Stereochemical aspects of the biosynthesis of the side chain of 9beta, 19-cyclopropyl sterols in maize seedlings treated with tridemorph

9β, 19-Cyclopropyl sterols such as 24-methyl pollinastanol accumulate dramatically in maize (Zea mays L. var LG 11) seedlings treated with Tridemorph (2,6-dimethyl-N-tridecyl-morpholine), a systemic fungicide (M. Bladocha, P. Benveniste, Plant Physiol 1983 41: 756-762). In contrast to the situation in control plants where 24-ethyl sterols predominate largely, 24-methyl sterols were more than 98% of total cyclopropyl sterols. In addition, 24-methyl cyclopropyl sterols were a mixture of (24-R)- and (24-S)-24-methyl epimers and are similar in that respect to the 24-methyl cholesterol of control plants. The presence of two epimers at C-24 has been previously explained by the operation of two routes (M. Zakelj, L. J. Goad, Phytochemistry 1983 22: 1931-1936). One may proceed via Δ²⁴⁽²⁸⁾- and Δ²⁴⁽²⁵⁾-sterols to produce the (24-R)-24-methyl epimer. The other route may involve reduction of either a Δ²⁴⁽²⁸⁾-, a Δ²³-, or a Δ²⁵-sterol intermediate to give the (24-S)-24-methyl epimer. Such intermediates have been searched for in excised Zea mays axes grown aseptically in the presence of Tridemorph and either [5-¹⁴C]mevalonic acid, or [Me-¹⁴C]-l-methionine. Whereas Δ²⁴⁽²⁸⁾- and Δ²⁴⁽²⁵⁾-cyclopropyl sterols were found in relatively large amounts, only traces of radioactivity were associated with Δ²⁵-sterols. Gas chromatography/mass spectrometry analysis of the sterols from axes grown in the presence of [Me-²H₃]-l-methionine showed that Δ²⁴⁽²⁸⁾-cyclopropyl sterols contained only two ²H atoms at C-28 as expected and that the 24-methyl pollinastanol fraction contained species with two ²H atoms and no species with three ²H atoms. These results indicate that both (24-R)- and (24-S)-epimers originate from a common Δ²⁴⁽²⁸⁾ precursor. After incubation of the axis with [5-¹⁴C,(4-R)-4-³H₁]mevalonic acid, the 24-methyl pollinastanol had a ³H:¹⁴C atomic ratio of 4:6 which is consistent with the intermediacy of a Δ²⁴⁽²⁵⁾-sterol. All these data are in accordance with a pathway where Δ²⁴⁽²⁸⁾-cyclopropyl sterols are isomerized to give Δ²⁴⁽²⁵⁾-cyclopropyl sterols which in turn would be reduced nonregiospecifically to yield both (24-R)- and (24-S)-24-methyl pollinastanols. A plausible mechanism for the reduction step is discussed.     

Cyclopropyl sterol and phospholipid composition of membrane fractions from maize roots treated with fenpropimorph

Maize (Zea mays L.) caryopses were grown in the presence of fenpropimorph, a systemic fungicide, for 7 days in the dark. Membrane fractions enriched, respectively, in endoplasmic reticulum, plasma membrane, and mitochondria were isolated from control and treated maize roots and analyzed for their free sterol, phospholipid, and fatty acid composition. In treated plants, the intracellular distribution of free sterols was dramatically modified both qualitatively and quantitatively. The normally occurring Δ⁵-sterols disappeared almost completely and were replaced by 9β, 19-cyclopropyl sterols, mainly cycloeucalenol and 24-methyl pollinastanol. These new compounds were found to accumulate in all the membrane fractions in such a way that the endoplasmic reticulum-rich fraction became the richest one in free sterols instead of the plasma membrane. In contrast, the fenpropimorph treatment of maize roots was shown not to affect either the relative proportions or the amounts of the individual phospholipids, but an increase in the unsaturation index of phospholipid-fatty acyl chains of the endoplasmic reticulum-rich fraction was observed. The present data suggest that, in higher plant membranes, cyclopropyl sterols could play a structural role similar to that of the bulk of Δ⁵-sterols.     

