David J. Chitwood

The glycosylceramides of the nematodeCaenorhabditis elegans contain an unusual, branched-chain sphingoid base

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 aniso-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 straint-chain, saturated, 2-hydroxylated C20–C26 acids; someiso-branched analogs also occurred. The sphingomyelins ofC. elegans were also hydrolyzed, and the sameiso-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 entirelyiso-branched.

Sterol metabolism in the nematodeCaenorhabditis elegans

The metabolism of various dietary sterols and the effects of an azasteroid on sitosterol metabolism in the free-living nematodeCaenorhabditis elegans was investigated. The major unesterified sterols ofC. elegans in media supplemented with sitosterol, cholesterol or desmosterol included 7-dehydrocholesterol (66.5%, 40.5%, 31.2%, respectively), cholesterol (6.7%, 52.3%, 26.9%), lathosterol (4.4%, 3.6%, 1.7%) and 4α-methylcholest-8(14)-en-3β-ol (4.2%, 2.1%, 3.8%). Esterified sterols, representing less than 20% of the total sterols, were somewhat similar except for a significantly higher relative content of 4α-methylcholest-8(14)-en-3β-ol (23.3%, 23.4%, 10.6%). ThusC. elegans not only removes the substituent at C24 of dietary sitosterol but possesses the unusual ability to produce significant quantities of 4α-methylsterols. WhenC. elegans was propagated in medium supplemented with sitosterol plus 5 μg/ml of 25-azacoprostane hydrochloride, the azasteroid strongly interfered with reproduction and motility ofC. elegans and strongly inhibited the Δ24-sterol reductase enzyme system; excluding sitosterol, the major free sterols of azacoprostane-treatedC. elegans were cholesta-5, 7, 24-trien-3β-ol (47.9%), desmosterol (9.4%), fucosterol (2.1%) and cholesta-7,24-dien-3β-ol (2.0%). These 4 sterols are likely intermediates in the metabolism of sitosterol inC. elegans.

STEROLS OF SOME DIATOMS

Abstract Sterols were identified from seven species of axenically cultured diatoms which may be used for oyster food. Five species contained 24-methylenecholesterol as the major sterol. Stigmasterol was the principal sterol in Amphora coffaeformis while 24-ethylcholesterol was the major sterol in Navicula pelliculosa . Each of the four species of the order Centrales had 24-methylcholesterol, which was all 22-dihydrobrassicasterol (24β-methyl epimer). The 24-methylcholesterol from Nitzschia brevirostris (order Pennales) was all campesterol (24α-methyl epimer). From this and previously published works on diatom sterols, it appears that sterols from the order Centrales are 24β-oriented and those from the order Pennales are 24α-oriented.

Novel nuclear methylation of sterols by the nematode caenorhabditis elegans

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 in C. elegans.

Sterols and alkenones of Isochrysis

Abstract Sterols were identified and quantified in six marine microalgal strains identified as belonging to the prymnesiophyte genus Isochrysis , several of these strains are in wide use in the commerical mariculture industry. One strain contained only cholesterol, and another contained a complex mixture of Δ 5 -sterols and dihydroxysterols. Two strains contained brassicasterol and two others contained epibrassicasterol as the principal sterol. The strains containing brassicasterol or epibrassicasterol also contained long chain alkenones characteristic of some members of the Prymnesiophyceae; whereas, those without brassicasterol or epibrassicasterol contained no long chain alkenones. These qualitative biochemical differences appear to have taxonomic significance and may be important in the value of these algae as live feeds for rearing marine invertebrates.

