Chitwood DJ et al. (1991) Lipids "Metabolism of plant sterols by nematodes."

Lusby WR, Chitwood DJ

[Lipids, 1991]

Parasitic nematodes do not biosynthesize sterols de novo and therefore possess a nutritional requirement for sterol, which must be obtained from their hosts. Consequently, the metabolism of phytosterols by plant-parasitic nematodes is an important process with potential for selective exploitation. The sterol compositions of several species of plant-parasitic nematodes were determined by capillary gas chromatography-mass spectrometry and compared with the sterol compositions of their hosts. Saturation of the phytosterol nucleus was the major metabolic transformation performed by the root-knot nematodes Meloidogyne arenaria and M. incognita and the corn root lesion nematode, Pratylenchus agilis. In addition to saturation, the corn cyst nematode, Heterodera zeae, dealkylated its host sterols at C-24. Because free-living nematodes can be cultured in sterol-defined artificial medium, they have been successfully used as model organisms for investigation of sterol metabolism in plant-parasitic nematodes. Major pathways of phytosterol metabolism in Caenorhabditis elegans, Turbatrix aceti and Panagrellus redivivus included C-24 dealkylation and 4 alpha-methylation (a pathway unique to nematodes). C. elegans and T. aceti introduced double bonds at C-7, and T. aceti and P. redivivus saturated the sterol nucleus similarly to the plant-parasitic species examined. Several azasteroids and long-chain dimethylalkylamines inhibited growth and development of C. elegans and also the delta 24-sterol reductase enzyme system involved in the nematode C-24 dealkylation pathway.

Chitwood DJ et al. (1986) J Nematol "Recent developments in nematode steroid biochemistry."

Chitwood DJ, Lozano R, Lusby WR

[J Nematol, 1986]

Current knowledge of steroid nutrition, metabolism, and function in free-living, plant-parasitic and animal-parasitic nematodes is reviewed, with emphasis upon recent investigation of Caenorhabditis elegans. A number of 4-desmethylsterols with a trans-A/B ring configuration can satisfy the steroid nutritional requirement in C. elegans, but sterols with a cis-A/B ring configuration or trans-A/B sterols with a 4-methyl group cannot. C. elegans removes methyl or ethyl substituents at C-24 of the plant sterols sitosterol, campesterol, stigmasterol, stigmastanol, and 24-methylene-cholesterol to produce various sterols with structures partially dependent upon that of the dietary sterol. Additional metabolic steps in C. elegans include reduction of Delta(2)(2)- and Delta-bonds, C-7 dehydrogenation, isomerization of a Delta-bond to a Delta(1)-bond, and 4alpha-methylation. An azasteroid and several long-chain alkyl amines interfere with the dealkylation pathway in C. elegans by inhibiting the Delta(2)-sterol reductase; these compounds also inhibit growth and reproduction in various plant-parasitic and animal-parasitic nematodes. A possible hormonal role for various steroids identified in nematodes is discussed.

Chitwood DJ et al. (1987) Comparative Biochemistry and Physiology "Sterol metabolism in the nematodes Panagrellus redivivus, Turbatrix aceti and C. elegans."

Salt TA, Lusby WR, Chitwood DJ

[Comparative Biochemistry and Physiology, 1987]

1. Panagrellus redivivus, Turbatrix aceti and Caenorhabditis elegans were sterilely propagated in semidefined media containing sitosterol or cholesterol, and sterols were isolated and identifed by capillary gas-liquid chromatography-mass spectometry. 2. Each species was capable of removal of the C-24 ethyl substituent of sitosterol and production of 4a-methylsterols. 3. Other modifications of the sterol nucleus varied among the species, as only T. aceti and C. elegans introduced *7- and *8(14)-bonds significantly. 4. P. redivivus and, to a much lesser extent, T. aceti reduced *5-bonds to produce substantial quantities of cholesterol and 4a-methylcholestanol.

Lozano R et al. (1985) Lipids "Dealkylation of various 24-alkylsterols by the nematode C. elegans."