Sterols and related metabolites from five species of sponges

Free sterol fractions were isolated from the marine sponges Phyllospongia madagascarensis, Scalarispongia sp., Oceanapia sp., Monanchora clathrata and studied by GLC, GLC–MS, and spectroscopy NMR. P. madagascarensis and Scalarispongia sp. contained common Δ⁵-sterols; cholesterol was shown to be a main sterol of both the sponges. Oceanapia sp. contained stanols and minor Δ⁵-sterols with 24R-24,25-methylene-5α-cholestan-3β-ol as a main constituent. Many free sterols from M. clathrata were Δ⁷-series compounds, and latosterol was a main sterol. Δ⁴-3-Ketosteroids and Δ⁵-sterol esters were found in the Antarctic sponge Haliclona sp., but free sterols were practically absent except for trace amount of cholesterol. A chemotaxonomic application of sterols in relation to the genera Phyllospongia, Oceanapia and the family Crambeidae is provided. The known cases of the absence of sterols in sponges and probable reasons of the phenomenon are discussed.     

Effects of Abnormal-Sterol Accumulation on Ustilago maydis Plasma Membrane H+-ATPase Stoichiometry and Polypeptide Pattern

Accumulation of 14α-methylated sterols or Δ⁸-sterols in Ustilago maydis affected three aspects of the plasma membrane H⁺-ATPase. Proton transport was reduced in Δ⁸-sterol-accumulating samples, due to an altered H⁺/ATP stoichiometry. ATP hydrolytic activity was increased, but no direct correlation with the extent or type of abnormal sterol accumulated could be drawn. Finally, Western blot analysis with antibodies against yeast PMA1 revealed a second lighter band (99-kDa band) in all samples from abnormal-sterol-accumulating sporidia. The conclusions are that the 99-kDa band and a reduced stoichiometry are directly linked to the presence of abnormal sterols, while changes in hydrolytic activity are linked only indirectly.     

Functional importance for developmental regulation of sterol biosynthesis in Acanthamoeba castellanii

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ?^8,14-sterol (<95%). The combination of [²H₃-methyl]methionine incubation to intact cultures showing C₂₈-ergosterol incorporates 2-²H atoms and C₂₉-7-dehydroporiferasterol incorporates 5 ²H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ?²⁴⁽²⁸⁾- and ?²⁵⁽²⁷⁾-olefin pathways to C₂₈- and C₂₉-sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C₂₈/C₂₉-?^5,7-sterols, viable encysted cells (mature cyst) possess mostly C₂₉-?⁵-sterol and non-viable encysted cells possess C₂₈/C₂₉-?^5,7-sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ?^5,7-sterol biosynthesis (to ?^5,7) - metabolism (to ?⁵ or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections.     

Recent Developments in Nematode Steroid Biochemistry

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 Δ²²- and Δ⁵-bonds, C-7 dehydrogenation, isomerization of a Δ⁷-bond to a Δ⁸⁽¹⁴⁾-bond, and 4α-methylation. An azasteroid and several long-chain alkyl amines interfere with the dealkylation pathway in C. elegans by inhibiting the Δ²⁴-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.     

Manipulation by tridemorph, a systemic fungicide, of the sterol composition of maize leaves and roots

The roots of maize (Zea mays L. var LG11) seedlings were watered with a solution of Tridemorph (2,6-dimethyl-N-tridecyl-morpholine), a systemic fungicide, for 3 to 4 weeks from the onset of germination. Very few ⁵-sterols, the major sterols of the control, were detected in the treated plants, and 9β,19-cyclopropyl sterols accumulated dramatically in both roots and leaves. ⁸-sterols were also found when low concentrations of the drug were used. The time course of the accumulation of the new sterols has been studied in plants treated with various concentrations (1-20 milligrams per liter) of Tridemorph. We found that: (a) cycloeucalenol-obtusifoliol isomerase, an enzyme opening the cyclopropane ring of cyclopropyl sterols, was strongly inhibited by the drug; and (b) the drug diffused readily from the roots to the whole plant and reached its enzymic targets in most of the leaf cells. The data obtained offer an opportunity to evaluate the physiological and biochemical consequence of the almost complete replacement of ⁵-sterols by cyclopropyl sterols in higher plant cells.     

