling requirements. In this study, we demonstrate that FGF-1 internalization and subsequent processing correlates with the mitogenic potential of the growth factor on NIH 3T3 cells. Using site-directed mutants of FGF-1 and inhibitors of the endocytic and degradative pathways, we provide evidence for growth factor internalization and exposure to an acidic environment as necessary components of FGF-1-induced mitogenesis. In addition, a protease-sensitive event(s) appears critical for a complete mitogenic response to FGF-1, whereas, this protease sensitivity was not detected under the same conditions for serum-stimulated mitogenesis. Therefore, proteolytic modification of internalized FGF-1 may result in the activation of additional, intracellular signaling events.

3T3 Cells↗
PubMed1991

Evidence for the presence of a growth factor in Dictyostelium discoideum.

Polypeptide hormones, recognized for their ability to regulate cell growth and differentiation, have been classified as growth factors. These growth factors have been extensively described in higher eukaryotic organisms and cell lines [Hedin and Westermark, Cell 37:9-20, 1984]. Here we report the identification and partial characterization of a putative growth factor present in vegetative amoebae of the cellular slime mold Dictyostelium discoideum. A mutant was selected and found to be temperature sensitive due to the absence of an extracellular protein suggestive of a growth factor. The putative growth factor (DGF) is a protein resistant to both heat and strong detergent treatment but sensitive to reducing agents. The physiological significance of DGF is as yet unknown. DGF is of interest both in relation to understanding the events which control cell proliferation in Dictyostelium and in its relationship to other known growth factors.

Dictyostelium↗
PubMed1979

Direct evidence for formation of hybrid vesicles by fusion of plasma and outer acrosomal membranes during the acrosome reaction in boar spermatozoa.

A variety of treatment procedures was utilized to identify the origin and composition of the vesicles formed during the acrosome reaction of boar spermatozoa. Whether the acrosome reaction occurred spontaneously or was induced chemically the vesicles were hybrid vesicles composed of roughly equal proportions of plasma and outer acrosomal membranes.

Acrosome↗
PubMed1999

Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation.

We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO(3), all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO-(4) to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO-(4) and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO-(3) is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO-(3) or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO-(3) effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.

Acrosome↗
PubMed2000

Mechanism of cAMP-induced H(+)-efflux of Dictyostelium cells: a role for fatty acids.

Aggregating Dictyostelium cells release protons when stimulated with cAMP. To find out whether the protons are generated by acidic vesicles or in the cytosol, we permeabilized the cells and found that this did not alter the cAMP-response. Proton efflux in intact cells was inhibited by preincubation with the V-type H(+) ATPase inhibitor concanamycin A and with the plasma membrane H(+) ATPase blocker miconazole. Surprisingly, miconazole also inhibited efflux in permeabilized cells, indicating that this type of H(+) ATPase is present on intracellular vesicles as well. Vesicular acidification was inhibited by miconazole and by concanamycin A, suggesting that the acidic vesicles contain both V-type and P-type H(+) ATPases. Moreover, concanamycin A and miconazole acted in concert, both in intact cells and in vesicles. The mechanism of cAMP-induced Ca2(+)-fluxes involves phospholipase A2 activity. Fatty acids circumvent the plasma membrane and stimulate vesicular Ca2(+)-efflux. Here we show that arachidonic acid elicited H(+)-efflux not only from intact cells but also from acidic vesicles. The target of regulation by arachidonic acid seemed to be the vesicular Ca2(+)-release channel.

4-Chloro-7-nitrobenzofurazan↗