Abstract:We have studied the lipid composition of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF). Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC). The total lipid concentration was approximately 220 micrograms/mg of protein, and the concentration of neutral glycolipids was 1.6-1.8 microgram/mg of protein for both NGF-treated and untreated cells. The neutral glycoli… Show more
“…After washing twice with TME solution to remove remaining Triton X-100, the sample was used for the lipid analysis. The isolation procedures for total lipids and for neutral and acidic lipids were described previously (13,34). Briefly, total lipids were extracted with chloroform/methanol ( (36).…”
A membrane microdomain called raft has been under extensive study since the assembly of various signaltransducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl--cyclodextrin was found to be effective to solubilize NAP-22, a neuronenriched Ca 2؉ -dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dosedependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.
“…After washing twice with TME solution to remove remaining Triton X-100, the sample was used for the lipid analysis. The isolation procedures for total lipids and for neutral and acidic lipids were described previously (13,34). Briefly, total lipids were extracted with chloroform/methanol ( (36).…”
A membrane microdomain called raft has been under extensive study since the assembly of various signaltransducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl--cyclodextrin was found to be effective to solubilize NAP-22, a neuronenriched Ca 2؉ -dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dosedependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.
“…Lipid Analyses-Lipids were extracted and purified from either whole cells or Triton X-100-insoluble domains using the method of Ariga et al (28). Cells (2.5 ϫ 10 8 ) or the appropriate fraction (isolated from 2.5 ϫ 10 8 cells) was sequentially extracted with chloroform/methanol (2:1, v/v), chloroform/methanol (1:1, v/v), and chloroform/methanol/ water (30:60:8, v/v).…”
The folate receptor, like many glycosylphosphatidylinositol-anchored proteins, is found associated with membrane domains that are insoluble in Triton X-100 at low temperature and that are enriched in cholesterol and sphingolipids. Depletion of cellular cholesterol has been shown to inhibit vitamin uptake by this receptor (Chang, W.-J., Rothberg, K. G., Kamen, B. A., and Anderson, R. G. W. (1993) J. Cell Biol. 118, 63-69), suggesting that these domains regulate this process. In this study, the importance of sphingolipids for folate receptor function was investigated in Caco-2 cells using fumonisin B 1 , a mycotoxin that inhibits the biosynthesis of these lipids. The folate receptor-mediated transport of 5-methyltetrahydrofolate was almost completely blocked in cells in which sphingolipids had been reduced by ϳ40%. This inhibition was dependent on the concentration and duration of the treatment with the mycotoxin and was mediated by the sphingolipid decrease. Neither receptor-mediated nor facilitative transport was inhibited by fumonisin B 1 treatment, indicating that the effect of sphingolipid depletion was specific for folate receptor-mediated vitamin uptake. A concurrent loss in the total amount of folate binding capacity in the cells was seen as sphingolipids were depleted, suggesting a causal relationship between folate receptor number and vitamin uptake. These findings suggest that dietary exposure to fumonisin B 1 could adversely affect folate uptake and potentially compromise cellular processes dependent on this vitamin. Furthermore, because folate deficiency causes neural tube defects, some birth defects unexplained by other known risk factors may be caused by exposure to fumonisin B 1 .
“…The isolation procedures for the total lipids, and neutral and acidic glycolipids from thyroid membranes and lymphocytes of Graves' disease were as described by Ariga et al (1988). Briefly, total lipids were extracted with chloroform: methanol (2: 1 and I: 1, v/v) and chloroform: methanol: water (30:60: 8, v/v) and filtered.…”
Section: Methodsmentioning
confidence: 99%
“…High performance thin-layer chromatography immunostaining HPTLC immunostaining was carried out according to the method of Ariga et al (1988). Glycolipids were chromatographed on HPTLC plate with the developing solvent system of chloroform: methanol: water (60:40:8, v/v).…”
Section: Preparation Of Moabs Against Thyrotropin (Tsh) Receptormentioning
SUMMARY
Sera from patients with Graves’ disease and Hashimoto's thyroiditis have been shown to react with the Forssman glycolipid antigen (Gb5) using the techniques of high performance thin‐layer chromatography (HPTLC) immunostaining and ELISA. Human monoclonal antibodies (MoAbs) have been prepared by fusion of human myeloma with peripheral lymphocytes from patients with Graves’ disease. A MoAb, TRMo‐4, reacted strongly and specifically with Gb5. These results suggest that anti‐Forssman antibody may be involved in the pathogenesis of these autoimmune diseases. The detection of anti‐Forssman glycolipid antibody may provide a useful means for clinical diagnosis and therapy.
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