An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a Δ7-sterol-C-5-desaturase from yeast

The mutant STE 1 was isolated by screening an ethylmethane sulfonate (EMS)-mutagenized population of Arabidopsis thaliana which consisted of 22 000 M₂ plants divided into 1100 pools of 20 plants by gas chromatography of sterols extracted from small leaf samples. STE 1 was characterized by the accumulation of three Δ⁷-sterols concomitantly with the decrease of the three corresponding Δ⁵-sterols which are the end products of the sterol pathway in wild-type leaves. The structure of these Δ⁷-sterols was determined after two steps of purification on HPLC, by gas chromatography coupled with mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectrometry (¹H-NMR). The accumulation of Δ⁷-sterols suggested that the mutant is deficient in the activity of the Δ⁷-sterol-C-5-desaturase. Genetic analysis showed that the accumulation of Δ⁷-sterols was due to a single recessive nuclear mutation. The mutant line STE 1 was backcrossed four times to the wild-type. The resulting STE 1 plants had wild-type morphology and set seeds normally, suggesting that the Δ⁷-sterols in STE 1 are good surrogates of physiologically active Δ⁵-sterols to sustain normal development. STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the Δ⁷-sterol-C-5-desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root-derived calli showed an increase in Δ⁵-sterols and a concomitant decrease in Δ⁷-sterols in comparison with STE 1 untransformed root-derived calli. Northern blot analysis using the ERG 3 probe showed a strong expression of ERG 3 in three of the seven transgenic calli. These results suggest that the accumulation of Δ⁷-sterols in the STE 1 mutant is due to a deficiency of the Δ⁷-sterol-C-5-desaturation step in the plant sterol biosynthesis pathway.     

Comparative study of the inhibition of sterol biosynthesis in Rubus fruticosus suspension cultures and zea mays seedlings by N-(1,5,9-trimethyldecyl)-4α,10-dimethyl-8-aza-trans-decal-3β-ol and derivatives

The nitrogen substituents present in tridemorph and fenpropimorph, which are systemic fungicides, have been linked to an 8-aza-bicyclic skeleton leading to N-(1,5,9-trimethyldecyl)-4α,10-dimethyl-8-aza-trans-decal-3β-ol and N-(3-(4-tert-butylphenyl-)2-methyl)-propyl-8-aza-4α,10-dimethyl-trans-decal-3β-ol respectively. The latter two compounds present in a stable molecule key structural elements of unstable C-8 and C-9 carbocationic high-energy intermediates which occur during the reactions catalysed by the Δ⁸ → Δ⁷-sterol isomerase and the cycloeucalenol-obtusifoliol isomerase, respectively. When given to either bramble cell suspension cultures or maize seedlings, they led to a spectacular accumulation of 9β,19-cyclopropyl sterols and were in that respect much more efficient than any known molecules and in particular than the N-benzyl decalin previously described which led to accumulation of Δ⁸-sterols. Surprisingly, treatment of the plant cells by the N-oxide derivatives of the N-benzyl decalin resulted in dramatic accumulation of Δ^8,14-sterols.     

Utilization of Δ5,7 - and Δ8-sterols by larvae of Heliothis zea

Larvae from two populations of Heliothis zea were reared on artificial diets containing various sterols, which supported suboptimal growth, and their tissue sterols were characterized in order to determine how these dietary sterols are utilized by this insect. The sterols studied included Δ^5,7-sterols (7-dehydrocholesterol or ergosterol), Δ⁸-sterols (lanosterol and/or 24-dihydrolanosterol), and a Δ⁵-sterol (4,4-dimethylcholesterol). Although larvae did not develop on 4,4-dimethylcholesterol, those fed primarily Δ⁸-4,4,14-trimethylsterols developed to the third instar. When the latter sterols were spared with cholesterol, the larvae reached the sixth instar and contained 4,4,14-trimethylsterols as well as cholesterol in their tissues. When larvae were fed 7-dehydrocholesterol, <1% of the larvae from one population developed to the sixth instar and these larvae contained 7-dehydrocholesterol as their principal sterol. The other larvae successfully completed their larval stage when they were transferred from the diet containing 7-dehydrocholesterol (or no sterol) to a diet containing cholesterol within at least 9 days. The sterol composition of larvae transferred from a diet containing cholesterol to a diet containing 7-dehydrocholesterol, after they had reached 60% of their final weight, was 54% cholesterol and 46% 7-dehydrocholesterol. The major sterol isolated from the tissues of the larvae fed ergosterol was also 7-dehydrocholesterol. Therefore, although the larva of H. zea can dealkylate and saturate the side chain of the Δ^5,7,22-24β-methylsterol, it carries out little metabolism of the B ring of the nucleus. These studies demonstrate that, when Δ^5,7- or Δ⁸-sterols are the principal sterols in the diet of H. zea, they are absorbed and incorporated into its tissues, although they slow the rate of growth and may prevent complete development of the larva.     

