The metabolism of plant sterols is disturbed in postmenopausal women with coronary artery disease.

Science.gov (United States)

Gylling, Helena; Hallikainen, Maarit; Rajaratnam, Radhakrishnan A; Simonen, Piia; Pihlajamäki, Jussi; Laakso, Markku; Miettinen, Tatu A

2009-03-01

In postmenopausal coronary artery disease (CAD) women, serum plant sterols are elevated. Thus, we investigated further whether serum plant sterols reflect absolute cholesterol metabolism in CAD as in other populations and whether the ABCG5 and ABCG8 genes, associated with plant sterol metabolism, were related to the risk of CAD. In free-living postmenopausal women with (n = 47) and without (n = 62) CAD, serum noncholesterol sterols including plant sterols were analyzed with gas-liquid chromatography, cholesterol absorption with peroral isotopes, absolute cholesterol synthesis with sterol balance technique, and bile acid synthesis with quantitating fecal bile acids. In CAD women, serum plant sterol ratios to cholesterol were 21% to 26% (P synthesis were reduced. Only in controls were serum plant sterols related to cholesterol absorption (eg, sitosterol; in controls: r = 0.533, P synthesis marker) and lathosterol-cholestanol (relative synthesis-absorption marker) were related to absolute synthesis and absorption percentage (P range from .05 to sterol metabolism is disturbed in CAD women; so serum plant sterols only tended to reflect absolute cholesterol absorption. Other relative markers of cholesterol metabolism were related to the absolute ones in both groups. ABCG5 and ABCG8 genes were not associated with the risk of CAD.

Phylogenetic distribution of fungal sterols.

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John D Weete

Full Text Available BACKGROUND: Ergosterol has been considered the "fungal sterol" for almost 125 years; however, additional sterol data superimposed on a recent molecular phylogeny of kingdom Fungi reveals a different and more complex situation. METHODOLOGY/PRINCIPAL FINDINGS: The interpretation of sterol distribution data in a modern phylogenetic context indicates that there is a clear trend from cholesterol and other Delta(5 sterols in the earliest diverging fungal species to ergosterol in later diverging fungi. There are, however, deviations from this pattern in certain clades. Sterols of the diverse zoosporic and zygosporic forms exhibit structural diversity with cholesterol and 24-ethyl -Delta(5 sterols in zoosporic taxa, and 24-methyl sterols in zygosporic fungi. For example, each of the three monophyletic lineages of zygosporic fungi has distinctive major sterols, ergosterol in Mucorales, 22-dihydroergosterol in Dimargaritales, Harpellales, and Kickxellales (DHK clade, and 24-methyl cholesterol in Entomophthorales. Other departures from ergosterol as the dominant sterol include: 24-ethyl cholesterol in Glomeromycota, 24-ethyl cholest-7-enol and 24-ethyl-cholesta-7,24(28-dienol in rust fungi, brassicasterol in Taphrinales and hypogeous pezizalean species, and cholesterol in Pneumocystis. CONCLUSIONS/SIGNIFICANCE: Five dominant end products of sterol biosynthesis (cholesterol, ergosterol, 24-methyl cholesterol, 24-ethyl cholesterol, brassicasterol, and intermediates in the formation of 24-ethyl cholesterol, are major sterols in 175 species of Fungi. Although most fungi in the most speciose clades have ergosterol as a major sterol, sterols are more varied than currently understood, and their distribution supports certain clades of Fungi in current fungal phylogenies. In addition to the intellectual importance of understanding evolution of sterol synthesis in fungi, there is practical importance because certain antifungal drugs (e.g., azoles target reactions in

Role of membrane sterols and cortical microtubules in gravity resistance in plants

Science.gov (United States)

Hoson, T.; Koizumi, T.; Matsumoto, S.; Kumasaki, S.; Soga, K.; Wakabayashi, K.; Sakaki, T.

Resistance to the gravitational force is a principal graviresponse in plants comparable to gravitropism Nevertheless only limited information has been obtained for this graviresponse We have examined mechanisms of signal perception transformation and transduction of the perceived signal and response to the transduced signal in gravity resistance using hypergravity conditions produced by centrifugation In Arabidopsis hypocotyls hypergravity treatment greatly increased the expression level of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase HMGR which catalyzes a reaction producing mevalonic acid a key precursor of terpenoids such as membrane sterols Geranyl diphosphate synthase gene was also up-regulated by hypergravity whereas the expression of other genes involved in membrane lipid metabolism was not influenced Hypergravity caused an increase in sterol content in azuki bean epicotyls but not in phospholipid glycolipid or fatty acid content Also hypergravity did not influence fatty acid composition in any lipid class Thus the effect of hypergravity on membrane lipid metabolism was specific for sterol synthesis On the other hand alpha- and beta-tubulin genes were up-regulated by hypergravity treatment in Arabidopsis hypocotyls Hypergravity also induced reorientation of cortical microtubules in azuki epicotyls the percentage of epidermal cells with transverse microtubles was decreased whereas that with longitudinal microtubules was increased Inhibitors of HMGR action and microtubule-disrupting agents completely prevented the gravity resistance

The Evolution of Sterol Biosynthesis in Bacteria: In Situ Fluorescence Localization of Sterols in the Nucleoid Bacterium Gemmata obscuriglobus

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Budin, M.; Jorgenson, T. L.; Pearson, A.

2004-12-01

acquired the ability to produce sterols for a specialized function related to their nuclear membrane. 1. Brocks, J.J., et al., Science 285:1033-36 (1999). 2. Bird, C.W., et al., Nature 230:473-74 (1971). 3. Bode, H.B., et al., Mol. Microbiol. 47:471-81 (2003). 4. Pearson, A., et al., Proc. Natl. Acad. Sci. USA 100:15352-57 (2003). 5. Fuerst, J.A. and R.I. Webb, Proc. Natl. Acad. Sci. USA 88:8184-88 (1991).

Depot sterols in comparisons with structural sterols in Cancer pagurus and Eriocheir sinensis

NARCIS (Netherlands)

Zandee, D.I.; Kruitwagen, E.C.J.

The differences in sterol content and sterol composition between the midgut gland and remaining parts (structural lipids) of male and female specimens of Cancer pagurus and Eriocheir sinensis are investigated. There are no differences in sterol content in the structural lipids between male and

Changes in Intestinal Gene Expression of Zebrafish (Danio rerio Related to Sterol Uptake and Excretion upon β-Sitosterol Administration

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Mai Takase

2018-01-01

Full Text Available Replacement of fishmeal with plant ingredients will introduce not only plant oil and protein but also phytosterol to the fish diet. Mammals strictly restrict the uptake of phytosterol at intestinal epithelial cells by regulating the gene expressions of sterol uptake and excretion proteins; however, phytosterol is found in the fish muscle and other organs. In order to assess the ability of phytosterol uptake by the intestinal epithelial cells of fish, no-sterol diet, cholesterol-, and β-sitosterol-containing diet was separately administered to zebrafish, and the relative mRNA expressions related to sterol uptake and excretion were evaluated. Gene expression of Niemann-Pick C1-like protein 1 in the sitosterol-fed group was significantly higher than that of the cholesterol-fed group (p < 0.05. The expression of apolipoprotein A-I gene was also higher in the sitosterol-fed group than that in the no-sterol and cholesterol-fed groups. The expressions of ATP-binding cassette, sub-family G, member 5 and 8, were significantly higher in the sitosterol-fed group, compared to the no-sterol group. Regarding the gene expression of ATP-binding cassette sub-family A, member 1, the sitosterol-fed group showed higher expression level compared to the other groups (p < 0.01. These results suggest that fish should be tolerant to phytosterols in contrast to mammals.

Biosynthesis and composition of sterols and sterol esters in the land snail Cepaea nemoralis (L.) (gastropoda, pulmonata, stylommatophora)

NARCIS (Netherlands)

Horst, D.J. van der; Voogt, P.A.

1972-01-01

1. 1. The biosynthesis and composition of sterols and sterol esters were studied in the land snail Cepaea nemoralis after injection of Na-1-14C-acetate. 2. 2. Free and esterified sterols appeared to be synthesized by the animals, whilst the specific radioactivity of the sterols from the esters

Neutral Sterols of Cephalic Glands of Stingless Bees and Their Correlation with Sterols from Pollen

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Maria Juliana Ferreira-Caliman

2012-01-01

de novo and, thus, all phytophagous insects depend on an exogenous source of sterols for growth, development, and reproduction. The sterol requirements of social bees are not fully known due to the fact that there is no well-defined diet available throughout the year with regard to floral resources. Our study aimed to characterize the sterols present in pollen stored in Melipona marginata and Melipona scutellaris colonies, as well as evaluating their presence in the mandibular, hypopharyngeal, and cephalic salivary gland secretions. We analyzed the chemical composition of pollen stored in the colonies and the composition of the cephalic glands of workers in three adult functional phases (newly emerged, nurses, and foragers by gas chromatography and mass spectrometry. The results showed that the pollen analyzed contained campesterol, stigmasterol, sitosterol, isofucosterol, lanosterol, and small amounts of cholesterol. The glands showed the same compounds found in the pollen analyzed, except lanosterol that was not found in M. scutellaris glands. Surprisingly, cholesterol was found in some glands with relative ratios greater than those found in pollen.

Sterol Synthesis in Diverse Bacteria

OpenAIRE

Wei, Jeremy H.; Yin, Xinchi; Welander, Paula V.

2016-01-01

Sterols are essential components of eukaryotic cells whose biosynthesis and function has been studied extensively. Sterols are also recognized as the diagenetic precursors of steranes preserved in sedimentary rocks where they can function as geological proxies for eukaryotic organisms and/or aerobic metabolisms and environments. However, production of these lipids is not restricted to the eukaryotic domain as a few bacterial species also synthesize sterols. Phylogenomic studies have identifie...

Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans.

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Dahlin, Paul; Srivastava, Vaibhav; Ekengren, Sophia; McKee, Lauren S; Bulone, Vincent

2017-01-01

The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heterotrophic Phytophthora infestans. We first present a comprehensive reconstruction of a likely sterol synthesis pathway for S. parasitica, causative agent of the disease saprolegniasis in fish. This pathway shows multiple potential routes of sterol synthesis, and draws on several avenues of new evidence: bioinformatic mining for genes with sterol-related functions, expression analysis of these genes, and analysis of the sterol profiles in mycelium grown in different media. Additionally, we explore the extent to which P. infestans, which causes the late blight in potato, can modify exogenously provided sterols. We consider whether the two very different approaches to sterol acquisition taken by these pathogens represent any specific survival advantages or potential drug targets.

Cholesterol and related sterols autoxidation.

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Zerbinati, Chiara; Iuliano, Luigi

2017-10-01

Cholesterol is a unique lipid molecule providing the building block for membranes, hormones, vitamin D and bile acid synthesis. Metabolism of cholesterol involves several enzymes acting on the sterol nucleus or the isooctyl tail. In the recent years, research interest has been focused on oxysterols, cholesterol derivatives generated by the addition of oxygen to the cholesterol backbone. Oxysterols can be produced enzymatically or by autoxidation. Autoxidation of cholesterol proceeds through type I or type II mechanisms. Type I autoxidation is initiated by free radical species, such as those arising from the superoxide/hydrogen peroxide/hydroxyl radical system. Type II autoxidation occurs stoichiometrically by non-radical highly reactive oxygen species such as singlet oxygen, HOCl, and ozone. The vulnerability of cholesterol towards high reactive species has raised considerable interest for mechanistic studies and for the potential biological activity of oxysterols, as well as for the use of oxysterols as biomarkers for the non-invasive study of oxidative stress in vivo. Copyright © 2017. Published by Elsevier Inc.

Structure-activity relationships between sterols and their thermal stability in oil matrix.

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Hu, Yinzhou; Xu, Junli; Huang, Weisu; Zhao, Yajing; Li, Maiquan; Wang, Mengmeng; Zheng, Lufei; Lu, Baiyi

2018-08-30

Structure-activity relationships between 20 sterols and their thermal stabilities were studied in a model oil system. All sterol degradations were found to be consistent with a first-order kinetic model with determination of coefficient (R 2 ) higher than 0.9444. The number of double bonds in the sterol structure was negatively correlated with the thermal stability of sterol, whereas the length of the branch chain was positively correlated with the thermal stability of sterol. A quantitative structure-activity relationship (QSAR) model to predict thermal stability of sterol was developed by using partial least squares regression (PLSR) combined with genetic algorithm (GA). A regression model was built with R 2 of 0.806. Almost all sterol degradation constants can be predicted accurately with R 2 of cross-validation equals to 0.680. Four important variables were selected in optimal QSAR model and the selected variables were observed to be related with information indices, RDF descriptors, and 3D-MoRSE descriptors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on sterol synthesis in various human cell types

NARCIS (Netherlands)

Vliet, A.K. van; Thiel, G.C.F. van; Huisman, R.H.; Moshage, H.; Yap, S.H.; Cohen, L.H.

1995-01-01

The three vastatins examined, lovastatin, simvastatin and pravastatin, are equally strong inhibitors of the sterol synthesis in human hepatocytes in culture with IC50-values of 4.1, 8.0 and 2.0 nM, respectively. However, in the human extrahepatic cells: umbilical vascular endothelial cells, retinal

Inability to fully suppress sterol synthesis rates with exogenous sterol in embryonic and extraembyronic fetal tissues

OpenAIRE

Yao, Lihang; Jenkins, Katie; Horn, Paul S.; Lichtenberg, M. Hayden; Woollett, Laura A.

2007-01-01

The requirement for cholesterol is greater in developing tissues (fetus, placenta, and yolk sac) as compared to adult tissues. Here, we compared cholesterol-induced suppression of sterol synthesis rates in the adult liver to the fetal liver, fetal body, placenta, and yolk sac of the Golden Syrian hamster. Sterol synthesis rates were suppressed maximally in non-pregnant adult livers when cholesterol concentrations were increased. In contrast, sterol synthesis rates were suppressed only margina...

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

Science.gov (United States)

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.

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses.

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Manzano, David; Andrade, Paola; Caudepón, Daniel; Altabella, Teresa; Arró, Montserrat; Ferrer, Albert

2016-09-01

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. © 2016

Sterols indicate water quality and wastewater treatment efficiency.

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Reichwaldt, Elke S; Ho, Wei Y; Zhou, Wenxu; Ghadouani, Anas

2017-01-01

As the world's population continues to grow, water pollution is presenting one of the biggest challenges worldwide. More wastewater is being generated and the demand for clean water is increasing. To ensure the safety and health of humans and the environment, highly efficient wastewater treatment systems, and a reliable assessment of water quality and pollutants are required. The advance of holistic approaches to water quality management and the increasing use of ecological water treatment technologies, such as constructed wetlands and waste stabilisation ponds (WSPs), challenge the appropriateness of commonly used water quality indicators. Instead, additional indicators, which are direct measures of the processes involved in the stabilisation of human waste, have to be established to provide an in-depth understanding of system performance. In this study we identified the sterol composition of wastewater treated in WSPs and assessed the suitability of human sterol levels as a bioindicator of treatment efficiency of wastewater in WSPs. As treatment progressed in WSPs, the relative abundance of human faecal sterols, such as coprostanol, epicoprostanol, 24-ethylcoprostanol, and sitostanol decreased significantly and the sterol composition in wastewater changed significantly. Furthermore, sterol levels were found to be correlated with commonly used wastewater quality indicators, such as BOD, TSS and E. coli. Three of the seven sterol ratios that have previously been used to track sewage pollution in the environment, detected a faecal signal in the effluent of WSPs, however, the others were influenced by high prevalence of sterols originating from algal and fungal activities. This finding poses a concern for environmental assessment studies, because environmental pollution from waste stabilisation ponds can go unnoticed. In conclusion, faecal sterols and their ratios can be used as reliable indicators of treatment efficiency and water quality during wastewater

Variation and sources of sterols in Kuala Selangor, Selangor

International Nuclear Information System (INIS)

Masni Mohd Ali; Norfariza Humrawali; Mohd Talib Latif

2010-01-01

This study explores the role of sterols as lipid bio markers to assess organic matter variations and their sources in surface sediments of Kuala Selangor, Selangor which involved extraction procedures and sterol compounds analyzed using GC-MS. Ten sterol compounds were found in the samples with phytosterols being the principal compounds which accounted 79 % of total sterols. This was followed by cholesterol and fecal sterols, each constitutes 6 % of total sterols while the rest are in the ranged of 1-5 %. Sterol Source Index (SSI) also reflected phytosterols predominant at all sampling stations but in different degree based on phytosterols compounds. Another issue was sewage contamination assessment using coprostanol/ cholesterol, coprostanol/ (coprostanol + cholestanol) and epi coprostanol/ coprostanol ratio. No sewage contamination occurred in the study area even though fecal sterols have been quantified. This analytical study indicates that the sediments in the study area consisted of a mixture of sterols from various sources even though dominated by phytosterols originated from terrestrial plants. (author)

Casein kinase 1 regulates sterol regulatory element-binding protein (SREBP) to control sterol homeostasis.

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Brookheart, Rita T; Lee, Chih-Yung S; Espenshade, Peter J

2014-01-31

Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

Dynamics of sterol synthesis during development of Leishmania spp. parasites to their virulent form.

Science.gov (United States)

Yao, Chaoqun; Wilson, Mary E

2016-04-12

The Leishmania spp. protozoa, the causative agents of the "neglected" tropical disease leishmaniasis, are transmitted to mammals by sand fly vectors. Within the sand fly, parasites transform from amastigotes to procyclic promastigotes, followed by development of virulent (metacyclic) promastigote forms. The latter are infectious to mammalian hosts. Biochemical components localized in the parasite plasma membrane such as proteins and sterols play a pivotal role in Leishmania pathogenesis. Leishmania spp. lack the enzymes for cholesterol synthesis, and the dynamics of sterol acquisition and biosynthesis in parasite developmental stages are not understood. We hypothesized that dynamic changes in sterol composition during metacyclogenesis contribute to the virulence of metacyclic promastigotes. Sterols were extracted from logarithmic phase or metacyclic promastigotes grown in liquid culture with or without cholesterol, and analyzed qualitatively and quantitatively by gas chromatograph-mass spectrometry (GC-MS). TriTrypDB was searched for identification of genes involved in Leishmania sterol biosynthetic pathways. In total nine sterols were identified. There were dynamic changes in sterols during promastigote metacyclogenesis. Cholesterol in the culture medium affected sterol composition in different parasite stages. There were qualitative and relative quantitative differences between the sterol content of virulent versus avirulent parasite strains. A tentative sterol biosynthetic pathway in Leishmania spp. promastigotes was identified. Significant differences in sterol composition were observed between promastigote stages, and between parasites exposed to different extracellular cholesterol in the environment. These data lay the foundation for further investigating the role of sterols in the pathogenesis of Leishmania spp. infections.

Sterol composition from inflorescences of Hieracium pilosella L.

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Tadeusz Krzaczek

2011-01-01

Full Text Available The fraction of sterol acetates from the inflorescences of Hieracium pilosella has been isolated in the typical way from petroleum ether extract. By means of the weight method the total amount of sterols was determined (0.2659%. The mixtures of sterol acetates and free sterols were investigated using GC-MS techniques. The occurrence of about 18 sterols has been observed. Cholesterol, cholest-8(14-en-3b-ol, cholesta-5.7-dien-3b-ol, cholest-7-en-3b-ol, ergosta-5.24-dien-3b-ol, campesterol, stigmasterol, b-sitosterol, fucosterol, 5a-stigmast-7-en-3a-ol were identified. The probable structures of lophenol, isofucosterol, 5a-stigmasta-7.24-dien-3b-ol, lanosta-9(11.24-dien-3b-ol and 24-ethylidene lophenol were stated on the basis of literature data. The last 4 sterols occur in a vestigial quantity, which made its identification impossible. Sitos erol and cholesterol are remarkably dominating sterols in the fraction.

Cerebral Accumulation of Dietary Derivable Plant Sterols does not Interfere with Memory and Anxiety Related Behavior in Abcg5-/- Mice

NARCIS (Netherlands)

Vanmierlo, Tim; Rutten, Kris; van Vark-van der Zee, Leonie C.; Friedrichs, Silvia; Bloks, Vincent W.; Blokland, Arjan; Ramaekers, Frans C.; Sijbrands, Eric; Steinbusch, Harry; Prickaerts, Jos; Kuipers, Folkert; Luetjohann, Dieter; Mulder, Monique

Plant sterols such as sitosterol and campesterol are frequently applied as functional food in the prevention of atherosclerosis. Recently, it became clear that plasma derived plant sterols accumulate in murine brains. We questioned whether plant sterols in the brain are associated with alterations

Characterisation of a desmosterol reductase involved in phytosterol dealkylation in the silkworm, Bombyx mori.

Directory of Open Access Journals (Sweden)

Leonora F Ciufo

Full Text Available Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C(29 and C(28 yielding cholesterol (C(27. The final step of this dealkylation pathway involves desmosterol reductase (DHCR24-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735. Following PCR-based cloning of the cDNA (1.6 kb and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD-dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250 kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.

Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

Science.gov (United States)

Ciufo, Leonora F.; Murray, Patricia A.; Thompson, Anu; Rigden, Daniel J.; Rees, Huw H.

2011-01-01

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction. Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation. PMID:21738635

Cerebral Accumulation of Dietary Derivable Plant Sterols does not Interfere with Memory and Anxiety Related Behavior in Abcg5-/- Mice

NARCIS (Netherlands)

T. Vanmierlo (Tim); K. Rutten (Kris); L.C. van Vark-van der Zee (Leonie); S. Friedrichs (Silvia); V.W. Bloks (Vincent ); A. Blokland (Arjan); F.C.S. Ramaekers (Franks); E.J.G. Sijbrands (Eric); H. Steinbusch; J. Prickaerts (Jos); F. Kuipers (Folkert); D. Lütjohann; M.T. Mulder (Monique)

2011-01-01

textabstractPlant sterols such as sitosterol and campesterol are frequently applied as functional food in the prevention of atherosclerosis. Recently, it became clear that plasma derived plant sterols accumulate in murine brains. We questioned whether plant sterols in the brain are associ/+ mice for

Speed Limits for Nonvesicular Intracellular Sterol Transport.

Science.gov (United States)

Dittman, Jeremy S; Menon, Anant K

2017-02-01

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by nonvesicular mechanisms requiring sterol transport proteins (STPs). Here we examine the idea that transport is enhanced at membrane contact sites where the ER is closely apposed to the PM. We conclude that sterol desorption from the membrane, rather than STP-mediated diffusion, is rate limiting in the cellular context, so there is no apparent kinetic benefit to having STP-mediated sterol transfer occur at contact sites. Contact sites may instead compartmentalize lipid synthesis or transport machinery, providing opportunities for regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantitative charge-tags for sterol and oxysterol analysis.

Science.gov (United States)

Crick, Peter J; William Bentley, T; Abdel-Khalik, Jonas; Matthews, Ian; Clayton, Peter T; Morris, Andrew A; Bigger, Brian W; Zerbinati, Chiara; Tritapepe, Luigi; Iuliano, Luigi; Wang, Yuqin; Griffiths, William J

2015-02-01

Global sterol analysis is challenging owing to the extreme diversity of sterol natural products, the tendency of cholesterol to dominate in abundance over all other sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative charge-tags for the LC-MS analysis of sterols including oxysterols. Sterols/oxysterols in plasma were extracted in ethanol containing deuterated internal standards, separated by C18 solid-phase extraction, and derivatized with GP, with or without prior oxidation of 3β-hydroxy to 3-oxo groups. By use of different isotope-labeled GPs, it was possible to analyze in a single LC-MS analysis both sterols/oxysterols that naturally possess a 3-oxo group and those with a 3β-hydroxy group. Intra- and interassay CVs were sterols/oxysterols in a single analytical run and can be used to identify inborn errors of cholesterol synthesis and metabolism. © 2014 American Association for Clinical Chemistry.

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms

Science.gov (United States)

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 sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth. PMID:22176028

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses1[OPEN

Science.gov (United States)

Andrade, Paola; Caudepón, Daniel; Arró, Montserrat

2016-01-01

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. PMID

Hepatic nuclear sterol regulatory binding element protein 2 abundance is decreased and that of ABCG5 increased in male hamsters fed plant sterols.

Science.gov (United States)

Harding, Scott V; Rideout, Todd C; Jones, Peter J H

2010-07-01

The effect of dietary plant sterols on cholesterol homeostasis has been well characterized in the intestine, but how plant sterols affect lipid metabolism in other lipid-rich tissues is not known. Changes in hepatic cholesterol homeostasis in response to high dietary intakes of plant sterols were determined in male golden Syrian hamsters fed hypercholesterolemia-inducing diets with and without 2% plant sterols (wt:wt; Reducol, Forbes Meditech) for 28 d. Plasma and hepatic cholesterol concentrations, cholesterol biosynthesis and absorption, and changes in the expression of sterol response element binding protein 2 (SREBP2) and liver X receptor-beta (LXRbeta) and their target genes were measured. Plant sterol feeding reduced plasma total cholesterol, non-HDL cholesterol, and HDL cholesterol concentrations 43% (P 6-fold (P = 0.029) and >2-fold (P sterol-fed hamsters compared with controls. Plant sterol feeding also increased fractional cholesterol synthesis >2-fold (P sterol feeding increased hepatic protein expression of cytosolic (inactive) SREBP2, decreased nuclear (active) SREBP2, and tended to increase LXRbeta (P = 0.06) and ATP binding cassette transporter G5, indicating a differential modulation of the expression of proteins central to cholesterol metabolism. In conclusion, high-dose plant sterol feeding of hamsters changes hepatic protein abundance in favor of cholesterol excretion despite lower hepatic cholesterol concentrations and higher cholesterol fractional synthesis.

Composition and Sources of Sterols in Pulau Tinggi, Johor, Malaysia

International Nuclear Information System (INIS)

Masni Mohd Ali; Norfariza Humrawali; Mohd Talib Latif; Mohamad Pauzi Zakaria

2011-01-01

This study explores the role of sterols as lipid bio markers to indicate their input which originates from various sources in the marine environment. Sterols and their ratios were investigated in sediments taken from sixteen sampling stations at Pulau Tinggi, Johor in order to assess the sources of organic matter. The compounds extracted from the sediments were quantified using a gas chromatography-mass spectrometry (GC-MS). The distributions of sterols indicated that organic matter at all sampling stations originated from a mixture of marine source and terrestrial origins at different proportions. A total of eleven sterols were quantified, with the major compounds being phytosterols (44 % of total sterols), cholesterol (11 %), brassica sterol (11 %) and fecal sterols (12 %). (author)

Fluorescent Sterols and Cholesteryl Esters as Probes for Intracellular Cholesterol Transport

Science.gov (United States)

Solanko, Katarzyna A.; Modzel, Maciej; Solanko, Lukasz M.; Wüstner, Daniel

2015-01-01

Cholesterol transport between cellular organelles comprised vesicular trafficking and nonvesicular exchange; these processes are often studied by quantitative fluorescence microscopy. A major challenge for using this approach is producing analogs of cholesterol with suitable brightness and structural and chemical properties comparable with those of cholesterol. This review surveys currently used fluorescent sterols with respect to their behavior in model membranes, their photophysical properties, as well as their transport and metabolism in cells. In the first part, several intrinsically fluorescent sterols, such as dehydroergosterol or cholestatrienol, are discussed. These polyene sterols (P-sterols) contain three conjugated double bonds in the steroid ring system, giving them slight fluorescence in ultraviolet light. We discuss the properties of P-sterols relative to cholesterol, outline their chemical synthesis, and explain how to image them in living cells and organisms. In particular, we show that P-sterol esters inserted into low-density lipoprotein can be tracked in the fibroblasts of Niemann–Pick disease using high-resolution deconvolution microscopy. We also describe fluorophore-tagged cholesterol probes, such as BODIPY-, NBD-, Dansyl-, or Pyrene-tagged cholesterol, and eventual esters of these analogs. Finally, we survey the latest developments in the synthesis and use of alkyne cholesterol analogs to be labeled with fluorophores by click chemistry and discuss the potential of all approaches for future applications. PMID:27330304

Inhaled tobacco sterols: uptake by the lungs and disposition to selected organs of rats

International Nuclear Information System (INIS)

Holden, W.E.; Maier, J.M.; Liebler, J.M.; Malinow, M.R.

1988-01-01

Tobacco sterols (cholesterol, beta-sitosterol, campesterol, and stigmasterol) are present in tobacco smoke and appear in plasma of mammals exposed to cigarette smoke. Because tobacco sterols may be important in the pathogenesis of smoking-induced lung and vascular diseases, we studied the pattern of deposition of cigarette sterols in the lungs and appearance of cigarette sterols in plasma and body organs of rats. After exposure to twenty 5 ml puffs of smoke from tobacco labeled with [4- 14 C]cholesterol or beta-[4- 14 C]sitosterol, rats were killed just after exposure (day 0) and on days 2, 5, 8, 11, 15, and 30, and the lungs and selected body organs analyzed for activity. We found that cigarette sterols are associated with particulates in cigarette smoke, deposited mostly in distal airspaces and parenchyma of the lungs, and appear in plasma and several body organs for more than 30 days after this single exposure to cigarette smoke. Bronchoalveolar lavage fluid contained relatively small amounts of radiolabel for only the first few days, suggesting that most of the sterols were rapidly incorporated in lung parenchyma. Because disorders of sterol metabolism have been implicated in a variety of diseases including atherosclerosis and cancer, the significance of tobacco sterols to human smoking-induced diseases deserves further study

Quality of deli-style turkey enriched with plant sterols.

Science.gov (United States)

Grasso, S; Brunton, N P; Lyng, J G; Harrison, S M; Monahan, F J

2016-12-01

Low-fat meat products could be excellent carriers for plant sterols, known for their cholesterol-lowering properties. In this study, we developed a protocol for the manufacture of a deli-style turkey enriched with plant sterols (S) at a level sufficient to deliver the maximum plant sterols amount recommended for cholesterol reduction by the European Food Safety Authority (3 g of plant sterols per day) in a 70 g portion. We investigated the stability of the plant sterols and the effects of their addition on the product quality. Plant sterols remained stable during the seven-day storage period. The addition of plant sterols significantly affected some texture parameters, shear force, lipid oxidation, L values and water-holding capacity compared with control (C). Sensory analysis was carried out by an untrained panel (32) using the difference-from-control test between C and S samples to evaluate first the extent of the overall sensory difference and then the extent of sensory difference on colour, texture and flavour. Results indicated that panellists considered the intensity of the difference between C and S samples to be 'small'. Plant sterols could be used as a potential health-promoting meat ingredient with no effect on plant sterol stability but with some effects on texture and sensory characteristics. © The Author(s) 2016.

Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues.

Science.gov (United States)

Schulz, Timothy A; Choi, Mal-Gi; Raychaudhuri, Sumana; Mears, Jason A; Ghirlando, Rodolfo; Hinshaw, Jenny E; Prinz, William A

2009-12-14

Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein-related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.

Impact of ice melting on distribution of particulate sterols in glacial fjords of Chilean Patagonia

Science.gov (United States)

Gutiérrez, Marcelo H.; Riquelme, Pablo; Pantoja, Silvio

2016-04-01

We analyzed variability in abundance and composition of sterols in waters of the fjord adjacent to glacier Jorge Montt, one of the fastest retreated glaciers in Patagonian Icefields. The study was carried out between August 2012 and November 2013 under different meltwater scenarios. Distribution of sterols in surface and bottom waters was determined by Gas Chromatography coupled to Mass Spectrometry. Sterol concentration ranged from 18 to 1726 ng/L in surface and bottom waters and was positive correlated with chlorophyll-a concentration. Under high melting conditions in austral summer, surface meltwaters showed high concentrations of sterols and were dominated by methylene-cholesterol, a representative sterol of centric diatoms. In the area near open ocean and in austral autumn, winter and spring in proglacial fjord, lower sterol concentrations in surface waters were accompanied by other microalgae sterols and an increase in relative abundance of plant sterols, evidencing a different source of organic matter. In autumn, when high meltwater flux was also evidenced, presence of stanols and an uncommon tri-unsaturated sterol suggests influence of meltwaters in composition of sterols in the downstream fjord. We conclude that ice melting can modify sterol composition by setting conditions for development of a singular phytoplankton population able to thrive in surface meltwater and by carrying glacier organic matter into Patagonian glacial fjords. In projected ice melting scenario, these changes in organic matter quantity and quality can potentially affect availability of organic substrates for heterotrophic activity and trophic status of glacial fjords. This research was funded by COPAS Sur-Austral (PFB-31)

Marine metabolites: The sterols of soft coral

Digital Repository Service at National Institute of Oceanography (India)

Sarma, N.S.; Krishna, M.S.; Pasha, Sk.G.; Rao, T.S.P.; Venkateswarlu, Y.; Parameswaran, P.S.

Sterols constitute a major group of secondary metabolites of soft corals. Several of these compounds have the 'usual' 3 beta-hydroxy, delta sup(5) (or delta sup(0)) cholestane skeleton, a large number of these metabolites are polar sterols...

Relative abundance of Delta(5)-sterols in plasma membrane lipids of root-tip cells correlates with aluminum tolerance of rice.

Science.gov (United States)

Khan, M Shahadat Hossain; Tawaraya, Keitarou; Sekimoto, Hiroshi; Koyama, Hiroyuki; Kobayashi, Yuriko; Murayama, Tetsuya; Chuba, Masaru; Kambayashi, Mihoko; Shiono, Yoshihito; Uemura, Matsuo; Ishikawa, Satoru; Wagatsuma, Tadao

2009-01-01

We investigated variations in aluminum (Al) tolerance among rice plants, using ancestor cultivars from the family line of the Al-tolerant and widely cultivated Japonica cultivar, Sasanishiki. The cultivar Rikuu-20 was Al sensitive, whereas a closely related cultivar that is a descendant of Rikuu-20, Rikuu-132, was Al tolerant. These two cultivars were compared to determine mechanisms underlying variations in Al tolerance. The sensitive cultivar Rikuu-20 showed increased permeability of the plasma membrane (PM) and greater Al uptake within 1 h of Al treatment. This could not be explained by organic acid release. Lipid composition of the PM differed between these cultivars, and may account for the difference in Al tolerance. The tolerant cultivar Rikuu-132 had a lower ratio of phospholipids to Delta(5)-sterols than the sensitive cultivar Rikuu-20, suggesting that the PM of Rikuu-132 is less negatively charged and less permeabilized than that of Rikuu-20. We used inhibitors of Delta(5)-sterol synthesis to alter the ratio of phospholipids to Delta(5)-sterols in both cultivars. These inhibitors reduced Al tolerance in Rikuu-132 and its Al-tolerant ancestor cultivars Kamenoo and Kyoku. In addition, Rikuu-132 showed a similar level of Al sensitivity when the ratio of phospholipids to Delta(5)-sterols was increased to match that of Rikuu-20 after treatment with uniconazole-P, an inhibitor of obtusifoliol-14alpha-demethylase. These results indicate that PM lipid composition is a factor underlying variations in Al tolerance among rice cultivars.

Plant sterol ester diet supplementation increases serum plant sterols and markers of cholesterol synthesis, but has no effect on total cholesterol levels.

Science.gov (United States)

Weingärtner, Oliver; Bogeski, Ivan; Kummerow, Carsten; Schirmer, Stephan H; Husche, Constanze; Vanmierlo, Tim; Wagenpfeil, Gudrun; Hoth, Markus; Böhm, Michael; Lütjohann, Dieter; Laufs, Ulrich

2017-05-01

This double-blind, randomized, placebo-controlled, cross-over intervention-study was conducted in healthy volunteers to evaluate the effects of plant sterol ester supplemented margarine on cholesterol, non-cholesterol sterols and oxidative stress in serum and monocytes. Sixteen volunteers, average age 34 years, with no or mild hypercholesterolemia were subjected to a 4 week period of daily intake of 3g plant sterols per day supplied via a supplemented margarine on top of regular eating habits. After a wash-out period of one week, volunteers switched groups. Compared to placebo, a diet supplementation with plant sterols increased serum levels of plant sterols such as campesterol (+0.16±0.19mg/dL, p=0.005) and sitosterol (+0.27±0.18mg/dL, psynthesis such as desmosterol (+0.05±0.07mg/dL, p=0.006) as well as lathosterol (+0.11±0.16mg/dL, p=0.012). Cholesterol serum levels, however, were not changed significantly (+18.68±32.6mg/dL, p=0.052). These findings could not be verified in isolated circulating monocytes. Moreover, there was no effect on monocyte activation and no differences with regard to redox state after plant sterol supplemented diet. Therefore, in a population of healthy volunteers with no or mild hypercholesterolemia, consumption of plant sterol ester supplemented margarine results in increased concentrations of plant sterols and cholesterol synthesis markers without affecting total cholesterol in the serum, activation of circulating monocytes or redox state. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis of hydroxylated sterols in transgenic Arabidopsis plants alters growth and steroid metabolism.

Science.gov (United States)

Beste, Lisa; Nahar, Nurun; Dalman, Kerstin; Fujioka, Shozo; Jonsson, Lisbeth; Dutta, Paresh C; Sitbon, Folke

2011-09-01

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and β forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.

SUPLEMENTASI STEROL LEMBAGA GANDUM (Triticum sp. PADA MARGARIN (Supplementation of Margarine with Wheat Germ Sterol

Directory of Open Access Journals (Sweden)

Sri Anna Marliyati1*

2010-06-01

Full Text Available Margarine is a water in oil (w/o emulsion product which is widely used for household cooking and baking industry. Consuming of margarine, which contains trans fatty acid may cause health problem due to the increase of LDL cholesterol. Since margarine is also a good carrier of phytosterol which prevent the absorption of cholesterol, there is a possibility to formulate a healthier margarine. In this research formulation and characteristics of products was investigated. The research work consisted of two steps: (1 supplementation of wheat germ sterol into margarine (two methods and (2 analysis of physical, chemical characteristics and hedonic score. Parameters of physical characteristics were melting point and emulsion stability, whereas chemical characteristics were water and oil contents. The hedonic test was carried out based on product’s color, odor, taste, texture, and spreadability. Results showed that method II of supplementation produced better margarine than method I, in which the concentration of sterol in the margarine was higher with a melting point similar to that of control, better emulsion stability, and higher hedonic score. Supplementation process was carried out by mixing sterol into fat phase melted at 50 0C, followed by mixing with aqueous phase at 4 0C. Sterol used for method II was extracted using mixed solvent of hexane and ethanol at the ratio of 1:2 (v/v, which was resulted from previous experimentation.

Genetics Home Reference: Greenberg dysplasia

Science.gov (United States)

... of certain hormones and digestive acids. During cholesterol synthesis, the sterol reductase function of the lamin B receptor allows ... this loss causes the condition. Absence of the sterol reductase function disrupts the normal synthesis of cholesterol within cells. This absence may also ...

Multicolor bleach-rate imaging enlightens in vivo sterol transport

DEFF Research Database (Denmark)

Wüstner, Daniel; Sage, Daniel

2011-01-01

, dehydroergosterol (DHE) in the genetically tractable model organism Caenorhabditis elegans (C. elegans). DHE is structurally very similar to cholesterol and ergosterol, two sterols used by the sterol-auxotroph nematode. We developed a new computational method measuring fluorophore bleaching kinetics at every pixel...... with a lysosomal marker, GFP-LMP1. Our new methods hold great promise for further studies on endosomal sterol transport in C. elegans....

The dynamin chemical inhibitor dynasore impairs cholesterol trafficking and sterol-sensitive genes transcription in human HeLa cells and macrophages.

Directory of Open Access Journals (Sweden)

Emmanuelle Girard

Full Text Available Intracellular transport of cholesterol contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we characterized the impact of dynasore, a recently described drug that specifically inhibits the enzymatic activity of dynamin, a GTPase regulating receptor endocytosis and cholesterol trafficking. Dynasore strongly inhibited the uptake of low-density lipoprotein (LDL in HeLa cells, and to a lower extent in human macrophages. In both cell types, dynasore treatment led to the abnormal accumulation of LDL and free cholesterol (FC within the endolysosomal network. The measure of cholesterol esters (CE further showed that the delivery of regulatory cholesterol to the endoplasmic reticulum (ER was deficient. This resulted in the inhibition of the transcriptional control of the three major sterol-sensitive genes, sterol-regulatory element binding protein 2 (SREBP-2, 3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR, and low-density lipoprotein receptor (LDLR. The sequestration of cholesterol in the endolysosomal compartment impaired both the active and passive cholesterol efflux in HMDM. Our data further illustrate the importance of membrane trafficking in cholesterol homeostasis and validate dynasore as a new pharmacological tool to study the intracellular transport of cholesterol.

Plant sterol or stanol esters retard lesion formation in LDL receptor-deficient mice independent of changes in serum plant sterols

NARCIS (Netherlands)

Plat, Jogchum; Beugels, Ilona; Gijbels, Marion J. J.; de Winther, Menno P. J.; Mensink, Ronald P.

2006-01-01

Statins do not always decrease coronary heart disease mortality, which was speculated based on increased serum plant sterols observed during statin treatment. To evaluate plant sterol atherogenicity, we fed low density lipoprotein-receptor deficient (LDLr(+/-)) mice for 35 weeks with Western diets

Identification of ergosterol and inhibition of sterol synthesis by Δ5-sterols in GL7, an auxotrophic mutant of yeast

International Nuclear Information System (INIS)

Dhanuka, I.C.

1988-01-01

Synthesis of ergosterol was demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the exogenous sterol was 5α-cholestan-3β-ol, 5α-cholest-8(14)-en-3β-ol, or 24β-methyl-5α-cholest-8(14)-en-3β-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol. Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labelling from [ 3 H]acetate. Except for some cholest-5-en-3β-ol (cholesterol) which was derived from the 5α-cholestan-3β-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the exogenous sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5α-cholest-8(9)-en-3β-ol and 5α-cholest-7-en-3β-ol (lathosterol) were converted to cholesta-5,7-dien-3β-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol

Plant sterol intakes and colorectal cancer risk in the Netherlands : cohort study on diet and cancer

NARCIS (Netherlands)

Normén, A.L.; Brants, H.A.M.; Voorrips, L.E.; Andersson, H.A.; Brandt, P.A. van den

2001-01-01

Background: Plant sterols in vegetable foods might prevent colorectal cancer. Objective: The objective was to study plant sterol intakes in relation to colorectal cancer risk in an epidemiologic study. Design: The study was performed within the framework of the Netherlands Cohort Study on Diet and

Vesicle fluctuation analysis of the effects of sterols on membrane bending rigidity

DEFF Research Database (Denmark)

Henriksen, Jonas Rosager; Rowat, Amy C.; Ipsen, John H.

2004-01-01

Sterols are regulators of both biological function and structure. The role of cholesterol in promoting the structural and mechanical stability of membranes is widely recognized. Knowledge of how the related sterols, lanosterol and ergosterol, affect membrane mechanical properties is sparse. This ...... on vesicle behaviour are also discussed. These recent modifications render vesicle fluctuation analysis an efficient and accurate method for determining how cholesterol, lanosterol, and ergosterol increase membrane bending rigidity....

Identifying avian sources of faecal contamination using sterol analysis.

Science.gov (United States)

Devane, Megan L; Wood, David; Chappell, Andrew; Robson, Beth; Webster-Brown, Jenny; Gilpin, Brent J

2015-10-01

Discrimination of the source of faecal pollution in water bodies is an important step in the assessment and mitigation of public health risk. One tool for faecal source tracking is the analysis of faecal sterols which are present in faeces of animals in a range of distinctive ratios. Published ratios are able to discriminate between human and herbivore mammal faecal inputs but are of less value for identifying pollution from wildfowl, which can be a common cause of elevated bacterial indicators in rivers and streams. In this study, the sterol profiles of 50 avian-derived faecal specimens (seagulls, ducks and chickens) were examined alongside those of 57 ruminant faeces and previously published sterol profiles of human wastewater, chicken effluent and animal meatwork effluent. Two novel sterol ratios were identified as specific to avian faecal scats, which, when incorporated into a decision tree with human and herbivore mammal indicative ratios, were able to identify sterols from avian-polluted waterways. For samples where the sterol profile was not consistent with herbivore mammal or human pollution, avian pollution is indicated when the ratio of 24-ethylcholestanol/(24-ethylcholestanol + 24-ethylcoprostanol + 24-ethylepicoprostanol) is ≥0.4 (avian ratio 1) and the ratio of cholestanol/(cholestanol + coprostanol + epicoprostanol) is ≥0.5 (avian ratio 2). When avian pollution is indicated, further confirmation by targeted PCR specific markers can be employed if greater confidence in the pollution source is required. A 66% concordance between sterol ratios and current avian PCR markers was achieved when 56 water samples from polluted waterways were analysed.

Overturning dogma: tolerance of insects to mixed-sterol diets is not universal.

Science.gov (United States)

Behmer, Spencer T

2017-10-01

Insects cannot synthesize sterols de novo, but like all eukaryotes they use them as cell membrane inserts where they influence membrane fluidity and rigidity. They also use a small amount for metabolic purposes, most notably as essential precursors for steroid hormones. It has been a long-held view that most insects require a small amount of specific sterol (often cholesterol) for metabolic purposes, but for membrane purposes (where the bulk of sterols are used) specificity in sterol structure was less important. Under this model, it was assumed that insects could tolerate mixed-sterol diets as long as a small amount of cholesterol was available. In the current paper this dogma is overturned, using data from plant-feeding insects that were fed mixed-sterol diets with different amounts and ratios of dietary sterols. Copyright © 2017 Elsevier Inc. All rights reserved.

