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At least 829 records · Page 46Linked to original sources
PubMed2001

Oxidative stress impairs insulin internalization in endothelial cells in vitro.

AIMS/HYPOTHESIS: Because oxidative stress has been suggested to be a significant contributing factor in the development of endothelial dysfunction and insulin resistance, we investigated whether reactive oxygen species contribute to insulin resistance by impairing insulin uptake through an effect on endothelial insulin receptor function. METHODS: Following a 2-h pro-oxidant challenge with xanthine oxidase, we examined the temporal pattern of insulin processing in the human umbilical endothelial cell line Ea.Hy926 and bovine aortic endothelial cells equilibrated with [125I]-insulin. Insulin receptor mRNA concentrations were analysed by RT-PCR and insulin receptor tyrosine phosphorylation and protein concentrations were estimated by western blotting. RESULTS: Xanthine oxidase exposure resulted in a major reduction in total insulin receptor-mediated [125I]-insulin internalization over a 1-h period in both Ea.Hy926 and bovine aortic endothelial cells. After 15 min, untreated bovine aortic endothelial cells internalized fivefold more cell-bound [125I]-insulin than pro-oxidant treated cells. The [125I]-insulin disappeared from the cell surface at a similar rate in both pro-oxidant and untreated cells, with relatively more [125I]-insulin being released into the medium in pro-oxidant treated cells. Although xanthine oxidase reduced insulin receptor mRNA and protein concentrations, cell surface insulin binding capacity was not affected. Following 5 min insulin exposure, insulin receptor auto-phosphorylation was considerably reduced in cells challenged with xanthine oxidase for 2 h, which could be important for insulin receptor activation and internalization. CONCLUSION/INTERPRETATION: Oxidative stress impairs insulin endocytosis in both arterial and venous endothelial cell lines. This was not a consequence of modified insulin binding capacity but could involve insufficient insulin receptor activation.

Animals↗
PubMed2001

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy.

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.

Animals↗
PubMed1990

Type C Niemann-Pick disease: documentation of abnormal LDL processing in lymphocytes.

Type C Niemann-Pick disease (NPC) is an autosomal recessive neurovisceral storage disorder in which defective intracellular cholesterol processing has been demonstrated in fibroblasts from NPC patients and obligate heterozygotes. In the present paper, the ability to esterify LDL-cholesterol was examined in cultured lymphocytes from 8 NPC patients, 8 obligate heterozygotes and 8 controls. Cholesteryl ester synthesis was 8% (+/- 5%) and 45% (+/- 16%) of controls in homozygous and heterozygous cell lines, respectively. Histochemical and electron microscopic examinations confirmed that this biochemical lesion was associated with abnormal intracellular accumulation of unesterified cholesterol in mutant lymphocytes. These results demonstrate that measurement of cholesterol esterification in cultured lymphocytes offers a quick and reliable means of confirming the diagnosis of NPC and that these cells may be useful for probing the primary molecular lesion of NPC.

Cell Compartmentation↗
PubMed1984

Possible exposure of leucine aminopeptidase on the cell surface of rabbit blood neutrophils by digitonin treatment.

The sensitivity of leucine aminopeptidase to diazotized sulfanilic acid (DSA) was compared between neutrophils from blood and peritoneal exudates of rabbit. The leucine aminopeptidase activity of peritoneal neutrophils was inhibited about 40% by DSA, whereas that of blood neutrophils was not inhibited at all by the reagent. However, pretreatment of blood neutrophils with digitonin in the presence and in the absence of divalent cations rendered leucine aminopeptidase sensitive to DSA to the same extent as peritoneal neutrophils, without affecting the cell viability and lactate dehydrogenase activity. These findings seem to indicate that the leucine aminopeptidase of blood neutrophils, which is normally inaccessible to DSA, was exposed on the cell surface by digitonin treatment.

Animals↗
PubMed1985

Cholesterol and the cell membrane.

Recent studies concerning cholesterol, its behavior and its roles in cell growth provide important new clues to the role of this fascinating molecule in normal and pathological states.

Animals↗
PubMed1986

How do the polyene macrolide antibiotics affect the cellular membrane properties?

In the 1970's great strides were made in understanding the mechanism of action of amphotericin B and nystatin: the formation of transmembrane pores was clearly demonstrated in planar lipid monolayers, in multilamellar phospholipid vesicles and in Acholeplasma laidlawii cells and the importance of the presence and of the nature of the membrane sterol was analyzed. For polyene antibiotics with shorter chains, a mechanism of membrane disruption was proposed. However, recently obtained data on unilamellar vesicles have complicated the situation. It has been shown that: membranes in the gel state (which is not common in cells), even if they do not contain sterols may be made permeable by polyene antibiotics, several mechanisms may operate, simultaneously or sequentially, depending on the antibiotic/lipid ratio, the time elapsed after mixing and the mode of addition of the antibiotic, there is a rapid exchange of the antibiotic molecules between the vesicles. Although pore formation is apparently involved in the toxicity of amphotericin B and nystatin, it is not the sole factor which contributes to cell death, since K+ leakage induced by these antibiotics is separate from their lethal action. The peroxidation of membrane lipids, which has been demonstrated for erythrocytes and Candida albicans cells in the presence of amphotericin B, may play a determining role in toxicity concurrently with colloid osmotic effect. On the other hand, it has been shown that the action of polyene antibiotics on cells is not always detrimental: at sub-lethal concentrations these drugs stimulate either the activity of some membrane enzymes or cellular metabolism. In particular, some cells of the immune system are stimulated. Furthermore, polyene antibiotics may act synergistically with other drugs, such as antitumor or antifungal compounds. This may occur either by an increased incorporation of the drug, under the influence of a polyene antibiotic-induced change of membrane potential, for example, or by a direct interaction of both drugs. That fungal membranes contain ergosterol while mammalian cell membranes contain cholesterol, has generally been considered the basis for the selective toxicity of amphotericin B and nystatin for fungi. Actually, in vitro studies have not always borne out this assumption, thereby casting doubt on the use of polyene antibiotics as antifungal agents in mammalian cell culture media.(ABSTRACT TRUNCATED AT 400 WORDS)

Amphotericin B↗
PubMed2005

Role of fission yeast myosin I in organization of sterol-rich membrane domains.