Differences in the sterol composition of Heliothis zea fed Zea mays versus Medicago sativa

Larvae of Heliothis zea were fed Zea mays or Medicago sativa in order to determine how these plants affected the development and sterol composition of the insect. The larvae fed corn kernels grew more rapidly than those fed alfalfa leaves. The corn kernels contained Δ⁵-24-alkylsterols whereas the leaves as well as the sprouts and flowers of alfalfa contained Δ⁷-24-alkylsterols. The corn-fed larvae dealkylated the dietary Δ⁵-sterols and utilized primarily cholesterol in their tissues. In contrast, although the larvae fed alfalfa dealkylated the Δ⁷-sterols present in the leaves, they were unable to metabolize the B-ring of these sterols and so both the prepupae and adults utilized large quantities of lathosterol in their tissues. Interestingly, H. zea, a generalized feeder, shared its inability to metabolize Δ⁷-sterols to Δ⁵-sterols with Hypera postica, a specialized feeder on alfalfa. This coleopteran also dealkylated the Δ⁷-24-alkylsterols and contained lathosterol, but not cholesterol, in its tissues. Therefore, the ability of H. zea to utilize either Δ⁵- or Δ⁷-sterols in its tissues may help to explain its ability to complete its development on a variety of different plants, although it may prefer a host, such as corn, that produces Δ⁵-sterols and supports a more-rapid rate of growth.     

Effect of ammonium ions on †5,7-sterol synthesis inSaccharomyces cerevisiae

The effect of ammonium concentration in the medium on †^5,7-sterol synthesis was examined. Higher concentrations of this nitrogen source in the medium decreased sterol synthesis and accumulation during growth. An intermittent supply with ammonium resulted in a proportional synthesis of †^5,7-sterols and biomass. The carbon to nitrogen molar ratio of ≧ 40 allowed the maximum accumulation of †^5,7-sterols with our strain of baker’s yeast.     

Proton motive force during growth of Streptococcus lactis cells

Experiments with the aerotolerant anaerobe Streptococcus lactis provide the opportunity for determining the proton motive force (Δp) in dividing cells. The two components of Δp, ΔΨ (the transmembrane potential) and ΔpH (the chemical gradient of H⁺), were determined by the accumulation of radiolabeled tetraphenylphosphonium (TPP⁺) and benzoate ions. The ΔΨ was calibrated with the K⁺ diffusion potential in starved, valinomycin-treated cells. With resting, glycolyzing cells, the Δp was measured also by the accumulation of the non-metabolizable sugar thiomethyl-β-galactoside (TMG). In resting cells the Δp, calculated either by adding ΔΨ and ZΔpH or from the levels of TMG, was relatively constant between pH 5 to 7, decreasing from 160 to 150 mV and decreasing further to 100 mV at pH 8.0. With the TPP⁺ probe for ΔΨ, we confirmed our previous finding that the K⁺ ions dissipate ΔΨ and increase ΔpH, whereas Na⁺ ions have little effect on ΔΨ and no effect on ΔpH. [³H]TPP⁺ and [¹⁴C]benzoate were added during exponential phase to S. lactis cells growing at pH 5 to 7 at 28°C in a defined medium with glucose as energy source. As with resting cells, the ΔpH and ΔΨ were dependent on the pH of the medium. At pH 5.1, the ΔpH was equivalent to 60 mV (alkaline inside) and decreased to 25 mV at pH 6.8. The ΔΨ increased from 83 mV (negative inside) at pH 5.1 to 108 mV at pH 6.8. The Δp, therefore, was fairly constant between pH 5 and 7, decreasing from 143 to 133 mV. The values for Δp in growing cells, just as in resting cells, are consistent with a system in which the net efflux of H⁺ ions is effected by a membrane-bound adenosine triphosphatase and glycolytically generated adenosine triphosphate. The data suggest that in both growing and resting cells the pH of the medium and its K⁺ concentration are the two principal factors that determine the relative contribution of ΔpH and ΔΨ to the proton motive force.     