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana.

Directory of Open Access Journals (Sweden)

Roy Mwenechanya

2017-06-01

Full Text Available Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites' genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51. The mutation, N176I, is found outside of the enzyme's active site, consistent with the fact that the resistant line continues to produce the enzyme's product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself.

Sterol Profile for Natural Juices Authentification by GC-MS

Science.gov (United States)

Culea, M.

2007-04-01

A GC-MS analytical method is described for some natural juices analysis. The fingerprint of sterols was used to characterize the natural juice. A rapid liquid-liquid extraction method was used. The sterols were separated on a Rtx-5MS capillary column, 15m×0.25mm, 0.25μm film thickness, in a temperature program from 50°C for 1 min, then ramped at 15°C/min to 300°C and held for 15 min. Identification of sterols and their patterns were used for juice characterization. The sterol profile is a useful approach for confirming the presence of juices of orange, grapefruit, pineapple and passion fruit in compounded beverages and for detecting of adulteration of fruit juices.

Sterol Profile for Natural Juices Authentification by GC-MS

International Nuclear Information System (INIS)

Culea, M.

2007-01-01

A GC-MS analytical method is described for some natural juices analysis. The fingerprint of sterols was used to characterize the natural juice. A rapid liquid-liquid extraction method was used. The sterols were separated on a Rtx-5MS capillary column, 15mx0.25mm, 0.25μm film thickness, in a temperature program from 50 deg. C for 1 min, then ramped at 15 deg. C/min to 300 deg. C and held for 15 min. Identification of sterols and their patterns were used for juice characterization. The sterol profile is a useful approach for confirming the presence of juices of orange, grapefruit, pineapple and passion fruit in compounded beverages and for detecting of adulteration of fruit juices

Increased plant sterol and stanol levels in brain of Watanabe rabbits fed rapeseed oil derived plant sterol or stanol esters

DEFF Research Database (Denmark)

Fricke, Christiane B.; Schrøder, Malene; Poulsen, Morten

2007-01-01

. Cholesterol synthesis in brain, indicated by lathosterol, a local surrogate cholesterol synthesis marker, does not seem to be affected by plant sterol or stanol ester feeding. We conclude that high dose intake of plant sterol and stanol esters in Watanabe rabbits results in elevated concentrations...... of these components not only in the periphery but also in the central nervous system....... of these components in brain tissue of homozygous and heterozygous Watanabe rabbits, an animal model for familial hypercholesterolemia. Homozygous animals received either a standard diet, RSO stanol or RSO sterol ester while heterozygous animals were additionally fed with 2 g cholesterol/kg to the respective diet...

Simultaneous effects of light intensity and phosphorus supply on the sterol content of phytoplankton.

Directory of Open Access Journals (Sweden)

Maike Piepho

Full Text Available Sterol profiles of microalgae and their change with environmental conditions are of great interest in ecological food web research and taxonomic studies alike. Here, we investigated effects of light intensity and phosphorus supply on the sterol content of phytoplankton and assessed potential interactive effects of these important environmental factors on the sterol composition of algae. We identified sterol contents of four common phytoplankton genera, Scenedesmus, Chlamydomonas, Cryptomonas and Cyclotella, and analysed the change in sterol content with varying light intensities in both a high-phosphorus and a low-phosphorus approach. Sterol contents increased significantly with increasing light in three out of four species. Phosphorus-limitation reversed the change of sterol content with light intensity, i.e., sterol content decreased with increasing light at low phosphorus supply. Generally sterol contents were lower in low-phosphorus cultures. In conclusion, both light and phosphorus conditions strongly affect the sterol composition of algae and hence should be considered in ecological and taxonomic studies investigating the biochemical composition of algae. Data suggest a possible sterol limitation of growth and reproduction of herbivorous crustacean zooplankton during summer when high light intensities and low phosphorus supply decrease sterol contents of algae.

Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9.

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Xinwei Liu

Full Text Available Oxysterol binding protein (OSBP and OSBP-related proteins (ORPS have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE were poor ligands for OSBP. In contrast, both long (ORP9L and short (ORP9S variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9).

Science.gov (United States)

Liu, Xinwei; Ridgway, Neale D

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway.

Science.gov (United States)

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.

Biofuels. Altered sterol composition renders yeast thermotolerant

DEFF Research Database (Denmark)

Caspeta, Luis; Chen, Yun; Ghiaci, Payam

2014-01-01

adaptive laboratory evolution to select yeast strains with improved growth and ethanol production at ≥40°C. Sequencing of the whole genome, genome-wide gene expression, and metabolic-flux analyses revealed a change in sterol composition, from ergosterol to fecosterol, caused by mutations in the C-5 sterol......Ethanol production for use as a biofuel is mainly achieved through simultaneous saccharification and fermentation by yeast. Operating at ≥40°C would be beneficial in terms of increasing efficiency of the process and reducing costs, but yeast does not grow efficiently at those temperatures. We used...... desaturase gene, and increased expression of genes involved in sterol biosynthesis. Additionally, large chromosome III rearrangements and mutations in genes associated with DNA damage and respiration were found, but contributed less to the thermotolerant phenotype....

Transport of sterols to the plasma membrane of leek seedlings

International Nuclear Information System (INIS)

Moreau, P.; Hartmann, M.A.; Perret, A.M.; Sturbois-Balcerazak, B.; Cassagne, C.

1998-01-01

To investigate the intracellular transport of sterols in etiolated leek (Allium porrum L.) seedlings, in vivo pulse-chase experiments with [1-14C]acetate were performed. Then, endoplasmic reticulum-, Golgi-, and plasma membrane (PM)-enriched fractions were prepared and analyzed for the radioactivity incorporated into free sterols. In leek seedlings sterols are present as a mixture in which (24R)-24-ethylcholest-5-en-3beta-ol is by far the major compound (around 60%). The other sterols are represented by cholest-5-en-3beta-ol, 24-methyl-cholest-5-en-3beta-ol, (24S)-24-ethylcholesta-5,22E-dien-3beta-ol, and stigmasta-5,24(24(1))Z-dien-3Beta-ol. These compounds are shown to reside mainly in the PM. Our results clearly indicate that free sterols are actively transported from the endoplasmic reticulum to the PM during the first 60 min of chase, with kinetics very similar to that of phosphatidylserine. Such a transport was found to be decreased at low temperature (12 degrees C) and following treatment with monensin and brefeldin A. These data are consistent with a membrane-mediated process for the intracellular transport of sterols to the PM, which likely involves the Golgi apparatus

STEROLS AS BIOMARKERS IN GYMNODINIUM BREVE DISTRIBUTION IN DINOFLAGELLATES

Science.gov (United States)

The sterol composition of marine microalgae has been shown to be a chemotaxonomic property potentially of value in distinguishing members of different algal classes. For example, members of the class Dinophyceae display sterol compositions ranging from as few as two (cholesterol ...

Plant ecdysteroids: plant sterols with intriguing distributions, biological effects and relations to plant hormones.

Science.gov (United States)

Tarkowská, Danuše; Strnad, Miroslav

2016-09-01

The present review summarises current knowledge of phytoecdysteroids' biosynthesis, distribution within plants, biological importance and relations to plant hormones. Plant ecdysteroids (phytoecdysteroids) are natural polyhydroxylated compounds that have a four-ringed skeleton, usually composed of either 27 carbon atoms or 28-29 carbon atoms (biosynthetically derived from cholesterol or other plant sterols, respectively). Their physiological roles in plants have not yet been confirmed and their occurrence is not universal. Nevertheless, they are present at high concentrations in various plant species, including commonly consumed vegetables, and have a broad spectrum of pharmacological and medicinal properties in mammals, including hepatoprotective and hypoglycaemic effects, and anabolic effects on skeletal muscle, without androgenic side-effects. Furthermore, phytoecdysteroids can enhance stress resistance by promoting vitality and enhancing physical performance; thus, they are considered adaptogens. This review summarises current knowledge of phytoecdysteroids' biosynthesis, distribution within plants, biological importance and relations to plant hormones.

Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast

DEFF Research Database (Denmark)

Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D.

2014-01-01

Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been...

The role of serum non-cholesterol sterols as surrogate markers of absolute cholesterol synthesis and absorption.

Science.gov (United States)

Miettinen, T A; Gylling, H; Nissinen, M J

2011-10-01

To study the whole-body cholesterol metabolism in man, cholesterol synthesis and absorption need to be measured. Because of the complicated methods of the measurements, new approaches were developed including the analysis of serum non-cholesterol sterols. In current lipidologic papers and even in intervention studies, serum non-cholesterol sterols are frequently used as surrogate markers of cholesterol metabolism without any validation to the absolute metabolic variables. The present review compares serum non-cholesterol sterols with absolute measurements of cholesterol synthesis and absorption in published papers to find out whether the serum markers are valid indicators of cholesterol metabolism in various conditions. During statin treatment, during interventions of dietary fat, and in type 2 diabetes the relative and absolute variables of cholesterol synthesis and absorption were frequently but not constantly correlated with each other. In some occasions, especially in subjects with apolipoprotein E3/4 and E4/4 phenotypes, the relative metabolic markers were even more sensitive than the absolute ones to reflect changes in cholesterol metabolism during dietary interventions. Even in general population at very high absorption the homeostasis of cholesterol metabolism is disturbed damaging the validity of the serum markers. It is worth using several instead of only one precursor and absorption sterol marker for making conclusions of altered synthesis or absorption of cholesterol, and even then the presence of at least some absolute measurement is valuable. During consumption of plant sterol-enriched diets and in situations of interfered cholesterol homeostasis the relative markers do not adequately reflect cholesterol metabolism. Accordingly, the validity of the relative markers of cholesterol metabolism should not be considered as self-evident. Copyright © 2011 Elsevier B.V. All rights reserved.

Sterol biosynthesis is required for heat resistance but not extracellular survival in leishmania.

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Wei Xu

2014-10-01

Full Text Available Sterol biosynthesis is a crucial pathway in eukaryotes leading to the production of cholesterol in animals and various C24-alkyl sterols (ergostane-based sterols in fungi, plants, and trypanosomatid protozoa. Sterols are important membrane components and precursors for the synthesis of powerful bioactive molecules, including steroid hormones in mammals. Their functions in pathogenic protozoa are not well characterized, which limits the development of sterol synthesis inhibitors as drugs. Here we investigated the role of sterol C14α-demethylase (C14DM in Leishmania parasites. C14DM is a cytochrome P450 enzyme and the primary target of azole drugs. In Leishmania, genetic or chemical inactivation of C14DM led to a complete loss of ergostane-based sterols and accumulation of 14-methylated sterols. Despite the drastic change in lipid composition, C14DM-null mutants (c14dm(- were surprisingly viable and replicative in culture. They did exhibit remarkable defects including increased membrane fluidity, failure to maintain detergent resistant membrane fraction, and hypersensitivity to heat stress. These c14dm(- mutants showed severely reduced virulence in mice but were highly resistant to itraconazole and amphotericin B, two drugs targeting sterol synthesis. Our findings suggest that the accumulation of toxic sterol intermediates in c14dm(- causes strong membrane perturbation and significant vulnerability to stress. The new knowledge may help improve the efficacy of current drugs against pathogenic protozoa by exploiting the fitness loss associated with drug resistance.

Sterol biosynthesis is required for heat resistance but not extracellular survival in leishmania.

Science.gov (United States)

Xu, Wei; Hsu, Fong-Fu; Baykal, Eda; Huang, Juyang; Zhang, Kai

2014-10-01

Sterol biosynthesis is a crucial pathway in eukaryotes leading to the production of cholesterol in animals and various C24-alkyl sterols (ergostane-based sterols) in fungi, plants, and trypanosomatid protozoa. Sterols are important membrane components and precursors for the synthesis of powerful bioactive molecules, including steroid hormones in mammals. Their functions in pathogenic protozoa are not well characterized, which limits the development of sterol synthesis inhibitors as drugs. Here we investigated the role of sterol C14α-demethylase (C14DM) in Leishmania parasites. C14DM is a cytochrome P450 enzyme and the primary target of azole drugs. In Leishmania, genetic or chemical inactivation of C14DM led to a complete loss of ergostane-based sterols and accumulation of 14-methylated sterols. Despite the drastic change in lipid composition, C14DM-null mutants (c14dm(-)) were surprisingly viable and replicative in culture. They did exhibit remarkable defects including increased membrane fluidity, failure to maintain detergent resistant membrane fraction, and hypersensitivity to heat stress. These c14dm(-) mutants showed severely reduced virulence in mice but were highly resistant to itraconazole and amphotericin B, two drugs targeting sterol synthesis. Our findings suggest that the accumulation of toxic sterol intermediates in c14dm(-) causes strong membrane perturbation and significant vulnerability to stress. The new knowledge may help improve the efficacy of current drugs against pathogenic protozoa by exploiting the fitness loss associated with drug resistance.

Lipid-lowering Activity of Natural and Semi-Synthetic Sterols and Stanols.

Science.gov (United States)

Taha, Dhiaa A; Wasan, Ellen K; Wasan, Kishor M; Gershkovich, Pavel

2015-01-01

Consumption of plant sterols/ stanols has long been demonstrated to reduce plasma cholesterol levels. The objective of this review is to demonstrate the lipid-lowering activity and anti-atherogenic effects of natural and semi-synthetic plant sterols/ stanols based on evidence from cell-culture studies, animal studies and clinical trials. Additionally, this review highlights certain molecular mechanisms by which plant sterols/ stanols lower plasma cholesterol levels with a special emphasis on factors that affect the cholesterol-lowering activity of plant sterols/stanols. The crystalline nature and the poor oil solubility of these natural products could be important factors that limit their cholesterol-lowering efficiency. Several attempts have been made to improve the cholesterol-lowering activity by enhancing the bioavailability of crystalline sterols and stanols. Approaches involved reduction of the crystal size and/or esterification with fatty acids from vegetable or fish oils. However, the most promising approach in this context is the chemical modification of plant sterols /stanols into water soluble disodium ascorbyl phytostanyl phosphates analogue by esterification with ascorbic acid. This novel semi-synthetic stanol derivative has improved efficacy over natural plant sterols/ stanols and can provide additional benefits by combining the cholesterol-lowering properties of plant stanols with the antioxidant potential of ascorbic acid. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Sterol composition of Cryptococcus neoformans in the presence and absence of fluconazole.

OpenAIRE

Ghannoum, M A; Spellberg, B J; Ibrahim, A S; Ritchie, J A; Currie, B; Spitzer, E D; Edwards, J E; Casadevall, A

1994-01-01

Analysis of the sterol compositions of 13 clinical isolates of the pathogenic yeast Cryptococcus neoformans obtained from five patients with recurring cryptococcal meningitis showed that, unlike Candida albicans, the major sterols synthesized by this yeast were obtusifoliol (range, 21.1 to 68.2%) and ergosterol (range, 0.0 to 46.5%). There was considerable variation in the sterol contents among the 13 isolates, with total sterol contents ranging from 0.31 to 5.9% of dry weight. The isolates f...

Biosynthesis of sterols from mevalonate in a starfish, Coscinasterias acutispina

International Nuclear Information System (INIS)

Teshima, Shin-ichi; Kanazawa, Akio

1976-01-01

This study deals with the biosynthesis of sterols from mevalonate in a starfish, Coscinasterias acutispina. After injection of mevalonate-2- 14 C, the metabolites were investigated by using thin-layer, column, and gas-liquid chromatographic techniques. The detailed investigation of radioactive desmethylsterols showed that radioactivity was mainly associated with cholest-7-enol. However, there was no evidence for the incorporation of mevalonate-2- 14 C into C 26 -, C 28 -, and C 29 -sterols besides cholestanol and cholesterol. The results indicated that the starfish, C. acutispina, is capable of synthesizing at least cholest-7-enol from mevalonate via probably squalene and lanosterol etc. But not sterols other than C 27 -sterols. Also, it was suggested that the conversion of cholest-7-enol to cholesterol may not proceed in this starfish. (auth.)

Ketopantoyl lactone reductase is a conjugated polyketone reductase.

Science.gov (United States)

Hata, H; Shimizu, S; Hattori, S; Yamada, H

1989-03-01

Ketopantoyl lactone reductase (EC 1.1.1.168) of Saccharomyces cerevisiae was found to catalyze the reduction of a variety of natural and unnatural conjugated polyketone compounds and quinones, such as isatin, ninhydrin, camphorquinone and beta-naphthoquinone in the presence of NADPH. 5-Bromoisatin is the best substrate for the enzyme (Km = 3.1 mM; Vmax = 650 mumol/min/mg). The enzyme is inhibited by quercetin, and several polyketones. These results suggest that ketopantoyl lactone reductase is a carbonyl reductase which specifically catalyzes the reduction of conjugated polyketones.

A Novel Fibrosis Index Comprising a Non-Cholesterol Sterol Accurately Predicts HCV-Related Liver Cirrhosis

DEFF Research Database (Denmark)

Ydreborg, Magdalena; Lisovskaja, Vera; Lagging, Martin

2014-01-01

of the present study was to create a model for accurate prediction of liver cirrhosis based on patient characteristics and biomarkers of liver fibrosis, including a panel of non-cholesterol sterols reflecting cholesterol synthesis and absorption and secretion. We evaluated variables with potential predictive...

Synthesis of Hydroxylated Sterols in Transgenic Arabidopsis Plants Alters Growth and Steroid Metabolism1[C][W][OA

Science.gov (United States)

Beste, Lisa; Nahar, Nurun; Dalman, Kerstin; Fujioka, Shozo; Jonsson, Lisbeth; Dutta, Paresh C.; Sitbon, Folke

2011-01-01

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and β forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels. PMID:21746809

Plant sterols and plant stanols in the management of dyslipidaemia and prevention of cardiovascular disease.

Science.gov (United States)

Gylling, Helena; Plat, Jogchum; Turley, Stephen; Ginsberg, Henry N; Ellegård, Lars; Jessup, Wendy; Jones, Peter J; Lütjohann, Dieter; Maerz, Winfried; Masana, Luis; Silbernagel, Günther; Staels, Bart; Borén, Jan; Catapano, Alberico L; De Backer, Guy; Deanfield, John; Descamps, Olivier S; Kovanen, Petri T; Riccardi, Gabriele; Tokgözoglu, Lale; Chapman, M John

2014-02-01

This EAS Consensus Panel critically appraised evidence relevant to the benefit to risk relationship of functional foods with added plant sterols and/or plant stanols, as components of a healthy lifestyle, to reduce plasma low-density lipoprotein-cholesterol (LDL-C) levels, and thereby lower cardiovascular risk. Plant sterols/stanols (when taken at 2 g/day) cause significant inhibition of cholesterol absorption and lower LDL-C levels by between 8 and 10%. The relative proportions of cholesterol versus sterol/stanol levels are similar in both plasma and tissue, with levels of sterols/stanols being 500-/10,000-fold lower than those of cholesterol, suggesting they are handled similarly to cholesterol in most cells. Despite possible atherogenicity of marked elevations in circulating levels of plant sterols/stanols, protective effects have been observed in some animal models of atherosclerosis. Higher plasma levels of plant sterols/stanols associated with intakes of 2 g/day in man have not been linked to adverse effects on health in long-term human studies. Importantly, at this dose, plant sterol/stanol-mediated LDL-C lowering is additive to that of statins in dyslipidaemic subjects, equivalent to doubling the dose of statin. The reported 6-9% lowering of plasma triglyceride by 2 g/day in hypertriglyceridaemic patients warrants further evaluation. Based on LDL-C lowering and the absence of adverse signals, this EAS Consensus Panel concludes that functional foods with plant sterols/stanols may be considered 1) in individuals with high cholesterol levels at intermediate or low global cardiovascular risk who do not qualify for pharmacotherapy, 2) as an adjunct to pharmacologic therapy in high and very high risk patients who fail to achieve LDL-C targets on statins or are statin- intolerant, 3) and in adults and children (>6 years) with familial hypercholesterolaemia, in line with current guidance. However, it must be acknowledged that there are no randomised, controlled

Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

Science.gov (United States)

Hata, H; Shimizu, S; Hattori, S; Yamada, H

1989-02-24

Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

Yeast metabolic engineering--targeting sterol metabolism and terpenoid formation.

Science.gov (United States)

Wriessnegger, Tamara; Pichler, Harald

2013-07-01

Terpenoids comprise various structures conferring versatile functions to eukaryotes, for example in the form of prenyl-anchors they attach proteins to membranes. The physiology of eukaryotic membranes is fine-tuned by another terpenoid class, namely sterols. Evidence is accumulating that numerous membrane proteins require specific sterol structural features for function. Moreover, sterols are intermediates in the synthesis of steroids serving as hormones in higher eukaryotes. Like steroids many compounds of the terpenoid family do not contribute to membrane architecture, but serve as signalling, protective or attractant/repellent molecules. Particularly plants have developed a plenitude of terpenoid biosynthetic routes branching off early in the sterol biosynthesis pathway and, thereby, forming one of the largest groups of naturally occurring organic compounds. Many of these aromatic and volatile molecules are interesting for industrial application ranging from foods to pharmaceuticals. Combining the fortunate situation that sterol biosynthesis is highly conserved in eukaryotes with the amenability of yeasts to genetic and metabolic engineering, basically all naturally occurring terpenoids might be produced involving yeasts. Such engineered yeasts are useful for the study of biological functions and molecular interactions of terpenoids as well as for the large-scale production of high-value compounds, which are unavailable in sufficient amounts from natural sources due to their low abundance. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans

OpenAIRE

Dahlin, Paul; Srivastava, Vaibhav; Ekengren, Sophia; McKee, Lauren S.; Bulone, Vincent

2017-01-01

The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heter...

Steryl ester synthesis, storage and hydrolysis: A contribution to sterol homeostasis.

Science.gov (United States)

Korber, Martina; Klein, Isabella; Daum, Günther

2017-12-01

Sterols are essential lipids of all eukaryotic cells, appearing either as free sterols or steryl esters. Besides other regulatory mechanisms, esterification of sterols and hydrolysis of steryl esters serve to buffer both an excess and a lack of free sterols. In this review, the esterification process, the storage of steryl esters and their mobilization will be described. Several model organisms are discussed but the focus was set on mammals and the yeast Saccharomyces cerevisiae. The contribution of imbalanced cholesterol homeostasis to several human diseases, namely Wolman disease, cholesteryl ester storage disease, atherosclerosis and Alzheimer's disease, Niemann-Pick type C and Tangier disease is described. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasma sterol evidence for decreased absorption and increased synthesis of cholesterol in insulin resistance and obesity.

Science.gov (United States)

Paramsothy, Pathmaja; Knopp, Robert H; Kahn, Steven E; Retzlaff, Barbara M; Fish, Brian; Ma, Lina; Ostlund, Richard E

2011-11-01

The rise in LDL with egg feeding in lean insulin-sensitive (LIS) participants is 2- and 3-fold greater than in lean insulin-resistant (LIR) and obese insulin-resistant (OIR) participants, respectively. We determined whether differences in cholesterol absorption, synthesis, or both could be responsible for these differences by measuring plasma sterols as indexes of cholesterol absorption and endogenous synthesis. Plasma sterols were measured by gas chromatography-mass spectrometry in a random subset of 34 LIS, 37 LIR, and 37 OIR participants defined by the insulin sensitivity index (S(I)) and by BMI criteria selected from a parent group of 197 participants. Cholestanol and plant sterols provide a measure of cholesterol absorption, and lathosterol provides a measure of cholesterol synthesis. The mean (±SD) ratio of plasma total absorption biomarker sterols to cholesterol was 4.48 ± 1.74 in LIS, 3.25 ± 1.06 in LIR, and 2.82 ± 1.08 in OIR participants. After adjustment for age and sex, the relations of the absorption sterol-cholesterol ratios were as follows: LIS > OIR (P LIR (P OIR (P = 0.11). Lathosterol-cholesterol ratios were 0.71 ± 0.32 in the LIS participants, 0.95 ± 0.47 in the LIR participants, and 1.29 ± 0.55 in the OIR participants. After adjustment for age and sex, the relations of lathosterol-cholesterol ratios were as follows: LIS sterol concentrations were positively associated with S(I) and negatively associated with obesity, whereas lathosterol correlations were the opposite. Cholesterol absorption was highest in the LIS participants, whereas cholesterol synthesis was highest in the LIR and OIR participants. Therapeutic diets for hyperlipidemia should emphasize low-cholesterol diets in LIS persons and weight loss to improve S(I) and to decrease cholesterol overproduction in LIR and OIR persons.

Plant sterols for adults with hypercholesterolemia treated with or without medication (statins

Directory of Open Access Journals (Sweden)

Raquel Bernácer

2015-07-01

Full Text Available Hypercholesterolemia is the most common coronary risk factor among the Spanish population; 37.4% of the Spanish adult population have cholesterol levels between 190 and 240 mg/dl. Foods enriched with plant sterols (PS can effectively reduce plasma cholesterol in patients with high levels. However, its effectiveness and safety in adults with moderate hypercholesterolemia who are on medication (statins or not has been less studied. The aim of this review is to establish the possible role of plant sterols in the control of hypercholesterolemia, as well as how safe they are for people with moderate hypercholesterolemia treated with statins. The main studies were looked at, regardless of design, language or publication date which studied the connection between “plant sterols” and “hypercholesterolemia”, using Pubmed/Medline, SCOPUS and Google Scholar databases. The studies brought together in this review show that an intake of between 2 and 3g/day of plant sterols effectively reduces plasma cholesterol levels in patients with hypercholesterolemia. Both clinical studies and available meta-analyses do not indicate any problems related to the drug-nutrient interaction associated with the use of plant sterol-enriched foods. In patients with moderate hypercholesterolemia where the use of statins is not justified a healthy diet, exercise and foods high in PS can provide the best therapeutic approach.

Plant Sterols: Chemical and Enzymatic Structural Modifications and Effects on Their Cholesterol-Lowering Activity.

Science.gov (United States)

He, Wen-Sen; Zhu, Hanyue; Chen, Zhen-Yu

2018-03-28

Plant sterols have attracted increasing attention due to their excellent cholesterol-lowering activity. However, free plant sterols have some characteristics of low oil solubility, water insolubility, high melting point, and low bioavailability, which greatly limit their application in foods. Numerous studies have been undertaken to modify their chemical structures to improve their chemical and physical properties in meeting the needs of various applications. The present review is to summarize the literature and update the progress on structural modifications of plant sterols in the following aspects: (i) synthesis of plant sterol esters by esterification and transesterification with hydrophobic fatty acids and triacylglycerols to improve their oil solubility, (ii) synthesis of plant sterol derivatives by coupling with various hydrophilic moieties to enhance their water solubility, and (iii) mechanisms by which plant sterols reduce plasma cholesterol and the effect of structural modifications on plasma cholesterol-lowering activity of plant sterols.

Sterol composition of shellfish species commonly consumed in the United States

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Katherine M. Phillips

2012-10-01

Full Text Available Background: Shellfish can be a component of a healthy diet due to a low fat and high protein content, but the cholesterol content of some species is often cited as a reason to limit their consumption. Data on levels of non-cholesterol sterols in commonly consumed species are lacking. Objective: Shellfish were sampled and analyzed to update sterol data in the United States Department of Agriculture (USDA National Nutrient Database for Standard Reference. Design: Using a nationwide sampling plan, raw shrimp and sea scallops, canned clams, and steamed oysters, blue crab, and lobster were sampled from 12 statistically selected supermarkets across the United States in 2007-08. For each species, four composites were analyzed, each comprised of samples from three locations; shrimp and scallops from six single locations were also analyzed separately. Using validated analytical methodology, 14 sterols were determined in total lipid extracts after saponification and derivatization to trimethylsilyethers, using gas chromatography for quantitation and mass spectrometry for confirmation of components. Results: Crab, lobster, and shrimp contained significant cholesterol (96.2–27 mg/100 g; scallops and clams had the lowest concentrations (23.4–30.1 mg/100 g. Variability in cholesterol among single-location samples of shrimp was low. The major sterols in the mollusks were brassicasterol (12.6–45.6 mg/100 g and 24-methylenecholesterol (16.7–41.9 mg/100 g, with the highest concentrations in oysters. Total non-cholesterol sterols were 46.5–75.6 mg/100 g in five single-location scallops samples, but 107 mg/100 g in the sixth, with cholesterol also higher in that sample. Other prominent non-cholesterol sterols in mollusks were 22-dehydrocholesterol, isofucosterol, clionasterol, campesterol, and 24-norcholesta-5,22-diene-3β-ol (4–21 mg/100 g. Conclusions: The presence of a wide range of sterols, including isomeric forms, in shellfish makes the analysis

Biocatalysis with thermostable enzymes: structure and properties of a thermophilic 'ene'-reductase related to old yellow enzyme.

Science.gov (United States)

Adalbjörnsson, Björn V; Toogood, Helen S; Fryszkowska, Anna; Pudney, Christopher R; Jowitt, Thomas A; Leys, David; Scrutton, Nigel S

2010-01-25

We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

Effects of seaweed sterols fucosterol and desmosterol on lipid membranes

DEFF Research Database (Denmark)

Mouritsen, Ole G.; Bagatolli, Luis A.; Duelund, Lars

2017-01-01

Higher sterols are universally present in large amounts (20–30%) in the plasma membranes of all eukaryotes whereas they are universally absent in prokaryotes. It is remarkable that each kingdom of the eukaryotes has chosen, during the course of evolution, its preferred sterol: cholesterol...

Cholesterol metabolism and serum non-cholesterol sterols: summary of 13 plant stanol ester interventions.

Science.gov (United States)

Hallikainen, Maarit; Simonen, Piia; Gylling, Helena

2014-04-27

The efficacy and safety of plant stanols added to food products as serum cholesterol lowering agents have been demonstrated convincingly, but their effects on cholesterol metabolism and on serum non-cholesterol sterols is less evaluated. The aim of this study was to assess the validity of serum non-cholesterol sterols and squalene as bioindices of cholesterol synthesis and absorption, and to examine how the individual serum non-cholesterol sterols respond to consumption of plant stanols. We collected all randomized, controlled plant stanol ester (STAEST) interventions in which serum cholestanol, plant sterols campesterol and sitosterol, and at least two serum cholesterol precursors had been analysed. According to these criteria, there was a total of 13 studies (total 868 subjects without lipid-lowering medication; plant stanol doses varied from 0.8 to 8.8 g/d added in esterified form; the duration of the studies varied from 4 to 52 weeks). Serum non-cholesterol sterols were assayed with gas-liquid chromatography, cholesterol synthesis with the sterol balance technique, and fractional cholesterol absorption with the dual continuous isotope feeding method. The results demonstrated that during the control and the STAEST periods, the serum plant sterol/cholesterol- and the cholestanol/cholesterol-ratios reflected fractional cholesterol absorption, and the precursor sterol/cholesterol-ratios reflected cholesterol synthesis. Plant sterol levels were dose-dependently reduced by STAEST so that 2 g of plant stanols reduced serum campesterol/cholesterol-ratio on average by 32%. Serum cholestanol/cholesterol-ratio was reduced less frequently than those of the plant sterols by STAEST, and the cholesterol precursor sterol ratios did not change consistently in the individual studies emphasizing the importance of monitoring more than one surrogate serum marker. Serum non-cholesterol sterols are valid markers of cholesterol absorption and synthesis even during cholesterol

Fatty acid and sterol composition of fenugreek seed (Trigonella foenum-graecum L.

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Mustafa Kıralan

2017-12-01

Full Text Available Oil content, fatty acid and sterol composition of fenugreek seeds obtained from three different provinces were investigated. Oil was obtained from fenugreek seeds by solvent extraction and oil content was determined between 7.01-8.82%. Fenugreek seed oils were determined to be rich of unsaturated fatty acids according to gas chromatography results. Especially, linoleic acid was the most important of the fatty acids and varied between 45.10-46.19%. Total sterol content of oils varied from 8 681.54 to 9 591.70 ppm. The major sterol was β- sitosterol, and it was found to be between 59.94-68.24% of the total sterols.

Sterol partitioning by HMGR and DXR for routing intermediates toward withanolide biosynthesis.

Science.gov (United States)

Singh, Shefali; Pal, Shaifali; Shanker, Karuna; Chanotiya, Chandan Singh; Gupta, Madan Mohan; Dwivedi, Upendra Nath; Shasany, Ajit Kumar

2014-12-01

Withanolides biosynthesis in the plant Withania somnifera (L.) Dunal is hypothesized to be diverged from sterol pathway at the level of 24-methylene cholesterol. The conversion and translocation of intermediates for sterols and withanolides are yet to be characterized in this plant. To understand the influence of mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways on sterols and withanolides biosynthesis in planta, we overexpressed the WsHMGR2 and WsDXR2 in tobacco, analyzed the effect of transient suppression through RNAi, inhibited MVA and MEP pathways and fed the leaf tissue with different sterols. Overexpression of WsHMGR2 increased cycloartenol, sitosterol, stigmasterol and campesterol compared to WsDXR2 transgene lines. Increase in cholesterol was, however, marginally higher in WsDXR2 transgenic lines. This was further validated through transient suppression analysis, and pathway inhibition where cholesterol reduction was found higher due to WsDXR2 suppression and all other sterols were affected predominantly by WsHMGR2 suppression in leaf. The transcript abundance and enzyme analysis data also correlate with sterol accumulation. Cholesterol feeding did not increase the withanolide content compared to cycloartenol, sitosterol, stigmasterol and campesterol. Hence, a preferential translocation of carbon from MVA and MEP pathways was found differentiating the sterols types. Overall results suggested that MVA pathway was predominant in contributing intermediates for withanolides synthesis mainly through the campesterol/stigmasterol route in planta. © 2014 Scandinavian Plant Physiology Society.

Solution structure of an arsenate reductase-related protein, YffB, from Brucella melitensis, the etiological agent responsible for brucellosis

International Nuclear Information System (INIS)

Buchko, Garry W.; Hewitt, Stephen N.; Napuli, Alberto J.; Van Voorhis, Wesley C.; Myler, Peter J.

2011-01-01

B. melitensis is a NIAID Category B microorganism that is responsible for brucellosis and is a potential agent for biological warfare. Here, the solution structure of the 116-residue arsenate reductase-related protein Bm-YffB (BR0369) from this organism is reported. Brucella melitensis is the etiological agent responsible for brucellosis. Present in the B. melitensis genome is a 116-residue protein related to arsenate reductases (Bm-YffB; BR0369). Arsenate reductases (ArsC) convert arsenate ion (H 2 AsO 4 − ), a compound that is toxic to bacteria, to arsenite ion (AsO 2 − ), a product that may be efficiently exported out of the cell. Consequently, Bm-YffB is a potential drug target because if arsenate reduction is the protein’s major biological function then disabling the cell’s ability to reduce arsenate would make these cells more sensitive to the deleterious effects of arsenate. Size-exclusion chromatography and NMR spectroscopy indicate that Bm-YffB is a monomer in solution. The solution structure of Bm-YffB shows that the protein consists of two domains: a four-stranded mixed β-sheet flanked by two α-helices on one side and an α-helical bundle. The α/β domain is characteristic of the fold of thioredoxin-like proteins and the overall structure is generally similar to those of known arsenate reductases despite the marginal sequence similarity. Chemical shift perturbation studies with 15 N-labeled Bm-YffB show that the protein binds reduced glutathione at a site adjacent to a region similar to the HX 3 CX 3 R catalytic sequence motif that is important for arsenic detoxification activity in the classical arsenate-reductase family of proteins. The latter observation supports the hypothesis that the ArsC-YffB family of proteins may function as glutathione-dependent thiol reductases. However, comparison of the structure of Bm-YffB with the structures of proteins from the classical ArsC family suggest that the mechanism and possibly the function of Bm

Insect molting hormone and sterol biosynthesis in spinach

International Nuclear Information System (INIS)

Grebenok, R.J.; Adler, J.H.

1990-01-01

Insect molting hormones, which are produced by plants and are effective molecules in the control of insect crop pests, are biosynthesized in developing spinach leaves (Spinacia oleracea L.). The major sterols biosynthesized by spinach are avenasterol (24α-ethyl-5α-cholesta-7,24(28)-dien-3β-ol), spinasterol (24α-ethyl-5α-cholesta-7,22-dien-3β-ol), and 22-dihydrospinasterol (24α-ethyl-5α-cholest-7-en-3β-ol). The major ecdysteroids biosynthesized are ecdysterone (2β,3β,14α,20R,22R,25-hexahydroxy-5β-cholest-7-en-6-one) and polypodine B (2β,3β,5β,14α,20R,22R,25-heptahycroxycholest-7-en-6-one) and polypodine B (2β,3β,5β,14α,20R,22R,25-heptahydroxycholest-7-en-6-one). When labeled 2- 14 C-mevalonic acid was incorporated into young leaves isolated squalene, sterols and ecdysteroids contained the label. During a short (16 h) incorporation period in intact young leaves of 100 day old plants, the avenasterol has the highest specific activity in counts per minute per μg of sterol followed by 22-dihydrospinasterol which is more highly labeled than spinasterol. The ecdysteroids synthesized, on an entire plant basis, account for 20% of the total steroid (sterol and ecdysteroid) isolated from the plant

Fecal sterols, seasonal variability, and probable sources along the ring of cenotes, Yucatan, Mexico

Science.gov (United States)

Arcega-Cabrera, F.; Velázquez-Tavera, N.; Fargher, L.; Derrien, M.; Noreña-Barroso, E.

2014-11-01

Rapid development in Yucatan has had a dramatic impact on the environment, especially the water supply. Groundwater is the only source of water in Yucatan, since surface water is virtually absent due to the karstic nature of the soil. The ring of cenotes (RC) is a geological feature which functions as a source of water and as nodes in the underground river system that canalizes water towards the coast. Numerous productive and domestic activities take place around the RC in the absence of wastewater treatment or sewage systems. Consequently, a number of researchers have hypothesized that pollutants could migrate from the land surface to the underlying aquifer and, eventually, to the coast. Therefore, the present study investigates the relationship among sources of fecal sterols and their levels in cenotes, using the expected levels of fecal sterols obtained by a spatial analysis of the sources and a Pollution Source Index. Accordingly, expected levels are compared with the detected levels of fecal sterols in 5 areas around the RC. Regarding levels, observed during a sampling campaign carried out along the RC during September 2011 (rainy season) and May 2012 (dry season), varied from low to high concentrations of sterols (0.5-2396.42 μg g- 1) and fecal sterols (0.3-1690.18 μg g- 1). These concentrations showed no relationship between neighboring cenotes, where similar fecal sterol concentrations or gradients were expected. When comparing expected fecal sterols levels with the detected ones, only two of the five analyzed areas concur, suggesting that no clear relationship exists among sources and fecal sterols levels at the regional scale. Multivariate analysis showed that fecal sterols were associated with sterols and fine grain particulates during the rainy season, which suggests co-transport. During the dry season, fecal sterols associated with fine grain particulate and organic matter, which indicates a change to a deposition phenomenon. These findings indicate

The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

Science.gov (United States)

Wang, Jinling; de Montellano, Paul R Ortiz

2003-05-30

Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis.

Science.gov (United States)

Lung, Shiu-Cheung; Liao, Pan; Yeung, Edward C; Hsiao, An-Shan; Xue, Yan; Chye, Mee-Len

2017-07-01

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis ( Arabidopsis thaliana ) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1 Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 ( GL2 ), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1 , smo1-1 , and ACBP1 +/- smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1 +/- smo1-1 Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. © 2017 American Society of Plant Biologists. All Rights Reserved.

Sterols from the soft coral Lobophytum strictum (Alcyonarian) from Lakshadweep sea

Digital Repository Service at National Institute of Oceanography (India)

Parameswaran, P.S.; Naik, C.G.; Das, B.; Kamat, S.Y.

Two polyhydroxy sterols 24(Xi)-methylcholestane-3(beta), 5a, 6(beta), 25-tetrol, 25-monoacetate, and 24S-methylcholestane-3(beta), 4(beta), 5(beta), 25-tetrol-6-one, 25-monoacetate (lo-bosterol), six monohydroxy sterols as well as batyl alcohol...

Traditional herbal medicines: potential degradation of sterols and sterolins by microbial contaminants

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S. Govender

2010-01-01

Full Text Available Medicinal plants with a high content of sterols and sterolins, such as Bulbine natalensis (rooiwortel and Hypoxis hemerocallidea (African potato, are commonly and inappropriately used in South Africa for the treatment of HIV/AIDS due to the inaccessibility of antiretroviral drugs. This study investigated the presence of active compounds, such as sterols and sterolins, in the herbal medicines. The research was carried out in the Nelson Mandela Metropole area. The effect of microbial contaminants isolated from the medicines on sterols and sterolins of rooiwortel extracts was assessed. Sterols and sterolins were detected in rooiwortel, raw African potatoes and one ready-made mixture. Co-incubation of rooiwortel with bacteria (Bacillus spp. and Pseudomonas putida and fungi (Aspergillus spp., Penicillium spp. and Mucor spp. that were isolated from these samples increased the rate of degradation of sterols and sterolins over time, with slower degradation at 4°C than at 28°C.

Sterol biosynthesis from acetate and the fate of dietary cholesterol and desmosterol in crabs

International Nuclear Information System (INIS)

Teshima, Shin-ichi; Kanazawa, Akio; Okamoto, Haruhito

1976-01-01

This paper deals with the sterol-synthesizing ability and the fate of dietary sterols, cholesterol and desmosterol, in the crabs, Sesarma dehaani and Helice tridens. Injected acetate-1- 14 C was not incorporated into either squalene or sterols in the above crabs. This suggested that the sterol-synthesizing ability from acetate is absent or weak in the crabs, S. dehaani and H. tridens. The apparent percentage absorptions of dietary cholesterol and desmosterol from the digestive tracts were 91.9 and 90.9, respectively. The ingested cholesterol and desmosterol were metabolized to steryl esters and polar compounds but only slightly to water-soluble sterols. Also, it was shown that the crab, S. dehaani, is capable of converting desmosterol to cholesterol. (auth.)

Live-cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport

DEFF Research Database (Denmark)

Modzel, M.; Solanko, K. A.; Szomek, M.

2018-01-01

brightness, significant photobleaching and excitation/emission in the ultraviolet region. Thus, special equipment is required to image such sterols. Here, we describe synthesis, characterization and intracellular imaging of new polyene sterols containing four conjugated double bonds in the sterol ring system....... We show that such analogues have red-shifted excitation and emission by ∼20 nm compared to DHE or CTL. The red shift was even more pronounced when preventing keto-enol tautomer equilibration by protecting the 3'-hydroxy group with acetate. We show that the latter analogue can be imaged...... on a conventional wide field microscope with a DAPI/filipin filter cube. The new polyene sterols show reduced photobleaching compared to DHE or CTL allowing for improved deconvolution microscopy of sterol containing cellular membranes....

Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

Science.gov (United States)

Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J; Schaller, Hubert

2014-01-01

The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

Plasma sterols and depressive symptom severity in a population-based cohort.

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Basar Cenik

Full Text Available Convergent evidence strongly suggests major depressive disorder is heterogeneous in its etiology and clinical characteristics. Depression biomarkers hold potential for identifying etiological subtypes, improving diagnostic accuracy, predicting treatment response, and personalization of treatment. Human plasma contains numerous sterols that have not been systematically studied. Changes in cholesterol concentrations have been implicated in suicide and depression, suggesting plasma sterols may be depression biomarkers. Here, we investigated associations between plasma levels of 34 sterols (measured by mass spectrometry and scores on the Quick Inventory of Depressive Symptomatology-Self Report (QIDS-SR16 scale in 3117 adult participants in the Dallas Heart Study, an ethnically diverse, population-based cohort. We built a random forest model using feature selection from a pool of 43 variables including demographics, general health indicators, and sterol concentrations. This model comprised 19 variables, 13 of which were sterol concentrations, and explained 15.5% of the variation in depressive symptoms. Desmosterol concentrations below the fifth percentile (1.9 ng/mL, OR 1.9, 95% CI 1.2-2.9 were significantly associated with depressive symptoms of at least moderate severity (QIDS-SR16 score ≥10.5. This is the first study reporting a novel association between plasma concentrations cholesterol precursors and depressive symptom severity.

Parameters for Martini sterols and hopanoids based on a virtual-site description

NARCIS (Netherlands)

Melo, M. N.; Ingolfsson, H. I.; Marrink, S. J.

2015-01-01

Sterols play an essential role in modulating bilayer structure and dynamics. Coarse-grained molecular dynamics parameters for cholesterol and related molecules are available for the Martini force field and have been successfully used in multiple lipid bilayer studies. In this work, we focus on the

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

Science.gov (United States)

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.

Competition between ergosterol and cholesterol in sterol uptake and intracellular trafficking in the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Valachovic, M.; Hronska, L.; Hapala, I.

