A simple, sensitive method for lipid phosphorus

Lowry, Robert R.; Tinsley, Ian J.

doi:10.1007/bf02534277

Cited by 158 publications

(57 citation statements)

References 3 publications

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“…The resulting succinylated and biotinylated PEG was reacted with 1, 2, 2, and 3 eq, respectively, of phosphatidylethanolamine, dicyclohexylcarbodiimide, diisopropylethylamine, and N-hydroxysuccinimide under argon for 16 h at 25°C in 10:1 methylene chloride/dimethylformamide, and the PE-PEG-biotin product was purified by preparative TLC in 70:20:1.5:1.5 methylene chloride/methanol/water/ammonium hydroxide. PE-PEG-biotin stock solutions in methylene chloride/methanol were standardized by phosphorus determination as described previously (21).…”

Section: Methodsmentioning

confidence: 99%

Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association

Wang¹,

Leventis

Silvius

2005

Journal of Biological Chemistry

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We have incorporated artificial lipid-anchored streptavidin conjugates with fully saturated or polyunsaturated lipid anchors into the plasma membranes of Jurkat T-lymphocytes to assess previous conclusions that the activation of signaling processes induced in these cells by clustering of endogenous glycosylphosphatidylinositol-anchored proteins or ganglioside GM1 depends specifically on the association of these membrane components with lipid rafts. Lipid-anchored streptavidin conjugates could be incorporated into Jurkat or other mammalian cell surfaces by inserting biotinylated phosphatidylethanolamine-polyethyleneglycols (PE-PEGs) and subsequently binding streptavidin to the cell-incorporated PE-PEGs. Saturated dipalmitoyl-PE-PEG-streptavidin conjugates prepared in this manner partitioned substantially into the detergent-insoluble membrane fraction isolated from Jurkat or fibroblast cells, whereas polyunsaturated dilinoleoyl-PE-PEG-anchored conjugates were wholly excluded from this fraction, consistent with the differences in the affinities of the two types of lipid anchors for liquidordered membrane domains. Remarkably, however, antibody-mediated cross-linking of either dipalmitoylor dilinoleoyl-PE-PEG-anchored streptavidin conjugates in Jurkat cells induced elevation of cytoplasmic calcium levels and tyrosine phosphorylation of the scaffolding protein linker of T-cell activation in a manner similar to that observed upon cross-linking of endogenous CD59 or ganglioside GM1. The amplitude of the cross-linking-stimulated elevation of cytoplasmic calcium moreover showed an essentially identical dependence on the level of incorporated streptavidin conjugate for either type of lipid anchor. Confocal fluorescence microscopy revealed that PE-PEG-streptavidin conjugates with saturated versus polyunsaturated anchors showed very similar surface distributions vis à vis GM1 or CD59 under conditions where one or both species were cross-linked. These results indicate that crosslinking of diverse proteins anchored only to the outer leaflet of the plasma membrane can induce activation of Jurkat T-cell-signaling responses, but they appear to contradict previous suggestions that this phenomenon rests specifically on the association of such species with lipid rafts.

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Section: Methodsmentioning

confidence: 99%

Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association

Wang¹,

Leventis

Silvius

2005

Journal of Biological Chemistry

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“…The amount of each of the major classes of PL was quantified by cutting out the individual spots and assaying for lipid phosphate using the method of Lowry and Tinsley [24] using adenosine monophosphate as standard. Following digestion with perchloric and nitric acid, the upper phase containing phosphomolybdate was read against fresh butyl acetate (Beckman DU Spectrophotometer, Fullerton, CA) at 310 nm.…”

