Sterol composition of Aureoumbra lagunensis, the Texas brown tide alga

Aureoumbra lagunensis, the alga responsible for the "Texas brown tide", contains (E)-24-propylidenecholesterol (35.7% of total sterols) as its dominant sterol, in common with other members of the Pelagophyceae. Other major sterols are stigmasterol (22.2%), sitosterol (19.2%), cholesterol (14.1%), and (24R)-24-propylcholesterol (5.2%). Trace amounts of 24-methylenecholesterol, crinosterol, clerosterol, campesterol, dihydrobrassicasterol, and 24-isopropylcholesterol were also detected.     

The biosynthetic incorporation of the intact leucine skeleton into sterol by the trypanosomatid Leishmania mexicana

The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-(13)C]leucine with L. mexicana promastigotes in the presence of ketoconazole gave 14alpha-methylergosta-8,24(24(1))-3beta-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of (13)C per molecule. (13)C NMR analysis of the 14alpha-methylergosta-8,24(24(1))-3beta-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-(14)C]acetate into sterol but had no inhibitory effect on [U-(14)C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-(14)C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-(14)C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.     

Sterols of marine microalgae Pyramimonas cf. cordata (Prasinophyta), Attheya ussurensis sp. nov. (Bacillariophyta) and a spring diatom bloom from Lake Baikal

The free sterol compositions of two marine microalgal species Pyramimonas cf. cordata (Prasinophyta), Attheya ussurensis sp. nov. (Bacillariophyta), and diatom bloom samples from Lake Baikal were determined by gas chromatography, gas chromatography-mass spectrometry and (for some sterol constituents) using nuclear magnetic resonance spectra. A variety of sterol profiles were found. The principal sterol in the prasinophyte P. cf. cordata, collected in the Sea of Japan near Vladivostok, was 24(R)-ethylcholesta-5,22E-dien-3beta-ol (poriferasterol), but not 24-ethyl-5,24(28)Z-dien-3beta-ol, as reported earlier in the related species Pyramimonas cordata. The principal sterol in the marine diatom A. ussurensis sp. nov. was identified as 24-ethylcholest-5-en-3beta-ol. The sample of diatom bloom caused by Stephanodiscus meyerii with admixtures of several other diatom species, contained cholesterol and 24-methylcholesta-5,24(28)-dien-3beta-ol as main sterol constituents.     

BODIPY-cholesterol: a new tool to visualize sterol trafficking in living cells and organisms

Analysis of sterol distribution and transport in living cells has been hampered by the lack of bright, photostable fluorescent sterol derivatives that closely resemble cholesterol. In this study, we employed atomistic simulations and experiments to characterize a cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon-24 (BODIPY-cholesterol). This probe packed in the membrane and behaved similarly to cholesterol both in normal and in cholesterol-storage disease cells and with trace amounts allowed the visualization of sterol movement in living systems. Upon injection into the yolk sac, BODIPY-cholesterol did not disturb zebrafish development and was targeted to sterol-enriched brain regions in live fish. We conclude that this new probe closely mimics the membrane partitioning and trafficking of cholesterol and, because of its excellent fluorescent properties, enables the direct monitoring of sterol movement by time-lapse imaging using trace amounts of the probe. This is, to our knowledge, the first cholesterol probe that fulfills these prerequisites.     

Synthesis of polyhydroxysterols (I): synthesis of 24-methylenecholest-4-en-3beta,6beta-diol, a cytotoxic natural hydroxylated sterol

Starting from stigmasterol (2), 24-methylenecholest-4-en-3beta, 6beta-diol (1), a cytotoxic natural dihydroxylated sterol, was synthesized via 10 steps in 20% overall yield. The introduction of a side-chain of sterol was achieved by solid-liquid phase-transfer Wittig reaction using (3-methyl-2-oxo)butyltriphenylarsonium bromide (12) and K(2)CO(3). Construction of the steroidal nucleus was finished by the addition of 3beta-acetoxycholest-5,6-en-24-one (7) with NBA in dioxane under ambient temperature and by the elimination of 3beta, 6beta-diacetoxy-5a-bromocholestane-24-one (9). The spectral data of the synthetic product (1) are completely consistent with those of the natural compound (1).     