1998-01-01

The fate of internal cholesterol was evaluated in cells grown under various conditions with respect to the amount and the nature of sterols supplemented to the cells. Steryl esters accumulate in stationary phase-yeast cells and they are rapidly hydrolyzed in cells during exponential growth or ergosterol depletion. Cholesterol and other 'unnatural' sterols are esterified more efficiently that native ergosterol and it was speculated that esterification could protect cellular membranes from accumulation of these less optimal sterols. We tested this idea by monitoring the mobility of 14 C-cholesterol between free and esterified fractions in cell supplemented with cholesterol or ergosterol. It was found that cells grown on cholesterol to the stationary phase accumulated up to 80 % of label in the steryl ester fraction. Subsequent growth in sterol-free media caused sterol-depletion of plasma membrane and induced hydrolysis of 14 C- cholesteryl esters and accumulation of the label in free membranous sterol pool.Supplementation of cells with external sterols resulted in a shift in sterol trafficking and in a new accumulation of 14 C-cholesteryl esters. This indicates that the absence of an efficient proof-reading mechanism in plasma membrane that would be able to remove preferentially cholesterol from the free sterol pool in plasma membrane to steryl esters in lipidic particles. The mobility of cholesterol molecules in non-growing cells wa negligible suggesting that active growth or membrane proliferation are required for shifts of sterol molecules between these pools. (authors)

Cloning and characterization of a nitrite reductase gene related to ...

African Journals Online (AJOL)

STORAGESEVER

2010-03-01

Mar 1, 2010 ... Alexander et al., 2005) and heme-type nitrite reductase gene (Smith and ... owing to a genotype-dependent response (Zhang et al.,. 1991; Sakhanokho et al., ..... Improvement of cell culture conditions for rice. Jpn. Agric. Res.

Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

Science.gov (United States)

Xu, Kai; Nagy, Peter D

2017-04-01

Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are

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

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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.

Identification and Characterization of Sterol Acyltransferases Responsible for Steryl Ester Biosynthesis in Tomato

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Juan A. Lara

2018-05-01

Full Text Available Steryl esters (SEs serve as a storage pool of sterols that helps to maintain proper levels of free sterols (FSs in cell membranes throughout plant growth and development, and participates in the recycling of FSs and fatty acids released from cell membranes in aging tissues. SEs are synthesized by sterol acyltransferases, a family of enzymes that catalyze the transfer of fatty acil groups to the hydroxyl group at C-3 position of the sterol backbone. Sterol acyltransferases are categorized into acyl-CoA:sterol acyltransferases (ASAT and phospholipid:sterol acyltransferases (PSAT depending on whether the fatty acyl donor substrate is a long-chain acyl-CoA or a phospolipid. Until now, only Arabidopsis ASAT and PSAT enzymes (AtASAT1 and AtPSAT1 have been cloned and characterized in plants. Here we report the identification, cloning, and functional characterization of the tomato (Solanum lycopersicum cv. Micro-Tom orthologs. SlPSAT1 and SlASAT1 were able to restore SE to wild type levels in the Arabidopsis psat1-2 and asat1-1 knock-out mutants, respectively. Expression of SlPSAT1 in the psat1-2 background also prevented the toxicity caused by an external supply of mevalonate and the early senescence phenotype observed in detached leaves of this mutant, whereas expression of SlASAT1 in the asat1-1 mutant revealed a clear substrate preference of the tomato enzyme for the sterol precursors cycloartenol and 24-methylene cycloartanol. Subcellular localization studies using fluorescently tagged SlPSAT1 and SlASAT1 proteins revealed that SlPSAT1 localize in cytoplasmic lipid droplets (LDs while, in contrast to the endoplasmic reticulum (ER localization of AtASAT1, SlASAT1 resides in the plasma membrane (PM. The possibility that PM-localized SlASAT1 may act catalytically in trans on their sterol substrates, which are presumably embedded in the ER membrane, is discussed. The widespread expression of SlPSAT1 and SlASAT1 genes in different tomato organs together

Free Sterols of the red alga Chondria armata (Kutz.) Okamura

Digital Repository Service at National Institute of Oceanography (India)

Govenkar, M.B.; Wahidullah, S.

. Results and Discussion The analysis of the sterol fraction by 1 H and 13 C NMR indicated it to be a mixture of four major D 5 3b-hydroxy sterols 2 cholest 5-en-3b-ol (choles- terol), 24j-methyl cholest-5,22-diene-3b-ol, 24j-ethyl cholest-5,22-diene-3b-ol....388 9 % 400 23j-Methyl 5a-cholestan-3b-ol C 28 H 50 O 30.759 6.7 % 402 24b-Ethyl cholest-5,22-diene-3b-ol C 29 H 48 O 31.21 4 % 412 24b-Ethyl cholest-5-en-3b-ol C 29 H 50 O 31.790 18.02 % 414 Table II. Mass spectroscopic characteristic of sterol acetates...

Bioinformatics approach of three partial polyprenol reductase genes in Kandelia obovata

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Basyuni, M.; Wati, R.; Sagami, H.; Oku, H.; Baba, S.

2018-03-01

This present study describesthe bioinformatics approach to analyze three partial polyprenol reductase genes from mangrove plant, Kandeliaobovataas well aspredictedphysical and chemical properties, potential peptide, subcellular localization, and phylogenetic. The diversity was noted in the physical and chemical properties of three partial polyprenol reductase genes. The values of chloroplast were relatively high, showed that chloroplast transit peptide occurred in mangrove polyprenol reductase. The target peptide value of mitochondria varied from 0.088 to 0.198 indicated it was possible to be present. These results suggested the importance of understanding the diversity of physicochemical properties of the different amino acids in polyprenol reductase. The subcellular localization of two partial genes located in the plasma membrane. To confirm the homology among the polyprenol reductase in the database, a dendrogram was drawn. The phylogenetic tree depicts that there are three clusters, the partial genes of K. obovata joined the largest one: C23157 was close to Ricinus communis polyprenol reductase. Whereas, C23901 and C24171 were grouped with Ipomoea nil polyprenol reductase, suggested that these polyprenol reductase genes form distinct separation into tropical habitat plants.

Genetic, anatomic, and clinical determinants of human serum sterol and vitamin D levels.

Science.gov (United States)

Stiles, Ashlee R; Kozlitina, Julia; Thompson, Bonne M; McDonald, Jeffrey G; King, Kevin S; Russell, David W

2014-09-23

An unknown fraction of the genome participates in the metabolism of sterols and vitamin D, two classes of lipids with diverse physiological and pathophysiological roles. Here, we used mass spectrometry to measure the abundance of >60 sterol and vitamin D derivatives in 3,230 serum samples from a well-phenotyped patient population. Twenty-nine of these lipids were detected in a majority of samples at levels that varied over thousands of fold in different individuals. Pairwise correlations between sterol and vitamin D levels revealed evidence for shared metabolic pathways, additional substrates for known enzymes, and transcriptional regulatory networks. Serum levels of multiple sterols and vitamin D metabolites varied significantly by sex, ethnicity, and age. A genome-wide association study identified 16 loci that were associated with levels of 19 sterols and 25-hydroxylated derivatives of vitamin D (P < 10(-7)). Resequencing, expression analysis, and biochemical experiments focused on one such locus (CYP39A1), revealed multiple loss-of-function alleles with additive effects on serum levels of the oxysterol, 24S-hydroxycholesterol, a substrate of the encoded enzyme. Body mass index, serum lipid levels, and hematocrit were strong phenotypic correlates of interindividual variation in multiple sterols and vitamin D metabolites. We conclude that correlating population-based analytical measurements with genotype and phenotype provides productive insight into human intermediary metabolism.

Plasma sterol evidence for decreased absorption and increased synthesis of cholesterol in insulin resistance and obesity1234

Science.gov (United States)

Knopp, Robert H; Kahn, Steven E; Retzlaff, Barbara M; Fish, Brian; Ma, Lina; Ostlund, Richard E

2011-01-01

Background: The rise in LDL with egg feeding in lean insulin-sensitive (LIS) participants is 2- and 3-fold greater than in lean insulin-resistant (LIR) and obese insulin-resistant (OIR) participants, respectively. Objective: We determined whether differences in cholesterol absorption, synthesis, or both could be responsible for these differences by measuring plasma sterols as indexes of cholesterol absorption and endogenous synthesis. Design: Plasma sterols were measured by gas chromatography–mass spectrometry in a random subset of 34 LIS, 37 LIR, and 37 OIR participants defined by the insulin sensitivity index (SI) and by BMI criteria selected from a parent group of 197 participants. Cholestanol and plant sterols provide a measure of cholesterol absorption, and lathosterol provides a measure of cholesterol synthesis. Results: The mean (±SD) ratio of plasma total absorption biomarker sterols to cholesterol was 4.48 ± 1.74 in LIS, 3.25 ± 1.06 in LIR, and 2.82 ± 1.08 in OIR participants. After adjustment for age and sex, the relations of the absorption sterol–cholesterol ratios were as follows: LIS > OIR (P LIR (P OIR (P = 0.11). Lathosterol-cholesterol ratios were 0.71 ± 0.32 in the LIS participants, 0.95 ± 0.47 in the LIR participants, and 1.29 ± 0.55 in the OIR participants. After adjustment for age and sex, the relations of lathosterol-cholesterol ratios were as follows: LIS sterol concentrations were positively associated with SI and negatively associated with obesity, whereas lathosterol correlations were the opposite. Conclusions: Cholesterol absorption was highest in the LIS participants, whereas cholesterol synthesis was highest in the LIR and OIR participants. Therapeutic diets for hyperlipidemia should emphasize low-cholesterol diets in LIS persons and weight loss to improve SI and to decrease cholesterol overproduction in LIR and OIR persons. PMID:21940599

Zinc finger transcription factors displaced SREBP proteins as the major Sterol regulators during Saccharomycotina evolution.

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Sarah L Maguire

2014-01-01

Full Text Available In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs, which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1 and C. albicans (Cph2 have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1 and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.

Zinc Finger Transcription Factors Displaced SREBP Proteins as the Major Sterol Regulators during Saccharomycotina Evolution

Science.gov (United States)

Maguire, Sarah L.; Wang, Can; Holland, Linda M.; Brunel, François; Neuvéglise, Cécile; Nicaud, Jean-Marc; Zavrel, Martin; White, Theodore C.; Wolfe, Kenneth H.; Butler, Geraldine

2014-01-01

In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina. PMID:24453983

In Vitro and In Vivo Anticancer Effects of Sterol Fraction from Red Algae Porphyra dentata

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Katarzyna Kazłowska

2013-01-01

Full Text Available Porphyra dentata, an edible red macroalgae, is used as a folk medicine in Asia. This study evaluated in vitro and in vivo the protective effect of a sterol fraction from P. dentata against breast cancer linked to tumor-induced myeloid derived-suppressor cells (MDSCs. A sterol fraction containing cholesterol, β-sitosterol, and campesterol was prepared by solvent fractionation of methanol extract of P. dentata  in silica gel column chromatography. This sterol fraction in vitro significantly inhibited cell growth and induced apoptosis in 4T1 cancer cells. Intraperitoneal injection of this sterol fraction at 10 and 25 mg/kg body weight into 4T1 cell-implanted tumor BALB/c mice significantly inhibited the growth of tumor nodules and increased the survival rate of mice. This sterol fraction significantly decreased the reactive oxygen species (ROS and arginase activity of MDSCs in tumor-bearing mice. Therefore, the sterol fraction from P. dentata showed potential for protecting an organism from 4T1 cell-based tumor genesis.

Effect of ketoconazole in combination with other inhibitors of sterol synthesis on fungal growth.

OpenAIRE

Sud, I J; Feingold, D S

1985-01-01

The effect of combination of ketoconazole with other sterol synthesis inhibitors on fungal growth was tested against a variety of fungi selected for resistance to ketoconazole. All of the sterol inhibitors, at concentrations lower than their MICs, caused an increase greater than fourfold in the ketoconazole susceptibility of some fungi. Some of the sterol synthesis inhibitors showed this effect with ketoconazole at levels that may be achieved clinically.

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

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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.

Effect of plant sterols and tannins on Phytophthora ramorum growth and sporulation

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The acquisition of plant sterols, mediated via elicitins, is required for growth and sporulation of Phytophthora spp. In this paper, we looked at the interaction between elicitins, sterols, and tannins. When ground leaf tissue was added to growth media, P. ramorum growth and sporulation was greates...

Effects of host cell sterol composition upon internalization of Yersinia pseudotuberculosis and clustered β1 integrin.

Science.gov (United States)

Kim, JiHyun; Fukuto, Hana S; Brown, Deborah A; Bliska, James B; London, Erwin

2018-01-26

Yersinia pseudotuberculosis is a foodborne pathogenic bacterium that causes acute gastrointestinal illness, but its mechanisms of infection are incompletely described. We examined how host cell sterol composition affected Y. pseudotuberculosis uptake. To do this, we depleted or substituted cholesterol in human MDA-MB-231 epithelial cells with various alternative sterols. Decreasing host cell cholesterol significantly reduced pathogen internalization. When host cell cholesterol was substituted with various sterols, only desmosterol and 7-dehydrocholesterol supported internalization. This specificity was not due to sterol dependence of bacterial attachment to host cells, which was similar with all sterols studied. Because a key step in Y. pseudotuberculosis internalization is interaction of the bacterial adhesins invasin and YadA with host cell β1 integrin, we compared the sterol dependence of wildtype Y. pseudotuberculosis internalization with that of Δ inv , Δ yadA , and Δ inv Δ yadA mutant strains. YadA deletion decreased bacterial adherence to host cells, whereas invasin deletion had no effect. Nevertheless, host cell sterol substitution had a similar effect on internalization of these bacterial deletion strains as on the wildtype bacteria. The Δ inv Δ yadA double mutant adhered least to cells and so was not significantly internalized. The sterol structure dependence of Y. pseudotuberculosis internalization differed from that of endocytosis, as monitored using antibody-clustered β1 integrin and previous studies on other proteins, which had a more permissive sterol dependence. This study suggests that agents could be designed to interfere with internalization of Yersinia without disturbing endocytosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Building Synthetic Sterols Computationally - Unlocking the Secrets of Evolution?

DEFF Research Database (Denmark)

Róg, Tomasz; Pöyry, Sanja; Vattulainen, Ilpo

2015-01-01

Cholesterol is vital in regulating the physical properties of animal cell membranes. While it remains unclear what renders cholesterol so unique, it is known that other sterols are less capable in modulating membrane properties, and there are membrane proteins whose function is dependent on chole......Cholesterol is vital in regulating the physical properties of animal cell membranes. While it remains unclear what renders cholesterol so unique, it is known that other sterols are less capable in modulating membrane properties, and there are membrane proteins whose function is dependent...

Significance of sterol structural specificity : desmosterol cannot replace cholesterol in lipid rafts

NARCIS (Netherlands)

Vainio, S.; Jansen, Maurice; Koivusalo, M.; Róg, T.; Karttunen, M.E.J.; Vattulainen, I.; Ikonen, E.

2006-01-01

Desmosterol is an immediate precursor of cholesterol in the Bloch pathway of sterol synthesis and an abundant membrane lipid in specific cell types. The significance of the difference between the two sterols, an additional double bond at position C24 in the tail of desmosterol, is not known. Here,

Divergent changes in serum sterols during a strict uncooked vegan diet in patients with rheumatoid arthritis.

Science.gov (United States)

Agren, J J; Tvrzicka, E; Nenonen, M T; Helve, T; Hänninen, O

2001-02-01

The effects of a strict uncooked vegan diet on serum lipid and sterol concentrations were studied in patients with rheumatoid arthritis. The subjects were randomized into a vegan diet group (n 16), who consumed a vegan diet for 2-3 months, or into a control group (n 13), who continued their usual omnivorous diets. Serum total and LDL-cholesterol and -phospholipid concentrations were significantly decreased by the vegan diet. The levels of serum cholestanol and lathosterol also decreased, but serum cholestanol:total cholesterol and lathosterol:total cholesterol did not change. The effect of a vegan diet on serum plant sterols was divergent as the concentration of campesterol decreased while that of sitosterol increased. This effect resulted in a significantly greater sitosterol:campesterol value in the vegan diet group than in the control group (1.48 (SD 0.39) v. 0.72 (SD 0.14); P vegan diet changes the relative absorption rates of these sterols and/or their biliary clearance.

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis1[OPEN

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Hsiao, An-Shan; Xue, Yan

2017-01-01

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1. Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 (GL2), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1, smo1-1, and ACBP1+/−smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1+/−smo1-1. Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. PMID:28500265

Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

NARCIS (Netherlands)

Kremer, D.R.; Veenhuis, M.; Fauque, G.; Peck Jr., H.D.; LeGall, J.; Lampreia, J.; Moura, J.J.G.; Hansen, T.A.

1988-01-01

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were

Gender-related difference in altered gene expression of a sterol regulatory element binding protein, SREBP-2, by lead nitrate in rats: correlation with development of hypercholesterolemia.

Science.gov (United States)

Kojima, Misaki; Degawa, Masakuni

2006-01-01

Changes in gene expression levels of hepatic sterol regulatory element binding protein-2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) after a single i.v. injection of lead nitrate (LN, 100 micromol kg(-1) body weight) were examined comparatively by real time reverse transcriptase-polymerase chain reaction (RT-PCR) in male and female rats. Significant increases in the gene expression level of SREBP-2, a transcription factor for the HMGR gene, occurred at 6-12 h in male and at 24-36 h in female rats after LN-treatment. The gene expression level of HMGR, a rate-limiting enzyme for cholesterol biosynthesis, significantly increased at 3-48 h in male rats and 12-48 h in female rats. Subsequently, significant increases in the amount of hepatic total cholesterol in male and female rats were also observed at 3-48 h and 24-48 h, respectively. The present findings demonstrate that increases in gene expressions of hepatic SREBP-2 and HMGR and the amount of hepatic total cholesterol by LN occur earlier in male rats than in the females, and that increases in the gene expression level of HMGR and the amount of hepatic total cholesterol occur prior to the increase in the gene expression level of SREBP-2 in either sex of rats. Copyright (c) 2006 John Wiley & Sons, Ltd.

Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

Science.gov (United States)

Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

2014-01-01

The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

Relationship between the rate of hepatic sterol synthesis and the incorporation of [3H]water

International Nuclear Information System (INIS)

Pullinger, C.R.; Gibbons, G.F.

1983-01-01

The true rate of sterol synthesis in liver cells was determined by measurement of the weight of desmosterol produced over a given time period during incubations in the presence of triparanol. The simultaneous presence of tritiated water ( 3 H 2 O) during the incubations permitted a direct observation of the weight of tritium incorporated into a given mass of newly synthesized sterol. The incorporation of tritium per atom of sterol carbon (H/C ratio) was lower than some previously reported values and suggests that a sizeable proportion of the reducing equivalents (NADPH) required for sterol synthesis arises via the pentose phosphate pathway. The H/C ratio changed significantly with length of the incubation period. The value of the ratio was also dependent upon whether the acetyl-CoA units utilized for sterol synthesis were derived predominantly from a carbohydrate or a fatty acid source

Molecular evidence of fungal signatures in the marine protist Corallochytrium limacisporum and its implications in the evolution of animals and fungi

Digital Repository Service at National Institute of Oceanography (India)

Manohar, C; Raghukumar, S.; Kasbekar, D.P.; Raghukumar, C

and ergosterol, in one of the isolates. These features, as well as the sterol C-14 reductase gene involved in the sterol pathway of animals and fungi were detected in the organism. Phylogenetic trees based on the α-AAR gene suggested that Corallochytrium...

Cholesterol biosynthesis by the cornea. Comparison of rates of sterol synthesis with accumulation during early development

International Nuclear Information System (INIS)

Cenedella, R.J.; Fleschner, C.R.

1989-01-01

The origin of the cholesterol needed by the cornea for growth and cell turnover was addressed by comparing absolute rates of sterol synthesis with rates of sterol accumulation during early development of the rabbit. Linearity of incorporation of 3 H 2 O and [ 14 C]mevalonate into digitonin-precipitable sterols with time of incubation in vitro and a lack of accumulation of 14 C in intermediates of sterol biosynthesis indicated that tritiated water can validly be used to measure rates of sterol synthesis by the cornea. The rate of sterol synthesis per unit weight of rabbit cornea was constant between 14 and 60 days of age at an average 1.03 nmol of 3 H of 3 H 2 O incorporated/mg dry cornea per 8 h. Essentially all of the synthesized cholesterol and most of the cholesterol mass was present in corneal epithelium. The cumulative sterol synthesized over the 46-day period studied exceeded the observed rate of cholesterol accumulation by sixfold. Cholesterol synthesized in excess of the growth requirement was likely used to support turnover of the epithelium which was estimated at 9 days. Removal of cholesterol from the cornea by excretion into tear fluid and clearance by high density lipoproteins are also considered

Genomic Influence in the Prevention of Cardiovascular Diseases with a Sterol-Based Treatment

Directory of Open Access Journals (Sweden)

Ismael San Mauro Martín

2018-04-01

Full Text Available Raised serum cholesterol concentration is a well-established risk factor in cardiovascular disease. In addition, genetic load may have an indirect influence on cardiovascular risk. Plant-based sterol-supplemented foods are recommended to help reduce the serum low-density lipoprotein cholesterol level. The objective was to analyse the influence of different polymorphisms in hypercholesterolemia patients following a dietary treatment with plant sterols. A randomised double-blind cross-over controlled clinical trial was carried out in 45 people (25 women. Commercial milk, containing 2.24 g of sterols, was ingested daily during a 3-week period, and then the same amount of skim milk, without sterols, was consumed daily during the 3-week placebo phase. Both phases were separated by a washout period of 2 weeks. At the beginning and end of each phase, blood draws were performed. Genes LIPC C-514T and APOA5 C56G are Ser19Trp carriers and greatly benefit from sterol intake in the diet. LIPC C-514T TT homozygous carriers had lower low-density lipoprotein cholesterol (LDL-c levels than CC homozygote and CT heterozygote carriers after the ingestion of plant sterols (p = 0.001. These two genes also showed statistically significant changes in total cholesterol levels (p = 0.025; p = 0.005, and no significant changes in high-density lipoprotein (HDL cholesterol levels (p = 0.032; p = 0.003, respectively. No statistically significant differences were observed for other genes. Further studies are needed to establish which genotype combinations would be the most protective against hypercholesterolemia.

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

Science.gov (United States)

Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K; Ejsing, Christer S; Carvalho, Pedro

2013-01-01

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway. DOI: http://dx.doi.org/10.7554/eLife.00953.001 PMID:23898401

Changes in the sterol compositions of milk thistle oil (Silybium marianum L.) during seed maturation

Energy Technology Data Exchange (ETDEWEB)

Harrabi, S.; Curtis, S.; Hayet, F.; Mayer, P.M.

2016-07-01

In this study, the total lipid content and sterol compositions were determined during the development of milk thistle seeds. The oil content increased to a maximum value of 36±1.7% and then declined to reach a value of 30.5±0.9% at full maturity. The sterol content of milk thistle seeds was affected by the ripening degree of the seeds. At the early stages of seed maturation, Δ7 -stigmastenol was the most abundant sterol followed by β-sitosterol. However, at full maturity, β-sitosterol was the most predominant sterol (46.50±0.8%). As the seed developed, campesterol and stigmasterol amounts increased, while Δ7 -avenasterol content decreased. It can be concluded that milk thistle seed oil has a characteristic sterol pattern comparable to the ones elucidated for olive oil and corn oil. The extracted oil from milk thistle seeds is rich in phytosterols and could be used in foodpreparation and human nutrition. (Author)

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

DEFF Research Database (Denmark)

Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K

2013-01-01

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate......-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin...... ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together...

In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

International Nuclear Information System (INIS)

Peters, J.

1977-01-01

Some experimental work is described showing that near-U.V. irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-U.V., and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex. (U.K.)

In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

Energy Technology Data Exchange (ETDEWEB)

Peters, J [California Univ., Irvine (USA)

1977-06-09

Some experimental work is described showing that near-uv irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-uv, and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex.

Sterol patterns of cultured zooxanthellae isolated from marine invertebrates: Synthesis of gorgosterol and 23-desmethylgorgosterol by aposymbiotic algae.

Science.gov (United States)

Withers, N W; Kokke, W C; Fenical, W; Djerassi, C

1982-06-01

QUANTITATIVE STEROL COMPOSITIONS OF CULTURED ZOOXANTHELLAE ISOLATED FROM VARIOUS PACIFIC AND ATLANTIC INVERTEBRATE HOSTS: Zoanthus sociatus (a zoanthid), Oculina diffusa (a scleractian coral), Tridacna gigas (a giant clam), Melibe pilosa (a nudibranch), and Aiptasia pulchella (a sea anemone) are reported. The results clearly demonstrate large differences in sterol patterns of zooxanthellae and that there is no obvious relationship between the taxonomic affiliation of the host and the sterol pattern of its isolated symbiont. The sterols of the zooxanthellae of O. diffusa (Cnidaria) and T. gigas (Mollusca) are qualitatively equivalent. Based on the structures of the two major free sterols synthesized by each alga, the zooxanthellae from different hosts were separated into three distinct groups. It was also found that an aposymbiotic alga can synthesize the unique marine sterols gorgosterol and 23-desmethylgorgosterol. Most of the sterols were identified by using mass spectroscopy and 360-MHz proton magnetic resonance. Spectroscopic data are reported for four novel sterols-(23,24R)-dimethyl-5alpha-cholest-(22E)-en-3beta-o l, 23-methyl-5alpha-cholest-22E-en-3beta-ol, cholesta-5,14-dien-3beta-ol, and 4alpha-methyl-5alpha-cholesta-8(14)-24-dien-3beta-ol.

Plant Sterols as Anticancer Nutrients: Evidence for Their Role in Breast Cancer

Directory of Open Access Journals (Sweden)

Bruce J. Grattan

2013-01-01

Full Text Available While many factors are involved in the etiology of cancer, it has been clearly established that diet significantly impacts one’s risk for this disease. More recently, specific food components have been identified which are uniquely beneficial in mitigating the risk of specific cancer subtypes. Plant sterols are well known for their effects on blood cholesterol levels, however research into their potential role in mitigating cancer risk remains in its infancy. As outlined in this review, the cholesterol modulating actions of plant sterols may overlap with their anti-cancer actions. Breast cancer is the most common malignancy affecting women and there remains a need for effective adjuvant therapies for this disease, for which plant sterols may play a distinctive role.

Sterols of Pneumocystis carinii hominis organisms isolated from human lungs

DEFF Research Database (Denmark)

Kaneshiro, E S; Amit, Z; Chandra, Jan Suresh

1999-01-01

in conjunction with analyses of chemically synthesized authentic standards. The sterol composition of isolated P. carinii hominis organisms has yet to be reported. If P. carinii from animal models is to be used for identifying potential drug targets and for developing chemotherapeutic approaches to clear human...... infections, it is important to determine whether the 24-alkylsterols of organisms found in rats are also present in organisms in humans. In the present study, sterol analyses of P. carinii hominis organisms isolated from cryopreserved human P. carinii-infected lungs and from bronchoalveolar lavage fluid were...

Effects of Aloe Sterol Supplementation on Skin Elasticity, Hydration, and Collagen Score: A 12-Week Double-Blind, Randomized, Controlled Trial.

Science.gov (United States)

Tanaka, Miyuki; Yamamoto, Yuki; Misawa, Eriko; Nabeshima, Kazumi; Saito, Marie; Yamauchi, Koji; Abe, Fumiaki; Furukawa, Fukumi

2016-01-01

Our previous study confirmed that Aloe sterol stimulates collagen and hyaluronic acid production in human dermal fibroblasts. This study aims to investigate whether Aloe sterol intake affects skin conditions. We performed a 12-week, randomized, double-blind, placebo-controlled study to evaluate the effects of oral Aloe sterol supplementation on skin elasticity, hydration, and the collagen score in 64 healthy women (age range 30-59 years; average 44.3 years) who were randomly assigned to receive either a placebo or an Aloe sterol-supplemented yogurt. Skin parameters were measured and ultrasound analysis of the forearm was performed. ANCOVA revealed statistical differences in skin moisture, transepidermal water loss, skin elasticity, and collagen score between the Aloe sterol and placebo groups. The gross elasticity (R2), net elasticity (R5), and biological elasticity (R7) scores of the Aloe sterol group significantly increased with time. In addition, skin fatigue area F3, which is known to decrease with age and fatigue, also increased with Aloe sterol intake. Ultrasound echogenicity revealed that the collagen content in the dermis increased with Aloe sterol intake. The results suggest that continued Aloe sterol ingestion contributes to maintaining healthy skin. © 2017 S. Karger AG, Basel.

Increased sesquiterpenoid biosynthesis and an apparent decrease in sterol biosynthesis in elicitor-treated tobacco cell suspension cultures

International Nuclear Information System (INIS)

Voegeli, U.; Bhatt, P.N.; Chappell, J.

1987-01-01

Addition of fungel elicitor prepared from Phytophthora parasitica to tobacco cell suspension cultures leads to an increased production of the phytoalexin capsidiol. Capsidiol is a sesquiterpenoid which is most likely synthesized from farnesylpyrophosphat (FPP) by a bicyclic cyclase reaction. Because FPP is also a substrate for squalene synthetase and therefore a precursor of sterol biosynthesis, the question arises whether or not the accumulation of capsidiol in elicitor-treated cells occurs at the expense of sterol biosynthesis. ( 14 C]-acetate was given to elicitor-treated and control (no treatment) cell cultures and incorporation into sterols and capsidiol determined. No labeled capsidiol was detected in control cells. In elicitor-treated cells about 12-15% of the radioactivity taken up by the cells was incorporated into capsidiol. In contrast, control cells incorporated 4 times more radioactivity into sterols than elicitor-treated cells. Similar results were obtained using ( 3 H)-mevalonate as a precursor of capsidiol and sterol biosynthesis. Likely explanations for the apparently decline in sterol biosynthesis in elicitor-treated cells include: (1) inhibition of squalene synthetase; (2) induction of capsidiol synthesizing enzymes; and (3) metabolic channeling of FPP into capsidiol versus sterols. These possibilities will be discussed further together with other results

Building synthetic sterols computationally – unlocking the secrets of evolution?

Directory of Open Access Journals (Sweden)

Tomasz eRog

2015-08-01

Full Text Available Cholesterol is vital in regulating the physical properties of animal cell membranes. While it remains unclear what renders cholesterol so unique, it is known that other sterols are less capable in modulating membrane properties, and there are membrane proteins whose function is dependent on cholesterol. Practical applications of cholesterol include e.g. its use in liposomes in drug delivery and cosmetics, cholesterol-based detergents in membrane protein crystallography, and its fluorescent analogs in studies of cholesterol transport in cells and tissues. Clearly, in spite of their difficult synthesis, producing the synthetic analogs of cholesterol is of great commercial and scientific interest. In this article, we discuss how synthetic sterols nonexistent in nature can be used to elucidate the roles of cholesterol's structural elements. To this end, we discuss recent atomistic molecular dynamics simulation studies that have predicted new synthetic sterols with properties comparable to those of cholesterol. We also discuss more recent experimental studies that have vindicated these predictions. The paper highlights the strength of computational simulations in making predictions for synthetic biology, thereby guiding experiments.

Dietary sources of aldose reductase inhibitors: prospects for alleviating diabetic complications.

Science.gov (United States)

Saraswat, Megha; Muthenna, P; Suryanarayana, P; Petrash, J Mark; Reddy, G Bhanuprakash

2008-01-01

Activation of polyol pathway due to increased aldose reductase activity is one of the several mechanisms that have been implicated in the development of various secondary complications of diabetes. Though numerous synthetic aldose reductase inhibitors have been tested, these have not been very successful clinically. Therefore, a number of common plant/ natural products used in Indian culinary have been evaluated for their aldose reductase inhibitory potential in the present study. The aqueous extracts of 22 plant-derived materials were prepared and evaluated for the inhibitory property against rat lens and human recombinant aldose reductase. Specificity of these extracts towards aldose reductase was established by testing their ability to inhibit a closely related enzyme viz, aldehyde reductase. The ex vivo incubation of erythrocytes in high glucose containing medium was used to underscore the significance in terms of prevention of intracellular sorbitol accumulation. Among the 22 dietary sources tested, 10 showed considerable inhibitory potential against both rat lens and human recombinant aldose reductase. Prominent inhibitory property was found in spinach, cumin, fennel, lemon, basil and black pepper with an approximate IC50 of 0.2 mg/mL with an excellent selectivity towards aldose reductase. As against this, 10 to 20 times higher concentrations were required for 50% inhibition of aldehyde reductase. Reduction in the accumulation of intracellular sorbitol by the dietary extracts further substantiated their in vivo efficacy. The findings reported here indicate the scope of adapting life-style modifications in the form of inclusion of certain common sources in the diet for the management of diabetic complications.

Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts

DEFF Research Database (Denmark)

Berzina, Zane; Solanko, Lukasz M; Mehadi, Ahmed S

2018-01-01

/LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous...... but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM...

Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

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Justin R. Prigge

2017-06-01

Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

Effect of parenteral serum plant sterols on liver enzymes and cholesterol metabolism in a patient with short bowel syndrome.

Science.gov (United States)

Hallikainen, Maarit; Huikko, Laura; Kontra, Kirsi; Nissinen, Markku; Piironen, Vieno; Miettinen, Tatu; Gylling, Helena

2008-01-01

Hepatobiliary complications are common during parenteral nutrition. Lipid moiety in commercially available solutions contains plant sterols. It is not known whether plant sterols in parenteral nutrition interfere with hepatic function in adults. We detected how different amounts of plant sterols in parenteral nutrition solution affected serum plant sterol concentrations and liver enzymes during a 1.5-year follow-up in a patient with short bowel syndrome. Serum lipid, plant sterol, and liver enzyme levels were measured regularly during the transition from Intralipid (100% soy-based intravenous fat emulsion) to ClinOleic (an olive oil-based intravenous fat emulsion with 80% olive oil, 20% soy oil and lower plant sterols); the lipid supply was also gradually increased from 20 to 35 g/d. Plant sterols in parenteral nutrition solution and serum were measured with gas-liquid chromatography. During infusion of soy-based intravenous fat emulsion (30 g/d, total plant sterols 87 mg/d), the concentrations of sitosterol, campesterol, and stigmasterol were 4361, 1387, and 378 microg/dL, respectively, and serum liver enzyme values were >or= 2.5 times above upper limit of normal. After changing to olive oil-based intravenous fat emulsion (20-35 g/d, plant sterols 37-65 mg/d), concentrations decreased to 2148 to 2251 microg/dL for sitosterol, 569-297 microg/dL for campesterol, and 95-55 microg/dL for stigmasterol. Concomitantly, liver enzyme values decreased to 1.4 to 1.8 times above upper limit of normal at the end of follow-up. The nutrition status of the patient improved. The amount of plant sterols in lipid emulsion affects serum liver enzyme levels more than the amount of lipid.

Sterol synthesis and cell size distribution under oscillatory growth conditions in Saccharomyces cerevisiae scale-down cultivations.

Science.gov (United States)

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.

Non-Cholesterol Sterol Levels Predict Hyperglycemia and Conversion to Type 2 Diabetes in Finnish Men

Science.gov (United States)

Cederberg, Henna; Gylling, Helena; Miettinen, Tatu A.; Paananen, Jussi; Vangipurapu, Jagadish; Pihlajamäki, Jussi; Kuulasmaa, Teemu; Stančáková, Alena; Smith, Ulf; Kuusisto, Johanna; Laakso, Markku

2013-01-01

We investigated the levels of non-cholesterol sterols as predictors for the development of hyperglycemia (an increase in the glucose area under the curve in an oral glucose tolerance test) and incident type 2 diabetes in a 5-year follow-up study of a population-based cohort of Finnish men (METSIM Study, N = 1,050) having non-cholesterol sterols measured at baseline. Additionally we determined the association of 538,265 single nucleotide polymorphisms (SNP) with non-cholesterol sterol levels in a cross-sectional cohort of non-diabetic offspring of type 2 diabetes (the Kuopio cohort of the EUGENE2 Study, N = 273). We found that in a cross-sectional METSIM Study the levels of sterols indicating cholesterol absorption were reduced as a function of increasing fasting glucose levels, whereas the levels of sterols indicating cholesterol synthesis were increased as a function of increasing 2-hour glucose levels. A cholesterol synthesis marker desmosterol significantly predicted an increase, and two absorption markers (campesterol and avenasterol) a decrease in the risk of hyperglycemia and incident type 2 diabetes in a 5-year follow-up of the METSIM cohort, mainly attributable to insulin sensitivity. A SNP of ABCG8 was associated with fasting plasma glucose levels in a cross-sectional study but did not predict hyperglycemia or incident type 2 diabetes. In conclusion, the levels of some, but not all non-cholesterol sterols are markers of the worsening of hyperglycemia and type 2 diabetes. PMID:23840693

A data mining approach to dinoflagellate clustering according to sterol composition: Correlations with evolutionary history.

Science.gov (United States)

This study examined the sterol compositions of 102 dinoflagellates (including several previously unexamined species) using clustering techniques as a means of determining the relatedness of the organisms. In addition, dinoflagellate sterol-based relationships were compared statistically to dinoflag...

Enzyme phylogenies as markers for the oxidation state of the environment: the case of respiratory arsenate reductase and related enzymes.

Science.gov (United States)

Duval, Simon; Ducluzeau, Anne-Lise; Nitschke, Wolfgang; Schoepp-Cothenet, Barbara

2008-07-16

Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far. We performed a genomic search of genes related to arrA coding for the molybdopterin subunit. The multiple alignment of the retrieved sequences served to reconstruct a neighbor-joining phylogeny of Arr and closely related enzymes. Our analysis confirmed the previously proposed proximity of Arr to the cluster of polysulfide/thiosulfate reductases but also unravels a hitherto unrecognized clade even more closely related to Arr. The obtained phylogeny strongly suggests that Arr originated after the Bacteria/Archaea divergence in the domain Bacteria, and was subsequently laterally distributed within this domain. It further more indicates that, as a result of accumulation of arsenate in the environment, an enzyme related to polysulfide reductase and not to arsenite oxidase has evolved into Arr. These findings are paleogeochemically rationalized by the fact that the accumulation of arsenate over arsenite required the increase in oxidation state of the environment brought about by oxygenic photosynthesis.

Structural Features and Potent Antidepressant Effects of Total Sterols and β-sitosterol Extracted from Sargassum horneri

Directory of Open Access Journals (Sweden)

Donghai Zhao

2016-06-01

Full Text Available The purified total sterols and β-sitosterol extracted from Sargassum horneri were evaluated for their antidepressant-like activity using the forced swim test (FST and tail suspension test (TST in mice. Total sterols and β-sitosterol significantly reduced the immobility time in the FST and TST. Total sterols were administered orally for 7 days at doses of 50, 100, and 200 mg/kg, and β-sitosterol was administered intraperitoneally at doses of 10, 20, and 30 mg/kg. β-sitosterol had no effect on locomotor activity in the open field test. In addition, total sterols and β-sitosterol significantly increased NE, 5-HT, and the metabolite 5-HIAA in the mouse brain, suggesting that the antidepressant-like activity may be mediated through these neurotransmitters.

Quantitative and qualitative analysis of sterols/sterolins and ...

African Journals Online (AJOL)

STORAGESEVER

2008-06-03

Jun 3, 2008 ... Most research has been carried out on ... method was developed to identify and quantify sterols (especially β-sitosterol) in chloroform extracts of .... Corms of the three Hypoxis spp. were planted in the same soil type.

Traditional herbal medicines: potential degradation of sterols and sterolins by microbial contaminants

OpenAIRE

S. Govender; M. van de Venter; D. du Plessis-Stoman; T. G. Downing

2010-01-01

Medicinal plants with a high content of sterols and sterolins, such as Bulbine natalensis (rooiwortel) and Hypoxis hemerocallidea (African potato), are commonly and inappropriately used in South Africa for the treatment of HIV/AIDS due to the inaccessibility of antiretroviral drugs. This study investigated the presence of active compounds, such as sterols and sterolins, in the herbal medicines. The research was carried out in the Nelson Mandela Metrop...

Multicomponent synthesis of 4,4-dimethyl sterol analogues and their effect on eukaryotic cells.

Science.gov (United States)

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.

Effect of dietary cholesterol and plant sterol consumption on plasma lipid responsiveness and cholesterol trafficking in healthy individuals.

Science.gov (United States)

Alphonse, Peter A S; Ramprasath, Vanu; Jones, Peter J H

2017-01-01

Dietary cholesterol and plant sterols differentially modulate cholesterol kinetics and circulating cholesterol. Understanding how healthy individuals with their inherent variabilities in cholesterol trafficking respond to such dietary sterols will aid in improving strategies for effective cholesterol lowering and alleviation of CVD risk. The objectives of this study were to assess plasma lipid responsiveness to dietary cholesterol v. plant sterol consumption, and to determine the response in rates of cholesterol absorption and synthesis to each sterol using stable isotope approaches in healthy individuals. A randomised, double-blinded, crossover, placebo-controlled clinical trial (n 49) with three treatment phases of 4-week duration were conducted in a Manitoba Hutterite population. During each phase, participants consumed one of the three treatments as a milkshake containing 600 mg/d dietary cholesterol, 2 g/d plant sterols or a control after breakfast meal. Plasma lipid profile was determined and cholesterol absorption and synthesis were measured by oral administration of [3, 4-13C] cholesterol and 2H-labelled water, respectively. Dietary cholesterol consumption increased total (0·16 (sem 0·06) mmol/l, P=0·0179) and HDL-cholesterol (0·08 (sem 0·03) mmol/l, P=0·0216) concentrations with no changes in cholesterol absorption or synthesis. Plant sterol consumption failed to reduce LDL-cholesterol concentrations despite showing a reduction (6 %, P=0·0004) in cholesterol absorption. An over-compensatory reciprocal increase in cholesterol synthesis (36 %, P=0·0026) corresponding to a small reduction in absorption was observed with plant sterol consumption, possibly resulting in reduced LDL-cholesterol lowering efficacy of plant sterols. These data suggest that inter-individual variability in cholesterol trafficking mechanisms may profoundly impact plasma lipid responses to dietary sterols in healthy individuals.

Brain Cholesterol Synthesis and Metabolism is Progressively Disturbed in the R6/1 Mouse Model of Huntington's Disease: A Targeted GC-MS/MS Sterol Analysis.

Science.gov (United States)

Kreilaus, Fabian; Spiro, Adena S; Hannan, Anthony J; Garner, Brett; Jenner, Andrew M

2015-01-01

Cholesterol has essential functions in neurological processes that require tight regulation of synthesis and metabolism. Perturbed cholesterol homeostasis has been demonstrated in Huntington's disease, however the exact role of these changes in disease pathogenesis is not fully understood. This study aimed to comprehensively examine changes in cholesterol biosynthetic precursors, metabolites and oxidation products in the striatum and cortex of the R6/1 transgenic mouse model of Huntington's disease. We also aimed to characterise the progression of the physical phenotype in these mice. GC-MS/MS was used to quantify a broad range of sterols in the striatum and cortex of R6/1 and wild type mice at 6, 12, 20, 24 and 28 weeks of age. Motor dysfunction was assessed over 28 weeks using the RotaRod and the hind-paw clasping tests. 24(S)-Hydroxycholesterol and 27-hydroxycholesterol were the major cholesterol metabolites that significantly changed in R6/1 mice. These changes were specifically localised to the striatum and were detected at the end stages of the disease. Cholesterol synthetic precursors (lathosterol and lanosterol) were significantly reduced in the cortex and striatum by 6 weeks of age, prior to the onset of motor dysfunction, as well as the cognitive and affective abnormalities previously reported. Elevated levels of desmosterol, a substrate of delta(24)-sterol reductase (DHCR24), were also detected in R6/1 mice at the end time-point. Female R6/1 mice exhibited a milder weight loss and hind paw clasping phenotype compared to male R6/1 mice, however, no difference in the brain sterol profile was detected between sexes. Several steps in cholesterol biosynthetic and metabolic pathways are differentially altered in the R6/1 mouse brain as the disease progresses and this is most severe in the striatum. This provides further insights into early molecular mediators of HD onset and disease progression and identifies candidate molecular targets for novel therapeutic

The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

Science.gov (United States)

Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

2002-07-16

Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

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Lund Frederik W

2012-10-01

Full Text Available Abstract Background Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE, an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport. Results We found that BChol is very photostable under two-photon (2P-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 μm2/s. Number and brightness (N&B analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent α ~ 0.63 and an anomalous diffusion constant of Dα = 1.95 x 10-3 μm2/sα. On a longer time scale (t > ~5 s, a transition to superdiffusion consistent with slow directed transport with an average velocity of v ~ 6 x 10-3 μm/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle

Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

Science.gov (United States)

Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

2009-01-01

Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

Distribution and evolution of sterols and aliphatic hydrocarbons in dated marine sediment cores from the Cabo Frio upwelling region, SW Atlantic, Brazil.

