Showing papers on "Lanosterol published in 2010"

Azole Binding Properties of Candida albicans Sterol 14-α Demethylase (CaCYP51)

Abstract: Purified Candida albicans sterol 14-α demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with K(s) values of 11 and 25 μM, respectively, and a K(m) value of 6 μM for lanosterol. Azole binding to CaCYP51 was "tight" with both the type II spectral intensity (ΔA(max)) and the azole concentration required to obtain a half-ΔA(max) being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with K(d) values of 10 to 26 μM under oxidative conditions, compared with 47 μM for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4- and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min(-1)), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.

Expression, Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B

Abstract: Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol ( K s , 8.6 μM) and eburicol ( K s , 22.6 μM). Membrane suspensions of AF51A bound to both lanosterol ( K s , 3.1 μM) and eburicol ( K s , 4.1 μM). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the K d (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 μM, respectively, in comparison with a K d value of 4 μM for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with K d values ranging from 1 μM for itraconazole to 11.9 μM for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus .

Production of individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum.

Abstract: Two-stage culture was efficient in enhancing total ganoderic acid (GA) production by Ganoderma lucidum (Fang and Zhong, Biotechnol Prog 18:51-54, 2002). As different GAs have different bioactivities, it is critical to understand the kinetics of individual GA production during fermentation, but no related information is yet available. To understand the regulation of GA biosynthesis, investigation of the accumulation of intermediate (lanosterol) and by-product (ergosterol) and of the expression of three important biosynthetic genes was also conducted in liquid shaking and static cultures of G. lucidum. The results showed that the content of individual GAs increased rapidly in the liquid static culture, and their maximum value was 6- to 25-fold that of shaking culture while lanosterol content in the former was lower than the latter. The transcript of squalene synthase (SQS), lanosterol synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase in liquid static culture was 4.3-, 2.1-, and 1.9-fold that of the shaking culture, respectively. Higher GA content in liquid static culture was related to increased transcription of those genes especially SQS. The work is helpful to the production of individual GAs and provided an insight into why the liquid static culture was superior to the shaking culture in view of biosynthetic gene expression.

Solubility limits of cholesterol, lanosterol, ergosterol, stigmasterol, and β-sitosterol in electroformed lipid vesicles

Abstract: Here we use nuclear magnetic resonance to measure the solubility limit of several biologically relevant sterols in electroformed giant unilamellar vesicle membranes containing phosphatidylcholine (PC) lipids in ratios of 1:1:X of DOPC:DPPC:sterol. We find solubility limits of cholesterol, lanosterol, ergosterol, stigmasterol, and β-sitosterol to be 65–70 mol%, ∼35 mol%, 30–35 mol%, 20–25 mol%, and ∼40 mol%, respectively. The low solubilities of stigmasterol and β-sitosterol, which differ from cholesterol only in their alkyl tails, show that subtle differences in tail structure can strongly affect sterol solubility. Below the solubility limits, the fraction of sterol to PC-lipid in electroformed vesicles linearly reflects the fraction in the original stock solutions used in the electroformation process.

Improved Model of Lanosterol 14α-Demethylase by Ligand-Supported Homology Modeling: Validation by Virtual Screening and Azole Optimization

Abstract: Lanosterol 14alpha-demethylase (CYP51) is an important target for antifungal drugs. An improved three-dimensional model of CYP51 from Candida albicans (CACYP51) was constructed by ligand-supported homology modeling and molecular dynamics simulations. The accuracy of the constructed model was evaluated by its performance in a small-scale virtual screen. The results show that known CYP51 inhibitors were efficiently discriminated by the model, and it performed better than our previous CACYP51 model. The active site of CACYP51 was characterized by multiple copy simultaneous search (MCSS) calculations. On the basis of the MCSS results, a series of novel azoles were designed and synthesized, and they showed good in vitro antifungal activity with a broad spectrum. The MIC(80) value of four of these compounds against C. albicans is 0.001 microg mL(-1), indicating that they are promising leads for the discovery of novel antifungal agents.

