Ergosterol reduction impairs mitochondrial DNA maintenance in S. cerevisiae

Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae, the enzymes of the ergosterol biosynthetic pathway (Erg proteins) are localized in different cellular compartments. With the aim of studying organelle interactions, we discovered that Erg27p resides mainly in Lipid Droplets (LDs) in respiratory competent cells, while in absence of respiration, is found mostly in the ER. The results presented in this paper demonstrate an interplay between the mitochondrial respiration and ergosterol production: on the one hand, rho° cells show lower ergosterol content when compared with wild type respiratory competent cells, on the other hand, the ergosterol biosynthetic pathway influences the mitochondrial status, since treatment with ketoconazole, which blocks the ergosterol pathway, or the absence of the ERG27 gene, induced rho° production in S. cerevisiae. The loss of mitochondrial DNA in the ∆erg27 strain is fully suppressed by exogenous addition of ergosterol. These data suggest the notion that ergosterol is essential for maintaining the mitochondrial DNA attached to the inner mitochondrial membrane

Plant-based beverages as good sources of free and glycosidic plant sterols

To address the ever-growing group of health-conscious consumers, more and more nutritional and health claims are being used on food products. Nevertheless, only very few food constituents, including plant sterols, have been appointed an approved health claim (European Commission and Food and Drugs Administration). Plant sterols are part of those limited lists of approved compounds for their cholesterol-lowering properties but have been praised for their anti-inflammatory and anti-carcinogenic properties as well. Despite this indisputable reputation, direct quantitative data is still lacking for naturally present (conjugated) plant sterols in beverages. This study aimed to fill this gap by applying a validated extraction and UPLC-MS/MS detection method to a diverse range of everyday plant-based beverages. B-sitosterol--D-glucoside (BSSG) showed to be by far the most abundant sterol in all beverages studied, with concentrations up to 60–90 mg per 100 mL in plant-based milk alternatives and fresh fruit juices. Ergosterol (provitamin D2) could be found in beers (0.8–6.1 g per 100 mL, from the yeast) and occasionally in juices (17–29 g per 100 mL). Overall, the results demonstrated that the concentrations of water-soluble sterol conjugates have been underestimated significantly and that specific plant-based beverages can be good, low-fat sources of these plant sterols

Anti-Candida targets and cytotoxicity of casuarinin isolated from Plinia cauliflora leaves in a bioactivity-guided study

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Quantifying carbon allocation to mycorrhizal fungi by temperate forest tree species across a nitrogen availability gradient

Terrestrial ecosystems make up the largest carbon pool with a major portion of that being forests. With carbon being a major concern due to global climate change, being able to make accurate models is increasingly important. Studies have shown that trees may allocate up to 50% of their photosynthetically fixed carbon underground; however these values haven’t been accurately quantified and underground carbon allocation has been historically overlooked. Mycorrhizal fungi may be a large portion of underground carbon allocation, as they have a symbiotic relationship with trees where they provide the plant with water and nutrients in return for sugars (carbon). New methods and knowledge will allow us to quantify carbon allocation and fungal biomass. Ergosterol is a biomarker that is the human equivalent of cholesterol for fungi which can be used to measure fungal biomass. Since both free-living and mycorrhizal fungi have ergosterol, a series of open and closed cores located at Bartlett Experimental Forest will separate the amount of ergosterol due to free-living versus mycorrhizal fungi. This is one of the first studies that will quantify fungal biomass and carbon allocation under a variation of natural settings and compare two different methods to estimate these values

Synergistic Anti-Candida Activity of Bengazole A in the Presence of Bengamide A †.

Bengazoles A⁻G from the marine sponge Jaspis sp. exhibit potent in vitro antifungal activity against Candida spp. and other pathogenic fungi. The mechanism of action (MOA) of bengazole A was explored in Candida albicans under both liquid culture and surface culture on Mueller-Hinton agar. Pronounced dose-dependent synergistic antifungal activity was observed with bengazole A in the presence of bengamide A, which is also a natural product from Jaspis sp. The MOA of bengazole A was further explored by monitoring the sterol composition of C. albicans in the presence of sub-lethal concentrations of bengazole A. The GCMS of solvent extracts prepared from liquid cultures of C. albicans in the presence of clotrimazole-a clinically approved azole antifungal drug that suppresses ergosterol biosynthesis by the inhibition of 14α-demethylase-showed reduced cellular ergosterol content and increased concentrations of lanosterol and 24-methylenedihydrolanosterol (a shunt metabolite of ergosterol biosynthesis). No change in relative sterol composition was observed when C. albicans was cultured with bengazole A. These results eliminate an azole-like MOA for the bengazoles, and suggest that another as-yet unidentified mechanism is operative

Biologically active compounds from selected aphyllophorales mycelial cultures

The obtained results confirm the hypothesis that mycelial cultures of domestic species of aphyllophorales are able to accumulate biologically active metabolites.Uzyskane wyniki wskazują, że przebadane kultury mycelialne krajowych gatunków grzybów afylloforoidalnych są zdolne do akumulacji metabolitów aktywnych biologicznie

Ras hyperactivation versus overexpression : Lessons from Ras dynamics in Candida albicans