Lozano R, Chitwood DJ, Svoboda JA, Lusby WR

[Lipids, 1985]

The metabolism of 4 dietary 24-alkylsterols was investigated in the free-living nematode Caenorhabditis elegans. The major unesterified sterols of C. elegans in media supplemented with either campesterol, 22-dihydrobrassicasterol or stigmasterol included cholesta-5,7-dienol, cholesterol, cholest-7-enol, and 4a-methylcholest-8(14)-enol. Dietary stigmastanol yielded cholest-7-enol, cholestanol, cholest-8(14)-enol, and 4a-methylcholest-8(14)-enol as major unesterified sterols. Esterified sterols comprised less than 22% of the total sterol. Removal of a C-24 ethyl substituent of sterols was neither hindered by the presence of a delta22-bond in the sterol side chain nor was it dependent on unsaturation in ring B of the steroid nucleus. C. elegans reduced a delta22-bond during its metabolism of stigmasterol; it did not introduce a delta5-bond during stigmastanol metabolism. C. elegans was capable of removing a C-24 methyl substituent regardless of it stereochemical orientation. Metabolic processes involving the steroid ring system of cholesterol (C-7 dehydrogenation, delta5-reduction, 4a-methylation, delta8(14)-isomerization) in C. elegans were not hindered by the presence of a 24-methyl group; various 24-methylsterol metabolites from campesterol were detected, mostly 24-methylcholesta-5,7-dienol. In contrast, no 24-ethylsterol metabolites from the dietary ethylsterols were found. More dietary 24-methylsterol remained unmetabolized than did dietary 24-ethylsterol. A 24a-ethyl group and a 24B-methyl group were dealkylated to a greater extent by C. elegans than was a 24a-methyl group, perhaps reflecting the substrate specificity of the dealkylation enzyme system, or suggesting different enzymes

Lozano R et al. (1987) Lipids "Metabolism of sterols of varying ring unsaturation and methylation by Caenorhabditis elegans."

Lusby WR, Chitwood DJ, Lozano R, Salt TA

[Lipids, 1987]

The metabolism of three dietary 4,4-desmethylsterols and two 4 alpha- methylsterols was investigated in the free-living nematode Caenorhabditis elegans. Dietary cholestanol was converted mostly to lathosterol. Dietary lathosterol, 7-dehydrocholesterol, 4 alpha- methylcholest-7-enol and 4 alpha-methylcholest-8(14)-enol each remained largely unchanged. An absolute requirement for a substantial quantity of 7-dehydrocholesterol in C. elegans did not exist. C. elegans was unable to remove a 4 alpha-methyl group or introduce a double bond at C-5 and also demonstrated the lack of a delta 7- reductase. Its nutritional sterol requirement was satisfied by cholestanol, lathosterol or 7-dehydrocholesterol; growth was comparable to that obtained previously in media containing delta 5- sterols. However, the two 4 alpha-methylsterols appeared to be unsatisfactory sterol nutrients. The possible physiological importance of 4 alpha-methylsterols is discussed briefly.

Salt TA et al. (1986) Steroids "24-Methyl-23-dehydrocholesterol: a new sterol intermediate in C-24 demethylation from the nematodes Panagrellus redivivus and Caenorhabditis elegans?"

Thompson MJ, Lusby WR, Lozano R, Salt TA, Chitwood DJ

[Steroids, 1986]

Panagrellus redivivus produced 24-methyl-23-dehydrocholesterol as 4.0% of the 4-desmethylsterols when propagated in a medium containing campesterol as the dietary sterol. The re-examination of previous data revealed that Caenorhabditis elegans produced 1.8% 24-methyl-23-dehydrocholesterol when propagated in medium containing campesterol. 24-Methyl-23-dehydrocholesterol was not detected when the nematodes were propagated in medium containing 22-dihydrobrassicasterol or 24-methylenecholesterol. This may be a result of the greater efficiency of dealkylation of the latter two sterols. This is the first report of the natural occurrence of this sterol in a non-photosynthetic organism, and the first report in organisms that dealkylate 24-alkylsterols.

Chitwood DJ et al. (1983) Steroids "Novel nuclear methylation of sterols by the nematode Caenorhabditis elegans."

Chitwood DJ, Lozano R, Svoboda JA, Thompson MJ, Lusby WR

[Steroids, 1983]

Caenorhabditis elegans possesses a unique sterol methylation pathway not reported to occur in any other organism and also removes the C-24 ethyl group of sitosterol (a plant sterol). This nematode produced substantial quantities of 4 alpha-methyl-5 alpha-cholest-8(14)-en-3 beta-ol and smaller amounts of lophenol from dietary cholesterol, desmosterol or sitosterol. When C. elegans was propagated in media containing sitosterol plus 25-azacoprostane hydrochloride (25-aza-5 beta-cholestane hydrochloride), an inhibitor of delta 24-sterol reductase in insects, its 4 alpha-methylsterol fraction largely consisted of equal amounts of 4 alpha-methyl-5 alpha-cholesta-7,24- dien-3 beta-ol and 4 alpha-methyl-5 alpha-cholesta-8(14),24-dien-3 beta-ol. Thus 25-azacoprostane hydrochloride inhibited both a delta 24-sterol reductase and a delta 7-sterol isomerase

Chitwood DJ et al. (1995) Lipids "The glycosylceramides of the nematode Caenorhabditis elegans contain an unusual, branched-chain sphingoid base."