Synthesis and In Vitro Evaluation of Novel 1, 2, 4-Triazole Derivatives as Antifungal Agents

Abstract: Despite the advances in medicine and the emergence of new antifungal agents, fungal infections remain a significant cause of morbidity and mortality. Azoles are widely used as antifungal agents. Azoles interfere with the conversion of lanosterol to ergosterol by inhibiting a fungal cytochrome P450enzyme, lanosterol 14
-demethylase. Resistance to azoles, particularly fluconazole, is emerging to Candida albicans , after long-term suppressive therapy. Thus, there is an urgent need for newer potent antifungals to combat resistance developed against widely used azoles. In present work, we report synthesis of novel triazole derivatives of 7-hydroxy-4-methylcoumarin using various substituted aromatic aldehydes and evaluated for their in vitro fungicidal activity against Candida albicans at various con-centrations to obtain minimum inhibitory concentration (MIC). Keyword: Azole, Antifungal, Minimum inhibitory concentration, Coumarin, Triazole. INTRODUCTION Fungal infections remain a significant cause of morbidity and mortality despite advances in medicinal chemistry. Antifungal drug discovery has identified three classes of natural products (griseo-fulvin [1], polyenes [2] and echinocandins [3]) and four classes of synthetic chemicals (azoles [4], allylamines [5], flucytosine [6] and phenylmorpholines [7]) with clinical value against fungal infec-tions. The azoles class of antifungal agent is chemically either an imidazole or a triazole group joined to an asymmetric carbon atom as their functional pharmacophore. They all work by blocking the active site of an enzyme variously known as lanosterol 14
-demethylase or cytochrome P450

Functional Characterization of the Chlamydomonas reinhardtii ERG3 Ortholog, a Gene Involved in the Biosynthesis of Ergosterol

Abstract: Background The predominant sterol in the membranes of the alga Chlamydomonas reinhardtii is ergosterol, which is commonly found in the membranes of fungi, but is rarely found in higher plants. Higher plants and fungi synthesize sterols by different pathways, with plants producing cycloartenol as a precursor to end-product sterols, while non-photosynthesizing organisms like yeast and humans produce lanosterol as a precursor. Analysis of the C. reinhardtii genome sequence reveals that this algae is also likely to synthesize sterols using a pathway resembling the higher plant pathway, indicating that its sterols are synthesized somewhat differently than in fungi. The work presented here seeks to establish experimental evidence to support the annotated molecular function of one of the sterol biosynthetic genes in the Chlamydomonas genome. Methodology/Principal Findings A gene with homology to the yeast sterol C-5 desaturase, ERG3, is present in the Chlamydomonas genome. To test whether the ERG3 ortholog of C. reinhardtii encodes a sterol C-5 desaturase, Saccharomyces cerevisiae ERG3 knockout strains were created and complemented with a plasmid expressing the Chlamydomonas ERG3. Expression of C. reinhardtii ERG3 cDNA in erg3 null yeast was able to restore ergosterol biosynthesis and reverse phenotypes associated with lack of ERG3 function. Conclusions/Significance Complementation of the yeast erg3 null phenotypes strongly suggests that the gene annotated as ERG3 in C. reinhardtii functions as a sterol C-5 desaturase.

Changes in cholesterol biosynthetic and transport pathways after excitotoxicity.

Abstract: The present study was carried out to elucidate changes in the gene expression and activity of cholesterol biosynthetic enzymes and transporters in the rat hippocampus after kainate excitotoxicity. Significantly increased cholesterol level was detected in the degenerating hippocampus, reaching double normal levels at 1 week after kainate injury. RT-PCR analyses of hippocampal homogenates showed significantly decreased mRNA expression of the transcription factor controlling cholesterol biosynthesis SREBP-2, and the rate-controlling enzyme HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase at all time points after kainate injection; and decreased lanosterol synthase and CYP51 at 1 and 2 weeks post-kainate injection respectively. GC-MS analyses showed a significant increase in cholesterol biosynthetic precursors lanosterol, desmosterol and 7-dehydrocholesterol at 1 day after kainate injection presumably reflecting biosysnthesis in injured neurons, and significant decreases in precursors at 1 and 2 weeks post-kainate injection, at time of gliosis in the degenerating hippocampus. Levels of cholesterol autooxidation including 7 ketocholesterol and cholesterol epoxides were elevated in the kainate lesioned hippocampus. Furthermore, loss of expression of the cholesterol transporter, ABCA1 was detected in neurons, but increased expression in astrocytes was detected after kainate lesions. The results suggest that increased cholesterol biosynthesis and loss of ABCA1 expression in injured neurons might result in increase in cholesterol in the degenerating hippocampus. The increased cholesterol might predispose to increased formation of cholesterol oxidation products which have been shown to be toxic to neurons.