Expression of a cystatin transgene can confer resistance to root lesion nematodes in Lilium longiflorum cv. ‘Nellie White’

AbstractLilium longiflorum cv. ‘Nellie White’ assumes a great economic importance as cut flowers, being one of the most valuable species (annual pot plants value above

Dealkylation of various 14-alkylsterols by the nematodeCaenorhabditis elegans

20,000,000) in terms of wholesales in the US. The root lesion nematode Pratylenchus penetrans (RLN) constitutes one of the main pests for lily producers due to the significant root damage it causes. Our efforts have focused on the generation of soybean hairy roots (as a transient test model) and stable transgenic lilies overexpressing a modified rice cystatin (Oc-IΔD86) transgene and challenged with root lesion nematodes. Lily transformation was achieved by gene gun co-bombardment using both a pBluescript-based vector containing the cystatin gene and pDM307 that contains a bar gene for phosphinothricin selection. Both soybean hairy roots and lilies overexpressing the OcIΔD86 transgene exhibited enhanced resistance to RLN infection by means of nematode reduction up to 75xa0±xa05xa0% on the total number of nematodes. In addition, lily plants overexpressing OcIΔD86 displayed an increase of plant mass and better growth performance in comparison to wild-type plants, thereby demonstrating an alternative strategy for increasing the yield and reducing nematode damage to this important floral crop.n

Inhibition of C28 and C29 phytosterol metabolism by N, N-dimethyldodecanamine in the nematodecaenorhabditis elegans

The metabolism of 4 dietary 24-alkylsterols was investigated in the free-living nematodeCaenorhabditis elegans. The major unesterified sterols ofC. elegans in media supplemented with either campesterol, 22-dihydrobrassicasterol or stigmasterol included cholesta-5,7-dienol, cholesterol, cholest-7-enol, and 4α-methylcholest-8(14)-enol. Dietary stigmastanol yielded cholest-7-enol, cholestanol, cholest-8(14)-enol, and 4α-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 Δ22-bond in the sterol side chain nor was it depedent on unsaturation in ring B of the steroid nucleus.C. elegans reduced a Δ22-bond during its metabolism of stigmasterol; it did not introduce a Δ22-bond during stigmastanol metabolism.C. elegans was capable of removing a C-24 methyl substituent regardless of its stereochemical orientation. Metabolic processes involving the steroid ring system of cholesterol (C-7 dehydrogenation, Δ5-bond, 4α-methylation, Δ8(14)-isomerization inC. 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 24α-ethyl group and a 24β-methyl group were dealkylated to a greater extent byC. elegans than was a 24α-methyl group, perhaps reflecting the substrate specificity of the dealkylation enzyme system, or suggesting different enzymes altogether.

Sterols of Tetraselmis (Prasinophyceae)

Effects on the metabolism of campesterol and stigmasterol inCaenorhabditis elegans were investigated using N,N-dimethyldodecanamine, a known inhibitor of growth, reproduction and the Δ24-sterol reductase of this nematode. 7-Dehydrocholesterol was the predominant sterol (51%) ofC. 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 Δ24-sterols (e.g., cholesta-5,7,24-trienol) and Δ22,24-sterols (e.g., cholesta-5,7,22, 24-tetraenol) but yielded no Δ22-sterols. Dealkylation of stigmasterol byC. elegans proceeded in the presence of the Δ22-bond; reduction of the Δ22-bond occurred prior to Δ24-reduction. Addition of 25 ppm amine to campesterol-supplemented media altered the sterol composition ofC. 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 Δ24-sterols (6%).C. elegans also was shown to be capable of dealkylating a Δ24(28)-sterol as it converted 24-methyl-enecholesterol to mostly 7-dehydrocholesterol. The proposed role of 24-methylenecholesterol as an intermediate between campesterol and 7-dehydrocholesterol was supported by the results.

Poly(T) variation in heteroderid nematode mitochondrial genomes is predominantly an artefact of amplification

Abstract 1. 1. Sterols were identified from 11 isolates of Tetraselmis , a unicellular Prasinophyte alga used frequently as food in mariculture. 2. 2. The principal sterol in eight isolates was either 24-methylenecholesterol or 24-methylcholesterol; the latter was determined to be campesterol in all cases. 3. 3. Campesterol is the first 24α sterol to be reported in the Prasinophyceae. 4. 4. In the remaining three isolates, cholesterol was the principal sterol with smaller amounts of 24-methylenecholesterol and campesterol present; in two of these strains total sterol approached 3% of dry weight. 5. 5. This is the first report of cholesterol as the principal sterol of a Prasinophyte; the C28 sterols found in Tetraselmis are the dominant sterols in most Prasinophyceae studied to date.