Same host-plant, different sterols: variation in sterol metabolism in an insect herbivore community.

PubMed

Janson, Eric M; Grebenok, Robert J; Behmer, Spencer T; Abbot, Patrick

2009-11-01

Insects lack the ability to synthesize sterols de novo, which are required as cell membrane inserts and as precursors for steroid hormones. Herbivorous insects typically utilize cholesterol as their primary sterol. However, plants rarely contain cholesterol, and herbivorous insects must, therefore, produce cholesterol by metabolizing plant sterols. Previous studies have shown that insects generally display diversity in phytosterol metabolism. Despite the biological importance of sterols, there has been no investigation of their metabolism in a naturally occurring herbivorous insect community. Therefore, we determined the neutral sterol profile of Solidago altissima L., six taxonomically and ecologically diverse herbivorous insect associates, and the fungal symbiont of one herbivore. Our results demonstrated that S. altissima contained Delta(7)-sterols (spinasterol, 22-dihydrospinasterol, avenasterol, and 24-epifungisterol), and that 85% of the sterol pool existed in a conjugated form. Despite feeding on a shared host plant, we observed significant variation among herbivores in terms of their qualitative tissue sterol profiles and significant variation in the cholesterol content. Cholesterol was absent in two dipteran gall-formers and present at extremely low levels in a beetle. Cholesterol content was highly variable in three hemipteran phloem feeders; even species of the same genus showed substantial differences in their cholesterol contents. The fungal ectosymbiont of a dipteran gall former contained primarily ergosterol and two ergosterol precursors. The larvae and pupae of the symbiotic gall-former lacked phytosterols, phytosterol metabolites, or cholesterol, instead containing an ergosterol metabolite in addition to unmetabolized ergosterol and erogsterol precursors, thus demonstrating the crucial role that a fungal symbiont plays in their nutritional ecology. These data are discussed in the context of sterol physiology and metabolism in insects, and the potential ecological and evolutionary implications.

Synergistic activity of magnolol with azoles and its possible antifungal mechanism against Candida albicans.

PubMed

Sun, L-M; Liao, K; Liang, S; Yu, P-H; Wang, D-Y

2015-04-01

The goal of this study was to investigate the synergic effects between magnolol and azoles, and the potential antifungal mechanisms. Microdilution checkerboard, time-kill and agar diffusion assay were employed to evaluate the synergic effects between magnolol and fluconazole (FLC). Magnolol significantly decreased the efflux of rhodamine 123 (Rh123), leading to greater intracellular accumulation of Rh123 in Candida albicans cells. Compared to the Candida drug resistance (cdr) 2 or multidrug resistance (mdr) 1 deletion mutant, the growth of cdr1 strain was most sensitive to magnolol exposure. In the presence of magnolol, MDR1 overexpressing cells were sensitive to FLC, whereas CDR1 and CDR2 overexpressing cells displayed tolerance to FLC. Magnolol treatment correlated with up-regulation of transporter and ergosterol biosynthesis pathway genes, analyzed by real-time reverse transcription-polymerase chain reaction. The ergosterol content of C. albicansSC5314 was significantly decreased after magnolol exposure. Magnolol synergizes with azoles for targeting of C. albicans by inducing a higher intracellular content of antifungals, by tapping into the competitive effect of ABC transporter Cdr1p substrates, and enhancing the effect by targeting of the ergosterol biosynthesis pathway. Our results provide the first evidence that magnolol may function as a Cdr1p substrate and as an inhibitor of ergosterol biosynthesis. This function can thus be exploited in combination with azoles to reverse multidrug resistance of C. albicans strains. © 2014 The Society for Applied Microbiology.

A natural food ingredient based on ergosterol: optimization of the extraction from Agaricus blazei, evaluation of bioactive properties and incorporation in yogurts.

PubMed

Corrêa, Rúbia C G; Barros, Lillian; Fernandes, Ângela; Sokovic, Marina; Bracht, Adelar; Peralta, Rosane M; Ferreira, Isabel C F R

2018-03-01

In recent years, mycosterols have emerged as potential functional ingredients for the development of sterol-enriched food products and dietary supplements. Agaricus blazei is a mushroom rich in bioactive compounds. For commercial purposes, their fruiting bodies must obey rigid morphological criteria. Those not conforming to these criteria are usually discarded, although this does not mean impairment of their content in bioactives. The aim of the present work was to propose the use of commercially discarded A. blazei fruiting bodies for obtaining an extract rich in ergosterol as a fortifier ingredient for yogurts. For extraction, the Soxhlet technology was used and the highest ergosterol yield (around 12%) was achieved in the 5 th cycle, yielding 58.53 ± 1.72 µg of ergosterol per 100 g of mushroom (dry weight). The ergosterol rich extract presented notable antioxidant and antimicrobial properties, besides showing no hepatotoxicity. When added to the yogurts it significantly enhanced their antioxidant properties. Furthermore, it did not significantly alter the nutritional or the individual fatty acid profiles of the final dairy products. Thus, A. blazei fruiting bodies that do not conform to the commercial requirements of the market and are normally discarded could be exploited for obtaining a natural high added-value food additive, following the circular bioeconomy concept.

Application of MCPA herbicide on soils amended with biostimulants: short-time effects on soil biological properties.

PubMed

Tejada, Manuel; García-Martínez, Ana M; Gómez, Isidoro; Parrado, Juan

2010-08-01

In this paper we studied in the laboratory the effect of MCPA herbicide at a rate of 1.5lha(-1) (manufactures rate recommended) on biological properties of a Plagic Antrosol amended with four biostimulants (WCDS, wheat condensed distillers soluble; PA-HE, hydrolyzed poultry feathers; CGHE, carob germ enzymatic extract; and RB, rice bran extract). Seven hundred grams of soil were mixed with WCDS at a rate of 10%, CGHE at a rate of 4.7%, PA-HE at a rate of 4.3%, and RB at a rate of 4.4%, respectively, in order to applying the same amount of organic matter to the soil (16.38 g organic matter). An unamended polluted and amended non-polluted soil were used as control. For all treatments, the soil ergosterol, dehydrogenase, urease, and phosphatase activities were measured at two incubation times (0 and 60 d). The 16S rDNA-DGGE profiles in all treatments were determined at the beginning and end of the incubation period. The results indicated that at the end of the incubation period and compared with the control soil, the dehydrogenase, urease and phosphatase activities and ergosterol decreased 39.3%, 20%, 15.7% and 56.5%, respectively in the non-organic amended polluted soil. The application of organic matter to unpolluted soil increased the enzymatic activities and ergosterol. However, this stimulation was higher in the soil amended with RB, followed by PA-HE, WCDS and CGHE. The application of herbicide in organic-amended soils decreased the enzymatic activities and ergosterol content. However, this decrease was lower than for the non-amended herbicide polluted soil. Possibly the low molecular weight protein content easily assimilated by soil microorganisms and the adsorption capacity of humic substances are responsible for less inhibition of these enzyme activities and soil ergosterol. The 16S rDNA-DGGE profiles indicated that herbicide did not negatively affect soil bacterial biodiversity. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

[Inhibition effect of Ag-antibiotic 702 on plant pathogenic fungi and related mechanisms].

PubMed

Wei, Sai-Jin; Du, Ya-Nan; Ni, Guo-Rong; Zhang, Hui-Wen; Tu, Guo-Quan; Pan, Xiao-Hua

2012-12-01

To explore the practical application value and action mechanisms of Ag-antibiotic 702 against pathogenic fungi, the inhibition spectrum of Ag-antibiotic 702 was studied by measuring the mycelium growth rate of pathogenic fungi, and the effects of Ag-antibiotic 702 on the membrane permeability of Rhizoctonia solani, a typical pathogenic fungus, were investigated, with the variations of mycelium electrolyte leakage and protein, nucleic acid, and Mg2+ and K+ contents under the action of Ag-antibiotic 702 determined, and the effects of Ag-antibiotic 702 on the cell membrane ergosterol biosynthesis and ultramicrostructure observed. The results showed that the active products of Ag-antibiotic 702 had stronger inhibition effect on 13 test pathogens, among which, Sclerotinia sclerotiorum was most sensitive, with the EC50 being 0.23 microg x mL(-1). As compared with the control, the relative electric conductivity of R. solani treated with Ag-antibiotic 702 was increased by 72.2%, the contents of protein, nucleic acid, and Mg2+ and K+ leaked from the R. solani cells were all increased, while the ergosterol content was decreased by 92.0%. The cell membrane outline was not clear, organelles were badly damaged, and vacuole appeared. It was suggested that the inhibition of ergosterol biosynthesis and the increase of membrane permeability could be the main action mechanisms of Ag-antibiotic 702 against pathogenic fungi.

Toward an Increased Functionality in Oyster ( Pleurotus) Mushrooms Produced on Grape Marc or Olive Mill Wastes Serving as Sources of Bioactive Compounds.

PubMed

Koutrotsios, Georgios; Kalogeropoulos, Nick; Kaliora, Andriana C; Zervakis, Georgios I

2018-06-20

Pleurotus ostreatus, P. eryngii, and P. nebrodensis were cultivated on nonconventional substrates containing grape marc (GMC) or olive mill byproducts (OMB); wheat straw (WHS) served as control. GMC-based media demonstrated equal/better mushroom productivity than WHS for P. eryngii and P. nebrodensis, while the cultivation performance of P. eryngii was improved in OMB-based media. Both GMC and OMB substrates led to large increase of fruit-bodies content in phenolic acids, resveratrol, triterpenic compounds, and ergosterol; in particular, P. eryngii mushrooms presented significantly more total phenolics and exhibited much higher antioxidant activity (2- to 8-fold increase). Furthermore, substrates containing GMC or OMB presented up to 27% increase in mushroom β-glucans. Overall, Pleurotus species responded in a different and mostly substrate-specific manner by selectively absorbing organic compounds. Phenolics and squalene content of substrates correlated very well with mushrooms antioxidant activity and ergosterol, respectively; the same was observed for triterpenics' content of substrates and mushrooms.

Anti-tumor and anti-angiogenic ergosterols from Ganoderma lucidum

NASA Astrophysics Data System (ADS)

Chen, Shaodan; Yong, Tianqiao; Zhang, Yifang; Su, Jiyan; Jiao, Chunwei; Xie, Yizhen

2017-10-01

This study was carried out to isolate chemical constituents from the lipid enriched fraction of Ganoderma lucidum extract and to evaluate their anti-proliferative effect on cancer cell lines and human umbilical vein endothelial cells. Ergosterol derivatives (1-14) were isolated from the lipid enriched fraction of G. lucidum. Their structures were established on the basis of spectroscopic analyses or by comparison of mass and NMR spectral data with those reported previously. Amongst, compound 1 was isolated and identified as a new compound. All the compounds were evaluated for their inhibitory effect on tumor cells and human umbilical vein endothelial cells in vitro. Compounds 9-13 displayed inhibitory activity against two tumor cell lines and human umbilical vein endothelial cells, which indicated that these four compounds had both anti-tumor and anti-angiogenesis activities. Compound 2 had significant selective inhibition against two tumor cell lines, while 3 exhibited selective inhibition against human umbilical vein endothelial cells. The structure–activity relationships for inhibiting human HepG2 cells were revealed by 3D-QASR. Ergosterol content in different parts of the raw material and products of G. lucidum was quantified. This study provides a basis for further development and utilization of ergosterol derivatives as natural nutraceuticals and functional food ingredients, or as source of new potential antitumor or anti-angiogenesis chemotherapy agent.

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane.

PubMed

Solanko, Lukasz M; Sullivan, David P; Sere, Yves Y; Szomek, Maria; Lunding, Anita; Solanko, Katarzyna A; Pizovic, Azra; Stanchev, Lyubomir D; Pomorski, Thomas Günther; Menon, Anant K; Wüstner, Daniel

2018-03-01

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE-containing cells were exposed to membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Determination of microbial colonisation in water-damaged buildings using chemical marker analysis by gas chromatography-mass spectrometry.

PubMed

Szponar, B; Larsson, L

2000-03-01

Gas chromatography-mass spectrometry was used to determine the microbial contents of building materials subjected to water damage in a laboratory experiment and of materials collected from houses affected by water during the flood in Klodzko in south-western Poland, July 1997. The samples were examined for 3-hydroxy fatty acids, markers of bacterial endotoxin, and ergosterol, marker of fungal biomass. The amounts of both 3-hydroxy fatty acids and ergosterol were higher in materials that had been exposed to water than in unexposed ones. All markers were stable in the building materials for at least 6 weeks at room temperature and could thus be used to reveal microbial contamination even when cultivation results for bacteria and fungi were negative. Direct measurement of 3-hydroxy fatty acids and ergosterol in human environments could be a useful method, e.g. in monitoring indoor air as regards presence of potentially harmful microorganisms and microbial constituents.

Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells.

PubMed

Vatsa, Parul; Chiltz, Annick; Luini, Estelle; Vandelle, Elodie; Pugin, Alain; Roblin, Gabriel

2011-07-01

The typical fungal membrane component ergosterol was previously shown to trigger defence responses and protect plants against pathogens. Most of the elicitors mobilize the second messenger calcium, to trigger plant defences. We checked the involvement of calcium in response to ergosterol using Nicotiana plumbaginifolia and Nicotiana tabacum cv Xanthi cells expressing apoaequorin in the cytosol. First, it was verified if ergosterol was efficient in these cells inducing modifications of proton fluxes and increased expression of defence-related genes. Then, it was shown that ergosterol induced a rapid and transient biphasic increase of free [Ca²⁺](cyt) which intensity depends on ergosterol concentration in the range 0.002-10 μM. Among sterols, this calcium mobilization was specific for ergosterol and, ergosterol-induced pH and [Ca²⁺](cyt) changes were specifically desensitized after two subsequent applications of ergosterol. Specific modulators allowed elucidating some events in the signalling pathway triggered by ergosterol. The action of BAPTA, LaCl₃, nifedipine, verapamil, neomycin, U73122 and ruthenium red suggested that the first phase was linked to calcium influx from external medium which subsequently triggered the second phase linked to calcium release from internal stores. The calcium influx and the [Ca²⁺](cyt) increase depended on upstream protein phosphorylation. The extracellular alkalinization and ROS production depended on calcium influx but, the ergosterol-induced MAPK activation was calcium-independent. ROS were not involved in cytosolic calcium rise as described in other models, indicating that ROS do not systematically participate in the amplification of calcium signalling. Interestingly, ergosterol-induced ROS production is not linked to cell death and ergosterol does not induce any calcium elevation in the nucleus. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Ochratoxin A and citrinin loads in stored wheat grains: impact of grain dust and possible prediction using ergosterol measurement.

PubMed

Tangni, E K; Pussemier, L

2006-02-01

Crop storage should be carried out under hygienic conditions to ensure safe products, but sometimes grain dust which has settled from previous storage may be left over and incorporated to the following stored grains. This paper describes the results obtained using a lab model developed in order to assess the impact of grain dust incorporation for its direct contribution as a contaminant but also as an inoculum in stored wheat. Settled grain dust (4 samples) released from Belgian grain storages were collected and analysed by HPLC for ergosterol, ochratoxin A (OTA) and citrinin (CIT) content. For OTA and for ergosterol, there was a high degree of variability in concentrations found in the dust samples (from 17.3-318 ng g(-1) and from 39-823 microg g(-1), respectively) whilst for CIT, the range was less significant (from 137-344 ng g(-1)). Incorporation of grain dust into wheat storage contributed to an increase in the concentrations of mycotoxins in the stored grain. Dust acts as a contaminant and as an inoculum. According to these two ways, patterns of mycotoxin generation vary with the nature of the mycotoxin, the mycotoxigenic potential of dust and the water activity of the wheat. OTA and CIT showed a very versatile image when considering the amounts of toxins produced under the selected experimental conditions. The development of a robust tool to forecast the mycotoxigenicity of dust was based on the determination of ergosterol content as a general marker of fungal biomass. Present results suggest that this predictive tool would only be valid for predicting the contamination level of CIT and OTA at reasonable moisture content (14-20%). The potential risk of having highly contaminated batches from stock to stock may thus occur and this paper discusses possible pathways leading to OTA and CIT contamination either under wet or dry storage conditions. We therefore, recommend taking precautionary measures not only by controlling and maintaining moisture at a reasonable level during storage of the raw materials but also by paying more attention to the cleaning of the stores before loading in the new harvests.

Contamination of pine and birch wood dust with microscopic fungi and determination of its sterol contents.

PubMed

Stuper-Szablewska, Kinga; Rogoziński, Tomasz; Perkowski, Juliusz

2017-06-27

Wood compounds, especially sterols, are connected with the level of contamination with microscopic fungi. Within this study, tests were conducted on wood dust samples collected at various work stations in a pine and birch timber conversion plant. Their contamination with mycobiota was measured as the concentration of ergosterol (ERG) by ultra performance liquid chromatography (UPLC). Another aim of this study was to assess the effect of contamination with microscopic fungi on the sterol contents in wood dusts. Analyses were conducted on five sterols: desmosterol, cholesterol, lanosterol, stigmasterol, and β-sitosterol using UPLC and their presence was confirmed using gas chromatography/mass spectrometry (GC/MS). The results of chemical analyses showed the greatest contamination with mycobiota in birch wood dust. We also observed varied contents of individual sterols depending on the wood dust type. Their highest concentration was detected in birch dust. The discriminant analysis covering all tested compounds as predictors showed complete separation of all tested wood dust types. The greatest discriminatory power was found for stigmasterol, desmosterol, and ergosterol.

Endogenous sterol biosynthesis is important for mitochondrial function and cell morphology in procyclic forms of Trypanosoma brucei.

PubMed

Pérez-Moreno, Guiomar; Sealey-Cardona, Marco; Rodrigues-Poveda, Carlos; Gelb, Michael H; Ruiz-Pérez, Luis Miguel; Castillo-Acosta, Víctor; Urbina, Julio A; González-Pacanowska, Dolores

2012-10-01

Sterol biosynthesis inhibitors are promising entities for the treatment of trypanosomal diseases. Insect forms of Trypanosoma brucei, the causative agent of sleeping sickness, synthesize ergosterol and other 24-alkylated sterols, yet also incorporate cholesterol from the medium. While sterol function has been investigated by pharmacological manipulation of sterol biosynthesis, molecular mechanisms by which endogenous sterols influence cellular processes remain largely unknown in trypanosomes. Here we analyse by RNA interference, the effects of a perturbation of three specific steps of endogenous sterol biosynthesis in order to dissect the role of specific intermediates in proliferation, mitochondrial function and cellular morphology in procyclic cells. A decrease in the levels of squalene synthase and squalene epoxidase resulted in a depletion of cellular sterol intermediates and end products, impaired cell growth and led to aberrant morphologies, DNA fragmentation and a profound modification of mitochondrial structure and function. In contrast, cells deficient in sterol methyl transferase, the enzyme involved in 24-alkylation, exhibited a normal growth phenotype in spite of a complete abolition of the synthesis and content of 24-alkyl sterols. Thus, the data provided indicates that while the depletion of squalene and post-squalene endogenous sterol metabolites results in profound cellular defects, bulk 24-alkyl sterols are not strictly required to support growth in insect forms of T. brucei in vitro. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Transcriptional profiling analysis of Penicillium digitatum, the causal agent of citrus green mold, unravels an inhibited ergosterol biosynthesis pathway in response to citral.

PubMed

OuYang, Qiuli; Tao, Nengguo; Jing, Guoxing

2016-08-11

Green mold caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Previously, we have observed that citral dose-dependently inhibited the mycelial growth of P. digitatum, with the minimum inhibitory concentration (MIC) of 1.78 mg/mL, but the underlying molecular mechanism is barely understood. In this study, the transcriptional profiling of the control and 1/2MIC-citral treated P. digitatum mycelia after 30 min of exposure were analyzed by RNA-Seq. A total of 6355 genes, including 2322 up-regulated and 4033 down-regulated genes, were found to be responsive to citral. These genes were mapped to 155 KEGG pathways, mainly concerning mRNA surveillance, RNA polymerase, RNA transport, aminoacyl-tRNA biosynthesis, ABC transporter, glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation, sulfur metabolism, nitrogen metabolism, inositol phosphate metabolism, fatty acid biosynthesis, unsaturated fatty acids biosynthesis, fatty acid metabolism, and steroid biosynthesis. Particularly, citral exposure affected the expression levels of five ergosterol biosynthetic genes (e.g. ERG7, ERG11, ERG6, ERG3 and ERG5), which corresponds well with the GC-MS results, the reduction in ergosterol content, and accumulation of massive lanosterol. In addition, ERG11, the gene responsible for lanosterol 14α-demethylase, was observed to be the key down-regulated gene in response to citral. Our present finding suggests that citral could exhibit its antifungal activity against P. digitatum by the down-regulation of ergosterol biosynthesis.

Impact of zinc supplementation on the improvement of ethanol tolerance and yield of self-flocculating yeast in continuous ethanol fermentation.

PubMed

Zhao, X Q; Xue, C; Ge, X M; Yuan, W J; Wang, J Y; Bai, F W

2009-01-01

The effects of zinc supplementation were investigated in the continuous ethanol fermentation using self-flocculating yeast. Zinc sulfate was added at the concentrations of 0.01, 0.05 and 0.1 g l(-1), respectively. Reduced average floc sizes were observed in all the zinc-supplemented cultures. Both the ethanol tolerance and thermal tolerance were significantly improved by zinc supplements, which correlated well with the increased ergosterol and trehalose contents in the yeast flocs. The highest ethanol concentration by 0.05 g l(-1) zinc sulfate supplementation attained 114.5 g l(-1), in contrast to 104.1 g l(-1) in the control culture. Glycerol production was decreased by zinc supplementations, with the lowest level 3.21 g l(-1), about 58% of the control. Zinc content in yeast cells was about 1.4 microMol g(-1) dry cell weight, about sixfold higher than that of control in all the zinc-supplemented cultures, and close correlation of zinc content in yeast cells with the cell viability against ethanol and heat shock treatment was observed. These studies suggest that exogenous zinc addition led to a reprogramming of cellular metabolic network, resulting in enhanced ethanol tolerance and ethanol production.

Effect of Fatty Acyl Group and Sterol Composition on Sensitivity of Lecithin Liposomes to Imidazole Antimycotics

PubMed Central

Yamaguchi, Hideyo; Iwata, Kazuo

1979-01-01

The specific affinity for membrane lipids and the membrane selectivity of three imidazole derivatives, clotrimazole, miconazole, and econazole, were studied using various types of liposomes with respect to the lecithin fatty acyl group composition and the liposome content and composition of sterol as membrane models. The sensitivity of liposomes to these drugs was primarily dependent upon the lecithin fatty acyl group composition. With sterol-free liposome systems, each imidazole induced maximum release of trapped glucose as a marker from the unsaturated dioleoyl lecithin liposomes, minimum release from the saturated dipalmitoyl lecithin liposomes, and intermediate release from egg lecithin liposomes. The sensitivity of the dipalmitoyl lecithin liposomes to any imidazole drug was not influenced by the incorporation of cholesterol or ergosterol. On the other hand, clotrimazole-induced permeability changes of liposomes prepared from unsaturated dioleoyl lecithin or egg lecithin were greatly enhanced by the incorporation of ergosterol, whereas they were suppressed by cholesterol incorporation. The sensitivity of liposomes prepared from these unsaturated lecithins to miconazole and econazole was also augmented by ergosterol incorporation, although it was scarcely altered by cholesterol incorporation. Negatively charged liposomes were more sensitive to the three imidazole drugs than positively charged liposomes. PMID:525988

Effect of fatty acyl group and sterol composition on sensitivity of lecithin liposomes to imidazole antimycotics.

PubMed

Yamaguchi, H; Iwata, K

1979-05-01

The specific affinity for membrane lipids and the membrane selectivity of three imidazole derivatives, clotrimazole, miconazole, and econazole, were studied using various types of liposomes with respect to the lecithin fatty acyl group composition and the liposome content and composition of sterol as membrane models. The sensitivity of liposomes to these drugs was primarily dependent upon the lecithin fatty acyl group composition. With sterol-free liposome systems, each imidazole induced maximum release of trapped glucose as a marker from the unsaturated dioleoyl lecithin liposomes, minimum release from the saturated dipalmitoyl lecithin liposomes, and intermediate release from egg lecithin liposomes. The sensitivity of the dipalmitoyl lecithin liposomes to any imidazole drug was not influenced by the incorporation of cholesterol or ergosterol. On the other hand, clotrimazole-induced permeability changes of liposomes prepared from unsaturated dioleoyl lecithin or egg lecithin were greatly enhanced by the incorporation of ergosterol, whereas they were suppressed by cholesterol incorporation. The sensitivity of liposomes prepared from these unsaturated lecithins to miconazole and econazole was also augmented by ergosterol incorporation, although it was scarcely altered by cholesterol incorporation. Negatively charged liposomes were more sensitive to the three imidazole drugs than positively charged liposomes.

Benzoylation of Ergosterol through Nucleophilic Acyl Substitution and Subsequent Formation of Ergosterol Benzoate Endoperoxide by Reaction with Singlet Oxygen Generated by Photosensitization

ERIC Educational Resources Information Center

Roslaniec, Mary C.; Sanford, Elizabeth M.

2011-01-01

Reactive oxygen species such as singlet oxygen have been a major focus of research in medicine. The effect of singlet oxygen on sterols within biological membranes is becoming increasingly more important. Ergosterol, a vitamin D precursor, is one such sterol. The benzoylation of ergosterol and subsequent reaction with singlet oxygen to form an…

Facultative Sterol Uptake in an Ergosterol-Deficient Clinical Isolate of Candida glabrata Harboring a Missense Mutation in ERG11 and Exhibiting Cross-Resistance to Azoles and Amphotericin B

PubMed Central

Hull, Claire M.; Parker, Josie E.; Bader, Oliver; Weig, Michael; Gross, Uwe; Warrilow, Andrew G. S.; Kelly, Diane E.

2012-01-01

We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 μg ml−1, respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14α-demethylase (Erg11p) mutant, wherein 14α-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14α-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium (glcYM). However, when grown on sterol-supplemented glcYM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, Δ7-cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11) that became less sensitive to FLC and VRC when cultured on sterol-supplemented glcYM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using glcYM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using glcYM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 μg AMB ml−1, respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata. PMID:22615281

Creation, characterization and utilization of Cryptococcus neoformans mutants sensitive to micafungin.

PubMed

Toh-E, Akio; Ohkusu, Misako; Shimizu, Kiminori; Yamaguchi, Masashi; Ishiwada, Naruhiko; Watanabe, Akira; Kamei, Katsuhiko

2017-12-01

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding β-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of β-1, 3-glucan synthesis in C. neoformans; the inhibition of β-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized β-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.

[Antifungals cellular targets and mechanisms of resistance].

PubMed

Accoceberry, Isabelle; Noël, Thierry

2006-01-01

Antifungals of systemic use for the treatment of invasive fungal infections belong to four main chemical families which have globally three cellular targets in fungal cells: fluorinated pyrimidines act on deoxyribonucleic acid (DNA) replication and protein synthesis; polyenes and azoles are toxic for ergosterol and its biosynthetic pathway; lipopeptides inhibit the synthesis of cell wall beta glucans. The resistance mechanisms that are developed by some fungi begin to be well understood particularly in Candida yeasts. The underlying bases of these mechanisms are either mutations that modify the antifungal target, or that block access to the target, and, on the other hand, the overexpression of genes encoding the target, or some membrane proteins involved in the active efflux of antifungal drugs.

Ergosterol content of fungi associated with Dendroctonus ponderosae and Dendroctonus rufipennis (Coleoptera: Curculionidae, Scolytinae)

Treesearch

Barbara J. Bentz; Diana L. Six

2006-01-01

Insects require sterols for normal growth, metamorphosis, and reproduction, yet they are unable to synthesize these organic compounds and are therefore dependent upon a dietary source. For phloephagous species, such as Dendroctonus bark beetles, whose food does not necessarily contain appropriate types or adequate quantities of sterols, fungal...

Phenolic Compounds, Antioxidant Activity and Lipid Profile of Huitlacoche Mushroom (Ustilago maydis) Produced in Several Maize Genotypes at Different Stages of Development.

PubMed

Valdez-Morales, Maribel; Carlos, L Céspedes; Valverde, María Elena; Ramírez-Chávez, Enrique; Paredes-López, Octavio

2016-12-01

Huitlacoche mushroom (composed by the fruiting bodies growing on the maize ears from the basidiomycete Ustilago maydis) is a culinary delicacy with a great economic and nutraceutical value. In this work, phenolic content, antioxidant activity, ergosterol and fatty acids profile from huitlacoche produced in 15 creole and in one hybrid maize genotypes, and harvested at different stages of development were determined. The hybrid crop was studied in raw and cooked samples. Total phenolic content ranged from 415.6 to 921.8.0 mg gallic acid equivalents per 100 g of flour. Samples exhibited attractive antioxidant activities: 75 % of antiradical activity on average by DPPH methodology, and ORAC values up to 7661.3 μmol Trolox equivalents /100  g. Important quantities of ferulic acid, quercetin, ergosterol, linoleic and oleic acids were observed. Stage of development and cooking process had an effect on evaluated compounds, sometimes negative and sometimes positive. Results suggest that huitlacoche is an attractive food source of phenolics with excellent antioxidant potential and interesting lipidic compounds.

Inhibitory activity of isoniazid and ethionamide against Cryptococcus biofilms.

PubMed

Cordeiro, Rossana de Aguiar; Serpa, Rosana; Marques, Francisca Jakelyne de Farias; de Melo, Charlline Vládia Silva; Evangelista, Antonio José de Jesus; Mota, Valquíria Ferreira; Brilhante, Raimunda Sâmia Nogueira; Bandeira, Tereza de Jesus Pinheiro Gomes; Rocha, Marcos Fábio Gadelha; Sidrim, José Júlio Costa

2015-11-01

In recent years, the search for drugs to treat systemic and opportunistic mycoses has attracted great interest from the scientific community. This study evaluated the in vitro inhibitory effect of the antituberculosis drugs isoniazid and ethionamide alone and combined with itraconazole and fluconazole against biofilms of Cryptococcus neoformans and Cryptococcus gattii. Antimicrobials were tested at defined concentrations after susceptibility assays with Cryptococcus planktonic cells. In addition, we investigated the synergistic interaction of antituberculosis drugs and azole derivatives against Cryptococcus planktonic cells, as well as the influence of isoniazid and ethionamide on ergosterol content and cell membrane permeability. Isoniazid and ethionamide inhibited both biofilm formation and viability of mature biofilms. Combinations formed by antituberculosis drugs and azoles proved synergic against both planktonic and sessile cells, showing an ability to reduce Cryptococcus biofilms by approximately 50%. Furthermore, isoniazid and ethionamide reduced the content of ergosterol in Cryptococcus spp. planktonic cells and destabilized or permeabilized the fungal cell membrane, leading to leakage of macromolecules. Owing to the paucity of drugs able to inhibit Cryptococcus biofilms, we believe that the results presented here might be of interest in the designing of new antifungal compounds.

Method Development for the Determination of Free and Esterified Sterols in Button Mushrooms (Agaricus bisporus).

PubMed

Hammann, Simon; Vetter, Walter

2016-05-04

Ergosterol is the major sterol in button mushrooms (Agaricus bisporus) and can occur as free alcohol or esterified with fatty acids (ergosteryl esters). In this study, gas chromatography with mass spectrometry in the selected ion monitoring mode (GC/MS-SIM) was used to determine ergosterol and ergosteryl esters as well as other sterols and steryl esters in button mushrooms. Different quality control measures were established and sample preparation procedures were compared to prevent the formation of artifacts and the degradation of ergosteryl esters. The final method was then used for the determination of ergosterol (443 ± 44 mg/100 g dry matter (d.m.)) and esterified ergosterol (12 ± 6 mg/100 g d.m.) in button mushroom samples (n = 4). While the free sterol fraction was vastly dominated by ergosterol (∼90% of five sterols in total), the steryl ester fraction was more diversified (nine sterols in total, ergosterol ∼55%) and consisted primarily of linoleic acid esters.

Short-term effects of propiconazole on hypothalamic-pituitary-gonadal function in the fathead minnows (Pimephales promelas)

EPA Science Inventory

Propiconazole is an ergosterol inhibitor commonly used in agriculture and has been detected in aquatic environments. Ergosterol inhibitors decrease fungal growth through effects on 14á-demethylase, a cytochrome P450 (CYP), isoform important for ergosterol biosynthesis. In higher ...

Rapid detection of kernel rots and mycotoxins in maize by near-infrared reflectance spectroscopy.

PubMed

Berardo, Nicola; Pisacane, Vincenza; Battilani, Paola; Scandolara, Andrea; Pietri, Amedeo; Marocco, Adriano

2005-10-19

Near-infrared (NIR) spectroscopy is a practical spectroscopic procedure for the detection of organic compounds in matter. It is particularly useful because of its nondestructiveness, accuracy, rapid response, and easy operation. This work assesses the applicability of NIR for the rapid identification of micotoxigenic fungi and their toxic metabolites produced in naturally and artificially contaminated products. Two hundred and eighty maize samples were collected both from naturally contaminated maize crops grown in 16 areas in north-central Italy and from ears artificially inoculated with Fusarium verticillioides. All samples were analyzed for fungi infection, ergosterol, and fumonisin B1 content. The results obtained indicated that NIR could accurately predict the incidence of kernels infected by fungi, and by F. verticillioides in particular, as well as the quantity of ergosterol and fumonisin B1 in the meal. The statistics of the calibration and of the cross-validation for mold infection and for ergosterol and fumonisin B1 contents were significant. The best predictive ability for the percentage of global fungal infection and F. verticillioides was obtained using a calibration model utilizing maize kernels (r2 = 0.75 and SECV = 7.43) and maize meals (r2 = 0.79 and SECV = 10.95), respectively. This predictive performance was confirmed by the scatter plot of measured F. verticillioides infection versus NIR-predicted values in maize kernel samples (r2 = 0.80). The NIR methodology can be applied for monitoring mold contamination in postharvest maize, in particular F. verticilliodes and fumonisin presence, to distinguish contaminated lots from clean ones, and to avoid cross-contamination with other material during storage and may become a powerful tool for monitoring the safety of the food supply.

Ergosterol from the soilborne fungus Ganoderma boninense.

PubMed

Toh Choon, R L; Sariah, M; Siti Mariam, M N

2012-10-01

Ergosterol is the main component of the fungal membrane and is not found in plants or other microbial cells. Therefore, it can be a useful biomarker for the quantification of fungal biomass. We are now reporting the first isolation and characterisation of ergosterol from the mycelium of G. boninense. The ergosterol structure was detected by Thin Liquid Chromatography (TLC) and Ultra Performance Liquid Chromatography (UPLC) and confirmed with Gas Chromatography coupled with Mass Spectrometry (GCMS) and Nuclear Magnetic Resonance (NMR) analysis. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A New Method for the Isolation of Ergosterol and Peroxyergosterol as Active Compounds of Hygrophoropsis aurantiaca and in Vitro Antiproliferative Activity of Isolated Ergosterol Peroxide.

PubMed

Nowak, Renata; Drozd, Marta; Mendyk, Ewaryst; Lemieszek, Marta; Krakowiak, Olga; Kisiel, Wanda; Rzeski, Wojciech; Szewczyk, Katarzyna

2016-07-21

In the present study, ergosterol peroxide and ergosterol were isolated for the first time from fresh fruit bodies of Hygrophoropsis aurantiaca (False Chanterelle). The substances were characterized mainly by spectroscopic methods (¹H-NMR, (13)C-NMR, DEPT-45, DEPT-90, DEPT-135, 2D-NMR). In our study, a new specific thin layer chromatographic method was developed for determination of ergosterol and ergosterol peroxide in H. aurantiaca extract. The method is based on the separation of n-hexane extract on silica gel (Silica Gel G) TLC plates using the optimized solvent system toluene/ethyl acetate (3:1; v/v). The main advantages of the developed method are the simplicity of operation and the low cost. The in vitro study results revealed the antiproliferative properties of ergosterol peroxide against LS180 human colon cancer cells. The described effect was attributed both to altered mitochondrial activity and decreased DNA synthesis. Additionally, in the same concentration range the investigated compound was not toxic to CCD 841 CoTr human colon epithelial cells. The present study suggests that fruit bodies of H. aurantiaca have great potential for producing substances and extracts with potential applications in medicine.

Transcriptional profile of Paracoccidioides spp. in response to itraconazole

PubMed Central

2014-01-01

Background Itraconazole is currently used to treat paracoccidioidomycosis. The mechanism of action of azoles has been elucidated in some fungi, although little is known regarding its mechanism of action in Paracoccidioides spp. The present work focused on identification of regulated transcripts using representational difference analysis of Paracoccidioides spp. yeast cells treated with itraconazole for 1 and 2 h. Results Paracoccidioides Pb01 genes up-regulated by itraconazole included genes involved in cellular transport, metabolism/energy, transcription, cell rescue, defense and virulence. ERG11, ERG6, ERG3, ERG5 and ERG25 were up-regulated at multiple time points. In vivo infection experiments in mice corroborated the in vitro results. Ergosterol levels and distribution were evaluated in Paracoccidioides Pb18 yeast cells, and the results demonstrate that both factors were changed in the fungus treated with itraconazole. Conclusion To our knowledge, this is the first transcriptional analysis of Paracoccidioides spp. exposed to a triazole drug. Here acetyl seems to be intensively produced from different metabolic pathways to produce ergosterol by the action of ergosterol synthesis related enzymes, which were also affected in other fungi. Among the genes affected, we identified genes in common with other fungi, as well as genes unique to Paracoccidioides Pb01. Those genes could be considered target to new drugs. Voltage-gated Ca2+ alpha subunit (CAV), Tetracycline resistance protein (TETA) and Hemolisyn-iii channel protein (HLYiii) were found only here and a probably involvement with resistence to itraconazole could be investigated in the future. However our findings do not permit inference to current clinical practice. PMID:24690401

Functional analysis of Paracoccidioides brasiliensis 14-3-3 adhesin expressed in Saccharomyces cerevisiae.

PubMed

Assato, Patricia Akemi; da Silva, Julhiany de Fátima; de Oliveira, Haroldo Cesar; Marcos, Caroline Maria; Rossi, Danuza; Valentini, Sandro Roberto; Mendes-Giannini, Maria José Soares; Zanelli, Cleslei Fernando; Fusco-Almeida, Ana Marisa

2015-11-04

14-3-3 proteins comprise a family of eukaryotic multifunctional proteins involved in several cellular processes. The Pb14-3-3 of Paracoccidioides brasiliensis seems to play an important role in the Paracoccidioides-host interaction. Paracoccidioides brasiliensis is an etiological agent of paracoccidioidomycosis, which is a systemic mycosis that is endemic in Latin America. In the initial steps of the infection, Paracoccidioides spp. synthetizes adhesins that allow it to adhere and invade host cells. Therefore, the aim of this work was to perform a functional analysis of Pb14-3-3 using Saccharomyces cerevisiae as a model. The functional analysis of Pb14-3-3 was performed in S. cerevisiae, and it was found that Pb14-3-3 partially complemented S. cerevisiae proteins Bmh1p and Bmh2p, which are recognized as two yeast 14-3-3 homologues. When we evaluated the adhesion profile of S. cerevisiae transformants, Pb14-3-3 acted as an adhesin in S. cerevisiae; however, Bmh1p did not show this function. The influence of Pb14-3-3 in S. cerevisiae ergosterol pathway was also evaluated and our results showed that Pb14-3-3 up-regulates genes involved in ergosterol biosynthesis. Our data showed that Pb14-3-3 was able to partially complement Bmh1p and Bmh2p proteins in S. cerevisiae; however, we suggest that Pb14-3-3 has a differential role as an adhesin. In addition, Pb-14-3-3 may be involved in Paracoccidioides spp. ergosterol biosynthesis which makes it an interest as a therapeutic target.

Ergosterol peroxide from Chaga mushroom (Inonotus obliquus) exhibits anti-cancer activity by down-regulation of the β-catenin pathway in colorectal cancer.

PubMed

Kang, Ju-Hee; Jang, Jeong-Eun; Mishra, Siddhartha Kumar; Lee, Hee-Ju; Nho, Chu Won; Shin, Dongyun; Jin, Mirim; Kim, Mi Kyung; Choi, Changsun; Oh, Seung Hyun

2015-09-15

In this study, we examined the effect of different fractions and components of Chaga mushroom (Inonotus Obliquus) on viability and apoptosis of colon cancer cells. Among them, one component showed the most effective growth inhibition and was identified as ergosterol peroxide by NMR analysis. We investigated the anti-proliferative and apoptosis mechanisms of ergosterol peroxide associated with its anti-cancer activities in human colorectal cancer (CRC) cell lines and tested its anti-tumor effect on colitis-induced CRC developed by Azoxymethane (AOM)/Dextran sulfate sodium (DSS) in a mouse model. We used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, flow cytometry assays, Western blot analysis, colony formation assays, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and AOM/DSS mouse models to study the molecular mechanism of metastatic activities in CRC cells. Ergosterol peroxide inhibited cell proliferation and also suppressed clonogenic colony formation in HCT116, HT-29, SW620 and DLD-1 CRC cell lines. The growth inhibition observed in these CRC cell lines was the result of apoptosis, which was confirmed by FACS analysis and Western blotting. Ergosterol peroxide inhibited the nuclear levels of β-catenin, which ultimately resulted in reduced transcription of c-Myc, cyclin D1, and CDK-8. Ergosterol peroxide administration showed a tendency to suppress tumor growth in the colon of AOM/DSS-treated mice, and quantification of the IHC staining showed a dramatic decrease in the Ki67-positive staining and an increase in the TUNEL staining of colonic epithelial cells in AOM/DSS-treated mice by ergosterol peroxide for both prevention and therapy. Our data suggest that ergosterol peroxide suppresses the proliferation of CRC cell lines and effectively inhibits colitis-associated colon cancer in AOM/DSS-treated mice. Ergosterol peroxide down-regulated β-catenin signaling, which exerted anti-proliferative and pro-apoptotic activities in CRC cells. These properties of ergosterol peroxide advocate its use as a supplement in colon cancer chemoprevention. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The Amoebicidal Effect of Ergosterol Peroxide Isolated from Pleurotus ostreatus.

PubMed

Meza-Menchaca, Thuluz; Suárez-Medellín, Jorge; Del Ángel-Piña, Christian; Trigos, Ángel

2015-12-01

Dysentery is an inflammation of the intestine caused by the protozoan parasite Entamoeba histolytica and is a recurrent health problem affecting millions of people worldwide. Because of the magnitude of this disease, finding novel strategies for treatment that does not affect human cells is necessary. Ergosterol peroxide is a sterol particularly known as a major cytotoxic agent with a wide spectrum of biological activities produced by edible and medicinal mushrooms. The aim of this report is to evaluate the amoebicidal activity of ergosterol peroxide (5α, 8α-epidioxy-22E-ergosta-6,22-dien-3β-ol isolated from 5α, 8α-epidioxy-22E-ergosta-6,22-dien-3β-ol) (Jacq.) P. Kumm. f. sp. Florida. Our results show that ergosterol peroxide produced a strong cytotoxic effect against amoebic growth. The inhibitory concentration IC50 of ergosterol peroxide was evaluated. The interaction between E. histolytica and ergosterol peroxide in vitro resulted in strong amoebicidal activity (IC50  = 4.23 nM) that may be due to the oxidatory effect on the parasitic membrane. We also tested selective toxicity of ergosterol peroxide using a cell line CCL-241, a human epithelial cell line isolated from normal human fetal intestinal tissue. To the best of our knowledge, this is the first report on the cytotoxicity of ergosterol peroxide against E. histolytica, which uncovers a new biological property of the lipidic compound isolated from Pleurotus ostreatus (Jacq.) P. Kumm. f. sp. Florida. Copyright © 2015 John Wiley & Sons, Ltd.

Altered sterol metabolism in budding yeast affects mitochondrial iron-sulfur (Fe-S) cluster synthesis.

PubMed

Ward, Diane M; Chen, Opal S; Li, Liangtao; Kaplan, Jerry; Bhuiyan, Shah Alam; Natarajan, Selvamuthu K; Bard, Martin; Cox, James E

2018-05-17

Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho- or Rho0) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron-sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial Fe metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29. Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increase mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Solid-state fermentation of cornmeal with the basidiomycete Ganoderma lucidum for degrading starch and upgrading nutritional value.

PubMed

Han, J R; An, C H; Yuan, J M

2005-01-01

The objective of this research was to study the ability of the basidiomycete Ganoderma lucidum to degrade starch and upgrade nutritional value of cornmeal during solid-state fermentation (SSF). On the basal medium that consisted of cornmeal and salt solution, alpha-amylase activity of G. lucidum reached its maximum value of 267 U g(-1) of culture on day 20 after inoculation. Prolongation of fermentation time from 10 to 25 days increased significantly the degradation rate of starch and ergosterol yield (a kind of physiologically active substances of G. lucidum, also as an indicator of mycelial biomass) (P < 0.01). Supplementation of glucose, sucrose or maltose to the basal medium also caused a significant increase in either the degradation rate of starch or the ergosterol yield as compared with control (P < 0.01). Among five kinds of nitrogen sources supplemented, yeast extract, casamino acid and peptone were more effective than (NH4)2SO4 and NH4NO3, and yeast extract gave the highest degradation rate of starch and ergosterol yield, followed by peptone. Through orthogonal experiments, the theoretical optimum culture medium for SSF of this fungus was the following: 100 g cornmeal, ground to 30-mesh powder, moistened with 67 ml of nutrient salt solution supplemented with 3 g yeast extract and 7.5 g glucose per litre. Under the optimum culture condition, the degradation rate of starch reached its maximum values of 70.4%; the starch content of the fermented product decreased from 64.5 to 25.3%, while the reducing sugar content increased from 4.2 to 20.6%. SSF also produced a significant increase (P < 0.01) from 11.0 to 16.5% in protein content. After SSF by G. lucidum, the digesting and absorbing ratio of cornmeal was strikingly increased and some active substances originated from G. lucidum remained in the fermented product. This implied that cornmeal could be processed into many kinds of special functional foods by SSF of G. lucidum.

Human cytochrome P450scc (CYP11A1) catalyzes epoxide formation with ergosterol.

PubMed

Tuckey, Robert C; Nguyen, Minh N; Chen, Jianjun; Slominski, Andrzej T; Baldisseri, Donna M; Tieu, Elaine W; Zjawiony, Jordan K; Li, Wei

2012-03-01

Cytochrome P450scc (P450scc) catalyzes the cleavage of the side chain of both cholesterol and the vitamin D(3) precursor, 7-dehydrocholesterol. The aim of this study was to test the ability of human P450scc to metabolize ergosterol, the vitamin D(2) precursor, and define the structure of the major products. P450scc incorporated into the bilayer of phospholipid vesicles converted ergosterol to two major and four minor products with a k(cat) of 53 mol · min(-1) · mol P450scc(-1) and a K(m) of 0.18 mol ergosterol/mol phospholipid, similar to the values observed for cholesterol metabolism. The reaction of ergosterol with P450scc was scaled up to make enough of the two major products for structural analysis. From mass spectrometry, NMR, and comparison of the NMR data to that for similar molecules, we determined the structures of the two major products as 20-hydroxy-22,23-epoxy-22,23-dihydroergosterol and 22-keto-23-hydroxy-22,23-dihydroergosterol. Molecular modeling and nuclear Overhauser effect (or enhancement) spectroscopy spectra analysis helped to establish the configurations at C20, C22, and C23 and determine the final structures of major products as 22R,23S-epoxyergosta-5,7-diene-3β,20α-diol and 3β,23S-dihydroxyergosta-5,7-dien-22-one. It is likely that the formation of the second product is through a 22,23-epoxy (oxirane) intermediate followed by C22 hydroxylation with the formation of strained 22-hydroxy-22,23-epoxide (oxiranol), which is immediately transformed to the more stable α-hydroxyketone. Molecular modeling of ergosterol into the P450scc crystal structure positioned the ergosterol side chain consistent with formation of the above products. Thus, we have shown that P450scc efficiently catalyzes epoxide formation with ergosterol giving rise to novel epoxy, hydroxy, and keto derivatives, without causing cleavage of the side chain.

Development of nutraceutical formulations based on the mycelium of Pleurotus ostreatus and Agaricus bisporus.

PubMed

Cardoso, Rossana V C; Fernandes, Ângela; Oliveira, M Beatriz P P; Calhelha, Ricardo C; Barros, Lillian; Martins, Anabela; Ferreira, Isabel C F R

2017-06-21

The present work is aimed at developing nutraceutical formulations based on the mycelium of Agaricus bisporus and Pleurotus ostreatus, highlighting the potential of in vitro culture as a tool to improve the production of bioactive compounds, namely phenolic acids and ergosterol. The mycelia of both species were cultured in different solid and liquid media in order to compare the growth rate and yielded biomass. Fruiting bodies, mycelia and culture media were compared regarding the antioxidant activity, anti-inflammatory effects in RAW264.7 cells and cytotoxicity in human tumor cell lines and non-tumor porcine liver cells. P. ostreatus mycelia showed higher contents of ergosterol and phenolic compounds, and stronger antioxidant activity than the corresponding fruiting body. P. ostreatus and A. bisporus did not show anti-inflammatory activity, and P. ostreatus was the only one showing cytotoxicity in tumor cell lines. The results show that these mushrooms provide compounds with antioxidant and cytotoxic capacities, with variations among species.

Development of a method for the determination of Fusarium fungi on corn using mid-infrared spectroscopy with attenuated total reflection and chemometrics.

PubMed

Kos, Gregor; Lohninger, Hans; Krska, Rudolf

2003-03-01

A novel method, which enables the determination of fungal infection with Fusarium graminearum on corn within minutes, is presented. The ground sample was sieved and the particle size fraction between >250 and 100 microm was used for mid-infrared/attenuated total reflection (ATR) measurements. The sample was pressed onto the ATR crystal, and reproducible pressure was applied. After the spectra were recorded, they were subjected to principle component analysis (PCA) and classified using cluster analysis. Observed changes in the spectra reflected changes in protein, carbohydrate, and lipid contents. Ergosterol (for the total fungal biomass) and the toxin deoxynivalenol (DON; a secondary metabolite) of Fusarium fungi served as reference parameters, because of their relevance for the examination of corn based food and feed. The repeatability was highly improved by sieving prior to recording the spectra, resulting in a better clustering in PCA score/score plots. The developed method enabled the separation of samples with a toxin content of as low as 310 microg/kg from noncontaminated (blank) samples. Investigated concentration ranges were 880-3600 microg/kg for ergosterol and 310-2596 microg/kg for DON. The percentage of correctly classified samples was up to 100% for individual samples compared with a number of blank samples.

Rapid and pervasive occupation of fallen mangrove leaves by a marine zoosporic fungus.

PubMed

Newell, S Y; Miller, J D; Fell, J W

1987-10-01

Samples of leaves of red mangrove (Rhizophora mangle) were incubated on an agar medium selective for pythiaceous oomycetes. Leaves on trees above the water did not contain oomycetes. Marine oomycetes, principally Phytophthora vesicula, had colonized leaves within 2 h of leaf submergence, probably finding them by chemotaxis. The frequency of occurrence of P. vesicula in submerged leaves reached 100% within 30 h of submergence. By 43 h most, if not all, parts of leaves were occupied, and surface treatment with a biocide indicated that leaves were occupied internally. Frequencies of P. vesicula remained near 100% through about 2 weeks of submergence and then declined to about 60% in older (>/=4 weeks) leaves. Leaves of white mangrove (Laguncularia racemosa) were also extensively occupied by P. vesicula after falling into the water column, but decaying leaves of turtlegrass (Thalassia testudinum) were not colonized by oomycetes. Ergosterol analysis indicated that the standing crop of living, non-oomycete (ergosterol-containing) fungal mass in submerged red-mangrove leaves did not rise above that which had been present in senescent leaves on the tree; decaying turtlegrass leaves had an ergosterol content that was only about 2% of the maximum concentration detected for red-mangrove leaves. These results suggest that oomycetes are the predominant mycelial eucaryotic saprotrophs of mangrove leaves that fall into the water column and that for turtlegrass leaves which live, die, and decompose under submerged conditions, mycelial eucaryotes make no substantial contribution to decomposition.

[Applications of near infrared reflectance spectroscopy in detecting chitin, ergosterol and mycotoxins].

PubMed

Yi, Yong-Yan; Li, De-Rong; Zhang, Yun-Wei; Yang, Fu-Yu

2009-07-01

The invasion extent and harmfulness of fungi can be determined by chitin, ergosterol and mycotoxins. It is important to monitor chitin, ergosterol and mycotoxins changes to prevent contamination of forage and feed products, and effectively control the sustainable development of the mildew. Predication of these chemical materials was often completed by laboratory analysis, which was time-consuming and cumbersome and could not reflect the results in time in the past. Near infrared reflectance spectroscopy (NIRS) is a rapid, convenient, highly efficient, nondestructive and low-cost analytical technique, which has been widely used in various fields such as food field and feed field for quantitative and qualitative analysis. It has a great potentiality of application in quality analysis. In this paper, the principle and the characteristic of NIRS and its applications in food, forage, feed and other agriculture products quality analysis were introduced. Its applications in fungal biomass (chitin, ergosterol) and mycotoxins were mainly reviewed. NIRS was used to quantify chitin, ergosterol and mycotoxins. Calibration equations and validation equations for these materials were developed. It is also expected that NIRS will play a more and more important role in the field of fungi with the establishment of calibration equation and improvement of model database.

Mechanism of the synergistic effect of amiodarone and fluconazole in Candida albicans.

PubMed

Gamarra, Soledad; Rocha, Elousa Maria F; Zhang, Yong-Qiang; Park, Steven; Rao, Rajini; Perlin, David S

2010-05-01

The antiarrhythmic drug amiodarone has been found to have fungicidal activity. In Saccharomyces cerevisiae, its antifungal activity is mediated by calcium overload stress, which leads to a rapid nuclear accumulation of the calcineurin-regulated transcription factor CRZ1. In addition, low doses of amiodarone have been reported to be synergistic with fluconazole in fluconazole-resistant Candida albicans. To establish its mechanism of toxicity in C. albicans, we used expression profiling of key pathway genes to examine cellular responses to amiodarone alone and in combination with fluconazole. Gene expression profiling of 59 genes was done in five C. albicans strains (three fluconazole-susceptible strains and two fluconazole-resistant strains) after amiodarone and/or fluconazole exposure. Of the 59 genes, 27 analyzed showed a significant change (>2-fold) in expression levels after amiodarone exposure. The up- or downregulated genes included genes involved in Ca(2+) homeostasis, cell wall synthesis, vacuolar/lysosomal transport, diverse pathway regulation, stress response, and pseudohyphal morphogenesis. As expected, fluconazole induces an increase in ergosterol pathway genes expression levels. The combination treatment significantly dampened the transcriptional response to either drug, suggesting that synergism was due to an inhibition of compensatory response pathways. This dampening resulted in a decrease in total ergosterol levels and decreased pseudohyphal formation, a finding consistent with decreased virulence in a murine candidiasis model.

Mobilization of steryl esters from lipid particles of the yeast Saccharomyces cerevisiae.

PubMed

Wagner, Andrea; Grillitsch, Karlheinz; Leitner, Erich; Daum, Günther

2009-02-01

In the yeast as in other eukaryotes, formation and hydrolysis of steryl esters (SE) are processes linked to lipid storage. In Saccharomyces cerevisiae, the three SE hydrolases Tgl1p, Yeh1p and Yeh2p contribute to SE mobilization from their site of storage, the lipid particles/droplets. Here, we provide evidence for enzymatic and cellular properties of these three hydrolytic enzymes. Using the respective single, double and triple deletion mutants and strains overexpressing the three enzymes, we demonstrate that each SE hydrolase exhibits certain substrate specificity. Interestingly, disturbance in SE mobilization also affects sterol biosynthesis in a type of feedback regulation. Sterol intermediates stored in SE and set free by SE hydrolases are recycled to the sterol biosynthetic pathway and converted to the final product, ergosterol. This recycling implies that the vast majority of sterol precursors are transported from lipid particles to the endoplasmic reticulum, where sterol biosynthesis is completed. Ergosterol formed through this route is then supplied to its subcellular destinations, especially the plasma membrane. Only a minor amount of sterol precursors are randomly distributed within the cell after cleavage from SE. Conclusively, SE storage and mobilization although being dispensable for yeast viability contribute markedly to sterol homeostasis and distribution.

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols.

PubMed

Hsueh, Ya-Wei; Chen, Mei-Ting; Patty, Philipus J; Code, Christian; Cheng, John; Frisken, Barbara J; Zuckermann, Martin; Thewalt, Jenifer

2007-03-01

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance ((2)H NMR) and vesicle extrusion. For the (2)H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from -2 to at least 31 degrees C). Adding ergosterol to a concentration of 25 mol % increases POPC-d(31) chain ordering as measured by the NMR spectral first moment M(1) and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25 degrees C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M(1). This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.

Ergosterol in POPC Membranes: Physical Properties and Comparison with Structurally Similar Sterols

PubMed Central

Hsueh, Ya-Wei; Chen, Mei-Ting; Patty, Philipus J.; Code, Christian; Cheng, John; Frisken, Barbara J.; Zuckermann, Martin; Thewalt, Jenifer

2007-01-01

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (2H NMR) and vesicle extrusion. For the 2H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d31 chain ordering as measured by the NMR spectral first moment M1 and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M1. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane. PMID:17142279

Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.

PubMed

Bottoms, Scott; Dickinson, Quinn; McGee, Mick; Hinchman, Li; Higbee, Alan; Hebert, Alex; Serate, Jose; Xie, Dan; Zhang, Yaoping; Coon, Joshua J; Myers, Chad L; Landick, Robert; Piotrowski, Jeff S

2018-01-12

Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast. Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain. We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates.

Relationships between Fungal Biomass and Nitrous Oxide Emission in Upland Rice Soils under No Tillage and Cover Cropping Systems.

PubMed

Zhaorigetu; Komatsuzaki, Masakazu; Sato, Yoshinori; Ohta, Hiroyuki

2008-01-01

The relationships between soil microbial properties and nitrous oxide emission were examined in upland soil under different tillage systems [no tillage (NT), rotary and plow tillage] and cover crop systems (fallow, cereal rye, and hairy vetch) in 2004 and 2005. Microbiological analyses included the determination of soil ergosterol as an indicator of fungal biomass, bacterial plate counting, and MPN estimations of ammonia oxidizers and denitrifiers. The combined practice of NT with rye-cover crop treatment increased fungal biomass but not bacterial populations in 0-10 cm deep soils. Such increase in fungal biomass was not found in 10-20 cm and 20-30 cm deep cover-cropped NT soil. The combined practice of NT with rye-cover cropping resulted in higher in situ N(2)O emission rates compared with rotary- and plow-till treatments. N(2)O flux was positively correlated with soil ergosterol content but not with denitrifier MPN and other soil chemical properties. These results suggested a significant contribution of fungi to N(2)O emission in cover-cropped NT soils.

The synthesis, regulation, and functions of sterols in Candida albicans: Well-known but still lots to learn.

PubMed

Lv, Quan-Zhen; Yan, Lan; Jiang, Yuan-Ying

2016-08-17

Sterols are the basal components of the membranes of the fungal pathogen Candida albicans, and these membranes determine the susceptibility of C. albicans cells to a variety of stresses, such as ionic, osmotic and oxidative pressures, and treatment with antifungal drugs. The common antifungal azoles in clinical use are targeted to the biosynthesis of ergosterol. In the past years, the synthesis, storage and metabolism of ergosterol in Saccharomyces cerevisiae has been characterized in some detail; however, these processes has not been as well investigated in the human opportunistic pathogen C. albicans. In this review, we summarize the genes involved in ergosterol synthesis and regulation in C. albicans. As well, genes in S. cerevisiae implicated in ergosterol storage and conversions with other lipids are noted, as these provide us clues and directions for the study of the homologous genes in C. albicans. In this report we have particularly focused on the essential roles of ergosterol in the dynamic process of cell biology and its fundamental status in the biological membrane system that includes lipid rafts, lipid droplets, vacuoles and mitochondria. We believe that a thorough understanding of this classic and essential pathway will give us new ideas about drug resistance and morphological switching in C. albicans.

The synthesis, regulation, and functions of sterols in Candida albicans: Well-known but still lots to learn

PubMed Central

Lv, Quan-zhen; Yan, Lan; Jiang, Yuan-ying

2016-01-01

ABSTRACT Sterols are the basal components of the membranes of the fungal pathogen Candida albicans, and these membranes determine the susceptibility of C. albicans cells to a variety of stresses, such as ionic, osmotic and oxidative pressures, and treatment with antifungal drugs. The common antifungal azoles in clinical use are targeted to the biosynthesis of ergosterol. In the past years, the synthesis, storage and metabolism of ergosterol in Saccharomyces cerevisiae has been characterized in some detail; however, these processes has not been as well investigated in the human opportunistic pathogen C. albicans. In this review, we summarize the genes involved in ergosterol synthesis and regulation in C. albicans. As well, genes in S. cerevisiae implicated in ergosterol storage and conversions with other lipids are noted, as these provide us clues and directions for the study of the homologous genes in C. albicans. In this report we have particularly focused on the essential roles of ergosterol in the dynamic process of cell biology and its fundamental status in the biological membrane system that includes lipid rafts, lipid droplets, vacuoles and mitochondria. We believe that a thorough understanding of this classic and essential pathway will give us new ideas about drug resistance and morphological switching in C. albicans. PMID:27221657

Ergosterol Peroxide Isolated from Ganoderma lucidum Abolishes MicroRNA miR-378-Mediated Tumor Cells on Chemoresistance

PubMed Central

Li, Xiang-Min; Yang, Weining; Jiao, Chun-Wei; Fang, Ling; Li, Sen-Zhu; Pan, Hong-Hui; Yee, Albert J.; Lee, Daniel Y.; Li, Chong; Zhang, Zhi; Guo, Jun; Yang, Burton B.

2012-01-01

Due to an altered expression of oncogenic factors and tumor suppressors, aggressive cancer cells have an intrinsic or acquired resistance to chemotherapeutic agents. This typically contributes to cancer recurrence after chemotherapy. microRNAs are short non-coding RNAs that are involved in both cell self-renewal and cancer development. Here we report that tumor cells transfected with miR-378 acquired properties of aggressive cancer cells. Overexpression of miR-378 enhanced both cell survival and colony formation, and contributed to multiple drug resistance. Higher concentrations of chemotherapeutic drugs were needed to induce death of miR-378-transfected cells than to induce death of control cells. We found that the biologically active component isolated from Ganoderma lucidum could overcome the drug-resistance conferred by miR-378. We purified and identified the biologically active component of Ganoderma lucidum as ergosterol peroxide. We demonstrated that ergosterol peroxide produced greater activity in inducing death of miR-378 cells than the GFP cells. Lower concentrations of ergosterol peroxide were needed to induce death of the miR-378-transfected cells than in the control cells. With further clinical development, ergosterol peroxide represents a promising new reagent that can overcome the drug-resistance of tumor cells. PMID:22952996

Optimization of ultrasound-assisted extraction to obtain mycosterols from Agaricus bisporus L. by response surface methodology and comparison with conventional Soxhlet extraction.

PubMed

Heleno, Sandrina A; Diz, Patrícia; Prieto, M A; Barros, Lillian; Rodrigues, Alírio; Barreiro, Maria Filomena; Ferreira, Isabel C F R

2016-04-15

Ergosterol, a molecule with high commercial value, is the most abundant mycosterol in Agaricus bisporus L. To replace common conventional extraction techniques (e.g. Soxhlet), the present study reports the optimal ultrasound-assisted extraction conditions for ergosterol. After preliminary tests, the results showed that solvents, time and ultrasound power altered the extraction efficiency. Using response surface methodology, models were developed to investigate the favourable experimental conditions that maximize the extraction efficiency. All statistical criteria demonstrated the validity of the proposed models. Overall, ultrasound-assisted extraction with ethanol at 375 W during 15 min proved to be as efficient as the Soxhlet extraction, yielding 671.5 ± 0.5mg ergosterol/100 g dw. However, with n-hexane extracts with higher purity (mg ergosterol/g extract) were obtained. Finally, it was proposed for the removal of the saponification step, which simplifies the extraction process and makes it more feasible for its industrial transference. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mechanism of the Synergistic Effect of Amiodarone and Fluconazole in Candida albicans▿ †

PubMed Central

Gamarra, Soledad; Rocha, Elousa Maria F.; Zhang, Yong-Qiang; Park, Steven; Rao, Rajini; Perlin, David S.

2010-01-01

The antiarrhythmic drug amiodarone has been found to have fungicidal activity. In Saccharomyces cerevisiae, its antifungal activity is mediated by calcium overload stress, which leads to a rapid nuclear accumulation of the calcineurin-regulated transcription factor CRZ1. In addition, low doses of amiodarone have been reported to be synergistic with fluconazole in fluconazole-resistant Candida albicans. To establish its mechanism of toxicity in C. albicans, we used expression profiling of key pathway genes to examine cellular responses to amiodarone alone and in combination with fluconazole. Gene expression profiling of 59 genes was done in five C. albicans strains (three fluconazole-susceptible strains and two fluconazole-resistant strains) after amiodarone and/or fluconazole exposure. Of the 59 genes, 27 analyzed showed a significant change (>2-fold) in expression levels after amiodarone exposure. The up- or downregulated genes included genes involved in Ca2+ homeostasis, cell wall synthesis, vacuolar/lysosomal transport, diverse pathway regulation, stress response, and pseudohyphal morphogenesis. As expected, fluconazole induces an increase in ergosterol pathway genes expression levels. The combination treatment significantly dampened the transcriptional response to either drug, suggesting that synergism was due to an inhibition of compensatory response pathways. This dampening resulted in a decrease in total ergosterol levels and decreased pseudohyphal formation, a finding consistent with decreased virulence in a murine candidiasis model. PMID:20194694

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

PubMed

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D; Schulz, Stefan; Fleißner, André

2016-10-18

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion

PubMed Central

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D.; Schulz, Stefan; Fleißner, André

2016-01-01

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion. PMID:27708165

Testing Experimental Compounds against American Cutaneous and Mucocutaneous Leishmaniasis

DTIC Science & Technology

1982-06-01

synthesize large amounts of plasma membrane ergosterol in serum-free media (26). Ketoconazole and miconazole actively disrupt ergosterol synthesis in a...walls cross- * react with L. donovani (40), it was thought that immunostimula- tion of a htst with BCG prior to infection with L. mexicana ama- zonensis

Vitamin D and sterol composition of ten types of mushrooms from retail suppliers in the United States

USDA-ARS?s Scientific Manuscript database

Vitamin D, ergosterol, ergosterol metabolites, and phytosterols were analyzed in ten mushroom types sampled nationwide in the U.S. to update the USDA Nutrient Database for Standard Reference. Sterols were analyzed by GC-FID with mass spectrometric confirmation of components. Vitamin D was assayed ...

Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes.

PubMed

Yamamoto, Keisuke; Hara, Kiyotaka Y; Morita, Toshihiko; Nishimura, Akira; Sasaki, Daisuke; Ishii, Jun; Ogino, Chiaki; Kizaki, Noriyuki; Kondo, Akihiko

2016-09-13

Red yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed. Xanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain. In this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common substrate for isoprenoid biosynthesis. The resulting decrease in ergosterol biosynthesis increased astaxanthin production. The efficient method for deleting diploid genes developed in this study has the potential to improve industrial production of various isoprenoids in X. dendrorhous.

A Genetic and Pharmacological Analysis of Isoprenoid Pathway by LC-MS/MS in Fission Yeast

PubMed Central

Takami, Tomonori; Fang, Yue; Zhou, Xin; Jaiseng, Wurentuya; Ma, Yan; Kuno, Takayoshi

2012-01-01

Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level. PMID:23145048

Seasonal ectomycorrhizal fungal biomass development on loblolly pine (Pinus taeda L.) seedlings

Treesearch

Shi-Jean S. Sung; L.M. White; D.H. Marx; W.J. Otrosina

1995-01-01

Ergosterol, a membrane sterol found in fungi but not in plants, was used to estimate live mycelia biomass in ectomycorrhizae. Loblolly pine (Pinus taeda L.) seeds were sown in April 1993 and grown with standard nursery culture ractices. Correlations between total seedling ergosterol and visual assessment of mycorrhizal colonization were high during...

Med15B Regulates Acid Stress Response and Tolerance in Candida glabrata by Altering Membrane Lipid Composition

PubMed Central

Qi, Yanli; Liu, Hui; Yu, Jiayin; Chen, Xiulai

2017-01-01

ABSTRACT Candida glabrata is a promising producer of organic acids. To elucidate the physiological function of the Mediator tail subunit Med15B in the response to low-pH stress, we constructed a deletion strain, C. glabrata med15BΔ, and an overexpression strain, C. glabrata HTUΔ/CgMED15B. Deletion of MED15B caused biomass production, glucose consumption rate, and cell viability to decrease by 28.3%, 31.7%, and 26.5%, respectively, compared with those of the parent (HTUΔ) strain at pH 2.0. Expression of lipid metabolism-related genes was significantly downregulated in the med15BΔ strain, whereas key genes of ergosterol biosynthesis showed abnormal upregulation. This caused the proportion of C18:1 fatty acids, the ratio of unsaturated to saturated fatty acids (UFA/SFA), and the total phospholipid content to decrease by 11.6%, 27.4%, and 37.6%, respectively. Cells failed to synthesize fecosterol and ergosterol, leading to the accumulation and a 60.3-fold increase in the concentration of zymosterol. Additionally, cells showed reductions of 69.2%, 11.6%, and 21.8% in membrane integrity, fluidity, and H+-ATPase activity, respectively. In contrast, overexpression of Med15B increased the C18:1 levels, total phospholipids, ergosterol content, and UFA/SFA by 18.6%, 143.5%, 94.5%, and 18.7%, respectively. Membrane integrity, fluidity, and H+-ATPase activity also increased by 30.2%, 6.9%, and 51.8%, respectively. Furthermore, in the absence of pH buffering, dry weight of cells and pyruvate concentrations were 29.3% and 61.2% higher, respectively, than those of the parent strain. These results indicated that in C. glabrata, Med15B regulates tolerance toward low pH via transcriptional regulation of acid stress response genes and alteration in lipid composition. IMPORTANCE This study explored the role of the Mediator tail subunit Med15B in the metabolism of Candida glabrata under acidic conditions. Overexpression of MED15B enhanced yeast tolerance to low pH and improved biomass production, cell viability, and pyruvate yield. Membrane lipid composition data indicated that Med15B might play a critical role in membrane integrity, fluidity, and H+-ATPase activity homeostasis at low pH. Thus, controlling membrane composition may serve to increase C. glabrata productivity at low pH. PMID:28710262

Structure of Yeast OSBP-Related Protein Osh1 Reveals Key Determinants for Lipid Transport and Protein Targeting at the Nucleus-Vacuole Junction.