Specialized membrane domains containing lipid rafts are thought to be important for membrane processes such as signaling and trafficking. An unconventional type I myosin has been shown to reside in lipid rafts and function to target a disaccharidase to rafts in brush borders of intestinal mammalian cells. In the fission yeast Schizosaccharomyces pombe, distinct sterol-rich membrane domains are formed at the cell division site and sites of polarized cell growth at cell tips. Here, we show that the sole S. pombe myosin I, myo1p, is required for proper organization of these membrane domains. myo1 mutants lacking the TH1 domain exhibit a uniform distribution of sterol-rich membranes all over the plasma membrane throughout the cell cycle. These effects are independent of endocytosis because myo1 mutants exhibit no endocytic defects. Conversely, overexpression of myo1p induces ectopic sterol-rich membrane domains. Myo1p localizes to nonmotile foci that cluster in sterol-rich plasma membrane domains and fractionates with detergent-resistant membranes. Because the myo1p TH1 domain may bind directly to acidic phospholipids, these findings suggest a model for how type I myosin contributes to the organization of specialized membrane domains.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed2003

Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils.

The integrity of lipid microdomains is disrupted after cell treatment with cholesterol-depleting reagents, such as methyl-beta-cyclodextrin (MCD). We investigated the roles of lipid microdomains in the regulation of intracellular signaling events and functional responses in isolated human neutrophils. Treatment of neutrophils with MCD caused inhibition of intracellular calcium increase evoked by interleukin-8 (IL-8) or low concentrations of formyl-Met-Leu-Phe (fMLP). No significant decrease of the initial peak of the calcium response was measured when neutrophils were stimulated with 100 nM or higher concentrations of fMLP. MCD inhibited the phosphorylation of extracellular signal-regulated kinase (Erk) induced by IL-8 or lower concentrations of fMLP. However, Erk phosphorylation evoked by higher concentrations of fMLP was only slightly affected. MCD treatment increased phosphorylation of p38 MAP kinase and caused strong up-regulation of both CD11b and CD66b in resting neutrophils. Cholesterol depletion greatly inhibited IL-8-induced elastase release but had little effect of fMLP-induced degranulation. Our study brings evidence suggesting that lipid microdomains are critically required for the signaling events triggered by IL-8. Calcium mobilization and elastase release induced by WKYMVM, a selective agonist for formyl peptide receptor-like 1 (FPRL1), were significantly inhibited by MCD, suggesting that the resistance of fMLP-mediated responses to MCD is not related to the partition of receptor subtypes to lipid microdomains. It is more probable that cholesterol depletion interferes with the ability of different G proteins to couple to their corresponding receptors and this might account for the differential effects of MCD treatment on chemoattractant-induced effects in human neutrophils.

Antigens, CD↗
PubMed2004

Increased iron staining in the cerebral cortex of cholesterol fed rabbits.

The link between hypercholesterolemia and neuronal damage is not clear. In the present study, we studied some of the possible effects of hypercholesterolemia on the brain, using the cholesterol fed New Zealand White rabbit as a model. An increase in the number of iron positive cells (i.e. oligodendrocytes) was observed in the brain parenchyma, in rabbits treated with a high cholesterol diet for 8 weeks. At this time, no neuronal death was observed, indicating that the increased iron did not occur as a consequence of neuronal injury. No heme oxygenase-1 (HO-1) or bilirubin immunoreactivity was observed in the brains in these rabbits, indicating that the iron accumulation did not occur as a consequence of increased breakdown of heme. It is postulated that cholesterol could have subtly damaged brain endothelial cells, resulting in increased iron transport across brain endothelial cells. Hypercholesterolaemia is known to be associated with increased plasma lipid peroxidation which might contribute to such damage.

Animals↗
PubMed2006

Trafficking of Streptococcus uberis in bovine mammary epithelial cells.

Results from our laboratory showed that Streptococcus uberis internalized bovine mammary epithelial cells by exploiting host cell cytoskeleton and signal transduction mechanisms. It was also shown that S. uberis survived intracellularly for up to 120 h and capable of transcytose bovine mammary epithelial cells. To define mechanisms and strategies used by S. uberis to move through host cells and survive intracellularly, internalization studies using specific inhibitors, double immunofluorescence labeling and confocal laser microscopy were conducted. When bovine mammary epithelial cells were treated with inhibitors of endocytic vesicle acidification, the number of intracellular S. uberis was similar to untreated controls. When selective inhibitors of lipid rafts/caveolae or receptor-mediated endocytosis were used, a significantly lower number of intracellular S. uberis was detected compared with untreated controls. However, when the effect of inhibitors of receptor-mediated endocytosis and lipid rafts/caveolae were compared, the latter induced the lowest S. uberis internalization values suggesting a preferential exploitation of caveolae-mediated endocytosis. Since caveloae-dependent intracellular trafficking does not include intravesicular acidification or lysosome fusion; these results suggest that by exploiting preferential intracellular trafficking pathways in bovine mammary epithelial cells, S. uberis avoids intracellular bactericidal mechanisms. Such a strategy would allow S. uberis to persist intracellularly and may explain how persistent intramammary infections occur.

Animals↗