Relief of temperature sensitivity in a concanavalin A resistant Chinese hamster ovary cell line auxotrophic for cholesterol.

The concanavalin A resistant, glycosylation-deficient, Chinese hamster ovary cell variant CR-7 is auxotrophic for cholesterol owing to an inability to adequately convert lanosterol to cholesterol. It is also temperature sensitive for growth, being unable to proliferate at 39 degrees C. Temperature sensitivity was relieved by addition of mevalonolactone, dolichol, or dolichyl-P to the growth medium, provided that cholesterol was also present in amounts sufficient to overcome cholesterol auxotrophy at 34 degrees C. Other metabolites of mevalonolactone (squalene, ubiquinone, lanosterol, and isopentenyladenine) were inactive in this regard. Measurement of dolichol levels in CR-7 and wild-type cells at 34 degrees C and after exposure to 39 degrees C showed that dolichol increased at 39 degrees C to an approximately equal extent in both cell types. Dolichol, dolichyl-P, ubiquinone, and isopentenyladenine had no effect on the sensitivity of either wild-type or CR-7 cells to the cytotoxic effects of concanavalin A. Mevalonolactone or lanosterol markedly increased the resistance of CR-7 to the lectin, but had no effect on wild-type cells. This raises the possibility that the presence of unusually large amounts of lanosterol, coupled with low amounts of cholesterol, in the membranes of CR-7 may be related to its concanavalin A resistance and other characteristic phenotypic abnormalities.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant Chinese hamster ovary cell line.

Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (CR-7), and revertant (RCR-7) cells. In contrast to WT and RCR-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of R-[2-14C]mevalonolactone revealed that CR-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but CR-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of 125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of 125I-LDL were also similar, suggesting that CR-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1-2 micrograms/ml) strongly and reversibly suppressed reductase activity in WT and RCR-7. CR-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of 3H-labeled mannose incorporation into cellular protein, but 3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.

Insensitivity of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity to low-density lipoprotein in concanavalin-A-resistant Chinese-hamster ovary cells.

A concanavalin-A-resistant mutant of Chinese-hamster ovary cells has been shown to have altered cholesterol content and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity compared to wild-type and spontaneous-revertant cell lines. These changes are associated with insensitivity of the mutant reductase activity to suppression by low-density lipoprotein and impaired receptor-mediated binding and uptake of 125I-labelled low-density lipoprotein.

The effects of experimental inflammation on turnover of fatty acid synthetase and levels of fatty acid synthetase messenger RNA in rat liver.

Suppression, by experimental inflammation, induced by subcutaneous injection of oil of turpentine, of the usual increase in liver fatty acid synthetase (FAS) activity resulting from fat-free feeding following starvation (adaptive synthesis) was shown to result entirely from lowered hepatic content of FAS protein. Comparison of changes in the relative rate of synthesis of FAS, determined radioimmunochemically during adaptive synthesis with and without inflammation, with concomitant changes in FAS activity, revealed that inflammation partically suppressed the increased rate of synthesis characteristic of adaptive synthesis, but insufficiently to account entirely for the suppression of enzyme activity. Inflammation accelerated the relative rate of degradation of FAS, causing a 50% decrease in enzyme half-life and a corresponding increase in kd and turnover index. Levels of translatable FAS mRNA rose only fivefold after 24 h of adaptive synthesis, while the relative rate of FAS synthesis increased 12-fold indicating the operation of both transcriptional and translational control. Inflammation, induced at the start of adaptive synthesis, caused a 65% lowering of the relative rate of FAS synthesis after 24 h and a 60% decrease in mRNA translatable as FAS, but was without effect on the total translational activity of the mRNA although alterations in the size distribution of RNA species in the mRNA fraction were noted.

Effects of oxygen tension on the lipid composition of Azotobacter chroococcum.

Lipid content and composition were determined in Azotobacter chroococcum grown, under nitrogen-fixing conditions, in continuous culture with intense aeration under atmospheres containing between 5 and 40% O2. Total lipid content remained almost constant at approximately 9% of dry weight. Phospholipid content was maximal at 20% O2 where it accounted for 92% of total lipid, and was minimal at 40% O2. Phosphatidylethanolamine was the only species of phosphatide detected in this fraction. Neutral lipid content was minimal at 20% O2 and maximal at 40% O2 where it represented approximately 30% of the total lipid. Glycolipid remained between 2 and 13% of total lipid throughout. The principal fatty acids of A. chroococcum were hexadecanoic (C16:0), hexadecenoic (C16:1), and octadecenoic acid (C18:1) at all O2 tensions, but C18:1 increased at the expense of C16:1 at higher O2 tensions, particularly in free fatty acid and phospholipid fractions. [U-14C]acetate was readily incorporated into lipid at both 20 and 40% O2, but total incorporation was much greater at 20% O2.

Lipid metabolism in Achlya: studies of lipid turnover during development.

The life cycle of Achlya involves germination of spores to form coenocytic somatic hyphae, followed by differentiation of hyphal tips into sporangia. From germination to release of new spores occupies 27-30 h. Total lipid made up 10% of dry weight in ungerminated spores. After germination, total lipid fell to 6% of dry weight in 15 h, then rose to 7.7% at the time of sporangium formation. Half of the initial loss of lipid took place within 2 h of germination. The ability of Achlya to incorporate [1-14C]acetate into lipid was maximal at the time of sporangium formation, and glycerides were the principal component of total lipid to become 14C-labelled at all stages of the life cycle. Fatty acid synthetase activity measured in cell-free extracts was low in spores and in actively elongating mycelium, but increased during idfferentiation to a level 15-fold greater than that in spores. Fatty acid oxidation, as estimated by the release of 14CO2 from 1-14C-labelled fatty acids, was also maximal at the time of sporangium formation.

Lipid metabolism in Achlya: changes in lipid composition during development.

Fractionation of total lipid extracted from Achlya sp. at various stages of its developmental cycle revealed that in spores total lipid was composed of 62% neutral lipid, 13% phospholipid, and 25% glycolipid. After germination, the proportion of neutral lipid rose slightly after 2 h then fell sharply to 10% after 8 h, whereupon it rose to 55% of total lipid after 30 h of growth, when sporulation was completed. Conversely, phospholipid rose to 77% of total lipid after 8 h, then declined to 40% after 30 h. Glycolipid was maintained at 10-20% of total lipid throughout the life cycle after spore germination. Quantitative determination of neutral lipid components by photo-reflectometry showed that triglycerides accounted for 20% of neutral lipid in spores, and free fatty acids made up 50%. During growth, the absolute levels of both components fell precipitously on germination, remained at low levels throughout vegetative growth, and rose at the time of sporulation. The fatty acid composition of total lipid, phospholipid, neutral lipid, and free fatty acid fractions extracted from vegetative and sporulating Achlya cells was determined. The principal fatty acids present in all fractions of both stages of the life cycle were hexadecanoic and octadecanoic acids. Hydroxyhexadecanoic acid, eicosatetraenoic acid, and an unidentified long-chain acid were completely absent from the phospholipids of vegetative cells, although they were found in significant quantities in lipid fractions from other stages of growth.