PubMed

Manik, Mohammad Kawsar; Yang, Huiseon; Tong, Junsen; Im, Young Jun

2017-04-04

Yeast Osh1 belongs to the oxysterol-binding protein (OSBP) family of proteins and contains multiple targeting modules optimized for lipid transport at the nucleus-vacuole junction (NVJ). The key determinants for NVJ targeting and the role of Osh1 at NVJs have remained elusive because of unknown lipid specificities. In this study, we determined the structures of the ankyrin repeat domain (ANK), and OSBP-related domain (ORD) of Osh1, in complex with Nvj1 and ergosterol, respectively. The Osh1 ANK forms a unique bi-lobed structure that recognizes a cytosolic helical segment of Nvj1. We discovered that Osh1 ORD binds ergosterol and phosphatidylinositol 4-phosphate PI(4)P in a competitive manner, suggesting counter-transport function of the two lipids. Ergosterol is bound to the hydrophobic pocket in a head-down orientation, and the structure of the PI(4)P-binding site in Osh1 is well conserved. Our results suggest that Osh1 performs non-vesicular transport of ergosterol and PI(4)P at the NVJ. Copyright © 2017 Elsevier Ltd. All rights reserved.

Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi.

PubMed

Zhang, De-Huai; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

2017-02-01

Ganoderic acids (GAs) in Ganoderma lingzhi exhibit anticancer and antimetastatic activities. GA yields can be potentially improved by manipulating G. lingzhi through genetic engineering. In this study, a putative lanosterol synthase (LS) gene was cloned and overexpressed in G. lingzhi. Results showed that its overexpression (OE) increased the ganoderic acid (GA) content and the accumulation of lanosterol and ergosterol in a submerged G. lingzhi culture. The maximum contents of GA-O, GA-Mk, GA-T, GA-S, GA-Mf, and GA-Me in transgenic strains were 46.6 ± 4.8, 24.3 ± 3.5, 69.8 ± 8.2, 28.9 ± 1.4, 15.4 ± 1.2, and 26.7 ± 3.1 μg/100 mg dry weight, respectively, these values being 6.1-, 2.2-, 3.2-, 4.8-, 2.0-, and 1.9-times higher than those in wild-type strains. In addition, accumulated amounts of lanosterol and ergosterol in transgenic strains were 2.3 and 1.4-fold higher than those in the control strains, respectively. The transcription level of LS was also increased by more than five times in the presence of the G. lingzhi glyceraldehyde-3-phosphate dehydrogenase gene promoter, whereas transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A enzyme and squalene synthase did not change significantly in transgenic strains. This study demonstrated that OE of the homologous LS gene can enhance lanosterol accumulation. A large precursor supply promotes GA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification and Characterization of Four Azole-Resistant erg3 Mutants of Candida albicans▿

PubMed Central

Martel, Claire M.; Parker, Josie E.; Bader, Oliver; Weig, Michael; Gross, Uwe; Warrilow, Andrew G. S.; Rolley, Nicola; Kelly, Diane E.; Kelly, Steven L.

2010-01-01

Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol Δ5,6-desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, ≥256, 16, 16, 8, and 1 μg ml−1, respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, <0.25 μg ml−1); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 μg ml−1). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14α-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted. PMID:20733039

Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC.

PubMed

Muniroh, M S; Sariah, M; Zainal Abidin, M A; Lima, N; Paterson, R R M

2014-05-01

Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of Heracleum persicum ethyl acetate extract on the growth, hyphal ultrastructure and aflatoxin biosynthesis in Aspergillus parasiticus.

PubMed

Razzaghi-Abyaneh, Mehdi; Saberi, Reza; Sharifan, Anosheh; Rezaee, Mohammad-Bagher; Seifili, Roghayeh; Hosseini, Seyed-Ibrahim; Shams-Ghahfarokhi, Masoomeh; Nikkhah, Mehdi; Saberi, Ismail; Amani, Akram

2013-11-01

The ethyl acetate extract of leaves, seeds and flowers of Heracleum persicum, a medicinal plant of Iran (family Apiaceae) inhibited growth and aflatoxin (AF) production of Aspergillus parasiticus. On the basis of total dry weight growth inhibition by the leaf extract ranged from 17.1 to 36.9 %, by the flower extract from 32.2 to 75.6 %, and by the seed extract from 27.5 to 74.9 %. Production of AFB1 and AFG1 was inhibited in a dose-dependent manner, with a reduction of 88.5-100 % at the highest concentration of 8,000 μg/ml tested. The flower extract decreased ergosterol content of hyphae most significantly. Electron microscopy further revealed structural defects in the treated A. parasiticus including disruption of cytoplasmic membranous compartments, detachment of plasma membrane from the cell wall, and disorganization of hyphal compartments. Collapsed hyphae without conidiation, shorter branches and undifferentiated hyphal tips were also evident. The results indicate that H. persicum extract exerts antifungal and anti-AF activities by disrupting plasma membrane integrity and permeability mainly through interference with ergosterol biosynthesis. These results show that H. persicum can serve as a potent and safe alternative for inhibiting toxigenic aspergilli growth and thus preventing AF contamination of foods and feeds.

Sterol Composition of Clinically Relevant Mucorales and Changes Resulting from Posaconazole Treatment.

PubMed

Müller, Christoph; Neugebauer, Thomas; Zill, Patrizia; Lass-Flörl, Cornelia; Bracher, Franz; Binder, Ulrike

2018-05-19

Mucorales are fungi with increasing importance in the clinics. Infections take a rapidly progressive course resulting in high mortality rates. The ergosterol biosynthesis pathway and sterol composition are of interest, since they are targeted by currently applied antifungal drugs. Nevertheless, Mucorales often exhibit resistance to these drugs, resulting in therapeutic failure. Here, sterol patterns of six clinically relevant Mucorales ( Lichtheimia corymbifera , Lichtheimia ramosa , Mucor circinelloides , Rhizomucor pusillus , Rhizopus arrhizus , and Rhizopus microsporus ) were analysed in a targeted metabolomics fashion after derivatization by gas chromatography-mass spectrometry. Additionally, the effect of posaconazole (POS) treatment on the sterol pattern of R. arrhizus was evaluated. Overall, fifteen different sterols were detected with species dependent variations in the total and relative sterol amount. Sterol analysis from R. arrhizus hyphae confronted with sublethal concentrations of posaconazole revealed the accumulation of 14-methylergosta-8,24-diene-3,6-diol, which is a toxic sterol that was previously only detected in yeasts. Sterol content and composition were further compared to the well-characterized pathogenic mold Aspergillus fumigatus . This work contributes to a better understanding of the ergosterol biosynthesis pathway of Mucorales, which is essential to improve antifungal efficacy, the identification of targets for novel drug design, and to investigate the combinatorial effects of drugs targeting this pathway.

Ergosterol, arabitol and manitol as tracers for biological aerosols

NASA Astrophysics Data System (ADS)

Rudich, Y.; Burshtein, N.; Lang-Yona, N.

2010-12-01

Airborne fungi can cause a wide array of adverse responses in humans depending on the type and quantity present. Since dose and human response is highly individual, the sensitivity of a person exposed is also an important consideration. The abundance of bioaerosols in the ambient air and their health impacts depend on the season and on the environmental conditions. In order to quantify and identify fungi bioaerosols’ contribution to atmospheric aerosols and the impact to public health, it has been suggested to use chemicals that are typical of bioaerosols as biomarkers in chemical analysis of collected aerosols. An often used biomarker for determining the fungal biomass is ergosterol. Recently, Bauer et al. (2008) found that mannitol and arabitol concentrations are correlated with the fungal spore counts in atmospheric PM10. In this study, ergosterol, arabitol and mannitol were quantified in ambient aerosols collected in the Eastern Mediterranean region for 12 months in order to understand their annual and seasonal behavior and to test whether arabitol and mannitol are good predictors of fungi. Finally, correlations between ergosterol abundances with inorganic ions, humidity, temperature, and synoptic data in order to identify dominant sources of fungal spores were also studied. We will report on the measurements and the observed correlations between the different tracers.

Aerobic kinetoplastid flagellate Phytomonas does not require heme for viability

PubMed Central

Kořený, Luděk; Sobotka, Roman; Kovářová, Julie; Gnipová, Anna; Flegontov, Pavel; Horváth, Anton; Oborník, Miroslav; Ayala, Francisco J.; Lukeš, Julius

2012-01-01

Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme. PMID:22355128

Aerobic kinetoplastid flagellate Phytomonas does not require heme for viability.

PubMed

Kořený, Luděk; Sobotka, Roman; Kovářová, Julie; Gnipová, Anna; Flegontov, Pavel; Horváth, Anton; Oborník, Miroslav; Ayala, Francisco J; Lukeš, Julius

2012-03-06

Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme.

Chemical constituents of Hericium erinaceum associated with the inhibitory activity against cellular senescence in human umbilical vascular endothelial cells.

PubMed

Noh, Hyung Jun; Yang, Hyo Hyun; Kim, Geum Soog; Lee, Seung Eun; Lee, Dae Young; Choi, Je Hun; Kim, Seung Yu; Lee, Eun Suk; Ji, Seung Heon; Kang, Ki Sung; Park, Hye-Jin; Kim, Jae-Ryong; Kim, Ki Hyun

2015-12-01

Hericium erinaceum is an edible and medicinal mushroom widely used in Korea, Japan, and China. On the search for biologically active compounds supporting the medicinal usage, the MeOH extract of the fruiting bodies of H. erinaceum was investigated for its chemical constituents. Six compounds were isolated and identified as hericenone D (1), (22E,24R)-5α,8α-epidioxyergosta-6,22-dien-3β-ol (2), erinacerin B (3), hericenone E (4), hericenone F (5) and isohericerin (6) by comparing their spectroscopic data with previously reported values. The inhibitory effects on adriamycin-induced cellular senescence in human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) of the isolates (1-6) were studied. Among the isolated compounds, ergosterol peroxide (2) reduced senescence associated β-galactosidase (SA-β-gal) activity increased in HUVECs treated with adriamycin. According to experimental data obtained, the active compound may inspire the development of a new pharmacologically useful substance to be used in the treatment and prevention of age-related diseases.

Pinus pinaster seedlings and their fungal symbionts show high plasticity in phosphorus acquisition in acidic soils.

PubMed

Ali, M A; Louche, J; Legname, E; Duchemin, M; Plassard, C

2009-12-01

Young seedlings of maritime pine (Pinus pinaster Soland in Aït.) were grown in rhizoboxes using intact spodosol soil samples from the southwest of France, in Landes of Gascogne, presenting a large variation of phosphorus (P) availability. Soils were collected from a 93-year-old unfertilized stand and a 13-year-old P. pinaster stand with regular annual fertilization of either only P or P and nitrogen (N). After 6 months of culture in controlled conditions, different morphotypes of ectomycorrhiza (ECM) were used for the measurements of acid phosphatase activity and molecular identification of fungal species using amplification of the ITS region. Total biomass, N and P contents were measured in roots and shoots of plants. Bicarbonate- and NaOH-available inorganic P (Pi), organic P (Po) and ergosterol concentrations were measured in bulk and rhizosphere soil. The results showed that bulk soil from the 93-year-old forest stand presented the highest Po levels, but relatively higher bicarbonate-extractable Pi levels compared to 13-year-old unfertilized stand. Fertilizers significantly increased the concentrations of inorganic P fractions in bulk soil. Ergosterol contents in rhizosphere soil were increased by fertilizer application. The dominant fungal species was Rhizopogon luteolus forming 66.6% of analysed ECM tips. Acid phosphatase activity was highly variable and varied inversely with bicarbonate-extractable Pi levels in the rhizosphere soil. Total P or total N in plants was linearly correlated with total plant biomass, but the slope was steep only between total P and biomass in fertilized soil samples. In spite of high phosphatase activity in ECM tips, P availability remained a limiting nutrient in soil samples from unfertilized stands. Nevertheless young P. pinaster seedlings showed a high plasticity for biomass production at low P availability in soils.

Effect of Plasterboard Composition on Stachybotrys chartarum Growth and Biological Activity of Spores

PubMed Central

Murtoniemi, Timo; Nevalainen, Aino; Hirvonen, Maija-Riitta

2003-01-01

The effects of plasterboard composition on the growth and sporulation of Stachybotrys chartarum as well as on the inflammatory potential of the spores were studied. S. chartarum was grown on 13 modified plasterboards under saturated humidity conditions. The biomass was estimated by measuring the ergosterol content of the S. chartarum culture while the spore-induced cytotoxicity and production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin-6 in mouse macrophages was used to illustrate the bioactivity of spores. The ergosterol content of S. chartarum correlated with the number of spores collected from plasterboards. The growth and sporulation decreased compared to that of the reference board in those cases where (i) the liner was treated with biocide, (ii) starch was removed from the plasterboard, or (iii) desulfurization gypsum was used in the core. Spores collected from all the plasterboards were toxic to the macrophages. The biocide added to the core did not reduce the growth; in fact, the spores collected from that board evoked the highest cytotoxicity. The conventional additives used in the core had inhibitory effects on growth. Recycled plasterboards used in the core and the board lacking the starch triggered spore-induced TNF-α production in macrophages. In summary, this study shows that the growth of a strain of S. chartarum on plasterboard and the subsequent bioactivity of spores were affected by minor changes to the composition of the core or liners, but it could not be totally prevented without resorting to the use of biocides. However, incomplete prevention of microbial growth by biocides even increased the cytotoxic potential of the spores. PMID:12839740

Supplementation of Male Pheromone on Rock Substrates Attracts Female Rock Lizards to the Territories of Males: A Field Experiment

PubMed Central

Martín, José; López, Pilar

2012-01-01

Background Many animals produce elaborated sexual signals to attract mates, among them are common chemical sexual signals (pheromones) with an attracting function. Lizards produce chemical secretions for scent marking that may have a role in sexual selection. In the laboratory, female rock lizards (Iberolacerta cyreni) prefer the scent of males with more ergosterol in their femoral secretions. However, it is not known whether the scent-marks of male rock lizards may actually attract females to male territories in the field. Methodology/Principal Findings In the field, we added ergosterol to rocks inside the territories of male lizards, and found that this manipulation resulted in increased relative densities of females in these territories. Furthermore, a higher number of females were observed associated to males in manipulated plots, which probably increased mating opportunities for males in these areas. Conclusions/Significance These and previous laboratory results suggest that female rock lizards may select to settle in home ranges based on the characteristics of scent-marks from conspecific males. Therefore, male rock lizards might attract more females and obtain more matings by increasing the proportion of ergosterol when scent-marking their territories. However, previous studies suggest that the allocation of ergosterol to secretions may be costly and only high quality males could afford it, thus, allowing the evolution of scent-marks as an honest sexual display. PMID:22253895

Contents of microscopic fungi in dusts coming from cereal analysis laboratories.

PubMed

Szwajkowska-Michalek, Lidia; Stuper, Kinga; Lakomy, Piotr; Matysiak, Anna; Perkowski, Juliusz

2010-01-01

Microscopic fungi - components of bioaerosol found in the workplace environment of individuals employed in the agricultural sector - constitute a considerable hazard for their health. This study includes quantitative and qualitative analyses of mycobionta contained in 20 samples of dusts collected from laboratories conducting analyses of cereals. A total of 27 species of viable microscopic fungi were isolated. The most frequently isolated genera Penicillium and Aspergillus, accounting for 27 percent and 26 percent of analyzed isolates. The content of fungal biomass was determined quantitatively using a fungal marker, ergosterol (ERG). Concentrations of this metabolite for all samples ranged from 0.48 mg/kg-212.36 mg/kg. Based on the analyses, it may be stated that the concentration of microfungi in settled dust from laboratories conducting analyses of cereals was varied, and in several cases markedly exceeded admissible concentration levels.

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress.

PubMed

Diniz, Raphael Hermano Santos; Villada, Juan C; Alvim, Mariana Caroline Tocantins; Vidigal, Pedro Marcus Pereira; Vieira, Nívea Moreira; Lamas-Maceiras, Mónica; Cerdán, María Esperanza; González-Siso, María-Isabel; Lahtvee, Petri-Jaan; da Silveira, Wendel Batista

2017-09-01

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.

Mechanism of action of coumarin and silver(I)-coumarin complexes against the pathogenic yeast Candida albicans.

PubMed

Thati, Bhumika; Noble, Andy; Rowan, Raymond; Creaven, Bernadette S; Walsh, Maureen; McCann, Malachy; Egan, Denise; Kavanagh, Kevin

2007-08-01

The anti-fungal activity and mode of action of a range of silver(I)-coumarin complexes was examined. The most potent silver(I)-coumarin complexes, namely 7-hydroxycoumarin-3-carboxylatosilver(I), 6-hydroxycoumarin-3-carboxylatosilver(I) and 4-oxy-3-nitrocoumarinbis(1,10-phenanthroline)silver(I), had MIC80 values of between 69.1 and 4.6 microM against the pathogenic yeast Candida albicans. These compounds also reduced respiration, lowered the ergosterol content of cells and increased the trans-membrane leakage of amino acids. A number of the complexes disrupted cytochrome synthesis in the cell and induced the appearance of morphological features consistent with cell death by apoptosis. These compounds appear to act by disrupting the synthesis of cytochromes which directly affects the cell's ability to respire. A reduction in respiration leads to a depletion in ergosterol biosynthesis and a consequent disruption of the integrity of the cell membrane. Disruption of cytochrome biosynthesis may induce the onset of apoptosis which has been shown previously to be triggered by alteration in the location of cytochrome c. Silver(I)-coumarin complexes demonstrate good anti-fungal activity and manifest a mode of action distinct to that of the conventional azole and polyene drugs thus raising the possibility of their use when resistance to conventional drug has emerged or in combination with such drugs.

Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species.

PubMed

Pinto, Eugénia; Vale-Silva, Luís; Cavaleiro, Carlos; Salgueiro, Lígia

2009-11-01

The composition and antifungal activity of clove essential oil (EO), obtained from Syzygium aromaticum, were studied. Clove oil was obtained commercially and analysed by GC and GC-MS. The EO analysed showed a high content of eugenol (85.3 %). MICs, determined according to Clinical and Laboratory Standards Institute protocols, and minimum fungicidal concentration were used to evaluate the antifungal activity of the clove oil and its main component, eugenol, against Candida, Aspergillus and dermatophyte clinical and American Type Culture Collection strains. The EO and eugenol showed inhibitory activity against all the tested strains. To clarify its mechanism of action on yeasts and filamentous fungi, flow cytometric and inhibition of ergosterol synthesis studies were performed. Propidium iodide rapidly penetrated the majority of the yeast cells when the cells were treated with concentrations just over the MICs, meaning that the fungicidal effect resulted from an extensive lesion of the cell membrane. Clove oil and eugenol also caused a considerable reduction in the quantity of ergosterol, a specific fungal cell membrane component. Germ tube formation by Candida albicans was completely or almost completely inhibited by oil and eugenol concentrations below the MIC values. The present study indicates that clove oil and eugenol have considerable antifungal activity against clinically relevant fungi, including fluconazole-resistant strains, deserving further investigation for clinical application in the treatment of fungal infections.

Utilization of hydrothermally pretreated wheat straw for production of bioethanol and carotene-enriched biomass.

PubMed

Petrik, Siniša; Kádár, Zsófia; Márová, Ivana

2013-04-01

In this work hydrothermally pretreated wheat straw was used for production of bioethanol by Saccharomyces cerevisiae and carotene-enriched biomass by red yeasts Rhodotorula glutinis, Cystofilobasidium capitatum and Sporobolomyces roseus. To evaluate the convertibility of pretreated wheat straw into ethanol, simultaneous saccharification and fermentation of S. cerevisiae was performed under semi-anaerobic conditions. The highest ethanol production efficiency of 65-66% was obtained following pretreatment at 200°C without the catalytic action of acetic acid, and at 195 and 200°C respectively in the presence of catalyst. Red yeast strain S. roseus produced 1.73-2.22 mg g(-1) of ergosterol on the filter cake, 1.15-4.17 mg g(-1) of ergosterol and 1.23-1.56 mg g(-1) of β-carotene on pretreated wheat straw hydrolysates and also the highest amount of carotenoids and ergosterol on untreated wheat straw (1.70 and 4.17 mg g(-1), respectively). Copyright © 2013 Elsevier Ltd. All rights reserved.

Nutritional changes in powdered red pepper upon in vitro infection of Aspergillus flavus

PubMed Central

Tripathi, Smita; Mishra, H.N.

2009-01-01

Quantitative losses in various biochemical constituents like capsaicin, carotenes, ascorbic acid, polyphenols, mineral matter, sugars (soluble and insoluble), protein and fat were estimated after the successful growth of Aspergillus flavus for 30 days on powdered red pepper. The fungal biomass was measured by ergosterol content and Aflatoxin B1 by HPLC. Amongst the various nutritional constituents evaluated for nutritional losses and changes the highest nutritional loss was reported in total carotenoids (88.55%) followed by total sugars (85.5%). The protein content of the infected sample increased from 18.01% to 23%. The nutritional profile of chilli powder (Capsicum annum var. sannam L.) shows highest share of total soluble sugars (32.89%) and fiber content (21.05%), followed by protein (18.01%) and fat (13.32%) making it an ideal solid- substrate for mould growth. At the end of incubation the fungal biomass was 192. 25 mg / 100 gram powder, total plate count 17.5 X 10 4 CFU/g and Aflatoxin B1 content was 30.06 μg / kg. PMID:24031333

Steroid toxicity and detoxification in ascomycetous fungi.

PubMed

Cvelbar, Damjana; Zist, Vanja; Kobal, Katja; Zigon, Dušan; Zakelj-Mavrič, Marija

2013-02-25

In the last couple of decades fungal infections have become a significant clinical problem. A major interest into fungal steroid action has been provoked since research has proven that steroid hormones are toxic to fungi and affect the host/fungus relationship. Steroid hormones were found to differ in their antifungal activity in ascomycetous fungi Hortaea werneckii, Saccharomyces cerevisiae and Aspergillus oryzae. Dehydroepiandrosterone was shown to be the strongest inhibitor of growth in all three varieties of fungi followed by androstenedione and testosterone. For their protection, fungi use several mechanisms to lower the toxic effects of steroids. The efficiency of biotransformation in detoxification depended on the microorganism and steroid substrate used. Biotransformation was a relatively slow process as it also depended on the growth phase of the fungus. In addition to biotransformation, steroid extrusion out of the cells contributed to the lowering of the active intracellular steroid concentration. Plasma membrane Pdr5 transporter was found to be the most effective, followed by Snq2 transporter and vacuolar transporters Ybt1 and Ycf1. Proteins Aus1 and Dan1 were not found to be involved in steroid import. The research of possible targets of steroid hormone action in fungi suggests that steroid hormones inhibit ergosterol biosynthesis in S. cerevisiae and H. werneckii. Results of this inhibition caused changes in the sterol content of the cellular membrane. The presence of steroid hormones most probably causes the degradation of the Tat2 permease and impairment of tryptophan import. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

trans-Cinnamic and Chlorogenic Acids Affect the Secondary Metabolic Profiles and Ergosterol Biosynthesis by Fusarium culmorum and F. graminearum Sensu Stricto

PubMed Central

Kulik, Tomasz; Stuper-Szablewska, Kinga; Bilska, Katarzyna; Buśko, Maciej; Ostrowska-Kołodziejczak, Anna; Załuski, Dariusz; Perkowski, Juliusz

2017-01-01

Plant-derived compounds limiting mycotoxin contamination are currently of major interest in food and feed production. However, their potential application requires an evaluation of their effects on fungal secondary metabolism and membrane effects. In this study, different strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to trans-cinnamic and chlorogenic acids on solid YES media. Fusaria produced phenolic acids, whose accumulation was lowered by exogenous phenolic compounds. In addition, fungi reduced exogenous phenolic acids, leading either to their conversion or degradation. trans-Cinnamic acid was converted to caffeic and ferulic acids, while chlorogenic acid was degraded to caffeic acid. The latter underwent further degradation to protocatechuic acid. Fungal-derived trans-cinnamic acid, as the first intermediate of the shikimate pathway, increased after chlorogenic acid treatment, presumably due to the further inhibition of the conversion of trans-cinnamic acid. Exogenous trans-cinnamic and chlorogenic acid displayed the inhibition of mycotoxin production by Fusaria, which appeared to be largely dependent on the phenolic compound and its concentration and the assayed strain. Exogenous phenolic acids showed different effects on ergosterol biosynthesis by fungi. It was found that the production of this membrane sterol was stimulated by trans-cinnamic acid, while chlorogenic acid negatively impacted ergosterol biosynthesis, suggesting that phenolic acids with stronger antifungal activities may upregulate ergosterol biosynthesis by Fusaria. This paper reports on the production of phenolic acids by Fusaria for the first time. PMID:28640190

trans-Cinnamic and Chlorogenic Acids Affect the Secondary Metabolic Profiles and Ergosterol Biosynthesis by Fusarium culmorum and F. graminearum Sensu Stricto.

PubMed

Kulik, Tomasz; Stuper-Szablewska, Kinga; Bilska, Katarzyna; Buśko, Maciej; Ostrowska-Kołodziejczak, Anna; Załuski, Dariusz; Perkowski, Juliusz

2017-06-22

Plant-derived compounds limiting mycotoxin contamination are currently of major interest in food and feed production. However, their potential application requires an evaluation of their effects on fungal secondary metabolism and membrane effects. In this study, different strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to trans -cinnamic and chlorogenic acids on solid YES media. Fusaria produced phenolic acids, whose accumulation was lowered by exogenous phenolic compounds. In addition, fungi reduced exogenous phenolic acids, leading either to their conversion or degradation. trans -Cinnamic acid was converted to caffeic and ferulic acids, while chlorogenic acid was degraded to caffeic acid. The latter underwent further degradation to protocatechuic acid. Fungal-derived trans -cinnamic acid, as the first intermediate of the shikimate pathway, increased after chlorogenic acid treatment, presumably due to the further inhibition of the conversion of trans -cinnamic acid. Exogenous trans -cinnamic and chlorogenic acid displayed the inhibition of mycotoxin production by Fusaria, which appeared to be largely dependent on the phenolic compound and its concentration and the assayed strain. Exogenous phenolic acids showed different effects on ergosterol biosynthesis by fungi. It was found that the production of this membrane sterol was stimulated by trans -cinnamic acid, while chlorogenic acid negatively impacted ergosterol biosynthesis, suggesting that phenolic acids with stronger antifungal activities may upregulate ergosterol biosynthesis by Fusaria. This paper reports on the production of phenolic acids by Fusaria for the first time.

Residential Culturable Fungi, (1–3, 1–6)-β-D-glucan, and Ergosterol Concentrations in Dust Are Not Associated with Asthma, Rhinitis or Eczema Diagnoses in Children

PubMed Central

Choi, Hyunok; Byrne, Sam; Larsen, Lisbeth Suldrup; Sigsgaard, Torben; Thorne, Peter S.; Larsson, Lennart; Sebastian, Aleksandra; Bornehag, Carl-Gustaf

2014-01-01

Background Qualitative reporting of home indoor moisture problems predicts respiratory diseases. However, causal agents underlying such qualitative markers remain unknown. Methods In the homes of 198 multiple allergic case children and 202 controls in Sweden, we cultivated culturable fungi by directly plating dust, and quantified(1–3, 1–6)-β-D-glucan, and ergosterol in dust samples from the child’s bedroom. We examined the relationship between these fungal agents and degree of parent or inspector reported home indoor dampness, and microbiological laboratory’s mold index. We also compared the concentrations of these agents between multiple allergic cases and healthy controls, as well as IgE-sensitization among cases. Results The concentrations of culturable fungal agents were comparable between houses with parent and inspector reported mold issues and those without. There were no differences in concentrations of the individual or the total summed culturable fungi, (1–3, 1–6)-β-D-glucan, and ergosterol between the controls and the multiple allergic case children, or individual diagnosis of asthma, rhinitis or eczema. Conclusion Culturable fungi, (1–3, 1–6)-β-D-glucan, and ergosterol in dust were not associated with qualitative markers of indoor dampness or mold or indoor humidity. Furthermore, these agents in dust samples were not associated with any health outcomes in the children. PMID:24016225

Source identification analysis for the airborne bacteria and fungi using a biomarker approach

NASA Astrophysics Data System (ADS)

Lee, Alex K. Y.; Lau, Arthur P. S.; Cheng, Jessica Y. W.; Fang, Ming; Chan, Chak K.

Our recent studies have reported the feasibility of employing the 3-hydoxy fatty acids (3-OH FAs) and ergosterol as biomarkers to determine the loading of the airborne endotoxin from the Gram-negative bacteria and fungal biomass in atmospheric aerosols, respectively [Lee, A.K.Y., Chan, C.K., Fang, K., Lau, A.P.S., 2004. The 3-hydroxy fatty acids as biomarkers for quantification and characterization of endotoxins and Gram-negative bacteria in atmospheric aerosols in Hong Kong. Atmospheric Environment 38, 6807-6317; Lau, A.P.S., Lee, A.K.Y., Chan, C.K., Fang, K., 2006. Ergosterol as a biomarker for the quantification of the fungal biomass in atmospheric aerosols. Atmospheric Environment 40, 249-259]. These quantified biomarkers do not, however, provide information on their sources. In this study, the year-long dataset of the endotoxin and ergosterol measured in Hong Kong was integrated with the common water-soluble inorganic ions for source identification through the principal component analysis (PCA) and backward air mass trajectory analysis. In the coarse particles (PM 2.5-10), the bacterial endotoxin is loaded in the same factor group with Ca 2+ and accounted for about 20% of the total variance of the PCA. This implies the crustal origin for the airborne bacterial assemblage. The fungal ergosterol in the coarse particles (PM 2.5-10) had by itself loaded in a factor group of 10.8% of the total variance in one of the sampling sites with large area of natural vegetative coverage. This suggests the single entity nature of the fungal spores and their independent emission to the ambient air upon maturation of their vegetative growth. In the fine particles (2.0) in the fine particles of the continental air masses imply the microbial source from activities related to biomass burning and industries from the north. The low K +/Na + ratios (1.2-1.4) in the fine particles of the mixed air masses suggest microbial sources from the local and regional indoor environment through kitchen emissions and the re-suspension of the road dust due to vehicular exhausts.

Toxicity of Diclofenac in the Fern Azolla filiculoides and the Lichen Xanthoria parietina.

PubMed

Vannini, Andrea; Paoli, Luca; Vichi, Marco; Bačkor, Martin; Bačkorová, Miriam; Loppi, Stefano

2018-03-01

This study investigated the occurrence of toxicity, expressed as damage to the photosynthetic apparatus, in the aquatic fern Azolla filiculoides and the lichen Xanthoria parietina following treatments with diclofenac at different concentrations (0.1, 1, 10 and 100 mg/L) and different exposure times (24, 48, 72 and 240 h). Measurements of photosynthetic efficiency, chlorophyll content and chlorophyll degradation indicated dose- and time-dependent toxicity, since significant differences with control samples as well as among treatments, emerged mainly for the highest concentration (100 mg/L) and the longest time (240 h). In addition, also the mycobiont of the lichen X. parietina showed similar toxic effects, expressed as ergosterol content. The absence of relevant alterations at the lowest concentration (0.1 mg/L) suggested a very limited susceptibility of these species to environmentally relevant levels of this pharmaceutical.

Phloretin-induced reduction in dipole potential of sterol-containing bilayers.

PubMed

Ostroumova, Olga S; Efimova, Svetlana S; Schagina, Ludmila V

2013-12-01

The phloretin-induced reduction in the dipole potential of planar lipid bilayers containing cholesterol, ergosterol, stigmasterol, 7-dehydrocholesterol and 5α-androstan-3β-ol was investigated. It is shown that effects depend on the type and concentration of membrane sterol. It is supposed that the effectiveness of phloretin in reducing the dipole potential of the bilayers that contain cholesterol, ergosterol and 7-dehydrocholesterol correlates with the ordering and condensing effects. The role of the concentration-dependent ability of different sterols to promote lateral heterogeneity in membranes is also discussed.

Chemical constituents of Aspergillus sp EJC08 isolated as endophyte from Bauhinia guianensis and their antimicrobial activity.

PubMed

Pinheiro, Eduardo A A; Carvalho, Josiwander M; Santos, Diellem C P dos; Feitosa, André O; Marinho, Patrícia S B; Guilhon, Giselle Maria S P; Santos, Lourivaldo S; Souza, Afonso L D de; Marinho, Andrey M R

2013-01-01

The present work reports the isolation of five compounds from Aspergillus sp EJC08 isolated as endophytic from Bauhinia guianensis, a tipical plant of the Amazon. The compounds ergosterol (1), ergosterol peroxide (2), mevalolactone (3), monomethylsulochrin (4) and trypacidin A (5) were isolated by chromatographic procedures and identified by spectral methods of 1D and 2D NMR and MS. Compounds 3, 4 and 5 were tested against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus and showed good activity.

Cryptic antifungal compounds active by synergism with polyene antibiotics.

PubMed

Kinoshita, Hiroshi; Yoshioka, Mariko; Ihara, Fumio; Nihira, Takuya

2016-04-01

The majority of antifungal compounds reported so far target the cell wall or cell membrane of fungi, suggesting that other types of antibiotics cannot exert their activity because they cannot penetrate into the cells. Therefore, if the permeability of the cell membrane could be enhanced, many antibiotics might be found to have antifungal activity. We here used the polyene antibiotic nystatin, which binds to ergosterol and forms pores at the cell membrane, to enhance the cellular permeability. In the presence of nystatin, many culture extracts from entomopathogenic fungi displayed antifungal activity. Among all the active extracts, two active components were purified and identified as helvolic acid and terramide A. Because the minimum inhibitory concentration of either compound was reduced four-fold in the presence of nystatin, it can be concluded that this screening method is useful for detecting novel antifungal activity. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Functional characterization of the Dsc E3 ligase complex in the citrus postharvest pathogen Penicillium digitatum.

PubMed

Ruan, Ruoxin; Chung, Kuang-Ren; Li, Hongye

2017-12-01

Sterol regulatory element binding proteins (SREBPs) are required for sterol homeostasis in eukaryotes. Activation of SREBPs is regulated by the Dsc E3 ligase complex in Schizosaccharomyces pombe and Aspergillus spp. Previous studies indicated that an SREBP-coding gene PdsreA is required for fungicide resistance and ergosterol biosynthesis in the citrus postharvest pathogen Penicillium digitatum. In this study, five genes, designated PddscA, PddscB, PddscC, PddscD, and PddscE encoding the Dsc E3 ligase complex were characterized to be required for fungicide resistance, ergosterol biosynthesis and CoCl 2 tolerance in P. digitatum. Each of the dsc genes was inactivated by target gene disruption and the resulted phenotypes were analyzed and compared. Genetic analysis reveals that, of five Dsc complex components, PddscB is the core subunit gene in P. digitatum. Although the resultant dsc mutants were able to infect citrus fruit and induce maceration lesions as the wild-type, the mutants rarely produced aerial mycelia on affected citrus fruit peels. P. digitatum Dsc proteins regulated not only the expression of genes involved in ergosterol biosynthesis but also that of PdsreA. Yeast two-hybrid assays revealed a direct interaction between the PdSreA protein and the Dsc proteins. Ectopic expression of the PdSreA N-terminus restored fungicide resistance in the dsc mutants. Our results provide important evidence to understand the mechanisms underlying SREBP activation and regulation of ergosterol biosynthesis in plant pathogenic fungi. Copyright © 2017 Elsevier GmbH. All rights reserved.

Annual distribution of allergenic fungal spores in atmospheric particulate matter in the Eastern Mediterranean; a comparative study between ergosterol and quantitative PCR analysis

NASA Astrophysics Data System (ADS)

Lang-Yona, N.; Dannemiller, K.; Yamamoto, N.; Burshtein, N.; Peccia, J.; Yarden, O.; Rudich, Y.

2012-03-01

Airborne fungal spores are an important fraction of atmospheric particulate matter and are major causative agents of allergenic and infectious diseases. Predicting the variability and species of allergy-causing fungal spores requires detailed and reliable methods for identification and quantification. There are diverse methods for their detection in the atmosphere and in the indoor environments; yet, it is important to optimize suitable methods for characterization of fungal spores in atmospheric samples. In this study we sampled and characterized total and specific airborne fungal spores from PM10 samples collected in Rehovot, Israel over an entire year. The total fungal spore concentrations vary throughout the year although the species variability was nearly the same. Seasonal equivalent spore concentrations analyzed by real-time quantitative-PCR-based methods were fall > winter > spring > summer. Reported concentrations based on ergosterol analysis for the same samples were and fall > spring > winter > summer. Correlation between the two analytical methods was found only for the spring season. These poor associations may be due to the per-spore ergosterol variations that arise from both varying production rates, as well as molecular degradation of ergosterol. While conversion of genome copies to spore concentration is not yet straightforward, the potential for improving this conversion and the ability of qPCR to identify groups of fungi or specific species makes this method preferable for environmental spore quantification. Identifying tools for establishing the relation between the presence of species and the actual ability to induce allergies is still needed in order to predict the effect on human health.

Anti-Candida albicans effectiveness of citral and investigation of mode of action.

PubMed

Lima, Igara Oliveira; de Medeiros Nóbrega, Fernanda; de Oliveira, Wylly Araújo; de Oliveira Lima, Edeltrudes; Albuquerque Menezes, Everardo; Cunha, Francisco Afrânio; Formiga Melo Diniz, Margareth de Fátima

2012-12-01

Candidiasis is a mycosis caused by Candida species, which is of clinical importance due to the increase in resistant yeasts. Candida infection has been a serious health problem due to the inappropriate use of antibiotics. Therefore, it is necessary to study molecules with an antifungal action. Citral is a monoterpene with known pharmacological properties, including antimicrobial action. The aim of this work was to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of citral and the probable mode of action. The MIC of citral was determined by the broth microdilution method using Sabouraud dextrose medium. Additionally, the interference of citral in cell wall (sorbitol assay) and the binding of citral to ergosterol and cholesterol were studied, carried out by broth microdilution method. The MIC and MFC of citral were 512 and 1024 µg/mL, respectively. The MIC of amphotericin B was 1 µg/mL. The mechanism of action did not involve either the cell wall or ergosterol. However, the presence of cholesterol increased the MIC of citral to 1024 µg/mL, indicating there is some interaction between citral and cholesterol. Amphotericin B was used as the positive control, and it showed a high MIC in the presence of ergosterol (32 µg/mL), while in the presence of cholesterol MIC increased to 4 µg/mL. Citral inhibits the growth of C. albicans. The probable mechanism of action did not involve the cell wall or ergosterol. Citral is able to interact with cholesterol. More studies are necessary to describe their effects completely.

Ergosterol, arabitol and mannitol as tracers for biogenic aerosols in the Eastern Mediterranean

NASA Astrophysics Data System (ADS)

Burshtein, N.; Yona, N. Lang; Rudich, Y.

2010-11-01

Aerosols containing biological components can have a significant effect on human health by causing primarily irritation, infection and allergies. Specifically, airborne fungi can cause a wide array of adverse responses in humans depending on the type and quantity present. In this study we used chemical biomarkers for analyzing fungi-containing aerosols in the eastern Mediterranean region during the year 2009 in order to quantify annual fungal abundances. The prime marker for fungi used in this study was ergosterol, and its concentrations were compared with those of mannitol and arabitol, which were recently suggested to also correlate with fungal spores concentrations (Bauer et al., 2008a). Back trajectory analysis, inorganic ions, humidity and temperature were used in an attempt to identify sources as well as the dependence on seasonal and environmental conditions. We found that the ambient concentrations of ergosterol, arabitol and mannitol range between 0 and 2.73 ng m-3, 1.85 and 58.27 ng m-3, 5.57 and 138.03 ng m-3, respectively. The highest levels for all biomarkers were during the autumn, probably from local terrestrial sources, as deduced from the inorganic ions and back trajectory analysis. Significant correlations were observed between arabitol and mannitol during the entire year except for the winter months. Both sugars correlated with ergosterol only during the spring and autumn. We conclude that mannitol and arabitol might not be specific biomarkers for fungi and that the observed correlations during spring and autumn may be attributed to high levels of vegetation during spring blossoms and autumn decomposition.

Ergosterol, arabitol and mannitol as tracers for biogenic aerosols in the eastern Mediterranean

NASA Astrophysics Data System (ADS)

Burshtein, N.; Lang-Yona, N.; Rudich, Y.

2011-01-01

Aerosols containing biological components can have a significant effect on human health by causing primarily irritation, infection and allergies. Specifically, airborne fungi can cause a wide array of adverse responses in humans depending on the type and quantity present. In this study we used chemical biomarkers for analyzing fungi-containing aerosols in the eastern Mediterranean region during the year 2009 in order to quantify annual fungal abundances. The prime marker for fungi used in this study was ergosterol, and its concentrations were compared with those of mannitol and arabitol which were recently suggested to also correlate with fungal spores concentrations (Bauer et al., 2008a). Back trajectory analysis, inorganic ions, humidity and temperature were used in an attempt to identify sources as well as the dependence on seasonal and environmental conditions. We found that the ambient concentrations of ergosterol, arabitol and mannitol range between 0 and 2.73 ng m-3, 1.85 and 58.27 ng m-3, 5.57 and 138.03 ng m-3, respectively. The highest levels for all biomarkers were during the autumn, probably from local terrestrial sources, as deduced from the inorganic ions and back trajectory analysis. Significant correlations were observed between arabitol and mannitol during the entire year except for the winter months. Both sugars correlated with ergosterol only during the spring and autumn. We conclude that mannitol and arabitol might not be specific biomarkers for fungi and that the observed correlations during spring and autumn may be attributed to high levels of vegetation during spring blossoms and autumn decomposing.

Annual distribution of allergenic fungal spores in atmospheric particulate matter in the eastern mediterranean; a comparative study between ergosterol and quantitative PCR analysis

NASA Astrophysics Data System (ADS)

Lang-Yona, N.; Dannemiller, K.; Yamamoto, N.; Burshtein, N.; Peccia, J.; Yarden, O.; Rudich, Y.

2011-10-01

Airborne fungal spores are an important fraction of atmospheric particulate matter and are major causative agents of allergenic and infectious diseases. Predicting the variability and species of allergy-causing fungal spores requires detailed and reliable methods for identification and quantification. There are diverse methods for their detection in the atmosphere and in the indoor environments; yet, it is important to optimize suitable methods for characterization of fungal spores in atmospheric samples. In this study we sampled and characterized total and specific airborne fungal spores from PM10 samples collected in Rohovot, Israel over an entire year. The total fungal spore concentrations vary throughout the year although the species variability was nearly the same. Seasonal equivalent spore concentrations analyzed by real-time quantitative-PCR-based methods were fall > winter > spring > summer. Reported concentrations based on ergosterol analysis for the same samples were and fall > spring > winter > summer. Correlation between the two analytical methods was found only for the spring season. These poor associations may be due to the per-spore ergosterol variations that arise from both varying production rates, as well as molecular degradation of ergosterol. While conversion of genome copies to spore concentration is not yet straightforward, the potential for improving this conversion and the ability of qPCR to identify groups of fungi or specific species makes this method preferable for environmental spore quantification. Identifying tools for establishing the relation between the presence of species and the actual ability to induce allergies is still needed in order to predict the effect on human health.

Determination of non-certified levoglucosan, sugar polyols and ergosterol in NIST Standard Reference Material 1649a

NASA Astrophysics Data System (ADS)

Pomata, Donatella; Di Filippo, Patrizia; Riccardi, Carmela; Buiarelli, Francesca; Gallo, Valentina

2014-02-01

Organic component of airborne particulate matter originates from both natural and anthropogenic sources whose contributions can be identified through the analysis of chemical markers. The validation of analytical methods for analysis of compounds used as chemical markers is of great importance especially if they must be determined in rather complex matrices. Currently, standard reference materials (SRM) with certified values for all those analytes are not available. In this paper, we report a method for the simultaneous determination of levoglucosan and xylitol as tracers for biomass burning emissions, and arabitol, mannitol and ergosterol as biomarkers for airborne fungi in SRM 1649a, by GC/MS. Their quantitative analysis in SRM 1649a was carried out using both internal standard calibration curves and standard addition method. A matrix effect was observed for all analytes, minor for levoglucosan and major for polyols and ergosterol. The results related to levoglucosan around 160 μg g-1 agreed with those reported by other authors, while no comparison was possible for xylitol (120 μg g-1), arabitol (15 μg g-1), mannitol (18 μg g-1), and ergosterol (0.5 μg g-1). The analytical method used for SRM 1649a was also applied to PM10 samples collected in Rome during four seasonal sampling campaigns. The ratios between annual analyte concentrations in PM10 samples and in SRM 1649a were of the same order of magnitude although particulate matter samples analyzed were collected in two different sites and periods.

Antifungal activity of geraniol and citronellol, two monoterpenes alcohols, against Trichophyton rubrum involves inhibition of ergosterol biosynthesis.

PubMed

Pereira, Fillipe de Oliveira; Mendes, Juliana Moura; Lima, Igara Oliveira; Mota, Kelly Samara de Lira; Oliveira, Wylly Araújo de; Lima, Edeltrudes de Oliveira

2015-02-01

Trichophyton rubrum is the most common fungus causing chronic dermatophytosis in humans. Antifungal activity of promising agents is of great interest. Geraniol and citronellol are monoterpenes with antimicrobial properties. This study aimed to investigate the inhibitory effects and possible mechanism of antifungal activity of geraniol and citronellol against strains of T. rubrum. The minimum inhibitory concentration (MIC) of each drug against 14 strains was determined by broth microdilution. The effects of the drugs on dry mycelial weight, conidial germination, infectivity on human nail fragments, and morphogenesis of T. rubrum were analyzed. The effects on the cell wall (test with sorbitol) and cell membrane (release of intracellular material and ergosterol biosynthesis) were investigated. MIC values of geraniol ranged between 16 and 256 µg/mL while citronellol showed MIC values from 8 to 1024 µg/mL. The drugs (MIC and 2 × MIC) inhibited the mycelial growth, conidia germination, and fungal growth on nail fragments. The drugs (half of MIC) induced the formation of wide, short, and crooked hyphae in T. rubrum morphology. With sorbitol, geraniol MIC was increased by 64-fold and citronellol by 32-fold. The drugs caused leakage of intracellular material and inhibited ergosterol biosynthesis. The results suggest that the drugs damage cell wall and cell membrane of T. rubrum through a mechanism that seems to involve the inhibition of the ergosterol biosynthesis. This study confirms that geraniol and citronellol can be regarded as potential drugs for controlling T. rubrum growth, with great potential against agents of dermatophytosis.

Isolation and Characterization of Bioactive Metabolites from Fruiting Bodies and Mycelial Culture of Ganoderma oerstedii (Higher Basidiomycetes) from Mexico.

PubMed

Mendoza, Guillermo; Suárez-Medellín, Jorge; Espinoza, César; Ramos-Ligonio, Angel; Fernández, José J; Norte, Manuel; Trigos, Ángel

2015-01-01

Various species of the genus Ganoderma have been used for centuries according to oriental tradition as a source of medicines and nutrients. A chemical study of the fruiting bodies and mycelial culture of G. oerstedii was carried out with the idea of isolating and characterizing active natural components present to make use of their potential pharmaceutical application in Mexico. The fruiting bodies and mycelial culture of G. oesrtedii were lyophylized and extracted one after the other with hexane, chloroform, and methanol. Following this process, each substance was extracted separately by using column chromatography. From fruiting bodies eight metabolites, five sterols (ergosta-7,22-dien-3β-ol, ergosterol peroxide, ergosterol, cerevisterol, and ergosta-7,22-dien-3-one) as well as three terpene compounds (ganodermanondiol, ganoderic acid Sz, and ganoderitriol M) were obtained from fruiting bodies. From the mycelial culture three metabolites, two sterols (ergosterol and cerevisterol), and a new terpene compound (ganoderic acetate from the acid) were obtained. These structures were established based on a spectroscopic analysis mainly using nuclear magnetic resonance and a comparison with data already established.

Differences in carbon source utilisation by orchid mycorrhizal fungi from common and endangered species of Caladenia (Orchidaceae).

PubMed

Mehra, S; Morrison, P D; Coates, F; Lawrie, A C

2017-02-01

Terrestrial orchids depend on orchid mycorrhizal fungi (OMF) as symbionts for their survival, growth and nutrition. The ability of OMF from endangered orchid species to compete for available resources with OMF from common species may affect the distribution, abundance and therefore conservation status of their orchid hosts. Eight symbiotically effective OMF from endangered and more common Caladenia species were tested for their ability to utilise complex insoluble and simple soluble carbon sources produced during litter degradation by growth with different carbon sources in liquid medium to measure the degree of OMF variation with host conservation status or taxonomy. On simple carbon sources, fungal growth was assessed by biomass. On insoluble substrates, ergosterol content was assessed using ultra-performance liquid chromatography (UPLC). The OMF grew on all natural materials and complex carbon sources, but produced the greatest biomass on xylan and starch and the least on bark and chitin. On simple carbon sources, the greatest OMF biomass was measured on most hexoses and disaccharides and the least on galactose and arabinose. Only some OMF used sucrose, the most common sugar in green plants, with possible implications for symbiosis. OMF from common orchids produced more ergosterol and biomass than those from endangered orchids in the Dilatata and Reticulata groups but not in the Patersonii and Finger orchids. This suggests that differences in carbon source utilisation may contribute to differences in the distribution of some orchids, if these differences are retained on site.

(4S)-4,8-dihydroxy-1-tetralone and other chemical constituents from Pestalotiopsis sp. EJC07, endophytic fromBauhinia guianensis.

PubMed

de Souza, Eleane M C; da Silva, Ellon L; Marinho, Andrey M R; Marinho, Patrícia S B

2016-03-01

The present work reports the isolation of eight compounds fromPestalotiopsis sp. EJC07 isolated as endophytic fromBauhinia guianensis, a tipical plant of the Amazon. The compounds (4S)-4,8-dihydroxy-1-tetralone (1), uracil (2), uridin (3), p-hydroxybenzoic acid (4), ergosterol (5), ergosterol peroxide (6), cerevisterol (7) and ducitol (8) were isolated by chromatographic procedures and identified by spectral methods of 1D and 2D NMR and MS. The compound 1 is being reported for the first time in the genusPestalotiopsis.

Dietary sterol preference in the silkworm, Bombyx mori.

PubMed

Nagata, Shinji; Omori, Yukie; Nagasawa, Hiromichi

2006-12-01

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that beta-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: beta-sitosterol > ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouth part might be different from the sterol recognition mechanism present in sterol metabolism.

Glycyrrhizic acid attenuates growth of Leishmania donovani by depleting ergosterol levels.

PubMed

Dinesh, Neeradi; Neelagiri, Soumya; Kumar, Vinay; Singh, Sushma

2017-05-01

In the present study, glycyrrhizic acid (GA) the main component of Glycyrrhiza glabra was evaluated for its efficacy as antileishmanial agent and its mode of action explored. GA inhibits promastigotes and intracellular amastigotes in a dose dependent manner at an IC 50 value of 34 ± 3.0 μM and 20 ± 4.2 μM respectively. GA was non-toxic against THP-1 macrophage host cell line. GA was found to inhibit recombinant Leishmania donovani HMG-CoA reductase (LdHMGR) enzyme at the half-maximum inhibitory concentration of 24 ± 4.3 μM indicating the sensitivity and specificity of GA towards the enzyme. However, GA could cause only 30% reduction in HMGR activity when measured in Leishmania promastigotes treated with 34 μM of GA. Interestingly western blot analysis revealed fivefold reduced HMGR expression in GLA treated promastigotes. To further study the mode of action of GA, we used transgenic parasites overexpressing LdHMGR. Results indicated that ∼2 fold resistance was exhibited by LdHMGR overexpressing promastigotes to GA with an IC 50 value of 74 μM compared to the wild type parasite. This explained the specific binding of GA to LdHMGR enzyme. There was ∼2 fold depletion in ergosterol levels in wild type promastigotes compared to the HMGR overexpressors. This data was further validated by exogenous supplementation of GA treated cells with ergosterol and 40% reversal of growth inhibition was observed. The results obtained suggested that GA kills the parasite by affecting sterol biosynthetic pathway, especially by inhibiting the L. donovani HMGR and altering ergosterol levels. The finding from the current study shows that GA is a potential antileishmanial chemotherapeutic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

The 3-hydroxy-3-methylglutaryl coenzyme-A reductases from fungi: a proposal as a therapeutic target and as a study model.

PubMed

Andrade-Pavón, Dulce; Sánchez-Sandoval, Eugenia; Rosales-Acosta, Blanca; Ibarra, José Antonio; Tamariz, Joaquín; Hernández-Rodríguez, César; Villa-Tanaca, Lourdes

2014-01-01

The enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) catalyzes the conversion of HMG-Co-A into mevalonate. This step is the limiting point for the synthesis of cholesterol in mammals and ergosterol in fungi. We describe in this article the genome organization of HMGR coding genes and those deduced from different fungi, recount the evidence showing statins as HMGR inhibitors for ergosterol synthesis and its effect in yeast viability, and propose fungal HMGR (HMGRf) as a model to study the use of pharmaceutical compounds to inhibit cholesterol and ergosterol synthesis. Bibliographical search and bioinformatic analyses were performed and discussed. HMGRfs belong to the class I with a high homology in the catalytic region. The sterol biosynthetic pathway in humans and fungi share many enzymes in the initial steps (such as the HMGR enzyme), but in the last steps enzymes are different rendering the two final products: cholesterol in mammals and ergosterol in fungi. With regards to inhibitors such as statins and other compounds, these affect also fungal viability. Since HMGR from Schizosaccharomyces pombe and Ustilago maydis are very similar to the human HMGR in the catalytic regions, we propose that fungal enzymes can be used to test inhibitors for a potential use in humans. We consider that HMGRf is a good therapeutic target to design and test new antifungal compounds. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Lovastatin synergizes with itraconazole against planktonic cells and biofilms of Candida albicans through the regulation on ergosterol biosynthesis pathway.

PubMed

Zhou, Yujie; Yang, Hong; Zhou, Xuedong; Luo, Hongke; Tang, Fan; Yang, Jin; Alterovitz, Gil; Cheng, Lei; Ren, Biao

2018-06-01

The increase of fungal infectious diseases and lack of safe and efficacious antifungal drugs result in the urgent need of new therapeutic strategies. Here, we repurposed the lovastatin (LOV) as a synergistic antifungal potentiator to itraconazole (ITZ) against Candida albicans planktonic cells and biofilms in vitro for the first time. Mutants from ergosterol biosynthesis pathway were employed and key gene expression profiles of ergosterol pathway were also measured. LOV single treatment was unable to inhibit C. albicans strains except the ERG3 and ERG11 double mutant. LOV and ITZ combination was capable of inhibiting the C. albicans planktonic cells and biofilms synergistically including the ITZ resistant mutants. The synergistic antifungal ability was stronger in either ERG11 or ERG3 dysfunctional mutants compared to wild type. The combination lost the synergistic activities in the ERG11 and ERG3 double mutant, while it was sensitive to LOV single treatment. The expression of HMG1, encoding HMG-CoA the target of LOV, was significantly upregulated in ERG11 and ERG3 double mutant strain by the treatment of the combination at 1.5 and 3 h. The combination also significantly increased the HMG1 expression in mutants from ergosterol pathway compared with wild type. The ERG11 and ERG3 gene expressions were upregulated by ITZ and its combination with LOV, but seemingly not by LOV single treatment after 1.5 and 3 h. The combination of LOV and ITZ on C. albicans planktonic cells and biofilms highlights its potential clinical practice especially against the azole drug-resistant mutants.

Effects of two 6-quinolinyl chalcones on the integrity of plasma membrane of Paracoccidioides brasiliensis.

PubMed

de Sá, Nívea P; Cisalpino, Patrícia S; Tavares, Luciana C; Espíndola, Leandro; Borelli, Beatriz M; Barbeira, Paulo Js; Cardoso Perdigão, Gabriele de Matos; Souza-Fagundes, Elaine M; Rosa, Carlos A; Pizzolatti, Moacir G; Johann, Susana

2017-03-01

Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, yet few therapeutic options exist. Our aim was to search for new compounds with high efficacy, low toxicity, shorter treatment time and affordable cost. We studied two synthetic 6-quinolinyl chalcones, 3b and 3e, to determine their effects on VERO cells, antifungal activity, survival curve, interaction with other drugs and phenotypic effects against several isolates of Paracoccidioides spp. In this study, we verified that the compounds were not toxic, exhibited superior in vitro activity compared with that shown by trimethoprim-sulfamethoxazole, and after 5 days of treatment, decreased the fungal cell viability by approximately 70%. Additionally, no interactions were observed between the tested compounds and other drugs. We also found that these compounds induced morphological changes, such as shriveling of cells, fragmentation of the plasma membrane and cytoplasmic disorganization in vitro. The changes observed by microscopy assays corroborate the observation made with propidium iodide, where the number of cells stained with the compounds was higher than that observed after amphotericin B treatment. We observed an increase in the efflux of K + and a loss of intracellular contents in cells treated with 3b and 3e, confirming their effects on fungal membranes. However, damage to the membrane was not associated with a decrease in membrane ergosterol levels. The experimental evidences showed no direct indications of cellular wall damage caused by these compounds. Thus, these results confirm the antifungal potential of 3b and 3e against Paracoccidioides spp. with possible action on the membrane.

Evaluation of Antifungal Activity and Mechanism of Action of Citral against Candida albicans.

PubMed

Leite, Maria Clerya Alvino; Bezerra, André Parente de Brito; de Sousa, Janiere Pereira; Guerra, Felipe Queiroga Sarmento; Lima, Edeltrudes de Oliveira

2014-01-01

Candida albicans is a yeast that commensally inhabits the human body and can cause opportunistic or pathogenic infections. Objective. To investigate the antifungal activity of citral against C. albicans. Methodology. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were determined by the broth microdilution techniques. We also investigated possible citral action on cell walls (0.8 M sorbitol), cell membranes (citral to ergosterol binding), the time-kill curve, and biological activity on the yeast's morphology. Results. The MIC and MFC of citral were, respectively, 64 µg/mL and 256 µg/mL. Involvement with the cell wall and ergosterol binding were excluded as possible mechanisms of action. In the morphological interference assay, it was observed that the product inhibited pseudohyphae and chlamydoconidia formation. The MIC and the MFC of citral required only 4 hours of exposure to effectively kill 99.9% of the inoculum. Conclusion. Citral showed in vitro antifungal potential against strains of C. albicans. Citral's mechanism of action does not involve the cell wall or ergosterol, and further study is needed to completely describe its effects before being used in the future as a component of new antifungals.

Sterol metabolism in the filasterean Capsaspora owczarzaki has features that resemble both fungi and animals

PubMed Central

Molina, María Celeste; Ruiz-Trillo, Iñaki; Uttaro, Antonio D.

2016-01-01

Sterols are essential for several physiological processes in most eukaryotes. Sterols regulate membrane homeostasis and participate in different signalling pathways not only as precursors of steroid hormones and vitamins, but also through its role in the formation of lipid rafts. Two major types of sterols, cholesterol and ergosterol, have been described so far in the opisthokonts, the clade that comprise animals, fungi and their unicellular relatives. Cholesterol predominates in derived bilaterians, whereas ergosterol is what generally defines fungi. We here characterize, by a combination of bioinformatic and biochemical analyses, the sterol metabolism in the filasterean Capsaspora owczarzaki, a close unicellular relative of animals that is becoming a model organism. We found that C. owczarzaki sterol metabolism combines enzymatic activities that are usually considered either characteristic of fungi or exclusive to metazoans. Moreover, we observe a differential transcriptional regulation of this metabolism across its life cycle. Thus, C. owczarzaki alternates between synthesizing 7-dehydrocholesterol de novo, which happens at the cystic stage, and the partial conversion—via a novel pathway—of incorporated cholesterol into ergosterol, the characteristic fungal sterol, in the filopodial and aggregative stages. PMID:27383626

Sterol metabolism in the filasterean Capsaspora owczarzaki has features that resemble both fungi and animals.

PubMed

Najle, Sebastián R; Molina, María Celeste; Ruiz-Trillo, Iñaki; Uttaro, Antonio D

2016-07-01

Sterols are essential for several physiological processes in most eukaryotes. Sterols regulate membrane homeostasis and participate in different signalling pathways not only as precursors of steroid hormones and vitamins, but also through its role in the formation of lipid rafts. Two major types of sterols, cholesterol and ergosterol, have been described so far in the opisthokonts, the clade that comprise animals, fungi and their unicellular relatives. Cholesterol predominates in derived bilaterians, whereas ergosterol is what generally defines fungi. We here characterize, by a combination of bioinformatic and biochemical analyses, the sterol metabolism in the filasterean Capsaspora owczarzaki, a close unicellular relative of animals that is becoming a model organism. We found that C. owczarzaki sterol metabolism combines enzymatic activities that are usually considered either characteristic of fungi or exclusive to metazoans. Moreover, we observe a differential transcriptional regulation of this metabolism across its life cycle. Thus, C. owczarzaki alternates between synthesizing 7-dehydrocholesterol de novo, which happens at the cystic stage, and the partial conversion-via a novel pathway-of incorporated cholesterol into ergosterol, the characteristic fungal sterol, in the filopodial and aggregative stages. © 2016 The Authors.

A New Ergosterol Analog, a New Bis-Anthraquinone and Anti-Obesity Activity of Anthraquinones from the Marine Sponge-Associated Fungus Talaromyces stipitatus KUFA 0207

PubMed Central

Noinart, Jidapa; Buttachon, Suradet; Dethoup, Tida; Gales, Luís; Pereira, José A.; Urbatzka, Ralph; Freitas, Sara; Lee, Michael; Silva, Artur M. S.; Pinto, Madalena M. M.; Vasconcelos, Vítor; Kijjoa, Anake

2017-01-01

A new ergosterol analog, talarosterone (1) and a new bis-anthraquinone derivative (3) were isolated, together with ten known compounds including palmitic acid, ergosta-4,6,8(14),22-tetraen-3-one, ergosterol-5,8-endoperoxide, cyathisterone (2), emodin (4a), questinol (4b), citreorosein (4c), fallacinol (4d), rheoemodin (4e) and secalonic acid A (5), from the ethyl acetate extract of the culture of the marine sponge-associated fungus Talaromyces stipitatus KUFA 0207. The structures of the new compounds were established based on extensive 1D and 2D spectral analysis, and in the case of talarosterone (1), the absolute configurations of its stereogenic carbons were determined by X-ray crystallographic analysis. The structure and stereochemistry of cyathisterone (2) was also confirmed by X-ray analysis. The anthraquinones 4a–e and secalonic acid A (5) were tested for their anti-obesity activity using the zebrafish Nile red assay. Only citreorosein (4c) and questinol (4b) exhibited significant anti-obesity activity, while emodin (4a) and secalonic acid A (5) caused toxicity (death) for all exposed zebrafish larvae after 24 h. PMID:28509846

Uptake and toxicity of glyphosate in the lichen Xanthoria parietina (L.) Th. Fr.

PubMed

Vannini, Andrea; Guarnieri, Massimo; Bačkor, Martin; Bilová, Ivana; Loppi, Stefano

2015-12-01

This study investigated if treatment of the lichen Xanthoria parietina (L.) Th. Fr. with glyphosate caused uptake of this herbicide as well as physiological alterations. Samples were treated with Glifene SL®, a common commercial glyphosate-based herbicide, at the lowest recommended doses (3.6g/L) as well as with doses slightly higher than the highest suggested (36 g/L). The results clearly showed glyphosate uptake in X. parietina proportionally to the dose provided. Adverse physiological effects were evident on the photosynthetic apparatus (photosynthetic efficiency, chlorophyll a content, chlorophyll degradation) as well as on the fungal respiration rates and cell membrane integrity (ergosterol content, dehydrogenase activity) already after 24h from treatment, also at the low application dose. It is concluded that lichens are suitable organisms for monitoring unwanted biological effects from the application of glyphosate-based herbicides, as well as for detecting the accumulation of this compound in the biota, thus screening for its environmental fate. Copyright © 2015 Elsevier Inc. All rights reserved.

Singlet Oxygen Detection Using Red Wine Extracts as Photosensitizers.

PubMed

Lagunes, Irene; Vázquez-Ortega, Fernanda; Trigos, Ángel

2017-09-01

Moderate consumption of red wine provides beneficial effects to health. This is attributed to polyphenol compounds present in wine such as resveratrol, quercetin, gallic acid, rutin, and vanillic acid. The amount of these antioxidants is variable; nevertheless, the main beneficial effects of red wine are attributed to resveratrol. However, it has been found that resveratrol and quercetin are able to photosensitize singlet oxygen generation and conversely, gallic acid acts as quencher. Therefore, and since resveratrol and quercetin are some of the most important antioxidants reported in red wines, the aim of this research was to evaluate the photosensitizing ability of 12 red wine extracts through photo-oxidation of ergosterol. The presence of 1 O 2 was detected by ergosterol conversion into peroxide of ergosterol through 1 H NMR analysis. Our results showed that 10 wine extracts were able to act as photosensitizers in the generation of singlet oxygen. The presence of 1 O 2 can damage other compounds of red wine and cause possible organoleptic alterations. Finally, although the reaction conditions employed in this research do not resemble the inherent conditions in wine making processing or storing, or even during its consumption, this knowledge could be useful to prevent possible pro-oxidant effects and avoid detrimental effects in red wines. © 2017 Institute of Food Technologists®.

Understanding colonization and proliferation potential of endophytes and pathogen in planta via plating, polymerase chain reaction and ergosterol assay.

PubMed

Chow, Yiing Yng; Rahman, Sadequr; Ting, Adeline Su Yien

2017-01-01

This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb) introduced into oil palm ramets (host model). The endophytes selected were Diaporthe phaseolorum (WAA02), Trichoderma asperellum (T2), and Penicillium citrinum (BTF08). Ramets were first inoculated with 100 mL of fungal cells (10 6  cfu mL - 1 ) via soil drenching. For the next 7 days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta . Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7 days after inoculation. Polymerase Chain Reaction (PCR) detected and identified the isolates from the plant tissues. The ergosterol assay (via high performance liquid chromatography, HPLC) confirmed the presence of endophytes and Gb in planta . The increase in ergosterol levels throughout 49 days was however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta . This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.

Endotoxin, ergosterol, fungal DNA and allergens in dust from schools in Johor Bahru, Malaysia- associations with asthma and respiratory infections in pupils.

PubMed

Norbäck, Dan; Markowicz, Pawel; Cai, Gui-Hong; Hashim, Zailina; Ali, Faridah; Zheng, Yi-Wu; Lai, Xu-Xin; Spangfort, Michael Dho; Larsson, Lennart; Hashim, Jamal Hisham

2014-01-01

There are few studies on associations between respiratory health and allergens, fungal and bacterial compounds in schools in tropical countries. The aim was to study associations between respiratory symptoms in pupils and ethnicity, chemical microbial markers, allergens and fungal DNA in settled dust in schools in Malaysia. Totally 462 pupils (96%) from 8 randomly selected secondary schools in Johor Bahru, Malaysia, participated. Dust was vacuumed from 32 classrooms and analysed for levels of different types of endotoxin as 3-hydroxy fatty acids (3-OH), muramic acid, ergosterol, allergens and five fungal DNA sequences. Multiple logistic regression was applied. Totally 13.1% pupils reported doctor's diagnosed asthma, 10.3% wheeze and 21.1% pollen or pet allergy. Indian and Chinese children had less atopy and asthma than Malay. Carbon dioxide levels were low (380-690 ppm). No cat (Fel d1), dog (Can f 1) or horse allergens (Ecu cx) were detected. The levels of Bloomia tropicalis (Blo t), house dust mite allergens (Der p 1, Der f 1, Der m 1) and cockroach allergens (Per a 1 and Bla g 1) were low. There were positive associations between levels of Aspergillus versicolor DNA and daytime breathlessness, between C14 3-OH and respiratory infections and between ergosterol and doctors diagnosed asthma. There were negative (protective) associations between levels of C10 3-OH and wheeze, between C16 3-OH and day time and night time breathlessness, between cockroach allergens and doctors diagnosed asthma. Moreover there were negative associations between amount of fine dust, total endotoxin (LPS) and respiratory infections. In conclusion, endotoxin at school seems to be mainly protective for respiratory illness but different types of endotoxin could have different effects. Fungal contamination measured as ergosterol and Aspergillus versicolor DNA can be risk factors for respiratory illness. The ethnical differences for atopy and asthma deserve further attention.

Personal exposure of dairy workers to dust, endotoxin, muramic acid, ergosterol, and ammonia on large-scale dairies in the high plains Western United States.

PubMed

Davidson, Margaret E; Schaeffer, Joshua; Clark, Maggie L; Magzamen, Sheryl; Brooks, Elizabeth J; Keefe, Thomas J; Bradford, Mary; Roman-Muniz, Noa; Mehaffy, John; Dooley, Gregory; Poole, Jill A; Mitloehner, Frank M; Reed, Sue; Schenker, Marc B; Reynolds, Stephen J

2018-03-01

Dairy workers experience a high degree of bioaerosol exposure, composed of an array of biological and chemical constituents, which have been tied to adverse health effects. A better understanding of the variation in the magnitude and composition of exposures by task is needed to inform worker protection strategies. To characterize the levels and types of exposures, 115 dairy workers grouped into three task categories on nine farms in the high plains Western United States underwent personal monitoring for inhalable dust, endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, ergosterol, and ammonia through one work shift. Eighty-nine percent of dairy workers were exposed to endotoxin at concentrations exceeding the recommended exposure guidelines (adjusted for a long work shift). The proportion of workers with exposures exceeding recommended guidelines was lower for inhalable dust (12%), and ammonia (1%). Ergosterol exposures were only measurable on 28% of samples, primarily among medical workers and feed handlers. Milking parlor workers were exposed to significantly higher inhalable dust, endotoxin, 3-OHFA, ammonia, and muramic acid concentrations compared to workers performing other tasks. Development of large modern dairies has successfully made progress in reducing worker exposures and lung disease prevalence. However, exposure to endotoxin, dust, and ammonia continues to present a significant risk to worker health on North American dairies, especially for workers in milking parlors. This study was among the first to concurrently evaluate occupational exposure to assayable endotoxin (lipid A), 3-hydroxy fatty acids or 3-OHFA (a chemical measure of cell bound and noncell-bound endotoxins), muramic acid, ergosterol, and ammonia among workers on Western U.S. dairies. There remains a need for cost-effective, culturally acceptable intervention strategies integrated in OHS Risk Management and production systems to further optimize worker health and farm productivity.