24-methylenepollinastanol and 24-dehydropollinastanol—two sterols of pollen

The esterified and unesterified sterol fractions of bee-gathered mixed pollens were examined, and total sterol composition was determined. Two new sterols of pollens, 14α-methyl-9β,19-cyclo-5α-cholest-24-en-3β-ol (24-dehydropollinastanol) and 14α-methyl-5α-ergost-24(28)-en-3β-ol (24-methylenepollinastanol) were isolated and identified. Both sterols were found primarily in the esterified sterol fraction, and 24-methylenepollinastanol accounted for 43% of the sterols of this fraction. 24-Dehydropollinastanol and four other sterols which also contain a 9β,19-cyclopropane ring were found only in the esterified sterol fraction. 24-Methylenecholesterol was the major sterol of the unesterified sterol fraction.     

The isolation of 2,3-oxidosqualene from the liver of rats treated with 1-dodecylimidazole, a novel hypocholesterolaemic agent

1. Non-saponifiable lipid from the livers of rats treated with 1-dodecylimidazole contained an unidentified compound that was not present in the livers from untreated animals. 2. Treated rats had lower serum cholesterol concentrations than control rats. 3. 1-Dodecylimidazole, when added to rat liver slices, inhibited the incorporation of [1-(14)C]acetate and [2-(14)C]mevalonate into digitonin-precipitable sterols and resulted in the accumulation of a labelled compound, which was chromatographically identical with the unknown compound described in 1 above. 4. Rats treated with 1-dodecylimidazole incorporated less [(14)C]mevalonate into liver digitonin-precipitable sterols than untreated animals and accumulated the unknown compound as a labelled intermediate. 5. The unknown intermediate had the same chromatographic properties, n.m.r. and mass spectra as authentic 2,3-oxidosqualene. 6. The identity of the intermediate as 2,3-oxidosqualene was further established by showing that it was incorporated into sterols by rat liver homogenates under anaerobic conditions. In addition, incubation of [(14)C]squalene with rat liver homogenates resulted in trapping of the radioactivity by the added intermediate. 7. It is suggested that the hypocholesterolaemic activity of 1-dodecylimidazole results in part from the inhibition of cholesterol biosynthesis at the level of 2,3-oxidosqualene sterol cyclase.     

Evolutionarily conserved Delta(25(27))-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii

Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ(24(28))-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24β-methyl) and 7-dehydroporiferasterol (C24β-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ(25(27))-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ(24(28))-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24β-methyl/ethyl groups and Δ(22(23))E and Δ(25(27))-double bond constructions. When incubated with [methyl-(2)H(3)]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24β-alkyl sterols, consistent only with a Δ(25(27))-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol.     

Difference in cytotoxicity of (22R)-cholest-5-ene-3 beta,7 alpha,22-triol and (22R)-cholest-5-ene-3 beta,7 beta,22-triol is not explained by different patterns of metabolites

Ehrlich ascites tumor cells in suspension culture were incubated with the plant-derived sterol isomers (22R)-cholest-5-ene-3 beta,7 alpha,22-triol and (22R)-cholest-5-ene-3 beta,7 beta,22-triol. Both sterols were 7-dehydroxylated by the neoplastic cells, and the product was identified as (22R)-22-hydroxycholesta-4,6-dien-3-one. At sub-toxic sterol concentrations the conversion of the 7 alpha-hydroxy compound was about 5 times higher than that of the 7 beta-isomer. At higher sterol concentrations the 7 beta-hydroxy compound caused growth inhibition of the Ehrlich ascites cells, whereas the 7 alpha-hydroxylated sterol was ineffective. The rate of 7 alpha-dehydroxylation was, however, too low to be considered a likely pathway for detoxification. No other lipid-extractable products were detected, and no water-soluble products with influence on cell proliferation were present. Thus, the cytotoxicity is probably attributed to a property of the 7 beta-hydroxyl group of the (22R)-cholest-5-ene-3 beta,7 beta,22-triol.     

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms

Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control<[2-(13)C]leucine<[2-(13)C]acetate<[1-(13)C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.     

Occurrence of (24S)-24-methylcholesta-5, 22E-dien-3β-ol in the diatom Phaeodactylum tricornutum

The principal sterol of the marine diatom Phaedactylum tricornutum was identified as (24S)-24-methylcholesta-5,22E-dien-3β-ol. Two deuterium atoms were incorporated into this sterol when the diatom was cultured in the presence of [CD₃]methionine indicating a 24-methylene intermediate.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols

Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-[2-(diethylamino)-ethoxy]androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy[3H]anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase (Saucier et al. 1985. J. Biol. Chem. 260: 14571-14579). In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. U18666A had the unusual effect of potentiating the inhibitory effect of 25-hydroxylanostene-3-one but did not influence the effect of other oxylanosterols. All the oxylanosterols, with the exception of 25-hydroxylanostene-3-one, enhanced intracellular esterification of cholesterol. The foregoing observations support consideration of oxylanosterols as playing an important role in the biological formation of regulatory oxysterols that modulate sterol biosynthesis at the level of HMG-CoA reductase.     

