Josephine R. Landrey

Desmosterol as the major sterol in L-cell mouse fibroblasts grown in sterol-free culture medium.

The principal sterol synthesized by L-cell mouse fibroblasts is desmosterol. Cholesterol was not detected in these cells when they were grown in a sterol-free culture medium. These findings indicate that, in cells, cholesterol can be replaced by desmosterol. Sterol analyses of six other tissue culture cell lines revealed cholesterol synthesis.

Ergosterol replacement of tetrahymanol in Tetrahymena membranes

Abstract The naturally occurring pentacyclic triterpene alcohol of Tetrahymena pyriformis , tetrahymanol, is replaced by ergosterol as a result of supplementation of the growth medium with that sterol. Since tetrahymanol is not metabolized by the ciliate, loss of tetrahymanol from sterol-supplemented cells appears to occur through dilution during growth. Ergosterol-grown cells are at least 20 times more sensitive to polyene antibiotics than normal cells. The substitution of ergosterol for tetrahymanol observed in whole cells is also reflected in isolated cilia and ciliary membrane preparations. In these membranes, the phospholipid/tetrahymanol ratio in normal cells is similar to the phospholipid/ergosterol ration in ergosterol-grown cells.

The configuration of Δ5,7,22-sterols in a tracheophyte

Abstract The epimer of ergosterol at C-24 was formed by the metabolism of 24α-methyl-cholesterol in the protozoan, Tetrahymena pyriformis . This is the first time 24-epiergosterol has been obtained in any manner. Its n.m.r. spectrum at 220 MHz was distinguishable from that of ergosterol. From the primitive tracheophyte, Lycopodium complanatum L., a 24-methyl-Δ5,7,22-sterol was isolated which proved to be ergosterol possibly containing its C-24 epimer as a minor constituent.

Dealkylation of 24-ethylsterols byTetrahymena pyriformis

WhenTetrahymena pyriformis was incubated with sitosterol ([24R]-24-ethylcholest-5-en-3β-ol]) or itstrans-Δ22-derivative (stigmasterol), the C-24-dealkylated product, cholesta-5,7,trans-22-trien-3β-ol, was obtained in both cases. 24(S)-24-Ethylcholesta-5,7,trans-22-trien-3β-ol also was found to be a metabolite. When sitosterol was the substrate, 24(R)-24-ethylcholesta-5,7-dien-3β-ol was obtained as a third product. Identifications were made by mass spectroscopy, quantitative chromatography, labeling with14C, and by other means. The dealkylated product (cholestratrienol) represented 30% of the sterols isolable after incubation. The administration of sterols to this organism did not induce sterol biosynthesis, since 2-14C-mevalonate failed to yield radioactive sterol in the presence of added stigmasterol.

The steric requirements for sterol inhibition of tetrahymanol biosynthesis

Many naturally occurring sterols are accumulated and metabolized byTetrahymena pyriformis. In most cases, the sterols are desaturated to giveΔ5,7,22-derivatives. Compounds with an ethyl, but not with a methyl, substituent at C-24 are dealkylated. Exposure of the ciliates to the appropriate sterol sharply curtails the synthesis of the native pentacyclic triterpenoid alcohols, tetrahymanol and diplopterol. An analysis with modified sterols has revealed several additional features that are required for desaturation at C-7,8 and C-22,23 and for inhibition of tetrahymanol biosynthesis. The presence of atrans-17(20)-double bond, which eliminates free rotation at C-20 and fixes C-22 to the right of the nucleus, does not interfere with desaturation, while thecis- or left-handed isomer is not metabolized. Thecis-Δ17(20)-isomer is, however, an effective inhibitor of tetrahymanol biosynthesis, although less so that thetrans-counterpart. When a methyl or hydroxyl group at C-20 protrudes to the front of the molecule in the right-handed conformation, metabolism is reduced or abolished. Shortening (by one C-atom) or lengthening of the sterol side chain has little effect on the ability of the compounds to inhibit tetrahymanol biosynthesis or to support growth, as long as the overall length of the side chain does not exceed seven carbons from C-20. The presence of a 7α-, 7β-, 20α-, 20β-, or a 25-hydroxy group in the cholesterol molecule sharply inhibits desaturation and curtails the effectiveness of the compound as an inhibitor of tetrahymanol biosynthesis. The 7- or 22-keto derivatives seem to act in a fashion similar to the hydroxy derivatives, but these compounds show greater inhibition of growth. 20-Methylcholesterol, however, is a potent inhibitor of synthesis, which suggests that the polarity of the substituent of C-20 is more important than bulk. Many sterols can effectively replace tetrahymanol in the membranes of these ciliates. However, several of the compounds, which inhibit synthesis, appear to be physiologically inappropriate, and poor growth results. An example of the latter class is 20-methylcholesterol. Finally, a class of sterols, represented by 20α-hydroxycholesterol and 7-ketocholesterol, does not severly inhibit tetrahymanol synthesis but leads to growth inhibition and surface abnormalities. These sterols apparently lead to a disordered membrane, even in the presence of tetrahymanol.

Isovaleric acid as a precursor of odd numbered iso fatty acids in Tetrahymena

Tris isovalerate-supplementedTetrahymena pyriformis W showed no qualitative change in fatty acid composition; however, an increase in polar lipids that contain odd numbered iso acids (C13, C15, C17, C19) occurred. This change was accompanied by a decrease in the proportional amount of even numbered normal acids (C14, C16, C18). The neutral and polar lipids from cells incubated with [1-14C] isovaleric acid were found to contain radioactivity. The methyl esters of the saturated fatty acids obtained from the polar lipids by alkaline methanolysis were separated by reversed phase chromatography, the identities confirmed by gas chromatography-mass spectrometry, and the specific activities determined. Iso acids were found to be the most heavily labeled materials. In addition to ceramide, two sphingolipid components were detected. One yielded saturated fatty acids after acidic methanolysis, while the other contained >93% α-hydroxy fatty acids. Radioactivity was noted in the long chain base fraction derived from the sphingolipids. Progressive growth inhibition occurred as the isovalerate concentration was increased in the culture medium; however, the ciliates were morphologically indistinguishable from unsupplemented cells.