Science.gov (United States)

Lourenço, Rafael André; Martins, César C; Taniguchi, Satie; Mahiques, Michel Michaelovitch; Montone, Rosalinda Carmela; Magalhães, Caio Augusto; Bícego, Márcia Caruso

2017-08-01

We report the distribution of selected lipid biomarkers specifically sterols and aliphatic hydrocarbons in sediment cores from Cabo Frio, SW Atlantic continental shelf, Brazil, corresponding approximately to the last 700 years. In the Cabo Frio region, a costal upwelling occurs as a quasi-seasonal phenomenon characterized by nutrient-rich bottom waters that intrude on the continental shelf and promote relatively high biological productivity compared to other Brazilian continental shelf areas. The results for sterols indicate the predominance of organic matter (OM) inputs related to marine organisms, mainly plankton, in all of the cores along the time scale studied. Principal component analyses show three different groups of variables, which may be associated with (i) the more effective intrusion of the nutrient-rich South Atlantic Central Water, resulting in the increase of marine lipid biomarkers such as sterols and short-chain n-alkanes; (ii) the influence of the Coastal Water with higher surface water temperature and subsequently lower primary productivity; and (iii) OM characterized by high total organic carbon and long-chain n-alkanes related to an allochthonous source. Relatively high concentrations of sterols and n-alkanes between 1450 and 1700 AD, chronologically associated with the Little Ice Age, suggest a period associated with changes in the local input of specific sources of these compounds. The concentrations of lipid biomarkers vary over core depth, but this does not suggest a notably high or low intensity of upwelling processes. It is possible that the climatic and sea surface temperature changes reported in previous studies did not affect the input of the sedimentary lipid biomarkers analyzed here.

Homocysteine and the methylenetetrahydrofolate reductase 677C-->T polymorphism in relation to muscle mass and strength, physical performance and postural sway

NARCIS (Netherlands)

Swart, K. M. A.; Enneman, A. W.; van Wijngaarden, J. P.; van Dijk, S. C.; Brouwer-Brolsma, E. M.; Ham, A. C.; Dhonukshe-Rutten, R. A. M.; van der Velde, N.; Brug, J.; van Meurs, J. B. J.; de Groot, L. C. P. G. M.; Uitterlinden, A. G.; Lips, P.; van Schoor, N. M.

2013-01-01

Elevated plasma homocysteine has been linked to reduced mobility and muscle functioning in the elderly. The relation of methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism with these associations has not yet been studied. This study aimed to investigate (1) the association of plasma

The major cellular sterol regulatory pathway is required for Andes virus infection.

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Josiah Petersen

2014-02-01

Full Text Available The Bunyaviridae comprise a large family of RNA viruses with worldwide distribution and includes the pathogenic New World hantavirus, Andes virus (ANDV. Host factors needed for hantavirus entry remain largely enigmatic and therapeutics are unavailable. To identify cellular requirements for ANDV infection, we performed two parallel genetic screens. Analysis of a large library of insertionally mutagenized human haploid cells and a siRNA genomic screen converged on components (SREBP-2, SCAP, S1P and S2P of the sterol regulatory pathway as critically important for infection by ANDV. The significance of this pathway was confirmed using functionally deficient cells, TALEN-mediated gene disruption, RNA interference and pharmacologic inhibition. Disruption of sterol regulatory complex function impaired ANDV internalization without affecting virus binding. Pharmacologic manipulation of cholesterol levels demonstrated that ANDV entry is sensitive to changes in cellular cholesterol and raises the possibility that clinically approved regulators of sterol synthesis may prove useful for combating ANDV infection.

Preservation of genes involved in sterol metabolism in cholesterol auxotrophs: facts and hypotheses.

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Giovanna Vinci

Full Text Available BACKGROUND: It is known that primary sequences of enzymes involved in sterol biosynthesis are well conserved in organisms that produce sterols de novo. However, we provide evidence for a preservation of the corresponding genes in two animals unable to synthesize cholesterol (auxotrophs: Drosophila melanogaster and Caenorhabditis elegans. PRINCIPAL FINDINGS: We have been able to detect bona fide orthologs of several ERG genes in both organisms using a series of complementary approaches. We have detected strong sequence divergence between the orthologs of the nematode and of the fruitfly; they are also very divergent with respect to the orthologs in organisms able to synthesize sterols de novo (prototrophs. Interestingly, the orthologs in both the nematode and the fruitfly are still under selective pressure. It is possible that these genes, which are not involved in cholesterol synthesis anymore, have been recruited to perform different new functions. We propose a more parsimonious way to explain their accelerated evolution and subsequent stabilization. The products of ERG genes in prototrophs might be involved in several biological roles, in addition to sterol synthesis. In the case of the nematode and the fruitfly, the relevant genes would have lost their ancestral function in cholesterogenesis but would have retained the other function(s, which keep them under pressure. CONCLUSIONS: By exploiting microarray data we have noticed a strong expressional correlation between the orthologs of ERG24 and ERG25 in D. melanogaster and genes encoding factors involved in intracellular protein trafficking and folding and with Start1 involved in ecdysteroid synthesis. These potential functional connections are worth being explored not only in Drosophila, but also in Caenorhabditis as well as in sterol prototrophs.

Expression and site-directed mutagenesis of human dihydrofolate reductase

Energy Technology Data Exchange (ETDEWEB)

Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

1988-05-17

A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

Expression and site-directed mutagenesis of human dihydrofolate reductase

International Nuclear Information System (INIS)

Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

1988-01-01

A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 → Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by α-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme

A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

Science.gov (United States)

Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

2014-05-01

The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

Sterol-specific membrane interactions with the toxins from ...

African Journals Online (AJOL)

The lipophilic toxins from Karlodinium micrum, KmTX, have negative effects on several co-occurring phytoplankton species, yet appear to have no effect on K. micrum itself. One of these compounds, KmTX2, has differing toxicity towards eukaryotic membranes with differing sterol compositions (vertebrate > fungal ...

Cholesterol lowering effect of a soy drink enriched with plant sterols in a French population with moderate hypercholesterolemia

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Bard Jean-Marie

2008-10-01

Full Text Available Abstract Background Plant sterols are an established non-pharmacological means to reduce total and LDL blood cholesterol concentrations and are therefore recommended for cholesterol management by worldwide-renown health care institutions. Their efficacy has been proven in many types of foods with the majority of trials conducted in spreads or dairy products. As an alternative to dairy products, soy based foods are common throughout the world. Yet, there is little evidence supporting the efficacy of plant sterols in soy-based foods. The objective of this study was to investigate the effect of a soy drink enriched with plant sterols on blood lipid profiles in moderately hypercholesterolemic subjects. Methods In a randomized, placebo-controlled double-blind mono-centric study, 50 subjects were assigned to 200 ml of soy drink either enriched with 2.6 g plant sterol esters (1.6 g/d free plant sterol equivalents or without plant sterols (control for 8 weeks. Subjects were instructed to maintain stable diet pattern and physical activity. Plasma concentrations of lipids were measured at initial visit, after 4 weeks and after 8 weeks. The primary measurement was the change in LDL cholesterol (LDL-C. Secondary measurements were changes in total cholesterol (TC, non-HDL cholesterol (non-HDL-C, HDL cholesterol (HDL-C and triglycerides. Results Regular consumption of the soy drink enriched with plant sterols for 8 weeks significantly reduced LDL- C by 0.29 mmol/l or 7% compared to baseline (p 96%, and products were well tolerated. Conclusion Daily consumption of a plant sterol-enriched soy drink significantly decreased total, non-HDL and LDL cholesterol and is therefore an interesting and convenient aid in managing mild to moderate hypercholesterolemia.

Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors.

Science.gov (United States)

Burr, Risa; Stewart, Emerson V; Espenshade, Peter J

2017-03-31

The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Co-suppression of sterol-regulatory element binding protein ...

African Journals Online (AJOL)

Administrator

2011-06-22

Jun 22, 2011 ... In Arabidopsis,. At5g35220 gene being sterol regulatory element-binding protein site 2, protease and metalloendopeptidase activity were required for chloroplast development and play a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-.

Sterols from the Lakshadweep sponge, Ircinia ramosa (Killer)

Digital Repository Service at National Institute of Oceanography (India)

Parameswaran, P.S.; Naik, C.G.; Das, B.; Kamat, S.Y.

Four monohydroxy sterols, viz, (22E,24S)-24-methylcholest-5,22-dien-3(beta)-ol (3), cholesterol (4), 24(Xi)-ethylcholesterol (8) and the corresponding Delta super(4)-3 ketones, viz. (22E,24S)-24-methylcholest-4,22-dien-3-one (1), cholest-4-en-3-one...

Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis[W

Science.gov (United States)

Meguro, Miki; Ito, Hisashi; Takabayashi, Atsushi; Tanaka, Ryouichi; Tanaka, Ayumi

2011-01-01

The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation. PMID:21934147

Trypanosoma cruzi response to sterol biosynthesis inhibitors: morphophysiological alterations leading to cell death.

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Rafael Luis Kessler

Full Text Available The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC(50/72 h or killing all cells within 24 hours (EC(100/24 h. Incubation with inhibitors at the EC(50/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC(50/72 h. By contrast, treatment with SBIs at the EC(100/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP, culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the "point of no return" in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite.

Tomato UDP-Glucose Sterol Glycosyltransferases: A Family of Developmental and Stress Regulated Genes that Encode Cytosolic and Membrane-Associated Forms of the Enzyme

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Karla Ramirez-Estrada

2017-06-01

Full Text Available Sterol glycosyltransferases (SGTs catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato (Solanum lycopersicum cv. Micro-Tom SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic

Effect of rapeseed oil derived plant sterol and stanol esters on atherosclerosis parameters in cholesterol challenged heterozygous Watanabe Heritable Hyperlipidemic rabbits

DEFF Research Database (Denmark)

Schrøder, Malene; Fricke, Christiane; Pilegaard, Kirsten

2009-01-01

Watanabe heritable hyperlipidaemic (Hh-WHHL) rabbits. Four groups (n 18 per group) received a cholesterol-added (2 g/kg) standard chow or this diet with added RSO stanol esters (17 g/kg), RSO stanol esters (34 g/kg) or RSO sterol esters (34 g/kg) for 18 weeks. Feeding RSO stanol esters increased plasma...... campestanol (P Feeding RSO sterol esters increased concentrations of plasma campesterol (P ... of the RSO stanol ester groups and in one in the RSO sterol ester group. Aortic cholesterol was decreased in the treated groups (P response to lowering of plasma cholesterol induced by RSO sterol and stanol esters. In conclusion, RSO stanol and sterol esters with a high concentration...

The aldo-keto reductase superfamily homepage.

Science.gov (United States)

Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

2003-02-01

The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme

International Nuclear Information System (INIS)

Hinojosa-Leon, M.; Dubourdieu, M.; Sanchez-Crispin, J.A.; Chippaux, M.

1986-01-01

Use of radioactive molybdenum demonstrates that the tetrathionate reductase of Salmonella typhimurium is a molydenum containing enzyme. It is proposed that this enzyme shares with other molybdo-proteins, such as nitrate reductase, a common molybdenum containing cofactor the defect of which leads to the loss of the tetrathionate reductase and nitrate reductase activities

Minor sterols from the sponge Ircinia ramosa (Killer)

Digital Repository Service at National Institute of Oceanography (India)

Parameswaran, P.S.; Naik, C.G.; Das, B.; Kamat, S.Y.

Three sterols, isolated from the lipid fraction of the sponge Ircinia ramosa were characterised as cholest-5-en-3 beta-ol-7-one (7-oxo cholesterol, 1), cholest 5-23-dien-b beta ol-7-one (7-oxo demosterol, 2) and 24E-ethyl cholest-5-en-3 beta -ol-7...

Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism.

Science.gov (United States)

Xu, Li-Qin; Liu, Yong-Jun; Yao, Kang; Liu, Hao-Hao; Tao, Xin-Yi; Wang, Feng-Qing; Wei, Dong-Zhi

2016-02-22

The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17β-hydroxysteroid dehydrogenase and β-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively.

EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of a health claim related to a combination of plant sterols and Cholesternorm®mix and reduction of blood LDL-cholesterol concentrations pursuant to Article 14 of Regulation (EC) No 1924/2006

DEFF Research Database (Denmark)

Tetens, Inge

Following an application from Health Concern B.V., submitted for authorisation of a claim pursuant to Article 14 of Regulation (EC) No 1924/2006 via the Competent Authority of the Netherlands, the Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the sci......Following an application from Health Concern B.V., submitted for authorisation of a claim pursuant to Article 14 of Regulation (EC) No 1924/2006 via the Competent Authority of the Netherlands, the Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion...... on the scientific substantiation of a health claim related to a combination of plant sterols and Cholesternorm®mix and reduction of blood LDL-cholesterol concentrations. The food which is the subject of the health claim is a combination of plant sterols (free and in esterified form) and Cholesternorm......®mix and provides at the levels of the proposed conditions of use around 0.52 g plant sterols, 0.95 g linoleic acid, 0.13 g alpha-linolenic acid and 0.13 g pectins per day. The combination of plant sterols and Cholesternorm®mix, which is the subject of the claim, is sufficiently characterised in relation...

The effect of plant sterol-enriched turkey meat on cholesterol bio-accessibility during in vitro digestion and Caco-2 cell uptake.

Science.gov (United States)

Grasso, S; Harrison, S M; Monahan, F J; Brayden, D; Brunton, N P

2018-03-01

This study evaluated the effect of a plant sterol-enriched turkey product on cholesterol bio-accessibility during in vitro digestion and cholesterol uptake by Caco-2 monolayers. Turkey products, one plant sterol-enriched (PS) and one plant sterol-free (C), were produced in an industrial pilot plant. Before simulated digestion, matrices were spiked with cholesterol (1:5 weight ratio of cholesterol to plant sterol). Plant sterols were included at a concentration equivalent to the minimum daily intake recommended by the European Food Safety Authority (EFSA) for cholesterol lowering. After simulated digestion, the percentage of cholesterol micellarization and uptake by Caco-2 cells in the presence of PS meat were measured. Compared to C meat, PS meat significantly inhibited cholesterol micellarization on average by 24% and Caco-2 cell accumulation by 10%. This study suggests that plant sterols in meat can reduce cholesterol uptake by intestinal epithelia and it encourages efforts to make new PS-based functional foods.

Formation of Plant Sterol Oxidation Products in Foods during Baking and Cooking Using Margarine without and with Added Plant Sterol Esters

NARCIS (Netherlands)

Lin, Y.; Knol, D.; Menéndez-Carreño, M.; Blom, W.A.M.; Matthee, J.; Janssen, H.G.; Trautwein, E.A.

2016-01-01

Plant sterols (PS) in foods are subject to thermal oxidation to form PS oxidation products (POP). This study measured POP contents of 19 foods prepared by typical household baking and cooking methods using margarines without (control) and with 7.5% added PS (as 12.5% PS-esters, PS-margarine). Median

Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

DEFF Research Database (Denmark)

Lund, F. W.; Lomholt, M. A.; Solanko, L. M.

2012-01-01

to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter...

Tracing the Temporal and Spatial Variations in the Origin of Fecal Material in Three Oklahoma Watersheds Using Sterol Fingerprints

Science.gov (United States)

Lu, Y.; Philp, P. R.

2014-12-01

Organic wastes, in particular fecal material, are qualified as one of the major causes of water quality deterioration. Their accumulation in water bodies may increase algal proliferation and eutrophication and the number of pathogenic organisms, which are responsible for many intestinal diseases especially when the water is used for recreational activities and/or as a supply for drinking water. In order to estimate the risk level associated with primary body contact in recreational water bodies, enumeration of some specific micro-organisms, such as Enterococci and Escherichia coli, are commonly used. Sterol distributions can provide some relevant information on the origin of fecal material in water system, since they are ubiquitous organic compounds and their distributions in many warm-blooded animal feces can be used as evidence for their source. In this study, we monitored fecal material contamination in three Oklahoma watersheds based on sterol fingerprints over a one-year period (2012 ~ 2013). The sterols from sediments and water samples (sterols associated to suspended particles as well as free sterols in water) were recovered using sonication and solid phase extraction (SPE), respectively, using different organic solvents. They were then identified and quantified by gas chromatography - mass spectrometry (GC-MS) using an internal standard. The GC-MS was previously calibrated with a sterol mixture injected at different concentrations. Our primary results show that the concentration of total sterols generally increases from the Upper Canadian contamination and provide a better understanding on the ability of using sterol fingerprints to determine the origin of the fecal contamination. Additionally, such a sampling strategy, over a one-year period at regular intervals, enable us to track the water contamination by feces according to the seasonal climatic variations such as drought or heavy rainfall events.

Effects of plant sterol esters in skimmed milk and vegetable-fat-enriched milk on serum lipids and non-cholesterol sterols in hypercholesterolaemic subjects: a randomised, placebo-controlled, crossover study.

Science.gov (United States)

Casas-Agustench, Patricia; Serra, Mercè; Pérez-Heras, Ana; Cofán, Montserrat; Pintó, Xavier; Trautwein, Elke A; Ros, Emilio

2012-06-01

Plant sterol (PS)-supplemented foods are recommended to help in lowering serum LDL-cholesterol (LDL-C). Few studies have examined the efficacy of PS-enriched skimmed milk (SM) or semi-SM enriched with vegetable fat (PS-VFM). There is also insufficient information on factors predictive of LDL-C responses to PS. We examined the effects of PS-SM (0·1 % dairy fat) and PS-VFM (0·1 % dairy fat plus 1·5 % vegetable fat) on serum lipids and non-cholesterol sterols in hypercholesterolaemic individuals. In a placebo-controlled, crossover study, forty-three subjects with LDL-C>1300 mg/l were randomly assigned to three 4-week treatment periods: control SM, PS-SM and PS-VFM, with 500 ml milk with or without 3·4 g PS esters (2 g free PS). Serum concentrations of lipids and non-cholesterol sterols were measured. Compared to control, LDL-C decreased by 8·0 and 7·4 % (P synthesis and high cholesterol absorption predicted improved LDL-C responses to PS.

Following Intracellular Cholesterol Transport by Linear and Non-Linear Optical Microscopy of Intrinsically Fluorescent Sterols

DEFF Research Database (Denmark)

Wustner, D.

2012-01-01

Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the development of new technical approaches to follow...... is on recent developments in imaging technology to follow the intracellular fate of intrinsically fluorescent sterols as faithful cholesterol markers. In particular, UV-sensitive wide field and multiphoton microscopy of the sterol dehydroergosterol, DHE, is explained and new methods of quantitative image...... analysis like pixel-wise bleach rate fitting and multiphoton image correlation spectroscopy are introduced. Several applications of the new technology including observation of vectorial sterol trafficking in polarized human hepatoma cells for investigation of reverse cholesterol transport are presented....

Preferential campesterol incorporation into various tissues in apolipoprotein E*3-Leiden mice consuming plant sterols or stanols

NARCIS (Netherlands)

Plat, J.; Jong, A.de; Volger, O.L.; Princen, H.M.G.; Mensink, R.P.

2008-01-01

Intestinal absorption of plant sterols and stanols is much lower as compared with that of cholesterol; and therefore, serum concentrations are low. Circulating plant sterols and stanols are incorporated into tissues. However, hardly any data are available about tissue distributions of individual

Sterol Methyl Oxidases Affect Embryo Development via Auxin-Associated Mechanisms.

Science.gov (United States)

Zhang, Xia; Sun, Shuangli; Nie, Xiang; Boutté, Yohann; Grison, Magali; Li, Panpan; Kuang, Susu; Men, Shuzhen

2016-05-01

Sterols are essential molecules for multiple biological processes, including embryogenesis, cell elongation, and endocytosis. The plant sterol biosynthetic pathway is unique in the involvement of two distinct sterol 4α-methyl oxidase (SMO) families, SMO1 and SMO2, which contain three and two isoforms, respectively, and are involved in sequential removal of the two methyl groups at C-4. In this study, we characterized the biological functions of members of the SMO2 gene family. SMO2-1 was strongly expressed in most tissues during Arabidopsis (Arabidopsis thaliana) development, whereas SMO2-2 showed a more specific expression pattern. Although single smo2 mutants displayed no obvious phenotype, the smo2-1 smo2-2 double mutant was embryonic lethal, and the smo2-1 smo2-2/+ mutant was dwarf, whereas the smo2-1/+ smo2-2 mutant exhibited a moderate phenotype. The phenotypes of the smo2 mutants resembled those of auxin-defective mutants. Indeed, the expression of DR5rev:GFP, an auxin-responsive reporter, was reduced and abnormal in smo2-1 smo2-2 embryos. Furthermore, the expression and subcellular localization of the PIN1 auxin efflux facilitator also were altered. Consistent with these observations, either the exogenous application of auxin or endogenous auxin overproduction (YUCCA9 overexpression) partially rescued the smo2-1 smo2-2 embryonic lethality. Surprisingly, the dwarf phenotype of smo2-1 smo2-2/+ was completely rescued by YUCCA9 overexpression. Gas chromatography-mass spectrometry analysis revealed a substantial accumulation of 4α-methylsterols, substrates of SMO2, in smo2 heterozygous double mutants. Together, our data suggest that SMO2s are important for correct sterol composition and function partially through effects on auxin accumulation, auxin response, and PIN1 expression to regulate Arabidopsis embryogenesis and postembryonic development. © 2016 American Society of Plant Biologists. All Rights Reserved.

Comparison of bile acid synthesis determined by isotope dilution versus fecal acidic sterol output in human subjects

International Nuclear Information System (INIS)

Duane, W.C.; Holloway, D.E.; Hutton, S.W.; Corcoran, P.J.; Haas, N.A.

1982-01-01

Fecal acidic sterol output has been found to be much lower than bile acid synthesis determined by isotope dilution. Because of this confusing discrepancy, we compared these 2 measurements done simultaneously on 13 occasions in 5 normal volunteers. In contrast to previous findings, bile acid synthesis by the Lindstedt isotope dilution method averaged 16.3% lower than synthesis simultaneously determined by fecal acidic sterol output (95% confidence limit for the difference - 22.2 to -10.4%). When one-sample determinations of bile acid pools were substituted for Lindstedt pools, bile acid synthesis by isotope dilution averaged 5.6% higher than synthesis by fecal acidic sterol output (95% confidence limits -4.9 to 16.1%). These data indicate that the 2 methods yield values in reasonably close agreement with one another. If anything, fecal acidic sterol outputs are slightly higher than synthesis by isotope dilution

Sterol Methyl Oxidases Affect Embryo Development via Auxin-Associated Mechanisms1

Science.gov (United States)

Zhang, Xia; Sun, Shuangli; Nie, Xiang; Boutté, Yohann; Grison, Magali; Li, Panpan; Kuang, Susu

2016-01-01

Sterols are essential molecules for multiple biological processes, including embryogenesis, cell elongation, and endocytosis. The plant sterol biosynthetic pathway is unique in the involvement of two distinct sterol 4α-methyl oxidase (SMO) families, SMO1 and SMO2, which contain three and two isoforms, respectively, and are involved in sequential removal of the two methyl groups at C-4. In this study, we characterized the biological functions of members of the SMO2 gene family. SMO2-1 was strongly expressed in most tissues during Arabidopsis (Arabidopsis thaliana) development, whereas SMO2-2 showed a more specific expression pattern. Although single smo2 mutants displayed no obvious phenotype, the smo2-1 smo2-2 double mutant was embryonic lethal, and the smo2-1 smo2-2/+ mutant was dwarf, whereas the smo2-1/+ smo2-2 mutant exhibited a moderate phenotype. The phenotypes of the smo2 mutants resembled those of auxin-defective mutants. Indeed, the expression of DR5rev:GFP, an auxin-responsive reporter, was reduced and abnormal in smo2-1 smo2-2 embryos. Furthermore, the expression and subcellular localization of the PIN1 auxin efflux facilitator also were altered. Consistent with these observations, either the exogenous application of auxin or endogenous auxin overproduction (YUCCA9 overexpression) partially rescued the smo2-1 smo2-2 embryonic lethality. Surprisingly, the dwarf phenotype of smo2-1 smo2-2/+ was completely rescued by YUCCA9 overexpression. Gas chromatography-mass spectrometry analysis revealed a substantial accumulation of 4α-methylsterols, substrates of SMO2, in smo2 heterozygous double mutants. Together, our data suggest that SMO2s are important for correct sterol composition and function partially through effects on auxin accumulation, auxin response, and PIN1 expression to regulate Arabidopsis embryogenesis and postembryonic development. PMID:27006488

Absence of anti-HMG-CoA reductase autoantibodies in severe self-limited statin-related myopathy.

Science.gov (United States)

Floyd, James S; Brody, Jennifer A; Tiniakou, Eleni; Psaty, Bruce M; Mammen, Andrew

2016-06-01

Patients with self-limited statin-related myopathy improve spontaneously when statins are stopped. In contrast, patients with statin-associated autoimmune myopathy have autoantibodies recognizing 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and usually require immunosuppressive therapy to control their disease. On initial presentation, it can sometimes be difficult to distinguish between these 2 diseases, as both present with muscle pain, weakness, and elevated serum creatine kinase (CK) levels. The goal of this study was to determine whether patients with severe self-limited statin-related myopathy also make anti-HMGCR autoantibodies. We screened 101 subjects with severe self-limited cerivastatin-related myopathy for anti-HMGCR autoantibodies. No patient with severe self-limited cerivastatin-related myopathy had anti-HMGCR autoantibodies. Anti-HMGCR autoantibody testing can be used to help differentiate whether a patient has self-limited myopathy due to cerivastatin or autoimmune statin-associated myopathy; these findings may apply to other statins as well. Muscle Nerve 54: 142-144, 2016. © 2016 Wiley Periodicals, Inc.

The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study

DEFF Research Database (Denmark)

Kohut, Peter; Wüstner, Daniel; Hronska, L

2011-01-01

of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps--Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols......Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We...... applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry...

Oxygen and xenobiotic reductase activities of cytochrome P450.

NARCIS (Netherlands)

Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

1995-01-01

The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

Structure and mechanism of dimethylsulfoxide reductase, a molybdopterin-containing enzyme of DMSO reductase family

International Nuclear Information System (INIS)

McEwan, A.G.; Ridge, J.P.; McDevitt, C.A.; Hanson, G.R.

2001-01-01

Full text: Apart from nitrogenase, enzymes containing molybdenum are members of a superfamily, the molybdopterin-containing enzymes. Most of these enzymes catalyse an oxygen atom transfer and two electron transfer reaction. During catalysis the Mo at the active site cycles between the Mo(VI) and Mo(IV) states. The DMSO reductase family of molybdopterin-containing enzymes all contain a bis(molybdopterin guanine dinucleotide)Mo cofactor and over thirty examples have now been described. Over the last five years crystal structures of dimethylsulfoxide (DMSO) reductase and four other enzymes of the DMSO reductase family have revealed that enzymes of this family have a similar tertiary structure. The Mo atom at the active site is coordinated by four thiolate ligands provided by the dithiolene side chains of the two MGD molecules of the bis(MGD)Mo cofactor as well as a ligand provided by an amino acid side chain. In addition, an oxygen atom in the form of an oxo, hydroxo or aqua group is also coordinated to the Mo atom. In the case of dimethylsulfoxide reductase X-ray crystallography of the product-reduced species and Raman spectroscopy has demonstrated that the enzyme contains a single exchangeable oxo group that is H-bonded to W116

A Study of the Reactivity of Polyhydroxylated Sterol Derivatives

Czech Academy of Sciences Publication Activity Database

Marek, Aleš; Klepetářová, Blanka; Elbert, Tomáš

2015-01-01

Roč. 4, č. 8 (2015), s. 808-817 ISSN 2193-5807 R&D Projects: GA AV ČR IAA400550801 Institutional support: RVO:61388963 Keywords : alpha-hydroxyketones * polyhydroxylated compounds * regiospecific reactions * silylation * sterols Subject RIV: CC - Organic Chemistry Impact factor: 3.275, year: 2015

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

Science.gov (United States)

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.

In vivo identification of promoter elements and transcription factors mediating activation of hepatic HMG-CoA reductase by T{sub 3}

Energy Technology Data Exchange (ETDEWEB)

Boone, Lindsey R.; Niesen, Melissa I. [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL (United States); Jaroszeski, Mark [Department of Chemical and Biomedical Engineering, College of Engineering, University of South Florida, Tampa, FL (United States); Ness, Gene C., E-mail: gness@hsc.usf.edu [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL (United States)

2009-07-31

The promoter elements and transcription factors necessary for triiodothyronine (T{sub 3}) induction of hepatic HMG-CoA reductase (HMGR) were investigated by transfecting rat livers with wild type and mutant HMGR promoter-luciferase constructs using in vivo electroporation. Mutations in the sterol response element (SRE), nuclear factor-y (NF-Y) site, and the newly identified upstream transcription factor-2 (USF-2) site essentially abolished the T{sub 3} response. Chromatin immunoprecipitation (ChIP) analysis demonstrated that T{sub 3} treatment caused a 4-fold increase in in vivo binding of USF-2 to the HMGR promoter. Co-transfection of the wild type HMGR promoter with siRNAs to USF-2, SREBP-2, or NF-Y nearly abolished the T{sub 3} induction, as measured by promoter activity. These data provide in vivo evidence for functional roles for USF-2, SREBP-2, and NF-Y in mediating the T{sub 3}-induction of hepatic HMGR transcription.

Rapeseed oil, olive oil, plant sterols, and cholesterol metabolism: an ileostomy study.

Science.gov (United States)

Ellegård, L; Andersson, H; Bosaeus, I

2005-12-01

To study whether olive oil and rapeseed oil have different effects on cholesterol metabolism. Short-term experimental study, with controlled diets. Outpatients at a metabolic-ward kitchen. A total of nine volunteers with conventional ileostomies. Two 3-day diet periods; controlled diet including 75 g of rapeseed oil or olive oil. Cholesterol absorption, ileal excretion of cholesterol, and bile acids. Serum levels of cholesterol and bile acid metabolites. Differences between diets evaluated with Wilcoxon's signed rank sum test. Rapeseed oil diet contained 326 mg more plant sterols than the olive oil diet. Rapeseed oil tended to decrease cholesterol absorption by 11% (P = 0.050), and increased excretion of cholesterol, bile acids, and their sum as sterols by 9% (P = 0.021), 32% (P = 0.038), and 51% (P = 0.011) compared to olive oil. A serum marker for bile acid synthesis (7alpha-hydroxy-4-cholesten-3-one) increased by 28% (P = 0.038) within 10 h of consumption, and serum cholesterol levels decreased by 7% (P = 0.024), whereas a serum marker for cholesterol synthesis (lathosterol) as well as serum levels of plant sterols remained unchanged. Rapeseed oil and olive oil have different effects on cholesterol metabolism. Rapeseed oil, tends to decrease cholesterol absorption, increases excretion of cholesterol and bile acids, increases serum marker of bile acid synthesis, and decreases serum levels of cholesterol compared to olive oil. This could in part be explained by different concentrations of natural plant sterols. Supported by the Göteborg Medical Society, the Swedish Medical Society, the Swedish Board for Agricultural Research (SJFR) grant 50.0444/98 and by University of Göteborg.

Regulation of steroid 5-{alpha} reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

Energy Technology Data Exchange (ETDEWEB)

Seo, Young-Kyo [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Zhu, Bing [Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0144 (United States); Jeon, Tae-Il [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Osborne, Timothy F., E-mail: tfosborn@uci.edu [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States)

2009-11-01

In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

Regulation of steroid 5-α reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

International Nuclear Information System (INIS)

Seo, Young-Kyo; Zhu, Bing; Jeon, Tae-Il; Osborne, Timothy F.

2009-01-01

In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

Effects of 2,3-iminosqualene in cultured cells

International Nuclear Information System (INIS)

Popjak, G.; Meenan, A.; Nes, W.D.

1987-01-01

2,3-Iminosqualene added to culture media 10 ug/ml) of rat hepatoma (H4-II-E-C3) or Chinese hamster ovary (CHO) cells irreversibly inactivates the squalene-oxide: lanosterol cyclase, but it does not inhibit general polyprenyl synthesis either from [ 14 C]acetate or [ 14 C]mevalonate. Isq added to lipoprotein-containing media of H4 cells causes in 24 hr an over twofold rise in HMG-CoA reductase and abolishes the repressive effect of mevalonate (MVA) on the reductase. H4 cells synthesize from [2- 14 C]-MVA labelled squalene, squalene-2,3-oxide, squalene-2,3-22,23-dioxide, but very little sterol. The conversion of MVA to these polyprenyls in the presence of Isq is as efficient as its conversion to squalene and cholesterol in control cells. They conclude that the repressor of HMG-CoA reductase derived from MVA is a sterol - whatever might be the nature of that sterol - and not a nonsteroidal derivative of MVA metabolism. H4 cells exposed to Isq in lipid-depleted media die in 48-72 hr, but can be rescued by LDL, but not by free cholesterol or MVA. CHO cells are more resistant than H4 cells and succumb only after 8-9 days' exposure to Isq

Scap is required for sterol synthesis and crypt growth in intestinal mucosa.

Science.gov (United States)

McFarlane, Matthew R; Cantoria, Mary Jo; Linden, Albert G; January, Brandon A; Liang, Guosheng; Engelking, Luke J

2015-08-01

SREBP cleavage-activating protein (Scap) is an endoplasmic reticulum membrane protein required for cleavage and activation of sterol regulatory element-binding proteins (SREBPs), which activate the transcription of genes in sterol and fatty acid biosynthesis. Liver-specific loss of Scap is well tolerated; hepatic synthesis of sterols and fatty acids is reduced, but mice are otherwise healthy. To determine whether Scap loss is tolerated in the intestine, we generated a mouse model (Vil-Scap(-)) in which tamoxifen-inducible Cre-ER(T2), a fusion protein of Cre recombinase with a mutated ligand binding domain of the human estrogen receptor, ablates Scap in intestinal mucosa. After 4 days of tamoxifen, Vil-Scap(-) mice succumb with a severe enteropathy and near-complete collapse of intestinal mucosa. Organoids grown ex vivo from intestinal crypts of Vil-Scap(-) mice are readily killed when Scap is deleted by 4-hydroxytamoxifen. Death is prevented when culture medium is supplemented with cholesterol and oleate. These data show that, unlike the liver, the intestine requires Scap to sustain tissue integrity by maintaining the high levels of lipid synthesis necessary for proliferation of intestinal crypts. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

∆24-sterol methyltransferase plays an important role in the growth and development of Sporothrix schenckii and Sporothrix brasiliensis

Directory of Open Access Journals (Sweden)

Luana Pereira Borba-Santos

2016-03-01

Full Text Available Inhibition of ∆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-3-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 14-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 14-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 towards 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

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

Science.gov (United States)

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

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

Science.gov (United States)

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

Using fecal sterols to assess dynamics of sewage input in sediments along a human-impacted river-estuary system in eastern China.

Science.gov (United States)

He, Ding; Zhang, Kai; Tang, Jianhui; Cui, Xingqian; Sun, Yongge

2018-05-01

Sedimentary fecal sterols and other sterol biomarkers, combined with bulk total organic carbon (TOC) and its stable carbon isotope were applied to characterize the sewage contamination across a ca. 280 km transect from the Xiaoqing River to the Laizhou Bay, a typical river-estuary system subjected to extensive anthropogenic stress due to rapid regional urbanization and industrialization in eastern China. Two sampling events were performed in both spring and summer seasons in the Laizhou Bay adjacent to the Xiaoqing River in order to assess the potential seasonal variation. Fecal sterols such as coprostanol and epicoprostanol, which are typical indicators of anthropogenic sewage input, displayed high concentrations of up to 63.2 μg g -1 dry weight (dw) and 13.1 μg g -1 dw, respectively. Results suggested that most of the stations along the Xiaoqing River were severely contaminated by fecal inputs with a decreasing trend from the river to the estuary that was mainly explained by the increasing distance from the diffuse sewage sources and the gradual dilution by sea water. Although there was no significant difference in fecal sterol concentrations between spring and summer in the Laizhou Bay, suggestive of no significant difference in sewage abundance, significantly higher average epicoprostanol/coprostanol and lower coprostanol/epicoprostanol ratios were observed in spring than summer, indicative of different sewage sources (e.g., human vs. non-human). Seasonal discharge and land-runoff, air temperature related to microbial activity differences and different extend of animal manure irrigation during agricultural planting could be additional reasons and need further investigation. Nevertheless, fecal sterol concentrations, distributions and diagnostic ratios should all be taken into consideration to better understand sewage inputs and source dynamics in river-estuary ecosystems. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel Synthesis of Phytosterol Ester from Soybean Sterol and Acetic Anhydride.

Science.gov (United States)

Yang, Fuming; Oyeyinka, Samson A; Ma, Ying

2016-07-01

Phytosterols are important bioactive compounds which have several health benefits including reduction of serum cholesterol and preventing cardiovascular diseases. The most widely used method in the synthesis of its ester analogous form is the use of catalysts and solvents. These methods have been found to present some safety and health concern. In this paper, an alternative method of synthesizing phytosterol ester from soybean sterol and acetic anhydride was investigated. Process parameters such as mole ratio, temperature and time were optimized. The structure and physicochemical properties of phytosterol acetic ester were analyzed. By the use of gas chromatography, the mole ratio of soybean sterol and acetic anhydride needed for optimum esterification rate of 99.4% was 1:1 at 135 °C for 1.5 h. FTIR spectra confirmed the formation of phytosterol ester with strong absorption peaks at 1732 and 1250 cm(-1) , which corresponds to the stretching vibration of C=O and C-O-C, respectively. These peaks could be attributed to the formation of ester links which resulted from the reaction between the hydroxyl group of soybean sterol and the carbonyl group of acetic anhydride. This paper provides a better alternative to the synthesis of phytosterol ester without catalyst and solvent residues, which may have potential application in the food, health-care food, and pharmaceutical industries. © 2016 Institute of Food Technologists®

Free-cholesterol loading does not trigger phase separation of the fluorescent sterol dehydroergosterol in the plasma membrane of macrophages

DEFF Research Database (Denmark)

Wüstner, Daniel

2008-01-01

membrane distribution of the fluorescent cholesterol-mimicking sterol dehydroergosterol (DHE) was investigated in FC-loaded J774 macrophages. Wide field fluorescence and deconvolution microscopy were combined with quantitative assessment of sterol distribution in straightened plasma membrane image segments...

X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

OpenAIRE

Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.; Joachimiak, A.

2009-01-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determi...

Effects of lovastatin (mevinolin) on sterol levels and on activity of azoles in Saccharomyces cerevisiae.

OpenAIRE

Lorenz, R T; Parks, L W

1990-01-01

The hypocholesterolemic drug lovastatin (mevinolin) was found to be very effective in lowering the sterol levels of the wild-type yeast Saccharomyces cerevisiae. Lovastatin dramatically decreased the steryl ester content from 2.62 to 0.8 micrograms/mg (dry weight), whereas the free sterol content decreased only from 2.79 to 2.24 micrograms/mg (dry weight) when lovastatin was present in the medium at 10 micrograms/ml. At higher concentrations (100 micrograms/ml), lovastatin nearly abolished th...

Characterization of the sterol 14α-demethylases of Fusarium graminearum identifies a novel genus-specific CYP51 function.

Science.gov (United States)

Fan, Jieru; Urban, Martin; Parker, Josie E; Brewer, Helen C; Kelly, Steven L; Hammond-Kosack, Kim E; Fraaije, Bart A; Liu, Xili; Cools, Hans J

2013-05-01

CYP51 encodes the cytochrome P450 sterol 14α-demethylase, an enzyme essential for sterol biosynthesis and the target of azole fungicides. In Fusarium species, including pathogens of humans and plants, three CYP51 paralogues have been identified with one unique to the genus. Currently, the functions of these three genes and the rationale for their conservation within the genus Fusarium are unknown. Three Fusarium graminearum CYP51s (FgCYP51s) were heterologously expressed in Saccharomyces cerevisiae. Single and double FgCYP51 deletion mutants were generated and the functions of the FgCYP51s were characterized in vitro and in planta. FgCYP51A and FgCYP51B can complement yeast CYP51 function, whereas FgCYP51C cannot. FgCYP51A deletion increases the sensitivity of F. graminearum to the tested azoles. In ΔFgCYP51B and ΔFgCYP51BC mutants, ascospore formation is blocked, and eburicol and two additional 14-methylated sterols accumulate. FgCYP51C deletion reduces virulence on host wheat ears. FgCYP51B encodes the enzyme primarily responsible for sterol 14α-demethylation, and plays an essential role in ascospore formation. FgCYP51A encodes an additional sterol 14α-demethylase, induced on ergosterol depletion and responsible for the intrinsic variation in azole sensitivity. FgCYP51C does not encode a sterol 14α-demethylase, but is required for full virulence on host wheat ears. This is the first example of the functional diversification of a fungal CYP51. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

Science.gov (United States)

Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

2001-04-03

Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

DEFF Research Database (Denmark)

Hartwig Petersen, Nicole; Færgeman, Nils J; Yu, Liqing

2008-01-01

fluorescent protein (NPC1L1-EGFP) and cholesterol analogues in hepatoma cells. At steady state about 42% of NPC1L1 resided in the transferrin (Tf) positive, sterol enriched endocytic recycling compartment (ERC), while time-lapse microscopy demonstrated NPC1L1 traffic between plasma membrane and ERC...... the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells NPC1L1 resided almost...... exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1 mediated cellular sterol uptake....

Sneaking under the toxin surveillance radar: parasitism and sterol ...

African Journals Online (AJOL)

This was not simply a reflection of retaining host lipid content because K. micrum contains octadecapentaenoic acid (18:5n3), largely in galactolipids of the chloroplast, whereas Amoebophrya sp. contained little to no 18:5n3. By having a sterol content similar to its host, Amoebophrya sp. is able to avoid cell lysis caused by ...

Possible regulation of sterol biosynthesis by phenolic acids

International Nuclear Information System (INIS)

Ranganathan, S.; Ramasarma, T.

1974-01-01

To test whether the phenolic acids, metabolites of tyrosine, regulate the biosynthesis of cholesterol, influence of phenolic acids on the incorporation of mevalonate-2- 14 C into sterols by rat liver and brain homogenate systems has been investigated in vitro. Results show that the combined presence of the aromatic ring and the carboxyl group in the compound under investigation inhibited the incorporation of labelled mevalonate. (M.G.B.)

Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

DEFF Research Database (Denmark)

Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie

2010-01-01

The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

Peroxo-Type Intermediates in Class I Ribonucleotide Reductase and Related Binuclear Non-Heme Iron Enzymes

DEFF Research Database (Denmark)

Kepp, Kasper Planeta; Bell, Caleb B.; Clay, MIchael D.

2009-01-01

We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class la ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O-O str...

Antitubercular activity and inhibitory effect on Epstein-Barr virus activation of sterols and polyisoprenepolyols from an edible mushroom, Hypsizigus marmoreus.

Science.gov (United States)

Akihisa, Toshihiro; Franzblau, Scott Gary; Tokuda, Harukuni; Tagata, Masaaki; Ukiya, Motohiko; Matsuzawa, Tsunetomo; Metori, Koichi; Kimura, Yumiko; Suzuki, Takashi; Yasukawa, Ken

2005-06-01

Seven sterols (1-7) and eight polyisoprenepolyols (8-15), isolated from the non-saponifiable lipid fraction of the dichloromethane extract of an edible mushroom, Hypsizigus marmoreus (Buna-shimeji), were tested for their antitubercular activity against Mycobacterium tuberculosis strain H37Rv using the Microplate Alamar Blue Assay (MABA). Six sterols (2-7) and two polyisoprenepolyols (8, 12) showed a minimum inhibitory concentration (MIC) in the range of 1-51 microg/ml, while the others (1, 9-11, 13-15) were inactive (MIC>128 microg/ml). The seven sterols (1-7) and three polyisoprenepolyols (8, 10, 12) were further evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Sterols 6 and 7 showed potent inhibitory effects while preserving the high viability of Raji cells.

Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

Science.gov (United States)

Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki , Gang; David R. , Sarkanen; Simo , Ford; Joshua D.

2003-10-21

Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB.

Science.gov (United States)

Roussel, Benoit D; Newton, Timothy M; Malzer, Elke; Simecek, Nikol; Haq, Imran; Thomas, Sally E; Burr, Marian L; Lehner, Paul J; Crowther, Damian C; Marciniak, Stefan J; Lomas, David A

2013-11-15

Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR.

Substrate Preferences and Catalytic Parameters Determined by Structural Characteristics of Sterol 14[alpha]-Demethylase (CYP51) from Leishmania infantum

Energy Technology Data Exchange (ETDEWEB)

Hargrove, Tatiana Y.; Wawrzak, Zdzislaw; Liu, Jialin; Nes, W. David; Waterman, Michael R.; Lepesheva, Galina I. (Vanderbilt); (TTU); (NWU)

2012-05-14

Leishmaniasis is a major health problem that affects populations of {approx}90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/function characterization of sterol 14{alpha}-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14{alpha}-methyl group from sterol precursors. The reaction is essential for membrane biogenesis and therefore has great potential to become a target for antileishmanial chemotherapy. Although L. infantum CYP51 prefers C4-monomethylated sterol substrates such as C4-norlanosterol and obtusifoliol (V{sub max} of {approx}10 and 8 min{sup -1}, respectively), it is also found to 14{alpha}-demethylate C4-dimethylated lanosterol (V{sub max} = 0.9 min{sup -1}) and C4-desmethylated 14{alpha}-methylzymosterol (V{sub max} = 1.9 min{sup -1}). Binding parameters with six sterols were tested, with K{sub d} values ranging from 0.25 to 1.4 {mu}m. Thus, L. infantum CYP51 is the first example of a plant-like sterol 14{alpha}-demethylase, where requirements toward the composition of the C4 atom substituents are not strict, indicative of possible branching in the postsqualene portion of sterol biosynthesis in the parasite. Comparative analysis of three CYP51 substrate binding cavities (Trypanosoma brucei, Trypanosoma cruzi, and L. infantum) suggests that substrate preferences of plant- and fungal-like protozoan CYP51s largely depend on the differences in the enzyme active site topology. These minor structural differences are also likely to underlie CYP51 catalytic rates and drug susceptibility and can be used to design potent and specific inhibitors.