Feeding conditions control the expression of genes involved in sterol metabolism in peripheral blood mononuclear cells of normoweight and diet-induced (cafeteria) obese rats

Peripheral blood mononuclear cells (PBMC) are easily obtainable cells from blood whose gene expression profiles have been proven to be highly robust in distinguishing a disease state from healthy state. Sterol metabolism is of physiological importance, and although its nutritional response in liver has been described, it is poorly studied in PBMC. To examine if PBMC sterol metabolism reflects diet-induced physiological responses, we analysed the whole genome gene expression response of PBMC and focused on sterol metabolism-related genes affected by different feeding conditions (ad libitum feeding, fasting, and refeeding) in normoweight (control) rats and in diet-induced (cafeteria) obese rats.Our results of microarray analysis show that, in control rats, 21 genes involved in sterol metabolism were regulated by the different feeding conditions, whereas in cafeteria-obese rats, only seven genes showed a changed expression. Most of the genes identified were classified into three pathways: sterol biosynthesis, cholesterol transport and uptake and sterol signaling. The expression profile of these genes was similar to that previously described for liver, decreasing in response to fasting conditions and recovering the levels found in fed animals after 6-h-refeeding. In addition, our data and the comparable expression pattern of sterol metabolism-related genes between PBMC and liver suggest similar sterol regulatory element-binding protein-mediated regulatory mechanisms in response to feeding conditions in both tissues.In conclusion, the expression of genes involved in sterol metabolism is highly controlled by feeding conditions in PBMC of control rats, but this control is impaired in cafeteria-obese animals. The pathophysiological significance of this impairment requires further investigation.     

Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length

It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-β-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15 mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37 °C addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. At 23 °C the 18:1-ceramide was not as effective at inhibiting sterol partitioning into the vesicles as its saturated equivalent, but at 37 °C the additional double bond had no effect. The longer 24:1-ceramide behaved as 24:0-ceramide at both temperatures. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with sphingomyelin.     

Sterol biosynthesis via cycloartenol and other biochemical features related to photosynthetic phyla in the amoeba Naegleria lovaniensis and Naegleria gruberi

The sterols and sterol precursors of two amoebae of the genus Naegleria, Naegleria lovaniensis and Naegleria gruberi were investigated. Cycloartenol, the sterol precursor in photosynthetic organisms, is present in both amoebae. In N. lovaniesis, it is accompanied by lanosterol and parkeol, as well as by the 24,25-dihydro derivatives of these triterpenes. One of the most striking features of these amoebae is the accumulation of 4 alpha-methylsterols which are present in similar amounts as those of 4,4-desmethylsterols (3-5 mg/g, dry weight). 4 alpha-Methylergosta-7,22-dienol was identified as a new compound. Ergosterol was the major 4,4-desmethylsterol, accompanied by small amounts of C27 and other C28 sterols. Treatment of N. lovaniensis with fenpropimorph modified the sterol pattern of this amoeba and inhibited its growth. This fungicide, known to inhibit steps of sterol biosynthesis in fungi and plants, induced the disappearance of 4 alpha-methyl-delta 7-sterols and the appearance of the unusual delta 6,8,22-ergostatrienol as in A. polyphaga. These results might be explained by a partial inhibition of the delta 8----delta 7 isomerase, the small amounts of delta 7-sterols formed being converted into ergosterol which is still present in fenpropimorph-exposed cells. De novo sterol biosynthesis in N. lovaniensis was shown by incorporation of [1-14C]acetate into sterols and sterol precursors, especially cycloartenol. Lanosterol and parkeol were not significantly labelled. Furthermore, [3-3H]squalene epoxide was efficiently cyclized by a cell-free system of this amoeba into cycloartenol, and again no significant radioactivity was detected in lanosterol and parkeol. This shows that cycloartenol, the sterol precursor in plants and algae, is also the sterol precursor in Naegleria species, and that these amoebae, like A. polyphaga, are related by some biosynthetic pathways to photosynthetic phyla. Lanosterol, the sterol precursor in non-photosynthetic phyla (animal and fungi) and parkeol are more likely dead-ends of this biosynthetic pathway. The peculiar phylogenetic position of these protozoa was further emphasized by the action of indole acetic acid and other auxine-like compounds on their growth. Indeed amoebic growth was enhanced in the presence of these higher plant growth hormones. The differences in the sterol composition of the protozoa we have hitherto examined is related to their sensitivity toward polyene macrolide antibiotics.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitors of ergosterol biosynthesis and growth of the trypanosomatid protozoan Crithidia fasciculata