Endotoxin, Ergosterol, Fungal DNA and Allergens in Dust from Schools in Johor Bahru, Malaysia- Associations with Asthma and Respiratory Infections in Pupils

PubMed Central

Norbäck, Dan; Markowicz, Pawel; Cai, Gui-Hong; Hashim, Zailina; Ali, Faridah; Zheng, Yi-Wu; Lai, Xu-Xin; Spangfort, Michael Dho; Larsson, Lennart; Hashim, Jamal Hisham

2014-01-01

There are few studies on associations between respiratory health and allergens, fungal and bacterial compounds in schools in tropical countries. The aim was to study associations between respiratory symptoms in pupils and ethnicity, chemical microbial markers, allergens and fungal DNA in settled dust in schools in Malaysia. Totally 462 pupils (96%) from 8 randomly selected secondary schools in Johor Bahru, Malaysia, participated. Dust was vacuumed from 32 classrooms and analysed for levels of different types of endotoxin as 3-hydroxy fatty acids (3-OH), muramic acid, ergosterol, allergens and five fungal DNA sequences. Multiple logistic regression was applied. Totally 13.1% pupils reported doctor’s diagnosed asthma, 10.3% wheeze and 21.1% pollen or pet allergy. Indian and Chinese children had less atopy and asthma than Malay. Carbon dioxide levels were low (380–690 ppm). No cat (Fel d1), dog (Can f 1) or horse allergens (Ecu cx) were detected. The levels of Bloomia tropicalis (Blo t), house dust mite allergens (Der p 1, Der f 1, Der m 1) and cockroach allergens (Per a 1 and Bla g 1) were low. There were positive associations between levels of Aspergillus versicolor DNA and daytime breathlessness, between C14 3-OH and respiratory infections and between ergosterol and doctors diagnosed asthma. There were negative (protective) associations between levels of C10 3-OH and wheeze, between C16 3-OH and day time and night time breathlessness, between cockroach allergens and doctors diagnosed asthma. Moreover there were negative associations between amount of fine dust, total endotoxin (LPS) and respiratory infections. In conclusion, endotoxin at school seems to be mainly protective for respiratory illness but different types of endotoxin could have different effects. Fungal contamination measured as ergosterol and Aspergillus versicolor DNA can be risk factors for respiratory illness. The ethnical differences for atopy and asthma deserve further attention. PMID:24523884

Characterization of the Microbial Community in Indoor Environments: a Chemical-Analytical Approach

PubMed Central

Sebastian, Aleksandra; Larsson, Lennart

2003-01-01

An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of 13C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples. PMID:12788704

Autumnal Biomass and Potential Productivity of Salt Marsh Fungi from 29° to 43° North Latitude along the United States Atlantic Coast†

PubMed Central

Newell, Steven Y.; Blum, Linda K.; Crawford, Richard E.; Dai, Ting; Dionne, Michele

2000-01-01

It has been established that substantial amounts of fungal mass accumulate in standing decaying smooth cordgrass (Spartina alterniflora) marshes in the southeastern United States (e.g., in standing decaying leaf blades with a total fungal organic mass that accounts for about 20% of the decay system organic mass), but it has been hypothesized that in marshes farther north this is not true. We obtained samples of autumnal standing decaying smooth cordgrass from sites in Florida to Maine over a 3-year period. The variation in latitude could not explain any of the variation in the living fungal standing crop (as determined by ergosterol content) or in the instantaneous rates of fungal growth (as determined by acetate incorporation into ergosterol at a standard temperature, 20°C), which led to the conclusion that the potential levels of fungal production per unit of naturally decaying grass are not different in northern and southern marshes. Twenty-one percent of the variation in the size of the living fungal standing crop could be explained by variation in the C/N ratio (the higher the C/N ratio the smaller the fungal crop), but the C/P ratio was not related to the size of the fungal crop. Instantaneous rates of fungal growth were negatively related to the size of the living fungal crop (r = −0.35), but these rates were not correlated with C/nutrient ratios. The same two predominant species of ascomycetes (one Phaeosphaeria species and one Mycosphaerella species) were found ejecting ascospores from standing decaying smooth cordgrass blades at all of the sites examined from Florida to Maine. PMID:10618221

The Effectiveness of Voriconazole in Therapy of Candida glabrata's Biofilms Oral Infections and Its Influence on the Matrix Composition and Gene Expression.

PubMed

Rodrigues, Célia F; Gonçalves, Bruna; Rodrigues, Maria Elisa; Silva, Sónia; Azeredo, Joana; Henriques, Mariana

2017-08-01

Candida glabrata is one of most prevalent yeast in fungal infections, especially in immunocompromised patients. Its azole resistance results in a low therapeutic response, particularly when associated with biofilms. The main goal of this work was to study the effectiveness of voriconazole (Vcz) against C. glabrata biofilms oral pathologies, as esophageal or oropharyngeal candidiasis. Antifungal susceptibilities were determined in pre-formed 24-h-biofilms and ERG genes expression was determined by qRT-PCR. Protein quantification was performed using BCA ® Kit, carbohydrate was estimated according to the Dubois assay and β-1,3 glucans concentration were determined using Glucatell ® kit. Finally, ergosterol, Vcz, and fluconazole (Flu) concentrations within the biofilm matrices were determined by RP-HPLC. Results showed that C. glabrata biofilms were more susceptible to Vcz than to Flu and that ERG genes expression evidenced an overexpression of the three ERG genes in the presence of both azoles. The matrix content presented a remarked decrease in proteins and an increase in carbohydrates, namely β-1,3 glucans. Ergosterol was successfully detected and quantified in the biofilm matrices, with no differences in all the considered conditions. Vcz demonstrated better diffusion through the biofilms and better cell penetration capacities, than Flu, indicating that the structure of the drug molecule fully influences its dissemination through the biofilm matrices. This work showed that Vcz is notably more effective than Flu for the treatment of resistant C. glabrata oral biofilms, which demonstrates a clinical relevance in its future use for the treatment of oropharyngeal/esophageal candidiasis caused by this species.

Assessment of microbiological contamination in the work environments of museums, archives and libraries.

PubMed

Skóra, Justyna; Gutarowska, Beata; Pielech-Przybylska, Katarzyna; Stępień, Łukasz; Pietrzak, Katarzyna; Piotrowska, Małgorzata; Pietrowski, Piotr

Museums, archives and libraries have large working environments. The goal of this study was to determine microbial contamination in these work places and estimate the influence of microclimatic parameters and total dust content on microbial contamination. In addition, research included evaluation of ergosterol concentration and fungal bioaerosol particle size distribution. Numbers of micro-organisms in the air and on the surfaces in museums were higher (2.1 × 10 2 -7.0 × 10 3  cfu/m 3 and 1.4 × 10 2 -1.7 × 10 4  cfu/100 cm 2 , respectively) than in archives and libraries (3.2 × 10 2 -7.2 × 10 2  cfu/m 3 and 8.4 × 10 2 -8.8 × 10 2  cfu/100 cm 2 , respectively). The numbers of micro-organisms detected in the tested museums, archives and libraries did not exceed occupational exposure limits proposed by Polish Committee for the Highest Permissible Concentrations and Intensities of Noxious Agents at the Workplace. The concentrations of respirable and suspended dust in museum storerooms were 2-4 times higher than the WHO-recommended limits. We found a correlation between microclimatic conditions and numbers of micro-organisms in the air in the tested working environments. In addition, a correlation was also found between ergosterol concentration and the number of fungi in the air. Fungi were the dominant micro-organisms in the working environments tested. Particles within the dominant fractions of culturable fungal aerosols sampled from museum storerooms had aerodynamic diameters between 1.1 and 2.1 µm.

Quantification of sterol-specific response in human macrophages using automated imaged-based analysis.

PubMed

Gater, Deborah L; Widatalla, Namareq; Islam, Kinza; AlRaeesi, Maryam; Teo, Jeremy C M; Pearson, Yanthe E

2017-12-13

The transformation of normal macrophage cells into lipid-laden foam cells is an important step in the progression of atherosclerosis. One major contributor to foam cell formation in vivo is the intracellular accumulation of cholesterol. Here, we report the effects of various combinations of low-density lipoprotein, sterols, lipids and other factors on human macrophages, using an automated image analysis program to quantitatively compare single cell properties, such as cell size and lipid content, in different conditions. We observed that the addition of cholesterol caused an increase in average cell lipid content across a range of conditions. All of the sterol-lipid mixtures examined were capable of inducing increases in average cell lipid content, with variations in the distribution of the response, in cytotoxicity and in how the sterol-lipid combination interacted with other activating factors. For example, cholesterol and lipopolysaccharide acted synergistically to increase cell lipid content while also increasing cell survival compared with the addition of lipopolysaccharide alone. Additionally, ergosterol and cholesteryl hemisuccinate caused similar increases in lipid content but also exhibited considerably greater cytotoxicity than cholesterol. The use of automated image analysis enables us to assess not only changes in average cell size and content, but also to rapidly and automatically compare population distributions based on simple fluorescence images. Our observations add to increasing understanding of the complex and multifactorial nature of foam-cell formation and provide a novel approach to assessing the heterogeneity of macrophage response to a variety of factors.

21 CFR 184.1950 - Vitamin D.

Code of Federal Regulations, 2010 CFR

2010-04-01

... irradiation of ergosterol isolated from yeast and related fungi and is purified by crystallization. (2.... It is also manufactured by ultraviolet irradiation of 7-dehydrocholesterol produced from cholesterol...

21 CFR 184.1950 - Vitamin D.

Code of Federal Regulations, 2011 CFR

2011-04-01

... irradiation of ergosterol isolated from yeast and related fungi and is purified by crystallization. (2.... It is also manufactured by ultraviolet irradiation of 7-dehydrocholesterol produced from cholesterol...

21 CFR 184.1950 - Vitamin D.

Code of Federal Regulations, 2012 CFR

2012-04-01

... irradiation of ergosterol isolated from yeast and related fungi and is purified by crystallization. (2.... It is also manufactured by ultraviolet irradiation of 7-dehydrocholesterol produced from cholesterol...

21 CFR 184.1950 - Vitamin D.

Code of Federal Regulations, 2013 CFR

2013-04-01

... irradiation of ergosterol isolated from yeast and related fungi and is purified by crystallization. (2.... It is also manufactured by ultraviolet irradiation of 7-dehydrocholesterol produced from cholesterol...

Overexpression of erg1 gene in Trichoderma harzianum CECT 2413: effect on the induction of tomato defence-related genes.

PubMed

Cardoza, R E; Malmierca, M G; Gutiérrez, S

2014-09-01

To investigate the effect of the overexpression of erg1 gene of Trichoderma harzianum CECT 2413 (T34) on the Trichoderma-plant interactions and in the biocontrol ability of this fungus. Transformants of T34 strain overexpressing erg1 gene did not show effect on the ergosterol level, although a drastic decrease in the squalene level was observed in the transformants at 96 h of growth. During interaction with plants, the erg1 overexpression resulted in a reduction of the priming ability of several tomato defence-related genes belonging to the salicylate pathway, and also of the TomLoxA gene, which is related to the jasmonate pathway. Interestingly, other jasmonate-related genes, such as PINI and PINII, were slightly induced. The erg1 overexpressed transformants also showed a reduced ability to colonize tomato roots. The ergosterol biosynthetic pathway might play an important role in regulating Trichoderma-plant interactions, although this role does not seem to be restricted to the final product; instead, other intermediates such as squalene, whose role in the Trichoderma-plant interaction has not been characterized, would also play an important role. The functional analysis of genes involved in the synthesis of ergosterol could provide additional strategies to improve the ability of biocontrol of the Trichoderma strains and their interaction with plants. © 2014 The Society for Applied Microbiology.

Trichodiene Production in a Trichoderma harzianum erg1-Silenced Strain Provides Evidence of the Importance of the Sterol Biosynthetic Pathway in Inducing Plant Defense-Related Gene Expression.

PubMed

Malmierca, M G; McCormick, S P; Cardoza, R E; Monte, E; Alexander, N J; Gutiérrez, S

2015-11-01

Trichoderma species are often used as biocontrol agents against plant-pathogenic fungi. A complex molecular interaction occurs among the biocontrol agent, the antagonistic fungus, and the plant. Terpenes and sterols produced by the biocontrol fungus have been found to affect gene expression in both the antagonistic fungus and the plant. The terpene trichodiene (TD) elicits the expression of genes related to tomato defense and to Botrytis virulence. We show here that TD itself is able to induce the expression of Botrytis genes involved in the synthesis of botrydial (BOT) and also induces terpene gene expression in Trichoderma spp. The terpene ergosterol, in addition to its role as a structural component of the fungal cell membranes, acts as an elicitor of defense response in plants. In the present work, using a transformant of T. harzianum, which is silenced in the erg1 gene and accumulates high levels of squalene, we show that this ergosterol precursor also acts as an important elicitor molecule of tomato defense-related genes and induces Botrytis genes involved in BOT biosynthesis, in both cases, in a concentration-dependent manner. Our data emphasize the importance of a balance of squalene and ergosterol in fungal interactions as well as in the biocontrol activity of Trichoderma spp.

Natural occurrence of fumonisins and ochratoxin A in some herbs and spices commercialized in Poland analyzed by UPLC-MS/MS method.

PubMed

Waśkiewicz, Agnieszka; Beszterda, Monika; Bocianowski, Jan; Goliński, Piotr

2013-12-01

Unsanitary conditions during harvesting, drying, packing and storage stages in production and processing of spices and herbs could introduce mycotoxin contamination. The occurrence of ochratoxin A and fumonisins in popular spices and herbs was studied, using liquid chromatography-electrospray-mass spectrometry. Apart from mycotoxins, ergosterol as a factor indicating fungal development was also analysed. A total of 79 different samples commercialized in Poland were randomly purchased from popular markets were tested for mycotoxins. The frequency of samples with fumonisins was lower (31%) than ochratoxin A (49%). Free from mycotoxins were samples of bay leaf and white mustard. ERG content - in spice samples with high concentration level of mycotoxins - was also significantly higher than in samples with little to no mycotoxins. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cytotoxic Effect on Human Myeloma Cells and Leukemic Cells by the Agaricus blazei Murill Based Mushroom Extract, Andosan™

PubMed Central

Holien, Toril; Mirlashari, Mohammad Reza; Misund, Kristine

2017-01-01

Agaricus blazei Murill is an edible mushroom of the Basidiomycetes family, which has been found to contain a number of compounds with antitumor properties, such as proteoglycans and ergosterol. In the present investigation, we show that the commercial mushroom product Andosan, which contains 82.4% Agaricus blazei Murill, together with medicinal mushrooms Hericium erinaceus (14.7%) and Grifola frondosa (2.9%), has a cytotoxic effect on primary myeloma cells, other myeloma cell lines, and leukemia cell lines in vitro. Although the exact content and hence the mechanisms of action of the Andosan extract are unknown, we have found in this investigation indications of cell cycle arrest when myeloma cell lines are cultivated with Andosan. This may be one of the possible explanations for the cytotoxic effects of Andosan. PMID:29238712

Cytotoxic Effect on Human Myeloma Cells and Leukemic Cells by the Agaricus blazei Murill Based Mushroom Extract, Andosan™.

PubMed

Tangen, Jon-Magnus; Holien, Toril; Mirlashari, Mohammad Reza; Misund, Kristine; Hetland, Geir

2017-01-01

Agaricus blazei Murill is an edible mushroom of the Basidiomycetes family, which has been found to contain a number of compounds with antitumor properties, such as proteoglycans and ergosterol. In the present investigation, we show that the commercial mushroom product Andosan, which contains 82.4% Agaricus blazei Murill, together with medicinal mushrooms Hericium erinaceus (14.7%) and Grifola frondosa (2.9%), has a cytotoxic effect on primary myeloma cells, other myeloma cell lines, and leukemia cell lines in vitro. Although the exact content and hence the mechanisms of action of the Andosan extract are unknown, we have found in this investigation indications of cell cycle arrest when myeloma cell lines are cultivated with Andosan. This may be one of the possible explanations for the cytotoxic effects of Andosan.

Phytotoxic components produced by pathogenic Fusarium against morning glory.

PubMed

Shimizu, Bun-ichi; Saito, Fukuko; Miyagawa, Hisahi; Watanabe, Ken; Ueno, Tamio; Sakata, Kanzo; Ogawa, Kei

2005-01-01

A pathogenic isolate of Fusarium, F. oxysporum f. sp. batatas O-17 (PF), causes wilt disease in leaf etiolation in sweet potato (Ipomoea batatas) and morning glory (Ipomoea tricolor). Extracts from PF cultures were screened for phytotoxic components using a growth inhibition assay with morning glory seedlings. The extracts were fractionated using differential solvent extraction and two active compounds, ergosterol and fusalanipyrone, were isolated from the less-polar fraction. Growth inhibition of morning glory seedlings showed a sigmoidal dose-response relationship, with fusalanipyrone exhibiting a two order of magnitude higher EC50 value than ergosterol (18 nM and 1.6 microM, respectively). Both compounds showed lower growth inhibition activity towards lettuce seedlings (Lactuca sativa). This study provides information on the phytotoxic components of PF and discusses the mechanism behind PFf-induced phytotoxicity.

Lethality of cytochalasin B and other compounds isolated from fungus Aspergillus sp. (Trichocomaceae) endophyte of Bauhinia guianensis (Fabaceae).

PubMed

Feitosa, André de O; Dias, Amanda Cristina S; Ramos, Gisele da C; Bitencourt, Heriberto R; Siqueira, José Edson S; Marinho, Patrícia Santana B; Barison, Andersson; Ocampos, Fernanda M M; Marinho, Andrey Moacir do R

Endophytic fungi are fungi that colonize internal tissues of plants; several biologically active compounds have been isolated from these fungi. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, this study aimed the isolation and structural identification of ergosterol (1), ergosterol peroxide (2), mevalonolactone (3), cytochalasin B (4) and cytochalasin H (5) from Aspergillus sp. EJC 04, an endophytic fungus from Bauhinia guianensis. The cytochalasin B (4) and the diacetate derivative of cytochalasin B (4a) showed high lethality in the brine shrimp assay. This is the first occurrence of cytochalasins in Amazonian endophytic fungi from B. guianensis. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

In silico Identification of Ergosterol as a Novel Fungal Metabolite Enhancing RuBisCO Activity in Lycopersicum esculentum.

PubMed

Mitra, Joyeeta; Narad, Priyanka; Sengupta, Abhishek; Sharma, P D; Paul, P K

2016-09-01

RuBisCO (EC 4.1.1.39), a key enzyme found in stroma of chloroplast, is important for fixing atmospheric CO2 in plants. Alterations in the activity of RuBisCO could influence photosynthetic yield. Therefore, to understand the activity of the protein, knowledge about its structure is pertinent. Though the structure of Nicotiana RuBisCO has been modeled, the structure of tomato RuBisCO is still unknown. RuBisCO extracted from chloroplasts of tomato leaves was subjected to MALDI-TOF-TOF followed by Mascot Search. The protein sequence based on gene identification numbers was subjected to in silico model construction, characterization and docking studies. The primary structure analysis revealed that protein was stable, neutral, hydrophilic and has an acidic pI. The result though indicates a 90 % homology with other members of Solanaceae but differs from the structure of Arabidopsis RuBisCO. Different ligands were docked to assess the activity of RuBisCO against these metabolite components. Out of the number of modulators tested, ergosterol had the maximum affinity (E = -248.08) with RuBisCO. Ergosterol is a major cell wall component of fungi and has not been reported to be naturally found in plants. It is a known immune elicitor in plants. The current study throws light on its role in affecting RuBisCO activity in plants, thereby bringing changes in the photosynthetic rate.

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms

PubMed Central

Nes, Craigen R.; Singha, Ujjal K.; Liu, Jialin; Ganapathy, Kulothungan; Villalta, Fernando; Waterman, Michael R.; Lepesheva, Galina I.; Chaudhuri, Minu; Nes, W. David

2012-01-01

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 C14-demethylase) 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 <[2-13C]leucine<[2-13C]acetate<[1-13C]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. PMID:22176028

Cultivated strains of Agaricus bisporus and A. brasiliensis: chemical characterization and evaluation of antioxidant and antimicrobial properties for the final healthy product--natural preservatives in yoghurt.

PubMed

Stojković, Dejan; Reis, Filipa S; Glamočlija, Jasmina; Ćirić, Ana; Barros, Lillian; Van Griensven, Leo J L D; Ferreira, Isabel C F R; Soković, Marina

2014-07-25

Agaricus bisporus (J. E. Lange) Emil J. Imbach and Agaricus brasiliensis Wasser, M. Didukh, Amazonas & Stamets are edible mushrooms. We chemically characterized these mushrooms for nutritional value, hydrophilic and lipophilic compounds. The antioxidant and antimicrobial activities of methanolic and ethanolic extracts were assessed. Hepatotoxicity was also evaluated. The ethanolic extract of both species was tested for inhibition of Listeria monocytogenes growth in yoghurt. Both species proved to be a good source of bioactive compounds. A. brasiliensis was richer in polyunsaturated fatty acids and revealed the highest concentration of phenolic acids, and tocopherols. A. bisporus showed the highest monounsaturated fatty acids and ergosterol contents. A. brasiliensis revealed the highest antioxidant potential, and its ethanolic extract displayed the highest antibacterial potential; the methanolic extract of A. bisporus revealed the highest antifungal activity. A. brasiliensis possessed better preserving properties in yoghurt.

Synthesis, structure and antimicrobial evaluation of a new gossypol triazole conjugates functionalized with aliphatic chains and benzyloxy groups.

PubMed

Pyta, Krystian; Blecha, Marietta; Janas, Anna; Klich, Katarzyna; Pecyna, Paulina; Gajecka, Marzena; Przybylski, Piotr

2016-09-01

Synthetic limitations in the copper-catalyzed azide alkyne cycloaddition (CuAAC) on gossypol's skeleton functionalized with alkyne (2) or azide (3) groups have been indicated. Modified approach to the synthesis of new gossypol-triazole conjugates yielded new compounds (24-31) being potential fungicides. Spectroscopic studies of triazole conjugates 24-31 have revealed their structures in solution, i.e., the presence of enamine-enamine tautomeric forms and π-π stacking intramolecular interactions between triazole arms. Biological evaluation of the new gossypol-triazole conjugates revealed the potency of 30 and 31 derivatives, having triazole-benzyloxy moieties, comparable with that of miconazole against Fusarium oxysporum. The results of HPLC evaluation of ergosterol content in different fungi strains upon treatment of gossypol and its derivatives enabled to propose a mechanism of antifungal activity of these compounds. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sterols of the fungi - Distribution and biosynthesis

NASA Technical Reports Server (NTRS)

Weete, J. D.

1973-01-01

The importance of sterols in the growth and reproduction in fungi is becoming increasingly apparent. This article concerns the composition and biosynthesis of ergosterol in these organisms. Comparison to plant and animal sterol formation are made.

Sterols of the fungi - Distribution and biosynthesis.

NASA Technical Reports Server (NTRS)

Weete, J. D.

1973-01-01

The importance of sterols in the growth and reproduction in fungi is becoming increasingly apparent. This article concerns the composition and biosynthesis of ergosterol in these organisms. Comparison to plant and animal sterol formation are made.

Short-term effects of natural and NH4+-enriched chabazite zeolitite amendments to soil microbial biomass

NASA Astrophysics Data System (ADS)

Ferretti, Giacomo; Keiblinger, Katharina Maria; Di Giuseppe, Dario; Faccini, Barbara; Colombani, Nicolò; Zechmeister-Boltenstern, Sophie; Coltorti, Massimo; Mastrocicco, Micòl

2017-04-01

Natural zeolite-bearing rocks (zeolitites) are known to be a suitable material for agricultural purposes by improving soil physicochemical properties and nitrogen use efficiency (NUE). However, little is known about their effects on soil microbial biomass. Aim of this work is to evaluate short-term effects of different chabazite-zeolitite amendments on soil microbial biomass (and activity). To this purpose a silty-clay agricultural soil was amended in three different ways, by the addition of 5 and 15 wt% of natural chabazite zeolitites (NZ) and 10 wt% of NH4+-enriched chabazite zeolitites (CZ). Soil pH, water content, dissolved organic carbon (C), total dissolved N, NH4+, NO3-, NO2-, microbial biomass C and N and ergosterol were periodically measured over a time course of 16 days in a laboratory incubation experiment. In order to verify the immobilization of N derived from CZ into microbial biomass, the δ15N signature of microorganisms was evaluated by the Extraction-Fumigation-Extraction method followed by EA-IRMS analysis. This latter investigation was possible because zeolitites were enriched with NH4+ derived from pig-slurry, which have a very high 15N natural abundance that allow to trace microbial incorporation. Soil amended with 5 wt% of NZ showed increased ergosterol content as well as microbial C/N ratio starting from day 9 of incubation, suggesting that fungal biomass was probably favored, although the same behavior was not found in the soil amended with 15 wt% of the same material. On the other hand, the NH4+-enriched CZ showed strong interactions with soil microbial biomass N. Isotopic measurements supported microbial assimilation of the N introduced with CZ since the second day of incubation. The high dissolved organic C and microbial biomass N suggested an increase of mineralization and immobilization processes. In addition, in CZ amended soil, microbial biomass N was related to NO3- production over time and inversely related to NH4+, suggesting high nitrification processes especially from day 7 of incubation. Low microbial C/N ratio support bacterial prevalence in the soil amended with CZ for N-assimilation and ammonia oxidation. This confirm that CZ is an efficient soil amendment providing an immediately available N pool to soil microbial biomass.

A new cytotoxic sesquiterpene quinone produced by Penicillium sp. F00120 isolated from a deep sea sediment sample.

PubMed

Lin, Xiuping; Zhou, Xuefeng; Wang, Fazuo; Liu, Kaisheng; Yang, Bin; Yang, Xianwen; Peng, Yan; Liu, Juan; Ren, Zhe; Liu, Yonghong

2012-01-01

A new fungal strain, displaying strong toxic activity against brine shrimp larvae, was isolated from a deep sea sediment sample collected at a depth of 1300 m. The strain, designated as F00120, was identified as a member of the genus Penicillium on the basis of morphology and ITS sequence analysis. One new sesquiterpene quinone, named penicilliumin A (1), along with two known compounds ergosterol (2) and ergosterol peroxide (3), were isolated and purified from the cultures of F00120 by silica gel column, Sephadex LH-20 column, and preparative thin layer chromatography. Their structures were elucidated by detailed nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analysis as well as comparison with literature data. The new compound penicilliumin A inhibited in vitro proliferation of mouse melanoma (B16), human melanoma (A375), and human cervical carcinoma (Hela) cell lines moderately.

Fatty acid and sterol composition of three phytomonas species.

PubMed

Nakamura, C V; Waldow, L; Pelegrinello, S R; Ueda-Nakamura, T; Filho, B A; Filho, B P

1999-01-01

Fatty acid and sterol analysis were performed on Phytomonas serpens and Phytomonas sp. grown in chemically defined and complex medium, and P. françai cultivated in complex medium. The three species of the genus Phytomonas had qualitatively identical fatty acid patterns. Oleic, linoleic, and linolenic were the major unsaturated fatty acids. Miristic and stearic were the major saturated fatty acids. Ergosterol was the only sterol isolated from Phytmonas sp. and P. serpens grown in a sterol-free medium, indicating that it was synthesized de novo. When P. françai that does not grow in defined medium was cultivated in a complex medium, cholesterol was the only sterol detected. The fatty acids and sterol isolated from Phytomonas sp. and P. serpens grown in a chemically defined lipid-free medium indicated that they were able to biosynthesize fatty acids and ergosterol from acetate or from acetate precursors such as glucose or threonine.

STEROL PROFILES IN 18 MACROALGAE OF THE PORTUGUESE COAST(1).

PubMed

Lopes, Graciliana; Sousa, Carla; Bernardo, João; Andrade, Paula B; Valentão, Patrícia; Ferreres, Federico; Mouga, Teresa

2011-10-01

The sterol profiles of dominant macroalgae occurring in the western Portuguese coast were evaluated. An analytical procedure, involving alkaline hydrolysis and extraction followed by separation by reversed-phase HPLC-diode array detection (HPLC-DAD), was optimized for the study of their sterols composition. The validated methodology is short in analysis time (as the compounds are determined in <20 min), sensitive, reproducible, and accurate. It was then successfully applied to the determination of campesterol, cholesterol, desmosterol, ergosterol, fucosterol, stigmasterol, and β-sitosterol in 18 species (three Chlorophyta, five Rhodophyta, and 10 Phaeophyta). The profiles obtained for the several macroalgal species were considerably different. C29 sterols were predominant in Phaeophyta and Chlorophyta (71%-95% of total sterol content), while in Rhodophyta cholesterol content is significantly higher (34%-87%). Among the studied species, Asparagopsis armata Harv. contained the lowest sterol amount (555 mg · kg(-1) dry weight), and Cystoseira tamariscifolia (Huds.) Papenf. the highest one (6,502 mg · kg(-1) dry weight). Data obtained may be helpful in identifying suitable marine sources of sterols, with potential applications in the food and pharmaceutical industries. © 2011 Phycological Society of America.

Influence of Rhizoctonia solani and Trichoderma spp. in growth of bean (Phaseolus vulgaris L.) and in the induction of plant defense-related genes.

PubMed

Mayo, Sara; Gutiérrez, Santiago; Malmierca, Monica G; Lorenzana, Alicia; Campelo, M Piedad; Hermosa, Rosa; Casquero, Pedro A

2015-01-01

Many Trichoderma species are well-known for their ability to promote plant growth and defense. We study how the interaction of bean plants with R. solani and/or Trichoderma affect the plants growth and the level of expression of defense-related genes. Trichoderma isolates were evaluated in vitro for their potential to antagonize R. solani. Bioassays were performed in climatic chambers and development of the plants was evaluated. The effect of Trichoderma treatment and/or R. solani infection on the expression of bean defense-related genes was analyzed by real-time PCR and the production of ergosterol and squalene was quantified. In vitro growth inhibition of R. solani was between 86 and 58%. In in vivo assays, the bean plants treated with Trichoderma harzianum T019 always had an increased size respect to control and the plants treated with this isolate did not decrease their size in presence of R. solani. The interaction of plants with R. solani and/or Trichoderma affects the level of expression of seven defense-related genes. Squalene and ergosterol production differences were found among the Trichoderma isolates, T019 showing the highest values for both compounds. T. harzianum T019 shows a positive effect on the level of resistance of bean plants to R. solani. This strain induces the expression of plant defense-related genes and produces a higher level of ergosterol, indicating its ability to grow at a higher rate in the soil, which would explain its positive effects on plant growth and defense in the presence of the pathogen.

A comparative study of mid-infrared diffuse reflection (DR) and attenuated total reflection (ATR) spectroscopy for the detection of fungal infection on RWA2-corn.

PubMed

Kos, Gregor; Krska, Rudolf; Lohninger, Hans; Griffiths, Peter R

2004-01-01

An investigation into the rapid detection of mycotoxin-producing fungi on corn by two mid-infrared spectroscopic techniques was undertaken. Corn samples from a single genotype (RWA2, blanks, and contaminated with Fusarium graminearum) were ground, sieved and, after appropriate sample preparation, subjected to mid-infrared spectroscopy using two different accessories (diffuse reflection and attenuated total reflection). The measured spectra were evaluated with principal component analysis (PCA) and the blank and contaminated samples were classified by cluster analysis. Reference data for fungal metabolites were obtained with conventional methods. After extraction and clean-up, each sample was analyzed for the toxin deoxynivalenol (DON) by gas chromatography with electron capture detection (GC-ECD) and ergosterol (a parameter for the total fungal biomass) by high-performance liquid chromatography with diode array detection (HPLC-DAD). The concentration ranges for contaminated samples were 880-3600 microg/kg for ergosterol and 300-2600 microg/kg for DON. Classification efficiency was 100% for ATR spectra. DR spectra did not show as obvious a clustering of contaminated and blank samples. Results and trends were also observed in single spectra plots. Quantification using a PLS1 regression algorithm showed good correlation with DON reference data, but a rather high standard error of prediction (SEP) with 600 microg/kg (DR) and 490 microg/kg (ATR), respectively, for ergosterol. Comparing measurement procedures and results showed advantages for the ATR technique, mainly owing to its ease of use and the easier interpretation of results that were better with respect to classification and quantification.

Miltefosine has post-antifungal effect and induces apoptosis in Cryptococcus yeasts.

PubMed

Spadari, Cristina de Castro; Vila, Taissa; Rozental, Sonia; Ishida, Kelly

2018-05-29

Cryptococcus spp. are common opportunistic fungal pathogens, particularly in HIV patients. The approved drug miltefosine (MFS) has potential as an alternative antifungal against cryptococcosis; however, the mechanism of action of MFS in Cryptococcus is poorly understood. Here, we examined the effects of MFS on C. neoformans and C. gattii yeasts (planktonic and biofilm lifestyles), to clarify its mechanism of action. MFS presented inhibitory and fungicidal effects against planktonic Cryptococcus cells, with similar activity against dispersion biofilm cells, while sessile biofilm cells were less sensitive to MFS. Interestingly, MFS had post-antifungal effect on Cryptococcus , with a proliferation delay of up to 8.15 h after short exposure to fungicidal doses. MFS at fungicidal concentrations increased plasma membrane permeability, likely due to direct interaction with ergosterol, as suggested by competition assays with exogenous ergosterol. Moreover, MFS reduced the mitochondrial membrane potential, increased ROS production, and induced DNA fragmentation and condensation, all of which are hallmarks of apoptosis. Transmission electron microscopy analysis showed that MFS-treated yeasts had a reduced mucopolysaccharide capsule (confirmed by morphometry in light microscopy), plasma membrane irregularities, mitochondrial swelling and a less conspicuous cell wall. Our results suggest that MFS increases plasma membrane permeability in Cryptococcus via interaction with ergosterol, and also affects the mitochondrial membrane, eventually leading to apoptosis, in line with its fungicidal activity. These findings confirm the potential of MFS as an antifungal against C. neoformans and C. gattii, and warrants further studies to establish clinical protocols for MFS use against cryptococcosis. Copyright © 2018 American Society for Microbiology.

An improved HPLC-DAD method for quantitative comparisons of triterpenes in Ganoderma lucidum and its five related species originating from Vietnam.

PubMed

Ha, Do Thi; Loan, Le Thi; Hung, Tran Manh; Han, Le Vu Ngoc; Khoi, Nguyen Minh; Dung, Le Viet; Min, Byung Sun; Nguyen, Nguyen Phuong Dai

2015-01-09

An HPLC-DAD method for the quality control of wild and cultivated Ganoderma lucidum (Linhzhi) and related species samples was developed and validated. The quantitative determination of G. lucidum and its related species using 14 triterpene constituents, including nine ganoderma acids (compounds 4-12), four alcohols (compounds 13-16), and one sterol (ergosterol, 17) were reported. The standard curves were linear over the concentration range of 7.5-180 µg/mL. The LOD and LOQ values for the analyses varied from 0.34 to 1.41 µg/mL and from 1.01 to 4.23 µg/mL, respectively. The percentage recovery of each reference compound was found to be from 97.09% to 100.79%, and the RSD (%) was less than 2.35%. The precision and accuracy ranged from 0.81%-3.20% and 95.38%-102.19% for intra-day, and from 0.43%-3.67% and 96.63%-103.09% for inter-day, respectively. The study disclosed in detail significant differences between the quantities of analyzed compounds in different samples. The total triterpenes in wild Linhzhi samples were significantly higher than in cultivated ones. The total constituent contents of the five related Linhzhi samples were considerably lower than that in the G. lucidum specimens, except for G. australe as its constituent content outweighed wild Linhzhi's content by 4:1.

Chemical Characterization and Antioxidant Potential of Wild Ganoderma Species from Ghana.

PubMed

Obodai, Mary; Mensah, Deborah L Narh; Fernandes, Ângela; Kortei, Nii Korley; Dzomeku, Matilda; Teegarden, Matthew; Schwartz, Steven J; Barros, Lillian; Prempeh, Juanita; Takli, Richard K; Ferreira, Isabel C F R

2017-01-25

The chemical characterization and antioxidant potential of twelve wild strains of Ganoderma sp. from Ghana, nine (LS1-LS9) of which were found growing wild simultaneously on the same dying Delonix regia tree, were evaluated. Parameters evaluated included the nutritional value, composition in sugars, fatty acids, phenolic and other organic compounds and some vitamins and vitamin precursors. Antioxidant potential was evaluated by investigating reducing power, radical scavenging activity and lipid peroxidation inhibition using five in vitro assays. Protein, carbohydrate, fat, ash and energy contents ranged between 15.7-24.5 g/100 g·dw, 73.31-81.90 g/100 g, 0.48-1.40 g/100 g, 0.68-2.12 g/100 g ash and 396.1-402.02 kcal/100 g, respectively. Fatty acids such as linoleic, oleic and palmitic acids were relatively abundant. Free sugars included rhamnose, fructose, mannitol, sucrose and trehalose. Total tocopherols, organic acids and phenolic compounds' content ranged between 741-3191 µg/100 g, 77-1003 mg/100 g and 7.6-489 µg/100 g, respectively. There were variations in the β-glucans, ergosterol and vitamin D₂ contents. The three major minerals in decreasing order were K > P > S. Ganoderma sp. strain AM1 showed the highest antioxidant activity. This study reveals, for the first time, chemical characteristics of Ganoderma spp. which grew simultaneously on the same tree.

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

PubMed

Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

2018-01-30

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

Antifungal mechanism of the combination of Cinnamomum verum and Pelargonium graveolens essential oils with fluconazole against pathogenic Candida strains.

PubMed

Essid, Rym; Hammami, Majdi; Gharbi, Dorra; Karkouch, Ines; Hamouda, Thouraya Ben; Elkahoui, Salem; Limam, Ferid; Tabbene, Olfa

2017-09-01

The present study aimed to investigate the anti-Candida activity of ten essential oils (EOs) and to evaluate their potential synergism with conventional drugs. The effect on secreted aspartic protease (SAP) activity and the mechanism of action were also explored. The antifungal properties of essential oils were investigated using standard micro-broth dilution assay. Only Cinnamomum verum, Thymus capitatus, Syzygium aromaticum, and Pelargonium graveolens exhibited a broad spectrum of activity against a variety of pathogenic Candida strains. Chemical composition of active essential oils was performed by gas chromatography-mass spectrometry (GC-MS). Synergistic effect was observed with the combinations C. verum/fluconazole and P. graveolens/fluconazole, with FIC value 0.37. Investigation of the mechanism of action revealed that C. verum EO reduced the quantity of ergosterol to 83%. A total inhibition was observed for the combination C. verum/fluconazole. However, P. graveolens EO may disturb the permeability barrier of the fungal cell wall. An increase of MIC values of P. graveolens EO and the combination with fluconazole was observed with osmoprotectants (sorbitol and PEG6000). Furthermore, the combination with fluconazole may affect ergosterol biosynthesis and disturb fatty acid homeostasis in C. albicans cells as the quantity of ergosterol and oleic acid was reduced to 52.33 and 72%, respectively. The combination of P. graveolens and C. verum EOs with fluconazole inhibited 78.31 and 64.72% SAP activity, respectively. To our knowledge, this is the first report underlying the mechanism of action and the inhibitory effect of SAP activity of essential oils in synergy with fluconazole. Naturally occurring phytochemicals C. verum and P. graveolens could be effective candidate to enhance the efficacy of fluconazole-based therapy of C. albicans infections.

Influence of Rhizoctonia solani and Trichoderma spp. in growth of bean (Phaseolus vulgaris L.) and in the induction of plant defense-related genes

PubMed Central

Mayo, Sara; Gutiérrez, Santiago; Malmierca, Monica G.; Lorenzana, Alicia; Campelo, M. Piedad; Hermosa, Rosa; Casquero, Pedro A.

2015-01-01

Many Trichoderma species are well-known for their ability to promote plant growth and defense. We study how the interaction of bean plants with R. solani and/or Trichoderma affect the plants growth and the level of expression of defense-related genes. Trichoderma isolates were evaluated in vitro for their potential to antagonize R. solani. Bioassays were performed in climatic chambers and development of the plants was evaluated. The effect of Trichoderma treatment and/or R. solani infection on the expression of bean defense-related genes was analyzed by real-time PCR and the production of ergosterol and squalene was quantified. In vitro growth inhibition of R. solani was between 86 and 58%. In in vivo assays, the bean plants treated with Trichoderma harzianum T019 always had an increased size respect to control and the plants treated with this isolate did not decrease their size in presence of R. solani. The interaction of plants with R. solani and/or Trichoderma affects the level of expression of seven defense-related genes. Squalene and ergosterol production differences were found among the Trichoderma isolates, T019 showing the highest values for both compounds. T. harzianum T019 shows a positive effect on the level of resistance of bean plants to R. solani. This strain induces the expression of plant defense-related genes and produces a higher level of ergosterol, indicating its ability to grow at a higher rate in the soil, which would explain its positive effects on plant growth and defense in the presence of the pathogen. PMID:26442006

Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

PubMed Central

Georgiev, Alexander; Sullivan, David P.; Kersting, Michael C.; Dittman, Jeremy S.; Beh, Christopher T.; Menon, Anant K.

2011-01-01

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologues of the mammalian oxysterol-binding protein (Osh1–7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1ts) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in nonvesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM. PMID:21689253

The Yeast Anaerobic Response Element AR1b Regulates Aerobic Antifungal Drug-dependent Sterol Gene Expression*

PubMed Central

Gallo-Ebert, Christina; Donigan, Melissa; Liu, Hsing-Yin; Pascual, Florencia; Manners, Melissa; Pandya, Devanshi; Swanson, Robert; Gallagher, Denise; Chen, WeiWei; Carman, George M.; Nickels, Joseph T.

2013-01-01

Saccharomyces cerevisiae ergosterol biosynthesis, like cholesterol biosynthesis in mammals, is regulated at the transcriptional level by a sterol feedback mechanism. Yeast studies defined a 7-bp consensus sterol-response element (SRE) common to genes involved in sterol biosynthesis and two transcription factors, Upc2 and Ecm22, which direct transcription of sterol biosynthetic genes. The 7-bp consensus SRE is identical to the anaerobic response element, AR1c. Data indicate that Upc2 and Ecm22 function through binding to this SRE site. We now show that it is two novel anaerobic AR1b elements in the UPC2 promoter that direct global ERG gene expression in response to a block in de novo ergosterol biosynthesis, brought about by antifungal drug treatment. The AR1b elements are absolutely required for auto-induction of UPC2 gene expression and protein and require Upc2 and Ecm22 for function. We further demonstrate the direct binding of recombinant expressed S. cerevisiae ScUpc2 and pathogenic Candida albicans CaUpc2 and Candida glabrata CgUpc2 to AR1b and SRE/AR1c elements. Recombinant endogenous promoter studies show that the UPC2 anaerobic AR1b elements act in trans to regulate ergosterol gene expression. Our results indicate that Upc2 must occupy UPC2 AR1b elements in order for ERG gene expression induction to take place. Thus, the two UPC2-AR1b elements drive expression of all ERG genes necessary for maintaining normal antifungal susceptibility, as wild type cells lacking these elements have increased susceptibility to azole antifungal drugs. Therefore, targeting these specific sites for antifungal therapy represents a novel approach to treat systemic fungal infections. PMID:24163365

Investigating the antifungal activity and mechanism(s) of geraniol against Candida albicans strains.

PubMed

Leite, Maria Clerya Alvino; de Brito Bezerra, André Parente; de Sousa, Janiere Pereira; de Oliveira Lima, Edeltrudes

2015-04-01

Candida albicans can be a yeast that is a commensal on the human body but can cause opportunistic or pathogenic infections. Candida infections may create serious health problems and as a result has initiated a search for new drugs with an antifungal action. Geraniol is an acyclic monoterpene alcohol with known pharmacological properties, including antimicrobial activity. The aim of this work was to evaluate the antifungal activity and mechanism(s) of geraniol against C. albicans strains. The minimum inhibitory concentration (MIC) was determined through broth microdilution techniques. We investigated possible geraniol activity on the fungal cell wall (sorbitol protect effect), cell membrane (geraniol to ergosterol binding), the time-kill curve, and its biological activity on the yeast's morphology. Amphotericin B was used as control, and all tests were performed in duplicate. The MIC of geraniol was 16 μg/ml (for 90% of isolates) but its probable mechanism of action did not involve the cell wall and ergosterol binding. In the morphological interference assay, we observed that the product inhibited pseudohyphae and chlamydoconidia formation. Time-dependent kill curve assay demonstrated that the fungicidal activity for MIC × 2 started at 2 h for the ATCC 76485 strain, and at 4 h for the LM-70 strain. Geraniol showed in vitro antifungal potential against strains of C. albicans but did not involve action on the cell wall or ergosterol. This study contributes to the development of new antifungal drugs, especially against Candida spp. © The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Photo-oxidation of ergosterol: indirect detection of antioxidants photosensitizers or quenchers of singlet oxygen.

PubMed

Lagunes, Irene; Trigos, Ángel

2015-04-01

Consumption of antioxidant supplements is associated to prevention of several diseases. However, recent studies suggest that antioxidants, besides scavenge free radicals could lead development of tumors. Due to conflicting reports on the antioxidant benefits, the capacity to photosensitize the generation of singlet oxygen of seven natural antioxidants was evaluated through photo-oxidation of ergosterol which proved to be an efficient method of indirect detection of singlet oxygen. Our results showed that curcumin, resveratrol and quercetin have pro-oxidant activity due they act as photosensitizers in generation of singlet oxygen. In addition, we observed that genistein, naringenin, β-carotene and gallic acid besides their antioxidant activity against ROS radicals, are capable of quenching ROS non-radicals as singlet oxygen. Finally, our results allow us to propose a new approach in classification of natural antioxidants scavengers of free radicals, based on their activity as quenchers of singlet oxygen or as photosensitizers in singlet oxygen generation. Copyright © 2015. Published by Elsevier B.V.

Some aspects of interrelations between fungi and other biota in forest soil.

PubMed

Krivtsov, Vladimir; Griffiths, Bryan S; Salmond, Ross; Liddell, Keith; Garside, Adam; Bezginova, Tanya; Thompson, Jacqueline A; Staines, Harry J; Watling, Roy; Palfreyman, John W

2004-08-01

Interrelations of fungal mycelium with other soil biota are of paramount importance in forestry and soil ecology. Here we present the results of statistical analysis of a comprehensive data set collected in the first (and the only) British fungus sanctuary over a period of four months. The variables studied included a number of soil properties, bacteria, protozoan flagellates, ciliates and amoebae, microbial and plant feeding nematodes, various microarthropods, and two fungal biomarkers--glomalin and ergosterol. One way ANOVA showed that the dynamics of the microbiota studied was influenced by seasonal changes. Superimposed on these changes, however, was variability due to biological interactions and habitat characteristics. Two fungal biomarkers, ergosterol and glomalin, were differently influenced by other biota and abiotic variables. The results indicate that the dynamics of soil fungi is influenced not only by soil microarthropods, but also by those found in forest litter. The overall outcome, therefore, is likely to be very complex and will depend upon specific conditions of any particular ecosystem.

Naproxen degradation test to monitor Trametes versicolor activity in solid-state bioremediation processes.

PubMed

Rodríguez-Rodríguez, Carlos E; Marco-Urrea, Ernest; Caminal, Gloria

2010-07-15

The white-rot fungus Trametes versicolor has been studied as a potential agent for the removal of environmental pollutants. For long-time solid-phase bioremediation systems a test is required to monitor the metabolic status of T. versicolor and its degradation capability at different stages. A biodegradation test based on the percentage of degradation of a spiked model pharmaceutical (anti-inflammatory naproxen) in 24 h (ND24) is proposed to monitor the removal of pharmaceuticals and personal care products in sewage sludge. ND24 is intended to act as a test complementary to ergosterol quantification as specific fungal biomarker, and laccase activity as extracellular oxidative capacity of T. versicolor. For samples collected over 45 d, ND24 values did not necessarily correlate with ergosterol or laccase amounts but in most cases, they were over 30% degradation, indicating that T. versicolor may be suitable for bioremediation of sewage sludge in the studied period. 2010 Elsevier B.V. All rights reserved.

Quality of Bread Supplemented with Antrodia
salmonea-Fermented Grains

PubMed Central

Chien, Rao-Chi; Ulziijargal, Enkhjargal

2016-01-01

Summary Fermented grains of buckwheat, oat, embryo rice and wheat, which were prepared by solid-state fermentation with Antrodia salmonea, and the mycelium was used to substitute 7% of wheat flour to make bread. No difference in proximate composition, texture profile and contents of non-volatile taste components was observed among bread samples. White bread and bread supplemented with mycelium and fermented grains looked different. Bread supplemented with fermented grains had similar thermal properties, which differed from those of white bread and bread supplemented with mycelium. Bread supplemented with fermented grains contained substantial mass fractions (on dry mass basis) of adenosine (0.92–1.96 µg/g), ergosterol (24.53–30.12 µg/g), ergothioneine (2.16–3.18 µg/g) and γ-aminobutyric acid (2.20–2.45 µg/g). In addition, bread supplemented with mycelium contained lovastatin (0.43 µg/g). White bread and bread supplemented with fermented grains had similar sensory results. Overall, fermented grains could be incorporated into bread to provide beneficial effects. PMID:27904408

Prey-dependent retention of dimethylsulfoniopropionate (DMSP) by mixotrophic dinoflagellates.

PubMed

Lee, Hyunwoo; Park, Ki-Tae; Lee, Kitack; Jeong, Hae Jin; Yoo, Yeong Du

2012-03-01

We investigated the retention of dimethylsulfoniopropionate (DMSP) in phototrophic dinoflagellates arising from mixotrophy by estimating the cellular content of DMSP in Karlodinium veneficum (mixotrophic growth) fed for 7-10 days on either DMSP-rich Amphidinium carterae (phototrophic growth only) or DMSP-poor Teleaulax sp. (phototrophic growth only). In K. veneficum fed on DMSP-poor prey, the cellular content of DMSP remained almost unchanged regardless of the rate of feeding, whereas the cellular content of DMSP in cells of K. veneficum fed on DMSP-rich prey increased by as much as 21 times the cellular concentration derived exclusively from phototrophic growth. In both cases, significant fractions (10-32% in the former case and 55-65% in the latter) of the total DMSP ingested by K. veneficum were transformed into dimethylsulfide and other biochemical compounds. The results may indicate that the DMSP content of prey species affects temporal variations in the cellular DMSP content of mixotrophic dinoflagellates, and that mixotrophic dinoflagellates produce DMS through grazing on DMSP-rich preys. Additional studies should be performed to examine the universality of our finding in other mixotrophic dinoflagellates feeding on diverse prey species. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

TRIADIMEFON INDUCES RAT THYROID TUMORS THROUGH A NON-TSH MEDIATED MODE OF ACTION

EPA Science Inventory

Conazoles are a class of fungicides used as agricultural and pharmaceutical products which inhibit ergosterol biosynthesis. Members of this class are hepatotoxic and cause mouse hepatocellular tumors and/or rat thyroid follicular cell tumors. Triadimefon-induced rat thyroid tumor...

TOXICITY PROFILES IN MICE TREATED WITH HEPATOTUMORIGENIC AND NON-HEPATOTUMORIGENIC TRIAZOLE CONAZOLE FUNGICIDES: PROPICONAZOLE, TRIADIMEFON, AND MYCLOBUTANIL

EPA Science Inventory

Conazoles comprise a class of fungicides used in agriculture and as pharmaceutical products. The fungicidal properties of conazoles are due to their inhibition of ergosterol biosynthesis. Certain conazoles are tumorigenic in rodents; both propiconazole and triadimefon are hepatot...

MYCLOBUTANIL AND TRIADIMEFON METHABOLISM BY RAT CYP ISOFORMS AND LIVER MICROSOMES

EPA Science Inventory

The mode of action of conazole fungicides is to inhibit cytochrome P450 (CYP) 51 activity and thus the biosynthesis of ergosterol by fungi. Conazoles can also modulate other CYP activities in vertebrate species including humans. Myclobutanil (MCL) and triadimefon (TRD) are ag...

Bioactive compounds and antioxidant activity exhibit high intraspecific variability in Pleurotus ostreatus mushrooms and correlate well with cultivation performance parameters.

PubMed

Koutrotsios, Georgios; Kalogeropoulos, Nick; Stathopoulos, Pantelis; Kaliora, Andriana C; Zervakis, Georgios I

2017-05-01

Experimental data related with oyster mushroom production and nutritional properties usually derive from the examination of only one strain, and hence their representativeness/usefulness is questionable. This work aims at assessing intraspecific variability in Pleurotus ostreatus by studying 16 strains, under the same conditions, in respect to essential cultivation and mushroom quality aspects, and by defining the impact of intrinsic/genetic factors on such parameters. Hence, mushroom yield, earliness, crop length, biological efficiency, productivity, and their content in selected macro and microconstituents (e.g. fatty acids, sterols, individual phenolic compounds, terpenic acids, glucans) as well as their antioxidant properties (i.e., antiradical activity, ferric reducing potential, inhibition of serum oxidation) were assayed. The effect of intrinsic/genetic factors was evident, especially as regards earliness, yield of each production flush and mushroom weight, whereas biological efficiency was not particularly influenced by the cultivated strain. Moreover, phenolics, ergosterol and antiradical activity demonstrated significant variability among strains in contrast to what was observed for fatty acids, β-glucans and ferric reducing potential. The observed heterogeneity reveals the limitations of using a low number of strains for evaluating mushroom production and/or their content in bioactive compounds, and as evidenced, it is valuable for breeding and commercial purposes.

Growth and cellular ion content of a salt-sensitive symbiotic system Azolla pinnata-Anabaena azollae under NaCl stress.

PubMed

Rai, Vandna; Sharma, Naveen Kumar; Rai, Ashwani K

2006-09-01

Salinity, at a concentration of 10 mM NaCl affected the growth of Azolla pinnata-Anabaena azollae association and became lethal at 40 mM. Plants exposed up to 30 mM NaCl exhibited longer roots than the control, especially during the beginning of incubation. Average root number in plants exposed to 10 and 20 mM NaCl remained almost the same as in control. A further rise in NaCl concentration to 30 mM reduced the root number, and roots shed off at 40 mM NaCl. Presence of NaCl in the nutrient solution increased the cellular Na+ of the intact association exhibiting differential accumulation by individual partners, while it reduced the cellular Ca2+ level. However, cellular K+ content did not show significant change. Cellular Na+ based on fresh weight of respective individual partners (host tissues and cyanobiont) remained higher in the host tissues than the cyanobiont, while reverse was true for K+ and Ca2+ contents. The contribution of A. azollae in the total cellular ion content of the association was a little because of meagre contribution of the cyanobiont mass (19-21%). High salt sensitivity of Azolla-Anabaena complex is due to an inability of the association to maintain low Na+ and high Ca2+ cellular level.

COMPARATIVE LIVER P450 ENZYME ACTIVITY AND HISTOPATHOLOGY IN MICE TREATED WITH THE CONAZOLE FUNGICIDES: MYCLOBUTANIL, PROPICONAZOLE AND TRIADIMETON

EPA Science Inventory

Conazoles used in agriculture and pharmaceutical products comprise a class of chemicals which inhibit ergosterol biosynthesis to act as fungicides. Both propiconazole and triadimefon are hepatotoxic and hepatotumorigenic in mice, while myclobutanil is not a mouse liver tumorigen....

Assessment of the Endocrine Toxicity of the Fungicide Prochloraz using the Larval Amphibian Growth and Development Assay

EPA Science Inventory

Prochloraz is a broad spectrum fungicide that acts by inhibiting ergosterol biosynthesis in target species. Toxicity results in non-target vertebrate species suggest this toxicant acts as an endocrine disruptor that inhibits aromatase, the enzyme responsible for the conversion of...

TOXICITY PROFILES IN RATS TREATED WITH TUMORIGENIC AND NONTUMORIGENIC TRIAZOLE CONAZOLE FUNGICIDES: PROPICONAZOLE, TRIADIMEFON, AND MYCLOBUTANIL

EPA Science Inventory

Conazoles are a class of azole based fungicides used in agriculture and as pharmaceutical products. They have a common mode of antifungal action through inhibition of ergosterol biosynthesis. Some members of this class have been shown to be hepatotoxic and will induce mouse hepa...

Analysis of the ergosterol biosynthesis pathway cloning, molecular characterization and phylogeny of lanosterol 14 α-demethylase (ERG11) gene of Moniliophthora perniciosa.

PubMed

de Oliveira Ceita, Geruza; Vilas-Boas, Laurival Antônio; Castilho, Marcelo Santos; Carazzolle, Marcelo Falsarella; Pirovani, Carlos Priminho; Selbach-Schnadelbach, Alessandra; Gramacho, Karina Peres; Ramos, Pablo Ivan Pereira; Barbosa, Luciana Veiga; Pereira, Gonçalo Amarante Guimarães; Góes-Neto, Aristóteles

2014-10-01

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.

Analysis of the ergosterol biosynthesis pathway cloning, molecular characterization and phylogeny of lanosterol 14 α-demethylase (ERG11) gene of Moniliophthora perniciosa

PubMed Central

de Oliveira Ceita, Geruza; Vilas-Boas, Laurival Antônio; Castilho, Marcelo Santos; Carazzolle, Marcelo Falsarella; Pirovani, Carlos Priminho; Selbach-Schnadelbach, Alessandra; Gramacho, Karina Peres; Ramos, Pablo Ivan Pereira; Barbosa, Luciana Veiga; Pereira, Gonçalo Amarante Guimarães; Góes-Neto, Aristóteles

2014-01-01

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches’ broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea. PMID:25505843

Steroids and triterpenes from the fruit bodies of Ganoderma lucidum and their anti-complement activity.

PubMed

Seo, Hyo Won; Hung, Tran Manh; Na, MinKyun; Jung, Hyun Ju; Kim, Jin Cheol; Choi, Jae Sue; Kim, Jung Hee; Lee, Hyeong-Kyu; Lee, IkSoo; Bae, KiHwan; Hattori, Masao; Min, Byung Sun

2009-11-01

To determine the anti-complement activity of natural triterpenes, chromatographic separation of the EtOAc-soluble fraction from the fruiting body of Ganoderma lucidum led to the isolation of three steroids and five triterpenoids. They were identified as ergosterol peroxide (1), ergosterol (2), genoderic acid Sz (3), stella sterol (4), ganoderic aic C1 (5), ganoderic acid A (6), methyl ganoderate A (7), and lucidenic acid A (8) based on spectroscopic evidence and physicochemical properties. These compounds were examined for their anti-complement activity against the classical pathway of the complement system. Compounds 2 and 3 showed potent anti-complement activity with IC50 values of 52.0 and 44.6 microM, respectively. Compound 1 exhibited significant inhibitory activity with an IC50 value of 126.8 microM, whereas compounds 4-8 were inactive. Our findings suggested that in addition to the ketone group at C-3, the delta7(8), delta9(11)-lanostadiene type triterpene also plays an important role in inhibiting the hemolytic activity of human serum against erythrocytes.

The in-vitro anti-leishmanial activity of inhibitors of ergosterol biosynthesis.

PubMed

Gebre-Hiwot, A; Frommel, D

1993-12-01

The in-vitro activity of a group of antifungal compounds known to inhibit ergosterol synthesis was investigated against Leishmania donovani grown as intracellular amastigotes in the human leukaemia monocyte cell line, THP-1. Toxicity on the host cells was assessed using the colorimetric MTT assay. Compounds inhibiting 2,3 oxidosqualene lanosterol cyclase; RO 43-3815, RO 43-5955, RO 43-8208, RO 42-6589 and RO 43-0688 displayed high activity with a median effective dose (ED50) of 0.6, 0.9, 3.5, 2.2 and 0.7 mg/L respectively. Of the azole compounds, oxiconazole had an ED50 value of 3.3 mg/L while ketoconazole showed the least activity. The delta-14-reductase and delta-8-delta-7 isomerase inhibitor, amorolfine, gave the highest therapeutic index with an ED50 value of 1.6 mg/L. Most compounds tested had a lower ED50 value than the standard antileishmanial drugs, sodium stibogluconate (5.5 mg Sbv/L) and meglumine antimoniate (3.0 mg Sbv/L) indicating the clean potential of these antifungal compounds in treating leishmaniasis.

Inhibitory potential of Grifola frondosa bioactive fractions on α-amylase and α-glucosidase for management of hyperglycemia.

PubMed

Su, Chun-Han; Lu, Tzy-Ming; Lai, Min-Nan; Ng, Lean-Teik

2013-01-01

This study examined the inhibitory effects of Grifola frondosa (GF), a medicinal mushroom popularly consumed in traditional medicine and health food, on digestive enzymes related to type 2 diabetes; chemical profiles and inhibitory kinetics of its bioactive fractions were also analyzed. Results showed that all GF extracts showed weak anti-α-amylase activity; however, strong anti-α-glucosidase activity was noted on GF n-hexane extract (GF-H). Further fractionation confirmed that compared with acarbose (a commercial α-glucosidase inhibitor), the nonpolar fraction of GF possessed a stronger anti-α-glucosidase activity but a weaker anti-α-amylase activity. These activities were not derived from ergosterol and ergosterol peroxide, two major compounds of this fraction. The inhibitory kinetics of GF-H on α-glucosidase was competitive inhibition. GF-H was as good as acarbose in inhibiting the starch digestion in vitro. Oleic acid and linoleic acid could be the major active constituents that have contributed to the potency of GF in inhibiting α-glucosidase activity. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

[Study on secondary metabolites of marine fungus Penicillium sp. FS60 from the South China Sea].

PubMed

Zhang, Ling; Li, Dong-Li; Chen, Yu-Chan; Tao, Mei-Hua; Zhang, Wei-Min

2012-07-01

To study the secondary metabolites of the marine fungus Penicillium sp. FS60 from the South China Sea and their cytotoxicities. The compounds were isolated from the culture of strain FS60 by various chromatographic methods (silica gel, reverse silica gel, Sephadex-LH20, preparative TLC, HPLC and PTLC) and recrystallization. Their structures were identified by extensive analysis of their spectroscopic data. Compounds were tested for their cytotoxicities against SF-268, MCF-7, and NCI-H460 cell lines by SRB method. While, Compounds were tested for their antibacterial activities against S. aureus, E. coli and P. aeruginosa. Seven compounds were isolated from the culture and identified as methyl 2,4-dihydroxy-3,5,6-trimethylbenzoate (1), 4-hydroxyacetophenone (2), 5-hydroxymethyl-furoic acid (3), isochromophilones VIII (4), ergosterol (5), ergosterol peroxide (6), and cerevisterol (7). Compound 1 is isolated from the genus Penicillium for the first time. Compound 3 is demonstrated to have significant inhibition against S. aureus and P. aeruginosa. Compound 4 is demonstrated to have significant inhibition against the three cell lines.

Development of Mushroom-Based Cosmeceutical Formulations with Anti-Inflammatory, Anti-Tyrosinase, Antioxidant, and Antibacterial Properties.

PubMed

Taofiq, Oludemi; Heleno, Sandrina A; Calhelha, Ricardo C; Alves, Maria José; Barros, Lillian; Barreiro, Maria Filomena; González-Paramás, Ana M; Ferreira, Isabel C F R

2016-10-14

The cosmetic industry is in a constant search for natural compounds or extracts with relevant bioactive properties, which became valuable ingredients to design cosmeceutical formulations. Mushrooms have been markedly studied in terms of nutritional value and medicinal properties. However, there is still slow progress in the biotechnological application of mushroom extracts in cosmetic formulations, either as antioxidants, anti-aging, antimicrobial, and anti-inflammatory agents or as hyperpigmentation correctors. In the present work, the cosmeceutical potential of ethanolic extracts prepared from Agaricus bisporus , Pleurotus ostreatus , and Lentinula edodes was analyzed in terms of anti-inflammatory, anti-tyrosinase, antioxidant, and antibacterial activities. The extracts were characterized in terms of phenolic acids and ergosterol composition, and further incorporated in a base cosmetic cream to achieve the same bioactive purposes. From the results obtained, the final cosmeceutical formulations presented 85%-100% of the phenolic acids and ergosterol levels found in the mushroom extracts, suggesting that there was no significant loss of bioactive compounds. The final cosmeceutical formulation also displayed all the ascribed bioactivities and as such, mushrooms can further be exploited as natural cosmeceutical ingredients.

Membrane of Candida albicans as a target of berberine.

PubMed

Zorić, Nataša; Kosalec, Ivan; Tomić, Siniša; Bobnjarić, Ivan; Jug, Mario; Vlainić, Toni; Vlainić, Josipa

2017-05-17

We investigated the mechanisms of anti-Candida action of isoquinoline alkaloid berberine, active constituent of medically important plants of Barberry species. The effects on membrane, morphological transition, synthesis of ergosterol and the consequent changes in membrane permeability have been studied. Polarization and lipid peroxidation level of the membrane following berberine treatment have been addressed. Minimal inhibitory concentration (MIC) of berberine against C. albicans was 17.75 μg/mL. Cytotoxic effect of berberine was concentration dependent, and in sub-MIC concentrations inhibit morphological transition of C. albicans cells to its filamentous form. Results showed that berberine affects synthesis of membrane ergosterol dose-dependently and induces increased membrane permeability causing loss of intracellular material to the outer space (DNA/protein leakage). Berberine also caused membrane depolarization and lipid peroxidation of membrane constituents indicating its direct effect on the membrane. Moreover, ROS levels were also increased following berberine treatment indicating further the possibility of membrane damage. Based on the obtained results it seems that berberine achieves its anti-Candida activity by affecting the cell membrane.

Mycochemical Investigation of the Turkey Tail Medicinal Mushroom Trametes versicolor (Higher Basidiomycetes): A Potential Application of the Isolated Compounds in Documented Pharmacological Studies.

PubMed

Habibi, Emran; Sadat-Ebrahimi, Seyed Esmaeel; Mousazadeh, Saeed Ali; Amanzadeh, Yaghoub

2015-01-01

The purpose of this study was to elucidate the chemical properties of the n-hexane, chloroform, and ethyl acetate extracts of the fruiting body of the medicinal mushroom Trametes versicolor. The study led to the isolation of 5 sterols, 2 triterpene derivatives, 1 hydroquinone-derived aromatic compound, and, finally, 1 cerebroside and 1 triglyceride derivative. These compounds were identified for first time in T. versicolor and were named as follows: 4-isobutoxyphenyl palmitate (5), N-D-2'-hydroxyheptanoic-1-O-β-D-glucopyranosyl-9-methyl-4,8-sphinga-dienine(cerebroside) (6), 3β-linoleyloxyergosta-7,22-diene (7), 3β-linoleyloxyergosta-7-ene (8), and betulinic acid (9). Other compounds elucidated in our study were ergosterol (1), ergosterol peroxide (2), trilinolein (3), ergosta-7, 22-dien-3β-ol (4), and betuline (10). These compounds were obtained via column or thin-layer chromatography before being identified by nuclear magnetic resonance spectroscopic analyses and infrared data. In addition, the beneficial pharmacological effects of the compounds are described here.

Contamination of wheat grain with microscopic fungi and their metabolites in Poland in 2006-2009.

PubMed

Stuper-Szablewska, Kinga; Perkowski, Juliusz

2014-01-01

Microscopic fungi are microorganisms commonly found in cereal products. Pathogens of cereals colonising kernels are responsible, among other things, for deterioration of the technological value of grain. However, the greatest threat is posed by mycotoxins produced by toxin-forming strains of these microorganisms. The aim of the present study was to determine the level of contamination with microscopic fungi and mycotoxins from the group of trichothecenes in wheat grain from Poland in a 4-year cycle. In the period 2006-2009, studies were conducted on the content of fungal metabolites (ergosterol [ERG] and type A and B trichothecenes) and the content of microscopic fungi expressed in colony-forming units (CFU) in wheat grain. A total of 129 grain samples were examined. Analysed wheat samples had similar contents of both the investigated fungal metabolites and levels of microscopic fungi. Contents of microscopic fungi were low. Concentration of ERG, on average, was 2.64 mg/kg, while in colony forming units this value ranged from 10(1) CFU/g to over 10(3) CFU/g. The total concentration of type A and B trichothecenes was also low and within the 4 years of the investigation did not exceed 0.062 mg/kg. Concentration of DON did not exceed 1,250 µg/kg, established as safe in grain for human consumption, in any of the tested samples. For the results collected in the years 2006-2009 and presented in this paper, correlations were calculated between the amount of mycoflora and analysed metabolites in 3 possible combinations: 0.7096 for ERG/total toxin concentration, 0.6086 for ERG/log CFU/g, and 0.4016 for the concentration of total toxins/log CFU/g. Highly significant correlations between the content of trichothecenes and the concentration of ERG indicate that the level of this metabolite is closely related to the content of mycotoxins in grain.

Impact on Vitamin D2, Vitamin D4 and Agaritine in Agaricus bisporus Mushrooms after Artificial and Natural Solar UV Light Exposure.

PubMed

Urbain, Paul; Valverde, Juan; Jakobsen, Jette

2016-09-01

Commercial mushroom production can expose mushrooms post-harvest to UV light for purposes of vitamin D2 enrichment by converting the naturally occurring provitamin D2 (ergosterol). The objectives of the present study were to artificially simulate solar UV-B doses occurring naturally in Central Europe and to investigate vitamin D2 and vitamin D4 production in sliced Agaricus bisporus (button mushrooms) and to analyse and compare the agaritine content of naturally and artificially UV-irradiated mushrooms. Agaritine was measured for safety aspects even though there is no rationale for a link between UV light exposure and agaritine content. The artificial UV-B dose of 0.53 J/cm(2) raised the vitamin D2 content to significantly (P < 0.001) higher levels of 67.1 ± 9.9 μg/g dry weight (DW) than sun exposure (3.9 ± 0.8 μg/g dry DW). We observed a positive correlation between vitamin D4 and vitamin D2 production (r(2) = 0.96, P < 0.001) after artificial UV irradiation, with vitamin D4 levels ranging from 0 to 20.9 μg/g DW. The agaritine content varied widely but remained within normal ranges in all samples. Irrespective of the irradiation source, agaritine dropped dramatically in conjunction with all UV-B doses both artificial and natural solar, probably due to its known instability. The biological action of vitamin D from UV-exposed mushrooms reflects the activity of these two major vitamin D analogues (D2, D4). Vitamin D4 should be analysed and agaritine disregarded in future studies of UV-exposed mushrooms.

Toxicological Tipping Points: Learning Boolean Networks from High-Content Imaging Data. (BOSC meeting)

EPA Science Inventory

The objective of this work is to elucidate biological networks underlying cellular tipping points using time-course data. We discretized the high-content imaging (HCI) data and inferred Boolean networks (BNs) that could accurately predict dynamic cellular trajectories. We found t...

The natural compound magnolol affects growth, biofilm formation, and ultrastructure of oral Candida isolates.