Metabolism of Δ0-, Δ5-, and Δ7-Sterols by Larvae of Heliothis zea

Heliothis zea was reared on artificial diets containing Δ⁵-sterols (cholesterol, campesterol, or sitosterol), Δ⁷-sterols (lathosterol, epifungisterol, or spinasterol), or Δ⁰-sterols (cholestanol, epicoprostanol, campestanol, or sitostanol) in order to determine how different dietary sterols affect the type of sterols present in the tissues of the late-sixth-instar larva. Although all of the dietary sterols (except epicoprostanol) supported the growth of the larvae, not all of the sterols were metabolized to the same end products. In each case, at least 80% of the sterols in the tissues of the larvae retained the same nucleus as that of the dietary sterol, indicating that H. zea carries out very little metabolism of ring B of Δ⁵-, Δ⁷-, and Δ⁰-sterols. The larvae dealkylated the Δ⁵-, Δ⁷-, and Δ⁰-alkylsterols to 24-desalkylsterols, but a greater percentage of the Δ⁵-alkylsterols were metabolized in this manner. The sterols present as free sterols in the larva were also present as esterifed sterols which accounted for 2–4% of the total sterols. Therefore, the sterol composition of the tissues of H. zea can be altered by varying the dietary sterols.     

In Vivo Nitrate Reduction in Roots and Shoots of Barley (Hordeum vulgare L.) Seedlings in Light and Darkness

In vivo NO₃⁻ reduction in roots and shoots of intact barley (Hordeum vulgare L. var Numar) seedlings was estimated in light and darkness. Seedlings were placed in darkness for 24 hours to make them carbohydrate-deficient. During darkness, the leaves lost 75% of their soluble carbohydrates, whereas the roots lost only 15%. Detached leaves from these plants reduced only 7% of the NO₃⁻ absorbed in darkness. By contrast, detached roots from the seedlings reduced the same proportion of absorbed NO₃⁻, as did roots from normal light-grown plants. The rate of NO₃⁻ reduction in the roots accounted for that found in the intact dark-treated carbohydrate-deficient seedlings. The rates of NO₃⁻ reduction in roots of intact plants were the same for approximately 12 hours, both in light and darkness, after which the NO₃⁻ reduction rate in roots of plants placed in darkness slowly declined. In the dark, approximately 40% of the NO₃⁻ reduction occurred in the roots, whereas in light only 20% of the total NO₃⁻ reduction occurred in roots. A lesser proportion was reduced in roots because the leaves reduced more nitrate in light than in darkness.     

Effect of Steric and Nuclear Changes in Steroids and Triterpenoids on Sexual Reproduction in Phytophthora cactorum

The comparative biological activity of 21 naturally occurring or synthetically derived steroids, 7 tetracyclic and pentacylic triterpenoids, and antheridiol incubated with cultures of Phytophthora cactorum has been examined. There was greater dependence on precise steric features of the sterol side chain than on the extent of nuclear unsaturation in inducing oospore formation. There was no significant effect on oospore formation by changing nuclear unsaturation in ring B from Δ⁵ to Δ⁷ or to Δ^5,7. Converting the unsaturated sterol to its corresponding stanol resulted in a significant reduction in the number of oospores produced. The effectiveness of sterols bearing different side chains in inducing oospores was found to be in the following relative order: 24α-ethyl = trans-Δ²²-24α-ethyl > trans-Δ²²-24β-ethyl = 24α-E-ethylidene = 24α-methyl > 24β-methyl = trans-Δ²²-24β-methyl = 26-methyl = saturated C₇ side chain and C-20 R (17-αH, 20-αH, right-handed conformer) = cis-Δ²²-C₇ side chain and C-20 R > saturated C₇ side chain and C-20 S (17-αH, 20-βH, right-handed conformer) > no sterol = 29-hydroxyporiferasterol = 20α-hydroxycholesterol = 24ξ-hydroxy-24-vinylcholesterol. Of the sterols examined the most significant stereochemical criterion for the induction of oospore formation was absence of bulk on the front face of C-20. This follows from the observation that 20-isocholesterol and 20α-hydroxycholesterol, in which a methyl and hydroxy group, respectively, project to the front in the right handed conformation, were inactive in stimulating production of oospores. None of the triterpenoids studied induced oospore formation to any significant degree. Oospore formation was not induced by antheridiol nor 29-hydroxyporiferasterol in combination or added separately to growing cultures of P. cactorum in the concentration range 0.01 - 10.0 milligrams per liter.     

