Manipulation of the Phospholipid Composition of Tissue Culture Cells

Gläser, Michael; Ferguson, Karen A.; Vagelos, P. Roy

doi:10.1073/pnas.71.10.4072

Cited by 98 publications

(50 citation statements)

References 23 publications

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“…Additional phosphatidylcholine mutants might be found by screening 5-10 times the number of colonies (i.e., 100,000 colonies or approximately 500 plates). A specific selection for phosphatidylcholine mutants would be invaluable, but unfortunately our results demonstrate that DNA and protein synthesis continued almost to the point of cell lysis (30)(31)(32)(33)(34)(35)(36) hr. Table 1).…”

Section: Discussionmentioning

confidence: 94%

“…These strains are defective either in CDP-diglyceridedependent phosphatidylserine sypthesis (26) or in phosphatidylethanolamine methylation (27,28), but neither of these pa.thways apparently plays a significant role in CHO cells. In this regard, most tnammalian lines, including CHO cells, require choline for growth (29,30 (Tables 1 and 2), the synthesis of other phospholipids continued for about 20 hr. Because sphingomyelin was also formed under conditions of CDP-choline depletion, the phosphorylcholine moiety of sphingomyelin may not be derived primarily from CDP-choline (31 …”

Section: Discussionmentioning

confidence: 99%

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Autoradiographic detection of animal cell membrane mutants altered in phosphatidylcholine synthesis.

Esko¹,

Raetz²

1980

Proc. Natl. Acad. Sci. U.S.A.

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We have screened approximately 20,000 colonies of Chinese hamster ovary cells immobilized on filter paper [Esko, J. D. & Raetz, C. R. H. (1978) Proc NatL Acad. Sci. USA 75, 1190USA 75, -1193 for strains unable to incorporate [methl-"'CJcholine into trichloroacetic acid-precipitable phospholipid at 40'C. Mutant 58, identified in this way, was specifically defective in choline incorporation, and other isolates were also blocked in thymidine and leucine incorporation into DNA and protein, respectively. Further analysis of mutant 58 revealed that the strain grew almost normally at 330C, the permissive temperature, but divided only once at 40'C, the restrictive temperature. After a 20-hr incubation at 400C, the phosphatidylcholine level dropped from 41% to 20% in the mutant whereas other phospholipids, including sphingomyelin, continued to accumulate. Wild-type cells contained approximately 50% phosphatidyicholine at both temperatures. Anion-exchange chromatography of the water-soluble choline metabolites extracted from mutant 58 revealed that phosphorylcholine accumulation increased from 6 nmol/mg of protein at 330C to 42 nmol/mg of protein at 40'C whereas CDP-choline decreased from 0.42 nmol to less than 0.07 nmol per mg of protein. Phosphorylcholine also increased in wild-type cells shifed from 330C to 40°C (from 1.8 nmol to 16 nmol per mg of protein), but the level of CDP-choline was not altered (from 0.52 nmol to 0.58 nmol per mg of protein). Enzymatic assays of extracts prepared from mutant and wild-type cells revealed a reduction of CTP: phosphorylcholine cytidylyltransferase (EC 2.7.7.15) activity (CDP-choline synthetase) in the mutant to 1/40th that in the wild type, and mixing experiments excluded the production of antagonists to CDP-choline synthesis in the mutant. Thus, the inability of the mutant to generate normal amounts of phosphatidylcholine in vivo was correlated with an enzymatic lesion in the biosynthesis of CDP-choline in vitro.Phosphatidylcholine is a major structural component of most cells possessing internal membranes (1, 2), representing about half of the membrane phospholipid. Its synthesis appears to be localized on the cytoplasmic face of the endoplasmic reticulum (3, 4), but little is known about the biochemical and genetic factors that control the relative amount and distribution of this lipid. Most subcellular membranes contain different amounts of phosphatidylcholine (1) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Autoradiographic detection of animal cell membrane mutants altered in phosphatidylcholine synthesis.

Esko¹,

Raetz²

1980

Proc. Natl. Acad. Sci. U.S.A.