PubMed

Behbehani, Jawad; Shreaz, Sheikh; Irshad, Mohammad; Karched, Maribassapa

2017-12-01

The incidence of oral candidosis has increased in recent years due to the escalation in HIV-infection, cancer treatments, organ transplantation, and diabetes. In addition, corticosteroid use, dentures, and broad-spectrum antibiotic use have also contributed to the problem. Treatment of oral candidosis has continued to be problematic because of the potential toxicity of antifungals in clinical use, and, above all, development of drug resistance among patients. In this study, the antifungal effect of magnolol was investigated against 64 strains of Candida spp. (four standard and 60 oral isolates) through minimum inhibitory concentration (MIC) and growth curve assays. Insight into the mechanisms of the antifungal action has been gained through ultrastructural studies using confocal scanning laser microscopy (CSLM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Molecular docking was done for predicting the interactions of magnolol with ergosterol at supramolecular level. The toxicity of magnolol on human erythrocytes was measured by in vitro hemolytic assay. MIC values of magnolol ranged from 16-64 μg/ml, respectively. All tested isolates showed a marked sensitivity towards magnolol in growth curve assays. Biofilm results suggested that magnolol showed strong anti-biofilm activity. The results obtained for four different Candida spp. demonstrated that MBIC values of magnolol showed the average biofilm inhibition by 69.5%, respectively. CLSM experiments showed that cells exposed to magnolol (MIC) exhibited cell membrane disruption. SEM analysis of magnolol treated cells resulted in deformed cells. TEM micrographs showed rupturing of the cell wall and plasma membrane, releasing the intracellular content, and swelling of the cell wall. Hemolytic activity of magnolol is 11.9% at its highest MIC compared to an activity level of 25.4% shown by amphotericin B (Amp B) at 1 μg/ml. Lipinski's parameters calculated for magnolol suggested its good oral bioavailability. Docking studies indicated that magnolol might be interacting with ergosterol in the fungal cell membranes. Together, the present study provides enough evidence for further work on magnolol so that better strategies could be employed to treat oral candidosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Environmental Controls of Microbial Resource Partitioning in Soils

NASA Astrophysics Data System (ADS)

Kandeler, Ellen; Poll, Christian; Kramer, Susanne; Mueller, Karolin; Marhan, Sven

2015-04-01

The mineralization and flow of plant-derived carbon in soils is relevant to global carbon cycling. Current models of organismic carbon fluxes in soil assume that separate bacterial and fungal energy channels exist in soil. Recent studies disentangle the herbivore and detritivore pathways of microbial resource use, identify the key players contributing to these two different pathways, and determine to what extent microbial substrate use is affected by environmental controls. To follow the kinetics of litter and root decomposition and to quantify the contribution of key players, it is necessary to use isotopic approaches like PLFA-SIP and ergosterol-SIP. It was shown that bacteria and sugar consuming fungi initiated litter decomposition in an incubation experiment during the first two weeks, whereas higher fungi started to grow after the depletion of low molecular weight substrates. Analyses of PLFA-SIP revealed, for example, that fungi assimilated C directly from the litter, whereas bacteria took up substrates in the soil and therefore depended more on external transport processes than fungi. In addition, we will present data from a field experiment showing the incorporation of root and shoot litter C into organic and microbial C pools under field conditions over a period of two years. Similar amounts of C derived from the two resources differing in substrate quality and amount were incorporated into microbial C and ergosterol pools over time, indicating the importance of root-derived C for the soil food web. High incorporation of maize C (up to 76%) into ergosterol suggests fast and high assimilation of maize C into fungal biomass. Nevertheless, there is still a debate whether bacteria, archaea and fungi start feeding on new substrates at the same time or if their activity occurs at different successional stages. This presentation gives a summery of current knowledge on microbial resource partitioning under lab and field conditions.

Chlorhexidine: beta-cyclodextrin inhibits yeast growth by extraction of ergosterol.

PubMed

Teixeira, K I R; Araújo, P V; Sinisterra, R D; Cortés, M E

2012-04-01

Chlorhexidine (Cx) augmented with beta-cyclodextrin (β-cd) inclusion compounds, termed Cx:β-cd complexes, have been developed for use as antiseptic agents. The aim of this study was to examine the interactions of Cx:β-cd complexes, prepared at different molecular ratios, with sterol and yeast membranes. The Minimal Inhibitory Concentration (MIC) against the yeast Candida albicans (C.a.) was determined for each complex; the MICs were found to range from 0.5 to 2 μg/mL. To confirm the MIC data, quantitative analysis of viable cells was performed using trypan blue staining. Mechanistic characterization of the interactions that the Cx:β-cd complexes have with the yeast membrane and assessment of membrane morphology following exposure to Cx:β-cd complexes were performed using Sterol Quantification Method analysis (SQM) and scanning electron microscopy (SEM). SQM revealed that sterol extraction increased with increasing β-cd concentrations (1.71 ×10(3); 1.4 ×10(3); 3.45 ×10(3), and 3.74 ×10(3) CFU for 1:1, 1:2, 1:3, and 1:4, respectively), likely as a consequence of membrane ergosterol solubilization. SEM images demonstrated that cell membrane damage is a visible and significant mechanism that contributes to the antimicrobial effects of Cx:β-cd complexes. Cell disorganization increased significantly as the proportion of β-cyclodextrin present in the complex increased. Morphology of cells exposed to complexes with 1:3 and 1:4 molar ratios of Cx:β-cd were observed to have large aggregates mixed with yeast remains, representing more membrane disruption than that observed in cells treated with Cx alone. In conclusion, nanoaggregates of Cx:β-cd complexes block yeast growth via ergosterol extraction, permeabilizing the membrane by creating cluster-like structures within the cell membrane, possibly due to high amounts of hydrogen bonding.

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

PubMed

Grillitsch, Karlheinz; Tarazona, Pablo; Klug, Lisa; Wriessnegger, Tamara; Zellnig, Günther; Leitner, Erich; Feussner, Ivo; Daum, Günther

2014-07-01

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production. Copyright © 2014 Elsevier B.V. All rights reserved.

Propiconazole enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras famesylation

EPA Science Inventory

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic ...

MODULATION OF CYTOCHROME P-450S AND OTHER XENOBIOTIC METABOLIZING ENZYMES (XME) LEADING TO COMMON MODE OF ACTION FOR MULTIPLE TOXICITIES: CONAZOLE RESEARCH

EPA Science Inventory

Conazoles are triazole containing azole fungicides used to protect fruits, grains, and grasses. They have broad antifungal activity and can prevent as well as treat fungal infections. Their antifungal characteristic is due to their ability to block the synthesis of ergosterol w...

Comparison of lipid profiles of Malassezia pachydermatis strains isolated from dogs with otitis externa and without clinical symptoms of disease.

PubMed

Czyzewska, Urszula; Siemieniuk, Magdalena; Pyrkowska, Aleksandra; Nowakiewicz, Aneta; Bieganska, Malgorzata; Dabrowska, Iwona; Bartoszewicz, Marek; Dobrzyn, Pawel; Tylicki, Adam

2016-01-01

Malassezia pachydermatis can cause infections of the skin and mucous membranes, especially in animals. It becomes a problem also in medicine. It is considered that metabolic disorders as well as hormonal and immunological status of the host promote diseases caused by M. pachydermatis. Here we consider whether specific features of fungi could also favour infections. We checked whether there are differences in lipid profiles between strains obtained from dogs with otitis externa and strains obtained from healthy dogs. Lipid profiles were determined using thin layer chromatography and gas chromatography-mass spectrometry. All analyses were carried out on 32 strains derived from dogs with otitis externa and 31 strains isolated from dogs without symptoms of disease. The results show that strains isolated from dogs without symptoms of otitis externa are characterised by a higher content of fatty acids. They contain significantly more behenic and lignoceric acids on medium without addition of lipids, and more oleic acid and total monounsaturated fatty acids on medium with lipids supplementation. These strains have also a higher content of esters of ergosterol and triglycerides. Data obtained show differences which may be specific features of M. pachydermatis-specific strains related to the ability of infection, which could be not directly related of the host condition. © 2015 Blackwell Verlag GmbH.

Positive and negative aspects of green coffee consumption - antioxidant activity versus mycotoxins.

PubMed

Jeszka-Skowron, Magdalena; Zgoła-Grześkowiak, Agnieszka; Waśkiewicz, Agnieszka; Stępień, Łukasz; Stanisz, Ewa

2017-09-01

The quality of coffee depends not only on the contents of healthy compounds but also on its contamination with microorganisms that can produce mycotoxins during development, harvesting, preparation, transport and storage. The antioxidant activity of green coffee brews measured in this study by ABTS, DPPH and Folin-Ciocalteu assays showed that coffee extracts from Robusta beans possessed higher activity in all assays than extracts from Arabica beans. The occurrence of ochratoxin A and aflatoxins (B1, B2, G1 and G2) in green coffee beans was studied using liquid chromatography/mass spectrometry. Apart from mycotoxins, the content of ergosterol as a marker indicating fungal occurrence was also determined. Among aflatoxins, aflatoxin B1 was the dominant mycotoxin in coffee bean samples, with the highest level at 17.45 ng g -1 . Ochratoxin A was detected in four samples at levels ranging from 1.27 to 4.34 ng g -1 , and fungi potentially producing this toxin, namely Aspergillus oryzae, Alternaria sp., Aspergillus foetidus, Aspergillus tamarii and Penicillium citrinum, were isolated. Steaming and decaffeination of coffee beans increased antioxidant activities of brews in comparison with those prepared from unprocessed beans. Although toxins can be quantified in green coffee beans and novel fungi were isolated, their concentrations are acceptable according to legal limits. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Fungal Transformation of Tree Stumps into a Suitable Resource for Xylophagous Beetles via Changes in Elemental Ratios

PubMed Central

Filipiak, Michał; Sobczyk, Łukasz; Weiner, January

2016-01-01

The elements present in dead pine stumps inhabited by larvae of wood-boring beetles (Stictoleptura rubra, Arhopalus rusticus and Chalcophora mariana) were analyzed over the initial (first 5 years; a chronosequence) stages of wood decay. The quantities of N, P, K, Ca, Mg, Fe, Zn, Mn, Cu and Na (but not S) increased with increases in the content of ergosterol (used as a proxy for the amount of fungal tissue). In fact, the amounts of P, N, K, Fe and Cu presented marked increases. These findings show that fungi stoichiometrically rearrange dead wood by importing externally occurring nutrients to decaying stumps. During the first years of wood decay, the ratios of C to other elements decrease substantially, but differently, for various elements, whereas the N:Fe, N:Cu, N:P and N:K ratios remain relatively stable. Therefore, the stoichiometric mismatch between xylophages and their food is greatly reduced. By changing the nutritional stoichiometry of dead wood, fungi create a nutritional niche for wood-eaters, and these changes enable the development of xylophages.

Elimination of quiescent slow-cycling cells via reducing quiescence depth by natural compounds purified from Ganoderma lucidum

PubMed Central

Dai, Jian; Miller, Matthew A.; Everetts, Nicholas J.; Wang, Xia; Li, Peng; Li, Ye; Xu, Jian-Hua; Yao, Guang

2017-01-01

The medical mushroom Ganoderma lucidum has long been used in traditional Chinese medicine and shown effective in the treatment of many diseases including cancer. Here we studied the cytotoxic effects of two natural compounds purified from Ganoderma lucidum, ergosterol peroxide and ganodermanondiol. We found that these two compounds exhibited cytotoxicity not only against fast proliferating cells, but on quiescent, slow-cycling cells. Using a fibroblast cell-quiescence model, we found that the cytotoxicity on quiescent cells was due to induced apoptosis, and was associated with a shallower quiescent state in compound-treated cells, resultant from the increased basal activity of an Rb-E2F bistable switch that controls quiescence exit. Accordingly, we showed that quiescent breast cancer cells (MCF7), compared to its non-transformed counterpart (MCF10A), were preferentially killed by ergosterol peroxide and ganodermanondiol treatment presumably due to their already less stable quiescent state. The cytotoxic effect of natural Ganoderma lucidum compounds against quiescent cells, preferentially on quiescent cancer cells vs. non-cancer cells, may help future antitumor development against the slow-cycling cancer cell subpopulations including cancer stem and progenitor cells. PMID:28099150

Antifungal activity of synthetic naphthoquinones against dermatophytes and opportunistic fungi: preliminary mechanism-of-action tests.

PubMed

Ferreira, Maria do Perpetuo Socorro Borges Carriço; Cardoso, Mariana Filomena do Carmo; da Silva, Fernando de Carvalho; Ferreira, Vitor Francisco; Lima, Emerson Silva; Souza, João Vicente Braga

2014-07-06

This study evaluated the antifungal activities of synthetic naphthoquinones against opportunistic and dermatophytic fungi and their preliminary mechanisms of action. The minimum inhibitory concentrations (MICs) of four synthetic naphthoquinones for 89 microorganisms, including opportunistic yeast agents, dermatophytes and opportunistic filamentous fungi, were determined. The compound that exhibited the best activity was assessed for its action against the cell wall (sorbitol test), for interference associated with ergosterol interaction, for osmotic balance (K+ efflux) and for membrane leakage of substances that absorb at the wavelength of 260 nm. All tested naphthoquinones exhibited antifungal activity, and compound IVS320 (3a,10b-dihydro-1H-cyclopenta [b] naphtho [2,3-d] furan-5,10-dione)-dione) demonstrated the lowest MICs across the tested species. The MIC of IVS320 was particularly low for dermatophytes (values ranging from 5-28 μg/mL) and Cryptococcus spp. (3-5 μg/mL). In preliminary mechanism-of-action tests, IVS320 did not alter the fungal cell wall but did cause problems in terms of cell membrane permeability (efflux of K+ and leakage of substances that absorb at 260 nm). This last effect was unrelated to ergosterol interactions with the membrane.

Elimination of the last reactions in ergosterol biosynthesis alters the resistance of Saccharomyces cerevisiae to multiple stresses.

PubMed

Liu, Guodong; Chen, Yun; Færgeman, Nils J; Nielsen, Jens

2017-09-01

The sterol composition of membranes is known to influence many phenotypes of yeast. However, a systematic study of the relationship between sterol composition and stress resistances has not been conducted. Here, we therefore constructed single or double gene deletion mutants of the last four enzymes in ergosterol biosynthesis in a prototrophic genetic background of Saccharomyces cerevisiae. Identification of the sterol composition of these mutants revealed a high flexibility of the sterol-processing steps instead of the previously proposed sequential conversion. Compared with the wild type, the mutants showed altered resistances to different exogenous stresses regarding the specific growth rate and duration of lag phase. The erg5 deletion mutant whose sterol has a saturated side chain exhibited overall robust growth under the tested stress conditions. The thermotolerant phenotype of erg5 deletion mutant was reproduced in filamentous fungus Penicillium oxalicum. These results highlight the important role of sterols in the response of yeast cells to environmental stresses, and suggest the possibility of improving the robustness of industrial yeast strains by engineering their sterol composition. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Elimination of quiescent slow-cycling cells via reducing quiescence depth by natural compounds purified from Ganoderma lucidum.

PubMed

Dai, Jian; Miller, Matthew A; Everetts, Nicholas J; Wang, Xia; Li, Peng; Li, Ye; Xu, Jian-Hua; Yao, Guang

2017-02-21

The medical mushroom Ganoderma lucidum has long been used in traditional Chinese medicine and shown effective in the treatment of many diseases including cancer. Here we studied the cytotoxic effects of two natural compounds purified from Ganoderma lucidum, ergosterol peroxide and ganodermanondiol. We found that these two compounds exhibited cytotoxicity not only against fast proliferating cells, but on quiescent, slow-cycling cells. Using a fibroblast cell-quiescence model, we found that the cytotoxicity on quiescent cells was due to induced apoptosis, and was associated with a shallower quiescent state in compound-treated cells, resultant from the increased basal activity of an Rb-E2F bistable switch that controls quiescence exit. Accordingly, we showed that quiescent breast cancer cells (MCF7), compared to its non-transformed counterpart (MCF10A), were preferentially killed by ergosterol peroxide and ganodermanondiol treatment presumably due to their already less stable quiescent state. The cytotoxic effect of natural Ganoderma lucidum compounds against quiescent cells, preferentially on quiescent cancer cells vs. non-cancer cells, may help future antitumor development against the slow-cycling cancer cell subpopulations including cancer stem and progenitor cells.

The influence of microwave radiation from cellular phone on fetal rat brain.

PubMed

Jing, Ji; Yuhua, Zhang; Xiao-qian, Yang; Rongping, Jiang; Dong-mei, Guo; Xi, Cui

2012-03-01

The increasing use of cellular phones in our society has brought focus on the potential detrimental effects to human health by microwave radiation. The aim of our study was to evaluate the intensity of oxidative stress and the level of neurotransmitters in the brains of fetal rats chronically exposed to cellular phones. The experiment was performed on pregnant rats exposed to different intensities of microwave radiation from cellular phones. Thirty-two pregnant rats were randomly divided into four groups: CG, GL, GM, and GH. CG accepted no microwave radiation, GL group radiated 10 min each time, GM group radiated 30 min, and GH group radiated 60 min. The 3 experimental groups were radiated 3 times a day from the first pregnant day for consecutively 20 days, and on the 21st day, the fetal rats were taken and then the contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), noradrenaline (NE), dopamine (DA), and 5-hydroxyindole acetic acid (5-HT) in the brain were assayed. Compared with CG, there were significant differences (P<0.05) found in the contents of SOD, GSH-Px, and MDA in GM and GH; the contents of SOD and GSH-Px decreased and the content of MDA increased. The significant content differences of NE and DA were found in fetal rat brains in GL and GH groups, with the GL group increased and the GH group decreased. Through this study, we concluded that receiving a certain period of microwave radiation from cellular phones during pregnancy has certain harm on fetal rat brains.

Δ24-Sterol Methyltransferase Plays an Important Role in the Growth and Development of Sporothrix schenckii and Sporothrix brasiliensis

PubMed Central

Borba-Santos, Luana P.; Visbal, Gonzalo; Gagini, Thalita; Rodrigues, Anderson M.; de Camargo, Zoilo P.; Lopes-Bezerra, Leila M.; Ishida, Kelly; de Souza, Wanderley; Rozental, Sonia

2016-01-01

Inhibition of Δ24-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol-5-ene-3β-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker® Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole. PMID:27014234

Δ(24)-Sterol Methyltransferase Plays an Important Role in the Growth and Development of Sporothrix schenckii and Sporothrix brasiliensis.

PubMed

Borba-Santos, Luana P; Visbal, Gonzalo; Gagini, Thalita; Rodrigues, Anderson M; de Camargo, Zoilo P; Lopes-Bezerra, Leila M; Ishida, Kelly; de Souza, Wanderley; Rozental, Sonia

2016-01-01

Inhibition of Δ(24)-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol-5-ene-3β-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker(®) Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole.

Arabidopsis ERG28 Tethers the Sterol C4-Demethylation Complex to Prevent Accumulation of a Biosynthetic Intermediate That Interferes with Polar Auxin Transport[C][W

PubMed Central

Mialoundama, Alexis Samba; Jadid, Nurul; Brunel, Julien; Di Pascoli, Thomas; Heintz, Dimitri; Erhardt, Mathieu; Mutterer, Jérôme; Bergdoll, Marc; Ayoub, Daniel; Van Dorsselaer, Alain; Rahier, Alain; Nkeng, Paul; Geoffroy, Philippe; Miesch, Michel; Camara, Bilal; Bouvier, Florence

2013-01-01

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28). Here, using nonlethal loss-of-function strategies focused on Arabidopsis thaliana ERG28, we found that the previously undetected SBI 4-carboxy-4-methyl-24-methylenecycloartanol (CMMC) inhibits polar auxin transport (PAT), a key mechanism by which the phytohormone auxin regulates several aspects of plant growth, including development and responses to environmental factors. The induced accumulation of CMMC in Arabidopsis erg28 plants was associated with diagnostic hallmarks of altered PAT, including the differentiation of pin-like inflorescence, loss of apical dominance, leaf fusion, and reduced root growth. PAT inhibition by CMMC occurs in a brassinosteroid-independent manner. The data presented show that ERG28 is required for PAT in plants. Furthermore, it is accumulation of an atypical SBI that may act to negatively regulate PAT in plants. Hence, the sterol pathway offers further prospects for mining new target molecules that could regulate plant development. PMID:24326590

Configuration of a high-content imaging platform for hit identification and pharmacological assessment of JMJD3 demethylase enzyme inhibitors.

PubMed

Mulji, Alpa; Haslam, Carl; Brown, Fiona; Randle, Rebecca; Karamshi, Bhumika; Smith, Julia; Eagle, Robert; Munoz-Muriedas, Jordi; Taylor, Joanna; Sheikh, Arshad; Bridges, Angela; Gill, Kirsty; Jepras, Rob; Smee, Penny; Barker, Mike; Woodrow, Mike; Liddle, John; Thomas, Pamela; Jones, Emma; Gordon, Laurie; Tanner, Rob; Leveridge, Melanie; Hutchinson, Sue; Martin, Margaret; Brown, Murray; Kruidenier, Laurens; Katso, Roy

2012-01-01

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.

Genomic Prediction of Seed Quality Traits Using Advanced Barley Breeding Lines.

PubMed

Nielsen, Nanna Hellum; Jahoor, Ahmed; Jensen, Jens Due; Orabi, Jihad; Cericola, Fabio; Edriss, Vahid; Jensen, Just

2016-01-01

Genomic selection was recently introduced in plant breeding. The objective of this study was to develop genomic prediction for important seed quality parameters in spring barley. The aim was to predict breeding values without expensive phenotyping of large sets of lines. A total number of 309 advanced spring barley lines tested at two locations each with three replicates were phenotyped and each line was genotyped by Illumina iSelect 9Kbarley chip. The population originated from two different breeding sets, which were phenotyped in two different years. Phenotypic measurements considered were: seed size, protein content, protein yield, test weight and ergosterol content. A leave-one-out cross-validation strategy revealed high prediction accuracies ranging between 0.40 and 0.83. Prediction across breeding sets resulted in reduced accuracies compared to the leave-one-out strategy. Furthermore, predicting across full and half-sib-families resulted in reduced prediction accuracies. Additionally, predictions were performed using reduced marker sets and reduced training population sets. In conclusion, using less than 200 lines in the training set can result in low prediction accuracy, and the accuracy will then be highly dependent on the family structure of the selected training set. However, the results also indicate that relatively small training sets (200 lines) are sufficient for genomic prediction in commercial barley breeding. In addition, our results indicate a minimum marker set of 1,000 to decrease the risk of low prediction accuracy for some traits or some families.

Genomic Prediction of Seed Quality Traits Using Advanced Barley Breeding Lines

PubMed Central

Nielsen, Nanna Hellum; Jahoor, Ahmed; Jensen, Jens Due; Orabi, Jihad; Cericola, Fabio; Edriss, Vahid; Jensen, Just

2016-01-01

Genomic selection was recently introduced in plant breeding. The objective of this study was to develop genomic prediction for important seed quality parameters in spring barley. The aim was to predict breeding values without expensive phenotyping of large sets of lines. A total number of 309 advanced spring barley lines tested at two locations each with three replicates were phenotyped and each line was genotyped by Illumina iSelect 9Kbarley chip. The population originated from two different breeding sets, which were phenotyped in two different years. Phenotypic measurements considered were: seed size, protein content, protein yield, test weight and ergosterol content. A leave-one-out cross-validation strategy revealed high prediction accuracies ranging between 0.40 and 0.83. Prediction across breeding sets resulted in reduced accuracies compared to the leave-one-out strategy. Furthermore, predicting across full and half-sib-families resulted in reduced prediction accuracies. Additionally, predictions were performed using reduced marker sets and reduced training population sets. In conclusion, using less than 200 lines in the training set can result in low prediction accuracy, and the accuracy will then be highly dependent on the family structure of the selected training set. However, the results also indicate that relatively small training sets (200 lines) are sufficient for genomic prediction in commercial barley breeding. In addition, our results indicate a minimum marker set of 1,000 to decrease the risk of low prediction accuracy for some traits or some families. PMID:27783639

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk; Flatt, Peter R.; McClenaghan, Neville H.

2010-08-20

Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mMmore » glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.« less

Research of epidermal cellular vegetal cycle of intravascular low level laser irradiation in treatment of psoriasis

NASA Astrophysics Data System (ADS)

Zhu, Jing; Bao, Xiaoqing; Zhang, Mei-Jue

2005-07-01

Objective: To research epidermal cellular vegetal cycle and the difference of DNA content between pre and post Intravascular Low Level Laser Irradiation treatment of psoriasis. Method: 15 patients suffered from psoriasis were treated by intravascular low level laser irradiation (output power: 4-5mw, 1 hour per day, a course of treatment is 10 days). We checked the different DNA content of epidermal cell between pre and post treatment of psoriasis and 8 natural human. Then the percentage of each phase among the whole cellular cycle was calculated and the statistical analysis was made. Results: The mean value of G1/S phase is obviously down while G2+M phase increased obviously. T test P<0.05.The related statistical analysis showed significant difference between pre and post treatments. Conclusions: The Intravascular Low Level Laser Irradiation (ILLLI) in treatment of psoriasis is effective according to the research of epidermal cellular vegetal cycle and the difference DNA content of Intravascular Low Level Laser Irradiation between pre and post treatment of psoriasis

Effects of subsoiling on lateral roots, sucrose metabolizing enzymes, and soil ergosterol in two Jeffrey pine stands

Treesearch

W.J. Otrosina; Shi-Jean S. Sung; L.M. White

1996-01-01

We determined the effects of subsoiling on woody lateral roots and enzyme activities involved in stem carbon metabolism of 90- to 100-year-old Jeffrey pine (Pinus jeffreyi Grev. And Balf.) growing on the eastern side of the California Sierra Nevada Range.Twelve 1.0-ha plots were established on each of two sites. Four site treatments thinning and subsoiling entire...

Stabilization of the yeast desaturase system by low levels of oxygen

NASA Technical Reports Server (NTRS)

Volkmann, C. M.; Klein, H. P.

1983-01-01

The stability of particulate palmitoyl-CoA desaturase preparations from anaerobically grown yeast cells was increased by exposure to low levels of oxygen. The stabilizing effect of oxygen may be based upon the increased amounts of palmitoleic acid and ergosterol that become available to the cells. These results suggest the evolutinary appearance of this system at a time when atmospheric oxygen was at a low level.

Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

PubMed Central

Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

2007-01-01

Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment. PMID:17875991

Saccharomyces cerevisiae and metabolic activators: HXT3 gene expression and fructose/glucose discrepancy in sluggish fermentation conditions.

PubMed

Díaz-Hellín, Patricia; Naranjo, Victoria; Úbeda, Juan; Briones, Ana

2016-12-01

When exposed to mixtures of glucose and fructose, as occurs during the fermentation of grape juice into wine, Saccharomyces cerevisiae uses these sugars at different rates. Moreover, glucose and fructose are transported by the same hexose transporters (HXT), which present a greater affinity for glucose, so that late in fermentation, fructose becomes the predominant sugar. Only a few commercial fermentation activators are available to optimally solve the problems this entails. The aim of this study was to investigate the relation between HXT3 gene expression and fructose/glucose discrepancy in two different media inoculated with a commercial wine strain of S. cerevisiae in the presence of three metabolic activators. Fermentation kinetics, vitality and major metabolites were also measured. Rehydration with ergosterol improved the area under the curve and the growth rate (µ max ) in both studied media. Also, the fructose/glucose discrepancy values were improved with all activator treatments, highlighting rehydration in the presence of ascorbic acid. The yeast rehydration process was demonstrated to influence HXT3 expression under the studied conditions. Tetrahydrofolic acid treatment greatly influenced HXT3 gene expression, especially on the 12th day of the fermentation process. To a lesser extent, ergosterol and ascorbic acid also improved this parameter.

Antifungal mechanism of essential oil from Anethum graveolens seeds against Candida albicans.

PubMed

Chen, Yuxin; Zeng, Hong; Tian, Jun; Ban, Xiaoquan; Ma, Bingxin; Wang, Youwei

2013-08-01

This work studied the antifungal mechanism of dill seed essential oil (DSEO) against Candida albicans. Flow cytometric analysis and inhibition of ergosterol synthesis were performed to clarify the mechanism of action of DSEO on C. albicans. Upon treatment of cells with DSEO, propidium iodide penetrated C. albicans through a lesion in its plasma membrane. DSEO also significantly reduced the amount of ergosterol. These findings indicate that the plasma membrane of C. albicans was damaged by DSEO. The effect of DSEO on the functions of the mitochondria in C. albicans was also studied. We assayed the mitochondrial membrane potential (mtΔψ) using rhodamine 123 and determined the production of mitochondrial dysfunction-induced reactive oxygen species (ROS) via flow cytometry. The effects of the antioxidant l-cysteine (Cys) on DSEO-induced ROS production and the antifungal effect of DSEO on C. albicans were investigated. Exposure to DSEO increased mtΔψ. Dysfunctions in the mitochondria caused ROS accumulation in C. albicans. This increase in the level of ROS production and DSEO-induced decrease in cell viability were prevented by the addition of Cys, indicating that ROS are an important mediator of the antifungal action of DSEO. These findings indicate that the cytoplasmic membrane and mitochondria are the main anti-Candida targets of DSEO.

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack.

PubMed

Fügi, Matthias A; Gunasekera, Kapila; Ochsenreiter, Torsten; Guan, Xueli; Wenk, Markus R; Mäser, Pascal

2014-05-01

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.

Carbon and nitrogen distribution in the green algal lichens Hypogymnia physodes and Platismatia glauca in relation to nutrient supply.

PubMed

Dahlman, Lena; Persson, Jörgen; Näsholm, Torgny; Palmqvist, Kristin

2003-05-01

With the aim of understanding how some lichens can survive intensive fertilization we investigated two green algal ( Trebouxia) lichens, Hypogymnia physodes (L.) Nyl. and Platismatia glauca (L.) W. Culb., and compared control (Ctr), and intensively fertilized (F) thalli. We measured total N, proteins and amino acids to assess lichen N status. Chlorophyll a indicated photosynthetic capacity and photobiont mass, ergosterol the metabolic demands of the fungus, and chitin the fungal biomass. For carbon status we measured glucose, the photobiont ( Trebouxia) export product ribitol, and the mycobiont-specific carbohydrates arabitol and mannitol. The F-thalli had 2-3 times higher protein and N concentrations, 5-10 times higher chlorophyll a concentrations, while ergosterol and chitin were doubled. The ribitol concentrations were 4-5 times higher in the F-thalli, while the fungal carbohydrates did not increase to the same extent. The amino acid arginine had increased 60-fold. The F-thalli also had a relatively higher N investment in the photobiont in relation to mycobiont tissue compared to the Ctr-thalli, probably resulting in an increased capacity for carbon assimilation, most possibly required for maintaining the higher nutrient status of the F-thalli. Arginine accumulation possibly avoided toxic effects of accumulated NH4+, albeit binding a significant fraction of assimilated carbon.

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack

PubMed Central

Fügi, Matthias A.; Gunasekera, Kapila; Ochsenreiter, Torsten; Guan, Xueli; Wenk, Markus R.; Mäser, Pascal

2014-01-01

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas’s disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile. PMID:24627128

The laforin-malin complex negatively regulates glycogen synthesis by modulating cellular glucose uptake via glucose transporters.

PubMed

Singh, Pankaj Kumar; Singh, Sweta; Ganesh, Subramaniam

2012-02-01

Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.

Antifungal activity of Rubus chingii extract combined with fluconazole against fluconazole-resistant Candida albicans.

PubMed

Han, Bing; Chen, Jia; Yu, Yi-qun; Cao, Yong-bing; Jiang, Yuan-ying

2016-02-01

This study aimed to investigate the antifungal activity of Rubus chingii extract in combination with fluconazole (FLC) against FLC-resistant Candida albicans 100 in vitro. A R. chingii extract and FLC-resistant C. albicans fungus suspension were prepared. The minimum inhibitory concentration and fractional inhibitory concentration index of R. chingii extract combined with FLC against C. albicans were determined, after which growth curves for C. albicans treated with R. chingii extract, FLC alone and a combination of these preparations were constructed. Additionally, the mechanisms of drug combination against C. albicans were explored by flow cytometry, gas chromatographic mass spectrometry and drug efflux pump function detection. R. chingii extract combined with FLC showed significant synergy. Flow cytometry suggested that C. albicans cells mainly arrest in G1 and S phases when they have been treated with the drug combination. The drug combination resulted in a marked decrease in the ergosterol content of the cell membrane. Additionally, efflux of Rhodamine 6G decreased with increasing concentrations of R. chingii extract. R. chingii extract combined with FLC has antifungal activity against FLC-resistant C. albicans. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

Effect of light and atmosphere on the cultivation of the golden oyster culinary-medicinal mushroom, Pleurotus citrinopileatus (higher Basidiomycetes).

PubMed

Hu, Shu-Hui; Wu, Chiu-Yeh; Chen, Yu-Kuei; Wang, Jinn-Chyi; Chang, Sue-Joan

2013-01-01

With an aim to explore the productivity and quality of the fruiting body of culinary-medicinal golden oyster mushroom Pleurotus citrinopileatus, the carbon dioxide (CO₂) concentration of the ambient atmosphere was adjusted and a light-emitting diode panel was used to illuminate the colonized mycelium at different wavelengths. Biological efficiency and yield were higher at CO₂ levels of 0.05 and 0.1% than other tested CO₂ levels, and the mature fruiting body showed the highest yellow value at a CO₂ level of 0.1% (of all tested CO₂ levels). The highest biological efficiency and yield was obtained at the 720-nm wavelength. The ergosterol content of the pileus of the fruiting body was higher than that of the stipe in any flush time at a 720-nm wavelength of light and a CO₂ concentration of 0.1%. The decreased percentages of cellulose and lignin at the appearance of primordia were larger than those of mycelial growth duration. The fruiting quality of P. citrinopileatus might thus be enhanced by 720-nm illumination and an atmosphere with a CO₂ concentration of 0.1 to 0.15%.

Trophic ecology of two cold-water coral species from the Mediterranean Sea revealed by lipid biomarkers and compound-specific isotope analyses

NASA Astrophysics Data System (ADS)

Naumann, Malik S.; Tolosa, Imma; Taviani, Marco; Grover, Renaud; Ferrier-Pagès, Christine

2015-12-01

Scleractinian cold-water corals (CWC) act as key ecosystem engineers in deep-sea reef environments worldwide. However, our current understanding of their trophic ecology is still limited, particularly in understudied temperate oceanic regions such as the Mediterranean Sea. Hence, this study investigated the trophic ecology of the CWC Desmophyllum dianthus and Madrepora oculata by employing lipid biomarker techniques and compound-specific isotope analyses on coral tissues, suspended particulate organic matter (sPOM), and surface sediment sampled in a Mediterranean CWC habitat. CWC exhibited high contents of poly- and monounsaturated fatty acids (FA) (≥49 and 32 % of FA, respectively) and cholesterol (≥67 % of sterols), while sPOM and sediment samples were enriched in saturated FA (≥44 % of FA) and sitosterol (≥35 % of sterols). CWC contained some rare very long-chained polyunsaturated FA (>C22) and ergosterol absent in sPOM and sediment samples. Our results indicate that Mediterranean CWC mainly consume living food items, rather than detrital sPOM or resuspended sediment, and provide evidence for preferred feeding on omnivorous and carnivorous zooplankton. Overall, these findings provide new insights to the trophic ecology of two common CWC from the Mediterranean Sea.

Functional analysis of two sterol regulatory element binding proteins in Penicillium digitatum

PubMed Central

Ruan, Ruoxin; Wang, Mingshuang; Liu, Xin; Sun, Xuepeng; Chung, Kuang-Ren

2017-01-01

The sterol regulatory element binding proteins (SREBPs) are key regulators for sterol homeostasis in most fungi. In the citrus postharvest pathogen Penicillium digitatum, the SREBP homolog is required for fungicide resistance and regulation of CYP51 expression. In this study, we identified another SREBP transcription factor PdSreB in P. digitatum, and the biological functions of both SREBPs were characterized and compared. Inactivation of PdsreA, PdsreB or both genes in P. digitatum reduced ergosterol contents and increased sensitivities to sterol 14-α-demethylation inhibitors (DMIs) and cobalt chloride. Fungal strains impaired at PdsreA but not PdsreB increased sensitivity to tridemorph and an iron chelator 2,2’-dipyridyl. Virulence assays on citrus fruit revealed that fungal strains impaired at PdsreA, PdsreB or both induce maceration lesions similar to those induced by wild-type. However, ΔPdsreA, ΔPdsreB or the double mutant strain rarely produce aerial mycelia on infected citrus fruit peels. RNA-Seq analysis showed the broad regulatory functions of both SREBPs in biosynthesis, transmembrane transportation and stress responses. Our results provide new insights into the conserved and differentiated regulatory functions of SREBP homologs in plant pathogenic fungi. PMID:28467453

Cellular content and biosynthesis of polyamines during rooster spermatogenesis.

PubMed Central

Oliva, R; Vidal, S; Mezquita, C

1982-01-01

The natural polyamines spermine and spermidine, and the diamine putrescine, were extracted from rooster testis cells separated by sedimentation at unit gravity, and from vas-deferens spermatozoa. The ratios spermine/DNA and spermidine/DNA were kept relatively constant throughout spermatogenesis, whereas the ratio putrescine/DNA rose in elongated spermatids. The cellular content of spermine, spermidine and putrescine decreased markedly in mature spermatozoa. Two rate-limiting enzymes in the biosynthetic pathway of polyamines, ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, showed their highest activities at the end of spermiogenesis and were not detectable in vas-deferens spermatozoa. A marked reduction in cell volume during spermiogenesis without a parallel decrease in the cellular content of polyamines suggests the possibility that the marked changes in chromatin composition and structure occurring in rooster late spermatids could take place in an ambience of high polyamine concentration. Images PLATE 1 PMID:7159401

Increase in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase function

PubMed Central

He, Yue; Yam, Candice; Pomraning, Kyle; Chin, Jacqueline S. R.; Yew, Joanne Y.; Freitag, Michael; Oliferenko, Snezhana

2014-01-01

Excess fatty acids and sterols are stored as triacylglycerols and sterol esters in specialized cellular organelles, called lipid droplets. Understanding what determines the cellular amount of neutral lipids and their packaging into lipid droplets is of fundamental and applied interest. Using two species of fission yeast, we show that cycling cells deficient in the function of the ER-resident CDP-DG synthase Cds1 exhibit markedly increased triacylglycerol content and assemble large lipid droplets closely associated with the ER membranes. We demonstrate that these unusual structures recruit the triacylglycerol synthesis machinery and grow by expansion rather than by fusion. Our results suggest that interfering with the CDP-DG route of phosphatidic acid utilization rewires cellular metabolism to adopt a triacylglycerol-rich lifestyle reliant on the Kennedy pathway. PMID:25318672

Cellular content of biomolecules in sub-seafloor microbial communities

NASA Astrophysics Data System (ADS)

Braun, Stefan; Morono, Yuki; Becker, Kevin W.; Hinrichs, Kai-Uwe; Kjeldsen, Kasper U.; Jørgensen, Bo B.; Lomstein, Bente Aa.

2016-09-01

Microbial biomolecules, typically from the cell envelope, can provide crucial information about distribution, activity, and adaptations of sub-seafloor microbial communities. However, when cells die these molecules can be preserved in the sediment on timescales that are likely longer than the lifetime of their microbial sources. Here we provide for the first time measurements of the cellular content of biomolecules in sedimentary microbial cells. We separated intact cells from sediment matrices in samples from surficial, deeply buried, organic-rich, and organic-lean marine sediments by density centrifugation. Amino acids, amino sugars, muramic acid, and intact polar lipids were analyzed in both whole sediment and cell extract, and cell separation was optimized and evaluated in terms of purity, separation efficiency, taxonomic resemblance, and compatibility to high-performance liquid chromatography and mass spectrometry for biomolecule analyses. Because cell extracts from density centrifugation still contained considerable amounts of detrital particles and non-cellular biomolecules, we further purified cells from two samples by fluorescence-activated cell sorting (FACS). Cells from these highly purified cell extracts had an average content of amino acids and lipids of 23-28 fg cell-1 and 2.3 fg cell-1, respectively, with an estimated carbon content of 19-24 fg cell-1. In the sediment, the amount of biomolecules associated with vegetative cells was up to 70-fold lower than the total biomolecule content. We find that the cellular content of biomolecules in the marine subsurface is up to four times lower than previous estimates. Our approach will facilitate and improve the use of biomolecules as proxies for microbial abundance in environmental samples and ultimately provide better global estimates of microbial biomass.

Information content and cross-talk in biological signal transduction: An information theory study

NASA Astrophysics Data System (ADS)

Prasad, Ashok; Lyons, Samanthe

2014-03-01

Biological cells respond to chemical cues provided by extra-cellular chemical signals, but many of these chemical signals and the pathways they activate interfere and overlap with one another. How well cells can distinguish between interfering extra-cellular signals is thus an important question in cellular signal transduction. Here we use information theory with stochastic simulations of networks to address the question of what happens to total information content when signals interfere. We find that both total information transmitted by the biological pathway, as well as its theoretical capacity to discriminate between overlapping signals, are relatively insensitive to cross-talk between the extracellular signals, until significantly high levels of cross-talk have been reached. This robustness of information content against cross-talk requires that the average amplitude of the signals are large. We predict that smaller systems, as exemplified by simple phosphorylation relays (two-component systems) in bacteria, should be significantly much less robust against cross-talk. Our results suggest that mammalian signal transduction can tolerate a high amount of cross-talk without degrading information content, while smaller bacterial systems cannot.

Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging

PubMed Central

Quinto-Su, Pedro A.; Lai, Hsuan-Hong; Yoon, Helen H.; Sims, Christopher E.; Allbritton, Nancy L.; Venugopalan, Vasan

2008-01-01

We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at λ = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications. PMID:18305858

Impact of uranium (U) on the cellular glutathione pool and resultant consequences for the redox status of U.

PubMed

Viehweger, Katrin; Geipel, Gerhard; Bernhard, Gert

2011-12-01

Uranium (U) as a redox-active heavy metal can cause various redox imbalances in plant cells. Measurements of the cellular glutathione/glutathione disulfide (GSH/GSSG) by HPLC after cellular U contact revealed an interference with this essential redox couple. The GSH content remained unaffected by 10 μM U whereas the GSSG level immediately increased. In contrast, higher U concentrations (50 μM) drastically raised both forms. Using the Nernst equation, it was possible to calculate the half-cell reduction potential of 2GSH/GSSG. In case of lower U contents the cellular redox environment shifted towards more oxidizing conditions whereas the opposite effect was obtained by higher U contents. This indicates that U contact causes a consumption of reduced redox equivalents. Artificial depletion of GSH by chlorodinitrobenzene and measuring the cellular reducing capacity by tetrazolium salt reduction underlined the strong requirement of reduced redox equivalents. An additional element of cellular U detoxification mechanisms is the complex formation between the heavy metal and carboxylic functionalities of GSH. Because two GSH molecules catalyze electron transfers each with one electron forming a dimer (GSSG) two UO(2) (2+) are reduced to each UO(2) (+) by unbound redox sensitive sulfhydryl moieties. UO(2) (+) subsequently disproportionates to UO(2) (2+) and U(4+). This explains that in vitro experiments revealed a reduction to U(IV) of only around 33% of initial U(VI). Cellular U(IV) was transiently detected with the highest level after 2 h of U contact. Hence, it can be proposed that these reducing processes are an important element of defense reactions induced by this heavy metal.

Abiotic and biotic factors responsible for antimonite oxidation in Agrobacterium tumefaciens GW4

PubMed Central

Li, Jingxin; Yang, Birong; Shi, Manman; Yuan, Kai; Guo, Wei; Wang, Qian; Wang, Gejiao

2017-01-01

Antimonite [Sb(III)]-oxidizing bacteria can transform the toxic Sb(III) into the less toxic antimonate [Sb(V)]. Recently, the cytoplasmic Sb(III)-oxidase AnoA and the periplasmic arsenite [As(III)] oxidase AioAB were shown to responsible for bacterial Sb(III) oxidation, however, disruption of each gene only partially decreased Sb(III) oxidation efficiency. This study showed that in Agrobacterium tumefaciens GW4, Sb(III) induced cellular H2O2 content and H2O2 degradation gene katA. Gene knock-out/complementation of katA, anoA, aioA and anoA/aioA and Sb(III) oxidation and growth experiments showed that katA, anoA and aioA were essential for Sb(III) oxidation and resistance and katA was also essential for H2O2 resistance. Furthermore, linear correlations were observed between cellular H2O2 and Sb(V) content in vivo and chemical H2O2 and Sb(V) content in vitro (R2 = 0.93 and 0.94, respectively). These results indicate that besides the biotic factors, the cellular H2O2 induced by Sb(III) also catalyzes bacterial Sb(III) oxidation as an abiotic oxidant. The data reveal a novel mechanism that bacterial Sb(III) oxidation is associated with abiotic (cellular H2O2) and biotic (AnoA and AioAB) factors and Sb(III) oxidation process consumes cellular H2O2 which contributes to microbial detoxification of both Sb(III) and cellular H2O2. PMID:28252030

Abiotic and biotic factors responsible for antimonite oxidation in Agrobacterium tumefaciens GW4

NASA Astrophysics Data System (ADS)

Li, Jingxin; Yang, Birong; Shi, Manman; Yuan, Kai; Guo, Wei; Wang, Qian; Wang, Gejiao

2017-03-01

Antimonite [Sb(III)]-oxidizing bacteria can transform the toxic Sb(III) into the less toxic antimonate [Sb(V)]. Recently, the cytoplasmic Sb(III)-oxidase AnoA and the periplasmic arsenite [As(III)] oxidase AioAB were shown to responsible for bacterial Sb(III) oxidation, however, disruption of each gene only partially decreased Sb(III) oxidation efficiency. This study showed that in Agrobacterium tumefaciens GW4, Sb(III) induced cellular H2O2 content and H2O2 degradation gene katA. Gene knock-out/complementation of katA, anoA, aioA and anoA/aioA and Sb(III) oxidation and growth experiments showed that katA, anoA and aioA were essential for Sb(III) oxidation and resistance and katA was also essential for H2O2 resistance. Furthermore, linear correlations were observed between cellular H2O2 and Sb(V) content in vivo and chemical H2O2 and Sb(V) content in vitro (R2 = 0.93 and 0.94, respectively). These results indicate that besides the biotic factors, the cellular H2O2 induced by Sb(III) also catalyzes bacterial Sb(III) oxidation as an abiotic oxidant. The data reveal a novel mechanism that bacterial Sb(III) oxidation is associated with abiotic (cellular H2O2) and biotic (AnoA and AioAB) factors and Sb(III) oxidation process consumes cellular H2O2 which contributes to microbial detoxification of both Sb(III) and cellular H2O2.

Computer vision in cell biology.

PubMed

Danuser, Gaudenz

2011-11-23

Computer vision refers to the theory and implementation of artificial systems that extract information from images to understand their content. Although computers are widely used by cell biologists for visualization and measurement, interpretation of image content, i.e., the selection of events worth observing and the definition of what they mean in terms of cellular mechanisms, is mostly left to human intuition. This Essay attempts to outline roles computer vision may play and should play in image-based studies of cellular life. Copyright © 2011 Elsevier Inc. All rights reserved.

RF Microalgal lipid content characterization

PubMed Central

Ahmad, Mahmoud Al; Al-Zuhair, Sulaiman; Taher, Hanifa; Hilal-Alnaqbi, Ali

2014-01-01

Most conventional techniques for the determination of microalgae lipid content are time consuming and in most cases are indirect and require excessive sample preparations. This work presents a new technique that utilizes radio frequency (RF) for rapid lipid quantification, without the need for sample preparation. Tests showed that a shift in the resonance frequency of a RF open-ended coaxial resonator and a gradual increase in its resonance magnitude may occur as the lipids content of microalgae cells increases. These response parameters can be then calibrated against actual cellular lipid contents and used for rapid determination of the cellular lipids. The average duration of lipid quantification using the proposed technique was of about 1 minute, which is significantly less than all other conventional techniques, and was achieved without the need for any time consuming treatment steps. PMID:24870372

Advances in Predictive Toxicology for Discovery Safety through High Content Screening.

PubMed

Persson, Mikael; Hornberg, Jorrit J

2016-12-19

High content screening enables parallel acquisition of multiple molecular and cellular readouts. In particular the predictive toxicology field has progressed from the advances in high content screening, as more refined end points that report on cellular health can be studied in combination, at the single cell level, and in relatively high throughput. Here, we discuss how high content screening has become an essential tool for Discovery Safety, the discipline that integrates safety and toxicology in the drug discovery process to identify and mitigate safety concerns with the aim to design drug candidates with a superior safety profile. In addition to customized mechanistic assays to evaluate target safety, routine screening assays can be applied to identify risk factors for frequently occurring organ toxicities. We discuss the current state of high content screening assays for hepatotoxicity, cardiotoxicity, neurotoxicity, nephrotoxicity, and genotoxicity, including recent developments and current advances.

Modelling of growth of aflatoxigenic A. flavus isolates from red chilli powder as a function of water availability.

PubMed

Marín, Sonia; Colom, Carmen; Sanchis, Vicente; Ramos, Antonio J

2009-01-15

The aim of the present work was to apply mathematical models for the prediction of growth of aflatoxigenic moulds in powdered Capsicum fruits as a function of its water availability. As prevention of fungal growth effectively conduces to prevention of mycotoxin accumulation, the development of models for prediction of growth of mycotoxigenic fungi becomes a key step in risk management. Two aflatoxigenic A. flavus from chilli powder were grown on 3% chilli powder extract agar at different water activity levels and their growth was evaluated over time in terms of colony sizes and ergosterol accumulation. Both variables were modelled over time, and the resulting parameters (growth rates and lag phases) were modelled as a function of water availability using the Rosso cardinal model. Linear logistic regression was also applied to predict the probability of growth over storage time. Both isolates showed a similar pattern of behaviour, with decreasing growth rates and increasing lag phases with decreasing water activity level. While estimation of optimum a(w) for growth was consistently around 0.97-0.99, the minimum estimated a(w) varied from 0.82 to 0.88 depending on the isolate and on the parameters used for predictions. Comparing growth rates obtained for colony size and ergosterol accumulation, a linear relationship between them could be observed. The rate of root square ergosterol/colony diameter/unit of time was 0.25-0.27. Probabilities of growth before 10 days over 90% were estimated at a(w) 0.91, while the safe period could be extended to more than 20 days (22-29 days) if water activity was decreased to an a(w)=0.87. Finally, the probability of growth is always under 50% when water availability is under a(w) 0.85, and almost null for A. flavus UdLTA 3.147. It was concluded that for safe production, storage and transport, chillies and chilli powder must be kept under 31% mc (db) (probability of growth <50%). However, growth is unlikely to occur if chilli is kept at approx. 34% for less than 10 days, or at approx. 33% for less than 20 days. Careful hazard analysis and critical control point (HACCP) techniques during raw material production and the subsequent stages of drying, transportation, elaboration and storage are indispensable.

Endotoxin, ergosterol, muramic acid and fungal DNA in dust from schools in Johor Bahru, Malaysia--Associations with rhinitis and sick building syndrome (SBS) in junior high school students.

PubMed

Norbäck, Dan; Hashim, Jamal Hisham; Markowicz, Pawel; Cai, Gui-Hong; Hashim, Zailina; Ali, Faridah; Larsson, Lennart

2016-03-01

This paper studied associations between ocular symptoms, rhinitis, throat and dermal symptoms, headache and fatigue in students by ethnicity and in relation to exposure to chemical microbial markers and fungal DNA in vacuumed dust in schools in Malaysia. A total of 462 students from 8 randomly selected secondary schools in Johor Bahru, Malaysia, participated (96% response rate). Dust was vacuumed from 32 classrooms and analysed for levels of five types of endotoxin as 3-hydroxy fatty acids (C10, C12, C14, C16 and C18 3-OH), muramic acid, ergosterol and five sequences of fungal DNA. Multiple logistic regression was applied. Totally 11.9% reported weekly ocular symptoms, 18.8% rhinitis, 15.6% throat and 11.1% dermal symptoms, 20.6% headache and 22.1% tiredness. Totally 21.1% reported pollen or furry pet allergy (atopy) and 22.0% parental asthma or allergy. Chinese students had less headache than Malay and Indian had less rhinitis and less tiredness than Malay. Parental asthma/allergy was a risk factor for ocular (odds ratio=3.79) and rhinitis symptoms (OR=3.48). Atopy was a risk factor for throat symptoms (OR=2.66), headache (OR=2.13) and tiredness (OR=2.02). There were positive associations between amount of fine dust in the dust samples and ocular symptoms (p<0.001) and rhinitis (p=0.006). There were positive associations between C14 3-OH and rhinitis (p<0.001) and between C18 3-OH and dermal symptoms (p=0.007). There were negative (protective) associations between levels of total endotoxin (LPS) (p=0.004) and levels of ergosterol (p=0.03) and rhinitis and between C12 3-OH and throat symptoms (p=0.004). In conclusion, the amount of fine dust in the classroom was associated with rhinitis and other SBS symptoms and improved cleaning of the schools is important. Endotoxin in the school dust seems to be mainly protective for rhinitis and throat symptoms but different types of endotoxin could have different effects. The ethnic differences in symptoms among the students deserve further attention. Copyright © 2015 Elsevier B.V. All rights reserved.

Extracts from Lentinula edodes (Shiitake) Edible Mushrooms Enriched with Vitamin D Exert an Anti-Inflammatory Hepatoprotective Effect.

PubMed

Drori, Ariel; Shabat, Yehudit; Ben Ya'acov, Ami; Danay, Ofer; Levanon, Dan; Zolotarov, Lidya; Ilan, Yaron

2016-04-01

Vitamin D has been known for its anti-inflammatory properties. Extracts derived from Lentinula edodes (Shiitake) edible mushroom exert an anti-inflammatory effect. These extracts contain high levels of ergosterol, which converts into ergocalciferol (vitamin D2) following exposure to ultraviolet light, followed by absorption and hydroxylation into the active form 25-hydroxyvitamin D [25(OH)D]. To determine the anti-inflammatory effect of overexpression of vitamin D in edible mushrooms, L. edodes mushrooms were exposed to ultraviolet-B light, freeze-dried, followed by measurement of vitamin D2 contents, in their dry weight. C57B1/6 mice were orally treated with vitamin D2-enriched or nonenriched mushroom extract prior and during concanavalin A-immune-mediated liver injury. Exposure to ultraviolet light increased vitamin D2 content in Shiitake edible mushrooms. Following feeding of vitamin D-enriched mushroom extracts to mice with immune-mediated hepatitis, a significant decrease in liver damage was noted. This was shown by a decrease in alanine aminotransferase and aspartate aminotransferase serum levels, a decrease in proportion of mice with severe liver injury, and by improvement in liver histology. These effects were associated with a decrease in serum interferon gamma levels. A synergistic effect was noted between the anti-inflammatory effect of the mushroom extracts and that of vitamin D. Oral administration of vitamin D-enriched L. edodes edible mushroom exerts a synergistic anti-inflammatory effect in the immune-mediated hepatitis. The data support its potential use as safe immunomodulatory adjuvant for the treatment of HCV and nonalcoholic steatohepatitis.

Chemically characterized Mentha cardiaca L. essential oil as plant based preservative in view of efficacy against biodeteriorating fungi of dry fruits, aflatoxin secretion, lipid peroxidation and safety profile assessment.

PubMed

Dwivedy, Abhishek Kumar; Prakash, Bhanu; Chanotiya, Chandan Singh; Bisht, Deepa; Dubey, Nawal Kishore

2017-08-01

The study reports Mentha cardiaca essential oil (EO) as plant based preservative against fungal and aflatoxin contamination of stored dry fruits. Mycoflora analysis of the dry fruits revealed Aspergillus favus LHP-PV-1 as the most aflatoxigenic isolate with highest Aflatoxin B 1 content. M. cardiaca EO showed broad fungitoxic spectrum inhibiting the tested moulds contaminating dry fruits. It's minimum inhibitory concentration (MIC), minimum aflatoxin inhibitory concentration (MAIC) and minimum fungicidal concentration (MFC) against A. favus LHP-PV-1 were recorded to be 1.25, 1.0 and 2.25 µL/mL respectively. The EO caused decrease in ergosterol content and enhanced leakage of Ca 2+ , K + and Mg 2+ ions from treated fungal cells, depicting fungal plasma membrane as the site of antifungal action. The EO showed promising DPPH free radical scavenging activity (IC 50 value:15.89 µL/mL) and favourable safety profile with LD 50 value (7133.70 mg/kg body wt.) when estimated through acute oral toxicity on mice. Carvone (61.62%) was recorded as the major component of the oil during chemical characterisation through GC-MS. Based on strong antifungal, antiaflatoxigenic and antioxidant potential, the chemically characterised M. cardiaca EO may be recommended as safe plant based preservative and shelf life enhancer of food items. This is the first report on antifungal and antiaflatoxigenic activity of M. cardiaca EO. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of Cellular DNA Content by Flow Cytometry.

PubMed

Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-10-02

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Analysis of Cellular DNA Content by Flow Cytometry.

PubMed

Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-11-01

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Contribution of high-content imaging technologies to the development of anti-infective drugs.

PubMed

Ang, Michelle Lay Teng; Pethe, Kevin

2016-08-01

Originally developed to study fundamental aspects of cellular biology, high-content imaging (HCI) was rapidly adapted to study host-pathogen interactions at the cellular level and adopted as a technology of choice to unravel disease biology. HCI platforms allow for the visualization and quantification of discrete phenotypes that cannot be captured using classical screening approaches. A key advantage of high-content screening technologies lies in the possibility to develop and interrogate physiologically significant, predictive ex vivo disease models that reproduce complex conditions relevant for infection. Here we review and discuss recent advances in HCI technologies and chemical biology approaches that are contributing to an increased understanding of the intricate host-pathogen interrelationship on the cellular level, and which will foster the development of novel therapeutic approaches for the treatment of human bacterial and protozoan infections. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

Internal and Surface-Localized Major Surface Proteases of Leishmania spp. and Their Differential Release from Promastigotes▿

PubMed Central

Yao, Chaoqun; Donelson, John E.; Wilson, Mary E.

2007-01-01

Major surface protease (MSP), also called GP63, is a virulence factor of Leishmania spp. protozoa. There are three pools of MSP, located either internally within the parasite, anchored to the surface membrane, or released into the extracellular environment. The regulation and biological functions of these MSP pools are unknown. We investigated here the trafficking and extrusion of surface versus internal MSPs. Virulent Leishmania chagasi undergo a growth-associated lengthening in the t1/2 of surface-localized MSP, but this did not occur in the attenuated L5 strain. The release of surface-localized MSP was enhanced in a dose-dependent manner by MβCD, which chelates membrane cholesterol-ergosterol. Furthermore, incubation of promastigotes at 37°C with Matrigel matrix, a soluble basement membrane extract of Engelbreth-Holm-Swarm tumor cells, stimulated the release of internal MSP but not of surface-located MSP. Taken together, these data indicate that MSP subpopulations in distinct cellular locations are released from the parasite under different environmental conditions. We hypothesize that the internal MSP with its lengthy t1/2 does not serve as a pool for promastigote surface MSP in the sand fly vector but that it instead functions as an MSP pool ready for quick release upon inoculation of metacyclic promastigotes into mammals. We present a model in which these different MSP pools are released under distinct life cycle-specific conditions. PMID:17693594

Ganoderin A, a novel 9,11-secosterol from Ganoderma lucidum spores oil.

PubMed

Ge, Fa-Huan; Duan, Ming-Hui; Li, Jing; Shi, Qing-Long

2017-12-01

In this study, four sterols were isolated from the Ganoderma lucidum spores oil obtained via supercritical CO 2 extraction. Four chemical constituents were ganoderin A (1), chaxine B (2), ergosterol, (3) and stellasterol (4). All the separated ingredients were characterized using spectral data interpretation and by comparing with reported data. Noticeably, stellasterol and chaxine B were both firstly isolated from Ganoderma lucidum spores oil and ganoderin A was shown to bear an unprecedented skeleton.

Anti-Candida activity of geraniol involves disruption of cell membrane integrity and function.

PubMed

Sharma, Y; Khan, L A; Manzoor, N

2016-09-01

Candidiasis is a major problem in immunocompromised patients. Candida, an opportunistic fungal pathogen, is a major health concern today as conventional drugs are highly toxic with undesirable side effects. Their fungistatic nature is responsible for drug resistance in continuously evolving strains. Geraniol, an acyclic monoterpene alcohol, is a component of several plant essential oils. In the present study, an attempt has been made to understand the antifungal activity of geraniol at the cell membrane level in three Candida species. With an MIC of 30-130μg/mL, this natural compound was fungicidal at concentrations 2×MIC. There was complete suppression of fungal growth at MIC values (growth curves) and encouragingly geraniol is non-toxic even at the concentrations approaching 5×MIC (hemolysis assay). Exposed cells showed altered morphology, wherein the cells appeared either broken or shrivelled up (SEM studies). Significant reduction was seen in ergosterol levels at sub-MIC and glucose-induced H(+) efflux at concentrations>MIC values. Our results suggest that geraniol disrupts cell membrane integrity by interfering with ergosterol biosynthesis and inhibiting the very crucial PM-ATPase. It may hence be used in the management and treatment of both superficial and invasive candidiasis but further studies are required to elaborate its mode of action. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Quantitative determination of steroids in the fruiting bodies and submerged-cultured mycelia of Inonotus obliquus.

PubMed

Gao, Yuan; Xu, Hongyu; Lu, Zhenming; Xu, Zhenghong

2009-11-01

This study describes the method of quantitative determination of betulin, ergosterol, cholesterol, lanosterol, stigmasterol and sitosterol in the fruiting bodies and submerged-cultured mycelia of Inonotus obliquus. A high performance liquid chromatographic (HPLC) method was applied to separate these steroids. The procedure was carried out on a reversed-phase C, column, using a stepwise gradient of water-methanol as mobile phase with the following profile: 0-10 min, 10% water, 90% methanol; 10-40 min, 3% water, 97% methanol. The flow rate was 1.4 mL/min and the detection wavelength was 202 nm. The analysis was completed within 40 min. The results showed that this method has good reproducibility and satisfactory recoveries for the determination of steroids. The relative standard deviations of the peak areas were less than 2.94% (n = 5) for intraday assays. A good linear correlation was obtained in a range of 0.4-4.8 microg. The recoveries of betulin, ergosterol, cholesterol, lanosterol, stigmasterol, and sitosterol were 100.05%-100.72%, 99.31%-101.04%, 97.52%-101.63%, 96.61%-100.08%, 96.21%-100.76% and 100.04%-100.51%, respectively. This method can be applied to evaluate real samples, and it is rapid, accurate and suitable for the quantitative determination of steroids in the fruiting bodies and submerged-cultured mycelia of Inonotus obliquus.

Synergistic mortality between a neonicotinoid insecticide and an ergosterol-biosynthesis-inhibiting fungicide in three bee species.

PubMed

Sgolastra, Fabio; Medrzycki, Piotr; Bortolotti, Laura; Renzi, Maria Teresa; Tosi, Simone; Bogo, Gherardo; Teper, Dariusz; Porrini, Claudio; Molowny-Horas, Roberto; Bosch, Jordi

2017-06-01

Neonicotinoid insecticides have been identified as an important factor contributing to bee diversity declines. Nonetheless, uncertainties remain about their impact under field conditions. Most studies have been conducted on Apis mellifera and tested single compounds. However, in agricultural environments, bees are often exposed to multiple pesticides. We explore the synergistic mortality between a neonicotinoid (clothianidin) and an ergosterol-biosynthesis-inhibiting fungicide (propiconazole) in three bee species (A. mellifera, Bombus terrestris, Osmia bicornis) following oral exposure in the laboratory. We developed a new approach based on the binomial proportion test to analyse synergistic interactions. We estimated uptake of clothianidin per foraging bout in honey bees foraging on seed-coated rapeseed fields. We found significant synergistic mortality in all three bee species exposed to non-lethal doses of propiconazole and their respective LD 10 of clothianidin. Significant synergism was only found at the first assessment times in A. mellifera (4 and 24 h) and B. terrestris (4 h), but persisted throughout the experiment (96 h) in O. bicornis. O. bicornis was also the most sensitive species to clothianidin. Our results underscore the importance to test pesticide combinations likely to occur in agricultural environments, and to include several bee species in environmental risk assessment schemes. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Synergism Effect of the Essential Oil from Ocimum basilicum var. Maria Bonita and Its Major Components with Fluconazole and Its Influence on Ergosterol Biosynthesis

PubMed Central

Cardoso, Nathalia N. R.; Alviano, Celuta S.; Blank, Arie F.; Romanos, Maria Teresa V.; Fonseca, Beatriz B.; Rozental, Sonia; Rodrigues, Igor A.; Alviano, Daniela S.

2016-01-01

The aim of this study was to evaluate the activity of the EO and its major components of Ocimum basilicum var. Maria Bonita, a genetically improved cultivar, against the fluconazole sensitive and resistant strains of Candida albicans and Cryptococcus neoformans. Geraniol presented better results than the EO, with a low MIC (76 μg/mL against C. neoformans and 152 μg/mL against both Candida strains). The combination of EO, linalool, or geraniol with fluconazole enhanced their antifungal activity, especially against the resistant strain (MIC reduced to 156, 197, and 38 μg/mL, resp.). The ergosterol assay showed that subinhibitory concentrations of the substances were able to reduce the amount of sterol extracted. The substances tested were able to reduce the capsule size which suggests they have an important mechanism of action. Transmission electron microscopy demonstrated cell wall destruction of C. neoformans after treatment with subinhibitory concentrations. In C. albicans ultrastructure alterations such as irregularities in the membrane, presence of vesicles, and cell wall thickening were observed. The biofilm formation was inhibited in both C. albicans strains at MIC and twice MIC. These results provide further support for the use of O. basilicum EO and its major components as a potential source of antifungal agents. PMID:27274752

Molds and mycotoxins in indoor environments--a survey in water-damaged buildings.

PubMed

Bloom, Erica; Nyman, Eva; Must, Aime; Pehrson, Christina; Larsson, Lennart

2009-11-01

Mycotoxins are toxic, secondary metabolites frequently produced by molds in water-damaged indoor environments. We studied the prevalence of selected, potent mycotoxins and levels of fungal biomass in samples collected from water-damaged indoor environments in Sweden during a 1-year period. One hundred samples of building materials, 18 samples of settled dust, and 37 samples of cultured dust were analyzed for: (a) mycoflora by microscopy and culture; (b) fungal chemical marker ergosterol and hydrolysis products of macrocyclic trichothecenes and trichodermin (verrucarol and trichodermol) by gas chromatography-tandem mass spectrometry; and (c) sterigmatocystin, gliotoxin, aflatoxin B(1), and satratoxin G and H by high performance liquid chromatography-tandem mass spectrometry. Sixty-six percent of the analyzed building materials samples, 11% of the settled dust samples, and 51% of the cultured dust samples were positive for at least one of the studied mycotoxins. In addition, except in the case of gliotoxin, mycotoxin-positive building material samples contained 2-6 times more ergosterol than mycotoxin-negative samples. We show that (a) molds growing on a range of different materials indoors in water-damaged buildings generally produce mycotoxins, and (b) mycotoxin-containing particles in mold-contaminated environments may settle on surfaces above floor level. The mass spectrometry methods used in this study are valuable tools in further research to survey mycotoxin exposure and investigate potential links with health effects.

Control of Basal Stem Rot Disease in Oil Palm by Supplementation of Calcium, Copper, and Salicylic Acid.

PubMed

Bivi, M Shahul Hamid Rahamah; Paiko, Adamu Saidu; Khairulmazmi, Ahmad; Akhtar, M S; Idris, Abu Seman

2016-10-01

Continuous supplementation of mineral nutrients and salicylic acid (SA) as foliar application could improve efficacy in controlling basal stem rot (BSR) disease in oil palm seedling. It is revealed from the results that the highest disease severity index (58.3%) was recorded in T8 treatments at 9 months after inoculation. The best disease control was achieved by T7 treatments (calcium/copper/SA [Ca/Cu/SA]) (5.0%) followed by T1 (5.5%), T5 (5.8%), T3 (8.3%), T6 (8.3%), T4 (13.3%), and T2 (15.8%) treatments. Continuous supplementation of Ca/Cu/SA was found to be the most effective in controlling the disease and the high performance liquid chromatography results showed the detection of ergosterol at very low concentration in the treated samples. Moreover, the transmission electron microscopy analysis results clearly indicated that T7 treatment was also enhancing lignification, which was responsible for the thickness of the secondary cell walls and middle lamella compared to untreated samples. It was therefore, concluded that continuous supplementation of minerals nutrients and SA could effectively suppress disease severity by reducing ergosterol activity and also improve the process of lignification in the treated plants. Furthermore, this treatment also managed to delay the onset of BSR symptoms and promote the growth of the seedlings and eventually suppress the BSR disease.

Control of Basal Stem Rot Disease in Oil Palm by Supplementation of Calcium, Copper, and Salicylic Acid

PubMed Central

Bivi, M. Shahul Hamid Rahamah; Paiko, Adamu Saidu; Khairulmazmi, Ahmad; Akhtar, M. S.; Idris, Abu Seman

2016-01-01

Continuous supplementation of mineral nutrients and salicylic acid (SA) as foliar application could improve efficacy in controlling basal stem rot (BSR) disease in oil palm seedling. It is revealed from the results that the highest disease severity index (58.3%) was recorded in T8 treatments at 9 months after inoculation. The best disease control was achieved by T7 treatments (calcium/copper/SA [Ca/Cu/SA]) (5.0%) followed by T1 (5.5%), T5 (5.8%), T3 (8.3%), T6 (8.3%), T4 (13.3%), and T2 (15.8%) treatments. Continuous supplementation of Ca/Cu/SA was found to be the most effective in controlling the disease and the high performance liquid chromatography results showed the detection of ergosterol at very low concentration in the treated samples. Moreover, the transmission electron microscopy analysis results clearly indicated that T7 treatment was also enhancing lignification, which was responsible for the thickness of the secondary cell walls and middle lamella compared to untreated samples. It was therefore, concluded that continuous supplementation of minerals nutrients and SA could effectively suppress disease severity by reducing ergosterol activity and also improve the process of lignification in the treated plants. Furthermore, this treatment also managed to delay the onset of BSR symptoms and promote the growth of the seedlings and eventually suppress the BSR disease. PMID:27721689

Degradation of naproxen and carbamazepine in spiked sludge by slurry and solid-phase Trametes versicolor systems.