Cloning and Characterization of the Lanosterol 14α-Demethylase Gene from Antrodia cinnamomea

Abstract: Sterol 14α-demethylase (CYP51) is one of the key enzymes for sterol biosynthesis in fungi; it is widely distributed in all members of the cytochrome P450 superfamily. In this study, AcCyp51, encodi...

Production of non-yeast sterols by yeast

Abstract: This invention relates to the production of 7-dehydrocholesterol, 25-hydroxy-7-dehydrocholesterol, and 25-hydroxy ergosterol in yeast such as Saccharomyces cerevisiae. It also related to various enzymes catalyzing the reduction of the double bond at position 24 of lanosterol, dimethyl zymosterol, methyl zymosterol, zymosterol, cholesta-7, 24-dienol, or cholesta-5,7,24-trienol; or the hydroxylation at position 25 of ergosterol, 7-dehydrocholesterol, cholesta-8-enol, and cholesta-7-enol. It also relates to various nucleic acids encoding cholesterol C25 -hydroxylases and sterol Δ24-reductases and their use to produce and hydroxylate 7-dehydrocholesterol or ergosterol. It also relates to the yeast strains so produced, and methods of making these sterols comprising the steps of cultivation a transformed yeast cell, and harvesting the resulting sterol(s).

Functional characterization and localization of Pneumocystis carinii lanosterol synthase.

Abstract: Organisms in the genus Pneumocystis are ubiquitous, opportunistic pathogenic fungi capable of causing a lethal pneumonia in immunocompromised mammalian hosts. Pneumocystis spp. are unique members of the fungal kingdom due to the absence of ergosterol in their cellular membranes. Although these organisms were thought to obtain cholesterol by scavenging, transcriptional analyses indicate that Pneumocystis carinii encodes gene homologs involved in sterol biosynthesis. To better understand the sterol pathway in these uncultivable fungi, yeast deletion strains were used to interrogate the function and localization of P. carinii lanosterol synthase (ERG7). The expression of PcErg7p in an ERG7-null mutant of the yeast Saccharomyces cerevisiae did not alter its growth rate and produced a functional lanosterol synthase, as evidenced by the presence of lanosterol detected by gas chromatographic analysis in levels comparable to that produced by the yeast enzyme. Western blotting and fluorescence microscopy revealed that, like the S. cerevisiae Erg7p, the PcErg7p localized to lipid particles in yeast. Using fluorescence microscopy, we show for the first time the presence of apparent lipid particles in P. carinii and the localization of PcErg7p to lipid particles in P. carinii. The detection of lipid particles in P. carinii and their association with PcErg7p therein provide strong evidence that the enzyme serves a similar function in P. carinii. Moreover, the yeast heterologous system should be a useful tool for further analysis of the P. carinii sterol pathway.

Standardization of chaga tincture and befungin

Abstract: Methods for standardizing the formulations Chaga Tincture and Befungin were developed, consisting of extraction of the non-phenol fraction predominantly containing tetracyclic triterpenes, including lanosterol and ergosterol, followed by spectrophotometry of colored complexes with vanillin in acidic conditions. The contents of tetracyclic triterpenes expressed as lanosterol ranged from 0.01% to 0.035% in Chaga Tincture and from 0.01% to 0.02% in Befungin.

