David H. Beach

Dehydrodinosterol, dinosterone and related sterols of a non-photosynthetic dinoflagellate, Crypthecodinium cohnii

Abstract The heterotrophic dinoflagellate Crypthecodinium cohnii contained the 4α-methyl sterols, dinosterol, dehydrodinosterol (4α,23,24-trimethylcholesta-5,22-dien-3β-ol) and the tentatively identified 4α,24-dimethyl-cholestan-3β-ol and 4α,24-dimethylcholest-5-en-3β-ol. The major 4-demethyl sterol was cholesta-5,7-dien-3β-ol which was accompanied by a smaller amount of cholesterol and traces of several other C 27 ,C 28 and C 29 sterols. In addition, a 3-oxo-steroid fraction was isolated and the major component identified as dinosterone (4α,23,24-trimethylcholest-22-en-3-one). The possible biosynthetic relationships of these compounds are discussed.

Sterols of Leishmania species, implications for biosynthesis

The major sterol of promastigotes of stocks of Leishmania tropica, L. donovani and 3 subspecies of L. mexicana has been identified as ergosta-5,7,24(28)-trien-3 beta-ol; and of an L. major stock as ergosta-7,24(28)-dien-3 beta-ol. 24-Methylcholesta-5,7,22-trien-3 beta-ol and 24-ethylcholesta-5,7,22-trien-3 beta-ol were minor constituents, and traces of ergosta-5,7,22,24(28)-tetraen-3 beta-ol and a C27-diene were also recognized in some species. Lanosterol and 4,4-dimethylcholesta-8,24-dien-3 beta-ol were detected in all species studied, and squalene was identified in a stock of L. tropica. The sterol composition of members of the genus Leishmania and the sterol biosynthetic pathways it implies are characteristic of yeast and other fungi.

Effects of ketoconazole on growth and sterol biosynthesis of Leishmania mexicana promastigotes in culture

Ketoconazole, a clinically effective antimycotic agent active in vitro against the amastigote stage of Leishmania mexicana Walter Reed 227 in human monocyte-derived macrophages, was found to inhibit growth and impair sterol biosynthesis of the cultured promastigote stage by approx. 50% at a concentration of approx. 10(-8)M. Sterol biosynthesis was interfered with at the level of the removal of the 14 alpha-methyl group of lanosterol, as judged by changes in the distribution of [2-14C]mevalonate radioactivity among desmethyl sterol and methyl sterol thin-layer chromatography fractions, by the loss of 4-desmethyl sterols (mainly 5-dehydroepisterol), and by the accumulation of 14 alpha-methyl sterols. The growth inhibition and sterol changes were evident in promastigotes cultured in a cholesterol-rich medium and in a cholesterol-poor medium, even though promastigotes incorporated cholesterol. The mechanism of action of ketoconazole against promastigotes may be that postulated for Candida albicans: interference with membrane permeability secondary to loss of desmethyl sterols and accumulation of 14 alpha-methyl sterols.

Sterols of ketoconazole-inhibited Leishmania mexicana mexicana promastigotes.

Leishmania mexicana mexicana promastigotes grown with cholesterol, supplied in natural products as the free sterol and as cholesteryl esters, were exposed to [2-14C]mevalonate and to the antimycotic drug ketoconazole. Growth was inhibited and cholesterol and 14 alpha-methyl sterols accumulated in free and esterified forms (cholesterol much greater than 4 alpha,14 alpha-dimethylcholesta-8,24-dien-3 beta-ol much greater than 14 alpha-methylcholesta-8,24-dien-3 beta-ol congruent to 14 alpha-methylergosta-8,24(28)-dien-3 beta-ol much greater than 4 alpha,14 alpha-dimethylergosta-8,24(28)-dien-3 beta-ol; identified by capillary gas chromatography/mass spectrometry, and by 1H and 13C nuclear magnetic resonance spectrometry). The 14 alpha-methyl sterols were preferentially labelled with 14C. The cholesterol was unlabelled and substituted for a substantial fraction of the major product of sterol biosynthesis, ergosta-5,7, 24(28)-trien-3 beta-ol (5-dehydroepisterol), but did not replace it and did not offer remarkable protection against either growth inhibition or alteration of sterol biosynthesis. Promastigotes grown with [6-2H]cholesterol or [4-14C]cholesterol did not contain labelled forms of Leishmania sterols, or other sterols. The chromatographic and spectrometric sterol analyses and the isotopic tracer findings suggested that ketoconazole impaired the cytochrome P-450 dependent 14 alpha-demethylation of lanosterol, that cholesterol was neither biosynthesized nor metabolized, and that the physiological functions of 5-dehydroepisterol had sterol structural requirements not entirely met by cholesterol. In all these studies, L. mexicana mexicana demonstrated a sterol biochemistry remarkably similar to that of fungi. This recommends an increase in interest in antimycotic drugs as chemotherapeutic agents for leishmanial infections.