We thank Prof. Neta Dean for the CIp10ADH1-Cherry plasmid and Prof. Aaron Mitchell for the BWP17 strain. We gratefully acknowledge Prof. Sudipta Maiti, TIFR, Mumbai, India for providing the data acquisition software. We also appreciate the feedback and discussions with Dr. Rohini Muthuswami, SLS, JNU as well as from the Protein Society group, New Delhi while this study was taking shape. We thank Prof. Alok Bhattacharya for Cytochalasin D. The GC-MS and fluorescence lifetime measurements were carried out at the Advanced Instrumentation Research Facility (AIRF), JNU. Confocal images were recorded either at the central instrumentation facility (CIF), SLS, JNU or at AIRF, JNU. This work was supported by project grants from Department of Biotechnology (DBT, Project grant no. BT/PR20410/BRB/10/1542/2016) and Department of Science and Technology (DST, Project grant no. SB/SO/BB-011/2014), India to S.S.K; and project grants from Department of Information Technology, (DIT, Project grant no. 12(4)/2007-PDD), India to S.S. for FCS setup. In addition, both S.S. and S.S.K. thank DBT-BUILDER for funding support (Project grant no. BT/PR5006/INF/153/2012). S.S.K. also acknowledges funding support from UGC Resource Networking grant to the School of Life Sciences. We thank DST-PURSE and JNU for assistance with funding for publication. G.S.V. and S.C.S. received a fellowship from UGC; V.A.P., B.Y., P.J., N.P., M.F.K. acknowledge CSIR for fellowships. S.L.S. received a fellowship from ICMR. D.T.H. and M.F.K. thank DBT-BUILDER for funding.Peer reviewedPublisher PD

The zinc cluster proteins Upc2 and Ecm22 promote filamentation in Saccharomyces cerevisiae by sterol biosynthesis-dependent and -independent pathways.

The transition between a unicellular yeast form to multicellular filaments is crucial for budding yeast foraging and the pathogenesis of many fungal pathogens such as Candida albicans. Here, we examine the role of the related transcription factors Ecm22 and Upc2 in Saccharomyces cerevisiae filamentation. Overexpression of either ECM22 or UPC2 leads to increased filamentation, whereas cells lacking both ECM22 and UPC2 do not exhibit filamentous growth. Ecm22 and Upc2 positively control the expression of FHN1, NPR1, PRR2 and sterol biosynthesis genes. These genes all play a positive role in filamentatous growth and their expression is upregulated during filamentation in an Ecm22/Upc2-dependent manner. Furthermore ergosterol content increases during filamentous growth. UPC2 expression also increases during filamentation and is inhibited by the transcription factors Sut1 and Sut2. The expression of SUT1 and SUT2 in turn is under negative control of the transcription factor Ste12. We suggest that during filamentation Ste12 becomes activated and reduces SUT1/SUT2 expression levels. This would result in increased UPC2 levels and as a consequence to transcriptional activation of FHN1, NPR1, PRR2 and sterol biosynthesis genes. Higher ergosterol levels in combination with the proteins Fhn1, Npr1 and Prr2 would then mediate the transition to filamentous growth.The project was supported by the Deutsche Forschungsgemeinschaft (DFG) grant HO 2098/5

Sterol 3β-glucosyltransferase biocatalysts with a range of selectivities, including selectivity for testosterone

The main objectives of this work were to characterise a range of purified recombinant sterol 3β-glucosyltransferases and show that rational sampling of the diversity that exists within sterol 3β-glucosyltransferase sequence space can result in a range of enzyme selectivities. In our study the catalytically active domain of the Saccharomyces cerevisiae 3β-glucosyltransferase was used to mine putative sterol 3β-glucosyltransferases from the databases. Selected diverse sequences were expressed in and purified from Escherichia coli and shown to have different selectivities for the 3β-hydroxysteroids ergosterol and cholesterol. Surprisingly, three enzymes were also selective for testosterone, a 17β-hydroxysteroid. This study therefore reports for the first time sterol 3β-glucosyltransferases with selectivity for both 3β- and 17β-hydroxysteroids and is also the first report of recombinant 3β-glucosyltransferases with selectivity for steroids with a hydroxyl group at positions other than C-3. These enzymes could therefore find utility in the pharmaceutical industry for the green synthesis of a range of glycosylated compounds of medicinal interest

Comparison of two extraction methods for ergosterol determination in vegetal feeds

Ergosterol is the principal sterol of fungi in which it plays an essential role in cell membrane and other cellular constituents. This sterol is considered as a good marker of fungal contamination and of mycotoxin production. After validation of ergosterol quantification by HPLC-UV system (linearity range: 0.2 to 20.0 mg/ml, repeatability: 3.27%, between day precision: 4.75%), 2 extraction methods of ergosterol from 3 vegetal matrixes (maize, barley and wheat) were compared: the first one, normalized by the AFNOR, is based on solid phase extraction (SPE), while the other is based on liquid/liquid extraction (LLE). The LLE procedure allowed ergosterol extraction gains of around 20% for high initial sterol contents (3 to 5 mg/kg) in naturally contaminated matrixes or in spiked samples, and of 86% for low initial sterol contents (1-2 mg/kg) in maize. Moreover, the precision of ergosterol determination was comparable for the 2 methods even if it was slightly lower using LLE and was more affected by the initial ergosterol contents in vegetal matrix than by its nature. These results suggest that ergosterol contents in vegetal feeds would be underestimated with the official method (SPE) and emphasize the importance of the extraction step