Ergosterol biosynthesis in novel melanized fungi from hypersaline environments.
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Halotolerant and halophilic melanized fungi were recently described in hypersaline waters. A close study of the sterol composition of such fungi, namely Hortaea werneckii, Alternaria alternata, Cladosporium sphaerospermum, Cladosporium sp., and Aureobasidium pullulans revealed the dominance of ergosterol and the presence of 29 intermediates of its biosynthesis pathway. The presence or absence of intermediates from distinct synthesis routes gave insight into the operative synthetic pathways from 4,4,14-trimethylcholesta-8,24-dien-3 beta-ol (lanosterol) to ergosterol in melanized fungi and in Saccharomyces cerevisiae, a reference yeast cultured in parallel. In all studied melanized fungi, initial methylation at C-24 took place before C-14 and C-4 demethylation, involving a different reaction sequence from that observed in S. cerevisiae. Further transformation was observed to occur through various routes. In A. alternata, isomerization at C-7 takes place prior to desaturation at C-5 and C-22, and methylene reduction at C-24. In addition to these pathways in Cladosporium spp., H. werneckii, and A. pullulans, ergosterol may also be synthesized through reduction of the C-24 methylene group before desaturation at C-5 and C-22 or vice versa. Moreover, in all studied melanized fungi except A. alternata, ergosterol biosynthesis may also proceed through C-24 methylene reduction prior to C-4 demethylation. -- Mejanelle, L., J. F. Lopez, N. Gunde-Cimerman, and J. O. Grimalt. Ergosterol biosynthesis in novel melanized fungi from hypersaline environments. J. Lipid Res. 2001. 42: 352--358. (+info)
Molecular dynamics simulation of the structure of dimyristoylphosphatidylcholine bilayers with cholesterol, ergosterol, and lanosterol.
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Five molecular dynamics computer simulations were performed on different phospholipid:sterol membrane systems in order to study the influence of sterol structure on membrane properties. Three of these simulated bilayer systems were composed of a 1:8 sterol:phospholipid ratio, each of which employed one of the sterol molecules: cholesterol, ergosterol, and lanosterol. The two other simulations were of a bilayer with a 1:1 sterol:phospholipid ratio. These simulations employed cholesterol and lanosterol, respectively, as their sterol components. The observed differences in simulations with cholesterol and lanosterol may have their implication on the form of the phospholipid/sterol phase diagram. (+info)
A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids.
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Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e. mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy. Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane. This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol. As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast [Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F. & Daum, G. (1995) Biochim. Biophys. Acta 1234, 214-220], although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several-fold the activity in MAM and also in the bulk endoplasmic reticulum. In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and steroldelta24-methyltransferase (Erg6p), are highly enriched in PAM. A possible role of PAM in the supply of lipids to the plasma membrane is discussed. (+info)
Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes.
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Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 microg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 microg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r(2) = 0.83 to 0.98) and highly significant (P << 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments. (+info)
Effect of aggregation of amphotericin B on lysophosphatidylcholine micelles as related to its complex formation with cholesterol or ergosterol.
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The effect of aggregation of amphotericin B (AmB), as well as the complex formation of AmB with cholesterol or ergosterol, was investigated in micelles and vesicles. AmB in lysophosphatidylcholine (LPC) micelles adopted a more favorable monomeric form than that in other drug formulations. At an LPC/AmB ratio of 200, AmB existed only in monomeric form. Such monomeric behavior is likely dependent upon the fluidity and size of the micelles. In LPC micelles composed of 90% monomeric AmB, AmB-ergosterol complex formation occurred with an increase in the sterol concentration, but the complex formation of AmB-cholesterol was slight. On the other hand, in LPC micelles composed of 40% monomeric AmB, the complex formation of AmB-cholesterol as well as AmB-ergosterol was extensive. These results suggest that the complex formation of AmB with both sterols is highly dependent upon the aggregated state of AmB. In addition, using monolayers, mixtures of AmB/LPC/ergosterol were became more stable with rising temperature, while the stability of mixtures of AmB/LPC/cholesterol remained unchanged, implying that complex formation of AmB with cholesterol is different from that of AmB with ergosterol. (+info)
Isolation of an antitumor compound from Agaricus blazei Murill and its mechanism of action.