Section: Methodsmentioning

confidence: 99%

Phospholipid composition of articular cartilage boundary lubricant

Sarma

Powell

LaBerge

2001

Journal Orthopaedic Research

151

143

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The mechanism of lubrication in normal human joints depends on loading and velocity conditions. Boundary lubrication, a mechanism in which layers of molecules separate opposing surfaces, occurs under severe loading. This study was aimed at characterizing the phospholipid composition of the adsorbed molecular layer on the surface of normal cartilage that performs as a boundary lubricant. The different types of phospholipid adsorbed onto the surface of cartilage were isolated by extraction and identified by chromatography on silica gel paper and mass spectroscopy. The main phospholipid classes identified were quantified by a phosphate assay. Gas chromatography and electrospray ionization mass spectrometry were used to further characterize the fatty acyl chains in each major phospholipid component and to identify the molecular species present. Phosphatidylcholine (41%), phosphatidylethanolamine (27%) and sphingomyelin (32%) were the major components of the lipid layer on the normal cartilage surface. For each lipid type, a mixture of fatty acids was detected, with a higher percentage of unsaturated species compared to saturated species. The most abundant fatty acid observed with all three lipid types was oleic acid (C18:l). Additional work to further quantify the molecular species using electrospray ionization mass spectrometry is recommended.

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“…Esterified and free cholesterol were assayed by the direct Lieberman-Burchard reaction (10). Phospholipid phosphorus was quantified by the procedure of Lowry et al (15).…”

Section: Chemical Analysis Of Pulmonary Lavage Samplesmentioning

confidence: 99%

The Chronically Reserpinized Rat as an Animal Model for Cystic Fibrosis: I. Acute Effect of Isoproterenol and Pilocarpine upon Pulmonary Lavage Fluid

et al. 1979

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SummaryLung lavage samples from rats treated in a chronic fashion with reserpine had mean increases of 133, 170, and 120% in the total protein, lipid, and carbohydrate contents, respectively, when compared with those of untreated control animals, when these values were expressed in terms of body weight. An acute single ip injection of the /I-adrenergic agent isoproterenol(10 mg) increased the glycoprotein content of pulmonary lavage fluid from control rats approximately 15-19%, but only 9-10% in those from reserpine tieated rats. By contrast, pilocarpine (10 mg), administered in the same manner, caused a 15% increase in the total protein, carbohydrate, and lipid content of lavage samples from control rats and increased these same constituents in the lavage samples of reserpine treated rats 98, 102, and 80%, respectively. The increased total carbohydrate content in the lavage samples of the treated animals was not associated with changes in the percent distribution of neutral sugars, amino sugars, or sialic acid. The. ratio of these various sugar components did not change in the lavage samples of control or reserpine treated rats upon stimulation with either pilocarpine or isoproterenol. The increased total lipid content fround in the lavage samples from the treated rats probably results from an increase in phospholipids. A decrease in phospholipid content qccurred in the lavage samples of reserpine treated rats upon stimulation, while the opposite was observed in those of control animals. Chronic treatment of rats with reserpine. thus, appears to induce an enhanced production of glycoproieins in the airwavs and to interfere with phospholi~id metabolism in m m -the lung. In addition, the drug treatment enhances the secretory response to pilocarpine in comparison with the responses of control animals. The enhanced response or hypersecretion of glycoproteins is a quantitative one and does not seem to involve alterations in the ratios or distribution of the various sugar components. This disturbance in the secretory function of the respiratory tract, a target organ which is prominently involved in cystic fibrosis (CF) together with alterations in other exocrine glands which resemble those of CF patients, makes the reserpine treated rat a useful model for the further study of possible pathogenetic mechanisms in CF.Speculation enhanced in the treated animals when compared to that of untreated control rats. It therefore appears that chronic reserpine administration causes a hyperse&tion of glycoproteins and changes in lipid metabolism in the respiratorv tract of the rat and -that, in conjunction with previous findings in other exocrine glands, these effects on lung function make the reserpine treated rat a useful tool for the study of the pathologic disturbance seen in the major exocrine glands affected in CF.An animal model for CF developed by the chronic administration of reserpine to rats shows not only morphologic and secretory changes in the salivary glands (16,17,18,19) and pancreas (20), but also alterations ...

show abstract

A simple, sensitive method for lipid phosphorus

Cited by 158 publications

References 3 publications

Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association

Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association

Phospholipid composition of articular cartilage boundary lubricant

The Chronically Reserpinized Rat as an Animal Model for Cystic Fibrosis: I. Acute Effect of Isoproterenol and Pilocarpine upon Pulmonary Lavage Fluid

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