PubMed

Rodríguez-Rodríguez, Carlos E; Marco-Urrea, Ernest; Caminal, Gloria

2010-04-01

Growth and activity of the white-rot fungus Trametes versicolor on sewage sludge were assessed in bioslurry and solid-phase systems. Bioslurry cultures with different loads of sludge (10%, 25% and 38%, w/v) were performed. A lag phase of at least 2 d appeared in the 25 and 38%-cultures, however, the total fungal biomass was higher for the latter and lower for the 10%-culture after 30 d, as revealed by ergosterol determination. Detectable laccase activity levels were found in the 10 and 25%-cultures (up to 1308 and 2588 AUL(-1), respectively) while it was negligible in the 38%-culture. Important levels of ergosterol and laccase were obtained over a 60 d period in sludge solid-phase cultures amended with different concentrations of wheat straw pellets as lignocellulosic bulking material. Degradation experiments in 25%-bioslurry cultures spiked with naproxene (NAP, analgesic) and carbamazepine (CBZ, antiepileptic) showed depletion of around 47% and 57% within 24h, respectively. Complete depletion of NAP and around 48% for CBZ were achieved within 72 h in sludge solid cultures with 38% bulking material. CBZ degradation is especially remarkable due to its high persistence in wastewater treatment plants. Results showed that T. versicolor may be an interesting bioremediation agent for elimination of emerging pollutants in sewage sludge. Copyright 2009 Elsevier Ltd. All rights reserved.

Synergistic interactions between a variety of insecticides and an ergosterol biosynthesis inhibitor fungicide in dietary exposures of bumble bees (Bombus terrestris L.).

PubMed

Raimets, Risto; Karise, Reet; Mänd, Marika; Kaart, Tanel; Ponting, Sally; Song, Jimao; Cresswell, James E

2018-03-01

In recent years, concern has been raised over honey bee colony losses, and also among wild bees there is evidence for extinctions and range contractions in Europe and North America. Pesticides have been proposed as a potential cause of this decline. Bees are exposed simultaneously to a variety of agrochemicals, which may cause synergistically detrimental impacts, which are incompletely understood. We investigated the toxicity of the fungicide imazalil in mixture with four common insecticides: fipronil (phenylpyrazoid), cypermethrin (pyrethroid), thiamethoxam, and imidacloprid (neonicotinoids). Ergosterol biosynthesis inhibitor (EBI) fungicides like imazalil can inhibit P450 detoxification systems in insects and therefore fungicide - insecticide co-occurrence might produce synergistic toxicity in bees. We assessed the impact of dietary fungicide - insecticide mixtures on the mortality and feeding rates of laboratory bumble bees (Bombus terrestris L.). Regarding mortality, imazalil synergised the toxicity of fipronil, cypermethrin and thiamethoxam, but not imidacloprid. We found no synergistic effects on feeding rates. Our findings suggest that P450-based detoxification processes are differentially important in mitigating the toxicity of certain insecticides, even those of the same chemical class. Our evidence that cocktail effects can arise in bumble bees should extend concern about the potential impacts of agrochemical mixtures to include wild bee species in farmland. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Phytosterols from Dunaliella tertiolecta Reduce Cell Proliferation in Sheep Fed Flaxseed during Post Partum

PubMed Central

Ciliberti, Maria Giovanna; Francavilla, Matteo; Intini, Simona; Albenzio, Marzia; Marino, Rosaria; Santillo, Antonella; Caroprese, Mariangela

2017-01-01

The post partum period is characterized by immunosuppression and increased disease susceptibility. Both phytosterols from microalga Dunaniella tertiolecta and dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) influence cell proliferation and cytokine release during inflammation. The objective of this paper was the evaluation of the effects of physterols, extracted and purified from D. tertiolecta, on the in vitro immune responses of ewes supplemented with flaxseed during post partum. Twenty Comisana parturient ewes were divided in two balanced groups, and supplemented with flaxseed (FS, 250 g/day) or fed with a conventional diet (CON). Blood samples (15 mL) were collected for five weeks, starting from lambing, in order to isolate peripheral blood mononuclear cells (PBMC). Stimulated PBMC were treated with a total sterols fraction from D. tertiolecta (TS), a mix of ergosterol and 7-dehydroporiferasterol (purified extract, PE), and a mix of acetylated ergosterol and 7-dehydroporiferasterol (acetylated purified extract, AcPE), extracted and purified from D. tertiolecta at two concentrations (0.4 and 0.8 mg/mL). Results of the experiment demonstrated that n-3 PUFA from flaxseed induced an anti-inflammatory cytokine profile, with an increase of both IL-10, IL-6 and a decrease of IL-1β. TS, PE, and AcPE purified from D. tertiolecta showed an anti-proliferative effect on sheep PBMC regardless their chemical composition and concentration. PMID:28684702

Identification and Characterization of Major Lipid Particle Proteins of the Yeast Saccharomyces cerevisiae

PubMed Central

Athenstaedt, Karin; Zweytick, Dagmar; Jandrositz, Anita; Kohlwein, Sepp Dieter; Daum, Günther

1999-01-01

Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism. PMID:10515935

Trophic effect of bombesin on the rat pancreas: is it mediated by the release of gastrin or cholecystokinin?

PubMed

Lhoste, E; Aprahamian, M; Pousse, A; Hoeltzel, A; Stock-Damge, C

1985-01-01

This work investigates the effect, on the rat pancreas, of a chronic administration of bombesin in function of the dose and duration of treatment and examines whether this effect may be mediated by the release of endogenous gastrin or cholecystokinin. Bombesin, administered three times daily for 5 or 15 days, induced a marked increase in pancreatic weight, its protein, RNA and enzyme contents with the dose of 10 micrograms/kg body weight; the ratios of pancreatic weight, protein and RNA contents to DNA contents increased significantly after a 5 day treatment, suggesting cellular hypertrophy. Pancreatic DNA content was markedly enhanced after a 15 day treatment, suggesting cellular hyperplasia. Antrectomy decreased plasma gastrin levels, but did not alter the pancreatico-trophic action of a 10 micrograms/kg bombesin treatment for 5 days. Proglumide, an inhibitor of cholecystokinin and gastrin in the pancreas, did not affect the growth of the pancreas induced by a 10 micrograms/kg bombesin treatment for 5 days. It is concluded that chronic bombesin induces, in the rat pancreas, cellular hypertrophy or hyperplasia depending on the duration of treatment. Pancreatic hypertrophy is not mediated by the release of endogenous gastrin or cholecystokinin.

Cellular metabolic rates from primary dermal fibroblast cells isolated from birds of different body masses.

PubMed

Jimenez, Ana Gabriela; Williams, Joseph B

2014-10-01

The rate of metabolism is the speed at which organisms use energy, an integration of energy transformations within the body; it governs biological processes that influence rates of growth and reproduction. Progress at understanding functional linkages between whole organism metabolic rate and underlying mechanisms that influence its magnitude has been slow despite the central role this issue plays in evolutionary and physiological ecology. Previous studies that have attempted to relate how cellular processes translate into whole-organism physiology have done so over a range of body masses of subjects. However, the data still remains controversial when observing metabolic rates at the cellular level. To bridge the gap between these ideas, we examined cellular metabolic rate of primary dermal fibroblasts isolated from 49 species of birds representing a 32,000-fold range in body masses to test the hypothesis that metabolic rate of cultured cells scales with body size. We used a Seahorse XF-96 Extracellular flux analyzer to measure cellular respiration in fibroblasts. Additionally, we measured fibroblast size and mitochondrial content. We found no significant correlation between cellular metabolic rate, cell size, or mitochondrial content and body mass. Additionally, there was a significant relationship between cellular basal metabolic rate and proton leak in these cells. We conclude that metabolic rate of cells isolated in culture does not scale with body mass, but cellular metabolic rate is correlated to growth rate in birds. Copyright © 2014 Elsevier Inc. All rights reserved.

High content image analysis for human H4 neuroglioma cells exposed to CuO nanoparticles.

PubMed

Li, Fuhai; Zhou, Xiaobo; Zhu, Jinmin; Ma, Jinwen; Huang, Xudong; Wong, Stephen T C

2007-10-09

High content screening (HCS)-based image analysis is becoming an important and widely used research tool. Capitalizing this technology, ample cellular information can be extracted from the high content cellular images. In this study, an automated, reliable and quantitative cellular image analysis system developed in house has been employed to quantify the toxic responses of human H4 neuroglioma cells exposed to metal oxide nanoparticles. This system has been proved to be an essential tool in our study. The cellular images of H4 neuroglioma cells exposed to different concentrations of CuO nanoparticles were sampled using IN Cell Analyzer 1000. A fully automated cellular image analysis system has been developed to perform the image analysis for cell viability. A multiple adaptive thresholding method was used to classify the pixels of the nuclei image into three classes: bright nuclei, dark nuclei, and background. During the development of our image analysis methodology, we have achieved the followings: (1) The Gaussian filtering with proper scale has been applied to the cellular images for generation of a local intensity maximum inside each nucleus; (2) a novel local intensity maxima detection method based on the gradient vector field has been established; and (3) a statistical model based splitting method was proposed to overcome the under segmentation problem. Computational results indicate that 95.9% nuclei can be detected and segmented correctly by the proposed image analysis system. The proposed automated image analysis system can effectively segment the images of human H4 neuroglioma cells exposed to CuO nanoparticles. The computational results confirmed our biological finding that human H4 neuroglioma cells had a dose-dependent toxic response to the insult of CuO nanoparticles.

Meatal Swabs Contain Less Cellular Material and Are Associated with a Decrease in Gram Stain Smear Quality Compared to Urethral Swabs in Men.

PubMed

Jordan, Stephen J; Schwebke, Jane R; Aaron, Kristal J; Van Der Pol, Barbara; Hook, Edward W

2017-07-01

Urethral swabs are the samples of choice for point-of-care Gram stain testing to diagnose Neisseria gonorrhoeae infection and nongonococcal urethritis (NGU) in men. As an alternative to urethral swabs, meatal swabs have been recommended for the collection of urethral discharge to diagnose N. gonorrhoeae and Chlamydia trachomatis infection in certain populations by nucleic acid amplification testing (NAAT), as they involve a less invasive collection method. However, as meatal swabs could be sampling a reduced surface area and result in fewer collected epithelial cells compared to urethral swabs, the adequacy of meatal swab specimens to collect sufficient cellular material for Gram stain testing remains unknown. We enrolled 66 men who underwent either urethral or meatal swabbing and compared the cellular content and Gram stain failure rate. We measured the difference in swab cellular content using the Cepheid Xpert CT/NG sample adequacy control crossing threshold (SAC CT ) and determined the failure rate of Gram stain smears (GSS) due to insufficient cellular material. In the absence of discharge, meatal smears were associated with a significant reduction in cellular content ( P = 0.0118), which corresponded with a GSS failure rate significantly higher than that for urethral swabs (45% versus 3%, respectively; P < 0.0001). When discharge was present, there was no difference among results from urethral and meatal swabs. Therefore, if GSS testing is being considered for point-of-care diagnosis of N. gonorrhoeae infection or NGU in men, meatal swabs should be avoided in the absence of a visible discharge. Copyright © 2017 American Society for Microbiology.

Evaluation of the Efficiency of the Reticulocyte Hemoglobin Content on Diagnosis for Iron Deficiency Anemia in Chinese Adults.

PubMed

Cai, Jie; Wu, Meng; Ren, Jie; Du, Yali; Long, Zhangbiao; Li, Guoxun; Han, Bing; Yang, Lichen

2017-05-02

Our aim was to evaluate the cut-off value and efficiency of using reticulocyte hemoglobin content as a marker to diagnose iron deficiency anemia in Chinese adults. 140 adults who needed bone marrow aspiration for diagnosis at the hematology department of the Peking Union Medical College Hospital were enrolled according to the inclusive and exclusive criteria. Venous blood samples were collected to detect complete blood count, including hemoglobin, reticulocyte hemoglobin content, hematocrit, mean cellular volume, corpuscular hemoglobin concentration, hemoglobin content, free erythrocyte protoporphyrin; iron indexes of serum ferritin, serum transferrin receptor, and unsaturated iron-binding capacity; and inflammation markers of C-reactive protein and α-acid glycoprotein. Bone marrow samples were obtained for the bone marrow iron staining, which was used as the standard for the evaluation of iron status in this study. Subjects were divided into three groups according to hemoglobin levels and bone marrow iron staining results: the IDA (iron deficiency anemia) group, the NIDA (non-iron deficiency anemia) group, and the control group. The differences of the above-mentioned indexes were compared among the three groups and the effect of inflammation was also considered. The cut-off value of reticulocyte hemoglobin content was determined by receiver operation curves. The IDA group ( n = 56) had significantly lower reticulocyte hemoglobin content, mean cellular volume, corpuscular hemoglobin concentration, hemoglobin content, and serum ferritin; and higher free erythrocyte protoporphyrin, unsaturated iron-binding capacity, and serum transferrin receptor ( p < 0.05) compared with the NIDA group ( n = 38) and control group ( n = 46). Hematocrit, serum ferritin, and unsaturated iron-binding capacity were significantly affected by inflammation while reticulocyte hemoglobin content and other parameters were not. The cut-off value of reticulocyte hemoglobin content for diagnosing iron deficiency anemia was 27.2 pg, with a sensitivity of 87.5% and a specificity of 92.9%. The cut-off values for mean cellular volume, serum ferritin, and serum transferrin receptor were 76.6, 12.9, and 4.89 mg/L, respectively. Reticulocyte hemoglobin content had the largest area under the curve of 0.929, while those for mean cellular volume, serum ferritin, serum transferrin receptor were 0.922, 0.887, and 0.900, respectively. Reticulocyte hemoglobin content has a high sensitivity and specificity in the diagnosis of iron deficiency anemia, and its comprehensive diagnostic efficacy is better than other traditional indicators-such as serum ferritin and serum transferrin receptor.

Efinaconazole in the treatment of onychomycosis

PubMed Central

Lipner, Shari R; Scher, Richard K

2015-01-01

Efinaconazole 10% topical solution is a new triazole recently approved for the treatment of onychomycosis. It inhibits fungal lanosterol 14α-demethylase in the ergosterol biosynthesis pathway, has potent antifungal activity against dermatophytes, as well as activity against Candida spp. and non-dermatophyte molds, and showed promising results in clinical trials. This review summarizes the mechanism of action, in vitro and in vivo data, clinical trials, safety, and quality-of-life data of efinaconazole as it applies to the treatment of onychomycosis. PMID:26082652

Distribution of sterols in the fungi. I - Fungal spores

NASA Technical Reports Server (NTRS)

Weete, J. D.; Laseter, J. L.

1974-01-01

Mass spectrometry was used to examine freely extractable sterols from spores of several species of fungi. Ergosterol was the most common sterol produced by any individual species, but it was completely absent from two species belonging to apparently distantly related groups of fungi: the aquatic Phycomycetes and the rust fungi. This fact could have taxonomic or phylogenetic implications. The use of glass capillary columns in the resolution of the sterols is shown to eliminate some of the difficulty inherent in this process.

Salt-induced changes in lipid composition and membrane fluidity of halophilic yeast-like melanized fungi.

PubMed

Turk, Martina; Méjanelle, Laurence; Sentjurc, Marjeta; Grimalt, Joan O; Gunde-Cimerman, Nina; Plemenitas, Ana

2004-02-01

The halophilic melanized yeast-like fungi Hortaea werneckii, Phaeotheca triangularis, and the halotolerant Aureobasidium pullulans, isolated from salterns as their natural environment, were grown at different NaCl concentrations and their membrane lipid composition and fluidity were examined. Among sterols, besides ergosterol, which was the predominant one, 23 additional sterols were identified. Their total content did not change consistently or significantly in response to raised NaCl concentrations in studied melanized fungi. The major phospholipid classes were phosphatidylcholine and phosphatidylethanolamine, followed by anionic phospholipids. The most abundant fatty acids in phospholipids contained C16 and C18 chain lengths with a high percentage of C18:2Delta9,12. Salt stress caused an increase in the fatty acid unsaturation in the halophilic H. werneckii and halotolerant A. pullulans but a slight decrease in halophilic P. triangularis. All the halophilic fungi maintained their sterol-to-phospholipid ratio at a significantly lower level than did the salt-sensitive Saccharomyces cerevisiae and halotolerant A. pullulans. Electron paramagnetic resonance (EPR) spectroscopy measurements showed that the membranes of all halophilic fungi were more fluid than those of the halotolerant A. pullulans and salt-sensitive S. cerevisiae, which is in good agreement with the lipid composition observed in this study.

Vitamin D2 Stability During the Refrigerated Storage of Ultraviolet B-Treated Cultivated Culinary-Medicinal Mushrooms.

PubMed

Slawinska, Aneta; Fornal, Emilia; Radzki, Wojciech; Jablonska-Rys, Ewa; Parfieniuk, Ewa

2017-01-01

The effects of ultraviolet B (UVB) irradiation on the synthesis of vitamin D2 and its stability during refrigerated storage was determined in fresh cultivated culinary-medicinal mushrooms (Agaricus bisporus, Pleurotus ostreatus, and Lentinus edodes) after harvest. The irradiated mushrooms were stored at 4°C for up to 10 days. The concentrations of vitamin D2 and ergosterol were determined using ultrahigh-performance liquid chromatography/tandem mass spectrometry. The cultivated mushrooms not treated with UVB were devoid of vitamin D2. After UVB irradiation, we obtained mushrooms with a large amount of ergocalciferol. A. bisporus showed the lowest vitamin D2 content (3.55 ± 0.11 μg D2/g dry weight); P. ostreatus contained 58.96 ± 1.15 μg D2/g dry weight, and L. edodes contained 29.46 ± 2.21 μg/g dry weight. During storage at 4°C, the amount of vitamin D2 was gradually decreased in P. ostreatus and L. edodes, whereas in A. bisporus vitamin D2 gradually increased until the sixth day, then decreased. Mushrooms exposed to UVB radiation contain a significant amount of vitamin D2 and are therefore an excellent food source of vitamin D.

Sterol limitation in a pollen-fed omnivorous lady beetle (Coleoptera: Coccinellidae).

PubMed

Pilorget, Lucia; Buckner, James; Lundgren, Jonathan G

2010-01-01

Nutritional constraints of non-prey foods for entomophagous arthropods are seldom investigated, yet are crucial to understanding their nutritional ecology and function within natural and managed environments. We investigated whether pollen from five maize hybrids was of variable quality for the lady beetle, Coleomegilla maculata, whether suitability of these pollens was related with their sterol profiles, and how augmenting sterols (beta-sitosterol, cholesterol, or ergosterol) affected the fitness and performance of C. maculata. Preimaginal survival, development rates, the duration of the pre-oviposition period, post-mortem adult dry weight, adult hind tibial length, sex ratio, fecundity, cohort generation time (T(c)), net replacement rate (R(0)) and intrinsic rate of increase (r) were measured. Individual sterols in the pollens were quantified using GC-MS. Pollens were of variable suitability for C. maculata; the development rate was positively correlated with the amount of 24-methylene-cholesterol and r was positively correlated with episterol and 24-methylene-lophenol found in the pollens. Performance of C. maculata was entirely unaffected by augmenting pollen meals with sterols. This research shows that pollens clearly vary in their sterol contents intraspecifically, which affects their suitability for omnivores that rely on pollen. However, sterols appear to be only one of the limiting nutrients in pollens.

Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications.

PubMed

Otero, José Manuel; Vongsangnak, Wanwipa; Asadollahi, Mohammad A; Olivares-Hernandes, Roberto; Maury, Jérôme; Farinelli, Laurent; Barlocher, Loïc; Osterås, Magne; Schalk, Michel; Clark, Anthony; Nielsen, Jens

2010-12-22

The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,596 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10) and ergosterol biosynthetic pathway (ERG8, ERG9). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function. With an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at http://www.sysbio.se/cenpk.

Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications

PubMed Central

2010-01-01

Background The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. Results In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,596 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10) and ergosterol biosynthetic pathway (ERG8, ERG9). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function. Conclusions With an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at http://www.sysbio.se/cenpk. PMID:21176163

Carvacrol and eugenol effectively inhibit Rhizopus stolonifer and control postharvest soft rot decay in peaches.

PubMed

Zhou, D; Wang, Z; Li, M; Xing, M; Xian, T; Tu, K

2018-01-01

This study aimed to investigate the antifungal mechanism of carvacrol and eugenol to inhibit Rhizopus stolonifer and the control of postharvest soft rot decay in peaches. To investigate the antifungal mechanism, the effects of carvacrol and eugenol on the mycelium growth, leakages of cytoplasmic contents, mycelium morphology, cell membrane and membrane composition of R. stolonifer were studied. Carvacrol and eugenol both exhibited dose-dependent antifungal activity against R. stolonifer, carvacrol at a concentration of 2 μl per plant and eugenol at a concentration of 4 μl per plant inhibited fungal growth completely. The two essential oils (EOs) increased cell membrane penetrability and caused the leakage of cytoplasm, nucleic acid and protein content. The observation using scanning electron microscopy and fluorescent microscopy showed modification of the hyphal morphology and breakage of the cell plasma membrane. Decreased ergosterol contents confirmed that the two EOs could destroy the membrane of R. stolonifer. For the in vivo test, the inhibition of soft rot disease and the induction of defence-related enzymes were investigated. Carvacrol and eugenol significantly reduced the incidence and severity of soft rot decay in inoculated peaches. The best treatments for controlling soft rot decay were obtained at 0·5 μl l -1 for carvacrol and 1 μl l -1 for eugenol. The activities of defence-related enzymes in peaches were also enhanced by fumigation with two EOs. This study showed that carvacrol and eugenol could effectively inhibit the growth of R. stolonifer in vitro and successfully control the incidence of soft rot decay in honey peaches. The above findings may be the main antifungal mechanism of carvacrol and eugenol on R. stolonifer. Furthermore, carvacrol and eugenol are helpful for their commercial application on the preservation of fresh fruit. © 2017 The Society for Applied Microbiology.

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

NASA Astrophysics Data System (ADS)

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-04-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

PubMed Central

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-01-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue. PMID:27063397

Biochemical and physiological responses of halophilic nanophytoplankton (Dunaliella salina) from exposure to xeno-estrogen 17α-ethinylestradiol.

PubMed

Belhaj, Dalel; Athmouni, Khaled; Frikha, Doniez; Kallel, Monem; El Feki, Abdelfattah; Maalej, Sami; Zhou, John L; Ayadi, Habib

2017-03-01

The environmental impacts of various pollutants on the entire levels of organisms are under investigation. Among these pollutants, endocrine-disrupting compounds (EDCs) present a serious hazard, even though the environmental significance of these compounds remains basically unknown. To drop some light on this field, we assessed the effects of a 11-day exposure of 17α-ethinylestradiol (EE2) on the growth, metabolic content, antioxidant response, oxidative stress, and genetic damage of Dunaliella salina, isolated from Tunisian biotopes. The results showed that at 10 ng L -1 , EE2 could stimulate the growth of D. salina and increase its cellular content of photosynthetic pigments and metabolites; however, it did not significantly increase the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) or the level of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ). In contrast, exposure to high levels of EE2 concentrations significantly inhibited the growth of D. salina (P < 0.05), decreased the cellular content of photosynthetic pigments, increased the cellular content of all of the metabolites and the SOD activity, and inhibited CAT and GPx activities. Nevertheless, the balance between oxidant and antioxidant enzymes was disrupted because H 2 O 2 content along with MDA content simultaneously increased. Contrary to expected results, DNA damage (strand breaks) decreased after the exposure of algae to EE2. The results of this study suggest that EE2 toxicity could result in environmental impacts with consequences on the whole aquatic community. Graphical abstract.

Antifungal Properties of Crude Extracts, Fractions, and Purified Compounds from Bark of Curatella americana L. (Dilleniaceae) against Candida Species

PubMed Central

Mendes de Toledo, Cleyton Eduardo; Santos, Patrícia Regina; Palazzo de Mello, João Carlos; Dias Filho, Benedito Prado; Ueda-Nakamura, Tânia

2015-01-01

The ethnomedicinal plant Curatella americana L. (Dilleniaceae) is a common shrub in the Brazilian cerrado, in which crude extract showed antifungal activity in a preliminary study. In this work, the antifungal and cytotoxic properties of the crude extract, fractions, and isolated compounds from C. americana were evaluated against the standard yeast strains Candida albicans, C. tropicalis, and C. parapsilosis, clinical isolates, and fluconazole-resistant strains. The combinatory effects between subfractions and isolated compounds and effects on cell morphology, virulence factors, and exogenous ergosterol were also evaluated. The MIC obtained against the Candida species including fluconazole-resistant strain ranged from 15.3 to 31.3 µg/mL for crude extract, 3.9 to 15.6 µg/mL for ethyl acetate fraction, and 7.8 to 31.3 µg/mL for subfractions. The isolated compounds identified as 4′-O-methyl-catechin, epicatechin-3-O-gallate, and 4′-O-methyl-catechin-3-O-gallate showed lower antifungal activity than the crude extract and fractions (MIC ranging from 31.3 to 125.0 µg/mL). The addition of exogenous ergosterol to yeast culture did not interfere in the antifungal activity of the extract and its fractions. Synergistic antifungal activity was observed between subfractions and isolated compounds. The effects on virulence factors and the different mechanisms of action compared to fluconazole and nystatin suggest that this ethnomedicinal plant may be an effective alternative treatment for candidiasis. PMID:26347790

Antifungal activity and mode of action of thymol and its synergism with nystatin against Candida species involved with infections in the oral cavity: an in vitro study.

PubMed

de Castro, Ricardo Dias; de Souza, Trícia Murielly Pereira Andrade; Bezerra, Louise Morais Dornelas; Ferreira, Gabriela Lacet Silva; Costa, Edja Maria Melo de Brito; Cavalcanti, Alessandro Leite

2015-11-24

Limitations of antifungal agents used in the treatment of oral candidiasis, as the development of resistant strains, are known by the scientific community. In this context, the aim of this study was to evaluate the antifungal activity of thymol against Candida albicans, Candida tropicalis and Candida krusei strains and to determine its mode of action and synergistic effect when combined with the synthetic antifungal nystatin. The minimum inhibitory concentration (MIC) was determined using a microdilution technique, and the minimum fungicidal concentration (MFC) was determined via subculture sowing. The mode of action of thymol was established by verifying fungal growth in the presence of sorbitol or ergosterol. The fractional inhibitory concentration index (FIC) was determined using the checkerboard method. Thymol presented an antifungal effect, with MICs of 39 μg/mL for C. albicans and C. krusei and 78 μg/mL for C. tropicalis. The results of the antifungal test remained unchanged in the presence of sorbitol; however, the MIC value of thymol against C. albicans increased eight times (from 39.0 to 312.5 μg/mL) in presence of exogenous ergosterol. The combination of thymol and nystatin reduced the MIC values of both products by 87.4%, generating an FIC index of 0.25. Thymol was found to have a fungicidal effect on Candida species and a synergistic effect when combined with nystatin.

Synergistic Interactions of Eugenol-tosylate and Its Congeners with Fluconazole against Candida albicans.

PubMed

Ahmad, Aijaz; Wani, Mohmmad Younus; Khan, Amber; Manzoor, Nikhat; Molepo, Julitha

2015-01-01

We previously reported the antifungal properties of a monoterpene phenol "Eugenol" against different Candida strains and have observed that the addition of methyl group to eugenol drastically increased its antimicrobial potency. Based on the results and the importance of medicinal synthetic chemistry, we synthesized eugenol-tosylate and its congeners (E1-E6) and tested their antifungal activity against different clinical fluconazole (FLC)- susceptible and FLC- resistant C. albicans isolates alone and in combination with FLC by determining fractional inhibitory concentration indices (FICIs) and isobolograms calculated from microdilution assays. Minimum inhibitory concentration (MIC) results confirmed that all the tested C. albicans strains were variably susceptible to the semi-synthetic derivatives E1-E6, with MIC values ranging from 1-62 μg/ml. The test compounds in combination with FLC exhibited either synergy (36%), additive (41%) or indifferent (23%) interactions, however, no antagonistic interactions were observed. The MICs of FLC decreased 2-9 fold when used in combination with the test compounds. Like their precursor eugenol, all the derivatives showed significant impairment of ergosterol biosynthesis in all C. albicans strains coupled with down regulation of the important ergosterol biosynthesis pathway gene-ERG11. The results were further validated by docking studies, which revealed that the inhibitors snugly fitting the active site of the target enzyme, mimicking fluconazole, may well explain their excellent inhibitory activity. Our results suggest that these compounds have a great potential as antifungals, which can be used as chemosensitizing agents with the known antifungal drugs.

Agarol, an ergosterol derivative from Agaricus blazei, induces caspase-independent apoptosis in human cancer cells.

PubMed

Shimizu, Takamitsu; Kawai, Junya; Ouchi, Kenji; Kikuchi, Haruhisa; Osima, Yoshiteru; Hidemi, Rikiishi

2016-04-01

Agaricus blazei (A. blazei) is a mushroom with many biological effects and active ingredients. We purified a tumoricidal substance from A. blazei, an ergosterol derivative, and named it 'Agarol'. Cytotoxic effects of Agarol were determined by the MTT assay using A549, MKN45, HSC-3, and HSC-4 human carcinoma cell lines treated with Agarol. Apoptosis was detected by flow cytometry analysis. Reactive oxygen species (ROS) levels and mitochondria membrane potential (∆ψm) were also determined by flow cytometry. Western blot analysis was used to quantify the expression of apoptosis-related proteins. Agarol predominantly induced apoptosis in two p53-wild cell lines (A549 and MKN45) compared to the other p53-mutant cell lines (HSC-3 and HSC-4). Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of ROS, reduced ∆ψm, release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol, upregulation of Bax, and downregulation of Bcl-2. Caspase-3 activities did not increase, and z-VAD-fmk, a caspase inhibitor, did not inhibit the Agarol-induced apoptosis. These findings indicate that Agarol induces caspase-independent apoptosis in human carcinoma cells through a mitochondrial pathway. The in vivo anticancer activity of Agarol was confirmed in a xenograft murine model. This study suggests a molecular mechanism by which Agarol induces apoptosis in human carcinoma cells and indicates the potential use of Agarol as an anticancer agent.

The mechanism of antifungal action of essential oil from dill (Anethum graveolens L.) on Aspergillus flavus.

PubMed

Tian, Jun; Ban, Xiaoquan; Zeng, Hong; He, Jingsheng; Chen, Yuxin; Wang, Youwei

2012-01-01

The essential oil extracted from the seeds of dill (Anethum graveolens L.) was demonstrated in this study as a potential source of an eco-friendly antifungal agent. To elucidate the mechanism of the antifungal action further, the effect of the essential oil on the plasma membrane and mitochondria of Aspergillus flavus was investigated. The lesion in the plasma membrane was detected through flow cytometry and further verified through the inhibition of ergosterol synthesis. The essential oil caused morphological changes in the cells of A. flavus and a reduction in the ergosterol quantity. Moreover, mitochondrial membrane potential (MMP), acidification of external medium, and mitochondrial ATPase and dehydrogenase activities were detected. The reactive oxygen species (ROS) accumulation was also examined through fluorometric assay. Exposure to dill oil resulted in an elevation of MMP, and in the suppression of the glucose-induced decrease in external pH at 4 µl/ml. Decreased ATPase and dehydrogenase activities in A. flavus cells were also observed in a dose-dependent manner. The above dysfunctions of the mitochondria caused ROS accumulation in A. flavus. A reduction in cell viability was prevented through the addition of L-cysteine, which indicates that ROS is an important mediator of the antifungal action of dill oil. In summary, the antifungal activity of dill oil results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ROS accumulation in A. flavus.

Synthesis, liquid crystallinity, and chiroptical properties of sterol-containing polyacetylenes

NASA Astrophysics Data System (ADS)

Lam, Jacky Wing Yip; Lai, Lo Ming; Tang, Ben Zhong

2006-08-01

Poly(phenylacetylene)s and poly(1-alkyne)s containing chiral sterol pendant groups with molecular structures of -[HC=C-C 6H 4-CO II-R] n-, -[HC=C-C 6H 4-O(CH II) 10-CO II-R] n- and -[HC=C(CH II) mCO II-R] n-, (where R = cholesterol, stigmasterol, ergosterol and m = 2, 3, 8} are designed and synthesized. The monomers are prepared by esterifications of acetylenic acids with cholesterol, stigmasterol, and ergosterol and exhibit cholestericity at high temperatures. Polymerizations of the monomers are effected by WCl 6-Ph 4Sn, MoCl 5-Ph 4Sn, and organorhodium catalysts, giving high molecular weight (M w up to 8.0 × 10 5) polymers in high yields (up to 99%). The structures and properties of the polymers are characterized and evaluated by IR, NMR, TGA, DSC, POM, X-ray, UV, and CD analyses. All the polymers are thermally stable (greater than or equal to 300 °C). Polymers with long flexible alkyl chains form smectic and cholesteric phases at elevated temperatures. With an increase in the spacer length in poly(1-alkyne)s, the packing arrangements of the mesogenic pendants in the mesophases change from bilayer or mixed mono- and bilayer into homogeneous monolayer structures. Few poly(phenylacetylene)s show CD bands in the absorption region of the polyacetylene backbones, revealing that the main chains are helically rotating with a preferred screw sense.

UV-dependent production of 25-hydroxyvitamin D{sub 2} in the recombinant yeast cells expressing human CYP2R1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi

Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D{sub 3} or vitamin D{sub 2} was added to the cell suspension of CYP2R1-expressing yeast cells in amore » buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D{sub 2} was produced without additional vitamin D{sub 2}. Endogenous ergosterol was likely converted into vitamin D{sub 2} by UV irradiation and thermal isomerization, and then the resulting vitamin D{sub 2} was converted to 25-hydroxyvitamin D{sub 2} by CYP2R1. This novel method for producing 25-hydroxyvitamin D{sub 2} without a substrate could be useful for practical purposes.« less

The influence of chemical protection on the content of heavy metals in wheat (Triticum aestivum L.) growing on the soil enriched with granular sludge.

PubMed

Wołejko, E; Łozowicka, B; Kaczyński, P; Konecki, R; Grobela, M

2017-08-01

The presence of heavy metals in Triticum aestivum L. growing on the soil enriched with granular sludge after chemical protection was observed. The five variants of treatments using herbicide (Chwastox Turbo 340SL) and four fungicides (Topsin M 500SC, Amistar 250SC, Artea 330EC, and Falcon 460EC) were performed. On control and experimental plots, the concentration of Ni, Pb, Cr, and Cu in wheat leaves were in the range 0.32-0.99, 0.92-1.57, 0.89-6.31, and 7.08-12.59 mg/kg and in grains 0.03 to 0.11, 0.14-0.25, 0.11-0.76, and 1.06-1.46 mg/kg, respectively. The concentration of Pb in grain protected by MCPA and 2,4-D with thiophanate-methyl and azoxystrobin was higher than the maximum levels of 0.20 mg/kg D.M. The bioconcentration factor (BCF) differed and depended on chemical protection. The highest value of BCF was achieved for Cd. The statistical analysis showed a significant correlation between concentration of metals and quality parameters of wheat. One observed significant negative correlations between Ni/Zeleny sedimentation value (r = -0.51) and between Pb/starch content (r = -0.57). Positive correlations were observed between Cd/yield, the number of grains/ergosterol concentration (respectively, r = 0.41, r = 0.55, r = 0.56), and Zn/thousand grain weight (r = 0.50) at a p ≤ 0.05.

A comparative study to evaluate natural attenuation, mycoaugmentation, phytoremediation, and microbial-assisted phytoremediation strategies for the bioremediation of an aged PAH-polluted soil.

PubMed

García-Sánchez, Mercedes; Košnář, Zdeněk; Mercl, Filip; Aranda, Elisabet; Tlustoš, Pavel

2018-01-01

Biological treatments are considered an environmentally option to clean-up polluted soil with polycyclic aromatic hydrocarbons (PAHs). A pot experiment was conducted to comparatively evaluate four different strategies, including natural attenuation (NA), mycoaugmentation (M) by using Crucibulum leave, phytoremediation (P) using maize plants, and microbial-assisted phytoremediation (MAP) for the bioremediation of an aged PAH-polluted soil at 180 days. The P treatment had higher affinity degrading 2-3 and 4 ring compounds than NA and M treatments, respectively. However, M and P treatments were more efficient in regards to naphthalene, indeno[l,2,3-c,d]pyrene and benzo[g,h,i]perylene degradation respect to NA. However, 4, 5-6 rings undergo a strong decline during the microbe-assisted phytoremediation, being the treatment which determined the highest rates of PAHs degradation. Sixteen PAH compounds, except fluorene and dibenzo[a,h]anthracene, were found in maize roots, whereas the naphthalene, phenanthrene, anthracene, fluoranthene, and pyrene were accumulated in the shoots, in both P and MAP treatments. However, higher PAH content in maize biomass was achieved during the MAP treatment respect to P treatment. The bioconversion and translocation factors were less than 1, indicating that phystabilization/phytodegradation processes occurred rather than phytoextraction. The microbial biomass, activity and ergosterol content were significantly boosted in the MAP treatment respect to the other treatments at 180 days. Ours results demonstrated that maize-C. laeve association was the most profitable technique for the treatment of an aged PAH-polluted soil when compared to other bioremediation approaches. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of Human Induced Pluripotent Stem Cell-Derived Neurons and Rat Primary CorticalNeurons as In Vitro Models of Neurite Outgrowth

EPA Science Inventory

High-throughput assays that can quantify chemical-induced changes at the cellular and molecular level have been recommended for use in chemical safety assessment. High-throughput, high content imaging assays for the key cellular events of neurodevelopment have been proposed to ra...

Relation of murine thoracic aortic structural and cellular changes with aging to passive and active mechanical properties.

PubMed

Wheeler, Jason B; Mukherjee, Rupak; Stroud, Robert E; Jones, Jeffrey A; Ikonomidis, John S

2015-02-25

Maintenance of the structure and mechanical properties of the thoracic aorta contributes to aortic function and is dependent on the composition of the extracellular matrix and the cellular content within the aortic wall. Age-related alterations in the aorta include changes in cellular content and composition of the extracellular matrix; however, the precise roles of these age-related changes in altering aortic mechanical function are not well understood. Thoracic aortic rings from the descending segment were harvested from C57BL/6 mice aged 6 and 21 months. Thoracic aortic diameter and wall thickness were higher in the old mice. Cellular density was reduced in the medial layer of aortas from the old mice; concomitantly, collagen content was higher in old mice, but elastin content was similar between young and old mice. Stress relaxation, an index of compliance, was reduced in aortas from old mice and correlated with collagen fraction. Contractility of the aortic rings following potassium stimulation was reduced in old versus young mice. Furthermore, collagen gel contraction by aortic smooth muscle cells was reduced with age. These results demonstrate that numerous age-related structural changes occurred in the thoracic aorta and were related to alterations in mechanical properties. Aortic contractility decreased with age, likely because of a reduction in medial cell number in addition to a smooth muscle contractile deficit. Together, these unique findings provide evidence that the age-related changes in structure and mechanical function coalesce to provide an aortic substrate that may be predisposed to aortopathies. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Reorganization of plasma membrane lipid domains during conidial germination.

PubMed

Santos, Filipa C; Fernandes, Andreia S; Antunes, Catarina A C; Moreira, Filipe P; Videira, Arnaldo; Marinho, H Susana; de Almeida, Rodrigo F M

2017-02-01

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Cellular therapy injections in today's orthopedic market: A social media analysis.

PubMed

Ramkumar, Prem N; Navarro, Sergio M; Haeberle, Heather S; Chughtai, Morad; Demetriades, Christopher; Piuzzi, Nicolas S; Mont, Michael A; Bauer, Thomas W; Muschler, George F

2017-12-01

The current state of cellular therapy for musculoskeletal conditions is at a crossroads. Marketing efforts are often outpacing clinical evidence and regulatory control. This study was an effort to describe the marketing of cellular therapy in musculoskeletal medicine by evaluating the content in popular social media channels. Specifically, media posts were evaluated for the following: (1) perspective, (2) tone, (3) content and (4) visibility. Social media content related to cell therapy for musculoskeletal conditions was assessed in a search using 28 hashtags on the public domains of Instagram and Twitter over a 2-year period (2014-2016) that resulted in analysis of 698 posts. Supplemental analyses of LinkedIn and Facebook domains were also conducted. A categorical scoring system was used to analyze perspective (patient, family or friend, business or organization), tone (positive, negative), content (education, advertisement, research, media coverage or patient experience) and visibility (number of hashtags per post). Sub-analyses of the advertisement content from various perspectives (patients, physicians and businesses) were performed. The media perspective was most frequently from a business or organization (83%; n = 575). A total of 94% of the posts had a positive tone and only 6% had a negative tone, and the only negative posts came from patients (60% positive and 40% negative). The most common content of social media posts were advertisements, representing 68% (n = 477) of all posts; this was confirmed in the Facebook analysis. The mean number of hashtags was five per post. Sub-analyses revealed approximately half of the advertising posts originated from a single business that recruited physicians to market their cell-based therapies on social media, which was confirmed in the LinkedIn analysis. The market messages related to cell-based therapies for musculoskeletal conditions available on social media are dominated by businesses that seem to use a network of physicians, apply several hashtags to enhance visibility and advertise these largely unproven modalities. The posts portray an almost exclusively positive tone, without providing a "fair balance" on the risks, benefits and limitations. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Evolutionary tradeoffs in cellular composition across diverse bacteria

PubMed Central

Kempes, Christopher P; Wang, Lawrence; Amend, Jan P; Doyle, John; Hoehler, Tori

2016-01-01

One of the most important classic and contemporary interests in biology is the connection between cellular composition and physiological function. Decades of research have allowed us to understand the detailed relationship between various cellular components and processes for individual species, and have uncovered common functionality across diverse species. However, there still remains the need for frameworks that can mechanistically predict the tradeoffs between cellular functions and elucidate and interpret average trends across species. Here we provide a comprehensive analysis of how cellular composition changes across the diversity of bacteria as connected with physiological function and metabolism, spanning five orders of magnitude in body size. We present an analysis of the trends with cell volume that covers shifts in genomic, protein, cellular envelope, RNA and ribosomal content. We show that trends in protein content are more complex than a simple proportionality with the overall genome size, and that the number of ribosomes is simply explained by cross-species shifts in biosynthesis requirements. Furthermore, we show that the largest and smallest bacteria are limited by physical space requirements. At the lower end of size, cell volume is dominated by DNA and protein content—the requirement for which predicts a lower limit on cell size that is in good agreement with the smallest observed bacteria. At the upper end of bacterial size, we have identified a point at which the number of ribosomes required for biosynthesis exceeds available cell volume. Between these limits we are able to discuss systematic and dramatic shifts in cellular composition. Much of our analysis is connected with the basic energetics of cells where we show that the scaling of metabolic rate is surprisingly superlinear with all cellular components. PMID:27046336

Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygen-controlled fermenter

PubMed Central

2012-01-01

Background Aspergillus fumigatus is a mold responsible for the majority of cases of aspergillosis in humans. To survive in the human body, A. fumigatus must adapt to microenvironments that are often characterized by low nutrient and oxygen availability. Recent research suggests that the ability of A. fumigatus and other pathogenic fungi to adapt to hypoxia contributes to their virulence. However, molecular mechanisms of A. fumigatus hypoxia adaptation are poorly understood. Thus, to better understand how A. fumigatus adapts to hypoxic microenvironments found in vivo during human fungal pathogenesis, the dynamic changes of the fungal transcriptome and proteome in hypoxia were investigated over a period of 24 hours utilizing an oxygen-controlled fermenter system. Results Significant increases in transcripts associated with iron and sterol metabolism, the cell wall, the GABA shunt, and transcriptional regulators were observed in response to hypoxia. A concomitant reduction in transcripts was observed with ribosome and terpenoid backbone biosynthesis, TCA cycle, amino acid metabolism and RNA degradation. Analysis of changes in transcription factor mRNA abundance shows that hypoxia induces significant positive and negative changes that may be important for regulating the hypoxia response in this pathogenic mold. Growth in hypoxia resulted in changes in the protein levels of several glycolytic enzymes, but these changes were not always reflected by the corresponding transcriptional profiling data. However, a good correlation overall (R2 = 0.2, p < 0.05) existed between the transcriptomic and proteomics datasets for all time points. The lack of correlation between some transcript levels and their subsequent protein levels suggests another regulatory layer of the hypoxia response in A. fumigatus. Conclusions Taken together, our data suggest a robust cellular response that is likely regulated both at the transcriptional and post-transcriptional level in response to hypoxia by the human pathogenic mold A. fumigatus. As with other pathogenic fungi, the induction of glycolysis and transcriptional down-regulation of the TCA cycle and oxidative phosphorylation appear to major components of the hypoxia response in this pathogenic mold. In addition, a significant induction of the transcripts involved in ergosterol biosynthesis is consistent with previous observations in the pathogenic yeasts Candida albicans and Cryptococcus neoformans indicating conservation of this response to hypoxia in pathogenic fungi. Because ergosterol biosynthesis enzymes also require iron as a co-factor, the increase in iron uptake transcripts is consistent with an increased need for iron under hypoxia. However, unlike C. albicans and C. neoformans, the GABA shunt appears to play an important role in reducing NADH levels in response to hypoxia in A. fumigatus and it will be intriguing to determine whether this is critical for fungal virulence. Overall, regulatory mechanisms of the A. fumigatus hypoxia response appear to involve both transcriptional and post-transcriptional control of transcript and protein levels and thus provide candidate genes for future analysis of their role in hypoxia adaptation and fungal virulence. PMID:22309491

Molecular and cellular neurocardiology: development, and cellular and molecular adaptations to heart disease

PubMed Central

Anderson, Mark E.; Birren, Susan J.; Fukuda, Keiichi; Herring, Neil; Hoover, Donald B.; Kanazawa, Hideaki; Paterson, David J.; Ripplinger, Crystal M.

2016-01-01

Abstract The nervous system and cardiovascular system develop in concert and are functionally interconnected in both health and disease. This white paper focuses on the cellular and molecular mechanisms that underlie neural–cardiac interactions during development, during normal physiological function in the mature system, and during pathological remodelling in cardiovascular disease. The content on each subject was contributed by experts, and we hope that this will provide a useful resource for newcomers to neurocardiology as well as aficionados. PMID:27060296

The Use of a Sensory Model to Facilitate the Study of the Biochemistry of Adhesion in Marine-Fouling Diatoms

DTIC Science & Technology

1993-07-30

transported to the cell membrane in the area of the raphe canal and, following vesicular fusion with the cellular membrane, their contents are released into...the external raphe canal and become free to interact with the substratum. This leads to cellular adhesion. Continued synthesis and secretion of polymer... cellular adhesion is measured as a function of an extracellular chemical signal. Results (a) Sensory Biology. The overall question in our research

Biochemical changes to fibroblast cells subjected to ionizing radiation.

PubMed

Jones, Pamala; Benghuzzi, Hamed; Tucci, Michelle; Richards, Latoya; Harrison, George; Patel, Ramesh

2008-01-01

High energy X-rays are capable of interacting with biological membranes to cause both functional and structural modifications. The goal of the present study was to investigate the effects human fibroblast cells exposed multiple times to 10 Gy over time. Following exposures of 2, 3, or 4 times to 10 Gy/10min the cells were evaluated for cell number changes, membrane damage, and intracellular glutathione content after 24, 48 and 72 hours. Twenty-four hours following exposure the cell numbers were reduced and increased levels of cellular membrane damage was evident. This trend was observed for the duration of the study. Interestingly, there was not an exposure dependent increase in cell damage or cell loss with time. Intracellular antioxidant systems were activated as indicated by anincrease in total cellular glutathione content. Additional studies are needed to determine if the cellular reduction is caused by a direct effect of the X-rays targeting the DNA or an indirect effect of the X-ray targeting the cellular membrane, which then generates radicals that target cell cycle checkpoints or DNA damage. In conclusion, fibroblast cells can be used to determine early and late events of cellular function following exposure to harmful levels of radiation exposure and results of exposure can be seen within twenty four hours.

PF1163A and B, new antifungal antibiotics produced by Penicillium sp. I. Taxonomy of producing strain, fermentation, isolation and biological activities.

PubMed

Nose, H; Seki, A; Yaguchi, T; Hosoya, A; Sasaki, T; Hoshiko, S; Shomura, T

2000-01-01

Two novel antifungal antibiotics, PF1163A and B, were isolated from the fermentation broth of Penicillium sp. They were purified from the solid cultures of rice media using ethyl acetate extraction, silica gel and Sephadex LH-20 column chromatographies. PF1163A and B showed potent growth inhibitory activity against pathogenic fungal strain Candida albicans but did not show cytotoxic activity against mammalian cells. These compounds inhibited the ergosterol biosynthesis in Candida albicans.

Lipid composition and sensitivity of Prototheca wickerhamii to membrane-active antimicrobial agents.

PubMed Central

Sud, I J; Feingold, D S

1979-01-01

The lipid composition of Prototheca wickerhamii ATCC 16529 is presented and discussed in relation to the unique susceptibility of the organism to drugs of three membrane-active antimicrobial classes: the polyenes, the polymyxins, and the imidazoles. The presence of ergosterol in the neutral lipid fraction of the membrane is likely responsible for the exquisite susceptibility to amphotericin B. The presence of a large quantity of free fatty acids in the membrane appears responsible for imidazole susceptibility. The membrane determinants of polymyxin B susceptibility are less well defined. PMID:518077

Treatment of Articular Cartilage Defects in the Goat with Frozen Versus Fresh Osteochondral Allografts: Effects on Cartilage Stiffness, Zonal Composition, and Structure at Six Months

PubMed Central

Pallante, Andrea L.; Görtz, Simon; Chen, Albert C.; Healey, Robert M.; Chase, Derek C.; Ball, Scott T.; Amiel, David; Sah, Robert L.; Bugbee, William D.

2012-01-01

Background: Understanding the effectiveness of frozen as compared with fresh osteochondral allografts at six months after surgery and the resultant consequences of traditional freezing may facilitate in vivo maintenance of cartilage integrity. Our hypothesis was that the state of the allograft at implantation affects its performance after six months in vivo. Methods: The effect of frozen as compared with fresh storage on in vivo allograft performance was determined for osteochondral allografts that were transplanted into seven recipient goats and analyzed at six months. Allograft performance was assessed by examining osteochondral structure (cartilage thickness, fill, surface location, surface degeneration, and bone-cartilage interface location), zonal cartilage composition (cellularity, matrix content), and cartilage biomechanical function (stiffness). Relationships between cartilage stiffness or cartilage composition and surface degeneration were assessed with use of linear regression. Results: Fresh allografts maintained cartilage load-bearing function, while also maintaining zonal organization of cartilage cellularity and matrix content, compared with frozen allografts. Overall, allograft performance was similar between fresh allografts and nonoperative controls. However, cartilage stiffness was approximately 80% lower (95% confidence interval [CI], 73% to 87%) in the frozen allografts than in the nonoperative controls or fresh allografts. Concomitantly, in frozen allografts, matrix content and cellularity were approximately 55% (95% CI, 22% to 92%) and approximately 96% (95% CI, 94% to 99%) lower, respectively, than those in the nonoperative controls and fresh allografts. Cartilage stiffness correlated positively with cartilage cellularity and matrix content, and negatively with surface degeneration. Conclusions: Maintenance of cartilage load-bearing function in allografts is associated with zonal maintenance of cartilage cellularity and matrix content. In this animal model, frozen allografts displayed signs of failure at six months, with cartilage softening, loss of cells and matrix, and/or graft subsidence, supporting the importance of maintaining cell viability during allograft storage and suggesting that outcomes at six months may be indicative of long-term (dys)function. Clinical Relevance: Fresh versus frozen allografts represent the “best versus worst” conditions with respect to chondrocyte viability, but “difficult versus simple” with respect to acquisition and distribution. The outcomes described from these two conditions expand the current understanding of in vivo cartilage remodeling and describe structural properties (initial graft subsidence), which may have implications for impending graft failure. PMID:23138239

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

PubMed

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

2011-10-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay

PubMed Central

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M.

2011-01-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin. PMID:22006542

Subcellular Localized Chemical Imaging of Benthic Algal Nutritional Content via HgCdTe Array FT-IR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetzel, D.; Murdock, J; Dodds, W

2008-01-01

Algae respond rapidly and uniquely to changes in nutrient availability by adjusting pigment, storage product, and organelle content and quality. Cellular and subcellular variability of the relative abundance of macromolecular pools (e.g. protein, lipid, carbohydrate, and phosphodiesters) within the benthic (bottom dwelling) alga Cladophora glomerata (a common nuisance species in fresh and saline waters) was revealed by FT-IR microspectroscopic imaging. Nutrient heterogeneity was compared at the filament, cellular, and subcellular level, and localized nutrient uptake kinetics were studied by detecting the gradual incorporation of isotopically labeled nitrogen (N) (as K15NO3) from surrounding water into cellular proteins. Nutritional content differed substantiallymore » among filament cells, with differences driven by protein and lipid abundance. Whole cell imaging showed high subcellular macromolecular variability in all cells, including adjacent cells on a filament that developed clonally. N uptake was also very heterogeneous, both within and among cells, and did not appear to coincide with subcellular protein distribution. Despite high intercellular variability, some patterns emerged. Cells acquired more 15N the further they were away from the filament attachment point, and 15N incorporation was more closely correlated with phosphodiester content than protein, lipid, or carbohydrate content. Benthic algae are subject to substantial environmental heterogeneity induced by microscale hydrodynamic factors and spatial variability in nutrient availability. Species specific responses to nutrient heterogeneity are central to understanding this key component of aquatic ecosystems. FT-IR microspectroscopy, modified for benthic algae, allows determination of algal physiological responses at scales not available using current techniques.« less

Recovery of phenotypes obtained by adaptive evolution through inverse metabolic engineering.

PubMed

Hong, Kuk-Ki; Nielsen, Jens

2012-11-01

In a previous study, system level analysis of adaptively evolved yeast mutants showing improved galactose utilization revealed relevant mutations. The governing mutations were suggested to be in the Ras/PKA signaling pathway and ergosterol metabolism. Here, site-directed mutants having one of the mutations RAS2(Lys77), RAS2(Tyr112), and ERG5(Pro370) were constructed and evaluated. The mutants were also combined with overexpression of PGM2, earlier proved as a beneficial target for galactose utilization. The constructed strains were analyzed for their gross phenotype, transcriptome and targeted metabolites, and the results were compared to those obtained from reference strains and the evolved strains. The RAS2(Lys77) mutation resulted in the highest specific galactose uptake rate among all of the strains with an increased maximum specific growth rate on galactose. The RAS2(Tyr112) mutation also improved the specific galactose uptake rate and also resulted in many transcriptional changes, including ergosterol metabolism. The ERG5(Pro370) mutation only showed a small improvement, but when it was combined with PGM2 overexpression, the phenotype was almost the same as that of the evolved mutants. Combination of the RAS2 mutations with PGM2 overexpression also led to a complete recovery of the adaptive phenotype in galactose utilization. Recovery of the gross phenotype by the reconstructed mutants was achieved with much fewer changes in the genome and transcriptome than for the evolved mutants. Our study demonstrates how the identification of specific mutations by systems biology can direct new metabolic engineering strategies for improving galactose utilization by yeast.

In vitro antitumor activity and structure characterization of ethanol extracts from wild and cultivated Chaga medicinal mushroom, Inonotus obliquus (Pers.:Fr.) Pilát (Aphyllophoromycetideae).

PubMed

Sun, Yong; Yin, Ting; Chen, Xian-Hui; Zhang, Gong; Curtis, Rempel B; Lu, Zhan-Hui; Jiang, Ji-Hong

2011-01-01

Inonotus obliquus (Pers.:Fr.) Pilát has been traditionally used as a folk remedy for treatment of cancers, cardiovascular disease and diabetes in Russia, Poland, and most of the Baltic countries, but natural reserves of this fungus have nearly been exhausted. This study was designed to investigate the artificial cultivation of I. obliquus and the antitumor activity of its tissues. The ethanol extract of cultivated sclerotium had the highest cell growth inhibitory rate (74.6%) as determined by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 78% of the bags produced sclerotia and only 6.17 g/bag of sclerotium was obtained. Extracts of the cultivated fruiting body showed 44.2% inhibitory activity against tumor cells. However, the yield was as high as 18.24 g/bag, and 98% of the bags produced fruiting body. The results of gas chromatography-mass spectroscopy (GC-MS) showed that similar compounds were extracted from the wild and cultivated samples. The principal compounds observed were lanosterol, inotodiol, and ergosterol. Their percentages of the mass fraction were 86.1, 59.9, and 71.8% of the total, for the wild sclerotium, cultivated sclerotium, and cultivated fruiting body, respectively. Ergosterol was found to be much higher (27.32%) in cultivated fruiting body. We conclude that cultivated fruiting body of I. obliquus obtained by inoculation of the substrate with spawn mycelium of the fifth generation could serve as an ideal substitute for the wild I. obliquus.

Mode of action and synergistic effect of valinomycin and cereulide with amphotericin B against Candida albicans and Cryptococcus albidus.

PubMed

Makarasen, A; Reukngam, N; Khlaychan, P; Chuysinuan, P; Isobe, M; Techasakul, S

2018-03-01

Both valinomycin and cereulide are cyclic depsipeptides and are known K + ion-selective ionophores. Valinomycin and cereulide feature low minimum inhibitory concentration (MIC) values against Candida albicans and Cryptococcus albidus. This study aims at investigating the mode of action and verifying the efficacy of valinomycin or cereulide alone and in combination with amphotericin B (AmB) in vitro against both microorganisms. Based on the results from membrane permeability and fluidity assays for detection of plasma membrane permeabilization and membrane dynamics, the present study demonstrated that valinomycin and cereulide exhibit antifungal activity against C. albicans and C. albidus by interrupting membrane-associated function. The mode of action of both valinomycin and cereulide are similar with that of AmB. Time-kill kinetics assay showed that valinomycin and cereulide exhibit fungistatic activity, whereas AmB features fungicidal activity. Additionally, the combination of compounds between each cyclic peptide and AmB reached maximal fungicidal activity more rapidly than AmB alone. This result corresponded with findings of scanning electron microscopy, fractional inhibitory concentration index and minimum fungicidal concentration (MFC)/MIC ratio, indicating that combinations of the drugs show synergistic effects for inhibiting the growth of these fungal strains. Sorbitol and ergosterol assays showed that both cyclic peptides affected cell wall and membrane components due to increases in MIC value, as observed in medium with sorbitol and ergosterol. Valinomycin and cereulide may promote permeability of fungal cell wall and cell membrane when used in combination with AmB. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Transcriptional and proteomic analysis of the Aspergillus fumigatus ΔprtT protease-deficient mutant.

PubMed

Hagag, Shelly; Kubitschek-Barreira, Paula; Neves, Gabriela W P; Amar, David; Nierman, William; Shalit, Itamar; Shamir, Ron; Lopes-Bezerra, Leila; Osherov, Nir

2012-01-01

Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, infecting immunocompromised patients. The fungus invades the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the host structural barriers. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. Unexpectedly, microarray analysis revealed that in addition to the expected decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of prtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of toxic pseurotin A. Proteomic analysis identified reduced levels of three secreted proteases (ALP1 protease, TppA, AFUA_2G01250) and increased levels of three secreted polysaccharide-degrading enzymes in the ΔprtT mutant possibly in response to its inability to derive sufficient nourishment from protein breakdown. This report highlights the complexity of gene regulation by PrtT, and suggests a potential novel link between the regulation of protease secretion and the control of iron uptake, ergosterol biosynthesis and secondary metabolite production in A. fumigatus.

Amauroderma rugosum (Blume & T. Nees) Torrend: Nutritional Composition and Antioxidant and Potential Anti-Inflammatory Properties

PubMed Central

Chan, Pui-Mun; Kanagasabapathy, Gowri; Tan, Yee-Shin; Sabaratnam, Vikineswary; Kuppusamy, Umah Rani

2013-01-01

Amauroderma rugosum is a wild mushroom that is worn as a necklace by the indigenous communities in Malaysia to prevent fits and incessant crying by babies. The aim of this study was to investigate the nutritive composition and antioxidant potential and anti-inflammatory effects of A. rugosum extracts on LPS-stimulated RAW264.7 cells. Nutritional analysis of freeze-dried mycelia of A. rugosum (KUM 61131) from submerged culture indicated a predominant presence of carbohydrates, proteins, dietary fibre, phosphorus, potassium, and sodium. The ethanol crude extract (EE), its hexane (HF), ethyl acetate (EAF), and aqueous (AF) fractions of mycelia of A. rugosum grown in submerged culture were evaluated for antioxidant potential and anti-inflammatory effects. EAF exhibited the highest total phenolic content and the strongest antioxidant activity based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. HF showed dose-dependent inhibition of NO production in LPS-stimulated RAW264.7 cells and NO radical scavenging activity. Gas chromatographic analysis of HF revealed the presence of ethyl linoleate and ergosterol, compounds with known anti-inflammatory properties. In conclusion, the nutritive compositions and significant antioxidant potential and anti-inflammatory effects of mycelia extracts of A. rugosum have the potential to serve as a therapeutic agent or adjuvant in the management of inflammatory disorders. PMID:24371454

Observational scores of dampness and mold associated with measurements of microbial agents and moisture in three public schools

PubMed Central

Cho, S. J.; Cox-Ganser, J. M.; Park, J.-H.

2015-01-01

We examined associations between observational dampness scores and measurements of microbial agents and moisture in three public schools. A dampness score was created for each room from 4-point-scale scores (0–3) of water damage, water stains, visible mold, moldy odor, and wetness for each of 8 room components (ceiling, walls, windows, floor, ventilation, furniture, floor trench, and pipes), when present. We created mixed microbial exposure indices (MMEIs) for each of 121 rooms by summing decile ranks of 8 analytes (total culturable fungi; total, Gram-negative, and Gram-positive culturable bacteria; ergosterol; (1→3)-β-D-glucan; muramic acid; and endotoxin) in floor dust. We found significant (P ≤ 0.01) linear associations between the dampness score and culturable bacteria (total, Gram-positive, and Gram-negative) and the MMEIs. Rooms with dampness scores greater than 0.25 (median) had significantly (P < 0.05) higher levels of most microbial agents, MMEIs, and relative moisture content than those with lower scores (≤0.25). Rooms with reported recent water leaks had significantly (P < 0.05) higher dampness scores than those with historical or no reported water leaks. This study suggests that observational assessment of dampness and mold using a standardized form may be valuable for identifying and documenting water damage and associated microbial contamination. PMID:25650175

Finding Toxicological Tipping Points from High-Content Imaging Data (WC10)

EPA Science Inventory

A key challenge to using in vitro data in risk assessment is differentiating between chemical-induced adaptive versus adverse cellular responses. To further investigate this issue, we studied the effects of hundreds of chemicals in HepG2 cells using high-content imaging (HCI). HC...

Increase in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase function.

PubMed

He, Yue; Yam, Candice; Pomraning, Kyle; Chin, Jacqueline S R; Yew, Joanne Y; Freitag, Michael; Oliferenko, Snezhana

2014-12-15

Excess fatty acids and sterols are stored as triacylglycerols and sterol esters in specialized cellular organelles, called lipid droplets. Understanding what determines the cellular amount of neutral lipids and their packaging into lipid droplets is of fundamental and applied interest. Using two species of fission yeast, we show that cycling cells deficient in the function of the ER-resident CDP-DG synthase Cds1 exhibit markedly increased triacylglycerol content and assemble large lipid droplets closely associated with the ER membranes. We demonstrate that these unusual structures recruit the triacylglycerol synthesis machinery and grow by expansion rather than by fusion. Our results suggest that interfering with the CDP-DG route of phosphatidic acid utilization rewires cellular metabolism to adopt a triacylglycerol-rich lifestyle reliant on the Kennedy pathway. © 2014 He, Yam, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Correlation analyses of phytochemical composition, chemical, and cellular measures of antioxidant activity of broccoli (Brassica oleracea L. Var. italica).

PubMed

Eberhardt, Marian V; Kobira, Kanta; Keck, Anna-Sigrid; Juvik, John A; Jeffery, Elizabeth H

2005-09-21

Chemical measures of antioxidant activity within the plant, such as the oxygen radical absorbance capacity (ORAC) assay, have been reported for many plant-based foods. However, the extent to which chemical measures relate to cellular measures of oxidative stress is unclear. The natural variation in the phytochemical content of 22 broccoli genotypes was used to determine correlations among chemical composition (carotenoids, tocopherols and polyphenolics), chemical antioxidant activity (ORAC), and measures of cellular antioxidation [prevention of DNA oxidative damage and of oxidation of the biomarker dichlorofluorescein (DCFH) in HepG2 cells] using hydrophilic and lipophilic extracts of broccoli. For lipophilic extracts, ORAC (ORAC-L) correlated with inhibition of cellular oxidation of DCFH (DCFH-L, r = 0.596, p = 0.006). Also, DNA damage in the presence of the lipophilic extract was negatively correlated with both chemical and cellular measures of antioxidant activity as measured by ORAC-L (r = -0.705, p = 0.015) and DCFH-L (r = -0.671, p = 0.048), respectively. However, no correlations were observed for hydrophilic (-H) extracts, except between polyphenol content and ORAC (ORAC-H; r = 0.778, p < 0.001). Inhibition of cellular oxidation by hydrophilic extracts (DCFH-H) and ORAC-H were approximately 8- and 4-fold greater than DCFH-L and ORAC-L, respectively. Whether ORAC-H has more biological relevance than ORAC-L because of its magnitude or whether ORAC-L bears more biological relevance because it relates to cellular estimates of antioxidant activity remains to be determined. Chemical estimates of antioxidant capacity within the plant may not accurately reflect the complex nature of the full antioxidant activity of broccoli extracts within cells.

Effect of Nitrogen on Cellular Production and Release of the Neurotoxin Anatoxin-A in a Nitrogen-Fixing Cyanobacterium

PubMed Central

Gagnon, Alexis; Pick, Frances R.

2012-01-01

Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and implicated in lethal poisonings of domesticated animals and wildlife. The factors leading to its production in nature and in culture are not well understood. Resource availability may influence its cellular production as suggested by the carbon-nutrient hypothesis, which links the amount of secondary metabolites produced by plants or microbes to the relative abundance of nutrients. We tested the effects of nitrogen supply (as 1, 5, and 100% N of standard cyanobacterial medium corresponding to 15, 75, and 1500 mg L−1 of NaNO3 respectively) on ANTX production and release in a toxic strain of the planktonic cyanobacterium Aphanizomenon issatschenkoi (Nostocales). We hypothesized that nitrogen deficiency might constrain the production of ANTX. However, the total concentration and more significantly the cellular content of anatoxin-a peaked (max. 146 μg/L and 1683 μg g−1 dry weight) at intermediate levels of nitrogen supply when N-deficiency was evident based on phycocyanin to chlorophyll a and carbon to nitrogen ratios. The results suggest that the cellular production of anatoxin-a may be stimulated by moderate nitrogen stress. Maximal cellular contents of other cyanotoxins have recently been reported under severe stress conditions in another Nostocales species. PMID:22701451

Suppressive effects of carotenoids on the antigen-induced degranulation in RBL-2H3 rat basophilic leukemia cells.

PubMed

Manabe, Yuki; Hirata, Takashi; Sugawara, Tatsuya

2014-01-01

In this study, the anti-degranulation effects of fifteen carotenoids were evaluated using RBL-2H3 rat basophilic leukemia cell line as a mast cell model. Nine carotenoids, fucoxanthin, zeaxanthin, β-carotene, astaxanthin, 3-hydroxyechinenone, fucoxanthinol, lycopene, β-cryptoxanthin, and siphonaxanthin significantly suppressed antigen-induced mast cell degranulation. Under the same conditions, the cellular carotenoid contents were quantified using high performance liquid chromatography-photodiode array (HPLC-PDA). There was no correlation between the cellular carotenoid contents and their anti-degranulation activities. These results indicate that the differences in the anti-degranulation activities of carotenoids were not related to their uptake by the cells.

Automated microscopy for high-content RNAi screening

PubMed Central

2010-01-01

Fluorescence microscopy is one of the most powerful tools to investigate complex cellular processes such as cell division, cell motility, or intracellular trafficking. The availability of RNA interference (RNAi) technology and automated microscopy has opened the possibility to perform cellular imaging in functional genomics and other large-scale applications. Although imaging often dramatically increases the content of a screening assay, it poses new challenges to achieve accurate quantitative annotation and therefore needs to be carefully adjusted to the specific needs of individual screening applications. In this review, we discuss principles of assay design, large-scale RNAi, microscope automation, and computational data analysis. We highlight strategies for imaging-based RNAi screening adapted to different library and assay designs. PMID:20176920

Distinction of broken cellular wall Ganoderma lucidum spores and G. lucidum spores using FTIR microspectroscopy

NASA Astrophysics Data System (ADS)

Chen, Xianliang; Liu, Xingcun; Sheng, Daping; Huang, Dake; Li, Weizu; Wang, Xin

2012-11-01

In this paper, FTIR microspectroscopy was used to identify broken cellular wall Ganoderma lucidum spores and G. lucidum spores. For IR spectra, broken cellular wall G. lucidum spores and G. lucidum spores were mainly different in the regions of 3000-2800, 1660-1600, 1400-1200 and 1100-1000 cm-1. For curve fitting, the results showed the differences in the protein secondary structures and the polysaccharide structures/content between broken cellular wall G. lucidum spores and G. lucidum spores. Moreover, the value of A1078/A1741 might be a potentially useful factor to distinguish broken cellular wall G. lucidum spores from G. lucidum spores. Additionally, FTIR microspectroscopy could identify broken cellular wall G. lucidum spores and G. lucidum spores accurately when it was combined with hierarchical cluster analysis. The result suggests FTIR microspectroscopy is very simple and efficient for distinction of broken cellular wall G. lucidum spores and G. lucidum spores. The result also indicates FTIR microspectroscopy may be useful for TCM identification.