Triglycerides, fatty acids, sterols, mono- and disaccharides and sugar alcohols in human milk and current types of infant formula milk

NARCIS (Netherlands)

Huisman, M; vanBeusekom, CM; Nijeboer, HJ; Muskiet, FAJ; Boersma, ER

Objective: To investigate differences in the fatty acid composition, sterols, minor carbohydrates and sugar alcohols between human and formula milk. Design: We analyzed the concentrations of triglycerides, sterols, di- and monosaccharides and sugar alcohols, as well as the fatty acid composition of

Effects of plant sterols derived from Aloe vera gel on human dermal fibroblasts in vitro and on skin condition in Japanese women

Directory of Open Access Journals (Sweden)

Tanaka M

2015-02-01

Full Text Available Miyuki Tanaka,1 Eriko Misawa,1 Koji Yamauchi,1 Fumiaki Abe,1 Chiaki Ishizaki2 1Functional Food Research Department, Food Science and Technology Institute, Morinaga Milk Industry Co, Ltd, Zama, Kanagawa, 2Ebisu Skin Research Center, Inforward, Inc., Tokyo, Japan Background: Aloe is known for its topical use for treating wounds and burns. Many previous studies reported the healing effects of Aloe vera. However, there are few clinical studies on the effect of orally administered A. vera gel on the skin. Aloe sterols are a type of plant sterols that have the capability to regulate the metabolism of glucose and lipids. In a recent study, we confirmed that ingested Aloe sterols reached the peripheral tissues through the bloodstream. However, their influence on dermal fibroblasts has not been investigated. Methods: First, we investigated the capability of Aloe sterols (cycloartenol and lophenol to stimulate human dermal fibroblasts in vitro. Then, we investigated the effect of intake of Aloe vera gel powder (AVGP containing 40 µg Aloe sterols on the skin conditions in Japanese women with dry skin in a randomized, double-blind, placebo-controlled trial. Results: After cocultivation with Aloe sterols, the production of collagen and hyaluronic acid increased by approximately two-fold and 1.5-fold, and gene expression levels of these enzymes responsible for their synthesis were also observed in human dermal fibroblasts. An increase in arm skin hydration was observed at 8 weeks in the AVGP group, whereas a slight decrease in arm skin hydration was noted in the placebo group. However, there was no statistical difference between AVGP and placebo groups in skin moisture. In subgroup analysis, the change in the mean wrinkle depth was significantly lower in the AVGP group than in the control group. In addition, percent body fat after 8 weeks was significantly lower in the AVGP group. No AVGP intake-dependent harmful phenomenon was observed during the intake

Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

DEFF Research Database (Denmark)

Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

1992-01-01

can replace light in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. We show further that sucrose alone is sufficient for the full expression of nitrate reductase genes in etiolated Arabidopsis plants. Finally, using a reporter gene, we show......Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression....... Located in the cytosol, nitrate reductase obtains its reductant not from photosynthesis but from carbohydrate catabolism. This relationship prompted us to investigate the indirect role that light might play, via photosynthesis, in the regulation of nitrate reductase gene expression. We show that sucrose...

Sebaceous lipid profiling of bat integumentary tissues: quantitative analysis of free Fatty acids, monoacylglycerides, squalene, and sterols.

Science.gov (United States)

Pannkuk, Evan L; Gilmore, David F; Fuller, Nathan W; Savary, Brett J; Risch, Thomas S

2013-12-01

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans and is devastating North American bat populations. Sebaceous lipids secreted from host integumentary tissues are implicated in the initial attachment and recognition of host tissues by pathogenic fungi. We are interested in determining if ratios of lipid classes in sebum can be used as biomarkers to diagnose severity of fungal infection in bats. To first establish lipid compositions in bats, we isolated secreted and integral lipid fractions from the hair and wing tissues of three species: big brown bats (Eptesicus fuscus), Eastern red bats (Lasiurus borealis), and evening bats (Nycticeius humeralis). Sterols, FFAs, MAGs, and squalene were derivatized as trimethylsilyl esters, separated by gas chromatography, and identified by mass spectrometry. Ratios of sterol to squalene in different tissues were determined, and cholesterol as a disease biomarker was assessed. Free sterol was the dominant lipid class of bat integument. Squalene/sterol ratio is highest in wing sebum. Secreted wing lipid contained higher proportions of saturated FFAs and MAGs than integral wing or secreted hair lipid. These compounds are targets for investigating responses of P. destructans to specific host lipid compounds and as biomarkers to diagnose WNS. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

The influence of sterol metabolism upon radiation-induced aneuploidy of Drosophila melanogaster in the yeast-drosophila system

International Nuclear Information System (INIS)

Savitsij, V.V.; Luchnikova, E.M.; Inge-Vechtomov, S.I.

1985-01-01

The influence of sterol metabolism upon induced Drosophila melanogaster mutagenesis in an ecology-genetic yeast-drosophila system has been studied. The sterol deficit in fly organism has been created for account of using as food substrate for fremales of biomass of saccharomyces cerevisiae living cells of 9-2-PZ12 train with nyssup(r1) locus mutation which blocks the ergosterol synthesis. It has been found that the Drosophila females content on mutant yeast increases the frequency of losses and non discrepancy of X-chromosomes induced by X-radiation (1000 R). Addition into yeast biomass of 0.1 % cholesterol solution in 10 %-ethanol reduces the oocytes resistance to X-radiation up to control level. Possible hormonal and membrane mechanisms of increasing radiation-induced aneuploidy of Drosophila and the role of sterol metabolism in organism resistance to damaging factors are discussed

Role of aldose reductase C-106T polymorphism among diabetic Egyptian patients with different microvascular complications

Directory of Open Access Journals (Sweden)

Nermine Hossam Zakaria

2014-04-01

Full Text Available The aldose reductase pathway proves that elevated blood glucose promotes cellular dysfunction. The polyol pathway converts excess intracellular glucose into alcohols via activity of the aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol which triggers variety of intracellular changes in the tissues. Among diabetes, activity is drastically increased in association with three main consequences inside the cells. The aim of this study was to detect the association of the C-106 T polymorphism of the aldose reductase gene and its frequency among a sample of 150 Egyptian adults with type 2 diabetic patients having diabetic microvascular. The detection of the aldose reductase C-106 T polymorphism gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. The genotype distribution of the C-106 T polymorphism showed that CC genotype was statistically significantly higher among patients with retinopathy compared to nephropathy. Patients with nephropathy had significant association with the TT genotype when compared with diabetic retinopathy patients. Follow up study after the genotype detection among recently diagnosed diabetic patients in order to give a prophylactic aldose reductase inhibitors; studying the microvascular complications and its relation to the genotype polymorphisms. The study may include multiple gene polymorphisms to make the relation between the gene and the occurrence of these complications more evident.

BIOLOGICAL ROLE OF ALDO-KETO REDUCTASES IN RETINOIC ACID BIOSYNTHESIS AND SIGNALING

Directory of Open Access Journals (Sweden)

F. Xavier eRuiz

2012-04-01

Full Text Available Several aldo-keto reductase (AKR enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3, as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance.

Sterol content in the artificial diet of Mythimna separata affects the metabolomics of Arma chinensis (Fallou) as determined by proton nuclear magnetic resonance spectroscopy.

Science.gov (United States)

Guo, Yi; Liu, Chen-Xi; Zhang, Li-Sheng; Wang, Meng-Qing; Chen, Hong-Yin

2017-12-01

Insects cannot synthesize sterols and must obtain them from plants. Therefore, reducing plant sterol content or changing sterol type might be an effective pest control strategy. However, the impacts of these changes on pests' natural predators remain unknown. Here, we fed artificial diets with reduced sterol content to Mythimna separata (Walker) (Lepidoptera: Noctuidae) and investigated the effects on its natural predator, Arma chinensis (Fallou) (Hemiptera: Pentatomidae). Reduced sterol content in M. separata (MS1, MS2, and MS5) was achieved by feeding them artificial diets prepared from a feed base subjected to one, two, or five cycles of sterol extractions, respectively. The content of most substances increased in A. chinensis (AC) groups feeding on MS2 and MS5. The content of eight substances (alanine, betaine, dimethylamine, fumarate, glutamine, glycine, methylamine, and sarcosine) differed significantly between the control (AC0) and treated (AC1, AC2, and AC5) groups. Metabolic profiling revealed that only AC5 was significantly distinct from AC0; the major substances contributing to this difference were maltose, glucose, tyrosine, proline, O-phosphocholine, glutamine, allantoin, lysine, valine, and glutamate. Furthermore, only two metabolic pathways, that is, nicotinate and nicotinamide metabolism and ubiquinone and other terpenoid-quinone biosynthesis, differed significantly between AC1 and AC5 and the control, albeit with an impact value of zero. Thus, the sterol content in the artificial diet fed to M. separata only minimally affected the metabolites and metabolic pathways of its predator A. chinensis, suggesting that A. chinensis has good metabolic self-regulation with high resistance to sterol content changes. © 2017 Wiley Periodicals, Inc.

Cloning and nitrate induction of nitrate reductase mRNA

OpenAIRE

Cheng, Chi-Lien; Dewdney, Julia; Kleinhofs, Andris; Goodman, Howard M.

1986-01-01

Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate in vivo labeled protein and in vitro translation products, demonstrating that nitrate induction increases nitrate reductase p...

Reduced biliary sterol output with no change in total faecal excretion in mice expressing a human apolipoprotein A-I variant.

Science.gov (United States)

Parolini, Cinzia; Caligari, Silvia; Gilio, Donatella; Manzini, Stefano; Busnelli, Marco; Montagnani, Marco; Locatelli, Marcello; Diani, Erika; Giavarini, Flavio; Caruso, Donatella; Roda, Enrico; Roda, Aldo; Sirtori, Cesare R; Chiesa, Giulia

2012-10-01

Apolipoprotein (apo)A-I(M) (ilano), is a molecular variant of apoA-I(wild-type), associated with dramatically low HDL-cholesterol levels, but no increased risk for cardiovascular disease. In view of the present uncertainties on the role of apoA-I in liver cholesterol removal by way of bile acids and neutral sterols, and of the greater capacity of apoA-I(M) (ilano) to remove arterial cholesterol, biliary sterol metabolism was evaluated in transgenic mice expressing apoA-I(M) (ilano). ApoA-I(M) (ilano) mice were fed a high-cholesterol/high-fat diet, and compared with human apoA-I(wild-type) mice. Plasma lipid levels, hepatic bile flow and composition, hepatic and intestinal cholesterol and bile acid content, and faecal sterol content were measured. Moreover, the expression of hepatic ABCA1, SR-B1 and that of hepatic and intestinal genes involved in bile acid metabolism were evaluated. The dietary treatment led to a strong elevation in HDL-cholesterol levels in A-I(M) (ilano) mice, associated with an increased expression of hepatic ABCA1. ApoA-I(M) (ilano) mice showed lower cholesterol output from the liver compared with apoA-I(wild-type) mice, in the absence of liver sterol accumulation. Faecal excretion of neutral sterols and bile acids was similar in the two mouse lines. In spite of a different response to the dietary challenge, with an increased ABCA1 expression and a lower hepatic cholesterol output in apoA-I(M) (ilano) mice, the net sterol excretion is comparable in the two transgenic lines. © 2012 John Wiley & Sons A/S.

Aloe sterol supplementation improves skin elasticity in Japanese men with sunlight-exposed skin: a 12-week double-blind, randomized controlled trial

Directory of Open Access Journals (Sweden)

Tanaka M

2016-11-01

Full Text Available Miyuki Tanaka,1 Yuki Yamamoto,2 Eriko Misawa,1 Kazumi Nabeshima,1 Marie Saito,1 Koji Yamauchi,1 Fumiaki Abe,1 Fukumi Furukawa2 1Functional Food Ingredients Department, Food Ingredients & Technology Institute, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa, 2Department of Dermatology, Wakayama Medical University, Kimiidera, Wakayama, Japan Background/objective: Recently, it was confirmed that the daily oral intake of plant sterols of Aloe vera gel (Aloe sterol significantly increases the skin barrier function, moisture, and elasticity in photoprotected skin. This study aimed to investigate whether Aloe sterol intake affected skin conditions following sunlight exposure in Japanese men. Methods: We performed a 12-week, randomized, double-blind, placebo-controlled study to evaluate the effects of oral Aloe sterol supplementation on skin conditions in 48 apparently healthy men (age range: 30–59 years; average: 45 years. The subjects were instructed to expose the measurement position of the arms to the sunlight outdoors every day for 12 weeks. The skin parameters were measured at 0 (baseline, 4, 8, and 12 weeks. Results: Depending on the time for the revelation of the sunlight, the b* value and melanin index increased and the skin moisture decreased. After taking an Aloe sterol tablet daily for 12 weeks, the skin elasticity index (R2, R5, and R7 levels were significantly higher than the baseline value. There were no differences between the groups in these skin elasticity values. In the subgroup analysis of subjects aged <46 years, the change in the R5 and R7 was significantly higher in the Aloe group than in the placebo group at 8 weeks (P=0.0412 and P=0.0410, respectively. There was a difference in the quantity of sun exposure between each subject, and an additional clinical study that standardizes the amount of ultraviolet rays is warranted. No Aloe sterol intake-dependent harmful phenomenon was observed during the intake period

14CO2-fixation and nitrate reductase activity in vivo in relation to hybrid vigour in maize

International Nuclear Information System (INIS)

Balasubramanian, V.; Shanthakumari, P.; Sinha, S.K.

1977-01-01

Dry matter accumulation in maize shoots, leaf area, 14 CO 2 -fixation and nitrate reductase activity in vivo were measured in the field grown heterotic hybrid CM 400x CM 300 and its inbred parents CM 300 and CM 400 from seedling to maturity. Rates of dry matter accumulation and leaf area development were higher in the hybrid during the initial vegetative phase than in the inbreds. The hybrid had more absolute level of 14 CO 2 -fixation and nitrate reductase activity, although the rates of these processes on unit weight basis were not higher than those of inbreds. It is concluded that the rapid development of leaf area in hybrids during the early stages of vegetative growth is probably important for hybrid vigour. (author)

5α-reductase activity in rat adipose tissue

International Nuclear Information System (INIS)

Zyirek, M.; Flood, C.; Longcope, C.

1987-01-01

We measured the 5 α-reductase activity in isolated cell preparations of rat adipose tissue using the formation of [ 3 H] dihydrotestosterone from [ 3 H] testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5α-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10 -8 M), when added to the medium, caused a 90% decrease in 5α-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5α-reductase activity in each tissue studied

Characterization of mitochondrial thioredoxin reductase from C. elegans

International Nuclear Information System (INIS)

Lacey, Brian M.; Hondal, Robert J.

2006-01-01

Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a k cat of 610 min -1 and a K m of 610 μM using E. coli thioredoxin as substrate. The reported k cat is 25% of the k cat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate

Oxidative demethylation of lanosterol in cholesterol biosynthesis: accumulation of sterol intermediates

International Nuclear Information System (INIS)

Shafiee, A.; Trzaskos, J.M.; Paik, Y.K.; Gaylor, J.L.

1986-01-01

With [ 3 H-24,25]-dihydrolanosterol as substrate, large-scale metabolic formation of intermediates of lanosterol demethylation was carried out to identify all compounds in the metabolic process. Utilizing knowledge of electron transport of lanosterol demethylation, we interrupted the demethylation reaction allowing accumulation and confirmation of the structure of the oxygenated intermediates lanost-8-en-3 beta,32-diol and 3 beta-hydroxylanost-8-en-32-al, as well as the demethylation product 4,4-dimethyl-cholesta-8,14-dien-3 beta-ol. Further metabolism of the delta 8.14-diene intermediate to a single product 4,4-dimethyl-cholest-8-en-3 beta-ol occurs under interruption conditions in the presence of 0.5 mM CN-1. With authentic compounds, each intermediate has been rigorously characterized by high performance liquid chromatography and gas-liquid chromatography plus mass spectral analysis of isolated and derivatized sterols. Intermediates that accumulated in greater abundance were further characterized by ultraviolet, 1 H-NMR, and infrared spectroscopy of the isolated sterols

[Comparison of Physico-chemical Aspects between E. coli and Human Dihydrofolate Reductase: an Equilibrium Unfolding Study].

Science.gov (United States)

Thapliyal, Charu; Jain, Neha; Chaudhuri, Pratima

2015-01-01

A protein, differing in origin, may exhibit variable physicochemical behaviour, difference in sequence homology, fold and function. Thus studying structure-function relationship of proteins from altered sources is meaningful in the sense that it may give rise to comparative aspects of their sequence-structure-function relationship. Dihydrofolate reductase is an enzyme involved in cell cycle regulation. It is a significant enzyme as.a target for developing anticancer drugs. Hence, detailed understanding of structure-function relationships of wide variants of the enzyme dihydrofolate reductase would be important for developing an inhibitor or an antagonist against the enzyme involved in the cellular developmental processes. In this communication, we have reported the comparative structure-function relationship between E. coli and human dihydrofolate reductase. The differences in the unfolding behaviour of these two proteins have been investigated to understand various properties of these two proteins like relative' stability differences and variation in conformational changes under identical denaturing conditions. The equilibrium unfolding mechanism of dihydrofolate reductase proteins using guanidine hydrochloride as a denaturant in the presence of various types of osmolytes has been monitored using loss in enzymatic activity, intrinsic tryptophan fluorescence and an extrinsic fluorophore 8-anilino-1-naphthalene-sulfonic acid as probes. It has been observed that osmolytes, such as 1M sucrose, and 30% glycerol, provided enhanced stability to both variants of dihydrofolate reductase. Their level of stabilisation has been observed to be dependent on intrinsic protein stability. It was observed that 100 mM proline does not show any 'significant stabilisation to either of dihydrofolate reductases. In the present study, it has been observed that the human protein is relatively less stable than the E.coli counterpart.

5α-reductases in human physiology: an unfolding story.

Science.gov (United States)

Traish, Abdulmaged M

2012-01-01

5α-reductases are a family of isozymes expressed in a wide host of tissues including the central nervous system (CNS) and play a pivotal role in male sexual differentiation, development and physiology. A comprehensive literature search from 1970 to 2011 was made through PubMed and the relevant information was summarized. 5α reductases convert testosterone, progesterone, deoxycorticosterone, aldosterone and corticosterone into their respective 5α-dihydro-derivatives, which serve as substrates for 3α-hydroxysteroid dehydrogenase enzymes. The latter transforms these 5α-reduced metabolites into a subclass of neuroactive steroid hormones with distinct physiological functions. The neuroactive steroid hormones modulate a multitude of functions in human physiology encompassing regulation of sexual differentiation, neuroprotection, memory enhancement, anxiety, sleep and stress, among others. In addition, 5α -reductase type 3 is also implicated in the N-glycosylation of proteins via formation of dolichol phosphate. The family of 5α-reductases was targeted for drug development to treat pathophysiological conditions, such as benign prostatic hyperplasia and androgenetic alopecia. While the clinical use of 5α-reductase inhibitors was well established, the scope and the magnitude of the adverse side effects of such drugs, especially on the CNS, is still unrecognized due to lack of knowledge of the various physiological functions of this family of enzymes, especially in the CNS. There is an urgent need to better understand the function of 5α-reductases and the role of neuroactive steroids in human physiology in order to minimize the potential adverse side effects of inhibitors targeting 5α-reductases to treat benign prostatic hyperplasia and androgenic alopecia.

Rapid fingerprinting of sterols and related compounds in vegetable and animal oils and phytosterol enriched- margarines by transmission mode direct analysis in real time mass spectrometry.

Science.gov (United States)

Alberici, Rosana M; Fernandes, Gabriel D; Porcari, Andréia M; Eberlin, Marcos N; Barrera-Arellano, Daniel; Fernández, Facundo M

2016-11-15

Plant-derived sterols, often referred to as phytosterols, are important constituents of plant membranes where they assist in maintaining phospholipid bilayer stability. Consumption of phytosterols has been suggested to positively affect human health by reducing cholesterol levels in blood via inhibition of its absorption in the small intestine, thus protecting against heart attack and stroke. Sterols are challenging analytes for mass spectrometry, since their low polarity makes them difficult to ionize by both electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), typically requiring derivatization steps to overcome their low ionization efficiencies. We present a fast and reliable method to characterize the composition of phytosterols in vegetable oils and enriched margarines. The method requires no derivatization steps or sample extraction procedures thanks to the use of transmission mode direct analysis in real time mass spectrometry (TM-DART-MS). Copyright © 2016 Elsevier Ltd. All rights reserved.

Sterol transfer between cyclodextrin and membranes: similar but not identical mechanism to NPC2-mediated cholesterol transfer.

Science.gov (United States)

McCauliff, Leslie A; Xu, Zhi; Storch, Judith

2011-08-30

Niemann--Pick C disease is an inherited disorder in which cholesterol and other lipids accumulate in the late endosomal/lysosomal compartment. Recently, cyclodextrins (CD) have been shown to reduce symptoms and extend lifespan in animal models of the disease. In the present studies we examined the mechanism of sterol transport by CD using in vitro model systems and fluorescence spectroscopy and NPC2-deficient fibroblasts. We demonstrate that cholesterol transport from the lysosomal cholesterol-binding protein NPC2 to CD occurs via aqueous diffusional transfer and is very slow; the rate-limiting step appears to be dissociation of cholesterol from NPC2, suggesting that specific interactions between NPC2 and CD do not occur. In contrast, the transfer rate of the fluorescent cholesterol analogue dehydroergosterol (DHE) from CD to phospholipid membranes is very rapid and is directly proportional to the acceptor membrane concentration, as is DHE transfer from membranes to CD. Moreover, CD dramatically increases the rate of sterol transfer between membranes, with rates that can approach those mediated by NPC2. The results suggest that sterol transfer from CD to membranes occurs by a collisional transfer mechanism involving direct interaction of CD with membranes, similar to that shown previously for NPC2. For CD, however, absolute rates are slower compared to NPC2 for a given concentration, and the lysosomal phospholipid lysobisphosphatidic acid (LBPA) does not stimulate rates of sterol transfer between membranes and CD. As expected from the apparent absence of interaction between CD and NPC2, the addition of CD to NPC2-deficient fibroblasts rapidly rescued the cholesterol accumulation phenotype. Thus, the recent observations of CD efficacy in mouse models of NPC disease are likely the result of CD enhancement of cholesterol transport between membranes, with rapid sterol transfer occurring during CD--membrane interactions.

Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

Institute of Scientific and Technical Information of China (English)

无

2001-01-01

Objective　Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. Methods　The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results　 The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. Conclusion　The activity of the NBT-reductase seems to be dependent on the glutathione contents.

A randomized placebo-controlled trial of fluvastatin for prevention of restenosis after successful coronary balloon angioplasty; final results of the fluvastatin angiographic restenosis (FLARE) trial

NARCIS (Netherlands)

J. Shepherd; H. Suryapranata (Harry); P.J. Pfister; P.J. de Feyter (Pim); G.A. van Es (Gerrit Anne); R. Melkert (Rein); G. Jackson (Graham); J.J.R.M. Bonnier (Hans); C.M. Miguel (Carlos); M.C. Vrolix (Mathias); A. Branzi (Angelo); P.W.J.C. Serruys (Patrick); D.P. Foley (David)

1999-01-01

textabstractBACKGROUND: The 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors competitively inhibit biosynthesis of mevalonate, a precursor of non-sterol compounds involved in cell proliferation. Experimental evidence suggests that fluvastatin may, independent of

Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate-based image segmentation

DEFF Research Database (Denmark)

Wüstner, Daniel; Landt Larsen, Ane; Færgeman, Nils J.

2010-01-01

The nematode Caenorhabditis elegans is a genetically tractable model organism to investigate sterol transport. In vivo imaging of the fluorescent sterol, dehydroergosterol (DHE), is challenged by C. elegans' high autofluorescence in the same spectral region as emission of DHE. We present a method....... Bleach-rate constants were determined for DHE in vivo and confirmed in model membranes. Using this method, we could detect enrichment of DHE in specific tissues like the nerve ring, the spermateca and oocytes. We confirm these results in C. elegans gut-granule-loss (glo) mutants with reduced...... homologues of Niemann-Pick C disease proteins. Our approach is generally useful for identifying fluorescent probes in the presence of high cellular autofluorescence....

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

Science.gov (United States)

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

Inoculation of the nonlegume Capsicum annuum L. with Rhizobium strains. 2. Changes in sterols, triterpenes, fatty acids, and volatile compounds.

Science.gov (United States)

Silva, Luís R; Azevedo, Jessica; Pereira, Maria J; Carro, Lorena; Velazquez, Encarna; Peix, Alvaro; Valentão, Patrícia; Andrade, Paula B

2014-01-22

Peppers (Capsicum spp.) are consumed worldwide, imparting flavor, aroma, and color to foods, additionally containing high concentrations of biofunctional compounds. This is the first report about the effect of the inoculation of two Rhizobium strains on sterols, triterpenes, fatty acids, and volatile compounds of leaves and fruits of pepper (Capsicum annuum L.) plants. Generally, inoculation with strain TVP08 led to the major changes, being observed a decrease of sterols and triterpenes and an increase of fatty acids, which are related to higher biomass, growth, and ripening of pepper fruits. The increase of volatile compounds may reflect the elicitation of plant defense after inoculation, since the content on methyl salicylate was significantly increased in inoculated material. The findings suggest that inoculation with Rhizobium strains may be employed to manipulate the content of interesting metabolites in pepper leaves and fruits, increasing potential health benefits and defense abilities of inoculated plants.

Crystallization and diffraction analysis of thioredoxin reductase from Streptomyces coelicolor

International Nuclear Information System (INIS)

Koháryová, Michaela; Brynda, Jiří; Řezáčová, Pavlína; Kollárová, Marta

2011-01-01

Thioredoxin reductase from S. coelicolor was crystallized and diffraction data were collected to 2.4 Å resolution. Thioredoxin reductases are homodimeric flavoenzymes that catalyze the transfer of electrons from NADPH to oxidized thioredoxin substrate. Bacterial thioredoxin reductases represent a promising target for the development of new antibiotics. Recombinant thioredoxin reductase TrxB from Streptomyces coelicolor was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from cryocooled crystals to 2.4 Å resolution using a synchrotron-radiation source. The crystals belonged to the primitive monoclinic space group P2 1 , with unit-cell parameters a = 82.9, b = 60.6, c = 135.4 Å, α = γ = 90.0, β = 96.5°

JS-K, a Nitric Oxide Prodrug, Has Enhanced Cytotoxicity in Colon Cancer Cells with Knockdown of Thioredoxin Reductase 1

Science.gov (United States)

Edes, Kornelia; Cassidy, Pamela; Shami, Paul J.; Moos, Philip J.

2010-01-01

Background The selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted. Methodology The role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured. Conclusions/Significance In cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner. PMID:20098717

Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle

NARCIS (Netherlands)

Vliet, A.K. van; Nègre-Arrariou, P.; Thiel, G.C.F. van; Bolhuis, P.A.; Cohen, L.H.

1996-01-01

Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 ± 6 nM and 4.0 ± 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 ± 38 nM). Through

Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

Science.gov (United States)

Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

2017-05-01

Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression was significantly positively correlated with triglyceride in both polycystic ovary syndrome and endometrial cancer (p = 0.028 and p = 0.027, respectively). Quantitative serum sterol regulatory element

Light Sensitivity of Lactococcus lactis Thioredoxin Reductase

DEFF Research Database (Denmark)

Skjoldager, Nicklas

The thioredoxin system has evolved in all kingdoms of life acting as a key antioxidant system in the defense against oxidative stress. The thioredoxin system utilizes reducing equivalents from NADPH to reduce protein disulfide targets. The reducing equivalents are shuttled via a flavin and redox...... active dithiol motif in thioredoxin reductase (TrxR) to reduce the small ubiquitous thioredoxin (Trx). Trx in turn regulates the protein dithiol/disulfide balance by reduction of protein disulfide targets in e.g. ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductase. The glutathione......, thus expected to rely mainly on the Trx system for thiol-disulfide control. L. lactis is an important industrial microorganism used as starter culture in the dairy production of cheese, buttermilk etc. and known to be sensitive to oxidative stress. The L. lactis TrxR (LlTrxR) is a homodimeric...

A sterol and spiroditerpenoids from a Penicillium sp. isolated from a deep sea sediment sample.

Science.gov (United States)

Li, Yan; Ye, Dezan; Shao, Zongze; Cui, Chengbin; Che, Yongsheng

2012-02-01

A new polyoxygenated sterol, sterolic acid (1), three new breviane spiroditerpenoids, breviones I-K (2-4), and the known breviones (5-8), were isolated from the crude extract of a Penicillium sp. obtained from a deep sea sediment sample that was collected at a depth of 5115 m. The structures of 1-4 were elucidated primarily by NMR experiments, and 1 was further confirmed by X-ray crystallography. The absolute configurations of 2 and 3 were deduced by comparison of their CD spectra with those of the model compounds. Compounds 2 and 5 showed significant cytotoxicity against MCF-7 cells, which is comparable to the positive control cisplatin.

Investigation of oxidation attack sites in sterols: Thermodynamics of hydrogen atom transfer

Czech Academy of Sciences Publication Activity Database

Škorňa, P.; Lengyel, Jozef; Rimarčík, J.; Klein, E.

2014-01-01

Roč. 1038, JUN 2014 (2014), s. 26-32 ISSN 2210-271X R&D Projects: GA ČR(CZ) GA14-27047S Institutional support: RVO:61388955 Keywords : sterol * steroid * oxidation Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.545, year: 2014

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

Science.gov (United States)

Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

2002-03-01

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

Comparison of sterols and fatty acids in two species of Ganoderma

Science.gov (United States)

2012-01-01

Background Two species of Ganoderma, G. sinense and G. lucidum, are used as Lingzhi in China. Howerver, the content of triterpenoids and polysaccharides, main actives compounds, are significant different, though the extracts of both G. lucidum and G. sinense have antitumoral proliferation effect. It is suspected that other compounds contribute to their antitumoral activity. Sterols and fatty acids have obvious bioactivity. Therefore, determination and comparison of sterols and fatty acids is helpful to elucidate the active components of Lingzhi. Results Ergosterol, a specific component of fungal cell membrane, was rich in G. lucidum and G. sinense. But its content in G. lucidum (median content 705.0 μg·g-1, range 189.1-1453.3 μg·g-1, n = 19) was much higher than that in G. sinense (median content 80.1 μg·g-1, range 16.0-409.8 μg·g-1, n = 13). Hierarchical clustering analysis based on the content of ergosterol showed that 32 tested samples of Ganoderma were grouped into two main clusters, G. lucidum and G. sinense. Hierarchical clustering analysis based on the contents of ten fatty acids showed that two species of Ganoderma had no significant difference though two groups were also obtained. The similarity of two species of Ganoderma in fatty acids may be related to their antitumoral proliferation effect. Conclusions The content of ergosterol is much higher in G. lucidum than in G. sinense. Palmitic acid, linoleic acid, oleic acid, stearic acid are main fatty acids in Ganoderma and their content had no significant difference between G. lucidum and G. sinense, which may contribute to their antitumoral proliferation effect. PMID:22293530

The role of biliverdin reductase in colorectal cancer

International Nuclear Information System (INIS)

Bauer, M.

2010-01-01

In recent years, the effects of biliverdin and bilirubin have been studied extensively, and an inhibitory effect of bile pigments in cancer progression has been proposed. In this study we focused on the effects of biliverdin reductase, the enzyme that converts biliverdin to bilirubin, in colorectal cancer. For in vitro experiments we used a human colorectal carcinoma cell line and transfected it with an expression construct of shRNA specific for biliverdin reductase, to create cells with stable knock-down of enzyme expression. Cell proliferation was analyzed using the CASY model TT cell counting device. Western blot protein analysis was performed to study intracellular signaling cascades. Samples of human colorectal cancer were analyzed using immunohistochemistry. We were able to confirm the antiproliferative effects of bile pigments on cancer cells in vitro. However, this effect was attenuated in biliverdin reductase knock down cells. ERK and Akt activation seen under biliverdin and bilirubin treatment was also reduced in biliverdin reductase deficient cells. Immunohistochemical analysis of tumor samples from patients with colorectal cancer showed elevated biliverdin reductase levels. High enzyme expression was associated with lower overall and disease free patient survival. We conclude that BVR is required for bile pigment mediated effects regarding cancer cell proliferation and modulation of intracellular signaling cascades. The role of BVR overexpression in vivo and its exact influence on cancer progression and patient survival need to be further investigated. (author) [de

Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

International Nuclear Information System (INIS)

Hintzpeter, Jan; Seliger, Jan Moritz; Hofman, Jakub; Martin, Hans-Joerg; Wsol, Vladimir; Maser, Edmund

2016-01-01

The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC 50 - and K i -values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin reductases. �

The Nox/Ferric reductase/Ferric reductase-like families of Eumycetes.

Science.gov (United States)

Grissa, Ibtissem; Bidard, Frédérique; Grognet, Pierre; Grossetete, Sandrine; Silar, Philippe

2010-09-01

Reactive Oxygen Species (ROS) are involved in plant biomass degradation by fungi and development of fungal structures. While the ROS-generating NADPH oxidases from filamentous fungi are under strong scrutiny, much less is known about the related integral Membrane (or Ferric) Reductases (IMRs). Here, we present a survey of these enzymes in 29 fungal genomes covering the entire available range of fungal diversity. IMRs are present in all fungal genomes. They can be classified into at least 24 families, underscoring the high diversity of these enzymes. Some are differentially regulated during colony or fruiting body development, as well as by the nature of the carbon source of the growth medium. Importantly, functional characterization of IMRs has been made on proteins belonging to only two families, while nothing or very little is known about the proteins of the other 22 families. Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Purification and properties of a mitochondrial lipoprotein inhibitor of sterol synthesis

International Nuclear Information System (INIS)

Madhosingh, C.; Migicovsky, B.B.; Starratt, A.N.

1976-01-01

A lipoprotein inhibitor of hydroxymethylglutaryl CoA reductase (EC 1.1.1.34) and of cholesterol synthesis by rat liver homogenates, was isolated from the mitochondria of starved rats' livers. The isolated lipoprotein complex contained a low molecular weight protein and fatty acids. The fatty acids identified were arachidonic, linoleic, oleic, stearic and palmitic. The saturated fatty acids and oleic acid did not inhibit. Inhibition of the enzyme was to a large extent related to the degree of fatty acid unsaturation. (auth.)

Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport

DEFF Research Database (Denmark)

Modzel, Maciej; Lund, Frederik W.; Wüstner, Daniel

2017-01-01

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol...... as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal...... fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first...

The effect of a combination of plant sterol-enriched foods in mildly hypercholesterolemic subjects.

Science.gov (United States)

Madsen, Martin B; Jensen, Anne-Mette; Schmidt, Erik B

2007-12-01

The purpose of this study was to evaluate the effect of low-fat products enriched with plant sterols in addition to a National Cholesterol Education Program step 1 diet on serum lipids and lipoproteins. This study was a double-blind, randomised, placebo-controlled cross-over design with a run-in period and 2 intervention periods, each lasting 4 weeks. A total of 46 mildly hypercholesterolemic subjects (age 50.6+/-9.8) completed the trial. The study products consisted of 20 g low-fat margarine (35% fat) and 250 ml low-fat milk (0.7% fat), in total delivering 2.3g plant sterols/d. Serum total and low-density lipoprotein cholesterol were significantly reduced by 5.5% (pUnilever Denmark A/S.

A LONG CHAIN ALCOHOL AND TWO STEROL COMPOUNDS FROM THE HEXANE EXTRACT OF STEM BARK OF Aglaia odorata Lour. (Meliaceae

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Tukiran Tukiran

2010-06-01

Full Text Available A long chain alcohol, 1-eicosanol together with two sterols, β-sitosterol and stigmasterol had been isolated from hexane extract of stem bark of pacar cina (Aglaia odorata Lour (Meliaceae. These structures had been established based on spectroscopic data (IR and NMR and by comparison to those of standard compounds.   Keywords: Aglaia odorata Lour, Alcohol, Meliaceae, Sterol

Kinetics of carbonyl reductase from human brain.

OpenAIRE

Bohren, K M; von Wartburg, J P; Wermuth, B

1987-01-01

Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. D...

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

Science.gov (United States)

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.

Respiratory arsenate reductase as a bidirectional enzyme

Science.gov (United States)

Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

2009-01-01

The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

The Hypoxic Regulator of Sterol Synthesis Nro1 Is a Nuclear Import Adaptor

Energy Technology Data Exchange (ETDEWEB)

T Yeh; C Lee; L Amzel; P Espenshade; M Bianchet

2011-12-31

Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 {angstrom} resolution shows an all-{alpha}-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.

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

Science.gov (United States)

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 liver plays a key role in whole body sterol accretion of the neonatal Golden Syrian hamster

OpenAIRE

Yao, Lihang; Horn, Paul S.; Heubi, James E.; Woollett, Laura A.

2007-01-01

Neonates have a significant requirement for cholesterol. From −1 to 25 days of age, the liver accrues 6.9 mg cholesterol and the extra-hepatic tissues accrue 107.7 mg cholesterol in the hamster. It is currently unknown if each of these body compartments synthesizes their own cholesterol or if they have alternative source(s) of sterol. Using 3H2O, in vivo hepatic sterol synthesis rates (per g liver per animal) increased between −1 and 5 days of age, decreased by 10 days of age, and increased a...

[Aldose reductase gene polymorphism and rate of appearance of retinopathy in non insulin dependent diabetics].

Science.gov (United States)

Olmos, P; Acosta, A M; Schiaffino, R; Díaz, R; Alvarado, D; O'Brien, A; Muñoz, X; Arriagada, P; Claro, J C; Vega, R; Vollrath, V; Velasco, S; Emmerich, M; Maiz, A

1999-04-01

Recent studies suggest that polymorphisms associated to the aldose reductase gene could be related to early retinopathy in noninsulin dependent diabetics (NIDDM). There is also new interest on the genetic modulation of coagulation factors in relation to this complication. To look for a possible relationship between the rate of appearance of retinopathy and the genotype of (AC)n polymorphic marker associated to aldose reductase gene. A random sample of 27 NIDDM, aged 68.1 +/- 10.6 years, with a mean diabetes duration of 20.7 +/- 4.8 years and a mean glycosilated hemoglobin of 10.6 +/- 1.6%, was studied. The genotype of the (AC)n, polymorphic marker associated to the 5' end of the aldose reductase (ALR2) gene was determined by 32P-PCR plus sequenciation. Mutations of the factor XIII-A gene were studied by single stranded conformational polymorphism, sequenciation and restriction fragment length polymorphism. Four patients lacked the (AC)24 and had a higher rate of appearance of retinopathy than patients with the (AC)24 allele (0.0167 and 0.0907 score points per year respectively, p = 0.047). Both groups had similar glycosilated hemoglobin (11.7 +/- 0.2 and 10.5 +/- 1.6% respectively). Factor XIII gene mutations were not related to the rate of appearance of retinopathy. Our data suggest that the absence of the (AC)24 allele of the (AC)n polymorphic marker associated to the 5' end of the aldose reductase gene, is associated to a five fold reduction of retinopathy appearance rate.

Structural complex of sterol 14[alpha]-demethylase (CYP51) with 14[alpha]-methylenecyclopropyl-[delta]7-24, 25-dihydrolanosterol[S

Energy Technology Data Exchange (ETDEWEB)

Hargrove, Tatiana Y.; Wawrzak, Zdzislaw; Liu, Jialin; Waterman, Michael R.; Nes, W. David; Lepesheva, Galina I. (Vanderbilt); (TTU); (NWU)

2012-06-28

Sterol 14{alpha}-demethylase (CYP51) that catalyzes the removal of the 14{alpha}-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14{alpha}-methylenecyclopropyl-{Delta}7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.

Corn fiber oil lowers plasma cholesterol levels and increases cholesterol excretion greater than corn oil and similar to diets containing soy sterols and soy stanols in hamsters.

Science.gov (United States)

Wilson, T A; DeSimone, A P; Romano, C A; Nicolosi, R J

2000-09-01

The aims of this study were to compare the cholesterol-lowering properties of corn fiber oil (CFO) to corn oil (CO), whether the addition of soy stanols or soy sterols to CO at similar levels in CFO would increase CO's cholesterol-lowering properties, and the mechanism(s) of action of these dietary ingredients. Fifty male Golden Syrian hamsters were divided into 5 groups of 10 hamsters each, based on similar plasma total cholesterol (TC) levels. The first group of hamsters was fed a chow-based hypercholesterolemic diet containing either 5% coconut oil + 0.24% cholesterol (coconut oil), 5% CO, 5% CFO, 5% CO + 0.6% soy sterols (sterol), or 5% CO + 0.6% soy stanols (stanol) in place of the coconut oil for 4 weeks. The stanol diet significantly inhibited the elevation of plasma TC compared to all other dietary treatments. Also, the CFO and sterol diets significantly inhibited the elevation of plasma TC compared to the CO and coconut oil diets. The CFO, sterol, and stanol diets significantly inhibited the elevation of plasma non-high density lipoprotein cholesterol compared to the CO and coconut oil diets. The stanol diet significantly inhibited the elevation of plasma high density lipoprotein cholesterol (HDL-C) compared to all other dietary treatments. The sterol diet significantly inhibited the elevation of plasma HDL-C compared to the CO and coconut oil diets, whereas the CFO diet significantly inhibited the elevation of plasma HDL-C compared to the coconut oil diet only. No differences were observed between the CFO and CO for plasma HDL-C. There were no differences observed between groups for plasma triglycerides. The CO and CFO diets had significantly less hepatic TC compared to the coconut oil, sterol, and stanol diets. The CO and CFO diets had significantly less hepatic free cholesterol compared to the sterol and stanol diets but not compared to the coconut oil diet; whereas the coconut oil and sterol diets had significantly less hepatic free cholesterol

Nitrate reductase activity and its relationship with applied nitrogen in soybean

International Nuclear Information System (INIS)

Ge Wenting; Jin Xijun; Ma Chunmei; Dong Shoukun; Gong Zhenping; Zhang Lei

2011-01-01

Field experiments were conducted to study the nitrate reductase activity and its relationship to nitrogen by using frame tests (pot without bottom), sand culture and 15 N-urea at transplanting in soybean variety Suinong 14. Results showed that the activity of nitrate reductase in leaf changed as a signal peak curve with the soybean growth, lower in vegetative growth phase, higher in reproductive growth period and reached the peak in blooming period, then decreased gradually. Nitrogen application showed obvious effect on the nitrate reductase activity. The activities of nitrate reductase in leaves followed the order of N 135 > N 90 > N 45 > N 0 in vegetative growth stage, no clear regularity was found during the whole reproductive growth period. The activities of nitrate reductase in leaves were accorded with the order of upper leaves > mid leaves > lower leaves, and it was very significant differences (P 15 N labeling method during beginning seed stage and full seed stage shown that 15 N abundance in various organs at different node position also followed the same order, suggesting that high level of nitrate reductase activity at upper leaves of soybean promoted the assimilation of NO 3 - . (authors)

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala.

Directory of Open Access Journals (Sweden)

Alberto Guevara-Flores

Full Text Available A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR, thioredoxin-glutathione reductase (TGR, and a putative thioredoxin reductase (TrxR was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

Methyl sterol and cyclopropane fatty acid composition of Methylococcus capsulatus grown at low oxygen tensions

Science.gov (United States)

Jahnke, L. L.; Nichols, P. D.

1986-01-01

The sterol and fatty acid concentrations for M. capsulatus grown in fed-batch cultures over a wide range of oxygen tensions (0.1-10.6 percent) and at a constant methane level are evaluated. The analyses reveal that the biomass decreases as oxygen levels are lowered; the sterol concentration increases when the oxygen range is between 0.5-1.1 percent and decreases when the oxygen range is below 0.5 percent; and the amount of monounsaturated C16 decreases and the concentration of cyclopropane fatty acids increases after oxygen is reduced. It is noted that growth and membrane synthesis occur at low oxygen concentrations and that the synthesis of membrane lipids responds to growth conditions.

RNA-Seq analysis uncovers non-coding small RNA system of Mycobacterium neoaurum in the metabolism of sterols to accumulate steroid intermediates.