Effect of Delta-9-Tetrahydrocannabinol on Mouse Resistance to Systemic Candida albicans Infection

Delta-9-tetrahydrocannabinol (Δ⁹-THC), the psychoactive component of marijuana, is known to suppress the immune responses to bacterial, viral and protozoan infections, but its effects on fungal infections have not been studied. Therefore, we investigated the effects of chronic Δ⁹-THC treatment on mouse resistance to systemic Candida albicans (C. albicans) infection. To determine the outcome of chronic Δ⁹-THC treatment on primary, acute systemic candidiasis, c57BL/6 mice were given vehicle or Δ⁹-THC (16 mg/kg) in vehicle on days 1–4, 8–11 and 15–18. On day 19, mice were infected with 5×10⁵ C. albicans. We also determined the effect of chronic Δ⁹-THC (4–64 mg/kg) treatment on mice infected with a non-lethal dose of 7.5×10⁴ C. albicans on day 2, followed by a higher challenge with 5×10⁵ C. albicans on day 19. Mouse resistance to the infection was assessed by survival and tissue fungal load. Serum cytokine levels were determine to evaluate the immune responses. In the acute infection, chronic Δ⁹-THC treatment had no effect on mouse survival or tissue fungal load when compared to vehicle treated mice. However, Δ⁹-THC significantly suppressed IL-12p70 and IL-12p40 as well as marginally suppressed IL-17 versus vehicle treated mice. In comparison, when mice were given a secondary yeast infection, Δ⁹-THC significantly decreased survival, increased tissue fungal burden and suppressed serum IFN-γ and IL-12p40 levels compared to vehicle treated mice. The data showed that chronic Δ⁹-THC treatment decreased the efficacy of the memory immune response to candida infection, which correlated with a decrease in IFN-γ that was only observed after the secondary candida challenge.     

Identification of a Sterol Δ7 Reductase Gene Involved in Desmosterol Biosynthesis in Mortierella alpina 1S-4

Molecular cloning of the gene encoding sterol Δ7 reductase from the filamentous fungus Mortierella alpina 1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated MoΔ7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoΔ7SR showed highest homology of 51% with that of sterol Δ7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the MoΔ7SR gene in yeast Saccharomyces cerevisiae revealed that MoΔ7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Δ7 reductase. In addition, with gene silencing of MoΔ7SR gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that MoΔ7SR is involved in desmosterol biosynthesis in M. alpina 1S-4. This study is the first report on characterization of sterol Δ7 reductase from a microorganism.     

Accumulation of 9β,19-cyclopropyl sterols in suspension cultures of bramble cells cultured with tridemorph

The addition of tridemorph, a systemic fungicide, to the medium of suspension cultures of bramble cells resulted after 4 weeks of growth in a strong accumulation of 9β,19-cyclopropyl sterols (90% of total sterols in treated cells) and in the disappearance of δ⁵-sterols (98% of total sterols in control cells). Cycloeucalenol and 24-methylene pollinastanol (both together constitute 70% of total sterols) are the major sterols of treated cells. Tridemorph probably inhibits the cyclocucalenol-obtusifoliol isomerase. As the fungicide impairs only slightly the growth of the cells, the possibility that 9β,19-cyclopropyl sterols substitute for δ⁵-sterols in the membranes of the treated cells is considered.