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The Basidiomycete fungus Agaricus blazei Murill has traditionally been used as a health food for the prevention of cancer, diabetes, hyperlipidemia, arteriosclerosis and chronic hepatitis. In the present study, we examined the antitumor activities of various substances isolated from the lipid fraction of A. blazei. Tumor growth was retarded by the oral administration of the lipid fraction extracted from A. blazei with a chloroform/methanol mixture in sarcoma 180-bearing mice. The substance with the antitumor activity in the lipid fraction was isolated via silica gel column chromatography, eluted with an acetonitrile/methanol (3:2) mixture and identified as ergosterol by direct comparison of the (1)H NMR and mass spectrometry spectral data of an authentic sample. The oral administration of ergosterol to sarcoma 180-bearing mice significantly reduced tumor growth at doses of 400 and 800 mg/kg administered for 20 d without side effects, such as the decreases in body, epididymal adipose tissue, thymus, and spleen weights and leukocyte numbers induced by cancer chemotherapy drugs. Ergosterol had no cytotoxicity against tumor cells. To clarify the antitumor activity of ergosterol, we examined the effects of ergosterol on tumor-induced angiogenesis using two in vivo models. Intraperitoneal administration of ergosterol at doses of 5, 10 and 20 mg/kg for 5 consecutive d inhibited the neovascularization induced by Lewis lung carcinoma cell-packed chambers, suggesting that either ergosterol or its metabolites may be involved in the inhibition of tumor-induced neovascularization. Therefore, we further examined the inhibitory effects of ergosterol on Matrigel-induced neovascularization. Female C57BL/6 mice were subcutaneously inoculated with Matrigel containing acidic fibroblast growth factor and heparin with or without ergosterol. Ergosterol inhibited the Matrigel-induced neovascularization, suggesting that ergosterol directly inhibits Matrigel-induced neovascularization. From these results, it seems likely that the antitumor activity of ergosterol might be due to direct inhibition of angiogenesis induced by solid tumors. This is the first report of ergosterol as an antiangiogenic substance. (+info)
Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae.
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To identify regulatory cis-elements in the proximal promoter of the yeast ERG9 squalene synthase gene, promoter deletion analysis was performed. This approach identified two regulatory elements, one an upstream repressing cis-element (URS), and the other an upstream activating cis-element (UAS). Electromobility shift assays (EMSAs) demonstrated that distinct proteins bind each element. Genetic screens were performed to identify yeast mutants that altered expression of ERG9 promoter-reporter gene fusions. Three non-ergosterol biosynthetic pathway genes were identified. A mutation in TPO1(YLL028W) led to a 5.5-fold increase in ERG9 expression while mutations in YER064C and SLK19 (YOR195W) led to a 3.1- and 5.6-fold decrease, respectively. Deletion analysis of these genes demonstrated that TPO1 and SLK19 specifically regulated ERG9 expression when tested with several different promoter-reporter gene fusions. Additionally, EMSAs demonstrated that extracts derived from the TPO1 deletion strain was unable to shift the repressing cis-element while protein extracts from the SLK19 deletion strain had a reduced shift of the activating cis-element. Furthermore, these two mutants showed quantitative differences in sterols and antifungal drug susceptibilities consistent with their role in regulating ERG9 expression. (+info)
A photophysical study of the polyene antibiotic filipin. Self-aggregation and filipin--ergosterol interaction.
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Filipin, a macrolide polyene antibiotic, is known to interact selectively with ergosterol, a constituent of fungi membranes. In this work, the fluorescence resonance energy transfer (FRET) between a fluorescent analog of ergosterol, dehydroergosterol (DHE), and filipin was measured in small unilamellar vesicles of dipalmitoylphosphatidylcholine at 25 degrees C. The time-resolved FRET results were rationalized in the framework of the mean concentration model, and were complemented with steady-state fluorescence intensity, anisotropy and absorption measurements. The results point to the formation of both DHE--filipin aggregates (evidence from static quenching of DHE fluorescence by filipin) and filipin--filipin aggregates (evidence from: (i) the FRET acceptor concentration distributions; (ii) spectral changes of filipin absorption in the vesicles, the excitonic interaction suggesting a stack arrangement; (iii) filipin fluorescence self-quenching), even in presence of DHE and low antibiotic mole fractions (<1 mol%). These results point out that apparently contradictory biochemical models for the action of filipin (some based on the presence of sterols, others not) can be equally valid. Moreover, since results (ii) and (iii) are also observed when a sterol is present, both models of action can actually coexist in membranes with a low sterol content. (+info)