Science.gov (United States)

Liu, Min; Zhu, Zhan-Tao; Tao, Xin-Yi; Wang, Feng-Qing; Wei, Dong-Zhi

2016-04-25

Understanding the metabolic mechanism of sterols to produce valuable steroid intermediates in mycobacterium by a noncoding small RNA (sRNA) view is still limited. In the work, RNA-seq was implemented to investigate the noncoding transcriptome of Mycobacterium neoaurum (Mn) in the transformation process of sterols to valuable steroid intermediates, including 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA). A total of 263 sRNA candidates were predicted from the intergenic regions in Mn. Differential expression of sRNA candidates was explored in the wide type Mn with vs without sterol addition, and the steroid intermediate producing Mn strains vs wide type Mn with sterol addition, respectively. Generally, sRNA candidates were differentially expressed in various strains, but there were still some shared candidates with outstandingly upregulated or downregulated expression in these steroid producing strains. Accordingly, four regulatory networks were constructed to reveal the direct and/or indirect interactions between sRNA candidates and their target genes in four groups, including wide type Mn with vs without sterol addition, 9OHAD, ADD, and BNA producing strains vs wide type Mn with sterol addition, respectively. Based on these constructed networks, several highly focused sRNA candidates were discovered to be prevalent in the networks, which showed comprehensive regulatory roles in various cellular processes, including lipid transport and metabolism, amino acid transport and metabolism, signal transduction, cell envelope biosynthesis and ATP synthesis. To explore the functional role of sRNA candidates in Mn cells, we manipulated the overexpression of candidates 131 and 138 in strain Mn-9OHAD, which led to enhanced production of 9OHAD from 1.5- to 2.3-fold during 6 d' fermentation and a slight effect on growth rate. This study revealed the complex and important regulatory

The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.

Science.gov (United States)

Botella, Jose A; Ulschmid, Julia K; Gruenewald, Christoph; Moehle, Christoph; Kretzschmar, Doris; Becker, Katja; Schneuwly, Stephan

2004-05-04

A growing body of evidence suggests that oxidative stress is a common underlying mechanism in the pathogenesis of neurodegenerative disorders such as Alzheimer's, Huntington's, Creutzfeld-Jakob and Parkinson's diseases. Despite the increasing number of reports finding a causal relation between oxidative stress and neurodegeneration, little is known about the genetic elements that confer protection against the deleterious effects of oxidation in neurons. We have isolated and characterized the Drosophila melanogaster gene sniffer, whose function is essential for preventing age-related neurodegeneration. In addition, we demonstrate that oxidative stress is a direct cause of neurodegeneration in the Drosophila central nervous system and that reduction of sniffer activity leads to neuronal cell death. The overexpression of the gene confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness. Sniffer belongs to the family of short-chain dehydrogenase/reductase (SDR) enzymes and exhibits carbonyl reductase activity. This is the first in vivo evidence of the direct and important implication of this enzyme as a neuroprotective agent in the cellular defense mechanisms against oxidative stress.

Effects of plant sterols and olive oil phenols on serum lipoproteins in humans

NARCIS (Netherlands)

Vissers, M.N.

2001-01-01

The studies described in this thesis investigated whether minor components from vegetable oils can improve health by decreasing cholesterol concentrations or oxidative modification of low-density-lipoprotein (LDL) particles.

The plant sterolsβ-sitosterol and sitostanol are

Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

Directory of Open Access Journals (Sweden)

Jolanta Marciniak

2014-01-01

Full Text Available Soluble nitrate reductase from cucumber roots (Cucumis sativus L. was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR from cotyledon roots and leaves.

Enhancement of carotenoid production by disrupting the C22-sterol desaturase gene (CYP61 in Xanthophyllomyces dendrorhous

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Loto Iris

2012-10-01

Full Text Available Abstract Background Xanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, which is a carotenoid with a great biotechnological impact. The ergosterol and carotenoid synthesis pathways are derived from the mevalonate pathway, and in both pathways, cytochrome P450 enzymes are involved. Results In this study, we isolated and described the X. dendrorhous CYP61 gene, which encodes a cytochrome P450 involved in ergosterol biosynthesis. This gene is composed of nine exons and encodes a 526 amino acid polypeptide that shares significant percentages of identity and similitude with the C22-sterol desaturase, CYP61, from other fungi. Mutants derived from different parental strains were obtained by disrupting the CYP61 gene with an antibiotic selection marker. These mutants were not able to produce ergosterol and accumulated ergosta-5,8,22-trien-3-ol and ergosta-5,8-dien-3-ol. Interestingly, all of the mutants had a more intense red color phenotype than their respective parental strains. The carotenoid composition was qualitatively and quantitatively analyzed by RP-HPLC, revealing that the carotenoid content was higher in the mutant strains without major changes in their composition. The expression of the HMGR gene, which encodes an enzyme involved in the mevalonate pathway (3-hydroxy-3-methylglutaryl-CoA reductase, was analyzed by RT-qPCR showing that its transcript levels are higher in the CYP61 mutants. Conclusions These results suggest that in X. dendrorhous, ergosterol regulates HMGR gene expression by a negative feedback mechanism and in this way; it contributes in the regulation of the carotenoid biosynthesis.

Serum plant sterols as surrogate markers of dietary compliance in familial dyslipidemias.

Science.gov (United States)

Mateo-Gallego, Rocío; Baila-Rueda, Lucía; Mouratidou, Theodora; De Castro-Orós, Isabel; Bea, Ana M; Perez-Calahorra, Sofía; Cenarro, Ana; Moreno, Luis A; Civeira, Fernando

2015-06-01

A well-balanced diet is the first-line treatment in hyperlipidemia. The objective was to study the association between serum phytosterols and dietary patterns to use them as surrogate markers of dietary compliance in primary dyslipidemias. 288 patients with primary hyperlipidemias (192 autosomal dominant hypercholesterolemia (ADH) and 96 familial combined hyperlipidemia (FCHL)) were included. Principal factor analysis identified 2 major dietary patterns using a 137-item food frequency questionnaire. "Vegetable & Fruits pattern" was characterized by higher intake of fruits, green beans, nuts, tomatoes, roasted or boiled potatoes, lettuce and chard and lower of processed baked goods, pizza and beer. "Western pattern" was positively characterized by hamburgers, pasta, sunflower oil, rice, chickpeas, whole milk, veal, red beans and negatively with white fish. Serum non-cholesterol sterols were determined by HPLC-MS/MS. Plant sterols to-total cholesterol (TC) levels were lower with a higher adherence to a "Vegetable & Fruits pattern" (P = 0.009), mainly in ADH subjects (R(2) = 0.019). Their concentration was greater with higher compliance to "Western pattern" especially in FCHL (P = 0.014). Higher levels of synthesis markers-to-TC with a greater adherence to "Vegetable & Fruits pattern" were found (P = 0.001) (R(2) = 0.033 and R(2) = 0.109 in ADH and FCHL respectively). In subjects with primary dislipidemia, dietary patterns associate with serum absorption and synthesis markers, but no with lipid concentrations. The influence of diet on non-cholesterol sterols levels is not powerful enough to use them as subrogate markers. Copyright © 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Determination of Main Plant Sterols in Turkish Bread Wheat (Triticum aestivum L. by GC-MS

Directory of Open Access Journals (Sweden)

Halil Erdem

2017-07-01

Full Text Available Plant sterols are belong to triterpenes family of natural products which includes more than 200 different types of plant sterols and more than 4000 other types of triterpenes. The optimization of method, specially the derivatization step as well as the corresponding analytical validation, is the main goal of this study. The optimum temperature, time and reagent volume of derivatization step were obtained at 60°C, 60 minutes and 50 µL, respectively. A rapid and sensitive gas chromatographic–mass spectrometric method was developed and validated for quantitative analysis of the most common plant sterols (β-sitosterol, campesterol and stigmasterol in 20 Turkish bread wheat cultivars using GC-MS-SIM. Separation of β-cholestanol (I.S, campesterol, stigmasterol and β-sitosterol was achieved on Rxi (5Sil MS column (60 m×0.25 mm. The limits of detection for β-sitosterol, campesterol and stigmasterol were 0.074, 0.054 and 0.064 mg kg-1, respectively with RSD ≤ 0.66%. The obtained concentrations of campesterol, stigmasterol and β-sitosterol from 20 Turkish bread wheat cultivars ranged from: 15.30 to 76.02, 4.27 to 23.23 and 303.21 to 682.66 mg kg-1, respectively.

Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa.

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Lijun Wang

Full Text Available Proanthocyanidins (PAs contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR are two key enzymes of the PA biosynthesis that produce the main subunits: (+-catechin and (--epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05 in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.

Lead nitrate-induced development of hypercholesterolemia in rats: sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis.

Science.gov (United States)

Kojima, Misaki; Masui, Toshimitsu; Nemoto, Kiyomitsu; Degawa, Masakuni

2004-12-01

Changes in the gene expressions of hepatic enzymes responsible for cholesterol homeostasis were examined during the process of lead nitrate (LN)-induced development of hypercholesterolemia in male rats. Total cholesterol levels in the liver and serum were significantly increased at 3-72 h and 12-72 h, respectively, after LN-treatment (100 micromol/kg, i.v.). Despite the development of hypercholesterolemia, the genes for hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and other enzymes (FPPS, farnesyl diphosphate synthase; SQS, squalene synthase; CYP51, lanosterol 14alpha-demethylase) responsible for cholesterol biosynthesis were activated at 3-24 h and 12-18 h, respectively. On the other hand, the gene expression of cholesterol 7alpha-hydroxylase (CYP7A1), a catabolic enzyme of cholesterol, was remarkably suppressed at 3-72 h. The gene expression levels of cytokines interleukin-1beta (IL-1beta) and TNF-alpha, which activate the HMGR gene and suppress the CYP7A1 gene, were significantly increased at 1-3 h and 3-24 h, respectively. Furthermore, gene activation of SREBP-2, a gene activator of several cholesterogenic enzymes, occurred before the gene activations of FPPS, SQS and CYP51. This is the first report demonstrating sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis in LN-treated male rats. The mechanisms for the altered-gene expressions of hepatic enzymes in LN-treated rats are discussed.

Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

Energy Technology Data Exchange (ETDEWEB)

Hintzpeter, Jan, E-mail: hintzpeter@toxi.uni-kiel.de [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Seliger, Jan Moritz [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Hofman, Jakub [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Martin, Hans-Joerg [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Wsol, Vladimir [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Maser, Edmund [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany)

2016-02-15

The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC{sub 50}- and K{sub i}-values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin

Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean.

Science.gov (United States)

Cheng, Qun; Li, Ninghui; Dong, Lidong; Zhang, Dayong; Fan, Sujie; Jiang, Liangyu; Wang, Xin; Xu, Pengfei; Zhang, Shuzhen

2015-01-01

Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.

Insig proteins mediate feedback inhibition of cholesterol synthesis in the intestine.

Science.gov (United States)

McFarlane, Matthew R; Liang, Guosheng; Engelking, Luke J

2014-01-24

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.

Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine*

Science.gov (United States)

McFarlane, Matthew R.; Liang, Guosheng; Engelking, Luke J.

2014-01-01

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes. PMID:24337570

Determining Antifungal Target Sites in the Sterol Pathway of the Yeast Candida and Saccharomyces

National Research Council Canada - National Science Library

Bard, Martin

1998-01-01

... as in topical infections which lead to significant losses in work-place productivity. The work reported here seeks to identify new target sites in the sterol biosynthetic pathway against which new antifungal compounds might be developed...

Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice.

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Sara H Windahl

Full Text Available Androgens are important regulators of bone mass but the relative importance of testosterone (T versus dihydrotestosterone (DHT for the activation of the androgen receptor (AR in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2, encoded by separate genes (Srd5a1 and Srd5a2. 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1⁻/⁻ mice. Four-month-old male Srd5a1⁻/⁻ mice had reduced trabecular bone mineral density (-36%, p<0.05 and cortical bone mineral content (-15%, p<0.05 but unchanged serum androgen levels compared with wild type (WT mice. The cortical bone dimensions were reduced in the male Srd5a1⁻/⁻ mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05 in orchidectomized WT mice but not in orchidectomized Srd5a1⁻/⁻ mice. Male Srd5a1⁻/⁻ mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05. Female Srd5a1⁻/⁻ mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1⁻/⁻ mice, is an indirect effect mediated by elevated circulating androgen levels.

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

DEFF Research Database (Denmark)

Wüstner, Daniel; Faergeman, Nils J

2008-01-01

proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other...

Studies on the Utilization, Metabolism and Function of Sterols in the House-Fly, Musca Domestica; Utilisation. Metabolisme et fonctions des sterols chez la mouche domestique (Musca Domestica); Izuchenie usvoeniya, metabolizma i funktsii sterinov v organizme domashnej mukhi Musca Domestica; Estudios sobre la asimilacion, el metabolismo y la funcion de los esteroles in la mosca comun (Musca Domestica)

Energy Technology Data Exchange (ETDEWEB)

Robbins, W. E. [United States Department of Agriculture, Agricultural Research Service, Entomology Research Division, Beltsville, MD (United States)

1963-09-15

Insects generally have been found to require a dietary source of sterol for normal larval growth and metamorphosis. Our work has pointed to two additional physiological roles for sterols in the housefly, Musca domestica L.: (1) A dietary source of sterol is essential for sustained viable egg production in the female fly; on a sterol-deficient diet eggs are produced but hatch and viability are low. (2) Cholesterol is also involved in the mobilization and utilization of nutrient reserves associated with the initiation of ovarian maturation in the female fly. The quantitative sterol requirements for the above physiological processes and the metabolic conversions that occur during growth, metamorphosis and reproduction have been studied in this insect, using C{sup 14}- and H{sup 3}-labelled sterols in conjunction with a variety of analytical tools, including reverse isotope dilution, gasliquid chromatography and spectroscopy, and employing aseptic rearing techniques and semi-defined larval and Adult diets. Both C{sup 14}-cholesterol and H{sup 3}-{beta}- sitosterol have been used as a source df sterol in either the larval or the adult diet of the house fly, and the pattern of utilization and metabolism was found to be almost identical for these two sterols. However, there was no detectable conversion of {beta}-sitosterol to cholesterol. Sub-minimal quantities of cholesterol have also been used in the larval diet in combination with ''sparing sterols'' such as choies tanol, which will fulfill in part but not entirely the sterol requirement of this insect. The utilization and fate of the 'sparing sterol' has been investigated using C{sup 14} cholestanol, and the metabolism of the minute quantity of essential cholesterol is currently under study using high-specific-activity C{sup 14} cholesterol. - Other species of insects, including the German cockroach (Blattella germanica), have been examined in relation to the patterns of utilization and the metabolic pathways for

Fatty acid and sterol contents during tulip leaf senescence induced by methyl jasmonate

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Marian Saniewski

2013-12-01

Full Text Available It has been shown previously that methyl jasmonate (JA-Me applied in lanolin paste on the bottom surface of intact tulip leaves causes a rapid and intense its senescence. The aim of this work was to study the effect of JA-Me on free and bound fatty acid and sterol contents during tulip leaf senescence. The main free and bound fatty acids of tulip leaf, in decreasing order of their abundance, were linolenic, linoleic, palmitic, oleic, stearic and myristic acids. Only the content of free linolenic acid decreased after treatment with JA-Me during visible stage of senescence. ß-Sitosterol (highest concentration, campesterol, stigmasterol and cholesterol were identified in tulip leaf. Methyl jasmonate evidently increased the level of ß-sitosterol, campesterol and stigmasterol during induced senescence. It is suggested that the increase in sterol concentrations under the influence of methyl jasmonate induced changes in membrane fluidity and permeability, which may be responsible for senescence.

Association between methylenetetrahydrofolate reductase (MTHFR ...

African Journals Online (AJOL)

Association between methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and risk of ischemic stroke in North Indian population: A hospital based case–control study. Amit Kumar, Shubham Misra, Anjali Hazarika, Pradeep Kumar, Ram Sagar, Abhishek Pathak, Kamalesh Chakravarty, Kameshwar ...

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

Science.gov (United States)

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.

Cholesterol-producing transgenic Caenorhabditis elegans lives longer due to newly acquired enhanced stress resistance

International Nuclear Information System (INIS)

Lee, Eun-Young; Shim, Yhong-Hee; Chitwood, David J.; Hwang, Soon Baek; Lee, Junho; Paik, Young-Ki

2005-01-01

Because Caenorhabditis elegans lacks several components of the de novo sterol biosynthetic pathway, it requires sterol as an essential nutrient. Supplemented cholesterol undergoes extensive enzymatic modification in C. elegans to form other sterols of unknown function. 7-Dehydrocholesterol reductase (DHCR) catalyzes the reduction of the Δ 7 double bond of sterols and is suspected to be defective in C. elegans, in which the major endogenous sterol is 7-dehydrocholesterol (7DHC). We microinjected a human DHCR expression vector into C. elegans, which was then incorporated into chromosome by γ-radiation. This transgenic C. elegans was named cholegans, i.e., cholesterol-producing C. elegans, because it was able to convert 7DHC into cholesterol. We investigated the effects of changes in sterol composition on longevity and stress resistance by examining brood size, mean life span, UV resistance, and thermotolerance. Cholegans contained 80% more cholesterol than the wild-type control. The brood size of cholegans was reduced by 40% compared to the wild-type control, although the growth rate was not significantly changed. The mean life span of cholegans was increased up to 131% in sterol-deficient medium as compared to wild-type. The biochemical basis for life span extension of cholegans appears to partly result from its acquired resistance against both UV irradiation and thermal stress

Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

DEFF Research Database (Denmark)

Björnberg, Olof; Viennet, Thibault; Skjoldager, Nicklas

2015-01-01

Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two...... enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli Trx......-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass...

An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway.

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Kevin A Robertson

2016-03-01

Full Text Available In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1. Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway.

An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway

Science.gov (United States)

Robertson, Kevin A.; Hsieh, Wei Yuan; Forster, Thorsten; Blanc, Mathieu; Lu, Hongjin; Crick, Peter J.; Yutuc, Eylan; Watterson, Steven; Martin, Kimberly; Griffiths, Samantha J.; Enright, Anton J.; Yamamoto, Mami; Pradeepa, Madapura M.; Lennox, Kimberly A.; Behlke, Mark A.; Talbot, Simon; Haas, Jürgen; Dölken, Lars; Griffiths, William J.; Wang, Yuqin; Angulo, Ana; Ghazal, Peter

2016-01-01

In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway. PMID:26938778

The effect of ionic and non-ionic surfactants on the growth, nitrate reductase and nitrite reductase activities of Spirodela polyrrhiza (L. Schleiden

Directory of Open Access Journals (Sweden)

Józef Buczek

2014-01-01

Full Text Available Inclusion into the medium of 5 mg•dm-3 of non-ionic (ENF or ionic (DBST surfactant caused 50-60% inhibition of nitrite reductase MR activity in S. polyrrhiza. At the same time, increased accumulation of NO2- in the plant tissues and lowering of the total and soluble protein contents were found. DBST also lowered the nitrate reductase (NR activity and the dry mass of the plants.

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

Energy Technology Data Exchange (ETDEWEB)

Rosenthal, Cindy [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Mueller, Uwe [Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung mbH, Albert-Einstein-Strasse 15, D-12489 Berlin (Germany); Panjikar, Santosh [European Molecular Biology Laboratory Hamburg, Outstation Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Sun, Lianli [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Ruppert, Martin [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Zhao, Yu [Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Stöckigt, Joachim [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China)

2006-12-01

Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222{sub 1} and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

International Nuclear Information System (INIS)

Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

2006-01-01

Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222 1 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å

A Cytotoxic Hydroperoxy Sterol from the Brown Alga, Nizamuddinia Zanardinii

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Abdolhossein Rustaiyan

2013-03-01

Full Text Available Background:The marine environment is a unique source of bioactive natural products, of which Nizamuddinia zanardinii is an important brown algae distributed in Oman Sea. Literature revealed that there is no report on phytochemistry and pharmacology of this valuable algae.Methods:Bioguided fractionation of the methanolic extract of Nizamuddinia zanardinii, collected from Oman Sea, led to the isolation of a hydroperoxy sterol. Its structure was determined by analysis of the spectroscopic data as 24-hydroperoxy-24-vinyl cholesterol (HVC. In vitro cytotoxic activity of this compound was evaluated against HT29, MCF7, A549, HepG2 and MDBK cell lines.Results:Although 24(R-hydroproxy-24-vinylcholesterol has been previously reported from Sargassum and Padina species, it is the first report on the presence of this compound from N. zanardinii. This compound exhibited cytotoxicity in all cell lines (IC50, 3.62, 9.09, 17.96, 32.31 and 37.31 μg/mL respectively. HVC was also evaluated for apoptotic activity and demonstrated positive results in terminal deoxynucleotidyl transferase dUTP Nick End labeling (TUNEL assay suggesting it a candidate for further apoptotic studies.Conclusions:Nizamuddinia zanardinii, a remarkable brown algae of Oman Sea, is a good source of hydroproxy sterols with promising cytotoxic on various cell lines particularly human colon adenocarcinoma.

Inhibition of steroid 5 alpha-reductase by specific aliphatic unsaturated fatty acids.

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Liang, T; Liao, S

1992-01-01

Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells. PMID:1637346

Conformational analysis of 9β,19-cyclopropyl sterols: Detection of the pseudoplanar conformer by nuclear Overhauser effects and its functional implications

International Nuclear Information System (INIS)

Nes, W.D.; Benson, M.; Lundin, R.E.; Le, P.H.

1988-01-01

Nuclear Overhauser difference spectroscopy and variable temperature studies of the 9β,19-cyclopropyl sterols 24,25-dehydropollinastanol (4,4-desmethyl-5α-cycloart-24-en-3β-ol) and cyclolaudenol [(24S)-24-methyl-5α-cycloart-25(27)-en-3β-ol] have shown the solution conformation of the B/C rings to be twist-chair/twist-boat rather than boat/chair as suggested in the literature. This is very similar to the known crystal structure conformation of 9β,19-cyclopropyl sterols. The effect of these conformations on the molecular shape is highly significant; the first conformation orients into a pseudoplanar or flat shape analogous to lanosterol, whereas the latter conformation exhibits a bent shape. The results are interpreted to imply that, for conformational reasons, cyclopropyl sterols can be expected to maintain the pseudoplanar shape in membrane bilayers

Genome sequence analysis of predicted polyprenol reductase gene from mangrove plant kandelia obovata

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Basyuni, M.; Sagami, H.; Baba, S.; Oku, H.

2018-03-01

It has been previously reported that dolichols but not polyprenols were predominated in mangrove leaves and roots. Therefore, the occurrence of larger amounts of dolichol in leaves of mangrove plants implies that polyprenol reductase is responsible for the conversion of polyprenol to dolichol may be active in mangrove leaves. Here we report the early assessment of probably polyprenol reductase gene from genome sequence of mangrove plant Kandelia obovata. The functional assignment of the gene was based on a homology search of the sequences against the non-redundant (nr) peptide database of NCBI using Blastx. The degree of sequence identity between DNA sequence and known polyprenol reductase was confirmed using the Blastx probability E-value, total score, and identity. The genome sequence data resulted in three partial sequences, termed c23157 (700 bp), c23901 (960 bp), and c24171 (531 bp). The c23157 gene showed the highest similarity (61%) to predicted polyprenol reductase 2- like from Gossypium raimondii with E-value 2e-100. The second gene was c23901 to exhibit high similarity (78%) to the steroid 5-alpha-reductase Det2 from J. curcas with E-value 2e-140. Furthermore, the c24171 gene depicted highest similarity (79%) to the polyprenol reductase 2 isoform X1 from Jatropha curcas with E- value 7e-21.The present study suggested that the c23157, c23901, and c24171, genes may encode predicted polyprenol reductase. The c23157, c23901, c24171 are therefore the new type of predicted polyprenol reductase from K. obovata.

Shotgun lipidomic analysis of chemically sulfated sterols compromises analytical sensitivity

DEFF Research Database (Denmark)

Casanovas, Albert; Hannibal-Bach, Hans Kristian; Jensen, Ole Nørregaard

2014-01-01

Shotgun lipidomics affords comprehensive and quantitative analysis of lipid species in cells and tissues at high-throughput [1 5]. The methodology is based on direct infusion of lipid extracts by electrospray ionization (ESI) combined with tandem mass spectrometry (MS/MS) and/or high resolution F...... low ionization efficiency in ESI [7]. For this reason, chemical derivatization procedures including acetylation [8] or sulfation [9] are commonly implemented to facilitate ionization, detection and quantification of sterols for global lipidome analysis [1-3, 10]....

Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

DEFF Research Database (Denmark)

Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

1992-01-01

Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression. ...

Sterols from Hericium erinaceum and their inhibition of TNF-α and NO production in lipopolysaccharide-induced RAW 264.7 cells.

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Li, Wei; Zhou, Wei; Cha, Ji Yun; Kwon, Se Uk; Baek, Kwang-Hyun; Shim, Sang Hee; Lee, Young Mi; Kim, Young Ho

2015-07-01

Erinarols G-J and 10 known ergostane-type sterols were isolated from a methanol extract of the dried fruiting bodies of Hericium erinaceum. Their chemical structures were elucidated using extensive spectroscopic analyses including 1D and 2D NMR experiments and HR-ESI-MS analysis, as well as through comparison with previously reported data. Anti-inflammatory effects of the isolated compounds were evaluated in terms of inhibition of tumor necrosis factor α (TNF-α) and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cells. The results showed that erinarols H and J, as well as 2 of the ergostane-type sterols exhibited inhibitory activity against TNF-α secretion, with inhibition values ranging from 33.7% to 43.3% at 10 μM. Erinarols J and three ergostane-type sterols exhibited significant inhibitory effects against NO production, with inhibition values ranging from 38.4% to 71.5% at 10 μM. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bioinformatics analysis of the predicted polyprenol reductase genes in higher plants

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Basyuni, M.; Wati, R.

2018-03-01

The present study evaluates the bioinformatics methods to analyze twenty-four predicted polyprenol reductase genes from higher plants on GenBank as well as predicted the structure, composition, similarity, subcellular localization, and phylogenetic. The physicochemical properties of plant polyprenol showed diversity among the observed genes. The percentage of the secondary structure of plant polyprenol genes followed the ratio order of α helix > random coil > extended chain structure. The values of chloroplast but not signal peptide were too low, indicated that few chloroplast transit peptide in plant polyprenol reductase genes. The possibility of the potential transit peptide showed variation among the plant polyprenol reductase, suggested the importance of understanding the variety of peptide components of plant polyprenol genes. To clarify this finding, a phylogenetic tree was drawn. The phylogenetic tree shows several branches in the tree, suggested that plant polyprenol reductase genes grouped into divergent clusters in the tree.

Serum sterol responses to increasing plant sterol intake from natural foods in the Mediterranean diet.

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Escurriol, Verónica; Cofán, Montserrat; Serra, Mercè; Bulló, Mónica; Basora, Josep; Salas-Salvadó, Jordi; Corella, Dolores; Zazpe, Itziar; Martínez-González, Miguel A; Ruiz-Gutiérrez, Valentina; Estruch, Ramón; Ros, Emilio

2009-09-01

Phytosterols in natural foods are thought to inhibit cholesterol absorption. The Mediterranean diet is rich in phytosterol-containing plant foods. To assess whether increasing phytosterol intake from natural foods was associated with a cholesterol-lowering effect in a substudy of a randomized trial of nutritional intervention with Mediterranean diets for primary cardiovascular prevention (PREDIMED study). One hundred and six high cardiovascular risk subjects assigned to two Mediterranean diets supplemented with virgin olive oil (VOO) or nuts, which are phytosterol-rich foods, or advice on a low-fat diet. Outcomes were 1-year changes in nutrient intake and serum levels of lipids and non-cholesterol sterols. Average phytosterol intake increased by 76, 158 and 15 mg/day in participants assigned VOO, nuts and low-fat diets, respectively. Compared to participants in the low-fat diet group, changes in outcome variables were observed only in those in the Mediterranean diet with nuts group, with increases in intake of fibre, polyunsaturated fatty acids and phytosterols (P natural foods appear to be bioactive in cholesterol lowering.

X-ray crystal structure of GarR-tartronate semialdehyde reductase from Salmonella typhimurium.

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Osipiuk, J; Zhou, M; Moy, S; Collart, F; Joachimiak, A

2009-09-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related beta-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 A resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.

Effect of sterol metabolism in the yeast-Drosophila system on the frequency of radiation-induced aneuploidy in the Drosophila melanogaster oocytes

International Nuclear Information System (INIS)

Savitskii, V.V.; Luchnikova, E.M.; Inge-Vechtomov, S.G.

1986-01-01

The effect of sterol metabolism on induced mutagenesis of Drosophila melanogaster was studied in the ecogenetic system of yeast-Drosophila. Sterol deficiency was created in Drosophila by using the biomass of live cells of Saccharomyces cerevisiae strain 9-2-P712 till mutation in locus nys/sup r1/ blocking the synthesis of ergosterol as the food. It was found that rearing of Drosophila females on the mutant yeast increases the frequency of loss and nondisjunction of X chromosomes induced in mature oocytes by X rays (1000 R). Addition of 0.1% of cholesterol solution in 10% ethanol to the yeast biomass restores the resistance of oocyte to X irradiation to the control level. The possible hormonal effect on membrane leading to increased radiation-induced aneuploidy in Drosophila and the role of sterol metabolism in determining the resistance to various damaging factors are discussed

Synthesis of steryl ferulates with various sterol structures and comparison of their antioxidant activity

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Steryl ferulates extracted from corn and rice differ in the structures of the phytosterol head groups, which had a significant impact on their activity as antioxidants in soybean oil used for frying. An improved method was used to synthesize steryl ferulates from commercial sterols to better underst...

Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species.

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Poonnachit, U; Darnell, R

2004-04-01

Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.

Antioxidant and Anti-Osteoporotic Activities of Aromatic Compounds and Sterols from Hericium erinaceum.

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Li, Wei; Lee, Sang Hyun; Jang, Hae Dong; Ma, Jin Yeul; Kim, Young Ho

2017-01-11

Hericium erinaceum , commonly called lion's mane mushroom, is a traditional edible mushroom widely used in culinary applications and herbal medicines in East Asian countries. In this study, a new sterol, cerevisterol 6-cinnamate ( 6 ), was isolated from the fruiting bodies of H. erinaceum together with five aromatic compounds 1 - 5 and five sterols 7 - 11 . The chemical structures of these compounds were elucidated using chemical and physical methods and comparison of HRESIMS, ¹D-NMR (¹H, 13 C, and DEPT) and 2D-NMR (COSY, HMQC, HMBC, and NOESY) spectra with previously reported data. The antioxidant and anti-osteoporotic activities of extracts and the isolated compounds 1 - 11 were investigated. All compounds exhibited peroxyl radical-scavenging capacity but only compounds 1 , 3 , and 4 showed potent reducing capacity. Moreover, compounds 1 , 2 , 4 , and 5 showed moderate effects on cellular antioxidant activity and inhibited the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastic differentiation. These results suggested that H. erinaceum could be utilized in the development of natural antioxidant and anti-osteoporotic nutraceuticals and functional foods.

The antibacterial efficacy of an aceraceous plant [Shantung maple (Acer truncatum Bunge)] may be related to inhibition of bacterial beta-oxoacyl-acyl carrier protein reductase (FabG).

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Zhang, Feng; Luo, Shi-Yun; Ye, Yan-Bin; Zhao, Wen-Hua; Sun, Xu-Guang; Wang, Zhi-Qun; Li, Ran; Sun, Ying-Hui; Tian, Wei-Xi; Zhang, Ying-Xia

2008-10-01

Polyphenols, including flavonoids, are the major components of the extracts from aceraceous plants. They possess remarkable antibacterial and antitumour activity. Our study focused on whether the inhibition of the bacterial type II fatty acid synthesis system is the mechanism for the antibacterial effect of the related plant polyphenols. Extracts obtained from the fallen leaves of the Shantung maple (Acer truncatum Bunge) using different solvents, and the related pure compound PGG (1,2,3,4,6-penta-O-galloyl-beta-D-glucose), potently inhibited the FabG (beta-oxoacyl-ACP reductase) steps in the fatty-acid-elongation cycle with the IC(50) values between 0.9 and 7.2 microg/ml. An ethyl acetate extract appeared to inhibit FabG reductase in a mixed manner with NADPH, as did PGG with NADPH, demonstrating that they interfered with the binding of the cofactor to the enzyme. Gram-positive and Gram-negative bacteria and some fungi were used to evaluate the antibacterial abilities of different extract samples. The experiments showed that a higher polyphenol content of the extracts led to a more potent inhibitory capacity against FabG, thus enhancing the antibacterial efficacy.

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

DEFF Research Database (Denmark)

Wustner, D.; Solanko, L.; Sokol, Olena

2011-01-01

Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehythoergosterol (DHE), a well-established marker for cholesterol, by introducing simultan......Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehythoergosterol (DHE), a well-established marker for cholesterol, by introducing...... and followed a stretched exponential decay, while the fluorescence lifetime of BCh2 was comparable in various cellular regions. Our results indicate that BCh2 is suitable for analyzing sterol uptake pathways and inter-organelle sterol flux in living cells. The BODIPY-moiety affects lipid phase preference...

Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

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Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

2004-04-01

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

Identification of 5α-reductase isoenzymes in canine skin.

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Bernardi de Souza, Lucilene; Paradis, Manon; Zamberlam, Gustavo; Benoit-Biancamano, Marie-Odile; Price, Christopher

2015-10-01

Alopecia X in dogs is a noninflammatory alopecia that may be caused by a hormonal dysfunction. It may be similar to androgenic alopecia in men that is caused by the effect of dihydrotestosterone (DHT). The 5α-reductase isoenzymes, 5αR1 and 5αR2, and a recently described 5αR3, are responsible for the conversion of testosterone into DHT. However, which 5α-reductases are present in canine skin has not yet been described. The main objective of this study was to determine the pattern of expression of 5α-reductase genes in canine skin. Skin biopsies were obtained from healthy, intact young-mature beagles (three males, four females) at three anatomical sites normally affected by alopecia X (dorsal neck, back of thighs and base of tail) and two sites generally unaffected (dorsal head and ventral thorax). Prostate samples (n = 3) were collected as positive controls for 5α-reductase mRNA abundance measurement by real-time PCR. We detected mRNA encoding 5αR1 and 5αR3 but not 5αR2. There were no significant differences in 5αR1 and 5αR3 mRNA levels between the different anatomical sites, irrespective of gender (P > 0.05). Moreover, the mean mRNA abundance in each anatomical site did not differ between males and females (P > 0.05). To the best of the authors' knowledge, this is the first study demonstrating the expression of 5α-reductases in canine skin and the expression of 5αR3 in this tissue. These results may help to elucidate the pathogenesis of alopecia X and to determine more appropriate treatments for this disorder. © 2015 ESVD and ACVD.

Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

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Plancarte, Agustin; Nava, Gabriela

2015-02-01

Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase

Gamma-irradiation activates biochemical systems: induction of nitrate reductase activity in plant callus.

OpenAIRE

Pandey, K N; Sabharwal, P S

1982-01-01

Gamma-irradiation induced high levels of nitrate reductase activity (NADH:nitrate oxidoreductase, EC 1.6.6.1) in callus of Haworthia mirabilis Haworth. Subcultures of gamma-irradiated tissues showed autonomous growth on minimal medium. We were able to mimic the effects of gamma-irradiation by inducing nitrate reductase activity in unirradiated callus with exogenous auxin and kinetin. These results revealed that induction of nitrate reductase activity by gamma-irradiation is mediated through i...

X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

Science.gov (United States)

Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.

2009-01-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme. PMID:19184529

Measurement of hepatic sterol synthesis in the Mongolian gerbil in vivo using [3H]water: diurnal variation and effect of type of dietary fat

International Nuclear Information System (INIS)

Mercer, N.J.; Holub, B.J.

1981-01-01

The hepatic synthesis of sterol was measured in the male Mongolian gerbil (Meriones unguiculatus) in vivo following the administration of [ 3 H]water by monitoring the incorporation of radioactivity into digitonin-precipitable sterol. A diurnal rhythm in cholesterol synthesis was exhibited under conditions of ad libitum feeding with alternating 12-hour periods of light (0200 to 1400 hr) and dark (1400 to 0200 hr). The zenith was reached between 1500 and 2100 hr and the nadir approximately 10-12 hours later between 0200 and 0400 hr, which provided a zenith/nadir ratio of 9.6 to 1.0. The in vivo rates of hepatic sterol synthesis and plasma cholesterol levels were measured in gerbils fed semi-purified diets containing either 19.5% beef tallow + 0.5% safflower, 20% lard, or 20% safflower oil and widely differing ratios of polyunsaturated: saturated fatty acids. All diets were equalized to contain 0.01% cholesterol and 0.05% plant sterol. After 3 days on the experimental diets, the mean rates of cholesterol synthesis (nmol/g liver per hr) were 41.5, 26.6, and 13.8 for animals fed the diets containing beef tallow, lard, and safflower oil, respectively. After 7 and 14 days, synthetic rates were lowest in the gerbils fed safflower oil as were also the plasma cholesterol levels. These results indicate that the type of dietary lipid can significantly influence the in vivo rate of sterol biosynthesis in gerbil liver. This response may contribute, at least in part, to the observed differences in plasma cholesterol levels

Synthesis of Molecularly Imprinted Polymer for Sterol Separation

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Yuangsawad Ratanaporn

2016-01-01

Full Text Available Molecular imprinted polymer (MIP was prepared by bulk polymerization in acetone using acrylamide as a functional monomer, ethylene glycol dimethacrylate as a crosslinker, stigmasterol as a template and benzoyl peroxide as an initiator. The obtained MIPs were characterized using a scanning electron microscope and a fourier transform infrared spectrophotometer. Performance in sterol adsorption of MIPs prepared under various conditions was investigated using a model solution of phytosterols in heptane, comparing with a nonimprinted polymer (NIP. Statistical analysis revealed that the amounts of crosslinker and template strongly affected the performance of MIP while the amount of solvent slightly affected the performance of MIP. MIP synthesized under the optimal condition had adsorption capacity of 1.28 mgsterols/gads which were 1.13 times of NIP.

Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

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Yeon Bok Kim

2014-01-01

Full Text Available Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

Comparative modelling and molecular docking of nitrate reductase from Bacillus weihenstephanensis (DS45

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R. Seenivasagan

2016-07-01

Full Text Available Nitrate reductase catalyses the oxidation of NAD(PH and the reduction of nitrate to nitrite. NR serves as a central point for the integration of metabolic pathways by governing the flux of reduced nitrogen through several regulatory mechanisms in plants, algae and fungi. Bacteria express nitrate reductases that convert nitrate to nitrite, but mammals lack these specific enzymes. The microbial nitrate reductase reduces toxic compounds to nontoxic compounds with the help of NAD(PH. In the present study, our results revealed that Bacillus weihenstephanensis expresses a nitrate reductase enzyme, which was made to generate the 3D structure of the enzyme. Six different modelling servers, namely Phyre2, RaptorX, M4T Server, HHpred, SWISS MODEL and Mod Web, were used for comparative modelling of the structure. The model was validated with standard parameters (PROCHECK and Verify 3D. This study will be useful in the functional characterization of the nitrate reductase enzyme and its docking with nitrate molecules, as well as for use with autodocking.

Proper Sterol Distribution Is Required for Candida albicans Hyphal Formation and Virulence

Science.gov (United States)

McCourt, Paula; Liu, Hsing-Yin; Parker, Josie E.; Gallo-Ebert, Christina; Donigan, Melissa; Bata, Adam; Giordano, Caroline; Kelly, Steven L.; Nickels, Joseph T.

2016-01-01

Candida albicans is an opportunistic fungus responsible for the majority of systemic fungal infections. Multiple factors contribute to C. albicans pathogenicity. C. albicans strains lacking CaArv1 are avirulent. Arv1 has a conserved Arv1 homology domain (AHD) that has a zinc-binding domain containing two cysteine clusters. Here, we explored the role of the CaAHD and zinc-binding motif in CaArv1-dependent virulence. Overall, we found that the CaAHD was necessary but not sufficient for cells to be virulent, whereas the zinc-binding domain was essential, as Caarv1/Caarv1 cells expressing the full-length zinc-binding domain mutants, Caarv1C3S and Caarv1C28S, were avirulent. Phenotypically, we found a direct correlation between the avirulence of Caarv1/Caarv1, Caarrv1AHD, Caarv1C3S, and Caarv1C28S cells and defects in bud site selection, septa formation and localization, and hyphal formation and elongation. Importantly, all avirulent mutant strains lacked the ability to maintain proper sterol distribution. Overall, our results have established the importance of the AHD and zinc-binding domain in fungal invasion, and have correlated an avirulent phenotype with the inability to maintain proper sterol distribution. PMID:27587298

Proper Sterol Distribution Is Required for Candida albicans Hyphal Formation and Virulence

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Paula McCourt

2016-11-01

Full Text Available Candida albicans is an opportunistic fungus responsible for the majority of systemic fungal infections. Multiple factors contribute to C. albicans pathogenicity. C. albicans strains lacking CaArv1 are avirulent. Arv1 has a conserved Arv1 homology domain (AHD that has a zinc-binding domain containing two cysteine clusters. Here, we explored the role of the CaAHD and zinc-binding motif in CaArv1-dependent virulence. Overall, we found that the CaAHD was necessary but not sufficient for cells to be virulent, whereas the zinc-binding domain was essential, as Caarv1/Caarv1 cells expressing the full-length zinc-binding domain mutants, Caarv1C3S and Caarv1C28S, were avirulent. Phenotypically, we found a direct correlation between the avirulence of Caarv1/Caarv1, Caarrv1AHD, Caarv1C3S, and Caarv1C28S cells and defects in bud site selection, septa formation and localization, and hyphal formation and elongation. Importantly, all avirulent mutant strains lacked the ability to maintain proper sterol distribution. Overall, our results have established the importance of the AHD and zinc-binding domain in fungal invasion, and have correlated an avirulent phenotype with the inability to maintain proper sterol distribution.

A comparison of accelerated solvent extraction, Soxhlet extraction, and ultrasonic-assisted extraction for analysis of terpenoids and sterols in tobacco.

Science.gov (United States)

Shen, Jinchao; Shao, Xueguang

2005-11-01

The performance of accelerated solvent extraction in the analysis of terpenoids and sterols in tobacco samples was investigated and compared with those of Soxhlet extraction and ultrasonically assisted extraction with respect to yield, extraction time, reproducibility and solvent consumption. The results indicate that although the highest yield was achieved by Soxhlet extraction, ASE appears to be a promising alternative to classical methods since it is faster and uses less solvent, especially when applied to the investigation of large batch tobacco samples. However, Soxhlet extraction is still the preferred method for analyzing sterols since it gives a higher extraction efficiency than other methods.

Glutathione reductase: solvent equilibrium and kinetic isotope effects

International Nuclear Information System (INIS)

Wong, K.K.; Vanoni, M.A.; Blanchard, J.S.

1988-01-01

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D 2 O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456

Antioxidant and Anti-Osteoporotic Activities of Aromatic Compounds and Sterols from Hericium erinaceum

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Wei Li

2017-01-01

Full Text Available Hericium erinaceum, commonly called lion’s mane mushroom, is a traditional edible mushroom widely used in culinary applications and herbal medicines in East Asian countries. In this study, a new sterol, cerevisterol 6-cinnamate (6, was isolated from the fruiting bodies of H. erinaceum together with five aromatic compounds 1–5 and five sterols 7–11. The chemical structures of these compounds were elucidated using chemical and physical methods and comparison of HRESIMS, 1D-NMR (1H, 13C, and DEPT and 2D-NMR (COSY, HMQC, HMBC, and NOESY spectra with previously reported data. The antioxidant and anti-osteoporotic activities of extracts and the isolated compounds 1–11 were investigated. All compounds exhibited peroxyl radical-scavenging capacity but only compounds 1, 3, and 4 showed potent reducing capacity. Moreover, compounds 1, 2, 4, and 5 showed moderate effects on cellular antioxidant activity and inhibited the receptor activator of nuclear factor κB ligand (RANKL-induced osteoclastic differentiation. These results suggested that H. erinaceum could be utilized in the development of natural antioxidant and anti-osteoporotic nutraceuticals and functional foods.

Measurement of rates of cholesterol synthesis using tritiated water

International Nuclear Information System (INIS)

Dietschy, J.M.; Spady, D.K.

1984-01-01

Rates of sterol synthesis in various tissues commonly are assessed by assaying levels of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase on isolated microsomes or by measuring the rates of incorporation of various 14 C-labeled substrates or [ 3 H]water into cholesterol by whole cell preparations in vitro or by the tissues of the whole animal in vivo. While measurement of activities of HMG-CoA reductase or rates of incorporation of 14 C-labeled substrates into cholesterol give useful relative rates of sterol production, neither method yields absolute rates of cholesterol synthesis. The use of [ 3 H]water circumvents the problem of variable and unknown dilution of the specific activity of the precursor pool encountered when 14 C-labeled substrates are used and does yield absolute rates of cholesterol synthesis provided that the 3 H/C incorporation ratio is known for a particular tissue. In 12 different experimental situations it has been found that from 21 to 27 micrograms atoms of 3 H are incorporated into cholesterol from [ 3 H]water in different tissues of several animal species, so that the 3 H/C incorporation ratio is similar under nearly all experimental conditions and varies from 0.78 to 1.00. When administered in vivo, [ 3 H]water rapidly equilibrates with intracellular water and is incorporated into sterols within the various organs at rates that are linear with respect to time. From such data it is possible to obtain absolute rates of cholesterol synthesis in the whole animal and in the various organs of the animal. Current data suggest, therefore, that use of [ 3 H]water yields the most accurate rates of cholesterol synthesis both in vitro and in vivo

The Biological Activity of alpha-Mangostin, a Larvicidal Botanic Mosquito Sterol Carrier Protein-2 Inhibitor

Science.gov (United States)

2010-01-01

it is known that esterase aids in the detoxiÞcation of or- ganophosphates ( Hemingway and Ransom 2000). In- terestingly, we found that -mangostin...Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism. J. Biol. Chem. 276: 48058Ð48065. Hemingway

Effects of sterol regulatory element-binding protein (SREBP in chickens

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Alipour Fahimeh

2012-02-01

Full Text Available Abstract Sterol regulatory element binding protein- 1 and -2 (SREBP-1 and -2 are key transcription factors involved in the biosynthesis of cholesterol and fatty acids. The SREBP have mostly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, though, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. Since a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we review Size and Tissue expression Pattern of SREBP and role of this protein in chickens.