Chitwood DJ, Lusby WR, Thompson MJ, Howarth OW, Kochansky JP

[Lipids, 1995]

Caenorhabditis elegans was cultured in semi-defined medium containing yeast extract, soy peptone, glucose, hemoglobin, Tween 80, and sitosterol. Monoglycosylceramides were chromatographically purified from nematode extracts. Their structures were elucidated with mass spectrometry, nuclear magnetic resonance spectroscopy, and analysis of methanolysis products of the parent cerebrosides. The glycosylceramides were unusual in that the only long-chain sphingoid base detected was an iso-branched compound with a C-4 double bond (i.e., 15-methyl-2-aminohexadec-4-en-1,3-diol). Glucose was the only sugar moiety detected. The fatty acids consisted of a series of primarily straight-chain, saturated, 2-hydroxylated C20-C26 acids; some iso-branched analogs also occurred. The sphingomyelins of C. elegans were also hydrolyzed, and the same iso-branched C17 compound was the only sphingoid base detected. This is the first structural analysis of a nematode glycosphingolipid and the first report of an organism in which the long-chain sphingoid bases are entirely iso-branched.

Lozano R et al. (1985) Lipids "Inhibition of C28 and C29 phytosterol metabolism by N,N-dimethyldodecanamine in the nematode Caenorhabditis elegans."

Svoboda JA, Lozano R, Lusby WR, Chitwood DJ, Thompson MJ

[Lipids, 1985]

Effects on the metabolism of campesterol and stigmasterol in Caenorhabditis elegans were investigated using N,N- dimethyldodecanamine, a known inhibitor of growth, reproduction and the delta 24-sterol reductase of this nematode. 7-Dehydrocholesterol was the predominant sterol (51%) of C. elegans grown in stigmasterol- supplemented media, whereas addition of 25 ppm amine resulted in a large decrease in the relative percentage of 7-dehydrocholesterol (23%) and the accumulation of a substantial proportion (33%) of delta 24-sterols (e.g., cholesta-5,7,24-trienol) and delta 22,24-sterols (e.g., cholesta-5,7,22, 24-tetraenol) but yielded no delta 22- sterols. Dealkylation of stigmasterol by C. elegans proceeded in the presence of the delta 22-bond; reduction of the delta 22-bond occurred prior to delta 24-reduction. Addition of 25 ppm amine to campesterol-supplemented media altered the sterol composition of C. elegans by increasing the percentage of unmetabolized dietary campesterol from 39 to 60%, decreasing the percentage of 7- dehydrocholesterol from 26 to 12%, and causing the accumulation of several delta 24-sterols (6%). C. elegans also was shown to be capable of dealkylating a delta 24 (28)-sterol as it converted 24- methylenecholesterol to mostly 7-dehydrocholesterol. The proposed role of 24-methylenecholesterol as an intermediate between campesterol and 7-dehydrocholesterol was supported by the

Chitwood DJ et al. (1987) "Metabolism and function of sterols in nematodes."

Lozano R, Thompson MJ, Chitwood DJ, Lusby WR, Svoboda JA

[1987]

Current knowledge of sterol biochemistry and physiology in nematodes is reviewed. Nematodes possess a nutritional requirement for sterol because they lack the capacity for de novo sterol biosynthesis. The free-living nematode Caenorhabditis elegans has recently been used as a model organism for investigation of nematode sterol metabolism. C. elegans is capable of removal of the C-24 alkyl substituent of plant sterols such as sitosterol and also possesses the remarkable ability to attach a methyl group at C-4 on the sterol nucleus. An azasteroid and several long-chain alkyl amines disrupt the phytosterol dealkylation pathway in C. elegans by inhibiting its *24-sterol reductase. These compounds inhibit growth and reproduction in certain parasitic nematodes and provide model compounds for development of novel nematode control