Preparation of delipidized serum protein for use in cell culture systems

Rothblat, George H.; Arbogast, Loretta Y.; Ouellette, Lucille A.; Howard, Barbara V.

doi:10.1007/bf02797438

Cited by 194 publications

(55 citation statements)

References 15 publications

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“…The cells were maintained at 37 Њ C in a 5% CO 2 incubator during all experiments, in 2.5 ml DMEM/F12 with 5% delipidated FCS, with media changed every 24 h. Delipidated FCS was prepared according to the method of Rothblat et al (28), and the absence of n-6 and n-3 fatty acid checked by gas liquid chromatography (GLC) (21,29). Fatty acids, 0-50 m, as their sodium salts, were complexed with fatty acid free bovine serum albumen at a molar ratio of 2:1.…”

Section: Methodsmentioning

confidence: 99%

Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic acid

Innis

Dyer

2002

Journal of Lipid Research

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The phospholipids, particularly phosphatidylethanolamine, of brain gray matter are enriched with docosahexaenoic acid (22:6n-3). The importance of uptake of preformed 22:6n-3 from plasma compared with synthesis from the ␣ -linolenic acid (18:3n-3) precursor in brain is not known. Deficiency of 18:3n-3 results in a compensatory increase in the n-6 docosapentaenoic acid (22:5n-6) in brain, which could be formed from the precursor linoleic acid (18:2n-6) in liver or brain. We studied n-3 and n-6 fatty acid incorporation in brain astrocytes cultured in chemically defined medium using delipidated serum supplemented with specific fatty acids. High performance liquid chromatography with evaporative light scattering detection and gas liquid chromatography were used to separate and quantify cell and media lipids and fatty acids. Although astrocytes are able to form 22:6n-3, incubation with 18:3n-3 or eicosapentaenoic acid (20:5n-3) resulted in a time and concentration dependent accumulation of 22:5n-3 and decrease in 22:6n-3 g/g cell fatty acids. Astrocytes cultured with 18:2n-6 failed to accumulate 22:5n-6. Astrocytes secreted cholesterol esters (CE) and phosphatidylethanolamine containing saturated and monounsaturated fatty acids, and arachidonic acid (20:4n-6) and 22:6n-3. These studies suggest conversion of 22:5n-3 limits 22:6n-3 synthesis, and show astrocytes release fatty acids in CE. Brain gray matter is highly enriched in the long chain polyunsaturated fatty acids docosahexaenoic acid (22:6n-3) and arachidonic acid (20:4n-6) (1). Docosahexaenoic acid is formed from 18:3n-3 by ⌬ 6 and ⌬ 5 desaturation and elongation which gives rise to 20:5n-3 and 22:5n-3 in endoplasmic reticulum; 22:5n-3 is then elongated to 24:5n-3 and undergoes a second ⌬ 6 desaturation to 24:6n-3 and is translocated to the peroxisomes where it undergoes one cycle of ␤ -oxidation to form 22:6n-3 ( Fig.

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Section: Methodsmentioning

confidence: 99%

Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic acid

Innis

Dyer

2002

Journal of Lipid Research

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“…Lipid-poor serum (LPS) was prepared by solvent extraction as described previously by Rothblat et al (14). Compactin was added to cells grown in MEM, 5% LPS to increase expression of HMG-CoA reductase.…”

Section: Methodsmentioning

confidence: 99%

Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Endoplasmic Reticulum Membranes Is Accelerated as a Result of Increased Susceptibility to Proteolysis

McGee

Cheng

Kumagai

et al. 1996

Journal of Biological Chemistry

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The endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to regulated degradation when cells are presented with an excess of sterols or mevalonate. In this report, we demonstrate the degradation of HMGCoA reductase in ER membranes prepared from cells which have been pretreated with mevalonate or sterols prior to membrane purification. Degradation of HMGCoA reductase in membranes prepared from pretreated cells is more rapid than in membranes prepared from cells which have received no regulatory molecules. In vitro degradation is blocked by protease inhibitors previously shown to inhibit reductase degradation in vivo and is specific for intact HMG-CoA reductase. The lumenal contents of the ER membranes are dispensible for the regulated proteolysis and the proteases responsible for reductase degradation are stably associated with the ER membrane. Regulated proteolysis of HMG-CoA reductase is inhibited by lactacystin, a newly defined inhibitor of the multicatalytic protease, the proteasome, and in vitro degradation of reductase correlates with the presence of proteasome subunits in purified ER membranes. The ubiquitin system for protein degradation, which has recently been shown to be required for the degradation of several ER membrane proteins, is not required for the degradation of HMG-CoA reductase. Finally, we conclude that the regulated proteolysis of HMG-CoA reductase in response to regulatory molecules such as mevalonate or sterols is mediated by increased susceptibility of the reductase to ER proteases, rather than the induction of a new proteolytic activity.

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“…Since DMSO is a potent inducer of murine erythroleukemia (MEL) cells (6, 7), a number of other MEL cell inducers (10,16) (24), and OlAl cells were tested for their response to DMSO in medium supplemented only with this delipidized serum. According to Fuchs and Green (8), this procedure removes all detectable vitamin A from the medium, so it should eliminate the DMSO response completely if the above hypothesis is correct.…”

Section: Methodsmentioning

confidence: 99%

Induced muscle differentiation in an embryonal carcinoma cell line.

Edwards¹,

Harris²,

McBurney³

1983

Mol. Cell. Biol.

174

107

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Cells of the teratocarcinoma-derived line P19S1801A1 (01A1) are pluripotent embryonal carcinoma cells and can be induced to differentiate when aggregated and exposed to dimethyl sulfoxide. Many nonneural cell types appear in dimethyl sulfoxide-treated cultures, cardiac and skeletal muscle being the most easily identified. We have used immunofluorescence procedures with monoclonal antibodies directed against muscle myosin to confirm and quantitate the number of muscle cells formed. A monoclonal antibody reactive with an embryonal carcinoma-specific surface antigen was used to confirm the disappearance of undifferentiated cells after dimethyl sulfoxide treatment. Cardiac muscle cells developed within 4 to 5 days of drug exposure, but skeletal muscle cells did not become evident until 7 to 8 days. We have isolated a mutant cell line (D3) which appears to be incapable of muscle development but which does form neurons and glial cells when exposed to high retinoic acid concentrations. We propose that this system will be useful for investigation of the means by which pluripotent cells become committed to development along the striated muscle lineages.

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Preparation of delipidized serum protein for use in cell culture systems

Cited by 194 publications

References 15 publications

Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic acid

Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic acid

Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Endoplasmic Reticulum Membranes Is Accelerated as a Result of Increased Susceptibility to Proteolysis

Induced muscle differentiation in an embryonal carcinoma cell line.

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