The origin of fetal sterols in second-trimester amniotic fluid : endogenous synthesis or maternal-fetal transport?

NARCIS (Netherlands)

Baardman, Maria E.; Erwich, Jan Jaap H. M.; Berger, Rolf M. F.; Hofstra, Robert M. W.; Kerstjens-Frederikse, Wilhelmina S.; Luetjohann, Dieter; Plosch, Torsten; Lutjohann, D.

OBJECTIVE: Cholesterol is crucial for fetal development. To gain more insight into the origin of the fetal cholesterol pool in early human pregnancy, we determined cholesterol and its precursors in the amniotic fluid of uncomplicated, singleton human pregnancies. STUDY DESIGN: Total sterols were

Substrate and cofactor binding to nitrile reductase : A mass spectrometry based study

NARCIS (Netherlands)

Gjonaj, L.; Pinkse, M.W.H.; Fernandez Fueyo, E.; Hollmann, F.; Hanefeld, U.

2016-01-01

Nitrile reductases catalyse a two-step reduction of nitriles to amines. This requires the binding of two NADPH molecules during one catalytic cycle. For the nitrile reductase from E. coli (EcoNR) mass spectrometry studies of the catalytic mechanism were performed. EcoNR is dimeric and has no Rossman

Forward genetic screening for regulators involved in cholesterol synthesis using validation-based insertional mutagenesis.

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Wei Jiang

Full Text Available Somatic cell genetics is a powerful approach for unraveling the regulatory mechanism of cholesterol metabolism. However, it is difficult to identify the mutant gene(s due to cells are usually mutagenized chemically or physically. To identify important genes controlling cholesterol biosynthesis, an unbiased forward genetics approach named validation-based insertional mutagenesis (VBIM system was used to isolate and characterize the 25-hydroxycholesterol (25-HC-resistant and SR-12813-resistant mutants. Here we report that five mutant cell lines were isolated. Among which, four sterol-resistant mutants either contain a truncated NH2-terminal domain of sterol regulatory element-binding protein (SREBP-2 terminating at amino acids (aa 400, or harbor an overexpressed SREBP cleavage-activating protein (SCAP. Besides, one SR-12813 resistant mutant was identified to contain a truncated COOH-terminal catalytic domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase. This study demonstrates that the VBIM system can be a powerful tool to screen novel regulatory genes in cholesterol biosynthesis.

Astragalus polysaccharides lowers plasma cholesterol through mechanisms distinct from statins.

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Yunjiu Cheng

Full Text Available To determine the efficacy and underlying mechanism of Astragalus polysaccharides (APS on plasma lipids in hypercholesterolemia hamsters. The effect of APS (0.25 g/kg/d on plasma and liver lipids, fecal bile acids and neutral sterol, cholesterol absorption and synthesis, HMG-CoA reductase activity, and gene and protein expressions in the liver and small intestine was investigated in twenty-four hypercholesterolemia hamsters. Treatment periods lasted for three months. APS significantly lowered plasma total cholesterol by 45.8%, triglycerides by 30%, and low-density lipoprotein-cholesterol by 47.4%, comparable to simvastatin. Further examinations revealed that APS reduced total cholesterol and triglycerides in the liver, increased fecal bile acid and neutral sterol excretion, inhibited cholesterol absorption, and by contrast, increased hepatic cholesterol synthesis and HMG-CoA reductase activity. Plasma total cholesterol or low-density lipoprotein-cholesterol levels were significantly correlated with cholesterol absorption rates. APS up-regulated cholesterol-7α-hydroxylase and LDL-receptor gene expressions. These new findings identify APS as a potential natural cholesterol lowering agent, working through mechanisms distinct from statins.

EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of a health claim related to 3 g/day plant sterols/stanols and lowering blood LDL-cholesterol and reduced risk of (coronary) heart disease pursuant to Article 19 of Regulation (EC) No 1924/2006

DEFF Research Database (Denmark)

Tetens, Inge

/2010 (yellow fat spreads, dairy products, mayonnaise and salad dressings) have a similar efficacy on blood LDL-cholesterol lowering, that plant sterols and stanol esters at a daily intake of 3 g (range 2.6 g to 3.4 g) plant sterols/stanols in matrices approved by Regulation (EC) No 376/2010 lower LDL...

Cell-free transfer of sterols by plant fractions

International Nuclear Information System (INIS)

Morre, D.J.; Wilkinson, F.E.; Morre, D.M.; Moreau, P.; Sandelius, A.S.; Penel, C.; Greppin, H.

1990-01-01

Microsomes from etiolated hypocotyls of soybean or leaves of light-grown spinach radiolabeled in vivo with [ 3 H]acetate or in vitro with [ 3 H]squalene or [ 3 H]cholesterol as donor transferred radioactivity to unlabeled acceptor membranes immobilized on nitrocellulose. Most efficient transfer was with plasma membrane or tonoplast as the acceptor. The latter were highly purified by aqueous two-phase partition (plasma membrane) and preparative free-flow electrophoresis (tonoplast and plasma membrane). Plasma membrane- and tonoplast-free microsomes and purified mitochondria were less efficient acceptors. Sterol transfer was verified by thin-layer chromatography of extracted lipids. Transfer was time- and temperature-dependent, required ATP but was not promoted by cytosol. The nature of the donor (endoplasmic reticulum, Golgi apparatus or both) and of the transfer mechanism is under investigation

Trace analysis of selected hormones and sterols in river sediments by liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Science.gov (United States)

Matić, Ivana; Grujić, Svetlana; Jauković, Zorica; Laušević, Mila

2014-10-17

In this paper, development and optimization of new LC-MS method for determination of twenty selected hormones, human/animal and plant sterols in river sediments were described. Sediment samples were prepared using ultrasonic extraction and clean up with silica gel/anhydrous sodium sulphate cartridge. Extracts were analyzed by liquid chromatography-linear ion trap-tandem mass spectrometry, with atmospheric pressure chemical ionization. The optimized extraction parameters were extraction solvent (methanol), weight of the sediment (2 g) and time of ultrasonic extraction (3× 10 min). Successful chromatographic separation of hormones (estriol and estrone, 17α- and 17β-estradiol) and four human/animal sterols (epicoprostanol, coprostanol, α-cholestanol and β-cholestanol) that have identical fragmentation reactions was achieved. The developed and optimized method provided high recoveries (73-118%), low limits of detection (0.8-18 ng g(-1)) and quantification (2.5-60 ng g(-1)) with the RSDs generally lower than 20%. Applicability of the developed method was confirmed by analysis of six river sediment samples. A widespread occurrence of human/animal and plant sterols was found. The only detected hormone was mestranol in just one sediment sample. Copyright © 2014 Elsevier B.V. All rights reserved.

N-terminus determines activity and specificity of styrene monooxygenase reductases.

Science.gov (United States)

Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

2017-12-01

Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

OpenAIRE

de Saint-Jean, Maud; Delfosse, Vanessa; Douguet, Dominique; Chicanne, Gaetan; Payrastre, Bernard; Bourguet, William; Antonny, Bruno; Drin, Guillaume

2011-01-01

Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Os...

Prevalence of methylenetetrahydrofolate reductase ( MTHFR ) and ...

African Journals Online (AJOL)

Methylenetetrahydrofolate reductase (MTHFR) and Cytosolic serine hydroxymethyltransferase (cSHMT) are enzymes involve in folate regulation in human. The C to T transition of the cSHMT and MTHFR genes at the 1420 as well as 677 nucleotides both carries TT genotype respectively. These enzymes have direct and ...

Determination of fatty acid, tocopherol and phyto sterol contents of the oils of various poppy (Papaver somniferum L.) seeds.

Energy Technology Data Exchange (ETDEWEB)

Enric, H.; Tekin, A.; Musa Ozcan, M.

2009-07-01

The fatty acid, tocopherol and sterol contents of the oils of several poppy seeds were investigated. The main fatty acids in poppy seed oils were linoleic (687.6-739.2 g kg{sup -}1), oleic (141.3-192.8 g kg{sup -}1) and palmitic (76.8-92.8 g kg{sup -}1). The oils contained an appreciable amount of {gamma}-tocopherol (195.37-280.85 mg kg{sup -}1), with a mean value of 261.31 mg kg-1 and {alpha}-tocopherol (21.99-45.83 mg kg{sup -}1), with a mean value of 33.03 mg kg{sup -}1. The concentrations of total sterol ranged from 1099.84 mg kg{sup -}1 (K.pembe) to 4816.10 mg kg-1 (2. sinif beyaz), with a mean value of 2916.20 mg kg{sup -}1. The major sterols were {beta}-sitosterol, ranging from 663.91 to 3244.39 mg kg{sup -}1; campesterol, ranging from 228.59 to 736.50 mg kg{sup -}1; and {delta}{sup 5}-avenasterol, ranging from 103.90 to 425.02 mg kg{sup -}1. The studied varieties of poppy seeds from Turkey were found to be a potential source of valuable oil. (Author) 31 refs.

Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase.

Science.gov (United States)

Gandin, Valentina; Fernandes, Aristi Potamitou; Rigobello, Maria Pia; Dani, Barbara; Sorrentino, Francesca; Tisato, Francesco; Björnstedt, Mikael; Bindoli, Alberto; Sturaro, Alberto; Rella, Rocco; Marzano, Cristina

2010-01-15

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, 'auranofin-like' gold(I) complexes all containing the [Au(PEt(3))](+) synthon and the ligands: Cl(-), Br(-), cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.

Methemoglobin reductase activity in intact fish red blood cells

DEFF Research Database (Denmark)

Jensen, Frank B; Nielsen, Karsten

2018-01-01

RBCs in physiological saline at normal Pco2 and pH. After initial loading of oxygenated RBCs with nitrite (partly oxidizing Hb to metHb), the nitrite is removed by three washes of the RBCs in nitrite-free physiological saline to enable the detection of RBC metHb reductase activity in the absence......Hb reductase activity in fish offsets their higher Hb autoxidation and higher likelihood of encountering elevated nitrite. Deoxygenation significantly raised the rates of RBC metHb reduction, and more so in rainbow trout than in carp. The temperature sensitivity of metHb reduction in rainbow trout RBCs...

HMG-CoA reductase inhibitory activity and phytocomponent investigation of Basella alba leaf extract as a treatment for hypercholesterolemia

Directory of Open Access Journals (Sweden)

Baskaran G

2015-01-01

Full Text Available Gunasekaran Baskaran,1 Shamala Salvamani,1 Siti Aqlima Ahmad,1 Noor Azmi Shaharuddin,1 Parveen Devi Pattiram,2 Mohd Yunus Shukor1 1Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, 2Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, Selangor, Malaysia Abstract: The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl, 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and a-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases. Keywords: HMG-CoA reductase, Basella alba, phytochemical, GC-MS/MS, RP-HPLC, hypercholesterolemia

Molecular mechanisms of drug resistance and tumor promotion involving mammalian ribonucleotide reductase

Energy Technology Data Exchange (ETDEWEB)

Choy, B.B.K.

1991-01-01

Mammalian ribonucleotide reductase is a highly regulated, rate-limiting activity responsible for converting ribonucleoside diphosphates to the deoxyribonucleotide precursors of DNA. The enzyme consists of two nonidentical proteins called M1 and M2, both of which are required for activity. Hydroxyurea is an antitumor agent which inhibits ribonucleotide reductase by interacting with the M2 component specifically at a unique tyrosyl free radical. Studies were conducted on a series of drug resistant mouse cell lines, selected by a step-wise procedure for increasing levels of resistance to the cytotoxic effects of hydroxyurea. Each successive drug selection step leading to the isolation of highly resistant cells was accompanied by stable elevations in cellular resistance and ribonucleotide reductase activity. The drug resistant cell lines exhibited gene amplification of the M2 gene, elevated M2 mRNA, and M2 protein. In addition to M2 gene amplification, posttranscriptional modulation also occurred during the drug selection. Studies of the biosynthesis rates with exogenously added iron suggest a role for iron in regulating the level of M2 protein when cells are cultured in the presence of hydroxyurea. The hydroxyurea-inactivated ribonucleotide reductase protein M2 has a destabilized iron centre, which readily releases iron. Altered expression of ferritin appears to be required for the development of hydroxyurea resistance in nammalian cells. The results show an interesting relationship between the expressions of ribonucleotide reductase and ferritin. The phorbol ester tumor promoter, TPA, is also able to alter the expression of M2. TPA was able to induce M2 mRNA levels transiently up to 18-fold within 1/2 hour. This rapid and large elevation of ribonucleotide reductase suggests that the enzyme may play a role in tumor promotion. Studies of the M2 promoter region were undertaken to better understand the mechanism of TPA induction of M2.

Identification of Multiple Soluble Fe(III Reductases in Gram-Positive Thermophilic Bacterium Thermoanaerobacter indiensis BSB-33

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Subrata Pal

2014-01-01

Full Text Available Thermoanaerobacter indiensis BSB-33 has been earlier shown to reduce Fe(III and Cr(VI anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI reduction activity in both the membrane and cytoplasm. The soluble fraction prepared from T. indiensis cells grown at 60°C was found to contain the majority of Fe(III reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS with the help of T. indiensis proteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs, thioredoxin reductase (Trx, NADP(H-nitrite reductase (Ntr, and thioredoxin disulfide reductase (Tdr were determined to be responsible for Fe(III reductase activity. Amino acid sequence and three-dimensional (3D structural similarity analyses of the T. indiensis Fe(III reductases were carried out with Cr(VI reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI reducing dihydrolipoamide dehydrogenase from Thermus scotoductus and thioredoxin disulfide reductase from Desulfovibrio desulfuricans. It appears that in addition to their iron reducing activity T. indiensis LPDs and Tdr are possibly involved in Cr(VI reduction as well.

Sterols and fatty acids analysis at the Llobregat River for a wastewater dumping episode; Analisis de esteroles y acidos grasos en el rio Llobrgat por un vertido de aguas residuales

Energy Technology Data Exchange (ETDEWEB)

Romero-Lacal, J.; Garcia-Mendi, C.; Vega, A.; Pujadas, M.

2006-07-01

The study by gas chromatography (HRGC) and mass spectrometry (MS) of fatty acids and sterols of water samples from Anoia and Llobregat River, allowed to rule out the hypothesis of the Anoia River as the cause of dumping episode and even to determine that the episode was not associated to the faecal or residual contamination. Nevertheless, it has permitted us to have a better knowledge of these families of organic compounds in the basin of the Llobregat River. In the acidic fraction, saturated and unsaturated fatty acids of 14, 16 and 18 atoms of carbon from oil fish, animal fat or hydrogenated oils with industrial and domestic use were found. The application of different sterols indices (coprostanol, cholesterol, cholestanol, stigmasterol, stigmastadienol and sitosterol) permitted us to study the role of the sterols and the significance of the contamination from animal sterols, fitosterols in the studied samples. (Author) 19 refs.

Pregna-5,17(20)-dien-21-oyl amides affecting sterol and triglyceride biosynthesis in Hep G2 cells.

Science.gov (United States)

Stulov, Sergey V; Mankevich, Olga V; Dugin, Nikita O; Novikov, Roman A; Timofeev, Vladimir P; Misharin, Alexander Yu

2013-04-01

Synthesis of series [17(20)Z]- and [17(20)E]-pregna-5,17(20)-dien-21-oyl amides, containing polar substituents in amide moiety, based on rearrangement of 17α-bromo-21-iodo-3β-acetoxypregn-5-en-20-one caused by amines, is presented. The titled compounds were evaluated for their potency to regulate sterol and triglyceride biosynthesis in human hepatoma Hep G2 cells in comparison with 25-hydroxycholesterol. Three [17(20)E]-pregna-5,17(20)-dien-21-oyl amides at a concentrations of 5 μM inhibited sterol biosynthesis and stimulated triglyceride biosynthesis; their regulatory potency was dependent on the structure of amide moiety; the isomeric [17(20)Z]-pregna-5,17(20)-dien-21-oyl amides were inactive. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synthesis, Spectroscopic and Theoretical Studies of New Quaternary N,N-Dimethyl-3-phthalimidopropylammonium Conjugates of Sterols and Bile Acids

Directory of Open Access Journals (Sweden)

Bogumil Brycki

2014-04-01

Full Text Available New quaternary 3-phthalimidopropylammonium conjugates of steroids were obtained by reaction of sterols (ergosterol, cholesterol, cholestanol and bile acids (lithocholic, deoxycholic, cholic with bromoacetic acid bromide to give sterol 3β-bromoacetates and bile acid 3α-bromoacetates, respectively. These intermediates were subjected to nuclephilic substitution with N,N-dimethyl-3-phthalimidopropylamine to give the final quaternary ammonium salts. The structures of products were confirmed by spectral (1H-NMR, 13C-NMR, and FT-IR analysis, mass spectrometry (ESI-MS, MALDI as well as PM5 semiempirical methods and B3LYP ab initio methods. Estimation of the pharmacotherapeutic potential has been accomplished for synthesized compounds on the basis of Prediction of Activity Spectra for Substances (PASS.

Streptococcus sanguinis Class Ib Ribonucleotide Reductase

Science.gov (United States)

Makhlynets, Olga; Boal, Amie K.; Rhodes, DeLacy V.; Kitten, Todd; Rosenzweig, Amy C.; Stubbe, JoAnne

2014-01-01

Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (MnIII2-Y•) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with FeII and O2 can self-assemble a diferric-tyrosyl radical (FeIII2-Y•) cofactor (1.2 Y•/β2) and with the help of NrdI can assemble a MnIII2-Y• cofactor (0.9 Y•/β2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and MnII2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μm) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR. PMID:24381172

In silico docking studies of aldose reductase inhibitory activity of commercially available flavonoids

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Arumugam Madeswaran

2012-12-01

Full Text Available The primary objective of this study was to investigate the aldose reductase inhibitory activity of flavonoids using in silico docking studies. In this perspective, flavonoids like biochanin, butein, esculatin, fisetin and herbacetin were selected. Epalrestat, a known aldose reductase inhibitor was used as the standard. In silico docking studies were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. The results showed that all the selected flavonoids showed binding energy ranging between -9.33 kcal/mol to -7.23 kcal/mol when compared with that of the standard (-8.73 kcal/mol. Inhibition constant (144.13 µM to 4.98 µM and intermolecular energy (-11.42 kcal/mol to -7.83 kcal/mol of the flavonoids also coincide with the binding energy. All the selected flavonoids contributed aldose reductase inhibitory activity because of its structural properties. These molecular docking analyses could lead to the further development of potent aldose reductase inhibitors for the treatment of diabetes.

Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

Energy Technology Data Exchange (ETDEWEB)

Kiyota, Eduardo [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Sousa, Sylvia Morais de [Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Santos, Marcelo Leite dos; Costa Lima, Aline da [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Menossi, Marcelo [Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Campinas-SP (Brazil); Yunes, José Andrés [Laboratório de Biologia Molecular, Centro Infantil Boldrini, Campinas-SP (Brazil); Aparicio, Ricardo, E-mail: aparicio@iqm.unicamp.br [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil)

2007-11-01

Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR.

Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

International Nuclear Information System (INIS)

Kiyota, Eduardo; Sousa, Sylvia Morais de; Santos, Marcelo Leite dos; Costa Lima, Aline da; Menossi, Marcelo; Yunes, José Andrés; Aparicio, Ricardo

2007-01-01

Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR

Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

Science.gov (United States)

Chen, Wei; Tuladhar, Anupama; Rolle, Shantelle; Lai, Yanhao; Rodriguez Del Rey, Freddy; Zavala, Cristian E; Liu, Yuan; Rein, Kathleen S

2017-08-15

Karenia brevis, the Florida red tide dinoflagellate produces a suite of neurotoxins known as the brevetoxins. The most abundant of the brevetoxins PbTx-2, was found to inhibit the thioredoxin-thioredoxin reductase system, whereas the PbTx-3 has no effect on this system. On the other hand, PbTx-2 activates the reduction of small disulfides such as 5,5'-dithio-bis-(2-nitrobenzoic acid) by thioredoxin reductase. PbTx-2 has an α, β-unsaturated aldehyde moiety which functions as an efficient electrophile and selenocysteine conjugates are readily formed. PbTx-2 blocks the inhibition of TrxR by the inhibitor curcumin, whereas curcumin blocks PbTx-2 activation of TrxR. It is proposed that the mechanism of inhibition of thioredoxin reduction is via the formation of a Michael adduct between selenocysteine and the α, β-unsaturated aldehyde moiety of PbTx-2. PbTx-2 had no effect on the rates of reactions catalyzed by related enzymes such as glutathione reductase, glutathione peroxidase or glutaredoxin. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibitory effects of various oxygenated sterols on the differentiation and function of tumor-specific cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Spangrude, G.J.; Sherris, D.; Daynes, R.A.

1982-01-01

Irradiation of skin with ultraviolet light (UVL) is capable of causing many biological and biochemical changes in this complex organ. One early consequence is the oxidation of epidermal plasma membrane cholesterol, causing the induction of a wide variety of photoproducts. It is well recognized that some oxygenated sterols possess potent biological activity on mammalian cells by their ability to inhibit endogeneous mevalonate and cholesterol biosynthesis. In the few immunological systems that have been studied, there is general agreement that lymphocyte function is lacking, as both afferent and efferent blockades have been suggested. These studies were undertaken to determine the effect of various oxygenated sterols (representing a number of known cholesterol-derived photoproducts) on the generation (afferent) and function (efferent) of cytotoxic T lymphocytes (CTLs). Cell-mediated immune responses which result in the generation of both alloantigen-specific and syngeneic tumor-specific CTLs were evaluated

Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies.

Science.gov (United States)

Moureaux, T; Leydecker, M T; Meyer, C

1989-02-15

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

Science.gov (United States)

Kaye, C; Crawford, N M; Malmberg, R L

1997-04-01

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

Overexpression of chloroplast NADPH-dependent thioredoxin reductase in Arabidopsis enhances leaf growth and elucidates in-vivo function of reductase and thioredoxin domains

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Jouni eToivola

2013-10-01

Full Text Available Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC contains both reductase (NTRd and thioredoxin (TRXd domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive thioredoxin domain, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modelling of the 3-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protected pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for

Sterol-derived hormone(s controls entry into diapause in Caenorhabditis elegans by consecutive activation of DAF-12 and DAF-16.

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Vitali Matyash

2004-10-01

Full Text Available Upon starvation or overcrowding, Caenorhabditis elegans interrupts its reproductive cycle and forms a specialised larva called dauer (enduring. This process is regulated by TGF-beta and insulin-signalling pathways and is connected with the control of life span through the insulin pathway components DAF-2 and DAF-16. We found that replacing cholesterol with its methylated metabolite lophenol induced worms to form dauer larvae in the presence of food and low population density. Our data indicate that methylated sterols do not actively induce the dauer formation but rather that the reproductive growth requires a cholesterol-derived hormone that cannot be produced from methylated sterols. Using the effect of lophenol on growth, we have partially purified activity, named gamravali, which promotes the reproduction. In addition, the effect of lophenol allowed us to determine the role of sterols during dauer larva formation and longevity. In the absence of gamravali, the nuclear hormone receptor DAF-12 is activated and thereby initiates the dauer formation program. Active DAF-12 triggers in neurons the nuclear import of DAF-16, a forkhead domain transcription factor that contributes to dauer differentiation. This hormonal control of DAF-16 activation is, however, independent of insulin signalling and has no influence on life span.

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome.

Science.gov (United States)

Balajthy, András; Somodi, Sándor; Pethő, Zoltán; Péter, Mária; Varga, Zoltán; Szabó, Gabriella P; Paragh, György; Vígh, László; Panyi, György; Hajdu, Péter

2016-08-01

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

A novel fibrosis index comprising a non-cholesterol sterol accurately predicts HCV-related liver cirrhosis.

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Magdalena Ydreborg

Full Text Available Diagnosis of liver cirrhosis is essential in the management of chronic hepatitis C virus (HCV infection. Liver biopsy is invasive and thus entails a risk of complications as well as a potential risk of sampling error. Therefore, non-invasive diagnostic tools are preferential. The aim of the present study was to create a model for accurate prediction of liver cirrhosis based on patient characteristics and biomarkers of liver fibrosis, including a panel of non-cholesterol sterols reflecting cholesterol synthesis and absorption and secretion. We evaluated variables with potential predictive significance for liver fibrosis in 278 patients originally included in a multicenter phase III treatment trial for chronic HCV infection. A stepwise multivariate logistic model selection was performed with liver cirrhosis, defined as Ishak fibrosis stage 5-6, as the outcome variable. A new index, referred to as Nordic Liver Index (NoLI in the paper, was based on the model: Log-odds (predicting cirrhosis = -12.17+ (age × 0.11 + (BMI (kg/m(2 × 0.23 + (D7-lathosterol (μg/100 mg cholesterol×(-0.013 + (Platelet count (x10(9/L × (-0.018 + (Prothrombin-INR × 3.69. The area under the ROC curve (AUROC for prediction of cirrhosis was 0.91 (95% CI 0.86-0.96. The index was validated in a separate cohort of 83 patients and the AUROC for this cohort was similar (0.90; 95% CI: 0.82-0.98. In conclusion, the new index may complement other methods in diagnosing cirrhosis in patients with chronic HCV infection.

The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

DEFF Research Database (Denmark)

Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

2017-01-01

The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...

Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase

DEFF Research Database (Denmark)

Montalvetti, A; Pena Diaz, Javier; Hurtado, R

2000-01-01

In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from L...

Methylenetetrahydrofolate reductase gene polymorphism in type 1 ...

African Journals Online (AJOL)

In patients with type-I diabetes mellitus folate deficiency is associated with endothelial dysfunction. So, polymorphism in genes involved in folate metabolism may have a role in vascular disease. This study was designed to evaluate the relationship between methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

Characterization of human warfarin reductase

OpenAIRE

Sokolová, Simona

2016-01-01

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Simona Sokolová Supervisor: PharmDr. Petra Malátková, Ph.D. Title of diploma thesis: Characterization of human warfarin reductase Warfarin is widely used anticoagulant drug. Considering the narrow therapeutic window of warfarin, it is important to fully understand its metabolism in human body. Oxidative, reductive and conjugation reactions are involved in warfarin metabolism. Howev...

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

International Nuclear Information System (INIS)

Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2014-01-01

Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

Energy Technology Data Exchange (ETDEWEB)

Hou, Feng; Miyakawa, Takuya [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kataoka, Michihiko [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Takeshita, Daijiro [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kumashiro, Shoko [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Uzura, Atsuko [Research and Development Center, Nagase and Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241 (Japan); Urano, Nobuyuki [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Nagata, Koji [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shimizu, Sakayu [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

2014-04-18

Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

Heterogeneous expression of cholesterol 7α-hydroxylase and sterol 27- hydroxylase genes in the rat liver lobulus

NARCIS (Netherlands)

Twisk, J.; Hoekman, M.F.M.; Mager, W.H.; Moorman, A.F.M.; Boer, P.A.J. de; Scheja, L.; Princen, H.M.G.; Gebhardt, R.

1995-01-01

We investigated the lobular localization and molecular level of expression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene

Inhibitory effect of rhetsinine isolated from Evodia rutaecarpa on aldose reductase activity.

Science.gov (United States)

Kato, A; Yasuko, H; Goto, H; Hollinshead, J; Nash, R J; Adachi, I

2009-03-01

Aldose reductase inhibitors have considerable potential for the treatment of diabetic complications, without increased risk of hypoglycemia. Search for components inhibiting aldose reductase led to the discovery of active compounds contained in Evodia rutaecarpa Bentham (Rutaceae), which is the one of the component of Kampo-herbal medicine. The hot water extract from the E. rutaecarpa was subjected to distribution or gel filtration chromatography to give an active compound, N2-(2-methylaminobenzoyl)tetrahydro-1H-pyrido[3,4-b]indol-1-one (rhetsinine). It inhibited aldose reductase with IC(50) values of 24.1 microM. Furthermore, rhetsinine inhibited sorbitol accumulation by 79.3% at 100 microM. These results suggested that the E. rutaecarpa derived component, rhetsinine, would be potentially useful in the treatment of diabetic complications.

Phytosterol stearate esters elicit similar responses on plasma lipids and cholesterol absorption but different responses on fecal neutral sterol excretion and hepatic free cholesterol in male Syrian hamsters.

Science.gov (United States)

Ash, Mark M; Hang, Jiliang; Dussault, Patrick H; Carr, Timothy P

2011-07-01

The dietary impact of specific phytosterols incorporated into phytosterol fatty acid esters has not been elucidated. Therefore, we tested the hypothesis that phytosterol esters containing different sterol moieties (sitosterol, sitostanol, or stigmasterol) but the same fatty acid moiety (stearic acid) produce different effects on cholesterol metabolism. Male Syrian hamsters were fed sitosterol, sitostanol, and stigmasterol stearate esters (25 g/kg diet) in an atherogenic diet containing cholesterol (1.2 g/kg) and coconut oil (80 g/kg). The phytosterol stearates produced no decrease in cholesterol absorption or plasma non-high-density lipoprotein cholesterol despite a reduction in liver free cholesterol in hamsters fed both sitosterol and sitostanol stearate diets. In addition, sitosterol stearate significantly increased fecal esterified and total neutral sterol excretion. Stigmasterol stearate did not differ from control in neutral sterol excretion, plasma lipids, or hepatic lipid concentration. Sitosterol stearate demonstrated the highest level of net intestinal hydrolysis, whereas sitostanol and stigmasterol stearate equivalently demonstrated the lowest. The cholesterol-lowering effect in liver-but not plasma-and the limited presence of fecal free sterols indicate that intact (unhydrolyzed) phytosterol stearates may impact cholesterol metabolism by mechanisms unrelated to the role of free phytosterols. The consumption of phytosterol esters at 2.5% of the diet elicited only modest impacts on cholesterol metabolism, although sitosterol stearate had a slightly greater therapeutic impact by lowering liver free cholesterol and increasing esterified and total neutral sterol fecal excretion, possibly due to a greater level of intestinal hydrolysis. Copyright © 2011 Elsevier Inc. All rights reserved.

21 CFR 864.7375 - Glutathione reductase assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

Energy Technology Data Exchange (ETDEWEB)

Pegan, Scott D.; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A.; Mesecar, Andrew D. (IdRS); (Purdue); (Colorado); (UIC)

2011-09-06

Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of human QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC{sub 50} values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.

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.

Science.gov (United States)

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.

The SUD1 gene encodes a putative E3 ubiquitin ligase and is a positive regulator of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in Arabidopsis.

Science.gov (United States)

Doblas, Verónica G; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M; Botella, Miguel A

2013-02-01

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum-associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

Colour formation in fermented sausages by meat-associated staphylococci with different nitrite- and nitrate-reductase activities

DEFF Research Database (Denmark)

Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne

2008-01-01

nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation......Three Staphylococcus strains, S. carnosus, S. simulans and S. saprophyticus, selected due to their varying nitrite and/or nitrate-reductase activities, were used to initiate colour formation during sausage fermentation. During fermentation of sausages with either nitrite or nitrate added, colour...... with hexanal content, and may be used as predictive tools. Overall, nitrite- and nitrate-reductase activities of Staphylococcus strains in nitrite-cured sausages were of limited importance regarding colour development, while in nitrate-cured sausages strains with higher nitrate reductase activity were crucial...

Aldose Reductase Inhibitory and Antiglycation Activities of Four ...

African Journals Online (AJOL)

Aldose Reductase Inhibitory and Antiglycation Activities of Four Medicinal Plant Standardized Extracts and Their Main Constituents for the Prevention of ... levels in galactosemic condition by using reverse phase high pressure liquid chromatography (RP-HPLC) and gas liquid chromatography (GLC) was determined.

Xylose reductase from the thermophilic fungus Talaromyces emersonii

Indian Academy of Sciences (India)

Prakash

Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene .... proteins with reversed coenzyme preference from NADPH to NADH ..... 399–404. Hasper A A, Visser J and de Graaff L H 2000 The Aspergillus.

Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

Science.gov (United States)

Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

1987-01-01

From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

Regulation of schistosome egg production by HMG CoA reductase

International Nuclear Information System (INIS)

VandeWaa, E.A.; Bennett, J.L.

1986-01-01

Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of 14 C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production

Preliminary study on health-related lipid components of bakery products.

Science.gov (United States)

Cercaci, Luisito; Conchillo, Ana; Rodriguez-Estrada, Maria Teresa; Ansorena, Diana; Astiasarán, Iciar; Lercker, Giovanni

2006-06-01

The purpose of this study was to evaluate the presence of health-related lipid components, in particular trans fatty acids and sterol oxidation products, in four bakery products. Both types of components are known for their adverse biological effects, especially the increase of atherogenic risk, and therefore it is advisable to monitor their presence in food products. Trans fatty acids were determined by silver-ion thin-layer chromatography-gas chromatography, whereas sterol oxidation was assessed by gas chromatography and gas chromatography-mass spectrometry determination of 7-keto derivatives (tracers of sterol oxidation). The amount of trans fatty acids (0.02 to 3.13 g/100 g of product), sterols (34.9 to 128.3 mg/100 g of product), and 7-keto derivatives of sterols (1.88 to 3.14 mg/kg of product) varied considerably among samples. The supply of phytosterols (22.5 to 64.0 mg/100 g of product) was not significant, and the extent of oxidation of most phytosterols to its corresponding 7-keto derivative was low (0.29 to 0.84%), except for that of brassicasterol (2.01 to 3.11%). The quality of ingredients and raw materials seems to have greatly influenced the fatty acid profile, stability, safety, and quality of the final product; these ingredients should be chosen with extreme care to decrease their potential negative health effects and to increase safety of these products.

Inhibition of aldose reductase activity by Cannabis sativa chemotypes extracts with high content of cannabidiol or cannabigerol.

Science.gov (United States)

Smeriglio, Antonella; Giofrè, Salvatore V; Galati, Enza M; Monforte, Maria T; Cicero, Nicola; D'Angelo, Valeria; Grassi, Gianpaolo; Circosta, Clara

2018-02-07

Aldose reductase (ALR2) is a key enzyme involved in diabetic complications and the search for new aldose reductase inhibitors (ARIs) is currently very important. The synthetic ARIs are often associated with deleterious side effects and medicinal and edible plants, containing compounds with aldose reductase inhibitory activity, could be useful for prevention and therapy of diabetic complications. Non-psychotropic phytocannabinoids exert multiple pharmacological effects with therapeutic potential in many diseases such as inflammation, cancer, diabetes. Here, we have investigated the inhibitory effects of extracts and their fractions from two Cannabis sativa L. chemotypes with high content of cannabidiol (CBD)/cannabidiolic acid (CBDA) and cannabigerol (CBG)/cannabigerolic acid (CBGA), respectively, on human recombinant and pig kidney aldose reductase activity in vitro. A molecular docking study was performed to evaluate the interaction of these cannabinoids with the active site of ALR2 compared to known ARIs. The extracts showed significant dose-dependent aldose reductase inhibitory activity (>70%) and higher than fractions. The inhibitory activity of the fractions was greater for acidic cannabinoid-rich fractions. Comparative molecular docking results have shown a higher stability of the ALR2-cannabinoid acids complex than the other inhibitors. The extracts of Cannabis with high content of non-psychotropic cannabinoids CBD/CBDA or CBG/CBGA significantly inhibit aldose reductase activity. These results may have some relevance for the possible use of C. sativa chemotypes based preparations as aldose reductase inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

The effect of growth phase on the lipid class, fatty acid and sterol composition in the marine dinoflagellate, Gymnodinium sp. in batch culture.

Science.gov (United States)

Mansour, Maged P; Volkman, John K; Blackburn, Susan I

2003-05-01

We have studied the effects of growth phase on the lipid composition in batch cultures of Gymnodinium sp. CS-380/3 over 43 days of culturing. The lipid content increased two fold, from late logarithmic (day 6) to linear growth phase (day 22) then decreased at stationary phase (day 43) while the lipid yield (mg l(-1)) increased 30-fold from day 6 to 30 mg l(-1) at day 43. Changes in fatty acid content mirrored those observed for the total lipid, while the sterol content continued to increase with culture age through to stationary phase. The largest changes occurred in the lipid classes, especially the polar lipids and triacylglycerols (oil). The proportion of triacylglycerols increased from 8% (of total lipids) at day 6 to 30% at day 43, with a concomitant decrease in the polar lipid fraction. The proportions of 16:0 and DHA [22:6(n-3)] increased while those of 18:5(n-3) and EPA [20:5(n-3)] decreased with increasing culture age. The proportion of the major sterol, dinosterol, decreased from 41% (day 6) to 29% (day 43), while the major dinostanol epimer (23R,24R) increased from 33% (day 6) to 38% (day 22). Despite small changes in the proportion of the main sterols, the same sterols were present at all stages of growth, indicating their value as a chemotaxonomic tool for distinguishing between strains within the same genus. Growth phase could be a useful variable for optimising the oil and DHA content with potential for aquaculture feeds and a source of DHA-rich oils for nutraceuticals.

The use of sterol distributions combined with compound specific isotope analyses as a tool to identify the origin of fecal contamination in rivers.

Science.gov (United States)

Biache, Coralie; Philp, R Paul

2013-03-01

The sterol distributions of 9 sediment samples from the Illinois River Basin (OK and AR, USA) were examined in order to identify the source of fecal contamination. The samples were extracted with organic solvent using sonication and the fractions containing the sterols were isolated and analyzed by gas chromatography-mass spectrometry. The sterol distributions of the Illinois River samples were dominated by phytosterols. They were compared to those of different animal feces and manures using a principal component analysis and correspondence appeared between the sediments and one group of chicken feces samples. Gas chromatography-isotope ratio mass spectrometry analyses were also performed to determine the δ(13)C values for the phytosterols and to get an indication of their origin based on the C(3)/C(4) plant signatures. The δ(13)C values obtained ranged from -30.6 ‰ to -17.4 ‰ (VPDB) corresponding to a mixed signature between C(3) and C(4) plants, indicating a C(4) plant contribution to the C(3) plant natural background. These observations indicate that a proportion of the phytosterols originated from chicken feces. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthesis of deuterium-labeled plant sterols and analysis of their side-chain mobility by solid state deuterium NMR

International Nuclear Information System (INIS)

Marsan, M.P.; Muller, I.; Milon, A.

1996-01-01

Sitosterol and stigmasterol, plant sterols, were deuterated at specific positions. Orientation and mobility of the deuterated sitosterol and stigmasterol (and two of their diasteromers) on oriented lipid bilayers were analyzed by deuterium NMR spectroscopy. Orientation and mobility of the side chains was revealed by these studies

Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells.

Science.gov (United States)

Sakai, Chika; Tomitsuka, Eriko; Esumi, Hiroyasu; Harada, Shigeharu; Kita, Kiyoshi

2012-05-01

Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010. Copyright © 2011 Elsevier B.V. All rights reserved.

DISP3, a sterol-sensing domain-containing protein that links thyroid hormone action and cholesterol metabolism

Czech Academy of Sciences Publication Activity Database

Zíková, Martina; Corlett, Alicia; Bendová, Zdeňka; Pajer, Petr; Bartůněk, Petr

2009-01-01

Roč. 23, č. 4 (2009), s. 520-528 ISSN 0888-8809 R&D Projects: GA AV ČR IAA500520705 Grant - others:EC(XE) LSHM-CT-2005-018652 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50110509 Keywords : thyroid hormone receptor * cholesterol metabolism * sterol-sensing domain Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.257, year: 2009

Cloning and sequence of the human adrenodoxin reductase gene

International Nuclear Information System (INIS)

Lin, Dong; Shi, Y.; Miller, W.L.

1990-01-01

Adrenodoxin reductase is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. The authors cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G+C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of housekeeping genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon

Determination of plant stanols and plant sterols in phytosterol enriched foods with a gas chromatographic-flame ionization detection method: NMKL collaborative study.

Science.gov (United States)

Laakso, Päivi H

2014-01-01

This collaborative study with nine participating laboratories was conducted to determine the total plant sterol and/or plant stanol contents in phytosterol fortified foods with a gas chromatographic method. Four practice and 12 test samples representing mainly commercially available foodstuffs were analyzed as known replicates. Twelve samples were enriched with phytosterols, whereas four samples contained only natural contents of phytosterols. The analytical procedure consisted of two alternative approaches: hot saponification method, and acid hydrolysis treatment prior to hot saponification. As a result, sterol/stanol compositions and contents in the samples were measured. The amounts of total plant sterols and total plant stanols varying from 0.005 to 8.04 g/100 g product were statistically evaluated after outliers were eliminated. The repeatability RSD (RSDr) varied from 1.34 to 17.13%. The reproducibility RSD (RSDR) ranged from 3.03 to 17.70%, with HorRat values ranging from 0.8 to 2.1. When only phytosterol enriched food test samples are considered, the RSDr ranged from 1.48 to 6.13%, the RSD, ranged from 3.03 to 7.74%, and HorRat values ranged from 0.8 to 2.1. Based on the results of this collaborative study, the study coordinator concludes the method is fit for its purpose.

Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases

International Nuclear Information System (INIS)

Andersson, S.; Russell, D.W.

1990-01-01

The microsomal enzyme steroid 5α-reductase is responsible for the conversion of testosterone into the more potent androgen dihydrotestosterone. In man, this steroid acts on a variety of androgen-responsive target tissues to mediate such diverse endocrine processes as male sexual differentiation in the fetus and prostatic growth in men. Here we describe the isolation, structure, and expression of a cDNA encoding the human steroid 5α-reductase. A rat cDNA was used as a hybridization probe to screen a human prostate cDNA library. A 2.1-kilobase cDNA was identified and DNA sequence analysis indicated that the human steroid 5α-reductase was a hydrophobic protein of 259 amino acids with a predicted molecular weight of 29,462. A comparison of the human and rat protein sequences revealed a 60% identity. Transfection of expression vectors containing the human and rat cDNAs into simian COS cells resulted in the synthesis of high levels of steroid 5α-reductase enzyme activity. Both enzymes expressed in COS cells showed similar substrate specificities for naturally occurring steroid hormones. However, synthetic 4-azasteroids demonstrated marked differences in their abilities to inhibit the human and rat steroid 5α-reductases

Overview of Catalytic Properties of Fungal Xylose Reductases and Molecular Engineering Approaches for Improved Xylose Utilisation in Yeast

Directory of Open Access Journals (Sweden)

Sk Amir Hossain

2018-03-01

Full Text Available Background and Objective: Xylose reductases belong to the aldo-keto reductase family of enzymes, which catalyse the conversion of xylose to xylitol. Yeast xylose reductases have been intensively studied in the last two decades due to their significance in biotechnological production of ethanol and xylitol from xylose. Due to its GRAS status and pronounced tolerance to harsh conditions, Saccharomyces cerevisiae is the ideal organism for industrial production of both xylitol and ethanol. However, Saccharomyces cerevisiae is unable to use xylose as the sole carbon source due to the lack of xylose specific transporters and insufficient activity of metabolic pathways for xylose utilisation. The aim of this paper is to give an overview of attempts in increasing biotechnological potential of xylose reductases and to highlight the prospective of this application. Results and Conclusion: In order to create strains with improved xylose utilization, different approaches were attempted including simultaneous overexpression of xylitol dehydrogenase, xylose reductase and pentose phosphate pathway enzymes, heterologous expression of putative xylose transporters or heterologous expression of genes coding for enzymes included in the xylose metabolism, respectively. Furthermore, number of attempts to genetically modify different xylose reductases is increasing. This review presents current knowledge about yeast xylose reductases and the different approaches applied in order to improve xylose metabolism in yeast.Conflict of interest: The authors declare no conflict of interest.

Aldose Reductase Inhibitory Activity of Compounds from  Zea mays L.

Science.gov (United States)

Kim, Tae Hyeon; Kim, Jin Kyu; Kang, Young-Hee; Lee, Jae-Yong; Kang, Il Jun; Lim, Soon Sung

2013-01-01

Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1–7) and 5 anthocyanins (compound 8–12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC50, 4.78 μM). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. PMID:23586057

Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

Science.gov (United States)

Waddell, D; Ullman, B

1983-04-10

From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

BIOCHEMISTRY OF DINOFLAGELLATE LIPIDS, WITH PARTICULAR REFERENCE TO THE FATTY ACID AND STEROL COMPOSITION OF A KARENIA BREVIS BLOOM

Science.gov (United States)

Leblond, Jeffrey D., Terence J. Evens and Peter J. Chapman. 2003. Biochemistry of Dinoflagellate Lipids, with Particular Reference to the Fatty Acid and Sterol Composition of a Karenia brevis Bloom. Phycologia. 42(4):324-331. (ERL,GB 1160). The harmful marine dinoflagella...

Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides

DEFF Research Database (Denmark)

Rattray, Fergal P; Myling-Petersen, Dorte; Larsen, Dianna

2003-01-01

A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin...