Six nitrogen-, sulfur- and cyclopropane-containing derivatives of cholestanol were examined as inhibitors of growth and sterol biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The concentrations of inhibitors in the culture medium required for 50% inhibition of growth were 0.32 microM for 24-thia-5 alpha,20 xi-cholestan-3 beta-ol (2), 0.009 microM for 24-methyl-24-aza-5 alpha,20 xi-cholestan-3 beta-ol (3), 0.95 microM for (20,21),(24,-25)-bis-(methylene)-5 alpha,20 xi-cholestan-3 beta-ol (4), 0.13 microM for 22-aza-5 alpha,20 xi-cholestan-3 beta-ol (5), and 0.3 microM for 23-azacholestan-3-ol (7). 23-Thia-5 alpha-cholestan-3 beta-ol (6) had no effect on protozoan growth at concentrations as high as 20 microM. Ergosterol was the major sterol observed in untreated C. fasciculata, but significant amounts of ergost-7-en-3 beta-ol, ergosta-7,24(28)-dien-3 beta-ol, ergosta-5,7,22,24(28)-tetraen-e beta-ol, cholesta-8,24-dien-3 beta-ol, and, in an unusual finding, 14 alpha-methyl-cholesta-8,24-dien-3 beta-ol were also present. When C. fasciculata was cultured in the presence of compounds 2 and 3, ergosterol synthesis was suppressed, and the principal sterol observed was cholesta-5,7,24-trien-3 beta-ol, a sterol which is not observed in untreated cultures. The presence of this trienol strongly suggests that 2 and 3 specifically inhibit the S-adenosylmethionine:sterol C-24 methyltransferase but do not interfere with the normal enzymatic processing of the sterol nucleus. When C. fasciculata was cultured in the presence of compounds 5 and 7, the levels of ergosterol and ergost-7-en-3 beta-ol were suppressed, but the amounts of the presumed immediate precursors of these sterols, ergosta-5,7,22,24(28)-tetraen-3 beta-ol and ergosta-7,24-(28)-dien-3 beta-ol, respectively, were correspondingly increased. These findings suggest that 5 and 7 specifically inhibit the reduction of the delta 24(28) side chain double bond. When C. fasciculata was cultured in the presence of compound 4, ergosterol synthesis was suppressed, but the sterol distribution in these cells was complex and not easily interpreted. Compound 6 had no significant effect on sterol synthesis in C. fasciculata.     

The formation and reduction of the 14,15-double bond in cholesterol biosynthesis

It was shown that 100mug quantities of 4,4'-dimethyl[2-(3)H(2)]cholesta-8,14-dien-3beta-ol (IIIa), tritiated cholesta-8,14-dien-3beta-ol, 4,4'-dimethyl[2-(3)H(2)]cholesta-7,14-dien-3beta-ol, dihydro[2-(3)H(2)]lanosterol and [24-(3)H]lanosterol were converted by a 10000g supernatant of rat liver homogenate into cholesterol in 17%, 54%, 6%, 9.5% and 24% yields respectively. From an incubation of dihydro[3alpha-(3)H]lanosterol with a rat liver homogenate in the presence of a trap up to 38% of the radioactivity was found to be associated with a fraction that was unambiguously shown to be 4,4'-dimethylcholesta-8,14-dien-3beta-ol. Another related compound, 4,4'-dimethylcholesta-7,14-dien-3beta-ol was also shown to be equally effective in its ability to trap compound (IIIa) from an incubation of dihydro[3alpha-(3)H]lanosterol. The mechanism of the further conversion of the compound (IIIa) into cholesterol occurred by the reduction of the 14,15-double bond and involved the addition of a hydrogen atom from the medium to C-15 and another from the 4-position of NADPH to C-14. Two possible mechanisms for the removal of the 14alpha-methyl group in sterol biosynthesis are discussed.     