Contribution of soil fauna to soil functioning in degraded environments: a multidisciplinary approach

NASA Astrophysics Data System (ADS)

Gargiulo, Laura; Mele, Giacomo; Moradi, Jabbar; Kukla, Jaroslav; Jandová, Kateřina; Frouz, Jan

2016-04-01

The restoration of the soil functions is essential for the recovery of highly degraded sites and, consequently, the study of the soil fauna role in the soil development in such environments has great potential from a practical point of view. The soils of the post-mining sites represent unique models for the study of the natural ecological succession because mining creates similar environments characterized by the same substrate, but by different ages according to the year of closure of mines. The aim of this work was to assess the contribution of different species of macrofauna on the evolution of soil structure and on the composition and activity of the microbial community in soil samples subjected to ecological restoration or characterized by spontaneous ecological succession. For this purpose, an experimental test was carried out in two sites characterized by different post-mining conditions: 1) natural succession, 2) reclamation with planting trees. These sites are located in the post-mining area of Sokolov (Czech Republic). For the experimental test repacked soil cores were prepared in laboratory with sieved soil sampled from the two sites. The soil cores were prepared maintaining the sequence of soil horizons present in the field. These samples were inoculated separately with two genera of earthworms (Lumbricus and Aporrectodea) and two of centipedes (Julida and Polydesmus). In particular, based on their body size, were inoculated for each cylinder 2 individuals of millipedes, 1 individual of Lumbricus and 4 individuals of Aporrectodea. For each treatment and for control samples 5 replicates were prepared and all samples were incubated in field for 1 month in the two original sampling sites. After the incubation the samples were removed from the field and transported in laboratory in order to perform the analysis of microbial respiration, of PLFA (phospholipid-derived fatty acids) and ergosterol contents and finally for the characterization of soil structure. All replicates were subjected to soil respiration measurement by means of chemical titration method. Then some replicates were destructively analyzed for PLFA and ergosterol and others were used for the 3D soil image analysis of the soil pore system. The soil cores were imaged using X-ray microtomography and three-dimensional image processing was performed in order to obtain 3D reconstructions and preliminary analysis of the identified biopores. The experimental approach used in this multidisciplinary study showed a promising potential to provide new useful information about the widely differentiated contribution of many types of macrofauna to the formation of the soil pore system and to the development of the soil microbial functions in different types of environments.

Dropping macadamia nuts-in-shell reduces kernel roasting quality.

PubMed

Walton, David A; Wallace, Helen M

2010-10-01

Macadamia nuts ('nuts-in-shell') are subjected to many impacts from dropping during postharvest handling, resulting in damage to the raw kernel. The effect of dropping on roasted kernel quality is unknown. Macadamia nuts-in-shell were dropped in various combinations of moisture content, number of drops and receiving surface in three experiments. After dropping, samples from each treatment and undropped controls were dry oven-roasted for 20 min at 130 °C, and kernels were assessed for colour, mottled colour and surface damage. Dropping nuts-in-shell onto a bed of nuts-in-shell at 3% moisture content or 20% moisture content increased the percentage of dark roasted kernels. Kernels from nuts dropped first at 20%, then 10% moisture content, onto a metal plate had increased mottled colour. Dropping nuts-in-shell at 3% moisture content onto nuts-in-shell significantly increased surface damage. Similarly, surface damage increased for kernels dropped onto a metal plate at 20%, then at 10% moisture content. Postharvest dropping of macadamia nuts-in-shell causes concealed cellular damage to kernels, the effects not evident until roasting. This damage provides the reagents needed for non-enzymatic browning reactions. Improvements in handling, such as reducing the number of drops and improving handling equipment, will reduce cellular damage and after-roast darkening. Copyright © 2010 Society of Chemical Industry.

G protein-coupled receptor internalization assays in the high-content screening format.

PubMed

Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

2006-01-01

High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

Intelligent Interfaces for Mining Large-Scale RNAi-HCS Image Databases

PubMed Central

Lin, Chen; Mak, Wayne; Hong, Pengyu; Sepp, Katharine; Perrimon, Norbert

2010-01-01

Recently, High-content screening (HCS) has been combined with RNA interference (RNAi) to become an essential image-based high-throughput method for studying genes and biological networks through RNAi-induced cellular phenotype analyses. However, a genome-wide RNAi-HCS screen typically generates tens of thousands of images, most of which remain uncategorized due to the inadequacies of existing HCS image analysis tools. Until now, it still requires highly trained scientists to browse a prohibitively large RNAi-HCS image database and produce only a handful of qualitative results regarding cellular morphological phenotypes. For this reason we have developed intelligent interfaces to facilitate the application of the HCS technology in biomedical research. Our new interfaces empower biologists with computational power not only to effectively and efficiently explore large-scale RNAi-HCS image databases, but also to apply their knowledge and experience to interactive mining of cellular phenotypes using Content-Based Image Retrieval (CBIR) with Relevance Feedback (RF) techniques. PMID:21278820

Oxygen requirements for formation and activity of the squalene expoxidase in Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Jahnke, L.; Klein, H. P.

1983-01-01

The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the 'mitochondrial' fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the 'microsomal' fraction. At 0.03 percent (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent K sub m for oxygen of 0.38 percent (vol/vol) was determined from a crude particulate preparation for the epoxidase.

β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine derivatives eradicate C. albicans in an ex vivo human dentinal tubule model by inhibiting sterol 14-α demethylase and cAMP pathway.

PubMed

Gowri, Meiyazhagan; Sofi Beaula, Winfred; Biswal, Jayashree; Dhamodharan, Prabhu; Saiharish, Raghavan; Rohan prasad, Surabi; Pitani, Ravishankar; Kandaswamy, Deivanayagam; Raghunathan, Ragavachary; Jeyakanthan, Jeyaraman; Rayala, Suresh K; Venkatraman, Ganesh

2016-04-01

Further quest for new anti-fungal compounds with proven mechanisms of action arises due to resistance and dose limiting toxicity of existing agents. Among the human fungal pathogens C. albicans predominate by infecting several sites in the body and in particular oral cavity and root canals of human tooth. In the present study, we screened a library of β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine compounds against Candida sp. Detailed molecular studies were carried out with the active compound 3 on C. albicans. Morphological damage and antibiofilm activity of compound 3 on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. In silico docking studies were carried out to elucidate the mechanism of action of compound 3. Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model. Screening data showed that several new compounds were active against Candida sp. Among them, Compound 3 was most potent and exerted time kill effect at 4h, post antifungal effect up to 6h. When used in combination with fluconazole or nystatin, compound 3 revealed an minimum inhibitory concentration (MIC) decrease by 4 fold for both drugs used. In-depth molecular studies with compound 3 on C. albicans showed that this compound inhibited yeast to hyphae (Y-H) conversion and this involved the cAMP pathway. Further, SEM images of C. albicans showed that compound 3 caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, in silico studies revealed that compound 3 docks with the active site of the key enzyme 14-α-demethylase and this might inhibit ergosterol synthesis. In support of this, ergosterol levels were found to be decreased by 32 fold in compound 3 treated samples as analyzed by high performance liquid chromatography (HPLC). Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed 83% eradication of C. albicans and a 6 log reduction in colony forming unit (CFU) after 24h treatment in the infected tooth samples in this model. Compound 3 was found to be very effective in eradicating C. albicans by inhibiting cAMP pathway and ergosterol biosynthesis. The results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection. Copyright © 2015 Elsevier B.V. All rights reserved.

Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"

ERIC Educational Resources Information Center

Deutch, Charles E.; Marshall, Pamela A.

2008-01-01

In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…

Impact of Sampling and Cellular Separation on Amino Acid Determinations in Drosophila Hemolymph.

PubMed

Cabay, Marissa R; Harris, Jasmine C; Shippy, Scott A

2018-04-03

The fruit fly is a frequently used model system with a high degree of human disease-related genetic homology. The quantitative chemical analysis of fruit fly tissues and hemolymph uniquely brings chemical signaling and compositional information to fly experimentation. The work here explores the impact of measured chemical content of hemolymph with three aspects of sample collection and preparation. Cellular content of hemolymph was quantitated and removed to determine hemolymph composition changes for seven primary amine analytes. Hemolymph sampling methods were adapted to determine differences in primary amine composition of hemolymph collected from the head, antenna, and abdomen. Also, three types of anesthesia were employed with hemolymph collection to quantitate effects on measured amino acid content. Cell content was found to be 45.4 ± 22.1 cells/nL of hemolymph collected from both adult and larvae flies. Cell-concentrated fractions of adult, but not larvae, hemolymph were found to have higher and more variable amine content. There were amino acid content differences found between all three areas indicating a robust method to characterize chemical markers from specific regions of a fly, and these appear related to physiological activity. Methods of anesthesia have an impact on hemolymph amino acid composition related to overall physiological impact to fly including higher amino acid content variability and oxygen deprivation effects. Together, these analyses identify potential complications with Drosophila hemolymph analysis and opportunities for future studies to relate hemolymph content with model physiological activity.

Impact of Air Pollutants on Oxidative Stress in Common Autophagy-Mediated Aging Diseases

PubMed Central

Numan, Mohamed Saber; Brown, Jacques P.; Michou, Laëtitia

2015-01-01

Atmospheric pollution-induced cellular oxidative stress is probably one of the pathogenic mechanisms involved in most of the common autophagy-mediated aging diseases, including neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s, disease, as well as Paget’s disease of bone with or without frontotemporal dementia and inclusion body myopathy. Oxidative stress has serious damaging effects on the cellular contents: DNA, RNA, cellular proteins, and cellular organelles. Autophagy has a pivotal role in recycling these damaged non-functional organelles and misfolded or unfolded proteins. In this paper, we highlight, through a narrative review of the literature, that when autophagy processes are impaired during aging, in presence of cumulative air pollution-induced cellular oxidative stress and due to a direct effect on air pollutant, autophagy-mediated aging diseases may occur. PMID:25690002

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion*

PubMed Central

Hung, Chien-Wen; Klein, Tobias; Cassidy, Liam; Linke, Dennis; Lange, Sabrina; Anders, Uwe; Bureik, Matthias; Heinzle, Elmar; Schneider, Konstantin; Tholey, Andreas

2016-01-01

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe. Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016. PMID:27477394

In Vitro and In Vivo Activities of Pterostilbene against Candida albicans Biofilms

PubMed Central

Li, De-Dong; Zhao, Lan-Xue; Mylonakis, Eleftherios; Hu, Gan-Hai; Zou, Yong; Huang, Tong-Kun; Yan, Lan

2014-01-01

Pterostilbene (PTE) is a stilbene-derived phytoalexin that originates from several natural plant sources. In this study, we evaluated the activity of PTE against Candida albicans biofilms and explored the underlying mechanisms. In 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assays, biofilm biomass measurement, confocal laser scanning microscopy, and scanning electron microscopy, we found that ≤16 μg/ml PTE had a significant effect against C. albicans biofilms in vitro, while it had no fungicidal effect on planktonic C. albicans cells, which suggested a unique antibiofilm effect of PTE. Then we found that PTE could inhibit biofilm formation and destroy the maintenance of mature biofilms. At 4 μg/ml, PTE decreased cellular surface hydrophobicity (CSH) and suppressed hyphal formation. Gene expression microarrays and real-time reverse transcription-PCR showed that exposure of C. albicans to 16 μg/ml PTE altered the expression of genes that function in morphological transition, ergosterol biosynthesis, oxidoreductase activity, and cell surface and protein unfolding processes (heat shock proteins). Filamentation-related genes, especially those regulated by the Ras/cyclic AMP (cAMP) pathway, including ECE1, ALS3, HWP1, HGC1, and RAS1 itself, were downregulated upon PTE treatment, indicating that the antibiofilm effect of PTE was related to the Ras/cAMP pathway. Then, we found that the addition of exogenous cAMP reverted the PTE-induced filamentous growth defect. Finally, with a rat central venous catheter infection model, we confirmed the in vivo activity of PTE against C. albicans biofilms. Collectively, PTE had strong activities against C. albicans biofilms both in vitro and in vivo, and these activities were associated with the Ras/cAMP pathway. PMID:24514088

Ethanol Production and Maximum Cell Growth Are Highly Correlated with Membrane Lipid Composition during Fermentation as Determined by Lipidomic Analysis of 22 Saccharomyces cerevisiae Strains

PubMed Central

Henderson, Clark M.; Lozada-Contreras, Michelle; Jiranek, Vladimir; Longo, Marjorie L.

2013-01-01

Optimizing ethanol yield during fermentation is important for efficient production of fuel alcohol, as well as wine and other alcoholic beverages. However, increasing ethanol concentrations during fermentation can create problems that result in arrested or sluggish sugar-to-ethanol conversion. The fundamental cellular basis for these problem fermentations, however, is not well understood. Small-scale fermentations were performed in a synthetic grape must using 22 industrial Saccharomyces cerevisiae strains (primarily wine strains) with various degrees of ethanol tolerance to assess the correlation between lipid composition and fermentation kinetic parameters. Lipids were extracted at several fermentation time points representing different growth phases of the yeast to quantitatively analyze phospholipids and ergosterol utilizing atmospheric pressure ionization-mass spectrometry methods. Lipid profiling of individual fermentations indicated that yeast lipid class profiles do not shift dramatically in composition over the course of fermentation. Multivariate statistical analysis of the data was performed using partial least-squares linear regression modeling to correlate lipid composition data with fermentation kinetic data. The results indicate a strong correlation (R2 = 0.91) between the overall lipid composition and the final ethanol concentration (wt/wt), an indicator of strain ethanol tolerance. One potential component of ethanol tolerance, the maximum yeast cell concentration, was also found to be a strong function of lipid composition (R2 = 0.97). Specifically, strains unable to complete fermentation were associated with high phosphatidylinositol levels early in fermentation. Yeast strains that achieved the highest cell densities and ethanol concentrations were positively correlated with phosphatidylcholine species similar to those known to decrease the perturbing effects of ethanol in model membrane systems. PMID:23064336

Ethanol production and maximum cell growth are highly correlated with membrane lipid composition during fermentation as determined by lipidomic analysis of 22 Saccharomyces cerevisiae strains.

PubMed

Henderson, Clark M; Lozada-Contreras, Michelle; Jiranek, Vladimir; Longo, Marjorie L; Block, David E

2013-01-01

Optimizing ethanol yield during fermentation is important for efficient production of fuel alcohol, as well as wine and other alcoholic beverages. However, increasing ethanol concentrations during fermentation can create problems that result in arrested or sluggish sugar-to-ethanol conversion. The fundamental cellular basis for these problem fermentations, however, is not well understood. Small-scale fermentations were performed in a synthetic grape must using 22 industrial Saccharomyces cerevisiae strains (primarily wine strains) with various degrees of ethanol tolerance to assess the correlation between lipid composition and fermentation kinetic parameters. Lipids were extracted at several fermentation time points representing different growth phases of the yeast to quantitatively analyze phospholipids and ergosterol utilizing atmospheric pressure ionization-mass spectrometry methods. Lipid profiling of individual fermentations indicated that yeast lipid class profiles do not shift dramatically in composition over the course of fermentation. Multivariate statistical analysis of the data was performed using partial least-squares linear regression modeling to correlate lipid composition data with fermentation kinetic data. The results indicate a strong correlation (R(2) = 0.91) between the overall lipid composition and the final ethanol concentration (wt/wt), an indicator of strain ethanol tolerance. One potential component of ethanol tolerance, the maximum yeast cell concentration, was also found to be a strong function of lipid composition (R(2) = 0.97). Specifically, strains unable to complete fermentation were associated with high phosphatidylinositol levels early in fermentation. Yeast strains that achieved the highest cell densities and ethanol concentrations were positively correlated with phosphatidylcholine species similar to those known to decrease the perturbing effects of ethanol in model membrane systems.

Antifungal mechanisms supporting boric acid therapy of Candida vaginitis.

PubMed

De Seta, Francesco; Schmidt, Martin; Vu, Bao; Essmann, Michael; Larsen, Bryan

2009-02-01

Boric acid is a commonly cited treatment for recurrent and resistant yeast vaginitis, but data about the extent and mechanism of its antifungal activity are lacking. The aim of this study was to use in vitro methods to understand the spectrum and mechanism of boric acid as a potential treatment for vaginal infection. Yeast and bacterial isolates were tested by agar dilution to determine the intrinsic antimicrobial activity of boric acid. Established microbial physiology methods illuminated the mechanism of the action of boric acid against Candida albicans. C. albicans strains (including fluconazole-resistant strains) were inhibited at concentrations attainable intravaginally; as were bacteria. Broth dilution MICs were between 1563 and 6250 mg/L and boric acid proved fungistatic (also reflected by a decrease in CO(2) generation); prolonged culture at 50,000 mg/L was fungicidal. Several organic acids in yeast nitrogen broth yielded a lower pH than equimolar boric acid and sodium borate but were less inhibitory. Cold or anaerobic incubation protected yeast at high boric acid concentrations. Cells maintained integrity for 6 h in boric acid at 37 degrees C, but after 24 h modest intrusion of propidium iodide occurred; loss of plate count viability preceded uptake of vital stain. Growth at sub-MIC concentrations of boric acid decreased cellular ergosterol. The drug efflux pump CDR1 did not protect Candida as CDR1 expression was abrogated by boric acid. Boric acid interfered with the development of biofilm and hyphal transformation. Boric acid is fungistatic to fungicidal depending on concentration and temperature. Inhibition of oxidative metabolism appears to be a key antifungal mechanism, but inhibition of virulence probably contributes to therapeutic efficacy in vivo.

Distance-Based Opportunistic Mobile Data Offloading

PubMed Central

Lu, Xiaofeng; Lio, Pietro; Hui, Pan

2016-01-01

Cellular network data traffic can be offload onto opportunistic networks. This paper proposes a Distance-based Opportunistic Publish/Subscribe (DOPS) content dissemination model, which is composed of three layers: application layer, decision-making layer and network layer. When a user wants new content, he/she subscribes on a subscribing server. Users having the contents decide whether to deliver the contents to the subscriber based on the distance information. If in the meantime a content owner has traveled further in the immediate past time than the distance between the owner and the subscriber, the content owner will send the content to the subscriber through opportunistic routing. Simulations provide an evaluation of the data traffic offloading efficiency of DOPS. PMID:27314361

Distance-Based Opportunistic Mobile Data Offloading.

PubMed

Lu, Xiaofeng; Lio, Pietro; Hui, Pan

2016-06-15

Cellular network data traffic can be offload onto opportunistic networks. This paper proposes a Distance-based Opportunistic Publish/Subscribe (DOPS) content dissemination model, which is composed of three layers: application layer, decision-making layer and network layer. When a user wants new content, he/she subscribes on a subscribing server. Users having the contents decide whether to deliver the contents to the subscriber based on the distance information. If in the meantime a content owner has traveled further in the immediate past time than the distance between the owner and the subscriber, the content owner will send the content to the subscriber through opportunistic routing. Simulations provide an evaluation of the data traffic offloading efficiency of DOPS.

3-bromopyruvate inhibits glycolysis, depletes cellular glutathione, and compromises the viability of cultured primary rat astrocytes.

PubMed

Ehrke, Eric; Arend, Christian; Dringen, Ralf

2015-07-01

The pyruvate analogue 3-bromopyruvate (3-BP) is an electrophilic alkylator that is considered a promising anticancer drug because it has been shown to kill cancer cells efficiently while having little toxic effect on nontumor cells. To test for potential adverse effects of 3-BP on brain cells, we exposed cultured primary rat astrocytes to 3-BP and investigated the effects of this compound on cell viability, glucose metabolism, and glutathione (GSH) content. The presence of 3-BP severely compromised cell viability and slowed cellular glucose consumption and lactate production in a time- and concentration-dependent manner, with half-maximal effects observed at about 100 µM 3-BP after 4 hr of incubation. The cellular hexokinase activity was not affected in 3-BP-treated astrocytes, whereas within 30 min after application of 3-BP the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inhibited, and cellular GSH content was depleted in a concentration-dependent manner, with half-maximal effects observed at about 30 µM 3-BP. The depletion of cellular GSH after exposure to 100 µM 3-BP was not prevented by the presence of 10 mM of the monocarboxylates lactate or pyruvate, suggesting that 3-BP is not taken up into astrocytes predominantly by monocarboxylate transporters. The data suggest that inhibition of glycolysis by inactivation of GAPDH and GSH depletion contributes to the toxicity that was observed for 3-BP-treated cultured astrocytes. © 2014 Wiley Periodicals, Inc.

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties.

PubMed

Farhat, Walid A; Chen, Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Sherman, Christopher; Derwin, Kathleen; Yeger, Herman

2008-06-01

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

Loss of selenium-binding protein 1 decreases sensitivity to clastogens and intracellular selenium content in HeLa cells

USDA-ARS?s Scientific Manuscript database

Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesized that loss of SBP1 modulates cellular selenium content and the response of ...

Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata

Treesearch

Rakesh Minocha; Dale R. Smith; Cathie Reeves; Kevin D. Steele; Subhash C. Minocha

1999-01-01

Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don. During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no...

A Comparison of Biologic Content in Three Elementary-School Science Curriculum Projects: ESS, S-APA, SCIS

ERIC Educational Resources Information Center

Simpson, Ronald D.

1974-01-01

Three elementary school science curriculum projects, Elementary Science Study (ESS), Science - A Process Approach (S-APA), and Science Curriculum Improvement Study (SCIS), are compared concerning the biologic content each project contains. The reviewer found a lack of activities designed to represent functions at the cellular level. Two projects…

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis

PubMed Central

Luna-Tapia, Arturo; Peters, Brian M.; Eberle, Karen E.; Kerns, Morgan E.; Foster, Timothy P.; Marrero, Luis; Noverr, Mairi C.; Fidel, Paul L.

2015-01-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. PMID:26231054

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

PubMed

Luna-Tapia, Arturo; Peters, Brian M; Eberle, Karen E; Kerns, Morgan E; Foster, Timothy P; Marrero, Luis; Noverr, Mairi C; Fidel, Paul L; Palmer, Glen E

2015-10-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric

Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneousmore » leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.« less

Inhibition of 3-Hydroxy-3-Methylglutaryl–Coenzyme A Reductase and Application of Statins as a Novel Effective Therapeutic Approach against Acanthamoeba Infections

PubMed Central

Lorenzo-Morales, Jacob; Machin, Rubén P.; López-Arencibia, Atteneri; García-Castellano, José Manuel; de Fuentes, Isabel; Loftus, Brendan; Maciver, Sutherland K.; Valladares, Basilio; Piñero, José E.

2013-01-01

Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease. PMID:23114753

Impact of nutrient imbalance on wine alcoholic fermentations: nitrogen excess enhances yeast cell death in lipid-limited must.

PubMed

Tesnière, Catherine; Delobel, Pierre; Pradal, Martine; Blondin, Bruno

2013-01-01

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations.

In Vitro Effect of Malachite Green on Candida albicans Involves Multiple Pathways and Transcriptional Regulators UPC2 and STP2

PubMed Central

Dhamgaye, Sanjiveeni; Devaux, Frederic; Manoharlal, Raman; Vandeputte, Patrick; Shah, Abdul Haseeb; Singh, Ashutosh; Blugeon, Corinne; Sanglard, Dominique

2012-01-01

In this study, we show that a chemical dye, malachite green (MG), which is commonly used in the fish industry as an antifungal, antiparasitic, and antibacterial agent, could effectively kill Candida albicans and non-C. albicans species. We have demonstrated that Candida cells are susceptible to MG at a very low concentration (MIC that reduces growth by 50% [MIC50], 100 ng ml−1) and that the effect of MG is independent of known antifungal targets, such as ergosterol metabolism and major drug efflux pump proteins. Transcriptional profiling in response to MG treatment of C. albicans cells revealed that of a total of 207 responsive genes, 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, heat shock, amino acid metabolism, etc., were upregulated, while 37 genes involved in iron acquisition, filamentous growth, mitochondrial respiration, etc., were downregulated. We confirmed experimentally that Candida cells exposed to MG resort to a fermentative mode of metabolism, perhaps due to defective respiration. In addition, we showed that MG triggers depletion of intracellular iron pools and enhances reactive oxygen species (ROS) levels. These effects could be reversed by the addition of iron or antioxidants, respectively. We provided evidence that the antifungal effect of MG is exerted through the transcription regulators UPC2 (regulating ergosterol biosynthesis and azole resistance) and STP2 (regulating amino acid permease genes). Taken together, our transcriptome, genetic, and biochemical results allowed us to decipher the multiple mechanisms by which MG exerts its anti-Candida effects, leading to a metabolic shift toward fermentation, increased generation of ROS, labile iron deprivation, and cell necrosis. PMID:22006003

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S-Adenosylmethionine:Sterol C-24 Methyltransferase.

PubMed

Kaneshiro, Edna S; Johnston, Laura Q; Nkinin, Stephenson W; Romero, Becky I; Giner, José-Luis

2015-01-01

The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild-type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy ((1)H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ(24(28)) -sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

Mechanisms of sterol uptake and transport in yeast.

PubMed

Jacquier, Nicolas; Schneiter, Roger

2012-03-01

Sterols are essential lipid components of eukaryotic membranes. Here we summarize recent advances in understanding how sterols are transported between different membranes. Baker's yeast is a particularly attractive organism to dissect this lipid transport pathway, because cells can synthesize their own major sterol, ergosterol, in the membrane of the endoplasmic reticulum from where it is then transported to the plasma membrane. However, Saccharomyces cerevisiae is also a facultative anaerobic organism, which becomes sterol auxotroph in the absence of oxygen. Under these conditions, cells take up sterol from the environment and transport the lipid back into the membrane of the endoplasmic reticulum, where the free sterol becomes esterified and is then stored in lipid droplets. Steryl ester formation is thus a reliable readout to assess the back-transport of exogenously provided sterols from the plasma membrane to the endoplasmic reticulum. Structure/function analysis has revealed that the bulk membrane function of the fungal ergosterol can be provided by structurally related sterols, including the mammalian cholesterol. Foreign sterols, however, are subject to a lipid quality control cycle in which the sterol is reversibly acetylated. Because acetylated sterols are efficiently excreted from cells, the substrate specificity of the deacetylating enzymes determines which sterols are retained. Membrane-bound acetylated sterols are excreted by the secretory pathway, more soluble acetylated sterol derivatives such as the steroid precursor pregnenolone, on the other hand, are excreted by a pathway that is independent of vesicle formation and fusion. Further analysis of this lipid quality control cycle is likely to reveal novel insight into the mechanisms that ensure sterol homeostasis in eukaryotic cells. Article from a special issue on Steroids and Microorganisms. Copyright © 2010. Published by Elsevier Ltd.

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S-Adenosylmethionine:Sterol C-24 Methyltransferase (SAM:SMT)

PubMed Central

Kaneshiro, Edna S.; Johnston, Laura Q.; Nkinin, Stephenson W.; Romero, Becky I.; Giner, José-Luis

2014-01-01

The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The P. carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography (HPLC) and proton nuclear magnetic resonance spectroscopy (1H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ24(28)-sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii. PMID:25230683

Impact of Nutrient Imbalance on Wine Alcoholic Fermentations: Nitrogen Excess Enhances Yeast Cell Death in Lipid-Limited Must

PubMed Central

Tesnière, Catherine; Delobel, Pierre; Pradal, Martine; Blondin, Bruno

2013-01-01

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations. PMID:23658613

Competitive Fitness of Fluconazole-Resistant Clinical Candida albicans Strains.

PubMed

Popp, Christina; Hampe, Irene A I; Hertlein, Tobias; Ohlsen, Knut; Rogers, P David; Morschhäuser, Joachim

2017-07-01

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1 and Tac1, which result in constitutive overexpression of multidrug efflux pumps, and Upc2, which result in constitutive overexpression of ergosterol biosynthesis genes. However, the deregulated gene expression that is caused by hyperactive forms of these transcription factors also reduces the fitness of the cells in the absence of the drug. To investigate whether fluconazole-resistant clinical C. albicans isolates have overcome the fitness costs of drug resistance, we assessed the relative fitness of C. albicans isolates containing resistance mutations in these transcription factors in competition with matched drug-susceptible isolates from the same patients. Most of the fluconazole-resistant isolates were outcompeted by the corresponding drug-susceptible isolates when grown in rich medium without fluconazole. On the other hand, some resistant isolates with gain-of-function mutations in MRR1 did not exhibit reduced fitness under these conditions. In a mouse model of disseminated candidiasis, three out of four tested fluconazole-resistant clinical isolates did not exhibit a significant fitness defect. However, all four fluconazole-resistant isolates were outcompeted by the matched susceptible isolates in a mouse model of gastrointestinal colonization, demonstrating that the effects of drug resistance on in vivo fitness depend on the host niche. Collectively, our results indicate that the fitness costs of drug resistance in C. albicans are not easily remediated, especially when proper control of gene expression is required for successful adaptation to life within a mammalian host. Copyright © 2017 American Society for Microbiology.

The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

PubMed Central

Poklepovich, Tomas J.; Rinaldi, Mauro A.; Tomazic, Mariela L.; Favale, Nicolas O.; Turkewitz, Aaron P.; Nudel, Clara B.; Nusblat, Alejandro D.

2012-01-01

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. PMID:22982564

Pesticide Residues and Bees – A Risk Assessment

PubMed Central

Sanchez-Bayo, Francisco; Goka, Koichi

2014-01-01

Bees are essential pollinators of many plants in natural ecosystems and agricultural crops alike. In recent years the decline and disappearance of bee species in the wild and the collapse of honey bee colonies have concerned ecologists and apiculturalists, who search for causes and solutions to this problem. Whilst biological factors such as viral diseases, mite and parasite infections are undoubtedly involved, it is also evident that pesticides applied to agricultural crops have a negative impact on bees. Most risk assessments have focused on direct acute exposure of bees to agrochemicals from spray drift. However, the large number of pesticide residues found in pollen and honey demand a thorough evaluation of all residual compounds so as to identify those of highest risk to bees. Using data from recent residue surveys and toxicity of pesticides to honey and bumble bees, a comprehensive evaluation of risks under current exposure conditions is presented here. Standard risk assessments are complemented with new approaches that take into account time-cumulative effects over time, especially with dietary exposures. Whilst overall risks appear to be low, our analysis indicates that residues of pyrethroid and neonicotinoid insecticides pose the highest risk by contact exposure of bees with contaminated pollen. However, the synergism of ergosterol inhibiting fungicides with those two classes of insecticides results in much higher risks in spite of the low prevalence of their combined residues. Risks by ingestion of contaminated pollen and honey are of some concern for systemic insecticides, particularly imidacloprid and thiamethoxam, chlorpyrifos and the mixtures of cyhalothrin and ergosterol inhibiting fungicides. More attention should be paid to specific residue mixtures that may result in synergistic toxicity to bees. PMID:24718419

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway.

PubMed

Xu, Yong-Jie; Singh, Amanpreet; Alter, Gerald M

2016-11-01

Hydroxyurea (HU) has been used for the treatment of multiple diseases, such as cancer. The therapeutic effect is generally believed to be due to the suppression of ribonucleotide reductase (RNR), which slows DNA polymerase movement at replication forks and induces an S phase cell cycle arrest in proliferating cells. Although aberrant mitosis and DNA damage generated at collapsed forks are the likely causes of cell death in the mutants with defects in replication stress response, the mechanism underlying the cytotoxicity of HU in wild-type cells remains poorly understood. While screening for new fission yeast mutants that are sensitive to replication stress, we identified a novel mutation in the erg11 gene encoding the enzyme sterol-14α-demethylase in the ergosterol biosynthesis pathway that dramatically sensitizes the cells to chronic HU treatment. Surprisingly, HU mainly arrests the erg11 mutant cells in cytokinesis, not in S phase. Unlike the reversible S phase arrest in wild-type cells, the cytokinesis arrest induced by HU is relatively stable and occurs at low doses of the drug, which likely explains the remarkable sensitivity of the mutant to HU. We also show that the mutation causes sterol deficiency, which may predispose the cells to the cytokinesis arrest and lead to cell death. We hypothesize that in addition to the RNR, HU may have a secondary unknown target(s) inside cells. Identification of such a target(s) may greatly improve the chemotherapies that employ HU or help to expand the clinical usage of this drug for additional pathological conditions. Copyright © 2016 by the Genetics Society of America.

Inhibitory Effect of Essential Oils on Aspergillus ochraceus Growth and Ochratoxin A Production

PubMed Central

Selvaraj, Jonathan Nimal; Wang, Yan; Zhao, Yueju; Zhou, Lu; Liu, Xiao; Liu, Yang

2014-01-01

Ochratoxin A (OTA) is a mycotoxin which is a common contaminant in grains during storage. Aspergillus ochraceus is the most common producer of OTA. Essential oils play a crucial role as a biocontrol in the reduction of fungal contamination. Essential oils namely natural cinnamaldehyde, cinnamon oil, synthetic cinnamaldehyde, Litsea citrate oil, citral, eugenol, peppermint, eucalyptus, anise and camphor oils, were tested for their efficacy against A. ochraceus growth and OTA production by fumigation and contact assays. Natural cinnamaldehyde proved to be the most effective against A. ochraceus when compared to other oils. Complete fungal growth inhibition was obtained at 150–250 µL/L with fumigation and 250–500 µL/L with contact assays for cinnamon oil, natural and synthetic cinnamaldehyde, L. citrate oil and citral. Essential oils had an impact on the ergosterol biosynthesis and OTA production. Complete inhibition of ergosterol biosynthesis was observed at ≥100 µg/mL of natural cinnamaldehyde and at 200 µg/mL of citral, but total inhibition was not observed at 200 µg/mL of eugenol. But, citral and eugenol could inhibit the OTA production at ≥75 µg/mL and ≥150 µg/mL respectively, while natural cinnamaldehyde couldn’t fully inhibit OTA production at ≤200 µg/mL. The inhibition of OTA by natural cinnamaldehyde is mainly due to the reduction in fungal biomass. However, citral and eugenol could significant inhibit the OTA biosynthetic pathway. Also, we observed that cinnamaldehyde was converted to cinnamic alcohol by A. ochraceus, suggesting that the antimicrobial activity of cinnamaldehyde was mainly attributed to its carbonyl aldehyde group. The study concludes that natural cinnamaldehyde, citral and eugenol could be potential biocontrol agents against OTA contamination in storage grains. PMID:25255251

[Fatty acids composition of cellular lipids of the collected and newly isolated Pseudomonas lupini strains].

PubMed

Hvozdiak, R I; Dankevych, L A; Votselko, S K; Holubets', O V

2005-01-01

Fatty acid composition of cellular lipids of 23 Pseudomonas lupini strains (Beltjukova et Koroljova 1968) has been investigated. Cellular fatty acids which contained from C10 to C19 carbon atoms have been identified. Basic fatty acid of those Pseudomonas cells are hexadecanoic, hexadecenoic and octadecanoic acids. The 3-hydroxydecanoic (C10:0 3OH), 3-hydroxydodecanoic (C12:0 3OH), 2-hydroxydodecanoic (C12:0 2OH) and cyclopropane fatty acids which contain 17 and 19 carbon atoms have been detected in cellular lipids. The cellular fatty acids spectra of 22 P. lupini strains are similar to cellular fatty acids spectrum of the type strain Pseudomonas syringae pv. syringae 8511. Pathogenic isolate 2, which fatty acid content of cell lipids significantly differ from lipids of cell fatty acids from P. lupini strains and cell lipids of fatty acids of typical strains Pseudomonas syringae pv. syringae 8511 and Pseudomonas savastanoi pv. phaseolicola 9066 is the exception.

Field-Based Experiential Learning Using Mobile Devices

NASA Astrophysics Data System (ADS)

Hilley, G. E.

2015-12-01

Technologies such as GPS and cellular triangulation allow location-specific content to be delivered by mobile devices, but no mechanism currently exists to associate content shared between locations in a way that guarantees the delivery of coherent and non-redundant information at every location. Thus, experiential learning via mobile devices must currently take place along a predefined path, as in the case of a self-guided tour. I developed a mobile-device-based system that allows a person to move through a space along a path of their choosing, while receiving information in a way that guarantees delivery of appropriate background and location-specific information without producing redundancy of content between locations. This is accomplished by coupling content to knowledge-concept tags that are noted as fulfilled when users take prescribed actions. Similarly, the presentation of the content is related to the fulfillment of these knowledge-concept tags through logic statements that control the presentation. Content delivery is triggered by mobile-device geolocation including GPS/cellular navigation, and sensing of low-power Bluetooth proximity beacons. Together, these features implement a process that guarantees a coherent, non-redundant educational experience throughout a space, regardless of a learner's chosen path. The app that runs on the mobile device works in tandem with a server-side database and file-serving system that can be configured through a web-based GUI, and so content creators can easily populate and configure content with the system. Once the database has been updated, the new content is immediately available to the mobile devices when they arrive at the location at which content is required. Such a system serves as a platform for the development of field-based geoscience educational experiences, in which students can organically learn about core concepts at particular locations while individually exploring a space.

Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC-MS and electronic nose.

PubMed

Olsson, J; Börjesson, T; Lundstedt, T; Schnürer, J

2002-02-05

Mycotoxin contamination of cereal grains can be detected and quantified using complex extraction procedures and analytical techniques. Normally, the grain odour, i.e. the presence of non-grain volatile metabolites, is used for quality classification of grain. We have investigated the possibility of using fungal volatile metabolites as indicators of mycotoxins in grain. Ten barley samples with normal odour, and 30 with some kind of off-odour were selected from Swedish granaries. The samples were evaluated with regard to moisture content, fungal contamination, ergosterol content, and levels of ochratoxin A (OA) and deoxynivalenol (DON). Volatile compounds were also analysed using both an electronic nose and gas chromatography combined with mass spectrometry (GC-MS). Samples with normal odour had no detectable ochratoxin A and average DON contents of 16 microg kg(-1) (range 0-80), while samples with off-odour had average OA contents of 76 microg kg(-1) (range 0-934) and DON contents of 69 microg kg(-1) (range 0-857). Data were evaluated by multivariate data analysis using projection methods such as principal component analysis (PCA) and partial least squares (PLS). The results show that it was possible to classify the OA level as below or above the maximum limit of 5 microg kg(-1) cereal grain established by the Swedish National Food Administration, and that the DON level could be estimated using PLS. Samples with OA levels below 5 microg kg(-1) had higher concentration of aldehydes (nonanal, 2-hexenal) and alcohols (1-penten-3-ol, 1-octanol). Samples with OA levels above 5 microg kg(-1) had higher concentrations of ketones (2-hexanone, 3-octanone). The GC-MS system predicted OA concentrations with a higher accuracy than the electronic nose, since the GC-MS misclassified only 3 of 37 samples and the electronic nose 7 of 37 samples. No correlation was found between odour and OA level, as samples with pronounced or strong off-odours had OA levels both below and above 5 microg kg(-1). We were able to predict DON levels in the naturally contaminated barley samples using the volatile compounds detected and quantified by either GC-MS or the electronic nose. Pentane, methylpyrazine, 3-pentanone, 3-octene-2-ol and isooctylacetate showed a positive correlation with DON, while ethylhexanol, pentadecane, toluene, 1-octanol, 1-nonanol, and 1-heptanol showed a negative correlation with DON. The root mean square error of estimation values for prediction of DON based on GC-MS and electronic nose data were 16 and 25 microg kg(-1), respectively.

Metabolic Cooperative Control of Electrolyte Levels by Adenosine Triphosphate in the Frog Muscle

PubMed Central

Gulati, J.; Ochsenfeld, M. M.; Ling, G. N.

1971-01-01

This study examines the effects of metabolic inhibitors on the content of cellular K, Na, and adenosine triphosphate (ATP). ATP and K are seen to fall in the inhibited tissues. The ATP content is correlated with the K content. The role of ATP is examined according to a recent biophysical approach. It is suggested that ATP may control the electrolyte levels by inducing conformational changes in the cytoplasmic proteins. PMID:5316285

Distinction of broken cellular wall Ganoderma lucidum spores and G. lucidum spores using FTIR microspectroscopy.

PubMed

Chen, Xianliang; Liu, Xingcun; Sheng, Daping; Huang, Dake; Li, Weizu; Wang, Xin

2012-11-01

In this paper, FTIR microspectroscopy was used to identify broken cellular wall Ganoderma lucidum spores and G. lucidum spores. For IR spectra, broken cellular wall G. lucidum spores and G. lucidum spores were mainly different in the regions of 3000-2800, 1660-1600, 1400-1200 and 1100-1000 cm(-1). For curve fitting, the results showed the differences in the protein secondary structures and the polysaccharide structures/content between broken cellular wall G. lucidum spores and G. lucidum spores. Moreover, the value of A1078/A1741 might be a potentially useful factor to distinguish broken cellular wall G. lucidum spores from G. lucidum spores. Additionally, FTIR microspectroscopy could identify broken cellular wall G. lucidum spores and G. lucidum spores accurately when it was combined with hierarchical cluster analysis. The result suggests FTIR microspectroscopy is very simple and efficient for distinction of broken cellular wall G. lucidum spores and G. lucidum spores. The result also indicates FTIR microspectroscopy may be useful for TCM identification. Copyright © 2012 Elsevier B.V. All rights reserved.

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

PubMed Central

Friedrich, Ralf P; Janko, Christina; Poettler, Marina; Tripal, Philipp; Zaloga, Jan; Cicha, Iwona; Dürr, Stephan; Nowak, Johannes; Odenbach, Stefan; Slabu, Ioana; Liebl, Maik; Trahms, Lutz; Stapf, Marcus; Hilger, Ingrid; Lyer, Stefan; Alexiou, Christoph

2015-01-01

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products. PMID:26170658

Bioaccessibility, uptake, and transport of carotenoids from peppers (Capsicum spp.) using the coupled in vitro digestion and human intestinal Caco-2 cell model.

PubMed

O'Sullivan, Laurie; Jiwan, Marvin A; Daly, Trevor; O'Brien, Nora M; Aherne, S Aisling

2010-05-12

Spanish bell peppers (Capsicum annuum L.) and chili peppers sourced from Kenya and Turkey were analyzed for their carotenoid content, bioaccessibility, and bioavailability. The order of total carotenoid content in peppers and their respective micelles was red > green > yellow. In terms of cellular carotenoid transport as a percentage of original food and micelle content, the order was yellow peppers > green > red; however, the opposite trend was seen for the actual amount of total carotenoids transported by Caco-2 cells. Although lutein was generally the most abundant carotenoid in the micelles (496.3-1565.7 microg 100 g(-1)), cellular uptake and transport of beta-carotene were the highest, 8.3-31.6 and 16.8-42.7%, respectively. Hence, the actual amount of carotenoids present in the original food and respective micelles seems to reflect the amount transported by Caco-2 cells. Therefore, color influenced the carotenoid profile, bioaccessibility, and bioavailability of carotenoids rather than pepper type.

Phenolic contents and cellular antioxidant activity of Chinese hawthorn "Crataegus pinnatifida".

PubMed

Wen, Lingrong; Guo, Xingbo; Liu, Rui Hai; You, Lijun; Abbasi, Arshad Mehmood; Fu, Xiong

2015-11-01

It is evident from various epidemiological studies that consumption of fruits and vegetables is essential to maintain health and in the disease prevention. Present study was designed to examine phenolic contents and antioxidant properties of three varieties of Crataegus pinnatifida (Chinese hawthorn). Shanlihong variety exhibited elevated levels of total phenolics and flavonoid contents, including free and bond phenolics. Procyanidin B2 was most abundant phenolic compound in all samples, followed by epicatechin, chlorogenic acid, hyperoside, and isoquercitrin. The free ORAC values, and free hydro-PSC values were 398.3-555.8 μmol TE/g DW, and 299.1-370.9 μmol VCE/g DW, respectively. Moreover, the free cellular antioxidant activity (CAA) values were 678-1200 μmol of QE/100 g DW in the no PBS wash protocol, and 345.9-532.9 μmol of QE/100 g DW in the PBS wash protocol. C. pinnatifida fruit could be valuable to promote consumer health. Copyright © 2015 Elsevier Ltd. All rights reserved.

Alpha-ketoglutarate and N-acetyl cysteine protect PC12 cells from cyanide-induced cytotoxicity and altered energy metabolism.

PubMed

Satpute, R M; Hariharakrishnan, J; Bhattacharya, R

2008-01-01

Cyanide is a rapidly acting neurotoxin that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia. This results in the dissipation of mitochondrial membrane potential (MMP) accompanied by decreased cellular ATP content which in turn is responsible for increased levels of intracellular calcium ions ([Ca(2+)](i)) and total lactic acid content of the cells. Rat pheochromocytoma (PC12) cells possess much of the biochemical machinery associated with synaptic neurons. In the present study, we evaluated the cytoprotective effects of alpha-ketoglutarate (A-KG) and N-acetylcysteine (NAC) against cyanide-induced cytotoxicity and altered energy metabolism in PC12 cells. Cyanide-antagonism by A-KG is attributed to cyanohydrin formation whereas NAC is known for its antioxidant properties. Data on leakage of intracellular lactate dehydrogenase and mitochondrial function (MTT assay) revealed that simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM) significantly prevented the cytotoxicity of cyanide. Also, cellular ATP content was found to improve, followed by restoration of MMP, intracellular calcium [Ca(2+)](i) and lactic acid levels. Treatment with A-KG and NAC also attenuated the levels of peroxides generated by cyanide. The study indicates that combined administration of A-KG and NAC protected the cyanide-challenged PC12 cells by resolving the altered energy metabolism. The results have implications in the development of new treatment regimen for cyanide poisoning.

High-Content, High-Throughput Screening for the Identification of Cytotoxic Compounds Based on Cell Morphology and Cell Proliferation Markers

PubMed Central

Martin, Heather L.; Adams, Matthew; Higgins, Julie; Bond, Jacquelyn; Morrison, Ewan E.; Bell, Sandra M.; Warriner, Stuart; Nelson, Adam; Tomlinson, Darren C.

2014-01-01

Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays. PMID:24505478

Understanding the cancer cell phenotype beyond the limitations of current omics analyses.

PubMed

Moreno-Sánchez, Rafael; Saavedra, Emma; Gallardo-Pérez, Juan Carlos; Rumjanek, Franklin D; Rodríguez-Enríquez, Sara

2016-01-01

Efforts to understand the mechanistic principles driving cancer metabolism and proliferation have been lately governed by genomic, transcriptomic and proteomic studies. This paper analyzes the caveats of these approaches. As molecular biology's central dogma proposes a unidirectional flux of information from genes to mRNA to proteins, it has frequently been assumed that monitoring the changes in the gene sequences and in mRNA and protein contents is sufficient to explain complex cellular processes. Such a stance commonly disregards that post-translational modifications can alter the protein function/activity and also that regulatory mechanisms enter into action, to coordinate the protein activities of pathways/cellular processes, in order to keep the cellular homeostasis. Hence, the actual protein activities (as enzymes/transporters/receptors) and their regulatory mechanisms ultimately dictate the final outcomes of a pathway/cellular process. In this regard, it is here documented that the mRNA levels of many metabolic enzymes and transcriptional factors have no correlation with the respective protein contents and activities. The validity of current clinical mRNA-based tests and proposed metabolite biomarkers for cancer detection/prognosis is also discussed. Therefore, it is proposed that, to achieve a thorough understanding of the modifications undergone by proliferating cancer cells, it is mandatory to experimentally analyze the cellular processes at the functional level. This could be achieved (a) locally, by examining the actual protein activities in the cell and their kinetic properties (or at least kinetically characterize the most controlling steps of the pathway/cellular process); (b) systemically, by analyzing the main fluxes of the pathway/cellular process, and how they are modulated by metabolites, all which should contribute to comprehending the regulatory mechanisms that have been altered in cancer cells. By adopting a more holistic approach it may become possible to improve the design of therapeutic strategies that would target cancer cells more specifically. © 2015 FEBS.

Sparse feature selection methods identify unexpected global cellular response to strontium-containing materials

PubMed Central

Autefage, Hélène; Littmann, Elena; Hedegaard, Martin A. B.; Von Erlach, Thomas; O’Donnell, Matthew; Burden, Frank R.; Winkler, David A.; Stevens, Molly M.

2015-01-01

Despite the increasing sophistication of biomaterials design and functional characterization studies, little is known regarding cells’ global response to biomaterials. Here, we combined nontargeted holistic biological and physical science techniques to evaluate how simple strontium ion incorporation within the well-described biomaterial 45S5 bioactive glass (BG) influences the global response of human mesenchymal stem cells. Our objective analyses of whole gene-expression profiles, confirmed by standard molecular biology techniques, revealed that strontium-substituted BG up-regulated the isoprenoid pathway, suggesting an influence on both sterol metabolite synthesis and protein prenylation processes. This up-regulation was accompanied by increases in cellular and membrane cholesterol and lipid raft contents as determined by Raman spectroscopy mapping and total internal reflection fluorescence microscopy analyses and by an increase in cellular content of phosphorylated myosin II light chain. Our unexpected findings of this strong metabolic pathway regulation as a response to biomaterial composition highlight the benefits of discovery-driven nonreductionist approaches to gain a deeper understanding of global cell–material interactions and suggest alternative research routes for evaluating biomaterials to improve their design. PMID:25831522

Membrane order in the plasma membrane and endocytic recycling compartment.

PubMed

Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

Mercury-induced biochemical and proteomic changes in rice roots.

PubMed

Chen, Yun-An; Chi, Wen-Chang; Huang, Tsai-Lien; Lin, Chung-Yi; Quynh Nguyeh, Thi Thuy; Hsiung, Yu-Chywan; Chia, Li-Chiao; Huang, Hao-Jen

2012-06-01

Mercury (Hg) is a serious environmental pollution threats to the planet. Accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. We investigated cellular, biochemical and proteomic changes in rice roots under Hg stress. Root growth rate was decreased and Hg, reactive oxygen species (ROS), and malondialdehyde (MDA) content and lipoxygenase activity were increased significantly with increasing Hg concentration in roots. We revealed a time-dependent alteration in total glutathione content and enzymatic activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) during Hg stress. 2-D electrophoresis revealed differential expression of 25 spots with Hg treatment of roots: 14 spots were upregulated and 11 spots downregulated. These differentially expressed proteins were identified by ESI-MS/MS to be involved in cellular functions including redox and hormone homeostasis, chaperone activity, metabolism, and transcription regulation. These results may provide new insights into the molecular basis of the Hg stress response in plants. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms.

PubMed

Simões, Manuel; Pereira, Maria O; Sillankorva, Sanna; Azeredo, Joana; Vieira, Maria J

2007-01-01

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm(2), and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.

Membrane order in the plasma membrane and endocytic recycling compartment

PubMed Central

Iaea, David B.; Maxfield, Frederick R.

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles. PMID:29125865

Semi-Degradable Scaffold for Articular Cartilage Replacement

PubMed Central

Charlton, DC; Peterson, MGE; Spiller, K; Lowman, A; Torzilli, PA; Maher, SA

2009-01-01

The challenge of designing a construct for the repair of focal cartilage defects such that it mimics the mechanical properties of and can integrate with native cartilage has not been met by existing technologies. Herein we describe a novel construct consisting of a non-degradable poly-vinyl alcohol scaffold to provide long-term mechanical stability, interconnected pores to allow for the infiltration of chondrocytes and poly-lactic glycolic acid microspheres for the incorporation of growth factors to enhance cellular migration. The objective of this study was to characterize the morphological features and mechanical properties of our porous PVA-PLGA construct as a function of PLGA content. Varying the PLGA content was found to have a significant effect on the morphological features of the construct. As PLGA content increased from 10 – 75%, samples exhibited a six-fold increase in average percent porosity, an increase in average microsphere diameter from 8 – 34 µm, and an increase in average pore diameter from 29 – 111 µm. The effect of PLGA content on Aggregate Modulus and Permeability was less profound. Our findings suggest that that morphology of the construct can be tailored to optimize cellular infiltration and the dynamic mechanical response. PMID:18333818

Impact of initial lipid content and oxygen supply on alcoholic fermentation in champagne-like musts.

PubMed

Ochando, Thomas; Mouret, Jean-Roch; Humbert-Goffard, Anne; Sablayrolles, Jean-Marie; Farines, Vincent

2017-08-01

Available nitrogen, lipids, or oxygen are nutrients with major impact on the kinetics of winemaking fermentation. Assimilable nitrogen is usually the growth-limiting nutrient which availability determines the fermentation rate and therefore the fermentation duration. In some particular cases, as in Champagne, grape musts have high available nitrogen content and low turbidity, i.e., below 50 Nephelometric Turbidity Unit (NTU). In the case of low turbidity, the availability of lipids, particularly phytosterols, becomes limiting. In this situation, control of oxygenation, which is necessary for lipid synthesis by yeast, is particularly crucial during fermentation. To mimic and understand these situations, a synthetic medium simulating the average composition of a Champagne must was used. This medium contained phytosterol (mainly β-sitosterol) concentrations ranging from 0 to 8mg/L corresponding to turbidity between 10 and 90 NTU. Population reached during the stationary phase and the maximum fermentation rate are conditioned by the initial phytosterol concentration determining the amount of nitrogen consumption. An early loss of viability was observed when the lipid concentrations were very low. For example, the viability continuously decreased during the stationary phase to a final value of 50% for an initial phytosterol concentration of 1mg/L. In some fermentations, 10mg/L oxygen were added at the end of the growth phase to combine the effects of initial content of phytosterols in the musts and the de novo synthesis of ergosterol and unsaturated fatty acids induced by oxygen addition. Effect of oxygen supply on the fermentation kinetics was particularly significant for media with low phytosterol contents. For example, the maximum fermentation rate was increased by 1.4-fold and the fermentation time was 70h shorter with oxygen addition in the medium containing 2mg/L of phytosterols. As a consequence of the oxygen supply, for the media containing 3, 5 and 8mg/L of phytosterols, the assimilable nitrogen was completely exhausted and the fermentation kinetics, as well as the final populations and viabilities (greater than 90%), were identical for the 3 conditions. The impacts of the lipid content and additional oxygen on acetate, glycerol and succinate synthesis were also studied. The phytosterols decreased the acetate and increased the succinate synthesis, and oxygenation resulted in a decrease in succinate formation. This work highlights the similarities and differences between the effects of lipids and oxygen on fermentation kinetics and yeast metabolism. This research highlights the need for an optimal combined management of lipid content in the must via turbidity and oxygenation, particularly in nitrogen-rich musts. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Study on the chemical constituents from Cyathea spinulosa].

PubMed

Jiang, Jian-Shuang; Zhan, Zhi-Lai; Feng, Zi-Ming; Yang, Ya-Nan; Zhang, Pei-Cheng

2012-04-01

To study the chemical constituents from Cyathea spinulosa. Compounds were isolated by chromatographic techniques. Their structures were elucidated by spectral methods. Eight compounds were isolated from the ethanol extract of Cyathea spinulosa and identified as stigmast-4-ene-3,6-dione (1), stigmast-3,6-dione (2), ergosterol (3), protocatechuic aldehyde (4), 1-O-beta-D-glucopyranosyl-(2S,3R,4E,8Z)-2-[(2-hydroxyoctadecanoyl) amido]-4,8- octadecadiene-1,3-diol (5), (2S,3S, 4R)-2-[(2'R) -2'-hydroxytetracosanoylamino]-1,3,4-octadecanetriol (6), beta-sitosterol (7), daucosterol (8). Compounds 1-6 are isolated from this plant for the first time.

Improvement of the ethanol productivity in a high gravity brewing at pilot plant scale.

PubMed

Dragone, Giuliano; Silva, Daniel P; de Almeida e Silva, João Batista; de Almeida Lima, Urgel

2003-07-01

A 23 full factorial design was used to study the influence of different experimental variables, namely wort gravity, fermentation temperature and nutrient supplementation, on ethanol productivity from high gravity wort fermentation by Saccharomyces cerevisiae (lager strain), under pilot plant conditions. The highest ethanol productivity (0.69 g l(-1) h(-1)) was obtained at 20 degrees P [degrees P is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20 degrees C], 15 degrees C, with the addition of 0.8% (w/v) yeast extract, 24 mg l(-1) ergosterol and 0.24% (v/v) Tween 80.

Thielavin B methyl ester: a cytotoxic benzoate trimer from an unidentified fungus (MSX 55526) from the Order Sordariales.

PubMed

Ayers, Sloan; Ehrmann, Brandie M; Adcock, Audrey F; Kroll, David J; Wani, Mansukh C; Pearce, Cedric J; Oberlies, Nicholas H

2011-11-02

As part of our ongoing investigation of filamentous fungi for anticancer leads, an active fungal extract was identified from the Mycosynthetix library (MSX 55526; from the Order Sordariales). Bioactivity-directed fractionation yielded the known ergosterol peroxide (2) and 5α,8α-epidioxyergosta-6,9(11),22-trien-3β-ol(3), and a new benzoate trimer, termed thielavin B methyl ester (1). The structure elucidation of 1 was facilitated by the use of HRMS coupled to an APPI (atmospheric pressure photoionization) source. Compound 1 proved to be moderately active against a panel of three cancer cell lines.

Nanoencapsulated Illicium verum Hook.f. essential oil as an effective novel plant-based preservative against aflatoxin B1 production and free radical generation.

PubMed

Dwivedy, Abhishek Kumar; Singh, Vipin Kumar; Prakash, Bhanu; Dubey, Nawal Kishore

2018-01-01

The study reports efficacy of Illicium verum essential oil (IvEO) against food borne moudls and its nanoencapsulation for enhancing antifungal and antiaflatoxigenic potency. Chemical characterization of the IvEO showed anethole (89.12%) as major compound followed by estragole (4.859%). The IvEO showed broad fungitoxic spectrum against common food borne moulds. It's minimum inhibitory concentration (MIC) and minimum aflatoxin B 1 inhibitory concentration (MAIC) against aflatoxigenic strain Aspergillus flavus LHP-PV-1 were 0.7, and 0.5 μL/mL respectively. Morphological observations of treatment sets by SEM and TEM along with decrease in ergosterol content and enhanced leakage of Ca 2+ , K + and Mg 2+ ions denoted fungal cell membrane as site of action. The IvEO showed promising free radical scavenging activity and favourable safety profile with high LD 50 value on mice. The IvEO also exhibited considerable protection of Pistacia vera from fungal contamination and complete protection from aflatoxin B 1 contamination in storage containers. Nanoencapsulated IvEO in gel form and lyophilized form exhibited enhanced efficacy as fungal inhibitor and aflatoxin suppressor. The chemically characterised IvEO may be recommended as plant based preservative having favourable safety and its nanocapsules may be of industrial significance as shelf life enhancer of food items. This is the first report on in situ antiaflatoxigenic efficacy and nanoencapsulation of IvEO. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antifungal and antiaflatoxigenic properties of Cuminum cyminum (L.) seed essential oil and its efficacy as a preservative in stored commodities.

PubMed

Kedia, Akash; Prakash, Bhanu; Mishra, Prashant K; Dubey, N K

2014-01-03

The study reports potential of Cuminum cyminum (cumin) seed essential oil (EO) as a plant based shelf life enhancer against fungal and aflatoxin contamination and lipid peroxidation. The EO showed efficacy as a preservative in food systems (stored wheat and chickpeas). A total of 1230 fungal isolates were obtained from food samples, with Aspergillus flavus LHP(C)-D6 identified as the highest aflatoxin producer. Cumin seed EO was chemically characterized through GC-MS where cymene (47.08%) was found as the major component. The minimum inhibitory concentration and minimum aflatoxin inhibitory concentration of EO were 0.6 and 0.5 μl/ml respectively. The EO showed toxicity against a broad spectrum of food borne fungi. The antifungal action of EO on ergosterol content in the plasma membrane of A. flavus was determined. The EO showed strong antioxidant potential having IC50 0.092 μl/ml. As a fumigant in food systems, the EO provided sufficient protection of food samples against fungal association without affecting seed germination. In view of the antifungal and antiaflatoxigenic nature, free radical scavenging potential and efficacy in food system, cumin seed EO may be able to provide protection of food commodities against quantitative and qualitative losses, thereby enhancing their shelf life. The present investigation comprises the first report on antifungal mode of action of cumin seed EO and its efficacy as fumigant in food systems. © 2013.

Observational scores of dampness and mold associated with measurements of microbial agents and moisture in three public schools.

PubMed

Cho, S J; Cox-Ganser, J M; Park, J-H

2016-04-01

We examined associations between observational dampness scores and measurements of microbial agents and moisture in three public schools. A dampness score was created for each room from 4-point-scale scores (0-3) of water damage, water stains, visible mold, moldy odor, and wetness for each of 8 room components (ceiling, walls, windows, floor, ventilation, furniture, floor trench, and pipes), when present. We created mixed microbial exposure indices (MMEIs) for each of 121 rooms by summing decile ranks of 8 analytes (total culturable fungi; total, Gram-negative, and Gram-positive culturable bacteria; ergosterol; (1→3)-β-D-glucan; muramic acid; and endotoxin) in floor dust. We found significant (P ≤ 0.01) linear associations between the dampness score and culturable bacteria (total, Gram-positive, and Gram-negative) and the MMEIs. Rooms with dampness scores greater than 0.25 (median) had significantly (P < 0.05) higher levels of most microbial agents, MMEIs, and relative moisture content than those with lower scores (≤0.25). Rooms with reported recent water leaks had significantly (P < 0.05) higher dampness scores than those with historical or no reported water leaks. This study suggests that observational assessment of dampness and mold using a standardized form may be valuable for identifying and documenting water damage and associated microbial contamination. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Aluminium-induced excessive ROS causes cellular damage and metabolic shifts in black gram Vigna mungo (L.) Hepper.

PubMed

Chowra, Umakanta; Yanase, Emiko; Koyama, Hiroyuki; Panda, Sanjib Kumar

2017-01-01

Aluminium-induced oxidative damage caused by excessive ROS production was evaluated in black gram pulse crop. Black gram plants were treated with different aluminium (Al 3+ ) concentrations (10, 50 and 100 μM with pH 4.7) and further the effects of Al 3+ were characterised by means of root growth inhibition, histochemical assay, ROS content analysis, protein carbonylation quantification and 1 H-NMR analysis. The results showed that aluminium induces excessive ROS production which leads to cellular damage, root injury, stunt root growth and other metabolic shifts. In black gram, Al 3+ induces cellular damage at the earliest stage of stress which was characterised from histochemical analysis. From this study, it was observed that prolonged stress can activate certain aluminium detoxification defence mechanism. Probably excessive ROS triggers such defence mechanism in black gram. Al 3+ can induce excessive ROS initially in the root region then transported to other parts of the plant. As much as the Al 3+ concentration increases, the rate of cellular injury and ROS production also increases. But after 72 h of stress, plants showed a lowered ROS level and cellular damage which indicates the upregulation of defensive mechanisms. Metabolic shift analysis also showed that the black gram plant under stress has less metabolic content after 24 h of treatment, but gradually, it was increased after 72 h of treatment. It was assumed that ROS played the most important role as a signalling molecule for aluminium stress in black gram.

Quercetin attenuates toosendanin-induced hepatotoxicity through inducing the Nrf2/GCL/GSH antioxidant signaling pathway.

PubMed

Jin, Yao; Huang, Zhen-Lin; Li, Li; Yang, Yang; Wang, Chang-Hong; Wang, Zheng-Tao; Ji, Li-Li

2018-06-19

Toosendanin (TSN) is the main active compound in Toosendan Fructus and Meliae Cortex, two commonly used traditional Chinese medicines. TSN has been reported to induce hepatotoxicity, but its mechanism remains unclear. In this study, we demonstrated the critical role of nuclear factor erythroid 2-related factor 2 (Nrf2) in protecting against TSN-induced hepatotoxicity in mice and human normal liver L-02 cells. In mice, administration of TSN (10 mg/kg)-induced acute liver injury evidenced by increased serum alanine/aspartate aminotransferase (ALT/AST) and alkaline phosphatase (ALP) activities, and total bilirubin (TBiL) content as well as the histological changes. Furthermore, TSN markedly increased liver reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and decreased liver glutathione (GSH) content and Nrf2 expression. In L-02 cells, TSN (2 μM) time-dependently reduced glutamate-cysteine ligase (GCL) activity and cellular expression of the catalytic/modify subunit of GCL (GCLC/GCLM). Moreover, TSN reduced cellular GSH content and the increased ROS formation, and time-dependently decreased Nrf2 expression and increased the expression of the Nrf2 inhibitor protein kelch-like ECH-associated protein-1 (Keap1). Pre-administration of quercetin (40, 80 mg/kg) effectively inhibited TSN-induced liver oxidative injury and reversed the decreased expression of Nrf2 and GCLC/GCLM in vivo and in vitro. In addition, the quercetin-provided protection against TSN-induced hepatotoxicity was diminished in Nrf2 knock-out mice. In conclusion, TSN decreases cellular GSH content by reducing Nrf2-mediated GCLC/GCLM expression via decreasing Nrf2 expression. Quercetin attenuates TSN-induced hepatotoxicity by inducing the Nrf2/GCL/GSH antioxidant signaling pathway. This study implies that inducing Nrf2 activation may be an effective strategy to prevent TSN-induced hepatotoxicity.

Honey Extracted Polyphenolics Reduce Experimental Hypoxia in Human Keratinocytes Culture.

PubMed

Chaudhary, Amrita; Bag, Swarnendu; Banerjee, Provas; Chatterjee, Jyotirmoy

2017-05-03

Hypoxic assault affects fundamental cellular processes and generates oxidative stress on healthy cells/molecules. Honey extracted polyphenolics (HEP) as a natural antioxidant reduced hypoxic cytotoxicity in this study. Different honey samples were physicochemically characterized to identify preferred (jamun) honey [pH 3.55 ± 0.04, conductivity (μs/cm) = 6.66 ± 0.14, water content % (w/w) = 14.70 ± 0.35, total solid content % (w/w) = 85.30 ± 0.35, phenol content (mg GAE/100 g) = 403.55 ± 0.35, flavonoid content (mg QE/100 g) = 276.76 ± 4.10, radical scavenging activity (% 500 μL) = 147.75 ± 3.13, catalase activity (absorbance at 620 nm) = 0.226 ± 0.01]. HEP was tested in different doses on hypoxic and normoxic cells (HaCaT) using viability and antioxidant assays. Cardinal molecular expressions such as cadherin-catenin-cytoskeleton complex (namely, E-cadherin, β-catenin, and F-actin), hypoxia marker (Hif 1 α), proliferation marker (Ki67), and epithelial master regulator (p63) were studied by immuno-cytochemisty (ICC) and qRT-PCR. The 0.063 mg/mL HEP demonstrated better vitality and functionality of HaCaT cells as per viability assay (*, P < 0.01) even under hypoxia. ICC and qRT-PCR observations indicated restoration of cellular survival and homeostasis under 0.063 mg/mL HEP after hypoxic assault. Furthermore, major spectral changes for nucleic acid and membrane phospholipid reorganizations by Fourier transform infrared spectroscopy illustrated a positive impact of 0.063 mg/mL HEP on hypoxic cells considering proliferation and cellular integrity. It was concluded that a specific dose of jamun HEP reduces hypoxic cytotoxicity.

Robust Classification of Small-Molecule Mechanism of Action Using a Minimalist High-Content Microscopy Screen and Multidimensional Phenotypic Trajectory Analysis

PubMed Central

Twarog, Nathaniel R.; Low, Jonathan A.; Currier, Duane G.; Miller, Greg; Chen, Taosheng; Shelat, Anang A.

2016-01-01

Phenotypic screening through high-content automated microscopy is a powerful tool for evaluating the mechanism of action of candidate therapeutics. Despite more than a decade of development, however, high content assays have yielded mixed results, identifying robust phenotypes in only a small subset of compound classes. This has led to a combinatorial explosion of assay techniques, analyzing cellular phenotypes across dozens of assays with hundreds of measurements. Here, using a minimalist three-stain assay and only 23 basic cellular measurements, we developed an analytical approach that leverages informative dimensions extracted by linear discriminant analysis to evaluate similarity between the phenotypic trajectories of different compounds in response to a range of doses. This method enabled us to visualize biologically-interpretable phenotypic tracks populated by compounds of similar mechanism of action, cluster compounds according to phenotypic similarity, and classify novel compounds by comparing them to phenotypically active exemplars. Hierarchical clustering applied to 154 compounds from over a dozen different mechanistic classes demonstrated tight agreement with published compound mechanism classification. Using 11 phenotypically active mechanism classes, classification was performed on all 154 compounds: 78% were correctly identified as belonging to one of the 11 exemplar classes or to a different unspecified class, with accuracy increasing to 89% when less phenotypically active compounds were excluded. Importantly, several apparent clustering and classification failures, including rigosertib and 5-fluoro-2’-deoxycytidine, instead revealed more complex mechanisms or off-target effects verified by more recent publications. These results show that a simple, easily replicated, minimalist high-content assay can reveal subtle variations in the cellular phenotype induced by compounds and can correctly predict mechanism of action, as long as the appropriate analytical tools are used. PMID:26886014

Robust Classification of Small-Molecule Mechanism of Action Using a Minimalist High-Content Microscopy Screen and Multidimensional Phenotypic Trajectory Analysis.

PubMed

Twarog, Nathaniel R; Low, Jonathan A; Currier, Duane G; Miller, Greg; Chen, Taosheng; Shelat, Anang A

2016-01-01

Phenotypic screening through high-content automated microscopy is a powerful tool for evaluating the mechanism of action of candidate therapeutics. Despite more than a decade of development, however, high content assays have yielded mixed results, identifying robust phenotypes in only a small subset of compound classes. This has led to a combinatorial explosion of assay techniques, analyzing cellular phenotypes across dozens of assays with hundreds of measurements. Here, using a minimalist three-stain assay and only 23 basic cellular measurements, we developed an analytical approach that leverages informative dimensions extracted by linear discriminant analysis to evaluate similarity between the phenotypic trajectories of different compounds in response to a range of doses. This method enabled us to visualize biologically-interpretable phenotypic tracks populated by compounds of similar mechanism of action, cluster compounds according to phenotypic similarity, and classify novel compounds by comparing them to phenotypically active exemplars. Hierarchical clustering applied to 154 compounds from over a dozen different mechanistic classes demonstrated tight agreement with published compound mechanism classification. Using 11 phenotypically active mechanism classes, classification was performed on all 154 compounds: 78% were correctly identified as belonging to one of the 11 exemplar classes or to a different unspecified class, with accuracy increasing to 89% when less phenotypically active compounds were excluded. Importantly, several apparent clustering and classification failures, including rigosertib and 5-fluoro-2'-deoxycytidine, instead revealed more complex mechanisms or off-target effects verified by more recent publications. These results show that a simple, easily replicated, minimalist high-content assay can reveal subtle variations in the cellular phenotype induced by compounds and can correctly predict mechanism of action, as long as the appropriate analytical tools are used.

A simple and fast method for fixation of cultured cell lines that preserves cellular structures containing gamma-tubulin.

PubMed

Alvarado-Kristensson, Maria

2018-01-01

When using fluorescence microscope techniques to study cells, it is essential that the cell structure and contents are preserved after preparation of the samples, and that the preparation method employed does not create artefacts that can be perceived as cellular structure/components. γ-Tubulin forms filaments that in some cases are immunostained with an anti-γ-tubulin antibody, but this immunostaining is not reproducible [[1], [2

Using cancer to make cellular reproduction rigorous and relevant

NASA Astrophysics Data System (ADS)

Duncan, Cynthia F.