Evidence for the Presence of “Metabolic Sterols” in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols

Mixed life cycle stages of rat‐derived Pneumocystis carinii were isolated from host lungs and their sterols were compared with those present in lungs from normal and immunosuppressed uninfected rats. Gas‐liquid chromatography consistently detected, resolved, and quantified 9, 10, and 20 sterol components in the total nonsaponifiable neutral lipid fraction of lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii preparations, respectively. In all samples, cholesterol was the most abundant sterol present, comprising 97%, 93%, and 78% of total sterols in lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii, respectively. Tentative identifications of several rat lung and P. carinii minor sterols were made based on gas‐liquid chromatogram retention times and fragmentation patterns from mass spectral analyses. Campesterol (ergost‐5‐en‐3‐ol), cholest‐5‐en‐3‐one, and β‐sitosterol (stigmast‐5‐en‐3‐ol) were among the minor components present in both types of lung controls, and were also components of P. carinii sterols. In contrast to lung controls, the sterols of P. carinii were enriched in C28 and C29 sterols with one or two double bonds, and a hydroxyl group at C‐3 (ergost‐5‐en‐3‐ol, ergost‐7‐en‐3‐ol, ergosta‐dien‐3‐ol, stigmast‐5‐en‐3‐ol, stigmast‐7‐en‐3‐ol and stigmasta‐dien‐3‐ol). Steryl esters of P. carinii, probably stored in cytoplasmic lipid droplets, were dominated by those present in the host lung. In separate studies. 3‐hydroxy‐3‐methylglutaryl coenzyme A activity, a key enzyme in the regulation of sterol biosynthesis, was detected in purified P. carinii preparations and incorporation of radiolabeled squalene and mevalonate was observed. Together, these results suggest that the parasite readily takes up and incorporates host sterols, and that the organism synthesizes some of its own “metabolic sterols”

The cyclopropane fatty acid of trypanosomatids

Cis-9,10-Methyleneoctadecanoic acid, one of a group of cyclopropane fatty acids commonly found in bacteria but not in eukaryotic cells, has been identified in the phosphatidylethanolamines of 27 isolates representing 5 genera of trypanosomatid flagellates (Crithidia, Leptomonas, Herpetomonas, Phytomonas, Leishmania). Its presence did not appear to be associated with endosymbiotic or other microbiol associates. It was absent from 12 isolates of the genera Blastocrithidia, Endotrypanum and Trypanosoma.

Some Phytomonas and Herpetomonas species form unique iso-branched polyunsaturated fatty acids.

Four trypanosomatid flagellates of the genera Phytomonas and Herpetomonas have been found to carry out the de novo biosynthesis of a variety of iso-branched, C18, C20 and C22, polyunsaturated fatty acids, with 2-5 methylene-interrupted double bonds, which have not been described heretofore from natural materials; iso-C18 delta 6,9, iso-C18 delta 9,12, iso-C20 delta 8,11,14, iso-C 20 delta 5,8,11,14, iso-C22 delta 4,7,10,13,16. Identifications were based upon combinations of chromatographic, chemical degradative, mass spectrometric and proton nuclear magnetic resonance spectrometric techniques. Under appropriate culture conditions, 85% of the total fatty acids of the organisms were branched. The subject trypanosomatids are recommended as model organisms with which to investigate influences of the physical properties of phospholipid fatty acyl groups on eukaryotic cell membrane functions.

Benzoquinones in stages of the life-cycle of the cestode Spirometra mansonoides.

Ubiquinone-10 and rhodoquinone-10 were detected in stages of the life-cycle of a pseudophyllidean cestode, Spirometra mansonoides, by chromatographic, UV spectrophotometric, proton nuclear magnetic resonance spectrometric and electron impact mass spectrometric methods. Ubiquinone-10 was identified in 1-day-old eggs and coracidia, and rhodoquinone-10 in coracidia, plerocercoids and adult tapeworms. Tentative identification were also made of ubiquinone-10 in procercoids and rhodoquinone-10 in 10-day-old eggs. The roles of benzoquinones in helminth aerobic and anaerobic metabolism are discussed in relation to their distribution in stages of the S. mansonoides life-cycle.

Biosynthesis of the novel fatty acid, 17-methyl-cis-9,10-methyleneoctadecanoic acid, by the parasitic protozoan, Herpetomonas megaseliae

Herpetomonas megaseliae, a flagellate protozoan parasite of the gut of a dipteran,Megaselia scalaris, is shown by chromatographic, spectrometric and radiotracer methods to synthesize de novo aniso-branched chain cyclopropane fatty acid, 17-methyl-cis-9,10-methyleneoctadecanoic acid.