INVESTIGATION OF 4-METHYL STEROLS FROM CULTURED DINOFLAGELLATE ALGAL STRAINS

The marine dinoflagellates Prorocentrum micans, Gonyaulax polyedra, Gymnodinium sp., and Alexandrium tamarense, collected from the Adriatic Sea during red-tide blooms, were cultured to investigate the 4-methyl sterol constituents. To ascertain a possible influence of cell age on the 4-methyl sterol content, for one strain (Gymnodinium sp.)we investigated the composition of these constituents at exponential and stationary growing phases. The lipid material extracted with acetone from the lyophilized algal samples was fractionated by thin-layer chromatography. The 4-methyl sterols recovered from the layer were converted into the corresponding OTMS derivatives. Nine of 11 constituents were identified by gas chromatography and gas chromatography-mass spectrometry; only two minor constituents were characterized by their gas chromatographic parameters. All free methyl sterols identified in the algal samples had been detected previously in various dinoflagellates. The 4-methyl sterol fractions generally contained very few constituents. Except for the Gymnodinium sp. sample, collected at the exponential growing phase (GyD2 exp), which contains 4,24-dimethylcholestan-3-ol as a unique constituent, dinosterol was the major component. Moreover, 4,24-ethylcholestan-3-ol was also an important constituent of both Prorocentrum and Gonyaulax strains, whereas considerable amounts of dinostanol characterized all the Gymnodinium sp. strains. In addition, the latter contained several minor constituents such as 4-methylcholestan-3-ol, 4,24-dimethylcholesta-22-en-3-ol, and 4-methyl-24-ethylcholestan-3-ol. 4-Methyl-24-methylene-cholestan-3-ol was a constituent of the Gymnodinium sp. sample, collected at the stationary growing phase (GyD2 stat)only, whereas 4-methylgorgostanol was identified only in the Alexandrium tamarense Gt4 strain. Except for 4-methyl-24-ethylcholesta-8(14)-en-3-ol, all the methyl sterol constituents from our algae show a saturated polynuclear system. The pathways by which side-chain modifications occur in dinoflagellate 4-methyl sterols are considered, and a map of the fragmentation pattern of the trimethylsilyl-4-methyl sterols under electronic impact is also reported.     

Origin of sterols in uredospores of Uromyces phaseoli

The sterols from healthy bean leaves are β-sitosterol, stigmasterol, campesterol and 28-isofucosterol. An additional sterol observed in bean leaves infected with Uromyces phaseoli was identified as 7,(Z)-24(28)-stigmastadien-3β-ol, which is the major sterol of the uredospores of the fungus. The fungus appears to stimulate sterol synthesis, but most of the increased sterol content of infected leaves can be attributed to the sterol of the uredospores.     

The structure of a Burkholderia pseudomallei immunophilin-inhibitor complex reveals new approaches to antimicrobial development

TbSMT [Trypanosoma brucei 24-SMT (sterol C-24-methyltransferase)] synthesizes an unconventional 24-alkyl sterol product set consisting of Δ24(25)-, Δ24(28)- and Δ25(27)-olefins. The C-methylation reaction requires Si(β)-face C-24-methyl addition coupled to reversible migration of positive charge from C-24 to C-25. The hydride shifts responsible for charge migration in formation of multiple ergostane olefin isomers catalysed by TbSMT were examined by incubation of a series of sterol acceptors paired with AdoMet (S-adenosyl-L-methionine). Results obtained with zymosterol compared with the corresponding 24-2H and 27-13C derivatives revealed isotopic-sensitive branching in the hydride transfer reaction on the path to form a 24-methyl-Δ24(25)-olefin product (kinetic isotope effect, kH/kD=1.20), and stereospecific CH3→CH2 elimination at the C28 branch and C27 cis-terminal methyl to form Δ24(28) and Δ25(27) products respectively. Cholesta-5,7,22,24-tetraenol converted into ergosta-5,7,22,24(28)-tetraenol and 24β-hydroxy ergosta-5,7,23-trienol (new compound), whereas ergosta-5,24-dienol converted into 24-dimethyl ergosta-5,25(27)-dienol and cholesta-5,7,24-trienol converted into ergosta-5,7,25(27)trienol, ergosta-5,7,24(28)-trienol, ergosta-5,7,24-trienol and 24 dimethyl ergosta-5,7,25(27)-trienol. We made use of our prior research and molecular modelling of 24-SMT to identify contact amino acids that might affect catalysis. Conserved tyrosine residues at positions 66, 177 and 208 in TbSMT were replaced with phenylalanine residues. The substitutions generated variable loss of activity during the course of the first C-1-transfer reaction, which differs from the corresponding Erg6p mutants that afforded a gain in C-2-transfer activity. The results show that differences exist among 24-SMTs in control of C-1- and C-2-transfer activities by interactions of intermediate and aromatic residues in the activated complex and provide an opportunity for rational drug design of a parasite enzyme not synthesized by the human host.