The 1983 report Nation at Risk highlighted the fact that test scores of American students were far below that of competing nations and educational standards were being lowered. This trend has continued and studies have also shown that students are not entering college ready for success. This trend can be reversed. Students can better understand and retain biology content expectations if they are taught in a way that is both rigorous and relevant. In the past, students have learned the details of cellular reproduction with little knowledge of why it is important to their everyday lives. This material is learned only for the test. Knowing the details of cellular reproduction is crucial for understanding cancer. Cancer is a topic that will likely affect all of my students at some point in their lives. Students used hands on activities, including simulations, labs, and models to learn about cellular reproduction with cancer as a theme throughout. Students were challenged to learn how to use the rigorous biology content expectations to think about cancer, including stem cell research. Students that will some day be college students, voting citizens, and parents, will become better learners. Students were assessed before and after the completion of the unit to determine if learning occurs. Students did learn the material and became more critical thinkers. Statistical analysis was completed to insure confidence in the results.

Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laszlo, A.; Radke, K.; Chin, S.

1981-10-01

We have investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the synthesis and modification of polypeptides in normal avian cells and cells infected by wild-type and temperature-sensitive Rous sarcoma virus (RSV). Using two-dimensional gel electrophoresis, we have detected alterations in both the abundance of cellular polypeptides and in their phosphorylation that seem unique to TPA treatment. However, the state of phosphorylation of the major putative substrate for the action of the src gene-associated protein kinase, the 34- to 36-kilodalton protein, was not altered. Moreover, examination of the phosphorylated amino acid content of total cellular phosphoproteins revealed that the response tomore » TPA was not associated with detectable increases in their phosphotyrosine content. These results make it unlikely that TPA acts by the activation of the phosphorylating activity of the cellular proto-src gene or by the activation of other cellular phosphotyrosine-specific kinases. We have shown previously that temperature-sensitive RSV-infected cells at nonpermissive temperature demonstrate an increased sensitivity to TPA treatment (Bissell, M.J., Hatie, C. and Calfin, M. (1979) Proc. Natl. Acad. Sci. USA 76, 348-352). Our present results indicate that this is not due to reactivation of the phosphorylating activity of the defective src gene product or to its leakiness, and they lend support to the notion of multistep viral carcinogenesis.« less

In vitro evaluation of cellular responses induced by ZnO nanoparticles, zinc ions and bulk ZnO in fish cells.

PubMed

Fernández, Dolores; García-Gómez, Concepción; Babín, Mar

2013-05-01

Zinc oxide nanoparticles (ZnO-NPs) are inevitably released into the environment and are potentially dangerous for aquatic life. However, the potential mechanisms of cytotoxicity of zinc nanoparticles remain unclear. Studying the toxicity of ZnO-NPs with In vitro systems will help to determine their interactions with cellular biomolecules. The aim of this study was to evaluate the cytotoxic potentials of ZnO-NPs in established fish cell lines (RTG-2, RTH-149 and RTL-W1) and compare them with those of bulk ZnO and Zn(2+) ions. Membrane function (CFDA-AM assay), mitochondrial function (MTT assay), cell growth (KBP assay), cellular stress (β-galactosidase assay), reductase enzyme activity (AB assay), reactive oxygen species (ROS), total glutathione cellular content (tGSH assay) and glutathione S-transferase (GST) activities were assessed for all cell lines. ZnO-NPs cytotoxicity was greater than those of bulk ZnO and Zn(2+). ZnO-NPs induced oxidative stress is dependent on their dose. Low cost tests, such as CFDA-AM, ROS, GST activity and tGSH cell content test that use fish cell lines, may be used to detect oxidative stress and redox status changes. Particle dissolution of the ZnO-NPs did not appear to play an important role in the observed toxicity in this study. Published by Elsevier B.V.

Mechanisms for Variation of Cellular P Stoichiometry: Diverse Cellular Phosphorus Allocation Strategies Across Microbial Groups from the Sargasso Sea

NASA Astrophysics Data System (ADS)

Popendorf, K.; Duhamel, S.

2016-02-01

Phosphorus is the least abundant of the three major macronutrients that define the canonical Redfield ratio, but its place in the backbone of nucleic acids and as an energy trafficking molecule lays a lower bound of cellular phosphorus content that is essential for all life. In addition to forming DNA, RNA, and adenosine triphosphate (ATP), significant amounts of cellular phosphorus may also be allocated to the production of phospholipids and polyphosphate. These latter two biochemicals in particular may occur in significant but highly variable amounts across different microbial groups, and the variation in cellular allocation to these biochemicals may be a contributing factor in defining the elemental stoichiometry of microbes. We investigated this variation in cellular phosphorus allocation across the most abundant microbial groups in the P-depleted Sargasso Sea: Prochlorococcus, Synechococcus, and heterotrophic bacteria. By coupling radioisotope tracing of phosphate and ATP with cell sorting flow cytometry and subsequent biochemical extractions, we made novel measurements of the P allocation to DNA, phospholipids, and polyphosphate in individual microbial groups from environmental populations. These results provide new insights into the cellular mechanisms of variation in stoichiometry and different microbial strategies for adaptation to low-P environments.

Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

NASA Astrophysics Data System (ADS)

Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

2016-03-01

Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint.

PubMed

Yamamori, Tohru; Yasui, Hironobu; Yamazumi, Masayuki; Wada, Yusuke; Nakamura, Yoshinari; Nakamura, Hideo; Inanami, Osamu

2012-07-15

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase. Copyright © 2012 Elsevier Inc. All rights reserved.

Acute β-Hydroxy-β-Methyl Butyrate Suppresses Regulators of Mitochondrial Biogenesis and Lipid Oxidation While Increasing Lipid Content in Myotubes.

PubMed

Schnuck, Jamie K; Johnson, Michele A; Gould, Lacey M; Gannon, Nicholas P; Vaughan, Roger A

2016-10-01

Leucine modulates synthetic and degradative pathways in muscle, possibly providing metabolic benefits for both athletes and diseased populations. Leucine has become popular among athletes for improving performance and body composition, however little is known about the metabolic effects of the commonly consumed leucine-derived metabolite β-hydroxy-β-methyl butyrate (HMB). Our work measured the effects of HMB on metabolic protein expression, mitochondrial content and metabolism, as well as lipid content in skeletal muscle cells. Specifically, cultured C2C12 myotubes were treated with either a control or HMB ranging from 6.25 to 25 μM for 24 h and mRNA and/or protein expression, oxygen consumption, glucose uptake, and lipid content were measured. Contrary to leucine's stimulatory effect on metabolism, HMB-treated cells exhibited significantly reduced regulators of lipid oxidation including peroxisome proliferator-activated receptor alpha (PPARα) and PPARβ/δ, as well as downstream target carnitine palmitoyl transferase, without alterations in glucose or palmitate oxidation. Furthermore, HMB significantly inhibited activation of the master regulator of energetics, AMP-activated protein kinase. As a result, HMB-treated cells also displayed reduced total mitochondrial content compared with true control or cells equivocally treated with leucine. Additionally, HMB treatment amplified markers of lipid biosynthesis (PPARγ and fatty acid synthase) as well as consistently promoted elevated total lipid content versus control cells. Collectively, our results demonstrate that HMB did not improve mitochondrial metabolism or content, and may promote elevated cellular lipid content possibly through heightened PPARγ expression. These observations suggest that HMB may be most beneficial for populations interested in stimulating anabolic cellular processes.

Comparison of cellular toxicity between multi-walled carbon nanotubes and onion-like shell-shaped carbon nanoparticles

NASA Astrophysics Data System (ADS)

Kang, Seunghyon; Kim, Ji-Eun; Kim, Daegyu; Woo, Chang Gyu; Pikhitsa, Peter V.; Cho, Myung-Haing; Choi, Mansoo

2015-09-01

The cellular toxicity of multi-walled carbon nanotubes (MWCNTs) and onion-like shell-shaped carbon nanoparticles (SCNPs) was investigated by analyzing the comparative cell viability. For the reasonable comparison, physicochemical characteristics were controlled thoroughly such as crystallinity, carbon bonding characteristic, hydrodynamic diameter, and metal contents of the particles. To understand relation between cellular toxicity of the particles and generation of reactive oxygen species (ROS), we measured unpaired singlet electrons of the particles and intracellular ROS, and analyzed cellular toxicity with/without the antioxidant N-acetylcysteine (NAC). Regardless of the presence of NAC, the cellular toxicity of SCNPs was found to be lower than that of MWCNTs. Since both particles show similar crystallinity, hydrodynamic size, and Raman signal with negligible contribution of remnant metal particles, the difference in cell viability would be ascribed to the difference in morphology, i.e., spherical shape (aspect ratio of one) for SCNP and elongated shape (high aspect ratio) for MWCNT.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries.

PubMed

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-09-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries

PubMed Central

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-01-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development. PMID:18544560

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, F.A.; Gray, J.W.

1983-10-18

A method for the simultaneous flow cylometric measurement of total cellular DNA content and of the uptake of DNA precursors as a measure of DNA synthesis during various phases of the cell cycle in normal and malignant cells in vitro and in vivo is described. The method comprises reacting cells with labelled halodeoxyuridine (HdU), partially denaturing cellular DNA, adding to the reaction medium monoclonal antibodies (mabs) reactive with HdU, reacting the bound mabs with a second labelled antibody, incubating the mixture with a DNA stain, and measuring simultaneously the intensity of the DNA stain as a measure of the total cellular DNA and the HdU incorporated as a measure of DNA synthesis. (ACR)

Compartmental genomics in living cells revealed by single-cell nanobiopsy.

PubMed

Actis, Paolo; Maalouf, Michelle M; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R Adam; Pourmand, Nader

2014-01-28

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

Effect of omega-3 polyunsaturated fatty acids on the cytoskeleton: an open-label intervention study.

PubMed

Schmidt, Simone; Willers, Janina; Riecker, Sabine; Möller, Katharina; Schuchardt, Jan Philipp; Hahn, Andreas

2015-02-14

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) show beneficial effects on cardiovascular health and cognitive functions, but the underlying molecular mechanisms are not completely understood. Because of the fact that cytoskeleton dynamics affect almost every cellular process, the regulation of cytoskeletal dynamics could be a new pathway by which n-3 PUFAs exert their effects on cellular level. A 12-week open-label intervention study with 12 healthy men was conducted to determine the effects of 2.7 g/d n-3 PUFA on changes in mRNA expression of cytoskeleton-associated genes by quantitative real-time PCR in whole blood. Furthermore, the actin content in red blood cells was analyzed by immunofluorescence imaging. N-3 PUFA supplementation resulted in a significant down-regulation of cytoskeleton-associated genes, in particular three GTPases (RAC1, RHOA, CDC42), three kinases (ROCK1, PAK2, LIMK), two Wiskott-Aldrich syndrome proteins (WASL, WASF2) as well as actin related protein 2/3 complex (ARPC2, ARPC3) and cofilin (CFL1). Variability in F-actin content between subjects was high; reduced actin content was only reduced within group evaluation. Reduced cytoskeleton-associated gene expression after n-3 PUFA supplementation suggests that regulation of cytoskeleton dynamics might be an additional way by which n-3 PUFAs exert their cellular effects. Concerning F-actin, this analysis did not reveal unmistakable results impeding a generalized conclusion.

Multiscale modelling of Flow-Induced Blood Cell Damage

NASA Astrophysics Data System (ADS)

Liu, Yaling; Sohrabi, Salman

2017-11-01

We study red blood cell (RBC) damage and hemolysis at cellular level. Under high shear rates, pores form on RBC membranes through which hemoglobin (Hb) leaks out and increases free Hb content of plasma leading to hemolysis. By coupling lattice Boltzmann and spring connected network models through immersed boundary method, we estimate hemolysis of a single RBC under various shear rates. The developed cellular damage model can be used as a predictive tool for hydrodynamic and hematologic design optimization of blood-wetting medical devices.

Hypoxia decreases creatine uptake in cardiomyocytes, while creatine supplementation enhances HIF activation.

PubMed

Santacruz, Lucia; Arciniegas, Antonio Jose Luis; Darrabie, Marcus; Mantilla, Jose G; Baron, Rebecca M; Bowles, Dawn E; Mishra, Rajashree; Jacobs, Danny O

2017-08-01

Creatine (Cr), phosphocreatine (PCr), and creatine kinases (CK) comprise an energy shuttle linking ATP production in mitochondria with cellular consumption sites. Myocytes cannot synthesize Cr: these cells depend on uptake across the cell membrane by a specialized creatine transporter (CrT) to maintain intracellular Cr levels. Hypoxia interferes with energy metabolism, including the activity of the creatine energy shuttle, and therefore affects intracellular ATP and PCr levels. Here, we report that exposing cultured cardiomyocytes to low oxygen levels rapidly diminishes Cr transport by decreasing V max and K m Pharmacological activation of AMP-activated kinase (AMPK) abrogated the reduction in Cr transport caused by hypoxia. Cr supplementation increases ATP and PCr content in cardiomyocytes subjected to hypoxia, while also significantly augmenting the cellular adaptive response to hypoxia mediated by HIF-1 activation. Our results indicate that: (1) hypoxia reduces Cr transport in cardiomyocytes in culture, (2) the cytoprotective effects of Cr supplementation are related to enhanced adaptive physiological responses to hypoxia mediated by HIF-1, and (3) Cr supplementation increases the cellular ATP and PCr content in RNCMs exposed to hypoxia. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

MicroRNA Profiling of Sendai Virus-Infected A549 Cells Identifies miR-203 as an Interferon-Inducible Regulator of IFIT1/ISG56

PubMed Central

Buggele, William A.

2013-01-01

The mammalian type I interferon (IFN) response is a primary barrier for virus infection and is essential for complete innate and adaptive immunity. Both IFN production and IFN-mediated antiviral signaling are the result of differential cellular gene expression, a process that is tightly controlled at transcriptional and translational levels. To determine the potential for microRNA (miRNA)-mediated regulation of the antiviral response, small-RNA profiling was used to analyze the miRNA content of human A549 cells at steady state and following infection with the Cantell strain of Sendai virus, a potent inducer of IFN and cellular antiviral responses. While the miRNA content of the cells was largely unaltered by infection, specific changes in miRNA abundance were identified during Sendai virus infection. One miRNA, miR-203, was found to accumulate in infected cells and in response to IFN treatment. Results indicate that miR-203 is an IFN-inducible miRNA that can negatively regulate a number of cellular mRNAs, including an IFN-stimulated gene target, IFIT1/ISG56, by destabilizing its mRNA transcript. PMID:23785202

Energy metabolism of intervertebral disc under mechanical loading.

PubMed

Wang, Chong; Gonzales, Silvia; Levene, Howard; Gu, Weiyong; Huang, Chun-Yuh Charles

2013-11-01

Intervertebral disc (IVD) degeneration is closely associated with low back pain (LBP), which is a major health concern in the U.S. Cellular biosynthesis of extracellular matrix (ECM), which is important for maintaining tissue integrity and preventing tissue degeneration, is an energy demanding process. Due to impaired nutrient support in avascular IVD, adenosine triphosphate (ATP) supply could be a limiting factor for maintaining normal ECM synthesis. Therefore, the objective of this study was to investigate the energy metabolism in the annulus fibrosus (AF) and nucleus pulposus (NP) of porcine IVD under static and dynamic compressions. Under compression, pH decreased and the contents of lactate and ATP increased significantly in both AF and NP regions, suggesting that compression can promote ATP production via glycolysis and reduce pH by increasing lactate accumulation. A high level of extracellular ATP content was detected in the NP region and regulated by compressive loading. Since ATP can serve not only as an intra-cellular energy currency, but also as a regulator of a variety of cellular activities extracellularly through the purinergic signaling pathway, our findings suggest that compression-mediated ATP metabolism could be a novel mechanobiological pathway for regulating IVD metabolism. © 2013 Orthopaedic Research Society.

Comparative cyto-histological study of needle tip aspirates and entry sites after intravitreal injection using different needle types

PubMed Central

Lytvynchuk, Lyubomyr; Sergienko, Andrij; Savytska, Iryna; Albert, Réka; Glittenberg, Carl; Binder, Susanne; Petrovski, Goran

2017-01-01

A comparison of the cellular content of needle tip aspirates and entry sites after transconjunctival intravitreal injection (IVI) using different needle types was performed. White outbred rats and human cadaver eyes were used for IVI by hypodermic 27 gauge (G) and 30G needles, and spinal anesthesia Pencan 27G needles. Aspiration of vitreous for quantitative morphological and cell cultivation analysis, as well as cyto-histological analysis of aspirates and entry sites were carried out. The most common cells in the aspirates from all needle types were conjunctival epithelial-, ciliary body non-pigmented epithelial- and sclerocyte-like cells and granular proteins. Crystallized vitreous specimens were present in each aspirate. The entry sites of hypodermic needles showed marked trauma in all wall layers of rat and human eyes accompanied by cellular destruction and hemorrhages. Pencan 27G needle caused less tissue trauma with partial reposition of sclerocytes. Transconjunctival IVIs with hypodermic 27G and 30G, and Pencan 27G needles result in trauma of all layers of the eyeball. The possible consequences of cellular content being cut and injected into the eye, as well as the entry site wound shape deserve future consideration and improvements. PMID:28692684

Comparative effect of chronic bombesin, gastrin-releasing peptide and caerulein on the rat pancreas.

PubMed

Damgé, C; Hajri, A; Lhoste, E; Aprahamian, M

1988-02-01

This study was designed to compare, on a molar basis, the effect of chronic bombesin, gastrin-releasing peptide (GRP) and caerulein on pancreatic growth in the rat. These 3 peptides were administered s.c. 3 times daily for 4 days at the following concentrations: 0.036, 0.36, 3.6 and 7.2 nmol/kg of body weight. Bombesin and GRP induced pancreatic growth in a dose-dependent manner from 3.6 nmol/kg. This growth was characterized by an increase in pancreatic weight, its protein and RNA contents but not in DNA content suggesting cellular hypertrophy. Caerulein exerted a biphasic effect on pancreatic growth, inducing cellular hypertrophy at low doses since 0.36 nmol/kg and atrophy with the highest dose (7.2 nmol/kg). Bombesin and caerulein (until 3.6 nmol/kg) increased the pancreatic content in chymotrypsin more than in amylase. The 7.2 nmol/kg caerulein treatment depressed all enzyme activities while the same dose of GRP increased pancreatic lipase content. It is concluded that (1) bombesin and GRP are equipotent trophic factors for the pancreas; (2) caerulein is the most potent factor and exerts a biphasic effect on pancreatic growth; (3) pancreatic growth and synthesis and/or secretion of enzymes are not regulated through the same mechanism.

The development of high-content screening (HCS) technology and its importance to drug discovery.

PubMed

Fraietta, Ivan; Gasparri, Fabio

2016-01-01

High-content screening (HCS) was introduced about twenty years ago as a promising analytical approach to facilitate some critical aspects of drug discovery. Its application has spread progressively within the pharmaceutical industry and academia to the point that it today represents a fundamental tool in supporting drug discovery and development. Here, the authors review some of significant progress in the HCS field in terms of biological models and assay readouts. They highlight the importance of high-content screening in drug discovery, as testified by its numerous applications in a variety of therapeutic areas: oncology, infective diseases, cardiovascular and neurodegenerative diseases. They also dissect the role of HCS technology in different phases of the drug discovery pipeline: target identification, primary compound screening, secondary assays, mechanism of action studies and in vitro toxicology. Recent advances in cellular assay technologies, such as the introduction of three-dimensional (3D) cultures, induced pluripotent stem cells (iPSCs) and genome editing technologies (e.g., CRISPR/Cas9), have tremendously expanded the potential of high-content assays to contribute to the drug discovery process. Increasingly predictive cellular models and readouts, together with the development of more sophisticated and affordable HCS readers, will further consolidate the role of HCS technology in drug discovery.

Mild intermittent hypoxemia in neonatal mice causes permanent neurofunctional deficit and white matter hypomyelination.

PubMed

Juliano, Courtney; Sosunov, Sergey; Niatsetskaya, Zoya; Isler, Joseph A; Utkina-Sosunova, Irina; Jang, Isaac; Ratner, Veniamin; Ten, Vadim

2015-02-01

Very low birth weight (VLBW) premature infants experience numerous, often self-limited non-bradycardic episodes of intermittent hypoxemia (IH). We hypothesized that these episodes of IH affect postnatal white matter (WM) development causing hypomyelination and neurological handicap in the absence of cellular degeneration. Based on clinical data from ten VLBW neonates; a severity, daily duration and frequency of non-bradycardic IH episodes were reproduced in neonatal mice. Changes in heart rate and cerebral blood flow during IH were recorded. A short-term and long-term neurofunctional performance, cerebral content of myelin basic protein (MBP), 2'3' cyclic-nucleotide 3-phosphodiesterase (CNPase), electron microscopy of axonal myelination and the extent of cellular degeneration were examined. Neonatal mice exposed to IH exhibited no signs of cellular degeneration, yet demonstrated significantly poorer olfactory discrimination, wire holding, beam and bridge crossing, and walking-initiation tests performance compared to controls. In adulthood, IH-mice demonstrated no alteration in navigational memory. However, sensorimotor performance on rota-rod, wire-holding and beam tests was significantly worse compared to naive littermates. Both short- and long-term neurofunctional deficits were coupled with decreased MBP, CNPase content and poorer axonal myelination compared to controls. In neonatal mice mild, non-ischemic IH stress, mimicking that in VLBW preterm infants, replicates a key phenotype of non-cystic WM injury: permanent hypomyelination and sensorimotor deficits. Because this phenotype has developed in the absence of cellular degeneration, our data suggest that cellular mechanisms of WM injury induced by mild IH differ from that of cystic periventricular leukomalacia where the loss of myelin-producing cells and axons is the major mechanism of injury. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of age and the use of hands-free cellular phones on driving behavior and task performance.

PubMed

Liu, Yung-Ching; Ou, Yang-Kun

2011-12-01

This study used a driving simulator to investigate the effect of using a Bluetooth hands-free cellular phone earpiece on the driving behavior of two age groups. Forty-eight participants (24 aged 20-26 and 24 aged 65-73) were examined to assess their performance on the following divided-attention tasks under 2 driving load conditions (high and low): (1) attempting to maintain the speed limit and (2) using a cellular phone while driving. The length of the call conversation (long vs. short) and the conversational content (complex vs. simple) were manipulated as within-subject independent variables. The driving behavior of the participants, their task reaction times and accuracy, and subjective ratings were collected as dependent variables. The results indicate that under low driving loads, short talk times, and simple conversational content, the driving behavior of the participants showed low variance in the vehicle's mean speed. In contrast, complex conversation had a significantly negative impact on driving behavior. Notably, under a low driving load, motorists' driving behaviors, measured in lateral acceleration, caused significantly smaller variance in complex conversations compared to no call and simple conversations. The use of a hands-free cellular phone affected the performance (acceleration, lane deviation, reaction time, and accuracy) of older drivers significantly more than younger drivers. While performing divided attention tasks, the accuracy of the older drivers was 66.3 percent and that of the younger drivers was 96.3 percent. Although this study did not find a clear impact of cellular phone use on the driving behavior of younger drivers, their divided-attention task reaction times and accuracy were better under no-call than calling conditions. This study indicates that the use of hands-free cellular phones could significantly affect the safety of driving among the older and present risks, although lesser, for younger drivers.

Isolation and analysis of linker histones across cellular compartments

PubMed Central

Harshman, Sean W.; Chen, Michael M.; Branson, Owen E.; Jacob, Naduparambil K.; Johnson, Amy J.; Byrd, John C.; Freitas, Michael A.

2013-01-01

Analysis of histones, especially histone H1, is severely limited by immunological reagent availability. This paper describes the application of cellular fractionation with LC-MS for profiling histones in the cytosol and upon chromatin. First, we show that linker histones enriched by cellular fractionation gives less nuclear contamination and higher histone content than when prepared by nuclei isolation. Second, we profiled the soluble linker histones throughout the cell cycle revealing phosphorylation increases as cells reach mitosis. Finally, we monitored histone H1.2–H1.5 translocation to the cytosol in response to the CDK inhibitor flavopiridol in primary CLL cells treated ex vivo. Data shows all H1 variants translocate in response to drug treatment with no specific order to their cytosolic appearance. The results illustrate the utility of cellular fractionation in conjunction with LC-MS for the analysis of histone H1 throughout the cell. PMID:24013129

Multicomponent synthesis of 4,4-dimethyl sterol analogues and their effect on eukaryotic cells.

PubMed

Alonso, Fernando; Cirigliano, Adriana M; Dávola, María Eugenia; Cabrera, Gabriela M; García Liñares, Guadalupe E; Labriola, Carlos; Barquero, Andrea A; Ramírez, Javier A

2014-06-01

Most sterols, such as cholesterol and ergosterol, become functional only after the removal of the two methyl groups at C-4 from their biosynthetic precursors. Nevertheless, some findings suggest that 4,4-dimethyl sterols might be involved in specific physiological processes. In this paper we present the synthesis of a collection of analogues of 4,4-dimethyl sterols with a diamide side chain and a preliminary analysis of their in vitro activity on selected biological systems. The key step for the synthesis involves an Ugi condensation, a versatile multicomponent reaction. Some of the new compounds showed antifungal and cytotoxic activity. Copyright © 2014 Elsevier Inc. All rights reserved.

Mitochondrial dysfunction in blood cells from amyotrophic lateral sclerosis patients.

PubMed

Ehinger, Johannes K; Morota, Saori; Hansson, Magnus J; Paul, Gesine; Elmér, Eskil

2015-06-01

Mitochondrial dysfunction is implicated in amyotrophic lateral sclerosis, where the progressive degeneration of motor neurons results in muscle atrophy, paralysis and death. Abnormalities in both central nervous system and muscle mitochondria have previously been demonstrated in patient samples, indicating systemic disease. In this case-control study, venous blood samples were acquired from 24 amyotrophic lateral sclerosis patients and 21 age-matched controls. Platelets and peripheral blood mononuclear cells were isolated and mitochondrial oxygen consumption measured in intact and permeabilized cells with additions of mitochondrial substrates, inhibitors and titration of an uncoupler. Respiratory values were normalized to cell count and for two markers of cellular mitochondrial content, citrate synthase activity and mitochondrial DNA, respectively. Mitochondrial function was correlated with clinical staging of disease severity. Complex IV (cytochrome c-oxidase)-activity normalized to mitochondrial content was decreased in platelets from amyotrophic lateral sclerosis patients both when normalized to citrate synthase activity and mitochondrial DNA copy number. In mononuclear cells, complex IV-activity was decreased when normalized to citrate synthase activity. Mitochondrial content was increased in amyotrophic lateral sclerosis patient platelets. In mononuclear cells, complex I activity declined and mitochondrial content increased progressively with advancing disease stage. The findings are, however, based on small subsets of patients and need to be confirmed. We conclude that when normalized to mitochondria-specific content, complex IV-activity is reduced in blood cells from amyotrophic lateral sclerosis patients and that there is an apparent compensatory increase in cellular mitochondrial content. This supports systemic involvement in amyotrophic lateral sclerosis and suggests further study of mitochondrial function in blood cells as a future biomarker for the disease.

Toxicity potentials from waste cellular phones, and a waste management policy integrating consumer, corporate, and government responsibilities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Seong-Rin; Schoenung, Julie M., E-mail: jmschoenung@ucdavis.ed

Cellular phones have high environmental impact potentials because of their heavy metal content and current consumer attitudes toward purchasing new phones with higher functionality and neglecting to return waste phones into proper take-back systems. This study evaluates human health and ecological toxicity potentials from waste cellular phones; highlights consumer, corporate, and government responsibilities for effective waste management; and identifies key elements needed for an effective waste management strategy. The toxicity potentials are evaluated by using heavy metal content, respective characterization factors, and a pathway and impact model for heavy metals that considers end-of-life disposal in landfills or by incineration. Cancermore » potentials derive primarily from Pb and As; non-cancer potentials primarily from Cu and Pb; and ecotoxicity potentials primarily from Cu and Hg. These results are not completely in agreement with previous work in which leachability thresholds were the metric used to establish priority, thereby indicating the need for multiple or revised metrics. The triple bottom line of consumer, corporate, and government responsibilities is emphasized in terms of consumer attitudes, design for environment (DfE), and establishment and implementation of waste management systems including recycling streams, respectively. The key strategic elements for effective waste management include environmental taxation and a deposit-refund system to motivate consumer responsibility, which is linked and integrated with corporate and government responsibilities. The results of this study can contribute to DfE and waste management policy for cellular phones.« less

Toxicity potentials from waste cellular phones, and a waste management policy integrating consumer, corporate, and government responsibilities.

PubMed

Lim, Seong-Rin; Schoenung, Julie M

2010-01-01

Cellular phones have high environmental impact potentials because of their heavy metal content and current consumer attitudes toward purchasing new phones with higher functionality and neglecting to return waste phones into proper take-back systems. This study evaluates human health and ecological toxicity potentials from waste cellular phones; highlights consumer, corporate, and government responsibilities for effective waste management; and identifies key elements needed for an effective waste management strategy. The toxicity potentials are evaluated by using heavy metal content, respective characterization factors, and a pathway and impact model for heavy metals that considers end-of-life disposal in landfills or by incineration. Cancer potentials derive primarily from Pb and As; non-cancer potentials primarily from Cu and Pb; and ecotoxicity potentials primarily from Cu and Hg. These results are not completely in agreement with previous work in which leachability thresholds were the metric used to establish priority, thereby indicating the need for multiple or revised metrics. The triple bottom line of consumer, corporate, and government responsibilities is emphasized in terms of consumer attitudes, design for environment (DfE), and establishment and implementation of waste management systems including recycling streams, respectively. The key strategic elements for effective waste management include environmental taxation and a deposit-refund system to motivate consumer responsibility, which is linked and integrated with corporate and government responsibilities. The results of this study can contribute to DfE and waste management policy for cellular phones. 2010 Elsevier Ltd. All rights reserved.

GABAergic neurons in cerebellar interposed nucleus modulate cellular and humoral immunity via hypothalamic and sympathetic pathways.

PubMed

Lu, Jian-Hua; Wang, Xiao-Qin; Huang, Yan; Qiu, Yi-Hua; Peng, Yu-Ping

2015-06-15

Our previous work has shown that cerebellar interposed nucleus (IN) modulates immune function. Herein, we reveal mechanism underlying the immunomodulation. Treatment of bilateral cerebellar IN of rats with 3-mercaptopropionic acid (3-MP), a glutamic acid decarboxylase antagonist that reduces γ-aminobutyric acid (GABA) synthesis, enhanced cellular and humoral immune responses to bovine serum albumin, whereas injection of vigabatrin, a GABA-transaminase inhibitor that inhibits GABA degradation, in bilateral cerebellar IN attenuated the immune responses. The 3-MP or vigabatrin administrations in the cerebellar IN decreased or increased hypothalamic GABA content and lymphoid tissues' norepinephrine content, respectively, but did not alter adrenocortical or thyroid hormone levels in serum. In addition, a direct GABAergic projection from cerebellar IN to hypothalamus was found. These findings suggest that GABAergic neurons in cerebellar IN regulate immune system via hypothalamic and sympathetic pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

Sewage sludge dewatering using flowing liquid metals

DOEpatents

Carlson, Larry W.

1986-01-01

A method and apparatus for reducing the moisture content of a moist sewage sludge having a moisture content of about 50% to 80% and formed of small cellular micro-organism bodies having internally confined water is provided. A hot liquid metal is circulated in a circulation loop and the moist sewage sludge is injected in the circulation loop under conditions of temperature and pressure such that the confined water vaporizes and ruptures the cellular bodies. The vapor produced, the dried sludge, and the liquid metal are then separated. Preferably, the moist sewage sludge is injected into the hot liquid metal adjacent the upstream side of a venturi which serves to thoroughly mix the hot liquid metal and the moist sewage sludge. The venturi and the drying zone after the venturi are preferably vertically oriented. The dried sewage sludge recovered is available as a fuel and is preferably used for heating the hot liquid metal.

Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

PubMed Central

Sauge-Merle, Sandrine; Cuiné, Stéphan; Carrier, Patrick; Lecomte-Pradines, Catherine; Luu, Doan-Trung; Peltier, Gilles

2003-01-01

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes. PMID:12514032

Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response.

PubMed Central

Eady, J. J.; Orta, T.; Dennis, M. F.; Stratford, M. R.; Peacock, J. H.

1995-01-01

Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-U1 and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines. PMID:7577452

Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy

PubMed Central

Actis, Paolo; Maalouf, Michelle; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R. Adam; Pourmand, Nader

2014-01-01

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis. PMID:24279711

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond.

PubMed

Zhang, Hongkang; Cohen, Adam E

2017-07-01

Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, in particular tools for all-optical electrophysiology, now provide a means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline - from target identification and validation, to target-based and phenotypic screens, to clinical trials. Copyright © 2017 Elsevier Ltd. All rights reserved.

Accumulation of intra-cellular polyphosphate in Chlorella vulgaris cells is related to indole-3-acetic acid produced by Azospirillum brasilense.

PubMed

Meza, Beatriz; de-Bashan, Luz E; Hernandez, Juan-Pablo; Bashan, Yoav

2015-06-01

Accumulation of intra-cellular phosphate, as polyphosphate, was measured when the microalga Chlorella vulgaris was immobilized in alginate with either of two wild-type strains of the microalgae growth-promoting bacterium Azospirillum brasilense or their corresponding IAA-attenuated mutants. Wild type strains of A. brasilense induced higher amounts of intra-cellular phosphate in Chlorella than their respective mutants. Calculations comparing intra-cellular phosphate accumulation by culture or net accumulation by the cell and the amount of IAA that was produced by each of these strains revealed that higher IAA was linked to higher accumulations of intra-cellular phosphate. Application of four levels of exogenous IAA reported for A. brasilense and their IAA-attenuated mutants to cultures of C. vulgaris enhanced accumulation of intra-cellular phosphate; the higher the content of IAA per culture or per single cell, the higher was the amount of accumulated phosphate. When an IAA-attenuated mutant was complemented with exogenous IAA, accumulation of intra-cellular phosphate at the culture level was even higher than phosphate accumulation with the respective wild type strains. When calculating the net accumulation of intra-cellular phosphate in the complementation experiment, net intra-cellular phosphate induced by the IAA-attenuated mutant was completely restored and was similar to the wild strains. We propose that IAA produced by A. brasilense is linked to polyphosphate accumulation in C. vulgaris. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome.

PubMed

Soubere Mahamoud, Yonis; Aite, Meziane; Martin, Catherine; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves; Habauzit, Denis

2016-01-01

Millimeter Waves (MMW) will be used in the next-generation of high-speed wireless technologies, especially in future Ultra-Broadband small cells in 5G cellular networks. Therefore, their biocompatibilities must be evaluated prior to their massive deployment. Using a microarray-based approach, we analyzed modifications to the whole genome of a human keratinocyte model that was exposed at 60.4 GHz-MMW at an incident power density (IPD) of 20 mW/cm2 for 3 hours in athermic conditions. No keratinocyte transcriptome modifications were observed. We tested the effects of MMWs on cell metabolism by co-treating MMW-exposed cells with a glycolysis inhibitor, 2-deoxyglucose (2dG, 20 mM for 3 hours), and whole genome expression was evaluated along with the ATP content. We found that the 2dG treatment decreased the cellular ATP content and induced a high modification in the transcriptome (632 coding genes). The affected genes were associated with transcriptional repression, cellular communication and endoplasmic reticulum homeostasis. The MMW/2dG co-treatment did not alter the keratinocyte ATP content, but it did slightly alter the transcriptome, which reflected the capacity of MMW to interfere with the bioenergetic stress response. The RT-PCR-based validation confirmed 6 MMW-sensitive genes (SOCS3, SPRY2, TRIB1, FAM46A, CSRNP1 and PPP1R15A) during the 2dG treatment. These 6 genes encoded transcription factors or inhibitors of cytokine pathways, which raised questions regarding the potential impact of long-term or chronic MMW exposure on metabolically stressed cells.

Potential of New Isolates of Dunaliella Salina for Natural β-Carotene Production.

PubMed

Xu, Yanan; Ibrahim, Iskander M; Wosu, Chiziezi I; Ben-Amotz, Ami; Harvey, Patricia J

2018-02-01

The halotolerant microalga Dunaliella salina has been widely studied for natural β-carotene production. This work shows biochemical characterization of three newly isolated Dunaliella salina strains, DF15, DF17, and DF40, compared with D. salina CCAP 19/30 and D. salina UTEX 2538 (also known as D. bardawil ). Although all three new strains have been genetically characterized as Dunaliella salina strains, their ability to accumulate carotenoids and their capacity for photoprotection against high light stress are different. DF15 and UTEX 2538 reveal great potential for producing a large amount of β-carotene and maintained a high rate of photosynthesis under light of high intensity; however, DF17, DF40, and CCAP 19/30 showed increasing photoinhibition with increasing light intensity, and reduced contents of carotenoids, in particular β-carotene, suggesting that the capacity of photoprotection is dependent on the cellular content of carotenoids, in particular β-carotene. Strong positive correlations were found between the cellular content of all-trans β-carotene, 9- cis β-carotene, all-trans α-carotene and zeaxanthin but not lutein in the D. salina strains. Lutein was strongly correlated with respiration in photosynthetic cells and strongly related to photosynthesis, chlorophyll and respiration, suggesting an important and not hitherto identified role for lutein in coordinated control of the cellular functions of photosynthesis and respiration in response to changes in light conditions, which is broadly conserved in Dunaliella strains. Statistical analysis based on biochemical data revealed a different grouping strategy from the genetic classification of the strains. The significance of these data for strain selection for commercial carotenoid production is discussed.

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

PubMed Central

Rhein, Cosima; Tripal, Philipp; Seebahn, Angela; Konrad, Alice; Kramer, Marcel; Nagel, Christine; Kemper, Jonas; Bode, Jens; Mühle, Christiane; Gulbins, Erich; Reichel, Martin; Becker, Cord-Michael; Kornhuber, Johannes

2012-01-01

Background Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. Methodology/Principal Findings We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. Conclusions/Significance These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity. PMID:22558155

Sterol synthesis and cell size distribution under oscillatory growth conditions in Saccharomyces cerevisiae scale-down cultivations.

PubMed

Marbà-Ardébol, Anna-Maria; Bockisch, Anika; Neubauer, Peter; Junne, Stefan

2018-02-01

Physiological responses of yeast to oscillatory environments as they appear in the liquid phase in large-scale bioreactors have been the subject of past studies. So far, however, the impact on the sterol content and intracellular regulation remains to be investigated. Since oxygen is a cofactor in several reaction steps within sterol metabolism, changes in oxygen availability, as occurs in production-scale aerated bioreactors, might have an influence on the regulation and incorporation of free sterols into the cell lipid layer. Therefore, sterol and fatty acid synthesis in two- and three-compartment scale-down Saccharomyces cerevisiae cultivation were studied and compared with typical values obtained in homogeneous lab-scale cultivations. While cells were exposed to oscillating substrate and oxygen availability in the scale-down cultivations, growth was reduced and accumulation of carboxylic acids was increased. Sterol synthesis was elevated to ergosterol at the same time. The higher fluxes led to increased concentrations of esterified sterols. The cells thus seem to utilize the increased availability of precursors to fill their sterol reservoirs; however, this seems to be limited in the three-compartment reactor cultivation due to a prolonged exposure to oxygen limitation. Besides, a larger heterogeneity within the single-cell size distribution was observed under oscillatory growth conditions with three-dimensional holographic microscopy. Hence the impact of gradients is also observable at the morphological level. The consideration of such a single-cell-based analysis provides useful information about the homogeneity of responses among the population. Copyright © 2017 John Wiley & Sons, Ltd.

Enhanced oral bioavailability of a sterol-loaded microemulsion formulation of Flammulina velutipes, a potential antitumor drug

PubMed Central

Yi, Chengxue; Zhong, Hui; Tong, Shanshan; Cao, Xia; Firempong, Caleb K; Liu, Hongfei; Fu, Min; Yang, Yan; Feng, Yingshu; Zhang, Huiyun; Xu, Ximing; Yu, Jiangnan

2012-01-01

Purpose To investigate the growth inhibition activity of Flammulina velutipes sterol (FVS) against certain human cancer cell lines (gastric SGC and colon LoVo) and to evaluate the optimum microemulsion prescription, as well as the pharmacokinetics of encapsulated FVS. Methods Molecules present in the FVS isolate were identified by gas chromatography/mass spectrometry analysis. The cell viability of FVS was assessed with methyl thiazolyl tetrazolium (MTT) bioassay. Based on the solubility study, phase diagram and stability tests, the optimum prescription of F. velutipes sterol microemulsions (FVSMs) were determined, followed by FVSMs characterization, and its in vivo pharmacokinetic study in rats. Results The chemical composition of FVS was mainly ergosterol (54.8%) and 22,23-dihydroergosterol (27.9%). After 72 hours of treatment, both the FVS (half-maximal inhibitory concentration [IC50] = 11.99 μg · mL−1) and the standard anticancer drug, 5-fluorouracil (IC50 = 0.88 μg · mL−1) exhibited strong in vitro antiproliferative activity against SGC cells, with IC50 > 30.0 μg · mL−1; but the FVS performed poorly against LoVo cells (IC50 > 40.0 μg · mL−1). The optimal FVSMs prescription consisted of 3.0% medium chain triglycerides, 5.0% ethanol, 21.0% Cremophor EL and 71.0% water (w/w) with associated solubility of FVS being 0.680 mg · mL−1 as compared to free FVS (0.67 μg · mL−1). The relative oral bioavailability (area-under-the-curve values of ergosterol and 22,23-dihydroergosterol showed a 2.56-fold and 4.50-fold increase, respectively) of FVSMs (mean diameter ~ 22.9 nm) as against free FVS were greatly enhanced. Conclusion These results indicate that the FVS could be a potential candidate for the development of an anticancer drug and it is readily bioavailable via microemulsion formulations. PMID:23049254

Identification of a novel dehydroergosterol enhancing microglial anti-inflammatory activity in a dairy product fermented with Penicillium candidum.

PubMed

Ano, Yasuhisa; Kutsukake, Toshiko; Hoshi, Ayaka; Yoshida, Aruto; Nakayama, Hiroyuki

2015-01-01

Despite the ever-increasing number of dementia patients worldwide, fundamental therapeutic approaches to treat this disease remain to be established. Preventive approaches such as diet, exercise and learning attract attention. Several epidemiological studies suggest that ingestion of fermented dairy products prevents cognitive decline in the elderly. These reports indicate that specific ingredients in the fermented dairy products elicit an anti-inflammatory or anti-oxidative activity that facilitates neuroprotection. The responsible components remain to be investigated. A number of studies have shown that inflammation caused by microglia is closely related to exaggeration of the pathology and cognitive decline seen in the elderly. Many researchers have proposed that controlling microglial activities could be effective in preventing and possibly curing dementia. In the present study, to elucidate specific compounds that regulate microglial activity from dairy products, repeated purification by HPLC, combined with evaluation using primary microglia, facilitated the identification of dehydroergosterol (DHE) as a novel component of the extract that enhances microglial anti-inflammatory activity. DHE contains three conjugated double bonds in a steroid ring system and is an analogue of ergosterol. Despite their related chemical structures, the anti-inflammatory activity of DHE is markedly stronger than that of ergosterol. P. candidum for camembert cheese produces DHE, but P. Roqueforti for blue cheese and Aspergillus do not. DHE also induces CD11b-positive microglia cells into CD206-positive M2 type microglia. Neurotoxicity and neuronal cell death induced by excessively activated microglia is suppressed by treatment with DHE. Thus, this is the first report to demonstrate that DHE, identified as a responsible compound in dairy products, can induce microglia into a preferable phenotype for our brain environment and can be safely introduced into the body by consumption of dairy products. We believe the uptake of DHE might help to prevent the onset of dementia.

Identification of a Novel Dehydroergosterol Enhancing Microglial Anti-Inflammatory Activity in a Dairy Product Fermented with Penicillium candidum

PubMed Central

Ano, Yasuhisa; Kutsukake, Toshiko; Hoshi, Ayaka; Yoshida, Aruto; Nakayama, Hiroyuki

2015-01-01

Despite the ever-increasing number of dementia patients worldwide, fundamental therapeutic approaches to treat this disease remain to be established. Preventive approaches such as diet, exercise and learning attract attention. Several epidemiological studies suggest that ingestion of fermented dairy products prevents cognitive decline in the elderly. These reports indicate that specific ingredients in the fermented dairy products elicit an anti-inflammatory or anti-oxidative activity that facilitates neuroprotection. The responsible components remain to be investigated. A number of studies have shown that inflammation caused by microglia is closely related to exaggeration of the pathology and cognitive decline seen in the elderly. Many researchers have proposed that controlling microglial activities could be effective in preventing and possibly curing dementia. In the present study, to elucidate specific compounds that regulate microglial activity from dairy products, repeated purification by HPLC, combined with evaluation using primary microglia, facilitated the identification of dehydroergosterol (DHE) as a novel component of the extract that enhances microglial anti-inflammatory activity. DHE contains three conjugated double bonds in a steroid ring system and is an analogue of ergosterol. Despite their related chemical structures, the anti-inflammatory activity of DHE is markedly stronger than that of ergosterol. P. candidum for camembert cheese produces DHE, but P. Roqueforti for blue cheese and Aspergillus do not. DHE also induces CD11b-positive microglia cells into CD206-positive M2 type microglia. Neurotoxicity and neuronal cell death induced by excessively activated microglia is suppressed by treatment with DHE. Thus, this is the first report to demonstrate that DHE, identified as a responsible compound in dairy products, can induce microglia into a preferable phenotype for our brain environment and can be safely introduced into the body by consumption of dairy products. We believe the uptake of DHE might help to prevent the onset of dementia. PMID:25760331

Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants▿

PubMed Central

Ruckenstuhl, Christoph; Lang, Silvia; Poschenel, Andrea; Eidenberger, Armin; Baral, Pravas Kumar; Kohút, Peter; Hapala, Ivan; Gruber, Karl; Turnowsky, Friederike

2007-01-01

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G25S, D335X (W, F, P), and G210A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G30S and L37P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R269G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines. PMID:17043127

Ergosteryl 2-naphthoate, An Ergosterol Derivative, Exhibits Antidepressant Effects Mediated by the Modification of GABAergic and Glutamatergic Systems.

PubMed

Lin, Mingzhu; Li, Haijun; Zhao, Yan; Cai, Enbo; Zhu, Hongyan; Gao, Yugang; Liu, Shuangli; Yang, He; Zhang, Lianxue; Tang, Guosheng; Wang, Ruiqing

2017-03-31

Phytosterols are a kind of natural component including sitosterol, campesterol, avenasterol, ergosterol (Er) and others. Their main natural sources are vegetable oils and their processed products, followed by grains, by-products of cereals and nuts, and small amounts of fruits, vegetables and mushrooms. In this study, three new Er monoester derivatives were obtained from the reflux reaction with Er: organic acids (furoic acid, salicylic acid and 2-naphthoic acid), 1-Ethylethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) in dichloromethane. Their chemical structures were defined by IR and NMR. The present study was also undertaken to investigate the antidepressant-like effects of Er and its derivatives in male adult mice models of depression, and their probable involvement of GABAergic and glutamatergic systems by the forced swim test (FST). The results indicated that Er and its derivatives display antidepressant effects. Moreover, one derivative of Er, ergosteryl 2-naphthoate (ErN), exhibited stronger antidepressant activity in vivo compared to Er. Acute administration of ErN (5 mg/kg, i.p.) and a combination of ErN (0.5 mg/kg, i.p.), reboxetine (2.5 mg/kg, i.p.), and tianeptine (15 mg/kg, i.p.) reduced the immobility time in the FST. Pretreatment with bicuculline (a competitive γ-aminobutyric acid (GABA) antagonist, 4 mg/kg, i.p.) and N -methyl-d-aspartic acid (NMDA, an agonist at the glutamate site, 75 mg/kg, i.p.) effectively reversed the antidepressant-like effect of ErN (5 mg/kg, i.p.). However, prazosin (a α1-adrenoceptor antagonist, 1 mg/kg, i.p.) and haloperidol (a non-selective D2 receptor antagonist, 0.2 mg/kg, i.p.) did not eliminate the reduced immobility time. Altogether, these results indicated that ErN produced antidepressant-like activity, which might be mediated by GABAergic and glutamatergic systems.

Glycotriazole-peptides derived from the peptide HSP1: synergistic effect of triazole and saccharide rings on the antifungal activity.

PubMed

Junior, Eduardo F C; Guimarães, Carlos F R C; Franco, Lucas L; Alves, Ricardo J; Kato, Kelly C; Martins, Helen R; de Souza Filho, José D; Bemquerer, Marcelo P; Munhoz, Victor H O; Resende, Jarbas M; Verly, Rodrigo M

2017-08-01

This work proposes a strategy that uses solid-phase peptide synthesis associated with copper(I)-catalyzed azide alkyne cycloaddition reaction to promote the glycosylation of an antimicrobial peptide (HSP1) containing a carboxyamidated C-terminus (HSP1-NH 2 ). Two glycotriazole-peptides, namely [p-Glc-trz-G 1 ]HSP1-NH 2 and [p-GlcNAc-trz-G 1 ]HSP1-NH 2 , were prepared using per-O-acetylated azide derivatives of glucose and N-acetylglucosamine in the presence of copper(II) sulfate pentahydrate (CuSO 4 ·5H 2 O) and sodium ascorbate as a reducing agent. In order to investigate the synergistic action of the carbohydrate motif linked to the triazole-peptide structure, a triazole derivative [trz-G 1 ]HSP1-NH 2 was also prepared. A set of biophysical approaches such as DLS, Zeta Potential, SPR and carboxyfluorescein leakage from phospholipid vesicles confirmed higher membrane disruption and lytic activities as well as stronger peptide-LUVs interactions for the glycotriazole-peptides when compared to HSP1-NH 2 and to its triazole derivative, which is in accordance with the performed biological assays: whereas HSP1-NH 2 presents relatively low and [trz-G 1 ]HSP1-NH 2 just moderate fungicidal activity, the glycotriazole-peptides are significantly more effective antifungal agents. In addition, the glycotriazole-peptides and the triazole derivative present strong inhibition effects on ergosterol biosynthesis in Candida albicans, when compared to HSP1-NH 2 alone. In conclusion, the increased fungicidal activity of the glycotriazole-peptides seems to be the result of (A) more pronounced membrane-disruptive properties, which is related to the presence of a saccharide ring, together with (B) the inhibition of ergosterol biosynthesis, which seems to be related to the presence of both the monosaccharide and the triazole rings.

Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility.

PubMed Central

Geber, A; Hitchcock, C A; Swartz, J E; Pullen, F S; Marsden, K E; Kwon-Chung, K J; Bennett, J E

1995-01-01

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae. PMID:8593007

Genetic and Genomic Architecture of the Evolution of Resistance to Antifungal Drug Combinations

PubMed Central

Hill, Jessica A.; Ammar, Ron; Torti, Dax; Nislow, Corey; Cowen, Leah E.

2013-01-01

The evolution of drug resistance in fungal pathogens compromises the efficacy of the limited number of antifungal drugs. Drug combinations have emerged as a powerful strategy to enhance antifungal efficacy and abrogate drug resistance, but the impact on the evolution of drug resistance remains largely unexplored. Targeting the molecular chaperone Hsp90 or its downstream effector, the protein phosphatase calcineurin, abrogates resistance to the most widely deployed antifungals, the azoles, which inhibit ergosterol biosynthesis. Here, we evolved experimental populations of the model yeast Saccharomyces cerevisiae and the leading human fungal pathogen Candida albicans with azole and an inhibitor of Hsp90, geldanamycin, or calcineurin, FK506. To recapitulate a clinical context where Hsp90 or calcineurin inhibitors could be utilized in combination with azoles to render resistant pathogens responsive to treatment, the evolution experiment was initiated with strains that are resistant to azoles in a manner that depends on Hsp90 and calcineurin. Of the 290 lineages initiated, most went extinct, yet 14 evolved resistance to the drug combination. Drug target mutations that conferred resistance to geldanamycin or FK506 were identified and validated in five evolved lineages. Whole-genome sequencing identified mutations in a gene encoding a transcriptional activator of drug efflux pumps, PDR1, and a gene encoding a transcriptional repressor of ergosterol biosynthesis genes, MOT3, that transformed azole resistance of two lineages from dependent on calcineurin to independent of this regulator. Resistance also arose by mutation that truncated the catalytic subunit of calcineurin, and by mutation in LCB1, encoding a sphingolipid biosynthetic enzyme. Genome analysis revealed extensive aneuploidy in four of the C. albicans lineages. Thus, we identify molecular determinants of the transition of azole resistance from calcineurin dependence to independence and establish multiple mechanisms by which resistance to drug combinations evolves, providing a foundation for predicting and preventing the evolution of drug resistance. PMID:23593013

Current status of antifungal susceptibility testing methods.

PubMed

Arikan, Sevtap

2007-11-01

Antifungal susceptibility testing is a very dynamic field of medical mycology. Standardization of in vitro susceptibility tests by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST), and current availability of reference methods constituted the major remarkable steps in the field. Based on the established minimum inhibitory concentration (MIC) breakpoints, it is now possible to determine the susceptibilities of Candida strains to fluconazole, itraconazole, voriconazole, and flucytosine. Moreover, utility of fluconazole antifungal susceptibility tests as an adjunct in optimizing treatment of candidiasis has now been validated. While the MIC breakpoints and clinical significance of susceptibility testing for the remaining fungi and antifungal drugs remain yet unclear, modifications of the available methods as well as other methodologies are being intensively studied to overcome the present drawbacks and limitations. Among the other methods under investigation are Etest, colorimetric microdilution, agar dilution, determination of fungicidal activity, flow cytometry, and ergosterol quantitation. Etest offers the advantage of practical application and favorable agreement rates with the reference methods that are frequently above acceptable limits. However, MIC breakpoints for Etest remain to be evaluated and established. Development of commercially available, standardized colorimetric panels that are based on CLSI method parameters has added more to the antifungal susceptibility testing armamentarium. Flow cytometry, on the other hand, appears to offer rapid susceptibility testing but requires specified equipment and further evaluation for reproducibility and standardization. Ergosterol quantitation is another novel approach, which appears potentially beneficial particularly in discrimination of azole-resistant isolates from heavy trailers. The method is yet investigational and requires to be further studied. Developments in methodology and applications of antifungal susceptibility testing will hopefully provide enhanced utility in clinical guidance of antifungal therapy. However, and particularly in immunosuppressed host, in vitro susceptibility is and will remain only one of several factors that influence clinical outcome.

Lipid Biomarkers and Carbon Isotope Ratios of Lipids Isolated from Acid Mine Drainage Biofilms: Dual Biosignatures for Eukaryotic Evolution and Oxygenation of Primitive Earth

NASA Astrophysics Data System (ADS)

Dasgupta, S.; Fang, J.; Zhang, L.; Li, J.

2012-12-01

Lipid analysis and carbon isotope ratios (δ13C) of lipids in biofilms in an acid mine drainage site (AMD) site in western Indiana revealed unique biogeochemical signatures of microeukaryotes, never recorded before. Dominance of photosynthetic microeukaryote Euglena was indicated by the detection of abundant phytadiene, phytol, phytanol, polyunsaturated n-alkenes, polyunsaturated fatty acids, short-chain (C25-32) wax esters (WE), ergosterol, and tocopherols. The WE were probably synthesized in mitochondria under anoxic conditions by the reverse β-oxidation pathway, whereas the sterols (ergosterol and ergosta-7,22-dien-3β-ol) were likely synthesized in the cytosol in the presence of molecular oxygen. The dual aerobic and anaerobic biosynthetic pathways of Euglena may be a response to survive the recurring anoxic and oxic conditions in primitive Earth, whereby microeukaryotes retained this mechanism of conserved compartmentalization within their physiology to evolve and diversify in extreme conditions. Hydrocarbons, including n-alkenes, phytadienes, and wax esters showed heavy δ13C values than usual. The primary cause for the 13C-enrichment can be attributed to a CO2-limiting system that exists in the AMD, which is further regulated by the pH of the AMD. Floating biofilms BF2, 4, and 6 showed more depleted δ13C values for phytadienes and n-alkenes (average of -23.6‰) as compared to benthic biofilm BF5 (average of -20.8‰), indicating that physiology plays an important role in isotopic discrimination. 13C-enriched values of the esters could result from kinetic isotope effects at two branch points (pyruvate and/or acetyl CoA) in the biosynthetic pathway. Our understanding of biogeochemical conditions in this AMD environment would allow us to identify unique sets of biosignatures that can act as a proxy in deciphering the links between eukaryotic evolutions, oxygenation of the early atmosphere, formation of BIF, and possibly iron-rich extraterrestrial environments.

New Funding Opportunity from the Human Biomolecular Atlas Program (HuBMAP)! | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

The NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) aims to develop a framework for functional mapping the human body with cellular resolution to enhance our understanding of cellular organization-function. HuBMAP will accelerate the development of the next generation of tools and techniques to generate 3D tissue maps using validated high-content, high-throughput imaging and omics assays, and establish an open data platform for integrating, visualizing data to build multi-dimensional maps.

Early changes in staurosporine-induced differentiated RGC-5 cells indicate cellular injury response to nonlethal blue light exposure.

PubMed

Zhang, Pei; Huang, Chen; Wang, Wei; Wang, Minshu

2015-06-01

Blue light has been previously demonstrated to induce injury of retinal cells. The cellular responses to nonlethal blue light exposure for each type of retinal cell are of particular interest but remain undetermined. Based on the doses of blue light reported in previous research to be nonlethal to retinal pigment epithelial cells, here we investigated whether and to what extent such doses of blue light are cytotoxic to staurosporine-differentiated RGC-5 cells. RGC-5 cells were differentiated for 24 hours using 200 nM staurosporine. The resulting cells were cultured and exposed to blue light at three different energy levels (1, 10, and 50 J cm(-2)). Cellular morphologies were investigated with an inverted microscope and cell viability was assessed with a Cell Counting Kit-8 (CCK-8) assay. The generation of intracellular reactive oxygen species (ROS) was evaluated by H2DCFDA. After loading of MitoTracker Green FM dye, the mitochondrial contents were analyzed using flow cytometry. The lactate dehydrogenase (LDH) activities in the media were also measured. The level of lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA). Treatment of the cells for 24 hours with 200 nM staurosporine successfully induced the differentiation of RGC-5 cells. No morphological changes were observed in the ssdRGC-5 cells exposed to blue light at 50 J cm(-2), which was the highest energy level tested. Exposure of the ssdRGC-5 cells to this energy level of blue light did, however, decrease their numbers by approximately 72.1% compared to the numbers of such cells found after being left in the dark. Remarkably, the levels of ROS generation and mitochondrial contents were, respectively, increased to 142% and 118% of those of the control by a 10 J cm(-2) exposure of blue light. The LDH activities and MDA levels exhibited no obvious changes in the blue light-exposed ssdRGC-5 cells compared to the control cells. In vitro nonlethal blue light exposure led to cellular damage of staurosporine-differentiated RGC-5 cells. These increases in oxidative stress and mitochondrial content were the early steps of the cellular response to the exposure of relatively low doses (10 J cm(-2)) of blue light.

Antioxidant potential of polyphenols and tannins from burs of Castanea mollissima Blume.

PubMed

Zhao, Shan; Liu, Jie Yuan; Chen, Si Yu; Shi, Ling Ling; Liu, Yu Jun; Ma, Chao

2011-10-12

Spiny burs of Castanea mollissima Blume (Chinese chestnut) are usually discarded as industrial waste during post-harvesting processing. The objective of this study was to establish an extraction and isolation procedure for tannins from chestnut burs, and to assess their potential antioxidant activity. Aqueous ethanol solution was used as extraction solvent, and HPD 100 macroporous resin column was applied for isolation. The influence of solvent concentration in the extraction and elution process on extraction yield, tannins and polyphenols content, as well as antioxidant potential, including DPPH and ABTS radical scavenging ability, reducing power ability and cellular antioxidant ability were assessed. In both the extraction and isolation process, 50% aqueous ethanol led to superior total tannins and polyphenols content as well as significantly higher antioxidant activity. In addition, the antioxidant activity and the total tannins content in extracts and fractions had a positive linear correlation, and the predominant components responsible for antioxidant activities were characterized as hydrolysable tannins. To the best of our knowledge, this is the first report on the enrichment of tannins from burs of C. mollissim using macroporous resin chromatography, and to assess the cellular antioxidant activity of them.

Mammalian Polyamine Metabolism and Function

PubMed Central

Pegg, Anthony E.

2009-01-01

Summary Polyamines are ubiquitous small basic molecules that play multiple essential roles in mammalian physiology. Their cellular content is highly regulated and there is convincing evidence that altered metabolism is involvement in many disease states. Drugs altering polyamine levels may therefore have a variety of important targets. This review will summarize the current state of understanding of polyamine metabolism and function, the regulation of polyamine content, and heritable pathological conditions that may be derived from altered polyamine metabolism. PMID:19603518

Cytotoxicity, genotoxicity, and metal release in patients with fixed orthodontic appliances: a longitudinal in-vivo study.

PubMed

Hafez, Hend Salah; Selim, Essam Mohamed Nassef; Kamel Eid, Faten Hussein; Tawfik, Wael Attia; Al-Ashkar, Emad A; Mostafa, Yehya Ahmed

2011-09-01

Treatment with fixed orthodontic appliances in the corrosive environment of the oral cavity warrants in-vivo investigations of biocompatibility. Eighteen control and 28 treated subjects were evaluated longitudinally. Four combinations of brackets and archwires were tested. Buccal mucosa cell samples were collected before treatment, and 3 and 6 months after appliance placement. The cells were processed for cytotoxicity, genotoxicity, and nickel and chromium contents. In the treatment group, buccal mucosa cell viability values were 8.1% at pretreatment, and 6.4% and 4.5% at 3 and 6 months, respectively. The composite score, a calculated DNA damage value, decreased from 125.6 to 98.8 at 6 months. Nickel cellular content increased from 0.52 to 0.68 and 0.78 ng per milliliter, and chromium increased from 0.31 to 0.41 and 0.78 ng per milliliter at 3 and 6 months, respectively. Compared with the control group, the treated subjects showed significant differences for DNA damage and chromium content at 3 months only. Fixed orthodontic appliances decreased cellular viability, induced DNA damage, and increased the nickel and chromium contents of the buccal mucosa cells. Compared to the control group, these changes were not evident at 6 months, possibly indicating tolerance for or repair of the cells and the DNA. Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Reduction of antiproliferative capacities, cell-based antioxidant capacities and phytochemical contents of common beans and soybeans upon thermal processing.

PubMed

Xu, Baojun; Chang, Sam K C

2011-12-01

The effects of boiling and steaming processes on the antiproliferative and cellular antioxidant properties, as well as phytochemicals, of two types of common beans (pinto and black beans) and two types of soybeans (yellow and black) were investigated. All thermal-processing methods caused significant (p<0.05) decreases in total phenolic content (TPC), total saponin content (TSC) and phytic acid content (PAC) values in all bean types (except for TPC values in pressure-steamed yellow soybeans) as compared to those of the raw beans. All types of uncooked raw beans exhibited cellular antioxidant activities (CAA) in dose-dependent manners. Black soybeans exhibited the greatest CAA, followed by black beans, pinto beans and yellow soybeans. The CAA of cooked beans were generally diminished or eliminated by thermal processing. The hydrophilic extracts from raw pinto beans, black beans and black soybeans exhibited antiproliferation capacities against human gastric (AGS) and colorectal (SW480) cancer cells in dose-dependent manners. The raw yellow soybeans exhibited dose-dependent antiproliferation activities against the SW480 cells. Most of the cooked beans lost their antiproliferation capacities as observed in the raw beans. These results indicate that different processing methods may have various effects on phytochemical profiles and bioactivities. Overall, thermal processing caused a significant reduction of the health-promotion effects of beans. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of a Dunaliella tertiolecta Strain with Increased Zeaxanthin Content Using Random Mutagenesis

PubMed Central

Kim, Minjae; Ahn, Junhak; Jeon, Hancheol; Jin, EonSeon

2017-01-01

Zeaxanthin is a xanthophyll pigment that is regarded as one of the best carotenoids for the prevention and treatment of degenerative diseases. In the worldwide natural products market, consumers prefer pigments that have been produced from biological sources. In this study, a Dunaliella tertiolecta strain that has 10–15% higher cellular zeaxanthin content than the parent strain (zea1), was obtained by random mutagenesis using ethyl methanesulfonate (EMS) as a mutagen. This mutant, mp3, was grown under various salinities and light intensities to optimize culture conditions for zeaxanthin production. The highest cellular zeaxanthin content was observed at 1.5 M NaCl and 65–85 μmol photons·m−2·s−1, and the highest daily zeaxanthin productivity was observed at 0.6 M NaCl and 140–160 μmol photons·m−2·s−1. The maximal yield of zeaxanthin from mp3 in fed-batch culture was 8 mg·L−1, which was obtained at 0.6 M NaCl and 140–160 μmol photons·m−2·s−1. These results suggest that random mutagenesis with EMS is useful for generating D. tertiolecta strains with increased zeaxanthin content, and also suggest optimal culture conditions for the enhancement of biomass and zeaxanthin production by the zeaxanthin accumulating mutant strains. PMID:28635662

Development of a Dunaliella tertiolecta Strain with Increased Zeaxanthin Content Using Random Mutagenesis.

PubMed

Kim, Minjae; Ahn, Junhak; Jeon, Hancheol; Jin, EonSeon

2017-06-21

Zeaxanthin is a xanthophyll pigment that is regarded as one of the best carotenoids for the prevention and treatment of degenerative diseases. In the worldwide natural products market, consumers prefer pigments that have been produced from biological sources. In this study, a Dunaliella tertiolecta strain that has 10-15% higher cellular zeaxanthin content than the parent strain ( zea1 ), was obtained by random mutagenesis using ethyl methanesulfonate (EMS) as a mutagen. This mutant, mp3 , was grown under various salinities and light intensities to optimize culture conditions for zeaxanthin production. The highest cellular zeaxanthin content was observed at 1.5 M NaCl and 65-85 μmol photons·m -2 ·s -1 , and the highest daily zeaxanthin productivity was observed at 0.6 M NaCl and 140-160 μmol photons·m -2 ·s -1 . The maximal yield of zeaxanthin from mp3 in fed-batch culture was 8 mg·L -1 , which was obtained at 0.6 M NaCl and 140-160 μmol photons·m -2 ·s -1 . These results suggest that random mutagenesis with EMS is useful for generating D. tertiolecta strains with increased zeaxanthin content, and also suggest optimal culture conditions for the enhancement of biomass and zeaxanthin production by the zeaxanthin accumulating mutant strains.

Sewage sludge dewatering using flowing liquid metals

DOEpatents

Carlson, L.W.

1985-08-30

This invention relates generally to the dewatering of sludge, and more particularly to the dewatering of a sewage sludge having a moisture content of about 50 to 80% in the form of small cellular micro-organism bodies having internally confined water.

The suppression of radiation-induced NF-{kappa}B activity by dexamethasone correlates with increased cell death in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nam, Seon Young; Chung, Hee-Yong

2005-10-21

In this study, we show that dexamethasone treatment increases ionizing radiation-induced cell death by inducing the inhibitory {kappa}B{alpha} (I{kappa}B{alpha}) pathway in mice. The effect of dexamethasone on radiation-induced cell death was assessed by changes in total spleen cellularity and bone marrow colony-forming unit-granulocyte-macrophage (CFU-GM) contents after total body irradiation. While in vivo treatment of mice with dexamethasone alone (1 mg/kg/day, for 2 days) failed to elicit cell death in spleen cells, the combined treatment with dexamethasone (1 mg/kg/day, for 2 days) and {gamma}-rays (1 or 5 Gy) caused a 50-80% reduction in total cellularity in spleen and CFU-GM contents inmore » bone marrow. These results demonstrate that dexamethasone has a synergistic effect on radiation-induced cellular damages in vivo. Immunoblot analysis showed that dexamethasone treatment significantly increases I{kappa}B{alpha} expression in the spleens of irradiated mice. In addition, the dexamethasone treatment significantly reduced radiation-induced nuclear translocation of the nucleus factor-{kappa}B in the spleens of irradiated mice. These results indicate that dexamethasone treatment in vivo may increase radiation-induced cell damages by increasing I{kappa}B{alpha} expression in hematopoietic organs such as spleen and bone marrow.« less

Mechanisms of ozone toxicity in cultured cells. I. Reduced clonogenic ability of polyunsaturated fatty acid-supplemented fibroblasts. Effect of vitamin E

DOE Office of Scientific and Technical Information (OSTI.GOV)

Konings, A.W.

1986-01-01

The direct action of ozone on viability and survival of normal and modified mouse lung fibroblasts has been studied. By cell manipulation of fibroblasts in culture, the content of polyunsaturated fatty acids (PUFA) in the phospholipids was increased from about 6% to about 40%. The cellular content of alpha-tocopherol (alpha-T) (vitamin E) could be drastically enhanced. Vitamin E supplementation to the cell did not influence the PUFA manipulation. Normal, PUFA, and PUFA(alpha-T) fibroblasts were exposed to ozone by bubbling 10 ppm through the cell suspensions for different periods of time (0-6 h). No significant effects of the ozone exposure couldmore » be established when normal fibroblasts were used. The PUFA fibroblasts, however, were very vulnerable to ozone toxicity, both in terms of dye uptake (Trypan blue) and cell death (clonogenic ability). When alpha-tocopherol was present in the cell (200 ng/10(6) cells), a clear protection against ozone toxicity was found. It is concluded that ozone toxicity might be higher under conditions of a relative high amount of polyunsaturated fatty acids in the membrane phospholipids of the cell and a low cellular antioxidant capacity. Cellular membranes are probably an important target for ozone-induced cell death.« less

Ocean acidification stimulates particulate organic carbon accumulation in two Antarctic diatom species under moderate and high natural solar radiation.

PubMed

Heiden, Jasmin P; Thoms, Silke; Bischof, Kai; Trimborn, Scarlett

2018-05-23

Impacts of rising atmospheric CO 2 concentrations and increased daily irradiances from enhanced surface water stratification on phytoplankton physiology in the coastal Southern Ocean remain still unclear. Therefore, in the two Antarctic diatoms Fragilariopsis curta and Odontella weissflogii the effects of moderate and high natural solar radiation combined with either ambient or future pCO 2 on cellular particulate organic carbon (POC) contents and photophysiology were investigated. Results showed that increasing CO 2 concentrations had greater impacts on diatom physiology than exposure to increasing solar radiation. Irrespective of the applied solar radiation regime, cellular POC quotas increased with future pCO 2 in both diatoms. Lowered maximum quantum yields of photochemistry in PSII (F v /F m ) indicated a higher photosensitivity under these conditions, being counteracted by increased cellular concentrations of functional photosynthetic reaction centers. Overall, our results suggest that both bloom-forming Antarctic coastal diatoms might increase carbon contents under future pCO 2 conditions despite reduced physiological fitness. This indicates a higher potential for primary productivity by the two diatom species with important implications for the CO 2 sequestration potential of diatom communities in the future coastal Southern Ocean. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Highlights in Endocytosis of Nanostructured Systems.