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“…The pattern of incorporation of labeled acetate into the nonsaponifiable fraction was different from that observed for the fatty acid fraction of the total lipid extract from potato disks. When DME was added at zero time and the slices were pulsed at 2-hr intervals during aging, label incorporation was consistently inhibited from 30 to 40% compared with that of the control (Table VIII), similar to the case in mammalian cells (7).…”

Section: Resultsmentioning

confidence: 70%

“…Cerulenin (2S) (3R) 2,3 expoxy-4-oxo-7,10 dodecadienoylamide, an antibiotic produced by the fungus Cephalosporium coerulens, specifically inhibits fatty acid synthetase (1,3). Dimethylaminoethanol serves as an analogue of choline and in that role is incorporated into an analogue of lecithin (7,20). In what follows, it is shown that both of these agents inhibit the development of wound-induced respiration in a time-restricted manner, and it is deduced accordingly that phospholipid, and by extension membrane, biosynthesis are central elements in respiratory development in aging slices.…”

mentioning

confidence: 97%

Inhibition of the Development of Induced Respiration and Cyanide-insensitive Respiration in Potato Tuber Slices by Cerulenin and Dimethylaminoethanol

Waring¹,

Laties²

1977

Plant Physiol.

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The interdependence of the development of wound-induced respiration and membrane-related phospholipid biosynthesis in potato tuber (Solanum tuberosum var. Russet) slices was establisbed by the ue of agents which selectively affect lipid and pbospbolipid synthesis. Cerulenin, a specific inbibitor of de novo fatty acid synthesis, inhibited the ultimate development of wound-induced respiration and of cyanide resistance only when given in the critical first 10 to 12 hours of dice ag. Similarly, wben slices were exposed to the choline analogue dimethylaminoethanol within the first 10 hours, the phospholipid composition of the membrane lipids was drasticafly altered, the wound-induced respiration in a 24-hr period was substantialy curtaied, and the development of cyanide insensitivity was sharply inhibited. These observations indicate that time-restricted membrane-related phospholipid synthesis is prerequisite to the development of wound-induced respiration and concurrent cyanide insensitivity.Fresh slices of a variety of bulky storage organs undergo a time-dependent respiratory rise-the wound-induced, or induced, respiration. The development of induced respiration through a 24-hr period is suppressed by actinomycin, an inhibitor of DNA-directed RNA synthesis, in a time-restricted manner. That is, actinomycin is inhibitory only when given in the first 8 to 10 hr of the 24-hr period following cutting. In the preceding paper (21), it was demonstrated that the time-restricted characteristic of actinomycin action applied also to the effect of actinomycin on choline incorporation into phospholipid, and selectively to the proliferation of three enzymes of lecithin biosynthesis, viz. phosphorylcholine-cytidyl transferase, phosphorylcholine-glyceride transferase, and phosphatidylphosphatase. The view was developed that membrane-related phospholipid synthesis is central to the development of induced respiration, and that the respiratory changes are a consequence of membrane biosynthesis and membrane intussusception into existing mitochondria.In this paper, we examine the effect of two specific and sensitive inhibitors of normal lipid biosynthesis within the framework of the time-restricted concept (see ref. 21). The intent is to distinguish between events which are prerequisite to woundinduced respiration and those which are merely coincident with it. The rationale is that metabolic elements which are inhibited in a time-restricted way -as is respiration by actinomycin -are

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“…For example, its fragility, antioxidative activity, and activities of receptors and enzymes in the membrane will be affected, resulting in cessation of cell growth followed by death [14].…”

Section: Resultsmentioning

confidence: 99%

Metabolic Disorder of Phospholipid and Membrane Damage in Keshan Disease Model

Guan

Zou

et al. 1986

J. Clin. Biochem. Nutr.

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SummaryYoung guinea pigs were fed a semipurified diet low in selenium (Se) and phospholipid. The blood Se and tissue glutathione peroxidase activity were lowered, electrocardiogram and ultrastructure of the myocardium appeared abnormal, and tissue chromolipids were elevated in guinea pigs fed with this basal diet. Diminished changes, however, occurred in groups receiving supplemental Se. Supplementation of Se resulted in maintenance of the normal level of total phospholipid and the ratio between different phospholipids in the cartilage (Acta Pharmacol. Sinica, 5 (1984) 264-267), but in the heart the ratio was improved only when adequate phospholipid (especially endogenous phospholipid) was given simultaneously. Activity of the membrane binding enzyme was slightly restored by Se supplementation. Only when both phospholipid and Se were given sufficiently did the cytochrome oxidase activity in myocardium and CAMP level in plasma increase markedly, even more than that of the control group.

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Manipulation of the Phospholipid Composition of Tissue Culture Cells

Abstract: ABSTRACT

Cited by 98 publications

References 23 publications

Autoradiographic detection of animal cell membrane mutants altered in phosphatidylcholine synthesis.

Autoradiographic detection of animal cell membrane mutants altered in phosphatidylcholine synthesis.

Inhibition of the Development of Induced Respiration and Cyanide-insensitive Respiration in Potato Tuber Slices by Cerulenin and Dimethylaminoethanol

Metabolic Disorder of Phospholipid and Membrane Damage in Keshan Disease Model

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