Method for separating and purifying crude lanosterol product

Abstract: The invention discloses a method for separating and purifying a crude lanosterol product in the technical field of the purification of organic substances, which comprises the steps of: dissolving the crude lanosterol product into an organic solvent at a temperature of between 25 and 40 DEG C through ultrasonic treatment to obtain a lanosterol solution; and filtering the lanosterol solution by a filter membrane, then using preparative high performance liquid chromatography to adopt overlapping injection for separation and purification treatment to obtain purified lanosterol. The method adopts reversed-phase high performance liquid chromatography, can ensure the characteristics of good resolution, high yield, high recovery rate, good purity and the like, can directly separate to obtain the lanosterol with the purity over 97 percent, and can be used for various sterol-related diseases and the research and development of biosynthetic pathways of various natural products. The method applies technology such as overlapping injection and linear amplification to overcome the disadvantage of low solubility caused by using reversed-phase chromatography to separate the lanosterol, uses a preparation column with the inner diameter of 20mm and the column length of 250mm, ensures that the daily preparation amount can reach near 250mg, and can meet various scientific and research requirements.

Peruvianosides A and B, Novel Triterpene Glycosides from the Bulbs of Scilla peruviana.

Abstract: Two new rearranged lanosterol trisaccharides, peruvianosides A (1) and B (2), were isolated from fresh bulbs of Scilla peruviana. Their structures were established mainly by extensive 2D NMR analysis. Peruvianoside A (1) showed an inhibitory activity on cyclic adenosine monophosphate (AMP) phosphodiesterase.

Method for extracting cholesterine in lanosterol by supercritical CO2

Abstract: A method for extracting cholesterol from lanosterol by supercritical CO2 is carried out by putting lanosterol and laminating agent into extractor, delivering CO2 into extractor by compressing pump, adjusting CO2 extractor pressure and temperature, controlling CO2 flow speed, extracting to obtain CO2 with solute, decompressing CO2 with solute, separating to obtain cholesterol and lanosterol, dissolving for cholesterol by solvent, lowering temperature and crystallizing to obtain high-content final product It's simple and inodorous, has better purity and light color

Modulators of lanosterol synthetase for treating acne or hyperseborrhea

Abstract: An in vitro method for screening candidate compounds for the preventive or curative treatment of acne, includes the determination of the capacity of a compound to modulate the expression or the activity of lanosterol synthetase (LSS), and the use of modulators of the expression or activity of this enzyme for the treatment of acne or skin disorders associated with a hyperseborrhea; methods for the in vitro diagnosis or prognosis of these pathologies are also described.

H03 Cerebral and extracerebral cholesterol biosynthesis is impaired in Huntington's disease patients

Abstract: Background Cholesterol in the CNS is fundamental for membrane trafficking, signal transduction and synaptogenesis and almost all the brain cholesterol is synthetised locally. Homeostasis is maintained through a neuronal specific cholesterol 24-hydroxylase which converts cholesterol into 24-hydroxycholesterol (24OHC). The levels of 24OHC in blood thus reflect the number of active neurons and the volume of the grey matter. We previously found that plasma levels of 24OHC were progressively reduced in early Huntington9s disease (HD)1 and that cholesterol biosynthesis was significantly impaired in the brain of multiple mouse HD models, and in fibroblasts of HD patients.2 Methods We analysed by mass spectrometry plasma levels of the cholesterol precursor lathosterol and lanosterol (marker of cholesterol biosynthesis), 24OHC and bile acid precursor 27-hydroxycholesterol, in 130 HD gene positive subjects (from presymptomatic to advanced stage of disease). Results We found that plasma levels of lathosterol and lanosterol, together with 24OHC and 27OHC, were reduced in all HD patients compared with healthy controls (p Conclusion Whole body cholesterol synthesis and brain derived 24OHC were significantly decreased following the disease burden. We suggest that the significant decrease in plasma 24OHC levels we reported in early HD1 are associated with two concurrent mechanisms, that is, the progressive loss in grey matter linked to the neurodegenerative process and the impairment in brain cholesterol biosynthesis, in agreement with our previous findings across multiple rodent models of HD2.