PubMed

Voltan, Aline R; Alarcon, Kaila M; Fusco-Almeida, Ana M; Soares, Christiane P; Mendes-Giannini, Maria J S; Chorilli, Marlus

2017-01-01

The focus of this review is the cellular internalisation mechanism of nanostructured systems (NSs) and their endosomal escape for targeted drug delivery. Endocytosis is a cellular process of internalisation of different molecules and foreign microorganisms. It is currently being studied for drug delivery through nanostructured systems. The most commonly studied routes of cellular uptake are phagocytosis, macro-pinocytosis, clathrinmediated endocytosis, caveolin-mediated endocytosis, and clathrin and caveolinindependent endocytosis. The mechanism utilised by NSs for cellular entry depends on factors such as cell type and its physicochemical properties. Currently, with the development of drugs-loaded onto NSs, it has been possible to increase the therapeutic index against few diseases. The NSs can deliver the active drug at locations that conventional drugs cannot, thereby minimising unwanted side effects. On cellular entry of NSs, there is a possibility of an endosomal escape of the contents into the cytoplasm, a mechanism that can be exploited so that NSs can migrate intra-cellularly and deliver the drug to the target of interest. Designing endolysosomal escape strategy is not an easy task, but it is critical for the optimal pharmacological action on the target tissue. The cellular uptake of drugs is a very important factor in therapy. Although NSs have emerged as effective drug delivery vehicle for treatment of diseases, it is crucial to understand the mechanism of NSs endocytosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Preparation of individual human diploid fibroblasts and study of ion transport.

PubMed

Abraham, E H; Breslow, J L; Epstein, J; Chang-Sing, P; Lechene, C

1985-01-01

A method for analyzing individual mammalian cells with electron probe microanalysis has been developed using human diploid fibroblasts. Cells were grown on the same support that is used for experimental manipulations and analysis. Steady-state cation and anion concentrations and kinetic processes during experimental perturbations could be measured on populations of less than 1,000 cells. Human diploid fibroblasts in normal tissue culture medium had the following intracellular concentrations (in mM): K, 168; Na, 25.0; Cl, 51.2; P, 84.1; S, 16.5; Ca, 6.04; and Mg, 10.0. The ratios of K to Na were equivalent when measured in the nuclear or cytoplasmic area of the cells. Serum in the incubation medium was found to increase the cellular effective permeability to Na by a factor of 2.5, while leaving the effective permeability to K unchanged. When returned to control medium after 7 h of incubation in K-free medium, the cells recovered normal K/Na in less than 1 h. In some experiments the coupling ratio of the ouabain-inhibitable cellular transport of Na to K was 3:2 and the ratio of Cl to K was 1:2. The sum of intracellular content (Na + K) (an estimate of cellular volume) did not change when the cells were placed in K-free medium and increased by less than 30% after ouabain treatment. After 5-7 h of ouabain treatment or of incubation in K-free medium, long after the intracellular K had been replaced by Na, the cellular chloride content had not changed significantly.

Identification of cellular and molecular factors determining the response of cancer cells to six ergot alkaloids.

PubMed

Mrusek, Marco; Seo, Ean-Jeong; Greten, Henry Johannes; Simon, Michael; Efferth, Thomas

2015-02-01

Ergot alkaloids are psychoactive and vasoconstricting agents of the fungus Claviceps purpurea causing poisoning such as ergotism in medieval times (St. Anthony's Fire). This class of substances also inhibits tumor growth in vitro and in vivo, though the underlying mechanisms are unclear as yet. We investigated six ergot alkaloids (agroclavine, ergosterol, ergocornin E, ergotamine, dihydroergocristine, and 1-propylagroclavine tartrate) for their cytotoxicity towards tumor cell lines of the National Cancer Institute, USA. 1-Propylagroclavine tartrate (1-PAT) revealed the strongest cytotoxicity. Out of 76 clinically established anticancer drugs, cross-resistance was found between the ergot alkaloids and 6/7 anti-hormonal drugs (=85.7 %) and 5/15 DNA-alkylating drugs (=33.3 %). The IC50 values for the six alkaloids were not correlated to well-known determinants of drug resistance, such as proliferative activity (as measured by cell doubling times, PCNA expression, and cell cycle distribution), the multidrug resistance-mediating P-glycoprotein/MDR1 and expression or mutations of oncogenes and tumor suppressor genes (EGFR, RAS, TP53). While resistance of control drugs (daunorubicin, cisplatin, erlotinib) correlated with these classical resistance mechanisms, ergot alkaloids did not. Furthermore, COMPARE and hierarchical cluster analyses were performed of mRNA microarray data to identify genes correlating with sensitivity or resistance to 1-PAT. Twenty-three genes were found with different biological functions (signal transducers, RNA metabolism, ribosome constituents, cell cycle and apoptosis regulators etc.). The expression of only 3/66 neuroreceptor genes correlated with the IC50 values for 1-PAT, suggesting that the psychoactive effects of ergot alkaloids may not play a major role for the cytotoxic activity against cancer cells. In conclusion, the cytotoxicity of ergot alkaloids is not involved in classical mechanisms of drug resistance opening the possibility to bypass resistance and to treat otherwise drug-resistant and refractory tumors. The modes of action are multifactorial, which is a typical feature of many natural compounds.

Role of glycerol 3-phosphate and glycerophosphate acyltransferase in the nutritional control of hepatic triacylglycerol synthesis

PubMed Central

Declercq, Peter E.; Debeer, Luc J.; Mannaerts, Guy P.

1982-01-01

1. Glycerol 3-phosphate content of isolated hepatocytes from starved rats and of glycogen-depleted hepatocytes from fed rats was low and severely limited triacylglycerol synthesis. 2. Raising the glycerol 3-phosphate content by addition of precursors to the cells resulted in a hyperbolic-like relationship between triacylglycerol synthesis and cellular glycerol 3-phosphate content. Statistical analysis of the curves showed no significant differences between the nutritional states either at saturating or at subsaturating glycerol 3-phosphate content. 3. Vmax. of glycerophosphate acyltransferase measured in homogenized hepatocytes was decreased by 30–40% in starvation. There was no change in apparent Km for glycerol 3-phosphate. Since at saturating glycerol 3-phosphate content esterification rates in hepatocytes of both nutritional states were identical, the enzyme is not limiting esterification under this condition. 4. At subsaturating glycerol 3-phosphate content the flux through glycerophosphate acyltransferase necessarily limits esterification. Therefore one would expect a decrease in esterification in starvation under this condition. This was the case when triacylglycerol synthesis was plotted against intracellular glycerol 3-phosphate concentration, calculated from the cellular glycerol 3-phosphate content and the intracellular water space, which was smaller in hepatocytes from starved rats. 5. The data obtained in hepatocytes were extrapolated to the intact liver by using the number of parenchymal cells per g of liver as determined from marker-enzyme analysis and the liver weight per 100g body weight. The extrapolation suggested that glycerol 3-phosphate is limiting esterification in vivo for contents below 0.3–0.4 and 0.5–0.65μmol/g for livers from fed and starved animals respectively. Also for a given fatty acid load and a glycerol 3-phosphate content below 0.3μmol/g the liver may esterify less in the starved state. However, at the glycerol 3-phosphate contents measured in freeze-clamped livers (0.30 and 0.44μmol/g for the fed and starved state respectively), livers in both nutritional states seemed capable of esterifying similar amounts of fatty acids. PMID:7115324

Exosomes and their role in the micro-/macro-environment: a comprehensive review

PubMed Central

Javeed, Naureen; Mukhopadhyay, Debabrata

2017-01-01

The importance of extracellular vesicles (EVs) in cell-cell communication has long been recognized due to their ability to transfer important cellular cargoes such as DNA, mRNA, miRNAs, and proteins to target cells. Compelling evidence supports the role of EVs in the horizontal transfer of cellular material which has the potential to influence normal cellular physiology and promote various disease states. Of the different types of EVs, exosomes have garnered much attention in the past decade due to their abundance in various biological fluids and ability to affect multiple organ systems. The main focus of this review will be on cancer and how cancer-derived exosomes are important mediators of metastasis, angiogenesis, immune modulation, and the tumor macro-/microenvironment. We will also discuss exosomes as potential biomarkers for cancers due to their abundance in biological fluids, ease of uptake, and cellular content. Exosome use in diagnosis, prognosis, and in establishing treatment regimens has enormous potential to revolutionize patient care. PMID:28290182

Exosomes and their role in the micro-/macro-environment: a comprehensive review.

PubMed

Javeed, Naureen; Mukhopadhyay, Debabrata

2017-09-26

The importance of extracellular vesicles (EVs) in cell-cell communication has long been recognized due to their ability to transfer important cellular cargoes such as DNA, mRNA, miRNAs, and proteins to target cells. Compelling evidence supports the role of EVs in the horizontal transfer of cellular material which has the potential to influence normal cellular physiology and promote various disease states. Of the different types of EVs, exosomes have garnered much attention in the past decade due to their abundance in various biological fluids and ability to affect multiple organ systems. The main focus of this review will be on cancer and how cancer-derived exosomes are important mediators of metastasis, angiogenesis, immune modulation, and the tumor macro-/microenvironment. We will also discuss exosomes as potential biomarkers for cancers due to their abundance in biological fluids, ease of uptake, and cellular content. Exosome use in diagnosis, prognosis, and in establishing treatment regimens has enormous potential to revolutionize patient care.

Determination of cellular strains by combined atomic force microscopy and finite element modeling.

PubMed Central

Charras, Guillaume T; Horton, Mike A

2002-01-01

Many organs adapt to their mechanical environment as a result of physiological change or disease. Cells are both the detectors and effectors of this process. Though many studies have been performed in vitro to investigate the mechanisms of detection and adaptation to mechanical strains, the cellular strains remain unknown and results from different stimulation techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by atomic force microscopy with finite element modeling and computational fluid dynamics, we report the cellular strain distributions exerted by common whole-cell straining techniques and from micromanipulation techniques, hence enabling their comparison. Using data from our own analyses and experiments performed by others, we examine the threshold of activation for different signal transduction processes and the strain components that they may detect. We show that modulating cell elasticity, by increasing the F-actin content of the cytoskeleton, or cellular Poisson ratio are good strategies to resist fluid shear or hydrostatic pressure. We report that stray fluid flow in some substrate-stretch systems elicits significant cellular strains. In conclusion, this technique shows promise in furthering our understanding of the interplay among mechanical forces, strain detection, gene expression, and cellular adaptation in physiology and disease. PMID:12124270

Effects of reactive oxygen species on cellular wall disassembly of banana fruit during ripening.

PubMed

Cheng, Guiping; Duan, Xuewu; Shi, John; Lu, Wangjin; Luo, Yunbo; Jiang, Weibo; Jiang, Yueming

2008-07-15

Fruit softening is generally attributed to cell wall disassembly. Experiments were conducted to investigate effects of various reactive oxygen species (ROS) on in vitro cellular wall disassembly of harvested banana fruit. The alcohol-extracted insoluble residue (AEIR) was obtained from the pulp tissues of banana fruit at various ripening stages and then used to examine the disassembly of cellular wall polysaccharides in the presence of superoxide anion (O2(-)), hydrogen peroxide (H2O2) or hydroxyl radical (OH) and their scavengers. The presence of OH accelerated significantly disassembly of cellular wall polysaccharides in terms of the increase in contents of total sugars released and uronic acid, and the decrease in molecular mass of soluble polysaccharides, using gel permeation chromatography. However, the treatment with H2O2 or O2(-) showed no significant effect on the disassembly of cellular wall polysaccharides. Furthermore, the degradation of the de-esterified AEIR was more susceptible to OH attack than the esterified AEIR. In addition, the effect of OH could be inhibited in the presence of OH scavenger. This study suggests that disassembly of cellular wall polysaccharides could be initiated by OH as the solublisation of the polysaccharides increased, which, in turn, accelerated fruit softening. Copyright © 2008 Elsevier Ltd. All rights reserved.

Content dependent selection of image enhancement parameters for mobile displays

NASA Astrophysics Data System (ADS)

Lee, Yoon-Gyoo; Kang, Yoo-Jin; Kim, Han-Eol; Kim, Ka-Hee; Kim, Choon-Woo

2011-01-01

Mobile devices such as cellular phones and portable multimedia player with capability of playing terrestrial digital multimedia broadcasting (T-DMB) contents have been introduced into consumer market. In this paper, content dependent image quality enhancement method for sharpness and colorfulness and noise reduction is presented to improve perceived image quality on mobile displays. Human visual experiments are performed to analyze viewers' preference. Relationship between the objective measures and the optimal values of image control parameters are modeled by simple lookup tables based on the results of human visual experiments. Content dependent values of image control parameters are determined based on the calculated measures and predetermined lookup tables. Experimental results indicate that dynamic selection of image control parameters yields better image quality.

Effect of restricted mobility on RNA content and nucleotide composition and on protein content in motoneurons of spinal cord anterior horns

NASA Technical Reports Server (NTRS)

Gorbunova, A. V.

1980-01-01

An investigation into the effect of hypokinesia on the ribonucleic acid (RNA) content, the nucleotide composition, and dynamics of protein content in the motoneuron of the rat spinal cord anterior horns is described. Methodology and findings are presented. The study results showed that the nucleotide composition of the total cellular RNA at all the studied periods of hypokinesia remained unchanged and is characteristic for the cytoplasmic, high polymer ribosomal RNA. This means that with a change in the functional state of the neuron the newly formed RNA of the nerve cell has the same composition of bases as the original RNA that belongs to the ribosomal type.

Acetyl-CoA carboxylase in Reuber hepatoma cells: variation in enzyme activity, insulin regulation, and cellular lipid content.

PubMed

Bianchi, A; Evans, J L; Nordlund, A C; Watts, T D; Witters, L A

1992-01-01

Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.

Pathway Profiling and Tissue Modeling Using ToxCast HTS Data

EPA Science Inventory

High-throughput screening (HTS) and high-content screening (HCS) assays are providing data-rich studies to probe and profile the direct cellular effects of thousands of chemical compounds in commerce or potentially entering the environment. In vitro profiling may compare unknown ...

Comparison of structural, architectural and mechanical aspects of cellular and acellular bone in two teleost fish.

PubMed

Cohen, Liat; Dean, Mason; Shipov, Anna; Atkins, Ayelet; Monsonego-Ornan, Efrat; Shahar, Ron

2012-06-01

The histological diversity of the skeletal tissues of fishes is impressive compared with that of other vertebrate groups, yet our understanding of the functional consequences of this diversity is limited. In particular, although it has been known since the mid-1800s that a large number of fish species possess acellular bones, the mechanical advantages and consequences of this structural characteristic - and therefore the nature of the evolution of this feature - remain unclear. Although several studies have examined the material properties of fish bone, these have used a variety of techniques and there have been no direct contrasts of acellular and cellular bone. We report on a comparison of the structural and mechanical properties of the ribs and opercula between two freshwater fish - the common carp Cyprinus carpio (a fish with cellular bone) and the tilapia Oreochromis aureus (a fish with acellular bone). We used light microscopy to show that the bones in both fish species exhibit poor blood supply and possess discrete tissue zones, with visible layering suggesting differences in the underlying collagen architecture. We performed identical micromechanical testing protocols on samples of the two bone types to determine the mechanical properties of the bone material of opercula and ribs. Our data support the consensus of literature values, indicating that Young's moduli of cellular and acellular bones are in the same range, and lower than Young's moduli of the bones of mammals and birds. Despite these similarities in mechanical properties between the bone tissues of the fish species tested here, cellular bone had significantly lower mineral content than acellular bone; furthermore, the percentage ash content and bone mineral density values (derived from micro-CT scans) show that the bone of these fishes is less mineralized than amniote bone. Although we cannot generalize from our data to the numerous remaining teleost species, the results presented here suggest that while cellular and acellular fish bone may perform similarly from a mechanical standpoint, there are previously unappreciated differences in the structure and composition of these bone types.

Potential of New Isolates of Dunaliella Salina for Natural β-Carotene Production

PubMed Central

Xu, Yanan; Wosu, Chiziezi I.; Ben-Amotz, Ami

2018-01-01

The halotolerant microalga Dunaliella salina has been widely studied for natural β-carotene production. This work shows biochemical characterization of three newly isolated Dunaliella salina strains, DF15, DF17, and DF40, compared with D. salina CCAP 19/30 and D. salina UTEX 2538 (also known as D. bardawil). Although all three new strains have been genetically characterized as Dunaliella salina strains, their ability to accumulate carotenoids and their capacity for photoprotection against high light stress are different. DF15 and UTEX 2538 reveal great potential for producing a large amount of β-carotene and maintained a high rate of photosynthesis under light of high intensity; however, DF17, DF40, and CCAP 19/30 showed increasing photoinhibition with increasing light intensity, and reduced contents of carotenoids, in particular β-carotene, suggesting that the capacity of photoprotection is dependent on the cellular content of carotenoids, in particular β-carotene. Strong positive correlations were found between the cellular content of all-trans β-carotene, 9-cis β-carotene, all-trans α-carotene and zeaxanthin but not lutein in the D. salina strains. Lutein was strongly correlated with respiration in photosynthetic cells and strongly related to photosynthesis, chlorophyll and respiration, suggesting an important and not hitherto identified role for lutein in coordinated control of the cellular functions of photosynthesis and respiration in response to changes in light conditions, which is broadly conserved in Dunaliella strains. Statistical analysis based on biochemical data revealed a different grouping strategy from the genetic classification of the strains. The significance of these data for strain selection for commercial carotenoid production is discussed. PMID:29389891

Cytoprotective Effect of Hydroalcoholic Extract of Pinus eldarica Bark against H2O2-Induced Oxidative Stress in Human Endothelial Cells

PubMed Central

Babaee, Fatemeh; Safaeian, Leila; Zolfaghari, Behzad; Haghjoo Javanmard, Shaghayegh

2016-01-01

Background: Pinus eldarica is a widely growing pine in Iran consisting of biologically active constituents with antioxidant properties. This study investigates the effect of hydroalcoholic extract of P. eldarica bark against oxidative damage induced by hydrogen peroxide (H2O2) in human umbilical vein endothelial cells (HUVECs). Methods: The total phenolic content of P. eldarica extract was determined using Folin-Ciocalteu method. The cytotoxicity of P. eldarica extract (25-1000 µg/ml) on HUVECs was assessed using 3-(4,5- Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method. Cytoprotective effect of P. eldarica extract (25-500 µg/ml) on H2O2-induced oxidative stress was also evaluated by MTT assay. The intra- and extra-cellular hydroperoxides concentration and ferric reducing antioxidant power (FRAP) were measured in pretreated cells. Results: The total phenolic content of P. eldarica extract was estimated as 37.04±1.8% gallic acid equivalent. P. eldarica extract (25-1000 µg/ml) had no cytotoxic effect on HUVECs viability. The pretreatment of HUVECs with P. eldarica extract at the concentrations of 50-500 µg/ml significantly reduced the cytotoxicity of H2O2. P. eldarica extract decreased hydroperoxides concentration and increased FRAP value in intra-cellular fluid at the concentration range of 100-500 µg/ml and in extra-cellular fluid at the concentration range of 25-500 µg/ml. Conclusions: This study revealed the antioxidant and cytoprotective effects of P. eldarica extract against H2O2-induced oxidative stress in HUVECs. Concerning the high content of phenolic compounds in P. eldarica, more research is needed to evaluate its clinical value in endothelial dysfunction and in other oxidative conditions. PMID:26931383

The omnivorous Tyrolean Iceman: colon contents (meat, cereals, pollen, moss and whipworm) and stable isotope analyses.

PubMed Central

Dickson, J H; Oeggl, K; Holden, T G; Handley, L L; O'Connell, T C; Preston, T

2000-01-01

The contents of the colon of the Tyrolean Iceman who lived ca. 5300 years ago include muscle fibres, cereal remains, a diversity of pollen, and most notably that of the hop hornbeam (Ostrya carpinifolia) retaining cellular contents, as well as a moss leaf (Neckera complanata) and eggs of the parasitic whipworm (Trichuris trichiura). Based almost solely on stable isotope analyses and ignoring the work on the colon contents, two recently published papers on the Iceman's diet draw ill-founded conclusions about vegetarianism and even veganism. Neither the pollen nor the moss is likely to have been deliberately consumed as food by the Iceman. All the available evidence concerning the Iceman's broad-based diet is reviewed and the significance of the colon contents for matters other than assessment of food intake is outlined. PMID:11205345

Particulate matter from both heavy fuel oil and diesel fuel shipping emissions show strong biological effects on human lung cells at realistic and comparable in vitro exposure conditions.

PubMed

Oeder, Sebastian; Kanashova, Tamara; Sippula, Olli; Sapcariu, Sean C; Streibel, Thorsten; Arteaga-Salas, Jose Manuel; Passig, Johannes; Dilger, Marco; Paur, Hanns-Rudolf; Schlager, Christoph; Mülhopt, Sonja; Diabaté, Silvia; Weiss, Carsten; Stengel, Benjamin; Rabe, Rom; Harndorf, Horst; Torvela, Tiina; Jokiniemi, Jorma K; Hirvonen, Maija-Riitta; Schmidt-Weber, Carsten; Traidl-Hoffmann, Claudia; BéruBé, Kelly A; Wlodarczyk, Anna J; Prytherch, Zoë; Michalke, Bernhard; Krebs, Tobias; Prévôt, André S H; Kelbg, Michael; Tiggesbäumker, Josef; Karg, Erwin; Jakobi, Gert; Scholtes, Sorana; Schnelle-Kreis, Jürgen; Lintelmann, Jutta; Matuschek, Georg; Sklorz, Martin; Klingbeil, Sophie; Orasche, Jürgen; Richthammer, Patrick; Müller, Laarnie; Elsasser, Michael; Reda, Ahmed; Gröger, Thomas; Weggler, Benedikt; Schwemer, Theo; Czech, Hendryk; Rüger, Christopher P; Abbaszade, Gülcin; Radischat, Christian; Hiller, Karsten; Buters, Jeroen T M; Dittmar, Gunnar; Zimmermann, Ralf

2015-01-01

Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon ("soot"). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.

Biochemical and Cellular Assessment of Acetabular Chondral Flaps Identified During Hip Arthroscopy.

PubMed

Hariri, Sanaz; Truntzer, Jeremy; Smith, Robert Lane; Safran, Marc R

2015-06-01

To analyze chondral flaps debrided during hip arthroscopy to determine their biochemical and cellular composition. Thirty-one full-thickness acetabular chondral flaps were collected during hip arthroscopy. Biochemical analysis was undertaken in 21 flaps from 20 patients, and cellular viability was determined in 10 flaps from 10 patients. Biochemical analysis included concentrations of (1) DNA (an indicator of chondrocyte content), (2) hydroxyproline (an indicator of collagen content), and (3) glycosaminoglycan (an indicator of chondrocyte biosynthesis). Higher values for these parameters indicated more healthy tissue. The flaps were examined to determine the percentage of viable chondrocytes. The percentage of acetabular chondral flap specimens that had concentrations within 1 SD of the mean values reported in previous normal cartilage studies was 38% for DNA, 0% for glycosaminoglycan, and 43% for hydroxyproline. The average cellular viability of our acetabular chondral flap specimens was 39% (SD, 14%). Only 2 of the 10 specimens had more than half the cells still viable. There was no correlation between (1) the gross examination of the joint or knowledge of the patient's demographic characteristics and symptoms and (2) biochemical properties and cell viability of the flap, with one exception: a degenerative appearance of the surrounding cartilage correlated with a higher hydroxyproline concentration. Although full-thickness acetabular chondral flaps can appear normal grossly, the biochemical properties and percentage of live chondrocytes in full-thickness chondral flaps encountered in hip arthroscopy show that this tissue is not normal. There has been recent interest in repairing chondral flaps encountered during hip arthroscopy. These data suggest that acetabular chondral flaps are not biochemically and cellularly normal. Although these flaps may still be valuable mechanically and/or as a scaffold in some conductive or inductive capacity, further study is required to assess the clinical benefit of repair. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Particulate Matter from Both Heavy Fuel Oil and Diesel Fuel Shipping Emissions Show Strong Biological Effects on Human Lung Cells at Realistic and Comparable In Vitro Exposure Conditions

PubMed Central

Dilger, Marco; Paur, Hanns-Rudolf; Schlager, Christoph; Mülhopt, Sonja; Diabaté, Silvia; Weiss, Carsten; Stengel, Benjamin; Rabe, Rom; Harndorf, Horst; Torvela, Tiina; Jokiniemi, Jorma K.; Hirvonen, Maija-Riitta; Schmidt-Weber, Carsten; Traidl-Hoffmann, Claudia; BéruBé, Kelly A.; Wlodarczyk, Anna J.; Prytherch, Zoë; Michalke, Bernhard; Krebs, Tobias; Prévôt, André S. H.; Kelbg, Michael; Tiggesbäumker, Josef; Karg, Erwin; Jakobi, Gert; Scholtes, Sorana; Schnelle-Kreis, Jürgen; Lintelmann, Jutta; Matuschek, Georg; Sklorz, Martin; Klingbeil, Sophie; Orasche, Jürgen; Richthammer, Patrick; Müller, Laarnie; Elsasser, Michael; Reda, Ahmed; Gröger, Thomas; Weggler, Benedikt; Schwemer, Theo; Czech, Hendryk; Rüger, Christopher P.; Abbaszade, Gülcin; Radischat, Christian; Hiller, Karsten; Buters, Jeroen T. M.; Dittmar, Gunnar; Zimmermann, Ralf

2015-01-01

Background Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. Objectives To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. Methods Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. Results The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon (“soot”). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. Conclusions Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices. PMID:26039251

Vacuolar amino acid transporter Avt5p is responsible for lithium uptake in Schizosaccharomyces pombe.

PubMed

Iwaki, Tomoko; Sekito, Takayuki; Kakinuma, Yoshimi

2010-01-01

The fission yeast Schizosaccharomyces pombe was sensitive to salinity; cell growth was stopped by 0.5 M NaCl and by 10 mM LiCl. The avt5+ gene encodes a vacuolar transporter with a broad specificity for amino acids. We found that the avt5Delta mutant became highly tolerant of Li+ and Na+ in growth. Concanamycin A-sensitive Li+ uptake as well as cellular Li+ content was lower in the avt5 mutant, suggesting a role of Avt5p in cellular uptake of toxic Li+.

The effects of media on sleep.

PubMed

Van den Bulck, Jan

2010-12-01

The media are an important part of young people's lives, but television, computer games, Internet use, cellular phone use, and even book reading threaten healthy sleep. Adults do not fully comprehend the ways in which young people use various media. Media use is a type of behavior that may displace sleep time or shorten it. Media content may lead to overexcitement or cause recurring nightmares. The cellular telephone is a particular threat. Parents may also use media excessively, establishing an unhealthy environment that may lead to sleep dysfunction in children and adolescents. Therefore, anticipatory guidance for healthy behavioral changes should be focused on the family.

Cellular and molecular responses of Neurospora crassa to non-thermal plasma at atmospheric pressure

NASA Astrophysics Data System (ADS)

Park, Gyungsoon; Ryu, Young H.; Hong, Young J.; Choi, Eun H.; Uhm, Han S.

2012-02-01

Filamentous fungi have been rarely explored in terms of plasma treatments. This letter presents the cellular and molecular responses of the filamentous fungus Neurospora crassa to an argon plasma jet at atmospheric pressure. The viability and cell morphology of N. crassa spores exposed to plasma were both significantly reduced depending on the exposure time when treated in water. The intracellular genomic DNA content was dramatically reduced in fungal tissues after a plasma treatment and the transcription factor tah-3 was found to be required for fungal tolerance to a harsh plasma environment.

Developing the conceptual instructional design with inquiry-based instruction model of secondary students at the 10th grade level on digestion system and cellular degradation issue

NASA Astrophysics Data System (ADS)

Rotjanakunnatam, Boonthida; Chayaburakul, Kanokporn

2018-01-01

The aims of this research study was to develop the conceptual instructional design with the Inquiry-Based Instruction Model (IBIM) of secondary students at the 10th grade level on Digestion System and Cellular Degradation issue using both oxygen and oxygen-degrading cellular nutrients were designed instructional model with a sample size of 45 secondary students at the 10th Grade level. Data were collected by asking students to do a questionnaire pre and post learning processes. The questionnaire consists of two main parts that composed of students' perception questionnaire and the questionnaire that asked the question answer concept for the selected questionnaire. The 10-item Conceptual Thinking Test (CTT) was assessed students' conceptual thinking evaluation that it was covered in two main concepts, namely; Oxygen degradation nutrients and degradation nutrients without oxygen. The data by classifying students' answers into 5 groups and measuring them in frequency and a percentage of students' performances of their learning pre and post activities with the Inquiry-Based Instruction Model were analyzed as a tutorial. The results of this research found that: After the learning activities with the IBIM, most students developed concepts of both oxygen and oxygen-degrading cellular nutrients in the correct, complete and correct concept, and there are a number of students who have conceptual ideas in the wrong concept, and no concept was clearly reduced. However, the results are still found that; some students have some misconceptions, such as; the concept of direction of electron motion and formation of the ATP of bioactivities of life. This cause may come from the nature of the content, the complexity, the continuity, the movement, and the time constraints only in the classroom. Based on this research, it is suggested that some students may take some time, and the limited time in the classroom to their learning activity with content creation content binding and dramatic storytelling increases in a relaxed classroom learning environment.

Multifunctional High Drug Loading Nanocarriers for Cancer Drug Delivery

NASA Astrophysics Data System (ADS)

Jin, Erlei

2011-12-01

Most anticancer drugs have poor water-solubility, rapid blood clearance, low tumor-selectivity and severe systemic toxicity to healthy tissues. Thus, polymeric nanocarriers have been widely explored for anticancer drugs to solve these problems. However, polymer nanocarriers developed to date still suffer drawbacks including low drug loading contents, premature drug release, slow cellular internalization, slow intracellular drug release and thereby low therapeutic efficiency in cancer thermotherapy. Accordingly, in this dissertation, functional nanocapsules and nanoparticles including high drug loading liposome-like nanocapsules, high drug loading phospholipid-mimic nanocapsules with fast intracellular drug release, high drug loading charge-reversal nanocapsules, TAT based long blood circulation nanoparticles and charge-reversal nuclear targeted nanoparticles are designed and synthesized. These functional carriers have advantages such as high drug loading contents without premature drug release, fast cellular internalization and intracellular drug release, nuclear targeted delivery and long blood circulation. As a result, all these drug carriers show much higher in vitro and in vivo anti-cancer activities.

Measurement of cellular copper levels in Bacillus megaterium during exponential growth and sporulation.

PubMed

Krueger, W B; Kolodziej, B J

1976-01-01

Both atomic absorption spectrophotometry (AAS) and neutron activation analysis have been utilized to determine cellular Cu levels in Bacillus megaterium ATCC 19213. Both methods were selected for their sensitivity to detection of nanogram quantities of Cu. Data from both methods demonstrated identical patterms of Cu uptake during exponenetial growth and sporulation. Late exponential phase cells contained less Cu than postexponential t2 cells while t5 cells contained amounts equivalent to exponential cells. The t11 phase-bright forespore containing cells had a higher Cu content than those of earlier time periods, and the free spores had the highest Cu content. Analysis of the culture medium by AAS corroborated these data by showing concomitant Cu uptake during exponential growth and into t2 postexponential phase of sporulation. From t2 to t4, Cu egressed from the cells followed by a secondary uptake during the maturation of phase-dark forespores into phase-bright forespores (t6--t9).

Effects of sodium fluoride on blood cellular and humoral immunity in mice.

PubMed

Guo, Hongrui; Kuang, Ping; Luo, Qin; Cui, Hengmin; Deng, Huidan; Liu, Huan; Lu, Yujiao; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Li, Yinglun; Wang, Xun; Zhao, Ling

2017-10-17

Exposure to high fluorine can cause toxicity in human and animals. Currently, there are no systematic studies on effects of high fluorine on blood cellular immunity and humoral immunity in mice. We evaluated the alterations of blood cellular immunity and humoral immunity in mice by using flow cytometry and ELISA. In the cellular immunity, we found that sodium fluoride (NaF) in excess of 12 mg/Kg resulted in a significant decrease in the percentages of CD3 + , CD3 + CD4 + , CD3 + CD8 + T lymphocytes in the peripheral blood. Meanwhile, serum T helper type 1 (Th1) cytokines including interleukin (IL)-2, interferon (IFN)-γ, tumor necrosis factor (TNF), and Th2 cytokines including IL-4, IL-6, IL-10, and Th17 cytokine (IL-17A) contents were decreased. In the humoral immunity, NaF reduced the peripheral blood percentages of CD19 + B lymphocytes and serum immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM). The above results show that NaF can reduce blood cellular and humoral immune function in mice, providing an excellent animal model for clinical studies on immunotoxicity-related fluorosis.

Optical scatter imaging of cellular and mitochondrial swelling in brain tissue models of stroke

NASA Astrophysics Data System (ADS)

Johnson, Lee James

2001-08-01

The severity of brain edema resulting from a stroke can determine a patient's survival and the extent of their recovery. Cellular swelling is the microscopic source of a significant part of brain edema. Mitochondrial swelling also appears to be a determining event in the death or survival of the cells that are injured during a stroke. Therapies for reducing brain edema are not effective in many cases and current treatments of stroke do not address mitochondrial swelling at all. This dissertation is motivated by the lack of a complete understanding of cellular swelling resulting from stroke and the lack of a good method to begin to study mitochondrial swelling resulting from stroke in living brain tissue. In this dissertation, a novel method of detecting mitochondrial and cellular swelling in living hippocampal slices is developed and validated. The system is used to obtain spatial and temporal information about cellular and mitochondrial swelling resulting from various models of stroke. The effect of changes in water content on light scatter and absorption are examined in two models of brain edema. The results of this study demonstrate that optical techniques can be used to detect changes in water content. Mie scatter theory, the theoretical basis of the dual- angle scatter ratio imaging system, is presented. Computer simulations based on Mie scatter theory are used to determine the optimal angles for imaging. A detailed account of the early systems is presented to explain the motivations for the system design, especially polarization, wavelength and light path. Mitochondrial sized latex particles are used to determine the system response to changes in scattering particle size and concentration. The dual-angle scatter ratio imaging system is used to distinguish between osmotic and excitotoxic models of stroke injury. Such distinction cannot be achieved using the current techniques to study cellular swelling in hippocampal slices. The change in the scatter ratio is then shown to correlate to mitochondrial swelling, as observed with electron microscopy. The system is finally used to study mitochondrial and cellular swelling. Evidence of the susceptibility of certain hippocampal regions, CA1 and the dentate gyrus, to exhibit mitochondrial swelling as the result of oxygen and glucose deprivation is presented. In addition, for the first time, the time course of mitochondrial swelling is seen. Finally, experiments with scatter imaging and measurement of nitric oxide with carbon fiber electrodes demonstrate a clear link between nitric oxide and cellular swelling. A potential mechanism of the action of nitric oxide is evaluated. Nitric oxide appears to act to cause cellular swelling without the release of glutamate. The use of targeted nitric oxide inhibitors may be useful for the reduction of edema.

Polysaccharide production by a reduced pigmentation mutant of Aureobasidium pullulans NYS-1.

PubMed

West, T P; Strohfus, B

2001-08-01

To isolate a reduced pigmentation mutant of Aureobasidium pullulans NYS-1 and characterize its cellular pigmentation plus its polysaccharide and biomass production relative to carbon source. Cellular pigmentation, polysaccharide levels and biomass production by the isolated mutant NYSRP-1 were analysed relative to carbon source. Cellular pigmentation of the mutant was lower than its parent strain using either carbon source. The mutant elaborated higher polysaccharide levels on sucrose than on corn syrup. The pullulan content of the polysaccharide synthesized and biomass production by the mutant rose as the carbon source concentration was increased. It is feasible to isolate a reduced pigmentation mutant from strain NYS-1 that exhibits elevated polysaccharide production using corn syrup as a carbon source. The mutant provides an advantage for commercial pullulan production because of its reduced pigmentation and enhanced polysaccharide synthesis.

Lipid Content and Cryotolerance of Bakers' Yeast in Frozen Doughs †

PubMed Central

Gélinas, Pierre; Fiset, Gisèle; Willemot, Claude; Goulet, Jacques

1991-01-01

The relationship between lipid content and tolerance to freezing at −50°C was studied in Saccharomyces cerevisiae grown under batch or fed-batch mode and various aeration and temperature conditions. A higher free-sterol-to-phospholipid ratio as well as higher free sterol and phospholipid contents correlated with the superior cryoresistance in dough or in water of the fed-batch-grown compared with the batch-grown cells. For both growth modes, the presence of excess dissolved oxygen in the culture medium greatly improved yeast cryoresistance and trehalose content (P. Gélinas, G. Fiset, A. LeDuy, and J. Goulet, Appl. Environ. Microbiol. 26:2453-2459, 1989) without significantly changing the lipid profile. Under the batch or fed-batch modes, no correlation was found between the cryotolerance of bakers' yeast and the total cellular lipid content, the total sterol content, the phospholipid unsaturation index, the phosphate or crude protein content, or the yeast cell morphology (volume and roundness). PMID:16348412

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

PubMed Central

Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

2015-01-01

NAD is an essential metabolite that exists in NAD+ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+ and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ. PMID:25730862

The arginine metabolite agmatine protects mitochondrial function and confers resistance to cellular apoptosis

PubMed Central

Arndt, Mary Ann; Battaglia, Valentina; Parisi, Eva; Lortie, Mark J.; Isome, Masato; Baskerville, Christopher; Pizzo, Donald P.; Ientile, Riccardo; Colombatto, Sebastiano; Toninello, Antonio; Satriano, Joseph

2009-01-01

Agmatine, an endogenous metabolite of arginine, selectively suppresses growth in cells with high proliferative kinetics, such as transformed cells, through depletion of intracellular polyamine levels. In the present study, we depleted intracellular polyamine content with agmatine to determine if attrition by cell death contributes to the growth-suppressive effects. We did not observe an increase in necrosis, DNA fragmentation, or chromatin condensation in Ha-Ras-transformed NIH-3T3 cells administered agmatine. In response to Ca2+-induced oxidative stress in kidney mitochondrial preparations, agmatine demonstrated attributes of a free radical scavenger by protecting against the oxidation of sulfhydryl groups and decreasing hydrogen peroxide content. The functional outcome was a protective effect against Ca2+-induced mitochondrial swelling and mitochondrial membrane potential collapse. We also observed decreased expression of proapoptotic Bcl-2 family members and of execution caspase-3, implying antiapoptotic potential. Indeed, we found that apoptosis induced by camptothecin or 5-fluorourocil was attenuated in cells administered agmatine. Agmatine may offer an alternative to the ornithine decarboxylase inhibitor difluoromethyl ornithine for depletion of intracellular polyamine content while avoiding the complications of increasing polyamine import and reducing the intracellular free radical scavenger capacity of polyamines. Depletion of intracellular polyamine content with agmatine suppressed cell growth, yet its antioxidant capacity afforded protection from mitochondrial insult and resistance to cellular apoptosis. These results could explain the beneficial outcomes observed with agmatine in models of injury and disease. PMID:19321739

Quantifying Trophic and Demographic Rates of Plankton-Rich Layers in East Sound, Orcas Island, Washington

DTIC Science & Technology

2008-01-01

ratios (Welschmeyer & Lorenzen, 1984), heterotrophic protist grazing rates (Montagnes & Lessard, 1999), and cellular carbon content (Putt & Stoecker 1989...dinoflagellates, diatoms, and other protist plankton. Limnology and Oceanography 45: 569-579 Menden-Deuer S, EJ Lessard & J Satterberg. 2001. Effect of

EFFECT OF FLUID SHEAR AND IRRADIANCE ON POPULATION GROWTH AND CELLULAR TOXIN CONTENT OF THE DINOFLAGELLATE ALEXANDRIUM FUNDYENSE.

EPA Science Inventory

The potential for in situ turbulence to inhibit dinoflagellate population growth has been demonstrated by experimentally exposing dinoflagellate cultures to quantified shear flow. However, despite interest in understanding environmental factors that affect the growth of toxic din...

Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae

PubMed Central

Arslan, Defne; Legendre, Matthieu; Seltzer, Virginie; Abergel, Chantal; Claverie, Jean-Michel

2011-01-01

Mimivirus, a DNA virus infecting acanthamoeba, was for a long time the largest known virus both in terms of particle size and gene content. Its genome encodes 979 proteins, including the first four aminoacyl tRNA synthetases (ArgRS, CysRS, MetRS, and TyrRS) ever found outside of cellular organisms. The discovery that Mimivirus encoded trademark cellular functions prompted a wealth of theoretical studies revisiting the concept of virus and associated large DNA viruses with the emergence of early eukaryotes. However, the evolutionary significance of these unique features remained impossible to assess in absence of a Mimivirus relative exhibiting a suitable evolutionary divergence. Here, we present Megavirus chilensis, a giant virus isolated off the coast of Chile, but capable of replicating in fresh water acanthamoeba. Its 1,259,197-bp genome is the largest viral genome fully sequenced so far. It encodes 1,120 putative proteins, of which 258 (23%) have no Mimivirus homologs. The 594 Megavirus/Mimivirus orthologs share an average of 50% of identical residues. Despite this divergence, Megavirus retained all of the genomic features characteristic of Mimivirus, including its cellular-like genes. Moreover, Megavirus exhibits three additional aminoacyl-tRNA synthetase genes (IleRS, TrpRS, and AsnRS) adding strong support to the previous suggestion that the Mimivirus/Megavirus lineage evolved from an ancestral cellular genome by reductive evolution. The main differences in gene content between Mimivirus and Megavirus genomes are due to (i) lineages specific gains or losses of genes, (ii) lineage specific gene family expansion or deletion, and (iii) the insertion/migration of mobile elements (intron, intein). PMID:21987820

Glutamine's protection against cellular injury is dependent on heat shock factor-1.

PubMed

Morrison, Angela L; Dinges, Martin; Singleton, Kristen D; Odoms, Kelli; Wong, Hector R; Wischmeyer, Paul E

2006-06-01

Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1(+/+)) and knockout (HSF-1(-/-)) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1(+/+) cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1(-/-) cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1(-/-) cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation.

Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems

PubMed Central

Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

2016-01-01

Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

Cellular manganese content is developmentally regulated in human dopaminergic neurons

NASA Astrophysics Data System (ADS)

Kumar, Kevin K.; Lowe, Edward W., Jr.; Aboud, Asad A.; Neely, M. Diana; Redha, Rey; Bauer, Joshua A.; Odak, Mihir; Weaver, C. David; Meiler, Jens; Aschner, Michael; Bowman, Aaron B.

2014-10-01

Manganese (Mn) is both an essential biological cofactor and neurotoxicant. Disruption of Mn biology in the basal ganglia has been implicated in the pathogenesis of neurodegenerative disorders, such as parkinsonism and Huntington's disease. Handling of other essential metals (e.g. iron and zinc) occurs via complex intracellular signaling networks that link metal detection and transport systems. However, beyond several non-selective transporters, little is known about the intracellular processes regulating neuronal Mn homeostasis. We hypothesized that small molecules that modulate intracellular Mn could provide insight into cell-level Mn regulatory mechanisms. We performed a high throughput screen of 40,167 small molecules for modifiers of cellular Mn content in a mouse striatal neuron cell line. Following stringent validation assays and chemical informatics, we obtained a chemical `toolbox' of 41 small molecules with diverse structure-activity relationships that can alter intracellular Mn levels under biologically relevant Mn exposures. We utilized this toolbox to test for differential regulation of Mn handling in human floor-plate lineage dopaminergic neurons, a lineage especially vulnerable to environmental Mn exposure. We report differential Mn accumulation between developmental stages and stage-specific differences in the Mn-altering activity of individual small molecules. This work demonstrates cell-level regulation of Mn content across neuronal differentiation.

Exosomes as divine messengers: are they the Hermes of modern molecular oncology?

PubMed Central

Braicu, C; Tomuleasa, C; Monroig, P; Cucuianu, A; Berindan-Neagoe, I; Calin, G A

2015-01-01

Exosomes are cell-derived vesicles that convey key elements with the potential to modulate intercellular communication. They are known to be secreted from all types of cells, and are crucial messengers that can regulate cellular processes by ‘trafficking' molecules from cells of one tissue to another. The exosomal content has been shown to be broad, composed of different types of cytokines, growth factors, proteins, or nucleic acids. Besides messenger RNA (mRNA) they can also contain noncoding transcripts such as microRNAs (miRNAs), which are small endogenous cellular regulators of protein expression. In diseases such as cancer, exosomes can facilitate tumor progression by altering their vesicular content and supplying the tumor niche with molecules that favor the progression of oncogenic processes such as proliferation, invasion and metastasis, or even drug resistance. The packaging of their molecular content is known to be tissue specific, a fact that makes them interesting tools in clinical diagnostics and ideal candidates for biomarkers. In the current report, we describe the main properties of exosomes and explain their involvement in processes such as cell differentiation and cell death. Furthermore, we emphasize the need of developing patient-targeted treatments by applying the conceptualization of exosomal-derived miRNA-based therapeutics. PMID:25236394

Acetyl-L-carnitine activates the peroxisome proliferator-activated receptor-γ coactivators PGC-1α/PGC-1β-dependent signaling cascade of mitochondrial biogenesis and decreases the oxidized peroxiredoxins content in old rat liver.

PubMed

Pesce, Vito; Nicassio, Luigi; Fracasso, Flavio; Musicco, Clara; Cantatore, Palmiro; Gadaleta, Maria Nicola

2012-04-01

The behavior of the peroxisome proliferator-activated receptor-γ coactivators PGC-1α/PGC-β-dependent mitochondrial biogenesis signaling pathway, as well as the level of some antioxidant enzymes and proteins involved in mitochondrial dynamics in the liver of old rats before and after 2 months of acetyl-L-carnitine (ALCAR) supplementation, was tested. The results reveal that ALCAR treatment is able to reverse the age-associated decline of PGC-1α, PGC-1β, nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 (ND1), and cytochrome c oxidase subunit IV (COX IV) protein levels, of mitochondrial DNA (mtDNA) content, and of citrate synthase activity. Moreover, it partially reverses the mitochondrial superoxide dismutase 2 (SOD2) decline and reduces the cellular content of oxidized peroxiredoxins. These data demonstrate that ALCAR treatment is able to promote in the old rat liver a new mitochondrial population that can contribute to the cellular oxidative stress reduction. Furthermore, a remarkable decline of Drp1 and of Mfn2 proteins is reported here for the first time, suggesting a reduced mitochondrial dynamics in aging liver with no effect of ALCAR treatment.

Impaired Photosynthesis in Phosphatidylglycerol-Deficient Mutant of Cyanobacterium Anabaena sp. PCC7120 with a Disrupted Gene Encoding a Putative Phosphatidylglycerophosphatase1

PubMed Central

Wu, Feng; Yang, Zhenle; Kuang, Tingyun

2006-01-01

Phosphatidylglycerol (PG) is a ubiquitous phospholipid in thylakoid membranes of cyanobacteria and chloroplasts and plays an important role in the structure and function of photosynthetic membranes. The last step of the PG biosynthesis is dephosphorylation of phosphatidylglycerophosphate (PGP) catalyzed by PGP phosphatase. However, the gene-encoding PGP phosphatase has not been identified and cloned from cyanobacteria or higher plants. In this study, we constructed a PG-deficient mutant from cyanobacterium Anabaena sp. PCC7120 with a disrupted gene (alr1715, a gene for Alr1715 protein, GenBank accession no. BAB78081) encoding a putative PGP phosphatase. The obtained mutant showed an approximately 30% reduction in the cellular content of PG. Following the reduction in the PG content, the photoautotrophical growth of the mutant was restrained, and the cellular content of chlorophyll was decreased. The decreases in net photosynthetic and photosystem II (PSII) activities on a cell basis also occurred in this mutant. Simultaneously, the photochemical efficiency of PSII was considerably declined, and less excitation energy was transferred toward PSII. These findings demonstrate that the alr1715 gene of Anabaena sp. PCC7120 is involved in the biosynthesis of PG and essential for photosynthesis. PMID:16815953

Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.

PubMed

Hühner, Jens; Ingles-Prieto, Álvaro; Neusüß, Christian; Lämmerhofer, Michael; Janovjak, Harald

2015-02-01

Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HDL cholesterol transport during inflammation.

PubMed

van der Westhuyzen, Deneys R; de Beer, Frederick C; Webb, Nancy R

2007-04-01

The aim of this article is to review recent advances made towards understanding how inflammation and acute phase proteins, particularly serum amyloid A and group IIa secretory phospholipase A2, may alter reverse cholesterol transport by HDL during inflammation and the acute phase response. Findings suggest that the decreased apoA-I content and markedly increased serum amyloid A content in HDL during the acute phase response result from reciprocal and coordinate transcriptional regulation of these proteins as well as HDL remodeling by group IIa secretory phospholipase A2. Serum amyloid A functions efficiently in a lipid-free or lipid-poor form to promote cholesterol efflux by ATP binding cassette protein ABCA1, evidently by functioning directly as an acceptor for cholesterol efflux as well as by increasing the availability of cellular free cholesterol. Serum amyloid A increases the ability of acute phase HDL to serve as an acceptor for SR-BI-dependent cellular cholesterol efflux. Altered remodeling of HDL by group IIa secretory phospholipase A2 in concert with cholesterol ester transfer protein may contribute to the generation of lipid-poor apoA-I and serum amyloid A acceptors for cholesterol efflux. Current data support a model for the acute phase response in which serum amyloid A and sPLA2-IIa, present at sites of inflammation and tissue damage, play a protective role by enhancing cellular cholesterol efflux, thereby promoting the removal of excess cholesterol from macrophages.

Whole Tumor Histogram-profiling of Diffusion-Weighted Magnetic Resonance Images Reflects Tumorbiological Features of Primary Central Nervous System Lymphoma.

PubMed

Schob, Stefan; Münch, Benno; Dieckow, Julia; Quäschling, Ulf; Hoffmann, Karl-Titus; Richter, Cindy; Garnov, Nikita; Frydrychowicz, Clara; Krause, Matthias; Meyer, Hans-Jonas; Surov, Alexey

2018-04-01

Diffusion weighted imaging (DWI) quantifies motion of hydrogen nuclei in biological tissues and hereby has been used to assess the underlying tissue microarchitecture. Histogram-profiling of DWI provides more detailed information on diffusion characteristics of a lesion than the standardly calculated values of the apparent diffusion coefficient (ADC)-minimum, mean and maximum. Hence, the aim of our study was to investigate, which parameters of histogram-profiling of DWI in primary central nervous system lymphoma can be used to specifically predict features like cellular density, chromatin content and proliferative activity. Pre-treatment ADC maps of 21 PCNSL patients (8 female, 13 male, 28-89 years) from a 1.5T system were used for Matlab-based histogram profiling. Results of histopathology (H&E staining) and immunohistochemistry (Ki-67 expression) were quantified. Correlations between histogram-profiling parameters and neuropathologic examination were calculated using SPSS 23.0. The lower percentiles (p10 and p25) showed significant correlations with structural parameters of the neuropathologic examination (cellular density, chromatin content). The highest percentile, p90, correlated significantly with Ki-67 expression, resembling proliferative activity. Kurtosis of the ADC histogram correlated significantly with cellular density. Histogram-profiling of DWI in PCNSL provides a comprehensible set of parameters, which reflect distinct tumor-architectural and tumor-biological features, and hence, are promising biomarkers for treatment response and prognosis. Copyright © 2018. Published by Elsevier Inc.

Vitamin B12 deficiency results in severe oxidative stress, leading to memory retention impairment in Caenorhabditis elegans.

PubMed

Bito, Tomohiro; Misaki, Taihei; Yabuta, Yukinori; Ishikawa, Takahiro; Kawano, Tsuyoshi; Watanabe, Fumio

2017-04-01

Oxidative stress is implicated in various human diseases and conditions, such as a neurodegeneration, which is the major symptom of vitamin B 12 deficiency, although the underlying disease mechanisms associated with vitamin B 12 deficiency are poorly understood. Vitamin B 12 deficiency was found to significantly increase cellular H 2 O 2 and NO content in Caenorhabditis elegans and significantly decrease low molecular antioxidant [reduced glutathione (GSH) and L-ascorbic acid] levels and antioxidant enzyme (superoxide dismutase and catalase) activities, indicating that vitamin B 12 deficiency induces severe oxidative stress leading to oxidative damage of various cellular components in worms. An NaCl chemotaxis associative learning assay indicated that vitamin B 12 deficiency did not affect learning ability but impaired memory retention ability, which decreased to approximately 58% of the control value. When worms were treated with 1mmol/L GSH, L-ascorbic acid, or vitamin E for three generations during vitamin B 12 deficiency, cellular malondialdehyde content as an index of oxidative stress decreased to the control level, but the impairment of memory retention ability was not completely reversed (up to approximately 50%). These results suggest that memory retention impairment formed during vitamin B 12 deficiency is partially attributable to oxidative stress. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Characterization of Thermophilic Halotolerant Aeribacillus pallidus TD1 from Tao Dam Hot Spring, Thailand

PubMed Central

Yasawong, Montri; Areekit, Supatra; Pakpitchareon, Arda; Santiwatanakul, Somchai; Chansiri, Kosum

2011-01-01

The bacterial strain TD1 was isolated from Tao Dam hot spring in Thailand. Strain TD1 was Gram positive, rod-shaped, aerobic, motile, and endospore forming. The cell was 2.0–40 μm in length and about 0.4 μm in diameter. The optimum growth occurred at 55–60 °C and at pH 7–8. Strain TD1 was able to grow on medium containing up to 10% NaCl. The DNA G+C content was 38.9 mol%. The cellular fatty acid content was mainly C16:0, which comprised 25.04% of the total amount of cellular fatty acid. 16S rDNA showed 99% identity to Aeribacillus pallidus DSM 3670T. Bayesian tree analysis strongly supported the idea that strain TD1 is affiliated with genus Aeribacillus, as Aeribacillus pallidus strain TD1. Although the 16S rDNA of A. pallidus strain TD1 is similar to that of A. pallidus DSM 3670T, some physiological properties and the cellular fatty acid profiles differ significantly. A. pallidus strain TD1 can produce extracellular pectate lyase, which has not been reported elsewhere for other bacterial strains in the genus Aeribacillus. A. pallidus strain TD1 may be a good candidate as a pectate lyase producer, which may have useful industrial applications. PMID:21954359

Comparing the effects of nano-sized sugarcane fiber with cellulose and psyllium on hepatic cellular signaling in mice

PubMed Central

Wang, Zhong Q; Yu, Yongmei; Zhang, Xian H; Floyd, Z Elizabeth; Boudreau, Anik; Lian, Kun; Cefalu, William T

2012-01-01

Aim To compare the effects of dietary fibers on hepatic cellular signaling in mice. Methods Mice were randomly divided into four groups (n = 9/group): high-fat diet (HFD) control, cellulose, psyllium, and sugarcane fiber (SCF) groups. All mice were fed a HFD with or without 10% dietary fiber (w/w) for 12 weeks. Body weight, food intake, fasting glucose, and fasting insulin levels were measured. At the end of the study, hepatic fibroblast growth factor (FGF) 21, AMP-activated protein kinase (AMPK) and insulin signaling protein content were determined. Results Hepatic FGF21 content was significantly lowered, but βKlotho, fibroblast growth factor receptor 1, fibroblast growth factor receptor 3, and peroxisome proliferator-activated receptor alpha proteins were significantly increased in the SCF group compared with those in the HFD group (P < 0.01). SCF supplementation also significantly enhanced insulin and AMPK signaling, as well as decreased hepatic triglyceride and cholesterol in comparison with the HFD mice. The study has shown that dietary fiber, especially SCF, significantly attenuates lipid accumulation in the liver by enhancing hepatic FGF21, insulin, and AMPK signaling in mice fed a HFD. Conclusion This study suggests that the modulation of gastrointestinal factors by dietary fibers may play a key role in both enhancing hepatic multiple cellular signaling and reducing lipid accumulation. PMID:22787396

The relationship between in vitro cellular aging and in vivo human age.

PubMed Central

Schneider, E L; Mitsui, Y

1976-01-01

Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging. PMID:1068470

Physical and chemical characteristics of mucin secreted by pseudomyxoma peritonei (PMP)

PubMed Central

Pillai, Krishna; Akhter, Javed; Mekkawy, Ahmed; Chua, Terence C; Morris, David L

2017-01-01

Background: Pseudomyxoma peritonei (PMP) is a rare disease with excess intraperitoneal mucin secretion. Treatment involves laparotomy, cytoreduction and chemotherapy that is very invasive with patients often acquiring numerous compromises. Hence a mucolytic comprising of bromelain and N-acetyl cystein has been developed to solubilise mucin in situ for removal by catherization. Owing to differences in mucin appearance and hardness, dissolution varies. Therefore the current study investigates the inter-mucin physical and chemical characteristics, in order to reformulate an effective mucolytic for all mucin. Method: PMP mucin, from the three categories (soft, semi hard and hard mucin) was solubilised and then various physical characteristics such as turbidity, density, kinematic viscosity were measured. The water content and the density of solid mucin were also determined. This was followed by the determination of sialic acid, glucose, lipid, Thiol (S-S and S-H) content of the samples. Lastly, the distribution of MUC2, MUC5B and MUC5AC was determined using western blot technique. Results: Both turbidity and kinematic viscosity and sialic acid content increased linearly as the hardness of mucin increased. However, density, hydration, protein, glucose, lipid and sulfhydryl and disulphide content decreased linearly as hardness of mucin increased. The distribution ratio of mucins (MUC2:MUC5B:MUC5AC) in soft mucin is 2.25:1.5:1.0, semi hard mucin is 1:1:1 and hard mucin is 3:2:1. Conclusion: The difference in texture and hardness of mucin may be due to cellular content, hydration, glucose, protein, lipids, thiol and MUC distribution. Soft mucin is solely made of glycoprotein whilst the others contained cellular materials. PMID:28138305

Influence of increasing temperature and salinity on herbicide toxicity in estuarine phytoplankton.

PubMed

DeLorenzo, Marie E; Danese, Loren E; Baird, Thomas D

2013-07-01

Ecological risk assessments are, in part, based on results of toxicity tests conducted under standard exposure conditions. Global climate change will have a wide range of effects on estuarine habitats, including potentially increasing water temperature and salinity, which may alter the risk assessment of estuarine pollutants. We examined the effects of increasing temperature and salinity on the toxicity of common herbicides (irgarol, diuron, atrazine, and ametryn) to the phytoplankton species Dunaliella tertiolecta. Static 96-h algal bioassays were conducted for each herbicide under four exposure scenarios: standard temperature and salinity (25°C, 20 ppt), standard temperature and elevated salinity (25°C, 40 ppt), elevated temperature and standard salinity (35°C, 20 ppt), and elevated temperature and elevated salinity (35°C, 40 ppt). The endpoints assessed were algal cell density at 96 h, growth rate, chlorophyll a content, lipid content, and starch content. Increasing exposure temperature reduced growth rate and 96-h cell density but increased the cellular chlorophyll and lipid concentrations of the control algae. Exposure condition did not alter starch content of control algae. Herbicides were found to decrease growth rate, 96 h cell density, and cellular chlorophyll and lipid concentrations, while starch concentrations increased with herbicide exposure. Herbicide effects under standard test conditions were then compared with those observed under elevated temperature and salinity. Herbicide effects on growth rate, cell density, and starch content were more pronounced under elevated salinity and temperature conditions. To encompass the natural variability in estuarine temperature and salinity, and to account for future changes in climate, toxicity tests should be conducted under a wider range of environmental conditions. Copyright © 2011 Wiley Periodicals, Inc.

Phenolic Profiles, Antioxidant Activities, and Neuroprotective Properties of Mulberry (Morus atropurpurea Roxb.) Fruit Extracts from Different Ripening Stages.

PubMed

Yang, Jiufang; Liu, Xuanjun; Zhang, Xiaoxu; Jin, Qing; Li, Jingming

2016-10-01

The present work investigated the phenolic profiles (including nonanthocyanin and anthocyanin phenolics), antioxidant activities, and neuroprotective potential of mulberry fruit (MF) (Morus atropurpurea Roxb.) grown in China at different ripening stages. High-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) was used to identify and quantify the phenolic compounds. The antioxidant capacity, total phenolic content (TPC), total flavonoid content (TFC), and total monomeric anthocyanin content (TAC) were determined using spectrophotometric methods. The neuroprotective effects of MFs at different ripening stages were investigated using Aβ 25-35 -treated PC12 cells as the cellular model of Alzheimer's disease. Of the 19 phenolic compounds characterized from the MF extracts, the contents of rutin and anthocyanins increased and that of chlorogenic acid decreased significantly with maturity. At the fully ripened stage, MF extracts showed the highest amounts of TPC (11.23 mg gallic acid equivalents/g fresh weight), TFC (15.1 mg rutin equivalents/g fresh weight), and TAC (1177 mg cyanidin 3-O-glucoside equivalents/100 g fresh weight). Meanwhile, antioxidant activity of MF extracts at this stage was highest according to ABTS (an IC50 value of 4.11 μg/mL) and DPPH (an IC50 value of 10.08 μg/mL) assays. Cellular assays revealed increased cell viability in cells treated with the ripe MF extracts; compared with the control groups, the ripening fruits also increased the antioxidant enzyme levels in PC12 cells. Together, these results suggest that the antioxidant activities and neuroprotective properties of ripening MFs are related to the contents and types of phenolic compounds that are present in the fruits. © 2016 Institute of Food Technologists®.

High-fat diet-induced hepatic steatosis reduces glucagon receptor content in rat hepatocytes: potential interaction with acute exercise

PubMed Central

Charbonneau, Alexandre; Unson, Cecilia G; Lavoie, Jean-Marc

2007-01-01

Studies have revealed that high-fat (HF) diets promote hyperglycaemia, whole-body insulin resistance and non-alcoholic fatty liver disease (NAFLD). Recently, hepatic glucagon resistance has been shown to occur in rats fed a HF diet. More precisely, diet-induced obesity (DIO) reduces the number of hepatic plasma membrane glucagon receptors (GR), which results in a diminished response to glucagon during a hyperglucagonaemic clamp. The present study was undertaken to test the hypothesis that a HF-DIO is associated with a desensitization and destruction of the hepatic GR. We also hypothesized that a single bout of endurance exercise would modify the GR cellular distribution under our DIO model. Male rats were either fed a standard (sd) or a HF diet for two weeks. Each group was subdivided into a non-exercised (Rest) and an acute exercised (EX) group. The HF diet resulted in a reduction of total hepatic GR (55%) and hepatic plasma membrane GR protein content (20%). These changes were accompanied by a significant increase in endosomal and lysosomal GR content with the feeding of a HF diet. The reduction of GR plasma membrane as well as the increase in endosomal GR was strongly correlated with an increase of PKC-α, suggesting a role of PKC-α in GR desensitization. EX increased significantly PKC-α protein content in both diets, suggesting a role of PKC-α in EX-induced GR desensitization. The present results suggest that liver lipid infiltration plays a role in reducing glucagon action in the liver through a reduction in total cellular and plasma membrane GR content. Furthermore, the GR desensitization observed in our in vivo model of HF diet-induced hepatic steatosis and in EX individuals may be regulated by PKC-α. PMID:17053032

The effect of particle size and amount of inoculum on fungal treatment of wheat straw and wood chips.

PubMed

van Kuijk, Sandra J A; Sonnenberg, Anton S M; Baars, Johan J P; Hendriks, Wouter H; Cone, John W

2016-01-01

The aim of this study was to optimize the fungal treatment of lignocellulosic biomass by stimulating the colonization. Wheat straw and wood chips were treated with Ceriporiopsis subvermispora and Lentinula edodes with various amounts of colonized millet grains (0.5, 1.5 or 3.0 % per g of wet weight of substrate) added to the substrates. Also, wheat straw and wood chips were chopped to either 0.5 or 2 cm. Effectiveness of the fungal treatment after 0, 2, 4, 6, or 8 wk of incubation was determined by changes in chemical composition, in vitro gas production (IVGP) as a measure for rumen degradability, and ergosterol content as a measure of fungal biomass. Incomplete colonization was observed for C. subvermispora treated wheat straw and L. edodes treated wood chips. The different particle sizes and amounts of inoculum tested, had no significant effects on the chemical composition and the IVGP of C. subvermispora treated wood chips. Particle size did influence L. edodes treatment of wheat straw. The L. edodes treatment of 2 cm wheat straw resulted in a more selective delignification and a higher IVGP than the smaller particles. Addition of 1.5 % or 3 % L. edodes inoculum to wheat straw resulted in more selective delignification and a higher IVGP than addition of 0.5 % inoculum. Particle size and amount of inoculum did not have an effect on C. subvermispora treatment of wood chips. At least 1.5 % L. edodes colonized millet grains should be added to 2 cm wheat straw to result in an increased IVGP and acid detergent lignin (ADL) degradation.

Introduction for Diffusion Chamber Culture Symposium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carsten, A. L.

The diffusion-chamber system has been applied to studies of cell kinetics, progenitor cell quantitation, humoral effects, immunological effects, cytogenetics, organogenesis, and the cellular effects of drugs and physical factors such as radiation, hypoxia, etc. Chamber contents have been analyzed by clot dissolution with measuring of cell content, limiting dilution evaluation, radionuclide utilization (tritiated thymidine labeling), growth of colony number, size and type, CFU-S or CFU-C content, or proliferation by secondary culture in mice or in vitro systems, and chromosome changes. Cell types ranging from embryonal tissues to adult normal and neoplastic tissues have been grown in hosts across species barriers.more » Advantages and disadvantages of this system are discussed.« less

Properties of rigid polyurethane foams filled with glass microspheres

NASA Astrophysics Data System (ADS)

Yakushin, V.; Bel'kova, L.; Sevastyanova, I.

2012-11-01

The effect of hollow glass microspheres with a density of 125 kg/m3 on the properties of low-density (54-90 kg/m3) rigid polyurethane foams is investigated. The thermal expansion coefficient of the foams and their properties in tension and compression in relation to the content of the microspheres (0.5-5 wt.%) are determined. An increase in the characteristics of the material in compression in the foam rise direction with increasing content of filler is revealed. The limiting content of the microspheres above which the mechanical characteristics of the filled foams begin to decrease is found. The distribution of the microspheres in elements of the cellular structure of the polyurethane foams is examined.

Anti-Helicobacter pylori metabolites from Rhizoctonia sp. Cy064, an endophytic fungus in Cynodon dactylon.

PubMed

Ma, Y M; Li, Y; Liu, J Y; Song, Y C; Tan, R X

2004-07-01

A new benzophenone, named rhizoctonic acid (1), together with three known compounds monomethylsulochrin (2), ergosterol (3) and 3beta,5alpha,6beta-trihydroxyergosta-7,22-diene (4) were isolated through bioassay-guided fractionations from the culture of Rhizoctonia sp. (Cy064), an endophytic fungus in the leaf of Cynodon dactylon. The structure of the new acid 1 was elucidated to be 5-hydroxy-2-(2-hydroxy-6-methoxy-4-methylbenzoyl)-3-methoxybenzoic acid by a combination of spectral analyses. Furthermore, the structure of monomethylsulochrin 2 was confirmed by 13C-NMR analysis. All four metabolites were subjected to a more detailed in vitro assessment of their antibacterial action against five clinically isolated and one reference (ATCC 43504) Helicobacter pylori strains.

Sterol biosynthesis de nova via cycloartenol by the soil amoeba Acanthamoeba polyphaga.

PubMed Central

Raederstorff, D; Rohmer, M

1985-01-01

The soil amoeba Acanthamoeba polyphaga is capable of synthesizing its sterols de novo from acetate. The major sterols are ergosterol and poriferasta-5,7,22-trienol. Furthermore C28 and C29 sterols of still unknown structure with an aromatic B-ring are also synthesized by the amoeba. The first cyclic sterol precursor is cycloartenol, which is the sterol precursor in all photosynthetic phyla. No trace of lanosterol, which is the sterol precursor in animals and fungi, could be detected. These results show that at least some of the biochemical processes of Acanthamoeba polyphaga might be phylogenetically related to those of unicellular algae. Addition of exogenous sterols to the culture medium does not influence the sterol biosynthesis and the sterol composition of the cells. PMID:4074326

Induction by carrot allelochemicals of insecticide-metabolising enzymes in the southern armyworm (Spodoptera eridania).

PubMed

Brattsten, L B; Evans, C K; Bonetti, S; Zalkow, L H

1984-01-01

Carrot foliage monoterpenes induce cytochrome P-450 up to 2.9-fold, NADPH cytochrome c (P-450) reductase up to 1.6-fold, NADPH-oxidation up to 3.8-fold, aldrin epoxidation up to 1.5-fold in southern armyworm larval midgut tissues when incorporated in their diet at 0.2% for 3 days. Stigmasterol and ergosterol did not substantially induce microsomal oxidase activities and significantly inhibited GSH S-aryltransferase activity and sulfotransferase activity. Coumarin did not substantially affect microsomal oxidase and sulfotransferase activity but is the most potent inducer of GSH S-aryltransferase activity, increasing this activity 7-fold. None of the chemicals is acutely toxic to the sixth instar larvae or affect the larval weight gain except coumarin which significantly depressed the maximal body weight attained.

Stingless Bee Larvae Require Fungal Steroid to Pupate.

PubMed

Paludo, Camila R; Menezes, Cristiano; Silva-Junior, Eduardo A; Vollet-Neto, Ayrton; Andrade-Dominguez, Andres; Pishchany, Gleb; Khadempour, Lily; do Nascimento, Fabio S; Currie, Cameron R; Kolter, Roberto; Clardy, Jon; Pupo, Mônica T

2018-01-18

The larval stage of the stingless bee Scaptotrigona depilis must consume a specific brood cell fungus in order to continue development. Here we show that this fungus is a member of the genus Zygosaccharomyces and provides essential steroid precursors to the developing bee. Insect pupation requires ecdysteroid hormones, and as insects cannot synthesize sterols de novo, they must obtain steroids in their diet. Larval in vitro culturing assays demonstrated that consuming ergosterol recapitulates the developmental effects on S. depilis as ingestion of Zygosaccharomyces sp. cells. Thus, we determined the molecular underpinning of this intimate mutualistic symbiosis. Phylogenetic analyses showed that similar cases of bee-Zygosaccharomyces symbiosis may exist. This unprecedented case of bee-fungus symbiosis driven by steroid requirement brings new perspectives regarding pollinator-microbiota interaction and preservation.

Naval Law Review. Volume 48

DTIC Science & Technology

2001-01-01

from airports and hotels, Internet cafes , libraries, and even cellular phones. This unmatched versatility has made e-mail the preferred method of...8217 contention, however, was not that the Franchise Tax Board applied the wrong section of the code; it was that the code “unfairly taxed the wife’s

Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

EPA Science Inventory

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...