Photoaffinity labeling of the oxysterol binding protein

International Nuclear Information System (INIS)

Taylor, F.R.; Kandutsch, A.A.; Anzalone, L.; Spencer, T.A.

1986-01-01

A cytosolic receptor protein for oxygenated sterols, that is thought to be involved in the regulation of HMG-CoA reductase and cholesterol biosynthesis, can be labeled covalently by the photoactivated affinity compound [5,6- 3 H]-7,7'-azocholestane-3β,25-diol (I). Several other compounds were tested including 25-hydroxycholesta-4,6-dien-3-one, 25-azido-27-norcholest-5-en-3β-ol,3β,25-dihydroxycholest-5-en-7-one and 3β-hydroxycholesta-8(14),9(11)-dien-15-one. However, these sterols either did not bind to the receptor with adequate affinity or did not react covalently with the receptor during photolysis. Compound I binds to the receptor with very high affinity (K/sub d/ = 30 nM). After activation with long wavelength UV, two tritium labeled proteins, M/sub r/ approximately 95K and 65K daltons, are found upon SDS gel electrophoresis. No labeling occurs when the binding reaction is carried out in the presence of a large excess of 25-hydroxycholesterol. It is possible that the smaller polypeptide is a degradation product. Under the reaction conditions investigated so far labeling is relatively inefficient (< 1% of bound sterol). These results are generally consistent with previous information suggesting that the M/sub r/ of the receptor subunit is 97,000. Covalent labeling of the receptor should greatly facilitate its further purification and characterization

Optimization and modeling for the synthesis of sterol esters from deodorizer distillate by lipase-catalyzed esterification.

Science.gov (United States)

Zhang, Xinyu; Yu, Jiang; Zeng, Aiwu

2017-03-01

In this paper, cotton seed oil deodorizer distillate (CSODD), was recovered to obtain fatty acid sterol ester (FASE), which is one of the biological activated substances added as human therapeutic to lower cholesterol. Esterification reactions were carried out using Candida rugosa lipase as a catalyst, and the conversion of phytosterol was optimized using response surface methodology. The highest conversion (90.8 ± 0.4%) was reached at 0.84 wt% enzyme load, 1:25 solvent/CSODD mass ratio, and 44.2 °C after 12 H reaction. A kinetic model based on the reaction rate equation was developed to describe the reaction process. The activation energy of the reaction was calculated to be 56.9 kJ/mol and the derived kinetic parameters provided indispensable basics for further study. The optimization and kinetic research of synthesizing FASE from deodorizer distillate provided necessary information for the industrial applications in the near future. Experimental results showed that the proposed process is a promising alternative to recycle sterol esters from vegetable oil deodorizer distillates in a mild, efficient, and environmental friendly method. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Effect of cystamine on rat tissue GSH level and glutathione reductase activity

International Nuclear Information System (INIS)

Kovarova, H.; Pulpanova, J.

1979-01-01

Reduced glutathione (GSH) level and glutathione reductase activity were determined by means of the spectrophotometric method in various rat tissues after i.p. administration of cystamine (50 mg/kg and 20 mg/kg). GSH amount dropped in the spleen and kidney at 10 and 20 min; following this interval, an increase of GSH level was observed in the liver at 20-30 min, in the spleen and kidney at 60 min after the treatment with a radioprotective cystamine dose (50 mg/kg). The changes in GSH level induced by a non-radioprotective cystamine dose (20 mg/kg) had an opposite tendency. The activity of glutathione reductase was decreased in all tissues studied. As to the mechanism of the radioprotective action, both the inactivation of glutathione reductase activity and the changes in GSH level seem to be the factors contributing to the radioprotective effect of cystamine by strengthening the cellular radioresistance. (orig.) 891 MG/orig. 892 RKD [de

Expression, purification, crystallization and preliminary X-ray diffraction analysis of carbonyl reductase from Candida parapsilosis ATCC 7330

International Nuclear Information System (INIS)

Aggarwal, Nidhi; Mandal, P. K.; Gautham, Namasivayam; Chadha, Anju

2013-01-01

The expression, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies on C. parapsilosis carbonyl reductase are reported. The NAD(P)H-dependent carbonyl reductase from Candida parapsilosis ATCC 7330 catalyses the asymmetric reduction of ethyl 4-phenyl-2-oxobutanoate to ethyl (R)-4-phenyl-2-hydroxybutanoate, a precursor of angiotensin-converting enzyme inhibitors such as Cilazapril and Benazepril. The carbonyl reductase was expressed in Escherichia coli and purified by GST-affinity and size-exclusion chromatography. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.86 Å resolution. The asymmetric unit contained two molecules of carbonyl reductase, with a solvent content of 48%. The structure was solved by molecular replacement using cinnamyl alcohol dehydrogenase from Saccharomyces cerevisiae as a search model

Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase.

Science.gov (United States)

Liu, Yi; Shi, Zi; Maximova, Siela; Payne, Mark J; Guiltinan, Mark J

2013-12-05

The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo.

Sagunja-Tang Improves Lipid Related Disease in a Postmenopausal Rat Model and HepG2 Cells

Directory of Open Access Journals (Sweden)

Hiroe Go

2015-01-01

Full Text Available The present study was conducted to investigate the effect of Sagunja-tang on the lipid related disease in a rat model of menopausal hyperlipidemia and lipid accumulation in methyl-β-cyclodextrin-induced HepG2 cells. In in vivo study using menopausal hyperlipidemia rats, Sagunja-tang reduced retroperitoneal and perirenal fat, serum lipids, atherogenic index, cardiac risk factor, media thickness, and nonalcoholic steatohepatitis score, when compared to menopausal hyperlipidemia control rats. In HepG2 cells, Sagunja-tang significantly decreased the lipid accumulation, total cholesterol levels, and low-density/very-low-density lipoprotein levels. Moreover, Sagunja-tang reversed the methyl-β-cyclodextrin-induced decrease in the protein levels of critical molecule involved in cholesterol synthesis, sterol regulatory element binding protein-2, and low-density lipoprotein receptor and inhibited protein levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase as well as activity. Phosphorylation level of AMP-activated protein kinase was stimulated by Sagunja-tang. These results suggest that Sagunja-tang has effect on inhibiting hepatic lipid accumulation through regulation of cholesterol synthesis and AMPK activity in vitro. These observations support the idea that Sagunja-tang is bioavailable both in vivo and in vitro and could be developed as a preventive and therapeutic agent of hyperlipidemia in postmenopausal females.

Sterol Regulation of Voltage-Gated K+ Channels.

Science.gov (United States)

Balajthy, Andras; Hajdu, Peter; Panyi, Gyorgy; Varga, Zoltan

2017-01-01

Cholesterol is an essential lipid building block of the cellular plasma membrane. In addition to its structural role, it regulates the fluidity and raft structure of the membrane and influences the course of numerous membrane-linked signaling pathways and the function of transmembrane proteins, including ion channels. This is supported by a vast body of scientific data, which demonstrates the modulation of ion channels with a great variety of ion selectivity, gating, and tissue distribution by changes in membrane cholesterol. Here, we review what is currently known about the modulation of voltage-gated K + (Kv) channels by changes in membrane cholesterol content, considering raft association of the channels, the roles of cholesterol recognition sites, and those of adaptor proteins in cholesterol-Kv channel interactions. We specifically focus on Kv1.3, the dominant K + channel of human T cells. Effects of cholesterol depletion and enrichment and 7-dehydrocholesterol enrichment on Kv1.3 gating are discussed in the context of the immunological synapse and the comparison of the in vitro effects of sterol modifications on Kv1.3 function with ex vivo effects on cells from hypercholesterolemic and Smith-Lemli-Opitz patients. © 2017 Elsevier Inc. All rights reserved.

Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes

International Nuclear Information System (INIS)

Liang, T.; Cheung, A.H.; Reynolds, G.F.; Rasmusson, G.H.

1985-01-01

21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a K/sub i/ value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [ 3 H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([ 3 H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2- 3 H]Diazo-MAPD binds to a single high affinity site in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

Science.gov (United States)

Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

2016-11-01

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Nitrate reductase gene involvement in hexachlorobiphenyl dechlorination by Phanerochaete chrysosporium

International Nuclear Information System (INIS)

De, Supriyo; Perkins, Michael; Dutta, Sisir K.

2006-01-01

Polychlorobiphenyl (PCB) degradation usually occurs through reductive dechlorination under anaerobic conditions and phenolic ring cleavage under aerobic conditions. In this paper, we provide evidence of nitrate reductase (NaR) mediated dechlorination of hexachlorobiphenyl (PCB-153) in Phanerochaete chrysosporium under non-ligninolytic condition and the gene involved. The NaR enzyme and its cofactor, molybdenum (Mo), were found to mediate reductive dechlorination of PCBs even in aerobic condition. Tungsten (W), a competitive inhibitor of this enzyme, was found to suppress this dechlorination. Chlorine release assay provided further evidence of this nitrate reductase mediated dechlorination. Commercially available pure NaR enzyme from Aspergillus was used to confirm these results. Through homology search using TBLASTN program, NaR gene was identified, primers were designed and the RT-PCR product was sequenced. The NaR gene was then annotated in the P. chrysosporium genome (GenBank accession no. AY700576). This is the first report regarding the presence of nitrate reductase gene in this fungus with the explanation why this fungus can dechlorinate PCBs even in aerobic condition. These fungal inoculums are used commercially as pellets in sawdust for enhanced bioremediation of PCBs at the risk of depleting soil nitrates. Hence, the addition of nitrates to the pellets will reduce this risk as well as enhance its activity

A new cytotoxic sterol methoxymethyl ether from a deep water marine sponge Scleritoderma sp. cf. paccardi.

Science.gov (United States)

Gunasekera, S P; Kelly-Borges, M; Longley, R E

1996-02-01

24(R)-Methyl-5 alpha-cholest-7-enyl 3 beta-methoxymethyl ether (1), a new sterol ether, has been isolated from a deep-water marine sponge Scleritoderma sp. cf. paccardi. Compound 1 exhibited in vitro cytotoxicity against the cultured murine P-388 tumor cell line with an IC50 of 2.3 micrograms/mL. The isolation and structure elucidation of 1 by NMR spectroscopy is described.

Isolation and primary structural analysis of two conjugated polyketone reductases from Candida parapsilosis.

Science.gov (United States)

Hidalgo, A R; Akond, M A; Kita, K; Kataoka, M; Shimizu, S

2001-12-01

Two conjugated polyketone reductases (CPRs) were isolated from Candida parapsilosis IFO 0708. The primary structures of CPRs (C1 and C2) were analyzed by amino acid sequencing. The amino acid sequences of both enzymes had high similarity to those of several proteins of the aldo-keto-reductase (AKR) superfamily. However, several amino acid residues in the putative active sites of AKRs were not conserved in CPRs-C1 and -C2.

Molecular Cloning and Expression of Bacterial Mercuric Reductase ...

African Journals Online (AJOL)

In order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr) Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and finally mer operon ...

Molecular Cloning and Expression of Bacterial Mercuric Reductase ...

African Journals Online (AJOL)

USER

2010-06-21

Jun 21, 2010 ... In order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr). Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and ...

Combined quantification of faecal sterols, stanols, stanones and bile acids in soils and terrestrial sediments by gas chromatography-mass spectrometry.

Science.gov (United States)

Birk, Jago Jonathan; Dippold, Michaela; Wiesenberg, Guido L B; Glaser, Bruno

2012-06-15

Faeces incorporation can alter the concentration patterns of stanols, stanones, Δ(5)-sterols and bile acids in soils and terrestrial sediments. A joint quantification of these substances would give robust and specific information about the faecal input. Therefore, a method was developed for their purification and determination via gas chromatography-mass spectrometry (GC-MS) based on a total lipid extract (TLE) of soils and terrestrial sediments. Stanols, stanones, Δ(5)-steroles and bile acids were extracted by a single Soxhlet extraction yielding a TLE. The TLE was saponified with KOH in methanol. Sequential liquid-liquid extraction was applied to recover the biomarkers from the saponified extract and to separate the bile acids from the neutral stanoles, stanones and Δ(5)-steroles. The neutral fraction was directly purified using solid phase extraction (SPE) columns packed with 5% deactivated silica gel. The bile acids were methylated in dry HCl in methanol and purified on SPE columns packed with activated silica gel. A mixture of hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS) and pyridine was used to silylate the hydroxyl groups of the stanols and Δ(5)-sterols avoiding a silylation of the keto groups of the stanones in their enol-form. Silylation of the bile acids was carried out with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing N-trimethylsilylimidazole (TSIM). TLEs from a set of soils with different physico-chemical properties were used for method evaluation and for comparison of amounts of faecal biomarkers analysed with saponification and without saponification of the TLE. Therefore, a Regosol, a Podzol and a Ferralsol were sampled. To proof the applicability of the method for faecal biomarker analyses in archaeological soils and sediments, additional samples were taken from pre-Columbian Anthrosols in Amazonia and an Anthrosol from a site in central Europe settled since the Neolithic. The comparison of the amounts of steroids

Contribution of thermolabile methylenetetrahydrofolate reductase variant to total plasma homocysteine levels in healthy men and women. Inter99 (2)

DEFF Research Database (Denmark)

Husemoen, Lise Lotte N; Thomsen, Troels F; Fenger, Mogens

2003-01-01

Elevation in plasma total homocysteine (tHcy) is believed to be causally related to cardiovascular disease. Like age and sex, the thermolabile variant of methylenetetrahydrofolate reductase (MTHFR(C677T)) is an important nonmodifiable determinant of tHcy, which may be considered when describing...

Potency of a novel saw palmetto ethanol extract, SPET-085, for inhibition of 5alpha-reductase II.

Science.gov (United States)

Pais, Pilar

2010-08-01

The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent membrane protein 5alpha-reductase irreversibly catalyses the conversion of testosterone to the most potent androgen, 5alpha-dihydrotestosterone (DHT). In humans, two 5alpha-reductase isoenyzmes are expressed: type I and type II. Type II is found primarily in prostate tissue. Saw palmetto extract (SPE) has been widely used for the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The mechanisms of the pharmacological effects of SPE include the inhibition of 5alpha-reductase, among other actions. Clinical studies of SPE have been equivocal, with some showing significant results and others not. These inconsistent results may be due, in part, to varying bioactivities of the SPE used in the studies. The aim of the present study was to determine the in vitro potency of a novel saw palmetto ethanol extract (SPET-085), an inhibitor of the 5alpha-reductase isoenzyme type II, in a cell-free test system. On the basis of the enzymatic conversion of the substrate androstenedione to the 5alpha-reduced product 5alpha-androstanedione, the inhibitory potency was measured and compared to those of finasteride, an approved 5alpha-reductase inhibitor. SPET-085 concentration-dependently inhibited 5alpha-reductase type II in vitro (IC(50)=2.88+/-0.45 microg/mL). The approved 5alpha-reductase inhibitor, finasteride, tested as positive control, led to 61% inhibition of 5alpha-reductase type II. SPET-085 effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is very low compared to data reported for other extracts. It can be concluded from data in the literature that SPET-085 is as effective as a hexane extract of saw palmetto that exhibited the highest levels of bioactivity, and is more effective than other SPEs tested. This study confirmed that SPET-085 has prostate health-promoting bioactivity that also corresponds favorably to

Effect of a plant sterol, fish oil and B vitamin combination on cardiovascular risk factors in hypercholesterolemic children and adolescents: a pilot study

Directory of Open Access Journals (Sweden)

Garaiova Iveta

2013-01-01

Full Text Available Abstract Background Assessment of cardiovascular disease (CVD risk factors can predict clinical manifestations of atherosclerosis in adulthood. In this pilot study with hypercholesterolemic children and adolescents, we investigated the effects of a combination of plant sterols, fish oil and B vitamins on the levels of four independent risk factors for CVD; LDL-cholesterol, triacylglycerols, C-reactive protein and homocysteine. Methods Twenty five participants (mean age 16 y, BMI 23 kg/m2 received daily for a period of 16 weeks an emulsified preparation comprising plant sterols esters (1300 mg, fish oil (providing 1000 mg eicosapentaenoic acid (EPA plus docosahexaenoic acid (DHA and vitamins B12 (50 μg, B6 (2.5 mg, folic acid (800 μg and coenzyme Q10 (3 mg. Atherogenic and inflammatory risk factors, plasma lipophilic vitamins, provitamins and fatty acids were measured at baseline, week 8 and 16. Results The serum total cholesterol, LDL- cholesterol, VLDL-cholesterol, subfractions LDL-2, IDL-1, IDL-2 and plasma homocysteine levels were significantly reduced at the end of the intervention period (pp Conclusions Daily intake of a combination of plant sterols, fish oil and B vitamins may modulate the lipid profile of hypercholesterolemic children and adolescents. Trial registration Current Controlled Trials ISRCTN89549017

Intraethnic variation in steroid-5-alpha-reductase polymorphisms in ...

Indian Academy of Sciences (India)

2015-06-01

Jun 1, 2015 ... in prostate cancer patients: a potential factor implicated ... reductase alpha polypeptides 1 and 2 in a set of 601 prostate cancer patients from four ..... tion in the key androgen-regulating genes androgen receptor, cytochrome ...

Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2.

Science.gov (United States)

Vasconcelos, Marta; Eckert, Helene; Arahana, Venancio; Graef, George; Grusak, Michael A; Clemente, Tom

2006-10-01

Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe(+3) reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 microM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 microM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.

Research progress on the roles of aldose reductase in diabetic retinopathy

Directory of Open Access Journals (Sweden)

Hong-Zhe Li

2015-07-01

Full Text Available Aldose reductase(ARbelonging to nicotinamide-adenine dinucleotide phosphate(NADPH-dependent aldehyde-keto reductase superfamily, is the key rate-limiting enzyme in the polyol pathway which plays an important role in the body's high-sugar metabolism. AR is widely present in the kidneys, blood vessels, lens, retina, heart, skeletal muscle and other tissues and organs, converts glucose to sorbitol which easy permeability of cell membranes, cause cell swelling, degeneration, necrosis, and have a close relationship with the development of chronic complications of diabetes mellitus. Diabetic retinopathy(DRis a multifactorial disease, the exact cause is currently unknown, but polyol pathway has been demonstrated to play an important role in the pathogenesis of DR. Clinical risk factors such as blood sugar control, blood pressure and other treatments for DR only play a part effect of remission or invalid, if we can find out DR genes associated with the disease, this will contribute to a better understanding of the pathological mechanisms and contribute to the development of new treatments and drugs. The current research progress of AR, AR gene polymorphism, Aldose reductase inhibitors to DR was reviewed in this article.

New method for recognition of sterol signalling molecules: Methinium salts as receptors for sulphated steroids

Czech Academy of Sciences Publication Activity Database

Kejík, Z.; Bříza, T.; Králová, Jarmila; Mikula, I.; Poučková, P.; Martásek, P.; Král, V.

2015-01-01

Roč. 94, February 2015 (2015), s. 15-20 ISSN 1878-5867 R&D Projects: GA TA ČR(CZ) TE01020028; GA ČR(CZ) GAP303/11/1291; GA MŠk(CZ) LH14008; GA MŠk(CZ) CZ.1.07/2.300/30.0060; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : Polymethinium salts * Sulphated sterols * Molecular recognition * Synthetic receptors Subject RIV: EB - Genetics ; Molecular Biology

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

Science.gov (United States)

Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

2006-01-01

Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å. PMID:17142919

Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility.

Science.gov (United States)

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

The effect of plant sterols and different low doses of omega-3 fatty acids from fish oil on lipoprotein subclasses

NARCIS (Netherlands)

Jacobs, D.M.; Mihaleva, V.V.; Schalkwijk, D.B. van; Graaf, A.A. de; Vervoort, J.; Dorsten, F.A. van; Ras, R.T.; Demonty, I.; Trautwein, E.A.; Duynhoven, J. van

2015-01-01

Scope: Consumption of a low-fat spread enriched with plant sterols (PS) and different low doses (<2 g/day) of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil reduces serum triglycerides (TGs) and low-density lipoprotein-cholesterol (LDL-Chol) and thus beneficially affects

Zymosterol is located in the plasma membrane of cultured human fibroblasts

International Nuclear Information System (INIS)

Echevarria, F.; Norton, R.A.; Nes, W.D.; Lange, Y.

1990-01-01

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. (1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. (2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. (3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool

Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

International Nuclear Information System (INIS)

Nascimento, Alessandro S.; Ferrarezi, Thiago; Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A.; Polikarpov, Igor

2006-01-01

Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP + reductase. Ferredoxin-NADP + reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source

Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

Energy Technology Data Exchange (ETDEWEB)

Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

2006-07-01

Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

The Drosophila carbonyl reductase sniffer is an efficient 4-oxonon-2-enal (4ONE) reductase.

Science.gov (United States)

Martin, Hans-Jörg; Ziemba, Marta; Kisiela, Michael; Botella, José A; Schneuwly, Stephan; Maser, Edmund

2011-05-30

Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 μM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the Sauromatum guttatum inflorescence.

Science.gov (United States)

Skubatz, Hanna; Howald, William N

2013-03-01

A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.

Crystallization of purple nitrous oxide reductase from Pseudomonas stutzeri

International Nuclear Information System (INIS)

Pomowski, Anja; Zumft, Walter G.; Kroneck, Peter M. H.; Einsle, Oliver

2010-01-01

The physiologically active form of nitrous oxide reductase was isolated and crystallized under strict exclusion of dioxygen and diffraction data were collected from crystals belonging to two different space groups. Nitrous oxide reductase (N 2 OR) from Pseudomonas stutzeri catalyzes the final step in denitrification: the two-electron reduction of nitrous oxide to molecular dinitrogen. Crystals of the enzyme were grown under strict exclusion of dioxygen by sitting-drop vapour diffusion using 2R,3R-butanediol as a cryoprotectant. N 2 OR crystallized in either space group P1 or P6 5 . Interestingly, the key determinant for the resulting space group was the crystallization temperature. Crystals belonging to space group P1 contained four 130 kDa dimers in the asymmetric unit, while crystals belonging to space group P6 5 contained a single dimer in the asymmetric unit. Diffraction data were collected to resolutions better than 2 Å

Acute sterol o-acyltransferase 2 (SOAT2 knockdown rapidly mobilizes hepatic cholesterol for fecal excretion.

Directory of Open Access Journals (Sweden)

Stephanie M Marshall

Full Text Available The primary risk factor for atherosclerotic cardiovascular disease is LDL cholesterol, which can be reduced by increasing cholesterol excretion from the body. Fecal cholesterol excretion can be driven by a hepatobiliary as well as a non-biliary pathway known as transintestinal cholesterol efflux (TICE. We previously showed that chronic knockdown of the hepatic cholesterol esterifying enzyme sterol O-acyltransferase 2 (SOAT2 increased fecal cholesterol loss via TICE. To elucidate the initial events that stimulate TICE, C57Bl/6 mice were fed a high cholesterol diet to induce hepatic cholesterol accumulation and were then treated for 1 or 2 weeks with an antisense oligonucleotide targeting SOAT2. Within 2 weeks of hepatic SOAT2 knockdown (SOAT2HKD, the concentration of cholesteryl ester in the liver was reduced by 70% without a reciprocal increase in hepatic free cholesterol. The rapid mobilization of hepatic cholesterol stores resulted in a ∼ 2-fold increase in fecal neutral sterol loss but no change in biliary cholesterol concentration. Acute SOAT2HKD increased plasma cholesterol carried primarily in lipoproteins enriched in apoB and apoE. Collectively, our data suggest that acutely reducing SOAT2 causes hepatic cholesterol to be swiftly mobilized and packaged onto nascent lipoproteins that feed cholesterol into the TICE pathway for fecal excretion.

Evaluation of constitutive iron reductase (AtFRO2 expression on mineral accumulation and distribution in soybean (Glycine max. L

Directory of Open Access Journals (Sweden)

Marta Wilton Vasconcelos

2014-04-01

Full Text Available Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene's expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg and Mo, pod walls (Fe, K, P, Cu and Ni, leaves (Fe, P, Cu, Ca, Ni and Mg and seeds (Fe, Zn, Cu and Ni. Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and

Nitrate reductase and nitrous oxide production by Fusarium oxysporum 11dn1 under aerobic and anaerobic conditions.

Science.gov (United States)

Kurakov, A V; Nosikov, A N; Skrynnikova, E V; L'vov, N P

2000-08-01

The fungus Fusarium oxysporum 11dn1 was found to be able to grow and produce nitrous oxide on nitrate-containing medium in anaerobic conditions. The rate of nitrous oxide formation was three to six orders of magnitude lower than the rates of molecular nitrogen production by common denitrifying bacteria. Acetylene and ammonia did not affect the release of nitrous oxide release. It was shown that under anaerobic conditions fast increase of nitrate reductase activity occurred, caused by the synthesis of enzyme de novo and protein dephosphorylation. Reverse transfer of the mycelium to aerobic conditions led to a decline in nitrate reductase activity and stopped nitrous oxide production. The presence of two nitrate reductases was shown, which differed in molecular mass, location, temperature optima, and activity in nitrate- and ammonium-containing media. Two enzymes represent assimilatory and dissimilatory nitrate reductases, which are active in aerobic and anaerobic conditions, respectively.

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

Science.gov (United States)

Crankshaw, D L; Hetnarski, K; Wilkinson, C F

1979-09-01

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

Transcriptional modulation of genes encoding nitrate reductase in ...

African Journals Online (AJOL)

The free aluminum (Al) content in soil can reach levels that are toxic to plants, and this has frequently limited increased productivity of cultures. Four genes encoding nitrate reductase (NR) were identified, named ZmNR1–4. With the aim of evaluating NR activity and the transcriptional modulation of the ZmNR1, ZmNR2, ...

Sex hormones reduce NNK detoxification through inhibition of short-chain dehydrogenases/reductases and aldo-keto reductases in vitro.

Science.gov (United States)

Stapelfeld, Claudia; Maser, Edmund

2017-10-01

Carbonyl reduction is an important metabolic pathway for endogenous and xenobiotic substances. The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, nicotine-derived nitrosamine ketone) is classified as carcinogenic to humans (IARC, Group 1) and considered to play the most important role in tobacco-related lung carcinogenesis. Detoxification of NNK through carbonyl reduction is catalyzed by members of the AKR- and the SDR-superfamilies which include AKR1B10, AKR1C1, AKR1C2, AKR1C4, 11β-HSD1 and CBR1. Because some reductases are also involved in steroid metabolism, five different hormones were tested for their inhibitory effect on NNK carbonyl reduction. Two of those hormones were estrogens (estradiol and ethinylestradiol), another two hormones belong to the gestagen group (progesterone and drospirenone) and the last tested hormone was an androgen (testosterone). Furthermore, one of the estrogens (ethinylestradiol) and one of the gestagens (drospirenone) are synthetic hormones, used as hormonal contraceptives. Five of six NNK reducing enzymes (AKR1B10, AKR1C1, AKR1C2, AKR1C4 and 11β-HSD1) were significantly inhibited by the tested sex hormones. Only NNK reduction catalyzed by CBR1 was not significantly impaired. In the case of the other five reductases, gestagens had remarkably stronger inhibitory effects at a concentration of 25 μM (progesterone: 66-88% inhibition; drospirenone: 26-87% inhibition) in comparison to estrogens (estradiol: 17-51% inhibition; ethinylestradiol: 14-79% inhibition) and androgens (14-78% inhibition). Moreover, in most cases the synthetic hormones showed a greater ability to inhibit NNK reduction than the physiologic derivatives. These results demonstrate that male and female sex hormones have different inhibitory potentials, thus indicating that there is a varying detoxification capacity of NNK in men and women which could result in a different risk for developing lung cancer. Copyright © 2017 Elsevier B

DNA damage induction of ribonucleotide reductase.

OpenAIRE

Elledge, S J; Davis, R W

1989-01-01

RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidas...

Nitrous oxide reductase genes (nosZ) of denitrifying populations in soil and the earthworm gut are phylogenetically similar

DEFF Research Database (Denmark)

Horn, Marcus A.; Drake, Harold L.; Schramm, Andreas

2006-01-01

-derived sequences, or were related to N2O reductases of the genera Bradyrhizobium, Brucella, Dechloromonas, Flavobacterium, Pseudomonas, Ralstonia, and Sinorhizobium. Although the numbers of estimators for genotype richness of sequence data from the gut were higher than those of soil, only one gut-derived nos...

Cell death by SecTRAPs: thioredoxin reductase as a prooxidant killer of cells.

Directory of Open Access Journals (Sweden)

Karin Anestål

Full Text Available BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins can be formed from the selenoprotein thioredoxin reductase (TrxR by targeting of its selenocysteine (Sec residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity.

Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

International Nuclear Information System (INIS)

Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2009-01-01

The NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra was expressed, purified, and crystallized and X-ray diffraction data of this crystal were collected to 2.2 Å resolution. (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%

Enhanced oral bioavailability of a sterol-loaded microemulsion formulation of Flammulina velutipes, a potential antitumor drug

Science.gov (United States)

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

Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis.

Science.gov (United States)

Gao, Yansong; Zhou, Yulian; Goldstein, Joseph L; Brown, Michael S; Radhakrishnan, Arun

2017-05-26

Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases cholesterol synthesis. Previously, we provided evidence that cholesterol inhibition is initiated when cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of cholesterol synthesis. These insights suggest a new structural model for cholesterol-mediated regulation of Scap activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Biological basis for statin resistance in naturally statin-producing organisms

DEFF Research Database (Denmark)

Rems, Ana; Frandsen, Rasmus John Normand

to lovastatin compared to the wild-type strain. Furthermore, we investigated if MlcD confers the resistance by functional complementation of the endogenous HMG-CoA reductases in S. cerevisiae. There are two isozymes of HMG-CoA reductase in yeast, HMG1 and HMG2, both involved in the sterol biosynthetic pathway......, which leads to the synthesis of ergosterol. Following deletion of HMG1 and HMG2 genes in S. cerevisiae, we inserted the mlcD gene into the knockout mutants, and tested the resulted strains for sensitivity to lovastatin. The HMG1 and HMG2 knockout mutants were unable to grow on minimal media and had...... an increased sensitivity to lovastatin on rich media. However, insertion of the mlcD gene restored the growth of the yeast mutants and increased their resistance to lovastatin. These results show that MlcD complements the activity of the deleted HMG-CoA reductases, enabling synthesis of ergosterol in yeast...

Relationship between nitrate reductase and nitrate uptake in phytoplankton in the Peru upwelling region

International Nuclear Information System (INIS)

Blasco, D.; MacIsaac, J.J.; Packard, T.T.; Dugdale, R.C.

1984-01-01

Nitrate reductase (NR) activity and 15 NO 3 - uptake in phytoplankton were compared under different environmental conditions on two cruises in the upwelling region off Peru. The NR activity and NO 3 - uptake rates responded differently to light and nutrients and the differences led to variations in the uptake: reductase ratio. Analysis of these variations suggests that the re-equilibration time of the two processes in response to environmental perturbation is an important source of variability. The nitrate uptake system responds faster than the nitrate reductase system. Considering these differences in response time the basic differences in the two processes, and the differences in their measurement, the authors conclude that the Nr activity measures the current nitrate-reducing potential, which reflects NO 3 - assimilation before the sampling time, while 15 NO 3 - uptake measures NO 3 - assimilation in the 6-h period following sampling

Absence of regulation of tumor cholesterogenesis in cell-free synthesizing systems

International Nuclear Information System (INIS)

Azrolan, N.; Coleman, P.S.

1986-01-01

In tumors, cholesterol synthesis de novo is deregulated relative to normal tissues. But no previous study has demonstrated the decontrol of tumor cholesterogenesis with cell-free cytosolic systems. They have utilized a lipid synthesizing, post-mitochondrial supernatant system (PMS), with 14 C-citrate as substrate, to characterize the cholesterogenic pathway in Morris Hepatoma 3924A and normal rat liver. The rate of cholesterogenesis in the hepatoma PMS was 6-fold higher than that in the liver system on a per cell basis. The ratio of sterol-to-fatty acid synthesis was also significantly greater in the tumor versus the liver PMS. The authors determined the steady-state carbon flux through the early intermediates of the lipogenic pathways. Whereas the liver system displayed a metabolic crossover point at the HMG-CoA reductase reaction, the hepatoma system showed no evidence of control at this rate-limiting site of sterol synthesis. Furthermore, acetyl-CoA formation from added citrate (via ATP-citrate lyase) exhibited rates of 42% and 88% in excess of that required for lipidogenesis by liver and tumor PMS systems, respectively. Clearly, a cell-free PMS system from tumor tissue displays the property of deregulated lipidogenesis, especially cholesterol biosynthesis. The authors suggest that deregulated and continuously operating cholesterogenesis would provide for an increased level of a mevalonate-derived sterol pathway intermediate proposed as a trigger for DNA synthesis and cell proliferation in tumors

Expression, purification and molecular structure modeling of thioredoxin (Trx) and thioredoxin reductase (TrxR) from Acidithiobacillus ferrooxidans.

Science.gov (United States)

Wang, Yiping; Zhang, Xiaojian; Liu, Qing; Ai, Chenbing; Mo, Hongyu; Zeng, Jia

2009-07-01

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.

Activity of cycloartane-type triterpenes and sterols isolated from Musa paradisiaca fruit peel against Leishmania infantum chagasi.

Science.gov (United States)

Silva, A A S; Morais, S M; Falcão, M J C; Vieira, I G P; Ribeiro, L M; Viana, S M; Teixeira, M J; Barreto, F S; Carvalho, C A; Cardoso, R P A; Andrade-Junior, H F

2014-09-25

The aim of the study was to evaluate in vitro the antileishmanial activity of triterpenes and sterols isolated from Musa paradisiaca (banana) fruit peel used traditionally to treat leishmaniasis. The compounds were isolated from the ethanolic extract of the peel of the banana fruit by column chromatography. The chemical structure of compounds was determined by (1)H and (13)C - nuclear magnetic resonance spectroscopy. The cytotoxicity was measured in RAW 264.7 cells and LLC-MK2. Leishmanicidal activity against L. infantum chagasi promastigotes was performed by the MTT colorimetric method and activity against amastigotes was assayed in mammalian cells using in situ ELISA method. Five compounds were identified, consisting of three triterpenes: cycloeucalenone, 31-norcyclolaudenone and 24-methylene-cicloartanol and a mixture of two sterols: beta-sitosterol and stigmasterol. With the exception of cycloeucalenone, all compounds showed statistically similar activity against promastigote to pentamidine. While, acting against amastigotes, excluding 31-norcyclolaudenone, other compounds showed activity similar to amphotericin B. All compounds showed low cytotoxicity in mammalian cells. This study partially confirms the use of Musa paradisiaca in folk medicine against leishmaniasis. Further in vivo studies are necessary to evaluate the efficacy. Copyright © 2014 Elsevier GmbH. All rights reserved.

The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

International Nuclear Information System (INIS)

Sueoka, Keigo; Yamazaki, Teruaki; Hiyama, Tetsuo; Nakamoto, Hitoshi

2009-01-01

An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H 2 O 2 . These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.

Alpha 1-blockers vs 5 alpha-reductase inhibitors in benign prostatic hyperplasia. A comparative review

DEFF Research Database (Denmark)

Andersen, J T

1995-01-01

During recent years, pharmacological treatment of symptomatic benign prostatic hyperplasia (BPH) has become the primary treatment choice for an increasing number of patients. The 2 principal drug classes employed are alpha 1-blockers and 5 alpha-reductase inhibitors. Current information from...... of patients who will respond well to alpha 1-blockers have yet to be identified, and data concerning the long term effects of these drugs are not yet available. 5 alpha-Reductase inhibitors have a slow onset of effect, but treatment leads to improvement in symptoms, reduction of the size of the prostate gland...... and improvement in objective parameters for bladder outflow obstruction. Approximately 30 to 50% of patients will respond to treatment with 5 alpha-reductase inhibitors. The definitive role of pharmacological treatment in symptomatic BPH remains to be established, although it seems that patients unfit...

Reconstitution of FMN-free NADPH-cytochrome P-450 reductase with a phosphorothioate analog of FMN: 31P NMR studies of the reconstituted protein

International Nuclear Information System (INIS)

Krum, D.P.; Otvos, J.D.; Calhoun, J.P.; Miziorko, H.M.; Masters, B.S.S.

1987-01-01

A phosphorothioate analog of FMN (FMNS) has been synthesized and shown to be completely competent in reconstituting the FMN-free form of NADPH-cytochrome P-450 reductase as evidenced by flavin determinations and cytochrome c reductase activity assays. The FMNS-reconstituted FMN-free reductase gives rise to an air-stable semiquinone, and the fluorescence of FMNS is quenched upon addition of FMN-free reductase. 31 P NMR spectra of the FMN-free reductase reveal only two resonances (-7.3 and -11.3 ppm), which are attributable to FAD. This result confirms the assignments of Otvos et al, and demonstrates unequivocally that there are no phosphate residues other than those of FMN and FAD attached to the steapsin-solubilized reductase. The addition of FMN to the FMN-free reductase resulted in the appearance of one additional resonance at 3.9 ppm. Addition of FMNS to the FMN-free reductase caused no change, surprisingly, in the 31 P NMR spectrum until Mn(II) was added, after which a peak centered at ∼ 45 ppm was observed. This unexpected result may be explained if the T 1 for the phosphate of FMNS is significantly longer than that of FMN, and suggests that the sulfur atom of FMNS may perturb the interaction of the phosphate with its protein environment. These results demonstrate the utility of phosphorothioate analogs as mechanistic probes for proteins containing nucleotide cofactors

Glutathione oxidation in response to intracellular H2O2: Key but overlapping roles for dehydroascorbate reductases.

Science.gov (United States)

Rahantaniaina, Marie-Sylviane; Li, Shengchun; Chatel-Innocenti, Gilles; Tuzet, Andrée; Mhamdi, Amna; Vanacker, Hélène; Noctor, Graham

2017-08-03

Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H 2 O 2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H 2 O 2 metabolism.

Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: Elucidation of the kinetic mechanism

International Nuclear Information System (INIS)

Levy, M.A.; Brandt, M.; Greway, A.T.

1990-01-01

A solubilized preparation of steroid 5 alpha-reductase from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase

Evaluation of the conserve flavin reductase gene from three ...

African Journals Online (AJOL)

STORAGESEVER

2009-12-15

Dec 15, 2009 ... means of PCR technique. The nucleic acid sequences of the PCR primers were designed using conserved nucleic acid sequences of the flavin reductase enzyme from. Rhodococcus sp. strain IGTS8. The oligonucleotide primers were as follows: 5'-GAA TTC ATG TCT GAC. AAG CCG AAT GCC-3' (forward) ...

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

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Hahn-Hägerdal Bärbel

2008-10-01

Full Text Available Abstract Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells-1 h-1 compared with 0.01 g (g cells-1 h-1

Binding of Fidarestat Stereoisomers with Aldose Reductase

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Dae-Sil Lee

2006-11-01

Full Text Available The stereospecificity in binding to aldose reductase (ALR2 of two fidarestat {6-fluoro-2',5'-dioxospiro[chroman-4,4'-imidazolidine]-2-carboxamide} stereoisomers [(2S,4Sand (2R,4S] has been investigated by means of molecular dynamics simulations using freeenergy integration techniques. The difference in the free energy of binding was found to be2.0 ± 1.7 kJ/mol in favour of the (2S,4S-form, in agreement with the experimentalinhibition data. The relative mobilities of the fidarestats complexed with ALR2 indicate alarger entropic penalty for hydrophobic binding of (2R,4S-fidarestat compared to (2S,4S-fidarestat, partially explaining its lower binding affinity. The two stereoisomers differmainly in the orientation of the carbamoyl moiety with respect to the active site and rotationof the bond joining the carbamoyl substituent to the ring. The detailed structural andenergetic insights obtained from out simulations allow for a better understanding of thefactors determining stereospecific inhibitor-ALR2 binding in the EPF charges model.

Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur.

Science.gov (United States)

Frederiksen, Trine-Maria; Finster, Kai

2003-06-01

The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.

Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

Science.gov (United States)

Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

2016-04-01

Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

Science.gov (United States)

Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

2004-10-01

In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

Suppression of cytochrome P450 reductase (POR) expression in hepatoma cells replicates the hepatic lipidosis observed in hepatic POR-null mice.

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Porter, Todd D; Banerjee, Subhashis; Stolarczyk, Elzbieta I; Zou, Ling

2011-06-01

Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis.

Investigating the effects of statins on cellular lipid metabolism using a yeast expression system.

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Agata Leszczynska

Full Text Available In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR, the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity with introduced wild-type or mutant form of human HMGR (hHMGR gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids.

Genetic variation of Aflatoxin B(1) aldehyde reductase genes (AFAR) in human tumour cells

DEFF Research Database (Denmark)

Praml, Christian; Schulz, Wolfgang; Claas, Andreas

2008-01-01

AFAR genes play a key role in the detoxification of the carcinogen Aflatoxin B(1) (AFB(1)). In the rat, Afar1 induction can prevent AFB(1)-induced liver cancer. It has been proposed that AFAR enzymes can metabolise endogenous diketones and dialdehydes that may be cytotoxic and/or genotoxic. Furth...... many aldo-keto reductases. This polarity change may have an effect on the proposed substrate binding amino acids nearby (Met(47), Tyr(48), Asp(50)). Further population analyses and functional studies of the nine variants detected may show if these variants are disease-related....

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

Science.gov (United States)

Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

Sterol regulatory element binding protein 2 overexpression is associated with reduced adipogenesis and ectopic fat accumulation in transgenic spontaneously hypertensive rats

Czech Academy of Sciences Publication Activity Database

Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šimáková, Miroslava; Šilhavý, Jan; Trnovská, J.; Kazdová, L.; Pravenec, Michal

2014-01-01

Roč. 63, č. 5 (2014), s. 587-590 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) LH12061 Institutional support: RVO:67985823 Keywords : sterol regulatory element binding protein 2 * transgenic * spontaneously hypertensive rat * lipid metabolism Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.293, year: 2014

Ubiquinol-cytochrome c reductase (Complex III) electrochemistry at multi-walled carbon nanotubes/Nafion modified glassy carbon electrodes

International Nuclear Information System (INIS)

Pelster, Lindsey N.; Minteer, Shelley D.

2012-01-01

Highlights: ► The electron transport chain is important to the understanding of metabolism in the living cell. ► Ubiquinol-cytochrome c reductase is a membrane bound complex of the electron transport chain (Complex III). ► The paper details the first bioelectrochemical characterization of ubiquinol-cytochrome c reductase at an electrode. - Abstract: Electron transport chain complexes are critical to metabolism in living cells. Ubiquinol-cytochrome c reductase (Complex III) is responsible for carrying electrons from ubiquinol to cytochrome c, but the complex has not been evaluated electrochemically. This work details the bioelectrochemistry of ubiquinol-cytochrome c reductase of the electron transport chain of tuber mitochondria. The characterization of the electrochemistry of this enzyme is investigated in carboxylated multi-walled carbon nanotube/tetrabutyl ammonium bromide-modified Nafion ® modified glassy carbon electrodes by cyclic voltammetry. Increasing concentrations of cytochrome c result in a catalytic response from the active enzyme in the nanotube sandwich. The experiments show that the enzyme followed Michaelis–Menten kinetics with a K m for the immobilized enzyme of 2.97 (±0.11) × 10 −6 M and a V max of 6.31 (±0.82) × 10 −3 μmol min −1 at the electrode, but the K m and V max values decreased compared to the free enzyme in solution, which is expected for immobilized redox proteins. This is the first evidence of ubiquinol-cytochrome c reductase bioelectrocatalysis.

Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene

DEFF Research Database (Denmark)

Edman, J C; Edman, U; Cao, Mi-Mi

1989-01-01

Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi...

The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from ...

African Journals Online (AJOL)

The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from Taxus media: Cloning, characterization and functional identification. Y Sun, M Chen, J Tang, W Liu, C Yang, Y Yang, X Lan, M Hsieh, Z Liao ...

Atomic Structure of Salutaridine Reductase from the Opium Poppy (Papaver somniferum)

Energy Technology Data Exchange (ETDEWEB)

Higashi, Yasuhiro; Kutchan, Toni M.; Smith, Thomas J. (Danforth)

2011-11-18

The opium poppy (Papaver somniferum L.) is one of the oldest known medicinal plants. In the biosynthetic pathway for morphine and codeine, salutaridine is reduced to salutaridinol by salutaridine reductase (SalR; EC 1.1.1.248) using NADPH as coenzyme. Here, we report the atomic structure of SalR to a resolution of {approx}1.9 {angstrom} in the presence of NADPH. The core structure is highly homologous to other members of the short chain dehydrogenase/reductase family. The major difference is that the nicotinamide moiety and the substrate-binding pocket are covered by a loop (residues 265-279), on top of which lies a large 'flap'-like domain (residues 105-140). This configuration appears to be a combination of the two common structural themes found in other members of the short chain dehydrogenase/reductase family. Previous modeling studies suggested that substrate inhibition is due to mutually exclusive productive and nonproductive modes of substrate binding in the active site. This model was tested via site-directed mutagenesis, and a number of these mutations abrogated substrate inhibition. However, the atomic structure of SalR shows that these mutated residues are instead distributed over a wide area of the enzyme, and many are not in the active site. To explain how residues distal to the active site might affect catalysis, a model is presented whereby SalR may undergo significant conformational changes during catalytic turnover.